Distinctive tumor biology of oral squamous cell carcinoma associated with oral submucous fibrosis and its suppressive immune microenvironment

Abstract

Background

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies in Southern and Southeastern Asia, and in the Hunan region of China, where betel nut chewing is prevalent. The prognosis of OSCC with oral submucous fibrosis (OSF), caused by areca nut use, remains debated. This study aimed to determine whether OSCC associated with OSF exhibits distinct tumor biological behaviors compared to OSCC without OSF, and to explore the tumor immune microenvironment, specifically focusing on the expression of Th17/Treg cells and the PD-1/PD-L1 axis.

Methods

OSCC patients with and without OSF (Jan 2013–Oct 2023) were recruited from Xiangya Stomatological Hospital, grouped by history and clinical features. Patients were followed every 3 months for recurrence and complications. SPSS 27 was used for descriptive and inferential statistics (Kaplan–Meier, Cox regression, χ²-test); survival/log survival/hazard functions were plotted to compare survival probabilities, stratified by sex, age, tumor location, stage (early/advanced, TNM), and habits (betel quid, smoking, alcohol). A subgroup of 88 patients (44 each) had immunohistochemical analysis for Th17/Treg and PD-1/PD-L1 expression.

Results

OSCC with OSF primarily affected the tongue and buccal mucosa, occurring in younger patients (median age 48 vs. 55 years). Recurrence was higher in OSF-associated OSCC (27.56% vs. 19.73%). Overall Survival (OS) rates for OSCC with OSF were 94.25% (1-year OS), 49.82% (3-year OS), and 41.23% (5-year OS), compared to 94.82% (1-year OS), 56.58% (3-year OS), and 47.92% (5-year OS) in non-OSF cases. A subgroup of 88 patients (44 each) had immunohistochemical analysis for the expression of IL-17 (a marker of Th17 cells) and Foxp3 (a marker of Treg cells), as well as PD-1/PD-L. Immune profiling showed elevated IL-17, Treg cells, PD-L1, and PD-1 in OSF-associated OSCC.

Conclusion

OSCC with OSF displays more aggressive tumor biological behavior and worse prognosis, which contributed by a more suppressive immune microenvironment marked by elevated levels of IL-17, Treg cells, and PD-1/PD-L1 expression. These results highlight the need for tailored treatment approaches in managing OSCC in patients with OSF.

Introduction

Oral submucous fibrosis (OSF) is a chronic, progressive disease that primarily affects the oral mucosa, leading to fibrosis and reduced elasticity of the tissues [1]. This condition, which is most prevalent in South and Southeast Asia, is strongly associated with the consumption of areca nut, a key component of betel quid [2]. Areca nut contains alkaloids such as arecoline, which promote collagen synthesis and contribute to tissue fibrosis [3]. Over time, OSF leads to severe limitations in mouth opening (trismus), difficulty swallowing, loss of taste sensation, and impaired speech, significantly affecting the patient's quality of life [4]. The disease is recognized as a pre-cancerous condition, with a malignant transformation rate of approximately 1.2%−3.7% in China, and up to 7.6% in India [5, 6]. The progression from OSF to oral squamous cell carcinoma (OSCC) is complex and is associated with multiple factors, including persistent irritation, genetic mutations, and immune dysregulation [7].

The clinical management of OSF remains challenging, and while cessation of betel nut use, medications, and surgical interventions are often employed, there is no definitive treatment available to reverse the fibrosis or prevent malignant transformation [8, 9]. Notably, OSCC in patients with OSF often presents differently compared to those without OSF. Studies show that OSF patients are typically younger and have higher recurrence rates of OSCC [10]. However, the prognosis of OSCC in the background of OSF remains controversial. For instance, Acharya et al. [10] conducted a retrospective study of clinicopathological features and reported higher recurrence rates in OSF-associated OSCC but did not find a significant difference in overall survival compared to non-OSF OSCC. In contrast, Divya et al. [11] performed a systematic review and concluded that OSCC arising from OSF is associated with a worse prognosis, including shorter survival time and higher metastatic potential. This discrepancy highlights the need for a deeper understanding of the tumor biological behaviors and immune features specific to OSCC in OSF patients to resolve conflicting findings.

