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The Expression of G250/MN/CA9 Antigen by Flow Cytometry: Its Possible Implication for Detection of Micrometastatic Renal Cancer Cells¹

Departments of Urology [G. L., F. B., J. T.] and Pathology [A. G-P., J-F. M.], and Clinical Immunology Laboratory [K. P-F., C. L., C. G.], North Hospital, CHU of Saint-Etienne, 42055 Saint-Etienne Cedex 2, France, and Department of Urology, University Hospital of Nijmegen, 6500HB Nijmegen, the Netherlands [E. O.]

	ABSTRACT

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Monoclonal antibody (mAb) G250 is a well characterized and specific mAb to renal cell carcinoma (RCC). The gene G250 was recently cloned and was proved to be homologous to MN/CA9. The G250/MN/CA9 antigen was recently explored as a potential marker for RCC. Flow cytometry (FCM) allows quantitative analysis of cells. The present study describes a flow cytometric method to detect this antigen in human cell lines and in malignant and normal renal tissues. Twelve human carcinoma cell lines (HeLa, Colo205, HT29, BxPC3, OVCAR3, SKOV3, ACHN, A704, CAKI-2, SKRC-59, SKRC-10, and SKRC-52), 10 specimens of normal peripheral blood mononuclear cells, and 38 malignant and 36 adjacent normal renal tissues were studied. The malignant and normal renal tissues were disaggregated mechanically into a single-cell suspension, stained by mAb G250, and analyzed by FCM. All 22 of the clear cell carcinomas, 6 of 8 mixed cell carcinomas, and 3 of 6 granular cell carcinomas were positive for G250/MN/CA9 antigen. SKRC-52 and SKRC-10 were strongly positive for G250/MN/CA9. The G250/MN/CA9 antigen could also be detected in HeLa, SKOV3, HT29, and A704 cells. One chromophobic, one chromophilic cell carcinoma, the normal renal tissues, and normal peripheral blood mononuclear cells were considered as negative. Our results further confirmed that the G250/MN/CA9 antigen was an ideal marker for RCC, especially for clear cell carcinomas, and that this antigen was present in several types of malignant cells. FCM may serve as a fast tool of immunocytochemical detection of renal cancer cells. Flow cytometric detection of renal cancer cells by using mAb G250 should be further explored.

	INTRODUCTION

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The incidence of RCC³ has increased steadily during recent years. Although the radiological image has revolutionized the diagnosis of RCC, nearly one-third of patients present metastatic disease at the time of diagnosis. Besides, as many as 40% of patients with local tumors will ultimately relapse with metastatic disease after surgery (1) . These clinical facts suggest that new diagnostic and monitoring methods are needed.

mAb G250 is a well characterized and specific mouse IgG2a to RCC. Previous immunohistochemical studies have shown that mAb G250 recognizes a RCC-associated antigen expressed on the surface of almost all RCCs but not on normal renal tissues and that its expression in normal organs is found to be restricted to the gastric mucosal cells and the large bile ducts (2) . Recent clinical trials with ¹³¹I-labeled mAb G250 as well as ¹³¹I-labeled chimeric mAb G250 for radiodiagnosis have further proved its tumor specificity (3 , 4) . Since the first report of mAb G250, a considerable effort has been made to explore its immunotherapeutic potential for RCC patients (5, 6, 7) . The mAb G250 has become thus far the most studied and promising mAb to RCC. Recently, the gene G250 was cloned and proved to be homologous to MN/CA9 (8) . Moreover, recent studies have demonstrated that antigen G250/MN/CA9 is a potential marker for RCC (9, 10, 11) .

FCM allows quantitative and multiparametric analysis of a large number of cells. Use of mAbs by means of FCM to detect surface antigens that are expressed by malignant cells can be fast and reliable (12, 13, 14, 15) . Obviously, FCM has been successfully applied in detecting the abnormal cells in hematological malignancies (16 , 17) . But as far as solid tumors are concerned, reports on antigen detection by FCM are very limited. One of the major reasons is the lack of specific mAbs for malignant cells of solid tumors. To our knowledge, mAbs that label malignant cells but not normal tissue cells are rare. The potential use of mAb G250 by FCM has not been explored. We have investigated G250/MN/CA9 antigen expression in human carcinoma cell lines and different cell types by FCM to broaden its diagnostic application. Here we report our results, which may provide some useful information.

