Clinical significance of macrophage heterogeneity in human malignant tumors

The fact that various immune cells, including macrophages, can be found in tumor tissue has long been known. With the recent introduction of the novel concept of macrophage differentiation into a classically activated phenotype (M1) and an alternatively activated phenotype (M2), the role of tumor‐associated macrophages (TAMs) is gradually beginning to be elucidated. Specifically, in human malignant tumors, TAMs that have differentiated into M2 macrophages act as “protumoral macrophages” and contribute to the progression of disease. Based on recent basic and preclinical research, TAMs that have differentiated into protumoral or M2 macrophages are believed to be intimately involved in the angiogenesis, immunosuppression, and activation of tumor cells. In this paper, we specifically discuss both the role of TAMs in human malignant tumors and the cell–cell interactions between TAMs and tumor cells.     

M-CSF cooperating with NFκB induces macrophage transformation from M1 to M2 by upregulating c-Jun

Increasing evidence suggests tumor-associated macrophages (TAMs) are polarized M2 subtype of macrophage that exerts pro-tumor effects and promote the malignancy of some cancers, but the concrete mechanism is not well defined. Our previous research exhibited that proto-oncogene AP-1 regulated IL-6 expression in macrophages and promoted the formation of M2 macrophages. In this study, we investigate whether extra-cellular stimulus M-CSF help this process or nuclear factor NFκB has a synergistic role in the activation state of polarized M2 subtype of macrophage. RAW 264.7 macrophage and 4T1 mouse breast cancer cells were co-cultured to reconstruct tumor microenvironment. Being co-cultured with 4T1 or its supernatant, the expression of c-Jun, the member of AP-1 family, has a dramatically increase both on the level of gene and on the protein in RAW 264.7 macrophages, but the expression of c-Fos does not increase neither on the level of gene nor on the protein. After co-cultured with 4T1, RAW 264.7 has a higher consumption of M-CSF than RAW 264.7 macrophages alone. With the stimulation of M-CSF, the mRNA of c-Jun increased significantly, but decreased remarkably after adding the anti-M-CSF. And at the same time, p50, the member of NFκB family, has a similar tendency to c-Jun. WB results suggest that with the stimulation of M-CSF, p-Jun in nuclear increases heavily but decreases after the neutralizing antibody added. Coimmunoprecipitation and immunoblotting techniques confirmed that c-Jun and p50 NFκB coprecipitated, and c-Jun protein expression is properly enhanced with rM-CSF effect. In conclusion, M-CSF induces macrophage transformation by upregulating c-Jun with a certain synergy of NFκB. Our study may present a novel therapeutic strategy against cancer.     

Depletion of tumor-associated macrophages inhibits lung cancer growth and enhances the antitumor effect of cisplatin

In lung cancer, tumor-associated macrophages (TAMs), especially M2-like TAMs, represent the main tumor progression components in the tumor microenvironment (TME). Therefore, M2-like TAMs may serve as a therapeutic target. The purpose of this study was to investigate the effect of M2-like TAM depletion in the TME on tumor growth and chemotherapy response in lung cancer. The levels of secreted monocyte chemoattractant protein (MCP-1) and prostaglandin E2 (PGE2) in the supernatants of lung cancer cell lines A549 and LLC were evaluated via ELISA. Cell migration assays were performed to assess the recruitment ability of macrophage cell lines THP-1 and J774-1 cells. Differentiation of macrophages was assessed via flow cytometry. Immunohistochemical staining was performed to visualize M2-like TAMs in transplanted lung cancer in mouse. We used the COX-2 inhibitor nimesulide to inhibit the secretion of MCP-1 and PGE2, which promotes macrophage migration and M2-like differentiation. Nimesulide treatment decreased the secretion of MCP-1 and PGE2 from lung cancer cells. Nimesulide treatment suppressed the migration of macrophages by blocking MCP-1. Lung cancer supernatant induced the differentiation of macrophages toward the M2-like phenotype, and nimesulide treatment inhibited M2-like differentiation by blocking MCP-1 and PGE2. In the lung cancer mouse model, treatment with nimesulide depleted M2-like TAMs in the TME and enhanced the tumor inhibitory effect of cisplatin. Our results indicated that blocking the secretion of MCP-1 and PGE2 from tumor cells depleted M2-like TAMs in the TME and the combination therapy with cisplatin considerably suppressed tumor growth in the LLC mouse model.     

