Early-life dietary exposures mediate persistent shifts in the gut microbiome and visceral fat metabolism.

In utero dietary exposures are linked to the development of metabolic syndrome in adult offspring. These dietary exposures can potentially impact gut microbial composition and offspring metabolic health. Female BALB/c mice were administered a lard, lard + flaxseed oil, high sugar, or control diet 4 wk before mating, throughout mating, pregnancy, and lactation. Female offspring were offered low-fat control diet at weaning. Fecal 16S sequencing was performed. Untargeted metabolomics was performed on visceral adipose tissue (VAT) of adult female offspring. Immunohistochemistry was used to determine adipocyte size, VAT collagen deposition, and macrophage content. Hippurate was administered via weekly intraperitoneal injections to low-fat and high-fat diet-fed female mice and VAT fibrosis and collagen 1A (COL1A) were assessed by immunohistochemistry. Lard diet exposure was associated with elevated body and VAT weight and dysregulated glucose metabolism. Lard + flaxseed oil attenuated these effects. Lard diet exposures were associated with increased adipocyte diameter and VAT macrophage count. Lard + flaxseed oil reduced adipocyte diameter and fibrosis compared with the lard diet. Hippurate-associated bacteria were influenced by lard versus lard + flax exposures that persisted to adulthood. VAT hippurate was increased in lard + flaxseed oil compared with lard diet. Hippurate supplementation mitigated VAT fibrosis pathology. Maternal high-fat lard diet consumption resulted in long-term metabolic and gut microbiome programming in offspring, impacting VAT inflammation and fibrosis, and was associated with reduced VAT hippurate content. These traits were not observed in maternal high-fat lard + flaxseed oil diet-exposed offspring. Hippurate supplementation reduced VAT fibrosis. These data suggest that detrimental effects of early-life high-fat lard diet exposure can be attenuated by dietary omega-3 polyunsaturated fatty acid supplementation.

Investigating the role of endogenous estrogens, hormone replacement therapy, and blockade of estrogen receptor-α activity on breast metabolic signaling.

PURPOSE: Menopause is associated with an increased risk of estrogen receptor-positive (ER +) breast cancer. To characterize the metabolic shifts associated with reduced estrogen bioavailability on breast tissue, metabolomics was performed from ovary-intact and ovariectomized (OVX) female non-human primates (NHP). The effects of exogenous estrogen administration or estrogen receptor blockade (tamoxifen treatment) on menopause-induced metabolic changes were also investigated. METHODS: Bilateral ovariectomies were performed on female cynomolgus macaques (Macaca fascicularis) to model menopause. OVX NHP were then divided into untreated (n = 13), conjugated equine estrogen (CEE)-treated (n= 13), or tamoxifen-treated (n = 13) subgroups and followed for 3 years. Aged-matched ovary-intact female NHP (n = 12) were used as a premenopausal comparison group. Metabolomics was performed on snap-frozen breast tissue. RESULTS: Changes in several different metabolic biochemicals were noted, particularly in glucose and fatty acid metabolism. Specifically, glycolytic, Krebs cycle, acylcarnitines, and phospholipid metabolites were elevated in breast tissue from ovary-intact NHP and OVX + CEE in relation to the OVX and OVX + tamoxifen group. In contrast, treatment with CEE and tamoxifen decreased several cholesterol metabolites, compared to the ovary-intact and OVX NHP. These changes were accompanied by elevated bile acid metabolites in the ovary-intact group. CONCLUSION: Alterations in estrogen bioavailability are associated with changes in the mammary tissue metabolome, particularly in glucose and fatty acid metabolism. Changes in these pathways may represent a bioenergetic shift in gland metabolism at menopause that may affect breast cancer risk.

Functions of Thrombospondin-1 in the Tumor Microenvironment.

