Prognostic impact of tumor-associated macrophage-related markers in patients with adenocarcinoma of the lung

doi:10.21203/rs.3.rs-1960878/v1

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Prognostic impact of tumor-associated macrophage-related markers in patients with adenocarcinoma of the lung

https://doi.org/10.21203/rs.3.rs-1960878/v1

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Macrophage polarization is an important pathogenetic factor in neoplastic diseases and is regulated by transcription factors, i.e., phosphorylated signal transducer and activator of transcription 1 (phospho-STAT1) for the M1 phenotype and c-Maf for the M2 phenotype. However, the role of macrophage phenotype in lung adenocarcinoma (LAD) remains unclear. Here, we examined whether the density of M1 and M2 macrophages was associated with prognosis in patients with LAD using double-labeling immunohistochemistry for the detection of macrophage markers. Additionally, programmed death ligand 1 (PD-L1) expression was investigated. Immune cells co-expressing CD68 and phospho-STAT1 were considered M1 macrophages, whereas those co-expressing CD68 and c-Maf were recognized as M2 macrophages. Three hundred seven patients with LAD were divided into two cohorts (N = 100 and 207 cases, respectively) to evaluate the associations of M1 and M2 phenotypes with prognosis in patients with LAD. We determined the cut-off values of CD68/phospho-STAT1-positive cells (5 or less) and CD68/c-Maf-positive cells (more than 11) to assess correlations with survival using receiver operating characteristic curve analysis in the first cohort. According to the cut-off values, high expression of CD68/c-Maf was identified as an independent prognostic marker, whereas CD68/Phospho-STAT1 expression was inversely correlated with patient outcomes. Moreover, the M1/M2 ratio (0.19 or less) was a poor prognostic factor for LAD. However, PD-L1 expression was not correlated with patient outcomes. Overall, these findings suggested that double staining of markers identifying M1 and M2 macrophages, including phospho-STAT1 and c-Maf, can be used as prognostic indicators for patients with LAD.

Lung cancer is the most common cancer type worldwide and is the leading cause of cancer-related death^1,2. The underlying molecular mechanisms of lung carcinogenesis are complex^3,4. The tumor microenvironment has attracted much attention in the field of solid cancers^4,5, and tumor cells and surrounding stromal cells play important roles in tumor progression and cancer metastasis^5,6. The link between tumor cells and stromal cells may be associated with tumor growth and progression^5,6. The surrounding cells forming the stroma include fibroblasts, vessels, and immune cells^5,6; and common immune cells, including lymphocytes (T cells, B cells, and natural killer cells), neutrophils, and monocytes/macrophages, are also involved in the tumor microenvironment^5,6. Among these cells, macrophages are major players in tumor progression and metastasis^7–11. Macrophages show heterogeneous phenotypes and functions and are important stromal cells. Notably, macrophages can be polarized into different phenotypes, i.e., pro-inflammatory M1 macrophages (classical type) or anti-inflammatory M2 macrophages (alternative type)^11,12. Both M1 and M2 macrophages are involved in tumor-related inflammation, whereas M2 macrophages promote angiogenesis, wound healing, and tumor growth^11,12. In the absence of M1 macrophage-orienting signals, M2 macrophages promote tumor cell proliferation in vitro and in experimental murine models; evaluation of distinct protumor and antitumor macrophage subsets is a challenging research topic in human cancers^13,14.

Previous studies have provided conflicting evidence regarding the significance of macrophages in cancer^15,16. Infiltrating macrophages have pro-inflammatory properties and consequently play antitumor roles in colorectal tumors^15,16. Additionally, macrophages are associated with good prognosis^15,16. However, other clinical studies have shown that macrophages promote tumor progression and are associated with poor prognosis in many tumors, including lung, cervical, ovarian, esophageal, and breast cancers as well as melanoma^17,18. After recruitment to the tumor site, macrophages are polarized into a specific phenotype (tumoricidal or tumorigenic) via exposure to tumor microenvironment-derived factors, such as cytokines, growth factors, and hypoxia^15–18. According to this theory of the tumor microenvironment, immune clearance of solid tumors, which is induced by macrophages, is altered by various factors^15–18.

