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Volume 31,Issue 10,2024 Table of Contents
2024, 31(10):943-950.
Abstract:
Immunotherapy products, represented by chimeric antigen receptor-modified T lymphocytes (CAR-T cells), have achieved significant progress in hematologic malignancies such as leukemia. The raw materials used in the production of cell therapy products are diverse and complex, with gene modification vectors and other raw materials that endow cells with specific functionalities or influence their qualities usually classified as crucial raw materials. These crucial raw materials often have significant impacts on the quality attributes and in vivo efficacy of cell therapy products, making them important concerns in the evaluation of cell therapy products. This article summarizes several crucial raw materials that are commonly used in cell therapy products (including lentiviral vectors and γ-retroviral vectors, CRISPR/Cas gene editing system, transposon system, immunomagnetic separation beads, and feeder layer cells) and proposes the current evaluation considerations and risk control strategies with the aim of advancing the clinical transformation and application of such products.
Immunotherapy products, represented by chimeric antigen receptor-modified T lymphocytes (CAR-T cells), have achieved significant progress in hematologic malignancies such as leukemia. The raw materials used in the production of cell therapy products are diverse and complex, with gene modification vectors and other raw materials that endow cells with specific functionalities or influence their qualities usually classified as crucial raw materials. These crucial raw materials often have significant impacts on the quality attributes and in vivo efficacy of cell therapy products, making them important concerns in the evaluation of cell therapy products. This article summarizes several crucial raw materials that are commonly used in cell therapy products (including lentiviral vectors and γ-retroviral vectors, CRISPR/Cas gene editing system, transposon system, immunomagnetic separation beads, and feeder layer cells) and proposes the current evaluation considerations and risk control strategies with the aim of advancing the clinical transformation and application of such products.
2024, 31(10):951-956.
Abstract:
Objective: To explore the effects of induced IL-7 expression on proliferation and in vitro antitumor activities of glypican-3 (GPC3)- specific chimeric antigen receptor gene modified-T (CAR-T) cells. Methods: The GPC3 CAR sequence fragment was inserted into the BamHⅠ/EcoRⅠs ite of the GV400 vector using seamless cloning, constructing second-generation CAR lentiviral vectors GPC3-BBZ and GPC3-BBZ-NFAT-IL-7. The lentiviruses were packaged with 293T cells and transfected into healthy human T cells to prepare CAR-T cells， which were divided into non-transduced T cell (NT) group, GPC3-BBZ CAR-T cell group and GPC3-BBZ-NFAT-IL-7 CAR-T cell group. The expression of CAR in CAR-T cells of each group was determined by flow cytometry. IL-7 mRNA expression level in CAR-T cells activated by GPC3 protein was determined by qPCR. The proliferation ability of CAR-T cells under GPC3 antigen stimulation was evaluated by cell counting. The secretion levels of IL-7, IFN-γ, and TNF-α by CAR-T cells after stimulation with tumor cells was determined using ELISA. The cytotoxicity of CAR-T cells against human hepatocellular carcinoma Huh-7 cells was tested by real-time cell analyzer (RTCA). Results: Lentiviral vectors GPC3-BBZ and GPC3-BBZ-NFAT-IL-7 were successfully constructed, and GPC3-specific CAR-T cells were prepared. After activation with GPC3 antigen, GPC3-BBZ-NFAT-IL-7 CAR-T cells effectively expressed IL-7 mRNA (P < 0.01) and exhibited stronger proliferation capacity (P < 0.05). Compared with GPC3-BBZ CAR-T cells, GPC3-BBZ-NFAT-IL-7 CAR-T cells co-cultured with GPC3-positive Huh-7 cells secreted higher levels of IL-7, IFN-γ, and TNF-α (P < 0.01 or P < 0.001). The RTCA results showed that the cytotoxicity of GPC3-BBZ-NFAT-IL-7 CAR-T cells against GPC3-positive Huh-7 cells was significantly higher than that of GPC3-BBZ CAR-T cells (P < 0.05). Conclusion: GPC3-specific CAR-T cells with inducible IL-7 expression were successfully prepared, which exhibited immune activity and tumor cell killing capacity in vitro.
Objective: To explore the effects of induced IL-7 expression on proliferation and in vitro antitumor activities of glypican-3 (GPC3)- specific chimeric antigen receptor gene modified-T (CAR-T) cells. Methods: The GPC3 CAR sequence fragment was inserted into the BamHⅠ/EcoRⅠs ite of the GV400 vector using seamless cloning, constructing second-generation CAR lentiviral vectors GPC3-BBZ and GPC3-BBZ-NFAT-IL-7. The lentiviruses were packaged with 293T cells and transfected into healthy human T cells to prepare CAR-T cells， which were divided into non-transduced T cell (NT) group, GPC3-BBZ CAR-T cell group and GPC3-BBZ-NFAT-IL-7 CAR-T cell group. The expression of CAR in CAR-T cells of each group was determined by flow cytometry. IL-7 mRNA expression level in CAR-T cells activated by GPC3 protein was determined by qPCR. The proliferation ability of CAR-T cells under GPC3 antigen stimulation was evaluated by cell counting. The secretion levels of IL-7, IFN-γ, and TNF-α by CAR-T cells after stimulation with tumor cells was determined using ELISA. The cytotoxicity of CAR-T cells against human hepatocellular carcinoma Huh-7 cells was tested by real-time cell analyzer (RTCA). Results: Lentiviral vectors GPC3-BBZ and GPC3-BBZ-NFAT-IL-7 were successfully constructed, and GPC3-specific CAR-T cells were prepared. After activation with GPC3 antigen, GPC3-BBZ-NFAT-IL-7 CAR-T cells effectively expressed IL-7 mRNA (P < 0.01) and exhibited stronger proliferation capacity (P < 0.05). Compared with GPC3-BBZ CAR-T cells, GPC3-BBZ-NFAT-IL-7 CAR-T cells co-cultured with GPC3-positive Huh-7 cells secreted higher levels of IL-7, IFN-γ, and TNF-α (P < 0.01 or P < 0.001). The RTCA results showed that the cytotoxicity of GPC3-BBZ-NFAT-IL-7 CAR-T cells against GPC3-positive Huh-7 cells was significantly higher than that of GPC3-BBZ CAR-T cells (P < 0.05). Conclusion: GPC3-specific CAR-T cells with inducible IL-7 expression were successfully prepared, which exhibited immune activity and tumor cell killing capacity in vitro.
