Subcutaneous co-injection model

Subcutaneous injections were performed as described (22). 2×10⁵ KPC cells alone or together with 1×10⁶ PSCs were resuspended in 100 μL PBS and injected subcutaneously into the flanks of 8–10 weeks old female syngeneic C57BL/6 mice (JAX, 000664). At the beginning of each experiment, and at the onset of treatment with monoclonal antibodies, mice were randomly assigned to the different groups. No estimation of sample size was performed before the experiments. Treatment with monoclonal antibodies was started one week after tumor inoculation and performed as described (23). Mice were injected intraperitoneally every three days over two weeks with anti-IL-6 (200 μg; MP5–20F3, BioXCell), anti-VEGFR2 (800 μg; DC101, BioXCell), or IgG1 isotype control (800 μg; MOPC-21, BioXCell). Mice were monitored daily, and tumor volume was measured by calipers. Measurements were taken in two dimensions, and tumor volume was calculated as length × width² × π/6. At the end of the experiment, mice were euthanized with CO₂, and tumors were collected and aliquoted for 10% formalin fixation for immunohistochemistry and digestion for flow cytometry analysis.