Mouse Model Of Pancreatic Adenocarcinoma Research Articles

Abstract 5140: Combined administration of poly(I:C) and resiquimod triggers effective antitumoral response in mouse models of pancreatic adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest malignancies worldwide. The lack of clinical symptoms in the early stages of the disease and the poor immunogenicity of PDAC are two major causes of late diagnosis and inefficient therapies. The tumor microenvironment of PDAC is rich in stromal cells inducing desmoplasia, complicating the arrival of medical compounds, as well as in immune cells, that are suppressed by several mediators produced by cancer cells. In this setting, it has been shown that some medications can restore the antitumoral activity of innate immune cells that, in turn, re-activate also adaptive immune cells. In this study, we present the combination of the TLR agonists poly(I:C) (pIC - TLR3) and resiquimod (R848 - TLR7/8) as an immunotherapeutic tool in PDAC. In a subcutaneous model of PDAC (K8484 cells, isolated from KPC mice that spontaneously develop PDAC and recapitulate the features of human pancreatic cancer), the intratumoral administration of pIC+R848 for 5 times every 2-3 days led to the complete regression of the tumor in 100% of the treated mice. Moreover, all mice were protected from a second and a third rechallenge with the same cell line, performed months after the first treatments. Mechanistic studies performed on the tumors with flow cytometry demonstrated an increase of MHC-II positive cells in the monocytic (Ly6C+) compartment, as well as an increase in the number of mature CD8+ T cells and NK cells. Similar findings were demonstrated also in another heterotopic model obtained injecting Panc02 cells, another cell line with a sarcomatous phenotype. Consistently with the flow cytometry data, the depletion of CD4+ and CD8+ T cells abolished the antitumoral efficacy of pIC+R848, while the depletion of NK cells and CSF-1R+ cells was not sufficient to abolish the antitumoral response, suggesting a strong involvement of the adaptive immunity. In line with these findings, the intratumoral administration of pIC+R848 in mice unable to produce IFN-γ failed to reduce the tumor growth. In conclusion, our work clearly demonstrates that pIC+R848 are an effective treatment for PDAC when injected intratumorally and that this activity strongly relies on IFN-γ and adaptive immunity. Further investigations are ongoing to assess their potential in orthotopic models using different routes of administration, in a setting closer to the real clinical situation. Citation Format: Aldo Ummarino, Clément Anfray, Andrea Mariancini, Domenico Supino, Cecilia Garlanda, Fernando Torres Andòn, Alberto Mantovani, Paola Allavena. Combined administration of poly(I:C) and resiquimod triggers effective antitumoral response in mouse models of pancreatic adenocarcinoma. [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 5140.

ECM-targeting bacteria enhance chemotherapeutic drug efficacy by lowering IFP in tumor mouse models.

Bacterial cancer therapies aim to manipulate bacteria to effectively deploy therapeutic payloads to tumors. Attenuated bacteria alone often cannot eradicate solid tumors. Attenuated Salmonella can be engineered to deliver cytotoxic drugs to either trigger an immune response or increase antitumor efficacy when combined with chemotherapeutic drugs. However, the extracellular matrix (ECM) surrounding cancer cells forms a barrier that often limits the ability of chemotherapeutic and cytotoxic drugs to penetrate and eliminate tumors. To overcome this limitation, we developed a strategy to combine chemotherapy with an attenuated Salmonella typhimurium strain engineered to secrete HysA protein (from Staphylococcus aureus; Hyaluronidase, HAase) in tumors. The engineered Salmonella effectively degraded hyaluronan (HA), which is a major ECM constituent in tumors, and suppressed tumor growth in mouse models of pancreatic adenocarcinoma (ASPC-1) and breast cancer (4T1). Furthermore, it prolonged survival when combined with chemotherapeutic drugs (doxorubicin or gemcitabine). Upon bacterial colonization, the HAase-mediated ECM degradation decreased interstitial fluid pressure (IFP) in the tumor microenvironment. Additionally, HA degradation using HAase-expressing bacteria in vivo led to decreased binding to the receptor, CD44, expressed in tumors. This may modulate proliferation- and apoptosis-related signal pathways. Therefore, ECM-targeting bacteria can be used as a synergistic anticancer therapeutic agent to maximize chemotherapeutic drug delivery into highly invasive tumors.

