Despite the remarkable clinical efficacy achieved by immune checkpoint blockade (ICB) and adoptive T cell therapy, the majority of patients with solid tumors still fail to achieve long-term responses to immunotherapy. One important reason is the complex metabolic patterns and inhibitory signals within the tumor microenvironment (TME), which induce metabolic reprogramming in immune cells and thereby impair their anti-tumor efficacy. This review summarizes the metabolic preferences of CD8+ T cells across various differentiation states, explores the metabolic changes that occur during their interaction with tumor cells and the TME, and discusses how these metabolic changes affect differentiation, function, and stemness of CD8+ T cells. Additionally, strategies that target metabolic molecules or pathways are highlighted to enhance the antitumor ability of CD8+ T cells, thereby improving the efficacy of chimeric antigen receptor gene-modified T lymphocyte (CAR-T cell) immunotherapy and ICB therapy.

ObjectiveTo investigate the mechanisms by which calpain small subunit 1 (CAPN4) regulates cisplatin resistance and cancer stem cell (CSC) stemness in lung adenocarcinoma, and to provide experimental evidence for reversing drug resistance through targeting CSC stemness.MethodsTissue samples were collected from 10 lung adenocarcinoma patients who underwent surgical resection at Fujian Cancer Hospital from January 2023 to January 2024. Immunohistochemistry (IHC) stining was used to detect the differential expression of CAPN4 in five cisplatin-resistant and five cisplatin-sensitive lung adenocarcinoma tissues, followed with a histological scoring (H-score). CAPN4 gene expression-related survival analysis in lung cancer patients was conducted using The Cancer Genome Atlas (TCGA) database and Gene Expression Profiling Interactive Analysis (GEPIA) platform. Additionally, tissue samples from two cisplatin-resistant and two cisplatin-sensitive lung adenocarcinoma cases were collected to establish lung adenocarcinoma organoid (PDO) models. H-E and IHC staining were used to assess the morphological consistency between PDOs and the primary tumors. CAPN4 gene expression was silenced using lentivirus-mediated shRNA transduction. The expression levels of stem cell markers ALDH1A1, CD133, Nanog, and SOX9 were detected at both the gene and protein levels using quantitative polymerase chain reaction (qPCR) and Western blotting (WB), respectively. The sensitivity of CAPN4-knockdown PDOs to cisplatin was evaluated using the adenosine triphosphate (ATP) assay, and the apoptosis was assessed using the caspase-3 assay.ResultsIHC results showed that CAPN4 protein expression was significantly upregulated in cisplatin-resistant lung adenocarcinoma tissues (P < 0.05). TCGA cohort analysis revealed that high CAPN4 expression was significantly associated with poor prognosis (reduced OS) in lung adenocarcinoma patients (HR = 1.4, P < 0.05). PDOs derived from cisplatin-resistant patients exhibited significant upregulation in CAPN4 protein and stemness markers at both gene and protein levels (all P < 0.05). Cisplatin sensitivity assays demonstrated that PDOs derived from cisplatin-resistant patients had significantly higher IC50 values than those from cisplatin-sensitive patients(P < 0.05). After CAPN4 knockdown, the expression of stem cell makers in PDOs derived from cisplatin-resistant patients were significantly reduced, and their sensitivity to cisplatin was enhanced (P < 0.05).ConclusionKnockdown of CAPN4 reduces stem cell marker expression and enhances cisplatin sensitivity in lung adenocarcinoma PDOs, providing a potential therapeutic target for reversing cisplatin resistance in lung cancer.

