Pdx-1 Cre Research Articles

Abstract 3095: Promotion of pancreatic cancer by perfluorooctanoic acid (PFOA)

Abstract Pancreatic cancer is the fourth leading cause of cancer deaths and one of the most lethal forms of cancer diagnosed in the United States. While the exact cause of pancreatic cancer is unknown, established risk factors for pancreatic cancer include smoking, alcohol consumption and pancreatitis; all of which share the ability to generate oxidative stress - a condition known to promote cancer progression. Perfluorooctanoic acid (PFOA), a chemical widely used in consumer and industrial applications, has been shown to induce pancreatic acinar cell tumors in rodents through a yet to be determined mechanism. In humans, epidemiologic studies have linked PFOA exposure to adverse chronic health effects including several types of cancer. PFOA has been detected in essentially all of the American population, with mean serum levels close to 4 ng/ml and a predicted half-life of ~ 4 yrs. We have previously shown that exposure of mice to PFOA for 7 days triggered oxidative stress in the pancreas, which was associated with focal ductal hyperplasia and inflammation. The purpose of this study was to determine if PFOA exposure promotes the progression of pancreatic cancer in a well-characterized mouse model of pancreatic cancer, the LSL-KRasG12D;Pdx-1 Cre model. For our studies, LSL-KRasG12D;Pdx-1Cre mice received either tap water or tap water supplemented with 5 ppm PFOA, starting at 2 months of age. Mice were sacrificed at 6 and 9 months of age, corresponding to 4 and 7 months of PFOA treatment, at which time serum and tissues were collected. Pancreata were processed for histologic examination (H&E stain), immunohistochemistry for CK19, used as a marker to identify neoplastic lesion area, and RNA isolation for gene expression. Serum was utilized for quantitation of amylase, lipase, PFOA and inflammatory cytokine levels. Our preliminary results show that PFOA accumulates in the serum and pancreas of treated LSL-KRasG12D;Pdx-1 Cre mice. In addition, exposure to PFOA increased the CK19+ lesion area in LSL-KRasG12D;Pdx-1 Cre mice indicating an acceleration of pancreatic cancer progression. qPCR analysis of pancreata verified increased expression of both CK19 and the early lesion marker Sox9 in PFOA-treated mice. Analysis of mRNA expression in the pancreas revealed an increase in oxidative stress, evidenced by elevated Sod1 expression, and an increase in inflammatory markers (Tnfα and IL1α). Together, these results demonstrate that PFOA leads to expansion of the pancreatic lesion area in the LSL-KRasG12D;Pdx-1 Cre mouse model which is accompanied by oxidative stress and an inflammatory response. These results, coupled with the widespread human exposure to PFOA and its biological persistence, suggest that it could be a prominent agent promoting pancreatic cancer progession. Citation Format: Barbara A. Hocevar, Lisa M. Kamendulis. Promotion of pancreatic cancer by perfluorooctanoic acid (PFOA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3095.

Abstract 4940: Nonredundant roles for immune checkpoint blockade and agonistic CD40 in mediating T-cell responses in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) is a highly aggressive cancer that is resistant to most treatments, with a five-year survival rate of ~10%. Immune checkpoint blockade (ICB) has had dramatic effects in many tumor histologies, but is ineffective in PDA. CD40 lies upstream of the immune checkpoints in the T-cell activation pathway, presenting a unique opportunity to intercede with T-cell activation independently of ICB. We hypothesized that administering agonistic CD40 antibody would prime effector T-cell populations, synergizing with ICB to provide therapeutic benefit in PDA. We administered agonistic CD40 monoclonal antibody (clone FGK45, anti-CD40) combined with anti-PD1 and anti-CTLA4 monoclonal antibodies (ICB) to mice bearing subcutaneous PDA tumors established using syngeneic tumor cell lines derived from the KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) genetically engineered mouse model of PDA. While tumor regressions were rare in mice treated with ICB or anti-CD40 alone (1/7 mice in each group), 100% of mice treated with ICB + anti-CD40 experienced tumor regressions (7/7 mice). Cell depletion experiments showed early tumor regressions depended primarily on CD4 T cells, although both CD4 and CD8 T cells were required for long-term remission. 50% of mice (4/8) treated with ICB + anti-CD40 remained tumor-free (median survival 112 days) and 3/4 mice resisted tumor rechallenge, indicative of a long-lived memory T-cell response against PDA. Treatment with ICB + anti-CD40, but neither alone, increased the total intratumoral CD3+ T-cell population 3.8-fold (p<0.01), with increases in both the CD4+ and CD8+ T-cell compartments (6.7-fold and 3.3-fold, respectively, p <0.016). CD40 stimulation, but not ICB, modulated the CD4 compartment in the TME, increasing the proportion of CD43+CD11a+ antigen-specific CD4 T helpers (41 - 43% v. 10%, +/-ICB v. control, p <0.03) and reducing FoxP3+ CD4+ T regulatory cells (Tregs) 2.7 to 3.7-fold (+/- ICB, p < 0.0001). This resulted in an increased ratio of CD8 T effector cells to Tregs (7.5 to 10.7 CD8/Treg, anti-CD40 +/-ICB, p<0.01) specifically after treatment with anti-CD40, regardless of ICB addition. ICB alone increased the proportion of proliferating CD8 T cells in the tumor draining lymph node that were ‘"reinvigorated" Eomes+Tbet-Ki67+ (17-19% +/- CD40 v. 8% control, p <0.01), but was unable to increase the same CD8 T-cell population in the tumor. However, the addition of CD40 to ICB reduced the frequency of PD1+ CD8 T effectors in the tumor (25% v. 52% control, p < 0.05), suggesting a tumor-specific benefit of ICB on the exhausted T-cell compartment. These studies reveal the nonredundant roles that agonistic CD40 and ICB play in generating a robust T-cell response against PDA, and highlight the clinical potential of combining CD40 activation with ICB as a novel therapeutic approach in ICB-resistant tumors. Citation Format: Alexander H. Morrison, Katelyn T. Byrne, Robert H. Vonderheide. Nonredundant roles for immune checkpoint blockade and agonistic CD40 in mediating T-cell responses in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4940.

