Pancreas Cancer Model Research Articles

Metabolic modulation unleashes immune vigor in murine pancreas tumors

Abstract Our study investigates the transformation of the pancreatic ductal adenocarcinoma tumor microenvironment from an immune-excluded to an immune-inflamed state using metabolic inhibitors metformin and mitochondria-selective conjugates, MMe and pCF3-MMe. In murine models of pancreas cancer, MMe and pCF3-MMe exhibited potent inhibition of tumor growth. Flow cytometry indicated increased levels of total T cells, activated CD8+, and NK T cells in MMe and pCF3-MMe treated mice. The anti-tumor effects of these metabolic inhibitors were reversed upon CD8+ T cell immunodepletion. In vitro experiments demonstrated elevated levels of pro-inflammatory chemokines in MMe-treated tumor cells, aligned with increased CD8+ T cell infiltration. Furthermore, metabolic inhibitor treatment activated both helper CD4+ and cytolytic CD8+ T cells, with the latter demonstrating increased glycolysis and acidification rates indicative of enhanced activation. Flow cytometry corroborated these findings, revealing heightened proliferation and activation markers on treated T cells. In addition to the direct effects on T cells, immunofluorescence microscopy suggested the activation of the mitochondrial antiviral-signaling protein (MAVS) pathway in tumor cells and an increase in apoptosis as indicated by the significant increase in caspase 3/7. Our findings highlight the potential of metabolic inhibitors to activate anti-tumor immune responses directly and indirectly in murine pancreas tumors.

A proteomic-based approach for tumour neoantigen discovery on pre-clinical model of pancreas cancer.

A proteomic-based approach for tumour neoantigen discovery on pre-clinical model of pancreas cancer.

A proteomic-based approach for tumour neoantigen discovery on pre-clinical model of pancreas cancer

A proteomic-based approach for tumour neoantigen discovery on pre-clinical model of pancreas cancer

A Pilot Study of Paricalcitol plus Nanoliposomal Irinotecan and 5-FU/LV in Advanced Pancreatic Cancer Patients after Progression on Gemcitabine-Based Therapy.

Vitamin D analogues remodel the desmoplastic stroma, and improve vascularity and efficacy of chemotherapy in preclinical pancreas cancer models. We conducted a pilot study to evaluate the safety and preliminary efficacy of the vitamin D analogue paricalcitol in combination with nanoliposomal irinotecan (Nal-iri) plus 5-fluorouracil/leucovorin (5-FU/LV) in patients with advanced pancreatic cancer who had progressed on gemcitabine-based therapy. Two dose levels (DL) of paricalcitol were tested: fixed dose weekly (75 mcg, DL1) and weight-based weekly (7 mcg/kg, /DL2). The primary endpoint was safety, and secondary endpoints included overall response rate, progression-free survival (PFS), and overall survival (OS). Correlative objectives aimed to identify molecular predictors of response and alterations in the tumor stroma. Twenty patients (10 each in DL1 and DL2) enrolled between March 2019 and May 2021. No grade 3/4 adverse events related to paricalcitol were observed. The most common toxicities were nausea, diarrhea and fatigue, which were similar in both cohorts. Three patients discontinued study after one cycle and were not radiographically evaluable. Of the remaining 17 evaluable patients, 2 had partial response and 12 had stable disease. The median PFS for response-evaluable patients in DL1 was 4.14 months, for DL2 was 4.83 months. Intent-to-treat median OS was 6.15 and 6.66 months for DL1 and DL2, respectively. Correlative studies showed increased tumor vascularity in posttreatment samples in patients receiving the higher dose of paricalcitol (DL2). Paricalcitol at 7 mcg/kg/week in combination with Nal-iri/ 5-FU/LV is safely tolerated, may increase tumor vascularity and warrants further investigation.

The circadian clock is disrupted in pancreatic cancer.