PTEN inhibits macrophage polarization from M1 to M2 through CCL2 and VEGF-A reduction and NHERF-1 synergism

PTEN has been studied in several tumor models as a tumor suppressor. In this study, we explored the role of PTEN in the inhibition state of polarized M2 subtype of macrophage in tumor microenvironment (TME) and the underlying mechanisms. To elucidate the potential effect in TME, RAW 264.7 macrophages and 4T1 mouse breast cancer cells were co-cultured to reconstruct tumor microenvironment. After PTEN was down-regulated with shRNA, the expression of CCL2 and VEGF-A, which are definited to promote the formation of M2 macrophages, have a dramatically increase on the level of both gene and protein in co-cultured RAW 264.7 macrophages. And at the same time, NHERF-1 (Na⁺/H⁺ exchanger regulating factor-1), another tumor suppressor has a similar tendency to PTEN. Q-PCR and WB results suggested that PTEN and NHERF-1 were consistent with one another no matter at mRNA or protein level when exposed to the same stimulus. Coimmunoprecipitation and immunofluorescence techniques confirmed that PTEN and NHERF-1 were coprecipitated, and NHERF-1 protein expression was properly reduced with rCCL2 effect. In addition, cell immunofluorescence images revealed a profound transferance, in co-cultured RAW 264.7 macrophages, an up-regulation of NHERF-1 could promote the PTEN marked expression on the cell membrane, and this form for the interaction was not negligible. These observations illustrate PTEN with a certain synergy of NHERF-1, as well as down-regulation of CCL2 suppressing M2 macrophage transformation pathway. The results suggest that the activation of PTEN and NHERF-1 may impede the evolution of macrophages beyond the M1 into M2 phenotype in tumor microenvironment.     

TGF-β-induced IRAK-M expression in tumor-associated macrophages regulates lung tumor growth

Tumor-associated macrophages (TAMs) constitute a major component of the immune cell infiltrate observed in the tumor microenvironment (TME). Factors present in the TME, including tumor growth factor-β (TGF-β), allow tumors to circumvent host-mediated immune responses to promote tumor progression. However, the molecular mechanism(s) involved are not clear. Toll-like receptors (TLRs) are important mediators of innate immune responses by immune cells, whose activation triggers the production of molecules required for anti-tumoral responses. Interleukin (IL) receptor-associated kinase (IRAK)-M is an inactive serine/threonine kinase, predominantly expressed in macrophages and is a potent negative regulator of TLR signaling. In this study, we show that TAMs express significantly higher levels of IRAK-M compared with peritoneal macrophages in a syngeneic mouse model of lung cancer. Subcutaneous implantation of Lewis lung carcinoma cells in IRAK-M(-/-) mice resulted in a five-fold reduction in tumor growth as compared with tumors in wild-type (WT) animals. Furthermore, compared with WT TAMs, TAMs isolated from IRAK-M(-/-) mice displayed features of a classically activated (M1) rather than alternatively activated (M2) phenotype, as manifest by greater expression of IL-12, interferon-γ (IFN-γ) and inducible nitric oxide synthase. Human lung cancer cells induced IRAK-M expression in human peripheral blood mononuclear cells (PBMCs) when co-cultured together. Tumor cell-induced expression of IRAK-M was dependent on the activation of TGF-β pathway. Similarly, treatment of human PBMCs or mouse macrophage cell line, RAW 264.4, with TGF-β, induced IRAK-M expression. Interestingly, IRAK-M gene expression in 439 human lung adenocarcinoma tumors correlated with poor survival in patients with lung cancer. Together, our data demonstrates that TGF-β-dependent induction of IRAK-M expression is an important, clinically relevant mechanism by which tumors may circumvent anti-tumor responses of macrophages.     

