Establishment and characterization of five new human renal tumor xenografts.

Ten different human renal cell carcinoma (RCC) primary tumors were xenografted into BALB/c nu/nu mice. Five of the tumors (NU-10, NU-12, NU-20, NU-22, and NU-28) gave rise to serially transplantable tumors that were further characterized. Histology, DNA index, immunohistochemical characteristics, growth rate, and clonogenic potential were followed from primary tumor to the 5th to 15th transplant passage. Only one of the tumors (NU-20) showed remarkable instability for all tested parameters in the first five transplant passages. Histology of the other tumors was essentially the same to the histology of the primary tumors, although differences between human and host-derived vessels were apparent. DNA index values in general showed a trend toward an aneuploid character of the xenografts. Immunohistochemical analyses showed a loss of intensity of staining but a concomitant rise in the fraction of positively staining cells with antibodies against cytokeratins, vimentin, tumor-associated antigens, and human leukocyte antigen (HLA) class I antigens. Human leukocyte antigen class II antigen expression showed a loss of intensity as well as a decrease in the fraction of positive cells. Tumor doubling time was lowest in transplant passage number 0, and stable growth was noticed in transplant passages 1 through 4. Clonogenic potential of four of the lines was higher for the xenografts than for the primary tumors. The authors conclude that, on xenografting, histologic characteristics of the primary tumor are essentially conserved. Progression in the first transplant passages, however, results in tumors with a more aggressive character.     

Xenotransplantation of testicular tissue into nude mice can be used for detecting leukemic cell contamination

BACKGROUND: Xeno-grafting of testicular tissue may allow viable gamete maturation. This would be beneficial for prepubertal cancer patients in that it may allow restoration of fertility without the risk of a cancer relapse. However it is unknown whether cancer cells in the testicular graft can transmit the malignancy into the host animal and also if gametes can be retrieved from testicular grafts that are contaminated with malignant cells. METHODS: Rat T-cell leukemia was employed as the source of leukemic lymphoblasts and testicular tissue. This was injected i.p. (lymphoblasts) or grafted s.c. (fresh or cryopreserved testicular tissue) into the back skin of intact nude mice. To simulate clinical autografting, testicular tissue was also transplanted into healthy piebald variegated (PVG) rats. RESULTS: 50-70% of the mice, receiving 200 or 6000 leukemic lymphoblasts, developed terminal leukemia. All mice, grafted with either fresh or cryopreserved testicular tissue from leukemic donor, developed generalized leukemia and/or local tumors. All syngenic PVG rats, treated in the same manner, died of generalized leukemia. In all of the retrieved leukemic grafts, rat spermatogenesis was destroyed and only leukemic infiltration was detected. CONCLUSIONS: Grafting testicular tissue contaminated with leukemic cells led to tumor growth at the injection site without potential to differentiate germline stem cells into gametes. Xenografting could provide a novel functional strategy for simultaneous detection of malignant cell contamination and spermatogonial potential in testicular xenografts collected for fertility preservation.     

Antigenicity of cryopreserved arterial allografts: comparison with fresh and glutaraldehyde treated grafts

The use of cryopreserved aortic allografts in cardiovascular surgery is widespread and has resulted in excellent outcomes. However, it is controversial whether cryopreservation suppresses the antigenicity of tissue. We designed experimental models to study whether the cryopreservation process alters antigenicity in comparison with that found in fresh and glutaraldehyde treated tissues. Fresh, cryopreserved, and glutaraldehyde treated thoracic aorta from Brown Norway rats were subcutaneously implanted into Lewis rats. Inflammatory cells infiltrating around the grafts were measured on days 7, 14, 28, and 56 after implantation. The glutaraldehyde treated grafts showed significantly less infiltration than the fresh or cryopreserved grafts (p < 0.005). No significant difference was detected between the fresh and cryopreserved grafts. Another study examined the effect of modifications of the aortic allograft on subsequent allogeneic skin graft antigenicity. Subcutaneous implantation of fresh, cryopreserved, and glutaraldehyde treated aortic grafts from Brown Norway into Lewis rats resulted in subsequent skin graft rejection at 4.4+/-0.7, 5.1+/-0.8, and 6.6+/-2.1 days, respectively. There was no significant difference between the fresh and cryopreserved groups; whereas skin grafts in the glutaraldehyde group survived longer than those in the cryopreserved group. These results indicate that cryopreservation had no significant influence on antigenic suppression of arterial allografts.     

