Figure 5. The aged microenvironment induces therapy resistance.

Yumm 1.7 tumors in young (8 weeks, n=10 mice/treatment) or aged (52 weeks, n=10 mice/treatment) mice fed PLX4720 (417 mg/kg) chow. Tumors responded in young (p=6 ×10⁻⁵, two way paired t-test) (a), but not aged mice (p=0.361). Dotted lines indicate untreated controls (b). Melanoma spheroids were treated with 100nM H₂O₂ (48h), embedded in collagen and treated with young media containing 1µM PLX4720 (ANOVA, p<0.0001) (c). WM35 spheroids were embedded in collagen and treated with aged media containing 1µM PLX4720 and/or 20mM NAC. Invasion was measured after 48h (representative images, magnification 40×, one-way ANOVA, p=0.02). 20mM NAC (unpaired t-test, p=0.03) and 1µM PLX4720 (two-tailed unpaired t-test, p=0.006) compared to control (d). Live-dead staining of spheroids from (d). ANOVA, p<0.0001; Holm-Sidak’s multiple comparisons test indicated (p<0.0001) after pre-treatment with 20mM NAC in presence of either control or 1µM PLX4720 (e). β-catenin staining of biopsies (magnification 200×) from patients undergoing vemurafenib treatment (percentage indicates percent RECIST response) (f). Tabulation of patient samples correlating H-score (intensity of stain per field of cells) to RECIST response (two-tailed paired t-test, p=0.035). 30% is considered a responder by RECIST criteria (g). Young mice (n=10/group) with Yumm1.7 tumors were treated with rsFRP2 (200ng/mL) bi-weekly. PLX4720 (417/mg/kg) was administered once the tumor reached 500mm³. rsFRP2-treated (red line) vs control (blue line) young mice (ANOVA, p=0.009; Holm-Sidak corrected multiple comparisons, p<0.05 after day 12, mean±s.e.m.) (h). Yumm1.7 tumors were injected in aged mice (52 weeks, n=5/group) pre-treated with α-sFRP2 antibody (1mg/kg, once weekly). Mice were administered control or 417mg/kg PLX4720-laced chow. Tumor volume at day 25 is shown (unpaired t-test with Welch’s correction, p=0.048). (i). RECIST response in patients under 65 vs. over 65. Two sample Wilcoxon rank-sum (Mann Whitney) test indicated statistical significance (p=0.016) (j). Data represented as mean±s.d. (a,b,c,e,i,j).