Journal of Experimental & Clinical Cancer Research最新文献

Tumor endothelial cells (TECs) play a critical role in regulating immune responses within the tumor microenvironment (TME). However, the mechanisms by which TECs modulate immune cell population remain unclear, particularly in non-small cell lung cancer (NSCLC). Here, we investigated how NSCLC cells tweak normal endothelial cells (NECs) into TECs and the subsequent effects on immune regulation. NECs were cocultured with various NSCLC cell lines, using 2D and 3D coculture models to evaluate TEC-mediated effects on immune cells. We show that direct coculture led to significant transcriptomic, proteomic and kinomic alterations in TECs, especially in pro-inflammatory pathways. We identified a downregulation of the co-stimulatory molecule OX40L in TECs compared to NECs, suggesting impaired T-cell proliferation support. While TECs showed a limited effect on CD8⁺ T-cell activation, TECs supported CD4⁺ T-cells polarization into Treg and Th22 subsets. Moreover, TECs also promoted M2-like macrophages polarization, thereby potentially contributing to the TME immunosuppression. State-of-the-art single-cell RNA sequencing of 3D multicellular tumor spheroids (MCTS) revealed formidable heterogeneity in the tumor cells and cancer-associated fibroblast compartments. It also unveiled distinct TEC subpopulations, including an inflammatory subset with an unfolded-protein response signature. This TEC cluster was absent in 2D-cultured NECs but present in freshly isolated and 2D-cultured TECs from NSCLC patients. Importantly, we identified a perivascular M2-like macrophage subset within MCTS that is in close contact with TECs, and is predicted to interact with them through MIF signaling. In conclusion, TECs in NSCLC tumors play a pivotal role in remodeling the TME immune landscape by promoting immune suppression. This study highlights the complex immunoregulatory functions of TECs within different in vitro models that mimic aspects of the TME. Our data may provide new insights into potential therapeutic strategies targeting TECs or regulatory signaling to improve the efficacy of immunotherapy in NSCLC.

Background: Dendritic cells (DCs) play a predominant role in antitumor immunity. As professional antigen-presenting cells (APCs), DCs can be functionally matured by TLR2 ligand binding to enhance innate immune response and subsequent T cell-dependent adaptive immunity. DC function is often suppressed by the tumor microenvironment, while current TLR2 agonists exhibit suboptimal stability and diminished efficacy in vivo. Therefore, reactivation of suppressed DCs could be a promising strategy for enhancing the efficacy of cancer immunotherapy.

Methods: To investigate the antitumor immunity induced by the novel Toll-like receptor 2 (TLR2) agonist SUP3 with better stability, we established murine melanoma, colon cancer and breast cancer tumor models. The hematopoietic growth factor Flt3L-dependent dendritic cells (FLDCs) were generated and utilized to examine their capacities of antigen processing and cross-presentation, and migration to the tumor-draining lymph nodes (TdLNs) in response to SUP3 treatment. To further improve the antitumor response of SUP3 by increasing the abundance and activation of DCs, Flt3L was administrated in vivo in combination with immune checkpoint blockade.

Results: SUP3 exhibited stronger inhibition of tumor growth and metastasis than classical TLR2 agonist, Pam3. SUP3 could increase cDC1 antigen cross-presentation and TdLN migration, promoting the proliferation, activation and cytotoxicity of antigen-specific cytotoxic T lymphocytes (CTL). SUP3 promoted the intracellular accumulation of antigens and facilitated the process of antigen cross-presentation, the processe regulated by the small GTPase Rab7. SUP3 induced PD-L1 expression by DCs via an interferon-γ-independent pathway. The combination of SUP3 treatment with immune checkpoint blockade by anti-PD-L1 further improved the antitumor response. Moreover, Flt3L increased DC proliferation and infiltration into the tumor tissues that further enhanced the effects of antitumor immunotherapy when used in combination with SUP3 and anti-PD-L1.

Conclusions: This study demonstrated that the modified and more stable TLR2 agonist SUP3 provided an optimal strategy for promoting antitumor immunity via activation of cDC1. SUP3 enhanced antigen cross-presentation by cDC1 and subsequent activation of CTLs. The antitumor effect was further enhanced when SUP3 and Flt3L synergized with PD-L1 blockade. Therefore, reactivation of suppressed DCs in tumor microenvironment would be a promising strategy for designing effective antitumor immunotherapy.

