Abstract
Objectives
Hypopharyngeal cancer (HPC), originating from the hypopharyngeal mucosa, is associated with a poor prognosis. Extracellular matrix (ECM) stiffness is closely linked to tumor progression and patient outcomes. This study aimed to investigate the effects of matrix stiffness and to identify molecular markers relevant to HPC prognosis, with the goal of improving clinical outcomes.
Methods
Immunohistochemical analysis and cervical enhanced computed tomography (CT) data were used to evaluate correlations among CT values, matrix stiffness, and prognosis, and to develop a prognostic model for HPC. Cell culture models with varying matrix stiffness were established using polypropylene hydrogel. Western blotting, colony formation, EDU incorporation, and Transwell assays were employed to assess the effects of matrix stiffness on proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). High-throughput sequencing identified differentially expressed genes in HPC cells cultured on matrices of differing stiffness. Gene editing, in vivo subcutaneous tumorigenesis studies, and Western blotting were performed to elucidate the molecular mechanisms by which high matrix stiffness influences HPC progression and the role of ephrin A2 (EFNA2) in proliferation, migration, and EMT.
Results
Arterial phase CT values were positively correlated with matrix stiffness. Increased matrix stiffness was associated with lymph node metastasis, diminished therapeutic response, and poorer prognosis. Furthermore, metastatic lymph nodes in HPC patients exhibited higher CT values than those in patients with nasopharyngeal carcinoma. A high-stiffness ECM promoted proliferation, migration, and EMT in HPC cells. Mechanistically, a stiff ECM enhanced EFNA2 expression, thereby promoting proliferation, migration, EMT, and tumor growth in vivo.
Conclusion
EFNA2 and elevated matrix stiffness jointly contribute to the malignant phenotype in HPC. EFNA2 may represent a potential therapeutic target for managing HPC progression induced by high matrix stiffness.
Keywords: Hypopharyngeal Carcinoma, Matrix Stiffness, EFNA2, Epithelial-Mesenchymal Transition
INTRODUCTION
Head and neck tumors rank as the seventh most common malignancies worldwide [1]. Hypopharyngeal carcinoma (HPC) is a particularly aggressive form of head and neck cancer. Despite advances in research and treatment options—including surgery, radiotherapy, and targeted therapy—the 5-year survival rate for HPC patients remains low, at approximately 30%–35% [2].
The stiffness of the extracellular matrix (ECM) is a defining physical and mechanical characteristic of solid tumors. As tumors progress, the ECM undergoes increased deposition, modification, and remodeling, leading to a highly fibrotic microenvironment with enhanced rigidity [3-5]. This increased stiffness is strongly associated with poor patient outcomes [6]. Moreover, ECM stiffness significantly influences cellular morphology, growth, and differentiation [7-10] and affects the synthesis, secretion, and metabolism of proteins and cytokines by activating multiple mechanotransduction pathways—some of which remain uncharacterized [11-13].
Ephrin A2 (EFNA2), a member of the ephrin family, is anchored to the cell membrane via glycosylphosphatidylinositol bonds and functions as a ligand for various Eph receptor tyrosine kinases. EFNA2 plays roles in tissue and organ development, neovascularization, cell adhesion and separation, as well as tumor progression [14]. Notably, EFNA2 expression is elevated in several cancers—including liver, breast, renal cell, and prostate cancers—compared to normal tissues. In prostate cancer, EFNA2 promotes migration and invasion by inducing epithelial-mesenchymal transition (EMT) and angiogenesis, while in liver cancer it inhibits apoptosis through the Rac1/Akt/NF-κB signaling pathway, thereby facilitating tumor growth [15-18]. However, the effects of EFNA2 on HPC’s biological behavior remain underexplored.
This study highlights a correlation between high matrix stiffness in tumor tissues, elevated computed tomography (CT) values, and poor prognosis in HPC patients. Notably, metastatic lymph nodes in HPC patients exhibit higher CT values than those in nasopharyngeal carcinoma (NPC) patients. Mechanistically, high matrix stiffness may drive HPC progression by upregulating EFNA2 expression. These findings offer valuable insights into the molecular mechanisms linking matrix stiffness to HPC development and prognosis, and they highlight the potential for identifying molecular markers and therapeutic targets to improve HPC outcomes.
MATERIALS AND METHODS
This study was approved by the Institutional Review Board of the Affiliated Hospital of Nantong University (No. 2022-L040) with written informed consent from patients with hypopharyngeal carcinoma and patients with NPC.
Animal study
The Laboratory Animal Center of Nantong University’s Ethics Committee authorized the methods for animal care and use. All relevant institutional and governmental legislation regarding the ethical use of animals were adhered to approval ID: SYXK (SU) 2022-0046.
Collection of human hypopharyngeal carcinoma and NPC samples
Tumor tissues were collected from the Pathology Departments of the Affiliated Hospital of Nantong University (China), and were stored in Clinical Biobank of Department of Otorhinolaryngology-Head and Neck Surgery of Affiliated Hospital of Nantong University. All patients were pathologically confirmed to have hypopharyngeal carcinoma or NPC, provided informed consent, and did not receive any anti-tumor therapy before biopsy or surgery.
