Host-derived Interleukin 1α induces an immunosuppressive tumor microenvironment via regulating monocyte-to-macrophage differentiation.

Tumor-associated macrophages exhibit high heterogeneity and contribute to the establishment of an immunosuppressive tumor microenvironment (TME). Although numerous studies have demonstrated that extracellular factors promote macrophage proliferation and polarization, the regulatory mechanisms governing the differentiation process to generate phenotypically, and functionally diverse macrophage subpopulations remain largely unexplored. In this study, we examined the influence of interleukin 1α (IL-1α) on the development of an immunosuppressive TME using orthotopic transplantation murine models of breast cancer. Deletion of host Il1α led to the rejection of inoculated congenic tumors. Single-cell sequencing analysis revealed that CX3CR1+ macrophage cells were the primary sources of IL-1α in the TME. The absence of IL-1α reprogrammed the monocyte-to-macrophage differentiation process within the TME, characterized by a notable decrease in the subset of CX3CR+ ductal-like macrophages and an increase in iNOS-expressing inflammatory cells. Comparative analysis of gene signatures in both human and mouse macrophage subsets suggested that IL-1α deficiency shifted the macrophage polarization from M2 to M1 phenotypes, leading to enhanced cytotoxic T lymphocyte activity in the TME. Importantly, elevated levels of IL-1α in human cancers were associated with worse prognosis following immunotherapy. These findings underscore the pivotal role of IL-1α in shaping an immune-suppressive TME through the regulation of macrophage differentiation and activity, highlighting IL-1α as a potential target for breast cancer treatment.

The complex heterogeneity of immune cell signatures across wasting tissues with C26 and 5-fluorouracil-induced cachexia.

Immune cell-driven pathways are linked to cancer cachexia. Tumor presence is associated with immune cell infiltration whereas cytotoxic chemotherapies reduce immune cell counts. Despite these paradoxical effects, both cancer and chemotherapy can cause cachexia; however, our understanding of immune responses in the cachexia condition with cancer and chemotherapy is largely unknown. We sought to advance our understanding of the immunology underlying cancer and cancer with chemotherapy-induced cachexia. CD2F1 mice were given 106 C26 cells, followed by five doses of 5-fluorouracil (5FU; 30 mg/kg LM, ip) or PBS. Indices of cachexia and tumor (TUM), skeletal muscle (SKM), and adipose tissue (AT) immune cell populations were examined using high-parameter flow cytometry. Although 5FU was able to stunt tumor growth, % body weight loss and muscle mass were not different between C26 and C26 + 5FU. C26 increased CD11b+Ly6g+ and CD11b+Ly6cInt inflammatory myeloid cells in SKM and AT; however, both populations were reduced with C26 + 5FU. tSNE analysis revealed 24 SKM macrophage subsets wherein 8 were changed with C26 or C26 + 5FU. C26 + 5FU increased SKM CD11b-CD11c+ dendritic cells, CD11b-NK1.1+ NK-cells, and CD11b-B220+ B-cells, and reduced Ly6cHiCX3CR1+CD206+CD163IntCD11c-MHCII- infiltrated macrophages and other CD11b+Ly6cHi myeloid cells compared with C26. Both C26 and C26 + 5FU had elevated CD11b+F480+CD206+MHCII- or more specifically Ly6cLoCX3CR1+CD206+CD163IntCD11c-MHCII- profibrotic macrophages. 5FU suppressed tumor growth and decreased SKM and AT inflammatory immune cells without protecting against cachexia suggesting that these cells are not required for wasting. However, profibrotic cells and muscle inflammatory/atrophic signaling appear consistent with cancer- and cancer with chemotherapy-induced wasting and remain potential therapeutic targets.NEW & NOTEWORTHY Despite being an immune-driven condition, our understanding of skeletal muscle and adipose tissue immune cells with cachexia is limited. Here, we identified immune cell populations in tumors, skeletal muscle, and adipose tissue in C26 tumor-bearing mice with/without 5-fluorouracil (5FU). C26 and C26 + 5FU had increased skeletal muscle profibrotic macrophages, but 5FU reduced inflammatory myeloid cells without sparing mass. Tumor presence and chemotherapy have contrasting effects on certain immune cells, which appeared not necessary for wasting.

Obesity worsens mitochondrial quality control and does not protect against skeletal muscle wasting in murine cancer cachexia.

