Tongxie Yaofang Regulates Expression of NKG2DL to Enhance Anti-tumor Effect of NK Cells in Colon Cancer under Chronic Stress / 中国实验方剂学杂志

ObjectiveTo observe the effect of Tongxie Yaofang on the function of tumor-related natural killer （NK） cells under chronic stress and explore the possible molecular mechanism. MethodFifty SPF-grade BABL/C male mice were randomized into normal， model， and low-， medium-， and high-dose （6.825， 13.65， and 27.3 g·kg-1， respectively） Tongxie Yaofang groups， with 10 mice in each group. Other groups except the blank group were subjected to 7 days of chronic restraint stress， and then forced swimming and tail suspension tests were carried out to evaluate the modeling performance. After the successful modeling， rats in Tongxie Yaofang groups were administrated with low-， medium-， and high-doses of Tongxie Yaofang by gavage， while those in the other groups were administrated with normal saline by gavage. After 14 days， each group of mice was inoculated with subcutaneous colon cancer to establish the model of colon cancer under chronic stress. The pathological changes of the tumor tissue in each group of mice were observed using hematoxylin-eosin （HE） staining. The content of CD49b-positive cells in the peripheral blood and tumor tissue of mice was measured by flow cytometry. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the content of molecules associated with NK cell activation in the peripheral blood. Western blot was employed to determine the protein levels of major histocompatibility complex class Ⅰ polypeptide-related sequences A and B （MICA+MICB） and UL-16-binding protein 1 （ULBP1） in the tumor tissue. ResultCompared with the normal group， the model group showed a decrease in 5-hydroxytryptamine （5-HT） content and an increase in corticosterone （CORT） content in the serum （P<0.05）. Compared with the model group， Tongxie Yaofang increased the 5-HT content and decreased the CORT content （P<0.05， P<0.01）. Compared with the normal group， the modeling increased the tumor volume and weight （P<0.05）， while Tongxie Yaofang inhibited such increases with no statistical significance. The tumor cells in the model group presented neat arrangement， irregular shape， uneven size， obvious atypia， common nuclear division， and small necrotic area， and blood vessels were abundant surrounding the tumor cells. Compared with the model group， Tongxie Yaofang groups showed sparse arrangement of tumor cells， different degrees of patchy necrosis areas in the tumor， and karyorrhexis， dissolution， and nuclear debris in the necrotic part. Compared with the normal group， the model group showed reduced CD49b-positive cells in the peripheral blood and tumor tissue （P<0.01）. Compared with the model group， Tongxie Yaofang increased CD49b-positive cells （medium dose P<0.01， high dose P<0.05， P<0.01）. Compared with the normal group， the modeling lowered the serum levels of granzymes-B （Gzms-B）， perforin （PF）， interferon （IFN）-γ， and tumor necrosis factor （TNF）-α （P<0.05， P<0.01）. Compared with the model group， low-dose Tongxie Yaofang elevated the serum levels of PF， Gzms-B， and TNF-α （P<0.05， P<0.01）， and medium-dose Tongxie Yaofang elevated the serum levels of Gzms-B， PF， IFN-γ， and TNF-α （P<0.05， P<0.01）. In addition， high-dose Tongxie Yaofang elevated the serum levels of PF， IFN-γ， and TNF-α （P<0.01）. Compared with the normal group， the model group presented down-regulated protein level of ULBP1 （P<0.05）. Compared with the model group， low-， medium-， and high-dose Tongxie Yaofang up-regulated the protein level of ULBP1 （P<0.05， P<0.01）， and medium- and high-dose Tongxie Yaofang up-regulated the protein level of MICA+MICB （P<0.05， P<0.01）. ConclusionTongxie Yaofang may promote NK cell activation by up-regulating the expression of MICA+MICB and ULBP1， thereby delaying the progression of colon cancer under chronic stress.

Effects of apatinib on quality of life and immune function in older adult patients with advanced non-small cell lung cancer / 中国基层医药

Objective:To analyze the effects of apatinib on quality of life and immune function in older adult patients with advanced non-small cell lung cancer.Methods:A total of 187 older adult patients with advanced non-small cell lung cancer admitted to Taizhou Cancer Hospital from January 2017 to January 2021 were included in this study. They were divided into the control group ( n = 93) and the observation group ( n = 94). The control group was treated with carboplatin combined with pemetrexed and the observation group was treated with apatinib based on carboplatin and pemetrexed. Sign and symptoms remission was compared between the observation and control groups. The levels of tumor markers, immune function, and quality of life score were compared between the two groups before and after treatment. Results:Total remission rate in the observation group was significantly higher than that in the control group (88.30% vs. 69.89%, χ2 = 9.59, P < 0.05). After treatment, carbohydrate antigen 125, carbohydrate antigen 50, and carcinoembryonic antigen in the observation group were (16.25 ± 5.47) μg/L, (15.23 ± 3.27) μg/L and (5.91 ± 2.66) mg/L, respectively, which were significantly lower than (21.49 ± 6.61) μg/L, (19.11 ± 3.48) μg/L and (10.14 ± 2.73) mg/L in the control group ( t = 5.91, 7.86, 10.73, all P < 0.05). The percentage of CD3 + and CD4 + cells, and the ratio of CD4 +/CD8 + cells in the observation group were (69.34 ± 8.85)%, (38.15 ± 6.52)%, (1.40 ± 0.33), respectively, which were significantly higher than (64.51 ± 8.74)%, (33.55 ± 6.33)%, (1.23 ± 0.25) in the control group ( t = -3.75, -5.36, -3.97, all P < 0.05). Quality of life score was increased in each group ( P < 0.001). The amplitude of increase in quality of life score was greater in the observation group compared with the control group ( P < 0.001). Conclusion:Apatinib can effectively reduce the level of tumor markers and improve immune function in older adult patients with advanced non-small cell lung cancer and improve quality of life.

