Immunochemical and immunohistological analyses of tumor-associated antigens defined by murine monoclonal antibodies against human pancreatic carcinoma cells

Two murine monoclonal antibodies, SK-930 (isotype IgG2a) and SK-117 (isotype IgG1), were produced from spleen cells of mice immunized against human pancreatic carcinoma cell lines, MIA-PaCa 2 and Panc-1. With the use of the avidin-biotin-immunoperoxidase technique, the SK-930 and SK-117 antibodies detected an antigen found in 24 and 23 formalin-fixed tissue sections, respectively, of tumors obtained from 30 different patients with pancreatic carcinoma. Reactivity was also frequently found with tumors of the gallbladder, bile duct, stomach, colon and esophagus, while a large panel of normal human tissues, including normal pancreatic tissues, displayed little reactivity. These observations suggest that SK-930 and SK-117 are of value in identifying tumor-associated antigen (TAA) expressed in pancreatic carcinoma and other carcinomas of the digestive system. SK-930 antibody immunoprecipitated a 134 kilodalton molecule from extracts of 125I- or [35S]methionine- or [3H]glucosamine-labeled tumor cells. The SK-117-defined antigen corresponds to 152/137 kilodalton molecules. Moreover, cytofluorometric analyses showed that cells treated with periodic acid exhibited greatly decreased reactivity to the two antibodies, but cells treated with neuraminidase, trypsin or pronase showed unchanged reactivity. The findings suggest that the epitopes of the novel TAA expressed on pancreatic carcinoma cells are carbohydrate moieties.     

Immunochemical and Immunohistological Analyses of Tumor-associated Antigens Defined by Murine Monoclonal Antibodies against Human Pancreatic Carcinoma Cells

Two murine monoclonal antibodies, SK-930 (isotype IgG2a) and SK-117 (isotype IgG1), were produced from spleen cells of mice immunized against human pancreatic carcinoma cell lines, MIA-PaCa 2 and Panc-1. With the use of the avidin-biotin-immunoperoxidase technique, the SK-930 and SK-117 antibodies detected an antigen found in 24 and 23 formalin-fixed tissue sections, respectively, of tumors obtained from 30 different patients with pancreatic carcinoma. Reactivity was also frequently found with tumors of the gallbladder, bile duct, stomach, colon and esophagus, while a large panel of normal human tissues, including normal pancreatic tissues, displayed little reactivity. These observations suggest that SK-930 and SK-117 are of value in identifying tumor-associated antigen (TAA) expressed in pancreatic carcinoma and other carcinomas of the digestive system. SK-930 antibody immunoprecipitated a 134 kilodalton molecule from extracts of ¹²⁵I- or [³⁵S]methionine- or [³H]glucosamine-labeled tumor cells. The SK-117-defined antigen corresponds to 152/137 kilodalton molecules. Moreover, cytofluorometric analyses showed that cells treated with periodic acid exhibited greatly decreased reactivity to the two antibodies, but cells treated with neuraminidase, trypsin or pronase showed unchanged reactivity. The findings suggest that the epitopes of the novel TAA expressed on pancreatic carcinoma cells are carbohydrate moieties.     

Selectivity of the micro-leukocyte adherence inhibition assay in pancreatic cancer.

A modified leukocyte adherence inhibition assay was performed on white blood cells from patients with ductal pancreatic cancer, other malignancies, benign gastrointestinal diseases including pancreatitis, and healthy controls, using four different ductal pancreatic cancer membrane preparations and similar preparations from gastric and colorectal cancers. A mean adherence index of less than or equal to 0.2 was evidence that the leukocytes "recognized" the antigen(s). In 9 of 10 patients with localized pancreatic cancer, 13 of 15 leukocyte populations "recognized" the pancreatic cancer antigen(s) and not other tested antigen(s). Leukocytes from only 11 of 18 patients (17 of 29 assays) with metastatic pancreatic cancer "recognized" the pancreatic tumor antigen (and no other antigen). The inability to recognize the pancreatic tumor antigen(s) was not related to nutritional, biochemical or therapeutic status of the patient, but was related to the demonstration of a response to skin test antigens. In contrast, 3 of 35 leukocyte populations in 2 of 31 patients with malignancies other than pancreatic, 1 of 28 with benign gastrointestinal disease, and one of 38 healthy control populations "recognized" the antigen. The LAI is worthy of further study in the differential diagnosis of pancreatic cancer.     

