Rhodamine-RCA in vivo labeling guided laser capture microdissection of cancer functional angiogenic vessels in a murine squamous cell carcinoma mouse model.

BACKGROUND: Cancer growth, invasion and metastasis are highly related to tumor-associated neovasculature. The presence and progression of endothelial cells in cancer is chaotic, unorganized, and angiogenic vessels are less functional. Therefore, not all markers appearing on the chaotic endothelial cells are accessible if a drug is given through the vascular route. Identifying endothelial cell markers from functional cancer angiogenic vessels will indicate the accessibility and potential efficacy of vascular targeted therapies. RESULTS: In order to quickly and effectively identify endothelial cell markers on the functional and accessible tumor vessels, we developed a novel technique by which tumor angiogenic vessels are labeled in vivo followed by Laser Capture Microdissection of microscopically isolated endothelial cells for genomic screening. Female C3H mice (N = 5) with established SCCVII tumors were treated with Rhodamine-RCA lectin by tail vein injection, and after fluorescence microscopy showed a successful vasculature staining, LCM was then performed on frozen section tissue using the PixCell II instrument with CapSure HS caps under the Rhodamine filter. By this approach, the fluorescent angiogenic endothelial cells were successfully picked up. As a result, the total RNA concentration increased from an average of 33.4 ng/ul +/- 24.3 (mean +/- S.D.) to 1913.4 ng/ul +/- 164. Relatively pure RNA was retrieved from both endothelial and epithelial cells as indicated by the 260/280 ratios (range 2.22-2.47). RT-PCR and gene electrophoresis successfully detected CD31 and Beta-Actin molecules with minimal Keratin 19 expression, which served as the negative control. CONCLUSION: Our present study demonstrates that in vivo Rhodamine RCA angiogenic vessel labeling provided a practical approach to effectively guide functional endothelial cell isolation by laser capture microdissection with fluorescent microscopy, resulting in high quality RNA and pure samples of endothelial cells pooled for detecting genomic expression.

Pulsed high-intensity focused ultrasound enhances systemic administration of naked DNA in squamous cell carcinoma model: initial experience.

PURPOSE: To determine whether exposures to pulsed high-intensity focused ultrasound can enhance local delivery and expression of a reporter gene, administered with systemic injection of naked DNA, in tumors in mice. MATERIALS AND METHODS: The study was performed according to an approved animal protocol and in compliance with guidelines of the institutional animal care and use committee. Squamous cell carcinoma (SCC7) tumors were induced subcutaneously in both flanks of female C3H mice (n = 3) and allowed to grow to average size of 0.4 cm(3). In each mouse, one tumor was exposed to pulsed high-intensity focused ultrasound while a second tumor served as a control. Immediately after ultrasound exposure, a solution containing a cytomegalovirus-green fluorescent protein (GFP) reporter gene construct was injected intravenously via the tail vein. The mouse was sacrificed 24 hours later. Tissue specimens were viewed with fluorescence microscopy to determine the presence of GFP expression, and Western blot analysis was performed, at which signal intensities of expressed GFP were quantitated. A paired Student t test was used to compare mean values in controls with those in treated tumors. Histologic analyses were performed with specific techniques (hematoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) to determine whether tumor cells had been damaged by ultrasound exposure. RESULTS: GFP expression was present in all sections of tumors that received ultrasound exposure but not in control tumors. Results of signal intensity measurement at Western blot analysis showed expressed GFP to be nine times greater in ultrasound-exposed tumors (160.2 +/- 24.5 [standard deviation]) than in controls (17.4 +/- 11.8) (P = .004, paired Student t test). Comparison of histologic sections from treated tumors with those from controls revealed no destructive effects from ultrasound exposure. CONCLUSION: Local exposure to pulsed high-intensity focused ultrasound in tumors can enhance the delivery and expression of systemically injected naked DNA.