Background: Mortality in cancer patients is predominantly caused by metastasis. Understanding mechanisms that lead to cancer cells gaining metastatic potential may lead to novel therapies that could prevent metastasis and improve overall survival. Increased cellular plasticity in adopting different modes of migration has been identified as an important factor in efficient metastasis. Although it is predominantly known as a major regulator of angiogenesis, Vascular Endothelial Growth Factor A (VEGFA) has also been shown to regulate cell migration and thus metastatic potential. VEGFA encodes for several isoforms through alternate mRNA splicing which exerts differences in signalling and biological activity. Using fibrosarcomas derived from mouse embryonic fibroblasts genetically engineered to express a single VEGFA isoform, previous research I contributed to showed there was an increased number of metastases to the lung from cells expressing only VEGFA120 (fs120 cells) compared to cells expressing VEGFA188 (fs188 cells) suggesting fs120 cells may have better metastatic potential. These studies also showed metastasis to the lung was selectively inhibited by anti-VEGFA antibodies in mice with fs120 tumours and this acted in part through inhibition of migration (English et al., 2017). During my MSc studies, I extended these studies of migration and found fs120 cells could migrate more efficiently in a custom non-adherent migration chamber than fs188 cells and this was inhibited by anti-VEGFA antibodies. As these suggested VEGFA120-expressing cells have greater plasticity in modes of cell migration and can adapt to more varied environments than VEGFA188-expressing cells, we hypothesised increased metastatic potential is VEGFA isoform-dependent and linked to an ability to adopt different migration modes in response to different microenvironments. However, as there are differences in embryonic development between mice expressing VEGFA120 and VEGFA188, and the mice were not isogenic, these findings needed to be confirmed in a new model with identical developmental and genetic backgrounds.
Objectives: To create new fibrosarcoma cell lines expressing single VEGFA isoforms to characterise differences in cell migration in comparison with embryonic-derived fibrosarcomas and to develop novel engineered microenvironments to test the plasticity of cell migration.
Methodology: Fibrosarcomas expressing only VEGFA120 or VEGFA188 were derived from Vegfa knockout (KO) adult mouse fibroblasts by stably introducing a VEGFA expression cassette using a transposon. Cellular functions, including cell proliferation, and the expression of cell migration-related proteins were characterized by MTT assay and western blotting in selected clones. PCL electrospinning of fibre scaffolds was used to mimic fibrillar collagen structures seen after imaging fs120 and fs188 mouse tumour sections by second-harmonic generation microscopy to generate a novel environment to characterise plasticity in migration. Cell migration capacity of adult- versus embryonic-derived fibrosarcomas expressing a single VEGFA isoform was characterised using in vitro platforms by single-cell live imaging. Altered expression of cell migration-related proteins across cell lines on treatment with inhibitors targeting VEGFA signal transduction (VEGFA by antibody B20.4.1.1, VEGFR1 by antibody MF-1 and VEGFR/PDGFR/FGFR by receptor tyrosine kinase inhibitor pazopanib) were characterized by Western blotting.
Results: The expression of single VEGFA isoforms in new, fibrosarcoma cell lines, derived from Vegfa KO fibroblasts, were successfully generated to express VEGFA at comparable levels to the fs120 and fs188 cells as determined by ELISA and QRT-PCR. There was no difference in the expression of VEGFR1 across any of the cell lines, but the upregulated expression of NRP1 was seen in fs188 cells compared to other cell lines. Increased phosphorylated Akt was observed in all fibrosarcoma cells generated from embryonic fibroblasts and in adult Vegfa KO cells expressing VEGFA188. Moreover, fs120 cells expressed a higher level of SOX2 than all other cell lines. The migration capacity of cells on 2D surfaces coated with fibronectin or collagen and on 3D-aligned fibre scaffolds was similar between VEGFA120 and VEGFA188 expressing cells, and independent of their origin. Interestingly, suppressed migration capacity was detected only in cells expressing VEGFA120 on treatment with the anti-VEGFA antibody, B20.4.1.1, indicating VEGFA isoform selective response independent of cell origin. The anti-VEGFR1 antibody MF-1 did not inhibit migration. Increased phosphorylation of VEGFR1 in VEGFA120-expressing fibrosarcoma cells was observed on treatment with B20.4.1.1 and pazopanib, but not MF-1. However, increases in phosphorylated Akt were repressed only by pazopanib treatment.
Conclusions: Fibrosarcoma cells expressing different VEGFA derived from different origins share similar characteristics, suggesting VEGFA isoform-independent changes between models. Downregulated migration capacity in fibrosarcoma cells expressing VEGFA120 in response to anti-VEGFA antibody, B20.4.1.1, was also independent of cell origin. Fibrosarcoma cells expressing VEGFA120 and VEGFA188 have comparable capacity in 2D and fibrillar migration. The differential expression of a VEGFA single isoform may not contribute to the modulation of plasticity in cell migration modes. However, in previous results from the MSc thesis, the motility of fs188 cells was abolished in the confined non-adherent chamber compared with fs120 cells. This discrepancy suggests that the varying expression of VEGFA isoforms in fibrosarcoma cells may play roles in regulating the adaptation of amoeboid mode that is integrin-independent, as opposed to migrating on 1D fibrillar structures. Nonetheless, confirming this idea would necessitate evaluating the migratory abilities of new cell lines within the confined non-adherent chamber. Upregulation of p-VEGFR1 was observed in VEGFA120-expressing cells treated with anti-VEGFA antibody and pazopanib, whereas downregulation of p-Akt was observed only with the pazopanib treatment. These suggest another receptor also interacting with VEGFA120 participates in regulating cell migration. In conclusion, the expression of VEGFA120 may potentially increase cell plasticity through another receptor, besides VEGFR, interacting with VEGFA. This signalling pathway is affected explicitly by the anti-VEGFA antibody and pazopanib. These outcomes open up a novel target for further treatment to prevent metastasis.
