Establishment and characterization of immortalized human vocal fold fibroblast cell lines

Vocal fold scarring is abnormal scar tissue in the lamina propria layer of the vocal fold. To facilitate investigation of vocal fold scarring, we established and characterized immortalized human vocal fold fibroblast (iHVFF) cell lines. Human vocal fold fibroblasts were immortalized by introducing Simian virus 40 large T antigen (SV40TAg) by transfection. Successfully transfected fibroblasts were sorted using flow cytometry. Immunofluorescence cytochemistry and western blot were applied to analyze the expression of fibronectin, vimentin, alpha-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP). Cell proliferation rate was measured by CCK-8 assay. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the mRNA expression level. The iHVFFs continued to proliferate for more than 30 generations and appeared spindle-shaped. The expression of Vimentin and α-SMA were detected in both iHVFFs and primary fibroblasts, and enhanced expression of FAP was observed in iHVFFs. Furthermore, iHVFFs exhibited an increased proliferative capability compared with the primary fibroblasts. RT-qPCR results suggested that collagen type III alpha 1 chain (COL3A1), interleukin-6, cyclooxygenase 2 (COX2), hyaluronan synthase 2 (HAS2), hepatocyte growth factor (HGF) in the iHVFFs significantly increased, whereas transforming growth factor-β1 (TGF-β1), elastin and matrix metallopeptidase-1 (MMP-1) expression significantly downregulated. No differences in mRNA expression of α-SMA, fibronectin and collagen type I alpha 2 chain (COL1A2) were noted between iHVFFs and primary fibroblasts. iHVFFs can be used as a novel tool cell for future researches on the mechanisms of pathogenesis and treatment of vocal fold scarring.


Introduction
Vocal fold scar is one of the most common benign laryngeal diseases. It is a pathological condition characterized by reduced flexibility of the mucosal vocal fold, which includes vocal fold atrophy, sulcus vocalis, anterior glottis web, traumatic or iatrogenic scarring, and so on (Hantzakos et al. 2019). Vocal fold scar may cause a series of symptoms comprising Yinying Chu and Yi Fang have contributed equally to this work. hoarseness, voice changes, dysphonia, exercise intolerance, and even airway obstruction. Diagnosis of this disease primarily relies on laryngeal examination using laryngoscope or stroboscope while the therapeutic methods remain challenging for laryngologists. Thus, unraveling the mechanisms underlying the pathogenesis of glottic scarring is necessary.
Vocal fold fibroblasts (VFFs) are recognized to play a pivotal role in the vocal fold scar formation. The VFFs would proliferate rapidly and secrete excessive collagen matrix proteins to would surface when the vocal fold was injured, resulting in the formation of fibrosis and scar (Li et al. 2014). Therefore, the analysis and research on VFFs are able to provide a theoretical basis for the understanding and treatment of vocal fold scars. However, the primary culture of VFFs is arduous, and research results from VFFs cultured from different labs vary widely.
As firstly discovered in 1960, the simian virus SV40 can encode the large and small tumor antigens involved in tumorigenesis (Srinivasan et al. 1997). The large tumor (large T) antigen is able to bind to a variety of cell cycle-related proteins to escape cell cycle arrest and apoptosis (Pipas 2009). Therefore, the SV40 large T antigen is often applied for genome integration resulting in cell immortalization. So far, a series of cells from different origins have been successfully immortalized into cell lines using SV40 large T antigen through all kinds of deliveries (Sun et al. 2020;Uprety et al. 2019;Mitani et al. 2019).
In the present study, we aimed to establish immortalized VFF cell lines by introducing EF1α promoterdriven SV40 large T antigen into primary human vocal fold fibroblasts and observed their morphologic characterizations, proliferation abilities, and expressions of fibroblast-related genes and proteins.

Human vocal fold fibroblasts isolation and culture
The research was submitted and approved by the ethics committee of the Affiliated Eye and ENT Hospital of Fudan University (No. 2020014-1). All patients were informed in detail and signed consent forms to allow access to their clinic and ward information. Primary human vocal fold fibroblasts were isolated from surgical resected normal vocal fold of patients with hypopharyngeal carcinoma enrolled in the study No. 2021155-1.
Briefly, the tissues were lavaged in sterile distilled water three times, minced manually to fragments < 1mm 3 and digested with 0.25% Trypsin-EDTA and Collagenase Type I for 1 h at 37 °C. The digested solution was filtered through 70 µm cell strainer and centrifuged at 1000 rpm for 5 min. Precipitated cells were resuspended in highglucose Dulbecco's modified eagle medium (DMEM, Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, USA) and 1% penicillin-streptomycin, seeded in the T75 cell culture flasks and maintained in the 37 °C humidified incubator with 5% CO 2 .

