Androgen loss weakens anti-tumor immunity and accelerates brain tumor growth.

Many cancers, including glioblastoma (GBM), have a male-biased sex difference in incidence and outcome. The underlying reasons for this sex bias are unclear but likely involve differences in tumor cell state and immune response. This effect is further amplified by sex hormones, including androgens, which have been shown to inhibit anti-tumor T cell immunity. Here, we show that androgens drive anti-tumor immunity in brain tumors, in contrast to its effect in other tumor types. Upon castration, tumor growth was accelerated with attenuated T cell function in GBM and brain tumor models, but the opposite was observed when tumors were located outside the brain. Activity of the hypothalamus-pituitary-adrenal gland (HPA) axis was increased in castrated mice, particularly in those with brain tumors. Blockade of glucocorticoid receptors reversed the accelerated tumor growth in castrated mice, indicating that the effect of castration was mediated by elevated glucocorticoid signaling. Furthermore, this mechanism was not GBM specific, but brain specific, as hyperactivation of the HPA axis was observed with intracranial implantation of non-GBM tumors in the brain. Together, our findings establish that brain tumors drive distinct endocrine-mediated mechanisms in the androgen-deprived setting and highlight the importance of organ-specific effects on anti-tumor immunity.

miR-644a is a tumor cell-intrinsic mediator of sex bias in glioblastoma.

Background: Biological sex is an important risk factor for glioblastoma (GBM), with males having a higher incidence and poorer prognosis. The mechanisms for this sex bias are thought to be both tumor intrinsic and tumor extrinsic. MicroRNAs (miRNAs), key post-transcriptional regulators of gene expression, have been previously linked to sex differences in various cell types and diseases, but their role in the sex bias of GBM remains unknown. Methods: We leveraged previously published paired miRNA and mRNA sequencing of 39 GBM patients (22 male, 17 female) to identify sex-biased miRNAs. We further interrogated a separate single-cell RNA sequencing dataset of 110 GBM patients to examine whether differences in miRNA target gene expression were tumor cell intrinsic or tumor cell extrinsic. Results were validated in a panel of patient-derived cell models. Results: We identified 10 sex-biased miRNAs (adjusted < 0.1), of which 3 were more highly expressed in males and 7 more highly expressed in females. Of these, miR-644a was higher in females, and increased expression of miR-644a target genes was significantly associated with decreased overall survival (HR 1.3, p = 0.02). Furthermore, analysis of an independent single-cell RNA sequencing dataset confirmed sex-specific expression of miR-644a target genes in tumor cells (p < 10-15). Among patient derived models, miR-644a was expressed a median of 4.8-fold higher in females compared to males. Conclusions: Our findings implicate miR-644a as a candidate tumor cell-intrinsic regulator of sex-biased gene expression in GBM.

Use of amniotic membrane in hard-to-heal wounds: a multicentre retrospective study.

OBJECTIVE: Hard-to-heal (chronic) wounds negatively impact patients and are a source of significant strain on the healthcare system and economy. These wounds are often resistant to standard of care (SoC) wound healing approaches due to a diversity of underlying pathologies. Cellular, acellular, and matrix-like products, such as amniotic membranes (AM), are a potential solution to these challenges. A growing body of evidence suggests that AM may be useful for treatment-resistant wounds; however, limited information is available regarding the efficacy of dehydrated amniotic membrane (DHAM) on multi-aetiology, hard-to-heal wounds. Therefore, we analysed the efficacy of DHAM treatment in reducing the size of hard-to-heal diabetic and venous leg ulcers (VLUs) that had failed to improve after SoC-based treatments. METHOD: In this multicentre retrospective study, we analysed wound size during clinic visits for patients being treated for either diabetic or VLUs. During each visit, the treatment consisted of debridement followed by application of DHAM. Each wound was measured after debridement and prior to DHAM application, and wound volumes over time or number of DHAM applications were compared. RESULTS: A total of 18 wounds in 11 patients were analysed as part of this study. Wounds showed a significant reduction in volume after a single DHAM application, and a 50% reduction in wound size was observed after approximately two DHAM applications. These findings are consistent with reports investigating DHAM treatment of diabetic ulcers that were not necessarily resistant to treatment. CONCLUSION: To our knowledge, this study is the first to directly compare the efficacy of standalone DHAM application to hard-to-heal diabetic and venous leg ulcers, and our findings indicate that DHAM is an effective intervention for resolving these types of wounds. This suggests that implementing this approach could lead to fewer clinic visits, cost savings and improved patient quality of life. DECLARATION OF INTEREST: This research was supported in part by Merakris Therapeutics, US, and facilitated access to deidentified patient datasets, which may represent a perceived conflict of interest; however, the primary data analysis was performed by FSB who is unaffiliated with Merakris Therapeutics. TCB is a founder, employee of and shareholder in Merakris Therapeutics; WSF is a co-founder of, consultant for, and shareholder in Merakris Therapeutics, and was also supported by the National Institutes of Health National Center for Advancing Translational Sciences Clinical and Translational Science Awards Grant KL2 Scholars Program (KL2TR001441). The research was also supported through endowments to WSF from the University of Texas Medical Branch Mimmie and Hallie Smith Endowed Chair of Transplant Research and the John L Hern University Chair in Transplant Surgery.

