Fig. 5.

Evaluation of mAb150 efficacy in A4 xenograft cell sub-populations following treatment (3 doses, 18 mg/kg; 1-week recovery between and after doses). A-i, ii. Schematic and corresponding dot blots in flow cytometry data for resolution of cell fractions- Top panel: label-chase identifies Pkh^hi (CSCs), PKH^lo (progenitors and aneuploid cells), PKH^neg (differentiated cells). Middle panel: PI based-DNA content resolves Host, Euploid and Aneuploid populations. Lower panel: Hoechst-Pyronin Y-PI based DNA-RNA quantification resolves 5 fractions viz. 1: Euploid G0, 2: Aneuploid G0, 3: Euploid G1, 4: Euploid S-G2/M and Aneuploid G1, 5: Aneuploid S − G2/M populations, A-iii. Fold-reduction following mAb150 treatment of the cell populations described in 3A-i; B. Heatmap representations of consolidated fold-change in the frequency of individual fractions between 8 treated xenografts and appropriate controls across - i. PKH regenerative hierarchy-based, ii. Ploidy-based and, iii. Ploidy-Cell cycle phase-based fractions; C. Evaluation of residual regeneration in mAb150 treated A4 xenografts assayed for- i. spheroid formation, ii. adherent colony formation, iii. soft agar colony formation, iv. Wound healing assay; D. Limiting dilution assays (3D – 3 doses of mAb150, 6D – 6 doses of mAb150 as described in methods), i. Tumor regenerative potential 0f 5000, 10,000 and 20,000 residual cells of sorted fractions, ii. Tumor initiating frequencies of residual cells of sorted fractions. 1:PKH^negHost, 2:PKH^negEu, 3:PKH^negAneu, 4:PKH^loEu, 5:PKH^loAneu, 6:PKH^hi. *P<0.05; **P<0.01; ***P<0.001.