Mark J. Bucsek

Housing temperature-induced stress drives therapeutic resistance in murine tumour models through β2-adrenergic receptor activation

Cancer research relies heavily on murine models for evaluating the anti-tumour efficacy of therapies. Here we show that the sensitivity of several pancreatic tumour models to cytotoxic therapies is significantly increased when mice are housed at a thermoneutral ambient temperature of 30 °C compared with the standard temperature of 22 °C. Further, we find that baseline levels of norepinephrine as well as the levels of several anti-apoptotic molecules are elevated in tumours from mice housed at 22 °C. The sensitivity of tumours to cytotoxic therapies is also enhanced by administering a β-adrenergic receptor antagonist to mice housed at 22 °C. These data demonstrate that standard housing causes a degree of cold stress sufficient to impact the signalling pathways related to tumour-cell survival and affect the outcome of pre-clinical experiments. Furthermore, these data highlight the significant role of host physiological factors in regulating the sensitivity of tumours to therapy.

β-Adrenergic Signaling in Mice Housed at Standard Temperatures Suppresses an Effector Phenotype in CD8+ T Cells and Undermines Checkpoint Inhibitor Therapy

The immune context of tumors has significant prognostic value and is predictive of responsiveness to several forms of therapy, including immunotherapy. We report here that CD8+ T-cell frequency and functional orientation within the tumor microenvironment is regulated by β2-adrenergic receptor (β-AR) signaling in host immune cells. We used three strategies-physiologic (manipulation of ambient thermal environment), pharmacologic (β-blockers), and genetic (β2-AR knockout mice) to reduce adrenergic stress signaling in two widely studied preclinical mouse tumor models. Reducing β-AR signaling facilitated conversion of tumors to an immunologically active tumor microenvironment with increased intratumoral frequency of CD8+ T cells with an effector phenotype and decreased expression of programmed death receptor-1 (PD-1), in addition to an elevated effector CD8+ T-cell to CD4+ regulatory T-cell ratio (IFNγ+CD8+:Treg). Moreover, this conversion significantly increased the efficacy of anti-PD-1 checkpoint blockade. These data highlight the potential of adrenergic stress and norepinephrine-driven β-AR signaling to regulate the immune status of the tumor microenvironment and support the strategic use of clinically available β-blockers in patients to improve responses to immunotherapy. Cancer Res; 77(20); 5639-51. ©2017 AACR.

Immune Adjuvant Activity of Pre-Resectional Radiofrequency Ablation Protects against Local and Systemic Recurrence in Aggressive Murine Colorectal Cancer

Purpose While surgical resection is a cornerstone of cancer treatment, local and distant recurrences continue to adversely affect outcome in a significant proportion of patients. Evidence that an alternative debulking strategy involving radiofrequency ablation (RFA) induces antitumor immunity prompted the current investigation of the efficacy of performing RFA prior to surgical resection (pre-resectional RFA) in a preclinical mouse model. Experimental Design Therapeutic efficacy and systemic immune responses were assessed following pre-resectional RFA treatment of murine CT26 colon adenocarcinoma. Results Treatment with pre-resectional RFA significantly delayed tumor growth and improved overall survival compared to sham surgery, RFA, or resection alone. Mice in the pre-resectional RFA group that achieved a complete response demonstrated durable antitumor immunity upon tumor re-challenge. Failure to achieve a therapeutic benefit in immunodeficient mice confirmed that tumor control by pre-resectional RFA depends on an intact adaptive immune response rather than changes in physical parameters that make ablated tumors more amenable to a complete surgical excision. RFA causes a marked increase in intratumoral CD8+ T lymphocyte infiltration, thus substantially enhancing the ratio of CD8+ effector T cells: FoxP3+ regulatory T cells. Importantly, pre-resectional RFA significantly increases the number of antigen-specific CD8+ T cells within the tumor microenvironment and tumor-draining lymph node but had no impact on infiltration by myeloid-derived suppressor cells, M1 macrophages or M2 macrophages at tumor sites or in peripheral lymphoid organs (i.e., spleen). Finally, pre-resectional RFA of primary tumors delayed growth of distant tumors through a mechanism that depends on systemic CD8+ T cell-mediated antitumor immunity. Conclusion Improved survival and antitumor systemic immunity elicited by pre-resectional RFA support the translational potential of this neoadjuvant treatment for cancer patients with high-risk of local and systemic recurrence.

