multiplex detection of surface molecules on colorectal cancers

RESEARCH ARTICLE

Multiplex detection of surface molecules on

colorectal cancers

Peter Ellmark1, Larissa Belov1, Pauline Huang1, C. Soon Lee2, Michael J. Solomon3,Daniel K. Morgan1 and Richard I. Christopherson1

1 School of Molecular and Microbial Biosciences G08, University of Sydney, Sydney, NSW, Australia2 Department of Anatomical Pathology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia3 Department of Colorectal Surgery, Royal Prince Alfred Hospital, Camperdown, NSW, Australia

A technique of fluorescence multiplexing is described for analysis of the plasma membraneproteome of colorectal cancer cells from surgically resected specimens, enabling detection andimmunophenotyping when the cancer cells are in the minority. A single-cell suspension wasprepared from a colorectal tumour, and the mixed population of cells was captured on a CDantibody microarray. The cancer cells were detected using a fluorescently tagged antibody forcarcinoembryonic antigen (CEA-Alexa647) or epithelial cell adhesion marker (EpCAM-Alexa488). Using this multiplexing procedure, dot patterns from colorectal cancers were distinctfrom those of adjacent normal tissue. Subtraction of the expression levels for each antigen fromnormal tissue from those for the cancer shows differential expression in the cancer of CD66c,CD15s, CD55, CD45, CD71, CD45RO, CD11b and CEA, in descending order. Cells captured onthe same microarray were also labelled with fluorescent CD3-phycoerythrin antibody revealingthe presence of tumour-infiltrating lymphocytes. The immunophenotypes of T lymphocytesfrom the tumour samples showed differential expression of HLA-DR, TCR a/b, CD49d, CD52,CD49e, CD5, CD95, CD28, CD38 and CD71, in descending order. Fluorescence multiplexing ofmixed cell populations captured on a single antibody microarray enables expression profiling ofmultiple sub-populations of cells within a tumour sample.

Received: June 24, 2005Revised: August 23, 2005

Accepted: September 13, 2005

Keywords:

Antibody microarrays / Cluster of differentiation antigens / Colorectal carcinoma /Immunophenotyping / Multiplexing

Proteomics 2006, 6, 1791–1802 1791

1 Introduction

Colorectal carcinoma is a common cause of cancer deaths inWestern countries [1]. The majority of colorectal cancers areadenocarcinomas that have arisen from benign adenomasdue to accumulation of multiple mutations [2, 3]. An efficient

and reliable staging system is critical for prognosis and clin-ical decisions for post-operative adjuvant treatment. Thecurrent staging methods rely on modifications of the Dukesclassification system [4, 5] based on clinical, radiological andhistopathological assessments. An alternative and moreaccurate staging system for the cancer could be based uponan extensive immunophenotype (partial membrane pro-teome) determined using an antibody microarray, wherepatterns of protein expression can be correlated with sub-types of the cancer. Such an immunophenotype would reflectthe genetic program of the malignant cells.

A cluster of differentiation (CD) antibody microarray(DotScan) has been developed that enables identification of 82surface antigens on leukaemia cells, providing an extensiveimmunophenotype from a single analysis [6, 7]. Immunophe-notypes have been obtained for leukaemias from more than

Correspondence: Professor Richard I. Christopherson, School ofMolecular and Microbial Biosciences, University of Sydney, NSW2006, AustraliaE-mail: [email protected]: 161-2-9351-4726

Abbreviations: ALL, acute lymphocytic leukaemia; AML, acutemyeloid leukaemia; CD, cluster of differentiation; CEA, carci-noembryonic antigen; CLL, chronic lymphocytic leukaemia;DotScan, a microarray of antibodies for cell capture; EpCAM, epi-thelial cell adhesion marker; TIL, tumour-infiltrating lymphocytes

