HPV6‐Induced Gene Expression Signature in Recurrent Respiratory Papillomatosis

ABSTRACT

Objectives

Recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus (HPV) types 6 and 11. However, the cellular and molecular mechanisms underlying its pathogenesis remain to be elucidated. Recently, the conditional reprogramming (CR) method, which reprograms epithelial cells to an undifferentiated stem cell state, has been shown to be effective for studying RRP. Here, we investigated the relationship between viral and host gene expression in CR cells and explored the molecular pathogenesis of RRP.

Methods

We evaluated the passage capacity and growth rate of CR cells from fresh RRP tissues and adjacent normal tissues from patients with RRP. Furthermore, we performed RNA‐seq analysis of CR cells across multiple passages to characterize the gene expression profiles associated with RRP.

Results

RRP‐derived CR cells exhibited greater passage capacity and proliferation rates than adjacent normal tissue‐derived CR cells. HPV6 gene was expressed in all RRP‐derived CR cells and its expression decreased with each passage. We identified three categories of genes correlated with HPV6: inflammatory genes (e.g., CTSS, CFB), oncogenic genes (e.g., CEACAM5, CEACAM6), and glycosylation‐related genes (e.g., ST6GALNAC1, FUT2).

Conclusion

HPV6‐induced gene expression signature in infected epithelial cells may play a role in RRP pathogenesis. Further molecular investigations are necessary to determine whether controlling these genes and related factors can control the progression of RRP.

Level of Evidence

N/A.

Using the conditional reprogramming method, we established a cell culture model of recurrent respiratory papillomatosis and analyzed gene expression. Genes related to human papillomavirus 6 expression were identified, including inflammatory genes (e.g., CTSS, CFB), oncogenic genes (e.g., CEACAM5, CEACAM6), and glycosylation‐associated genes (e.g., ST6GALNAC1, FUT2).

1. Introduction

Recurrent respiratory papillomatosis (RRP) is a rare disease caused by human papillomavirus (HPV), mainly HPV types 6 and 11. This leads to the growth of benign tumors in the respiratory tract, particularly in the upper airway. It primarily affects laryngeal tissues, with a propensity for high recurrence rates and rarely malignant transformation [1]. The histopathological features include exophytic projections or multiple papillary formations of stratified squamous epithelium overlying a central fibrovascular core [1]. Koilocytosis, characterized by distinct perinuclear halos in the squamous epithelial cells, suggests HPV infection [2].

The virus infects the basal epithelial cells through microabrasions and persists as episomal DNA without genomic integration [3]. Unlike high‐risk HPV types, these low‐risk HPVs rarely cause malignancy, but instead drive persistent epithelial proliferation and disease recurrence. The viral proteins E1 and E2 support episomal maintenance and replication, while E5, E6, and E7 modify host cell functions to create a favorable environment for viral persistence [3, 4]. In particular, the E5 protein enhances EGFR signaling and promotes epithelial proliferation without malignancy [5, 6]. HPV evades immune detection by downregulating MHC class I expression and altering the immune response, leading to a Th2‐skewed cytokine profile and dendritic cell dysfunction [1, 5]. Persistent HPV infection induces chronic inflammation and oxidative stress, which further drives disease recurrence [4]. Additionally, HPV DNA can remain latent in clinically normal mucosa, reactivating under immune suppression or stress, which explains the recurrent nature of RRP [7, 8].

Despite these findings, the precise molecular mechanisms that regulate HPV latency, immune evasion, and epithelial proliferation in RRP remain poorly understood. In particular, the mechanism by which HPV infection alters gene expression in epithelial cells and contributes to disease progression has not yet been fully elucidated. Understanding these transcriptional changes could provide key insights into the mechanisms driving RRP and identify potential therapeutic targets. Previous studies have reported gene expression analyses of RRP tissues. However, these data reflect the influence of the surrounding tissue microenvironment, making it difficult to directly assess the impact of HPV infection on the epithelial cells. To overcome this limitation, an experimental system that allows the isolation and stable culture of HPV‐infected epithelial cells is essential.