2024, 31(10):957-962.
Abstract:
Objective: To investigate the anti-tumor effect of dendritic cells (DCs) vaccines loaded with shikonin (SK) and tumor cell lysate (TCL). Methods: DC vaccines of normal mouse origin loaded with SK and TCL were prepared in vitro. Fluorescence intensity of CD80 and CD86 on the surface of DCs was detected by flow cytometry. The expression of T-bet and RORγt in normal mouse splenic T cells co-cultured DCs that stimulated by SK + TCL was determined by flow cytometry, and the contents of IFN-γ, IL-12P70, and TNF-α in the co-culture supernatant were detected using ELISA. A Lewis lung cancer 3LL cell-bearing mouse model was established, and the mice were randomized into PBS + TCL group (PBS [1 mL] + TCL [5 × 105 cell/100 μL]), SK-L + TCL group (low SK concentration [1.25 mg/kg] + TCL), SK-M + TCL group (medium SK concentration [2.5 mg/kg] + TCL), and SK-H + TCL group (high SK concentration [5 mg/kg] + TCL), and paclitaxel (PTX) + TCL vaccine group (PTX [2 mg/kg] + TCL). Ten days after the end of vaccination, the solid tumor volume and survival rate of the mice were observed, and the killing capacity of splenic cytotoxic T lymphocytes (CTLs) was assessed by LDH assay. Results: DC vaccines loaded with SK + TCL showed high levels of CD80 and CD86 expression (both P < 0.01). The levels of IL-12P70, IFN- γ, and TNF- α in the DC-T cell co-culture supernatants were significantly increased (all P < 0.01), and the expression of T-bet and RORγt (both P < 0.01) in T cells were significantly elevated. A successful Lewis lung carcinoma mouse model was established, and the DC vaccines loaded with SK-H + TCL significantly delayed the growth of transplanted tumors and increased the mouse survival rate, while strongly inducing the cytotoxic activity of splenic CTLs (all P < 0.01). Conclusion: The SK + TCL-loaded DC vaccine can activate DCs to a mature state, up-regulate the expression of T-bet and RORγt in T cells, and initiate Th1 effector cells. SK shows promising antitumor effects in the treatment of Lewis lung carcinoma in mice.
Objective: To investigate the anti-tumor effect of dendritic cells (DCs) vaccines loaded with shikonin (SK) and tumor cell lysate (TCL). Methods: DC vaccines of normal mouse origin loaded with SK and TCL were prepared in vitro. Fluorescence intensity of CD80 and CD86 on the surface of DCs was detected by flow cytometry. The expression of T-bet and RORγt in normal mouse splenic T cells co-cultured DCs that stimulated by SK + TCL was determined by flow cytometry, and the contents of IFN-γ, IL-12P70, and TNF-α in the co-culture supernatant were detected using ELISA. A Lewis lung cancer 3LL cell-bearing mouse model was established, and the mice were randomized into PBS + TCL group (PBS [1 mL] + TCL [5 × 105 cell/100 μL]), SK-L + TCL group (low SK concentration [1.25 mg/kg] + TCL), SK-M + TCL group (medium SK concentration [2.5 mg/kg] + TCL), and SK-H + TCL group (high SK concentration [5 mg/kg] + TCL), and paclitaxel (PTX) + TCL vaccine group (PTX [2 mg/kg] + TCL). Ten days after the end of vaccination, the solid tumor volume and survival rate of the mice were observed, and the killing capacity of splenic cytotoxic T lymphocytes (CTLs) was assessed by LDH assay. Results: DC vaccines loaded with SK + TCL showed high levels of CD80 and CD86 expression (both P < 0.01). The levels of IL-12P70, IFN- γ, and TNF- α in the DC-T cell co-culture supernatants were significantly increased (all P < 0.01), and the expression of T-bet and RORγt (both P < 0.01) in T cells were significantly elevated. A successful Lewis lung carcinoma mouse model was established, and the DC vaccines loaded with SK-H + TCL significantly delayed the growth of transplanted tumors and increased the mouse survival rate, while strongly inducing the cytotoxic activity of splenic CTLs (all P < 0.01). Conclusion: The SK + TCL-loaded DC vaccine can activate DCs to a mature state, up-regulate the expression of T-bet and RORγt in T cells, and initiate Th1 effector cells. SK shows promising antitumor effects in the treatment of Lewis lung carcinoma in mice.
2024, 31(10):963-969.
Abstract:
Objective: To develop a neoantigen peptide vaccine for personalized treatment of colorectal cancer (CRC), and to explore the feasibility and effectiveness of neoantigen peptide and its induced neoantigen reactive T cells (NRT) therapy for CRC. Methods: DNA and RNA were extracted from mouse CRC cell line CT26, followed by whole-exome and transcriptome sequencing to analyze tumor gene mutations and expression. Peptides with high immunogenicity were screened and synthesized through a machine learning based neoantigen prediction system. Mice were subcutaneously immunized with synthesized peptides, and the interferon (IFN)-γ level of splenocytes from immunized mice was determined using flow cytometry to screen peptides with strong immunogenicity. Afterwards, bone marrow-derived dendritic cells (BMDCs) loaded with immunogenic peptides were used to immunize mice bearing CRC model. The IFN-γ secretion ability by effector cells was determined by ELISPOT assay, and the cytotoxicity of γ secretion ability by effector cells was determined by ELISPOT assay, and the cytotoxicity of splenocytes from immunized mice was examined by time-resolved fluorescence immunoassay. In addition, the tumor growth and survival period of tumor-bearing mice were observed. Results: The neoantigen Glud1-V546I induced stronger IFN-γ secretion by NRT cells (P < 0.000 1). Compared with the wild peptide (Glud1-WT), Glud1-V546I induced higher IFN-γ secretion by NRT cells (P < 0.000 1) and stronger cytotoxicity (P < 0.000 1) in tumor-bearing mice. Meanwhile, Glud1-V546I significantly inhibited tumor growth (P < 0.001) and prolonged the survival of tumor-bearing mice (P < 0.01). Conclusion: The neoantigen peptide Glud1-V546I from mouse CT26 cells demonstrates effective anti-tumor responses in tumor-bearing mice, suggesting the potential for developing neoantigenbased personalized immunotherapies in CRC.