Our study aims to clarify whether OSCC associated with OSF exhibits distinct clinical and biological characteristics compared to OSCC without OSF. We focus on evaluating differences in tumor location, patient demographics, recurrence rates, survival outcomes, and the immune microenvironment. We specifically selected Th17/Treg cells and the PD-1/PD-L1 axis as key immune targets for investigation based on their well-established roles in oral oncology and OSF pathogenesis. Th17 cells secrete IL-17 to promote chronic inflammation and angiogenesis, while Treg cells (marked by Foxp3) suppress anti-tumor immune responses both pathways are known to be dysregulated in OSF-related chronic inflammation and malignant transformation [12–14]. The PD-1/PD-L1 axis mediates immune checkpoint inhibition, enabling tumor cells to evade cytotoxic T-cell attack; our previous work demonstrated differential PD-1/PD-L1 expression in OSF-associated OSCC [15], and targeting this axis has shown therapeutic potential in OSCC [16]. By examining these two critical immune pathways, we hope to uncover underlying immunopathogenic mechanisms that contribute to the progression of OSCC in the context of OSF. This research is critical for developing more targeted treatment strategies for OSCC patients with OSF and improving their clinical management.

Materials and methods

Retrospective analysis

This retrospective cohort study included 746 OSCC patients, comprising 381 with oral submucous fibrosis (OSF) and 365 without OSF, recruited between January 2013 and September 2023 from Central South University Xiangya Stomatology Hospital, Hunan Province, China. Hunan, known for its high areca nut consumption, has seen a rising OSCC incidence over the past decade.

To ensure cohort consistency and reduce confounding, strict inclusion and exclusion criteria were applied. For inclusion: patients had pathologically confirmed primary OSCC (per WHO 2017 histological criteria); those in the OSF group required additional pathological confirmation of OSF (submucosal collagen hyperplasia/hyalinization, with or without epithelial atrophy/dysplasia) [5, 6]; complete data (demographics, tumor characteristics staged per AJCC 8th edition, baseline habits) were available; and follow-up lasted ≥ 3 months.

Exclusion applied to patients with metastatic OSCC at diagnosis (distant metastasis to lung/liver/bone, confirmed via imaging/pathology), other malignancies within 5 years of OSCC, severe organ dysfunction, incomplete clinical follow-up data, or loss to follow-up within 3 months.

Patient data were obtained from hospital records, including demographics (age, sex, occupation, address), clinical details (mouth opening, tumor site and size, TNM stage, recurrence, follow-up status), and habits (areca nut, tobacco, and alcohol use). Smoking history was categorized by daily cigarette consumption and duration, while areca nut and alcohol use were classified by frequency and duration. Data collection followed a structured questionnaire.

The cohort included 369 males and 12 females with OSCC and OSF, and 320 males and 45 females without OSF, with age ranges of 23–73 years and 16–96 years, respectively. Follow-up from 2013 to 2023 was conducted via hospital visits or telephone to document recurrence, progression, and outcomes. Statistical analyses summarized data and identified associations, with results presented as frequencies, percentages, and means ± standard deviations.

Immune phenotyping analysis by IHC

Eighty-eight OSCC patients (half with OSF) were analyzed by immunohistochemistry (IHC) for the expression of Th17/Treg and PD1/PD-L1. All diagnoses were pathologically confirmed, with clinical staging per the AJCC 8th edition and histological grading per WHO criteria.

Tumor tissue sections were cut to 4-μm thickness. The sections were deparaffinized in xylene and hydrated using an alcohol series. The following antibodies were used: PD-1 (ab52587, Abcam, Cambridge) at a dilution of 1:50, PD-L1 (ab213524, Abcam, Cambridge) at 1:50, IL-17 (13082–1-AP, PTG, Wuhan), and Foxp3 (bs-10211r, Bioss, Beijing) at 1:100. Anti-rabbit, mouse-IgG antibody-HRP polymer was added and incubated for 30 min at 37 °C. Staining was visualized with diaminobenzidine (DAB, Dako, Glostrup, Denmark), and counterstaining with hematoxylin was performed to visualize the nuclei. Immunostaining was evaluated by two investigators, and five high-power fields were selected. If there were differences, the inconsistent samples were reevaluated, and the consensus score was adopted.

