b16 bearing mice  (R&D Systems)

Journal: Scientific Reports

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

doi: 10.1038/s41598-021-00734-4

Figure Lengend Snippet: Hsp70 is released from tumor cells in soluble and EV-bound forms. ( a ) B16 cells were incubated with rHsp70-Alexa647 (red) and were then stained with cmHsp70.1 antibody labeled with FITC (green). Nuclei were stained with DAPI (blue). Scale bar: 5 μm; confocal microscopy data. ( d ) Cartoon illustrating the separation of EV-bound and soluble exo- and cell-Hsp70. To discriminate between exogenously introduced Hsp70 and the cellular chaperone, rHsp70 was labeled with biotin and introduced into B16 cell culture for 6 h and then the cells were washed and allowed to export Hsp70 to the extracellular medium for 90 min. Medium was fractionated into EVs and soluble fractions. Both fractions were first incubated with NeutrAvidin-agarose to trap biotinylated Hsp70, and the unbound material was mixed with ATP-agarose to collect the remaining chaperone. The same procedure was employed with the medium of B16 cells without Hsp70 administration. ( b ) EVs were separated as described in Materials and Methods, and analyzed using an Exo-FACS ready-to-use kit for analysis of CD9 positive EVs with flow cytometry. ( c ) EVs were visualized via electron microscopy. For the detection of CD63 and Hsp70 in EVs, TEM microscopy was used with appropriate antibodies. Scale bars: 200 nm. ( e ) Western blotting of separated fractions, as shown in ( d ). NeutrAvidin-precipitated samples were probed with Avidin-peroxidase (upper panel), then ATP-precipitated—with anti-Hsp70 antibody (lower panel). A full-length blot images used to generate the panels are shown in Suppl Fig. . ( f ) Paths of exoHsp70 transport inside cells. Upon administration to cell culture, Hsp70 penetrates tumor cells using an endocytosis mechanism and (1) firstly occurs in early Rab4 and Rab11 endosomes, and (2) part of the Hsp70 appears in the extracellular milieu as a result of recirculation. Other Hsp70-containing endosomes mature into Rab5 and Rab7 endosomes (3) and at this stage some stimuli, such as acidification, can cause the release of Hsp70 into the cytoplasm. (4) Occurring in the cytoplasm together with cellular Hsp70, formerly exoHsp70 could be trapped by multivesicular bodies, due to invagination of the membrane of the late endosome (5). Both exo- and cell-Hsp70 in the cytoplasm could be anchored to the plasma membrane and then due to plasma membrane blebbing, could form Hsp70 containing vesicles that cannot be considered as exosomes (6). We cannot exclude the capacity of Hsp70 (exo- or cell-) to be released from cells forming channels (7).

Article Snippet: To reveal the possible infiltration of B16 tumors with pro-tumor M2 macrophages we prepared serial 7 μm frozen section from tumors isolated from B16-bearing mice and probed the slices with an antibody to arginase-1 (AF5868, R&DSystems, USA) followed by anti-sheep antibody (ab6747, Abcam, UK) and then anti-rabbit antibody conjugated with Alexa488 (Invitrogen, USA).

Techniques: Incubation, Staining, Labeling, Confocal Microscopy, Cell Culture, Flow Cytometry, Electron Microscopy, Microscopy, Western Blot, Avidin-Biotin Assay

Journal: Scientific Reports

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

doi: 10.1038/s41598-021-00734-4

Figure Lengend Snippet: Both soluble Hsp70 and EVs Hsp70 pull out intracellular (endo) Hsp70 to the cell surface and sensitize B16 cells to cytotoxic lymphocytes. ( a ) B16 cells were incubated with rHsp70 for 6 h and were subjected to the CTL test with lymphocytes from a healthy mouse. Cell death was recorded in real time with the aid of xCELLigence. ( b ) EVs and soluble fractions from B16 cells incubated with rHsp70 were collected and subjected to ( b ) Western blotting or ( c ) Hsp70-ELISA. Data from three independent experiments are presented. A full-length blot images used to generate the panel is shown in Suppl Fig. ( d ) B16 cells were incubated with rHsp70-Alexa647, EVs Hsp70 and soluble Hsp70, and subjected to flow cytometry. Representative data of three independent experiments is presented. ( e ) B16 cells were incubated with the soluble fraction or with EVs-Hsp70 for 3 h and lymphocytes of an untreated mouse were added. rHsp70 (50 μg/mL) was used as a positive control. The CTL assay was employed with the aid of xCELLigence. Representative data of three independent experiments is presented.