Disruption of the circadian clock is linked to cancer development and progression. Establishing this connection has proven beneficial for understanding cancer pathogenesis, determining prognosis, and uncovering novel therapeutic targets. However, barriers to characterizing the circadian clock in human pancreas and human pancreatic cancer-one of the deadliest malignancies-have hindered an appreciation of its role in this cancer. Here, we employed normalized coefficient of variation (nCV) and clock correlation analysis in human population-level data to determine the functioning of the circadian clock in pancreas cancer and adjacent normal tissue. We found a substantially attenuated clock in the pancreatic cancer tissue. Then we exploited our existing mouse pancreatic transcriptome data to perform an analysis of the human normal and pancreas cancer samples using a machine learning method, cyclic ordering by periodic structure (CYCLOPS). Through CYCLOPS ordering, we confirmed the nCV and clock correlation findings of an intact circadian clock in normal pancreas with robust cycling of several core clock genes. However, in pancreas cancer, there was a loss of rhythmicity of many core clock genes with an inability to effectively order the cancer samples, providing substantive evidence of a dysregulated clock. The implications of clock disruption were further assessed with a Bmal1 knockout pancreas cancer model, which revealed that an arrhythmic clock caused accelerated cancer growth and worse survival, accompanied by chemoresistance and enrichment of key cancer-related pathways. These findings provide strong evidence for clock disruption in human pancreas cancer and demonstrate a link between circadian disruption and pancreas cancer progression.

Mitochondria targeted metabolic inhibitors promote lymphocyte infiltration into murine pancreatic tumors

Abstract Pancreatic ductal adenocarcinoma is a malignant carcinoma with a 5-year survival rate of 11% characterized with poor prognosis, rising incidence, and resistance to chemo- and immune-therapies. Cancer cell evasion and suppression of anti-tumor immune responses in pancreas cancer are regulated in part by metabolic reprogramming within cancer and tumor accessory cells. We sought to determine if targeted inhibition of mitochondrial oxidative phosphorylation by mito-metformin (MMe) and newly developed variant pCF3-MMe influence the tumor immune microenvironment. Our results showed that MMe strongly inhibited tumor growth in murine models of pancreas cancer. Flow cytometry revealed a significant increase in total CD45+ leukocytes, with elevated cytolytic granzyme B+ and CD44+ CD8+ T cells. Immunodepleting CD8+ T cells, reversed the anti-tumor effects of MMe. Immunofluorescence microscopy and immunoblot analysis indicated that MMe stimulated oligomerization of mitochondrial antiviral-signaling protein (MAVS) and phosphorylation of IRF3 and IRF7. Further, levels of the IRF3/7 target chemokines CXCL9, CXCL10, and CXCL11 were increased in MMe-treated tumor cells, consistent with a role in CD8+ T cell infiltration. These data indicate OXPHOS inhibition stimulated virus-independent MAVS oligomerization, activation of downstream proinflammatory signaling, and reignition of the suppressed pancreatic tumor immune microenvironment. Funding Provided by NIH (2211376.1-5 R01 CA226279-01.04) and Center for Immunology MCW

Abstract 1644: Dual inhibition of MAT2A and PRMT5 delivers synergistic anti-tumor responses in preclinical models of MTAP-deleted cancer

Abstract Approximately 15% of all tumors harbor deletions in the 9p21 locus that encompass CDKN2A and MTAP. MTAP fuels the adenine and methionine salvage pathways by converting MTA into precursors of building blocks for protein and DNA production. In MTAP-deleted (MTAPdel) tumors, cellular MTA accumulates and partially inhibits the essential protein methyl transferase, PRMT5, through direct displacement of the cofactor S-adenosyl methionine (SAM) from the PRMT5 active site. This context provides therapeutic opportunities, currently under clinical evaluation, to selectively extinguish PRMT5 activity in tumor cells with MAT2A inhibitors that limit SAM synthesis or with direct PRMT5 inhibitors that are MTA-cooperative. Preclinical modeling of these synthetic lethal relationships, and elucidation of their cellular mechanisms of action, suggests efficacy can vary depending upon the extent of MTA accumulation and/or adaptive compensation to PRMT5 inhibition. To potentially maximize therapeutic activity among mechanistically heterogenous MTAPdel tumors, we evaluated combinatorial pathway suppression with MAT2A and PRMT5 inhibitors in MTAPWT and MTAPdel tumor models in vitro and in vivo. We noted synergistic antiproliferative effects when the potent and selective MAT2A inhibitor IDE397 was combined with any of multiple MTA-cooperative PRMT5 inhibitors in MTAPdel cell lines that exhibit sensitivity to each single agent. In vivo, the combination of IDE397 and MTA-cooperative PRMT5 inhibitors were well tolerated, and induced durable tumor regressions, including complete responses, at dose levels well below the maximally efficacious preclinical dose of each individual agent in MTAPdel lung adenocarcinoma and pancreas cancer models H838 and BXPC3. Pharmacodynamic target engagement in the tumor was evaluated by quantitative assessment of symmetric dimethyl arginine (SDMA), as detected by IHC, and indicated earlier onset and greater extent of PRMT5 inhibition by combination treatment as compared to either agent alone. The potentiation of the antitumor response with the combination did not occur in the MTAPWT setting. Thus, combined inhibition of MAT2A and PRMT5 potentially offers a compelling dual synthetic lethal opportunity to address unmet need for the many patients afflicted with MTAPdel cancers. Citation Format: Marcus M. Fischer, Kimberline Gerrick, Brian Belmontes, Katherine Slemmons, Yevgeniy Freyman, Jay Jain, Steve Federowicz, Isaac Bishof, Arjun A. Rao, Melissa Fleury, Zineb Mounir, Mark R. Lackner, Paul E. Hughes, Mike White, Claire L. Neilan. Dual inhibition of MAT2A and PRMT5 delivers synergistic anti-tumor responses in preclinical models of MTAP-deleted cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1644.

