Inhibition of M2 tumor-associated macrophages polarization by modulating the Wnt/β-catenin pathway as a possible liver cancer therapy method

Liver cancer remains a significant global health challenge, characterized by high incidence and mortality rates. Despite advancements in medical treatments, the prognosis for liver cancer patients remains poor, highlighting the urgent need for novel therapeutic approaches. Traditional Chinese medicine (TCM), particularly Calculus bovis (CB), has shown promise in addressing this need due to its multi-target therapeutic mechanisms. CB refers to natural or synthetic gallstones, traditionally sourced from cattle, and used in TCM for their anti-inflammatory, detoxifying, and therapeutic properties. In modern practice, synthetic CB is often utilized to ensure consistent supply and safety. This article aims to discuss the findings of Huang et al, who investigated the anti-liver cancer properties of CB, focusing on its ability to inhibit M2 tumor-associated macrophage (TAM) polarization via modulation of the Wnt/β-catenin pathway. Huang et al employed a comprehensive approach integrating chemical analysis, animal model testing, and advanced bioinformatics. They identified active components of CB using UPLC-Q-TOF-MS, evaluated its anti-neoplastic effects in a nude mouse model, and elucidated the underlying mechanisms through network pharmacology, transcriptomics, and molecular docking studies. The study demonstrated that CB significantly inhibited liver tumor growth in vivo, as evidenced by reduced tumor size and weight in treated mice. Histological analyses confirmed signs of tumor regression. CB was found to modulate the tumor microenvironment by inhibiting the polarization of M2 phenotype-TAMs, as shown by reduced expression of M2 markers and downregulation of mRNA levels of C-C motif chemokine 22, arginase-1, transforming growth factor-beta 2, and interleukin-10. The study further revealed that CB's antineoplastic activity involved the downregulation of Wnt5B and β-catenin and upregulation of Axin2, thus inhibiting the Wnt/β-catenin signaling pathway. These findings highlight the therapeutic potential of CB in liver cancer treatment through its modulation of the Wnt/β-catenin pathway and suppression of M2 phenotype-TAM polarization. This study underscores the value of integrating TCM with modern therapeutic strategies to develop novel effective treatments for liver cancer.

Tumor-associated macrophages (TAMs) are present in and are important components of the tumor microenvironment (TME). TAMs differentiate into 2 functionally distinct morphologies, classically activated (M1)-type TAMs and alternatively activated (M2)-type TAMs, when stimulated by different cytokines. The 2 types of TAMs exhibit distinct properties and functions. M1 TAMs secrete high levels of pro-inflammatory and chemotactic factors, exerting proinflammatory, antitumor effects. Conversely, M2 TAMs alter the extracellular matrix, facilitate cellular immune escape, and stimulate tumor angiogenesis, thereby promoting anti-inflammatory responses and tumor growth. The ratio of M1 TAMs to M2 TAMs in the TME is closely related to the prognosis of the tumor. Tumor cells and other cells in the TME can regulate the polarization of TAMs and thus promote tumor progression through the secretion of various substances; however, polarized TAMs can also act on various cells in the TME through the secretion of exosomes, thus forming a positive feedback loop. Therefore, modulating the phenotype of TAMs in the TME or blocking the polarization of M2 TAMs might be a new approach for cancer treatment. However, the intracellular signaling pathways involved in the polarization of TAMs are poorly understood. The AKT signaling pathway is an important signaling pathway involved in the polarization, growth, proliferation, recruitment, and apoptosis of TAMs, as well as the action of TAMs on other cells within the TME. This paper reviews the AKT signaling pathway in the polarization of TAMs and the regulation of the TME and provides new ideas for tumor immunotherapy.

In this article, we comment on the work published by Huang et al, which explores the mechanisms by which Calculus bovis (CB) modulates the liver cancer immune microenvironment via the Wnt/β-catenin signalling pathway. The study demonstrates that active components in CB effectively inhibit the activation of the Wnt/β-catenin pathway, significantly reducing the polarization of M2 tumor-associated macrophages. Both in vivo and in vitro experiments have validated the anti-tumour effects of CB, revealing its complex mechanisms of action through the modulation of immune cell functions within the tumour microenvironment. This article highlights CB's therapeutic potential in liver cancer treatment and calls for further investigations into its mechanisms and clinical applications to develop safer, more effective options for patients. The study also revealed that key components of CB, such as bilirubin and bile acids, inhibit tumour cell proliferation and promote apoptosis through multiple pathways. Future research should explore the mechanisms of action of CB and its potential integration with existing treatments to improve the therapeutic outcomes of liver cancer patients. With multidisciplinary collaboration and advanced research, CB could become a key component of comprehensive liver cancer treatment, offering new hope for patients.

