As an early step in tumor metastasis, targeting carcinoma cell motility and invasion within the primary tumor microenvironment presents a unique opportunity to impede tumor progression. Previous clinical data have indicated that high density of TAMs within a tumor microenvironment correlates with poor prognosis (55
). Furthermore, recent experimental data has provided compelling evidence indicating TAMs in the direct facilitation of tumor invasion (15
). The studies herein describe the in vivo functional isolation of a subpopulation of TAMs caught in the act of promoting carcinoma cell motility; their gene expression profile, compared with a GT population, isolated indiscriminately to function by flow cytometry using the macrophage known macropinocytic capacity and cell-surface expression of F4/80 as markers. It has previously been shown that phagocytosis of dextran does not compromise a macrophage’s motility or its ability to secrete EGF in response to CSF-1 (15
). Furthermore, it has previously been shown that invasive TAMs are a uniform population that express F4/80 and are macropinocytic (15
). Therefore, these experiments are specific for comparing the gene expression of those TAMs actively promoting tumor cell invasion versus the vast majority of TAMs that are not.
Previously, gene expression studies of invasive tumor cells collected through the in vivo invasion assay have yielded mechanistic insight into the cell biology–namely motility (26
) and viability (28
)–of this subpopulation of tumor cells. Considering an inherent requirement for TAMs, we hypothesized that gene expression studies of invasive TAMs would also provide useful insight into the physiology of TAM promotion of tumor progression. The invasive TAM arrays indicated a considerable number of transcripts as differentially regulated, despite use of highly stringent gene selection criteria. Therefore, we interrogated possible artifactual explanations that could account for these differences. In-depth examination of the array data failed to identify consistent biases or deviations from the norm; therefore, technical artifacts could not account for why so many genes were differentially regulated (Supplemental Fig. 1
). Moreover, QRT-PCR on subsequent biologic repeats confirmed all tested results from the gene expression arrays (). We were also concerned that the large number of differences would be due to an irreducible contamination of carcinoma cells, but significant overlap in expression data was not found when the results were compared with invasive carcinoma cell expression data. Taken together, these results indicate that this invasive macrophage population has a unique transcriptome significantly different from the GT population. Useful data can be extracted from the microarray data; however, given that some of these differences may be caused by the experimental perturbation required to collect these cells, caution must be exercised and mechanistic experiments will be needed to confirm that the results are physiologically relevant.
IPA 6.3 was used to embark on a bioinformatic analysis of the enriched functional categories within the stringently identified 1457 transcripts called as differentially regulated by SAM. Physiologic system development and function categories were ranked by significance of enrichment for clues as to how this invasive TAM population was affecting the tumor microenvironment (). It was anticipated that, similar to invasive carcinoma cells (25
), invasive TAMs would distinctly demonstrate enrichment in molecules involved in the paracrine loop required for tumor cell migration and invasion–EGF and CSF-1R in macrophages (19
)– and those mediating macrophage motility to be specifically enriched in invasive TAMs. In fact, the opposite was found wherein Csf1r
transcript expression, whose biochemical function is critical to the paracrine loop expression, was diminished, as were molecules critical to normal macrophage migration (). Unlike tumor cells, macrophages are nongenomically transformed immune cells in which exquisite transcriptional, translational, and posttranslational control is maintained to prevent a shift in balance of immune activity to an overly exaggerated inflammatory response. Therefore, although the results were not anticipated, we do not suggest that they conflict with the established protein requirement for these molecules in the paracrine loop and macrophage motility. The CSF-1R, while not only being on the cell surface, is maintained in a large cryptic intracellular pool from where it can be rapidly mobilized to the cell surface (57
). Furthermore, it has recently been shown that Csf1r
expression is decreased in macrophages following stimulation by lipopolysaccharide (58
), and it is feasible that other molecules communicating between TAMs and tumor cells during their active comigration may produce a similar effect.
Conversely, embryonic and tissue development were enriched ontologic designations of genes overexpressed in invasive TAMs. This finding was interesting, considering that macrophages are well known to have essential tissue trophic functions during development (1
), and especially during mammary gland development (21
). TAMs may be recapitulating a number of these tissue trophic functions executed during development to promote tumor progression (63
). The annotation of transcriptional profiles to function was further explored using GSEA (46
), an analytical method that examines the distribution of functionally related genes in an ordered gene expression dataset without the selection of upregulated or downregulated genes. This method was used to examine the KEGG pathways that are present in humans and enriched in the invasive TAM dataset. Notably, the Wnt-signaling pathway, a pathway critical to normal tissue development was identified as highly enriched (, ). Although a KEGG pathway does not exist specifically for breast cancer, it was noted that the invasive TAM gene set was enriched for molecules frequently overexpressed in human basal cell carcinoma (). Except for Wnt-signaling being a common denominator, the significance of this association is unclear. However, through population studies it is known that the diagnosis of numerous skin cancers, including basal cell carcinoma, confers an increased relative risk of the development of breast cancer (65
). Moreover, in human basal cell carcinoma, increased TAM density correlates with increased depth of invasion and microvessel density (67
). This finding suggests that perhaps the KEGG gene set attributed to basal cell carcinoma derives in part from the TAMs present within the tumor micronenvironment.
