br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion The success of CAR-T cell therapy is based on two major facts: one is that cancer cells express tumor-associated antigens (TAAs) on their surface that can be detected by the human immune system; the other is that the CAR molecules target these TAAs on cancer cells can be genetically engineered to be expressed in T cells. To overcome the obstacles facing CAR-T cell therapies in the treatment of solid tumors, it is crucial to identify novel TAAs and design unique CAR structures. Although multiple targets, such as Her2, mesothelin, and TEM8, have been used for CAR-T cell applications in pancreatic cancer and TNBC [36], [37], novel TAAs still need to be identified to develop a CAR-T cell therapy specific to solid tumors. The receptor tyrosine kinase AXL has been described as a relevant molecular marker and a key player in cancerous invasiveness, especially in TNBC [38]. To evaluate the potential of AXL as an anti-tumor target, we detected AXL expression in tumor cell lines and patient tissue and found aberrant overexpression of AXL in various solid tumors, such as breast cancer, RCC, and pancreatic cancer cells. Among the tumor cell lines and breast cancer patient tissue samples, the TNBC cell line and tissue samples expressed the highest level of AXL. All these data suggest that AXL is a candidate target for CAR-T therapy of solid tumors, especially in TNBC. AXL/GAS6 signaling has been shown to mediate tumor angiogenesis and promote growth [39]. Other than small molecular inhibitors, Protease Inhibitor Library targeting AXL have significantly improved survival rates in preclinical tumor xenograft models and have therefore advanced into clinical trials [40]. YW327.6S2 showed high anti-tumor activity in A549 non-small-cell lung cancer (NSCLC) and MDA-MB-231 breast cancer models [41]. Another anti-AXL antibody 20G7-D9 showed inhibition of tumor growth in TNBC patient-derived xenograft (PDX) models and suppressed the AXL-dependent Epithelial-Mesenchymal Transition (EMT) and metastasis formation [38]. Compared to antibody therapy, CAR-T cell therapy has the advantage of anti-tumor activity at a very low effector-to-target ratio. In order to obtain the same level of cytotoxicity as CAR-T cells, much higher doses of antibodies would need to be administered, which would cause adverse off-target effects. From the positive results observed with AXL antibody therapy, we predict a promising future for the AXL-CAR-T cell treatment against solid tumors. In this study, we used a third-generation CAR, which includes CD28 and CD137 (4/1BB) co-stimulatory intracellular signaling domains, in order to obtain improved persistence and cytotoxicity [42]. Our data from the AXL-negative tumor cell line MCF-7 showed that CAR-T cells have stronger cytotoxicity than non-transduced T cells. Although CAR-T cells do not have a target to bind in MCF-7 cells, third-generation CAR molecules still endowed greater anti-tumor potency. Traditionally, radioactive chromium (Cr51) release assays [43] and lactose dehydrogenase (LDH) release assays [44] are the most common methods to evaluate the cytotoxicity of CAR-T cells. However, the Cr51 release assay presents disadvantages, like the harmful effects of radioactivity to the scientist and the environment, additional cost of radioactive waste handling, and special research equipment requirements. For these reasons, the LDH assay was preferred for cytotoxicity measurements. However, since both target cells and CAR-T cells secrete LDH, complicated control groups must be included in every experiment. Furthermore, the sensitivity of the assay is poor. In this study, we introduced luciferase into target tumor cell lines to measure cytotoxicity in vitro. In the co-culture system, only target cells express luciferase, allowing us to simply measure and compare the luminescence to accurately evaluate viable target cells. The sensitivity of the luciferase assay is also much higher than that of the LDH assay. This simple and sensitive luciferase assay system for cytotoxicity measurements lays the foundation for high throughput CAR-T cell screening. Furthermore, the luciferase-based assay can detect other types of cytotoxicity induced by immune-therapeutic cells or agents. The same concepts have been addressed in a recently published article by Matta H [45].