The role of macrophages in lung adenocarcinoma (LAD), the most common histological type of lung cancer, remains unclear. Therefore, in this study, we evaluated stroma-infiltrating macrophages (M1 and M2 phenotype) and investigated the associations of M1/M2 macrophages with prognosis in patients with LAD. Additionally, programmed death ligand 1 (PD-L1) expression in the tumor and surrounding immune cells was examined.

Patients

In total, 307 patients with LAD attending Iwate Medical University were enrolled in this study. Histological classifications were determined according to the World Health Organization (WHO) classification criteria^3,4. In addition, tumors were graded (low, intermediate, or high) based on International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society (IASLC/ATS/ERS) classifications¹⁹. The classification is according to the predominant histologic pattern of the LAD which often co-exists with less dominant elements of other histologic patterns²⁰. Moreover, we defined tumor spread through air spaces (STAS) as micropapillary or solid clusters of single tumor cells free floating within air spaces beyond the edge of the tumor, as previously reported²¹. Finally, tumor-infiltrating lymphocytes were defined as described elsewhere^22,23. Based on the 8th edition of the American Joint Committee on Cancer Staging Manual, the pathological stage was diagnosed as stage I in 197 patients and stages II and III in 110 patients. Histopathological variables, including lymphatic invasion, venous invasion, pleural invasion, and STAS, are shown in Table 1.

Table 1

Clinicopathological findings in patients with lung adenocarcinoma.
Factor	Cohort 1		Cohort 2		P value
Total	100		207
Age, median (range) (years)	70	(45–88)	70	(40–88)	N.S.
Sex					N.S.
Male	47	(47.0)	97	(46.9)
Female	53	(53.0)	110	(53.1)
Smoke					N.S.
Smoker	53	(53.0)	100	(48.3)
Never	47	(47.0)	107	(51.7)
pStage					N.S.
I	71	(71.0)	126	(60.9)
II	16	(16.0)	32	(15.5)
III	13	(13.0)	49	(23.6)
IASLC/ATS/ERS classification subtypes					N.S.
Low group	6	(6.0)	15	(7.3)
Intermediate group	74	(74.0)	152	(73.4)
High group	20	(20.0)	40	(19.3)
Lymphatic invasion					N.S.
Positive	23	(23.0)	36	(17.4)
Negative	77	(77.0)	171	(82.6)
Venous invasion					N.S.
Positive	21	(21.0)	42	(20.3)
Negative	79	(79.0)	165	(79.7)
Pleural invasion					N.S.
Positive	31	(31.0)	54	(26.1)
Negative	69	(69.0)	153	(73.9)
STAS					N.S.
Positive	31	(31.0)	67	(32.4)
Negative	69	(69.0)	140	(67.6)
Recurrence					N.S.
Positive	37	(37.0)	88	(42.5)
Negative	62	(62.0)	113	(54.6)
Unknown	1	(1.0)	6	(2.9)
Death					N.S.
Alive	66	(66.0)	131	(63.3)
Dead	34	(34.0)	76	(36.7)
Disease free survival, median (range) (days)	1825	(87–1825)	1651	(53–1825)	N.S.
Overall survival, median (range) (days)	1825	(87–1825)	1800	(95–1825)	N.S.
N.S., not significant; pStage, pathological Stage; IASLC, International Association for the Study of Lung Cancer; ATS, American Thoracic Society; ERS, European Respiratory Society; STAS, spread through air spaces.

The 307 patients with LAD were divided into two cohorts, including the first and second cohorts (validation). There were no significant differences in clinicopathological findings between the two cohorts. Clinicopathological variables are summarized in Table 1.

The Ethics Committee of Iwate Medical University School of Medicine approved this study (approval no. MH2021-068). All patients provided written informed consent to participate in this study, according to our institutional guidelines. All study protocols were performed according to the standards set by the Declaration of Helsinki.

Determination of overall survival and disease-free survival

Lung cancer-specific survival, defined as cause of death from lung cancer, was used to assess overall survival. In addition, recurrence-free survival, which excluded secondary cancers, was adopted to measure disease-free survival. The duration of disease-free survival was determined based on the presence/absence of metastasis, evaluated during the follow-up period (3–4 times/year) using computed tomography.

Sample size determination

The sample size required to identify patient outcomes was determined to be at least 100 cases (for cohort 1) using the Freedman method (R 4.2.0 software). The statistical power (detection power) was set to 0.8, which is commonly used in medical studies.