Plasmid construction and cell transfection
The pWPI-LargeT-internal ribosome entry site (IRES)-green fluorescent protein (GFP) plasmids consisting of EF1α promoter and the Large T antigen were kindly shared by Dr. Pengyu Huang from Shanghai Tech University. 293FT cells were transfected with the pWPI-LargeT-IRES-GFP plasmids and packaging plasmids for recombinant lentivirus packaging. Viruses were collected, titred, and used for subsequent transfection on primary human vocal fold fibroblasts. The infection efficiency was the highest at an MOI of 100. Cell medium containing viruses was changed to fresh medium 72 h after the transfection.

Quantitative real-time polymerase chain reaction analysis
Fibroblasts were lysed with TRIzol (Invitrogen, CA, USA) and total RNA was extracted. Prime-Script RT reagent Kit (TaKaRa, Tokyo, Japan) was applied to convert the total RNA into corresponding complementary DNA by reverse transcription. Real-time Polymerase chain reaction (RT-PCR) was performed using TB Green Premix ExTaq (TaKaRa, Tokyo, Japan) on the Applied Biosystems 7500 Fast Real-Time PCR System (BioRad, Richmond, CA, USA). The reaction system was comprised of 10 μL TB Green Premix Ex Taq (2x), 0.4 μL ROX Reference Dye II (50 ×), 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), 2 μL cDNA template, and 6.8 μL sterile ddH2O. The PCR cycling program was 95 °C for 30 s and the next 40 cycles of 95 °C for 5 s, 60 °C for 34 s. Melt curve at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. The relative expression levels of target genes were normalized to GAPDH and analyzed by the comparative 2 − ΔΔCT method. PCR samples were visualized by agarose gel electrophoresis. Primers used for PCR were listed as follows in Table 1.

Cell sorting
A Fluorescence Activated Cell Sorting (FACS) Ariall cell sorter (BD Biosciences) was used for cell sorting. Briefly, cell samples were resuspended into FACS buffer after trypsin digestion and phosphate buffered saline (PBS) washing. Single cell suspensions were prepared and the GFP positive cells were evaluated and sorted ultimately. Data were analyzed using FlowJo Software.

Cell proliferation assay
Cell proliferative ability was quantified with the CCK-8 assays kit (Dojindo, Tokyo, Japan) according to the manufacturer's instructions. In brief, fibroblasts were seeded in a 96-well plate at 2 × 10 3 cells per well.
After attachment, at 0, 24, 48, 72, 96, 120 h, cells were treated with 10 μL of CCK-8 reagent and incubated for 1 h at 37 °C. Absorbance was measured at a wavelength of 450 nm using Microplate Reader (Bio-Rad, Richmond, USA). Cells of each group were analyzed for 5 replicates.

Establishment of immortalized human vocal fold fibroblast cell lines by a transfer plasmid containing SV40 large T genes
Primary fibroblasts were successfully isolated from the vocal fold specimens of three donors and termed VFF1, VFF2, VFF3. As shown in Fig. 1A, the extracted primary cells exhibited typical fibroblastic morphology with stellate-shaped or spindle-shaped.
To overcome the paucity of the limited life span of primary cells, we established an immortalized fibroblast cell line iHVFF by a transfer plasmid containing SV40 Large T genes into the primary human vocal fold fibroblasts. The structure of the plasmid is illustrated in Fig. 1B. Subsequently, the GFP positive iHVFFs were sorted using FACS (Fig. 1C). The introduced large T genes in iHVFFs were successfully validated by PCR amplification and agarose gel electrogenesis (Fig. 1D).

