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human metastatic triple negative breast cancer cell lines tnbc cl mda mb 231 (ATCC)

Name: human metastatic triple negative breast cancer cell lines tnbc cl mda mb 231
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ATCC human metastatic triple negative breast cancer cell lines tnbc cl mda mb 231
$Fig. 2 Characterization of sEV isolated from plasma of <t>TNBC-patients</t> or healthy donors (HDs). a, b Representative particle distribution images (NTA) of sEV derived from plasma of a HD and TNBC Pt #1. c sEV numbers/µg protein in fraction #4 for Pts and HDs. d Representative TEM images of sEV from plasma of a HD and TNBC-Pt #1. e Western blots of sEV (fraction #4) isolated from plasma of a HD and TNBC-Pt #1. f On bead ﬂow cytometry of sEV from plasma of TNBC Pts and HDs. Shown are RFI values for immunosuppressive proteins on the sEV surface (upper row) and for immunostimulatory proteins (lower row). Data are presented as means ± SD. Mann-Whitney tests were used to evaluate differences between TNBC Pts and HDs. ns = no signiﬁcant difference. g The RFI scores for immunosuppressive or immunostimulatory proteins and the ratios of stim/supp proteins calculated for all the above listed sEV as described in the text. Note the higher suppressive and stimulatory scores for TNBC Pt’s sEV than for HD’s sEV and the signiﬁcantly higher stim/supp ratio for sEV of HDs than for TNBC Pts. h Apoptosis (%) induced in activated primary human CD8 + T cells by sEV from plasma of HDs (n = 5) and TNBC-Pts (n = 5). Data were analyzed by an unpaired t-test and are presented as means ± SD. i Flow cytometry images of apoptosis induced in CD8 + T cells by increasing doses of sEV from a representative HD or TNBC Pt.$
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1) Product Images from "Small EV in plasma of triple negative breast cancer patients induce intrinsic apoptosis in activated T cells."

Article Title: Small EV in plasma of triple negative breast cancer patients induce intrinsic apoptosis in activated T cells.

Journal: Communications biology

doi: 10.1038/s42003-023-05169-3

$Fig. 2 Characterization of sEV isolated from plasma of TNBC-patients or healthy donors (HDs). a, b Representative particle distribution images (NTA) of sEV derived from plasma of a HD and TNBC Pt #1. c sEV numbers/µg protein in fraction #4 for Pts and HDs. d Representative TEM images of sEV from plasma of a HD and TNBC-Pt #1. e Western blots of sEV (fraction #4) isolated from plasma of a HD and TNBC-Pt #1. f On bead ﬂow cytometry of sEV from plasma of TNBC Pts and HDs. Shown are RFI values for immunosuppressive proteins on the sEV surface (upper row) and for immunostimulatory proteins (lower row). Data are presented as means ± SD. Mann-Whitney tests were used to evaluate differences between TNBC Pts and HDs. ns = no signiﬁcant difference. g The RFI scores for immunosuppressive or immunostimulatory proteins and the ratios of stim/supp proteins calculated for all the above listed sEV as described in the text. Note the higher suppressive and stimulatory scores for TNBC Pt’s sEV than for HD’s sEV and the signiﬁcantly higher stim/supp ratio for sEV of HDs than for TNBC Pts. h Apoptosis (%) induced in activated primary human CD8 + T cells by sEV from plasma of HDs (n = 5) and TNBC-Pts (n = 5). Data were analyzed by an unpaired t-test and are presented as means ± SD. i Flow cytometry images of apoptosis induced in CD8 + T cells by increasing doses of sEV from a representative HD or TNBC Pt.$
Figure Legend Snippet: Fig. 2 Characterization of sEV isolated from plasma of TNBC-patients or healthy donors (HDs). a, b Representative particle distribution images (NTA) of sEV derived from plasma of a HD and TNBC Pt #1. c sEV numbers/µg protein in fraction #4 for Pts and HDs. d Representative TEM images of sEV from plasma of a HD and TNBC-Pt #1. e Western blots of sEV (fraction #4) isolated from plasma of a HD and TNBC-Pt #1. f On bead ﬂow cytometry of sEV from plasma of TNBC Pts and HDs. Shown are RFI values for immunosuppressive proteins on the sEV surface (upper row) and for immunostimulatory proteins (lower row). Data are presented as means ± SD. Mann-Whitney tests were used to evaluate differences between TNBC Pts and HDs. ns = no signiﬁcant difference. g The RFI scores for immunosuppressive or immunostimulatory proteins and the ratios of stim/supp proteins calculated for all the above listed sEV as described in the text. Note the higher suppressive and stimulatory scores for TNBC Pt’s sEV than for HD’s sEV and the signiﬁcantly higher stim/supp ratio for sEV of HDs than for TNBC Pts. h Apoptosis (%) induced in activated primary human CD8 + T cells by sEV from plasma of HDs (n = 5) and TNBC-Pts (n = 5). Data were analyzed by an unpaired t-test and are presented as means ± SD. i Flow cytometry images of apoptosis induced in CD8 + T cells by increasing doses of sEV from a representative HD or TNBC Pt.

