ALDH, Aldehyde dehydrogenase; CSC, Cancer Stem Cells; GC, Gastric cancer, HSC, hematopoietic stem cells; BMDC, Bone Marrow derived Cells

Transcription

1 Co-expression of CD34 and ALDH br in Human Gastric Cancer cells A potential marker for Cancer Stem Cells. Arachimani Anand Kumar 1*@, Marappagounder Dhanasekaran 1 and Sankaran Raj Kumar 2. * To whom the correspondence should be addressed. 1 Department of Stem Cells, 2 Department of Gastroenterology, Lifeline Institute of Regenerative Medicine, RIGID Health Care Pvt Ltd, 5/639, Rajiv Gandhi Salai, Perungudi, Chennai , India. Ph ext 338, Fax: E.mail: Present Address Department of Stem Cells and Regenerative Medicine, Dr.Kamakshi Memorial Hospital, No.1, Radial Road, Pallikaranai, Chennai , India. Ph ext 112, Fax: Running title: Gastric cancer cells and CD34, ALDH expression. Abbreviations. ALDH, Aldehyde dehydrogenase; CSC, Cancer Stem Cells; GC, Gastric cancer, HSC, hematopoietic stem cells; BMDC, Bone Marrow derived Cells

2 ABSTRACT Background & Objectives The origin of gastric cancer from bone marrow derived stem cells has been postulated in animal models. In this study we sought to investigate the expression of ALDH and CD34 markers in the cells of normal and gastric cancer tissue using Flowcytometry. Materials and Methods Five gastric cancer resected specimens with adjoining normal tissues were taken for the study. Single cell suspensions were made from these samples and were incubated with CD34, CD45 antibodies conjugated with PE and FITC respectively and with Aldeflour reagent for FACS analysis. Fifty thousand events were obtained for analysis of the cells. Results The percentage CD34 cells in normal gastric tissue were higher than in cancer tissue. The ALDH expressing cells was similarly decreased in cancer tissues, though the CD34 + ALDH + cells constituted ~ 0.4% of cancer mass. Interpretation and Conclusion Increased CD34 + cells in normal gastric tissue adjoining the tumor reflects altered homeostasis and a critical role in angiogenic sustenance of the tumor mass. In the cancer tissues, CD34 + ALDH bright populations represented only a very small population of cells, and given the characteristics of ALDH + cells, this population may constitute the cancer stem cell population. Key words Gastric carcinoma, Cancer stem cells, ALDH, CD34 cells

3 INTRODUCTION Gastric neoplastic transformation is a complex multistep process with definite etiological and progressive phases (1). Chronic inflammatory destruction of the gastric epithelium and the subsequent compensatory proliferation of the tissue drives the pathological course that eventually recruit bone marrow derived stem cells (BMDCs) or hematopoietic stem cells (HSCs) for regenerative repair (2). Although the concept that cancers originate from stem cells (SC) is becoming scientifically accepted, mechanisms by which SC contribute to gastric cancer initiation and progression are largely unknown (3). For Gastric cancer, investigation of this problem has been hindered by the complexity of the pathophysiological process and by a paucity of specific markers for identification and isolation of SC from normal and malignant gastric epithelium. Furthermore, the identification of transforming stem cell, whether it is a gastric stem cell or HSC or both is challenging. Previous results from our study revealed an association of CD34 + Oct4 + expressing HSC population in the contribution of neoplastic transformation of gastric tissue (Unpublished data). In this study, we sought to isolate and characterise stem cell population using Flow Cytometry based on the enzymatic activity of Aldehyde dehydrogenase (Aldefluor assay) and CD34 marker expression. Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme, and its expression in the sera of the gastric cancer patients was investigated earlier, but with no apparent changes (4). Similarly the metabolic association between ALDH and various cancers were also investigated (5). Except for the association of ALDH bright cells with the cancer stem cells and metastatic efficiency (6,7), no other credible metabolic significance for the cancer has been reported. Given these instances, we try to investigate the expression of ALDH positive cells in normal and gastric cancer tissue and its association with CD34 + cells.

