Collagenase d and dnase 1

The human breast cancer patient-derived xenograft HER2+ ER− xenograft model BC_004 was purchased from OncoTest (Freiburg, Germany). Tumor fragments were digested with Collagenase D and DNase I (Roche), counted and 2 × 10⁶ BC004 cells were injected in total volume of 20 µl PBS into the mammary fat pad. Treatment was started once tumors reached an average volume of approximately 200 mm³.

Stromal cells were isolated as previously described (Irla et al, 2008) by enzymatic digestion with collagenase D and DNase I (Roche) and depletion of hematopoietic cells using anti‐CD45 magnetic beads and AutoMACS (Miltenyi Biotec). TECs, endothelial cells, and fibroblasts were cell‐sorted with EpCAM, CD31, and gp38 markers, respectively, with a FACSAriaIII cell sorter (BD).

The human ovarian carcinoma cell line OVCAR-5 was purchased from the National Cancer Institute (Cat. Nr. 0507676). The cell line SK-OV-3 is an internal cell line, which was tested and authenticated to be SK-OV-3 after a full match with the reference databases of ATCC, JCRB, RIKEN, KCLB, and DSMZ. All cell lines were confirmed to be free of murine pathogens and murine viruses (Biomedical diagnostics, Hannover, Germany). To generate human xenograft tumors 3 × 10⁶ OVCAR-5 or 5 × 10⁶ SK-OV-3 tumor cells were injected in 100 μl PBS into the right flank of isoflurane anesthetized humanized mice. The human breast cancer patient-derived xenograft BC_038, a triple negative breast cancer, was obtained from Oncotest and transplanted into non-humanized NOG mice for three rounds in vivo before use in this study. Tumor fragments were digested with Collagenase D and DNase I (Roche), counted and injected into the mammary fat pad of humanized mice. Tumor growth was monitored twice a week by perpendicular caliper measurement and tumor volume was calculated using the following formula: volume = 0.5 × length² × width.