RESUMO
Tumor-associated macrophages (TAMs) have recently emerged as potentially crucial therapeutic targets for cancer. Thus, the development of macrophage-mediated phagocytosis assays is vital for preclinical drug screening of different tumor cells. This assay can be used to evaluate the effect of anti-cancer therapy, such as immunotherapy, radiotherapy, and chemotherapy, on different tumor cells. Here, we describe the in-vitro phagocytosis assay in detail. As an example of immunotherapy treatment, we used a monoclonal antibody to block an anti-phagocytic signal (CD47) to evaluate the assay using human brain tumor cells and monocyte-derived macrophages. We also demonstrated that this assay can be used to evaluate the effect of different irradiation doses on the phagocytosis of brain tumor cells. This functional assay is fast, accurate, and highly reproducible. Furthermore, the results successfully demonstrate that anti-CD47 antibodies and irradiation can enhance the macrophage-mediated phagocytosis of brain tumors.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/terapia , Antígeno CD47/imunologia , Glioblastoma/terapia , Temozolomida/uso terapêutico , Animais , Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/radioterapia , Linhagem Celular Tumoral , Terapia Combinada , Glioblastoma/tratamento farmacológico , Glioblastoma/imunologia , Glioblastoma/radioterapia , Humanos , Imunoterapia , Macrófagos/efeitos dos fármacos , Macrófagos/efeitos da radiação , Camundongos , Fagocitose/efeitos dos fármacos , Fagocitose/efeitos da radiação , Temozolomida/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recent studies hint that endogenous dsRNA plays an unexpected role in cellular signaling. However, a complete understanding of endogenous dsRNA signaling is hindered by an incomplete annotation of dsRNA-producing genes. To identify dsRNAs expressed in Caenorhabditis elegans, we developed a bioinformatics pipeline that identifies dsRNA by detecting clustered RNA editing sites, which are strictly limited to long dsRNA substrates of Adenosine Deaminases that act on RNA (ADAR). We compared two alignment algorithms for mapping both unique and repetitive reads and detected as many as 664 editing-enriched regions (EERs) indicative of dsRNA loci. EERs are visually enriched on the distal arms of autosomes and are predicted to possess strong internal secondary structures as well as sequence complementarity with other EERs, indicative of both intramolecular and intermolecular duplexes. Most EERs were associated with protein-coding genes, with â¼1.7% of all C. elegans mRNAs containing an EER, located primarily in very long introns and in annotated, as well as unannotated, 3' UTRs. In addition to numerous EERs associated with coding genes, we identified a population of prospective noncoding EERs that were distant from protein-coding genes and that had little or no coding potential. Finally, subsets of EERs are differentially expressed during development as well as during starvation and infection with bacterial or fungal pathogens. By combining RNA-seq with freely available bioinformatics tools, our workflow provides an easily accessible approach for the identification of dsRNAs, and more importantly, a catalog of the C. elegans dsRNAome.