Obesity induces PD-1 on macrophages to suppress anti-tumour immunity.

Obesity is a leading risk factor for progression and metastasis of many cancers1,2, yet can in some cases enhance survival3-5 and responses to immune checkpoint blockade therapies, including anti-PD-1, which targets PD-1 (encoded by PDCD1), an inhibitory receptor expressed on immune cells6-8. Although obesity promotes chronic inflammation, the role of the immune system in the obesity-cancer connection and immunotherapy remains unclear. It has been shown that in addition to T cells, macrophages can express PD-19-12. Here we found that obesity selectively induced PD-1 expression on tumour-associated macrophages (TAMs). Type I inflammatory cytokines and molecules linked to obesity, including interferon-γ, tumour necrosis factor, leptin, insulin and palmitate, induced macrophage PD-1 expression in an mTORC1- and glycolysis-dependent manner. PD-1 then provided negative feedback to TAMs that suppressed glycolysis, phagocytosis and T cell stimulatory potential. Conversely, PD-1 blockade increased the level of macrophage glycolysis, which was essential for PD-1 inhibition to augment TAM expression of CD86 and major histocompatibility complex I and II molecules and ability to activate T cells. Myeloid-specific PD-1 deficiency slowed tumour growth, enhanced TAM glycolysis and antigen-presentation capability, and led to increased CD8+ T cell activity with a reduced level of markers of exhaustion. These findings show that obesity-associated metabolic signalling and inflammatory cues cause TAMs to induce PD-1 expression, which then drives a TAM-specific feedback mechanism that impairs tumour immune surveillance. This may contribute to increased cancer risk yet improved response to PD-1 immunotherapy in obesity.

VHL loss reprograms the immune landscape to promote an inflammatory myeloid microenvironment in renal tumorigenesis.

Clear cell renal cell carcinoma (ccRCC) is characterized by dysregulated hypoxia signaling and a tumor microenvironment (TME) highly enriched in myeloid and lymphoid cells. Loss of the von Hippel Lindau (VHL) gene is a critical early event in ccRCC pathogenesis and promotes stabilization of HIF. Whether VHL loss in cancer cells affects immune cells in the TME remains unclear. Using Vhl WT and Vhl-KO in vivo murine kidney cancer Renca models, we found that Vhl-KO tumors were more infiltrated by immune cells. Tumor-associated macrophages (TAMs) from Vhl-deficient tumors demonstrated enhanced in vivo glucose consumption, phagocytosis, and inflammatory transcriptional signatures, whereas lymphocytes from Vhl-KO tumors showed reduced activation and a lower response to anti-programmed cell death 1 (anti-PD-1) therapy in vivo. The chemokine CX3CL1 was highly expressed in human ccRCC tumors and was associated with Vhl deficiency. Deletion of Cx3cl1 in cancer cells decreased myeloid cell infiltration associated with Vhl loss to provide a mechanism by which Vhl loss may have contributed to the altered immune landscape. Here, we identify cancer cell-specific genetic features that drove environmental reprogramming and shaped the tumor immune landscape, with therapeutic implications for the treatment of ccRCC.

Differential Effects of Glutamine Inhibition Strategies on Antitumor CD8 T Cells.

