Jason W.-L. Eng

Baseline tumor growth and immune control in laboratory mice are significantly influenced by subthermoneutral housing temperature.

Significance We show that the mandated, subthermoneutral laboratory housing temperature, which is known to cause chronic, metabolic cold stress, induces suppression of the antitumor immune response and promotes tumor growth and metastasis. When mice are housed at thermoneutrality, there are fewer immunosuppressive cells with significantly enhanced CD8+ T cell-dependent control of tumor growth. These findings underscore the fact that investigating mouse models under a single set of environmental temperature conditions may lead to a misunderstanding of the antitumor immune potential. These data also highlight the need for additional study to determine how systemic metabolic stress modulates the functions of immune effector cells, particularly in tumor-bearing mice, and whether cancer therapies, including immunotherapy, are impacted by housing temperature. We show here that fundamental aspects of antitumor immunity in mice are significantly influenced by ambient housing temperature. Standard housing temperature for laboratory mice in research facilities is mandated to be between 20–26 °C; however, these subthermoneutral temperatures cause mild chronic cold stress, activating thermogenesis to maintain normal body temperature. When stress is alleviated by housing at thermoneutral ambient temperature (30–31 °C), we observe a striking reduction in tumor formation, growth rate and metastasis. This improved control of tumor growth is dependent upon the adaptive immune system. We observe significantly increased numbers of antigen-specific CD8+ T lymphocytes and CD8+ T cells with an activated phenotype in the tumor microenvironment at thermoneutrality. At the same time there is a significant reduction in numbers of immunosuppressive MDSCs and regulatory T lymphocytes. Notably, in temperature preference studies, tumor-bearing mice select a higher ambient temperature than non-tumor-bearing mice, suggesting that tumor-bearing mice experience a greater degree of cold-stress. Overall, our data raise the hypothesis that suppression of antitumor immunity is an outcome of cold stress-induced thermogenesis. Therefore, the common approach of studying immunity against tumors in mice housed only at standard room temperature may be limiting our understanding of the full potential of the antitumor immune response.

Nitric oxide and nanotechnology: A novel approach to inhibit neointimal hyperplasia

OBJECTIVE Nitric oxide (NO) has been shown to inhibit neointimal hyperplasia after arterial interventions in several animal models. To date, however, NO-based therapies have not been used in the clinical arena. Our objective was to combine nanofiber delivery vehicles with NO chemistry to create a novel, more potent NO-releasing therapy that can be used clinically. Thus, the aim of this study was to evaluate the perivascular application of spontaneously self-assembling NO-releasing nanofiber gels. Our hypothesis was that this application would prevent neointimal hyperplasia. METHODS Gels consisted of a peptide amphiphile, heparin, and a diazeniumdiolate NO donor (1-[N-(3-Aminopropyl)-N-(3-ammoniopropyl)]diazen-1-ium-1,2-diolate [DPTA/NO] or disodium 1-[(2-Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate [PROLI/NO]). Nitric oxide release from the gels was evaluated by the Griess reaction, and scanning electron microscopy confirmed nanofiber formation. Vascular smooth muscle cell (VSMC) proliferation and cell death were assessed in vitro by (3)H-thymidine incorporation and Personal Cell Analysis (PCA) system (Guava Technologies, Hayward, Calif). For the in vivo work, gels were modified by reducing the free-water content. Neointimal hyperplasia after periadventitial gel application was evaluated using the rat carotid artery injury model at 14 days (n = 6 per group). Inflammation and proliferation were examined in vivo with immunofluorescent staining against CD45, ED1, and Ki67 at 3 days (n = 2 per group), and graded by blinded observers. Endothelialization was assessed by Evans blue injection at 7 days (n = 3 per group). RESULTS Both DPTA/NO and PROLI/NO, combined with the peptide amphiphile and heparin, formed nanofiber gels and released NO for 4 days. In vitro, DPTA/NO inhibited VSMC proliferation and induced cell death to a greater extent than PROLI/NO. However, the DPTA/NO nanofiber gel only reduced neointimal hyperplasia by 45% (intima/media [I/M] area ratio, 0.45 +/- 0.07), whereas the PROLI/NO nanofiber gel reduced neointimal hyperplasia by 77% (I/M area ratio, 0.19 +/- 0.03, P < .05) vs control (injury alone I/M area ratio, 0.83 +/- 0.07; P < .05). Both DPTA/NO and PROLI/NO nanofiber gels significantly inhibited proliferation in vivo (1.06 +/- 0.30 and 0.19 +/- 0.11 vs injury alone, 2.02 +/- 0.20, P < .05), yet had minimal effect on apoptosis. Only the PROLI/NO nanofiber gel inhibited inflammation (monocytes and leukocytes). Both NO-releasing nanofiber gels stimulated re-endothelialization. CONCLUSIONS Perivascular application of NO-releasing self-assembling nanofiber gels is an effective and simple therapy to prevent neointimal hyperplasia after arterial injury. Our study demonstrates that the PROLI/NO nanofiber gel most effectively prevented neointimal hyperplasia and resulted in less inflammation than the DPTA/NO nanofiber gel. This therapy has great clinical potential to prevent neointimal hyperplasia after open vascular interventions in patients.

