The contribution of somatic mutations to metastasis of colorectal cancers is currently unknown. T... more The contribution of somatic mutations to metastasis of colorectal cancers is currently unknown. To find mutations involved in the colorectal cancer metastatic process, we performed deep mutational analysis of 676 genes in 107 stages II to IV primary colorectal cancer, of which half had metastasized. The mutation prevalence in the ephrin (EPH) family of tyrosine kinase receptors was 10-fold higher in primary tumors of metastatic colorectal than in nonmetastatic cases and preferentially occurred in stage III and IV tumors. Mutational analyses in situ confirmed expression of mutant EPH receptors. To enable functional studies of EPHB1 mutations, we demonstrated that DLD-1 colorectal cancer cells expressing EPHB1 form aggregates upon coculture with ephrin B1 expressing cells. When mutations in the fibronectin type III and kinase domains of EPHB1 were compared with wild-type EPHB1 in DLD-1 colorectal cancer cells, they decreased ephrin B1-induced compartmentalization. These observations p...
Recent molecular classifications of Colorectal Cancer (CRC) based on global gene expression profi... more Recent molecular classifications of Colorectal Cancer (CRC) based on global gene expression profiles have defined subtypes displaying resistance to therapy and poor prognosis. Upon evaluation of these classification systems we discovered that their predictive power arises from genes expressed by stromal cells rather than by epithelial tumor cells. Bioinformatic and immunohistochemical analyses identify stromal markers that associate robustly with disease relapse across the various classifications. Functional studies indicate that cancer-associated fibroblasts (CAFs) increase the frequency of tumor initiating cells, an effect that is dramatically enhanced by transforming growth factor beta (TGF-beta) signaling. Likewise, we find that all poor-prognosis CRC subtypes share a gene program induced by TGF-beta in tumor stromal cells. Using patient-derived tumor organoids and xenografts, we show that the use of TGF-beta signaling inhibitors to block the crosstalk between cancer cells and the microenvironment halts disease progression.
Background. Colorectal cancer (CRC) is the third most common cancer and remains a large unmet nee... more Background. Colorectal cancer (CRC) is the third most common cancer and remains a large unmet need. Dysregulation of Wnt and receptor tyrosine kinase (RTK) signalling pathways are important oncogenic driving events in CRC. Due to this dysregulation, Wnt target genes are expressed at higher levels in CRC particularly in tumor initiating cells. We previously performed an unbiased screen of bispecific antibodies (bAbs) targeting Wnt and RTK targets that resulted in the selection of MCLA-158. Methods. A cohort of 32 genetically and transcriptionally annotated patient-derived colorectal cancer and normal colon organoids were used to functionally characterize responses to antibodies based on morphological changes with high content 3D imaging. Binding affinity was measured by surface plasma resonance and cell based assays. The antibody binding epitopes were mapped by shotgun mutagenesis and FACS based screening. Ligand (R-spondin or EGF) blocking activity was measured in vitro by competition for ligand binding or functional inhibition of ligand dependent growth. In vivo activity was evaluated in xenograft models generated from organoids subcutaneously implanted into immunocompromised mice. Safety was evaluated via once weekly intravenous administration of MCLA-158 to cynomolgus monkeys for 4 weeks and monitoring for pathological changes. Results. MCLA-158, an ADCC enhanced common light chain IgG1 bispecific antibody, binds in domain III of EGFR and in the N-Cap/1st LRR of LGR5, both ligand binding regions, however, only EGF binding was blocked by MCLA-158. MCLA-158 demonstrated inhibitory activity in 74% of tumor organoids independent of KRAS mutational status but was not active on organoids of the cohort harboring both KRAS and PIK3CA mutations. MCLA-158 was significantly more active on organoids derived from tumors than from normal tissue in contrast to cetuximab, which demonstrated equivalent activity on both (range 20-100 fold, n=4). In vivo activity was evaluated against tumor organoids with different KRAS mutation status shown to be sensitive to MCLA-158 in vitro. In all cases, MCLA-158 significantly inhibited the growth of the tumor compared to both control and cetuximab treatment. Inhibitors of both the Wnt and EGFR pathways have shown significant toxicity in humans. An initial evaluation of MCLA-158 toxicity in cynomolgus monkeys did not demonstrate any pathological finding after repeated dosing at 25mg/kg. Conclusions. MCLA-158 demonstrates superior activity compared to reference antibodies in both in vitro and in vivo tumor organoid based assays regardless of KRAS status and was well tolerated in non-human primates. These preclinical data suggest MCLA-158 could benefit patients with metastatic CRC and warrant clinical evaluation. Citation Format: Rob Roovers, Bram Herpers, Mark James, Berina Eppink, Carme Cortina, David Maussang-Detaille, Ingrid Kolfschoten, Sylvia Boy, Marc van de Wetering, Wim De Lau, Robert Doornbos, Kuan Yan, Lucia Salinaro, Lex Bakker, john de Kruif, Hans Clevers, Robert Vries, Eduard Batlle, Leo Price, Mark Throsby. Preclinical evaluation of MCLA-158: A bispecific antibody targeting LGR5 and EGFR using patient-derived colon carcinoma organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 32. doi:10.1158/1538-7445.AM2017-32