Oocyte maturation,follicle rupture and luteinization in human cryopreserved ovarian tissue following xenografting

BACKGROUND: Previous studies have demonstrated development of antral follicles in cryopreserved human ovarian tissue after autografting and xenografting, thus indicating successful preservation of follicular function. The study aim was to assess whether these follicles could also undergo periovulatory changes in response to hCG. METHODS: Ovarian tissue from three patients were dehydrated in propanediol (PROH)/sucrose and cryopreserved using the slow cooling/rapid thaw procedure. Thawed tissue was placed under the kidney capsule in immunodeficient mice. Following growth (>20 weeks) in the presence of gonadotrophin, hCG was administered and ovarian tissue examined histologically. RESULTS: Thirty-two antral follicles (diameter range 0.6 to 5 mm) were examined. Histological evidence of a response to hCG was evident in all follicles. Disruption of the concentric layers of mural granulosa and theca cells was apparent in all antral cavities. In 17 (53%) follicles the exterior follicular wall had reduced to a few cells thick, and in eight (25%) the wall had ruptured. Mucified oocyte-cumulus cell complexes were present in 32 follicles, 17 of which had begun to detach from the pedicle. Resumption of meiosis had occurred in over half the oocytes (five metaphase II and seven metaphase I oocytes, eight germinal vesicle breakdown). Two corpora lutea were also detected. CONCLUSIONS: Follicles cryopreserved within human ovarian tissue using the PROH procedure, can develop to the antral stage and undergo periovulatory changes following xenografting and exposure to a luteinizing stimulus.     

Spermatogonial survival after cryopreservation and short-term orthotopic immature human cryptorchid testicular tissue grafting to immunodeficient mice

BACKGROUND: Fertility preservation has become an urgent clinical requisite for prepubertal male cancer patients undergoing gonadotoxic treatment. As these patients do not yet produce spermatozoa for freezing, only immature tissue is available for storage. We studied the survival and proliferative activity of spermatogonia and Sertoli cells after cryopreservation of cryptorchid testicular tissue pieces followed by xenografting for 21 days. METHODS AND RESULTS: Single pieces of tissue from cryptorchid testes (2-9 mm(3)) of young boys (2-12 years) were cryopreserved, thawed and transplanted into the scrotum of mice. Quantitative morphometric and immunohistochemical techniques were used to evaluate the integrity of the tissue, as well as the survival and proliferative capacity of spermatogonia and Sertoli cells before and after freezing/thawing/grafting. Three weeks after grafting, cryopreserved tissue was removed and analysed. Most of the tubules (88.3%) were intact and there was no fibrosis or sclerosis, 14.5% of the initial spermatogonial population remained, as identified by the MAGE A4 antibody, and 32% of these cells showed proliferative activity evidenced by Ki67, compared to 17.8% before cryopreservation and grafting. The number of Sertoli cells was unchanged and 5.1% were Ki67-positive, compared to none at all before freezing and grafting. CONCLUSIONS: Through our orthotopic xenografting model, we have demonstrated the survival and proliferative activity of spermatogonia and Sertoli cells in cryopreserved immature human cryptorchid tissue. Testicular tissue banking may thus prove to be a promising technique for the preservation of fertility in prepubertal boys undergoing oncological treatments. As the stem cell niche is maintained, the cryopreserved tissue can potentially be used for future autotransplantation. In addition, whole tissue freezing does not exclude alternative clinical uses, including isolated cell transplantation after dissociation, selection and enrichment. However, as this work was done on cryptorchid tissue, studies on normal immature testicular tissue, involving longer grafting periods, are needed to demonstrate a differentiation capacity before clinical implementation. Ethical and safety issues should also be addressed.     

