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. 2005 Jun 15;65(12):5238-47.
doi: 10.1158/0008-5472.CAN-04-3804.

Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells

Robert Gastpar  1 , Mathias Gehrmann, Maria A Bausero, Alexzander Asea, Catharina Gross, Josef A Schroeder, Gabriele Multhoff
Affiliations

Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells

Robert Gastpar et al. Cancer Res. .

Abstract

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo-/Colo+) and colon (CX-/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo-/CX- derived exosomes were Hsp70 membrane positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes.

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Figures

Figure 1
Figure 1
A, expression of Hsp70 and Bag-4 on the plasma membrane of human pancreas (Colo−/Colo+) and colon (CX−/CX+) carcinoma sublines. Nonpermeabilized, 7-AAD negatively gated, viable carcinoma sublines Colo−/Colo+ and CX−/CX+ with intact plasma membrane were stained either with Hsp70− (cmHsp70.1) or Bag-4 (IMG-152) specific antibodies and analyzed by flow cytometry. The histograms represent positively stained cells in white, corrected for nonspecific staining, using an isotype-matched control antibody marked in gray. Mean values ± SE of three independent were summarized in Table 1A. B, Hsp70 and Bag-4 were actively released in detergent-soluble vesicles by viable carcinoma sublines. The amount of Hsp70 protein released by tumor sublines Colo−/Colo+ and CX−/CX+ was quantified by ELISA. Cell-free supernatants of Colo×/Colo+ (top) and CX−/CX+ (bottom) cells, diluted either in PBS buffer (control, ●), or in buffer containing the detergent Triton X-100 (1% v/v, o), Lubrol WX (1% v/v, △), and Brij 98 (0.5% v/v, ▲) were analyzed. One representative kinetic of three independent experiments was illustrated.
Figure 2
Figure 2
A, biophysical characterization of vesicles released by Colo−/Colo+ and CX−/CX+ carcinoma sublines. Sucrose density gradient centrifugation of Colo+ (left) and CX+ (right) derived vesicles was done on a sucrose density gradient by ultracentrifugation in a density range of 1.08 to 1.24 g/mL. Fractions were collected from top to bottom and analyzed after separation on a 10% SDS-PAGE by silver stain (top). As a marker for exosomes, acetylcholinesterase activity was measured in each individual fraction by standard colorimetric assay. Maximum protein amount and acetylcholinesterase activity were detected at a density of 1.17 g/mL. One representative experiment of three was shown. B, biochemical characterization of exosomes released by Colo−/Colo+ and CX−/CX+ carcinoma sublines. Comparative analysis of the protein composition in whole cell lysates (lys) and exosomal (exos) fractions of tumor sublines Colo−/Colo+ and CX−/CX+, as determined by silver staining (top) and Western blot analysis (bottom), using specific antibodies directed against tubulin, Bag-4, Hsp70, Hsc70, Rab-4, Rab-7, Rab-9, Rab-11, Grp94, and Calnexin. One representative silver stain and Western blot out of five was shown. C, surface of exosomes derived from Colo+ and CX+ carcinoma sublines are strongly positive for Hsp70/Bag-4. Exosomes derived from Colo−/Colo+ (left) and CX−/CX+ (right) carcinoma sublines were covalently bound to aldehyde/sulfate latex beads with a diameter of 4 μm. Exosome-coated beads were stained with antibodies directed against Hsp70 (cmHsp70.1, top, white histograms), Bag-4 (IMG-152, bottom, white histograms), and isotype-matched control antibodies (gray histograms) and analyzed by flow cytometry. Only the population containing single beads was gated and analyzed. Mean values of three independent experiments ± SE were summarized in Table 1B. D, immunoelectron microscopic view of the Hsp70 localization on the exosomal surface of CX− (top) and CX+ (bottom) cells at a magnification of ∼ ×60.000; scale bar, 50 nm. Localization of Hsp70 was visualized by 10 nm gold particles. No staining was found on exosomes incubated with an isotype-matched negative control antibody (data not shown).
