Pancreatic Tumor-bearing Mice Research Articles

Development of gelatin nanoparticles for positron emission tomography diagnosis in pancreatic cancer

Abstract Pancreatic cancer remains difficult to diagnose using currently available imaging probes. Thus, this study aimed to develop a novel imaging agent for the diagnosis of pancreatic cancer using positron emission tomography (PET). Specifically, this study explores gelatin-based nanoparticles fabricated via radiation-induced crosslinking, as gelatin is known for its ability to produce biocompatible materials. Aqueous gelatin solutions were irradiated with γ-rays to produce nanoparticles with average diameters of 5–20 nm via a radiation crosslinking technique. The gelatin nanoparticles were labeled with 64Cu, exhibiting negative surface potentials. Furthermore, the nanoparticles were evaluated in vivo by injecting them into pancreatic tumor-bearing mice. Notably, the nanoparticles accumulated in the tumors. Hence, 64Cu-labeled gelatin nanoparticles show promise as a platform for next-generation PET imaging agents for pancreatic cancer.

Cardiac Atrophy, Dysfunction, and Metabolic Impairments: A Cancer-Induced Cardiomyopathy Phenotype

Muscle atrophy and weakness are prevalent features of cancer. Although extensive research has characterized skeletal muscle wasting in cancer cachexia, limited studies have investigated how cardiac structure and function are affected by therapy-naive cancer. Herein, orthotopic, syngeneic models of epithelial ovarian cancer and pancreatic ductal adenocarcinoma, and a patient-derived pancreatic xenograft model, were used to define the impact of malignancy on cardiac structure, function, and metabolism. Tumor-bearing mice developed cardiac atrophy and intrinsic systolic and diastolic dysfunction, with arterial hypotension and exercise intolerance. In hearts of ovarian tumor-bearing mice, fatty acid-supported mitochondrial respiration decreased, and carbohydrate-supported respiration increased-showcasing a substrate shift in cardiac metabolism that is characteristic of heart failure. Epithelial ovarian cancer decreased cytoskeletal and cardioprotective gene expression, which was paralleled by down-regulation of transcription factors that regulate cardiomyocyte size and function. Patient-derived pancreatic xenograft tumor-bearing mice show altered myosin heavy chain isoform expression-also a molecular phenotype of heart failure. Markers of autophagy and ubiquitin-proteasome system were upregulated by cancer, providing evidence of catabolic signaling that promotes cardiac wasting. Together, two cancer types were used to cross-validate evidence of the structural, functional, and metabolic cancer-induced cardiomyopathy, thus providing translational evidence that could impact future medical management strategies for improved cancer recovery in patients.

TSG-6+ cancer-associated fibroblasts modulate myeloid cell responses and impair anti-tumor response to immune checkpoint therapy in pancreatic cancer

Resistance to immune checkpoint therapy (ICT) presents a growing clinical challenge. The tumor microenvironment (TME) and its components, namely tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs), play a pivotal role in ICT resistance; however, the underlying mechanisms remain under investigation. In this study, we identify expression of TNF-Stimulated Factor 6 (TSG-6) in ICT-resistant pancreatic tumors, compared to ICT-sensitive melanoma tumors, both in mouse and human. TSG-6 is expressed by CAFs within the TME, where suppressive macrophages expressing Arg1, Mafb, and Mrc1, along with TSG-6 ligand Cd44, predominate. Furthermore, TSG-6 expressing CAFs co-localize with the CD44 expressing macrophages in the TME. TSG-6 inhibition in combination with ICT improves therapy response and survival in pancreatic tumor-bearing mice by reducing macrophages expressing immunosuppressive phenotypes and increasing CD8 T cells. Overall, our findings propose TSG-6 as a therapeutic target to enhance ICT response in non-responsive tumors.

Robust pancreatic tumor suppression by a novel combination treatment with anti-PD-1 immune checkpoint antibody and stroma modifying RNA oligonucleotide STNM01 in mice.

e14630 Background: Tumor stromal remodeling is an obstacle for immune checkpoint blockade therapies. STNM01 is a synthetic RNA oligonucleotide that represses carbohydrate sulfotransferase 15 (CHST15), which is responsible for tumor stromal remodeling. We have previously reported that STNM01 monotherapy by intratumoral injection repressed tumor stromal remodeling while increase tumor-infiltrating T lymphocytes (TILs) in pancreatic tumor-bearing mice. STNM01 was shown to increase TILs in patients with unresectable pancreatic cancer in a Phase I/IIa trial. In the present study, we investigated whether STNM01-mediated TIL enhancement improves anti-tumor effect of anti-PD-1 immune checkpoint antibody by combination treatment in murine models of pancreatic tumor. Methods: In mouse pancreatic cancer KPC and Pan02 subcutaneous syngeneic tumor models, mice were divided into 4 groups for treatment; 1) control, 2) STNM01 monotherapy, 3) anti-PD-1 antibody monotherapy, and 4) combination therapy with STNM01 and anti-PD-1 antibody. Intratumoral injections with 0.9-1.0 mg/mL of STNM01 or control and intraperitoneal administrations of anti-PD-1 antibody or control (5mg/kg) were then performed twice a week for 2 weeks. Mice were sacrificed after 2 week-treatment from baseline, and anti-tumor effects were evaluated by immunohistochemistry for KPC and flowcytometry for Pan02 model, respectively. Results: In the KPC-implanted syngeneic mouse model, combination treatment with intratumoral STNM01 and systemic anti-PD-1 antibody synergistically and robustly suppressed tumor growth as mean % tumor growth inhibition was over 80%. Tumor-infiltrating CD4+ and CD8+ T cells significantly increased in the anti-PD-1 plus STNM01 combination therapy group compared to anti-PD-1 monotherapy group. In the Pan02-implanted syngeneic mouse model, the combination treatment with STNM01 and anti-PD-1 significantly augmented necrosis area of tumor. Flowcytometry analyses showed that tumor-infiltrating CD3+, CD4+ and CD8+ T cells significantly increased in the anti-PD-1 plus STNM01 combination therapy group compared to control group. Although FoxP3+ Treg did not change, the ratio of CD8 to FoxP3 was significantly high only in the anti-PD-1 plus STNM01 combination therapy group. Notably, Ly6C+Ly6G+ granulocytic MDSCs dramatically diminished in the anti-PD-1 plus STNM01 combination therapy group, while anti-PD-1 monotherapy had no impact on tumoral MDSCs. Conclusions: The present work represents the first report of robust synergy between systemic anti-PD-1 antibody and single stroma modifying agent through intratumoral route of administration. Combination usage of STNM01 would provide a novel and clinically feasible therapeutic option to trigger remarkable effect of immune checkpoint inhibitors to this most hard-to-treat solid tumor.

