H 1 . M . CELL(Hum Cell) Copyright C ,3005 by The Japan Human Cell Society

Vol. 18 No. 1 Printed in Japan

Original Article Cell Line

Establishment and characterization of EB virus-free normal B-lymphocyte and interleukin-6-producing poorly differentiated

adenocarcinoma cell lines derived from gastric tumor tissue

Satoshi OHI’ ’, Hisashi HASHIMOTO’, Toshiaki TACHIBANA’, Isao TAE3EI’, Masako NAK&lIMA’, Kahei SATQ’, Katsuhiko YANAGA’ and Hiroshi ISHIKAWA’

ci\bstracb We successfully established two cell lines. an adenocarcinoma cell line (designated as HIGS) and Epstein-Barr virus-free normal B-lymphoc>-te cell line (designated as HIGSBL). derived from a moderately to poorly differentiated adenocarcinoma of the stomach. and examined their characteristics. The tumor delivered to our laboratory from an operating room was cut into small pieces and cultured on the dishes. HIGS and HIGSBL were established from each individual dish after the onset of primary culture. Although their culture methods were the same, the HIGS cell line was not established from the dishes growing HIGSBL cells. In addition, HIGSBL cells were scarcely observed in the HIGS cell dishes. Because of these factors. we have considered until now that HIGSBL cells may inhibit the growth of HIGS cells or cause damage to HIGS cells by unknown mechanisms. Injection of HIGSBL cells. other Rlymphocyte cell lines. or the conditioned media of HIGSBL cells into nude mice bearing HIGSgrafted tumors was performed individually. When HIGS and HIGSBL cells were cocultured in the same dishes. HIGSBL cells inhibited the proliferation of HIGS cells. The inhibition of grafted tumor growth was confirmed by the injection of not only the HIGSBL cells but also the B-lymphocytes. Furthermore, this inhibition was only observed when the conditioned medium of Blymphoqtes was injected into the nude mice. These results suggested that the secretory products by general B-Iymphoqtes (including HIGS-BL) have some ability to inhibit the proliferation of HIGS cells. In addition. susceptibility tests 10 anticancer drugs suggcstrd that HlGS cells were sensitive to CDDP, ADM and MMC. and HIGS-BL cells were sensitive to CDDP. If CDDP was used for chemotherapy in the patient. the drug produced atrophy of HIGSBL cells. The study about HIGS and HIGS-BL cells reported the necessity for novel therapeutic approaches in oncotherapy.

Keywords: establishment, poorly differentiated adenocarcinoma, stomach, B- lymphocyte, anti-cancer drugs

[HUMA!! CELL 18( 1 ) : 35 - 44.20051

1: Department of Anatomy 11, Jikei University of

2: Department of Applied Biological Science, Nihon Medicine

University of Bioresource Science 3: Department of Surgery, Jikei University School of

Medicine

Introduction

We know that normal Blymphocytes occasionally grow steadily on dishes cultured in order to establish the carcinoma cell line derived from tumor tissue from our experience. In most cases, these B-lymphocytes have Epstein-Barr virus (EBV). EBV-infected B-

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lymphocytes grow easily without t he intentional addition of cytokine(s) (interleukin (1L)-6, etc.). but the successful establishment of EBV-free normal B- lymphocytes is rare without the addition of cytokines. In addition. several tumors often secrete IL-6 as a B- lymphocyte growth factor. In these tumors. t h e invasion of B-lymphocytes is observed in the tissue. The role of IL-6 as a tumor growth inhibitor or tumor growth factor has been reported'"'. In the oncotherapy of patients, i t is very important to investigate the function of Blymphocytes in tumor tissue.

We recently succeeded in establishing two cell lines; a poorly differentiated adenocarcinonia cell line (HIGS) and a B-lymphocyte cell line (HIGS-RL). derived from the same gastric tumor tissue. We investigated the cell characteristics and the effects of HIGSBL cells on HIGS cells in t h o and in zitm.