ObjectiveTo identify HLA-G-binding proteins (HGBPs) by screening targeting ankyrin sequences from a phage display-based ankyrin protein library using human leukocyte antigen G (HLA-G) as the target, and to evaluate their functions.MethodsThe expression of HLA-G in tumor tissues and its correlation with clinical prognosis and immune infiltration were analyzed using bioinformatics tools such as TCGA and GTEx databses. The extracellular domain of HLA-G was subjected to biopanning with a phage-displayed ankyrin protein library, followed by random selection and sequencing of monoclonal phage clones. The functional properties of dominant phage clones were validated using ELISA and immunofluorescence staining. HGBPs were produced and purified using a prokaryotic expression system, and their affinity and tumor-specific binding ability were evaluated using ELISA, surface plasmon resonance (SPR), and immunofluorescence staining.ResultsBioinformatics analysis revealed that HLA-G is widely overexpressed in tumor tissues and is correlated with overall survival (OS) and immune cell infiltration (P < 0.05). After five rounds of biopanning, dominant clones were obtained. Both ELISA and immunofluorescence staining results showed that these dominant phages had a significantly higher affinity to HLA-G positive cells compared to HLA-G negative cells (P < 0.05, P < 0.001). The purified HGBPs exhibited an affinity of up to 17 nmol/L for HLA-G. ELISA results showed significant binding of HGBP to HLA-G (P < 0.05), and immunofluorescence staining confirmed that HGBP could specifically bind to HLA-G-positive cells (P < 0.01).ConclusionThe HGBPs identified via phage display exhibit high affinity and specificity to HLA-G on tumor cells.

ObjectiveTo investigate the molecular mechanism by which 2',4'-dihydroxychalcone (D2) inhibits proliferation, migration, and epithelial-mesenchymal transition (EMT) in colorectal cancer cells through miR-7-5p-mediated autophagy.MethodsHuman colorectal cancer cell lines HCT-15 and SW620 were treated with D2 at concentrations of 0, 12.5, 25, and 50 μmol/L. Cell proliferation and clonogenic capacity were evaluated using MTT and colony formation assays. Cell migration was assessed by wound healing and Transwell assays. WB assay was used to detect the expression of EMT-related proteins, autophagy-related proteins, and key components of the PI3K/AKT/mTOR pathway. Autophagosome formation was visualized by immunofluorescence staining. TCGA database and KEGG pathway analyses were performed to evaluate miR-7-5p expression and its association with colorectal cancer. RT-qPCR was used to quantify miR-7-5p expression, and lentiviral transduction was employed to establish stable miR-7-5p knockdown or overexpression cell lines.ResultsD2 significantly inhibited colorectal cancer cell proliferation, migration, and EMT (P < 0.05 or P < 0.01). TCGA and KEGG analyses revealed that miR-7-5p expression was downregulated in colorectal cancer and closely associated with disease progression. D2 treatment (12.5, 25, and 50 μmol/L) significantly upregulated miR-7-5p expression in HCT-15 and SW620 cells (P < 0.01). At 25 μmol/L, D2 increased the expression of autophagy-related proteins (LC3 and p-ULK1) and inhibited the PI3K/AKT/mTOR signaling pathway (P < 0.05), accompanied by increased autophagosome formation (P < 0.01). In miR-7-5p-knockdown cells treated with D2, the levels of LC3 and p-ULK1 were significantly reduced compared to D2-only treated cells (P < 0.05 or P < 0.01).ConclusionD2 upregulates miR-7-5p to induce autophagy, thereby inhibiting colorectal cancer cell proliferation, migration, and EMT, possibly through suppression of the PI3K/AKT/mTOR signaling pathway.