Gemcitabine-Lipid Conjugate and ONC201 Combination Therapy Effectively Treats Orthotopic Pancreatic Tumor-Bearing Mice.

Gemcitabine (GEM) is a nucleoside analogue approved as a first line of therapy for pancreatic ductal adenocarcinoma (PDAC). However, rapid metabolism by plasma cytidine deaminase leading to the short half-life, intricate intracellular metabolism, ineffective cell uptake, and swift development of chemoresistance downgrades the clinical efficacy of GEM. ONC201 is a small molecule that inhibits the Akt and ERK pathways and upregulates the TNF-related apoptosis-inducing ligand (TRAIL), which leads to the reversal of both intrinsic and acquired GEM resistance in PDAC treatment. Moreover, the pancreatic cancer cells that were able to bypass apoptosis after treatment of ONC201 get arrested in the G1-phase, which makes them highly sensitive to GEM. To enhance the in vivo stability of GEM, we first synthesized a disulfide bond containing stearate conjugated GEM (lipid-GEM), which makes it sensitive to the redox tumor microenvironment (TME) comprising high glutathione levels. In addition, with the help of colipids 1,2-dioleoyl-glycero-3-phosphocholine (DOPC), cholesterol, and 1,2-distearoyl-glycero-3-phosphoethanolamine-poly(ethylene glycol)-2000 (DSPE-PEG 2000), we were able to synthesize the lipid-GEM conjugate and ONC201 releasing liposomes. A cumulative drug release study confirmed that both ONC201 and GEM showed sustained release from the formulation. Since MUC1 is highly expressed in 70-90% PDAC, we conjugated a MUC1 binding peptide in the liposomes which showed higher cytotoxicity, apoptosis, and cellular internalization by MIA PaCa-2 cells. A biodistribution study further confirmed that the systemic delivery of the liposomes through the tail vein resulted in a higher accumulation of drugs in orthotopic PDAC tumors in NSG mice. The IHC of the excised tumor grafts further confirmed the higher apoptosis and lower metastasis and cell proliferation. Thus, our MUC1 targeting binary drug-releasing liposomal formulation showed higher drug payload, enhanced plasma stability, and accumulation of drugs in the pancreatic orthotopic tumor and thus is a promising therapeutic alternative for the treatment of PDAC.

Abstract A021: Nutrient competition in the tumor microenvironment alters NK cell metabolism in Pancreatic Cancer

Abstract Tumor cells present constant nutritional demand of macronutrients and micronutrients for their rapid growth needs. Often this nutrient limitation hampers the function of anti-tumor immune cells, which need basal energy levels to execute cytotoxic effects against malignant cells. Hence, we investigated nutrient limitation-induced metabolic alterations in NK cells that regulate the anti-tumor activity of NK cells. Using 2D cultures and PDAC organoid models, we demonstrate that pancreatic tumor cells exhaust vitamin B6 (VB6) in the co-culture milieu, causing reduced killing activity of NK cells. Additionally, we demonstrate that PDAC patients have significantly reduced plasma VB6 as compared to the healthy counterparts. This data aligns with the epidemiological studies showing that VB6 intake reduces the risk of pancreatic cancer incidence. Moreover, we noted a significant reduction in circulating VB6 in the pancreatic tumor-bearing mice as compared to healthy counterparts. Utilizing mass spectrometry-based metabolomics we noted that limitation of the VB6 prevents glycogen breakdown in NK cells. This was validated by follow up electron microscopy-based studies that demonstrated glycogen depots in NK cells that were very prominent upon inhibiting glycogenolysis. Metabolic tracing studies demonstrated that NK cells require VB6 for intracellular glycogen breakdown. Correspondingly, knocking down glycogen phosphorylase, a key enzyme involved in glycogen breakdown abrogated anti-tumoral NK cell function. Notably, supplementation of co-cultures in 2D and organoid models and in vivo orthotopic mouse models, with VB6 restored NK cell function against pancreatic tumor cells. In parallel, we observed dependence of tumor cell metabolism on VB6 for sustaining growth. Accordingly, we observed that tumor cells actively deplete VB6 in tumor microenvironments, as observed by comparing VB6 levels in circulation and in tumor interstitial fluids. Finally, we observed that VB6 supplementation in combination with inhibitors targeting VB6-driven metabolic pathway dependencies in tumor cells effectively enhances NK cell frequency and diminishes tumor burden in vivo. These studies demonstrate a novel role of glycogen breakdown as a critical energy source for activated NK cells and demonstrate, for the first time a key role of glycogenolysis in NK cell cytotoxic activities. Our results expand the understanding of the critical role of micronutrients (vitamin B6) in regulating cancer progression and anti-tumor immunity and open new avenues for developing NK cell-based immunotherapies for PDAC patients. Citation Format: Kamiya Mehla. Nutrient competition in the tumor microenvironment alters NK cell metabolism in Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A021.