	MATERIALS AND METHODS

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The mAb G250.
The murine mAb G250 (IgG2a) was used for immunocytochemical staining. The isolation and characterization of mAb G250 has been described elsewhere (2) .

Renal Carcinoma and Other Carcinoma Cell Lines and Culture.
The ACHN, A704, and CAKI-2 cell lines were provided by the European bank of cell lines. SKRC-52, SKRC-10, and SKRC-59 were a gift from Dr. Oosterwijk, Urology Research Laboratory, Nijmegen, the Netherlands. The cells were cultured in RPMI 1640. The following carcinoma cell lines were also obtained from the European bank of cell lines and treated in the same way: HeLa, Colo205, BxPC3, HT29, OVCAR3, and SKOV3. The confluent cells were harvested after the treatment with 0.25% trypsin solution and diluted with a PBS-BSA-EDTA solution for a final concentration of 0.5 x 10⁶/ml. The cells were then filtered through a 50 µm nylon mesh (Filcons; Dako) to isolate the cells into single-cell suspensions. Treated cells were ready for immunolabeling.

Tumor Specimens and Normal Renal Tissues.
The malignant renal tissues without necrotic areas and the matched normal renal cortex from a distant site were obtained from open or laparoscopic surgery. Thirty-eight consecutive specimens (from 24 men and 14 women) were analyzed. The age ranged from 38 to 84 years old with a mean of 62.4 ± 11.9 years. These tumors were staged according to the 1997 TNM classification (18) . Ten tumors were staged to pT_1a, 5 to pT_1b, 4 to pT₂, 11 to pT_3a, 6 to pT_3b, and 2 to pT_3c. There were 36 conventional carcinomas (22 clear cell carcinomas, 8 mixed cell carcinomas, and 6 granular cell carcinomas), one chromophobic cell carcinoma, and one chromophilic cell carcinoma. The renal tissues were mechanically disaggregated into a single cell suspension by using 50 µm mesh (Medicons; Dako, Copenhagen, Denmark). Briefly, a piece of 0.3 cm³ of tissue plus 1.5 ml of HBBS was put into Medicons chamber. The Medicons was inserted into a Medimachine and run for one min. The tissue suspension was obtained by syringe aspiration and treated with 2 ml of the erythrolysing solution (Biosource Europe, Fleurus, Belgium) for 10 min at room temperature. The tissue suspension was centrifuged for 10 min at 300 x g at room temperature. After removing the supernatant, PBS-BSA-ETDA solution was added to the cells for a concentration of 2 x 10⁶/ml. The cells were filtered with a 50 µm nylon mesh before the immunolabeling.

The Normal PBMCs.
Ten specimens of 5 ml of blood from normal donors were obtained. The blood specimens were subjected to a density gradient separation. The PBMCs were obtained and washed with HBSS. The pipetted cells were maintained in PBS-BSA-EDTA solution. Fifty µl of the suspension were used for the immunological staining.

The Immunological Staining.
Ten µl of mAb G250 (100 µg/ml) was added to a tube containing a 50-µl aliquot of cells. This appropriate working concentration was set by our preliminary tests on cell lines and renal tissues. Cells and antibody were gently mixed and incubated in the dark at room temperature for 30 min. For the malignant and normal renal cells, 50 µl of normal goat serum was added to the tube before adding the mAb for 10 min at room temperature. Ten µl of FITC-conjugated goat antimouse immunoglobulin antiserum (Dako) was added after washing with 2 ml of PBS solution. After another 30-min incubation in the dark, the cells were washed with 2 ml of PBS solution. Then the cell pellets were suspended in 200 µl of PBS-BSA-EDTA solution and fixed in 200 µl of 2% paraformaldehyde solution. The flow cytometric analysis was performed within 24 h. An isotype IgG2a (Dako) was used as a negative control.

