Early apoptotic responses in transgenic mouse mammary carcinoma for photodynamic therapy

SummaryBackgroundMale 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.MethodsDetection 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 96 h, 40 J/cm2 of 652 nm 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.ResultsM30 staining was significantly increased within 2 h following light treatment and persisted until 96 h after treatment. Flow cytometric analysis for the S-phase fraction (SPF) of tumour cells post-PDT showed a substantial decrease in SPF at 2 h post PDT, and recovery of SPF within 96 h.ConclusionsCytokeratin 18 cleavage seems to be both an early and ongoing event during the cellular response to PDT. Calculating the M30/SPF ratio at both 2 h and 96 h 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.