Characteristics of immortalized human vocal fold fibroblast cell lines
To shed light on whether the immortalized VFFs had similar properties compared with primary VFFs, we observed the characteristics of the immortalized fibroblasts. As shown in Fig. 2A and B, approximate levels of Vimentin, α-SMA and fibronectin were expressed after the immortalization, indicating that immortalized VFFs keep demonstrating fibroblastlike characteristics. However, as shown in Fig. 2B, FAP overexpression was observed in all immortalized fibroblasts, whereas most of primary VFFs exhibited limited expression of FAP. This suggests that immortalized fibroblasts were constantly kept in an activated state, which might contribute to their persistent capacity to replicate.
The phenotype of immortalized human vocal fold fibroblast cell lines Ultimately, we assessed the proliferation capabilities of immortalized cell lines. The results pinpointed that the immortalized VFF cell lines obtained a stronger capacity of proliferation versus primary cells (Fig. 3A). Next, the relative gene expression level was tested and analyzed using the 2 −ΔΔCT method ( Fig. 3B). Real-time PCR demonstrated that immortalized fibroblasts exhibited a significantly higher level of collagen type III alpha 1 chain (COL3A1), Interleukin-6 (IL-6), cyclooxygenase (COX2), hyaluronan synthase 2 (HAS2) and hepatocyte growth factor (HGF) mRNA while a lower level of transforming growth factor-β1 (TGF-β1), elastin, and matrix metallopeptidase-1 (MMP-1) mRNA in comparison to the wild type group (P < 0.05). Meanwhile, there were no significant differences of α-SMA, fibronectin and collagen type I alpha 2 chain (COL1A2) mRNA levels between the immortalized cell lines and primary fibroblasts (P > 0.05).