The conditional reprogramming (CR) method is a cell culture technique that enables the efficient expansion of primary epithelial cells while preserving their normal phenotype, differentiation potential, and genomic characteristics [9, 10, 11]. This method involves co‐culturing primary cells with irradiated mouse fibroblast feeder cells and treating them with a Rho‐associated kinase (ROCK) inhibitor (Y‐27632) [10]. The CR method allows the generation of stable epithelial cultures from limited tissue samples, including surgical specimens and biopsies, making it a powerful tool for studying epithelial pathologies, including RRP and conducting gene expression analysis.

To overcome RRP, it is indispensable to elucidate the molecular mechanisms of RRP, such as the regulation of HPV latency, immune evasion, and epithelial proliferation. In this study, to identify sets of related genes, we conducted gene expression analysis of CR cells derived from both RRP and adjacent normal tissues.

2. Methods

2.1. Patient Characteristics

Five patients (median age, 61 years; range, 29–77) with a history of RRP were recruited for this prospective study at the University of Tokyo Hospital with the approval of the Ethics Committee (2020120G). Biopsy samples of RRP lesions and adjacent normal tissue (non‐papilloma, NP) were obtained from patients undergoing surgery, immediately placed in saline on ice, and processed. Tissue samples were collected from various laryngeal regions, including true vocal folds, false vocal folds, and arytenoid areas. Papillomas were defined as exophytic, cauliflower‐like lesions exhibiting a central vascular core in each papilla or a symmetrical distribution of intraepithelial papillary capillary loops on laryngoscopy with narrow‐band imaging (NBI) [1, 12]. Tissue samples of the normal mucosa were collected from regions with no visible abnormalities on both laryngoscopy and NBI. The Derkay score, a validated clinical staging system that assesses the extent of disease in 25 anatomical subsites of the aerodigestive tract, was determined from recorded laryngoscopic images to assess the severity of RRP [13]. The clinical data of the patients are summarized in Table 1.

TABLE 1.

Characteristics of patients analyzed in this study.

ID	Age (years)	Sex	Surgery	Derkay score	HPV type
1	61	M	2nd	7	6a
2	77	F	6th	12	6vc
3	29	M	7th	9	6vc
4	29	M	2nd	8	6a
5	63	M	4th	8	6a

Abbreviation: HPV, human papillomavirus.

2.2. Tissue Processing and Cell Culture Using the Conditional Reprogramming

Epithelial cells derived from RRP and NP tissues were cultured using the CR method [10, 14]. Briefly, tissues were washed with PBS, minced with scissors, and digested with 0.05% trypsin–EDTA (Nacalai, Kyoto, Japan) at 37°C for 15 min. After dissociation by pipetting, the cells were washed once with DMEM containing FBS and then resuspended in F‐medium (Table S1). 3 T3‐J2 mouse fibroblast cell line, used as a feeder, was inactivated by irradiation at 30 Gy. Tissue‐derived cells and 2.5–5 × 10⁵ irradiated 3 T3‐J2 cells were seeded onto a 6 cm dish and cultured in F‐media supplemented with Y‐27632 (MCE, Monmouth Junction, NJ, USA). Cells were passaged upon reaching 80%–90% confluency, and the cells collected from the primary culture were designated as passage 0 (P0). Cells were cryopreserved using CELLBANKER 1 (Takara, Shiga, Japan), as needed, during passaging. The reagents used for cell culture are listed in Table S1. The concentration of propidium iodide‐negative viable cells was determined using Flow‐Count fluorospheres (Beckman) and analyzed using a CytoFLEX S flow cytometer (Beckman).