Immunohistochemical staining
The paraffin sections were dried at 60 °C for 6 hours and deparaffinized with xylene. After repairing antigens and inhibiting endogenous peroxidase activity, tissue sections were incubated with primary antibody (COL1A1, Proteintech, 67288-1-Ig, dilution rate: 1:500) at 4 °C overnight. The slides were then incubated with secondary antibody (enhanced enzyme-labeled goat anti-mouse/rabbit IgG polymer, ZSGB-BIO, PV-9000) at room temperature for 30 minutes, counterstained with diaminobenzidine (DAB, Sigma, 7411-49-6) and hematoxylin (Sigma, H3136). The staining area was scored as 1 for <10%, 2 for 10%–25%, 3 for 25%–50%, and 4 for >50%. The staining intensity was scored as 1, 2, 3, or 4 for negative, weak, moderate, or strong staining, respectively. The staining score was obtained by multiplying the staining area score with the staining intensity score. The detailed clinicopathological features of the patients are listed in Supplementary Table 1.
Cell culture
The HPC cell line (FaDu) was obtained from ATCC, and it was grown at 37 °C in high-glucose Dulbecco’s modified eagle medium (DMEM) with 10% fetal bovine serum. FaDu cells were cultured with two kinds of matrix with different hardness. Firstly, the gel with different hardness was irradiated under ultraviolet light and washed with proper amount of aseptic water. FaDu cells in good condition were digested and cultured on polyacrylamide hydrogel with different hardness for 3 days.
Preparation of polyacrylamide gels substrates
The short plate of the Western blot glass and the long plate of thick glass (1.0 mm specification) were washed, placed in a high-pressure sterilizer, dried, and irradiated under ultraviolet light for 1 hour. Acrylamide (AM), N,N’-methylenebisacrylamide (Bis), ammonium persulfate, TEMED, and distilled water were prepared under sterile conditions. In the ultra-clean worktable, the glass plates were clamped with a glue rack, and the solution is mixed in different proportions and added to the gap between the glass plates according to the research of TseJR [19]. To simulate the low matrix stiffness (SOFT, 1 kPa) or the high matrix stiffness (STIFF, 20 kPa), the ratios of AM:Bis were adjusted to 5%: 0.03% (SOFT) and 8%: 0.264% (STIFF), respectively. After solidification, the gel was cut with a sterile tube about 34 mm in diameter, and the cut gel was put into a six-hole plate with sterile water and incubated at 4 °C for 24 hours. The gel was then exposed to ultraviolet light for 30 minutes, and the liquid was discarded. The gel surface was fully covered with the crosslinking agent Sulfo-SANPAH (0.2 mg/mL) under sterile conditions, and exposed to ultraviolet light for 30 minutes. Any unbound sulfonamides were washed off with sterile water. Finally, 20 μL of aseptic collagen I (0.2 mg/mL) was added to each well. The gels were stored at 4 °C and set aside.
Transfection
The EFNA2 interference plasmid (shEFNA2), the empty plasmid (Vector), and the EFNA2 overexpression plasmid (OE EFNA2) purchased from Shanghai Genechem Ltd. were introduced into FaDu cells according to the transfection method of Liposome TM3000 (Invitrogen). All sequences are listed in Supplementary Table 2.
Quantitative real-time polymerase chain reaction
Total RNA was extracted from FaDu cells referring to the protocol of the TRIzol method RNA extraction kit (Thermo Fisher Scientific, 15596018). Complementary DNA (cDNA) synthesis was performed with 1 μg of total RNA using the Reverse Transcription Kit (Biosharp, BL696A), which includes 5× Reaction Mix and Enzyme Mix, under the following conditions: 25 °C for 10 minutes, 55 °C for 15 minutes, 85 °C for 5 minutes, and 4 °C hold. For quantitative real-time polymerase chain reaction (qRT-PCR), specific primers for EFNA2 and the reference gene GAPDH were designed and synthesized. The sequences of the primers are listed in Supplementary Table 3. The qRT-PCR reaction was performed in a 20 μL reaction volume containing: 10 μL of 2× SYBR Green Master Mix (intercalator dye used for DNA binding), 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 2 μL of cDNA, and 7.2 μL of RNase-free water, under the following conditions: 95 °C for 10 minutes, and 40 cycles of 95 °C for 15 seconds and 60 °C for 1 minute. The cycle threshold (Ct) values were quantified using the 2^-ΔΔCt method. The formula for calculating the relative expression level of the target gene is: 2^-ΔΔCt, where ΔΔCt=(Ct, target gene−Ct, reference gene) sample−(Ct, target gene−Ct, reference gene)control.
Western blotting
Cell protein was extracted with prepared RIPA lysate on ice, and the protein concentration was determined by Bicinchoninic Acid Assay. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to electrophorese 20 μg of proteins, which were subsequently transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% defatted milk at room temperature for 1 hour, incubated with primary antibodies at 4 °C overnight, and then with secondary antibodies (Proteintech, SA00001-1, SA00001-2) at room temperature for 1 hour. Using an ECL Plus Kit, the immunoreactive protein bands were discovered (Zsbio). All the antibody information is presented in Supplementary Table 4.
Transwell assays
In vitro, FaDu cells (5×104 cells) were resuspended in 200 μL of serum-free DMEM, and the cell suspensions were inoculated into Transwell chambers (8 μm pores; BD Biosciences), and the lower layer was added with 500 μL of complete medium containing 10% serum. After 24 hours of incubation, the chambers were removed, fixed in 4% paraformaldehyde, stained with 1% crystalline violet, photographed under microscope.
