Housing temperature plays a critical role in determining gut microbiome composition in research mice: Implications for experimental reproducibility.

Preclinical mouse models are widely used for studying mechanisms of disease and responses to therapeutics, however there is concern about the lack of experimental reproducibility and failure to predict translational success. The gut microbiome has emerged as a regulator of metabolism and immunological processes in health and disease. The gut microbiome of mice differs by supplier and this affects experimental outcomes. We have previously reported that the mandated, mildly cool housing temperature for research mice (22°-26 °C) induces chronic adrenergic stress which suppresses anti-tumor immunity and promotes tumor growth compared to thermoneutral housing (30 °C). Therefore, we wondered how housing temperature affects the microbiome. Here, we demonstrate that the gut microbiome of BALB/c mice is easily modulated by a few degrees difference in temperature. Our results reveal significant differences between the gut microbiome of mice housed at 22°-23 °C vs. 30 °C. Although the genera vary, we consistently observed an enrichment of members of the family Lachnospiraceae when mice are housed at 22°-23 °C. These findings demonstrate that adrenergic stress and need for increased energy harvest to support thermogenesis, in addition to other factors such as diet, modulates the gut microbiome and this could be one mechanism by which housing temperature affects experimental outcomes. Additionally, tumor growth in mice housed at 30 °C also increases the proportion of Lachnospiraceae. The idea that stress can alter the gut microbiome and cause differences in experimental outcomes is applicable to mouse studies in general and is a variable that has significant potential to affect experimental reproducibility.

Using Mice to Model Human Disease: Understanding the Roles of Baseline Housing-Induced and Experimentally Imposed Stresses in Animal Welfare and Experimental Reproducibility.

Mice are the most common animal used to study disease, but there are real concerns about the reproducibility of many of these experiments. This review discusses how several different sources of chronic stress can directly impact experimental outcomes. Mandated housing conditions induce an underappreciated level of chronic stress but are not usually considered or reported as part of the experimental design. Since chronic stress plays a critical role in the development and progression of many somatic diseases including cancer, obesity, and auto-immune diseases, this baseline stress can directly affect outcomes of such experiments. To study the role of stress in both physical and psychiatric diseases, there has been a proliferation of protocols for imposing chronic stress on mice. For somatic diseases, biomarkers can be used to compare the models with the disease in patients, but to evaluate the validity of psychiatric models, behavioral tests are carried out to assess changes in behavior and these tests may themselves cause an underappreciated degree of additional stress. Therefore, it is important for animal welfare to reduce baseline stress and select the most humane protocols for inducing and assessing chronic stress to obtain the most reliable outcomes.

Chronic Adrenergic Stress Contributes to Metabolic Dysfunction and an Exhausted Phenotype in T Cells in the Tumor Microenvironment.

Metabolic dysfunction and exhaustion in tumor-infiltrating T cells have been linked to ineffectual antitumor immunity and the failure of immune checkpoint inhibitor therapy. We report here that chronic stress plays a previously unrecognized role in regulating the state of T cells in the tumor microenvironment (TME). Using two mouse tumor models, we found that blocking chronic adrenergic stress signaling using the pan ß-blocker propranolol or by using mice lacking the ß2-adrenergic receptor (ß2-AR) results in reduced tumor growth rates with significantly fewer tumor-infiltrating T cells that express markers of exhaustion, with a concomitant increase in progenitor exhausted T cells. We also report that blocking ß-AR signaling in mice increases glycolysis and oxidative phosphorylation in tumor-infiltrating lymphocytes (TIL), which associated with increased expression of the costimulatory molecule CD28 and increased antitumor effector functions, including increased cytokine production. Using T cells from Nur77-GFP reporter mice to monitor T-cell activation, we observed that stress-induced ß-AR signaling suppresses T-cell receptor (TCR) signaling. Together, these data suggest that chronic stress-induced adrenergic receptor signaling serves as a "checkpoint" of immune responses and contributes to immunosuppression in the TME by promoting T-cell metabolic dysfunction and exhaustion. These results also support the possibility that chronic stress, which unfortunately is increased in many patients with cancer following their diagnoses, could be exerting a major negative influence on the outcome of therapies that depend upon the status of TILs and support the use of strategies to reduce stress or ß-AR signaling in combination with immunotherapy.

