Research ArticleExperimental Studies
Open Access

Transcriptome of Oral Cancer Cells Adapted to Suspension Culture Is Potentially Related to Cancer Progressive Phenotypes

KOUHEI SAKURAI, TATSUYA ANDO, YUKA IDETA, YUICHIRO HAYASHI, JUNICHI BABA, KENJI MITSUDO and HIROYASU ITO
Anticancer Research September 2023, 43 (9) 3905-3911; DOI: https://doi.org/10.21873/anticanres.16578

Abstract

Background/Aim: Cervical lymph node metastasis worsens oral cancer prognosis. Cancer cells with high metastatic ability can delay or resist apoptosis and survive in the floating condition during circulation. The involved genes and pathways in this process remain largely unknown. This study aimed to establish an oral cancer cell line adapted to suspension culture by in vitro selection and perform gene expression analysis. Materials and Methods: The oral cancer cell subline adapted to suspension culture was isolated by in vitro selection from the oral cancer cell line, HSC-3. The transcriptome profiles of HSC-3 and its subline were compared using gene expression microarrays. Gene Ontology (GO) enrichment analysis, Gene Set Enrichment Analysis (GSEA), and Ingenuity Pathway Analysis (IPA) were performed to predict the involved pathways and molecules in cancer progression. Results: The subline was designated as HSC-3S5. The cellular viability of HSC-3S5 cells at the suspension culture was higher than that of HSC-3 cells. A total of 961 genes were differentially expressed between HSC-3 and HSC-3S5 cells under the threshold cut-off (FDR-adjusted p-value of <0.05 and absolute fold change of >1.5). GO terms, such as growth regulation, were enriched in the DEGs. GSEA revealed the association between the DEGs and significant gene sets, including metastasis and stemness. IPA predicted that the proliferation-related pathways were enhanced while the apoptotic pathway was inhibited in HSC-3S5 cells compared to HSC-3 cells. Conclusion: Our transcriptome analysis revealed several potentially activated pathways and molecules in the floating-adapted oral cancer cells and indicated molecular implications for cancer progression.

Key Words:

The most common malignancy in the head and neck region is oral cancer (1, 2). Squamous cell carcinoma is its predominant histological type. The survival rate is significantly lower in advanced stages although the 5-year survival rate in the early stages is >80% (3). Generally, cervical lymph node metastasis is one of the most important prognostic factors of oral cancer. A study analyzing 14,554 patients revealed a poorer prognosis in patients with cervical lymph node metastasis than those without it (4). Furthermore, the mortality risk escalated with the increasing number of metastatic lymph nodes (4). Therefore, the molecular mechanisms of oral cancer metastasis should be elucidated to improve the prognosis.

The gene expression signature is dramatically changed during cancer metastasis. Several stimuli, such as hypoxia and cytokine in the tumor microenvironment, alter gene expression in cancer cells and enhance the metastatic phenotype (5). The transcriptome analysis using head and neck cancer samples from the primary site and its corresponding metastatic lymph node revealed PI3K-AKT signaling activation and high EGFR expression in metastatic cancer cells (6). We previously analyzed the transcriptome between oral cancer cell line HSC-3 cells and its metastatic subline HSC-3-M3 cells (7). HSC-3-M3 cells were established by in vivo selection from the parental HSC-3 cell line (8). Our analysis showed a potential association between the gene expression profile of HSC-3-M3 cells and the NF-Embedded ImageB pathway and the cancer progressive phenotypes, such as invasion and metastasis (7). Subsequently, Li et al. reanalyzed our transcriptome data and suggested that fibroproliferation-related biological processes were activated in HSC-3-M3 cells (9). An understanding of gene expression profiles in highly metastatic oral cancer cells would help identify novel diagnostic and therapeutic targets.

Metastasis is a multistep process, including invasion, circulation, and colonization of distant organs (10). Cancer cells that enter into circulation at the primary site lose their attachment to the scaffold, such as the extracellular matrix (ECM). ECM detachment triggers apoptotic pathways in cells, leading to cellular death, also known as “anoikis” (11, 12). However, cancer cells with high metastatic potential overcome the stress of the anchorage-independent condition (13, 14). Various pathways, including FAK-Src-PI3K-AKT and RAC1-JNK, were activated to enhance anchorage-independent survival and growth (15, 16).