Chemotherapeutic treatment after surgery for LAD

Following surgery, tegafur uracil was administered to 85 of 165 patients with LAD, tegafur/gimeracil/oteracil was administered to 1 of 165 patients with LAD, and platinum-doublet chemotherapy, including carboplatin plus gemcitabine, tegafur/gimeracil/oteracil, or paclitaxel, was administered to 62 of 165 patients with LAD. Radiotherapy was performed in 12 patients with LAD, and tyrosine kinase inhibitors were administered to 5 patients. The other 142 patients did not receive additional chemotherapy following surgery.

Construction of tissue microarrays (TMAs)

The paraffin-embedded tissues used for construction of TMAs were stored at room temperature. We created TMAs using a manual tissue array (Azumaya Co., Tokyo, Japan). We selected invasive areas with a strong desmoplastic area for assessment of immunohistochemical expression. Such areas were identified by expert pathologists (N.Y. and T.S.). Three-millimeter-thick tissue cores were obtained from target lesions and placed into recipient blocks containing 12 cores, including 10 cancer tissues and 2 control tissues. After construction, we prepared 3-µm-thick sections stained with hematoxylin and eosin using the initial slides to confirm the histological diagnosis. Serial sections were cut from TMA blocks for immunohistochemical staining.

Immunohistochemistry for PD-L1

Three-micron-thick tissue sections were placed on charged slides, dried, and melted in an oven at 42°C for 180 min. After deparaffinization and rehydration, the sections were heated in Envision FLEX target retrieval solution (pH 6.0 or 9.0; Dako) for 20 min and washed twice for 5 min in phosphate-buffered saline. Hydrogen peroxide (3%) was used to block endogenous peroxidase activity for 5 min. Immunohistochemistry was performed using the DAKO Envision + system. The specimens were heated in citrate buffer (pH 6.0) for 20 min each using PT Link (Dako). The antigen-antibody reaction was visualized using an enhanced polymer-based detection system. Hematoxylin was used as the counterstain. Desmoplastic tissues surrounding invasive colorectal cancer and interstitial lung disease were used as positive controls.

Double staining immunohistochemistry

For immunohistochemistry, blocks were sectioned at a thickness of 3 µm. Antibodies targeting CD68 (clone PG-M1; Dako), phosphorylated signal transducer and activator of transcription 1 (Phospho-STAT1; monoclonal, clone, 58D6; Cell Signaling Technology, Danvers, MA, USA), and c-Maf (clone EPR16484; Abcam, Cambridge, UK) were used for analyses. Immunohistochemistry for double staining was performed using a Dako Envision + system with dextran polymers conjugated with horseradish peroxidase (Dako), as previously described¹². First, sections were stained with anti-CD68 antibodies for 30 min at room temperature, generating a brown color. Denaturing Solution (BioCare Medical-CA, USA) was added for 5 min at room temperature for elution during double staining. Antigen retrieval was performed by heat treatment for 45 min with HIER T-EDTA Buffer (Dako). After incubation, sections were reacted with phospho-STAT1- or c-Maf-specific reagents using dextran polymers conjugated with horseradish peroxidase (Dako) overnight at 4°C using a Vina Green Chromogen Kit (BioCare Medical-CA), producing green staining. Finally, slides were washed in Wash Buffer (Dako) for 3 min. The sections were counterstained with hematoxylin.

The antibodies used in this study are listed in Supplementary Table 1.

Assessment of immunohistochemical results

The immunopositivity of TAMs was examined for CD68/c-Maf and CD163/phospho-STAT1. Inflammatory cells were carefully excluded from the analysis. Only nuclear positivity for c-Maf and phospho-STAT was considered significant, whereas only cytoplasmic expression of CD68 was regarded as positive. Ten fields were determined to evaluate the positivity of the immunostained cells by visual assessment. The immunostaining intensity (weak, moderate, and strong) was evaluated, and positive cells were defined as those showing greater than moderate intensity. Positive cells were counted under the selected 10 fields, and the total positive cells were determined. In addition, sections showing greater than 1% PD-L1 immunohistochemical expression in the tumor and surrounding immune cells were considered positive. Determination of positive cells was performed by expert diagnostic pathologists (S.S., N.Y., and T.S.) who were blinded to the study endpoint. If results among the pathologists were discordant, a consensus was reached through discussion.

