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Abstract
Aims: To microscopically analyze the chemotherapeutic response of tumors using in vivo staining based on an annexinV-Cy5.5 probe and independently asses their apoptotic count using quantitative histological analysis. Methods: Lewis Lung Carcinomas cells, that are sensitive (CS-LLC) and resistant (CR-LLC) to chemotherapy were implanted in nude mice and grown to tumours. Mice were treated with cyclophosphamide and injected with a Cy5.5-annexinV fluorescent probe. In vivo imaging was performed using Fluorescence Molecular Tomography. Subsequently tumours were excised and prepared for histology. The histological tumour sections were stained for apoptosis using a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. A minimum of ten tissue sections were analyzed per tumour for apoptosis quantification by TUNEL staining and corresponding Cy5.5 distribution. Results: We detected higher levels of apoptosis and corresponding higher levels of Cy5.5 fluorescence in the CS-LLC vs. the CR-LLC tumours. The cell count rate on CS-LLC sections over CR-LLC was found to be ∼2 :1 where the corresponding area observed on Cy5.5 distribution measurements revealed a ∼1.7 :1 ratio of CS-LLC over CR-LLC. These observations are consistent with the higher apoptotic index expected from the CS-LLC cell line. Conclusions: Quantitative analysis of histological slices revealed higher fluorescence and higher apoptotic count in the CS-LLC tumour images compared to the CR-LLC tumour images. These observations demonstrate that the annexinV-Cy5.5 probe sensed the chemotherapeutic effect of cyclophospamide and further confirmed in vivo FMT measurements.
Highlights
Non-invasive fluorescence imaging has emerged in recent years as an important in vivo method for smallanimal research
The p-values for the mean number of objects found for mouse 1 (M1) Cyclophosphamide-sensitive Lewis lung carcinoma (CS-LLC) vs. cyclophosphamide-resistant Lewis lung carcinoma (CR-LLC) was p < 0.0009, for mouse 2 (M2) CS-LLC vs. CR-LLC it was p < 0.0001, and for mouse 3 (M3) CS-LLC vs. CR-LLC it was p < 0.0001 in the Cy5.5 channel and M1: p < 0.4625, M2: p < 0.0001, and M3: p < 0.0001 for the Cy3 channel
The results indicate significantly higher fluorescence both distribution and intensity from the CS-LLC compared to the CR-LLC as follows; On an average there were significantly higher number of objects observed (840 vs. 174) and higher total area observed (1.66 : 1) between the CS-LLC and CR-LLC respectively
Summary
Non-invasive fluorescence imaging has emerged in recent years as an important in vivo method for smallanimal research This development has followed after the emergence of bio-compatible fluorochromes with molecular specificity that serve as reporter molecules in vivo in a variety of biotechnology applications [13, 14,27]. In-order to accurately correlate FMT findings with the underlying micro-distribution it is important to obtain multiple sections through the volume of interest and extract average fluorescence values. Besides using this approach for validating FMT observations with the underlying histology, average histological analysis can be used to summarize the histological appearance of different lesions for comparison purposes and yield concise descriptive measures of the underlying biology.
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