WO2023069652A1 - Fibroblastes associés au cancer répétant des phénotypes et des fonctions à travers des types de tumeurs et des espèces - Google Patents

Fibroblastes associés au cancer répétant des phénotypes et des fonctions à travers des types de tumeurs et des espèces Download PDF

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WO2023069652A1
WO2023069652A1 PCT/US2022/047323 US2022047323W WO2023069652A1 WO 2023069652 A1 WO2023069652 A1 WO 2023069652A1 US 2022047323 W US2022047323 W US 2022047323W WO 2023069652 A1 WO2023069652 A1 WO 2023069652A1
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caf
cell
cafs
cells
cancer
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Deshka S. FOSTER
Jeffrey A. NORTON
Howard Y. Chang
Michael Januszyk
Michael T. Longaker
Daniel James DELITTO
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Leland Stanford Junior University
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G01N33/5759—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds localised on the membrane of tumour or cancer cells

Definitions

  • Solid tumors contribute to over 80% of cancer deaths. For most solid tumor types, treatment involves a combination of chemotherapy, radiation, and surgery. Multi-pronged approaches using agents that target multiple cell types in the tumor microenvironment (TME) improve therapeutic efficacy.
  • TME tumor microenvironment
  • the TME is composed of transformed cancer cells along with nontransformed stromal cells including immune cells, endothelial cells, pericytes, and cancer associated fibroblasts (CAFs).
  • CAFs cancer associated fibroblasts
  • solid tumors are usually identified by their firm texture compared with benign parenchyma. This is primarily attributable to the contribution of CAFs, which secrete extracellular matrix (ECM) components (e.g., collagens), generating a dense fibrotic network (desmoplasia) within the tumor.
  • ECM extracellular matrix
  • the binding of tumor ECM proteins to integrin receptors on local cells supports their recruitment to the TME. Further, the ECM serves as a scaffold for cancer cell proliferation and invasion.
  • CAFs have yet to become a target in mainstream cancer therapy. This is primarily because CAF heterogeneity and CAF functions in solid tumors remain incompletely understood. It is clear that CAFs can facilitate tumor proliferation, invasion, and metastasis, and are often associated with a poor prognosis. However, when CAFs were depleted from PDAC-bearing mice, for example, survival was decreased secondary to increased tumor proliferation. Such paradoxical observations of CAF function support the need for further investigation of this complex cell population.
  • CAF heterogeneity is perhaps best characterized in terms of cell surface marker expression.
  • CAFs are known to express a set of canonical fibroblast-associated markers; however, there is no single marker that is shared by all CAFs.
  • CAF heterogeneity at the transcriptomic level is not as well characterized. It has yet to be determined whether individual CAF subpopulations are preserved across sites and species, although previous analyses hint at shared phenotypes. It has also yet to be determined if CAF subtype is related to location within a tumor. In order to effectively target CAFs therapeutically, it is becoming more and more pressing to elucidate common CAF phenotypes and their functional roles.
  • CAFs are largely derived from local tissue-resident cells that are activated in the context of neoplasia. Activation is thought to occur secondary to local factors such as hypoxia, oxidative stress, and growth factors released from other cells in the TME. For example, immune cells secrete cytokines triggering signaling pathways that lead to CAF activation.
  • Other CAF-activating molecules include TGFB, IL6, FAK, CXCL12, PDGF, and FGF.
  • Local tissue mechanics are known to influence CAF activation, heterogeneity, and functional roles within the tumor, including mechanotransduction signaling, such as the focal adhesion kinase (FAK) pathway. Additionally, changes in chromatin accessibility have also been implicated in CAF activation.
  • FAK focal adhesion kinase
  • Cancer-associated fibroblasts are non-transformed cells that provide a key component of the tumor microenvironment in solid tumors, including without limitation desmoplastic tumors of epithelial origin, which include breast cancer, pancreatic cancer, basal cell carcinoma, etc.
  • CAFs and CAF subtypes are identified herein as a therapeutic target to improve multi-modal chemotherapeutics and immunotherapies; and as a method of determining the effect of an agent on this population.
  • CAF heterogeneity is conserved across solid tumor types and species.
  • the identified subpopulations fall into three broad functional categories: steady statelike, mechanoresponsive, and immunomodulatory, each of which subset is shown to have characteristic modes of gene expression and chromatin accessibility.
  • Subsets may be referred to herein as clusters.
  • the clusters may be further characterized by spatial organization, differentiation, communication groups, and the like. Clusters are governed by specific changes in chromatin accessibility and are spatially-distinct.
  • Each cluster is comprised of distinct subtypes with functional relevance. It is shown herein that selective disruption of underlying immune or mechanical force signaling results in predictable shifts in cell subpopulation distributions with clinically meaningful results.
  • a method for profiling CAFs in a solid tumor which provides for clustering of the CAFs according to the number that are steady state-like, mechanoresponsive, and immunomodulatory.
  • the CAF profile is used in determining an appropriate therapeutic strategy.
  • CAFs from multiple distinct desmoplastic cancers are shown to cluster into these three sub-types. Characterization can be performed with scRNA-seq profiles, scATAC-seq, spatial transcriptomics, analysis of cell surface proteins by immunohistochemistry or comparable staining, flow cytometry, and the like as known in the art.
  • characterization is performed by simultaneous snRNA-seq and scATAC-seq, where the sequence data is clustered to determine CAF subsets.
  • Mechanoresponsive CAFs are characterized by elevated expression of genes associated with mechanotransduction and fibrosis. Spatially these CAFs are localized to regions relatively devoid of immune cells. Mechanoresponsive CAF subtypes, which deposit ECM proteins in the tumor stroma, can constrain cancer cell proliferation, invasion and tumor growth. In some embodiments, therapies that are sparing of FAK pathway signaling in these CAFs, either those that are protective at the primary tumor site or at sites of metastatic spread, are selected for treatment of highly desmoplastic and fibrotic tumors.
  • therapies that modulate FAK pathway signaling are co-administered with counter-agents at the primary tumor site and/or at sites of metastatic spread, to optimize the effect on these CAFs.
  • Pathways expressed in these subsets include mechanotransduction related pathways, including, for example, GAS, PERIOSTIN, THBS, and SPP1.
  • Specific genes include MGP, FOSB, GAS6, YAP, LRRC15, and ACTA2 and “Focal Adhesion” pathways, and fibrosis-related genes such as PDGFRB and vitamin-K-dependent matrix proteins such as POSTN.
  • Mgp, Gas6, and Postn are Vitamin K-dependent factors involved in activating and phosphorylating members of the FAK pathway including FAK itself, paxillin, and pp60-src.
  • Fosb is a member of the AP-1 axis, known to be involved in FAK pathway activation.
  • Steady state-like CAFs are characterized by PI16, DPP4, LY6A, and CD34, as well as BMP1, BMP3, WNT2, and DCN.
  • Immunomodulatory CAFs are characterized by elevated expression of genes associated with immune signaling, including IL1R1, MYD88, IL6ST, CXCL1, CXCL12, IL6, and CCL19 as well as other subtypes characterized by IFNy-associated genes, particularly those relevant to antigen presentation, including IFNGR1, B2M, CD74, H2-D1, and H2-K1. Spatially these immunomodulatory CAFs are largely in areas of the tumor where they can interact with immune cells in the paracrine regulation of immune cell migration and behavior within the tumor microenvironment. It is shown that in patients treated with an immune checkpoint inhibitor, e.g. an anti-PDL1 agent, the proportion of immuno-modulatory CAF subtypes are significantly diminished, whereas the proportion of mechano-responsive CAF subtypes are largely unaffected by this therapy.
  • an immune checkpoint inhibitor e.g. an anti-PDL1 agent
  • FIGS 1A-1 F Schematics illustrating that transformed cancer cells show distinct transcriptional and epigenomic programs in accordance with tumor type and tissue of origin (e.g., breast or colon) (left panel), whereas CAF subtypes may be conserved between tumor types (right panel).
  • B Schematic showing procedure for scRNA-seq of endogenous mouse breast tumor tissue.
  • C UMAP plot showing all tumor cells sequenced (left panel) in silica CAF selection UMAP plot demonstrating 6 distinct CAF clusters: dark grey circle indicates mechanoresponsive (MR) CAF clusters, medium grey circle indicates steady state-like (SSL) CAF clusters and light grey circle indicates immunomodulatory (IM) CAF clusters (right panel).
  • MR mechanoresponsive
  • SSL steady state-like
  • IM immunomodulatory
  • Violin plots showing differentially expressed genes characteristic of each mouse breast cancer CAF cluster. Colors correspond to the indicated CAF cluster in the UMAP above.
  • E UMAP plot showing the sample distribution by hash-oligo data for the endogenous mouse breast tumors sequenced. Mechanoresponsive, steady state-like, and immunomodulatory CAF cluster groups are circled as labelled in figure panel.
  • F PCA plot showing CytoTRACE analysis of MBr scRNA-seq CAF data. Mechanoresponsive and immunomodulatory CAF cluster groups are circled as labelled in figure panel.
  • FIGS. 2A-2I UMAP plot displaying mouse breast cancer scATAC-seq clusters (bottom panel) in silica fibroblast selection integration with MBr CAF scRNA-seq data (top panel).
  • B Anchor based label transfer of scRNA clusters into scATAC data (left panel) results in 4 integrated MBr CAF scRNA-ATAC clusters (right panel).
  • C Heatmap showing characteristic differential paired chromatin accessibility-gene expression for integrated MBr CAF scRNA-ATAC clusters.
  • UMAP plots showing integrated MBr CAF scRNA-ATAC data for key genes of interest characteristic of immunomodulatory and steady state-like CAF clusters (top row) and mechanoresponsive clusters (bottom row).
  • E Chromatin accessibility peaks for key genes of interest characteristic of immunomodulatory CAF clusters (Cxcl12 and 116) and mechanoresponsive (Gas6 and Thbsl).
  • F Schematic showing procedure for multiome sequencing of endogenous mouse breast tumor tissue. Data represent samples from nonmalignant breast tissue, early breast tumors and late breast tumors. Each time point included three biological replicates.
  • UMAP plot includes all cells that underwent multiome sequencing (left panel) in silica CAF selection UMAP plot demonstrating 7 distinct CAF clusters: dark grey circle indicates mechanoresponsive (MR) CAF clusters, medium grey circle indicates steady state-like (SSL) CAF clusters and light grey circle indicates immunomodulatory (IM) CAF clusters (right panel). Characteristic genes for each cluster are provided in figure labels. H. Violin plots indicate differentially expressed genes characteristic of each MBr multiome CAF cluster. I. Transcription factor (TF) motif analysis was performed on mouse breast tissue multiome data using the Signac and chromVAR packages. Feature plots indicate cells with highly accessible motifs for the indicated TF.
