The expression of two immunosuppressive SIGLEC family molecules in papillary thyroid cancer and their effect on prognosis

The thyroid cancer (THCA) subtype that occurs more frequently is papillary thyroid cancer (PTC). Despite a favorable postoperative outcome, traditional antitumor therapy does not offer ideal results for patients with metastasis, relapse, and radioiodine resistance. Recent studies demonstrated the remarkable effects of immune checkpoint inhibitors on solid tumors, of which the immunoglobulin superfamily member SIGLEC10 and SIGLEC15 act as novel immunotherapy targets for tumors. Nevertheless, their role in PTC prognosis is still indefinite. Immunohistochemistry was utilized to examine the expression of SIGLEC10 and SIGLEC15 in 244 PTC tissue specimens. Then the expression correlation between the two was analyzed in normal tissues (NT), tumor cells (TC), and tumor stroma (TS), respectively. Subsequently, the retrospective data on patients with PTC were collected to examine whether the two immunosuppressive SIGLEC family members could affect their prognosis. We confirmed that TC expressed higher levels of SIGLEC10 than NT. However, SIGLEC10 was down-regulated in TS and predicted poor outcomes. Meanwhile, down-regulation of SIGLEC15 expression was observed in both TC and TS, indicating a favorable prognosis. PTC patients with both SIGLEC10-SIGLEC15+ expression in TC and TS had a significantly higher recurrence risk. The expression of SIGLEC10 in TS and SIGLEC15 in TC or TS was an independent predictor of PFS, and a positive correlation was shown between SIGLEC10 and SIGLEC15 expression in TS. Therefore, our results indicate that SIGLEC10 and SIGLEC15 may be applied as significant prognostic markers for PTC and attractive targets for THCA immunotherapy.


Introduction
Thyroid cancer (THCA) remains the most frequent endocrine tumor, with the incidence exhibiting a continuously rising trend recently [1].The most frequent pathological type of this disease is papillary thyroid cancer (PTC), which accounts for 85% of all diagnosed cases [2].The first-line treatment for PTC is surgery, followed by radiotherapy and thyroidstimulating hormone suppression, which typically leads to favorable outcomes.Despite this, 10-15% of patients relapse, 5% of patients occur lung and bone metastasis, and even death developed in some cases [3].Therefore, it is crucial to examine new biomarkers to help predict THCA prognosis.
Several studies have shown that the tumor microenvironment (TME) is crucial to the formation, progression, and prognosis of tumors [4][5][6][7].However, it is common for TME to contain an elevated number of regulatory immunosuppressive T cells, which may be partly due to the key negative regulatory factors typically expressed in cancer cells, including programmed death ligand-1 (PD-L1).Consequently, the T-cell-induced immune regulation is inhibited, and anti-immune activity of the body is down-regulated or prevented, thus enabling tumors to escape by disabling the immune system [8,9].Based on this, as a fourth primary therapy option after surgery, radiotherapy, and chemotherapy for cancers, immunotherapy becomes increasingly popular.In particular, there are great clinical successes with immune checkpoint inhibitors in the application of various types of tumors [10].For anaplastic thyroid cancer (ATC) and advanced differentiated thyroid cancer, their treatment has also included immunotherapy, which deeply altered treatment paradigms [11,12].Nevertheless, as the most well-known immunotherapy with good clinical effects, anti-PD-1/PD-L1 treatment has an efficacy rate of 20%-30% in patients with solid tumors [13].Therefore, it is essential for tumor immune research to reveal the molecular mechanisms mediating immune escape continuously.
As a member of the immunoglobulin superfamily, Sialic acid-binding immunoglobulin-type lectin (SIGLEC) recognizes sialylated glycans specially and regulates the function of immune cells [14].Recently, SIGLEC members are increasingly discovered to act in a critical role in tumor immunosuppression [15][16][17].Among them, SIGLEC10 on macrophages could bind to a new "don't eat me" glycoprotein CD24 signal, thereby inhibiting tumor cells from phagocytosis [15].Recent studies reported that high SIGLEC10 expression predicted poor prognosis in human malignancies, including renal clear cell carcinoma, glioma, and hepatocellular carcinoma (HCC) [18][19][20].However, SIGLEC10 expression and its clinical relevance have not been well examined in PTC.Independent from PD-L1, SIGLEC15 was identified as a promising anti-tumor target [21].Tumor immunosuppression may be mediated by SIGLEC15 expression on tumor-associated macrophages (TAMs), which could cooperate with Syk and DAP12, thus increasing the secretion of TGF-β [22].A previous study demonstrated that SIGLEC15 expression is elevated in ATC and follicular thyroid cancer (FTC), and its expression levels were associated with THCA (26 PTC, 16 FTC, 31 ATC, and 13 thyroid adenomas) worse prognosis.Furthermore, it promotes THCA progression by activating the STAT1/STAT3 pathway, especially for ATC and FTC [23].In another study, this team also revealed the role of SIGLEC15 and considered it a target for the immunotherapy of THCA.They found that high expression of SIGLEC15 was detected in THCA, which could promote lymph node metastasis and extrathyroid extension.More importantly, patients with elevated SIGLEC15 expression suffered immune exhaustion [10].Despite this, fewer studies looked at the role of SIGLEC15 and its combination with other immune checkpoints in the prognosis of PTC, particularly, large-scale retrospective studies.
In our work, two immunosuppressive SIGLEC family members SIGLEC10 and SIGLEC15 were investigated as research subjects to analyze their expression and correlation with the prognosis of PTC.These results provide significant scientific evidence for SIGLEC10 and SIGLEC15 as promising prognostic and therapeutic targets of THCA.

