Elevated plasma concentrations of soluble VEGFA isoforms are associated with poor prognosis in parallel with improved response to treatment with the anti-VEGFA antibody bevacizumab. To uncover the underlying mechanism to these observations, we administered anti-VEGFA therapy to mice bearing luminescent mouse fibrosarcomas expressing single VEGFA isoforms or their wild-type counterparts expressing all isoforms (fs120, fs164, fs188, or fsWT). Expression of the more soluble isoforms conferred an advantage for lung metastasis from subcutaneous tumors (fs120/164 vs. fs188/WT); fs120 cells also produced more lung colonies than fs188 cells when injected intravenously. Metastasis from subcutaneous fs120 tumors was more sensitive than fs188 to treatment with the anti-VEGFA antibody B20-4.1.1. Despite elevated plasma levels of VEGFA in fs120 tumor-bearing mice and a dependence on VEGF receptor 1 activity for metastasis to the lung, B20-4

Micrometastasis is a barrier to the development of effective cancer therapies for prostate cancer metastasis to bone. The mechanisms remain incompletely characterised, primarily due to an inability to adequately monitor the initial metastatic events in vivo. This study aimed to establish a new model, allowing the tracking of prostate cancer cells homing to bone, and furthermore, to evaluate the response of this approach to therapeutic modulation, using the integrin antagonist GLPG0187. A single murine metatarsal was engrafted into a dorsal skinfold chamber implanted on a SCID mouse. Fluorescently-labeled human prostate (PC3-GFP) or oral (SCC4-GFP) cancer cells were administered via intracardiac (i.c) injection, with simultaneous daily GLPG0187 or vehicle-control treatment (i.p. 100 mg/kg/day) for the experimental duration. Metatarsal recordings were taken every 48 h for up to 4 weeks. Tissue was harvested and processed for microCT, multiphoton analysis, histology and immunohistochemistry. Cell viability, proliferation and migration in vitro were also quantified following treatment with GLPG0187. Metatarsals rapidly revascularised by inosculation with the host vasculature (day 5-7). PC3-GFP cells adhered to the microvascular endothelium and/or metatarsal matrix 3 days after administration, with adhesion maintained for the experimental duration. GLPG0187 treatment significantly (p < 0.05) reduced PC3 cell number within the metatarsal in vivo and reduced migration (p < 0.05) and proliferation (p < 0.05) but not cell viability in vitro. This new model allows evaluation of the early events of tumour-cell homing and localisation to the bone microenvironment, in addition to determining responses to therapeutic interventions.

<p>The supplementary data file contains the following data and figures: Fig S1- CD31 and CD34 co-localize to the vasculature in fs120-LS and fs188-LS subcutaneous tumors. Fig S2 - VEGFR1 activity has no effect on tumor growth or vascular density of subcutaneous fs120-LS or fs188-LS tumors. Fig S3 - fs120-LS and fs188-LS cells metastasize to the lung early during subcutaneous tumor growth. Fig S4 - fs120-LS cells express higher levels of PlGF2 than fs188-LS cells in vitro, with no difference in levels of PLGF2 detected in subcutaneous tumors. Fig S5 - Expression of laminin and collagen-I in lysates of fs120-LS and fs188-LS subcutaneous tumors. Fig S6 - Quantification of single cell migration using live video microscopy. Fig S7 - Localization of CD11b cells with respect to intravenously injected fibrosarcoma cells within the lung. Fig S8 - Summary of results showing key differences between fibrosarcomas expressing VEGF120 and VEGF188 and the effects of B20-4.1.1 and stromal VEGFR1 activity. Fig S9 - Effect of cediranib on survival of fs120-LS cells in the lung 48 h after iv injection.</p>

<p>The supplementary methods file contains additional detailed information regarding: 1. The production of cell lines stably expressing luciferase. 2. Mouse strains and pre-clinical models of tumor growth and metastasis. 3. Acquisition and analysis of bioluminescence data from preclinical and in vitro studies. 4. Acquisition and analysis of in vitro migration data. 5. Antibodies and immuno-techniques used within this study.</p>