Statistical analysis

Data were analyzed using JMP Pro 16.1 software (SAS). Fisher’s exact tests were used to compare differences in clinicopathological findings between cohorts. Age distributions were compared using Mann-Whitney U tests. Kaplan-Meier analyses were performed using log-rank tests for survival analyses. Results with p values less than 0.05 were considered significant. Univariate and multivariate analyses were conducted using Cox proportional hazards models to identify the variables that significantly predicted overall and disease-free survival. The level of significance was p < 0.05, and the confidence interval was determined at the 95% level.

Immunohistochemistry combining CD68 with antibodies specific for phospho-STAT1 or c-Maf displayed double-positive cells in all cases. The distinction between double-positive cells was observed easily with little or no background staining (Fig. 1). Heterogeneous expression in double-positive cells was observed in whole sections. Additionally, we identified areas with strong desmoplastic fibrosis and immune cells to some extent. Overall, we observed significantly higher numbers of CD68/c-Maf-positive cells (1–156; median, 19) than CD68/phospho-STAT1-positive cells (1–111; median, 6; p < 0.001).

Sensitivity and specificity of predominant CTA based on overall and disease-free survival in cohort 1 and cohort 2

We determined the cut-off values of positive cells to assess correlations with survival in patients using receiver operating characteristic (ROC) curve analysis based on the sensitivity and specificity of the relevant outcome for each positive cell count (Supplementary Fig. 1; cohort 1). If an ROC curve was generated from pairs of weighted mean sensitivities and mean specificities, discrimination of the program for survival was expressed by the area under the curve (Supplementary Fig. 1). According to our results, the cut-off values for CD68/phospho-STAT1- and CD68/c-Maf-positive cells were 5 or less and greater than 11, respectively, for determining overall survival (Supplementary Fig. 1-A and 1-B). Additionally, a cut-off value of 0.19 or less for the M1/M2 ratio was determined for overall survival (Supplementary Fig. 1-C). Detailed data are shown in supplementary Table 2. According to the findings we showed in supplementary Table 2, both cut-off values were generally appropriate.

Kaplan-Meier analyses were performed to determine the associations of disease-free and overall survival with each macrophage phenotype (cohort 2). Patients with low CD68/phospho-STAT1 expression had poorer disease-free and overall survival than patients with high CD68/phospho-STAT1 expression (p < 0.0001; Fig. 2A, B). Additionally, disease-free and overall survival were correlated with high CD68/c-Maf expression (p < 0.0001; Fig. 2C, D) compared with low CD68/c-Maf expression. Finally, patients with low M1/M2 ratios showed poor overall and disease-free survival (Fig. 2E, F).

Associations of clinicopathological findings and markers with survival in cohort 2

In disease-free survival, 8 factors (sex, smoking history, pathological stage, lymphatic invasion, venous invasion, STAS, M1-positive cells, and M2-positive cell) were observed, and 5 factors, i.e., pathological stage, lymphatic invasion, STAS, M1-positive cells, and M2-positive cell, were retained (Table 2). In overall survival, 9 factors, including sex, smoking history, age, pathological stage, lymphatic invasion, venous invasion, IASLC/ATS/ERS, M1-positive cells, and M2-positive cells, were found in univariate analysis. However, only pathological stage, M1-positive cells, and M2-positive cells were retained in multivariate analysis (Table 3).