Combination immunotherapy of glioblastoma with dendritic cell cancer vaccines,anti-PD-1 and poly I:C / 药物分析学报

Glioblastoma(GBM)is a lethal cancer with limited therapeutic options.Dendritic cell(DC)-based cancer vaccines provide a promising approach for GBM treatment.Clinical studies suggest that other immu-notherapeutic agents may be combined with DC vaccines to further enhance antitumor activity.Here,we report a GBM case with combination immunotherapy consisting of DC vaccines,anti-programmed death-1(anti-PD-1)and poly I:C as well as the chemotherapeutic agent cyclophosphamide that was integrated with standard chemoradiation therapy,and the patient remained disease-free for 69 months.The patient received DC vaccines loaded with multiple forms of tumor antigens,including mRNA-tumor associated antigens(TAA),mRNA-neoantigens,and hypochlorous acid(HOCl)-oxidized tumor lysates.Furthermore,mRNA-TAAAs were modified with a novel TriVac technology that fuses TAAs with a destabilization domain and inserts TAAs into full-length lysosomal associated membrane protein-1 to enhance major histo-compatibility complex(MHC)class Ⅰ and Ⅱ antigen presentation.The treatment consisted of 42 DC cancer vaccine infusions,26 anti-PD-1 antibody nivolumab administrations and 126 poly I:C injections for DC infusions.The patient also received 28 doses of cyclophosphamide for depletion of regulatory T cells.No immunotherapy-related adverse events were observed during the treatment.Robust antitumor CD4+and CD8+T-cell responses were detected.The patient remains free of disease progression.This is the first case report on the combination of the above three agents to treat glioblastoma patients.Our results suggest that integrated combination immunotherapy is safe and feasible for long-term treatment in this patient.A large-scale trial to validate these findings is warranted.

The effect of tumor-associated macrophage on proliferation of renal carcinoma cells and related mechanism / 中华泌尿外科杂志

Objective:To investigate the effect of tumor-associated macrophage(TAM) on proliferation of renal carcinoma cells and its related mechanism.Methods:The model of TAM was established by stimulating human monocytic leukemia cell line THP-1 with phorbol myristate acetate (PMA), bacterial endotoxin (LPS) and interferon-γ (IFN- γ). Then the TAM model was co-cultured with carcinoma cell lines ACHN and 786-O in vitro .The cytokines IL-6, TNF-α and IL-1β in TAM supernatant were detected by enzyme-linked immunosorbent assay (ELISA). MTT method was used to detect the proliferation of ACHN and 786-O cells treated with supernatant of TAM or TAM/Tocilizumab. Western blot was used to detect lactate dehydrogenase A (LDHA) expression of both renal cancer cells co-cultured with TAM or TAM/Tocilizumab. The ACHN and 786-O cells with LDHA-overexpression and LDHA-knockdown were cultured in TAM supernatant in vitro. The cell proliferation was detected by MTT and the relative proliferation rate was calculated.Results:THP-1 cells was differentiated into TAM through the treatment of 80 ng/ml PMA combined with 20 ng/ml LPS and 20 ng/ml IFN- γ.The expression rate of CD68, a cell surface marker on TAM, was (36.2 ±4.5)%. When TAM was co-cultured with ACHN cells, the results of ELISA showed that the secretion of IL-6 in the supernatant was significantly elevated compared with that in the supernatant when ACHN cells cultured alone [(138.0 ±12.4) pg/ml and (19.7±4.9) pg/ml], and the secretion of TNF- α [(122.5 ±14.2) pg/ml and (12.6 ±2.3) pg/ml] and IL-1 β [(89.2 ±6.4) pg/ml and (69.2 ±3.5) pg/ml] were also significantly increased. The secretion of IL-6 [(119.2 ±14.8) pg/ml and (17.1 ±3.3) pg/ml], TNF- α [(122.6 ±14.4) pg/ml and (45.7 ±7.2) pg/ml] and IL-1 β [(95.1 ±11.8) pg/ml and (88.2 ±12.7) pg/ml] in the supernatant were also significantly elevated when 786-O cells co-cultured with TAM compared with 786-O cells cultured alone. After treated with the supernatant of TAM for 72 hours, the relative proliferation rates of ACHN and 786-O cells [(128.6 ±21.4)% and (124.2 ±19.7)%] were significantly higher than that of the control group (100.0%). At the same time, the expression of LDHA in ACHN and 786-O cells increased significantly. After 72 hours of treatment with the supernatant of TAM combined with tocilizumab, the relative proliferation rates of ACHN and 786-O cells [(76.5±13.7)% and (74.8±12.5)%] were significantly lower than that of the control group(100.0%), and the expression of LDHA was also significantly decreased at the same time. The relative proliferation rates of ACHN and 786-O cells in LDHA overexpression group [(121.5 ±17.2)% and (122.7±21.6)%]were significantly higher than that in blank-vector-transfection group[(93.3±10.7)% and (89.8±11.2)%], while the relative proliferation rates in LDHA-knockdown group [(61.4±11.2)% and (58.0 ±10.6)% ]were significantly lower than that in blank-vector-transfection group.Conclusions:By secreting IL-6, TAM can up-regulate the expression of LDHA and promote the proliferation of renal cancer cells.