Abstract IA013: Preclinical testing of CD73 inhibitors for pancreatic cancer immunoprevention

Abstract Introduction: The all stages combined five-year survival rate for pancreatic adenocarcinoma (PDA) is 11%; however, the five-year survival rate for localized PDA is 42%. These statistics highlight the importance of early prevention strategies to prevent disease progression and metastatic dissemination. Through the NCI PREVENT program, this research program explores immunoprevention strategies for PDA by targeting CD73, a gatekeeper ectoenzyme responsible for production of extracellular adenosine. We have recently shown aggressive subtypes of pancreatic intraepithelial neoplasia (PanIN) and PDA arising in ductal pancreatic epithelium have elevated CD73 and intrapancreatic adenosine indicating adenosine generation may be an early trigger of immunosuppression. We hypothesize inhibition of CD73 and adenosine generation will promote a more robust anti-tumor immune response and prevent PanIN and PDA progression. Methods: We tested three small molecule CD73 inhibitors (APCP, OP-5244, and AB680) in a syngeneic PDA mouse model by injecting 100-200k murine PDA cells derived from KrasG12D;Trp53R172H/+;Pdx:Cre (KPC) mice in the flanks of C57BL/6 female mice. Tumor sizes were measured weekly and tumor volume and mass were recorded at time of death. Dosage: APCP oral gavage (3x/week at 20mg/kg) and intraperitoneal (IP) (3x/week at 20 mg/kg). OP-5244 oral (3x/week at 25mg/kg and 10mg/kg). AB680 oral gavage (3x/week at 10mg/kg). HPLC analysis was performed for each inhibitor to quantify adenosine levels. Results: IP delivery of APCP significantly reduced tumor growth and intratumoral adenosine levels; however oral gavage delivery did not reduce tumor growth. Similarly, oral gavage delivery of OP-5244 did not reduce tumor growth. AB680 significantly reduced tumor volume and intratumoral adenosine levels and CyTOF immunoprofiling showed activated CD8+ T cells, dendritic cells, and macrophages were significantly increased in the tumors from AB680 treated mice. Conclusion: APCP IP delivery is more effective than oral gavage delivery and OP-5244 oral gavage delivery does not significantly decrease tumor growth. AB680 oral gavage delivery significantly decreases tumor growth and tumor adenosine concentrations. We observed a significant increase in infiltration of activated CD8+ T cells. AB680 shows high translational potential for preclinical testing in spontaneous GEM models. Citation Format: Lincoln Ballew, Kanchan Singh, Vidhi Chandra, Tingting Mills, Erika Y. Faraoni, Victoria Mota, Trent Clark, Lana Vornik, Michelle I. Savage, Shizuko Sei, Altaf Mohammed, Holger K. Eltzschig, Powel H. Brown, Florencia McAllister, Jennifer M. Bailey-Lundberg. Preclinical testing of CD73 inhibitors for pancreatic cancer immunoprevention [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA013.

Influences of calorie restriction and lipopolysaccharide therapy on inflammation, cytokine response, and cell proliferation in pancreatic adenocarcinoma mouse model.

The study aimed to investigate the effects of lipopolysaccharide (LPS) alone and in combination with calorie restriction (CR) on the pancreatic tissues in C57BL/6 mice modeled with pancreatic ductal adenocarcinoma (PDAC). Forty male C57BL/6 mice (10-13 weeks old) were divided into five groups; LPS,LPS + CR, PDAC, PDAC + LPS, and PDAC + LPS + CR. Nuclear factor kappa B (NF-κβ), interleukin-6 (IL-6), and c-Jun N-terminal kinases (JNK) mRNA expression levels were measured in pancreatic tissues. NF-κβ, IL-6, JNK, and proliferating cell nuclear antigen(PCNA) peptide levels were determined by immunohistochemistry. Oxidative stress markers and antioxidant enzyme activities were determined spectrophotometrically. TH1/TH2 cytokine measurements were determined by a flow cytometer. It was detected that the number of PCNA immune + cells in the PDAC + LPS + CR group was significantly lower than in the PDAC and PDAC + LPS groups (p < 0.01, p < 0.05 respectively). PDAC + LPS + CR group's plasma interferon-gamma(IFN-γ), IL-6, IL-2, tumor necrosis factor-alpha, IL-3, and IL-4 levels were found to be significantly lower than the PDAC group (p < 0.01, p < 0.001, p < 0.01, p < 0.05, p < 0.01, and p < 0.05 respectively). According to our findings, the combination of low-dose LPS and 40% CR was found to be more effective in PDAC model mice.

