Abstract
BackgroundThe mechanisms by which primary cilia affect glioma pathogenesis are unclear. Depending on the glioma cell line, primary cilia can promote or inhibit tumor development. Here, we used piggyBac-mediated transgenesis to generate patient-derived glioblastoma (GBM) cell lines that stably express Arl13b:GFP in their cilia. This allowed us to visualize and analyze the behavior of cilia and ciliated cells during live GBM cell proliferation.ResultsTime-lapse imaging of Arl13b:GFP+ cilia revealed their dynamic behaviors, including distal tip excision into the extracellular milieu. Recent studies of non-cancerous cells indicate that this process occurs during the G0 phase, prior to cilia resorption and cell cycle re-entry, and requires ciliary recruitment of F-actin and actin regulators. Similarly, we observed ciliary buds associated with Ki67− cells as well as scattered F-actin+ cilia, suggesting that quiescent GBM cells may also utilize an actin network-based mechanism for ciliary tip excision. Notably, we found that the proliferation of ciliated GBM cells was promoted by exposing them to conditioned media obtained from ciliated cell cultures when compared to conditioned media collected from cilia-defective cell cultures (depleted in either KIF3A or IFT88 using CRISPR/Cas9). These results suggest that GBM cilia may release mitogenic vesicles carrying factors that promote tumor cell proliferation. Although Arl13b is implicated in tumor growth, our data suggest that Arl13b released from GBM cilia does not mediate tumor cell proliferation.ConclusionCollectively, our results indicate that ciliary vesicles may represent a novel mode of intercellular communication within tumors that contributes to GBM pathogenesis. The mitogenic capacity of GBM ciliary vesicles and the molecular mediators of this phenomenon requires further investigation.
Highlights
Gliomas are the most common primary malignancies of the central nervous system (CNS)
PiggyBac transposon‐mediated delivery of ADP-ribosylation factor-like protein 13b (Arl13b):GFP into patient‐derived GBM cells permits the visualization and tracking of GBM primary cilia and ciliated tumor cells We previously reported that cilia were detectable in patient-derived GBM cell lines and biopsies [11]
We examined additional GBM biopsies and found A RL13B+ cilia colocalized with acetylated alpha-tubulin, a tubulin concentrated in the ciliary axoneme (Fig. 1a–d), confirming our previous findings that ARL13B is present in human GBM primary cilia
Summary
Gliomas are the most common primary malignancies of the central nervous system (CNS). We used piggyBac-mediated transgen‐ esis to generate patient-derived glioblastoma (GBM) cell lines that stably express Arl13b:GFP in their cilia. This allowed us to visualize and analyze the behavior of cilia and ciliated cells during live GBM cell proliferation. We found that the prolifera‐ tion of ciliated GBM cells was promoted by exposing them to conditioned media obtained from ciliated cell cultures when compared to conditioned media collected from cilia-defective cell cultures (depleted in either KIF3A or IFT88 using CRISPR/Cas9) These results suggest that GBM cilia may release mitogenic vesicles carrying factors that promote tumor cell proliferation. Arl13b is implicated in tumor growth, our data suggest that Arl13b released from GBM cilia does not mediate tumor cell proliferation
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