ALDH + /CD44 + /CD24 − expression in cells from body cavity fluids

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Background:Enhanced expression of aldehyde dehydrogenase 1 (ALDH1) and phenotypic markers (CD44+/CD24−) in stem cells from breast tumors has been reported. This study was undertaken to monitor expression of these markers in cells from body cavity

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  Cytometry Part B (Clinical Cytometry) 78B:176–182 (2010)  ALDH 1 /CD44 1 /CD24 2 Expression in Cells from Body Cavity Fluids  Awtar Krishan, 1 *  Deepti Sharma, 1 Siddharth Sharma, 1 Ronald M. Hamelik, 1 Parvin Ganjei-Azar, 2 and Mehrdad Nadji  2 1 Department of Pathology (R-71), Miller School of Medicine, University of Miami, Miami, Florida 33101 2 Department of Pathology (R-5), Miller School of Medicine, University of Miami, Miami, Florida 33101 Background: Enhanced expression of aldehyde dehydrogenase 1 (ALDH1) and phenotypic markers(CD44 1  /CD24 2 ) in stem cells from breast tumors has been reported. This study was undertaken to moni-tor expression of these markers in cells from body cavity fluids of female patients suspected to have amalignancy.Methods: Cells from peritoneal and pleural fluids of 100 female patients were examined by diagnosticcytology and analyzed by laser flow cytometry for enhanced ALDH1 expression. Cells from 36 body cavityfluids with ALDH1 bright fluorescence were then analyzed for the expression of CD44 and CD24 markers.Results: In samples positive for malignancy, ALDH1 bright cells with both SSC low and SSC high were seen.In 15 body cavity fluids positive for malignancy, the percentage of ALDH1 bright cells ranged from 0.26 to6.34% of the total cells. The percentage of ALDH1 bright cells with CD44 1  /CD24 2 expression in thesesamples ranged from 0.02 to 3.66%. ALDH1 bright cells with CD44 1  /CD24 2 expression were also presentin body cavity fluids of patients in whom diagnostic cytology could not detect any malignancy. However,the percentage of ALDH1 bright and CD44 1  /CD24 2 cells amongst the 21 body cavity fluids with negativecytology was lower than that of samples with malignancy.Conclusions: Expression of ALDH1 bright and the CD44 1  /CD24 2 phenotype in body cavity fluids in whichdiagnostic cytology could not find any malignant cells suggests that this phenotype may not be restrictedto the putative breast tumor stem cells. It is possible that only subsets of cells with this phenotype arethe putative breast tumor stem cells. V C  2009 Clinical Cytometry SocietyKey terms: aldehyde dehydrogenase; body cavity fluids; flow cytometry; stem cell markers How to cite this article: Krishan A, Sharma D, Sharma S, Hamelik RM, Ganjei-Azar P, Nadji M. ALDH þ  /CD44 þ  / CD24  expression in cells from body cavity fluids. Cytometry Part B 2010; 78B: 176–182.Several recent studies have focused on the expressionof stem cell markers in human solid tumors and in meta-static tumor cells in bone marrow (1–4). In one of theearliest studies, Al-Hajj et al. (5) identified and isolatedthe tumorigenic breast tumor stem cells as CD44 þ  / CD24   /Lineage - in eight of nine breast cancer patients.In Brca1 positive breast tumors, distinct CD44 þ  /CD24   / CD133 þ cells with cancer stem cell characteristics andincreased expression of stem cell associated genes (e.g.,Oct4, Notch1, ALDH1, Fgfr1, and Sox1) has beenreported by Wright et al. (6). Honeth et al. (7) reportedthat CD44 þ  /CD24  cells (ranging from only a few toclose to 100% of tumor cells) were detected in 31% of basal-like breast tumors.Besides the CD44 þ  /CD24   /CD133 þ markers, over-expression of aldehyde dehydrogenase 1 (ALDH1) hasbeen reported in hematopoietic and solid tumor stemcells (8). Ginestier et al. (2) have shown that normaland malignant human mammary epithelial cells with increased ALDH1 activity have stem/progenitor likeproperties and ALDH1 expression correlated with poor