Objective: To develop a neoantigen peptide vaccine for personalized treatment of colorectal cancer (CRC), and to explore the feasibility and effectiveness of neoantigen peptide and its induced neoantigen reactive T cells (NRT) therapy for CRC. Methods: DNA and RNA were extracted from mouse CRC cell line CT26, followed by whole-exome and transcriptome sequencing to analyze tumor gene mutations and expression. Peptides with high immunogenicity were screened and synthesized through a machine learning based neoantigen prediction system. Mice were subcutaneously immunized with synthesized peptides, and the interferon (IFN)-γ level of splenocytes from immunized mice was determined using flow cytometry to screen peptides with strong immunogenicity. Afterwards, bone marrow-derived dendritic cells (BMDCs) loaded with immunogenic peptides were used to immunize mice bearing CRC model. The IFN-γ secretion ability by effector cells was determined by ELISPOT assay, and the cytotoxicity of γ secretion ability by effector cells was determined by ELISPOT assay, and the cytotoxicity of splenocytes from immunized mice was examined by time-resolved fluorescence immunoassay. In addition, the tumor growth and survival period of tumor-bearing mice were observed. Results: The neoantigen Glud1-V546I induced stronger IFN-γ secretion by NRT cells (P < 0.000 1). Compared with the wild peptide (Glud1-WT), Glud1-V546I induced higher IFN-γ secretion by NRT cells (P < 0.000 1) and stronger cytotoxicity (P < 0.000 1) in tumor-bearing mice. Meanwhile, Glud1-V546I significantly inhibited tumor growth (P < 0.001) and prolonged the survival of tumor-bearing mice (P < 0.01). Conclusion: The neoantigen peptide Glud1-V546I from mouse CT26 cells demonstrates effective anti-tumor responses in tumor-bearing mice, suggesting the potential for developing neoantigenbased personalized immunotherapies in CRC.
WU Wenqing
,
JIANG Longwei
,
JIA Shaochang
,
HU Jianhua
,
KE Chuanqing
,
ZHANG Haoli
,
FU Huiying
,
XIONG Xie
2024, 31(10):970-975.
Abstract:
Objective: To explore the therapeutic effects and prognostic value of dendritic cell and cytokine-induced killer cell (DC-CIK) infusion in different routes in the treatment of intermediate and advanced primary liver cancer (PLC). Methods: A retrospective study was conducted on the clinical data of 69 patients with intermediate and advanced PLC treated with DC-CIK at the Oncology Department of the General Hospital of the Eastern Theater of Operations between October 2018 and September 2021. The patients were divided into a hepatic arterial infusion (HAI) group (n = 29) and an intravenous infusion (IV) group (n = 40) according to the different infusion modes used for DC-CIK treatment. The changes in peripheral blood T lymphocyte subsets (CD3+ , CD4+ , CD8+ T cells and CD4+ /CD8+ T cell ratio), cytokines (IL-2, IL-6, IFN-γ, and TNF-α), and alpha-fetoprotein (AFP) before and after treatment, treatment efficacy, overall survival (OS), and the incidence of adverse reactions were compared between the two groups. Results: After DC-CIK treatment, the objective remission rate (ORR) was 0%, and the disease control rate (DCR) was 75.8% in the HAI group; the ORR was 0%, and the DCR was 72.5% in the IV group (all P > 0.05). There were no significant changes in T-lymphocyte subpopulation between the two groups before and after treatment (all P > 0.05). The mean levels of peripheral blood IL-2, IL-6, IFN- γ and TNF- α after treatment were significantly higher than those before treatment in both groups (all P < 0.01), but with no significant difference between the two groups (all P > 0.05). The mean OS in HAI group was 48.17 months, and the OS in IV group was 39.65 months, with no significant difference between the two groups (P > 0.05). No severe adverse reactions occurred during the treatment. Conclusion: Autologous DC-CIK treatment via HAI is safe and effective for PLC. Compared to IV treatment, it tends to further improve the clinical benefit, which is worthy of clinical reference.
Objective: To explore the therapeutic effects and prognostic value of dendritic cell and cytokine-induced killer cell (DC-CIK) infusion in different routes in the treatment of intermediate and advanced primary liver cancer (PLC). Methods: A retrospective study was conducted on the clinical data of 69 patients with intermediate and advanced PLC treated with DC-CIK at the Oncology Department of the General Hospital of the Eastern Theater of Operations between October 2018 and September 2021. The patients were divided into a hepatic arterial infusion (HAI) group (n = 29) and an intravenous infusion (IV) group (n = 40) according to the different infusion modes used for DC-CIK treatment. The changes in peripheral blood T lymphocyte subsets (CD3+ , CD4+ , CD8+ T cells and CD4+ /CD8+ T cell ratio), cytokines (IL-2, IL-6, IFN-γ, and TNF-α), and alpha-fetoprotein (AFP) before and after treatment, treatment efficacy, overall survival (OS), and the incidence of adverse reactions were compared between the two groups. Results: After DC-CIK treatment, the objective remission rate (ORR) was 0%, and the disease control rate (DCR) was 75.8% in the HAI group; the ORR was 0%, and the DCR was 72.5% in the IV group (all P > 0.05). There were no significant changes in T-lymphocyte subpopulation between the two groups before and after treatment (all P > 0.05). The mean levels of peripheral blood IL-2, IL-6, IFN- γ and TNF- α after treatment were significantly higher than those before treatment in both groups (all P < 0.01), but with no significant difference between the two groups (all P > 0.05). The mean OS in HAI group was 48.17 months, and the OS in IV group was 39.65 months, with no significant difference between the two groups (P > 0.05). No severe adverse reactions occurred during the treatment. Conclusion: Autologous DC-CIK treatment via HAI is safe and effective for PLC. Compared to IV treatment, it tends to further improve the clinical benefit, which is worthy of clinical reference.
