Growth and metastatic behavior of human tumor cells implanted into nude and beige nude mice

The growth and metastatic behavior of several human tumor lines grown in adult nude mice, nude mice pretreated with antiserum against asialo GM1 glycoprotein, and beige nude mice were studied. The cell lines were all injected s.c. and i.v. A human colon carcinoma line was also injected into the spleen, and two human renal carcinoma lines were injected into renal subcapsule. All the tumor lines grew as fast or faster in adult nude mice compared with beige nude mice. There were no discernible differences in the production of experimental lung metastasis among the three groups of mice, but human colorectal carcinoma cells and human renal carcinoma cells produced more metastases in nude mice than in beige nude mice after intrasplenic or renal subcapsule injection, respectively. In vitro cytotoxicity assays confirmed that adult nude mice had high levels of natural killer (NK) cell activity whereas nude mice pretreated with anti-asialo GM1 serum and beige nude mice did not. The in vitro NK cell activity of nude mice was demonstrable against mouse lymphoma cells but not against human leukemia cells which were sensitive to lysis by human NK cells. These results suggest that the implantation of human tumor cells into beige nude mice, which are deficient in NK cell activity does not provide an advantageous model for the study of growth and metastasis of human neoplasms.Abbreviations NK natural killer - HCC human colon carcinoma - HRCC human renal cell carcinoma - anti-asGM1 anti-asialo GM1 - i.s. intrasplenic - RSC renal subcapsule - HBSS Ca²⁺- and Mg²⁺-free Hanks' balanced salt solution     

Selection of more aggressive variants of the GI101A human breast cancer cell line: A model for analyzing the metastatic phenotype of breast cancer

In vivo models utilizing orthotopic injection of tumor cells into nude mice have proven valuable for the study of metastasis. However, breast cancers are among the more difficult of human tumors to grow in immunodeficient mice, with a relatively low tumor take. Fewer still develop spontaneous metastases. The injection of GI101A breast cancer cells into the mammary fatpad (mfp) produced lung metastases in 25% of tumor-bearing mice. Selecting cells from the lung metastases and recycling in vivo resulted in the isolation of a series of variant cell lines. These cell lines were tested for tumorigenicity and metastasis in nude mice following mfp injection compared with the original cell line, and in vitro expression of factors associated with the metastatic phenotype measured. The in vivo selected cell lines were more aggressive, with higher tumor take, faster local growth rate and increased incidence (≥85%) and extent of lung metastasis. However, the metastasis-selected variants showed no increases in expression of the growth factor receptors EGFR or HER-2, and the pro-angiogenic factors VEGF-A and IL-8. Immunohistochemistry of mfp tumors revealed no differences in microvessel density (counting CD-31 positive structures) and cell proliferation (PCNA-positive cells) comparing the GI101A line with selected variants. No TUNEL-positive cells were detected in the tumors of the metastasis-derived variant, with a small number of cells undergoing apoptosis detected in sections of GI101A tumors. In vitro, the metastasis-derived variants were found to have a more robust expression of phosphorylated PKB/Akt, with or without EGF or serum stimulation, suggesting an association between Akt activation and metastatic ability. This new series of isogenic cell lines may be valuable for identifying molecular mechanisms involved in the metastatic progression of breast cancer. This revised version was published online in July 2006 with corrections to the Cover Date.     

α-Difluoromethylornithine inhibits liver metastasis produced by intrasplenic injection of human tumor cells into nude mice

The purpose of these studies was to examine metastatic potentials of a human colon tumor xenograft (T6) and three different human tumor cell lines (LS174T, HT29 and A549) using the intrasplenic-nude mouse model system (ISMS model system). A further objective was to study the activity of-difluoromethylornithine (DFMO) against primary and metastatic growth of the xenograft and the three cell lines. DFMO is an irreversible inhibitor of ornithine decarboxylase, a rate-limiting step in polyamine biosynthesis. Tumor burdens in the liver of nude mice were observed 6 weeks after the intrasplenic injection with LS174T and 12–14 weeks after intrasplenic injections with T6, HT29 and A549. Most of the mice developed primary tumor growth in the spleens. DFMO showed significant activity against liver metastases but had little or no activity against primary tumor growth in the spleens of the ISMS model and against s.c. growth of the xenograft. The studies demonstrated that the ISMS model system is an excellent system for studying metastatic behavior of human tumors and for studying the antimetastatic activity of experimental drugs.     

