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Mesenchymal stem cell-mediated adipogenic transformation: a key driver of oral squamous cell carcinoma progression

Stem Cell Research & Therapy volume 16, Article number: 12 (2025) Cite this article

Abstract

Background

Interaction between mesenchymal stem cells (MSCs) and oral squamous cell carcinoma (OSCC) cells plays a major role in OSCC progression. However, little is known about adipogenic differentiation alteration in OSCC-derived MSCs (OSCC-MSCs) and how these alterations affect OSCC growth.

Methods

MSCs were successfully isolated and cultured from normal gingival tissue, OSCC peritumoral tissue, and OSCC tissue. This included gingiva-derived MSCs (GMSCs), OSCC adjacent noncancerous tissues-derived MSCs (OSCCN-MSCs), and OSCC-MSCs. The adipogenic and osteogenic differentiation capabilities of these cells were evaluated using Oil Red O and Alizarin Red S staining, respectively. OSCC cells were then co-cultured with either OSCC-MSCs or GMSCs to assess the impact on OSCC cell proliferation and migration. Subcutaneous xenograft experiments were conducted in BALB/c-nu mice to further investigate the effects in vivo. Additionally, immunohistochemical staining was performed on clinical samples to determine the expression levels of fatty acid synthase (FASN) and the proliferation marker Ki67.

Results

OSCC-MSCs exhibited enhanced adipogenic differentiation and reduced osteogenic differentiation compared to GMSCs. OSCC-MSCs significantly increased the proliferation and migration of OSCC cells relative to GMSCs and promoted tumor growth in mouse xenografts. Lipid droplet accumulation in the stroma was significantly more pronounced in OSCC + OSCC-MSCs xenografts compared to OSCC + GMSCs xenografts. Free fatty acids (FFAs) levels were elevated in OSCC tissues compared to normal gingival tissues. Moreover, OSCC-MSCs consistently secreted higher levels of FFAs in condition medium than GMSCs. Knockdown of FASN in OSCC-MSCs reduced their adipogenic potential and inhibited their ability to promote OSCC cell proliferation and migration. Clinical sample analysis confirmed higher FASN expression in OSCC stroma, correlating with larger tumor size and increased Ki67 expression in cancer tissues, and was associated with poorer overall survival.

Conclusions

OSCC-MSCs promoted OSCC proliferation and migration by upregulating FASN expression and facilitating FFAs secretion. Our results provide new insight into the mechanism of OSCC progression and suggest that the FASN of OSCC-MSCs may be potential targets of OSCC in the future.

Introduction

Oral squamous cell carcinoma (OSCC) is a cancer originating from the squamous epithelium of the oral mucosa, commonly affecting the gingiva, alveolar ridges, buccal mucosa, floor of the mouth, palate, and tongue. OSCC accounts for about 90% of head and neck squamous cell carcinoma (HNSCC) and is the most common malignancy in the oral and maxillofacial region [1]. According to 2018 global cancer statistics, there were 890,000 new cases and 450,000 deaths due to OSCC worldwide [2]. The incidence of OSCC is increasing, with projections indicating a 30% rise by 2030, leading to 1.08 million new cases annually [1, 2]. Treatments primarily include surgery, radiation, and chemotherapy. Despite advancements in targeted therapies and immunotherapies, such as PD1 and/or PDL1 inhibitors, the five-year survival rate remains around 50%, with no significant improvement in prognosis. The tumor’s location significantly affects patients’ quality of life due to physical and psychological impacts. Thus, finding more effective treatments for OSCC remains a key research focus [3, 4].

Mesenchymal stem cells (MSCs) are adult stem cells known for their self-renewal and multipotent differentiation potential [5, 6]. First isolated from bone marrow by Friedenstein in the early 1970s, MSCs were shown to form colony-forming unit-fibroblasts (CFU-F) [7]. In the 1990s, MSCs were found to differentiate into osteogenic, adipogenic, chondrogenic, and neurogenic lineages [8,9,10]. They have since been isolated from various tissues, including dental pulp, gingiva, umbilical cord, adipose tissue, and muscle [11,12,13].

Cancer-associated MSCs (CA-MSCs) are a subset of MSCs that reside within the tumor microenvironment and exhibit multipotent differentiation potential and high plasticity. CA-MSCs interact closely with cancer cells over long periods and are significantly influenced by these cancer cells. As a crucial component of the tumor microenvironment, CA-MSCs play a vital role in cancer development and progression [14, 15]. Compared to MSCs from adjacent non-cancerous tissues, MSCs derived from gastric cancer tissues secrete large amounts of IL-8, promoting gastric cancer growth [16]. Similarly, MSCs from colorectal cancer tissues enhance colorectal cancer growth through the IL-6/JAK2/STAT3 signaling pathway [17]. Beyond gastrointestinal cancers, studies in brain tumors, breast cancer, liver cancer, lung cancer, melanoma, osteosarcoma, ovarian cancer, and prostate cancer have shown that CA-MSCs primarily facilitate cancer cell growth and metastasis by creating an immunosuppressive microenvironment, activating related signaling pathways, and promoting angiogenesis in tumor tissues [4].

Investigating the effects of MSCs from normal tissues on the biological behavior of OSCC cells and mouse xenografts is currently a hot topic in this field, with numerous studies yielding inconsistent and sometimes contradictory results [18,19,20,21,22]. For example, Liu et al. found that injecting human-exfoliated deciduous tooth MSCs into CAL27 OSCC xenografts in BALB/c-nu mice suppressed tumor growth by downregulating VEGF-A expression through miR-100-5p and miR-1246, thereby reducing microvascular density around the tumors [19]. In contrast, Raj et al. reported that human dental pulp MSCs secreted inflammatory factors that promoted the growth of OSCC cells AW13516 [20]. However, research on OSCC-derived MSCs (OSCC-MSCs) and their role in OSCC is still very limited. The specific effects of OSCC-MSCs on OSCC behavior remain unclear and require further investigation.

In this study, MSCs were successfully isolated and cultured from normal gingival tissue, OSCC peritumoral tissue, and OSCC tissue, including gingiva-derived MSCs (GMSCs), OSCC adjacent noncancerous tissues-derived MSC (OSCCN-MSCs), and OSCC-MSCs. We found that compared to GMSCs, OSCC-MSCs showed significantly enhanced adipogenic differentiation ability and significantly reduced osteogenic differentiation ability. OSCC-MSCs promoted the proliferation and migration of OSCC cells by upregulating FASN expression and facilitating free fatty acids (FFAs) secretion. Clinically, patients with high FASN expression in the OSCC stroma have a poor prognosis. In summary, these findings highlight the potential clinical implications of targeting MSC adipogenic transformation in managing OSCC development.

