Blocking LTB(4) signaling-mediated TAMs recruitment by Rhizoma Coptidis sensitizes lung cancer to immunotherapy.

Immune checkpoint blockade (ICB) induces durable immune responses across a spectrum of advanced cancers and revolutionizes the oncology field. However, only a subset of patients achieves long-lasting clinical benefits. Tumor-associated macrophages (TAMs) usually secrete immunosuppressive cytokines and contribute to the failure of ICB therapy. Therefore, it is crucial to mechanically manipulate the abundance and function of TAMs in the tumor microenvironment (TME), which can offer a promising molecular basis to improve the clinical response efficacy of ICB in cancer patients. This study aims to investigate TAMs in the immunosuppressive microenvironment to identify new therapeutic targets, improve the ability to predict and guide responses to clinical immunotherapy, and develop new strategies for immunotherapy of lung tumors. Lewis lung carcinoma (LLC) xenograft-bearing mouse models were established to analyze the antitumor activity of Rhizoma Coptidis (RC) in vivo. A systems pharmacology strategy was used to predict the correlation between RC and M2 macrophages. The effect of RC on the abundance of M2 macrophages was analyzed by flow cytometry of murine samples. Western blot was performed to analyze the expression of Leukotriene A4 hydrolase (LTA4H) and LTB4 receptor 1 (BLT1) in harvested lung cancer tissues. The impact of blocking leukotriene B4 (LTB4) signaling by RC on the recruitment of M2 macrophages was assessed in vitro and in vivo. Transwell migration assays were conducted to clarify the inhibition of macrophage migration by blocking LTB4. Lta4h-/- mice were used to investigate the sensitivity of immunotherapy to lung cancer by blocking the LTB4 signaling. Here, we report that RC, an herbal medicine from the family Ranunculaceae, suppresses the recruitment and immunosuppressive function of TAMs, which in turn sensitizes lung cancer to ICB therapy. Firstly, a systems pharmacology strategy was proposed to identify combinatorial drugs for ICB therapy with a systems biology perspective of drug-target-pathway-TME phenotype. We predicted and verified that RC significantly inhibits tumor growth and the infiltration of M2-TAMs into TME of LLC tumor-bearing mice. Then, RC inhibits the recruitment of macrophages to the tumor TME via blocking LTB4 signaling, and suppresses the expression of immunosuppressive factors (IL-10, TGF-β and VEGF). As a result, RC enables CD8+ T cells to retain their proliferative and infiltrative abilities within the TME. Ultimately, these events promote cytotoxic T-cell-mediated clearance of tumor cells, which is further enhanced by the addition of anti-PD-L1 therapy. Furthermore, we employed LTA4H deficient mice (Lta4h-/- mice) to evaluate the antitumor efficiency, the results showed that the efficacy of immunotherapy was enhanced due to the synergistic effect of LTB4 signaling blockage and ICB inhibition, leading to remarkable inhibition of tumor growth in a mouse model of lung adenocarcinoma. Taken together, these findings suggest that RC enhances antitumor immunity, providing a rationale for combining RC with immunotherapies as a potential anti-cancer treatment strategy.

Yan J ，Zhu J ，Li X ，Yang R ，Xiao W ，Huang C ，Zheng C ... -  《-》

ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation.