Adrenergic stress constrains the development of anti-tumor immunity and abscopal responses following local radiation.

The abscopal effect following ionizing radiation therapy (RT) is considered to be a rare event. This effect does occur more frequently when combined with other therapies, including immunotherapy. Here we demonstrate that the frequency of abscopal events following RT alone is highly dependent upon the degree of adrenergic stress in the tumor-bearing host. Using a combination of physiologic, pharmacologic and genetic strategies, we observe improvements in the control of both irradiated and non-irradiated distant tumors, including metastatic tumors, when adrenergic stress or signaling through ß-adrenergic receptor is reduced. Further, we observe cellular and molecular evidence of improved, antigen-specific, anti-tumor immune responses which also depend upon T cell egress from draining lymph nodes. These data suggest that blockade of ß2 adrenergic stress signaling could be a useful, safe, and feasible strategy to improve efficacy in cancer patients undergoing radiation therapy.

Temperature as a modulator of the gut microbiome: what are the implications and opportunities for thermal medicine?

There is substantial research being conducted on the relationships between the gut microbiome, the immune response and health and disease. Environmental temperature and heat stress are known to modify the gut microbiome. Changes in core temperature have been linked, in multiple phyla, to altered microbiome composition and function. This raises the question of whether local/regional or whole body thermal therapies which target tumors in the abdomen, peritoneal cavity, or pelvis influence the gut microbiome. To date, there is little information on whether thermal therapy exerts positive or negative effects on the microbiome. This is an intriguing question since there is growing interest in the immunological impact of various thermal therapies. The goal of this brief review is to highlight research on how environmental conditions, particularly temperature (internal as well as external temperatures) influences the gut microbiome. Given the potential for temperature shifts to modulate gut microbe function and composition, it is likely that various forms of thermal therapy, including hyperthermic intraperitoneal chemotherapy (HIPEC), deep regional, and whole body hyperthermia influence the microbiome in ways that are currently not appreciated. More research is needed to determine whether thermal therapy induced changes in the microbiome occur, and whether they are beneficial or detrimental to the host. Currently, although approaches to microbiome modification such as dietary intervention, fecal transfer, probiotics and prebiotics are being developed, the potential of temperature manipulation has, as yet, not been explored. Therefore, new research could reveal whether perturbations of the microbiome composition that have negative health consequences (dysbiosis) could be an important target for treatment by thermal medicine.

ß2 adrenergic receptor-mediated signaling regulates the immunosuppressive potential of myeloid-derived suppressor cells.

Catecholamines released by sympathetic nerves can activate adrenergic receptors present on nearly every cell type, including myeloid-derived suppressor cells (MDSCs). Using in vitro systems, murine tumor models in wild-type and genetically modified (ß2-AR-/-) mice, and adoptive transfer approaches, we found that the degree of ß2-AR signaling significantly influences MDSC frequency and survival in tumors and other tissues. It also modulates their expression of immunosuppressive molecules such as arginase-I and PD-L1 and alters their ability to suppress the proliferation of T cells. The regulatory functions of ß2-AR signaling in MDSCs were also found to be dependent upon STAT3 phosphorylation. Moreover, we observed that the ß2-AR-mediated increase in MDSC survival is dependent upon Fas-FasL interactions, and this is consistent with gene expression analyses, which reveal a greater expression of apoptosis-related genes in ß2-AR-/- MDSCs. Our data reveal the potential of ß2-AR signaling to increase the generation of MDSCs from both murine and human peripheral blood cells and that the immunosuppressive function of MDSCs can be mitigated by treatment with ß-AR antagonists, or enhanced by ß-AR agonists. This strongly supports the possibility that reducing stress-induced activation of ß2-ARs could help to overcome immune suppression and enhance the efficacy of immunotherapy and other cancer therapies.

Adrenergic Receptor Signaling Regulates the Response of Tumors to Ionizing Radiation.

