CD206+ macrophage is the major TAMs in LSCC tumors

In LSCC tumor, the interaction of a nest of tumor cells with adjacent stroma cells is critical for tumor progression. To visualize the localization of different types of TAMs in LSCC tumor, we analyzed the FFPE sample of 80 LSCC patients who received only surgical treatment without pre- and post-operative adjuvant therapies. The demographic and clinicopathological characteristics of the patients are summarized in Table 1. The median age at diagnosis was 62.6 years (ranging from 46.0 to 80.0), and 78/80 (97.5%) of the patients were men. Fifty-six patients (70.0%) had a history of smoking while 43 (53.7%) had a history of alcohol drinking. According to the 8th Edition of the AJCC guidelines, 7 (8.8%) patients had stage I disease, 6 (7.5%) patients stage II, 32 (40.0%) patients stage III, and 35 (43.8%) patients stage IV.

Table 1 Demographic and clinicopathological characteristics of the patients with LSCC (tissue microarray of 80 patients)

We first visualized the localization of three subtypes of TAMs using double-immunofluorescence staining of pan macrophage marker CD68 combined with iNOS (M1-like), CD163 (M2-like), and CD206 (M2-like), respectively, on patient tissue microarray (TMA). The results showed low level of iNOS+ macrophages infiltration in the tumor microenvironment of LSCC. iNOS+ TAMs was observed in only 16.3% (13/80) of LSCC tumors and all of them were detected in the tumor stroma (TS) (Fig. 1A), with no existence in tumor nest (TN) or adjacent normal tissue (ANT). On the other hand, CD206+ macrophages were more commonly detected in the TME (Fig. 1B) with 66.3% (53/80) in TS and 35.0% (28/80) in TN. Interestingly, a small amount of CD206+ macrophage were also found in the ANT region of 15 (18.8%) tumors. Surprisingly, we did not detect CD68+CD163+ macrophage in the serial sections of all 80 tumors, though CD163 was commonly expressed by macrophage (Fig. 1C). Statistically, paired t-test showed that the infiltration level of CD206+ macrophages was significantly higher than that of iNOS+ M1-like macrophages in patients with LSCC (p-value < 0.0001, Fig. 1D). This result suggested that CD206+ macrophage is the major M2-like TAMs in LSCC.

Fig. 1

CD206+ M2-like macrophage was the dominnat TAM population in the tumor microenvironment of LSCC. A Immunofluorescence staining of CD68+iNOS+ M1-like TAMs on LSCC tumor sections. iNOS (green), CD68 (red) and DAPI (blue). The corresponding HE staining was shown in the lower right-hand panel. B and C Double-immunofluorescence staining of CD68+CD206+ and CD68+CD163+ M2-like TAMs. B CD206 (green) and CD68 (red) staining of LSCC sections. Total nuclei were co-stained with DAPI (blue); The corresponding HE staining was shown in the lower right hand panel. C Two representative double-immunofluorescence staining of CD68+CD206+ and CD68+CD163+ TAMs on LSCC tissues. Upper panels showed CD68+(red) and CD206+ (green) and lower panels showed CD68+ (green) and CD163+ (red). D The infiltrating level of CD68+CD206+ TAMs was significantly higher than that of CD68+iNOS+ in LSCC tissue, p-value < 0.0001; E Representative dot plots displayed CD206+ M2 and iNOS+ M1 in fresh tumor tissues of LSCC patients using flow-cytometric analysis. The corresponding statistics analysis was shown in F using Paired sample t test, with statistical significance indicated as follows: ****p < 0.0001; G Multiplex immunofluorescence staining of CD68, CD206 and iNOS of LSCC tissue

We further confirmed the enrichment of CD206+ macrophages in the TME by analyzing the composition of TAMs from fresh tumor tissue samples of LSCC patients by flow cytometry. Consistently, the infiltration of CD206 + TAMs was significantly higher than that of iNOS + TAMs (p-value < 0.0001, Fig. 1E, F). Therefore we concluded that CD206+ macrophage is the major TAMs in the TME of LSCC (Fig. 1G, Additional file 3: Fig. S3).

Infiltration of CD206+ macrophages was higher in tumor stroma region compared to tumor nest

We next examined the localization of CD206+ macrophages in more detail using double-immunofluorescence staining and found CD206+ macrophage infiltration in the TS region in 53 of the 80 LSCC tumors, ranging from 0 to 55.67 while only 28 tumors showed infiltration in the TN region, ranging from 0 to 17.0. Paired t-test confirmed that the infiltration of CD206+ macrophages was significantly higher in the TS regions than in the TN regions (p-value < 0.0001, Fig. 2A–D). Moreover, we could barely detect CD206+ macrophages in the ANT regions, with only 15 tumors showed positive CD206+ infiltration and all less than 6.0.

