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. 2019 Nov 8;199(2):172–181. doi: 10.1111/cei.13389

Oral cancer‐associated tertiary lymphoid structures: gene expression profile and prognostic value

K Li 1,2,3, Q Guo 4, X Zhang 5, X Dong 5, W Liu 6, A Zhang 6, Y Li 5, J Yan 5, G Jia 5, Z Zheng 5, W Tang 6, L Pan 6, M An 7, B Zhang 2,3, S Liu 1,✉, B Fu 6,✉
PMCID: PMC6954676  PMID: 31652350

Summary

Tertiary lymphoid structure (TLS) provides a local and critical microenvironment for both cellular and humoral immunity and supports effective antigen presentation and lymphocyte activation. However, the gene expression profile and prognostic significance of TLS in oral cancer remain largely unrevealed. In this study, we found the presence of both intratumoral and peritumoral TLSs in a series of 65 patients with oral cancer treated by surgical resection, with positive detection rates of 33.8 and 75.4%, respectively. The presence of intratumoral TLSs, but not peritumoral TLSs, was significantly associated with decreased P53 and Ki67 scores (P = 0·027 and 0·047, respectively). The survival analyses revealed that oral cancer patients with higher grades of TLSs was associated with improved disease‐free survival (DFS) and overall survival (OS) (P = 0·037 and 0·031, respectively). Gene expression profiling analysis of the cytokines and chemokines responsible for lymph‐node neogenesis identified a three‐up‐regulated‐gene set, i.e. IL7, LTB and CXCL13, which was shown to be correlated with human oral cancer‐associated TLSs. This study provides a framework for better understanding of oral cancer‐associated TLSs and for delineating future innovative prognostic biomarkers and immune therapeutic strategies for oral cancer.

Keywords: oral cancer, prognostic biomarker, tertiary lymphoid structure, therapeutic target

Tertiary lymphoid structure (TLS) provides a local and critical microenvironment for both cellular and humoral immunity and supports effective antigen presentation and lymphocyte activation. Here, we found that oral cancer patients with higher grade TLSs signified improved disease‐free survival (DFS) and overall survival (OS). Gene expression profiling analysis of the cytokines and chemokines responsible for lymph‐node neogenesis identified a 3‐upregulated‐gene set, i.e. IL7, LTB and CXCL13, which was indicated correlated with oral cancer‐associated TLSs.

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Introduction

Lymphoid organs and lymphoid cells are the basis for immune responses 1. Lymphoid organs are generally classified into primary and secondary lymphoid organs (SLOs). Primary lymphoid organs, such as thymus and bone marrow, are responsible for producing lymphocytes from immature progenitors 2. In the periphery, SLOs such as lymph nodes and spleen provide sites for B and T cell priming, clonal expansion, somatic hypermutation, affinity maturation and immunoglobulin class‐switching 3, 4. Similar to SLOs’ stereoscopic structure, ectopic or tertiary lymphoid structures (TLSs) are often defined as an accumulation of lymphoid and stromal cells that form at ectopic sites under pathological conditions, including autoimmune disease, infection, graft rejection and cancer 5.

TLSs provide a local and critical microenvironment for generating anti‐tumor cellular and humoral immune responses. In human cancers, spontaneous TLSs have been detected in various tumor types, such as hepatocellular carcinoma 6, bladder cancer 7, colorectal cancer 8, melanoma 9 and breast cancer 10. Mounting evidence indicates that TLSs are bases for T cell priming, B cell activation and antibody production by forming a favorable immune microenvironment to control tumor development in most solid tumors 11.

Oral cancer is one of the most common cancers and generally diagnosed at advanced stages 12. It could arise at multiple sites in the oral cavity, including the lip, oral floor, tongue, gingiva, palate and buccal mucosa. Despite advances in clinical diagnosis and management of oral cancer, the morbidity and mortality of oral squamous cell carcinomas (OSCC) patients has remained high during the last decades, with an overall 5‐year survival rate of approximately 50% 13. The unavailability of reliable prognostic markers and therapeutic strategies have been a bottleneck in oral cancer diagnosis and treatment. Growing evidence supports the importance of the immune microenvironment in cancer development 14. Tumor microenvironment and immune surveillance status represent crucial prognostic hallmarks of oral cancer. The manipulation of TLSs neogenesis and maintenance may lead to long‐lasting adaptive anti‐tumor responses, which may represent an efficient therapeutic strategy for oral cancer.

