The Consensus Molecular Subtype 4 (CMS4) of colorectal cancer (CRC) has the worst prognosis and the highest frequency of hepatic metastases. It is characterized by abundant cancer-associated fibroblasts (CAFs) in the tumor microenvironment and active TGFβ signaling, but the molecular drivers of metastasis remain unclear. Here, we show that TGFβ signaling in CRC patient-derived CAFs from the primary tumor induces production of IL-6 family cytokines, particularly IL-6 and IL-11. These cytokines stimulate hepatocytes to express myeloid chemoattractants, including SAA1, through gp130-dependent JAK/STAT signaling. This promotes neutrophil recruitment to the liver, potentially creating a pro-metastatic niche. This IL-6 family-JAK/STAT stromal signaling axis is active in both a murine model of CMS4 as well as in patients with human CRC in vivo. Combined, our data reveal that TGFβ-driven CAF signaling actively contributes to the formation of a neutrophil-dependent, pre-metastatic hepatic niche in the metastatic phenotype of CMS4 CRC.

Oncolytic virus (OV) therapy is a promising treatment for various tumors. However, in pancreatic ductal adenocarcinoma (PDAC), the high abundance of cancer-associated fibroblasts (CAFs) can limit OV therapy efficacy by impairing viral spread and anti-tumor immunity. We have previously shown that oncolytic reovirus infection of CAFs depends on the expression of the reovirus entry receptor junctional adhesion molecule A (JAM-A), which is not or lowly expressed in most PDAC CAFs. We propose that increasing JAM-A expression on CAFs will boost viral spread in a tumor. However, there are currently no known regulators of JAM-A expression. Therefore, we performed a genome-wide CRISPR-Cas9 knockout screen to identify novel regulators of JAM-A expression. Ablation of the top negative regulator, zinc finger E-box binding homeobox 1 (ZEB1), in pancreatic fibroblasts led to strong JAM-A upregulation. We show that ZEB1 directly regulates JAM-A expression by binding to the enhancer-box (E-box) regions located within the JAM-A promoter. Importantly, ZEB1 ablation increased the sensitivity of fibroblasts to reovirus infection and subsequent cell death. Our work provides a novel overview of genes regulating JAM-A expression and provides a rational approach of combining ZEB1 inhibition with reovirus therapy to target both CAFs and tumor cells in stroma-rich tumors such as PDAC.