Activated T cells undergo metabolic reprogramming to meet anabolic, differentiation, and functional demands. Glutamine supports many processes in activated T cells, and inhibition of glutamine metabolism alters T cell function in autoimmune disease and cancer. Multiple glutamine-targeting molecules are under investigation, yet the precise mechanisms of glutamine-dependent CD8 T cell differentiation remain unclear. We show that distinct strategies of glutamine inhibition by glutaminase-specific inhibition with small molecule CB-839, pan-glutamine inhibition with 6-diazo-5-oxo-l-norleucine (DON), or by glutamine-depleted conditions (No Q) produce distinct metabolic differentiation trajectories in murine CD8 T cells. T cell activation with CB-839 treatment had a milder effect than did DON or No Q treatment. A key difference was that CB-839-treated cells compensated with increased glycolytic metabolism, whereas DON and No Q-treated cells increased oxidative metabolism. However, all glutamine treatment strategies elevated CD8 T cell dependence on glucose metabolism, and No Q treatment caused adaptation toward reduced glutamine dependence. DON treatment reduced histone modifications and numbers of persisting cells in adoptive transfer studies, but those T cells that remained could expand normally upon secondary Ag encounter. In contrast, No Q-treated cells persisted well yet demonstrated decreased secondary expansion. Consistent with reduced persistence, CD8 T cells activated in the presence of DON had reduced ability to control tumor growth and reduced tumor infiltration in adoptive cell therapy. Overall, each approach to inhibit glutamine metabolism confers distinct effects on CD8 T cells and highlights that targeting the same pathway in different ways can elicit opposing metabolic and functional outcomes.

Acly Deficiency Enhances Myelopoiesis through Acetyl Coenzyme A and Metabolic-Epigenetic Cross-Talk.

Hematopoiesis integrates cytokine signaling, metabolism, and epigenetic modifications to regulate blood cell generation. These processes are linked, as metabolites provide essential substrates for epigenetic marks. In this study, we demonstrate that ATP citrate lyase (Acly), which metabolizes citrate to generate cytosolic acetyl-CoA and is of clinical interest, can regulate chromatin accessibility to limit myeloid differentiation. Acly was tested for a role in murine hematopoiesis by small-molecule inhibition or genetic deletion in lineage-depleted, c-Kit-enriched hematopoietic stem and progenitor cells from Mus musculus. Treatments increased the abundance of cell populations that expressed the myeloid integrin CD11b and other markers of myeloid differentiation. When single-cell RNA sequencing was performed, we found that Acly inhibitor-treated hematopoietic stem and progenitor cells exhibited greater gene expression signatures for macrophages and enrichment of these populations. Similarly, the single-cell assay for transposase-accessible chromatin sequencing showed increased chromatin accessibility at genes associated with myeloid differentiation, including CD11b, CD11c, and IRF8. Mechanistically, Acly deficiency altered chromatin accessibility and expression of multiple C/EBP family transcription factors known to regulate myeloid differentiation and cell metabolism, with increased Cebpe and decreased Cebpa and Cebpb. This effect of Acly deficiency was accompanied by altered mitochondrial metabolism with decreased mitochondrial polarization but increased mitochondrial content and production of reactive oxygen species. The bias to myeloid differentiation appeared due to insufficient generation of acetyl-CoA, as exogenous acetate to support alternate compensatory pathways to produce acetyl-CoA reversed this phenotype. Acly inhibition thus can promote myelopoiesis through deprivation of acetyl-CoA and altered histone acetylome to regulate C/EBP transcription factor family activity for myeloid differentiation.

Ojeok-san ameliorates visceral and somatic nociception in a mouse model of colitis induced colorectal cancer.

Cancer patients can develop visceral, somatic, and neuropathic pain, largely due to the malignancy itself and its treatments. Often cancer patients and survivors turn to the use of complementary and alternative medicine (CAM) to alleviate pain and fatigue. Thus, it is necessary to investigate how CAM therapies work as novel analgesics to treat cancer pain. Ojeok-san (OJS) is an herbal formula consisting of seventeen herbs. This herbal formula has been shown to possess anti-inflammatory, immunoregulatory, and analgesic properties. In this study, we examined the potential beneficial effects and mechanism of action of OJS in a preclinical model of colitis-associated colorectal cancer. Male and female C57BL/6J mice were exposed to the carcinogen, azoxymethane (AOM, 10 mg/kg) and a chemical inflammatory driver, dextran sulfate sodium (DSS1-2%), to promote tumorigenesis in the colorectum. OJS was given orally (500, 1000, and 2000 mg/kg) to determine its influence on disease activity, tumor burden, nociception, sedation, Erk signaling, and behavioral and metabolic outcomes. In addition, in vitro studies were performed to assess CT-26 cell viability, dorsal root ganglia (DRG) activation, and bone-marrow-derived macrophage (BMDM) inflammatory response to lipopolysaccharide stimulation after OJS treatment. We found that administration of 2000 mg/kg of OJS was able to mitigate mechanical somatic and visceral nociception via Erk signaling without affecting symptom score and polyp number. Moreover, we discovered that OJS has sedative properties and elicits prolonged total sleeping time in AOM/DSS mice. Our in vitro experiments showed that OJS has the capacity to reduce TNFα gene expression in LPS-stimulated BMDM, but no changes were observed in DRG spike number and CT-26 cell proliferation. Taken together, these data suggest that OJS ameliorates nociception in mice and warrants further examination as a potential CAM therapy to promote analgesia.

