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

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.


Figure S1
Figure S1. Western blot demonstrating specificity of home-made anti-Hsp70 antibody to Hsp70. Human lung carcinoma A549 cells, mouse melanoma B16 and colon cancer carcinoma CT-26 cells were heat shocked (43 o C, 6 hrs of recovery) and then subjected to western blotting with polyclonal anti-Hsp70 (upper panel) antibody the same that were used for Hsp70-ELISA in the manuscript. B16 lysates were also used for western blotting with monoclonal anti-Hsp70 antibody (Clone 8D1) the same that was used for detection of Hsp70 in B16 EVs.

Figure S2.
Introduction: To check which transport mechanism is employed by the exogenous chaperone in B16 cells we introduced Hsp70 labeled with Alexa-488 in concentration 50 µg/ml and analysed cells using flow cytometry assay. Exo Hsp70 incorporated into 100% of B16 cells already 15 min after administration and its amount inside cells grew with time reaching maximum after 6 hrs of incubation (Fig. S2a).
To explore the presumable mechanism of intracellular transport of exoHsp70 we employed the variety of inhibitors of intracellular transport and found that clathrindependent endocytosis (dynasore and chlorpromazine) most probably was used to , suppress caveolin-dependent endocytosis (filipin) or to destroy lipid rafts as well as to suppress caveolin-dependent endocytosis using methyl β-cyclodextrin (MβCD).
Dynasore and chlorpromazine decreased exo-Hsp70 incorporation until 41.5% and 72.5% respectively whereas both filipin and MβCD did not prevent Hsp70 penetration into B16 cells (Fig.S2b). One could conclude that exo-Hsp70 uses clathrin-dependent endocytosis pathway. Figure S2. Exogenous Hsp70 penetrates tumor cells using predominantly endocytosis (a) Flow cytometry experiment data demonstrating that rHsp70 labelled with Alexa488 accumulates inside tumor cells in time; (b) B16 cells were incubated with rHsp70 labeled with Alexa488 in presence of various intracellular transport inhibitors and then the cells were analyzed using a flow cytometry. Data of four independent experiments summarized. (c) B16 cells were transiently transfected with rab4-, rab5-, rab7-and rab11-rfp plasmids (red) and then incubated with rHsp70 labelled with Alexa488 (green) for 6 hours. Lysosomes were detected with the aid of LysoTracker (red), nuclei stained with DAPI.
To confirm that we traced the way of exo-Hsp70 in vesicular structures of B16 cells and added chaperone labeled with Alexa-488 to cells previously transfected with plasmids bearing Rab4, Rab11, Rab5 or Rab7 for 6 hrs. Non-transfected cells were also stained with LysoTracker®. Cells were studied with the aid of confocal microscopy. The highest amount of exo-Hsp70 was detected in early Rab4 and Rab11 endosomes which usually are recycled Fig.S2c). Remarkable amount of chaperone molecules was noticed in non-recycled early Rab5 and late Rab7 endosomes and only a little part of exo-Hsp70 was found to be co-localized with lysosomes.
To reveal endo-Hsp70 on a surface of B16 cells, cells were incubated with Hsp70 labeled with Alexa-555 for 6 hours, washed three times with ice-cold PBS, stained with the cmHsp70.1-FITC antibody (kindly provided by Professor G. Multhoff, Technical University of Munchen, Germany), and fixed with 4% paraformaldehyde. Nuclei were stained with DAPI.
Fluorescence images were captured using a Leica TCS SP2 confocal microscope (Leica, Germany). To avoid possible cross-interference of various fluorochromes, images for DAPI, Alexa-555, and FITC were acquired using the sequential image recording method. Table S1. Nanoparticle tracking analysis (NTA) of EVs isolated from conditioned culture medium. NTA of particle mode size and concentration. Each sample was measured in triplicate. Standard deviation (SD) is shown in parentheses.

Figure S10
Figure S10. CTL assay of CT-26 cells in presence of EVs-CNTR and EVs-Hsp70. As effector cells splenocytes isolated from spleen of naïve C3HA mice. The CTL assay was performed with the aid of xCELLigence technique, which allows to trace the cell populations' behavior in real time. Importantly, lymphocytes did not cause any changes in Cell Index, so a signal change related only to adherent tumor cells.