Table 2

Association of clinicopathological variables, PD-L1, M1 and M2 with disease-free survival in univariate and multivariate analyses.
		Univariate analysis			Multivariate analysis
Factor		HR	(95%CI)	p-value	HR	(95%CI)	p-value
Sex	Male vs. female	1.7	(1.17–2.48)	0.0054	1.23	(0.77–1.97)	0.3923
Smoke	Smoker vs. never	1.76	(1.21–2.56)	0.0034	1.18	(0.72–1.94)	0.5033
Age	≥ 70 years vs. <70 years	1.06	(0.73–1.54)	0.7575
pStage	II and III vs I	4.12	(2.80–6.06)	< 0.0001	2.66	(1.68–4.23)	< 0.0001
Lymphatic invasion	Positive vs. negative	4.05	(2.65–6.17)	< 0.0001	1.98	(1.20–3.25)	0.0074
Venous invasion	Positive vs. negative	2.59	(1.71–3.93)	< 0.0001	1.28	(0.80–2.04)	0.3026
STAS	Positive vs. negative	2.29	(1.57–3.34)	< 0.0001	1.65	(1.10–2.48)	0.0153
IASLC/ATS/ERS classification subtypes	High group vs. low and intermediate group	1.54	(0.99–2.4)	0.0582
PD-L1 (TC)	Positive vs. negative	1.07	(0.65–1.76)	0.7848
PD-L1 (IC)	Positive vs. negative	1.25	(0.76–2.05)	0.3755
CD68+/p-STAT1+(M1)	Low vs. high	4.41	(2.89–6.73)	< 0.0001	4.68	(3.00-7.31)	< 0.0001
CD68+/c-Maf+(M2)	High vs, low	1.78	(1.17–2.71)	0.0069	2.38	(1.54–3.70)	0.0001
PD-L1, Programmed cell Death 1-Ligand 1; M1, M1 macrophage; M2, M2 macrophage; HR, hazard ratio; 95%CI, 95% confidence interval; pStage, pathological Stage; STAS, Spread through air spaces; IASLC, International Association for the Study of Lung Cancer; ATS, American Thoracic Society; ERS, European Respiratory Society; TC, Tumor cells; IC, Tumor-infiltrating immune cells; p-STAT1, phospho-STAT1.

Table 3

Association of clinicopathological variables, PD-L1, M1 and M2 with overall survival in univariate and multivariate analyses.
		Univariate analysis			Multivariate analysis
Factor		HR	(95%CI)	p-value	HR	(95%CI)	p-value
Sex	Male vs. female	2.08	(1.31–3.30)	0.0019	1.48	(0.81–2.69)	0.2021
Smoke	Smoker vs. never	2.02	(1.27–3.21)	0.003	1.29	(0.69–2.44)	0.4234
Age	≥ 70 years vs. <70 years	1.67	(1.05–2.65)	0.0303	1.39	(0.85–2.27)	0.1893
pStage	II and III vs I	3.66	(2.30–5.83)	< 0.0001	1.95	(1.12–3.39)	0.0177
Lymphatic invasion	Positive vs. negative	3.7	(2.28-6.00)	< 0.0001	1.32	(0.68–2.55)	0.405
Venous invasion	Positive vs. negative	2.01	(1.21–3.33)	0.007	1.06	(059 − 1.90)	0.8541
STAS	Positive vs. negative	1.44	(0.90–2.28)	0.1246
IASLC/ATS/ERS classification subtypes	High group vs. low and intermediate group	2.49	(1.54–4.02)	0.0002	1.66	(0.93–2.96)	0.0846
PD-L1 (TC)	Positive vs. negative	1.19	(0.67–2.12)	0.5654
PD-L1 (IC)	Positive vs. negative	1.23	(0.69–2.20)	0.4811
CD68+/p-STAT1+(M1)	Low vs. high	11.67	(5.60-24.31)	< 0.0001	10.81	(5.07–23.03)	< 0.0001
CD68+/c-Maf+(M2)	High vs, low	2.07	(1.22–3.51)	0.0072	2.46	(1.42–4.26)	0.0013
PD-L1, Programmed cell Death 1-Ligand 1; M1, M1 macrophage; M2, M2 macrophage; HR, hazard ratio; 95%CI, 95% confidence interval; pStage, pathological Stage; STAS, Spread through air spaces; IASLC, International Association for the Study of Lung Cancer; ATS, American Thoracic Society; ERS, European Respiratory Society; TC, Tumor cells; IC, Tumor-infiltrating immune cells; p-STAT1, phospho-STAT1.

Association of clinicopathological findings and markers with survival in cohort 2

Next, we examined the associations of clinicopathological findings with disease-free survival in LAD. Univariate analysis identified 7 factors, i.e., sex, smoking history, pathological stage, lymphatic invasion, venous invasion, STAS, and M1/M2 ratio (Table 4). Four factors, i.e., pathological stage, lymphatic invasion, STAS, and M1/M2 ratio, were retained in multivariate analysis using a Cox proportional hazards model (Table 4). Similarly, 8 factors, i.e., sex, smoking history, age, pathological stage, lymphatic invasion, venous invasion, IASLC/ATS/ERS, and M1/M2 ratio, were identified, and four factors, i.e., pathological stage, lymphatic invasion, STAS, and M1/M2 ratio, were associated with overall survival in univariate analysis (Table 5). Three factors (pathological stage, lymphatic invasion, and M1/M2 ratio) were also retained in multivariate analysis (Table 5).