Research advances in tumor-associated macrophages in hepatocellular carcinoma microenvironment / 临床肝胆病杂志

Since there is a lack of obvious clinical symptoms in the early stage of hepatocellular carcinoma (HCC), most patients have progressed to the advanced stage at the time of confirmed diagnosis. There are limited treatment options for HCC patients who miss the opportunity for surgery, so it is of great importance to find new therapeutic targets. Tumor-associated macrophages (TAMs) are a group of macrophages existing in the tumor immune microenvironment and affect the malignant behaviors of HCC cells and the state of immune escape within the tumor. This article introduces the origin and classification of TAM, summarizes the role and mechanism of TAMs in vascular proliferation, invasion and metastasis, formation and maintenance of stemness, and anti-tumor immunity in HCC, and briefly describes the current research advances in therapeutic targets for TAM, and it is pointed out that targeting TAM may be a promising direction for clinical treatment.

Effect Mechanism of Modified Baitouwengtang Treating Colorectal Cancer by Regulating CSF1R/STING/TBK1 Signaling to Polarize Phenotype of Tumor-associated Macrophages （TAMs） / 中国实验方剂学杂志

ObjectiveThis study aims to investigate the effect of modified Baitouwengtang （MBTWD） on tumor growth and the number of tumor-associated macrophages （TAMs） in tumor tissue of MC38 cell tumor-bearing mice with colorectal cancer and explores whether MBTWD mediates the remodeling of TAM phenotype to play an immunologically antitumor effect. MethodFirstly， The C57BL/6 mouse tumor model grafted subcutaneously was established， and then model mice were classified into a model group， positive control group（3 mg·kg-1）， and MBTWD groups with high and low dosages（23.43、46.86 g·kg-1）， with 10 mice in each group. In addition， 10 healthy mice were set as the blank group， and the changes in body weight， tumor volume， and survival status of mice in each group were observed. Tumor tissue， spleen， and peripheral blood were collected to calculate the tumor volume change， tumor inhibition rate， and spleen mass. Hematoxylin-eosin （HE） staining was used to observe the morphological changes of tumor tissue， and an immunofluorescence assay was used to detect the expression levels of CD4， CD8， and CD206 in tumor tissues of tumor-bearing mice. The secretion levels of transforming growth factor （TGF）-β， interleukin （IL）-6， and chemokine （C-C Motif） ligand 2 （CCL2） in peripheral serum were measured by using enzyme-linked immunosorbent assay （ELISA）. Secondly， a co-culture model induced by IL-4 in vitro of MC38 cells and murine monocytic macrophage RAW264.7 cells was established. Cell proliferation and activity assay （CCK-8） was used to detect the inhibitory effect of MBTWD containing serum on cell proliferation. A transwell experiment was used to detect the effect of IL-4-induced M2 macrophages on the invasion of MC38 cells. Flow cytometry was used to detect the expression of CD86 on the membrane of M2 macrophages induced by IL-4 with MBTWD containing serum. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the effect of MBTWD containing serum on the mRNA expression levels of M1 macrophage-related polarization factors CD86， nitric oxide synthase （iNOS）， and IL-12， as well as M2 macrophage-related polarization factors CD206， CD163， and IL-10 after co-cultivation. Finally， the protein expression levels of colony-stimulating factor 1 receptor （CSF1R）， stimulator of interferon genes （STING）， and TANK binding kinase 1 （TBK1） in tumor tissues of tumor-bearing mice were detected by Western blot. ResultIn vivo experimental results show that compared with the model group， the MBTWD can significantly inhibit the tumor growth of tumor-bearing mice. Immunofluorescence experiments show that the MBTWD can increase the number of CD8+ T cell infiltration in tumor tissue of tumor-bearing mice， reduce the number of CD206+ TAMs infiltration， and down-regulate the secretion levels of cytokines IL-6， TGF-β， and CCL2 in peripheral blood of tumor-bearing mice. The results of in vitro experiments show that the MBTWD containing serum has no obvious inhibitory effect on cell proliferation， but the cell supernatant after co-cultivation with RAW264.7 cells can inhibit the proliferation activity of MC38 cells， and the invasion ability of MC38 cells is enhanced by IL-4-induced M2 macrophages. However， this effect can be inhibited in a concentration-dependent manner by the MBTWD containing serum. At the same time， the results of Real-time PCR show that the MBTWD containing serum can up-regulate the mRNA expression levels of M1 macrophage-related polarization factors CD86， iNOS， and IL-12 and down-regulate those of M2 macrophage-related polarization factors CD206， CD163， and IL-10. Flow cytometry results also confirm that the MBTWD containing serum can increase the number of repolarized CD86+ M1 macrophages， indicating that MBTWD can induce M2 macrophages to repolarized M1 macrophages to play an anti-tumor growth role. Finally， Western blot results show that MBTWD can down-regulate the expression of CSF1R protein and up-regulate that of STING and TBK1 proteins in tumor tissue of tumor-bearing mice. ConclusionMBTWD can down-regulate the infiltration number of CD206+ TAMs and increase the infiltration of CD8+ T cells， thereby playing an immunologically antitumor effect on the growth inhibition of colorectal cancer， which may be related to regulating CSF1R signaling and then activating STING/TBK1 signaling pathway to induce phenotypic remodeling of TAMs.

