Microglia mediate the in vivo effects of CpG-C.

(a-e) Chronic in vivo two-photon imaging in CX3CR1GFP/+ mice indicated that microglia cells (green) have dynamic relations with tdTomato-labeled D122 tumor cells (red; 15-μm stacks, with 1-μm z-steps) and that CpG-C treatment increases tumor internalization by microglia. (a) A microglia cell (arrow) interacting with a tumor cell (arrowhead; two hours post–tumor cells inoculation), phagocytizing it (one day later), and dismantling it (day 2 post–tumor cell injection, inset shows an engulfed tdTomato-positive cell or part of it). (b) Different levels of interaction between microglia and tumor cells—(i) no interaction; (ii) contact; and (iii) microglia phagocytized a tumor cell. (c) Partial reconstitution of a 15-μm stack with 1-μm z-steps demonstrating the microglia-tumor cells’ “battlefield” four hours after tumor cell injection. (d) Representative images and quantification (e) of microglia-tumor interactions in control and CpG-C–treated mice four hours and one day following tumor cell inoculation (arrows for contact and arrowheads for internalization). CpG-C treatment resulted in increased contacts four hours following tumor inoculation (n = 3; F(1,4) = 2.875, p = 0.0218) and in microglia internalization of tumor cells/debris 4 (p = 0.0372) and 24 hours (n = 3; F(1,4) = 3.400, p = 0.0041) following tumor inoculation. Scale bar for (a-d) is 20 μm. (f-h) CX3CR1GFP/+ mice were treated with a single systemic prophylactic CpG-C treatment, injected mCherry-labeled D122 tumor cells using the aECAi approach, and brains were analyzed using ImageStream FACS. While CpG-C treatment did not affect the number of microglia cells (n = 5; two-tailed Mann–Whitney U = 10, p = 0.6905; f) or capacity of tumor cell infiltration (indicated by total mCherry area in perfused brains; two-tailed Mann–Whitney U = 9, p = 0.5476; g), it resulted in increased phagocytosis of tumor cells by microglia (two-tailed Student t test for unpaired samples, t(4) = 3.8850, p = 0.0178; h). Scale bar for (e-g) is 5 μm. Data in (e) are presented as mean (±SEM) and box plot whiskers represent minimum–maximum range (f-h). The underlying data for this figure can be found in S1 Data. aECAi, assisted external carotid artery inoculation; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; n.s., nonsignificant; ROI, region of interest.