Primary glioblastoma multiforme tumors and recurrence : Comparative analysis of the danger signals HMGB1, HSP70, and calreticulin.

PURPOSE: Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor. Despite improved multimodal therapies, the tumor recurs in most cases. Diverging patient survival suggests great tumor heterogeneity and different therapy responses. Danger signals such as high-mobility group box protein 1 (HMGB1), heat shock protein 70 (HSP70), and calreticulin (CRT) are biomarker candidates, due to their association with tumor progression versus induction of antitumor immune responses. Overexpression of these danger signals has been reported for various types of tumors; however, their role in GBM is still elusive. A direct comparison of their expression in the primary tumor versus the corresponding relapse is still lacking for most tumor entities. PATIENTS AND METHODS: We therefore performed an expression analysis by immunohistochemistry of the danger signals HMGB1, HSP70, and CRT in primary tumors and the corresponding relapses of 9 patients with de novo GBM. RESULTS: HMGB1 was highly expressed in primary tumors with a significant reduction in the respective relapse. The extracellular HSP70 expression was significantly increased in the relapse compared to the primary tumor. CRT was generally highly expressed in the primary tumor, with a slight increase in the relapse. CONCLUSION: The combination of a decreased expression of HMGB1, an increased expression of extracellular HSP70, and an increased expression of CRT in the relapse seems to be beneficial for patient survival. HMGB1, extracellular HSP70, and CRT could be taken into concerted consideration as potential biomarkers for the prognosis of patients with GBM.

Fractionated radiotherapy is the main stimulus for the induction of cell death and of Hsp70 release of p53 mutated glioblastoma cell lines.

BACKGROUND: Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Despite a multimodal therapy consisting of resection followed by fractionated radiotherapy (RT) combined with the chemotherapeutic agent (CT) temozolomide (TMZ), its recurrence is almost inevitable. Since the immune system is capable of eliminating small tumor masses, a therapy should also aim to stimulate anti-tumor immune responses by induction of immunogenic cell death forms. The histone deacetylase inhibitor valproic acid (VPA) might foster this. METHODS: Reflecting therapy standards, we applied in our in vitro model fractionated RT with a single dose of 2Gy and clinically relevant concentrations of CT. Not only the impact of RT and/or CT with TMZ and/or VPA on the clonogenic potential and cell cycle of the glioblastoma cell lines T98G, U251MG, and U87MG was analyzed, but also the resulting cell death forms and release of danger signals such as heat-shock protein70 (Hsp70) and high-mobility group protein B1 (HMGB1). RESULTS: The clonogenic assays revealed that T98G and U251MG, having mutated tumor suppressor protein p53, are more resistant to RT and CT than U87MG with wild type (WT) p53. In all glioblastoma cells lines, fractionated RT induced a G2 cell cycle arrest, but only in the case of U87MG, TMZ and/or VPA alone resulted in this cell cycle block. Further, fractionated RT significantly increased the number of apoptotic and necrotic tumor cells in all three cell lines. However, only in U87MG, the treatment with TMZ and/or VPA alone, or in combination with fractionated RT, induced significantly more cell death compared to untreated or irradiated controls. While necrotic glioblastoma cells were present after VPA, TMZ especially led to significantly increased amounts of U87MG cells in the radiosensitive G2 cell cycle phase. While CT did not impact on the release of Hsp70, fractionated RT resulted in significantly increased extracellular concentrations of Hsp70 in p53 mutated and WT glioblastoma cells. CONCLUSIONS: Our results indicate that fractionated RT is the main stimulus for induction of glioblastoma cell death forms with immunogenic potential. The generated tumor cell microenvironment might be beneficial to include immune therapies for GBM in the future.