EDU staining
A total of 104 cells were inoculated in each well, the medium was removed and 100 μL EDU solution (50 μM) was added. After 2 hours of incubation, the medium was discarded, fixed in 4% paraformaldehyde for 30 minutes. 50 μL glycine (2 mg/mL) was added, incubated for 5 minutes. 100 μL 1×Apollo staining solution and 100 μL 1×Hoechst 33342 was added and incubated for 30 minutes at room temperature. The cells were photographed under fluorescent microscope and the percentage of EDU positive cells was calculated (EDU positive rate=number of purple cells/numbers of blue cells×100%).
Wound-healing assay
FaDu cells were seeded into 6-well plates. When cells density reached 90%, they were scratched using a 200 μL pipette tip. Cells were cultured with serum-free DMEM and photographed at 0 and 36 hours under microscope.
Colony formation assay
Inoculate 6-well plates with a cell density of 103 cells/well and remove the medium after 14 days of incubation, wash with PBS and fix with 4% paraformaldehyde, then stain with 0.1% crystalline violet, wash with tap water and air dry for photographs.
Phalloidin assay
Cells were washed with pre-warmed PBS, fixed with 4% paraformaldehyde for 10 minutes; permeabilized with 0.5% Triton X-100 solution for 5 minutes; cells were covered with the prepared ghost pen cyclopeptide working solution at room temperature for 30 minutes, washed with PBS; nuclei were re-stained using 4´,6-diamidino-2-phenylindole (DAPI) solution, photographed under fluorescent microscope.
Mouse xenograft
Six-week-old male BALB/c nude mice were subcutaneously injected with 2×106 FaDu cells into their flanks, then sacrificed 3 weeks after the inoculation, and tumor xenografts were collected.
Statistical analysis
Statistical was analyzed by SPSS version 23.0 (IBM Corp.) and GraphPad prism. Data were presented as mean±standard deviation. Student t-test, one-way analysis of variance (ANOVA), and two-way ANOVA were used to assess the differences between individual groups, where P<0.05, P<0.01, P<0.001, and P<0.0001 were considered significant. Details of the data are given in the figure legends.
RESULTS
Matrix stiffness and CT values as prognostic markers in patients with HPC
Previous studies have demonstrated that tumor tissue is typically stiffer than normal tissue in solid tumors [20]. In this study, paraffin-embedded specimens were collected from HPC patients treated at Nantong University Hospital between January 2014 and October 2021, along with comprehensive pathological and clinical data. Collagen, which provides structural support to the ECM, is represented by collagen type I (COL1A1), a relatively stiff collagen [21]. Therefore, COL1A1 was used as a marker to assess tissue stiffness. Optimal threshold points for COL1A1 immunohistochemical (IHC) staining scores in primary tumors were determined using X-tile software, dividing samples into low stiffness (scores 0–8) and high stiffness (scores 9–12) groups. Survival analysis revealed that HPC patients with high ECM stiffness had a significantly worse prognosis than those with low ECM stiffness (P=0.006) (Fig. 1A and B). Thus, tumor tissue stiffness —as indicated by COL1A1 expression—may serve as a biological marker for predicting HPC prognosis.
Fig. 1.
COL1A1 expression and arterial phase computed tomography (CT) values correlate with the prognosis of patients with hypopharyngeal cancer (HPC). (A) Representative images of immunohistochemical (IHC) for COL1A1 in HPC tumor tissues. Scale bar: 20 μm. (B) Kaplan-Meier analysis (log-rank test) of the overall survival in patients with HPC and different matrix stiffness values. (C) Pearson correlation analysis between matrix stiffness of tumor tissue and arterial phase CT values of tumor tissue under enhanced CT in HPC patients. (D) Pearson correlation analysis between matrix stiffness of tumor tissue and CT values of tumor tissue in the venous phase under enhanced CT in HPC patients. (E) Kaplan-Meier analysis (log-rank test) of the overall survival in patients with HPC and different CT values of tumor tissue in the arterial phase of enhanced CT. (F) Kaplan-Meier analysis (log-rank test) of the overall survival in patients with HPC and different matrix stiffness with different arterial phase CT values. (G) Representative images of IHC for COL1A1 in lymph nodes from patients with HPC and nasopharyngeal carcinoma (NPC). Scale bar: 20 μm. (H) Quantification of IHC staining scores of COL1A1 expression. Student t-test, mean±standard error of the mean (SEM), *P<0.05 and ****P<0.0001. (I) Quantification of arterial phase CT values of metastatic cervical lymph nodes from patients with HPC and NPC. Student t-test, mean±SEM, ***P<0.001.
