Murine Pancreatic Cancer Cells Research Articles

Dendritic cell maturation is induced by p53-armed oncolytic adenovirus via tumor-derived exosomes enhancing systemic antitumor immunity.

Dendritic cells (DCs) are crucial in cancer immunity, because they activate cytotoxic T cells by presenting tumor antigens. Recently, oncolytic virus therapy has been recognized as a systemic immune stimulator. We previously developed a telomerase-specific oncolytic adenovirus (OBP-301) and a p53-armed OBP-301 (OBP-702), demonstrating that these viruses strongly activate systemic antitumor immunity. However, their effects on DCs remained unclear. In the present study, the aim was to elucidate the mechanisms of DC activation by OBP-702, focusing particularly on tumor-derived exosomes. Exosomes (Exo53, Exo301, or Exo702) were isolated from conditioned media of human or murine pancreatic cancer cell lines (Panc-1, MiaPaCa-2, and PAN02) after treatment with Ad-p53, OBP-301, or OBP-702. Exo702 derived from Panc-1 and MiaPaCa-2 cells significantly upregulated CD86, CD80, CD83 (markers of DC maturation), and IFN-γ in DCs in vitro. Similarly, Exo702 derived from PAN02 cells upregulated CD86 and IFN-γ in bone marrow-derived DCs in a bilateral PAN02 subcutaneous tumor model. This DC maturation was inhibited by GW4869, an inhibitor of exosome release, and anti-CD63, an antibody targeting the exosome marker. Intratumoral injection of OBP-702 into PAN02 subcutaneous tumors significantly increased the presence of mature DCs and CD8-positive T cells in draining lymph nodes, leading to long-lasting antitumor effects through the durable activation of systemic antitumor immunity. In conclusion, tumor-derived exosomes play a significant role in DC maturation following OBP-702 treatment and are critical for the systemic activation of antitumor immunity, leading to the abscopal effect.

Pancreatic cancer cell-derived migrasomes promote cancer progression by fostering an immunosuppressive tumor microenvironment

Pancreatic cancer is distinguished by an immunosuppressive tumor microenvironment (TME) that facilitates cancer progression. The assembly of the TME involves numerous contributing factors. Migrasomes, recently identified as cellular organelles in migrating cells, play a pivotal role in intercellular signaling. However, research into their involvement in cancers remains nascent. Thus far, whether pancreatic cancer cells generate migrasomes and their potential role in TME formation remains unexplored. In this study, it was found that both murine and human pancreatic cancer cells could indeed generate migrasomes, termed pancreatic cancer cell-derived migrasomes (PCDMs), which actively promote cancer progression. Moreover, utilizing chemokine antibody arrays and quantitative mass spectrometry analysis, we observed significant differences between the chemokines, cytokines, and proteins present in PCDMs compared to their originating cell bodies. Notably, PCDMs exhibited an enrichment of immunosuppression-inducing factors. Furthermore, macrophages could directly uptake PCDMs, leading to the expression of high levels of M2-like markers and secretion of tumor-promoting factors. PCDM-induced macrophages played a pivotal role in inhibiting T cell proliferation and activation partially through ARG-1. In summary, this study provides compelling evidence that pancreatic cancer cells generate migrasomes, which play a crucial role in promoting tumor progression by contributing to an immunosuppressive TME. The exploration of migrasomes as a therapeutic target could pave the way for the development of tailored immunotherapies for pancreatic cancer.

Adipose tissue-derived extracellular vesicles from obese mice suppressed splenocyte-mediated pancreatic cancer cell death.

Obesity is a risk factor for pancreatic cancer and negatively contributes to the immune system. However, the mechanisms by which obesity mediates these actions are still poorly understood. Recent studies have demonstrated that extracellular vesicles (EVs) are key mediators of communication between cells and may influence various aspects of cancer progression. We aim to explore the influence of EVs derived from adipose tissue of obese mice on cytokine production within the interactions between cancer cells and immune cells. We isolated EVs from the adipose tissue of both C57BL6/J mice and Ob/Ob mice. Subsequently, we treated EVs with Panc02 cells, the murine ductal pancreatic cancer cell line, which were co-cultured with splenocytes. Viability and SMAD4 gene expression were examined in Panc02 cells, and cytokine concentrations of IL-6, IL-4, IL-12, and IL-12p70 were measured in the cultured medium. Interestingly, we observed a significant reduction in splenocyte-mediated Panc02 cell death when treated with EVs derived from the adipose tissue of Ob/Ob mice, compared to those from C57BL6/J mice. Additionally, EVs from Ob/Ob mice-derived adipose tissue significantly increased the levels of IL-4, IL-2, and IL-12p70 in the culture media of Panc02 cells co-cultured with splenocytes, compared to EVs from C57BL6/J mice-derived adipose tissue. Adipose tissue-derived EVs from obese mice suppressed splenocyte-mediated Panc02 cell death and upregulated IL-4, IL-2, and IL-12p70 in cultured medium.

