Complexity of VEGF Responses in Skin Carcinogenesis Revealed through Ex Vivo Assays Based on a VEGF-A Null Mouse Keratinocyte Cell Line  Isabel Mirones, Claudio J. Conti, Jesús Martínez, Marta Garcia, Fernando Larcher  Journal of Investigative Dermatology  Volume 129, Issue 3, Pages 730-741 (March 2009) DOI: 10.1038/jid.2008.292 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Generation of VEGF-null mouse keratinocytes. Appearance (phase contrast micrography) of cultured VEGF wild-type (WT) polyclonal VMIK (a) and VEGF-null (b) mouse clone 42A8F5 (passage 21) in low calcium medium. Scale bar=100μm. (c) Schematic representation of the WT-floxed VEGF allele, and recombined, VEGF-null allele. A 2kb Sac I–Sac I fragment results from excision of the VEGF exon 3. Black boxes represent exons. The hybrid probe is represented by a closed box over the gene. (d) Southern blot analysis of genomic DNA, digested with Sac I and hybridized to a 422pb probe, showing the 4 and 2kb Sac I fragments of the WT and deleted in exon 3 VEGF allele (Δ), respectively. Each lane corresponds to different representative VEGF-null (42A8F5) or WT (8A1, 8B1 and 8C1) clones. Journal of Investigative Dermatology 2009 129, 730-741DOI: (10.1038/jid.2008.292) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 VEGF protein expression in VEGF-WT and -null clones. (a) Establishment of different mouse keratinocyte clones from primary VEGF-LoxP cells. (b) VEGF-A protein levels assessed by ELISA in the supernatant of VEGF-WT clones (from clone 42A8E12 to 42A8E8) and adeno Cre-treated keratinocytes clones (from clone 42A8F5 to clone B7). Black arrows indicate the clones selected for the study and colored arrows indicate the ras-transformed clones used for the study on tumor growth and metastasis formation. Journal of Investigative Dermatology 2009 129, 730-741DOI: (10.1038/jid.2008.292) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Growth, differentiation, and lack of tumorigenicity of VEGF-WT and -null clones. (a) Analysis of proliferation of VEGF-WT (8A1 clone) and VEGF-null (42A8F5 clone) keratinocytes. Primary mouse keratinocytes cultures were used as control. Cells were plated at a density of 105 per dish in the complete keratinocyte medium (low Ca2+). Cells were counted daily for 3 days. Asterisks indicate a P-value <0.01. (b) Immunofluorescence analysis of K5 keratin expression (green) in 42A8F5 VEGF-null clone. DAPI (blue) was used to stain nuclei. Scale bar=25μm. (c) Induction of differentiation markers after calcium switch. Double immunofluorescence against keratin K1 (green) and K10 (red) in primary, VMIK (8A1 clone), and VEGF-null (42A8F5 clone) keratinocytes after 24 and 72hours of Ca2+-induced differentiation. DAPI (blue) was used to stain nuclei. Scale bar=25μm (d) Absence of tumorigenesis in untransformed VEGF-WT and -null keratinocyte cell lines. The mouse on the right (no tumor development) was injected with clone 8A1. The mouse on the left (tumor surrounded by dotted line) was injected with VMIK cells transformed by retroviral transduction of the Hras oncogene (used as positive control for tumor development). Journal of Investigative Dermatology 2009 129, 730-741DOI: (10.1038/jid.2008.292) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Subcutaneous tumor development in Hras-transformed VEGF-WT and -null keratinocyte cell lines. (a) Hras-transduced VEGF-WT (5A11 and 5C10 clones) and VEGF-null (6C12 and 6D9 clones) cells form undifferentiated carcinomas in immunodeficient mice. Tumor volume of VEGF-WT-Hras and VEGF-null-Hras keratinocyte clones after subcutaneous injection became highly divergent from day 21. Results (mean±SE) are from six mice in each group. (b) In vivo fluorescence imaging of representative tumor-bearing mice 8 weeks after subcutaneous cell injection. Representative animals injected either with clone 5C10 (left) or with VEGF-null clone 6C12 (right)) were illuminated with white light (left panel) or blue (GFP exciting) light (right panel). Note the large tumor burden in the animal injected with the VEGF-WT clone as compared with the animal injected with VEGF-null cells. The VEGF-null tumor is indicated with a white arrow. (c) Histopathology of subcutaneous tumors. Left panels: GFP expression (immunoperoxidase staining). Inset (upper panel left) shows macroscopic GFP direct fluorescence. Right panels: H&E staining. Upper panels: VEGF-WT-Hras tumor. Lower panels: VEGF-null-Hras tumor. Arrowheads indicate lacunar blood vessels. Scale bar=100μm. (d) Proliferation assessed by BrdU incorporation (upper panels). Scale bar=25μm. Tumor apoptosis assessed by TUNEL assay (lower panels). (e and f) Quantitative data of BrdU- and TUNEL-positive nuclei from five different fields from each tumor section (mean±SE). Journal of Investigative Dermatology 2009 129, 730-741DOI: (10.1038/jid.2008.292) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Angiogenic performance of VEGF-WT and VEGF-null skin tumors. (a) Analysis of tumor vasculature in microvessels by CD31 immunofluorescence. CD31-positive capillaries are visualized in green (FITC label). Scale bar=100μm (b) Bar graph shows the number of CD31-positive vessels staining per field. (c) Quantitative Evans blue permeability assay in tumors derived from clones 5C10 and 6C12 (mean±SE). (d) Southern blot analysis of DNA from cultured cells reestablished from VEGF-WT-Hras tumors (lane 3), VEGF-null-Hras tumors (lane 2), and polyclonal VMIK cells (lane 1). (e) Northern blot analysis of PlGF in clones 5C10, 8A1, 6C12, and 42A8F5 (lanes marked as 1, 2, 3, and 4 respectively). Membranes were probed for PlGF and 7S RNA probe to verify that equal amounts of mRNA were loaded and transferred. Journal of Investigative Dermatology 2009 129, 730-741DOI: (10.1038/jid.2008.292) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Metastatic ability of VEGF-null and -WT cells. (a) Macroscopic appearance and GFP fluorescence of lung metastatic nodules 50 and 100 days after tail vein injection of VEGF-WT-Hras cells (clone 5C10) and unaffected lungs after injection of VEGF-null-Hras cells (clone 6C12). Scale bar=1cm. (b) H&E staining of micrometastasis (indicated by arrows and dotted lines) corresponding to VEGF-WT injected mice lungs at 50 days after injection (upper panel left), 100 days after injection (lower panel left), and unaffected lung from mice that received injection of VEGF-null cells (lower panel right). Scale bar=200μm. Quantitative data of micrometastasis from five different fields from each section at 100 days after injection (mean±SE; upper panel right). Journal of Investigative Dermatology 2009 129, 730-741DOI: (10.1038/jid.2008.292) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions