Cancers likely originate in progenitor zones containing stem cells and perivascular stromal cells. Much evidence suggests stromal cells play a central role in tumor initiation and progression. Brain perivascular cells (pericytes) are contractile and function normally to regulate vessel tone and morphology, have stem cell properties, are interconvertible with macrophages and are involved in new vessel formation during angiogenesis. Nevertheless, how pericytes contribute to brain tumor infiltration is not known. In this study we have investigated the underlying mechanism by which the most lethal brain cancer, Glioblastoma Multiforme (GBM) interacts with pre-existing blood vessels (co-option) to promote tumor initiation and progression. Here, using mouse xenografts and laminin-coated silicone substrates, we show that GBM malignancy proceeds via specific and previously unknown interactions of tumor cells with brain pericytes. Two-photon and confocal live imaging revealed that GBM cells employ novel, Cdc42-dependent and actin-based cytoplasmic extensions, that we call flectopodia, to modify the normal contractile activity of pericytes. This results in the co-option of modified pre-existing blood vessels that support the expansion of the tumor margin. Furthermore, our data provide evidence for GBM cell/pericyte fusion-hybrids, some of which are located on abnormally constricted vessels ahead of the tumor and linked to tumor-promoting hypoxia. Remarkably, inhibiting Cdc42 function impairs vessel co-option and converts pericytes to a phagocytic/macrophage-like phenotype, thus favoring an innate immune response against the tumor. Our work, therefore, identifies for the first time a key GBM contact-dependent interaction that switches pericyte function from tumor-suppressor to tumor-promoter, indicating that GBM may harbor the seeds of its own destruction. These data support the development of therapeutic strategies directed against co-option (preventing incorporation and modification of pre-existing blood vessels), possibly in combination with anti-angiogenesis (blocking new vessel formation), which could lead to improved vascular targeting not only in Glioblastoma but also for other cancers.

Funding: This work was funded by Spanish Ministry of Science and Innovation: FEDER (BFU-2010-27326) and Consolider (CSD2007-00023); Health Institute Carlos III: Red TERCEL (RD12/0019/0024); P.H.C was also supported by Marie Curie Intra-European Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2014 Caspani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Glioblastoma Multiforme (GBM) is a highly invasive brain cancer, with prominent vascular involvement, characterized by twisted blood vessel [1] and infiltration along external vessel walls [2] , which makes it resistant to treatment. Evidence from a rat GBM model has shown that early tumor vasculature forms by co-option of pre-existing brain blood vessels and precedes new vessel formation (angiogenesis) [3] . Vessel co-option also occurs during metastasis of other tumors, as recently demonstrated for the spread of breast cancer into the brain [4] . Furthermore, co-option is also responsible for tumor recurrence and metastasis following anti-angiogenic therapies, both in GBM and in other types of cancer [5] - [8] . Therefore, vessel co-option is likely to be a principle cause of malignancy, which occurs during tumor initiation/progression, metastasis and re-initiation after treatment. However, in contrast to angiogenesis that is well understood, the cellular and molecular bases of vessel co-option in tumors are currently unknown. The normal brain microvasculature is made up of narrow tubes (capillaries), consisting of endothelial cells surrounded by contractile pericytes, which function normally to regulate vessel tone and morphology [9] , [10] . Because pericytes are located on the abluminal wall of blood vessels, they are good candidates for a role in mediating vessel co-option by tumor cells. Brain pericytes are pluripotential cells with stem cell properties [11] - [13] , similar if not identical to the mesenchymal stem cells that occupy an equivalent perivascular location in bone marrow. There is a growing realization that, in addition to their critical role in maintaining blood vessel integrity and controlling blood flow, pericytes are also key players in other aspects of brain homeostasis and disease. For example, evidence suggests that they are regulators of innate immunity and, depending on the context, can mediate not only pro-inflammatory functions associated with host defense [14] , but also the anti-inflammatory response to malignant tumors such as human GBM, which includes the inhibition of T cell function and local immunosuppression [15] . Consistent with a role in normal cerebral immunity, purified brain pericytes have been shown to be interconvertible with macrophages [16] and to behave as macrophage-like cells in culture, by phagocytosing plastic beads [17] and by secreting inflammatory cytokines such as IL-1β, TNF-α and IL-6. Moreover, pericytes play an additional role in maintaining a proper function of the brain-immune interface, by controlling the migration of leukocytes in response to inflammatory mediators [18] . Given that immune cells contribute to tumor progression [19] , pericytes could therefore provide a critical node for local control of both vessel co-option and immune system modulation. Within established tumors, blood vessels are often dysmorphic, with abnormal pericyte coverage and either atypical or absent endothelium [20] . Recent research, aimed to understand the possible function of pericytes in tumor progression, has emphasized their role in new vessel formation during angiogenesis [21] . In co-culture experiments, pericytes have been shown to modulate the angiogenic response of endothelial cells to glioma cells [22] . Furthermore, the recent discovery that GBM stem cells can trans-differentiate into tumor pericytes during the process of angiogenesis [23] further emphasizes the contribution of perivascular cells to tumor growth. While these findings underline the role of pericytes in the establishment of new vessels, very little is known about pericyte function in tumor infiltration. It is now recognized, for example, that perivascular tumor invasion occurs in some types of cancers [24] . Recent discoveries in breast and colon carcinomas have proposed that paracrine crosstalk between tumor and stromal cells is able to promote tumor growth and motility [25] , [26] . Nevertheless, up to now no information exists about the cell biology of tumor cell/pericyte interaction, either in GBM or in cancer in general.