A Physiochemical, In Vitro, and In Vivo Comparative Analysis of Verteporfin-Lipid Conjugate Formulations: Solid Lipid Nanoparticles and Liposomes.

VisudyneⓇ, a liposomal formulation of verteporfin (benzoporphyrin derivative; BPD), is the only nanomedicine approved to date for photodynamic therapy (PDT). We have previously demonstrated that BPD conjugated to the lysophospholipid 1-arachidoyl-2-hydroxy-sn-glycero-3-phosphocholine (BPD-PC) exhibits the greatest physical stability in liposomes, while maintaining cancer cell phototoxicity, from a panel of BPD lipid conjugates evaluated. In this study, we prepared 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-based solid lipid nanoparticles (LNPs) that stably entrap BPD-PC, which resemble the composition of the SpikevaxⓇ Moderna COVID-19 vaccine, and compared them to a DPPC based liposomal formulation (Lipo BPD-PC). We evaluated the photochemical, optical, and phototherapeutic properties of both formulations. We also investigated the in vivo distribution and tumor microdistribution of both formulations. Our results demonstrated that Lipo BPD-PC is able to generate 17% more singlet oxygen than LNP BPD-PC, while interestingly, LNP BPD-PC is able to produce 76% more hydroxyl radicals and/or peroxynitrite anion. Importantly, only 28% of BPD-PC leaches out of the LNP BPD-PC formulation during 7 days of incubation in serum at 37 °C, while 100% of BPD-PC leaches out of the Lipo BPD-PC formulation under the same conditions. Despite these differences, there was no significant difference in cellular uptake of BPD-PC or phototoxicity in CT1BA5 murine pancreatic cancer cells (derived from a genetically engineered mouse model). Interestingly, PDT using LNP BPD-PC was more efficient at inducing immunogenic cell death (calreticulin membrane translocation) than Lipo BPD-PC when using IC25 and IC50 PDT doses. In vivo studies revealed that CT1BA5 tumor fluorescence signals from BPD-PC were 2.41-fold higher with Lipo BPD-PC than with LNP BPD-PC; however, no significant difference was observed in tumor tissue selectivity or tumor penetration. As such, we present LNP BPD-PC as a unique and more stable nanoplatform to carry BPD lipid conjugates, such as BPD-PC, with a potential for future photodynamic immune priming studies and multiagent drug delivery.

Effects of triggers of senescence and senolysis in murine pancreatic cancer cells

BackgroundThe combination of senescence triggers with senolytic drugs is considered a promising new approach to cancer therapy. Here, we studied the efficacy of the genotoxic agent etoposide (Eto) and irradiation in inducing senescence of Panc02 pancreatic cancer cells, and the capability of the Bcl-2 inhibitor navitoclax (ABT-263; Nav) to trigger senolysis. MethodsPanc02 cells were treated with Eto or irradiated with 5–20 Gy before exposure to Nav. Cell survival, proliferation, and senescence were assessed by trypan blue staining, quantification of DNA synthesis, and staining of senescence-associated β-galactosidase (SA-β-Gal)-positive cells, respectively. Levels of mRNA were determined by real-time polymerase chain reaction, and protein expression was analyzed by immunoblotting. Panc02 cells were also grown as pancreatic tumors in mice, which were subsequently treated with Eto and Nav. ResultsEto and irradiation had an antiproliferative effect on Panc02 cells that was significantly or tendentially enhanced by Nav. In vivo, Eto and Nav together, but not Eto alone, significantly reduced the proportion of proliferating cells. The expression of the senescence marker γH2AX and tumor infiltration with T-cells were not affected by the treatment. In vitro, almost all Eto-exposed cells and a significant proportion of cells irradiated with 20 Gy were SA-β-Gal-positive. Application of Nav reduced the percentage of SA-β-Gal-positive cells after irradiation but not after pretreatment with Eto. In response to triggers of senescence, cultured Panc02 cells showed increased protein levels of γH2AX and the autophagy marker LC3B-II, and higher mRNA levels of Cdkn1a, Mdm2, and PAI-1, while the effects of Nav were variable. ConclusionsIn vitro and in vivo, the combination of senescence triggers with Nav inhibited tumor cell growth more effectively than the triggers alone. Our data also provide some evidence for senolytic effects of Nav in vitro.

