Ct26 wt

This study was approved by the Special Committee on Scientific Research and Academic Ethics of Inner Mongolia Agricultural University (No. 2020-049). Specific pathogen-free (SPF) BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Six- to 8-week-old mice were raised and housed under SPF conditions and received sterilized feed and water. The mouse colorectal cancer cell line, CT26.WT (ATCC^® CRL-2638), was cultured in RPMI-1640 medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) in a humidified incubator (Thermo, USA) at 37°C, 5% CO₂. The probiotic strain, Probio-M9, was provided by the Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, China.

The human colorectal adenocarcinoma cell line HT-29 (ATCC HT-38™) and the murine colorectal carcinoma cell line CT26.WT (ATCC CRL-2638™) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were subcultured 2–3 times a week in McCoy’s 5a medium (Sigma-Aldrich, St. Louis, MO, USA) and RPMI-1640 (Sigma-Aldrich), respectively. The culture media were supplied with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin and 100 U/mL streptomycin (Sigma-Aldrich). Sunitinib-resistant HT-29 cells, named HT-29/SR, were generated by continuous exposure to 2 µM sunitinib for up to 5 months and the resistance was routinely verified during this time frame. Untreated parental HT-29 cells were cultured in parallel with the HT-29/SR cells. The cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO₂.

The cell lines used in this study were NIH3T3 (ATCC CRL-1658), MEF (ATCC SCRC-1040) and CT26.WT (ATCC CRL-2638). Dulbecco’s Modified Eagle Medium (DMEM) High Glucose (Invitrogen, CA, United States) with added supplements was used for cell culture. Cells were cultured in DMEM with 10% Fetal Bovine Serum (Invitrogen) and 1% penicillin-streptomycin combination antibiotic (Invitrogen). Cells were seeded at 80% confluence and transiently transfected with 75 nM pre-designed Stealth RNAi™ siRNA targeting mouse zyxin (Invitrogen, siRNA ID: MSS238956) using Lipofectamine 3,000 (Thermo Fisher Scientific) according to manufacturer’s instruction. To test zyxin knockdown efficiency, zyxin protein level was determined by western blot at 72 h.

All cell culture reagents were purchased from Biowest and Serana. Tumor cell lines ZR-75-1, MCF-7, HSC-4, CaoV-3 and T-47D as well as the human NK cell line KHYG-1 and murine T cell line CTLL-2 were obtained from DSMZ or ATCC and cultured in the respective recommended medium under standard conditions. KHYG-1 and CTLL-2 were cultured in presence of 10 ng/mL IL-2 (PeproTech, Rocky Hill, NJ, USA).
For in vivo studies, the murine tumor cell lines B16.F10 and CT26.wt (ATCC) were stably transfected by electroporation (Amaxa) with a plasmid coding for human MUC1 (40 tandem repeats) and confirmed for TA-MUC1 expression in vitro and in vivo in mice by flow cytometry and immunohistochemistry using GT-00A. Cells were cultured in standard medium +100 nM methotrexate (Hexal, Holzkirchen, Germany) as selection pressure. For the metastasis model, hMUC1-B16.F10 cells were further transduced to stably express firefly luciferase (110 RLU/cell) enabling detection of metastasis. Origin, TA-MUC1 and IL-15R expression status of cell lines are indicated in Table S1.
Peripheral blood mononuclear cells (PBMCs) were prepared from commercially available buffy coats (DRK Berlin) or leukapheresates (Charité Berlin) of healthy donors by Biocoll separation (Biochrom, Cambridge, UK) and density gradient centrifugation. PBMCs from leukapheresate products were stored frozen in liquid nitrogen.

The human and mouse colon adenocarcinoma cell lines SW480 and CT26.WT (ATCC), respectively, were employed. Cells were plated in DMEM/F12 or RPMI-1640 (both from Gibco), for SW480 and CT26.WT, respectively, supplemented with 10% fetal bovine serum (Corning) and 1% streptomycin/amphotericin (Gibco) (complete medium), at 37 ºC in a 5% CO₂ incubator.