In vivo tumor delivery of the green fluorescent protein geneto report future occurrence of metastasisSatoshi Hasegawa,1–3 Meng Yang,1–3 Takashi Chishima,3 Yohei Miyagi,4 Hiroshi Shimada,3

A. R. Moossa,2 and Robert M. Hoffman1,2

1AntiCancer, Inc., San Diego, California 92111; 2Department of Surgery, University of California, San Diego,California 92103; and Departments of 3Surgery and 4Pathology, Yokohama City University School ofMedicine, Yokohama, Japan.

The green fluorescent protein (GFP) gene was administered to intraperitoneally (i.p.) growing human stomach cancer in nude miceto visualize future regional and distant metastases. GFP retroviral supernatants were injected i.p. from day 4 to day 10 after i.p.implantation of the cancer cells. Tumor and metastasis fluorescence was visualized every other week with the use of fluorescenceoptics via a laparotomy on the tumor-bearing animals. At 2 weeks after retroviral GFP delivery, GFP-expressing tumor cells wereobserved in gonadal fat, greater omentum, and intestine, indicating that these primary i.p. growing tumors were efficientlytransduced by the GFP gene and could be visualized by its expression. At the second and third laparotomies, GFP-expressing tumorcells were observed to have spread to lymph nodes in the mesentery and other regional sites. At the fourth laparotomy, widespreadtumor growth was visualized by GFP expression, inducing liver metastasis. No normal tissues were found to be transduced by theGFP retrovirus. Thus, reporter gene transduction of the primary tumor enabled detection of its subsequent metastasis. This genetherapy model could be applied to primary tumors before resection or other treatment to have a fluorescent early detection systemfor metastasis and recurrence. Cancer Gene Therapy (2000) 7, 1336–1340

Key words: metastasis; green fluorescent protein; reporter gene therapy; early detection.

Anumber of approaches have been taken to labeltumor cells to visualize and track them in vivo.

Previous attempts to genetically label tumor cells fortracking purposes used the Escherichia coli b-galactosi-dase (lacZ) gene to detect micrometastases.1,2 However,detection of lacZ requires extensive histological prepa-ration, with sacrifice of the tissue and/or animal; there-fore, it was not possible to image, visualize, and studytumor cells in real-time in viable fresh tissue or in thelive animal.

The ability to confer real-time visualization and imag-ing of tumor growth and progression in viable freshtissue and in the live animal would be an importantfactor in the development of a real-time reporter genefor metastasis and recurrence. Several approaches havebeen developed with this goal in mind: Fukumura et al3

and Chambers et al4 labeled tumor tissue with fluores-cent dyes. However, these methods are not suitable forlong-term metastasis studies. Weissleder et al5 haveinfused tumor-bearing animals with protease-activatednear-infrared fluorescent probes. Tumors with appropri-ate proteases could activate the probes and could be

imaged externally. The limits to such a system include amuch higher liver to tumor background precluding livermetastasis imaging, which is among the most importantmetastatic sites; the stated time limit of 96 hours, whichprecludes growth and efficacy studies; the requirementof appropriate tumor protease activity; and the require-ment of selective tumor delivery of the probes.

Another attempt involved insertion of the luciferasegene into tumor cells such that they emit light.6 How-ever, luciferase enzymes transferred to mammalian cellsrequire the exogenous injected delivery of their luciferinsubstrate, an invasive and impractical requirement in anintact animal. The image resolution of this approach isslow, requiring anesthesia due to the long periodsneeded for image acquisition. Also, it is not knownwhether luciferase genes can function stably over signif-icant time periods in tumors and in the metastasesderived from them.

It became clear that higher specificity, resolution, andphysiological conditions were necessary to report the nat-ural course of tumor progression and metastasis on areal-time basis. The green fluorescent protein (GFP) gene,cloned from the bioluminescent jellyfish Aequorea victoria,7

was chosen to satisfy these conditions because it hasdemonstrated its great potential for use as a cellularmarker.8,9 GFP cDNA encodes a 283-aa monomericpolypeptide with a molecular mass of 27 kDa10,11 that

Received December 28, 1999; accepted June 30, 2000.Address correspondence and reprint requests to Dr. Robert M. Hoff-

man, AntiCancer, Inc., 7917 Ostrow Street, San Diego, CA 92111. E-mailaddress: all@anticancer.com

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requires no other Aequorea proteins, substrates, or cofac-tors to fluoresce.12 Recently, GFP gene gain-of-functionbright mutants have been generated by various tech-niques13–16 that have been humanized for high expres-sion.17

