Targeted Regulation and Cellular Imaging of Tumor-Associated Macrophages in Triple-Negative Breast Cancer: From New Mechanistic Insights to Candidate Translational Applications

Abstract

The complex interplay between immune cells and tumor cells within the tumor microenvironment (TME) can lead to disease progression. Specifically, signals generated in the TME can cause immunosuppression, promoting angiogenesis and immune evasion, which leads to tumor development. The interplay of M1 and M2 macrophage populations that coincide with these tumor markers is particularly important in the TME. Triple-negative breast cancer (TNBC) often presents as advanced disease, and these tumors are also often bereft of recognized molecular targets that can be found in other subtypes, limiting their therapeutic options. However, tumor-associated macrophages (TAMs) infiltration in TNBC is frequently observed. Moreover, a high density of TAMs, particularly M2 macrophages, is associated with poorer outcomes in various cancers, including TNBC. This provides a strong basis for exploiting TAMs as potential therapeutic targets. Specifically, efforts to increase M2 to M1 repolarization are promising therapeutic approaches in TNBC, and four recent studies wherein divergent approaches to target the M2-rich macrophage population and reverse immune subversion are described. These and similar efforts may yield promising diagnostic or therapeutic options for TNBC, a great clinical need.

1. Introduction

The cellular microenvironment of metastatic solid tumors is composed of heterogeneous malignant cells and other supporting nonmalignant cells such as cancer-associated fibroblasts, angiogenic endothelial cells, mesenchymal stem cells, and pericytes, along with lymphoid and myeloid immune cells. The latter includes B- and T-cells, dendritic cells, and tumor-associated macrophages (TAMs).

TAMs form a major component of the tumor microenvironment (TME) and likely are the most abundant cell [1, 2, 3]. Macrophages, which are differentiated from monocytes, are heterogeneous and belong to the inherent myeloid cells present in TME. The roles and phenotypes of macrophages depend on their homeostatic and pathological microenvironment. Macrophages can aid in enhancing immunity by clearing cellular debris and tumor cells, as well as boosting adaptive immunity [4, 5]. In contrast, continued or prolonged activation of macrophages can result in a dysregulated host immune defense, ultimately resulting in pathogenetic outcomes [4].

Macrophages can release soluble factors such as cytokines and stimulate the complement system, contributing to inflammation [6]. In the case of a large tumor volume, the microenvironment can have low oxygen tension and acidic pH to create conditions that are identical to tissue damage seen in inflammatory conditions [7, 8]. In turn, the macrophages within an inflammatory microenvironment can initiate mechanisms to repair the “damaged tissue.” These include triggers to initiate neoangiogenesis, tissue remodeling, and removal of dead and damaged cells as well as promoting immunosuppression [8, 9, 10, 11]. In this regard, tumor growth can behave as an aberrant but complex interaction between tumor cells, immune system, and stromal cells in which proliferating and dying cells coexist similar to a wound [8, 9, 12].

The prognostic value of TAM infiltration in several different kinds of cancer, such as breast [13], lung [14], prostate [15], and gastric [16], has been demonstrated in various studies. Other studies reported on a correlation between TAM density and poor prognosis [17, 18]. Zhao et al. have also demonstrated that increased penetration of TAMs correlated with poor prognosis and reduced patient survival in breast cancer [19]. Further subset analysis has shown differential prognostic association between M1 and M2 macrophage phenotypes. A high M1 phenotype density has been shown to correlate with better prognosis due to the pro-inflammatory role of this subtype. In contrast, predominance of M2 phenotype correlated with a poorer overall survival as found in gastric cancer, partly due to their anti-inflammatory property, including increased regulatory T-cells [20].

Malignant transformation is associated with an “angiogenic switch,” marked by an increase in the number of new blood vessels [21, 22]. Macrophages also appear to be important players in angiogenesis. TAMs may stimulate the tumor neovascularization by producing angiogenic factors. Leek et al. have shown a positive relationship between high vascular grade and increased macrophage infiltration in breast carcinoma, which leads to reduced disease-free survival and overall survival [23]. TAMs residing in hypoxic tumor areas have increased expression of vascular endothelial growth factor A (VEGFA) [24]. This correlates with TAM-mediated induction of metalloproteinases (MMPs), contributing to increased tumor angiogenesis, which is consistent with findings of TAMs as major source of MMP9 in a mouse model of human ovarian cancer [25]. Chen et al. have shown that a hypoxic TME can be crucial for preferential polarization of recruited macrophages into M2 subtype [26]. These recruited M2 macrophages significantly enhance tumor neovascularization while protecting the cancer cells of the immune system.