Homeostatic iron regulatory protein drives glioblastoma growth via tumor cell-intrinsic and sex-specific responses.

Background: Glioblastoma (GBM) displays alterations in iron that drive proliferation and tumor growth. Iron regulation is complex and involves many regulatory mechanisms, including the homeostatic iron regulator (HFE) gene, which encodes the homeostatic iron regulatory protein. While HFE is upregulated in GBM and correlates with poor survival outcomes, the function of HFE in GBM remains unclear. Methods: We interrogated the impact of cell-intrinsic Hfe expression on proliferation and survival of intracranially implanted animals through genetic gain- and loss-of-function approaches in syngeneic mouse glioma models, along with in vivo immune assessments. We also determined the expression of iron-associated genes and their relationship to survival in GBM using public data sets and used transcriptional profiling to identify differentially expressed pathways in control compared to Hfe-knockdown cells. Results: Overexpression of Hfe accelerated GBM proliferation and reduced animal survival, whereas suppression of Hfe induced apoptotic cell death and extended survival, which was more pronounced in females and associated with attenuation of natural killer cells and CD8+ T cell activity. Analysis of iron gene signatures in Hfe-knockdown cells revealed alterations in the expression of several iron-associated genes, suggesting global disruption of intracellular iron homeostasis. Further analysis of differentially expressed pathways revealed oxidative stress as the top pathway upregulated following Hfe loss. Hfe knockdown indeed resulted in enhanced 55Fe uptake and generation of reactive oxygen species. Conclusions: These findings reveal an essential function for HFE in GBM cell growth and survival, as well as a sex-specific interaction with the immune response.

Tumor cell-derived spermidine promotes a pro-tumorigenic immune microenvironment in glioblastoma via CD8+ T cell inhibition.

The glioblastoma microenvironment is enriched in immunosuppressive factors that potently interfere with the function of cytotoxic T lymphocytes. Cancer cells can directly impact the immune system, but the mechanisms driving these interactions are not completely clear. Here we demonstrate that the polyamine metabolite spermidine is elevated in the glioblastoma tumor microenvironment. Exogenous administration of spermidine drives tumor aggressiveness in an immune-dependent manner in pre-clinical mouse models via reduction of CD8+ T cell frequency and phenotype. Knockdown of ornithine decarboxylase, the rate-limiting enzyme in spermidine synthesis, did not impact cancer cell growth in vitro but did result in extended survival. Furthermore, glioblastoma patients with a more favorable outcome had a significant reduction in spermidine compared to patients with a poor prognosis. Our results demonstrate that spermidine functions as a cancer cell-derived metabolite that drives tumor progression by reducing CD8+T cell number and function.

Let the Guard Down: cAMP Activators Can Improve Immunotherapy in GBM.

Enhancing T-cell infiltration into glioblastoma (GBM) tumors has proven challenging yet remains crucial for improving the efficacy of immunotherapy for patients with this deadly cancer. In this issue, Qin, Huang, Li, and colleagues find that inhibiting vasculature formation driven by cancer stem cells is a promising target to enhance immunotherapy in GBM. See related article by Qin, Huang, Li, et al., p. 1351 (2).

Metaorganismal choline metabolism shapes olfactory perception.

