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Regulated delivery of molecular cargo to invasive tumour-derived microvesicles.

Clancy JW, Sedgwick A, Rosse C, Muralidharan-Chari V, Raposo G, Method M, Chavrier P, D'Souza-Schorey C - Nat Commun (2015)

Bottom Line: Here we demonstrate that in amoeboid-like invasive tumour cell lines, the v-SNARE, VAMP3, regulates delivery of microvesicle cargo such as the membrane-type 1 matrix metalloprotease (MT1-MMP) to shedding microvesicles.MT1-MMP delivery to nascent microvesicles depends on the association of VAMP3 with the tetraspanin CD9 and facilitates the maintenance of amoeboid cell invasion.Together these studies demonstrate the importance of microvesicle cargo sorting in matrix degradation and disease progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, USA.

ABSTRACT
Cells release multiple, distinct forms of extracellular vesicles including structures known as microvesicles, which are known to alter the extracellular environment. Despite growing understanding of microvesicle biogenesis, function and contents, mechanisms regulating cargo delivery and enrichment remain largely unknown. Here we demonstrate that in amoeboid-like invasive tumour cell lines, the v-SNARE, VAMP3, regulates delivery of microvesicle cargo such as the membrane-type 1 matrix metalloprotease (MT1-MMP) to shedding microvesicles. MT1-MMP delivery to nascent microvesicles depends on the association of VAMP3 with the tetraspanin CD9 and facilitates the maintenance of amoeboid cell invasion. VAMP3-shRNA expression depletes shed vesicles of MT1-MMP and decreases cell invasiveness when embedded in cross-linked collagen matrices. Finally, we describe functionally similar microvesicles isolated from bodily fluids of ovarian cancer patients. Together these studies demonstrate the importance of microvesicle cargo sorting in matrix degradation and disease progression.

No MeSH data available.


Related in: MedlinePlus

Invasive microvesicles can be isolated from peripheral bodily fluids of patients with diagnosed abdomino-pelvic mass(A) Unfractionated ascites or isolated TMVs were resuspended in sterile, filtered 1X PBS and subjected to nano particle tracking analysis using a NanoSight LM10 as per the manufacturers protocol. Measurements of concentration (upper panel) and percentage undersized (lower panel) vs. particle diameter (nm) shown represent the mean of 10 individual acquisitions for each sample type. The curves presented are representative of the patient population studied. (B) Microvesicles from patients with Stage IIB serous adenocarcinoma were fixed and examined by whole mount transmission electron microscopy as described in Methods. Scale bar = 500 nm. (C) Equal amounts of unfractionated ascites fluid and isolated microvesicles (determined using BCA assay) from patient samples, were probed by western blotting as indicated. Note, protein is equal within but not between patients. Data shown is from patients later diagnosed with serous cystadenocarcinoma of the ovary (28), high-grade serous ovarian carcinoma (32), poorly-differentiated ovarian carcinoma (33). Unfractionated fluid in parallel with isolated TMVs, were resuspended in sterile, filtered, 1X PBS (D) or 1X PBS +NSC405020 (E) prior to mixing with TMV-free complete cell culture media. The mixture was overlaid onto FITC-gelatin coated coverslips and allowed to degrade matrix for a period of 14 hours. TMVs were fixed, stained as indicated, and subjected to confocal microscopy to examine levels of matrix degradation. Scale bar = 50 μm. (F) Equal amounts of protein from unfractionated serum and isolated TMVs (determined using BCA assay) were probed by western blot as indicated. In parallel, microvesicles were mixed with 1X PBS prior to addition to TMV-free complete cell culture media and incubation with FITC-conjugated gelatin-coated coverslips for a period of 14 hours. Scale bar = 10 μm. Data shown is from a patient later diagnosed with serous ovarian carcinoma.
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Figure 4: Invasive microvesicles can be isolated from peripheral bodily fluids of patients with diagnosed abdomino-pelvic mass(A) Unfractionated ascites or isolated TMVs were resuspended in sterile, filtered 1X PBS and subjected to nano particle tracking analysis using a NanoSight LM10 as per the manufacturers protocol. Measurements of concentration (upper panel) and percentage undersized (lower panel) vs. particle diameter (nm) shown represent the mean of 10 individual acquisitions for each sample type. The curves presented are representative of the patient population studied. (B) Microvesicles from patients with Stage IIB serous adenocarcinoma were fixed and examined by whole mount transmission electron microscopy as described in Methods. Scale bar = 500 nm. (C) Equal amounts of unfractionated ascites fluid and isolated microvesicles (determined using BCA assay) from patient samples, were probed by western blotting as indicated. Note, protein is equal within but not between patients. Data shown is from patients later diagnosed with serous cystadenocarcinoma of the ovary (28), high-grade serous ovarian carcinoma (32), poorly-differentiated ovarian carcinoma (33). Unfractionated fluid in parallel with isolated TMVs, were resuspended in sterile, filtered, 1X PBS (D) or 1X PBS +NSC405020 (E) prior to mixing with TMV-free complete cell culture media. The mixture was overlaid onto FITC-gelatin coated coverslips and allowed to degrade matrix for a period of 14 hours. TMVs were fixed, stained as indicated, and subjected to confocal microscopy to examine levels of matrix degradation. Scale bar = 50 μm. (F) Equal amounts of protein from unfractionated serum and isolated TMVs (determined using BCA assay) were probed by western blot as indicated. In parallel, microvesicles were mixed with 1X PBS prior to addition to TMV-free complete cell culture media and incubation with FITC-conjugated gelatin-coated coverslips for a period of 14 hours. Scale bar = 10 μm. Data shown is from a patient later diagnosed with serous ovarian carcinoma.