Figure 3
Figure 3
CD94+ NK cells specifically migrate toward Hsp70/Bag-4–positive exosomes derived from Colo+ and CX+ carcinoma sublines. A, VivaSpin treated (2-fold concentrated) supernatants (SN*) collected after a 24-hour cultivation period in serum-free medium, derived from Colo−/Colo+ (left) and CX−/CX+ (right) carcinoma sublines were used as positive controls for the migratory capacity of CD94+ NK cells. Exosome-depleted and exosome-enriched fractions derived from the same supernatants by ultracentrifugation at 150,000 × g were also used as attractants for NK cells. The transwell system consisted of two compartments separated by a polycarbonate membrane with a pore size of 5 μm. The different attractants, unseparated supernatant (SN*), exosome-depleted, and exosome-enriched fractions were placed in the lower compartment in a total volume of 600 μL; 0.5 × 106 51Cr-labeled CD94+ NK cells were placed in the upper compartment. After a 4-hour coincubation period at 37°C, the percentage of specifically migrating cells was determined in a γ-counter. B, migratory capacity of CD94+ NK cells was also tested toward Hsp70 peptide TKD (right) and exosomes derived from Colo+ carcinoma cells (left) either untreated or after preincubation with an MHC class I–specific (W6/32) or with an Hsp70-specific (cmHsp70.1) antibody. NK cells migrated specifically toward TKD and exosomes derived from Colo+ carcinoma cells. The MHC class I–specific antibody W6/32 did not affect migratory capacity; however, the Hsp70-specific antibody completely abrogated migratory capacity of NK cells toward TKD and Colo+ exosomes. Columns, mean of three independent experiments; bars, SE.
Figure 4
Figure 4
Hsp70/Bag-4–positive exosomes derived from Colo+ and CX+ carcinoma sublines stimulate cytolytic activity of CD94+ NK cells against Hsp70/Bag-4–positive tumor targets. CD94+ NK cells were incubated either with IL-2 alone (control), with IL-2 plus TKD (TKD), or with IL-2 plus exosomes (10 μg protein/mL) derived from Colo−/Colo+ or CX−/CX+ carcinoma sublines for 4 days. A, cytolytic activity was measured using Colo+/Colo− and CX+/CX− carcinoma sublines as target cells in a standard 51Cr release assay. A comparable, strong lysis was detected against Colo+ and CX+ carcinoma sublines if NK cells were stimulated either with IL-2 plus TKD (TKD) or with exosomes derived from Colo+ and CX+ carcinoma sublines. After stimulation with IL-2 alone or with exosomes derived from Colo− and CX− carcinoma sublines, lytic activity of Hsp70/Bag-4–positive tumor target cells was weaker. Mean values of three to four independent experiments ± SE, at one distinct E/T ratio of 10:1, were summarized in Table 2A. B, lytic activity of CD94+ NK cells against CX+/CX− tumor target cells was also measured after stimulation with exosomes derived from Colo−/Colo+ carcinoma sublines. Again, lysis of CX+ tumor targets was significantly enhanced by stimulation with exosomes derived from Colo+ but not of Colo− exosomes. These data revealed that Hsp70 reactivity in NK cells is inducible by Hsp70/Bag-4 surface-positive exosomes, independent of the tumor cell origin. Mean values of four independent experiments ±SE, at one distinct E/T ratio of 10:1, were summarized in Table 2B. C, Hsp70/Bag-4–positive exosomes derived from Colo+ and CX+ carcinoma sublines stimulate secretion of granzyme B by CD94+ NK cells. CD94+ NK cells (2 × 106 cells/mL) were incubated with different concentrations (0.1, 1, and 10 μg protein/mL) of Hsp70/Bag-4 surface-negative and surface-positive exosomes, generated from Colo− and Colo+ carcinoma sublines. As a control, NK cells were incubated with low-dose IL-2 (100 IU/mL) alone. After 4 days, the cell-free culture supernatants were harvested and the amount of released granzyme B was quantified by ELISA. Columns, mean values of three independent experiments; bars, SE. *Significantly different values from control levels (P < 0.05).
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