Enhanced CT imaging of the neck is commonly used to evaluate regional lesions in patients with head and neck tumors [22]. CT images offer accessible and well-preserved information; the grayscale intensity reflects tissue density and is associated with peri-tumoral lymphocyte infiltration and cervical lymph node metastasis [23]. In this study, clinical data from enhanced CT scans of the neck in HPC patients were analyzed, revealing a significant positive correlation between relative matrix stiffness and the CT values of both the arterial and venous phases of tumor tissue (r=0.426, P=0.019) (Fig. 1C and D). Additionally, using X-tile software, cut-off values for arterial phase CT values in HPC tumor tissues were established, dividing the cohort into a low expression group (39.5–97 HU) and a high expression group (97–110 HU). Survival analysis indicated a significant negative correlation between arterial CT values and patient prognosis (P=0.005) (Fig. 1E). Based on IHC results and CT measurements, patients were classified into four groups: high matrix stiffness with high CT, high matrix stiffness with low CT, low matrix stiffness with high CT, and low matrix stiffness with low CT. Survival analysis of these groups demonstrated that patients with both high matrix stiffness and high arterial phase CT values experienced a significant reduction in overall survival (Fig. 1F).
Additionally, metastatic lymph nodes are generally stiffer than surrounding soft tissue or benign lymph nodes [24]. In a separate cohort of HPC and NPC patients from Nantong University Hospital, results showed that both COL1A1 expression and arterial CT values were significantly higher in cervical lymph nodes with HPC metastasis compared to non-metastatic nodes (Fig. 1G-I), indicating increased matrix stiffness in metastatic lymph nodes. Furthermore, metastatic cervical lymph nodes in HPC patients exhibited significantly higher COL1A1 expression and arterial CT values than those in NPC patients (Fig. 1G-I). This difference may explain why metastatic cervical lymph nodes in NPC patients tend to shrink or disappear after radiotherapy—a response rarely seen in HPC patients. These findings suggest that high ECM stiffness is linked to poor prognosis, and that COL1A1 expression and enhanced CT imaging can serve as markers for prognosis and therapeutic response in HPC patients.
High matrix stiffness promotes FaDu cell proliferation, migration and EMT
FaDu cells cultured on higher-stiffness substrates displayed larger cell areas, irregular morphologies, and enhanced spreading, whereas cells grown on lower-stiffness substrates appeared more rounded and had smaller areas (Fig. 2A). These observations suggest that substrate stiffness may influence the motility and migration of HPC cells [25,26].
Fig. 2.
Effect of different matrix stiffness on FaDu cell proliferation, migration, and epithelial-mesenchymal transition (EMT) processes. (A) Phalloidin staining of FaDu cells on different matrix stiffness, blue: 4´,6-diamidino-2-phenylindole (DAPI); red: ghost pen loop peptide; scale bar: 20 μm, the rightmost image is a merged image magnified four times. (B, C) The proliferation capacity of FaDu cells on different matrix stiffness using a colony formation assay, and quantification of the colony number. Student t-test, mean±standard error of the mean (SEM) (n=3), *P<0.05. (D, E) The proliferation capacity of FaDu cells on different matrix stiffness using an EDU staining assay, and quantification of the positive cells. Scale bar: 20 μm. Student t-test, mean±SEM (n=3), *P<0.05. (F, G) The migration capacity of FaDu cells on different matrix stiffness using a Transwell assay, and quantification of the number of cells that underwent migration. Scale bar: 100 μm. Student t-test, mean±SEM (n=3), ***P<0.001. (H, I) Western blot analysis of EMT-related genes and gray value analysis. Two-way analysis of variance, mean±SEM (n=3); *P<0.05 and ***P<0.001.
Using colony formation, EDU, and Transwell migration assays, we observed that FaDu cells cultured on high-stiffness matrices exhibited increased clonogenicity (Fig. 2B and C), enhanced proliferation (Fig. 2D and E), and greater migration (Fig. 2F and G) compared to cells cultured on low-stiffness matrices. These results indicate that high matrix stiffness promotes the proliferation and migration of HPC cells.
The EMT is a key event in tumor invasion and metastasis. Factors such as ECM components, hypoxia, and matrix stiffness can induce a mesenchymal shift in tumor cells, thereby driving metastasis. EMT progression was evaluated in FaDu cells cultured on substrates of varying stiffness using immunoblotting assays. The results showed increased expression of mesenchymal markers such as Vimentin and N-cadherin, and a significant decrease in the epithelial marker E-cadherin in cells cultured on high-stiffness matrices (Fig. 2H and I), suggesting that a high matrix stiffness environment induces EMT in HPC cells.
Differentially expressed genes in FaDu cells on different matrix stiffness
To investigate gene expression differences in FaDu cells cultured on substrates of varying stiffness, RNA was extracted and subjected to high-throughput sequencing (RNA-seq). In total, 1901 differentially expressed genes (DEGs) were identified, with 999 genes significantly upregulated and 912 genes significantly downregulated in cells cultured on high-stiffness matrices compared to those on low-stiffness matrices (Fig. 3A). To further understand the molecular effects of matrix stiffness on HPC cell progression, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the DEGs. The analyses revealed significant enrichment in pathways and processes, including the PI3K-Akt signaling path-way, axon guidance, and plasma membrane localization (Fig. 3B). Notably, EFNA2 was enriched in all three of these pathways (Fig. 3C). Additionally, the study found marked differences in EFNA2 expression levels among hypopharyngeal carcinoma cells cultured on substrates with different stiffness. Subsequent qRT-PCR and Western blot experiments confirmed that increased matrix rigidity substantially elevated both mRNA and protein levels of EFNA2 (Fig. 3D-F).
Fig. 3.
