Multicellular gastric cancer spheroids recapitulate growth pattern and differentiation phenotype of human gastric carcinomas

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Multicellular Gastric Cancer Spheroids Recapitulate Growth Pattern and Differentiation Phenotype of Human Gastric Carcinomas BARBARA MAYER,* GIANNOULA KLEMENT,* MAYUMI KANEKO,‡ SHAN MAN,* SERGE JOTHY,‡ JANUSZ RAK,§ and ROBERT S. KERBEL* *Molecular and Cellular Biology Research, and ‡Department of Laboratory Medicine and Pathobiology, University of Toronto, Sunnybrook and Women’s College Health Sciences Centre, Toronto, Ontario, Canada; and §Hamilton Civic Hospitals Research Centre, McMaster University, Hamilton, Ontario, Canada

Background & Aims: Advanced gastric cancer has a poor prognosis and is largely unresponsive to currently available chemotherapeutic drugs. The development of more effective therapies would be aided by better preclinical models. Methods: An in vitro multicellular gastric cancer spheroid model was established using the liquid overlay technique and compared with the corresponding xenografts in immunodeficient mice. Results: Twelve of 17 (71%) gastric cancer cell lines reflected growth characteristics of their parental gastric carcinomas in threedimensional culture. Thus, cell lines derived from peritoneal and pleural carcinomatosis grew as single cells (HSC-39, KATO-II, KATO-III) and cell aggregates (SNU-5, SNU-16). Cell lines representing adenosquamous (MKN-1) and tubular differentiation (MKN-28, MKN-74, N87) formed partly compact multicellular spheroids recapitulating the tumor architecture of the respective original tumor. The differentiated phenotype was lost after subcutaneous implantation of the in vitro spheroids in mice. The degree of morphologic differentiation was reflected by the levels of mucin and constitutive E-cadherin expression. Heterogeneous changes of other adhesion molecules (EpCAM, ␣2␤1, CD44s, Lex, sLex) were observed. In contrast, cell lines derived from poorly differentiated gastric carcinomas (Hs-746T, RF-1, RF-48) formed fully compact spheroids mimicking the poorly differentiated phenotype, were E-cadherin negative, and showed only CD44s up-regulation. Conclusions: Recapitulating some complexity of their in vivo counterparts, multicellular gastric cancer spheroids may represent a physiologically valid model for studying the biology of this cancer, and testing new therapeutic strategies.

he majority of patients with gastric cancer in western countries are diagnosed at an advanced tumor stage. Despite significant progress in surgical treatment often resulting in complete local tumor resection, the 5-year survival rate of advanced gastric cancer patients remains less than 30%.1 Adjuvant treatment, including

T

combination chemotherapy, has had essentially no impact on improving prognosis.2 A number of new therapeutic targets in combination with conventional chemotherapy have been proposed.3–5 However, the quest for more selective targets and less toxic regimens has been hindered by a number of factors including lack of reliable preclinical models, particularly ones avoiding difficult and expensive animal experiments. It is of considerable interest that various tumor characteristics, such as growth, metastasis, and intrinsic resistance to various types of anti-cancer therapy, are often dictated by the collective properties of tumor cell populations rather than by intrinsic features of individual tumor cells. This multicellular “community effect” is mediated by direct and indirect interactions of the tumor cells with each other and with their surrounding microenvironment resulting in a variety of molecular changes in the tumor cells.6,7 Some of these complex features of in situ solid tumors can be recapitulated in three-dimensional (3D) multicellular spheroid culture, but frequently not in the traditional monolayer and unicellular suspension cultures.8,9 According to their in vivo–like characteristics, multicellular tumor spheroids have been proposed to represent a more physiologically relevant in vitro model for avascular microregions of larger tumors and small tumor nodules such as occult micrometastases.10,11 The most striking from a therapeutic point of view is that cell interactions in tumor spheroids mediate “multicellular” drug resistance to an array of different chemotherapeutic agents similarly to that observed in the clinic.12–16 In many of these instances, the compactAbbreviations used in this paper: CAM, cell adhesion molecule; cMC, corrected mean channel; 2D, two-dimensional; 3D, three-dimensional; EpCAM, epithelial cell adhesion molecule; FACS, fluorescence-activated cell sorter. © 2001 by the American Gastroenterological Association 0016-5085/01/$35.00 doi:10.1053/gast.2001.27989

