Abstract B72: Environmental temperature-induced chronic stress drives therapeutic resistance in murine tumor models through β2-adrenergic receptor activation
Authors: Eng Jason W-L , Reed Chelsey B. , Kokolus Kathleen , Bucsek Mark , Pitoniak Rose , Utley Adam T. , Ma WenWee , Repasky Elizabeth A. , Hylander Bonnie L. 
Published: October 2015

Abstract
Pre-clinical evaluation of novel therapies primarily relies on mouse models for predicting efficacy and toxicity. Unfortunately, when taken into the clinic, promising therapies often fail to achieve expected results in patients or unexpected toxicities are encountered. Emerging evidence now suggests that laboratory housing conditions, including ad libitum feeding and limited physical activity, can lead to metabolic abnormalities which in turn directly affect experimental outcomes in models of diseases such as obesity. We are particularly interested in the ramifications of housing temperature in tumor-bearing mice. IACUC guidelines dictate that all animal facilities must select a single temperature within the range of 20-26˚C (standard temperature, ST) to maintain mice, even though the thermoneutral temperature of mice ranges from 29-31˚C (TT). Because mice are able to maintain a normal body temperature of 37˚C, investigators have not been concerned, but in fact mice must generate a significant amount of heat through adaptive thermogenesis, and this is a norepinephrine (NE) driven process. The tumor promoting effects of catecholamines (epinephrine and norepinephrine) are currently under investigation in several labs. We have previously reported that at ST, the anti-tumor immune response is severely suppressed and when cold stress is alleviated by housing at TT, significantly higher numbers of tumor specific CD8 T-cells and fewer suppressor cells (MDSC, Tregs) are seen, correlating with significantly improved tumor control. Now we have observed that housing mice at TT instead of ST also significantly improves the response of pancreatic tumors to apoptosis inducing therapies in both immunocompetant and SCID mouse models. We found that a syngeneic tumor, Pan02, in C57BL/6 mice showed increased sensitivity to cisplatin when mice were placed at 30˚C vs. 22˚C. Similarly, human cell lines (MiaPaca2, BxPC3) and patient derived xenografts also demonstrated improved responses to both cisplatin and a second therapy, Apo2L/TRAIL, when mice were placed at thermoneutrality. Examination of the tumors revealed that the norepinephrine levels and the numbers of tyrosine hydroxylase-positive macrophages, a major source of norepinephrine, decreased in the tumors of mice housed at TT compared with those at ST. Thus, we hypothesized that tumor cells respond to the elevated levels of norepinephrine in the microenvironment in chronically cold stressed animals by increasing the expression of key survival molecules. Subsequent analysis of tumors from mice housed at TT revealed that the expression of anti-apoptotic molecules Bcl-2, Bcl-XL, Mcl-1 and phosphorylated BAD112 were decreased compared to those housed at ST. Tumor cells were found to express β1,2 adrenergic receptors and in vitro treatment demonstrated that adrenergic signaling can directly induce the expression of Bcl-2 and Bcl-XL in a time dependent manner in human pancreatic tumor cell lines, and that this increased protein expression correlates with increased resistance to both chemotherapeutic and death receptor-mediated apoptosis. In vivo, treatment of tumor-bearing mice with the β1, 2-blocker, propranolol, increased the response of tumors in mice housed at 22˚C to Apo2L/TRAIL, but not mice housed at 30˚C. Resistance to apoptotic therapy at ST was lost by shRNA knock down of β2-AR (MiaPaca2). Altogether, these findings have important implications for the study of immunotherapies which depend on both activation of the anti-tumor immune response and the sensitivity of tumors to induction of apoptosis. Our results support the idea that adrenergic signaling in the tumor microenvironment (at ST) can greatly impact the testing of novel therapies and suggests strategies for improving their efficacy both in mouse models and in the clinic where patients experience a variety of stresses. Supported by the Roswell Park Alliance Foundation and NIH R01 CA135368.
Citation Format: Jason W-L Eng, Chelsey B. Reed, Kathleen Kokolus, Mark Bucsek, Rose Pitoniak, Adam T. Utley, WenWee Ma, Elizabeth A. Repasky, Bonnie L. Hylander. Environmental temperature-induced chronic stress drives therapeutic resistance in murine tumor models through β2-adrenergic receptor activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B72.