Clinical significance of CD163+ tumor‐associated macrophages in patients with adult T‐cell leukemia/lymphoma

In several malignant tumors including lymphoma, macrophages that infiltrate tumor tissues are called tumor‐associated macrophages (TAMs). We discovered that TAMs, especially the CD163⁺ alternatively activated phenotype (M2), were closely involved with progression of adult T‐cell leukemia/lymphoma (ATLL). We used CD68 (a pan‐macrophage marker) and CD163 (an M2 marker) to immunostain 58 ATLL samples. Statistical analyses showed that a high number of CD68⁺ TAMs and an increased percentage of CD163⁺ cells among the TAMs were associated with a worse clinical prognosis; multivariate analysis indicated that the percentage of CD163⁺ cells was an independent prognostic factor. We also carried out in vitro coculture experiments with ATLL cell lines (ATN‐1 and TL‐Mor) and monocyte‐derived macrophages and found that direct coculture with M2 macrophages significantly increased BrdU incorporation into ATLL cell lines. A cytokine array analysis showed that macrophage‐derived soluble factors including C5a, tumor necrosis factor‐α, growth‐related oncogene‐α, CCL1/I‐309, and interleukin‐6 stimulated ATLL cell lines. CD163 expression in macrophages was strongly induced by direct contact with ATN‐1 cells, and downregulation of CD163 in macrophages significantly suppressed growth of cocultured ATN‐1 cells. These results suggest that interaction between M2 macrophages and lymphoma cells may be an appropriate target in treatment of patients with ATLL.     

Depletion of tumor‐associated macrophages inhibits lung cancer growth and enhances the antitumor effect of cisplatin

In lung cancer, tumor‐associated macrophages (TAMs), especially M2‐like TAMs, represent the main tumor progression components in the tumor microenvironment (TME). Therefore, M2‐like TAMs may serve as a therapeutic target. The purpose of this study was to investigate the effect of M2‐like TAM depletion in the TME on tumor growth and chemotherapy response in lung cancer. The levels of secreted monocyte chemoattractant protein (MCP‐1) and prostaglandin E2 (PGE2) in the supernatants of lung cancer cell lines A549 and LLC were evaluated via ELISA. Cell migration assays were performed to assess the recruitment ability of macrophage cell lines THP‐1 and J774‐1 cells. Differentiation of macrophages was assessed via flow cytometry. Immunohistochemical staining was performed to visualize M2‐like TAMs in transplanted lung cancer in mouse. We used the COX‐2 inhibitor nimesulide to inhibit the secretion of MCP‐1 and PGE2, which promotes macrophage migration and M2‐like differentiation. Nimesulide treatment decreased the secretion of MCP‐1 and PGE2 from lung cancer cells. Nimesulide treatment suppressed the migration of macrophages by blocking MCP‐1. Lung cancer supernatant induced the differentiation of macrophages toward the M2‐like phenotype, and nimesulide treatment inhibited M2‐like differentiation by blocking MCP‐1 and PGE2. In the lung cancer mouse model, treatment with nimesulide depleted M2‐like TAMs in the TME and enhanced the tumor inhibitory effect of cisplatin. Our results indicated that blocking the secretion of MCP‐1 and PGE2 from tumor cells depleted M2‐like TAMs in the TME and the combination therapy with cisplatin considerably suppressed tumor growth in the LLC mouse model.     

TAMs为靶点的抗肿瘤治疗研究进展

肿瘤微环境与肿瘤细胞通过分子和细胞间的相互作用,在肿瘤的发生发展和转移扩散中具有重要意义。肿瘤相关巨噬细胞（TAMs）作为肿瘤微环境中数量最多的炎症细胞群之一,在肿瘤进展中起到重要作用。肿瘤细胞通过释放多种趋化因子、细胞因子和生长因子招募巨噬细胞,并使其向M2型巨噬细胞类似的特性发展。同时,巨噬细胞释放多种因子,促进肿瘤细胞的生长、血管新生、迁移、侵袭、侵入血管并最终形成远处转移。TAMs在肿瘤组织中的密度与肿瘤患者治疗失败和不良预后密切相关,以TAMs为靶点的抗肿瘤治疗相关研究近年来取得重大进展。在肿瘤发生发展中根据TAMs的作用机制,以TAMs为靶点的抗肿瘤治疗策略是抑制肿瘤微环境中巨噬细胞招募、TAMs生存能力、TAMs表型即由M2型转化为M1型的重塑。本文就TAMs为靶点的抗肿瘤治疗最新进展进行综述。      