Fig. 2

The infiltration pattern of CD68+CD206+ M2-like macrophages in the TME. A–C The infiltrating level of CD68 + CD206 + M2-like macrophages in tumor stroma A, tumor nest B, and corresponding adjacent normal tissue C. D Patients with LSCC exhibited different infiltrating levels of CD68+iNOS+ M1 and CD68+CD206+ M2 TAMs; Paired sample t test was used and statistical significance indicated as follows: ****p < 0.0001. E Immunofluorescent staining of patient tumor samples in cancer tissue and ANT region. Patients with positive E1 and negative E3 TS CD68+iNOS+ M1 TAM infiltration, and the CD68+iNOS+ M1 infiltration E2, E4; F Patients with positive F1 and negative F3 TN CD68+CD206+ M2 TAM infiltration, and the CD68+CD206+ M2 infiltration of their corresponding ANT F2, F4. G Patients with high G1 and low G3 levels of TS CD68+CD206+ M2 TAM infiltration, and the CD68+CD206+ M2 infiltration of their corresponding ANT G2, G4. LSCC, Laryngeal squamous cell carcinoma; TN, Tumor nest; TS, Tumor stroma; ANT, Adjacent normal tissue

Correlation between iNOS+ and CD206+ TAMs infiltration and the clinicopathological characteristics of LSCC

To evaluated the correlation between TAMs and clinicopathological characteristics of LSCC tumors, we stratified LSCC patients by TS infiltrated CD206+ TAMs. Since the median number of TS CD206+ TAMs was 8.0 for all patients and 14.0 for patients with positive TS infiltration, we defined patients with at least 14.0 TS infiltration value of TS CD206+ TAMs as high infiltration group, and those with value less than 14.0 as low infiltration group (Fig. 2E–G). In addition, each group was further divided into positive-infiltration or no infiltration groups based on the total presence of TS iNOS+ and TN CD206+ TAMs (Fig. 4).

We then analyzed the correlation between the infiltration of TS iNOS+ M1-like macrophages, TS and TN M2-like macrophages and the clinicopathological features and prognosis of LSCC patients (Table 2). The results indicated that patients with TS iNOS+ TAMs infiltration showed a significantly lower probability of tumor recurrence in the long-term follow-up compared to those with no TS iNOS+ TAMs infiltration (p-value = 0.037). On the contrary, TN CD206+ TAMs infiltrations was significantly correlated with worse recurrence and long-term survival outcome (both p-value < 0.001), and a similar correlation was found for TS CD206+ TAMs (p-value = 0.016 and 0.034, respectively). Interestingly, among the 53 patients with positive TS CD206+ TAMs infiltration, those with higher CD206+ TAMs infiltration exhibited unsatisfactory recurrence and survival outcomes compared to those with low TS CD206+ infiltration (p-value < 0.001, respectively). In addition, patients with pathological stage IV disease were more likely to show positive TS CD206+ infiltration than those with stage I-III disease (80.0% (28 in 35) vs 55.6% (25 in 45), p-value = 0.022). Among all patients that exhibited positive TS CD206+ infiltration, patients with stage IV disease showed comparable infiltrating levels to those with stage I-III disease (high level of TS CD206+ TAMs rate: 53.6% (15 in 28) and 48.0% (12 in 25) for stage IV and stage I-III, respectively, p-value = 0.685).

Table 2 Correlations between the M1 and M2 TAMs infiltration levels and the clinicopathologic characteristics of LSCC (intra-groups analysis)Prognostic value of iNOS+ and CD206+ TAM infiltration in LSCC patients

We performed Kaplan–Meier analysis and the log-rank test to evaluate the correlation between iNOS+ or CD206+ TAMs infiltration and patient survival. Only 1 (3.8%) of the 13 patients with positive TS iNOS+ TAMs infiltration exhibited tumor recurrence at 28 months and died at 40.0 months after initial treatment. The analysis showed that positive TS iNOS+ TAMs infiltration indicated a significantly better recurrence-free survival (RFS) (p-value = 0.0303) and overall survival (p-value = 0.0585, Fig. 3A, B) compared to those with no TS iNOS+ TAMs infiltration. In contrast, patients with positive TN CD206+ TAMs infiltration showed significantly worse recurrence and survival outcomes than those with no TN CD206+ infiltration (p-value < 0.0001, Fig. 3C, D). We did not find a statistical significant correlation between TS CD206+ TAMS and patient RFS or OS. However, a subgroup of patients with high TS CD206 + TAMs infiltration showed markedly worse RFS and OS survival compared to those with low or negative CD206 + TAM infiltration (p-value < 0.0001, Fig. 3E, F).