DOI 10.1002/pmic.200500468

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

1792 P. Ellmark et al. Proteomics 2006, 6, 1791–1802

1100 patients using the DotScan technology in which capturedleukocytes are imaged directly without staining using an opticalscanner [7]. This technique enables immunophenotyping ofhigh-level leukaemias in peripheral blood when the total leuko-cyte count is greater than 106109 cells/L, with normal periph-eral blood leukocytes constituting approximately 3.66109 cells/L. We have found that an extensive immunophenotype is suffi-cient to diagnose the major sub-types of leukaemia: acute mye-loid leukaemia (AML), acute lymphocytic leukaemia (ALL) andchronic lymphocytic leukaemia (CLL) ([6, 7], and manuscript inpreparation). The immunophenotype of cancer cells may alsoprovide prognostic information and identify highly expressedknown CD antigens as targets for therapeutic antibodies. Forexample, Rituximab binds to CD20 and is used to treat non-Hodgkin’s lymphoma, Mylotarg binds to CD33 and is used forAML, and Campath-1H binds to CD52 and is used for B cell CLL[8]. Therapeutic antibodies may be cytostatic by blocking a cyto-kine receptor, or cytotoxic if the antibody carries a drug, toxin orradioisotope. Alternatively, the Fc domain of a therapeutic anti-body bound to a target cell may induce antibody-dependent cel-lular cytotoxicity (ADCC) or complement-dependent cytotoxi-city (CDC) [8]. Today, 20% of all biopharmaceuticals in clinicaltrials are therapeutic antibodies [9].

In contrast to high-level leukaemias, cell suspensionsmade from biopsies of colorectal cancers may contain aminority of cancer cells and a variety of other cell types, suchas normal columnar mucosal cells, goblet cells, Argentaffincells, Paneth cells, fibroblasts, tumour-infiltrating lympho-cytes (TIL), leukocytes from lymph nodes, and even smoothmuscle cells. The resulting immunophenotype from anoptical scan of cells captured on an antibody microarraywould reflect the heterogeneity of cells in the biopsy. In thisstudy, we have visualized the epithelial cells captured on theDotScan microarray by labelling these cells with fluores-cently labelled antibodies against surface molecules foundpredominantly on colorectal cancer cells (carcinoembryonicantigen, CEA, or epithelial cell adhesion molecule, EpCAM),and T lymphocytes (CD3 or CD4). These fluorescently label-led cells on the microarray were visualised with a laser scan-ner to provide three immunophenotypes (dot patterns) ofsub-populations of cells captured on a single microarray. Theimmunophenotypes of colorectal cancer cells obtained usingthis fluorescence multiplexing technique were distinct fromnormal adjacent tissue, and multiple cell populations in atumour sample could be analysed simultaneously. Withminor modifications, this technique would be applicable toimmunophenotyping cancer cells from other solid tumours.

2 Materials and methods

2.1 Antibodies

An mAb against EpCAM was obtained from Becton Dick-inson (Franklin Lakes, NJ, USA) and against CEA (akaCD66e) from Serotec (obtained from Australian Laboratory

Services, Sydney, Australia). These colorectal cancer-specificantibodies were labelled with Alexa488 and Alexa647,respectively, using the Alexa Fluor antibody labelling kit(Molecular Probes, Eugene, OR, USA). The fluorophore-conjugated antibodies, CD3-phycoerythrin (PE), CD4-PE andCD21-FITC, were obtained from Beckman Coulter (Glades-ville, NSW, Australia).

2.2 Tumour samples and cell lines

Tissue samples were obtained from Royal Prince AlfredHospital, (Camperdown, NSW, Australia). Samples wereobtained with informed consent under the approved ProtocolNo. X03–0343. The human cell lines, CCRF-CEM, LIM,SW480, CaCo and Raji cells were grown in RPMI 1640 me-dium (Sigma-Aldrich Pty. Ltd., Sydney, Australia) supple-mented with 10% foetal calf serum (FCS; Invitrogen, Carls-bad, CA, USA), 2 mM glutamine (Invitrogen), and 50 mg/mLgentamicin sulphate (Invitrogen). T84 cells were grown inDMEM HAMS/F12 medium (Invitrogen) with the sameadditives.