2024, 31(10):976-983.
Abstract:
Objective: To investigate the effects of four-and-a-half LIM domains 2 (FHL2) on glioma cell proliferation and its molecular mechanisms. Methods: The relationship between FHL2 mRNA expression levels in gliomas and patient prognosis was analyzed using TCGA and CGGA databases. The protein expression levels of FHL2 in collected human glioma tissue samples and human glioma cell lines (U87, T98G, U251, SNB19, GSC23, A172, LN229, G267), and astrocyte NHA were analyzed using Western blot (WB). Lentiviral vecors were used to construct U87 cells with stable FHL2 knockdown and SNB19 cells overexpressing FHL2, namely U87-shGFP, U87-shFHL2-1#, U87-shFHL2-4#, and SNB19-3flag, SNB19-3flag-FHL2 groups. The effects of FHL2 knockdown and overexpression on cell proliferation were assessed using CCK-8 assays and colony formation assay. Coimmunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC/MS) were employed to identify proteins interacting with FHL2 in glioma cells, and Co-IP and immunofluorescence were used to verify their binding and co-localization. Changes in intracellular lactate production and lactate dehydrogenase (LDH) activity following FHL2 knockdown and overexpression were measured using a microplate reader. WB was used to analyze the protein expression levels of FHL2, LDHA, and p-LDHA in normal brain tissues and glioma tissues, as well as their relationships. The small molecule inhibitor of LDHA, AT-101, was used to treat SNB19 cells overexpressing FHL2. The role of FHL2 in glioma lactate metabolism and the potential therapeutic effect of AT-101 in glioma were validated using CCK-8 assays and colorimetric assays with a microplate reader. Results: Co-IP and LC/MS analyses revealed an interaction between FHL2 and LDHA in glioma cells. Overexpression of FHL2 increased LDHA activity and lactate production (all P < 0.001), thereby promoting glioma cell proliferation (P < 0.001 or P < 0.001). Conversely, knockdown of FHL2 reduced LDHA activity and lactate production (P < 0.001, P < 0.05) and inhibited cell growth (P < 0.01). AT101 inhibited LDHA activity and significantly suppressed FHL2-induced glioma cell proliferation while restoring phosphorylated LDHA (Y10) levels (P < 0.01, P < 0.001). Conclusion: FHL2 interacts with LDHA protein, and FHL2 promotes LDHA activity and lactic acid production by activating the expression of p-LDHA (Y10), thus facilitating the proliferation of glioma cells. Targeting this interaction may become a potential strategy for treating gliomas.
Objective: To investigate the effects of four-and-a-half LIM domains 2 (FHL2) on glioma cell proliferation and its molecular mechanisms. Methods: The relationship between FHL2 mRNA expression levels in gliomas and patient prognosis was analyzed using TCGA and CGGA databases. The protein expression levels of FHL2 in collected human glioma tissue samples and human glioma cell lines (U87, T98G, U251, SNB19, GSC23, A172, LN229, G267), and astrocyte NHA were analyzed using Western blot (WB). Lentiviral vecors were used to construct U87 cells with stable FHL2 knockdown and SNB19 cells overexpressing FHL2, namely U87-shGFP, U87-shFHL2-1#, U87-shFHL2-4#, and SNB19-3flag, SNB19-3flag-FHL2 groups. The effects of FHL2 knockdown and overexpression on cell proliferation were assessed using CCK-8 assays and colony formation assay. Coimmunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC/MS) were employed to identify proteins interacting with FHL2 in glioma cells, and Co-IP and immunofluorescence were used to verify their binding and co-localization. Changes in intracellular lactate production and lactate dehydrogenase (LDH) activity following FHL2 knockdown and overexpression were measured using a microplate reader. WB was used to analyze the protein expression levels of FHL2, LDHA, and p-LDHA in normal brain tissues and glioma tissues, as well as their relationships. The small molecule inhibitor of LDHA, AT-101, was used to treat SNB19 cells overexpressing FHL2. The role of FHL2 in glioma lactate metabolism and the potential therapeutic effect of AT-101 in glioma were validated using CCK-8 assays and colorimetric assays with a microplate reader. Results: Co-IP and LC/MS analyses revealed an interaction between FHL2 and LDHA in glioma cells. Overexpression of FHL2 increased LDHA activity and lactate production (all P < 0.001), thereby promoting glioma cell proliferation (P < 0.001 or P < 0.001). Conversely, knockdown of FHL2 reduced LDHA activity and lactate production (P < 0.001, P < 0.05) and inhibited cell growth (P < 0.01). AT101 inhibited LDHA activity and significantly suppressed FHL2-induced glioma cell proliferation while restoring phosphorylated LDHA (Y10) levels (P < 0.01, P < 0.001). Conclusion: FHL2 interacts with LDHA protein, and FHL2 promotes LDHA activity and lactic acid production by activating the expression of p-LDHA (Y10), thus facilitating the proliferation of glioma cells. Targeting this interaction may become a potential strategy for treating gliomas.
2024, 31(10):984-990.
Abstract:
Objectives: To investigate the expression of ubiquitin-specific protease 21 (USP21) in cholangiocarcinoma (CCA) and its effect on the proliferation and migration of CCA cells, as well as the underlying mechanisms. Methods: The expression of USP21 in CCA tissues and cells was detected using bioinformatics approaches, immunohistochemistry, and Western blotting (WB). The effects of USP21 knockdown on the proliferation and migration of CCA cell lines (QBC939 and RBE) were assessed by in vitro colony formation, EdU, and Transwell assays. The oncogenic mechanism of USP21 was explored using RNA sequencing, mass spectrometry, co-immunoprecipitation (CO-IP), and WB. Results: Analysis of databases such as TCGA revealed that USP21 mRNA expression was significantly elevated in CCA tissues (P < 0.05) and USP21 protein expression was highly expressed in both CCA tissues and cells (P < 0.05, P < 0.01, or P < 0.001). Knockdown of USP21 led to a significant decrease in the proliferative and migratory abilities of QBC939 and RBE cells (P < 0.01 or P < 0.001). RNA sequencing results indicated that USP21 knockdown inhibited the proliferative and migratory abilities of CCA cells by suppressing the PI3K/AKT signaling pathway (P < 0.05). Mass spectrometry identified a binding between USP21 and insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1). Co-IP and WB results demonstrated that USP21 interacts with IGF2BP1 and regulates its protein expression through the ubiquitination pathway (P < 0.001 or P < 0.000 1). Conclusion: USP21 is highly expressed in both CCA tissues and cells and enhances the proliferative and migratory abilities of CCA through the IGF2BP1/PI3K/AKT signaling pathway.
