Tumor cell clusters with increased tumorigenesis, metastasis, and CD44 expression. Tumor cell clusters with increased tumorigenesis, metastasis, and CD44.

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Tumor cell clusters with increased tumorigenesis, metastasis, and CD44 expression. Tumor cell clusters with increased tumorigenesis, metastasis, and CD44 expression. A, Representative bioluminescence images of single cells (S) and clusters (C) of TN1 PDX tumor cells in parallel during tumorigenic monitoring upon orthotopic implantation (1,000 cells per mammary fat pad), on days 0 (D0) and 18 (D18). B, Quantitative bioluminescence signals (total flux, p/s; left) and fold change (right) of tumorigenesis mediated by TN1 tumor cells in singles and clusters during the 18-day monitoring in A (n = 5). t test **, P < 0.01. C, Representative bioluminescence images of single cells (S) and clusters (C) of TN2 PDX tumor cells in parallel during tumorigenic monitoring upon orthotopic implantation (5,000 cells per mammary fat pad), on days 0 (D0) and 24 (D24). D, Quantitative bioluminescence signals (total flux, p/s; left) and fold change (right) of tumorigenesis mediated by TN2 tumor cells in singles and clusters during the 24-day monitoring in C (n = 8). t test *, P < 0.05; **, P < 0.01; ***, P < 0.001. E, Images of mammospheres derived from single and clustered tumor cells of TN2 PDXs. Scale bars, 50 μm. F, Quantitated bar graph of mammospheres derived from single cells and clusters. N = 6 biological replicates. t test ***, P = 0.0008. G, Bioluminescence imaging (BLI) images of lung colonization mediated by single cells and clusters of L2T-labeled TN1 PDX cells (5 × 105) at day 0 (D0) and weeks 1, 2, and 8 (Wk1, 2, and 8) after tail-vein infusion. H, Quantitated BLI signal (total flux, p/s) of the TN1 single cell– and cluster-mediated lung metastases described in G (n = 5). t test *, P = 0.012; ***, P = 0.00012. I, CD44 (brown) and CD31 (pink) IHC staining of the TN1 PDX-bearing mouse lung sections (slide 228) showing a CD44+ CTC cluster within CD31+ vasculature. Scale bar, 25 μm. J, Proportion of CD44+ CTCs in the events of single and cluster CTCs counted within the in situ vasculature of human breast tumors (n = 5) and metastases (n = 3), and PDX lung metastases (n = 3; Supplementary Table S3). t test ****, P < 0.0001. Xia Liu et al. Cancer Discov 2019;9:96-113 ©2019 by American Association for Cancer Research