中文摘要

研究背景

口腔鳞状细胞癌（oral squamous cell carcinoma, OSCC）是最常见的口腔颌面部恶性肿瘤，具有发病率高，生存率低，易转移复发的临床特征。大多数口腔鳞状细胞癌由具有癌变潜能的口腔黏膜潜在恶性疾患（Oral potentially malignant disorders, OPMD）发展而来，例如口腔白斑（Oral leukoplakia, OLK）、红斑和粘膜下纤维化等。OSCC的发病通常先是由遗传因素、精神因素、局部刺激等引起口腔黏膜损伤和/或慢性炎症，从而导致OLK等OPMD的发病和进展，并最终发生癌前病变向OSCC的演变。

目前有观点认为OLK的免疫微环境处于低水平慢性炎症的免疫调控状态，而进展为OSCC后则转变为免疫抑制的肿瘤免疫微环境，但是，由OLK向OSCC恶性转化过程中的免疫调控机制仍未被阐明。调节性T细胞（Regulatory T cells, Treg cells, 即Treg细胞）可以通过分泌抑制性细胞因子如白细胞介素-10（Interleukin-10, IL-10）、颗粒酶等直接杀伤效应T细胞或抗原呈递细胞（Antigen-presenting cells, APCs），也可以通过表达CD25消耗白细胞介素-2（Interleukin-2, IL-2），或者通过免疫检查点分子负向调控APCs的成熟和功能。在OSCC癌前到癌的发展过程中，Treg细胞被发现在疾病微环境中增多，但是Treg细胞增多的调控机制、临床意义和靶向价值，目前仍不清楚，既有研究认为其能改善临床预后，也有研究认为其会降低患者生存率。另外，通过诱导肿瘤微环境中的Treg细胞耗竭从而增强抗肿瘤免疫反应也是现阶段肿瘤免疫治疗的热点研究策略之一，且在某些肿瘤中已取得了较好的实验治疗效果。然而，也有多项研究发现，单纯诱导总Treg细胞耗竭的策略，往往治疗效果不够理想，甚至可能会加速肿瘤进展。

基于上述研究背景及研究现状，本课题提出如下研究假说：

（1）从OPMD向OSCC进展过程中，Treg细胞不断增多，调控免疫抑制微环境，介导了癌前病变的恶性转化和肿瘤细胞的免疫逃逸。

（2）Treg细胞可能在OPMD恶变为OSCC的不同阶段具有不同的来源和招募机制，并且可能发挥着不同的调控作用。

（3）不同靶向策略耗竭不同来源的Treg细胞，改善肿瘤免疫抑制微环境，从而降低OPMD恶变风险以及抑制OSCC的肿瘤进展。

总之，研究OSCC发生发展过程中Treg细胞来源和功能的动态变化，揭示其变化的机制，并研究和验证其靶向价值，是实现靶向Treg细胞治疗OSCC亟需解决的问题。

研究目的

探究OSCC发生发展中Treg细胞动态变化的原因，揭示口腔病变微环境不同阶段中Treg细胞的来源，并深入研究不同阶段靶向病损组织或肿瘤浸润Treg细胞的治疗方式，以及评估不同阶段靶向治疗的价值，为癌前病变的防治和开发OSCC新型免疫疗法提供理论基础。

研究方法

本研究利用OSCC原发诱导模型4-NQO饲水（4-硝基喹啉-N-氧化物）和原位移植瘤模型4MOSC1细胞株原位注射，结合正常舌组织，OLK轻度增生，OLK异常增生和OSCC癌组织的临床患者单细胞转录组测序数据和空间转录组分析，并主要采用多色流式细胞染色，删除抗体或小分子抑制剂治疗和成纤维细胞-T细胞体外共培养等多种实验手段，探究OPMD及OSCC免疫微环境的变化，揭示 Treg细胞在口腔癌前病损到口腔鳞癌进展过程中的变化特征、主要来源和调控机制。

