PLCXD2 expression relates to the immune microenvironment and prognosis of head and neck squamous cell carcinoma

doi:10.21203/rs.3.rs-4330330/v1

Objective — Despite the advances in oncology, the prognosis of head and neck squamous cell carcinoma (HNSC) patients remains dismal. In this study, we aimed to determine the relevance of PLCXD2 expression in the tumor microenvironment to the HNSC patient clinicopathological features.

Methods — Gene expression analysis and multicolor immunofluorescence histochemistry with HNSC tissuemicroarrays were conducted to examine the relation between PLCXD2 expression and patient outcomes. Additionally, Spearman correlation analysis was used to assess the relationship between PLCXD2 protein expression and tumor immune infiltrating cells (TIICs), as well as immune checkpoints (PD-1, PD-L1, and CTLA-4) in HNSC tissue, while Chi-square test and Cox proportional-hazards models were employed to validate the correlation between PLCXD2 protein levels and clinicopathological characteristics with patient survival.

Results — Our findings revealed higher PLCXD2 expression in HNSC tissue compared to control benign tissues. Additionally, we observed a distinct association between the presence of PLCXD2 protein in cancer nests and various TIICs, including CD4+ T cells, CD8+ T cells, dendritic cells, as well as CTLA-4+ cells in HNSC tissues. Furthermore, we demonstrated a correlation between PLCXD2 protein expression in cancer cells and advanced TNM stage, as well as a poorer prognosis.

Conclusion — Taken together, this study supports PLCXD2 as an independent prognostic marker and a potentially promising target for immunotherapy in HNSC.

Biological sciences/Cancer

Biological sciences/Immunology

Earth and environmental sciences/Biogeochemistry

Health sciences/Oncology

PLCXD2

head and neck squamous cell carcinoma

tumor immune infiltrating cells

CTLA-4

prognosis

Head and neck cancers rank seventh among malignant tumors worldwide [1, 2], with over 90% of these malignancies being head and neck squamous cell carcinomas (HNSC) originating from the mucosal surfaces in this region [1, 2]. While surgery, radiotherapy and chemotherapy remain the primary treatment modalities as they can enhance patient prognosis in clinical settings, advanced cases still exhibit a 50% recurrence rate with life-threatening consequences [3]. This high recurrence rate can be attributed, in part, to the complex tumor microenvironment (TME), which comprises various tumor immune infiltrating cells (TIICs) and other components alongside cancer cells and stromal cells [4]. Based on research on the TME in recent literature, emerging immune checkpoint inhibitors (ICIs) have shown promise in improving outcomes for select patients [3]. However, the limitation remains that only a minority of patients derive benefit from ICIs, necessitating the identification of more effective therapeutic targets in clinical practice [5, 6].

The Cancer Genome Atlas (TCGA) utilizes high-throughput next-genome sequencing (NGS) and bioinformatics to provide gene expression data for 33 tumor types [7], including HNSC [8]. This freely accessible data resource has significantly enhanced the capabilities of the tumor research community in diagnosing, treating, and predicting outcomes for cancer patients [7]. Additionally, the Genotype-Tissue Expression (GTEx) portal serves as a comprehensive web-based platform for researchers to investigate cell- and tissue-specific gene expression patterns [9, 10]. Using these two databases, our preliminary assessments revealed significantly elevated phosphatidylinositol-specific phospholipase C X domain containing 2 (PLCXD2) mRNA expression in HNSC tissue compared to non-tumor tissues and was associated with patient prognosis.

PLCXD2, a protein-coding gene located on chromosome 3q13.2, was found to exhibit higher mRNA expression in HNSC tissue compared to non-tumor tissues and was associated with patient prognosis. Functionally, PLCXD2 is predicted to facilitate phosphoric diester hydrolase activity and participate in lipid catabolic processes and signal transduction pathways [11]. Notably, PLCXD2 protein expression was predominantly detected in the cell nucleus of HeLa cell lines [12]. Despite these insights, PLCXD2 clinical significance in HNSC cancer tissues remains poorly understood. While many research teams have relied on gene expression data to infer protein expression changes [13], it is widely recognized that the correlation between mRNA and protein expression levels is often weak [13, 14].

In this present study, we employed multicolor immunofluorescence histochemistry (mIHC) utilizing HNSC tissue microarray (TMA) specimens and clinical data to assess PLCXD2 protein expression in the TME and examine its correlation with immune checkpoint molecules. Collectively, the results emphasize PLCXD2 as an indicator for prognosis and a promising candidate for immunotherapeutic interventions in HNSC.