The final density of each sample was calculated as the average number of positive cells in the five high-power fields (HPFs) for IL-17/Foxp3. For PD-1/PD-L1, the final immunoreactivity score (IRS) was determined by the staining intensity and extent scores. The staining intensity was scored as: 0 (none), 1 (weak), 2 (moderate), or 3 (strong). The extent of staining was scored as: 0 (< 5%), 1 (5–25%), 2 (26–50%), 3 (51–75%), and 4 (> 75%), with the stained area relating to the entire cancer area. The staining intensity and the percentage of cell-positive percentages were then multiplied to generate the IRS for each case, ranging from 0 to 12. Tissues with a final staining score of > 4 were considered high. PD-1 was considered high when > 10% of the membrane was moderately or strongly stained.

Statistical analyses

Statistical analyses including Mann–Whitney U, t-tests, chi-square/Fisher’s exact tests, Kaplan–Meier, and Cox regression, were performed using SPSS 23.0.

Results

OSCC with OSF occurred at a significantly earlier age and had favorable onset sites

The demographic data shows that OSCC in OSF patients occurred significantly earlier than in non-OSF cases (Table 1). Patients with OSCC and OSF were predominantly younger, peaking in the 41–50 age group (37.3%), while OSCC without OSF was more common in older patients (51–60 age group, 35.6%). This age difference was significant (p = 0.000), suggesting OSF may contribute to earlier malignancy onset. Additionally, OSCC with OSF more frequently involved the buccal mucosa (43.6%) and tongue (47.2%), whereas non-OSF cases had higher tongue involvement (56.2%) and lower buccal mucosa tumors (25.8%) (Table 1), indicating OSF may influence tumor location.

Table 1.

Clinicopathological characteristics and analysis of OSCC with and without OSF patients

Categories		Cases				P-value	χ2-test
		OSCC with OSF		OSCC without OSF
		Cases	Percentage %	Cases	Percentage %
Sex	Male	369	96.9	320	87.7	0.000	22.257
	Female	12	3.1	45	12.3
Age Group	11–20	0	0	1	0.3	0.000*	94.797
	21–30	13	3.4	5	1.4
	31–40	73	19.2	28	7.7
	41–50	142	37.3	79	21.6
	51–60	117	30.7	130	35.6
	61–70	32	8.4	81	22.2
	71–80	4	1	36	9.9
	81–90	0	0	4	1.1
	91–100	0	0	1	0.3
Median Age		48			55
Location	Tongue	180	47.2	205	56.2	0.000*	36.007
	Buccal	166	43.6	94	25.8
	Gingiva	15	3.9	26	7.1
	Maxilla & Mandible	4	1	16	4.4
	Floor of the mouth	7	1.8	10	2.7
	Lips	6	1.6	5	1.4
	Oropharyngeal	2	0.5	6	1.6
	Glands	0	0	2	0.5
	Palatal	1	0.3	1	0.3
N of Cigarette Smoking/Day	No Smoking	51	13.4	133	36.4	0.000*	54.299
	1–10	98	25.7	75	20.5
	11–20	151	39.6	108	29.6
	> 20	81	21.3	49	13.4
Alcohol/Frequency	Non Drinker	123	32.3	199	54.5	0.000*	37.746
	Daily	122	32	75	20.5
	Weekly	136	35.7	91	24.9

Abbreviations: OSCC oral squamous cell carcinoma, OSF oral submucous fibrosis

Total sample size: OSCC with OSF, n = 381; OSCC without OSF, n = 365. All percentages are calculated relative to the total sample size of each group (OSF or non-OSF)

*P < 0.001, indicating a statistically significant difference between the OSCC with OSF and OSCC without OSF groups

Lower survival rate in OSCC with OSF compared to OSCC without OSF even at the same tumor size and neck lymph metastasis in early-stage

Comparison of survival outcomes between OSCC patients with and without OSF, stratified by TNM stage, revealed lower survival rates in OSF patients even when controlling for tumor size, lymph node metastasis, age, and location (Table 2, Fig. 1A-D).

Table 2.

