Naturally occurring mouse hepatitis virus infection in the nude mouse.

A group of athymic nude mice developed an unusual chronic wasting disease within 1-3 months after their arrival into the laboratory. Affected nude mice had severe, acute-to-chronic, active hepatitis with multinucleated giant hepatocytes and fibrosis. Vascular and central nervous system lesions were frequently present, giant cell peritonitis, ascites, and multinucleated giant cells in the intestinal epithelial villi were less frequently observed. Mouse hepatitis virus was isolated from the livers of three mice with lesions. The virus, when inoculated into nude mice, produced lesions similar to those observed in the natural outbreak.     

Effects of granulocyte-colony-stimulating factor and interleukin-2 on ascites formation and the survival time of nude mice bearing human ovarian cancer cells

 The aim of this study was to elucidate the effect of intraperitoneal (i.p.) instillations of granulocyte-colony-stimulating factor (G-CSF) and/or interleukin-2 (IL-2) on ascites formation and the survival time of nude mice with malignant ascites, produced by i.p. inoculation of human ovarian cancer cells. When the nude mice were treated with medium alone, ascites was observed in all mice 28 days after tumor inoculation. When the mice were treated with cis-diamminedichloroplatinum(II) (cisplatin) alone, G-CSF alone or IL-2 alone, it took 35 days for the ascites to form in all mice. When cisplatin was combined with G-CSF or IL-2, one of ten mice did not form ascites during the observation period. Surprisingly, when G-CSF and IL-2 were simultaneously administered, ascites formation was not observed in any mice. Although i.p. treatment with cisplatin alone significantly prolonged the survival time, compared to medium alone, the lytic activity of spleen cells against HRA cells was significantly suppressed. When G-CSF or IL-2 was combined with cisplatin, the suppression by cisplatin was eliminated and subsequently resulted in a prolongation of the survival time. When G-CSF was combined with IL-2, both the peritoneal and splenic macrophages/monocytes were stimulated and the splenic lytic activity was about double that following treatment with G-CSF alone on IL-2 alone, suggesting that complete inhibition of ascites formation results not only from a significant increase of the peritoneal macrophages but also from enhancement of the lytic activity. Two mice, died from dissemination of tumor in the abdominal cavity, but eight mice survived without tumor for more than 90 days. As confirmed by monitoring body weight and hematocrit, G-CSF and IL-2 seemed to have no adverse effect. From these results, we conclude that a combination therapy with G-CSF and IL-2 might be of clinical use for inhibiting large amounts of ascites, which may inhibit therapeutic effects for ovarian cancer patients. Received: 20 May 1996 / Accepted: 19 September 1996     

Essential Role of T Cells in the Postexposure Prophylaxis of Rabies in Mice

Athymic nude mice injected intramuscularly with a street strain of rabies virus were not protected against rabies by postexposure administration of beta-propiolactone-inactivated rabies vaccine. In contrast, their normal littermates were completely protected from death by the same vaccination regimens. Nude mice did not produce IgG antibody as a result of the vaccine during the test period of 15 days, whereas normal littermates produced IgG antibody from day 5 after vaccination. However, passive immunization with antirabies hyperimmune mouse ascites showed that antibody was completely ineffective in protecting either nude mice or their normal littermates against rabies when given later than 2 days after infection. No significant difference in the induction of circulating interferon by the vaccination was noted in these mice. Passive transfer of immune spleen cells to nude mice immediately after infection resulted in 30 to 37.5% protection of the mice. Passively transferred spleen cells did not produce detectable amounts of neutralizing antibody in the recipient mice except on day 2 after the transfer, when a low level of antibody was detected. These observations demonstrate the essential role of T cells in the postexposure prophylaxis of rabies in mice. The mechanisms of the failure of postexposure vaccination in nude mice are discussed.     

Human pancreatic adenocarcinoma: In vitro and in vivo morphology of a new tumor line established from ascites

Summary A human pancreatic tumor cell line has been established from the ascites of a patient with histopathologically confirmed adenocarcinoma of the head of the pancreas and maintained for more than 12 months in the laboratory. Epitheloid tumor cell colonies, which resulted from primary tissue cultures of the ascitic cell component, were mechanically isolated by needle micromanipulation. Tumorigenicity was proven in athymic nude mice. Morphologically the pancreatic tumor epithelial cells grew to confluency with moderately tight adhesion to the culture plastic surface and with free-floating cells in the medium. Upon re-establishment of the tumoral xenograft in tissue culture, the epithelial cells retained their original morphology. Histologically the tumor grown in nude mice exhibited prototypic characteristics of the primary adenocarcinoma in the patient, producing abundant mucin and displaying a broad spectrum of glandular differentiation, which ranged from well to poorly differentiated adenocarcinomas with occasionally localized lymphocytic infiltrations. Furthermore, the tumor expressed carcinoembryonic antigen and human pancreas cancer associated antigen. This tumor line, designated AsPC-1, has been cultured for at least 10 passages in vitro and 3 in vivo. It represents a new model for human pancreatic cancer. This work was supported in part by Research Grant CA-18410 awarded by the National Cancer Institute through the National Pancreatic Cancer Project.     