Intraperitoneal administration of an I125 labelled monoclonal antibody for localization of human colorectal cancer in the nude mouse

The monoclonal antibody (MoAb) HMGF-1 was evaluated in the radioimmunodetection of human colonic cancer transplanted intraperitoneally (i.p.) into athymic nude mice. This antibody reacts with a component of the human milkfat globule, as well as a wide range of epithelial cells and adenocarcinomas of various origins. Purified MoAb was iodinated with 125I and administered i.p. into nu/nu mice bearing (i.p.) xenografts of human colonic adenocarcinoma (X56). Differential tissue counts of radioactivity demonstrated preferential localization of the antibody in i.p. and subcutaneous (s.c.) tumor tissue as compared to normal tissues. Maximum per cent dose per g of tumor (25.17 +/- 1.37), maximum tumor: blood ratio (4.45 +/- 0.14) and maximum tumor: tissue ratios (34.2 +/- 0.12) were obtained at the optimal labelling time of 5 days after antibody injection. Selective localization to tumor was confirmed with a control anti-hepatitis virus MoAb of the same isotype and by localization studies in non-tumor bearing athymic mice. Half lives of the persistence of the iodine 125 in the tumor bearing and non tumor bearing mice were 5 and 7 days, respectively, indicating approximate antibody half lives. Whole body scans showed distinct tumor images without the use of subtraction techniques. This pilot experimental study demonstrates the feasibility of i.p. administration of labelled antitumor MoAb in the imaging of i.p. tumors in an athymic mouse system. Whether or not these observations are applicable to the human situation remains to be carefully established.     

Diagnostic assessment of the developmental potential of human cryopreserved ovarian tissue from multiple patients using xenografting

BACKGROUND: Although ovarian tissue cryopreservation for women at risk of losing ovarian function is offered by many clinics, there is a lack of evidence relating to the developmental potential of the stored tissue and, therefore, its clinical potential. This study was designed to use xenografting of cryopreserved tissue from multiple patients to assess the reproducibility of preservating developmental potential, the variation in developing follicle profile and the relationship between pre-freeze histology and post-thaw development. METHODS: Using previously published methods, cryopreserved ovarian cortex from nine patients was thawed and grafted under the kidney capsules of immunodeficient mice. Development of follicles was assessed after 26 weeks and compared to histology prior to freezing. RESULTS: Multiple growing follicles including antral stages were observed in multiple grafts of tissue from all patients. Metaphase II oocytes (n=9) were observed in follicles in grafts from five patients. There was no relationship between pre-freeze histology and developing follicle profile in xenografts. CONCLUSIONS: The propanediol freezing method used in this study is capable of reproducibly preserving the developmental potential of human ovarian follicles. The developing follicle profile after cryopreservation cannot be accurately predicted from pre-freeze histology. Xenografting provides a powerful tool for assessing the potential of human cryopreserved ovarian tissue.     

Control of differentiation in a rectal adenocarcinoma cell line: the role of diffusable and cell-associated factors

The rectal adenocarcinoma cell line, HRA-19a1.1, was cultured in a three-dimensional type I collagen gel and then xenografted in nu/nu mice to determine whether the in vivo environment could induce further morphological differentiation of the in vitro collagen gel cultures. Three phenotypic changes were observed. Type I collagen induces glandular differentiation in vitro in which well polarized cells are organized around a central lumen, as has been previously reported. Seven days after xenografting this structure in nu/nu mice, the glandular structures appeared to have 'ballooned' forming cyst-like structures lined by a monolayer of flattened cells. There were no stromal cells associated with the graft at this stage, but with time stromal cells invaded the collagen. At points where these cells were closely associated with the HRA-19 cells there was a marked phenotypic change, with the flattened lining cells seen at day 7 becoming columnar. By 21 days the stromal cells had replaced the collagen and the histology of the graft now resembled that of an adenocarcinoma. Placing this cell-collagen culture in a Millipore chamber prior to grafting resulted in cyst-like structures only. Here we provide conclusive evidence that heterologous connective tissue cells can induce differentiation of a rectal adenocarcinoma cell line by a non- or poorly diffusible factor(s). Furthermore, we show that this cell-collagen xenograft method has certain advantages over conventional xenograft methods: notably, a consistent 100 per cent take rate; considerably fewer cells are required to form a tumour; and the time taken to form a tumour is dramatically reduced.     

Testicular sperm retrieval and cryopreservation prior to initiating ovarian stimulation as the first line approach in patients with non-obstructive azoospermia.