Leptin Augments Antitumor Immunity in Obesity by Repolarizing Tumor-Associated Macrophages.

Although obesity can promote cancer, it may also increase immunotherapy efficacy in what has been termed the obesity-immunotherapy paradox. Mechanisms of this effect are unclear, although obesity alters key inflammatory cytokines and can promote an inflammatory state that may modify tumor-infiltrating lymphocytes and tumor-associated macrophage populations. To identify mechanisms by which obesity affects antitumor immunity, we examined changes in cell populations and the role of the proinflammatory adipokine leptin in immunotherapy. Single-cell RNAseq demonstrated that obesity decreased tumor-infiltrating lymphocyte frequencies, and flow cytometry confirmed altered macrophage phenotypes with lower expression of inducible NO synthase and MHC class II in tumors of obese animals. When treated with anti-programmed cell death protein 1 (PD-1) Abs, however, obese mice had a greater absolute decrease in tumor burden than lean mice and a repolarization of the macrophages to inflammatory M1-like phenotypes. Mechanistically, leptin is a proinflammatory adipokine that is induced in obesity and may mediate enhanced antitumor immunity in obesity. To directly test the effect of leptin on tumor growth and antitumor immunity, we treated lean mice with leptin and observed tumors over time. Treatment with leptin, acute or chronic, was sufficient to enhance antitumor efficacy similar to anti-PD-1 checkpoint therapy. Further, leptin and anti-PD-1 cotreatment may enhance antitumor effects consistent with an increase in M1-like tumor-associated macrophage frequency compared with non-leptin-treated mice. These data demonstrate that obesity has dual effects in cancer through promotion of tumor growth while simultaneously enhancing antitumor immunity through leptin-mediated macrophage reprogramming.

A guide to interrogating immunometabolism.

The metabolic charts memorized in early biochemistry courses, and then later forgotten, have come back to haunt many immunologists with new recognition of the importance of these pathways. Metabolites and the activity of metabolic pathways drive energy production, macromolecule synthesis, intracellular signalling, post-translational modifications and cell survival. Immunologists who identify a metabolic phenotype in their system are often left wondering where to begin and what does it mean? Here, we provide a framework for navigating and selecting the appropriate biochemical techniques to explore immunometabolism. We offer recommendations for initial approaches to develop and test metabolic hypotheses and how to avoid common mistakes. We then discuss how to take things to the next level with metabolomic approaches, such as isotope tracing and genetic approaches. By proposing strategies and evaluating the strengths and weaknesses of different methodologies, we aim to provide insight, note important considerations and discuss ways to avoid common misconceptions. Furthermore, we highlight recent studies demonstrating the power of these metabolic approaches to uncover the role of metabolism in immunology. By following the framework in this Review, neophytes and seasoned investigators alike can venture into the emerging realm of cellular metabolism and immunity with confidence and rigour.

Cell-programmed nutrient partitioning in the tumour microenvironment.