2.1. Gene Expression Profiling Interactive Analysis (GEPIA)

GEPIA2 (http://gepia2.cancer-pku.cn/#index) can be used to analyze bulk gene expression profiles of tumors and benign tissues using data from the TCGA and GTEx datasets [15]. In this study, this bioinformatics tool was used to compare PLCXD2 mRNA expression levels in HNSC tissues (n = 519) with those in benign control tissues (n = 44). Additionally, we assessed the relationship of PLCXD2 mRNA expression with HNSC patients' clinical outcomes.

2.2 Clinical samples and information

TMAs comprising samples from various sites within the head and neck region, including 113 tongue cancer tissues, 86 buccal mucosa cancerous tissues, 76 larynx carcinoma tissues, 55 matched non-cancerous tissues from surgical margins and 6 squamous papillomae, and the clinical and follow-up data of these corresponding patients were obtained from the Affiliated Hospital of Nantong University for the period from year 2004 to 2013. The TMA cores contained tissues with a diameter of 2 mm, and the section thickness of the TMA was 4 microns, as previously described [16, 17]. Clinical data, such as gender, age, tumor location, differentiation grade and tumor stage according to the 8th edition of the American Joint Committee on Cancer (AJCC) classification [18], were collected from the patients' medical records. Importantly, none of the included patients in the study had undergone radiotherapy, chemotherapy or immunotherapy prior to surgery. The overall survival (OS) time of patients was defined as the period from the initial pathological diagnosis to death. The study protocol was approved by the Human Research Ethics Committee of the Affiliated Hospital of Nantong University (2018 - K020).

2.3 mIHC staining and scoring

We conducted mIHC staining using the Opal 7-Color mIHC Kit (NEL810001KT; Akoya Biosciences, USA). Antigen retrieval on the TMA slides was achieved through microwave heating, following the kit's instructions. After incubating the primary antibodies (refer to Table S1) and subsequent washing steps, the sections were further treated with Opal™ secondary-HRP and underwent additional washes, and this cycle was repeated as necessary, following which nuclear staining was performed on the TMA sections using DAPI (F6057, Sigma, USA) according to established protocols [19, 20].

The multicolor immunofluorescence-stained TMA sections were scanned using Multispectral Imaging and Whole Slide Scanning (Vectra 3.0, PerkinElmer, USA). The differential positive fluorescence signals of various proteins (including PLCXD2 and other immune markers) in each tissue core were individually assessed within the tumor and stroma using matched software (inForm 2.6, PerkinElmer, USA). The final score, ranging from 0 to 100, was calculated as the ratio of stained cells to the number of Cytokeratin or DAPI multiplied by 100%.

2.4 Statistical analysis

Wilcoxon rank-sum test and Wilcoxon Signed Ranks test were used to compare PLCXD2 protein expression levels in the TME of HNSC and non-tumorous tissues. Pearson's test was utilized for assessing the correlation between PLCXD2 protein levels and other immune markers. The X-tile tool (Yale University, USA) was used to determine the optimal cutoff point for PLCXD2 protein expression based on patients' OS rates. The association between PLCXD2 protein expression and clinicopathological features was assessed using Chi-square test. Prognostic factors for HNSC were determined using Cox proportional-hazards models and the Kaplan–Meier (K-M) method. Statistical analyses were performed using SPSS 24.0 (IBM, USA), and significance level was set at P < 0.05. The significance threshold of R was > 0.2.

3.1 PLCXD2 mRNA expression in HNSC tissues

Here, we assessed the feasibility of PLCXD2 as a biomarker for HNSC. As anticipated, we found that PLCXD2 mRNA expressions in HNSC tissue were significantly elevated compared to non-cancerous tissues, with a notable statistical difference (log2FC < 1, P < 0.01) (Figure 1A). Additionally, high PLCXD2 mRNA expressions were strongly associated with poorer prognoses (HR = 1.3, P = 0.036) in patients with HNSC (Figure 1B).

3.2 Association between PLCXD2 protein levels and immune markers in HNSC tissues TME

We conducted mIHC staining on HNSC TMA sections to analyze the relationship between PLCXD2 protein expression and various TIICs, including CD4+ T cells, CD8+ T cells, CD56+ NK cells, CD66b+ neutrophils, CD68+CD86+ M1-like macrophages, CD68+CD163+ M2-like macrophages and Lamp3+ dendritic cells, and also examined their correlation with commonly used immune checkpoints PD-1, PD-L1, and CTLA-4.