Lactobacillus casei combined with Lactobacillus reuteri alleviate pancreatic cancer by inhibiting TLR4 to promote macrophage M1 polarization and regulate gut microbial homeostasis

BackgroundPancreatic cancer is a highly lethal disease with no effective treatments. Lactobacillus casei (L. casei) and Lactobacillus reuteri (L. reuteri) exhibited therapeutic effects on several cancers, but their roles in pancreatic cancer are unknown. This study aims to explore how L. casei & L. reuteri influence pancreatic cancer and the underlying mechanisms.MethodsPancreatic cancer cells were treated with L. casei & L. reuteri and co-cultured with macrophages in a transwell system in vitro. Pancreatic cancer xenograft model was established and L. casei & L. reuteri was used to treat mice in vivo. MTT, CCK-8 assay or immunohistochemical staining were used to determine the proliferation of pancreatic cancer cells or tumor tissues. Transwell assay was applied to test the migration and invasion of pancreatic cells. RT-qPCR was utilized to assess TLR4 and MyD88 expressions in pancreatic cells or tumor tissues. WB, immunofluorescence staining, or flow cytometry was used to evaluate the M1/M2 polarization of macrophages. Besides, the composition of gut microbiota of tumor-bearing mice was determined by 16 S rRNA sequencing, and ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) untargeted metabolomics was used to evaluate the metabolic profiles of feces.ResultsL. casei & L. reuteri inhibited the proliferation, migration, invasion of pancreatic cancer cells and pancreatic cancer cell-induced M2 polarization of macrophages by suppressing TLR4. Meanwhile, L. casei & L. reuteri repressed pancreatic cancer growth and promoted M1 macrophage polarization. Besides, L. casei & L. reuteri reduced fecal Alloprevotella and increased fecal azelate and glutamate in nude mice, while TLR4 inhibitor TAK-242 increased Clostridia UCG-014, azelate, uridine, methionine sulfoxide, oxypurinol, and decreased glyceryl monoester in the feces of pancreatic tumor-bearing mice. Fecal oxypurinol and glyceryl monoester levels were positively or negatively associated with gut Clostridia UCG-014 abundance, respectively.ConclusionL. casei & L. reuteri alleviate pancreatic cancer by inhibiting TLR4 to promote macrophage M1 polarization and regulate gut microbial homeostasis.

Automated Synthesis and Preclinical Evaluation of Optimized Integrin α6-Targeted Positron Emission Tomography Imaging of Pancreatic Cancer.

Integrin α6 has been considered a promising biomarker, is overexpressed in many tumors, and plays a vital role in tumor formation, recurrence, and metastasis. In this study, we identified a novel high-affinity integrin α6-targeted peptide named RD2 (Arg-Trp-Tyr-Asp-PEG4)2-Lys-Lys and developed a 18F-radiolabeled peptide tracer ([18F]-AlF-NOTA-RD2) and evaluated its potential application in positron emission tomography (PET) imaging of pancreatic cancer. [18F]-AlF-NOTA-RD2 was produced using GMP (Good Manufacturing Practice of Medical Products)-compliant automatic radiosynthesis on a single GE FASTLab2 cassette-type synthesis module. The stability of [18F]-AlF-NOTA-RD2 was analyzed in phosphate-buffered saline (PBS) and fetal bovine serum (FBS). The cell uptake assay of the tracer was assessed using PANC-1 cells. In addition, small-animal PET imaging and biodistribution studies of [18F]-AlF-NOTA-RD2 were performed in pancreatic cancer subcutaneous tumor-bearing mice. The PET tracer [18F]-AlF-NOTA-RD2 was obtained with a radiochemical yield of 23.7 ± 4.7%, radiochemical purity of >99%, and molar activity of 165.7 ± 59.1 GBq/μmol. [18F]-AlF-NOTA-RD2 exhibited good in vitro stability in PBS and FBS. LogP octanol water value for the tracer was -2.28 ± 0.05 (n = 3). The binding affinity of RD2 to the integrin α6 protein (Kd = 0.13 ± 3.65 μM, n = 3) was significantly higher than that of the RWY (CRWYDENAC) (Kd = 6.97 ± 1.44 μM, n = 3). Small-animal PET imaging and biodistribution also revealed that [18F]-AlF-NOTA-RD2 displayed rapid and good tumor uptake and lower liver background uptake in PANC-1 tumor-bearing mice. [18F]-AlF-NOTA-RD2 showed significant radioactivity accumulation in tumors and was successfully blocked by NOTA-RD2. Compared with [18F]-FDG, [18F]-AlF-NOTA-RD2 PET imaging and biodistribution studies in PANC-1 xenograft tumor-bearing mice confirmed a good tumor-to-muscle ratio (8.69 ± 2.03 vs 1.41 ± 0.23, respectively) at 0.5 h and (2.99 ± 3.02 vs 1.43 ± 0.17, respectively) at 1 h post injection. Autoradiography of human pancreatic cancer tumor tissues further confirmed high accumulation of [18F]-AlF-NOTA-RD2. In summary, we developed an optimized integrin α6-targeted imaging tracer and obtained high radioactivity products with a cassette-type synthesis module; moreover, the tracer exhibited good binding affinity with integrin α6 and good target specificity for PANC-1 cells in xenograft pancreatic tumor-bearing mice, demonstrating its promising application as a noninvasive PET radiotracer of integrin α6 expression in pancreatic cancer.