prognosis. In colon cancer patients, Huang et al. (9)reported that progression from normal to mutant epithe-lium and adenoma was accompanied by significant *Correspondence to: Awtar Krishan, Department of Pathology(R-71), Miller School of Medicine, University of Miami, PO Box016960, Miami, FL 33101. E-mail: akrishan@med.miami.eduReceived 4 June 2009; Revision 20 November 2009; Accepted 25November 2009Published online 30 December 2009 in Wiley InterScience (www.interscience.wiley.com).DOI: 10.1002/cyto.b.20509 V C  2009 Clinical Cytometry Society  increase in the number of ALDH1 bright  / CD133 þ  / CD44 þ cells. Although expression of these markers has beendescribed in both hematopoietic and solid tumor stemcells, there are no published reports on their expressionin cells from body cavity fluids of patients suspected tohave a malignancy. In the present study, we have ana-lyzed cells from body cavity fluids of female patients for the coexpression of ALDH1, CD44 and CD24 markers. MATERIALS AND METHODSProcessing of Body Cavity Fluids Residual peritoneal and pleural fluids were collectedunder University of Miami and Jackson Medical Centreapproved IRB protocols. After coarse filtration thrugauze, cell pellets were prepared by centrifugation (rela-tive centrifugal force x 220, 5 min at 4  C) and washedonce with 5 ml of phosphate buffered saline (PBS). Try-pan blue was used to count the number of dye exclud-ing cells. Most of the samples were analyzed within 24hours of collection and storage in a refrigerator. A totalof 100 samples were collected and out of these, 36 haddistinct ALDH1 bright cells as determined by comparisonof cells incubated with or without the ALDH1 inhibitor,diethylaminobenzaldehyde (DEAB). Fifteen out of the 36samples were confirmed by diagnostic cytology to havemalignant cells. Seven of the 36 samples were describedby diagnostic cytology to contain atypical epithelial or inflammatory cells while the remaining 14 samples didnot contain any recognizable malignant cells.Slides prepared from cells in the body cavity fluids were stained and examined by the diagnostic pathologistfor the presence of malignant cells. Where ever feasible,immunohistochemisty was used to stain the cells for thepresence of markers such as estrogen and thyroid tran-scription factor-1. Aldefluor V R Staining  Aldefluor  V R  staining was performed according to theprotocol described by Storms et al. (10). An aliquotof 2    10 6 cells was centrifuged and washed with 2 mlof PBS. The cell pellet was resuspended in 1 ml of  Aldefluor  V R  buffer containing 5  l l of activated Aldefluor  V R  reagent (StemCell Technology, Durham, NC). One half of the sample was transferred to a second tube containing5  l l of the ALDH1 inhibitor, DEAB. Samples were mixedand incubated in a 37  C water bath for 1 h with inter-mittent shaking. CD44/CD24 Staining  After 60 min of incubation with the Aldefluor  V R  rea-gent, 20  l l of CD44-RPE (catalogue no 555479, clone-G44-26, BD Pharmingen TM , San Jose, CA) and 5  l l of CD24-PE-Alexa Fluor  V R  610 conjugate, (catalogue no. MHCD2422, clone SN3, Invitrogen, Camarillo, CA.) wereadded to the incubation mixture for 20 min at roomtemperature in the dark. After centrifugation and wash-ing, the cell pellets were suspended in 400  l l of  Aldefluor  V R  buffer.Samples were analyzed on a Beckman Coulter XL flow cytometer. Threshold and electronic gates were used inForward Scatter versus Side Scatter plots to exclude de-bris and red blood cells. Electronic gates were used toidentify cells with ALDH1 positive expression by com-paring dot plots of cells incubated with Aldefluor  V R   with or without the ALDH1 inhibitor, DEAB. Cells with  ALDH1 positive expression and high and low side scat-ter were subsequently analyzed for CD44 and CD24expression. List mode data analysis and color compensa-tion was performed with WinList software (Verity Soft- ware, Topsham, ME). RESULTS