GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy.

BACKGROUND: Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary treatment that has become a mainstay of advanced cancer treatment. Conventional glypican-3 (GPC3)-CAR-T cells have not produced ideal clinical outcomes in advanced hepatocellular carcinoma (HCC), and the mechanism is unclear. This study aims to investigate the clinical utility of novel GPC3-7-19-CAR-T cells constructed by our team and to explore the mechanisms underlying their antitumor effects. METHODS: We engineered a novel GPC3-targeting CAR including an anti-GPC3 scFv, CD3ζ, CD28 and 4-1BB that induces co-expression of IL-7 at a moderate level (500 pg/mL) and CCL19 at a high level (15000 pg /mL) and transduced it into human T cells. In vitro, cell killing efficacy was validated by the xCELLigence RTCA system, LDH nonradioactive cytotoxicity assay and was confirmed in primary HCC organoid models employing a 3D microfluid chip. In vivo, the antitumor capacity was assessed in a humanized NSG mouse xenograft model. Finally, we initiated a phase I clinical trial to evaluate the safety and effect of GPC3-7-19-CAR-T cells in the clinic. RESULTS: GPC3-7-19-CAR-T cells had 1.5-2 times higher killing efficiency than GPC3-CAR-T cells. The tumor formation rates in GPC3-7-19-CAR-T cells treated model were reduced (3/5vs.5/5), and the average tumor volumes were 0.74 cm3 ± 1.17 vs. 0.34 cm3 ± 0.25. Of note, increased proportion of CD4+ TEM and CD8+ TCM cells was infiltrated in GPC3-7-19-CAR-T cells group. GPC3-7-19-CAR-T cells obviously reversed the immunosuppressive tumor microenvironment (TME) by reducing polymorphonuclear (PMN)-myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells infiltration and recruiting more dendritic cells (DCs) to HCC xenograft tumor tissues. In one patient with advanced HCC, GPC3-7-19-CAR-T-cell treatment resulted in tumor reduction 56 days after intravenous infusion. CONCLUSIONS: In conclusion, GPC3-7-19-CAR-T cells achieved antitumor effects superior to those of conventional GPC3-CAR-T cells by reconstructing the TME induced by the dominant CD4+ TEM and CD8+ TCM cell subsets. Most importantly, GPC3-7-19-CAR-T cells exhibited good safety and antitumor efficacy in HCC patients in the clinic. ► Novel GPC3-7-19-CAR-T cells designed with mediate level of IL-7 secretion and high level of CCL19 secretion, which could recruit more mature DCs to assist killing on GPC3+HCCs. ►DC cells recruited by CCL19 could interact with CD4+ T cells and promote the differentiation of CD4+TEFF cells into CD4+TEM and CD8+TCM subsets, leading a better anti-tumor effect on GPC3+HCCs. ►Compared with conventional GPC3-CAR-T, GPC3-7-CCL19-CAR-T cells could reverse tumor immunosuppressive microenvironment by reducing PMN-MDSC and Treg cell infiltration.

Lipidomics and Transcriptomics Differ Liposarcoma Differentiation Characteristics That Can Be Altered by Pentose Phosphate Pathway Intervention.

