Background: Pancreatic ductal adenocarcinoma (PDAC) recently became the third-deadliest cancer due to resistance to chemotherapy and immunotherapies. The tumor microenvironment (TME) in PDAC is thought to contribute to this resistance, with up to 80% of the tumor bulk consisting of stroma. We investigated the role of two major stromal cell types, pancreatic stellate cells (PSCs) and macrophages, in the production of the immunosuppressive proteoglycan versican (VCAN) as a precursor to the identification of novel mechanisms that may enhance therapeutic response. Methods: B6 KPC mice were utilized as a murine model of PDAC. A human PDAC tissue microarray (TMA) was developed representing normal and neoplastic pancreatic tissue across 131 patients. Immunohistochemistry (IHC) was used to determine levels of VCAN and CD8+ T cells. Bone marrow-derived macrophages (BMDMs) were differentiated from BALB/c mouse femurs and polarized to M1 (antitumor) or M2 (protumor) status. Additionally, PDAC organotypic spheroids were derived from both human and murine tissues whereas PSCs were derived solely from human tissue. Results: IHC analysis of KPC tumors revealed elevated levels of stromal VCAN compared to normal pancreatic tissue (p<0.001, n=20). VCAN accumulation was increased even in the earliest stages of acinar-to-ductal metaplasia in KPC mice. Areas of intense stromal VCAN staining trended toward reduced CD8+ T cell infiltration (4.9 vs 7.3 cells/high power field (hpf), p=0.3). IHC analysis of human PDAC revealed elevated VCAN accumulation across all stages compared to the normal adjacent tissue (n=231, p<0.001). Areas of high VCAN accumulation demonstrated reduced CD8+ T cells compared to areas of low VCAN (0.6 vs 2.9 cells/hpf, p<0.001). To investigate cell types responsible for enhanced VCAN accumulation in the tumor microenvironment, relative expression (RE) of VCAN was compared in vitro to M0 macrophages. Organotypic cancer spheroids demonstrated increased expression of VCAN from KPC mice (RE=49, n=2) and patient-derived PDAC tissue (RE=14, p=0.01, n=3). M1-polarized BMDMs had increased expression of VCAN (RE=24) compared to M2 BMDMs (RE=8) (p<0.001, n=3). Interestingly, M1 BMDMs cultured in PDAC-conditioned media had reduced RE of M1 markers: TNFα (395 vs 37, p=0.03) and iNOS (24723 vs 4813, p=0.003), and increased M2 markers: Arg-1 (127 versus 1049, p=0.02) and YM-1 (0.5 vs 3, p=0.02). PDAC-conditioned media also reduced VCAN expression of M1 BMDMs (24 vs 9, p=0.02). PSCs derived from human PDAC also demonstrated enhanced RE of VCAN compared to negative controls (RE=68, p=0.017) with no significant change in the presence of PDAC conditioned media (p=0.4). Conclusions: The accumulation of VCAN is common in PDAC and correlates with CD8+ T cell exclusion. Epithelial and stromal components are responsible for VCAN production. VCAN deserves further investigation as a target for therapeutic interventions for PDAC. Citation Format: Hanna R. Rainiero, Philip B. Emmerich, Chelsie K. Sievers, Connor J. Maloney, Rosabella T. Pitera, Susan N. Payne, Mitchell G. Depke, Cheri A. Pasch, Linda Clipson, Jillian K. Johnson, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Versican production is driven by both epithelial and stromal cells in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1904.

