Introduction
Oral squamous cell carcinoma (OSCC) is the most prevalent malignant form of oral cancers, with an incidence of 377,713 and 117,757 deaths reported in 2020 [1]. Despite advancements in therapeutic approaches, radiotherapy and cisplatinbased chemotherapy remain the main noninvasive treatments for patients with OSCC [2,3]. However, a substantial number of patients respond poorly to these therapies [4] and the overall 5-year survival rate for OSCCs is approximately 50% [5,6]. This underscores the urgent need for the development of novel therapeutic approaches. To achieve this, physiologically relevant models are required for testing potential drug treatments.
Traditionally, cell culture systems involve the growth of cells in a two-dimensional (2D) manner on tissue culture plastics. Although 2D cultures offer advantages such as simplicity and cost-effectiveness, they significantly alter gene expression profiles and fail to reflect the intricate cell-cell interactions and tumor microenvironments observed in cancerous tissues [7]. Consequently, drugs optimized in 2D cultures face challenges in their translation to clinical settings. Animal models are valuable tools in cancer research; however, they have inherent limitations, such as high cost, time-consuming processes, and challenges in translating findings to human clinical studies [8]. Therefore, three-dimensional (3D) cell culture models have emerged as promising models. In 3D models, tumor cells organize into a solid architecture, resulting in the mechanical and functional heterogeneity of cells [9]. Furthermore, they can be exposed to diverse biochemical signals from different gradients, leading to the creation of more realistic and physiologically relevant environments. These models significantly enhance cell-based drug screening by identifying toxic and ineffective substances and mitigate the drawbacks of 2D cell cultures and animal models [10]. Therefore, this shift toward 3D models represents a novel approach for investigating drug candidates in preclinical studies.
In this study, we investigated the efficacy of biological chemicals on well-established OSCC 3D culture models.
Materials and Methods
1. Reagents
The pharmacological inhibitors, Y27632 and latrunculin A (Lat. A), obtained from Enzo Life Sciences, Inc., were used to inhibit RhoA downstream kinase (ROCK) and actin polymerization. Verteporfin (VP) was purchased from Sigma-Aldrich. For the invasion assay, type I collagen (Cellmatrix type 1-A) was purchased from Nitta Gelatin.
2. Cell cultures
Human gingival fibroblasts immortalized by hTERT transfection (hTERT-hNOFs) were utilized as previously described [11,12]. Cancer-associated fibroblasts (CAFs) were obtained from patients who underwent wisdom tooth extraction from Yonsei University of College of Dentistry (IRB 2-2012-0027) and primary cells were used for CAF studies [12,13]. Both fibroblasts were cultured in F medium, a mixture of Dulbecco’s modified Eagles medium (Gibco BRL) and F-12 Ham’s medium (Ham’s F12; Gibco BRL) in a 3:1 ratio, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. OSCC cells (YD10B and YD38) were cultured in F medium with additional supplements: 1 × 10–10 M cholera toxin, 0.4 mg/mL hydrocortisone, 5 μg/mL insulin, 5 μg/mL transferrin, and 2 × 10–11 M triiodothyronine (T3). Human normal epidermal keratinocytes, obtained from the Yonsei University College of Dentistry, were used below the 9th passage (IRB-2-2009-0002), as described previously [12], and cultured in keratinocyte growth medium (#CC-3107; Lonza). All cells were maintained in an incubator at 37℃ with 5% CO2, and the culture medium was refreshed every 3 days.
3. 3D raft culture
A collagen mixture was prepared by combining 8 volumes of type I collagen solution (Nitta Gelatin) with one volume of 10 × medium, consisting of Dulbecco’s modified Eagles medium (Gibco) and F-12 Ham’s medium (Ham’s F12) in a 3:1 ratio. The pH was adjusted by adding reconstitution buffer (0.022 g/μL NaHCO3, 0.0477 g/μL Hepes, and 0.05 N NaOH). Fibroblasts (hTERT-hNOFs and CAFs) at a concentration of 1.5 × 105 were mixed with 250 μL collagen mixture. The mixture with cell and collagen was seeded into a Mill-cell (3.0 μm pore size, 12 mm diameter; Merck Millipore) and allowed to incubated at 37℃ for 24 hours. YD10B and YD38 cells were seeded on top of the collagen mixtures with or without fibroblasts (3 × 105 cells). The outer medium, including cholera toxin, hydrocortisone, insulin, transferrin, and T3, around the inserts were added with and without biological inhibitors. The rafts were incubated for 4 days and subsequently transitioned to the air-liquid interface (ALI) for an additional 14 days.
