Early apoptotic responses in transgenic mouse mammary carcinoma for photodynamic therapy
Heinrich Walt1, Marius Nap2, Ann M Dorward3
1Research Division of Gynaecology, Department of Obstetrics and Gynaecology, University Hospital, Zurich, CH-8091, Switzerland.
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/cm(2) 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.
Insights
Photodynamic therapy (PDT) induces early and ongoing apoptosis in RAS-driven breast tumors, as detected by M30 staining. The M30/SPF ratio serves as a tumor dynamic index (TDI) to monitor PDT response.
Area of Science:
- Oncology
- Biomedical Engineering
- Cell Biology
Background:
- Male transgenic mice expressing the human RAS gene develop mammary adenocarcinoma.
- This model is utilized to study photodynamic therapy (PDT) responses.
Purpose of the Study:
- To investigate apoptotic responses in a mouse mammary tumor model following PDT.
- To evaluate the utility of M30 antibody and S-phase fraction (SPF) in monitoring PDT effects.
Main Methods:
- Apoptosis detection using M30 antibody against caspase-cleaved cytokeratin 18.
- PDT administered with a chlorin-based photosensitizer and laser light.
- Immunohistochemistry and flow cytometry for M30 expression and cell cycle analysis (SPF).
Main Results:
- M30 staining significantly increased within 2 hours and persisted up to 96 hours post-PDT.
- S-phase fraction (SPF) substantially decreased at 2 hours and recovered by 96 hours post-PDT.
- Distinct cellular dynamics observed at early and late time points post-PDT.
Conclusions:
- Cytokeratin 18 cleavage is an early and ongoing event during the cellular response to PDT.
- The M30/SPF ratio can serve as a tumor dynamic index (TDI) to monitor events post-PDT.
- This index offers insights into cellular dynamics following therapeutic interventions.
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