The gene expression change is crucial in detached cancer cell survival and proliferation, such as breast, lung, colon, and cervical cancer cells (17–19). Our previous finding revealed that highly metastatic oral cancer cells detached from the scaffold shifted their transcriptome to adapt to the floating condition and form anchorage-independent cellular clusters (20). Conversely, oral cancer cells with low metastatic capacity died by apoptosis in similar conditions. However, genes and pathways that contribute to the survival of floating oral cancer cells remain largely unknown.

We hypothesize that specific pathways and expression of genes are activated in floating oral cancer cells. Therefore, the current study established an oral cancer cell line adapted to suspension culture by in vitro selection and performed the gene expression analysis. Our result indicated the potential association of the gene expression profile with diverse cancer progressive phenotypes.

Materials and Methods

Cell culture. HSC-3 and HSC-3-M3 cell lines were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan), and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal calf serum (Thermo Fisher Scientific).

HSC-3 subline establishment. The subline adapted to suspension culture was established as previously reported with modifications (21). HSC-3 cells at 1.0×105 were mixed into 3 ml of high glucose DMEM supplemented with 10% FCS, and seeded at Nunclon™ Sphera™ 6 well low-attachment plate (Thermo Fisher Scientific). Cells were transferred to the standard plate after culturing at 37°C for 72 h, and the surviving cells attached to the plate were named HSC-3S1. Cells were expanded at the standard plate and transferred to the low-attachment plate similarly. This procedure was repeated five times, and the resultant cells were designated as HSC-3S5. All assays were performed within five passages after its establishment.

Cell viability assay. Cells of 3.0×103 were seeded at the Nunclon™ Sphera™ 96 well low-attachment plate (Thermo Fisher Scientific). The suspended cells were subjected to cell viability assay with WST-8 (Dojindo laboratories, Kumamoto, Japan) according to the manufacturer’s instructions at the designated time point. The 450-nm absorbance of samples was measured with Sunrise Rainbow (Tecan, Männedorf, Switzerland). Student’s t-test was used to calculate statistical significance.

Gene expression microarrays. Total RNA was isolated from HSC-3 (n=4) and HSC-3S5 (n=4) cultured at a standard plate using TRIzol reagent (Thermo Fisher Scientific). RNA integrity number was assessed by an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). A SurePrint G3 Human Gene Expression v3 8×60 K Microarray (Agilent) and quantile normalization were performed. The expression dataset was deposited into the National Center for Biotechnology Information Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/) and is accessible through GEO Series accession number GSE235020.

Gene Ontology (GO) enrichment analysis. GO enrichment analysis was performed with Metascape https://metascape.org/gp/index.html#/main/step1 (22). The list of differentially expressed genes (DEGs) [false discovery rate (FDR) adjusted at p<0.05 and absolute fold change at >1.5] obtained from the microarray analysis was used.

Gene set enrichment analysis (GSEA). The fold change in all genes between HSC-3 and HSC-3S5 was analyzed and the gene list was used as the input data. GSEA (v4.2.0) was run following the default parameters (23).

Ingenuity Pathway Analysis (IPA). IPA (version 52912811) (QIAGEN, Venlo, Netherlands) was used to find significant pathways and activated states of predicted molecules (7, 20). DEGs were used for IPA Upstream Regulator Analysis and Regulator Effect Analysis, which were visualized following the manufacturer’s instructions.

Results

Establishment of HSC-3 cells adapted to the suspension culture. We aimed to establish the subline, which can advantageously grow in the floating condition, from an oral cancer cell line, HSC-3 (Figure 1A). HSC-3 cells were seeded at the low-attachment plate for suspension culture, and incubated for 72 h. A few surviving cells attached to the plate and continued to grow when seeded at the standard plate again. We repeated this procedure five times and designated the resultant cells as HSC-3S5 cells. The cellular viability of HSC-3 and HSC-3S5 cells were comparable at the attached condition using the standard plate (data not shown). However, the viability of HSC-3S5 and HSC-3-M3 (a metastatic subline) (8) cells at the low-attachment plate were slightly but significantly higher than that of the parental HSC-3 cells at 24 h (Figure 2A). This result indicated that HSC-3S5 and HSC-3-M3 cells can advantageously grow at suspension culture compared to HSC-3 cells.

Figure 1.
Figure 1.

Establishment of the HSC-3 subline adapted to suspension culture. (A) Schematic representation showing the procedure of establishing the HSC-3 subline, HSC-3S5. Anoikis-sensitive cells were excluded by repeating the suspension culture using a low-attachment plate. (B) Cell viability of HSC-3, HSC-3S5, and HSC-3-M3 (a metastatic subline (8)) cells at the low-attachment plate. The viability at 0 h in each cell was set as 1. *p<0.05 (Student’s t-test).