Quantitative and qualitative differences in growth, invasion and lung colonization of an anaplastic and a papillary human thyroid cancer cell linein vitro andin vivo

Invasion and metastasis remain major reasons for failure of anti-cancer therapy. Cell lines derived from human carcinomas are frequently used to investigate the molecular mechanisms that underlie invasion and metastasis. Unfortunately many of these cell lines do not retain the malignant characteristics of their parental tumors. We therefore conducted a series of experimentsin vivo andin vitro to identify which aspects of malignancy of a papillary (NPA'87) and an anaplastic (DR090-1) thyroid carcinoma were consistent with the pathology of the parental tumor types. We evaluated tumor growth, invasion and metastasis of DRO90-1 and NPA'87in vivo following inoculation of the tumor cells under the dermis, under the renal capsule and into the lateral tail vein of nude mice. This evaluationin vivo showed that the anaplastic carcinoma had a faster growth rate compared with the papillary carcinoma. Furthermore, the papillary carcinoma cells could destroy and infiltrate surrounding tissue but were not capable of extravasation and colonization of lung tissue. The anaplastic cells formed lung nodules following injection into the tail vein of nude mice. This lung colonizing capability of DR090-1 correlated with their capacity to secrete an active 62 kDa gelatinase and to migrate through reconstituted basement membranein vitro.     

Clonal dominance detected in metastases but not primary tumors of retrovirally marked human breast carcinoma injected into nude mice

Human breast cancer cell lines which grow in athymic (nude) mice provide a model of tumor cell growth and metastasis. Marking transplanted tumor cell populations with retroviral vectors provides a means of studying the dynamics of tumor cell growthin vivo. We evaluated three human breast cancer cell lines, MDA-MB-435, MDA-MB-231 and MCF-7, and found the cells were highly susceptible to retroviral gene transfer after a single 2-h exposure (90.9%, 62.7% and 72.3%, respectively). MDA-MB-435 cells (5×10⁵) marked with a retroviral vector containing the-galactosidase gene (approximately 10⁴ uniquely marked clones) were injected into the mammary fat pad of athymic mice to study clonal dominance. Primary tumors resected 10 weeks after injection expressed-galactosidase, demonstrating persistent vector expressionin vivo. Southern blot analysis did not reveal clonal dominance in the primary tumors of the five mice studied. In contrast, pulmonary metastases in each animal were monoclonal or biclonal. These results demonstrate clonal dominance in pulmonary metastases but not primary tumors of retrovirally marked MDA-MB-435 cells. Our findings suggest that this model may also be used to introduce retroviral vectors expressing oncogenes, and anti-sense oncogenes, to determine their effect on tumor cell proliferation and metastasisin vivo.     

Dissemination of human solid tumor cells in the bloodstream of immunodeficient mice

Seven human solid tumor cell lines transplanted into hereditarily immunodeficient mice (nude and beige/nude) were typed for tumor-associated surface antigens and glycoconjugates using fluorescent conjugates of 7 monoclonal antibodies, 5 lectins, and 2 ligands. With this set of 14 selected tumor markers, peripheral blood samples from mice bearing the respective tumors were examined by flow cytofluorimetry for the presence of tumor cells disseminated in their circulation. Tumor cells were detected in the blood of mice carrying a uterine tumor, indicating that the metastatic process can be followed intravitally in immunodeficient animals bearing human solid tumors. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 12, pp. 615–618, December, 1995     