Oncolytic vaccinia viruses (OVV) demonstrate the capacity to inflame the tumor microenvironment (TME) and elicit infiltrating tumor-specific T cells responses. However, OVV treatment paradoxically alters the cancer-immune equilibrium within tumors, attenuating anti-tumor immunity and necessitating a deeper understanding of the viral-induced immune landscape to optimize therapeutic potential. This study investigates the impact of single-domain antibody-armed OVs targeting TIM-3 on TME remodeling, aiming to overcome localized immunosuppression and enhance tumor responsiveness to immunotherapeutic interventions. We engineered a tumor-selective OVV vector encoding single-domain antibodies against murine (mNbTIM3) or human (hNbTIM3) TIM-3. The therapeutic efficacy of OVV-mNbTIM3 was evaluated across multiple murine cancer models, demonstrating that localized V_HHTIM3 delivery via OVV-mNbTIM3 promotes systemic anti-tumor immunity in established cancers. Mechanistically, OVV-mNbTIM3 treatment enhances DCs maturation and tumor-specific CD8⁺ T cells activation. Notably, intratumoral expression of hNbTIM3 also conferred therapeutic benefit in humanized mice bearing patient-derived cancer xenografts. These findings provide mechanistic insights for enhancing OVV combination therapies and inform the rational design of next-generation oncolytic viruses with engineered immunomodulatory properties.

Background: Aberrant glycogen metabolism drives lipid accumulation and adaptive lipid homeostasis reprogramming, a metabolic adaptation critical for sustaining malignant progression and chemoresistance in cholangiocarcinoma (CCA). While our prior study highlighted glycogen degradation as pivotal for CCA tumorigenesis, the molecular mechanisms governing lipogenesis and its therapeutic exploitation remain elusive.

Methods: We performed single-cell RNA sequencing to explore metabolic status in CCA. A high-throughput screening of 994 bioactive compound library was performed to identify pharmacological agents capable of inhibiting CCA and targeting this metabolic vulnerability. The drug efficacy was demonstrated through in vitro and in vivo experiments. Additionally, a biotinylated WA derivative was synthesized and its target was investigated using liquid chromatography-tandem mass spectrometry. Validating the clinical potential of the compound for targeted antitumor therapy in combination with gemcitabine in vivo.

Result: Through integrated multi-omics analysis, we identified pronounced lipid droplet accumulation in CCA tissues. Subsequent high-throughput screening of bioactive compounds revealed Withaferin A (WA) as a potent dual suppressor of lipid deposition and cholangiocarcinogenesis. Mechanistically, WA directly binds acetyl-CoA carboxylase 1 (ACC1), inhibiting its catalytic conversion of acetyl-CoA to malonyl-CoA. Notably, resultant malonyl-CoA depletion abolished ACC1 auto-malonylation, thereby enhancing SQSTM1/p62-mediated cargo recognition and triggering selective autophagic degradation, consequently disrupting de novo lipogenesis and lipid droplet accumulation. Therapeutically, WA synergized with gemcitabine to enhance antitumor efficacy and prolong survival in preclinical models.

Conclusion: Our study confirms that pharmacological blockade of ACC1 significantly inhibits de novo lipogenesis and CCA tumorigenesis, suggesting that WA may serve as a potential small-molecule inhibitor targeting lipid metabolism for CCA treatment.

Background: Hepatocellular carcinoma (HCC) is among the leading causes of cancer-related mortality worldwide. The high recurrence rate and resistance to chemotherapy of HCC contribute to poor clinical outcomes, necessitating the development of novel therapeutic strategies. Glutathione S-transferase kappa 1 (GSTK1) is specifically localized to mitochondria and peroxisomes, participates in adiponectin secretion and insulin resistance, and inhibits the progression of non-alcoholic fatty liver disease. However, the role of GSTK1 in HCC is unknown. We aimed to determine the role of GSTK1 in HCC progression.

Methods: N-nitrosodiethylamine (DEN)/ carbon tetrachloride and DEN/high-fat, high-fructose, high-cholesterol diet models were used in hepatocyte-specific Gstk1 knockout and control mice to establish a murine HCC model. Human HCC cell lines with GSTK1 overexpression or knockdown were used to determine GSTK1 function in tumor growth and migration in vitro. Non-target metabolomics analysis, RNA-sequence, transmission electron microscope (TEM), immunoprecipitation (IP), liquid chromatography, and high-throughput mass spectrometry (LC-MS/MS) were used to determine the mechanism by which GSTK1 participates in HCC.

