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lournol of Immunological Methods, 147 (1992) 57-64

e

1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00

JIM 06188

A rapid method for separating tumour infiltrating cells and tumour cells from colorectal tumours L.G. Durrant Cancer Research Campaign Laboratories, University of Nottingham, Nottingham NG/4 7BN, UK (Received 4 September 1990, revised received 2 August 1991, accepted 4 October 1991)

As solid tumours are composed of a heterogeneous mixture of cells the role of any particular cell type is difficult to analyse. A rapid method of sorting freshly disaggregated highly viable single cell suspensions of colorectal tumours has therefore been developed. Infiltrating cells were initially sorted from tumour cell suspensions using magnetic beads coated with a cocktail of monoclonal antibodies recognising leucocyte common antigen, CD45 (lymphocytes), CDw14 (macrophages and granulocytes) and Thy-l antigen (stromal cells). Between 4-10 X 10 6 cells, 70-87% pure, were rapidly sorted. The tumour cells were then sorted from the remaining cell suspension using a cocktail of monoclonal antibodies which recognise 100% of colorectal tumours. Between 2-7 X 10 6 tumour cells, 70-87% pure, were rapidly sorted. Both populations of cells were 95% viable and able to grow in vitro. Key words: Colorectal tumor; Tumor infiltrating lymphocyte; Cell sorting

Introduction

Solid tumours are composed of mixture of both tumour cells and infiltrating cells. Understanding the individual roles of each cell type and the interactions between them is extremely important and would be facilitated if highly viable populations of each cell type could be rapidly produced.

Correspondence to: LG. Durrant, Cancer Research Campaign Laboratories, University of Nottingham. Nottingham NG7 2RD, UK. Abbreviations: PWM, pokeweed mitogen; IGF-I, insulin like growth factor 1.

Previous studies have isolated tumour infiltrating lymphocytes by density gradient separation on Ficoll (Vose et aI., 1984), Percol or Nycodenz gradients. Although these methods produce large numbers of highly viable lymphocytes the recovery of tumour cells is very poor. Furthermore the technique is time consuming. Tumour cells have also been sorted by flow cytometry (Durrant et aI., 1986a) and although viable tumour cells can be recovered the yields are low. Isolation of neuroblastoma cells from bone marrow has been achieved using magnetic microspheres labelled with monoclonal antibodies (Kemshead et aI., 1986) and we have used a similar approach to sort both tumour cells and infiltrating cells from colorectal tumours. This method rapidly yields large numbers of highly viable infiltrating and tumour cells.

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Material and methods

eel/lines

CI46 cells, derived from a colorectal adenoma (Durrant et aI., 1986b) and bearing the epitope defined by the monoclonal antibody NCRC37 (Durrant et aI., 1989a, b) but not expressing CEA, were used in these studies. MKN45 cells (Hojo et aI., 1977), expressing CEA antigen but not staining with NCRC37 monoclonal antibody were also used. Both cell lines were routinely grown in DMEM supplemented with 10% newborn calf serum (Gibco, Paisley, Fife).

Lymphocytes Peripheral blood lymphocytes were isolated on Lymphoprep separation medium (Flow Labs, Irving, Fife) as previously described (Durrant et aI., 1984).

Tumours Solid tumours were finely minced and disaggregated in 0.05% collagenase and 0.05% elastase as previously described (Durrant et aI., 1986a, b). Cells were washed three times in medium containing 10% FCS and 0.02% EDTA and then filtered through a narrow gauge gauze. Only single cell suspensions which were > 90% viable were sorted.

Monoclonal antibodies Anti-infiltrating antibodies.

F15-42 reactive with human Thy-l (McKenzie and Fabre, 1981), FlO-89-4 reactive with human leucocyte common antigen, CD45 (Dalchau et al., 1980) and Bear-l reactive with CDw14 were obtained from Serotech (Bicester, UK). Anti-tumour antibodies. NCRC37 is an IgG 1 antibody which recognises an antigen on the cell surface of colorectal tumour cells (Durrant et aI., 1989). NCRC23 is an IgGI antibody which recognises CEA but not NCA (Price et al., 1987). NCRC34 specifically recognises cytokeratin 18, which is expressed by all colorectal tumour cells, and bas been produced in our laboratory.

Mitogenicity studies Single cell suspensions of unsorted or sorted cells were plated into 96 well tissue culture plates

in a 1: 1 mixture of HamsF10: MEM medium containing 0.05% BSA at a density of 10 5 cells per well. They were then pulsed for 2-3 days with 1-100 ng/ml of recombinant insulin like growth factor I OGF-I, Bachem) or gastrin (G-17, Sigma, Poole, Dorset) or with 10 ILg/ml of pokeweed mitogen (PWM). During the final 12 b of the study [ 75 Se]selenomethionine was added to monitor protein synthesis. This has previously been shown to correlate with cell growth (Durrant et al., 1991) in colorectal tumours which have very diverse intracellular thymidine pools and therefore cannot be assessed for proliferation using this label. For direct comparison the lymphocytes were also assessed for cell activation by selenomethionine incorporation. Labelled cells were counted directly using a gamma counter. The results were expressed as mean ± SO of six wells and were analysed for significance using Student's t test.

