Distinct macrophage subsets have been linked with either protective or pathogenic roles in cancer. A protective role has been described for M1 macrophages, which activate tumour-killing mechanisms and antagonise the suppressive activity of TAMs. In contrast, TAMs suppress adaptive tumour-specific immune responses and promote tumour growth, invasion, metastasis, stroma remodelling and angiogenesis. TAMs have a suppressive M2-like phenotype. Accumulating evidence from many tumour models suggests that macrophages contribute to tumour progression, with increasing numbers of TAMs correlating with poor outcomes. Here we investigated the interplay between canine mammary carcinoma cells and macrophages. We have shown that cancer cells and macrophages have mutual effects on each other, and that the cytokines CSF-1 and CCL2 are important mediators of this interaction. Furthermore, we have identified that CSF-1 is likely to signal via the transcription factor Twist-1 to exert its effects on macrophage activation.
Our data corroborates previous studies. Wasserman et al. (2012) demonstrated the capacity of several canine cancer cells to inhibit macrophage MHC II expression, therefore driving TAMs into the alternative M2-activation pathway [32]. Król et al. (2012) [16] showed that LPS-induced activation of macrophages was inhibited by co-culturing macrophages with canine mammary cancer cells. Here we utilised canine mammary carcinoma cells and showed by cellular granularity and MHC II expression of macrophages that REM134 cells can inhibit LPS-induced activation of macrophages.
There is currently controversy regarding the dogma of classic and alternative macrophage activation, termed M1 and M2, respectively. As research into macrophage biology has evolved, so has the growing amount of information regarding recognition receptors, cytokines, and the signalling and genetic programs behind them that control an increasing number of functions of macrophages. Therefore there is a need to recognise a broader functional repertoire of macrophages that may not fit into the distinct M1 and M2 classifications [20]. Furthermore, tumour microenvironments, compared to a healthy tissue, are haphazard and may contain areas of hypoxia, higher lactate, extracellular acidosis and glucose starvation [8]. Within a tumour multiple and different M1 and M2 stimuli may act on macrophages, and in this context, macrophages may not form distinct activation subsets nor clonally expand, leading to a spectrum of macrophage phenotypes. As a marker of M2-activation we used CD301. In contrast to the granularity and MHC II expression data, expression of CD301 increased after LPS-activation but was unaffected by the presence of cancer cells, indicating that, in these culture conditions, macrophages under the influence of cancer cells are in an activation state between the extremes of the M1 or M2 spectrum.
CSF-1 and CCL2 have well-characterised roles in macrophage activation including inducing macrophage survival and recruitment [17, 29]. Expression of both CSF-1 and CCL2 have been independently correlated with cancer progression in several tumour types [28]. Our studies show that blocking the receptor of CCL2, CCR2, with a small molecule inhibitor could increase macrophage activation, and this supports previous studies where RAW264.7 macrophages are able to produce this cytokine [22] and, by blocking this autocrine signalling, induce cellular activation. This may be mediated through Activin A, which can simultaneously alter the expression of CCR2 and CCL2 in macrophages, depending on their previous activation state [29]. Blocking CCR2 signalling is expected to induce the expression of Activin A, which is known to drive MHC II expression in macrophages, as well as phagocytosis and other M1 characteristics [11]. We showed that in the presence of LPS the effects of blocking CCR2 were more pronounced. Here, blocking CCR2 in macrophages allowed for a marked increase in cellular activation. The addition of rhCCL2 could not reverse this effect, demonstrating that blockade of the receptor was complete. Interestingly, it has been shown that LPS treatment alone can induce CCL2 expression in RAW264.7 macrophages as a negative feedback, since its expression is controlled by STAT-3, an immune regulator [22, 31]. The effect of CCL2 on macrophages was confirmed by CD301 expression. Adding recombinant CCL2 increased expression of this marker. Overall, the results with CCL2 indicate a shift towards an M2 activation phenotype in the presence of CCL2/CCR2 signalling. Another work found that co-culture of macrophages and canine cancer cells reduced the relative expression of CCL2 by the macrophages, but at the same time induced its expression by cancer cells [16], therefore supporting the hypothesis that macrophages will be directed towards an M2 phenotype in the presence of cancer cells. However, CCR2 blockade also had the opposite effect of reducing the percentage of highly-granular macrophages. These highly-granular cells represent only a small fraction of the total macrophage population, but are likely to represent an activated subset of the cellular population [24]. Therefore, the reduction in the highly-granular subgroup of macrophages in the absence of CCR2 signalling points away from the more accepted role of CCR2 inducing M2 polarization of TAMs.
