Proportion Of M2 Macrophages Research Articles

Effect of extracellular vesicles derived from inflammatory H9C2 cardiomyocytes on the polarization of macrophages and its mechanism

Abstract Objective The aim of this study was to investigate the effect of Extracellular vesicles (EVs) derived from inflammatory cardiomyocytes on the polarization of macrophages and its related mechanism. Methods EVs derived from the inflammatory and normal cardiomyocyte cell line H9C2 were extracted by differential centrifugation respectively. The extracted EVs were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western blot analysis (WB). The RAW264.7 macrophages in the inflammatory environment were treated with EVs, the proliferative activities were detected by CCK8, the expression of inflammatory factors were detected by qRT-PCR, and the differentiation of RAW264.7 macrophages into M1 and M2 was analyzed by flow cytometry. In vivo experiments, the animal model of viral myocarditis was established in Balb/c mice with intraperitoneal injection of CVB3. EVs derived from inflammatory H9C2 cardiomyocytes and medium were injected into mice by tail vein injection, respectively. The mice myocardium was stained by immunohistochemistry, and the total RNA of myocardial tissue was extracted for the detection of inflammatory factors by RT-PCR. In terms of mechanism research, mRNA expression profiles of RAW264.7 cells after EVs treatment were analyzed by RNA sequencing for functional annotation and mechanism study. At the same time, the extracted EVs derived from H9C2 normal group and the inflammatory group were subjected to 4D-Lable Free technology for quantitative proteomic analysis, and the results were analyzed bioinformatics, and then by WB analysis.Results We found that EVs derived from inflammatory H9C2 cardiomyocytes decreased the proliferative activity of RAW264.7 macrophages, the expression of pro-inflammatory cytokines IL-6, IL-1β, TNF-ɑ, and the proportion of M1 macrophages in the inflammatory state, while the proportion of M2 macrophages, which mainly inhibit inflammation, increased, and the expression levels of anti-inflammatory cytokines IL-10 and CD-206 increased. Results of immunohistochemical staining and RT-PCR showed that the EVs of inflammatory H9C2 regulated the heterogeneity of infiltrating macrophages and the expression of several inflammatory cytokines in mouse myocardium inflammatory tissue. Meanwhile, in vitro studies have found that the regulatory effect of EVs derived from inflammatory H9C2 cardiomyocytes on macrophage heterogeneity is mediated by MAPK signaling pathway. Conclusion EVs derived from inflammatory H9C2 cardiomyocytes can regulate the polarization of macrophages, change the expression of inflammatory factors, and regulate the myocardial inflammatory microenvironment, which may be achieved by mediating the p38 MAPK pathway through the delivery of PP2A.

Exosomal miR-106a-5p from highly metastatic colorectal cancer cells drives liver metastasis by inducing macrophage M2 polarization in the tumor microenvironment

BackgroundThe tumor microenvironment (TME) is a dynamic system orchestrated by intricate cell-to-cell crosstalk. Specifically, macrophages within the TME play a crucial role in driving tumor progression. Exosomes are key mediators of communication between tumor cells and the TME. However, the mechanisms underlying exosome-driven crosstalk between tumor cells and macrophages during colorectal cancer (CRC) progression remain incompletely elucidated.MethodsSingle-cell RNA sequencing were analyzed using the Seurat package. Exosomes were isolated using ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blot. miRNAs differentially expressed in exosomes were analyzed using the limma package. CD206 expression in CRC tissues, exosomes tracing, and exosomal miR-106a-5p transport were observed through immunofluorescence. Macrophage polarization was assessed via qRT-PCR, ELISA, and flow cytometry. The interactions between miR-106a-5p, hnRNPA1, and SOCS6 were evaluated using miRNA pull-down, RIP, and dual-luciferase reporter assays. Transwell assays and liver metastasis model explored the role of exosomal miR-106a-5p-induced M2 macrophages in promoting CRC liver metastasis.ResultThe proportion of M2 macrophages is increased in CRC with liver metastasis compared to those without. Highly metastatic CRC cells release exosomes enriched with miR-106a-5p, which promote macrophages M2 polarization by suppressing SOCS6 and activating JAK2/STAT3 pathway. These M2 macrophages reciprocally enhance CRC liver metastasis. hnRNPA1 regulate the transport of miR-106a-5p into exosomes. Clinically, elevated miR-106a-5p in plasma exosomes correlated with liver metastasis and poor prognosis.ConclusionCRC-derived exosomal miR-106a-5p plays a critical role in promoting liver metastasis and is a potential biomarker for the prevention and treatment of CRC liver metastasis.

"Comparative study of adipose-derived stem cells from localized scleroderma patients and healthy donors in treating skin fibrosis".