The identification of thrombospondin-1 as an angiogenesis inhibitor in 1990 prompted interest in its role in cancer biology and potential as a therapeutic target. Decreased thrombospondin-1 mRNA and protein expression are associated with progression in several cancers, while expression by nonmalignant cells in the tumor microenvironment and circulating levels in cancer patients can be elevated. THBS1 is not a tumor suppressor gene, but the regulation of its expression in malignant cells by oncogenes and tumor suppressor genes mediates some of their effects on carcinogenesis, tumor progression, and metastasis. In addition to regulating angiogenesis and perfusion of the tumor vasculature, thrombospondin-1 limits antitumor immunity by CD47-dependent regulation of innate and adaptive immune cells. Conversely, thrombospondin-1 is a component of particles released by immune cells that mediate tumor cell killing. Thrombospondin-1 differentially regulates the sensitivity of malignant and nonmalignant cells to genotoxic stress caused by radiotherapy and chemotherapy. The diverse activities of thrombospondin-1 to regulate autophagy, senescence, stem cell maintenance, extracellular vesicle function, and metabolic responses to ischemic and genotoxic stress are mediated by several cell surface receptors and by regulating the functions of several secreted proteins. This review highlights progress in understanding thrombospondin-1 functions in cancer and the challenges that remain in harnessing its therapeutic potential.

Thrombospondin-1 interactions regulate eicosanoid metabolism and signaling in cancer-related inflammation.

The metabolism of arachidonic acid and other polyunsaturated fatty acids produces eicosanoids, a family of biologically active lipids that are implicated in homeostasis and in several pathologies that involve inflammation. Inflammatory processes mediated by eicosanoids promote carcinogenesis by exerting direct effects on cancer cells and by affecting the tumor microenvironment. Therefore, understanding how eicosanoids mediate cancer progression may lead to better approaches and chemopreventive strategies for the treatment of cancer. The matricellular protein thrombospondin-1 is involved in processes that profoundly regulate inflammatory pathways that contribute to carcinogenesis and metastatic spread. This review focuses on interactions of thrombospondin-1 and eicosanoids in the microenvironment that promote carcinogenesis and how the microenvironment can be targeted for cancer prevention to increase curative responses of cancer patients.

Endoplasmic Reticulum Stress Pathway, the Unfolded Protein Response, Modulates Immune Function in the Tumor Microenvironment to Impact Tumor Progression and Therapeutic Response.

Despite advances in cancer therapy, several persistent issues remain. These include cancer recurrence, effective targeting of aggressive or therapy-resistant cancers, and selective treatments for transformed cells. This review evaluates the current findings and highlights the potential of targeting the unfolded protein response to treat cancer. The unfolded protein response, an evolutionarily conserved pathway in all eukaryotes, is initiated in response to misfolded proteins accumulating within the lumen of the endoplasmic reticulum. This pathway is initially cytoprotective, allowing cells to survive stressful events; however, prolonged activation of the unfolded protein response also activates apoptotic responses. This balance is key in successful mammalian immune response and inducing cell death in malignant cells. We discuss how the unfolded protein response affects cancer progression, survival, and immune response to cancer cells. The literature shows that targeting the unfolded protein response as a monotherapy or in combination with chemotherapy or immunotherapies increases the efficacy of these drugs; however, systemic unfolded protein response targeting may yield deleterious effects on immune cell function and should be taken into consideration. The material in this review shows the promise of both approaches, each of which merits further research.

Doxorubicin-Induced Myocardial Fibrosis Involves the Neurokinin-1 Receptor and Direct Effects on Cardiac Fibroblasts.

BACKGROUND: Cancer patients receiving anthracycline-based chemotherapy (Anth-bC) may experience early cardiac fibrosis, which could be an important contributing mechanism to the development of impaired left ventricular (LV) function. Substance P, a neuropeptide that predominantly acts via the neurokinin 1 receptor (NK-1R), contributes to adverse myocardial remodelling and fibrosis in other cardiomyopathies. We sought to determine if NK-1R blockade is effective against doxorubicin (Dox - a frequently used Anth-bC)-induced cardiac fibrosis and cardiomyocyte apoptosis. In addition, we explored the direct effects of Dox on cardiac fibroblasts. METHODS: Male Sprague-Dawley rats were randomised to receive saline, six cycles of Dox (1.5mg Dox/kg/cycle) or Dox with an NK-1R antagonist (L732138, 5mg/kg/daily through Dox treatment). At 8 weeks after the initial dose of Dox, LV function and histopathological myocardial fibrosis and cell apoptosis were assessed. Collagen secretion was measured in vitro to test direct Dox activation of cardiac fibroblasts. RESULTS: Rats undergoing Dox treatment (9mg/kg cumulative dose) developed cardiac fibrosis and cardiomyocyte apoptosis. NK-1R blockade partially mitigated cardiac fibrosis while completely preventing cardiomyocyte apoptosis. This resulted in improved diastolic function. Furthermore, we found that Dox had direct effects on cardiac fibroblasts to cause increased collagen production and enhanced cell survival. CONCLUSIONS: This study demonstrates that cardiac fibrosis induced by Anth-bC can be reduced by NK-1R blockade. The residual fibrotic response is likely due to direct Dox effects on cardiac fibroblasts to produce collagen.

