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

Yue S, Mu W, Erb U, Zöller M - Oncotarget (2015)

Bottom Line: Approaching to elaborate the underlying mechanism, we compared ASMLwt, -CD151kd and/or Tspan8kd clones.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Cell Biology, University Hospital of Surgery, Heidelberg, Germany.

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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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.
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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.

Mentions: Exosome binding to matrix proteins is accompanied by matrix degradation, which is affected in ASML-CD151kd, -Tspan8kd and -CD151/Tspan8kd exosomes. ASML-CD151kd exosomes did not degrade coll I and FN and poorly degraded coll II and coll IV. Coll II, FN and more pronounced LN332 degradation was reduced in the presence of ASML-Tspan8kd compared to ASMLwt exosomes (Fig.3A). Reduced matrix degradation corresponds to reduced protease expression in exosomes. A CD151kd mostly affects expression of MMP2 and MMP3, a Tspan8kd affects MMP9, but promotes MMP14 and TACE expression. Furthermore, while exosomal MMP9 and MMP14 expression corresponds to cellular expression, MMP2, CD13 and TACE expression is reduced in ASML-Tspan8kd exosomes (Fig.3B,3C). Notably, recovery in exosomes correlates with the tetraspanin association. Thus, after mild exosome lysis, CD13 (mostly), MMP9 and TACE co-immunoprecipitate with Tspan8, but not CD151. MMP2 and MMP14 co-immunoprecipitate with CD151, but not Tspan8 and exosomal MMP13 does not co-immunoprecipitate with CD151 or Tspan8, which was confirmed in the reverse setting in cell and exosome lysates for MMP2 and MMP9 (Fig.3D,3E). Furthermore, MMP2 was not recovered in light sucrose gradient fractions of ASML-CD151kd exosomes, and MMP9, CD13 and TACE were not or poorly recovered in light sucrose gradient fractions of ASML-Tspan8kd exosomes (Fig.3F). Thus, changes in the exosomal versus the cellular protease profile are due to the selective association of CD151 with MMP2 and MMP14 and of Tspan8 with CD13, MMP9 and TACE. Zymography confirmed the absence of active MMP2 in CD151kd exosomes, strongly reduced MMP9 activity in ASML-Tspan8kd exosomes and poor recovery of active MMP2 and MMP9 in CD151/Tspan8kd exosomes (Fig.3G). The impact of CD151- and/or Tspan8-associated exosomal proteases also was apparent in degradation of the natural matrix of LnStr and LuFb. ASML-CD151kd exosomes did not degrade coll I and LN111 and ASML-Tspan8kd exosomes did not degrade LN332 (Fig.3H). Confirming the contribution of Tspan8-associated MMP9 and MMP13 to coll II, FN and LN332 modulation, degradation was abolished in the presence of an MMP9/MMP13 inhibitor, while degradation of coll I, coll IV and FN, which require CD151-associated MMP2, was not seen in the presence of an MMP2 inhibitor. LN332, coll IV and FN degradation was also impaired in the presence of the TACE inhibitor TAPI (Fig.3I).


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

Yue S, Mu W, Erb U, Zöller M - Oncotarget (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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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.
Mentions: Exosome binding to matrix proteins is accompanied by matrix degradation, which is affected in ASML-CD151kd, -Tspan8kd and -CD151/Tspan8kd exosomes. ASML-CD151kd exosomes did not degrade coll I and FN and poorly degraded coll II and coll IV. Coll II, FN and more pronounced LN332 degradation was reduced in the presence of ASML-Tspan8kd compared to ASMLwt exosomes (Fig.3A). Reduced matrix degradation corresponds to reduced protease expression in exosomes. A CD151kd mostly affects expression of MMP2 and MMP3, a Tspan8kd affects MMP9, but promotes MMP14 and TACE expression. Furthermore, while exosomal MMP9 and MMP14 expression corresponds to cellular expression, MMP2, CD13 and TACE expression is reduced in ASML-Tspan8kd exosomes (Fig.3B,3C). Notably, recovery in exosomes correlates with the tetraspanin association. Thus, after mild exosome lysis, CD13 (mostly), MMP9 and TACE co-immunoprecipitate with Tspan8, but not CD151. MMP2 and MMP14 co-immunoprecipitate with CD151, but not Tspan8 and exosomal MMP13 does not co-immunoprecipitate with CD151 or Tspan8, which was confirmed in the reverse setting in cell and exosome lysates for MMP2 and MMP9 (Fig.3D,3E). Furthermore, MMP2 was not recovered in light sucrose gradient fractions of ASML-CD151kd exosomes, and MMP9, CD13 and TACE were not or poorly recovered in light sucrose gradient fractions of ASML-Tspan8kd exosomes (Fig.3F). Thus, changes in the exosomal versus the cellular protease profile are due to the selective association of CD151 with MMP2 and MMP14 and of Tspan8 with CD13, MMP9 and TACE. Zymography confirmed the absence of active MMP2 in CD151kd exosomes, strongly reduced MMP9 activity in ASML-Tspan8kd exosomes and poor recovery of active MMP2 and MMP9 in CD151/Tspan8kd exosomes (Fig.3G). The impact of CD151- and/or Tspan8-associated exosomal proteases also was apparent in degradation of the natural matrix of LnStr and LuFb. ASML-CD151kd exosomes did not degrade coll I and LN111 and ASML-Tspan8kd exosomes did not degrade LN332 (Fig.3H). Confirming the contribution of Tspan8-associated MMP9 and MMP13 to coll II, FN and LN332 modulation, degradation was abolished in the presence of an MMP9/MMP13 inhibitor, while degradation of coll I, coll IV and FN, which require CD151-associated MMP2, was not seen in the presence of an MMP2 inhibitor. LN332, coll IV and FN degradation was also impaired in the presence of the TACE inhibitor TAPI (Fig.3I).

Bottom Line: Approaching to elaborate the underlying mechanism, we compared ASMLwt, -CD151kd and/or Tspan8kd clones.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Cell Biology, University Hospital of Surgery, Heidelberg, Germany.

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.

Show MeSH
Related in: MedlinePlus