Results

First, we challenged human GBM cells (U87) with mouse brain slices where blood vessels were pre-labeled with black ink (Figure 1A, B). This assay revealed a remarkable ability of GBM cells to pull pre-existing blood vessels into the graft. Vessel co-option happens quickly (within 15 hours) with conversion of normal capillaries into highly twisted structures, demonstrating a capacity for rapid vascular network acquisition, even in the absence of new vessel formation. Next, we used GFP-actin labeling and 2-photon live imaging to identify the intrinsic GBM cellular mechanisms involved in vessel-co-option (Figure S1; Figure 1C–F). We found that, 6 hours after seeding, tumor cells in contact with blood vessels can convert straight segments (up to ∼60–70 µm long) into hairpin bends (Figure 1C and Movie S1). Vessel re-organization is linked to the appearance of tumor cell protrusions, that we called ‘flectopodia’ (flectere, to bend, and podós, foot), characterized by an unusual, discontinuous (moniliform) organization of actin, with highly dynamic beads (0.6–3.0 µm in diameter), moving bidirectionally. Flectopodia are 7–30 µm long, present in 9% of GFP-actin+-cells on vessels (n = 266), with the ability to elongate for up to 20 µm or more, at a rate of 14–99 µm/hour, and to form a bent vessel segment in 30 minutes. A gap of approximately 10 µm separates the GFP-actin labeling from the vessel lumen, suggesting that flectopodia are in close contact with perivascular cells. Flectopodia-associated bending often involves a pair of GFP-actin labeled cells, cooperating via a cytoplasmic bridge. During the process, while the leading cell performs the re-arrangement, the lagging cell translocates the bent segment ahead of the first cell (Figure 1D–F; Movie S1). This suggests that flectopodia mediate the ongoing ratchet-like recruitment of host vessels at the co-option front of the tumor.

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larger image TIFF original image Download: 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). https://doi.org/10.1371/journal.pone.0101402.g001

Next, to validate and extend our ex-vivo observations, we established a xenograft model that recapitulates human GBM in mice (Figure 1G) and used it to confirm the presence of flectopodia and vessel co-option in situ. High resolution-microscopy of sections from GFP-actin-labeled xenografts identified flectopodia-like extensions as early as 2 days (Figure 1H). In situ hybridization at 7 days for the activated mouse (m)-pericyte marker Rgs5, a gene controlling tumor vasculature remodeling [27], [28], showed the presence of m-Rgs5+-cells surrounding abnormally dilated vessels throughout the graft (Figure 1I–K). m-Rgs5+-pericytes are also detected in the infiltrating margin of 1-month xenografts (Figure 1L–N). Taken together these data strongly suggest that host brain perivascular cells are a target cell type for GBM vessel co-option and modification throughout tumor progression.

To confirm that GBM cells interact with pericytes, GFP-actin labeled U373 and U87 cells were co-cultured with brain slices pre-labeled in situ for pericytes, using either a DsRed-transgene reporter for the pericyte marker NG2 [9] or a fluorescent Dextran tracer (Methods). Our results show that tumor cells on vessels interact with Dextran (Dextran Labeled Pericytes, DLPs) or NG2-DsRed labeled perivascular cells (Figure 2A, B; Figure S2 A–G). Unexpectedly, we also found tumor-derived cytoplasm within the cortex of the target pericyte (Figure 2B and Figure S2 G), implying a role for cytoplasmic transfer in the co-option process (see also Movie S2).