Spectrally selective bSSFP using off-resonant RF excitations permits high spatiotemporal resolution 3D metabolic imaging of hyperpolarized [1-13 C]Pyruvate-to-[1-13 C]lactate conversion.

To develop a high spatiotemporal resolution 3D dynamic pulse sequence for preclinical imaging of hyperpolarized [1-13 C]pyruvate-to-[1-13 C]lactate metabolism at 7T. A standard 3D balanced SSFP (bSSFP) sequence was modified to enable alternating-frequency excitations. RF pulses with 2.33 ms duration and 900 Hz FWHM were placed off-resonance of the target metabolites, [1-13 C]pyruvate (by approximately -245 Hz) and [1-13 C]lactate (by approximately 735 Hz), to selectively excite those resonances. Relatively broad bandwidth (compared to those metabolites' chemical shift offset) permits a short TR of 6.29 ms, enabling higher spatiotemporal resolution. Bloch equation simulations of the bSSFP response profile guided the sequence parameter selection to minimize spectral contamination between metabolites and preserve magnetization over time. Bloch equation simulations, phantom studies, and in vivo studies demonstrated that the two target resonances could be cleanly imaged without substantial bSSFP banding artifacts and with little spectral contamination between lactate and pyruvate and from pyruvate hydrate. High spatiotemporal resolution 3D images were acquired of in vivo pyruvate-lactate metabolism in healthy wild-type and endogenous pancreatic tumor-bearing mice, with 1.212 s acquisition time per single-metabolite image and (1.75 mm)3 isotropic voxels with full mouse abdomen 56 × 28 × 21 mm3 FOV and fully-sampled k-space. Kidney and tumor lactate/pyruvate ratios of two consecutive measurements in one animal, 1 h apart, were consistent. Spectrally selective bSSFP using off-resonant RF excitations can provide high spatio-temporal resolution 3D dynamic images of pyruvate-lactate metabolic conversion.

Abstract 2676: Gender-associated improvement in gemcitabine’s therapeutic outcome by Palmatine

Abstract Despite a plethora of therapeutic strategies, improved survival is only modest for patients diagnosed with pancreatic ductal adenocarcinoma (PDAC). Additionally, between 2000 and 2018, pancreatic cancer incidence significantly increased with the greatest increase among women ages 15-35 years. We identified a protoberberine alkaloid, Palmatine (PMT), that was effective in slowing tumor growth and improving therapeutic outcome of gemcitabine (GEM) in pancreatic tumor bearing mice. Interestingly, the improved therapeutic outcome with the combination was observed in a sex-associated manner in mice. Intriguingly, PMT reduced proliferation of cells isolated from surgically resected tumor samples in 71% of female and 33% of male pancreatic cancer patients. We also observed that the correlation between PMT effects on cancer-related biological functions was associated with downregulation of STAT3 signaling. RNA-seq data from STAT3-KD pancreatic cancer cells revealed enrichment of genes associated with fatty acid metabolism. Of these, mRNA expression of ELOVL2 (Elongation of Very Long Chain Fatty Acids-Like 2) was most significantly increased suggesting a negative regulatory role for STAT3 in ELOVL2 expression. In silico analysis of the pancreatic cancer UalcanPan database shows that ELOVL2 is significantly reduced in human pancreatic tumors relative to normal pancreas and that high expression of ELOVL2 is associated with increased survival. The fatty acid elongase, ELOVL2, is involved in the production of docahexaenoic acid (DHA). Our experimental data show that PMT increased ELOVL2 expression. Taken together, these findings suggest the potential utility of PMT in therapeutic sensitization of GEM through deregulation of STAT3-mediated ELOVL2 signaling. These observations further suggest a sex-associated role for PMT in improving outcomes that may in part involve ELOVL2 function. Supported in part through VA Merit Award BX003876 and Owens Foundation pilot (APK). Citation Format: Meagan Marie Ybarra, Xiaoyu Yang, Amanda Munoz, Michelle Villarreal, Joel Michalek, Li-Ju Wang, Yidong Chen, Ikeno Yuji, Glenn Halff, Susan Weintraub, Rita Gosh, Addanki P. Kumar. Gender-associated improvement in gemcitabine’s therapeutic outcome by Palmatine [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 2676.