Abstract A056: Epithelial hyaluronan synthase 2 expression promotes pancreas cancer metastasis

Abstract A defining characteristic of pancreatic ductal adenocarcinoma (PDA) is its robust desmoplastic reaction that promotes tumor progression and protects PDA from treatment. As a major component of the extreme microenvironment, hyaluronan (HA) is synthesized by the hyaluronan synthase (Has) family of enzymes and secreted in excess by both epithelial cells and cancer-associated fibroblasts (CAFs). We have previously described the role of HA in binding water and swelling to create vessel-crushing interstitial gel-fluid pressures and its function as a biophysical barrier. However, HA also acts by signaling through a wide range of specific receptors that can drive proliferation, migration and invasion. In an effort to understand the role of HA in PDA development and pathogenesis, we developed a KPC mouse model of autochthonous pancreas cancer in which Has2 is conditionally deleted in tumor epithelial cells (TECs). We demonstrate that Has2-deficient epithelial cells produce little to no HA and altered the dense microenvironment. We also found that deletion of Has2 leads to an increase in overall survival and a striking reduction in metastasis. We have further investigated the cell autonomous functions of Has2, associated with 3D invasion and activation of oncogenic signaling pathways in TECs. These data implicate a potential Achilles’ heel in PDA, whereby HAS2 is required to maintain a highly metastatic phenotype involving both non-cell and cell autonomous mechanisms. Citation Format: Bo Tu, Heather Wright T. Wright, Christopher C. Dufort, Martin C. Whittle, Libing Feng, Shelley M. Thorsen, Sunil R. Hingorani. Epithelial hyaluronan synthase 2 expression promotes pancreas cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A056.

Abstract IA018: ATP production in pancreas cancer and tumor infiltrating lymphocytes

Abstract Diet can influence both the microbiome and (at least in mice) the outcomes of pancreas cancer therapy. For example, ketogenic diet can synergize with either current standard of care therapy ( gemcitabine + nab-paclitaxel) or PI3K inhibition in mouse models of pancreas cancer. I will discuss our recent work exploring how diet interacts with the microbiome, and how such interactions may influence pancreas cancer chemotherapy response. A simple hypothesis for how diet influences microbiome composition is that certain microbes prefer certain nutrients, and diets high in those nutrients lead to outgrowth of those microbes. To enable testing of this hypothesis, we developed isotope-tracer strategies that directly measure the nutrient preferences of different members of the gut microbiome. I will describe this new technology and its application to support the above hypothesis. I will further describe experiments manipulating diet and/or microbiome to influence the response of pancreas cancer to PI3K inhibition. The longterm goal is to understand, at a biochemical level, the links between diet, microbiome, and the effectiveness of cancer therapy, and to develop translatable strategies for manipulating metabolism via diet to improve therapeutic outcomes. Citation Format: Joshua D. Rabinowitz. ATP production in pancreas cancer and tumor infiltrating lymphocytes [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr IA018.