Results: GSTK1 was shown to suppress HCC in vivo and in vitro. Non-target metabolomics analysis indicated that GSTK1 participates in L-carnitine metabolism. L-carnitine supplementation inhibited proliferation and promoted apoptosis of HCC cells in vivo and in vitro. This effect was enhanced by GSTK1 overexpression. Mechanically, TEM and western blot showed that GSTK1 influences mitochondrial quality control (MQC) by promoting mitochondrial biosynthesis and mitochondrial fusion. GSTK1 was shown to inhibit mitochondrial fission and mitophagy, which was consistent with the immunofluorescence results. IP and LC-MS/LMS indicated that GSTK1 combines with PGAM5 and competes with DRP1. Additionally, GSTK1 was shown to be regulated by transcription factors (PPARα/RXRα) and the RXRα agonist, bexarotene, inhibited HCC cell proliferation.

Conclusions: GSTK1 was shown to be a tumor suppressor via its role in MQC and L-carnitine metabolism. Bexarotene and L-carnitine supplementation may serve as potential therapeutic strategies for HCC treatment.

Background: One of the main challenges in cancer treatment is addressing the metabolic reprogramming of tumor cells, which require more energy and biomolecules than healthy cells. Cancer cells alter their metabolism by switching between glycolysis and oxidative phosphorylation (OXPHOS). These processes depend on transmembrane proteins that respond to the extracellular environment. Our research identified the transmembrane form of Chloride Intracellular Channel 1 (tmCLIC1) as a marker of malignancy and a potential therapeutic target. tmCLIC1 levels are increased in several solid tumors, supporting cancer growth and progression, whereas they are mostly absent in healthy cells. We confirmed that tmCLIC1 is the specific target of the antidiabetic drug metformin, an OXPHOS inhibitor in cancer cells.

Methods: tmCLIC1 is the primary target of metformin in glioblastoma-initiating cells, as shown by single-channel patch-clamp recordings and NMR experiments. Various patient-derived glioblastoma cells with different genetic backgrounds were used to demonstrate that CLIC1 CRISPR-Cas9 knockout and/or its point mutation at arginine 29 removes metformin's antitumor effects. Functional assays were used to assess the effects on proliferation, mitochondrial metabolism, and tumor growth in vitro and in vivo, using zebrafish and murine xenograft models.

Results: Metformin inhibits the function of tmCLIC1 through direct and specific binding involving arginine 29 in the tmCLIC1 sequence. Additionally, during hypoglycemia, metformin promotes glioblastoma cell apoptosis by inhibiting the Cancerous Inhibitor of Protein Phosphatase 2 A (CIP2A) and activating the PP2A B56δ subunit. This leads to the dephosphorylation of Glycogen Synthase Kinase 3 Beta (GSK3β), resulting in the breakdown of the pro-survival protein MCL-1 and subsequent cell death. Inhibition of tmCLIC1 is crucial for this metformin-driven antineoplastic effect, mainly through regulating the PP2A-GSK3β-MCL-1 pathway under hypoglycemic conditions. The chronic presence of metformin within the tumors impairs in vivo growth at nanomolar concentrations.

Conclusions: The therapeutic role of metformin to treat brain tumors remains debated. Our findings show that drug delivery is essential, as in vivo, tumor growth decreases at concentrations below 10 nanomolar. We propose that sustained CNS metformin levels may improve tmCLIC1 inhibition, providing a basis for optimizing interactions with metformin or related compounds to enhance therapeutic efficacy.

Background: Lactylation, a recently identified post-translational modification derived from lactate, has emerged as a regulator of tumor metabolism. However, its functional relevance and molecular targets in bladder cancer (BLCA) remain unclear.

Methods: We performed immunohistochemistry on patient tissues, global lactylation proteomics using LC-MS/MS, and in vitro and in vivo functional assays. Gene editing via CRISPR/Cas9, overexpression systems, and pharmacological interventions were employed to study P300-mediated HNRNPA1-K350 lactylation in driving BLCA cell aggression. Metabolomics and glycolytic flux assays were used to assess the metabolic consequences of HNRNPA1 lactylation. Molecular characterization was validated through gene expression and splicing analyses. Small-molecule drug screening was conducted via molecular docking to identify potential inhibitors targeting HNRNPA1.