BackgroundCrosstalk between pancreatic cancer cells and tumor-associated macrophages (TAMs) is a critical driver of malignant progression, and plays an important role in the low response rate to immunotherapy in patients with for pancreatic cancer. Although it is known that cancer cells induce TAM infiltration and M2 polarization, the underlying mechanisms remain elusive. Herein, we identified matrix metalloproteinase 28 (MMP28), a highly expressed protein, as a key regulator of this process.MethodsImmunohistochemical staining and qRT-PCR were used to validate MMP28 as a potential marker for the prognosis of patients with pancreatic cancer. We evaluated the tumor-promoting effect of MMP28 in vitro with CCK-8, Transwell, and EdU assay and Western blotting and explored the potential mechanism of MMP28-induced M2 polarization of TAMs with a coculture system, immunofluorescence staining and flow cytometry. A subcutaneous graft tumor model was constructed to assess the tumor-promoting effect of MMP28 and its ability to induce M2 TAM infiltration.ResultsThe relevant results of this study revealed a strong correlation between MMP28 expression and TAM infiltration, with a predominance of M2-polarized TAMs in pancreatic cancer tissues. Mechanistic investigations demonstrated that MMP28 promotes the secretion of multiple cytokines, including IL-8 and VEGFA through the activation of the MAPK/JNK signaling pathway. These cytokines act as potent chemoattractants and polarizing factors for TAMs. Additionally, we discovered an interaction between MMP28 and ANXA2, which contributes to the regulation of TAM recruitment and polarization. In vivo studies confirmed the critical role of MMP28 in tumor growth and TAM infiltration. Depletion of macrophages, inhibition of JNK, or neutralization of IL-8 and VEGFA significantly suppressed tumor progression. Transcriptomic analysis suggested that IL-8 and VEGFA induce M2 TAM polarization by modulating TAM amino acid metabolism.ConclusionsCollectively, our findings elucidate a novel mechanism by which pancreatic cancer cells manipulate the tumor microenvironment through MMP28-dependent cytokine secretion, promoting TAM infiltration and M2 polarization. These results highlight MMP28 as a promising therapeutic target for pancreatic cancer.Graphical Schematic overview of the mechanisms by which MMP28 promotes the migration and polarization of TAMs. High levels of MMP28 promote the secretion of IL-8 and VEGFA by cancer cells by mediating the phosphorylation of the MAPK/JNK signalling pathway and then recruiting TAMs. IL-8 and VEGFA subsequently induce amino acid metabolism alterations in TAMs by binding to relevant receptors on TAMs, which ultimately promote the polarization of TAMs to the M2 phenotype. In addition, ANXA2 increases MMP28-mediated M2 TAM infiltration by interacting with MMP28.

Dysregulated expression of ribosomal protein S3A (RPS3A) is associated with the tissue infiltration of immune-related cells in a variety of cancers. However, the role of RPS3A in immune cell infiltration in glioma remains unclear. This study aimed to explore the role of RPS3A in the glioma immune microenvironment.RPS3A expression was detected in tumor tissues from patients with glioma. U251 cells were transfected with RPS3A shRNA (sh-RPS3A) and overexpression vector (pcDNA-RPS3A) and then co-cultured with PMA-induced THP-1 cells. Cell viability, invasion, and apoptosis were detected by Edu staining, Transwell, and flow cytometry, respectively. The expression of tumor-associated macrophage (TAM) M1 and M2 markers was detected with RT-qPCR. Next, the interaction between RPS3A and E4 transcription factor 1 (E4F1) was verified by Co-IP analysis, and the binding of E4F1 to colony-stimulating factor 1 (CSF1) promoter was verified by ChIP analysis. Overexpression vectors of CSF1 and E4F1 were used to treat sh-RPS3A-transfected U251 cells for reversal experiments. Finally, U251 cells transfected with sh-RPS3A adenovirus vectors were subcutaneously injected into nude mice to construct a xenograft tumor model, and the growth and metastasis of glioma in vivo were monitored.RPS3A was significantly upregulated in glioma tissues. Overexpression of RPS3A promoted glioma cell proliferation and invasion and inhibited apoptosis. Moreover, overexpression of RPS3A promoted TAM proliferation, invasion, and M2 polarization. Silencing RPS3A had the opposite effect. Silencing RPS3A inhibited autophagy in U251 cells, whereas rapamycin, an activator of autophagy, reversed the inhibitory effect of RPS3A silencing on TAM M2 polarization. Meanwhile, RPS3A promoted its expression by interacting with E4F1, and E4F1 promoted CSF1 transcriptional activation. Overexpression of CSF1 promoted the proliferation and invasion of U251 cells and reversed the inhibitory effect of RPS3A silencing on TAM proliferation and invasion, but had no effect on TAM M2 polarization. The results of in vivo experiments showed that knockdown of RPS3A significantly inhibited glioma tumor growth and metastasis in vivo.This study revealed that RPS3A recruited TAMs by upregulating E4F1-mediated transcription activation of CSF1, and promoted the M2 polarization of TAMs through autophagy, promoting glioma cell malignant growth and tumor progression.