Localized scleroderma (LoS) is an autoimmune disease characterized by fibrosis of the skin and atrophy of the subcutaneous fat tissue. Adipose-derived mesenchymal stem cells (ASCs) is a promising treatment approach for LoS. However, ASCs from scleroderma patients (LoS-ASCs) have been shown to exhibit altered characteristics compared to ASCs from healthy donors (healthy-ASCs). This study aimed to compare the abilities of LoS-ASCs and healthy-ASCs in treating skin fibrosis. The paracrine ability of ASCs was tested with cytokine array. Bleomycin-challenged mice models received subcutaneous injection of LoS-ASCs and healthy-ASCs. Pathological staining and western blotting of COL1, α-SMA was performed. Fibroblasts derived from LoS lesions (LoS-FB) was co-cultured with ASCs, and subjected to RNA sequencing to further explore the similarities and differences in the treatment mechanism. In vivo comparison revealed that healthy ASCs had a stronger proliferation ability and secreted higher levels of growth factors and cytokines, including VEGFA, PDGFB, and IL-10. Pathological staining of the skin in mice models treated with ASCs demonstrated that healthy ASCs were more effective in reducing dermal thickness and collagen deposition, while increasing microvessel density and the proportion of M2 macrophages. Co-culture with both healthy-ASCs and LoS-ASCs reduced the proliferation and migration abilities of LoS-FB, as well as the protein expression of α-SMA and COL1. RNA sequencing and validation revealed potential difference in the canonical Wnt pathway. Healthy-ASCs exhibited stronger proliferation, paracrine, anti-fibrosis, pro-angiogenesis, and immunomodulation abilities in treating skin fibrosis in scleroderma mouse models. Allogenic ASCs obtained from healthy donors are more efficient in treating skin fibrosis, and could serve as a potential alternative for patients who are not suitable candidates for liposuction surgery in the future.

Differential regulation of radio-sensitivity/macrophage polarization in intestinal tissue and colorectal cancer for the optimized preoperative radiotherapy

Surgery is the preferred treatment for colorectal cancer, and the preoperative radiotherapy (pre-RT) improves surgical success and postoperative quality of life for patients. However, the therapeutic effect of pre-RT is limited by radiation resistance of tumor and radiation susceptibility of intestine, probably resulting in poor tumor suppression and postoperative anastomotic injury. Herein, we select carboxymethyl chitosan (CMCS) to complex with cerium oxide (CeO2) and calcium ion (Ca2+), obtaining CMCS/CeO2&Ca nano-enema for the optimized pre-RT of colorectal cancer. CMCS/CeO2 and CMCS/CeO2&Ca can mop up various kinds of reactive oxygen species (ROS) and decomposes H2O2 that largely exists in tumor tissue into OH and oxygen (some OH in acidic environment and main oxygen in neutral environment), enhancing free radical clearance in normal tissue and radiation sensitivity of tumor. Meanwhile, RT-induced M1 macrophage in intestinal tissue and the proportion of M2 macrophages in X-rays-treated tumor could be reduced, owing to CMCS/CeO2&Ca-induced change of ROS level. Moreover, CMCS/CeO2&Ca could also promote the proliferation of stem cells in intestine. Therefore, CMCS/CeO2&Ca improves the radio-immunotherapy outcome of colorectal cancer and reduces RT-induced intestinal inflammation/fibrosis, providing an optimal scheme and an effective enema for pre-RT for colorectal cancer.

Natural Derivatives of Selective HDAC8 Inhibitors with Potent in Vivo Antitumor Efficacy against Breast Cancer.

HDAC8 is a therapeutic target with great promise for breast cancer. Here, we reported a novel compound corallorazine D from Nocardiopsis sp. XZB108, selectively inhibited HDAC8 (IC50 = 0.90 ± 0.014 μM), suggesting that it may be a promising nonhydroxamate HDAC8 inhibitor. Upon additional modifications of corallorazine D, a candidate compound 5k, demonstrated remarkable inhibitory potency against HDAC8 (IC50 = 0.12 ± 0.01 nM), 89-fold superior to PCI-34051. The selectivity of 5k was at least 439-fold, superior to corallorazine D, confirming the efficacy of our modifications. In an orthotopic mouse model of breast cancer, 5k displayed nearly 4-fold superior antitumor activity than SAHA. Furthermore, 5k triggered antitumor immunity by activating T cells. Treatment with 5k significantly increased the proportion of M1 macrophages and decreased the proportion of M2 macrophages (M1/M2 ratio = 2.67 ± 0.25). 5k represents a promising compound for further investigation as a potential treatment for breast cancer.

