Activation of Toll-like receptors signaling in non-small cell lung cancer cell line induced by tumor-associated macrophages

Background： Inflammation is often linked with the progress and poor outcome of lung cancer. The understanding of the relationship between tumor-associated macrophages （TAMs） and lung cancer cells involves in the underlying mechanism of inflammatory cytokine production. Toll-like receptors （TLRs） are engaged in promoting the production of pro-inflammatory cytokines and play an important role in tumor immunology. Methods： To investigate the mechanisms by which TAMs influence the production of pro-inflammatory cytoldnes in lung cancer cells, we established an in vitro coculture system using TAMs and human non- small cell lung cancer （NSCLC） cell line SPC-A1. Levels of interleukin （IL）-113, IL-6 and IL-8 in SPC-A1 were evaluated by RT-PCR and cytometric bead array assay after being cocultured with TAMs. Expression changes of TLRs and TLRs signaling pathway proteins in SPC-Al were further confirmed by RT-PCR and western blot. The level changes of IL-1β, IL-6 and IL-8 in SPC-Al were also detected after the stimulation of TLRs agonists. Results： We found that the phenotype markers of TAMs were highly expressed after stimulating human monocyte cell line THP-1 by phorbol-12-myristate-β-acetate （PMA）. Higher mRNA and supernate secretion levels of IL-1β, IL-6 and IL-8 were detected in SPC-A1 after being eocultured with TAMs. We also found that TLR1, TLR6 and TLR7 were up-regulated in SPC-A1 in the coculture system with TAMs. Meanwhile, TLRs signaling pathway proteins were also significantly activated. Moreover, pre-treatment with agonist ligands for TLR1, TLR6 and TLR7 could dramatically promote inductions of IL-1β, IL-6 and IL-8. Conclusions： These findings demonstrated that TAMs may enhance IL-1β, IL-6 and IL-8 expressions via TLRs signaling pathway. We conclude that TAMs contribute to maintain the inflammation microenvironment and ultimately promote the development and progression of lung cancer.

2-Hexyl-4-Pentylenic Acid(HPTA) Stimulates the Radiotherapy-induced Abscopal Effect on Distal Tumor through Polarization of Tumor-associated Macrophages

Objective The aim of this study was to explore the effects of 2-hexyl-4-pentylenic acid(HPTA)in combination with radiotherapy(RT)on distant unirradiated breast tumors.Methods Using a rat model of chemical carcinogen(7,12-dimethylbenz[a]anthracene,DMBA)-induced breast cancer,tumor volume was monitored and treatment response was evaluated by performing HE staining,immunohistochemistry,immunofluorescence,q RT-PCR,and western blot analyses.Results The results demonstrated that HPTA in combination with RT significantly delayed the growth of distant,unirradiated breast tumors.The mechanism of action included tumor-associated macrophage(TAM)infiltration into distant tumor tissues,M1 polarization,and inhibition of tumor angiogenesis by IFN-γ.Conclusion The results suggest that the combination of HPTA with RT has an abscopal effect on distant tumors via M1-polarized TAMs,and HPTA may be considered as a new therapeutic for amplifying the efficacy of local RT for non-targeted breast tumors.

A bioactive nanocomposite integrated specific TAMs target and synergistic TAMs repolarization for effective cancer immunotherapy

Reactive oxygen species(ROS)generated from photosensitizers exhibit great potential for repolarizing immunosuppressive tumor-associated macrophages(TAMs)toward the anti-tumor M1 phenotype,representing a promising cancer immunotherapy strategy.Nevertheless,their effectiveness in eliminating solid tumors is generally limited by the instability and inadequate TAMs-specific targeting of photosensitizers.Here,a novel core-shell integrated nano platform is proposed to achieve a coordinated strategy of repolarizing TAMs for potentiating cancer immunotherapy.Colloidal mesoporous silica nanoparticles(CMSN)are fabricated to encapsulate photosensitizer-Indocyanine Green(ICG)to improve their stability.Then ginseng-derived exosome(GsE)was coated on the surface of ICG/CMSN for targeting TAMs,as well as repolarizing TAMs concurrently,named ICG/CMSN@GsE.As expected,with the synergism of ICG and GsE,ICG/CMSN@GsE exhibited better stability,mild generation of ROS,favorable specificity toward M2-like macrophages,enhancing drug retention in tumors and superior TAMs repolarization potency,then exerted a potent antitumor effect.In vivo,experiment results also confirm the synergistic suppression of tumor growth accompanied by the increased presence of anti-tumor M1-like macrophages and maximal tumor damage.Taken together,by integrating the superiorities of TAMs targeting specificity and synergistic TAMs repolarization effect into a single nanoplatform,ICG/CMSN@GsE can readily serve as a safe and high-performance nanoplatform for enhanced cancer immunotherapy.