Abstract 1011: Tumor cell intrinsic factors dictate T cell infiltration and therapeutic response in pancreatic ductal adenocarcinoma

Abstract The establishment of immune heterogeneity in the tumor microenvironment (TME) is poorly understood, despite recent data that the success of immunotherapies is dictated by the immune environment of the tumor site. Pancreatic ductal adenocarcinoma (PDA) is characteristically devoid of CD8 T cells and resistant to therapeutic intervention. However, a small subset of patients (15%) have tumors highly infiltrated by CD8 T cells, correlating with improved overall survival. To better elucidate the determinants of immune heterogeneity in the PDA TME, we generated clones from spontaneous tumors harvested from KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mice, a genetically engineered mouse model of PDA. Using a panel of 17 tumor clones, we found the clones segregated in to two groups with differential immune cell infiltration upon implantation in congenic C57BL/6 mice. 7/17 tumor clones were categorized as ‘T cell high,' with an immune infiltrate comprising CD8 T cells, CD103+ dendritic cells (DCs), and a dearth of myeloid derived suppressor cells (MDSCs). In contrast, the remaining 10/17 tumor clones were categorized as ‘T cell low' lines, with TME dominated by myeloid cells and macrophages, especially granulocytic MDSCs. Hypothesizing that increased T cell infiltrate would render PDA sensitive to therapy, we treated two T cell high and two T cell low tumor clones with combination immunotherapy and chemotherapy. Mice bearing T cell high clones responded to therapy (7/7 and 4/7 mice cured) and formed protective memory responses against secondary tumor challenge, while none of the mice bearing T cell low tumors responded to treatment (0/7 and 0/7 mice cured). At baseline, T cell high tumors had similar proportions of functional CD8 T cells as in T cell low tumors (62.9% vs. 50.2% IFN-g+ in T cell high vs low, p=0.06). However, the proportion of activated CD44hiPD-1+ CD8 T cells was significantly increased in T cell high tumors (62.2% vs. 35.1% in T cell low clones, p<0.0001), and predicted response to therapy across the panel of tumor clones. CD44hiPD-1+ CD8 T cells trafficked in to the tumor site independently of CXCR3 (53.8% in control vs. 50.0% in anti-CXCR3 treated tumors, p=0.8), in contrast to the bulk CD8 T cell population which required CXCR3 to enter the TME (7.5% in control vs 0.53% in anti-CXCR3 treated tumors, p<0.01). Furthermore, in Batf3 KO mice, T cell high tumors lacked CD8 T cells and resembled T cell low tumors, indicating the requirement for CD103+ DCs in the TME for T cell infiltration. These data reveal tumor cell intrinsic factors as major determinants of immune heterogeneity in the TME, and highlight the use of this panel of clonal tumor lines as a tool to probe the immune heterogeneity of the TME. Future studies will identify the specific factors regulating T cell infiltration and mechanisms to convert T cell low tumors to T cell high tumors, thereby improving responses to immunotherapy. Citation Format: Katelyn T. Byrne, Jinyang Li, Robert H. Vonderheide, Ben Z. Stanger. Tumor cell intrinsic factors dictate T cell infiltration and therapeutic response in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1011.

Abstract 4708: Imprime (beta-1,3/1,6 glucan) synergizes with a CD40 agonist to stimulate T cell-dependent antitumor activity in a poorly immunogenic model of pancreatic carcinoma

Abstract Introduction: While some solid tumors show remarkable sensitivity to immunotherapy, pancreatic ductal adenocarcinoma (PDAC) has demonstrated impressive resistance. This poor responsiveness to immunotherapy may reflect multiple mechanisms necessary for generating productive T-cell immunosurveillance, including ineffective tumor antigen presentation by myeloid cells. Here, we hypothesized that combining two immune agonists capable of “licensing” myeloid cells with enhanced T-cell stimulatory properties would synergize to reverse the poorly immunogenic state of pancreatic cancer and, in doing so, drive productive T cell-dependent antitumor immunity. Methods: C57BL/6 mice were challenged subcutaneously with a syngeneic PDAC cell line (7940B.PDA) derived from a tumor arising spontaneously in the KrasG12D/+; Trp53R172H/+; Pdx-1 Cre (KPC) mouse model. Mice with tumors measuring approximately 5 mm3 in diameter were treated with defined combinations of gemcitabine chemotherapy, a CD40 agonist (FGK45), and Imprime (a yeast β-1,3/1,6 glucan that coordinately activates innate and adaptive immune responses through pattern recognition receptors). CD4 and CD8 T cells were depleted in vivo using GK1.5 and 2.43 antibodies, respectively. Tumor growth curves and overall survival were determined. Results: Monotherapy with Imprime or a CD40 agonist only modestly delayed tumor outgrowth compared to chemotherapy alone. Delivering chemotherapy 48 hours prior to Imprime administration produced a two-fold increase in the median overall survival, but responses were not durable and all mice relapsed. In contrast, concurrent single administration of Imprime and a CD40 agonist induced complete and durable regressions in 60% of mice. Tumor-free mice resisted tumor rechallenge and therapeutic efficacy was completely abrogated with CD4/CD8 cell depletion. Conclusions: The combination of two myeloid-directed immune agonists (Imprime and a CD40 agonist) shows promising activity for stimulating T cell-dependent antitumor immunity against PDAC, a poorly immunogenic cancer. Citation Format: Mingen Liu, Kathleen Graham, Anissa S. Chan, Nadine R. Ottoson, Nandita Bose, Gregory L. Beatty. Imprime (beta-1,3/1,6 glucan) synergizes with a CD40 agonist to stimulate T cell-dependent antitumor activity in a poorly immunogenic model of pancreatic carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4708.

Abstract 627: Antitumor effect of an oligosaccharide API in a genetically engineered mouse-derived allograft (GEDA)

