Quantitative analysis of chemotherapeutic effects in tumors using in vivo staining and correlative histology.

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 approximately 2 :1 where the corresponding area observed on Cy5.5 distribution measurements revealed a approximately 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.

Imaging of differential protease expression in breast cancers for detection of aggressive tumor phenotypes.

PURPOSE: To determine if different expression levels of tumor cathepsin-B activity in well differentiated and undifferentiated breast cancers could be revealed in vivo with optical imaging. MATERIALS AND METHODS: A well differentiated human breast cancer (BT20, n = 8) and a highly invasive metastatic human breast cancer (DU4475, n = 8) were implanted orthotopically in athymic nude mice. Tumor-bearing animals were examined in vivo with near-infrared fluorescence (NIRF) imaging 24 hours after intravenous injection of an enzyme-sensing imaging probe. Immunohistochemistry, Western blotting (on cells and whole tumor samples), and correlative fluorescence microscopy were performed. RESULTS: Both types of breast cancers activated the NIRF probe so that tumors became readily detectable. However, in tumors of equal size, there was a 1.5-fold higher fluorescence signal in the highly invasive breast cancer (861 arbitrary units +/- 88) compared with the well differentiated lesion (566 arbitrary units +/- 36, P <.01). Western blotting confirmed a higher cathepsin-B protein content in the highly invasive breast cancer (DU4475) of about 1.4-fold (whole tumor samples) to 1.7-fold (cells). Immunohistochemistry and fluorescence microscopy findings confirmed the imaging findings. CONCLUSION: Cathepsin-B enzyme activity can be determined in vivo with NIRF optical imaging, while differences in tumoral expression may correlate with tumor aggressiveness.

Steady-state blood volume measurements in experimental tumors with different angiogenic burdens a study in mice.

PURPOSE: To experimentally validate the effectiveness of magnetic resonance (MR) imaging enhanced with long-circulating iron oxide for measurement of vascular volume fractions (VVFs) as indicators of angiogenesis in different experimental tumor models. MATERIALS AND METHODS: Tumors with differing degrees of angiogenesis-9L rodent gliosarcoma, DU4475 human mammary adenocarcinoma, HT1080 human fibrosarcoma, and EOMA hemangioendothelioma--were implanted in nude mice. Tumoral VVFs were measured at submillimeter voxel resolutions by using 1.5-T MR imaging. A technetium-labeled intravascular radiotracer was injected into a subset of the animals to validate the MR imaging measurements. Microvessel density and vascular endothelial growth factor (VEGF) also were measured. Statistical analysis was performed with analysis of variance. RESULTS: High-resolution multisection MR maps of tumor blood volume were obtained in all tumor models. Mean tumoral VVF differed significantly among the different tumors: 2.1% +/- 0.3 (standard error of mean) for 9L gliosarcoma, 3.1% +/- 0.4 for DU4475 mammary adenocarcinoma, 5.5% +/- 0.8 for HT1080 fibrosarcoma, and 6.6% +/- 0.9 for EOMA hemangioendothelioma (P <.01). There was a strong correlation between the MR imaging and radiotracer measurements. There was considerable intra- and intertumoral heterogeneity among the VVFs. MR imaging measurements were in accordance with conventional measurements of angiogenesis, such as microvessel density count and VEGF. CONCLUSION: Measurements of tumoral VVF at high-resolution MR imaging with long-circulating iron oxide are feasible and correlate with angiogenic burden in experimental tumor models.