11 Tumor evolution of brain-specific tropism in metastatic renal cell carcinoma

Abstract Background Brain metastases (BMets) pose a clinical challenge in the management of patients (pts) with metastatic renal cell carcinoma (RCC), leading to significant morbidity and mortality. Herein, we sought to comprehensively characterize the molecular landscape of BMets in RCC. Methods We performed panel-based DNA (DNAseq) and whole transcriptome (RNAseq) sequencing to analyze BMets, matched non-Brain metastases (nBMets), and matched primary renal tumors (PRT) from pts who underwent surgical resection of BMet(s) from RCC at our institution. Results Our cohort consisted of 95 samples from 53 patients (BMets = 54, nBMets = 14, PRT = 27) with clear cell histology in 45 pts (84.9%). DNAseq was available on 85 samples (50 pts). Patient-level mutations revealed recurring mutations in VHL (18 pts, 36%), TP53 (n = 12 pts, 24%), PBRM1 (12 pts, 24%), SETD2 (12 pts, 24%), and BAP1 (4 pts, 8%). Mutations within the MTOR signaling pathway were enriched, with 25 pts (50%) having at least one mutation in PTEN (12 pts, 24%), TSC1 (4 pts, 8%), TSC2 (5 pts, 10%), or MTOR (9 pts, 18%). Copy number analyses revealed frequent deletions in chr14q21 (34 pts, 68%) and chr9p21 (35 pts, 70%) and gains in chr20q13 (n = 31, 62%) and chr7p15 (n = 31, 62%). At the sample-level, PTEN mutations were more common in BMets (11 pts, 22.9%) vs nBMets (0 pts) and PRTs (3 pts, 12%, p = 0.124), as were deletions in chr13q22 (BMets = 13pts [26.5%], nBMet = 1pts [8.3%], PRT = 1 [4%]; p = 0.036) and gains in chr20q13 (BMets = 30pts [61.2%], nBMet = 4pts [33.3%], PRT = 8 [32%]; p = 0.03). Deletions in chr9p21 were enriched in BMets (32, 65.3%) and nBMets (8, 66.7%) relative to PRTs (7, 28%, p = 0.006), whereas other copy number alterations and somatic mutations exhibited similar proportions across specimen sites. Differential expression analysis performed on 86 samples (51 pts) identified 806 differentially expressed genes (DEGs) between BMets and PRT (adjusted [adj.] p < 0.05) and 399 DEGs between BMet and nBMets (adj. p < 0.05). Gene set enrichment analysis of MSigDB Hallmark gene sets revealed upregulation of MTORC1 signaling, glycolysis, and MYC targets in BMets comparted to PRT and nBMETs (adj. p <0.0001). In contrast, immune-related gene sets, such as interferon-alpha, interferon-gamma, and tumor necrosis factor-alpha, were enriched in PRT relative to BMets (adj. p <0.0001), but not nBMETs (adj. p > 0.05). CIBERSORT cellular deconvolution analysis comparing BMets with PRT revealed decreased proportions of M1 macrophages (p = 0.0003) and CD8+ T-cells (p = 0.0106), but an increased proportion of M2 macrophages (p = 0.0009). Conclusions RCC with brain metastases are characterized by distinct copy number alterations, enrichment of MTOR pathway mutations, MTOR pathway hyperactivation, and an immunosuppressive tumor milieu. These findings may hold therapeutic implications of MTOR pathway inhibition and immune modulators in treating RCC BMets.

Aucubin suppresses TLR4/NF-κB signalling to shift macrophages toward M2 phenotype in glucocorticoid-associated osteonecrosis of the femoral head.

In this study, we investigated whether the ability of aucubin to mitigate the pathology of GONFH involves suppression of TLR4/NF-κB signalling and promotion of macrophage polarization to an M2 phenotype. In necrotic bone tissues from GONFH patients, we compared levels of pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages as well as levels of TLR4/NF-κB signalling. In a rat model of GONFH, we examined the effects of aucubin on these parameters. We further explored its mechanism of action in a cell culture model of M1 macrophages. Necrotic bone tissues from GONFH patients contained a significantly increased macrophage M1/M2 ratio, and higher levels of TLR4, MYD88 and NF-κB p65 than bone tissues from patients with hip osteoarthritis. Treating GONFH rats with aucubin mitigated bone necrosis and demineralization as well as destruction of trabecular bone and marrow in a dose-dependent manner, based on micro-computed tomography. These therapeutic effects were associated with a decrease in the overall number of macrophages, decrease in the proportion of M1 macrophages, increase in the proportion of M2 macrophages, and downregulation of TLR4, MYD88 and NF-κB p65. These effects invivo were confirmed by treating cultures of M1 macrophage-like cells with aucubin. Aucubin mitigates bone pathology in GONFH by suppressing TLR4/NF-κB signalling to shift macrophages from a pro- to anti-inflammatory phenotype.

The correlation between TRIM28 expression and immune checkpoints in CRPC.