The potency for fertilization and successful implantation was compared between fresh and cryopreserved testicular spermatozoa obtained from the same patient with non-obstructive azoospermia. Spermatozoa cryopreserved at the outset were also evaluated. Non-obstructive azoospermic men (n = 55) underwent testicular sperm extraction (TESE); mature spermatozoa were found in 33 (60%) of them. Of 57 intracytoplasmic sperm injection (ICSI) cycles in 25 patients, 15 used fresh spermatozoa (14 patients, group 1), 24 used the excess spermatozoa cryopreserved after 'fresh' ICSI (11 couples who did not conceive in the 'fresh' cycle, group 2) and 18 cycles used cryopreserved spermatozoa at the outset (11 other patients, group 3). Fertilization, cleavage, embryo quality, implantation and take home baby rates were not significantly different in groups 1 and 2, and 6/14 couples ultimately had healthy babies (42.8% cumulative take home baby rate per TESE). In group 3, neither the fertilization rate, embryo development, pregnancy nor implantation rates per embryo transfer were significantly different from groups 1 and 2. The cumulative delivery and ongoing pregnancy rate in this group was 36. 4%. Cryopreservation did not impair the availability of motile spermatozoa for ICSI. When immotile spermatozoa were injected, however, fertilization rate decreased dramatically. Since criteria for predicting the presence of spermatozoa in the testicular tissue of patients with non-obstructive azoospermia are inadequate, it is suggested that TESE be performed prior to initiating ovarian stimulation.     

Patterns of specific genomic alterations associated with poor prognosis in high-grade renal cell carcinomas

A series of 13 sporadic renal cell carcinomas was analyzed for the specific chromosome rearrangements after serial xenografting into immunodeficient mice. Seven tumors displayed genetic traits of the conventional subtype and 5 showed genetic features of the papillary subtype. In all the xenografted conventional tumors, we observed loss of 3p, as well as loss of the 9p21 region and of the long arm of chromosome 14, both considered as markers of a poor prognosis. In the xenografted papillary tumors, a duplication of chromosome arm 8q was observed concomitant with the duplication of the 7q31 region. The association of the 7q31 and 8q22qter duplicated regions was also observed for one conventional tumor. The latency of tumor take was found to be reduced and the median time to passage statistically shorter for all tumors which presented the associated duplication of the 7q31 and 8q22qter regions. The proto-oncogene NOV (nephroblastoma overexpressed gene) maps to 8q24.1 and is overexpressed in some Wilms tumors. It could be an interesting candidate gene, since its level of expression and release in the culture medium was found to be increased in all of the fast growing tumors analyzed.     

Pituitary Folliculo-Stellate-Like Cells Stimulate Somatotroic Pituitary Tumor Growth in Nude Mice

A new cell line (TtT/GF) established from a murine pituitary thyrotropic tumor having characteristics similar to those of pituitary folliculo-stellate cell (FS cell) was implanted into nude mice together with cells from a rat pituitary somatotrophic tumor cell line (MtT/S) to determine whether the former enhances pituitary tumor growth. For as long as 2-3 mo after implantation, MtT/S cells implanted either alone or together with fibroblasts formed either no tumors or only very small tumors in the nude mice. In contrast all of the nude mice that had received MtT/S cells implanted together with TtT/GF cells developed large tumors. Furthermore, the mice bearing the MtT/S and TtT/GF implants showed a significantly higher body weight and serum growth hormone level than those bearing only MtT/S cells or a combination of MtT/S cells and fibroblasts. The TtT/GF cell line itself had no tumorigenicity during the experimental period. Therefore, the TtT/GF cell line as a model of FS cells enhanced pituitary endocrine cell tumor formation. Additionally, immunocytochemistry showed that TtT/GF cells positive for glial fibrillary acidic protein (GFAP) or S-100 protein were present in the parenchymatous tissue elements or connective tissue surrounding the tumor nests. In the parenchymatous tissue, the TtT/GF cells exhibited a stellate appearance and surrounded neighboring tumor cells with their long cell processes. These results suggest that TtT/GF cells can serve as a model for pituitary FS cells, and are capable of stimulating pituitary tumor growth either by modifying the microenvironment or producing growth factors.     