Objectives: To investigate the expression of ubiquitin-specific protease 21 (USP21) in cholangiocarcinoma (CCA) and its effect on the proliferation and migration of CCA cells, as well as the underlying mechanisms. Methods: The expression of USP21 in CCA tissues and cells was detected using bioinformatics approaches, immunohistochemistry, and Western blotting (WB). The effects of USP21 knockdown on the proliferation and migration of CCA cell lines (QBC939 and RBE) were assessed by in vitro colony formation, EdU, and Transwell assays. The oncogenic mechanism of USP21 was explored using RNA sequencing, mass spectrometry, co-immunoprecipitation (CO-IP), and WB. Results: Analysis of databases such as TCGA revealed that USP21 mRNA expression was significantly elevated in CCA tissues (P < 0.05) and USP21 protein expression was highly expressed in both CCA tissues and cells (P < 0.05, P < 0.01, or P < 0.001). Knockdown of USP21 led to a significant decrease in the proliferative and migratory abilities of QBC939 and RBE cells (P < 0.01 or P < 0.001). RNA sequencing results indicated that USP21 knockdown inhibited the proliferative and migratory abilities of CCA cells by suppressing the PI3K/AKT signaling pathway (P < 0.05). Mass spectrometry identified a binding between USP21 and insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1). Co-IP and WB results demonstrated that USP21 interacts with IGF2BP1 and regulates its protein expression through the ubiquitination pathway (P < 0.001 or P < 0.000 1). Conclusion: USP21 is highly expressed in both CCA tissues and cells and enhances the proliferative and migratory abilities of CCA through the IGF2BP1/PI3K/AKT signaling pathway.
2024, 31(10):991-996.
Abstract:
Objective: To evaluate the effect and mechanism of CD36 interference on platelet activation mediated by leukemia cell culture supernatant. Methods: Platelets were cultured with supernatant from L1210 murine leukemia cells for 4, 6, 12, and 24 hours, with platelets cultured in regular medium as the control. To determine the optimal time for supernatant-mediated platelet activation, flow cytometry was used to detect the expression of the platelet activation marker P-selectin (CD62P), and Western blot (WB) was used to detect CD36 expression. A CD36 interference vector was constructed and transfected into activated platelets. The cells were divided into the following groups: control group, model group, CD36 interference empty vector group (si-CD36 NC), CD36 interference group (si-CD36), inhibitor group (iCRT3), and inhibitor + CD36 interference group (iCRT3 + si-CD36). CCK-8 assay was used to detect platelet viability, flow cytometry was used to detect CD62P expression in platelets, and WB was used to detect the expression of PECAM-1, CD36, and β -catenin proteins in platelets. Results: The optimal time for platelet activation mediated by L1210 murine leukemia cell supernatant was 12 hours. Compared with the control group, the platelet viability, CD62P expression, and protein expression of PECAM-1, CD36, and β-catenin were significantly increased in the model group (all P < 0.01). Compared with the model group, platelet viability, CD62P expression, and protein expression of PECAM-1, CD36, and β-catenin were significantly decreased in the si-CD36 and iCRT3 groups (all P < 0.01). The changes were more pronounced in the iCRT3 + si-CD36 group compared to the iCRT3 group. Conclusion: CD36 interference inhibits β-catenin protein expression and, in combination with a Wnt/β-catenin pathway inhibitor, further inhibits murine leukemia cell supernatant-mediated platelet activation.
Objective: To evaluate the effect and mechanism of CD36 interference on platelet activation mediated by leukemia cell culture supernatant. Methods: Platelets were cultured with supernatant from L1210 murine leukemia cells for 4, 6, 12, and 24 hours, with platelets cultured in regular medium as the control. To determine the optimal time for supernatant-mediated platelet activation, flow cytometry was used to detect the expression of the platelet activation marker P-selectin (CD62P), and Western blot (WB) was used to detect CD36 expression. A CD36 interference vector was constructed and transfected into activated platelets. The cells were divided into the following groups: control group, model group, CD36 interference empty vector group (si-CD36 NC), CD36 interference group (si-CD36), inhibitor group (iCRT3), and inhibitor + CD36 interference group (iCRT3 + si-CD36). CCK-8 assay was used to detect platelet viability, flow cytometry was used to detect CD62P expression in platelets, and WB was used to detect the expression of PECAM-1, CD36, and β -catenin proteins in platelets. Results: The optimal time for platelet activation mediated by L1210 murine leukemia cell supernatant was 12 hours. Compared with the control group, the platelet viability, CD62P expression, and protein expression of PECAM-1, CD36, and β-catenin were significantly increased in the model group (all P < 0.01). Compared with the model group, platelet viability, CD62P expression, and protein expression of PECAM-1, CD36, and β-catenin were significantly decreased in the si-CD36 and iCRT3 groups (all P < 0.01). The changes were more pronounced in the iCRT3 + si-CD36 group compared to the iCRT3 group. Conclusion: CD36 interference inhibits β-catenin protein expression and, in combination with a Wnt/β-catenin pathway inhibitor, further inhibits murine leukemia cell supernatant-mediated platelet activation.
ZHOU Yusen
,
JIA Peng
,
LIAN Yixiang
,
FANG Yuting
,
CHEN Ting
,
FAN Tianyu
,
PENG Gaoyang
,
HU Lijun
,
YIN Jiangliu
2024, 31(10):997-1007.
