Chimeric antigen receptor (CAR)-modified natural killer (NK) cells represent a promising immunotherapeutic modality for cancer treatment. However, their potential utilities have not been explored in hepatocellular carcinoma (HCC). Glypian-3 (GPC3) is a rational immunotherapeutic target for HCC. In this study, we developed GPC3-specific NK cells and explored their potential in the treatment of HCC. The NK-92/9.28.z cell line was established by engineering NK-92, a highly cytotoxic NK cell line with second-generation GPC3-specific CAR. Exposure of GPC3 HCC cells to this engineered cell line resulted in significant in vitro cytotoxicity and cytokine production. In addition, soluble GPC3 and TGF-β did not significantly inhibit the cytotoxicity of NK-92/9.28.z cells in vitro, and no significant difference in anti-tumor activities was observed in hypoxic (1%) conditions. Potent anti-tumor activities of NK-92/9.28.z cells were observed in multiple HCC xenografts with both high and low GPC3 expression, but not in those without GPC3 expression. Obvious infiltration of NK-92/9.28.z cells, decreased tumor proliferation, and increased tumor apoptosis were observed in the GPC3 HCC xenografts. Similarly, efficient retargeting on primary NK cells was achieved. These results justified clinical translation of this GPC3-specific, NK cell-based therapeutic as a novel treatment option for patients with GPC3 HCC.

Our previous study indicated that GPC3-targeted chimeric antigen receptor (CAR) T cell therapy has a high safety profile in patients with hepatocellular carcinoma (HCC). However, the response rate requires further improvement. Here, we analyzed the combined effect of GPC3-CAR T cells and sorafenib in both immunocompetent and immunodeficient mouse models of hepatocellular carcinoma. In immunocompetent mouse model, mouse CAR (mCAR) T cells induced regression of small tumors (approximately 130 mm 3 tumor volume) but had no effect on large, established tumors (approximately 400 mm 3 tumor volume). Sorafenib, at a subpharmacologic but not a pharmacologic dose, augmented the antitumor effects of mCAR T cells, in part by promoting IL12 secretion in tumorassociated macrophages (TAMs) and cancer cell apoptosis. In an immunodeficient mouse model, both subpharmacologic and pharmacologic doses of sorafenib had limited impacts on the function of human CAR (huCAR) T cells in vitro and showed synergistic effects with huCAR T cells in vivo, which can at least partially be ascribed to the upregulated tumor cell apoptosis induced by the combined treatment. Thus, this study applied two of the most commonly used mouse models for CAR T cell research and demonstrated the clinical potential of combining sorafenib with GPC3-targeted CAR T cells against HCC.

Adoptive immunotherapy based on chimeric antigen receptor–modified T (CAR-T) cells has been demonstrated as one of the most promising therapeutic strategies in the treatment of malignancies. However, CAR-T cell therapy has shown limited efficacy for the treatment of solid tumors. This is, in part, because of tumor heterogeneity and a hostile tumor microenvironment, which could suppress adoptively transferred T cell activity. In this study, we, respectively, engineered human- or murine-derived–armored glypican-3 (GPC3)–specific CAR-T cells capable of inducibly expressing IL-12 (GPC3-28Z-NFAT-IL-12) T cells. The results showed that GPC3-28Z-NFAT-IL-12 T cells could lyse GPC3+ tumor cells specifically and increase cytokine secretion compared with GPC3-28Z T cells in vitro. In vivo, GPC3-28Z-NFAT-IL-12 T cells augmented the antitumor effect when encountering GPC3+ large tumor burdens, which could be attributed to IL-12 increasing IFN-γ production, favoring T cells infiltration and persistence. Furthermore, in immunocompetent hosts, low doses of GPC3-m28Z-mNFAT-mIL-12 T cells exerted superior antitumor efficacy without prior conditioning in comparison with GPC3-m28Z T cells. Also, mIL-12 secretion decreased regulatory T cell infiltration in established tumors. In conclusion, these findings demonstrated that the inducible expression of IL-12 could boost CAR-T function with less potential side effects, both in immunodeficient and immunocompetent hosts. The inducibly expressed IL-12–armored GPC3–CAR-T cells could broaden the application of CAR-T–based immunotherapy to patients intolerant of lymphodepletion chemotherapy and might provide an alternative therapeutic strategy for patients with GPC3+ cancers.

Chimeric antigen receptor modified T cells (CAR-T) therapy is an emerging immunotherapy against malignancies. However, only limited success was obtained in solid tumors. Polyinosinic-polycytidylic acid (poly I:C), ligand of TLR3, mediates innate immune and adaptive immune and shows broad antitumor effect on many types of cancer. In the present study, we combined EGFRvIII-targeted CAR-T cells with poly I:C treatment and evaluated the synergic antitumor effect in vitro and in immunocompetent mice bearing subcutaneous colon or orthotopic breast cancer xenografts. Poly I:C significantly promoted more IL-2 and IFN γ production as well as higher lytic activity of CAR-T cells. Upon systemic administration in vivo , CAR-T cells obviously suppressed tumor growth, and poly I:C significantly enhanced the suppression. Further study showed that poly I:C exerted antitumor effect dependent on type I IFNs. In addition, poly I:C decreased myeloid-derived suppressor cells (MDSC) number in peripheral blood and spleen, and attenuated the immunosuppressive activity of MDSC on proliferation and cytolytic function of CAR-T. Depletion of MDSC with anti-Gr1 Ab further increased the antitumor effect of CAR-T cells plus poly I:C treatment. In conclusion, CAR-T treatment combined with intratumoral delivery of poly I:C resulted in synergistic antitumor activity. We thus provide a rationale to translate this immunotherapeutic strategy to solid tumors.