4 MATERIALS AND METHODS Selection of Patients Patients admitted for gastric cancer resection in Lifeline Hospitals, Perungudi, Chennai were chosen for this study. Prior to operation, a written consent from the patients were obtained for the use of resected human tissue for research. This study was approved by the Institutional Committee for Stem Cell Research and Therapy (IC-SCRT) and Institutional Ethics Committee (IEC) as per the Indian Council of Medical Research (ICMR) Stem Cell Guidelines Isolation and processing of Gastric cells Five complete gastric resected samples with cancer proper and adjoining normal tissues were collected for the study. Normal tissue portions were carefully dissected out from the specimen leaving 2mm margins from cancer proper and separately processed. Briefly, a 100mg of gastric tissue was rinsed in ice cold Dulbecco phosphate buffered saline (DPBS) (without calcium and magnesium) until the blood stains were removed. Tumor/normal tissue was minced into tiny fragments (2 mm 3 ), followed by enzymatic digestion with 3.0 mg/ml Collagenase (Stem Cell Technologies; Vancouver, BC) for 1-2 hour at 37 0 C with intermittent pipetting to disperse the cells. Cells were then filtered sequentially through 100µ and 40 µfilters, and washed twice in DPBS at 400g for 10 minutes. The resultant pellet was added with RBC lysing solution (0.7%ammonium chloride) and incubated for 2 minutes. The lysing was arrested by adding 0.9% ice cold sodium chloride and the cells were washed and finally resuspended in required volume. FACS Characterisation of cells To a 100 µl processed single cell suspension (1 X 10 6 cells/ml), 20 µl of CD34 and 20 µl of CD45 antibodies conjugated with PE and FITC respectively (BD Bioscience) were added and incubated for 15 minutes at room temperature in the dark. After incubation,

5 900 µl of PBS was added to the stained cells and mixed well. To this mixture, 5 µl of the 7-Amino Actinomycin D (7-AAD) dye was added and again incubated in dark for 10 minutes at room temperature. The cells were vortexed and aliquated for characterisation in FACS Area (BD). For ALDH assay, cell suspension containing 1 X 10 6 cells/ml, was added with 5µl of Aldefluor reagent and incubated for 30 minutes at 37 0 C. Fifty thousand events were acquired for analysis of the cellular composition of the sample. The total viability was assessed by 7-AAD method. RESULTS Table 1 and Fig 3 summarised the expression of various markers in the normal and cancer tissues. The ALDH expressing cells constitute 0.18% of the normal gastric tissue (Fig 1D, 3) whereas the CD34 expressing population represents 2.7% (Fig 3, 1C). The number of CD45 cells in the normal gastric tissue was insignificant and constituted less than 0.1% of the total (Fig 1C). In the gastric cancer tissue the ALDH expressing cells constituted 0.14% of total cells, less than their normal counterparts (Fig 2B, 3). Similarly the CD34 population also significantly decreased to 0.36% (Fig 2C, 3). The double positive CD34 + ALDH bright population constituted 0.38% of the population (Fig 2D, 3). DISCUSSION Normal gastric epithelium surrounding the cancer mass had showed a disproportionate number of cells expressing CD34 marker (Fig1). Though this increase in CD34 + expressing cells may be partly explained as increased angiogenic and/or inflammatory manifestations, it nevertheless signifies an altered homeostasis in the gastric tissue surrounding the cancer proper. Further, the inflammatory infiltration represented by the cells expressing CD45 antigen is low (Fig 1C), indicating a change in the vascular bed or in the characteristics of the surrounding gastric stroma. Though CD34 positive stromal cells are essential for the maintenance of gastrointestinal mesenchymal elements including smooth muscles and vessels, they are not required or form integral part of normal gastrointestinal epithelial cell maturation and proliferation (8). Moreover, this

