Sensory neuro-tumor crosstalk: Therapeutic opportunities and emerging frontiers in cancer neuroscience.

The tumor microenvironment is defined by nutrient starvation, waste product accumulation, hypoxia, and acidosis, which collectively contribute towards immune exhaustion and suppression. Models that mimic the tumor microenvironment will be instrumental to develop new therapies that improve immune response. Microfluidic models can mimic the tumor metabolic microenvironment to elucidate mechanisms that lead to weakened tumor immunity and to new tumor immunotherapies. We developed microfluidic models where cancer cells were cultured as a dense mass embedded in a 3D matrix. The microfluidic platform has lateral channels that are lined with endothelial cells to mimic the tumor vasculature. These vessels can be perfused with immune cells or drugs that extravasate into the tumor mass. Immune cells were isolated from the tumor mass within these microfluidic models and gene expression was analyzed to quantify changes in immune cell function under tumor metabolic microenvironment conditions. Optical metabolic imaging of NADH and FAD autofluorescence with 2-photon microscopy monitored metabolic dynamics during tumor-immune cell interactions. Immunotherapy drugs were also perfused through the microfluidic devices to measure drug efficacy. Changes in immune cell function after drug exposure were analyzed by optical metabolic imaging to examine metabolic dynamics, alongside fluorescence microscopy to visualize cytotoxicity changes. Natural killer cells exhibited directional migration towards the tumor, which indicates that natural killer cells can detect the presence of the tumor several hundreds of microns away. Real-time microscopy revealed that natural killer cells destroyed tumor cells at the tumor periphery and notably at the innermost tumor core. Gene expression analysis of immune cells and tumor cells cultured in the microfluidic model reveals that the tumor cells have created an environment consistent with immunosuppression, immune exhaustion, and nutrient starvation. Metabolic imaging reveals that immune cells exposed to the tumor microenvironment have an altered metabolic profile, specifically decreased redox ratio that persists even under normal culture conditions. The microfluidic model provides a system to examine immune cytotoxicity, metabolic dynamics during tumor-immune interactions, and the impact of immunotherapies in enhancing immune cytotoxicity. Optical metabolic imaging and microfluidic models provide novel insight into metabolic dynamics with tumor cell and immune cell interactions within the tumor metabolic microenvironment. Future studies will expand this technology towards studying immune exhaustion and dysfunction in other immune therapies. Citation Format: Shujah H. Rehman, Jose M. Ayuso, Maria Maria Virumbrales-Munoz, Patrick H. McMinn, David J. Beebe, Melissa C. Skala. Monitoring immune-tumor cell interactions in 3D microfluidic models with optical metabolic imaging and molecular profiling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3874.

Endurance exercise can cause immunosuppression and increase the risk of upper respiratory illness. The present study examined changes in the secretion of T helper (Th) cell cytokines after endurance exercise. Ten highly trained road cyclists [mean±SEM: age 24.2±1.7 years; height 1.82±0.02 m; body mass 73.8±2.0 kg; peak oxygen uptake 65.9±2.3 mL/(kg•min)] performed 2 h of cycling exercise at 90% of the second ventilatory threshold. Peripheral blood mononuclear cells were isolated and stimulated with phytohemagglutinin. Plasma cortisol concentrations and the concentration of Th1/Th2/Th17 cell cytokines were examined. Data were analyzed using both traditional statistics and magnitude-based inferences. Results revealed a significant decrease in plasma cortisol at 4-24 h postexercise compared with pre-exercise values. Qualitative analysis revealed postexercise changes in concentrations of plasma cortisol, IL-2, TNF, IL-4, IL-6, IL-10, and IL-17A compared with pre-exercise values. A Th1/Th2 shift was evident immediately postexercise. Furthermore, for multiple cytokines, including IL-2 and TNF (Th1), IL-6 and IL-10 (Th2), and IL-17 (Th17), no meaningful change in concentration occurred until more than 4 h postexercise, highlighting the duration of exercise-induced changes in immune function. These results demonstrate the importance of considering "clinically" significant versus statistically significant changes in immune cell function after exercise.

