BackgroundThe risk of pancreatic cancer, the 4th deadliest cancer for both men and women in the United States, is increased by obesity. Calorie restriction (CR) is a well-known dietary regimen that prevents or reverses obesity and suppresses tumorigenesis in a variety of animal models, at least in part via inhibition of mammalian target of rapamycin (mTOR) signaling. Branched-chain amino acids (BCAA), especially leucine, activate mTOR and enhance growth and proliferation of myocytes and epithelial cells, which is why leucine is a popular supplement among athletes. Leucine is also increasingly being used as a treatment for pancreatic cancer cachexia, but the effects of leucine supplementation on pancreatic tumor growth have not been elucidated.ResultsSupplementation with leucine increased pancreatic tumor growth in both lean (104 ± 17 mm3 versus 46 ± 13 mm3; P <0.05) and overweight (367 ± 45 mm3 versus 230 ± 39 mm3; P <0.01) mice, but tumor enhancement was associated with different biological outcomes depending on the diet. In the lean mice, leucine increased phosphorylation of mTOR and downstream effector S6 ribosomal protein, but in the overweight mice, leucine reduced glucose clearance and thus increased the amount of circulating glucose available to the tumor.ConclusionsThese findings show that leucine supplementation enhances tumor growth in both lean and overweight mice through diet-dependent effects in a murine model of pancreatic cancer, suggesting caution against the clinical use of leucine supplementation for the purposes of skeletal muscle enhancement in cachectic patients.

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Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States, and effective therapies for PDAC are currently lacking. Moreover, PDAC is promoted and exacerbated by obesity, while cachexia and sarcopenia are exceptionally common comorbidities that predict both poor survival and treatment response. Managing PDAC with immunotherapies has thus far proven ineffective, partly due to the metabolically hostile tumor microenvironment. β-hydroxy-β-methylbutyrate (HMB), a metabolite of leucine commonly used as a dietary supplement to boost muscle growth and immune function, may be an attractive candidate to augment PDAC therapy. We therefore sought to test the hypothesis that HMB would enhance antitumor immunity while protecting mouse muscle mass. Control and diet-induced obese C57BL/6 male mice bearing subcutaneously injected Panc02 tumors were supplemented with 1% HMB and treated with or without 50 mg/kg gemcitabine (n = 15/group). HMB was associated with reduced muscle inflammation and increased muscle fiber size. HMB also reduced tumor growth and promoted antitumor immunity in obese, but not lean, mice, independent of the gemcitabine treatment. Separately, in lean tumor-bearing mice, HMB supplementation promoted an anti-PD1 immunotherapy response (n = 15/group). Digital cytometry implicated the decreased abundance of M2-like macrophages in PDAC tumors, an effect that was enhanced by anti-PD1 immunotherapy. We confirmed that HMB augments M1-like macrophage (antitumor) polarization. These preclinical findings suggest that HMB has muscle-sparing and antitumor activities against PDAC in the context of obesity, and that it may sensitize otherwise nonresponsive PDAC to immunotherapy.