Abstract The use of oligosaccharides as active pharmaceutical ingredients lags behind that of other biological molecules such as proteins and nucleic acids. However, there is now a growing understanding of the complex biological functions of saccharide structures. This has resulted in an increased interest in exploring the pharmaceutical applications of both oligosaccharides and glycoconjugates. Despite recent advances in the treatment of pancreatic adenocarcinoma (PDAC), the median survival remains <12 months. Patients typically present with late stage disease and are often unable to tolerate drug combination regimens due to the associated toxicity. Here we report a novel oligosaccharide drug candidate, RiXOVA (G-blocks). The term “G-blocks” refers to highly defined and specialized short oligomers comprising α-L-guluronate residues derived from alginates extracted from brown seaweed. The G-block oligomers are well tolerated at i.v./i.p. doses >100mg/kg in mice. These oligomers have previously demonstrated the ability to alter matrices of biological macromolecules including mucins, giving rise to the hypothesis that they may have similar effects on the dense desmoplastic tissue within tumours. We here describe the anti-tumour properties of RiXOVA in different PDAC mouse models. Initial efficacy studies were performed in a xenograft model using the CAPAN-2 cells. RiXOVA was found to be non-toxic and non-immunogenic (I.P, 25mg/kg Q3D10 ) in mice. Relative to the start of dosing, the tumour growth in RiXOVA treated mice was 160% +/-30 (n=10) compared to 240% +/- 61 (n=10, p value=0.001) in the vehicle treated mice. Further testing was carried out in a Genetically Engineered Mouse-Derived Allograft (GEDA). The GEDA model recapitulates the dense desmoplastic stroma of the original donor (KPC mouse), unlike cell line-based allografts. KPC (LSL-KrasG12D; LSL-Trp53R172H; Pdx1-cre) mouse tumour fragments (2 donors) were implanted subcutaneously in the flank of recipient LSL-Trp53R172H; Pdx1-cre (PC), immunocompetent mice. Relative to the start of dosing, the tumour growth in the RiXOVA treated group (25 mg/kg TIW) was 260% +/- 160 (n=10), compared to 540% +/- 210 (n=8) for the Vehicle (p = 0.001) and 130 % +/- 57 (n=9) for the combination of RiXOVA + gemcitabine (100 mg/kg BIW p <0.001) compared to the vehicle. Ex vivo analysis of tumour tissue revealed changes in the expression and distribution of ECM proteins. Collectively, these results indicate RiXOVA to be a non-toxic oligosaccharide, that acts at the level of the tumour microenviroment to inhibit tumour growth. Whilst these results are in a preliminary stage they indicate G-block oligomers may represent a novel treatment modality in PDAC. Citation Format: Shalini V. Rao, Tonje S. Steigedal, Aarthi Gopinathan, Synnøve N. Magnussen, Sabina S. Strand, Duncan Jodrell, Fran Richards, Kurt I. Draget, Catherine T. Nordgård. Antitumor effect of an oligosaccharide API in a genetically engineered mouse-derived allograft (GEDA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 627.

Gemcitabine-Induced TIMP1 Attenuates Therapy Response and Promotes Tumor Growth and Liver Metastasis in Pancreatic Cancer.

Gemcitabine constitutes one of the backbones for chemotherapy treatment in pancreatic ductal adenocarcinoma (PDAC), but patients often respond poorly to this agent. Molecular markers downstream of gemcitabine treatment in preclinical models may provide an insight into resistance mechanisms. Using cytokine arrays, we identified potential secretory biomarkers of gemcitabine resistance (response) in the transgenic KRasG12D; Trp53R172H; Pdx-1 Cre (KPC) mouse model of PDAC. We verified the oncogenic role of the cytokine tissue inhibitor of matrix metalloproteinases 1 (TIMP1) in primary pancreatic tumors and metastases using both in vitro techniques and animal models. We identified potential pathways affected downstream of TIMP1 using the Illumina Human H12 array. Our findings were validated in both primary and metastatic models of pancreatic cancer. Gemcitabine increased inflammatory cytokines including TIMP1 in the KPC mouse model. TIMP1 was upregulated in patients with pancreatic intraepithelial neoplasias grade 3 and PDAC lesions relative to matched normal pancreatic tissue. In addition, TIMP1 played a role in tumor clonogenic survival and vascular density, while TIMP1 inhibition resensitized tumors to gemcitabine and radiotherapy. We observed a linear relationship between TIMP-1 expression, liver metastatic burden, and infiltration by CD11b+Gr1+ myeloid cells and CD4+CD25+FOXP3+ Tregs, whereas the presence of tumor cells was required for immune cell infiltration. Overall, our results identify TIMP1 upregulation as a resistance mechanism to gemcitabine and provide a rationale for combining chemo/radiotherapy with TIMP1 inhibitors in PDAC. Cancer Res; 77(21); 5952-62. ©2017 AACR.

Tumor-Derived CCL2 Mediates Resistance to Radiotherapy in Pancreatic Ductal Adenocarcinoma.

Local tumor growth is a major cause of morbidity and mortality in nearly 30% of patients with pancreatic ductal adenocarcinoma (PDAC). Radiotherapy is commonly used for local disease control in PDAC, but its efficacy is limited. We studied the impact of selectively intervening on radiotherapy-induced inflammation as an approach to overcome resistance to radiotherapy in PDAC. PDAC cell lines derived from primary pancreatic tumors arising spontaneously in KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx-1 Cre mice were implanted into syngeneic mice and tumors were focally irradiated using the Small Animal Radiation Research Platform (SARRP). We determined the impact of depleting T cells and Ly6C+ monocytes as well as inhibiting the chemokine CCL2 on radiotherapy efficacy. Tumors were analyzed by flow cytometry and IHC to detect changes in leukocyte infiltration, tumor viability, and vascularity. Assays were performed on tumor tissues to detect cytokines and gene expression. Ablative radiotherapy alone had minimal impact on PDAC growth but led to a significant increase in CCL2 production by tumor cells and recruitment of Ly6C+CCR2+ monocytes. A neutralizing anti-CCL2 antibody selectively inhibited radiotherapy-dependent recruitment of monocytes/macrophages and delayed tumor growth but only in combination with radiotherapy (P < 0.001). This antitumor effect was associated with decreased tumor proliferation and vascularity. Genetic deletion of CCL2 in PDAC cells also improved radiotherapy efficacy. PDAC responds to radiotherapy by producing CCL2, which recruits Ly6C+CCR2+ monocytes to support tumor proliferation and neovascularization after radiotherapy. Disrupting the CCL2-CCR2 axis in combination with radiotherapy holds promise for improving radiotherapy efficacy in PDAC. Clin Cancer Res; 23(1); 137-48. ©2016 AACR.

Abstract B54: Genetically engineered mouse-derived allografts (GEDAs): an immunocompetent mouse model of PDAC for the evaluation of novel therapeutic strategies