Materials and Methods

Patient history On November 18. 2002. the patient (Japanese male,

51 years old) underwent surgical section of a gastric tumor. The serum alpha-fetoprotein (WP) level was 465ng/ml before surgery. The patient was diagnosed histopathologically as having a moderately to poorly differentiated adenocarcinoma producing AFP (Figure 1 4 . The helniinthic abscess, probably anisakis, and metastatic liver tumor (pT2? Y2, M1. stage IV) were observed.

Culturr mrdia. solutions and materials Growth medium ( G R I I ) : 1)uIbecco.s modified eagle

medium: nutrient mixture Ham's F-12 (1:l) (GIBCO. Lot. 1132337. VSA) supplemented with 20% fetal bovine serum (Morvgate batch: 83300124, i\US). 0.1% MEM non-essential amino acids solution (GIBCO, Lot. 1133557. LISA). 0.25 #tg/ml fungizone (GIBCO, Lot. 3074952. USA) and 50U penicillin-50 ;( g streptomycin/ ml (GIBCO. Lot. 1167961, LJSA).

Hanks solution: Hanks (NISSlil, Lot 08020831. JPN) supplemented with the sanie amounts of the above antibiotics without fungizone.

Trypsin-EDTA solution: Phosphate buffered saline (PBS) (-) (NISSUI. Lot. 133303, JP?;) supplemented with O._"k trypsin (Difco Laboratories. Lot. 2140531,

IISA) and O.(Y?% EDTA 6Omm and l0mm round dishes (FALCON. JPN)

were used for in zjitro culture. The 15ml and 50ml centrifugal tubes (FALCON. JPN), and 5ml and l0ml disposable pipettes (Coster, t 6A) were used for whole culture manipulations.

Establishment and Characterization of Two Cell Lines Primary culture

The gastric tumor ectoniized surgically from the stomach was delivered in G M with 50 nil of t he centrifugal tube at Of to our laboratory. The gastric tumor was washed with running Hanks solution and cut into small pieces in Hanks solution using lazar blades in inclined l0Onim tissue culture dishes while pouring Hanks solution. All segments were collected in a 50mI centrifugal tube and pipetted as strongly a s possible with a 5ml pipene. The tube was left for about

Fig. I : Orik~nal tumor tissue and HKStumor tissue (a) Many invasivc lymphocytes are obsc*rvvd in the tumor 1issuc. 14 i sl o pa I h o 1 o gi ca I d i if c rcn t i a t e d adenocarcinonia. H-E xl00 (b) Grafted HIGS-tumor tissue produccd in subcutis of nudc

d iagnos is is poor I y

mouse

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HUMAN CELLVol. 18 No. 1 (2005)

10 sec at room temperature. The supernatant containing small segments and isolated cells were collected into new 50 ml centrifugal tubes and centrifuged at 300xG for 5 min. Then, the supernatant was removed, and the pellet was suspended with a proper amount of fresh GM. The suspension was plated onto 60 mm tissue culture dishes and cultured in a COrincubator (37"C, 4.7% COZ in humidified air). The large segments were dissociated with the above mentioned digestive solution at 37°C for appropriate periods. After vigorous pipetting, the dissociated cells were collected and cultured in the COrincubator. The GM was changed twice a week. Subcultures were performed in a timely fashion. Two types of the cells, the adhesive type and floating type, grown on dishes were cultured with individual methods, respectively, until their establishment.

Transplantation into the nude mice After establishment, adhesive cells (HIGS), at

about 1 X lo7 cells/0.5ml of Hanks solution/mouse, were transplanted into the subcutis of nude mice (male, BALB/cA, 4-weeksald).

At about 8 weeks after the transplantation, the grafts were ectomized surgically and washed with Hanks solution. Some parts of the graft were immediately fixed with 10% buffered formalin, embedded in paraffin, and cut into sections 4 pm in thickness. Subsequently, the slices were stained with hematoxylin and eosin for light microscopic observation.

Electron microscopic observation The small parts of the graft, HIGS cells or HIGSBL

cells were iked with 2.5% glutaldehyde in 0.1M PBS for 1 hour at room temperature and post fEed with 1% OsOl in 0.1M PBS at 0°C for 30 min. The cells were rinsed with same buffer, then dehydrated with ethanol, immersed twice in absolute propylene oxide, and embedded in Quetol 812. Sections were cut at a thickness of 100nm with a diamond knife and mounted onto grids. Following staining with uranyl acetate and lead citrate, they were observed with a JEOL JEM- 12OOEX-IT electron microscope at 80kV.