Microbes living in the intestine can regulate key signaling processes in the central nervous system that directly impact brain health. This gut-brain signaling axis is partially mediated by microbe-host-dependent immune regulation, gut-innervating neuronal communication, and endocrine-like small molecule metabolites that originate from bacteria to ultimately cross the blood-brain barrier. Given the mounting evidence of gut-brain crosstalk, a new therapeutic approach of "psychobiotics" has emerged, whereby strategies designed to primarily modify the gut microbiome have been shown to improve mental health or slow neurodegenerative diseases. Diet is one of the most powerful determinants of gut microbiome community structure, and dietary habits are associated with brain health and disease. Recently, the metaorganismal (i.e., diet-microbe-host) trimethylamine N-oxide (TMAO) pathway has been linked to the development of several brain diseases including Alzheimer's, Parkinson's, and ischemic stroke. However, it is poorly understood how metaorganismal TMAO production influences brain function under normal physiological conditions. To address this, here we have reduced TMAO levels by inhibiting gut microbe-driven choline conversion to trimethylamine (TMA), and then performed comprehensive behavioral phenotyping in mice. Unexpectedly, we find that TMAO is particularly enriched in the murine olfactory bulb, and when TMAO production is blunted at the level of bacterial choline TMA lyase (CutC/D), olfactory perception is altered. Taken together, our studies demonstrate a previously underappreciated role for the TMAO pathway in olfactory-related behaviors.

Sex-Biased T-cell Exhaustion Drives Differential Immune Responses in Glioblastoma.

Sex differences in glioblastoma (GBM) incidence and outcome are well recognized, and emerging evidence suggests that these extend to genetic/epigenetic and cellular differences, including immune responses. However, the mechanisms driving immunologic sex differences are not fully understood. Here, we demonstrate that T cells play a critical role in driving GBM sex differences. Male mice exhibited accelerated tumor growth, with decreased frequency and increased exhaustion of CD8+ T cells in the tumor. Furthermore, a higher frequency of progenitor exhausted T cells was found in males, with improved responsiveness to anti-PD-1 treatment. Moreover, increased T-cell exhaustion was observed in male GBM patients. Bone marrow chimera and adoptive transfer models indicated that T cell-mediated tumor control was predominantly regulated in a cell-intrinsic manner, partially mediated by the X chromosome inactivation escape gene Kdm6a. These findings demonstrate that sex-biased predetermined behavior of T cells is critical for inducing sex differences in GBM progression and immunotherapy response. SIGNIFICANCE: Immunotherapies in patients with GBM have been unsuccessful due to a variety of factors, including the highly immunosuppressive tumor microenvironment in GBM. This study demonstrates that sex-biased T-cell behaviors are predominantly intrinsically regulated, further suggesting sex-specific approaches can be leveraged to potentially improve the therapeutic efficacy of immunotherapy in GBM. See related commentary by Alspach, p. 1966. This article is featured in Selected Articles from This Issue, p. 1949.

A subset of broadly responsive Type III taste cells contribute to the detection of bitter, sweet and umami stimuli.

Taste receptor cells use multiple signaling pathways to detect chemicals in potential food items. These cells are functionally grouped into different types: Type I cells act as support cells and have glial-like properties; Type II cells detect bitter, sweet, and umami taste stimuli; and Type III cells detect sour and salty stimuli. We have identified a new population of taste cells that are broadly tuned to multiple taste stimuli including bitter, sweet, sour, and umami. The goal of this study was to characterize these broadly responsive (BR) taste cells. We used an IP3R3-KO mouse (does not release calcium (Ca2+) from internal stores in Type II cells when stimulated with bitter, sweet, or umami stimuli) to characterize the BR cells without any potentially confounding input from Type II cells. Using live cell Ca2+ imaging in isolated taste cells from the IP3R3-KO mouse, we found that BR cells are a subset of Type III cells that respond to sour stimuli but also use a PLCß signaling pathway to respond to bitter, sweet, and umami stimuli. Unlike Type II cells, individual BR cells are broadly tuned and respond to multiple stimuli across different taste modalities. Live cell imaging in a PLCß3-KO mouse confirmed that BR cells use this signaling pathway to respond to bitter, sweet, and umami stimuli. Short term behavioral assays revealed that BR cells make significant contributions to taste driven behaviors and found that loss of either PLCß3 in BR cells or IP3R3 in Type II cells caused similar behavioral deficits to bitter, sweet, and umami stimuli. Analysis of c-Fos activity in the nucleus of the solitary tract (NTS) also demonstrated that functional Type II and BR cells are required for normal stimulus induced expression.

Temperature Is Sufficient to Condition a Flavor Preference for a Cold-Paired Solution in Rats.