Mentions: To investigate the physiological and clinical relevance of the TMVs characterized in these studies, we examined for their presence in excess of thirty clinical samples of body fluids. Abdominal ascites, or when ascites was absent, saline wash, was collected during exploratory laparoscopy, from fully consented patients with a prior identification of an ovarian mass. Representative nanoparticle tracking analysis (NTA) of isolated microvesicles showed an enriched population of larger particles relative to unfractionated ascites (Figure 4A). Median particle sizes were 264 nm and 410 nm in the unfractionated fluid and microvesicle fractions respectively. While serous epithelial ovarian cancer was the most common of the cases analyzed in this study, samples from patients diagnosed with mucinous, endometroid and clear cell ovarian tumors, exhibited similar fractionation profiles. These results were supported by electron microscopy of isolated microvesicle or exosome fractions from patient ascites including those diagnosed with Stage IIB serous adenocarcinoma, IIIC cystadenocarcinoma, or mixed epithelial cell carcinoma. Representative images of Stage IIB serous adenocarcinoma microvesicles are shown in Figure 4B and exosomes in Supplementary Figure 11. Isolated TMVs are enriched with VAMP3, MT1-MMP, and ARF6 relative to the unfractionated fluid as determined by western blotting (Figure 4C). These TMVs also contain additional cargo molecules that are currently used or being investigated for potential use as biomarkers. One commonly used biomarker for ovarian cancer, and identified on microvesicles, is the glycoprotein cancer antigen 125 (CA-125). CA-125 is significantly enriched in isolated microvesicles (Figure 4C). These microvesicles do not, however, contain elevated levels of human epididymis protein 4 (HE4), a marker currently undergoing further investigation as a biomarker for use along with CA-125. As we have previously identified functional MT1-MMP as a cargo component in TMVs, we used ascites derived microvesicles in an in vitro invasion assay. The unfractionated fluid showed areas of broad degradation with TMV and other membrane remnants (Figure 4D left) likely due to the presence of soluble proteases within the unfractionated fluid as well as TMV-associated proteases. In contrast, isolated TMVs showed areas of localized proteolytic matrix degradation (Figure 4D right). The invasive capacity of isolated ascites TMVs is dependent on functional MT1-MMP as incubation of isolated TMVs in the presence of NSC405020 greatly reduced vesicle associated gelatin degradation (Figure 4E). Additionally, serum collected from a subset of patients was also found to contain circulating TMVs that were enriched in ARF6, MT1-MMP, VAMP3 and CA-125 and are also capable of matrix degradation (Figure 4F).