Survival distributions for the different levels of TNM Stages (Early/Advanced stage) and recurrence rate

TNM stages		OSCC with OSF				OSCC without OSF				P-value	χ2-test
	Reccurance rate	Total N	N of cases (D)	N of cases (A)	Survival (%)	Total N	N of cases (D)	N of cases (A)	Survival (%)
Early Stage (T1-T2)		241 (63.30%)	135	106	44.00%	223 (61.10%)	95	128	57.40%	0.004	8.339
Stage I	No	91 (75.20)				92 (82.90)
	Re-occur	30 (24.80)				19 (17.10)
Stage II	No	82 (68.30)				91 (81.30)
	Re-occur	38 (31.70)				21 (18.80)
Advanced Stage (T3-T4)		140 (36.70%)	96	44	31.40%	142 (38.90%)	95	47	33.10%	0.764	0.09
Stage III	No	51 (83.60)				53 (88.30)
	Re-occur	10 (16.40)				7 (11.70)
Stage IV	No	52 (65.80)				57 (69.50)
	Re-occur	27 (34.20)				25 (30.50)
Total No		276 (72.40)				293 (80.30)				0.003
Total Re-occur		105 (27.60)				72 (19.70)
Total		381	231	150	39.40%	365	190	175	47.90%

Abbreviations: OSCC oral squamous cell carcinoma, OSF oral submucous fibrosis, TNM Tumor-Node-Metastasis (staged according to the AJCC 8th edition), D number of death patients, A number of alive patients

Fig. 1.

Survival Functions in OSCC with and without OSF

In early-stage (T1-T2) disease, OSCC with OSF (n = 241) showed significantly lower survival (44.0%) than non-OSF cases (57.4%), with 135 vs 95 deaths and 106 vs 128 survivors respectively (P = 0.004, χ² = 8.339, Table 2). For advanced-stage (T3-T4), survival rates were comparable (OSF: 31.4%, n = 140; non-OSF: 33.1%, n = 142), with similar mortality (OSF: n = 96; non-OSF: n = 95) and survival (OSF: n = 44; non-OSF: n = 47) counts (P = 0.764, χ² = 0.09, Table 2).

The survival disparity was most notable in early-stage tumors (T1-T2), where OSCC with OSF had lower survival (44.00%) than non-OSF cases (57.40%), indicating OSF's negative impact on early-stage prognosis (Table 2, Fig. 1D).

These findings demonstrate significantly worse survival in early-stage OSCC with OSF, though this difference lessens in advanced stages. This suggests OSF may promote more aggressive tumor behavior, reduce treatment efficacy, or delay diagnosis, particularly affecting early-stage outcomes (Table 2).

Higher recurrence rate in OSCC with OSF

OSCC with OSF showed higher recurrence rates across all stages than OSCC without OSF (Table 2). In Stage I, recurrence was 24.8% (with OSF) vs. 17.1% (without OSF); in Stage II, 31.7% vs. 18.8%; in Stage III, 16.4% vs. 11.7%; and in Stage IV, 34.2% vs. 30.5%. Overall, OSCC with OSF had a significantly higher total recurrence rate (27.6%) than non-OSF cases (19.7%) (Table 2), suggesting OSCC with OSF may be more aggressive and prone to recurrence at any stage.

More unfavorable outcomes in OSCC with OSF

OSCC patients with OSF showed significantly worse long-term survival than those without OSF, particularly at 3 and 5 years (Table 3). While 1-year survival was similar (94.25% with OSF vs. 94.82% without OSF), rates declined more sharply in OSF patients by 3 years (49.82% vs. 56.58%) and 5 years (41.23% vs. 47.92%) (Table 3).

Table 3.

