Abstract :
[en] Glioblastoma is an aggressive high-grade astrocytoma. Current standard treatments consist of maximal surgical resection followed by chemo-/radio- therapy. Unfortunately, this treatment protocol is impaired by incomplete resections and systematic relapses often leading to poor prognoses. The development of viro-/immuno- therapies gave uneven results mainly due to the pro-tumoral and immunologically cold glioblastoma microenvironment. This microenvironment is regulated by glioblastoma stem-like cells, M2-like macrophages/microglia, and Tregs, all these populations being themselves partly driven by CXCL12/CXCR4 pathway. This pathway indeed promotes tumor cells self-renewal, migration, angiogenesis, M2-like polarization, and Tregs recruitment. Moreover, high expression of CXCR4 positively correlates with tumor progression, recurrence, and low patient survival.
In this context, we have developed an attenuated oncolytic HSV-1 (oHSV) expressing a CXCL12/CXCR4 pathway inhibitor (oHSV/P2G) aiming at continuous production of the inhibitor at the tumor site. oHSV-P2G is expected to cause tumor cell lysis, disrupt the pro-tumoral microenvironment, trigger inflammation and adaptative immune response against virus and tumor antigens. This project aims at the characterization of the infiltration, the polarization and the activation of specific immune sub-populations, in the perspective of developing efficient treatments.
In vitro RT-qPCR and RNA sequencing were performed on HMC3 microglia cell line and on monocyte-derived macrophages (MDMs). In vivo assays were performed on an orthotopic syngeneic immunocompetent C57Bl6 mice model. Mice were engrafted by stereotaxic injection in the right striatum of the brain, with 100k GL261N4 primary murine glioblastoma cells expressing human Nectin (HSV-1 entry receptor). oHSV/P2G treatment (106 pfu in 2 µL) was injected in the same way 7 and 14 days post-engraftment (d .p.e.). Brains were collected and immune cells were isolated 21 d.p.e. for flow cytometry and 10x Genomics Single Cell protocol on fixed cells.
In vitro results performed on HMC3 and MDMs from patients, suggest that microglia and macrophages can be polarised, at the transcription level, towards a pro-inflamatory phenotype using conditionned media from infected glioblastoma cells. Besides, we showed in vivo that oHSV/P2G is able to increase CD45+ leucocytes invasion towards and inside the tumor mass, cell populations consisting mainly in macrophages, B and T cells. Moreover, microglia and macrophages were polarised towards a M1-like pro-inflamatory phenotype upon oHSV/P2G infection, B and T cells were activated and a decrease in regulatory T cells (Tregs) proportion was observed. Single-cell analysis was also performed on isolated immune cells after an in vivo assay to characterize precisely these populations and, potentially, sub-populations. The data from this experiment are currently being analysed and will be presented at the conference.
In conclusion, oHSV/P2G is able to increase immune cells invasion at the tumor site and to polarise sub-population of cells towards a pro-inflamatory phenotype. oHSV/P2G could therefore be an add-on to already existing therapies.
Name of the research project :
In-depth characterization of the glioblastoma microenvironment, and in particular of the innate immunity, in response to a CXCR4 antagonist delivered by virotherapy.