Rationale: Immune checkpoint inhibitors (ICIs) against the PD-1/PD-L1 pathway showed limited success in non-small cell lung cancer (NSCLC) patients, especially in those with activating epidermal growth factor receptor (EGFR) mutations. Elucidation of the mechanisms underlying EGFR-mediated tumor immune escape and the development of effective immune therapeutics are urgently needed. Immunoglobulin-like transcript (ILT) 4, a crucial immunosuppressive molecule initially identified in myeloid cells, is enriched in solid tumor cells and promotes the malignant behavior of NSCLC. However, the upstream regulation of ILT4 overexpression and its function in tumor immunity of NSCLC with EGFR activation remains unclear. Methods: ILT4 expression and EGFR phosphorylation in human NSCLC tissues and cell lines were analyzed using immunohistochemistry (IHC), real-time PCR, Western blotting, immunofluorescence, and flow cytometry. The molecular signaling for EGFR-regulated ILT4 expression was investigated using mRNA microarray and The Cancer Genome Atlas (TCGA) database analyses and then confirmed by Western blotting. The regulation of tumor cell proliferation and apoptosis by ILT4 was examined by CCK8 proliferation and apoptosis assays. The impact of ILT4 and PD-L1 on tumor-associated macrophage (TAM) recruitment and polarization was evaluated using Transwell migration assay, flow cytometry, enzyme linked immunosorbent assay (ELISA) and real-time PCR, while their impact on T cell survival and cytotoxicity was analyzed by CFSE proliferation assay, apoptotic assay, flow cytometry, ELISA and cytolytic assay. Tumor immunotherapy models targeting at paired Ig-like receptor B (PIR-B, an ortholog of ILT4 in mouse)/ILT4 and/or PD-L1 were established in C57BL/6 mice inoculated with stable EGFR- overexpressing Lewis lung carcinoma (LLC) cells and in humanized NSG mice inoculated with EGFR mutant, gefitinib-resistant PC9 (PC9-GR) or EGFR-overexpressing wild type H1299 cells. PIR-B and ILT4 inhibition was implemented by infection of specific knockdown lentivirus and PD-L1 was blocked using human/mouse neutralizing antibodies. The tumor growth model was established in NSG mice injected with PIR-B-downregulated LLC cells to evaluate the effect of PIR-B on tumor proliferation. The frequencies and phenotypes of macrophages and T cells in mouse spleens and blood were detected by flow cytometry while those in tumor tissues were determined by IHC and immunofluorescence. Results: We found that ILT4 expression in tumor cells was positively correlated with EGFR phosphorylation in human NSCLC tissues. Using NSCLC cell lines, we demonstrated that ILT4 was upregulated by both tyrosine kinase mutation-induced and epidermal growth factor (EGF)-dependent EGFR activation and subsequent AKT/ERK1/2 phosphorylation. Overexpressed ILT4 in EGFR-activated tumor cells induced TAM recruitment and M2-like polarization, which impaired T cell function. ILT4 also directly inhibited T cell proliferation, cytotoxicity, and IFN-γ expression and secretion. In EGFR-activated cell lines in vitro and in wild-type EGFR-activated C57BL/6 and humanized NSG immunotherapy models in vivo, either ILT4 (PIR-B) or PD-L1 inhibition enhanced anti-tumor immunity and suppressed tumor progression by counteracting TAM- and dysfunctional T cell- induced immuno-suppressive TME; the combined inhibition of both molecules showed the most dramatic tumor retraction. Surprisingly, in EGFR mutant, TKI resistant humanized NSG immunotherapy model, ILT4 inhibition alone rather than in combination with a PD-L1 inhibitor suppressed tumor growth and immune evasion. Conclusions: ILT4 was induced by activation of EGFR-AKT and ERK1/2 signaling in NSCLC cells. Overexpressed ILT4 suppressed tumor immunity by recruiting M2-like TAMs and impairing T cell response, while ILT4 inhibition prevented immunosuppression and tumor promotion. Furthermore, ILT4 inhibition enhanced the efficacy of PD-L1 inhibitor in EGFR wild-type but not in EGFR mutant NSCLC. Our study identified novel mechanisms for EGFR-mediated tumor immune escape, and provided promising immunotherapeutic strategies for patients with EGFR-activated NSCLC.

Chen X ，Gao A ，Zhang F ，Yang Z ，Wang S ，Fang Y ，Li J ，Wang J ，Shi W ，Wang L ，Zheng Y ，Sun Y ... -  《Theranostics》

Immunosuppressive TREM2(+) macrophages are associated with undesirable prognosis and responses to anti-PD-1 immunotherapy in non-small cell lung cancer.

Immune checkpoint blockade (ICB) has been improving patient outcomes of non-small cell lung cancer (NSCLC), but its effectiveness is highly subjective to individual tumor microenvironment. As dominant immune cells in NSCLC, tumor-associated macrophages (TAMs) display high diversity and plasticity. This study aims to find crucial TAM subtypes associated with ICB response in NSCLC. Large cohorts of NSCLC patients from The Cancer Genome Atlas and a single-cell sequencing dataset were integrated to illustrate immunosuppressive phenotypes of TAMs, followed by experimental verification. 341 NSCLC surgical samples and 40 tissue samples of NSCLC patients who received ICB treatment were collected to state the clinical importance of TAMs. We identified a TREM2 positive (+) TAM subtype in NSCLC stratifying patient responses to immunotherapy. NSCLC patients with high TREM2+ TAM infiltration exhibited advanced tumor progression, inferior prognosis and unique NSCLC molecular characteristics, especially mutations of EGFR. TREM2+ TAMs were induced in TME, but not existed in peripheral blood. TREM2+ TAMs were enriched with multiple anti-inflammatory cytokines, exhibiting a M2-like immunosuppressive phenotype, and potentiate T cell dysfunction including impaired anti-tumor activity of CD8+ T cells and enhanced differentiation towards FOXP3+ Tregs, thus facilitating immune evasion of NSCLC. Response rates to PD-1-based ICB were higher in patients with low TREM2+ TAMs (31.58%) compared to high TREM2+ TAMs (14.29%). Our findings implicated the immunosuppressive role of TREM2+ TAMs in NSCLC, and systematically reveal the clinical significance of TREM2+ TAMs as predictive and prognostic markers for NSCLC patients with ICB treatment.