Techniques Used: Isolation, Clinical Proteomics, Derivative Assay, Western Blot, Cytometry, MANN-WHITNEY, Flow Cytometry

Fig. 3 Uptake of TEX labeled with PKH26 by immune cells. TEX isolated from supernatants of TNBC cell lines were labeled with the PKH26 dye and were co-incubated with recipient immune cells for various time periods. a Percentages of CD8+ Jurkat cells up-taking labeled TEX during coincubation as measured by ﬂow cytometry. Data are mean values from two independent assays and are shown in the excel table in Supplementary Data 1. b Representative histograms for the uptake of labeled TEX presented as variables of the uptake time. c Microscopic images of CD8+ Jurkat cells co- incubated with PKH26 labeled TEX1. TEX = Red, Actin = green, DAPI stained nuclei = blue; scale bar = 20 µm. d Comparison of TEX1 uptake by activated primary CD4 + T and CD8 + T cells at different time points. e Flow cytometry images of apoptosis induced in activated primary CD4 + T, CD8 + T, B and NK cells following co-incubation with TEX1.

Figure Legend Snippet: Fig. 3 Uptake of TEX labeled with PKH26 by immune cells. TEX isolated from supernatants of TNBC cell lines were labeled with the PKH26 dye and were co-incubated with recipient immune cells for various time periods. a Percentages of CD8+ Jurkat cells up-taking labeled TEX during coincubation as measured by ﬂow cytometry. Data are mean values from two independent assays and are shown in the excel table in Supplementary Data 1. b Representative histograms for the uptake of labeled TEX presented as variables of the uptake time. c Microscopic images of CD8+ Jurkat cells co- incubated with PKH26 labeled TEX1. TEX = Red, Actin = green, DAPI stained nuclei = blue; scale bar = 20 µm. d Comparison of TEX1 uptake by activated primary CD4 + T and CD8 + T cells at different time points. e Flow cytometry images of apoptosis induced in activated primary CD4 + T, CD8 + T, B and NK cells following co-incubation with TEX1.

Techniques Used: Labeling, Isolation, Incubation, Cytometry, Staining, Comparison, Flow Cytometry

Fig. 4 Immunoregulatory proteins expressed on the surface of recipient immune cells and on TEX or sEV of malignant and non-malignant origins. a The heatmap presenting mean RFI values for proteins expressed on the surface of activated primary CD4 + T, CD8 + T, B and NK cells. b The heatmap presenting mean RFI values for proteins found on the surface of TEX1, TEX2, HaCaT sEV, TNBC Pt-sEV and HD-sEV. c An image of a T cell interacting with various TEX (shown as blue vesicles) that carry immunoregulatory proteins on the surface membrane. TEX binding to complementary receptors expressed by the T cell initiate immunoregulatory signals which result in immune downregulation [(-)Ireg] and/or immune stimulation. The sum of these simultaneously delivered signals will determine whether TEX mediate immune suppression or immune stimulation in a recipient T cell. Note that a single sEV might carry multiple signaling proteins on its surface membrane.

Figure Legend Snippet: Fig. 4 Immunoregulatory proteins expressed on the surface of recipient immune cells and on TEX or sEV of malignant and non-malignant origins. a The heatmap presenting mean RFI values for proteins expressed on the surface of activated primary CD4 + T, CD8 + T, B and NK cells. b The heatmap presenting mean RFI values for proteins found on the surface of TEX1, TEX2, HaCaT sEV, TNBC Pt-sEV and HD-sEV. c An image of a T cell interacting with various TEX (shown as blue vesicles) that carry immunoregulatory proteins on the surface membrane. TEX binding to complementary receptors expressed by the T cell initiate immunoregulatory signals which result in immune downregulation [(-)Ireg] and/or immune stimulation. The sum of these simultaneously delivered signals will determine whether TEX mediate immune suppression or immune stimulation in a recipient T cell. Note that a single sEV might carry multiple signaling proteins on its surface membrane.