Patients and tissue samples
A retrospective study was conducted of patients undergoing PTC primary surgery at Zhejiang Provincial People's Hospital from January 2006 to January 2010.Overall, 244 tumor samples from patients diagnosed with PTC were collected in this study.All pathological specimens were confirmed by three professional pathologists.The diagnosis was determined finally by routine histopathological examination, a few were confirmed by immunohistochemistry (IHC) as well.The study excluded patients who had other tumor types or had received preoperative antitumor treatments.The medical charts were reviewed for clinical and pathological characteristics and data regarding treatment, course, and outcome.The TNM staging of each case was classified based on the 8th American Joint Committee on Cancer (AJCC) criteria.Each patient signed written informed consent, and we obtained approval from the Ethics Committee of Zhejiang Provincial People's Hospital.Follow-up visits were conducted every three months for the first two years after surgery, followed by every six months.At every evaluation during follow-up, the patients underwent clinical evaluations, blood biochemistry tests (Tg, TgAb, T3, T4, etc.,) as well as ultrasound exams.In addition, chest radiography or computed tomography was obtained every year.Pathology and imaging confirmed all cases of relapse.In the current research, survival time was determined as the duration from the date of diagnosis to the date of relapse (or death) or the latest follow-up.We also collected an additional 38 adjacent normal tissues (NT) and 40 PTC tissues to further analyze the expression of SIGLEC10 and SIGLEC15.

Tissue microarray (TMAs) construction
For the construction of TMAs, hematoxylin and eosin (H&E) stained slides were reviewed, and 280 PTC and 38 NT tissues were confirmed independently by three experienced pathologists.We carefully selected representative areas from paraffin-embedded tissue blocks and extracted them with the TM-1 Tissue Microarray Kit.To construct the TMAs, they were embedded in blank paraffin blocks.For better fixation of the paraffin-embedded TMAs, it was incubated at 60 °C for about 30 min and cooled down to 25 °C.We sliced 3-μm thick sections from the TMAs blocks and stained them with IHC.

Immunohistochemistry
For IHC, xylene was employed to deparaffinized paraffinembedded thyroid segments, and ethanol was utilized to rehydrate it.The antigen was then recovered from the segments utilizing 1 mM EDTA, and non-specific binding was reduced by preincubating the segments in TBS with 5% goat serum.The sections were then treated with either the SIGLEC10 (TA367254, OriGene, Wuxi, China) or SIGLEC15 (A20773, Wuhan, China) antibodies.Thyroid tissue samples were examined using DAB (Beyotime, Hangzhou, China) and counterstained with hematoxylin after treatment with horseradish peroxidase secondary antibodies.
Every specimen's immune-positive rate and staining levels were incorporated into the IHC score.Three pathologists examined all the slices' protein-expressing scores in the current investigation.The immune-positive rate was graded from 0 to 4 as follows: Less than 5% of stained cells received a score of 0, 6%-25% received a score of 1, 26-50% received a score of 2, 51-75% received a score of 3, and 76%-100% received a score of 4. The staining levels were rated on a scale of 0 to 3, with 0 being negative, 1 being weak, 2 being moderate, and 3 being strong.SIGLEC10 and SIGLEC15's immunoreactivity score (IRS), which ranged from 0 to 12, was determined as the intensity and positive rate product.In this study, NT, tumor cells (TC), and tumor stroma (TS) were scored.