Abstract Despite recent advances in the treatment of patients with pancreatic adenocarcinoma (PDAC), survival remains low. Pre-clinical models of human malignancies often fail to capture the complexity of tumors and as such lack clinical predictive power. Patient-derived tumor xenografts (PDXs) are an emerging pre-clinical platform, capable of retaining the molecular heterogeneity of their originating sample and able to predict the response to therapies in some cases. However, the lack of immune system of the mouse PDX host is a major drawback for a preclinical model, especially with the advent of promising cancer immunotherapies (e.g. anti-PD-1/PD-L1, CXCR4 antagonist). Genetically Engineered Mouse models (GEMMs) carry mutations in genes that are associated with the human disease and can thus mimic the genetic, phenotypic and physiological aspect of the human disease, in an immunocompetent background. The Lox-Stop-Lox (LSL)-Kras(G12D); LSL-Trp53(R172H); Pdx1-cre (KPC) mouse is a favoured model of PDA, recapitulating the desmoplastic tumor histology, immune profile and drug resistance of human PDAC. However, KPC tumor development speed is variable and tumor monitoring requires ultrasound imaging, so therapeutic studies are long, complex and expensive. We describe here a new model, Genetically Engineered mouse-Derived Allograft (GEDA), that applies the methods developed for PDXs to KPC tumors. Pieces of KPC tumor were implanted subcutaneously in the flank of recipient LSL-Trp53 (R172H); Pdx1-cre (PC), immunocompetent mice. At primary passage, 10 mice were implanted from a single primary tumor, and the engraftment rate is 90%. Tumors are palpable 2 weeks after the tumor implantation and can be monitored by calliper measurement for up to 10 weeks, with a tumor doubling time of 14.4 ± 1.5 days. The incidence of metastases was similar to KPC mice, but organ distribution was different, with 57% of 45 recipient mice developing lung metastases, and 0% liver metastases. Histologically the GEDAs appeared more like primary KPC tumors than subcutaneous allograft of KPC cell lines, with intratumoral heterogeneity including desmoplastic regions identified by the presence of collagen fibres and αSMA-expressing cells. Implantation of GEDAs onto GFP-mice revealed that the original donor stroma disappeared within 2 weeks and was replaced by host stroma, but the stromal composition remained comparable even over 5 passages in vivo, with ±15% of variation in αSMA-expressing area between donor and recipients. Typically, around 20% of the GEDA cells were positive for Ki67, remaining stable over 5 passages, suggesting that aggressiveness of the tumor was stable. Qualitatively, by CD3 IHC, the T cell distribution is similar to that reported in KPC tumors (Feig et al, PNAS 2013) with T cell exclusion from tumor cell nests. A therapeutic study was performed with gemcitabine. Tumor growth was not inhibited by gemcitabine (100 mg/kg IP, twice per week); tumor volume change at day 60 (%): 1544 ± 263 (treated) vs. 1350 ± 156 (control), n= 6 per group, p=0.25 ). Few KPC primary tumors show significant response to gemcitabine, so the PDA GEDA was more like KPC than subcutaneous allografts of KPC cell lines which tend to be gemcitabine sensitive. A biobank of KPC GEDAs is now being generated, which will be tested to determine the heterogeneity of therapeutic response to gemcitabine and other agents.In conclusion, we have generated PDAC GEDAs, which combine advantages of both GEMMs and PDXs, cost-effective generation of cohorts of mice for therapeutic studies. The stromal content and intact immune system should enable the evaluation of immunomodulatory therapies and also agents targeting the stromal components of PDAC. Citation Format: Séverine Mollard, Kristopher K. Frese, Aarthi Gopinathan, Frances M. Richards, Duncan I. Jodrell.{Authors}. Genetically engineered mouse-derived allografts (GEDAs): an immunocompetent mouse model of PDAC for the evaluation of novel therapeutic strategies. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B54.

Abstract A07: Identification of endocrine/metabolic biomarkers associated to early PDC in a transgenic mouse model

Abstract Background: Pancreatic ductal adenocarcinoma (PDC) is a deadly disease, even surgery contributes little to survival if PDC is not diagnosed at an initial stage. Glucose homeostasis alteration associated to PDC was described to identify an high risk population for screening. Moreover the weight loss often manifests several months before cachexia. The crosstalk between metabolism and cancer could be a key to early diagnosis. Our aim is to find endocrine/metabolic biomarkers of early PDC development in a gold standard murine model. Materials and Methods: Candidate markers were investigated using LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) mouse, a genetically-engineered model that develops PDC with 100% penetrance. KPC has been fed with standard diet (SD) or high fat diet (HFD) introducing a further risk factor for diabetes and metabolic alterations. To overcome the problem of asynchronous development of PDC in KPC, 7T-MRI was used to classify mice in actual pathological stage: normal (S1); cystic degeneration (S2); small tumor (S3) and advanced pancreatic cancer (S4). We performed metabolic test (OGTT) and serum endocrine/metabolic profile (xMAP technology and clinical biochemistry) in n=27 KPC (and control mice PDX-1 Cre) fed with HFD vs n=26 KPC (and control mice PDX-1 Cre) fed with SD. To prioritize markers we computed the ROC curve analysis. Results: i) Glucose metabolism: KPC were normoglycemic regardless of the diet; the AUC in OGTT increases in KPC fed with HFD (HFD vs SD: S1 p&amp;lt;0,001, S2 p&amp;lt;0,001, S3 p=0,002, Cre p&amp;lt;0,001) ii) Serum levels of 29 endocrine/metabolic biomarkers were assessed during disease progression. The more relevant results are: increase of PYY (p&amp;lt;0,003) and decrease of leptin (p&amp;lt;0,02), GIP (p&amp;lt;0.005) albumin (p&amp;lt;0.001) both in HFD and SD fed KPC (Cre vs S2); increase of amylase in stage 1 HFD-fed KPC (p=0,037). Moreover PYY was able to discriminate control from stage 2 KPC with an AUC=0,929 in the ROC curve analysis (p&amp;lt;0,001). Conclusions: Unlike human, in the mouse model the development of PDC is not associated with an hyperglycemic status, regardless of the diet. However KPC fed with HFD experience a mild impairment of glucose metabolism (as well as in control mice) and a mild pancreatic damage. Our results identify Leptin, PYY, GIP and albumin as markers of early stage PDC in a preclinical model with a diagnostic potential to be validated in human patients. Citation Format: Valentina Pasquale, Erica Dugnani, Daniela Liberati, Paolo Marra, Tamara Canu, Antonio Esposito, Lorenzo Piemonti1.{Authors}. Identification of endocrine/metabolic biomarkers associated to early PDC in a transgenic mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A07.