Abstract C042: Deletion of Gα13 promotes inflammation in the KPC mouse model of pancreatic cancer

Abstract G protein-coupled receptor (GPCR) signaling regulates many aspects of tumor biology, including inflammation, angiogenesis, and metastasis. However, the tumor-specific functions of G proteins, immediate downstream signaling partners of GPCRs, are not well understood. Counter to previous reports in epithelial cancer cell lines, we recently showed that Gα13, a member of the G12 family of heterotrimeric G proteins, is a tumor suppressor in the genetically engineered KPC (Pdx1-Cre; KrasG12D/+; Tp53R172H/+) mouse model of pancreas cancer (Shields et al., Cell Rep, 2022). Mechanistically, we found that Gα13 loss increases mTOR signaling in mouse and human pancreas tumors and that Gα13-deficient tumors are susceptible to rapamycin. We now show that Gα13 loss in the KPC mouse model is also associated with increased expression of inflammatory cytokines. Moreover, loss of Gα13 also promotes NF-κB signaling and inflammatory cytokines expression in cerulein-driven pancreatitis. Consistently, we find that loss of Gα13 increases the recruitment of macrophages and promotes acinar-ductal metaplasia in cerulein-driven pancreatitis. In our ongoing studies, we are evaluating the role of mTOR signaling in mediating the increased inflammation seen following the loss of Gα13 in our mouse models. Together, our studies show that the tumor-suppressive function of Gα13 is related to the regulation of mTOR signaling and inflammation in pancreas cancer. Citation Format: Mario A. Shields, Anastasia E. Metropulos, Christina Spaulding, Thao ND Pham, Hidayatullah G. Munshi. Deletion of Gα13 promotes inflammation in the KPC mouse model of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C042.

T Cell-Mediated Antitumor Immunity Cooperatively Induced By TGFβR1 Antagonism and Gemcitabine Counteracts Reformation of the Stromal Barrier in Pancreatic Cancer.

The desmoplastic stroma of pancreatic cancers forms a physical barrier that impedes intratumoral drug delivery. Attempts to modulate the desmoplastic stroma to increase delivery of administered chemotherapy have not shown positive clinical results thus far, and preclinical reports in which chemotherapeutic drugs were coadministered with antistromal therapies did not universally demonstrate increased genotoxicity despite increased intratumoral drug levels. In this study, we tested whether TGFβ antagonism can break the stromal barrier, enhance perfusion and tumoral drug delivery, and interrogated cellular and molecular mechanisms by which the tumor prevents synergism with coadministered gemcitabine. TGFβ inhibition in genetically engineered murine models (GEMM) of pancreas cancer enhanced tumoral perfusion and increased intratumoral gemcitabine levels. However, tumors rapidly adapted to TGFβ-dependent stromal modulation, and intratumoral perfusion returned to pre-treatment levels upon extended TGFβ inhibition. Perfusion was governed by the phenotypic identity and distribution of cancer-associated fibroblasts (CAF) with the myelofibroblastic phenotype (myCAFs), and myCAFs which harbored unique genomic signatures rapidly escaped the restricting effects of TGFβ inhibition. Despite the reformation of the stromal barrier and reversal of initially increased intratumoral exposure levels, TGFβ inhibition in cooperation with gemcitabine effectively suppressed tumor growth via cooperative reprogramming of T regulatory cells and stimulation of CD8 T cell-mediated antitumor activity. The antitumor activity was further improved by the addition of anti-PD-L1 immune checkpoint blockade to offset adaptive PD-L1 upregulation induced by TGFβ inhibition. These findings support the development of combined antistroma anticancer therapies capable of impacting the tumor beyond the disruption of the desmoplastic stroma as a physical barrier to improve drug delivery.

Abstract 1502: Pre-conditioning modifies the tumor microenvironment to enhance solid tumor CAR T cell efficacy and endogenous immunity

Abstract Chimeric antigen receptor (CAR) T cell therapy has led to impressive clinical responses in patients with hematological malignancies; however, its utility in patients with solid tumors has been limited. While CAR T cells for the treatment of advanced prostate cancer are being clinically evaluated and are anticipated to show bioactivity, their safety and the impact of the immunosuppressive tumor microenvironment (TME) have not been faithfully explored preclinically. Using a novel human prostate stem cell antigen knock-in (hPSCA-KI) immunocompetent mouse model and syngeneic murine PSCA CAR T cells, we performed analyses of normal and tumor tissues by flow cytometry, immunohistochemistry, and/or RNA sequencing. We further assessed the beneficial impact of cyclophosphamide (Cy) pre-conditioning on modifications to the immunosuppressive TME and impact on PSCA-CAR T cell safety and efficacy. We observed an in vivo requirement of Cy pre-conditioning in uncovering the efficacy of PSCA-CAR T cells in prostate and pancreas cancer models, with no observed toxicities in normal tissues with endogenous PSCA expression. This combination also dampened the immunosuppressive TME, generated pro-inflammatory myeloid and T cell signatures in tumors, and enhanced the recruitment of antigen-presenting cells, and endogenous as well as adoptively-transferred CAR T cells, resulting in long-term anti-tumor immunity. Citation Format: John P. Murad, Dileshni Tilakawardane, Anthony K. Park, Kelly T. Kennewick, Lupita S. Lopez, Hee J. Lee, Brenna J. Gittins, Wen-Chung Chang, Chau P. Tran, Catalina Martinez, Anna M. Wu, Robert E. Reiter, Tanya B. Dorff, Stephen J. Forman, Saul J. Priceman. Pre-conditioning modifies the tumor microenvironment to enhance solid tumor CAR T cell efficacy and endogenous immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1502.