Combination of anthracyclines and anti-CD47 therapy inhibit invasive breast cancer growth while preventing cardiac toxicity by regulation of autophagy.

BACKGROUND: A perennial challenge in systemic cytotoxic cancer therapy is to eradicate primary tumors and metastatic disease while sparing normal tissue from off-target effects of chemotherapy. Anthracyclines such as doxorubicin are effective chemotherapeutic agents for which dosing is limited by development of cardiotoxicity. Our published evidence shows that targeting CD47 enhances radiation-induced growth delay of tumors while remarkably protecting soft tissues. The protection of cell viability observed with CD47 is mediated autonomously by activation of protective autophagy. However, whether CD47 protects cancer cells from cytotoxic chemotherapy is unknown. METHODS: We tested the effect of CD47 blockade on cancer cell survival using a 2-dimensional high-throughput cell proliferation assay in 4T1 breast cancer cell lines. To evaluate blockade of CD47 in combination with chemotherapy in vivo, we employed the 4T1 breast cancer model and examined tumor and cardiac tissue viability as well as autophagic flux. RESULTS: Our high-throughput screen revealed that blockade of CD47 does not interfere with the cytotoxic activity of anthracyclines against 4T1 breast cancer cells. Targeting CD47 enhanced the effect of doxorubicin chemotherapy in vivo by reducing tumor growth and metastatic spread by activation of an anti-tumor innate immune response. Moreover, systemic suppression of CD47 protected cardiac tissue viability and function in mice treated with doxorubicin. CONCLUSIONS: Our experiments indicate that the protective effects observed with CD47 blockade are mediated through upregulation of autophagic flux. However, the absence of CD47 in did not elicit a protective effect in cancer cells, but it enhanced macrophage-mediated cancer cell cytolysis. Therefore, the differential responses observed with CD47 blockade are due to autonomous activation of protective autophagy in normal tissue and enhancement immune cytotoxicity against cancer cells.

Mitochondrial autophagosomes as a mechanism of drug resistance in breast carcinoma.

We have previously described the process by which mitochondria donate their membranes for the formation of autophagosomes, and in this study we show that the same process could be involved in drug sequestration and exocytosis resulting in multidrug-resistant cancerous cells. We examine the implications of mitochondrial vesicle formation of mitoautophagosomes (MAPS) in response to the cytotoxic drug MKT-077, which targets mortalin, in a drug-resistant breast carcinoma cell line overexpressing P-glycoprotein (P-gp). The breast cancer cell line MCF-7Adr is derived from MCF-7, but differs from its ancestral line in tolerance of MKT-077-induced mitochondrial toxicity. Our ultrastructural observations suggest that autophagy in the MCF-7Adr cells entails regional sequestration of MKT077 in multilamellar LC3-labeled MAPS, which then separate from their mitochondria, and fuse with or engulf each other. MAPS appeared to be migrating through the cytoplasm and fusing with the plasma membrane, thus carrying out exocytotic secretion. This mechanism, which seems ineffective in the ancestral cell line, provides a resistance mechanism for MKT-077 by enhancing the efflux process of the cells. After 8 hr of MKT-077 exposure, a fraction of the resistant cells appeared viable and contained larger number of smaller sized mitochondria. Mitoautophagosomes, therefore, provide a potentially novel model for multidrug resistance in cancerous cells and may contribute to the P-gp efflux process.

CD47 signaling pathways controlling cellular differentiation and responses to stress.