However, until now, oral cancer‐associated TLSs have been largely unknown. Only one study has reported that the presence of TLSs in OSCC was correlated with decreased disease‐specific mortality 15. The prognostic value of TLSs for oral cancer remains to be further explored. In addition, the gene expression profile of oral cancer‐associated TLSs is still unclear. Identification of the key parameters in TLSs is essential to effectuate the TLSs‐based therapeutic strategies. In the present study, we inquired into the presence of intratumoral and peritumoral TLSs in oral cancer, investigated the association between intratumoral and peritumoral TLSs and clinicopathological characteristics of the enrolled patients and further evaluated the prognostic value of TLSs for oral cancer. We also identified and verified the differentially expressed genes between TLSs‐positive and TLSs‐negative oral cancer tissues. The results of this study lay the foundation for future exploration of innovative prognostic markers and immune therapeutic strategies for oral cancer.

Materials and methods

Ethics, consent and permissions

The study was approved by the Ethics Committee of Liaocheng People’s Hospital. All procedures involving human participants followed the ethical standards of the institutional and/or national research committee. As a retrospective study, an exemption of informed consent from the subjects was granted.

Patients and samples

This study included 65 subjects who were pathologically diagnosed as oral cancer and treated in the Department of Oral and Maxillofacial Surgery at Liaocheng People’s Hospital between January 2011 and December 2016. The patients’ inclusive criteria were as follows: (1) the primary tumor was located in the lip, oral floor, tongue, gingiva, palate or buccal mucosa; (2) they had been diagnosed as oral cancer and undergone curative surgery; and (3) they showed no evidence of distant metastasis. The paraffin‐embedded tissue specimens from the subjects were collected. The demographic and clinicopathological characteristics of the enrolled subjects were extracted from the electronic medical records. The tumor–node–metastasis (TNM) staging of oral cancer patients was determined according to the 7th edition of the American Joint Committee on Cancer (AJCC) cancer staging manual 16. Follow‐up information, including disease‐free survival (DFS) and overall survival (OS), were collected. DFS was calculated as the time from surgery to the first present of local recurrence or metastasis. OS was calculated as the time from surgery to death. Recurrences or metastases were determined based on diagnostic tests [X‐ray, computed tomography (CT), magnetic resonance imaging (MRI) and/or positron emission tomography (PET) scan] and confirmed by tissue pathology when available. Patients who died from causes other than oral cancer were defined as survivors.

Immunohistochemistry (IHC) staining

The formalin‐fixed paraffin‐embedded (FFPE) oral cancer tissue specimens were cut as serial 5‐µm sections. The sections were deparaffinized in xylene and hydrated through an ethanol series. The sections were then boiled for 5 min in citrate buffer (pH 6.0) and left for self‐cooling to retrieve epitopes. After quenching endogenous peroxidase with 3% hydrogen peroxide for 15 min, non‐specific binding was blocked by incubating the sections in 10% horse serum, followed by incubation with primary antibodies at 4°C overnight. The primary antibodies are listed in Supporting information, Table S1. The sections were then rinsed and incubated with appropriate biotinylated secondary antibodies for 30 min at 37°C, then rinsed again and incubated with peroxidase‐conjugated streptavidin for 30 min at 37°C. Finally, the sections were reacted with diaminobenzidine (DAB) solution for 5 min, followed by counterstaining with hematoxylin. The sections were viewed and photographed with a light microscope (Nikon, Tokyo, Japan).

Hematoxylin and eosin (H&E) staining

The paraffin‐embedded human oral cancer tissue specimens were cut into 5‐µm serial sections. The sections were stained with Harris modified hematoxylin and rinsed in running tapwater, and then restained with eosin Y, dehydrated, cleared, slide‐mounted and visualized by a light microscope (Nikon).