4. Hematoxylin and eosin staining
The rafts were fixed in paraformaldehyde, embedded in paraffin, sectioned, and subsequently stained with hematoxylin and eosin.
Results
1. 3D raft culture models using fibroblasts for drug screening
To establish a 3D model for OSCC, we initially investigated the importance of fibroblasts in forming the epithelial layer for setting up a 3D culture model. We established 3D culture models with or without two types of fibroblasts, hTERThNOFs, and CAFs (Fig. 1A). The hTERT-hNOFs exhibited features resembling CAF-like phenotypes when co-cultured with OSCC cells [14]. CAFs were isolated and primary cultured from a patient with OSCC. The results revealed the formation of a continuous layer of tightly packed cells when 3D culture models were established with these two types of fibroblasts (Fig. 1C) compared to models without fibroblasts (Fig. 1B). Therefore, we conducted experiments using epithelial cells cultured with fibroblasts to establish 3D culture models.
2. VP inhibits cell growth and invasiveness in OSCC 3D culture models
Next, we investigated the effects of biological chemicals, including Y27632 (a ROCK inhibitor), Lat.A (an inhibitor of actin polymerization), and VP (an inhibitor of YAP), on the OSCC 3D culture model. For 3D culture models, two types of OSCC cells were employed. YD10B, derived from tongue SCC, features a frameshift mutation at codon 236 of exon 7 in the p53 gene. In contrast, YD38, derived from the lower gingiva, harbors the wild-type p53 gene [15]. Previous studies have demonstrated that fibroblasts play a key role in OSCC invasiveness, and that ROCK-YAP signaling contributes to aggressive OSCC phenotypes [14]. Therefore, we selected three pharmacological inhibitors to target the ROCK-YAP signaling pathway.
In the OSCC 3D culture model using hTERT-hNOFs and CAFs, the YD10B and YD38 OSCC cells invaded the stroma (Fig. 2A and 2E). After treatment with Y27632 and Lat.A, the invasiveness of OSCC cells weakly decreased (Fig. 2B, 2C, 2F and 2G). VP not only inhibited the invasion of OSCC cells, but also the formation of the epithelial layer (Fig. 2D and 2H).
We further investigated the effects of pharmacological inhibitors in a OSCC 3D cultured model using CAFs isolated from patients with OSCC. Consistent with the results of the OSCC model using hTERT-hNOFs, the model using primary cultured CAFs showed that OSCC cells invaded the stroma (Fig. 3A and 3E). Furthermore, there was a weak inhibition of invasion in Y27632- and Lat.A-treated OSCC 3D culture models (Fig. 3B, 3C, 3F and 3G). Finally, VP effectively inhibited epithelial layer formation and invasion (Fig. 3D and 3H).
Collectively, we demonstrated that OSCC 3D models cultured with fibroblasts are suitable for use as drug screening models for patients with OSCC. Furthermore, we found that VP effectively inhibited the formation of the epithelial layer as well as invasion in OSCC 3D culture models.
Discussion
Chemotherapy has been employed as an adjuvant treatment for OSCCs before surgical excision to augment tumor resectability and improve the survival rate of patients with OSCC [2,3]. Despite the discovery of various chemotherapeutic drugs, such as platinum (Cisplatin, Carboplatin, and Oxaliplatin), molecular-targeted drugs (Cetuximab and Gefitinib), and immune checkpoint inhibitors [16], the 5-year survival rate of patients with OSCC has remained almost unchanged for several decades [16]. Moreover, a considerable proportion of patients with OSCC develop drug resistance and experience relapsed after undergoing chemotherapy [4]. Therefore, there is an imperative need to develop novel therapeutic approaches and physiologically relevant models for drug screening in clinical settings. As a foundational step, we established OSCC 3D models using fibroblasts and identified the efficacy of various biological inhibitors using these models.