Table 4

Association of clinicopathological variables, PD-L1, and M1/M2 ratio with disease-free survival in univariate and multivariate analyses.
		Univariate analysis
Factor		HR	(95%CI)	p-value	HR	(95%CI)	p-value
Sex	Male vs. female	1.7	(1.17–2.48)	0.0054	1.21	(0.73-2.00)	0.4701
Smoke	Smoker vs. never	1.76	(1.21–2.56)	0.0034	1.2	(0.71–2.02)	0.4902
Age	≥ 70 years vs. <70 years	1.06	(0.73–1.54)	0.7575
pStage	II and III vs I	4.12	(2.80–6.06)	< 0.0001	2.77	(1.75–4.38)	< 0.0001
Lymphatic invasion	Positive vs. negative	4.05	(2.65–6.17)	< 0.0001	2.48	(1.51–4.07)	0.0003
Venous invasion	Positive vs. negative	2.59	(1.71–3.93)	< 0.0001	1.15	(0.73–1.81)	0.5402
STAS	Positive vs. negative	2.29	(1.57–3.34)	< 0.0001	1.56	(1.04–2.36)	0.0326
IASLC/ATS/ERS classification subtypes	High group vs. low and intermediate group	1.54	(0.99–2.4)	0.0582
PD-L1 (TC)	Positive vs. negative	1.07	(0.65–1.76)	0.7848
PD-L1 (IC)	Positive vs. negative	1.25	(0.76–2.05)	0.3755
M1/M2 ratio	Low vs. high	2.74	(1.88–3.99)	< 0.0001	2.78	(1.87–4.11)	< 0.0001
PD-L1, Programmed cell Death 1-Ligand 1; M1, M1 macrophage; M2, M2 macrophage; HR, hazard ratio; 95%CI, 95% confidence interval; pStage, pathological Stage; STAS, Spread through air spaces; IASLC, International Association for the Study of Lung Cancer; ATS, American Thoracic Society; ERS, European Respiratory Society; TC, Tumor cells; IC, Tumor-infiltrating immune cells.

Table 5

Association of clinicopathological variables, PD-L1, and M1/M2 ratio with overall survival in univariate and multivariate analyses.
		Univariate analysis			Multivariate analysis
Factor		HR	(95%CI)	p-value	HR	(95%CI)	p-value
Sex	Male vs. female	2.08	(1.31–3.30)	0.0019	1.62	(0.85–3.09)	0.1418
Smoke	Smoker vs. never	2.02	(1.27–3.21)	0.003	1.27	(0.65–2.50)	0.4855
Age	≥ 70 years vs. <70 years	1.67	(1.05–2.65)	0.0303	1.51	(0.94–2.44)	0.091
pStage	II and III vs I	3.66	(2.30–5.83)	< 0.0001	2.53	(1.46–4.40)	0.001
Lymphatic invasion	Positive vs. negative	3.7	(2.28-6.00)	< 0.0001	2.34	(1.26–4.33)	0.007
Venous invasion	Positive vs. negative	2.01	(1.21–3.33)	0.007	0.98	(0.57–1.69)	0.9468
STAS	Positive vs. negative	1.44	(0.90–2.28)	0.1246
IASLC/ATS/ERS classification subtypes	High group vs. low and intermediate group	2.49	(1.54–4.02)	0.0002	1.06	(0.60–1.85)	0.8462
PD-L1 (TC)	Positive vs. negative	1.19	(0.67–2.12)	0.5654
PD-L1 (IC)	Positive vs. negative	1.23	(0.69–2.20)	0.4811
M1/M2 ratio	Low vs. high	3.95	(2.48–6.28)	< 0.0001	3.86	(2.37–6.29)	< 0.0001
PD-L1, Programmed cell Death 1-Ligand 1; M1, M1 macrophage; M2, M2 macrophage; HR, hazard ratio; 95%CI, 95% confidence interval; pStage, pathological Stage; STAS, Spread through air spaces; IASLC, International Association for the Study of Lung Cancer; ATS, American Thoracic Society; ERS, European Respiratory Society; TC, Tumor cells; IC, Tumor-infiltrating immune cells.