Abstract:
Objective: To investigate the expression and biological processes of chitinase-3-like protein 2 (CHI3L2) in brain gliomas and its impact on clinical prognosis of patients based on bioinformatics methods. Methods: With Chinese Glioma Genome Atlas (CGGA) serving as the training set (n = 325) and The Cancer Genome Atlas (TCGA) as the validation set (n = 702), the relationship between CHI3L2 and clinicopathologic features of brain glioma patients, as well as its prognostic value and biological processes were analyzed and cross-validated. Kaplan-Meier method was used for survival analysis. Cox regression model was employed to analyze the association between CHI3L2 expression and relevant clinicopathological features as well as prognosis of brain glioma patients. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic value of CHI3L2 for brain glioma. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Sene Set Variation Analysis (GSEA) were conducted to assess the potential biological processes associated with CHI3L2 in brain glioma. A Nomogram incorporating CHI3L2 and other relevant factors was constructed, and calibration curve and C-Index value were used to evaluate the Nomogram’s accuracy in prognosis prediction. The protein and mRNA expression levels of CHI3L2 in normal astrocyte HA1800, as well as glioma U215 and U87 cells, were assessed using Western blotting and qPCR, respectively. Immunohistochemistry was performed on 7 normal brain samples, 5 lowgrade glioma samples (LGG, WHO Ⅰ - Ⅱ), and 6 glioblastoma sample (GBM, WHO Ⅳ) that collected from the Department of Pathology at Changsha Central Hospital, with an aim to verify the expression of CHI3L2 in normal brain tissues and glioma tissues of different grades. Results: CHI3L2 was highly expressed in patients with GBM (P < 0.000 1), non-1p/19q coding glioma (P < 0.000 1), IDH-wild type glioma (P < 0.000 1), and non-MGMT methylation glioma (P < 0.01), showing certain predictive value for GBM. Additionally, CHI3L2 was identified as an independent prognostic factor for overall survival (OS) in glioma patients (P < 0.001). The constructed nomogram exhibited good predictive accuracy for patient prognosis. Furthermore, CHI3L2 was significantly associated with immune cell infiltration level, tumor immune microenvironment, and immune cells in LGG and GBM. Elevated CHI3L2 protein (P < 0.05) and mRNA (P < 0.000 1) levels in gliomas were correlated with higher malignant grade, as further confirmed by immunohistochemistry results. Conclusion: CHI3L2 expression is intricately associated with the malignancy, clinicopathological characteristics, and prognosis of brain glioma. It actively participates in the tumor microenvironment and immune infiltration within glioma, thereby representing a promising therapeutic target for glioma treatment.
Objective: To investigate the expression and biological processes of chitinase-3-like protein 2 (CHI3L2) in brain gliomas and its impact on clinical prognosis of patients based on bioinformatics methods. Methods: With Chinese Glioma Genome Atlas (CGGA) serving as the training set (n = 325) and The Cancer Genome Atlas (TCGA) as the validation set (n = 702), the relationship between CHI3L2 and clinicopathologic features of brain glioma patients, as well as its prognostic value and biological processes were analyzed and cross-validated. Kaplan-Meier method was used for survival analysis. Cox regression model was employed to analyze the association between CHI3L2 expression and relevant clinicopathological features as well as prognosis of brain glioma patients. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic value of CHI3L2 for brain glioma. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Sene Set Variation Analysis (GSEA) were conducted to assess the potential biological processes associated with CHI3L2 in brain glioma. A Nomogram incorporating CHI3L2 and other relevant factors was constructed, and calibration curve and C-Index value were used to evaluate the Nomogram’s accuracy in prognosis prediction. The protein and mRNA expression levels of CHI3L2 in normal astrocyte HA1800, as well as glioma U215 and U87 cells, were assessed using Western blotting and qPCR, respectively. Immunohistochemistry was performed on 7 normal brain samples, 5 lowgrade glioma samples (LGG, WHO Ⅰ - Ⅱ), and 6 glioblastoma sample (GBM, WHO Ⅳ) that collected from the Department of Pathology at Changsha Central Hospital, with an aim to verify the expression of CHI3L2 in normal brain tissues and glioma tissues of different grades. Results: CHI3L2 was highly expressed in patients with GBM (P < 0.000 1), non-1p/19q coding glioma (P < 0.000 1), IDH-wild type glioma (P < 0.000 1), and non-MGMT methylation glioma (P < 0.01), showing certain predictive value for GBM. Additionally, CHI3L2 was identified as an independent prognostic factor for overall survival (OS) in glioma patients (P < 0.001). The constructed nomogram exhibited good predictive accuracy for patient prognosis. Furthermore, CHI3L2 was significantly associated with immune cell infiltration level, tumor immune microenvironment, and immune cells in LGG and GBM. Elevated CHI3L2 protein (P < 0.05) and mRNA (P < 0.000 1) levels in gliomas were correlated with higher malignant grade, as further confirmed by immunohistochemistry results. Conclusion: CHI3L2 expression is intricately associated with the malignancy, clinicopathological characteristics, and prognosis of brain glioma. It actively participates in the tumor microenvironment and immune infiltration within glioma, thereby representing a promising therapeutic target for glioma treatment.
2024, 31(10):1008-1016.