Gold nanoparticle-directed autophagy intervention for antitumor immunotherapy via inhibiting tumor-associated macrophage M2 polarization

1-Ethoxycarbonyl-beta-carboline inhibits the M2 polarization of tumor-associated macrophages: A study based on network pharmacology and molecular docking analyses

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with a high degree of malignancy and poor prognosis. Tumor-associated macrophages (TAMs) have been identified as significant contributors to the growth and metastasis of TNBC through the secretion of various growth factors and chemokines. Salvianolic acid A (SAA) has been shown to have anti-cancer activities. However, the potential activity of SAA on re-polarized TAMs remains unclear. As there is a correlation between the TAMs and TNBC, this study investigates the effect of SAA on TAMs in the TNBC microenvironment. For that purpose, M2 TAM polarization was induced by two kinds of TNBC-conditioned medium (TNBC-TCM) in the absence or presence of SAA. The gene and protein expression of TAM markers were analyzed by qPCR, FCM, IF, ELISA, and Western blot. The protein expression levels of ERK and p-ERK in M2-like TAMs were analyzed by Western blot. The migration and invasion properties of M2-like TAMs were analyzed by Transwell assays. Here, we demonstrated that SAA increased the expression levels of CD86, IL-1β, and iNOS in M2-like TAMs and, conversely, decreased the expression levels of Arg-1 and CD206. Moreover, SAA inhibited the migration and invasion properties of M2-like TAMs effectively and decreased the protein expression of TGF-β1 and p-ERK in a concentration-dependent manner, as well as TGF-β1 gene expression and secretion. Our current findings for the first time demonstrated that SAA inhibits macrophage polarization to M2-like TAMs by inhibiting the ERK pathway and promotes M2-like TAM re-polarization to the M1 TAMs, which may exert its anti-tumor effect by regulating M1/M2 TAM polarization. These findings highlight SAA as a potential regulator of M2 TAMs and the possibility of utilizing SAA to reprogram M2 TAMs offers promising insights for the clinical management of TNBC.

15P - Using a multiplexed immunofluorescence assay to detect immunosuppressive cells and their mechanisms in the pancreatic tumor microenvironment

