Figure 1.
GBM cells co-opt and modify blood vessels in-vivo.
A, Scheme showing seeding of human-GBM cells (grey spots) onto mouse brain slices. B, White arrowheads point to abnormal blood vessels (black-Ink), co-opted by fluorescence-labeled cells (MiRu+, red). Asterisks indicate agglomerated co-opted vessels in the body of the graft. C, Maximum projection (top) and 4D-reconstruction (bottom)-video frames (respectively) showing GFP-actin human GBM cells (green) re-arranging blood vessels. Frames have been selected to visualize flectopodia with GFP-actin-beads (green, white arrows) bending (yellow arrowheads) a previously straight vessel (DiI-red; blue arrowheads). D–F, A highly schematic cartoon of vessel structure before (D), during (E) and after (F) flectopodia-mediated co-option (based on Movie S1). 1 and 2, co-operating tumor cells (green), linked by cytoplasmic bridge; dashed-lines, recruited/modified vessel segment; black and blue arcs, which show the advance of GBM cells on the vessel, are analogous to the expanding tumor margin. G, Scheme of GBM-hanging drop xenografts. H, GFP-actin-U373 cell (green) in striatum of 2 day-xenograft, contacting host vessel (DiI-red) through a flectopodium (arrow) with moniliform-actin (white arrowhead). I, arrowheads point to Ink-filled, dilated vessels in 7 day-U87 graft. Activated perivascular cells (ap, Rgs5+, blue) are visible on co-opted, modified vessels (cv, black-Ink) at the expanding edge (red dashed-outlines) of both 7day- (J–K) and 1 month- (M–N) U87-xenografts. Note the difference in diameter between normal (black arrowhead) and co-opted (red arrowhead) vessels in K. Scale bars: 30 µm (B), 10 µm (C, H), 200 µm (J, L), 40 µm (K), 25 µm (N).
Figure 2.
GBM cells target pericytes and modify their contractility.
GFP-actin-GBM cells (green) contacting an NG2-DsRed+-pericyte (A, red iso-surface in magnified box) and a DLP (B, blue; confocal section) through flectopodia (arrowheads indicate moniliform actin). Note the presence of GFP-actin within the DLP (merged channels, inset in B). v, vessels (DiD-blue in A; Ink-filled-grey in B). C, Scheme showing pericyte (colored cells) in-vivo (top; BV, blood vessel), in-vitro (middle) and on silicone-substrate (bottom). Wrinkling is associated with high αSMA-expression (red-color). D and E (boxed area in G), DIC-optic video-frames of the same field before (D) and after (E) GBM cell addition to pre-plated pericytes. Pericytes alone produce stable drifting wrinkles (red arrows) that are de-stabilized by GBM cells. White and yellow arrowheads indicate the appearance and disappearance of wrinkles, respectively. Dashed line marks the upper-limit of GBM cell population, transposed from F and G, which show the low magnification of FR Dextran-labeled GBM cells (white false-color in F and magenta in G), plated on cultured pericytes. Time in minutes. H, Traces of two wrinkles, produced before (i) and after (ii) U87-GBM cell-addition, revealing the spatial evolution and colored to indicate lengthening (violet to green) or shortening (green to red) for each time-frame (numbers). I, 3D-plot summarizing the wrinkling behavior of pericytes, either alone (red points, n = 40) or with U87-GBM cells (green points, n = 23). Note the lack of green points in clusters 1 and 2. E1, E2 and C: track-straightness of the ends (E) and center (C) of each wrinkle. Scale bars: 10 µm (A, B), 30 µm (D), 100 µm (G).
Figure 3.
GBM cell/pericyte interaction involves flectopodia and cytoplasmic mixing.
A, A flectopodia-like extension (arrow) from a FR labeled-GBM cell (magenta) contacts wrinkling pericytes (arrowhead) on silicone-laminin substrate (grey, DIC-optics). B, GFP-actin-beads in a presumptive flectopodia correlate with varicosities (arrowheads in insets). C, A beaded GFP-actin-extension (U87 cell, white arrowhead) induces altered wrinkling of pericytes (red arrowhead). D, Cdc42 protein (green, white arrowhead in the magnification) partially co-localizes with FR-dextran (FR, magenta, yellow arrowhead) as dots (0.5 µm in diameter) in the extension of a GBM cell. Fixed co-cultures show human CD44 protein in GBM cell flectopodia-like extensions (E–F, cyan, arrows) and in cytoplasmic particles (asterisks and arrowhead in magnification) in target pericytes (phalloidin, red). G–L, Time-lapse analysis of a U87 cell extending and retracting flectopodia (red and white dashed-arrows, respectively) and shedding terminal varicosities (asterisks, and magnifications in H, L and L’). The cell of interest was outlined and filled with a transparent yellow color using Photoshop. M, Double-labeled GDH cells (arrowheads) on constricted (co) and dilated (di) vessel segments (7 day-xenograft). N, Stepwise fusion-like process of a GBM cell (magenta, arrowheads) with a pericyte (arrows), resulting in a migratory cell-derivative (asterisk). Co-cultured GBM cells (MiRu+, red) and pericytes (FlEm+, green) show partial (O, arrow) or complete (P, white arrowheads) co-labeled cytoplasm, 12 or 48 hours after replating, respectively. Time in minutes. Scale bars: 30 µm (A, E), 10 µm (B, C, G, I’, N, O), 5 µm (D).
Figure 4.
Cdc42-inhibition in GBM cells blocks flectopodia-mediated co-option and activates innate immunity.
A, Cdc42 is present in U373 cell-flectopodia in brain slices (arrows). B, GFP-actin-labeled tumor cell (green; red color indicates transfection of the oligonucleotide siRNA control) co-opting a bent vessel (red arrowheads). C, A non-polarized, iCdc42-treated, GFP-actin+-U373 cell (yellow, arrowhead, indicates double labeling of green [GFP] and red [negative control for transfection]), on a straight vessel (Ink-filled, black-filled-white arrowheads). D–E, Graphs of the effects of iCdc42-treatment on the length of GBM cell extensions and the angle of vessel bending (asterisk, see Methods for length/angle-grouping). D, n = 15 (iRNA and iCdc42); controls, n = 22. E, n = 13 (controls and iCdc42). F, Two juxtaposed U373-grafts, with wild-type (MiRu+, red) or iCdc42 (FlEm+, green) cells. Magnifications display 3D rendering of Ink-filled co-opted convoluted (1, arrows) and non-co-opted straight (2, arrowheads) vessels, respectively. G–H, Quantitative analysis of graft/host margin interaction in short-term slice implants, incorporating both individual and juxtaposed grafts; n = 15 (all controls); n = 11 (iCdc42, G), n = 7 (iCdc42, H). I, Video-frames illustrating two macrophage-like cells (white arrowheads and arrow, respectively) pursuing and destroying a GBM cell (yellow arrows) (see also Movie S8). Time in minutes. U373-wild type (J, 7-day) or iCdc42 (K, 3-day and L, 7-day) xenografts analyzed for the indicated markers. Tc and dtc, core and degenerating tumor-core; white arrows and arrowheads show infiltrating and smooth margin, respectively; dashed outlines mark the original graft. Scale bars: 10 µm (A–C, F1, I), 100 µm (F, J).
Figure 5.
Two-signal model for GBM progression explains the effect of wild-type (green) and iCdc42 (light-green) GBM cells on inter-convertible, contractile pericytes (the white, wrinkling cell in the center). Blue, macrophage-like pericyte; pink and white, activated and co-opted pericytes, respectively.