Macrophages are highly versatile and have the capacity to differentiate into Kupffer cells, osteoclasts and/or pneumocytes in resident tissues or into inflammatory Mø subtypes in microbial infections (M1 type) or after tissue injury (M2 type). TAMs mediate a host response to the tumorigenic process, and can induce anti-tumor immunity (M1 type TAMs) or, paradoxically, enhance tumorigenesis (M2 type TAMs) 
. Most correlative data from human cancers suggest that TAMs are protumorigenic, including in thyroid cancer 
. In PyMT induced breast cancers, TAMs facilitate tumor angiogenesis and lung metastases but have no effect on primary tumor growth 
. In RIP1-Tag induced pancreatic neuroendocrine tumors (PNET), CSF-1 dependent TAMs facilitate the transition from hyperplastic angiogenic islets to PNET, but have no effect on the subsequent tumor phenotype 
. In mutant APC intestinal polyposis models, CSF-1-dependent TAMs promote polyp growth. However, their role in malignant transformation could not be evaluated in this model of benign disease 
. Whereas these studies implicate TAMs as tumor promoters, the severely dysmorphic phenotype of the Csf-1
knock mouse used in these studies, and the fact that this model does not allow TAMs to be depleted during defined stages of tumor development, limit the conclusions that can be derived from these experiments. Moreover, because oncogenes may induce distinct inflammatory signals which differentially impact the phenotype of TAMs, cancer models induced by oncoproteins known to be involved in disease pathogenesis may more faithfully recapitulate the function of TAMs in human cancer.
-induced thyroid cancers are ideally suited for studying the biology of TAMs in cancer progression because 1) human thyroid cancer progression to PDTCs and ATCs is accompanied by an increased infiltration of TAMs which comprise nearly ~50% of the tumor volume in ATCs 
; 2) BRAF
promotes progression of human WDPTCs to PDTCs and ATCs 
and 3) activation of the BRAF-MAPK pathway in vitro in thyroid cells induces the expression of TAM chemoattractants 
. In this study we used murine models of BRAF-induced PTCs/PDTCs to examine the role of TAMs in thyroid cancer progression. Conditional activation of BRAF in murine thyroids is associated with a significant increase in the major TAM chemoattractants, Csf-1
. This is accompanied by a dense infiltration of TAMs and the development of PTCs/PDTCs with a short latency 
. To examine whether there is a causal relationship between TAM infiltration and PTC development, we conditionally depleted CCR2-dependent TAMs during BRAF induction using a Ccr2-DTR
approach. The trafficking of monocytes from the marrow into the circulation and tissues requires signaling through the CCL2/CCR2 pathway and monocytes lacking CCR2 expression become trapped in the BM 
. Once in the circulation and/or tissues, CCR2 expression on monocytes/Mø is down-regulated during resting states but remains upregulated during inflammation 
. The DT/Ccr2-DTR
model is therefore a robust approach to achieve TAM depletion since TAMs are targeted directly through M
expression of CCR2 in tumors and/or indirectly by the depletion of BM-derived CCR2+
monocytes. Indeed, treatment of mice with DT during BRAF induction resulted in a nearly complete depletion of TAMs and unexpectedly, of αSMA+
CAFs. In mouse models, CAFs facilitate malignant transformation 
, stimulate tumor angiogenesis and remodel the extracellular matrix to enhance tumor cell invasion 
and metastases 
. TAMs and CAFs frequently co-localize within the stroma, yet there is little known about their interrelationship. In inflammation, increased M
recruitment stimulates myofibroblast expansion 
. A similar link between TAMs and CAFs has not been established for any malignancy, and is suggested by the findings described above. This suggests that at least some of the functions of M2 type Mø within neoplastic and inflamed tissues are conserved and that non-neoplastic models of inflammation may be useful in understanding how TAMs may remodel the tumor niche.
CCR2 expression may be shared by non-myelomonocytes, including cancer cells 
, endothelial cells, 
, NK cells 
and/or myofibroblast precursors 
. The effects we observed are most likely mediated by CCR2-dependent TAMs because Ccr2
expression in PTCs is restricted to Cd45+
myelomonocytes and because pharmacotherapy with GW2580, a selective c-FMS/CSF-1R kinase inhibitor, phenocopied the effects of DT treatment in PTCs of CCR2-DTR mice.
Selective depletion of CCR2-dependent TAMs in established PTCs resulted in smaller tumors and decreased proliferation. We also saw fewer tall cells and decreased foci of PDTC. This is potentially significant since patients with tall cell variant PTCs and PDTC have more frequent distant metastases and a higher mortality 
. Our results are in contrast to those observed by others using CCL2/CCR2 targeted cancer models 
. In murine cervical cancers, the depletion of CCR2-dependent TAMs resulted in a compensatory recruitment of pro-tumorigenic tumor infiltrating neutrophils (TINs), that when depleted, impaired the tumor phenotype 
. Although we also observed an increase in TINs in PTCs following the depletion of CCR2-dependent TAMs, these did not rescue the PTC phenotype. This was not simply due to an insufficient exposure to TINs since chronic depletion of TAMs in PTCs of Csf-1
knockout mice also impaired the tumor phenotype. Thus, our data confirm that TAM depletion evokes a compensatory surge in TINs but at least in thyroid, this does not prevent the dampening effect on tumor phenotype.
The mouse models used in this study do not have a high enough frequency of distant metastases to allow us to explore the contribution of TAMs to the metastatic process. This is consequential, because recruitment of CCR2 positive inflammatory Mø has been shown to occur preferentially in the lung pre-metastatic niche rather than in the primary mammary tumors of late-stage PyMT-induced breast cancers, which instead recruit CCR2-negative, CSF-1 dependent TAMs 
. Besides the differences between tumor types, these data suggest that the versatility of TAM subpopulations and function may also be tumor stage specific.
Following BRAF activation, Csf-1
is markedly overexpressed in the murine cancers. Moreover, the TAMs recruited in the early stages of tumorigenesis express high levels of c-fms
. Depletion of CSF-1 reduced TAM infiltration and induced smaller PTCs with a more differentiated phenotype. One Csf-1
knock out mouse did not show significant TAM depletion in the tumor specimens. This has been previously reported in a model of mouse PNETs 
and ascribed to potential c-FMS/CSF-1R-dependent and independent rescue pathways of TAM recruitment. Interestingly, the phenotype of PTCs in the outlier Csf-1
knockout mice with high TAM density was similar to that of control PTCs, suggesting that compensatory signals may allow a minority of PTCs to circumvent CSF-1 and recruit pro-tumorigenic TAMs.
As TAMs promote PTC progression, these cells may be a rational therapeutic target for patients with refractory advanced PTCs, particularly PDTCs and ATCs. We show that the c-FMS/CSF-1R kinase inhibitor GW2580 phenocopied the antitumorigenic effects of genetically depleting TAMs. The PTCs from GW2580-treated mice were smaller, had fewer TAMs and CAFs and exhibited a more differentiated PTC phenotype. Our study is the first to demonstrate that targeting TAMs alone with a small molecule inhibitor impairs primary tumor progression for any cancer type. By contrast, in breast cancer 
and glioblastoma 
mouse models, PLX3397, another C-FMS/CSF-1R kinase inhibitor, did not affect primary tumor phenotype, but did improve the efficacy of cytotoxic chemotherapies and decreased tumor invasion, respectively. The data in thyroid models is potentially significant, particularly in view of the remarkable extent of TAM infiltration seen in patients with advanced thyroid cancers