1.1 TAM polarization

Macrophages that express high levels of tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS), or major histocompatibility complex (MHC) class II molecules have been considered antitumorigenic. Expression of high levels of arginase-1 (ARG1), interleukin (IL)-10, CD163, CD204, or CD206 by macrophages has been associated with pro-tumorgenic behavior [27]. Macrophage polarization is an area of intense immunological research [28, 29, 30]. In a conventional dualistic approach, M1 macrophages refer to macrophages activated through lipopolysaccharide (LPS) or the polarizing cytokine interferon gamma (INFγ) [31, 32]. These are identified by the expression of “M1 marker genes” such as NOS2, IL12, and MHC class II transactivator (CIITA) [28]. M2 macrophages are IL-4 stimulated and typically express “M2 marker genes” such as ARG1, among other signal transducer and activator of transcription 6 (STAT6)-induced markers [8, 28, 29, 30, 32].

However, macrophages can express both ARG1 and NOS2 simultaneously, suggesting the inadequacy of the strict dichotomous model to address layers of complexity. This dualistic model has been replaced by a spectrum model, wherein M1 and M2 are thought to represent ends of a continuum. Heterogeneity in the microenvironment along with other development factors must be considered for TAM phenotyping. Metabolic changes within the TME have significant potential to change polarization of macrophages. It has been shown that oxidative pathways result in M2 polarized macrophages, whereas M1 macrophages depend more on glycolysis [6, 32].

2. Triple-negative breast cancer: an unmet need in contemporary cancer medicine

Breast cancer ranks as the second most common cancer type worldwide with higher incidence being seen in African Americans and Hispanics. Lifetime risk of breast cancer remains at one in every eight women equating to about 13% of American women developing breast cancer in their lifetime. It is estimated that in 2022, approximately 288,000 new cases of invasive breast cancer will be diagnosed in women within the United States and over 43,000 will die from their breast cancer (SEER database; http://cancer.gov).

Triple-negative breast cancer (TNBC) refers to tumors lacking expression of estrogen receptor (ER), progesterone receptor (PR), and receptor tyrosine-protein kinase erbB-2 (HER2), all of which are molecular targets of therapeutic agents, ensuring TNBC remains difficult to treat. Chemotherapy remains the mainstay for standard of care treatment of TNBC, with preferential use of platinum compounds in BRCA1/2 mutated breast cancer that are triple-negative. About 10–20% of all diagnosed breast cancers are triple-negative. TNBCs will often present with higher grade tumors that clinically correlate with a poorer outcome as compared to other breast cancer subtypes. In particular, patients with TNBC tend to present with clinically more advanced disease in the form of larger tumors and a higher burden of nodal involvement. This is reflective of their inherently aggressive nature [33]. Despite responses to treatment, these cancers can present with earlier relapses involving the visceral sites [34, 35, 36, 37, 38, 39]. Despite multi-agent systemic treatment, fewer that 30% of patients with metastatic breast cancer survive longer than 5 years and virtually no patient with metastatic TNBC will be alive after that [40, 41]. Despite a higher risk of recurrence for TNBC, better clinical and pathological initial response to chemotherapy has been seen in TNBC compared to other breast tumors, an interesting but paradoxical contrast [41, 42]. In a large majority of residual TNBCs that persist after initial chemotherapy, there may be targetable pathway alterations that could serve as therapeutic targets [41, 43]. Use of poly ADP ribose polymerase (PARP) inhibitors, phosphoinositide 3-kinase (PI3K) inhibitors, mitogen-activated protein (MAP) kinase (MEK) inhibitors, heat-shock protein 90 (HSP90) inhibitors, histone deacetylase inhibitors, etc., are notable examples [41]. Some of these interventions have been approved by the US Food and Drug Administration (FDA) and others remain investigational. The standard of care for TNBC remains multiagent chemotherapy; however, use of PARP inhibitors (such as olaparib) and immune checkpoint inhibitors (such as pembrolizumab) have recently been incorporated as part of adjuvant or neoadjuvant potentially curative options approved in certain settings [39, 44, 45, 46, 47]. Unfortunately, TNBC patients display remarkable clinical diversity, making treatment decisions challenging, as seen with the recent voluntary withdrawal of the expedited approval of atezolizumab in combination with chemotherapy against metastatic TNBC, despite initial approval for use in this setting. In summary, TNBC remains a major unmet need in contemporary cancer medicine.

