Molecular mechanisms underlying radioresistance of glioblastoma initiating cells

Abstract

Glioblastoma (GBM) is the most frequent and malignant primary brain tumor. The current standard of care for adult patient with diagnosed GBM is surgery followed by radiotherapy (RT) plus concomitant and adjuvant temozolomide (TMZ) chemotherapy. Despite intense patient management, conventional therapies are not able to achieve long-term remissions and eventually almost every tumor recurs. The impossibility of extensive tumor debulking, the marked heterogeneity of lesions, the poor drug delivery in the brain and the presence of cancer cells with stem features (Cancer Stem Cells, CSCs) within the bulk of the tumor contribute significantly to the lack of effective treatment options.

We ought to develop an in-vitro model to investigate molecular mechanisms underlying GBM resistance based on two major cornerstones: (i) the key duality between Glioblastoma Initiating Cells (GICs) and the bulk of the tumor; and (ii) the intratumoral heterogeneity. Consequently, we conceived a paired model where both GICs and differentiated GBM cells depicting the bulk of the tumor were represented. Both culture models were derived from the same GBM post- surgical specimen, but were established and maintained in different culturing conditions. Moreover, we aimed to design a model that could preserve as much as possible the intratumoral heterogeneity of GBM, within the known limits of in-vitro cultures. Consequently, cultures obtained from GBM specimens were not sorted for expression of putative cancer stem cells markers.

Six different GBM patients’ samples were processed and established in-vitro as both Differentiated Glial Cells (DGC) and GICs cultures. Established GICs cultures and corresponding tumor-of-origin were analysed according to the molecular subtypes defined on the basis of transcriptomic signature and both were classified as predominantly Mesenchymal. DGC and GICs deriving from the same patient and growing in cultures as monolayer and neurospheres respectively, were compared side-by-side and stem’s functional features and markers expression were investigated. Neurosphere cultures demonstrated to be enriched in GICs, whereas monolayer cultures were not, as indicated by their poor clonogenic capacity and absent CSCs markers expression. In addition, CSCs markers’ expression patterns highlighted the heterogeneous nature of GICs cultures. Consequently, we demonstated that the neurosphere culture method is a proper approach to isolate GICs within the GBM tumor mass, preserving GICs heterogenic nature.

Radiosensitivity of four established culture pairs was investigated by means of clonogenic assay and all established unsorted GICs-enriched cultures ended up being more radioresistant than their differentiated counterparts. Importantly, radiation response of irradiated GICs, but not of DGC, correlates with patient’s outcome, thus supporting the GICs leading role in defining patient treatment response. In conclusion, we propose a quick and affordable method to faithfully determine cancer cells’ treatment response and potentially predict patient outcome based on empirical data.

Following clinically relevant fractionated radiotherapy we detected, by means of transcriptomic analysis, marked activation of inflammatory-related pathways, ECM remodeling, cell migration and intercellular crosstalk in GICs. Strikingly, several genes pointed to epithelial/mesenchymal transition processes via IL6/JAK/STAT3 and TNF-α/NFkβ pathways. A small signature of radiation-induced Mes-associated genes was defined in GICs: ICAM1, COX2, CTGF, IL6, LIF and NNMT.

In addition, the possible involvement of ITGA6 in GICs response to ionizing radiation was investigated. The knock-down of ITGA6 in GICs-enriched culture enhanced their radiosensitivity, potentially improving tumor radiocurability, and reported decreased capacity to retain stemness after radiotherapy.

El Glioblastoma (GBM) es el tumor cerebral primario maligno más frecuente en adultos. El tratamiento actual, consiste en cirugía seguida de radioterapia (RT) más quimioterapia, no evita las recidivas a largo plazo.

Para investigar los mecanismos moleculares que subyacen a la resistencia de GBM a la RT, se ha desarrollado un modelo in-vitro basado en dos pilares fundamentales: (i) la dualidad entre las Glioblastoma Initiating Cells (GICs) y el resto de células neoplásicas (células diferenciadas, DGC); y (ii) la heterogeneidad intratumoral. Los cultivos de GICs y las muestras de tumor homólogas se clasificaron como de tipo mesenquimal. Se compararon los cultivos DGC y GICs por sus características funcionales y metabólicas, la expresión de marcadores de células madres tumorales y la respuesta a la RT. Los cultivos GICs demostraron estar enriquecidos en CSCs, y el patrón de expresión de marcadores de CSCs evidenció su heterogeneidad, a diferencia de lo observado en DGC. Además, todos los cultivos enriquecidos en GICs fueron, a largo plazo, más resistentes a la RT en comparación con sus homólogos diferenciados. Es importante destacar que la radioresistencia de las GICs, pero no de las DGC, se correlaciona con el pronóstico de los pacientes, apoyando así el papel de las GICs en la respuesta al tratamiento. En conclusión, se propone un método rápido y económico para determinar fielmente la respuesta al tratamiento con RT de las células tumorales y potencialmente predecir la evolución del paciente basado en datos empíricos.

Para entender mejor el fenómeno de la resistencia a la RT de las GICs se realizó un análisis transcriptómico de DGC y GICs postirradiación. Exclusivamente en las GICs se detectó una activación significativa de las vías relacionadas con la inflamación, remodelación de la matriz extracelular, migración celular, interacción célula-célula y transición epitelio- mesénquima mediado por STAT3 y NF-κβ. Se identificó un grupo de genes asociados al perfil mesenquimal e inducidos por la radiación en GICs: ICAM1, COX2, CTGF, IL-6, LIF y NNMT.

Finalmente, se investigó la posible implicación de ITGA6, previamente descrito como marcador de CSCs en GBM, en la respuesta de GICs a la RT. La inhibición de ITGA6 en los cultivos enriquecidos en GICs aumentó la sensibilidad a la RT, mejorando potencialmente la respuesta al tratamiento.