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Clinical Cancer Research Vol. 7, 1627-1632, June 2001
© 2001 American Association for Cancer Research

Regular Articles

Tumor Angiogenesis and Its Possible Role in Intravasation of Colorectal Epithelial Cells

Yu-Wen Tien, King-Jen Chang1, Yung-Ming Jeng, Po-Huang Lee, Ming-Shi Wu, Jaw-Town Lin and Su-Ming Hsu

Departments of Surgery [Y. W. T., K. J. C., P. H. L.], Pathology [Y. M. J., S. M. H.], and Gastroenterology [M. S. W., J. T. L.], National Taiwan University, Taipei 10002, Taiwan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: To determine whether an increase in tumor angiogenesis facilitates intravasation of colorectal epithelial cells, we compared intratumoral microvessel counts with the presence of circulating colorectal epithelial cells in the portal venous blood from patients with colorectal carcinomas.

Experimental Design: Circulating colorectal epithelial cells were detected by a reverse transcription-PCR assay to amplify guanylyl cyclase C (GCC) transcripts. The extent of tumor vascularization was quantitatively assessed by immunohistochemical staining with anti-CD31 antibody.

Results: Colorectal epithelial cells (as measured by GCC mRNA expression) were detected in the portal venous blood in 30 of 58 patients (52%). The mean (± SD) microvessel count in the tumors from patients with expression of GCC mRNA in their portal venous blood was 82.74 ± 24.97. The corresponding values in the tumors from patients without expression of GCC mRNA in portal venous blood was 65.96 ± 19. For each 10-microvessel increase per x200 field, the risk of colorectal epithelial cell presence in the portal venous blood increased 1.52-fold (95% confidence interval, 1.19–2.12; P = 0.005).

Conclusion: High intratumoral vessel count was noted to be a valuable factor for predicting the presence of colorectal epithelial cells in the portal venous blood.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Much experimental evidence indicates that tumor growth depends on angiogenesis (1) . Moreover, the extent of vascularization (as assessed by microvessel quantification immunohistochemically) in human colorectal tumor also was reported to be a significant predictor of an increased risk of hematogenous metastatic disease (2, 3, 4) . Most of these studies took (as end points) metastases in specific organs distant from the primary tumor, and the step(s) in the metastatic process (intravasation or steps after intravasation) that might be facilitated by increased tumor angiogenesis was not investigated.

To determine whether an increase in tumor vascularity increases the chance of colorectal epithelial cell entry into the blood stream, we looked for a correlation between the intratumoral microvessel count and the presence of circulating colorectal epithelial cells in the portal venous blood from patients with colorectal carcinomas.


   PATIENTS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients.
Informed consent was obtained from all patients, and the ethics committee of the National Taiwan University approved the study protocol.

From July 1998 to August 1999, 58 patients (30 males and 28 females; ages 28–89 years; mean age, 65 years) with histologically confirmed colorectal adenocarcinoma (31 patients with colon carcinoma and 27 patients with rectal carcinoma) treated at National Taiwan University Hospital were included. Tumor stage and grading were classified by the Astler-Coller system.

Blood and Tumor Sample Collection.
Immediately after entering the peritoneal cavity, before manipulation of the tumor, 10 ml of portal venous blood were collected through a catheter placed in the portal vein via the right gastroepiploic vein. The position of the catheter was confirmed with an intraoperative fluoroscope to avoid mispositioning of the catheter into the superior mesenteric vein. A piece at least 2 cm3 of freshly harvested tumor tissue from each resected specimen was snap frozen in liquid nitrogen at the time of operation and stored at -70°C.

Portal venous blood samples from 6 patients undergoing colorectal resection for benign disease were obtained as control. In addition, peripheral blood samples from 11 healthy volunteers were analyzed.

RNA Extraction.
Blood samples were diluted with 10 ml of PBS. After density centrifugation through Ficoll-Paque (Pharmacia; 30 min at 400 x g), peripheral blood mononuclear cells and any possible colorectal epithelial or tumor cells were harvested from the interface and washed twice in PBS.

RNA extractions of cultured colon cancer cells, tumor tissue, and peripheral blood mononuclear cells were performed with commercially available RNA extraction kits (Qiagen Bioscience Corp., Valencia, CA) in accordance with the recommendations of the manufacturer. The extracted RNA was treated with RNase-free DNase I for 15 min at 25°C to eliminate DNA contamination.

To monitor the RNA quality and performance of reverse transcription of all analyzed samples, we also ran a RT-PCR2 for actin with 1 ml of the mixture.