BACKGROUND: More than 650 million people are obese (BMI > 30) worldwide, which increases their risk for several metabolic diseases and cancer. While cachexia and obesity are at opposite ends of the weight spectrum, leading many to suggest a protective effect of obesity against cachexia, mechanistic support for obesity's benefit is lacking. Given that obesity and cachexia are both accompanied by metabolic dysregulation, we sought to investigate the impact of obesity on skeletal muscle mass loss and mitochondrial dysfunction in murine cancer cachexia. METHODS: Male C57BL/6 mice were given a purified high fat or standard diet for 16 weeks before being implanted with 106 Lewis lung carcinoma (LLC) cells. Mice were monitored for 25 days, and hindlimb muscles were collected for cachexia indices and mitochondrial assessment via western blotting, high-resolution respirometry and transmission electron microscopy (TEM). RESULTS: Obese LLC mice experienced significant tumour-free body weight loss similar to lean (-12.8% vs. -11.8%, P = 0.0001) but had reduced survival (33.3% vs. 6.67%, χ2  = 10.04, P = 0.0182). Obese LLC mice had reduced muscle weights (-24%, P < 0.0354) and mCSA (-16%, P = 0.0004) with similar activation of muscle p65 (P = 0.0337), and p38 (P = 0.0008). ADP-dependent coupled respiration was reduced in both Obese and Obese LLC muscle (-30%, P = 0.0072) consistent with reductions in volitional cage activity (-39%, P < 0.0001) and grip strength (-41%, P < 0.0001). TEM revealed stepwise reductions in intermyofibrillar and subsarcolemmal mitochondrial size with Obese (IMF: -37%, P = 0.0009; SS: -21%, P = 0.0101) and LLC (IMF: -40%, P = 0.0019; SS: -27%, P = 0.0383) mice. Obese LLC mice had increased pAMPK (T172; P = 0.0103) and reduced FIS1 (P = 0.0029) and DRP1 (P < 0.0001) mitochondrial fission proteins, which were each unchanged in Lean LLC. Further, mitochondrial TEM analysis revealed that Obese LLC mice had an accumulation of damaged and dysfunctional mitochondria (IMF: 357%, P = 0.0395; SS: 138%, P = 0.0174) in concert with an accumulation of p62 (P = 0.0328) suggesting impaired autophagy and clearance of damaged mitochondria. Moreover, we observed increases in electron lucent vacuoles only in Obese LLC muscle (IMF: 421%, P = 0.0260; SS: 392%, P = 0.0192), further supporting an accumulation of damaged materials that cannot be properly cleared in the obese cachectic muscle. CONCLUSIONS: Taken together, these results demonstrate that obesity is not protective against cachexia and suggest exacerbated impairments to mitochondrial function and quality control with a particular disruption in the removal of damaged mitochondria. Our findings highlight the need for consideration of the severity of obesity and pre-existing metabolic conditions when determining the impact of weight status on cancer-induced cachexia and functional mitochondrial deficits.

Genome-wide search algorithms for identifying dynamic gene co-expression via Bayesian variable selection.

A wealth of gene expression data generated by high-throughput techniques provides exciting opportunities for studying gene-gene interactions systematically. Gene-gene interactions in a biological system are tightly regulated and are often highly dynamic. The interactions can change flexibly under various internal cellular signals or external stimuli. Previous studies have developed statistical methods to examine these dynamic changes in gene-gene interactions. However, due to the massive number of possible gene combinations that need to be considered in a typical genomic dataset, intensive computation is a common challenge for exploring gene-gene interactions. On the other hand, oftentimes only a small proportion of gene combinations exhibit dynamic co-expression changes. To solve this problem, we propose Bayesian variable selection approaches based on spike-and-slab priors. The proposed algorithms reduce the computational intensity by focusing on identifying subsets of promising gene combinations in the search space. We also adopt a Bayesian multiple hypothesis testing procedure to identify strong dynamic gene co-expression changes. Simulation studies are performed to compare the proposed approaches with existing exhaustive search heuristics. We demonstrate the implementation of our proposed approach to study the association between gene co-expression patterns and overall survival using the RNA-sequencing dataset from The Cancer Genome Atlas breast cancer BRCA-US project.

Emodin reduces surgical wounding-accelerated tumor growth and metastasis via macrophage suppression in a murine triple-negative breast cancer model.