Silencing the hsp25 Gene Eliminates Migration Capability of the Highly Metastatic Murine 4T1 Breast Adenocarcinoma Cell

The 25-kDa heat shock protein (Hsp25) is associated with various malignancies and is expressed at high levels in biopsies as well as circulating in the serum of breast cancer patients. In this study, we used RNA interference technology to silence the hsp25 gene in 4T1 breast adenocarcinoma cells, known as a poorly immunogenic, highly metastatic cell line. We demonstrate that transfection of 4T1 cells with short interference RNA-Hsp25 dramatically inhibits proliferation as compared with control transfected cells. In addition, we show that 4T1 cells transfected with short interference RNA-Hsp25 abrogates tumor migration potential by a mechanism that is in part due to the repression of matrix metalloproteinase 9 expression and a concomitant upregulation of its antagonist, tissue inhibitor metalloproteinase 1. Taken together, these findings provide a model system for the study of metastatic potential of tumors and are suggestive of an earlier unrecognized role for Hsp25 in tumor migration.

Nitric oxide regulates the 26S proteasome in vascular smooth muscle cells

It is well established that nitric oxide (NO) inhibits vascular smooth muscle cell (VSMC) proliferation by modulating cell cycle proteins. The 26S proteasome is integral to protein degradation and tightly regulates cell cycle proteins. Therefore, we hypothesized that NO directly inhibits the activity of the 26S proteasome. The three enzymatic activities (chymotrypsin-like, trypsin-like and caspase-like) of the 26S proteasome were examined in VSMC. At baseline, caspase-like activity was approximately 3.5-fold greater than chymotrypsin- and trypsin-like activities. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly inhibited all three catalytically active sites in a time- and concentration-dependent manner (P<0.05). Caspase-like activity was inhibited to a greater degree (77.2% P<0.05). cGMP and cAMP analogs and inhibitors had no statistically significant effect on basal or NO-mediated inhibition of proteasome activity. Dithiothreitol, a reducing agent, prevented and reversed the NO-mediated inhibition of the 26S proteasome. Nitroso-cysteine analysis following S-nitrosoglutathione exposure revealed that the 20S catalytic core of the 26S proteasome contains 10 cysteines which were S-nitrosylated by NO. Evaluation of 26S proteasome subunit protein expression revealed differential regulation of the alpha and beta subunits in VSMC following exposure to NO. Finally, immunohistochemical analysis of subunit expression revealed distinct intracellular localization of the 26S proteasomal subunits at baseline and confirmed upregulation of distinct subunits following NO exposure. In conclusion, NO reversibly inhibits the catalytic activity of the 26S proteasome through S-nitrosylation and differentially regulates proteasomal subunit expression. This may be one mechanism by which NO exerts its effects on the cell cycle and inhibits cellular proliferation in the vasculature.

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.

Housing Temperature-Induced Stress Is Suppressing Murine Graft-versus-Host Disease through β2-Adrenergic Receptor Signaling.

Graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic cell transplantation, a potentially curative therapy for hematologic diseases. It has long been thought that murine bone marrow–derived T cells do not mediate severe GVHD because of their quantity and/or phenotype. During the course of experiments testing the impact of housing temperatures on GVHD, we discovered that this apparent resistance is a function of the relatively cool ambient housing temperature. Murine bone marrow–derived T cells have the ability to mediate severe GVHD in mice housed at a thermoneutral temperature. Specifically, mice housed at Institutional Animal Care and Use Committee–mandated, cool standard temperatures (∼22°C) are more resistant to developing GVHD than are mice housed at thermoneutral temperatures (∼30°C). We learned that the mechanism underlying this housing-dependent immunosuppression is associated with increased norepinephrine production and excessive signaling through β-adrenergic receptor signaling, which is increased when mice are cold stressed. Treatment of mice housed at 22°C with a β2-adrenergic antagonist reverses the norepinephrine-driven suppression of GVHD and yields similar disease to mice housed at 30°C. Conversely, administering a β2-adrenergic agonist decreases GVHD in mice housed at 30°C. In further mechanistic studies using β2-adrenergic receptor–deficient (β2-AR−/−) mice, we found that it is host cell β2-AR signaling that is essential for decreasing GVHD. These data reveal how baseline levels of β-adrenergic receptor signaling can influence murine GVHD and point to the feasibility of manipulation of β2-AR signaling to ameliorate GVHD in the clinical setting.

Mild cold-stress depresses immune responses: Implications for cancer models involving laboratory mice

Physiologically accurate mouse models of cancer are critical in the pre‐clinical development of novel cancer therapies. However, current standardized animal‐housing temperatures elicit chronic cold‐associated stress in mice, which is further increased in the presence of tumor. This cold‐stress significantly impacts experimental outcomes. Data from our lab and others suggest standard housing fundamentally alters murine physiology, and this can produce altered immune baselines in tumor and other disease models. Researchers may thus underestimate the efficacy of therapies that are benefitted by immune responses. A potential mediator, norepinephrine, also underlies stress pathways common in mice and humans. Therefore, research into mechanisms connecting cold‐stress and norepinephrine signaling with immune depression in mice could highlight new combination therapies for humans to simultaneously target stress while stimulating anti‐tumor immunity.

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.

Housing Temperature Influences the Pattern of Heat Shock Protein Induction in Mice following Mild Whole Body Hyperthermia

Abstract Purpose: Researchers studying the murine response to stress generally use mice housed under standard, nationally mandated conditions as controls. Few investigators are concerned whether basic physical aspects of mouse housing could be an additional source of stress, capable of influencing the subsequent impact of an experimentally applied stressor. We have recently become aware of the potential for housing conditions to impact important physiological and immunological properties in mice. Materials and methods: Here we sought to determine whether housing mice at standard temperature (ST; 22 °C) vs. thermoneutral temperature (TT; 30 °C) influences baseline expression of heat shock proteins (HSPs) and their typical induction following a whole body heating. Results: There were no significant differences in baseline expression of HSPs at ST and TT. However, in several cases, the induction of Hsp70, Hsp110 and Hsp90 in tissues of mice maintained at ST was greater than at TT following 6 h of heating (which elevated core body temperature to 39.5 °C). This loss of HSP induction was also seen when mice housed at ST were treated with propranolol, a β-adrenergic receptor antagonist, used clinically to treat hypertension and stress. Conclusions: Taken together, these data show that housing temperature significantly influences the expression of HSPs in mice after whole body heating and thus should be considered when stress responses are studied in mice.

Stress, metabolism and cancer: integrated pathways contributing to immune suppression.

AbstractThe potential for immune cells to control cancers has been recognized for many decades, but only recently has real excitement begun to spread through the oncology community following clear evidence that therapeutic blockade of specific immune-suppressive mechanisms is enough to make a real difference in survival for patients with several different advanced cancers. However, impressive and encouraging as these new clinical data are, it is clear that more effort should be devoted toward understanding the full spectrum of factors within cancer patients, which have the potential to block or weaken antitumor activity by immune cells. The goal of this brief review is to highlight recent literature revealing interactive stress and metabolic pathways, particularly those mediated by the sympathetic nervous system, which may conspire to block immune cells from unleashing their full killing potential. There is exciting new information regarding the role of neurogenesis by tumors and adrenergic signaling in cancer progression (including metabolic changes associated with cachexia and lipolysis) and in regulation of immune cell function and differentiation. However, much more work is needed to fully understand how the systemic metabolic effects mediated by the brain and nervous system can be targeted for therapeutic efficacy in the setting of immunotherapy and other cancer therapies.