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larger image TIFF original image Download: 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). https://doi.org/10.1371/journal.pone.0101402.g002

Our identification of pericytes as a specific GBM cell target raised the possibility that the altered blood vessel morphology in tumors could be caused by deregulated pericyte contraction. To test this, we established an in vitro system using isolated mouse brain pericytes (Methods) cultured on deformable silicone substrates [29], with the novel coating of human-laminin to reproduce the blood vessel basal lamina that houses perivascular cells in vivo (Figure 2C). Pericytes in vitro express NG2 and display attributes consistent with stem (CD44; Vimentin; Nestin), contractile (α-smooth muscle actin, αSMA) and macrophage (CD68 and phagocytosis) potential (Figure S2 H). Two days after plating, cells generate compression forces (indicative of vasoconstriction activity [29]) visible, using Differential Interference Contrast (DIC) imaging, as wrinkles in the silicone sheet (Figure 2D). These are organized around local nodes of higher contractility (Movie S3), correlated with the expression of αSMA protein, a key determinant of pericyte contraction (Figure S2 I–K’). Remarkably, αSMA is also enriched in DLPs in vivo, suggesting that they may represent strategic nodes for the regulation of brain vessel tone (Figure S2 L–N’). Time-lapse confocal analysis showed that individual wrinkles change in position and length over time (between 20 and 200 µm) and cycle with a period of approximately 25/40 minutes (Movie S3; Figure S2 O–O’ and data not shown).

We then investigated if pericyte wrinkling-activity was affected by GBM cell addition. Although neither U373 nor U87 cells alone deform the substrate, in co-cultures they induce nearby pericytes to generate both new wrinkles and destabilize pre-existing ones (Figure 2E–G; Movie S4; Figure S2 P–P’). Quantification of the contractile activity with and without GBM cells, by tracking the behavior of identified wrinkles (Figure S2 O”, P”), revealed a difference in the way the wrinkles move in space and time (E2–E1, P = 0.02, where E1 and E2 are defined as track-straightness of each wrinkle end, Methods). Moreover and interestingly, including the track-straightness of the wrinkle center (C) and plotting all the wrinkle data in a 3D-scatter graph (Figure 2I), showed that GBM cells abolish the formation of wrinkles that drift laterally or drift laterally and pivot, typical of pericytes in nodes and inter-nodes (point clusters 1 and 2, respectively), leaving only the less organized activity characteristic of anti-nodes (point cluster 3; Movie S3). In conclusion, therefore, our data provide strong evidence that tumor cells can corrupt the intrinsic contractility of brain pericytes.

Next, we investigated the cellular mechanisms employed by GBM cells to induce pericyte dysfunction. Live imaging of U373 and U87 GBM cell/pericyte co-cultures on silicone-laminin substrates identified long extensions (maximum length 81±32 µm) (Figure 3A; Figure S2 P, P’), characterized by a discontinuous distribution of both cytoplasmic varicosities and GFP-actin (Figure 3B, C). Moreover, co-transfection assays in vitro showed that the small GTPase Cdc42, a principle regulator of cell polarity and actin cytoskeletal organization [30], is co-localized with the actin beads within cytoplasmic varicosities (Figure S3 A) and the native protein is visible within the GBM cell extensions on silicone substrate (Figure 3D). Importantly, analysis of human CD44, a cancer-associated cell surface adhesion molecule [31], revealed that the edges and tips of GBM cell elongations contact the target pericyte (Figure 3E, F). Remarkably, tumor cell projections predict the locations where the wrinkling pattern is changing (Figure S4 A-C”; Movie S5 and Figure 3C). Taken together, these converging lines of evidence strongly suggest that the cellular extensions seen on silicone substrates are similar, if not identical, to the flectopodia described above (Figure 1, 2). Flectopodia, therefore, are robust GBM cellular specializations that deregulate pericyte contractility.