ObjectiveTo investigate the effects of enolase 1 (ENO1) on the proliferation, migration, and invasion of gastric cancer cells and its underlying molecular mechanisms.MethodsThe expression levels of ENO1 in human gastric cancer cell lines (HGC27, MKN-45, N-87, MGC803, BGC-823) and human gastric mucosal epithelial cells (GES-1) were detected using WB assay. Gene editing tools such as CRISPR and overexpression system were used to construct ENO1 knockdown and knockdown-rescue cell lines. Both MKN-45 and BGC-823 cells were grouped into control (Ctrl) group, ENO1 knockdown (ENO1 KD) group, and ENO1 knockdown-rescue (ENO1 KD-OE) group. The effects of ENO1 knockdown or ENO1 knockdown-rescue on the proliferation, migration, invasion, and apoptosis of gastric cancer cells were evaluated using colony formation assay, EdU staining, scratch wound healing assay, Transwell chamber assay and flow cytometry. Additionally, a xenograft model was established in nude mice, and the effects of ENO1 on tumor growth were monitored using small animal in vivo imaging and tumor tissue block measurement. ENO1 was silenced in MKN-45 cells employing RNA interference technology, and the downstream target genes of ENO1 were identified using RNA co-immunoprecipitation sequencing (RIP-seq) and bioinformatics analysis. The molecular mechanisms by which ENO1 regulates the proliferation, migration and invasion of gastric cancer cells was also analyzed.ResultsENO1 was significantly upregulated in gastric cancer cell lines (P < 0.01 or P < 0.001). ENO1 knockdown significantly inhibited proliferation, migration, and invasion while promoting apoptosis in MKN-45 and BGC-823 cells (P < 0.001, P < 0.000 1). Rescue experiments showed that restoring ENO1 expression significantly enhanced cell proliferation, migration, invasion, and inhibited apoptosis (P < 0.05, P < 0.01, P < 0.001, P < 0.000 1). In vivo experiments demonstrated that ENO1 knockdown significantly inhibited tumor growth in nude mice (P < 0.000 1). The differentially expressed genes interacting with ENO1 protein were primarily enriched in pathways related to RNA splicing. Additionally, ENO1 protein was found to interact with the PKM gene, and their expressions showed a positive correlation in gastric cancer tissues (r = 0.886).ConclusionENO1 is highly expressed in gastric cancer cells. ENO1 interacts with precursor mRNA of PKM to influence its RNA splicing process, thereby regulating PKM2 expression and promoting gastric cancer progression.

ObjectiveTo investigate the molecular mechanisms of bexarotene in treating double hit lymphoma (DHL) based on Weighted Gene Co-expression Network Analysis (WGCNA), thereby providing potential targets and experimental evidence for DHL treatment.MethodsThe gene expression datasets GSE44164 and GSE43677 were downloaded from the Gene Expression Omnibus(GEO) database, and differentially expressed genes (DEGs) were identified. WGCNA was employed to identify gene modules associated with DHL. A protein-protein interaction (PPI) network was constructed to screen for key hub genes. Drug-gene association analysis was conducted using the EpiMed platform to identify potential targeted drugs for DHL. The effects of bexarotene on DHL cell proliferation and key protein expression were evaluated using the CCK-8 assay and Western blotting (WB), and its effects on cell apoptosis was evaluated using flow cytometry.ResultsWGCNA identified a turquoise module highly associated with DHL, and 10 hub genes (COL1A2, COL3A1, MMP2, COL5A2, DCN, BGN, FN1, MMP9, FBN1, and LUM) were screened from the PPI network. Drug association analysis nominated bexarotene as a potential therapeutic agent. In vitro validation demonstrated that bexarotene significantly inhibited U2932 cell viability (P < 0.05), promoted cell apoptosis (P < 0.001), and downregulated c-Myc and COL1A2 expression (P < 0.05).ConclusionBexarotene may exert anti-DHL effects by suppressing the c-Myc signaling pathway and modulating extracellular matrix-related genes. Further studies are warranted to validate its in vivo efficacy and potential for combination therapy.

ObjectiveTo investigate the effects of toosendanin (TSN) on the proliferation, apoptosis, migration and invasion of esophageal squamous cell carcinoma (ESCC) KYSE150 cells, and to elucidate its underlying molecular mechanisms.MethodsCCK-8 assay, colony formation assay, and EdU assay were used to assess the effects of varying TSN concentrations (0.062 5, 0.125, and 0.25 mol/L) on KYSE150 cell proliferation. The impacts of TSN on the apoptosis, migration, and invasion of KYSE150 cells were evaluated using flow cytometry, wound healing assay, and Transwell chamber assay, respectively. The expression of hypoxia-inducible factor 1 alpha (HIF1A) in esophageal cancer tissues was analyzed using the GEPIA database. qPCR was used to detect the expression level of HIF1A mRNA in human esophageal epithelial Het-1A and KYSE150 cells, and in TSN-treated KYSE150 cells. Western blot(WB) was performed to detect the effects of TSN on the upstream signaling pathway AKT/mTOR of HIF1A and the expression of downstream proteins related to cell migration, invasion, and apoptosis.ResultsTSN of varying concentrations significantly inhibited proliferation, migration, and invasion of KYSE150 cells and promoted apoptosis in a dose-dependent manner (P < 0.05 or P < 0.01). HIF1A mRNA was highly expressed in KYSE150 cells, and its expression was significantly downregulated after TSN treatment(P < 0.05 or P < 0.01). TSN markedly downregulated the expression of HIF1A and key upstream signaling proteins p-AKT and p-mTOR. In addition, TSN significantly suppressed the expression of downstream proteins associated with cell migration, invasion, and apoptosis, including N-cadherin, vimentin, Bcl-2, and caspase-3, while upregulating the expression of E-cadherin(P < 0.05 or P < 0.01).ConclusionTSN inhibits the proliferation, migration, and invasion, and induces apoptosis in ESCC KYSE150 cells by down-regulating HIF1A expression through suppression of the AKT/mTOR signaling pathway.