Analysis and validation of differentially expressed genes (DEGs) of FaDu cells cultured on different substrate hardness. (A) Volcano maps showing the DEGs between FaDu cells cultured on different matrix stiffness, as identified using RNA-seq. Red: upregulated DEGs; blue: downregulated DEGs. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs. (C) Venn diagram showing gene overlap in the PI3K-Akt signaling pathway, axon guidance pathway, and plasma membrane cellular localization. (D, E) Western blot analysis of ephrin A2 (EFNA2) and gray value analysis. Student t-test, mean± standard error of the mean (SEM) (n=3), **P<0.01. (F) Quantitative real-time polymerase chain reaction of EFNA2 in FaDu cells cultured on different matrix stiffness. Student t-test, mean±SEM (n=3), ***P<0.001.
EFNA2 promotes FaDu cell proliferation, migration and EMT processes
To explore the biological functions of EFNA2 in HPC, FaDu cells were transfected with either an EFNA2 overexpression construct (OE EFNA2) or short hairpin RNA targeting EFNA2 (shEFNA2), along with their corresponding control vectors. EFNA2 mRNA and protein levels were measured by qRT-PCR and Western blotting across different groups. The results demonstrated that EFNA2 expression was significantly increased in the OE EFNA2 group and decreased in the shEFNA2 group relative to the Vector control, with shEFNA2-1 showing the most pronounced inhibition (Fig. 4A-C). The stable FaDu cell line with EFNA2 knockdown (shEFNA2-1) was selected for further experiments.
Fig. 4.
Ephrin A2 (EFNA2) promotes FaDu cell proliferation and migration and the epithelial-mesenchymal transition (EMT) process. (A, B) Western blot analysis of EFNA2 in FaDu stable cell lines overexpressing and knocking down EFNA2, and gray value analysis. One-way analysis of variance (ANOVA), mean± standard error of the mean (SEM) (n=3), ***P<0.001. (C) Quantitative real-time polymerase chain reaction of EFNA2 mRNA expression in FaDu stable cell lines overexpressing and knocking down EFNA2. One-way ANOVA, mean±SEM (n=3), ****P<0.0001. (D, E) The migration capacity of vector, OE EFNA2, and shEFNA2 cells using a Transwell assay, and quantification of the number of cells that underwent migration in three groups. Scale bar: 100 μm. One-way ANOVA, mean±SEM (n=3), ***P<0.001 and ****P<0.0001. (F, G) The migration capacity of vector, OE EFNA2, and shEFNA2 cells using a cellular wound healing assay, and quantification of the wound healing rate in three groups. Scale bar: 100 μm. One-way ANOVA, mean±SEM (n=3), ***P<0.001 and ****P<0.0001. (H, I) The proliferation capacity of vector, OE EFNA2, and shEFNA2 cells using a EDU staining assay, and quantification of the positive cells in three groups. Scale bar: 100 μm. One-way ANOVA, mean±SEM (n=3), *P<0.05. (J, K) Western blot analysis of EFNA2 and EMT-related genes, and gray value analysis. Two-way ANOVA, mean±SEM (n=3), *P<0.05, **P<0.01, and ***P<0.001. (L) Representative nasopharyngeal carcinoma xenografts in the indicated group of mice (n=3 per group). (M, N) Tumor weight and volume were measured after the tumors were surgically dissected. Student t-test, mean±SEM, **P<0.01 and ***P<0.001.
Transwell and wound-healing assays revealed that the migratory capacity of FaDu cells was significantly reduced following EFNA2 knockdown, whereas EFNA2 overexpression enhanced migration (Fig. 4D-G). A similar trend was observed in cell proliferation, as assessed by the EDU assay (Fig. 4H and I). To assess the impact of EFNA2 on EMT progression, Western blot analysis of EMT-related proteins was performed, showing that EFNA2 overexpression promoted EMT, while its knockdown inhibited EMT in FaDu cells (Fig. 4J and K).
Based on the in vitro effects of EFNA2 on HPC cell proliferation, a subcutaneous tumorigenesis assay was performed in nude mice. The results indicated that both the volume and weight of subcutaneous tumors were significantly reduced in the shEFNA2 group compared to the control group (Fig. 4L-N), suggesting that EFNA2 knockdown effectively inhibits tumor growth in vivo. Together, these findings provide evidence that EFNA2 promotes proliferation, migration, and EMT in HPC.
Knockdown of EFNA2 inhibits EMT in FaDu cells by high matrix stiffness
To assess the combined effects of matrix stiffness and EFNA2 on HPC cells, vector control cells were cultured on both low- and high-stiffness substrates, while shEFNA2 cells were cultured on high-stiffness substrates for 3 days. Western blot analysis of EMT-related proteins revealed that high matrix stiffness promotes EMT relative to low stiffness, and that EFNA2 knockdown significantly inhibited EMT in FaDu cells on high-stiffness substrates (Fig. 5). These findings suggest that high matrix stiffness promotes EMT in HPC cells via EFNA2.
Fig. 5.
Knockdown of ephrin A2 (EFNA2) inhibits the epithelial-mesenchymal transition (EMT) process induced by higher matrix stiffness in FaDu cells. (A, B) Western blot analysis of EFNA2 and EMT-related genes in FaDu-vector cells cultured on different matrix stiffness and FaDu-shEFNA2 cells cultured on high matrix stiffness, and gray value analysis. Two-way analysis of variance, mean±standard error of the mean (n=3); *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.
