Long-term characterization of activated microglia/macrophages facilitating the development of experimental brain metastasis through intravital microscopic imaging

Abstract

BackgroundMicroglia/macrophages (M/Ms) with multiple functions derived from distinct activation states are key surveillants maintaining brain homeostasis. However, their activation status and role during the brain metastasis of malignant tumors have been poorly characterized.MethodsHeterozygous CX3CR1-GFP transgenic mice were used to visualize the dynamic changes of M/Ms during the development of experimental brain metastasis through long-term intravital imaging equipped with redesigned bilateral cranial windows. The occurrence of experimental brain metastasis was evaluated after M/Ms were depleted with PLX3397, a CSF-1R inhibitor. The possible mediators of M/Ms in facilitating the brain metastasis were determined using reverse transcription-PCR, immunofluorescence, correlational analysis, and MMP inhibition.ResultsHere, we showed that M/Ms were persistently activated and facilitated the formation of melanoma brain metastasis in vivo. We observed that M/Ms gradually and massively accumulated in the metastasis, with a 2.89-fold increase. To precisely depict the dynamic changes in the activation state of M/Ms, we defined the branching parameter to quantify their morphological alterations. The quantitative data showed that the extent of activation of M/Ms in metastatic foci was enhanced, with a 2.27-fold increase from day 1 to day 21. Along with the activation, the M/Ms increased their moving velocity (4.15-fold) and established a rapid, confined, and discontinuous motility behavior. The occurrence of melanoma brain metastasis was significantly hindered under M/M elimination, indicating the key role of M/Ms in the experimental brain metastasis. Interestingly, we found that M/Ms highly expressed matrix metalloproteinase 3 (MMP3), which were strongly correlated with M/M activation and the decrease of tight junction protein zonula occludens-1 (ZO-1). An MMP inhibitor moderately decreased the occurrence of melanoma brain metastasis, suggesting that MMP3 secreted by M/Ms may facilitate melanoma cell growth.ConclusionsOur results indicated that the activated M/Ms were essential in the development of melanoma brain metastasis, suggesting that M/Ms are a potential therapeutic target for tumor brain metastasis.Electronic supplementary materialThe online version of this article (10.1186/s12974-018-1389-9) contains supplementary material, which is available to authorized users.

Microenvironmental Regulation of Tumor Progression and Therapeutic Response in Brain Metastasis

“…However, Chuang et al demonstrated that tumor cells block pro-apoptotic functions of microglia and exploit tissue damage responses to increase their invasive capacity (86). The role of microglia in tumor cell extravasation was further confirmed by Qiao et al using a CSF1R inhibitor to deplete microglia in prevention trial settings in a mouse model for melanoma BrM (84). The authors also found that Mmp3 expression by microglia was negatively correlated with ZO-1 expression on endothelial cells.…”

“…Although the BrM field currently lacks detailed insight into the molecular identity of disease-associated macrophages/microglia compared to neurodegenerative diseases or primary brain tumors, a series of pre-clinical studies shed light into tumor-associated macrophage (TAM) functions during distinct steps of the metastatic cascade. Invasion of metastasizing tumor cells is rapidly sensed by microglia and the presence of single tumor cells is sufficient to recruit and to activate microglia (84, 85). Given the role of microglia in immune surveillance and host defense, it is tempting to speculate that the initial contact between tumor cells and microglia at sites of extravasation leads to clearance of invading tumor cells.…”

“…The authors also found that Mmp3 expression by microglia was negatively correlated with ZO-1 expression on endothelial cells. Moreover, the incidence of melanoma BrM was decreased by Mmp3 inhibition (84). Co-option of microglial functions and adoption of leukocytic characteristics to increase the capacity of tumor cells to colonize the brain parenchyma was previously proposed (5) (Figure 1; Box 1).…”

“…CSF1R inhibition can be achieved by CSF1R blocking antibodies (e.g., RG7155) (159) or ATP competitive small molecule inhibitors (e.g., BLZ945, PLX3397, or PLX5622) (91, 160) (Figure 2; Box 1). Qiao et al employed PLX3397 in a prevention trial setting and demonstrated that microglia depletion reduced tumor cell transmigration potential of melanoma brain metastatic cells (84). This is in line with previous findings that demonstrated that clodronate liposome mediated microglia depletion resulted in a reduction of the BrM burden (98).…”

Immune Microenvironment of Brain Metastases—Are Microglia and Other Brain Macrophages Little Helpers?

“…Depletion of microglia and macrophages by treatment with PLX3397, an inhibitor of colony stimulating factor-1 receptor (CSF-1R), reduced the total number and mean size of the brain metastases by 83 and 65%, respectively. Microglia and macrophages from metastatic brains expressed MMP3 and treatment with PD166793, an MMP inhibitor, reduced the total number and mean size of the brain metastases by 50 and 53%, respectively (82). In a transplantable model of B16 melanoma cells the presence of microglia (CD11b+F4/80+CD45 low ) infiltration into intracranial B16 melanoma tumors increased following combined PD-1/CTLA-4 blockade and the increase in microglia correlated with intracranial therapeutic efficacy.…”