Lovo cells

SW480, LS174T, RKO, LOVO cells (DMEM, 10% FBS), HT29, NCM460 (McCoy's 5A, 10% FBS), HCT116, and DLD1 (RPMI‐1640, 10% FBS) were purchased from ATCC and maintained at 37℃ with 5% CO₂.
All clinical samples utilized in this study, including primary CRC tissues and paired‐adjacent noncancerous colon tissues further than 5 cm, were collected from patients undergoing radical colon resection at the Department of Gastroenterology, Shenzhen Hospital, Southern Medical University (Guangdong, China). Fresh samples were frozen in liquid nitrogen immediately after resection and stored at −80℃. Samples were histologically stained with hematoxylin and eosin, and evaluated by experienced gastrointestinal pathologists for histological grade of cancers based on criteria set by the World Health Organization. Normal colorectal mucosa was defined as all straight, nonbranching crypts with histopathologically normal cells. All protocols were approved by the Ethic Committee of Southern Medical University (NYSZYYEC20190013) after obtaining patients’ informed consent. Samples details were summarized in Table S1.

RAW264.7 and LoVo cells were purchased from ATCC. Cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotic and antimycotic solutions. LPS, TNF-α, and IFN-γ were purchased from Peprotech, Cranbury, NJ, USA. Kynurenine was purchased from Sigma Aldrich, St. Louis, MO, USA.

A panel of KRAS mutant CRC cell lines was cultured in 1:1 DMEM/Ham's F12 media with 10% fetal bovine serum, 1% penicillin/streptomycin, and 2 mM L-glutamine. Palbociclib/PD0332991 (Selleck Chemicals) and MEK162 (Selleck Chemicals) were dissolved in dimethyl sulfoxide (DMSO) as 10 mM and 30 mM stocks, respectively. Lovo cells were obtained from ATCC. HCT116, SW480, SKCO1, SW403, SW837, LS174T, SW948, SW1116, LS1034, and T84 cell identity was verified by short tandem repeat (STR) analysis.

Human colon epithelial CCD 841 CoN cells (CRL-1790), human colorectal adenocarcinoma HCT 116 (CCL-247), HT-29 (HTB-38), SW480 (CCL-228) and LoVo cells (CCL-229) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). CCD 841 CoN cells were grown in Eagle’s minimum essential medium (EMEM, 30-2003, ATCC, Manassas, VA, USA), HCT 116 and HT-29 cells were grown in McCoy’s 5A medium (30-2007, ATCC, Manassas, VA, USA). SW480 were maintained in Leibovitz’s L-15 medium (L-15, 30-2008, ATCC, Manassas, VA, USA). LoVo cells were grown in F-12K medium (30-2004, ATCC, Manassas, VA, USA). Cells were grown as a monolayer in a humidified incubator, at 37 °C in a humidified atmosphere with 5% (v/v) CO₂, in specific culture medium supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin and 10% fetal bovine serum (FBS, 30-2020, ATCC, Manassas, VA, USA). Medium was changed 2–3 times/week. Cells were seeded into multi-well plates the day before treatments to allow cell attachment. Treatments were performed by culturing cells in complete medium with 3 kDa whey extracts (up to 40% v/v) up to 72 h, given the high efficiency in inhibiting colon cancer cell viability obtained from 3 kDa milk extracts [6 (link)]. Control (Ctr) cells were treated with corresponding volume (% v/v) of Hanks’ balanced salt solution (HBSS)-10 mM Hepes.

The human colorectal cancer cell lines HT-29 (grade 1) and LoVo (grade 4) as well as CCD-18Co cells representing the normal colonic epithelium were obtained from ATCC (Manassas, VA, USA). HT-29 cells were cultured with McCoy’s 5A (ATCC), LoVo cells with F12-K media (ATCC), CCD18-Co with Eagle’s minimum essential medium (Lonza, Basel, Switzerland) with 1% sodium pyruvate and 1% nonessential amino acids (all from Sigma-Aldrich, St. Louis, MO, USA). All media contained 1% l-glutamine and penicillin-streptomycin solution and 10% fetal bovine serum (Sigma-Aldrich). Cell cultures were provided in 5% CO 2 at 37 °C and 95% humidity. The medium was changed twice a week, and cells were passaged at approximately 70% confluence and trypsinized with 0.25% trypsin–EDTA solution (Sigma-Aldrich).

HCT116 cells, LOVO cells, SW480 cells, and NCM460 cells were purchased from ATCC (American type culture collection, Manassas, VA, USA) and deposited in the Central Laboratory of basic research of integrated traditional Chinese and Western medicine, School of Basic Medical Sciences, Guangzhou University of Chinese Medicine.

Four human CRC cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). HCT-116 cells (ATCC No. CCL-247) were maintained in McCoy’s 5A medium, LoVo cells (ATCC No. CCL-229) were maintained in F-12 K medium (Kaighn’s Modification of Ham’s F-12 Medium), and SW480 cells (ATCC No. CCL-228) and SW1116 cells (ATCC No. CCL-233) were maintained in Leibovitz’s l-15 medium. The media were supplemented with 10% fetal bovine serum, and the cells were incubated in a humidified atmosphere of 95% air and 5% CO₂ at 37°C.