Type 1 collagen

A 3D culture matrix comprising type I collagen gel and matrigel was produced as follows: 25 mL of type I collagen (BD Bioscience; 3.7 mg/mL) and matrigel (BD Bioscience) were mixed in a 1:1 ratio, dropped onto 16-mm glass cover slides in six-well culture plates, and 100 mL of absolute ethanol was added to each well until the gel polymerized at room temperature. After culture for 4 days without changing the culture media, cells were fixed with 4% formaldehyde in PBS for 10 min. The formation of vasculogenic-like structures was assessed by the presence of Periodic acid-Schiff (PAS)-positive ECM surrounded by cancer cells. Vascular mimicry formation capacity was evaluated by counting the number of vascular mimicking tubules.

Palmitic acid (P9767), low endotoxin and fatty acid free‐bovine serum albumin (BSA) (A8806), LPS (L2630), and IL‐1β (I5271) were purchased from Sigma (St. Louis, MO). Recombinant murine interferon‐gamma (CTK‐358‐2PS) was from MoBiTec (Goettingen, Germany). Type‐1 collagen was from BD Biosciences. Annexin V (A23204) and propidium iodide (P3566) were from Invitrogen (Eugene, Oregon).
Anakinra (Kineret^®) was purchased from SOBI (Swedish Orphan Biovitrum AB). TAK‐242 (tlrl‐cli95) was from InvivoGen (Distributed by LabForce AG, Switzerland). Murinized Anti‐IL‐1β Ab is a noncommercialized product provided by Novartis. Bovine FetA was kindly provided by Prof. Jahnen‐Dechent, Aachen University, Germany, and the levels of endotoxins were tested in his laboratory with Endosafe ultrasensitive PTS, Charles River. Murine FetA was donated by Prof. Bhattacharya, Visva‐Bharati University, West Bengal, India. Experiments shown in Figure 1C and D were performed with murine FetA. All other experiments were done with bovine FetA.

C2BBe1 cells were cultured in the apical chamber of transwells (Corning) coated with type 1 collagen (BD Biosciences) and placed in the ECIS transwell array. Cells were cultured in DMEM containing 10% FBS, and the next day (day 1), the medium was replaced with that containing 0.5% serum. On day 2, media was changed and test agents were added in duplicate in media containing 0.5% serum. The experiment was concluded 48 hours later (day 4). Barrier resistance was measured on the ECIS instrument. The plots shown are at 75 Hz beginning at day 2 post-addition of the test agents with resistance values normalized to those observed just prior to the addition of test agents. For barrier disruption and reformation studies, cells were cultured in transwells in the ECIS array as described above in the presence of 10% serum for one day and 0.5% serum for another day. On day 2, the media was replaced with media containing 0.5% serum and 2.5 mM EGTA (in apical and basolateral chambers). The EGTA was washed off 5 hours later and replaced with media containing 0.5% serum and test agents. Cells were monitored over the next 48 hours for barrier reformation and the plots shown are at 75 Hz with resistance values normalized to those observed just prior to the addition of test agents.

An in vitro adhesion assay was performed by resuspending 40,000 cells in complete media and seeding them on 96-well plates coated with 10 µg/ml type 1 collagen (BD Bioscience: 354246) or 10 µg/ml fibronectin (Sigma: F1141) for 30 min at 37 °C. Cells were fixed using 3.7% formaldehyde in PBS for 15 min, washed twice with washing buffer (0.1% BSA in RPMI), and stained with a crystal violet solution. After washing the excess dye out, the plates were allowed to dry for 1 h. Then the crystal violet stain absorbed by the cell nuclei was solubilized with 10% acetic acid and the optical density was measured at 570 nm^{8 (link)}.