Early apoptotic responses in transgenic mouse mammary carcinoma for photodynamic therapy

Walt, Heinrich; Nap, Marius; Dorward, Ann M.; Leers, Mathie P.G.; Tennent, Barbara J.; Varga, Zsuzsanna; Stallmach, Thomas; Björklund, Viveka; Beamer, Wesley G.

doi:10.1016/j.pdpdt.2006.07.002

« Previous Next »Photodiagnosis and Photodynamic Therapy
Volume 3, Issue 4 , Pages 227-233, December 2006

Early apoptotic responses in transgenic mouse mammary carcinoma for photodynamic therapy

Heinrich Walt, PhD
- Research Division of Gynaecology, Department of Obstetrics and Gynaecology, University Hospital, Zurich, CH-8091, Switzerland
- Corresponding author. Tel.: +41 1 255 5372; fax: +41 1 255 4553.
Marius Nap
- Department of Pathology, Atrium Heerlen Medical Centre, Heerlen, CX, 4446 6401, The Netherlands
Ann M. Dorward
- The Jackson Laboratory, Bar Harbor, ME 04609, USA
Mathie P.G. Leers
- Department of Pathology, Atrium Heerlen Medical Centre, Heerlen, CX, 4446 6401, The Netherlands
Barbara J. Tennent
- The Jackson Laboratory, Bar Harbor, ME 04609, USA
Zsuzsanna Varga
- Department of Pathology, University Hospital, Zurich, CH-8091, Switzerland
Thomas Stallmach
- Department of Pathology, University Hospital, Zurich, CH-8091, Switzerland
Viveka Björklund
- Cancer Council, Bromma, SE 16762, Sweden
Wesley G. Beamer
- The Jackson Laboratory, Bar Harbor, ME 04609, USA

published online 18 September 2006.

Summary

Background

Male transgenic mice expressing the human RAS gene on an FVB strain background develop adenocarcinoma of the breast between 7 and 8 weeks of age. We have utilized this mammary tumour model to investigate apoptotic responses following photodynamic therapy (PDT) with a chlorin-based, water-soluble photosensitizer.

Methods

Detection of apoptosis was accomplished by use of the antibody M30 against a neo-epitope of caspase-cleaved cytokeratin 18 that becomes available at an early stage of the apoptotic cascade. Mice bearing multiple tumours were injected with the photosensitizer intraperitoneally, and following a drug-light interval of 96h, 40J/cm² of 652nm laser light was applied to one tumour per animal, while the other tumours were protected from light to serve as host controls. The M30 antibody was used for standard immunohistochemistry of tumour sections and flow cytometric detection of epitope expression coupled to cell cycle analysis in tumour cell populations retrieved from paraffin blocks.

Results

M30 staining was significantly increased within 2h following light treatment and persisted until 96h after treatment. Flow cytometric analysis for the S-phase fraction (SPF) of tumour cells post-PDT showed a substantial decrease in SPF at 2h post PDT, and recovery of SPF within 96h.

Conclusions

Cytokeratin 18 cleavage seems to be both an early and ongoing event during the cellular response to PDT. Calculating the M30/SPF ratio at both 2h and 96h suggested distinct cellular dynamics at early and late time points, and we propose the M30/SPF ratio as a tumour dynamic index (TDI) to monitor events post PDT.

Keywords: Photodynamic therapy, Apoptosis, Multiparameter flow cytometry, Tumour dynamic index, Mouse mammary tumours