2.3. RNA Sequencing Analysis

RNA‐seq libraries were prepared by on‐bead reverse transcription with template switching. PolyA RNA was captured on oligo‐dT‐conjugated magnetic beads, followed by reverse transcription with SuperScript IV (Thermo Fisher Scientific, Waltham, MA, USA) and a template‐switching oligo. After washing, cDNA was amplified through two rounds of PCR using the KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Basel, Switzerland) and purified. Barcoded libraries were pooled, fragmented, adapter‐ligated, and indexed using the NEBNext Ultra II FS Kit (New England Biolabs, Ipswich, MA, USA). The final libraries were selected and sequenced on an Illumina NovaSeq 6000 (47 bp read 1 and 175 bp read 2).

2.4. Transcriptome Data Analysis

Transcriptome data analysis was performed according to a previous report [15]. In brief, adapter trimming and quality filtering of sequencing data were performed using Cutadpat‐v2.10. The filtered reads were mapped to reference RNA (GRCh38: HPV genome HPV6a, 6b, 6vc, 11) using STAR‐2.7.10b [16], and read counts of each gene symbol were calculated using featureCounts v2.0.2 [17] between‐sample normalization was performed against raw count data using R 4.4.2 (https://cran.r‐project.org/) and TCC package (EEE‐E method). To normalize gene expression in individual cases of RRP and NP, differentially expressed genes (DEGs) were defined as those with a fold change of ≥ 5 in RRP‐derived CR cells and ≥ 4 in NP‐derived CR cells, respectively. Correlated gene modules among DEGs were detected using the WGCNA package. Gene expression values were log2‐transformed after adding a pseudo‐count of one to avoid log (0). DEG analysis in RRP‐ and NP‐derived CR cells at P0 was performed using the R package DESeq2 with a log2 fold change > 1, an adjusted p‐value < 0.05. Gene ontology (GO) analysis was performed using the R package clusterProfiler with gene‐level count data, applying significance thresholds of p < 0.05 and q < 0.05. Correlation analysis between host gene expression and HPV6 gene expression was conducted using Spearman's correlation analysis in R. Genes with a correlation coefficient of ≥ 0.6 and p < 0.05 with HPV6 were significantly correlated.

2.5. Statistical Analyses

All statistical analyses were performed using a two‐tailed paired Student's t‐test (for pairwise comparisons) in GraphPad Prism8 software (GraphPad Software). All statistical analyses were conducted at a significance level of p < 0.05.

3. Results

3.1. Proliferation Characteristics of CR Cells Derived From RRP and NP Tissues

We established CR cells from RRP tissue in all five cases (ID1‐5) and from NP tissue in three of the five patients (ID 1, 4, and 5) who provided informed consent. CR cells were serially passaged and cultured for up to 30 days or until cell proliferation plateaued. In RRP‐derived CR cells, approximately 10 mg of tissue produced more than 1 × 10⁸ cells, whereas in NP‐derived CR cells, the same amount of tissue yielded around 1 × 10⁷ cells (Figure 1A). When comparing the early proliferation rate (divisions/day) between RRP‐derived and NP‐derived CR cells, RRP‐derived CR cells showed higher proliferation rates than NP‐derived CR cells at both stages, with a significantly higher rate from P1 to P2 (Figure 1B). No significant correlations were found between proliferation characteristics and patient background factors, including Derkay score and HPV subtype (data not shown). These results indicate that RRP‐derived CR cells have higher proliferative potential than NP‐derived CR cells.

FIGURE 1.

Cell proliferation of cultured cells using the conditional reprogramming method. (A) Primary culture derived from RRP and NP tissues was collected when the cells reached 80%–90% confluency and designated as P0. Approximately 1 × 10⁵ cells of P0 were passaged and cultured using the conditional reprogramming method, and the number of live cells was analyzed at each passage. (B) The proliferation rate (number of cell divisions per day) from P0 to P1 and P1 to P2 was measured. RRP, Recurrent respiratory papillomatosis; NP, Non‐papilloma; ns, not significant, *: p < 0.05 by two‐tailed, paired Student's t‐test. [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]