3D Ultrasound-Guided Photoacoustic Imaging to Monitor the Effects of Suboptimal Tyrosine Kinase Inhibitor Therapy in Pancreatic Tumors.

Pancreatic cancer is a disease with an incredibly poor survival rate. As only about 20% of patients are eligible for surgical resection, neoadjuvant treatments that can relieve symptoms and shrink tumors for surgical resection become critical. Many forms of treatments rely on increased vulnerability of cancerous cells, but tumors or regions within the tumors that may be hypoxic could be drug resistant. Particularly for neoadjuvant therapies such as the tyrosine kinase inhibitors utilized to shrink tumors, it is critical to monitor changes in vascular function and hypoxia to predict treatment efficacy. Current clinical imaging modalities used to obtain structural and functional information regarding hypoxia or oxygen saturation (StO2) do not provide sufficient depth penetration or require the use of exogenous contrast agents. Recently, ultrasound-guided photoacoustic imaging (US-PAI) has garnered significant popularity, as it can noninvasively provide multiparametric information on tumor vasculature and function without the need for contrast agents. Here, we built upon existing literature on US-PAI and demonstrate the importance of changes in StO2 values to predict treatment response, particularly tumor growth rate, when the outcomes are suboptimal. Specifically, we image xenograft mouse models of pancreatic adenocarcinoma treated with suboptimal doses of a tyrosine kinase inhibitor cabozantinib. We utilize the US-PAI data to develop a multivariate regression model that demonstrates that a therapy-induced reduction in tumor growth rate can be predicted with 100% positive predictive power and a moderate (58.33%) negative predictive power when a combination of pretreatment tumor volume and changes in StO2 values pretreatment and immediately posttreatment was employed. Overall, our study indicates that US-PAI has the potential to provide label-free surrogate imaging biomarkers that can predict tumor growth rate in suboptimal therapy.

Abstract 935: Preclinical activity of seribantumab in gastrointestinal cancers with NRG1 fusions