CD47 is a widely expressed integral membrane protein that serves as the counter-receptor for the inhibitory phagocyte receptor signal-regulatory protein-α (SIRPα) and as a signaling receptor for the secreted matricellular protein thrombospondin-1. Recent studies employing mice and somatic cells lacking CD47 have revealed important pathophysiological functions of CD47 in cardiovascular homeostasis, immune regulation, resistance of cells and tissues to stress and chronic diseases of aging including cancer. With the emergence of experimental therapeutics targeting CD47, a more thorough understanding of CD47 signal transduction is essential. CD47 lacks a substantial cytoplasmic signaling domain, but several cytoplasmic binding partners have been identified, and lateral interactions of CD47 with other membrane receptors play important roles in mediating signaling resulting from the binding of thrombospondin-1. This review addresses recent advances in identifying the lateral binding partners, signal transduction pathways and downstream transcription networks regulated through CD47 in specific cell lineages. Major pathways regulated by CD47 signaling include calcium homeostasis, cyclic nucleotide signaling, nitric oxide and hydrogen sulfide biosynthesis and signaling and stem cell transcription factors. These pathways and other undefined proximal mediators of CD47 signaling regulate cell death and protective autophagy responses, mitochondrial biogenesis, cell adhesion and motility and stem cell self-renewal. Although thrombospondin-1 is the best characterized agonist of CD47, the potential roles of other members of the thrombospondin family, SIRPα and SIRPγ binding and homotypic CD47 interactions as agonists or antagonists of signaling through CD47 should also be considered.

CD47 Receptor Globally Regulates Metabolic Pathways That Control Resistance to Ionizing Radiation.

Modulating tissue responses to stress is an important therapeutic objective. Oxidative and genotoxic stresses caused by ionizing radiation are detrimental to healthy tissues but beneficial for treatment of cancer. CD47 is a signaling receptor for thrombospondin-1 and an attractive therapeutic target because blocking CD47 signaling protects normal tissues while sensitizing tumors to ionizing radiation. Here we utilized a metabolomic approach to define molecular mechanisms underlying this radioprotective activity. CD47-deficient cells and cd47-null mice exhibited global advantages in preserving metabolite levels after irradiation. Metabolic pathways required for controlling oxidative stress and mediating DNA repair were enhanced. Some cellular energetics pathways differed basally in CD47-deficient cells, and the global declines in the glycolytic and tricarboxylic acid cycle metabolites characteristic of normal cell and tissue responses to irradiation were prevented in the absence of CD47. Thus, CD47 mediates signaling from the extracellular matrix that coordinately regulates basal metabolism and cytoprotective responses to radiation injury.

CD47 signaling regulates the immunosuppressive activity of VEGF in T cells.

Thrombospondin-1 (TSP1) inhibits angiogenesis, in part, by interacting with the ubiquitous cell-surface receptor CD47. In endothelial cells, CD47 interacts directly with vascular endothelial growth factor receptor (VEGFR)-2, and TSP1 inhibits VEGFR2 phosphorylation and signaling by disrupting this association. We show that CD47 similarly associates with and regulates VEGFR2 in T cells. TSP1 inhibits phosphorylation of VEGFR2 and its downstream target Src in wild type but not in CD47-deficient human Jurkat and primary murine T cells. VEGFR2 signaling inhibits proliferation and TCR signaling in wild type T cells. However, ligation of CD47 by TSP1 or loss of CD47 expression reverses some inhibitory effects of VEGF on proliferation and T cell activation. We further found that VEGF and VEGFR2 expression are upregulated in CD47-deficient murine CD4(+) and human Jurkat T cells, and the resulting autocrine VEGFR2 signaling enhances proliferation and some TCR responses in the absence of CD47. Thus, CD47 signaling modulates the ability of VEGF to regulate proliferation and TCR signaling, and autocrine production of VEGF by T cells contributes to this regulation. This provides a mechanism to understand the context-dependent effects of TSP1 and VEGF on T cell activation, and reveals an important role for CD47 signaling in regulating T cell production of the major angiogenic factor VEGF.

Tipping off endothelial tubes: nitric oxide drives tip cells.