Differentiation and gene expression profile of tumor-associated macrophages

Tumor microenvironment is composed of proliferating neoplastic cells, a vascular network of endothelial cells, extra cellular matrix produced by fibroblasts, cellular compartments of adaptive immunity like lymphocytes and dendritic cells as well as cells of innate immunity, e.g., natural killer cells and macrophages. Many pre-clinical and clinical studies demonstrate an inversed correlation between macrophage infiltrate and patients' prognosis indicating a macrophage supporting role for tumor progression as producers of growth and angiogenic factors and as regulators of tissue remodelling. Based on in vitro models, macrophages have been classified in pro-inflammatory, classically activated macrophages (M1; stimulated by IFN-γ or LPS) and anti-inflammatory, alternatively activated macrophages (M2; stimulated by either IL-4/IL-13, IL-1β/LPS in combination with immune complexes or by IL-10/TGFβ/glucocorticoids). Tumor escape has been linked with a switch from M1 activation in the early tumor initiation process towards M2-like phenotype during tumor progression, a process that highlights the heterogeneity and plasticity of macrophage activation and which offers a possible therapeutic target directed against reversing the TAM phenotype in the tumor. Here, we review different tumor-environmental stimuli and signalling cascades involved in this switch in differentiation and the so connected gene regulation in TAMs. In addition, therapeutic applications deducted from this differentiation and gene regulatory processes are presented. Data from pre-clinical as well as clinical studies clearly support the notion, that TAMs are excellent novel therapeutic targets for the fight against cancer.     

Tumor‐associated macrophages exhibit pro‐ and anti‐inflammatory properties by which they impact on pancreatic tumorigenesis

Pancreatic ductal adenocarcinoma (PDAC) still ranking 4th in the order of fatal tumor diseases is characterized by a profound tumor stroma with high numbers of tumor‐associated macrophages (TAMs). Driven by environmental factors, monocytes differentiate into M1‐ or M2‐macrophages, the latter commonly regarded as being protumorigenic. Because a detailed analysis of TAMs in human PDAC development is still lacking, freshly isolated PDAC‐derived TAMs were analyzed for their phenotype and impact on epithelial‐mesenchymal‐transition (EMT) of benign (H6c7) and malignant (Colo357) pancreatic ductal epithelial cells. TAMs exhibited characteristics of M1‐macrophages (expression of HLA‐DR, IL‐1β, or TNF‐α) and M2‐macrophages (expression of CD163 and IL‐10). In the presence of TAMs, H6c7, and Colo357 cells showed an elongated cell shape along with an increased expression of mesenchymal markers such as vimentin and reduced expression of epithelial E‐cadherin. Similar to TAMs, in vitro generated M1‐ and M2‐macrophages both mediated EMT in H6c7 and Colo357 cells. M1‐macrophages acquired M2‐characteristics during coculture that could be prevented by GM‐CSF treatment. However, M1‐macrophages still potently induced EMT in H6c7 and Colo357 cells although lacking M2‐characteristics. Overall, these data demonstrate that TAMs exhibit anti‐ as well as proinflammatory properties that equally contribute to EMT induction in PDAC initiation and development.     

Tumor versus tumor-associated macrophages: how hot is the link?

One of the functions of macrophages is to provide a defense mechanism against tumor cells. In contrast, tumor-associated macrophages (TAMs), which represent the major inflammatory component of the stroma of many tumors, are associated with tumor progression and metastasis. TAMs, in contrast with normal macrophages, exhibit the M2 phenotype, and thereby exhibit pro-tumoral functions, including angiogenesis and matrix remodeling. This review will discuss the role of TAMs in tumor progression and provide an overview of their significant part in tumor metastasis and angiogenesis.     

