Immunogenicity of 56 degrees C and UVC-treated prostate cancer is associated with release of HSP70 and HMGB1 from necrotic cells.

BACKGROUND: Prostate hyperthermia and photodynamic therapy can be delivered by a variety of procedures which result in a wide range of temperatures and light energy and cause different kinds of cell death. METHODS: We have addressed the immunogenic effect of heating and UVC irradiation on the prostate cancer (PCa) cell line LNCaP, by studying the release of Danger Associated Molecule Pattern (DAMP) molecules HSP70 and HMGB1 and the dendritic cell (DC) antigen-presenting efficiency. RESULTS: Intracellular upmodulation and extracellular release of HSP70 were inversely correlated. Mild temperatures (43-47 degrees C) induced an early increase of intracellular HSP70, whereas the highest temperature (56 degrees C) induced its extrusion from the cell. Likewise, UVC caused an immediate migration of HSP70 into the cell medium in the absence of any intracellular modulation. 56 degrees C and UVC also induced a robust release of HMGB1. The release of DAMP molecules was closely associated with post-apoptotic membrane damage, as shown by double Annexin V/propidium iodide staining, whereas beta-tubulin, a structural component of cell membranes, was specifically induced by 56 degrees C heating. Tumor uptake strongly impaired the cytokine-driven maturation of DCs and 56 degrees C heating led to a significant recovery of CD83 and CCR7 DC maturation markers, but did not influence the antigen cross-presentation activity. On the contrary, UVC-treated LNCaP had negligible effects on DC maturation, but increased the cross-priming of tumor specific CTL. CONCLUSIONS: These data may be of use in the design of effective non-surgical PCa ablations that combine tumor destruction with long lasting immunity.

Post-apoptotic tumors are more palatable to dendritic cells and enhance their antigen cross-presentation activity.

Critical issues for cytotoxic lymphocyte (CTL) cross-priming are (a) the maturation state of dendritic cells (DC), (b) the source of the tumor-associated antigens (TAA) and (c) the context in which they are delivered to DCs. Drug-induced apoptosis has recently been implicated in CTL cross-priming. However, since drug-treatment produces in vivo more tumor cells than the DC default apoptotic clearance program can cope with, they are expected to proceed to secondary necrosis and change their molecular pattern. Here we have addressed this issue on renal carcinoma cells (RCC) by using different apoptotic stimuli. UVC, but not gamma-irradiation or anthracyclins, induced after 4h treatment of the RCC cell line K1 a combination of apoptotic (phosphatydilserine and calreticulin plasma membrane mobilization) and necrotic (membrane incompetence) features. Heat shock protein (Hsp)-70 and chromatin-bound high mobility box 1 HMGB1 protein, typical of necrosis, were released during the further 20h and thus made accessible to co-cultured monocyte-derived immature (i) DC. UVC-treated, secondary necrotic RCC cell lines were cross-presented with higher efficiency by cytokine-matured (m) DC than their early apoptotic (i.e. gamma-irradiated) counterpart. Upstream events such as increased tumor uptake, activation of genes involved in the antigen-processing machinery, and increased expression of costimulatory and maturation molecules were also observed after loading iDC with secondary necrotic, but not apoptotic, tumor cells. These data offer a description of the molecular and immunogenic characteristics of post-apoptotic tumors which can be exploited to increase the efficiency of in vivo and ex vivo TAA delivery to the DC cross-presentation pathway.