Objectives: The more aggressive behavior of type 2 than type 1 endometrial cancers may be due to differences in their tumor microenvironments. In other solid cancers, tumor associated macrophages (TAMs) play a pivotal role in orchestrating the microenvironment. TAMs differentially polarize into M1 or M2 macrophages with distinct cytokine secretion patterns and actions. The aim of our work is to characterize the density, subtype and distribution of TAMs in endometrial hyperplasia and cancer. Methods: Five patients with complex atypical hyperplasia (hyperplasia), 5 patients with grade 1, non-invasive endometrioid adenocarcinoma (type 1) and 5 patients with uterine papillary serous carcinoma (type 2) were included. Formalin-fixed, paraffin-embedded sections from each specimen were stained with anti-CD68 and anti-CD163 monoclonal antibodies as markers for total TAMs and M2 TAMs, respectively. M1 macrophages were estimated by subtraction of CD163+ from CD68+ densities. Macrophages were counted at 40x magnification in 10 fields per slide by 4 observers. Repeated measure models, accounting for intra- and inter-observer correlation were contstructed and the compound symmetry covariance method was used to determine the relationship between macrophages and cancer. To establish an in vitro model, immortalized histiocytic lymphoma (U937) cells were differentiated to macrophages and treated with LPS to induce a M1 phenotype. Supernatants were applied to Cytokine C1000 microarray (RayBiotech) to screen for differentially expressed cytokines. Results: The correlation between the 4 observers was robust (r=0.71). Most TAMs were located in the stromal (mean=41.0/field) compared to epithelial (mean=11.0) or luminal (mean=11.6) compartments (p<0.0001). The mean total TAM density was highest in patients with type 2 cancers (mean=90.1/field), followed by type 1 cancers (mean=51.4) and hyperplasias (mean=41.1, p<0.0001). The calculated density of M1 macrophages was higher in type 2 cancers (mean=71.7) compared to type 1 cancers (mean =36.4, p<0.0001). This difference was only observed in the stromal compartment (p<0.0001). In the in vitro M1 macrophage model, ENA-78, a neutrophil chemo-attractant, and IL-6 were expressed at the highest concentrations. Conclusions: Type 2 cancers have nearly twice the TAM density of type 1 cancers. This difference is due to M1 macrophage predominance in the stroma of type 2 cancers. We postulate that cytokines produced by M1 TAMs within the tumor stroma contribute to the aggressive biology of these cancers. Future experiments will determine how well the in vitro model mimics M1 TAMs in the tumor microenvironment. Citation Format: Michael G. Kelly, Antonio M.C. Francisco, Adela Cimic, Anne Wofford, Nora Fitzgerald, Jie Yu, Robert N. Taylor. Type 2 endometrial cancer is associated with an M1 subtype, tumor associated macrophage polarization in the stromal compartment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1090. doi:10.1158/1538-7445.AM2014-1090

Tumor-associated macrophages (TAMs) differentiate into two main types based on signals from their microenvironment: the classically activated M1 type and the alternatively activated M2 type. An increased presence of M2-type TAMs has been identified in colorectal cancer patients, and the laminin alpha-1 chain (LAMA1), a key structural component of the extracellular matrix, plays a pivotal role in tumor metastasis. However, the exact mechanism by which LAMA1 promotes the polarization of TAMs towards the M2 phenotype and induces colorectal cancer remains elusive. In this work, we investigated the levels of LAMA1 and M2 TAMs in peripheral serum and tissue specimens from patients with colorectal cancer by utilizing Western Blotting, ELISA, and multicolor immunofluorescence. Peripheral blood samples from 20 patients were collected for ELISA analysis, with samples from 5 healthy individuals serving as controls. Additionally, tissue samples from 5 tumor patients and 5 normal controls were used to assess LAMA1 protein levels. Furthermore, statistical methods were employed to analyze the relationship between LAMA1 levels, M2 TAMs infiltration, and the pathological characteristics of colorectal cancer. Then we utilized Western Blotting, qPCR, and immunofluorescence to detect M2 TAMs markers and polarization mechanism after treatment with exogenous LAMA1 protein. Moreover, we established a mouse subcutaneous tumor model and utilized Western Blotting, immunohistochemistry and multicolor immunofluorescence to assess LAMA1 levels, tumor proliferation and proportion of M2 TAMs. Our analysis revealed that LAMA1 is overexpressed in the tumor microenvironment (TME) of colorectal cancer patients, which correlates with increased infiltration of M2-type TAMs. Furthermore, we found that the EGFR/AKT/CREB signaling pathway contributes to the polarization of TAMs toward the M2 subtype. Both in vitro and in vivo experiments demonstrated that LAMA1 promotes M2 macrophage polarization and facilitates tumor growth. Overall, these findings highlight a central role for LAMA1 in regulating macrophage polarization through the EGFR/AKT/CREB signaling pathway. Consequently, this process contributes to immune suppression and promotes tumor progression.

