Ex vivo Dynamics of Human Glioblastoma Cells in a Microvasculature-on-a-Chip System Correlates with Tumor Heterogeneity and Subtypes

The perivascular niche (PVN) plays an essential role in brain tumor stem-like cell (BTSC) fate control, tumor invasion, and therapeutic resistance. Here, a microvasculature-on-a-chip system as a PVN model is used to evaluate the ex vivo dynamics of BTSCs from ten glioblastoma patients. BTSCs are found to preferentially localize in the perivascular zone, where they exhibit either the lowest motility, as in quiescent cells, or the highest motility, as in the invasive phenotype, with migration over long distance. These results indicate that PVN is a niche for BTSCs, while the microvascular tracks may serve as a path for tumor cell migration. The degree of colocalization between tumor cells and microvessels varies significantly across patients. To validate these results, single-cell transcriptome sequencing (10 patients and 21 750 single cells in total) is performed to identify tumor cell subtypes. The colocalization coefficient is found to positively correlate with proneural (stem-like) or mesenchymal (invasive) but not classical (proliferative) tumor cells. Furthermore, a gene signature profile including PDGFRA correlates strongly with the “homing” of tumor cells to the PVN. These findings demonstrate that the model can recapitulate in vivo tumor cell dynamics and heterogeneity, representing a new route to study patient-specific tumor cell functions

Impaired Inflammatory Responses in Murine Lrrk2-Knockdown Brain Microglia

LRRK2, a Parkinson's disease associated gene, is highly expressed in microglia in addition to neurons; however, its function in microglia has not been evaluated. Using Lrrk2 knockdown (Lrrk2-KD) murine microglia prepared by lentiviral-mediated transfer of Lrrk2-specific small inhibitory hairpin RNA (shRNA), we found that Lrrk2 deficiency attenuated lipopolysaccharide (LPS)-induced mRNA and/or protein expression of inducible nitric oxide synthase, TNF-α, IL-1β and IL-6. LPS-induced phosphorylation of p38 mitogen-activated protein kinase and stimulation of NF-κB-responsive luciferase reporter activity was also decreased in Lrrk2-KD cells. Interestingly, the decrease in NF-κB transcriptional activity measured by luciferase assays appeared to reflect increased binding of the inhibitory NF-κB homodimer, p50/p50, to DNA. In LPS-responsive HEK293T cells, overexpression of the human LRRK2 pathologic, kinase-active mutant G2019S increased basal and LPS-induced levels of phosphorylated p38 and JNK, whereas wild-type and other pathologic (R1441C and G2385R) or artificial kinase-dead (D1994A) LRRK2 mutants either enhanced or did not change basal and LPS-induced p38 and JNK phosphorylation levels. However, wild-type LRRK2 and all LRRK2 mutant variants equally enhanced NF-κB transcriptional activity. Taken together, these results suggest that LRRK2 is a positive regulator of inflammation in murine microglia, and LRRK2 mutations may alter the microenvironment of the brain to favor neuroinflammation

LPS-induced p38 phosphorylation is specifically inhibited in Lrrk2-KD cells.

<p>(<b>A, C, D</b>) Cells were incubated with LPS (100 ng/mL) for the indicated times (A, C) or 30 min (D), and the levels of phosphorylated p38 (p-p38), JNK (p-JNK), ERK (p-ERK), and total p38 (A) or total and phosphorylated MKK3/6 (C, D) were determined by Western blotting. α-tubulin was used as an internal control. (<b>B, E</b>) Band intensities of p-p38 (B) and p-MKK3/6 (D) were quantified using a densitometer. Values are means ± SEMs of three independent experiments (*<i>p</i><0.05, **, <i>p</i><0.01 vs. control). Data are representative of three independent experiments.</p

List of primer sequences used for qRT-PCR.¹

1<p><b>Fwd</b>, Forward primer; <b>Rev</b>, Reverse primer.</p

Effect of hLRRK2 overexpression on the NF-κB signaling pathway and NF-κB activity in HEK293T cells.

<p>(<b>A</b>) HEK293T cells (parental or TCM-HEK) were transfected with a 5× NF-κB–luciferase reporter construct, empty vector (mock), or hLRRK2 expression vector. One unit is the basal NF-κB activity detected in untreated parental HEK293T cells. <i>Left:</i> NF-κB activity was measured by luciferase assay. <i>Right:</i> LRRK2 expression was confirmed by Western blotting for c-myc. β-actin was used as a loading control. Values are means ± SEMs of three independent experiments. (**<i>p</i><0.01 vs. mock). (<b>B</b>) LPS induced a dose-dependent increase in NF-κB activity in TCM-HEK cells with or without LRRK2. Cells were treated with the indicated amount of LPS for 6 h. The values shown are fold-induction relative to the values of NF-κB activity in unstimulated TCM-HEK cells, expressed as means ± SEMs of three independent experiments (*<i>p</i><0.05, **<i>p</i><0.01 vs. mock). (<b>C</b>) <i>Upper panel:</i> TCM-HEK cells were transfected with LRRK2-WT and each LRRK2 mutant, and NF-κB activity was analyzed after treating with LPS (10 ng/mL) for 6 h. <i>Lower panel:</i> Expression of LRRK2 mutants was detected by Western blotting. Values are means ± SEMs of three independent experiments. (<b>D</b>) TCM-HEK cells were transfected with mock, G2019S (100, 250 and 750 ng), and G2385R (100 and 500 ng) hLRRK2 expression vector. Values are means ± SEMs of three independent experiments. <i>Upper panel:</i> NF-κB activity measured by luciferase assay. <i>Lower panel:</i> LRRK2 levels determined by Western blotting (*p<0.05; <i>ns</i>, not significant). (<b>E</b>) NF-κB DNA-binding activity was analyzed by EMSA. Nuclear extracts were obtained from mock and LRRK2-WT-overexpressing TCM-HEK cells stimulated with LPS (10 ng/mL) for the indicated times. (<b>F</b>) A supershift assay was performed using nuclear extracts obtained 1 h after LPS treatments and preincubated with NF-κB p50 and p65 antibodies. Arrowheads indicate p50/p65 complex. Specific binding (arrowhead) was analyzed using an excess (20×) of unlabeled (Cold) DNA.</p

Attenuation of inflammatory responses in Lrrk2-KD microglia.

<p>(<b>A</b>) BV-2 microglia were infected with lentivirus expressing non-targeted (Con) or Lrrk2-targeted shRNA (KD). Two stable clones of each group were selected. Expression levels of Lrrk2 mRNA and protein were analyzed by qRT-PCR (left) and Western blotting (right), respectively. Parental (−), con, and KD cells were treated with or without 100 ng/mL LPS (B–E), 10 µg/mL LTA (F), 500 ng/mL CL097 (G), or 500 ng/ml ODN1668 (H) for indicated times (B, D, E), 12 h (C), 24 h (F), or 48 h (G, H). (<b>B</b>) TNF-α and IL-6 secretion into the culture medium were analyzed by ELISA. (<b>C, D, F–H</b>) iNOS protein expression was assayed by Western blotting (C), and NO release was measured using the Griess reagent, as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034693#s2" target="_blank">Methods</a> (D, F–H). (<b>E</b>) TNF-α, IL-1β, IL-6 and iNOS mRNA levels were analyzed by qRT-PCR. Gapdh mRNA and α-tubulin protein levels were analyzed as internal controls for qRT-PCR and Western blotting, respectively. Values are means ± SEMs (*p<0.05, **<i>p</i><0.01 vs. control). Data are representative of at least three independent experiments unless indicated otherwise.</p