It has been suspected that tumor resection surgery itself may accelerate breast cancer (BC) lung metastasis in some patients. Emodin, a natural anthraquinone found in the roots and rhizomes of various plants, exhibits anticancer activity. We examined the perioperative use of emodin in our established surgery wounding murine BC model. Emodin reduced primary BC tumor growth and metastasis in the lungs in both sham and surgical wounded mice, consistent with a reduction in proliferation and enhanced apoptosis (primary tumor and lungs). Further, emodin reduced systemic inflammation, most notably the number of monocytes in the peripheral blood and reduced pro-tumoral M2 macrophages in the primary tumor and the lungs. Consistently, we show that emodin reduces gene expression of select macrophage markers and associated cytokines in the primary tumor and lungs of wounded mice. Overall, we demonstrate that emodin is beneficial in mitigating surgical wounding accelerated lung metastasis in a model of triple-negative BC, which appears to be mediated, at least in part, by its actions on macrophages. These data support the development of emodin as a safe, low-cost, and effective agent to be used perioperatively to alleviate the surgery triggered inflammatory response and consequential metastasis of BC to the lungs.

Panaxynol alleviates colorectal cancer in a murine model via suppressing macrophages and inflammation.

Currently available colorectal cancer (CRC) therapies have limited efficacy and severe adverse effects that may be overcome with the alternative use of natural compounds. We previously reported that panaxynol (PA), a bioactive component in American ginseng, possesses anticancer properties in vitro and suppresses murine colitis through its proapoptotic and anti-inflammatory properties. Because colitis is a predisposing factor of CRC and inflammation is a major driver of CRC, we sought to evaluate the therapeutic potential of PA in CRC. Azoxymethane-dextran sodium sulfate (AOM/DSS) mice (C57BL/6) were administered 2.5 mg/kg PA or vehicle 3 times/wk via oral gavage over 12 wk. PA improved clinical symptoms (P ≤ 0.05) and reduced tumorigenesis (P ≤ 0.05). This improvement may be reflective of PA's restorative effect on intestinal barrier function; PA upregulated the expression of essential tight junction and mucin genes (P ≤ 0.05) and increased the abundance of mucin-producing goblet cells (P ≤ 0.05). Given that macrophages play a substantial role in the pathogenesis of CRC and that we previously demonstrated that PA targets macrophages in colitis, we next assessed macrophages. We show that PA reduces the relative abundance of colonic macrophages within the lamina propria (P ≤ 0.05), and this was consistent with a reduction in the expression of important markers of macrophages and inflammation (P ≤ 0.05). We further confirmed PA's inhibitory effects on macrophages in vitro under CRC conditions (P ≤ 0.05). These results suggest that PA is a promising therapeutic compound to treat CRC and improve clinical symptoms given its ability to inhibit macrophages and modulate the inflammatory environment in the colon.NEW & NOTEWORTHY We report that panaxynol (PA) reduces colorectal cancer (CRC) by improving the colonic and tumor environment. Specifically, we demonstrate that PA improves crypt morphology, upregulates crucial tight junction and mucin genes, and promotes the abundance of mucin-producing goblet cells. Furthermore, PA reduces macrophages and associated inflammation, important drivers of CRC, in the colonic environment. This present study provides novel insights into the potential of PA as a therapeutic agent to ameliorate CRC tumorigenesis.

Pyruvate dehydrogenase kinase supports macrophage NLRP3 inflammasome activation during acute inflammation.

Activating the macrophage NLRP3 inflammasome can promote excessive inflammation with severe cell and tissue damage and organ dysfunction. Here, we show that pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) significantly attenuates NLRP3 inflammasome activation in murine and human macrophages and septic mice by lowering caspase-1 cleavage and interleukin-1ß (IL-1ß) secretion. Inhibiting PDHK reverses NLRP3 inflammasome-induced metabolic reprogramming, enhances autophagy, promotes mitochondrial fusion over fission, preserves crista ultrastructure, and attenuates mitochondrial reactive oxygen species (ROS) production. The suppressive effect of PDHK inhibition on the NLRP3 inflammasome is independent of its canonical role as a pyruvate dehydrogenase regulator. Our study suggests a non-canonical role of mitochondrial PDHK in promoting mitochondrial stress and supporting NLRP3 inflammasome activation during acute inflammation.

Trim-Away in adult animals through Nano-ERASER and its application in cancer therapy.

