Mesothelin-based CAR-T cells exhibit potent antitumor activity against ovarian cancer.

BACKGROUND: Ovarian cancer (OC) is characterized by its rapid growth and spread which, accompanied by a low 5-year survival rate, necessitates the development of improved treatments. In ovarian cancer, the selective overexpression of Mucin-16 (MUC16, CA125) in tumor cells highlights its potential as a promising target for developing anti-tumor therapies. However, the potential effectiveness of CAR-T cell therapy that targets MUC16 in ovarian cancer cells is unknown. METHODS: The expression of MUC16 in viable OC cells was detected using immunofluorescence and flow cytometry techniques. A MSLN-CAR construct, comprising the MUC16-binding polypeptide region of mesothelin (MSLN), a CD8 hinge spacer and transmembrane domain, 4-1BB, and CD3ζ endo-domains; was synthesized and introduced into T cells using lentiviral particles. The cytotoxicity of the resultant CAR-T cells was evaluated in vitro using luciferase assays. Cytokine release by CAR-T cells was measured using enzyme-linked immunosorbent assays. The anti-tumor efficacy of the CAR-T cells was subsequently assessed in mice through both systemic and local administration protocols. RESULTS: MSLN-CAR T cells exhibited potent cytotoxicity towards OVCAR3 cells and their stem-like cells that express high levels of MUC16. Also, MSLN-CAR T cells were inefficient at killing SKOV3 cells that express low levels of MUC16, but were potently cytotoxic to such cells overexpressing MUC16. Moreover, MSLN-CAR T cells delivered via tail vein or peritoneal injection could shrink OVCAR3 xenograft tumors in vivo, with sustained remission observed following peritoneal delivery of MSLN-CAR T cells. CONCLUSIONS: Collectively, these results suggested that MSLN-CAR T cells could potently eliminate MUC16- positive ovarian cancer tumor cells both in vitro and in vivo, thereby providing a promising therapeutic intervention for MUC16-positive patients.

CD166-specific CAR-T cells potently target colorectal cancer cells.

Chimeric antigen receptor (CAR) T-cell therapy is emerging as an effective cancer treatment, such as for hematological malignancies, however its effectiveness as an approach to treat solid tumors, such as in colorectal cancer (CRC), remains to be better developed. One area of intense development has been in the identification and characterization of novel cancer-related ligand receptors for CAR design and evaluation. It is known that the CD6 receptors CD166 and CD318 are highly expressed in CRC, and several CAR-Ts have also been explored in preclinical and clinical studies for the treatment of CRC, with promising safety and efficacy findings. Here, we constructed a CAR based on the extracellular domain of CD6 and demonstrate its cytotoxic effect in target positive human CRC cell lines. Unexpectedly, we found that CD6-CAR-T cells targeted CD166 instead of CD318. Furthermore, CD6-CAR-T cells show robust cytotoxicity to CD166-positive cell lines in a dose-dependent manner with cytokine IFN-γ significantly released. Particularly, CD6-CAR-T cells show potent cytotoxicity targeting CRC cancer stem cells (CSCs), highlighting that CD6-CAR-T is a promising approach for the therapy of CRC.

The Multi-Omics Analysis of Key Genes Regulating EGFR-TKI Resistance, Immune Infiltration, SCLC Transformation in EGFR-Mutant NSCLC.

BACKGROUND: Lung cancer is a high-risk malignancy worldwide. The harboring of epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) makes EGFR-tyrosine kinase inhibitor (EGFR-TKI) an attractive therapeutic option. However, patients usually suffer the primary and secondary resistance to EGFR-TKI. And the molecular alteration is still not fully clear and needs further study. METHODS: The GEO database was utilized to find the differentially expressed genes (DEGs) in NSCLC profiles resistant to the 1st or 2nd generation EGFR-TKI. We analyzed the expression and pathway enrichment of hub genes, and the prognosis of EGFR mutant/wild-type lung adenocarcinoma (LUAD). Moreover, small cell lung cancer (SCLC) and TKI-resistant profiles were used to find common DEGs, and construct miRNA regulatory network. Analysis was performed of hub genes' related immune infiltration, drug sensitivity, and methylation. Further, we analyzed hub gene expression in EGFR-mutant LUAD and paracancerous tissue by qRT-PCR. RESULTS: A total of 107 DEGs were found related to TKI resistance. Eleven hub genes were obtained after visualization, of which 5 hub genes were co-expressed in SCLC with common miRNAs. Lower expression of SPP1 (hub gene) was associated with better survival in NSCLC. The immune infiltration analysis showed more CD4+ T cells in the resistant group with high expression of SPP1. SPP1 and CD44 promoters' methylations were independent prognostic factors of LUAD. And the expression level of SPP1 related to the sensitivity of EGFR-TKIs in multiple cancer cell lines. qRT-PCR validated the higher expression of SPP1 in EGFR-mutant LUAD than in normal tissue. CONCLUSION: Our study suggested that the upregulation of SPP1 might induce resistance to the 1st and 2nd generation EGFR-TKI, and influence tumor immune infiltration, resulting in poor survival. ZEB1, SPP1, MUC1, CD44, and ESRP1 might be molecular drivers of SCLC transformation of TKI resistance.