2.1 PDL-1 and triple-negative breast cancer

Immunotherapy has been of interest and a focus for the development of therapeutics for many years. Clinical trials have yielded mixed results. Vaccine trials have exploited the idea of increasing immune system engagement by increasing tumor recognition by the immune system but without consistent results. Active engagement of the immune system using immunotherapies continues to be both of clinical and investigational interest [41].

TNBCs are known to have genomic instability and have been shown to have higher degree of tumor-infiltrating lymphocytes [48] along with increased expression of programmed cell death ligand 1 (PD-L1) in comparison with other breast tumor types [49, 50]. Immune tolerance regulation has been linked to PD-L1 and its receptor programmed cell death protein 1 (PD-1). PD-1 is a receptor expressed on the surface of cells, like T-cells in the adaptive immune environment. These cells can then bind to either PD-L1 or PD-L2 which can be found on both tumor cells and tumor-infiltrating cells. This can in turn induce inhibition and depletion of T-cells which limits the tumor cell clearance and allows the tumor cells to evade innate and adaptive immune mechanisms [51, 52, 53]. Therefore, inhibition of immune checkpoints can reverse the immunosuppressive environment to promote an effective local immune response [54, 55].

TNBC has been shown to harbor mutations and tumor-infiltrating lymphocytes, along with a higher expression of PD-L1, making immunotherapy an attractive therapeutic approach [56, 57, 58]. In KEYNOTE-012, a phase 1b study, the PD-1 inhibitor pembrolizumab was evaluated as monotherapy for TNBC patients whose tumors expressed PD-L1. An overall response rate of 19% was seen in the study with one complete response. There were four partial responses seen, and 29% of patients had stable disease on treatment [59]. KEYNOTE-086 further explored the use of pembrolizumab in patients with metastatic TNBC. This study characterized patients by treatment history for their metastatic disease as well as PD-L1 expression on tumor cells [53, 60]. Progression-free survival (PFS) was similar with an overall response of 4.7% in both PD-L1 positive and negative previously treated patients of Cohort A in this study. Cohort B consisted of untreated patients with positive PD-L1 expression and showed a higher overall response rate of 23.1% [53]. KEYNOTE-119 evaluated advanced TNBC patients with 1:1 randomization to single-agent pembrolizumab vs. physician choice of chemotherapy and failed to meet its primary endpoint [61]. KEYNOTE-522, a prospective randomized trial evaluating neoadjuvant and adjuvant pembrolizumab for patients with TNBC assigned to pembrolizumab plus chemotherapy and placebo plus chemotherapy in a 2:1 ratio, showed a pathological complete response of 64.8% vs. 51.2% between the two groups. Patients with early TNBC who had received pembrolizumab with neoadjuvant chemotherapy had a significantly higher complete pathological response compared to the group that received placebo with neoadjuvant chemotherapy [62, 63]. Atezolizumab, a monoclonal antibody targeting PD-L1, was the first immune checkpoint inhibitor to be approved in combination with Nab-paclitaxel for unresectable locally advanced and metastatic TNBC expressing PD-L1 [64]. Approval was withdrawn when results of IMpassion131 showed failure to meet the primary endpoint of PFS superiority compared to the frontline treatment. Additionally, there was no survival advantage seen in the PD-L1 positive population nor in the intention to treat population. In fact, the study investigators observed a negative trend for overall survival [65], highlighting the urgent need for new treatment options.

3. Targeted preclinical imaging and therapy of tumor-associated macrophages in models of triple-negative breast cancer

As immunotherapies targeting co-stimulatory blockade move to the forefront of cancer therapeutics, it becomes increasingly important to understand the contribution of inflammatory cells to tumor progression and their potential use for targeted therapy. As discussed earlier, TAMs are critical components of the TME in many solid tumors, including breast cancers, and play key roles in facilitating tumor progression and metastases [30]. This pro-tumor effect of TAM appears to be mediated by increased proliferation of tumor cells, angiogenesis, matrix remodeling, and the sustained release of growth factors and cytokines within the TME. Although both phenotypic and functional heterogeneity are well documented for the macrophage lineage, and the activation state can be clearly defined as a spectrum (see Section 1.2.), here we will utilize two distinct states of polarized activation to demonstrate macrophage targeting in translational experiments. Specifically, the classically activated (M1) macrophage acts in response to IFNγ and/or LPS, and the release of IL-12, IL-23, and tumor necrosis factor (TNF), resulting in efficient antigen presentation and antitumor activity. The alternatively activated (M2) macrophage was originally discovered to respond to IL-4 [66] and can be characterized by low IL-12 and high IL-10 expression, dampened inflammation, increased parasite clearance, tissue remodeling, and promotion of tumor progression [30].