Trim-Away is a versatile intracellular protein degradation pathway that has been extensively explored in vitro. However, the in vivo application of Trim-Away is limited at oocyte and zygote stages due to the lack of an in vivo practical approach for intracellular antibody delivery. To broaden the application of Trim-Away, especially for clinical use, we developed a nanogel-based Nano-ERASER system. Here, we demonstrated that the intracellular delivery of anti-programmed cell death ligand 1 (PD-L1) antibody through Nano-ERASER could effectively deplete PD-L1 in triple negative breast cancer (TNBC) cells and induce cancer cell death. Furthermore, with the help of a tumor tissue-targeted nanogel, anti-PD-L1 antibody-loaded Nano-ERASER effectively inhibited tumor progression in a TNBC mouse model. These results confirmed that Nano-ERASER realized Trim-Away in adult animals for the first time, which could be an effective tool for disease treatment and studying gene/protein function both in vitro and in vivo.

Use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in atherosclerosis.

Dysfunction in the macrophage lysosomal system including reduced acidity and diminished degradative capacity is a hallmark of atherosclerosis, leading to blunted clearance of excess cellular debris and lipids in plaques and contributing to lesion progression. Devising strategies to rescue this macrophage lysosomal dysfunction is a novel therapeutic measure. Nanoparticles have emerged as an effective platform to both target specific tissues and serve as drug delivery vehicles. In most cases, administered nanoparticles are taken up non-selectively by the mononuclear phagocyte system including monocytes/macrophages leading to the undesirable degradation of cargo in lysosomes. We took advantage of this default route to target macrophage lysosomes to rectify their acidity in disease states such as atherosclerosis. Herein, we develop and test two commonly used acidic nanoparticles, poly-lactide-co-glycolic acid (PLGA) and polylactic acid (PLA), both in vitro and in vivo. Our results in cultured macrophages indicate that the PLGA-based nanoparticles are the most effective at trafficking to and enhancing acidification of lysosomes. PLGA nanoparticles also provide functional benefits including enhanced lysosomal degradation, promotion of macroautophagy/autophagy and protein aggregate removal, and reduced apoptosis and inflammasome activation. We demonstrate the utility of this system in vivo, showing nanoparticle accumulation in, and lysosomal acidification of, macrophages in atherosclerotic plaques. Long-term administration of PLGA nanoparticles results in significant reductions in surrogates of plaque complexity with reduced apoptosis, necrotic core formation, and cytotoxic protein aggregates and increased fibrous cap formation. Taken together, our data support the use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in the treatment of atherosclerosis.Abbreviations: BCA: brachiocephalic arteries; FACS: fluorescence activated cell sorting; FITC: fluorescein-5-isothiocyanatel; IL1B: interleukin 1 beta; LAMP: lysosomal associated membrane protein; LIPA/LAL: lipase A, lysosomal acid type; LSDs: lysosomal storage disorders; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence intensity; MPS: mononuclear phagocyte system; PEGHDE: polyethylene glycol hexadecyl ether; PLA: polylactic acid; PLGA: poly-lactide-co-glycolic acid; SQSTM1/p62: sequestosome 1.

Surgical wounding enhances pro-tumor macrophage responses and accelerates tumor growth and lung metastasis in a triple negative breast cancer mouse model.

Approximately one-third of all breast cancer mortality results from metastatic recurrence after initial success of surgery and/or therapy. Although primary tumor removal is widely accepted as beneficial, it has long been suspected that surgery itself contributes to accelerated metastatic recurrence. We investigated surgical wounding's impact on tumor progression and lung metastasis in a murine model of triple negative breast cancer (TNBC). Ten-week-old female mice were inoculated with 4 T1 cells (week 0) and were either subjected to a 2 cm long cutaneous contralateral incision (wounded) or control (non-wounded) on week 2 and monitored for 3 weeks (week 5). Mice with surgical wounding displayed significantly accelerated tumor growth observable as early as 1-week post wounding. This was confirmed by increased tumor volume and tumor weight, post-mortem. Further, surgical wounding increased metastasis to the lungs, as detected by IVIS imaging, in vivo and ex vivo (week 5). As expected then, wounded mice displayed decreased apoptosis and increased proliferation in both the primary tumor and in the lungs. Flow cytometry revealed that primary tumors from wounded mice exhibited increased tumor associated macrophages and specifically M2-like macrophages, which are important in promoting tumor development, maintenance, and metastasis. Immunofluorescence staining and gene expression data further confirms an increase in macrophages in both the primary tumor and the lungs of wounded mice. Our data suggests that surgical wounding accelerates tumor progression and lung metastasis in a mouse model of TNBC, which is likely mediated, at least in part by an increase in macrophages.