We have developed technology that has enabled thestable transduction of the GFP gene into a large series ofhuman tumor cell lines in vitro.18–25 The tumor cell lineswere able to stably express GFP at high levels both invitro and in vivo. We have previously demonstrated theimportant parameter that GFP-expressing cancer cellscould be directly visualized in fresh tissues of trans-planted animals at a very high resolution down to thesingle-cell level.18–22 With this technology, we were ableto visualize tumor cells that had seeded with or withoutsubsequent colonization in all the major organs, includ-ing the liver, lung, brain, spinal cord, axial skeleton, andlymph nodes.18–22 Our recent results include the devel-opment of orthotopic GFP metastatic models of lungcancer,23 prostate cancer,24 melanoma,25 and colon can-cer.26 These results demonstrated that GFP gene-trans-fected tumor cells represent a new tool to study tumorcell growth, dissemination, invasion, metastasis, andprogression through all stages.

In this study, a GFP-gene tumor transduction system wasdeveloped as a potential clinical application to report theoccurrence and recurrence of metastasis in real-time. Ret-roviral gene transfer leads to stable integration in the targetcell genome that is limited only to dividing cells.27 In thisstudy, we have targeted GFP to primary cancer cells in vivousing a retroviral vector. We demonstrate here the GFPtransduction of the primary tumor that results in GFPexpression in subsequent metastases.

MATERIALS AND METHODS

GFP retroviral vector

pLEIN, a retroviral vector, was purchased from Clontech (PaloAlto, Calif). pLEIN expresses enhanced GFP and the neomy-cin resistance gene on the same bicistronic message.23

Cell culture

PT67, an NIH 3T3-based packaging cell line expressing the10A1 viral envelope, was purchased from Clontech. PT67 cellswere cultured in Dulbecco’s modified Eagle’s medium (IrvineScientific, Santa Ana, Calif) supplemented with 10% heat-inactivated fetal bovine sera (Gemini Bio-Products, Calabasas,Calif), 100 U/mL penicillin, and 100 mg/mL streptomycin.23

NUGC-4 cells were a kind gift of Dr. Narita (Laboratory ofExperimental Pathology, Aichi Cancer Center Research Insti-tute, Nagoya, Japan).28 NUGC-4 cells were cultured in RPMI1640 (Life Technologies, Grand Island, NY) supplementedwith 10% heat-inactivated fetal bovine sera, 100 U/mL peni-cillin, and 100 mg/mL streptomycin.

Retroviral transduction of packaging cells

PT67 packaging cells were plated at a density of 1 3 105 cellsin a 6-well plate 24 hours before transfection. The cells were70–80% confluent at the time of GFP retrovirus transduction.A total of 2.5 mg of plasmid and 15 mL of N-(1-[2,3-dioleoy-

loxy]propyl)-N,N,N-trimethylammonium methylsulfate re-agent (Boehringer Mannheim, Indianapolis, Ind) were mixedaccording to the manufacturer’s protocol. The cells wereexamined by fluorescence microscopy 48 hours after transfec-tion. For selection, the cells were cultured in the presence of0.5–2.0 mg/mL G418 (Life Technologies) for 14 days.

Preparation of retroviral supernatant

Retroviral GFP producer packaging cells were cultured at 80%confluence for 24 hours to generate retroviral supernatants.Supernatants were harvested, passed through a 0.45-mm filter,and stored at 280°C.29

Viral titer determination

NIH 3T3 cells were plated at a density of 1 3 105 cells in 10-cmdishes 24 hours before infection. The cells, at 70% confluenceat the time of infection, were incubated in 5 mL of undilutedand diluted retroviral supernatants. Selection in G418 (1.0mg/mL) began at 48 hours postinfection. After 14 days, cellswere stained with 0.5% methylene blue dissolved in 50%methanol; G418-resistant colonies were counted.29

Tumor transplantation

A total of 1 3 107 NUGC-4 cells in 1 mL of RPMI 1640 wereinoculated subcutaneously into 6- to 8-week-old BALB/c nu/nufemale mice. At 5 weeks postinoculation, the subcutaneoustumor was excised and cut into pieces that could pass throughan 18-gauge needle. Tumor pieces were suspended in RPMI1640, and 1 mL of suspension was injected with an 18-gaugeneedle into the peritoneal cavity. All mice were engrafted atthe same time.