Abstract Background: Colorectal cancer (CRC) is characterized by a metastatic pattern that shows tropism towards the liver which is the major cause of CRC-related deaths. The current CRC classification, Consensus Molecular Subtypes (CMS), suggests an association between transforming growth factor (TGF)-β signaling, cancer-associated fibroblasts (CAFs), and the risk of liver metastasis. However, the key downstream effectors of TGFβ signaling in CAFs and their role in hepatic metastases remain poorly understood. Methods: The aim of this study was to investigate the TGF-β/CAF dependent cross talk with the hepatocytes in pre-metastatic niche formation. CAFs from 18 primary CRC tissues and 7 liver-metastasized CRC were stimulated with TGFβ1 and qPCR arrays/ELISAs were performed to identify differentially expressed genes. Results were cross-referenced with RNAseq expression data of CMS-classified CRC and in fresh samples from CRC patients. To investigate CAF-liver crosstalk hepatocytes were stimulated with the TGFβ primed CAF-derived cytokines and the effect on pro-inflammatory gene expression and neutrophil migration was determined. Subsequently, blockade of these pathways via chemical inhibition and CRISPR/Cas9 genetic ablation was performed to study the molecular mechanism involved in the induction of this hepatic pro-inflammatory program. Results: TGFβ signaling in primary CRC-derived CAFs leads to increased expression of different IL-6 cytokine family members, which was also reflected in human CRC samples. CAF-derived IL6 family members induce upregulation of myeloid chemoattractants, including SAA1, in hepatocytes and increased neutrophil-to-hepatocyte migration in vitro. Chemical blockade and genetic ablation of the IL-6 family cytokine signaling pathway showed the critical role of the gp130 co-receptor in the regulation of this inflammatory response in hepatocytes and the potential formation of a premetastatic niche. Conclusion: TGFβ signaling in CAFs actively contributes to the formation of a neutrophil-dependent, pre-metastatic hepatic niche, and this mechanism might play a role in CMS4 subtype CRC. Citation Format: Subinuer Abudukelimu, Tom J. Harryvan, Stef G. Janson, Imke Stouten, Els M. Verdegaal, Lukas JAC Hawinkels. TGFβ signaling in cancer-associated fibroblasts drives a hepatic gp130-dependent pro-metastatic inflammatory program in CMS4 colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1498.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy which shows unparalleled therapeutic resistance due to its genetic and cellular heterogeneity, dense stromal tissue, and immune‐suppressive tumour microenvironment. Oncolytic virotherapy has emerged as a new treatment modality which uses tumour‐specific viruses to eliminate cancerous cells. Non‐human primate adenoviruses of the human adenovirus B (HAdV‐B) species have demonstrated considerable lytic potential in human cancer cells as well as limited preexisting neutralizing immunity in humans. Previously, we have generated a new oncolytic derivative of the gorilla‐derived HAdV‐B AdV‐lumc007 named ‘GoraVir’. Here, we show that GoraVir displays oncolytic efficacy in pancreatic cancer cells and pancreatic‐cancer‐associated fibroblasts. Moreover, it retains its lytic potential in monoculture and co‐culture spheroids. In addition, we established the ubiquitously expressed complement receptor CD46 as the main entry receptor for GoraVir. Finally, a single intratumoural dose of GoraVir was shown to delay tumour growth in a BxPC‐3 xenograft model at 10 days post‐treatment. Collectively, these data demonstrate that the new gorilla‐derived oncolytic adenovirus is a potent oncolytic vector candidate that targets both pancreatic cancer cells and tumour‐adjacent stroma.

Abstract Pancreatic tumors display an abundance of cancer-associated fibroblasts (CAFs), which negatively affect prognosis and therapy response. Oncolytic virotherapy exploits viruses that preferentially lyse epithelial cancer cells as opposed to normal cells. Interestingly, we have observed that oncolytic reoviruses are able to infect and lyse CAFs, in addition to epithelial cancer cells. Targeting CAFs, in addition to cancer cells, could be advantageous to increase therapy effectiveness. It could serve as a conduit for viral spread and simultaneously disrupt the desmoplastic barrier around tumors, thereby also accelerating the influx of other therapeutics and immune cells. We previously found that the proneness of CAFs to lysis by oncolytic reovirus correlates with the cell surface expression levels of the reovirus entry receptor junction adhesion molecule A (JAM-A). However, most pancreatic CAFs do not express JAM-A. Therefore, a genome-wide CRISPR/Cas9 screen was employed to identify the genes regulating JAM-A expression on fibroblasts, which can subsequently be targeted to sensitize CAFs to reovirus. Pancreatic stellate cells with a moderate JAM-A expression level were transduced with a gRNA library making a knockout of one gene per cell. The highest and lowest JAM-A expressing cells were sorted and sequenced to identify the gRNAs that regulate JAM-A expression. Clonal CRISPR/Cas9-generated knockouts of a top negative regulator were generated and infected with reovirus, followed by cell viability assays to quantify their susceptibilities to reovirus-induced cell death. F11R, the gene encoding JAM-A, was identified as the top positive regulator of JAM-A expression in the CRISPR/Cas9 screen, verifying the validity of the screen. The top negative regulators identified were Fibroblast Growth Factor Receptor 1 (FGFR1) and Zinc finger E-box Binding homeobox 1 (Zeb1), thereby serving as potential therapeutic targets to sensitize CAFs to reovirus treatment. Using clonal Zeb1 knock-outs, Zeb1 was confirmed as a strong regulator of JAM-A expression. Zeb1 knockout in JAM-A negative pancreatic fibroblasts caused a robust upregulation of JAM-A and sensitized these inherently resistant fibroblasts to reovirus-directed cytolysis. Additionally, the clinically approved drug Mocetinostat, previously described to inhibit Zeb1, upregulated JAM-A expression on CAFs and increased cell lysis by reovirus. Altogether, our data show that Zeb1 is a strong negative regulator of JAM-A expression on fibroblasts and that Zeb1 inhibition can sensitize CAFs to reovirus-induced cell death. This research provides a rationale for combining Zeb1 inhibitory drugs with oncolytic reovirus treatment to improve killing of CAFs, which in turn could boost overall tumor eradication. Citation Format: Nicole Dam, Tom J. Harryvan, Bernhard Schmierer, Lukas J.A.C. Hawinkels, Vera Kemp. Zeb1 downregulation sensitizes pancreatic cancer-associated fibroblasts to killing by oncolytic reovirus through upregulation of the reovirus receptor junction adhesion molecule A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2025.