Sub-chronic oral toxicity screening of quercetin in mice.

BACKGROUND: Quercetin is an organic flavonoid present in several fruits and vegetables. The anti-inflammatory, antiviral, antioxidant, cardio-protective, anti-carcinogenic and neuroprotective properties demonstrated by this dietary supplement endorses it as a possible treatment for inflammatory diseases and cancer. Unfortunately, conflicting research has cast uncertainties on the toxicity of quercetin. The main purpose of this study was to determine if quercetin has any toxic properties in mice at doses that have shown efficacy in pre-clinical studies regarding cancer, cancer therapy, and their off-target effects. METHODS: A sub-chronic toxicity study of quercetin was examined in male and female CD2F1 mice. Three different doses of quercetin (62, 125, and 250 mg/kg of diet) were infused into the AIN-76A purified diet and administered to mice ad libitum for 98 days. Body weight (BW), food consumption, water intake, body composition, blood count, behavior, and metabolic phenotype were assessed at various timepoints during the course of the experiment. Tissue and organs were evaluated for gross pathological changes and plasma was used to measure alkaline phosphatase (AP), aspartate transaminase (AST), and alanine transaminase (ALT). RESULTS: We found that low (62 mg/kg of diet), medium (125 mg/kg of diet), and high (250 mg/kg of diet) quercetin feeding had no discernible effect on body composition, organ function, behavior or metabolism. CONCLUSIONS: In summary, our study establishes that quercetin is safe for use in both female and male CD2F1 mice when given at ~ 12.5, 25, or 50 mg/kg of BW daily doses for 14 weeks (i.e. 98 days). Further studies will need to be conducted to determine any potential toxicity of quercetin following chronic ingestion.

miR155 deficiency reduces breast tumor burden in the MMTV-PyMT mouse model.

miRNA155 (miR155) has emerged as an important regulator of breast cancer (BrCa) development. Studies have consistently noted an increase in miR155 levels in serum and/or tissues in patients with BrCa. However, what is less clear is whether this increase in miR155 is a reflection of oncogenic or tumor suppressive properties. To study the effects of miR155 in a transgenic model of BrCA, we developed an MMTV-PyMT mouse deficient in miR155 (miR155-/- PyMT). miR155-/- mice (n = 11) exhibited reduced tumor number and volume palpations at ∼14-18 wk of age compared with miR155 sufficient littermates (n = 12). At 19 wk, mammary glands were excised from tumors for RT-PCR, and tumors were counted, measured, and weighed. miR155-/- PyMT mice exhibited reduced tumor volume, number, and weight, which was confirmed by histopathological analysis. There was an increase in apoptosis with miR155 deficiency and a decrease in proliferation. As expected, miR155 deficiency resulted in upregulated gene expression of suppressor of cytokine signaling 1 (Socs1)-its direct target. There was a reduction in gene expression of macrophage markers (CD68, Adgre1, Itgax, Mrc1) with miR-155-/- and this was confirmed with immunofluorescence staining for F4/80. miR155-/- increased expression of M1 macrophage marker Nos2 and reduced expression of M2 macrophage markers IL-10, IL-4, Arg1, and MMP9. Overall, miR155 deficiency reduced BrCA and improved the tumor microenvironment through the reduction of genes associated with protumorigenic processes. However, given the inconsistencies in the literature, additional studies are needed before any attempts are made to harness miR155 as a potential oncogenic or tumor suppressive miRNA.NEW & NOTEWORTHY To examine the effects of miR155 in a transgenic model of breast cancer, we developed an MMTV-PyMT mouse-deficient in miR155. We demonstrate that global loss of miR155 resulted in blunted tumor growth through modulating the tumor microenvironment. Specifically, miR155-deficient mice had smaller and less invasive tumors, an increase in apoptosis and a decrease in proliferation, a reduction in tumor-associated macrophages, and the expression of genes associated with protumoral processes.

Obesity reduced survival with 5-fluorouracil and did not protect against chemotherapy-induced cachexia or immune cell cytotoxicity in mice.