Fig. 3

Kaplan–Meier curves for OS and RFS of LSCC cohort (n = 80). A and B Patients with positive TS CD68+iNOS+ M1 TAM showed higher OS (p-value = 0.0585) and RFS (p-value = 0.0303) than those with negative TS CD68+iNOS+ M1 infiltration; C and D Patients with positive TN CD68+CD206+ M2 TAM showed significantly worse OS (p-value < 0.0001) and RFS (p-value < 0.0001) outcome than those with no TN CD68+CD206+ M2 infiltration; E and F Patients with high TS CD68+CD206+ M2 TAM infiltrating level showed worst OS and RFS outcome among all patients, significantly worse than those of no and low TS CD68+CD206+ M2 infiltration (p-value < 0.0001, respectively); G and H Validation cohort using maximum IHC section of whole LSCC tumor tissue. G Representative CD68 and CD206, CD68 and iNOS IHC staining image in IHC serial section of LSCC tissues with different recurrence-free survival outcomes; H Patients who suffered from tumor recurrence within one year showed significantly higher CD68+CD206+ M2 high-infiltration rate than those who showed no tumor recurrence five years after initial surgery (p-value = 0.035). OS, Overall survival; RFS, Recurrence-free survival; TS, Tumor stroma; TN, Tumor nest; IHC, Immunohistochemical. OS and RFS rates were calculated according to the Kaplan–Meier method, and statistical differences between the different groups were calculated using the log-rank test

Maximum immunohistochemical (IHC) section of whole LSCC tumor tissue in validation cohort confirmed the prognostic value of CD206 + TAMs

To validate the correlation between the infiltration of TAMs subgroups and the prognosis of LSCC patients, we reviewed the whole medical history and pathological section materials of 20 LSCC patients who underwent initial tumor resection without any postoperative adjuvant radiotherapy or chemotherapy at a single clinical center in 2015. Thirteen (65.0%) of the 20 patients showed tumor recurrence within one year after primary surgical treatment, however, the other 7 (35.0%) showed no tumor recurrence over five years after initial surgery, indicating a significantly better survival outcome. Next, we examined the infiltration of TAMs on the pathological section materials of whole tumor tissue using immunohistochemical staining. The result indicated that most patients with no recurrence showed no or low CD206+ TAMs infiltration and only 2 (28.6%) patients in this group exhibited high CD206+ TAMs infiltrating levels. However, 10 (76.9%) in the 13 patients in the one-year recurrence group showed a high level of CD206+ TAMs infiltration. In addition, the rate of high CD206+ TAMs infiltration in the one-year recurrence group was significantly higher than that in no recurrence group (p-value = 0.035, Fig. 3G, H). We also tested the infiltration of iNOS+ TAMs and found that 4 and 2 patients in the no recurrence group and one-year recurrence group showed positive iNOS+ TAMs infiltration, respectively. Consistently, patients with satisfactory long-term recurrence-free survival generally showed positive iNOS+ TAMs infiltration (57.1% and 15.4%, respectively, p-value = 0.052, Fig. 3G, H).

Molecular phenotypic characteristics analysis of iNOS+ TAMs and CD206+ TAMs using flow cytometry

Next, we investigated the immune cell composition in the TME in fresh tumor tissue samples to gain insights into how TAMs interact with other cells. Flow cytometry results revealed that the infiltration of total CD68+ TAMs was not associated with CD3+ tumor-infiltrating T cells (TILs) including CD4+ TILs, CD8+ TILs, and CD4 + /CD8 + TILs (Fig. 4, panels A1-A4). However, the CD206+ TAM subgroup showed strong correlation with CD4+ TILs (R square = 0.3007, p-value = 0.0343, Fig. 4, panels B1-B4). Considering the interaction of major histocompatibility complex class II (MHC-II) with CD4+ T cell receptors (TCRs) plays a central role in CD4+ T cell immunity, we divided CD206+ TAMs into HLA-DRhigh and HLA-DRlow subgroups and analyzed the correlation of each with CD4+ TILs. Interestingly, HLA-DRhigh CD206+ TAMs showed strong correlation with both CD4+ TILs and CD4 + /CD8 + ratio (R square = 0.4044 and 0.6190, p-value = 0.0108 and 0.0005, respectively, Fig. 4, panels C1-C4). In contrast, no correlation was found between HLA-DRlow subgroup with CD3 T cell and its subtypes (CD4 + , CD8 + and CD4 + /CD8 + ratio, p-value > 0.05, Fig. 4, panels D1-D4).