Methods

Patient samples and clinical data

A total of 86 surgical resection specimens of OSCC from the Third Affiliated Hospital of Sun Yat-sen University, collected between 2014 and 2021, were included in this study. The specimens were processed undergoing formalin fixation, paraffin embedding, and subsequent sectioning for hematoxylin-eosin (H&E) staining. These specimens were further analyzed to assess the expression levels of FASN and Ki67 expression. Inclusion criteria for OSCC included histological diagnosis by two experienced pathologists and no prior chemotherapy or preoperative radiotherapy. Clinical and pathological characteristics of the patients were recorded, encompassing parameters such as age, gender, tumor dimensions, tumor localization, differentiation grade, TNM staging, and more. All patients provided informed consent, acknowledging their participation in the study. The study protocol was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University.

Normal gingival fresh tissues (3 cases) and OSCC fresh tissues (3 cases) were obtained from the Third Affiliated Hospital of Sun Yat-sen University. Normal gingival tissues were collected from individuals undergoing mandibular third molar extraction. Informed consent was obtained from all patients, and the study was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University. The fresh tissues were used for MSCs isolation, and Oil Red O staining, and part of the tissues were fixed, and then used for Perilipin-1 immunohistochemical (IHC) staining.

Isolation and culture of GMSCs, OSCCN-MSCs and OSCC-MSCs

The sampling standard for adjacent tissues in OSCC was defined as follows: the distance from tumors was greater than 1 cm but less than 2 cm. Tissue samples, including normal gingival tissues, OSCC tissues, and peritumoral tissues, were cut into approximately 1 mm3 fragments and washed with PBS. The fragments were digested sequentially with dispase (2 mg/mL) and collagenase I (1 mg/mL), followed by culture in α-MEM (Gibco, Grand Island, NY, USA) complete medium at 37 °C with 5% CO2.

Identification of GMSCs, OSCCN-MSCs and OSCC-MSCs

The characterization of MSCs adhered to the Minimal Criteria for Defining Multipotent Mesenchymal Stromal Cells [23]. Flow cytometry analysis utilized antibodies targeting CD105, CD90, CD73, CD146, CD45, and CD34 to assess the immunophenotype of MSCs. Isotype-matched control IgG or IgM antibodies were used as controls. The capacity for self-renewal was determined using CFU-F assay. MSCs were cultured for 21 days in adipogenic or osteogenic induction media, and their differentiation potential into adipocytes and osteocytes was determined using Oil Red O (Sigma Aldrich, St.Louis, MO, USA) and Alizarin Red S (Sigma Aldrich) staining, respectively.

Tumor cell line culture

The OSCC cell lines CAL27 and HSC3 were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco). The cells were maintained in a humidified incubator at 37 °C with 5% CO2.

Co-culture assay

Prepare aseptic 12-well plates and dispense 1 mL of CAL27 or HSC3 single-cell suspensions (5 × 10^5 cells/mL) into each well. Next, insert a 0.4 μm membrane chamber into each well and introduce 0.5 mL of single-cell suspensions (1 × 10^5 cells/mL) of GMSCs, OSCC-MSCs, or OSCC-MSCs transfected with different interference chains into the upper chamber. Following a 72-hour co-cultivation period, cells were retrieved from the lower chamber (CAL27 or HSC3) to conduct cell proliferation, scratch, and Transwell assays.

Cell proliferation assay

Cell-counting kit 8 (CCK-8) cell proliferation assay was performed by cell counting kit-8 (CCK8; Dojindo, Kumamoto, Japan) for 4 days. Initially, the cells subjected to different treatments were prepared as single-cell suspensions at a concentration of 1 × 10^5 cells/mL. In 96-well plates, 100 µL of cell suspension was added to each well, with each cell group having five replicate wells. After 12 h and on days 1, 2, 3, and 4, the previous culture medium in the 96-well plates was carefully removed. Following this, 100 µL of complete culture medium containing 10% CCK-8 was introduced into each well. After a 3-hour incubation period, the optical density (OD) value at 450 nm was assessed.

A 5-ethynyl-2’-deoxyuridine (EdU) cell proliferation assay was conducted using the EdU assay kit (Beyotime, Shanghai, China). Different treated cells (1 × 10^5 cells/well) were seeded in 12-well plates for 24 h. Cell proliferation was assessed using the EdU assay kit following the manufacturer’s instructions. Microscopy (Olympus, Tokyo, Japan) was employed to capture images, and the cell proliferation activity was evaluated based on the average ratio of EdU-stained cells (red) to DAPI-stained cells (blue).

Scratch assay

For the scratch assay, an initial cell seeding of 5 × 10^5 cells was carried out in 12-well plates. Once the cells had reached confluence, a 10 µL sterile pipette tip was utilized to generate a scratch in the monolayer of cells. Following this, the assessment of cell migration into the scratched area was performed 12–16 h after the scratch was created using Image J software.

Transwell migration

A total of 1 × 10^5 cells, following various treatments, were suspended in a serum-free medium and seeded into the upper chamber of a Transwell insert with 8 μm pores. Subsequently, 600 µl of medium containing 20% FBS was added to the lower chamber. After 48 h of incubation at 37 °C, the cells were fixed using 4% paraformaldehyde and then stained with 0.5% crystal violet. Non-migratory cells on the upper side of the Transwell membrane were gently removed using a cotton swab. Finally, the migrated cells were photographed and quantified.

RNA interference

For RNA interference experiments, siRNAs for knocking down FASN were synthesized by RiboBio (Guangzhou, China). siRNAs were transfected using Lipo3000 (Invitrogen, Life Technologies, Carlsbad, CA, USA) following the protocols provided by the manufacturer.

siFASN#1: 5′-UGGAGCGUAUCUGUGAGAAUU-3′.

siFASN#2: 5′-UGGAGCGUAUCUGUGAGAA-3′.

siFASN#3: 5′-AACCCUGAGAUCCCAGCGCUG-3′.

