A Novel Pancreatic Cancer Mini-tumor Model to Study Desmoplasia and Myofibroblastic Cancer-Associated Fibroblast Differentiation (2022)

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