Tables 1 and 2 list the diagnostic cytology evaluationand percentage of ALDH1 bright cells with CD44 þ  /CD24  phenotype in the 36 body cavity fluids (19 peritonealand 17 pleural fluids) with ALDH1 bright cells. Body Cavity Fluids with Malignant Cells Figure 1 shows dot plots of cells from body cavity fluid of a patient with primary breast adenocarcinoma in which diagnostic cytology confirmed the presence of estrogen receptor positive malignant cells (Table 2, PL-63). In Side Scatter vs. Forward Scatter plots, thresholdand electronic gates were used to exclude debris (datanot shown). Dot plots 1A and 1B are respectively of cells incubated with Aldefluor  V R  and Aldefluor  V R  plus ALDH1 blocker (DEAB). Comparison of these two plotsidentifies the cells with high ALDH1 expression in gatesR2 and R3 of SSC low  and SSC high  , respectively. In dotplot 1B of cells incubated with the ALDH1 inhibitor (DEAB), very few cells with ALDH1 bright expression were seen in these two gates. Dot plots 1C and 1Dshow the expression of CD44 and CD24 in ALDH1 bright cells (gates R2 and R3 in Fig. 1A) with SSC low  andSSC high  . Amongst the ALDH1 bright  /SSC low  , (2.66% out of 4.1%) or 64% were CD44 þ  /CD24  (Fig. 1C). In contrast30.7% (1.3% out of 4.1%) of the ALDH1 bright  /SSC low   were double positive for the expression of CD44 andCD24. CD44 þ  /CD24  and CD44 þ  /CD24 þ cells amongstthe ALDH1 bright  /SSC high  cells were 44.6% and 52.6%(1.0% and 1.18% out of 2.24%), respectively as shown inFigure 1D.In contrast to data shown in Figure 1 where the dotplot1A shows two distinct populations of ALDH1 bright cells with low and high side scatter, some of the samplesfrom patients with malignant cells had a diffuse cluster of  ALDH1 bright cells (Fig. 2A, gate R2). These cells were notpresent in the sample incubated with the DEAB inhibitor and thus were presumed to be ALDH1 bright cells. Figure2B shows that most of the ALDH1 bright cells in thissample had CD44 þ  /CD24  expression.In Figure 2C, cells from body cavity fluid of a patient with metastatic breast carcinoma shows the presence of  ALDH1 bright cells with low (gate R2) and high sidescatter. In Figure 2D, gate R3 shows that most of the MARKER EXPRESSION IN CELLS FROM BODY FLUIDS  177 Cytometry Part B: Clinical Cytometry   ALDH1 bright cells with low sided scatter in this samplehad CD44 þ  /CD24  expression. A small population(0.57%) were CD44 þ  with mid level CD24 expression(gate R4). Body Cavity Fluids with Atypical or Inflammatory Cells Figure 3 shows two examples of body cavity fluids in which diagnostic cytology did not find any malignantcells. In dot plot 3A (pleural fluid with atypical, epithe-lial cells), 1.8% of the cells had ALDH1 bright expressionas determined by comparison with dot plots of cellsincubated with ALDH1 inhibitor, DEAB (data notshown). Gated analysis of the ALDH1 bright cells in thissample shown in Figure 3B shows that out of the 1.8% ALDH1 bright cells, 1.17% had CD44 þ  /CD24  expression.In cells from another diagnostic cytology negativesample (pleural fluid with inflammatory cells) shown inFigures 3C and 3D, a distinct population of ALDH1 bright cells (1.66%) was seen. As shown in Figure 3D, most of these cells (0.93%) had positive expression of CD44and CD24. A small population (0.67%) of the ALDH1-bright cells was CD44 þ  /CD24  as shown in gate R3 of Figure 3D. As shown in Table 3, the percentage of ALDH1 bright cells in the 15 body cavity fluids in which presence of malignant cells was confirmed by diagnostic cytology,ranged from 0.26 to 6.34% of the total cells. The Table 1 Characteristics of the 19 Peritoneal (ascites) Fluids Analyzed  Lab ID Cytology diagnosis ALDH1 bright ALDH1 bright  /CD44 þ  /CD24  ASC-27 Atypical cells of epithelial origin 2.24% 2.06%ASC-28 Adenocarcinoma a 2.15% 0.36%ASC-31 Reactive mesothelium, inflammation 1.17% 0.10%ASC-38 Adenocarcinoma, ovarian primary a 1.18% 0.99%ASC-42 No malignant cells detected. 