Cancer cells characteristically consume glucose through Warburg metabolism1, a process that forms the basis of tumour imaging by positron emission tomography (PET). Tumour-infiltrating immune cells also rely on glucose, and impaired immune cell metabolism in the tumour microenvironment (TME) contributes to immune evasion by tumour cells2-4. However, whether the metabolism of immune cells is dysregulated in the TME by cell-intrinsic programs or by competition with cancer cells for limited nutrients remains unclear. Here we used PET tracers to measure the access to and uptake of glucose and glutamine by specific cell subsets in the TME. Notably, myeloid cells had the greatest capacity to take up intratumoral glucose, followed by T cells and cancer cells, across a range of cancer models. By contrast, cancer cells showed the highest uptake of glutamine. This distinct nutrient partitioning was programmed in a cell-intrinsic manner through mTORC1 signalling and the expression of genes related to the metabolism of glucose and glutamine. Inhibiting glutamine uptake enhanced glucose uptake across tumour-resident cell types, showing that glutamine metabolism suppresses glucose uptake without glucose being a limiting factor in the TME. Thus, cell-intrinsic programs drive the preferential acquisition of glucose and glutamine by immune and cancer cells, respectively. Cell-selective partitioning of these nutrients could be exploited to develop therapies and imaging strategies to enhance or monitor the metabolic programs and activities of specific cell populations in the TME.

Impact of weight loss and partial weight regain on immune cell and inflammatory markers in adipose tissue in male mice.

Weight fluctuations are common among individuals with obesity and are associated with increased morbidity. We examined adipose tissue immune and inflammatory markers in mice following weight loss and partial weight regain. Male C57BL/6 mice were randomized into four groups (n = 8-10/group): low-fat diet for 32 wk (LFD), high-fat diet for 32 wk (HFD), LFD for 28 wk and then changed to a HFD for 4 wk (LFD→H), and HFD for 21 wk and then changed to LFD for 7 wk and then changed to HFD for 4 wk (HFD→L→H). LFD→H and HFD→L→H mice did not differ in body weight, fat mass, or fat percentage; however, these parameters were greater than in LFD (P < 0.05) but lower than in HFD (P < 0.05). HFD→L→H mice had smaller adipocytes than HFD and LFD→H (P < 0.05) but not LFD mice. Expressions of CD11c and CD8a genes were elevated in epididymal fat of HFD→L→H compared with LFD→H and LFD (P < 0.05)mice. However, CD11c was lower in HFD→L→H than in HFD mice (P < 0.05), but there was no difference in CD8a between these groups. TNFα and IFNγ expressions were increased in HFD→L→H compared with LFD and LFD→H mice (P < 0.05), although HFD→L→H had lower expression of these cytokines than HFD (P < 0.05). IL-1ß was greater in HFD→L→H compared with LFD (P < 0.05) but was not different from LFD→H or HFD mice. Monocyte chemoattractant protein-1 was lower (P < 0.05) in HFD→L→H than in LFD→H. These data reinforce the importance of maintaining a body weight in the range that is recommended for optimal health to reduce immune and inflammatory perturbations associated with obesity.NEW & NOTEWORTHY We examined the immune and inflammatory status of adipose tissue in mice after they underwent weight loss followed by partial weight regain. We show an increase in selected immune cells and inflammatory mediators, in high-fat diet-fed mice that had prior exposure to a high-fat diet. Although weight fluctuations appear to exacerbate immune cell abundance and inflammation in adipose tissue, severity is less than in mice that were exposed to sustained high-fat diet feedings.

Targeting Metabolism to Improve the Tumor Microenvironment for Cancer Immunotherapy.