Magnetic bead sorting Initially sheep anti-mouse Ig coated Dynabeads M-450 (Oynal, Oslo, Norway) were coated with either a combination of anti-Thy-l, anti-LCA and anti-COw14 or a combination of NCRC23 and NCRC37. 50 ILl of beads and 6 ILg of each antibody were incubated together at 4°C in the dark for 4 h. The beads were washed several times in PBS containing 1% newborn calf serum prior to adding to cells. 2 X 10 7 beads were added to 2 X 107 cells in 5 ml of PBS: NBCS. Cells attached to magnetic beads were removed and washed using magnetic separation (MPC 1, Dynal). The cells were more efficiently sorted following coating with the appropriate monoclonal antibodies (10 IL g of each antibody for 30 min) and then incubating with sheep anti-mouse Ig coated dynabeads M-450 and magnetic separation. Results The predominant infiltrating population in colorectal tumours are lymphocytes and although the proportion varies between individual tumours at least 50% of the cells within a tumour are of lymphoid origin (Durrant et al., 1987). Initial

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experiments were therefore carried out on 10 7 cells of a colorectal cell line C146 admixed with 107 lymphocytes. Magnetic beads were coated with a combination of anti-colorectal tumour monoclonal antibodies, NCRC23 and NCRC37. The number of beads added to the tumour: lymphocyte mixture ranged from 2 X 10 7 to 8 X 10 7(Table I). There was no improvement in yield following the addition of more beads. After the initial separations a new aliquot of coated beads was added to the original suspension but the recoveries of cells from these second separations were only 40-70% of the initial recovery. In the final experiment a third aliquot of beads was added to the original suspension but only a further 13% of cells were recovered. In a separate experiment, cells were precoated with monoclonal antibodies and sorted on beads coated with high affinity rabbit anti-mouse Ig. The efficiency of sorting was increased from 34 + 4% to 92% (Table I). There is a large size difference between colorectal tumour cells and lymphocytes and therefore it is relatively simple to count the number of

each cell type in a population on a haemocytometer. However to confirm the purity of the sorts obtained in the present studies the cells were stained with the anti-tumour monoclonal antibodies, NCRC23 and NCRC37, used for sorting and with an anti-cytokeratin monoclonal antibody, NCRC34, which stains all color ectal tumour cells. C146 cells bound NCRC37 but not NCRC23. The original populations were composed of 55-72% NCRC37 and 48-65% NCRC34 positive cells. Following magnetic bead sorting the cells were 78-97% NCRC37 and 96-97% NCRC34 positive. However between 26-40% of the tumour cells remained unsorted (Table 11). Sorted cells grew at the same rate as unsorted cells (Fig. 1) despite the beads remaining attached to cells for several days. Unlike cell lines, tumours are very heterogeneous. They are composed of cells from different lineages and even the tumour cells express very diverse antigens. To ensure that the cocktail of monoclonal antibodies could sort all cells equally two different colorectal cell lines expressing different tumour associated antigens were admixed

TABLE I SORTING OF Cl46 COLORECTAL TUMOUR CELLS FROM LYMPHOCYTES BY MAGNETIC BEADS COATED WITH THE ANTI·COLORECTAL TUMOUR MONOCLONAL ANTIBODIES NCRC23 AND NCRC37 Sort

Initial no. of Cl46 cells

Bead no.

Sorting with beads precooted with monoclonlll antibodies 1 10 7 2x 107

2

3

10

10

7

7

4xl0

8x10

7

7

Sorting with cells precooled with monoclonal antibodies 4 107 2x 10'

No.of sorts

No. of cells recovered

Separation efficiency

1 2 Total

2.5xI0 6 1.3 x 10 6 3.8x10 6

38

1 2 Total

6

2.3x10 1.0 x 10 6

1 2 Total

6

(l

3.3x 10 6

1.9 x 10 1.3 X 10 6 3.2X 10 6

33

32

(l

1 2 3 Total

6.0X 10 6

2.4x 10 6

0.8x 106 9.2x 10 6

92

Cells were sorted by two methods, either precoating beads with monoclonal antibodies and allowing these antibodies to recognise the cells or by precoating the cells with specific monoclonal antibodies and then binding the rabbit anti-mouse beads to the bound monoclonal antibodies.

a

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TABLE II

TABLE III

PHENOTYPIC ANALYSIS OF CI46 CELLS SORTED BY MAGNETIC BEADS COATED WITH MONOCLONAL ANTIBODIES NCRC23 AND NCRC31

SORTING OF COLORECTAL (NCRC37+ve) AND GASTRIC (NCRC23 + vel TUMOUR CELLS FROM LYMPHOCYTES BY MAGNETIC BEADS COATED WITH ANTICOLORECTAL TUMOUR MONOCWNAL ANTIBODIES NCRC23 AND NCRC37

Sort no.