Mouse subcutaneous xenograft tumor model

In summary, four-week-old female BALB/c-nu mice were maintained in aseptic conditions within laminar flow cabinets, with free access to food and water. The room temperature was maintained at 22 ± 2 ℃, and the humidity was controlled at 50 ± 10%. The mice were randomly allocated into four groups using a random number generator to ensure unbiased group assignment. During the injection of tumor cells, mice were anesthetized with isoflurane (2%) via inhalation, and the depth of anesthesia was ensured by continuously monitoring respiratory rate using a respiratory monitor and body temperature with a rectal thermometer. A heating pad was used to maintain the body temperature of the mice during anesthesia. In two groups, 3 × 10^6 GMSCs and 3 × 10^6 OSCC cells (CAL27 or HSC3) were injected at the thigh root, while in the other two groups, 3 × 10^6 OSCC-MSCs and 3 × 10^6 OSCC cells (CAL27 or HSC3) were injected. Subcutaneous tumor formation in the mice was observed, and the longest (L) and widest (W) diameters of the tumors were recorded using a vernier caliper by an investigator blinded to the group allocation. Tumor volume was calculated using the formula: V = L×W2 × 0.4 [24]. Following 4 weeks of tumor growth, all mice were euthanized by lethal intraperitoneal injection of sodium pentobarbital (100 mg/kg), and death was confirmed by the absence of heartbeat and respiratory arrest. The tumors were excised, photographed, and weighed. Tissue samples were either snap-frozen or paraffin-embedded for subsequent experiments. This animal study was approved by the Experimental Animal Ethics Committee of the Third Affiliated Hospital, Sun Yat-Sen University. All experimental procedures were approved by the Institutional Animal Ethics Committee and were conducted in strict accordance with the ARRIVE 2.0 guidelines.

Oil red O staining for tissues

To prepare the Oil Red O staining solution, 50 mg of Oil Red O was dissolved in 50 ml of 99.5% isopropanol. The solution was stirred continuously until the dye was fully dissolved. Subsequently, the solution was diluted with pure water to achieve a final concentration of 60%. The mixture was then filtered through filter paper to remove any undissolved particles, ensuring a clear and homogeneous staining solution.

Frozen tissue sections were cut to a thickness of 10 μm. The sections were fixed in 10% formalin for 10 min to preserve tissue integrity. Following fixation, the sections were rinsed three times with pure water to remove the fixative. The sections were then allowed to air-dry at room temperature for 5 min. Next, the sections were immersed in 60% isopropanol for 2–5 min to facilitate lipid penetration. After this, the sections were stained by immersing them in the prepared Oil Red O solution for 15 min. The stained sections were differentiated by soaking them in 60% isopropanol for 2 min, and the color development was observed under a microscope. To remove excess stain, the sections were rinsed twice with pure water. For counterstaining, the sections were stained with hematoxylin for 30 s and then blued under running tap water. Finally, the stained sections were mounted using an aqueous mounting medium. The scoring criteria for the ratio of positive area to stromal area in tissue sections are as follows: areas with < 5% positive area are scored as 0, areas with 5–25% positive area are scored as 1, areas with 25–50% positive area are scored as 2, and areas with ≥ 50% positive area are scored as 3.

Immunohistochemistry (IHC)

Formalin-fixed, paraffin-embedded blocks were sectioned into slices with a thickness of 4 μm. IHC experiments were conducted using the Novolink Polymer Detection System kit (Leica Biosystems, UK) following the manufacturer’s instructions. The scoring criteria for FASN and Perilipin-1 IHC results involve assessment by two experienced pathologists based on staining intensity and the percentage of positive cells in the tissue sections. The staining intensity scoring method was the same as that used in a previous study [25]. The scoring criteria for staining intensity are as follows: areas with no staining are scored as 0, yellow staining is scored as 1, light brown staining is scored as 2, and dark brown staining is scored as 3. The scoring criteria for the percentage of positive cells in the tissue sections are as follows: areas with < 10% positive cells are scored as 0, 10–40% positive cells are scored as 1, 40–70% positive cells are scored as 2, and ≥ 70% positive cells are scored as 3. To obtain the final score, the staining intensity score is multiplied by the percentage of positive cells score. The product is then categorized into the following final scores: a product of 0 to 1 is scored as “-“, a product of 2 to 3 is scored as “+”, a product of 4 to 6 is scored as “++”, and a product greater than 6 is scored as “+++”. The area-based scoring is applied to Ki67. The FASN, Perilipin-1 and Ki67 expression was classified into two groups: “++” and “+++” were categorized as high expression group, while “-” and “+” were considered low expression group. The details of FASN, Perilipin-1 and Ki67 antibody are in Table S1.

FFAs quantification

The determination of FFAs in tumor cells, cell supernatants, and OSCC tissues was carried out following the instructions provided in the Free Fatty Acid assay kit (Abcam, Cambridge, UK).

RNA preparation, cDNA synthesis, and quantitative real-time PCR (RT-qPCR)

Total RNA was extracted from the cells following the steps outlined in the AG RNAex Pro Reagent manual (AG, Changsha, China). Subsequently, the extracted RNA was reverse transcribed using the Evo M-MLV RT Kit with gDNA Clean (AG) following the manufacturer’s instructions. RT-qPCR was conducted utilizing the SYBR Green Premix Pro Taq HS qPCR Kit (AG), strictly adhering to the manufacturer’s guidelines. The assays were executed on a LightCycler 480 (Roche, Basel, Switzerland). Each sample was analyzed for RT-qPCR in a total of three biological replicates, and within each biological replicate, three technical replicates were performed for robustness and accuracy. The RT-qPCR data analysis employed the relative quantification method (2−ΔΔCt), with GAPDH serving as the internal reference for normalization. Primer sequences are detailed in Supplementary Table S2.

Western blotting

Protein expression was assessed using Western blotting analysis. Briefly, following specific treatments, cells were rinsed twice with ice-cold PBS. Protein extraction was conducted using RIPA lysis and extraction buffer (KeyGen Biotechnology, Nanjing, China). Equal amounts of proteins (30 µg per lane) were separated on an 8–12% SDS-PAGE gel and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). The PVDF membrane was incubated sequentially with primary antibodies and secondary antibodies. Antibody dilutions were optimized as detailed in Supplementary Table S1. Finally, protein visualization was achieved using enhanced chemiluminescence (Millipore).

TCGA and GEO data analysis

The UCSC Xena Browser (https://xenabrowser.net/) provided the standardized Pan-Cancer dataset (TCGA TARGET GTEx, PANCAN, N = 19131, G = 60499). Cancer types with fewer than three samples were excluded, resulting in data from 34 cancers. FASN (ENSG00000169710) expression data were extracted from various sample types and log2 transformed (log2(x + 0.001)). High-quality TCGA prognostic datasets were sourced from Cell [26] and TARGET follow-up data from UCSC Cancer Browser, excluding samples with follow-up times under 30 days. R software (version 3.6.4) and the survival package (version 3.2-7) were used to identify the FASN expression and the survival analysis of HNSCC patients from TCGA-HNSC dataset. Single-cell sequencing data (GSE103322) [27] from 5902 cells across 18 HNSCC samples (Smart-seq2) were analyzed. The t-SNE plot visualized single-cell clustering, with different colors representing various cell types. A bar chart depicted the expression abundance of FASN in different cells.