Angiogenesis, the formation of new blood vessels from pre-existing vessels, is a complex process that warrants cell migration, proliferation, tip cell formation, ring formation, and finally tube formation. Angiogenesis is initiated by a single leader endothelial cell called "tip cell," followed by vessel elongation by "stalk cells." Tip cells are characterized by their long filopodial extensions and expression of vascular endothelial growth factor receptor-2 and endocan. Although nitric oxide (NO) is an important modulator of angiogenesis, its role in angiogenic sprouting and specifically in tip cell formation is poorly understood. The present study tested the role of endothelial nitric oxide synthase (eNOS)/NO/cyclic GMP (cGMP) signaling in tip cell formation. In primary endothelial cell culture, about 40% of the tip cells showed characteristic sub-cellular localization of eNOS toward the anterior progressive end of the tip cells, and eNOS became phosphorylated at serine 1177. Loss of eNOS suppressed tip cell formation. Live cell NO imaging demonstrated approximately 35% more NO in tip cells compared with stalk cells. Tip cells showed increased level of cGMP relative to stalk cells. Further, the dissection of NO downstream signaling using pharmacological inhibitors and inducers indicates that NO uses the sGC/cGMP pathway in tip cells to lead angiogenesis. Taken together, the present study confirms that eNOS/NO/cGMP signaling defines the direction of tip cell migration and thereby initiates new blood vessel formation.

Diet modulates the gut microbiome, metabolism, and mammary gland inflammation to influence breast cancer risk.

Several studies have indicated a strong link between obesity and the risk of breast cancer. Obesity decreases gut microbial biodiversity and modulates Bacteroidetes-to-Firmicutes proportional abundance, suggesting that increased energy-harvesting capacity from indigestible dietary fibers and elevated lipopolysaccharide bioavailability may promote inflammation. To address the limited evidence linking diet-mediated changes in the gut microbiota to breast cancer risk, we aimed to determine how diet affects the microbiome and breast cancer risk. Female 3-week-old BALB/c mice were fed six different diets (control, high-sugar, lard, coconut oil, lard+flaxseed oil, and lard+safflower oil) for 10 weeks. Fecal 16s sequencing was performed for each group. Diet shifted fecal microbiome populations and modulated mammary gland macrophage infiltration. Fecal conditioned media shifted macrophage polarity and inflammation. In our DMBA-induced breast cancer model, diet differentially modulated tumor and mammary gland metabolism. We demonstrated how dietary patterns change metabolic outcomes, and gut microbiota, which may contribute to breast tumor risk. Furthermore, we showed the influence of diet on metabolism, inflammation, and macrophage polarity. This study suggests that dietary-microbiome interactions are key mediators of breast cancer risk.

Study design and methods for the pilot study of muscadine grape extract supplement to improve fatigue among older adult cancer survivors (FOCUS) trial.

INTRODUCTION: Fatigue is a prevalent symptom among both cancer survivors and older adults. Negative consequences of fatigue include increased sedentary behavior, decreased physical activity and function, and lower quality of life. Few pharmacologic interventions improve fatigue. Our preclinical and clinical data show promising effects of a muscadine grape extract supplement (MGES) on oxidative stress, mitochondrial bioenergetics, the microbiome, and the symptom of fatigue. This pilot study seeks to translate these observations to cancer survivorship by testing the preliminary effect of MGE supplementation on older adult cancer survivors with self-reported fatigue. MATERIALS AND METHODS: We designed a double-blinded placebo-controlled pilot study to evaluate preliminary efficacy of MGE supplementation versus placebo on fatigue among older adult cancer survivors (aged ≥65 years) who report baseline fatigue. Sixty-four participants will be enrolled and randomized 1:1 to twice daily MGES (four tablets twice daily) versus placebo for 12 weeks. The primary outcome is change in Patient-Reported Outcomes Measurement Information System (PROMIS) Fatigue score from baseline to 12 weeks. Secondary outcomes are change in self-reported physical function, physical fitness (6-min walk test), self-reported physical activity, global quality of life (QOL), and the Fried frailty index. Correlative biomarker assays will assess changes in 8-hydroxy-2 deoxyguanosine, peripheral blood mitochondrial function, inflammatory markers, and the gut microbiome. DISCUSSION: This pilot study builds on preclinical and clinical observations to estimate effects of MGE supplementation on fatigue, physical function, QOL, and biologic correlates in older adult cancer survivors. Trial registration #: CT.govNCT04495751; IND 152908.