While ionizing radiation is a major form of cancer therapy, radioresistance remains a therapeutic obstacle. We have previously shown that the mandated housing temperature for laboratory mice (∼22°C) induces mild, but chronic, cold stress resulting in increased circulating norepinephrine, which binds to, and triggers activation of, beta-adrenergic receptors (ß-AR) on tumor and immune cells. This adrenergic signaling increases tumor cell intrinsic resistance to chemotherapy and suppression of the anti-tumor immune response. These findings led us to hypothesize that adrenergic stress signaling increases radioresistance in tumor cells in addition to suppressing T-cell-mediated anti-tumor immunity, thus suppressing the overall sensitivity of tumors to radiation. We used three strategies to test the effect of adrenergic signaling on responsiveness to radiation. For one strategy, mice implanted with CT26 murine colon adenocarcinoma were housed at either 22°C or at thermoneutrality (30°C), which reduces physiological adrenergic stress. For a second strategy, we used a ß-AR antagonist ("beta blocker") to block adrenergic signaling in mice housed at 22°C. In either case, tumors were then irradiated with a single 6 Gy dose and the response was compared to mice whose adrenergic stress signaling was not reduced. For the third strategy, we used an in vitro approach in which several different tumor cell lines were treated with a ß-AR agonist and irradiated, and cell survival was then assessed by clonogenic assay. Overall, we found that adrenergic stress significantly impaired the anti-tumor efficacy of radiation by inducing tumor cell resistance to radiation-induced cell killing and by suppression of anti-tumor immunity. Treatment using beta blockers is a promising strategy for increasing the anti-tumor efficacy of radiotherapy.

Depression Stresses the Immune Response and Promotes Prostate Cancer Growth.

Depression induces secretion of neuropeptide Y from prostate cancer cells, which, in turn, recruits myeloid-derived suppressor cells (MDSC) to the tumor; tumor cells and MDSCs secrete IL6, which activates STAT3 within cancer cells. Prostate cancer samples from depressed patients reveal a similar phenotype, suggesting new treatment strategies based upon blockade of ß2-adrenergic receptors and/or neuropeptide Y.See related article by Cheng et al., p. 2621.

Manipulation of Ambient Housing Temperature To Study the Impact of Chronic Stress on Immunity and Cancer in Mice.

Mice are the preeminent research organism in which to model human diseases and study the involvement of the immune response. Rapidly accumulating evidence indicates a significant involvement of stress hormones in cancer progression, resistance to therapies, and suppression of immune responses. As a result, there has been a concerted effort to model human stress in mice. In this article, we discuss recent literature showing how mice in research facilities are chronically stressed at baseline because of environmental factors. Focusing on housing temperature, we suggest that the stress of cool housing temperatures contributes to the impact of other imposed experimental stressors and therefore has a confounding effect on mouse stress models. Furthermore, we propose that manipulation of housing temperature is a useful approach for studying the impact of chronic stress on disease and the immune response and for testing therapeutic methods of reducing the negative effects of chronic stress.

ß-Adrenergic signaling blocks murine CD8+ T-cell metabolic reprogramming during activation: a mechanism for immunosuppression by adrenergic stress.

Primary and secondary lymphoid organs are heavily innervated by the autonomic nervous system. Norepinephrine, the primary neurotransmitter secreted by post-ganglionic sympathetic neurons, binds to and activates ß-adrenergic receptors expressed on the surface of immune cells and regulates the functions of these cells. While it is known that both activated and memory CD8+ T-cells primarily express the ß2-adrenergic receptor (ß2-AR) and that signaling through this receptor can inhibit CD8+ T-cell effector function, the mechanism(s) underlying this suppression is not understood. Under normal activation conditions, T-cells increase glucose uptake and undergo metabolic reprogramming. In this study, we show that treatment of murine CD8+ T-cells with the pan ß-AR agonist isoproterenol (ISO) was associated with a reduced expression of glucose transporter 1 following activation, as well as decreased glucose uptake and glycolysis compared to CD8+ T-cells activated in the absence of ISO. The effect of ISO was specifically dependent upon ß2-AR, since it was not seen in adrb2-/- CD8+ T-cells and was blocked by the ß-AR antagonist propranolol. In addition, we found that mitochondrial function in CD8+ T-cells was also impaired by ß2-AR signaling. This study demonstrates that one mechanism by which ß2-AR signaling can inhibit CD8+ T-cell activation is by suppressing the required metabolic reprogramming events which accompany activation of these immune cells and thus reveals a new mechanism by which adrenergic stress can suppress the effector activity of immune cells.

