In this study, we evaluated multiple immune cell fractions in both breast cancer tissues and matched blood samples, and then observed a partial correlation in the composition of immune cells at the tumor site and in the peripheral blood. In the comprehensive analysis of the association between each immune cell lineage fraction with hTIL and hPD-L1, we found for the first time that innate immune responses were poor or had disappeared in patients with clinically diagnosed breast cancers, especially in cases with higher hTIL or positive hPD-L1, and that acquired immune responses were active. We also found that non-B-cell antigen-presenting cell fractions were involved primarily in the PD-L1 pathway in breast cancer microenvironments.
To the best of our knowledge, most of the analyses of the immune cell compositions of breast cancer tissues using a multicolor FCM had 10 colors or less [32, 37], and there were only two studies with more than 11 colors [38, 39]. Although the reactivities of the labeled antibodies were not always the same, and a direct comparison was not possible, a similar distribution of the leukocyte infiltrations was observed in the tumor tissue in our study and a previous study with a distribution median of 218 CD45+ TIL/mg of tumor tissue (interquartile range: 85–445 CD45+ TIL/mg) . Although there are very few reports of systematic examinations of leukocyte compositions in breast cancer tissue, studies have reported the ratio of total T to be 86% (mean)  or 75% (median)  of the leukocytes (CD45+ cells) in breast cancer tissues, suggesting that T cells account for the majority of TILs . In our study, the proportion of total T cells in the leukocytes in the tumors was 57.3% (mean), which was slightly lower than that in previous reports, probably owing to the difference in the antibody used and gating strategy. In three previous studies, the proportions of CD19+ B cells in CD45+ TIL were found to be 8% (mean), 4.58% (median), and approximately 10% (mean), respectively [32, 37, 38], which were similar to our results. With regard to other lineages, the findings of a previous study showed that the proportions of CD14+/CD40+/CD163+ M2 macrophages were 0.06% (median), CD11b+/CD15+/HLA-DR-MDSCs were 1.19% (median), and CD56+ NK were 2.33% (median) . However, the studies were few, and the definitions of each lineage did not match those in our study; therefore, valid comparisons could not be made. No comparable reports were found for the remaining lineages.
This is the first study to show an association between the immune cell composition of blood and that of breast cancer tissues. The immune cell composition of blood showed a partial correlation with the tumor tissues and the percentages of the immune cell fractions showed certain differences between the tumor tissues and blood. Although this suggested that the composition of tumor-infiltrating immune cells may be estimated using blood samples, there were significant differences between them.
Although there was a significant difference between the subtypes, hTIL was observed to be correlated with certain clinicopathological factors, including subtypes [3–5], prognoses, and responses to chemotherapy [4, 6, 7], in breast cancer. In our study, higher hTIL scores were associated with high-grade tumors, ER negativity, higher Ki67 positive ratios, and hPD-L1 positivity (Online Resource Table S6). Numerous studies have also reported that ER-positive breast cancer is the least immune-infiltrated subtype, which is consistent with our results [2, 5]. However, there are certain controversies regarding other clinicopathological factors, and results vary among different studies [2, 4, 5, 41]. No studies have systematically assessed the relationship between hTIL and the immune cell fraction using FCM. In this study, we demonstrated that hTIL was associated with the degree of leukocyte infiltration in the tumor tissues and leukocyte composition. We found that although there were positive correlations between the hTIL scores and percentages of total T, CD4+ T, and CD8+ T, there were negative correlations between the hTIL scores and NK and NKT. Therefore, we speculate that a higher hTIL reflects the amount of immune cell infiltration, and the state in which acquired immunity is activated, relative to innate immunity in clinically diagnosed breast cancers.
As mentioned previously, PD-L1 plays a significant role in immune tolerance mechanisms [8, 9], and its expression is suggested to reflect ongoing (or active) immune responses in addition to immunosuppression via the PD-1/PD-L1 pathway . hPD-L1 was shown to correlate with certain clinicopathological factors, including subtypes . It is also a clinically approved predictive marker for atezolizumab in triple-negative advanced breast cancer . In the present study, hPD-L1 positivity was associated with ER negativity and relatively high hTIL scores but not other factors, probably owing to the small cohort size (Online Resource Table S7). Although PD-L1 expression in multiple types of immune cells or tumor cells has been reported [8, 9], there is no consensus as to which immune cell fraction is responsible for the substantial function of the PD-L1 pathway in breast cancer. The findings of only one report that evaluated PD-L1 expression in CD4 + T, CD8 + T, and B cells showed that the overall proportion of the PD-L1 positive TILs was very low and could only be detected in a small number of tumors . In the present study, we found that a substantial proportion of PD-L1 positive immune cells consisted of non-B-cell antigen-presenting cell fractions, such as Mo/Mφ, CD16+ Mo, MDSC, DC, and mDC fractions, and that the PD-L1 positive ratios were significantly higher in tumor tissues than in blood, suggesting that these fractions were involved primarily in the PD-L1 pathway in breast cancer tissue. Additionally, we found that hPD-L1 positive tumors exhibited increased leukocyte infiltration in tumor tissues, and that hPD-L1 reflected PD-L1 expression in Mo/Mφ, CD16+ Mo, DC, and mDCs. These results suggested that hPD-L1 expression indicates the activation status of the immune tolerance mechanism that occurs in non-B-cell antigen-presenting cells in response to increased immune cell infiltration, mainly effector cells that secrete interferon-gamma to induce PD-L1 expression on various cells, into the breast cancer microenvironment.
The FCM findings will be useful in the exploration of new immune-related factors in breast cancer. Briefly, by evaluating the expression of candidate proteins related to tumor immunity by IHC and analyzing them along with these data, the function of candidate proteins may be verified. We are now focusing on some candidate proteins as immuno-regulatory factors in breast cancer and planning further analyses of the FCM data to validate their immunological functions.
This study had several limitations. First, the number of patients enrolled was relatively small. Second, a pilot study empirically found that the number of cells required for FCM was not sufficient in cases of ER-positive breast cancer, especially in cases with lower Ki67s. Third, cases of small tumor sizes and post-N-acetylcysteine with pathological complete response were excluded owing to technical problems in the collection of the tumor tissues. Therefore, there was an inevitable bias in the enrollment of the cases; it differed from the general breast cancer cohort in terms of larger invasive tumor sizes, more ER-negative cases, and higher Ki67 cases (Online Resource Table S2). Finally, although, as mentioned above, the significance of the TIL is suggested to vary between subtypes, a subgroup analysis could not be performed because of the small sample size. In future studies, the collection of more samples and performance of more detailed analyses are recommended.