International Journal of Pancreatology, vol. 26, no. 1, 33–41, August 1999 © Copyright 1999 by Humana Press Inc. All rights of any nature whatsoever reserved. 0169-4197/99/26:33–41/$12.25
Human Pancreatic Leiomyosarcoma (PZX-7) Growing as a Serially Transplantable Xenograft in Immunosuppressed Mice Attila Zalatnai,* József Bocsi, Tibor Csákány, Tamás Fekete, and Jovánka Lásztity 1st Institute of Pathology and Experimental Medicine, Budapest, Hungary
Summary Background. Several human leiomyosarcoma xenografts have been established, but pancreatic smooth muscle sarcomas have never been serially transplanted and investigated. Method. Immunosuppression of CBA/CA mice was achieved by thymectomy, whole-body irradiation, and bone marrow reconstruction. Tumor fragments were subcutaneously implanted from a Grade III pancreatic leiomyosarcoma and serially passaged for more than 24 mo. The xenografted tumors were characterized by morphological, morphometrical, biochemical, and flow cytometric methods. Results. The tumor has retained its characteristic morphology and no further differentiation occurred. The mitotic counts and the amount of the connective tissue all remained constant. The calculated volume doubling time was 11.3 d. Immunohistochemically, the tumor proved to be p53-negative, but the strong expression of the bcl-2 remained as a constant feature throughout successive transplantations. The DNA index and the proliferation indices did not change significantly with the time (mean DI: 1.65, range: 1.561–1.70; mean PI: 17.9%, range: 15.3–20.7%). Lactose dehydrogenase (LDH) isoenzyme electrophoresis evidenced a retained human pattern of the tumor even after 32 mo of transplantations. Conclusion. The first human pancreatic leiomyosarcoma xenograft (PZX-7) growing in immunosuppressed mice is described and characterized. Key Words: Pancreatic neoplasms; leiomyosarcoma; xenografts; human tumors; lactate dehydrogenase.
To achieve this goal, the given xenograft systems must be stable, retaining their fundamental biological behavior, morphological properties, and proliferative activities. Smooth muscle sarcomas have rarely been subjects of xenotransplantation (1–7), and pancreatic leiomyosarcomas have never been investigated in experimental systems. Indeed, primary leiomyosarcoma of the pancreas is an extremely rare finding; to date only 27 such cases have been reported. Recently, we described an additional case occurring in an elderly male (8), and this tumor was also implanted into immunosuppressed mice in order
Introduction Malignant neoplasms growing as serially transplantable tumors may serve as additional tools for testing the potentially promising anticancer drugs. Received July 29, 1998; Revised February 19, 1999; Accepted April 19, 1999. *Author to whom all correspondence and reprint requests should be addressed: 1st Institute of Pathology and Experimental Cancer Research, Semmelweis University of Medicine, H-1085 Budapest, Üllöi út 26. Hungary. E-mail: [email protected]
34 to establish a serially transplantable line. It was successfully passaged over 7 generations for more than 2 yr. This represents the first human pancreatic leiomyosarcoma xenograft system.
Zalatnai et al. tumor has been designated as PZX-7, but the successively transplanted ones as PZX-7/1, PZX-7/2, and so forth. The volume doubling time (VDT) was calculated according to Broström et al. (12):
Materials and Methods
Primary Tumor A Grade III leiomyosarcoma of the pancreas was operated on in 1995. Its detailed characteristics have been published elsewhere (8). Briefly, the tumor was located in the pancreatic head of a 57-yr-old male, and its smooth muscle origin was confirmed immunohistochemically. The tumor had infiltrated the surrounding tissues, and the patient expired 7 mo later.
Host Animals Male and female inbred CBA/CA mice were used. Immunosuppression has been achieved by the standard method used at our institute (9) originally introduced by Pickard et al. (10). Briefly, thymectomy in young animals was followed by a whole-body irradiation and bone marrow reconstruction. Mice were kept in an air-conditioned animal facility (10/cage) with 55% relative humidity and at a temperature of 25°C. Tap water and pelleted rodent chow were provided ad libitum. During the whole experiment, the ethical standards of the National Health Scientific Board were followed.
