Background
The recent success of immunotherapies has increased interest in the immune status of breast cancer [
1,
2]. Tumor-infiltrating lymphocytes (TILs) represent a mechanism for assessment of immune status. Studies have shown that TILs are prognostic, particularly for estrogen receptor–negative (ER
−) and highly proliferative ER
+ cancers [
3‐
6]. Despite their prognostic value, high TILs counts are found in only a small subset of breast carcinomas [
6‐
9] whereas macrophages are the most common immune cells [
10]. Tumor-associated macrophages (TAMs) are pleiotropic regulators of tumor cells and microenvironment, modulating tumor growth, activation, and response to therapy [
11‐
16]. Novel immunomodulatory agents specifically targeting TAM proteins, such as colony-stimulating factor 1 receptor (CSF-1R) and matrix metalloproteinase-9 (MMP-9), are currently in the pipeline and/or under clinical testing as mono-therapy or in combination with conventional established therapies and/or immune checkpoint inhibitors. However, TAM biomarkers’ potential in companion diagnostics remains unclear. Unlike TILs, TAMs cannot be assessed by standardized methods on hematoxylin/eosin (H&E) slides. Although they can be seen, they are largely ignored with respect to morphologic diagnostics. Similarly, their molecular assessment has been shown to be highly variable and highly heterogeneous, resulting in the lack of adequate cell models and discrepancies between murine models and human macrophage biologic features [
17].
Conventionally, TAMs have been divided into M1 and M2 subtypes to define their polarization status. In general, M1-polarized macrophages mediate resistance to intracellular pathogens and tumors (Th1-driven responses) whereas M2-polarized macrophages mediate resistance to parasites, immunoregulation, tissue repair, and immuno-tolerance against tumors. However, this conventional M1/M2 dichotomy is controversial and not consistently representative of the TAM functional continuum [
14]. Previous reports have associated TAMs with outcome of breast cancer patients but with contradictory results [
18‐
23]. In most cases, their prognostic assessment is limited by
in situ single-marker, semi-quantitative chromogenic detection of “traditional” biomarkers and their M1/M2-like features (for example, CD68, CD163, metalloproteinases, and arginase) or within high-throughput genomic data, lacking key spatial context, neither of which has seen adoption in the clinical setting.
Within proteins that are differentially expressed in M1- and M2-like TAM subtypes, we were particularly interested in MMP-9, a member of MMP family, because it has been shown to play a role in extracellular matrix remodeling and invasion in breast cancer. Specific MMP-9 inhibitors, such as GS-5745 (Andecaliximab) [
24,
25], are being tested in clinical trials in combination with chemotherapy or immune checkpoint inhibitors in order to block paracrine signaling and metastasis and to alter the immune microenvironment within the tumor. In preclinical models, inhibition of MMP-9 has been shown to inhibit immune-suppressive myeloid cell polarization, regulatory T cells, desmoplastic reaction, and effector T-cell trafficking. These data suggest that MMP-9 inhibition could modulate immune suppression. In breast cancer, however, MMP-9 has been traditionally studied as a tumor cell–derived peptidase and, to a lesser extent, as an immune cell protein, participating in regulation of tumor microenvironment and immune cell infiltrate.
Here, we have objectively and simultaneously measured
in situ the expression of TAM biomarkers (CD68, CD163, and the druggable target MMP-9) in two distinct breast cancer cohorts by using the validated quantitative immunofluorescence (QIF) AQUA method [
26]. We compared our results with mRNA expression data from the largest available breast cancer series (METABRIC) to evaluate whether protein expression combined with spatial distribution would be more informative as biomarker. Our objectives were to subclassify TAMs as the most prevalent breast cancer immune cells and to determine how their polarization is associated with breast tumors’ molecular phenotype and patients’ outcome and how these features could be further exploited in pharmacologic modulation of macrophage function.
Discussion
Breast cancer intervention strategies have been traditionally tumor cell–centered. Recent approaches endorse a paradigm shift encompassing interactions between tumor cells and microenvironment aiming to overcome resistance to treatment but also improve efficiency and long-term effects of therapeutic approaches. Introduction of immune-related markers to breast cancer management, such as TILs, has been proven to be a useful predictive tool, especially for achievement of pathologic complete response following treatment [
3‐
5,
9,
29‐
31]. Immunotherapy (especially PD-axis targeting) has revolutionized the management of many solid tumors, and recent data from early and advanced stage breast cancer trials are encouraging [
1,
2]. Hence, in breast cancer, compared with other neoplasms, PD-L1 expression levels are relatively low (about 15–30% of cases) [
7], and lymphocyte infiltration in most breast tumors is modest [
6,
9]. Therefore, in addition to manipulation of the adaptive immune system, inclusion of the innate arm of the immune system, where TAMs play an important role, might result in better tumor management.