Statistics analysis

Statistical analysis was performed using GraphPad Prism 10.1.2 software. Each set of data represents 3 independent replicates and is expressed as mean ± SD, unless otherwise indicated. Comparisons between groups were made using a two-tailed Student’s t-test (two groups), a Mann-Whitney test (two groups), or one-way ANOVA followed by post hoc multiple comparison tests (multiple groups). The chi-square test or Fisher’s test was used to compare the correlation between FASN, Ki67 expression, and the clinical data. The Kaplan-Meier log-rank test was used for the analysis of overall survival (OS). The significance level was set as p < 0.05 (ns, not significant; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Results

Identification of GMSCs, OSCCN-MSCs, and OSCC-MSCs

To compare the characteristics of the three types of MSCs (GMSCs, OSCCN-MSCs, and OSCC-MSCs), they were isolated from different sources: normal gingival tissues, OSCC peritumoral tissues, and OSCC tissues. All three cell types exhibited similar spindle-shaped adherent growth under microscopic analysis (Fig. 1A). Flow cytometry revealed characteristic MSC surface markers. GMSCs, OSCCN-MSCs, and OSCC-MSCs were positive for CD105, CD90, CD73, and CD146, and negative for CD45 and CD34 (Fig. 1B). OSCC-MSCs exhibited the highest self-renewal potential and greatest proliferation rate, followed by OSCCN-MSCs and GMSCs (Fig. 1C-D). In multilineage differentiation experiments, it was observed that GMSCs, OSCCN-MSCs, and OSCC-MSCs exhibited the ability to mineralize and form nodules under osteo inductive conditions (Fig. 1E). Additionally, these cells were found to generate lipid droplets when induced towards adipogenic differentiation (Fig. 1F). We observed that, compared to GMSCs, OSCC-MSCs exhibited a significant decrease in osteogenic ability with reduced expression of osteogenic markers (RUNX2, ALP) (Fig. 1E). However, their adipogenic ability was notably enhanced with increased expression of adipogenic markers (PPARγ, LPL) (Fig. 1F).

Fig. 1
figure 1

OSCC-MSCs display enhanced capacity to differentiate into adipocytes when cultured under adipogenic inductive conditions. (A) Phenotypic characterization of GMSCs, OSCCN-MSCs, and OSCC-MSCs. Scale bar, 100 μm. (B) Flow cytometric analysis was performed to determine the expression of CD105, CD90, CD73, CD146, CD45, and CD34 on GMSCs, OSCCN-MSCs, and OSCC-MSCs, respectively. (C) Crystal violet staining to detect the colony-forming ability of GMSCs, OSCCN-MSCs, and OSCC-MSCs. (D) EdU staining to compare the proliferation of GMSCs, OSCCN-MSCs, and OSCC-MSCs. Scale bar, 100 μm. (E) The osteogenic differentiation potential of GMSCs, OSCCN-MSCs, and OSCC-MSCs was evaluated by quantifying the percentage of Alizarin red S-positive staining area, as well as analyzing the expression levels of Runx2 and ALP. (F) The adipogenic differentiation potential of GMSCs, OSCCN-MSCs, and OSCC-MSCs was assessed by quantifying the percentage of Oil Red O-positive staining area, as well as evaluating the expression levels of PPARγ and LPL. Scale bar, 200 μm. Full-length blots are presented in Appendix 2 Fig. 1

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OSCC-MSCs enhance the proliferation and migration of OSCC cells and promote tumor growth in mouse xenografts

We established a dual-cell co-culture system using a Transwell chamber (Fig. 2A). In this setup, GMSCs or OSCC-MSCs were placed in the upper chamber, while CAL27 or HSC3 cells were seeded in the lower chamber, with no direct contact between the cells in the two compartments. After a 72-hour co-cultivation period, cells were collected from the lower chamber (CAL27 or HSC3) for cell proliferation assays (EdU and CCK8) and migration assays (scratch and Transwell). The results in Fig. 2B-E illustrated a significant enhancement in the proliferation capacity of CAL27 and HSC3 cells when co-cultured with OSCC-MSCs, as compared to co-culture with GMSCs. Concurrently, co-cultivation with OSCC-MSCs promoted the migration of CAL27 and HSC3 cells compared to co-cultivation with GMSCs (Fig. 2F-I). These results indicate that OSCC-MSCs promote the proliferation and migration of OSCC cells.

Fig. 2
figure 2

OSCC-MSCs promote the proliferation and migration of OSCC cells in vitro. (A) Schematic drawing of OSCC cell lines (CAL27 and HSC3) co-cultured with GMSCs or OSCC-MSCs through indirect cell-cell contact. (B-C) CCK-8 assay was used to determine the proliferation of CAL27 and HSC3 after co-culturing with GMSCs or OSCC-MSCs, respectively. (D-E) EdU assay was used to determine the proliferation of CAL27 and HSC3 after co-culturing with GMSCs or OSCC-MSCs, respectively. Scale bar, 100 μm. (F-G) Scratch assay was performed to analyze the migration ability of CAL27 and HSC3 after co-culturing with GMSCs or OSCC-MSCs, respectively. Scale bar, 200 μm. (H-I) Transwell assay was performed to analyze the migration ability of CAL27 and HSC3 after co-culturing with GMSCs or OSCC-MSCs, respectively. Scale bar, 200 μm

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Subsequently, we investigated whether OSCC-MSCs could effectively enhance OSCC cell proliferation in vivo. GMSCs and OSCC cells (CAL27 or HSC3), or OSCC-MSCs and OSCC cells (CAL27 or HSC3) were injected into the thigh root of BALB/c-nu mice. Notably, OSCC-MSCs promoted the growth of OSCC xenografts in comparison to GMSCs (Fig. 3A-C and E-G). Furthermore, Fig. 3D and H presented a typical histopathological feature and Ki67 expression of the xenografts using H&E staining and IHC staining. The tumor cells in the CAL27 + OSCC-MSCs group displayed a notable increase in the Ki67 expression compared to the CAL27 + GMSCs group (Fig. 3D), a similar observation was made in the HSC3 + GMSCs group and the HSC3 + OSCC-MSCs group (Fig. 3H). Collectively, these results demonstrate that OSCC-MSCs significantly enhance the proliferation of OSCC cells in both in vitro and in vivo when compared to GMSCs.

