Chun-Ping Wu

The Composition of Microbiome in Larynx and the Throat Biodiversity between Laryngeal Squamous Cell Carcinoma Patients and Control Population

The throat is an ecological assemblage involved human cells and microbiota, and the colonizing bacteria are important factors in balancing this environment. However, this bacterial community profile has thus been poorly investigated. The purpose of this study was to investigate the microbial biology of the larynx and to analyze the throat biodiversity in laryngeal carcinoma patients compared to a control population in a case-control study. Barcoded pyrosequencing analysis of the 16S rRNA gene was used. We collected tissue samples from 29 patients with laryngeal carcinoma and 31 control patients with vocal cord polyps. The findings of high-quality sequence datasets revealed 218 genera from 13 phyla in the laryngeal mucosa. The predominant communities of phyla in the larynx were Firmicutes (54%), Fusobacteria (17%), Bacteroidetes (15%), Proteobacteria (11%), and Actinobacteria (3%). The leading genera were Streptococcus (36%), Fusobacterium (15%), Prevotella (12%), Neisseria (6%), and Gemella (4%). The throat bacterial compositions were highly different between laryngeal carcinoma subjects and control population (p = 0.006). The abundance of the 26 genera was significantly different between the laryngeal cancer and control groups by metastats analysis (p<0.05). Fifteen genera may be associated with laryngeal carcinoma by partial least squares discriminant analysis (p<0.001). In summary, this study revealed the microbiota profiles in laryngeal mucosa from tissue specimens. The compositions of bacteria community in throat were different between laryngeal cancer patients and controls, and probably were related with this carcinoma. The disruption of this bio-ecological niche might be a risk factor for laryngeal carcinoma.

Hypoxia promotes stem-like properties of laryngeal cancer cell lines by increasing the CD133+ stem cell fraction.

Evidence indicates that a hypoxic micro-environment plays an essential role in the regulation of cancer stem cells (CSCs). However, whether hypoxia is able to regulate the stem-like biological properties of laryngeal cancer cells remains unknown. In this study, we investigated the influence of hypoxia on the stemness of two laryngeal cancer cell lines, Hep-2 and AMC-HN-8. We cultured the two cell lines under hypoxia and normoxia and examined the influence of hypoxia on the expression of hypoxia-inducible factors (HIFs) and the cancer stem-like properties of these cells, including cell cycle distribution, expression of stem cell genes (OCT4, SOX2 and NANOG) and laryngeal CSC surface marker (CD133), proliferation, invasion, colony formation and sphere formation capacity. We determined that both of these cell lines, when maintained under hypoxic conditions, showed expanded cells in the G0/G1 phase, exhibited preferential expression of stem cell genes and CD133, and manifested upregulation of HIFs. When treated with hypoxia followed by normoxia exposure, the two cell lines exhibited enhanced capacities for proliferation, invasion, and sphere and colony formation compared with cells maintained consistently under normoxia. Our findings indicate that a hypoxic microenvironment may upgrade the stem-like biological properties of laryngeal cancer cell lines by the expansion of the CD133(+) stem cell fraction.

Microbiota in the Throat and Risk Factors for Laryngeal Carcinoma

ABSTRACT The compositions and abundances of the microbiota in the ecological niche of the human throat and the possible relationship between the microbiota and laryngeal cancer are poorly understood. To obtain insight into this, we enrolled 27 laryngeal carcinoma patients and 28 subjects with vocal cord polyps as controls. For each subject, we simultaneously collected swab samples from the upper throat near the epiglottis (site I) and tissue samples from the vestibulum laryngis to the subglottic region (site II). The microbiota of the throat were fully characterized by pyrosequencing of barcoded 16S rRNA genes. We found 14 phyla, 20 classes, 38 orders, 85 families, and 218 genera in the throats of enrolled subjects. The main phyla were Firmicutes (54.7%), Fusobacteria (14.8%), Bacteroidetes (12.7%), and Proteobacteria (10.6%). Streptococcus (37.3%), Fusobacterium (11.3%), and Prevotella (10.6%) were identified as the three most predominant genera in the throat. The relative abundances of 23 bacterial genera in site I were significantly different from those in site II (P < 0.05). The relative proportions of 12 genera largely varied between laryngeal cancer patients and control subjects (P < 0.05). Collectively, this study outlined the spatial structure of microbial communities in the human throat. The spatial structure of bacterial communities significantly varied in two anatomical sites of the throat. The bacterial profiles of the throat of laryngeal cancer patients were strongly different from those of control subjects, and several of these microorganisms may be related to laryngeal carcinoma.