高转移人卵巢癌裸鼠皮下移植瘤模型的建立及其生物学特性

用人卵巢癌细胞系HO－8910移植探鼠，建立高转移人卵巢癌探鼠皮下移植瘤模型命名为（NSMO），已传代23次，仍保持高转移的特性。共接种BALB/c棵小鼠57只，皮下成瘤率为100％，平均带瘤存活时间为159．9天。在解剖47只裸鼠中有转移瘤鼠42只（89．4％）。最早出现转移的时间为56天。移植瘤组织学和超微结构保持原来人卵巢分化差的浆液性乳头状囊腺癌的形态特征和分泌功能。流式细胞术分析显示DNA指数为1．4，染色体分析显示众数为54（属于超二倍体），显示人类癌症的特征。移植瘤相关标记物检测显示大多数癌细胞雌激素受体和孕激素受体阳性。该模型为转移机制的研究和寻找抗转移药物提供了理想的工具。     

Inhibition of establishment of primary and micrometastatic tumors by a urokinase plasminogen activator receptor antagonist

Tumor establishment and metastasis are dependent on extracellular matrix proteolysis, tumor cell migration, and angiogenesis. Urokinase plasminogen activator (uPA) and its receptor are essential mediators of these processes. The purpose of this study was to investigate the effect of a recombinant human uPAR antagonist on growth, establishment, and metastasis of tumors derived from human cancer cell lines. A noncatalytic recombinant protein, consisting of amino acids 1-137 of human uPA and the CH2 and CH3 regions of mouse IgG₁ (uPA-IgG), was expressed, purified, and shown to bind specifically to human uPAR and to saturate the surface of human tumor cells which express uPAR. Daily i.p. administration of uPA-IgG to nude mice extended latencies of unstaged tumors derived from Lox melanoma and SW48 colon carcinoma cells by 7.7 and 5.5 days, respectively. uPA-IgG treatment did not affect the growth of Lox or KB tumors staged to 200 mg before antagonist treatment commenced. The effect of uPA-IgG on the establishment of micrometastases was assessed in SCID mice. KB head/neck tumor cells were injected in the tail vein and allowed to seed for 48 h before initiation of daily i.p. injections of uPA-IgG for 24 days. The number of lung colonies ranged between 5 and 30% of vehicle-treated mice in two separate experiments. Furthermore, a single 800 g dose of uPA-IgG administered 1 h prior to tail vein injection of KB cells reduced lung colony formation to just 3.5% of vehicle-treated SCID mice. These data demonstrate that antagonism of uPAR arrested metastasis and inhibited the establishment of primary tumors and micrometastases. Thus, small molecule uPAR antagonists may serve as useful adjuvant agents in combination with existing cancer chemotherapy. © Rapid Science 1998     

Differential experimental micrometastasis to lung, liver, and bone with lacZ-tagged CWR22R prostate carcinoma cells

LacZ-tagged human prostate carcinoma CWR22Rv1 cells metastasize spontaneously to lung, liver, and bone from subcutaneous primary tumors in athymic nude mice; these organs are ‘natural’ targets of metastasis for the human disease. To evaluate the mechanism(s) of metastasis to these organs, an experimental metastasis model was used by taking advantage of the ultrasensitive detection of lacZ. Within 1 h after tail vein injection, micrometastases were forming in lung, liver, bone, kidney, and brain with very different quantitative levels. The kinetics of loss of unstable micrometastases and retention of stable ones were also very different in these organs. After injecting suspensions of single cells, both whole-organ and serial-section staining for lacZ revealed considerable heterogeneity in cell number of individual lung micrometastases while micrometastases in liver contained only 1 or 2 cells. The size of individual bone micrometastases also suggested only 1 or 2 cells. Tumor cells could also be detected in the small blood vessels of the lung within minutes after injection. These studies indicate that lacZ-tagged CWR22Rv1 cells after tissue culturing contain subsets of cells capable of establishing transient micrometastases in lung, liver, and bone after direct injection into the animal’s circulation. Moreover, the quantitative and qualitative properties of the micrometastases in the three organs differ significantly, suggesting different mechanisms for stabilization and fates of micrometastases in these organs.     