Macrophages are activated into M1 (classically activated) or M2 (alternatively activated) phenotypes^8,12. When examining the roles of the macrophage phenotype in tissue samples, immunostaining of the phenotype is often used. Nitric oxide synthase 2, Toll-like receptor (TLR) 2, TLR4, CD80, and CD86 have been used as M1 markers, whereas CD115, CD206, arginase 1, CD163, and CD301 have been recognized as M2 markers^17,24. However, it is still difficult to definitively distinguish M1 from M2 macrophages²⁴ because these markers can also be expressed in other cells, including lymphocytes and leukocytes^14,15,23. Moreover, macrophages can express some markers simultaneously owing to the continuum of phenotypes between M1 and M2^14,15,24. To overcome this problem, double immunostaining for specific phenotype markers and pan macrophage markers is necessary for evaluation of the macrophage phenotype in tissue samples¹². Identification of M1 and M2 macrophages relies on the accurate selection of markers to differentiate M1 from M2 macrophages¹². In this study, we used CD68/phospho-STAT1 for M1 macrophages and CD68/c-Maf for M2 macrophages.

In response to interferon, STAT1 is upregulated, and its phosphorylated form binds to the promoter region of interferon-stimulated genes²⁵. A predominance of STAT1 activation promotes the M1 polarization of TAMs by increasing pro-inflammatory cytokines^7,24, suggesting that STAT1 may be an M1 marker. TAMs isolated from STAT1-knockout mice fail to suppress T-cell responses^7,24,25. These cells lack arginase I activity, which reduces nitric oxide production from inducible nitric oxide synthase (iNOS)^{25, 26}. Taken together, these data indicate that STAT1 activation in TAMs may be responsible for upregulation of iNOS and arginase I activity, thereby resulting in T-cell suppression²⁵. However, it is unclear what factors of the tumor microenvironment are responsible for STAT1 upregulation in TAMs²⁵. In this study, low expression of the CD68/phospho STAT1-positive TAM phenotype was found to be a poor prognostic factor and an independent factor predicting prognosis. In a previous study, STAT1 expression in macrophages identified patients with improved survival and an intact tumor immune system, which may benefit from immunotherapy²⁵. Thus, our current findings contributed to elucidation of the role of TAM1 (M1) infiltration in the invasive tumor area.

c-Maf is essential for macrophage self-renewal but is also expressed in T cells, including Th2 and Th17 cells^27–29. Liu et al. identified c-Maf as an essential regulator for immunosuppressive macrophage polarization²⁷ and showed that c-Maf is predominantly expressed in M2-like macrophages in both mice and humans²⁷. Furthermore, inhibition of c-Maf in macrophages results in an M1-like phenotype with diminished immunosuppressive function and promotes antitumor T-cell immunity, leading to significantly reduced tumor progression²⁷. Thus, c-Maf is a core molecule in immunosuppressive macrophage polarization. In this study, we showed for the first time that high expression of CD68/c-Maf -positive macrophages was correlated with prognosis in patients with LAD, similar to the findings of a previous report in cervical cancer²⁸.

In this study, PD-L1 expression in cancer cells and surrounding immune cells was not correlated with survival in patients with LAD, suggesting that PD-L1 expression cannot predict outcomes in these patients. Although cancer immunotherapy using anti-PD-L1 immunocheckpoint inhibitors has been widely applied in patients with non-small cell lung cancer, many patients are resistant to such treatment^29–33. Inhibition of c-Maf may contribute to overcoming such resistance²⁷. Indeed, anti-PD-1 therapy combined with c-Maf inhibition significantly reduces tumor progression²⁷. Additionally, immunomodulators that can specifically target c-Maf in macrophages may be promising because c-Maf is the critical transcription factor for many immune cell subsets²⁷. Targeting patients with high expression of c-Maf-positive macrophages may offer a novel strategy to reinforce current cancer immunotherapies, such as immunocheckpoint-inhibitor therapy.