3D cell culture models for OSCCs can be constructed using various techniques, such as scaffold-based systems, spheroid cultures, and organoids [17,18]. Scaffold-based systems involve culturing oral cancer cells on biocompatible scaffolds that mimic the extracellular matrix found in tissues [17]. These scaffolds provide structural support and allow cells to grow in a 3D configuration that closely resembles the tumor architecture and environment. Spheroid cultures involve growing cancer cells as spherical aggregates, which can mimic the spatial organization [18]. However, spheroid culture, being an aggregates of single tumor cells, has the limitation that for studying interaction with surrounding microenvironment cells, additional co-culture with cells from TME is necessary. Recently, patientderived organoids (PDOs) have attracted increasing interest. PDOs are derived directly from tumor samples and offer personalized models that faithfully represent the unique characteristics of patients. PDOs are valuable tools for studying tumor biology, assessing drug responses, and exploring personalized medical approaches by evaluating the effectiveness of different treatments on a patient-specific basis [19]. Despite their potential, PDOs have several limitations. Establishing PDOs is demanding in terms of technical expertise and time. The success rate of generating PDOs varies among patient samples, and not all samples may yield viable organoids. Moreover, the availability of patient tissues for PDOs generation is a limiting factor. Obtaining sufficient amounts of tumor tissue for viable organoid establishment in the oral cavity is particularly challenging. Acquiring tissues from patients with OSCC is especially difficult given its relatively low incidence rate in Korea (4.4% in 2020) [20]. As an alternative, in vitro 3D culture models using primary cultured cells is not only more efficient and scalable but also holds significant value as a primary model for drug testing. Moreover, the model created in this study, using an ALI method that considers the characteristics of OSCC occurring in the respiratory tract, can be considered to better reflect in vivo features compared to other OSCC 3D culture models.
Previously, we reported that modification of the tumor microenvironment, such as cytoskeletal changes and matrix remodeling via RhoA-ROCK-YAP in CAFs, modulates OSCC invasion [14]. Therefore, we selected several biological inhibitors for drug screening in 3D models that can block RhoAROCK- YAP signaling, namely Y27632, Lat.A and VP. Consistent with our previous study [14], there is supporting evidence. The potent ROCK inhibitor Y27632 induces changes in tumor cell morphology, decreasing cell proliferation and invasiveness in vitro, thereby limiting tumor growth in vivo [21]. The actin polymerization inhibitor Lat.A induced acute cell injury and programmed cell death by activating the caspase-3/7 pathway in gastric cancer [22]. Recently, VP has been shown to inhibit the YAP-TEAD complex, preventing YAP-induced oncogenic growth and their anticancer activity has been reported in various cancers including ovarian [23], pancreatic [24], thyroid [25], and breast [26]. In particular, VP in head and neck squamous cell carcinoma cells mediates the attenuation of epithelialmesenchymal transition (EMT) and stemness, as well as the expression of the immunosuppressive protein programmed death-ligand 1 (PD-L1) [27]. In this study, we investigated the anti-cancer effects of these three inhibitors. Y27632 and Lat. A weakly inhibited the invasion of OSCC cells, whereas VP was more effective in inhibiting the invasion and growth of tumor cells in OSCC 3D models. Furthermore, compared to other inhibitors, VP visually inhibits both the growth cancer cells the cellularity of CAFs in the stromal region. A key distinction in our results from previous studies is that while previous studies have confirmed the anticancer effects of Y27632, Lat.A, and VP by maintaining tumor cells in 2D culture systems, we screened the effects of inhibitors in 3D culture, taking into consideration the interaction between cancer cells and fibroblasts [28]. Although these findings did not elucidate the detailed mechanism, we anticipate that VP exerts effects on the cancer cells themselves, such as inhibiting EMT or stemness and suppressing the YAP activity of fibroblasts. This in turn induces cytoskeletal alterations, thereby inhibiting OSCC invasion.
In conclusion, our 3D culture models demonstrate the potential for drug screening, with VP showing particularly promising efficacy. Further research on VP is necessary for facilitating its use in the treatment of OSCCs.
