Introduction

Human extranodal nasal type NK/T-cell lymphoma is a predominant extranodal lymphoma characterized by distinctive morphology, immunophenotype and biological behavior. It is more prevalent in Asia, Mexico, and Central and South America, few in western countries and areas,^{1, 2} and is almost consistently associated with Epstein-Barr virus (EBV).^{1, 2, 3, 4} There are about 60 newly diagnosed cases of human extranodal nasal type NK/T-cell lymphoma in West China Hospital of Sichuan University every year, accounting for 16% of non-Hodgkin's lymphomas in the same period.^{5, 6}

Human extranodal nasal type NK/T-cell lymphoma mainly involved mid-facial areas such as nose, sinonasal regions, nasopharynx and palate. Because of its special distribution in anatomy, the biopsy tissues are usually too small to be used anymore after establishment of the diagnosis. Insufficient supply of the tumor materials is a serious problem, which has obviously restricted the deep research for this special tumor. In the last decade, several groups all over the world have done some work in this field, and so far, a few reports could be found in the literature. HANK1, the first cell line of human extranodal nasal type NK/T-cell lymphoma, was reported by Kagami et al.⁷ In the same year, NK-YS, another cell line of the tumor was reported by Tsuchiyama et al.⁸ In 2001, Nagata et al.⁹ successfully established two cell lines of SNK-6 and SNT-8. However, until now no report has been found in establishment of an animal model for human extranodal nasal type NK/T-cell lymphoma by means of subcutaneously implanting the tumor tissues into nude mice. It is known that animal models of the tumors occupy more advantages for research in vivo than that of cell lines but the related work is more difficult, especially for human extranodal nasal type NK/T-cell lymphoma. Immunodeficient nude mice have been widely used as xenograft recipients for human tumors including various kinds of hematological malignancies,^{10, 11} which encourage us to make attempt in this field.

Materials and methods

Patient

A 48-year-old Chinese male complained of persistent mycteroxerosis, headache, nasal obstruction and hemorrhage for 3 months. The nasal biopsy was taken and the diagnosis of nasal NK/T-cell lymphoma was established based on histopathology, immunophenotype and in situ hybridization analysis (positive for CD45RO, cytoplasmic CD3, CD56, granzyme B and EB virus encoded RNA 1/2 (EBER1/2), but negative for surface CD3 and CD20) in May 2000. The patient was discharged from the hospital with complete remission after having received local radiation therapy and two courses of chemotherapy of CHOP (cyclophosphamide, adriamycin, vincristine and prednisolone). Two year later, the patient was presented with fever and stomachache for 1 month, and the gastroscopy showed that a huge ulcerative lesion was presented in the sinus ventriculi. The biopsy was taken and the diagnosis of secondary extranodal nasal type NK/T-cell lymphoma of stomach was established based on the similarity of morphology and the immunophenotypes to the primary tumor. The patient was admitted to hospital. The palliative resection of stomach and gastric-intestinal anastomosis was performed because of widespread adhesion of the organs in abdominal cavity and medium dose of ascites. The ulcerative neoplasm was located in the sinus ventriculi, with the size of 5 cm 4 cm 3 cm, involving in duodenal bulb, middle part of mesocolon transversum, arteria colica media and caput pancreatic and so on. The patient died of multiorgan failure 1 week later after the operation.

Nude mice model establishing

Three- to four-week-old male or female BALB/c (nu/nu) nude mice were provided by West China Animal Center of Sichuan University, weighed approximately 15–20 g, fed in the specific pathogen free (SPF) barrier system and provided food and water ad libitum. All procedures were performed under aseptic condition.

Pieces of fresh tumor sample without necrosis were obtained from the stomach during the operation, it was washed repeatedly in Hank's liquid, minced into 0.5–1 mm³ bits in RPMI1640 culture media with 100 U ml^-1 penicillin and 100 g ml^-1 streptomycin and subcutaneously implanted into the right axillary region of nude mice anesthetized with 0.6% pentobarbital via intraperitoneal (i.p.) injection. The general condition and the appearance changes of the lymphoma-bearing nude mice were observed every day, and the size of transplanted tumor was measured in three dimensions twice a week. As soon as the transplanted tumor reached about 1000 mm³ in size, or the animal showed distress, the mouse was killed. A part of the tumor tissues was used for continuous transplantation, the remaining part was divided into two parts, one for routine morphologic observation and the other preserved in liquid nitrogen for further use.