Overexpression of interleukin-17 in tumor-associated macrophages is correlated with the differentiation and angiogenesis of laryngeal squamous cell carcinoma

Background Interleukin-l7 （IL-17）, which exerts strong pro-inflammatory effects, has emerged as an important mediator in inflammation-associated cancer. The aim of this study was to clarify the relationship between IL-17 and tumor associated macrophages （TAMs）, and the correlation of the microvessel density in the development of laryngeal squamous cell carcinoma （LSCC）.Methods Histopathological observations and immunohistochemistry staining for IL-17, CD68, and CD34 were performed on 72 specimens （32 cases of LSCC, 20 cases of adjacent tissues of carcinoma as controls, and 20 cases of chronic hypertrophic laryngitis）. Double immunohistochemical staining was done to determine which cells expressed IL-17. Real-time quantitative PCR determined the mRNA expression of IL-17. ELISA was used to detect the expression of the serum level of IL-17 in the three groups.Results The inflammation response had increased in LSCC. Overexpression of IL-17 and CD68 protein were seen in LSCC （P 〈0.01）. The expression of IL-17 was different between well and poorly differentiated LSCC （P 〈0.01）. The IL-17 expressing cells were mainly located in macrophages （CD68＋/IL17＋） as demonstrated by double immunohistochemical staining. IL-17 expression significantly correlated with high microvessel density （CD34＋） in LSCC （P 〈0.05）. Relatively higher mRNA expression levels of IL-17 were seen in LSCC compared to the controls （P 〈0.05）. The serum expression of IL-17 was similar among the three groups （P 〉0.05）.Conclusion IL-17 was expressed by TAMs, and IL-17 may significantly correlate to the differentiation and angiogenesis in the development of LSCC.

口腔鳞癌血管生成中MMP-2与巨噬细胞的关系

目的:探讨人类口腔鳞状细胞癌(OSCC)中基质金属蛋白酶-2(MMP-2)的表达并与肿瘤相关巨噬细胞(TAMs)以及微血管密度(MVD)间的关系。方法:采用免疫组化SABC法分别检测在42例口腔鳞癌和10例口腔正常组织中MMP-2的表达情况和CD68标记的巨噬细胞及CD34标记的MVD值。结果:口腔鳞癌中MMP-2的表达与淋巴结转移、TNM分期有关(P<0.05);MMP-2的表达与MVD值之间存在相关性(r=0.641,P<0.001),同时也与肿瘤内浸润的巨噬细胞相关(r=0.433,P<0.01)。结论:OSCC中的TAMs与MMP-2密切相关,TAMs可能通过MMP-2发挥促血管生成作用,促进肿瘤生长、发展和转移。

卵巢上皮源性肿瘤动态对比增强磁共振成像半定量参数与肿瘤相关性巨噬细胞密度关系的研究

目的:研究探讨卵巢上皮源性肿瘤动态对比增强磁共振成像(DCE-MRI)半定量参数与肿瘤组织微环境中肿瘤相关性巨噬细胞(TAMs)密度之间的关系。方法:选择在医院治疗的80例卵巢上皮源性肿瘤患者,所有患者术前均行DCE-MRI检查,计算DCE-MRI半定量参数,患者手术时均行TAMs病理学检查,观察DCE-MRI半定量参数与TAMs密度之间的关系。结果:DCE-MRI半定量参数中达峰时间(TTP)值随着肿瘤的恶性程度增加而降低,最大浓度(Cmax)、浓度-时间曲线下面积(AUC)及最大斜率(MS)值随肿瘤的恶性程度增加逐渐增加,差异均有统计学意义(F=5.210,F=3.284,F=2.587,F=6.301;P<0.05);以病理诊断结果为“金标准”绘制受试者工作特征(ROC)曲线显示,MS的AUC最大,其灵敏度、特异度及准确率分别为70.86%、77.15%和72.89%;不同肿瘤恶性程度的TAMs比较,差异有统计学意义(F=35.787,P<0.05);DCE-MRI半定量参数TTP、Cmax、AUC及MS与TAMs密度关系之间无统计学意义。结论:DCE-MRI半定量参数AUC、Cmax及MS对卵巢上皮源性肿瘤有较高的鉴别诊断价值,而MS值诊断价值最高,且可间接判断肿瘤组织的TAMs密度,有利于判断肿瘤的性质。