DISCUSSION
Recent studies have demonstrated that matrix stiffness contributes to the progression of various cancers, including pancreatic, colorectal, liver, and lung cancers [27-29]. Tumor development is heavily influenced by stromal sclerosis. As research advances, several mechanisms have been identified through which matrix stiffness affects tumor progression, thereby informing the development of therapeutic targets and drugs aimed at modulating matrix stiffness [6,20]. Given the extremely poor prognosis of HPC, it is critical to further elucidate its developmental mechanisms, identify early detection markers, and develop novel treatment strategies. Previous research has shown that solid tumor tissue is generally stiffer than normal tissue [20], and that metastatic lymph nodes are often stiffer than adjacent soft tissue or benign lymph nodes [21]. Type I collagen, a major ECM component that provides structural strength, is relatively stiff [22]. In this study, COL1A1 IHC staining was performed on tumor and lymph node tissues from HPC patients to evaluate matrix stiffness. Collagen deposition was significantly higher in cervical lymph nodes with HPC metastases compared to non-metastatic nodes, indicating that metastatic nodes are stiffer than benign ones—a finding consistent with previous studies [21]. Moreover, HPC patients with high tumor matrix stiffness typically have a poor prognosis, suggesting that relative matrix stiffness is a valuable prognostic biomarker. In clinical practice, enhanced neck CT is commonly used to assess lesion extent in HPC patients [23]. CT data are readily available and well-preserved, and CT values reflect tissue density and correlate with peri-tumoral lymphocyte infiltration and cervical lymph node metastasis [24]. Analysis of enhanced CT data in HPC patients revealed a positive correlation between tumor tissue CT values (in both arterial and venous phases) and relative matrix stiffness. In the arterial phase, higher CT values were associated with poorer prognosis, further validating their prognostic significance. Combining relative tumor matrix stiffness with arterial phase CT values enables a more comprehensive prognostic assessment. Furthermore, our results indicate that matrix stiffness in metastatic lymph nodes is higher in HPC than in NPC, which may explain the differential responses to radiotherapy observed between these cancers. Future studies should investigate the relationship and underlying molecular mechanisms between matrix stiffness and radiotherapy response across different cancers.
The findings of this study indicate that matrix stiffness contributes to HPC progression. To further explore this, two polyacrylamide gel–based cell culture models with varying stiffness were used to culture HPC cells. The results showed that higher matrix stiffness promoted malignant behaviors in HPC cells. High-throughput sequencing was employed to elucidate the molecular mechanisms underlying the effects of matrix stiffness, revealing 1901 DEGs—999 upregulated and 912 downregulated —in cells cultured on stiffer substrates. Further analysis showed that EFNA2 was enriched in all three pathways related to these genes, and its expression was significantly upregulated in response to increased matrix stiffness. Given that EFNA2 responds to changes in matrix stiffness and that increased stiffness promotes HPC progression, it was hypothesized that EFNA2 is correlated with HPC progression. This hypothesis was confirmed by experiments showing that high EFNA2 expression enhanced HPC cell proliferation, migration, and EMT, whereas its knockdown inhibited these malignant behaviors and suppressed the EMT induced by high matrix stiffness.
Numerous studies have demonstrated that matrix stiffness significantly influences tumor development, progression, and patient prognosis [6,27]. However, few studies have examined the effects of matrix stiffness on HPC. Detailed analysis of CT imaging findings has also been shown to aid in disease assessment and prognostication [30-32]. This study aimed to investigate the impact of matrix stiffness on HPC and integrate CT values for a comprehensive, noninvasive evaluation of patient status without incurring additional costs. It should be noted that the incidence of HPC is relatively low, and this study included only a small sample of patients from our hospital. To improve the accuracy of CT value–based assessments for HPC and enhance patient risk stratification, future studies should expand the sample size through multi-center collaboration. Moreover, given the limited research on the role of EFNA2 in HPC, this study also investigated the combined effects of matrix stiffness and EFNA2 on HPC, with the goal of identifying potential therapeutic targets for high matrix stiffness–induced HPC progression.
HIGHLIGHTS
▪ Elevated matrix stiffness in tumor tissue and high computed tomography (CT) values on enhanced CT scans are linked to poor prognosis in hypopharyngeal cancer (HPC) patients. Combining measurements of tumor tissue matrix stiffness and enhanced arterial-phase CT values improves the prognostic assessment in HPC patients.
▪ Metastatic lymph nodes in HPC patients exhibit higher CT values than those in patients with nasopharyngeal carcinoma (NPC). This disparity may explain why metastatic cervical lymph nodes in NPC patients often shrink or disappear after radiotherapy—a response rarely observed in HPC patients.
▪ Increased matrix stiffness promotes proliferation, migration, and epithelial–mesenchymal transition in HPC cells.
▪ Ephrin A2 (EFNA2), which is upregulated in HPC cells cultured on high-stiffness matrices compared to those grown on low-stiffness matrices, is critical for mediating the malignant phenotypes induced by high matrix stiffness.
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (grant no. 82372977, 82371130, 82103435 and 82203799), Jiangsu Provincial Medical Key Discipline (Laboratory) Cultivation Unit (grant no. JSDW202244), Jiangsu Provincial Research Hospital (grant no. YJXYY202204).
Footnotes
No potential conflict of interest relevant to this article was reported.