This study delves into the unexplored realm of castration-resistant prostate cancer (CRPC) by investigating the role of TRIM28 and its intricate molecular mechanisms using high-throughput single-cell transcriptome sequencing and advanced bioinformatics analysis. Our comprehensive examination unveiled dynamic TRIM28 expression changes, particularly in immune cells such as macrophages and CD8+ T cells within CRPC. Correlation analyses with TCGA data highlighted the connection between TRIM28 and immune checkpoint expression and emphasized its pivotal influence on the quantity and functionality of immune cells. Using TRIM28 knockout mouse models, we identified differentially expressed genes and enriched pathways, unraveling the potential regulatory involvement of TRIM28 in the cGAS-STING pathway. Invitro, experiments further illuminated that TRIM28 knockout in prostate cancer cells induced a notable anti-tumor immune effect by inhibiting M2 macrophage polarization and enhancing CD8+ T cell activity. This impactful discovery was validated in an insitu transplant tumor model, where TRIM28 knockout exhibited a deceleration in tumor growth, reduced proportions of M2 macrophages, and enhanced infiltration of CD8+ T cells. In summary, this study elucidates the hitherto unknown anti-tumor immune role of TRIM28 in CRPC and unravels its potential regulatory mechanism via the cGAS-STING signaling pathway. These findings provide novel insights into the immune landscape of CRPC, offering promising directions for developing innovative therapeutic strategies.

Direct Current Electrical Stimulation Shifts THP-1-Derived Macrophage Polarization towards Pro-Regenerative M2 Phenotype.

A macrophage shift from the M1 to the M2 phenotype is relevant for promoting tissue repair and regeneration. In a previous in vivo study, we found that direct current (DC) electrical stimulation (EStim) increased the proportion of M2 macrophages in healing tissues and directed the balance of the injury response away from healing/scarring towards regeneration. These observations led us to hypothesize that DC EStim regulates macrophage polarization towards an M2 phenotype. THP-1-derived M0, M1 (IFN-γ and LPS), and M2 (IL-4 and IL-13) macrophages were exposed (or not: control group) to 100 mV/mm of DC EStim, 1 h/day for three days. Macrophage polarization was assessed through gene and surface marker expressions and cytokine secretion profiles. Following DC EStim treatment, M0 cells exhibited an upregulation of M2 marker genes IL10, CD163, and PPARG. In M1 cells, DC EStim upregulated the gene expressions of M2 markers IL10, TGM2, and CD206 and downregulated M1 marker gene CD86. EStim treatment also reduced the surface expression of CD86 and secretion of pro-inflammatory cytokines IL-1β and IL-6. Our results suggest that DC EStim differentially exerts pro-M2 effects depending on the macrophage phenotype: it upregulates typical M2 genes in M0 and M1 cells while inhibiting M1 marker CD86 at the nuclear and protein levels and the secretion of pro-inflammatory interleukins in M1 cells. Conversely, M2 cells appear to be less responsive to the EStim treatment employed in this study.

Downregulation of Setdb2 promotes alternative activation of macrophages via the PI3K/Akt pathway to attenuate NAFLD after sleeve gastrectomy

Non-alcoholic fatty liver disease (NAFLD) stands as the most prevalent hepatic disorder, with bariatric surgery emerging as the most effective intervention for NAFLD remission. Sleeve gastrectomy (SG) has notably ascended as the predominant procedure due to its comparative simplicity and consistent surgical outcomes. Nonetheless, the underlying mechanisms remain unclear. In this study, we probed the therapeutic potential of SG for NAFLD induced by a high-fat diet (HFD) in mice, with a focus on its impact on liver lipid accumulation, macrophage polarization, and the role of the histone methyltransferase Setdb2. SG prompted significant weight loss, diminished liver size and liver-to-body weight ratio, and enhanced liver function, evidenced by reduced serum levels of triglycerides (TG), total cholesterol (T-CHO), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Histological examination confirmed a reduction in liver lipid accumulation. Additionally, flow cytometry unveiled an increased proportion of M2 macrophages and a decrease in Setdb2 expression was shown in the SG group, suggesting an association between Setdb2 levels and postsurgical macrophage polarization. Furthermore, the conditional knockout of Setdb2 in mice further mitigated HFD-induced steatosis and promoted the M2 macrophage phenotype. Mechanistically, Setdb2 knockout in bone marrow-derived macrophages (BMDMs) favored M2 polarization, with RNA sequencing and western blotting analyses corroborating the upregulation of the PI3K/Akt signaling pathway. The effects of Setdb2 on macrophage activation were nullified by the PI3K inhibitor LY294002, suggesting that Setdb2 facilitates alternative macrophage activation through the PI3K/Akt signaling pathway. These comprehensive findings underscore the potential of SG as a therapeutic intervention for NAFLD by regulating the critical function of Setdb2 in macrophage polarization and activation, thereby offering novel insights into NAFLD pathogenesis and therapeutic targets.

Tumor evolution of brain-specific tropism in metastatic renal cell carcinoma.

