In vivo selection and characterization of metastatic variants from human pancreatic adenocarcinoma by using orthotopic implantation in nude mice

We determined whether the implantation of human pancreatic cancer cells into the pancreas of nude mice can be used to select variants with increasing metastatic potential. COLO 357 line fast-growing cells were injected into the spleen or pancreas of nude mice. Hepatic metastases were harvested, and tumor cells were reinjected into the spleen or pancreas. This cycle was repeated several times to yield cell lines L3.6sl (spleen to liver) and L3.6pl (pancreas to liver). The variant cells produced significantly higher incidence and number of lymph node and liver metastases than the parental cells. Their increased metastatic potential was associated with increased expression (mRNA and protein) of the proangiogenic molecules basic fibroblast growth factor, vascular endothelial growth factor, and interleukin-8. The metastatic cells also exhibited increased motility and invasiveness, which were associated with increased expression of collagenase type IV (MMP-9) and decreased expression of E-cadherin. Collectively, the data show that the orthotopic implantation of human pancreatic cancer cells in nude mice is a relevant model with which to study the biology of pancreatic cancer metastasis and to select variant cell lines with enhanced metastatic potential.     

NF-kappaB activity blockade impairs the angiogenic potential of human pancreatic cancer cells

The effect of blockade of NF-kappaB activity on human pancreatic cancer angiogenesis was determined in an orthotopic xenograft model. Highly metastatic L3.3 human pancreatic cancer cells, which expressed an elevated level of constitutive NF-kappaB activity, were transfected with a mutated IkappaBalpha (IkappaBalphaM). After implantation in the pancreas of nude mice, parental (L3.3) and control vector-transfected (L3.3-Neo) cells produced rapidly growing tumors and liver metastases, whereas IkappaBalphaM-transfected (L3.3-IkappaBalphaM) cells had decreased tumorigenicity and metastatic potential. NF-kappaB signaling blockade significantly inhibited the in vitro and in vivo expression of the major proangiogenic molecules vascular endothelial growth factor and interleukin-8 and decreased tumor vascular formation. These events were correlated with retarded tumor growth and suppression of metastasis. Collectively, these data suggest that suppression of tumorigenicity and metastasis by NF-kappaB blockade is due to impaired angiogenic potential of tumor cells.     

Early tumor growth in metastatic organs influenced by the microenvironment is an important factor which provides organ specificity of colon cancer metastasis

We have previously demonstrated that liver metastases in nude mice and lung metastases in nude rats occurred specifically, when KM12SM human colon carcinoma cells were inoculated orthotopically into the cecal wall of nude mice and rats. To clarify the relationship between the tumor growth potential in the metastatic organs and the metastatic organ preference in these two metastatic models, we have evaluated the in vitro cell growth activities affected by the organ conditioned medium (CM) from the liver and lung, and the in vivo growth activities of the ectopic implanted tumors in the liver and lung. The tumorigenicity of the ectopic implanted tumors was 100% in mouse liver, 33% in rat liver, 50% in mouse lung, and 75% in rat lung. The crude liver CM of the animals showed inhibitory activities for KM12SM cell growth in a dosage-dependent manner, and the crude lung CM stimulated KM12SM cell growth. The liver CM of nude mice inhibited the KM12SM cell growth more strongly compared with the CM of nude rats, and the lung CM of nude rats was more strongly stimulated compared with the CM of nude mice. The liver CM of nude mice had non-heparin binding factors, which stimulated or inhibited KM12SM cell growth, in a molecular weight range of 50 to 100 kDa. By contrast, the liver CM of nude rats showed no growth stimulating activity for KM12SM cells. These results suggest that the metastatic organ specificity of KM12SM cells may depend on the early tumor growth influenced by the microenvironment in metastatic organs.     

