Figure 3.
EOMA-derived EVs containing miR-126 are responsible for macrophage conversion to LYVE+ tumorigenic TAMs cells
(A) Representative images of mice shows 6- to 8-week-old female 129P3/J mice received subcutaneous injection of EOMA cells pre-treated with GW4869 (2.5 μg/mL × 48 h) or vehicle control. (B) Tumor volume was decreased on d10 compared with control group and tumor progression was slow, as control group mice died on day17. ∗p < 0.05, n = 11, Student’s t test. (C) Kaplan-Meier survival curves show GW4869 pre-treatment of cells significantly increased life span of HE-affected mice. Volume quantified using calipers (length ×·width ×·height). (D) Mice (129P3/J) injected with a combination of EOMA cells and MφEOMA EV. Sham cells were used from pair-matched mφ from the same isolation as for EVEOMA-treated cells except that Mφ were treated with EVMAE. Here, (i) threshold dose of EOMA cells caused tumor, (ii) sub-threshold of EOMA cells failed to cause tumor, (iii) sub-threshold dose caused tumor in presence of MφEOMA EV, (iv) sub-threshold dose failed to cause tumor in presence of sham cells. (E) Flipped skin shows subcutaneous tumor. (F) Tumor volumes were measured until d10 after injecting the above-mentioned cell quantities. ∗p < 0.05, n = 3 and 4), one-way ANOVA. (G) Schematic diagram demonstrating the study design of macrophage isolation and treatment with EVEOMA after delivering the antagomir-126 or scrambled oligonucleotides in EOMA cells by nano-electroporation (NEP). (H) Representative images of isolated wound macrophages on d7 using CD11b magnetic sorting was stained with LYVE1, F4/80, and CDH5, eNOS after incubation with EVEOMA (107/mL) for 7 days. (I) Percentage of colocalization of fluorescence intensity of different endothelial markers. Images were taken on three individual repeats. Results are expressed as mean ± SD, ∗p < 0.05, n = 6, two-way ANOVA. (J and K) d7 wound macrophages treated with EVEOMA+ NEP α-miR126 showed less intensity of endothelial markers compared with macrophages treated with EVEOMA+ NEP ci. Quantification of Pearson colocalization coefficient (K) shows significant decrease in endothelial marker intensity in treated group compared with control group. Results are expressed as mean ± SD, ∗p < 0.05, n = 6, two-way ANOVA. (L and M) Macrophages isolated from HE tumor after delivering the antagomir-126 by TNT showed a similar pattern in expressing endothelial and tumor markers to NEP, and colocalization quantification (M) confirms it. Results are expressed as mean ± SD, ∗p < 0.05, n = 6, two-way ANOVA. Data presented as mean ± SEM (B,F) or mean ± SD (I,K,M).