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EB House Austria, Research Program for the Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, AustriaLaboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan
EB House Austria, Research Program for the Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria
EB House Austria, Research Program for the Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria
EB House Austria, Research Program for the Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria
EB House Austria, Research Program for the Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria
EB House Austria, Research Program for the Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria
EB House Austria, Research Program for the Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria
EB House Austria, Research Program for the Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria
Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, JapanDepartment of Pharmacology, Graduate School of Medicine, Chiba University, Chiba, Japan
11 These authors contributed equally to this work.
Christina Guttmann-Gruber
Footnotes
11 These authors contributed equally to this work.
Affiliations
EB House Austria, Research Program for the Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria
11 These authors contributed equally to this work.
Affiliations
EB House Austria, Research Program for the Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria
Extracellular vesicles (EVs) are cell-derived membrane-bound vesicles that are important mediators of intercellular communication, and critical orchestrators of both physiological and pathologic processes. They are found in various biological fluids, including serum/plasma, breast milk, and urine (
). EVs include both microvesicles, generated by outward budding from the plasma membrane, and exosomes, formed by inward budding into endosomes and released into the extracellular environment by subsequent fusion with the plasma membrane (
). The molecular content of EVs includes proteins, lipids, DNA, and RNA, and collectively represents a fingerprint of the cell from which they originated. In this respect, and in the context of cancer, tumor-derived EVs that circulate in the blood represent a source of specific biomarkers that can be utilized for cancer detection and diagnosis, as well as to gain insight into the biology of the tumor (
). Recently, EVs have been implicated in the crosstalk between stromal fibroblasts and tumor cells, enhancing tumor-relevant signaling cascades in oral squamous cell carcinoma (SCC) cells (
). As such, deciphering the bioactive cargo of tumor-derived EVs and their impact on shaping the tumor microenvironment may offer fresh insight into pathophysiological tumor mechanisms and uncover previously unrecognized targets for therapy.
Patients with recessive dystrophic epidermolysis bullosa (RDEB) carry loss-of-function mutations in COL7A1, which is essential for maintaining the integrity of the basement membrane zone in the skin and mucous membranes (
). Owing to repeated cycles of wounding, infection, and chronic inflammation, patients with RDEB develop highly aggressive SCC at sites of persistent wounds, the pathomechanisms of which are poorly understood. Arising tumors resemble nonhealing wounds or exuberant granulation tissue (
), requiring invasive biopsies and histological analyses to confirm diagnosis. Furthermore, tumors that have metastasized to internal tissues are not readily detected in regular patient checkups. Here, we investigate the feasibility of utilizing tumor-derived EVs as liquid biopsies for the detection of a recently described tumor marker gene Ct-SLCO1B3 (also known as Ct-OATP1B3 mRNA). Gene-level expression profiling of RDEB-SCC cells compared with nontumor keratinocytes consistently identified SLCO1B3 to be one of the most differentially expressed genes between the two groups (Supplementary Figure S1 online). SLCO1B3, previously reported to be overexpressed in RDEB-SCC (
Type VII collagen regulates expression of OATP1B3, promotes front-to-rear polarity and increases structural organisation in 3D spheroid cultures of RDEB tumour keratinocytes.
), encodes a member of the organic anion transporting polypeptide superfamily whose expression is normally restricted to the liver. Two variant transcripts of SLCO1B3 are known to exist, the normal liver-type (Lt) transcript and a cancer-type (Ct) transcript that has been identified in several solid tumors and is transcribed from an alternate exon (Ct-exon 1) (
A cancer-specific variant of the SLCO1B3 gene encodes a novel human organic anion transporting polypeptide 1B3 (OATP1B3) localized mainly in the cytoplasm of colon and pancreatic cancer cells.
) (Figure 1a). Using primer pairs capable of distinguishing between the two variants, semiquantitative real-time PCR demonstrated that although the expression level varied greatly across the different tumor cell lines, Ct-SLCO1B3 transcripts were specifically detected in all patient-derived RDEB-SCC cell lines investigated (Figure 1b–d) as well as in several tumor biopsies taken from patients (Figure 1e). Importantly, we did not detect Ct-SLCO1B3 expression in either nontumor RDEB keratinocyte or normal human keratinocyte lines (Figure 1b–d). Lt-SLCO1B3 transcripts were not detected in any nonmalignant keratinocyte line tested and only marginally in several RDEB-SCC lines tested (Supplementary Figure S1). These data demonstrate that Ct-SLCO1B3 is a tumor marker gene in RDEB-SCC.