Role of 1q trisomy in tumorigenicity, growth, and metastasis of human leukemic B-cell clones in nude mice

In order to investigate the association between various karyotypes of human tumor cells and biological behavior of tumors such as tumorigenicity, rate of growth, and the capacity to form metastasis, six chromosomally distinctive clones were isolated from an Epstein-Barr virus-transformed human chronic lymphocytic leukemic B-cell line which progressively grew and metastasized in irradiated nude mice. When inoculated into nude mice one clone (D10-1) with the karyotype of 46,XY,dup(1)(q11----q32) was more tumorigenic, grew faster, and produced more metastases than the other five clones. When mixtures of different clone-derived cells were grown in vitro or inoculated s.c. into nude mice the proportion of D10-1 cells was higher than their expected numbers in the cultures, s.c. tumors, and splenic and lymph nodal metastases. The growth and metastatic potential of the D10-1 clone were inhibited when cells from this clone were mixed with one or more clones that had slower growth. Duplication of 1q has been observed as a secondary aberration in human hematological malignancies and solid cancers. Our results demonstrate that duplication of the chromosome segment of 1q11----1q32 is associated with advantages in proliferation and metastasis formation.     

Effect of the vascular endothelial growth factor receptor-2 antibody DC101 plus gemcitabine on growth,metastasis and angiogenesis of human pancreatic cancer growing orthotopically in nude mice

Vascular endothelial growth factor (VEGF) is the major pro-angiogenic factor for most tumors. VEGF expression has been shown to be associated with a poor prognosis in human pancreatic cancer. The purpose of our study was to determine the effect of blockade of VEGF receptor-2 activity with or without gemcitabine on tumor growth and metastasis in an orthotopic model of human pancreatic cancer in nude mice. Therapy with gemcitabine or DC101, a VEGF receptor-2 antibody, resulted in a significant reduction of primary pancreatic tumor growth compared to untreated controls. The combination of DC101 and gemcitabine inhibited primary pancreatic tumor growth and lymphatic metastasis to a greater degree than either agent alone. Treatment with DC101 decreased vessel counts and increased the area of hypoxic tumor tissue compared to controls. Immunofluorescent double staining for apoptotic endothelial cells demonstrated a significant increase in the number apoptotic endothelial cells 24 days after initiation of therapy with DC101 plus gemcitabine. DC101 plus gemcitabine also increased tumor cell death and decreased tumor cell proliferation in pancreatic tumors. These findings indicate that blockade of VEGF receptor activation interferes with the survival of tumor endothelial cells, resulting in a reduction of primary pancreatic tumor growth in nude mice. Furthermore, the data demonstrate that anti-VEGF receptor-2 therapy potentiates the tumoricidal effect of gemcitabine in this model. Anti-VEGF receptor-2 therapy in combination with gemcitabine may be a novel therapeutic approach for advanced pancreatic cancer.     

Establishment of a new human pancreatic cancer cell line, NOR-P1, with high angiogenic activity and metastatic potential

We present here a new cell line, NOR-P1, established from a metastatic subcutaneous tumor of a patient with pancreatic cancer. The cells show rapid growth in culture with a doubling time of 16 h and high migration activity. Genetic and molecular analyses revealed high telomerase activity and a mutation in the K-ras oncogene. Of particular interest, the cells express markedly elevated mRNA levels of angiogenic factors, vascular endothelial growth factor and platelet-derived growth factor, as well as other tumor growth-related factors. Subcutaneous transplantation of the NOR-P1 cells into nude mice formed solid, hemorrhagic tumors which were histologically diagnosed as adenocarcinoma with dense blood vessels and severe extravasation of blood. Furthermore, when NOR-P1 cell suspension was injected directly into the pancreas of nude mice, the cells grew rapidly to form intra-pancreatic tumors associated with liver metastases and peritoneal dissemination that resulted in cachexia and subsequent death. These properties suggest that NOR-P1 is an aggressive pancreatic cancer cell line with a high metastatic potential and may serve as a useful experimental model for studying tumor angiogenesis and metastasis of pancreatic cancer.     