Hepatic signal transducer and activator of transcription-3 signalling drives early-stage pancreatic cancer cachexia via suppressed ketogenesis.

Patients with pancreatic ductal adenocarcinoma (PDAC) often suffer from cachexia, a wasting syndrome that significantly reduces both quality of life and survival. Although advanced cachexia is associated with inflammatory signalling and elevated muscle catabolism, the early events driving wasting are poorly defined. During periods of nutritional scarcity, the body relies on hepatic ketogenesis to generate ketone bodies, and lipid metabolism via ketogenesis is thought to protect muscle from catabolizing during nutritional scarcity. We developed an orthotopic mouse model of early PDAC cachexia in 12-week-old C57BL/6J mice. Murine pancreatic cancer cells (KPC) were orthotopically implanted into the pancreas of wild-type, IL-6-/-, and hepatocyte STAT3-/- male and female mice. Mice were subject to fasting, 50% food restriction, ad libitum feeding or ketogenic diet interventions. We measured longitudinal body composition by EchoMRI, body mass and food intake. At the endpoint, we measured tissue mass, tissue gene expression by quantitative real-time polymerase chain reaction, whole-body calorimetry, circulating hormone levels, faecal protein and lipid content, hepatic lipid content and ketogenic response to medium-chain fatty acid bolus. We assessed muscle atrophy in vivo and C2C12 myotube atrophy in vitro. Pre-cachectic PDAC mice did not preserve gastrocnemius muscle mass during 3-day food restriction (-13.1±7.7% relative to food-restricted sham, P=0.0117) and displayed impaired fatty acid oxidation during fasting, resulting in a hypoketotic state (ketogenic response to octanoate bolus, -83.0±17.3%, P=0.0328; Hmgcs2 expression, -28.3±7.6%, P=0.0004). PDAC human patients display impaired fasting ketones (-46.9±7.1%, P<0.0001) and elevated circulating interleukin-6 (IL-6) (12.4±16.5-fold increase, P=0.0001). IL-6-/- PDAC mice had improved muscle mass (+35.0±3.9%, P=0.0031) and ketogenic response (+129.4±44.4%, P=0.0033) relative to wild-type PDAC mice. Hepatocyte-specific signal transducer and activator of transcription 3 (STAT3) deletion prevented muscle loss (+9.3±4.0%, P=0.009) and improved fasting ketone levels (+52.0±43.3%, P=0.018) in PDAC mice. Without affecting tumour growth, a carbohydrate-free diet improved tibialis anterior myofibre diameter (+16.5±3.5%, P=0.0089), circulating ketone bodies (+333.0±117.6%, P<0.0001) and Hmgcs2 expression (+106.5±36.1%, P<0.0001) in PDAC mice. Ketone supplementation protected muscle against PDAC-induced atrophy in vitro (+111.0±17.6%, P<0.0001 myofibre diameter). In early PDAC cachexia, muscle vulnerability to wasting is dependent on inflammation-driven metabolic reprogramming in the liver. PDAC suppresses lipid β-oxidation and impairs ketogenesis in the liver, which is reversed in genetically modified mouse models deficient in IL-6/STAT3 signalling or through ketogenic diet supplementation. This work establishes a direct link between skeletal muscle homeostasis and hepatic metabolism. Dietary and anti-inflammatory interventions that restore ketogenesis may be a viable preventative approach for pre-cachectic patients with pancreatic cancer.