Flow-cytometric DNA analysis of hematopoietic and lymphoid proliferations: a comparison of fresh, formalin-fixed and B5-fixed tissues

Flow-cytometric DNA analysis of human tumors using paraffin-embedded tissue samples is becoming an increasingly popular method of determining ploidy and proliferative rate. Particularly with hematopoietic/lymphoid proliferations, little is known about how these data compare with data from fresh suspension studies, or how B5-fixed tissues compare with those fixed in formalin. For these reasons, flow-cytometric DNA ploidy and cell cycle analysis was performed on 16 hyperplastic tonsils and 28 lymphoid/hematopoietic neoplasms using fresh (or fresh-frozen) cell suspensions (FR), B5-fixed paraffin-embedded (B5), and formalin-fixed paraffin-embedded (FO) tissue. Ploidy analysis showed all tonsil specimens to be diploid regardless of preparative method; however, for the neoplastic cases the FO and B5 preparations agreed with the FR preparations in 50% and 61% of the cases, respectively. Complete agreement between all three tissue preparation methods was present in only 36% of the neoplastic cases. Percent S or S + G2M phase fractions showed relatively poor correlation between the three preparative methods, with the best correlations found between the FR and B5 samples (S phase, r = 0.44; S + G2M, r = 0.43). In conclusion, while potentially useful data can be obtained from flow-cytometric DNA analysis of fixed, paraffin-embedded lymphoid/hematopoietic tissues, the specific limitations of such analyses must be recognized. Ploidy and proliferative phase data from fixed tissue preparations are not equivalent to information obtained from fresh suspension studies. B5-fixed tissue provides an acceptable alternative to formalin-fixed tissue for such analyses.     

Experimental Sertoli cell tumors in the mouse and their progression into a mixed germ cell-sex cord proliferation.

Males of transgenic families where the large T protein of polyoma virus is expressed in the seminiferous epithelium of the testis (Sertoli and germ cells) develop bilateral testicular tumors when they become old (15 to 18 months). The histological features of these tumors revealed a neoplastic proliferation of Sertoli cell origin. Occasional isolated germ cells arrested at premeiotic stages were seen in the tumor. They did not participate in tumoral proliferation and their malignant character could not at first be established. Tumor cells injected in athymic (nu/nu) mice generated secondary tumors. In this case, a proliferative component of non-Sertoli origin was clearly evident. Its ultrastructural characteristics and the expression of genes that are transcribed in vivo in male germ cells (c-kit, LDH-X, and Hox a-4) suggest the progression of an initial, apparently pure Sertoli cell tumor into a mixed proliferation.     

Development of antral follicles in human cryopreserved ovarian tissue following xenografting

This study reports the first gross morphological and histological evidence of antral follicle development in human ovarian tissue following cryopreservation. Human ovarian tissue was cryopreserved using propanediol and sucrose and grafted under the renal capsule of bilaterally oophorectomized severe combined immunodeficient (SCID) mice. Follicles at all stages of development were observed in the grafted tissue whereas, prior to grafting, only primary and primordial follicles were present. Antral follicles were rarely observed on grafts examined <20 weeks after grafting either non-frozen tissue (fresh, 1/7) or cryopreserved tissue (0/11). In contrast, development of at least one antral follicle was evident on the majority of sites > or = 20 weeks after grafting (fresh 7/9, cryopreserved 18/24). Antral follicle diameters ranged from 0.1 to 5.0 mm. Histological examination of these antral follicles indicated normal follicular morphology, i.e. antral cavities encapsulated by concentric layers of theca and granulosa cells. Pedicles containing germinal vesicle oocytes were observed protruding from the granulosa cell layers. The development and morphology of the cryopreserved and fresh tissue following grafting was similar.     

Atypical fibroxanthoma developing on a pacemaker pocket mimicking a pyogenic granuloma

Malignant tumors at the site of implantation of a pacemaker generator, although rare, have been reported in the literature. We present a case of an 89-year-old man with atypical fibroxanthoma in a pacemaker pocket. The device had been implanted for more than 4 years. An exophytic tumor had developed in this place and was clinically interpreted as a pyogenic granuloma. An excisional biopsy revealed the nature of the tumor. To our knowledge, the association of atypical fibroxanthoma arising from a pacemaker pocket has not been previously reported. A review of the literature has revealed four malignant soft tissue tumors previously reported at the pacemaker site. Routine examination in all patients with implanted pacemaker generators should be practiced at follow-up visits. This would allow an early diagnosis of a malignant associated neoplasm. Pathologists should become familiar with this type of devices and their potential neoplastic complications.     