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ness or cohesion of the spontaneous cell aggregates was found to have a direct impact on cell cycle progression and multicellular resistance, which seemed to be mediated by adhesive events.17,18 Consequently, a better understanding of the expression and function of cell adhesion molecules (CAMs) mediating intercellular and cell-matrix adhesion may be crucial to understanding such community effects, and the impact they can have on drug sensitivity or resistance. Indeed, altered CAM expression has profound effects on cytoskeletal organization and signal transduction resulting in changes in cell shape, differentiation, survival, proliferation, and migration.19 This helps explain observations such as “CAM-DR,” i.e., cell adhesion mediated– drug resistance, by Dalton et al., who showed that ␣5␤1 integrin interacting with fibronectin mediates cell aggregation and intrinsic drug resistance of human multiple myeloma cells.20,21 In gastric cancer, multiple changes in CAM expression have been described in carcinogenesis and tumor progression. Differences in the CAM expression profile have been recognized between the main gastric cancer types depending on their differentiation phenotype.22 Moreover, changes in the expression profile of certain adhesion receptors, such as E-cadherin,23–25 distinct CD44 isoforms,26 –28 and various Lewis carbohydrates,29 –31 have been identified as independent prognostic factors in gastric cancer that are associated with metastatic disease and poor outcome. Because cell adhesion molecules may play a pivotal role in both tumor progression and therapeutic resistance,17,18,21,32 they represent a rational and novel target in anticancer therapy. Evaluation of the CAM profile may provide a useful tool to prospectively select the subset of gastric cancer patients that will benefit from particular types of therapy. Using a large panel of gastric cancer cell lines representative of different histotypes and stages of disease progression, the aim of the present study was to establish and characterize a multicellular gastric cancer spheroid model with special emphasis on the expression profile of biologically or therapeutically relevant cell adhesion molecules. The results suggest multicellular gastric cancer spheroids, but not their respective monolayer or suspension cultures, recapitulate to a remarkable degree the morphologic and functional tumor differentiation of their tissue of origin. Most importantly, implantation of these multicellular tumor spheroids into the subcutis of nude mice resulted in a substantial loss of the previously differentiated tumor architecture, similar to that found for the respective xenografts after subcutaneous injection of single cell suspensions, i.e., the standard animal model for human tumor xenografting. Spheroid formation was

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accompanied by heterogeneous changes in the expression of various cell adhesion molecules mimicking heterogeneity in CAM expression observed in primary and metastatic gastric cancer in vivo. These findings indicate that multicellular gastric cancer spheroids reflect their in vivo counterparts, at least in the case of tumor architecture and cell adhesion molecule expression. As such, they should be considered as a possible valid in vitro model for initial investigations of anticancer drugs designed to target gastric cancer, especially differentiation agents and cell adhesion– based therapeutic strategies.

Materials and Methods Human Gastric Cancer Cell Lines Seventeen gastric cancer cell lines of primary and metastatic origin were tested for their ability to form multicellular tumor spheroids. The cell lines AGS (CRL-1739), RF-1 (CRL1864), SNU-1 (CRL-5971), NCI-N87 (CRL-5822), RF-48 (CRL-1863), SNU-5 (CRL-5973), SNU-16 (CRL-5974), KATO-III (HTB-103), and Hs 746T (HTB-135) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). The cell lines TMK-1, MKN-1, MKN-7, MKN-28, MKN-45, MKN-74, HSC-39, and KATO-II were donated by Dr. R. Lotan (M.D. Anderson Cancer Center, Houston, TX). All cell lines were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco BRL, Grand Island, NY) at 37°C in a humidified atmosphere containing 5% CO2. The cell lines were routinely tested for mycoplasma contamination using a commercial kit (Gibco BRL). Organ site and histologic differentiation of the parental gastric carcinomas from which the cell lines were established as well as the growth characteristics of the cell lines in two-dimensional (2D) and 3D cultures are summarized in Table 1.

Multicellular Tumor Spheroid Culture 3D cell culture was performed using the liquid overlay technique as described previously.33 Briefly, 24-well tissue culture plates (Nunc Brand Products, Roskilde, Denmark) were covered with 1% SeaPlaque agarose (FMC BioProducts, Rockland, ME) diluted in serum-free RPMI-1640 medium to prevent attachment of the tumor cells to the plastic dish. Multicellular tumor spheroids were initiated from confluent monolayer cultures or dense suspension cultures, which were treated with 1 mmol/L EDTA (Sigma Chemical Co., St. Louis, MO), pH 7.2, at 37°C to prepare single-cell suspensions. After washing twice, the viability of the tumor cells was determined using the trypan blue exclusion test, and 1 ⫻ 105 viable tumor cells in 1 mL RPMI-1640 medium supplemented with 10% fetal bovine serum were plated in each well. The 24-well plates were gently agitated to allow cell-cell contact and incubated for 48 hours at 37°C in a humidified atmosphere containing 5% CO2. Using this approach, a single multicellular aggregate was obtained in each well. The diameter of 10 multicellular

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Table 1. Origin and Growth Characteristics of Human Gastric Cancer Cell Lines Parental tumor Cell line