Results: Protein lactylation levels were significantly elevated in BLCA tissues, correlating with poor prognosis. HNRNPA1 was identified as a central lactylation target. Glycolysis-induced lactate production promoted P300-mediated lactylation of HNRNPA1 at lysine 350, which facilitated PKM pre-mRNA splicing toward the PKM2 isoform, enhancing glycolytic flux and supporting tumor growth. Inhibition of glycolysis or LDHA knockdown reduced HNRNPA1 lactylation, suppressed PKM2 expression, and impaired BLCA cell proliferation, migration, and invasion. Metabolomic profiling linked HNRNPA1-K350 lactylation with increased aerobic glycolysis in BLCA cells. A small-molecule inhibitor, identified through molecular docking, attenuated cell proliferation by binding to HNRNPA1 and suppressing PKM2 expression.

Conclusions: This study reveals a lactate-driven mechanism coupling alternative splicing to metabolic reprogramming via HNRNPA1 lactylation, identifying HNRNPA1-K350 lactylation as a key driver of glycolysis-dependent tumor progression. A therapeutic approach targeting HNRNPA1 in BLCA is proposed.

Cholangiocarcinoma (CCA) is an aggressive malignancy with limited therapeutic options. Increasing evidence suggests that a subset of CCA patients can benefit from multiple tyrosine kinase inhibitors (mTKIs) such as surufatinib. Biomarker studies that can identify potential responders to mTKI therapy are crucial for improving treatment outcomes. In this study, we examined the expression of protein tyrosine phosphatase non-receptor type 9 (PTPN9) in CCA patients treated with surufatinib and analyzed its association with treatment response. Immunoprecipitation coupled with mass spectrometry (IP-MS) was performed to identify novel PTPN9 substrates. Orthotopic mouse models, biochemical assays, structural analyses, and functional experiments were conducted to validate these novel findings. Single-cell RNA sequencing, ELISA, and immunohistochemistry were utilized to investigate microenvironmental tumor-promoting factors.CCA patients with surufatinib non-response exhibited significantly decreased PTPN9 expression compared to that response to surufatinib. PTPN9 knockdown in orthotopic models led to reduced sensitivity to surufatinib. IGF1R was identified as a new substrate of PTPN9, which preferably to dephosphorylate IGF1R at Y1166. In clinical tissues, PTPN9 expression was inversely correlated with IGF1R Y1165/1166 phosphorylation. Establishment of a surufatinib-resistant CCA cell line further confirmed decreased PTPN9 expression and elevated IGF1R signaling. In vivo blockade of IGF1R signaling significantly enhanced surufatinib sensitivity. Mechanistically, crystal structure analysis revealed Tyr333 and Asp335 as key PTPN9 residues interacting with IGF1R; mutation of these residues restored IGF1R signaling and abolished the tumor-suppressive effect of PTPN9. Furthermore, cancer-associated fibroblasts (CAFs) were identified as the major source of IGF1 in CCA microenvironment, essential for IGF1R-driven tumor progression.In summary, the PTPN9-IGF1R axis plays a pivotal role in modulating mTKI sensitivity and tumor progression in CCA. This axis serves as a promising biomarker for identifying potential mTKI beneficiaries and represents a potential therapeutic target to enhance mTKI efficacy and overcome resistance.

Background: T cell acute lymphoblastic leukemia is a fatal hematological malignancy. Despite the treatment progress, no targeted therapy is available currently, which urges to deepen the understanding of the underlying mechanism of T-ALL cell growth/survival. Autophagy is a conserved cellular process, which plays a dual role in human cancers. Nevertheless, many aspects of the involvement of autophagy in T-ALL are not fully understood.

Methods: T-ALL patient cells and normal control cells were subjected to RT‒qPCR analysis. Gene silence and overexpression was used to study the function of ATG4B and sestrin 3 (SESN3) in T-ALL cells. Atg4b deficient mice were used to study the role of Atg4b in normal hematopoietic cells and T cell development. The efficacy of S130, an ATG4B inhibitor to suppress T-ALL cell growth was evaluated in xenograft models.

Results: The results showed that the expression of several autophagy-related genes (especially ATG4B) was significantly higher in T-ALL patient cells than control cells. ATG4B ablation decreased autophagic flux and inhibited T-ALL cell growth. In contrast, Atg4b depletion had mild effects on normal hematopoiesis and T cell development. RNA-seq data and subsequent studies revealed a novel regulatory circuit of ATG4B and SESN3, and the results indicated that SESN3 hampered T-ALL cell growth via the inhibition of both mTOR/S6K/protein synthesis pathway and autophagy. Importantly, S130 exhibited anti-leukemia activity in xenograft models.

Conclusions: The present study demonstrates that a novel ATG4B-SESN3 regulatory circuit plays a crucial role in T cell leukemogenesis, which suggests that targeting ATG4B is a promising strategy for T-ALL treatment.