Research progress in drugs targeting tumor associated macrophage / 药学学报

Tumor brings great threat to human public health. In recent years, incidence rate and mortality of tumor were rapidly increased in the world. Anti-tumor therapies have undergone the development of cytotoxic therapy, targeted therapy, and immunotherapy. Among them, tumor immunotherapy is rapidly developed and becomes an important anti-tumor therapy in recent years, although it also brings some related side effects. Tumor microenvironment (TME) is composed of immune cells, vascular vessels, fibroblasts, the extracellular matrix, etc. TME significantly affects the efficacy of immunotherapy. Macrophages in the TME are named as tumor associated macrophages (TAMs). Recently, increasing studies have shown that TAMs play an important role in the regulation of tumor immunity, especially in tumor immune surveillance and immune escape. Currently, more and more anti-tumor immunotherapy strategies targeting TAMs are at the development stage. Based on the important role of TAMs in the TME and their potential as therapeutic targets in tumor immunotherapy, we first reviewed the subtypes and functions of TAMs, as well as the roles of TAMs in tumors. Furthermore, we summarized the research progress on anti-tumor strategies targeting TAMs and the current status of drug targeting TAMs. The current review will provide new ideas and novel insights for tumor immunotherapy.

Progress in antitumor effect of tumor-associated macrophages in co-culture system / 中华微生物学和免疫学杂志

Macrophages are important cells of the immune system. Tumor-associated macrophages are enriched macrophages near tumor cells or tissues. Their role is mainly to promote the construction of tumor inflammatory microenvironment and inhibit tumor immune response. Cell co-culture system is a symbiotic culture system formed by mimicking the internal environment of the body in vitro. The co-culture condition is relatively consistent with the environment in vivo, enabling better information exchange and material exchange between cells, which is a supplement to the monolayer cell culture and animal experiments. Tumor-associated macrophages and tumor cells co-exist in the tumor microenvironment. Thus, constructing a co-culture system for tumor-associated macrophages and tumor cells would be conducive to studying the antitumor effect of tumor-associated macrophages and developing new immunotherapy drugs. The co-culture system would provide a new direction for treating malignant tumors. This article mainly reviewed the co-culture patterns of macrophages and the antitumor effects of different phenotypes of macrophages, and highlighted the importance of using immunotherapy to treat malignant tumors in the tumor microenvironment.

Preparation and polarization activity research of astragalus polysaccharide-superparamagnetic iron oxide nanocomposite / 药学学报

Size and surface modification are the two key factors affecting the effect of macrophages polarization induced by superparamagnetic iron oxide nanoparticles (SPIONs). The smaller the particle size, the better the polarization effect of SPIONs. Besides, the reasonable SPIONs surface modification method can also be used to enhance the polarization effect. In this study, SPIONs was prepared by solvothermal method and optimized by Box-Benhnken center combination design and response surface method. Furthermore, astragalus polysaccharide-superparamagnetic iron oxide nanocomplex (APS-SPIONs) was successfully constructed by EDC/NHS esterification method. The structure of APS-SPIONs was confirmed by dynamic light scatter and infrared spectrometer, and the contents of iron and polysaccharide were characterized by spectrophotometry. The effect of APS-SPIONs on inducing mouse macrophages RAW264.7 polarization was investigated by flow cytometry. The RAW264.7 macrophages-HepG2 human hepatoma cancer cells Transwell co-culture system was established to investigate APS-SPIONs improve anti-tumor function of macrophages in vitro, and the proliferation activity of APS-SPIONs on RAW264.7 detected by cell counting kit-8 (CCK-8) method. The results showed that the average particle size and zeta potential of APS-SPIONs were (82.93 ± 1.47) nm and (-24.00 ± 0.47) mV. Polysaccharide and Fe content were 8.69% and 7.04%, respectively. APS-SPIONs effectively induced the polarization of RAW264.7 into M1 type in vitro, improving the anti-tumor ability of macrophages in a co-culture system, without effecting the proliferation of macrophages. Our study provides a drug development strategy and preliminary research results to educate macrophages and reshape the tumor immune microenvironment to achieve tumor-killing effects.