We were compelled to further study the significant over-expression of Wnt7b in TAMs because of its known production by macrophages (48
) and the known role of Wnt signaling in development (68
) and cancer (70
). The Wnt7b ligand is a notoriously low-abundant transcript, and it was necessary to use nested primers for semiquantitative PCR to detect message (48
). Genetic tools to transgenically label cells producing Wnt7b, described in studies of macrophage contribution to vascular remodeling of hyaloid vessels in the developing mouse eye (48
), will be useful in future tumor studies although conclusive studies may be hampered when transitioning these models from the relatively hypocellular developing mouse eye to the hypercellular tumor microenvironment. Therefore, although our gene expression studies originally identified Wnt7b as being increased in invasive TAMs compared with GTs, we aimed to provide evidence that in general, TAMs express Wnt7b.
Semiquantitative PCR confirmed that Wnt7b is minimally enriched in TAMs and further enriched in invasive TAMs (). We considered that if carcinoma cells produced the majority of Wnt7b, a profile similar to that seen in would be produced, because flow cytometric sorting generally isolates a 97–99% pure population on postsort, whereas cell separation following the in vivo invasion assay yields a slightly higher contamination (25
). Therefore, a second validated (50
) macrophage isolation step following flow cytometry was used to more purely study the Wnt7b expression in adherent TAMs. After isolation of TAMs and splenic macrophages by flow cytometry, they were allowed to adhere to petri dishes. RNA was subsequently extracted from both pure macrophage populations and Wnt7b expression was only detected in TAMs.
As mentioned above, in the developing mouse eye, macrophages direct vascular remodeling of hyaloid vessels by producing Wnt7b (48
). Interestingly, recent work (49
) has proposed a mechanism in this model whereby macrophages produce Wnt7b that stimulates the canonical Wnt signaling pathway in vascular endothelial cells (VECs), which express Wnt coreceptors Lrp5 and Frizzled, in a paracrine fashion. This signaling cascade eventually results in the stabilization of β-catenin and VEC entry into cell cycle. Furthermore, in the developing mouse eye, angiopoietin-2 production by vessel-related pericytes provides an apoptotic signal to VECs, leading to their death and overall vascular remodeling. This would suggest that in the absence of an apoptotic signal to VECs, this model could provide a mechanism for macrophage stimulation of VEC proliferation, such as in tumor angiogenesis.
Perivascular TAM clusters are indicated to most efficiently promote carcinoma cell motility in an EGF/CSF-1 paracrine loop dependent manner in PyMT mammary tumors. Consequently, these perivascular TAM clusters have been proposed to be an invasive hotspot (15
). In human breast cancer tissue sections, triple staining for juxtaposed clusters of macrophages, vascular endothelial cells, and a specific protein marker found in invasive carcinoma cells indicated that more of these clusters correlated with human metastatic disease (71
). We propose that these macrophages located along the tumor vasculature and associated with invasive tumor cells are the invasive TAM subpopulation and that these TAMs function, in part, through modulating Wnt-signaling in the local microenvironment. It would not be insignificant that TAM-derived Wnt7b may also have a role in vasculogenesis, as seen in the developing eye (48
) because there is considerable evidence of extensive angiogenesis at the sites of leukocytic invasion in human breast tumors (6
). Furthermore, it is believed that these angiogenic beds are also the sites of tumor cell invasion (3
). Our results linking Wnt7b and tumor cell invasion are consistent with this hypothesis. Not insignificantly, human breast tumors that have higher expression levels of WNT7B
are associated with advanced disease as defined by tumor-positive lymph nodes ().
This article describes the gene expression signature of a unique invasive TAM subpopulation that putatively has roles in tumor progression through promotion of tumor metastasis and angiogenesis, with the role of Wnt signaling as a common denominator via TAM-derived Wnt7b. Moreover, we submit that with careful mining, the results from the gene expression arrays described in this study will provide a useful tool for further insight into mechanisms of TAM promotion of tumor progression.