2.3 Preparation of single-cell suspensions from

colorectal tissue

The tissues used were obtained from patients having aresection for colorectal cancer; the normal tissue wasobtained from the same resection specimen adjacent to thetumour. Tissue samples were placed immediately in coldHanks’ buffer (Sigma-Aldrich) to maintain cell viabilityessential for cell capture. The samples were cut into 2-mmstrips and incubated with 2% collagenase in RPMI 1640 at377C for 1 h. The semi-digested tissue was then forcedthrough a fine wire mesh tea strainer to obtain discrete viablecells. Larger cell aggregates were removed with a 50-mm Fil-con filter membrane (Becton Dickinson). The cells were thentreated with 0.1% DNase for 20 min at room temperature todigest DNA released from lysed cells. If contamination witherythrocytes was significant, they were separated from othercells by centrifugation through Histopaque. Alternatively,erythrocytes were removed using lysis buffer (0.15 Mammonium chloride, 10 mM potassium hydrogen carbon-ate, 0.1 mM EDTA).

2.4 Capture of cells on DotScan microarrays

The CD antibody microarrays were constructed as duplicatedots (10 nL) on NC FAST slides (Schleicher and Schuell,Keene, NH, USA), as described previously [7]. Cells weresuspended at a density of 0.66107–1.36107/mL in 230 mL ofgrowth medium (RPMI 1640 containing 10% FCS and 2%heat-inactivated human AB serum), and incubated on themicroarray at 377C for 1 h. Unbound cells were gentlywashed off with PBS, and captured cells were fixed to themicroarrays by incubation in PBS containing 3.7% formalinat 377C for 15 min.


Proteomics 2006, 6, 1791–1802 Protein Arrays 1793

2.5 Fluorescence multiplexing to immunophenotype

sub-populations of cells

An optical image of the dot pattern of captured cells wasobtained with a DotScan array reader (Medsaic Pty. Ltd., Eve-leigh, NSW, Australia). Microarrays were then incubated withblocking buffer (PBS containing 1% BSA and 2% heat-inacti-vated human AB serum) for 20 min at room temperature. Theblocking buffer was carefully drained from the microarrayand fluorophore-labelled antibodies (3.3–5 mg/mL in 150 mLblocking buffer) were incubated with the captured cells for30 min at room temperature. The microarray was washedwith PBS and scanned at 488, 532 and 647 nm using a Molec-ular Imager FX Pro (Bio-Rad, Hercules, CA, USA) or Typhoon8600 Variable Mode Imager (Amersham-Pharmacia, CastleHill, NSW, Australia). The optical and fluorescent imageswere analysed using DotScan software (Medsaic). Data be-tween different tissue samples were normalized using aninternal control (see below). CD29 expression for cancer andadjacent normal tissue was the same for colorectal biopsiesstained with fluorescent EpCAM and CEA. Thus, normal-ization of fluorescence was done relative to expression of

CD29, used as a “house-keeping” antigen. For fluorescencescans obtained using CD3 staining, CD44 expression wasused as a house-keeping antigen, the data were normalizedrelative to that antigen. Fluorescence intensities for each CDantibody are the average values from duplicate dots in themicroarray. The background value for the corresponding iso-type control antibody was subtracted from the intensity valuefor each dot in the microarray. Hierarchical cluster analysiswas performed using Spotfire software (version 8.0, SpotfireAB). Unsupervised clustering was performed on the normal-ized and background adjusted data, using the Un-weightedPair-Group Method with Arithmetic mean. Euclidean dis-tance was used for similarity measure, and the antigens weresorted based on the average values of the rows.

3 Results

3.1 Immunophenotypes of colorectal cancer cell lines

To investigate the potential of the DotScan microarray foranalysis of colorectal cancer tissue, five different colorectalcancer cell lines were analysed (Fig. 1). Although the micro-

Figure 1. Immunophenotypic analysis of colorectal cancer cell lines. (A) Addresses of the CD antibodies for onemicroarray of duplicates on a slide without the CD44 alignment dots (see square in B). Immunophenotypes areshown as dot patterns of captured cells for (B) HT29, (C) SW480, (D) CaCo, (E) LIM, (F) T84.



arrays were designed for leukocytes, 7–12 distinct spots ofcaptured cells could be detected with the colorectal cancercell lines. The following CD antigens were positive for HT29:CD9, CD15, CD24, CD29, CD44, CD66c, and CD71; forSW480: CD9, CD15, CD29, CD44, CD71, and CD95; forCaCo: CD9, CD15, CD29, CD44, CD66c, and CD71; for LIM:CD9, CD10, CD15, CD24, CD29, CD33, CD44, CD49e,CD66c, CD71, CD77, and CD95; for T84: CD9, CD13, CD15,CD24, CD29, CD31, CD44, CD66c, CD71, and CD95. CDantigens common to all these cell lines were CD9, CD15,CD29, CD44 and CD71. CD66c (CEA family) and CD95enabled discrimination between the cell lines.