Identilication of the cells Floating cells (HIGSBL) on dishes were confirmed

as to whether they were B-lymphocytes using flow cytometry (Leukemia lymphoma analysis a), CD45 gating). In order to c o b whether the floating cells were infected with Epstein-Barr virus (EBV), the infection was investigated using the polymerase chain- reaction (PCR) of EBVs deoxyribonucleic acid (DNA).

Karyotype analysis of HIGS and HIGSBL The two cell lines were cultured in GM

supplemented with 0.2 p g/ml of colcemid for 48 hr in a COrincubator at 37"C, respectively. Each of them was collected into a 15ml centrifugal tube using centrifugation at 300xG for 5 min and treated with 0.075M KCl solution for 20 min at 37°C; subsequently, each of the cells were fured with methanol: acetate (3:l) solution at 0°C for 10 min. After fixation, HIGS and HIGS-BL cells were stained with 4% Giemsa solution. Then, 80 and 41 cells each were selected randomly, and these karyotypes were analyzed.

Assay of tumor markers and cytokines in the conditioned media of HIGS cells

The amounts of AFP, CAl9-9, IL2, IL-3, IL-4, IL5, IL6, G-CSF and GM-CSF in the conditioned media (used for 3 days culture) and in the control GM (without cells, also incubated at 37°C for 3 days) were measured using chemiluminescent immunoassay (CLIA) , radioimmnoassay (RIA), chemiluminescent enzyme immunoassay (CLEIA), enzyme immunoassay (EIA) or enzyme linked immunosorbent assay

WJW.

Assay of cytokines in the conditioned media of HIGS BL cells

The amounts of IL2, IL3 and IL5 were measured using ELISA, and that of IL4 and IL-6 were measured using CLEIA in the conditioned media (used for 3 days culture).

Susceptibility tests to anti-cancer drugs Anti-cancer drugs (CPT-11, 5FU, CDDP, MMC,

ADM, VP-16, CPA, and TXL) were tested for their effect on HIGS cells, and HIGSBL cells, CPT-l1,5FU,

37

CDDP and MMC were tested. These tests were performed using an oxygen electrode-apparatus (Daikin Dox-10, Tsukuba Japan)”. The HIGS or HIGS BL cell-suspensions were centrifuged at 300xG for 5 min, and each pellet was re-suspended with 0.1M HEPESbuffered (HE-) GM, respectively. Previously, the tubes containing HE-GM and 1Opg each anti- cancer drug/ml (drug-GM) were prepared. Subsequently, the drug-GM was added to the cells (1 X lo6 cells/ml, finally) and suspended gently with a pipette. Then, the cell-suspensions were plated onto the oxygen electrode-apparatus at 37C, and the susceptibility of the anti-cancer drugs was analyzed for 10 or 15 hr.

Results

Effect of HIGS-BL cells to HIGS cells Coculture of HIGS and HIGSBL cells

HIGS cells in confluence on the bottom of 60 m dishes were cocultured with HIGSBL cells. HIGSBL cells (about 1 X lo7) were seeded onto a confluent sheet of the HIGS cells on the 60 mm dish. The co- culture was continued for about 4 weeks. These cells on the dishes were observed with light and electron microscopes.

Transplantation of B-lymphocytes into nude mice bearing grafted (HIGS) tumors

KIGSBL, HAGM-BL and HASC-BL cells (about 1 X lo8) (be published elsewhere) were respectively transplanted into the subcutis of nude mice bearing the grafted HIGS tumor. HAGM-BL and HASC-BL cells: normal B-lymphocytes previously established from a gastric and a S-colon tumor of the other patients, respectively. The tumor volumes were measured once a week for four weeks from the onset of the transplantation.

Injection of the conditioned media by HIGSBL cells into nude mice bearing grafted HIGS tumors

One ml of the conditioned media (used for 3 days cultured) by HIGSBL was injected into the subcutis of nude mice bearing the HIGS grafts. The tumor volumes were measured once every two days for 10 days.

