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© 2005 American Society for Nutrition J. Nutr. 135:2719-2722, November 2005

Symposium

Exfoliated Colonic Epithelial Cells: Surrogate Targets for Evaluation of Bioactive Food Components in Cancer Prevention1,2

Alka Kamra*,{dagger}, George Kessie*, June-Home Chen*, Shilpa Kalavapudi*, Robert Shores*,{dagger}, Ibtihal McElroy*, T. Gireesh**, P. R. Sudhakaran**, Sudhir K. Dutta{dagger} and Padmanabhan P. Nair*,{ddagger},3

* NonInvasive Technologies, Elkridge, MD 21075; {dagger} Division of GI Research, Sinai Hospital, Baltimore, MD 21215; ** Department of Biochemistry, University of Kerala, Kariavattom, Trivandrum, 695581, Kerala, India; and {ddagger} Division of Human Nutrition, Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205

3To whom correspondence should be addressed. E-mail: ppn{at}noninvasivetech.com.

ABSTRACT

There is significant evidence supporting the hypotheses that lifestyle, diet, and bioactive components in foods are important modifiers of cancer risk. However, our ability to assess host response noninvasively is limited. To overcome this, we have developed a technology to isolate several million viable exfoliated somatic colonic cells from a small sample of stool (0.5–1.0 g) by a procedure known as somatic cell sampling and recovery (SCSR). Orally administered carotenoids appear in these cells several days after consuming the supplement, usually showing a peak concentration between 5–7 d after their ingestion. The time lag observed for the appearance of orally administered carotenoids in SCSR cells corresponds to the turnover rate of the colonic mucosa. This is an example of how changes in cell turnover rates can be carefully assessed using the SCSR system. The specific mechanisms by which individual constituents of diet affect the cancer process are not fully understood. However, host response to dietary constituents may be investigated, noninvasively, by following the modulation of tumor-associated molecular markers in these exfoliated SCSR cells. We have demonstrated the feasibility of using SCSR cells to detect the expression of carcinoembryonic antigen, CD44, and its splice variants, c-myc, c-erbb2, and mutations in the p53 gene. In this regard, SCSR cells are a readily available surrogate cellular target that may serve to monitor changes in cell turnover, differentiation, and expression of cancer-associated biomarkers that are likely to be modulated by bioactive food components.

KEY WORDS: • exfoliated colonocytes • cancer prevention • diet • RT-PCR

There is a large body of evidence to support hypotheses relating diet and dietary components in the modulation of cancer risk. Bioactive components from dietary sources may be involved in modulating numerous biological processes related to carcinogenesis, including the modification of epigenetic events leading to malignant transformation. To monitor their action at the cellular level in interventional studies, it is essential to have a system for the serial sampling of viable somatic cells. This is essential to assess the absorption, retention, and bioactivity of dietary components on genetic elements associated with control of cellular proliferation.

Exfoliated cells from the colonic mucosa as surrogate cellular targets

The epithelium of the gastrointestinal tract undergoes constant and rapid renewal (1). These terminally differentiated cells that are exfoliated into the fecal stream represent an important source of informational macromolecules providing a pathophysiological profile of the colonic epithelium. Their life span of 3 to 4 d and a mean generation time of about 1 d accounts for the rapid turnover of this cell population (2,3). It is estimated that the normal colonic epithelium contains 5 x 1010 cells and that one-sixth to one-third of these cells are shed every 24 h, giving rise to ~1010 exfoliated cells per day (4,5). A tumor of 1 cm (3) may contain more that 109 cells, and they turnover at similar or accelerated rates compared with normal cells (6,7). More than 1% of the epithelial cells shed from the colon into the stools is estimated to be derived from tumors of this size. These cells are thought to be more resistant to degradative processes such as apoptosis, which degrade DNA in normal terminally differentiated epithelial cells (8).