  • TF Transcription factor
  • FIGS 3A- 3L Schematic showing 10X Genomics Visium spatial transcriptomic analysis of endogenous mouse breast tumors.
  • B H&E staining of a representative section of an endogenous mouse breast tumor used for Visium analysis (left panel). Visium spatial transcriptomic sequencing panels showing cell type represented representation in the representative tumor section (right panels).
  • C Representative Visium sections showing CAF representation within normal breast parenchyma, early and late endogenous breast tumors.
  • E E.
  • FIGS 4A-4P Schematic showing scRNA-seq of human breast tumor tissue.
  • B UMAP plot showing all tumor cells sequenced (top panel) in silica CAF selection UMAP plot showing 6 human breast cancer (HBr) scRNA-seq CAF clusters. Clusters as labelled in figure panel (bottom panel).
  • C Heatmap showing characteristic differential gene expression for HBr scRNA-seq CAF clusters.
  • D Violin plots showing differentially expressed genes characteristic of each human breast cancer CAF cluster. Colors represent clusters visualized in B.
  • E UMAP plot showing hash- oligo data for the human breast tumors sequenced. Each human sample was stained with two hash-oligos for validation as indicated by the colored circles in the labels.
  • F Label transfer projection of mouse breast CAF scRNA-seq clusters onto human breast CAF scRNA-seq clusters. Clusters as labeled in figure panel. CAF clusters indicated with colors corresponding to MBr CAF scRNA-seq data.
  • G Schematic showing scRNA-seq of human breast and pancreas tumor tissue.
  • H In silica CAF selection. I. Heatmap showing characteristic differential gene expression for human breast scRNA-seq CAF clusters.
  • J UMAP plot showing 6 human breast cancer (HBr) scRNA-seq CAF clusters as in B (right panel).
  • K Heatmap showing characteristic differential gene expression for human pancreas scRNA-seq CAF clusters.
  • L UMAP plot showing 7 human pancreas cancer (HPanc) scRNA-seq CAF clusters.
  • M UMAP plot showing integrated human breast-pancreas CAF data (7 clusters). Green shading indicates mechano-responsive CAF clusters whereas gold shading indicates immuno-modulatory CAF clusters.
  • N UMAP plot showing integrated human breast-pancreas CAF data in terms of organ of origin.
  • O Violin plots showing selected highest-differentially expressed genes characteristic of integrated human breast-pancreas CAF clusters. One cluster is only represented by HPanc CAFs, as indicated (blue box).
  • UMAP plot showing eight transcriptionally-defined CAF clusters for mouse allograft breast cancer specimens from FAK-intact and Col1 a2CreERT2::FAKfl/fl mice (data represent three biological replicates per group, hash-oligos incorporated to distinguish biological replicates) (top panel).
  • UMAP plot grouped by CAF origin (FAK-intact in red vs Col1 a2CreERT2::FAKfl/fl in green).
  • Primary clusters (5 and 6) lost with fibroblast-specific FAK knockout are highlighted with orange circles (bottom panel).
  • Violin plots illustrating expression of genes of interest between CAFs (FAK-intact in red vs Col1 a2CreERT2::FAKfl/fl in green).
  • MR and IM CAF clusters of interest highlighted with grey boxes as labelled in the figure panel.
  • D UMAP plot showing transcriptionally-defined clusters for human BCC scRNA-seq CAF.
  • E UMAP plot from CAF-specific scRNA-seq data colored according to pre- vs post- immune checkpoint blockade for human BCC.
  • F Label transfer projection of mouse breast CAF scRNA-seq clusters on human BCC CAF scRNA-seq clusters.
  • Immunomodulatory CAF cluster 0 is highly represented in post-therapy CAFs, while SSL cluster 3 is almost entirely found in pre-therapy samples. These patterns are highlighted with yellow shading. Feature plot of LRRC15 expression shown in inset corresponds closely with MBr cluster 2 (MR1 ) correlation as anticipated. G. Schematic summarizing CAF subpopulation perturbations observed with FAK knockout in the context of mouse breast cancer compared with immunotherapy in the context of human BCC.
  • FIGS 6A-6D A. EnrichR Gene Ontology (GO) analysis plots showing top 10 features for each of the mouse breast (mBr) scRNA-seq clusters, order in terms of master clusters (MR - left, SSL - middle, IM - right).
  • B. CellChat analysis delineates Pattern 1 CAF subtypes (mechanoresponsive, MR; Clusters mBr RNA -2, 4, and 5) versus Pattern 2 CAF subtypes (steady state-like and immunomodulatory, SSL/IM; Clusters mBr RNA -0, 1 , and 3).
  • C. THBS signaling pathway network which characterizes Pattern 1 (MR) CAFs primarily.
  • D. MIF signaling pathway network which characterizes Pattern 2 (SSL/IM) CAFs primarily.
  • FIG. 7 Mouse endogenous breast cancer scATAC-seq transcription factor footprinting showing loci characteristic for CAF Clusters mBr ATAC -0 and 1 .
  • FIGS. 8A-8F A. UMAP plot showing endogenous mouse breast OAF multiome timecourse sequencing, color indicates timepoint (non-tumor breast parenchyma vs early tumor vs late tumor). Dark grey circle indicates mechanoresponsive (MR) OAF clusters whereas medium grey circle indicates steady state-like (SSL) OAF clusters and light grey circle indicates immunomodulatory (IM) OAF clusters.
  • B Percentage of OAF subclusters relative to timecourse as indicated at right.
  • Feature plots of mouse multiome data showing expression of example genes characterizing the OAF clusters. Relevant clusters highlighted with colors in each panel. Circled regions indicate CAF superclusters (MR, SSL, or IM) as indicated in the figure legend.
  • D Heatmap showing characteristic differential gene expression and paired chromatin accessibility for mouse breast CAF multiome clusters.
  • E Mouse multiome ATAC-seq data showing chromatin accessibility proximal to genes of interest defining the CAF sub-types.
  • F UMAP plot showing CytoTRACE analysis of mouse breast CAF multiome sequencing data. Circled regions indicate CAF superclusters (MR, SSL, or IM) as indicated in the figure legend.
  • FIGS. 9A-9B A. CellChat analysis of mouse breast multiome (mBr Mul,i ) CAFs wTSS wMeta. Relative to signaling pathway networks noted in left panels and cell/cluster type as noted in right panels. B. Label transfer projection of scRNA-seq data from Buecher et al., 2021 mapped onto mouse breast CAF multiome clusters. Top left panel copied from Buecher et al., 2021 as indicated in the figure panel. Circled regions indicate CAF super clusters (MR, SSL, or IM) as indicated in the figure legend.
  • FIGS. 10A-10D A. Representative Visium slices for non-cancer mouse breast parenchyma, early tumor, and late tumor with representation of mouse breast CAF multiome subtypes shown at right.
  • C Mouse breast CAF scRNA-seq UMAP plot (from Figure 1 ).
  • FIG. 1 1 Schematic (left), and confocal imaging data (right) of allograft tumors using syngenic pancreas cancer cells injected into the pancreas of aSMA CreEFtT2 ::Rosa26 VT2/GK3 (Rainbow) mice.
  • FIGS. 12A-12C A. Representative example of rainbow confocal imaging of a section of endogenous mouse breast tumor (aSMA CreEFtT2 ::Rosa26 VT2/GK3 ::MMTV-PyMT) (left panel) with example CAF clones in each of the primary rainbow colors (right panels), scale bars indicate 25um.
  • C Additional CODEX feature plots illustrating localization of cell type-specific markers (Ly6G, CD68, CD8) on the right side of the manifold.
  • FIGS. 13A-13G A. Example human breast tumors - gross tissue pictures showing examples of fibrotic (desmoplastic) tumors.
  • B Schematic showing human breast tumor multiome sequencing.
  • C UMAP plot showing all tumor cells sequenced (left panel) for human breast tumor multiome sequencing from three unique tumors sequenced independently in silica CAF selection UMAP plot showing 6 human breast (hBr) CAF multiome clusters.
  • D Heatmap showing characteristic differential gene expression and paired chromatin accessibility for human breast CAF multiome clusters.
  • E UMAP plot showing CytoTRACE analysis of human breast CAF multiome data.
  • F Human multiome AT AC data showing chromatin accessibility proximal to characteristic genes.
  • G Label transfer projection of human breast CAF multiome clusters onto mouse breast mult-iome CAF clusters. Relevant super clusters indicated with grey outlines as labeled in figure panel (numbers identify human multiome CAF clusters).
  • FIGS. 14A-14B A. Immunocytochemistry of freshly isolated human pancreas cancer CAFs shows strong aSMA and COL1 expression, DAPI (blue) for nuclear staining. Scale bars, 25um.
  • FIGS. 15A-15E A. FACS isolation of allograft mouse breast CAFs and phospho-flow analysis for FAK-WT (FAK +/+ , wildtype) control. B. FACS isolation of allograft mouse breast CAFs and phospho-flow analysis for FAK-knockout (FAK /_ , KO) mice. C. Phospho-flow cytometric analysis of CAFs isolated Rainbow mouse endogenous (MMTV-PyMT) breast tumors with genetic FAK knockout versus control for proteins of interest related to MR, SSL, and IM CAF subtypes (phospho (p)-FAK, MGP, CD26 (DPP4), as indicated). D.
  • the subject methods are used for prophylactic or therapeutic purposes.
  • the term "treating" is used to refer to both prevention of relapses, and treatment of pre-existing conditions.
  • the prevention of inflammatory disease can be accomplished by administration of the agent prior to development of a relapse.
  • the treatment of ongoing disease, where the treatment stabilizes or improves the clinical symptoms of the patient, is of particular interest.
  • the terms "subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human.
  • Mammalian species that provide samples for analysis include canines; felines; equines; bovines; ovines; etc. and primates, particularly humans.
  • Animal models, particularly small mammals, e.g. murine, lagomorpha, etc. can be used for experimental investigations.
  • the methods of the invention can be applied for veterinary purposes.
  • the term “theranosis” refers to the use of results obtained from a diagnostic method to direct the selection of, maintenance of, or changes to a therapeutic regimen, including but not limited to the choice of one or more therapeutic agents, changes in dose level, changes in dose schedule, changes in mode of administration, and changes in formulation. Diagnostic methods used to inform a theranosis can include any that provides information on the state of a disease, condition, or symptom.
  • diagnosis is used herein to refer to the identification of a molecular or pathological state, disease or condition in a subject, individual, or patient.
  • prognosis is used herein to refer to the prediction of the likelihood of death or disease progression, including recurrence, spread, and drug resistance, in a subject, individual, or patient.
  • prediction is used herein to refer to the act of foretelling or estimating, based on observation, experience, or scientific reasoning, the likelihood of a subject, individual, or patient experiencing a particular event or clinical outcome. In one example, a physician may attempt to predict the likelihood that a patient will survive.