Shuyuwan Regulates M1/M2 Polarization of Tumor-associated Macrophages to Inhibit Progression of Colorectal Cancer / 中国实验方剂学杂志

ObjectiveTo explore the molecular mechanism of Shuyuwan regulating polarization of tumor-associated macrophages （TAMs） to inhibit the progression of colorectal cancer （CRC）. MethodThe nude mouse model of orthotopic transplantation of colon cancer was established. Male BALB/c-nu nude mice （n=28， 4 weeks old） were randomly assigned into 4 groups （n=7）： Model group （normal saline） and low-， medium-， and high-dose （1.725， 2.310， 2.895 g·kg-1·d-1， respectively） Shuyuwan groups. On day 9 after the tumor block was inoculated， the mice were administrated by gavage with corresponding agents at a dose of 15 mL·kg-1 once a day， 6 days a week， and no agent on the 7th day. After two consecutive weeks of intervention， the nude mice were sacrificed and the tumor samples were collected. A part of the colon tissue and the tumor tissue was used to prepare sections， and hematoxylin-eosin （HE） staining was performed for pathological observation. The expression of inducible nitric oxide synthase （iNOS） and arginase-1 （Arg-1） in the tumor tissue was detected by immunohistochemistry （IHC）. The mRNA levels of interleukin-12 （IL-12）， epidermal growth factor （EGF）， and transforming growth factor-β1 （TGF-β1） in the tumor tissue were determined by Real-time polymerase chain reaction （PCR）. Western blot was employed to determine the protein levels of iNOS， IL-12， EGF， and TGF-β1 in the tumor tissue. ResultCompared with the model group， Shuyuwan inhibited the growth of colon cancer cells in nude mice and caused the tumor cell necrosis in different degrees. The high-dose Shuyuwan group had the strongest inhibitory effect on the growth of tumor cells， which basically lost the normal morphology. Furthermore， Shuyuwan up-regulated the expression of iNOS and IL-12 in M1-type macrophages （P<0.05） and down-regulated the expression of Arg-1， EGF， and TGF-β1 in M2-type macrophages （P<0.05）， which indicated the weakened polarization of macrophages toward M2 type and the enhanced polarization toward M1 type after treatment with Shuyuwan. ConclusionShuyuwan can inhibit the growth of orthotopically transplanted colon tumor by blocking the polarization of TAMs to M2 type and promoting the polarization of TAMs to M1 type.

Nanoparticles (NPs)-mediated Siglec15 silencing and macrophage repolarization for enhanced cancer immunotherapy

T cell infiltration and proliferation in tumor tissues are the main factors that significantly affect the therapeutic outcomes of cancer immunotherapy. Emerging evidence has shown that interferon-gamma (IFNγ) could enhance CXCL9 secretion from macrophages to recruit T cells, but Siglec15 expressed on TAMs can attenuate T cell proliferation. Therefore, targeted regulation of macrophage function could be a promising strategy to enhance cancer immunotherapy via concurrently promoting the infiltration and proliferation of T cells in tumor tissues. We herein developed reduction-responsive nanoparticles (NPs) made with poly (disulfide amide) (PDSA) and lipid-poly (ethylene glycol) (lipid-PEG) for systemic delivery of Siglec15 siRNA (siSiglec15) and IFNγ for enhanced cancer immunotherapy. After intravenous administration, these cargo-loaded could highly accumulate in the tumor tissues and be efficiently internalized by tumor-associated macrophages (TAMs). With the highly concentrated glutathione (GSH) in the cytoplasm to destroy the nanostructure, the loaded IFNγ and siSiglec15 could be rapidly released, which could respectively repolarize macrophage phenotype to enhance CXCL9 secretion for T cell infiltration and silence Siglec15 expression to promote T cell proliferation, leading to significant inhibition of hepatocellular carcinoma (HCC) growth when combining with the immune checkpoint inhibitor. The strategy developed herein could be used as an effective tool to enhance cancer immunotherapy.

Immunosuppressive tumor microenvironment contributes to tumor progression in diffuse large B-cell lymphoma upon anti-CD19 chimeric antigen receptor T therapy / 医学前沿

Anti-CD19 chimeric antigen receptor (CAR)-T cell therapy has achieved 40%-50% long-term complete response in relapsed or refractory diffuse large B-cell lymphoma (DLBCL) patients. However, the underlying mechanism of alterations in the tumor microenvironments resulting in CAR-T cell therapy failure needs further investigation. A multi-center phase I/II trial of anti-CD19 CD28z CAR-T (FKC876, ChiCTR1800019661) was conducted. Among 22 evaluable DLBCL patients, seven achieved complete remission, 10 experienced partial remissions, while four had stable disease by day 29. Single-cell RNA sequencing results were obtained from core needle biopsy tumor samples collected from long-term complete remission and early-progressed patients, and compared at different stages of treatment. M2-subtype macrophages were significantly involved in both in vivo and in vitro anti-tumor functions of CAR-T cells, leading to CAR-T cell therapy failure and disease progression in DLBCL. Immunosuppressive tumor microenvironments persisted before CAR-T cell therapy, during both cell expansion and disease progression, which could not be altered by infiltrating CAR-T cells. Aberrant metabolism profile of M2-subtype macrophages and those of dysfunctional T cells also contributed to the immunosuppressive tumor microenvironments. Thus, our findings provided a clinical rationale for targeting tumor microenvironments and reprogramming immune cell metabolism as effective therapeutic strategies to prevent lymphoma relapse in future designs of CAR-T cell therapy.