1.63% 1.01%ASC-47 No malignant cells detected. (history of hepatitis) 0.67% 0.06%ASC-48 Adenocarcinoma, ER þ  (history of endocervical neoplasm) a 0.63% 0.02%ASC-52 No malignant cells detected (history of recurrent ovarian cancer) 0.81% 0.57%ASC-59 Adenocarcinoma, (history of ovarian cancer) a 1.95% 1.30%ASC-60 Atypical cells of epithelial origin. 2.03% 1.01%ASC-65 Reactive mesothelium, inflammation 2.59% 0.02%ASC-71 Adenocarcinoma (history of ovarian cancer) a 1.00% 0.74%ASC-73 No malignant cells detected. Acute inflammation (history of cholangiocarcinoma) 0.33% 0.02%ASC-75 Metastatic breast adenocarcinoma, ER þ a 1.52% 1.26%ASC-86 Adenocarcinoma, breast primary, ER þ a 3.10% 0.44%ASC-87 Atypical cells 0.40% 0.11%ASC-89 No malignant cells detected. 2.60% 1.70%ASC-94 No malignant cells detected. 0.43% 0.33%ASC-95 No malignant cells detected (history of chronic liver disease) 1.30% 1.09%ER, estrogen receptor. a Cytopathology diagnosis of malignancy.Table 2 Characteristics of the 17 Pleural Fluids Analyzed  Lab ID Cytology diagnosis ALDH1 bright ALDH1 bright  /CD44 þ  /CD24  PL-26 Atypical epithelial cells 2.09% 0.63%PL-33 Adenocarcinoma a 0.78% 0.73%PL-35 Adenocarcinoma, ER þ , TTF1  a 0.59% 0.42%PL-40 No malignant cells detected 2.04% 0.51%PL-56 Atypical cells of epithelial origin 1.66% 0.67%PL-57 Abnormal large lymphoid cells, consistent with malignant lymphoma a 0.26% 0.10%PL-62 Atypical cells of epithelial origin, inflammation and reactive process 1.48% 0.80%PL-63 Primary breast adenocarcinoma, ER þ a 4.1% (SSC low ) 2.66% (SSC low )2.24% (SSC high ) 1.0% (SSC high )PL-68 No malignant cells detected, reactive mesothelium 0.06% 0.02%PL-74 Atypical cells of epithelial origin, (history of breast cancer) 1.80% 1.17%PL-76 Adenocarcinoma, HER2 þ , ER  , TTF1  a 0.46% 0.35%PL-79 Adenocarcinoma, ER þ  TTF1  a 0.94% 0.68%PL-80 Adenocarcinoma, ER þ , HER2 þ , TTF1  a 1.38% 0.30%PL-81 No tumor cells detected 1.30% 0.14%PL-90 Adenocarcinoma of colon a 1.83% 1.68%PL-91 No tumor cells detected, inflammation 0.17% 0.15%PL-98 No tumor cells detected, (history of squamous cell carcinoma) 1.51% 1.25%ER, estrogen receptor; HER2, HER2/neu; TTF1, thyroid transcription factor 1. a Cytopathology diagnosis of malignancy. 178  KRISHAN ET AL. Cytometry Part B: Clinical Cytometry  percentage of ALDH1 bright  / cells with CD44 þ  /CD24  expression ranged from 0.02 to 3.66%. Amongst the21 body cavity fluids in which diagnostic cytology didnot detect any malignant cells, the percentage of  ALDH1 bright cells was 0.06–2.6% and the percentage of CD44 þ  /CD24  cells was 0.02–1.7%. DISCUSSION Detection and characterization of stem cells in solidtumors offers the possibility that therapeutic modalitiesto selectively target tumor stem cells can be developedresulting in possible control of tumor growth and metas-tasis. However, it is important that markers specific totumor stem cells be identified so as to provide a ration-ale for selective destruction of the tumor stem cells while sparing those in the normal tissues. Expression of cell surface markers (e.g., CD44, CD133), certain detoxi-fying enzymes such as ALDH1 and drug efflux character-istics of stem cells [for identification of side population(SP) phenotype] have been used to identify and sort pu-tative tumor stem cells for further study in vitro and for testing of tumorigenicity in immunodeficient mice(1,6,11–15). Aldehyde dehydrogenases are involved in metabolismof aldehydes to their corresponding carboxylic acids. ALDH1 may have a role in early stem cell differentiationby oxidizing retinol to retinoic acid (16). Increased ALDH1 activity has been reported in murine and humanhematopoietic and cord blood stem cells and in solid tu-mor such as those of pancreas and lung (17–22).Flow cytometric detection of ALDH1 in single cells was made possible by development of a fluorescent sub-strate, BODIPY  V R  aminoacetaldehyde (BAAA), which after intracellular diffusion is converted by ALDH1 intoBODIPY  V R  aminoacetate (BAA), a green fluorescentmarker (10). By blocking ALDH1 with DEAB and com-paring cells incubated with and without the blocker andstained with the Aldefluor  V R  dye, one can rapidly identify the ALDH1 bright cells.Several studies have used the Aldefluor  V R  stainingmethod for identification and sorting of the ALDH1 bright cells and shown that ALDH1 bright cells with low side F IG . 