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Cancer cells exert mastery over the local tumor-associated stroma (TAS) to configure protective immunity within the tumor microenvironment. The immunomodulatory character of pancreatic lysates of patients with cancer differs from those with pancreatitis. In this study, we evaluated the cross-talk between pancreatic cancer and its TAS in primary human cell culture models. Upon exposure of TAS to pancreatic cancer cell-conditioned media, we documented robust secretion of IL6 and IL8. This TAS response was MyD88-dependent and sufficient to directly suppress both CD4+ and CD8+ T-cell proliferation, inducing Th17 polarization at the expense of Th1. We found that patients possessed a similar shift in circulating effector memory Th17:Th1 ratios compared with healthy controls. The TAS response also directly suppressed CD8+ T-cell–mediated cytotoxicity. Overall, our results demonstrate how TAS contributes to the production of an immunosuppressive tumor microenvironment in pancreatic cancer. Cancer Res; 77(3); 672–83. ©2016 AACR.

Introduction: Pancreatic cancer (PC) is associated with a high rate of cachexia, which specifically diminishes quality of life, prohibits effective therapies, and subsequently contributes to morbidity and mortality. Unfortunately, mechanisms underlying this cancer-induced muscle wasting in the human disease remain incompletely described, in part due to limited translational models. Therefore, we hypothesize that the development of more representative models of PC cachexia will allow for development of therapeutic targets for cachexia. To test our hypothesis, we propose to 1) establish the first patient-derived xenograft (PDX) cancer cachexia models to identify the muscle associated with PC induced cachexia and 2) support these results by examining the corresponding skeletal muscle of PC patients whom contributed to the PDX models. Methods: Rectus abdominis muscle was biopsied from surgically resected PC patients and matched non-cancer controls. After surgical harvest of PC specimen, PDX models were derived in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice and skeletal muscle was subsequently harvested for histologic investigations on ultrastructural disorganization and qRT-PCR for atrophy-related transcription factors differentially regulated in PC patients. Systemic cytokine expression profiles were analyzed with luminex technology and confirmed by ELISA. Results: Rectus biopsies from patients with resected PC displayed marked muscle fiber atrophy, increased extracellular space, greater variation in fiber size and shape and more centralized nuclei compared to controls. These architectural abnormalities were also present in mice bearing xenografts from corresponding PC patients. Despite the absence of an adaptive immune system, PDX mice demonstrated high levels of systemic TNFα, IL-1β, IL6 and KC (IL8) with a concomitant decrease in anti-inflammatory cytokine IL10 when compared to matched controls. Further, skeletal muscle from both patients with PC and mice bearing PDX tumors demonstrated increased expression of the Forkhead boxO1 (FoxO1) transcription factor and FoxO target gene and E3 ubiquitin ligase, MuRF1, both of which have been directly implicated in the regulation of muscle mass. Conclusions: Preoperative muscle wasting in PC is associated with characteristic architectural abnormalities and elevated FoxO1-MuRF1 levels. Mice bearing PDX demonstrate comparable elevations in circulating pro-inflammatory cytokines, muscle pathology and FoxO1-MuRF1 levels. These results provide a valid translational model of cachexia which suggests a central role for FoxO1 and MuRF1 in PC-associated muscle wasting. Citation Format: Daniel Delitto, Sarah M. Judge, Rachel L. Nosacka, Andrea Knowlton, Fernanda G. Rocha, Kevin E. Behrns, Steven J. Hughes, Shannon M. Wallet, Andrew R. Judge, Jose G. Treviño. Pro-inflammatory cytokine secretion and gene networks associate with pancreatic cancer induced cachexia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1017.

Background: The systemic treatment of pancreatic cancer (PC) is hindered by the rapid development of chemoresistance to current cytotoxic therapies. Mechanisms governing the development of chemoresistance remain poorly characterized, particularly with respect to contributions from the tumor microenvironment. Thus, the goal of this study was to identify novel mechanisms acting within the tumor microenvironment which lead to PC chemoresistance. Methods: Intratumoral soluble mediator concentrations from resected PC specimens (n = 26) as well as supernatants from co-cultures of primary tumor-associated pancreatic stellate cells (PSCs) and PC cells (n = 12) were evaluated using a panel of 41 growth factors, chemokines and cytokines. The effect of CXCL10, a highly expressed soluble mediator during co-culture, on viability, proliferation, and apoptosis of PC cells was evaluated with and without gemcitabine treatment. In addition, the contribution of CXCL10 on migration patterns of peripheral blood mononuclear cells (PBMCs) was assessed. Results: Co-culture of tumor-associated PSCs with PC cells revealed increased CXCL10 levels compared to either cell type cultured alone. In addition, high intratumoral CXCL10 concentrations correlated with reduced overall survival (HR 6.9; P = .006). While CXCL10 treatment had a small effect on the viability of PC cells, it led to significantly increased PC cell viability in the presence of gemcitabine. Further, gemcitabine treatment induced the expression of the CXCL10 receptor, CXCR3, and this induction of CXCR3 was associated with the absence of apoptotic markers in PC cells. Finally, constitutive expression of CXCL10 by PC cells preferentially led to the migration of regulatory immune cell subsets. Conclusion: Paracrine CXCL10 signaling between stromal, PC and immune cells may be responsible not only for chemoresistance to gemcitabine, but also the recruitment and potential polarization of regulatory immune cell subsets in the pancreatic cancer microenvironment. Citation Format: Daniel Delitto, Chelsey Perez, Brian S. Black, Heather L. Sorenson, Andrea E. Knowlton, Song Han, Dongyu Zhang, George A. Sarosi, Lyle L. Moldawer, Kevin E. Behrns, Chen Liu, Thomas J. George, Ryan M. Thomas, Jose G. Trevino, Shannon M. Wallet, Steven J. Hughes. CXCL10 within the tumor microenvironment induces gemcitabine resistance in pancreatic cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5028. doi:10.1158/1538-7445.AM2015-5028

