Ht 29

Human CRC cell lines Caco-2 and HT-29 (Bioresource Collection and Research Center, HsinChu, Taiwan) were grown in Dulbecco’s modified Eagle’s medium (Gibco BRL, Grand Island, NY, USA) containing 2 mM L-glutamine and 1.5 g/L sodium bicarbonate, supplemented with 10% FBS and 2% penicillin–streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin). The cells were cultured in a humidified incubator at 37 °C with 5% CO₂.

Five human colorectal cancer cell lines (HCT116, HT29, SW480, Caco-2, and Colo205) were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Dulbecco’s Modified Eagle’s Medium containing 10% foetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin served as the cell culture medium throughout all experiments. The cells were maintained at 37 °C in a humidified atmosphere of 5% CO₂ in air and passaged every 2 days to obtain exponential growth. All five cell lines were maintained in culture no more than 15 passages. All culture reagents were sourced from Gibco (Invitrogen, Carlsbad, CA, USA).

There were 4 cell lines used in this study including SW480, SW620, HCT 116, and HT-29 cells (Bioresource Collection and Research Center, Hsinchu, Taiwan). SW480 and SW620 cells were established from a same Caucasian male patient with colon adenocarcinoma, and SW480 was isolated from the primary colon tumor site, SW620 was isolated from a lymph node metastasis site. SW480, but not HT-29, secrets granulocyte-macrophage colony-stimulating factor (GM-CSF) that stimulates the proliferation of acute megakaryoblastic leukemia M-07e [21 (link)]. SW480 and SW620 synthesize small quantities of carcinoembryonic antigen (CEA) and belong to Broders' grade 4 cancer [22 (link)]. By Northern blot analysis, the oncogenes c‐myc, H‐ras, K‐ras, N‐ras, myb, fos, and p53 were all expressed in SW480 and SW620 cells [23 (link)]. HT-29 cells were isolated from a Caucasian female patient with colorectal adenocarcinoma (ATCC data). HT-29 and SW480, but not SW620, express vitamin D receptor whose activation by 1,25-dihydroxyvitamin D inhibits cell proliferation and clonogenic growth in soft agar [24 (link)]. HT 116 has a mutation in codon 13 of the ras protooncogene and expresses transforming growth factor beta 1 and beta 2. SW480 and SW620 cells are cultured in Leibovitz's L-15 medium with 10% FBS, and HT-29 and HCT 116 cells are cultured in McCoy's 5a medium with 10% FBS.

The CRC cell lines SW1116, SW480, SW620, DLD1, HT29, and CaCO₂ were purchased from the Bioresource Collection and Research Center (BCRC; Hsinchu, Taiwan). SW1116, SW480 and SW620 cells were cultured in Leibovitz L-15 Medium (Life Technologies, Grand Island, NY, USA). HT29 and DLD1 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA). CaCO₂ cells were cultured in Eagle’s minimum essential medium (Invitrogen). Wild type HCT116 (p53^+/+) cells and isogenic p53 null cells (HCT116 (p53^−/−)) were purchased from Horizon Discovery Group (Cambridge, UK) [60 (link)] and cultured in RPMI 1640 medium (Invitrogen). All media were supplemented with 10% fetal bovine serum (FBS; Invitrogen) and antibiotics (100 U mL^-l penicillin and 100 mg mL^-l streptomycin) according to the manufacturer’s instructions.

Human colorectal carcinoma cell lines HCT-116 and HT-29, and mouse colon carcinoma cell line CT-26 were purchased from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). HCT-116 and HT-29 cells were grown in McCoy’s 5A (Sigma-Aldrich, Darmstadt, Germany) and CT-26 was grown in RPMI-1640 (Gibco, Dublin, Ireland) supplemented with 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate. All cells were maintained in humidified air containing 5% CO₂ incubator at 37 °C and cultured every 2–3 days.
EGFL6 recombinant protein was purchased from Sino Biological (Beijing, China). EGFL6 therapeutic antibody (EGFL6-E5-IgG) was generated and provided by Dr. Yu-Ching Lee using phage display technology. Bevacizumab (Avastin®) was purchased from Genentech (California, USA). 5-FU (Fluorouracil® Injection) was purchased from Pfizer (New York City, USA).