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.

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.

Abstract 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, B20-4.1.1 did not affect survival in the lung on intravenous injection. B20-4.1.1 inhibited subcutaneous tumor growth and decreased vascular density in both fs120 and fs188 tumors. However, migration of fs120, but not fs188 cells, in vitro was inhibited by B20-4.1.1. The greater survival of fs120 cells in the lung was associated with VEGFR1-dependent accumulation of CD11b-positive myeloid cells and higher expression of the VEGFR1 ligand, PlGF2, by the fs120 cells in vitro and in the plasma and lungs of fs120 tumor-bearing mice. We conclude that soluble VEGFA isoform expression increases fibrosarcoma metastasis through multiple mechanisms that vary in their sensitivity to anti-VEGF/VEGFR inhibition, with VEGFA-targeted therapy suppressing metastasis through effects on the primary tumor rather than the metastatic site. Cancer Res; 77(10); 2633–46. ©2017 AACR.

Urology Research Laboratory 3 , Department of Urology, University of Bern, Murtenstrasse 35, CH3010, Bern, Switzerland Department of Urology 4 , Leiden University Medical Center, J3-100, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Urology & Oncology 5 , Nuffield Department of Surgery, John Radcliffe Hospital University of Oxford, Oxford, OX3 9DU, UK Galapagos SASU 6 , 60, Avenue Gaston Roussel, 93230 Romainville, France

Abstract We aimed to determine the influence of differential tumor cell expression of vascular endothelial growth factor A (VEGF) isoforms on lung metastasis and response to radiotherapy. We hypothesized that vascular and other adaptations of the tumor microenvironment, in response to tumor cell expression of individual VEGF isoforms, would impact on tumor progression and treatment response. Mouse fibrosarcoma cells that exclusively express either VEGF120, 164 or 188 (fs120, fs164 and fs188 cells respectively) and cells expressing all three isoforms (fsWT) were grown as sub-cutaneous implants in SCID mice. Tumor sections were stained for vascularity (CD31), oxygenation status (pimonidazole protein adducts), activated fibroblasts/pericytes (FAPalpha/alpha-sma), necrosis (H&E), apoptosis (TUNEL) and extra-cellular matrix proteins (collagen-1, laminin, fibronectin). Interstitial fluid pressure (IFP) was measured by the ‘wick-in-needle’ technique. Parallel single VEGF isoform-expressing cell lines were generated to stably express luciferase2 and mStrawberry (LS) for analysis of lung metastasis from sub-cutaneous implants. Luminescence was measured in lung tissue lysates, using an IVIS Lumina II optical imaging system. Sub-cutaneous tumors were irradiated with a single or fractionated 20 Gy dose of X-rays and tumor volume response measured. All tumors were well-vascularized but IFP was lower in untreated fs188 tumors than in fs120 tumors, consistent with a lower vascular permeability, as previously reported. Fs188 tumors were also significantly less hypoxic (P < 0.05) than fs120 tumors (approximately 20 versus 30% pimonidazole staining). Perivascular alpha-SMA was most prevalent in fs188 and fsWT tumors. FAPalpha was lowest in fs120 tumours. Lungs from fs120-LS and fs164-LS but not fs188-LS and fsWT-LS tumor-bearing mice were significantly more luminescent than control lungs (910 ± 283 and 1107 ± 401 versus 8 ± 3 luminescence units respectively). Laminin staining was significantly greater in the more metastatic tumor types (fs120 and fs164) but no other matrix protein showed any correlation. Fs188 tumors were significantly more sensitive to both single and fractionated radiotherapy (growth and necrosis end-points) than fs120 tumors, despite similar radio-sensitivities of the cells irradiated in vitro. However, TUNEL staining in CD31+ cells was greater in fs120 tumors than in fs188 tumors, suggesting that fs120-associated vasculature was more radio-sensitive than fs188-associated vasculature. Results showed that tumor cell expression of individual VEGF isoforms had a profound effect on the tumor microenvironment. The more metastatic fs120 tumors had relatively high IFP and hypoxia, factors known to influence tumor progression. Less pericyte investiture in fs120 and fs164 tumors could also facilitate tumor cell escape into the circulation for these more metastatic types, which may also relate to high levels of laminin in the extra-cellular matrix. The radio-sensitivity of fs188 tumors is most likely explained by relatively low hypoxia, as oxygen is a classic radio-sensitizer. The fact that blood vessels in the fs120 tumors were actually more sensitive that those in fs188 tumors suggests that the response of blood vessels has only a minor influence on the overall tumor response to radio-therapy. These results suggest the potential for differential VEGF isoform expression (or down-stream consequences of expression) as prognostic and predictive tumor bio-markers. This work was supported by a Programme Grant from Cancer Research UK. Citation Format: William R. English, Sarah Jane Lunt, Matthew Fisher, Jack E. Hurrell, Diane V. Lefley, Debayan Mukherjee, Rachel Daniel, Scott K. Lyons, Chryso Kanthou, Gillian M. Tozer. Differential tumor cell expression of VEGF isoforms impacts on the tumor microenvironment, metastasis and radiation response in a mouse fibrosarcoma model. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A57. doi:10.1158/1538-7445.CHTME14-A57

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.