Chemotherapy-induced tumor immunogenicity is mediated in part by megakaryocyte-erythroid progenitors

Chemotherapy remains one of the main treatment modalities for cancer. While chemotherapy is mainly known for its ability to kill tumor cells directly, accumulating evidence indicates that it also acts indirectly by enhancing T cell-mediated anti-tumor immunity sometimes through immunogenic cell death. However, the role of immature immune cells in chemotherapy-induced immunomodulation has not been studied. Here, we utilized a mouse pancreatic cancer model to characterize the effects of gemcitabine chemotherapy on immature bone marrow cells in the context of tumor immunogenicity. Single cell RNA sequencing of hematopoietic stem and progenitor cells revealed a 3-fold increase in megakaryocyte-erythroid progenitors (MEPs) in the bone marrow of gemcitabine-treated mice in comparison to untreated control mice. Notably, adoptive transfer of MEPs to pancreatic tumor-bearing mice significantly reduced tumor growth and increased the levels of anti-tumor immune cells in tumors and peripheral blood. Furthermore, MEPs increased the tumor cell killing activity of CD8 + T cells and NK cells, an effect that was dependent on MEP-secreted CCL5 and CXCL16. Collectively, our findings demonstrate that chemotherapy-induced enrichment of MEPs in the bone marrow compartment contributes to anti-tumor immunity.

Thrombocytopenia in a Mouse Model of Pancreatic Cancer

Background: Pancreatic ductal adenocarcinoma (PDAC) is often associated with hypercoagulability and thrombocytosis. However, a subset of patients experiences thrombocytopenia and bleeding that is typically attributed to chemotherapy and/or anticoagulant treatment. Direct, pathophysiological effects of PDAC on platelet clearance, production, and function are poorly defined. Aim: Characterize platelet clearance, production, and function in a mouse model of PDAC associated with bleeding to better understand mechanisms that predispose cancer patients to coagulation dysfunction. Methods: C57BL/6J mice were orthotopically injected with pancreatic tumor cells derived from KrasG12D/+, p53R172H/+, ElasCreER/+ mice (KPC2 cells). Bleeding was assessed with a tail transection model. Complete blood cell counts were measured. Platelet clearance, hematopoietic stem and progenitor cell (HSPC) populations, and platelet activation were characterized by flow cytometry. CD41 in splenic tissue was detected by immunohistochemistry. Plasma thrombopoietin was quantified by ELISA.Results: Compared to control mice, KPC2 tumor-bearing mice exhibited increased tail bleeding, which correlated with decreased platelet count and increased mean platelet volume. Tumor-bearing mice had enhanced platelet clearance and increased spleen mass, and CD41 staining of spleens suggested splenic sequestration of platelets. Thrombopoietin levels were unchanged in tumor-bearing mice. Megakaryocyte progenitors including CD41+ hematopoietic stem cells (LSK, CD41+) were paradoxically increased, suggesting induction of the emergency hematopoiesis pathway. Platelets in tumor-bearing mice showed reduced activation following stimulation with ADP or convulxin. Conclusion: KPC2 pancreatic tumor-bearing mice had increased bleeding associated with thrombocytopenia, increased platelet clearance, altered hematopoiesis, and decreased platelet activation. Crosstalk between tumor cells, platelets, and bone marrow may contribute to coagulopathy in PDAC.

Obesity enriches for tumor protective microbial metabolites and treatment refractory cells to confer therapy resistance in PDAC

ABSTRACT Obesity causes chronic inflammation and changes in gut microbiome. However, how this contributes to poor survival and therapy resistance in patients with pancreatic cancer remain undetermined. Our current study shows that high fat diet-fed obese pancreatic tumor bearing mice do not respond to standard of care therapy with gemcitabine and paclitaxel when compared to corresponding control diet-fed mice. C57BL6 mice were put on control and high fat diet for 1 month following with pancreatic tumors were implanted in both groups. Microbiome of lean (control) and obese (high fat diet fed) mice was analyzed. Fecal matter transplant from control mice to obese mice sensitized tumors to chemotherapy and demonstrated extensive cell death. Analysis of gut microbiome showed an enrichment of queuosine (Q) producing bacteria in obese mice and an enrichment of S-adenosyl methionine (SAM) producing bacteria in control diet-fed mice. Further, supplementation of obese animals with SAM sensitized pancreatic tumors to chemotherapy. Treatment of pancreatic cancer cells with Q increased PRDX1 involved in oxidative stress protection. In parallel, tumors in obese mice showed increase in CD133+ treatment refractory tumor populations compared to control animals. These observations indicated that microbial metabolite Q accumulation in high fat diet-fed mice protected tumors from chemotherapy induced oxidative stress by upregulating PRDX1. This protection could be reversed by treatment with SAM. We conclude that relative concentration of SAM and queuosine in fecal samples of pancreatic cancer patients can be developed as a potential biomarker and therapeutic target in chemotherapy refractory pancreatic cancer.