Ultrasound-visualized nanocarriers with siRNA for targeted inhibition of M2-like TAM polarization to enhance photothermal therapy in NSCLC

Photothermal therapy(PTT)has received a lot of attention as a promising strategy for eliminating tumors quickly.However,the unavoidable inflammatory response during the treatment might result in a high concentration of M2-like tumor-associated macrophages(TAMs),increasing the risk of tumor recurrence and metastasis.To address this problem,gold-based nanocarriers(PGMP-small interfering RNA(siRNA)nanoparticles(NPs))containing STAT6siRNA,that inhibited M2-like TAM polarization,were designed and investigated for PTT and gene therapy of non-small cell lung cancer(NSCLC).In an NSCLC model,the nanocarriers demonstrated excellent siRNA delivery ability and a high gene transfection rate of up to 90%in macrophages,thus inhibiting the polarization of about 87%of M2-like TAMs and effectively suppressing the invasion and metastasis of NSCLC.Meanwhile,the unique gold nanosphere structure offered improved PTT and contrast-enhanced ultrasound imaging,thus contributing to the efficient elimination and real-time monitoring of the tumor tissues.These nanocarriers with combined gene and photothermal therapeutic capabilities improved the efficacy of single-modality treatment,and showed the potential to inhibit cancer cell recurrence and metastasis to ultimately cure NSCLC.

TGR5 deficiency activates antitumor immunity in non-small cell lung cancer via restraining M2 macrophage polarization

The bile acid-responsive G-protein-coupled receptor TGR5 is expressed in monocytes and macrophages,and plays a critical role in regulating inflammatory response.Our previous work has shown its role in promoting the progression of non-small cell lung cancer(NSCLC),yet the mechanism remains unclear.Here,using Tgr5-knockout mice,we show that TGR5 is required for M2 polarization of tumorassociated macrophages(TAMs)and suppresses antitumor immunity in NSCLC via involving TAMsmediated CD8+T cell suppression.Mechanistically,we demonstrate that TGR5 promotes TAMs into protumorigenic M2-like phenotypes via activating c AMP-STAT3/STAT6 signaling.Induction of c AMP production restores M2-like phenotypes in TGR5-deficient macrophages.In NSCLC tissues from human patients,the expression of TGR5 is associated with the infiltration of TAMs.The co-expression of TGR5 and high TAMs infiltration are associated with the prognosis and overall survival of NSCLC patients.Together,this study provides molecular mechanisms for the protumor function of TGR5 in NSCLC,highlighting its potential as a target for TAMs-centric immunotherapy in NSCLC.

Targeting UDP-α-D-glucose 6-dehydrogenase alters the CNS tumor immune microenvironment and inhibits glioblastoma growth

Glioblastoma(GBM,WHO grade IV glioma)is the most common and lethal malignant brain tumor in aduts with a dismal prognosis.The extracellular matrix(ECM)supports GBM progression by promoting tumor cell proliferation,migration,and immune escape.Uridine diphosphate(UDP)-glucose 6-dehydrogenase(UGDH)is the rate-limiting enzyme that catalyzes the biosynthesis of glycosaminoglycans that are the principal component of the CNS ECM.We investigated how targeting UGDH in GBM infuence$the GBM immune microenvironment,including tumor-associated microglia/macrophages(TAMs)and T cells.TAMs are the main im-mune effector cells in GBM and can directly target tumor cells if properly activated.In co-cultures of GBM cells and human primary macrophages,UGDH knockdown in GBM cells pro-moted macrophage phagocytosis and M-like polarization.In orthotropic human GBM xeno-grafts and syngeneic mouse glioma models,targeting UGDH decreased ECM deposition,increased TAM phagocytosis marker expression,reduced M2-like TAMS and inhibited tumor growth.UGDH knockdown in GBM cells also promoted cytotoxic T cell ifltration and activa-tion in orthotopic syngeneic mouse glioma models.The potent and in-human-use small mole-cule GAG synthesis inhibitor 4-methylumbelliferone(4-MU)was found to inhibit GBM cell proliferation and migration in vitro,mimic the macrophage and T-cell responses to UGDH knockdown in vitro and in vivo and inhibit growth of orthotopic murine GBM.Our study shows that UGDH supports GBM growth through multiple mechanisms and supports the development of ECM-based therapeutic strategies to simultaneously target tumor cells and their microenvi-ronment.