Research Progress on the Role of Tumor-Associated Macrophages in Multiple Myeloma --Review / 中国实验血液学杂志

Bone marrow microenvironment is a highly complex environment surrounding tumor, which plays an important role in the survival, proliferation, drug resistance and migration of multiple myeloma (MM) cells. As an important cellular component in tumor microenvironment, tumor-associated macrophages(TAM) has attracted attention due to its key role in tumor progression and drug resistance. Targeting TAM has shown potential therapeutic value in cancer treatment. In order to clarify the role of macrophages in MM progression, it is necessary to understand the differentiation of TAM and its characteristics of promoting MM. This paper reviews the research progress on how TAM is programmed in MM and the mechanism of TAM promoting tumor development and drug resistance.

Prevention and treatment of lung cancer by regulating tumor-associated macrophages with traditional Chinese medicine / 中国中药杂志

Lung cancer is one of the common malignant tumors in the world, and its incidence and mortality is increasing year by year. Interactions between tumor cells and immune cells in the tumor microenvironment(TME) affect tumor proliferation, infiltration, and metastasis. Tumor-associated macrophages(TAMs) are prominent components of TME, and they have dual regulation effects on malignant progression of lung cancer. The number, activity, and function of M2 macrophages are related to the poor prognosis of lung cancer, and M2 macrophages participate in tumor angiogenesis and immune escape. It has been proved that traditional Chinese medicines(TCMs) and their active ingredients can enhance the antitumor effects, reduce the toxicity of chemotherapy and radiotherapy, and prolong the survival rates of patients with cancer. This paper summarized the role of TAMs in the lung cancer initiation and progression, explored the molecular mechanism of TCM in regulating the recruitment, polarization phenotype, activity, and expression of related factors and proteins of TAMs, and discussed related signal pathways in the prevention and treatment of lung cancer based on the TCM theory of "reinforcing healthy qi and eliminating pathogen". This paper is expected to provide new ideas for the immunotherapy of targeted TAMs.

Progressin Tumor-Associated Macrophages in the Treatment of Pancreatic Cancer / 中国医学科学院学报

Pancreatic cancer is one of the digestive system tumors with a high degree of malignancy,and most of the patients are diagnosed in advanced stages.Because of limited available therapies,the mortality of this disease remains high.Tumor-associated macrophages(TAM),the main immune cells in the tumor microenvironment,are involved in the regulation of the occurrence and development of pancreatic cancer.Specifically,TAM are involved in the proliferation,invasion,immune escape,and chemoresistance of pancreatic cancer cells,demonstrating potential in the targeted therapy of pancreatic cancer.In this paper,we summarize the TAM-based therapies including consuming TAM,reprogramming TAM,dynamic imaging of TAM with nanoprobes,and regulating the phagocytic ability of TAM for pancreatic cancer,aiming to provide a theoretical basis for developing new therapies for pancreatic cancer.

The suppression of cervical cancer ferroptosis by macrophages: The attenuation of ALOX15 in cancer cells by macrophages-derived exosomes

Induction of cancer cell ferroptosis has been proposed as a potential treatment in several cancer types. Tumor-associated macrophages (TAMs) play a key role in promoting tumor malignant progression and therapy resistance. However, the roles and mechanisms of TAMs in regulating tumor ferroptosis is still unexplored and remains enigmatic. This study shows ferroptosis inducers has shown therapeutic outcomes in cervical cancer in vitro and in vivo. TAMs have been found to suppress cervical cancer cells ferroptosis. Mechanistically, macrophage-derived miRNA-660-5p packaged into exosomes are transported into cancer cells. In cancer cells, miRNA-660-5p attenuates ALOX15 expression to inhibit ferroptosis. Moreover, the upregulation of miRNA-660-5p in macrophages depends on autocrine IL4/IL13-activated STAT6 pathway. Importantly, in clinical cervical cancer cases, ALOX15 is negatively associated with macrophages infiltration, which also raises the possibility that macrophages reduce ALOX15 levels in cervical cancer. Moreover, both univariate and multivariate Cox analyses show ALOX15 expression is independent prognostic factor and positively associated with good prognosis in cervical cancer. Altogether, this study reveals the potential utility of targeting TAMs in ferroptosis-based treatment and ALOX15 as prognosis indicators for cervical cancer.

Pancreatic melatonin enhances anti-tumor immunity in pancreatic adenocarcinoma through regulating tumor-associated neutrophils infiltration and NETosis

Tumor microenvironment contributes to poor prognosis of pancreatic adenocarcinoma (PAAD) patients. Proper regulation could improve survival. Melatonin is an endogenous hormone that delivers multiple bioactivities. Here we showed that pancreatic melatonin level is associated with patients' survival. In PAAD mice models, melatonin supplementation suppressed tumor growth, while blockade of melatonin pathway exacerbated tumor progression. This anti-tumor effect was independent of cytotoxicity but associated with tumor-associated neutrophils (TANs), and TANs depletion reversed effects of melatonin. Melatonin induced TANs infiltration and activation, therefore induced cell apoptosis of PAAD cells. Cytokine arrays revealed that melatonin had minimal impact on neutrophils but induced secretion of Cxcl2 from tumor cells. Knockdown of Cxcl2 in tumor cells abolished neutrophil migration and activation. Melatonin-induced neutrophils presented an N1-like anti-tumor phenotype, with increased neutrophil extracellular traps (NETs) causing tumor cell apoptosis through cell-to-cell contact. Proteomics analysis revealed that this reactive oxygen species (ROS)-mediated inhibition was fueled by fatty acid oxidation (FAO) in neutrophils, while FAO inhibitor abolished the anti-tumor effect. Analysis of PAAD patient specimens revealed that CXCL2 expression was associated with neutrophil infiltration. CXCL2, or TANs, combined with NET marker, can better predict patients' prognosis. Collectively, we discovered an anti-tumor mechanism of melatonin through recruiting N1-neutrophils and beneficial NET formation.