Abstract 3213: Hypomethylation therapy leads to immune polarization and improved efficacy of immunotherapy in transgenic model of pancreatic cancer

Abstract BACKGROUND Despite significant activity of immunotherapies in several malignancies, their efficacy in pancreatic cancer has been limited. One possible explanation may be the absence of a tumor microenvironment and immune cells that are necessary. We have demonstrated that 5-aza-2-deoxycitidine (DAC) had a profound effect on pancreatic cancer associated stroma and up-regulated many immune pathways. Therefore we proposed that hypomethylating therapy may alter the microenvironment to allow immue checkpoint inhibitors greater therapeutic efficacy. METHODS Transgenic mice that spontaneously develop pancreatic cancer (Kras, Trp53, Pdx-1 Cre [KPC]) underwent weekly transabdominal ultrasound to detect mice initially with larger (6-8 mm) and subsequently with smaller (3-5 mm) tumors. PBS or DAC (5 mg/kg) and anti -PD1H or isotype control antibody were administered every 72 hours intraperitoneally for 3 doses. Tumor size was monitored biweekly and 3D tumor volume reconstruction was used to estimate size. The immune cell infiltrate was characterized by quantitative immunohistochemistry or by FACS. RESULTS Mice treated with DAC with 6-8 mm tumors demonstrated a significantly increased survival and tumor necrosis as well as polarization of the infiltrating macrophages towards an M1 (CD86, CD163) phenotype and a trend towards an increase in infiltrating CD8 cells compared with controls. The shift in the T cell population and the macrophage phenotypes was not seen in the spleen or peripheral blood of either wild type or tumor bearing animals treated with DAC compared with controls. We subsequently treated mice with 3 doses of DAC followed by 3 doses of anti -PD1H and compared them to anti-PD1H alone in KPC mice with smaller (3-5 mm) tumors. In DAC + anti-PD1H we noted a decrease in the growth rate of tumors, an increased survival and a significant increase in necrosis and CD8 infiltrates, as well as a significant decrease in Ki67 and increase in Tunel staining compared with anti-PD1H alone. CONCLUSIONS Our results suggest that DAC pre-treatment prior to checkpoint inhibitors may favorably polarize the tumor infiltrating immune cell population, lead to increased recruitment of CD8 positive T cells, result in marked tumor necrosis and slow tumor growth. These effects ultimately contribute to increased therapeutic efficacy of checkpoint inhibitors in pancreatic cancer. We plan to optimize the efficacy of epigenetic therapy and checkpoint inhibitors by using other monoclonal antibodies (anti-PD1, PD1L)and different dosing regimens. Citation Format: Tamas Gonda, Jarwei Fang, Catheine Do, Anna Zhukovskaya, Martha Salas, Benjamin Tycko. Hypomethylation therapy leads to immune polarization and improved efficacy of immunotherapy in transgenic model of pancreatic cancer. [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 3213.

Abstract A076: CD40 stimulation overrides role of innate immune sensors for priming of T cells in cancer

Abstract CD40 ligation is a critical step in the activation and maturation of CD40+ antigen-presenting cells (APCs) and facilitates robust T cell priming, but the interaction of CD40 activation with innate immune sensors — particularly in combination with anti-tumor chemotherapy — is unknown. To investigate this issue, we used the KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDA) to study the effect of chemotherapy with agonistic CD40 treatment. KPC mice bearing spontaneous PDA were diagnosed by ultrasound and treated with gemcitabine (Gem) and nab-paclitaxel (nP), with or without agonist CD40 antibody (FGK45). Gem/nP/CD40-treated mice exhibited a tumor response rate of 37.5% vs. 0% in the Gem/nP group; however, no regressions were observed in Gem/nP/CD40-treated KPC mice if they were first depleted of CD8 T cells, indicating T cell dependency. To understand the mechanism of Gem/nP/CD40 treatment, a PDA cell line was developed from a C57Bl/6 KPC mouse with a spontaneous tumor and injected subcutaneously into syngeneic mice. Mice bearing established tumors that were treated with Gem/nP/CD40 had increased rates of tumor regression (59.7%) compared to Gem/nP treatment (11.2%) or CD40 alone (15.3%), as well as significantly reduced tumor growth kinetics (p&amp;lt;0.0001), and improved overall survival (p&amp;lt;0.01) compared to either Gem/nP or CD40 alone. The anti-tumor effect was entirely lost in CD40 knockout (KO) mice or in IFN-γ KO mice, or in mice depleted of CD8 T cells. In the tumor site at day 12, while overall CD3+ T cell populations did not vary across cohorts, the proportion of CD4+FoxP3+ T regulatory (Treg) cells was reduced 6.9-fold after Gem/nP/CD40 and the proportion of activated CD44hiCD4+ T helper cells increased 1.8-fold compared to control treatment (p&amp;lt;0.01 in each case). The ratios of both CD4 T helper and CD8 T effector cells to Tregs were significantly increased (p&amp;lt;0.001 and p&amp;lt;0.05, respectively). Two days after CD40 therapy, APCs in the tumor had increased CD86 and MHCII (37% vs. 10% in control group, p&amp;lt;0.0001), and increased production of both TNF-α and IL-12p40. Despite this evidence for a robust CD40-dependent T cell anti-tumor response with Gem/nP/CD40, there was no impact on this phenotype when we studied KO mice deficient for classically described innate immune sensors: Gem/nP/CD40 treatment was fully effective in host mice genetically deficient in expression of MyD88, TLR4, TLR3, TRIF, IFNAR, STING, Caspase1, Caspase11, IL-1R, and TNF-α. Thus, despite the well-described role of innate immune sensors in potentiating T cell responses to tumors, agonist CD40 therapy is sufficient to override the requirement for any of these pathways. Our findings reveal that CD40 stimulation bypasses canonical mediators of immune activation, and suggests from a translational standpoint, that CD40 agonists may be critical and non-redundant pieces of a combination armamentarium for the treatment of highly immunosuppressive cancers. Citation Format: Katelyn T. Byrne, Robert H. Vonderheide. CD40 stimulation overrides role of innate immune sensors for priming of T cells in cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A076.

Abstract PR07: Agonistic CD40 antibody combined with chemotherapy drives TLR4/MyD88 independent regression of pancreatic tumors in a T cell dependent manner