AUTHOR CONTRIBUTIONS
Conceptualization: BY, ZZ. Methodology: SH. Formal analysis: MC. Formal analysis: MC, YS, HN. Investigation: MX, HL, YL, TX. Resources: BY. Project administration: QZ, SH. Funding acquisition: BY. Writing–original draft: YL. Writing–review & editing: QZ. Investigation: YL, TX. All authors read and agreed to the published version of the manuscript.
SUPPLEMENTARY MATERIALS
Supplementary materials can be found online at https://doi.org/10.21053/ceo.2024.00315.
Clinicopathological features of the hypopharyngeal cancer patients
Sequences of the small interfering RNAs (siRNAs)
Primers used in quantitative real-time polymerase chain reaction
Antibodies used for Western blot and immunohistochemistry
REFERENCES
- 1.Chow LQM. Head and neck cancer. N Engl J Med. 2020 Jan;382(1):60–72. doi: 10.1056/NEJMra1715715. [DOI] [PubMed] [Google Scholar]
- 2.Kwon DI, Miles BA, Education Committee of the American Head and Neck Society (AHNS) Hypopharyngeal carcinoma: do you know your guidelines. Head Neck. 2019 Mar;41(3):569–76. doi: 10.1002/hed.24752. [DOI] [PubMed] [Google Scholar]
- 3.Melica ME, La Regina G, Parri M, Peired AJ, Romagnani P, Lasagni L. Substrate stiffness modulates renal progenitor cell properties via a ROCK-mediated mechanotransduction mechanism. Cells. 2019 Dec;8(12):1561. doi: 10.3390/cells8121561. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Wells RG. The role of matrix stiffness in regulating cell behavior. Hepatology. 2008 Apr;47(4):1394–400. doi: 10.1002/hep.22193. [DOI] [PubMed] [Google Scholar]
- 5.Feng J, Tang Y, Xu Y, Sun Q, Liao F, Han D. Substrate stiffness influences the outcome of antitumor drug screening in vitro. Clin Hemorheol Microcirc. 2013;55(1):121–31. doi: 10.3233/CH-131696. [DOI] [PubMed] [Google Scholar]
- 6.Jiang Y, Zhang H, Wang J, Liu Y, Luo T, Hua H. Targeting extracellular matrix stiffness and mechanotransducers to improve cancer therapy. J Hematol Oncol. 2022 Mar;15(1):34. doi: 10.1186/s13045-022-01252-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Zhou C, Duan M, Guo D, Du X, Zhang D, Xie J. Microenvironmental stiffness mediates cytoskeleton re-organization in chondrocytes through laminin-FAK mechanotransduction. Int J Oral Sci. 2022 Mar;14(1):15. doi: 10.1038/s41368-022-00165-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Hasegawa K, Fujii S, Matsumoto S, Tajiri Y, Kikuchi A, Kiyoshima T. YAP signaling induces PIEZO1 to promote oral squamous cell carcinoma cell proliferation. J Pathol. 2021 Jan;253(1):80–93. doi: 10.1002/path.5553. [DOI] [PubMed] [Google Scholar]
- 9.Wu Y, Xu X, Liu F, Jing Z, Shen D, He P, et al. Three-dimensional matrix stiffness activates the piezo1-AMPK-autophagy axis to regulate the cellular osteogenic differentiation. ACS Biomater Sci Eng. 2023 Aug;9(8):4735–46. doi: 10.1021/acsbiomaterials.3c00419. [DOI] [PubMed] [Google Scholar]
- 10.Shukla VC, Higuita-Castro N, Nana-Sinkam P, Ghadiali SN. Substrate stiffness modulates lung cancer cell migration but not epithelial to mesenchymal transition. J Biomed Mater Res A. 2016 May;104(5):1182–93. doi: 10.1002/jbm.a.35655. [DOI] [PubMed] [Google Scholar]
- 11.Chaudhuri O, Cooper-White J, Janmey PA, Mooney DJ, Shenoy VB. Effects of extracellular matrix viscoelasticity on cellular behaviour. Nature. 2020 Aug;584(7822):535–46. doi: 10.1038/s41586-020-2612-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Patwardhan S, Mahadik P, Shetty O, Sen S. ECM stiffness-tuned exosomes drive breast cancer motility through thrombospondin-1. Biomaterials. 2021 Dec;279:121185. doi: 10.1016/j.biomaterials.2021.121185. [DOI] [PubMed] [Google Scholar]
- 13.Yoshida GJ. Regulation of heterogeneous cancer-associated fibroblasts: the molecular pathology of activated signaling pathways. J Exp Clin Cancer Res. 2020 Jun;39(1):112. doi: 10.1186/s13046-020-01611-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Jurney WM, Gallo G, Letourneau PC, McLoon SC. Rac1-mediated endocytosis during ephrin-A2- and semaphorin 3A-induced growth cone collapse. J Neurosci. 2002 Jul;22(14):6019–28. doi: 10.1523/JNEUROSCI.22-14-06019.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Feng YX, Zhao JS, Li JJ, Wang T, Cheng SQ, Yuan Y, et al. Liver cancer: EphrinA2 promotes tumorigenicity through Rac1/Akt/NF-kappaB signaling pathway. Hepatology. 2010 Feb;51(2):535–44. doi: 10.1002/hep.23313. [DOI] [PubMed] [Google Scholar]