Serial Transplantations The surgically removed tumor was divided into two parts: one had been fixed in neutral buffered formalin for further processing, and the remaining tissue was minced and paired samples of 3–4 mm in diameter were introduced subcutaneously into the back region of 4 previously immunosuppressed mice. When the xenotransplants reached a diameter of about 0.3 cm, their volume was determined weekly with microcaliper. The two largest diameters of the bilateral masses were measured separately and the volumes (V) were calculated by the following formula (11): V = length × width2/2. As soon as the tumors grew to about 1.5 cm in largest diameter, the animals were killed by neck dislocation and autopsied. Half of the neoplasms were processed for routine histology and immunohistochemistry, and the other parts were transplanted further. The original International Journal of Pancreatology
log 2 × (t1 − t0) log V1 − log V0
where V0 means the tumor volume when it first became palpable (0.3 cm in diameter) and V1 is its volume at time t1 when further transplantation was carried out (1.5 cm in diameter).
Histology Serial sections of 5-µm thickness were prepared from formalin-fixed, paraplast-embedded material, and were routinely stained by H&E. Mitotic figures in the nonnecrotic tumorous areas have been counted in high-powered fields (HPFs) (× 40 objective, × 10 ocular). The total area of each microscopic field was 0.036 mm2, and the final values were expressed as a number of mitoses/mm2. Mitotic activity was assessed by screening of 100–500 HPFs (depending on the tumor size and the necrotic areas). The connective tissue fibers were visualized by picrosiriusred staining (13).
Immunohistochemistry It was carried out by indirect method and visualization by streptavidin and biotinylated kit. In a deparaffinated, 5-µm thick section, the following primary antibodies were used: p53 (NOVOCASTRA, Newcastle, UK) and bcl-2 protein (BioGenex, San Ramon, CA) after a microwave antigen-retrieval procedure.
Morphometry The relative amount of the picrosirius red-stained collagenous fibers to the whole tumorous areas has been determined by using an Olympus Vanox microscope equipped with a Cue-2 image analyzer system (Tokyo, Japan).
Flow Cytometry The detailed process was described earlier (9). Briefly, the 50-µm paraplast sections were dewaxed and digested by 0.25% trypsin containing 0.025% RNase A, followed by an ultrasound treatment. The nuclei were stained with propidium iodide. AnalyVolume 26, 1999
Fig. 1. Microscopic appearance of the xenografted leiomyosarcoma. (A) Tumor from the first passage (HE, × 400); (B) tumor from the seventh passage (HE, × 250). The spindle-shaped sarcomatous elements are arranged in closely packed fascicles, and this pattern has remained as a constant feature throughout the serial transplantations without any sign of dedifferentiation.
sis was performed on a Becton-Dickinson FACStar flow cytometer. The DNA index and the synthetic S-phase fraction (SPF) were determined by using the Rabinovitch’s Multicycle Software. Analysis of variance with one criterion of classification was performed according to Adler and Roessler (14).
Lactate Dehydrogenase (LDH) Isoenzyme Measurement The slightly modified method originally described by Montes et al. (15) was applied. Tumor pieces (0.1 g) and control tissues (mouse liver, human heart, and liver) frozen in liquid nitrogen were crushed and suspended in 1 mL of 0.9% NaCl + 0.66 mM ethylenediamine tetra-acetic acid (EDTA) solution. After sonication for 15 s and incubation at 4°C for 30 min, the samples were centrifuged (16,250g for 20 min at 4°C). Sixty microliters of the supernatant mixed with 90 µL of 1.7% agarose gel have been loaded on a 1.7% agarose gel and separated by electrophoresis (25 mA/cm at 4°C) in 47 mM sodium veronal buffer (pH = 8.6). The isoenzymes were detected as purple bands after incubating the gel in the reagent buffer (5 mg tetrazolium blue, 5 mg potassium cyanide, 10 mg NAD, 0.25 mg phenoazinmethosulphate, 0.25 M lithium lactate in 6 mL phosphate-buffered saline [PBS]; pH = 7.6) for 60 min at 37°C. International Journal of Pancreatology
Results Macroscopy The tumors had first become palpable after 31 d and approx 110 d later they had reached a diameter of 1.4–1.5 cm. The firm nodules did not invade the neighboring tissues, and they did not result in any ulceration of the skin. The tumors were surrounded by a delicate connective tissue capsule, and no distant metastases were seen. Their cut surface was mainly homogenous, pale tan, with no cystic degeneration, but small necrotic foci did appear in the center.