3.2. HPV Gene Expression in CR Cells Derived From RRP and NP Tissues

To elucidate the molecular mechanisms underlying the high proliferative potential of RRP‐derived CR cells, we performed RNA sequencing analysis of RRP‐derived and NP‐derived CR cells at P0, P1, and P4. We assumed that the proliferation from P0 to P1 and P1 to P2 was primarily influenced by gene expression profiles at P0 and P1, respectively. Therefore, we analyzed gene expression at both time points. As references for HPV, we used subtypes a, b, and vc of HPV6 as well as HPV11. The subtype with the highest number of matching transcripts was assigned to the HPV type in each case (Table 1). HPV6 gene was detected in CR cells derived from RRP tissue in all cases, and its expression level decreased at each passage (P0, P1, and P4), while HPV11 gene was not detected (Figure 2A). In contrast, neither HPV6 nor HPV11 genes were detected in CR cells derived from NP tissues. Correlation analysis between HPV6 gene expression at P0 and patient background factors revealed no significant association with disease severity (Derkay score, r = 0.05, p = 0.93). In contrast, no significant associations were observed between HPV6 gene expression and proliferation rate (Figure 2B,C) or between HPV6 gene expression and HPV6 subtype (data not shown). These results suggest that, while HPV6 gene expression in CR cells at P0 is associated with disease severity and duration, it does not directly influence the proliferation rate of CR cells.

FIGURE 2.

HPV6 gene expression in cultured cells using the conditional reprogramming method. RNA sequencing analysis was performed on the RRP‐derived CR cells at P0, P1, and P4. (A) Number of HPV6‐derived transcripts per million transcripts. (B) Spearman correlation between HPV6‐derived transcripts at P0 and the proliferation rate (number of cell divisions per day) from P0 to P1. (C) Spearman correlation between HPV6‐derived transcripts at P1 and the proliferation rate (number of cell divisions per day) from P0 to P1. HPV6, Human papillomavirus 6; RRP, Recurrent respiratory papillomatosis; TPM, Transcripts per million transcripts. [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]

3.3. Transcriptomic Characteristics of RRP‐Derived CR Cells

To identify the transcriptomic characteristics of RRP‐derived CR cells, we first performed clustering analysis on the gene expression data from P0, P1, and P4 of RRP‐derived and NP‐derived CR cells from ID 1, 4, and 5. However, no gene groups specific to either RRP or NP have been identified. Therefore, we proceeded with DEG analysis of RRP‐derived and NP‐derived CR cells at P0 (Figure 3A,B; Tables S2 and S3). This analysis revealed 164 upregulated and 73 downregulated DEGs in RRP‐derived CR cells compared with NP‐derived CR cells. The genes upregulated in RRP included inflammatory genes (e.g., CTSS and CFB), oncogenic genes (e.g., CEACAM5 and CEACAM6), and glycosylation‐related genes (e.g., ST6GALNAC1 and FUT2), while downregulated genes included skin formation and maintenance‐related genes (e.g., KRT16 and KRT6C). GO analysis showed that the genes upregulated in RRP‐derived CR cells were involved in the development of sensory and reproductive systems, glands, ears, and extracellular matrix formation. In contrast, the genes downregulated in these cells were associated with the development of skin and epithelial tissues (Figure 3C, Tables S4 and S5). Contrary to our expectations, this analysis did not reveal any GO terms related to cell proliferation or the immune response, which are generally known to be related to the pathology of RRP.

FIGURE 3.