Abstract 235: Differential polyamine expression in human pancreatic cancer supports the use of polyamine blockade therapy to modulate the in vivo pancreatic tumor microenvironment

Abstract Pancreatic cancer has a poor five-year survival rate of less than 8% and is projected to be the second leading cause of cancer related deaths in the US by the year 2030. Polyamine metabolism is an underexplored therapeutic target in pancreatic ductal adenocarcinoma (PDAC). The current investigation profiled RNA expression of genes associated with polyamine metabolism, transport and homeostasis in clinical pancreatic cancer samples to reveal evidence for dysregulation of polyamine-based pathways in PDAC. Increased expression of select pro-polyamine genes was associated with poorer patient prognosis in data obtained from the TCGA dataset. Preclinical experimental data supported the use of polyamine blockade therapy (PBT) in pancreatic tumor bearing mice, and revealed that PBT increased survival and decreased tumor weights in comparison to control treatment. PBT therapeutic effects were associated with increased expression of CD86 T cell co-stimulatory marker present on antigen presenting cells in the tumor microenvironment. Collectively, polyamine dysregulation in human pancreatic cancer is associated with poorer patient prognosis, whereas PBT treatment resulted in improved in vivo therapeutic outcomes in part through immune modulation in the tumor microenvironment. Citation Format: Sai Preethi Nakkina, Sarah B. Gitto, Veethika Pandey, Jignesh Parikh, Dirk Geerts, Carlo Maurer, Kenneth P. Olive, Otto Phanstiel, Deborah A. Altomare. Differential polyamine expression in human pancreatic cancer supports the use of polyamine blockade therapy to modulate the in vivo pancreatic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 235.

Abstract 3546: DFMO based improvement in survival of pancreatic cancer-bearing mice is associated with modulation of immune suppression in the tumor microenvironment

Abstract Existing therapeutics have failed to improve pancreatic ductal adenocarcinoma (PDAC) patient outcomes, in part due to tumor associated immune suppression and upregulation of compensatory mechanisms. KRAS and c-MYC are important oncogenes in PDAC linked to tumor immune suppression. We describe the use of difluoromethylornithine (DFMO) and a c-RAF inhibitor (GW5074) to indirectly target c-MYC and KRAS, respectively. DFMO in combination with GW5074 was tested for ability to improve pancreatic cancer outcomes. Pancreatic cancer cells showed decreased cell viability and increased apoptosis in response to a combination of DFMO + GW5074. In vivo orthotopic models of pancreatic tumors had decreased tumor weight in DFMO + GW5074 treated mice, when compared to control group. However, only treatment with single agent DFMO resulted in improved survival of immunocompetent pancreatic tumor bearing mice with respect to control, in contrast to treatment with GW5074 alone or DFMO + GW5074. To understand why the in vivo data was contrary to in vitro results, immunohistochemical analysis of immune cells in the tumor microenvironment was used to reveal increased expression of markers associated with anti-tumor effects such as CD86, CD3, CD4 and CD8 in DFMO treated tumors. Tumors treated with DFMO also displayed decreased expression of MYC, suggesting that DFMO-associated MYC suppression could be linked to decreased immune suppression in the tumor microenvironment and improved survival. In contrast, GW5074 treatment maintained MYC expression in tumors. Overall, the present study points to DFMO being an immunomodulatory agent, and a need for further understanding of DFMO-based therapeutic strategies in PDAC. Citation Format: Sai Preethi Nakkina, Sarah B. Gitto, Jordan M. Beardsley, Veethika Pandey, Michael M. Rohr, Jignesh Parikh, Otto Phanstiel, Deborah A. Altomare. DFMO based improvement in survival of pancreatic cancer-bearing mice is associated with modulation of immune suppression in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3546.

Abstract 6349A: Mechanical stress activates autophagy to induce drug resistance in pancreatic cancer cells

Abstract INTRODUCTION: One of the most fundamental problems that oncologists are currently facing in pancreatic cancer patient care is the high resistance to all types of chemotherapies or targeted therapies. Thus, there is an urgent need for increasing patient response to treatment. A common characteristic among most pancreatic cancer patients that could serve as potential target for treatment, is the biomechanically abnormal tumor microenvironment (TME). This altered TME is characterized by an increased stiffness which among others, is responsible for the development of mechanical stresses in the tumor interior. Even though several studies have investigated the effect of mechanical stress on cancer cells and tumor progression, it has not yet defined whether it can affect drug sensitivity and what are the mechanisms involved. HYPOTHESIS: Based on our recent findings, obtained by a Reverse Phase Protein Assay, we observed that mechanical stress activates a resistance response, employing autophagy activation, in pancreatic cancer cells. To this end, we hypothesize that mechanical stress developed during tumor growth, can induce autophagy activation to eventually promote drug resistance in pancreatic tumors. Therefore, inhibition of this mechanism can improve therapy. METHODOLOGY: We use two pancreatic cancer cells lines, MIA PaCa-2 and PANC-1, to apply mechanical stress, either in a 2D experimental setup or by employing tumor spheroids in a 3D agarose matrix. In both cases, cells are exposed to the most common chemotherapeutic agents used for pancreatic cancer treatment, gemcitabine and 5-Fluorouracil (5FU). The cytotoxic effect of each drug, as well as autophagy- and signaling pathway- activation are then analyzed. Finally, in vitro assays and treatments, along with in vivo animal models of pancreatic tumors, are performed to test the efficacy of an autophagy inhibitor combined with chemotherapy and a mechanotherapeutic that modulates intratumoral mechanical stresses. RESULTS: Proteomic analysis, together with pharmacological inhibition and siRNA treatments, revealed that mechanical stress activates autophagy to impair cell apoptosis and promote cancer cell survival. This stress-adaptation mechanism in turn impairs the cytotoxic effect of both gemcitabine and 5FU. A combined treatment employing chemotherapy with an autophagy inhibitor, and a mechanotherapeutic agent, showed promising results in increasing the overall-survival of pancreatic tumor-bearing mice. CONCLUSION: Our results provide solid evidence that alleviation of intratumoral mechanical stresses with mechanotherapeutics combined with autophagy inhibitors can improve the efficacy of chemotherapeutic agents in pancreatic cancer. Citation Format: Maria Kalli, Ruxuan Li, Ioannis Zervantonakis, Triantafyllos Stylianopoulos. Mechanical stress activates autophagy to induce drug resistance in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6349A.