Macrophage dysregulation is central to the pathogenesis of human TNBC. Given that TAMs are influenced by their TME, it becomes important to explore how disease-specific changes in TNBC, specifically the large TAM population within the TME, can be selectively exploited for clinical applications. Thus, a main goal of this book chapter is to provide a few recent selected examples of basic and applied research programs that study TAM biology in the setting of TNBC, toward bringing discoveries and new mechanistic insights into translational applications.

Here, we have highlighted four specific examples of reversal of immune subversion in TNBC and targeted cellular imaging in vivo of TAMs in preclinical models of disease, namely:

• Attenuating TNBC with a lysosome-targeted DNA nanodevice [67] (Figure 1A).

• Ligand-directed targeting a vitamin D receptor in the cell surface of TAMs in a TNBC model for tumor ablation and immune subversion reversal [39] (Figure 1B).

• Magnetic resonance imaging (MRI) of superparamagnetic iron oxide nanoparticle (SPION)-loaded TAMs in vivo in an isogenic mouse model of TNBC (Figure 1C) [68].

• Controlling TNBC dormancy through differentially activated TAM-derived exosomes and their cargo (Figure 1D) [69].

Figure 1.

Selected examples of targeting TAMs in TNBC for therapeutic or imaging applications in pre-clinical models of disease. (A) Administration of E64, an inhibitor or cysteine proteases, conjugated to DNA, leads to selective autophagocytosis and lysosomal uptake, where cysteine protease activity is blocked selectively, enabling TAMs to display tumor antigens and activate CD8+ T cells for anti-tumor activity [67]. (B) PDIA3 is a novel biomarker of TNBC, and it can be selectively targeted with the CSSTRESAC peptide ligand. CSSTRESAC displayed on a hybrid AAV/phage engineered to deliver HSVtk specifically homes to TAMs in EF43.fgf4-derived mammary tumors. Administration of appropriate HSVtk substrates for imaging (radiolabeled reporters) or suicide gene therapy (GCV) enables tumor monitoring and treatment [39]. (C) Ferumoxytol-contrasted MRI enables TNBC imaging and quantification and localization of the iron-containing TAM population in the TME of a mouse model of TNBC [68]. (D) Breast cancer metastasized to the bone marrow (BM) can become dormant, rendering the site invulnerable to circulating chemotherapy. Activating TLR4, for example by LPS administration, can repolarize M2 macrophages to M1. These M1 TAMs can secrete exosomes that interact with cancer stem cells to reverse their quiescent state, enabling effective anti-tumor therapy [69]. Created with BioRender.Com.

3.1 Attenuating TNBC with a lysosome-targeted DNA nanodevice

Cui et al. recently reported a novel approach to exploit the known lysosomal trapping phenomenon of DNA-based agents [67]. Nucleic acid therapies seem to be preferentially trafficked to the lysosome via the endo-lysosomal pathway [70, 71]. Rather than reinvent the wheel—re-engineer the nucleic acid—for alternative organelle targeting, the authors identified lysosomal functions that could be co-opted for an antitumor effect, specifically that an increase in cysteine protease activity in lysosomes diminishes antigen presentation on M2 macrophages, avoiding T-cell activation and tumor immunity [67].

Cui and team [67] completed a proteomic analysis of M1 and M2 macrophages to identify distinct markers of tumorigenesis associated with M2 polarization. In addition to validating known mitochondrial and adhesion proteins, the M2 macrophage-specific profile included a panel of elevated lysosomal proteins, findings confirmed in TAM samples from TNBC patients. Specific deletion of transcription factor EB (TFEB), a regulator of the concerted network of lysosomal enzymes responsible for degrading proteins [72] and transcriptional regulation of autophagosome-lysosome fusion and function [73], resulted in delayed tumor growth and decreased lysosomal activity, likely caused by increased antigen presentation by TAMs and the recruitment of CD8⁺ T-cells [67].

The cysteine proteinase inhibitor E64 [74] was then selected to develop a therapeutic agent with the same effect as TFEB depletion [67]. Increased lysosomal cysteine protease activity is known to improve antigen cross-presentation [75], so E64, a specific inhibitor of cysteine proteases [76], was conjugated to a 38 base pair DNA sequence that could be picked up by scavenger receptors on TAMs for autophagolysosome processing [67]. The benefits of this approach would be twofold: 1) direct antitumor activity from immunomodulation [77] and 2) to sensitize cells to cancer drugs [78].