Correlation analysis
We analyzed the correlation between the IRS of SIGLEC10 and SIGLEC15 in NT, TC, and TS, respectively.Furthermore, Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancer-pku.cn)was also retrieved to conduct an expression correlation of SIGLEC10 and SIGLEC15 [24].In addition, we also applied the GEPIA database to investigate the correlation between PD-L1 expression and SIGLEC10 and SIGLEC15 expression, respectively.

Statistical analysis
Statistics analyses were employed by GraphPad Prism v8.0 and SPSS v25.0.Univariate and multivariate analyses were conducted through the chi-square criterion.Cox proportional-hazards regression models and the Kaplan-Meier method were used to perform the survival analysis.P < 0.05 was considered statistically significant.

Patient characteristics
The clinicopathological characteristics were presented in Table 1.The median age of 244 enrolled patients was 44

Expression of SIGLEC10 in tumor cells, tumor stroma, and normal tissues
The SIGLEC10 protein levels in tumor cells, tumor stroma, and normal tissues are unknown currently.It was observed that SIGLEC10 was expressed mainly on the membrane and in the cytoplasm in normal tissues, tumor cells, and tumor stroma (Fig. 1A-G).In addition, tumor cells showed the strongest staining intensity, while tumor stroma showed the weakest staining intensity (Fig. 1H).
In tumor cells, SIGLEC10 had a median IRS of 9 (range: 2-12).A cut-off value of high-or low-level SIGLEC10 expression in tumor cells was determined by the median score.The IRS > 9.0 represented a high SIGLEC10 expression whereas the IRS ≤ 9.0 represented a low SIGLEC10 expression.120 (49.18%) cases exhibited high SIGLEC10 expression levels, while 124 (50.82%) cases exhibited low SIGLEC10 expression levels in tumor cells.
In tumor stroma, SIGLEC10 had a median IRS of 2 (range: 0-12).A cut-off value of high-or low-level SIGLEC10 expression in tumor stroma was determined by the median score.The IRS > 2.0 represented a high SIGLEC10 expression whereas the IRS ≤ 2.0 represented a low SIGLEC10 expression.102 (41.80%) cases exhibited high SIGLEC10 expression levels, while 142 (58.20%) cases exhibited low SIGLEC10 expression levels in tumor stroma.

Expression of SIGLEC15 in tumor cells, tumor stroma, and normal tissues
The SIGLEC15 protein levels in tumor cells, tumor stroma, and normal tissues are unknown currently.It was observed that SIGLEC15 was expressed mainly on the membrane and  2A-G).In addition, normal tissues showed the strongest staining intensity, while there was no statistical difference in the staining intensity between tumor stroma and tumor cells (Fig. 2H).
In tumor cells, SIGLEC15 had a median IRS of 0 (range: 0-8).A cut-off value of high-or low-level SIGLEC15 expression in tumor cells was determined by the median score.The IRS > 0 represented a high SIGLEC15 expression whereas the IRS = 0 represented a low SIGLEC15 expression.152 (62.30%) cases exhibited high SIGLEC15 expression levels, while 92 (37.70%) cases exhibited low SIGLEC15 expression levels in tumor cells.
In tumor stroma, SIGLEC15 had a median IRS of 1 (range: 0-12).A cut-off value of high-or low-level SIGLEC15 expression in tumor stroma was determined by the median score.The IRS > 1.0 represented a high SIGLEC15 expression whereas the IRS ≤ 1.0 represented a low SIGLEC15 expression.80 (32.79%) cases exhibited high SIGLEC15 expression levels, while 164 (67.21%) cases exhibited low SIGLEC15 expression levels in tumor stroma.

Correlation analysis between SIGLEC10 and SIGLEC15 expression levels
The correlation between SIGLEC10 and SIGLEC15 in tumor cells was first conducted in the GEPIA database.While their expression levels did not show any significant correlation (Fig. 3A).Our data also confirmed this (Fig. 3B).However, statistical significance was discovered in both correlation analyses between SIGLEC10 and SIGLEC15 expression in tumor stroma (P < 0.0001) and normal tissues (P = 0.0164).In this study, a positive correlation was observed between SIGLEC10 and SIGLEC15 expression (Fig. 3C, D).In addition, both SIGLEC10 expression and SIGLEC15 expression correlated positively with PD-L1 expression as well (Fig. 3E, F).