Class I histone deacetylase-selective novel synthetic inhibitors potently inhibit human tumor proliferation.

We have developed previously a class of synthetic hybrid histone deacetylase (HDAC) inhibitors, which were built from hydroxamic acid of trichostatin A and pyridyl ring of MS-275. In this study we evaluated the antitumor effects of these novel hybrid synthetic HDAC inhibitors, SK-7041 and SK-7068, on human cancer cells. Both SK-7041 and SK-7068 effectively inhibited cellular HDAC activity at nanomolar concentrations and induced the time-dependent hyperacetylation of histones H3 and H4. These HDAC inhibitors preferentially inhibited the enzymatic activities of HDAC1 and HDAC2, as compared with the other HDAC isotypes, indicating that class I HDAC is the major target of SK-7041 and SK-7068. We found that these compounds exhibited potent antiproliferative activity against various human cancer cells in vitro. Growth inhibition effect of SK-7041 and SK-7068 was related with the induction of aberrant mitosis and apoptosis in human gastric cancer cells. Both compounds induced the accumulation of cells at mitosis after 6 h of treatment, which was demonstrated by accumulation of tetraploid cells, lack of G(2) cyclin/cyclin-dependent kinase inactivation, and higher mitotic index. After 12 h of treatment, apoptotic cells were increased through mitochondrial and caspase-mediated pathway. Finally, in vivo experiment showed that SK-7041 or SK-7068 was found to reduce the growth of implanted human tumors in nude mice. Therefore, based on isotype specificity and antitumor activity, SK-7041 and SK-7068 HDAC inhibitors are expected to be promising anticancer therapeutic agents and need additional clinical development.     

SK-7041, a new histone deacetylase inhibitor, induces G2-M cell cycle arrest and apoptosis in pancreatic cancer cell lines

A novel hybrid synthetic histone deacetylase inhibitor, SK-7041, was synthesized from hydroaxamic acid of trichostatin A (TSA) and pyridyl ring of MS-275. TSA and SK-7041 both induced apoptosis and G2-M cell cycle arrest in pancreatic cancer cell lines. The expressions of p21 and cyclin D2 were up-regulated and that of cyclin B1 was down-regulated by TSA or SK-7041. The expression levels of Mcl-1 and Bcl-XL but not those of Bcl-2, Bax, and Bak were suppressed by TSA or SK-7041 treatment. SK-7041 or TSA induced apoptosis and G2-M cell cycle arrest by up-regulating p21 and down-regulating cyclin B1, Mcl-1, and Bcl-XL.     

Cancer-associated stromal fibroblasts promote pancreatic tumor progression

Pancreatic adenocarcinoma is characterized by a dense background of tumor associated stroma originating from abundant pancreatic stellate cells. The aim of this study was to determine the effect of human pancreatic stellate cells (HPSC) on pancreatic tumor progression. HPSCs were isolated from resected pancreatic adenocarcinoma samples and immortalized with telomerase and SV40 large T antigen. Effects of HPSC conditioned medium (HPSC-CM) on in vitro proliferation, migration, invasion, soft-agar colony formation, and survival in the presence of gemcitabine or radiation therapy were measured in two pancreatic cancer cell lines. The effects of HPSCs on tumors were examined in an orthotopic murine model of pancreatic cancer by co-injecting them with cancer cells and analyzing growth and metastasis. HPSC-CM dose-dependently increased BxPC3 and Panc1 tumor cell proliferation, migration, invasion, and colony formation. Furthermore, gemcitabine and radiation therapy were less effective in tumor cells treated with HPSC-CM. HPSC-CM activated the mitogen-activated protein kinase and Akt pathways in tumor cells. Co-injection of tumor cells with HPSCs in an orthotopic model resulted in increased primary tumor incidence, size, and metastasis, which corresponded with the proportion of HPSCs. HPSCs produce soluble factors that stimulate signaling pathways related to proliferation and survival of pancreatic cancer cells, and the presence of HPSCs in tumors increases the growth and metastasis of these cells. These data indicate that stellate cells have an important role in supporting and promoting pancreatic cancer. Identification of HPSC-derived factors may lead to novel stroma-targeted therapies for pancreatic cancer.     