The growing field of immune metabolism has revealed promising indications for metabolic targets to modulate anti-cancer immunity. Combination therapies involving metabolic inhibitors with immune checkpoint blockade (ICB), chemotherapy, radiation, and/or diet now offer new approaches for cancer therapy. However, it remains uncertain how to best utilize these strategies in the context of the complex tumor microenvironment (TME). Oncogene-driven changes in tumor cell metabolism can impact the TME to limit immune responses and present barriers to cancer therapy. These changes also reveal opportunities to reshape the TME by targeting metabolic pathways to favor immunity. Here we explore current strategies that shift immune cell metabolism to pro-inflammatory states in the TME and highlight a need to better replicate physiologic conditions to select targets, clarify mechanisms, and optimize metabolic inhibitors. Unifying our understanding of these pathways and interactions within the heterogenous TME will be instrumental to advance this promising field and enhance immunotherapy.

Prolonged high-fat-diet feeding promotes non-alcoholic fatty liver disease and alters gut microbiota in mice.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) has become an epidemic largely due to the worldwide increase in obesity. While lifestyle modifications and pharmacotherapies have been used to alleviate NAFLD, successful treatment options are limited. One of the main barriers to finding safe and effective drugs for long-term use in NAFLD is the fast initiation and progression of disease in the available preclinical models. Therefore, we are in need of preclinical models that (1) mimic the human manifestation of NAFLD and (2) have a longer progression time to allow for the design of superior treatments. AIM: To characterize a model of prolonged high-fat diet (HFD) feeding for investigation of the long-term progression of NAFLD. METHODS: In this study, we utilized prolonged HFD feeding to examine NAFLD features in C57BL/6 male mice. We fed mice with a HFD (60% fat, 20% protein, and 20% carbohydrate) for 80 wk to promote obesity (Old-HFD group, n = 18). A low-fat diet (LFD) (14% fat, 32% protein, and 54% carbohydrate) was administered for the same duration to age-matched mice (Old-LFD group, n = 15). An additional group of mice was maintained on the LFD (Young-LFD, n = 20) for a shorter duration (6 wk) to distinguish between age-dependent and age-independent effects. Liver, colon, adipose tissue, and feces were collected for histological and molecular assessments. RESULTS: Prolonged HFD feeding led to obesity and insulin resistance. Histological analysis in the liver of HFD mice demonstrated steatosis, cell injury, portal and lobular inflammation and fibrosis. In addition, molecular analysis for markers of endoplasmic reticulum stress established that the liver tissue of HFD mice have increased phosphorylated Jnk and CHOP. Lastly, we evaluated the gut microbial composition of Old-LFD and Old-HFD. We observed that prolonged HFD feeding in mice increased the relative abundance of the Firmicutes phylum. At the genus level, we observed a significant increase in the abundance of Adercreutzia, Coprococcus, Dorea, and Ruminococcus and decreased relative abundance of Turicibacter and Anaeroplasma in HFD mice. CONCLUSION: Overall, these data suggest that chronic HFD consumption in mice can mimic pathophysiological and some microbial events observed in NAFLD patients.

Effects of high fat diet-induced obesity on mammary tumorigenesis in the PyMT/MMTV murine model.

Clinical studies provide strong evidence that obesity and associated adipose tissue (AT) inflammation are risk factors for breast cancer (BrCA); however, mechanistic knowledge of the interaction of obesity, BrCA, and menopausal status has proven to be not only lacking, but contradictory. Obesity-induced inflammation and elevated biosynthesis of estrogens, through aromatase-mediated metabolism of precursors, have been linked with hormone receptor positive (HP) postmenopausal BrCA but not previously associated with premenopausal BrCA risk. Thus, further delineation of the interaction of obesity, inflammation, and aromatase is required for the development of therapeutic treatment options. The purpose of this study was to examine the effect of high fat diet (HFD)-induced inflammation on tumorigenesis in a model of pre and postmenopausal HP BrCA. Female PyMT/MMTV ovary intact and ovariectomized mice were fed low and HFD diets to examine the role of obesity-induced inflammation and hormone production in the development of HP BrCA. Tumor statistics for number, volume, weight, histopathology scoring and gene expression of macrophage and inflammatory mediators were measured in the AT and mammary gland at sacrifice. HFD feedings of ovary intact mice resulted in increased adiposity and tumorigenesis, indicated by increased primary tumor volume, multiplicity, tumor burden, and increased tumor progression represented by histopathological scoring. HFD-induced obesity significantly upregulated aromatase and macrophage marker expression in the AT (F4/80 and CD11c) and mammary gland (Mertk) in a premenopausal model of BrCA. Conversely, HFD feedings had no significant effect on tumorigenesis in a postmenopausal model of BrCA despite large increases in adiposity in ovariectomized mice; however, limitations within the model may have precluded any significant findings. This data suggests that obesity-induced increases in inflammation and hormone production, via aromatase expression, is associated with increases in tumorigenesis in a model of premenopausal HP BrCA in the PyMT/MMTV strain.

