Pancreatic cancer (PC) is one of the deadliest diseases of human digestive malignancies. Despite the recent advances in surgery and chemotherapy, the 5-year survival rate of PC continues to be less than 10%. As a promising tumor therapy,Chimeric antigen receptor T cell (CAR-T), however, performed poorly in PC treatment and need to be further updated. In our study, on the basis of our previous research, we use anti-MSLN CAR-T as effector cell and explore the different effects and mechanism of gut microbiota (PC or healthy control) on anti-MSLN CAR-T treatment. Firstly, we detect the differences of gut microbiota and T cell cholesterol metabolism in PC and healthy control by means of 16S-rRNA,PCR, western blot and ELISA; explore the different effects of gut microbiota on the subtype of T cells; and analyze the relationships between intestinal flora composition and T cell cholesterol metabolism or subtype changes by means of Spearman's correlation. Secondly, we also explore the different effects of gut microbiota on the proliferation, migration, subtype, inflammatory cytokines expression and anti-tumor effector function of anti-MSLN CAR-T cells by means of flow cytometry and cytotoxicity assay. Thirdly, we discuss the different expression of cholesterol esterification enzyme 1 (ACAT-1) and other core genes of cholesterol metabolism in anti-MSLN CAR-T. Lastly, we evaluate the effects of different gut microbiota on the treatment of PC by anti-MSLN CAR-T cells in NSG mouse model of subcutaneous PC transplantation and liver metastasis. Through the above experiments, a new theoretical basis is provided in which gut microbiota regulates the subtype and anti-tumor function of anti-MSLN CAR-T by ACAT-1 expression. Furthermore, our findings, which demonstrate the relationship of gut microbiota and CAR-T cell, may be translatable for the treatment of other solid tumors like PC.

1. Inclusion criteria and exclusion criteria for the experimental group of this project

Inclusion Criteria:

1. the patients who are confirmed by imaging, pathology and body fluid biopsy to have metastasized pancreatic cancer and can not be cured by operation; or the patients who recover well one month after operation but still have residual focus, recurrence or metastasis.
2. age ≥ 30 and ≤ 75
3. the estimated life span is more than 1 month.
4. Karnofsky score ≥ 60; ECoG ≤ 2
5. the function of important organs: Echocardiography indicated that the ejection fraction of heart was ≥ 50%; ECG showed no obvious abnormality; creatinine clearance rate calculated by Cockcroft Gault formula was ≥ 40ml / min; ALT and AST were ≤ 3 times of normal value; total bilirubin was ≤ 2.0mg/dl; coagulation function: Pt and appt were < 2 times of normal value; arterial oxygen saturation (SpO2) > 92%;Blood routine test: Hgb ≥ 80g / L, ANC ≥ 1 × 109 / L, PLT ≥ 50 × 109 / L.
6. sign the informed consent.

Exclusion Criteria:

1. patients who used immunosuppressive drugs, hormones, antibiotics and probiotics one month before admission.
2. serious active infection.
3. HIV positive, active hepatitis infection.
4. previous history of other malignant tumors. Excluding: Patients with cured skin basal or squamous cell carcinoma and cervical carcinoma in situ at any time before the study; patients with other tumors not listed above but cured by operation only without other further treatment measures and disease-free survival period ≥ 5 years can be included in the study.

(6) patients participating in other clinical trials at the same time. (7) in the opinion of the researcher, the subjects are not suitable to be selected or cannot cooperate to participate in or complete the study.

(8) patients with congenital immunodeficiency. Exclusion criteria

1. patients who had used immunosuppressive drugs, hormones, antibiotics and probiotics in the first month before inclusion.
2. severe active infection.
3. human immunodeficiency virus (HIV) positive, active hepatitis infection.
4. previous history of other malignant tumors. Excluding: patients with cured basal or squamous cell carcinoma of the skin and carcinoma in situ of the cervix at any time prior to the study; Subjects with disease-free survival ≥5 years of other tumors not listed above, which have been cured by surgery only without other further treatment measures, can be included in the study.

(6) patients who also participate in other clinical trials. (7) the investigator considers that the subject is not suitable for inclusion or unable to cooperate in or complete the study.

(8) patients with congenital immune deficiency. (9) a history of myocardial infarction and severe arrhythmia within six months. 2. Inclusion criteria and exclusion criteria for the control group of this project

1. no history of digestive tract diseases, infectious diseases or immune diseases.
2. no history of smoking or drinking.
3. did not take antibiotics and other drugs and probiotics for 1 month before enrollment.

Pancreatic cancer patients and Healthy person.

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