Survival rate of OSCC with and without OSF (1,3 and 5 years)

	OSCC with OSF	Survival rate (%)	OSCC without OSF	Survival rate (%)	P-value
Overall Survival rate
1 year	(328/348)	94.25%	(311/328)	94.82%
3 years	(138/277)	49.82%	(159/281)	56.58%
5 years	(47/114)	41.23%	(69/144)	47.92%
Stage I
1 year	(114/117)	97.44%	(105/107)	98.13%	0.256
3 years	(47/87)	54.02%	(60/91)	65.93%
5 years	(16/37)	43.24%	(26/46)	56.52%
Stage II
1 year	(100/104)	96.15%	(86/89)	96.63%	0.221
3 years	(45/85)	52.94%	(45/76)	59.21%
5 years	(18/42)	42.85%	(23/47)	48.94%
Stage III
1 year	(47/52)	90.38%	(50/54)	92.59%	0.230
3 years	(21/43)	48.84%	(25/44)	56.82%
5 years	(7/17)	41.18%	(8/18)	44.44%
Stage IV
1 year	(67/75)	89.33%	(70/78)	89.74%	0.338
3 years	(25/62)	40.32%	(29/70)	41.43%
5 years	(6/18)	33.33%	(12/33)	36.36%

Abbreviations: OSCC oral squamous cell carcinoma, OSF oral submucous fibrosis

Survival rate = (Number of surviving patients/Number of patients with complete follow-up data) × 100%

Denominators in parentheses (e.g., 348 for 1-year OSCC with OSF survival) refer to the number of patients with complete follow-up data defined as patients who were followed up continuously without loss to follow-up until the corresponding time point (1-year, 3-year, or 5-year) or until death. Patients lost to follow-up before the target time point were excluded from the survival rate calculation for that specific time point to minimize bias

These findings demonstrate consistently lower survival rates for OSCC with OSF across all stages and time intervals, underscoring its more unfavorable prognosis.

More addictive to cigarette smoking and alcohol consumption in OSCC with OSF, and worsen the prognosis

OSCC patients with OSF showed higher rates of heavy smoking and frequent alcohol consumption than those without OSF (Table 4, Fig. 1E, F). Among OSCC with OSF patients, 39.6% smoked 11–20 cigarettes/day (vs 29.6% without OSF) and 21.3% smoked > 20/day (vs 13.4%). Heavy smokers (> 20/day) with OSF had lower survival (29.6%) than non-OSF counterparts (30.6%) (Table 4).

Table 4.

Survival distributions for different risk factors

Frequency	OSCC with OSF				OSCC without OSF				P-value
	Total N (%)	N of Cases (D)	N of Cases (A)	Survival (%)	Total N (%)	N of cases (D)	N of cases (A)	Survival (%)
No-Smoking	51 (13.4)	18	33	64.7	133 (36.4)	45	88	66.2	0.001
1–10	98 (25.7)	60	38	38.8	75 (20.5)	44	31	41.3
11–20	151 (39.6)	96	55	36.4	108 (29.6)	67	41	38
> 20	81 (21.3)	57	24	29.6	49 (13.4)	34	15	30.6
Non-Drinker	123 (32.3)	66	57	46.3	199 (54.5)	85	114	57.3	0.001
Weekly	136 (35.7)	85	51	37.5	91 (24.9)	56	35	38.5
Daily	122 (32.0)	80	42	34.4	75 (20.5)	49	26	34.7
Overall	381 (100)	231	150	39.4	365 (100)	190	175	47.9

Abbreviations: OSCC oral squamous cell carcinoma, OSF oral submucous fibrosis, D number of death, A number of alive patients

“Frequency” includes two subcategories: (1) Cigarette smoking frequency (No-Smoking, 1–10, 11–20, > 20 cigarettes per day); (2) Alcohol consumption frequency (Non-Drinker, Weekly, Daily)

Survival rate = (Number of surviving patients (A)/Total number of patients in each risk factor subgroup) × 100%

Total sample size: OSCC with OSF, n = 381; OSCC without OSF, n = 365

For alcohol, only 32.3% of OSCC with OSF patients were non-drinkers (vs 54.5% without OSF), while 35.7% drank weekly (vs 24.9%) and 32.0% daily (vs 20.5%). These patterns suggest greater alcohol addiction in OSCC with OSF patients, potentially contributing to their poorer prognosis.

The findings indicate that OSCC with OSF patients have higher tobacco and alcohol addiction rates, likely worsening their prognosis (Table 4).