Establishment of EGF, EGF-R and FN-R positive human adenocarcinoma cell line (GAC-1)]

A human adenocarcinoma cell line designated as GAC-1, was established from ascites of the 56-year old male patient with rapidly progressive gastric cancer. The doubling time was about 18.5 hours in vitro, and cell cycle analysis using flow cytometry showed marked increase of S phase (46.1%). Immunohistochemical demonstration of GAC-1 cells revealed positive staining of TGF-alpha, EGF, EGF-R, FN-R, laminin and negative staining of fibronectin. Histogram of them indicated aneuploidy with modal number 57 and they formed tumors in nude mice.     

Establishment and characterization of a human scirrhus type gastric cancer cell line, GCIY, producing CA19-9]

A human gastric cancer cell line, designated GCIY, was established from ascites of a patient with scirrhus type gastric cancer. Doubling time of this cell line was 55 hours. The karyotype indicated that these cells were human cells and the chromosome number varied widely, the mode being 57. GCIY cells were of moderate size and showed monolayer arrangement. They had a large nucleus and a clear nucleolus. Papanicolaou staining showed that they had the characteristics of adenomatous epithelia. They could be subcutaneously transplanted into nude mice, and they histologically resembled the original tumor. They were immunohistochemically recognized by anti-CA19-9 antibody and weakly by anti-CA125, CEA and alpha FP antibodies. Cultured medium also contained CA19-9, CA125, CEA and alpha FP.     

Establishment of a human ovarian clear cell carcinoma cell line (RMG-I) and its single cell cloning--with special reference to the stem cell of the tumor

A cell line, designated RMG-I, has been established from ascites of a patient with ovarian clear cell carcinoma. RMG-I cells have grown well for more than 6 years with mirror ball formation. Chromosome analysis revealed aneuploidy with a modal number of 47 and 3 marker chromosomes. The histological characteristics of the transplanted tumor were similar to those observed in the original tumor which was composed of dark cells, clear cells, and hobnail cells. Thus, the RMG-I cell line was identified to be an ovarian clear cell carcinoma. Immunohistochemically, basic fetoprotein (BFP) and ferritin were demonstrated in the original tumor, the cultured cells, and the transplanted tumor. Furthermore, in order to clarify whether these 3 kinds of cells originate from one stem cell or not, monoclonal cell population were transplanted into nude mice, and its histological investigation revealed that 3 types of cells existed in the transplanted tumor, proving that they originate from one stem cell.     

Smac mimetics induce inflammation and necrotic tumour cell death by modulating macrophage activity

Smac mimetics (SMs) comprise a class of small molecules that target members of the inhibitor of apoptosis family of pro-survival proteins, whose expression in cancer cells hinders the action of conventional chemotherapeutics. Herein, we describe the activity of SM83, a newly synthesised dimeric SM, in two cancer ascites models: athymic nude mice injected intraperitoneally with IGROV-1 human ovarian carcinoma cells and immunocompetent BALB/c mice injected with murine Meth A sarcoma cells. SM83 rapidly killed ascitic IGROV-1 and Meth A cells in vivo (prolonging mouse survival), but was ineffective against the same cells in vitro. IGROV-1 cells in nude mice were killed within the ascites by a non-apoptotic, tumour necrosis factor (TNF)-dependent mechanism. SM83 administration triggered a rapid inflammatory event characterised by host secretion of TNF, interleukin-1β and interferon-γ. This inflammatory response was associated with the reversion of the phenotype of tumour-associated macrophages from a pro-tumoural M2- to a pro-inflammatory M1-like state. SM83 treatment was also associated with a massive recruitment of neutrophils that, however, was not essential for the antitumoural activity of this compound. In BALB/c mice bearing Meth A ascites, SM83 treatment was in some cases curative, and these mice became resistant to a second injection of cancer cells, suggesting that they had developed an adaptive immune response. Altogether, these results indicate that, in vivo, SM83 modulates the immune system within the tumour microenvironment and, through its pro-inflammatory action, leads cancer cells to die by necrosis with the release of high-mobility group box-1. In conclusion, our work provides evidence that SMs could be more therapeutically active than expected by stimulating the immune system.     