Repeated clodronate-liposome treatment results in neutrophilia and is not effective in limiting obesity-linked metabolic impairments.

Depletion of macrophages is thought to be a therapeutic option for obesity-induced inflammation and metabolic dysfunction. However, whether the therapeutic effect is a direct result of reduced macrophage-derived inflammation or secondary to decreases in fat mass is controversial, as macrophage depletion has been shown to disrupt energy homeostasis. This study was designed to determine if macrophage depletion via clodronate-liposome (CLD) treatment could serve as an effective intervention to reduce obesity-driven inflammatory and metabolic impairments independent of changes in energy intake. After 16 wk on a high-fat diet (HFD) or the AIN-76A control (low-fat) diet (LFD) ( n = 30/diet treatment), male C57BL/6J mice were assigned to a CLD- or PBS-liposome treatment ( n = 15/group) for 4 wk. Liposomes were administered biweekly via intraperitoneal injections (8 administrations in total). PBS-liposome-treated groups were pair-fed to their CLD-treated dietary counterparts. Metabolic function was assessed before and after liposome treatment. Adipose tissue, as well as the liver, was investigated for macrophage infiltration and the presence of inflammatory mediators. Additionally, a complete blood count was performed. CLD treatment reduced energy intake. When controlling for energy intake, CLD treatment was unable to regress metabolic dysfunction or nonalcoholic fatty liver disease and impaired adipose tissue insulin action. Moreover, repeated CLD treatment induced neutrophilia and anemia, increased adipose tissue mRNA expression of the proinflammatory cytokines IL-6 and IL-1ß, and augmented circulating IL-6 and monocyte chemoattractant protein-1 concentrations ( P < 0.05). This study suggests that repeated intraperitoneal administration of CLD to deplete macrophages attenuates obesity by limiting energy intake. Moreover, after controlling for the benefits of weight loss, the accompanying detrimental side effects limit regular CLD treatment as an effective therapeutic strategy.

Macrophage depletion using clodronate liposomes decreases tumorigenesis and alters gut microbiota in the AOM/DSS mouse model of colon cancer.

