Yeku et al used CAR-T cells to autocrine IL-12 to make CAR-T cells have stronger proliferation capacity and persistence in the body.21 Adachi et al designed CAR-T cells that secrete IL-7 and CCL19 at the same time and proved that they not only make CAR-T cells more proliferative but also increase the tumor infiltration ability of CAR-T cells.22 However, these methods also have certain limitations. have long-lasting tumor killing and proliferative capabilities. Moreover, animal experiments verified that enhanced MUC1-CAR-T cells had significant antitumor function and a prolonged half-life by subcutaneous transplantation models of esophageal cancer and PDX models of esophageal cancer, in vivo. Conclusion These results indicated that enhanced MUC1-CAR-T CZ415 cells have a significant cytotoxic effect on esophageal cancer, and may likely to provide a novel strategy for the treatment of esophageal cancer. Keywords: JAK-STAT, MUC1, esophageal cancer, chimeric antigen receptor-T cells, CAR-T cell Background Esophageal LAMP3 cancer (EC) is one of the most common malignancies in China, and the main histological CZ415 category is esophageal squamous cell carcinoma (ESCC).1 Although esophagostomy is the most effective treatment for ESCC patients at present, the long-term survival is quite low and unsatisfactory with a relative overall five-year relative survival rate of only 20C30%.2 In addition, more than half of all postoperative patients relapse within 2C3 years, among which almost 80% eventually die due to cancer recurrence.3,4 Therefore, novel treatment strategies are of utmost importance to improve this condition. In recent years, chimeric antigen receptor-modified T cells (CAR-T) therapy have demonstrated remarkable efficacy against several malignancies, especially blood tumors, and represent a new type of cancer treatment.5 The FDA has approved two types of CAR-T cell products for the treatment of hematological tumors.6 Although CAR-T therapy performs well in CZ415 hematological tumors, they have not shown similar efficacy in the treatment of other cancers.7 Due to the suppressive tumor microenvironment, good infiltration and existence of CAR-T cells in solid tumors is a challenge, and on the other hand, the antitumor T cell function is insufficient activated.8,9 Studies have shown that, except for T cell receptor engagement (signal 1), and co-stimulation (signal 2), cytokine engagement (signal 3) also played a key role in optimal T cell activation for anti-tumor function, which was probably lost in second-generation CAR-T cells.10 The cytokines of the c family, such as IL-2, IL-7, IL-15 and IL-21, all transmit signals through the JAK-STAT pathway, and these cytokines are necessary for the activation and proliferation of T cells and NK cells. Kagoya et al recently developed new-generation CAR-T cells, which encode a truncated cytoplasmic domain from the interleukin (IL)-2 receptor and a STAT3-binding tyrosine-X-X-glutamine (YXXQ) motif at the C-terminus of the CAR structure, together with the first T cell receptor (TCR) and second so-stimulatory signaling domain. This new-generation CAR-T cells have proven to significantly enhance the anti-tumor activity of CAR-T cells.11 Based on the above-mentioned CAR-T cell design, we aimed to design enhanced CAR-T cells to effectively and specifically eliminate esophageal cancer. MUC1, a CZ415 200 kDa, complex glycoprotein, has two subtypes: a transmembrane type and a secretory type.12 MUC1 is always overexpressed in various malignancies including EC.13 In a previous study, carcinoma and pericarcinomatous tissues of 108 ESCC patients were analyzed via immunohistochemical staining, which showed that tissue of 70 ESCC patients showed high expression levels of MUC1. In addition, MUC1 was significantly higher expressed in ESCC tissue compared to para-carcinoma esophageal tissue (65.4%:10.0%, p <0.01).14 Therefore, we designed a type of CAR-T cells that not only targeted MUC1 but also activated cytokineCcytokine interaction signaling, and verified the efficiency of this enhanced CAR-T cell on EC. The results showed that enhanced MUC1-CAR-T cells had a significant antitumor ability against EC cells, and that the antitumor effects were much better when compared to traditional MUC1-CAR-T cells. Furthermore, in vivo, enhanced MUC1-CAR-T cells survived longer in mice, which improved the efficiency of treatment and reduced relapses. These findings showed that enhanced MUC1-CAR-T cells have significant antitumor activity against EC, can overcome the limitations of traditional CAR-T cells for solid tumors, and provide novel strategies for the treatment of EC. Methods Cell Lines and Culture Conditions After obtaining consent from Xinjiang Uygur Autonomous Region Peoples Hospital, fresh blood was obtained from healthy volunteers. Healthy volunteers provided written informed consent in compliance with the Declaration of Helsinki. Peripheral blood mononuclear cells (PBMC) were isolated from blood by isopycnic gradient centrifugation using Lymphoprep TM (Solarbio, Beijing, China), and then sorted T cells through magnetic beads of human T cell subtype CD3 + (Miltenyi Biotec Inc, Auburn, CA, USA). Isolated T cells were resuspended and cultured in XCVIVO15 medium (Lonza, Basel, Switzerland) supplemented with 5% human AB serum (Valley Biomedical Inc, Winchester, VA, USA), 10 mM N-acetyl L-cysteine (Sigma Aldrich, St. Louis, MO, USA) and 300 U/mL human IL-2 (PeproTech, Rocky Hill, CT, USA). EC cells (Eca-109, TE10, TE13, and OE19.