In vivo transduction of GFP

A total of 1 mL of the GFP retroviral supernatant, produced asdescribed above, was supplemented with 8.0 mg/mL polybreneand was administrated to the tumor-bearing mice once per dayintraperitoneally (i.p.) from day 4 to day 10 after tumortransplantation.30

Visualization of tumor growth, spread, and metastasisby GFP fluorescence

Mice were anesthetized by isoflurane inhalation and put in asupine position. A laparotomy was performed via a midlineincision. Fresh visceral organs were analyzed under a fluores-cence microscope with GFP filters (Chromatechnology Corp.,Brattleboro, Vt). After observation, the abdominal wall andthe skin were closed with 6–0 silk sutures.

Histological examination

For histological studies, GFP-expressing tissues were removedat the time of sacrifice and put into 10% buffered formalin. Allof the tissues were subsequently processed through alcoholdehydration, chlorate, and paraffinization. Tissues were em-bedded in paraffin and sectioned at 3 mm. All slides werestained by hematoxylin-eosin and examined microscopically.

RESULTS

Packaging cells and viral titer

The packaging cells were examined by fluorescence micros-copy 48 hours after transfection and were visualized to

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highly express GFP. After selection of the packaging cellsin G418 for 2 weeks, the retroviral supernatants wereharvested and titrated on NIH 3T3 cells. The supernatantviral titer was ;1.5 3 105 colony-forming units/mL. A totalof 12 mice were injected with 1 mL of this virus daily fromday 4 to day 10 after tumor transplantation. The experi-ment plan is outlined in Figure 1.

In vivo GFP transduction of i.p. tumor tissue

At 2 weeks after tumor transplantation, the first laparot-omy was performed on the tumor-bearing mice that hadbeen infected with GFP retroviral supernatants. The vis-ceral organs were analyzed during the surgical procedureby fluorescence microscopy. The tumors growing i.p. werevisualized to strongly express the GFP gene at the firstlaparotomy, mainly growing in gonadal fat, the greateromentum, and the intestine. The small disseminated tu-mors in gonadal fatty tissue could not be detected bybright-field microscopy (Table 1; Fig 2, a and b).

Visualization of tumor spread and metastasis by GFPexpression

At 4 weeks posttransplantation, the second laparotomyrevealed GFP-expressing tumors on the surface of the

colon (Fig. 2g). At the second laparotomy (4 weeksposttransplantation), GFP-expressing tumor cells werevisualized in lymph nodes in the mesentery (Table 1; Fig2, c–e). The second laparotomy demonstrated thatmesenteric lymph nodes, adjacent to GFP-expressing

Figure 1. Experimental plan.

Figure 2. Imaging of tumors after in vivo GFP: retroviral transduction.GFP fluorescence enabled visualization of small, disseminated tumorsin gonadal fatty tissue 2 weeks after implantation; these tumors couldnot be detected by bright-field microscopy. a: bright field; b: fluores-cence. Bar 5 500 mm. GFP-expressing tumor cells were visualized inlymphatic vessels and lymph nodes in the mesenterium at 4 weeksposttransplantation. c: bright field; d,e: fluorescence. Bar 5 500 mm. At4 weeks posttransplantation, GFP-expressing tumors were found onthe surface of the colon (g). Bar 5 1000 mm. After 8 weeks, mesentericlymph nodes were swelling with GFP-expressing tumors (h). Bar 51000 mm. At the time of death, multiple metastatic nodules expressingthe GFP gene were observed on the surface of the liver (f). Bar 5 1000mm. The experimental plan is outlined in Figure 1.

Table 1. GFP-Reported Time Course of Metastatic Spread of Peritoneal Human Stomach Cancer NUGC-4 in Nude Mice

Week

Number of positive mice out of a total of 12

OmentumGonadal

fat MesenteriumAbdominal

wall Liver Intestine Kidney Spleen

2 5 6 0 2 0 7 0 04 5 7 1 3 0 11 0 06 6 9 8 11 0 12 0 08 6 9 9 12 1 12 2 1

Tumors were transplanted i.p. and then treated with a GFP retrovirus from 4 to 10 days posttransplantation. Tumors were visualized byGFP expression in laparotomized mice under fluorescence optics at the indicated times. See Materials and Methods for details.

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tumors on the colon, were involved with GFP-expressingtumor cells (Table 1; Fig 2h). At 8 weeks, GFP-express-ing tumors were found on the intestines, mesentery, andperitoneum as well as on the surface of the kidney anddiaphragm (Table 1; Fig 3). In one mouse, multiplemetastatic nodules were observed on the surface of theliver (Table 1; Fig 2f; all visualized by GFP expression).