PLCXD2 protein was primarily localized in the nuclear and cytoplasmic compartments of both HNSC tissues and benign squamous epithelial tissues. Notably, PLCXD2 protein expression varied across different cell types, being observed not only in cancer cells and non-tumor squamous epithelial cells but also in stromal cells. Wilcoxon rank sum test revealed that PLCXD2 protein expression was significantly higher in cancer cells (10.58 ± 14.07) compared to squamous epithelial cells (4.16 ± 5.24) (Z = -3.890, P < 0.001). Additionally, PLCXD2 protein expression in cancer stromal cells (21.51 ± 21.39) was significantly higher than in benign stromal cells (12.14 ± 13.24) (Z = -3.890, P = 0.007) (Figure 2A -B). These results aligned with the PLCXD2 mRNA expression findings from the GEPIA data. Furthermore, the Wilcoxon Signed Ranks Test demonstrated that PLCXD2 protein expression in cancer cells was significantly lower than in cancer stromal cells (Z = -6.498, P < 0.001).

We analyzed PLCXD2 protein expression in the TME of HNSC using Pearson's test and observed a correlation between PLCXD2 protein levels in cancer nests and that in cancer stromal cells (r = 0.216, P < 0.001). Additionally, PLCXD2 protein levels in cancer cells were positively correlated with CD4+ T cells (CD3+CD4+) (r = 0.236, P < 0.001), CD8+ T cells (CD3+CD8+) in the cancer stroma (r = 0.313, P < 0.001), and dendritic cells (Lamp3+) in both tumor intratumoral (r = 0.204, P = 0.008) and stroma (r = 0.212, P = 0.005) (Figure 2C, Figure 3A, Table S2-1). Furthermore, we observed CTLA-4 protein expression not only in TIICs but also in some cancer cells. PLCXD2 protein levels in cancer cells were correlated with CTLA4+ cells in cancer nests (r = 0.262, P < 0.001). However, no significant correlation was found between PLCXD2 protein expression in cancer stromal cells and TIICs or immune checkpoints (PD-1, PD-L1, and CTLA-4) in the TME of HNSC (Figure 2D, Figure 3B, Table S2-2).

Higher expression and lower expression of PLCXD2 protein in HNSC (A) compared to non-tumor squamous epithelial tissue (B). PLCXD2 protein expression is shown in purple. C: Immunostaining for CD3+ cells, CD4+ cells, and CD8+ cells in HNSC tissue. D: Immunostaining for Lamp3+ cells and CTLA-4+ cells in HNSC tissue. Cytokeratin staining is shown in green, and nuclei are stained with DAPI (blue).

3.3 Association between PLCXD2 protein levels and clinicopathological characteristics of HNSC patients

Based on the OS of HNSC patients, we determined a cutoff point for PLCXD2 protein expression in cancer cells (score 0 – 91.97). Scores equal to or less than 8.57 were classified as low-expression (n = 167), while scores ranging from 8.57 to 100 were categorized as high-expression (n = 108) groups. Chi-square test results confirmed that PLCXD2 protein expression in cancer cells was associated with age (χ² = 4.331, P = 0.037), tumor location (χ² = 12.261, P = 0.002), depth of tumor invasion (χ² = 13.369, P = 0.010), lymph node metastasis (LNM) (χ² = 5.041, P = 0.025), and TNM classification (χ² = 15.543, P = 0.004) (Table 1).

However, no suitable cutoff value was identified to establish a relationship between the score of PLCXD2 protein expression in stromal cells (ranging from 0 to 92.59) and clinical outcomes. Even when considering the median or half of the population, no evidence was found to suggest a correlation between PLCXD2 protein expression in stromal cells and clinical features of HNSC (data not shown).

Table 1. The association between PLCXD2 protein expression in cancer cells and clinical characteristics of HNSC

Groups	n	PLCXD2 protein in cancer cells		χ²	p value
Groups	n	Low expression (%)	High expression (%)	χ²	p value
Total	275	167(60.73)	108(39.27)
Age(years)				4.331	0.037*
≤ 60	118	80 (67.80)	38 (32.20)
> 60	157	87 (55.41)	70 (44.59)
Gender				0.677	0.411
Male	105	67 (63.81)	38 (36.19)
Female	170	100 (58.82)	70 (41.18)
Location				12.261	0.002*
Tongue	113	81 (71.68)	32 (28.32)
Buccal mucosa	86	51 (59.30)	35 (40.70)
Larynx	76	35 (46.05)	41 (53.95)
Differentiation				1.134	0.567
Well	150	94 (62.67)	56 (37.33)
Middle	115	66 (57.39)	49 (42.61)
Poor	10	7 (70.00)	3 (30.00)
T stage				13.369	0.010*
Tis	14	12 (85.71)	2 (14.29)
I	74	51 (68.92)	23 (31.08)
II	123	72 (58.54)	51 (41.46)
III	45	24 (53.33)	21 (46.67)
IV	7	1 (14.29)	6 (85.71)
Unknown	12
Lymph node metastasis				5.041	0.025*
Yes	51	24 (47.06)	27 (52.94)
No	212	136 (64.15)	76 (35.85)
Unknown	12
TNM stage				15.543	0.004*
Stage 0	18	14 (77.78)	4 (22.22)
Stage I	60	42 (70.00)	18 (30.00)
Stage II	94	59 (62.77)	35 (37.23)
Stage III	82	44 (53.66)	38 (46.34)
Stage IV	9	1 (11.11)	8 (88.89)
Unknown	12