An overview of the role of sympathetic regulation of immune responses in infectious disease and autoimmunity

Abstract Stress in patients and pre-clinical research animals plays a critical role in disease progression Activation of the sympathetic nervous system (SNS) by stress results in secretion of the catecholamines epinephrine (Epi) and norepinephrine (NE) from the adrenal gland and sympathetic nerve endings. Adrenergic receptors for catecholamines are present on immune cells and their activity is affected by stress and the accompanying changes in levels of these neurotransmitters. In this short review, we discuss how this adrenergic stress impacts two categories of immune responses, infections and autoimmune diseases. Catecholamines signal primarily through the β2-adrenergic receptors present on innate and adaptive immune cells which are critical in responding to infections caused by pathogens. In general, this adrenergic input, particularly chronic stimulation, suppresses lymphocytes and allows infections to progress. On the other hand, insufficient adrenergic control of immune responses allows progression of several autoimmune diseases.

Adrenergic Signaling: A Targetable Checkpoint Limiting Development of the Antitumor Immune Response

An immune response must be tightly controlled so that it will be commensurate with the level of response needed to protect the organism without damaging normal tissue. The roles of cytokines and chemokines in orchestrating these processes are well known, but although stress has long been thought to also affect immune responses, the underlying mechanisms were not as well understood. Recently, the role of nerves and, specifically, the sympathetic nervous system, in regulating immune responses is being revealed. Generally, an acute stress response is beneficial but chronic stress is detrimental because it suppresses the activities of effector immune cells while increasing the activities of immunosuppressive cells. In this review, we first discuss the underlying biology of adrenergic signaling in cells of both the innate and adaptive immune system. We then focus on the effects of chronic adrenergic stress in promoting tumor growth, giving examples of effects on tumor cells and immune cells, explaining the methods commonly used to induce stress in preclinical mouse models. We highlight how this relates to our observations that mandated housing conditions impose baseline chronic stress on mouse models, which is sufficient to cause chronic immunosuppression. This problem is not commonly recognized, but it has been shown to impact conclusions of several studies of mouse physiology and mouse models of disease. Moreover, the fact that preclinical mouse models are chronically immunosuppressed has critical ramifications for analysis of any experiments with an immune component. Our group has found that reducing adrenergic stress by housing mice at thermoneutrality or treating mice housed at cooler temperatures with β-blockers reverses immunosuppression and significantly improves responses to checkpoint inhibitor immunotherapy. These observations are clinically relevant because there are numerous retrospective epidemiological studies concluding that cancer patients who were taking β-blockers have better outcomes. Clinical trials testing whether β-blockers can be repurposed to improve the efficacy of traditional and immunotherapies in patients are on the horizon.

The impact of β-adrenergic signaling on radioresistance and anti-tumor immunity

Our laboratory has demonstrated that mildly cool housing temperatures, (a stress known to be mediated by sympathetic nerve activity and β-adrenergic receptors) can significantly enhance the incidence, growth, and metastasis of tumors in murine models by suppressing anti-tumor immunity [1]. Furthermore, we have shown that mild cool stress also decreases the sensitivity of cancer cells to several types of chemo- and targeted therapeutics [2]. These data strongly suggest that baseline responses of mouse models of tumor growth and tumor immunology may be significantly influenced by housing temperature and the degree of adrenergic stress experienced by tumor bearing mice. We have now become interested in dissecting the mechanisms by which increased adrenergic stress signaling resulting from cool housing conditions impacts anti-tumor immunity and the response of tumors to therapeutics. To investigate the effects of increased adrenergic signaling on anti-tumor immunity, we used β-AR antagonists (β-blockers) to block β-AR signaling resulting from mild cold stress induced by housing temperatures. The addition of a β-blocker to these mice significantly delayed tumor growth, and recapitulated the increased tumor growth control observed in mice housed at thermoneutrality where cold stress is alleviated. However, β-AR blockade did not alter tumor growth in mice housed at thermoneutrality compared to vehicle controls, suggesting that β-AR signaling is responsible for the impairment of anti-tumor immunity in chronically cold stressed mice. To further investigate the effects of β-AR signaling on suppressing anti-tumor immunity, we repeated these experiments in immunodeficient SCID mice and demonstrated that β-blockers had no effect on tumor growth in mice regardless of housing temperature. We also examined the effects of adrenergic stress signaling on radio-resistance. We addressed this question by stimulating β-ARs on Pan02 (murine) and Mia-PACA2 (human) pancreatic tumor cells and performed clonogenic assays. Those cells treated with a β-agonist exhibited improved survival and proliferation at varying doses of radiation when compared to untreated controls similar to our earlier studies using chemotherapy. In summary, β-adrenergic receptor activity appears to play a fundamental role in the regulation of both anti-tumor immunity as well as intrinsic therapeutic sensitivity. Therefore, strategic combinations of β-receptor antagonists with chemo-radiation therapy, or immunotherapy may provide significantly improved tumor control. This work was supported by the Peter T. Rowley Breast Cancer Research Grant and the Harry J. Lloyd Charitable Trust.

Abstract B72: Environmental temperature-induced chronic stress drives therapeutic resistance in murine tumor models through β2-adrenergic receptor activation

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 BAD 112 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.