Organoids derived from neoadjuvant FOLFIRINOX patients recapitulate therapy resistance in pancreatic ductal adenocarcinoma

Purpose: We investigated whether organoids can be generated from resected tumors of patients who received eight cycles of neoadjuvant FOLFIRINOX chemotherapy before surgery, and evaluated the sensitivity/resistance of these surviving cancer cells to cancer therapy. Experimental Design: We generated a library of 10 PDAC organoid lines: five each from treatment-naive and FOLFIRINOX-treated patients. We, first, assessed the histological, genetic, and transcriptional characteristics of the organoids and their matched primary PDAC tissue. Next, the organoids' response to treatment with single agents - 5-FU, irinotecan, and oxaliplatin - of the FOLFIRINOX regimen as well as combined regimen was evaluated. Finally, global mRNA-seq analyses were performed to identify FOLFIRINOX resistance pathways. Results: All 10 patient-derived PDAC organoids recapitulate histological, genetic, and transcriptional characteristics of their primary tumor tissue. Neoadjuvant FOLFIRINOXtreated organoids display resistance to FOLFIRINOX (5/5), irinotecan (5/5) and oxaliplatin (4/5) when compared to treatment-naive organoids (FOLFIRINOX: 1/5, irinotecan: 2/5, oxaliplatin: 0/5). 5-FU treatment responses between naive and treated organoids were similar. Comparative global transcriptome analysis of treatment-naive and FOLFIRINOX samples - in both organoids and corresponding matched tumor tissues - uncovered modulated pathways mainly involved in genomic instability, energy metabolism, and innate immune system. Conclusion: Resistance development in neoadjuvant FOLFIRINOX organoids, recapitulating their primary tumor resistance, suggests continuation of FOLFIRINOX therapy as an adjuvant treatment may not be advantageous for these patients. Gene expression profiles of PDAC organoids identify targetable pathways involved in chemoresistance development upon neoadjuvant FOLFIRINOX treatment, thus opening up combination therapy possibilities.Genome Instability and Cance

Human pancreatic tumour organoid-derived factors enhance myogenic differentiation

Background: Most patients with pancreatic cancer develop cachexia, which is characterized by progressive muscle loss. The mechanisms underlying muscle loss in cancer cachexia remain elusive. Pancreatic tumour organoids are 3D cell culture models that retain key characteristics of the parent tumour. We aimed to investigate the effect of pancreatic tumour organoid-derived factors on processes that determine skeletal muscle mass, including the regulation of muscle protein turnover and myogenesis. Methods: Conditioned medium (CM) was collected from human pancreatic cancer cell lines (PK-45H, PANC-1, PK-1, and KLM-1), pancreatic tumour organoid cultures from a severely cachectic (PANCO-9a) and a non-cachectic patient (PANCO-12a), and a normal pancreas organoid culture. Differentiating C2C12 myoblasts and mature C2C12 myotubes were exposed to CM for 24 h or maintained in control medium. In myotubes, NF-kB activation was monitored using a NF-κB luciferase reporter construct, and mRNA expression of E3-ubiquitin ligases and REDD1 was analysed by RT-qPCR. C2C12 myoblast proliferation and differentiation were monitored by live cell imaging and myogenic markers and myosin heavy chain (MyHC) isoforms were assessed by RT-qPCR. Results: Whereas CM from PK-1 and KLM-1 cells significantly induced NF-κB activation in C2C12 myotubes (PK-1: 3.1-fold, P < 0.001; KLM-1: 2.1-fold, P = 0.01), Atrogin-1/MAFbx and MuRF1 mRNA were only minimally and inconsistently upregulated by the CM of pancreatic cancer cell lines. Similarly, E3-ubiquitin ligases and REDD1 mRNA expression in myotubes were not altered by exposure to pancreatic tumour organoid CM. Compared with the control condition, CM from both PANCO-9a and PANCO-12a tumour organoids increased proliferation of myoblasts, which was accompanied by significant downregulation of the satellite cell marker paired-box 7 (PAX7) (PANCO-9a: −2.1-fold, P < 0.001; PANCO-12a: −2.0-fold, P < 0.001) and myogenic factor 5 (MYF5) (PANCO-9a: −2.1-fold, P < 0.001; PANCO-12a: −1.8-fold, P < 0.001) after 48 h of differentiation. Live cell imaging revealed accelerated alignment and fusion of myoblasts exposed to CM from PANCO-9a and PANCO-12a, which was in line with significantly increased Myomaker mRNA expression levels (PANCO-9a: 2.4-fold, P = 0.001; PANCO-12a: 2.2-fold, P = 0.004). These morphological and transcriptional alterations were accompanied by increased expression of muscle differentiation markers such as MyHC-IIB (PANCO-9a: 2.5-fold, P = 0.04; PANCO-12a: 3.1-fold, P = 0.006). Although the impact of organoid CM on myogenesis was not associated with the cachexia phenotype of the donor patients, it was specific for tumour organoids, as CM of control pancreas organoids did not modulate myogenic fusion. Conclusions: These data show that pancreatic tumour organoid-derived factors alter the kinetics of myogenesis, which may eventually contribute to impaired muscle mass maintenance in cancer cachexia

Gastrointestinal cancer-associated fibroblasts expressing Junctional Adhesion Molecule-A are amenable to infection by oncolytic reovirus

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-Delta 24). 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.Therapeutic cell differentiatio

Gastrointestinal cancer-associated fibroblasts expressing Junctional Adhesion Molecule-A are amenable to infection by oncolytic reovirus

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-Delta 24). 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