Background: Clinical responses to checkpoint inhibitors in gastrointestinal cancers have largely been limited to MSI-high disease. Proteolysis of an immunosuppressive proteoglycan versican (VCAN) by ADAMTS proteases correlates with enhanced CD8+ T cell infiltration in colorectal cancer independent of mismatch repair status. Here we investigate the potential of this biomarker in pancreatic ductal adenocarcinoma (PDAC) as VCAN is abundant VCAN in this disease. Methods: KPC mice were used to generate mouse PDAC tissue and organotypic spheroids. A human PDAC tissue microarray (TMA) was developed representing normal and neoplastic pancreatic tissues across 131 patients. IHC was used to determine levels of VCAN, an immunostimulatory fragment versikine (Vkine), and CD8+ T cells. Bone marrow-derived macrophages (BMDMs) were derived from BALB/c mice. VCAN-specific ADAMTS protease expression profiles were compared between antitumor (M1) and protumor (M2) mouse macrophages, human pancreatic stellate cells (PSCs) and patient-derived PDAC-organotypic spheroids. Results: IHC analysis of normal murine pancreas demonstrated low VCAN and Vkine staining. VCAN is abundant in metaplastic/neoplastic KPC mouse pancreatic tissues. Rare VCAN proteolytic predominant areas (samples with both low VCAN and high Vkine) in KPC tumors had increased tumor-infiltrating CD8+ T cells/ high-powered field compared to weak areas (17 vs 4, p<0.001). Human PDACs had abundant VCAN expression compared to normal pancreatic tissues (p<0.001, n=118). Vkine was absent from normal tissues and present only at low levels in tumor cores (p<0.001, n=117). No human PDAC VCAN proteolytic-predominant samples were identified. M1 BMDMs demonstrated high relative expression (RE) of VCAN-specific ADAMTS proteases relative to unpolarized M0 BMDMs; however, these levels decreased under KPC-conditioned media (ADAMTS4, 101 vs 5, p=0.003; ADAMTS9, 80 vs 1, p=0.02; ADAMTS15, 103 versus 1.6, p<0.001; ADAMTS20, 881 vs 3.2, p<0.001). Similarly, M2 BMDMs had elevated ADAMTS-levels that were reduced under KPC-conditioned media, (ADAMTS4, 42 vs 0.4, p=0.0029; ADAMTS9, 40 vs 1, p=0.002; ADAMTS15, 23 vs 3, p=0.027; ADAMTS20, 310 vs 0.8, p<0.001; n=3). RE levels of ADAMTS proteases in KPC spheroids was consistently lower than reported above in macrophages. RE of ADAMTS proteases in human PSCs were between 18 and 304. No differences in ADAMTS expression were identified under PDAC-conditioned media in the human PSCs (p=0.3). Conclusions: The VCAN proteolysis predominant phenotype correlates with increased CD8+ T cell infiltration in PDAC though is rarely present in the human disease. PDAC epithelial cells influence the stromal cells to reduce VCAN proteolysis through decreased ADAMTS expression by macrophages. Future studies examining measures to enhance VCAN proteolysis could result in a more immune permissive microenvironment. Citation Format: Philip B. Emmerich, Chelsie Sievers, Hanna Rainiero, Rosabella Pitera, Connor Maloney, Susan Payne, Mitchell Depke, Cheri Pasch, Linda Clipson, Jillian K. Johnson, Kristina Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Stromal cell driven proteolysis of versican is limited by epithelial cells in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1909.

Background: Immune therapies have shown great promise for some cancers. To date, pancreatic ductal adenocarcinomas (PDAC) have been largely resistant to immunotherapeutic approaches in part due to their exclusion of tumor infiltrating lymphocytes. The mechanisms by which this exclusion occurs are not well-characterized but are thought to involve both innate and adaptive immune responses. Methods: The primary objective of this study was to investigate how dual anti-PD-1/anti-CSF1R therapy changes the tumor immune cell niche and tumor growth in an allograft version of the KPC mouse model of PDAC (Pdx-1-Cre; LSL-KrasG12D; LSL-Trp53 R172H). Anti-PD1 therapy in the form of monoclonal antibody from BioXCell® was dosed twice weekly via intraperitoneal injections at 200µg/mouse. Anti-CSF1R in the form of the small-molecule inhibitor BLZ945 from Selleckchem® was dosed daily via oral gavage at 200 mg/kg. Human THP-1 cells differentiated and polarized to macrophages were used for viability and gene expression analyses. Results: In B6 recipient mice, the combination anti-PD-1 plus BLZ945 resulted in reduced percent tumor volume change (11% ±17) versus vehicle treated controls (121% ±174) (p=0.02). Flow cytometry of the spleens of tumor-bearing mice demonstrated an increase in conventional dendritic cells with combination treatment compared to controls (15.3 ±2.3% vs 8.3 ±0.6%, respectively; p=0.02). Mice lacking conventional dendritic cells (B6.BATF3-/-) had an increased percent tumor volume change (84% ±78) compared to B6 wild type mice (11% ±16) (p=0.01) when receiving combination treatment. IHC staining of allografted tumors revealed a trend towards increased infiltrating CD8+ T cells/hpf in B6 wild type mice treated with anti-PD-1 or combination therapy (21±24/hpf) compared to control treated mice (11±18/hpf). A similar trend was observed in B6.BATF3-/- mice (2.9±4.4/hpf vs 0.5±0.50/hpf); however, the magnitude was greatly reduced compared to B6 wild type mice. Interestingly, BLZ945 treated tumors did not show evidence of statistically significant reduced macrophage recruitment in vivo (268 vs 241, p=0.16). Macrophage viability in vitro was also unaffected by BLZ945 treatment (fold change in viability 0.03, p=0.6). Preferential changes in macrophage polarization in vitro were observed, with significant reductions in M2 polarization markers Arg-1, YM-1, and CD206 (p= 0.0005, 0.0029, and 0.012, respectively). Conclusions: The combination of an anti-PD-1 agent in combination with inhibition of the macrophage-predominant cytokine, anti-CSF1R, slows PDAC tumor growth in a conventional dendritic cell-dependent manner. Further characterization of the interplay between innate and adaptive immune cells upon immunotherapy treatment should be investigated to increase the efficacy of these therapeutics. Citation Format: Chelsie K. Sievers, Philip B. Emmerich, Hanna R. Rainiero, Connor Maloney, Rosabella Pitera, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. BLZ945 and anti-PD-1 combination immunotherapy modulates the immune landscape in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3209.

