Additional file 3 of Cryoablation-induced neutrophil Ca2+ elevation and NET formation exacerbate immune escape in colorectal cancer liver metastasis
Creators: name: Tan, Hongtong
name: Jiang, Yiquan
name: Shen, Lujun
name: Nuerhashi, Gulijiayina
name: Wen, Chunyong
name: Gu, Ling
name: Wang, Yujia
name: Qi, Han
name: Cao, Fei
name: Huang, Tao
name: Liu, Ying
name: Xie, Weining
name: Deng, Wuguo
name: Fan, Weijun
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Supplementary Material 3: Figure S3. (A, B) Representative flow cytometry images and statistical results demonstrate the enhanced proliferation capacity of CD8 + T cells (CFSE) after cryoablation, indicating that cryoablation-treated SW480 colorectal cancer cells can stimulate CD8 + T cell proliferation in vitro. In this experiment, SW480 cells or cryoablation-treated SW480 cells were co-cultured with human PBMCs in 12-well plates for 7 days. The groups were as follows: control (PBMC only), SW480 + PBMC, and SW480 (cryoablation) + PBMC. Human PBMCs were isolated using Lymphoprep™ (StemCell) and labeled with CFSE dye (5 μM, Abcam), then co-cultured with SW480 or cryoablation-treated SW480 cells (rapid freezing for 4 min, rapid thawing at 50 °C for 3 min), with three replicates per group. During the culture, anti-CD3/CD28 (25 μl, StemCell) and IL-2 (10 ng/ml, Peprotech) were used to simulate CD8 + T cells. After 7 days, cells were collected, stained with a viability dye and anti-human CD8 antibody (PE-Cy7, BD), and analyzed by flow cytometry. (C, D) Representative flow cytometry images and statistical results show that neutrophils suppress CD8 + T cells proliferation after cryoablation. The groups were SW480 (cryoablation) + PBMC and SW480 (cryoablation) + PBMC + neutrophils. Human PBMCs were isolated using Lymphoprep™ (StemCell) and labeled with CFSE dye (5 μM, Abcam), then co-cultured with cryoablation-treated SW480 cells (rapid freezing for 4 min, rapid thawing at 50 °C for 3 min). For the SW480 (cryoablation) + PBMC + neutrophils group, an additional 1.0E + 06 neutrophils (purified using the Human Neutrophil Isolation Kit, StemCell) were added. Three replicates were set for each group. During the culture, anti-CD3/CD28 (25 μl, StemCell) and IL-2 (10 ng/ml, Peprotech) were used to simulate CD8 + T cells, and 1.0E + 06 neutrophils were added every other day. After 7 days, cells were collected, stained with a viability dye and anti-human CD8 antibody (PE-Cy7, BD), and analyzed by flow cytometry. (E, F) show that neutrophils enhance apoptosis in CD8 + T cells after cryoablation. The experiment consisted of two groups: SW480 (cryoablation) + PBMC and SW480 (cryoablation) + PBMC + neutrophils. Briefly, 300,000 cryoablation-treated SW480 cells (rapid freezing for 4 min, rapid thawing at 50 °C for 3 min) and 300,000 human PBMCs (with or without 500,000 human neutrophils) were added 6-well plates. Anti-CD3/CD28 (25 μl) and IL-2 (10 ng/ml) were included in the co-culture system. After 5 days of culture, cells were collected and stained with anti-Annexin V and PI dyes according to the manufacturer’s instructions (GOONIE, Cat# 100–102). Each group included 3 replicates, and significant differences were indicated by p-values. Human PBMCs (Cat# 07851) and neutrophils (Cat# 19666) were purified from human peripheral blood following the manufacturer’s instructions (StemCell).