- 16.Fox BP, Kandpal RP. Invasiveness of breast carcinoma cells and transcript profile: Eph receptors and ephrin ligands as molecular markers of potential diagnostic and prognostic application. Biochem Biophys Res Commun. 2004 Jun;318(4):882–92. doi: 10.1016/j.bbrc.2004.04.102. [DOI] [PubMed] [Google Scholar]
- 17.Hafner C, Schmitz G, Meyer S, Bataille F, Hau P, Langmann T, et al. Differential gene expression of Eph receptors and ephrins in benign human tissues and cancers. Clin Chem. 2004 Mar;50(3):490–9. doi: 10.1373/clinchem.2003.026849. [DOI] [PubMed] [Google Scholar]
- 18.Li S, Wu Z, Chen Y, Kang Z, Wang H, He P, et al. Diagnostic and prognostic value of tissue and circulating levels of Ephrin-A2 in prostate cancer. Tumour Biol. 2016 Apr;37(4):5365–74. doi: 10.1007/s13277-015-4398-7. [DOI] [PubMed] [Google Scholar]
- 19.Tse JR, Engler AJ. Preparation of hydrogel substrates with tunable mechanical properties. Curr Protoc Cell Biol. 2010 Jun;Chapter 10:Unit 10.16. doi: 10.1002/0471143030.cb1016s47. [DOI] [PubMed] [Google Scholar]
- 20.Huang J, Zhang L, Wan D, Zhou L, Zheng S, Lin S, et al. Extracellular matrix and its therapeutic potential for cancer treatment. Signal Transduct Target Ther. 2021 Apr;6(1):153. doi: 10.1038/s41392-021-00544-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Hynes RO, Naba A. Overview of the matrisome: an inventory of extracellular matrix constituents and functions. Cold Spring Harb Perspect Biol. 2012 Jan;4(1):a004903. doi: 10.1101/cshperspect.a004903. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Johnson DE, Burtness B, Leemans CR, Lui VW, Bauman JE, Grandis JR. Head and neck squamous cell carcinoma. Nat Rev Dis Primers. 2020 Nov;6(1):92. doi: 10.1038/s41572-020-00224-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Keberle M, Strobel P, Marx A, Hahn D, Hoppe F. CT determination of lymphocytic infiltration around head and neck squamous cell carcinomas may be a predictor of lymph node metastases. Eur Arch Otorhinolaryngol. 2003 Nov;260(10):558–64. doi: 10.1007/s00405-003-0640-9. [DOI] [PubMed] [Google Scholar]
- 24.Sigrist RM, Liau J, Kaffas AE, Chammas MC, Willmann JK. Ultrasound elastography: review of techniques and clinical applications. Theranostics. 2017 Mar;7(5):1303–29. doi: 10.7150/thno.18650. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Wei X, Lou H, Zhou D, Jia Y, Li H, Huang Q, et al. TAGLN mediated stiffness-regulated ovarian cancer progression via RhoA/ROCK pathway. J Exp Clin Cancer Res. 2021 Sep;40(1):292. doi: 10.1186/s13046-021-02091-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Deng B, Zhao Z, Kong W, Han C, Shen X, Zhou C. Biological role of matrix stiffness in tumor growth and treatment. J Transl Med. 2022 Nov;20(1):540. doi: 10.1186/s12967-022-03768-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Piersma B, Hayward MK, Weaver VM. Fibrosis and cancer: a strained relationship. Biochim Biophys Acta Rev Cancer. 2020 Apr;1873(2):188356. doi: 10.1016/j.bbcan.2020.188356. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Affo S, Yu LX, Schwabe RF. The role of cancer-associated fibroblasts and fibrosis in liver cancer. Annu Rev Pathol. 2017 Jan;12:153–86. doi: 10.1146/annurev-pathol-052016-100322. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Zhang W, Zhang S, Zhang W, Yue Y, Qian W, Wang Z. Matrix stiffness and its influence on pancreatic diseases. Biochim Biophys Acta Rev Cancer. 2021 Aug;1876(1):188583. doi: 10.1016/j.bbcan.2021.188583. [DOI] [PubMed] [Google Scholar]
- 30.Jiang Y, Jin C, Yu H, Wu J, Chen C, Yuan Q, et al. Development and validation of a deep learning CT signature to predict survival and chemotherapy benefit in gastric cancer: a multicenter, retrospective study. Ann Surg. 2021 Dec;274(6):e1153–61. doi: 10.1097/SLA.0000000000003778. [DOI] [PubMed] [Google Scholar]
- 31.Sandrasegaran K, Lin Y, Asare-Sawiri M, Taiyini T, Tann M. CT texture analysis of pancreatic cancer. Eur Radiol. 2019 Mar;29(3):1067–73. doi: 10.1007/s00330-018-5662-1. [DOI] [PubMed] [Google Scholar]
- 32.Dighe S, Swift I, Brown G. CT staging of colon cancer. Clin Radiol. 2008 Dec;63(12):1372–9. doi: 10.1016/j.crad.2008.04.021. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Clinicopathological features of the hypopharyngeal cancer patients
Sequences of the small interfering RNAs (siRNAs)
Primers used in quantitative real-time polymerase chain reaction
Antibodies used for Western blot and immunohistochemistry