Establishment of HIGS and HIGS-BL cell lines The colonies of the carcinoma cells were observed

on the bottom of the 60mm round dishes during about 5 months of primary culture. In addition, the floating small cells and small cell clusters were observed on the other dishes at the same time, but no adhesive cells or any other type of cells were observed there. The carcinoma cells and the floating cells were subcultured over 5 times until establishment. Established carcinoma and floating cells were designated as “HIGS (Figure 2-a, c-f) and “HIGSBL” (Figure 2-b, g, h), respectively. In the manipulation of the subcultures, the dispersion of HIGS cells on dishes was easily performed by gently pipetting, and did not to need trypsin or other enzymes.

Characterization of HIGS cell line Transplantaion of HIGS cells into nude mice

At about 8 weeks after transplantation, the HIGS cells produced an adenocarcinoma resembling to the original tumor in nude mice (Figure 1-b).

Assay of tumor markers and cytokines The amounts of AFP, CA19-9, IL-2, IL3, IL4, IL5,

IL6, G-CSF and GM-CSF in the conditioned medium were 4 .4ng/ml , <6U/ml, <0.8U/ml, <3lpg/ml, 7.8pg/ml, <5.0pg/ml, 8,150 pg/ml, 29pg/ml and 88pg/ml, respectively. In the control, the amounts of these agents were <0.4ng/ml, <6U/ml, <0.8U/ml, <3lpg/ml, 2.4pg/ml, <5pg/ml, 0.2pg/ml, <10pg/ml and <8pg/ml respectively.

Anti-cancer drug susceptibility test Figure 3 (a, b) shows the changes of the electric

current in the drug-GM with or without cells for 15 hr or 10 hr. These figures suggest CDDP, MMC and ADM were effective to HIGS cells, and CPT-11, 5FU, W-16, CPA and TXL were not effective to the cells.

Karyotype Figure 4-a shows the number of chromosomes in

HIGS cells randomly picked up. Figure 4-b shows the HIGS cell’s chromosome analysis (63, X, del(2) (43),

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Fig. 2: Micrographs of in iitro cultured HIGS and HIGSBL cells (a, b) The observation of HIGS cells (a) and HIGSBL cells (b) with inverted-phase contrast microscopy. (c-h) Electron microscopic observation of HlGS (c-0 and HIGSRL cells (R. h). (a) HIGS cells grew on a monolayer sheet with contact inhibition and were occasionally floating cells in growth medium. X I 0 0 (b) HIGSBL cells grew as floating cluster without ERV infection. Aggregations of cells were observed in the dishes. and this aggregation was easily dispersed by gently pipetting. XIS0 (c) Most cells have large and round nuclei and small amount of cytoplasm. The scale bar represents 5 , ~ m in length. (d) Microvilli between cells are shown by arrows. The scale bar represents 2 :l m in Imgth. (e) Arrows show desmosomes. and arrow heads show Golgi apparatuses. The scale bar represents 2OOnni in length. (0 Most cells investigated have several clusters of glycogen granules (arrows) and lipid droplets (arrow heads) in the cytoplasm. The scale bar represents S00nm in length. (n) Well- developed microvilli (arrows) and warped cell membranes and nuclei are observed. The scale bar represents 2 rim in length. (h) Mitochondria (arrows) and rough endoplasmic reticulum (arrow heads) are well-dcvchwd. The scale bar represents .XKInm in length.

39

del(5) (q2), + 9 mar + 2 dmin) . HIGS-HL cell's identification (Figure 5). Figure 4

suggests HIGSBL cells were normal Blymphocytes. Characterization of HIGS-BL cell line Flow cytometry

The LLA CD45 gating was accomplished for the

5000 - Cdls only

4500

5 F u

CDDP

MMC ~ 3000 ,

5 2500 'i - i E 1000

1500 --A=-- - . - 1000 Jj

SO0

0 0 60 110 I80 140 300 360 420 480 YO 600 6bO 710 'BO 840

T i m (mml

(a) : Anti-cancer drug susceptibility test of HIGS cells (1)