4530 Background: Brain metastases (BMets) pose a clinical challenge in the management of patients (pts) with metastatic renal cell carcinoma (RCC), leading to significant morbidity and mortality. Herein, we sought to comprehensively characterize the molecular landscape of BMets in RCC. Methods: We performed panel-based DNA (DNAseq) and whole transcriptome (RNAseq) sequencing to analyze BMets, matched non-Brain metastases (nBMets), and matched primary renal tumors (PRT) from pts who underwent surgical resection of BMet(s) from RCC at our institution. Results: Our cohort consisted of 95 samples from 53 patients (BMets = 54, nBMets = 14, PRT = 27) with clear cell histology in 45 pts (84.9%). DNAseq was available 85 samples (50 pts). Patient-level mutations revealed recurring mutations in VHL(18 pts, 36%), TP53 (n = 12 pts, 24%), PBRM1(12 pts, 24%), SETD2(12 pts, 24%), and BAP1(4 pts, 8%). Mutations within the MTOR signaling pathway were enriched, with 25 pts (50%) having at least one mutation in PTEN (12 pts, 24%), TSC1 (4 pts, 8%), TSC2 (5 pts, 10%), or MTOR (9 pts, 18%). Copy number analyses revealed frequent deletions in chr14q21 (34 pts, 68%) and chr9p21 (35 pts, 70%) and gains in chr20q13 (n = 31, 62%) and chr7p15 (n = 31, 62%). At the sample-level, PTEN mutations were more common in BMets (11 pts, 22.9%) vs nBMets (0 pts) and PRTs (3 pts, 12%, p = 0.124), as were deletions in chr13q22 (BMets = 13pts [26.5%], nBMet = 1pts [8.3%], PRT = 1 [4%]; p = 0.036) and gains in chr20q13 (BMets = 30pts [61.2%], nBMet = 4pts [33.3%], PRT = 8 [32%]; p = 0.03). Deletions in chr9p21 were enriched in BMets (32, 65.3%) and nBMets (8, 66.7%) relative to PRTs (7, 28%, p = 0.006), whereas other copy number alterations and somatic mutations exhibited similar proportions across specimen sites. Differential expression analysis performed on 86 samples (51 pts) identified 806 differentially expressed genes (DEGs) between BMets and PRT (adjusted [adj.] p < 0.05) and 399 DEGs between BMet and nBMets (adj. p < 0.05). Gene set enrichment analysis of MSigDB Hallmark gene sets revealed upregulation of MTORC1 signaling, glycolysis, and MYC targets in BMets comparted to PRT and nBMETs (adj. p <0.0001). In contrast, immune-related gene sets, such as interferon-alpha, interferon-gamma, and tumor necrosis factor-alpha, were enriched in PRT relative BMets (adj. p <0.0001), but not nBMETs (adj. p > 0.05). CIBERSORT cellular deconvolution analysis comparing BMets with PRT revealed decreased proportions of M1 macrophages (p = 0.0003) and CD8+ T-cells (p = 0.0106), but an increase proportion of M2 macrophages (p = 0.0009). Conclusions: RCC with brain metastases are characterized by distinct copy number alterations, enrichment of MTOR pathway mutations, MTOR pathway hyperactivation, and an immunosuppressive tumor milieu. These findings may hold therapeutic implications of MTOR pathway inhibition and immune modulators in treating RCC BMets.

IL-4 attenuates myocardial infarction injury by promoting M2 macrophage polarization

IL-4, an immunoregulatory cytokine, plays a role in various cellular pathways and is known to regulate M2 macrophage polarization. Numerous studies have suggested that promoting the polarization of macrophages toward the M2 phenotype is beneficial for myocardial infarction (MI) recovery. However, whether IL-4 can achieve therapeutic effects in MI by regulating M2 macrophage polarization remains unclear. In this study, the authors observed that IL-4 increased the proportion of M2 macrophages in the ischemic myocardium compared to the PBS group. Additionally, IL-4 reduced the infiltration of inflammatory cells and the expression of proinflammatory-related proteins, while enhancing the expression of genes associated with tissue repair. Furthermore, IL-4 facilitated the recovery of cardiac function and reduced fibrosis in the post-MI phase. Importantly, when macrophages were depleted, the therapeutic benefits of IL-4 mentioned above were attenuated. These findings provide evidence for the effectiveness of IL-4 in treating MI through the regulation of M2 macrophage polarization, thereby encouraging further development of this therapeutic approach.

Enhancement of Bone Tissue Regeneration with Multi-Functional Nanoparticles by Coordination of Immune, Osteogenic, and Angiogenic Responses.

