Stress signaling in breast cancer cells induces matrix comp. that promote chemoresistant metastasis (2018)
Figure 1

Figure 1. JNK signaling mediates mammary tumor growth and metastasis to lungs.

A Kaplan-Meier analysis of overall survival in breast cancer patients with recurrent lethal disease, classified according to p-JNK expression. p-JNK was determined by immunostaining of breast cancer samples in a tissue microarray; p-JNK low, n = 31; p-JNK high, n = 10. P value and hazard ratio (HR) were calculated by log-rank test.

B Immunostaining of p-c-Jun (arrows) in patient-matched breast tumor and lung metastasis. Scale bar, 50 μm.

C Quantification of p-c-Jun positive (p-c-Jun+) cells per tumor area in matched primary tumors and lung metastases. Boxes depict median from 12 patients with upper and lower quartiles. Whiskers show maximum and minimum. P value was determined by two tailed paired Student's t test.

D Quantification of p-c-Jun positive (p-c-Jun+) cancer cells within mammary tumors and matched metastases in mice injected with MDA231-LM2 cells. Values are mean from three mice per group ± SEM. P value was determined by a two-tailed paired Student's t-test.

E Immunofluorescence analysis of p-c-Jun in micrometastatic (day 7) and macrometastatic (day 21) nodules in a xenograft mouse model injected intravenously with MDA231-LM2 cells. DAPI was used to stain cell nuclei, and cancer cells express GFP as a marker. Scale bar, 50 μm.

F Quantification of p-c-Jun+ cancer cells in panel E showing mean from three mice per group ± SEM. P value was determined by two-tailed Mann-Whitney test.

G Experimental setup to address the role of JNK signaling in mammary tumor progression and metastasis. MDA231-LM2 or SUM159-LM1 cells were implanted bilaterally into the fourth mammary fat pads of NSG mice. In vivo treatment with the JNK inhibitor CC-401 (JNKi) commenced at day 5 post implantation and was repeated every three days thereafter until analysis.

H Mammary tumor growth curves from mice described in panel G. Each value represents the mean ± SEM. For MDA231-LM2, n = 12 tumors in 6 mice for each experimental group. For SUM159-LM1, n = 16 tumors in 8 mice for each experimental group.

I, J Lung metastatic burden in MDA231-LM2 or SUM159-LM1 tumor-bearing mice treated with JNKi. (I) Quantification of lung metastases. FOV, field of view; LS, lung section. Each value represents the mean ± SEM. For metastatic foci per FOV, 8-10 fields were quantified per mouse. MDA231-LM2 tumor-bearing mice: n = 6 mice per group. SUM159-LM1 tumor-bearing mice, vehicle n = 8 mice; JNKi n = 7 mice. (J) Representative histology examples of metastasis in lungs, detected by expression of human vimentin. Scale bar, 100 μm.

K Experimental setup to determine lung colonization of in vivo vehicle-treated and JNK-inhibited MDA231-LM2 cancer cells after intravenous injection. In vivo treatment with JNKi commenced at the time of implantation and was repeated every 3 days thereafter until analysis at day 17.

L Metastatic burden determined by photon flux in lungs of MDA231-LM2-injected NSG mice treated with vehicle and JNKi. Boxes show the median with upper and lower quartiles. Whiskers represent minimum and maximum values. Vehicle control, n = 8; JNKi-treated, n = 8.

M Examples of bioluminescence in mice that were quantified in panel L. P values were determined by a two-tailed Mann-Whitney test.

For panels H, I and L, *P < 0.05, **P < 0.01, ****P < 0.0001.