Fluorouracil/5-flourouracil (5FU) is a first-line chemotherapy drug for many cancer types; however, its associated toxicities contribute to poor quality of life and reduced dose intensities negatively impacting patient prognosis. While obesity remains a critical risk factor for most cancers, our understanding regarding how obesity may impact chemotherapy's toxicities is extremely limited. C56BL/6 mice were given high fat (Obese) or standard diets (Lean) for 4 months and then subjected to three cycles of 5FU (5d-40 mg/kg Lean Mass, 9d rest) or PBS vehicle control. Shockingly, only 60% of Obese survived 3 cycles compared to 100% of Lean, and Obese lost significantly more body weight. Dihydropyrimidine dehydrogenase (DPD), the enzyme responsible for 5FU catabolism, was reduced in obese livers. Total white blood cells, neutrophils, and lymphocytes were reduced in Obese 5FU compared to Lean 5FU and PBS controls. While adipocyte size was not affected by 5FU in Obese, skeletal muscle mass and myofibrillar cross section area were decreased following 5FU in Lean and Obese. Although adipose tissue inflammatory gene expression was not impacted by 5FU, distinct perturbations to skeletal muscle inflammatory gene expression and immune cell populations (CD45+ Immune cells, CD45+CD11b+CD68+ macrophages and CD45+CD11b+Ly6clo/int macrophage/monocytes) were observed in Obese only. Our evidence suggests that obesity induced liver pathologies and reduced DPD exacerbated 5FU toxicities. While obesity has been suggested to protect against cancer/chemotherapy-induced cachexia and other toxicities, our results demonstrate that obese mice are not protected, but rather show evidence of increased susceptibility to 5FU-induced cytotoxicity even when dosed for relative lean mass.

Breast cancer cell-derived microRNA-155 suppresses tumor progression via enhancing immune cell recruitment and antitumor function.

Evidence suggests that increased microRNA-155 (miR-155) expression in immune cells enhances antitumor immune responses. However, given the reported association of miR-155 with tumorigenesis in various cancers, a debate is provoked on whether miR-155 is oncogenic or tumor suppressive. We aimed to interrogate the impact of tumor miR-155 expression, particularly that of cancer cell-derived miR-155, on antitumor immunity in breast cancer. We performed bioinformatic analysis of human breast cancer databases, murine experiments, and human specimen examination. We revealed that higher tumor miR-155 levels correlate with a favorable antitumor immune profile and better patient outcomes. Murine experiments demonstrated that miR-155 overexpression in breast cancer cells enhanced T cell influx, delayed tumor growth, and sensitized the tumors to immune checkpoint blockade (ICB) therapy. Mechanistically, miR-155 overexpression in breast cancer cells upregulated their CXCL9/10/11 production, which was mediated by SOCS1 inhibition and increased phosphorylated STAT1 (p-STAT1)/p-STAT3 ratios. We further found that serum miR-155 levels in breast cancer patients correlated with tumor miR-155 levels and tumor immune status. Our findings suggest that high serum and tumor miR-155 levels may be a favorable prognostic marker for breast cancer patients and that therapeutic elevation of miR-155 in breast tumors may improve the efficacy of ICB therapy via remodeling the antitumor immune landscape.

5-Fluorouracil disrupts skeletal muscle immune cells and impairs skeletal muscle repair and remodeling.

5-Fluorouracil (5FU) remains a first-line chemotherapeutic for several cancers despite its established adverse side effects. Reduced blood counts with cytotoxic chemotherapies not only expose patients to infection and fatigue, but can disrupt tissue repair and remodeling, leading to lasting functional deficits. We sought to characterize the impact of 5FU-induced leukopenia on skeletal muscle in the context of remodeling. First, C57BL/6 mice were subjected to multiple dosing cycles of 5FU and skeletal muscle immune cells were assessed. Second, mice given 1 cycle of 5FU were subjected to 1.2% BaCl2 intramuscularly to induce muscle damage. One cycle of 5FU induced significant body weight loss, but only three dosing cycles of 5FU induced skeletal muscle mass loss. One cycle of 5FU reduced skeletal muscle CD45+ immune cells with a particular loss of infiltrating CD11b+Ly6cHi monocytes. Although CD45+ cells returned following three cycles, CD11b+CD68+ macrophages were reduced with three cycles and remained suppressed at 1 mo following 5FU administration. One cycle of 5FU blocked the increase in CD45+ immune cells 4 days following BaCl2; however, there was a dramatic increase in CD11b+Ly6g+ neutrophils and a loss of CD11b+Ly6cHi monocytes in damaged muscle with 5FU compared with PBS. These perturbations resulted in increased collagen production 14 and 28 days following BaCl2 and a reduction in centralized nuclei and myofibrillar cross-sectional area compared with PBS. Together, these results demonstrate that cytotoxic 5FU impairs muscle damage repair and remodeling concomitant with a loss of immune cells that persists beyond the cessation of treatment.NEW & NOTEWORTHY We examined the common chemotherapeutic 5-fluorouracil's (5FU) impact on skeletal muscle immune cells and skeletal muscle repair. 5FU monotherapy decreased body weight and muscle mass, and perturbed skeletal muscle immune cells. In addition, 5FU decreased skeletal muscle immune cells and impaired infiltration following damage contributing to disrupted muscle repair. Our results demonstrate 5FU's impact on skeletal muscle and provide a potential explanation for why some patients may be unable to properly repair damaged tissue.