Coprocytobiology: a novel concept

As an excretory product of the digestive system, stool is a mixture of cellular, microbial, protozootic, and viral components, mixed with remnants of the diet and secretory products from the gastrointestinal tract. On a dry weight basis, ~55% of the stool mass is contributed by microbial flora normally residing in the colon (9). Conservative estimates based on yields of alu-specific DNA (human), showed that cellular elements from the gastrointestinal tract accounted for ~10% of the dry weight of stools.

Contrary to conventional wisdom, we demonstrated that human stool contains a significant number of exfoliated epithelial cells recoverable in a viable state (trypan blue exclusion or propidium iodide exclusion by flow cytometry) (10). These cells can be isolated in a viable state (>85% viability) from a small sample (0.5–1.0 g) of human stool by a procedure known as somatic cell sampling and recovery (SCSR).4 These cells are exclusively of colonic origin and are of epithelial lineage, because they express cytokeratins (10). They are ideal surrogate targets for the noninvasive determination of the uptake and the turnover of dietary components and to conduct a variety of downstream molecular studies.

Isolating colonic cells from the contaminating materials from a small aliquot of stool makes the analysis of biomarkers more sensitive. This increases the likelihood of removing interference and thereby enhancing the signal strength of the biomarker. As an example of interference, the presence of PCR inhibitors in viral RNA extracts from stool has been shown to interfere with the amplification procedure (11–13). The ability to detect an aberrant cell population representing 0.1% of the total cellular milieu is beyond the capabilities of technologies using whole stool as a source material. This impediment has been overcome in studies of genetic variability for wildlife conservation by the use of coprocytobiology (CCB) technology in following tiger and lion populations (14), as well as in DNA typing from human feces in forensic medicine (15).

Isolation of somatic cells, noninvasively, by the SCSR technique and cell characteristics

SCSR cells are viable exfoliated colonic cells recovered from stool samples (10). This highly refined system involves the initial collection of 0.5- to 1.0-g aliquots of stool samples in a nontoxic preservative solution (SCSR transport; NonInvasive Technologies) and transporting it to the laboratory, without any refrigeration, within 7 d of collection. The stool suspension is thoroughly mixed and filtered sequentially through a 330-µM nylon mesh and a 40-µM filter cap, and then placed in a 50-mL centrifuge tube. The suspension is underlaid with 10 mL of a heavier cushion solution previously warmed up to room temperature (SCSR cushion; NonInvasive Technologies), and the tubes are centrifuged for 10 min at 200 x g in an unrefrigerated tabletop centrifuge. Light cells collect at the interface, and heavy cells migrate into the cushion. Each fraction is collected separately, washed with PBS at least 4 times, and counted in a phase contrast hemocytometer with trypan blue to distinguish viable cells from necrotic ones. Alternatively, the cells may be counted in a Coulter counter set (Z2 Coulter Particle Count and Size Analyzer; Beckman) to give size distribution histograms for cells.

A wet preparation of the final cell suspension when viewed under a phase-contrast microscope shows a large numbers of cells. The SCSR procedure generally yields 20–40 million cells per gram of stool, wet weight. Size distribution histograms show the existence of 2 distinct populations, one between 2–5 µm and a second one between 5–8 µm. The smaller cells appear to be a distinct population of quiescent cells, which exhibit autotrophism when held at ambient temperature (unpublished observation). In terms of density ({rho}), they are dispersed over a wide range, between 1.033 {rho} and 1.139 {rho}.

Characteristics of SCSR cells and expression of biomarkers

The expression of the blood group antigen in the adult is localized to cells shed from the proximal segments (cecum and proximal colon) of the colon (16,17). Flow cytometric studies on SCSR cells showed the presence of significant number of cells expressing the blood group phenotype of the donor. This demonstrated that cells originating in the proximal segment of the colon survive the transit through the entire large bowel and are recoverable intact in numbers proportionate to the surface area of their origin (18).