  • therapeutic agent refers to a molecule or compound that confers some beneficial effect upon administration to a subject.
  • the beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.
  • treatment or “treating,” or “palliating” or “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit.
  • therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment.
  • the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.
  • an effective amount or “therapeutically effective amount” refers to the amount of an agent that is sufficient to effect beneficial or desired results.
  • the therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the specific dose will vary depending on the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.
  • Suitable conditions shall have a meaning dependent on the context in which this term is used. That is, when used in connection with an antibody, the term shall mean conditions that permit an antibody to bind to its corresponding antigen. When used in connection with contacting an agent to a cell, this term shall mean conditions that permit an agent capable of doing so to enter a cell and perform its intended function. In one embodiment, the term “suitable conditions” as used herein means physiological conditions.
  • biomarker refers to, without limitation, proteins, RNA or DNA sequences, together with their related metabolites, mutations, variants, polymorphisms, modifications, fragments, subunits, degradation products, elements, and other analytes or sample-derived measures. Markers can include expression levels of a gene. Markers can also include combinations of any one or more of the foregoing measurements, including temporal trends and differences. Broadly used, a marker can also refer to a CAF cell subset.
  • To “analyze” includes determining a set of values associated with a sample by measurement of a marker (such as, e.g., presence or absence of a marker or constituent expression levels) in the sample and comparing the measurement against measurement in a sample or set of samples from the same subject or other control subject(s).
  • a marker such as, e.g., presence or absence of a marker or constituent expression levels
  • the markers of the present teachings can be analyzed by any of various conventional methods known in the art.
  • To “analyze” can include performing a statistical analysis, e.g. normalization of data, determination of statistical significance, determination of statistical correlations, clustering algorithms, and the like.
  • sample in the context of the present teachings refers to any biological sample that is isolated from a subject, generally a tumor sample, e.g. a solid tumor sample, comprising fibroblasts. Samples can be obtained from a subject by means including but not limited to venipuncture, biopsy, needle aspirate, lavage, scraping, surgical incision, or intervention or other means known in the art.
  • a “dataset” is a set of numerical values resulting from evaluation of a sample (or population of samples) with respect to CAF profiling.
  • the values of the dataset can be obtained, for example, by experimentally obtaining measures from a sample and constructing a dataset from these measurements; or alternatively, by obtaining a dataset from a service provider such as a laboratory, or from a database or a server on which the dataset has been stored.
  • the term “obtaining a dataset associated with a sample” encompasses obtaining a set of data determined from at least one sample.
  • Obtaining a dataset encompasses obtaining a sample, and processing the sample to experimentally determine the data, e.g., sequencing, measuring antibody binding, etc.
  • the phrase also encompasses receiving a set of data, e.g., from a third party that has processed the sample to experimentally determine the dataset.
  • Measurement refers to determining the presence, absence, quantity, amount, or effective amount of a substance in a clinical or subject-derived sample, including the presence, absence, or concentration levels of such substances, and/or evaluating the values or categorization of a subject's clinical parameters based on a control, e.g. baseline levels of the marker.
  • a method for profiling CAFs in a solid tumor which provides for clustering of the CAFs according to the number that are steady state-like, mechanoresponsive, and immunomodulatory.
  • the CAF profile is used in determining an appropriate therapeutic strategy. Using gene expression data, CAFs from multiple distinct desmoplastic cancers are shown to cluster into these three sub-types. Characterization can be performed with scRNA-seq profiles, scATAC-seq, spatial transcriptomics, analysis of cell surface proteins by immunohistochemistry or comparable staining, flow cytometry, and the like as known in the art.
  • methods are provided for profiling cancer associated fibroblasts (CAFs) in an individual.
  • the CAF population is clustered according to single cell mRNA expression patterns, e.g. by scRNAseq, which may be further integrated with analysis of chromosome structure (scATAC-seq), multiomic analysis, and the like.
  • simultaneous snRNAseq and scATAC-seq is performed.
  • tumor tissue specimens are dissociated into single cells.
  • fibroblasts are isolated, e.g. by flow cytometry, which may utilize staining for one or more fibroblast lineage markers. Alternatively, selection is performed in silico to screen for fibroblasts.
  • the cell suspensions from individual samples may be labeled with hashtag oligonucleotide-labeled antibodies.
  • Single-cell RNA-seq (scRNA-seq) is then performed for droplet-based microfluidics. Droplets of the cellular suspensions, reverse transcription master mix, and partitioning oil are mixed, loaded onto a single cell chip, and processed.
  • cDNA is amplified, with cDNA size selected. cDNA is then fragmented, followed by end repair and A-tailing. Sequencing adaptors are ligated to the cDN. cDNA was amplified using a sample-specific index oligo as primer. Final libraries are sequenced. Single-cell AT AC sequencing (scATAC-seq) and multiome sequencing may also be performed, using isolated nuclei for scATAC-seq of genomic DNA.
  • scATAC-seq Single-cell AT AC sequencing
  • multiome sequencing may also be performed, using isolated nuclei for scATAC-seq of genomic DNA.
  • a method for profiling CAFs comprising: (a) obtaining a cell sample comprising cancer associated fibroblasts; (b) optionally labeling the cells with hashtag oligonucleotide-labeled antibodies; (s) separating droplets comprising single cells; (d) lysing the cells; (e) reverse transcribing cellular mRNA; (f) amplifying cDNA; (g) sequencing the cDNA; (h) importing the cDNA sequence to a computer system configured for accepting sequence data, and (i) performing clustering algorithms on the sequence data to obtain analysis of CAF subsets present in the sample.
  • step (d) sequencing genomic sequences present in isolated nuclei in the sample by ATAC methods; (f2) importing the genomic DNA sequence to a computer system configured for accepting sequence data, and (g2) performing clustering algorithms on the sequence data to obtain analysis of CAF subsets present in the sample.
  • Cell barcodes representative of quality cells may be differentiated from apoptotic cell barcodes or background RNA based on a threshold of having at least 200 unique transcripts profiled, less than 100,000 total transcripts, and less than 10% of their transcriptome of mitochondrial origin.
  • Unique molecular identifiers (UMIs) from each cell barcode can be retained for downstream analysis, and may be normalized. Principal components of the aggregated data can be used for uniform manifold approximation and projection (UMAP) analysis.
  • Cell annotations may be ascribed a reference dataset.
  • Cell-type marker lists may also generated using a ROC test to assign predictive power to each gene.
  • Data generated using a multiome platform can comprise an ATAC matrix computer step of barcode processing, read trimming, read alignment, duplicate marking, peak calling, and peakbarcode matrix generation using a reference genome; (2) a gene expression (GEX) Matrix Computer step of read trimming, genome alignment, transcriptome alignment, UMI correction, and UMI counting; and joint cell calling.
  • Downstream secondary analysis may comprise dimensionality reduction, clustering, peak annotation, transcription factor analysis, differential expression analysis, differential accessibility analysis, and feature linkage.
  • Mechanoresponsive (MR) CAFs are characterized by elevated expression of genes associated with mechanosensitive signaling mediators and ECM components. ATAF analysis reveals accessibility of canonical fibrosis-related TF motifs such as TGFB-related Smad2::Smad3. Specific upregulated genes include MGP, FOSB, GAS6, YAP, LRRC15, and ACTA2 and “Focal Adhesion” pathways, and fibrosis-related genes such as PDGFRB and vitamin-K-dependent matrix proteins such as POSTN. These genes are associated with multiple mechanotransduction pathways including “Focal Adhesion” and “Focal Adhesion-PI3K-akt- mTOR-signaling”. Mgp, Gas6, and Postn are Vitamin K-dependent factors involved in activating and phosphorylating members of the FAK pathway including FAK itself, paxillin, and pp60-src.
  • MR clusters correspond to clusters identified herein as RNA2 (ATAC2), RNA4 (ATAC4) or RNA5(ATAC5).
  • This subtype of CAFs is associated with upregulation of Col4A, PDGFRB, Mgb, Gas6, Postn, and Fosb. Accessibility is found proximal to FAK (Ptk2) as well as integrins and specific mechanoresponsive matrix proteins such as Postn, Thbsl , Timp2, Fsp1 , and Pdgfra.
  • ATAC-2 lineage-specific transcription factors included Fos, Fosb, Junb, and Jund, all of which are related to mechanotransduction, as well as Runxl and Runx2.
  • MR CAFs express, for example, COL6A, POSTN, COL4A, PDGFRB, MGB and FOSB.
  • Steady state-like CAFs are characterized by PI16, DPP4, LY6A, and CD34, as well as BMP1, BMP3, WNT2, and DCN.
  • ATAC By ATAC, there are elevated chromatin accessibility proximal to SSL genes, including Dpp4, Ly6a, and Cd34; and the GATA1 locus.
  • cytokine expression e.g. CXCL12, DCN, STAT1 , SPON2.
  • Immunomodulatory CAFs are characterized by elevated expression of genes associated with immune signaling, including IL1R1, MYD88, IL6ST, CXCL1, CXCL12, IL6, and CCL19 as well as other subtypes characterized by IFNy-associated genes, particularly those relevant to antigen presentation, including IFNGR1, B2M, CD74, H2-D1, and H2-K1.
  • the human cluster is shown to express FAP and TIMP2.
  • an apparatus for profiling CAFs comprising a sequencing apparatus for single cell sequencing, configured to accept one or both of cDNA sequence information and genomic sequence information.
  • the apparatus is operably joined to a computer system configured for accepting sequence data, and performing clustering algorithms.
  • a method of preparing a sample for profiling CAFs comprising: (a) obtaining a cell sample comprising cancer associated fibroblasts; (b) optionally labeling the cells with hashtag oligonucleotide-labeled antibodies; (c) separating droplets comprising single cells; (d) lysing the cells; (e) reverse transcribing cellular mRNA; (f) amplifying cDNA; (g) sequencing the cDNA; (h) importing the cDNA sequence to a computer system configured for accepting sequence data, and (i) performing clustering algorithms on the sequence data.
  • step (d) sequencing genomic sequences from isolated nuclei in the sample by AT AC methods; (f2) importing the genomic DNA sequence to a computer system configured for accepting sequence data, and (g2) performing clustering algorithms on the sequence data.
  • Also described herein is a method for assessing prognosis relating to CAF subsets, e.g. response to a FAK inhibitor, response to an immune checkpoint inhibitor, etc. comprising: obtaining a sequencing dataset of scRNAseq, and/or scATACseq associated with a sample obtained from the subject, wherein the dataset comprises quantitiative data for these sequences; in silico fibroblast selection, and clustering the CAF phenotypes in the data, to obtain an analysis of the CAF subtypes present in the sample.
  • the data may be analyzed by a computer processor.
  • the processor may be communicatively coupled to a storage memory for analyzing the data.
  • a computer-readable storage medium storing computer-executable program code, the program code comprising: program code for storing and analyzing data obtained by the methods of the invention.
