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.

Identification of autophagy-related genes as potential biomarkers for type 1 diabetes mellitus.

Background: Type 1 diabetes mellitus (T1DM) is a metabolic disease in which the autoimmune destruction of pancreatic islet ß-cells occurs. This study sought to investigate the role of autophagy-related genes and immune cells in the development of T1DM. Methods: We acquired the raw gene expression profiles of 302 T1DM and 422 normal control peripheral blood samples from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified using the Limma package, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. The Search Tool for the Retrieval of Interacting Genes/Proteins (https://string-db.org/) and Cytoscape autophagy genes were intersected with the DEGs for the immune cell analysis and the correlation analysis. Results: A total of 568 DEGs were identified in the T1DM and normal samples, of which 301 were upregulated and 267 were downregulated. The results of the functional and pathway enrichment analyses showed that the DEGs were closely associated with autophagy and immunity. Member RAS oncogene family (RAB11A), protein tyrosine phosphatase non-receptor type 11, lamin A/C, heat shock protein70, heat shock protein family A member 4, cluster of differentiation 8A, caspase 3 (CASP3), exportin 1, proto-oncogene, non-receptor tyrosine kinase, SMAD family member 4, and sirtuin 1 (SIRT1) were located at the center of the protein-protein interaction network as the core genes. The peripheral blood T cells were more elevated in the T1DM subjects than the normal subjects. RAB11A, CASP3, and SIRT1 are autophagy-associated genes. RAB11A and CASP3 were positively correlated with most immune cells, while SIRT1 was negatively correlated with most immune cells. Conclusions: Autophagy-related genes (i.e., RAB11A, CASP3, and SIRT1) and immune cells (i.e., T and B cells) may play important regulatory roles in the development of T1DM. Our findings provide novel insights into and potential targets for T1DM prediction and treatment.

Reinforcement of transvaginal repair using polypropylene mesh functionalized with basic fibroblast growth factor.

Numerous modifications have been developed over the past two decades seeking to improve the transvaginal repair in the pelvic organ prolapse (POP) by using polypropylene (PP) mesh implants. The hydrophobicity of PP, however, presents a great hindrance for translating potential technologies into viable clinical applications. In this study, by manipulating self-polymerization and strong adhesive characteristics of dopamine, we developed a facile method to enhance the transvaginal repair by modifying PP meshes with polydopamine (PDA), which allowed easy grafting of basic fibroblast growth factor (bFGF) onto the surface of PP. Such surface modification of PP meshes with bFGF was found to efficiently promote bioactivity without changing the morphology or mechanical properties of the PP meshes. Additionally, bFGF-modified PP meshes significantly promoted cell viability and adhesion compared to the unmodified PP. Ultimately, after three months of implantation, the bFGF-modified PP meshes exhibited improved tissue repair with greater degree of organization of deposited collagen, increased tensile strength and reduced inflammatory response. Overall, the surface-modified PP meshes will be highly practical as templates for transvaginal repair in the POP treatment.

Tumor-Triggered Controlled Drug Release from Electrospun Fibers Using Inorganic Caps for Inhibiting Cancer Relapse.

A smart, tumor-trigged, controlled drug release using inorganic "caps" with CO3 (2-) functional groups in electrospun fibers is presented for inhibiting cancer relapse. When the drug-loaded intelligent electrospun fibers encounter pathological acidic environments, the inorganic gates react with the acids and produce CO2 gas, which enables water penetration into the core of the fibers to induce rapid drug release.

Optimization of intrinsic and extrinsic tendon healing through controllable water-soluble mitomycin-C release from electrospun fibers by mediating adhesion-related gene expression.

To balance intrinsic and extrinsic healing during tendon repair is challenging in tendon surgery. We hypothesized that by mediating apoptotic gene and collagen synthesis of exogenous fibroblasts, the adhesion formation induced by extrinsic healing could be inhibited. With the maintenance of intrinsic healing, the tendon could be healed with proper function with no adhesion. In this study, we loaded hydrophilic mitomycin-C (MMC) into hyaluronan (HA) hydrosols, which were then encapsulated in poly(L-lactic acid) (PLLA) fibers by micro-sol electrospinning. This strategy successfully provided a controlled release of MMC to inhibit adhesion formations with no detrimental effect on intrinsic healing. We found that micro-sol electrospinning was an effective and facile approach to incorporate and control hydrophilic drug release from hydrophobic polyester fibers. MMC exhibited an initially rapid, and gradually steadier release during 40 days, and the release rates could be tuned by its concentration. In vitro studies revealed that low concentrations of MMC could inhibit fibroblast adhesion and proliferation. When lacerate tendons were healed using the MMC-HA loaded PLLA fibers in vivo, they exhibited comparable mechanical strength to the naturally healed tendons but with no significant presence of adhesion formation. We further identified the up-regulation of apoptotic protein Bax expression and down-regulation of proteins Bcl2, collage I, collagen III and α-SMA during the healing process associated with minimum adhesion formations. This approach presented here leverages new advances in drug delivery and nanotechnology and offers a promising strategy to balance intrinsic and extrinsic tendon healing through modulating genes associated with fibroblast apoptosis and collagen synthesis.

Self-coated interfacial layer at organic/inorganic phase for temporally controlling dual-drug delivery from electrospun fibers.

Implantable tissue engineering scaffolds with temporally programmable multi-drug release are recognized as promising tools to improve therapeutic effects. A good example would be one that exhibits initial anti-inflammatory and long-term anti-tumor activities after tumor resection. In this study, a new strategy for self-coated interfacial layer on drug-loaded mesoporous silica nanoparticles (MSNs) based on mussel-mimetic catecholamine polymer (polydopamine, PDA) layer was developed between inorganic and organic matrix for controlling drug release. When the interface PDA coated MSNs were encapsulated in electrospun poly(L-lactide) (PLLA) fibers, the release rates of drugs located inside/outside the interfacial layer could be finely controlled, with short-term release of anti-inflammation ibuprofen (IBU) for 30 days in absence of interfacial interactions and sustained long-term release of doxorubicin (DOX) for 90 days in presence of interfacial interactions to inhibit potential tumor recurrence. The DOX@MSN-PDA/IBU/PLLA hybrid fibrous scaffolds were further found to inhibit proliferation of inflammatory macrophages and cancerous HeLa cells, while supporting the normal stromal fibroblast adhesion and proliferation at different release stages. These results have suggested that the interfacial obstruction layer at the organic/inorganic phase was able to control the release of drugs inside (slow)/outside (rapid) the interfacial layer in a programmable manner. We believe such interface polymer strategy will find applications in where temporally controlled multi-drug delivery is needed.