TLSs classification

TLS was assessed on H&E sections, as previously described 6, 17. On average, four H&E sections from different tumor blocks of each patient were analyzed. In the H&E sections, dense lymphocytic clusters were defined as TLSs. Based on the H&E section with the highest TLS density, oral cancer‐associated TLSs were classified into five categories (Fig. 1b), according to the locations and counts of TLSs, as previously described 18. The classified criteria are listed in Supporting information, Table S2: grade 0, no TLSs; grade 1, peritumoral TLS count ranged from 1 to 4 and no intratumoral TLSs; grade 2, peritumoral TLS count > 4 and no intratumoral TLSs; grade 3, intratumoral TLS count ranged from 1 to 4; grade 4, intratumoral TLS count > 4. No criteria were assigned to peritumoral TLSs in grades 3 and 4, although some specimens in grades 3 or 4 had peritumoral TLSs. All evaluations and classifications of TLSs were carried out by two pathologists who were blinded to each other and were not provided with any clinical information of the patients.

Figure 1.

Figure 1

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Tertiary lymphoid structures (TLSs) in human oral cancer tissues. (a) Hematoxylin and eosin (H&E) staining. The images show representative peritumoral and intratumoral TLSs. Arrowheads indicate TLSs. (b) TLS grading. Based on the location and count, TLS was classified into five categories (grades 0–4). (c) Positive rate of oral cancer‐associated TLSs with various grades (grades 0–4). (d) Immunohistochemical (IHC) staining of CD3 and CD20. The images show representative IHC staining on consecutive sections of the same oral cancer tissue sample for detection of T cells (CD3) and B cells (CD20) in oral cancer‐associated TLSs. (a,d) Images show the corresponding areas from white rectangles of the bottom left corner images. Magnification: main images ×400; bottom left corner images ×100.

RNA extraction, reverse transcription–real‐time quantitative polymerase chain reaction (RT–qPCR)

The total RNA was extracted using RNeasy FFPE Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Briefly, the tissue sections were deparaffinized using deparaffinization solution, followed by protease digestion, binding to a solid substrate, washing and elution. The cDNA was synthesized using PrimeScript RT Master Mix (Takara, Dalian, China). Gene expression levels were detected by RT–qPCR using TG Green Premix Ex Taq II kit (Takara) with a 7500 RT–PCR system (Applied Biosystems, Foster City, CA, USA), as previously described 19. The primer sequences are listed in Supporting information, Table S3. PCR amplifications were performed in triplicate with 2 μl cDNA, 10 μl 2 × master mix, 0·8 μl forward primer (10 μM), 0·8 μl reverse primer (10 μM), 0·4 μl ROX II and 6 μl ddH2O. The PCR program was set as follows: activation at 95°C for 30 s; 40 cycles at 95°C for 5 s and at 60°C for 34. Anti‐beta actin (ACTB) (β‐actin) served as an internal reference gene.

Statistical analysis

All statistical analyses were performed using spss version 18 software (SPSS Inc., Chicago, IL, USA). Relative gene expression was quantized as mean ± standard error of the mean (s.e.m.) and analyzed using the t‐test. For statistical analysis of gene expression, correction for multiple testing was performed using Benjamini–Hochberg false discovery rate procedure 20. Pearson’s χ2 test or Fisher’s exact test (when appropriate) was applied to evaluate the correlation between clinicopathological features and TLS status. Survival analysis was conducted using the Kaplan–Meier method. Univariate analysis was performed to evaluate the prognostic significance of TLSs using the log‐rank test. P < 0·05 was considered statistically significant.