Blockade of vascular endothelial growth factor receptor and epidermal growth factor receptor signaling for therapy of metastatic human pancreatic cancer

We determined whether concurrent blockage of vascular endothelial growth factor (VEGF) receptor and epidermal growth factor (EGF) receptor signaling by two novel tyrosine kinase inhibitors, PTK 787 and PKI 166, respectively, can inhibit angiogenesis and, hence, the growth and metastasis of human pancreatic carcinoma in nude mice. Highly metastatic human pancreatic carcinoma L3.6pl cells were injected into the pancreas of nude mice. Seven days later, groups of mice began receiving oral doses of PTK 787 and PKI 166 three times weekly. Some groups of mice also received i.p. injections of gemcitabine twice a week. The mice were necropsied when the control mice became moribund. Treatment with PTK 787 and PKI 166, with gemcitabine alone, or with the combination of PTK 787, PKI 166, and gemcitabine produced 69, 50, and 97% reduction in the volume of pancreatic tumors, respectively. Administration of protein tyrosine kinase inhibitors and gemcitabine also significantly decreased the incidence of lymph node and liver metastasis. The therapeutic efficacy directly correlated with a decrease in circulating proangiogenic molecules (VEGF, interleukin-8), a decrease in microvessel density, a decrease in proliferating cell nuclear antigen staining, and an increase in apoptosis of tumor cells and endothelial cells. Therapies produced by combining gemcitabine with either PKI 166 or PTK 787 were similar to those produced by combining gemcitabine with both PKI 166 and PTK 787. These results suggest that blockade of either epidermal growth factor receptor or VEGF receptor signaling combined with chemotherapy provides an effective approach to the therapy of pancreatic cancer.     

Characterization of a newly established human Burkitt's lymphoma cell line, OMA-BL-1.

Using culture techniques, we have been able to grow occult tumor cells from the bone marrow from cancer patients and have developed a new malignant lymphoid cell line, OMA-BL-1, from the bone marrow of a 17-year-old patient with recurrent Burkitt's lymphoma. The tumor cells grew rapidly in vitro in suspension culture, and very aggressively in vivo in athymic nude mice with metastases to the liver and abdominal cavity. The morphological, chromosomal, immunophenotypic and molecular biologic characteristics of fresh uncultured tumor cells from the patient and tumor cells grown in culture and in athymic nude mice were very similar. The cells were positive for Epstein-Barr virus-associated nuclear antigens (EBNA) and chromosome analysis of the cells revealed an atypical chromosomal abnormality of 45,X,-X,i(8q), HSR(18)(q21),t(8;14)(q24;q32). Southern analysis demonstrated that c-myc was rearranged and amplified in these cells. Immunophenotypic analysis of the cells using flow cytometry showed monoclonal B cells expressing a surface IgG-kappa isotype. The tumor cells grown in nude mice had a significant decrease in CD24 expression when compared to cultured tumor cells. Electron microscopy of the fresh and cultured cells revealed Herpes virus, most likely Epstein-Barr virus, particles. This cell line has been maintained in culture for over 18 months. The aggressive growth and metastatic properties of this cell line in athymic nude mice make it a potentially useful experimental model to study the biology of human lymphoma.     

Molecular mechanisms of pancreatic carcinogenesis

Pancreatic ductal adenocarcinoma is one of the most fatal malignancies. Intensive investigation of molecular pathogenesis might lead to identifying useful molecules for diagnosis and treatment of the disease. Pancreatic ductal adenocarcinoma harbors complicated aberrations of alleles including losses of 1p, 6q, 9p, 12q, 17p, 18q, and 21q, and gains of 8q and 20q. Pancreatic cancer is usually initiated by mutation of KRAS and aberrant expression of SHH. Overexpression of AURKA mapping on 20q13.2 may significantly enhance overt tumorigenesity. Aberrations of tumor suppressor genes synergistically accelerate progression of the carcinogenic pathway through pancreatic intraepithelial neoplasia (PanIN) to invasive ductal adenocarcinoma. Abrogation of CDKN2A occurs in low-grade/early PanIN, whereas aberrations of TP53 and SMAD4 occur in high-grade/late PanIN. SMAD4 may play suppressive roles in tumorigenesis by inhibition of angiogenesis. Loss of 18q precedes SMAD4 inactivation, and restoration of chromosome 18 in pancreatic cancer cells results in tumor suppressive phenotypes regardless of SMAD4 status, indicating the possible existence of a tumor suppressor gene(s) other than SMAD4 on 18q. DUSP6 at 12q21-q22 is frequently abrogated by loss of expression in invasive ductal adenocarcinomas despite fairly preserved expression in PanIN, which suggests that DUSP6 works as a tumor suppressor in pancreatic carcinogenesis. Restoration of chromosome 12 also suppresses growths of pancreatic cancer cells despite the recovery of expression of DUSP6; the existence of yet another tumor suppressor gene on 12q is strongly suggested. Understanding the molecular mechanisms of pancreatic carcinogenesis will likely provide novel clues for preventing, detecting, and ultimately curing this life-threatening disease.     