  • the method further comprises selecting a treatment regimen for the patient based on the analysis. In an embodiment, the method further comprises determining a treatment course for the subject based on the analysis.
  • Treatment regimens of interest can include administration of immune checkpoint inhibitors, administration of FAK-targeted therapies, administration of additional cancer therapies not associated with FAK inhibition of ICI, decisionmaking for proceeding with extended hospital stay, medication, extended care at an intermediate facility, increased follow-up, and the like.
  • the information obtained from the CAF clustering can be used to (a) determine type and level of therapeutic intervention warranted and (b) to optimize the selection of therapeutic agents.
  • therapeutic regimens can be individualized and tailored according to characterization of the cancer, thereby providing a regimen that is individually appropriate.
  • the data can be subjected to non-supervised hierarchical clustering to reveal relationships among profiles.
  • hierarchical clustering can be performed, where the Pearson correlation is employed as the clustering metric.
  • One approach is to consider a patient disease dataset as a “learning sample” in a problem of “supervised learning”.
  • CART is a standard in applications to medicine (Singer (1999) Recursive Partitioning in the Health Sciences, Springer), which can be modified by transforming any qualitative features to quantitative features; sorting them by attained significance levels, evaluated by sample reuse methods for Hotelling's T 2 statistic; and suitable application of the lasso method.
  • Problems in prediction are turned into problems in regression without losing sight of prediction, indeed by making suitable use of the Gini criterion for classification in evaluating the quality of regressions.
  • methods of clustering single cell data are known in the art and can be applied to the single cell sequence data.
  • SNN shared nearest neighbor
  • samples are obtained as a series, e.g., a series of tumor samples obtained during the course of treatment
  • the samples can be obtained at fixed intervals, at intervals determined by the status of the most recent sample or samples or by other characteristics of the individual, or some combination thereof. It will be appreciated that an interval may not be exact, according to an individual's availability for sampling and the availability of sampling facilities, thus approximate intervals corresponding to an intended interval scheme are encompassed by the invention.
  • One or more cells or cell types, or samples containing one or more cells or cell types can be isolated from body samples.
  • the cells can be separated from body samples by red cell lysis, centrifugation, elutriation, density gradient separation, apheresis, affinity selection, panning, FACS, centrifugation with Hypaque, solid supports (magnetic beads, beads in columns, or other surfaces) with attached antibodies, etc.
  • red cell lysis centrifugation, elutriation, density gradient separation, apheresis, affinity selection, panning, FACS, centrifugation with Hypaque, solid supports (magnetic beads, beads in columns, or other surfaces) with attached antibodies, etc.
  • a relatively homogeneous population of cells can be obtained.
  • a heterogeneous cell population can be used.
  • cells are dispersed into a single cell suspension, e.g. by enzymatic digestion with a suitable protease, e.g. collagenase, dispase, etc; and the like.
  • An appropriate solution is used for dispersion or suspension.
  • Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hanks balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM.
  • Convenient buffers include HEPES1 phosphate buffers, lactate buffers, etc.
  • the cells can be fixed, e.g.
  • the methods of the invention include the use of liquid handling components.
  • the liquid handling systems can include robotic systems comprising any number of components.
  • any or all of the steps outlined herein can be automated; thus, for example, the systems can be completely or partially automated.
  • Fully robotic or microfluidic systems include automated liquid-, particle-, cell- and organism-handling including high throughput pipetting to perform all steps of screening applications.
  • This includes liquid, particle, cell, and organism manipulations such as aspiration, dispensing, mixing, diluting, washing, accurate volumetric transfers; retrieving, and discarding of pipet tips; and repetitive pipetting of identical volumes for multiple deliveries from a single sample aspiration.
  • These manipulations are cross-contamination- free liquid, particle, cell, and organism transfers.
  • This instrument performs automated replication of microplate samples to filters, membranes, and/or daughter plates, high-density transfers, full-plate serial dilutions, and high capacity operation.
  • platforms for multi-well plates, multi-tubes, holders, cartridges, minitubes, deep-well plates, microfuge tubes, cryovials, square well plates, filters, chips, optic fibers, beads, and other solid-phase matrices or platform with various volumes are accommodated on an upgradable modular platform for additional capacity.
  • This modular platform includes a variable speed orbital shaker, and multi-position work decks for source samples, sample and reagent dilution, assay plates, sample and reagent reservoirs, pipette tips, and an active wash station.
  • the methods of the invention include the use of a plate reader.
  • interchangeable pipet heads with single or multiple magnetic probes, affinity probes, or pipetters robotically manipulate the liquid, particles, cells, and organisms.
  • Multi-well or multi-tube magnetic separators or platforms manipulate liquid, particles, cells, and organisms in single or multiple sample formats.
  • the instrumentation will include a detector, which can be a wide variety of different detectors, depending on the labels and assay.
  • useful detectors include a microscope(s) with multiple channels of fluorescence; plate readers to provide fluorescent, ultraviolet and visible spectrophotometric detection with single and dual wavelength endpoint and kinetics capability, fluorescence resonance energy transfer (FRET), luminescence, quenching, two-photon excitation, and intensity redistribution; CCD cameras to capture and transform data and images into quantifiable formats; and a computer workstation.
  • the robotic apparatus includes a central processing unit which communicates with a memory and a set of input/output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus. Again, as outlined below, this can be in addition to or in place of the CPU for the multiplexing devices of the invention.
  • a central processing unit which communicates with a memory and a set of input/output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus.
  • input/output devices e.g., keyboard, mouse, monitor, printer, etc.
  • this can be in addition to or in place of the CPU for the multiplexing devices of the invention.
  • the general interaction between a central processing unit, a memory, input/output devices, and a bus is known in the art. Thus, a variety of different procedures, depending on the experiments to be run, are stored in the CPU memory.
  • DNA sequencing may be accomplished using high-throughput DNA sequencing techniques.
  • next generation and high-throughput sequencing include, for example, massively parallel signature sequencing, polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing with HiSeq, MiSeq, and other platforms, SOLiD sequencing, ion semiconductor sequencing (Ion Torrent), DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real time (SMRT) sequencing, MassARRAY®, and Digital Analysis of Selected Regions (DANSRTM).
  • BMC Genomics 13 (1 ): 341 ; Liu, Lin; Li, Yinhu; Li, Siliang; Hu, Ni; He, Yimin; Pong, Ray; Lin, Danni; Lu, Lihua; Law, Maggie (1 January 2012).
  • high throughput sequencing generates at least 1 ,000, at least 5,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 100,000 or at least 500,000 sequence reads per hour; with each read being at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120 or at least 150 bases per read.
  • Sequencing can be performed using nucleic acids described herein such as genomic DNA, cDNA derived from RNA transcripts or RNA as a template. Sequencing may comprise massively parallel sequencing.
  • high-throughput sequencing involves the use of technology available by Helicos BioSciences Corporation (Cambridge, Massachusetts) such as the Single Molecule Sequencing by Synthesis (SMSS) method.
  • high-throughput sequencing involves the use of technology available by 454 Lifesciences, Inc. (Branford, Connecticut) such as the Pico Titer Plate device which includes a fiber optic plate that transmits chemiluminescent signal generated by the sequencing reaction to be recorded by a CCD camera in the instrument. This use of fiber optics allows for the detection of a minimum of 20 million base pairs in 4.5 hours.
  • high-throughput sequencing is performed using Clonal Single Molecule Array (Solexa, Inc.) or sequencing-by-synthesis (SBS) utilizing reversible terminator chemistry.
  • Solexa, Inc. Clonal Single Molecule Array
  • SBS sequencing-by-synthesis
  • high-throughput sequencing of RNA or DNA can take place using AnyDot. chips (Genovoxx, Germany), which allows for the monitoring of biological processes (e.g., miRNA expression or allele variability (SNP detection).
  • the AnyDot-chips allow for 10x - 50x enhancement of nucleotide fluorescence signal detection.
  • Other high-throughput sequencing systems include those disclosed in Venter, J., et al. Science 16 February 2001 ; Adams, M. et al, Science 24 March 2000; and M. J, Levene, et al. Science 299:682-686, January 2003; as well as US Publication Application No. 20030044781 and 2006/0078937.
  • the growing of the nucleic acid strand and identifying the added nucleotide analog may be repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.
  • the methods disclosed herein may comprise amplification of DNA.
  • Amplification may comprise PCR-based amplification.
  • amplification may comprise nonPCR-based amplification.
  • Amplification of DNA may comprise using bead amplification followed by fiber optics detection as described in Marguiles et al. "Genome sequencing in microfabricated high- density pricolitre reactors", Nature, doi: 10.1038/nature03959; and well as in US Publication Application Nos. 200200 12930; 20030058629; 20030 1001 02; 20030 148344 ; 20040248 161 ; 200500795 10,20050 124022; and 20060078909.
  • Amplification of the nucleic acid may comprise use of one or more polymerases.
  • the polymerase may be a DNA polymerase.
  • the polymerase may be a RNA polymerase.
  • the polymerase may be a high fidelity polymerase.
  • the polymerase may be KAPA HiFi DNA polymerase.
  • the polymerase may be Phusion DNA polymerase.
  • Amplification may comprise 20 or fewer amplification cycles. Amplification may comprise 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, or 9 or fewer amplification cycles. Amplification may comprise 18 or fewer amplification cycles. Amplification may comprise 16 or fewer amplification cycles. Amplification may comprise 15 or fewer amplification cycles. Data Analysis
  • a clustering pattern can be generated from a CAF sample using any convenient protocol, for example as described below.
  • the readout can be a mean, average, median or the variance or other statistically or mathematically-derived value associated with the measurement.
  • the readout information can be further refined by direct comparison with the corresponding reference or control pattern.
  • the absolute values obtained for each clustering under identical conditions will display a variability that is inherent in live biological systems and also reflects the variability inherent between individuals.
  • a reference or control pattern can be a pattern that is obtained from a sample of a patient known to have a condition of interest, or known to be free of a condition of interest, e.g. breast cancer, pancreatic cancer, etc.
  • the obtained pattern is compared to a single reference/control profile to obtain information regarding the phenotype of the patient being assayed.
  • the obtained signature pattern is compared to two or more different reference/control profiles to obtain more in depth information regarding the phenotype of the patient.
  • the obtained signature pattern can be compared to a positive and negative reference profile to obtain confirmed information regarding whether the patient has the phenotype of interest.
  • the analysis and database storage can be implemented in hardware or software, or a combination of both.
  • a machine-readable storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a any of the datasets and data comparisons of this invention.
  • Such data can be used for a variety of purposes, such as patient monitoring, initial diagnosis, and the like.
  • the invention is implemented in computer programs executing on programmable computers, comprising a processor, a data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device.
  • Program code is applied to input data to perform the functions described above and generate output information.
  • the output information is applied to one or more output devices, in known fashion.