1. Dot plots of cells from pleural fluid of a breast adenocarcinoma patient incubated with Aldefluor V R reagent with or without DEAB inhibitor( A ,  B ), respectively. Gates R2 and R3 show cells with ALDH1 bright  /SSC low (4.1%) and ALDH1 bright  /SSC high (2.24%) expression. Dot plot 1 C  showsthat amongst the ALDH1 bright  /SSC low cells in gate R2, 2.66% and 1.3% were CD44 þ  /CD24  and CD44 þ  /CD24 þ , respectively. ( D ) shows thatamongst the ALDH1 bright  /SSC high cells (gate R3), 1.00 and 1.18% cells were CD44 þ  /CD24 þ , CD44 þ  /CD24  , respectively. MARKER EXPRESSION IN CELLS FROM BODY FLUIDS  179 Cytometry Part B: Clinical Cytometry  scatter (SSC low   ) are capable of forming tumors inNOD-SCID mice (2,4,9). Hess et al. (23) showed that ALDH1 bright cells from human cord blood express primi-tive stem cell markers such as CD34 and CD133. Pearceand Bonnet (24) purified ALDH1 positive lineage negative cells from murine bone marrow and showed that they overlapped with the SP cells. Gentry et al. (25,26)showed that sorted ALDH1 bright cells from cord blood were highly enriched in hematopoietic colony formingcells and ALDH1 bright cells from human bone marrow formed endothelial and fibroblast colonies in culture.Ginestier et al. (2) reported that in normal human andbreast tumors 3–10% of the cells are ALDH1 positive andare more efficient in forming mammospheres in vitrothan the ALDH1 negative cells. In NOD-SCID mice implan-tation of the sorted ALDH1 positive cells (but not the ALDH1 negative cells) led to development of tumors.Besides ALDH1, several other studies have reportedon the expression of markers such as CD44, CD24 andCD133 in tumor cell lines and solid tumors. The impor-tance of these markers was shown by Al-Hajj et al. (5) who reported that as few as 100 CD44 þ  /CD24  andLin  cells isolated from eight out of nine breast tumor patients were capable of tumor formation in NOD-SCIDmice. Wright et al. (6) reported that expression of stemcell associated genes (Oct4, Notch1, ALDH1, Fgfr1, andSox1) was increased in CD44 þ  /CD24  and CD133 þ cells. Croker et al. (4) used breast tumor cell lines toshow that cells with ALDH1 high   /CD44 þ  /CD24  andCD133 þ expression demonstrated increased colony for-mation in vitro and tumsrcenicity and metastasis inNOD-SCID mice as compared to cells with low ALDH1and CD44 þ expression. Similarly, Huang et al. (9) re-ported that ALDH1 positive colorectal cells implanted inNOD-SCID mice generated tumors while the ALDH1 negative cells did not form tumors. In this study, coexpression of CD44 and CD133 with ALDH1 had a modest effect on tu-mor initiation.In contrast to these studies, other workers havereported heterogeneity in expression of these markersin normal and malignant tissues. For example, Honeth et al. (7) reported that 35 and 31% of breast tumors were CD44   /CD24  and CD44 þ  /CD24  , respectively. InHER2 þ tumors, the CD44 þ  /CD24  phenotype was F IG . 2. Dot plots of cells from an adenocarcinoma of colon ( A ) and ovarian carcinoma ( C ) showing the expression of ALDH1 bright cells. Out of the1.83% ALDH1 bright cells in A, 1.68% were CD44 þ  /CD24  as shown in ( B ). In the sample shown in C, out of the 1.95% cells with ALDH1 bright andlow side scatter, 1.3% were CD44 þ  /CD24  and 0.57% were CD44 þ  /CD24 þ ( D ). 180  KRISHAN ET AL. Cytometry Part B: Clinical Cytometry
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