Fig. 3
figure 3

OSCC-MSCs promote OSCC tumor growth in vivo. (A) Representative images of xenografts from two groups (CAL27 + GMSCs or CAL27 + OSCC-MSCs). n = 5 tumors. (B) Tumor volumes were calculated in CAL27 + GMSCs and CAL27 + OSCC-MSCs groups. n = 5 tumors. (C) Tumor weights were calculated in CAL27 + GMSCs and CAL27 + OSCC-MSCs groups. n = 5 tumors. (D) Representative H&E and IHC staining of Ki67 (left panel) in CAL27 + GMSCs and CAL27 + OSCC-MSCs xenografts. The bar graph (right panel) shows the quantification of Ki67 staining in the xenografts. Scale bar, 100 μm. n = 5 tumors. Data are presented as median with interquartile range. (E) Representative images of xenografts from two groups (HSC3 + GMSCs or HSC3 + OSCC-MSCs, left panel). n = 5 tumors. (F) Tumor volumes were calculated in HSC3 + GMSCs and HSC3 + OSCC-MSCs groups. n = 5 tumors. (G) Tumor weights were calculated in HSC3 + GMSCs and HSC3 + OSCC-MSCs groups. n = 5 tumors. (H) Representative H&E and IHC staining of Ki67 (left panel) in HSC3 + GMSCs and HSC3 + OSCC-MSCs xenografts. The bar graph (right panel) shows the quantification of Ki67 staining in the xenografts. Scale bar, 100 μm. n = 5 tumors. Data are presented as median with interquartile range

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Lipid droplet accumulation in OSCC stroma and elevated FFAs levels in conditioned medium of OSCC-MSCs

In Fig. 1F, we observed a significant enhancement in the adipogenic capability of OSCC-MSCs compared to GMSCs. Additionally, in our mouse xenograft model, we observed a significantly greater accumulation of lipid droplets in the stroma of the CAL27/HSC3 + OSCC-MSCs groups compared to the CAL27/HSC3 + GMSCs groups (Fig. 4A). Moreover, in our analysis of three pairs of fresh tissue specimens (Fig. 4B), we observed a notable presence of lipid droplets in the stroma of OSCC tissues, as evidenced by positive Oil Red O staining. In contrast, no positive staining was detected in the stroma of normal gingival tissues. Perilipin-1, a protein encoded by the PLIN1 gene and a key structural component located on the surface of lipid droplets within cells, exhibited higher expression in the stroma of OSCC tissues compared to normal gingival stroma. These preliminary findings (Fig. 4B) suggest a potential association between OSCC stroma and lipid droplet accumulation. However, further studies with larger cohorts are needed to confirm these observations and establish statistical significance.

Fig. 4
figure 4

Lipid droplets and free fatty acids are increased in OSCC tissues. (A) Representative images of Oil Red O staining in CAL27 + GMSCs and CAL27 + OSCC-MSCs xenografts, as well as HSC3 + GMSCs and HSC3 + OSCC-MSCs xenografts (left panel). The bar graph (right panel) shows the quantification of the Oil Red O score in the xenografts. Scale bar, 100 μm. n = 5 tumors. Data are presented as median with interquartile range. (B) Representative images of Oil Red O staining and Perilipin-1 IHC staining in healthy gingival tissues and OSCC tissues. n = 3, Scale bar, 100 μm. (C) The relative levels of FFAs in healthy gingival and OSCC tissues were quantified using ELISA. (D) The relative levels of FFAs in the conditioned medium of GMSCs and OSCC-MSCs were quantified using ELISA

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We quantified the relative levels of FFAs in three pairs of fresh tissues using ELISA, revealing higher FFAs levels in OSCC tissues compared to normal gingival tissues (Fig. 4C). Subsequently, we assessed the levels of FFAs in the conditioned medium of GMSCs and OSCC-MSCs. Remarkably, OSCC-MSCs consistently exhibited higher FFAs levels in their conditioned medium than GMSCs (Fig. 4D). These results demonstrate that OSCC-MSCs possess a markedly increased adipogenic capacity, as evidenced by the significant accumulation of lipid droplets and elevated levels of FFAs.

FFAs promote proliferation and migration of OSCC cells

To confirm the promotional effect of FFAs on the proliferation and migration of OSCC cells, we subjected CAL27 and HSC3 cells to exogenously added oleic acids treatment. The outcomes showed that exogenous FFAs significantly promoted the proliferation and migration of OSCC cells (Fig. 5). These findings suggest that OSCC-MSCs may facilitate the proliferation and migration of OSCC cells by releasing FFAs into the extracellular environment.

Fig. 5
figure 5

Exogenous addition of oleic acids promotes OSCC cells proliferation and migration. (A-B) EdU staining was performed to evaluate the effect of oleic acids on the proliferation of CAL27 and HSC3. Scale bar, 100 μm. (C-D) Scratch assay was used to determine the effect of oleic acids on the migration ability of CAL27 and HSC3. Scale bar, 200 μm. (E-F) Transwell assay was used to determine the effect of oleic acids on the migration ability of CAL27 and HSC3. Scale bar, 200 μm

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Knocking down FASN inhibits the adipogenic capability of OSCC-MSC cells

FASN is a crucial enzyme responsible for catalyzing the biosynthesis of fatty acids. When cells require fatty acid synthesis, FASN increases its activity, leading to an accumulation of intracellular lipids and the formation of lipid droplets. The heightened adipogenic ability observed in OSCC-MSCs, along with the increased lipid droplet accumulation in the stroma of OSCC tissue and the xenograft tumors of OSCC + OSCC-MSCs group, implies the potential involvement of FASN in this crucial process. We quantified FASN expression levels in both GMSCs and OSCC-MSCs using RT-qPCR and Western blotting. The results demonstrate significant upregulation of both FASN mRNA and protein levels in OSCC-MSCs compared to GMSCs (Fig. 6A-B).

Fig. 6
figure 6

Knocking down FASN of OSCC-MSCs inhibits the capacity to differentiate into adipocytes and reduces the secretion of FFAs. (A) The mRNA expression of FASN was quantified by RT-qPCR analysis in GMSCs and OSCC-MSCs. (B) Western blot analysis of FASN expression levels in GMSCs and OSCC-MSCs. (C) Western blot analysis of FASN expression levels in OSCC-MSCs transfected with silencing RNA targeting NC/FASN (siNC/siFASN). siFASN#1 was used for subsequent experiments. (D-E) The adipogenic differentiation potential of OSCC-MSCs after transfecting siNC or siFASN#1 was assessed by quantifying the percentage of Oil Red O-positive staining area, as well as evaluating the expression levels of PPARγ and LPL. Scale bar, 200 μm. (F) Relative amounts of free fatty acids in the conditioned medium of OSCC-MSCs after transfecting siNC or siFASN#1 were measured by ELISA assays. Full-length blots are presented in Appendix 2 Fig. 6

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To further investigate the influence of FASN in OSCC-MSCs on the proliferation and migration of OSCC cells, we initially designed three FASN interference sequences. Western blotting validation confirmed the efficacy of siFASN#1 and siFASN#3 in effectively knocking down FASN, with siFASN#1 showing the most pronounced effect (Fig. 6C). Therefore, siFASN#1 was used for subsequent experiments. Knocking down the expression of FASN in OSCC-MSCs significantly inhibited the adipogenic ability of the cells and led to a decrease in the expression levels of associated adipogenic markers (Fig. 6D-E). Following the transfection of siFASN#1 into OSCC-MSCs for 24 h, we collected conditioned medium from both the control group and the FASN knockdown group of OSCC-MSCs. ELISA analysis revealed that knocking down FASN resulted in a reduction in FFAs secretion in the conditioned medium of OSCC-MSCs (Fig. 6F). The above results indicate that the enhanced adipogenesis and increased FFAs secretion in OSCC-MSCs are closely associated with high expression of FASN.