Trafficking of the E64-DNA compound to lysosomes was confirmed with a fluorescent reporter [67]. E64-DNA inhibited cysteine protease activity specifically, which increased antigen presentation and CD8⁺ T-cell recruitment [77]. When tested in the E0771 TNBC tumor model, E64-DNA was able to selectively target TAMs, internalize via scavenger receptors, and localize to lysosomes. Further analyses revealed M2 macrophages were preferentially targeted. After E64-DNA administration, tumor growth was hampered, and the number of CD8⁺ T effector cells increased, as did markers of both T-cell proliferation and activation. Further investigation confirmed that E64-DNA acts on the M2 macrophage population to reduce cysteine protease activity, which facilitates antigen presentation on the TAM cells, leading to activation of CD8+ T-cells and slowed tumor growth [67].

Finally, in addition to this nascent antitumor immunomodulatory activity, E64-DNA enhanced chemosensitivity, and combination therapy with E64-DNA and cyclophosphamide led to longer-term efficacy and tumor regression. The increase in tumor cell death provides a ready supply of antigens TAM presentation leading to antitumor immunity [67]. This report exemplifies the potential of exploiting what some might originally write off as a negative (lysosomal trapping of nucleic acids) to counterintuitively engineer an organelle-specific DNA conjugate for affecting lysosomal functions toward immunotherapeutic applications.

3.2 Ligand-directed targeting a vitamin D receptor in the cell surface of TAMs in a TNBC model for tumor ablation and immune subversion reversal

3.2.1 Viruses as therapeutic agents

The use of oncolytic viruses to selectively target cancer cells is fairly widespread. Multiple viruses have been selected or engineered for specific purposes, often tumor cell destruction with minimal impact to nonmalignant tissues. Some of the most common—and clinically advanced—are adenoviruses [79, 80] and adeno-associated viruses [81]. AAVP is a unique hybrid AAV and bacteriophage (phage) vector first described in 2006 [82]. AAVP contains cis-elements from AAV within the single-stranded phage genome that facilitates tumor-targeted delivery of a transgene cassette for noninvasive tumor imaging and/or therapy [82]. Unlike mammalian viruses that are conventionally used for gene therapy, AAVP has been extensively characterized and has several safety features built into the vector design, such as: (i) targeting peptides to ensure receptor-mediated transduction and tumor-specific gene expression, (ii) well-characterized fate of the genome (concatemerization and integration of intact genomes) [82], and (iii) the ability to avoid neutralization, as proven in repeat dose studies using pet dogs with spontaneous tumors [83] and several mouse models, including transgenic tumor models with intact immune systems [82, 84]. Receptor-mediated AAVP internalization is required for transduction, eliminating off-target effects, even during phage particle clearance through the reticuloendothelial system (RES), sparing healthy tissues while a strong promoter drives the transgene expression of the within tumors. In the following section, we report one translational approach utilizing AAVP to selectively target the TAM population for theranostic gene delivery.

3.2.2 Novel molecular markers of TAMs in TNBC

A recently reported study describing the identification and validation of the CSSTRESAC (single letter amino acid code) peptide and its receptor, a novel TAM biomarker, is summarized here. Staquicini et al. devised a combinatorial peptide library-based screening that allowed the identification of peptides selectively targeting the TAM-rich TME of mammary tumors [39]. Assuming that peptides binding to mammary tumors in vivo, but not to the corresponding breast cancer cells (BCCs) in vitro, would selectively target the TME, an in vivo combinatorial selection was performed by injecting a naive CX₇C (C, cysteine; X, any residue) phage peptide library into EF43.fgf4 tumor bearing mice. These cells produce a rapidly growing, aggressive syngeneic model of TNBC that is highly infiltrated with F4/80⁺ TAMs. After 24 hours, tumor homing peptides were recovered by bacterial infection, amplified, and re-injected for two additional rounds of screening. To facilitate the selection of microenvironment-specific binders, peptides enriched in the tumor were selected based on negative binding to the cancer cells. The peptide CSSTRESAC targeted tumors in vivo but did not bind EF43.fgf4 breast cancer cells in vitro [85], suggesting specific targeting of the TME. Binding assays to cellular components of the TME showed that CSSTRESAC bound exclusively to F4/80⁺ TAMs [39].

Peptide affinity chromatography and mass spectrometry identified protein disulfide-isomerase A3 (PDIA3) and vitamin D-binding protein (DBP) as targets of CSSTRESAC on the surface of TAMs. Because the CSSTRESAC phage bound specifically to the CD11b⁺ F4/80⁺ TAM population, and because PDIA3 was validated as its receptor, PDIA3 expression on the surface of TAMs was investigated. TAMs were isolated from EF43.fgf4 tumors based on CD11b, IL-10, IL-12, and PDIA3 expression. Expression of PDIA3 on the surface of CD11b⁺ IL-10^high IL-12^low macrophages was confirmed by flow cytometry, suggesting PDIA3 is a novel surface marker of M2-polarized macrophage [39]. With a novel ligand/receptor interaction confirmed in TAMs in the TNBC model, the diagnostic and therapeutic utility of this finding was then investigated.