Flow Cytometric Analysis.
The samples were analyzed by FCM (XL Beckman). A typical cell area was gated and more than 1 x 10⁴ events were counted. An isotype negative control was used to define the threshold of the background staining. The result was expressed in percentage of the gated cells. The negative control was always less than 5%. Thus, the result of the sample that was more than 5% was considered as positive. For the carcinoma cell lines, the flow cytometric analysis was done in triplicate, and the results were expressed as an average ± SE.

	RESULTS

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Immunolabeling of Disaggregated Renal Cells.
The antigen G250/MN/CA9 was positive on all of the 22 clear cell carcinomas. Six of eight mixed cell carcinomas and three of six granular cell carcinomas were positive. No normal renal tissue specimen was considered as positive. A typical example of staining pattern of malignant and normal tissues can be seen in Fig. 1 . The average percentages of positive-staining neoplastic cells were different according to tumor cell types (Table 1) .

View larger version (12K): [in this window] [in a new window]   Fig. 1. A typical flow cytometric histogram of RCC and normal renal tissues. A RCC exhibited a strong positive staining by mAb G250, whereas the corresponding normal renal tissues showed negative.

View larger version (65K): [in this window] [in a new window]   Fig. 2. The average percentage of positive staining cells of different carcinoma cell lines. Six carcinoma cell lines, SKRC-10, SKRC-52, HeLa, A704, HT29, and SKOV3, exhibited the positive G250/MN/CA9 antigen as revealed by FCM. Results were expressed as average percentage of three different experiments.

	DISCUSSION

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The mAb G250 is thus far the most studied and specific mAb to RCC that recognizes an aberrantly expressed RCC antigen. On the basis of the initial immunological evidence, mAb G250 reacts with all clear cell carcinomas and the majority of the other types of RCC but not with the normal renal cells (2) . The G250 antigen was recently identified as MN/CA9 antigen (8) . The MN/CA9 antigen recognized by mAb M75 was first detected on the cell surface of a cervical carcinoma cell line, HeLa (19 , 20) . The MN/CA9 was found to be expressed in normal epithelial cells of gastric mucosal, cervical, and colorectal tumors (21 , 22) . The MN/CA9 antigen was recently explored in RCC by using mAb M75 immunohistochemical study and considered as a reliable biomarker (9) . A recent immunohistochemical study by mAb G250 characterized the antigen expression in RCC, which showed that 99% of clear cell, 65.5% of granular cell, and 76.5% of mixed cell carcinomas expressed G250/MN/CA9 (7) . Therefore, mAb G250 and mAb M75 recognized the same antigen and yielded very similar results (7 , 9) . After the cloning of the MN/CA9 gene, McKiernan et al. (10) first demonstrated, by RT-PCR assay using specific primer for the MN/CA9 gene, that all 12 of the clear cell carcinomas showed MN/CA9 expression and observed no expression in the remaining 5 granular or papillary cell carcinomas. But two more recent RT-PCR assays showed G250/MN/CA9 expression in some granular cell carcinomas (7 , 11) . A small number of tumor specimens studied in the first RT-PCR assay and different RT-PCR protocols among laboratories may explain this discrepancy. Here we first presented a flow cytometric detection of this antigen. Our results showed that G250/MN/CA9 antigen could be detected in all 22 clear cell, 6 of 8 mixed cell, and 3 of 6 granular cell carcinomas. Our results were comparable with those of the immunohistochemical studies (7 , 9) , which further confirmed that G250/MN/CA9 antigen was ideal marker for RCC, especially for clear cell carcinomas. We had one chromophobic and one chromophilic cell carcinoma that were negative for G250/MN/CA9 antigen. Chromophobic cell carcinoma may not express G250/MN/CA9. This was consistent with the immunohistochemical study (9) . mAb G250 may be used to distinguish the chromophobic cell carcinomas from other cell types of renal carcinomas. But it should be pointed out that only three chromophobic cell carcinomas were analyzed in literature. A further study on a large scale of chromophobic and chromophilic carcinomas is needed to ascertain its correctness.