具有高转移潜能的人肝癌细胞系的建立及其生物学特性

目的利用裸鼠人肝癌高转移模型（ＬＣＩ－Ｄ２０）的皮下移植瘤组织在体外建立一株具有高转移潜能的人肝癌细胞系（ＭＨＣＣ９７）,并对其一般生物学特性进行观察。方法将分离的瘤细胞制成细胞悬液,用１０％人ＡＢ型血清的高糖ＤＭＥＭ培养液建成该细胞系,采用流式细胞术和染色体Ｇ－显带方法,进行细胞遗传学分析;用ＡＢＣ免疫组化法,观察其肺转移灶中癌细胞甲胎蛋白（ＡＦＰ）表达情况。结果ＭＨＣＣ９７细胞为典型的上皮样细胞,符合一般上皮性恶性肿瘤细胞的病理学特征。该细胞经皮下和肝内接种均可使裸鼠致瘤,并发生肺部转移。肝内接种者,肺转移达１００％（１２／１２）。ＭＨＣＣ９７细胞为异倍体细胞,染色体均为超二倍体,ｉ（１）（ｑ）和ｄｅｒ（４）（ｐｔｅｒ→ｑ３５：：？）等为其标志染色体,未显示有完整Ｙ染色体存在。肺转移灶的癌细胞ＡＦＰ阳性。结论ＭＨＣＣ９７细胞具有与原移植瘤相似的生物学特性。染色体的畸变可能与其发生发展有关     

Differences in 20q13.2 copy number between colorectal cancers with and without liver metastasis.

Frequent gains of 20q have been identified recently in many neoplasias, including breast, bladder, ovarian, pancreatic, and colon cancers. A high level of 20q13.2 amplification is associated with poor prognosis in breast cancer. We investigated the copy number of the 20q13.2 region including the ZNF217 oncogene in 17 nonmetastatic colorectal cancers (CRCs), 18 primary CRC tumors with liver metastasis, and 18 metastatic lesions by two-color fluorescence in situ hybridization to evaluate the significance of an increased copy number of 20q13.2 in CRC, especially in those cases with liver metastasis. The frequency of increased relative copy number of the 20q13.2 region was higher in primary and liver metastatic lesions of CRC than in CRC lesions without liver metastasis. In particular, a high-level increase (>3.0-fold) in the relative copy number of 20q13.2 was observed in 2 of 18 (11%) primary CRC lesions with liver metastasis, 7 of 18 (39%) liver metastatic lesions, and in none of the cases of primary CRC without liver metastasis. The absolute and relative copy number of chromosome 20q13.2 was higher in CRCs with metastasis than in CRCs without metastasis. The percentage of cells with high-level 20q13.2 amplification was also higher in both lesions with metastasis per specimen than without metastasis. Our results suggest that the level of 20q13.2 amplification correlates with the metastatic potential and tumor progression of CRC. The results also suggest that 20q13.2 amplification with ZNF217 is associated with increased metastatic potential.     

Pancreatic cancer cell-derived vascular endothelial growth factor is biologically active in vitro and enhances tumorigenicity in vivo

Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulator that acts by binding to high-affinity transmembrane receptors. Although both VEGF and its receptors are overexpressed in human pancreatic ductal adenocarcinoma (PDAC), this malignancy is not generally considered to be highly vascular. It is not known, therefore, whether the abundance of VEGF in PDAC is biologically relevant. To address this issue, we measured the angiogenic effects of pancreatic cancer cell-derived VEGF in an in vitro endothelial cell proliferation assay and characterized the consequences of suppressing VEGF expression on pancreatic tumor growth in an athymic nude mouse model. We found that human pancreatic cancer cell lines secrete large quantities of biologically active VEGF into conditioned medium (CM). Stable transfection of an anti-sense VEGF(189) (AS-VEGF(189)) expression construct into PANC-1 pancreatic cancer cells resulted in decreased VEGF expression and secretion, a decreased capacity of the resultant CM to enhance endothelial cell proliferation and a significant attenuation of tumor cell proliferation in vitro. Furthermore, when injected into athymic nude mice, AS-VEGF(189)-expressing cells exhibited an 80% decrease in tumor growth compared with control cells. These results support the hypothesis that VEGF promotes pancreatic cancer growth in vivo and suggest that anti-VEGF therapy may be useful in the treatment of this disease.     

