RESUMEN
Cellular heterogeneity, for example, the intratumoral coexistence of cancer cells with and without stem cell characteristics, represents a potential root of therapeutic resistance and a significant challenge for modern drug development in glioblastoma (GBM). We propose here that activation of the innate immune system by stimulation of innate immune receptors involved in antiviral and antitumor responses can similarly target different malignant populations of glioma cells. We used short-term expanded patient-specific primary human GBM cells to study the stimulation of the cytosolic nucleic acid receptors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I). Specifically, we analyzed cells from the tumor core versus "residual GBM cells" derived from the tumor resection margin as well as stem cell-enriched primary cultures versus specimens without stem cell properties. A portfolio of human, nontumor neural cells was used as a control for these studies. The expression of RIG-I and MDA5 could be induced in all of these cells. Receptor stimulation with their respective ligands, p(I:C) and 3pRNA, led to in vitro evidence for an effective activation of the innate immune system. Most intriguingly, all investigated cancer cell populations additionally responded with a pronounced induction of apoptotic signaling cascades revealing a second, direct mechanism of antitumor activity. By contrast, p(I:C) and 3pRNA induced only little toxicity in human nonmalignant neural cells. Granted that the challenge of effective central nervous system (CNS) delivery can be overcome, targeting of RIG-I and MDA5 could thus become a quintessential strategy to encounter heterogeneous cancers in the sophisticated environments of the brain.
Asunto(s)
Antineoplásicos/farmacología , Citosol/inmunología , ARN Helicasas DEAD-box/inmunología , Glioblastoma/tratamiento farmacológico , Glioblastoma/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/inmunología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Citosol/efectos de los fármacos , Citosol/metabolismo , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Helicasa Inducida por Interferón IFIH1 , Ligandos , Receptores Inmunológicos , Transducción de Señal/efectos de los fármacos , Células Madre/efectos de los fármacos , Células Madre/inmunología , Células Madre/metabolismoRESUMEN
Microglia, the resident immune cells of the brain, are difficult to obtain in high numbers and purity using currently available methods; to date, microglia for experimental research are mainly isolated from the brain or from mixed glial cultures. In this paper, we describe a basic protocol for the in vitro differentiation of mouse embryonic stem (ES) cells into microglial precursor cells. Microglia are obtained by a protocol consisting of five stages: (i) cultivation of ES cells, (ii) formation and differentiation of embryoid bodies, (iii) differentiation into neuroectodermal lineage and isolation of myeloid precursor cells, (iv) differentiation into microglial precursor cells and (v) cultivation of ES cell-derived microglial precursors (ESdMs). The protocol can be completed in 60 d and results in stably proliferating ESdM lines, which show inducible transcription of inflammatory genes and cell marker expression comparable with primary microglia. Furthermore, ESdMs are capable of chemokine-directed migration and phagocytosis, which are major functional features of microglia.