CD163+ Tumor-Associated Macrophage Recruitment Predicts Papillary Thyroid Cancer Recurrence.

INTRODUCTION: Skewed immune response plays a pivotal role in tumor progression. Systemic inflammatory responses represented by combined peripheral leukocyte fractions are prognostic predictors of multiple cancers, including thyroid cancer. We previously reported the prognostic significance of lymphocyte-to-monocyte ratio (LMR) in curatively resected papillary thyroid cancer (PTC). Therefore, this study aimed to analyze immune cell profiles in the tumor microenvironment and their association with LMR in curatively resected PTC. MATERIALS AND METHODS: The immune cell profiles of primary tumors in 162 patients with curatively resected PTC were analyzed clinicopathologically. Immunohistochemistry of tumor-associated macrophages (TAMs), myeloid-derived suppressor cells, and lymphocytes was performed using CD163, CD33, and CD3 antibodies, respectively. Prognostic analysis and correlation assays were performed using the immunocyte profiles. The gene expression of tumor-derived chemokines was assessed using a The Cancer Genome Atlas database. RESULTS: Patients with a higher density of CD163+ TAMs exhibited a significantly worse prognosis than their counterparts (10-y recurrence-free survival: 80.9% versus 91.2%, P = 0.011). Multivariate prognostic analyses revealed that high CD163+ cell density (P = 0.011), low preoperative LMR (P = 0.003), pN1b (P = 0.005), and high thyroglobulin level (P = 0.038) were independent predictors of recurrence. High CD163+ cell density had a prognostic impact on stage II and III PTC. Interestingly, high CD163+ cell density correlated with low LMR and high monocyte fraction in peripheral blood. Indeed, the expression of TAM-inducible, tumor-derived chemokines is increased in the The Cancer Genome Atlas database. CONCLUSIONS: A high density of infiltrated CD163+ TAMs predicts recurrence in correlation with low LMR and circulating monocyte accumulation. Thus, TAMs should be considered when assessing advanced PTC.

The roles of glioma-associated macrophages/microglia and potential targets for anti-glioma therapy.

Glioblastoma (GBM) is the central nervous system tumor with the most aggressive behavior, and no definitive therapy has yet been found. The tumor microenvironment of GBM is immunosuppressive and is considered a 'cold tumor' with low lymphocytic infiltration, but is characterized by a high proportion of glioma-associated macrophages/microglia (GAMs). GAMs promote tumor growth and also affect treatment resistance in GBM. In this review, we describe the origin and classification of GAMs in humans and describe the mechanisms of their activation and the cell-cell interactions between tumor cells and GAMs. We also describe the history of GAM detection methods, especially immunohistochemistry, and discusses the merits and limitations of these techniques. In addition, we summarized chemotactic factors for GAMs and the therapies targeting these factors. Recent single-cell RNA analysis and spatial analysis add new insights to our previous knowledge of GAMs. Based on these studies, GBM therapies targeting GAMs are expected to be further developed.

Tumor-derived IL-6 promotes chordoma invasion by stimulating tumor-associated macrophages M2 polarization and TNFα secretion.

AIMS: Chordoma is a rare and aggressive bone tumor with high-recurrence and lack of effective treatment methods. Tumor associated macrophages (TAMs) are abundant in tumor microenvironment (TME) and polarize toward M2 in chordoma. It has been observed that the high proportion of M2 cells is associated with chordoma rapid progression. However, the mechanism of TAMs polarization and promotion to tumor progression in chordoma is still unclear. The is an urgent need for further research. MATERIALS AND METHODS: Flow cytometry and immunohistochemical staining was used to detect the degree of macrophages infiltration in chordoma. A co-culture model of chordoma cells and macrophages was established in vitro to investigate the effects of their interaction on cell function, cytokine secretion, and RNA transcriptome expression. KEY FINDINGS: In this study, we found M2 macrophage was predominantly abundant immune cell population in chordoma, and its proportion was associated with the degree of bone destruction. We demonstrated that interleukin 6 (IL-6) derived from chordoma cells could induce TAMs polarization by activating STAT3 phosphorylation, and TAMs could enhance chordoma cells migration and invasion through TNFα/NF-κB pathway. The interaction of chordoma cells and TAMs could promote the bone destruction-related factor Cathepsin B (CTSB) and inhibitory immune checkpoints expression. We also confirmed blocking IL-6/STAT3 pathway could significantly attenuate the M2 polarization of TAMs and decrease the secretion of TNFα. SIGNIFICANCE: This study illustrates the dynamics between chordoma cells and TAMs in promoting chordoma invasion and suggests that IL-6/STAT3 pathway is a potential therapeutic target to reduce TAM-induced chordoma invasion.