研究结果

4-NQO诱发的癌前病变和癌变进展的组织病理学与 HPV 阴性 OSCC 的发生发展和组织病理学非常相似，在该原发诱导模型中，我们发现免疫炎性细胞不断突破基底层，并且在OSCC阶段，有大量的异型细胞产生。同时，随着癌前向癌的疾病进展过程，脾脏、颈部引流淋巴结和舌头中Treg细胞的浸润比例逐渐升高，与此同时，我们还发现颈部引流淋巴结和舌组织中分泌干扰素-γ（Interferon-γ, IFN-γ）的CD4⁺ T细胞，即Th1细胞（Type 1 helper T cells, Th1 cells）比例升高，而在鳞癌阶段分泌IFN-γ的CD8⁺ T细胞，即Tc1细胞（Type 1 cytotoxic T cells, Tc1 cells）明显降低，证明在OSCC肿瘤微环境中形成了明显的免疫抑制微环境。为了排除4-NQO直接诱导Treg细胞的可能性，我们在体外培养CD4⁺ naive T细胞并诱导其向Treg细胞分化，并在培养条件中添加4-NQO，结果发现，4-NQO并不能直接诱导或促进Treg细胞分化。不仅如此，我们在OSCC原位移植瘤模型中同样发现Treg细胞的比例升高，并且，在引流淋巴结中Tc1细胞的比例明显降低。以上结果证明，在OSCC发生发展进程中，免疫抑制微环境形成伴随Treg细胞比例的逐渐升高，同时，在鳞癌免疫微环境中具有抗肿瘤功能的Tc1细胞的比例明显降低。

为了研究Treg细胞在OSCC癌前到癌的进展中发挥的作用，我们使用anti-CD25删除抗体对原位接种4MOSC1的荷瘤小鼠进行治疗。结果发现，在使用抗体清除Treg细胞后，可以明显降低肿瘤的体积和重量，同时，在引流淋巴结和肿瘤组织中CD8⁺ T细胞的比例和增殖能力明显升高，分泌IFN-γ的 Tc1细胞的浸润比例同样明显升高。不仅如此，M2型巨噬细胞的比例降低，树突状细胞（Dendritic Cells, DCs）细胞比例升高，这些变化都证实了肿瘤的抑制性免疫微环境得到了有效改善。然而，在4-NQO原发性肿瘤模型中，使用anti-CD25抗体并不能有效降低Treg细胞的比例，显示anti-CD25抗体在该模型中不能发挥作用，这可能与Treg细胞的补体逃逸和该模型本身的性质有关，具体机制尚待深入探究。

接下来我们收集了临床样本中正常舌组织，OLK轻度增生，OLK异常增生和OSCC癌组织进行单细胞测序，发现在口腔癌前向癌进展中，Treg细胞的比例不断升高。在空间转录组数据中，同样发现Treg细胞在鳞癌的固有层中浸润增加，与小鼠模型研究的结论一致。此外，Treg细胞活化相关基因的表达也随着OSCC进展明显升高。基于上述结论，我们进一步探究了Treg细胞在不同阶段的来源。我们发现，在OSCC癌前向癌的进展中，胸腺Treg（Thymus-derived Treg, tTreg）细胞的特征性靶点HELIOS表达呈现先下降后上升的趋势。我们推测，在OLK阶段口腔黏膜病损区域中Treg细胞的增多主要通过诱导外周来源的Treg（Peripherally derived Treg, pTreg）细胞分化为主；而在OSCC阶段，则伴随大量的tTreg细胞浸润。由于pTreg主要由转化生长因子-β（Transforming growth factor beta, TGF-β）诱导，我们检测了病损微环境中不同细胞间的TGF-β信号通路交流，结果发现，在OLK阶段中主要存在TGF-β阳性的肌成纤维细胞和CD4⁺ T细胞之间的信号交流，然后利用共表达网络分析TGF-β阳性的肌成纤维细胞中的枢纽基因，筛选出PLAUR^hi 的成纤维细胞可能是分泌TGF-β的主要细胞。同样，我们也研究了OSCC阶段募集tTreg细胞的关键趋化因子，发现在OSCC发生后Treg细胞中CXCR3、CCR4和CCR7呈现高表达，而CD4⁺ 细胞毒性T淋巴细胞（Cytotoxic T Lymphocyte, CTL）和CD8⁺ CTL主要升高的趋化因子受体是CXCR4。通过对趋化因子配体的表达进行分析，我们发现，随着OSCC的进展，CXCR3的配体CXCL9/10/11主要在肌成纤维细胞、成纤维细胞和髓系细胞中逐渐上升，而成纤维细胞和髓系细胞中CXCR4的配体CXCL12明显下降。上述结果显示，在OSCC阶段，肿瘤微环境通过募集CXCR3⁺ tTreg细胞促进肿瘤免疫抑制微环境的形成。