Organ site

Histology

Cell culture Differentiation

2D

3D

HSC-39 KATO-II KATO-III SNU-1

peritoneal fluida 65 pleural effusion66 pleural effusion66 primary stomach ca67

signet ring cell ca signet ring cell ca signet ring cell ca adenoca poor

Sus Sus Sus M/Sus

— — — —

MKN-7 AGS SNU-5 MKN-45 TMK-1 SNU-16

lymph node metastasis68 primary stomach ca69 peritoneal fluid67 liver metastasis68 lymph node metastasis70 peritoneal fluid67

adenoca adenoca adenoca adenoca adenocab adenoca

M M M/Sus M M M/Sus

Aloose Aloose Aloose Aloose Acluster Acluster

MKN-1 N87 MKN-74 MKN-28 RF-1 RF-48 Hs 746T

lymph node metastasis68 liver metastasis67 liver metastasis68 lymph node metastasis68 primary stomach ca71 peritoneal fluid72 limb metastasis73

adenoca adenoca adenoca adenoca adenoca adenoca

M M M M Sus Sus M

Spartly Spartly Spartly Spartly Sfull Sfull Sfull

well moderate/poor poor poor poor poor adenosquamous ca well moderate moderate poor poor poor

ca, carcinoma; M, monolayer; Sus, suspension of single cells and small cell clumps; Aloose, aggregate of cells loosely attached to each other; Acluster, aggregate containing a fraction of clustered cells; Spartly, partly compact spheroid; Sfull, fully compact spheroid. aNumbers refer to original reference. bEstablished from a xenotransplant in nude mice.

aggregates was measured with a calibrated ocular micrometer in an inverted microscope, respectively. The average diameter is given in Figure 1.

Subcutaneous Tumor Cell Implantation in Immunodeficient Mice MKN-1 and MKN-28 gastric cancer cells were injected subcutaneously into the right hind flank of 6 – 8-week-

Figure 1. Spontaneous cell aggregation patterns of gastric cancer cell lines in 3D culture using the liquid overlay technique (average diameter is given in parentheses). (A ) MKN-45, multicellular aggregate of loosely associated cells and easy to disperse by low-level mechanical forces (loose aggregate, 710 ␮m). (B) TMK-1, loose multicellular aggregate with a portion of cells forming clusters, but still easy to disrupt (clustered aggregate, 483 ␮m). In contrast, multicellular spheroids showed a higher degree of compaction and were resistant to mechanical disruption. (C ) MKN-1, multicellular spheroid with local areas of compaction (partly compact spheroid, 462 ␮m). (D) RF-1, fully compact multicellular spheroid (265 ␮m). Original magnification 40⫻.

old, female CD-1 nude mice (Charles River, St. Constant, Quebec, Canada), either as multicellular tumor spheroids preformed for 48 hours in vitro or as single-cell suspensions. Four mice were used for each implantation. Surgical procedure was done under inhalation anesthesia, using a mixture of 2% isoflurane (Janssen, Toronto, Canada) and oxygen. Spheroids were implanted through a 1-cm skin incision in the flanks of the mouse. To avoid the influence of acute inflammatory cells,

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a distant subcutaneous pocket was created using blunt dissection, and 20 multicellular tumor spheroids in 50 ␮L serumfree RPMI 1640 medium were placed in it. The incision was then closed using 2 interrupted 6-0 silk sutures. Cell suspension grafts were produced as follows: a confluent monolayer of cells was detached with 1 mmol/L EDTA, pH 7.2, at 37°C, washed twice, and 2 ⫻ 106 viable tumor cells suspended in 50 ␮L serum-free medium were injected using a 26G needle. The tumor diameter of the subcutaneous xenografts was measured weekly, and when the tumor volume reached ⬃1.5 cm3 volume (estimated according to the formula for ellipsoid (width2 ⫻ length)/2), the tumors were excised for histomorphological examination. All animal studies were performed according to the guidelines and approval of the institutional Animal Care and Use Committee.

Histologic and Morphologic Characterization Gastric cancer cells were grown on positively charged glass slides (Fisher Scientific, Nepean, Ontario, Canada) under sterile conditions. The confluent monolayer cultures were washed twice in phosphate-buffered saline (PBS), fixed in 95% ethanol containing 5% acetic acid for 5 minutes, and stained with standard H&E. Multicellular tumor spheroids and subcutaneous xenografts were fixed in 3.7% phosphate-buffered formalin (Fisher Scientific) overnight at 4°C and embedded in paraffin (Oxford, St. Louis, MO). Paraffin sections in 3-␮m thickness were prepared and stained with H&E. Mucin production in monolayers, spheroid cultures, and xenografts was evaluated using routine periodic acid–Schiff (PAS) technique and mucicarmine staining.

Monoclonal Antibodies The epithelial origin of the cancer cell lines was confirmed using the monoclonal antibody CK22 (immunoglobulin [Ig] G1; Biomeda, Foster City, CA), directed against a variety of cytokeratins. Epithelial CAM (EpCAM) expression was assessed with the monoclonal antibodies Ber-EP4 (IgG1; Dako, Glostrub, Denmark) and KS 1/4 (IgG2a, a gift from Dr. R.A. Reisfeld, The Scripps Research Institute, La Jolla, CA). E-cadherin expression was analyzed with the monoclonal antibodies HECD-1 (IgG1) and SHE78-7 (IgG2a; Takara Shuzo, Shiga, Japan). Expression of the integrins ␣2␤1, ␣V␤3, and ␣V␤5 was investigated with the monoclonal antibodies BHA2.1 (IgG1), LM609 (IgG1), and P1F6 (IgG1), respectively, all obtained from Chemicon (Temecula, CA). CD44 expression was detected with monoclonal antibody F10-44-2 (IgG2a; Novocastra Laboratory Inc., Newcastle upon Tyne, England). Expression of the 2B4 antigen, a Lewisx (CD15)related carbohydrate, was assessed with monoclonal antibody 2B4 (IgM)29 and that of sialyl-Lex (sCD15) with monoclonal antibody KM93 (IgM; Kamiya Biomedical Co., Seattle, WA). The myeloma proteins MOPC-21 (IgG1), UPC-10 (IgG2a), and MOPC-104E (IgM; all purchased from Sigma) were used as isotype controls. The monoclonal antibodies were used as

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undiluted cell culture supernatants or purified proteins in appropriate concentrations.