Microscopy The original tumor was classified as a Grade III leiomyosarcoma composed of closely packed spindle-shaped elements with occasional multinucleated giant cells and minute necrotic areas (Fig. 1A).
Serial Transplantations The tumor has been maintained by successive transplantations for more than 24 mo, but the morphological characteristics of the xenografted tumors did not change with the time. Even at the latest passage, all neoplastic nodules were well circumscribed. The animals were in good condition and free of disVolume 26, 1999
Fig. 2. Growth curve of the PZX-7 leiomyosarcoma after stabilization of the line. Individual values represent tumor volumes (mean ± SEM) of 8-8 neoplasms growing in 4-4 animals. The curve shows a slow progression. The calculated volume doubling time is 11.3 d.
tant metastases. Central necrotic areas were seen just in small foci, but no cystic degeneration, skin ulceration, or superinfection occurred. Figure 2 shows the typical growth curve seen after stabilization of the line. The tumors grew continuously without any sign of spontaneous regression. In the first 3 mo, there was no striking variation among the individual tumor volumes, but at the fourth month of transplantation, their diameters exhibited a wide range. The calculated VDT proved to be 11.3 d. Microscopically, the original histological pattern was well preserved throughout the xenograftings; the degree of differentiation remained constant, multinucleated tumorous giant cells were found in a limited number, no polymorphism developed, the extent of necrosis did not increase, there was no rhabdomyoblastic differentiation (16), and no myxoid or metaplastic areas appeared (Fig. 1B). Occasionally, foamy cells were noted in a scattered fashion. As in the original specimen, no inflammatory host reaction appeared, and no pronounced vascularization was noted.
Immunohistochemistry Neither the primary tumor nor the samples from the late passages expressed p53. However, a strong bcl-2 protein positivity was a constant finding in the majority of the tumor cells throughout the whole experiment. International Journal of Pancreatology
Zalatnai et al.
Fig. 3. Changes in the mitotic activity of the tumor samples during successive transplantations expressed as the number of mitotic figures per mm2. Abscissa indicates the order of xenograftings.
Mitotic Activity (Fig. 3) The original human tumor contained 22.3 mitoses/mm 2, and this number increased after implantation into the mice (33/mm2; +48%). This rise could be attributed to an adaptation response, because beginning with the first passage, the mitosis count had dropped to the original level. During transplantations, no increased mitotic activity was observable (the number of mitotic figures varied between 21.5 and 14/mm2), indicating that the proliferative activity had remained constant. Some tumors were left growing without further transplantation, and comparison was made between the mitotic activities counted at the time of xenografting and 5 mo later (PZX-7/7, 5 mo). We could not observe any increased proliferative activity between the younger and the older tumors.
Connective Tissue Morphometry The relative amount of the collageneous fibers during consecutive transplantations is shown in Fig. 4. In general, the stromal elements accounted for about 10–14% of the whole histological specimen, and this proportion has remained relatively constant.
Flow Cytometry Measurements (Table 1, Fig. 5) The original tumor proved to be aneuploid, the DNA index being 1.561. Throughout successive pasVolume 26, 1999
37 Table 1 Flow Cytometric DNA indices (DI) and Proliferation Indices (PI) of the Human Pancreatic Leiomyosarcoma (PZX-7) Samples During Serial Xenograftings into Immunosuppressed CBA/CA Micea Designation
Fig. 4. Quantitative morphometric measurements in PZX-7 human leiomyosarcoma xenografts. Results of the connective tissue morphometry during serial transplantations. Bars represent the percentage ratio of the picrosirius red-stained collagenous fibers in the given tumor.
sagings, this pattern has remained as a constant feature. The DNA indexes varied between 1.59 and 1.70, and there was no significant shift in the proliferative indices either (mean: 17.9%, range: 15.28–20.73%).
