Figure 6

Cooperativity between TNFα and hyper-activated Ras promotes the dissemination of tumor cells to lymph nodes. The scheme describes the "Experimental design" of in vivo mouse experiments, including cell preparation. *CM preparation: Ras^G12V (A) MCF-7 cells were stimulated by TNFα for 8 hr, CM were removed and replaced by fresh, serum-free non-TNFα-containing medium for additional 36 hr. **CM preparation: The same as in *, but no TNFα included at any stage. (B) Determination of tumor growth in the mammary fat pad of mice. All MCF-7 tumor cells expressed mCherry, to enable their detection by the Maestro device in intact mice. To provide accurate determination of tumor sizes, the Maestro device was used to quantify fluorescence in excised tumors, ex vivo, at the end of two experiments performed (termination of experiments was based on animal care regulations). The Figure shows combined results of these experiments, including n=7 in each of the mice groups. For more details on the results in group 4 – see "Results". *p<0.05, **p=0.01, ***p=0.001 for comparisons between the Cells^ControlCM^Control group and all other groups. (C) Kinetics of tumor cell dissemination to LN, followed by the Maestro device in intact mice in three independent experiments combined, including a total of n=10-12 in each of the mice groups. All tumor cells expressed mCherry, to enable their detection by the Maestro device in intact mice, p values are shown in the Figure. No statistical differences were obtained in comparisons between any of the other Cell-CM combinations.