The analysis of stem cell hierarchies in human cancers has been hampered by the impossibility of ... more The analysis of stem cell hierarchies in human cancers has been hampered by the impossibility of identifying or tracking tumor cell populations in an intact environment. To overcome this limitation, we devised a strategy based on editing the genomes of patientderived tumor organoids using CRISPR/Cas9 technology to integrate reporter cassettes at desired marker genes. As proof of concept, we engineered human colorectal cancer (CRC) organoids that carry EGFP and lineage-tracing cassettes knocked in the LGR5 locus. Analysis of LGR5-EGFP + cells isolated from organoid-derived xenografts demonstrated that these cells express a gene program similar to that of normal intestinal stem cells and that they propagate the disease to recipient mice very efficiently. Lineage-tracing experiments showed that LGR5 + CRC cells self-renew and generate progeny over long time periods that undergo differentiation toward mucosecreting-and absorptive-like phenotypes. These genetic experiments confirm that human CRCs adopt a hierarchical organization reminiscent of that of the normal colonic epithelium. The strategy described herein may have broad applications to study cell heterogeneity in human tumors.
Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations ... more Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations leading to the initiation of colorectal cancer (CRC). Here we screen for genes required for maintaining the tumour stem cell phenotype and identify the zinc-finger transcription factor GATA6 as a key regulator of the WNT and BMP pathways in CRC. GATA6 directly drives the expression of LGR5 in adenoma stem cells whereas it restricts BMP signalling to differentiated tumour cells. Genetic deletion of Gata6 from mouse colon adenomas increases the levels of BMP factors, which signal to block self-renewal of tumour stem cells. In human tumours, GATA6 competes with β-catenin/TCF4 for binding to a distal regulatory region of the BMP4 locus that has been linked to increased susceptibility to development of CRC. Hence, GATA6 creates an environment permissive for CRC initiation by lowering the threshold of BMP signalling required for tumour stem cell expansion.
Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations ... more Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations leading to the initiation of colorectal cancer (CRC). Here we screen for genes required for maintaining the tumour stem cell phenotype and identify the zinc-finger transcription factor GATA6 as a key regulator of the WNT and BMP pathways in CRC. GATA6 directly drives the expression of LGR5 in adenoma stem cells whereas it restricts BMP signalling to differentiated tumour cells. Genetic deletion of Gata6 from mouse colon adenomas increases the levels of BMP factors, which signal to block self-renewal of tumour stem cells. In human tumours, GATA6 competes with β-catenin/TCF4 for binding to a distal regulatory region of the BMP4 locus that has been linked to increased susceptibility to development of CRC. Hence, GATA6 creates an environment permissive for CRC initiation by lowering the threshold of BMP signalling required for tumour stem cell expansion.
Recent molecular classifications of colorectal cancer (CRC) based on global gene expression profi... more Recent molecular classifications of colorectal cancer (CRC) based on global gene expression profiles have defined subtypes
displaying resistance to therapy and poor prognosis. Upon evaluation of these classification systems, we discovered that
their predictive power arises from genes expressed by stromal cells rather than epithelial tumor cells. Bioinformatic and
immunohistochemical analyses identify stromal markers that associate robustly with disease relapse across the various
classifications. Functional studies indicate that cancer-associated fibroblasts (CAFs) increase the frequency of tumor-initiating
cells, an effect that is dramatically enhanced by transforming growth factor (TGF)-b signaling. Likewise, we find that all poorprognosis
CRC subtypes share a gene program induced by TGF-b in tumor stromal cells. Using patient-derived tumor organoids
and xenografts, we show that the use of TGF-b signaling inhibitors to block the cross-talk between cancer cells and the
microenvironment halts disease progression.
Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations ... more Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations leading to the initiation of colorectal cancer (CRC). Here we screen for genes required for maintaining the tumour stem cell phenotype and identify the zinc-finger transcription factor GATA6 as a key regulator of the WNT and BMP pathways in CRC. GATA6 directly drives the expression of LGR5 in adenoma stem cells whereas it restricts BMP signalling to differentiated tumour cells. Genetic deletion of Gata6 from mouse colon adenomas increases the levels of BMP factors, which signal to block self-renewal of tumour stem cells. In human tumours, GATA6 competes with β-catenin/TCF4 for binding to a distal regulatory region of the BMP4 locus that has been linked to increased susceptibility to development of CRC. Hence, GATA6 creates an environment permissive for CRC initiation by lowering the threshold of BMP signalling required for tumour stem cell expansion.
EphB-positive and ephrin-B-positive cells. Furthermore, genetic inhibition of ADAM10 activity in ... more EphB-positive and ephrin-B-positive cells. Furthermore, genetic inhibition of ADAM10 activity in the intestine of mice results in a lack of compartmentalization of Paneth cells within the crypt stem cell niche, a defect that phenocopies that of EphB3-null mice. These results provide important insights into the regulation of cell migration in the intestinal epithelium and may help in the understanding of the nature of the cell sorting process in other epithelial tissues where Eph-ephrin interactions play a central role.
The generation of human-induced pluripotent stem cells (iPS) has raised expectations for disease ... more The generation of human-induced pluripotent stem cells (iPS) has raised expectations for disease modeling, drug discovery, and cell therapy. Results: VP16-polycistronic vectors display enhanced reprogramming capacity. Conclusion: Primary tubular renal cells are amenable for iPSC reprogramming in the absence of oncogenes. Significance: Kidney-derived iPSCs provide a reliable cellular platform for the study of kidney pathology and drug discovery studies.
Since the initial discovery that OCT4, SOX2, KLF4, and c-MYC overexpression sufficed for the indu... more Since the initial discovery that OCT4, SOX2, KLF4, and c-MYC overexpression sufficed for the induction of pluripotency in somatic cells, methodologies replacing the original factors have enhanced our understanding of the reprogramming process. However, unlike in mouse, OCT4 has not been replaced successfully during reprogramming of human cells.
The genes encoding tyrosine kinase receptors EphB2 and EphB3 are b-catenin and Tcf4 target genes ... more The genes encoding tyrosine kinase receptors EphB2 and EphB3 are b-catenin and Tcf4 target genes in colorectal cancer (CRC) and in normal intestinal cells 1,2 . In the intestinal epithelium, EphB signaling controls the positioning of cell types along the crypt-villus axis 1 . In CRC, EphB activity suppresses tumor progression beyond the earliest stages 3,4 . Here we show that EphB receptors compartmentalize the expansion of CRC cells through a mechanism dependent on E-cadherin-mediated adhesion. We demonstrate that EphB-mediated compartmentalization restricts the spreading of EphBexpressing tumor cells into ephrin-B1-positive territories in vitro and in vivo. Our results indicate that CRC cells must silence EphB expression to avoid repulsive interactions imposed by normal ephrin-B1-expressing intestinal cells at the onset of tumorigenesis.
The contribution of somatic mutations to metastasis of colorectal cancers is currently unknown. T... more The contribution of somatic mutations to metastasis of colorectal cancers is currently unknown. To find mutations involved in the colorectal cancer metastatic process, we performed deep mutational analysis of 676 genes in 107 stages II to IV primary colorectal cancer, of which half had metastasized. The mutation prevalence in the ephrin (EPH) family of tyrosine kinase receptors was 10-fold higher in primary tumors of metastatic colorectal than in nonmetastatic cases and preferentially occurred in stage III and IV tumors. Mutational analyses in situ confirmed expression of mutant EPH receptors. To enable functional studies of EPHB1 mutations, we demonstrated that DLD-1 colorectal cancer cells expressing EPHB1 form aggregates upon coculture with ephrin B1 expressing cells. When mutations in the fibronectin type III and kinase domains of EPHB1 were compared with wild-type EPHB1 in DLD-1 colorectal cancer cells, they decreased ephrin B1-induced compartmentalization. These observations p...