Abstract 5624: Dec2, a circadian rhythm gene facilitates pancreatic cancer dormancy through evasion of antitumor immunity

Abstract Metastatic recurrence following curative intent oncologic surgery is a major clinical problem in patients with pancreatic ductal adenocarcinoma (PDAC). These recurrences are from disseminated tumor cells (DTCs) and can sometimes occur years after surgery indicating that DTCs undergo a period of dormancy. The mechanisms that drive cancer dormancy and its awakening are poorly understood in part due to a lack of animal models that recapitulate dormant tumor cell biology. We created a novel model of PDAC dormancy where mice undergo surgery to remove the primary tumor and are followed for evidence of metastatic recurrence. Dormant DTCs can be isolated from mice without clinical evidence of recurrence for molecular studies. Single cell RNAseq of dormant DTCs detected that the circadian rhythm gene, Dec2 is upregulated. We knocked out (KO) Dec2 from murine pancreatic cancer cells and when implanted into the model we found almost no mice developed metastatic recurrence. When immunodeficient or immunocompetent mice depleted of CD4 and CD8 T cells were used as hosts, we observed no difference in survival in mice engrafted with Dec WT vs. KO cells indicating the survival benefit in the Dec KO group was due to an immune mediated mechanism. We performed a differential expression analysis on bulk RNAseq data comparing the Dec2 WT vs. KO cells and found antigen presentation a top upregulated pathway. We then observed that surface MHC-I was significantly upregulated in Dec2 KO cells as compared to wild type cells. Further mechanistic studies revealed that proteosome activity was significantly higher in Dec2 KO cells and treatment with proteosome inhibitors decreased surface MHCI levels in Dec2 KO cells. Cut and tag experiments revealed that Dec2 binds the promoter of a number of genes that regulate the proteosome. In conclusion, Dec2 promotes cancer dormancy by down regulating cell surface MHCI which facilitates immune evasion. Citation Format: Lan Wang, Chris Harris, Orjola Prela, Wade C. Narrow, Darren R. Carpizo. Dec2, a circadian rhythm gene facilitates pancreatic cancer dormancy through evasion of antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5624.

Abstract 5358: The influence of extracellular vesicles on immune cells in PDAC

Abstract Introduction: Pancreatic Ductal Adenocarcinoma (PDAC) is bad. The tumor microenvironment consists of an abundance of immunosuppressive immune cells and anti-inflammatory cytokines that contribute to local and systemic immunosuppression in PDAC. Tumor cells can influence the tumor microenvironment and, with that, the present immune cells through the secretion of cytokines or chemokines or the secretion of extracellular vesicles (EVs). EVs are a media of communication among the tumor cells themselves, but also with other cells of the body, with the goal to promote tumor expansion and metastasis and decrease any anti-tumor-directed forces of the immune system. Our lab has shown that those EVs play a significant role in tumor growth and metastasis in PDAC. We have found MUC1 to be an important marker for tumor-derived EVs (tdEVs), associated with their activity and influence on cell proliferation and tumor growth in vitro and in vivo. Determining which immune cell populations are most targeted and impacted by tdEVs is essential to improving our understanding of the role of EVs in PDAC. Methods: TdEVs were isolated from murine pancreatic cancer cell lines through differential ultracentrifugation. EVs were then labeled with a membrane dye, which allows tracking of the EVs in the mouse. We designed an in vivo study to investigate the effects that tdEVs have on different immune cell populations, as well as tumor growth and metastasis. This study has two distinct phases: a 3-week “Pre-Education Phase”, with injection of either MUC1+ or MUC1KO EVs, is followed by orthotopic implantation of tumor cells in the pancreas. Data: We hypothesized that tdEVs impact effector immune cells in PDAC, leading to phenotypic, transcriptional, and functional changes that result in a decreased anti-tumor immune response. The highest uptake of EVs was found in macrophages and dendritic cells (DCs). Furthermore, most immune cells preferably take up MUC1+ as compared to MUC1KO EVs. After three weeks of EV education, RNA sequencing of the liver showed differential gene expression in a variety of genes related to processes associated with B cells. Treatment with MUC1+ EVs downregulated B cell-related processes in the liver, B cell differentiation, proliferation and activity are reduced. Several genes coding for immunoglobulin parts were affected as well. Immune cell profiling of the blood and spleen showed that MUC1+ EVs reduce NK and macrophage numbers in the spleen as compared to MUC1KO EV treatment after three weeks. Additionally, EV-treatment increased CD8+ T cell numbers in the blood but decreased CD8+ T cells in the spleen. Conclusion: We have shown that tdEVs induce phenotypic and transcriptional changes in immune cell populations and alter immune cell frequencies in vivo. Ongoing research in our lab involves analysis of phenotypic and transcriptional changes after tumor implantation as well as functional differences in EV-treated immune cells in vitro and in vivo. Citation Format: Clara S. Mundry, Ying Huang, Paul M. Grandgenett, Michael A. Hollingsworth. The influence of extracellular vesicles on immune cells in PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5358.