Inorganic nanoparticles are promising materials for bone tissue engineering due to their chemical resemblance to the native bone structure. However, most studies are unable to capture the entirety of the defective environment, providing limited bone regenerative abilities. Hence, this study aims to develop a multifunctional nanoparticle to collectively control the defective bone niche, including immune, angiogenic, and osteogenic systems. The nanoparticles, self-assembled by biomimetic mineralization and tannic acid (TA)-mediated metal-polyphenol network (MPN), are released sustainably after the incorporation within a gelatin cryogel. The released nanoparticles display a reduction in M1 macrophages by means of reactive oxygen species (ROS) elimination. Consequently, osteoclast maturation is also reduced, which is observed by the minimal formation of multinucleated cells (0.4%). Furthermore, the proportion of M2 macrophages, osteogenic differentiation, and angiogenic potential are consistently increased by the effects of magnesium from the nanoparticles. This orchestrated control of multiple systems influences the in vivo vascularized bone regeneration in which 80% of the critical-sized bone defect is regenerated with new bones with mature lamellar structure and arteriole-scale micro-vessels. Altogether, this study emphasizes the importance of the coordinated modulation of immune, osteogenic, and angiogenic systems at the bone defect site for robust bone regeneration.

Abstract PO4-24-12: Premenopausal breast cancers show higher proportion of immunologically cold tumors and are associated with distinct immune cell infiltrate

Abstract Background Breast cancers (BC) in the young women tend to have poor outcomes and higher proportion of aggressive subtypes. Immunotherapy has shown promising results lately, especially for aggressive categories like triple negative breast cancers (TNBC). Success of the immunotherapy is shown to be dependent on the tumor immune microenvironment (TIME) which plays important role in disease progression and response to therapy. Though numerous studies have demonstrated immune hot tumors are associated with better prognosis, mechanism of immunologically hot TIME is not yet deciphered. Material and methods Gene expression data derived from RNA sequencing (n=40) of primary BC was used to divide them into immune hot and cold tumors by using 15 gene signature (Wang et al., Sci. Adv. 2021). Average expression of the hot and cold (hot=12 and cold=3) genes was derived for each tumor and median value of their ratio was used to categorize them into immunologically hot and cold tumors. Association of the hot and cold tumors with clinical characteristics was examined. CIBERSORT algorithm was used to identify immune cell subtypes and Kaplan Meier survival analysis was performed to assess the prognostic significance. Similar analysis was replicated in TCGA (n=1083) dataset. Results Equal proportion of immune hot and cold tumor was observed in the cohort. Higher proportion of cold tumors were observed in younger and premenopausal (p=0.002) patients. Subtype analysis showed higher proportion of cold tumors in TNBC compared to hormone receptor positive tumors (p< 0.0001). No significant difference was observed in other clinicopathological characteristics between the groups. Further analysis of cold tumors for immune cell subtypes, categorized by the menopausal status showed higher proportion of plasma cells and CD4 memory resting cells and lower proportion of M2 macrophages and B memory cell in premenopausal (n=129, TCGA) compared postmenopausal (n=322, TCGA) tumors. In addition, premenopausal cold tumors were associated with better overall survival compared to postmenopausal group (p=0.001). Conclusion Younger premenopausal women with BC are likely to have more immunologically cold tumors. Though circulating levels of sex steroid hormones is the predominant difference between the pre and postmenopausal women, its influence on TIME and therapeutic significance must be explored. Citation Format: Vidya P Nimbalkar, Snijesh VP, Savitha Rajarajan, Annie Alexander, Rakesh Ramesh, B S Srinath, Jyothi S Prabhu. Premenopausal breast cancers show higher proportion of immunologically cold tumors and are associated with distinct immune cell infiltrate [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-12.

23-Hydroxybetulinic acid attenuates 5-fluorouracil resistance of colorectal cancer by modulating M2 macrophage polarization via STAT6 signaling

Macrophage polarization is closely associated with the inflammatory processes involved in the development and chemoresistance of colorectal cancer (CRC). M2 macrophages, the predominant subtype of tumor-associated macrophages (TAMs) in a wide variety of malignancies, have been demonstrated to promote the resistance of CRC to multiple chemotherapeutic drugs, such as 5-fluorouracil (5-FU). In our study, we investigated the potential of 23-Hydroxybetulinic Acid (23-HBA), a significant active component of Pulsatilla chinensis (P. chinensis), to inhibit the polarization of M2 macrophages induced by IL-4. Our results showed that 23-HBA reduced the expression of M2 specific marker CD206, while downregulating the mRNA levels of M2 related genes (CD206, Arg1, IL-10, and CCL2). Additionally, 23-HBA effectively attenuated the inhibitory effects of the conditioned medium from M2 macrophages on apoptosis in colorectal cancer SW480 cells. Mechanistically, 23-HBA prevented the phosphorylation and nuclear translocation of the STAT6 protein, resulting in the inhibition of IL-10 release in M2 macrophages. Moreover, it interfered with the activation of the IL-10/STAT3/Bcl-2 signaling pathway in SW480 cells, ultimately reducing M2 macrophage-induced resistance to 5-FU. Importantly, depleting STAT6 expression in macrophages abolished the suppressive effect of 23-HBA on M2 macrophage polarization, while also eliminating its ability to decrease M2 macrophage-induced 5-FU resistance in cancer cells. Furthermore, 23-HBA significantly diminished the proportion of M2 macrophages in the tumor tissues of colorectal cancer mice, simultaneously enhancing the anti-cancer efficacy of 5-FU. The findings presented in this study highlight the capacity of 23-HBA to inhibit M2 macrophage polarization, a process that contributes to reduced 5-FU resistance in colorectal cancer.