SIGLEC10 and SIGLEC15 expression in PTC and clinicopathological variables
As summarized in Table 2, no statistical significance was discovered between SIGLEC10 expression in tumor cells and clinicopathological features.However, SIGLEC10 expression in tumor stroma was significantly related to N staging (P = 0.030) and total thyroidectomy (P = 0.005).In contrast, other examined clinicopathological factors had no significant correlation with high SIGLEC10 expression in tumor stroma (P > 0.05).
As summarized in Table 3, a statistical significance was discovered between SIGLEC15 expression in tumor cells and N staging (P = 0.001).And SIGLEC15 expression in tumor stroma was significantly related to bilaterality (P = 0.026), N staging (P = 0.026), and total thyroidectomy (P = 0.011).In contrast, other examined clinicopathological factors had no significant correlation with high SIGLEC15 expression in tumor cells or tumor stroma (P > 0.05).
The univariate analyses showed significant correlations between relapse and SIGLEC10 expression in tumor stroma (P = 0.027), SIGLEC15 expression in tumor cells (P = 0.002), and SIGLEC15 expression in tumor stroma (P = 0.003).Furthermore, we also Fig. 3 Correlation analysis of SIGLEC10 and SIGLEC15 expression levels.A The correlation analysis of SIGLEC10 and SIGLEC15 expression levels in the GEPIA database.We utilized Pearson's correlation coefficients to determine the correlation between the two variables.B-D Correlation analysis between SIGLEC10 and SIGLEC15 expression levels in TC, TS, and NT, respectively.E The correlation analysis of SIGLEC10 and PD-L1 expression levels in the GEPIA database.F The correlation analysis of SIGLEC15 and PD-L1 expression levels in the GEPIA database identified other prognostic factors for PFS by univariate analyses as iodine radiotherapy (P < 0.001), maximal tumor diameter (P = 0.011), total thyroidectomy (P = 0.012), bilaterality (P = 0.012), and capsule invasion (P = 0.013) (Table 4).Following multivariate analysis (Table 4), both iodine radiotherapy (P < 0.001) and total thyroidectomy (P = 0.039) were determined as PFS predictors in PTC patients.However, neither SIGLEC10 nor SIGLEC15 expression was found to be independent prognostic indicators (Table 4).

Discussion
Despite favorable postoperative results and ideal prognoses for most patients with PTC, some well-differentiated THCA tumors remain aggressive [25].They are refractory to conventional treatments, including those which are inoperable, occur recurrence following surgery, and don't respond to radioactive iodine therapy [26].In addition, PTC is a very heterogeneous disorder, and the progression of cancers depends on a complex network consisting of multiple signaling mechanisms [27,28].Hence, it is particularly important to screen novel markers of PTC diagnosis and prognosis to formulate treatment and follow-up plans.The current study aimed to investigate the expression of two immunosuppressive SIGLEC family molecules as well as provide their clinical significance in PTC.SIGLEC10 is a cell surface protein belonging to the immunoglobulin superfamily [29], and its expression in immune cells has a negative regulatory relationship with their function [30].In our study, we observed that SIGLEC10 is significantly up-regulated in PTC tumor tissues, while the expression is not associated with the prognosis in PTC patients.However, low expression of SIGLEC10 in PTC stroma tissues is related to poor prognosis in PTC patients.These results are inconsistent with previous results that high expression of SIGLEC10 could be considered a clinical biomarker of poor prognosis in several cancers [18][19][20].Nevertheless, in aneurysmal subarachnoid hemorrhage (aSAH), an elevated level of SIGLEC10 in cerebrospinal fluid over the long term indicates a better prognosis.This may be attributed to SIGLEC10's anti-inflammatory properties, which could improve secondary damage and reduce excessive host defense [31].Therefore, exploring the other functions SIGLEC10 plays in PTC, such as its influence of immune modulation on the microenvironment of inflammation, will assist in revealing its role in the prognosis of less malignant PTC.In addition, the SIGLEC10 expression in TS is SIGLEC15 is also an immunosuppressive member of the SIGLEC family [32].Bioinformatics studies have revealed that SIGLEC15 up-regulation is widespread in cancers, including THCA [21,33].Interestingly, SIGLEC15 expression may be associated with distinct prognoses in various cancer types.For example, there was no correlation between the SIGLEC15 expression and the prognoses of gastric cancer and non-small cell lung cancer [34,35].However, its high expression could indicate a good prognosis for pancreatic ductal adenocarcinoma and a poor prognosis for renal carcinoma [21,36].SIGLEC15 has been shown to be up-regulated in THCA, and its expression in FTC and ATC samples is significantly elevated compared with matched surrounding PTC [10,23].However, a separate study to investigate the expression of SIGLEC15 in PTC has not been reported.In addition, SIGLEC15 is observed mainly in tumor cells as well as tumor-infiltrating immune cells [37].Therefore, in addition to tumor cells, it is also necessary to detect SIGLEC15 expression in the tumor stroma.In our research, compared with NT, the SIGLEC15 expression was downregulated in PTC tumor tissues and stroma tissues, and expression levels were not significantly different between the two types of tissues.Exactly, the differential expression of SIGLEC15 in PTC and ATC indicates that SIGLEC15 may promote the aggressiveness of THCA.Since PTC is considered an early stage of the transition to ATC [38], future research should investigate whether SIGLEC15 functions in this progression.Moreover, we also observed that high SIGLEC15 expression in both TC and TS is related to poor prognosis in PTC.Notably, consistent with SIGLEC10 in TS, the SIGLEC15 expression in TC or TS is also correlated with N staging.Meanwhile, the expression of SIGLEC15 in TS is correlated with bilaterality and total thyroidectomy, indicating that this indicator may guide the choice of the surgical procedures of PTC.
Since a combination of different immune checkpoint inhibitors might produce a better clinical outcome [39], we examined the significance of SIGLEC10 and SIGLEC15 in the prognosis of PTC simultaneously.As far as we know, it's the first time to explore together the role of both in tumor prognosis.In the current study, we showed that patients with PTC in the SIGLEC10-SIGLEC15+ group had a higher risk of recurrence than patients in the SIGLEC10 + SIGLEC15-or SIGLEC10-SIGLEC15-group, which applies to both TC and TS.However, no significant differences were observed between the SIGLEC10-SIGLEC15+ group and the SIGLEC10 + SIGLEC15+ group.Taken together, these results may provide a prognostic biomarker with high specificity for PTC.Furthermore, we noticed that SIGLEC10 expression significantly positively correlated with SIGLEC15 expression in TS and both SIGLEC10 and SIGLEC15 expressions exhibited a positive correlation with PD-L1 expression.Moreover, the univariate analyses also identified that SIGLEC10 expression in TS, and SIGLEC15 expression in TC and TS were independent prognostic factors of PFS.However, neither SIGLEC10 nor SIGLEC15 expression was found to be independent prognostic indicators.Collectively, these findings may provide the possibility of combined checkpoint blockade therapy for advanced THCA or ATC.However, there are still restrictions on our work.Firstly, it was retrospective research with bias in selecting variables such as clinicopathological characteristics, thus losing data accuracy.Secondly, a more in-depth study of SIGLEC15 and SIGLEC10's cellular mechanisms is needed in PTC, such as co-culture experiments of macrophages and PTC cells.Finally, the use of TMAs gave rise to tumor heterogeneity and poor staining quality on some images.