Zhang H ，Liu Z ，Wen H ，Guo Y ，Xu F ，Zhu Q ，Yuan W ，Luo R ，Lu C ，Liu R ，Gu J ，Ge D ... -  《-》

Modified Bushen Yiqi formula enhances antitumor immunity by reducing the chemotactic recruitment of M2-TAMs and PMN-MDSCs in Lewis lung cancer-bearing mice.

Modified Bushen Yiqi formula (MBYF) has shown efficacy as an herbal combination therapy with anti-PD-1 for lung cancer patients. However, the underlying mechanisms of its antitumor effects in lung cancer remain unclear. This study aims to observe the antitumor effect of MBYF and explore its synergistic mechanism in combination with anti-PD-1 based on the tumor immune microenvironment. The antitumor effect of MBYF was assessed in Lewis Lung Cancer (LLC)-bearing mice by evaluating tumor volume, weight, and histology in five groups (model control, MBYF 8.125 g/kg, MBYF 16.25 g/kg, MBYF 32.50 g/kg, anti-PD-1). Mechanisms were analyzed using pharmacology network and tumor RNA-sequencing. Tumor-infiltrating immune cells were measured by flow cytometry and immunohistochemistry. Targets and pathways were validated through qRT-PCR, immuno-histochemistry, and western blotting. The synergistic effect of MBYF in combination with anti-PD-1 was validated in three groups (model control, anti-PD-1, anti-PD-1+MBYF 16.25 g/kg). MBYF inhibited tumor growth and proliferation and demonstrated safety for the heart, liver, and kidney. Mechanistically, MBYF downregulated tumor proliferation by suppressing the expression of CCND1, CTNNB1, EGFR, and the PI3K-AKT/STAT3/ERK pathway. Furthermore, MBYF may upregulated the antitumor immunity (CD4+T cells, active CD8+ T cells, and NK cells) by reducing the infiltration of M2-TAMs and PMN-MDSCs. MBYF may inhibit the recruitment of M2-TAMs by downregulating the CCR5-CCLs axis and PMN-MDSCs by the CXCR2-CXCLs axis. In vivo study confirmed that MBYF enhanced the antitumor effect of anti-PD-1 therapy. Modified Bushen Yiqi formula enhances antitumor immunity in the treatment of lung cancer by reducing the chemotactic recruitment of M2-TAMs and PMN-MDSCs, suggesting its potential as an adjunct therapy to enhance anti-PD-1 responses and improve treatment outcomes. Further research and clinical studies are needed to validate and expand upon these promising findings.

Kong Q ，Zhu H ，Gong W ，Deng X ，Liu B ，Dong J ... -  《-》

SHISA3 Reprograms Tumor-Associated Macrophages Toward an Antitumoral Phenotype and Enhances Cancer Immunotherapy.

The main challenge for immune checkpoint blockade (ICB) therapy lies in immunosuppressive tumor microenvironment (TME). Repolarizing M2-like tumor-associated macrophages (TAMs) into inflammatory M1 phenotype is a promising strategy for cancer immunotherapy. Here, this study shows that the tumor suppressive protein SHISA3 regulates the antitumor functions of TAMs. Local delivery of mRNA encoding Shisa3 enables cancer immunotherapy by reprogramming TAMs toward an antitumoral phenotype, thus enhancing the efficacy of programmed cell death 1 (PD-1) antibody. Enforced expression of Shisa3 in TAMs increases their phagocytosis and antigen presentation abilities and promotes CD8+ T cell-mediated antitumor immunity. The expression of SHISA3 is induced by damage/pathogen-associated molecular patterns (DAMPs/PAMPs) in macrophages via nuclear factor-κB (NF-κB) transcription factors. Reciprocally, SHISA3 forms a complex with heat shock protein family A member 8 (HSPA8) to activate NF-κB signaling thus maintaining M1 polarization of macrophages. Knockout Shisa3 largely abolishes the antitumor efficacy of combination immunotherapy with Toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) and PD-1 antibody. It further found that higher expression of SHISA3 in antitumoral TAMs is associated with better overall survival in lung cancer patients. Taken together, the findings describe the role of SHISA3 in reprogramming TAMs that ameliorate cancer immunotherapy.

Zhang S ，Yu B ，Sheng C ，Yao C ，Liu Y ，Wang J ，Zeng Q ，Mao Y ，Bei J ，Zhu B ，Chen S ... -  《Advanced Science》