miR-489 Confines Uncontrolled Estrogen Signaling through a Negative Feedback Mechanism and Regulates Tamoxifen Resistance in Breast Cancer.

Approximately 75% of diagnosed breast cancer tumors are estrogen-receptor-positive tumors and are associated with a better prognosis due to response to hormonal therapies. However, around 40% of patients relapse after hormonal therapies. Genomic analysis of gene expression profiles in primary breast cancers and tamoxifen-resistant cell lines suggested the potential role of miR-489 in the regulation of estrogen signaling and development of tamoxifen resistance. Our in vitro analysis showed that loss of miR-489 expression promoted tamoxifen resistance, while overexpression of miR-489 in tamoxifen-resistant cells restored tamoxifen sensitivity. Mechanistically, we found that miR-489 is an estrogen-regulated miRNA that negatively regulates estrogen receptor signaling by using at least the following two mechanisms: (i) modulation of the ER phosphorylation status by inhibiting MAPK and AKT kinase activities; (ii) regulation of nuclear-to-cytosol translocation of estrogen receptor α (ERα) by decreasing p38 expression and consequently ER phosphorylation. In addition, miR-489 can break the positive feed-forward loop between the estrogen-Erα axis and p38 MAPK in breast cancer cells, which is necessary for its function as a transcription factor. Overall, our study unveiled the underlying molecular mechanism by which miR-489 regulates an estrogen signaling pathway through a negative feedback loop and uncovered its role in both the development of and overcoming of tamoxifen resistance in breast cancers.

Identification and Biosynthesis of Pro-Inflammatory Sulfonolipids from an Opportunistic Pathogen Chryseobacterium gleum.

Sulfonolipids (SoLs) are a unique class of sphingolipids featuring a sulfonate group compared to other sphingolipids. However, the biological functions and biosynthesis of SoLs in human microbiota have been poorly understood. Here, we report the discovery and isolation of SoLs from a human opportunistic pathogen Chryseobacterium gleum DSM16776. We show for the first time the pro-inflammatory activity of SoLs with mice primary macrophages. Furthermore, we used both in vivo heterologous expression and in vitro biochemical reconstitution to characterize two enzymes, cysteate synthase and cysteate fatty acyltransferase, that are specifically involved in the biosynthesis of SoLs rather than other sphingolipids. Based on these two SoL-specific enzymes, our bioinformatics analysis showed a wider distribution of SoL biosynthetic genes in microbes that had not been reported as SoL producers. We selected four of these strains and verified their cysteate synthase and cysteate fatty acyltransferase activities in SoL biosynthesis. Considering this wider distribution of SoL-specific biosynthetic enzymes in the context of SoLs' activity in mediating inflammation, a common and fundamental biological process, it may suggest a more comprehensive function of SoLs at play.

Modulation of the tumor microenvironment by armed vesicular stomatitis virus in a syngeneic pancreatic cancer model.