Tongxie Yaofang regulates tumor-associated macrophage polarization in colorectal cancer under chronic stress / 中国中药杂志

This study aims to investigate the intervention effect and mechanism of Tongxie Yaofang in regulating tumor-associated macrophage polarization on colorectal cancer under chronic stress. BALB/C mice were randomized into blank, control, model, mifepristone, and low-, medium-, and high-dose Tongxie Yaofang groups. The other groups except the blank and model groups were subjected to chronic restraint stress and subcutaneous implantation of colon cancer cells for the modeling of colon cancer under stress. Du-ring this period, the body mass and tumor size of each group of mice were recorded. The degree of depression in mice was assessed by behavioral changes. Enzyme-linked immunosorbent assay was employed to determine the levels of cortisol(CORT), 5-hydroxytryptamine(5-HT), norepinephrine(NE), M1-associated inflammatory cytokines [interleukin(IL)-1β, IL-12, and tumor necrosis factor(TNF)-α], and M2-associated inflammatory cytokines(IL-4 and IL-10) in the serum. The tumor growth of mice in each group was regularly monitored by in vivo imaging. The histopathological changes of tumors in each group of mice were observed by hematoxylin-eosin staining. The proportions of CD86 and CD206 in the tumor tissue were detected by flow cytometry and immunofluorescence staining. Western blot was employed to determine the protein levels of Janus kinase(JAK)1, JAK2, JAK3, signal transducer and activator of transcription(STAT)3, and STAT6 in the tumor tissue. The results showed that chronic stress increased the immobility time of mice, elevated the serum levels of CORT, IL-4, and IL-10, lowered the levels of 5-HT, NE, IL-1β, IL-12, and TNF-α, and promoted the growth of subcutaneous tumors. The tumor cells in the tumor tissue grew actively, with obvious atypia and up-regulated protein levels of CD206, JAK1, JAK2, JAK3, STAT3, and STAT6, and down-regulated protein level of CD86. The treatment with Tongxie Yaofang shortened the immobility time of mice, lowered the serum levels of CORT, IL-4, and IL-10, elevated the serum levels of 5-HT, NE, IL-1β, IL-12, and TNF-α, and inhibited the growth of subcutaneous tumors in mice. Moreover, the treatment caused different degrees of necrosis in the tumor tissues, down-regulated the protein levels of CD206, JAK1, JAK2, JAK3, STAT3, and STAT6, and up-regulated the protein level of CD86. In summary, Tongxie Yaofang can promote the transformation of M2 macrophages to M1 macrophages and change the tumor microenvironment under chronic stress to inhibit the development of colorectal cancer, which may be related to the JAK/STAT signaling pathway.

Expression Levels of the CA9, WT1, and PRAME genes and genotyping-associated antigens for the diagnosis and prognosis of colorectal cancer

Background: Colorectal cancer (CRC) is the third most prevalent type of cancer worldwide, and is one of the major health problems in Asia, Africa, Europe, and America. The tumor antigens recently are of interesting indicators as diagnostic and prognostic tools, The aim of the present study is to detect the expression levels of carbonic anhydrase IX (CA9), the Wilms tumor gene (WT1), and the preferentially expressed antigen in melanoma (PRAME) in the peripheral blood of CRC patients in comparison with healthy controls. Methods: A prospective case-control study of CRC patients was conducted. We included 25 newly-diagnosed CRC eligible patients and obtained peripheral blood samples of them as well as 10 blood samples from the control group. All samples were then submitted to deoxyribonucleic acid (DNA) extraction and a molecular study through real-time polymerase chain reaction (PCR). Results: The CRC group consisted of 15 (60%) female and 10 (40%) male patients with a mean age of 50.52 ± 9.8 years, while the control group included 4 (40%) female and 6 (60%) male patients with a mean age of 47.7 ± 7.9 years. The CRC group, 24 (96%) of patient samples were CA9-positive with strong statistically significant differences (p < 0.00001; sensitivity: 96%; specificity: 90%). Regarding the WT1 gene, there were 11 (44%) positive samples in the CRC group, with no statistically significant differences (p = 0.055; sensitivity: 44%; specificity: 90%). The PRAME gene was positive in 9 (36%) samples in the CRC group, with no statistically significant differences (p = 0.357; sensitivity: 36%; specificity: 80%. Among CA9 (24 patients; 96%) of patients with CRC expressed positive results, in WT1 11(91.6%) CRC patients expressed gene, and in PRAME gene, 9 patients with CRC (81.8%) expressed positive results. Conclusion: Overexpression of the CA9 gene in CRC of high sensitivity and specificity to be used as a tool to discriminate CRC from benign associate with high accuracy compare to WT1 and PRAME genes. (AU)