Endoglin is expressed by CAFs, does not correlate to stage-III CRC patient survival and TCGA database analysis

Quantification of pSmad western blots and endoglin konock down in endothelial cells

Tumor formation and angiogenesis after co-injection with WT/ENG KO MEFs

Endoglin is not expressed on NF in vivo, CAF marker protein expression, total Smad2 and endoglin expression on mCAFs

BACKGROUND & AIMS: Improving clinical management of early stage colorectal cancers (T1CRCs) requires a better understanding of their underlying biology. Accumulating evidence shows that cancer-associated fibroblasts (CAFs) are important determinants of tumor progression in advanced colorectal cancer (CRC), but their role in the initial stages of CRC tumorigenesis is unknown. Therefore, we investigated the contribution of T1CAFs to early CRC progression. METHODS: Primary T1CAFs and patient-matched normal fibroblasts (NFs) were isolated from endoscopic biopsy specimens of histologically confirmed T1CRCs and normal mucosa, respectively. The impact of T1CAFs and NFs on tumor behavior was studied using 3-dimensional co-culture systems with primary T1CRC organoids and extracellular matrix (ECM) remodeling assays. Whole-transcriptome sequencing and gene silencing were used to pinpoint mediators of T1CAF functions. RESULTS: In 3-dimensional multicellular cultures, matrix invasion of T1CRC organoids was induced by T1CAFs, but not by matched NFs. Enhanced T1CRC invasion was accompanied by T1CAF-induced ECM remodeling and up-regulation of CD44 in epithelial cells. RNA sequencing of 10 NF-T1CAF pairs revealed 404 differentially expressed genes, with significant enrichment for ECM-related pathways in T1CAFs. Cathepsin H, a cysteine-type protease that was specifically up-regulated in T1CAFs but not in fibroblasts from premalignant lesions or advanced CRCs, was identified as a key factor driving matrix remodeling by T1CAFs. Finally, we showed high abundance of cathepsin H-expressing T1CAFs at the invasive front of primary T1CRC sections. CONCLUSIONS: Already in the earliest stage of CRC, cancer cell invasion is promoted by CAFs via direct interactions with epithelial cancer cells and stage-specific, cathepsin H-dependent ECM remodeling. RNA sequencing data of the 10 NF-T1CAF pairs can be found under GEO accession number GSE200660.

Antibodies for immunohistochemistry, immunofluorescence and western blot.

Characterization of patient-derived NFs and CAFs

HEK293 invasion, BMP-9 expression by CRC cells and CAFs, invasion assays

Endoglin expression by CRC cells, HT29 proliferation and in vivo data HT29 only injections

