Semaphorin 3A mediated brain tumor stem cell proliferation and invasion in EGFRviii mutant gliomas

Abstract

Background: Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a median survival of approximately 15 months. Semaphorin 3A (Sema3A), known for its axon guidance and antiangiogenic properties, has been implicated in GBM growth. We hypothesized that Sema3A directly inhibits brain tumor stem cell (BTSC) proliferation and drives invasion via Neuropilin 1 (Nrp1) and Plexin A1 (PlxnA1) receptors.

Methods: GBM BTSC cell lines were assayed by immunostaining and PCR for levels of Semaphorin 3A (Sema3A) and its receptors Nrp1 and PlxnA1. Quantitative BrdU, cell cycle and propidium iodide labeling assays were performed following exogenous Sema3A treatment. Quantitative functional 2-D and 3-D invasion assays along with shRNA lentiviral knockdown of Nrp1 and PlxnA1 are also shown. In vivo flank studies comparing tumor growth of knockdown versus control BTSCs were performed. Statistics were performed using GraphPad Prism v7.

Results: Immunostaining and PCR analysis revealed that BTSCs highly express Sema3A and its receptors Nrp1 and PlxnA1, with expression of Nrp1 in the CD133 positive BTSCs, and absence in differentiated tumor cells. Treatment with exogenous Sema3A in quantitative BrdU, cell cycle, and propidium iodide labeling assays demonstrated that Sema3A significantly inhibited BTSC proliferation without inducing cell death. Quantitative functional 2-D and 3-D invasion assays showed that treatment with Sema3A resulted in increased invasion. Using shRNA lentiviruses, knockdown of either NRP1 or PlxnA1 receptors abrogated Sema3A antiproliferative and pro-invasive effects. Interestingly, loss of the receptors mimicked Sema3A effects, inhibiting BTSC proliferation and driving invasion. Furthermore, in vivo studies comparing tumor growth of knockdown and control infected BTSCs implanted into the flanks of nude mice confirmed the decrease in proliferation with receptor KD.

Conclusions: These findings demonstrate the importance of Sema3A signaling in GBM BTSC proliferation and invasion, and its potential as a therapeutic target.

Keywords: Brain tumor stem cells; Glioma; Neuropilin; Plexin; Semaphorin.

Fig. 1

GBM stem cells express Sema3A ligand and receptors. Immunocytochemistry demonstrating expression of stem cell markers CD133 (a) and Nestin (b) (scale bar = 50 μm). Phase-contrast microscopy of a tumorsphere (scale bar = 200 μm) (c). Differentiation of xenografts results in loss of CD133 (d), as shown by immunostaining (scale bar = 50 μm). PCR demonstrating expression of Nrp1, PlxnA1, and Sema3A in BTSCs (uncropped gels presented in Supp Fig. 8) (e). Immunostaining showing Nrp1 is expressed in BTSCs (f) but not differentiated (DDX) cells (g) (scale bar = 100 μm). Elevated Nrp1 expression is associated with CD133-positive BTSCs by flow cytometric analysis of CD133 sorted BTSCs demonstrating 10-fold higher CD133 expression in CD133-high (green) cells compared to CD133-low (blue) (h). 95% of CD133-high fraction cells are positive for CD133, compared to only 47% of CD133-low cells, which have low expression levels (i). qRT-PCR analysis of Nrp1 and PlxnA1 expression in CD133-high and low fractions demonstrating a significant increase in Nrp1 mRNA expression in CD133-high cells compared to CD133-low, but no change in PlxnA1 expression (n = 6; p < 0.05) (j)

Fig. 2

Sema3A drives invasion of BTSCs. a Gap migration assay demonstrating increased percentage of cells within initial boundaries (white dashed line), indicating increased invasive migration in Sema3A (100 ng/mL) treated BTSCs compared to Control over 24 h (green = phalloidin; scale bar = 100 μm). b Quantification of gap migration assays showing increased invasive index with Sema3A treatment (***p < 0.0005). c 3-dimensional Matrigel tumorsphere invasion assays comparing Control and Sema3A (100 ng/mL) treated tumorspheres for 12 h, showing increased process extension with treatment, indicating increased invasion (scale bar = 200 μm). d Quantification of 3-dimensional invasion assay showing increased invasion index with Sema3A treatment compared to Controls (*p < 0.05). e BrdU labeling of Control and Sema3A (10 ng/mL) treated BTSCs demonstrating decreased BrdU positive cells (green) as well as decreased total cells (blue) per field (scale bar = 50 μm). f Quantification of proliferation assays in mean BrdU positive labeled cells per field (mpf) (**p < 0.005). g PI labeling of Control and Sema3A treated BTSCs demonstrating no difference in percent positive cells, indicating no change in cell death

Fig. 3

Sema3A mediates antiproliferative effects via Nrp1 and PlxnA1. a Quantitation of proliferation assay comparing mean DAPI labeled cells per field in Control, Nrp1-KD, and PlxnA1-KD infected BTSCs in the absence and presence of Sema3A (10 ng/mL). Control non-targeting virus treated cells maintain the antiproliferative response to Sema3A. Nrp1-KD and PlxnA1-KD BTSCs demonstrate a decreased baseline proliferation, and show no difference between untreated and treated conditions. b Quantitation of the mean change in DAPI labeled cells per field over 24 h. In control non-targeting virus treated BTSCs, Sema3A abolishes proliferation, as there is no change in cell number between the start and end of the assay. Similarly, there is no change in Nrp1-KD BTSC proliferation in untreated or treated conditions. Here, the proliferation rate of PlxnA1-KD BTSCs was not significantly different than non-targeting virus treated BTSCs but showed loss of the Sema3A antiproliferative response (***p < 0.0005; NS, not significant)

Fig. 4

Sema3A pro-invasive effects are Nrp1 and PlxnA1 dependent. Gap migration assay with Control, Nrp1-KD and PlxnA1-KD BTSCs in the absence and presence of Sema3A (100 ng/mL). Control non-targeting virus treated BTSCs increase invasive migration in response to Sema3A. Nrp1-KD and PlxnA1-KD BTSCs are unresponsive to Sema3A. Nrp1-KD cells demonstrate an increased baseline invasive index (**p < 0.005; ***p < 0.0005; NS, not significant)

Fig. 5

Nrp1-KD and PlxnA1-KD tumors in athymic nude mice flanks. a Shows decreased mean tumor diameter in both receptor knockdowns. NT tumor diameter was statistically significantly greater than Nrp1-KD at all time points after week 3, while comparison with PlxnA1-KD approached significance following week 5. b Scatter plot of flank tumor diameters at week 7. c Mean tumor weight. d Sample images of tumors in mice. Black arrows point to tumors. n = 5 per arm (one NT mouse died of non-tumor related causes at the start of the experiment; **p < 0.005)