Generation of a Lung Squamous Cell Carcinoma Three-dimensional Culture Model with Keratinizing Structures

Overview

Journal Sci Rep

Specialty Science

Date 2021 Dec 22

PMID 34934075

Authors

Shigeto Kawai

Kiyotaka Nakano

Keiichi Tamai

Etsuko Fujii

Mimori Yamada

Hiroshi Komoda

Hirofumi Sakumoto

Osamu Natori

Masami Suzuki

Affiliations

Soon will be listed here.

Abstract

Tumor nests in lung squamous cell carcinoma (LUSC) have a hierarchical structure resembling squamous epithelium. The nests consist of basal-like cells on the periphery and layers of keratinocyte-like cells that differentiate towards the center of the nest, forming keratin pearls. Reproducing this spatial heterogeneity in in vitro models would be useful for understanding the biology of LUSC. Here, we established a three-dimensional (3D) culture model with a squamous epithelial structure using LUSC cell lines PLR327F-LD41 and MCC001F, established in-house. When PLR327F-LD41 cells were cultured in a mixture of Matrigel and collagen I, they generated 3D colonies (designated cancer organoids, or COs) with involucrin (IVL)-positive keratinizing cells in the center (IVL COs). COs with uniform size were generated by seeding PLR327F-LD41 cells in a form of small cell aggregates. Since Notch signaling induces the differentiation of squamous epithelium, we confirmed the effect of γ-secretase inhibitor in inhibiting Notch signaling in IVL COs. Surprisingly, γ-secretase inhibitor did not block induction of IVL-positive cells; however, cells residing between the CK5-positive basal-like layer and IVL-positive layer decreased significantly. Thus, our 3D culture model with uniform size and structure promises to be a useful tool for elucidating the biology of LUSC and for screening drug-candidates.

References

Egeblad M, Nakasone E, Werb Z . Tumors as organs: complex tissues that interface with the entire organism. Dev Cell. 2010; 18(6):884-901. PMC: 2905377. DOI: 10.1016/j.devcel.2010.05.012. View

Fujii E, Yamazaki M, Kawai S, Ohtani Y, Watanabe T, Kato A . A simple method for histopathological evaluation of organoids. J Toxicol Pathol. 2018; 31(1):81-85. PMC: 5820108. DOI: 10.1293/tox.2017-0060. View

Kawai S, Yamazaki M, Shibuya K, Yamazaki M, Fujii E, Nakano K . Three-dimensional culture models mimic colon cancer heterogeneity induced by different microenvironments. Sci Rep. 2020; 10(1):3156. PMC: 7035265. DOI: 10.1038/s41598-020-60145-9. View

Chen S, Giannakou A, Wyman S, Gruzas J, Golas J, Zhong W . Cancer-associated fibroblasts suppress SOX2-induced dysplasia in a lung squamous cancer coculture. Proc Natl Acad Sci U S A. 2018; 115(50):E11671-E11680. PMC: 6294935. DOI: 10.1073/pnas.1803718115. View

Ricci-Vitiani L, Lombardi D, Pilozzi E, Biffoni M, Todaro M, Peschle C . Identification and expansion of human colon-cancer-initiating cells. Nature. 2006; 445(7123):111-5. DOI: 10.1038/nature05384. View

Dotto G . Notch tumor suppressor function. Oncogene. 2008; 27(38):5115-23. PMC: 2747622. DOI: 10.1038/onc.2008.225. View

Direito I, Madeira A, Brito M, Soveral G . Aquaporin-5: from structure to function and dysfunction in cancer. Cell Mol Life Sci. 2016; 73(8):1623-40. PMC: 11108570. DOI: 10.1007/s00018-016-2142-0. View

Fujii E, Suzuki M, Matsubara K, Watanabe M, Chen Y, Adachi K . Establishment and characterization of in vivo human tumor models in the NOD/SCID/gamma(c)(null) mouse. Pathol Int. 2008; 58(9):559-67. DOI: 10.1111/j.1440-1827.2008.02271.x. View

Parsa R, Yang A, McKeon F, Green H . Association of p63 with proliferative potential in normal and neoplastic human keratinocytes. J Invest Dermatol. 1999; 113(6):1099-105. DOI: 10.1046/j.1523-1747.1999.00780.x. View

10.

Eramo A, Lotti F, Sette G, Pilozzi E, Biffoni M, Di Virgilio A . Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ. 2007; 15(3):504-14. DOI: 10.1038/sj.cdd.4402283. View

11.

Damelin M, Geles K, Follettie M, Yuan P, Baxter M, Golas J . Delineation of a cellular hierarchy in lung cancer reveals an oncofetal antigen expressed on tumor-initiating cells. Cancer Res. 2011; 71(12):4236-46. DOI: 10.1158/0008-5472.CAN-10-3919. View

12.

Varga J, Greten F . Cell plasticity in epithelial homeostasis and tumorigenesis. Nat Cell Biol. 2017; 19(10):1133-1141. DOI: 10.1038/ncb3611. View

13.

Valkenburg K, de Groot A, Pienta K . Targeting the tumour stroma to improve cancer therapy. Nat Rev Clin Oncol. 2018; 15(6):366-381. PMC: 5960434. DOI: 10.1038/s41571-018-0007-1. View

14.

Thomas A, Liu S, Subramaniam D, Giaccone G . Refining the treatment of NSCLC according to histological and molecular subtypes. Nat Rev Clin Oncol. 2015; 12(9):511-26. DOI: 10.1038/nrclinonc.2015.90. View

15.

Kijima T, Nakagawa H, Shimonosono M, Chandramouleeswaran P, Hara T, Sahu V . Three-Dimensional Organoids Reveal Therapy Resistance of Esophageal and Oropharyngeal Squamous Cell Carcinoma Cells. Cell Mol Gastroenterol Hepatol. 2018; 7(1):73-91. PMC: 6260338. DOI: 10.1016/j.jcmgh.2018.09.003. View

16.

Blanpain C, Lowry W, Pasolli H, Fuchs E . Canonical notch signaling functions as a commitment switch in the epidermal lineage. Genes Dev. 2006; 20(21):3022-35. PMC: 1620020. DOI: 10.1101/gad.1477606. View

17.

Bissell M, Radisky D . Putting tumours in context. Nat Rev Cancer. 2002; 1(1):46-54. PMC: 2975572. DOI: 10.1038/35094059. View

18.

Travis W, Brambilla E, Nicholson A, Yatabe Y, Austin J, Beasley M . The 2015 World Health Organization Classification of Lung Tumors: Impact of Genetic, Clinical and Radiologic Advances Since the 2004 Classification. J Thorac Oncol. 2015; 10(9):1243-1260. DOI: 10.1097/JTO.0000000000000630. View

19.

Ishimoto S, Wada K, Usami Y, Tanaka N, Aikawa T, Okura M . Differential expression of aquaporin 5 and aquaporin 3 in squamous cell carcinoma and adenoid cystic carcinoma. Int J Oncol. 2012; 41(1):67-75. DOI: 10.3892/ijo.2012.1445. View

20.

Fatehullah A, Tan S, Barker N . Organoids as an in vitro model of human development and disease. Nat Cell Biol. 2016; 18(3):246-54. DOI: 10.1038/ncb3312. View