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. 2015 Feb 10;6(4):2366-84.
doi: 10.18632/oncotarget.2958.

The tetraspanins CD151 and Tspan8 are essential exosome components for the crosstalk between cancer initiating cells and their surrounding

Shijing Yue  1 , Wei Mu  1 , Ulrike Erb  1 , Margot Zöller  1
Affiliations

The tetraspanins CD151 and Tspan8 are essential exosome components for the crosstalk between cancer initiating cells and their surrounding

Shijing Yue et al. Oncotarget. .

Abstract

Tspan8 and CD151 are metastasis-promoting tetraspanins and a knockdown (kd) of Tspan8 or CD151 and most pronounced of both tetraspanins affects the metastatic potential of the rat pancreatic adenocarcinoma line ASML. Approaching to elaborate the underlying mechanism, we compared ASMLwt, -CD151kd and/or Tspan8kd clones. We focused on tumor exosomes, as exosomes play a major role in tumor progression and tetraspanins are suggested to be engaged in exosome targeting. ASML-CD151/Tspan8kd cells poorly metastasize, but regain metastatic capacity, when rats are pretreated with ASMLwt, but not ASML-CD151kd and/or -Tspan8kd exosomes. Both exosomal CD151 and Tspan8 contribute to host matrix remodelling due to exosomal tetraspanin-integrin and tetraspanin-protease associations. ASMLwt exosomes also support stroma cell activation with upregulation of cytokines, cytokine receptors and proteases and promote inflammatory cytokine expression in hematopoietic cells. Finally, CD151-/Tspan8-competent exosomes support EMT gene expression in poorly-metastatic ASML-CD151/Tspan8kd cells. These effects are not seen or are weakened using ASML-CD151kd or -Tspan8kd exosomes, which is at least partly due to reduced binding/uptake of CD151- and/or Tspan8-deficient exosomes. Thus, CD151- and Tspan8-competent tumor exosomes support matrix degradation, reprogram stroma and hematopoietic cells and drive non-metastatic ASML-CD151/Tspan8kd cells towards a motile phenotype.

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Conflict of interest statement