In vivo and in vitro inhibition of pancreatic cancer growth by targeted alpha therapy using 213Bi-CHX.A"-C595

PURPOSE: The aim of this study was to investigate the effect of targeted alpha therapy for the control of in vitro pancreatic cancer cell clusters and micrometastatic cancer lesions in vivo. METHODS: The expression of tumor-associated antigen MUC-1 on three pancreatic cancer cell clusters and animal xenografts was detected by indirect immmunostaining. Monoclonal antibodies C595 (test) and A2 (non-specific control) were labeled with 213Bi using the chelator CHX.A" to form the alpha-immunoconjugate (AIC). Cell clusters were incubated with AIC and examined at 48 h. Apoptosis was documented using the TUNEL assay. In vivo, an antiproliferative effect for tumors was tested at two days post-subcutaneous cell inoculation. Mice were injected with different concentrations of AIC by regional or systemic administration. Changes in tumor progression were assessed by tumor size. RESULTS: MUC-1 is strongly expressed on CFPAC-1, PANC-1 and moderate expression was found CAPAN-1 cell clusters and tumor xenografts. The AICs can target and kill cancer cell clusters (100 mm) in vitro. Some 73-81 % of cells were TUNEL positive cells in the clusters after incubation with AIC. At two days post- cell inoculation in mice, a single local injection of 74 and 148 MBq/kg of AIC causes complete inhibition of tumor growth. Systemic injections of 111, 222 and 333 MBq/kg of AIC cause significant tumor growth delay after 16 weeks, compared with the nonspecific control providing 333 MBq/kg after 16 weeks. CONCLUSIONS: CFPAC-1, PANC-1 and CAPAN-1 pancreatic cancer cell clusters and pancreatic tumor xenografts show high expression of the MUC-1 target antigen. 213Bi-C595 can specifically target and regress pancreatic cancer cell clusters in vitro, and delay and inhibit tumor growth in vivo. 213Bi-C595 may be a useful agent for the treatment of micrometastatic pancreatic cancer with overexpression of MUC 1 antigen in post-surgical patients with minimal residual disease.     

Armed and targeted measles virus for chemovirotherapy of pancreatic cancer

No curative therapy is currently available for locally advanced or metastatic pancreatic cancer. Therefore, new therapeutic approaches must be considered. Measles virus (MV) vaccine strains have shown promising oncolytic activity against a variety of tumor entities. For specific therapy of pancreatic cancer, we generated a fully retargeted MV that enters cells exclusively through the prostate stem cell antigen (PSCA). Besides a high-membrane frequency on prostate cancer cells, this antigen is expressed on pancreatic adenocarcinoma, but not on non-neoplastic tissue. PSCA expression levels differ within heterogeneous tumor bulks and between human pancreatic cell lines, and we could show specific infection of pancreatic adenocarcinoma cell lines with both high- and low-level PSCA expression. Furthermore, we generated a fully retargeted and armed MV-PNP-anti-PSCA to express the prodrug convertase purine nucleoside phosphorylase (PNP). PNP, which activates the prodrug fludarabine effectively, enhanced the oncolytic efficacy of the virus on infected and bystander cells. Beneficial therapeutic effects were shown in a pancreatic cancer xenograft model. Moreover, in the treatment of gemcitabine-resistant pancreatic adenocarcinoma cells, no cross-resistance to both MV oncolysis and activated prodrug was detected.     

Tumor cell lysate-pulsed human dendritic cells induce a T-cell response against pancreatic carcinoma cells: an in vitro model for the assessment of tumor vaccines

Dendritic cells (DCs) are potent antigen-presenting cells and play a pivotal role in T cell-mediated immunity. DCs have been shown to induce strong antitumor immune responses in vitro and in vivo, and their efficacy is being investigated in clinical trials. Compared with vaccination strategies directed against a single tumor antigen, tumor-cell lysate as the source of antigen offers the potential advantage of inducing a broad T-cell response against multiple known, as well as unknown, tumor-associated antigens expressed by the individual tumor. We used pancreatic carcinoma cell lines to develop an in vitro model for monitoring T-cell responses induced by lysate-pulsed DCs. Monocyte-derived DCs of HLA-A2(+) donors were pulsed with lysate generated from the HLA-A2(+) pancreatic carcinoma cell line Panc-1. In some experiments, the immunogenic protein keyhole limpet hemocyanin (KLH) was added to the lysate. Subsequently, the antigen-loaded DCs were activated with tumor necrosis factor-alpha and prostaglandin E(2). Autologous mononuclear cells were cocultured with DCs in the presence of low-dose interleukin (IL)-2 and IL-7 and were restimulated weekly with new DCs. High levels of IL-12 and IFN-gamma could be detected in the supernatants, indicating a T-helper type 1-type immune response. This cytokine profile was associated with the expression of the activation marker CD69 on both T helper and CTLs and with an antigen-induced proliferative T-cell response. After 4 weeks, CTL-mediated cytotoxicity was assessed. Tumor cell lysis was specific for Panc-1 tumor cells and was MHC class I-restricted. Cytokine secretion, CD69 expression of T cells, and antigen-induced T-cell proliferation correlated with the cytotoxic activity and were more pronounced when KLH was added to the lysate. This is the first study to show that T cells specific for pancreatic carcinoma cells can be generated in vitro by lysate-pulsed DCs and that the T-cell response can be enhanced by KLH. This in vitro model can be applied to compare different strategies in the development of DC-based tumor vaccines.     