  • the computer can be, for example, a personal computer, microcomputer, or workstation of conventional design.
  • Each program is preferably implemented in a high level procedural or object oriented programming language to communicate with a computer system.
  • the programs can be implemented in assembly or machine language, if desired. In any case, the language can be a compiled or interpreted language.
  • Each such computer program is preferably stored on a storage media or device (e.g., ROM or magnetic diskette) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein.
  • the system can also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.
  • a variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention.
  • One format for an output means test datasets possessing varying degrees of similarity to a trusted profile. Such presentation provides a skilled artisan with a ranking of similarities and identifies the degree of similarity contained in the test pattern.
  • the clustering patterns and databases thereof can be provided in a variety of media to facilitate their use.
  • Media refers to a manufacture that contains the signature pattern information of the present invention.
  • the databases of the present invention can be recorded on computer readable media, e.g. any medium that can be read and accessed directly by a computer.
  • Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
  • magnetic storage media such as floppy discs, hard disc storage medium, and magnetic tape
  • optical storage media such as CD-ROM
  • electrical storage media such as RAM and ROM
  • hybrids of these categories such as magnetic/optical storage media.
  • Recorded refers to a process for storing information on computer readable medium, using any such methods as known in the art. Any convenient data storage structure can be chosen, based on the means used to access the stored information. A variety of data processor programs and formats can be used for storage, e.g. word processing text file, database format, etc.
  • kits for the classification, diagnosis, prognosis, theranosis, and/or prediction of an outcome in a subject may further comprise a software package for data analysis of the tumor CAF status, which may include reference profiles for comparison with the test profile and comparisons to other analyses as referred to above.
  • the kit may also include instructions for use for any of the above applications.
  • Kits provided by the invention may comprise one or more of the reagents described herein.
  • a kit may also include other reagents that are useful in the invention, such as modulators, fixatives, containers, plates, buffers, therapeutic agents, instructions, and the like.
  • Such kits may additionally comprise one or more therapeutic agents.
  • the kit may further comprise a software package for data analysis of the physiological status, which may include reference profiles for comparison with the test profile.
  • kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider. Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials. Kits described herein can be provided, marketed and/or promoted to health providers, including physicians, nurses, pharmacists, formulary officials, and the like. Kits may also, in some embodiments, be marketed directly to the consumer.
  • providing an evaluation of a subject for a classification, diagnosis, prognosis, theranosis, and/or prediction of an outcome includes generating a written report that includes the artisan’s assessment of the subject’s state of health i.e. a “diagnosis assessment”, of the subject’s prognosis, i.e. a “prognosis assessment”, and/or of possible treatment regimens, i.e. a “treatment assessment”.
  • a subject method may further include a step of generating or outputting a report providing the results of a diagnosis assessment, a prognosis assessment, or treatment assessment, which report can be provided in the form of an electronic medium (e.g., an electronic display on a computer monitor), or in the form of a tangible medium (e.g., a report printed on paper or other tangible medium).
  • an electronic medium e.g., an electronic display on a computer monitor
  • a tangible medium e.g., a report printed on paper or other tangible medium.
  • a “report,” as described herein, is an electronic or tangible document which includes report elements that provide information of interest relating to a diagnosis assessment, a prognosis assessment, and/or a treatment assessment and its results.
  • a subject report can be completely or partially electronically generated.
  • a subject report includes at least a diagnosis assessment, i.e. a diagnosis as to whether a subject will have a particular clinical responseduring pregnancy, and/or a suggested course of treatment to be followed.
  • a subject report can further include one or more of: 1) information regarding the testing facility; 2) service provider information; 3) subject data; 4) sample data; 5) an assessment report, which can include various information including: a) test data, where test data can include an analysis of cellular signaling responses to activation, b) reference values employed, if any.
  • the report may include information about the testing facility, which information is relevant to the hospital, clinic, or laboratory in which sample gathering and/or data generation was conducted.
  • This information can include one or more details relating to, for example, the name and location of the testing facility, the identity of the lab technician who conducted the assay and/or who entered the input data, the date and time the assay was conducted and/or analyzed, the location where the sample and/or result data is stored, the lot number of the reagents (e.g., kit, etc.) used in the assay, and the like.
  • Report fields with this information can generally be populated using information provided by the user.
  • the report may include information about the service provider, which may be located outside the healthcare facility at which the user is located, or within the healthcare facility. Examples of such information can include the name and location of the service provider, the name of the reviewer, and where necessary or desired the name of the individual who conducted sample gathering and/or data generation. Report fields with this information can generally be populated using data entered by the user, which can be selected from among pre-scripted selections (e.g., using a drop-down menu). Other service provider information in the report can include contact information for technical information about the result and/or about the interpretive report.
  • the report may include a subject data section, including subject medical history as well as administrative subject data (that is, data that are not essential to the diagnosis, prognosis, or treatment assessment) such as information to identify the subject (e.g., name, subject date of birth (DOB), gender, mailing and/or residence address, medical record number (MRN), room and/or bed number in a healthcare facility), insurance information, and the like), the name of the subject's physician or other health professional who ordered the susceptibility prediction and, if different from the ordering physician, the name of a staff physician who is responsible for the subject's care (e.g., primary care physician).
  • subject data that is, data that are not essential to the diagnosis, prognosis, or treatment assessment
  • information to identify the subject e.g., name, subject date of birth (DOB), gender, mailing and/or residence address, medical record number (MRN), room and/or bed number in a healthcare facility), insurance information, and the like
  • the report may include a sample data section, which may provide information about the biological sample analyzed, such as the source of biological sample obtained from the subject (e.g. blood, type of tissue, etc.), how the sample was handled (e.g. storage temperature, preparatory protocols) and the date and time collected. Report fields with this information can generally be populated using data entered by the user, some of which may be provided as prescripted selections (e.g., using a drop-down menu).
  • the source of biological sample obtained from the subject e.g. blood, type of tissue, etc.
  • how the sample was handled e.g. storage temperature, preparatory protocols
  • Report fields with this information can generally be populated using data entered by the user, some of which may be provided as prescripted selections (e.g., using a drop-down menu).
  • the report may include an assessment report section, which may include information generated after processing of the data as described herein.
  • the interpretive report can include, for example, results of the analysis, methods used to calculate the analysis, and interpretation, i.e. prognosis.
  • the assessment portion of the report can optionally also include a Recommendation(s).
  • the reports can include additional elements or modified elements.
  • the report can contain hyperlinks which point to internal or external databases which provide more detailed information about selected elements of the report.
  • the patient data element of the report can include a hyperlink to an electronic patient record, or a site for accessing such a patient record, which patient record is maintained in a confidential database. This latter embodiment may be of interest in an in-hospital system or in-clinic setting.
  • the report is recorded on a suitable physical medium, such as a computer readable medium, e.g., in a computer memory, zip drive, CD, DVD, etc.
  • the report can include all or some of the elements above, with the proviso that the report generally includes at least the elements sufficient to provide the analysis requested by the user (e.g., a diagnosis, a prognosis, or a prediction of responsiveness to a therapy).
  • the types of cancer that can be treated using the subject methods of the present invention include but are not limited to epithelial cancers, i.e. carcinomas, such as adenocarcinomas, squamous cell carcinomas, and the like, including, for example, adrenal cortical cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain cancers, breast cancer, cervical cancer, colon and rectum cancer, endometrial cancer, esophagus cancer, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, lung carcinoid tumors, male breast cancer, nasal cavity and paranasal cancer, nasopharyngeal cancer, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, salivary gland cancer, non-melanoma and melanoma skin
  • Dosage and frequency may vary depending on the half-life of the agent in the patient. It will be understood by one of skill in the art that such guidelines will be adjusted for the molecular weight of the active agent, the clearance from the blood, the mode of administration, and other pharmacokinetic parameters.
  • the dosage may also be varied for localized administration, e.g. intranasal, inhalation, etc., or for systemic administration, e.g. i.m., i.p., i.v., oral, and the like.
  • An active agent can be administered by any suitable means, including topical, oral, parenteral, intrapulmonary, intraperitoneal, and intranasal.
  • Parenteral infusions include intramuscular, intravenous (bolus or slow drip), intraarterial, intraperitoneal, intrathecal or subcutaneous administration.
  • An agent can be administered in any manner which is medically acceptable. This may include injections, by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intratumor, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophthalmic, rectal, or topical. Sustained release administration is also specifically included in the disclosure, by such means as depot injections or erodible implants.
  • an agent can be formulated with an a pharmaceutically acceptable carrier (one or more organic or inorganic ingredients, natural or synthetic, with which a subject agent is combined to facilitate its application).
  • a suitable carrier includes sterile saline although other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those of ordinary skill in the art.
  • An "effective amount” refers to that amount which is capable of ameliorating or delaying progression of the diseased, degenerative or damaged condition. An effective amount can be determined on an individual basis and will be based, in part, on consideration of the symptoms to be treated and results sought. An effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
  • compositions comprising a pharmaceutically acceptable excipient.
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • the compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • the diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • compounds which are "commercially available” may be obtained from commercial sources including but not limited to Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee Wl, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc.
  • the active agents of the invention and/or the compounds administered therewith are incorporated into a variety of formulations for therapeutic administration.
  • the agents are formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and are formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • administration of the active agents and/or other compounds can be achieved in various ways, usually by oral administration.
  • the active agents and/or other compounds may be systemic after administration or may be localized by virtue of the formulation, or by the use of an implant that acts to retain the active dose at the site of implantation.
  • the active agents and/or other compounds may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.
  • the agents may be combined, as previously described, to provide a cocktail of activities.
  • the following methods and excipients are exemplary and are not to be construed as limiting the invention.
  • the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • the agents can be used alone or prepared in a sustained release formulation involving liposomal, hydrogel, pegylated or other formulation.
  • Formulations are typically provided in a unit dosage form, where the term "unit dosage form,” refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active agent in an amount calculated sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active agent in an amount calculated sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for the unit dosage forms of the present invention depend on the particular complex employed and the effect to be achieved, and the pharmacodynamics associated with each complex in the host.
  • the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, are commercially available.
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are commercially available.
  • Any compound useful in the methods and compositions of the invention can be provided as a pharmaceutically acceptable base addition salt.
  • “Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid.
  • Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like.
  • Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like.
  • Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
  • the active agent may be administered in dosages of 0.01 mg to 500 mg /kg body weight per day, e.g. about 20 mg/day for an average person. Dosages will be appropriately adjusted for pediatric formulation.
  • compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
  • a carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group, and non-covalent associations.
  • Suitable covalent-bond carriers include proteins such as albumins, peptides, and polysaccharides such as aminodextran, each of which have multiple sites for the attachment of moieties.
  • the nature of the carrier can be either soluble or insoluble for purposes of the invention.
  • Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, his
  • the active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • the preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97- 119, 1997.