ObjectiveTo investigate whether lncRNA SOX2-OT inhibits the proliferation and migration of colorectal cancer (CRC) HCT-116 cells by regulating the miR-215-5p/NIN/RPN12 binding protein 1 homolog (NOB1) signaling pathway.MethodsCancerous and paired adjacent tissue samples from 29 CRC patients treated at Wuhan Jinyintan Hospital from June 2022 to May 2024 were collected, along with CRC cell lines (SW480, HCT-116, HP116, and LoVo) and normal human colon epithelial HCoApiC cells. The mRNA expression levels of SOX2-OT, miR-215-5p, and NOB1 in CRC tissues and cells were measured using qPCR method. HCT-116 cells were transfected with SOX2-OT knockdown or overexpression plasmids and corresponding negative control plasmids using RNA interference technology, dividing the cells into control group, si-NC group, si-SOX2-OT group, si-SOX2-OT + inhibitor (Inh) NC group, si-SOX2-OT + miR-215-5p Inh group, si-SOX2-OT + oe-NC group, and si-SOX2-OT + oe-NOB1 group. The mRNA expression levels of SOX2-OT, miR-215-5p, and NOB1 in each group of cells were detected using qPCR method. MTT assay, scratch wound healing assay, Transwell chamber assay, and flow cytometry were used to measure cell proliferation, migration, invasion, and apoptosis, respectively. Western blot was applied to detect protein expression levels of E-cadherin, N-cadherin, vimentin, Bcl-2, BAX, PCNA, MMP-9, and NOB1. The targeting relationship between miR-215-5p and SOX2-OT or NOB1 was validated using dualluciferase reporter gene assays.ResultsSOX2-OT and NOB1 mRNA were significantly upregulated, while miR-215-5p was downregulated in both CRC tissues and cells (all P < 0.05). In HCT-116 cells with SOX2-OT knockdown, the expression of SOX2-OT and NOB1 mRNA, cell proliferation, wound healing rate, invasive cell number, and protein levels of N-cadherin, vimentin, Bcl-2, NOB1, PCNA, and MMP-9 were significantly reduced (all P < 0.05), while miR-215-5p expression, apoptosis rate, and protein levels of E-cadherin and BAX were significantly increased (all P < 0.05). Both miR-215-5p knockdown and NOB1 overexpression reversed the inhibitory effects of SOX2-OT knockdown on HCT-116 cells (both P < 0.05). miR-215-5p was validated to target SOX2-OT and NOB1.ConclusionSOX2-OT knockdown upregulates miR-215-5p expression and downregulates NOB1 expression, further inhibiting the proliferation, migration, and invasion of HCT-116 cells and promoting apoptosis.

