The landscape and clinical impact of tumor-associated macrophages and PD-L1 in primary breast cancers and their brain metastases

Abstract

Background: Tumor-associated macrophages (TAMs) influence the tumor microenvironment and can contribute to tumor progression. They can polarize into M1 (classically activated) or M2 (alternatively activated) phenotype, which exhibit divergent functional characteristics. The comparison of TAMs between primary breast cancer (BC) and corresponding brain metastases (BMs) remains insufficiently explored and is the focus of this study.

Methods: This study aimed to compare the infiltration of TAMs and PD-L1 expression in primary breast cancer and their brain metastases, by analyzing 27 paired samples and 26 additional brain metastases. Immunohistochemical staining was performed for the following markers: CD68, CD86 (M1), CD163 (M2), and PD-L1.

Results: CD68 showed significantly higher expression levels in brain metastases compared to the corresponding primary breast cancers. In contrast, the expression of CD86 and CD163 showed comparable results between the primary tumors and their brain metastatic counterparts. Macrophages were consistently found to be more frequently present in the tumor stroma compared to the tumor nest. Survival analysis of the primary revealed that high expression of CD163 was associated with a recurrence-free survival. (RFS). Conversely, high expression of CD86 in brain metastases was associated with longer overall survival. Low expression of CD68 and CD163 in brain metastases correlated with the presence of meningeal carcinomatosis. The expression of PD-L1 in the primary tumor did not necessarily reflect the status of PD-L1 in the corresponding brain metastases.

Conclusions: Overall, this study highlights the complex influence of TAMs on the course of primary breast cancers and their brain metastases. The discordant expression of the immune checkpoint molecule PD-L1 underscores the importance of evaluating the PD-L1 status in cerebral metastases to guide potential immunotherapeutic approaches.

Keywords: CD163; CD68; CD86; PD-L1; TAMs; brain metastases; breast cancer; tumor-associated macrophages.

Figure 1

Staining intensity. Representative micrographs showing weak (1 point), medium (2 points) and strong (3 points) expression of (A) CD68, (B) CD163, (C) CD86. The IRS was subsequently calculated according to the formula: SI (staining intensity) × PP (percentage of positive cells).

Figure 2

Sankey diagram of PD-L1 expression in primary breast cancer and their brain metastases The flow of PD-L1 expression status (positive or negative) across different stages and subtypes of breast cancer progression: from primary tumors to subtypes in both primary tumors and brain metastases, culminating in the PD-L1 expression status in brain metastases.

Figure 3

Correlation of PD-L1 expression between primary breast cancer and brain metastasis. The graphs show scatter plots and corresponding regression lines to illustrate a linear relationship. (A) represents the correlation for the entire cohort. (B–D) show the subgroups: HER2 +, HR+/HER2 -, and Triple-negative. The strength of the correlation is represented by the coefficient of determination (R²), which is displayed along with the p-value and sample size (n) in the upper left corner of the plots.

Figure 4

Marker expression and the recurrence-free survival of breast cancer patients – univariate analysis. (A–G) The expression levels of the markers were dichotomized into ‘low’ and ‘high’ according to the median-split method. Kaplan-Meier curves were generated for the 150-months recurrence-free survival and statistical analysis was performed with the log-rank test. The p-values are indicated in the lower- left corner of each plot. Sample sizes are indicated in the upper-right corner of each plot. TN, tumor nest; TS, tumor stroma.

Figure 5

Marker expression and overall survival of breast cancer patients – univariate analysis. (A–G) The expression levels of the markers were dichotomized into ‘low’ and ‘high’ according to the median-split method. Kaplan-Meier curves were generated for overall survival and statistical analysis was performed with the log-rank test. The p-values are indicated in the lower- left corner of each plot. Sample sizes are indicated in the upper-right corner of each plot. TN, tumor nest; TS, tumor stroma.

Figure 6

Marker expression and brain metastasis survival (BM – Death) – univariate analysis. (A–G) The expression levels of the markers were dichotomized into ‘low’ and ‘high’ according to the median-split method. Kaplan-Meier curves were generated for overall survival and statistical analysis was performed with the log-rank test. The p-values are indicated in the lower- left corner of each plot. Sample sizes are indicated in the upper-right corner of each plot. TN, tumor nest; TS, tumor stroma.