Establishment and molecular characterization of HCB-541, a novel and aggressive human cutaneous squamous cell carcinoma cell line

Abstract

Cutaneous squamous cell carcinoma (cSCC) is a common type of skin cancer that can result in significant morbidity, although it is usually well-managed and rarely metastasizes. However, the lack of commercially available cSCC cell lines hinders our understanding of this disease. This study aims to establish and characterize a new metastatic cSCC cell line derived from a Brazilian patient. A tumor biopsy was taken from a metastatic cSCC patient, immortalized, and named HCB-541 after several passages. The cytokeratin expression profile, karyotypic alterations, mutational analysis, mRNA and protein differential expression, tumorigenic capacity in xenograft models, and drug sensitivity were analyzed. The HCB-541 cell line showed a doubling time between 20 and 30 h and high tumorigenic capacity in the xenograft mouse model. The HCB-541 cell line showed hypodiploid and hypotetraploidy populations. We found pathogenic mutations in TP53 p.(Arg248Leu), HRAS (Gln61His) and TERT promoter (C228T) and high-level microsatellite instability (MSI-H) in both tumor and cell line. We observed 37 cancer-related genes differentially expressed when compared with HACAT control cells. The HCB-541 cells exhibited high phosphorylated levels of EGFR, AXL, Tie, FGFR, and ROR2, and high sensitivity to cisplatin, carboplatin, and EGFR inhibitors. Our study successfully established HCB-541, a new cSCC cell line that could be useful as a valuable biological model for understanding the biology and therapy of metastatic skin cancer.

Keywords: Cell line establishment; Cutaneous squamous cell carcinoma; In vivo model; Molecular profile.

Fig. 1

Morphological and proliferative characterization of HCB-541 cell line. A Representative photomicrograph of the HCB-541 cell line at 10 × magnification, showing the establishment days. B Real-time cell analysis showing the doubling time of HCB-541 cells under different SFB concentrations, with bars representing time (hours) at 60 h. C Hematoxylin eosin staining and immunocytochemistry of HCB-541 cells for the expression of Ki67; vimentin and cytokeratin proteins profile (CK20, Cytokeratin A1/A3, CK17, CK8). ⁺Early-passage number (< 5); ⁺⁺ Late-passage number (> 20). Photomicrographs at × 100 magnification. *, ** represents intergroup statistical differences (p < 0.05)

Fig. 2

Immunohistochemical staining for comparative expression among the patient's tumor, HCB-541 cell line, and xenograft model. A Photomicrography at 100 × magnification HCB-541 passage number: 20. The protein markers studied were Cytokeratin 5/6 (CK5/6), protein 63 (p63), protein 40 (p40), and Common Acute Lymphoblastic Leukemia Antigen (CD10). B Subcutaneous inoculation of 1 × 10⁶ (n = 3) and 3 × 10⁶ (n = 3) cells resulted in tumor growth after 2 weeks in NSG mice. C Tumor volume versus tumor weight of the two amounts 1 × 10⁶ (n = 3) and 3 × 10.⁶ (n = 3) cells that were inoculated subcutaneously. * represents intergroup statistical differences (p < 0.05)

Fig. 3

TERT promoter mutation analysis, and gene expression analysis of a pan-cancer panel by NanoString™ and functional pathways analysis A TERT promoter gene analysis from patient’s tumor. B TERT promoter gene analysis from HCB-541 tumor cell line after establishment. C Heatmap of genes altered in HCB-541 compared with normal HACAT cells. D Upregulated gene in silico interaction network. E Downregulated gene in silico interaction network. Each circle represents a gene (node), and each connection represents a direct or indirect connection (edge). HCB-541 passage number: 25

Fig. 4

Protein profile of MAPK and RTK phosphorylated of HCB-541 cell line and sensibility to different antineoplastic agents. A RTK phosphorylated array of HCB-541 cell line; B Densitometric analyses of RTK detection array are represented by bar graphs; C RTK phosphorylated profile of HCB-541 by Western blot. D MAPK phosphorylated array of HCB-541 cell line. E Densitometric analyses of MAPK detection array are represented by bar graphs. F MAPK phosphorylated profile of HCB-541 by Western blot analysis. Each RTK and MAPK is duplicate in the arrays (two spots side by side), and three pairs of phosphotyrosine positive controls are in the corners of each array; G Afatinib–target-PanErbB, EGFR T90M; H Allitinib–anti-PanErbB, EGFR T90M; I Erlotinib–anti-EGFR L858R; D Cetuximab–monoclonal antibody against EGFR; K Lapatinib–anti-EGFR and HER2; L Carboplatin–DNA synthesis inhibitor; M Cisplatin–DNA synthesis inhibitor; N 5-Flouracil–DNA/RNA synthesis inhibitor; O Everolimus–mTOR inhibitor. Data presented as the mean of three independent experiments. *, **, *** represents intergroup statistical differences (p < 0.05). HCB-541 passage number: 25