人胰腺癌裸鼠移植瘤浸润和转移方式的研究

为了探讨胰头癌浸润及转移规律，我们用自建的三株人胰头癌裸鼠移植瘤，接咱在鼠背部、腹腔及抓垫皮下。结果表明：三株移植瘤背部接种均以局部浸润为主，可经淋巴疲乏转移至局部淋巴结（ＰＣＴ－３转移率为３３％；ＰＣＴ－４为４０％；ＰＣＴ－５为３３％），无远处淋巴结和（或）脏器转移；腹腔接种及抓垫接种与背部皮下接种结果相似。肿瘤的生长速度和浸润及转移的能力与肿瘤分化程度有一定关系。     

Tumor progression and metastasis in murine D2 hyperplastic alveolar nodule mammary tumor cell lines

We have examined tumor progression and metastatic properties of three clonal murine mammary tumor cell lines of recent origin (D2A1, D2.OR and D2.1). These lines were derived from spontaneous mammary tumors which originated from a D2 hyperplastic alveolar nodule (HAN) line. D2A1 cells were more malignant than D2.OR or D2.1 cells, whether measured by experimental metastasis assays after intravenous injection in nude mice or chick embryos,in vivo growth rate of primary tumors following mammary fat pad injection in nude mice, or spontaneous metastasis assay from primary tumors growing in mammary fat pads. D2A1 cells also were more invasivein vitro in a Matrigel invasion assay than D2.1 cells, while the D2.OR cells were non-invasive in this assay. The increased invasiveness and malignancy of D2A1 cells were associated with increased levels of mRNA for the cysteine proteinase cathepsin L. Levels of osteopontin (OPN), nm23, int-1 and int-2 mRNAs were also examined. Nm23 levels were highest in the most malignant cell line. These cell lines provide a model for studying the tumorigenic and metastatic ability of mammary tumor cells and offer several advantages: they were cloned from mammary tumors that originate from a common source of preneoplastic cells (D2HAN); they are of relatively recent origin; and they have spontaneously arrived at different stages of tumor progression.     

Metastatic behavior and cell surface properties of HT-29 human colon carcinoma variant cells selected for their differential expression of sialyl-dimeric Lex antigen

Immunochemical studies of human colorectal carcinoma with various monoclonal antibodies against Le^x-related carbohydrate antigens previously revealed that the amount of sialyl-dimeric Le^x antigen (NeuAc2–3Gal/1–4(Fucr1–3)GlcNAc1–3Gal1–4 (Fuc1–3)GlcNAc1-R: SLX) associated with metastatic lesions was greater than in the primary tumors. To assess whether an experimental model can be used to study the direct relationship between this carbohydrate antigen and the tumor cell's metastatic behavior, we selected variant cells with increased surface SLX from established human colon carcinoma cell line HT-29. The cells in the upper 5% or lower 5% population in fluorescence intensity after reacting with a monoclonal antibody, FH6, were retrieved separately by a fluorescence-activated cell sorter and propagated. After three- or four-times selection, we obtained stable cell lines with low and high cell surface SLX antigens (HT-29 M1 and HT-29 M2, respectively). Binding of monoclonal antibody FH6 was detected to glycolipids extracted from HT-29 M2 cells but not from HT-29 Ml cells. Glycoprotein components having reactivity with monoclonal antibody FH6 were below the detectable level. HT-29 M2 cells injected intrasplenically into nude mice showed a slightly reduced incidence of metastatis to lung, liver and lymph nodes than did HT-29 M1 cells. Subsequently we found that SLX antigen was not detectable by immunohistochemical examination of these tumor cells grown in nude mice. Re-established cell line from nude mice xenografts expressed SLX antigenin vitro.     