Increasing evidence suggests that stimulus temperature modifies taste signaling. However, understanding how temperature modifies taste-driven behavior is difficult to separate as we must first understand how temperature alone modifies behavior. Previous work has suggested that cold water is more rewarding and "satiating" than warm water, and water above orolingual temperature is avoided in brief-access testing. We explored the strength of cold water preference and warm water avoidance by asking: (1) if cold temperature alone was sufficient to condition a flavor preference and (2) if avoidance of warm stimuli is driven by novelty. We addressed these questions using custom-designed equipment that allows us to monitor and maintain solution temperatures. We conducted two-bottle preference tests, after pairing Kool-Aid flavors with 10 or 40 °C. Rats preferred the flavor paired with cold temperature, both while it was cold and for 1 day while solutions were presented at 22 °C. We then examined the role of novelty in avoidance of 40 °C. Rats were maintained on 10, 22, or 40 °C water in their home cage to increase familiarity with the temperatures. Rats were then subject to a series of brief-access taste tests to water or sucrose at 10 to 40 °C. Rats that had 40 °C experience licked more to 40 °C water, but not sucrose, during brief-access testing. In a series of two-bottle preference tests, rats maintained on 40 °C water had a decreased preference for 10 °C water when paired opposite 40 °C water. Together, these data contribute to our understanding of orosensory-driven behavior with water at different temperatures.

Altering salivary protein profile can decrease aversive oromotor responding to quinine in rats.

Bitter taste is often associated with toxins, but accepting some bitter foods, such as green vegetables, can be an important part of maintaining a healthy diet. It has previously been shown that animals exposed to quinine upregulate a set of salivary proteins (SPs), and those with upregulated SPs have increased rates of feeding on a quinine diet as well as increased brief-access licking to and higher detection thresholds for quinine. These studies suggest that SPs alter orosensory feedback; however, they rely on SPs upregulated by diet exposure and cannot control for the role of learning. Here, we use taste reactivity to determine if SPs can alter bitter taste in animals with no previous bitter diet experience. First, saliva with proteins stimulated by injections of isoproterenol and pilocarpine was collected from anesthetized rats; this "donor saliva" was analyzed for protein concentration and profile. Bitter-naïve rats were implanted with oral catheters and infused with taste stimuli dissolved in saliva that contained all of the SPs from the donors, saliva that was filtered of SPs, water, or artificial saliva. Their orofacial movements were recorded and quantified. We found that presence of quinine increased movements associated with aversive stimuli, but adding SPs to the infusion was sufficient to reduce aversive oromotor responding to quinine. The effect was dependent on the total protein concentration of the saliva, as protein concentration increased aversive responses decreased. Additionally, infusions of whole saliva altered aversive responding to quinine, but not other stimuli (citric acid, NaCl, sucrose). Our work suggests that effect of these SPs is specific and the presence of SPs is sufficient to decrease aversive orosensory feedback to bitter stimuli.

Rats are unable to discriminate quinine from diverse bitter stimuli.

Compounds described by humans as "bitter" are sensed by a family of type 2 taste receptors (T2Rs). Previous work suggested that diverse bitter stimuli activate distinct receptors, which might allow for perceptually distinct tastes. Alternatively, it has been shown that multiple T2Rs are expressed on the same taste cell, leading to the contrary suggestion that these stimuli produce a unitary perception. Behavioral work done to address this in rodent models is limited to Spector and Kopka (Spector AC, Kopka SL. J Neurosci 22: 1937-1941, 2002), who demonstrated that rats cannot discriminate quinine from denatonium. Supporting this finding, it has been shown that quinine and denatonium activate overlapping T2Rs and neurons in both the mouse and rat nucleus of the solitary tract (NTS). However, cycloheximide and 6-n-propylthiouracil (PROP) do not appear to overlap with quinine in the NTS, suggesting that these stimuli may be discriminable from quinine and the denatonium/quinine comparison is not generalizable. Using the same procedure as Spector and Kopka, we tasked animals with discriminating a range of stimuli (denatonium, cycloheximide, PROP, and sucrose octaacetate) from quinine. We replicated and expanded the findings of Spector and Kopka; rats could not discriminate quinine from denatonium, cycloheximide, or PROP. Rats showed a very weak ability to discriminate between quinine and sucrose octaacetate. All animals succeeded in discriminating quinine from KCl, demonstrating they were capable of the task. These data suggest that rats cannot discriminate this suite of stimuli, although they appear distinct by physiological measures.