Histopathology and immunophenotype

For histological examination, an autopsy was performed on each passage of nude mice. The samples were fixed in 10% neural-buffered formalin, embedded in paraffin. Four-micrometer-thick sections were made and stained with hematoxylin and eosin. Morphologic observation included architecture of the tumors and distribution of the tumor cells, the size and shapes of the tumor cells, the number of mitotic figures and other combined changes including necrosis, angiocentric invasion and so on. The antibodies used in present study were summarized in Supplementary Table 1.

DNA extraction and polymerase chain reaction (PCR)

Genomic DNA from paraffin-imbedded and fresh tissues of transplanted tumors was extracted by phenol–chloroform procedures. Successful DNA extraction was confirmed by amplification of 110 bp fragment of -globin. Detection of DNA for human sequence-tagged site SY14 and T-cell receptor (TCR-) gene rearrangement was performed (Supplementary Table 2). One microliter of 25 mol l^-1 of each primer (Takara, Tokyo, Japan, in supplementary data) was used in 25 l reaction. The PCR reaction mixture (Takara) contained 2.5 l of 10 PCR buffer, 1.5 l of 25 mmol l^-1 MgCl₂, 2 l of 2.5 mmol l^-1 of dNTP, 1.25 U of Taq DNA polymerase and 1 g template DNA. Five microliters of PCR products was electrophoresed in 1.5% agarose gels (110 V, 30 min). Gels were stained with Goldview (Saibaisheng, Beijing, China) and visualized with ultraviolet light using the Image Acquisition and Analysis Software (Bio-Rad, CA, USA). PCR product of human sequence-tagged site SY14 was directly sequenced by ABI 377 DNA Sequencer (Genecore Company, Shanghai, China), and the result was blasted in NCBI GenBank.

In situ hybridization

In situ hybridization (ISH) was performed with fluorescein-labeled oligonucleotide probe complementary to two EBV-encoded small RNAs, that is, EBER-1 and EBER-2 (EBER1/2) (Dako, Glostrup, Denmark). Rabbit anti-FITC antibody conjugated with alkaline phosphatase (Dako) was used to combine with the probe, while NBT/BCIP was used as a substrate. The dark blue purple hybridizing signal was located in cell nucleus.

Fluorescent in situ hybridization

Fluorescent in situ hybridization (FISH) was performed to detect the Homo sapiens Y chromosome in transplanted tumors using CEP Y probe (DYZ1, Vysis, Dowaes Grove, IL, USA) labeled with spectrum Orange according to the manufacturer's instructions. CEP Y probe was a DNA probe hybridized to satellite III sequence of the Y chromosome (band Yq12, locus DYZ1). Fluorescence signals were visualized by fluorescence microscope (Olympus, Tokyo, Japan) equipped with the appropriate filter set, and the images were captured by the DP Controller imaging system (Olympus).

Result

Growth and passage

The pieces of human extranodal nasal type NK/T-cell lymphoma tumor tissues were transplanted into one 4-week-old female BALB/c (nu/nu) nude mouse. The 1st passage of transplanted tumor was visible 12 weeks later after the transplantation. After undergoing about 10 weeks of slow-growth period, the transplanted tumor in the 1st passage reached the size of 1.38 cm 1.0 cm 1.1 cm and was implanted to the 2nd passage at the end of the 28th week. Then, subsequent passages were performed by serial transplantation of the tumor tissues. After the 15th passage, the transplanted tumor demonstrated a stable growth curve for about 3–4 weeks, which include 1 week for latent period and slow-growth period, respectively, 1–2 weeks for rapid-growth period. The nude mice often showed cachexia in the late stage. Up to now, the transplanted tumor had been passaged to the 32nd generation (Figures 1, 2a).

Figure 1.

Growth curve of transplanted tumors. The growth curves of the 1st, 4th, 8th and 12th passages of transplanted tumor indicated that the growth period became shorter with the continuous passage (a). The growth curves of transplanted tumor were stable after the 15th passage, about 3–4 weeks: 1 week for latent period, 1 week for slow-growth period, 1–2 weeks for rapid-growth period (b). ^*Passage of resuscitation.

Full figure and legend (111K)

Figure 2.