Nested Duplex RT-PCR.
Total RNA (2.0 µg) in a volume of 20 µl was denatured for 10 min at 70°C and quickly chilled on ice. The cDNA was synthesized in a total volume of 20 µl containing 4 µl of 5x first-strand buffer, 4 mM DTT, 400 units of Superscript II (all from Life Technologies, Inc.), 16 units of RNase inhibitor, 100 µM random hexamer, and 500 µmol/l of each deoxynucleotide triphosphate mix. The reaction mix was incubated for 60 min at 42°C. Reverse transcriptase was inactivated for 5 min at 95°C, and the mixture was stored at -80°C.

GCC primers designed by Carrithers et al. (5) were used to detect GCC mRNA. The first round of the nested PCR was performed using 4 µl of the cDNA from the reverse transcription. The PCR mix contained 0.2 µM each of GCC out primer (antisense, nucleotides 1197–1218; and sense, nucleotides 685–708), together with 10x PCR buffer and 200 µM deoxynucleotide. The PCR conditions were 95°C for 2 min, 1 cycle; 94°C for 30 s, 58°C for 30 s, and 72°C for 90 s, 35 cycles; and 72°C for 7 min, 1 cycle. The second PCR was performed with 10 µl of this reaction mixture, antisense (nucleotides 1000–1021) and sense (nucleotides 759–781) primers essentially as described above for the external PCR. RT-PCR products (30 µl) were analyzed by agarose gel (2.5%) electrophoresis and visualized by UV transillumination after staining with ethidium bromide (0.5 µg/ml). The nested GCC PCR yielded a 262-bp product. The amplified products were sequenced using the ABI Model 373A DNA Sequencer (Perkin-Elmer Biosystem, Foster City, CA) as specified by the manufacturer. The DNA sequences were aligned and analyzed using an Acer computer. For each sample, both negative and positive controls were included. The negative control contained all components of the RT-PCR reaction but no target RNA. The positive control was RNA extracted from CCL 220 cells.

Measurement of Sensitivity of Nested GCC RT-PCR.
The sensitivity of this technique was determined by supplementing normal peripheral blood samples with the colon cancer cell line (CCL 220). Tumor cells (0, 1, 10, 102, 103, and 104) were added to 10 ml of peripheral blood samples from healthy donors, and then these blood samples were analyzed by GCC RT-PCR assays.

Immunohistochemistry.
All tissue samples had been fixed in 10% buffered formalin and routinely processed for light microscopy. Sections (4-mm thick) were dewaxed in xylene and dehydrated in ethanol. The sections were predigested with protease for 20 min at 37°C and then immersed in 3% H2O2 for 30 min to inhibit endogenous peroxidase. After washing with PBS, they were incubated in normal rabbit serum for 30 min, followed by incubation overnight with anti-CD31 monoclonal antibody (Union Biotech, Inc.) at a 1:50 dilution. The sections were then incubated with biotinylated rabbit antimouse IgG for 15 min. Peroxidase-conjugated avidin was used at a dilution of 1:500. After washing in PBS, the slides were developed by immersing into 0.01% H2O2 and 0.05% diaminobenzidine tetrahydrochloride for 2 min. Normal mouse IgG was substituted for the primary antibody in the negative control. The sections were counterstained with hematoxylin. After staining, blood vessels appeared intensely brown in color, which facilitated identification and quantification. Representative sections of primary colorectal tumors stained for CD31 are seen in Fig. 1Citation .



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  		Fig. 1. Immunohistochemical staining for CD31 of primary colorectal carcinoma. Microvessels are represented by brown capillaries or clusters, which stand out sharply from the other tissue.

 
Vascular Counting.
Slides were examined at low power magnification (x40 and x100) to identify the areas of highest vessel density. For each slide, the three most vascular areas within the tumor mass were chosen. A x200 field in each of these three areas was counted. The average counts of the three fields were recorded. A vessel lumen was not required for identification of a microvessel; single cell or cell clusters were counted. Two pathologists, without knowledge of the corresponding clinicopathological data, each counted all of the immunostained slides.

Statistical Analysis.
The Statistical Analysis System for personal computers (SAS Institute, Inc.) was used for all analyses. P < 0.05 was considered statistically significant. Because the outcome of interest in our study, intravasation, was either present or absent, we used logistic regression as outlined by Cox. We looked for associations between the presence or the absence of GCC mRNA expression in portal venous blood and clinicopathological factors, such as microvessel count, tumor grade, tumor size, nodal status, lymphatic invasion, and vascular invasion. This method provides odds ratios or estimates of the relative risk of GCC mRNA expression in portal venous blood. We first examined univariate relations to determine which factors were related to intravasation and next performed stepwise multivariate logistic regression to determine whether some combination of variables provided a better estimate of the relative risk of presence of intravasated, GCC mRNA-positive cells than any single variable.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
No catheter-related complication was noted in any of the 58 patients.