  • the agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
  • the pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • Toxicity of the active agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index.
  • the data obtained from these cell culture assays and animal studies can be used in further optimizing and/or defining a therapeutic dosage range and/or a sub-therapeutic dosage range (e.g., for use in humans). The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
  • a "therapeutically effective amount” refers to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder.
  • a therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., to delay or minimize the growth and spread of cancer.
  • a therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.
  • a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.
  • the term “dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
  • a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
  • a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses.
  • all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts.
  • a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).
  • each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.
  • Concomitant administration means administration of one or more components, such as engineered proteins and cells, known therapeutic agents, etc. at such time that the combination will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of components. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration.
  • a first prophylactic or therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent to a subject with a disorder.
  • Chemotherapy may include Abitrexate (Methotrexate Injection), Abraxane (Paclitaxel Injection), Adcetris (Brentuximab Vedotin Injection), Adriamycin (Doxorubicin), Adrucil Injection (5-FU (fluorouracil)), Afinitor (Everolimus) , Afinitor Disperz (Everolimus) , Alimta (PEMET EXED), Alkeran Injection (Melphalan Injection), Alkeran Tablets (Melphalan), Aredia (Pamidronate), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arzerra (Ofatumumab Injection), Avastin (Bevacizumab), Bexxar (Tositumomab), BiCNU (Carmustine), Blenoxane (Bleomycin), Bosulif (Bosutinib), Bus
  • Immune checkpoint inhibitors are known and used in the art, and may include, without limitation, Nimotuzumab and immune checkpoint inhibitors such as nivolumab, pembrolizumab/MK-3475, pidilizumab and AMP-224 targeting PD-1 ; and BMS-935559, MEDI4736, MPDL3280A and MSB0010718C targeting PD-L1 and those targeting CTLA-4 such as ipilimumab.
  • Nimotuzumab and immune checkpoint inhibitors such as nivolumab, pembrolizumab/MK-3475, pidilizumab and AMP-224 targeting PD-1 ; and BMS-935559, MEDI4736, MPDL3280A and MSB0010718C targeting PD-L1 and those targeting CTLA-4 such as ipilimumab.
  • Antibiotics e.g. antibiotics with the classes of aminoglycosides; carbapenems; and the like; penicillins, e.g. penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc. penicillins in combination with [3-lactamase inhibitors, cephalosporins, e.g.
  • vancomycin examples include, for example, oritavancin and dalbavancin (both lipoglycopeptides).
  • Telavancin is a semi-synthetic lipoglycopeptide derivative of vancomycin (approved by FDA in 2009).
  • vancomycin analogs are disclosed, for example, in WO 2015022335 A1 and Chen et al. (2003) PNAS 100(10): 5658- 5663, each herein specifically incorporated by reference.
  • Non-limiting examples of antibiotics include vancomycin, linezolid, azithromycin, daptomycin, colistin, eperezolid, fusidic acid, rifampicin, tetracyclin, fidaxomicin, clindamycin, lincomycin, rifalazil, and clarithromycin.
  • Radiotherapy means the use of radiation, usually X-rays, to treat illness. X-rays were discovered in 1895 and since then radiation has been used in medicine for diagnosis and investigation (X-rays) and treatment (radiotherapy). Radiotherapy may be from outside the body as external radiotherapy, using X-rays, cobalt irradiation, electrons, and more rarely other particles such as protons. It may also be from within the body as internal radiotherapy, which uses radioactive metals or liquids (isotopes) to treat cancer.
  • FAK and FAK modulating therapies are a biological process in which eukaryotic cells can sense extracellular mechanical stimuli such as ECM stiffness, interstitial fluid pressure, and stretch and translate them into biochemical signals.
  • FAK acts as a homeostatic mechanosensor which spontaneously self-adjusts its activation status to match the ECM stiffness.
  • the ECM stiffness is thought to be a risk factor for tumor progression. It has been reported that ECM stiffness promotes cells toward an EMT-like phenomenon with higher invasive and metastatic activity in cancer cells. In addition to proliferation and cell motility, ECM stiffness- mediated FAK activation may promote the fibrotic and inflammatory events associated with pathological conditions. Therapeutic targeting of FAK activity is proposed for counteracting the deleterious effect of dysregulated mechanotransduction-related diseases.
  • therapies that are sparing of FAK pathway signaling in MR CAFs are selected for treatment of highly desmoplastic and fibrotic tumors.
  • therapies that modulate FAK pathway signaling are co-administered with counter-agents at the primary tumor site and/or at sites of metastatic spread, to optimize the effect on these CAFs.
  • FAK is highly expressed in numerous cancers, including ovarian, breast, pancreatic, lung, melanoma, prostate, colorectal, glioblastoma, and esophageal cancers, and FAK regulates the diverse tumorigenic pathways, making it a drug target for cancer therapy.
  • FAK inhibitors include, for example, BI-853520 (IN10018); GSK2256098; NVP-TAC544; PF-431396; PF-573228; TAE226; VS-4718 (PND-1186); VS-6062 (PF-00562271 ); VS-6063 (Defactinib); 1 H-Pyrrolo(2,3- b) pyridine; APG-2449; BI-853520 (IN10018); etc.
  • Most FAK inhibitors are small molecules targeting enzymatic or kinase-dependent actions of FAK, such as ATP competitive kinase inhibitors targeting the ATP-binding site domain to block FAK kinase catalytic activity.
  • the other type of inhibitors blocking FAK kinase activity, are allosteric inhibitors, which target other sites of FAK and induce conformation change to block kinase activity. These molecules are regarded as aKI type of inhibitors.
  • BI-853520 (IN10018) is a highly selective and potent FAK inhibitor that blunts FAK autophosphorylation on Tyr397 with IC50 of 1 nM in PC3 prostate cancer cells and suppressed PC-3 prostate adenocarcinoma xenografts growth in nude mice.
  • NVP-TAC544 is a potent and ATP- competitive inhibitor of FAK to inhibit FAK activity.
  • PF-431396 is an FAK/PYK2 dual inhibitor with an IC50 of 2 and 11 nM for FAK and PYK2, respectively.
  • VS-4718 (PND- 1186) is a selective, highly effective and reversible FAK inhibitor with IC50 of 1 .5 nM in vitro and with IC50 of -100 nM in breast carcinoma cells.
  • 1 H-Pyrrolo(2,3-b) pyridine is an allosteric FAK kinase inhibitor by induction of DFG- loop conformation, binding to the hinge region of FAK and suppressing the kinas
  • Chloropyramine hydrochloride is a small-molecule inhibitor interrupting the protein-protein interaction of FAK with VEGFR-3.
  • the IC50 value of C4 in various cancer cell lines ranged from 1 to 20 pM.
  • Roslin 2 is an identified compound targeting the protein-protein interface between FAK andp53.
  • LD2-LD3-LD4 is a polypeptide capable of disrupting interactions between the paxillin LD motifs and the FAK FAT domain by competing interactions with FAK. The overexpression of LD2-LD3-LD4 prevents FAK localization at FAs, failing to transmit the integrin- mediated signaling.
  • Multiomic analysis reveals conservation of cancer associated fibroblast phenotypes across species and tissue of origin
  • CAFs Cancer associated fibroblasts
  • SSL steady state-like
  • MR mechanoresponsive
  • IM immunomodulatory
  • CAF single-cell gene expression and chromatin accessibility (validated using a paired multiome sequencing approach) in conjunction with spatial transcriptomics across multiple solid tumor types and species, with the aim of identifying commonalities that provide therapeutic targets.
  • SSL steady state-like
  • MR mechanoresponsive
  • IM immunomodulatory
  • fibroblast-specific disruption of mechanotransduction causes early changes in the expression of immune genes, depletes a subpopulation of MR CAFs, and leads to more aggressive tumor biology.
  • Modularity-optimized clustering of the resulting gene expression dataset identified seven transcriptionally-defined populations, which were ascribed as B-cells, T-cells, Macrophages, Granulocytes, Epithelial Cells, Endothelial Cells, and CAFs ( Figure 1 C - left panel).
  • CAFs were re-clustered to delineate six mouse breast (MBr) CAF subgroups (Clusters 0-5) ( Figure 1 C - right panel, Table 1 ).
  • Cluster mBrRNA- 1 demonstrated expression of Pi16, Dpp4, Dpt, and Cd34, closely aligning with recent descriptions of a stemlike phenotype in steady-state tissue.
  • mBrRNA-3 exhibited similarly high expression of Pi16 and Dpp4 with lower expression of extracellular matrix (ECM) constituents, particularly collagen, compared to mBrRNA-1 ( Figure 1 D).
  • ECM extracellular matrix
  • mBrRNA-3 was associated with higher cytokine and growth factor expression, such as Bmp1 , Bmp3, and Wnt2 ( Figure 1 D). Given the overlap in Pi16 and Dpp4 expression, we termed these groups steady state-like (SSL) fibroblasts.
  • SSL steady state-like
  • Clusters mBrRNA-2, 4, and 5 demonstrated markedly elevated expression of mechanosensitive signaling mediators and ECM components.
  • mBrRNA-2 CAFs selectively expressed genes associated with mechanotransduction and focal adhesion kinase (FAK) pathway signaling, including Mgp, Gas6, Postn, and Fosb ( Figure 1 D).
  • FAK focal adhesion kinase
  • MBr-4 was associated with Lrrc15 and Spp1 expression, both of which have been implicated in OAF pathophysiology, as well as similarly high expression of ECM components (Figure 1 D).
  • mBrRNA-5 also demonstrated the expression of fibrosis-associated factors, including Thbs2, Fsp1 , Col6a1 , and Cdh11 ( Figure 1 D).
  • mBrRNA-0 CAFs were characterized by contributions to multiple inflammatory pathways. Interestingly, this included elements of both type II interferon and IL1 signaling. I L1 -associated genes, such as 111 r1 , Myd88, Il6st, and Cxcll , were consistent with other descriptions of inflammatory CAFs. However, mBrRNA-0 was also enriched for I FNy-associated genes, particularly those relevant to antigen presentation, including Ifngrl , B2m, Cd74, H2-D1 , and H2- K1. Finally, mBrRNA-0 was tightly associated with Cxcll 2 expression, implicated as a regulator of cancer cell growth and local immune response (Figure 1 D).
  • IM immunomodulatory
  • Pattern 1 highlights the MR fibroblast subgroups (mBrRNA-2, 4, and 5) that communicate with other fibroblasts, and Pattern 2 was associated with the IM and SSL subgroups (mBrRNA-0, 1 , and 3) that communicate with immune cells (FIG. 6B - left panel).
  • Mechanotransduction-related pathways including GAS, PERIOSTIN, THBS, and SPP1 , drive Pattern 1 CAFs, whereas cytokine signaling such as CCL, CXCL, and IL1 drive Pattern 2 (FIG.
