CD44 knockdown leads to prolonged OS as well as reduced metastasis formation in SCID mice. After lentiviral transduction with CD44 or control shRNA, HMC-1.2 cells were injected subcutaneously into 15 SCID mice per group. (A) Kaplan-Meier survival analysis for the CD44-knockdown (CD44 kd; red curve) and control (Co; blue curve) groups was performed. Median survival was significantly different between groups (CD44 knockdown, 110 days; control, 97 days; *P < .05, log-rank test). (B) Primary tumor weight in mice receiving HMC-1.2 CD44-knockdown cells (red box) compared with mice injected with control HMC-1.2 cells (Co; blue box) was slightly reduced (P > .05, Mann-Whitney U test). Results are shown as boxplots (Tukey) with median (black line), 25th to 75th percentile (boxes), and ranges without outliers (whiskers). (C) Bars represent the percentage of ulcerated primary tumors in mice receiving HMC-1.2 CD44-knockdown cells (red bar) compared with mice injected with control HMC-1.2 cells (Co; blue bar). CD44 knockdown did not significantly change the ulceration rate in these experiments (P > .05, Fisher exact test). (D) For control (blue boxes) and CD44-knockdown (red boxes) mice, the estimated numbers of observed MCs (human HMC-1.2 cells) per 10⁶ murine cells, determined by human Alu-sequence–specific qPCR in lung, BM, and blood, are shown as logarithmic boxplots (Tukey) with median (black line), 25th to 75th percentile (boxes), ranges (whiskers), and outliers (black dots). As visible, metastatic lung infiltration by disseminated human HMC-1.2 cells transduced with CD44 shRNA was significantly lower compared with controls (CD44 knockdown: median, 142.6; mean ± SD, 238.0 ± 292.9; control: median, 877.5; mean ± SD, 3550 ± 5933). *P < .05 (Mann-Whitney U test). Median number of circulating CD44-depleted HMC-1.2 cells in the animals’ blood was slightly lower compared with control HMC-1.2 cells (CD44 knockdown: median, 81.6; mean ± SD, 224.4 ± 268.5; control: median, 215.1; mean ± SD, 216.1 ± 200.4) (P > .05, Mann-Whitney U test). A similar trend could not be observed in the BM. (E) In a linear prediction model, the numbers of observed disseminated MCs (human HMC-1.2 cells) per 10⁶ murine cells in the lungs (left) and blood (right) in the CD44-knockdown (red curves) and control (blue curves) groups with 95% confidence intervals (colored areas) were related to the primary tumor weight. Interestingly, the slopes of the corresponding regression lines were significantly different (P < .05) when comparing the CD44-knockdown (red curves) and control (blue curves) groups.