Anti-CD47 immunotherapy as a therapeutic strategy for the treatment of breast cancer brain metastasis.

The presence of cell surface protein CD47 allows cancer cells to evade innate and adaptive immune surveillance resulting in metastatic spread. CD47 binds to and activates SIRPα on the surface of myeloid cells, inhibiting their phagocytic activity. On the other hand, CD47 binds the matricellular protein Thrombospondin-1, limiting T-cell activation. Thus, blocking CD47 is a potential therapeutic strategy for preventing brain metastasis. To test this hypothesis, breast cancer patient biopsies were stained with antibodies against CD47 to determine differences in protein expression. An anti-CD47 antibody was used in a syngeneic orthotopic triple-negative breast cancer model, and CD47 null mice were used in a breast cancer brain metastasis model by intracardiac injection of the E0771-Br-Luc cell line. Immunohistochemical staining of patient biopsies revealed an 89% increase in CD47 expression in metastatic brain tumors compared to normal adjacent tissue (p ≤ 0.05). Anti-CD47 treatment in mice bearing brain metastatic 4T1br3 orthotopic tumors reduced tumor volume and tumor weight by over 50% compared to control mice (p ≤ 0.05) and increased IBA1 macrophage/microglia marker 5-fold in tumors compared to control (p ≤ 0.05). Additionally, CD47 blockade increased the M1/M2 macrophage ratio in tumors 2.5-fold (p ≤ 0.05). CD47 null mice had an 89% decrease in metastatic brain burden (p ≤ 0.05) compared to control mice in a brain metastasis model. Additionally, RNA sequencing revealed several uniquely expressed genes and significantly enriched genes related to tissue development, cell death, and cell migration tumors treated with anti-CD47 antibodies. Thus, demonstrating that CD47 blockade affects cancer cell and tumor microenvironment signaling to limit metastatic spread and may be an effective therapeutic for triple-negative breast cancer brain metastasis.

In Vitro Cell Impedance Assay to Examine Antigen-Specific T-Cell-Mediated Melanoma Cell Killing to Support Cancer Immunotherapy Drug Discovery.

The design of cancer immunotherapy drugs is essential for the continued investigation of novel drug regimens to improve responses and increase the survival of cancer patients. Methods to examine the interaction of effector immune cells with target cancer cells are limited by labor-intensive labeling that can be examined at specific time points. In this report, we examine an antigen-dependent model of effector cytotoxic (CD8+) T-cell-mediated cytotoxicity of target murine melanoma cells using a real-time cell impedance assay. The real-time monitoring allows measurement of viability and kinetics, allowing for a better understanding of effector/target cell interactions to support drug discovery.

Mitigation of doxorubicin-induced cardiotoxicity with an H2O2-Activated, H2S-Donating hybrid prodrug.

Doxorubicin (DOX) is one of the most effective anticancer agents in clinical oncology. Its continued use, however, is severely limited by its dose-dependent cardiotoxicity which stems, in part, from its overproduction of reactive oxygen species (ROS) and often manifests itself as full-blown cardiomyopathy in patients, years after the cessation of treatment. Therefore, identifying DOX analogs, or prodrugs, with a diminished cardiotoxic profile is highly desirable. Herein, we describe a novel, H2O2-responsive DOX hybrid codrug (mutual prodrug) that has been rationally designed to concurrently liberate hydrogen sulfide (H2S), a purported cardioprotectant with anticancer activity, in an effort to maintain the antitumor effects of DOX while simultaneously reducing its cardiotoxic side effects. Experiments with cardiomyoblast cells in culture demonstrated a rapid accumulation of prodrug into the cells, but diminished apoptotic effects compared with DOX, dependent upon its release of H2S. Cells treated with the prodrug exhibited significantly higher Nrf2 activation relative to DOX-treated cells. Preliminary indications, using a mouse triple-negative breast cancer cell line sensitive to DOX treatment, are that the prodrug maintains considerable toxicity against the tumor-inducing cell line, suggesting significant promise for this prodrug as a cardioprotective chemotherapeutic to replace DOX.