Regulated delivery of molecular cargo to invasive tumour-derived microvesicles.

Clancy JW, Sedgwick A, Rosse C, Muralidharan-Chari V, Raposo G, Method M, Chavrier P, D'Souza-Schorey C - Nat Commun (2015)

Invasive microvesicles can be isolated from peripheral bodily fluids of patients with diagnosed abdomino-pelvic mass(A) Unfractionated ascites or isolated TMVs were resuspended in sterile, filtered 1X PBS and subjected to nano particle tracking analysis using a NanoSight LM10 as per the manufacturers protocol. Measurements of concentration (upper panel) and percentage undersized (lower panel) vs. particle diameter (nm) shown represent the mean of 10 individual acquisitions for each sample type. The curves presented are representative of the patient population studied. (B) Microvesicles from patients with Stage IIB serous adenocarcinoma were fixed and examined by whole mount transmission electron microscopy as described in Methods. Scale bar = 500 nm. (C) Equal amounts of unfractionated ascites fluid and isolated microvesicles (determined using BCA assay) from patient samples, were probed by western blotting as indicated. Note, protein is equal within but not between patients. Data shown is from patients later diagnosed with serous cystadenocarcinoma of the ovary (28), high-grade serous ovarian carcinoma (32), poorly-differentiated ovarian carcinoma (33). Unfractionated fluid in parallel with isolated TMVs, were resuspended in sterile, filtered, 1X PBS (D) or 1X PBS +NSC405020 (E) prior to mixing with TMV-free complete cell culture media. The mixture was overlaid onto FITC-gelatin coated coverslips and allowed to degrade matrix for a period of 14 hours. TMVs were fixed, stained as indicated, and subjected to confocal microscopy to examine levels of matrix degradation. Scale bar = 50 μm. (F) Equal amounts of protein from unfractionated serum and isolated TMVs (determined using BCA assay) were probed by western blot as indicated. In parallel, microvesicles were mixed with 1X PBS prior to addition to TMV-free complete cell culture media and incubation with FITC-conjugated gelatin-coated coverslips for a period of 14 hours. Scale bar = 10 μm. Data shown is from a patient later diagnosed with serous ovarian carcinoma.
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Figure 4: Invasive microvesicles can be isolated from peripheral bodily fluids of patients with diagnosed abdomino-pelvic mass(A) Unfractionated ascites or isolated TMVs were resuspended in sterile, filtered 1X PBS and subjected to nano particle tracking analysis using a NanoSight LM10 as per the manufacturers protocol. Measurements of concentration (upper panel) and percentage undersized (lower panel) vs. particle diameter (nm) shown represent the mean of 10 individual acquisitions for each sample type. The curves presented are representative of the patient population studied. (B) Microvesicles from patients with Stage IIB serous adenocarcinoma were fixed and examined by whole mount transmission electron microscopy as described in Methods. Scale bar = 500 nm. (C) Equal amounts of unfractionated ascites fluid and isolated microvesicles (determined using BCA assay) from patient samples, were probed by western blotting as indicated. Note, protein is equal within but not between patients. Data shown is from patients later diagnosed with serous cystadenocarcinoma of the ovary (28), high-grade serous ovarian carcinoma (32), poorly-differentiated ovarian carcinoma (33). Unfractionated fluid in parallel with isolated TMVs, were resuspended in sterile, filtered, 1X PBS (D) or 1X PBS +NSC405020 (E) prior to mixing with TMV-free complete cell culture media. The mixture was overlaid onto FITC-gelatin coated coverslips and allowed to degrade matrix for a period of 14 hours. TMVs were fixed, stained as indicated, and subjected to confocal microscopy to examine levels of matrix degradation. Scale bar = 50 μm. (F) Equal amounts of protein from unfractionated serum and isolated TMVs (determined using BCA assay) were probed by western blot as indicated. In parallel, microvesicles were mixed with 1X PBS prior to addition to TMV-free complete cell culture media and incubation with FITC-conjugated gelatin-coated coverslips for a period of 14 hours. Scale bar = 10 μm. Data shown is from a patient later diagnosed with serous ovarian carcinoma.
Mentions: To investigate the physiological and clinical relevance of the TMVs characterized in these studies, we examined for their presence in excess of thirty clinical samples of body fluids. Abdominal ascites, or when ascites was absent, saline wash, was collected during exploratory laparoscopy, from fully consented patients with a prior identification of an ovarian mass. Representative nanoparticle tracking analysis (NTA) of isolated microvesicles showed an enriched population of larger particles relative to unfractionated ascites (Figure 4A). Median particle sizes were 264 nm and 410 nm in the unfractionated fluid and microvesicle fractions respectively. While serous epithelial ovarian cancer was the most common of the cases analyzed in this study, samples from patients diagnosed with mucinous, endometroid and clear cell ovarian tumors, exhibited similar fractionation profiles. These results were supported by electron microscopy of isolated microvesicle or exosome fractions from patient ascites including those diagnosed with Stage IIB serous adenocarcinoma, IIIC cystadenocarcinoma, or mixed epithelial cell carcinoma. Representative images of Stage IIB serous adenocarcinoma microvesicles are shown in Figure 4B and exosomes in Supplementary Figure 11. Isolated TMVs are enriched with VAMP3, MT1-MMP, and ARF6 relative to the unfractionated fluid as determined by western blotting (Figure 4C). These TMVs also contain additional cargo molecules that are currently used or being investigated for potential use as biomarkers. One commonly used biomarker for ovarian cancer, and identified on microvesicles, is the glycoprotein cancer antigen 125 (CA-125). CA-125 is significantly enriched in isolated microvesicles (Figure 4C). These microvesicles do not, however, contain elevated levels of human epididymis protein 4 (HE4), a marker currently undergoing further investigation as a biomarker for use along with CA-125. As we have previously identified functional MT1-MMP as a cargo component in TMVs, we used ascites derived microvesicles in an in vitro invasion assay. The unfractionated fluid showed areas of broad degradation with TMV and other membrane remnants (Figure 4D left) likely due to the presence of soluble proteases within the unfractionated fluid as well as TMV-associated proteases. In contrast, isolated TMVs showed areas of localized proteolytic matrix degradation (Figure 4D right). The invasive capacity of isolated ascites TMVs is dependent on functional MT1-MMP as incubation of isolated TMVs in the presence of NSC405020 greatly reduced vesicle associated gelatin degradation (Figure 4E). Additionally, serum collected from a subset of patients was also found to contain circulating TMVs that were enriched in ARF6, MT1-MMP, VAMP3 and CA-125 and are also capable of matrix degradation (Figure 4F).