Results

TLSs in human oral cancer tissue specimens

We first performed H&E staining to assess the presence of TLSs in human oral cancer specimens. As shown in Fig. 1a, TLSs were observed within the tumor tissue (defined as intratumoral TLSs), around the tumor tissue or in the peritumoral area (defined as peritumoral TLSs). In 65 human oral cancer specimens, intratumoral TLSs were found in 33·8% (22 of 65) specimens, and peritumoral TLSs were observed in 75·4% (49 of 65) specimens (Table 1). No significant correlation was found between the intratumoral and peritumoral TLS counts (P = 0·963; Supporting information, Fig. S1). We further classified oral cancer‐associated TLSs into five categories based on their locations and counts (Fig. 1b and Supporting information, Table S2): 15·4% (10 of 65) of tumors were classified as grade 0, 38·5% (25 of 65) as grade 1, 12·3% (eight of 65) as grade 2, 27·7% (18 of 65) as grade 3 and 6·2% (four of 65) as grade 4 (Fig. 1c).

Table 1.

Correlations between the presence of intratumoral and peritumoral tertiary lymphoid structures (TLSs) and clinicopathological characteristics of the enrolled oral cancer patients

Characteristic N Intratumoral TLSs+ P‐value Peritumoral TLSs+ P‐value
n % n %
All cases 65 22 33·8   49 75·4  
Gender              
Male 44 14 31·8 0·617 35 79·5 0·260
Female 21 8 38·1   14 66·7  
Age (years)              
≤ 60 27 9 33·3 0·941 19 51·9 0·429
> 60 38 13 34·2   30 92·1  
Tumor size (cm)              
< 3 47 15 31·9 0·181 37 78·7 0·696
≥ 3 11 6 54·5   8 72·7  
Unknown 7 1 14·3   4 57·1  
Tumor location              
Lip 13 3 23·1 0·301 9 69·2 0·551
Oral floor 8 4 50·0   4 50·0  
Tongue 23 9 39·1   16 69·6  
Gingival 14 4 28·6   7 50·0  
Others 7 2 28·6   0 0·0  
Histological type              
Squamous 54 17 31·5 0·124 41 75·9 0·367
Adenocarcinoma 4 3 75·0   2 50·0  
Others 4 2 50·0   3 75·0  
Unknown 3 0 0·0   2 66·7  
Histological grade              
Low 9 4 44·4 0·345 8 88·9 0·120
Middle 9 3 33·3   4 44·4  
High 21 4 19·0   15 71·4  
Unknown 26 10 38·5   22 84·6  
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A previous report indicated that there were B and T cells in both peritumoral and intratumoral TLSs 18. As shown in Fig. 1d, positive expression of CD3 (T cells) and CD20 (B cells) was observed in both peritumoral and intratumoral TLSs, with a positive rate of 100%. This finding was consistent with the H&E data (Fig. 1a) regarding the presence of oral cancer‐associated TLSs. The positive rates of TLS, organized by a dense central B cell follicle with peripheral T cells in peritumoral and intratumoral TLSs, were 57·1% (28 of 49) and 63·6% (14 of 22), respectively.

Previous studies observed the accumulations of follicular dendritic cells (FDCs) in TLSs with two patterns: distinct meshwork or diffuse distribution 15. We performed immunohistochemistry staining of CD21 to detect FDCs in oral cancer‐associated TLSs (Fig. 2a). The proportion of peritumoral and intratumoral TLSs with continuous FDC meshwork was 42·9% (21 of 49) and 54·5% (12 of 22), respectively. The proportion of TLSs with continuous FDC meshwork was significantly higher in TLS grades 3 and 4 compared with those in TLS grades 1 and 2 (68·2 versus 36·4%, P = 0·021; Fig. 2b).

Figure 2.

Figure 2

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Follicular dendritic cells (FDCs) in oral cancer‐associated TLSs. (a) IHC staining of CD21 for detection of FDCs. Images show the corresponding areas from white rectangles of the bottom left corner images. Magnification: main images ×400; bottom left corner images ×100. (b) Correlation between the proportion of TLSs with continuous FDC meshwork and the TLS grades.