A new human cholangiocellular carcinoma cell line (HuCC-T1) producing carbohydrate antigen 19/9 in serum-free medium

Summary A human cholangiocellular carcinoma cell line, HuCC-T1, was established in vitro from the malignant cells of ascites of a 56-yr-old patient. Histologic findings of the primary liver tumor revealed a moderately differentiated adenocarcinoma. Tumor cells from the ascites have been cultured with RPMI 1640 medium containing 0.2% lactalbumin hydrolysate and the cultured cells grew as monolayers with a population doubling time of 74 h during exponential growth at Passage 25. They had an epithelial-like morphology and were positive for mucine staining. Ultrastructural studies revealed the presence of microvilli on the cell surface and poorly developed organelles in the cytoplasm. The HuCC-T1 cell was tumorigenic in nude mice. The number of chromosomes in HuCC-T1 ranged from 61 to 80. These human cholangiocellular carcinoma cells in serum-free medium secreted several tumor markers, including carbohydrate antigen 19/9, carbohydrate antigen 125, carcinoembryonic antigen, and tissue polypeptide antigen. The carbohydrate antigen 19/9 secretion level of HuCC-T1 cells cultured in PRMI 1640 medium with 1% fetal bovine serum was sixfold higher than that with 0.2% lactalbumin hydrolysate. These findings suggest that HuCC-T1 will provide useful information to clarify the mechanism of tumor marker secretion and tumor cell growth in the human cholangiocellular carcinoma.     

Establishment and characterization of human pancreatic adenocarcinoma cell line in tissue culture and the nude mouse

We established a human pancreatic carcinoma cell line, designated SPH, from cancerous ascites of a 57-year-old male patient with ductal adenocarcinoma of the pancreas. The cells have been cultured for 32 months with RPMI-1640 medium supplemental with 10% fetal calf serum. The population doubling time of this cell line was about 35 h, and the modal number of chromosomes was 85 at passage 20. The cells produced CA19-9, SPan-1, and DUPAN-2 in the conditioned medium and formed tumors in nude mice, the histology of which was similar to that of the primary tumor. Based on these findings, this cell line is considered to be a very useful model for studying many aspects of primary and metastatic pancreatic cancer cell biology.     

Anti-MUC1 monoclonal antibody (C595) and docetaxel markedly reduce tumor burden and ascites, and prolong survival in an in vivo ovarian cancer model

MUC1 is associated with cellular transformation and tumorigenicity and is considered as an important tumor-associated antigen (TAA) for cancer therapy. We previously reported that anti-MUC1 monoclonal antibody C595 (MAb C595) plus docetaxel (DTX) increased efficacy of DTX alone and caused cultured human epithelial ovarian cancer (EOC) cells to undergo apoptosis. To further study the mechanisms of this combination-mediated apoptosis, we investigated the effectiveness of this combination therapy in vivo in an intraperitoneal (i.p.) EOC mouse model. OVCAR-3 cells were implanted intraperitoneally in female athymic nude mice and allowed to grow tumor and ascites. Mice were then treated with single MAb C595, DTX, combination test (MAb C595 and DTX), combination control (negative MAb IgG(3) and DTX) or vehicle control i.p. for 3 weeks. Treated mice were killed 4 weeks post-treatment. Ascites volume, tumor weight, CA125 levels from ascites and survival of animals were assessed. The expression of MUC1, CD31, Ki-67, TUNEL and apoptotic proteins in tumor xenografts was evaluated by immunohistochemistry. MAb C595 alone inhibited i.p. tumor growth and ascites production in a dose-dependent manner but did not obviously prevent tumor development. However, combination test significantly reduced ascites volume, tumor growth and metastases, CA125 levels in ascites and improved survival of treated mice compared with single agent-treated mice, combination control or vehicle control-treated mice (P<0.05). The data was in a good agreement with that from cultured cells in vitro. The mechanisms behind the observed effects could be through targeting MUC1 antigens, inhibition of tumor angiogenesis, and induction of apoptosis. Our results suggest that this combination approach can effectively reduce tumor burden and ascites, prolong survival of animals through induction of tumor apoptosis and necrosis, and may provide a potential therapy for advanced metastatic EOC.     