Bitter-Induced Salivary Proteins Increase Detection Threshold of Quinine, But Not Sucrose.

Exposures to dietary tannic acid (TA, 3%) and quinine (0.375%) upregulate partially overlapping sets of salivary proteins which are concurrent with changes in taste-driven behaviors, such as rate of feeding and brief access licking to quinine. In addition, the presence of salivary proteins reduces chorda tympani responding to quinine. Together these data suggest that salivary proteins play a role in bitter taste. We hypothesized that salivary proteins altered orosensory feedback to bitter by decreasing sensitivity to the stimulus. To that end, we used diet exposure to alter salivary proteins, then assessed an animal's ability to detect quinine, using a 2-response operant task. Rats were asked to discriminate descending concentrations of quinine from water in a modified forced-choice paradigm, before and after exposure to diets that alter salivary protein expression in a similar way (0.375% quinine or 3% TA), or 1 of 2 control diets. Control animals received either a bitter diet that does not upregulate salivary proteins (4% sucrose octaacetate), or a nonbitter diet. The rats exposed to salivary protein-inducing diets significantly decreased their performance (had higher detection thresholds) after diet exposure, whereas rats in the control conditions did not alter performance after diet exposure. A fifth group of animals were trained to detect sucrose before and after they were maintained on the 3% TA diet. There was no significant difference in performance, suggesting that these shifts in threshold are stimulus specific rather than task specific. Taken together, these results suggest that salivary proteins reduce sensitivity to quinine.

Altering salivary protein profile can increase acceptance of a novel bitter diet.

Bitter taste is often associated with toxins, but accepting some bitter foods, such as green vegetables, can be an important part of maintaining a healthy diet. In rats and humans, repeated exposure to a bitter stimulus increases acceptance. Repeated exposure allows an individual the opportunity to learn about the food's orosensory and postingestive effects. It also alters the salivary protein (SP) profile, which in turn alters taste signaling. We have hypothesized that altering the salivary proteome plays a role in the increased acceptance after repeated exposure. Here we test this and attempt to disentangle the contribution of learning during dietary exposure from the contribution of SPs in increased acceptance of bitter diet. Dietary exposure to quinine or tannic acid and injection of isoproterenol (IPR) result in similar salivary protein profiles. Here we used either the bitter stimulus tannic acid or IPR injection to upregulate a subset of SPs before exposing animals to a novel diet containing quinine (0.375%). Control animals received either a control diet before being exposed to quinine, or a diet containing sucrose octaacetate, a compound that the animals avoid but does not alter SP profiles. The treatments that alter SP expression increased rate of feeding on the quinine diet compared to the control treatments. Additionally, tannic acid exposure altered intake and meal size of the quinine diet. These data suggest that SPs, not just learning about bitter food, increase acceptance of the bitter diet.

Ventral tegmental area orexin 1 receptors promote palatable food intake and oppose postingestive negative feedback.

Hypothalamic orexin neurons project to numerous brain areas, including the ventral tegmental area (VTA), which is involved in motivation and food-seeking behavior. Here we address how exogenously administered orexin-A and endogenous orexin 1 receptor (OX1R) activation in the VTA affects feeding behavior. We hypothesized that orexin-A and OX1R antagonist SB334867 delivered to the VTA, at doses that were subthreshold for effect when injected into the ventricle, would affect intake of palatable foods in multiple test situations. We first used a hedonic feeding model in which satiated rats selectively consume a high-fat diet (HFD). Intra-VTA orexin-A stimulated additional consumption of chow and increased HFD intake in this model. In ad libitum-fed rats given daily 30-min test sessions, intra-VTA orexin-A also increased intake of HFD and 0.1 M sucrose. Further analysis of licking patterns revealed that that VTA orexin-A increased meal size and licking burst size only toward the end of the meal. Consistent with this finding, a subthreshold dose of VTA orexin-A prevented intake suppression induced by gastrointestinal nutrient infusion. Surprisingly, intra-VTA orexin-A had no effect on operant responding for sucrose pellets on a progressive ratio schedule of reinforcement. A role for endogenous VTA OX1R stimulation is supported by our finding that bilateral VTA injection of the selective OX1R antagonist SB334867 suppressed 0.1 M sucrose intake. Together, our data suggest that OX1R activity in the VTA facilitates food intake, potentially by counteracting postingestive negative feedback that would normally suppress feeding later in a meal.