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Metastasis depends on a gene program expressed by the tumor microenvironment upon TGF-beta stimulation. CRC (Colorectal cancer) cell lines did not induce robust stromal TGF-beta responses when injected into nude mice as shown by lack of p-SMAD2 accumulation in tumor-associated stromal cells. To enforce high TGF-beta signaling in xenografts, we engineered CRC cell lines to secrete active TGF-beta. Subcutaneous tumors obtained from HT29-M6TGF-β, KM12L4aTGF-β cells and SW48TGF-β cells contained abundant p-SMAD2+ stromal cells.

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Immunostaining Protocol: P-Smad2 (Xenograft and Mice)

Cancer Biology > Invasion & metastasis > Animal models > Cell isolation and culture
Authors: Alexandre Calon
Alexandre CalonAffiliation: Oncology Programme, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1329
Elisa Espinet
Elisa EspinetAffiliation: Oncology Programme, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1330
Sergio Palomo-Ponce
Sergio Palomo-PonceAffiliation: Oncology Programme, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1331
Daniele V. F. Tauriello
Daniele V. F. TaurielloAffiliation: Oncology Programme, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1332
Mar Iglesias
Mar IglesiasAffiliation: Department of Pathology, Hospital Universitari del Mar, Barcelona, Spain
Bio-protocol author page: a1333
María Virtudes Céspedes
María Virtudes CéspedesAffiliation: IIB Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
Bio-protocol author page: a1334
Marta Sevillano
Marta SevillanoAffiliation: Oncology Programme, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1335
Cristina Nadal
Cristina NadalAffiliation: Institut de Malalties Hemato-Oncològiques, Hospital Clínic, Barcelona, Spain
Bio-protocol author page: a1336
Peter Jung
Peter JungAffiliation: Oncology Programme, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1337
Xiang H.-F. Zhang
Xiang H.-F. ZhangAffiliation: Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, USA
Bio-protocol author page: a1338
Daniel Byrom
Daniel ByromAffiliation: Chemistry and Molecular Pharmacology Programme, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1339
Antoni Riera
Antoni RieraAffiliation: Chemistry and Molecular Pharmacology Programme, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1340
David Rossell
David RossellAffiliation: Biostatistics and Bioinformatics Unit, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1341
Ramón Mangues
Ramón ManguesAffiliation: IIB Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
Bio-protocol author page: a1342
Joan Massague
Joan MassagueAffiliation: Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, USA
Bio-protocol author page: a1343
Elena Sancho
Elena SanchoAffiliation: Oncology Programme, Institute for Research in Biomedicine, Barcelona, Spain
For correspondence: elena.sancho@irbbarcelona.org
Bio-protocol author page: a1344
 and Eduard Batlle
Eduard BatlleAffiliation: Oncology Programme, Institute for Research in Biomedicine, Barcelona, Spain
For correspondence: eduard.batlle@irbbarcelona.org
Bio-protocol author page: a1345
Vol 4, Iss 9, 5/5/2014, 1914 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1119

[Abstract] Metastasis depends on a gene program expressed by the tumor microenvironment upon TGF-beta stimulation. CRC (Colorectal cancer) cell lines did not induce robust stromal TGF-beta responses when injected into nude mice as shown by lack of p-SMAD2 accumulation in tumor-associated stromal cells. To enforce high TGF-beta signaling in xenografts, we engineered CRC cell lines to secrete active TGF-beta. Subcutaneous tumors obtained from HT29-M6TGF-β, KM12L4aTGF-β cells and SW48TGF-β cells contained abundant p-SMAD2+ stromal cells.

Materials and Reagents

  1. Paraffin sections (subcutaneous tumors samples or liver metastasis from nude mice respectively injected subcutaneously or intrasplenic with CRC cells)
  2. XILOL
    Note: Xylol also referred to as xylene or dimethylbenzene is a solvent used in histology as a clearing agent to remove paraffin from dried microscope slides prior to staining.
  3. MilliQ H2O
  4. Wash buffer (Dako, catalog number: K800721)
  5. Rabbit anti-P-Smad2 (Cell Signaling Technology, catalog number: 3108)
  6. BrightVision poly-HRP anti- Rabbit (Immunologic, catalog number: DPVR-110HRP)
  7. Envision FLEX antibody diluent (Dako, catalog number: K8006)
  8. Peroxidase Blocking Solution (Dako, catalog number: S202386)
  9. ImmPACT DAB (Vector Laboratories, catalog number: SK-4105)
  10. DPX mounting media (Sigma-Aldrich, catalog number: 06522)
  11. Hematoxylin
  12. Citrate buffer (pH 6) (see Recipes)