Abstract Background. Oncogenic rearrangements of the neuregulin 1 gene (NRG1) consist of a 5' partner fused to a 3' NRG1 sequence that retains the EGF-like domain, and are found in 0.2% of solid tumors including lung, breast and gastrointestinal (GI) cancers. Carcinomas of GI origin, including pancreatic and cholangiocarcinoma, represent around 20% of solid tumors harboring NRG1 fusions and there is no approved therapy for this group of cancers. The chimeric NRG1 oncoproteins bind to HER3/ERBB3 leading to trans-activation of other ERBB family members and trigger a signaling cascade that culminates in oncogenesis. Although targeting HER3 represents a rational therapeutic strategy for cancers harboring NRG1 fusions, this has remained relatively unexplored for NRG1 fusion-positive GI malignancies. In this study we investigated the efficacy of the anti-HER3 monoclonal antibody seribantumab in preclinical models of NRG1-driven GI cancers. Methods. We developed models of isogenic pancreatic cancer cells with NRG1 fusions by lentiviral-mediated cDNA expression of ATP1B1-NRG1 and SLC3A2-NRG1 fusions in immortalized pancreatic ductal cells (H6c7). Seribantumab efficacy was evaluated in isogenic cell lines and in patient-derived xenograft (PDX) models of pancreatic adenocarcinoma (CTG-0943, APP-NRG1 fusion) and intrahepatic cholangiocarcinoma (CH-07-0068, RBPMS-NRG1 fusion). Western blotting analysis was used to evaluate protein phosphorylation. Expression of NRG1 fusions was confirmed by RT-PCR and NGS. Results. Expression of NRG1 fusions in H6c7 cells resulted in enhanced phosphorylation of HER3 and AKT and increased sensitivity to afatinib, as compared to empty vector control cells (H6c7-EV). Treatment of H6c7-SLC3A2-NRG1 cells with seribantumab resulted in a dose-dependent inhibition of HER3 and AKT phosphorylation. Seribantumab treatment of H6c7-ATP1B1-NRG1 and H6c7-SLC3A2-NRG1 cells resulted in dose-dependent inhibition of cell growth with IC50 values of 0.05 and 0.2 µM, respectively. In contrast, growth of H6c7-EV cells was much less sensitive to seribantumab (IC50 &amp;gt; 1µM). Tumor growth inhibition was observed after administration of seribantumab to PDX mouse models of pancreatic adenocarcinoma and intrahepatic cholangiocarcinoma. While seribantumab (5 mg and 10 mg per dose, BIW) was equally effective to the clinical equivalent dose of afatinib (5 mg/kg QD) in the cholangiocarcinoma PDX model, the two doses of seribantumab were more effective than afatinib in the pancreatic cancer PDX model, causing tumor shrinkage of up to 55% (23-77% range). There was no shrinkage of afatinib-treated pancreatic PDX tumors. Our results here suggest that seribantumab is effective at reducing tumor growth in preclinical models of gastrointestinal cancers with NRG1 fusions. These data support the use of seribantumab to treat GI and other cancers with NRG1 fusions in the ongoing phase 2 CRESTONE study (NCT#04383210). Citation Format: Igor Odintsov, Allan J. Lui, Paul R. Bloom, Morana Vojnic, Shawn Leland, Marc Ladanyi, Romel Somwar. Preclinical activity of seribantumab in gastrointestinal cancers with NRG1 fusions [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 935.

DCLK1-Isoform2 Alternative Splice Variant Promotes Pancreatic Tumor Immunosuppressive M2-Macrophage Polarization.

Tumor-associated M2-macrophages are one of the most abundant immunosuppressive cell types in the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). However, the molecular mechanisms responsible for the generation of M2-macrophages are unclear. Here, we demonstrated that overexpression of DCLK1-isoform2 in AsPC1 and MIA PaCa2 cells resulted in the polarization of M1-macrophages toward an M2 phenotype via secreted chemokines/cytokines. These M2-macrophages enhanced parental PDAC cell migration, invasion, and self-renewal, and this was associated with increased expression of Snail and Slug. We observed distinct expression of Dclk-isoform2, marked infiltration of M2-macrophages, and a marginal increase of CD8+ T cells in 20-week-old KPCY mice pancreas compared with 5 weeks old. Utilizing an autochthonous mouse model of pancreatic adenocarcinoma, we observed distinct immunoreactive Dclk1 and arginase1 in tissues where CD8+ T-cell infiltration was low and observed a paucity of DCLK1 and arginase1 staining where CD8+ T-cell infiltration was high. Finally, we found that DCLK1-isoform2 tumor-educated M2-macrophages inhibit CD8+ T-cell proliferation and granzyme-B activation. Inhibition of DCLK1 in an organoid coculture system enhanced CD8+ T-cell activation and associated organoid death. We conclude that DCLK1-isoform2 is a novel initiator of alternate macrophage activation that contributes to the immunosuppression observed in the PDAC TME. These data suggest that tumor DCLK1-isoform2 may be an attractive target for PDAC therapy, either alone or in conjunction with immunotherapeutic strategies.

Loss of Rnf43 to accelerate KRAS-mediated neoplasia in a clinically relevant genetically engineered mouse model of pancreatic adenocarcinoma.