Pre-conditioning modifies the TME to enhance solid tumor CAR T cell efficacy and endogenous protective immunity

Chimeric antigen receptor (CAR) T cell therapy has led to impressive clinical responses in patients with hematological malignancies; however, its effectiveness in patients with solid tumors has been limited. While CAR T cells for the treatment of advanced prostate and pancreas cancer, including those targeting prostate stem cell antigen (PSCA), are being clinically evaluated and are anticipated to show bioactivity, their safety and the impact of the immunosuppressive tumor microenvironment (TME) have not been faithfully explored preclinically. Using a novel human PSCA knockin (hPSCA-KI) immunocompetent mouse model, we evaluated the safety and therapeutic efficacy of PSCA-CAR T cells. We demonstrated that cyclophosphamide (Cy) pre-conditioning significantly modified the immunosuppressive TME and was required to uncover the efficacy of PSCA-CAR T cells in metastatic prostate and pancreas cancer models, with no observed toxicities in normal tissues with endogenous expression of PSCA. This combination dampened the immunosuppressive TME, generated pro-inflammatory myeloid and T cell signatures in tumors, and enhanced the recruitment of antigen-presenting cells, as well as endogenous and adoptively transferred T cells, resulting in long-term anti-tumor immunity.

Immune checkpoint inhibition in syngeneic mouse cancer models by a silicasome nanocarrier delivering a GSK3 inhibitor

Checkpoint blocking antibodies that interfere in the PD-1/PD-L1 axis provide effective cancer immunotherapy for tumors that are immune inflamed or induced to become “hot”. It has also been demonstrated that a small molecule inhibitor of the signaling hub kinase GSK3 can interfere in the PD-1/PD-L1 axis in T-cells by suppressing PD-1 expression. This provides an alternative approach to intervening in the PD-1/PD-L1 axis to provide cancer immunotherapy. In this communication, we demonstrate the remote loading of GSK3 inhibitor AZD1080 into the porous interior of mesoporous silica nanoparticles coated with a lipid bilayer (a.k.a. silicasomes). In a MC38 colon cancer model, intravenous injection (IV) of silicasome-encapsulated AZD1080 significantly improved biodistribution and drug delivery to the tumor site. The improved drug delivery was accompanied by cytotoxic MC38 tumor cell killing by perforin-releasing CD8+ T-cells, exhibiting reduced PD-1 expression. IV injection of encapsulated AZD1080 also resulted in significant tumor shrinkage in other syngeneic mouse tumor models, including another colorectal tumor (CT26), as well as pancreas (KPC) and lung (LLC) cancer models. Not only was the therapeutic efficacy of encapsulated AZD1080 similar or better than anti-PD-1 antibody, but the treatment was devoid of treatment toxicity. These results provide proof-of-principal demonstration of the feasibility of using encapsulated delivery of a GSK3 inhibitor to provide cancer immunotherapy, with the possibility to be used as a monotherapy or in combination with chemotherapy or other immunomodulatory agents.