'.DO0

4 500

4000

p 3500 3000

3 2500

- -

g 1000

I500

I 000

500

0

Y

I w16 CPA H

0 60 120 180 240 300 360 410 480 540 600

T m ( n n n )

(b) : Anti-cancer drug susceptibility test of HlGS cells (2)

roo0

4500

4000

3500

E 3000 2 6 2500

$ 2000

I500

1000

500

0

- CdLr only

CDDP

0 60 120 180 240 300 360 420 480 YO 600 660 720 780 840

Tmnr(mn)

(c) : Anti-cancer drug susceptibility test of HICS-BL cells

Fig. 3: Anticancer drug susceptibility trkts using the oxygen electrodeapparatus (Dox-10) Each inclination-graph shows the content mount of oxygen in the medium of each well during time. The cell activity respiration is suppressed with anti-cancer drugs. the degree of the inclination is slow

Anticancer drug susceptibility test Figure 3(c) suggests CDDP was effective toward

HIGSBL cells. and MMC, CPT-11 and 5FU were not effective to the cells.

Karyotype and EBV infection HIGS-BL cells have normal karyotype 46. XY

(Figure 4-b). And no EBV DNA was detected with PCR

Effect of HIGS-BL cells to HIGS cells Cosulture of HIGS and HIGSBL cells

The colonies of HIGS cells that were co-cultured in culture dishes with HIGSBL cells were thoroughly surrounded with HIGSBL cells. No more growth of the colonies was confirmed at least by inverted-phase contrast microscopic observation. Suppression of the colony's growth was confirmed by observation using an inverted-phase contrast microscope for two weeks (Figure 6).

Transplantation of B-lymphocytes into the nude mice bearing grafted (HIGS) tumors

Figure 7 shows the changes of the graft volume for 4 weeks from the onset of the Blymphocyte injection. T h e growth of t h e grafted tumor (HIGS) was suppressed clearly by injected B-lymphocytes (HIGS HL, HAGM-BL or HASC-BL) in all mice.

S o abnormalities were histologically observed in g r a f t e d B -1 y in p h o c p t e- transplantation. In the whole B-lymphocyte-grafted HIGS-tumors. however, t h e cell atrophies were observed in the inner tissue of the tumor (Figure 8).

H I G S- t u m o r s with o u t

Injection of the conditioned media by HIGSBL cells into nude mice bearing grafted HIGS tumor

T h e volumes of the grafts were increased by degrees until day 8. However, t he volume of the conditioned media-injected tumor was reduced at day 10 (Figure 9).

Discussion

The level of serum AFP was high (465ng/ml) in the

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HVMAN CELL Vol. 18 No. 1 (2005)

Fii. 4: Chromosome analysis

Fig. 5: LLA CD45 gating of HIGSBL cells

41

patient before surgery. However, the AFP level in the conditioned medium by HIGS was very low. This cause may be that the patient has a metastatic liver tumor. AFP is mostly secreted from the hepatic carcinoma or metastatic foci of the tumor. Therefore, we consider that a large amount of serum AFP (detected in the patient before surgery) was secre ted from t h e metastatic liver tumor, not the original gastric tumor. Electron microscopic observation of HIGS cells suggest the cells are poorly differentiated, because of immature developments of cell organelles, such as the Golgi apparatus, mitochondria and rough ER

HIGS-BL cells have normal karyotypes and microstructures, and are not accompanied by EBV infection. EBV is a DNA virus infecting various epithelial cells and/or lymphocytes (especially B- lymphocyte) of most healthy adults and children"'. This virus often induces lymphoproliferative disease,

Fig. 6 Co-culture of HIGS cells and HlGSBL cells HIGSBL cells were seeded onto dishes growing HIGS colonies. Whole colonies of HIGS cells were completely surrounded by HlGSBL cells. The growth of colonies was suppressed during cw

s *- HICS-BL / t i 1 I

t i 4 HACM-BL~ I

HASS BL

0 7 21 ?I Tune [dn)