Identification of cancer stem-like side population cells in purified primary cultured human laryngeal squamous cell carcinoma epithelia.

Cancer stem-like side population (SP) cells have been identified in many solid tumors; however, most of these investigations are performed using established cancer cell lines. Cancer cells in tumor tissue containing fibroblasts and many other types of cells are much more complex than any cancer cell line. Although SP cells were identified in the laryngeal squamous cell carcinoma (LSCC) cell line Hep-2 in our pilot study, it is unknown whether the LSCC tissue contains SP cells. In this study, LSCC cells (LSCCs) were primary cultured and purified from a surgically resected LSCC specimen derived from a well-differentiated epiglottic neoplasm of a Chinese male. This was followed by the verification of epithelium-specific characteristics, such as ultrastructure and biomarkers. A distinct SP subpopulation (4.45±1.07%) was isolated by Hoechst 33342 efflux analysis from cultured LSCCs by using a flow cytometer. Cancer stem cell (CSC)-associated assays, including expression of self-renewal and CSC marker genes, proliferation, differentiation, spheroid formation, chemotherapy resistance, and tumorigenicity were then conducted between SP and non-SP (NSP) LSCCs. In vitro and in vivo assays revealed that SP cells manifested preferential expression of self-renewal and CSC marker genes, higher capacity for proliferation, differentiation, and spheroid formation; enhanced resistance to chemotherapy; and greater xenograft tumorigenicity in immunodeficient mice compared with NSP cells. These findings suggest that the primary cultured and purified LSCCs contain cancer stem-like SP cells, which may serve as a valuable model for CSC research in LSCC.

Notch1 overexpression associates with poor prognosis in human laryngeal squamous cell carcinoma.

Objective: This study aimed to investigate the expression of Notch1 in human laryngeal squamous cell carcinoma (LSCC) tissues and its relationship to clinicopathologic characteristics as well as their prognostic value in LSCC. Methods: Samples from 106 patients with LSCC were analyzed for Notch1 expression by immunohistochemical staining. The relationship between Notch1 expression and clinicopathologic parameters was subsequently analyzed. Univariate analysis and multivariate analysis of patient survival were examined using the Kaplan-Meier method and Cox proportional hazards model, respectively. Results: We found that Notch1 had positive expression in 71 of 106 cases of LSCC (66.98%), which was obviously higher than laryngeal normal tissues (P < .01) and significantly correlated with the clinical stage, lymph node metastasis, and histological grade (all Ps < .05). Univariate analysis revealed that Notch1 expression tended to show an unfavorable influence on overall survival (OS) and disease-free survival (DFS) (both Ps < .01). Multivariate analysis demonstrated that Notch1 was an independent prognostic factor for patients with LSCC (P < .05). Conclusion: These results reveal that Notch1 expression is a potential prognostic factor for malignant progression, metastasis, and survival of LSCC patients. Furthermore, it has been demonstrated that high expression of Notch1 was associated with unfavorable OS and DFS in LSCC patients.

Cancer-associated fibroblasts in a human HEp-2 established laryngeal xenografted tumor are not derived from cancer cells through epithelial-mesenchymal transition, phenotypically activated but karyotypically normal.