Indirect Immunofluorescence Staining Surface expression of cell adhesion molecules was assessed using indirect immunofluorescence staining and quantified by flow cytometric analysis according to standard protocols. All 3 of the cell culture forms, i.e., suspension cells, confluent monolayers, and multicellular tumor spheroids, were treated with 1 mmol/L EDTA, pH 7.2, at 37°C, and cell suspensions were adjusted to 1 ⫻ 106 viable cells per sample and kept on ice at all times. After incubation with 10% rabbit serum (Dako) for 20 minutes, the cells were stained with the primary monoclonal antibody for 1 hour, washed twice with PBS (pH 7.4) and fluorescein-conjugated rabbit anti-mouse Ig (Jackson Immunoresearch, West Grove, PA), diluted 1:50 in PBS containing 10% fetal calf serum, and 0.1% NaN3 was added for 30 minutes. Cells were again washed twice, resuspended in 1 mL PBS, pH 7.4, and counterstained with 0.5 mg/mL propidium iodide (Sigma) for 10 minutes to exclude dead cells. Using FACScan flow cytometry (Becton Dickinson, San Jose, CA), 15,000 unfixed cells were acquired from each sample and analyzed with the CellQuest Version 3.2.1fls software program (Becton Dickinson). The corrected mean channel (cMC) values were calculated by subtracting the mean channel of the isotype control staining from the mean channel of the specific antibody staining, respectively. ⌬cMC reflects the difference between the cMC of the specific antibody staining in 2D culture and that in 3D culture. Two to 3 independent experiments were carried out. Differences between the average of the cMC in 2D culture and that in 3D culture for each specific stain were calculated using the unpaired Student t test, and P values ⬍ 0.05 were considered significant.

Results Spontaneous Cell Aggregation Patterns in Gastric Cancer Cell Lines 3D culture of 17 primary and metastatic human gastric cancer cell lines using the liquid overlay technique resulted in distinct patterns of spontaneous cell aggregation with different macroscopic appearance and degree of compaction (Table 1). Multicellular aggregates composed of tumor cells in loose relationship to each other and easily dispersed by gentle agitation were formed by MKN-45 (Figure 1A), AGS, MKN-7, and SNU-5 cells. Other cell lines, i.e., TMK-1 (Figure 1B) and SNU-16 cells, formed multicellular aggregates containing a subpopulation of tumor cells clustered into tighter formations, but were also disrupted by low level mechanical force. In contrast, multicellular tumor spheroids showed a higher degree of compaction than multicellular aggre-

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Figure 2. Growth characteristics of gastric cancer cell lines in 2D and 3D cell cultures. Cell lines forming partly compact multicellular spheroids: (A and B) MKN-1, (C and D) MKN-74, (E and F ) MKN-28, and (G and H ) N87. Cell lines forming fully compact multicellular spheroids: (I and J ) Hs746T and (K and L) RF-48. Arrows indicate cell clusters in 2D culture. Original magnification: A, C, E, G, and I, 200⫻; K, 100⫻; B, D, F, H, J, and L, 40⫻.

gates and were resistant to mechanical disruption. Two types of multicellular spheroids could be distinguished. Partly compact spheroids with areas of local compaction such as those formed by MKN-1 (Figures 1C and 2B), MKN-74 (Figure 2D), MKN-28 (Figure 2F ), and N87 (Figure 2H ) cells. Macroscopically, this type of spheroid appeared to be comprised of smaller, tightly associated multicellular clusters, which upon closer histologic examination revealed a clear tissue organization (see below). Fully compact spheroids with a well-rounded geometry were formed by RF-1 (Figure 1D), Hs746T

(Figure 2J), and RF-48 (Figure 2L) tumor cells. Finally, no spontaneous aggregation was found in 3D cultures of SNU-1, HSC-39, KATO-II, and its subline KATO-III. These cell lines grew as unattached single cells or small cell clumps reminiscent of those seen in the corresponding 2D cultures (results not shown). To some degree, the ability to form multicellular spheroids could be predicted from the growth characteristics seen in the dense 2D cultures of the respective cell line. For example, in confluent monolayer culture, MKN-28 cells piled up and formed small compact cell clusters loosely attached to

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Table 2. CAM Expression in Gastric Cancer Cell Lines Grown in 2D and 3D Conditions Cell line

Culture

Aggregates MKN-45

2D 3D TMK-1 2D 3D Partly compact spheroids MKN-1 2D 3D MKN-74 2D 3D MKN-28 2D 3D N87 2D 3D Fully compact spheroids Hs 746T 2D 3D RF-1 2D 3D RF-48 2D 3D

