Adipose-derived mesenchymal stem cells differentiate into heterogeneous cancer-associated fibroblasts in a stroma-rich xenograft model

Cancer-associated fibroblasts (CAFs) are the key components of the densely proliferated stroma in pancreatic ductal adenocarcinoma (PDAC) and contribute to tumor progression and drug resistance. CAFs comprise heterogeneous subpopulations playing unique and vital roles. However, the commonly used mouse models have not been able to fully reproduce the histological and functional characteristics of clinical human CAF. Here, we generated a human cell-derived stroma-rich CDX (Sr-CDX) model, to reproduce the clinical tumor microenvironment. By co-transplanting human adipose-derived mesenchymal stem cells (AD-MSCs) and a human PDAC cell line (Capan-1) into mice, the Sr-CDX model recapitulated the characteristics of clinical pancreatic cancer, such as accelerated tumor growth, abundant stromal proliferation, chemoresistance, and dense stroma formed from the heterogeneous CAFs. Global RNA sequencing, single-cell based RNA sequencing, and histological analysis of CAFs in the Sr-CDX model revealed that the CAFs of the Sr-CDX mice were derived from the transplanted AD-MSCs and composed of heterogeneous subpopulations of CAF, including known and unknown subtypes. These lines of evidences suggest that our new tumor-bearing mouse model has the potential to address an open question in CAF research, that is the mechanism of CAF differentiation.

P03.29 Characterization of treatment-induced adaptive immune responses in pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy marked by poor prognosis and profound drug resistance characterized in more than 90% of cases by KRAS mutations. To recapitulate central aspects of PDAC, we employed genetically engineered mouse models presenting KrasG12D pancreas specific expression. Through a high-throughput combination drug screen with trametinib as backbone we identified a high synergism with the multikinase inhibitor nintedanib, preferentially in mesenchymal PDAC, a subtype of this disease characterized by poor prognosis and therapeutic resistance. This combinatorial treatment, that led to the induction of apoptosis in vitro and disease regression in vivo, was accompanied by a strong tumor infiltration of CD8 positive T cells.Materials and MethodsTo characterize the treatment-induced adaptive immune cell infiltration in vivo, we performed orthotopic transplantations of KRAS-driven murine PDAC cell lines presenting mesenchymal and epithelial morphology. The derived control and nintedanib + trametinib treated PDAC tumors were analyzed by multi-color immunofluorescence stainings. We compared the findings to high parameter flow cytometry results.ResultsConfocal microscopy of the immunofluorescence stainings revealed an overall increase of tumor-infiltrating lymphocytes (TIL) in the tumors upon combinatorial treatment with substantial differences in quantity and spatial distribution. Tumors derived from a PDAC cell line of epithelial morphology were characterized by few TIL mainly located at the invasive margins of the tumors, while tumors derived from a mesenchymal PDAC cell line showed a strong increase of TIL even in the center of the tumor mass. Furthermore, an increased ratio of CD8 positive cytotoxic T cells to CD4 positive helper T cells as well as a decrease of Foxp3 and CD4 positive regulatory T cells could be observed for tumors derived from the mesenchymal PDAC cell line under combinatorial treatment. To investigate if the observed recruitment of T cells was indispensable for treatment efficacy of the combinatorial therapy, we orthotopically transplanted the mesenchymal PDAC cell line in immunodeficient CD3-Knockout (CD3ko) mice and applied an analogous combinatorial treatment scheme. In the CD3ko mice, the combinatorial treatment did not lead to an increased survival or tumor regression as observed in immunocompetent mice. However, flow cytometry and immunofluorescence stainings revealed an increase of B cells upon nintedanib + trametinib treatment.ConclusionsOur findings indicate a reduced efficacy of the combinatorial treatment in T cell deficient mice, underlining the importance of T cells in treatment-induced anti-tumor responses and enlarging the understanding of the role of TIL in PDAC.Disclosure InformationJ. Heetmeyer: None. C. Falcomatà: None. S. Bärthel: None. C. Schneeweis: None. A. Coluccio: None. C. Veltkamp: None. G. Schneider: None. D. Saur: None.

Application of a MYC degradation screen identifies sensitivity to CDK9 inhibitors in KRAS-mutant pancreatic cancer

Stabilization of the MYC oncoprotein by KRAS signaling critically promotes the growth of pancreatic ductal adenocarcinoma (PDAC). Thus, understanding how MYC protein stability is regulated may lead to effective therapies. Here, we used a previously developed, flow cytometry–based assay that screened a library of >800 protein kinase inhibitors and identified compounds that promoted either the stability or degradation of MYC in a KRAS-mutant PDAC cell line. We validated compounds that stabilized or destabilized MYC and then focused on one compound, UNC10112785, that induced the substantial loss of MYC protein in both two-dimensional (2D) and 3D cell cultures. We determined that this compound is a potent CDK9 inhibitor with a previously uncharacterized scaffold, caused MYC loss through both transcriptional and posttranslational mechanisms, and suppresses PDAC anchorage-dependent and anchorage-independent growth. We discovered that CDK9 enhanced MYC protein stability through a previously unknown, KRAS-independent mechanism involving direct phosphorylation of MYC at Ser62. Our study thus not only identifies a potential therapeutic target for patients with KRAS-mutant PDAC but also presents the application of a screening strategy that can be more broadly adapted to identify regulators of protein stability.