Knocking down FASN in OSCC-MSCs inhibits the proliferation and migration ability of OSCC cells

To assess the impact of FASN expression in OSCC-MSCs on the proliferation and migration of OSCC cells, we employed the co-culture system depicted in Fig. 7A. Specifically, OSCC-MSCs transfected with either the control or siFASN#1 were added to the upper chamber, while CAL27 or HSC3 cells were seeded in the lower chamber. We observed that knocking down FASN in OSCC-MSCs downregulated the FFAs levels within CAL27 and HSC3 cells (Fig. 7B-C), and inhibited the proliferation and migration of CAL27 and HSC3 cells (Fig. 7D-I). These results suggest that inhibiting FASN expression in OSCC-MSCs significantly decreased the proliferation and migration of OSCC cells.

Fig. 7
figure 7

Knockdown of FASN in OSCC-MSCs inhibits the proliferation and migration of OSCC cell lines. (A) Schematic drawing of OSCC cell lines (CAL27 and HSC3) co-cultured with OSCC-MSCs transfected with siNC or siFASN#1. (B-C) Relative amounts of FFAs in CAL27 and HSC3 were measured by ELISA assays after coculturing with OSCC-MSCs transfected with siNC or siFASN#1. (D-E) EdU staining was performed to evaluate the proliferation of CAL27 and HSC3 after coculturing with OSCC-MSCs transfected with siNC or siFASN#1. Scale bar, 100 μm. (F-G) Scratch assay was used to analyze the migration ability of CAL27 and HSC3 after coculturing with OSCC-MSCs transfected with siNC or siFASN#1. Scale bar, 200 μm. (H-I) Transwell assay was used to analyze the migration ability of CAL27 and HSC3 after coculturing with OSCC-MSCs transfected with siNC or siFASN#1. Scale bar, 200 μm

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FASN is highly expressed in OSCC stroma and is associated with poor prognosis

We collected 86 OSCC clinical samples, and Table S3 summarizes the clinical and pathological characteristics of the patients. FASN expression was evaluated in both cancer and adjacent tissue stroma using IHC. The results showed that FASN expression in the cancer stroma was higher compared to the peritumoral stroma (Fig. 8A and C, and Table 1). Based on the immunohistochemical intensity of FASN in cancer stroma, samples were divided into high and low expression groups. Correlation analysis revealed that FASN expression is closely associated with tumor size (Table 2). Additionally, a positive correlation was found between FASN expression in the cancer stroma and Ki67 expression in OSCC (Fig. 8B and D, and Table 3). High FASN expression in the cancer stroma was also associated with lower overall survival rates (Fig. 8E).

Fig. 8
figure 8

FASN expression in the OSCC stroma is positively correlated with Ki67 expression in cancer tissue and poor prognosis. (A) Representative images of IHC staining showing FASN expression levels in OSCC stroma and peritumoral stroma. Scale bar, 100 μm. (B) Representative images of IHC staining showing FASN expression levels in OSCC stroma and Ki67 expression in OSCC. Scale bar, 100 μm. (C) The bar graph depicts the distribution of FASN-low and FASN-high groups across peritumoral stroma and cancerous stroma categories. A chi-square test was employed to analyze the differential expression of FASN between peritumoral and cancerous stromal tissues in OSCC. (D) The bar graph illustrates the distribution of Ki67-low and Ki67-high groups across FASN-low and FASN-high categories. The correlation between FASN expression in the stromal compartment and Ki67 levels in tumor cells of 86 OSCC cases was evaluated using Chi-square test. (E) Kaplan-Meier survival curves of overall survival (OS) based on FASN expression in 86 OSCC patients. All patients were divided into two groups based on the expression of FASN. A significant level was calculated by log-rank test (n = 86)

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Table 1 Comparison of FASN expression levels in cancer stroma and peritumoral stroma
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Table 2 Relationship between FASN expression of OSCC stroma and clinicopathological features of OSCC
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Table 3 Correlation between FASN expression in OSCC stroma and Ki67 expression in OSCC
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High FASN expression correlates with poor survival in HNSCC

We further explored the TCGA database to evaluate the expression and prognostic implications of FASN in HNSCC. As shown in Fig. 9A, FASN mRNA expression is significantly higher in tumor tissues compared to peritumor tissues in HNSCC. Kaplan-Meier survival analysis reveals a significant association between high FASN expression and reduced overall survival (p = 0.03, HR = 1.37, 95% CI: 1.04, 1.80) (Fig. 9B). Analysis of single-cell sequencing data (GSE103322) from 5902 cells in 18 HNSCC samples highlights that stromal cells are a major component of HNSCC, with FASN expression in these cells second only to malignant tumor cells (Fig. 9C-D). Since OSCC accounts for about 90% of HNSCC, these findings further support our hypothesis that elevated FASN expression in the OSCC stroma may be associated with poorer tumor prognosis.