733 Background: The genetic heterogeneity of pancreatic ductal adenocarcinoma (PDAC) demands a personalized molecular-targeted treatment approach. While activating KRAS mutations are a near ubiquitous event in PDAC pathogenesis, 5-10% of cases display deleterious driver mutations in the Wnt-signaling negative regulator, ring finger 43 (RNF43). Despite this characterization there are no personalized treatment options for this subset of patients. Methods: We have developed a genetically engineered mouse model (GEMM) of PDAC, driven by an activating mutation in Kras and deletion of Rnf43 under control of a pancreas specific promoter (KRC). Mice were followed for survival outcomes and histological changes which were compared to a Kras driven PDAC GEMM (KC). Mice underwent serial magnetic resonance imaging (MRI), with and without dynamic contrast enhanced (DCE) imaging, to evaluate cystic tumor morphology and contrast enhancement during tumor progression. Single cell RNA sequencing (scRNAseq) was also performed to assess changes in single cell populations during tumor progression. Lastly, we established ex vivo cultures from KRC and KC tumors and performed bulk RNA-sequencing (RNAseq) and in vitro pharmacology studies. Results: KRC mice displayed a decrease in overall survival and higher incidence of both high grade pre-neoplastic lesions and invasive PDAC compared to KC mice. Serial MRI revealed increased cystic morphology of KRC mice during tumor progression with increasing DCE intensity. scRNAseq of KRC tumors from moribund mice displayed two distinct populations of both macrophages and fibroblasts, similar to our previous report of Kras/Tp53 and Kras/Ink4a driven GEMMs. Lastly, primary cultures from KRC tumors demonstrated increased expression of Wnt-related genes by RNAseq and increased sensitivity to small molecule porcupine inhibition relative to KC cell lines, demonstrating functional Wnt dependence of the KRC system. Conclusions: Rnf43 is a bona fide tumor suppressor gene in PDAC. This GEMM is a novel platform for drug discovery in RNF43-mutated PDAC with the eventual goal of implementing a precision oncology approach in these patients.

Development of multi-drug loaded PEGylated nanodiamonds to inhibit tumor growth and metastasis in genetically engineered mouse models of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. Nanomedicine, however, offers new opportunities to facilitate drug delivery in PDAC. Our previous work has shown that poly(ethylene glycol)-functionalized nanodiamond (ND) mediated drug delivery offered a considerable improvement over free drug in PDAC. Inspired by this result and guided by molecular simulations, we opted for simultaneous loading of irinotecan and curcumin in ultra-small PEGylated NDs (ND-IRT + CUR). We observed that ND-IRT + CUR was more efficacious in killing AsPC-1 and PANC-1 cells than NDs with single drugs. Using NDs functionalized with a near-infrared (NIR) dye, we demonstrated the preferential localization of the NDs in tumors and metastatic lesions. We further demonstrate that ND-IRT + CUR is capable of producing pronounced anti-tumor effects in two different clinically relevant, immune-competent genetic models of PDAC. Cytokine profiling indicated that NDs with or without drugs downregulated the expression of IL-10, a key modulator of the tumor microenvironment. Thus, using a combination of in silico, in vitro, and in vivo approaches, we show for the first time the remarkable anti-tumor efficacy of PEGylated NDs carrying a dual payload of irinotecan plus curcumin. These results highlight the potential use of such nano-carriers in the treatment of patients with pancreatic cancer.

Tumor Cell-Intrinsic Factors Underlie Heterogeneity of Immune Cell Infiltration and Response to Immunotherapy

The biological and functional heterogeneity between tumors-both across and within cancer types-poses a challenge for immunotherapy. To understand the factors underlying tumor immune heterogeneity and immunotherapy sensitivity, we established a library of congenic tumor cell clones from an autochthonous mouse model of pancreatic adenocarcinoma. These clones generated tumors that recapitulated Tcell-inflamed and non-T-cell-inflamed tumor microenvironments upon implantation in immunocompetent mice, with distinct patterns of infiltration by immune cell subsets. Co-injecting tumor cell clones revealed the non-T-cell-inflamed phenotype is dominant and that both quantitative and qualitative features of intratumoral CD8+ Tcells determine response to therapy. Transcriptomic and epigenetic analyses revealed tumor-cell-intrinsic production of the chemokine CXCL1 as a determinant of the non-T-cell-inflamed microenvironment, and ablation of CXCL1 promoted Tcell infiltration and sensitivity to a combination immunotherapy regimen. Thus, tumor cell-intrinsic factors shape the tumor immune microenvironment and influence the outcome of immunotherapy.

The combination of everolimus and zoledronic acid increase the efficacy of gemcitabine in a mouse model of pancreatic adenocarcinoma.