Mesenchymal stem cell-derived exosomes promote tissue repair injury in rats with liver trauma by regulating gut microbiota and metabolism

ObjectiveThe effects of mesenchymal stem cells (MSCs) and MSC-derived exosomes (MSC-exos) on serum metabolites and intestinal microbiota in rats after liver trauma were discussed. MethodsAdult Wistar Albino rats were assigned into control, model (liver trauma), MSCs, and MSC-exos groups (n = 6). The study examined changes in the inflammatory environment in liver tissues were analyzed by histological examination and analysis of macrophage phenotypes. Alterations in serum metabolites were determined by untargeted metabonomics, and gut microbiota composition was characterized by 16S rDNA sequencing. Correlations between specific gut microbiota, metabolites, and inflammatory response were calculated using Spearman correlation analysis. ResultsRats with liver trauma after MSCs and MSC-exos treatment exhibited attenuated inflammatory infiltration and necrosis in liver tissues. MSCs and MSC-exos treatment reduced the proportion of M1 macrophages, accompanied by a decrease in inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α) levels. Furthermore, MSCs and MSC-exos treatment expanded the proportion of M2 macrophages, accompanied by an increase in arginase-1 (Arg-1) and interleukin-10 (IL-10) levels. The beneficial effects of MSC-exo treatment on rats with liver trauma were superior to those of MSC treatment. The composition and abundance of the gut microbiota and metabolites were altered in pathological rats, whereas MSC and MSC-exo intervention partially restored specific gut microbiota and metabolite alterations. At the phylum level, alterations in Bacteroidota, Proteobacteria, and Verrucomicrobiota were observed after MSC and MSC-exo intervention. At the genus level, Intestinimonas, Alistipes, Aerococcus, Faecalibaculum, and Lachnospiraceae_ND3007_group were the main differential microbiota. 6-Methylnicotinamide, N-Methylnicotinamide, Glutathione, oxidized, ISOBUTYRATE, ASCORBATE, EICOSAPENTAENOATE, GLYCEROL 3-PHOSPHATE, and Ascorbate radical were selected as important differential metabolites. There was a clear correlation between Ascorbate, Intestinimonas/Faecalibaculum and inflammatory cytokines. ConclusionMSC-exos promoted the repair of tissue damage in rats with liver trauma by regulating serum metabolites and intestinal microbiota, providing new insights into how MSC-exos reduced inflammation in rats with liver trauma.

Analysis of the effect of CCR7 on the microenvironment of mouse oral squamous cell carcinoma by single-cell RNA sequencing technology

BackgroundStudies have shown that CCR7, an important inflammatory factor, can promote the proliferation and metastasis of oral squamous cell carcinoma (OSCC), but its role in the tumor microenvironment (TME) remains unclear. This paper explores the role of CCR7 in the TME of OSCC.MethodsIn this work, we constructed CCR7 gene knockout mice and OSCC mouse models. Single-cell RNA sequencing (scRNA-seq) and bioinformatics were used to analyze the differences in the OSCC microenvironment between three CCR7 gene knockout mice (KO) and three wild-type mice (WT). Immunohistochemistry, immunofluorescence staining, and flow cytometry were used to analyze the expression of key genes in significantly different cell types between the KO and WT groups. An in vitro experiment was used to verify the effect of CCR7 on M2 macrophage polarization.ResultsIn the mouse OSCC models, the tumor growth rate in the KO group was significantly lower than that in the WT group. Eight main cell types (including tumor cells, fibroblasts, macrophages, granulocytes, T cells, endothelial cells, monocytes, and B cells) were identified by Seurat analysis. The scRNA-seq results showed that the proportion of tumor cells was lower, but the proportion of inflammatory cells was significantly higher in the KO group than in the WT group. CellPhoneDB analysis results indicated a strong interaction relationship between tumor cells and macrophages, T cells, fibroblasts, and endothelial cells. Functional enrichment results indicated that the expression level of the Dusp1 gene in the KO group was generally higher than that in the WT group in various cell types. Macrophage subclustering results indicated that the proportion of M2 macrophages in the KO group was lower than that in the WT group. In vitro experimental results showed that CCR7 can promote M2 macrophage polarization, thus promoting the proliferation, invasion and migration of OSCC cells.ConclusionsCCR7 gene knockout can significantly inhibit the growth of mouse oral squamous cell carcinoma by promoting the polarization of M2 macrophages.