We examined the role of macrophages in inflammation associated with colorectal cancer (CRC). Given the emerging evidence on immune-microbiota interactions in CRC, we also sought to examine the interaction between macrophages and gut microbiota. To induce CRC, male C57BL/6 mice ( n = 32) received a single injection of azoxymethane (AOM), followed by three cycles of dextran sodium sulfate (DSS)-supplemented water in weeks 1, 4, and 7. Prior to the final DSS cycle ( week 7) and twice weekly until euthanasia, mice ( n = 16/group) received either 200 µl ip of clodronate-filled liposomes (CLD) or phosphate-buffered saline (PBS) encapsulated liposomes to deplete macrophages. Colon tissue was analyzed for polyp burden, macrophage markers, transcription factors, and inflammatory mediators. Stool samples were collected, and DNA was isolated and subsequently sequenced for 16S rRNA. Clodronate liposomes decreased tumor number by ∼36% and specifically large (≥1 mm) tumors by ∼36% ( P < 0.05). This was consistent with a decrease in gene expression of EMR1 in the colon tissue and polyp tissue as well as expression of select markers associated with M1 (IL-6) and M2 macrophages (IL-13, IL-10, TGFß, CCL17) in the colon tissue ( P < 0.05). Similarly, there was a decrease in STAT3 and p38 MAPK and ERK signaling in colon tissue. Clodronate liposomes increased the relative abundance of the Firmicutes phylum ( P < 0.05) and specifically Lactobacillaceae and Clostridiaceae families, which have been associated with reduced CRC risk. Overall, these data support the development of therapeutic strategies to target macrophages in CRC and provide support for further evaluation of immune-microbiota interactions in CRC. NEW & NOTEWORTHY We found that macrophage depletion during late-stage tumorigenesis is effective at reducing tumor growth. This was associated with a decrease in macrophage markers and chemokines in the colon tissue and a decrease in transcription factors that are linked to colorectal cancer. The macrophage-depleted group was found to have an increased abundance of Firmicutes, a phylum with documented anti-tumorigenic effects. Overall, these data support the development of therapeutic strategies to target macrophages in colorectal cancer.

miR155 deficiency aggravates high-fat diet-induced adipose tissue fibrosis in male mice.

Noncoding RNAs are emerging as regulators of inflammatory and metabolic processes. There is evidence to suggest that miRNA155 (miR155) may be linked to inflammation and processes associated with adipogenesis. We examined the impact of global miRNA-155 deletion (miR155-/-) on the development of high-fat diet (HFD)-induced obesity. We hypothesized that loss of miR155 would decrease adipose tissue inflammation and improve the metabolic profile following HFD feedings. Beginning at 4-5 weeks of age, male miR155-/- and wild-type (WT) mice (n = 13-14) on a C57BL/6 background were fed either a HFD or low-fat diet for 20 weeks. Body weight was monitored throughout the study. Baseline and terminal body composition was assessed by DEXA analysis. Adipose tissue mRNA expression (RT-qPCR) of macrophage markers (F4/80, CD11c, and CD206) and inflammatory mediators (MCP-1 and TNF-α) as well as adiponectin were measured along with activation of NFκB-p65 and JNK and PPAR-γ Adipose tissue fibrosis was assessed by picrosirius red staining and western blot analysis of Collagen I, III, and VI. Glucose metabolism and insulin resistance were assessed by Homeostatic Model Assessment - Insulin Resistance (HOMA-IR), and a glucose tolerance test. Compared to WT HFD mice, miR155-/- HFD mice displayed similar body weights, yet reduced visceral adipose tissue accumulation. However, miR155-/- HFD displayed exacerbated adipose tissue fibrosis and decreased PPAR-γ protein content. The loss of miR155 did not affect adipose tissue inflammation or glucose metabolism. In conclusion, miR155 deletion did not attenuate the development of the obese phenotype, but adipose tissue fibrosis was exacerbated, possibly through changes to adipogenic processes.

Loss of monocyte chemoattractant protein-1 expression delays mammary tumorigenesis and reduces localized inflammation in the C3(1)/SV40Tag triple negative breast cancer model.

Monocyte chemoattractant protein 1 (MCP-1) has been implicated as a major modulator in the progression of mammary tumorigenesis, largely due to its ability to recruit macrophages to the tumor microenvironment. Macrophages are key mediators in the connection between inflammation and cancer progression and have been shown to play an important role in tumorigenesis. Thus, MCP-1 may be a potential therapeutic target in inflammatory and difficult-to-treat cancers such as triple negative breast cancer (TNBC). We examined the effect of MCP-1 depletion on mammary tumorigenesis in a model of TNBC. Tumor measurements were conducted weekly (until 22 weeks of age) and at sacrifice (23 weeks of age) in female C3(1)/SV40Tag and C3(1)/SV40Tag MCP-1 deficient mice to determine tumor numbers and tumorvolumes. Histopathological scoring was performed at 12 weeks of age and 23 weeks of age. Gene expression of macrophage markers and inflammatory mediators were measured in the mammary gland and tumor microenvironment at sacrifice. As expected, MCP-1 depletion resulted in decreased tumorigenesis, indicated by reduced primary tumor volume and multiplicity, and a delay in tumor progression represented by histopathological scoring (12 weeks of age). Deficiency in MCP-1 significantly downregulated expression of macrophage markers in the mammary gland (Mertk and CD64) and the tumor microenvironment (CD64), and also reduced expression of inflammatory cytokines in the mammary gland (TNFα and IL-1ß) and the tumor microenvironment (IL-6). These data support the hypothesis that MCP-1 expression contributes to increased tumorigenesis in a model of TNBC via recruitment of macrophages and subsequent increase in inflammatory mediators.