Transplantation o f B-lymphocytes inlo the nude mice bearing HICStumor

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Fig. 8: Light microscopic observation of B-lymphocyte- transplanted HlGStumor (H-E stain) (a) HIGSBL-transplanted tumor tissue. S I(@ (b) HACM-BL-transplanttad tumor tissue. S 100 (c) HASC-BL-transplanted tumor tissue. X 100

- C m d i h d mcd!um o f IiIGS-BL Uus

- -

I - I 1

0 2 4 6 a 10

(dag)

Fig, 9: Injection of Ihe conditioned medium of HIGSBL cells into nude mice bearing HIGStumor

HUMAN CELLVol. 18 No. 1 (2005)

lymphomas, nasopharyngeal carcinoma, Hodgkin’s disease’”. *) and gastric carcinoma rarely”. lo’. An EBV- infected B-lymphocyte cell line is often established easily, because of the abnormally strong growth. However, the HIGS-BL cell line was successfully established without EBV-infection. A large amount of IL-6 secreted from HIGS and HIGS-BL cells was confirmed with CLEM. IL6 inhibits the growth of T lymphocytes and enhances that of B-lymphocytes. We considered that the IL6 secreted from those two cell lines steadily promoted the growth of the EBV-free HIGSBL cells steadily. Therefore, the most invasive lymphocytes observed in original tumor tissue are probably B-lymphocytes, not T-lymphocytes.

Many reports about IL-6-producing tumors or cultured carcinoma cells have been published at present, and they investigate the functions of IL-6. Takizawa et al., in 1993l’ and Yamaji et al., in 2004’’ reported the inhibitory functions of IL6 toward tumor proliferation. However, Miki et al., in 198g3’ and Watson JM in 1990” reported that tumor-producing IL-6 functions as a tumor growth factor. Therefore, it is very important to investigate the functions of the IL-6 produced by tumors.

In this study, we used the two cell lines for the immunological experiments. No HIGS cells were observed on HIGS-BL cell-growing dishes. We have until now guessed that HIGSBL cells may inhibit the growth (proliferation) of HIGS cells by unknown mechanisms. Therefore, we seeded HIGSBL cells onto colonies of HIGS cells in tissue culture dishes. As result, the growth of the (HIGS) colonies was inhibited for at least 2 weeks. On the dishes, the HIGSBL cells were surrounded completely by HIGS colonies. Although, we investigated whether the HIGSBL cells might interact directly with the HIGS cells, a direct interaction between HIGS cells and HIGS-BL cells were not observed using the electron microscope. However, when the conditioned medium by HIGSBL cells was injected into the HIGSgrafts carried by nude mice, the tumor growth was inhibited, and the volume was slightly reduced. These results suggest that the HIGS-BL cell-produced factor(s) functions as a paracrine inhibitory factor of HIGS cells in uitro and in vivo. If the paracrine inhibitory factor is IL6 produced

by HIGS-BL cells, IL-6 from the HIGS cells may function as an autocrine growth factor on HIGS cell growth. We guessed that IL6 from HIGS cells induced the proliferation of HIGSBL cells, and IL6 from HIGS BL cells inhibits that of the HIGS tumor.

The results of the anticancer drug’s susceptibility tests suggested both of HIGS and HIGSBL cell lines are sensitive to CDDP. Therefore, if CDDP was used for chemotherapy in the patient, they will affect to the function of B-lymphocytes (in the tumor) as tumor growth inhibitor and induce their atrophy. HIGS and HIGS-BL cells provided the essentiality of a novel therapeutic approach in patients with B-lymphocyte- invaded tumor.

References

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8) Rickinson AB and Kieff E: Epstein-Barr virus. Fields Virology, 4 th ed. Knipe DM, Howley PM, et al., eds. pp. 25752627, Lippincott-Williams and Wdkins, 2001

9) Tokunaga M, Land CE, Uemura Y, et al.: Epstein- Barr virus gastric carcinoma. Am. J. Pathol. 143:

125@1254,1993 10) Imai S, Koizumi S, Sugiura M, et al.: Gastric

carcinoma : monoclonal epithelial malignant cells expressing Epstein-Barr virus latent infection protein. Proc. Natl. Acad. Sci. USA. 91:9131-9135, 1994

Received 2005.5.7, Accepted 2005.5.23

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