Correlations between TLSs and clinicopathological characteristics

Having determined the presence of TLSs in oral cancer, we further sought to explore the correlations between TLSs and clinicopathological characteristics. As shown in Table 1, no correlations were found between the presence of intratumoral or peritumoral TLSs and gender, age, tumor size, tumor location, histological type and histological grade of the oral cancer subjects (P > 0·05). Additionally, no significant associations between the total TLS (intratumoral and peritumoral) counts and the clinicopathological characteristics were observed (Supporting information, Table S4). We next attempted to investigate the potential associations between TLSs and common molecular classifications of oral cancer. As shown in Fig. 3a, oral cancer patients with lower P53 scores (≤ 50%) showed a significantly higher positive rate of intratumoral TLSs than those with higher P53 scores (> 50%) (46.2 versus 21·1%, P = 0·027). The positive rate of intratumoral TLSs was higher in patients with lower Ki67 scores (≤ 50%) than those with higher Ki67 scores (> 50%) (48.4 versus 17·6%, P = 0·047; Fig. 3b). However, no significant associations were found between peritumoral TLSs and P53 or Ki67 scores (P > 0·05; Fig. 3a,b). Moreover, the follicular dendritic cell (FDC)‐positive TLS counts (P = 0·038 and P = 0·044, respectively; Supporting information, Fig. S2a, S2b) rather than the total TLS counts (P = 0·122 and P = 0·329, respectively; Fig. 3c,d) were correlated with P53 and Ki67 scores.

Figure 3.

Figure 3

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Correlations between the positive rates of intratumoral and peritumoral TLSs (a,b) or the total TLS counts (c,d) and P53 or Ki67 scores.

TLSs signify a favorable prognosis in human oral cancer

To assess the relationship between the presence of TLSs and prognosis of human oral cancer, we collected the follow‐up data of the involved patients. Among the 65 patients, three (4·6%) were excluded due to lack of follow‐up data. The median follow‐up time was 44 months (range = 1–83 months). During the follow‐up period, 38·7% (24 of 62) patients relapsed and 27·4% (17 of 62) died of oral cancer. Kaplan–Meier survival analysis showed that oral cancer patients with higher‐grade TLSs had longer DFS and OS (P = 0·037 and 0·031, respectively; Fig. 4a,b). We then combined the oral cancer patients into the following three categories and compared DFS and OS among them: patients having no TLSs, patients having only peritumoral TLSs (grades 1 and 2) and patients having intratumoral TLSs (grades 3 and 4). The results demonstrated a correlation between the presence of TLSs and longer DFS (P = 0·009; Fig. 4c). The DFS periods of patients having no TLSs, peritumoral TLSs and intratumoral TLSs were 36·3 ± 8·7 months, 52·3 ± 4·8 months and 65·6 ± 4·9 months, respectively. Moreover, we found the presence of TLSs was associated with improved OS (P = 0·006; Fig. 4d). The OS periods for patients having no TLSs, peritumoral TLSs and intratumoral TLSs were 45·9 ± 12·4 months, 59·2 ± 3·9 months and 76·1 ± 3·1 months, respectively. We further analyzed the correlation between the total TLS counts and survival of oral cancer patients. As shown in Supporting information, Fig. S3a, S3b, the total TLS counts and FDC‐positive TLS counts were relevant for survival (P = 0·011 and 0·034, respectively). Taken together, these results suggested that the presence of TLSs signified a favorable prognosis for oral cancer.

Figure 4.

Figure 4

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Prognostic value of TLSs in human oral cancer. Kaplan–Meier analysis of (a,c) disease‐free survival (DFS) and (b,d) overall survival (OS) for 65 oral cancer patients with various TLS grades. ‘Censored’ indicates that the participants did not experience recurrence or death before the end of the study. The P‐value shown in each figure was calculated using the log‐rank test and indicates the statistically significant among the survival curves. The log‐rank P‐values between different groups were as follows: (a) grades 1 versus 0, P = 0·161; grades 2 versus 0, P = 0·165; grades 3 versus 0, P = 0·016; grades 4 versus 0, P = 0·032. (b) Grades 1 versus 0, P = 0·424; grades 2 versus 0, P = 0·274; grades 3 versus 0, P = 0·022; grades 4 versus 0, P = 0·047. (c) Grades 1+2 versus 0, P = 0·088; grades 3+4 versus 0, P = 0·003. (d) Grades 1+2 versus 0, P = 0·344; grades 3+4 versus 0, P = 0·002.

