Successful Partnerships: Exploring the Potential of Immunogenic Signals Triggered by TMZ, CX-4945, and Combined Treatment in GL261 Glioblastoma Cells

Abstract

Background: The relevance of the cancer immune cycle in therapy response implies that successful treatment may trigger the exposure or the release of immunogenic signals. Previous results with the preclinical GL261 glioblastoma (GB) showed that combination treatment of temozolomide (TMZ) + CX-4945 (protein kinase CK2 inhibitor) outperformed single treatments, provided an immune-friendly schedule was followed. Our purpose was to study possible immunogenic signals released in vitro by GB cells. Methods: GL261 GB cells were treated with TMZ and CX-4945 at different concentrations (25 µM–4 mM) and time frames (12–72 h). Cell viability was measured with Trypan Blue and propidium iodide. Calreticulin exposure was assessed with immunofluorescence, and ATP release was measured with bioluminescence. Results: TMZ showed cytostatic rather than cytotoxic effects, while CX-4945 showed remarkable cytotoxic effects already at low concentrations. Calreticulin exposure after 24h was detected with TMZ treatment, as well as TMZ/CX-4945 low concentration combined treatment. ATP release was significantly higher with CX-4945, especially at high concentrations, as well as with TMZ/CX-4945. Conclusions: combined treatment may produce the simultaneous release of two potent immunogenic signals, which can explain the outperformance over single treatments in vivo. A word of caution may be raised since in vitro conditions are not able to mimic pharmacokinetics observed in vivo fully.

Immune System-Related Changes in Preclinical GL261 Glioblastoma under TMZ Treatment: Explaining MRSI-Based Nosological Imaging Findings with RT-PCR Analyses

“…The beneficial effect of TMZ in preclinical/clinical settings was mainly attributed to its effect as DNA alkylating agent and activation of the apoptotic cascade [ 49 , 50 ]. However, it is worth noting that TMZ alone has a cytostatic rather than cytotoxic mechanism, when added to GL261 cultured cells at concentrations similar to the ones used in preclinical studies, as described in [ 9 , 51 , 52 , 53 ]. Thus, the main beneficial effects observed with TMZ therapy may have a different explanation.…”

“…At the therapy starting point, GL261 GB tumours display an essentially protumoural microglia/macrophage phenotype, supported by qPCR results and encoded in the red colour over the tumour mass of the nosological images. TMZ therapy may lead to release/exposure of immunogenic signals [ 53 ], which set the cancer immunity cycle [ 59 ]. In the meanwhile, both M1 and M2 populations increase (see also Figure 2 A): M1 microglia/macrophages will participate in tumour cell killing [ 9 ], and M2 microglia/macrophages are waiting for M1 polarisation.…”

Far-Red Fluorescent Murine Glioma Model for Accurate Assessment of Brain Tumor Progression

“…GL261 cells are moderately immunogenic and suitable for orthotopic implantation in syngeneic and immunocompetent animals to study tumor immunology [ 39 , 41 ]. For instance, glioma GL261 cells were used to study the mechanisms of immunogenicity of cell death in response to various anti-cancer therapeutic agents, including to chemotherapeutics and photodynamic therapy [ 42 , 43 , 44 , 45 ].…”

Taxifolin Targets PI3K and mTOR and Inhibits Glioblastoma Multiforme

“…Autophagy can lead to type II programmed cell death and evidence suggests that autophagy may be important in the treatment of GBM [ 60 , 61 ]. Autophagy promoting drugs, such as temozolomide, can selectively kill apoptosis-resistant glioblastoma cells [ 62 , 63 ]. In this study, we found that treatment with taxifolin increased the expression of autophagy-related proteins including LC3B-II, Atg7, atg12, and Beclin-1 suggesting that the autophagic pathway was activated in our tumor model.…”