Equipment

  1. Oven
  2. Immunostaining apparatus
  3. Autoclave

Procedure

  1. Stove samples at 65 °C just before starting the immunostaining technique. Remove the samples from the oven when the wax present in sections is completely undone.
  2. De-waxing and rehydration: Place slides in a rack to perform following washes (bath).
    1. XILOL: 10 min
    2. XILOL: 10 min
    3. XILOL: 5 min
    4. 100% EtOH: 10 min 
    5. 100% EtOH: 5 min
    6. 96% EtOH: 5 min
    7. 90% EtOH: 10-15 times
    8. 80% EtOH: 10-15 times
    9. 70% EtOH: 10-15 times
    10. 50% EtOH: 10-15 times
    11. 25% EtOH: 10-15 times
    12. H2O MilliQ: 10-15 times
  3. Antigen retrieval.
    1. Citrate Buffer (pH 6)
    2. Time: 20 min autoclave (121 °C)
  4. 3 washes 5 min with 1 ml 1x wash buffer.
  5. Blocking endogenous peroxidase.
    1.  200 ml Peroxidase Blocking Solution
    2. Time: 10 min
  6. 3 washes 5 min with 1ml 1x wash buffer.
  7. Incubation with primary antibody.
    1. Antibody: Rabbit anti-P-Smad2
    2. Dilution 1/200 in Envision FLEX antibody diluent
    3. 200 µl/sample
    4. O/N 4 °C
  8. 3 washes 5 min with 1 ml 1x wash buffer.
  9. Incubation with antibody BrightVision.
    1.  Antiboby: BrightVision anti-Rabbit
    2. 150 µl/sample
    3. Time: 45 min at room temperature
  10. 3 washes 5 min with 1 ml 1x wash buffer.
  11. Revealed with ImmPACT DAB.
    1.  200 µl/sample
    2. Time: 10 min
  12. 3 washes 5 min with distilled water.
  13. Hematoxilin (1 ml) counterstaining, time: 2 min.
  14. Rinse in distilled water bath.
  15. Rinse in TAP water bath.
  16. Dehydration and mounting with DPX.

Representative data



Figure 1. p-SMAD2 staining (arrowhead) in liver metastasis generated after intrasplenic injection of CRC cells. E: epithelial cells, Str: stromal cells. Scale bars = 10 μm

Recipes

  1. Citrate buffer (pH 6)
    Citrate 5.8 g sodium citrate
    Set pH 6 with citric acid
    2 L MilliQ

Acknowledgments

A.C. and D.V.F.T hold a Juan de la Cierva postdoctoral fellowship, E.E. an FPI PhD fellowship (both from Spanish Ministry of Science and Innovation). This work has been supported by grants from Instituto de Salud Carlos III FEDER (RD09/0076/00036) and the “Xarxa de Bancs de tumors sponsored by Pla Director d’Oncologia de Catalunya (XBTC), to E.B. from the European Research Council (Starting grant - 208488) and Consolider programmes (MICINN), to E.S. and A.C. by the Spanish Ministry of Science and Innovation, to JM by NIH grant CA34610 and to RM by grants PS09/00965 (MICINN) and NanoCoMets (CIBERBBN).

References

  1. Calon, A., Espinet, E., Palomo-Ponce, S., Tauriello, D. V., Iglesias, M., Cespedes, M. V., Sevillano, M., Nadal, C., Jung, P., Zhang, X. H., Byrom, D., Riera, A., Rossell, D., Mangues, R., Massague, J., Sancho, E. and Batlle, E. (2012). Dependency of colorectal cancer on a TGF-beta-driven program in stromal cells for metastasis initiation. Cancer Cell 22(5): 571-584.


How to cite: Calon, A., Espinet, E., Palomo-Ponce, S., Tauriello, D. V., Iglesias, M., Céspedes, M. V., Sevillano, M., Nadal, C., Jung, P., Zhang, X. H., Byrom, D., Riera, A., Rossell, D., Mangues, R., Massague, J., Sancho, E. and Batlle, E. (2014). Immunostaining Protocol: P-Smad2 (Xenograft and Mice). Bio-protocol 4(9): e1119. DOI: 10.21769/BioProtoc.1119; Full Text



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