A novel method for isolating lymphatic endothelial cells from lymphatic malformations and detecting PIK3CA somatic mutat
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ORIGINAL ARTICLE
A novel method for isolating lymphatic endothelial cells from lymphatic malformations and detecting PIK3CA somatic mutation in these isolated cells Hidehito Usui1 · Yoshinori Tsurusaki2 · Hiroko Shimbo2 · Hirotomo Saitsu3 · Noriaki Harada4 · Norihiko Kitagawa1 · Kyoko Mochizuki1 · Munetaka Masuda5 · Kenji Kurosawa6 · Masato Shinkai1 Received: 30 April 2020 / Accepted: 17 July 2020 © Springer Nature Singapore Pte Ltd. 2020
Abstract Purpose Tissue disaggregation and the cell sorting technique by surface markers has played an important role in isolating lymphatic endothelial cells (LECs) from lymphatic malformation (LM). However, this technique may have the drawback of impurities or result in isolation failure because it is dependent on surface marker expressions, the heterogeneity of which has been found in the lymphatic system. We developed a novel method for isolating LM-LECs without using whole tissue disaggregation. Methods Seven LM surgical specimens were collected from seven patients with LMs. LM-LECs were detached from the LM cyst wall by “lumen digestion” and irrigating the cystic cavity with trypsin, and maintained in culture. Results The cells formed a monolayer with a cobblestone-like appearance. Immunohistochemistry and quantitative RT-PCR of these cells revealed high expression of lymphatic-specific genes, confirming their identity as LM-LECs. The whole-exome sequencing and PIK3CA sequencing of these cells revealed somatic mutations in PIK3CA in all cases. Conclusions We established a novel technique for isolating LM-LECs from LM tissue by “lumen digestion” without wholetissue disaggregation. The limited incorporation of non-LM LECs in the isolate in our method could make it an important tool for investigating the heterogeneity of gene expression as well as mutations in LM-LECs. Keywords Lymphangioma · Lymphatic malformations · Lymphatic endothelial cell · PIK3CA
Introduction * Hidehito Usui [email protected] 1
Department of Surgery, Kanagawa Children’s Medical Center, 2‑138‑4, Mutsukawa, Minami‑ku, Yokohama, Japan
2
Clinical Research Institute, Kanagawa Children’s Medical Center, 2‑138‑4, Mutsukawa, Minami‑ku, Yokohama, Japan
3
Department of Biochemistry, Hamamatsu University School of Medicine, 1‑20‑1, Hanndayama, Higashi‑ku, Hamamatsu, Japan
4
Department of Clinical Laboratory, Kanagawa Children’s Medical Center, 2‑138‑4, Mutsukawa, Minami‑ku, Yokohama, Japan
5
Department of Surgery, Yokohama City University, 3‑9, Fukuura, Kanazawa‑ku, Yokohama, Japan
6
Department of Medical Genetics, Kanagawa Children’s Medical Center, 2‑138‑4, Mutsukawa, Minami‑ku, Yokohama, Japan
Lymphatic malformations (LM), also known as lymphangioma, are low-flow vascular anomalies in the lymphatic system. LMs form a mass lesion composed of thin-walled irregularly shaped lymphatic cysts of varying size, lined with lymphatic endothelial cells (LECs) [1]. The International Society for the Study of Vascular Anomalies classifies LMs into macrocystic (diameter ≥ 1 cm), microcys
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