Sircol collagen assay kit

Collagen concentration was assessed on day 7 using the Sircol™ Collagen Assay Kit (Biocolor, Antrim, UK). For collagen isolation, 2 mL of 0.1 mg/mL pepsin in 0.5 M acetic acid was added to the cultivated media. Pepsin digestion was performed overnight at 4 °C with constant agitation of the medium. To concentrate the collagen sample, 100 μL of acid-neutralizing reagent was added to 1 mL of the samples and cell debris was removed by centrifugation. As a blank control, 100 μL of cold isolation and concentration reagent was added to all samples, incubated overnight at 4 °C, and centrifuged at 13,000×g for 10 min, after which the supernatant was discarded. One milliliter of Sircol dye reagent was added to the samples and incubated at room temperature for 30 min with constant agitation. After centrifugation, the supernatant was discarded, the pellet of each sample was dissolved in 1 mL of alkali reagent, and the absorbance was measured at 550 nm.

The tumor collagen content was measured using Sircol Collagen Assay Kit (Biocolor, UK) according to the manufacturer's instructions. PDX mice with tumors that reached a size of ≈1 cm in diameter were selected for solid stress measurement. The protocol of grouping and drug administration was the same as described above. 24 h after the last injection, the tumor tissues were harvested, washed with Hank's buffer, and measured using a caliper. Solid stress was measured using the tumor opening technique.^[30] The tumor was cut along its longest axis (≈80% of its shortest diameter) and then soaked in Hank's buffer for 10 min to diminish any transient, poroelastic responses. Next, the width of the opening at the surface of the tumor at the middle of the cut was measured. Finally, the width of the opening was divided by the diameter perpendicular to the cut to calculate the solid stress.