Figure 2.
Figure 2.

Transcriptome analysis of HSC-3 and HSC-3S5 cells. (A) Volcano plot showing differentially expressed genes (DEGs) between HSC-3 (n=4) and HSC-3S5 (n=4) cells. The dotted lines indicate the threshold cut-off (FDR-adjusted p-value of <0.05 and absolute fold change of >1.5). The red and blue dots show up-regulated and down-regulated genes in HSC-3S5 cells, respectively. (B) Gene Ontology (GO) enrichment analysis using DEGs.

Gene expression analysis by microarray. We isolated RNA from HSC-3 and HSC-3S5 cells cultured at a standard plate and performed the gene expression microarrays (Figure 2A). A total of 961 genes were found to be differentially expressed between HSC-3 and HSC-3S5 cells under the threshold cut-off (FDR-adjusted p-value of <0.05 and absolute fold change of >1.5). We performed GO enrichment analysis using the 961 DEGs, which revealed that GO terms, such as “growth regulation”, “collagen-containing ECM”, and “calcium ion binding” were enriched (Figure 2B).

Significantly enriched gene sets related to cancer phenotypes. Next, we performed GSEA and revealed that the gene sets, including “metastasis up”, “stemness up”, and “NF-Embedded ImageB targets” were enriched in the upregulated genes in HSC-3S5 cells (Figure 3A-C). Conversely, gene sets, such as “metastasis down”, “glioma stem cell down”, and “carcinoma-associated fibroblast down” were enriched in the down-regulated genes in HSC-3S5 cells (Figure 3D-F).

Figure 3.
Figure 3.

Gene set enrichment analysis showing the association of the gene expression profile with cancer phenotypes. (A-F) Enrichment plot for the gene set of (A) METASTASIS_UP, (B) STEMNESS_UP, (C) NF-Embedded ImageB_TARGETS, (D) METASTASIS_DOWN, (E) GLIOMA_STEM_CELL_DOWN, and (F) CARCINOMA_ASSOCIATED_FIBROBLAST_DOWN. NES; Normalized enrichment score.

Prediction of activated pathways and molecules in HSC-3S5 cells. We explored potentially activated pathways and molecules in HSC-3S5 cells using IPA. The IPA Upstream Regulator Analysis predicts the regulators controlling the DEGs with p-values and activation z-score. Z-scores of >2 or <2 are considered activated or inhibited states, respectively. Several regulators, such as tumor necrosis factor (TNF), NF-Embedded ImageB (complex), and RAC1 were predicted to be activated in HSC-3S5 cells (Figure 4A). We also performed the IPA Regulator Effect Analysis, which predicts regulatory networks of upstream regulators, DEGs, and biological functions. The top three ranked networks calculated by consistency score were shown in Table I, and the top-ranked network was visualized in Figure 4B. Proliferation-related pathways were activated while the apoptotic pathways were inhibited in HSC-3S5 cells through the upstream regulators and DEGs.

Figure 4.
Figure 4.

Prediction of pathways and molecules potentially activated in HSC-3S5 cells. (A) The IPA Upstream Regulator Analysis. A plot showing predicted upstream regulators in HSC-3 and HSC-3S5 cells. The dotted lines indicate the threshold cut-off (p-value of <0.05 and absolute activation z-score of >2.0). The regulators with z-scores of >2 (red dots) mean activated states in HSC-3S5 cells while the regulators with z-scores <2 (blue dots) mean inhibited states in HSC-3S5 cells. (B) The IPA Regulator Effect Analysis. The upstream regulators (top) were predicted to be associated with the diseases and functions (bottom) through DEGs (middle) in HSC-3S5 cells. The meanings of each color are shown at the bottom of the Figure.

View this table:
Table I.

Top 3 ranked regulator effects based on consistency score calculated by the Ingenuity Pathway Analysis (IPA).

Discussion

Highly metastatic cancer cells can delay or resist anoikis, which is apoptosis upon loss of attachment to the scaffold (11, 12). This study isolated HSC-3S5 cells from its parental cell line HSC-3 cells using a low-attachment plate. Anoikis-sensitive HSC-3 cells were excluded by repeating adherent and suspension cultures. Similarly, oral and prostate cancer cell lines adapted to suspension culture were isolated from their parental cells (21, 24). Kupferman et al. alternatively isolated floating-adapted oral cancer cells by rotating the culture plate, which prevented the cancer cells from attaching to the bottom of the plate (25). These isolation methods are relatively easy, and the resultant cell line is useful for analyzing the molecular mechanisms of cancer progression. Notably, the properties and gene expression profile of each resultant cell probably depend on the parental cancer cell type and the isolation method. Furthermore, the duration that the phenotypes and gene expression pattern are maintained after the isolation should be further analyzed.