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larger image TIFF original image Download: 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). https://doi.org/10.1371/journal.pone.0101402.g003

Detailed analysis showed that flectopodia are characterized by alternating phases of extension and retraction (Figure 3G–L). Surprisingly, we found that, during retraction, cytoplasmic fragments (3–8 µm in diameter), corresponding to varicosities located originally at the advancing tip (Figure 3I’–K’) can be transferred into the target pericyte (Figure 3H, L and L’; Movie S6; Figure S4 D). These data are compatible with the GBM cell-cytoplasmic fragments positive for h-CD44 identified inside the contacted pericyte (Figure 3E, F) and corroborate cytoplasmic transfer observed in brain slices (Figure 2B; Figure S2 E, G). Moreover, this is supported by the mixing of GBM cell-derived Cherry-tagged Cdc42 and host DLP cytoplasms in xenografts (Figure S3 D–G). Taken together, our data indicate that co-option involves pericyte up-take of cytoplasmic micro-domains released by flectopodia.

Unexpectedly, our data suggest that GBM cells can use pericytes also as fusion-partners. First, MiniRuby-labeled (MiRu+) U373 grafts into unlabeled hosts generate clusters of strongly MiRu+-cells, which express mouse Rgs5 but lack human centromeric (h-cen) DNA (Figure S5 A, B). Furthermore, grafting Dextran MiRu+-U373 or U87 cells, into mouse brains or slices harboring DLPs, resulted in a range of differentially double-labeled derivatives (Figure S5 C–R). Among these, a novel cell type, that we called GDH (GBM cell/DLP Hybrid), is particularly striking due to its intense double labeling and location on highly constricted/dilated vessels, far beyond the tumor margin (Figure 3M; Figure S5 D, E, I). Surprisingly, GDH cells lack GBM cell-specific markers, h-CD44, h-Nestin, and h-cen DNA (Figure S5 K, M, O), but maintain high levels of αSMA, characteristic of the parental DLP (Figure S6 A–F). Critically, these curious double-labeled derivatives are strongly associated with both the presence of Nitrotyrosine (Figure S6 G–J) and hypoxia (Pimonidazole-staining, Figure S6 K–M), indicating that vessel hyper-contractility is linked to oxidative/nitrative stress [32]. GDH cells retain their strategic position even in advanced GBM-xenografts (Figure S6 N–U) and, interestingly, shed Cdc42+-particles in the lumen of dilated vessels in 7-day grafts, which are also found in sinusoidal vessels in 1-month-tumours (Figure S6 X, Y).

Remarkably, time-lapse confocal imaging on silicone substrates showed that the double-labeled cytoplasm, characteristic of GDH cells, could result from a fusion-like process that leads to the merger of a cell pair. This process, involving the interaction of a round GBM cell with a raised pericyte, occurs in three steps over 2–3 hours (Figure 3N and Movie S7). A preliminary cell/cell contact-phase (1-hour) is followed by tumor cell-cytoplasmic transfer and coalescence of the cell bodies (1-hour), with final translocation of the presumptive hybrid-derivative. When both GBM cells and pericytes were loaded with different colored Dextrans and combined on glass, we found cohorts of differentially double-labeled progenies (Figure 3O, P; Figure S7), some with aberrantly sized nuclei, indicative of abnormal ploidy. Time-course analysis showed that loss of h-CD44 and h-Nestin is already complete 48 h after cytoplasmic mixing (data not shown), supporting our cell-hybrid data in mouse xenografts (Figure S5). Thus, these results identify pericytes, for the first time, as a specific GBM cell-target for the production of fusion-like hybrids, with the potential to generate novel malignant cell variants, such as the hyper-contractile GDH cells, strategically located to maintain a hypoxic penumbra at the invasive edge of the tumor.

Next, considering that flectopodia are actin-based extensions from highly polarized cells, we reasoned that inhibition of the actin GTPase Cdc42 might block vessel co-option. Immunohistochemistry on GBM cells seeded onto brain slices showed that Cdc42 is enriched in flectopodia (Figure 4A). Reducing Cdc42 in tumor cells, using either siRNA (iCdc42) or the specific Cdc42-inhibitor Secramine-A [33], results in shortened extensions to vessels and in reduced angle of vessel bending in brain slices (Figure 4B-E). Secramine-A also decreases the likelihood of a bend occurring where a tumor cell is attached to a blood vessel (Figure S8 A, B). To test Cdc42-function at the tumor/host margin, GBM cell-pellets, with and without inhibition for Cdc42, were grafted either separately or adjacent to each other into brain slices (Figure 4F). Importantly, while wild type-cells pull some vessels into the graft and use others for radial migration, iCdc42-treated cells show little affinity for blood vessel and no co-option (Figure 4F–H; Figure S8 C, C’). Co-opted vessels present abnormal constrictions, dilations and localized hairpin bends, while vessels adjacent to iCdc42-grafts maintain a straight morphology (Figure 4F1 and F2, respectively).