BackgroundGemcitabine is a standard treatment for pancreatic adenocarcinoma. Many mechanisms are involved in gemcitabine resistance, such as reduced expression of the human equilibrative nucleoside transporter 1 (hENT1) membrane transporter, deoxycytidine kinase deficiency, and changes in the signal transmission of mitogen-activity protein kinase (MAPK) and the phosphoinositide 3-kinase (PI3K) pathways.AimTo evaluate the anti-tumor efficiency of blocking signaling pathways using combined action of gemcitabine, everolimus and zoledronic acid versus gemcitabine alone in a mouse subcutaneous xenograft.MethodsImplantations of two human pancreatic adenocarcinoma cells lines (PANC1, K-ras mutated and gemcitabine-resistant; and BxPc3, wild-type K-ras and gemcitabine-sensitive) were performed on male athymic nude mice. The mice received different treatments: gemcitabine, gemcitabine plus everolimus, everolimus, gemcitabine plus zoledronic acid, everolimus plus zoledronic acid, or gemcitabine plus everolimus and zoledronic acid, for 28 days. We measured the tumor volume and researched the expression of the biomarkers involved in the signaling pathways or in gemcitabine resistance.ResultsIn wild-type K-ras tumors, the combinations of gemcitabine plus everolimus; zoledronic acid plus everolimus; and gemcitabine plus zoledronic acid and everolimus slowed tumor growth, probably due to caspase-3 overexpression and reduced Annexin II expression. In mutated K-ras tumors, gemcitabine plus everolimus and zoledronic acid, and the combination of zoledronic acid and everolimus, decreased tumor volume as compared to gemcitabine alone, inhibiting the ERK feedback loop induced by everolimus.ConclusionThe combination of zoledronic acid and everolimus has an antitumor effect and could increase gemcitabine efficacy.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Phosphorylation of Rab-coupling protein by LMTK3 controls Rab14-dependent EphA2 trafficking to promote cell:cell repulsion

The Rab GTPase effector, Rab-coupling protein (RCP) is known to promote invasive behaviour in vitro by controlling integrin and receptor tyrosine kinase (RTK) trafficking, but how RCP influences metastasis in vivo is unclear. Here we identify an RTK of the Eph family, EphA2, to be a cargo of an RCP-regulated endocytic pathway which controls cell:cell repulsion and metastasis in vivo. Phosphorylation of RCP at Ser435 by Lemur tyrosine kinase-3 (LMTK3) and of EphA2 at Ser897 by Akt are both necessary to promote Rab14-dependent (and Rab11-independent) trafficking of EphA2 which generates cell:cell repulsion events that drive tumour cells apart. Genetic disruption of RCP or EphA2 opposes cell:cell repulsion and metastasis in an autochthonous mouse model of pancreatic adenocarcinoma—whereas conditional knockout of another RCP cargo, α5 integrin, does not suppress pancreatic cancer metastasis—indicating a role for RCP-dependent trafficking of an Eph receptor to drive tumour dissemination in vivo.

CXCR2-Dependent Accumulation of Tumor-Associated Neutrophils Regulates T-cell Immunity in Pancreatic Ductal Adenocarcinoma.

Tumor-associated neutrophils are increasingly recognized for their ability to promote tumor progression, mediate resistance to therapy, and regulate immunosuppression. Evidence from various murine models has shown that the chemokine receptor CXCR2 attracts neutrophil into tumors and, therefore, represents a tractable therapeutic target. Here, we report prominent expression of a neutrophil gene signature in a subset of human pancreatic adenocarcinoma (PDA). CXCL5 was the most prominently expressed CXCR2 ligand in human PDA, and its expression was higher in PDA than in any other common tumor represented in The Cancer Genome Atlas. Using a genetically engineered mouse model of PDA, we found that tumor and stromal cells differentially expressed CXCR2 ligands, with Cxcl5 high in tumor and Cxcl2 high in stroma. Cxcl5 expression was associated with mutant Kras expression and regulated by NF-κB activation. Host CXCR2 inhibition by genetic ablation prevented neutrophil accumulation in pancreatic tumors and led to a T cell-dependent suppression of tumor growth. In the absence of neutrophils, activated and functional T cells infiltrated pancreatic tumors otherwise devoid of effector T cells. Thus, the CXCR2-ligand axis helps establish an immunosuppressive microenvironment in PDA, highlighting the potential utility of targeting this axis as a novel therapy for this deadly disease. Cancer Immunol Res; 4(11); 968-82. ©2016 AACR.