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

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 ß2-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 ß-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 ß-blockers have better outcomes. Clinical trials testing whether ß-blockers can be repurposed to improve the efficacy of traditional and immunotherapies in patients are on the horizon.

The Impact of Housing Temperature-Induced Chronic Stress on Preclinical Mouse Tumor Models and Therapeutic Responses: An Important Role for the Nervous System.

In the last 10-15 years, there has been a recognition that the catecholamines (norepinephrine, NE, and epinephrine, Epi) released by the sympathetic nervous system under stressful conditions promote tumor growth through a variety of mechanisms. Tumors recruit autonomic nerves during their development and NE is then released locally in the tumor microenvironment (TME). Acting through adrenergic receptors present on a variety of cells in the TME, NE and Epi induce proliferation, resistance to apoptosis, epithelial to mesenchymal transition, metastasis of tumor cells, angiogenesis, and inflammation in the TME. These pre-clinical studies have been conducted in mouse models whose care and housing parameters are outlined in "The Guide for the Care and Use of Laboratory Animals [1]. In particular, the Guide mandates that mice be housed at standardized sub-thermoneutral temperatures; however, this causes a state of chronic cold-stress and elevated levels of NE. Although mice are able to maintain a normal body temperature when kept at these cool temperatures, it is becoming clear that this cold-stress is sufficient to activate physiological changes which affect experimental outcomes. We find that when mice are housed under standard, sub-thermoneutral temperatures (~22 °C, ST), tumor growth is significantly greater than when mice are housed at thermoneutrality (~30 °C TT). We also find that the anti-tumor immune response is suppressed at ST and this immunosuppression can be reversed by housing mice at TT or by administration of propranolol (a ß-adrenergic receptor antagonist) to mice housed at ST. Furthermore, at ST tumors are more resistant to therapy and can also be sensitized to cytotoxic therapies by housing mice at TT or by treating mice with propranolol. The implications of these observations are particularly relevant to the way in which experiments conducted in preclinical models are interpreted and the findings implemented in the clinic. It may be that the disappointing failure of many new therapies to fulfill their promise in the clinic is related to an incomplete preclinical assessment in mouse models. Further, an expanded understanding of the efficacy of a therapy alone or in combination obtained by testing under a wider range of conditions would better predict how patients, who are under various levels of stress, might respond in a clinical setting. This may be particularly important to consider since we now appreciate that long term outcome of many therapies depends on eliciting an immune response.It is clear that the outcome of metabolic experiments, immunological investigations and therapeutic efficacy testing in tumors of mice housed at ST is restricted and expanding these experiments to include results obtained at TT may provide us with valuable information that would otherwise be overlooked.

ß-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 ß2-adrenergic receptor (ß-AR) signaling in host immune cells. We used three strategies-physiologic (manipulation of ambient thermal environment), pharmacologic (ß-blockers), and genetic (ß2-AR knockout mice) to reduce adrenergic stress signaling in two widely studied preclinical mouse tumor models. Reducing ß-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 ß-AR signaling to regulate the immune status of the tumor microenvironment and support the strategic use of clinically available ß-blockers in patients to improve responses to immunotherapy. Cancer Res; 77(20); 5639-51. ©2017 AACR.

Pancreatic cancer stem cells in patient pancreatic xenografts are sensitive to drozitumab, an agonistic antibody against DR5.