肿瘤相关巨噬细胞相关性miR-99a对子宫内膜癌细胞生长和侵袭的调控作用

背景与目的：肿瘤相关巨噬细胞（tumor-associated macrophage,TAM）浸润与肿瘤进展密切相关,但作用机制尚不明确,因此,探索miR-99a对单核巨噬细胞极化的影响及其对子宫内膜癌（endometrial cancer,EC）细胞生长、侵袭的影响。方法：检测EC组织中巨噬唾液酸蛋白（macrosialin）CD68表达并分析其与临床病理学特征之间的关系;运用人EC细胞系HEC-1B、RL95-2培养上清液诱导人单核细胞U937向TAM（M2型巨噬细胞）分化;将人工合成的miR-99a模拟物片段转染至诱导后的巨噬细胞,转染后运用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR）及流式细胞术检测巨噬细胞相关因子CD68、CD163以及巨噬细胞甘露糖受体（mcrophage mannose receptor）CD206表达量变化,并运用酶联免疫吸附法（enzyme-linked immunosorbent assay,ELISA）检测巨噬细胞分泌相关细胞因子IL-12、IL-4和IL-10分泌量变化;将转染miR-99a的诱导后巨噬细胞与EC细胞共培养,运用细胞计数试剂盒（cell counting kit-8,CCK-8）和transwell法检测其对EC细胞增殖和侵袭能力的影响,并初步分析其可能的作用机制。结果：EC组织CD68高表达并与肿瘤肌层浸润及血管生成呈正相关;肿瘤细胞培养上清液成功诱导单核细胞向M2型TAM极化。转染miR-99a后单核细胞组CD68及CD163表达较对照组下降（P＜0.01）,而CD206表达差异无统计学意义（P＞0.05）,流式细胞术进一步证实上述表达变化;ELISA结果发现,转染miR-99a诱导后巨噬细胞中IL-12分泌增多（P＜0.01）,而IL-4、IL-10分泌减少（P＜0.01）,提示巨噬细胞向M2型极化受抑制。将诱导后巨噬细胞与EC细胞共培养,共培养后EC细胞增殖侵袭能力较对照组增加,而转染miR-99a模拟片段至诱导后巨噬细胞能够抑制其对增殖（P＜0.01）及侵袭能力的促进作用（P＜0.05）。诱导后巨噬细胞中过表达miR-99a后细胞中mTOR及其通路受到抑制。结论：EC间质巨噬细胞浸润与肿瘤肌层浸润及血管新生相关,miR-99a能够逆转单核细胞向M2表型极化,并抑制EC细胞介导TAM的促EC细胞生长和侵袭作用,其作用可能通过调控mTOR通路产生。     

Metformin prevents cancer metastasis by inhibiting M2-like polarization of tumor associated macrophages

Accumulated evidence suggests that M2-like polarized tumor associated macrophages (TAMs) plays an important role in cancer progression and metastasis, establishing TAMs, especially M2-like TAMs as an appealing target for therapy intervention. Here we found that metformin significantly suppressed IL-13 induced M2-like polarization of macrophages, as illustrated by reduced expression of CD206, down-regulation of M2 marker mRNAs, and inhibition of M2-like macrophages promoted migration of cancer cells and endothelial cells. Metformin triggered AMPKα1 activation in macrophage and silencing of AMPKα1 partially abrogated the inhibitory effect of metformin in IL-13 induced M2-like polarization. Administration of AICAR, another activator of AMPK, also blocked the M2-like polarization of macrophages. Metformin greatly reduced the number of metastases of Lewis lung cancer without affecting tumor growth. In tumor tissues, the percentage of M2-like macrophage was decreased and the area of pericyte-coated vessels was increased. Further, the anti-metastatic effect of metformin was abolished when the animals were treated with macrophages eliminating agent clodronate liposome. These findings suggest that metformin is able to block the M2-like polarization of macrophages partially through AMPKα1, which plays an important role in metformin inhibited metastasis of Lewis lung cancer.     