Multi-color palette of fluorescent proteins for imaging the tumor microenvironment of orthotopic tumorgraft mouse models of clinical pancreatic cancer specimens

Pancreatic-cancer-patient tumor specimens were initially established subcutaneously in NOD/SCID mice immediately after surgery. The patient tumors were then harvested from NOD/SCID mice and passaged orthotopically in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). The primary patient tumors acquired RFP-expressing stroma. The RFP-expressing stroma included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Further passage to transgenic nude mice ubiquitously expressing green fluorescent protein (GFP) resulted in tumors that acquired GFP stroma in addition to their RFP stroma, including CAFs and TAMs as well as blood vessels. The RFP stroma persisted in the tumors growing in the GFP mice. Further passage to transgenic nude mice ubiquitously expressing cyan fluorescent protein (CFP) resulted in tumors acquiring CFP stroma in addition to persisting RFP and GFP stroma, including RFP- and GFP-expressing CAFs, TAMs and blood vessels. This model can be used to image progression of patient pancreatic tumors and to visually target stroma as well as cancer cells and to individualize patient therapy.     

人卵巢浆液性囊腺癌永生化细胞系BUPH∶OVCA-3的建立及其生物学特性

介绍人卵巢浆液性囊腺癌永生化细胞系的建立 ,研究其生物学特性 .以卵巢浆液性乳头状囊腺癌的腹水细胞为材料 ,进行体外培养 .将永生化基因———SV4 0T抗原基因转染第 2代细胞 ,得到永生化细胞系 .通过光学显微镜、生长曲线测定、染色体分析、双层软琼脂培养、裸鼠接种、免疫组化等 ,研究其生物学特性 ,并与其来源细胞的生物学特性进行比较 .建立了一株人卵巢浆液性囊腺癌永生化细胞系 ,命名为BUPH∶OVCA 3,现已传至 6 0余代 .其生物学特性为 ,细胞生长旺盛 ;具有人体恶性细胞的核型特征 ;细胞恶性度较低 ,不具有集落形成能力及裸鼠接种致瘤性 ;除较未永生化细胞生长速率增快 ,饱和密度增加外 ,仍保留上皮细胞的分化表型 .结果表明 ,BUPH∶OVCA 3为一株恶性度较低的人卵巢浆液性囊腺癌永生化细胞系 ,保留其来源细胞的生物学特性 ,可作为研究恶性度较低的卵巢上皮癌的体外模型     

Establishment and characterization of a human gall bladder carcinoma cell line NOZ

A human gall bladder carcinoma cell line was established from ascites of a patient of peritonitis carcinomatosa. The pathological diagnosis of this patient was adenocarcinoma tubular ++, moderately differentiated. This cell line was composed of polygonal, spindle and round shaped cells. Each cell types were cloned by single cell cloning technique and each cloned cell secreted CEA or Ferritin or none of them. The doubling time of cell number was 48 hours, and plating efficiency was 14-19%. NOZ cell was transplantable to nude mouse. The morphological feature of transplanted tumor was similar to the original one.     

Establishment of a primary human sarcoma model in athymic nude mice

Improvement of soft tissue sarcoma patient outcome requires well-characterized animal models in which to evaluate novel therapeutic options. Xenograft sarcoma models are frequently used, but commonly with established cell lines rather than with primary human sarcoma cells. The objective of the present study was to establish a reproducible xenograft model of primary human soft tissue sarcoma in athymic nude mice. Primary soft tissue sarcoma cells from four resected human sarcomas were isolated, cultured until the third passage and injected subcutaneously into athymic nude mice. The sarcoma xenograft was further analyzed by histological and immunohistochemical staining. In two out of four sarcomas tumor growth could successfully be established leading to solid tumors of up to 540 mm³ volume. Histological and immunohistochemical staining confirmed the mouse xenograft as identical sarcoma compared with the original patient’s tissue. In the present study a reproducible xenograft model of primary human soft tissue sarcoma in athymic nude mice was established. This animal model is of great interest for the study of sarcomogenesis and therapy.     

A human hybrid myeloma for production of human monoclonal antibodies

We produced somatic cell hybrids between human myeloma cells and a lymphoblastoid cell line that is hypoxanthine phosphoribosyl transferase-deficient and ouabain-resistant. These hybrids were phenotypically similar to the human myeloma parental cells and grew as well as the human lymphoblastoid parental cells. After counterselection in 6-thioguanine, mutants that were 6-thioguanine-and ouabain-resistant were obtained, one of which was used as a fusion partner with lymphoblastoid B cells that produce anti-tetanus toxoid (TT) antibodies. These hybrids secreted human anti-TT monoclonal antibodies in much larger amounts than the parental lymphoblastoid cells, and were stable for a period of over 10 mo until the present time. Thus, by hybridizing plasmacytomas with lymphoblastoid cells, we constructed a fusion partner that secretes large amounts of immunoglobulin (Ig), grows at a fast rate, has a high fusion frequency, and supports the production of monoclonal antibodies over long periods of time. Moreover, anti-TT antibody-producing hybrids have been grown as solid tumors in irradiated BALB/c nude mice and then adopted to ascites growth, producing 1 to 8 mg of human immunoglobulin per 1 ml of ascites fluid.     