Differential Expression of Polyamine Pathways in Human Pancreatic Tumor Progression and Effects of Polyamine Blockade on Tumor Microenvironment.

Simple SummaryPancreatic cancer has a five-year survival rate of less than 8% and is the fourth leading cause of cancer death in the United States. Existing therapeutics have failed to improve pancreatic ductal adenocarcinoma (PDAC) patient outcomes. There has been success with other tumor types in targeting aberrant polyamine upregulation as a therapeutic strategy. The present study identified dysregulation of polyamine pathways to be evident in human PDAC progression. Additionally, reduced survival of pancreatic cancer patients was associated with increased expression of specific polyamine-related genes. Polyamine blockade therapy significantly increased overall survival of pancreatic tumor-bearing mice, along with macrophage presence (F4/80) and significantly increased T-cell co-stimulatory marker (CD86) in the tumor microenvironment. Based on these findings, we hypothesized that a polyamine blockade therapy could potentially prime the tumor microenvironment to be more susceptible to existing therapeutics. Future studies which test polyamine blockade therapy with existing therapeutics could increase the molecular tools available to treat PDAC.Pancreatic cancer is the fourth leading cause of cancer death. Existing therapies only moderately improve pancreatic ductal adenocarcinoma (PDAC) patient prognosis. The present study investigates the importance of the polyamine metabolism in the pancreatic tumor microenvironment. Relative mRNA expression analysis identified differential expression of polyamine biosynthesis, homeostasis, and transport mediators in both pancreatic epithelial and stromal cells from low-grade pancreatic intraepithelial neoplasia (PanIN-1) or primary PDAC patient samples. We found dysregulated mRNA levels that encode for proteins associated with the polyamine pathway of PDAC tumors compared to early lesions. Next, bioinformatic databases were used to assess expression of select genes involved in polyamine metabolism and their impact on patient survival. Higher expression of pro-polyamine genes was associated with poor patient prognosis, supporting the use of a polyamine blockade therapy (PBT) strategy for inhibiting pancreatic tumor progression. Moreover, PBT treatment of syngeneic mice injected intra-pancreatic with PAN 02 tumor cells resulted in increased survival and decreased tumor weights of PDAC-bearing mice. Histological assessment of PBT-treated tumors revealed macrophage presence and significantly increased expression of CD86, a T cell co-stimulatory marker. Collectively, therapies which target polyamine metabolism can be used to disrupt tumor progression, modulate tumor microenvironment, and extend overall survival.

IgE-Based Therapeutic Combination Enhances Antitumor Response in Preclinical Models of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) represents 3% of all cancer cases and 7% of all cancer deaths in the United States. Late diagnosis and inadequate response to standard chemotherapies contribute to an unfavorable prognosis and an overall 5-year survival rate of less than 10% in PDAC. Despite recent advances in tumor immunology, tumor-induced immunosuppression attenuates the immunotherapy response in PDAC. To date, studies have focused on IgG-based therapeutic strategies in PDAC. With the recent interest in IgE-based therapies in multiple solid tumors, we explored the MUC1-targeted IgE potential against pancreatic cancer. Our study demonstrates the notable expression of FceRI (receptor for IgE antibody) in tumors from PDAC patients. Our study showed that administration of MUC1 targeted-IgE (mouse/human chimeric anti-MUC1.IgE) antibody at intermittent levels in combination with checkpoint inhibitor (anti-PD-L1) and TLR3 agonist (PolyICLC) induces a robust antitumor response that is dependent on NK and CD8 T cells in pancreatic tumor-bearing mice. Subsequently, our study showed that the antigen specificity of the IgE antibody plays a vital role in executing the antitumor response as nonspecific IgE, induced by ovalbumin (OVA), failed to restrict tumor growth in pancreatic tumor-bearing mice. Utilizing the OVA-induced allergic asthma-PDAC model, we demonstrate that allergic phenotype induced by OVA cannot restrain pancreatic tumor growth in orthotopic tumor-bearing mice. Together, our data demonstrate the novel tumor protective benefits of tumor antigen-specific IgE-based therapeutics in a preclinical model of pancreatic cancer, which can open new avenues for future clinical interventions.