Liposarcoma (LPS) is a rare and heterogeneous malignancy of adipocytic origin. Well-differentiated liposarcoma (WDLPS) and dedifferentiated liposarcoma (DDLPS) are two of the most common subtypes, showing similar genetic characterizations but distinct biological behaviors and clinical prognosis. Compared to WDLPS, DDLPS is more aggressive and has the potential of metastasis, as the malignant adipocytic tumor's metabolic changes may have taken place during the tumorigenesis of LPSs. Therefore, to investigate the lipid alterations between the two subtypes, high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) based untargeted lipidomic analysis was performed onto LPS tissues from 6 WDLPS and 7 DDLPS patients. The lipidomic analysis showed the upregulated phosphatidylcholines and phosphoethanolamines in DDLPS, and the upregulated triglycerides and diglycerides in WDLPS, which might be due to the uncompleted adipocytic dedifferentiation leading to such tumorigenesis. Such a finding was also confirmed by the similarity comparison of two LPS subtypes to the transcriptome of stromal vascular fraction at different differentiation stages. Transcriptomic analysis also demonstrated that metabolic pathways including the pentose phosphate pathway (PPP) were upregulated in WDLPS compared to DDLPS. Therefore, the cell line LPS853 was treated with the PPP inhibitor 6-aminonicotinamide ex vivo and the proliferation and invasion of LPS853 was significantly promoted by PPP inhibition, suggesting the potential role of PPP in the development and differentiation of LPS. In conclusion, this study described the altered lipid profiles of WDLPS and DDLPS for the first time, revealing the different differentiation stages of the two subtypes and providing a potential metabolic target for LPS treatment.

Immunotherapy for liposarcoma: emerging opportunities and challenges.

Liposarcoma (LPS) is a rare adipocyte-derived malignancy accounting for 20% of all soft tissue sarcomas. Although surgery and chemotherapy are the standard treatment for LPS, the large tumor burden and high recurrence rate make it difficult to treat, especially when the disease progresses. With the progress of immunotherapies in other tumors such as melanoma and lung cancer, interest has been risen in exploring immunotherapy for LPS. This review discusses the understanding of the tumor microenvironment of LPS; the current status of immunotherapy in LPS, including immune checkpoint inhibitors, adoptive cell therapy, cancer vaccines, oncolytic viruses and combination therapies; and the future directions for exploiting strategies to make the effect of immunotherapy stronger and more durable.

Liposarcoma is a rare type of malignant tumor with no effective treatment. Immunotherapy is a new kind of treatment that functions by activating the immune system to kill tumors cells. It has gained significant progress in other cancer types. This review discusses its exploration and application in liposarcoma.

Intratumoral CD73: An immune checkpoint shaping an inhibitory tumor microenvironment and implicating poor prognosis in Chinese melanoma cohorts.

Background: As a novel immune checkpoint, CD73 has been reported to play prominent roles in several malignancies. However, the significance of CD73 in melanoma remains ambiguous. This study sought to reveal the impact of CD73 on the tumor microenvironment (TME) and patients' prognosis, and to investigate whether CD73 could be a therapeutic target in Chinese melanomas, which were dominated by acral and mucosal subtypes. Methods: Two independent Chinese cohorts of 194 patients with melanoma were enrolled. CD73 and PD-L1 expression as well as CD8+ and CD56+ cell infiltrations were evaluated by immunohistochemistry in 194 resected melanoma samples. Clinical outcomes of patients were assessed utilizing the Kaplan-Meier plotter and Cox proportional hazard analysis. RNA-seq data was obtained from TCGA database. Gene set functional annotations were performed based on GO, KEGG and GSEA analysis. CIBERSORT, ssGSEA and TIMER were used to explore the association between CD73 and immune infiltration. These findings were validated by establishing tumor xenograft model, and functions of tumor-infiltrating immune cells were examined by flow cytometry and immunofluorescence. Results: High CD73 expression showed poorer clinical outcomes and was identified as an independent prognostic indicator for survival in two cohorts. Expression of CD73 was more prevalent than PD-L1 in Chinese melanoma cohorts (54.6% vs 23.2%). Co-expression of both immune checkpoints was infrequent (12.9%) in melanoma, and 54.4% of PD-L1 negative cases showed elevated expression of CD73. CD73high tumors showed a microenvironment with fewer CD8+ T cells and CD56+ NK cells infiltration, which displayed a dysfunctional phenotype. With the treatment of CD73 inhibitor APCP, the amount of CD8+ T cells and CD56+ NK cells infiltrated in tumors was elevated and the immunosuppressive effect of CD73 was eliminated. Conclusions: High CD73 expression was associated with an inhibitory TME and adverse clinical outcomes of melanoma. In comparison to PD-L1, CD73 was more prevalent and possessed more definite prognostic significance. Therefore, it may serve as a prognostic indicator and immunotherapeutic target next to PD-L1 in melanoma for Chinese population.

