Tinzaparin attenuates proliferation of melanoma cells in primary tumor and lymph nodes and attenuates tumor progression in ret transgenic mice. Immunofluorescence stainings of tumor cell proliferation with Ki67 (green) and DAPI (nuclei, blue). Cryosections of vehicle-treated tumors (A) show more proliferating melanoma cells compared with tinzaparin-treated tumors (B; scale bars = 50 µm). Quantification showed a significant reduction of Ki67-positive cells after treatment with tinzaparin (C). Analysis of Ki67-positive tumor cells in lymph nodes of vehicle-treated ret mice and tinzaparin-treated animals was assessed by flow cytometry. Results show that tinzaparin induces a significant reduction of proliferating melanoma cells (D). Bars show mean ± SD (n = 7-10). Tumor-derived VEGF was measured in tumor and lymph nodes of ret transgenic mice (E-F) by bio-plex assay. VEGF levels were decreased in tumor samples (E) and lymph nodes (F) after treatment with tinzaparin (n = 5-7 different mice each group). Consequently, a survival benefit (G) and a reduced tumor weight (H) compared with vehicle-treated control was observed after tinzaparin treatment (n = 13 of 2 independent experiments). Plot shows mean ± SD; *P < .05. See also supplemental Video 1.