SCSR cells express housekeeping genes [glyceraldehyde 3 phosphate dehydrogenase (GAPDH); Fig. 1c and ß-actin; Fig. 1e] and are of epithelial lineage, because they express cytokeratins 8, 18, and 19 as demonstrated by Western blot analysis (18) and by RT-PCR of cytokeratin 19 (Fig. 1a). Furthermore, flow cytometric analysis showed the absence of CD45, the leukocyte common antigen that is indicative of the presence of blood (10). We have demonstrated the presence of lymphoid cell markers of differentiation on these cells (19). The human insulin receptor has been cloned using a 484 bp amplicon of a segment of the receptor using these colonocytes (20). Collectively, these results demonstrate that SCSR cells express mammalian genes.



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  		FIGURE 1 Electrophoresis of PCR products of housekeeping genes; cytokeratin 19 (epithelial cells marker), GAPDH and ß-actin and colon cancer markers, c-myc, c-erbb-2, APC, CEA, Cox-2, and CD 44. 1a (Cytokeratin 19), Amplification is shown in 4 samples of exfoliated colonocytes (Lane1–4); 1b (c myc p67), Amplification is shown in 4 samples of exfoliated colonocytes (lane1–4); Lane 5 (LS-180; colon cancer cell line, positive control); Lane 6 (LS-180, -RT control); Lane 7 (LS 180, no template DNA, negative control); 1c (GAPDH), Amplification is shown in 4 samples of exfoliated colonocytes (Lane1–4); 1d (c-erbb-2), Amplification is shown in 4 samples of exfoliated colonocytes (lane1–4); Lane 5 (LS-180; colon cancer cell line, positive control), Lane 6 (LS-180, -RT control); Lane 7 (LS 180, no template DNA, negative control); 1e (ß-actin), Amplification is shown in 4 samples of exfoliated colonocytes (Lane1–4); 1f (APC), Amplification is shown in 4 samples of exfoliated colonocytes (Lane1–4); 1g (CEA), Amplification is shown in 4 samples of exfoliated colonocytes (Lane1–4); 1h (Cox-2), Amplification is shown in 5 samples of exfoliated colonocytes (Lane1–5); 1i (CD 44), Amplification is shown in 4 samples of exfoliated colonocytes (Lane1–4) and HT-29, colon cancer cell line (lane 5).

 
Carcinoembryonic antigen (CEA) is a family of glycosylated proteins found in the tissues of the primitive endoderm and certain variants are overexpressed in malignancies of the gastrointestinal tract (21). Benchimol et al. (22) have shown the presence of CEA on the luminal surface of normal colonic epithelial cells. We have demonstrated the expression of CEA in colonocytes using flow cytometry (18). These observations are consistent with previously reported observations (22). Aliquots of the synthesized first-strand cDNA were used with the appropriate primer pairs to amplify a 350-bp fragment of CEA (Fig. 1g) We have demonstrated the expression of human leukocyte antigen and clusters of differentiation (CD) markers on SCSR cells (23). SCSR cells are also known to exhibit cell-mediated cytotoxicity against tumor cells, e.g., LS-180 adenocarcinoma cell line (24). Plant lectin studies have shown Jacalin and wheat germ lectins to be useful in predicting the presence of colonic neoplasms (19). Using genomic DNA, we have successfully demonstrated the presence of mutations in exon 4 of the p53 gene in colorectal cancer (10). SCSR cells isolated from patients with inflammatory bowel disease and colon cancer showed the abnormal expression of Cox-2 (10), evidenced by the amplification of a 396 bp fragment of the Cox-2 gene using specific primers and the cDNA synthesized from the transcripts isolated these cells (Fig. 1h). Similarly, the expression of multidrug resistance gene has been demonstrated in SCSR cells by PCR amplification. Other tumor-associated markers that we have seen in colonocytes isolated from colon cancer patients include c-myc (25) (Fig. 1b) and c-erbb2 (Fig. 1d), the APC gene (Fig. 1f), and splice variants of CD44 (Fig. 1i). Exon specific amplification of Variant 1 (exon 5) through Variant 6 (exon 11) regions of CD44 cDNA resulted in variable expression of 5 variant isoforms ranging from 232 bp to 718 bp in a HT-29 (colon cancer cell line) and 1–4 different isoforms within the same range using colonocytes (Fig. 1i).