Abstract Pancreatic ductal adenocarcinoma (PDA) is a highly aggressive cancer that is resistant to most treatments, and more than 90% of patients with metastatic PDA die within five years of diagnosis. Recent clinical studies show that combining gemcitabine (Gem) with nanoparticle albumin-bound paclitaxel, Abraxane (Abrx), prolongs progression-free and overall survival as compared to Gem alone, but the objective response rate of the combination was only 23%, and nearly every patient eventually progressed. Given that CD40 activation can reverse tumor-induced immune suppression and promote anti-tumor T cell responses, we administered agonistic CD40 monoclonal antibody (FGK45) with combined Gem/Abrx chemotherapy to mice bearing subcutaneous PDA tumors established using syngeneic tumor cell lines derived from the KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) genetically engineered mouse model of PDA. We found only rare tumor regressions in mice treated with chemotherapy alone (Gem, Abrx, or Gem/Abrx) or FGK45 alone, whereas more than 50% of mice treated with Gem/Abrx/FGK45 experienced tumor regressions (p&amp;lt;0.0001). Mechanistic studies revealed that this effect was T cell dependent, and in some mice treated with Gem/Abrx/FGK45, tumors were completely rejected within 3 weeks and the mice remained tumor free for over 100 days, suggesting long-lived T cell memory directed against PDA. Although the overall CD3+ T cell population in the tumor after therapy did not vary across treatment cohorts, the proportion of FoxP3+CD4+ T regulatory cells (Tregs) was reduced 6.9-fold after treatment with Gem/Abrx/FGK45 (p&amp;lt;0.05, Gem/Abrx/FGK45 treatment versus chemotherapy or FGK45 alone), with a concurrent 1.75-fold increase in activated CD44hiCD4+ T helper cells (p&amp;lt;0.01). As a result, the ratio of CD4 Thelper or CD8 T effector cells to Tregs was increased intratumorally. The reduction of Tregs was observed early after therapy, and in the absence of Treg to Thelper conversion, suggesting a change in the priming of the CD4 compartment. Indeed, within 24 hours after Gem/Abrx/FGK45 treatment, antigen-presenting cells (APCs) in the tumor and draining lymph node had increased expression of both CD86 and MHCII (37% versus 10% in control group, p&amp;lt;0.0001 by ANOVA), as well as increased production of TNF-alpha (39% versus 24% in control group, p&amp;lt;0.0001 by ANOVA). These changes in the activation status of APCs in the Gem/Abrx/FGK45 treated-tumor microenvironment may help promote Thelper differentiation. Furthermore, responses to Gem/Abrx/FGK45 are independent of the TLR4 and MyD88 pathways, which have previously been reported as critical mediators of the anti-tumor immune response generated with chemotherapy. Spontaneous PDA tumors arising in KPC mice were also susceptible to Gem/Abrx/FGK45 therapy, exhibiting slowed growth rates and even tumor regression after treatment (37.5% stable disease or tumor regression, versus 0% in Gem/Abrx combined group), in a CD8 dependent manner. These studies not only highlight the clinical potential of adding CD40 activation to standard-of-care chemotherapy as a novel strategy for PDA, but also underscore an emerging hypothesis that T cell immune surveillance can be effective against this otherwise highly immunosuppressive tumor if triggered by robust vaccination. This abstract is also presented as Poster A40. Citation Format: Katelyn T. Byrne, Robert H. Vonderheide. Agonistic CD40 antibody combined with chemotherapy drives TLR4/MyD88 independent regression of pancreatic tumors in a T cell dependent manner. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr PR07.

Abstract LB-204: Reprogramming inflammatory monocytes to mediate anti-tumor activity in pancreatic carcinoma

Abstract In cancer, increased mobilization of inflammatory monocytes from the bone marrow into the peripheral blood correlates inversely with patient survival. Peripheral blood inflammatory monocytes, which express CCR2, are recruited to tumor tissue by the chemokine CCL2 where they then differentiate into macrophages and support tumor development, growth, and metastasis. While neutralization of CCL2 can block inflammatory monocyte recruitment and inhibit metastasis, this approach has recently been shown to be at risk for lethal outcomes if therapy is interrupted. An alternative approach to inhibiting inflammatory monocyte recruitment to tumors is to reprogram monocytes with anti-tumor activity. Using the KrasG12D/+; Trp53R172H/+; Pdx-1 Cre (KPC) genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC), we report that systemic immune activation induced with an agonist CD40 monoclonal antibody can redirect Gr-1+ inflammatory monocytes to infiltrate the tumor microenvironment and degrade tumor-associated fibrosis leading to tumor regression. Extra-tumoral macrophages were found to be necessary for mobilization of inflammatory monocytes from the bone marrow into the peripheral blood. In addition, systemic IFN-γ released in response to anti-CD40 therapy was necessary for reprogramming inflammatory monocytes with anti-tumor activity. Inflammatory monocytes responding to IFN-γ displayed a distinct matrix metalloproteinase gene expression profile necessary for selective degradation of extracellular matrix proteins, including type I collagen and fibronectin, which define fibrosis in PDAC. Therefore, although inflammatory monocytes are commonly associated with pro-tumor activity, our findings demonstrate that they can also be reprogrammed with potent anti-tumor properties. Citation Format: Kristen B. Long, Whitney L. Gladney, Graham M. Tooker, Gregory L. Beatty. Reprogramming inflammatory monocytes to mediate anti-tumor activity in pancreatic carcinoma. [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 LB-204. doi:10.1158/1538-7445.AM2015-LB-204

Abstract 3168: A role for tumor-derived CCL2 in directing leukocyte infiltration and stromal heterogeneity in pancreatic ductal adenocarcinoma

Abstract Tumor heterogeneity within solid tumors is a recognized barrier to the efficacy of standard therapies. In pancreatic ductal adenocarcinoma (PDAC) the microenvironment is characterized by varying degrees of cytokine/chemokine production, stromal extracellular matrix, and immune cell infiltrates (Hezel et al. 2006). To investigate stromal phenotypical differences in PDAC, we have used the KrasG12D-; Trp53R172H; Pdx-1 Cre (KPC) genetically engineered mouse model which recapitulates tumor heterogeneity seen in human PDAC (Hingorani et al. 2005). Gross histological analysis of KPC tumors allowed us to stratify tumors into 2 broad groups: stromal and non-stromal. We found that stromal tumors were comprised of a significant macrophage infiltrate, whereas non-stromal tumors were generally void of macrophages. We modeled this stromal heterogeneity using tumor cell lines derived from KPC tumors. We found that, when implanted under the skin of wild-type mice, some cell lines produced stromal tumors rich in macrophages, while others formed non-stromal tumors with few macrophages within the microenvironment. Supernatant cultured from these KPC tumor cell lines in vitro revealed elevated CCL2 production, specifically in cell lines from stromal tumors. As CCL2 is a known chemo-attractant for monocytes, we hypothesized that tumor-derived CCL2 contributes to monocyte recruitment and stromal heterogeneity within the tumor microenvironment. To test this hypothesis, we have used an in vitro trans-well migration assay. With this assay, we found that more bone marrow derived cells migrate toward supernatant from stromal tumors relative to non-stromal tumors. This finding was lost in the presence of a CCL2 inhibitor. Ongoing studies are examining a role for tumor-derived CCL2 in regulating tumor-associated stroma in PDAC. Our findings suggest the importance of tumor-immune cell interactions in defining stromal heterogeneity and may offer insight into mechanisms that regulate the stromal compartment in human PDAC. Citation Format: Graham M. Tooker, Whitney L. Gladney, Gregory Beatty. A role for tumor-derived CCL2 in directing leukocyte infiltration and stromal heterogeneity in pancreatic ductal adenocarcinoma. [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 3168. doi:10.1158/1538-7445.AM2015-3168

