Imperial College London
Portrait Picture

ProfessorIainMcNeish

Faculty of Medicine, Department of Surgery & Cancer

Chair in Oncology
 
 
 
//

Contact

 

+44 (0)20 7594 2185i.mcneish Website

 
 
//

Assistant

 

Ms Sophie Lions +44 (0)20 7594 2792

 
//

Location

 

G036Institute of Reproductive and Developmental BiologyHammersmith Campus

//

Summary

 

Publications

Citation

BibTex format

@article{Spear:2019:10.3791/60622,
author = {Spear, S and McNeish, IA and Capasso, M},
doi = {10.3791/60622},
journal = {Journal of Visualized Experiments},
pages = {1--11},
title = {Generation of orthotopic pancreatic tumors and ex vivo characterization of tumor-infiltrating T cell cytotoxicity.},
url = {http://dx.doi.org/10.3791/60622},
volume = {154},
year = {2019}
}
Download

RIS format (EndNote, RefMan)

TY  - JOUR
AB  - In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response. This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 µL basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFNγ and TNFα) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFNγ+, TNFα+ and CD107a+ CD4+ and CD8+
AU  - Spear,S
AU  - McNeish,IA
AU  - Capasso,M
DO  - 10.3791/60622
EP  - 11
PY  - 2019///
SN  - 1940-087X
SP  - 1
TI  - Generation of orthotopic pancreatic tumors and ex vivo characterization of tumor-infiltrating T cell cytotoxicity.
T2  - Journal of Visualized Experiments
UR  - http://dx.doi.org/10.3791/60622
UR  - https://www.ncbi.nlm.nih.gov/pubmed/31868177
UR  - https://www.jove.com/video/60622/generation-orthotopic-pancreatic-tumors-ex-vivo-characterization
UR  - http://hdl.handle.net/10044/1/75797
VL  - 154
ER  -
Download