Recent molecular classifications of Colorectal Cancer (CRC) based on global gene expression profi... more Recent molecular classifications of Colorectal Cancer (CRC) based on global gene expression profiles have defined subtypes displaying resistance to therapy and poor prognosis. Upon evaluation of these classification systems we discovered that their predictive power arises from genes expressed by stromal cells rather than by epithelial tumor cells. Bioinformatic and immunohistochemical analyses identify stromal markers that associate robustly with disease relapse across the various classifications. Functional studies indicate that cancer-associated fibroblasts (CAFs) increase the frequency of tumor initiating cells, an effect that is dramatically enhanced by transforming growth factor beta (TGF-beta) signaling. Likewise, we find that all poor-prognosis CRC subtypes share a gene program induced by TGF-beta in tumor stromal cells. Using patient-derived tumor organoids and xenografts, we show that the use of TGF-beta signaling inhibitors to block the crosstalk between cancer cells and the microenvironment halts disease progression.
Background. Colorectal cancer (CRC) is the third most common cancer and remains a large unmet nee... more Background. Colorectal cancer (CRC) is the third most common cancer and remains a large unmet need. Dysregulation of Wnt and receptor tyrosine kinase (RTK) signalling pathways are important oncogenic driving events in CRC. Due to this dysregulation, Wnt target genes are expressed at higher levels in CRC particularly in tumor initiating cells. We previously performed an unbiased screen of bispecific antibodies (bAbs) targeting Wnt and RTK targets that resulted in the selection of MCLA-158. Methods. A cohort of 32 genetically and transcriptionally annotated patient-derived colorectal cancer and normal colon organoids were used to functionally characterize responses to antibodies based on morphological changes with high content 3D imaging. Binding affinity was measured by surface plasma resonance and cell based assays. The antibody binding epitopes were mapped by shotgun mutagenesis and FACS based screening. Ligand (R-spondin or EGF) blocking activity was measured in vitro by competition for ligand binding or functional inhibition of ligand dependent growth. In vivo activity was evaluated in xenograft models generated from organoids subcutaneously implanted into immunocompromised mice. Safety was evaluated via once weekly intravenous administration of MCLA-158 to cynomolgus monkeys for 4 weeks and monitoring for pathological changes. Results. MCLA-158, an ADCC enhanced common light chain IgG1 bispecific antibody, binds in domain III of EGFR and in the N-Cap/1st LRR of LGR5, both ligand binding regions, however, only EGF binding was blocked by MCLA-158. MCLA-158 demonstrated inhibitory activity in 74% of tumor organoids independent of KRAS mutational status but was not active on organoids of the cohort harboring both KRAS and PIK3CA mutations. MCLA-158 was significantly more active on organoids derived from tumors than from normal tissue in contrast to cetuximab, which demonstrated equivalent activity on both (range 20-100 fold, n=4). In vivo activity was evaluated against tumor organoids with different KRAS mutation status shown to be sensitive to MCLA-158 in vitro. In all cases, MCLA-158 significantly inhibited the growth of the tumor compared to both control and cetuximab treatment. Inhibitors of both the Wnt and EGFR pathways have shown significant toxicity in humans. An initial evaluation of MCLA-158 toxicity in cynomolgus monkeys did not demonstrate any pathological finding after repeated dosing at 25mg/kg. Conclusions. MCLA-158 demonstrates superior activity compared to reference antibodies in both in vitro and in vivo tumor organoid based assays regardless of KRAS status and was well tolerated in non-human primates. These preclinical data suggest MCLA-158 could benefit patients with metastatic CRC and warrant clinical evaluation. Citation Format: Rob Roovers, Bram Herpers, Mark James, Berina Eppink, Carme Cortina, David Maussang-Detaille, Ingrid Kolfschoten, Sylvia Boy, Marc van de Wetering, Wim De Lau, Robert Doornbos, Kuan Yan, Lucia Salinaro, Lex Bakker, john de Kruif, Hans Clevers, Robert Vries, Eduard Batlle, Leo Price, Mark Throsby. Preclinical evaluation of MCLA-158: A bispecific antibody targeting LGR5 and EGFR using patient-derived colon carcinoma organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 32. doi:10.1158/1538-7445.AM2017-32
The analysis of stem cell hierarchies in human cancers has been hampered by the impossibility of ... more The analysis of stem cell hierarchies in human cancers has been hampered by the impossibility of identifying or tracking tumor cell populations in an intact environment. To overcome this limitation, we devised a strategy based on editing the genomes of patientderived tumor organoids using CRISPR/Cas9 technology to integrate reporter cassettes at desired marker genes. As proof of concept, we engineered human colorectal cancer (CRC) organoids that carry EGFP and lineage-tracing cassettes knocked in the LGR5 locus. Analysis of LGR5-EGFP + cells isolated from organoid-derived xenografts demonstrated that these cells express a gene program similar to that of normal intestinal stem cells and that they propagate the disease to recipient mice very efficiently. Lineage-tracing experiments showed that LGR5 + CRC cells self-renew and generate progeny over long time periods that undergo differentiation toward mucosecreting-and absorptive-like phenotypes. These genetic experiments confirm that human CRCs adopt a hierarchical organization reminiscent of that of the normal colonic epithelium. The strategy described herein may have broad applications to study cell heterogeneity in human tumors.
Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations ... more Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations leading to the initiation of colorectal cancer (CRC). Here we screen for genes required for maintaining the tumour stem cell phenotype and identify the zinc-finger transcription factor GATA6 as a key regulator of the WNT and BMP pathways in CRC. GATA6 directly drives the expression of LGR5 in adenoma stem cells whereas it restricts BMP signalling to differentiated tumour cells. Genetic deletion of Gata6 from mouse colon adenomas increases the levels of BMP factors, which signal to block self-renewal of tumour stem cells. In human tumours, GATA6 competes with β-catenin/TCF4 for binding to a distal regulatory region of the BMP4 locus that has been linked to increased susceptibility to development of CRC. Hence, GATA6 creates an environment permissive for CRC initiation by lowering the threshold of BMP signalling required for tumour stem cell expansion.
Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations ... more Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations leading to the initiation of colorectal cancer (CRC). Here we screen for genes required for maintaining the tumour stem cell phenotype and identify the zinc-finger transcription factor GATA6 as a key regulator of the WNT and BMP pathways in CRC. GATA6 directly drives the expression of LGR5 in adenoma stem cells whereas it restricts BMP signalling to differentiated tumour cells. Genetic deletion of Gata6 from mouse colon adenomas increases the levels of BMP factors, which signal to block self-renewal of tumour stem cells. In human tumours, GATA6 competes with β-catenin/TCF4 for binding to a distal regulatory region of the BMP4 locus that has been linked to increased susceptibility to development of CRC. Hence, GATA6 creates an environment permissive for CRC initiation by lowering the threshold of BMP signalling required for tumour stem cell expansion.
Recent molecular classifications of colorectal cancer (CRC) based on global gene expression profi... more Recent molecular classifications of colorectal cancer (CRC) based on global gene expression profiles have defined subtypes
displaying resistance to therapy and poor prognosis. Upon evaluation of these classification systems, we discovered that
their predictive power arises from genes expressed by stromal cells rather than epithelial tumor cells. Bioinformatic and
immunohistochemical analyses identify stromal markers that associate robustly with disease relapse across the various
classifications. Functional studies indicate that cancer-associated fibroblasts (CAFs) increase the frequency of tumor-initiating
cells, an effect that is dramatically enhanced by transforming growth factor (TGF)-b signaling. Likewise, we find that all poorprognosis
CRC subtypes share a gene program induced by TGF-b in tumor stromal cells. Using patient-derived tumor organoids
and xenografts, we show that the use of TGF-b signaling inhibitors to block the cross-talk between cancer cells and the
microenvironment halts disease progression.
Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations ... more Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations leading to the initiation of colorectal cancer (CRC). Here we screen for genes required for maintaining the tumour stem cell phenotype and identify the zinc-finger transcription factor GATA6 as a key regulator of the WNT and BMP pathways in CRC. GATA6 directly drives the expression of LGR5 in adenoma stem cells whereas it restricts BMP signalling to differentiated tumour cells. Genetic deletion of Gata6 from mouse colon adenomas increases the levels of BMP factors, which signal to block self-renewal of tumour stem cells. In human tumours, GATA6 competes with β-catenin/TCF4 for binding to a distal regulatory region of the BMP4 locus that has been linked to increased susceptibility to development of CRC. Hence, GATA6 creates an environment permissive for CRC initiation by lowering the threshold of BMP signalling required for tumour stem cell expansion.