Carotenoids, tocopherols, and retinol in SCSR cells

Micronutrients such as the tocopherols, retinol, and the carotenoids have a chemopreventive action against colonic carcinogenesis, and they may be essential for the functioning and structural integrity of the gastrointestinal epithelium. In our laboratory, we have determined the concentrations of tocopherols, retinol, and the carotenoids in human SCSR cells (26) (Table 1).


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  		TABLE 1 Micronutrients in SCSR cells

 
Human volunteers were placed on a carotenoid-poor diet for 3 wk, and SCSR cells were isolated daily and analyzed for ß-carotene (27). The levels of ß-carotene in SCSR cells dropped an average of 64% in 7 d, while retinol concentrations remained unaffected. After the depletion period, 2 groups of subjects received a carotenoid-rich diet while a third group continued on the original deficient diet. SCSR cells isolated from stool samples collected daily were analyzed for carotenoid concentrations. There was a sharp rise in ß-carotene, lycopene, and lutein concentrations, and the time to peak varied between 5 and 14 d, depending upon the carotenoid (Table 2). This lag in the kinetics of transfer of the dietary component has been repeatedly demonstrated in several experiments.


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  		TABLE 2 Kinetics of uptake of dietary bioactive components by SCSR cells

 
The study suggests that uptake of these micronutrients by the colonic mucosa occurs in the deep cryptal zone where the actively proliferating cells extract the nutrients from the systemic circulation and migrate along the crypt axis toward the luminal surface, a process that takes several days. These data demonstrate a potential model for studying extracorporeally, mucosal turnover rates, which indirectly give us an insight into the physiological host response to such factors as energy, dietary fiber intake, and bioactive food components and their interactions in different population and lifestyle groups. In addition, this technology presents us with the opportunity to assess, noninvasively, changes in cell turnover rates induced by bioactive food components. Similarly, this technique has been used to demonstrate the existence of distinct physiological compartments derived from dietary copper in human subjects (28).

Conclusion

We have demonstrated the feasibility of using exfoliated epithelial cells isolated from fecal material for the noninvasive detection and the assessment of the bioactivity in vivo of biologically important food components. It is now feasible to monitor and to determine the physiological effects of these dietary components on specific biomarkers acting as sentinels of cell growth and differentiation. The concept of using exfoliated cells as surrogate cellular targets for studying bioactive dietary components and their physiological activity in the intact organism advances our ability to further our knowledge of the role of diet in cancer prevention. Besides, the ability to measure the bioavailability, as well as kinetics of uptake and utilization of micronutrients using exfoliated colonocytes is a novel, noninvasive approach for evaluating the effects of bioactive food components.

FOOTNOTES

1 Presented as part of the symposium "Nutritional ‘Omics’ Technologies For Elucidating the Roles of Bioactive Food Components in Colon Cancer Prevention," held at the 2005 Experimental Biology meeting on April 5, 2005, San Diego, CA. The symposium was sponsored by the American Society for Nutritional Sciences and the Diet and Cancer and Dietary Bioactive Food Components Research Interest Sections. The proceedings are published as a supplement to The Journal of Nutrition. This supplement is the responsibility of the Guest Editors to whom the Editor of The Journal of Nutrition had delegated supervision of both technical conformity to the published regulation of The Journal of Nutrition and general oversight of the scientific merit of each article. The opinions expressed in this publication are those of the authors and are not attributable to the sponsors or the publisher, editor, or editorial board of The Journal of Nutrition. Guest editors for the supplement publication were Cindy D. Davis, National Cancer Institute, NIH; and Norman Hord, Department of Food Science and Human Nutrition, Michigan State University. Back

2 Supported by Grants from the National Cancer Institute, the National Institute of Digestive Diseases and Kidney Diseases, National Institutes of Health, SBIR R44 DK56567 and R44 CA 81799. Back

4 Abbreviations used: CCB, coprocytobiology; CD, clusters of differentiation; CEA, carcinoembryonic antigen; GAPDH, glyceraldehyde 3 phosphate dehydrogenase; SCSR, somatic cell sampling and recovery. Back

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