spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online May 28, 2004
doi: 10.1242/10.1242/jcs.01145

Journal of Cell Science 117, 2827-2839 (2004)
Published by The Company of Biologists 2004
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Peinado, H.
Right arrow Articles by Cano, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Peinado, H.
Right arrow Articles by Cano, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Snail and E47 repressors of E-cadherin induce distinct invasive and angiogenic properties in vivo

Hector Peinado1,3, Faustino Marin2, Eva Cubillo1, Hans-Juergen Stark3, Norbert Fusenig3, M. Angela Nieto2 and Amparo Cano1,*

1 Departamento de Bioquímica, UAM. Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC-UAM), Arturo Duperier 4, 28029 Madrid, Spain
2 Instituto Cajal CSIC, Doctor Arce, 37, 28002 Madrid, Spain
3 German Cancer Research Center, DKFZ, Im Neuenheimer Feld 280, Heidelberg 69120, Germany



View larger version (80K):

[in a new window]
  		Fig. 1. Analysis of organotypic cultures. Organotypic cultures of MDCK-CMV (CMV) (a-d), MDCK-Snail (Snail) (e-h) and MDCK-E47 (E47) cells (i-l) were prepared as described in Materials and Methods and grown for 3 weeks. Histological (a,e,i) and double immunofluorescence analyses for E-cadherin (red) and vimentin (green) (b,f,j) were performed on cryostat sections; nuclei were stained with DAPI (blue). In situ hybridization analysis for Snail (c,g,k) and E47 transcripts (d,h,l) were performed on vibratome sections. Note the infiltration of Snail-expressing cells into the collagen gel and the transdifferentiation to an epithelial non-invading phenotype of the upper layer of MDCK-Snail cultures which re-express E-cadherin and have lost vimentin and Snail expression (arrows in e, f and g). Bars, 50 µm.

 


View larger version (75K):

[in a new window]
  		Fig. 2. MDCK-Snail and MDCK-E47 cells exhibit invasive and migratory abilities in in vivo transplantation assays. (A) low power and (B) high power images of MDCK-CMV (CMV) (a,d), MDCK-Snail (Snail) (b,e) and MDCK-E47 (E47) cells (c,f) grown on collagen type I gel, transplanted onto the backs of nude mice and allowed to grow for 10 days. Cryostat sections of each type of transplant were analysed by histology (a-c) and double immunofluorescence for E-cadherin (red) and vimentin (green) (d-f); nuclei were stained with DAPI (blue). (A) The lower limit of the collagen gel in contact with the host stroma is indicated by dashed black (a-c) and yellow lines (d-f). MDCK-CMV cells form an organized epithelial multilayer with cyst like structures without infiltration into the collagen gel (black arrowheads in a), whereas MDCK-Snail cells are clearly infiltrating (black arrowheads in b). Note also vimentin-positive cells apparently migrating from the host stroma into the collagen gel in MDCK-Snail and MDCK-E47 transplants (red arrows in b, c, e and f), in contrast to the complete absence of cells in the lower part of the collagen gel in MDCK-CMV transplants (a,d). Bar, 50 µm. (B) High power images of the upper part of the indicated 10-day transplants showing the histology (a-c) and immunofluorescence analyses for E-cadherin (red) and vimentin (green) (d-f); nuclei were stained with DAPI (blue). Note the absence of E-cadherin and expression of vimentin in all cell layers of MDCK-Snail (e) and MDCK-E47 (f) transplants. Bar, 50 µm.

 


View larger version (101K):

[in a new window]
  		Fig. 3. Twenty day-transplants of MDCK-Snail and MDCK-E47 cells exhibit a high infiltration potential. Transplants of the indicated cells lines were allowed to grow on the backs of nude mice for 20 days and subsequently either (A) fixed in formaldehyde and processed for histology or (B) frozen in OCT and subjected to immunofluorescence staining after cryostat sectioning. (A) MDCK-CMV (CMV) (a,d) cells form a multilayer of highly differentiated epithelial cells with a clear basal membrane delimitation from the remaining collagen gel (dashed line in a, and yellow arrow in d). MDCK-Snail (b,e) and MDCK-E47 (c,f) cells fully infiltrate the collagen gel and adipose and muscle stromal tissues; the dashed lines in b and c indicate the hypothetical limit of the collagen gel. Panels d to f show magnified images of the lower part of the transplants. See also blood vessels in the lower part of MDCK-E47 transplants (c,f). Bar, 50 µm. (B) MDCK-CMV transplants maintain E-cadherin expression and absence of vimentin (a,d) in all cell layers, while no expression of E-cadherin and homogeneous expression of vimentin is observed in all cells of MDCK-E47 transplants (c,f). Invading cells of MDCK-Snail transplants do not express E-cadherin and are vimentin positive, while the upper non invading layers re-express E-cadherin and have lost vimentin (b,e). Nuclei were stained with DAPI (blue). Bar, 50 µm.