Smoking is an established risk factor for the development of pancreatic adenocarcinoma (PC). However, the relationship between smoking and PC tumor biology is incompletely defined. We report reduced overall survival (OS) in PC patients who continued smoking after surgical resection with curative intention (HR 1.93; P = .040). We further demonstrate augmented paracrine signaling via the hepatocyte growth factor (HGF)/c-Met pathway as a result of nicotine exposure in the PC microenvironment. Specifically, HGF, secreted by patient-derived PC tumor associated stroma (TAS), activates the c-Met receptor in PC cells. This paracrine activation subsequently leads to downstream induction of inhibitor of differentiation-1 (Id1) in PC cells, previously established as a mediator of chemoresistance. Further delineation of the signaling pathway demonstrates HGF-induced Id1 expression is abrogated by silencing of c-Met or pharmacologic inhibition. In patient-derived PC xenografts, nicotine treatment augmented tumor growth and metastasis; tumor lysates from nicotine-treated mice demonstrated elevated HGF expression and phospho-Met levels. Additionally, patients with high intratumoral phospho-Met levels exhibited reduced overall survival compared to those with low phospho-Met levels (Median OS 6.1 vs. 15.2 months, respectively; P = .028). Taken together, our data reveal that nicotine promotes the progression of PC via a microenvironment-dependent, paracrine signaling mechanism. Citation Format: Daniel Delitto, Dongyu Zhang, Song Han, Brian S. Black, Andrea E. Knowlton, Adrian C. Vlada, George A. Sarosi, Kevin E. Behrns, Ryan M. Thomas, Xiaomin Lu, Chen Liu, Thomas J. George, Steven J. Hughes, Shannon M. Wallet, Jose G. Trevino. Nicotine reduces survival via augmentation of paracrine HGF-MET signaling in the pancreatic cancer microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 470. doi:10.1158/1538-7445.AM2015-470

Direct implantation of viable surgical specimens provides a representative preclinical platform in pancreatic adenocarcinoma (PC). Patient-derived xenografts consistently demonstrate retained tumor morphology and genetic stability. However, the evolution of the tumor microenvironment over time remains poorly characterized in these models. This work specifically addresses the recruitment and incorporation of murine stromal elements into expanding patient-derived PC xenografts, establishing the rapidity by which murine cells are integrated into networks of invading cancer cells. In addition, we provide methodology and observations in the establishment and maintenance of a patient-derived PC xenograft model. A total of 25 histologically confirmed pancreatic adenocarcinoma specimens were implanted subcutaneously into NOD-SCID mice. Patient demographics, staging, pathologic analysis and outcomes were analyzed. After successful engraftment of tumors, histologic and immunofluorescent analyses were performed on explanted tumors. PC specimens were successfully engrafted in 15 of 25 (60%) of attempts. Successful engraftment does not appear to correlate with clinicopathologic factors or patient survival. Tumor morphology is conserved through multiple passages and tumors retain metastatic potential. Interestingly, despite morphologic similarity between passages, human stromal elements do not appear to expand with invading cancer cells. Rather, desmoplastic murine stroma dominates the xenograft microenvironment after the initial implantation. Recruitment of stromal elements in this manner to support and maintain tumor growth represents a novel avenue for investigation into tumor-stromal interactions. Citation Format: Daniel Delitto, Kien Pham, Adrian C. Vlada, George A. Sarosi, Ryan M. Thomas, Kevin E. Behrns, Chen Liu, Steven J. Hughes, Shannon M. Wallet, Jose G. Trevino. Patient-derived xenograft models for pancreatic adenocarcinoma demonstrate retention of tumor morphology through the incorporation of murine stromal elements. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1466. doi:10.1158/1538-7445.AM2015-1466