Techniques Used: Membrane, Binding Assay

Fig. 5 TEX-induced apoptosis of activated human CD8 + T cells is not blocked by neutralizing Abs or speciﬁc inhibitors. a Apoptosis (%) induced in CD8 + T cells by TEX1 or TEX2 (50 µg/mL) was not signiﬁcantly blocked by the neutralizing Abs used or the TGF-β inhibitor. b Representative ﬂow cytometry for apoptosis of activated CD8 + T cells co-incubated with TEX1 in the presence of blocking Abs. c Inhibition of apoptosis by various neutralizing Abs of activated CD8 + T cells co-incubated with sEV from TNBC Pts (25 µg/mL). The pretreatment of TNBC-pts sEV with protein kinase (PK) or heat (HI) signiﬁcantly reduced but did not eliminate T cell apoptosis. d The pretreatment of CD8 + T cells or activated primary CD4 + T cells with the mix of blocking Abs speciﬁc for PD1, TRAIL, Fas and anti-CTLA4 (used at the f.c. of 10, 10, 10 and 20 µg/mL, respectively) failed to reduce apoptosis induced by TEX 1 (50 µg/mL). In the blocking assays, primary human T cells were incubated with different blocking Abs and the TGF-β inhibitor. All Abs were used at the f.c. of 10 µg/mL, except for anti-CTLA4 Abs which were used at the f.c. of 20 µg/mL. TGF-β inhibitor was used at the f.c. of 50 nM. Blocking was performed for 30 min before co-incubation for 6 h with TEX or sEV from plasma of TNBC-Pts or HDs. Apoptosis was evaluated by Annexin-V binding assays. The data presented in (a) and (d) are means ± SD from 3 independent experiments.

Figure Legend Snippet: Fig. 5 TEX-induced apoptosis of activated human CD8 + T cells is not blocked by neutralizing Abs or speciﬁc inhibitors. a Apoptosis (%) induced in CD8 + T cells by TEX1 or TEX2 (50 µg/mL) was not signiﬁcantly blocked by the neutralizing Abs used or the TGF-β inhibitor. b Representative ﬂow cytometry for apoptosis of activated CD8 + T cells co-incubated with TEX1 in the presence of blocking Abs. c Inhibition of apoptosis by various neutralizing Abs of activated CD8 + T cells co-incubated with sEV from TNBC Pts (25 µg/mL). The pretreatment of TNBC-pts sEV with protein kinase (PK) or heat (HI) signiﬁcantly reduced but did not eliminate T cell apoptosis. d The pretreatment of CD8 + T cells or activated primary CD4 + T cells with the mix of blocking Abs speciﬁc for PD1, TRAIL, Fas and anti-CTLA4 (used at the f.c. of 10, 10, 10 and 20 µg/mL, respectively) failed to reduce apoptosis induced by TEX 1 (50 µg/mL). In the blocking assays, primary human T cells were incubated with different blocking Abs and the TGF-β inhibitor. All Abs were used at the f.c. of 10 µg/mL, except for anti-CTLA4 Abs which were used at the f.c. of 20 µg/mL. TGF-β inhibitor was used at the f.c. of 50 nM. Blocking was performed for 30 min before co-incubation for 6 h with TEX or sEV from plasma of TNBC-Pts or HDs. Apoptosis was evaluated by Annexin-V binding assays. The data presented in (a) and (d) are means ± SD from 3 independent experiments.

Techniques Used: Cytometry, Incubation, Blocking Assay, Inhibition, Clinical Proteomics, Binding Assay

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Journal: Communications biology

Article Title: Small EV in plasma of triple negative breast cancer patients induce intrinsic apoptosis in activated T cells.

doi: 10.1038/s42003-023-05169-3

Figure Lengend Snippet: Fig. 2 Characterization of sEV isolated from plasma of TNBC-patients or healthy donors (HDs). a, b Representative particle distribution images (NTA) of sEV derived from plasma of a HD and TNBC Pt #1. c sEV numbers/µg protein in fraction #4 for Pts and HDs. d Representative TEM images of sEV from plasma of a HD and TNBC-Pt #1. e Western blots of sEV (fraction #4) isolated from plasma of a HD and TNBC-Pt #1. f On bead ﬂow cytometry of sEV from plasma of TNBC Pts and HDs. Shown are RFI values for immunosuppressive proteins on the sEV surface (upper row) and for immunostimulatory proteins (lower row). Data are presented as means ± SD. Mann-Whitney tests were used to evaluate differences between TNBC Pts and HDs. ns = no signiﬁcant difference. g The RFI scores for immunosuppressive or immunostimulatory proteins and the ratios of stim/supp proteins calculated for all the above listed sEV as described in the text. Note the higher suppressive and stimulatory scores for TNBC Pt’s sEV than for HD’s sEV and the signiﬁcantly higher stim/supp ratio for sEV of HDs than for TNBC Pts. h Apoptosis (%) induced in activated primary human CD8 + T cells by sEV from plasma of HDs (n = 5) and TNBC-Pts (n = 5). Data were analyzed by an unpaired t-test and are presented as means ± SD. i Flow cytometry images of apoptosis induced in CD8 + T cells by increasing doses of sEV from a representative HD or TNBC Pt.