Crosstalk between colon cancer cells and macrophages via inflammatory mediators and CD47 promotes tumour cell migration

Tumour-associated macrophages (TAMs) of the M2 phenotype are present in the stroma of many tumours and are frequently associated with the progression of several types of cancer. We investigated the role of M2 macrophages in colon cancer progression and found that human colon cancer tissue had elevated numbers of CD68⁺ (macrophage marker) cells and CD206⁺ (M2 macrophage marker) cells and increased CD47 expression. To explore potential interplay between colon cancer cells and M2 macrophages, we differentiated the monocyte cell line THP-1 into M1 and M2 macrophages (CD206^high and Th2 cytokine-secreting cells), respectively. M2 macrophages migrated faster than M1 macrophages towards SW480-conditioned medium. Similarly, M2 macrophage-conditioned medium induced SW480 cell migration and CD47 expression. Factors released by macrophages were involved in this induction. In addition, SW480 cells migrated faster when co-cultured with M2 macrophages. Inhibition of CD47 with blocking antibodies or siRNA significantly reduced the migration of SW480 cells in the presence of M2 macrophages. This effect was further decreased via blocking antibodies against the CD47 ligand signal-regulatory protein α (SIRPα). Additionally, cancer cells also secreted significant levels of IL-10, thereby promoting M2 macrophage differentiation. These findings indicate that a TAM-enriched tumour microenvironment promotes colon cancer cell migration and metastasis.     

Tumor associated macrophage expressing CD204 is associated with tumor aggressiveness of esophageal squamous cell carcinoma

Tumor associated macrophages (TAMs) are the most abundant cancer stromal cells educated by tumor microenvironment to acquire trophic functions facilitating angiogenesis, matrix breakdown and cancer cell motility. Tumor associated macrophages have anti‐inflammatory properties or “alternatively” activated (M2) phenotype expressing CD204 and/or CD163. To know the role of TAMs in the growth and progression of esophageal squamous cell carcinomas (ESCCs), we calculated intratumoral CD204, CD163 or CD68 expressing macrophage count (M?C) and CD34‐positive microvessel density (MVD) by immunohistochemistry in 70 cases of surgically resected ESCCs and compared them with the clinicopathological factors and prognosis of patients. M?C had positive linear association with MVD. High CD204⁺ M?C were significantly correlated with more malignant phenotypes including depth of tumor invasion, lymph and blood vessel invasion, lymph node metastasis as well as clinical stages. On the other hand, CD163⁺ M?C did not associate with these clinicopathological factors with the exception of depth of tumor invasion and blood vessel invasion. Patients with high CD204⁺ M?C ESCCs showed poor disease‐free survival (P = 0.021). Conditioned media of five ESCC cell lines (TE‐8, ‐9, ‐10, ‐11 and ‐15) induced mRNA as well as protein expression of CD204 but not of CD163 with upregulation of vascular endothelial growth factor‐A mRNA in TPA treated human acute monocytic leukemia cell line THP‐1. These results overall indicate that CD204 is a useful marker for TAMs contributing to the angiogenesis, progression and prognosis of ESCCs whose specific tumor microenvironment may educate macrophages to be CD204⁺ M2 TAMs.     

肿瘤相关巨噬细胞在胃癌中的作用

巨噬细胞广泛参与固有免疫及适应性免疫,其表型和功能具有很大可塑性,在不同微环境中巨噬细胞可分化为经典活化（M1表型）和替代活化（M2表型）,其中M2表型有免疫调节作用,参与诱导Th2反应。肿瘤微环境浸润的巨噬细胞即肿瘤相关巨噬细胞（TAMs）多具M2表型,能促进肿瘤新生血管生成、肿瘤侵袭和转移。由此可见,TAMs对胃癌的发生发展具有重要的免疫调节作用,本文就此进行综述。      

Role of tumor-associated macrophages at the invasive front in human colorectal cancer progression