In vivo self-assembled albumin nanoparticle elicit antitumor immunity of PD-1 inhibitor by imaging and clearing tumor-associated macrophages.

Eliciting anti-tumor immune responses and improving the tumor microenvironment crucial for boosting the effectiveness of anti-PD-1 immunotherapy. Tumor-associated macrophages (TAMs), the primary types of immune cells infiltrating tumors, play a critical role in the formation of an immunosuppressive microenvironment. In this study, we constructed a novel Evans Blue (EB)-based in vivo self-assembled nanocarrier system, mUNO-EB-ICG-Fc@Alb nanoparticles (designated as MA NPs), for targeted imaging and clearance of M2-TAMs to elicit antitumor immunotherapy of PD-1 inhibitor. In vitro experiments demonstrated the specific fluorescence imaging and killing effect of MA NPs on M2-TAMs. In vivo experiments shown that MA NPs-induced chemodynamic therapy (CDT) successfully reversed the tumor immunosuppressive microenvironment (ITM), promoted intratumoral infiltration of T lymphocytes, and ultimately enhancing the anti-tumor immunotherapy effect of PD-1 inhibitors. This study might provide good inspiration for improving the therapeutic efficacy of cancer immunotherapy.

SPOP downregulation promotes bladder cancer progression based on cancer cell-macrophage crosstalk via STAT3/CCL2/IL-6 axis and is regulated by VEZF1.

Background: Cancer cells are intimately intertwined with tumor microenvironment (TME), fostering a symbiotic relationship propelling cancer progression. However, the interaction between cancer cells and tumor-associated macrophages (TAMs) in urothelial bladder cancer (UBC) remains poorly understood. Methods: UBC cell lines (5637, T24 and SW780), along with a monocytic cell line (U937) capable of differentiating into macrophage, were used in a co-culture system for cell proliferation and stemness by MTT, sphere formation assays. VEZF1/SPOP/STAT3/CCL2/ IL-6 axis was determined by luciferase reporter, ChIP, RNA-seq, co-IP, in vitro ubiquitination, RT-qPCR array and ELISA analyses. Results: We observed the frequent downregulation of SPOP, an E3 ubiquitin ligase, was positively associated with tumor progression and TAM infiltration in UBC patients and T24 xenografts. Cancer cell-TAM crosstalk promoting tumor aggressiveness was demonstrated dependent on SPOP deficiency: 1) In UBC cells, STAT3 was identified as a novel substrate of SPOP, and SPOP deficiency increased STAT3 protein stability, elevated chemokine CCL2 secretion, which induced chemotaxis and M2 polarization of macrophage; 2) In co-cultured macrophages, IL-6 secretion enhanced UBC cell proliferation and stemness. Additionally, transcription factor VEZF1 could directly activate SPOP transcription, and its overexpression suppressed the above effects in UBC cells. Conclusions: A pivotal role of SPOP in maintaining UBC stemness and remodeling immunosuppressive TME was revealed. Both the intrinsic signaling (dysregulated VEZF1/SPOP/STAT3 axis) and the extrinsic cues from TME (CCL2-IL-6 axis based on macrophages) promoted UBC progression. Targeting this crosstalk may offer a promising therapeutic strategy for UBC patients with SPOP deficiency.

Spatial resolved transcriptomics reveals distinct cross-talk between cancer cells and tumor-associated macrophages in intrahepatic cholangiocarcinoma.