Conclusions
In conclusion, this study provided, as far as we know, the first evidence of SIGLEC10 and SIGLEC15 to investigate their expression in TC and TS and prognostic value in PTC.We confirmed that SIGLEC10 expression was up-regulated in TC.However, SIGLEC10 was down-regulated in TS and correlated with poor prognosis.Similarly, SIGLEC15 expression was both down-regulated in TC and TS, while high expression of SIGLEC15 in TC or TS predicted poor prognosis.Both SIGLEC10-SIGLEC15+ expression in TC and TS had the highest risk of recurrence in patients with PTC.SIGLEC10 expression in TS and SIGLEC15 expression in TC or TS were independent prognostic factors of PFS, and there was a positive correlation between the expression of SIGLEC10 and SIGLEC15 in TS.Therefore, SIGLEC10 and SIGLEC15 might act as specific and valuable prognostic biomarkers for PTC and attractive targets for THCA immunotherapy.

Fig. 2
Fig. 2 SIGLEC15 expression in TC, TS and NT.A-C High-/ middle-/low-SIGLEC15 expression in TC.D-F High-/ middle-/low-SIGLEC15 expression in TS.G The representative IHC stained image of SIGLEC15 in NT.H IRS of SIGLEC15 in TC, TS, and NT.Mean ± SD. ***P < 0.001

Fig. 4
Fig.4Correlation of the SIGLEC10/SIGLEC15 expression level and PFS in PTC patients.A, B The Kaplan-Meier analysis was utilized to examine the PFS of SIGLEC10 expression in TC and TS, respectively.C, D The Kaplan-Meier analysis was utilized to examine the PFS of SIGLEC15 expression in TC and TS, respectively.E Relationship between the expression level of SIGLEC10/SIGLEC15 in TC and PFS in PTC patients.F Relationship between the expression level of SIGLEC10/ SIGLEC15 in TS and PFS in PTC patients

Table 2
Correlation between SIGLEC10 expressions and clinicopathological features of 244 patients with PTC

Table 3
Correlation between SIGLEC15 expressions and clinicopathological features of 244 patients with PTC