Mucina

E-cadb

EpCAM

␣2␤1

␣v␤3

␣v␤5

CD44s

Lex

sLex

nd nd nd nd

75.29c 79.77 130.22 152.21

102.06 100.49 105.35 95.81

144.35 164.71 28.3 29.93

0.74 0.1 0.05 0.09

11.17 11.73 7.6 9.03

nd nd nd nd

0.02 0.18 1.19 0.95

16.33 14.05 10.33 11.26

— — — ⫹ — ⫹ ⫾ ⫹⫹

40.78 31.19 33.86 30.94 169.95 165.01 418.79 593.84d

106.78 91.49 106.33 228.8d 538.68 712.59d 625.41 1003.44d

69.39 97.58 119.75 132.29 218.56 135.44d 149.57 161.78

21.54 24.66 4.03 8.91 0.57 0.33 5.07 8.09

24.68 37.69 22.02 23.31 54.56 31.33 18.54 23.17

30.08 131.86d 36.31 57.5 0.39 0.07 49.84 86.39d

0.28 0.08 1.54 2.03 25.60 91.41d 1.8 8.45

8.57 17.06 12.9 15.1 12.83 18.23 44.21 97.73d

203.66 207.55 0.11 0.31 0.34 0.30

40.9 47.15 29.72 39.59 14.85 17.48

47.67 46.27 0.61 2.29 0.55 0.92

108.47 141.55d 71.04 195.4d 73.24 204.05d

0.05 0.58 4.77 8.25 10.55 17.49

6.04 5.46 11.94 28.18 12.38 26.91

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.18 0.09 1.6 0.18 0.65 0.55

32.71 27.28 4.05 3.17 6.08 6.32

nd, not done. of mucin expression (⫺, not detectable; ⫾, weak cytoplasmic; ⫹, a few positive glands; ⫹⫹, many positive glands). bE-cadherin. cEach value represents the average of the corrected mean channel values of 2 or 3 independent FACS analyses. dSignificant difference between the average of the corrected mean channels in 2D and 3D cultures, P ⬍ 0.05. aExtent

the pavement-like monolayer (Figure 2E). Similarly, the confluent monolayer culture of the pleomorphic Hs 746T cells formed small cellular aggregates (Figure 2I ), and RF-1 cells and their metastatic counterpart RF-48 (Figure 2K ) aggregated into cell clusters in suspension cultures. Expression of Cell Adhesion Molecules in Multicellular Gastric Cancer Spheroids To assess whether the degree of compaction observed in the various types of multicellular aggregates reflects changes in cell adhesion induced upon transition from 2D to 3D cell culture, surface expression of a panel of cell adhesion molecules was compared between 2D and 3D cell cultures using fluorescence-activated cell sorter (FACS) analysis (Table 2). The cell adhesion molecules were selected on the basis of their prognostic relevance in human gastric cancer and their impact on drug resistance. Interestingly, in gastric cancer cell lines forming multicellular aggregates, such as MKN-45 and TMK-1 cells, no differences were seen in the expression pattern of the cell adhesion molecules tested between 2D and 3D cell cultures. In contrast, in gastric cancer cell lines forming partly compact multicellular spheroids, one or several cell adhesion molecules changed expression level in 3D cultures. Specifically, EpCAM expression was up-regu-

lated in 3D cultures of MKN-74 (⌬122.47; Figure 3A), MKN-28 (⌬173.91), and N87 (⌬378.03) cells, and an increased expression of CD44s was observed in MKN-1 (⌬101.78; Figure 3B) and N87 spheroids (⌬36.55). Upregulation of E-cadherin and the carbohydrate epitopes Lex and sialyl-Lex was restricted to the spheroid culture of individual cell lines, i.e., E-cadherin in N87 spheroids (⌬175.05; Figure 3C), Lex in MKN-28 spheroids (⌬65.81), and sialyl-Lex in N87 spheroids (⌬53.52). The integrin ␣2␤1 was down-regulated in spheroid culture of MKN-28 cells (⌬⫺83.12; Figure 3D) even though the integrins ␣V␤3 and ␣V␤5 did not change expression. In gastric cancer cell lines forming fully compact multicellular spheroids, comparison of 2D and 3D cultures showed an up-regulation of CD44s expression in spheroids (Hs 746T, ⌬33.08; RF-1, ⌬124.36; RF-48, ⌬130.81), whereas no changes in the expression level of the other cell adhesion molecules tested were noted (Table 2). Monophasic FACS histograms revealed that the alterations in CAM expression in spheroid culture primarily are caused by changes in the expression levels of the cell adhesion molecules, rather than changes in the number of positive tumor cells. In addition, none of the cell adhesion molecules tested showed either de novo expression or complete loss of expression in multicellular spheroids compared with the corresponding 2D cultures.