Complement component 1, q subcomponent binding protein (C1QBP) in lipid rafts mediates hepatic metastasis of pancreatic cancer by regulating IGF‐1/IGF‐1R signaling

Pancreatic cancer shows a remarkable predilection for hepatic metastasis. Complement component 1, q subcomponent binding protein (C1QBP) can mediate growth factor‐induced cancer cell chemotaxis and distant metastasis by activation of receptor tyrosine kinases. Coincidentally, insulin‐like growth factor‐1 (IGF‐1) derived from the liver and cancer cells itself has been recognized as a critical inducer of hepatic metastasis. However, the mechanism underlying IGF‐1‐dependent hepatic metastasis of pancreatic cancer, in which C1QBP may be involved, remains unknown. In the study, we demonstrated a significant association between C1QBP expression and hepatic metastasis in patients with pancreatic cancer. IGF‐1 induced the translocation of C1QBP from cytoplasm to lipid rafts and further drove the formation of CD44 variant 6 (CD44v6)/C1QBP complex in pancreatic cancer cells. C1QBP interacting with CD44v6 in lipid rafts promoted phosphorylation of IGF‐1R and thus activated downstream PI3K and MAPK signaling pathways which mediated metastatic potential of pancreatic cancer cells including proliferation, apoptosis, invasion, adhesion and energy metabolism. Furthermore, C1QBP knockdown suppressed hepatic metastasis of pancreatic cancer cells in nude mice. We therefore conclude that C1QBP in lipid rafts serves a key regulator of IGF‐1/IGF‐1R‐induced hepatic metastasis from pancreatic cancer. Our findings about C1QBP in lipid rafts provide a novel strategy to block IGF‐1/IGF‐1R signaling in pancreatic cancer and a reliable premise for more efficient combined modality therapies.     

Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma

MCAM/MUC18 expression correlates with tumor thickness and metastatic potential of human melanoma cells in nude mice. Moreover, ectopic expression of MUC18 in primary cutaneous melanoma cells leads to increased tumor growth and metastasis in vivo. Here we tested the effect of a fully human anti-MUC18 antibody, ABX-MA1, on angiogenesis, tumor growth, and metastasis. ABX-MA1 had no effect on melanoma cell proliferation rate in vitro. However, when cells of the metastatic melanoma lines A375SM and WM2664 (which express high levels of MUC18) were injected s.c. into nude mice and treated with ABX-MA1 (100 micro g, weekly, i.p. for 5 weeks), tumor growth was significantly inhibited compared with control IgG-treated mice. ABX-MA1 treatment also suppressed experimental lung metastasis of these melanoma cells. ABX-MA1 disrupted spheroid formation by melanoma cells expressing MUC18 (homotypic interaction) and the ability of these cells to attach to human vascular endothelial cells [HUVECs (MUC18 positive)] in vitro. ABX-MA1 treatment of melanoma cells in vitro significantly inhibited the promoter and collagenase activity of matrix metalloproteinase 2, resulting in decreased invasion through Matrigel-coated filters. Decreased expression of matrix metalloproteinase 2 was also observed in the implanted tumors in vivo. Moreover, because HUVECs also express MUC18, ABX-MA1 directly disrupted the tube-like formation by HUVECs in an in vitro vessel formation assay. Collectively, these results point to usefulness of ABX-MA1 as a modality to treat melanoma either alone or in combination with conventional chemotherapy or other antitumor agents.     