MiR-216 and miR-217 expression is reduced in transgenic mouse models of pancreatic adenocarcinoma, knockout of miR-216/miR-217 host gene is embryonic lethal

Mice harboring a G12D activating Kras mutation are among the most heavily studied models in the field of pancreatic adenocarcinoma (PDAC) research. miRNAs are differentially expressed in PDAC from patients and mouse models of PDAC. To better understand the relationship that Kras activation has on miRNA expression, we profiled the expression of 629 miRNAs in RNA isolated from the pancreas of control, young, and old P48+/Cre;LSL-KRASG12D as well as PDX-1-Cre;LSL-KRASG12D mice. One hundred of the differentially expressed miRNAs had increased expression in the advanced disease (old) P48+/Cre;LSL-KRASG12D compared to wild-type mice. Interestingly, the expression of three miRNAs, miR-216a, miR-216b, and miR-217, located within a ∼30-kbp region on 11qA3.3, decreased with age (and phenotype severity) in these mice. miR-216/-217 expression was also evaluated in another acinar-specific ELa-KrasG12D mouse model and was downregulated as well. As miR-216/-217 are acinar enriched, reduced in human PDAC and target KRAS, we hypothesized that they may maintain acinar differentiation or represent tumor suppressive miRNAs. To test this hypothesis, we deleted a 27.9-kbp region of 11qA3.3 containing the miR-216/-217 host gene in the mouse's germ line. We report that germ line deletion of this cluster is embryonic lethal in the mouse. We estimate that lethality occurs shortly after E9.5. qPCR analysis of the miR-216b and miR-217 expression in the heterozygous animals showed no difference in expression, suggesting haplosufficiency by some type of compensatory mechanism. We present the differential miRNA expression in KrasG12D transgenic mice and report lethality from deletion of the miR-216/-217 host gene in the mouse's germ line.

Abstract B10: Transcriptome analysis of mouse PDA subtypes

Abstract Patients with Pancreatic Adenocarcinoma (PDA) often present with advanced stage disease including widespread metastasis and have dismal 5-year survival rates. The molecular mechanism promoting PDA progression and metastasis remain poorly understood. We have previously demonstrated that tumor cells undergo Epithelial-to-Mesenchymal Transition (EMT) in mouse models of PDA [Rhim et al., 2012]. EMT is a developmental process where cells loss epithelial characteristics and transdifferentiate into a mesenchymal-like state that is highly associated with increased invasion and metastasis. To better understand molecular mechanisms of PDA development and metastatic disease, we subjected tumors cells from a lineage labeled mouse model of PDA to RNA sequencing to identify global transcriptional changes. Interestingly, we discovered that there are at least two distinct subtypes of PDA based on transcriptome profiling. Comparison of gene expression profiles from mouse PDA subtypes to established subtypes from human PDA datasets [Collisson et al., 2011] revealed that mouse PDA is transcriptionally similar to the human disease. Differences in transcriptional profiling between PDA subtypes may dictate response to chemotherapeutics and metastatic competency. Citation Format: David Balli, Nicole Aiello, Robert Norgard, Jingyang Li, Amine Sahmoud, Ben Stanger. Transcriptome analysis of mouse PDA subtypes. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B10.