Homing of CSSTRESAC phage to the tumors in two aggressive breast cancer models [86, 87] was robust and specific, and, when displayed on AAVP to deliver the Herpes simplex virus thymidine kinase (HSVtk) gene, markedly slowed tumor growth, at least partially due to ganciclovir (GCV)-mediated cell death via HSVtk suicide gene therapy (Figure 1B) [39]. HSVtk expression levels over time, as well as the response to GCV therapy, can be monitored with positron emission tomography (PET) using an HSVtk reporter probe such as [¹⁸F]-FEAU [82, 88, 89] or [¹²⁴I]-FIAU [90].

Importantly, upon binding, the CSSTRESAC peptide—alone or displayed on phage—induced the expression of pro-inflammatory cytokines IL-6, TNF, and IL-1β in CD11b⁺ F4/80⁺ TAMs, reverting from an M2-rich macrophage population toward an inflammatory TME reminiscent of classical M1 macrophages and further inhibiting tumor growth (Figure 2A) [39]. Collectively, these data confirm the binding specificity of the CSSTRESAC peptide to the TME, specifically the M2 macrophage population expressing PDIA3 on the cell surface, and the potential for an immunoregulatory response that shifts the cytokine profile toward an inflammatory M1 population and further induces antitumor activity.

Figure 2.

Immunomodulation roles of CSSTRESAC and ferumoxytol remain untapped. (A) The CSSTRESAC peptide alone or displayed on phage/AAVP functions in an immunoregulatory role in TAMs in an EF43.fgf4-derived mouse model of TNBC. After treatment with CSSTRESAC, the tumors decrease in volume, and the macrophages revert from an M2-rich population to an M1-polarized population with the expression of several pro-inflammatory cytokines [39]. (B) Similarly, iron agents like ferumoxytol can trigger re-polarization from M2 to M1 TAMs after phagocytosis; IL-10 levels drop while levels TNF and other inflammatory cytokines increase [91, 92, 93]. Created with BioRender.com.

Extrapolating from publicly available datasets, high PDIA3 transcript expression levels in TNBC patients were associated with markers of immune suppression and M2 polarity as well as angiogenic markers associated with poor prognosis, confirming the potential clinical utility of CSSTRESAC-based therapeutic agents [39]. With these promising data confirming the ability to target the TAM population of the TME specifically, the potential effects of CSSTRESAC-targeted agents in TNBC warrant further investigation, for both immunoregulatory and theranostic applications.

3.3 MRI of superparamagnetic iron oxide nanoparticle (SPION)-loaded TAMs in vivo in an isogenic mouse model of TNBC

Breast cancer patients undergo a series of imaging studies in order to diagnose disease, monitor disease progression, and evaluate responses to therapy. The presence of TAMs in the microenvironment of TNBC, in particular, promotes tumor growth and metastasis formation. Accordingly, a method of imaging this cell population specifically would be of clinical interest, particularly relating to response to immunotherapies utilized in these patients. Sillerud et al. recently reported one such approach, wherein an iron (Fe) nanoparticle is selectively phagocytosed by TAMs in mouse models of breast cancer for visualization by MRI [68]. A decrease in signal is detectable with T2-weighted MRI, and the spatial and temporal dynamics of particle uptake can be quantified and monitored [68].

Ferumoxytol is a superparamagnetic iron oxide nanoparticle (SPION) that has been approved by the FDA for patients with iron-deficient anemia. Its off-label use as a contrast agent for MRI has been studied for almost two decades in parallel with its role as an iron replacement therapy [91, 92, 94, 95, 96]. Importantly, a multicenter study found ferumoxytol was generally well tolerated and safe for administration [93].

More importantly, both M1 and M2 macrophages—but not tumor cells—can internalize ferumoxytol [97]. When combined with work done to validate ferumoxytol uptake and detection in lymphomas and sarcomas, known to have CD68⁺ and CD163⁺ TAM populations, ferumoxytol can function as an “imaging biomarker for TAM” [98]. When in a macrophage-rich TME, ferumoxytol might also induce some cytotoxicity and M1 macrophage polarization, making the tumor more susceptible to immunotherapeutic agents [99, 100, 101] (Figure 2B). Yet another application of clinical importance would be to use MRI to image ferumoxytol-containing TAMs as a surrogate for tumor localization. Specifically, on T2-weighted MRI, ferumoxytol produces a decrease in signal, darkening dramatically from a hyperintense image at baseline [68].