Abstract LB-052: Photochemistry mediated tumor priming potentiates cytoreduction of distant metastases to enable potential surgical cure in a late stage model of pancreas cancer

Abstract Pancreatic cancer is a devastating disease for which the 5 year survival rate remains < 10% despite years of concerted research efforts. Potentially curative surgical resection cannot even be offered to most patients due to the presence of distant metastases at the time of diagnosis. Here, we present a neoadjuvant strategy comprised of photochemistry mediated tumor modulation combined with systemic therapy to significantly cytoreduce, and in 50% of cases completely eliminate, distant metastases to create a window of potential resectability. In a late stage model of pancreas cancer in which distant metastases are present at the time of treatment, combined photodynamic therapy (PDT) and gemcitabine/abraxane led to a 10,000 fold decrease in the number of distant metastases compared to untreated controls. In comparison, gemcitabine + abraxane, the current standard of care for pancreas cancer, only led to a 100 fold cytoreduction. 50% of animals treated with PDT + gem/abraxane harbored no detectable metastases 51 days after treatment compared to 0% of gem/abraxane treated animals, suggesting that PDT plus cytoreduction could enable potentially curative surgery in a sizeable percentage of animals. The strategy presented here is rapidly translatable and holds significant promise to dramatically increase the percentage of patients that are currently eligible for curative therapy for this devastating disease. Citation Format: Sriram Anbil, Zhiming Mai, Michael Pigula, Tayyaba Hasan. Photochemistry mediated tumor priming potentiates cytoreduction of distant metastases to enable potential surgical cure in a late stage model of pancreas cancer [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 LB-052.

Sufficiency of CD40 activation and immune checkpoint blockade for T cell priming and tumor immunity

Innate immune receptors such as toll-like receptors (TLRs) provide critical molecular links between innate cells and adaptive immune responses. Here, we studied the CD40 pathway as an alternative bridge between dendritic cells (DCs) and adaptive immunity in cancer. Using an experimental design free of chemo- or radiotherapy, we found CD40 activation with agonistic antibodies (⍺CD40) produced complete tumor regressions in a therapy-resistant pancreas cancer model, but only when combined with immune checkpoint blockade (ICB). This effect, unachievable with ICB alone, was independent of TLR, STING, or IFNAR pathways. Mechanistically, αCD40/ICB primed durable T cell responses, and efficacy required DCs and host expression of CD40. Moreover, ICB drove optimal generation of polyfunctional T cells in this "cold" tumor model, instead of rescuing T cell exhaustion. Thus, immunostimulation via αCD40 is sufficient to synergize with ICB for priming. Clinically, combination αCD40/ICB may extend efficacy in patients with "cold" and checkpoint-refractory tumors.

SPARC dependent collagen deposition and gemcitabine delivery in a genetically engineered mouse model of pancreas cancer.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is characterised by extensive matrix deposition that has been implicated in impaired drug delivery and therapeutic resistance. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that regulates collagen deposition and is highly upregulated in the activated stroma subtype with poor prognosis in PDAC patients.MethodsKrasG12D;p48-Cre;SPARC−/− (KC-SPARC−/−) and KrasG12D;p48-Cre;SPARCWT (KC-SPARCWT) were generated and analysed at different stages of carcinogenesis by histological grading, immunohistochemistry for epithelial and stromal markers, survival and preclinical analysis. Pharmacokinetic and pharmacodynamic studies were conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunohistochemistry following gemcitabine treatment (100 mg/kg) in vivo.FindingsGlobal genetic ablation of SPARC in a KrasG12D driven mouse model resulted in significantly reduced overall and mature collagen deposition around early and advanced pancreatic intraepithelial neoplasia (PanIN) lesions and in invasive PDAC (p < .001). However, detailed pathological scoring and molecular analysis showed no effects on PanIN to PDAC progression, vessel density (CD31), tumour incidence, grading or metastatic frequency. Despite comparable tumour kinetics, ablation of SPARC resulted in a significantly shortened survival in KC-SPARC−/− mice (280 days versus 485 days, p < .03, log-rank-test). Using LC-MS/MS, we show that SPARC dependent collagen deposition does not affect intratumoural gemcitabine accumulation or immediate therapeutic response in tumour bearing KC-SPARCWT and KC-SPARC−/−mice.InterpretationGlobal SPARC ablation reduces the collagen-rich microenvironment in murine PDAC. Moreover, global SPARC depletion did not affect tumour growth kinetics, grading or metastatic frequency. Notably, the dense-collagen matrix did not restrict access of gemcitabine to the tumour. These findings may have direct translational implications in clinical trial design.