BACKGROUND: Multiple studies have shown that tumor-associated macrophages (TAMs) promote cancer initiation and progression. However, the reprogramming of macrophages in the tumor microenvironment (TME) and the cross-talk between TAMs and malignant subclones in intrahepatic cholangiocarcinoma (iCCA) has not been fully characterized, especially in a spatially resolved manner. Deciphering the spatial architecture of variable tissue cellular components in iCCA could contribute to the positional context of gene expression containing information pathological changes and cellular variability. METHODS: Here, we applied spatial transcriptomics (ST) and digital spatial profiler (DSP) technologies with tumor sections from patients with iCCA. RESULTS: The results reveal that spatial inter- and intra-tumor heterogeneities feature iCCA malignancy, and tumor subclones are mainly driven by physical proximity. Tumor cells with TME components shaped the intra-sectional heterogenetic spatial architecture. Macrophages are the most infiltrated TME component in iCCA. The protein trefoil factor 3 (TFF3) secreted by the malignant subclone can induce macrophages to reprogram to a tumor-promoting state, which in turn contributes to an immune-suppressive environment and boosts tumor progression. CONCLUSIONS: In conclusion, our description of the iCCA ecosystem in a spatially resolved manner provides novel insights into the spatial features and the immune suppressive landscapes of TME for iCCA.

Targeting SRSF10 might inhibit M2 macrophage polarization and potentiate anti-PD-1 therapy in hepatocellular carcinoma.

BACKGROUND: The efficacy of immune checkpoint blockade therapy in patients with hepatocellular carcinoma (HCC) remains poor. Although serine- and arginine-rich splicing factor (SRSF) family members play crucial roles in tumors, their impact on tumor immunology remains unclear. This study aimed to elucidate the role of SRSF10 in HCC immunotherapy. METHODS: To identify the key genes associated with immunotherapy resistance, we conducted single-nuclear RNA sequencing, multiplex immunofluorescence, and The Cancer Genome Atlas and Gene Expression Omnibus database analyses. We investigated the biological functions of SRSF10 in immune evasion using in vitro co-culture systems, flow cytometry, various tumor-bearing mouse models, and patient-derived organotypic tumor spheroids. RESULTS: SRSF10 was upregulated in various tumors and associated with poor prognosis. Moreover, SRSF10 positively regulated lactate production, and SRSF10/glycolysis/ histone H3 lysine 18 lactylation (H3K18la) formed a positive feedback loop in tumor cells. Increased lactate levels promoted M2 macrophage polarization, thereby inhibiting CD8+ T cell activity. Mechanistically, SRSF10 interacted with the 3'-untranslated region of MYB, enhancing MYB RNA stability, and subsequently upregulating key glycolysis-related enzymes including glucose transporter 1 (GLUT1), hexokinase 1 (HK1), lactate dehydrogenase A (LDHA), resulting in elevated intracellular and extracellular lactate levels. Lactate accumulation induced histone lactylation, which further upregulated SRSF10 expression. Additionally, lactate produced by tumors induced lactylation of the histone H3K18la site upon transport into macrophages, thereby activating transcription and enhancing pro-tumor macrophage activity. M2 macrophages, in turn, inhibited the enrichment of CD8+ T cells and the proportion of interferon-γ+CD8+ T cells in the tumor microenvironment (TME), thus creating an immunosuppressive TME. Clinically, SRSF10 could serve as a biomarker for assessing immunotherapy resistance in various solid tumors. Pharmacological targeting of SRSF10 with a selective inhibitor 1C8 enhanced the efficacy of programmed cell death 1 (PD-1) monoclonal antibodies (mAbs) in both murine and human preclinical models. CONCLUSIONS: The SRSF10/MYB/glycolysis/lactate axis is critical for triggering immune evasion and anti-PD-1 resistance. Inhibiting SRSF10 by 1C8 may overcome anti-PD-1 tolerance in HCC.

XIAOPI formula inhibits chemoresistance and metastasis of triple-negative breast cancer by suppressing extracellular vesicle/CXCL1-induced TAM/PD-L1 signaling.