The EF43.fgf4 mouse model of TNBC [87] introduced earlier was utilized for this work as well [68]. Initially, tumor and nonmalignant tissues were imaged at baseline, and then tumor and off-site ferumoxytol accumulation after administration was assessed. By 24 hours, dramatic contrast changes were evident in the MR signal from the tumor, while no significant change was evident in the control tissues (Figure 1C) [68]. This decrease was most evident in T2-weighted images, with about a 10-fold difference from baseline starting 1 day after ferumoxytol administration and remaining evident up to 4 days later, before returning to baseline by 7 days. The level of iron within the tumors can be quantified, revealing the distribution of TAMs in the tumor [68]. Higher levels of macrophages, and M2 macrophages in particular, are associated with metastatic potential, resistance to treatment, and an overall worse prognosis [102], information that would be relevant for clinicians and patients. Further elucidation of the role of ferumoxytol in M2 to M1 polarization [99] could broaden treatment options or otherwise combine Fe-based imaging with immunotherapies, chemotherapies, etc., for theranostic applications [103, 104].

3.4 Controlling TNBC dormancy through differentially activated TAM-derived exosomes and their cargo

3.4.1 Breast cancer dormancy in bone marrow

Breast cancer cells (BCCs) preferentially disseminate to the bone marrow (BM) [105]. The BM niche contributes to the survival of BCCs by allowing their transition into dormancy [106]. Dormant BCCs can remain undetected for extended periods by acquiring a cancer stem cell (CSC) phenotype [107]. Cancer stem cells (CSCs) share properties with nonmalignant stem cells such as self-renewal, cell cycle quiescence, and drug resistance [108]. Reactivation of dormant BCCs/CSCs in BM is associated with poor patient prognosis and results in cancer resurgence [109, 110]. Targeting dormant BCCs/CSCs is challenging because current treatments mostly target the active cycling cells [111]. Additionally, dormant BCCs home to the endosteal niche of the BM close to endogenous hematopoietic stem cells (HSCs) and utilize strategies similar to those of the endogenous HSCs to survive and remain dormant [105, 112]. More importantly, the method of survival includes the marrow microenvironment with macrophages comprising a major component of the supporting cells. Thus, any treatment aimed to eliminate the BCCs can also target the survival and/or function of the HSCs found within the same niche. This could result in disruption of hematopoietic activity, which will affect the individual’s immune system and other organ functions that require immune competence. Hence, it is imperative to elucidate the precise mechanisms of communication between cells in the BM microenvironment and BCCs to understand how dormancy is achieved and how the same microenvironment can reverse the dormant phase. Such understanding will lead to effective methods to selectively eliminate malignant stem cells without harm to the endogenous HSCs.

The BM is a complex organ composed of various niches that aid in hematopoietic activity and maintain dormancy [112, 113, 114]. The cellular component of the BM niche includes mesenchymal stem cells, fibroblasts, and macrophages, all contributing to the survival of BCC dormancy [69, 115, 116]. Intercellular communication between BM niche cells and BCCs is fundamental for BC dormancy [105]. The interaction between components of the BM niche and BCCs can be direct through intercellular communication such as gap junctions and/or contact-independent, which involves soluble and insoluble factors such as cytokines and microvesicles (i.e. exosomes). Both forms of the aforementioned interactions facilitate and maintain BC dormancy [116, 117]. Disrupting these interactions can reverse the dormant phenotype of BCCs resulting in cancer recurrence [115, 118, 119]. The next section addresses the role of macrophages in BC dormancy and provides evidence supporting that intercellular communication between the niche and BCCs is essential for dormancy.

3.4.2 Role of macrophages in breast cancer dormancy

BCCs recruit macrophages to the TME by upregulating chemokines such as C-C motif chemokine ligand 2 (CCL2) [120]. Recruited macrophages have been shown to modulate BC dormancy or reversal. For instance, Ma et al. demonstrated that a macrophage subset expressing high levels of CD204 and IL-4 receptor facilitated BC metastasis to the bone [121]. Depletion of this macrophage subset halted BCC proliferation [121]. Depletion of CD11b⁺ VEGFR^high CCR2^high macrophages prevented extravasation and growth of BCCs within the lungs [122]. This macrophage subset is significantly present in lungs with BC metastasis compared to healthy lungs [122]. Although these findings are related to BC metastasis to the lung, it is plausible that a similar mechanism is occurring upon BC metastasis to the BM or bone.