在组学数据分析的基础上，我们通过小鼠肿瘤模型进一步验证单细胞分析的结果。在4-NQO原发肿瘤模型中，我们发现，在OLK阶段（诱导16周）Helios⁺ Treg细胞比例显著下降，而在OSCC阶段（诱导24周）Helios⁺ Treg细胞比例有所回升，与临床样本的单细胞测序分析发现的tTreg细胞比例先下降后升高的趋势一致。而在4MOSC1原位移植瘤模型中，Helios⁺ Treg与WT组相比并没有差异，即tTreg细胞所占总Treg细胞的比例在鳞癌阶段没有发生明显变化，也与临床样本的分析结果一致。同时，我们使用RT-qPCR检测OLK和OSCC阶段的病损组织，结果发现TGF-β信号通路相关基因表达升高。为了验证OLK和OSCC阶段成纤维细胞中TGF-β的表达水平，我们通过RT-qPCR检测发现，随着口腔鳞癌进展，TGF-β的mRNA表达水平逐渐升高。不仅如此，在将OLK病损组织中的成纤维细胞与CD4⁺ naive T细胞共培养后，成纤维细胞以TGF-β依赖途径诱导了更多的Treg细胞的分化。此外，我们发现，分离的原代成纤维细胞中Plaur的mRNA表达水平明显升高，而且在人类成纤维细胞基因相关性分析中，Plaur与TGF-β1表达呈现正相关趋势。

最后，我们通过RT-qPCT分析了4MOSC1原位移植瘤模型的肿瘤组织，发现OSCC阶段Cxcl9/10/11-Cxcr3信号轴的表达显著增强，与单细胞测序数据提示的结果一致。基于上述结果，我们使用Cxcr3小分子抑制剂AMG487处理4MOSC1模型，发现AMG487可以有效抑制肿瘤生长体积和重量。对小鼠进行免疫学分析发现，AMG487处理显著抑制了小鼠颈部引流淋巴结和肿瘤组织中Treg细胞的比例；同时，增加了CD4⁺ 和CD8⁺ T细胞中IFN-γ 和TNF-α的分泌。以上结果证实，瘤内抑制Cxcr3可以明显抑制OSCC的肿瘤体积和Treg细胞的浸润比例，同时有效促进T细胞的抗肿瘤免疫反应。

研究结论

本课题首先解析了OSCC发生发展过程中Treg细胞增多的原因，发现在OLK阶段增加的Treg细胞是以TGF-β诱导的pTreg细胞为主，在OSCC阶段则伴随着大量tTreg细胞浸润。然后，通过机制研究，发现表达Plaur的成纤维细胞通过增强TGF-β信号通路，促进了癌前病变中pTreg细胞的诱导分化。最后，本课题发现并验证了在OSCC中，肿瘤微环境中的肌成纤维细胞、成纤维细胞和髓系细胞通过Cxcl9/10/11-Cxcr3信号轴招募了tTreg细胞向肿瘤组织的浸润，加剧了肿瘤免疫抑制微环境的形成，靶向该信号轴能有效抑制tTreg细胞的浸润并增强OSCC的抗肿瘤免疫反应。本研究阐明了Treg细胞在口腔癌前病损到口腔鳞癌进展过程中的变化特征、主要来源和调控机制，并证实靶向Plaur⁺ 成纤维细胞和Cxcr3⁺ Treg细胞作为OSCC的新型免疫治疗策略的潜在应用价值。

Abstract

Background

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the oral and maxillofacial region, characterized by high incidence, low survival rates, and a tendency to metastasize and recur. Most OSCC cases arise from oral potentially malignant disorders (OPMDs), such as oral leukoplakia (OLK), erythroplakia, and submucous fibrosis, which have cancerous potential. The onset of OSCC typically starts with oral mucosal injury and/or chronic inflammation induced by genetic factors, psychological factors, and local irritants, leading to the development and progression of OPMDs such as OLK, which then eventually progresses to precancerous lesions and OSCC.