Abstract 3727: Synergistic effect of CD47 blockade and EPHA3 treatment on recurrent head and neck cancers

Abstract CD47 acts as a “don’t eat me “signal and suppresses it through its interaction with signal regulatory protein (SIRP-a) on the macrophage. CD47 is expressed in many human cancer cells, including head and neck cancer. Additionally, we reported in a previous study that EphA3 is overexpressed in radioresistant head and neck cancer and may play an important role in the development of radioresistance in head and neck cancer. In our present study, we demonstrated that the blockade of EPHA3 and CD47 attenuates the function of M2-like macrophages and may contribute to macrophage polarization toward the M1 phenotype. Here, we combined a therapeutic strategy of an anti-EPHA3 and an anti-CD47 antibody. Using mRNA sequencing and fluorescence imaging, we showed that the combination treatment could significantly enhance the effectiveness of anti-EPHA3 and anti-CD47 antibodies, in reactivating macrophages and reversing macrophage polarization. In addition to using flow cytometry in vivo, combination treatments can alter the proportion of tumor-associated macrophages (TAMs). Collectively, our findings suggested that the combination treatment of anti-EphA3 and the anti-CD47 antibodies could significantly improve tumor suppression, increase the proportion of M1 macrophages, and decrease the proportion of M2 macrophages. Citation Format: Song Hee Kim, Myung Woul Han, Ji Won Kim, Seong Who Kim. Synergistic effect of CD47 blockade and EPHA3 treatment on recurrent head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3727.

The ERK-cPLA2-ACSL4 axis mediating M2 macrophages ferroptosis impedes mucosal healing in ulcerative colitis

Ulcerative colitis (UC) is a chronic gastrointestinal disease that can be managed with 5-aminosalicylic acid (5-ASA), the standard treatment for UC. However, the effectiveness of 5-ASA is not always optimal. Our study revealed that despite 5-ASA treatment, cells continued to experience excessive ferroptosis, which may hinder mucosal healing in UC and limit the success of this treatment approach in achieving disease remission. We found that combining 5-ASA with the ferroptosis inhibitor Fer-1 led to a significant inhibition of ferroptosis in macrophages present in the colon tissue, along with an increase in the proportion of M2 macrophages, suggesting that targeting ferroptosis in M2 macrophages could be a potential therapeutic strategy for alleviating UC. Our study also demonstrated that M2 macrophages are more susceptible to ferroptosis compared to M1 macrophages, and this susceptibility is associated with the activated arachidonic acid (AA) metabolism pathway mediated by ERK-cPLA2-ACSL4. Additionally, we found that the expression of cPLA2 gene pla2g4a was increased in the colon of UC patients compared to healthy controls. Furthermore, targeted metabolomics analysis revealed that the combination treatment group, as opposed to the 5-ASA treatment group, exhibited the ability to modulate AA metabolism. Overall, our findings emphasize the importance of addressing macrophage ferroptosis in order to enhance macrophage anti-inflammation, improve mucosal healing, and achieve better therapeutic outcomes for patients with UC.

Repositioning baloxavir marboxil as VISTA agonist that ameliorates experimental asthma

V-type immunoglobulin domain–containing suppressor of T-cell activation (VISTA), a novel negative checkpoint regulator, plays an essential role in allergic pulmonary inflammation in mice. Treatment with a VISTA agonistic antibody could significantly improve asthma symptoms. Thus, for allergic asthma treatment, VISTA targeting may be a compelling approach. In this study, we examined the functional mechanism of VISTA in allergic pulmonary inflammation and screened the FDA-approved drugs for VISTA agonists. By using mass cytometry (CyTOF), we found that VISTA deficiency primarily increased lung macrophage infiltration in the OVA-induced asthma model, accompanied by an increased proportion of M1 macrophages (CD11b+F4/80+CD86+) and a decreased proportion of M2 macrophages (CD11b+F4/80+CD206+). Further in vitro studies showed that VISTA deficiency promoted M1 polarization and inhibited M2 polarization of bone marrow–derived macrophages (BMDMs). Importantly, we discovered baloxavir marboxil (BXM) as a VISTA agonist by virtual screening of FDA-approved drugs. The surface plasmon resonance (SPR) assays revealed that BXM (KD = 1.07 µM) as well as its active form, baloxavir acid (BXA) (KD = 0.21 µM), could directly bind to VISTA with high affinity. Notably, treatment with BXM significantly ameliorated asthma symptoms, including less lung inflammation, mucus secretion, and the generation of Th2 cytokines (IL-5, IL-13, and IL-4), which were dramatically attenuated by anti-VISTA monoclonal antibody treatment. BXM administration also reduced the pulmonary infiltration of M1 macrophages and raised M2 macrophages. Collectively, our study indicates that VISTA regulates pulmonary inflammation in allergic asthma by regulating macrophage polarization and baloxavir marboxil, and an old drug might be a new treatment for allergic asthma through targeting VISTA.