BACKGROUND: The immunosuppressive microenvironment in pancreatic ductal adenocarcinoma is a major factor that limits the benefits of immunotherapy, especially immune checkpoint blockade. One viable strategy for reverting the immunosuppressive conditions is the use of an oncolytic virus (OV) in combination with other immunotherapy approaches. Infection of PDAC cells with a robust OV can change the tumor microenvironment and increase tumor antigen release by its lytic activities. These changes in the tumor may improve responses to immunotherapy, including immune checkpoint blockade. However, a more potent OV may be required for efficiently infecting pancreatic tumors that may be resistant to OV. METHODS: Vesicular stomatitis virus, a rapid replicating OV, was armed to express the Smac protein during virus infection (VSV-S). Adaptation by limited dilution largely increased the selective infection of pancreatic cancer cells by VSV-S. The engineered OV was propagated to a large quantity and evaluated for their antitumor activities in an animal model. RESULTS: In a syngeneic KPC model, intratumoral injection of VSV-S inhibited tumor growth, and induced increasing tumor infiltration of neutrophils and elimination of myeloid derived suppressor cells and macrophages in the tumor. More importantly, M2-like macrophages were eliminated preferentially over those with an M1 phenotype. Reduced levels of arginase 1, TGF-ß and IL-10 in the tumor also provided evidence for reversion of the immunosuppressive conditions by VSV-S infection. In several cases, tumors were completely cleared by VSV-S treatment, especially when combined with anti-PD-1 therapy. A long-term survival of 44% was achieved. CONCLUSIONS: The improved OV, VSV-S, was shown to drastically alter the immune suppressive tumor microenvironment when intratumorally injected. Our results suggest that the combination of potent OV treatment with immune checkpoint blockade may be a promising strategy to treat pancreatic cancer more effectively.

A novel circ_MACF1/miR-942-5p/TGFBR2 axis regulates the functional behaviors and drug sensitivity in gefitinib-resistant non-small cell lung cancer cells.

BACKGROUND: Resistance to gefitinib remains a major obstacle for the successful treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations. In this paper, we studied the precise actions of circular RNA (circRNA) microtubule actin crosslinking factor 1 (circ_MACF1) in gefitinib resistance. METHODS: We established gefitinib-resistant NSCLC cells (PC9/GR and A549/GR). The levels of circ_MACF1, microRNA (miR)-942-5p, and transforming growth factor beta receptor 2 (TGFBR2) were gauged by quantitative real-time PCR (qRT-PCR) or western blot. Subcellular fractionation and Ribonuclease R (RNase R) assays were done to characterize circ_MACF1. Cell survival, proliferation, colony formation, apoptosis, migration, and invasion were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-Ethynyl-2'-Deoxyuridine (EdU), colony formation, flow cytometry, and transwell assays, respectively. Dual-luciferase reporter assays were used to verify the direct relationship between miR-942-5p and circ_MACF1 or TGFBR2. The xenograft assays were used to assess the role of circ_MACF1 in vivo. RESULTS: Circ_MACF1 was down-regulated in A549/GR and PC9/GR cells. Overexpression of circ_MACF1 repressed proliferation, migration, invasion, and promoted apoptosis and gefitinib sensitivity of A549/GR and PC9/GR cells in vitro, as well as inhibited tumor growth under gefitinib in vivo. Circ_MACF1 directly targeted miR-942-5p, and miR-942-5p mediated the regulatory effects of circ_MACF1. TGFBR2 was identified as a direct and functional target of miR-942-5p. Circ_MACF1 modulated TGFBR2 expression through miR-942-5p. CONCLUSION: Our findings demonstrated that circ_MACF1 regulated cell functional behaviors and gefitinib sensitivity of A549/GR and PC9/GR cells at least partially by targeting miR-942-5p to induce TGFBR2 expression.

Therapeutic Potential of Emodin for Gastrointestinal Cancers.

Gastrointestinal (GI) cancers cause one-third of all cancer-related deaths worldwide. Natural compounds are emerging as alternative or adjuvant cancer therapies given their distinct advantage of manipulating multiple pathways to both suppress tumor growth and alleviate cancer comorbidities; however, concerns regarding efficacy, bioavailability, and safety are barriers to their development for clinical use. Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a Chinese herb-derived anthraquinone, has been shown to exert anti-tumor effects in colon, liver, and pancreatic cancers. While the mechanisms underlying emodin's tumoricidal effects continue to be unearthed, recent evidence highlights a role for mitochondrial mediated apoptosis, modulated stress and inflammatory signaling pathways, and blunted angiogenesis. The goals of this review are to (1) highlight emodin's anti-cancer properties within GI cancers, (2) discuss the known anti-cancer mechanisms of action of emodin, (3) address emodin's potential as a treatment complementary to standard chemotherapeutics, (4) assess the efficacy and bioavailability of emodin derivatives as they relate to cancer, and (5) evaluate the safety of emodin.