Histological examination

GFP-expressing tissues were examined after hematoxy-lin-eosin staining, and these tissues were identified aspoorly differentiated adenocarcinomas (Fig 4). All GFP-expressing tissue was found to be malignant, indicatingthat normal tissues were not transduced.

A total of 1 mL of retroviral supernatant of PT67 cellswas injected into the peritoneal cavity of five nontrans-planted nude mice once a day for 5 days. The nude micewere sacrificed 2 weeks after the last injection of retro-

viral supernatants; no GFP expression could be detectedin any organs and tissue.

DISCUSSION

This study demonstrated that the GFP gene was able totransduce primary i.p. growing tumor cells by injectionof GFP retroviral supernatants, resulting in subsequentlymphatic, liver and other metastasis.

In the early phase of tumor progression in this model,tumor nodules were visualized by GFP expression; thesetumors were observed to be growing on gonadal fat, onthe intestine, and on the abdominal wall, but not inmesenteric lymph nodes or on the liver, which occurredonly late in the course of the experiment. These exper-iments demonstrated that retroviral administration ofthe GFP gene to the primary tumors enabled thevisualization of subsequent metastasis; this visualizationdid not extend to cells that could have been accidentallyseeded at the time of transplantation. Although therehave been reports of GFP-induced immunogenicity,31

the aggressive tumor progression observed in thepresent experiment suggests that in the NUGC-4 modelin nude mice, the T-cell-deficient host does not mountan immunological defense against the GFP-expressingtumor. This is similar to the results we obtained withB16-GFP melanoma in C57BL/6 mice.25 Future experi-ments will compare NUGC-4 cells transduced both invitro and in vivo with nontransduced NUGC-4 cells todetermine whether GFP transduction affects the dissem-ination pattern of this tumor.

There are two major advantages to retroviral genetransfer compared with other gene delivery systems.First, retroviral gene transfer can lead to stable integra-tion in the target cell genome, thus providing thepossibility of long-term gene expression.27 The GFPgene was continuously expressed in tumor cells for atleast 7 weeks in the present study. Recently GFP on aherpes simplex virus-1 Epstein-Barr virus vector wasadministered to tumor-bearing animals. However, long-time GFP expression did not seem to be achieved.32

Second, retroviral-mediated gene transfer is limited tothe transduction of dividing cells. Retroviruses do nottransduce nondividing cells. It has been reported thatcancer cells attached to visceral organs begin to prolif-erate within 1 week of inoculation into the peritonealcavity.33 During this period in the present study, GFPretroviral-containing supernatants were injected. Mostcells, such as mesothelial cells, muscle cells, liver cells,and fibroblasts in the peritoneal cavity are not usuallydividing.27 Histological examination confirmed retrovi-ral GFP gene expression in cancer cells only. This resultwas confirmed by injecting non-tumor-bearing mice withGFP retrovirus, which resulted in no GFP transductionof normal tissue.

The use of GFP to visualize and track tumor growthand dissemination has advantages over other methodssuch as detection with lacZ and reverse transcriptasepolymerase chain reaction in that for GFP, no tissue or

Figure 3. Peritoneal tumor dissemination was visualized 8 weeksafter tumor implantation. D, tumor on diaphragm; K, tumor on thesurface of the kidney; M, tumor nodule in mesentery; T, tumor onintestine and peritoneum; arrowhead, tumor nodule on intestine;arrow, tumor on parietal peritoneum. Bar 5 5 mm.

Figure 4. Histological examination demonstrated that GFP-ex-pressing tissues were poorly differentiated adenocarcinomas. a: onthe intestine (340 magnification); T and arrowhead, tumor; M,mouse smooth muscle; I, mouse intestinal mucosa. b: in thelymphatic vessel of the mesentery of the intestine (340 magnifica-tion); T and arrowhead, tumor. c: in the diaphragm (3100 magnifi-cation); T and arrowhead, tumor; D, diaphragm. d: on parietalperitoneum (3200 magnification).

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cell preparation is needed. Therefore, GFP cancer cellscan be visualized in vivo in real time.26

The present study has demonstrated that GFP trans-duction of cancer cells in vivo can facilitate the detectionof future subclinical metastasis, which could eventuallybe applied clinically as a reporter of tumor progressionor recurrence.

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