BACKGROUND: Triple-negative breast cancer (TNBC) is challenged by the low chemotherapy response and poor prognosis. Emerging evidence suggests that cytotoxic chemotherapy may lead to the pro-metastatic tumor microenvironment (TME) by eliciting pro-tumor extracellular vesicles (EVs) from cancer cells. However, the precise mechanisms and therapeutic approaches remain inadequately understood. PURPOSE: This study aims to determine whether XIAOPI formula (Chinese name XIAOPI San, XPS), a nationally sanctioned medication for mammary hyperplasia, can chemosensitize TNBC by remodeling the TME via modulating EV signaling, and exploring its underlying mechanisms. METHODS: Multiple methodologies, such as EV isolation, transmission electron microscope, flow cytometry, dual-luciferase reporter assays, co-immunoprecipitation and in vivo breast cancer xenograft, were employed to elucidate the effect and molecular mechanisms of XPS on paclitaxel-induced EV signaling (EV-dead) of TNBC. RESULTS: XPS, at non-toxic concentrations, synergized with PTX to inhibit the invasion and chemoresistance of TNBC cells co-cultured with macrophages. Compared to EV-dead, XPS co-treatment-elicited EVs (EV-deadXPS) exhibited a decreased capacity to promote the invasion, chemoresistance and cancer stem cell subpopulation of the co-cultured TNBC cells. Mechanistically, XPS administration led to a reduction in CXCL1 cargo in EV-dead, and thereby attenuated its activation effect on macrophage polarization into M2 phenotype through the transcriptional downregulation of PD-L1 expression. Furthermore, XPS effectively reduced the number of EV-dead from TNBC cells by inhibiting CXCL1-mediated intraluminal vesicle (ILV) biogenesis in multivesicular bodies (MVBs). Moreover, molecular explorations revealed that XPS impaired ILV biogenesis by disrupting the RAB31/FLOT2 complex via suppressing the CXCL1/Myc signaling. Importantly, XPS significantly chemosensitized paclitaxel to inhibit TNBC growth and metastasis in vivo by suppressing EV-deadCXCL1-induced PD-L1 activation and M2 polarization of macrophages. CONCLUSION: This pioneering study not only sheds novel light on EV-deadCXCL1 as a potential therapeutic target to suppress TNBC chemoresistance and metastasis, but also provides XPS as a promising adjuvant formula to chemosensitize TNBC by remodeling EV-deadCXCL1-mediated immunosuppressive TME.

CD11b/CD86 involved in the microenvironment of colorectal cancer by promoting Wnt signaling activation.

BACKGROUND: Colorectal cancer (CRC) is a malignancy that arises within the gastrointestinal tract. Despite ongoing research, the etiology and pathogenesis of CRC remain elusive; particularly, the distribution and characteristics of tumor-associated macrophages is currently an active area of investigation in understanding the pathological progression and prevention of CRC. METHODS: This study utilized CRC patient surgical samples, mouse models of colitis-associated cancer, colonic organoid, and co-culture cell line to examine the changes in CD11b/CD86 at different pathological region and detect the Wnt signaling pathway activity. RESULTS: Our findings revealed a sensitive and increased expression of CD11b from the early to the advanced CRC tissues and correlated with poor prognosis, while CD86 expression was reduced in advanced CRC tissues. CD133 expression was also elevated in advanced CRC tissues and mainly co-localized with CD11b, suggesting a positive regulatory effect of CD11b and CD133 expression that may contribute to CRC progression. In AOM/DSS mouse models, activation of the Wnt signaling pathway was associated with increased CD133 and CD11b expression. In vitro, THP-1 cell was induced to high expression of CD11b, and the above conditional cultural medium enhanced HCT116 cell colony number and CD133 protein expression. Furthermore, colonic crypts from AOM/DSS mouse models were isolated to culture, and the colonic organoids exhibited dilation and significant increases expression of CD133 and ß-Catenin/N-P-B-Catenin. CONCLUSIONS: CD11b might be an important factor to participate the progress of CRC. And the high CD11b of CRC microenviroment might potentially promote CD133 expression and associate with Wnt signal activation.

Spatial Isoforms Reveal the Mechanisms of Metastasis.

In esophageal squamous cell carcinoma (ESCC), lymph node (LN) metastasis is associated with poor survival. Emerging evidence has demonstrated elevated CD8+ T-cell levels in metastatic LNs following immunotherapy and increased chemoresistance. However, the underlying regulatory mechanisms of CD8+ T cells in chemoresistant/metastatic patients have not been elucidated. Given that metastasis is linked to aberrant splicing patterns, transcripts with alternative splicing forms also play a critical role. With spatial transcriptomics (ST), spatial isoform transcriptomics (SiT), single-cell RNA sequencing (scRNA-seq), and T-cell receptor (TCR) sequencing, the spatial isoforms are analyzed quantitatively in human solid tumors and LNs. These isoforms are classified according to expression and spatial distribution patterns and proposed that the temporal heterogeneity in isoforms is attributed to isoform biogenesis dynamics. C1QC+ tumor-associated macrophages (TAMs) contribute to the formation of metastases in the context of both immunotherapy and chemotherapy. Additionally, CD74 isoforms can be targeted by mRNA drugs, such as antisense oligonucleotides (ASOs) and RNA interference (RNAi), to prevent chemoresistance and metastasis. Overall, this work suggests that C1QC+ TAMs interact with CD8+ CXCL13+ Tex cells via enrichment with the CD74 isoform in the ESCC 's metastatic lymph node.