As with CCL2, CSF-1 had a negative impact on LPS-induced macrophage activation. While adding recombinant CSF-1 reduced macrophage expression of MHC II following LPS activation, blocking it with a soluble recombinant CSF-1R allowed a marked activation of these cells. This indicates that by blocking CSF-1R signalling, an inflammatory phenotype can be obtained, even in the presence of cancer cells.
Both macrophages and canine mammary cancer cells are able to proliferate in the presence of rhCSF-1. While CCL2 itself induces some cancer cell proliferation, CCL2 conditioning of the macrophages increases the effect that these cells have on cancer cell proliferation. Macrophage conditioned media is also capable of inducing expression of CSF-1R and CCR2 by mammary carcinoma cells, indicating that there is a cycle whereby cancer cells induce macrophage proliferation and phenotypic change, and the presence of macrophages leads to cancer cell growth, with both processes occurring, at least partially, through CSF-1R/CCR2 signalling (Fig. 8). CSF-1R expressed by cancer cells does not have any mutations compared to the native receptor (data not shown), indicating that it must depend on either autocrine or paracrine signalling to support cancer expansion. CSF-1R has been associated with mammary cancer aggressiveness in dogs [14, 15], and the expression of the receptor has been shown to be increased in canine mammary adenocarcinoma cells in co-cultures with macrophages [16].
The effect of macrophage conditioned media on the cancer cells is biologically relevant since it increases cancer cell metabolism, as measured by cellular glucose uptake and cellular proliferation. Increased expression of CCL2 can induce insulin resistance [13], which is usually associated with hyperinsulinemia [25] and increased glucose uptake by cancer cells. Lactic acid production by tumour cells, generated as a consequence of glucose metabolism, has been shown to be central for the signalling that induces M2-macrophages (as measured by Arg-1 expression) and vascular endothelial growth factor expression [6]. Therefore, when in contact with macrophages, cancer cells will consume more glucose, thereby reinforcing an M2 phenotypic state on the macrophages.
CSF-1R signalling is relevant both for macrophages and cancer cells. However the role of CCL2 produced by the cancer cells, as described previously [16], appears to be confined to altering the macrophage phenotype, as cancer cells themselves were unaffected by inhibition of CCR2. The proliferative response of cancer cells to CCL2-treated macrophage CM is probably due to activation of other receptors in the cancer cells by soluble factors secreted from macrophages. This highlights the complexity of the tumour microenvironment and the importance of the secretome in the interplay between cancer cells and TAMs.
Twist-1 is a transcription factor that has been associated with macrophage activation and function and in the interaction between tyrosine kinase receptors (such as CSF-1R) and Toll-like receptors (such as TLR4, the receptor for LPS) [7]. To assess the importance of Twist-1 signalling in the M1-to-M2 transition, expression of Twist-1 was depleted in macrophages. A decrease in Twist-1 protein levels lead to increased macrophage activation, as measured by phagocytosis capacity, cell granularity and MHC II expression. However, when LPS was added, Twist-1 knock down only partially impeded macrophage activation by LPS. Therefore, depletion of Twist-1 had similar effects to treating macrophages with recombinant CSF-1, indicating that CSF-1 may signal through Twist-1 to mediate the M1-to-M2 transition.