More immunosuppressive environment within OSCC with OSF contribute to the unfavorable outcome

Higher expression of PD-1/PD-L1 in OSCC with OSF

PD-1 and PD-L1 expression was evaluated by IHC staining. Representative strong and weak staining are presented in Fig. 2. PD-L1 was in membrane and cytoplasm of tumor cells (Fig. 2E, F), and PD-1 was stained in lymphocytes scattered between tumor stroma (Fig. 2A, B). High PD-L1 expression was detected in 63.6% (28/44) of OSCC patients with OSF and in only 34.1% (15/44) of OSCC patients without OSF. High PD-1 expression was found in the cell membrane and cytoplasm of tumor-infiltrating lymphocytes (TILs) in 52.3% (23/44) of OSCC patients with OSF and in 45.5% (20/44) of OSCC patients without OSF (Fig. 2I).

Fig. 2.

Expression of PD-1/PD-L1 in OSCC with and without OSF

The rate of high PD-L1 expression per group was nearly twofold higher in OSCC patients with OSF than in OSCC patients without OSF (63.6%:34.1%, 28:15, Fig. 2I). These values indicate a significant upregulation of PD-L1 expression in OSCC patients with OSF compared to OSCC patients without OSF. Moreover, we performed three subgroup analyses to examine the association of PD-L1 with PD-1 expression and its relationship with OSF. All OSCC patients were divided into three subgroups as follows: group A: PDL1-high/PD-1-high; group B: PD-L1-high/PD-1-low or PD-L1-low/PD-1-high; and group C: PD-L1-low/PD-1-low; These results showed that the co-expression of PD-L1 and PD-1 was higher in the OSCC with OSF than in OSCC without OSF (36.4% and 13.6%, respectively, Fig. 2I). bar 250μm.

Elevated expression of Th17/Treg in OSCC with OSF

The representative immunohistochemistry staining is shown in the Fig. 3. The average expression of IL-17 in the microenvironment infiltration of OSF-OSCC and Non-OSF-OSCC was 33.75 and 26.77, respectively (Fig. 3A, B, E). Using the average number of IL-17 cell infiltration as the cutoff value, the samples were divided into high expression and low expression groups. In OSCC, the IL-17 expression in OSF-OSCC was slightly higher than that in Non-OSF-OSCC (Fig. 3E). The average expression of Foxp3 in the microenvironment infiltration of OSF-OSCC and Non-OSF-OSCC was 22.58 and 17.28, respectively (Fig. 3C, D, F). Using the average number of Foxp3 cell infiltration as the cutoff value, the samples were divided into high expression and low expression groups. In OSCC, the expression of Foxp3 in OSF-OSCC was significantly higher than that in Non-OSF-OSCC (P < 0.01, Fig. 3F).

Fig. 3 .

Differential expression of Th17, Treg in OSCC with and without OSF

Immunohistochemical analysis corroborated these findings, demonstrating significantly elevated expression of both Th17 and Treg cells within the tumor microenvironment in OSCC with OSF when compared to OSCC without OSF samples. Notably, the balance between Th17 and Treg cells, measured through the Th17/Treg ratio, shifted in two groups, with OSCC with OSF exhibiting a lower Th17/Treg ratio, indicating a more suppressive immune environment associated with OSCC with OSF (Fig. 3G).*, p<0.01; bar 250μm

These results collectively suggest that the immune microenvironment in OSCC with OSF, marked by a high presence of Th17 and Treg cells and a disrupted Th17/Treg balance, contributes to a more aggressive clinical course and poorer prognosis. This underscores the importance of immune profiling in understanding the progression of OSCC in the context of OSF and highlights potential avenues for targeted therapeutic strategies.

Discussion

Our study provides compelling evidence that OSCC in patients with OSF presents with distinct demographic, clinical, and prognostic characteristics when compared to OSCC without OSF. These findings underscore the significant impact of OSF on OSCC development, progression, and survival outcomes, emphasizing the need for tailored clinical management strategies for this subgroup of patients.

The results indicate that OSCC in patients with OSF occurs at a significantly younger age than OSCC without OSF, with a peak incidence in the 41–50 age group compared to 51–60 years in the non-OSF group. This earlier onset suggests that OSF serves as a predisposing factor for carcinogenesis, likely due to chronic inflammation, epithelial atrophy, and fibrosis leading to malignant transformation at an accelerated rate [17]. The strong association between OSF and betel quid consumption may also contribute to the earlier onset of OSCC, given the well-documented carcinogenic effects of areca nut components [3, 18].

