HKCs from individuals of Back versus White descent display greater oncogenic and self-renewal potential.Figure 2(A) Multiple HKC strains (passage 2,3) from individuals of the two ancestries were stably infected with a lentivirus expressing dominant negative mutant TP53 (TP53R248W) and superinfected with a H-RasV12 expressing virus, followed, 24 hours later, by parallel intradermal injections into the back of NOD-SCID female mice. 7 strains per ancestry, 4-5 mice per assays were used. Tumors were excised 15 days post-injection. Shown is size quantification of all individual tumors (dots) by digital caliper. **p<0.01. n(tumors by White HKCs) =28, n(tumors by Black HKCs)=31, n((tumors by Black HKCs with <20% genome admixture)=13. Two-tailed unpaired Mann-Whitney test. In the plot, bars represent mean +/- SD. (B) Representative immunohistochemical analysis with anti-pan keratin antibodies of a pair of tumor lesions formed by parallel injections of White versus Black HKC strains together with quantification of the pan-keratin positive areas (mm2) of individual lesions (dots) formed by 6 HKCs strains of the two ancestries. n(tumors per ancestry) = 8. *p<0.05. Horizontal line: median. Two-tailed unpaired t-test. Scale bar: 500μm. Double immunofluorescence analysis of tumors formed by six HKC strains, as in the previous panels, with antibodies against Ki67 (C) together with anti-pan-keratin antibodies for tumor cells identification. Shown are representative images and quantification of results of individual lesions (dots). Digitally acquired images were used to determine the % of Ki67 positive cells (C) in pan-keratin positive areas, examining 3-5 fields in each case. For Ki67, K10 and K1 staining: n(tumors per ancestry) = 24, 22 and 6, respectively. ****p<0.0001; **p<0.005, horizontal line: median, two-tailed unpaired t test. Scale bars: 100μm. Double immunofluorescence analysis of tumors formed by six HKC strains, as in the previous panels, with antibodies against keratin 10 and 1 (K10, K1) (D) together with anti-pan-keratin antibodies for tumor cells identification. Shown are representative images and quantification of results of individual lesions (dots). Digitally acquired images were used to determine the % of K1/K10 positivity (D) in pan-keratin positive areas, examining 3-5 fields in each case. For Ki67, K10 and K1 staining: n(tumors per ancestry) = 24, 22 and 6, respectively. ****p<0.0001; **p<0.005, horizontal line: median, two-tailed unpaired t test. Scale bars: 100μm. (E) Colony forming assay of multiple HKCs from White versus Black donors (passages 2,3) plated in triplicate dishes, at limited density (1000 cells per 60mm dish). At day 8, cells were fixed and stained by crystal violet and percentage of large colonies ("holoclones"), 10x size relative to total number of colonies in the dish, was determined by Fiji software. Shown are representative images and bar graph quantification in decreasing order of holoclone forming capability by all indicated HKC strains (three dishes per strain). Asterisks indicate Black strains with less than 20% admixture (Fig 1B). Inset: pooled values of all HKCs from White versus Black ancestries shown as individual dots together with mean +/- SD. n(White HKC strains)= 35; n(tumors by Black HKCs, total, < and > 20% genome admixture)= 32, 12 and 20 respectively. *** p<0.0005; ****p<0.0001, Two-tailed unpaired t-test. (F) Sphere forming assays of HKC strains from White and Black donors plated in Matrigel suspension (2000 cells per 8-wells chamber slide, 2-3 wells per stain) and cultured for 8 days. Quantification of large spheres (> 2000 pixels >=0.0095mm2) versus total number of spheres (>100 pixels >=0.00047mm2) formed by each strain was determined by Fiji software. Shown are representative images together with bar graph quantification in decreasing order of large sphere forming capability by all indicated HKC strains. Asterisks indicate Black strains with less than 20% admixture (Fig 1B) Inset: pooled values of all HKCs from White versus Black ancestries shown as individual dots together with mean +/- SD. n(White HKC strains)= 11; n(tumors by Black HKCs, total, < and > 20% genome admixture)= 11, 4 and 7. **p<0.01; *p<0.05. Two-tailed unpaired t-test. Scale bars: 300μm.