Abstract 1356: Chemotherapy and agonistic CD40 cooperate to regress pancreatic adenocarcinoma independently of TLR4 and MyD88

Abstract Pancreatic ductal adenocarcinoma (PDA) is an aggressive and lethal disease, with current standards of care extending overall survival by only a few months, as observed with the combination of nab-paclitaxel (nP) and gemcitabine (Gem). As such, efforts have begun to focus on the prospect of immune therapy has a new paradigm for patients in PDA. Given the potential synergy between chemotherapy and immune stimulation to create an anti-tumor vaccine, we hypothesized that agonistic CD40 monoclonal antibody with combined Gem/nP chemotherapy can activate the immune response and reverse immunosuppression in the PDA microenvironment. Using the KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) genetically engineered mouse model of PDA, we found that 37.5% of mice treated with Gem/nP/CD40 therapy had tumor regressions or stable disease, compared to only 9% of mice receiving Gem/nP without CD40 (p&amp;lt;0.03). Furthermore, when mice were depleted of CD8 T cells, the tumor regressions were completely ablated. To understand the mechanism by which chemotherapy potentiated CD40 agonist treatment, we injected PDA cell lines generated from KPC tumors subcutaneously into C57Bl/6 mice, and found that more than 50% of mice treated with Gem/nP/CD40 experienced T cell dependent tumor regressions compared to only rare regressions in Gem/nP or CD40 treated mice (p&amp;lt;0.0001). Tumor regressions were dependent on T cells and IFN-gamma, and accordingly, we found the proportions of both CD4 and CD8 T cells producing IFN-gamma to be increased 2- to 4-fold in mice treated with Gem/nP/CD40 as compared to other cohorts. Concurrent with increased effector T cells, the proportion of T regulatory cells in the tumor was reduced 6.9-fold after Gem/nP/CD40 (p&amp;lt;0.05, versus Gem/nP or CD40 alone). The loss of the immunosuppressive tumor microenvironment was detectable 24 hours after Gem/nP/CD40 treatment, when CD11b+ and CD11c+ cells in the tumor reduced production of IL-10 and TGF-beta by 1.3 to 5-fold (compared to Gem/nP treatment), and increased production of IL-12 (35% versus 23% in CD40 treated mice), concurrent with increased expression of both CD86 and MHCII (37% versus 10% in control group). Despite prior results linking MyD88 signaling with immune potentiating effects of chemotherapy, we observed that responses to Gem/nP/CD40 were independent of MyD88, and TLR4 pathways. Tumor regressions with Gem/nP/CD40 were also independent of TRIF, IFNAR, Caspase 1 and 4, TLR3, muMT, TNF-alpha, and Perforin. These studies reveal a novel MyD88- and TLR4-independent mechanism of chemotherapeutic activation of the immune system, and highlight the significant clinical potential of combining Gem/nP with agonistic CD40 stimulation. Citation Format: Katelyn T. Byrne, Robert H. Vonderheide. Chemotherapy and agonistic CD40 cooperate to regress pancreatic adenocarcinoma independently of TLR4 and MyD88. [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 1356. doi:10.1158/1538-7445.AM2015-1356

Abstract B09: Targeting macrophages to degrade fibrosis in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) is commonly resistant to chemotherapy due to extensive stromal fibrosis and poor vascularity which combine to inhibit chemotherapy diffusion into tumor tissue. Strategies that target tumor-associated fibrosis have shown promise in preclinical and early clinical trials. We have previously shown that treatment with an agonist CD40 monoclonal antibody (mAb) can induce macrophages to facilitate degradation of tumor-associated fibrosis in PDA, resulting in tumor regressions. Here, we investigated the mechanism by which macrophages facilitate stromal degradation using the KrasG12D; Trp53R172H; Pdx-1 Cre (KPC) genetically engineered mouse model of PDA. By immunofluorescence imaging, we first characterized the extracellular matrix that surrounds PDA tumors in the KPC model. We found high levels of fibronectin, hyaluronic acid and type I collagen present within the surrounding PDA stroma. Treatment with a CD40 mAb resulted in macrophage dependent degradation of both fibronectin and type I collagen, but had no effect on hyaluronic acid. To examine the ability of macrophages to degrade type I collagen, we developed an in vitro colorimetric collagen degradation assay. We found that neither CD40 activation nor classical activation with IFN-γ and LPS were sufficient to induce macrophages to degrade type I collagen. In contrast, classical activation of macrophages in the presence of tumor cells led to &amp;gt;50% collagen degradation in vitro. This effect was correlated with the ability of classically activated macrophages to eliminate tumor cells. Our findings suggest that macrophages acquire the capacity to degrade tumor-associated stromal fibrosis only after activation and successful elimination of tumor cells. This finding has important implications for the design of novel strategies that exploit the anti-fibrotic potential of macrophages for potentially improving drug delivery in PDA. Citation Format: Kristen B. Long, Santiago L. Luque, Gregory L. Beatty. Targeting macrophages to degrade fibrosis in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B09.

Abstract B119: Triptolide abrogates expression of the Met and epidermal growth factor receptors in pancreatic cancer

Abstract Background: Minnelide, a pro-drug of the Chinese herb triptolide, is currently in Phase I clinical trials against pancreatic cancer. Preclinical studies with Minnelide have demonstrated its efficacy in preventing tumor formation and causing tumor regression in multiple animal models of pancreatic cancer. However, its mechanism of action remains unclear. Pancreatic cancer cells overexpress several receptor tyrosine kinases including the Met and EGF receptors which drive proliferation, migration and invasion of these cells. Here, using human pancreatic cancer cells as well as the KRasG12D; Trp53R172H; Pdx-1 Cre (KPC) animal model, we show the ability of triptolide, or its water soluble pro-drug, Minnelide, to down-regulate the Met and EGF receptors, both in vitro and in vivo. Methods:Pancreatic cancer cells were treated with triptolide for varying times (0-24h) and receptor levels evaluated using western blotting or confocal microscopy. The KPC mouse model recapitulates human disease progression and is therefore the most relevant model for chemotherapeutic drug testing. KPC animals, which spontaneously form pancreatic tumors, were treated with Minnelide for one week and tumors harvested. mRNA expression was analyzed using real time PCR and protein expression assessed by immunohistochemistry. Results: Treatment with triptolide causes an increase in tyrosine phosphorylation at earlier time points (0.5 - 6h), accompanied by an increase in levels of the Met and EGF receptor tyrosine kinases. However, longer treatments (12-24h) resulted in marked decrease in levels of both Met and EGF receptors. Further investigation using tumors from KPC animals treated with Minnelide show a decrease in gene expression of both the Met and EGF receptors compared with tumors from saline injected animals. Furthermore, immunohistochemical analysis of formalin fixed paraffin embedded sections from these tumors show a decrease in levels of the Met receptor. Conclusion: Treatment of cells or KPC pancreatic tumor bearing animals with triptolide or Minnelide leads to decreased expression of the Met and EGF receptor tyrosine kinases. These results lend further insight into the mechanism of action of Minnelide, a pro-drug currently in clinical trials against pancreatic cancer. Citation Format: Veena Sangwan, Kelsey M. Jensen, Vikas Dudeja, Rohit Chugh, Sulagna Banerjee, Selwyn M. Vickers, Morag Park, Ashok K. Saluja. Triptolide abrogates expression of the Met and epidermal growth factor receptors in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B119.