The cellular viability of HSC-3S5 and HSC-3-M3 cells was slightly higher than that of HSC-3 cells in suspension culture. We performed a soft agar colony formation assay as previously reported to evaluate the anchorage-independent survival and growth for a longer period (>14 days) (20). Contrary to our expectation, HSC-3, HSC-3S5, and HSC-3-M3 cells did not form visible colonies (data not shown). Therefore, we need to recognize that HSC-3S5 cells, as well as HSC-3-M3 cells,0 can survive under the anchorage-independent condition for a relatively short time in vitro. However, a few surviving detached cancer cells reattaching soon after metastatic circulation could colonize distant organs in a patient. Further analysis will be needed to reveal how delaying anoikis contributes to cancer metastasis in vivo.

In this study, our transcriptome analysis identified 961 DEGs between HSC-3 and HSC-3S5 cells. These DEGs were associated with GO terms, including growth regulation, ECM, and calcium ion binding. Our previous gene expression analysis using HSC-3 and its metastatic subline HSC-3-M3 cells identified 1,018 DEGs and suggested the potential activation of NF-Embedded ImageB pathway in HSC-3-M3 cells (7). Thus, the integration of our transcriptome profiles of HSC-3S5 and HSC-3-M3 cells would elucidate commonly activated pathways and molecules of these sublines.

Moreover, the HSC-3S5 cell gene expression profile was associated with cancer phenotypes. Interestingly, GSEA revealed that the stemness-related gene set was significantly associated with the gene profiles of HSC-3 and HSC-3S5 cells. Morta et al. demonstrated that low adherent cancer cell subpopulation had cancer stem-like properties (26). A comparison of functions between the cancer stem cells and HSC-3S5 cells would be interesting to elucidate the crosstalk of stemness and anchorage-independent survival. GSEA also indicated the association of the gene expression profile with the data set of cancer-associated fibroblasts (CAFs). Generally, highly aggressive cancer cells activate CAFs, which promote cancer progression (9, 27). The contribution of the interaction of cancer cells and tumor microenvironment to anchorage-independent survival will be an important topic in the future.

The IPA suggested the hypothesis that the DEGs were related to cancer progressive phenotypes, such as HSC-3S5 cell proliferation and anti-apoptosis. We need to perform a functional analysis to demonstrate significant pathways and genes involved in these cancer progressive phenotypes. This study focused on transcriptome profiles but also should analyze proteome and metabolome to further understand the characteristics of cancer cells with anchorage-independent survival (28).

Conclusion

Our transcriptome analysis indicated the potential association of the gene expression profile of the floating-adapted oral cancer cells with various cancer phenotypes. Considering that cancer cell detachment from the scaffold is the first step of metastasis, a better understanding of this process would be important to suppress cancer progression. HSC-3S5 cells are a useful cell line model for elucidating the mechanisms of anchorage-independent survival and metastasis. Our result provides insight into oral cancer progression although further analysis is required to clarify the molecular mechanisms.

Acknowledgements

The Authors would like to thank Ms. Asuka Haga at Fujita Health University and Ms. Mineka Nakajo at Yokohama City University for their helpful assistances.

Footnotes

  • Authors’ Contributions

    K.S. conceived the study, performed the experiment, and wrote the manuscript. Y.I. contributed to IPA. T.A., Y.H., J.B., K.M. and H.I. participated in planning the study. All Authors read and approved the final manuscript.

  • Conflicts of Interest

    The Authors declare that they have no competing interests.

  • Funding

    This research was funded by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science (grant number 21K09862).

  • Received June 17, 2023.
  • Revision received July 25, 2023.
  • Accepted July 27, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Transcriptome of Oral Cancer Cells Adapted to Suspension Culture Is Potentially Related to Cancer Progressive Phenotypes
KOUHEI SAKURAI, TATSUYA ANDO, YUKA IDETA, YUICHIRO HAYASHI, JUNICHI BABA, KENJI MITSUDO, HIROYASU ITO
Anticancer Research Sep 2023, 43 (9) 3905-3911; DOI: 10.21873/anticanres.16578
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