Lymphoma-bearing nude mice of human extranodal nasal type NK/T-cell lymphoma. (a) Some passages of lymphoma-bearing nude mice were shown, including the 20th passage in resuscitation. The arrows pointed to the transplanted tumors. Enlargement of axillary nodes were observed in the 5th passage, and ulcer of the transplanted site appeared in the 11th passage. The transplanted tumor formed an ill-defined mass in the right axillary region of nude mouse (left in b), and the arrows pointed to the transplanted tumors. The cut surface was gray–white–red color, with tender and fish-flesh-like appearance (right in b).

Full figure and legend (142K)

Seven passages of frozen tissues and/or cells had been thawed out, including the 5th, 6th, 8th, 10th, 16th, 18th and 20th passages. The survival rate after resuscitation was 100%. The growth curve of resuscitative transplanted tumor and the condition of resuscitative-lymphoma-bearing nude mice were similar to the original transplanted tumor (Figure 1). The pieces of fresh transplanted tumor samples in 20th passage were taken from the nude mice, and transplanted directly into the right axillary region of 10 nude mice, respectively. The transplanted tumors had appeared in all of the 10 nude mice 1 week later. The survival rate after transplantation reached 100% as well.

Pathologic features

The transplanted tumor formed an ill-defined mass in the right axillary region of the nude mouse. The size was about 1.1 cm 1.0 cm 1.0 cm to 1.6 cm 2.3 cm 1.2 cm (Figure 2b). The cut surface was gray–white–red in color, with tender and fish-flesh-like appearance (Figure 2b). All of the lymphoma-bearing nude mice showed splenomegaly at different extents, which ranged from 3.2 cm 1.0 cm 0.8 cm to 3.8 cm 1.4 cm 1.1 cm. Ulceration in the transplantation sites and enlargement of axillary nodes were presented in some of the passages (Figure 2a). No obvious abnormalities were viewed in other organs of lymphoma-bearing nude mice, such as liver, lung, kidney, brain and so on.

The histopathological features of the transplanted tumors were almost similar to those of the original secondary extranodal nasal type NK/T-cell lymphoma of the stomach. The medium- to large-sized tumor cells were arranged in sheets with a little mesenchyma, and more or less coagulation necrosis also could be found in some of the samples. The cytoplasm of the tumor cells was mild-to-moderate amount, pale or slightly eosinophilic, and most of the tumor cells occupy a large, round–ovoid and pleomorphic nuclei containing homogeneously dispersed granular chromatin and one (rarely two) basophilic nucleolus. Mitotic figures were common, estimated at 3–10 per high-power field, and pathologic mitotic figures were also observed. The angiocentric and angiodestructive growth patterns were visible in some of samples, but the inflammatory components were less prominent (Figure 3). The tumor cells also diffusely infiltrated in soft tissues (skeleton muscles and fatty tissues) (Figures 4a, b). The skin ulcer was often found at the site of implantation.

Figure 3.

Histopathology of transplanted tumors cells in lymphoma-bearing nude mice. The histopathological features of transplanted tumor cells in the 1st (c, H&E 200; d, H&E 1000) and 30th (e, H&E 200; f, H&E 1000) passages were similar to those of the donor tumor cells (a, H&E 200; b, H&E 1000). The medium- to large-sized tumor cells were arranged in sheets with a little mesenchyma.

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Figure 4.

Infiltration and dissemination of transplanted tumor cells in lymphoma-bearing nude mice. The tumor cells diffusely infiltrated in muscle bundles and fatty tissues (a, H&E 400; b, H&E 200). The tumor cells disseminated to liver (c, H&E 400), spleen (d, H&E 200), lung (e, H&E 400) and kidney (f, H&E 400) of lymphoma-bearing nude mice. The arrows pointed to the transplanted tumor cells.

Full figure and legend (307K)

In the late stage, the tumor cells disseminated all over the bodies of the nude mice, the organs involved were spleen, lymph node, liver, kidney, lung and so on (Figures 4c–f). In the involved spleen of all passages, the distribution of the tumor cells mainly located in the white pulps and also infiltrated the red pulps. Twelve of 32 (37.5%) lymphoma-bearing nude mice passages presented with lymph nodes were involved. The architecture of lymph nodes had been destructed partially or completely. Liver dissemination was observed in all lymphoma-bearing nude mice passages except the 28th passage. The tumor cells mainly infiltrated in the portal area, sometimes tumor cells also could be found in sinusoid regions. Kidney dissemination was observed in about 40% (13 of 32) nude mice, and the tumor cells infiltrated in the interstitial region or surrounding the vessels near the hilus renals. Lung was involved in 12 of 14 passages (86%). Tumor cells infiltrated in the interstitial regions of the lung and formed in clusters nearby the small vessels. No tumor dissemination was found in the brains of 12 of 14 passages.

