Abstract A57: Differential tumor cell expression of VEGF isoforms impacts on the tumor microenvironment, metastasis and radiation response in a mouse fibrosarcoma model
Authors: English William R. , Lunt Sarah Jane , Fisher Matthew , Hurrell Jack E. , Lefley Diane V. , Mukherjee Debayan , Daniel Rachel , Lyons Scott K. , Kanthou Chryso , Tozer Gillian M. 
Published: May 2015

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 &lt; 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