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Figure 3. Expression of cell adhesion molecules in 2D and 3D cell cultures of various gastric cancer cell lines (FACS analysis). Dashed line represents isotype control staining in 2D culture, dotted line represents isotype control staining in 3D culture, thin continuous line represents specific antibody staining in 2D culture, and thick continuous line represents specific antibody staining in 3D culture. The ⌬cMC (calculated as described in Materials and Methods) are the following: A, MKN-74, ⌬122.47; B, MKN-1, ⌬101.78; C, N87, ⌬175.05; and D, MKN-28, ⌬⫺83.12. Differences in CAM expression between 2D and 3D, P ⬍ 0.05, respectively.

Differentiation Phenotype in Multicellular Gastric Cancer Spheroids In Vitro The most obvious difference between the group of fully compact spheroids and the group of partly compact spheroids was found in the expression of the cell adhesion molecule E-cadherin, which plays an important role in the establishment and maintenance of epithelial differentiation.34 E-cadherin expression in multicellular gastric cancer spheroids (Table 2) correlated with the differentiation phenotype of their respective parental tumors (Table 1). All E-cadherin negative, fully compact gastric cancer spheroids were derived from poorly differentiated gastric adenocarcinomas, whereas all E-cadherin positive, partly compact multicellular spheroids originated from moderately to well-differentiated gastric cancer. In addition, expression levels of several other cell adhesion molecules known to interact with the actin cytoskeleton, i.e., EpCAM,35,36 the integrin ␣2␤1 ,37 and CD44s,38,39 were significantly altered in the partly compact multicellular spheroids. These findings led to the assumption that multicellular spheroid formation is accompanied by reorganization of the cytoskeleton resulting in the re-expression of the original differentiated phenotype. Histologic and morphologic comparison of 2D and 3D cell cultures revealed that partly compact spheroids, but not their corresponding monolayers, recapitulated the differentiated tumor architecture of the parental carcinomas. MKN-1 cells were established from an adenosquamous gastric cancer and therefore would be expected to show both glandular and squamous differ-

entiation in spheroid culture. The multicellular spheroids of this cell line were composed of concentric cell nests with the typical swirl-like structure of squamous differentiation. The cell nests consisted of a peripheral layer of flattened tumor cells, larger round cells in the inner part, and occasional central keratinization. Trabecular structures were observed, although no glandular lumens were detected (Figures 4B). MKN-28 cells derived from a moderately differentiated gastric adenocarcinoma formed multicellular tumor spheroids showing the typical features of the moderately differentiated phenotype, i.e., tumor spots consisting of polymorphic cancer cells and tumor glands that sometimes were filled with mucin (Figures 4D). Similarly, MKN-74 cells, established from a moderately differentiated gastric adenocarcinoma, formed a spheroid-like structure consisting of only a few cell layers. The tumor cells were arranged in a trabecular-like pattern and formed irregular glands with occasional mucin production (results not shown). In comparison, N87 spheroids were organized in glandular structures with most of the well-differentiated glands showing a strong mucin reaction at the apical cell border and in the glandular lumen, comparable to the welldifferentiated gastric adenocarcinoma from which the cell line originated (Figure 4F ). Similar to the partly compact spheroids reflecting the differentiated phenotype of their respective parental tumors, the gastric cancer cell lines Hs-746T, RF-1, and RF-48, all derived from poorly differentiated adenocarcinomas, formed fully compact spheroids that also reca-

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pitulated the tumor architecture of their tissue of origin. The tumor cells were organized in a sheet-like fashion (Hs-746T; Figure 4H ) and the expression of mucin was localized in the cytoplasm of the cells and did not change upon transition from 2D to 3D cultures. The degree of histomorphological differentiation in multicellular gastric cancer spheroids was reflected by the extent of E-cadherin expression. MKN-1 spheroids characterized by squamous differentiation showed a weak E-cadherin expression (Figure 5A). In comparison, the moderately differentiated phenotype of MKN-28 spheroids was reflected by a more intense E-cadherin staining intensity (Figure 5C), and the highly differentiated N87 spheroids not only showed the strongest E-cadherin expression but also revealed the highest number of Ecadherin–positive tumor cells (Figure 5E). In contrast, poorly differentiated multicellular spheroids without glandular structures formed by Hs-746T (Figure 5G), RF-1, and RF-48 cells were all E-cadherin negative. Differentiation Phenotype in Multicellular Gastric Cancer Spheroids In Vivo MKN-1 gastric cancer cells established from an adenosquamous carcinoma have been found to recapitulate the typical differentiation characteristics of a squamous carcinoma but not of an adenocarcinoma when cultured as multicellular tumor spheroids. This observation suggested the need for a stromal component if full restoration of the original tumor architecture is to be achieved. To confirm this requirement, MKN-1 spheroids preformed in vitro were implanted subcutaneously into nude mice, and the differentiation phenotype was compared with that of spheroids grown in vitro. Interestingly, the resulting xenografts, in contrast to the parental MKN-1 spheroids in vitro (Figures 4B and 6A), exhibited only tumor cell nests and lost the swirl-like organization and central keratinization typical of the cancers in situ (observed in the patient) and the parental spheroids (Figure 6B). Even the trabecular formation observed in MKN-1 spheroids in vitro were absent after the subcutaneous implantation. MKN-1 xenografts established from subcutaneously injected single-cell suspensions, i.e., the current standard animal model for

Š Figure 4. Histomorphological characteristics of gastric cancer cell lines in (A, C, E, and G) 2D and (B, D, F, and H ) 3D cell cultures. Multicellular tumor spheroids recapitulate the differentiation phenotype similar to their parental tumors. (A and B) MKN-1, adenosquamous carcinoma; (C and D) MKN-28, moderately differentiated adenocarcinoma; (E and F ) N87, well-differentiated adenocarcinoma; (G and H ) Hs746T, poorly differentiated adenocarcinoma. Original magnification 400⫻; H&E staining.