Analysis of differentially expressed genes in cultured cells from RRP and NP tissues. (A) A volcano plot illustrates transcriptome data from P0 cultured cells derived from RRP and NP, excluding HPV6 gene. Differentially expressed genes were defined as those with an adjusted p < 0.05, a log2 fold change > 1. Red dots represent upregulated genes and blue dots represent downregulated genes, while gray dots indicate non‐significant genes. The names of genes of interest are displayed. (B) Heatmap of the top 20 genes upregulated in RRP‐derived CR cells. (C) Gene Ontology (GO) enrichment analysis of differentially expressed genes. GO terms significantly enriched in genes upregulated in RRP‐derived CR cells are shown on the left, while those enriched in NP‐derived CR cells are shown on the right. HPV6, Human papillomavirus 6; RRP, Recurrent respiratory papillomatosis; NP, Non‐papilloma. [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]

3.4. Host Genes Correlated With HPV6 Expression in RRP‐Derived CR Cells

Finally, we analyzed the correlation between HPV6 and host gene expression in RRP‐derived CR cells at P0, P1, and P4 from all cases to investigate how HPV infection alters gene expression in these cells. To control for gene expression changes associated with passage number rather than HPV infection, we performed the same analysis in NP‐derived CR cells and excluded genes that exhibited passage‐dependent expression. We identified 145 host genes significantly correlated with HPV6 (correlation coefficient ≥ 0.6 and p < 0.05). Subsequently, GO analysis was performed using genes with a correlation coefficient ≥ 0.6, which identified gene groups associated with the negative regulation of T cell activation and proliferation, positive regulation of viral entry into host cells, and glycosylation processes (Table S6). To identify genes specifically associated with RRP pathogenesis induced by HPV6, we compared genes differentially expressed upon HPV6 infection with those highly expressed in the RRP‐derived epithelium. Consequently, 35 common genes were identified (Figure 4A, Table 2). Among these, we focused on three categories of genes: inflammatory, oncogenic, and glycosylation‐related genes (Figure 4B). In the oncogenic category, genes belonging to the carcinoembryonic antigen (CEA)‐related cell adhesion molecule (CEACAM) family were highly expressed. We also analyzed genes that were downregulated in RRP‐derived CR cells and were negatively correlated with HPV6; however, no such gene was detected.

FIGURE 4.

Analysis of host genes correlated with HPV6 gene expression in cultured cells from RRP tissues. Correlation analysis between host gene expression and HPV6 gene expression was conducted in cultured cells from RRP tissues at P0, P1, and P4. Genes with a correlation coefficient of ≥ 0.6 and p < 0.05 with HPV6 were significantly considered correlated. (A) Venn diagram illustrating shared genes between upregulated DEGs in RRP and genes correlated with HPV6. (B) Scatter plot showing the correlation between HPV6 gene expression and the expression levels of three categorized representative HPV6‐correlated genes, as well as other genes of interest, assessed by Spearman's correlation analysis. HPV6, Human papillomavirus 6; RRP, Recurrent respiratory papillomatosis; DEG, Differentially expressed genes; TPM, Transcripts per million transcripts. [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]

TABLE 2.

HPV/RRP associated genes list.

Gene	Log2 fold change	Adjusted p‐value	Correlation coefficient
35 common genes
TCN1	2.60	1.8.E−07	0.87
ST6GALNAC1	1.48	1.0.E−02	0.86
CEACAM7	3.23	2.0.E−06	0.82
FER1L6	2.95	7.9.E−05	0.82
CTSS	2.66	3.8.E−09	0.79
BCAS1	2.70	3.4.E−06	0.78
ACKR3	2.04	8.4.E−04	0.76
PSCA	2.52	3.5.E−13	0.76
SAA2‐SAA4	1.66	1.4.E−02	0.76
ALG1L	5.13	1.3.E−06	0.75
SAA1	1.47	4.7.E−02	0.75
HSD17B11	1.86	8.9.E−04	0.71
STRA6	2.57	1.4.E−04	0.71
CFB	3.13	7.9.E−10	0.69
PLAC8	2.14	9.7.E−05	0.68
GCNT3	1.33	3.2.E−02	0.68
FUT2	1.92	3.1.E−04	0.68
WFDC2	3.38	1.0.E−09	0.68
IRAG2	2.07	4.0.E−02	0.67
B3GNT6	4.78	1.9.E−12	0.66
LOXL4	3.63	1.8.E−04	0.66
RARB	3.12	1.6.E−08	0.66
CEACAM5	2.02	2.4.E−06	0.66
ALDH5A1	2.09	1.3.E−02	0.65
EPHB2	2.79	7.6.E−07	0.64
GLUL	1.30	4.9.E−02	0.64
MDK	2.51	5.3.E−05	0.64
BCL2A1	5.24	3.1.E−10	0.63
SMIM22	2.49	3.2.E−04	0.63
CDH6	5.74	1.7.E−12	0.62
C4orf19	3.62	6.7.E−05	0.62
WDR72	4.49	2.1.E−15	0.61
CEACAM6	1.67	9.8.E−03	0.61
MYEOV	2.00	7.1.E−03	0.61
LYPD2	3.83	1.9.E−10	0.60

