CAR-T, Gut Microbiota, Pancreatic Cancer
Conditions
Keywords
pancreatic cancer, chimeric antigen receptor T cell, gut microbiota, Acyl-CoA, cholesterol acyltransferase 1, Mesothelin
Brief summary
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.
Interventions
None listed
Sponsors
First Affiliated Hospital of Harbin Medical University
Study design
Observational model
CASE_CONTROL
Time perspective
PROSPECTIVE
Eligibility
Sex/Gender
ALL
Age
30 Years to 75 Years
Healthy volunteers
Yes
Inclusion criteria
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.
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| In vivo experiment: comparison of intestinal microflora and T cell cholesterol metabolism and subtypes between pancreatic cancer patients and healthy people The difference of proportion and the correlation. | from January 2020 to December 2020. | Fecal and peripheral blood samples were collected from patients with pancreatic cancer and healthy people respectively: (1) through Illumina miseq High throughput sequencing technology was used to detect 16S rRNA to analyze the diversity of intestinal flora (α and β diversity), and bioinformatics methods such as random forest and lefse were used to analyze the diversity of flora composition; (2) the difference of cholesterol content in T cells was detected by ELISA; (3) the difference of ACAT-1 expression in T cells was detected by PCR and WB; (4) the difference of ACAT-1 expression in T cells was detected by flow cytometry. The proportion of CD8 +, TNF α +, Th1, Th2, Th17, Treg and other subtypes in T cells was detected by cell analyzer. Furthermore, the correlation between intestinal microflora composition and peripheral blood T cell cholesterol metabolism and subtype difference was determined by Spearman correlation analysis (statistical method). |
| In vitro experiment: to verify the regulation of intestinal flora on the biological behavior of anti msln car-t in vitro and the role of ACAT-1 in it. | from January 2021 to December 2021. | In vitro, intestinal flora supernatant of pancreatic cancer and healthy human were co-cultured with anti-msln car-t, respectively, to verify the effect of intestinal flora on biological behavior of anti-msln car-t.To analyze how the metabolism of cholesterol in anti-msln car-t cells, with acat-1 as the key enzyme, participates in the process of regulating the biological behavior of anti-msln car-t by intestinal flora, and preliminarily uncover the relevant mechanism. |
| Animal model experiment: to verify the ability of intestinal bacteria to control anti msln car-t targeted killing pancreatic cancer cells. | from January 2022 to December 2022. | Furthermore, the influence of differences in intestinal flora composition between pancreatic cancer and healthy human on anti-msln car-t function was verified in animal models of pancreatic cancer. |
Countries
China
Contacts
Primary ContactYunwei Wei
Backup ContactLei Zhao
Outcome results
None listed