  • Pattern 1 CAF subgroup cells interact more with each other and with ECM proteins (FIG. 6C), while Pattern 2 CAF subgroup cell interactions favor immune cells, exemplified by MIF signaling (FIG. 6D).
  • FIG. 6C ECM proteins
  • FIG. 6D MIF signaling
  • the clusters with fewer cells did not map clearly to the scATAC-seq data, potentially due to the presence of fewer transcription factors at a gene expression level compared to the other clusters.
  • these groups may be underrepresented in our scATAC dataset.
  • mBrATAC-4 showed significantly elevated accessibility proximal to Gas6, Yap, and Acta2 in keeping with a myofibroblast phenotype ( Figure 2C-E).
  • mBrATAC-2 was associated with accessibility proximal to FAK (Ptk2) as well as integrins and specific mechanoresponsive matrix proteins such as Postn, Thbsl , Timp2, Fsp1 , and Pdgfra.
  • mBrATAC-2 lineage-specific transcription factors included Fos, Fosb, Junb, and Jund, all of which are related to mechanotransduction, as well as Runxl and Runx2.
  • Multiome analysis follows parallel scRNA-seq/scATAC-seq derived fibroblast clusters throughout tumor development.
  • simultaneous measurements were obtained using the Chromium Single Cell Multiome platform (1 Ox Genomics) for three conditions: non-tumor mammary parenchyma, early mouse breast tumors and late breast tumor (Figure 2F).
  • Initial clustering revealed six transcriptionally and epigenomically distinct cell types: T-cells, B- cells, myeloid cells, epithelial cells, endothelial cells, and CAFs (Figure 2G - left panel). Following in silico fibroblast selection, eight CAF clusters were identified (Figure 2G - right panel).
  • Fibroblasts from nonmalignant mammary tissue were largely confined to mouse breast multiome (mBrMulti-) clusters 0, 2, and 4 (FIG. 8A-B).
  • the majority of cells in the remaining clusters were obtained from early breast tumors, while late tumors showed comparatively more epithelial cells with proportionately fewer CAFs, although these CAFs were similarly distributed across the 8 clusters (FIG. 8A-B).
  • mBrMulti-2 demonstrated a striking resemblance to the stemlike Pi16 signature, consistent with its presence in steady-state, nonmalignant breast tissue (Figure 2G-H, S3A-E).
  • mBrMulti 0 included both CAFs and steady-state breast tissue fibroblasts, and these cells were enriched for the production of basement membrane components, such as laminin and type IV collagens, as well as uniquely high expression of the disintegrin and metalloprotease domain (ADAM) family, suggesting a prominent role in migration and remodeling.
  • basement membrane components such as laminin and type IV collagens
  • ADAM disintegrin and metalloprotease domain
  • mBrMulti-3 Progression to malignancy introduced mBrMulti-3, which was associated with mechanotransduction, ECM production, and less diverse transcriptional programming (FIG. 8F).
  • mBrMulti-3 also aligned with the Lrrc15 signature, previously found to be associated with malignancy.
  • mBrMulti-6 demonstrated the highest expression of Trpsl , recently implicated in benign fibroblast regenerative behavior.
  • mBrMulti-1 demonstrated overlapping gene expression patterns that placed these cells on a spectrum between mBrMulti-2 and mBrMulti-3, particularly from the perspective of ECM production. However, these cells demonstrated several unique attributes that distinguished them from either group.
  • mBrMulti-1 cells expressed genes associated with hyaluronic acid synthesis (Has1 , Has2), as well as the homing receptor for hyaluronic acid, Cd44.
  • mBrMulti-1 was also unique in the expression of IL1 responsive genes, such as Cxcl1 , II6, Ccl2, and 111 r1 , often associated with traditional inflammatory CAFs.
  • high expression of ECM genes clearly separated these fibroblasts from the more immunomodulatory clusters.
  • mBrMulti-4 expressed genes traditionally associated with lymphocyte signaling, such as Lef1 , Dock2, Il7r, and Ptpn22. However, these genes have also been described in fibroblasts during wound healing and tissue repair. Remarkably, transcriptomic profiles of mBrMulti-5 strongly suggested a role in antigen presentation, demonstrating expression of Cd74, H2-Aa, Cd83, and Cd86; although the capacity of CAFs to function as effective antigen presenting cells remains debated.
  • the IM cluster mBrRNA-0 appeared to incorporate subpopulations of primarily mBrMulti-4, 5, and 7. This may be due to the larger size of mBrRNA-0 cluster compared to the other clusters, as additional subpopulations may be captured by the multiome analysis. This phenomenon is exemplified by comparable IL1 - associated signaling from a subpopulation of cluster mBrMulti-1 and IFNy-associated signaling from cluster mBrMulti-5, both of which were characterized within mBrRNA-0.
  • Sox6 is a TF known to regulate differentiation in a variety of tissue types, and recent knockdown studies have suggested potential profibrotic and proinflammatory roles for Sox6.
  • aSMA is a well-established marker of myofibroblasts and is strongly expressed by this subgroup in our spatial transcriptomic analysis (Figure 3H).
  • the Rainbow background was applied to our endogenous breast cancer transgenic mouse model (MMTV-PyMT::aSMACreERT2::Rosa26VT2/GK3) (Figure 3I - far left panel).
  • MMTV-PyMT::aSMACreERT2::Rosa26VT2/GK3 Figure 3I - far left panel.
  • This general region of the protein UMAP was also predominated by markers such as CD8 and Ly6G, supporting an immune-like phenotype (FIG. 12B-C). Additionally, Ly6C marked cell populations in this region of the manifold that most likely correspond to macrophages, with evident co-expression of CD68 (FIG. 12C). Non-specific cell populations were further represented in the left side of the manifold, which was defined primarily by DAPI expression as well as non-expression of the other CODEX markers.
  • CODEX data were subsequently mapped to Rainbow fluorescence imaging using a per- specimen alignment mask, in which each specimen's CODEX expression was directly overlaid on its corresponding Rainbow image ( Figure 3J).
  • individual phenotypic filters were applied for SSL and IM CAFs versus MR CAFs to restrict fluorescent GFP, mCerulean, mCherry, and mOrange channels to the areas co-localized with the respective CAF population.
  • Distributions of clonal expression for IM and SSL versus MR CAFs were then analyzed using kernel density plots. Overall, both CAF populations demonstrated strong poly- clonality, with heterogeneous clonal expression across all rainbow colors.
  • SSL and IM fibroblasts exhibited Rainbow fluorescence associated with aSMA lineage induced mCerulean, mCherry, and mOrange clonal populations, confirming lineage plasticity among aSMA-positive myofibroblasts.
  • CAFs from hBrRNA-1 and 2 were also associated with elevated expression of fibrosis-related genes such as PDGFRB and vitamin-K-dependent matrix proteins such as POSTN.
  • hBrRNA-0 cells were characterized by elevated expression of SSL genes such as FAP (which shares significant homology with DPP4 expressed in the SSL MBr clusters) and TIMP2, and CAFs from hBrRNA-4 and 5 were associated with expression of cytokines and other signaling elements such as CXCL12, STAT1 , as well as DCN consistent with IM identity (Figure 4D).
  • the distribution of the clusters was not significantly different among tumors from three different patients despite their differences in age, tumor type, and stage (Figure 4E, Table 2).
  • CAFs within hBrRNA-hPaRNA-0, 2, and 4 demonstrated increased expression of vitamin K-dependent matrix proteins such POSTN and mechanotransduction signaling features such as FOSB ( Figure 40 - top right panels).
  • CAFs from hBrRNA- hPaRNA-1 and 5 exhibited elevated expression of immune-related genes, including CXCL12 and DCN ( Figure 40 - top left panels).
  • this subpopulation may represent a lineage of mesothelium-derived CAFs.
  • TWIST1 expression is also associated with epithelial mesenchymal transition (EMT) and CAF transdifferentiation.
  • EMT epithelial mesenchymal transition
  • FAP+ IM CAFs appeared separate from MR CAFs (aSMA/COL1 +) (FIG. 14A-B).
  • CAFs are often the most numerous cell type in solid tumors. While current therapies focus largely on transformed cancer cells and more recently on immune cells, CAFs represent an understudied therapeutic target with tremendous potential to improve cancer treatment. In order to effectively target CAFs therapeutically, a more comprehensive understanding of the heterogeneity and function of these cells is warranted.
  • DOCK2 contributes to pulmonary fibrosis by promoting lung fibroblast to myofibroblast transition. Am J Physiol Cell Physiol.
  • Twistl is a key regulator of cancer-associated fibroblasts. Cancer Res 75, 73-85.
  • Circ_WBSCR17 aggravates inflammatory responses and fibrosis by targeting miR-185-5p/SOX6 regulatory axis in high glucose-induced human kidney tubular cells. Life Sci 259, 118269.
  • rPanglaoDB an R package to download and merge labeled single-cell RNA-seq data from the PanglaoDB database. Bioinformatics.
  • Cell lines used for allograft tumor experiments included Py8119 and Pan02. All cells were maintained under sterile conditions in a humidified incubator under 5% CO2 at 37 e C. A phasecontrast microscope (Leica) was used to image cells. Cells were grown to 70-80% confluence in DMEM-Glutamax after minimal passage prior to implantation in the mammary fat pad or pancreas of mice. 10,000 cells were implanted per site under sterile conditions.
  • mice Animal Models. The following mouse strains were purchased from Jackson Laboratories: Black/6 (C57BL/6J), Actin-CreERT2 (Tg(CAG-cre/Esr1 )5Amc/J), MMTV-PyMT (FVB/N- Tg(MMTV-PyVT)634Mul/J), Col1 -CreERT2 (Tg(Col1 a2-cre/ERT,-ALPP)7Cpd/J), and FAKfl/fl (B6.129P2(FVB)-Ptk2tm1 ,1 Guan/J) mice. aSMA-CreERT2 were courtesy of Dr. Ivo Kalajzic, University of Connecticut.
  • Tissue processing and histology 4% paraformaldehyde (Electron Microscopy Sciences) was used to fix human and mouse tumor samples for 20 h at 4 °C. Standard protocol was used to embed the tissues into paraffin. For cryopreservation, specimens were protected from cryofreeze in 30% sucrose (Sigma) then until saturation at 4 °C following fixation, followed by OCT until saturation at 4 °C, and then embedded in OCT. Representative tissue specimens were stained with hematoxylin and eosin (H&E, Sigma-Aldrich), Picrosirius Red Stain (Abeam), or Masson’s trichrome (Sigma-Aldrich) per manufacturer’s protocols.
  • H&E hematoxylin and eosin
  • Picrosirius Red Stain Abeam
  • Masson Masson’s trichrome (Sigma-Aldrich) per manufacturer’s protocols.