* p < 0.05

3.4 The relation between the PLCXD2 protein expression and HNSC patient prognosis

To determine the relationship between PLCXD2 protein status and 5-year OS in patients, Cox regression analysis and K-M curves with log-rank tests were conducted. Univariate analysis revealed that PLCXD2 protein expression (HR: 2.232, P = 0.001), tumor differentiation (HR: 1.532, P = 0.032), depth of tumor invasion (HR: 1.482, P = 0.005), LNM (HR: 3.314, P < 0.001) and TNM classification (HR: 1.754, P < 0.001) significantly influenced patient survival. In multivariate analyses, PLCXD2 protein expression (HR: 1.955, P = 0.010) and TNM classification (HR: 1.617, P = 0.001) were identified as independently associated with the 5-year OS of HNSC patients (Table 2, Figure 4).

Table 2.Univariate and multivariate analysis of factors associated with HNSC patient survival.

	Univariate analysis				Multivariate analysis
	HR	P >\|z\|	95% CI		HR	P >\|z\|	95% CI
PLCXD2 protein expression in cancer cells High vs low	2.332	0.001*	1.426	3.814	1.955	0.010*	1.175	3.253
Age (years) ≤60 vs >60	1.587	0.078	0.949	2.653
Gender Male vs Female	1.441	0.180	0.844	2.460
Location Tongue vs Buccal mucosa vs	1.125	0.432	0.839	1.508
Differentiation Well and Middle vs Poor	1.532	0.032*	1.038	2.261	1.475	0.054	0.994	2.188
T Tis vs T2 vs T3 vs T4	1.482	0.005*	1.125	1.953
Lymph node metastasis Yes vs No	3.314	<0.001*	1.993	5.510
TNM stage 0 vs I vs II vs III and IV	1.754	<0.001*	1.333	2.309	1.617	0.001*	1.219	2.146

*p<0.05;

This study utilized bioinformatics to investigate the association between PLCXD2 mRNA expression and patient prognosis in HNSC. Upon validation using mIHC, PLCXD2 protein expression was found to be correlated with tumor malignancy and the status of the TME. Further analysis of PLCXD2 at the protein expression level revealed its relevance to tumor progression and identified it as an independent prognostic factor in HNSC.

Previous studies investigating the relationship between the PLCXD2 gene and human cancer through Genome-wide association studies (GWAS) have demonstrated that PLCXD2 (specifically rs2399395 at 3q13.2) is associated with the risk of esophageal squamous cell carcinoma and lung cancer in the Chinese Han populations [21, 22]. However, these studies did not explore correlations between the TME and clinical characteristics at the PLCXD2 protein expression level. Given that mRNA expression by different tissue cells can result in the production of proteins with unique biological activities and diverse physiological functions [14, 23], we investigated PLCXD2 protein expression in the TME of HNSC tissues.

As anticipated, PLCXD2 protein expression was predominantly observed in the nucleus of both benign and tumor cells, with varying degrees of expression. Importantly, the presence of PLCXD2 protein in HNSC tissue was significantly elevated compared to non-cancerous tissues. Additionally, we found a correlation between PLCXD2 protein expression in cancer nests and that in cancer stromal cells. Considering our observation that high expression of PLCXD2 was closely associated with poor prognosis in HNSC patients, we speculate that PLCXD2 may play a role in promoting the occurrence and development of malignant tumors.

The complicated TME encompasses cancer cells, stromal cells (including TIICs, carcinoma-associated fibroblasts, and other cell types), and the extracellular matrix [24]. Understanding the TME is crucial for advancing cancer immunoprevention and immune interception strategies [25]. Thus, we conducted a correlation analysis between the score of PLCXD2 protein expression and the identified TIICs or immune checkpoints in the TMA of HNSC.