Despite the fact that the role of endoglin on endothelial cells has been extensively described, its expression and biological role on (epithelial) cancer cells is still debatable. Especially its function on squamous cell carcinoma (SCC) cells is largely unknown. Therefore, we investigated SCC endoglin expression and function in three types of SCCs; head and neck (HNSCC), esophageal (ESCC) and vulvar (VSCC) cancers. Endoglin expression was evaluated in tumor specimens and 14 patient-derived cell lines. Next to being expressed on angiogenic endothelial cells, endoglin is selectively expressed by individual SCC cells in tumor nests. Patient derived HNSCC, ESCC and VSCC cell lines express varying levels of endoglin with high interpatient variation. To assess the function of endoglin in signaling of TGF-β ligands, endoglin was overexpressed or knocked out or the signaling was blocked using TRC105, an endoglin neutralizing antibody. The endoglin ligand BMP-9 induced strong phosphorylation of SMAD1 independent of expression of the type-I receptor ALK1. Interestingly, we observed that endoglin overexpression leads to strongly increased soluble endoglin levels, which in turn decreases BMP-9 signaling. On the functional level, endoglin, both in a ligand dependent and independent manner, did not influence proliferation or migration of the SCC cells. In conclusion, these data show endoglin expression on individual cells in the tumor nests in SCCs and a role for (soluble) endoglin in paracrine signaling, without directly affecting proliferation or migration in an autocrine manner.

Mutations in Bone Morphogenetic Protein (BMP) Receptor (BMPR)1A and SMAD4 are detected in 50% of juvenile polyposis syndrome (JPS) patients, who develop stroma-rich hamartomatous polyps. The established role of stromal cells in regulating BMP activity in the intestine implies a role for stromal cells in polyp development. We used conditional Cre-LoxP mice to investigate how specific loss of BMPR1A in endothelial cells, fibroblasts, or myofibroblasts/smooth muscle cells affects intestinal homeostasis. Selective loss of BMPR1A in fibroblasts causes severe histological changes in the intestines with a significant increase in stromal cell content and epithelial cell hyperproliferation, leading to numerous serrated polyps. This phenotype suggests that crucial changes occur in the fibroblast secretome that influences polyp development. Analyses of publicly available RNA expression databases identified CXCL12 as a potential candidate. RNAscope in situ hybridization showed an evident increase of Cxcl12-expressing fibroblasts. In vitro, stimulation of fibroblasts with BMPs resulted in downregulation of CXCL12, while inhibition of the BMP pathway resulted in gradual upregulation of CXCL12 over time. Moreover, neutralization of CXCL12 in vivo in the fibroblast-specific BMPR1A KO mice resulted in a significant decrease in polyp formation. Finally, in CRC patient specimens, mRNA-expression data showed that patients with high GREMLIN1 and CXCL12 expression had a significantly poorer overall survival. Significantly higher GREMLIN1, NOGGIN, and CXCL12 expression were detected in the Consensus Molecular Subtype 4 (CMS4) colorectal cancers, which are thought to arise from serrated polyps. Taken together, these data imply that fibroblast-specific BMP signaling-CXCL12 interaction could have a role in the etiology of serrated polyp formation.

BACKGROUND:Cross-presentation of exogenous antigens in HLA-class I molecules by professional antigen presenting cells (APCs) is crucial for CD8+ T cell function. Recent murine studies show that several non-professional APCs, including cancer-associated fibroblasts (CAFs) also possess this capacity. Whether human CAFs are able to cross-present exogenous antigen, which molecular pathways are involved in this process and how this ultimately affects tumor-specific CD8+ T cell function is unknown. METHODS:In this study, we investigated the ability of human colorectal cancer (CRC)-derived CAFs to cross-present neoantigen-derived synthetic long peptides (SLPs), corresponding to tumor-derived mutant peptides, and how this affects tumor-specific T-cell function. Processing of the SLP was studied by targeting components of the cross-presentation machinery through CRISPR/Cas9 and siRNA-mediated genetic ablation to identify the key molecules involved in fibroblast-mediated cross-presentation. Multispectral flow cytometry and killing assays were performed to study the effect of fibroblast cross-presentation on T cell function. RESULTS:Here, we show that human CRC-derived CAFs display an enhanced capacity to cross-present neoantigen-derived SLPs when compared with normal colonic fibroblasts. Cross-presentation of antigens by fibroblasts involved the lysosomal protease cathepsin S. Cathepsin S expression by CAFs was detected in situ in human CRC tissue, was upregulated in ex vivo cultured CRC-derived CAFs and showed increased expression in normal fibroblasts after exposure to CRC-conditioned medium. Cognate interaction between CD8+ T cells and cross-presenting CAFs suppressed T cell function, reflected by decreased cytotoxicity, reduced activation (CD137) and increased exhaustion (TIM3, LAG3 and CD39) marker expression. CONCLUSION:These data indicate that CAFs may directly suppress tumor-specific T cell function in an antigen-dependent fashion in human CRC.