Conflict of Interest Disclosure

The authors declare that they have no competing interests

Figures

Figure 1
Figure 1. CD151 and Tspan8 requirement for metastasis formation and for exosome distribution
Clones 16 and 24 were used for further experiments. (B-F) BDX rats (5/group) received an ifp injection of 1×106 ASMLwt or -CD151kd and/or -Tspan8kd cells. (B) Tumor growth in the popliteal node; (C) survival time and survival rate; (D) mean survival time, indicating significant differences compared to ASMLwt-bearing rats and between ASML-CD151/Tspan8kd versus -CD151kd or Tpsan8kd bearing rats: s; (E) No of rats with small or large LN metastasis and of rats with no, few or >1000 lung metastases; significant differences compared to ASMLwt-bearing rats: *, and between ASML-CD151/Tspan8kd versus -CD151kd or Tpsan8kd bearing rats: s; (F) recovery of CD151 and Tspan8 in lung and LN lysates of control and tumor-bearing rats. (G) Rats (3/group) received a single injected of dye-labeled exosomes, iv. Rats were sacrificed after 48h; (H) rats (3/group) received three injected of dye-labeled exosomes in 3d intervals, ifp, and were sacrificed 48h after the last injection; (G,H) lymphatic organs were excised and the recovery of dye-labeled cells (exosome uptake) was evaluated by flow cytometry. The mean±SD of dye-labeled cells is shown; significant differences to the uptake of ASMLwt exosomes: *; (I) Rats (5/group) received 1×106 ASML-CD151/Tspan8kd cells ifp and starting at day -6 in 3d intervals, 100μg exosomes, ifp. Rats were scarified after 21d. Recovery of tumor cells in draining LN, lung and BM was evaluated by flow cytometry after staining for the ASML marker C4.4A; the mean No±SD of tumor cells / 103 cells is shown; significant differences to ASML-CD151/Tspan8kd bearing rats: *. A CD151kd or a Tspan8kd retards tumor growth. ASML-CD151/Tspan8kd cells rarely metastasize. ASML-CD151kd and/or ASML-Tspan8kd exosomes are poorly recovered in lymphoid organs, which is accompanied by ASML-Tspan8kd exosome retention at the injection site.
Figure 2
Figure 2. The impact of CD151 and Tspan8 on tetraspanin and adhesion molecule expression in ASML cells and exosomes
(A-D) ASML cells and exosomes were stained for expression of the indicated tetraspanins and adhesion molecules; (A,C) Mean±SD (3 assays) of the % stained cells and exosomes; significant differences to ASMLwt cells and exosomes: * and (B,D) WB. (E) Exosome lysates of ASMLwt and -CD151kd and/or Tspan8kd were precipitated with the indicated anti-integrin antibodies and were blotted with anti-CD151 and anti-Tspan8. (F,G) Dye-labeled exosomes were seeded on matrix-coated plates. Where indicated exosomes were preincubated with antibodies. After 2h incubation, plates were washed and remaining fluorescence was evaluated in an ELISA reader. The mean percent±SD (triplicates) of bound exosomes is shown, (F) differences to ASMLwt exosomes: *; (G) differences by antibody preincubation: *. Expression of additional tetraspanins and of integrins is not affected in ASML-CD151kd and/or -Tspan8kd cells. Instead, integrin expression in exosomes is reduced corresponding to the associating tetraspanin. Exosomal CD151 and Tspan8 slightly affect exosome adhesion to matrix proteins.
Figure 3
Figure 3. The association of exosomal CD151 and Tspan8 with proteases and the impact on matrix degradation and host cell invasiveness
(A) Matrix protein degradation (WB) by ASMLwt, -CD151kd and/or Tspan8kd exosomes; (B) Protease recovery in ASMLwt, -CD151kd and/or Tspan8kd cells and exosomes; mean±SD (3 assays) of the percent stained cells and exosome-coated beads; significant differences to ASMLwt cells/exosomes: *; significant differences between cells and exosomes: *; (C) protease recovery in ASMLwt and -CD151kd and/or -Tspan8kd cells and exosomes as revealed by WB; (D) Coimmunoprecipitation of exosomal CD151 and Tspan8 with proteases and (E) of MMP2 and MMP9 with CD151 and Tspan8; (F) recovery of proteases in light and heavy sucrose density fractions in ASMLwt, -CD151kd and/or -Tspan8kd exosomes; (G) gelatin (zymography) degradation by ASMLwt, -CD151kd and/or Tspan8kd exosomes and exosome-depleted conditioned medium; (H) native LnStr and LuFb matrix degradation (WB) by ASMLwt, -CD151kd and/or Tspan8kd exosomes and (I) inhibition of exosome-mediated matrix protein degradation by TACE, MMP2 and MMP9/MMP13 inhibitors (WB). Recovery of proteases in exosomes is mostly dictated by the association with CD151 and/or Tspan8 such that in the absence of CD151 mostly MMP2 and in the absence of Tspan8 mostly MMP9 expression / activity are strongly reduced. Exosomal TACE activity apparently depends on both Tspan8 and CD151. Reduced exosomal protease recovery has consequences on matrix protein, matrigel and native matrix degradation.
Figure 4
Figure 4. The impact of exosomal CD151 and Tspan8 on host cell adhesion, motility and invasiveness
(A) LnStr, LuFb and RAEC were seeded on their corresponding untreated or exosome-treated matrix; mean±SD of adherent cells; significant differences compared to the native matrix: *; (B) confocal microscopy of actin cytoskeleton organization in LnStr and LuFb depending on exosome treated stroma (scale bar: 10μm); (C) videomicroscopy of LuFb and LnStr that were seeded on their untreated or ASML exosome-treated matrix. The relative migration of cells during 12h incubation (mean of 20 individual cells) and a representative example are shown; significant differences in migration due to ASML exosome treatment are indicated; (D) Matrigel penetration and invasion of LnStr and RAEC in dependence on CD151- and/or Tspan8-competent exosomes; the mean±SD (triplicates) of penetrating cells; significant differences to untreated matrigel: *; significant difference between ASML-CD151/Tspan8kd versus -CD151kd or -Tspan8kd exosomes: s; and representative examples of invasion (scale bar: 250μm). Exosomal CD151 and Tspan8 slightly affect host cell adhesion to matrix proteins, but support stroma cell motility and invasiveness, which might be promoted by exosomal proteases.
Figure 5
Figure 5. The impact of exosomal CD151 and Tspan8 on host cell adhesion molecule and protease expression