Downregulation of IL-8 and IL-10 by LRRC8A Inhibition through the NOX2-Nrf2-CEBPB Transcriptional Axis in THP-1-Derived M2 Macrophages.

M2-polarized, tumor-associated macrophages (TAMs) produce pro-tumorigenic and angiogenic mediators, such as interleukin-8 (IL-8) and IL-10. Leucine-rich repeat-containing protein 8 members (LRRC8s) form volume-regulated anion channels and play an important role in macrophage functions by regulating cytokine and chemokine production. We herein examined the role of LRRC8A in IL-8 and IL-10 expression in THP-1-differentiated M2-like macrophages (M2-MACs), which are a useful tool for investigating TAMs. In M2-MACs, the pharmacological inhibition of LRRC8A led to hyperpolarizing responses after a transient depolarization phase, followed by a slight elevation in the intracellular concentration of Ca2+. Both the small interfering RNA-mediated and pharmacological inhibition of LRRC8A repressed the transcriptional expression of IL-8 and IL-10, resulting in a significant reduction in their secretion. The inhibition of LRRC8A decreased the nuclear translocation of phosphorylated nuclear factor-erythroid 2-related factor 2 (Nrf2), while the activation of Nrf2 reversed the LRRC8A inhibition-induced transcriptional repression of IL-8 and IL-10 in M2-MACs. We identified the CCAAT/enhancer-binding protein isoform B, CEBPB, as a downstream target of Nrf2 signaling in M2-MACs. Moreover, among several upstream candidates, the inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) suppressed the Nrf2-CEBPB transcriptional axis in M2-MACs. Collectively, the present results indicate that the inhibition of LRRC8A repressed IL-8 and IL-10 transcription in M2-MACs through the NOX2-Nrf2-CEBPB axis and suggest that LRRC8A inhibitors suppress the IL-10-mediated evasion of tumor immune surveillance and IL-8-mediated metastasis and neovascularization in TAMs.

Unveiling the contribution of tumor-associated macrophages in driving epithelial-mesenchymal transition: a review of mechanisms and therapeutic Strategies.

Tumor-associated macrophages (TAMs), fundamental constituents of the tumor microenvironment (TME), significantly influence cancer development, primarily by promoting epithelial-mesenchymal transition (EMT). EMT endows cancer cells with increased motility, invasiveness, and resistance to therapies, marking a pivotal juncture in cancer progression. The review begins with a detailed exposition on the origins of TAMs and their functional heterogeneity, providing a foundational understanding of TAM characteristics. Next, it delves into the specific molecular mechanisms through which TAMs induce EMT, including cytokines, chemokines and stromal cross-talking. Following this, the review explores TAM-induced EMT features in select cancer types with notable EMT characteristics, highlighting recent insights and the impact of TAMs on cancer progression. Finally, the review concludes with a discussion of potential therapeutic targets and strategies aimed at mitigating TAM infiltration and disrupting the EMT signaling network, thereby underscoring the potential of emerging treatments to combat TAM-mediated EMT in cancer. This comprehensive analysis reaffirms the necessity for continued exploration into TAMs' regulatory roles within cancer biology to refine therapeutic approaches and improve patient outcomes.

Breast cancer colonization by Malassezia globosa accelerates tumor growth.