 


View larger version (97K):

[in a new window]
  		Fig. 4. MDCK-Snail and MDCK-E47 cells induce an angiogenic response in the host stromal tissue. Cryostat sections of 10-day transplants of MDCK-CMV (a,d,g), MDCK-Snail (b,e,h) and MDCK-E47 (c,f,i) cells were subjected to histological (a-c) and immunofluorescence analysis (d-i) for CD31 (red) and vimentin (green). Yellow arrows indicate the border between the collagen gel and host stromal tissue, red arrows indicate the upper limit of CD31-positive cells. (g-i) Magnified images of CD31 positive cells (indicated as insets in d-f) are shown; white arrows indicate blood vessels. Bars, 50 µm.

 


View larger version (173K):

[in a new window]
  		Fig. 5. Angiogenic response of 20-day transplants. MDCK-CMV (CMV) (a,d), MDCK-Snail (b,e) and MDCK-E47 (c,f) transplants grown for 20 days on the backs of nude mice were fixed in formaldehyde and processed for histological analysis. (a-d) Images corresponding to the lower part of the transplants; yellow arrows indicate the limit within the basal membrane and the remaining collagen gel (a) or the hypothetical edge of the gel (b,c). (d-f) Magnified images of the lower (d) and upper part (e,f) of the transplants. Blood vessels (indicated by blue arrows) were detected in the stromal region of CMV transplants (a,d) while they extend all over the surface of Snail (b,e) and E47 (c,f) transplants. Bars, 50 µm.

 


View larger version (72K):

[in a new window]
  		Fig. 6. Analyses of xenografted tumours induced by MDCK-Snail and MCDK-E47 cells. (A) Growth behaviour of MDCK-CMV, MDCK-Snail and MDCK-E47 cells after injection in nude mice. (B) Proliferation of the three cell types in two-dimensional cultures. (C) Tumours induced into nude mice by MDCK-Snail (Snail) (a-e) and MDCK-E47 (E47) (f-j) cells were analysed by histology (a,f); immunostaining for E-cadherin (b,g); and in situ hybridization for E-cadherin (c,h), Snail (d,i) and E47 (e,j) transcripts. Note the absence of E-cadherin (c) and focal expression of E47 (e) transcripts in MDCK-Snail induced tumours; and the more heterogeneous expression of E-cadherin (h) and E47 (j) transcripts, but complete absence of E-cadherin protein (g) in MDCK-E47 induced tumours. Inset in b shows E-cadherin staining in the epidermis adjacent to the tumour. Cryostat sections were used for the histological and immunofluorescence analyses; vibratome sections were used in the ISH analyses. Bars, 50 µm.

 


View larger version (97K):

[in a new window]
  		Fig. 7. Analysis of angiogenesis in xenografted tumours induced by MDCK-Snail and MCDK-E47 cells. (A) Tumours induced in nude mice by MDCK-Snail (Snail) (a-d) and MDCK-E47 (E47) (e-h) cells were analysed by immunostaining for CD-31 (a,e) and endoglin (b,f); or immunohistochemistry for {alpha}-smooth muscle actin ({alpha}SMA) (c,d,g,h). Note the higher number of small blood vessels in MDCK-E47 derived tumours (e-h) than those induced by MDCK-Snail cells (a-d). (B) Expression of angiogenic factors in MDCK-Snail and MDCK-E47 induced tumours. Vibratome sections were processed either for TGFß1 mRNA (a,c) detection or for VEGF-A mRNA plus {alpha}SMA (b,d) detection. TGFß1 mRNA expression appears in both types of tumour with a scattered pattern covering the full extent of the tumour. VEGF-A expression appears only in Snail-induced tumours, in the form of isolated patches, which are also positive for {alpha}SMA. Bars, 50 µm.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2004