Fig. 9
figure 9

High FASN expression correlates with poor survival in HNSCC. (A) The violin plot shows the distribution of FASN mRNA expression in tumor (red) and peritumor (blue) samples across multiple cancer types from the TCGA dataset. Each pair of violins represents the distribution of FASN expression in tumor and peritumor samples for a specific cancer type. The red dashed box highlights the significantly elevated expression of FASN in tumor tissues compared to peritumor tissues in HNSCC. There were totally 518 HNSCC and 44 normal samples in TCGA-HNSC dataset. (B) The Kaplan-Meier plot compares overall survival probabilities between high FASN expression (red line) and low FASN expression (blue line) groups in HNSCC patients from TCGA-HNSC dataset. (C) The t-SNE plot illustrates the distribution of major cell lineages within HNSCC_GSE103322 based on gene expression profiles. Different cell types are color-coded, including CD4 T conv (blue), CD8 T (green), CD8 Tex (brown), Endothelial (pink), Stromal cells (purple), Malignant cells (dark blue), and others. (D) The bar plot presents the mean expression levels of FASN across various cell types derived from single-cell RNA sequencing data in HNSCC_GSE103322. Each bar represents the average FASN expression for a specific cell type, with cell types including malignant cells, stromal cells, myofibroblasts, CD8 T cells, endothelial cells, CD4 T cells, plasma cells, mast cells, monocytes/macrophages, and myocytes. The red dashed box emphasizes the high mean expression of FASN in stromal cells

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Discussion

MSCs play an indispensable role in tumor initiation and progression. It has been proposed that tumor-derived MSCs can promote tumor advancement [28]. However, few studies have investigated the impact of OSCC-MSCs on the biological behavior of OSCC. In this study, we successfully isolated GMSCs, OSCCN-MSCs, and OSCC-MSCs from normal gingival tissues, OSCC peritumoral tissues, and OSCC tissues, respectively. Our findings revealed that OSCC-MSCs exhibited significantly higher adipogenic differentiation and markedly lower osteogenic differentiation compared to GMSCs. OSCC-MSCs promoted the proliferation and migration of OSCC cells through high FASN expression, which enhanced FFAs secretion. Analysis of the TCGA-HNSC cohort revealed high FASN expression in HNSCC, correlating with poor prognosis. Single-cell data analysis further indicated high FASN expression in stromal cells within HNSCC. Clinically, elevated FASN expression in the OSCC stroma was associated with larger tumor sizes, increased Ki67 expression in OSCC, and poorer overall survival rates. Our study highlights the critical role of OSCC-MSCs in the tumor microenvironment, as they promote OSCC progression through mechanisms involving high FASN expression, underscoring their significance as potential therapeutic targets.

Here, we successfully isolated MSCs from distinct sources: normal gingival tissues (GMSCs), peritumoral tissues (OSCCN-MSCs), and OSCC tumor tissues (OSCC-MSCs). The plastic adherence, surface marker expression, and multipotent differentiation potential of these MSCs are all in accordance with the criteria set by the International Society for Cellular Therapy [23]. Unlike cancer-associated fibroblasts, which are tumor-activated fibroblasts, these MSCs retain multipotent differentiation capacity. GMSCs were first isolated and characterized by Zhang et al. in 2009 [29]. Since then, numerous studies have successfully isolated and identified GMSCs [30]. However, it was not until 2021 that Ji et al. isolated and characterized OSCC-MSCs and oral leukoplakia-derived MSCs (OLK-MSCs) [31]. In 2024, Guo et al. isolated and characterized OSCC-MSCs, normal oral mucosa-derived MSCs (OM-MSCs), and OLK-MSCs [32]. To date, only three studies, including ours, have conducted research related to OSCC-MSCs. All previous studies have identified the adipogenic and osteogenic differentiation capabilities of these MSCs but did not compare the differentiation abilities among these cell types. Our findings first demonstrated that OSCC-MSCs exhibited significantly higher adipogenic differentiation ability and lower osteogenic differentiation compared to GMSCs. This is further evidenced by the increased lipid droplets in both OSCC-MSCs xenograft and clinical OSCC tumor stroma, as well as the higher FFAs levels in OSCC tissues. In our study, OSCC-MSCs exhibited increased expression of PPARγ and decreased expression of Runx2 compared to GMSCs. PPARγ and Runx2 are two pivotal transcription factors that determine the differentiation pathway of MSCs. PPARγ promotes adipogenesis while simultaneously inhibiting osteogenesis, whereas Runx2 drives MSCs towards osteoblast differentiation [33,34,35]. These findings collectively confirm that OSCC-MSCs possess a higher adipogenic capacity. How does the enhanced adipogenic capacity of OSCC-MSCs affect the biological behavior of OSCC cells?

In this study, FFAs were significantly increased in the conditioned medium of OSCC-MSCs. Exogenous addition of an appropriate amount of FFAs was found to promote the proliferation and migration of OSCC cells, suggesting a new mechanism through which OSCC-MSCs may influence OSCC progression. Our research confirmed that OSCC-MSCs promote OSCC proliferation and migration by secreting FFAs as a result of enhanced adipogenic differentiation, in contrast to GMSCs. OSCC-MSCs, originating from normal MSCs that migrate to the tumor site, acquire distinct characteristics under the influence of the tumor microenvironment [36]. While there is extensive research on the role of normal tissue-derived MSCs in OSCC [18,19,20,21,22], studies investigating the specific interaction between OSCC-MSCs and OSCC cells remain limited. Kansy et al. first isolated HNSCC-MSCs and discovered that HNSCC-MSCs secrete CXCL8 and CXCL12, promoting xenograft growth in mice [37]. Liotta et al. found that HNSCC-MSCs inhibit CD4+ and CD8+ T lymphocytes, promoting HNSCC tumor growth [38]. Compared to OLK-MSCs, OSCC-MSCs highly express CPNE7, activating the NF-κB pathway and increasing CXCL8 secretion, which promotes OSCC metastasis [31]. The aforementioned study indicates that OSCC-MSCs can promote OSCC proliferation by secreting chemokines or regulating the immune microenvironment. However, our findings differ fundamentally: OSCC-MSCs promote OSCC progression not by secreting chemokines, but by enhancing adipogenic differentiation and secreting FFAs. This novel mechanism provides new insights into the role of OSCC-MSCs in OSCC progression, highlighting the potential of FFAs as a new class of factors involved in the progression of OSCC.

FASN is a key enzyme responsible for the endogenous synthesis of fatty acids, playing a crucial role in lipid metabolism. In radiotherapy-resistant HNSCC cells, FASN is highly expressed [39]. Inhibition of FASN expression in HNSCC cells through inhibitors reduces the proliferation and migration of HNSCC cells [40] and increases their sensitivity to chemotherapy drugs [41]. These studies indicate that the high expression of FASN in HNSCC cells is pivotal in tumor progression. In our study, analysis of the TCGA database demonstrated that FASN mRNA expression is significantly elevated in HNSCC tumor tissues compared to peritumor tissues, and this elevation correlates with poorer overall survival. However, in addition to tumor cells, there are also stromal cells within the tumor tissue. Single-cell sequencing data further highlighted that not only do tumor cells highly express FASN in HNSCC tumors, but stromal cells also exhibit high FASN expression. These findings suggest that the upregulation of FASN in the tumor microenvironment also plays a critical role in OSCC progression. Studies have already confirmed the importance of FASN in different tumor microenvironments [42,43,44]. Knocking down the expression of FASN in cancer-associated fibroblasts inhibits colorectal cancer migration through lipid metabolic reprogramming [42]. De novo fatty-acid synthesis by FASN is crucial for Treg cell maturation, and the loss of FASN in Treg cells inhibits melanoma and colorectal cancer growth [43]. Suppressing FASN activity in tumor-associated macrophages can markedly decrease the release of extracellular cytokines that promote tumor growth, thereby impeding tumor progression [44]. However, the role of FASN in OSCC tumor microenvironment has not yet been fully elucidated, which prompted us to explore its function in this specific context. In our findings, FASN was upregulated in OSCC-MSCs compared to GMSCs. Knocking down FASN in OSCC-MSCs inhibited their pro-proliferative and pro-migratory effects on OSCC cells. Further investigation with 86 clinical OSCC samples confirmed higher FASN expression in the tumor stroma than in adjacent normal tissues, correlating positively with tumor size and Ki67 expression in cancer tissues. Thus, our study provides novel insights into the contribution of FASN in OSCC-MSCs and emphasizes its potential as a therapeutic target in the tumor microenvironment.