Immuno-reactive cancer organoid model to assess effects of the microbiome on cancer immunotherapy.

Immune checkpoint blockade (ICB) therapy has demonstrated good efficacy in many cancer types. In cancers such as non-resectable advanced or metastatic triple-negative breast cancer (TNBC), it has recently been approved as a promising treatment. However, clinical data shows overall response rates (ORRs) from ~ 3-40% in breast cancer patients, depending on subtype, previous treatments, and mutation status. Composition of the host-microbiome has a significant role in cancer development and therapeutic responsiveness. Some bacterial families are conducive to oncogenesis and progression, while others aid innate and therapeutically induced anti-tumor immunity. Modeling microbiome effects on anti-tumor immunity in ex vivo systems is challenging, forcing the use of in vivo models, making it difficult to dissect direct effects on immune cells from combined effects on tumor and immune cells. We developed a novel immune-enhanced tumor organoid (iTO) system to study factors affecting ICB response. Using the 4T1 TNBC murine cell line and matched splenocytes, we demonstrated ICB-induced response. Further administration of bacterial-derived metabolites from species found in the immunomodulatory host-microbiome significantly increased ICB-induced apoptosis of tumor cells and altered immune cell receptor expression. These outcomes represent a method to isolate individual factors that alter ICB response and streamline the study of microbiome effects on ICB efficacy.

Metabolic Implications of Immune Checkpoint Proteins in Cancer.

Expression of immune checkpoint proteins restrict immunosurveillance in the tumor microenvironment; thus, FDA-approved checkpoint inhibitor drugs, specifically PD-1/PD-L1 and CTLA-4 inhibitors, promote a cytotoxic antitumor immune response. Aside from inflammatory signaling, immune checkpoint proteins invoke metabolic reprogramming that affects immune cell function, autonomous cancer cell bioenergetics, and patient response. Therefore, this review will focus on the metabolic alterations in immune and cancer cells regulated by currently approved immune checkpoint target proteins and the effect of costimulatory receptor signaling on immunometabolism. Additionally, we explore how diet and the microbiome impact immune checkpoint blockade therapy response. The metabolic reprogramming caused by targeting these proteins is essential in understanding immune-related adverse events and therapeutic resistance. This can provide valuable information for potential biomarkers or combination therapy strategies targeting metabolic pathways with immune checkpoint blockade to enhance patient response.

Intestinal Microbiota Influence Doxorubicin Responsiveness in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is highly aggressive with a poor 5-year survival rate. Targeted therapy options are limited and most TNBC patients are treated with chemotherapy. This study aimed to determine whether doxorubicin (Dox) shifts the gut microbiome and whether gut microbiome populations influence chemotherapeutic responsiveness. Female BALB/c mice (n = 115) were injected with 4T1-luciferase cells (a murine syngeneic TNBC model) and treated with Dox and/or antibiotics, high-fat diet-derived fecal microbiota transplant (HFD-FMT), or exogenous lipopolysaccharide (LPS). Metagenomic sequencing was performed on fecal DNA samples. Mice that received Dox were stratified into Dox responders or Dox nonresponders. Mice from the Dox responders and antibiotics + Dox groups displayed reduced tumor weight and metastatic burden. Metagenomic analysis showed that Dox was associated with increased Akkermansia muciniphila proportional abundance. Moreover, Dox responders showed an elevated proportional abundance of Akkermansia muciniphila prior to Dox treatment. HFD-FMT potentiated tumor growth and decreased Dox responsiveness. Indeed, lipopolysaccharide, a structural component of Gram-negative bacteria, was increased in the plasma of Dox nonresponders and FMT + Dox mice. Treatment with exogenous LPS increases intestinal inflammation, reduces Dox responsiveness, and increases lung metastasis. Taken together, we show that modulating the gut microbiota through antibiotics, HFD-FMT, or by administering LPS influenced TNBC chemotherapy responsiveness, lung metastasis, and intestinal inflammation.