Effects of suramin on metastatic ability,proliferation, and production of urokinase-type plasminogen activator and plasminogen activator inhibitor type 2 in human renal cell carcinoma cell line SN12C-PM6

Effects of suramin, a polysulfonated naphthylurea compound, on metastatic ability, proliferation, and production of plasminogen activators and plasminogen activator inhibitors were studied using the highly metastatic human renal cell carcinoma cell line, SN12C-PM6. After renal subscapular implantation of tumor cells in nude mice, suramin significantly inhibited metastasis of tumor cells to the lungs and liver. In vitro growth of tumour cells was inhibited by suramin in a dose-dependent manner, at relatively low doses (ID₅₀ = 105 µg/ml). Plasminogen activator inhibitor type 2 (PAI-2) production by tumor cells was enhanced by suramin (100 µg/ml), whereas urokinase-type plasminogen activator (uPA) production was suppressed. Thus, the increase in PAI-2 and the decrease in uPA production correlated with the inhibitory effects on tumour growth and metastasis by suramin. Therefore suramin may be beneficial for the treatment of patients with an early stage of renal cancer with potential risk of metastasis.     

Evaluation of metastatic ability at specific times during primary tumor growth: a novel spontaneous metastasis assay

A transformed NIH 3T3 fibroblast cell line, CI-e, normally does not produce spontaneous metastasis from subcutaneous or footpad tumors in nude mice. However, pulmonary tumor nodules are formed when more than 1 × 10³ cells are injected intravenously into nude mice. Co-injection of 1 × 10⁶ heavily irradiated and inactivated cells increases the clonogenic ability of the viable cells in that tumor colonies then occur with as few as 1 × 10² viable cells. Utilizing the action of these inactivated cells to enhance the lung colonizing ability of a relatively small number of viable tumor cells, we have developed a novel experimental model of spontaneous metastasis. In this model, a footpad tumor of the nude mouse metastasizes to the lungs following intravenous injection of 1 × 10⁶ inactivated cells at a specific time of tumor growth and following tumor foot amputation, whereas no spontaneous metastasis develops without injection of inactivated cells. This model enables us to detect metastatic ability which would otherwise be too low to detect using other assays. In addition, it allows us to evaluate metastatic ability at a specific time point during primary tumor growth, since no metastases can develop during the periods before inactivated cell injection and after tumor amputation. Using this model, we have determined that the metastatic ability of CI-e tumors in the footpad is constant throughout the exponential and stationary growth phases, even though cells isolated from exponentially growing tumors possess a 3.3-fold greater lung colonizing ability following intravenous injection than those from stationary tumors. This new experimental model may be applicable to other tumor cell lines and to other analyses where metastatic ability during a defined interval of tumor growth is of importance.     

Inverse expressions of the N-myc oncogene and β1 integrin in human neuroblastoma: relationships to disease progression in a nude mouse model system

A nude mouse model for human neuroblastoma has been developed to examine possible relationships between amplification/over-expression of the N-myc oncogene and altered regulation of expression of specific integrin subunits during tumor progression. Subcutaneous (ectopic) or intra-adrenal (orthotopic) injection of the neuroblastoma cell lines SK-N-SH or IMR-32 has generated a number of derivative tumor cell lines. Tumor cell lines derived from SK-N-SH cells (which do not express N-myc) or IMR-32 cells (which over-express N-myc) produce tumors at higher rates when re-injected into the subcutaneous space of nude mice. Moreover, cell lines derived from tumors initiated by IMR-32 cells exhibit shorter latent periods than do IMR-32 cells direct from tissue culture. With regard to integrin subunit expression, SK-N-SH and related cell lines express high levels of ₁ integrin, which is associated with the ₂ and ₃ integrin subunits (predominantly ₃). IMR-32 cells display reduced ₁ expression, and that which is produced is not associated with common a subunits. LaN1 cells, which express N-myc at even higher levels than do IMR-32 cells, express even less ₁. Interestingly, the tumor-derived cell lines (especially those from tumors initiated in adrenal glands) also exhibit reduced integrin expression compared with the parental cell lines; this reduction is associated with the enhanced tumor take rate observed when the cells are re-injected into nude mice. Our results raise the possibility of a relationship between over-expression of N-myc and down-regulation of ₁ integrin expression (possibly some a subunits also). In addition, the data suggest that human neuroblastoma-derived cell lines which exhibit reduced integrin expression display more aggressive tumor growth in nude mice.     

