Busulfan

To study the melanoma TAM origin, we used two strategies to track BM-derived cells and identify them through the expression of GFP and/or CD45.2⁺. The Myb^fl/flMx-1^cre/wt (CD45.1⁺) mice were administered 10 μg/g of Poly(I:C) (P1530, Sigma-Aldrich, MA, USA) i.p. every other day, for a total of 7 times, to induce the genetic depletion of BM precursors. The B6.UBQ-GFP (CD45.2⁺) mice were treated with 200 μg/g of Busulfan (cat#14843, Cayman Chemical, MI, USA) i.p. every day, for a total of 5 times, to induce the chemical ablation of BM precursors. For the adoptive transfer, 10 × 10⁶ BM cells, extracted from the femur and tibia of the B6.UBQ-GFP and B6 donor mice as previously described [28 (link)], were transferred retro-orbitally to the Myb^fl/flMx-1^cre/wt and B6.UBQ-GFP mice, respectively. Blood samples were extracted from the transplanted mice to evaluate their chimerism (Myb^fl/flMx-1^cre/wt every 4 weeks, B6.UBQ-GFP every week). YUMM1.7 tumors were injected after the transplant was stable (Myb^fl/flMx-1^cre/wt after 12 weeks, B6.UBQ-GFP after 8 weeks) and the tumors were processed after 2 weeks, along with blood and skin samples. B6.UBQ-GFP-transplanted Myb^fl/flMx-1^cre/wt mice were also used to assess the PDVC57 TAM infiltration.

Viable mast cells were counted by hemacytometer via trypan blue exclusion and plated at 1e6/ml, then exposed to 8.5 or 20 Gy single-dose radiation from a cesium source or chemotherapeutic agents. Busulfan and cyclophosphamide were purchased from Cayman Chemicals (14843 and 13849, Ann Arbor, MI) and resuspended in DMSO. Drugging time and conditions were chosen based on clinical usage where cells were drugged with 800 ng/ml Busulfan and/or 30 ug/ml cyclophosphamide, which corresponded to steady-state serum levels of the drug during clinical utilization (15 (link)). Cells were approximately 98% viable prior to irradiation. Every 24 h, cells were counted and assessed for viability by trypan blue exclusion out to 96 h post-irradiation or 72 h post-drugging.