Blockade of the epidermal growth factor receptor signaling by a novel tyrosine kinase inhibitor leads to apoptosis of endothelial cells and therapy of human pancreatic carcinoma

We determined whether down-regulation of the epidermal growth factor-receptor (EGF-R) signaling pathway by oral administration of a novel EGF-R tyrosine kinase inhibitor (PKI166) alone or in combination with gemcitabine (administered i.p.) can inhibit growth and metastasis of human pancreatic carcinoma cells implanted into the pancreas of nude mice. Therapy beginning 7 days after orthotopic injection of L3.6pl human pancreatic cancer cells reduced the volume of pancreatic tumors by 59% in mice treated with gemcitabine only, by 45% in those treated with PKI166 only, and by 85% in those given both drugs. The combination therapy also significantly inhibited lymph node and liver metastasis, which led to a significant increase in overall survival. EGF-R activation was significantly blocked by therapy with PKI166 and was associated with significant reduction in tumor cell production of VEGF and IL-8, which in turn correlated with a significant decrease in microvessel density and an increase in apoptotic endothelial cells. Collectively, our results demonstrate that oral administration of an EGF-R tyrosine kinase inhibitor decreased growth and metastasis of human pancreatic cancer growing orthotopically in nude mice and increased survival. The therapeutic effects were mediated in part by inhibition of tumor-induced angiogenesis attributable to a decrease in production of proangiogenic molecules by tumor cells and increased apoptosis of tumor-associated endothelial cells.     

Restoration of SMAD4 by gene therapy reverses the invasive phenotype in pancreatic adenocarcinoma cells

SMAD4 is a critical cofactor in signal transduction pathways activated in response to transforming growth factor-beta (TGF-beta)-related ligands, regulating cell growth and differentiation. The roles played by SMAD4 inactivation in tumours highlighted it as a tumour-suppressor gene. However, restoration of the TGF-beta antiproliferative pathway following SMAD4 gene transfer in null-tumour cell lines is controversial. Herein, we report the inhibitory effects of SMAD4 on pancreatic tumour invasion and angiogenesis. Adenoviral transfer of this gene in a panel of SMAD4 homozygous-deleted human pancreatic tumour cell lines restored SMAD4 protein expression and function. Although it did not affect proliferation significantly in vitro, SMAD4 inhibited in vivo tumour growth in immunodeficient mice. In this xenograft setting, differential suppression of tumour growth in vivo was mediated, at least in part, through downregulation of vascular endothelial growth factor and expression of gelatinases. We documented the reduced invasion and angiogenesis histologically and by intravital microscopy, and gained mechanistic insight at the messenger and protein level. Finally, we found a negative reciprocal regulation between SMAD4 and ETS-1. ETS-1 is considered a marker for tumour invasion. Upon SMAD4 deletion, we detected high expression levels of ETS-1 in pancreatic tumour cells, suggesting the shift of the pancreatic tumour toward an invasive phenotype.     

Vascular endothelial growth factor-trap suppresses tumorigenicity of multiple pancreatic cancer cell lines.

PURPOSE: Vascular endothelial growth factor A (VEGF-A) is a potent angiogenic agent that binds to two high affinity VEGF receptors (VEGFRs), a process facilitated by the low affinity neuropilin receptors. Although VEGF-A is overexpressed in pancreatic ductal adenocarcinoma, it is not known whether the in vivo growth of multiple pancreatic cancer cells can be efficiently blocked by VEGF-A sequestration. EXPERIMENTAL DESIGN: Four human pancreatic cancer cell lines were grown s.c. in athymic nude mice. One cell line also was used to generate an orthotopic model of metastatic pancreatic cancer. The consequences of VEGF-A sequestration on tumor growth and metastasis were examined by injecting the mice with a soluble VEGFR chimer (VEGF-Trap) that binds VEGF-A with high affinity. RESULTS: VEGF-Trap, initiated 2 days after tumor cell inoculation, suppressed the s.c. growth of four pancreatic cancer cell lines and markedly decreased tumor microvessel density. Analysis of RNA from tumors generated with T3M4 cells revealed that VEGF-Trap decreased the expression of VEGFR-1 and neuropilin-1 and -2. VEGF-Trap, initiated 3 weeks after tumor implantation, also attenuated intrapancreatic tumor growth and metastasis in an orthotopic model using PANC-1 cells. CONCLUSIONS: VEGF-Trap is a potent suppressor of pancreatic tumor growth and metastasis and also may act to attenuate neuropilin-1 and -2 and VEGFR-1 expression. Therefore, VEGF-Trap may represent an exceedingly useful therapeutic modality for pancreatic ductal adenocarcinoma.