Human Origin The xenograft retained its human character even after nine consecutive passages. Five tumor samples were implanted into nonimmunosuppressed CBA mice, but none of them yielded a tumor. Other evidence was given by LDH electrophoresis. As Fig. 6 shows, the mouse liver is characterized by a single, broad band, but all the human tissues (heart muscle, liver, and the leiomyosarcoma) displayed five differently migrating bands. The isoenzyme pattern of the tumor resembled that of the human liver and a human pancreatic cancer xenograft (PZX-2/14): mainly isoenzymes I and II were expressed.
Discussion The article describes and characterizes for the first time a human pancreatic leiomyosarcoma line main-tained as a serially transplantable xenograft in artificially immunosuppressed mice. In our earlier work, we have shown that human ductal pancreatic carcinoma xenograft can also be established in this host system (9). As a continuation of that study, a Grade III leiomyosarcoma was successfully transplanted and serially passaged in these animals for over 24 mo. International Journal of Pancreatology
Original tumor PZX-7/1 PZX-7/2 PZX-7/3 PZX-7/4 PZX-7/5 PZX-7/6 PZX-7/7 a
DI 1.56 ± 0.05 1.70 ± 0.06 1.69 ± 0.05 1.62 ± 0.15 1.68 ± 0.03 1.68 ± 0.07 1.65 ± 0.01 1.59 ± 0.02
PI, % 20.73 ± 1.02 18.89 ± 5.09 19.04 ± 4.42 16.59 ± 1.68 18.90 ± 6.50 15.28 ± 0.74 16.20 ± 0.42 17.45 ± 2.05
Values represent mean ± SD.
An important question is whether pancreatic leiomyosarcoma differs from other intra-abdominal smooth muscle malignancies. Surely we do not know, but the survey of the literature suggests that the biological behavior of these tumors may be quite different depending on the localization. In colorectal leiomyosarcoma cases, Friesen et al. reported a mean follow-up of 6.9 yr (17). In leiomyosarcomas of stomach, the 5-yr survival is about 19% (18), but in other reports, it may reach as high as 72% (19). Renal sarcomas after radical nephrectomy proved to be curable in about 1/3 of cases (20). Regarding hepatic leiomyosarcomas, Ebert reported an 8-mo survival (21), but Chen and Baur et al. presented cases with a tumor-free survival of 6.5 or 10 yr, respectively (22,23). Gates et al., analyzing 54 such cases from the world literature, could find a mean survival of 3.3 yr (24). Conversely, the retroperitoneal leiomyosarcomas exhibited a short median survival (25 mo) (25). Because the intraabdominal leiomyosarcomas differ significantly in their biological characteristics, it was conceivable that the pancreatic leiomyosarcoma would also behave alternatively. More precisely, we do not know how, because the majority of the case reports in the literature about this tumor do not regularly state the survival. Usually a very short course is mentioned, but there are data about 2 yr 9 mo survival. It is true that the patient from whom our xenografts were removed died 7 mo after the operation, but in this case, just a palliative intervention could be performed. Volume 26, 1999
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Fig. 5. Representative flow cytometric DNA histograms of PZX-7 leiomyosarcoma and the successive xenografted tumor samples. (A) original tumor; (B–H) tumor samples derived form the serial transplantations. The originally aneuploid pattern (DNA index: 1.561) has not been changed with the time. On the histograms, the first peak corresponds to G1 diploid cells that originated from normal tissues (stromal elements, blood vessels, and so forth). The second and the third peaks represent aneuploid tumor cells in G1 and G2 phases. Abscissa means the DNA content; ordinate shows the number of cells measured.
Fig. 6. LDH-isoenzyme electrophoresis after 32 mo of the primary transplantation. The first column exhibits the LDH pattern of the host tissue (liver), indicating one single broad band. The human tissues (heart and liver) display five differently migrating bands (LDH isoenzymes I–V). The xenografted leiomyosarcoma (PZX7/9) is also characterized by five isoenzymes. PZX-2/14: a human pancreatic adenocarcinoma xenograft (data are not shown).