Conventionally, macrophage subpopulations have been described as either classically activated (M1, pro-inflammatory, or tumoricidal) or alternatively activated (M2 specialized to suppress inflammation) [
15]. This M1/M2 subgrouping underrepresents the diverse functional spectrum acquired in response to changing environmental stimuli and is not strictly indicative of their anti-tumor or immune-suppressive role. Although both MMP-9 and CD163 have been traditionally related with M2-phenotype, here we show that they do not always correlate with worse prognosis, as previously reported in breast cancer [
20‐
22,
32‐
34]. So far, non-small cell lung, prostate, and colorectal cancer are the notable exemptions where intense TAM infiltration is associated with better outcome [
23]. We also show that ER status is an important determinant of the association of these TAM markers’ expression with outcome. Interestingly, although all three—CD68, CD163, and MMP-9—are preferentially expressed in ER
− and non-luminal tumors, both at protein and mRNA level, MMP-9 is associated with worse outcome only in ER
+ tumors. Although this initially appears to be a paradox, it could be indicative of bypass mechanisms that activate the expression of the protein in TAMs of some ER
+ tumors or induce the recruitment of certain subclasses of TAMs. Indeed, we show that, in ER
+ tumors, MMP-9 is found mostly in CD163
+ TAMs but that, in ER
− tumors, it was higher in all CD68
+ macrophages. This pattern could be indicative of recruitment of specific TAM subtypes or induction of TAM reprogramming (phenotypical and functional polarization) by different tumor cell subtypes, as previously shown in
in vitro [
35‐
38] and breast cancer tissue [
39] studies. It could also underline the importance of further exploration of how this TAM pattern could be affected by established treatment modalities (especially endocrine therapy in ER
+ tumors, chemotherapy/radiotherapy, or immune therapies) or could modulate response to them and how this could be exploited to optimize responses or manipulate resistance [
14‐
16]. We could not establish an association of CD68, CD163, or MMP-9 with HER2 status, which could be partially attributed to the low number of positive 3+ cases (
n = 20, 5%) and high number of unknown/equivocal cases (
n = 106, 26.6%).
In previous studies, TAMs have been evaluated subjectively by semi-quantitative chromogenic methods and several antibodies with different antigen retrieval, titer, and detection systems [
19‐
23]. Consequently, the quantitative approach we use in this work is not directly comparable to that of previous reports. Our discrepancy with previous reports could be attributed to the single biomarker methodology used by other groups and their limitations to detect the M1/M2 dichotomy to capture the net effect of TAM biomarkers on patient survival. Variability in definitions, outcomes, measurements, experimental procedure, antibody titration, validation, and concentration may contribute to heterogeneity between studies. Our data suggest that determination of expression levels of more than one TAM biomarker, identification of co-expression or mutual exclusivity, spatial context (co-localization), and hormone receptor status are important for investigation of their impact on patient prognosis. This could also partially explain the discrepancy we observed in survival evaluation of TAM marker expression between mRNA and QIF. The most representative example is the one of CD163, the hallmark of M2-like phenotype, which would conventionally be expected to represent a worse outcome prognosticator. However, this was not the case when assessed by QIF and we mostly attribute this to the fact that the levels of other proteins, such as MMP-9, should be co-assessed to better reflect the function of TAMs in certain tumors.
There are a number of limitations to this work. Perhaps most significantly, it is based on a retrospective assessment of two, small, single-institution, breast cancer cohorts, both of which are heterogeneously treated. We show only OS data since do not have adequate recurrence data to assess the predictive profile of these biomarkers. Another limitation is that we examined only two M2 markers (MMP-9 and CD163) of the many described that could be co-expressed in these specimens and affect outcome or subclassification. Finally, our cases were represented in TMA format, which may induce under- or over-representation of the marker levels because of tumor heterogeneity. However, the comparable results in most of the co-expression seen in the METABRIC dataset using mRNA measurements in whole-tissue section tumor samples support the validity of our findings.