Macrophages are an essential component of antitumor activity; however, the role of tumor-associated macrophages (TAMs) in colorectal cancer (CRC) remains controversial. Here, we elucidated the role of TAMs in CRC progression, especially at the early stage. We assessed the TAM number, phenotype, and distribution in 53 patients with colorectal neoplasia, including intramucosal neoplasia, submucosal invasive colorectal cancer (SM-CRC), and advanced cancer, using double immunofluorescence for CD68 and CD163. Next, we focused on the invasive front in SM-CRC and association between TAMs and clinicopathological features including lymph node metastasis, which were evaluated in 87 SM-CRC clinical specimens. The number of M2 macrophages increased with tumor progression and dynamic changes were observed with respect to the number and phenotype of TAMs at the invasive front, especially at the stage of submucosal invasion. A high M2 macrophage count at the invasive front was correlated with lymphovascular invasion, low histological differentiation, and lymph node metastasis; a low M1 macrophage count at the invasive front was correlated with lymph node metastasis. Furthermore, receiver operating characteristic curve analysis revealed that the M2/M1 ratio was a better predictor of the risk of lymph node metastasis than the pan-, M1, or M2 macrophage counts at the invasive front. These results suggested that TAMs at the invasive front might play a role in CRC progression, especially at the early stages. Therefore, evaluating the TAM phenotype, number, and distribution may be a potential predictor of metastasis, including lymph node metastasis, and TAMs may be a potential CRC therapeutic target.     

温阳散结汤含药血清通过NF-κB通路调控肿瘤相关巨噬细胞极化对Lewis肺癌的影响

目的:观察温阳散结汤对 Lewis 肺癌小鼠肿瘤生长及瘤组织中巨噬细胞M2型极化的影响,并探索其调控NF-κB信号通路抑制肿瘤相关巨噬细胞M2型极化,对Lewis肺癌细胞侵袭、迁移能力的影响。方法:建立C57BL/6小鼠 Lewis 肺癌移植瘤模型,温阳散结汤干预14 d 后观察小鼠瘤重和肺转移灶,计算肺转移抑瘤率,免疫组化染色检测小鼠瘤组织中巨噬细胞M2型表面标志物 CD206 的表达;温阳散结汤灌胃SD大鼠制备含药血清,采用IL-13干预RAW264.7小鼠巨噬细胞建立巨噬细胞M2型极化模型,CCK-8法检测温阳散结汤含药血清对巨噬细胞增殖的影响,流式细胞术和Western-blot检测 CD206的表达,RT-PCR检测巨噬细胞M2型标志基因的mRNA表达,ELISA法检测细胞上清中IL-1β、IL-10、TGF-β 和TNF-α 的水平变化,Western-blot检测氧化物酶体增殖物激活受体γ（PPARγ）、核因子-κB p65(NF-κB p65)和磷酸化核因子-κB p65（pNF-κB p65）的蛋白表达;收集温阳散结汤含药血清处理的M2型巨噬细胞条件培养基,将其作用于Lewis肺癌细胞,通过Transwell侵袭迁移实验检测Lewis 肺癌细胞的侵袭和运动迁移能力。结果:温阳散结汤干预后,明显减少了小鼠瘤重和肺转移灶数,并抑制了瘤组织CD206的阳性表达（P<0.05）;温阳散结汤含药血清干预后,IL-13诱导的M2型巨噬细胞中F4/80+CD206+的细胞比例和CD206蛋白表达明显减少,MRC1、Arg1、Ym1的mRNA表达水平显著降低,细胞中TNF-α、IL-1β的含量明显升高,IL-10、TGF-β的含量明显降低,同时明显下调了细胞PPARγ、NF-κB p65、pNF-κB p65的蛋白表达及pNF-κB p65/NF-κB p65比值（均P<0.05）;温阳散结汤含药血清处理的M2型巨噬细胞条件培养基干预后,Lewis肺癌细胞迁移和侵袭细胞数明显减少（P<0.05）。结论:温阳散结汤具有抑制肺癌肿瘤生长和转移的作用,其作用机制可能与调节NF-κB通路,抑制IL-13诱导的RAW264.7细胞M2型极化,从而抑制Lewis肺癌细胞侵袭和迁移能力相关。