Pancreatic ductal adenocarcinoma (PDAC) patients often present with irresectable or metastasized disease, resulting in an overall 5-year survival rate of 11%.1Siegel R.L. et al.CA Cancer J Clin. 2022; 72: 7-33Crossref PubMed Scopus (2437) Google Scholar To improve current therapeutic modalities, modeling the full complexity of PDAC in a personalized fashion is essential. Organoid technology enables close-to-patient models but currently lacks the typical desmoplastic tumor microenvironment.2Tuveson D. et al.Science. 2019; 364: 952-955Crossref PubMed Scopus (353) Google Scholar,3Lau H.C.H. et al.Nat Rev Gastroenterol Hepatol. 2020; 17: 203-222Crossref PubMed Scopus (73) Google Scholar Up to 80% of PDAC consists of stromal cells, predominantly cancer-associated fibroblasts (CAFs). CAF populations are heterogenous and crucially involved in tumor growth, chemoresistance, immune evasion, and metastasis.4Kobayashi H. et al.Nat Rev Gastroenterol Hepatol. 2019; 16: 282-295Crossref PubMed Scopus (233) Google Scholar, 5Helms E. et al.Cancer Discov. 2020; 10: 648-656Crossref PubMed Scopus (126) Google Scholar, 6Biffi G. et al.Physiol Rev. 2021; 101: 147-176Crossref PubMed Scopus (206) Google Scholar To provide basis for improved therapy, it is essential to integrate CAFs and recapitulate desmoplasia in organoid-based models.2Tuveson D. et al.Science. 2019; 364: 952-955Crossref PubMed Scopus (353) Google Scholar,3Lau H.C.H. et al.Nat Rev Gastroenterol Hepatol. 2020; 17: 203-222Crossref PubMed Scopus (73) Google Scholar In this study, we established a novel human, multicellular mini-tumor (MT) model containing both pancreatic tumor organoids and patient-derived CAFs from tumor resection material and fine-needle biopsies. We induced formation of MTs with heterogenous desmoplastic PDAC characteristics by modulating transforming growth factor β (TGFβ) and platelet-derived growth factor β signaling. MTs contain different archetypical CAF subsets, recapitulating patient CAF heterogeneity of human PDAC. Therefore, our model provides an important novel platform for both basic and preclinical research in a patient-specific fashion and can serve as a gateway for establishment of MTs from other stroma-dense gastrointestinal tumors. To ensure pathology resemblance of close-to-patient models, we designed a multiplex immunofluorescent panel to identify 2 major CAF subsets.5Helms E. et al.Cancer Discov. 2020; 10: 648-656Crossref PubMed Scopus (126) Google Scholar,6Biffi G. et al.Physiol Rev. 2021; 101: 147-176Crossref PubMed Scopus (206) Google Scholar Myofibroblastic CAFs (MyCAFs) and inflammatory CAFs were defined as being platelet-derived growth factor receptor (PDGFR)-β+/α− smooth muscle actin (αSMA)+/pSMAD2+ and PDGFRα+/αSMA−/pSMAD2−, respectively. The abundant presence of PDGFRβ+ CAFs was confirmed in all specimens investigated (n = 10), while PDGFRα+ CAFs were limited (Figure 1A, Supplemental Methods). To test if desmoplasia with heterogeneous CAF subsets can be recapitulated in an MT model, we cocultured pancreatic tumor-derived organoids (PDOs) and the pancreatic stellate cell line hPS17Froeling F.E.M. et al.Am J Pathol. 2009; 175: 636-648Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar that expresses canonical fibroblast and pancreatic stellate cell markers (Figure A1A–C). PDOs and hPS1 cells were mixed in a 1:8 ratio, reflecting the in vivo tumor-stroma ratio and aggregated in 3D Matrigel domes (Figure A1D and E). To stimulate CAF proliferation (PDGFRβ signaling) and differentiation (TGFβ signaling, a central driver of MyCAF differentiation5Helms E. et al.Cancer Discov. 