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larger image TIFF original image Download: 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). https://doi.org/10.1371/journal.pone.0101402.g004

We then showed that CD44, a GBM marker with fusogenic properties [34], is enriched at vessel contact sites and cooperates with Cdc42 in vessel co-option/modification in brain slices. Our data demonstrated that knocking down CD44 (by shDNA) in combination with Cdc42 increases the inhibitory effect of iCdc42 alone on flectopodia-length and the angle of vessel bending, with a reduction in the number of glomeruloid-like structures by 70% (Figure S8 D–H). Additionally, iCdc42 shifts tumor cell-phenotype from ensheathing/re-arranging vessels, to a loosely associated state, a tendency amplified when CD44 is also reduced (Figure S8 I, J). Taken together, these data suggest that Cdc42 and CD44 act synergistically during flectopodia-induced vessel co-option/modification.

Subsequently, we tested the effect of iCdc42-GBM cells on pericyte behavior on silicone/laminin substrates. In addition to the initial pericyte activation induced by wild-type GBM cells (Figure S9 A–B”, E–F”, M), confocal live imaging showed a further pericyte-transformation into hyper-activated macrophage/dendritic-like cell phenotype, capable of killing and engulfing iCdc42-treated tumor cells, with concomitant overall reduction in wrinkling activity (Figure 4I; Movies S8, S9; Figure S9 C–D”, G–H”, I–L). In summary, this study uncovers a switching role for Cdc42 not only in flectopodia-mediated vessel co-option, but also in suppressing the activation of pericytes into cytotoxic, macrophage-like phenotypes.

We next investigated whether iCdc42-treatment could block vessel co-option and promote an immune response in vivo. Seven-day xenografts of wild type-GBM cells show high levels of h-CD44 and h-Nestin, with m-Rgs5+-cells around dilated, co-opted vessels (Figure 4J). In contrast, iCdc42 tumors appear to be compromised. Three days after grafting, implants present only a thin h-CD44+-shell and an intense m-Rgs5+-core, with no evidence for h-Nestin+-cells or vessel co-option (Figure 4K; Figure S9 P). By 7-days, the h-CD44+-shell and the m-Rgs5+-core are reduced or even absent (Figure 4L), with an accumulation of vimentin+-microglia at the implantation site, demonstrating an increased host phagocytic response (Figure S9 Q). Taken together, our data indicate that Cdc42 activity in GBM cells favors tumor-establishment over clearance.

Targeting the Cdc42/CD44/actin/pericyte/hypoxia axis (demonstrated above), to block vessel co-option in patients, depends critically on the underlying mechanism being conserved from mouse to human. Strikingly, we found that 88% (7/8) of an unbiased sample of 8 primary human GBM tumors show abnormally elevated levels of CDC42 and CD44, restricted to perivascular locations on abnormal blood vessels at the boundary between tumor tissue and normal looking brain (Figure S10 A–C), a feature reproduced in an independent GBM biopsy (Figure S10 E). Interestingly, these two genes are part of a coordinately expressed group of genes (synexpression group), that may function together in tumorigenesis, including Hypoxia induced factor-1 alpha (HIF1Α), the actin binding protein Transgelin (TAGLN, SM22) and Platelet-derived growth factor receptor beta (PDGFRΒ), markers of abnormal tumor vessels [35] and pericytes [36], respectively (Figure S10 A–C). Notably, all the tumors (2/8) that recur 6 months after radio- and chemotherapies correlate to those cases where expression of these genes are greatest. Taken together, this validates the Cdc42/CD44/actin/pericyte/hypoxia axis as a desirable target for GBM therapy.

Overall, our results suggest a 2-signal model for GBM progression that involves two distinct tumor cell-derived signals, which act on contractile brain pericytes (Figure 5 and Figure S11 A–D). The first signal (signal-1) causes pericyte activation and conversion to phagocytic, macrophage-like cells. In contrast, signal-2, which is flectopodia-dependent and requires active Cdc42 function, promotes vessel co-option by engaging contractile pericytes. The combination of these events generates fusion-like hybrids. In the presence of both signals, the tumor expands by continuous co-option and diversification, while, in the absence of signal-2, it is cleared by the unrestricted generation of cytotoxic cells, derived from the activation of contractile pericytes.