Cell population

% of cells staining with the

following monoclonal antibodies: NCRC31

NCRC34

Sorting with beads precoated with monoclono/ antibodies Presort nd 1 48 Sorted nd 92 Unsorted nd 40 2

Presort Sorted Unsorted

SS 92 nd

61 91 39

3

Presort Sorted Unsorted

S5

61 91 27

78 nd

3 x 10 6 colorectal tumour cells and 3 x 106 gastric tumour cells were admixed with 6 x ]06 lymphocytes prior to magnetic bead sorting Q

Sorting with cells precooted with monoclono/ antibodies 4 Presort 72 65 Sorted 97 96 Unsorted nd 26 • Cells were sorted by two methods either precoating beads with monoclonal antibodies and allowing these antibodies to recognise the cells or by precoating the cells with specific monoclonal antibodies and then binding the rabbit anti-mouse beads to the bound monoclonal antibodies.

together and an equal number of lymphodytes added. They were sorted with the magnetic beads coated with the anti-colorectal tumour cocktail and 50% of the epithelial cells were recovered 10'

e TIME (DAYS)

Fi,. 1. Growth of CI46 cells prior (e) and post (0) sortin, with magnetic beads coated with monoclonal antibody NCRC37.

Number of cells

Percentage of cells staining with the following antibodies:

1.2 X 107

16 46 nd

Prior to sorting Sorted cells 5 Unsorted cells 1

x 10 6 x 106

NCRC37 NCRC23 NCR04

28 61 nd

42 97 23

97% pure (Table III). Analysis of the tumour associated antigen expression of the sorted cells showed that 46-61 % expressed each of the marker antigens, showing that both cell types had been efficiently selected.

Fresh tumours Tumours were disaggregated with collagenase (prescreened to ensure no residual trypsin activity) and washed with medium containing serum and EDTA to produce a highly viable single cell suspension. Initially a tumour which was 43% epithelial and 56% infiltrating cells was sorted with magnetic beads coated with anti-infiltrating monoclonal antibodies. 107 cells which were 78% pure were sorted. The remaining cells were then sorted on beads coated with anti-tumour monoclonal antibodies. 6.7 x 106 cells which were 76% pure were sorted (Table IV). As the purity was less than that observed for the tumour cell line /Iymphocyte sorts an alternative approach was adopted. The cells were presaturated with the monoclonal antibodies and then sorted with beads coated with sheep anti-mouse IgG antisera. The initial tumour sorted by this method was composed of 38% epithelial cells and 62% infiltrating cells. 4.5 x 106 infiltrating cells, 86% pure were sorted and 7.2 X 106 tumour cells, 86% pure were isolated. A second tumour which was composed of 59% epithelial cells yielded 4.5 X 106 infiltrating cells, 87% pure, and 6.2 X 106 tumour

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cells, 87% pure, following magnetic bead sorting (Table IV, Fig. 2). It was difficult to assess the phenotypic composition of the purified tumour population by immunocytochemical staining since the rabbit anti-mouse beads used to sort tumour cells coated with mouse monoclonal antibodies remained attached to the cells but still had sites available for binding other mouse antibodies. It may be possible to stain the sorted cells with rat monoclonal antibodies recognising antigens distinct from those used to sort the cells. The contaminating cells which were sorted due to clumping with the tumour cells were also difficult to stain since they remained firmly attached to the tumour cells. However, morphologically the cells associated with the large tumour cells with polymorphic nuclei were small round cells similar to resting lymphocytes and there were few mononuclear cells in evidence. Three more tumours have now been sorted and then screened for their mitogenic response to growth factors. Between 4-7 X 10 6 infiltrating cells were sorted with purities ranging from 7080%. Whereas a similar number of tumour cells with similar purity was sorted from two of the three tumours, the third tumour was only sorted for infiltrating cells (Table IV, Fig. 2). Unsorted tumour cultures all responded to pokeweed mitogen, reflecting the abundance of lymphocytes in these cultures. Similarly sorted infiltrating cells also responded to PWM. However the purified tumour cells failed to respond to the lymphocyte mitogen (Fig. 3a) since the predominant contaminating cell population appeared to be small resting lymphocytes it is as100 #

20

562

574

580

601

607

TUt.aJRS

Fig. 2. Sorting of colorectal tumours with anti-infiltrating or anti-tumour monoclonal antibodies. Open bars. unsorted infiltrating; black hatched bars, sorted infiltrating; grey bars, unsorted tumour; white hatched bars, sorted tumour.

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A rapid method for separating tumour infiltrating cells and tumour cells from colorectal tumours.

As solid tumours are composed of a heterogeneous mixture of cells the role of any particular cell type is difficult to analyse. A rapid method of sort...
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