Abstract 7463: Chromosomal instability elicits anti-tumor immunity in pancreatic cancer

Abstract Chromosomal instability (CIN) is a hallmark of solid cancers and an established driver of disease progression, therapy resistance, metastasis and on the whole poor patient outcome. Pancreatic cancer is particularly devastating, defined by dismal survival rates with many patients presenting with metastatic disease upon diagnosis, and 5-year survival only reaching ~10%. Despite recent efforts to improve prognosis for pancreatic cancer patients with immunotherapies alone and in combination with other therapeutics progress remains limited. Despite CIN’s established role as a driver of disease progression, we have made the paradoxical observation that chromosomally unstable, murine pancreatic cancer cells elicit an anti-tumor immune response. CIN was induced experimentally in a chromosomally stable, metastatic model of pancreatic cancer. Modeling metastatic dissemination by injection of cells into the tail vein of mice led to robust tumor formation in the lungs where CIN-low cells formed many small lesions while CIN-high cells formed fewer but larger tumors. We investigated if the tumors were infiltrated differentially by immune cells by immunofluorescence and found that CIN-high tumors were more readily infiltrated by effector T-cells. This observation motivated us to test immunotherapy response in CIN-low and CIN-high cells. Subcutaneously implanted tumor cells were treated with an immune checkpoint inhibitor and isotype control antibody which revealed that CIN-high cells responded to immunotherapy while CIN-low cells did not. To put forward potential mechanisms explaining CIN-driven anti-tumor immunity, we performed molecular analyses of CIN-low and CIN-high tumors. First, we found that inflammatory pathways were significantly up-regulated in CIN-high tumors compared with CIN-low tumors. These findings were validated by cytokine measurements and RNA in situ hybridization. To dissect inflammatory pathway activation mechanistically, we engineered CIN-high cells using CRISPR/Cas9 that were deficient for key inflammatory cytokines and transcription factors. These experiments suppressed cell-intrinsic and ligand-induced inflammation. Yet, we failed to determine singular cytokine expression as mediator of anti-tumor immunity. To rule out the contribution of ligand-induced inflammation in the tumor microenvironment, we probed immune checkpoint blockade in combination with antibodies blocking inflammatory ligand receptors in CIN-high cells. We found that immune checkpoint blockade was cancer cell-intrinsic and independent of blocking antibodies. In summary, our data implicates CIN as a determinant of therapeutic and genetic anti-tumor immunity in a preclinical model of pancreatic cancer. Citation Format: Daniel Bronder, Roshan K. Sriram, Mercedes Duran, Melody Di Bona, Sarina McElduff, Samuel F. Bakhoum. Chromosomal instability elicits anti-tumor immunity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7463.

Mucin-1-Targeted Chimeric Antigen Receptor T Cells Are Effective and Safe in Controlling Solid Tumors in Immunocompetent Host.

The chimeric antigen receptor (CAR) T-cell therapy in solid epithelial tumors has been explored, however, with limited success. As much of the preclinical work has relied on xenograft models in immunocompromised animals, the immune-related efficacies and toxicities may have been missed. In this study, we engineered syngeneic murine CAR T cells targeting the tumor form of human mucin-1 (tMUC1) and tested the MUC1 CAR T cells' efficacy and toxicity in the immunocompetent human MUC1-expressing mouse models. The MUC1 CAR T cells significantly eliminated murine pancreatic and breast cancer cell lines in vitro. In vivo, MUC1 CAR T cells significantly slowed the mammary gland tumor progression in the spontaneous PyVMT×MUC1.Tg (MMT) mice, prevented lung metastasis, and prolonged survival. Most importantly, there was minimal short or long-term toxicity with acceptable levels of transient liver toxicity but no kidney toxicity. In addition, the mice did not show any signs of weight loss or other behavioral changes with the treatment. We also report that a single dose of MUC1 CAR T-cell treatment modestly reduced the pancreatic tumor burden in a syngeneic orthotopic model of pancreatic ductal adenocarcinoma given at late stage of an established tumor. Taken together, these findings suggested the further development of tMUC1-targeted CAR T cells as an effective and relatively safe treatment modality for various tMUC1-expressing solid tumors.

