Background: Pancreatic Ductal Adenocarcinoma (PDAC) has a dismal five-year survival rate of 13% and is closely associated with cachexia. Cancer cachexia is a multifactorial syndrome characterized by irreversible wasting of skeletal muscles, fat loss and systemic inflammation. While cachexia is known to confer resistance to immune checkpoint inhibition in several cancers, the bidirectional relationship between cachexia and the immune system in PDAC remains unclear, necessitating the development of novel preclinical models. Our laboratory has characterized a novel pancreatic cancer cachexia model in C57BL/6J mice by utilizing the pancreatic cancer cell line called KPCL-4 derived from KPC-LSIY mice (Kras^LSL-G12D/+Tp53^LSL-R172H/+Pdx1-Cre/R26^LSL-LSIY). Methods: KPCL-4 cells were orthotopically injected into the pancreas of male and female C57BL/6J mice and hallmarks of cachexia were assessed at endpoint by measurement of tumor weight, terminal tumor-adjusted body weight, skeletal muscle, adipose tissue, liver and spleen masses, proteolytic markers and grip strength. Plasma cytokine and chemokine concentrations were quantified by Luminex assay and high-dimensional flow cytometry was used to investigate changes in tumor-infiltrating immune populations. Results: We observed a sex bias in cachexia presentation despite similar tumor weights in male and female mice, whereby males exhibited a >5% decrease in terminal tumor-adjusted body weight (p < 0.001), >50% fat loss (p < 0.001), upregulation of proteolytic markers in skeletal muscles (p < 0.01) and reduction in skeletal muscle mass (p < 0.05), function (p < 0.01) and cross-sectional area (p < 0.0001) whereas females demonstrated conserved skeletal muscle mass with 33% fat loss (p < 0.05), reduction in muscle cross-sectional area (p < 0.0001) and splenomegaly (p < 0.01). While intra-tumoral immune populations did not exhibit sex-specific differences, plasma cytokine concentrations were differentially upregulated in males and females, suggesting functional differences in immune cells as potent drivers of sex bias in KPCL-4-driven cachexia. Conclusions: The KPCL-4 orthotopic PDAC model exhibits prominent hallmarks of cachexia and serves as a novel platform for investigating the complex interplay between cancer cachexia and immunomodulation.

Background: Pancreatic ductal adenocarcinoma (PDAC), comprising of 90% pancreatic cancer malignancies, is a highly aggressive cancer with a five-year survival rate of 10.8%. Current therapies include surgical resection and chemotherapy, which are mostly ineffective because of metastasis and an immunosuppressive tumor microenvironment (TME). Tomatidine, a natural metabolite present in the tomato plant, has anti-cancer and anti-inflammatory properties. Further, tomatidine has been reported to possibly correlate with inhibition of Activating Transcription Factor 4 (ATF4)-dependent signaling in multiple diseases such as skeletal muscle atrophy and dengue virus infection. ATF4, a master regulator of cellular stress, has been implicated in different cancers including PDAC by promoting cell survival, inducing resistance towards commonly used chemotherapeutic agents as well as by affecting anti-tumor immunity. Hypothesis: We hypothesize that tomatidine can inhibit pancreatic cancer by regulating ATF4-dependent signaling. Methods: Pancreatic tumor cell lines were assayed for cell growth after tomatidine treatment. Subcutaneous tumor-bearing C57BL/6 mice and athymic mice were treated with 5 mg/kg daily i.p. injections of tomatidine or vehicle. Orthotopic tumor bearing C57BL/6 mice were treated with either 5 mg/kg tomatidine daily alone, 10 mg/kg gemcitabine twice per week alone or both in combination, respectively via i.p. injections and assessed in comparison to vehicle. To assess ATF4-dependent signaling, protein and RNA expression of ATF4 and related genes as well as binding activity of ATF4 was evaluated via immunoblotting, Q-PCR analysis and chromatin immunoprecipitation. Further, healthy donor PBMCs were treated with tomatidine in presence of cytokines to observe myeloid derived suppressor cell (MDSC) differentiation. Results: We found that tomatidine can inhibit pancreatic tumor growth in vitro and in vivo. Tomatidine also enhanced the gemcitabine chemosensitivity in an orthotopic model of pancreatic cancer in vivo. However, there was little effect of tomatidine on tumor growth when pancreatic cancer cells were implanted into athymic mice, suggesting tomatidine may affect anti-tumor immunity. Tomatidine reduced phosphorylation of 4EBP1 (downstream of ATF4) in vitro and reduced mRNA expression of ATF4 and eIF4EBP1 in MT5 tumor-bearing mice treated with tomatidine. Further, tomatidine reduced the transcriptional binding activity of ATF4 with downstream genes such as eIF4EBP1. Tomatidine affected the rate of MDSC differentiation and reduced expression of ATF4 and related gene expression in immune cells. Conclusion: This study sheds light on tomatidine, a novel plant derived anti-cancer treatment that targets an upregulated pathway (ATF4 dependent signaling) in pancreatic cancer and can therefore be used to develop a novel therapeutic against the pancreatic tumor as well as its microenvironment. Citation Format: Debasmita Mukherjee, Lena Bercz, Liliana D’Alesio, Jessica Wedig, Timothy Pfau, Molly Torok, Shrina Jasani, Thomas A. Mace. Tomatidine targets ATF4-dependent signaling to limit pancreatic cancer in the tumor microenvironment [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 B077.