Isolation and Characterization of Tumor Cells from the Ascites of Ovarian Cancer Patients: Molecular Phenotype of Chemoresistant Ovarian Tumors

Tumor cells in ascites are a major source of disease recurrence in ovarian cancer patients. In an attempt to identify and profile the population of ascites cells obtained from ovarian cancer patients, a novel method was developed to separate adherent (AD) and non-adherent (NAD) cells in culture. Twenty-five patients were recruited to this study; 11 chemonaive (CN) and 14 chemoresistant (CR). AD cells from both CN and CR patients exhibited mesenchymal morphology with an antigen profile of mesenchymal stem cells and fibroblasts. Conversely, NAD cells had an epithelial morphology with enhanced expression of cancer antigen 125 (CA125), epithelial cell adhesion molecule (EpCAM) and cytokeratin 7. NAD cells developed infiltrating tumors and ascites within 12–14 weeks after intraperitoneal (i.p.) injections into nude mice, whereas AD cells remained non-tumorigenic for up to 20 weeks. Subsequent comparison of selective epithelial, mesenchymal and cancer stem cell (CSC) markers between AD and NAD populations of CN and CR patients demonstrated an enhanced trend in mRNA expression of E-cadherin, EpCAM, STAT3 and Oct4 in the NAD population of CR patients. A similar trend of enhanced mRNA expression of CD44, MMP9 and Oct4 was observed in the AD population of CR patients. Hence, using a novel purification method we demonstrate for the first time a distinct separation of ascites cells into epithelial tumorigenic and mesenchymal non-tumorigenic populations. We also demonstrate that cells from the ascites of CR patients are predominantly epithelial and show a trend towards increased mRNA expression of genes associated with CSCs, compared to cells isolated from the ascites of CN patients. As the tumor cells in the ascites of ovarian cancer patients play a dominant role in disease recurrence, a thorough understanding of the biology of the ascites microenvironment from CR and CN patients is essential for effective therapeutic interventions.     

Productive ascites growth of heterohybridomas between Epstein-Barr virus-transformed human B cells and murine P3X63Ag8.653 myeloma cells

Heterohybridomas between Epstein-Barr virus-transformed human B cells and murine myeloma P3X63Ag8.653 cells were found to produce sizable quantities of human monoclonal antibodies in nude mouse ascites. Mice injected intraperitoneally with two of such heterohybridomas yielded several milliters of ascites fluid which contained nearly 100-fold higher anti-platelet activities than those in routine culture supernatants.     

Hollow spheroids in ascites of ovarian clear cell carcinoma: how are they formed and how do they behave?

N. Kato, K. Narutomi, M. Fukase and T. Motoyama Hollow spheroids in ascites of ovarian clear cell carcinoma: how are they formed and how do they behave? Objective: Although the multicellular aggregates (spheroids) in malignant ascites are usually solid throughout, they sometimes have acellular hollow spaces, especially in ascites of ovarian clear cell carcinoma. The purpose of this study is to analyse the origin and behaviour of hollow spheroids. Methods: Archival cytological and histological specimens of 32 ovarian carcinomas, including 12 clear cell carcinomas, were reviewed. HAC‐2, a clear cell carcinoma cell line, was injected into the abdominal cavity of nude mice for direct comparison of ascitic cytology and tumour histology. Spheroids that were collected from nude mice ascites were cultured in vitro to observe their behaviour. Results: Five of six clear cell carcinomas with hollow spheroids showed spherule‐like hyaluronan‐rich stroma in their tumour tissue, whereas those without hollow spheroids did not. After heterotransplantation, both ascites and tumour imprints showed small or large hollow spheroids. Hyaluronan was detected in the former but not in the latter. The abdominal tumours showed compact spherule‐like hyaluronan‐rich stroma, enlarged oedematous stroma or intermediate stroma. In both size and hyaluronan status, small and large hollow spheroids were approximately comparable to spherule‐like hyaluronan‐rich stroma and oedematous stroma, respectively. During culture in vitro, hollow spheroids were maintained as hollow spheroids in suspension, and produced daughter hollow spheroids. Conclusions: The hollow space in the spheroids originates from spherule‐like hyaluronan‐rich stroma, where water trapping by hyaluronan causes enlargement of the space. The matrix within the hollow space serves as a scaffold that regulates cell polarity and matrix production.