Metastasis is a major barrier to long-term survival and therapeutic options for aggressive, metastatic forms of breast cancer remain limited. Studies using patient samples have identified tumor-resident bacteria that preferentially associate with specific breast cancer types including highly aggressive TNBC. However, it is not yet understood how intratumoral bacteria directly contributes to disease progression and metastatic propensity independent of other prognostic factors. It is therefore the goal of the Dedhar and Finlay labs to identify how specific bacteria within metastatic breast cancer control immune and tumor cell functions to regulate metastatic potential and determine the outcome of disease progression. Using the syngenic, immunocompetent 4T1 and 67NR breast cancer models of metastatic and non-metastatic disease, we found microbiome depletion significantly reduces primary tumor growth highly metastatic 4T1 tumors specifically. We also found bacterial depletion reduces metastatic burden and extends survival time compared to microbiome-replete controls. Along with alterations in disease progression, microbiome depletion induces changes in immune cell function that occur specifically in the metastatic 4T1 tumors, revealing differential microbial-based regulation of metastatic versus non-metastatic disease. To identify bacteria that control metastasis in microbiome-replete controls, we plated surgically resected tumor suspensions on bacterial growth media and compared bacteria from the 4T1 and 67NR primary tumors. We identified several species of the Bacillus genus that were unique to 4T1 tumors and were present both within the primary tumor as well as metastatic nodules. To determine how these bacteria effect disease progression, we designed several in vivo model systems to directly test the ability of the isolated bacteria to promote metastasis. Using an orthotopic inoculation model with 4T1 or EMT6 cells, we found that following intratumoral injection, the 4T1- derived Bacillus species was actually able to augment metastasis when introduced directly back into primary tumors. To determine the specificity of this phenomenon, we then compared the effects of the 4T1 and 67NR-isolated bacteria on metastasis by injecting 4T1 cells that had been co-cultured with either bacteria prior to injection. Interestingly, we found that while the 67NR-derived bacteria had little effect on metastasis, the 4T1-derived Bacillus species significantly enhanced metastatic tumor burden compared to all other groups including those cultured with the 67NR-derived bacteria. These data demonstrate the ability of certain bacteria to promote metastatic disease. Based on these findings, we hypothesize specific bacteria play a causative role in augmenting metastatic propensity, and seek to determine functional differences between intratumoral bacteria to identify mechanistic targets for prevention of metastasis. We also seek to expand this work into clinical models to identify potential prognostic factors as well as mechanistic targets for disease treatment. Citation Format: Zachary J. Gerbec, Antonio Serapio-Palacios, Sarah E. Woodword, Jorge Pena Diaz, Brett Finlay, Shoukat Dedhar. Tumor-derived bacteria drive breast cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A047.

Exercise profoundly alters immunological processes to improve overall health and immunity. The link between immune cell metabolism and function has prompted study of immune cell bioenergetics following acute exercise and the role of muscle-resident immune cells in training adaptations. This review highlights recent work in the area and discusses potential dietary approaches for boosting exercise-induced immunometabolic benefits. Human studies highlight the ability of exercise to alter immune cell bioenergetics, with some also reporting accompanying changes in immune cell function. Rodent studies involving moderate exercise report improved innate and adaptive immune cell phenotypes that are accompanied by increased mitochondrial size and bioenergetic function. Various muscle resident immune cell subpopulations including macrophages, mast cells, and regulatory T cells also appear to be involved in the adaptive responses to exercise. Fasting, exogenous ketones, and mitochondrial enhancing compounds (e.g., sulforaphane, urolithin A) could theoretically potentiate the immunometabolic benefits of exercise based on their independent effects, but evidence for combined interventions is currently lacking. Exercise and dietary manipulations that independently alter immunometabolic pathways could be combined to maximize associated health benefits. This may benefit those who cannot meet physical activity guidelines or want to maximize exercise adaptation.

The present study tested the hypothesis that immune cell function is influenced by ambient photoperiod. The male Siberian hamster served as the experimental model because day length regulates a variety of seasonal adaptations in physiology. Adult hamsters were in long days (16 h of light daily), which sustains gonadal function, or transferred to short days (8 h) for >4 wk to induce testes regression. Blood was drawn from the ocular sinus or splenocytes obtained to assess basal indexes of immune cell function. In hamsters in short days, natural killer cell cytolytic capacity, as well as spontaneous blastogenesis in both whole blood and isolated lymphocytes, were enhanced compared with that in hamsters in long days. By contrast, phagocytosis and oxidative burst activity by both granulocytes and monocytes were suppressed in hamsters by exposure to short days versus long days. Selective changes in immune cell function coincided with short-day-induced gonadal atrophy. These findings raise the hypothesis that photoperiod regulation of physiological adaptations, including distinct immune cell functions, may help individuals anticipate seasonal challenges posed by opportunistic diseases or climate to facilitate survival.

The effect of resistance exercise on the immune cell function in humans: A systematic review.