2020; 10: 648-656Crossref PubMed Scopus (126) Google Scholar,6Biffi G. et al.Physiol Rev. 2021; 101: 147-176Crossref PubMed Scopus (206) Google Scholar) in MTs, we withdrew the TGFβR1 inhibitor A83-01 from the standard organoid growth medium and added PDGF-BB (MT medium). PDGF-BB induced hPS1 proliferation and invasion into the Matrigel, causing loss of cell-cell contacts between PDO and hPS1 cells (Figure A1E). To avoid this and resemble the physiological juxtaposed CAF localization to PDOs, we induced direct contact through aggregation in ultralow attachment plates. Direct contact cocultures showed higher expression of mesenchymal markers than PDO monoculture and homogenous coculture in Matrigel domes (Figure 1B). Moreover, a direct cell contact in the presence of PDGFRβ and TGFβ activity showed strong induction of mesenchymal genes (N-cadherin, vimentin, αSMA) and repressed stem-cell-related gene expression (LGR5, OLFM4) of the MTs, resembling genetic features of PDAC (Figure 1B and Figure A1F, Table A1). Knowing PDGF-BB induces desmoplasia and cell contact enhances mesenchymal gene expression, we sought to maintain cell contact in the MT model. Continuous MT suspension culture in ultralow-attachment plates prevented loss of cell-cell interaction in culture between PDO and hPS1 over time (Figure 1C). Culture conditions without PDGF-BB led to MTs with no desmoplasia, indicating that exogenous addition of PDGF-BB is crucial to stimulate MT desmoplasia. In accordance, endogenous expression of PDGF-BB in PDOs was negligible (Figure A1G). PDGF-BB stimulation induced desmoplasia but lacked typical CAF marker expression found in vivo when A83-01 was present in the medium (Figure 1D). Differentiation into a pSMAD2+/αSMA+ MyCAF-like state was only observed upon A83-01 removal (Figure 1D). In MT medium, the limited presence of inflammatory CAF-like cells was also observed by costaining of PDGFRα and pSTAT3 (Figure A1H), corresponding to the in vivo prevalence of this cell type compared to MyCAFs.6Biffi G. et al.Physiol Rev. 2021; 101: 147-176Crossref PubMed Scopus (206) Google Scholar Interestingly, in response to PDGF-BB, hPS1 can either encapsulate PDOs (PDO1 & PDO2) or form a desmoplastic core (PDO3; Figures 1D and 2A ), hinting at differential PDO-CAF crosstalk in determining spatial self-organization of MTs. In summary, direct cell-cell contact and PDGFRβ/TGFβ signaling resulted in a pathology resembling MTs with regard to desmoplasia and MyCAF differentiation. Having established this model with hPS1 cells, we investigated whether we could generate MTs using primary, patient-derived PDAC fibroblasts. Primary fibroblasts were mixed with PDO1 and cultured either in organoid growth medium (+TGFβi, −PDGF-BB) or MT medium (−TGFβi, +PDGF-BB). In line with our observations, desmoplastic MTs only form when grown in the MT medium (Figure 2B). As a proof of concept, we generated fully autologous MTs to study the feasibility of generating treatment-naïve models at the time of diagnosis. PDO (FNADO) and fibroblast (FNA-CAF) cultures were established from a single fine-needle biopsy (Figure A2A). The mutation status of FNADO was consistent with the majority of human PDACs, including KRASp.(Gly12Asp) and TP53p.(Arg196∗) mutations and loss of CDKN2A (Figure A2B). FNADO tumorigenicity was confirmed in mouse xenografts. MTs of FNADO and FNA-CAFs closely resembled a xenografted primary tumor when cultured in the MT medium (Figure 2C). Finally, we aimed to test the applicability of MTs in comparison to PDO monocultures with regard to drug sensitivity. Stroma-rich tumors often are resistant to chemotherapeutics, including oxaliplatin.8Ham I.H. et al.Cancers (Basel). 