The tumor cells, no matter presented in the transplanted tumors or disseminated in other organs, displayed the similar morphology and immunophenotypes. The tumor cells were positive for CD56, cytoplasmic CD3, granzyme B or TIA-1 and LMP1, sometimes for CD8 but negative for surface CD3, CD7, CD20 and CD1a. EBER1/2 was found. No TCR- gene rearrangement was detected in the transplanted tumors. It was almost similar to the donor tumor of secondary extranodal nasal type NK/T-cell lymphoma of the stomach (Table 1, Figure 5). However, some discrepancy was noted in the immunophenotype of tumor cells between donor tissues and transplanted tumors. Some of tumor cells in transplanted tumor were positive for CD5 and CD57 antigens (Figure 6), unlike those in donor tissue (negative for CD5 and CD57). On the other hand, the loss of CD45RO expression had been observed in the course of transplanted tumor passaging (Figure 6). We also found that CD56 and CD57 antigens were expressed differently in the transplanted tumors of the same passage (Table 1, Figure 6).

Figure 5.

Immunostaining of cytoplasmic CD3, CD56, granzyme B, LMP1 and EBER1/2, SP 1000. The immunophenotype of 1st and post-15th passages of transplanted tumor was similar to that of the human extranodal nasal type NK/T-cell lymphoma of stomach (donor).

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Figure 6.

Different immunostaining of CD5, CD57, CD56 and CD45RO in the transplanted tumor cells SP 1000. Unlike tumor cells in donor (negative expression), some of tumor cells in the transplanted tumors were positive for CD5 and CD57 antigens. On the other hand, CD45RO antigen was positive in the tumor cells of donor and the 1st passage of transplanted tumor, but negative in the 30th generation. In the 15th passage, expression of CD56 was positive, but CD57 was negative. In the 30th passage, both CD56 and CD57 positively expressed. CD56 and CD57 antigens were expressed differently in the transplanted tumors of the same passage.

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Table 1 - Summary of immunophenotype and in situ hybridization observations of primary and secondary original tumor and the transplanted tumor.

Full table

No bands of 190 bp had been observed in PCR analysis for TCR- gene rearrangement except positive control (Jurkat cell line, the data were not showed), which suggested that TCR- gene was in germline configuration in donor samples and transplanted tumors, and no clonal T-cell proliferation was presented.

Species identification

Two methods, detection of human sequence-tagged sites SY14 and Y chromosome by PCR and FISH, respectively, had been used to identify the species of the transplanted tumor in present study. The results showed that the tumor tissue of donor patient and transplanted tumor in nude mice had amplified the same special bands of 472 bp, same to the positive control, but no band had been observed in tissues of normal male nude mice (Figure 7a). The blast result demonstrated that there were no mutations of the SY14 DNA sequences between donor tumor and transplanted tumor tissues, and the homology was 100%. The result of Homo sapiens Y chromosome detection showed that an orange signal could be visualized in the nuclei of both transplanted tumor cells and SNK6 cell line (positive control) (Figure 7b), but not in the cells of negative and blank control.

Figure 7.

PCR and fluorescent in situ hybridization (FISH) analysis for species identification of transplanted tumor. The tumor tissue of donor and transplanted tumor in nude mice had amplified the same 472 bp special bands of human sequence-tagged sites SY14, same to the positive control, but no band had been observed in tissues of normal male nude mice (a). Positive control: reactive hyperplastic lymph node of a male. Negative control: normal male nude mice tissue. The orange fluorescence signal of Homo sapiens Y chromosome was visualized in nuclei of SNK6 cell line (positive control, developed from the peripheral blood of a male presented with human extranodal nasal type NK/T-cell lymphoma and kindly provided by Professor Nagata of Tokyo Medical and Dental University) (left in b) and transplanted tumor cells (right in b) (FISH 600).