Cancer-associated fibroblasts (CAFs) play a crucial role in cancer progression and even initiation. However, the origins of CAFs in various cancer types remain controversial, and one of the important hypothesized origins is through epithelial-mesenchymal transition (EMT) from cancer cells. In this study, we investigated whether the HEp-2 laryngeal cancer cells are able to generate CAFs via EMT during tumor formation, which is now still unknown. The laryngeal xenografted tumor model was established by inoculating the HEp-2 laryngeal cancer cell line in nude mice. Primary cultured CAFs from the tumor nodules and matched normal fibroblasts (NFs) from the adjacent connective tissues were subcultured, purified, and verified by immunofluorescence. Migration, invasion, and proliferation potentials were compared between the CAFs and NFs. A co-culture of CAFs with HEp-2 cells and a co-injection of CAFs with HEp-2 cells in nude mice were performed to examine the cancer-promoting potential of CAFs to further verify their identity. Karyotypic analyses of the CAFs, NFs, and HEp-2 cells were conducted. A co-culture of NFs with HEp-2 cells was also performed to examine the expression of activated markers of CAFs. A pathological examination confirmed that the laryngeal xenografted tumor model was successfully established, containing abundant CAFs. Immunocytochemical staining verified the purities and identities of the CAFs and NFs. Although the CAFs manifested higher migration, invasion, proliferation, and cancer-promoting capacities compared with the NFs, an analysis of chromosomes revealed that both the CAFs and NFs showed typical normal mouse karyotypes. In addition, the NFs co-cultured with HEp-2 cells did not show induced expressions of activated markers of CAFs. Our findings reveal that the CAFs in the HEp-2 established laryngeal xenografted tumor are not of laryngeal cancer origin but of mouse origin, indicating that the HEp-2 laryngeal cancer cells cannot generate their own CAFs via EMT in this model.

Establishment and characterization of a novel HPV-negative laryngeal squamous cell carcinoma cell line, FD-LSC-1, with missense and nonsense mutations of TP53 in the DNA-binding domain

Laryngeal squamous cell carcinoma (LSCC) is a common malignancy in China; however, publically available LSCC cell lines are few and not established from Chinese populations. Hence, novel and well-characterized LSCC cell lines of Chinese origin are urgently needed to provide researchers with a comprehensive database for LSCC research. From 40 cases of LSCC, we established a novel cell line that was maintained for more than 100 passages in vitro and was found to have typical epithelial morphology and ultrastructure. In-depth characterization analysis revealed polyploidy in DNA content; a doubling time of some 24h; high tumorigenicity in immunodeficient mice; higher invasive potential and more sensitive to radiation and cisplatin compared with HeLa cell line; upregulated Ki67, Notch1, EGFR, and CK5 protein levels; negative infection of human papillomavirus (HPV) and mycoplasma; expression of head and neck squamous cell carcinoma (HNSCC) biomarkers; mutations of TP53 in exons 5 and 8; a near-triploid karyotype with complex structural aberrations; and dozens of dysregulated genes and miRNAs. Cell authentication testing by the American Type Culture Collection (ATCC) confirmed the human origin of this cell line. Our findings indicate that a novel and well-differentiated LSCC cell line recapitulating the primary tumors malignant characteristics is established and well characterized. It does not match any cell lines within the ATCC database and helps to elucidate the molecular pathogenesis of LSCC.

Separation and cultivation of laryngeal carcinoma-associated fibroblasts and biological influence on a laryngeal carcinoma cell line

Abstract Conclusions: Carcinoma-associated fibroblasts (CAFs) can influence the biological characteristics of a laryngeal carcinoma cell line. These results could lay the foundation for further studies on the role of CAFs in the laryngeal tumor–host microenvironment. Objective: CAFs are important contributors to the microenvironment in determining the fate of tumors. The aim of this study was to separate, culture, and identify laryngeal CAFs and investigate their biological influence on the laryngeal carcinoma cell line. Methods: The primary CAFs and normal fibroblasts (NFs) of the larynx were obtained by tissue culture. The cells were verified according to immunohistochemical and immunofluorescence staining of certain proteins. Conditioned medium (CM) from CAFs and NFs was obtained. Functional assays were performed to test the influence of each CM on laryngeal carcinoma cell lines. Results: Third-passage purified laryngeal CAFs and NFs were successfully attained. The CAFs showed positive staining for vimentin, α-smooth muscle actin (α-SMA), and fibroblast activation protein (FAP). The migration ability of the CAFs increased significantly compared with that of NFs (p < 0.05). CM from CAFs (compared with CM from NFs) stimulated proliferation, migration, and invasion to a greater extent.

