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Abstract
Background Glioblastoma multiforme (GBM) is the most lethal form of brain cancer with a median survival of < 2 years despite treatment advances. Evidence indicates that GBM has been shown to reprogram immune responses to promote its progression. A predominant component of the immunosuppressive tumor microenvironment (TME) is glioblastoma-associated macrophages (GAM). GAM represents up to 50% of the bulk cell population in the GBM microenvironment and is crucial in tumor progression and persistence. We and others have shown the expression of the Ephrin A3 (EphA3) receptor in both tumor and GAM. We hypothesize that dual targeting of both the cancer cell and the immunosuppressive TME with EphA3-specific chimeric antigen receptor T (EphA3-CART) cells would enhance antitumor.
Methods We first investigated the expression of the EphA3 receptor on GBM patient-derived xenografts using immunohistochemistry (IHC) and Western blot. We then polarize primary monocyte or THP-1 monocytic cell line into M2-like macrophages and investigated the expression of the EphA3 receptor on both flow cytometry and then studied the effect of EphA3-CART against EphA3+ SNB-19 GBM cell line using the polarized macrophage. We also assessed the antitumor activity of the EphA3-CART in vivo. NSG mice were subcutaneously injected with 1 x 106 Luc+ SNB-19 GBM cell line and monitored for tumor growth using bioluminescence imaging (BLI) and/or tumor size using vernier calipers. At BLI of >1 x108 p/s and/or tumor volume of 200-300 mm3, mice were randomized and treated with 5 x 106 EphA3-CAR T cells. And were further monitored by both BLI and/or tumor size using vernier calipers.
Results Our immunohistochemical analysis data revealed EphA3 expression on 26.623% of the PDX representing the area of the human cancer tissue and was confirmed using the western blot analysis of the selected GBM PDX lines in our biobank (figure 1). EphA3-CART cells exhibited potent antitumor activity against EphA3+ GBM cell line SNB-19 (figure 2). M2 polarized macrophage marker expressing a high level of CD206 as well as EphA3 compared to M1 polarized macrophages. EphA3-CART cells were effective in eliminating M2 macrophages (figure 3) and ameliorated M2-induced CART cell inhibition. Finally, EphA3-CART cells exhibited superior tumor control in GBM xenografts compared to the control un-transduced T cells (UTD), (figure 4).
Conclusions In summary, we have developed EphA3-CART cells that target both the tumor cells and GAM within GBM TME which holds promise in improving CART antitumor activity within the immunosuppressive solid tumor milieu.
Results of Immunohistochemical analysis of patient derived xenograft (PDX) passaged in the nude mice. Photomicrograph (A) and the result of statical analayses (B) are shown. Immunohistochemical analyses demostrate high expression levels of GFAP, EphA3, CD11b, ARG1 and iNOS. The EphA3 expression was shown to occupy most area. Orginal magnification, x20; scale bar, 100 μm
Cytotoxicity assay; SNB-19 ZsG was cocultures with increasing ratios of either EphA3-CART cells or the un-transduced (UTD) T cell control for 72 hours. 2-way ANOVA, ****P<0.0001
Cytotoxicity assay: M2-polarised THP-1 monocyctic cells was cocultured with increasing ratios of either EphA3-CART cells or the un-transduced (UTD) T cell control 72 hours. 2-way ANOVA. ****P<0.0001
SNB-19 GBM cell line (1x106) was engrafted subcutaneously to into NSG mice and when turmoe burden reached 1x108 photon/second, mice were randomized to treatment of 5x106 cells EphA3-CART UTD. Turmors were monitored with bioluminescence imaging and vernier callipers. 2-way ANOVA. **P<0.05