Extracellular Vesicles as Biomarkers for the Detection of a Tumor Marker Gene in Epidermolysis Bullosa-Associated Squamous Cell Carcinoma Yuchen Sun,

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Extracellular Vesicles as Biomarkers for the Detection of a Tumor Marker Gene in Epidermolysis Bullosa-Associated Squamous Cell Carcinoma Yuchen Sun, Katharina Woess, Melanie Kienzl, Victoria M. Leb-Reichl, Andrea Feinle, Monika Wimmer, Roland Zauner, Verena Wally, Ursula Luetz-Meindl, Jemima E. Mellerio, Ignacia Fuentes, Andrew P. South, Johann W. Bauer, Julia Reichelt, Tomomi Furihata, Christina Guttmann-Gruber, Josefina Piñón Hofbauer Journal of Investigative Dermatology Volume 138, Issue 5, Pages 1197-1200 (May 2018) DOI: 10.1016/j.jid.2017.11.022 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Ct-SLCO1B3 is a tumor-specific marker gene in RDEB-SCC. (a) SLCO1B3 gene structure showing the transcription initiation sites of liver type (Lt) and cancer type (Ct) variants. (b) Relative expression (2ˆdCT values) of Ct-SLCO1B3 variant in normal human keratinocytes (NHK), RDEB keratinocytes (RDEB), and RDEB-SCC lines (SCC) by sqRT-PCR and normalized to GAPDH values. (c) The median expression levels (red line) of Ct-SLCO1B3 in each group were calculated. Ct-SLCO1B3 expression is significantly increased in RDEB-SCC cells (n = 7) compared with NHK (n = 3) (Mann-Whitney test, P = 0.0167) and nonmalignant RDEB Kc (n = 6) (Mann-Whitney test, P = 0.0012) (d) Agarose gel electrophoresis of sqRT-PCR amplified products after 50 cycles of amplification. (e) Ct-SLCO1B3 was also detected in total RNA isolated from several RDEB-SCC tumor biopsies by RT-PCR. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RDEB, recessive dystrophic epidermolysis bullosa; SCC, squamous cell carcinoma; sqRT-PCR, semiquantitative real-time reverse transcriptase-PCR. Journal of Investigative Dermatology 2018 138, 1197-1200DOI: (10.1016/j.jid.2017.11.022) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Ct-SLCO1B3 transcripts are detected in RDEB-SCC tumor-derived EVs. EVs isolated from serum-free conditioned medium using a differential centrifugation protocol (a) had a mean size of 176.3 nm as analyzed by dynamic light scattering (b) and confirmed by transmission electron microscopy (TEM) (c). (d) Western blotting detected CD81 in all, and TSG101 in two of four, EV preparations. Calnexin and GM130 were absent, demonstrating lack of contaminating cellular organelles. (e) Detection of Ct-SLCO1B3 transcripts in EVs derived from RDEB-SCC cells by RT-PCR. (f) Schematic of xenograft transplantation of RDEB-SCC tumor cells into CB17.Cg-PrkdcscidLystbg-J mice and isolation of EVs from the serum. (g) TEM analyses confirmed the size and morphology of isolated serum EVs. EVs from control mice are shown. (h) Ct-SLCO1B3 mRNA detected in serum EVs isolated from tumor-bearing mice by nested RT-PCR. Scale bar = 200 nm. EV, extracellular vesicle; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate buffered saline; RDEB, recessive dystrophic epidermolysis bullosa; RT-PCR, reverse transcriptase-PCR; SCC, squamous cell carcinoma; TEM, transmission electron microscopy. Journal of Investigative Dermatology 2018 138, 1197-1200DOI: (10.1016/j.jid.2017.11.022) Copyright © 2017 The Authors Terms and Conditions