BACKGROUND: Therapeutic resistance and tumor recurrence are two major hurdles in the treatment of pancreatic ductal adenocarcinoma. Recent findings suggest that both of these attributes are associated with a small subset of pancreatic tumor initiating cancer stem cells (CSCs). Here, we demonstrate that drozitumab, a human agonistic monoclonal antibody which binds the death receptor DR5, selectively eliminates CSCs, resulting in tumor growth inhibition and even regression of pancreatic tumors. METHODS: To examine the efficacy of drozitumab against pancreatic CSCs, we treated patient-derived pancreatic tumor xenografts (PDX) in immunocompromised SCID mice and evaluated tumor control. To assess apoptosis following drozitumab treatment, we identified the CSCs as CD24+, CD44+, and EpCAM+ by FACS analysis, and measured in vivo and in vitro levels of cleaved caspase-3. Lastly, in vitro evaluation of DR5 re-expression was performed using isolated patient pancreatic cancer xenograft cells along with the cell line, Panc-1. After treatment with drozitumab, the remaining DR5- cells were assessed by FACS analysis for DR5 expression at the cell surface at 8, 24 and 48 h post-treatment. All in vivo growth data was analyzed by 2-way Anova, incidence data was analyzed using Mantel-Cox, and in vitro studies statistics were performed with a t-test. RESULTS: We find that while 75-100 % of CSCs express DR5, only 25 % of bulk tumor cells express the death receptors at any one time. Consequently, drozitumab treatment of SCID mice bearing PDX kills higher percentages of CSCs than bulk tumor cells. Additionally, SCID mice implanted with isolated CSCs and then immediately treated with drozitumab fail to ever develop tumors. In vitro studies demonstrate that while drozitumab treatment reduces the DR5+ cell population, the remaining tumor cells begin to express DR5, suggesting a mechanism by which continuous administration of drozitumab can ultimately result in tumor regression despite the initially low percentage of DR5+ cells. CONCLUSIONS: Overall, our work reveals that treatment of pancreatic tumors with the drozitumab can lead to long-term tumor control by targeting both bulk cells and CSCs.

Thermoneutrality, Mice, and Cancer: A Heated Opinion.

The 'mild' cold stress caused by standard sub-thermoneutral housing temperatures used for laboratory mice in research institutes is sufficient to significantly bias conclusions drawn from murine models of several human diseases. We review the data leading to this conclusion, discuss the implications for research and suggest ways to reduce problems in reproducibility and experimental transparency caused by this housing variable. We have found that these cool temperatures suppress endogenous immune responses, skewing tumor growth data and the severity of graft versus host disease, and also increase the therapeutic resistance of tumors. Owing to the potential for ambient temperature to affect energy homeostasis as well as adrenergic stress, both of which could contribute to biased outcomes in murine cancer models, housing temperature should be reported in all publications and considered as a potential source of variability in results between laboratories. Researchers and regulatory agencies should work together to determine whether changes in housing parameters would enhance the use of mouse models in cancer research, as well as for other diseases. Finally, for many years agencies such as the National Cancer Institute (NCI) have encouraged the development of newer and more sophisticated mouse models for cancer research, but we believe that, without an appreciation of how basic murine physiology is affected by ambient temperature, even data from these models is likely to be compromised.

Tumor-Priming Smoothened Inhibitor Enhances Deposition and Efficacy of Cytotoxic Nanoparticles in a Pancreatic Cancer Model.

Most pancreatic adenocarcinoma patients present with unresectable disease and benefit little from chemotherapy. Poor tumor perfusion and vascular permeability limit drug deposition. Previous work showed that Smoothened inhibitors of hedgehog signaling (sHHI) promote neovascularization in spontaneous mouse models of pancreatic cancer (PaCA) and enhance tumor permeability to low-molecular weight compounds. Here, we tested the hypothesis that sHHI can enhance tumor deposition and efficacy of drug-containing nanoparticles consisting of 80 to 100 nm sterically-stabilized liposomes (SSL) containing doxorubicin (SSL-DXR). SCID mice bearing low-passage patient-derived PaCA xenografts (PDX) were pretreated p.o. for 10 days with 40 mg/kg/d NVP-LDE225 (erismodegib), followed by i.v. SSL-DXR. Microvessel density, permeability, perfusion, and morphology were compared with untreated controls, as was SSL deposition and therapeutic efficacy. The sHHI alone affected tumor growth minimally, but markedly increased extravasation of nanoparticles into adenocarcinoma cell-enriched regions of the tumor. Immunostaining showed that sHHI treatment decreased pericyte coverage (α-SMA(+)) of CD31(+) vascular endothelium structures, and increased the abundance of endothelium-poor (CD31(-)) basement membrane structures (collagen IV(+)), suggesting increased immature microvessels. SSL-DXR (15 mg/kg) administered after sHHI pretreatment arrested tumor volume progression and decreased tumor perfusion/permeability, suggesting an initial vascular pruning response. Compared with controls, one cycle of 10-day sHHI pretreatment followed by 6 mg/kg SSL-DXR doubled median tumor progression time. Three cycles of treatment with sHHI and SSL-DXR, with a 10-day between-cycle drug holiday, nearly tripled median tumor progression time. Based upon these data, short-term sHHI treatment sequenced with nanoparticulate drug carriers constitutes a potential strategy to enhance efficacy of pancreatic cancer therapy.

Tumor priming by Apo2L/TRAIL reduces interstitial fluid pressure and enhances efficacy of liposomal gemcitabine in a patient derived xenograft tumor model.

Interstitial fluid pressure (IFP) is elevated in tumors and high IFP, a negative cancer prognosticator, is known to limit the uptake and efficacy of anti-tumor therapeutics. Approaches that alter the tumor microenvironment and enhance uptake of therapeutics are collectively referred to as tumor "priming". Here we show that the cytotoxic biological therapy Apo2L/TRAIL can prime the tumor microenvironment and significantly lower IFP in three different human tumor xenograft models (Colo205, MiaPaca-2 and a patient gastrointestinal adenocarcinoma tumor xenograft). We found that a single dose of Apo2L/TRAIL resulted in a wave of apoptosis which reached a maximum at 8h post-treatment. Apoptotic debris subsequently disappeared concurrent with an increase in macrophage infiltration. By 24h post-treatment, treated tumors appeared less condensed with widening of the stromal areas which increased at 48 and 72h. Analysis of tumor vasculature demonstrated a significant increase in overall vessel size at 48 and 72h although the number of vessels did not change. Notably, IFP was significantly reduced in these tumors by 48h after Apo2L/TRAIL treatment. Administration of gemcitabine at this time resulted in increased tumor uptake of both gemcitabine and liposomal gemcitabine and significantly improved anti-tumor efficacy of liposomal gemcitabine. These results suggest that Apo2L/TRAIL has a potential as a tumor priming agent and provides a rationale for developing a sequencing schema for combination therapy such that an initial dose of Apo2L/TRAIL would precede administration of gemcitabine or other therapies.

Housing temperature-induced stress drives therapeutic resistance in murine tumour models through ß2-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 ß-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.

Defining immunological impact and therapeutic benefit of mild heating in a murine model of arthritis.

Traditional treatments, including a variety of thermal therapies have been known since ancient times to provide relief from rheumatoid arthritis (RA) symptoms. However, a general absence of information on how heating affects molecular or immunological targets relevant to RA has limited heat treatment (HT) to the category of treatments known as "alternative therapies". In this study, we evaluated the effectiveness of mild HT in a collagen-induced arthritis (CIA) model which has been used in many previous studies to evaluate newer pharmacological approaches for the treatment of RA, and tested whether inflammatory immune activity was altered. We also compared the effect of HT to methotrexate, a well characterized pharmacological treatment for RA. CIA mice were treated with either a single HT for several hours or daily 30 minute HT. Disease progression and macrophage infiltration were evaluated. We found that both HT regimens significantly reduced arthritis disease severity and macrophage infiltration into inflamed joints. Surprisingly, HT was as efficient as methotrexate in controlling disease progression. At the molecular level, HT suppressed TNF-α while increasing production of IL-10. We also observed an induction of HSP70 and a reduction in both NF-κB and HIF-1α in inflamed tissues. Additionally, using activated macrophages in vitro, we found that HT reduced production of pro-inflammatory cytokines, an effect which is correlated to induction of HSF-1 and HSP70 and inhibition of NF-κB and STAT activation. Our findings demonstrate a significant therapeutic benefit of HT in controlling arthritis progression in a clinically relevant mouse model, with an efficacy similar to methotrexate. Mechanistically, HT targets highly relevant anti-inflammatory pathways which strongly support its increased study for use in clinical trials for RA.