Quantifying the M1/M2 ratio in TAMs could be used to evaluate macrophage polarization for clinical applications³⁴. The M1/M2 ratio is a more biologically relevant indicator of cancer prognosis compared with M1 or M2 density alone^34,35. This ratio could represent either a positive or a negative impact on tumor growth. A lower M1/M2 ratio often indicates a poor prognosis in patients with cancer, whereas a better prognosis is associated with a higher M1/M2 ratio^34–36. In the current study, we found an M1/M2 ratio of 0.19, suggesting that at diagnosis the tumor microenvironment is mainly polarized toward an M2 phenotype³⁴. These data are consistent with previous studies suggesting that M2 macrophages are involved in cancer development in several human malignancies, including cervical cancer²⁸. Further studies are needed to evaluate the mechanisms related to this macrophage phenotype ratio.

There were some limitations to this study. First, histological heterogeneity of M1 and M2 phenotypes may make it difficult to evaluate the expression patterns of macrophage-specific proteins. The specialization of macrophages in microenvironments explains their heterogeneity. Moreover, the heterogeneous functional properties of macrophages could result from their locations in tumor tissues^{37, 38}. There are three typical locations, i.e., the tumor center, invasive front (interface between tumor cells and stroma), and tumor stroma³⁷. Notably, we found that the distribution pattern of macrophages could be an independent prognostic factor in gastric cancer³⁷. According to the relative macrophages’ densities in the tumor nest or tumor stroma, the examined gastric cancer cases could be divided into nest-dominant and stroma-dominant patterns³⁷. Patients with a stroma-dominant pattern tended to have poorer survival and higher malignancy, and macrophages accumulating in the tumor stroma may participate more actively in stroma activation³⁷. Second, we did not validate the findings in a second cohort because the first cohort was large. However, we believe that the current cohort was an adequate size for evaluating outcomes in patients with LAD.

In conclusion, our data suggested that high CD68/c-Maf expression was a worse predictor in patients with LAD and that low CD68/phospho-STAT1 expression was also a poor prognostic factor in LAD. Finally, a low M1/M2 ratio may contribute to prediction of outcomes, suggesting that a shift from the M2 to M1 phenotype in TAMs may be related to worse survival in patients with LAD. Double-staining with these markers may be suitable for identifying M1 and M2 phenotypes in histological samples.

Declarations

Ethical approval

and consent to participate

Informed consent

was obtained from each patient according to institutional guidelines, and the research protocols were approved by the ethics committee of Iwate Medical University Hospital (approval number MH2021-068).

Declarations

Conflict of interest statement

We declare no conflicts of interest.

Consent for publication

Not applicable.

Funding

No funding was received for this study.

Author contributions

S. Shikanai, who is first author, constructed the figures and tables and performed the statistical analyses. T. Sugai, who is the corresponding author, contributed to the preparation of the manuscript, including all aspects of the data collection and analysis. N. Yamada helped immunohistochemical staining. M. Osakabe assisted with the statistical analyses. N. Yanagawa and M. Sugai helped with interpretation of the pathological results. H. Saito and M. Maemondo provided clinical support during the preparation of the manuscript.

Acknowledgments

We gratefully acknowledge the technical assistance and support of members of the Department of Molecular Diagnostic Pathology, Iwate Medical University.

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There is NO conflict of interest to disclose.

SugaiTSupplementaryTablesM1M2LAD.docx
Supplementaryfigure1.tif
Supplementary Figure 1. ROC curves for CD68+/phospho-STAT1+ cells, CD68+/c-Maf+ cells, and M1/M2 ratios based on overall survival. A. ROC curve for CD68+/phospho-STAT1+ cells. B. ROC curve for CD68+/c-Maf+ cells. C. ROC curve for the M1/M2 ratio.

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Prognostic impact of tumor-associated macrophage-related markers in patients with adenocarcinoma of the lung

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Abstract

Figures

Introduction

Materials and Methods

Patients

Determination of overall survival and disease-free survival

Sample size determination

Chemotherapeutic treatment after surgery for LAD

Construction of tissue microarrays (TMAs)

Immunohistochemistry for PD-L1

Double staining immunohistochemistry

Assessment of immunohistochemical results

Statistical analysis

Results

Associations of clinicopathological findings and markers with survival in cohort 2

Association of clinicopathological findings and markers with survival in cohort 2

Discussion

Declarations

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Funding

Author contributions

Acknowledgments

References

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Supplementary Files

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