Rat tail type 1 collagen solution

To fluorescently tag collagen gels, the method by was modified (Doyle, 2018 (link)). Briefly, 5 ml of 3 mg/ml type I rat-tail collagen solution (Corning, 354249) was polymerized at room temperature for 30 min. Thereafter, collagen gels were incubated with 50 mM borate buffer solution (Fisher Bioreagent, BP168-500) (pH 9.0) for 15 min at RT. The solution was then aspirated, and 5 ml of Atto 488 NHS ester dye solution (Sigma-Aldrich, 41698) was added to the collagen gel and incubated at RT for 1 h. The dye solution was again aspirated and quenched in 10 ml of TRIS [tris(hydroxymethyl)aminomethane] buffer (Alfa Aesar, J62662) (pH 7.0) for 10 min at RT. Fluorescently labeled collagen gels were further washed six times in PBS (with Ca²⁺/Mg2⁺) over a 4-h period. The collagen gels were then depolymerized by adding 750 µl of 500 mM glacial acetic acid (Sigma, 45726) and placed on a rocker for 1 h at 4°C. Acidified collagen solution was then dialyzed (Thermo-Scientific, 87735) against 20 mM glacial acetic acid for 20 h at 4°C at a 1:1,000 ratio. The final labeled collagen concentration created was estimated by calculating the known starting and final volumes of the collagen solution (between 9 and 10 mg/ml). A total of 2–10% labeled collagen stock solution was then created by mixing with unlabeled type I collagen solution (calculations based on protein weight (Doyle, 2018 (link))).

3D Coll I matrices were reconstructed on poly(styrene-alt-maleic anhydride) (PSMA) (Sigma-Aldrich, Germany) coated 13 mm coverslips (Diagonal, Germany), resulting in covalently binding of collagen to PSMA-layer^{42 (link)}. Type I rat-tail collagen solution (Corning, New York, USA) was diluted with 250 mM phosphate buffered at pH 7.5 (Sigma-Aldrich) to achieve 2 mg/ml Coll I concentration, as described in^{8 (link)}. Subsequently, prepared Coll I solution were transferred onto the surface of PSMA-coated coverslips and were polymerized for 90 min 37 °C and 95% humidity and 5% CO₂ content. Reconstituted Coll I matrices were washed twice with PBS and kept in PBS prior to use.

3D cultures were generated as previously reported [30 (link)]. Briefly, a 1 mg/mL rat-tail collagen type I solution (Corning) was made according to the manufacturer’s “alternate gelation procedure” and stored on ice prior to use. Cells were detached with trypsin, pelleted at 1200 rpm × 3 min and then resuspended in 10 mL of MCF-12A medium and counted. 75,000 cells were seeded per gel per 1.5 mL of collagen solution in a 12-well plate. After 30 min at 37 °C, 2 mL of MCF-12A medium was added to each well and the gel was detached from the edges of the well using a sterile pipette tip. Culture medium was changed every 2–3 days and gels were harvested after 14 days. Gels were processed for paraffin embedding for histological analysis and whole mount microscopy as described in [10 (link)]. Gel diameter was measured using Axiovision (Zeiss) imaging software.

Gels were prepared from a rat tail collagen type I solution (Corning, New-York, USA) in Nunc LabTek II chambered coverglass 8 wells (Gibco, Saint Aubin, France). In order for the collagen gel to strongly adhere to the bottom glass surface, LabTeks were first treated with sodium hydroxide (NaOH 0.1N) for 1 min, and allowed to dry. Then 3-Aminopropyl-trimethoxysilane (APTMS) was added for 10 min. After this treatment, LabTeks were cleaned 3 times with distilled water and dried. Finally, glutaraldehyde (0.5% in PBS) was added for 30 min, and after the solution was removed, LabTeks were rinced 3 times with distilled water and allowed to dry before adding the collagen solution. All the following steps were then carried out in ice (around 4 °C) to prevent polymerization of the collagen before adding it into the LabTeks. Solutions containing collagen (0.95, 1.8 and 4.5 mg/mL), RPMI medium supplemented with glutamine, FBS, antibiotics and fibronectin (10 μg/mL) were prepared. After adding NaOH (0.1 M) to reach a pH of 7.4, freshly harvested cancer cells were included in the collagen mixture to obtain a final concentration of 2 × 10⁶ cells/mL. The solution was poured into the LabTeks and transfered to an incubator for gel polymerization at 37 °C and 5% CO₂ for 30 min.

Various concentrations of ldECM hydrogels were prepared following a previously published protocol but with slight modifications [20 (link)]. Lyophilized dECM was crushed into a powder using a freezer mill (6775 Freezer/Mill; SPEX SamplePrep, Metuchen, NJ, USA). ldECM powders (1, 2, and 3 w/v%) were solubilized using 0.5 M acetic acid with pepsin (1% w/w for ldECM) (Sigma-Aldrich, St. Louis, MO, USA) for 48 h. After complete solubilization of ldECM, 10X Phosphate-Buffered Saline (PBS) was added at a 1:10 ratio to the ldECM solution, and the pH was increased to 7.0, using 5M sodium hydroxide (Sigma-Aldrich). Tissue-mimetic hybrid hydrogels were prepared by mixing 1% ldECM hydrogel with 3 mg/mL of a high concentration of rat tail collagen type I solution (9.41 mg/mL; Corning, NY, USA). 1% ldECM and collagen solutions were varied in volume ratios of 25/75, 50/50, and 75/20 to optimize the tissue-mimetic hybrid hydrogel composition. Moreover, fibrinogen (3 mg/mL; Sigma-Aldrich) solution in PBS (Gibco) and the type I collagen solution were thoroughly mixed at a 1:1 ratio, and the collagen/fibrinogen solution was mixed with thrombin (50 U/mL, Sigma-Aldrich) at a volume ratio of 50:1 to use the collagen/fibrin hydrogel as a control group.