Bottom Line: Here we demonstrate that in amoeboid-like invasive tumour cell lines, the v-SNARE, VAMP3, regulates delivery of microvesicle cargo such as the membrane-type 1 matrix metalloprotease (MT1-MMP) to shedding microvesicles.MT1-MMP delivery to nascent microvesicles depends on the association of VAMP3 with the tetraspanin CD9 and facilitates the maintenance of amoeboid cell invasion.Together these studies demonstrate the importance of microvesicle cargo sorting in matrix degradation and disease progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, USA.

ABSTRACT
Cells release multiple, distinct forms of extracellular vesicles including structures known as microvesicles, which are known to alter the extracellular environment. Despite growing understanding of microvesicle biogenesis, function and contents, mechanisms regulating cargo delivery and enrichment remain largely unknown. Here we demonstrate that in amoeboid-like invasive tumour cell lines, the v-SNARE, VAMP3, regulates delivery of microvesicle cargo such as the membrane-type 1 matrix metalloprotease (MT1-MMP) to shedding microvesicles. MT1-MMP delivery to nascent microvesicles depends on the association of VAMP3 with the tetraspanin CD9 and facilitates the maintenance of amoeboid cell invasion. VAMP3-shRNA expression depletes shed vesicles of MT1-MMP and decreases cell invasiveness when embedded in cross-linked collagen matrices. Finally, we describe functionally similar microvesicles isolated from bodily fluids of ovarian cancer patients. Together these studies demonstrate the importance of microvesicle cargo sorting in matrix degradation and disease progression.

No MeSH data available.


Related in: MedlinePlus