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human tumor xenografting, also showed limited, vague squamous differentiation (Figure 6C). Likewise, multicellular MKN-28 spheroids (Figures 4D and 6D) recapitulated the moderately differentiated tumor architecture of their parental adenocarcinoma in situ, but lost their glandular structures and mucin secretion when implanted into the subcutis of mice (Figure 6E). The resulting tissue architecture was comparable to that obtained in subcutaneous xenografts produced after injection of MKN-28 suspension cells (Figure 6F ).

Discussion Advanced gastric cancer has a poor prognosis, and many chemotherapeutic drugs have been tried using different combinations and therapeutic regimens in clinical trials over the last 2 decades. However, current chemotherapy has failed to improve substantially the poor prognosis associated with advanced gastric cancer.2,3 The development of alternative treatment strategies and new drugs is therefore of great importance. The testing of such new drugs, whether alone or in combinations with conventional cytotoxic drugs, would be improved by the establishment of more reliable preclinical models, both in vitro and in vivo. In this regard, the present study evaluated the multicellular spheroid model with special emphasis on tumor differentiation and cell adhesion molecule expression, both of which may represent potential therapeutic targets per se, or targets for chemosensitization. Twelve of 17 (71%) gastric cancer cell lines recapitulated growth characteristics of their parental gastric carcinomas in 3D culture. All 6 gastric cancer cell lines derived from pleural or peritoneal carcinomatosis also grew as single cells or cell aggregates, whereas 6 of 11 gastric cancer cell lines, which originated from solid gastric cancers, formed multicellular tumor spheroids. It is unclear why some of the gastric cancer cell lines tested failed to reflect the aggregation pattern of the original solid carcinomas and did not form multicellular tumor spheroids, even though they demonstrated adhesive growth characteristics in monolayer cultures. Both tumor cell–associated and microenvironmental factors have been found to influence spheroid formation.40 Expression of nonfunctional cell adhesion molecules, the lack of cross talk between different adhesion receptors, and disruption of adhesion-mediated signal transduction might inhibit homotypic cell-to-cell interaction necessary for spheroid formation. Alternatively, direct and extracellular matrix-dependent interactions with stromal cells may be required for spheroid formation in these nonaggregating gastric cancer cell lines.

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Figure 6. Histomorphological characteristics of multicellular gastric cancer spheroids grown (A and D) in vitro and (B and E ) in vivo. (C and F ) Multicellular tumor spheroids lose their differentiated tumor architecture after implantation into the subcutis of immunodeficient mice and show a differentiation phenotype similar to that of xenografts after subcutaneous injection of the same cell line in single cell suspension. (A–C ) MKN-1, adenosquamous carcinoma; (D–F ) MKN-28, moderately differentiated adenocarcinoma; original magnification, 400⫻. Inserts localize subcutaneous tumor growth; original magnification, 200⫻. H&E staining.

Histomorphological comparison revealed that multicellular tumor spheroids, but not their corresponding monolayer and suspension cultures, recapitulate the differentiation phenotype of the tissue of origin, the in situ gastric carcinomas. Two types of multicellular gastric cancer spheroids were obtained. One type was partly compact multicellular spheroids composed of moderately to well-differentiated tumor glands, which recapitulated the degree of differentiation in the original tumors. The other type was fully compact multicellular spheroids, consisting of tumor cell sheets reflecting the poorly differentiated phenotype of the parental gastric carcinomas from which they were derived. The same recapitulation of the parental (in situ) tumor architecture in Š Figure 5. Expression of (A, C, E, and G) E-cadherin and (B, D, F, and H ) CD44s in multicellular gastric cancer spheroids with various degrees of histomorphological differentiation (FACS analysis). Dotted line represents isotype control staining, and thick continuous line represents specific antibody staining. (A and B) MKN-1, spheroid with squamous differentiation. (C and D) MKN-28, moderately differentiated spheroid. (E and F ) N87, moderately to well-differentiated spheroid. (G and H ) Hs-746T, poorly differentiated spheroid. cMC (calculated as described in Materials and Methods) are the following: A, 31.19; B, 131.86; C, 165.01; D, 0.07; E, 593.84; F, 86.39; G, 0.09; and H, 141.55.