Abstract A54: Ly6Chi inflammatory monocytes mediate tumor regression in a mouse model of spontaneously arising pancreatic ductal adenocarcinoma

Abstract Macrophage infiltration into solid malignancies most often portends a poor prognosis. In pancreatic ductal adenocarcinoma (PDAC), macrophages dominate the leukocyte infiltrate with effector T lymphocytes remaining scarce. Strategies to restore productive immunosurveillance in PDAC have largely focused on inducing anti-tumor T cell immunity. However, we have previously shown in patients and a mouse model of PDAC that immune modulation with a CD40 agonist can induce macrophages to facilitate tumor regression. Thus, strategies that restore innate immunosurveillance may hold promise in the treatment of PDAC. To further investigate the role of macrophages as targets for anti-tumor immunotherapy, we utilized the KrasG12D; Trp53R172H; Pdx-1 Cre (KPC) genetically engineered mouse model of PDAC. We previously reported a role for liposomal targeted macrophages in facilitating tumor regression induced with CD40 therapy. However, unexpectedly we found by immunohistochemistry that macrophages targeted by liposomes do not infiltrate PDAC tumors in response to CD40 therapy. This finding suggested a role for additional leukocyte subsets in mediating the anti-tumor effect. Using immunohistochemistry, we found that systemic CD40 activation induced the infiltration of Ly6C+ myeloid cells into KPC tumors. Based on this finding, we hypothesized that macrophages may regulate the infiltration of Ly6Chi inflammatory monocytes, which are the critical effector myeloid cells mobilized in the peripheral blood by CD40 agonist therapy. In support of this hypothesis, we found that tumor regression induced with a CD40 agonist was abrogated by treatment with the anti-Ly6C/Ly6G RB6-8C5 depleting antibody. Flow cytometric analysis of peripheral blood revealed that RB6-8C5 and liposomes target distinct myeloid subsets. Whereas RB6-8C5 was found to deplete Ly6ChiCCR2+ inflammatory monocytes, CEL depleted Ly6Cneg myeloid cells. Our findings suggest that CD40-induced tumor regression in PDAC is mediated by inflammatory monocytes which infiltrate tumor lesions under the regulation of extra-tumoral macrophages. Citation Format: Whitney L. Gladney, Graham M. Tooker, Gregory L. Beatty. Ly6Chi inflammatory monocytes mediate tumor regression in a mouse model of spontaneously arising pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A54.

Abstract PR4: Restoring T cell immuno-surveillance in pancreatic carcinoma using CD40 agonists.

Abstract A hallmark of the tumor microenvironment in pancreatic ductal adenocarcinoma (PDA) is a profound immune infiltrate dominated by myeloid cells with a scarcity of T cells. Immune suppression imposed by this early immune reaction to PDA represents a significant clinical challenge to T cell immunotherapy. To circumvent this immune suppression, we have studied CD40 agonists for their well-recognized ability to induce potent T cell dependent anti-tumor immunity. However, in our studies using the KPC mouse model of PDA (KrasG12D/+ Trp53R172H/+ Pdx-1 Cre), we have found that delivery of a CD40 agonist alone or in combination with chemotherapy is unable to induce productive T cell immunosurveillance. Here, we examine barriers to the development of anti-tumor T cell immunity in PDA. By transplanting PDA tumor cells or whole PDA tumor tissue obtained from tumor-bearing KPC mice into syngeneic littermates, we demonstrate that a CD40 agonist (FGK45) in combination with gemcitabine chemotherapy can synergize to induce productive anti-tumor immunity leading to complete and sustained regression of established subcutaneous tumors (IgG2a alone – 0%; gemcitabine alone – 0%; FGK45 alone – 38%; gemcitabine + FGK45 – 71%; p &amp;lt; 0.001). In contrast to our findings in the KPC model, depletion of either CD4 (GK1.5) or CD8 (2.43) T cells abolished this effect (IgG2a alone – 0%; gemcitabine + FGK45 – 75%; gemcitabine + FGK45 + GK1.5 – 0%; gemcitabine + FGK45 + 2.43 – 14%; p &amp;lt;0.001). This observation suggested that PDA cells retain their immunogenicity and are capable of being recognized and targeted by T cells. To examine the ability of combination therapy (gemcitabine + FGK45) to induce T cell dependent anti-tumor immunity against spontaneously arising PDA, KPC mice treated with combination therapy were challenged subcutaneously two weeks later with PDA cells. However, no immune protection to tumor challenge was observed suggesting a failure to induce tumor-specific T cell immunity. To determine whether systemic immune suppression within KPC mice may inhibit the ability to induce productive T cell immunity, we next transplanted PDA tumor cells subcutaneously into KPC mice with ultrasound-confirmed PDA tumors. We found that combination therapy (gemcitabine + FGK45) delivered to these KPC mice bearing two tumors (i.e. implanted tumor plus a spontaneously arising PDA tumor) could elicit an anti-tumor T cell immune response capable of inducing tumor regression of the subcutaneously implanted tumor (Control – 0%; gemcitabine + FGK45 – 100%; p &amp;lt;0.001). Moreover, by delivering combination therapy to KPC animals in this “two tumor” system, we observed by immunohistochemistry an unprecedented and robust T cell infiltrate, comprised of both CD4 and CD8 T cells, into the spontaneously arising PDA tumor. Our findings suggest that ineffective T cell priming rather than immunoediting may limit T cell immunosurveillance in PDA – a finding that has important implications into the design of cancer immunotherapy for this disease. This abstract is also presented as Poster B84. Citation Format: Gregory L. Beatty, Rafi Winograd, Rebecca Evans, Santiago Lombo Luque, Patrick Guirnalda, Robert H. Vonderheide. Restoring T cell immuno-surveillance in pancreatic carcinoma using CD40 agonists. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR4.