Weight loss following diet-induced obesity does not alter colon tumorigenesis in the AOM mouse model.

Obesity presents a significant public health concern given its association with increased cancer incidence, unfavorable prognosis, and metastasis. However, there is very little literature on the effects of weight loss, following obesity, on risk for colon cancer or liver cancer. Therefore, we sought to study whether intentional weight loss through diet manipulation was capable of mitigating colon and liver cancer in mice. We fed mice with a high-fat diet (HFD) comprised of 47% carbohydrates, 40% fat, and 13% protein for 20 wk to mimic human obesity. Subsequently, azoxymethane (AOM) was used to promote colon and liver carcinogenesis. A subset of obese mice was then switched to a low-fat diet (LFD) containing 67.5% carbohydrate, 12.2% fat, and 20% protein to promote intentional weight loss. Body weight loss and excess fat reduction did not protect mice from colon cancer progression and liver dysplastic lesion in the AOM-chemical-cancer model even though these mice had improved blood glucose and leptin levels. Intentional weight loss in AOM-treated mice actually produced histological changes that resemble dysplastic alterations in the liver and presented a higher percentage of F4/80+CD206+ macrophages and activated T cells (CD4+CD69+) in the spleen and lymph nodes, respectively. In addition, the liver of AOM-treated mice exposed to a HFD during the entire period of the experiment exhibited a marked increase in proliferation and pNF-κB activation. Altogether, these data suggest that intentional weight loss following chemical-induced carcinogenesis does not affect colon tumorigenesis but may in fact negatively impact liver repair mechanisms.

Intra- and inter-laboratory reproducibility and accuracy of the LuSens assay: A reporter gene-cell line to detect keratinocyte activation by skin sensitizers.

Several non-animal methods are now available to address the key events leading to skin sensitization as defined by the adverse outcome pathway. The KeratinoSens assay addresses the cellular event of keratinocyte activation and is a method accepted under OECD TG 442D. In this study, the results of an inter-laboratory evaluation of the "me-too" LuSens assay, a bioassay that uses a human keratinocyte cell line harboring a reporter gene construct composed of the rat antioxidant response element (ARE) of the NADPH:quinone oxidoreductase 1 gene and the luciferase gene, are described. Earlier in-house validation with 74 substances showed an accuracy of 82% in comparison to human data. When used in a battery of non-animal methods, even higher predictivity is achieved. To meet European validation criteria, a multicenter study was conducted in 5 laboratories. The study was divided into two phases, to assess 1) transferability of the method, and 2) reproducibility and accuracy. Phase I was performed by testing 8 non-coded test substances; the results showed a good transferability to naïve laboratories even without on-site training. Phase II was performed with 20 coded test substances (performance standards recommended by OECD, 2015). In this phase, the intra- and inter-laboratory reproducibility as well as accuracy of the method was evaluated. The data demonstrate a remarkable reproducibility of 100% and an accuracy of over 80% in identifying skin sensitizers, indicating a good concordance with in vivo data. These results demonstrate good transferability, reliability and accuracy of the method thereby achieving the standards necessary for use in a regulatory setting to detect skin sensitizers.