Endogenous BM-derived macrophages have a crucial role in BC dormancy and can be polarized into M1 or M2 phenotype depending on microenvironmental cues [69]. Classically activated macrophages (M1) exert antitumor response in BC, whereas alternatively activated (M2) macrophages employ a pro-tumorigenic effect [123]. However, with respect to dormancy, M2 macrophages maintain cycling quiescence whereas M1 macrophages can reverse dormancy [69]. Such polarization has been demonstrated when the toll-like receptor was activated, suggesting that infectious agents that activate these receptors could be a method to reverse dormancy.

M1 macrophages facilitate reversed dormancy by releasing microvesicles that promote NF-κB signaling in quiescent BCCs to mediate cell cycle activation (Figure 3) [69]. As a result, M1-derived microvesicles sensitized BCCs to chemotherapy by reducing BCC stemness. Conversely, M2 macrophages form gap junctions with CSCs to support dormancy and chemoresistance of BC in BM [69]. Mechanistically, the polarization of M2 macrophages into an M1 phenotype was shown to be mediated by LPS, which activated toll-like receptor 4 signaling. This study showed the role of contact-dependent and contact-independent interactions between BCCs and macrophages in BC dormancy.

Figure 3.

Left panel shows the bone marrow cavity and the predominant region for dormant breast cancer cells within the endosteal niche. The established dormant phenotype is highlighted in boxed region, which is enlarged. The latter shows M2 macrophage sustaining gap junctional-mediated dormancy. Activation of M2 macrophages to M1 type release microvesicles to reverse dormancy.

In TNBC cells, macrophage polarization toward an M2 phenotype is facilitated by the oncogene multiple copies in T-cell malignancy-1 (MCT-1) [124]. Silencing of MCT-1 in TNBC reduced overall tumor volume and the total number of M2 macrophages within the TME [124]. Interestingly, MCT-1 enhances mammosphere formation in TNBC cells through IL-6 signaling. Blockade of IL-6 signaling with the IL-6 receptor (IL-6R) monoclonal antibody tocilizumab reduced mammosphere formation and downregulated MCT-1 expression [124]. Another strategy to target MCT-1 expression was shown by miRNA-34a reducing stemness in TNBC cells and prevented M2 polarization. Macrophage polarization in the TME can also be mediated by oncometabolites such as lactate which enhance BCC proliferation through the ERK/STAT3 signaling pathway [125]. Pharmacological inhibition of ERK/STAT3 with selumetinib and stattic, respectively, abrogated lactate levels within the TME and prevented macrophage polarization to M2 phenotype [125]. Collectively, these studies provided evidence of the importance of macrophage polarization in BC dormancy and reversal.

As stated earlier, the chemokine CCL2 is crucial in macrophage recruitment to the TME. Thus, efforts to prevent macrophage recruitment and polarization in the TME aimed to target the chemoattractant CCL2. However, although preclinical studies showed promising results and effectively abrogated macrophage recruitment to the TME, anti-CCL2 antibodies failed in clinical studies [126]. Therefore, studies need to be conducted to develop strategies to target macrophage recruitment or polarization into M2 phenotype to inhibit tumor progression.

4. Conclusion

Macrophages are key to the behavior of tumors and metastatic dissemination. TNBC, while missing specific targetable markers amenable to treatment, is rife with a potentially vulnerable population of macrophages, M2 polarized macrophages in particular. This population in primary tumor sites can be specifically and selectively targeted to (1) induce antitumor immunity and drug sensitivity, (2) produce a theranostic gene for imaging and treatment of the TAM population, and (3) image TAMs in TNBC for diagnosis or assessing response to therapy. Furthermore, M2 macrophages interact with non-hematopoietic cells in BM to maintain cellular quiescence/dormancy, which can be selectively repolarized to an M1 type population to reverse tumor cell quiescence/dormancy and enable systemic therapy.

Acknowledgments

This work was supported by grant awards from Metavivor Foundation and New Jersey Commission on Cancer Research.

Conflict of interest

FIS, RP, and WA are inventors on a pending patent application related to the CSSTRESAC peptide and associated technology. They are entitled to royalty payments from licensing or commercialization. RP and WA are founders and equity stockholders of PhageNova Bio, which has licensed this IP. RP is the Chief Scientific Officer and a paid consultant for PhageNova Bio. These conflicts are managed by Rutgers, The State University of New Jersey. The remaining authors declare no conflicts of interest.