Expression of lysine demethylase 5a (Kdm5a) influences tumour aggressiveness in murine pancreatic cancer cells

Expression of lysine demethylase 5a (Kdm5a) influences tumour aggressiveness in murine pancreatic cancer cells

A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2 deficient pancreatic cancer

Pancreatic cancer is one of the deadliest diseases in human malignancies. Among total pancreatic cancer patients, ~10% of patients are categorized as familial pancreatic cancer (FPC) patients, carrying germline mutations of the genes involved in DNA repair pathways (e.g., BRCA2). Personalized medicine approaches tailored toward patients’ mutations would improve patients’ outcome. To identify novel vulnerabilities of BRCA2-deficient pancreatic cancer, we generated isogenic Brca2-deficient murine pancreatic cancer cell lines and performed high-throughput drug screens. High-throughput drug screening revealed that Brca2-deficient cells are sensitive to Bromodomain and Extraterminal Motif (BET) inhibitors, suggesting that BET inhibition might be a potential therapeutic approach. We found that BRCA2 deficiency increased autophagic flux, which was further enhanced by BET inhibition in Brca2-deficient pancreatic cancer cells, resulting in autophagy-dependent cell death. Our data suggests that BET inhibition can be a novel therapeutic strategy for BRCA2-deficient pancreatic cancer.

Optically induced electrothermal microfluidic tweezers in bio-relevant media

Non-contact micro-manipulation tools have enabled invasion-free studies of fragile synthetic particles and biological cells. Rapid electrokinetic patterning (REP) traps target particles/cells, suspended in an electrolyte, on an electrode surface. This entrapment is electrokinetic in nature and thus depends strongly on the suspension medium’s properties. REP has been well characterized for manipulating synthetic particles suspended in low concentration salt solutions (~ 2 mS/m). However, it is not studied as extensively for manipulating biological cells, which introduces an additional level of complexity due to their limited viability in hypotonic media. In this work, we discuss challenges posed by isotonic electrolytes and suggest solutions to enable REP manipulation in bio-relevant media. Various formulations of isotonic media (salt and sugar-based) are tested for their compatibility with REP. REP manipulation is observed in low concentration salt-based media such as 0.1× phosphate buffered saline (PBS) when the device electrodes are passivated with a dielectric layer. We also show manipulation of murine pancreatic cancer cells suspended in a sugar-based (8.5% w/v sucrose and 0.3% w/v dextrose) isotonic medium. The ability to trap mammalian cells and deposit them in custom patterns enables high-impact applications such as determining their biomechanical properties and 3D bioprinting for tissue scaffolding.

CCR4 Blockade Diminishes Intratumoral Macrophage Recruitment and Augments Survival of Syngeneic Pancreatic Cancer-Bearing Mice.

Pancreatic cancer is known for its tumor microenvironment (TME), which is rich in stromal and immune cells supporting cancer growth and therapy resistance. In particular, tumor-associated macrophages (TAMs) are known for their angiogenesis- and metastasis-promoting properties, which lead to the failure of conventional therapies for pancreatic cancer. Hence, treatment options targeting TAMs are needed. The C-C chemokine receptor type 4 (CCR4) is critical for immune cell recruitment into the TME, and in this paper we explore the effects of its genetic or immunotherapeutic blockade in pancreatic-cancer-bearing mice. Murine PDA6606 pancreatic cancer cells and murine peritoneal macrophages were used for in vitro migration assays. In vivo, a syngeneic, orthotropic pancreatic cancer model was established. Tumor growth and survival were monitored under prophylactic and therapeutic application of a CCR4 antagonist (AF-399/420/18025) in wildtype (CCR4wt) and CCR4-knockout (CCR4-/-) mice. Immune infiltration was monitored in tumor tissue sections and via flow cytometry of lysed tumors. PDA6606 cells induced less migration in CCR4-/- than in CCR4wt macrophages in vitro. Pancreatic TAM infiltration was higher, and survival was reduced in CCR4wt mice compared to CCR4-/- mice. Antagonizing CCR4 in wildtype mice revealed similar results as in CCR4-/- mice without antagonization. Prophylactic CCR4 antagonist application in wildtype mice was more efficient than therapeutic antagonization. CCR4 seems to be critically involved in TAM generation and tumor progression in pancreatic cancer. CCR4 blockade may help prolong the relapse-free period after curative surgery in pancreatic cancer and improve prognosis.