Tumor progression is the result of continuous cross-talk between tumor cells and the tumor microenvironment. In our study, we found that OSCC-MSCs can promote OSCC proliferation and migration by upregulating FASN expression. So, is the upregulation of FASN expression in OSCC-MSCs regulated by OSCC cells? OSCC cells can condition their microenvironment through complex signaling networks and intercellular interactions, thereby promoting malignant progression and therapeutic resistance. For example, HMGB1 released by OSCC cells induces the polarization of macrophages into the M1 phenotype. This polarization leads to the secretion of IL-6 through the activation of the NF-κB signaling pathway, thereby promoting the aggressive migration of OSCC cells [45]. Extracellular vesicles released by OSCC cells can stimulate oral fibroblasts, converting them into cancer-associated fibroblasts [46]. In our study, why do OSCC-MSCs exhibit high expression of FASN? This implies a potential regulatory mechanism through which OSCC cells may influence FASN expression in OSCC-MSCs, thereby contributing to tumor progression, and warranting further investigation.

In our co-culture experiments, OSCC cells and MSCs were grown in separate chambers of a Transwell system, which restricted the study to the effects of secreted factors (e.g., FFAs) from OSCC-MSCs on OSCC cell behavior. In a more physiological setting, MSCs and OSCC cells may interact directly via cell-cell contact in addition to secreting factors. The lack of direct cell-cell interaction in our experimental design is a limitation, and future studies should incorporate 3D co-culture systems or organotypic models to more closely mimic the in vivo tumor microenvironment. While we observed elevated FASN expression in OSCC-MSCs, the underlying mechanisms driving this upregulation remain unclear. We did not delve into the molecular pathways responsible for FASN expression in OSCC-MSCs. This is an important area for future research, as understanding how FASN is upregulated could open new avenues for targeted therapies.

Conclusions

Our study finds that adipogenic transformation of OSCC-MSCs promotes OSCC cell proliferation and migration, partially through high expression of FASN and enhanced FFAs secretion. Our results provide new insight into the mechanism of OSCC progression and suggest that the FASN of OSCC-MSCs may be potential targets of OSCC in the future.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

OSCC:

Oral squamous cell carcinoma

HNSCC:

Head and neck squamous cell carcinoma

MSCs:

Mesenchymal stem cells

OSCC-MSCs:

OSCC-derived MSCs

OSCCN-MSCs:

OSCC adjacent noncancerous tissues-derived MSCs

GMSCs:

Gingiva-derived MSCs

OLK-MSCs:

Oral leukoplakia-derived MSCs

OM-MSCs:

Oral mucosa-derived MSCs

CA-MSCs:

Cancer-associated MSCs

FASN:

Fatty acid synthase

FFAs:

Free Fatty Acids

OS:

Overall survival

IACUC:

Institutional Animal Care and Use Committee

CFU-F:

Colony-forming unit-fibroblasts

H&E:

Hematoxylin-eosin

CCK-8:

Cell-counting kit 8

OD:

Optical density

EdU:

5-ethynyl-2’-deoxyuridine

IHC:

Immunohistochemistry

RT-qPCR:

Quantitative real-time PCR

PVDF:

Polyvinylidene difluoride

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Acknowledgements

The authors declare that no Artificial Intelligence was used to generate data in this study, and Artificial Intelligence was only used for language editing.

Funding

This research was funded by the National Natural Science Foundation of China (No.82271021), the China Postdoctoral Science Foundation (No.2023M744005 and No.2022M713568), Science and Technology Projects in Guangzhou, China (No.2024A03J0098), and National Natural Science Foundation Cultivation Project of the Third Affiliated Hospital of Sun Yat-sen University (2024GZRPYMS06).

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Author notes

  1. Yiting Shao, Yu Du and Zheng Chen contributed equally to this work.

Authors and Affiliations

  1. Department of Stomatology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510630, China

    Yiting Shao, Zheng Chen, Shaoqin Tu, Yi Feng, Yuluan Hou & Hong Ai

  2. Department of Pathology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510630, China

    Yu Du

  3. Hospital of Stomatology, Guanghua School of Stomatology, South China Center of Craniofacial Stem Cell Research, Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, 510055, China

    Lei Xiang & Xiaoxing Kou

  4. Key Laboratory of Stem Cells and Tissue Engineering (Sun Yat-sen University), Ministry of Education, Guangzhou, 510080, China

    Xiaoxing Kou

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  1. Yiting Shao
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Contributions

YTS, XXK, and HA designed the study. YTS, YD, and ZC performed the experiments. LX, SQT, YF and YLH assisted with sample collection and provided guidance. YTS and YD wrote the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Xiaoxing Kou or Hong Ai.

Ethics declarations

Ethics approval and consent to participate

The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University (Title of the approved project: A Mechanistic Study on the Role of ZEB1-Induced Upregulation of FASN in Promoting Adipogenic Differentiation of Mesenchymal Stem Cells and the Progression of Oral Squamous Cell Carcinoma; Approval No: RG2024-056-01; Date: 2024-04-08). Written informed consent was obtained from individual or guardian participants. All experiments involving mice were approved by the Animal Ethics Committee of Sun Yat-sen University (Title of the approved project: Effect of Mesenchymal Stem Cells on the Tumorigenesis of Oral Squamous Cell Carcinoma; Approval No: IACUC-00344982; Date:2022-09-01).

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Not applicable.

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The authors declare no conflict of interest in this work.

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Shao, Y., Du, Y., Chen, Z. et al. Mesenchymal stem cell-mediated adipogenic transformation: a key driver of oral squamous cell carcinoma progression. Stem Cell Res Ther 16, 12 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04132-9

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04132-9

Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512