Myoferlin Contributes to the Metastatic Phenotype of Pancreatic Cancer Cells by Enhancing Their Migratory Capacity through the Control of Oxidative Phosphorylation

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies with an overall survival of 5% and is the second cause of death by cancer, mainly linked to its high metastatic aggressiveness. Accordingly, understanding the mechanisms sustaining the PDAC metastatic phenotype remains a priority. In this study, we generated and used a murine in vivo model to select clones from the human Panc-1 PDAC cell line that exhibit a high propensity to seed and metastasize into the liver. We showed that myoferlin, a protein previously reported to be overexpressed in PDAC, is significantly involved in the migratory abilities of the selected cells. We first report that highly metastatic Panc-1 clones expressed a significantly higher myoferlin level than the corresponding low metastatic ones. Using scratch wound and Boyden’s chamber assays, we show that cells expressing a high myoferlin level have higher migratory potential than cells characterized by a low myoferlin abundance. Moreover, we demonstrate that myoferlin silencing leads to a migration decrease associated with a reduction of mitochondrial respiration. Since mitochondrial oxidative phosphorylation has been shown to be implicated in the tumor progression and dissemination, our data identify myoferlin as a valid potential therapeutic target in PDAC.

Effect of macrophages on pancreatic cancer.

324 Background: Pancreatic Ductal Adenocarcinoma (PDAC) is expected to be the second leading cause of cancer related deaths by 2030. TGF-β is a well-studied PDAC mediator with a context dependent role as initially a tumor suppressor with potential to convert to a tumor promoter in later stages. Tumor associated macrophages and interleukins, such as the pro-inflammatory interleukin, IL23, are not well studied regarding PDAC. We hypothesized PDAC treated with TGF-β and macrophages would induce a more aggressive phenotype. Methods: We investigated aggressive behavior with a primary PDAC cell line in vivo and a metastatic PDAC cell line in vitro. A primary pancreatic cell line, Panc-1 cells, were pre-treated with PBS, IL23, macrophages (10:1 ratio of Panc-1 cells to macrophages), IL23 + macrophages, TGF-ß, TGF-ß + macrophages, or TGF-ß + macrophages + IL23. After treatment, cells were orthotopically implanted into the pancreas of NOD SCID gamma mice with 5 mice per group. Mice weights were recorded twice weekly for 4-weeks. Primary lesions and metastasis were investigated with ANOVA. AsPC-1 cells, a metastatic pancreatic cell line, were pre-treated with the same seven treatments. We investigated pSTAT3 expression and the streak closure in vitro. Results: Panc-1 cells treated with macrophages had the largest pancreatic tumor weight and diameter compared to PBS control, IL23 alone, and TGF-β alone (P < 0.001). When macrophages treatment included TGF-β, pancreatic tumor weights and diameters decreased as compared to macrophages alone and macrophages + IL23 (P < 0.001). Macrophage treatment induced higher liver weights and higher number of surface liver metastatic lesions suggesting higher metastatic disease burden (P < 0.03). AsPC-1 cells treated with combinations of macrophages and TGF-β increased pSTAT3 expression compared to PBS control. AsPC-1 cells treated with macrophages closed the gap in the scratch assay faster than PBS control 24 hours after treatment (P < 0.001). Conclusions: We demonstrated macrophages have a key role in converting primary pancreatic cancer into a more aggressive phenotype in vivo whereas they have less effect on metastatic pancreatic cancer in vitro.

Ruxolitinib and Polycation Combination Treatment Overcomes Multiple Mechanisms of Resistance of Pancreatic Cancer Cells to Oncolytic Vesicular Stomatitis Virus

ABSTRACT Vesicular stomatitis virus (VSV) is a promising oncolytic virus (OV). Although VSV is effective against a majority of pancreatic ductal adenocarcinoma cell (PDAC) cell lines, some PDAC cell lines are highly resistant to VSV, and the mechanisms of resistance are still unclear. JAK1/2 inhibitors (such as ruxolitinib and JAK inhibitor I) strongly stimulate VSV replication and oncolysis in all resistant cell lines but only partially improve the susceptibility of resistant PDACs to VSV. VSV tumor tropism is generally dependent on the permissiveness of malignant cells to viral replication rather than on receptor specificity, with several ubiquitously expressed cell surface molecules playing a role in VSV attachment to host cells. However, as VSV attachment to PDAC cells has never been tested before, here we examined if it was possibly inhibited in resistant PDAC cells. Our data show a dramatically weaker attachment of VSV to HPAF-II cells, the most resistant human PDAC cell line. Although sequence analysis of low-density lipoprotein (LDL) receptor (LDLR) mRNA did not reveal any amino acid substitutions in this cell line, HPAF-II cells displayed the lowest level of LDLR expression and dramatically lower LDL uptake. Treatment of cells with various statins strongly increased LDLR expression levels but did not improve VSV attachment or LDL uptake in HPAF-II cells. However, LDLR-independent attachment of VSV to HPAF-II cells was dramatically improved by treating cells with Polybrene or DEAE-dextran. Moreover, combining VSV with ruxolitinib and Polybrene or DEAE-dextran successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. IMPORTANCE Oncolytic virus (OV) therapy is an anticancer approach that uses viruses that selectively infect and kill cancer cells. This study focuses on oncolytic vesicular stomatitis virus (VSV) against pancreatic ductal adenocarcinoma (PDAC) cells. Although VSV is effective against most PDAC cells, some are highly resistant to VSV, and the mechanisms are still unclear. Here we examined if VSV attachment to cells was inhibited in resistant PDAC cells. Our data show very inefficient attachment of VSV to the most resistant human PDAC cell line, HPAF-II. However, VSV attachment to HPAF-II cells was dramatically improved by treating cells with polycations. Moreover, combining VSV with polycations and ruxolitinib (which inhibits antiviral signaling) successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. We envision that this novel triple-combination approach could be used in the future to treat PDAC tumors that are highly resistant to OV therapy.