A, FACS sorting of PDX tumor cells that are negative for mouse CD31, mouse CD45 and mouse MHC I expression (negative selection, “mMHC-I-”) and those that are further positive for human MHC I expression (positive selection, “hMHC-Ihi”). B, Viable cell numbers and TIC frequency of PDX tumors. Upper panel, The number of viable cells after positive (“hMHC-Ihi”) selections were compared to those with negative (“mMHC-I-”). Mean ± SE, n = 3–4, **, P < 0.01; ***, P < 0.001. Lower table, Table depicting TIC frequency of PDX tumors. Tumor tissues were digested by collagenase IV for either 20 minutes (20 min) or 2 hours (2 h), excluded of mouse stromal cells (CD31/CD45/MHC-I), and sorted using human-MHC I staining to provide hMHC-Ihi cells and hMHC-Ilo tumor cells. Twenty or 200 cells of MB929 PDX tumor and 10 or 100 cells of CRC042 PDX tumor were subcutaneously injected into NSG mice with high-protein matrigel, and monitored for tumor formation for 24 weeks. L-Calc Software was used to calculate the TIC frequency (with 95% confidence interval).