Malassezia globosa is a lipophilic basidiomycetous yeast that occurs abundantly in breast tumors and that may contribute to a shortened overall survival of breast cancer (BRAC) patients, suggesting that the yeast may participate in the carcinogenesis of BRAC. However, the mechanisms involved in the M. globosa-based acceleration of BRAC are unknown. Here, we show that M. globosa can colonize mammary tissue in 7,12-dimethylbenz[a] anthracene-induced mice. The abundance of M. globosa shortened the overall survival and increased the tumor incidence. Transcriptome data illustrated that IL-17A plays a key role in tumor growth due to M. globosa colonization, and tumor-associated macrophage infiltration was elevated during M. globosa colonization which triggers M2 polarization of macrophages via toll-like receptors 4/nuclear factor kappa-B (Nf-κB) signaling. Our results show that the expression of sphingosine kinase 1 (Sphk1) is increased in breast tumors after inoculation with M. globosa. Moreover, we discovered that Sphk1-specific small interfering RNA blocked the formation of lipid droplets, which can effectively alleviate the expression of the signal transducer and activator of the transcription 3 (STAT3)/Nf-κB pathway. Taken together, our results demonstrate that M. globosa could be a possible factor for the progression of BRAC. The mechanisms by which M. globosa promotes BRAC development involve the IL-17A/macrophage axis. Meanwhile, Sphk1 overexpression was induced by M. globosa infection, which also promoted the proliferation of MCF-7 cells.IMPORTANCELiterature has suggested that Malassezia globosa is associated with breast tumors; however, this association has not been confirmed. Here, we found that M. globosa colonizes in breast fat pads leading to tumor growth. As a lipophilic yeast, the expression of sphingosine kinase 1 (Sphk1) was upregulated to promote tumor growth after M. globosa colonization. Moreover, the IL-17A/macrophages axis plays a key role in mechanisms involved in the M. globosa-induced breast cancer acceleration from the tumor immune microenvironment perspective.

A novel EZH1/2 dual inhibitor inhibits GCB DLBCL through cell cycle regulation and M2 tumor-associated macrophage polarization.

The incidence of germinal center B-cell-like type diffuse large B-cell lymphoma (GCB DLBCL) is steadily increasing, with a known hereditary component. Although some molecular mechanisms in GCB DLBCL have been elucidated, understanding remains incomplete, limiting the effectiveness of targeted therapies. In GCB DLBCL patients, abnormally high expression of zeste homologs 2 (EZH2) is noted, and the compensatory effect of EZH1 following EZH2 inhibition contributes to poor prognosis. This highlights the potential of dual targeting of EZH1/2 as a promising strategy. In this study, we developed a novel inhibitor, EZH-1-P2, targeting EZH1/2 and evaluated its antitumor effects on DLBCL cells. Mechanistically, inhibition of EZH1/2 affects the epigenetic regulation of gene expression related to p53, impacting cell cycle progression and GCB DLBCL cell growth. Additionally, while EZH1/2 inhibition impacts NOTCH signaling, the precise mechanism by which it affects M2-type tumor-associated macrophage polarization and germinal center expansion requires further investigation. Our research introduces EZH-1-P2 as a novel inhibitor with potential as a candidate for GCB DLBCL therapy, although further studies are needed to fully elucidate its mechanisms.

Interleukin-34-orchestrated tumor-associated macrophage reprogramming is required for tumor immune escape driven by p53 inactivation.

As the most frequent genetic alteration in cancer, more than half of human cancers have p53 mutations that cause transcriptional inactivation. However, how p53 modulates the immune landscape to create a niche for immune escape remains elusive. We found that cancer stem cells (CSCs) established an interleukin-34 (IL-34)-orchestrated niche to promote tumorigenesis in p53-inactivated liver cancer. Mechanistically, we discovered that Il34 is a gene transcriptionally repressed by p53, and p53 loss resulted in IL-34 secretion by CSCs. IL-34 induced CD36-mediated elevations in fatty acid oxidative metabolism to drive M2-like polarization of foam-like tumor-associated macrophages (TAMs). These IL-34-orchestrated TAMs suppressed CD8+ T cell-mediated antitumor immunity to promote immune escape. Blockade of the IL-34-CD36 axis elicited antitumor immunity and synergized with anti-PD-1 immunotherapy, leading to a complete response. Our findings reveal the underlying mechanism of p53 modulation of the tumor immune microenvironment and provide a potential target for immunotherapy of cancer with p53 inactivation.

Calculus bovis inhibits M2 tumor-associated macrophage polarization via Wnt/ß-catenin pathway modulation to suppress liver cancer.