multicellular tumor spheroids has been reported for colorectal,41– 43 renal,33 and squamous carcinoma.44,45 In contrast, multicellular aggregates did not restore original tumor differentiation, suggesting that a certain degree of multicellular compaction allowing cell-to-cell interaction is required to establish the differentiation phenotype. However, the spatial arrangement of cancer cells seems to be a prerequisite for the expression of not only the morphologic differentiation phenotype, but also more importantly, the acquisition of a functional differentiation as evident from the ability of these glandular structures to secrete mucin. Morphologic and functional differentiation induced in 3D cultures was most impressive in multicellular tumor spheroids established from N87 cells, which originate from a well-differentiated liver metastasis. N87 spheroids contained polarized, mucin-secreting tumor glands, which showed an up-regulation of the mucin-bound carbohydrate epitope sLex and revealed an increased expression of E-cadherin. In fact, the degree of morphologic differentiation in multicellular gastric cancer spheroids could be correlated with the extent of E-cadherin expression. In moderately to well-differentiated gastric cancer spheroids, the level of E-cadherin expression increased in parallel to the

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degree of glandular differentiation, whereas poorly differentiated gastric cancer spheroids were E-cadherin negative. The correlation between E-cadherin expression and the degree of tumor differentiation is, in fact, remarkably similar to the correlation found in clinical gastric specimens.24,25,46 The advantage of 3D culture systems is also supported by recent reports in the field of tissue engineering showing that normal cells such as hepatocytes47 or bone cells48 can acquire histotypic reorganization and maintain organ-specific functionality in spheroid culture. It can be speculated that tumor-specific differentiation is part of the genetic composition of the epithelial cells, and that the differentiation phenotype is profoundly modulated by the organ microenvironment. Thus, multicellular gastric cancer spheroids in vitro closely recapitulated the differentiated tumor architecture of their parental tumors and lost both morphologic and functional differentiation after subcutaneous implantation in immunodeficient mice. The suppression of the differentiated phenotype by the stromal environment in the subcutis was further confirmed in xenografts produced after subcutaneous injection of the same cell line in suspension, i.e., the traditional animal model for growth of human tumor xenografts. Reciprocal stromal-epithelial interactions play a key role in the biology of the epithelium, including differentiation, and depend on the organ specificity49 and the nature of the stromal cells.50,51 Stromal cells and tumor cells communicate by an array of intercellular modalities, e.g., by direct cell-to-cell contact,50,51 via extracellular matrix molecules and soluble factors,52,53 all of which are able to substantially modulate the epithelial differentiation phenotype. The antigenic profile obtained in 3D culture was spheroid-type dependent. Gastric cancer spheroids showing a differentiated tumor architecture varied in the expression level of a number of cell adhesion molecules. EpCAM, E-cadherin, CD44s and ␣2␤1 all showed changes in their expression levels, suggesting that reexpression of the differentiated phenotype in multicellular tumor spheroids may be based on the reorganization of the cytoskeleton. In contrast, in poorly differentiated gastric cancer spheroids, the only alteration in CAM expression was restricted to the up-regulation of CD44s. Likewise, changes in CAM expression associated with multicellular spheroid formation have been reported for individual cell lines that originated from other types of tumors.43,54 –58 Interestingly, as was seen for the lack of tissue architecture, multicellular aggregates also did not exhibit any changes in CAM expression, suggesting that physical proximity of the tumor cells is not sufficient to induce alterations in CAM expression. In addition to the

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cytoskeletal changes, modifications in growth factors59 or extracellular matrix expression60,61 induced in spheroid culture may further modulate CAM expression profile, although these factors can also act as direct morphogens on cancer cells.52,62,63 Human gastric carcinomas are histologically divided in 2 main types differing in their differentiation phenotype, i.e., so-called “intestinal” and “diffuse.”64 From a molecular point of view, distinct genotypic and phenotypic characteristics have been found to be associated with these 2 types of gastric cancer.22 The present study shows that multicellular gastric cancer spheroids recapitulate both the histologic and antigenic heterogeneity, the hallmarks of patient tumors,6 and suggest this model may be representative of the complexity inherent to patient tumors. As a preclinical cancer model, it holds promise as an in vitro surrogate of in situ cancers and thus should be superior to traditional culture systems for testing the effectiveness of either conventional or newly designed anticancer drugs. In this regard, it will be of interest to assess the antitumor effects of potential drugs for the treatment of gastric cancer using monolayer versus multicellular spheroid culture systems, and compare the results to the same gastric carcinomas grown in vivo as either ectopic or orthotopic xenografts.

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Received March 13, 2001. Accepted June 13, 2001. Address requests for reprints to: Robert S. Kerbel, Ph.D., Molecular and Cellular Biology Research, Sunnybrook and Women’s College Health Sciences Centre, 2075 Bayview Avenue, S218, Toronto, Ontario, Canada, M4N 3M5. e-mail: [email protected]; fax: (416) 480-5703. Supported by a grant from the National Cancer Institute of Canada (NCIC) (to R.S.K.). Dr. Mayer is a research fellow of the Canadian Institutes of Health Research and Dr. Klement is a research fellow of the NCIC. The authors thank Juntao Liu for excellent support in FACS analysis, the colleagues in the Department of Laboratory Medicine and Pathobiology for technical advice, and Cassandra Cheng and Lynda Woodcock for excellent secretarial assistance.