ObjectiveTo investigate the role and molecular mechanism of dual-specificity phosphatase 26 (DUSP26) in the proliferation, migration and invasion of lung adenocarcinoma (LUAD) A549 cells.MethodsThe expression profile of DUSP26 was retrieved from the GEPIA2 tumor database, and its differential expression in LUAD tissues and normal human lung tissues were analyzed. Twelve pairs of LUAD tissue and paracancerous tissue surgically resected at Pingxiang People's Hospital between October 2022 and October 2023 were collected. The difference in DUSP26 expression between LUAD tissues and paracancerous tissues was analyzed using immunohistochemical (IHC) staining and Western blotting (WB). Additionally, the expression of DUSP26 in four LUAD cells (A549, SK-LU-1, Calu-3, H1299) and two normal bronchial epithelial cells (BEAS-2B, HBEC) was detected using WB method. A549 cells stably overexpressing DUSP26 (DUSP26-OE) or corresponding negative control (DUSP26-OENC) were constructed via lentiviral transfection. The effects of DUSP26 overexpression on cell proliferation, migration and invasion were detected using colony formation, scratch assay, and Transwell chamber assay, respectively. The expression levels of TGF-1/SMAD2/3 pathway-and epithelial-mesenchymal transition (EMT)-related proteins were detected using WB method, and the expression levels of Ki-67 and cyclin D1 in cells were detected by immunofluorescence staining. Rescue experiments were conducted by adding 5 ng/mL recombinant TGF-1. A nude mouse xenograft model was established using A549 cells to observe the effect of DUSP26 overexpression on the in vivo growth of transplanted tumors. The expression levels of TGF-1/SMAD2/3 pathway-and EMT-related proteins in transplanted tumor tissues were assessed using WB method, and the expression levels of Ki-67 and cyclin D1 in transplanted tumor tissues were detected using immunofluorescence staining.ResultsDUSP26 expression was downregulated in both LUAD tissues and cells (P < 0.05, P < 0.01, P < 0.001 or P < 0.000 1). Compared with the DUSP26-OENC group, the DUSP26-OE group showed significantly reduced proliferation, migration and invasion of A549 cells (P < 0.01 or P < 0.001). Furthermore, the protein levels of TGF-1, p-SMAD2/3, vimentin, N-cadherin, snail, Ki-67, and cyclin D1 were significantly reduced (P < 0.01, P < 0.001 or P < 0.000 1), while E-cadherin level was increased in the DUSP26-OE group (P < 0.000 1). The addition of 5 ng/mL TGF-1 recombinant protein partially reversed the effects of DUSP26 overexpression in vitro experiments. The nude mice A549 cell xenograft model was successfully constructed. The growth rate of transplanted tumors was significantly slower in the DUSP26-OE group, with reduced volume and mass (all P < 0.001). The protein levels of TGF-1, p-SMAD2/3, vimentin, N-cadherin, snail, Ki-67, and cyclin D1 in the transplanted tumor tissues were all reduced (P < 0.01 or P < 0.001), while E-cadherin level was increased (P < 0.000 1).ConclusionDUSP26 is downregulated in both LUAD tissues and cells. Upregulation of DUSP26 suppresses the proliferation, migration and invasion of A549 cells by inhibiting the TGF-1/SMAD2/3 signaling pathway.

ObjectiveTo analyze the clinicopathological characteristics and prognostic factors in gastric cancer patients with bone metastasis, and to evaluate the impact of different treatment regimens on survival in patients with synchronous and metachronous bone metastasis.MethodsA total of 120 gastric cancer patients with bone metastasis treated at Nanjing Drum Tower Hospital between 2015 and 2023 were enrolled, including 36 with synchronous bone metastasis and 84 with metachronous bone metastasis. Clinicopathological features were compared between the two groups using the 2 test. Cox proportional hazards regression model was employed to identify risk factors for overall survival after bone metastasis (OS-BM). The Kaplan-Meier method was used to analyze the effects of different treatments on OS-BM in both synchronous and metachronous groups.ResultsAmong the 120 patients, 104 (86.6%) had metastases to other organs. Comparative analysis revealed that synchronous bone metastasis patients exhibited elevated serum C-reactive protein and decreased serum albumin, whereas metachronous bone metastasis patients had reduced peripheral white blood cell and neutrophil counts (all P < 0.05). Metachronous bone metastasis (HR = 2.35, 95% CI [1.47, 3.74], P < 0.01), serum CA125 ≥ 30.2 U/mL (HR = 1.6,95% CI [1.03, 2.48], P = 0.036), white blood cell count ≥ 9.5 × 109/L (HR = 2.15, 95% CI [1.17, 3.92], P = 0.013), and absence of immunotherapy (HR = 2.26, 95% CI [1.5, 3.39], P < 0.01) were independent risk factors affecting patient prognosis. Combined immunotherapy significantly prolonged OS-BM in gastric cancer patients with bone metastasis compared to non-immunotherapy regimens (9.63 vs 4.53 months, P = 0.002). Patients with metachronous bone metastasis demonstrated better response to immunotherapy compared to those with synchronous metastases (median OS-BM: 10.8 vs 7.3 months, P = 0.004).ConclusionImmunotherapy is an independent protective factor for survival in gastric cancer patients with bone metastasis. Early combination therapy centered on immunotherapy alongside chemotherapy is recommended to prolong survival in such patients.