The effect of HOXC10 gene on biological behaviors of glioma cells and mechanism in tumor microenvironment / 中华肿瘤杂志

Objective: To study the effects of Homeobox C10 (HOXC10) on biological characteristics such as migration, invasion and proliferation of glioma cancer cells and to explore the role of HOXC10 gene in glioma microenvironment. Methods: The expression level of HOXC10 in high grade glioma (glioblastoma) and low grade glioma and its effect on patient survival were analyzed by using The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) database. Hoxc10-siRNA-1, HOXC10-siRNA-2 and siRNA negative control (NC) were transfected into U251 cells according to the operation instructions of HOXC10-siRNA transfection. 100 ng/ mL recombinant protein chemokine ligand 2 (reCCL2) was added into the transfection group, and was labeled as HOXC10-siRNA-1+ reCCL2 and HOXC10-siRNA-2+ reCCL2 groups. The expressions of HOXC10 mRNA and target protein in each group was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and western blot. The proliferation ability of cells in each group was detected by cell counting kit 8 (CCK8) method. The migration ability of cells was detected by Transwell assay and Nick assay, and cell apoptosis was detected by flow cytometry. The expression of chemokines in each group was detected by multiple factors. Co-incubation assays were performed to determine the role of HOXC10 and chemokine ligand 2 (CCL2) in recruiting and polarizing tumor-associated macrophages (M2-type macrophages). Results: The median expression level of HOXC10 in high grade gliomas was 8.51, higher than 1.00 in low grade gliomas (P<0.001) in TCGA database. The median expression level of HOXC10 in high grade gliomas was 0.83, higher than 0.00 in low grade gliomas (P=0.002) in CGGA database. The 5-year survival rate of patients with high HOXC10 expression in TCGA database was 28.2%, lower than 78.7% of those with low HOXC10 expression (P<0.001), and the 5-year survival rate of patients with high HOXC10 expression in CGGA database was 20.3%, lower than 58.0% of those with low HOXC10 expression (P<0.001). The numbers of cell migration in HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (45±3) and (69±4) respectively, lower than (159±3) in NC group (P<0.05). The cell mobility of HOXC10-siRNA-1 group and HOXC10-siRNA-2 group at 48 hours were (15±2)% and (28±4)% respectively, lower than (80±5)% of NC group (P<0.05). The expressions of vimentin in HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (141 740.00±34 024.56) and (94 655.00±5 687.97), N-cadherin were (76 810.00±14.14) and (94 254.00±701.45), β-catenin were (75 786.50±789.84) and (107 296.50±9 614.53), lower than (233 768.50±34 114.37), (237 154.50±24 715.50) and (192 449.50±24 178.10) of NC group (P<0.05). The A value of HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (0.44±0.05) and (0.32±0.02) at 96 hours, lower than 0.92±0.12 of NC group (P<0.05). The apoptosis rates of HOXC10-siRNA-1 group and HOXC10 siRNA-2 group were (10.23±1.24)% and (13.81±2.16)%, higher than (4.60±0.07)% of NC group (P<0.05). The expression levels of CCL2 in U251 cells in HOXC10-siRNA-1 and HOXC10-siRNA-2 groups were (271.63±44.27) and (371.66±50.21), lower than (933.93±29.84) in NC group (P<0.05). The expression levels of CCL5 (234.81±5.95 and 232.62±5.72), CXCL10 (544.13±48.14 and 500.87±15.65) and CXCL11 (215.75±15.30 and 176.18±16.49) in HOXC10-siRNA-1 and HOXC10-siRNA-2 groups were higher than those in NC group (9.98±0.71, 470.54±18.84 and 13.55±0.73, respectively, P<0.05). The recruited numbers of CD14(+) THP1 in HOXC10-siRNA-1 and HOXC10-siRNA-2 groups were (159.33±1.15) and (170.67±1.15), respectively, lower than (360.00±7.81) in NC group (P<0.05), while addition of reCCL2 promoted the recruitment of CD14(+) THP1 cells (287.00±3.61 and 280.67±2.31 in HOXC10-siRNA-1+ reCCL2 group and HOXC10-siRNA-2+ reCCL2 group, respectively, P<0.05). The expressions level of M2-type macrophage-related gene TGF-β in HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (0.30±0.02) and (0.28±0.02), respectively, lower than (1.06±0.10) in NC group (P<0.05). The expressions level of M1-related gene NOS2 in HOXC10-siRNA-1 and HOXC10-siRNA-2 were (11 413.95±1 911.85) and (5 894.00±945.21), respectively, higher than (13.39±4.32) in NC group (P<0.05). Conclusions: The expression of HOXC10 in glioma is high and positively correlated with the poor prognosis of glioma patients. Knockdown of HOXC10 can inhibit the proliferation, migration and metastasis of human glioma U251 cells. HOXC10 may play an immunosuppressive role in glioma microenvironment by promoting the expression of CCL2 and recruiting and polarizing tumor-associated macrophages (M2 macrophages).