Figure 5

Immunophenotyping of LysM-EGFP and CD11c-EYFP cells.

(a) Representative evaluation of the percentage of LysM-EGFP and CD11c-EYFP expressing fluorescent cells in each of the populations specified in Fig. 4a, in the brain of tumor bearing mice at D28. Values are expressed as cumulated percentages. (b) Distribution of the different immune populations in the whole EYFP⁺, EYFP⁺/EGFP⁺ and EGFP⁺ populations at D28. (c) Multicolor immunostaining of EYFP⁺ (yellow), Iba1⁺ (cyan) and MHCII⁺ (magenta) cells in sagittal sections of tumor bearing brains at D21 (top) and D28 (bottom). (m) meninges; (cc) corpus callosum. Dotted-lines highlight tumor margins. Dotted-squares: EYFP⁺ cells in the (cc). Arrow: Flow of EYFP⁺ cells arising for deep brain structures such as ventricles. Scale-bar: 200 μm. (d) Dura-mater specific morphologies as observed in-vivo by two-photon imaging of EYFP⁺ (yellow, arrow) and EGFP⁺ (green, arrowhead) cells above the tumor at D15 post-graft. Note the second-harmonic signal (blue) characteristic of the fibers of the meningeal layer. (e) Main morphologies of EYFP⁺ (yellow) cells imaged in vivo in peri-tumoral and tumor areas. Red: tumor cells; blue: vasculature. (f ) Distribution of the three main morphologies (star-like elongated and amoeboid) of EYFP⁺ cells observed in vivo by two photon imaging at D21 and D28. Scale bar: 20 μm. (g) In vivo two-photon image of a CD11c-EYFP⁺/LysM-EGFP⁺ cell taken at D26 post-graft (asterisk: CD11c-EYFP⁺/LysM-EGFP⁺ cell, arrowhead: CD11c-EYFP⁺ cell, arrow: LysM-EGFP⁺ cell). Scale-bar: 100 μm.