ObjectiveTo investigate epithelial cell heterogeneity in ovarian cancer (OC) and its impact on the tumor microenvironment (TME) by constructing OC epithelial subtypes using publicly available single-cell RNA sequencing (scRNA-seq) data and bioinformatics approaches.MethodsEight cell types and gene expression matrices were identified from the GSE118828 OC scRNA-seq dataset, and a deconvolution approach was used to estimate cellular composition in bulk RNA sequencing (bulk RNA-seq) data. OC samples were stratified into high-epithelial (C1) and low-epithelial (C2) subtypes based on epithelial cell abundance. Differences in overall survival (OS) and immune microenvironment features between patients with C1 and C2 subtypes were analyzed. Differentially expressed genes (DEGs) between C1 and C2 subtypes, as well as between early-stage (stage Ⅰ~Ⅱ) and late-stage (stage Ⅲ-Ⅳ) OC groups were identified to screen epithelial-related genes associated with OC progression, and their influence on the TME was analyzed. Protein expression of the epithelial-related genes in OC progression was validated using data from the Human Protein Atlas (HPA) database.ResultsPatients with the C1 subtype exhibited significantly better OS compared to those with C2 subtype(P < 0.05). The C1 and C2 subtypes demonstrated distinct immune microenvironment infiltration profiles. Infiltration levels of T helper(Th) cells, M1 macrophages, and activated dendritic cells (DCs) were significantly higher in the C1 subtype (all P < 0.05). In late-stage OC patients with C1 subtype, the expression of HAS1, DAPL1, and ADH1B was significantly upregulated (all P < 0.05). Furthermore, DAPL1 expression was positively correlated with the degree of macrophage and regulatory T cells (Treg) infiltration in OC (P < 0.05). HPA database analysis confirmed that DAPL1 protein expression was significantly upregulated in OC tissues.ConclusionThe high-epithelial subtype of ovarian cancer may exert anti-tumor effects through extensive infiltration of T helper (Th) cells, M1 macrophages, and activated dendritic cells within the tumor microenvironment. Conversely, upregulation of the epithelial-related gene DAPL1 may induce the infiltration of macrophages and Treg, thereby promoting OC progression.

ObjectiveTo explore the relationship between programmed death-1 (PD-1) inhibitors and cancer-related fatigue (CRF) in patients with malignant melanoma, and to identify associated influencing factors.MethodsA total of 100 patients with malignant melanoma treated at Jinan People's Hospital between April 2019 and April 2024 were included as study subjects. The Chinese version of the Piper Fatigue Scale was used to evaluate patients' fatigue levels within three months before and after the initiation of PD-1 inhibitor therapy.ResultsThere was a significant difference in CRF score before and after PD-1 inhibitor treatment (P < 0.001). Univariate analysis showed no significant association between fatigue severity and factors such as gender, smoking history, tumor site, or PD-1 inhibitor type (all P > 0.05). However, age, tumor stage, anemia, leukopenia, secondary hypothyroidism, secondary adrenal insufficiency (AI), and secondary adrenocorticotropic hormone deficiency (P < 0.05 or P < 0.01 or P < 0.001) were significantly associated with CRF. Multivariate regression analysis identified secondary hypothyroidism, secondary AI, anemia, and leukopenia as independent risk factors for severe CRF in patients with malignant melanoma (all P < 0.05).ConclusionAdverse reactions of PD-1 inhibitors, including secondary hypothyroidism, secondary AI, anemia, and leukopenia, are independent risk factors for CRF in patients with malignant melanoma.