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A novel method for isolating lymphatic endothelial cells from lymphatic malformations and detecting PIK3CA somatic mutation in these isolated cells

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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 whole-tissue 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.

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Funding

This study was funded by the Kanagawa Prefectural Hospital’s Child Health Care and Research Foundation (to H.U.), the Naito Foundation (to Y.T.), and the Yokohama Foundation for Advancement of Medical Science (to Y.T.).

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Authors and Affiliations

  1. Department of Surgery, Kanagawa Children’s Medical Center, 2-138-4, Mutsukawa, Minami-ku, Yokohama, Japan

    Hidehito Usui, Norihiko Kitagawa, Kyoko Mochizuki & Masato Shinkai

  2. Clinical Research Institute, Kanagawa Children’s Medical Center, 2-138-4, Mutsukawa, Minami-ku, Yokohama, Japan

    Yoshinori Tsurusaki & Hiroko Shimbo

  3. Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1, Hanndayama, Higashi-ku, Hamamatsu, Japan

    Hirotomo Saitsu

  4. Department of Clinical Laboratory, Kanagawa Children’s Medical Center, 2-138-4, Mutsukawa, Minami-ku, Yokohama, Japan

    Noriaki Harada

  5. Department of Surgery, Yokohama City University, 3-9, Fukuura, Kanazawa-ku, Yokohama, Japan

    Munetaka Masuda

  6. Department of Medical Genetics, Kanagawa Children’s Medical Center, 2-138-4, Mutsukawa, Minami-ku, Yokohama, Japan

    Kenji Kurosawa

Authors
  1. Hidehito Usui
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  2. Yoshinori Tsurusaki
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  3. Hiroko Shimbo
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  4. Hirotomo Saitsu
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  5. Noriaki Harada
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  6. Norihiko Kitagawa
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  7. Kyoko Mochizuki
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  8. Munetaka Masuda
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  9. Kenji Kurosawa
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  10. Masato Shinkai
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Correspondence to Hidehito Usui.

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Usui, H., Tsurusaki, Y., Shimbo, H. et al. A novel method for isolating lymphatic endothelial cells from lymphatic malformations and detecting PIK3CA somatic mutation in these isolated cells. Surg Today 51, 439–446 (2021). https://doi.org/10.1007/s00595-020-02122-3

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  • DOI: https://doi.org/10.1007/s00595-020-02122-3

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