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

Dominique M O Higgins,Brian D Milligan,Mark Schroeder,Irving L Weissman,Maisel Caliva,Pavan S Upadhyayula,Fredric B Meyer,Samuel H Cheshier,Jann N Sarkaria,Brett Carlson,John R Henley

doi:10.1186/s12885-020-07694-4

Abstract

BackgroundGlioblastoma 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.MethodsGBM 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.ResultsImmunostaining 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.ConclusionsThese findings demonstrate the importance of Sema3A signaling in GBM BTSC proliferation and invasion, and its potential as a therapeutic target.

Highlights

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a median survival of approximately 15 months
When the xenograft cells were plated in stem cell conditions in the absence of extracellular matrix, self-adherent balls of tumor cells known as tumorspheres formed, indicating the presence of brain tumor stem cell (BTSC) (Fig. 1c)
Downregulation of Semaphorin 3A (Sema3A) receptors inhibits GBM growth in vivo Based on our findings that Sema3A and cognate receptors can regulate cell growth in cell-based assays, we studied the effects of receptor knockdown on proliferation in vivo, injecting each non-targeting virus (NT), Neuropilin 1 (Nrp1)-KD, and Plexin A1 (PlxnA1)-KD BTSCs into the flank of athymic nude mice (n = 5 per group)

Summary

Introduction

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a median survival of approximately 15 months. One attribute responsible for the aggressiveness and refractory nature of these tumors is the presence of endogenous stem cell like GBM cells [7,8,9,10,11]. These subpopulations demonstrate increased resistance to chemotherapy and radiotherapy [12, 13]. Regulation of GBM invasion is at the core of successful treatment These migratory patterns closely resemble those of normal neural stem and progenitor cells [26]. This indicates that BTSCs may be responding to endogenous guidance factors directing invasion

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