Current viewpoints suggest that the immune microenvironment in OLK is in a state of low-level chronic inflammation, whereas, following progression to OSCC, it transitions into an immunosuppressive tumor immune microenvironment. However, the immune regulatory mechanisms involved in the malignant transformation from OLK to OSCC remain unclear. Regulatory T cells (Treg cells) play a crucial role in this process by secreting inhibitory cytokines such as interleukin-10 (IL-10) and granzyme, directly killing effector T cells or antigen-presenting cells (APCs), or by expressing CD25 to deplete interleukin-2 (IL-2), or through immune checkpoint molecules that negatively regulate the maturation and function of APCs. During the development from precancerous lesions to OSCC, an increased number of Treg cells has been observed in the disease microenvironment, but the regulatory mechanisms, clinical significance, and therapeutic targeting potential of Treg cell accumulation remain unclear. Some studies suggest that Treg cell accumulation can improve clinical prognosis, while others suggest it may reduce patient survival rates. Moreover, inducing Treg cell depletion in the tumor microenvironment to enhance antitumor immune responses has become a hot research strategy in cancer immunotherapy, and promising experimental therapeutic effects have been observed in some tumors. However, several studies have found that simple induction of total Treg cell depletion often yields suboptimal treatment outcomes and may even accelerate tumor progression.

Based on the above research background and current situation, this study proposes the following hypotheses:

(1) During the progression from OPMD to OSCC, Treg cells continuously accumulate, regulating the immunosuppressive microenvironment, mediating the malignant transformation of precancerous lesions, and facilitating immune escape of tumor cells.

(2) Treg cells may have different sources and recruitment mechanisms at various stages of OPMD malignant transformation to OSCC and may exert different regulatory roles.

(3) Targeting Treg cells from different sources at different stages of OSCC can improve the immunosuppressive microenvironment, reducing the risk of malignant transformation of OPMD and inhibiting OSCC tumor progression.

In conclusion, studying the dynamic changes in Treg cell sources and functions during the occurrence and progression of OSCC, revealing the underlying mechanisms, and exploring their targeting potential are crucial issues for developing targeted therapies for OSCC.

Objective

This study aims to explore the dynamic changes in Treg cells during the development of OSCC, revealing the sources of Treg cells at different stages of oral lesions, and to investigate therapeutic strategies targeting Treg cells infiltrating tumor tissues or lesions at various stages. It also evaluates the value of targeted therapies at different stages, providing a theoretical basis for the prevention and treatment of precancerous lesions and the development of new immunotherapies for OSCC.

Methods

The study utilizes an OSCC primary induction model with 4-NQO (4-nitroquinoline-1-oxide) in drinking water and a subcutaneous transplantation tumor model with the 4MOSC1 cell line. The analysis combines clinical single-cell transcriptome sequencing and spatial transcriptome data from normal tongue tissue, OLK mild hyperplasia, OLK atypical hyperplasia, and OSCC cancer tissues. The main experimental approaches include multi-color flow cytometry staining, antibody or small molecule inhibitor treatment, and fibroblast-T cell co-culture in vitro, to explore the immune microenvironment changes in OPMD and OSCC. The study will reveal the characteristics, sources, and regulatory mechanisms of Treg cells in the progression from oral precancerous lesions to oral squamous carcinoma.

