| BACKGROUNDGastric cancer (GC) is one of the most common malignant tumors worldwide, and threatens human beings' health and lives seriously. There is still lack of effective treatments for patients with advanced GC (AGC) to improve their survival time. Duo to the disadvantages caused by nonspecific antitumor activity, such as high recurrence rate and severe adverse events, the predominant therapeutics for AGC including chemotherapy and radiotherapy has been criticized in recent years. As the development of molecular biological technology, targeted therapy is becoming an increasingly hot spot in the research field of cancer treatment.Chimeric antigen receptor (CAR) T-cell immunotherapy is a new way of targeted therapy in which the CAR-redirected T cells, expressing engineered receptors specific to a particular antigen, are reintroduced into patients and elicit an effective antitumor immune response. CAR molecule is composed of four major parts: extracellular antigen-recognition domain, hinge region, transmembrane region and intracellular signaling moiety. To enhance the activation and proliferation of CAR T cell, second- and third-generation, with more costimulatory molecules have been developed. Although this strategy has been proven to be effective in treatment of hematological malignancies and presents many features, such as high specificity and MHC-independent effects, its potential clinical application in solid tumors is hampered by concerns over its safety and efficacy.Human epidermal growth factor receptor 2 (HER2) proteins are overexpressed in a high proportion of GC cases and affect the maintenance of cancer stem cell (CSC)subpopulations, which are used as targets for the clinical treatment of patients with HER2-positive GC. Positive clinical outcomes, obtained using the targeted agent trastuzumab, suggest that the surface antigen HER2 may serve as a target for CAR T cell therapy.OBJECTIVESIn this study, we generated a novel type of CAR protein, targeting antigen HER2, which are encoded by genes harboring HER2scFv-CD8-CD137-CD3? sequences. Then to observe the phenotypic features of CART-HER2 cells and to evaluate the antitumor activity of CART-HER2 cells against GC cells and gastric CSCs in vitro and xenotransplanted tumors in vivo.METHODS1. To transduce HER2scFv-CD8-CD137-CD3? transgene into T cells by lentivirus and to evaluate the transfection efficiency and phenotypic features by flow cytometry.2. To assess the surface expression of HER2 in GC cells by flow cytometry and to silence HER2 expression via transduction of a levtivirus-mediated short hairpin RNA.3. To verify the specific activity and cytotoxicity of CART-HER2 cell against GC cells in vitro using cytokines detection assays and lactate dehydrogenase (LDH) release assays,respectively.4. To obtain gastric CSCs by in vitro sphere-forming assays and to verify the killing activity of CART-HER2 cells against CSC subpopulations by co-culture assays.5. Use BALB/c nude mice and HER2 positive GC cells or gastric CSCs to establish the subcutaneous xenotransplanted tumor model.6. To evaluate the antitumor activity of CART-HER2 cells against xenografts by measuring the mean tumor volume (TV) and the mean tumor weight (TW).7. To determine the persistence and homing ability of CART-HER2 cells, we use qPCR to measure the copy numbers of CAR in the peripheral blood and tumor samples of mice and use immunohistochemical (IHC) staining for anti-CD3 to observe the number of infiltrating T cells under microscope, respectively.RESULTS1. The CAR transgene was successfully transduced into T cells, and the mean transfection efficiency of CART-HER2 cells was 34.22% ± 4.00%. CAR T cells mainly comprised a large population of CD8+CD56_ cells (72.55% ± 7.26%) and a small fraction of CD45RO+CD62L+CCR7+ cells (23.35% ±7.59%).2. HER2 was highly expressed in GC cell lines, with percentages ranging from 84.39% to 98.60%, however the primary GC cells exhibited a percentage of 56.80% and 2.48%,respectively. The expression positive rates of HER2 in N87-HER2KD cells and 7901-HER2KD cells were 41.01% and 56.00%, respectively.3. After the incubation, the levels of cytokines released by CART-HER2 cells, including IL-2, IFN-y, IL-4, GM-CSF, TNF-a, and Granzyme B, were significantly elevated in the supernatants of HER2 positive GC cells compared with those of mock T cells and NT T cells. CART-HER2 cells also exhibited higher average killing activity against HER2 positive GC cells and HER2KD cells than did control cells.4. We successfully obtained the gastric CSCs. The rates of the surface expression of HER2 and CD44 in separated spheres were 73.5% and 98.6%, respectively. After 48h of co-culture, most of the spheres in the CAR group were disintegrated into small pieces.5. The subcutaneous xenotransplanted tumor model of HER2 positive GC cells or gastric CSCs was successfully established in BALB/c nude mice.6. The rates of tumor growth were considerably inhibited by treatment with CART-HER2 cells. The mean TV and TW were significantly lower than control group.7. The copy numbers of CAR were detected at a high level in the blood and tumor samples obtained after the mice were sacrificed on day 33, and remained at a detectable level for at least 56 days in the blood of the remaining mice. The IHC staining showed considerable increases in the recruitment of human CD3+ T cells in CAR group.CONCLUSIONS1. CAR protein can be stably expressed on the surface of T cells.2. After expansion by in vitro culture, the rate of CART-HER2 cells expressing the central memory phenotypes are significantly increased.3. CART-HER2 cells can be specifically activated by HER2 positive GC cells and effectively kill these tumor cells ex vivo.4. CART-HER2 cells exhibit effective and persistent antitumor activity against xenografts derived from HER2 positive GC and gastric CSCs in mice.5. CART-HER2 cells can significantly inhibit the growth of tumor spheres and effectively kill HER2 positive gastric CSCs. |
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