Background: Pancreatic ductal adenocarcinoma (PDAC) recently became the third-deadliest cancer due to its resistance to effective therapies. The tumor microenvironment (TME) in PDAC is thought to contribute to this resistance, with up to 80% of the tumor bulk consisting of stroma including cancer associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Gemcitabine (gem) and nab-paclitaxel (nab-p) is standard of care for PDAC with modest efficacy. Immunotherapies, effective in other cancers, are often combined with chemotherapy in an attempt to augment tumor response. We investigated the impact of gem and nab-p on the TME and antigen presentation of PDAC. Methods: A cancer organoid line from KPC (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre) mice, was used as a murine model of PDAC. The organoids were treated in vitro with 50uM gem and 5.85uM nab-p for 12-24 hrs and subsequently tested for MHC class I expression via flow cytometry. A co-culture in Matrigel was created using KPC organoids, pro-tumor M2-polarized bone-marrow derived macrophages (BMDMs), and pancreatic stellate cells (PSCs). After 48 hrs the co-culture was treated with gem and nab-p for 24 hrs and collected to assess macrophage and fibroblast polarization markers via flow cytometry. KPC organoids were used to generate allografts in wild-type B6 mice randomized to receive 100 mg/kg gem (i.p.) and 30 mg/kg nab-p (i.v.) or control. After 96 hrs, tumors were processed for flow cytometry analyses. Results: The percentage of MHC I expressing KPC cells increased from baseline following 12 hrs of gem/nab-p treatment (32.7% vs 50.4%, p= 0.12 and declined at 24, 48 and 72 hour post-treatment time points (22.5%, p=0.3, 23.2%, p=0.31, and 13.1%, p=0.06, respectively). In the co-culture, gem and gem/nab-p significantly enhanced immunoregulatory inflammatory CAF (iCAF) subtype over control (17.5 vs 17.6 vs 10%, respectively, p<0.001). A decrease in myofibroblastic CAFs (myCAFs) was also observed. M1 macrophages in the co-culture decreased significantly with gem and gem/nab-p compared to controls (6.7% vs 6.9% vs 13.8%, respectively, p=0.02/p=0.004). There was also a subsequent increase in M2 macrophages. KPC allografts demonstrated no significant change in MHC I levels following a 96-hour gem/nab-p treatment compared to vehicle. The CAF subtypes showed trends similar to the in vitro co-culture, with more iCAFs (14.9% vs 26.3%, p=0.32) and less myCAFs (83.5% vs 71.3%, p=0.31). We were also able to identify an MHC II class of antigen-presenting CAFs (apCAFs), within both our in vitro and in vivo studies at levels less than 1% of all CAFs. However, no change was observed with treatment. Conclusion: Here we demonstrate reduced MHC class I expression, increased M2 TAM polarization, and increased iCAFs, all associated with an immunoregulatory phenotype. These finding suggest that further studies on alternative combinations of immunotherapies are warranted. Citation Format: Hanna R. Rainiero, Philip B. Emmerich, Nathaniel Verhagen, Rebecca Destefanis, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Dustin A. Deming. Gemcitabine and nab-paclitaxel increase immunosuppressive environment in PDAC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5102.