Results

The histopathology of premalignant lesions and cancer progression induced by 4-NQO is very similar to the occurrence, development, and histopathology of HPV-negative OSCC. In this primary induction model, we observed that immune-inflammatory cells continuously break through the basal layer, and during the OSCC stage, a large number of atypical cells are produced. As the disease progresses from premalignant to malignant, the infiltration of Treg cells in the spleen, cervical lymph nodes, and tongue tissue gradually increases. Additionally, we found that the proportion of IFN-γ-secreting CD4⁺ T cells, i.e., Th1 cells (Type 1 helper T cells), increased in the cervical lymph nodes and tongue tissues, while the proportion of IFN-γ-secreting CD8⁺ T cells, i.e., Tc1 cells (Type 1 cytotoxic T cells), significantly decreased at the squamous carcinoma stage, indicating the formation of a distinct immunosuppressive microenvironment in the OSCC tumor microenvironment.

To exclude the possibility of 4-NQO directly inducing Treg cells, we cultured CD4⁺ naive T cells in vitro and induced their differentiation into Treg cells in the presence of 4-NQO. The results showed that 4-NQO did not directly induce or promote the differentiation of Treg cells. Furthermore, we observed that the proportion of Treg cells increased in the OSCC subcutaneous transplantation tumor model, and the proportion of Tc1 cells in the draining lymph nodes significantly decreased. These findings suggest that during OSCC development, the immunosuppressive microenvironment is formed alongside a gradual increase in Treg cells, while the proportion of Tc1 cells with antitumor functions is significantly reduced in the squamous carcinoma immune microenvironment.

To investigate the role of Treg cells in the progression from precancerous to cancerous stages in OSCC, we treated tumor-bearing mice with anti-CD25 deletion antibodies after subcutaneous injection of 4MOSC1 cells. The results showed that after Treg cells were cleared by the antibody, the tumor volume and weight were significantly reduced, and the proportion and proliferation ability of CD8⁺ T cells in the draining lymph nodes and tumor tissues significantly increased, with an increased infiltration of IFN-γ-secreting Tc1 cells. Moreover, the proportion of M2 macrophages decreased, while the proportion of dendritic cells (DCs) increased, confirming that the inhibitory immune microenvironment in the tumor was effectively improved. However, in the 4-NQO primary tumor model, the use of anti-CD25 antibodies did not effectively reduce the proportion of Treg cells, suggesting that the anti-CD25 antibody does not function in this model. This may be related to complement escape by Treg cells and the intrinsic nature of the model, and the exact mechanism remains to be further explored.

Next, we collected clinical samples from normal tongue tissue, OLK mild hyperplasia, OLK dysplasia, and OSCC cancer tissues for single-cell sequencing. We found that the proportion of Treg cells increased progressively during the transition from oral precancerous lesions to cancer. In spatial transcriptomic data, we also observed that Treg cell infiltration increased in the dermal layer of the squamous carcinoma, consistent with the conclusions from the mouse model study. Furthermore, the expression of Treg cell activation-related genes was also significantly elevated as OSCC progressed. Based on these findings, we further explored the origin of Treg cells at different stages. We discovered that during the progression from precancerous to cancerous stages in OSCC, the characteristic marker HELIOS expression of thymus-derived Treg (tTreg) cells showed a trend of first decreasing and then increasing. We hypothesize that in the OLK stage, the increase in Treg cells in the oral mucosal lesions is mainly driven by the differentiation of peripherally derived Treg (pTreg) cells, while in the OSCC stage, there is an influx of tTreg cells. Since pTreg cells are primarily induced by transforming growth factor-beta (TGF-β), we examined the TGF-β signaling communication between different cells in the lesion microenvironment. The results revealed that during the OLK stage, the primary signal exchange occurred between TGF-β-positive myofibroblasts and CD4⁺ T cells. By using co-expression network analysis of hub genes in TGF-β-positive myofibroblasts, we identified that PLAURhi fibroblasts might be the major cells secreting TGF-β. Similarly, we studied the key chemokines that recruit tTreg cells in the OSCC stage, finding that after OSCC onset, Treg cells expressed high levels of CXCR3, CCR4, and CCR7, while the major chemokine receptors elevated in CD4⁺ cytotoxic T lymphocytes (CTLs) and CD8+ CTLs were CXCR4. Through chemokine ligand expression analysis, we found that as OSCC progressed, the ligands for CXCR3, namely CXCL9/10/11, gradually increased in myofibroblasts, fibroblasts, and myeloid cells, while the ligand for CXCR4, CXCL12, significantly decreased in fibroblasts and myeloid cells. These results show that in the OSCC stage, the tumor microenvironment promotes the formation of an immunosuppressive microenvironment by recruiting CXCR3⁺ tTreg cells.