At our institute, the xenografted human tumors are used for preclinical testing of newly synthesized antineoplastic drugs. At present, leiomyosarcomas are basically treated by surgical intervention, but after recurrence or distant metastases, chemotherapy is applied. Moreover, in some reports, hormonal sensitivity has also been stated. There are some concerns about the gonadotropin-releasing hormone (GnRH) treatment of uterine leiomyomas, namely, the possibility of sarcomatous transformation (26). Some leiomyosarcomas may contain estrogen and progesterone receptors, and these tumors are hormone-sensitive (27). Glycocorticoids may also significantly influence the growth of these tumors (3). It is worth mentioning that in a pilot experiment this PZX-7 tumor proved to be responsive to a somatostatin–analog therapy, and histological responsiveness was evidenced by increased apoptotic index, similarly to our ordinary pancreatic ductal carcinoma xenografts (data are not shown). Immunohistochemical expression of p53 in leiomyosarcomas is generally low, ranging from 20 to 43% (28–30). Our PZX-7 line was regularly negative throughout the xenograftings. Regarding bcl2 expression, our knowledge is very premature. Nakanishi et al. (29) reported that various soft tissue sarcomas showed 43% of positive staining for bclInternational Journal of Pancreatology
39 2 protein. However, all five leiomyosarcomas cases were negative. Our tumor exhibited a strong positive expression of this antiapoptotic protein. Soft tissue sarcomas frequently show instability during xenotransplantation. Shift in differentiation may reach as high as 30%, but leiomyosarcomas exhibit a more stable morphologic and flow cytometric features than other soft tissue sarcomas do (1). It is known that mesenchymal multipotential stem cells might undergo divergent differentiation, and this process could profoundly be modified by the microenvironment of the given tumor (7). In Roholl et al.’s study (2), which investigated two xenografted leiomyosarcomas, one line underwent an increased leiomyogenic differentiation as in study of Budach et al. (5). The genetic instability could be attributed to the frequent chromosomal abnormalities (31). This pancreatic leiomyosarcoma line proved to be a stable xenograft. The morphological picture, the degree of differentiation, and the mitotic activity all remained unchanged during 24 mo of consecutive transplantations. No desmoplastic reaction occurred. The relative amount of connective tissue remained constant. Flow cytometric studies revealed a narrow range of the aneuploid pattern indicating the genetic stability. An important question is whether the xenografted tumors retain their human origin. During repeated passagings, the host tissues may overgrow the original tumor cells, or ultimately, the whole neoplasm may be replaced by a mouse tumor. In carcinomas, the histologic picture serves a good orientation, because the epithelial elements represent the human tissues. Just the stromal components are derived from the mouse. In spindle-cell sarcoma xenografts, however, based on morphologic ground alone, such a distinction cannot be made. Another problem is that sometimes malignant host stromal tumors can develop during serial transplantations (32–34). Although the precise mechanism of this phenomenon remains to be elucidated, it seems highly possible that the transplanted human tumor cells trigger or facilitate this process. That is why discrimination of human and host malignancies during consecutive passagings is imperative. For differentiation of the human and mice tumors, several methods are available: antimouse antibodies, chromosome analysis, or detection of some human-type enzymes, like glucose-6-phosphate Volume 26, 1999
40 dehydrogenase or LDH. The LDH isoenzymes are frequently investigated in different xenograft systems (35–39). The human origin of pancreatic cancer xenografts has also been evidenced by this method (9,40). In the present work, we also applied the LDH-isoenzyme electrophoresis, and it was shown that the pancreatic leiomyosarcoma did retain its human character even after 32 mo of primary transplantation. More evidence of the human character was given by transplantation of the tumor into nonimmunosuppressed mice. The immunocompetent animals rapidly destroyed the leiomyosarcoma fragments, and no tumor has developed even after 5 mo. Taken together, a stable, transplantable human pancreatic leiomyosarcoma xenograft designated as PZX-7 has been presented and characterized for the first time in the literature. The tumor has retained its morphological-biological properties and human character after 24 mo of implantation, and it seems to be suitable for preclinical screening of potential antineoplastic drugs.
Acknowledgments The authors are grateful to Ildikó Bakonyi for her expert care of the animals as well as to Pal Szarvashegyi, Ildikó Felletar, and Anna Tamási for their excellent technical assistance. This work was granted by the Hungarian Scientific Research Fund (T 23697/1996., to A Z).
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