Figure 3.

Metformin treatment limits metastasis formation in previously irradiated lung tissue. (A) C57BL/6 mice were left untreated (Ctrl), received metformin (Met) treatment, 15Gy WTI or received WTI in combination with metformin (Met) treatment for first three weeks post WTI. (B) Seeding of circulating tumor cells into the lungs was initiated 21 days after irradiation. Therefore, 0.5 × 10(6) metastatic tumor cells were intravenously transplanted via the tail vein of C57BL/6 mice. Fourteen days after tumor cell injection animals were sacrificed, lungs were isolated and subjected for lung histopathology using hematoxylin and eosin staining. Areas of extravasated tumor cells were emphasized by asterisks (B1, B3). Arrows point towards formed metastases (B1, B4). Scale bar: 200 µm; magnifications: 40 µm. (C) Sites of vascular leakage (designated as sites of tumor cell extravasation/micrometastasis) and (D) subsequently formed macrometastasis were quantified in whole lung sections (0Gy Ctrl, 0Gy Met, 15Gy Met: n = 7 per group; 15Gy ctrl: n = 6). Data are shown as individual biological replicates with mean values ±SEM. p-values indicate: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001 by one-way ANOVA with post hoc Tukey’s multiple comparison test.