Quantifying Tumor-Induced Chemotaxis

MC38 tumors without IR and three days post-IR were excised from WT and Sirpα^−/− mice. After being weighed, tumors were minced followed by culturing in RPMI-1640 with 10% FBS. After 24 h, the culture supernatants (tumor-conditioned medium) were collected and tested for the presence of MCP-1 and KC chemokines by a multiplex assay (BioLegend), and the capability of driving monocyte/MDSC or PMN chemotaxis by in vitro chemotaxis assays^{49 (link),51 (link)}. Ly6C⁺ monocytes/MDSCs and Ly6G⁺ PMNs (1 × 10⁶ each) isolated from WT mouse bone marrow^{52 (link)} were labeled with CFSE and placed into the upper chamber of the transwell device in a 24-well plate. The collected tumor-conditioned medium (0.5 ml) was added into the lower chamber followed by incubation at 37 °C for 2 h. Chemotaxis of monocytes/MDSC and Ly6G⁺ PMN into the lower chamber was quantified by a SpectraMax iD5 Multi-Mode Microplate Reader with SoftMax Pro 7 Software (Molecular Devices) and calculated against the total cells loaded. Control experiments were performed using medium without culturing tumor tissues.