Furthermore, OSCC in patients with OSF exhibits a predilection for the buccal mucosa and tongue, with a notably higher proportion of tumors located in the buccal mucosa compared to OSCC without OSF. This site preference is driven by two key biological factors tied to OSF pathogenesis and areca nut exposure: First, the buccal mucosa and tongue are the primary sites of direct contact with betel quid during chewing, chronic mechanical friction and chemical damage from areca nut alkaloids (e.g., arecoline) in these regions induce more severe and persistent epithelial dysplasia, a precursor to OSCC [18, 19]. Second, the buccal mucosa has a dense submucosal tissue layer that is inherently more susceptible to collagen deposition and fibrosis in OSF [3, 19]; this fibrotic microenvironment disrupts normal tissue homeostasis, impairs immune surveillance, and creates a pro-carcinogenic niche that favors malignant transformation [6, 14]. In contrast, other oral sites (e.g., gingiva, floor of the mouth) experience less direct betel quid contact and have thinner submucosa, leading to lower OSF-related damage and subsequent OSCC risk. This localization pattern aligns with the pathophysiology of OSF, where chronic fibrosis and epithelial dysplasia primarily affect the buccal mucosa, making it more susceptible to malignant transformation. The preferential tumor location in OSCC with OSF may have significant implications for early detection, as careful examination of the buccal mucosa in high-risk individuals could aid in prompt diagnosis and intervention.

Our study demonstrates that OSCC associated with OSF shows significantly poorer clinical outcomes compared to OSCC without OSF. Across all tumor stages, patients with OSF exhibited lower survival rates, particularly in early-stage tumors (T1–T2), where the survival rate was 44.00% versus 57.40% in those without OSF. In advanced stages (T3–T4), this survival gap narrowed, likely due to the universally poor prognosis in late-stage OSCC, regardless of OSF status.

The consistently worse outcomes in the OSF group may stem from multiple factors, including restricted mouth opening and fibrotic tissue that can obscure early tumor detection and complicate surgical procedures. The dense stroma in OSF may also limit chemotherapeutic drug penetration, reducing treatment efficacy [20]. Additionally, OSCC with OSF showed higher recurrence rates across all stages (27.6% vs. 19.7%), especially in early-stage tumors (T1-T2), suggesting a more aggressive disease course. The fibrotic microenvironment may hinder complete tumor resection, increasing the likelihood of residual malignant or dysplastic cells and subsequent recurrence.

Survival disparities also became more evident over time, with lower 1-, 3-, and 5-year survival rates in OSCC with OSF. This suggests that OSF-related OSCC may follow a different progression pattern than conventional OSCC, necessitating more intensive treatment strategies, prolonged follow-up, and possibly novel therapeutic approaches to improve long-term outcomes [20].

The analysis of smoking and alcohol consumption patterns reveals that OSCC patients with OSF are more likely to be heavy smokers and alcohol consumers, both of which are well-established risk factors for OSCC. The detrimental effects of smoking were more pronounced in OSCC with OSF, where higher cigarette consumption correlated with significantly lower survival rates. Similarly, daily alcohol consumption was associated with the worst survival outcomes in OSCC with OSF, further compounding the poor prognosis of these patients.

The synergistic carcinogenic effects of betel quid, tobacco, and alcohol in OSCC with OSF likely accelerate tumor progression and worsen survival [21]. Chronic exposure to these carcinogens in a fibrotic environment may lead to persistent cellular damage, impaired immune surveillance, and enhanced tumor aggressiveness. These findings emphasize the importance of aggressive tobacco and alcohol cessation programs, particularly in regions with a high prevalence of OSF, to mitigate the burden of OSCC in this vulnerable population. Clinically, these data support integrating targeted cessation into OSF patient care: screen early for smoking/alcohol use (via standardized questionnaires), deliver personalized counseling on amplified OSCC/poor survival risks, offer adjuncts like nicotine replacement (smokers) or naltrexone (alcohol dependence) for severe addiction, and use monthly follow-up to monitor adherence. Multi-disciplinary teams (nurses, addiction specialists) can address barriers to cessation, cutting modifiable risks for better outcomes.