Sensitivity and Specificity of GCC RT-PCR.
A 262-bp PCR product was detected in samples containing 10 or more CCL 220 cells in 10 ml of blood (Fig. 2)Citation . This fragment had the expected size delineated from the position of the chosen primers.



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  		Fig. 2. Upper panel, GCC RT-PCR assay of 104, 103, 102, 10, 1, and 0 cancer cells of the CCL 220 cell line in 10 ml of peripheral blood obtained from a healthy volunteer. GCC mRNA of 10 cancer cells in 10 ml blood could be detected. As shown in the lower panel, for each sample, actin cDNA was amplified in parallel. Lane M, molecular weight marker.

 
The 262-bp products from 2 tumor, 2 portal venous blood, and 1 cell line specimens were sequenced and were identical with the GCC sequences published by de Sauvage et al. (6) . GCC transcripts were detected in all of the tumor specimens and thus confirmed the presence of GCC-expressing cells in all tumors. None of the peripheral blood samples from 11 healthy volunteers or portal venous blood samples from 6 patients undergoing colorectal resection for benign disease was positive for GCC mRNA.

Patient Specimens.
GCC transcripts were detected in portal venous blood in 34 of 58 patients (59%; Table 1Citation ). There was a significant difference in GCC RT-PCR-positive expression between patients with (10 of 11; 91%) and without (20 of 47; 43%; P = 0.001; Table 1Citation ) liver metastasis.


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  		Table 1 Correlation between GCC mRNA-positive cell detection rate in portal venous blood and colorectal tumor stage

 
MVD and Clinicopathological Findings.
Considerable intratumor heterogeneity was observed in the distribution of stained microvessels, and mean MVD for anti-CD31 antibody in this study was 75.79 ± 24.

Table 2Citation shows the correlation between MVD and major clinicopathological variables. There were no statistically significant associations between MVD and clinicopathological factors, such as sex, age, tumor size, histological type, lymphatic invasion, or depth of invasion. However, MVD was significantly related to lymph node metastasis, venous invasion, tumor stage, and liver metastasis (P = 0.007, P = 0.005, P = 0.002, and P = 0.008, respectively).


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  		Table 2 The clinicopathologic findings and MVD of colorectal cancer

 
MVD and Expression of GCC mRNA in Portal Venous Blood.
Table 3Citation shows that in the colorectal carcinomas of the patients with GCC mRNA expression in portal venous blood, the mean microvessel count (in areas of highest neovascularization) was 83 per x200 field. In the carcinomas of the patients without GCC mRNA expression, the corresponding value was 66 per x200 field. Univariate analysis revealed the differences between the groups to be statistically significant (P = 0.005). Furthermore, with each 10-microvessel increase in the count at x200, there was a 1.59-fold increase in the risk of expression of GCC mRNA in portal venous blood (95% confidence interval, 1.19–2.12; P = 0.005; Table 3Citation ). We plotted the percentage of patients with GCC mRNA expression in portal venous blood against the microvessel count as it progressively increased by 40-vessel increments (Fig. 3)Citation . The plot showed that the prevalence of GCC mRNA expression increased as the vessel count increased, reaching 77% among patients with carcinomas that had vessel counts of >80 per x200 field.


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  		Table 3 Comparison of clinicopathological features between patients with and without GCC mRNA-positive cells in portal venous blood

 


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  		Fig. 3. The prevalence of expression of GCC mRNA in portal venous blood increased as vessel counts of tumors increased.

 
We next used stepwise multivariate logistic regression to determine whether some combination of variables would provide a better estimate of relative risk of expression of GCC mRNA in portal venous blood than any single variable. By itself, microvessel count provided the best estimate.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The most fearsome aspect of cancer is metastasis. The metastatic spread of cancer cells from a primary tumor to distant sites in the body is responsible for most cancer patient morbidity and mortality. For a tumor cell to metastasize, it must breach a series of barriers (extracellular matrix and vessel wall) to gain access to the vasculature from the primary tumor, exit from the circulation (extravasation), and establish secondary growth (7 , 8) . Studies using the light microscope and immunohistochemistry have concluded that the number and density of microvessels in human colorectal cancer directly correlate with their potential to produce hematogenous metastasis (2, 3, 4) .

Explanations for this angiogenesis-enhanced metastatic mechanism included: (a) the increase in tumor vessels increases the chance for tumor cell entry into the circulation; (b) newly formed vessels or capillaries have leaky and weak basement membranes that tumor cells can penetrate more easily than those of mature vessels (9) ; (c) escape of tumor cells into the tumor neovasculature is facilitated by the degradative enzymes secreted by the endothelial cells at the tips of growing capillaries (10) ; and (d) angiogenic tumor cells, which have greater opportunity to survive and multiply at the site of metastasis, are more likely to arise from a highly angiogenic primary tumor (11 , 12) . Almost all of these conclusions were obtained by associating the number or the characteristics of newly formed vessels in the primary tumor with the risk of metastasis formation, but whether neovascularization enhances the intravasation of colorectal cancer cells remains unknown.