Down-regulation of RCC1 sensitizes immunotherapy by up-regulating PD-L1 via p27kip1 /CDK4 axis in non-small cell lung cancer.

In recent years, although Immune Checkpoint Inhibitors (ICIs) significantly improves survival both in local advanced stage and advanced stage of non-small cell lung cancer (NSCLC), the objective response rate of ICI monotherapy is still only about 20%. Thus, to identify the mechanisms of ICI resistance is critical to increase the efficacy of ICI treatments. By bioinformatics analysis, we found that the expression of regulator of chromosome condensation 1 (RCC1) in lung adenocarcinoma was significantly higher than that in normal lung tissue in TCGA and Oncomine databases. The survival analysis showed that high expression RCC1 was associated with the poor prognosis of NSCLC. And the expression of RCC1 was inversely related to the number of immune cell infiltration. In vitro, knockdown of RCC1 not only significantly inhibited the proliferation of lung adenocarcinoma cells but also increased the expression levels of p27kip1 and PD-L1, and decreased the expression level of CDK4 and p-Rb. In vivo, knockdown of RCC1 significantly slowed down the growth rate of tumour, and further reduced the volume and weight of tumour model after treated by PD-L1 monoclonal antibody. Therefore, RCC1 could up-regulate the expression level of PD-L1 by regulating p27kip1 /CDK4 pathway and decrease the resistance to ICIs. And this study might provide a new way to increase the efficacy of PD-L1 monoclonal antibody by inhibiting RCC1.

Effect of low dose of berberine on the radioresistance of cervical cancer cells via a PI3K/HIF-1 pathway under nutrient-deprived conditions.

Purpose: Radiotherapy (RT) is one of the major treatments of cervical cancer. Although the prognosis of clinical cervical cancer becomes better in recent years, some patients still suffer from the recurrence and metastasis. Insufficiency of glucose and oxygen supply could increase the radioresistance of cervical cancer cells through regulating hypoxia-inducible factor 1 (HIF-1) in tumor microenvironment and glucose metabolism. And, berberine can regulate HIF-1. However, how berberine regulates tumor microenvironment and radioresistance through HIF-1 remains to be elucidated.Materials and methods: The human HeLa cervical cancer cells were treated with berberine and radiation under the high and low concentrations of glucose and oxygen, respectively. The survival of cells was tested by CCK-8 assay and colony formation assay. We investigated the PI3K- and IDH3α-related pathway molecules that may regulate HIF-1α by qPCR and western blot. Differentially expressed genes (DEGs) were identified by integrating five related cohort profile datasets. Protein-protein interaction (PPI) network analyses of DEGs related to HIF-1α were conducted by using the STRING database and Cytoscape software.Results: Berberine dramatically damaged HeLa cells under hypoxic and low-glucose conditions compared with the normoxic and high-glucose conditions. The clonogenic assay indicated that the application of berberine decreased the number of colony counts compared to the negative control. Low doses of berberine might decrease the level of phospho-PI3K and HIF-1α under the nutrient-deprived conditions. Moreover, we found that most of the differentially expressed genes which were related to CDKN1B were the downstream molecules regulated by HIF-1α.Conclusion: The results indicated that berberine could dramatically overcome the low-glucose and hypoxia-induced radioresistance. And the regulation berberine on nutrition-deficient conditions might involve in PI3K/HIF-1 pathway. Thus, the interference of glucose metabolism by berberine might be an attractive method to eliminate radioresistant cells and improve radiotherapy efficacy.