Article Snippet: Human metastatic Triple Negative Breast Cancer Cell Lines (TNBC-CL) MDA-MB-231 (CL 1) and MDA-MB-436 (CL 2) as well as a non-malignant cell line, HaCaT (immortalized human keratinocytes), were obtained from ATCC including the authentication certificate (genomic profiling) and were cultured in Dulbecco’s modified Eagle’s medium (Gibco Fisher Scientific).

Techniques: Isolation, Clinical Proteomics, Derivative Assay, Western Blot, Cytometry, MANN-WHITNEY, Flow Cytometry

Journal: Communications biology

Article Title: Small EV in plasma of triple negative breast cancer patients induce intrinsic apoptosis in activated T cells.

doi: 10.1038/s42003-023-05169-3

Figure Lengend Snippet: Fig. 3 Uptake of TEX labeled with PKH26 by immune cells. TEX isolated from supernatants of TNBC cell lines were labeled with the PKH26 dye and were co-incubated with recipient immune cells for various time periods. a Percentages of CD8+ Jurkat cells up-taking labeled TEX during coincubation as measured by ﬂow cytometry. Data are mean values from two independent assays and are shown in the excel table in Supplementary Data 1. b Representative histograms for the uptake of labeled TEX presented as variables of the uptake time. c Microscopic images of CD8+ Jurkat cells co- incubated with PKH26 labeled TEX1. TEX = Red, Actin = green, DAPI stained nuclei = blue; scale bar = 20 µm. d Comparison of TEX1 uptake by activated primary CD4 + T and CD8 + T cells at different time points. e Flow cytometry images of apoptosis induced in activated primary CD4 + T, CD8 + T, B and NK cells following co-incubation with TEX1.

Techniques: Labeling, Isolation, Incubation, Cytometry, Staining, Comparison, Flow Cytometry

Journal: Communications biology

Article Title: Small EV in plasma of triple negative breast cancer patients induce intrinsic apoptosis in activated T cells.

doi: 10.1038/s42003-023-05169-3

Figure Lengend Snippet: Fig. 4 Immunoregulatory proteins expressed on the surface of recipient immune cells and on TEX or sEV of malignant and non-malignant origins. a The heatmap presenting mean RFI values for proteins expressed on the surface of activated primary CD4 + T, CD8 + T, B and NK cells. b The heatmap presenting mean RFI values for proteins found on the surface of TEX1, TEX2, HaCaT sEV, TNBC Pt-sEV and HD-sEV. c An image of a T cell interacting with various TEX (shown as blue vesicles) that carry immunoregulatory proteins on the surface membrane. TEX binding to complementary receptors expressed by the T cell initiate immunoregulatory signals which result in immune downregulation [(-)Ireg] and/or immune stimulation. The sum of these simultaneously delivered signals will determine whether TEX mediate immune suppression or immune stimulation in a recipient T cell. Note that a single sEV might carry multiple signaling proteins on its surface membrane.

Techniques: Membrane, Binding Assay

Journal: Communications biology

Article Title: Small EV in plasma of triple negative breast cancer patients induce intrinsic apoptosis in activated T cells.

doi: 10.1038/s42003-023-05169-3

Figure Lengend Snippet: Fig. 5 TEX-induced apoptosis of activated human CD8 + T cells is not blocked by neutralizing Abs or speciﬁc inhibitors. a Apoptosis (%) induced in CD8 + T cells by TEX1 or TEX2 (50 µg/mL) was not signiﬁcantly blocked by the neutralizing Abs used or the TGF-β inhibitor. b Representative ﬂow cytometry for apoptosis of activated CD8 + T cells co-incubated with TEX1 in the presence of blocking Abs. c Inhibition of apoptosis by various neutralizing Abs of activated CD8 + T cells co-incubated with sEV from TNBC Pts (25 µg/mL). The pretreatment of TNBC-pts sEV with protein kinase (PK) or heat (HI) signiﬁcantly reduced but did not eliminate T cell apoptosis. d The pretreatment of CD8 + T cells or activated primary CD4 + T cells with the mix of blocking Abs speciﬁc for PD1, TRAIL, Fas and anti-CTLA4 (used at the f.c. of 10, 10, 10 and 20 µg/mL, respectively) failed to reduce apoptosis induced by TEX 1 (50 µg/mL). In the blocking assays, primary human T cells were incubated with different blocking Abs and the TGF-β inhibitor. All Abs were used at the f.c. of 10 µg/mL, except for anti-CTLA4 Abs which were used at the f.c. of 20 µg/mL. TGF-β inhibitor was used at the f.c. of 50 nM. Blocking was performed for 30 min before co-incubation for 6 h with TEX or sEV from plasma of TNBC-Pts or HDs. Apoptosis was evaluated by Annexin-V binding assays. The data presented in (a) and (d) are means ± SD from 3 independent experiments.

Techniques: Cytometry, Incubation, Blocking Assay, Inhibition, Clinical Proteomics, Binding Assay

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