An elevated Th17/Treg ratio in OSCC with OSF fosters an immunosuppressive microenvironment driving tumor progression. While Th17 cells promote angiogenesis and chronic inflammation [12, 20], Tregs (FOXP3⁺) suppress cytotoxic T cells, NK cells, and dendritic cells, enabling immune evasion [13, 22]. Chronic areca nut exposure in OSF recruits Th17 and Tregs, sustaining inflammation and fibrosis [23].

This Th17/Treg imbalance reflects dysregulated immunity, where Treg dominance and IL-17-driven MDSC recruitment enhance immune tolerance [24, 25]. OSCC with OSF exhibits stronger immunosuppression than OSCC alone, with higher PD-1/PD-L1 expression and fibrosis-linked TGF-β, IL-6, and IL-1β driving Th17/Treg expansion [19, 26, 27].

The fibrotic OSF stroma creates a hypoxic niche that upregulates PD-1/PD-L1, inhibiting T-cell cytotoxicity [15, 16, 28]. OSCC without OSF lacks this fibrotic milieu, showing milder immune dysfunction. PD-1/PD-L1 co-expression and exhausted TILs further highlight OSF’s aggressive immunosuppression.

FOXP3⁺ Tregs dominate in OSF-associated OSCC, skewing the Th17/Treg ratio toward tolerance. These changes correlate with advanced disease and metastasis, suggesting immunotherapy resistance. Targeting PD-1/PD-L1 and restoring Th17/Treg balance could improve outcomes, guided by comprehensive immune profiling [10, 14, 29].

Conclusion

Our study underscores the critical role of OSF in modifying the clinical trajectory and prognosis of OSCC. OSCC with OSF is characterized by earlier onset, preferential buccal mucosa involvement, higher recurrence rates, and significantly poorer survival outcomes. The presence of OSF poses additional challenges in diagnosis, treatment, and long-term management, necessitating a multidisciplinary approach to improve clinical outcomes. More suppressive immune mircroenvironment could contribute to the worsen tumor biological behaviors within OSCC with OSF, and future research should focus on elucidating the molecular mechanisms underlying the suppressive immune microenvironment brought by OSF and exploring novel therapeutic strategies to enhance patient survival.

Abbreviations

OSCC

Oral Squamous Cell Carcinoma (core tumor type)

OSF

Oral Submucous Fibrosis (key associated condition)

Th17

T helper 17 cells (core immune cell type)

Treg

Regulatory T cells (core immune cell type)

PD-1

Programmed Death 1 (key immune checkpoint)

PD-L1

Programmed Death-Ligand 1 (key immune checkpoint)

IL-17

Interleukin-17 (key cytokine for Th17 function)

Authors’ contributions

Senjeet Sreekissoon contributed to Writing – original draft, Visualization, Project administration, Methodology, Investigation, Data curation, and Conceptualization; Dezhi Wang contributed to Visualization, Validation, Software, and Data curation; Yan Wang contributed to Writing – review & editing, Writing – original draft, and Visualization; Changxin Wang contributed to Validation, Software, and Data curation; Tereda Seifu Neda contributed to Visualization, Software, and Data curation; Mengbo Zhu contributed to Software, Methodology, Formal analysis, and Data curation; Xiaodan Fang contributed to Software, Methodology, and Formal analysis; Kun Li contributed to Writing – review & editing; Baisheng Wang contributed to Software, Resources, and Methodology; Yanjia Hu contributed to Software, Resources, and Methodology; Long Li contributed to Software, Resources, and Methodology; and Hongzhi Quan contributed to Writing – review & editing, Validation, Supervision, Software, Resources, Project administration, Methodology, Formal analysis, Data curation, and Conceptualization.

Funding

This work was supported by the Hunan Provincial Health Commission Scientific Research Project (20254697) and the Hunan Province Natural Science Foundation (2023JJ30814).

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

This work was approved by the Ethics Committee of Xiangya Stomatological Hospital & School of Stomatology, Central South University under the Reference Number 20220044 and was conducted in accordance with the principles of the Declaration of Helsinki. Informed consent to participate was obtained from all participants.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.