Anti-GPC3-CAR T Cells Suppress the Growth of Tumor Cells in Patient-Derived Xenografts of Hepatocellular Carcinoma

Abstract

Background: The lack of a general clinic-relevant model for human cancer is a major impediment to the acceleration of novel therapeutic approaches for clinical use. We propose to establish and characterize primary human hepatocellular carcinoma (HCC) xenografts that can be used to evaluate the cytotoxicity of adoptive chimeric antigen receptor (CAR) T cells and accelerate the clinical translation of CAR T cells used in HCC.

Methods: Primary HCCs were used to establish the xenografts. The morphology, immunological markers, and gene expression characteristics of xenografts were detected and compared to those of the corresponding primary tumors. CAR T cells were adoptively transplanted into patient-derived xenograft (PDX) models of HCC. The cytotoxicity of CAR T cells in vivo was evaluated.

Results: PDX1, PDX2, and PDX3 were established using primary tumors from three individual HCC patients. All three PDXs maintained original tumor characteristics in their morphology, immunological markers, and gene expression. Tumors in PDX1 grew relatively slower than that in PDX2 and PDX3. Glypican 3 (GPC3)-CAR T cells efficiently suppressed tumor growth in PDX3 and impressively eradicated tumor cells from PDX1 and PDX2, in which GPC3 proteins were highly expressed.

Conclusion: GPC3-CAR T cells were capable of effectively eliminating tumors in PDX model of HCC. Therefore, GPC3-CAR T cell therapy is a promising candidate for HCC treatment.

Keywords: CAR; PDX; T cells; cell therapy; hepatocellular carcinoma.

Figures

Figure 1

Characterization of patient-derived xenograft (PDX) model of hepatocellular carcinoma. (A) Patient-derived xenografts (PDXs) were established, and tumor growth was measured at a given time for each PDX xenograft. Tumor volume was calculated as (a × b2)/2, the length (a), and width (b) of tumor. (B,C) H&E and IHC staining tissue of primary tumor and PDXs. The xenograft demonstrates morphology, glypican 3, and AFP staining consistent with the original human tumor. Brown color indicates positive staining. IgG was used as a negative control. Scale bar is 50 μm. (D) mRNA expression level of tumor-related genes [MET (MET proto-oncogene, receptor tyrosine kinase), CTNNB1 (catenin beta 1), AXIN1 (axin 1), TP53 (tumor protein p53), RB1 (RB transcriptional corepressor 1), PTEN (phosphatase and tensin homolog), BCL2 (BCL2, apoptosis regulator), AFP (alpha fetoprotein), KRT19 (keratin 19), CDKN2A (cyclin-dependent kinase inhibitor 2A), CDKN1B (cyclin-dependent kinase inhibitor 1B), and CCND1 (cyclin D1)] in primary tumor and PDXs. Results represent mean ± SD of three individual experiments.

Figure 2

Construction of chimeric anti-glypican 3 (GPC3) vectors and generation of GPC3-chimeric antigen receptor (CAR) T cells. (A) Schematic representation of a lentiviral vector encoding the signal peptide, anti-GPC3 scFv, CD28 transmembrane domain, 4-1BB costimulatory endodomain, and CD3ζ signaling domain along with eGFP using 2A. (B) A representative of GPC3-CARs expression on human T cells transduced with lentivirus was analyzed using flow cytometry, which detected eGFP at days 7 and 14. (C) Transduction efficiency. Results represent mean ± SD of six individual experiments. No difference was detected between the percentages of GPC3-CAR T and Control CAR T. (D) The expansion of transduced T cells in vitro from day 0 to 21. Results represent mean ± SD of three individual experiments.

Figure 3

Phenotypic analysis and cytokines produced in glypican 3 (GPC3)-chimeric antigen receptor (CAR) T cells. (A) Flow cytometry comparison of the common surface phenotype of GPC3-CAR T cells (red line) at day 14 of culture with freshly isolated T cells (blue line). Black line represents the isotype control. Histogram overlays show 16 markers related to lymphocyte activation, differentiation, migration, adhesion, and exhaustion. (B) Interferon-γ and (C) interleukin-2 were secreted by the indicated modified CAR T cells co-cultured with hepatocellular carcinoma cell lines and A549 for 24 h. Results represent triplicates. ***P < 0.001 with T-test.

Figure 4

Cytotoxicity of glypican 3 (GPC3)-chimeric antigen receptor (CAR) T cells against targeted cells in vitro. (A) Cytotoxicity of GPC3-CAR T cells was assayed by coincubation with Huh-7-GL (GPC3+), HepG2-GL (GPC3+), and A549-GL (GPC3−) cells at E:T ratios ranging from 1:3 to 5:1. (B) Serial killing of GPC3-CAR cells against Huh-7-GL and HepG2-GL cells at E: T ratios of 1:1 in indicated durations. Results represent mean ± SEM.

Figure 5

Cytotoxicity of glypican 3 (GPC3)-chimeric antigen receptor (CAR) T cells against hepatocellular carcinoma cell lines in xenografts. (A,B) Growth curve of Huh-7 and HepG2 xenografts (n = 5) treated with the Control- or GPC3-CAR T cells at indicated time point (arrow). At the end of the experiment, the tumors treated with GPC3-CAR T cells were significantly smaller than those in the Control group. (C,D) Huh-7 and HepG2 tumor weights from the mice treated with CAR T cells at the end of the experiment, respectively. (E,F) GPC3-CAR T cells in tumors were significantly higher than Control-CAR T groups. Results represent mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 with T-test.

Figure 6

Glypican 3 (GPC3)-chimeric antigen receptor (CAR) T cells efficiently abolish growth of patient-derived xenografts (PDXs) of hepatocellular carcinoma. (A–C) Growth curve of PDX1, PDX2, and PDX3 (n = 5) treated with the Control- or GPC3-CAR T cells at indicated time point (arrow). At the end of the experiment, the tumors treated with GPC3-CAR T cells were significantly smaller than those in the Control group. (D–F) PDX1, PDX2, and PDX3 tumor weights from the mice treated with CAR T cells at the end of the experiment. (G–I) GPC3-CAR T cells in tumors were significantly higher than Control-CAR T groups. Results represent mean ± SD. ***P < 0.001 with T-test.