Background: Pancreatic ductal adenocarcinoma (PDAC) remains the 4th most deadly cancer in the US. Current chemotherapeutic regimens poorly control PDAC progression and no approved immunotherapeutic options currently exist. PDAC incidence and progression are increased by obesity, which promotes a potently protumor PDAC microenvironment characterized by inflammation, fibrosis, and immunosuppression. β Hydroxy β Methylbutyrate, found to be safe and well-tolerated in clinical trials over many years, blunts cancer cachexia; however it has not been tested in PDAC. Further, the role of HMB in modifying existing therapies has been understudied. We sought to determine if HMB treatment would alter therapeutic response of lean and obese mice to gemcitabine or anti-PD1 immunotherapy. Methods: Lean and obese C57BL/6 mice were treated with HMB alone or in combination with gemcitabine or anti-PD1 immunotherapy following subcutaneous injection of Panc02 PDAC cells. In addition, bone marrow-derived macrophages (BMDM) were treated with HMB in vitro with or without LPS. Tumor/macrophage transcriptomic analysis, followed by gene set enrichment analysis and enrichment mapping identified transcriptional responses. CIBERSORTx determined microenvironment immune cell composition. Results: a) HMB reduces obesity-associated PDAC progression; b) HMB treatment and gemcitabine together suppress PDAC growth in obese mice; c) HMB mitigates PDAC immunosuppression and promotes tumor immune surveillance; d) HMB enhances the efficacy of anti-PD1 immunotherapy; e) HMB promotes M1 polarization of BMDM. Conclusion: Overall, HMB evokes antitumor immune responses in the context of pro-inflammatory stimuli such as obesity or immunotherapy. Moreover, HMB promotes immune surveillance in PDAC, synergizing with both cytotoxic chemotherapy and immunotherapy. HMB has been found to be safe and well tolerated in clinical trials for many years. Hence, HMB-induced suppression of PDAC tumor growth and promotion of immune surveillance may offer significant synergy with chemotherapies or immunotherapies in PDAC. This research was supported by R35CA197627 to S. Hursting. Citation Format: Michael F. Coleman, Kristyn A. Liu, Suhas K. Etigunta, Alex J. Pfeil, Xiaohu Tang, Salvador Fabela, Laura M. Lashinger, Zhengrong Cui, Stephen D. Hursting. β Hydroxy β Methylbutyrate synergizes with cytotoxic chemotherapy and immunotherapy in a mouse model of pancreatic cancer [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 3151.

Background: Pancreatic ductal adenocarcinoma (PDAC) continues to represent a critical unmet therapeutic need in the US, and is exacerbated by obesity. Current chemotherapeutic regimens poorly control PDAC progression, and immunotherapy has yet to be widely successful against PDAC. Obesity promotes a potently protumor PDAC microenvironment including suppression of antitumor immune response. The purpose of our studies was to test whether dietary supplementation with β-hydroxy-β-methylbutyrate (HMB), shown to be safe and well tolerated in cancer patients, could offset obesity-mediated exacerbation of PDAC and promote antitumor immunosurveillance. Methods: Lean and obese C57BL/6 mice were treated with HMB alone or in combination with anti-PD1 immunotherapy following subcutaneous injection of Panc02 PDAC cells. Bone marrow-derived macrophages (BMDM) were treated with HMB in vitro with or without lipopolysaccharide (LPS). Tumor/macrophage transcriptomic analysis using Affymetrix arrays followed by gene set enrichment was performed, and tumor microenvironment immune cell composition was determined by mass cytometry. Results: a) HMB cooperates with obesity and immunotherapy to reduce PDAC progression, as indicated by reduced tumor growth with the combination regimen of HMB + anti-PD1 relative to obese or anti-PD1 immunotherapy treated mice without HMB supplementation; b) HMB mitigates PDAC immunosuppression and promotes tumor immune surveillance, as demonstrated by induction of an antitumor immune-related gene expression profile in response to HMB supplementation; c) HMB promotes pro-inflammatory polarization of tumor-associated macrophages and BMDM, as shown by increased CD38 positivity and reduced Arg1 positivity in tumor-associated macrophages from HMB supplemented mice relative to control. Further, BMDM transcriptional response to HMB alone or HMB in combination with LPS paralleled that of BMDM treated with LPS relative to untreated LPS. Conclusion: Overall, our findings suggest HMB is an adjuvant for antitumor immune responses in PDAC. Moreover, HMB promotes immune surveillance in PDAC, synergizing with immunotherapy. Hence, HMB-induced suppression of PDAC tumor growth and promotion of immune surveillance may offer significant synergy with chemotherapies or immunotherapies in PDAC. Funding: This work was supported by R35CA197627 to SDH. Citation Format: Michael F. Coleman, Kristyn A. Liu, Alexander J. Pfeil, Suhas K. Etigunta, Xiaohu Tang, Laura M. Lashinger, Zhengrong Cui, Stephen D. Hursting. β-Hydroxy-β-methylbutyrate promotes immunotherapy response and pro-inflammatory macrophage polarization in a mouse model of pancreatic cancer [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 1995.