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

Abstract

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.

Figure 1

RCA lectin in vivo labeling is an effective way to mark functional angiogenic vessels. SCCVII tumors were systemically labeled with Rhodamine-RCA Lectin and 7 μm tissue sections were counterstained with DAPI and observed under 40× objective upright microscope (Zeiss, USA) through Rhodamine and DAPI filters. (scale bar = 50 um) A: Endothelial cell nuclei staining pattern observed with bright blue DAPI counterstain in SCCVII neoplasm. B: Functional angiogenic vessels labeled by tail vein injection of Rd-RCA lectin. RCA-I is able to successfully stain endothelial cells due to the specific affinity to beta D-galactose which is found on the luminal surface of blood vessels. C: Merged signals of vessels and the cell nuclear staining by DAPI. The abundance of nuclei present in the tissue is not representative of the functional and accessible tumor vasculature in the merged image.

Figure 2

Successful Rd-RCA I lectin guided capture of endothelial cells under inverted 40× magnification. A: Fluorescent labeled angiogenic and functional vessels are distinguished from the surrounding epithelium prior to performing LCM. B: A low-powered infrared laser was pulsed over the targeted cells, which expanded the film on the cap and extracted the entire vessel. C: An intact vessel containing a homogeneous cell population generated for molecular analysis. The presence of RCA-Rhodamine labeled endothelial cells on the "cap" image and the absence of the targeted cells in the "after" microdissection image confirmed successful capture. A power setting of 40 mW and duration of 450 μs were utilized to obtain a 5.0–6.0 μm diameter laser spot size, which permitted precise selection of endothelium using the Pix Cell II Instrument.

Figure 3

Assessment of RNA integrity in frozen sections of mouse SCCVII tumor tissue. The Agilent BioAnalyzer system determined that the RNA from two-round amplification of endothelial and epithelial cell samples had comparable concentration of 1278.5 ng/μl. The spectra clearly indicated the presence of a marker peak and a broad hump in the electropherogram confirming good RNA quality. Further mRNA analysis demonstrated a high quality starting sample of total RNA by the 260/280 ratio for the five endothelial and epithelial cell samples. Moreover, the RNA quality and concentration was comparable to that of the control sample.

Figure 4

The purity of endothelial cell RNA pool is measured by RT-PCR amplification of endothelial cell marker CD31, squamous cell carcinoma epithelial specific gene Keratin 19 as well as the Beta-Actin housekeeping gene from LCM samples. Approximately 200 endothelial cells as well as control epithelial cells were microdissected and captured from each sample RNA was extracted and then amplified. In order to verify the quality and purity of the LCM endothelial RNA samples, custom designed primers for house keeping gene Beta-Actin, CD31D, and two pairs of Keratin 19 (KER19A, KER19 B) were applied to the pool by RT-PCR. The results indicated that the endothelial pool does not contain Keratin 19 mRNA, which is expressed in the epithelial pool.

Figure 5

RT-PCR amplification of housekeeping genes and CD31 endothelial cell markers from LCM derived cells. About 200 endothelial cells were microdissected and captured, the RNA was extracted and amplified. After first strand cDNA synthesis, primers designed for murine CD31 RT-PCR mCD31a: 5'TTCTGAACTCCAACAGCGAG-3', 5'-AGGCTCAAGGGAGGACACTT-3'; mCD31b: 5'-TGCCGAAGGCCCAAAGAAGA-3', 5'GCTCAGACCTTAGGAAACCG-3, GAPDH and β-Actin were detected by PCR.

Figure 6

Integrin Beta 3 and CD31 are highly expressed in the SCCVII tumor angiogenic vessels detected by RT-PCR from laser capture microdissected endothelial cells and confirmed by immunohistochemical staining of Integrin Beta-3. Integrin immunohistochemistry on paraffin embedded 7 μm sections were stained with mouse monoclonal antibodies from BD-Pharmingen, USA at a dilution of 1:200. Positive CD31 and Beta 3 showed dark brown and intense orange-brown staining, respectively. Each tumor section was counterstained with hemotoxylin.