NIH3T3 transfectant containing human K-ras oncogene shows enhanced metastatic activity after in vivo tumor growth or co-culture with fibroblasts

A clone of NIH3T3 transformant (H-3), obtained by transfecting genomic DNA of a human colon carcinoma cell line, contains human K-ras oncogene and yields metastatic pulmonary nodules after intravenous injection of the cells into nude mice. This metastatic ability was enhanced remarkably after in vivo tumor growth (subcutaneous tumor formation in nude mice) accompanied by increased mRNA expression and gene amplification of the human-derived K-ras oncogene, while it declined gradually as the passage number increased in vitro, with corresponding decreases of gene amplification and mRNA expression. Six subclones were randomly selected from H-3 cells which had been subcultured to passage 22. All of the clones in culture showed almost the same low level of metastatic ability and exhibited little K-ras oncogene amplification with correspondingly low mRNA expression. However, after they formed tumors in nude mice, every clone acquired high metastatic ability and the gene amplification increased, with elevated mRNA expression. These experimental facts indicated that acquisition of metastatic ability coupled with the function of K-ras oncogene was conditional in nature, being strongly affected by in vivo tumor circumstances. The low metastatic and G-418-resistant H-3 cells were co-cultured with BALB/c3T3 fibroblasts for 2–4 weeks. After removal of fibroblasts by exposure to G-418, the tumor cells exhibited increased metastatic ability and human K-ras oncogene mRNA, suggesting an intimate interaction between H-3 cells and fibroblasts influencing the function of transfected human K-ras oncogene. Fibroblasts of the host animal may thus have an important role in generating enhanced metastatic activity of H-3 cells.     

Influence of polyamines on in vitro and in vivo features of aggressive and metastatic behavior by human breast cancer cells

Increased cellular activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine (PA) synthesis, is an independent adverse prognostic factor for overall survival in human breast cancer [4], thus suggesting an important role for PA in tumor progression. The experiments presented here were designed to investigate the role of PA in invasion and metastasis, using the highly aggressive MDA-MB-435 and MDA-MB-231 human breast cancer cell lines. Administration of α-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, significantly reduced, in a dose-dependent manner, the invasiveness in matrigel of both MDA-MB-435 and MDA-MB-231 cells by ∼70%. DFMO treatment also inhibited (P<0.0001) `stellate' colony formation (an indicator of aggressive phenotype) by MDA-MB-435 cells plated in the matrigel outgrowth assay. Administration of DFMO (2% in drinking water) reduced the growth rate of both cell lines implanted orthotopically in nude mice. To evaluate metastasis while minimizing effects on proliferation, DFMO-treated mice were sacrificed later to allow their tumors to reach the same size of the tumors in the control mice. The most striking finding was that DFMO, while ineffective in reducing local invasion, nearly totally abolished (P=0.0152) pulmonary metastasis in mice bearing MDA-MB-435 xenografts. These results support a role of PA in promoting breast cancer aggressiveness, particularly with regard to the development of distant metastasis. Furthermore, the data suggest that PA involvement is distal to local invasion in the metastatic cascade. This revised version was published online in July 2006 with corrections to the Cover Date.     