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Discussion

Human extranodal nasal type NK/T-cell lymphoma is an aggressive neoplasm with a poor prognosis. The etiology and pathogenesis of the tumor is still unknown, and the research on this special lymphoma might be hampered by the limitative samples of the tumor. In the process of establishing the model, we had tried to transplant samples from six patients into nude mice. For the five failed cases, the tumor samples were all taken from the involved nasal cavities, and the nude mice had been infectious before the transplanted tumor was formed. Only the one taken from the secondary gastric NK/T-cell lymphoma was successfully transplanted. So the most difficult problem, which should be solved in establishing an animal model, is infection.

Our group had spent 3 years in attempting to establish the nude mice model of human extranodal nasal type NK/T-cell lymphoma by means of directly implanting small pieces of tumor tissue into the subcutaneous region of the BALB/c (nu/nu) nude mice. We could not successfully transplant the tumor tissue of patient to the nude mice by direct injection of single cell suspension. However, the tumor cells could be passaged by injection of single cell suspension from one nude mice to another, which suggested that the pieces of nasal type NK/T-cell lymphoma tissue might be easier as an allograft to grow in the nude mice than single cell suspension.

The present novel nude mice model of the tumor maintains the essential characters of human extranodal nasal type NK/T-cell lymphoma in histopathology, immunophenotype, genotype, status of EBV infection and so on. The comparison between the present nude mice model and the cell lines of human extranodal nasal type NK/T-cell lymphoma reported in the literatures is listed in Table 2.^{7, 8, 9} However, there are also some of the differences in above all aspects between the transplanted tumor of the nude mice and tumor samples of the donor patients along with the increased number of the transplanted generations. First, inflammatory cells intermingled are gradually decreased in number, the size of the tumor cell become larger and less polymorphism and the contents of the interstitial tissue are also decreased. Second, the expression of some antigens is sometimes more or less instable, which could also be found in cell lines and animal models of other tumors.^{12, 13, 14, 15} The exact reason is still unknown, and Lovejoy et al.¹⁶ considered that some variations in phenotype may be due to interspecies differences in gene function, but others arise as a result of differences in functional redundancy of a specific gene product. We hypothesize that these changes might be related to some suitable potential acquired by tumor cells to adapt the changed environment.

Table 2 - Comparing four cell lines and present novel nude mice model of the human extranodal nasal type NK/T-cell lymphoma.

Full table

It is known that some criteria as follows are required for the confirmation of an established heterogenic transplantation: firstly, it is continuously passaged for at least 15 generations with a relatively stable growth cycle. Secondly, the survival rates of both transplantation and resuscitation must be reaching 60–90%. Thirdly, it occupies the same species with that of the donor sample. And finally, it maintains the characteristics of the tumor in histopathology, immunophenotype and genotype and other related factors^{7, 8, 9, 17} The present novel nude mice model of human extranodal nasal type NK/T-cell lymphoma matches all the above criteria including the number of generation, with a stable growth cycles and 100% survival rate in both transplantation and resuscitation and so on.

The species identification is very important for heterogenic transplantation. It had been reported that the method for species identification included chromosome karyotype analysis, specific Homo sapiens origin gene detection, human leukocyte antigen and so on.^{7, 8, 9, 15, 18} Most researches adopted chromosome karyotype analysis to confirm the species origin, which was an easier method for ascites tumor or leukemia model, but was difficult for solid tumor.¹⁹ With the development of cytogenetics, some advanced technologies such as spectral karyotyping, FISH and comparative genomic hybridization had been adopted to analyze the species of transplanted tumor and cell line.^{20, 21} So far, FISH technique, which is convenient, sensitive and specific, had became one of the necessary chromosomal analysis methods.^{22, 23, 24, 25} In our experience of species identification, FISH is better than karyotype analysis in solid tumors. In addition, we also adopted PCR method to detect the human sequence-tagged site SY14, which was specific to human Y chromosome, to confirm the species of the present transplanted tumor in another aspect.

Up to now, we have established and identified the novel nude mice model of human extranodal nasal type NK/T-cell lymphoma, which maintained the essential characters of this special lymphoma and would be an ideal tool in vivo for not only understanding the pathogenesis and dissemination of the tumor, but also evaluating various novel treatments to supply effective therapeutic strategies against this lymphoma.

References

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Acknowledgements

We wish to thank Professor Norio Shimizu (Department of Virology, Tokyo Medical and Dental University, Japan) and Dr Yu Zhang (First Department of Internal Medicine, Tokyo Medical University, Japan) for providing us with human extranodal nasal type NK/T-cell lymphoma cell line, SNK6. This work was supported by National Natural Science Foundation of China (30470747 and 30570769).

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)