Age-Related Changes in Eosinophil Function in Human Subjects

Periodontitis has been traditionally regarded as a chronic inflammatory oral infection. However, recent studies indicate that this oral disease may have profound effects on systemic health. The search for cellular/molecular mechanisms linking periodontitis to changes in systemic health and systemic physiology has resulted in the evolution of a new area of lipid research establishing linkages between existing multidisciplinary biomedical literature, recent observations concerning the effects of serum lipids on immune cell phenotype/function, and a heightened interest in systemic responses to chronic localized infections. There appears to be more than a casual relationship between serum lipid levels and systemic health (particularly cardiovascular disease, diabetes, tissue repair capacity, and immune cell function), susceptibility to periodontitis, and serum levels of pro-inflammatory cytokines. In terms of the potential relationship between periodontitis and systemic disease, it is possible that periodontitis-induced changes in immune cell function cause metabolic dysregulation of lipid metabolism through mechanisms involving proinflammatory cytokines. Sustained elevations of serum lipids and/or pro-inflammatory cytokines may have a serious negative impact on systemic health. The purpose of this paper is to present the background, supporting data, and hypotheses related to this concept. As active participants in this emerging and exciting area of investigation, we hope to stimulate interest and awareness among biomedical scientists and practitioners.

ABSTRACTExcessive inflammation and impaired healing of cardiac tissue following a myocardial infarction (MI) can drive the development of heart failure. Cardiac repair begins immediately after the onset of MI and continues for months. The repair process can be divided into the following 3 overlapping phases, each having distinct functions and sequelae: the inflammatory phase, the proliferative phase, and the maturation phase. Macrophages, neutrophils, and lymphocytes are present in the myocardium throughout the repair process and govern the duration and function of each of these phases. However, changes in the functions of these cell types across each phase are poorly characterized. Numerous immunomodulatory therapies that specifically target inflammation have been developed for promoting cardiac repair and preventing heart failure after MI. However, these treatments have been largely unsuccessful in large‐scale clinical randomized controlled trials. A potential explanation for this failure is the lack of a thorough understanding of the time‐dependent evolution of the functions of immune cells after a major cardiovascular event. Failure to account for this temporal plasticity in cell function may reduce the efficacy of immunomodulatory approaches that target cardiac repair. This review is concerned with how the functions of different immune cells change with time following an MI. Improved understanding of the temporal changes in immune cell function is important for the future development of effective and targeted treatments for preventing heart failure after MI.

CREB and C/EBP β signaling pathways are modulated during inflammation and also targeted by Bacillus anthracis edema toxin (ET), but how these factors individually and jointly contribute to changes in immune cell function is poorly understood. Using CRISPR/Cas9 gene editing, macrophage cell lines lacking CREB and isoforms of C/EBP β were generated and analyzed for changes in responses to LPS, ET, and IL-4. Macrophages lacking C/EBP β suppressed induction of IL-10 and Arg1, while IL-6 was increased in these cells following exposure to LPS. Examination of C/EBP β isoforms indicated the 38 kDa isoform was necessary for the expression of IL-10 and Arg1. ChIP-Seq analysis of CREB and C/EBP β binding to targets on the chromosome of human PBMC identified several regions where both factors overlapped in their binding, suggesting similar gene targeting or cooperative effects. Based on the ChIP-Seq data, a panel of previously unknown targets of CREB and C/EBP β was identified and includes genes such as VNN2, GINS4, CTNNBL1, and SULF2. Isoforms of a transcriptional corepressor, transducin-like enhancer of Split (TLE), were also found to have CREB and C/EBP β binding their promoter and were up regulated by ET. Finally, we explore a possible layer of C/EBP β regulation by a protein complex consisting of adenomatous polyposis coli (APC) and PKA. Collectively, these data provide new insights into the role of CREB and C/EBP β as immunosignaling regulators and targets of an important bacterial virulence factor.

The increasing prevalence of childhood obesity is an urgent public health crisis. Children with obesity (≥ 95th percentile BMI for age and sex) are at an increased risk for developing asthma as well as cardiometabolic and autoimmune disorders. However, the mechanisms by which excess weight impacts immune function in children is incompletely understood. To address this gap in knowledge, we assessed changes in immune cell function in obese children before and after bariatric surgery, a procedure used to facilitate weight loss in patients with severe obesity. We integrated high dimensional spectral flow cytometry, in vitro functional assays, and single-cell RNA-sequencing of intra-operative visceral adipose tissue and PBMCs to find that CD8+ T cells from obese children, compared to age-matched controls, had increased expression of TOX and inhibitory receptors (PD-1, LAG-3). This dysfunctional phenotype was persistent 6-18 months after surgery, despite significant weight loss (average change in BMI of 17%). Moreover, baseline CD8+ T cells from obese patients exhibit impaired proliferation and altered metabolic profiles following TCR stimulation. Future work will determine functional differences in the peripheral T cells of bariatric surgery patients at baseline compared to 6-18 months post-surgery and assess the local immune landscape within visceral adipose depots from obese compared to non-obese children.