Abstract 396: Ibrutinib exerts potent antifibrotic activity in a mouse model of pancreatic adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a disease with a poor prognosis characterized by a dense stromal fibroinflammatory reaction that is a significant obstacle to effective therapy. The desmoplastic stroma comprises many inflammatory cells, in particular mast cells that are a key component of the PDAC microenvironment and whose infiltration often correlates with poor patient outcome. Reducing the stroma is of key importance for improving the standard of care. We have previously successfully utilized ibrutinib, a clinically-approved Bruton's Tyrosine Kinase inhibitor, as a mast cell blocker in a mouse model of β cell tumorigenesis. Here, we made use of a transgenic mouse model of PDAC and patient derived xenografts (PDXs) to assess ibrutinib's impact on the tumor microenvironment and animal survival. Across these various experimental pre-clinical models, we show that ibrutinib significantly diminishes tissue fibrosis, increases survival and improves the outcome of standard care. Our results suggest that ibrutinib could be deployed to one more type of cancer and open the way to new clinical trials assessing its therapeutic impact in PDAC patients. Citation Format: Daniel Massó-Vallés, Toni Jauset, Erika Serrano, Nicole M. Sodir, Kim Pedersen, Nesrine I. Affara, Jonathan R. Whitfield, Marie-Eve Beaulieu, Gerard I. Evan, Laurence Elias, Joaquín Arribas, Laura Soucek. Ibrutinib exerts potent antifibrotic activity in a mouse model of pancreatic 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 396. doi:10.1158/1538-7445.AM2015-396

Ibrutinib exerts potent antifibrotic and antitumor activities in mouse models of pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense stromal fibroinflammatory reaction that is a major obstacle to effective therapy. The desmoplastic stroma comprises many inflammatory cells, in particular mast cells as key components of the PDAC microenvironment, and such infiltration correlates with poor patient outcome. Indeed, it has been hypothesized that stromal ablation is critical to improve clinical response in patients with PDAC. Ibrutinib is a clinically approved Bruton's tyrosine kinase inhibitor that inhibits mast cells and tumor progression in a mouse model of β-cell tumorigenesis. Here, we show that ibrutinib is highly effective at limiting the growth of PDAC in both transgenic mouse and patient-derived xenograft models of the disease. In these various experimental settings, ibrutinib effectively diminished fibrosis, extended survival, and improved the response to clinical standard-of-care therapy. Our results offer a preclinical rationale to immediately evaluate the clinical efficacy of ibrutinib in patients with PDAC.

Circulating Tumor Cells as a Biomarker of Response to Treatment in Patient-Derived Xenograft Mouse Models of Pancreatic Adenocarcinoma

Circulating tumor cells (CTCs) are cells shed from solid tumors into circulation and have been shown to be prognostic in the setting of metastatic disease. These cells are obtained through a routine blood draw and may serve as an easily accessible marker for monitoring treatment effectiveness. Because of the rapid progression of pancreatic ductal adenocarcinoma (PDAC), early insight into treatment effectiveness may allow for necessary and timely changes in treatment regimens. The objective of this study was to evaluate CTC burden as a biomarker of response to treatment with a oral phosphatidylinositol-3-kinase inhibitor, BKM120, in patient-derived xenograft (PDX) mouse models of PDAC. PDX mice were randomized to receive vehicle or BKM120 treatment for 28 days and CTCs were enumerated from whole blood before and after treatment using a microfluidic chip that selected for EpCAM (epithelial cell adhesion molecule) positive cells. This microfluidic device allowed for the release of captured CTCs and enumeration of these cells via their electrical impedance signatures. Median CTC counts significantly decreased in the BKM120 group from pre- to post-treatment (26.61 to 2.21 CTCs/250 µL, p = 0.0207) while no significant change was observed in the vehicle group (23.26 to 11.89 CTCs/250 µL, p = 0.8081). This reduction in CTC burden in the treatment group correlated with tumor growth inhibition indicating CTC burden is a promising biomarker of response to treatment in preclinical models. Mutant enriched sequencing of isolated CTCs confirmed that they harbored KRAS G12V mutations, identical to the matched tumors. In the long-term, PDX mice are a useful preclinical model for furthering our understanding of CTCs. Clinically, mutational analysis of CTCs and serial monitoring of CTC burden may be used as a minimally invasive approach to predict and monitor treatment response to guide therapeutic regimens.

Circulating Tumor Cells as a Biomarker of Response to Treatment in Patient-Derived Xenograft Mouse Models of Pancreatic Adenocarcinoma

Circulating Tumor Cells as a Biomarker of Response to Treatment in Patient-Derived Xenograft Mouse Models of Pancreatic Adenocarcinoma