Based on the genomic data analysis, we further verified the single-cell analysis results using a mouse tumor model. In the 4-NQO primary tumor model, we found that during the OLK stage (16 weeks of induction), the proportion of Helios⁺ Treg cells significantly decreased, while in the OSCC stage (24 weeks of induction), the proportion of Helios⁺ Treg cells increased, which was consistent with the trend observed in the clinical sample single-cell sequencing analysis, where the proportion of tTreg cells first decreased and then increased. In the 4MOSC1 subcutaneous transplantation tumor model, there was no difference in the proportion of Helios⁺ Treg cells compared to the WT group, meaning that the proportion of tTreg cells did not change significantly during the squamous carcinoma stage, which was also consistent with the clinical sample analysis results. Meanwhile, we used RT-qPCR to detect the expression of TGF-β signaling pathway-related genes in OLK and OSCC stage lesion tissues, revealing elevated gene expression levels. To verify the expression of TGF-β in fibroblasts during the OLK and OSCC stages, we conducted RT-qPCR and found that TGF-β mRNA expression gradually increased as oral squamous carcinoma progressed. Furthermore, when fibroblasts from OLK lesion tissues were co-cultured with CD4⁺ naive T cells, the fibroblasts induced more Treg cell differentiation via a TGF-β-dependent pathway. Additionally, we found that the mRNA expression of Plaur in isolated primary fibroblasts was significantly elevated, and in human fibroblast gene correlation analysis, Plaur expression showed a positive correlation with TGF-β1 expression.

Finally, we analyzed the tumor tissues from the 4MOSC1 subcutaneous transplantation tumor model using RT-qPCR and found a significant increase in the expression of the Cxcl9/10/11-Cxcr3 signaling axis during the OSCC stage, consistent with the results suggested by the single-cell sequencing data. Based on these results, we treated the 4MOSC1 model with the small molecule CXCR3 inhibitor AMG487 and found that AMG487 could effectively inhibit tumor growth volume and weight. Immunological analysis of the mice revealed that AMG487 treatment significantly reduced the proportion of Treg cells in the draining lymph nodes and tumor tissues, while increasing the secretion of IFN-γ and TNF-α in CD4⁺ and CD8⁺ T cells. These results confirm that inhibiting Cxcr3 in the tumor microenvironment can significantly reduce tumor volume and Treg cell infiltration in OSCC while effectively promoting antitumor immune responses in T cells.

Conclusion

This study first investigates the reasons behind the increased number of Treg cells during the development of OSCC and finds that, in the OLK stage, the increased Treg cells are predominantly pTreg cells induced by TGF-β, while in the OSCC stage, there is a significant infiltration of tTreg cells. Subsequently, through mechanistic research, it was discovered that fibroblasts expressing Plaur enhance the TGF-β signaling pathway, thereby promoting the induction and differentiation of pTreg cells during the precancerous lesions. Finally, this study identifies and validates that in OSCC, myofibroblasts, fibroblasts, and myeloid cells in the tumor microenvironment recruit tTreg cells to infiltrate the tumor tissue via the Cxcl9/10/11-Cxcr3 signaling axis, which exacerbates the formation of an immunosuppressive tumor microenvironment. Targeting this signaling axis effectively inhibits the infiltration of tTreg cells and enhances the antitumor immune response in OSCC. This research elucidates the changing characteristics, primary sources, and regulatory mechanisms of Treg cells in the progression from oral precancerous lesions to oral squamous carcinoma, and confirms the potential therapeutic value of targeting Plaur⁺ fibroblasts and Cxcr3⁺ Treg cells as a novel immunotherapeutic strategy for OSCC.