BACKGROUND: Calculus bovis (CB), used in traditional Chinese medicine, exhibits anti-tumor effects in various cancer models. It also constitutes an integral component of a compound formulation known as Pien Tze Huang, which is indicated for the treatment of liver cancer. However, its impact on the liver cancer tumor microenvironment, particularly on tumor-associated macrophages (TAMs), is not well understood. AIM: To elucidate the anti-liver cancer effect of CB by inhibiting M2-TAM polarization via Wnt/ß-catenin pathway modulation. METHODS: This study identified the active components of CB using UPLC-Q-TOF-MS, evaluated its anti-neoplastic effects in a nude mouse model, and elucidated the underlying mechanisms via network pharmacology, transcriptomics, and molecular docking. In vitro assays were used to investigate the effects of CB-containing serum on HepG2 cells and M2-TAMs, and Wnt pathway modulation was validated by real-time reverse transcriptase-polymerase chain reaction and Western blot analysis. RESULTS: This study identified 22 active components in CB, 11 of which were detected in the bloodstream. Preclinical investigations have demonstrated the ability of CB to effectively inhibit liver tumor growth. An integrated approach employing network pharmacology, transcriptomics, and molecular docking implicated the Wnt signaling pathway as a target of the antineoplastic activity of CB by suppressing M2-TAM polarization. In vitro and in vivo experiments further confirmed that CB significantly hinders M2-TAM polarization and suppresses Wnt/ß-catenin pathway activation. The inhibitory effect of CB on M2-TAMs was reversed when treated with the Wnt agonist SKL2001, confirming its pathway specificity. CONCLUSION: This study demonstrated that CB mediates inhibition of M2-TAM polarization through the Wnt/ß-catenin pathway, contributing to the suppression of liver cancer growth.

EL4 Murine-Lymphoma-Stromal-Cell Fusion Hybrids Observed With Multiple Distinct Morphologies in the Primary Tumor and Metastatic Organs of a Syngeneic Mouse Model.

BACKGROUND/AIM: We and others have previously shown that cell fusion plays an important role in cancer metastasis. Color coding of cancer and stromal cells with spectrally-distinct fluorescent proteins is a powerful tool, as pioneered by our laboratory to detect cell fusion. We have previously reported color-coded cell fusion between cancer cells and stromal cells in metastatic sites by using color-coded EL4 murine lymphoma cells and host mice expressing spectrally-distinct fluorescent proteins. Cell fusion occurred between cancer cells or, between cancer cells and normal cells, such as macrophages, fibroblasts, and mesenchymal stem cells. In the present study, the aim was to morphologically classify the fusion-hybrid cells observed in the primary tumor and multiple metastases EL4 formed from cells expressing red fluorescent protein (RFP) in transgenic mice expressing green fluorescent protein (GFP), in a syngeneic model. MATERIALS AND METHODS: RFP-expressing EL4 murine lymphoma cells were cultured in vitro. EL4-RFP cells were harvested and injected intraperitoneally into immunocompetent transgenic C57/BL6-GFP mice to establish a syngeneic model. Two weeks later, mice were sacrificed and each organ was harvested, cultured, and observed using confocal microscopy. RESULTS: EL4 intraperitoneal tumors (primary) and metastases in the lung, liver, blood, and bone marrow were formed. All tumors were harvested and cultured. In all specimens, RFP-EL4 cells, GFP-stromal cells, and fused yellow-fluorescent hybrid cells were observed. The fused hybrid cells showed various morphologies. Immune cell-like round-shaped yellow-fluorescent fused cells had a tendency to decrease with time in liver metastases and circulating blood. In contrast fibroblast-like spindle-shaped yellow-fluorescent fused cells increased in the intraperitoneal primary tumor, lung metastases, and bone marrow. CONCLUSION: Cell fusion between EL4-RFP cells and GFP stromal cells occurred in primary tumors and all metastatic sites. The morphology of the fused hybrid cells varied in the primary and metastatic sites. The present results suggest that fused cancer and stromal hybrid cells of varying morphology may play an important role in cancer progression.

Reduced Abundance of Phocaeicola in Mucosa-associated Microbiota Is Associated with Distal Colorectal Cancer Metastases Possibly through an Altered Local Immune Environment.