The intense inhomogeneous magnetic fields acting on the diamagnetic materials naturally present in cells can generate strong magnetic forces. We have developed a superconducting magnet platform with large gradient high magnetic field (LG-HMF), which can produce three magnetic force fields of -1360, 0, and 1312 T(2)/m, and three corresponding apparent gravity levels, namely 0, 1, and 2-g for diamagnetic materials. In this study, the effects of different magnetic force fields on osteoblast-like cells (MG-63 and MC3T3-E1) viability, microtubule actin crosslinking factor 1 (MACF1) expression and its association with cytoskeleton were investigated. Results showed that cell viability increased to different degrees after exposure to 0 or 1-g conditions for 24 h, but it decreased by about 30% under 2-g conditions compared with control conditions. An increase in MACF1 expression at the RNA or protein level was observed in osteoblast-like cells under the magnetic force field of -1360 T(2)/m (0-g) relative to 1312 T(2)/m (2-g). Under control conditions, anti-MACF1 staining was scattered in the cytoplasm and partially colocalized with actin filaments (AFs) or microtubules (MTs) in the majority of osteoblast-like cells. Under 0-g conditions, MACF1 labeling was concentrated at perinuclear region and colocalization was not apparent. The patterns of anti-MACF1 labeling on MTs varied with MTs' changing under LG-HMF environment. In conclusion, LG-HMF affects osteoblast-like cell viability, MACF1 distribution, expression, and its association with cytoskeleton to some extent.

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Our recent clinical study demonstrated that glypican-3 (GPC3)-specific chimeric antigen receptor-modified T (CAR-T) cells are a promising treatment for hepatocellular carcinoma (HCC). However, the interaction of programmed cell death 1 (PD-1) and PD-L1-mediated T-cell inhibition is involved in immune evasion in a wide range of solid tumors, including HCC. To overcome this problem, we introduced a fusion protein composed of a PD-1 extracellular domain and CH3 from IgG4 into GPC3-specific CAR-T cells (GPC3-28Z) to block the PD-1/PD-L1 pathway. GPC3-specific CAR-T cells carrying the PD-1-CH3 fusion protein (sPD1) specifically recognized and lysed GPC3-positive HCC cells. The proliferation capacity of GPC3-28Z-sPD1 T cells after weekly stimulation with target cells was much higher than that of control GPC3-28Z T cells. Additionally, the coexpression of sPD1 could protect CAR-T cells from exhaustion when incubated with target cells, as phosphorylated AKT and Bcl-xL expression levels were higher in GPC3-28Z-sPD1 T cells than in GPC3-28Z cells. Importantly, in two HCC tumor xenograft models, GPC3-28Z-sPD1 T cells displayed a significantly higher tumor suppression capacity than GPC3-28Z T cells. In addition, an increased number of CD3 T cells in the circulation and tumors and increased granzyme B levels and decreased Ki67 expression levels in the tumors were observed in the mice treated with GPC3-28Z-sPD1 T cells. Together, these data indicated that GPC3-specific CAR-T cells carrying sPD1 show promise as a treatment for patients with HCC.

The diamagnetic levitation as a novel ground-based model for simulating a reduced gravity environment has been widely applied in many fields. In this study, a special designed superconducting magnet, which can produce three apparent gravity levels (0, 1, and 2 g), namely high magneto-gravitational environment (HMGE), was used to simulate space gravity environment. The effects of HMGE on osteoblast gene expression profile were investigated by microarray. Genes sensitive to diamagnetic levitation environment (0 g), gravity changes, and high magnetic field changes were sorted on the basis of typical cell functions. Cytoskeleton, as an intracellular load-bearing structure, plays an important role in gravity perception. Therefore, 13 cytoskeleton-related genes were chosen according to the results of microarray analysis, and the expressions of these genes were found to be altered under HMGE by real-time PCR. Based on the PCR results, the expressions of WASF2 (WAS protein family, member 2), WIPF1 (WAS/WASL interacting protein family, member 1), paxillin, and talin 1 were further identified by western blot assay. Results indicated that WASF2 and WIPF1 were more sensitive to altered gravity levels, and talin 1 and paxillin were sensitive to both magnetic field and gravity changes. Our findings demonstrated that HMGE can affect osteoblast gene expression profile and cytoskeleton-related genes expression. The identification of mechanosensitive genes may enhance our understandings to the mechanism of bone loss induced by microgravity and may provide some potential targets for preventing and treating bone loss or osteoporosis.