The primary growth of laryngeal squamous cell carcinoma cells in vitro is effectively supported by paired cancer-associated fibroblasts alone.

Most primarily cultured laryngeal squamous cell carcinoma cells are difficult to propagate in vitro and have a low survival rate. However, in our previous work to establish a laryngeal squamous cell carcinoma cell line, we found that laryngeal cancer-associated fibroblasts appeared to strongly inhibit the apoptosis of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In this study, we investigated whether paired laryngeal cancer-associated fibroblasts alone can effectively support the growth of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In all, 29 laryngeal squamous cell carcinoma specimens were collected and primarily cultured. The laryngeal squamous cell carcinoma cells were separated from cancer-associated fibroblasts by differential trypsinization and continuously subcultured. Morphological changes of the cultured laryngeal squamous cell carcinoma cells were observed. Immunocytofluorescence was used to authenticate the identity of the cancer-associated fibroblasts and laryngeal squamous cell carcinoma cells. Flow cytometry was used to quantify the proportion of apoptotic cells. Western blot was used to detect the protein levels of caspase-3. Enzyme-linked immunosorbent assay was used to detect the levels of chemokine (C-X-C motif) ligand 12, chemokine (C-X-C motif) ligand 7, hepatocyte growth factor, and fibroblast growth factor 1 in the supernatants of the laryngeal squamous cell carcinoma and control cells. AMD3100 (a chemokine (C-X-C motif) receptor 4 antagonist) and an anti–chemokine (C-X-C motif) ligand 7 antibody were used to block the tumor-supporting capacity of cancer-associated fibroblasts. Significant apoptotic changes were detected in the morphology of laryngeal squamous cell carcinoma cells detached from cancer-associated fibroblasts. The percentage of apoptotic laryngeal squamous cell carcinoma cells and the protein levels of caspase-3 increased gradually in subsequent subcultures. In contrast, no significant differences in the proliferation capacity of laryngeal squamous cell carcinoma cells cocultured with cancer-associated fibroblasts were detected during subculturing. High level of chemokine (C-X-C motif) ligand 12 was detected in the culture supernatant of cancer-associated fibroblasts. The tumor-supporting effect of cancer-associated fibroblasts was significantly inhibited by AMD3100. Our findings demonstrate that the paired laryngeal cancer-associated fibroblasts alone are sufficient to support the primary growth of laryngeal squamous cell carcinoma cells in vitro and that the chemokine (C-X-C motif) ligand 12/chemokine (C-X-C motif) receptor 4 axis is one of the major contributors.

Alterations of microbiota structure in the larynx relevant to laryngeal carcinoma

The microbial communities that inhabit the laryngeal mucosa build stable microenvironments and have the potential to influence the health of the human throat. However, the associations between the microbiota structure and laryngeal carcinoma remain uncertain. Here, we explored this question by comparing the laryngeal microbiota structure in laryngeal cancer patients with that in control subjects with vocal cord polyps through high-throughput pyrosequencing. Overall, the genera Streptococcus, Fusobacterium, and Prevotella were prevalent bacterial populations in the laryngeal niche. Tumor tissue samples and normal tissues adjacent to the tumor sites (NATs) were collected from 31 laryngeal cancer patients, and the bacterial communities in laryngeal cancer patients were compared with control samples from 32 subjects. A comparison of the laryngeal communities in the tumor tissues and the NATs showed higher α-diversity in cancer patients than in control subjects, and the relative abundances of seven bacterial genera differed among the three groups of samples. Furthermore, the relative abundances of ten bacterial genera in laryngeal cancer patients differed substantially from those in control subjects. These findings indicate that the laryngeal microbiota profiles are altered in laryngeal cancer patients, suggesting that a disturbance of the microbiota structure might be relevant to laryngeal cancer.