A highly metastatic Lewis lung carcinoma orthotopic green fluorescent protein model

The Lewis lung carcinoma has been widely used for many important studies. However, the subcutaneous transplant or orthotopic cell-suspension injection models have not allowed the expression of its full metastatic potential. A powerful new highly metastatic model of the widely-used Lewis lung carcinoma is reported here using surgical orthotopic implantation (SOI) of tumor fragments and enhanced green fluorescent protein (GFP) transduction of the tumor cells. To achieve this goal, we first developed in vitro a stable high-expression GFP transductant of the Lewis lung carcinoma with the pLEIN retroviral expression vector containing the enhanced Aequorea victoria GFP gene. Stable high-level expression of GFP was found maintained in vivo in subcutaneously-growing Lewis lung tumors. The in vivo GFP-expressing tumors were harvested and implanted as tissue fragments by SOI in the right lung of additional nude mice. This model resulted in rapid orthotopic growth and extensive metastasis visualized by GFP-expression. 100% of the animals had metastases on the ipsilateral diaphragmatic surface, contralateral diaphragmatic surface, contralateral lung parenchima, and in mediastinal lymph nodes. Heart metastases were visualized in 40%, and brain metastases were visualized in 30% of the SOI animals. Mice developed signs of respiratory distress between 10–15 days post-tumor implantation and were sacrificed. The use of GFP-transduced Lewis lung carcinoma transplanted by SOI reveals for the first time the high malignancy of this tumor and provides an important useful model for metastasis, angiogenesis and therapeutic studies.     

Gene transduction of NK4, HGF antagonist,inhibits in vitro invasion and in vivo growth of human pancreatic cancer

In this study, we investigated the therapeutic effects of adenovirally-mediated transfer of the sequence of NK4, an antagonist for hepatocyte growth factor (HGF), against human pancreatic carcinoma. HGF has been implicated to play an important role in invasion and metastasis of various human cancers through tumor-stromal interactions. Although NK4 has been shown to block the metastatic behavior of cancer cells, problems with cellular delivery of NK4 must be addressed before it can be used for clinical trials. The effects of NK4 gene transduction mediated by recombinant adenovirus (Ad-NK4) were evaluated in a human pancreatic cancer cell line (SUIT-2) by in vitro scattering assays, invasion assays, and subcutaneous transplantation in nude mice. NK4 transduction markedly inhibited scattering and invasion of SUIT-2 cells stimulated by HGF without affecting cell proliferation in vitro. Furthermore, Ad-NK4 significantly inhibited the growth of tumors transplanted to nude mice. The tumor reduction induced by Ad-NK4 was associated with a decreased number of blood vessels surrounding the tumors. These findings suggest that Ad-NK4 gene therapy may be a unique and promising strategy for the treatment of pancreatic cancer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of regional location of the inoculation site and dietary fat on the pathology of MDA-MB-435 human breast cancer cell-derived tumors grown in nude mice

The effects of inoculation site and dietary fat intake on the growth and metastasis of the MDA-MB-435 human breast cancer cell line were studied in athymic nude mice. The tumor cells, 1 × 10⁶, were injected into either a right-sided thoracic or inguinal mammary fat pad (mfp), and 1 week later mice were randomly assigned to a high-fat (HF), 23% corn oil, or a low-fat (LF), 5% corn oil, diet. There were 30 mice in the HF, and 30 in the LF subgroups from each of the two inoculation site groups. The experiment was terminated 15 weeks after the tumor cell inoculations. Within the thoracic mfp-injected group, a HF diet reduced latency, increased growth rate at the primary site, and enhanced metastasis to regional lymph nodes, lungs, and intra-abdominal sites. For mice inoculated into an inguinal mfp, fat intake affected neither primary nor metastatic tumor development and growth; in both subgroups lung metastasis was significantly less than in the HF-fed, thoracic mfp-injected subgroup. The histological features of the lung metastases were consistent with a vascular mode of spread, whereas the extensive intra-abdominal lymph node involvement observed in mice with inguinal mfp tumors was in keeping with lymphatic-borne metastases.