β-干扰素抑制裸鼠荷人肝癌切除术后复发和生长的实验研究

目的:研究β-干扰素(INF-β)对肝癌根治性切除后复发和生长的抑制作用。 方法:将人肝癌组织植入25只BALB/c裸鼠肝脏，建立人肝癌原位移植瘤模型。第10 d完整切除荷瘤后，随机分为3组：A组对照组（生理盐水皮下注射）；B组小剂量实验组（INF-β 3×105U/d皮下注射）;C组大剂量实验组（INF-β 6×105U/d皮下注射）。连续注射35 d后处死裸鼠，观察肿瘤复发情况，测量肿瘤大小和体积；免疫组织化学法检测肿瘤增殖核抗原(Ki-67)、诱导型一氧化氮合酶(iNOS)和半胱氨酸天冬氨酸蛋白酶-3(caspase-3)表达。 结果:A组肿瘤复发率为100%，B组为87.5%，C组为77.8%，3组比较无显著差异（P>0.05）。B组和C组抑瘤率分别为73.6%和94.3%，与A组比较差异显著（P<0.05）。B组和C组肿瘤组织iNOS、Ki-67的表达明显低于A组，而caspase3的表达明显高于A组（P<0.05）。 结论:β-干扰素可有效抑制肝癌切除术后复发肿瘤的生长。大剂量β-干扰素较小剂量对肝癌的生长抑制作用更强，其机制可能与肿瘤组织内iNOS、Ki-67和caspase3的表达有关。     

A method to isolate DNA from small archival tissue samples for p53 gene analysis

The tumor suppressor gene p53 is involved in predisposition to a variety of human cancers, including those from Li–Fraumeni cancer family syndrome patients. Studies of inheritance of p53 germline mutations require confirmation of the mutation in the tumors from family members. These studies as well as other retrospective studies of tumor specific mutations, are often hampered by a lack of available fresh or frozen tumor tissue samples for DNA extraction to confirm the suspected p53 mutation. Here we describe a simple technique for DNA isolation that permits mutational analysis of p53 from minimal amounts of paraffin-embedded archival tissue samples. © 1993 Wiley-Liss, Inc.     

Effects of Trypanosoma brucei gambiense infections in Microtus montanus on susceptibility to Ehrlich's tumors.

Trypanosoma brucei gambiense infections in the field vole Microtus montanus increased susceptibility to Ehrlich's tumor growth. Whereas uninfected voles were totally resistant to intraperitoneal Ehrlich's ascites tumor cell challenge, over 78% of the animals infected with the trypanosomes developed tumors after challenge. Likewise, when Ehrlich's ascites cells were injected subcutaneously to induce solid tumor formation, only 7% of uninfected controls developed tumors, whereas over 82% of trypanosome-infected animals exhibited malignancies after Ehrlich's cell challenge. Finally, when solid tumors grown in albino CD-1 mice were implanted subcutaneously into uninfected voles, the tumor mass rapidly diminished in size and could not be found when animals were examined 2 weeks postimplant. However, in trypanosome-infected voles, implanted tumors exhibited pronounced expansion, and viable, solid tumors were recovered from over 70% of the challenged voles at 2 weeks postimplant. The implications of trypanosome-induced immunosuppression, especially toward susceptibility to neoplastic growth, are discussed.     

Polyoma-induced thymic epithelial tumors: analysis by 2D gel electrophoresis of tumors upon labeling the entire tumor bearing host.

Injection of neonatal C3H/Bi mice with polyoma virus (PTA-5) results after 4 to 20 weeks in the development of thymic epithelial tumors in 2/3 of the mice. These tumors appear to be the result of a neoplastic proliferation or transformation of thymic epithelial cells. The tumors contain immature lymphoid infiltrates characteristic of normal tissue and express the same function as normal tissue by providing the microenvironment for T cell maturation. To establish more comprehensively the relation of neoplastic to normal thymic epithelial cells, tumor-bearing mice and their normal counterparts were labeled in vivo by injection of S35-L-methionine. The stroma and lymphoid cells were separated and analyzed by two dimensional gel electrophoresis followed by radiofluorography. It was found that the epithelial portion of the tumor differed in several structural polypeptide components from normal tissue while the polypeptide composition of the thymocyte population remained unaltered. It was concluded that the neoplasia is confined to the epithelium compartment and the function of the neoplastic organ is maintained.