Abbreviations: HPV, human papillomavirus; RRP, recurrent respiratory papillomatosis.

4. Discussion

The CR method enables the in vitro establishment of primary epithelial cells from healthy human tissues and human tumor samples. In this study, we used the CR method to investigate host gene alterations in HPV6‐infected epithelial cells. Although tissue‐based gene expression analysis is direct and feasible, it reflects gene expression from the non‐epithelial components of papillomas, such as infiltrating leukocytes, fibroblasts, and endothelial cells. To elucidate HPV6‐induced transcriptional changes specifically in epithelial cells, we utilized CR cultures to obtain purified epithelial populations and performed a correlation analysis between HPV6 and host gene expression. We successfully established CR cells from RRP and NP tissues. These cells were capable of multiple passages. Although RRP cells exhibited superior proliferation and serial passaging abilities compared with NP‐derived CR cells, notable variability was observed between individual cases. These results suggest that CR cells retain some of the characteristics of their original tissues. HPV6 gene expression was detected in all RRP‐derived CR cells, but the expression levels varied significantly across different cases. Several genes were highly expressed in RRP‐derived CR cells, and some were correlated with HPV6 gene expression. These findings suggest that the observed variability in RRP‐derived CR cells is likely driven by differences in HPV6 gene expression and the host gene expression associated with it. Based on these observations, the CR method could be a valuable tool for investigating HPV6‐induced epithelial transformation and the cellular and molecular mechanisms underlying RRP pathogenesis.

HPV6 gene expression in RRP‐derived CR cells decreased with each passage in our CR cultures, consistent with previous reports [18]. This suggests that the HPV6 genome persists in an episomal form, rather than being integrated into the host genome. Furthermore, the absence of widespread reinfection in CR culture suggests that viral production or infection targets, such as basal cells, are not sustained in the CR culture system. Single‐cell analysis of epithelial cell composition and gene expression in RRP‐derived primary CR cultures may provide new insights into the mechanisms underlying HPV6 persistent infection.

Analysis of genes highly expressed in RRP‐derived CR cells and those showing a strong correlation with HPV6 expression identified gene groups implicated in cell inflammation, proliferation, and glycosylation. Among the inflammatory genes identified in this study, Cathepsin S (CTSS) is an enzyme that regulates the maturation of neutrophil serine proteases essential for neutrophil activation [19]. CTSS also contributes to neutrophil recruitment. In RRP tissue, neutrophils are more abundant compared with NP tissue [20]. Areas of neutrophil accumulation in inflammatory tissues, including tumors, have fewer T cells, and an increase in neutrophils is associated with suppressed T cell responses [21, 22, 23]. Therefore, elevated Cathepsin S expression may be involved in the formation of a neutrophil‐dominant immunosuppressive microenvironment [23]. Another gene of interest is complement factor B (CFB), a serine protease that activates the alternative complement pathway, which is associated with inflammation, immunological regulation, and bacterial cytotoxicity [24]. CFB promotes airway hyper‐responsiveness and inflammation by inducing Th2 cytokines such as IL‐4, IL‐5, and IL‐13 [25]. Therefore, CFB may contribute to the Th2 microenvironment in RRP by potentially suppressing Th1‐mediated antiviral responses. Among the cell oncogenic genes, CEACAM5 and CEACAM6 function as cell adhesion molecules and are involved in tumor growth through cell–cell adhesion and signal transduction [26]. CEACAM6 can activate the ERK1/2/MAPK pathway directly or through EGFR, leading to stimulation of tumor proliferation [27]. These findings suggest that HPV6 infection upregulates proliferation‐associated genes, thereby contributing to RRP tumor progression.

