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Exploration of Pericyte-Derived Factors Implicated in Lung Cancer Brain Metastasis Protection: A Pilot Messenger RNA Sequencing Using the Blood–Brain Barrier In Vitro Model

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Abstract

Metastatic brain tumors have poor prognoses and pose unmet clinical problems for the patients. The blood–brain barrier (BBB) implication is supposed to play a major role in brain metastasis. However, the role of pericytes remains to be elucidated in the brain metastasis. This pilot study described the expression profile of interactions between pericytes, endothelial cells, and cancer cells. We applied an in vitro BBB model with rat primary cultured BBB-related cells (endothelial cells and pericytes), and performed the gene expression analyses of pericytes under the lung cancer cells coculture conditions. Pericytes demonstrated inhibition of the cancer cell proliferation significantly (p < 0.05). RNA was extracted from the pericytes, complementary DNA library was prepared, and RNA-seq was performed. The sequence read data were analyzed on the Management and Analysis System for Enormous Reads and Tag Count Comparison-Graphical User Interface platforms. No statistically or biologically significant differentially expressed genes (DEGs) were detected in the explanatory analyses. Lot-specific DEG detection demonstrated significant decreases in the expression of two genes (Wwtr1 and Acin1), and enrichment analyses using Metascape software revealed the inhibition of apoptotic processes in fibroblasts. Our results suggest that the expression profiles of brain pericytes are partially implicated in the prevention of lung cancer metastasis to the brain. Pericytes exerted an anti-metastatic effect in the BBB model, and their neurohumoral factors remain to be elucidated.

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Acknowledgements

Portions of this work were presented at the 37th Annual Meeting of the Japan Society for Neuro-Oncology, Nanao-City, Ishikawa, Japan, December 2, 2019 (Ujifuku et al. 2019). We referred to Online Mendelian Inheritance in Man (OMIM) (Amberger et al. 2011) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa and Goto 2000) in this study. Computations were partially performed on the NIG supercomputer at ROIS National Institute of Genetics. The authors would like to thank Enago (www.enago.jp) for the English language review.

Funding

This work was supported by JSPS and HAS under the Japan-Hungary Research Cooperative Program (to Y.M.) and, in part, Grants-in-Aid for Scientific Research from JSPS KAKENHI (C)17K10839 (to K.U.), (C)17K10869 (to T.M.), and (C) 17K10840 (to Y.M.), and Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research; BINDS) from AMED under Grant Number JP17am0101001.

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Authors and Affiliations

  1. Department of Neurosurgery, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan

    Kenta Ujifuku & Takayuki Matsuo

  2. Department of Neurosurgery, Nagasaki University Hospital, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan

    Takashi Fujimoto, Kei Sato & Yoichi Morofuji

  3. Biomedical Research Support Center, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki, 852-8523, Japan

    Hideki Muto & Hiroshi Masumoto

  4. Department of Medical Pharmacology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki, 852-8523, Japan

    Shinsuke Nakagawa

  5. BBB Laboratory, PharmaCo-Cell Company Ltd., Dai-ichi-senshu bldg. 2nd floor, 6-19 Chitose-machi, Nagasaki, 852-8135, Japan

    Masami Niwa

Authors
  1. Kenta Ujifuku
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  2. Takashi Fujimoto
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  4. Yoichi Morofuji
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  7. Shinsuke Nakagawa
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  9. Takayuki Matsuo
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Contributions

KU, TF, YM, and SN contributed to conception and design. KU, TF, and YM contributed to development of methodology. KU, TF, KS, and HM contributed to acquisition of data. KU and HM are involved in analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis). KU, YM, and SN are involved in writing, review, and/or revision of the manuscript. MN and TM contributed to study supervision.

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Correspondence to Kenta Ujifuku.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

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Supplementary file1 (PPTX 108 kb)

Supplementary file2 (XLSX 7798 kb)

List 1. Tag-count-datasheet for TCC-GUI analysis of this study.

Supplementary file3 (XLSX 5721 kb)

List 2. Original data outputs analyzed using Tophat2-CuffLinks2-CummRbund on Maser (List2ofLot1, List2ofLot2, and List2ofLot3). Abbreviations in the spreadsheet; C, control. K, KNS-62. A, A549. Numbers indicate batches of pericytes. For example, 2CK indicates the DEG comparison between control and KNS-62 in pericytes lot number 2.

Supplementary file4 (XLSX 7359 kb)

Supplementary file5 (CSV 6 kb)

Supplementary file6 (CSV 5 kb)

List 3. Datasheets for Metascape analysis. These were processed from the list 2 original data. Up-regulated genes are listed in List 3A and down-regulated genes are listed in List 3B.

Supplementary file7 (CSV 484 kb)

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Ujifuku, K., Fujimoto, T., Sato, K. et al. Exploration of Pericyte-Derived Factors Implicated in Lung Cancer Brain Metastasis Protection: A Pilot Messenger RNA Sequencing Using the Blood–Brain Barrier In Vitro Model. Cell Mol Neurobiol 42, 997–1004 (2022). https://doi.org/10.1007/s10571-020-00988-y

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