Abstract:
Objective：To investigate the expression of immunogenic cell death-related genes, autophagy associated gene 5 (ATG5) and protein disulfide isomerase A3 (PDIA3), in cervical cancer tissues, and to explore their correlation with clinicopathological features and prognosis of cervical cancer patients, as well as the related signaling pathways and drug sensitivity. Methods: A total of 60 cervical cancer tissue specimens from the patients treated at Affiliated Hospital of Qingdao University during January 2016 and December 2023 were collected as the experimental group. Additionally, 26 normal cervical specimens collected from patients with hysteromyoma and adenomyosis served as the control group. The data of two groups of patients were also collected. The differential protein expression of ATG5 and PDIA3 in cervical cancer tissues and normal cervical tissues was detected by immunohistochemistry, and their relationship with patients' clinicopathological parameters was also analyzed. The influence of ATG5 and PDIA3 expression in cervical cancer tissues on patient prognosis was analyzed using interactive analysis platform (GEPIA). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were utilized to explore their functional roles and signaling pathways associated with ATG5 and PDIA3 genes. The sensitivity of cervical cancer patients to chemotherapeutic drugs was analyzed using the pRRophetic package based on the high and low expression of ATG5 and PDIA3 in cancer tissues. Results: The positive expression rates of ATG5 and PDIA3 in cervical cancer tissues were significantly higher than those in normal cervical tissues (83.3% vs 11.5%, χ 2 = 39.538, P = 0.001; 75.0% vs 46.2%, χ 2 = 6.753, P = 0.009). The expression levels of ATG5 differed notably across patients with different tumor diameter, FIGO stage and lymph node metastasis (all P < 0.01), while the expression levels of PDIA3 differed greatly across patients with different tumor diameter, differentiation degree, FIGO stage and lymph node metastasis (P < 0.05 or P < 0.01). The expression levels of ATG5 and PDIA3 were positively correlated (r = 0.679, P < 0.001). Prognostic analysis via GEPIA showed that high ATG5 and PDIA3 expression correlated with poor prognosis in cervical cancer patients (all P < 0.05). Key enriched functions and signaling pathways for ATG5 and PDIA3 included cell proliferation and differentiation, antigen processing and presentation, P53 binding, Wnt signaling pathway, MAPK signaling pathway and mTOR signal pathway. Conclusion: ATG5 and PDIA3 are highly expressed in cervical cancer tissues, with their elevated expression is associated with poor prognosis. Both ATG5 and PDIA3 are involved in cell proliferation and differentiation, antigen processing and presentation, and various signaling pathways, making them potential targets for cervical cancer treatment.
Objective：To investigate the expression of immunogenic cell death-related genes, autophagy associated gene 5 (ATG5) and protein disulfide isomerase A3 (PDIA3), in cervical cancer tissues, and to explore their correlation with clinicopathological features and prognosis of cervical cancer patients, as well as the related signaling pathways and drug sensitivity. Methods: A total of 60 cervical cancer tissue specimens from the patients treated at Affiliated Hospital of Qingdao University during January 2016 and December 2023 were collected as the experimental group. Additionally, 26 normal cervical specimens collected from patients with hysteromyoma and adenomyosis served as the control group. The data of two groups of patients were also collected. The differential protein expression of ATG5 and PDIA3 in cervical cancer tissues and normal cervical tissues was detected by immunohistochemistry, and their relationship with patients' clinicopathological parameters was also analyzed. The influence of ATG5 and PDIA3 expression in cervical cancer tissues on patient prognosis was analyzed using interactive analysis platform (GEPIA). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were utilized to explore their functional roles and signaling pathways associated with ATG5 and PDIA3 genes. The sensitivity of cervical cancer patients to chemotherapeutic drugs was analyzed using the pRRophetic package based on the high and low expression of ATG5 and PDIA3 in cancer tissues. Results: The positive expression rates of ATG5 and PDIA3 in cervical cancer tissues were significantly higher than those in normal cervical tissues (83.3% vs 11.5%, χ 2 = 39.538, P = 0.001; 75.0% vs 46.2%, χ 2 = 6.753, P = 0.009). The expression levels of ATG5 differed notably across patients with different tumor diameter, FIGO stage and lymph node metastasis (all P < 0.01), while the expression levels of PDIA3 differed greatly across patients with different tumor diameter, differentiation degree, FIGO stage and lymph node metastasis (P < 0.05 or P < 0.01). The expression levels of ATG5 and PDIA3 were positively correlated (r = 0.679, P < 0.001). Prognostic analysis via GEPIA showed that high ATG5 and PDIA3 expression correlated with poor prognosis in cervical cancer patients (all P < 0.05). Key enriched functions and signaling pathways for ATG5 and PDIA3 included cell proliferation and differentiation, antigen processing and presentation, P53 binding, Wnt signaling pathway, MAPK signaling pathway and mTOR signal pathway. Conclusion: ATG5 and PDIA3 are highly expressed in cervical cancer tissues, with their elevated expression is associated with poor prognosis. Both ATG5 and PDIA3 are involved in cell proliferation and differentiation, antigen processing and presentation, and various signaling pathways, making them potential targets for cervical cancer treatment.
2024, 31(10):1017-1021.
Abstract:
钙黏素3（CDH3）是经典钙黏素家族中的一员，在特异性细胞黏附和复杂的细胞信号转导方面起重要作用。CDH3在 恶性肿瘤中的作用明显依赖于癌细胞种类，其既可以作为原癌因子，促进肿瘤细胞增殖和侵袭，也可以作为肿瘤抑制因子，抑制 肿瘤细胞的侵袭性。这些特征表明，CDH3可作为特异性恶性肿瘤风险评估和监测预后的新的分子生物学标志物。目前，针对 CDH3的单克隆抗体及放射免疫疗法已在动物模型中取得了初步疗效，其中一些已在开展临床试验，本文综述了近年来CDH3在 恶性肿瘤中作用及机制、临床应用的研究进展。
钙黏素3（CDH3）是经典钙黏素家族中的一员，在特异性细胞黏附和复杂的细胞信号转导方面起重要作用。CDH3在 恶性肿瘤中的作用明显依赖于癌细胞种类，其既可以作为原癌因子，促进肿瘤细胞增殖和侵袭，也可以作为肿瘤抑制因子，抑制 肿瘤细胞的侵袭性。这些特征表明，CDH3可作为特异性恶性肿瘤风险评估和监测预后的新的分子生物学标志物。目前，针对 CDH3的单克隆抗体及放射免疫疗法已在动物模型中取得了初步疗效，其中一些已在开展临床试验，本文综述了近年来CDH3在 恶性肿瘤中作用及机制、临床应用的研究进展。
2024, 31(10):1022-1028.