2021; 13: 1172Crossref PubMed Scopus (17) Google Scholar To this end, we tested oxaliplatin resistance in a treatment-naive PDO (PDO4) and MT model (PD04 + hPS1). Indeed, oxaliplatin induced significant cell death in monoculture conditions but not in MTs (Figure 2D). This indicates that MTs have a potential to faithfully recapitulate drug sensitivity observed in stroma-rich tumors. Here we report the creation of a human, multicellular MT model that in comparison to earlier efforts9Seino T. et al.Cell Stem Cell. 2018; 22: 454-467.e6Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar,10Tsai S. et al.BMC Cancer. 2018; 18: 335Crossref PubMed Scopus (186) Google Scholar now recapitulates both desmoplastic features of PDAC and differentiation toward the MyCAF subset. Importantly, these pathologies resembling MTs can be generated from both surgical resections as well as endoscopic biopsies, thus, for the first time, facilitating modeling the entire patient spectrum. Especially, endoscopic acquisition of patient material enables MT generation of treatment-naïve tumors and modeling of tumors not amenable to surgical resection. This model is easy to integrate into current organoid-based cell culture models and provides basis to study a plethora of fundamental aspects of PDAC, including the spatial architecture of PDAC tumor cells and associated stroma. For preclinical studies, MT cultures could be implemented in medium-throughput screening platforms. The next essential step to further improve this MT model is to include additional stromal cell types, for example, immune cells, endothelial cells, and adipocytes.2Tuveson D. et al.Science. 2019; 364: 952-955Crossref PubMed Scopus (353) Google Scholar,3Lau H.C.H. et al.Nat Rev Gastroenterol Hepatol. 2020; 17: 203-222Crossref PubMed Scopus (73) Google Scholar Finally, since the PDGF and TGFβ pathways also play key roles in other stroma-dense gastrointestinal tumors,4Kobayashi H. et al.Nat Rev Gastroenterol Hepatol. 2019; 16: 282-295Crossref PubMed Scopus (233) Google Scholar our model is expected to be extendable to, for example, colon, gastric, and esophageal cancers. The authors wish to thank the Hubrecht Organoid Technology (HUB) and CancerGenomicsCenter.nl for providing and funding PDOs and prof. Hemant Kocher (Queen Mary University London) for providing hPS1 cells used in this study. The authors also thank Dr A. Farshadi for providing cells (PDO4) and reagents (oxaliplatin). Illustrations in Figures 1B, 1C and A1D were made with BioRender (www.biorender.com). Download .pdf (.89 MB) Help with pdf files Figure A1(A) Representative brightfield image of PDO1 and hPS1. Scalebar 100 μm. (B) Relative mRNA expression of CYCG, FABP4, FAP, PLIN2, PDPN, VIM, CCL2, CXCL1, IL1α, IL6, PDGFRα, PDGFRβ, αSMA, and COL1A1 in hPS1. (C) PDGFRB and GAPDH protein levels in hPS1 cells treated with TGFβ receptor inhibitor A83-01 and TGFβ3 ligand for 24 hours; N = 3. (D) Schematic overview of MT 3D matrigel culture generation. (E) Representative brightfield images of PDO1 + hPS1 and PDO1-H2B-mCerulean3 (Cyan) + hPS1 MTs 3D matrigel cultures grown for 10 days in OG, OG + PDGF-BB, OG-TGFβ inhibitor, or OG + PDGF-BB-TGFβ inhibitor (MT) medium; n = 3, scalebar 100 μm. (F) Relative mRNA expression of FABP4, FAP, PLIN2, PDPN, VIM, CCL2, CXCL1, CTGF, IL6, PDGFRα, PDGFRβ, αSMA, and COL1A1 in hPS1 cultured in 2D or as aggregates in 3D Matrigel domes in medium ± TGFβ inhibitor an PDGF-BB for 5 days (n = 3). (G) Relative expression (2ˆ−ΔCT) of PDGFB in PDO1 and PDO3. The endothelial cell line ECRF was used as a positive control. (H) Multiplex staining of patient-derived PDAC resection material and MTs (PDO1 + hPS1) for PDGFRα and pSTAT3 Download .pdf (.06 MB) Help with pdf files Figure A2(A) Representative brightfield image of FNAPDO after 7 days culture in matrigel; scalebar 100 μm. (B) Overview table of cancer-related mutations in FNAPDO based on AmpliSeq Cancer Hotspot Panel V2 genome sequencing Download .docx (.02 MB) Help with docx files Supplemental Methods and Table A1