Discussion
The human vocal fold is a multi-layer architecture comprised of the epithelium layer, lamina propria, and muscular layer. Congenital sulcus vocalis, voice overuse, trauma, CO 2 laser surgical operations and chemicals could result in vocal fold scarring (Allen 2010). Vocal fold wound healing is considered as a dynamic process consisting of inflammation, cell proliferation and structure remodeling (Maxson et al. 2012). Fibroblasts activated by transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), fibroblast growth factor (FGF), also called myofibroblasts, are considered to be one of the main sources of extracellular matrix components such as collagen type I, collagen type III, elastin and proteoglycans (Hansen and Thibeault 2006). Myofibroblasts proliferate, migrate, secrete extracellular matrix and cytokines, and remodel the injured structure.
However, in previous studies, as the passage number of primary human vocal fold fibroblasts increased, the proliferation rate slowed down and the proliferation ability declined due to the cellular senescence. The consistency of repeated experiments is limited as well (Claydon and Owens 2008). Chen and Thibeault immortalized human vocal fold fibroblasts extracted from healthy human vocal cords through transducing IRES internal ribosome entry site, GFP green fluorescent protein, VF Vocal fold fibroblasts, IVF Immortalized vocal fold fibroblasts, PCR polymerase chain reaction the human telomerase reverse transcriptase (hTERT) gene. The cell lines maintained the similar spindleshaped morphologic characteristics, gene expression (COL1A1, COL1A2, COL6A3, fibronectin, elastin) and chromosome karyotyping compared with primary cells (Chen and Thibeault 2009). In this experimental research, we successfully utilized the SV40 large T antigen to immortalize the HVFFs obtained from patients with hypopharyngeal carcinoma, which we named iHVFFs and demonstrated that this immortalized cell line maintained similar morphology to primary cells under phase-contrast microscopy. Then, we evaluated the mRNA transcriptive level of fibroblast markers, including ECM proteins, proinflammatory and pro-fibrosis cytokines and cancer-associated fibroblast markers, such as α-SMA and FAP.
Activated fibroblasts, also called myofibroblasts, were considered to be the major element of wound healing response and vocal fold scars (Hinz 2016). It is widely accepted that Vimentin is expressed in both quiescent, resting fibroblasts and myofibroblasts whereas α-SMA and FAP are expressed in myofibroblasts (Kalluri 2016). Thus, α-SMA and FAP were known to be the markers of activated fibroblasts. To verify the fibroblast traits of SV40 large T transduced HVFFs, we examined the expression of Vimentin, α-SMA and FAP by immunofluorescence staining, western blot and RT-qPCR. In line with the results of the previous study (Sun et al. 2020), the primary fibroblasts we isolated maintained the characteristics of fibroblasts and were partially activated. Notably, the two of three immortalized cell lines exhibited enhanced FAP expression, which means after immortalization, iHVFFs may be further activated to some extent.
Next, we assessed the proliferative phenotype and the CCK8 results confirmed that immortalized cells have stronger proliferation ability compared with primary cells (P < 0.05). HVFF immortalization can lead to a higher proliferation rate. Enhanced proliferation rate has also been observed in other cell lines transduced with SV40 T antigen, such as human dental mesenchymal cells, human corneal epithelial cells (Huang et al. 2015;Kim et al. 2016). Another commonly used immortalization method hTERT expression also led to a higher proliferation rate in rat embryo dorsal root ganglia Schwann cells (SCs), primary porcine bronchial epithelial cells (PBECs), etc. (Jiang et al. 2012;Xie et al. 2018). Nevertheless, growth rates remained unchanged in hTERT transfected Scleroderma (SSc) fibroblast (Kapanadze et al. 2010).
Owing to myofibroblasts being one of the main sources of IL-6, fibronectin, collagens, MMPs, elastin and HGF, we measured the corresponding mRNA transcript level expression level in iHVFF cell lines and parental primary cells. IL-6 is a proinflammatory and pro-fibrosis cytokine secreted by tumor cells, cancer-associated fibroblasts, endothelial cells, and tumor-infiltrating immune cells (Kaur et al. 2020;Yang et al. 2020). Dysregulation of IL-6 has been considered to be related to chronic Fig. 2 Identify and characterization of iHVFFs with immunocytochemistry and western blot. A Immunofluorescence staining of the fibroblast markers, Vimentin and α-SMA, in iHVFFs and primary fibroblasts. B Protein bands of Vimentin, α-SMA, FAP, and fibronectin in iHVFFs and primary fibroblasts observed using western blot assay. α-SMA alpha-smooth muscle actin, FAP fibroblast activation protein, VF Vocal fold fibroblasts, IVF Immortalized vocal fold fibroblasts inflammation, fibrosis, immunity diseases (Tanaka et al. 2014). Our study confirmed that immortalization might enhance IL-6 transcript levels. Extracellular matrix (ECM) proteins mainly consist of fibronectin, collagen, elastin, hyaluronic acid (HA). Fibronectin (FN), secreted by activated fibroblasts, is a key component that participated in wound healing, fibrosis, cell migration, cell adhesion, wound healing, and carcinoma progression (Grinnell et al. 1981;Patten and Wang 2021;Pankov and Yamada 2002). Vocal scarring is characterized by disorganized collagen deposition. Hyaluronic acid (HA) is actively secreted during wound healing and inflammation. HA has a wide range of effects in anti-inflammatory, anti-proliferation, tissue regeneration, angiogenesis, etc. (Fraser et al. 1997;Litwiniuk et al. 2016). qPCR results demonstrated that the transcript level of fibronectin and collagen I in iHVFFs remained similar, whereas collagen III and HA increased in iHVFFs in comparison to primary cells. Furthermore, the transcript level of elastin decreased in immortalized cells. TGF-β is a pleiotropic factor participating in all stages of the wound healing process (Lichtman et al. 2016;Penn et al. 2012;Finnson et al. 2013). TGF-β1, one of the subtypes of the TGF-β family, plays a vital role in the regulation of inflammation, angiogenesis, re-epithelialization to accelerate the wound healing process and enhance the breaking strength of repaired tissue (Sweed et al. 2021;Beck et al. 1991). The iHVFF cell lines we created exhibited a lower transcript level of TGF-β1. The mechanism of these alterations remains further exploration. Fig. 3 Cell proliferation phenotype and relative gene expression levels of iHVFFs. A Cell proliferative capacities were tested using the CCK-8 assay. Three immortalized iHVFF cell lines all exhibited a significant higher proliferative capability during day 2-5 in comparison with primary fibroblasts (P < 0.01). B The mRNA expression levels of α-SMA, fibronectin, elastin, COL1A2, COL3A1, TGF-β1, MMP1, COX2, IL-6, HAS2, HGF in iHVFFs and untransfected primary cells. COL1A2 collagen type I alpha 2 chain, COL3A1 collagen type III alpha 1 chain, TGF-β1 transforming growth factor-β1, MMP-1 matrix metallopeptidase-1, COX2 cyclooxygenase 2, IL-6 interleukin-6, HAS2 hyaluronan synthase 2, HGF hepatocyte growth factor, VF Vocal fold fibroblasts, IVF Immortalized vocal fold fibroblasts

Conclusion
In this study, the iHVFFs cell lines were successfully established by introducing SV40 large T antigen into primary human vocal fold fibroblasts derived from normal vocal fold of hypopharyngeal carcinoma patients. Our study also indicated that iHVFFs have similar phenotypes and stronger proliferation capacity compared with primary VFFs. We profiled gene expressions of iHVFFs and confirmed that several genes related to ECM proteins, proinflammatory and pro-fibrosis cytokines were changed. The immortalized cell lines may serve as a valuable tool for further investigation of vocal fold diseases.