It has been well demonstrated in animal models that most radiolabeled tumor cells, injected into a vein, are trapped in the capillary beds of the first target organ, and few are detectable in the peripheral blood (13 , 14) . Theoretically, intravasated colorectal tumor cells would be more frequently detected in portal rather than peripheral veins. Thus, detection of circulating tumor cells in portal venous blood may give a more accurate indication of intravasated colorectal tumor cells and seemed to be more suitable for this study.

PCR-based assays of mutated DNA or tissue-specific RNA were reported to be highly sensitive methods for detecting circulating tumor cells. However, DNA-targeted PCR assay may detect DNA derived from degraded instead of viable tumor cells (15) . The advantage of RNA identification is that it implies that the RNA is intact (extracellular RNA is rapidly degraded if it is not within an intact cell) and functional (only viable cells produce the coded-for protein). Therefore, an RNA-targeted PCR assay was selected as the most appropriate assay to detect intravasated viable tumor cells with metastatic potential. Detection of CK-20 mRNA (16 , 17) or CEA mRNA (18 , 19) has been used for the detection of circulating viable colorectal tumor cells. Because of expression in the epidermal Merkel cells and granulocytes, CK-20 mRNA transcripts were reported to be detected in a significant portion of blood from healthy volunteers (20 , 21) . Similarly, application of CEA RT-PCR was also hindered by a wide variation in mRNA expression level and expression in blood cells from patients without cancers (22 , 23) . In contrast to CK-20 and CEA, GCC (a member of the guanylyl cyclase family of the receptors) is specifically expressed only in intestinal mucosa cells, and this expression persists once those cells undergo neoplastic transformation (5) . Therefore, a GCC RT-PCR assay was chosen to detect circulating colorectal tumor cells. This technique proved to be extremely sensitive, allowing detection of 10 cancer cells in 10 ml of blood, and gave evidence of GCC mRNA-positive cells in the portal venous blood of 30 of 58 patients (52%).

Monoclonal antibody against CD31 was used to identify blood vessels in this study. The quantification of blood vessels within colorectal tumors showed no significant association with tumor size, histological type, or depth of invasion. However, a significant correlation was found between MVD in histological sections of colorectal carcinoma stained for CD31 and the presence of GCC mRNA in portal venous blood. The mean microvessel count in the colorectal carcinomas of the patients with GCC mRNA expression in portal venous blood was significantly higher than that in the carcinomas of the patients without GCC mRNA expression in portal venous blood (P = 0.005). Among the five patients with a microvessel count of up to 40 per x200 field (i.e., carcinomas with slight neovascularization), 1 patient (20%) had GCC mRNA expression in portal venous blood. As the microvessel count increased, the prevalence of GCC mRNA-positive cells in portal venous blood increased. The prevalence of patients with GCC mRNA expression and counts of 41–80 microvessels and of those with GCC mRNA expression and counts of more than 80 microvessels was 39 and 77%, respectively. An increase in tumor vascularity thus does increase the GCC mRNA-positive cell detection in the portal venous blood.

The presence of intravasated cancer cells in portal venous blood does not necessarily predict subsequent liver metastasis. Experimental studies have shown that only ~0.01% of cancer cells injected into the circulation formed metastatic foci (13 , 14) . However, animal studies also showed that the risk of metastatic formation was proportional to the amount of injected tumor cells (14) . Our results did prove that circulating colorectal epithelial cells could be detected more frequently in portal venous blood from patients with tumors of high vascularity. However, the clinical significance of detection of circulating colorectal epithelial cells in portal venous blood awaits further longitudinal studies.

In conclusion, we have detected circulating colorectal epithelial cells in portal venous blood by a GCC-specific nested RT-PCR and investigated the correlation with tumor vascularity. The current result proved that circulating colorectal epithelial cells in portal venous blood are significantly more frequently detected in patients with hypervascular tumors than in patients with hypovascular tumors. Vascularity of tumor was noted to be the most valuable predictor of the presence of circulating colorectal epithelial cells in portal venous blood.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Surgery, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei 10002, Taiwan. Phone: 886-2-23123456, extension 5106; Fax: 886-2-23568810; E-mail: ywt5106{at}ha.mc.ntu.edu.tw Back

2 The abbreviations used are: RT-PCR, reverse transcription-PCR; GCC, guanylyl cyclase C; MVD, microvessel density; CK, cytokeratin; CEA, carcinoembryonic antigen. Back

Received 12/ 2/00; revised 2/20/01; accepted 3/ 6/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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