Figure 4. Organ localization of BC cells knockdown for CDH2, in vivo.
(A) Cartoon summarizes study design. (B) 5 × 105 cancer stem cells from MDA-MB-231-Oct4-GFP, knockdown for CDH2 (RFP) or Cx43 (RFP), or control with scramble shRNA (RFP) were injected intravenously into NSG mice. At day 10, the lungs, brain, and femurs were harvested. Cells from one femur/mouse were used to isolate total RNA for real time PCR with primers for human and mouse GAPDH. The results are presented as human GAPDH/Total GAPDH (human & murine) ±SD, n = 6. **P < 0.05 versus CDH2 and Cx43 knockdown. Fluorescence image: yellow arrow represents pan-cytokeratin positive cells (Texas red); white arrows: Ki67-Alexa-AF405–positive cycling cells (blue + red = pink). (B, C) Paraffin embedded from “(B)” were imaged for cancer stem cells (RFP for knockdown vector + GFP for Oct4A = yellow) with EVOS FL Auto 2. Shown are 200× magnification for each image, representing six mice/group. (D, E) Quantitation by ImageJ of BC cells in the femur, lungs, and brain from “(E),” mean of 10 fields/slide for six mice ± SD. ***P < 0.05 versus lungs or brain.