Abstract:
T细胞免疫球蛋白黏蛋白分子3（TIM-3）是一种免疫检查点分子，在T细胞、自然杀伤（NK）细胞和树突状细胞（DC）等 多种免疫细胞上表达，并通过与多种配体结合而发挥免疫抑制作用。作为一种免疫负调节分子，TIM-3可以通过抑制CD8+ T细 胞的作用，增强调节性T（Treg）细胞的免疫效应，从而影响机体抗肿瘤免疫反应。阻断TIM-3信号通路、联合阻断TIM-3与PD-1， 以及抑制TIM-3和半乳凝素9（Gal-9）之间的相互作用，均可增强免疫细胞的抗肿瘤作用。目前，TIM-3抗体和小分子抑制剂等药 物分子正处在临床前研究阶段，显现出巨大应用价值。本文综述了TIM-3的结构、与配体和细胞的相互作用，深入探索了TIM-3 在肿瘤进展中的作用及作为治疗靶点的潜力，发掘其在抗肿瘤免疫中的应用前景，为开发新的肿瘤治疗策略提供了理论基础和 潜在靶点。
T细胞免疫球蛋白黏蛋白分子3（TIM-3）是一种免疫检查点分子，在T细胞、自然杀伤（NK）细胞和树突状细胞（DC）等 多种免疫细胞上表达，并通过与多种配体结合而发挥免疫抑制作用。作为一种免疫负调节分子，TIM-3可以通过抑制CD8+ T细 胞的作用，增强调节性T（Treg）细胞的免疫效应，从而影响机体抗肿瘤免疫反应。阻断TIM-3信号通路、联合阻断TIM-3与PD-1， 以及抑制TIM-3和半乳凝素9（Gal-9）之间的相互作用，均可增强免疫细胞的抗肿瘤作用。目前，TIM-3抗体和小分子抑制剂等药 物分子正处在临床前研究阶段，显现出巨大应用价值。本文综述了TIM-3的结构、与配体和细胞的相互作用，深入探索了TIM-3 在肿瘤进展中的作用及作为治疗靶点的潜力，发掘其在抗肿瘤免疫中的应用前景，为开发新的肿瘤治疗策略提供了理论基础和 潜在靶点。
2024, 31(10):1029-1034.
Abstract:
肿瘤治疗一直是科研界研究的热点。目前，临床上常用的肿瘤治疗手段存在术后预后较差、药物不良反应多及耐药 性等局限性。因此，研发高效、不良反应少的抗肿瘤药物迫在眉睫。研究表明，瑞舒伐他汀是治疗高脂血症的一线药物，同时具 有抗肿瘤特性，主要包括：抑制肿瘤细胞增殖并诱导细胞凋亡，抑制肿瘤细胞转移和血管生成，诱导脂代谢重编程，以及免疫治疗 和协同抗肿瘤效应等。同时，瑞舒伐他汀与纳米递送系统、天然产物等联合应用有望解决其通透性较差、不良反应多的问题。因 此，开发瑞舒伐他汀作为抗肿瘤药物具有巨大的临床应用潜力和价值。本文论述了瑞舒伐他汀在肿瘤治疗中的临床应用现状及 其抗肿瘤的作用机制，为促进瑞舒伐他汀抗肿瘤机制的进一步研究和开发更有效安全的抗肿瘤药物提供了重要的理论基础。
肿瘤治疗一直是科研界研究的热点。目前，临床上常用的肿瘤治疗手段存在术后预后较差、药物不良反应多及耐药 性等局限性。因此，研发高效、不良反应少的抗肿瘤药物迫在眉睫。研究表明，瑞舒伐他汀是治疗高脂血症的一线药物，同时具 有抗肿瘤特性，主要包括：抑制肿瘤细胞增殖并诱导细胞凋亡，抑制肿瘤细胞转移和血管生成，诱导脂代谢重编程，以及免疫治疗 和协同抗肿瘤效应等。同时，瑞舒伐他汀与纳米递送系统、天然产物等联合应用有望解决其通透性较差、不良反应多的问题。因 此，开发瑞舒伐他汀作为抗肿瘤药物具有巨大的临床应用潜力和价值。本文论述了瑞舒伐他汀在肿瘤治疗中的临床应用现状及 其抗肿瘤的作用机制，为促进瑞舒伐他汀抗肿瘤机制的进一步研究和开发更有效安全的抗肿瘤药物提供了重要的理论基础。
2024, 31(10):1035-1039.
Abstract:
化脓性汗腺炎（HS）是一种皮肤汗腺的慢性化脓性疾病，长期持续的慢性炎症可导致严重的并发症，如皮肤鳞状细胞 癌（cSCC）。HS继发的cSCC具有侵袭性强、转移率和病死率高、预后差的特点。完整手术切除是HS继发cSCC首选的治疗方 案，但术后复发转移风险较高。本文报道1例确诊HS两年后继发cSCC的病例，确诊HS继发cSCC时病灶范围较广，并且有多发 淋巴结转移可能，完整手术切除难度大，术后复发转移风险高。患者先经化疗、免疫靶向新辅助治疗后，病灶完整手术切除，术后 行辅助免疫治疗，17个月余未见肿瘤复发与转移，获得较好的临床治疗效果并延长了患者生存期。
化脓性汗腺炎（HS）是一种皮肤汗腺的慢性化脓性疾病，长期持续的慢性炎症可导致严重的并发症，如皮肤鳞状细胞 癌（cSCC）。HS继发的cSCC具有侵袭性强、转移率和病死率高、预后差的特点。完整手术切除是HS继发cSCC首选的治疗方 案，但术后复发转移风险较高。本文报道1例确诊HS两年后继发cSCC的病例，确诊HS继发cSCC时病灶范围较广，并且有多发 淋巴结转移可能，完整手术切除难度大，术后复发转移风险高。患者先经化疗、免疫靶向新辅助治疗后，病灶完整手术切除，术后 行辅助免疫治疗，17个月余未见肿瘤复发与转移，获得较好的临床治疗效果并延长了患者生存期。
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- 《中国肿瘤生物治疗杂志》
- 1994年创刊
- 主办单位：Chinese Society of Immunology, Chinese Anti-cancer Association
- 邮编：200433
- 电话：021-81871002-22
- 电子邮箱：cjcb@biother.cn
- 网址:http://www.biother.cn
- 刊号：ISSN 1007-385X
- CN 31-1725/R
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Chinese Journal Of Cancer Biotheray ® 2025 All Rights Reserved
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Chinese Journal Of Cancer Biotheray ® 2025 All Rights Reserved
Supported by:Beijing E-Tiller Technology Development Co., Ltd.