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Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN
Contributor:Kamińska-Kaczmarek, Bożena (1961– ) : Supervisor
Publisher:Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN
Place of publishing: Date issued/created: Description:207 pages : illustrations ; 30 cm ; Bibliography ; Summary in English
Degree grantor:Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN
Type of object: Subject and Keywords:GAMs ; GBM ; Glioma ; Lactadherin ; Microenvironment ; Osteopontin
Abstract:Malignant gliomas are the most common CNS tumors and the most aggressive one is glioblastoma multiforme (GBM). The treatment of GBM includes tumor resection combined with radio- and chemotherapy, however due to diffuse growth surgical resection is difficult and the blood-brain barrier limits the penetration of many drugs. Thus, the effectiveness of therapy is very low and the average survival of GBM patients is 15 months. Large-scale analysis of gene expression based on data from TCGA database showed significant differences in transcriptional profiles in GBM. This led to identification of three molecular subtypes of GBM: proneural, mesenchymal and classical. Mes-GBM is characterized by the highest aggressiveness and the strongest infiltration of myeloid cells: microglia and monocytes/macrophages (so called GAMs), which in the tumor microenvironment (TME) undergo reprograming into tumor supportive cells. In Laboratory of Molecular Neurobiology IBD PAS, osteopontin (Spp1) and lactadherin (Mfg-E8) were identified as proteins potentially involved in this process. Both Spp1 and Mfg-E8 are integrin ligands and their expression is increased in many cancers. The aim of this study was to assess their role in the pathogenesis of malignant gliomas, in particular in modulation of TME. For this purpose, a rat C6 glioma model was used, in which intracranial tumors bear the histopathological similarities to human GBMs. To further determine a degree of similarity between the rat C6 gliomas and human GBMs, global changes in gene expression were analyzed and compared to signatures specific to human GBM subtypes, showing the greatest similarity to Mes-GBM. Analysis of genes expressed in microglia isolated from C6 gliomas confirmed the tumor-supportive properties of these cells. To verify the hypothesis about participation of Spp1 and Mfg-E8 in the pro-tumoral activation of GAMs and TME modulation, C6 glioma cells with stably silenced expression of Spp1 and Mfg-E8 were used. Significant inhibition of the growth of Spp1 and Mfg-E8 depleted gliomas in vivo was observed. In Mfg-E8 depleted tumors, also the migration of glioma cells into brain parenchyma was restricted. The analysis of the GAMs phenotype showed that both proteins are involved in the induction of the pro-tumoral, immunosuppressive activation. In Mfg-E8 depleted tumors, the percentage of CD3+ cells increased. In Spp1 depleted tumors, an increase in the percentage of cells expressing the pro-inflammatory interleukin 1β was observed, which may indicate the initiation of an efficient antitumor response. The analysis of Arg-1+ and Trem2+ cells in control tumors showed that the proteins are expressed in distinct populations of myeloid cells found in different tumor regions, confirming the heterogeneity of GAMs in glioblastoma. The RGD motif of Spp1 was identified as crucial for a tumor growth. A rescue experiment was performed, in which constructs encoding different variants of Spp1 were used. Reestablishing expression of the wild type Spp1 in glioma cells depleted of Spp1 (shSpp1), Spp1 with a mutation at the thrombin cleavage site or Spp1 with a deletion of the C-terminal region restored tumor growth. Expression of the Spp1 variant with a point mutation in the RGD motif did not restore tumor growth. However, the application of a short peptide containing the RGD motif, which could potentially act as a competitive inhibitor for Spp1 and Mfg-E8, did not affect tumor size and microglial activation in vivo compared to the control peptide (SCR). Altogether, this study demonstrated that Spp1 and Mfg-E8 are crucial for in vivo tumor growth and reprograming of the TME, especially for the activation of GAMs. Although Spp1 in cancer shows a pleiotropic effect and different regions of the protein are important in many aspects of tumor development (such as adhesion, migration or interaction with GSCs), the RGD motif and its interactions with target integrins play the key role in tumor growth in vivo.
Resource type: Detailed Resource Type: Source: Language: Language of abstract: Digitizing institution:Nencki Institute of Experimental Biology of the Polish Academy of Sciences
Original in:Library of the Nencki Institute of Experimental Biology PAS
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