6 increase in CD34 + cells in the surrounding normal tissue was not accompanied by appreciable increase in ALDH + cells (Fig 3, 1D, Table 1) and the level of ALDH expression is consistent with the level of ALDH activity normally associated with tissue such as Stomach, liver, oesophagus and colorectal tissues (5). Expression of CD34 + cells and ALDH + cells was significantly decreased in gastric carcinoma (Fig 2, 3). Nevertheless, a subset of gastric cancer cells appear to express both CD34 + ALDH + markers, representing ~ 0.4% of the cancer cells (Fig 2D, 3; Table 1). Cancers bearing ALDH + cells are always aggressive, possibly reflecting on the different rate of renewal of cancer stem cells and are 1.76 times more likely to develop metastasis than ALDH negative tumors (9). ALDH appear to be a universal factor in enhancing the abilities of cells or progenitors. Accordingly, the circulating progenitor cells that express CD34 + ALDH bright phenotype has been shown to effectively reconstitute hematopoiesis (10), and in colorectal cancers, the putative cancer stem cells expressing ESA + CD44 + markers are also positive for High ALDH activity, and these ALDH bright cells are tumerogenic in all cases investigated (11). Indeed, during the progression from normal colonic epithelium to adenoma-carcinoma sequence, ALDH + cells increased in number and became distributed farther up the crypt, and implantation of, as few as 25 ALDH bright cells from colorectal cancers to nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice generated xenograft tumors (12). Similar results have been documented in lung cancer (13), brest cancer (14), where ALDH bright cells reconstituted the cancer in a xenobiotic transplantation whereas Aldeflour negative cancer cells could not. Stem or progenitor cells of both normal and cancerous gastric tissues express high level of ALDH activity (7). However, during the pathophysiological course of the gastric carcinogenesis the resident gastric stem cell niche along with stem cells are destroyed and the loss of compensatory proliferation leads to gastric atrophy (15, 16). Subsequent influx of bone marrow derived stem cell (BMDC) population for regenerative repair becoming the consequential step leading to gastric cancer (15). Indeed, the human betagalactosidase positive BMDCs reconstituted the 90% of the gastric mucosa in lethally

7 irradiated mice, and with the persistence of Helicobacter infection, a proliferative zone forms deeper within the gastric mucosa, giving rise to metaplasia (17, 18). In a recent study, we have implicated the role of CD34 + Oct4 + expressing HSC population in contributing neoplastic transformation of gastric tissue (Unpublished data). These studies have demonstrated the association of CD34 expressing cells to the formation of intraglandular gastric epithelium (2) and in the genesis of gastric carcinoma (17). Since ALDH positivity has been implicated to CSC phenotype in various cancers (10), the gastric cancer cell population that expresses both CD34 + ALDH + markers might be a potential cancer stem cell. Further investigations are however required for elucidating the CD34 + ALDH + cells for its cancer reconstituting efficiency in animal models.

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11 Table 1 Expression of various markers in normal and cancer tissue Tissue Normal gastric tissue Gastric cancer tissue Type of CD34 + ALDH + cells CD34 + cells ALDH + CD34 + cell cells cells ALDH + cells Sample 1 2.4% 0.2% 0.3% 0.1% 0.4% Sample 2 3.2% 0.1% 0.5% 0.2% 0.4% Sample 3 2.8% 0.1% 0.4% 0.1% 0.5% Sample 4 2.9% 0.2% 0.3% 0.2% 0.3% Sample 5 2.3% 0.2% 0.3% 0.1% 0.3% Average 2.7% 0.18% 0.36% 0.14% 0.38%

12 Fig 1 Normal gastric cells CD34, CD45, ALDH expression

13 Fig 2 Gastric cancer cells CD34, CD45, ALDH expression

14 Fig 3 CD 34 and ALDH expression 3.50% 3.00% 2.50% 2.00% 1.50% 1.00% 0.50% 0.00% CD34+ cells ALDH+ cells Normal gastric tissue CD34+ cells ALDH+ cells CD34+ ALDH+ cells Gastric cancer tissue Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 %Total