Circular RNAs (circRNAs) are reported to be involved in the tumorigenesis of lung adenocarcinoma (LUAD). Here, this study focused on studying the function and mechanism of circHSPB6 in LUAD progression. Levels of genes and proteins were tested using qRT-PCR and western blotting analyses. The 5-ethynyl-2'-deoxyuridine (EdU), colony formation, flow cytometry, and transwell assays were adopted for in vitro assays. In vivo assay was conducted using mouse xenograft models. The binding between let-7a-2-3p and circHSPB6 or CCL2 was validated using RIP and dual-luciferase reporter assays. The M2 polarization of tumor-associated macrophages (TAMs) was analyzed by flow cytometry. LUAD tissues and cells showed high circHSPB6 expression, knockdown of circHSPB6-suppressed LUAD cell proliferation, migration, invasion, and induced cell apoptosis in vitro, as well as hindered tumor growth in vivo. Mechanistically, circHSPB6/let-7a-2-3p/CCL2 forms a feedback loop. CircHSPB6 could regulate CCL2 expression via sponging let-7a-2-3p. Further rescue assays showed that the effects of circHSPB6 silencing on LUAD cells were reversed by let-7a-2-3p inhibition or CCL2 overexpression. Moreover, circHSPB6 promoted the M2 polarization and infiltration of TAMs by CCL2. Functionally, circHSPB6 knockdown in A549 and H1299 cells inhibited TAM M2 polarization and then suppressed cell proliferation, migration, invasion, and emergency medical technicians (EMT) progression, while these effects were reversed by CCL2 up-regulation CircHSPB6 induced TAM M2 polarization to promote LUAD cell proliferation, migration, invasion, and EMT progression through let-7a-2-3p/CCL2 axis.

Tumor-associated macrophages (TAMs) usually display an antiinflammatory M2-like phenotype to facilitate tumor growth. However, what drives M2 polarization of TAMs and how TAMs suppress antitumor immunity within the tumor microenvironment (TME) remain largely undefined. Using several murine tumor models, we showed that hedgehog (Hh) signaling in myeloid cells is critical for TAM M2 polarization and tumor growth. We also found that tumor cells secrete sonic hedgehog (SHH), an Hh ligand, and that tumor-derived SHH drives TAM M2 polarization. Furthermore, Hh-induced functional polarization in TAMs suppresses CD8+ T cell recruitment to the TME through the inhibition of CXCL9 and CXCL10 production by TAMs. Last, we demonstrated that Krüppel-like factor 4 (Klf4) mediates Hh-dependent TAM M2 polarization and the immunosuppressive function. Collectively, these findings highlight a critical role for tumor-derived SHH in promoting TAM M2 polarization, a mechanism for TAM-mediated immunosuppression, and may provide insights into the design of new cancer immunotherapeutic strategies.

In this paper, the mechanism of the Wnt/β-catenin pathway is introduced, and the process and principle of the experiment conducted by Huang et al is explained. We discussed the reliability of the conclusion that Calculus bovis (C. bovis) inhibits M2 tumor-associated macrophage polarization via Wnt/β-catenin pathway modulation to suppress liver cancer. We also offer suggestions for further studies of the use of C. bovis in the treatment of liver cancer.

Plentiful studies have clarified that circular RNAs (circRNAs) are crucial in colorectal cancer (CRC)’s occurrence and development, but its function has not been fully elucidated. The purpose of this study was to investigate the biological functions of circPLCE1 on epithelial mesenchymal transformation (EMT) and glycolysis in CRC, and tumor-associated macrophage (TAM) polarization. The results affirmed augment of circPLCE1 and γ-Actin Gene (ACTG1) but decline of miR-485-5p in CRC. Knockdown circPCLE1 refrained CRC proliferation, glucose consumption, lactic acid and pyruvate production, M2 macrophage markers (IL-10, MRC1), N-cadherin, Snail, reduced the proportion of CD206+ and CD168+ macrophages, but expedited M1 macrophage markers (TNF-α, IL-6) and E-cadherin, while descending miR-485-5p expedited EMT, glycolysis in CRC and TAM M2 polarization . Additionally, it was affirmed that the repression or motivation of depressive or elevated circPCLE1 on EMT, glycolysis in CRC and TAM M2 polarization were reversed via facilitated ACTG1 and miR-485-5p, separately. Mechanism studies have clarified that circPCLE1 as a competitive endogenous RNA adsorbed miR-485-5p to mediate ACTG1. It was assured that refrained circPCLE1 constrained CRC tumor growth, EMT and TAM M2 polarization. In brief, circPCLE1 expedites EMT, glycolysis in CRC and TAM M2 polarization via modulating the miR-485-5p/ACTG1 axis, and is supposed to be a latent molecular target for CRC therapy later.