Our findings revealed a positive correlation between PLCXD2 protein expression in cancer cells and the presence of CD4 + T cells and CD8 + T cells in the cancer stroma, as well as dendritic cells in both the cancer intratumoral area and stroma. Furthermore, PLCXD2 protein expression in cancer cells was associated with CTLA4 + cells in nests. Taken together, these results indicate a positive linear dependence between PLCXD2 protein expression and the presence of these immune cell populations.

Within the TME, CD4 + T cells exhibit diverse functions in cancer surveillance and are linked to varying outcomes [26]. CD8 + T cells serve as cytotoxic T cells, binding to MHC-I antigens and exerting anti-cancer effects [27, 28]. Dendritic cells, as crucial antigen-presenting cells in the TME, integrate innate and adaptive immunity and activate T cells [29, 30]. CTLA-4, an important immune checkpoint, is upregulated in activated T cells and suppresses cancer immune responses [31, 32]. Recent studies have consistently shown enhanced CTLA-4 protein expression in tumor cells of various human cancer tissues [33–35], consistent with our findings and indicating a potential association with poor patient prognosis. Based on our correlation analysis results, increased PLCXD2 protein expression in cancer tissue may stimulate the secretion of specific cytokines and recruit TIICs such as CD4 + T cells, CD8 + T cells and dendritic cells. Concurrently, it may contribute to an increase in the ratio of CTLA-4 + cells within the TME.

The observed phenomenon suggests that the facilitating effect of PLCXD2 protein on cancer progression may outweigh the tumor immunity effect exerted by these three types of TIILs in the TME of HNSC. Given that clinical application of monoclonal antibody immunosuppressants targeting CTLA-4 has been shown to enhance the effectiveness of HNSC treatment [36], it could be beneficial to explore the use of anti-CTLA-4 monoclonal antibodies to improve treatment outcomes in cases where PLCXD2 protein expression is high in HNSC tissues.

HNSC, categorized as a solid tumor, exhibits TNM classifications closely associated with patient prognosis [37]. In this study, we investigated aberrant PLCXD2 protein expression in relation to clinical characteristics and TNM staging. High PLCXD2 protein expression in cancer cells has been commonly reported in older patients, with tumors located predominantly in the larynx, followed by the buccal mucosa and tongue. Additionally, elevated PLCXD2 protein expression correlated with increased tumor depth invasion, regional LNM, and advanced TNM classification, all of which were associated with poorer prognosis. This suggests that up-regulation of PLCXD2 protein in cancer cells may accelerate HNSC progression. Our findings suggest that PLCXD2 may serve as a candidate oncogene facilitating malignant tumor development and could potentially serve as a prognostic marker for HNSC patients.

Our research has several limitations. Firstly, further investigation into the role of PLCXD2 in tumorigenesis is warranted using cellular and animal models. Secondly, mechanistic experiments are needed to elucidate immune interactions within the TME of HNSC. Lastly, the lack of multicenter HNSC tissue samples limits the generalizability of our research findings.

In conclusion, our study confirms PLCXD2 protein expression in HNSC tissues as an independent prognostic marker and a potential target for immunotherapy in HNSC.

Declaration of Competing Interest

The authors of this article declare that there is no conflict of interest related to this article.

Acknowledgments

This work was supported by the Science and Technology Plan Project (JC2023082) Nantong, Jiangsu; the Research Project of Municipal Health Commission (MS2022047) Nantong, Jiangsu, China.

CRediT authorship contribution statement

Mingming Tang: Writing – original draft. Zihao Zhang: Methodology. Xinjiang Xu: Data curation. Qingwen Chen: Conceptualization. Yingze Wei: Investigation. Hongyan Qian: Methodology. Hao Wu: Resources. Liang Han: Writing – review, Funding acquisition.

Ethics approval and consent topaticipate

The human study protocol was approved by the Human Research Ethics Committee of the local hospital. Informed consent was obtained from all subjects and/or their legal guardian(s) (No.2024-A 03).

Human and animal rights

No animals were used in this research.All procedures performed in studies involving human participants were in accordance with the ethical standards of institutional and/or research committee and with the 1975 Declaration of Helsinki, as revised in 2013.

Data availability statement

All relevant data are within the manuscript and its Additional files.

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No competing interests reported.

PLCXD2 expression relates to the immune microenvironment and prognosis of head and neck squamous cell carcinoma

Status:

Version 1

Abstract

Figures

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1. Gene Expression Profiling Interactive Analysis (GEPIA)

2.2 Clinical samples and information

2.3 mIHC staining and scoring

2.4 Statistical analysis

3. RESULTS

4. DISCUSSION

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1