(A,B) Flow cytometry analysis of protease expression in LnStr and LuFb after coculture with ASMLwt, -CD151kd and/or -Tspan8kd exosomes; mean percent±SD (3 assays) of stained cells; significant differences to untreated cells: *; (C,D) immunohistology of protease and adhesion molecule expression in draining LN after repeated ifp application of ASMLwt or -CD151kd and/or -Tspan8kd exosomes (scale bar: 150μm). Short term in vitro coculture of stroma cells with ASML exosomes hardly affected protease and adhesion molecule (data not shown) expression. Instead, after repeated exosome application in vivo, ASMLwt exosomes particularly promoted MMP2, MMP9 and TACE as well as CD49c, CD49d and CD104 expression. CD104 and TACE upregulation were weakened in ASML-CD151/Tspan8kd exosome treated rats and CD49c, CD49d, MMP2 and MMP9 expression were not supported.
Figure 5
Figure 5. The impact of exosomal CD151 and Tspan8 on host cell adhesion molecule and protease expression
(A,B) Flow cytometry analysis of protease expression in LnStr and LuFb after coculture with ASMLwt, -CD151kd and/or -Tspan8kd exosomes; mean percent±SD (3 assays) of stained cells; significant differences to untreated cells: *; (C,D) immunohistology of protease and adhesion molecule expression in draining LN after repeated ifp application of ASMLwt or -CD151kd and/or -Tspan8kd exosomes (scale bar: 150μm). Short term in vitro coculture of stroma cells with ASML exosomes hardly affected protease and adhesion molecule (data not shown) expression. Instead, after repeated exosome application in vivo, ASMLwt exosomes particularly promoted MMP2, MMP9 and TACE as well as CD49c, CD49d and CD104 expression. CD104 and TACE upregulation were weakened in ASML-CD151/Tspan8kd exosome treated rats and CD49c, CD49d, MMP2 and MMP9 expression were not supported.
Figure 6
Figure 6. Stroma and endothelial cell responses to exosomal CD151 and Tspan8
(A-C) Flow cytometry analysis of LnStr, RAEC and LuFb after coculture with ASMLwt, -CD151kd and/or -Tspan8kd exosomes; mean percent±SD (3 assays) of stained cells; significant differences to untreated cells: *; (D) representative examples of SDF1, CXCR4, VEGFR1 and VEGFR3 expression in LnStr and RAEC after coculture with ASMLwt, -CD151kd and/or -Tspan8kd exosomes (scale bar: 10μm) and (E) in draining LN after repeated application of ASMLwt, -CD151kd and/or -Tspan8kd exosomes (scale bar: 150μm). Exosomal CD151 and Tspan8 promote upregulated expression of several growth factors and their receptors, which varies depending on the target cell. Upregulated expression of some markers, e.g. bFGF and VEGFR1 essentially depends on the presence of both CD151 and Tspan8, whereas SDF1 and FGFR expression is independent of exosomal CD151 and Tspan8. However, exosomal Tspan8 is essential for VEGFR3 upregulation.
Figure 6
Figure 6. Stroma and endothelial cell responses to exosomal CD151 and Tspan8
(A-C) Flow cytometry analysis of LnStr, RAEC and LuFb after coculture with ASMLwt, -CD151kd and/or -Tspan8kd exosomes; mean percent±SD (3 assays) of stained cells; significant differences to untreated cells: *; (D) representative examples of SDF1, CXCR4, VEGFR1 and VEGFR3 expression in LnStr and RAEC after coculture with ASMLwt, -CD151kd and/or -Tspan8kd exosomes (scale bar: 10μm) and (E) in draining LN after repeated application of ASMLwt, -CD151kd and/or -Tspan8kd exosomes (scale bar: 150μm). Exosomal CD151 and Tspan8 promote upregulated expression of several growth factors and their receptors, which varies depending on the target cell. Upregulated expression of some markers, e.g. bFGF and VEGFR1 essentially depends on the presence of both CD151 and Tspan8, whereas SDF1 and FGFR expression is independent of exosomal CD151 and Tspan8. However, exosomal Tspan8 is essential for VEGFR3 upregulation.
Figure 7
Figure 7. Leukocyte responses to exosomal CD151 and Tspan8
(A-F) BMC and LNC were cocultured with ASMLwt, -CD151kd and/or -Tspan8kd exosomes; (A,C,D,F) cytokine and activation marker expression as well as activation of the JAK/STAT signaling pathway and of NFAT and FoxP3 was evaluated by flow cytometry; mean percent±SD of stained cells; significant differences to BMC and LNC cultured in the absence of exosomes: * and representative examples; (B,E) representative examples of confocal microscopy (scale bar: 10μm). In BMC and LNC, ASML exosomes strengthen inflammatory cytokine expression; These effects are supported by, but not exclusively depending on exosomal CD151 and Tspan8.
Figure 7
Figure 7. Leukocyte responses to exosomal CD151 and Tspan8
(A-F) BMC and LNC were cocultured with ASMLwt, -CD151kd and/or -Tspan8kd exosomes; (A,C,D,F) cytokine and activation marker expression as well as activation of the JAK/STAT signaling pathway and of NFAT and FoxP3 was evaluated by flow cytometry; mean percent±SD of stained cells; significant differences to BMC and LNC cultured in the absence of exosomes: * and representative examples; (B,E) representative examples of confocal microscopy (scale bar: 10μm). In BMC and LNC, ASML exosomes strengthen inflammatory cytokine expression; These effects are supported by, but not exclusively depending on exosomal CD151 and Tspan8.
Figure 8
Figure 8. Metastasis-supporting exosomes and EMT: ASML-CD151/Tspan8kd cells were cocultured for 48h with ASMLwt, -CD151kd or -Tspan8kd exosomes
(A,B) ASMLwt, -CD151kd and/or -Tspan8kd cells and exosomes stained for the indicated EMT markers; mean percent±SD of stained cells / exosome-coated latex beads; significant differences to ASMLwt cells / exosomes: *. (C-E) EMT gene expression evaluated by flow cytometry (mean percent stained cells, 3 assays); significant differences to ASML-CD151/Tspan8kd cells cultured in the absence of exosomes: *, confocal microscopy (scale bar: 10μm) and WB. Expression of the EMT related vimentin protein as well as of the transcription factors Slug, Snail, Twist and, particularly, Notch, becomes upregulated, upregulation of vimentin and Snail predominantly depending on Tspan8 and of Notch on CD151 and Tspan8.
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