Prognostic Implication of M2 Macrophages Are Determined by the Proportional Balance of Tumor Associated Macrophages and Tumor Infiltrating Lymphocytes in Microsatellite-Unstable Gastric Carcinoma

Abstract

Tumor associated macrophages are major inflammatory cells that play an important role in the tumor microenvironment. In this study, we investigated the prognostic significance of tumor associated macrophages (TAMs) in MSI-high gastric cancers using immunohistochemistry. CD68 and CD163 were used as markers for total infiltrating macrophages and M2-polarized macrophages, respectively. The density of CD68+ or CD163+ TAMs in four different areas (epithelial and stromal compartments of both the tumor center and invasive front) were analyzed in 143 cases of MSI-high advanced gastric cancers using a computerized image analysis system. Gastric cancers were scored as "0" or "1" in each area when the density of CD68+ and CD163+ TAMs was below or above the median value. Low density of CD68+ or CD163+ macrophages in four combined areas was closely associated with more frequent low-grade histology and the intestinal type tumor of the Lauren classification. In survival analysis, the low density of CD163+ TAMs was significantly associated with poor disease-free survival. In multivariate survival analysis, CD163+ TAMs in four combined areas, stromal and epithelial compartments of both tumor center and invasive front were independent prognostic indicator in MSI-high gastric cancers. In addition, the density of CD163+ TAMs correlated with tumor infiltrating lymphocytes (TILs). Our results indicate that the high density of CD163+ TAMs is an independent prognostic marker heralding prolonged disease-free survival and that the prognostic implication of CD163+ TAMs might be determined by the proportional balance of TAMs and TILs in MSI-high gastric cancers.

Fig 1. Representative image of the IF and TC in MSI-H advanced GCs.

H&E section of GCs (original magnification, 12.5x) (top) showing each regions of the tumor: IF and TC. Immunohistochemical staining for CD68 and CD163 in each region (bottom). Abbreviations: IF, invasive front; TC, tumor center; MSI-H, microsatellite instability-high; GC, gastric cancer.

Fig 2. Immunohistochemical staining of CD68 and CD163 and measurement of the density of CD68+ and CD163+ TAMs.

Using the automatic image analysis system (ScanScope XT; Aperio) for positive pixel count v9 algorithm, the density of CD68+ or CD163+ TAM was measured separately in the epithelium (left) and stroma (right).

Fig 3. Density of CD68+ TAMs (a) stroma^high/epithelium^low, (b) stroma^low/epithelium^high, (c) stroma^low/epithelium^low and (d) stroma^high/epithelium^high.

Fig 4. Development of the scoring system.

The total score was determined by adding the scores of four different areas (S and E compartments of IF and TC regions). In addition to the total score, analysis of the compartments (S or E) and regions (IF or TC) was conducted by adding the scores of two paired areas, e.g., to determine the score of IF. We added the scores in S at IF and E at IF, which ranged from 0 to 2. For the S compartments, the scores in S at IF and S at TC were added. The same method was applied in scoring of the densities of TAMs of the other two combined areas–TC (STC + ETC) and E (EIF + ETC). Abbreviations: S, stroma; E, eithelium; IF, invasive front; TC, tumor center.

Fig 5. Box plots comparing the density of CD68+ or CD163+ TAMs according to different tumor areas (TC, IF, S and E)

(a) The density of CD68+ TAMs was significantly higher in IF and S rather than in TC and E, respectively. (b) The density of CD163+ TAMs tends to be higher in IF than in TC and is significantly higher in S than in E. Statistical significance was evaluated using a Wilcoxon signed-rank test. Abbreviations: TC, tumor center; IF, invasive front; S, stroma; E, epithelium.

Fig 6. Kaplan-Meier survival analysis with log-rank test of CD68+ TAMs.

(a) Survival curves of the low density (score 0) (n = 44) vs. high density (score 1 and 2) (n = 98) groups in IF (SIF + EIF). (b) Survival curves of the low density (score 0) (n = 36) vs. high density (score 1 and 2) (n = 95) groups in TC (STC + ETC). (c) Survival curves of the low density (score 0) (n = 47) vs. high density (score 1 and 2) (n = 89) groups in S (SIF+STC). (d) Survival curves of the low density (score 0) (n = 51) vs. high density (score 1 and 2) (n = 75) groups in E (EIF+ETC). (e) Survival curves of five subgroups determined by the total score in four combined areas (SIF + STC + EIF + ETC) (I, score 0 (n = 24); II, score 1 (n = 22); III, score 2 (n = 32); IV, score 3 (n = 25) and V, score 4 (n = 21)). (f) Survival curves of the low density (score 0) (n = 24) vs. high density (score 1–4) (n = 100) groups in four combined areas (SIF + STC + EIF + ETC). Abbreviations: TC, tumor center; IF, invasive front; S, stroma; E, epithelium.

Fig 7. Kaplan-Meier survival analysis with log-rank test of CD163+ TAMs.

(a) Survival curves of the low density (score 0) (n = 38) vs. high density (score 1 and 2) (n = 92) groups in IF (SIF + EIF). (b) Survival curves of the low density (score 0) (n = 51) vs. high density (score 1 and 2) (n = 85) groups in TC (STC + ETC). (c) Survival curves of the low density (score 0) (n = 41) vs. high density (score 1 and 2) (n = 92) groups in S (SIF+STC). (d) Survival curves of the low density (score 0) (n = 52) vs. high density (score 1 and 2) (n = 78) groups in E (EIF+ETC). (e) Survival curves of the five subgroups determined by the total score in four combined areas (SIF + STC + EIF + ETC) (I, score 0 (n = 25); II, score 1 (n = 25); III, score 2 (n = 26); IV, score 3 (n = 25) and V, score 4 (n = 27)). (f) Survival curves of the low density (score 0) (n = 25) vs. high density (score 1–4) (n = 103) groups in four combined areas (SIF + STC + EIF + ETC). Abbreviations: TC, tumor center; IF, invasive front; S, stroma; E, epithelium.

Fig 8. (a, b) Box plots illustrating densities of CD8+ or FoxP3+ TILs in dependence of CD163+ TAMs density in IF regions (Mann-Whitney U test). (c, d) Association between the density of CD8+ or FoxP3 TILs and the density of CD163+ TAMs (Spearman’s rank correlation test).

* The density of CD8+ and FoxP3+ TILs is shown as the number of infiltrated lymphocytes per unit area (mm²) in IF irrespective of S or E compartment. Abbreviations: TIL, tumor infiltrating lymphocyte; IF, invasive front; S, stroma; E, epithelium.

Fig 9. Kaplan-Meier survival analysis with log-rank test according to combinatory statuses of CD8+ or FoxP3+ TILs and CD163+ TAMs.

(a) Survival curves of the low density (n = 70) vs. high density (n = 73) groups of CD8+ TILs in IF. (b) Survival curves of the low density (n = 72) vs. high density (n = 71) groups of FoxP3+ TILs in IF. (c) Survival curves of CD8+^high /CD163+^high (n = 52) vs. CD8+^high / CD163+^low or CD8+^low /CD163+^high (n = 45) vs. CD8+^low /CD163+^low (n = 37) (d) Survival curves of FoxP3+^high /CD163+^high (n = 52) vs. FoxP3+^high / CD163+^low or FoxP3+^low /CD163+^high (n = 39) vs. FoxP3+^low /CD163+^low (n = 38). Abbreviations: TIL, tumor infiltrating lymphocyte; TC, tumor center; IF, invasive front; S, stroma; E, epithelium; H, high density; L, low density.