The role of G250/MN/CA9 in carcinogenesis is unclear. Its involvement in the development of RCC is suggested by the fact that it is detected in malignant renal cells but absent in normal renal cells. We have tested a panel of human carcinoma cell lines. Our results showed that SKRC-10 and SKRC-52 were strongly G250/MN/CA9 positive. Because MN/CA9 antigen was recently detected in ovarian, cervical, and colorectal tumor specimens (21 , 22) , it is interesting to observe the G250/MN/CA9 antigen expression in carcinoma cell lines of these organs by FCM. In the present study, the HeLa cells presented positive staining on the G250/MN/CA9 antigen. Among two colon carcinoma cell lines, HT29 and Colo205, the former was G250/MN/CA9 positive, whereas the latter was negative. An ovary carcinoma cell line, SKOV3, was also G250/MN/CA9 positive. These results confirmed that G250/MN/CA9 antigen existed in several types of malignant cells, which suggests that this antigen is a promising tumor biomarker.

Our flow cytometric detection of G250/MN/CA9 may have some clinical applications. Firstly, in view of the growing importance of this marker for RCC, a fast, reliable, and quantitative analysis of this antigen may be useful for diagnostic studies. The detection of G250/MN/CA9 antigen by FCM may provide such a technique for immunocytochemical diagnosis of RCC. These advantages of studying antigen expression by FCM over conventional immunohistochemical techniques were also observed by other researchers (23) . If further improved, this technique may be applied in the clinical laboratory as a supplementary tool. Secondly, FCM may be used to detect the circulating renal cancer cells. In the absence of an effective serum marker for RCC, detecting the circulating renal cancer cells may be a useful surrogate marker. The major techniques are RT-PCR, FCM, and immunocytochemistry. Very recently, the detection of circulating renal cancer cells has become possible by using the G250/MN/CA9 gene (7 , 24) . McKiernan et al. (24) found a percentage of 1.8% of MN/CA9 positive in normal controls by RT-PCR. But Uremura et al. (7) demonstrated that one-third of healthy donor samples were G250/MN/CA9 positive by using RT-PCR and suggested that a quantitative analysis was needed for the detection of the circulating renal cancer cells by RT-PCR. FCM was recently used to characterize the circulating cancer cells in solid tumors. Some technical problems have been resolved, and these studies seemed to prove that FCM could be used to detect the circulating cancer cells (25, 26, 27) . Whether FCM can be used for detecting the circulating renal cancer cells has not been explored. The first step is to select appropriate mAbs. Our results provided evidence that mAb G250 could be used for flow cytometric analysis of renal cancer cells. We think that mAb G250 was an optimal one to detect renal cancer cells by FCM. But many more exacting demands must be met for FCM to be used to detect renal cancer cells in blood. In fact, it is very difficult for FCM to show that there are no positive cells in normal PBMCs as the other researchers’ techniques have shown (25) . This may be caused by autofluorescent background staining. Nevertheless, FCM can be used quantitatively to compare the specimens, which means that a cutoff may be defined. Besides, PBMCs can be stained by other specific mAbs, such as CD45, to reduce the background staining. These advantages make us think that detection of circulating renal cells by FCM may be possible. We have previously proved that mAb G250 and mAb VU-1D9 have a higher affinity to renal cancer cells compared with other available mAbs (28) . It is reasonable to detect the micrometastatic renal cancer cells by a combination of mAbs, e.g., G250/VU-1D9/CD45, because FCM can simultaneously analyze three markers. We are currently setting up a three-color flow cytometric model study by using this panel to see whether a distinction between normal PBMCs and renal cancer cells can be defined.

In conclusion, we conducted a study of detecting G250/MN/CA9 antigen by FCM. Our results confirmed that G250/MN/CA9 antigen was an optimal marker for RCC, especially for clear cell carcinomas. FCM may be a supplementary tool to diagnose RCC by detecting G250/MN/CA9 antigen. Its potential application should be further explored.

	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 Supported by La Ligue Nationale Contre le Cancer de la Loire, France.

² To whom requests for reprints should be addressed, at Department of Urology, North Hospital, CHU of St-Etienne, 42055 St-Etienne Cedex 2, France.

³ The abbreviations used are: RCC, renal cell carcinoma; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cell; FCM, flow cytometry; RT-PCR, reverse transcription-PCR.

Received 7/ 8/00; revised 10/ 4/00; accepted 10/ 5/00.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, G. Articles by Tostain, J. Search for Related Content PubMed PubMed Citation Articles by Li, G. Articles by Tostain, J.