Gastrointestinal (GI) cancers are characterized by extensive tumor stroma that both promotes tumor progression and acts as a physical barrier for adjacent tumor cells, limiting the effect of current treatment modalities. Oncolytic virotherapy is currently investigated in clinical trials as a novel therapeutic agent for different malignancies of the GI tract, but it is largely unknown whether these viruses can also target the tumor stroma. Here, we investigated the tropism of two commonly studied OVs, adenovirus and reovirus, towards primary GI fibroblasts from human oesophageal, gastric, duodenal and pancreatic carcinomas ( N = 36). GI fibroblasts were susceptible to type 3 Dearing (T3D) strain R124 and bioselected mutant reovirus ( jin -3) infection but not oncolytic adenovirus (Ad5-Δ24). Efficient infection and apoptosis of human and mouse GI cancer-derived fibroblasts by these reoviruses was partially dependent on the expression of the reovirus entry receptor, Junctional Adhesion Molecule-A (JAM-A). Moreover, human GI cancer organoid-fibroblast co-cultures showed higher overall infectivity when containing JAM-A expressing fibroblasts as compared to JAM-A negative fibroblasts, indicating a potential role of JAM-A expressing fibroblasts for viral dissemination. We further show that JAM-A is not only necessary for efficient reovirus infection of fibroblasts but also partially mediates reovirus-induced apoptosis, dependent on signaling through the C-terminal PDZ-domain of JAM-A. Altogether, our data show the presence of JAM-A expressing fibroblasts in both human and murine GI cancers that are amenable to infection and induction of apoptosis by reovirus, extending the potential anti-cancer actions of reovirus with stromal targeting.

In intestinal homeostasis, continuous renewal of the epithelium is crucial to withstand the plethora of stimuli which can damage the structural integrity of the intestines. Fibroblasts contribute to this renewal by facilitating epithelial cell differentiation as well as providing the structural framework in which epithelial cells can regenerate. Upon dysregulation of intestinal homeostasis, (pre-) malignant neoplasms develop, a process which is accompanied by (epi) genetic alterations in epithelial cells as well as phenotypic changes in fibroblast populations. In the context of invasive carcinomas, these fibroblast populations are termed cancer-associated fibroblasts (CAFs). CAFs are the most abundant cell type in the tumor microenvironment of colorectal cancer (CRC) and consist of various functionally heterogeneous subsets which can promote or restrain cancer progression. Although most previous research has focused on the biology of epithelial cells, accumulating evidence shows that certain fibroblast subsets can also importantly contribute to tumor initiation and progression, thereby possibly providing avenues for improvement of clinical care for CRC patients. In this review, we summarized the current literature on the emerging role of fibroblasts in various stages of CRC development, ranging from adenoma initiation to the metastatic spread of cancer cells. In addition, we highlighted translational and therapeutic perspectives of fibroblasts in the different stages of intestinal tumor progression.