Gene expression profiling of the cytokines in oral cancer‐associated TLSs

Previous studies have identified gene expression profiling of TLSs in various tumor types 21, 22, but in oral cancer it was still veiled. To delineate the inflammatory network in TLSs, we compared the relative gene expression levels between oral cancer tissues with and without TLSs using RT–qPCR. As shown in Fig. 5a and Supporting information, Fig. S4, the results revealed that in TLS‐positive oral cancer tissues, the expression of only a few mRNA encoding inflammatory‐related genes, including IL‐7 (P = 0·034), LTB (P = 0·034) and CXCL13 (P = 0·043), were significantly up‐regulated compared with TLS‐negative tissues. No significant alternations were found in other genes encoding inflammatory cytokines and chemokines responsible for lymph‐node neogenesis, including IL1B, IL6, TNF, IFNG, CXCL1, CXCL9, CXCL10, CXCL11, CCL2, CCL3, CCL5, CCL19 and CCL21 (P > 0·05; Fig. 5a and Supporting information, Fig. S4). We further evaluated the protein expression levels of the three up‐regulated molecules [IL‐7, lymphotoxin beta (LTβ) and chemokine (C‐X‐C motif) ligand 13] in human oral cancer‐associated TLSs. As shown in Fig. 5b, immunohistochemistry staining revealed that IL‐7, LTβ and CXCL13 were expressed more intensively in TLSs‐positive oral cancer tissues compared with TLS‐negative tissues. The results were consistent with the gene expression data.

Figure 5.

Figure 5

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Gene and protein expression levels of the inflammatory cytokines and chemokines responsible for lymph‐node neogenesis in TLSs‐positive oral cancer tissues. (a) The relative gene expression levels in TLS‐positive tissues versus the TLS negative tissues (NC, negative control) by real‐time quantitative polymerase chain reaction (qPCR). NC, n = 8; TLSs+, n = 8. The P‐values were adjusted using the Benjamini–Hochberg false discovery rate procedure for multiple testing and show above each column. (b) The relative protein expression levels of interleukin (IL)‐7, lymphotoxin beta (LTβ) and chemokine (C‐X‐C motif) ligand 13 (CXCL13). TLSs+, n = 4; TLSs–, n = 4. Images show the corresponding areas from white rectangles of the bottom left corner images. Magnification: main images ×400; bottom left corner images ×100.

Discussion

Tumor immune microenvironment is associated with clinical outcomes 23. TLSs provide a local and important microenvironment for both cellular and humoral immune and support effective antigen presentation and lymphocyte activation 11, 24. In this study, we verified the presence of intratumoral and peritumoral TLSs in human oral cancer tissues, with positive rates of 33·8 and 75·4%, respectively. The presence of intratumoral TLSs, but not peritumoral TLSs, was associated with P53 and Ki67 scores. The survival analyses revealed that the presence of TLSs signified longer DFS and OS, suggesting a favorable prognosis for oral cancer. Gene expression profiling analysis of the cytokines and chemokines related to lymph‐node neogenesis identified a three‐up‐regulated gene set, including IL‐7, LTB and CXCL13, for human oral cancer‐associated TLSs. These findings provide new insights into the development of innovative prognostic markers and immune therapeutic strategies for oral cancer.

Mounting evidence reveals that the presence of TLSs is usually correlated with a better prognosis in cancer patients 6, 10, 25, 26, 27, although there are exceptions 28. For oral cancer, our data showed that TLSs also appeared to be a favorable prognostic indicator, both for tumor recurrence and patient survival. The results are consistent with the previous reports of Wirsing et al. 15. We found that the presence of intratumoral TLSs was associated with lower P53 and Ki67 scores in human oral cancer tissues. It has been shown that, in most cancers, higher P53 and Ki67 scores indicate more advanced cancer 29. Thus, this result further supports that TLSs represent a favorable prognosis for oral cancer.

