FIGURE 4.

Cancer-associated fibroblasts did not expand with CSC cells but facilitated CSC cell seeding and expansion in the left lung. Transplanted A223 and B931 CSCs were used in all experiments as indicated. (A,B) 10,000 GFP⁺ B931 cells were co-transplanted with 50,000 RFP⁺ CAFs directly to the left lung and 4–5 mice were sacrificed on days 0, 3, 6, 9, and 12 post-transplant and left lungs imaged as described in panel (F) to detect GFP and RFP positive lesions. Representative images of lungs at each time point are presented in panel (A) and quantification of GFP and RFP intensity is presented in panel (B). (C) RFP⁺ CAFs were imaged with an inverted fluorescent microscope using a 10× objective to demonstrate RFP positivity. (D–G) Representative images of left lung tumor, and quantification of left lung tumor volume in nude mice (D,E) and C57BL/6J mice (F,G) after 100 flow sorted side population (CSCs) of the indicated SCC cell lines were directly injected to the left lung with and without 5,000 CAFs or NAFs co-transplanted. Lungs were harvested and imaged from nude mice on day 19 and from C57BL/6J mice on day 25. (H) Brightfield and fluorescent imaging of gross tumors in the left lung tumor immediately after harvest was performed using a fluorescent dissecting microscope. SCCs were labeled with green fluorescence protein (GFP) and CAFs with red fluorescence protein (RFP). Only SCCs but not CAFs were detected. Scale bar: 1 mm. ^nsP > 0.05, *P < 0.05, ****P < 0.0001.