Abstract PO-120: Differential expression of polyamine pathways in human pancreatic tumor progression and effects of polyamine blockade therapy on the in vivo pancreatic tumor microenvironment

Abstract Pancreatic cancer is the fourth leading cause of cancer death in the United States, with a five-year survival rate of less than 8%. Existing therapies have failed to improve pancreatic ductal adenocarcinoma (PDAC) patient prognosis. The dense desmoplastic reaction which occurs in PDAC makes it challenging for drugs and immune cells to infiltrate the fibrotic barrier. There is a need to exploit lesser explored targets in PDAC that can influence both the tumor and its microenvironment. One such avenue could be via targeting polyamine metabolism which is upregulated in pancreatic tumors. Though aberrant polyamine upregulation in pancreatic tumors has been known for decades, there has been little progress in translating this information into a PDAC therapeutic strategy. Additionally, there is a dearth of information regarding the dysregulation of polyamine metabolism in human PDAC and its association with clinical outcomes. Thus far, preclinical studies targeting polyamines using polyamine blockade therapy (PBT) has improved survival of pancreatic tumor bearing mice. Literature shows effectiveness of PBT in eliciting an anti-tumor immune response in other tumor types. Whether these results translate to the immune-privileged PDAC microenvironment need to be determined. The present study explores polyamine gene expression in human PDAC samples by mRNA expression analysis of frozen PDAC and Pancreatic intraepithelial neoplasia (PanIN). The Cancer Genome Atlas in the public domain was used to identify clinical outcomes of PDAC patients associated with select polyamine gene expression. Further, the anti-tumor effects of PBT and associated tumor microenvironment changes were identified using in vivo PDAC models and histological assessment. Polyamine dysregulation was found to be evident in human PDAC progression. Also, increased expression of certain polyamine-related genes was associated with poorer survival of pancreatic cancer patients. When targeting polyamines using PBT in immunocompetent C57Bl/6 mice with Pan02 tumor cells injected in the pancreas, PBT significantly increased overall survival. PBT also resulted in an increase in the infiltration of macrophages (F4/80) and expression of T-cell co-stimulatory marker (CD86) as assessed by immunohistochemistry and further quantification of imaging. Based on these changes, we hypothesized that PBT could prime the tumor microenvironment to be more susceptible to existing therapeutics. In conclusion, targeting polyamines using PBT results in increased survival and immune modulation in PDAC. Citation Format: Sai Preethi Nakkina, Sarah B. Gitto, Veethika Pandey, Jignesh G. Parikh, Dirk Geerts, Kenneth P. Olive, Otto Phanstiel, Deborah A. Altomare, Carlo Maurer. Differential expression of polyamine pathways in human pancreatic tumor progression and effects of polyamine blockade therapy on the in vivo pancreatic tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-120.

Abstract 1878: Fc-enhanced anti-CTLA-4 antibody, AGEN1181: new mechanistic insights for potent antitumor immunity and combination potential in treatment-resistant solid tumors

Abstract The advent of immune checkpoint inhibitors (ICI) targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), or programmed death-ligand 1 (PD-L1) transformed the treatment landscape in a variety of human cancers. However, responses to currently approved ICI agents occur in small subsets of patients with immunogenic tumors, and resistance to ICI therapy continues to be a limiting factor to achieving optimal clinical benefits. AGEN1181 is a Fc-enhanced, clinical-stage anti-CTLA-4 antibody designed to expand therapeutic benefit to a broader patient population, including tumor types that are refractory to conventional ICI approaches. Here we show that AGEN1181 exploits a novel Fcy receptor-related mechanism of action to promote T cell-mediated antitumor immunity. In primary in vitro T cell and antigen presenting cell (APC) co-culture stimulation assays, AGEN1181 demonstrated superior T cell priming and activation that was independent of Treg depletion, and across all CD16 allele variants - in contrast to conventional IgG1 agents that showed limited activity. In PD-1 refractory tumor-bearing mouse models, a murine AGEN1181 surrogate demonstrated superior antitumor activity compared to an Fc-unmodified anti-CTLA-4 antibody. This enhanced activity was associated with improved T cell priming, intratumoral Treg depletion, increased frequency of CD8 effector memory precursor T cells, enhanced peripheral TCR clonality, and expansion of tumor-associated T cell clones. Next we evaluated the combination potential of the AGEN1181 surrogate with targeted therapies, cell therapy or checkpoint blockade in a series of treatment-resistant preclinical tumor models. Notably, this antibody induced robust tumor control and antitumor immunity when administered alongside anti-PD-1 and focal radiation, adoptive T cell therapy, or iNKT-activating therapy in the refractory B16F10 or B16F1.OVA melanoma models. In treatment-resistant KPC pancreatic ductal tumor-bearing mice, curative responses were seen in combination with chemotherapy (gemcitabine, nab-paclitaxel and cisplatin). Moreover, combinations with other ICI (e.g. anti-PD-1 or anti-TIGIT antibodies) deepened responsiveness and overcame limited activity seen with conventional ICI therapy. Importantly, antitumor immune responses could be further improved with optimal sequencing of combination therapies with AGEN1181. Together, these data demonstrate that the novel Fcy receptor-related mechanism of action employed by AGEN1181 enhances antitumor immunity and confers the potential to expand therapeutic benefit to tumors refractory to ICI therapy. Clinical responses support these findings and a phase II trial is underway in patients with a range of solid tumor types, including angiosarcoma and microsatellite stable tumors. Citation Format: Antoine J. Tanne, Claire Galand, Abdo Abou-Slaybi, Margaret Wilkens, Marilyn Marques, Serina Ng, Haiyong Han, Sylvia Dietrich, Jeremy Waight, Bishnu Joshi, Burcu Yigit, Xavier Michelet, Daniel Levey, David Savitsky, Jennifer Buell, Dhan Chand. Fc-enhanced anti-CTLA-4 antibody, AGEN1181: new mechanistic insights for potent antitumor immunity and combination potential in treatment-resistant solid tumors [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 1878.