3.2 Fluorescence multiplexing of a mixture of cells

The procedure for determining immunophenotypes of sub-populations of cells in a single analysis was developed usingan equal mixture of three human cell lines: T84 colorectalcancer, Raji B cell lymphoma and CCRF-CEM T cell leukae-mia. Figure 2 shows dot patterns from a single microarrayfor this mixture of cell lines. The optical scan (Fig. 2B)represents the collective immunophenotype of the mixed cellpopulation, while the dot patterns obtained for the threefluorescently labelled antibodies (CEA-Alexa647, CD4-PE,and CD21-FITC) are shown in Fig. 2C–E. The intensities of

Figure 2. Immunophenotypic analysis of a mixture of the human cell lines T84 (colorectal cancer), CCRF-CEM (T cell ALL), and Raji (B celllymphoma). Cells (136106/mL, 230 mL) were incubated on a single DotScan slide and the optical dot pattern for captured cells wasrecorded. These cells were then labelled with a mixture of antibodies tagged with fluorophores: anti-CEA-Alexa647, anti-CD4-PE, and anti-CD21-FITC. (A) Addresses of the CD antibodies for one microarray; (B) optical scan of captured cells showing the duplicate microarrays; (C)cells labelled with anti-CEA-Alexa647 (blue); (D) anti-CD4-PE (red); (E) anti-CD21-FITC (green); (F) composite image for the three fluores-cently labelled antibodies, where white dots are positive for all three antibodies, yellow dots are both CD41 and CD211, and purple dots areboth CD41 and CEA1cells.



Figure 3. Expression profiles forthe mixture of human T84,CCRF-CEM and Raji cell linesfrom Fig. 2. (A) Optical scan ofthe mixture of T84, CCRF-CEMand Raji cells (from Fig. 2B); flu-orescent scans to visualize cellscaptured on the same micro-array and stained with the anti-bodies (C) CEA-Alexa647 (fromFig. 2C), (E) CD4-PE (fromFig. 2D), (G) CD21-FITC (fromFig. 2E). For comparison, opticalscans are shown for each cellline analysed on individualmicroarrays (136106 cells/mL,230 mL). (B) T84, (D) CCRF-CEM,and (F) Raji. Dot intensities werequantified with ImageQuantsoftware and bar charts weregenerated using DotScan soft-ware, the data are not normal-ized.



Figure 4. Detection of T84 colo-rectal cancer cells diluted in aleukocyte mixture. (A) Opticalscan of T84 cells alone. (B–E)T84 cells diluted in equal num-bers of Raji and CCRF-CEM cellswith a constant total cell countof 136106/mL and detected byfluorescence multiplexing usingthe CEA-Alexa647 antibody. (B)T84 cells 20% of total cell count,(C) 10%, (D) 5%, (E) 2.5%(0.336106 cells/mL). The dataare not normalized.



the dots in Fig. 2B–E are quantified as bar charts in Fig. 3A,C, E, G, respectively. The bar charts for the three fluores-cence scans of the cell mixture (Fig. 3C, E, G) are almostidentical to those for optical scans of the individual cell lines(Fig. 3B, D, F), showing that this fluorescence multiplexingtechnique enables determination of the immunophenotypeof a particular sub-population of cells in a mixture.

