Heat Shock Protein (HSP) 72 Enters Early Endosomes Preparatory to CellRelease

Purpose: To establish the mechanism by which hyperthermia induces the release of Hsp72 in tumor specific peptides for delivery to antigen presenting cells (APC). Materials and methods: 4T1 scrb-shRNAGFP and Hsp72-shRNAGFP cells were injected into the breast pad of a female BALB/c mouse for temperature kinetics of mice exposed to HT using circulating water bath set and nanoshellmediated hyperthermia. Baseline and heat-induced intracellular Hsp72 expression was measured by immunoblotting with anti-Hsp72. Four hours post hyperthermia (HT) treatment Hsp72 colocalization was determined using inhibitors and exosomes were recovered from the supernatant and Hsp72 levels were measured. Tumor size was measured after hyperthermia. Results: In this study, we show that the released Hsp72 is found in two forms: 1) within highly immunologicallyactive exosomes, known to be enriched in major histocompatibility complex (MHC) class I and II complexes, costimulatory molecules (CD40, CD80, CD86) and specifically members of the HSP70 family (including Hsp72, Hsp73, Hsp75, Grp78) and HSP90 family (including Hsp82, Hsp90, Grp96, Grp98) and, 2) as Hsp72-PC, in which free Hsp72 is chaperoning tumor specific peptides. Further, we show that exposure of 4T1-bearing tumors to hyperthermia (41°C, 60 min) induces significant tumor regression (p<0.05) as compared to 4T1-bearing mice maintained at ambient temperature (25°C, 60 min) tumor. However, repeated injection of 4T1 tumor-bearing mice with anti-Hsp72 antibody, abrogated hyperthermia-induced tumor regression. Blood samples taken at various times confirmed that the blocking antibody significantly reduced plasma Hsp72 levels. Conclusions: Our results suggested that hyperthermia stimulated tumor regression, in part, by stimulating the release of Hsp72 from tumors which is taken up by APC preparatory to activating CD8+ CTL cytotoxic responses against tumors. Since HSP are found in all cellular organisms, it is likely be applicable to the prevention/treatment of diseases of agriculturally relevant animals. It is expected to contribute to the broader understanding of intracellular trafficking pathways as an approach to therapy.