EphB-positive and ephrin-B-positive cells. Furthermore, genetic inhibition of ADAM10 activity in ... more EphB-positive and ephrin-B-positive cells. Furthermore, genetic inhibition of ADAM10 activity in the intestine of mice results in a lack of compartmentalization of Paneth cells within the crypt stem cell niche, a defect that phenocopies that of EphB3-null mice. These results provide important insights into the regulation of cell migration in the intestinal epithelium and may help in the understanding of the nature of the cell sorting process in other epithelial tissues where Eph-ephrin interactions play a central role.
The generation of human-induced pluripotent stem cells (iPS) has raised expectations for disease ... more The generation of human-induced pluripotent stem cells (iPS) has raised expectations for disease modeling, drug discovery, and cell therapy. Results: VP16-polycistronic vectors display enhanced reprogramming capacity. Conclusion: Primary tubular renal cells are amenable for iPSC reprogramming in the absence of oncogenes. Significance: Kidney-derived iPSCs provide a reliable cellular platform for the study of kidney pathology and drug discovery studies.
Since the initial discovery that OCT4, SOX2, KLF4, and c-MYC overexpression sufficed for the indu... more Since the initial discovery that OCT4, SOX2, KLF4, and c-MYC overexpression sufficed for the induction of pluripotency in somatic cells, methodologies replacing the original factors have enhanced our understanding of the reprogramming process. However, unlike in mouse, OCT4 has not been replaced successfully during reprogramming of human cells.
The genes encoding tyrosine kinase receptors EphB2 and EphB3 are b-catenin and Tcf4 target genes ... more The genes encoding tyrosine kinase receptors EphB2 and EphB3 are b-catenin and Tcf4 target genes in colorectal cancer (CRC) and in normal intestinal cells 1,2 . In the intestinal epithelium, EphB signaling controls the positioning of cell types along the crypt-villus axis 1 . In CRC, EphB activity suppresses tumor progression beyond the earliest stages 3,4 . Here we show that EphB receptors compartmentalize the expansion of CRC cells through a mechanism dependent on E-cadherin-mediated adhesion. We demonstrate that EphB-mediated compartmentalization restricts the spreading of EphBexpressing tumor cells into ephrin-B1-positive territories in vitro and in vivo. Our results indicate that CRC cells must silence EphB expression to avoid repulsive interactions imposed by normal ephrin-B1-expressing intestinal cells at the onset of tumorigenesis.
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Papers by Carme Cortina
displaying resistance to therapy and poor prognosis. Upon evaluation of these classification systems, we discovered that
their predictive power arises from genes expressed by stromal cells rather than epithelial tumor cells. Bioinformatic and
immunohistochemical analyses identify stromal markers that associate robustly with disease relapse across the various
classifications. Functional studies indicate that cancer-associated fibroblasts (CAFs) increase the frequency of tumor-initiating
cells, an effect that is dramatically enhanced by transforming growth factor (TGF)-b signaling. Likewise, we find that all poorprognosis
CRC subtypes share a gene program induced by TGF-b in tumor stromal cells. Using patient-derived tumor organoids
and xenografts, we show that the use of TGF-b signaling inhibitors to block the cross-talk between cancer cells and the
microenvironment halts disease progression.
displaying resistance to therapy and poor prognosis. Upon evaluation of these classification systems, we discovered that
their predictive power arises from genes expressed by stromal cells rather than epithelial tumor cells. Bioinformatic and
immunohistochemical analyses identify stromal markers that associate robustly with disease relapse across the various
classifications. Functional studies indicate that cancer-associated fibroblasts (CAFs) increase the frequency of tumor-initiating
cells, an effect that is dramatically enhanced by transforming growth factor (TGF)-b signaling. Likewise, we find that all poorprognosis
CRC subtypes share a gene program induced by TGF-b in tumor stromal cells. Using patient-derived tumor organoids
and xenografts, we show that the use of TGF-b signaling inhibitors to block the cross-talk between cancer cells and the
microenvironment halts disease progression.