  • ICC Immunocytochemistry
  • Fibroblasts were seeded onto coverslips coated with 1% Embryomax gelatin (EMD Millipore). The following day, cells were fixed with 4% with paraformaldehyde for 10 minutes, permeabilized with 0.5% Triton-X-100 (Sigma) for 15 minutes, and then incubated with 1X Powerblock (Biogenex) for 1 hr. Cells were then stained with primary antibody at 4 °C overnight. Cells were then washed with 0.1% Tween-20 (PBST; Sigma-Aldrich), stained with secondary antibody for 1 h at room temperature, and then mounted using Prolong Gold Antifade Mountant with DAPI (Life Technologies).
  • IF Immunofluorescence
  • Cryopreserved specimens were cryosectioned at 8um onto Superfrost Plus microscope slides (FisherSci). Samples were permeabilized with 0.5% Triton-X- 100 (Sigma) for 15 minutes, and then incubated with 1X Powerblock (Biogenex) for 1 hr. Primary antibodies were applied to tissue specimens for 1 h at room temperature, and then rinsed three times with 0.1% Tween-20 (Sigma). Secondary antibodies were applied for one hour at room temperature. The antibody incubation and washing steps were repeated if multiple proteins were stained for in one specimen section. Slides were then mounted using Prolong Gold Antifade Mountant with DAPI (Life Technologies).
  • Tissue Clearing optimized to preserve expression of endogenous fluorophores as previously described by our laboratory (Foster et al., 2019) was pursued on selected Rainbow whole mount and sectioned wound specimens.
  • tertbutanol (FisherSci) was buffered to a pH 9.5 with triethylamine (FisherSci).
  • Fixed tissue specimens were placed into increasing gradients of tert-butanol (33%, 66%, and 100%) at room temperature for 30 minutes each and then left in 100% tert-butanol overnight.
  • mice Animal models as previously described received 5 days of 200ml (20mg/ml in corn oil) tamoxifen induction for activation of Cre recombinase (per the protocol provided by Jackson Labs). Mouse tumors were monitored and measured several times per week. Prior to tumor harvest, mice were sacrificed.
  • the digest was quenched with quench media (DMEM (Gibco DMEM, ThermoFisher) with 15% FBS), then centrifuged at 500 x g for 5 min at 4 °C, resuspended in quench media, and filtered through 100, 70, and 40 pm cell strainers (Falcon cell strainer, ThermoFisher). Red blood cell lysis was performed using Hybri-Max (Sigma) per the manufacturer's protocol. Histopaque was performed using Histopaque-1119 (Sigma-Aldrich), per the manufacturer's protocol.
  • quench media DMEM (Gibco DMEM, ThermoFisher) with 15% FBS
  • red blood cell lysis was performed using Hybri-Max (Sigma) per the manufacturer's protocol.
  • Histopaque was performed using Histopaque-1119 (Sigma-Aldrich), per the manufacturer's protocol.
  • Antibodies against the following cell surface markers primarily or secondarily conjugated to the same fluorophore were used for exclusion of “lineage” cells in mouse and human specimens in order to isolate fibroblasts in an unbiased manner: CD45, CD31 , Ter119, Tie2, CD324, and CD326. This approach has been previously validated by our laboratory in other fibrotic pathologies.
  • phospho-specific flow-cytometry analysis a single-cell suspension was prepared using manual tissue dispersion rather than enzymatic digestion to preserve phosphorylated signal, and then prepared using the BD Biosciences Cytofix/CytopermTM kit according to manufacturer’s instructions. Phosphorylated protein analysis was conducted using the FACS Aria II system.
  • Tumor Tissue Processing Tumor tissue specimens from mouse or human specimens was minced on ice. The tissue was then digested for 3x 30 minutes in a 37nC water bath agitator in 0.5 mg/mL collagenase (collagenase type IV, ThermoFisher) digest buffer in Medium 199 (HyClone, GE Healthcare) consisting of 5% fetal bovine serum (Gibco FBS, ThermoFisher), DNase I (Worthington), Poloxamer 188 (Cat. P5556-100ML, Sigma), HEPES, and CaCI2.
  • collagenase collagenase type IV, ThermoFisher
  • Medium 199 HyClone, GE Healthcare
  • fetal bovine serum Gibco FBS, ThermoFisher
  • DNase I Worthington
  • Poloxamer 188 Cat. P5556-100ML, Sigma
  • HEPES and CaCI2.
  • the digest was quenched with quench media (DMEM (Gibco DMEM, ThermoFisher) with 15% FBS), then centrifuged at 300 x G for 5 minutes at 4°C, resuspended in quench media, and filtered through 100, 70, and 40Dm cell strainers (Falcon cell strainer, ThermoFisher). Red blood cell lysis was performed using Hybri-Max (Sigma) per the manufacturer's protocol. Cells were counted and re-suspended for further processing.
  • quench media DMEM (Gibco DMEM, ThermoFisher) with 15% FBS
  • cDNA was amplified using a sample-specific index oligo as primer, followed by another round of double-sided size selection using SpriSelect beads. Final libraries were analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control purposes. Libraries were sequenced on a HiSeq 4000 Illumina platform targeting 50,000 reads per cell.
  • Base calls were converted to reads using the Cell Ranger (10X Genomics; version 3.1 ) implementation mkfastq and then aligned against either the Cell Ranger mm10 reference genome or GRCh38 v3.0.0 human reference genome, or using Cell Ranger’s count function with SC3Pv3 chemistry and 5,000 expected cells per sample.
  • Cell barcodes representative of quality cells were differentiated from apoptotic cell barcodes or background RNA based on a threshold of having at least 200 unique transcripts profiled, less than 100,000 total transcripts, and less than 10% of their transcriptome of mitochondrial origin.
  • UMIs Unique molecular identifiers
  • scATAC-seq and Multiome Data Preparation Single-cell ATAC sequencing (scATAC- seq) and multiome sequencing were performed at the Stanford Genomics Facility (SGF) using the 10x Genomics platform. The relevant protocols were followed per manufacturer’s guidelines. Nuclei isolation was pursued as for scATAC-seq and multiome sequencing using 0.1x lysis buffer with 3-minute incubation.
  • scATAC-seq Data Processing and Analysis Raw base call (BCL) files were demultiplexed to fastq files using the 10x Genomics Cell Ranger tool cellranger-atac mkfastq. These files were then aligned to the mouse genome (mm10) using cellranger-atac count with default parameters. Downstream analysis of scATAC-seq data were performed using ArchR, a novel tool developed by our collaborators (Granja et al., 2020) (Granja et al., 2021 ).
  • Multiome Data Analysis Single cell ATAC-seq data were integrated with scRNA-seq data using ArchR’s unconstrained implementation of Seurat’s label transfer algorithm, as previously described (Foster et al., 2021).
  • Multiome Data Analysis Data generated using the 10X Multiome platform were processed using 10X’s cellranger-arc toolkit in Linux per the manufacturer’s instructions. This included (1 ) an ATAC matrix computer step of barcode processing, read trimming, read alignment, duplicate marking, peak calling, and peak-barcode matrix generation using either the mm10 mouse or GRCh38 human reference genome; (2) a gene expression (GEX) Matrix Computer step of read trimming, genome alignment, transcriptome alignment, UMI correction, and UMI counting; and joint cell calling. Downstream secondary analysis for ATAC and GEX included dimensionality reduction, clustering, peak annotation, transcription factor analysis, differential expression analysis, differential accessibility analysis, and feature linkage as described above using the Seurat, Signac, and ArchR toolkits.
  • Tumor tissues were cryosectioned at -20 degrees onto gene expression slides.
  • the Gene Expression Slide & Reagent kit was followed per protocol, and used to produce sequencing libraries.
  • the libraries were then sequenced using NextSeq (Illumina), and Bel files were demultiplexed.
  • Raw FASTQ files and histology images were processed by sample with the Space Ranger software, which uses STAR v.2.5.1 b (Dobin et al., 2013) for genome alignment, against the Cell Ranger mm10 reference genome.
  • pairwise cell-cell neighbor interactions were quantified by extracting a feature matrix, f, of the probability of cell type representation in each Visium spot.
  • a pairing score was calculated as the dot product of individual cell type vectors in f with the adjacency matrix (i.e. f1 -j -f2) using AdjacencyScore in R (Govek et al., 2021 ). Scores were averaged at the group-level, then subtracted between groups to quantify the magnitude of differential cell pairings in early tumor vs. native breast tissue and late tumor vs. early tumor.
  • Raw imaging data were processed for graphical rendering and cell segmentation using the standardized Akoya Biosciences pipeline, then exported to RStudio for computational analysis.

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Abstract

L'invention concerne des méthodes pour l'établissement de profils et l'analyse de fibroblastes associés au cancer (CAF) dans une tumeur solide par mise en oeuvre d'une analyse de séquence monocellulaire pour déterminer le nombre et la distribution de CAF qui sont de type stable, mécanosensibles et immunomodulateurs. Des sous-populations spécifiquement définies sont ici fournies comme moyen pour améliorer des chimiothérapies et des immunothérapies multimodales.
PCT/US2022/047323 2021-10-21 2022-10-20 Fibroblastes associés au cancer répétant des phénotypes et des fonctions à travers des types de tumeurs et des espèces Ceased WO2023069652A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119001096A (zh) * 2024-08-29 2024-11-22 宁夏医科大学总医院 一种小鼠成纤维细胞亚群的鉴定试剂组合物及其使用方法
WO2025064116A1 (fr) * 2023-09-18 2025-03-27 Arizona Board Of Regents On Behalf Of The University Of Arizona Compositions et méthodes pour le traitement d'affections fibrotiques et du cancer

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2021081491A2 (fr) * 2019-10-25 2021-04-29 Dana-Farber Cancer Institute, Inc. Systèmes organoïdes adaptés au patient pour l'étude du cancer

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WO2021081491A2 (fr) * 2019-10-25 2021-04-29 Dana-Farber Cancer Institute, Inc. Systèmes organoïdes adaptés au patient pour l'étude du cancer

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SEBASTIAN AIMY, HUM NICHOLAS R., MARTIN KELLY A., GILMORE SEAN F., PERAN IVANA, BYERS STEPHEN W., WHEELER ELIZABETH K., COLEMAN MA: "Single-Cell Transcriptomic Analysis of Tumor-Derived Fibroblasts and Normal Tissue-Resident Fibroblasts Reveals Fibroblast Heterogeneity in Breast Cancer", CANCERS, vol. 12, no. 5, pages 1307, XP093064563, DOI: 10.3390/cancers12051307 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025064116A1 (fr) * 2023-09-18 2025-03-27 Arizona Board Of Regents On Behalf Of The University Of Arizona Compositions et méthodes pour le traitement d'affections fibrotiques et du cancer
CN119001096A (zh) * 2024-08-29 2024-11-22 宁夏医科大学总医院 一种小鼠成纤维细胞亚群的鉴定试剂组合物及其使用方法

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