The sensitivity of fluorescence multiplexing for immu-nophenotyping a minority of cancer cells in a colorectalbiopsy was tested with a series of dilutions of T84 cells, withequal numbers of Raji and CCRF-CEM cells and a constanttotal cell count of 136106/mL. Figure 4 shows expression ofthe same CD antigens for T84 cells diluted over the range4.36106–0.336106 cells/mL. When the T84 cells werediluted 1:40 to 0.336106 cells/mL (Fig. 4E), some of the CDantigens, notably CD24, CD31, and CD95, were not apparentin the fluorescent immunophenotype. The fluorescence ofthese three CD antigen spots diminished differentially, butother antigens characteristic of this colorectal cancer cellline, CD9, CD15, CD29 and CD66c, were still detectablewhen T84 cells were diluted 1:40. This result shows thatimmunophenotypes characteristic of colorectal cancer maystill be obtained from biopsies where the proportion ofmalignant cells is low. Cells captured on antibody dots visu-alized optically (Fig. 2B), but absent (CD24, CD31 and CD95)or depleted (CD4 and CD21) by fluorescence, may haveselective depletion of the antigen on the exposed surface ofcaptured cells. This effect, more obvious at lower densities ofcells in a mixture, may be due to capping of surface antigensin an active process on cells [10] to the contact area betweenthe cell and the antibody-microarray surface during the 60-min incubation. Evidence for such a time-dependent cappingprocess has been obtained from experiments where cellswere captured by CD antibodies immobilized on Biacorechips (L. Bransgrove and R. I. Christopherson, unpublishedresults).

3.3 Expression profiles of colorectal tissues

The surface antigens, EpCAM and CEA, are expressed oncolorectal cells and the dot patterns obtained after labellingthe same microarray with EpCAM-Alexa488 and CEA-Alexa647 are similar, as illustrated in the heat map ofFig. 5A, B. Fluorescent labelling of captured cells from colo-rectal biopsy samples with CD3-PE shows distinct T lym-phocyte patterns for normal and colorectal cancer tissues(Fig. 5C). Data from spots that corresponded to the sameantibody as the staining antibody (i.e., the CEA dot from theCEA staining, the CD3 dot from the CD3 staining and theEpCAM dot from the EpCAM staining) were disregardedbecause of the possibility of capping of that CD antigen to-ward the antibody dot with consequent depletion of the anti-gen on the exposed surface of the captured cell. Cells fromcancer tissue (Fig. 5) express high levels of CD9, CD15,CD29, CD66c, CD71, similar to the colorectal cell lines(Fig. 1).

To identify differential expression of surface antigens oncolorectal cancers (T–N), the expression levels of antigens fornormal colorectal cells (N) were subtracted from the corre-sponding values for cancer cells (T, Tables 1, 2). Only anti-gens with greater than 1.5-fold differential expression areincluded in the tables. Reproducibility of DotScan array datahas been tested using the human myeloid cell line, HL60 byWhite et al. [11]. Mean intensity values were calculated fromtriplicate arrays, each of which contained duplicate antibodydots, the error bars representing standard errors of these sixmeasurements were small (see Figs. 1 and 2 of [11]). The cut-off of 1.5-fold differential expression used here is certainlygreater than the inherent error in the measurement, al-though changes greater than 1.5-fold would have more sig-nificance.

Changes in dot intensities between cancer and normaltissue may represent up-regulation of a particular antigen oneach cancer cell, or an increasing population of a particularcancer cell type that expresses the antigen. For a monoclonalcancer, the former is true. Surface molecules differentiallyexpressed on colorectal cancer using the DotScan microarraywere CD66c, CD15s, CD55, CD45, CD71, CD45RO, CD11band CEA, in descending order. The results from the CEAstaining (Table 2) revealed fewer significant changes(p,0.05) between tumour and normal samples than theEpCAM staining (Table 1) attributed to differences inexpression levels, and/or the affinity of the antibody. Simi-larly, T lymphocytes from tumours (T) showed differentialexpression (T - N) of HLA-DR, TCR a/b, CD49d, CD52,CD49e, CD5, CD95, CD28, CD38 and CD71, in descendingorder (Table 3). Some additional surface antigens were up-regulated on a few tumours relative to normal tissue (datanot shown). Such variation may indicate different tumourtypes or stages of transformation. Staining of colorectal can-cers with EpCAM-Alexa488 and CEA-Alexa647 revealedsome expression of CD15s and CD55 that may be useful forsub-classification of colorectal cancer stages.

Unsupervised hierarchical cluster analysis of the sam-ples, using the antigens that differ significantly between thenormal and tumour samples (Fig. 6) shows that the tumoursamples preferentially cluster together, indicating thisapproach may be of diagnostic value. The data set from the Tcell staining (CD3) also cluster together, with one exception(Fig. 6C). Our results demonstrate the potential of array-based diagnosis, where patterns of antigen expression enablediscrimination between healthy and diseased samples.