Abstract 235: Preclinical activity of an antibody drug conjugate targeting tumor specificmuc1 structural peptide-glycotope

Abstract SAR566658 antibody drug conjugate (ADC) is a humanized DS6 (huDS6) antibody conjugated through a cleavable linker to the cytotoxic maytansinoid derivative DM4 that has been evaluated in the clinical setting. The purpose of our work was; 1) characterize the epitope targeted by anti DS6 on mucin1, 2) determine the prevalence of antigen expression in a patient population and 3) further explore preclinical activities of the ADC. Murine DS6 monoclonal antibody (muDS6) was generated from serous ovary adenocarcinoma immunization of immunocompetent mice. It specifically recognizes a MUC-1 tandem repeat domain in the context of cancer associated glycosylation. CA6-positive MUC-1 carries mucin-type O-linked glycans with α2,3-sialylated and β1,4-galactosylated termini, and antibody binding was abrogated by treating MUC-1 with specific glycosidases that remove either one of these glycan structures. However, the antibody did not bind to synthetic glycans modeled according to the major O-glycans of MUC-1. Our characterization of the peptide-glycotope leads us to conclude that tumor associated glycosylation is essential for the formation of the epitope on the peptide sequence of the MUC-1 tandem repeat domain. CA6 expression was evaluated by immunohistochemistry in paraffin embedded tumor tissue samples: 35.2% of breast cancer patients, 70,1% of ovarian cancer patients and 58,5% of bladder cancer patients have at least 30% of CA6 positive cells with an intensity of 2+/3+ in a multinational population-based study. In pre-clinical in vivo models, SAR566658 induced anti-tumor activity against CA6 positive tumor models of human pancreas, cervix and bladder cancer as well as and 3 Breast Patient-Derived Xenografts (PDX). Efficacy was correlated with MUC1-CA6 expression levels. In 3 additional models, SAR566658 showed anti-tumor activity that was more potent when compared to 3 conventional tubulin cytotoxic agents, docetaxel, vinorelbine and vinblastine. Citation Format: Marc Trombe, Anne Caron, Alexia Tellier, Chantal Carrez, Stephane Guérif, Severine Clavier, Nathalie Karst, Juhani Saarinen, Tero Satomaa, Virve Pitkänen, Olli Aitio, Annamari Heiskanen, Matteo Fassan, Jan Pinkas, Raffaele Baffa, Veronique Blanc, Celine Nicolazzi. Preclinical activity of an antibody drug conjugate targeting tumor specificmuc1 structural peptide-glycotope [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 235.

Obesity promotes tumor growth and induces transcriptomic and epigenomic changes in a murine pancreas cancer model

Obesity promotes tumor growth and induces transcriptomic and epigenomic changes in a murine pancreas cancer model

The CTLA-4 x OX40 bispecific antibody ATOR-1015 induces anti-tumor effects through tumor-directed immune activation

BackgroundThe CTLA-4 blocking antibody ipilimumab has demonstrated substantial and durable effects in patients with melanoma. While CTLA-4 therapy, both as monotherapy and in combination with PD-1 targeting therapies, has great potential in many indications, the toxicities of the current treatment regimens may limit their use. Thus, there is a medical need for new CTLA-4 targeting therapies with improved benefit-risk profile.MethodsATOR-1015 is a human CTLA-4 x OX40 targeting IgG1 bispecific antibody generated by linking an optimized version of the Ig-like V-type domain of human CD86, a natural CTLA-4 ligand, to an agonistic OX40 antibody. In vitro evaluation of T-cell activation and T regulatory cell (Treg) depletion was performed using purified cells from healthy human donors or cell lines. In vivo anti-tumor responses were studied using human OX40 transgenic (knock-in) mice with established syngeneic tumors. Tumors and spleens from treated mice were analyzed for CD8+ T cell and Treg frequencies, T-cell activation markers and tumor localization using flow cytometry.ResultsATOR-1015 induces T-cell activation and Treg depletion in vitro. Treatment with ATOR-1015 reduces tumor growth and improves survival in several syngeneic tumor models, including bladder, colon and pancreas cancer models. It is further demonstrated that ATOR-1015 induces tumor-specific and long-term immunological memory and enhances the response to PD-1 inhibition. Moreover, ATOR-1015 localizes to the tumor area where it reduces the frequency of Tregs and increases the number and activation of CD8+ T cells.ConclusionsBy targeting CTLA-4 and OX40 simultaneously, ATOR-1015 is directed to the tumor area where it induces enhanced immune activation, and thus has the potential to be a next generation CTLA-4 targeting therapy with improved clinical efficacy and reduced toxicity. ATOR-1015 is also expected to act synergistically with anti-PD-1/PD-L1 therapy. The pre-clinical data support clinical development of ATOR-1015, and a first-in-human trial has started (NCT03782467).