To our knowledge, this is the first study to investigate the gene expression profiling of lymph‐node neogenesis‐related cytokines and chemokines for oral cancer‐associated TLSs. A three‐gene set, including IL‐7, LTB and CXCL13, was found significantly up‐regulated in oral cancer‐associated TLSs, suggesting that these genes may be used to predict the presence of TLSs in oral cancer. A 12‐chemokine gene expression signature (GES), including CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21, CXCL9, CXCL10, CXCL11 and CXCL13, has demonstrated a positive association with the presence of TLSs or ectopic lymph node‐like structures in melanoma 22. Silina et al. reported that the up‐regulated expression of CXCL13, LTB, CCL21 and CXCL12 was correlated with higher TLS density in human lung squamous cell carcinoma (LSCC) 17. It appears that TLSs in different tumor types may share some lymphoid chemokines, such as CXCL13 and LTB. CXCL13 production in B cells is involved in lymphoid neogenesis via Toll‐like receptor/lymphotoxin receptor signaling 30. LTs play critical roles in lymphoid tissue organogenesis and maintenance 31. LTβ is one of the characteristic cytokines that can independently predict the presence of TLSs 32. It has been shown that CXCL13, LTβ and B cell‐activating factor (BAFF) regulate the recirculation of lymphocytes and dendritic cells homing into lymphoid structures 33. Apart from CXCL13 and LTβ, IL‐7 may exert essential roles in the development and maintenance of TLSs. It has been shown that lymphatic endothelial cell‐derived IL‐7 regulates lymphatic vessel remodeling and expansion in an autocrine manner 34, while fibroblast‐derived IL‐7 was critical for reconstruction and remodeling of the distinct lymph‐node microenvironment in a paracrine manner 35. Nayar et al. reported that IL‐7 appeared to be a key regulator in the early phases of TLSs‐associated lymphangiogenesis 36. These findings indicate IL‐7 exerts essential roles in lymph‐node organogenesis and lymphocyte development and homeostasis, suggesting that it may provide crucial signals for the development and maintenance of TLSs. Collectively, these three chemokines may profoundly affect the development and maintenance of oral cancer‐associated TLSs.

Tumor microenvironments are usually immunosuppressive and prevent effective lymphocyte priming 31, 37. Recent breakthroughs in cancer immunotherapy, such as immune checkpoint inhibitors, have shown unprecedented persistence responses in patients with various cancers 38, 39. However, the majority of patients are refractory to the current immunotherapy treatment 40. TLSs are inside or adjacent to tumor tissues and promote lymphocyte trafficking and infiltration, and have become an intriguing target for manipulating anti‐tumor immunity 11. On one hand, the induction and manipulation of cancer‐associated TLSs may open up a new pathway for tumor immunotherapy. So far, several treatments targeting TLSs have been developed and have shown promising anti‐tumor effects in various mouse models 41, 42. On the other hand, current cancer immunotherapies [i.e. anti‐ cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) and anti‐programmed death 1 (PD‐1)] might be more effective in combination with agents that induce TLSs neogenesis. For oral cancer, considering the significant correlation between the presence of TLSs and better prognosis, it has become an intriguing hypothesis that being able to induce TLSs formation might also be a potent strategy to induce anti‐tumor immunity. However, TLSs‐inducing reagents have not yet been developed. In the future, we will focus on the induction of oral cancer‐associated TLSs based on the expression levels of cytokines and chemokines combined with the usage of proper biomaterials as a scaffold to enhance the efficacy of anti‐tumor immunity.

In conclusion, we have verified the presence of intratumoral and peritumoral TLSs in human oral cancer tissues, and revealed that the presence of TLSs appeared to be a favorable prognostic indicator for oral cancer patients. For the first time, a three‐up‐regulated‐gene set, including IL‐7, LTB and CXCL13, was identified to be involved in oral cancer‐associated TLSs. This study provides a framework for better understanding of oral cancer‐associated TLSs, and for delineating future innovative prognostic biomarkers and immune therapeutic targets of clinical interest.

Disclosures

The authors declare that they have no conflicts of interest.