4 Discussion

Mixtures of cells captured on the DotScan microarray can belabelled with fluorescent antibodies to determine immuno-phenotypes of sub-populations of cells by fluorescence mul-tiplexing. The procedure was developed using a mixture ofthe T84 colorectal cancer cell line with T and B lymphoid celllines as the model system. While the immunophenotype of



Figure 5. Comparison of immunophenotypes ofcolorectal cancers and normal tissues. Capturedcells were stained with the fluorescent anti-bodies: (A) EpCAM-Alexa488; (B) CEA-Alexa647,and (C) CD3-PE. Heat maps show average inten-sities of antigen expression for colorectal cancerbiopsies at left (n = 12 for the EpCAM staining,n = 14 for the CEA staining, and n=9 for CD3staining,) and normal tissue at right (n = 11 forthe EpCAM staining, n = 7 for the CEA staining,and n=9 for CD3 staining,). Green indicates lowvalues, black middle values, and red high values.The number of samples stained with each anti-body varied because some samples onlyexpressed low levels of CEA or EpCAM, and awell-resolved dot pattern was not obtained.

the T84 cells could not be determined from the optical scanof the mixture (Figs. 2B, 3A), fluorescence multiplexingenabled immunophenotyping for the T84 cells to levels aslow as 2.5% (Fig. 4E). Although the relationship betweenfluorescence intensity and number of cells applied was notlinear for all antigens, this semi-quantitative method could

distinguish between cancer and normal tissue obtainedfrom patients having a resection for colorectal cancer(Figs. 5, 6; Tables 1, 2). The main focus of this method is todistinguish between normal and transformed cells and tofind strongly expressed antigens that may be therapeutictargets.



Figure 6. Hierarchical clustering analysis for samples of colo-rectal cancer and normal tissue from Fig. 5. Unsupervised hier-archical clustering analysis was performed using data for anti-gens that differ significantly between tumour and normal sam-ples (see Tables 1–3). Data are shown for all samples from Fig. 5stained with the fluorescent antibodies: (A) EpCAM-Alexa488, (B)CEA-Alexa647, and (C) CD3-PE.

A number of surface antigens were expressed at higherlevels on cells recovered from colorectal cancer than fromnormal tissue adjacent to the cancer; notably CD66c, CD15s,CD55, CD45, CD71, CD45RO, CD11b, CEA, CD44, CD13,

CD43, CD11a and CD24 (Figs. 5, 6; Tables 1, 2). Most ofthese antigens are already known to be overexpressed oncolorectal carcinomas. EpCAM is an adhesion moleculefound on normal epithelial cells [12], but often overexpressedby colorectal cancers. CEA (CD66e) and the closely relatedCD66c, both members of the carcinoembryonic antigenfamily, are expressed by epithelial cells and are up-regulatedon colorectal cancers [13]. CD71, the transferrin receptor, isup-regulated on proliferating cells [14] and is a commonneoplastic marker. CD43 is also expressed on colorectal can-cers, and may have an anti-apoptotic effect on cells. CD11b,normally only expressed on some types of leukocytes, isstrongly associated with metastasis and tumour growth andmay be useful as a prognostic and diagnostic marker [15].The Lewis antigen, CD15s (sialyl Lewis X), is expressed on ahigh proportion of colorectal cancers and is associated withmetastasis and poor prognosis. CD55 (decay acceleratingfactor, DAF) is a complement resistance factor that may limitcomplement-dependent cytotoxicity (CDC) induced by mAbbound to target cells. CD55 has been implicated in tumor-igenesis and is overexpressed on many colorectal cancers[16]. No significant down-regulation of antigens was detectedin the colorectal cancer samples relative to the normal sam-ples (Tables 1, 2), possibly due to the choice of antibodies onthe microarray. Some of the antibodies were specifically cho-sen for the known overexpression of the antigen on tumourcells, while other antibodies recognise antigens expressed oninfiltrating leukocytes or up-regulated upon inflammationassociated with tumour progression.