Long Non-Coding RNA LINC01410 Promoted Tumor Progression via the ErbB Signaling Pathway by Targeting STAT5 in Gallbladder Cancer.

OBJECTIVES: Long non-coding RNAs (lncRNAs) have been recently emerging as crucial molecules in multiple human cancers. However, their expression patterns, roles as well as the underlying mechanisms in gallbladder cancer (GBC) remain largely unclear. MATERIALS AND METHODS: The expression of lncRNAs in GBC was downloaded from GEO database. Quantitative real-time polymerase chain reaction (qRT-PCR) and RNA in situ hybridization (ISH) were used to detect the expression of lncRNAs in GBC tissues. The full-sequence of LINC01410 was determined by RACE assay. Subcellular distribution of LINC01410 was examined by nuclear/cytoplasmic RNA fractionation analysis. Loss- and gain-of-function experiments were conducted to explore the biological functions of LINC01410 in vitro and in vivo. RNA pull-down, RNA immune-precipitation (RIP), and Western blot assay were conducted to investigate the mechanisms underlying the biological function of LINC01410 in GBC. RESULTS: LINC01410 was significantly upregulated in the GBC tissues compared to adjacent non-tumor tissues. High LINC01410 expression was significantly associated with poor prognosis of GBC patients. We identified LINC01410 to be 2,877 bp in length and mainly localized in the cytoplasm of GBC cells. Overexpression of LINC01410 promoted GBC cell proliferation, migration, and invasion in vitro and GBC progression in vivo, whereas LINC01410 downregulation rescued these effects in vitro. From RNA pull-down and RIP assay, we identified that STAT5 was a critical downstream target of LINC01410. Furthermore, ErbB signaling pathway was involved in the malignant phenotypes of GBC mediated by LINC01410. CONCLUSIONS: Our results suggested that LINC01410 was an important lncRNA that promoted GBC progression via targeting STAT5 and activating ErbB signaling pathway.

Development and Validation of the Nomograms for Predicting Overall Survival and Cancer-Specific Survival in Patients With Synovial Sarcoma.

Background: The study aimed to build and validate practical nomograms to predict overall survival (OS) and cancer-specific survival (CSS) for patients with synovial sarcoma (SyS). Methods: A total of 893 eligible patients confirmed to have SyS between 2007 and 2015 were selected from the Surveillance, Epidemiology, and End Results (SEER) database. Patients were randomly divided into the training cohort (n = 448) and validation cohort (n = 445). Clinically independent prognostic and important factors were determined according to the Akaike information criterion in multivariate Cox regression models when developing the nomograms with the training cohort. The predictive accuracy of nomograms was bootstrapped validated internally and externally with the concordance index (C-index) and calibration curve. Decision curve analysis (DCA) was performed to compare the clinical usefulness between nomograms and American Joint Commission on Cancer (AJCC) staging system. Results: Two nomograms shared common indicators including age, insurance status, tumor site, tumor size, SEER stage, surgery, and radiation, while marital status and tumor site were only included into the OS nomogram. The C-index of nomograms for predicting OS and CSS was 0.819 (0.873-0.764) and 0.821 (0.876-0.766), respectively, suggesting satisfactory predictive performance. Internal and external calibration curves exhibited optimal agreement between the nomogram prediction and the actual survival. Additionally, DCA demonstrated that our nomograms had obvious superiority over the AJCC staging system with more clinical net benefits. Conclusions: Two nomograms predicting 3- and 5-year OS and CSS of SyS patients were successfully constructed and validated for the first time, with higher predictive accuracy and clinical values than the AJCC staging system regarding OS and CSS.