Objectives: The aim of this study was to identify the microbiota whose decrease in tumor area was associated with the metastatic process of distal colorectal cancer (CRC). Methods: Twenty-eight consecutive patients with distal CRC undergoing surgical resection in our hospital were enrolled. Microbiota in 28 specimens from surgically resected colorectal cancers were analyzed using 16S ribosomal ribonucleic acid gene amplicon sequencing and the relative abundance (RA) of microbiota was evaluated. The densities of tumor-infiltrating lymphocytes (TIL) and tumor associated macrophages (TAM) in the colorectal cancers were immunohistochemically evaluated. Results: Phocaeicola was the most abundant microbiota in normal mucosa. The RA of Phocaeicola in tumor tissues tended to be lower than that in normal mucosa although the difference was not significant (p=0.0732). The RA of Phocaeicola at tumor sites did not correlate either with depth of tumor invasion (pT-stage) or tumor size, however they were significantly reduced in patients with nodal metastases (p<0.05) and those with distant metastases (p<0.001). The RA of Phocaeicola at tumor sites showed positive correlation with the densities of CD3(+) or CD8(+) TIL. Since P. vulgatus was the most dominant species (47%) of the Phocaeicola, the RA of P. vulgatus and CRC metastasis and its association with TIL and TAM were also investigated. P. vulgatus showed a similar trend to genus Phocaeicola but was not statistically significant. Conclusions: A relative reduction of Phocaeicola attenuates the local anti-tumor immune response in distal CRC, which may facilitate metastatic spread.

Surface Molecularly Engineered Mitochondria Conduct Immunophenotype Repolarization of Tumor-Associated Macrophages to Potentiate Cancer Immunotherapy.

Reprogramming tumor-associated macrophages (TAMs) to an inflammatory phenotype effectively increases the potential of immune checkpoint blockade (ICB) therapy. Artificial mitochondrial transplantation, an emerging and safe strategy, has made brilliant achievements in regulating the function of recipient cells in preclinic and clinic, but its performance in reprogramming the immunophenotype of TAMs has not been reported. Here, the metabolism of M2 TAMs is proposed resetting from oxidative phosphorylation (OXPHOS) to glycolysis for polarizing M1 TAMs through targeted transplantation of mannosylated mitochondria (mPEI/M1mt). Mitochondria isolated from M1 macrophages are coated with mannosylated polyethyleneimine (mPEI) through electrostatic interaction to form mPEI/M1mt, which can be targeted uptake by M2 macrophages expressed a high level of mannose receptors. Mechanistically, mPEI/M1mt accelerates phosphorylation of NF-κB p65, MAPK p38 and JNK by glycolysis-mediated elevation of intracellular ROS, thus prompting M1 macrophage polarization. In vivo, the transplantation of mPEI/M1mt excellently potentiates therapeutic effects of anti-PD-L1 by resetting an antitumor proinflammatory tumor microenvironment and stimulating CD8 and CD4 T cells dependent immune response. Altogether, this work provides a novel platform for improving cancer immunotherapy, meanwhile, broadens the scope of mitochondrial transplantation technology in clinics in the future.

Implantable Microneedle-Mediated Eradication of Postoperative Tumor Foci Mitigates Glioblastoma Relapse.

Glioblastoma multiforme (GBM) remains incurable despite multimodal treatments after surgical debulking. Almost all patients with GBM relapse within a narrow margin (2-3 cm) of the initial resected lesion due to the unreachable residual cancerous cells. Here, a completely biodegradable microneedle for surgical cavity delivery glioblastoma-associated macrophages (GAMs)-activating immune nano-stimulator that mitigates glioblastoma relapse is reported. The residual tumor lesion-directed biocompatible microneedle releases the nano-stimulator and toll-like receptor 9 agonist in a controlled manner until the microneedles completely degrade over 1 week, efferently induce in situ phonotypic shifting of GAMs from anti- to pro-inflammatory and the tumor recurrence is obviously inhibited. The implantable microneedles offer a significant improvement over conventional transdermal ones, as they are 100% degradable, ensuring safe application within surgical cavities. It is also revealed that the T cells are recruited to the tumor niche as the GAMs initiate anti-tumor response and eradicate residual GBM cells. Taken together, this work provides a potential strategy for immunomodulating the postoperative tumor niche to mitigate tumor relapse in GBM patients, which may have broad applications in other malignancies with surgical intervention.