A comparison of T lymphocyte populations detected intumour biopsies and normal tissues using imaging withanti-CD3-PE, showed increased expression of the followingantigens in tumours: HLA-DR, TCR a/b, CD49d, CD52,CD49e, CD5, CD95, CD28, CD38, CD71, CD29, CD7, CD4and CD134 in descending order (Table 3). Of these surfaceantigens, CD38, CD71, CD134 and HLA-DR are known to beelevated on T lymphocytes in states of immune activation[17, 18]. These results could be attributed to a different pop-ulation of T lymphocytes and perhaps also to increasednumbers. For example, there is increased expression ofCD38, a T cell activation marker [19] and CD71, the transfer-rin receptor up-regulated on proliferating cells. There is alsoincreased expression of CD3 and TCRa/b, responsible forantigen recognition. CD2 is an accessory adhesion moleculealso important for T cell activation, CD28 is an important co-stimulatory molecule, whereas CD4 is a co-receptor [20].Expression levels of CD4 and CD8 may also be relevant toprognosis of colorectal cancer since it has been shown [18]that patients with a low CD4/CD8 ratio among CD31 cells (Tcells) have a significantly higher 5-year survival [18]. Theresults from the T cell staining, which displayed differencesin the infiltrating leukocyte population between tumour andnormal samples, demonstrate the advantage of simulta-neous analysis of multiple sub-populations captured on aDotScan microarray. The combined information from sev-eral of the sub-populations, (in addition to information



Table 1. Expression profiles of EpCAM-positive colorectal cancer cells and adjacent normal cells

Antigen Tumour(intensity, T)a)

Normal(intensity, N)a)

Differentialexpression (T–N)

Foldchange (T/N)

p valueb)

CD66c 136.9 4.5 132.4 30.6 0.0014CD15s 85.5 0.6 84.9 142.3 0.019CD55 67.0 1.1 65.9 59.7 0.027CD45 50.7 2.2 48.5 23.1 0.0015CD71 56.4 8.5 47.9 6.7 0.0065CD45RO 50.7 3.7 47.0 13.6 0.0097CD11b 47.3 6.0 41.3 7.9 0.0059CEA 49.9 12.8 37.1 3.9 0.035CD44 48.2 16.4 31.8 2.9 0.017CD13 36.7 6.2 30.5 6.0 0.014CD43 36.1 6.1 30.0 5.9 0.0091CD11a 35.7 7.5 28.2 4.7 0.0090CD24 31.5 5.0 26.6 6.3 0.0038

a) Values for fluorescence intensity for each sample of Figs. 5 and 6 were normalized against CD29 (constant fortumours and normals) on a scale from 0 to 255 and mean values for tumour (n = 12) and normal (n = 11) sam-ples are shown. Only antigens with an average difference .25, and fold change .1.5 are shown.

b) Two-sided Student’s t-test.

Table 2. Expression profiles of CEA-positive colorectal cancer cells and adjacent normal cells




Foldchange (T/N)

p valueb)

CD66c 232.9 27.7 205.2 8.4 0.0000044CD15s 189.8 4.9 184.9 38.7 0.0035401CD55 97.8 5.4 92.4 18.0 0.0033520CD71 93.2 16.2 77.0 5.7 0.0023340

a) Values for fluorescence intensity for each sample of Figs. 5 and 6 were normalized against CD29 (constant fortumours and normals) on a scale from 0 to 255 and mean values for tumour (n = 14) and normal (n = 7) samplesare shown. Only antigens with an average difference .35, and fold change .1.5 are shown.

b) Two-sided Student’s t-test.

Table 3. Expression profiles of CD31 T lymphocytes from colorectal cancers and adjacent normal tissue




Foldchange (T/N)

p valueb)

HLA-DR 146.6 32.5 114.2 4.5 0.00001TCR a/b 174.7 67.4 107.3 2.6 0.00005CD49d 167.2 65.4 101.8 2.6 0.00458CD52 236.9 136.1 100.9 1.7 0.02913CD49e 116.5 16.6 99.9 7 0.0008CD5 208.4 115.6 92.8 1.8 0.00645CD95 157.7 65.2 92.5 2.4 0.005CD28 123.9 32.8 91.1 3.8 0.00267CD38 148.8 60.3 88.6 2.5 0.00037CD71 99.6 14.6 85 6.8 0.0013CD29 225.9 146.7 79.2 1.5 0.0007CD7 171.9 100.7 71.2 1.7 0.04473CD4 83 34.3 48.7 2.4 0.03203CD134 53.1 7.7 45.3 6.9 0.00045

a) Values for fluorescence intensity for each sample of Figs. 5 and 6 were normalized against CD45 (constant for Tlymphocytes) on a scale from 0 to 255 and mean values for tumour (n = 9) and normal (n = 9) samples are shown.Only antigens with an average difference .35 and fold change .1.5 are shown.b) Two-sided Student’s t-test.



regarding lymph node status and presence of metastasis), ina particular tumour could provide a better prognosis for thepatient.