Author contributions

K. L., Q. G., W. L., A. Z., W. T., L. P. and M. A. performed the experiments and analyses. X. Z., X. D., Y. L., J. Y., G. J. and Z. Z. collected the samples from patients and contributed to data acquisition. B. Z., S. L. and B. F. conceived and designed the study and experiments. K. L., Q. G., A. Z. and B. F. wrote and edited the paper. All authors read and approved the final manuscript.

Supporting information

Fig. S1. Correlation between intratumoral and peritumoral TLS counts. The correlation was analyzed using Pearson’s correlation analysis. Pearson's correlation coefficient (r) is a measure of the strength of the association, where the value r 2 = 0.00003 means no linear correlation between the two variables.

Fig. S2. Correlations between the number of FDC+ TLS and (a) P53 or (b) Ki67 scores. Data are presented as mean ± standard error of mean (SEM) and analyzed using t‐test.

Fig. S3. (a)The total TLS counts and (b) FDC+ TLS counts correlate with survival of oral cancer patients. Data were analyzed using Kaplan‐Meier curve analysis. Significance was calculated using log‐rank test.

Fig. S4. qRT‐PCR analysis for transcripts of genes involved in lymphoid neogenesis in human oral cancer tissues. Relative expression (2Ct(reference gene)‐Ct(target gene)) was calculated in both TLS+ and NC (TLS‐) groups. TLSs+, n = 8; NC, n = 8. Data were statistically analyzed using the two‐tailed Wilcoxon rank‐sum test with Benjamini‐Hochberg correction for multiple testing.

Table S1. Primary antibodies

Table S2. The classified criteria of oral cancer‐associated tertiary lymphoid structures (TLSs).

Table S3. The primer sequences for detecting the candidate genes encoding human inflammatory cytokines and chemokines related to lymph‐node neogenesis.

Table S4. Correlations between the total TLS counts (intratumoural and peritumoural TLS counts) and clinicopathologic characteristics of the enrolled oral cancer patients. Comparison between groups was performed by ANOVA or two‐tailed t‐test as appropriate.

Click here for additional data file. (540.2KB, docx)

Acknowledgments

The authors are thankful for the financial support from the National Natural Science Foundation of China (no. 81472530 and 81702884), China Postdoctoral Science Foundation (no. 2017M612290) and Medicine and Health Science Technology Foundation of Shandong Province (no. 2015WS0381 and 2016WS0216).

Contributor Information

S. Liu, Email: shuwei_liu1@126.com.

B. Fu, Email: fubo.22@163.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1. Correlation between intratumoral and peritumoral TLS counts. The correlation was analyzed using Pearson’s correlation analysis. Pearson's correlation coefficient (r) is a measure of the strength of the association, where the value r 2 = 0.00003 means no linear correlation between the two variables.

Fig. S2. Correlations between the number of FDC+ TLS and (a) P53 or (b) Ki67 scores. Data are presented as mean ± standard error of mean (SEM) and analyzed using t‐test.

Fig. S3. (a)The total TLS counts and (b) FDC+ TLS counts correlate with survival of oral cancer patients. Data were analyzed using Kaplan‐Meier curve analysis. Significance was calculated using log‐rank test.

Fig. S4. qRT‐PCR analysis for transcripts of genes involved in lymphoid neogenesis in human oral cancer tissues. Relative expression (2Ct(reference gene)‐Ct(target gene)) was calculated in both TLS+ and NC (TLS‐) groups. TLSs+, n = 8; NC, n = 8. Data were statistically analyzed using the two‐tailed Wilcoxon rank‐sum test with Benjamini‐Hochberg correction for multiple testing.

Table S1. Primary antibodies

Table S2. The classified criteria of oral cancer‐associated tertiary lymphoid structures (TLSs).

Table S3. The primer sequences for detecting the candidate genes encoding human inflammatory cytokines and chemokines related to lymph‐node neogenesis.

Table S4. Correlations between the total TLS counts (intratumoural and peritumoural TLS counts) and clinicopathologic characteristics of the enrolled oral cancer patients. Comparison between groups was performed by ANOVA or two‐tailed t‐test as appropriate.

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