Abstract
The intratumor heterogeneity represents one of the most difficult challenges for the development of effective therapies to treat pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG). These brain tumors are composed of heterogeneous cell subpopulations that coexist and cooperate to build a functional network responsible for their aggressive phenotype. Understanding the cellular and molecular mechanisms sustaining such network will be crucial for the identification of new therapeutic strategies. To study more in-depth these mechanisms, we sought to apply the Multifluorescent Marking Technology. We generated multifluorescent pGBM and DIPG bulk cell lines randomly expressing six different fluorescent proteins and from which we derived stable optical barcoded single cell-derived clones. In this study, we focused on the application of the Multifluorescent Marking Technology in 2D and 3D in vitro/ex vivo culture systems. We discuss how we integrated different multimodal fluorescence analysis platforms, identifying their strengths and limitations, to establish the tools that will enable further studies on the intratumor heterogeneity and interclonal interactions in pGBM and DIPG.
Highlights
Pediatric Glioblastoma and Diffuse Intrinsic Pontine Glioma (DIPG), are amongst the most aggressive tumors of the central nervous system affecting children and young adults, for which there is no effective treatment [1,2]
We used the Lentiviral Gene Ontology (LeGO) vectors, previously described our patient primary derived cell lines are exclusively grown in stem cell‐like culture condition [12], the original protocol was modified in order to remove the serum from the viral preparation
The viral pellet was resuspended in phosphate‐buffered saline (PSB) and diluted in the fSotremtheCReGll BMmedairukming(FaingudrOe p1,ti2c.aLl Benatricvoidruinsgco[1n7c,e1n9]tr, aetxiponre)s. sing six different fluorescent proteins with distinTcthexcpiGtaBtiMon and eDmIiPsGsiopnrpimroapreyrtcieesl:l LlienGeOs,Gno2r(meGalFlPy),eLxepGanOd-Ved2a, Ls enGeOur-So2sp(Th-eSraeps,phwireer)e, LceuGltOur-emdOardahnegree2n(tmon-Olaramnignei2n),[2L0e]GtOo -eEnBsuFPre2a(EhBigFhPe2r) acenldl tLraeGnsOd-udcKtiaotnusehffkiaci2en(dcKyaatnudshtkoaf2a)c(iFliitgautereth1e, 1v.isLueanltiizvaitriuosnporfotdhuecttriaons).duced fluorescent proteins under a microscope
Summary
Pediatric Glioblastoma (pGBM) and Diffuse Intrinsic Pontine Glioma (DIPG), are amongst the most aggressive tumors of the central nervous system affecting children and young adults, for which there is no effective treatment [1,2]. A significant degree of genetic and phenotypic intratumor heterogeneity has been recently identified in pGBM and DIPG, which may represent one of the most challenging aspect in the effort to develop new effective therapeutic strategies for these diseases [8,9,10,11,12]. It has been recently demonstrated that pGBMs and DIPGs are characterized by a complex and heterogenous sub-clonal architecture, where distinct cell subpopulations coexist and co-operate, building a cellular network promoting tumorigenesis and responsible of the aggressive phenotype [12]. Evidence has clearly demonstrated the existence of direct interconnections between the neuronal compartment of the brain microenvironment wiring the glioma cells and vice versa [14,15]
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