The unsupervised cluster analysis (Fig. 6A, B) indicatesthat the microarray/multiplexing method described here canbe used to discriminate between diseased and normal cellsamples and may have use as a diagnostic tool. Althoughthere did not appear to be any clustering of different clinicalstages of the tumour samples, according to the Dukes clas-sification or to the International Union Against Cancer(UICC) classification (data not shown), the number of sam-ples of each sub-class was too small for detailed investiga-tion. Some surface antigens such as CD15s and CD55 wereonly detected on some tumours, and virtually absent in nor-mal samples. Addition to the microarray of colorectal-specif-ic antibodies or antibodies against other surface moleculesmay enable sub-classification of colorectal cancers.

An extensive immunophenotype of a colorectal cancercould also enable selection of a suitable therapeutic antibodyfor the patient. A number of therapeutic antibodies are underdevelopment for treating colorectal cancers. Edrecolomabtargets EpCAM [21], a human cell surface glycoproteinexpressed on some normal and most neoplastic epithelialcells. The therapeutic antibody, huAb A33, binds to A33, acell surface glycoprotein expressed in normal human colo-rectal and small bowel epithelium and on greater than 95%of human colorectal cancers [22]. A33 is absent from mostother human tissues and tumours. Cetuximab, a chimericmAb specific for EGFR, is effective in the treatment of somemetastatic colorectal cancers [23]. In the future, all availabletherapeutic antibodies could be spotted on the microarray,and suitable antibodies, or combinations of antibodies couldbe selected for therapy of individual patients.

In the present research, we have used three fluo-rophores and a laser scanner for imaging. Additional anti-bodies labelled with other fluorophores could be used todetermine immunophenotypes of other sub-populations ofcells in the biopsy sample. Antibody microarrays capturingintact cells provide an alternative procedure to methodswhere soluble proteins are analysed [24], or tumour anti-gens are arrayed and antibodies from patient serum arebound [25]. Cell capture enables easy detection of mem-brane proteins that may be difficult to solubilise and analyseby standard methods. The DotScan technology enablesdetermination of new patterns of expression of known CDantigens on cells, while 2-DE or multi-dimensional chro-matography enables complete analysis of the membraneproteome with the prospect of discovery of new proteins.These two approaches are complementary. This reportextends the use of a CD antibody microarray to obtainextensive immunophenotypes of multiple sub-populationsof cells in a colorectal biopsy specimen where the cells ofinterest may be in the minority. With some modification,fluorescence multiplexing of mixed populations of cellscaptured on a DotScan microarray could be used for analy-sis of other tissues and solid tumours.

We thank Professor John Wallace and Ms Briony Forbes ofthe Department of Molecular Biosciences, University of Ade-laide, Australia for providing several human colorectal cancercell lines. P.E. received a fellowship from the Bengt LindqvistMinnesfond. This study was supported in part by researchfunding from Medsaic Pty. Ltd. to R.I.C. The DotScan assay issubject to intellectual property rights and information may beobtained from Medsaic Pty Ltd, Suite 145, National InnovationCentre, Australian Technology Park, Garden Street, Eveleigh,NSW 1430, Australia. DotScan is a microarray of CD anti-bodies dedicated to the memory of Mrs. Lee Dixon. Note: Severalof the authors (L.B., P.H. and R.I.C.) have declared a financialinterest in a company (Medsaic Pty. Ltd.) whose product(DotScan) was studied in the present work. Several of theauthors (L.B. and P.H.) are employed by the company (Med-saic Pty. Ltd.) whose product was used in the present work. Oneof the authors (R.I.C.) holds a patent related to the work that isdescribed in the present study.

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multiplex detection of surface molecules on colorectal cancers

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