Blocking muscle wasting via deletion of the muscle-specific E3 ligase MuRF1 impedes pancreatic tumor growth

Cancer-induced muscle wasting reduces quality of life, complicates or precludes cancer treatments, and predicts early mortality. Herein, we investigate the requirement of the muscle-specific E3 ubiquitin ligase, MuRF1, for muscle wasting induced by pancreatic cancer. Murine pancreatic cancer (KPC) cells, or saline, were injected into the pancreas of WT and MuRF1-/- mice, and tissues analyzed throughout tumor progression. KPC tumors induces progressive wasting of skeletal muscle and systemic metabolic reprogramming in WT mice, but not MuRF1-/- mice. KPC tumors from MuRF1-/- mice also grow slower, and show an accumulation of metabolites normally depleted by rapidly growing tumors. Mechanistically, MuRF1 is necessary for the KPC-induced increases in cytoskeletal and muscle contractile protein ubiquitination, and the depression of proteins that support protein synthesis. Together, these data demonstrate that MuRF1 is required for KPC-induced skeletal muscle wasting, whose deletion reprograms the systemic and tumor metabolome and delays tumor growth.

Abstract 4830: Uncoupling protein 2 (UCP2) loss of function mediates PDAC tumor suppression by mitochondrial fusion

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a dependence on highly active mitochondria to grow and metastasize. We previously found that mitochondrial fusion suppressed PDAC growth and metastasis, leading to improved survival in a preclinical model. The tumor suppressive mechanisms of mitochondrial fusion remain unclear, and thus we explored the role of redox homeostasis through uncoupling protein 2 (UCP2). Methods/Results: Murine pancreatic cancer cells (KPC) with enhanced mitochondrial fusion (sgDrp1) exhibited higher levels of mitochondrial ROS compared to controls targeting GFP (sgGFP), suggesting altered redox homeostasis. We performed RNAseq on these two cell lines and found that sgDRP1 cells exhibited reduced levels of UCP2. The UCP2 protein is known to have indirect antioxidant effects by reducing mitochondrial reactive oxygen species via uncoupling of ATP production from the electron transport chain. We found that abrogating UCP2 in KPC cells reduced its capacity for OXPHOS. Moreover, rescue of UCP2 in sgDrp1 cells restored their mitochondrial activity. Conclusions: Mitochondrial fusion may suppress tumor growth and induce mitophagy by altering redox homeostasis. UCP2 may be a mediator of the tumor suppressive phenotype induced by mitochondrial fusion, thus linking mitochondrial dynamics and PDAC antioxidant strategies. Future Work: These results will be explored for their translation in vivo and the effects of pharmacological inhibition of UCP2, both in vitro and in vivo, via the specific inhibitor Genipin, will be explored. Citation Format: Ariana Carolina Acevedo-Diaz, Emily G. Caggiano, Meifang Yu, Cullen Taniguchi. Uncoupling protein 2 (UCP2) loss of function mediates PDAC tumor suppression by mitochondrial fusion. [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 4830.

Conditional ablation of heparan sulfate expression in stromal fibroblasts promotes tumor growth in vivo.

Heparan sulfate (HS) is a glycocalyx component present in the extracellular matrix and cell-surface HS proteoglycans (HSPGs). Although HSPGs are known to play functional roles in multiple aspects of tumor development and progression, the effect of HS expression in the tumor stroma on tumor growth in vivo remains unclear. We conditionally deleted Ext1, which encodes a glycosyltransferase essential for the biosynthesis of HS chains, using S100a4-Cre (S100a4-Cre; Ext1f/f) to investigate the role of HS in cancer-associated fibroblasts, which is the main component of the tumor microenvironment. Subcutaneous transplantation experiments with murine MC38 colon cancer and Pan02 pancreatic cancer cells demonstrated substantially larger subcutaneous tumors in S100a4-Cre; Ext1f/f mice. Additionally, the number of myofibroblasts observed in MC38 and Pan02 subcutaneous tumors of S100a4-Cre; Ext1f/f mice decreased. Furthermore, the number of intratumoral macrophages decreased in MC38 subcutaneous tumors in S100a4-Cre; Ext1f/f mice. Finally, the expression of matrix metalloproteinase-7 (MMP-7) markedly increased in Pan02 subcutaneous tumors in S100a4-Cre; Ext1f/f mice, suggesting that it may contribute to rapid growth. Therefore, our study demonstrates that the tumor microenvironment with HS-reduced fibroblasts provides a favorable environment for tumor growth by affecting the function and properties of cancer-associated fibroblasts, macrophages, and cancer cells.