GO analysis revealed a strong correlation between HPV6 and the glycosylation‐related genes. The upregulation of these genes in virus‐infected epithelial cells may have significant implications for viral entry and immune evasion, particularly through antigen epitope masking. For instance, glycosylation of viral surface proteins, such as the SARS‐CoV‐2 spike protein and Epstein–Barr virus (EBV) glycoproteins, enhances the viral ability to bind host cell receptors [28, 29]. Additionally, extensive glycosylation of viral glycoproteins can shield antigenic epitopes, preventing recognition by neutralizing antibodies and contributing to immune evasion [30, 31]. These findings suggest that targeting glycosylation pathways may offer new therapeutic strategies for the treatment of RRP, potentially improving immune response and viral clearance.

This study has several limitations. First, the small sample size and the diverse disease states of the patients limited the generalizability of the findings. Moreover, all patients had recurrent papillomatosis, and none were HPV11‐positive. Future comparative studies with a larger number of cases, including non‐recurrent cases and HPV11‐positive papillomatosis, would be valuable. Second, the higher proliferative capacity of RRP‐derived CR cells compared with NP‐derived CR cells may reflect fewer cell divisions at P0. This could be due to a higher proportion of cells with proliferative capacity in RRP tissues, requiring fewer divisions to achieve the same final cell count, even when starting from equivalent cell numbers or tissue amount. Third, clustering analysis of gene expression data from RRP‐derived and NP‐derived CR cells revealed no distinct separation into RRP and NP clusters. As a result, we were unable to identify the gene sets characteristic of each CR cell type, which may reflect potential influences from patient backgrounds and other confounding factors. Finally, the functional relevance of genes correlated with HPV6 remains unclear. To address this, future single‐cell RNA sequencing is essential to better understand the cellular heterogeneity of HPV6‐infected epithelium and to explore the potential functional roles of these correlated genes.

5. Conclusion

In this study, we highlighted the potential link between HPV6‐induced gene expression changes in epithelial cells and the pathogenesis of RRP through gene expression analysis of CR cells. The CR method served as an effective tool to investigate the cellular and molecular mechanisms involved in RRP pathogenesis. Further investigations are needed to fully elucidate the roles of these gene expression changes in RRP progression and to explore potential therapeutic strategies targeting these pathways.

Ethics Statement

The study protocol was approved by the Human Ethics Committee of the University of Tokyo (No. 2020120G) and Tokyo University of Science (No. 20011) and complied with the tenets of the amended Declaration of Helsinki.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1. Reagents used for cell culture. FBS, fetal bovine serum; EGF, epidermal growth factor.

Table S2. List of upregulated DEGs in RRP at P0. DEG, differentially expressed genes; RRP, recurrent respiratory papillomatosis.

Table S3. List of downregulated DEGs in RRP at P0. DEG, differentially expressed genes; RRP, recurrent respiratory papillomatosis.

Table S4. List of GO terms enriched in RRP at P0. GO, Gene Ontology; RRP, recurrent respiratory papillomatosis.

Table S5. List of GO terms enriched in NP at P0. GO, Gene Ontology; NP, non‐papilloma.

Table S6. List of GO terms enriched in genes correlated with HPV6. GO, Gene Ontology; HPV6, human papillomavirus 6.

Matsumoto N., Ueha S., Ueha R., et al., “ HPV6‐Induced Gene Expression Signature in Recurrent Respiratory Papillomatosis,” The Laryngoscope 135, no. 10 (2025): 3732–3739, 10.1002/lary.32325.