Fig. 4

The correlation between tumor-infiltrating T lymphocytes and TAMs of fresh LSCC tissue samples using flow-cytometric analysis (n = 15). A The correlation between CD68+ total TAM infiltration and total CD3+ T cell A1, CD4+ T cell in total CD3+A2, CD8+ T cell in total CD3+A3, and CD4+/CD8+ rate A4; (B) The correlation between CD68+CD206+ M2 TAM infiltration and total CD3+ T cell B1, CD4+ T cell in total CD3+B2, CD8+ T cell in total CD3+B3, and CD4+/CD8+ rate B4; C The correlation between CD68+HLA-DRhighCD206+ M2 subgroup infiltration and total CD3+ T cell C1, CD4+ T cell in total CD3+C2, CD8+ T cell in total CD3+C3, and CD4+/CD8+ rate C4; D The correlation between CD68+HLA-DRlow/−CD206+ M2 subgroup infiltration and total CD3+ T cell D1, CD4+ T cell in total CD3+D2, CD8+ T cell in total CD3+D3, and CD4+/CD8+ rate D4. The presence of costimulatory molecule for T-cell activation including CD40 (first column), CD80 (second column), CD86 (third column) and CD25 (last column) in CD68+HLA-DRlow/−CD206+ M2, CD68+HLA-DRhighCD206+ M2, and CD68+iNOS+ M1 subgroups E, and the scatter plot (with mean ± SD) comparing fractions of the above-mentioned surface antigens within the three TAM subsets F. TAMs, Tumor-associated macrophages. Linear regression analyses were used to test the correlation between different subgroups of cells

To investigate the functional role of HLA-DRhigh and HLA-DRlow/− TAMs in the TME of LSCC, we analyzed the expression of costimulatory molecule for T-cell activation including CD40, CD80 and CD86 in each subset (Fig. 4E). We discovered that HLA-DRhigh CD206+ TAMs showed significantly higher CD40 and CD86 expression than either HLA-DRlow/− CD206+ (p-value < 0.0001) or iNOS+ TAMs (p-value = 0.0257 and 0.0032, respectively) whereas the expression of CD80 of HLA-DRhigh CD206+ TAMs and iNOS+ TAMs was similar (p-value = 0.5485), and both were higher than that of HLA-DRlow/− TAMs (p-value = 0.0002 and 0.0022, respectively). Interestingly, we also found that CD25+ expressing TAMs was nearly absent in HLA-DRlow/−CD206+ TAMs, whereas it exited in HLA-DRhigh CD206+ with a range of 0% to 7.89% and iNOS+ TAMs with a range of 5.1% to 43.2%. The difference was statically significant (p-value = 0.0424 and 0.0117, respectively Fig. 4F).

Co-localization of HLA-DRhigh−CD206+ TAMs and CD4+ TILs in the tumor microenvironment of LSCC

To further explore the interaction between HLA-DRhighCD206+ TAMs and CD4+ TILs in the tumor microenvironment of LSCC, we examined the localization of both types of cells on serial tumor tissue section using immunohistochemical (IHC) staining for HLA-DR, CD206 and CD4. The result showed that in section with high CD206+ TAM infiltration was accompanied by the enrichment of CD4 + TILs whereas in area with less CD206 staining, there was also less CD4 + TILs (compare the left and right columns in Fig. 5).

Fig. 5

Representative immunohistochemical (IHC) of HLA-DR, CD206 and CD4 using serial LSCC tissue section. Corresponding HE staining and immunohistochemical staining of CD206+, HLA-DR+ M2-like TAMs and CD4+ T lymphocytes were shown as indicated

HLA-DRhigh−CD206+ and CD4+ TILs co-localize with apoptotic tumor cells in the tumor microenvironment of LSCC

Given that apoptotic tumor cells regulate the TME, including the modulation of macrophage polarization [21, 22], we used immunohistochemical and double-labeling immunofluorescence staining of serial tumor tissue section to investigate the localization of TAMs. We found that HLA-DRhigh CD206+ macrophages preferentially accumulated around areas with a large number of Cleaved Caspase-3+ apoptotic tumor cells (Fig. 6 and Additional file 4: Fig. S4).

Fig. 6

Representative image of co-localization of Cleaved-Caspase 3 + apoptotic tumor cells, HLA-DRhighCD206+ M2 TAMs, and CD4+ TILs in the tumor microenvironment of LSCC serial tissue section. A Immunohistochemical staining of Cleaved Caspase-3+ tumor cell and CD68 + TAMs; B Double-labeling immunofluorescence of HLA-DRhighCD206 + M2 TAMs; C Double-labeling immunofluorescence of CD4 + and CD8 + TILs using serial sections of LSCC