Figure 1Ct-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.
To determine whether Ct-SLCO1B3 could be detected in EVs and thus serve as a potential biomarker for SCC screening, we isolated EVs from conditioned cell culture medium. RDEB tumor and nontumor cells were grown to confluence, washed with phosphate buffered saline, and then cultivated for a further 4 days in serum-free medium (Figure 2a). Cell culture supernatants were harvested on days 2 and 4 after switching to serum-free conditions, pooled, filtered through a 0.22-μm membrane, and finally subjected to a differential centrifugation protocol to pellet the EVs (
) (Supplementary Methods online; see also Supplementary Tables S1–S3 online). Dynamic light scattering analyses (Zetasizer Nano, Malvern Instruments, Worcestershire, UK) revealed that isolated EVs had an average particle size of 176.3 nm (Figure 2b), which was confirmed by transmission electron microscopy (Figure 2c). Western blot analyses demonstrated that the EVs isolated from tumor cells were highly enriched for the exosome marker CD81, whereas TSG101 was not consistently expressed in all samples tested (Figure 2d). EV biogenesis and release is reported to be enhanced in cancer cells (
and references therein). Not surprisingly, reduced CD81 levels, which may be indicative of lower numbers of EVs per microliter sample, were observed in preparations derived from RDEB keratinocytes as compared with those derived from tumor cell lines. Importantly, all EV fractions were negative for the endoplasmic reticulum marker calnexin and the Golgi marker GM130. Notably, reverse transcription PCR analyses demonstrated the expression of Ct-SLCO1B3 exclusively in EVs isolated from the conditioned medium of RDEB-SCC lines and not in those derived from nontumor cells (Figure 2e). Finally, to demonstrate the utility of this detection method in an in vivo setting, we injected RDEB-SCC2 cells, previously shown to reliably produce xenograft tumors (
), intradermally into the flanks of CB17.Cg-PrkdcscidLystbg-J mice (Figure 2f). When tumor volume reached 500 mm3, mice were euthanized and blood was collected. Blood from mice that had not received tumor cells served as controls. Serum EVs were isolated using the ExoQuick kit (System Biosciences, Palo Alto, CA) following the manufacturer’s protocols. Transmission electron microscopy and dynamic light scattering analyses of EVs isolated from the serum of control mice demonstrated that the particles were predominantly <200 nm in size (Figure 2g and data not shown), confirming the success of the isolation protocol. Finally, reverse transcriptase PCR and subsequent nested PCR on the amplified products using primer sets specific for human Ct-SLCO1B3 confirmed the presence of Ct-SLCO1B3 transcripts in EVs isolated from the serum of RDEB tumor-bearing mice but not in those isolated from control serum (Figure 2h). Primers specific for GAPDH amplified a specific product in all serum-derived EV samples tested.
Figure 2Ct-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.
Collectively, our data establish Ct-SLCO1B3 as a robust and reliable tumor marker in RDEB-SCC that could be exploited as a biomarker for this cancer. Toward this end, we demonstrate the isolation of RDEB-SCC-derived EVs both in vitro and from the serum of tumor-bearing mice, and show that these contain Ct-SLCO1B3 transcripts, highlighting the feasibility of this minimally invasive method in the detection of RDEB-SCC particularly once it has metastasized beyond the skin.
All human tissue samples described in this study were taken for routine examination purposes at the Department of Dermatology, University Hospital Salzburg, with written informed of the patient in accordance with the Declaration of Helsinki and approval of institutional and local ethics committees. All animal experiments were performed with the approval of the local regulatory committees.
Acknowledgments
This project was funded by the Paracelsus Medical University Salzburg (E-14/19/098GRU) and DEBRA Austria. YS was supported by the Tobitate! Study Abroad Initiative and Chiba University’s Program to Support Sending Graduate Students Abroad. TF was supported by JSPS KAKENHI (15K14994). We thank Birgit Tockner, Anna Stierschneider, Anna Neumayer, and Ancuela Andosch for excellent technical assistance, and the group of Dr. Hannelore Breitenbach-Koller for the use of their ultracentrifuge.
Type VII collagen regulates expression of OATP1B3, promotes front-to-rear polarity and increases structural organisation in 3D spheroid cultures of RDEB tumour keratinocytes.
A cancer-specific variant of the SLCO1B3 gene encodes a novel human organic anion transporting polypeptide 1B3 (OATP1B3) localized mainly in the cytoplasm of colon and pancreatic cancer cells.
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