Hsp70-containing extracellular vesicles are capable of activating of adaptive immunity in models of mouse melanoma and colon carcinoma

Abstract

The release of Hsp70 chaperone from tumor cells is found to trigger the full-scale anti-cancer immune response. Such release and the proper immune reaction can be induced by the delivery of recombinant Hsp70 to a tumor and we sought to explore how the endogenous Hsp70 can be transported to extracellular space leading to the burst of anti-cancer activity. Hsp70 transport mechanisms were studied by analyzing its intracellular tracks with Rab proteins as well as by using specific inhibitors of membrane domains. To study Hsp70 forms released from cells we employed the assay consisting of two affinity chromatography methods. Hsp70 content in culture medium and extracellular vesicles (EVs) was measured with the aid of ELISA. The properties and composition of EVs were assessed using nanoparticle tracking analysis and immunoblotting. The activity of immune cells was studied using an assay of cytotoxic lymphocytes, and for in vivo studies we employed methods of affinity separation of lymphocyte fractions. Analyzing B16 melanoma cells treated with recombinant Hsp70 we found that the chaperone triggered extracellular transport of its endogenous analog in soluble and enclosed in EVs forms; both species efficiently penetrated adjacent cells and this secondary transport was corroborated with the strong increase of Natural Killer (NK) cell toxicity towards melanoma. When B16 and CT-26 colon cancer cells before their injection in animals were treated with Hsp70-enriched EVs, a powerful anti-cancer effect was observed as shown by a two-fold reduction in tumor growth rate and elevation of life span. We found that the immunomodulatory effect was due to the enhancement of the CD8-positive response and anti-tumor cytokine accumulation; supporting this there was no delay in CT-26 tumor growth when Hsp70-enriched EVs were grafted in nude mice. Importantly, pre-treatment of B16 cells with Hsp70-bearing EVs resulted in a decline of arginase-1-positive macrophages, showing no generation of tumor-associated macrophages. In conclusion, Hsp70-containing EVs generated by specifically treated cancer cells give a full-scale and effective pattern of anti-tumor immune responses.

Combined Cytotoxic Effect of Inhibitors of Proteostasis on Human Colon Cancer Cells

“…There is much data in the literature indicating a correlation between the intracellular and extracellular levels of Hsp70 and the degree of tumor aggressiveness [ 13 ], therefore, we evaluated the amount of (i) intracellular Hsp70, (ii) Hsp70 expressed on the cell surface, and (iii) extracellular Hsp70. To assess intracellular and extracellular Hsp70, we used a recently published Hsp70-ELISA [ 17 ]. Using this method, we showed that all HCC cells have high Hsp 70 levels but the most malignant HCC7 (IIIC stage) and HCC6 (IIIB stage) cell lines contain lower amounts of Hsp70 in their cell lysates (CL) and export larger amounts of the chaperone to the cell media.…”

“…After washing with Buffer A (20 mM Borate buffer [pH 8.0], 0.15 M NaCl, 0.05% Tween-20) the wells were loaded with 100 µL of the appropriate samples as indicated above and with the rHsp70 titers used for calibration; all probes were mixed with 100 µL of Buffer A. Plates were further incubated for 1 h at 37 °C on a shaker and, after washing in Buffer A, biotinylated anti-Hsp70 polyclonal antibody (0.01 µg/mL) was added and the plate incubated for 1 h at 37 °C on a shaker. After washing, StreptAvidin–Peroxidase was added for an additional 1 h. The staining reaction was performed using tetramethylbenzene in citrate buffer (pH 4.5) and the intensity of staining was measured with a Varioscan (Thermo Fisher, Waltham, MA, USA) [ 17 ]. All results were obtained in triplicate.…”

“…To visualize the amount of Hsp70 on the cell surface, HCC cells were plated on 24-well plates with pre-loaded coverslips. After they attached to the surface, the cells were washed in PBS and incubated with antibodies to surface HSP70 (Clone 8D1, [ 17 ]) on ice. After careful washing, cells were further incubated with secondary antibodies labeled with AlexaFluor555 fluorescent label (Goat anti-Mouse IgG, A28180, Invitrogen, Waltham, MA, USA).…”

Immunological aspects of heat shock protein functions and their significance in the development of cancer vaccines

“…The introduction of recombinant HSP70 to the tumor cel stimulates the transport of endogenous HSP70 to the extracellular space of the tumor, leading to a rapid activation of the immune response. The immunomodulatory effect of HSP70bearing exosomes was manifested by CD8(+) activation, the accumulation of antitumor cytokines and the activation of NK cells, which had a positive effect on the reduction in tumor growth rate and elevation of life span in mice [66].…”

Tumour Derived Extracellular Vesicles: Challenging Target to Blunt Tumour Immune Evasion

“…Meanwhile, the antitumour activity of TEV enriched in different HSP isoforms was also reported [ 93 , 94 , 95 , 96 , 97 ]. HSP70 + TEV secreted by human pancreatic and colon carcinoma cell lines induced NK [ 93 , 94 ] and Th1 reactivity [ 95 ], and converted Treg into Th17 cells [ 96 ].…”