In Vitro Measurement and Mathematical Modeling of Thermally-Induced Injury in Pancreatic Cancer Cells.

Thermal therapies are under investigation as part of multi-modality strategies for the treatment of pancreatic cancer. In the present study, we determined the kinetics of thermal injury to pancreatic cancer cells in vitro and evaluated predictive models for thermal injury. Cell viability was measured in two murine pancreatic cancer cell lines (KPC, Pan02) and a normal fibroblast (STO) cell line following in vitro heating in the range 42.5-50 °C for 3-60 min. Based on measured viability data, the kinetic parameters of thermal injury were used to predict the extent of heat-induced damage. Of the three thermal injury models considered in this study, the Arrhenius model with time delay provided the most accurate prediction (root mean square error = 8.48%) for all cell lines. Pan02 and STO cells were the most resistant and susceptible to hyperthermia treatments, respectively. The presented data may contribute to studies investigating the use of thermal therapies as part of pancreatic cancer treatment strategies and inform the design of treatment planning strategies.

Fluorinated triphenylphosphonium analogs improve cell selectivity and invivo detection of mito-metformin.

Triphenylphosphonium (TPP+) conjugated compounds selectively target cancercells by exploiting their hyperpolarized mitochondrial membrane potential. Todate, studies have focused on modifying either the linker or the cargo of TPP+-conjugated compounds. Here, we investigated the biological effects ofdirect modification to TPP+ to improve the efficacy and detection of mito-metformin (MMe), a TPP+-conjugated probe we have shown to have promising preclinical efficacy against solid cancer cells. We designed, synthesized, and tested trifluoromethyl and methoxy MMe analogs (pCF3-MMe, mCF3-MMe, and pMeO-MMe) against multiple distinct human cancer cells. pCF3-MMe showed enhanced selectivity toward cancer cells compared to MMe, while retaining thesame signaling mechanism. Importantly, pCF3-MMe allowed quantitative monitoring of cellular accumulation via 19F-NMR invitro and invivo. Furthermore, adding trifluoromethyl groups to TPP+ reduced toxicity invivo while retaining anti-tumor efficacy, opening an avenue to de-risk these next-generation TPP+-conjugated compounds.

Abstract PR023: Targeting gemcitabine resistance in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with a devastating 5-year survival rate of 11%. A lack of durable responses to standard-of-care chemotherapy combinations renders its treatment particularly challenging and largely contributes to the devastating prognosis. Gemcitabine, a pyrimidine anti-metabolite, is a cornerstone in PDAC therapy, but resistance remains a major hurdle. Multiple mechanisms of chemoresistance have been suggested to be mediated by rewired metabolism in PDAC cells. Accordingly, we hypothesize that metabolic reprogramming can be targeted to re-sensitize PDAC tumors to anti-metabolite chemotherapy. To define the spectrum of targetable metabolic and transcriptomic programs that drive cell-intrinsic resistance to gemcitabine, we established organoids from treatment-naïve PDAC patients. Gemcitabine high versus low responders, as assessed by dose response viability assays, were selected for metabolomic profiling and RNA sequencing. To define real-time reprogramming during the acquisition of resistance, we generated gemcitabine resistant murine pancreatic cancer cell lines and collected a time course series of metabolomic and transcriptomic datasets of sensitive, intermediate resistant and resistant cells. Integration of these dataset through a systems biology approach to define primary and de novo resistance is being used to characterize metabolic gemcitabine resistance mechanisms. These are functionally validated to provide novel approaches to re-sensitize resistant PDAC cells to gemcitabine in vitro and in vivo. The improvement of current chemotherapy combinations represents a promising approach, with potential to immediately translate into a clinical benefit and improve survival in this deadly disease. Citation Format: Alica Katrin Beutel, Rima Singh, Cecily Anaraki, Gregory Tong, Thomas Martinez, Alexander Kleger, Zeljka Jutric, Christopher James Halbrook. Targeting gemcitabine resistance in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR023.