Article Snippet: To reveal the possible infiltration of B16 tumors with pro-tumor M2 macrophages we prepared serial 7 μm frozen section from tumors isolated from B16-bearing mice and probed the slices with an antibody to arginase-1 (AF5868, R&DSystems, USA) followed by anti-sheep antibody (ab6747, Abcam, UK) and then anti-rabbit antibody conjugated with Alexa488 (Invitrogen, USA).

Techniques: Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Positive Control, CTL Assay

Journal: Scientific Reports

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

doi: 10.1038/s41598-021-00734-4

Figure Lengend Snippet: Hsp70-containing EVs do not affect B16 cell growth in vitro, but inhibit growth and increase survival rate of B16-bearing mice . The Hsp70 content in B16 cells, untreated and incubated with rHsp70 measured using ( a ) western blotting and ( b ) with a Hsp70-ELISA assay. A full-length blot images used to generate the panels on b are shown in Suppl Fig. . ( c ) EVs-CNTR or EVs-Hsp70 were added to B16 cells and their proliferation was measured using xCELLigence. B16 melanoma cells were used to inoculate C57Bl/6 mice, together with EVs-CNTR or EVs-Hsp70, after 19 days tumors were isolated, photographed ( d ) and weighed ( e ). *** p = 0.0006; *p = 0.0404, Dunnett’s multiple comparison test. ( f ) Cumulative proportion Kaplan–Meier curve. Survival was analyzed in the ’Untreated’ group (n = 10), ‘EVs-CNTR’ group (n = 10) and ‘EVs-Hsp70’ group (n = 10).

Article Snippet: To reveal the possible infiltration of B16 tumors with pro-tumor M2 macrophages we prepared serial 7 μm frozen section from tumors isolated from B16-bearing mice and probed the slices with an antibody to arginase-1 (AF5868, R&DSystems, USA) followed by anti-sheep antibody (ab6747, Abcam, UK) and then anti-rabbit antibody conjugated with Alexa488 (Invitrogen, USA).

Techniques: In Vitro, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Isolation

Journal: Scientific Reports

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

doi: 10.1038/s41598-021-00734-4

Figure Lengend Snippet: Hsp70-containing EVs cause the generation of a specific immune response. ( a ) Lymphocytes from B16 tumor-bearing mice were isolated from spleens, magnetically separated and the CD8 + cells were counted (** p < 0.0001, Dunnett’s multiple comparison test). ( b , c ) Lymphocytes from B16 tumor-bearing mice from three groups ( b ) and from CT-26-bearing mice ( c ) were used as effector cells in the CTL assay performed with the aid of xCELLigence (total fraction, upper panel) and from the CD8 + lymphocyte fraction (lower panel). ( d , e ) The concentration of three cytokines (IL-10, TNF-α and IFN-γ) were measured in sera of B16-bearing mice ( d ) and CT-26-bearing mice ( e ) on day 19 after tumor cell inoculation. ( *p = 0.0003; **p < 0.0001, Dunnett’s multiple comparison test).

Article Snippet: To reveal the possible infiltration of B16 tumors with pro-tumor M2 macrophages we prepared serial 7 μm frozen section from tumors isolated from B16-bearing mice and probed the slices with an antibody to arginase-1 (AF5868, R&DSystems, USA) followed by anti-sheep antibody (ab6747, Abcam, UK) and then anti-rabbit antibody conjugated with Alexa488 (Invitrogen, USA).

Techniques: Isolation, CTL Assay, Concentration Assay

Journal: Scientific Reports

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

doi: 10.1038/s41598-021-00734-4

Figure Lengend Snippet: Hsp70-containing EVs inhibit the pro-tumor maturation of macrophages. ( a ) Representative histological sections obtained from B16 cell tumors on day 19 after inoculation of cells, with or without EVs, then stained with anti-arginase-1 antibody; confocal microscopy. Scale bar 100 μm. ( b ) Number of Arginase-1 positive cells as calculated per area (100 × 100 μm), 100 sections were analyzed for each treatment (* p = 0.013, **p < 0.0001, Dunnett’s multiple comparisons test). ( c ) Western blotting of B16 tumor tissue probed with anti-arginase-1 antibody. A full-length blot images used to generate the panels are shown in Suppl Fig. .

Article Snippet: To reveal the possible infiltration of B16 tumors with pro-tumor M2 macrophages we prepared serial 7 μm frozen section from tumors isolated from B16-bearing mice and probed the slices with an antibody to arginase-1 (AF5868, R&DSystems, USA) followed by anti-sheep antibody (ab6747, Abcam, UK) and then anti-rabbit antibody conjugated with Alexa488 (Invitrogen, USA).

Techniques: Staining, Confocal Microscopy, Western Blot