RP-1 has been reported to provide protection against lethal gamma-irradiation in mice. The present study was undertaken to understand its mechanism of action, especially with respect to modulation of radiation-induced changes in immune cell function, plasma antioxidant potential, cell cycle perturbations, apoptosis in mouse bone marrow cells, and micronuclei frequency in mice reticulocytes. 2 Gy reduced mitogenic response of splenic lymphocytes significantly at 48 h. Pre-irradiation RP-1 treatment significantly countered the radiation-induced loss of splenocyte proliferation. RP-1 treatment, with or without radiation, suppressed macrophage activation as compared to control. Irradiation decreased plasma antioxidant status significantly (p < 0.05) at 1 and 2 h (4.8 +/- 0.224 and 4.9 +/- 0.057 mM Fe2+) as compared to control (6.29 +/- 0.733 mM Fe2+) that was countered by RP-1 pre-treatment significantly (p < 0.05). RP-1 and irradiation individually caused G2 delay in bone marrow cells. RP-1 pre-treatment augmented radiation-induced G2 delay and elicited significant (p < 0.05) recovery in S-phase fraction at 48 h in comparison to irradiated group. Radiation-induced apoptosis (3%) was significantly higher than the control. RP-1 pre-treatment further enhanced apoptosis frequency (7.2%) in bone marrow cells. RP-1 pre-treatment significantly (p < 0.05) reduced (1.23%) the radiation-induced MN frequency (2.9%) observed at 48 h post-irradiation interval. Since the radioprotective manifestation of RP-1 is mediated through multiple mechanisms, needs further investigation.

BackgroundCurrent needle-based vaccination for respiratory viruses is ineffective at producing sufficient, long-lasting local immunity in the elderly. Direct pulmonary delivery to the resident local pulmonary immune cells can create long-term mucosal responses. However, criteria for drug vehicle design rules that can overcome age-specific changes in immune cell functions have yet to be established.ResultsHere, in vivo charge-based nanoparticle (NP) uptake was compared in mice of two age groups (2- and 16-months) within the four notable pulmonary antigen presenting cell (APC) populations: alveolar macrophages (AM), interstitial macrophages (IM), CD103+ dendritic cells (DCs), and CD11b+ DCs. Both macrophage populations exhibited preferential uptake of anionic nanoparticles but showed inverse rates of phagocytosis between the AM and IM populations across age. DC populations demonstrated preferential uptake of cationic nanoparticles, which remarkably did not significantly change in the aged group. Further characterization of cell phenotypes post-NP internalization demonstrated unique surface marker expression and activation levels for each APC population, showcasing heightened DC inflammatory response to NP delivery in the aged group.ConclusionThe age of mice demonstrated significant preferences in the charge-based NP uptake in APCs that differed greatly between macrophages and DCs. Carefully balance of the targeting and activation of specific types of pulmonary APCs will be critical to produce efficient, age-based vaccines for the growing elderly population.Graphical

In a study of voluntary immunomodulation, 45 subjects were assigned either to a control group or one of two experimental groups. All groups had blood and saliva samples collected before and after either a 30 minute rest condition (Control group) or a 30 minute cyberphysiologic strategy (Experimental groups) to increase neutrophil adherence. These samples were analyzed on a range of immunologic measurements including neutrophil adherence. The second experimental group practiced a cyberphysiologic strategy two weeks prior to the experimental session. Subjects in each group returned to repeat their exercise in a second session the following week. Analysis of all immune measurements revealed statistical significance for changes in neutrophil adherence. These studies suggest that such strategies may be used to effect changes in immune cell functions. Analysis further revealed that those subjects with prior cyberphysiologic training were able, by the second session, to induce a significant increase in neutrophil adherence.

Type 2 diabetes (T2D) is an inflammatory disease promoted by changes in immune cell function. Monocytes and T cells play roles in T2D inflammation as evidenced, in part, by our demonstration of pro-inflammatory T cell subset skewing in T2D patients. However, the role of cross-talk amongst immune system cells in T2D is poorly unstudied. PBMC depletion studies showed that T2D patient T cells, unlike non-diabetic (ND) donor cells, required monocytes to maintain IL-17 hyper-secretion. In contrast, B cell depletion did not affect T2D Th17 function, but surprisingly, B cell depletion from non-diabetic (ND) mixed cell cultures significantly elevated Th17 function. Based on the role B cell IL-10 plays in inflammation resolution, we tested the hypothesis that IL-10 regulates immune cell cross-talk to block Th17 function. IL-10 addition to B cell-depleted PBMCs decreased IL-17 to “healthy” levels in both ND and T2D samples, indicating that both cell types are physiologically capable of interpreting the anti-inflammatory signals from B cells despite the presence of pro-inflammatory monocytes in T2D samples. Critically, unlike ND B cells, T2D B cells fail to secrete IL-10, thus lack of B cell IL-10 in T2D samples compounds elevated pro-Th17 cytokine production by monocytes. We conclude that IL-10 can block monocyte-mediated increases in Th17 function, and that lack of B cell IL-10 in T2D associates with IL-17-induced inflammation thus metabolic imbalance.