Unsupervised tSNE clustering based on the top 1000 highly variable genes suggested that interpatient heterogeneity was stronger than the global transcriptional state of between tumor cells within the same sample, which was consistent with a previous report.61 Open in a separate window Figure 5 Single\cell RNA\seq correlates on\chip colocalization to transcriptional signatures/subtypes. Furthermore, a gene signature profile including PDGFRA correlates strongly with the homing of tumor cells to the PVN. These findings demonstrate that this model can recapitulate in vivo tumor cell dynamics and heterogeneity, representing a new route to study patient\specific tumor cell functions. = 4, at day 6), which Fluopyram was comparable to previously reported results for in vitro microvessel models.32, 43, 45 This permeability coefficient is higher than that in the mural/stromal cell supported microvessels, suggesting that microvessels made of a mono\layer of endothelial cells are leakier in the absence of other vascular mural cells, such as fibroblasts and pericytes. Furthermore, a suspension of 10 m sized fluorescent polystyrene beads was perfused into the upper channel of a 3 d aged chip. We observed that this beads readily traveled through the microvascular network and joined the lower microchannel with minimal adherence to the microvessel wall (Figure ?(Figure1h1h and Movie S1, Supporting Information). Finally, the microvasculature hydrogel slab was retrieved, fixed, and dehydrated for scanning electron microscopy (SEM) to confirm the formation of 3D architecture of interconnected endothelial lumen network (Physique ?(Figure11i). 2.3. Preferential Localization of BTSCs in PVN The role of PVN in controlling BTSC fate has been reported in human GBM and validated with animal xenograft models.4, 5, 46, 47 Using tissue\engineered microvasculature models to determine whether BTSCs preferentially localize within the PVN, we quantified colocalization of microvessels and BTSCs (GS5) relative to a GBM cell line (U87). Tumor cells were prestained with lipophilic cell tracking dye Dil (Invitrogen), mixed with GFP\HUVECs, and loaded into the microfluidic chip to examine microvessel growth and tumor cell dynamics. After 7 d, we observed that BTSCs preferentially localized in the perivascular zone (Physique 2 a), specifically in the bifurcation region of the microvessel network. In contrast, U87 cells were overpopulated and did not colocalize (Physique ?(Figure2b).2b). In addition, we observed that incorporation of U87 cells led to fast microvessel remodeling and unstable microvessel network formation, whereas GS5 cells resulted in well\connected microvessel network in 4C5 d. Previous in vivo studies reported that U87 failed to accurately model human GBM compared to patient\derived tumor stem cells.30, 48 Researchers characterized multiple GBM cell lines and showed that U87 exhibits high mitotic figures (as PLXNC1 measured by Ki67) but low levels of neural stem cell markers, such as nestin, Sox2, and CD133.48, 49 Our result is concordant with previously reported studies that compare different cell sources for tumorigenic GBM models. In practice, pathologists diagnose GBM based on three golden standards: mitoses, microvascular proliferation, and necrosis.50 However, it is not fully characterized how proliferative GBM cells migrate and distribute in the brain relative to the vascular system.51 We observed that GS5\EC coculture system exhibited a more connected vessel network. GS5 cells resided in the region near microvessels, whereas U87 showed a different localization pattern in a similar device. We used ImageJ to determine the colocalization of tumor and microvessel signals in the same image by quantifying the Pearson’s correlation coefficient (see the Experimental Section). Quantitative analysis confirmed that patient derived BTSCs (GS5) showed a significantly Fluopyram higher Pearson’s correlation coefficient (0.44 0.02, = 11) than that of U87 cells (?0.03 0.02, = 10) (Figure ?(Physique2c)2c) 7 d after loading into the microchip. One popular hypothesis of tumor nutrient supply is usually that tumor cells can respond to the existing blood vessels (vessel co\option).8 As we could observe and evaluate the relative tumor\vessel location, our model may serve as a high\throughput platform to test anti\vessel\co\option drug in vitro. Open in a separate window Physique 2 Quantification of tumor cell localization relative to microvessels. a) Phase and Fluopyram fluorescent images of microvessels with BTSC GS5. BTSCs were incubated with the Dil membrane dye for 40 min prior to coculture with GFP\HUVECs. BSTCs localize more preferentially to the branching points of the microvessel network. Scale bar: 80 m. b) Fluorescent images of GBM cell line RFP\U87 cells loaded in a microvessel network. RFP\U87 cells randomly distributed in the gel space.