Relationship of carbohydrate antigen 19-9 and Lewis antigens in pancreatic cancer

Carbohydrate antigen (CA) 19-9 identified by a murine monoclonal antibody against a colorectal carcinoma antigen is thought to be a sialylated Lewis (Le)a blood group antigen and occurs in high concentration in serum of patients with pancreatic carcinoma. This study was designed to identify the relationship between Lewis antigens and CA 19-9 in patients with pancreatic cancer. The following analyses were performed in 20 pancreatic cancer patients: Lea and Leb antigen phenotype in saliva (modified enzyme-linked immunosorbent assay) or on red cells (hemagglutination); CA 19-9 levels (radioimmunoassay) in serum; and CA 19-9 and Lea and Leb expression (immunoperoxidase assay) on tumor tissue. Lea-b- patients based on salivary phenotype failed to express CA 19-9 in tumor tissue and had normal or low levels of CA 19-9 (less than 37 units/ml) in serum (P = 0.0011, versus Lea+b- and Lea-b+ patients). Eighty-eight % of Lea+b- and Lea-b+ patients had elevated serum CA 19-9 levels (greater than 37 units/ml). All Lea+b- and Lea-b+ patients expressed both Lea and Leb antigens in tumor tissue. These results support the view that Lea-b- pancreatic cancer patients cannot manufacture CA 19-9. Surprisingly, Lea-positive patients express Leb antigen in tumor tissue; in this subgroup, Leb antigen may be a tumor-specific biomarker.     

Apoptotic pancreatic tumor cells are superior to cell lysates in promoting cross-priming of cytotoxic T cells and activate NK and gammadelta T cells

Tumor vaccines using dendritic cells (DCs) have been shown to induce antitumor CTL responses. The choice of the tumor antigen preparation used for DC loading is still an unresolved issue. We compared DCs pulsed with cell lysates, whole apoptotic tumor cells or their supernatants of the HLA-A2(+) human pancreatic carcinoma cell line Panc-1 for their capacity to activate T cells. Monocyte-derived DCs from HLA-A2(+) donors were pulsed with tumor antigen, matured subsequently, and cocultured with autologeous peripheral blood mononuclear cells. After three weekly restimulations with DCs, T-cell activation was assessed by intracellular IFN-gamma staining and cytotoxicity assays. Compared with lysate, pulsing DCs with the supernatant of apoptotic tumor cells induced a higher frequency of activated CTLs and T-helper cells, as well as an enhanced MHC class I-restricted tumor cell lysis. No activation of natural killer (NK) or gammadelta T cells was detected. Pulsing DCs with whole apoptotic tumor cells induced an even more pronounced lytic effect. However, in this case, MHC class-I blocking was only partially effective, and unrelated cell lines were also killed. IFN-gamma staining revealed activation of CTLs and T-helper cells, as well as NK and gammadelta T cells. Trans-well cultures of NK cells, apoptotic tumor cells, and DCs showed that NK cell activation was dependent on direct cell-to-cell contact with tumor cells and the presence of interleukin-12 produced by DCs. These results indicate that the choice of antigen preparation is a critical determinant in the induction of antitumor immunity. Tumor vaccines consisting of DCs and apoptotic tumor cells may be able to activate CTLs, as well as effector cells of the innate immune system.     

K-ras 抗原致敏的DC诱导CTL 对胰腺癌体内杀伤活性的研究*

目的:研究K-ras多肽的致敏树突状细胞（DC）活化的特异性细胞毒性T 淋巴细胞（CTL）对胰腺癌的体内外杀伤作用。方法:联合应用粒细胞- 巨噬细胞集落刺激因子和白细胞介素-4 诱导培养外周血DC。表达K-ras突变体的胰腺癌细胞株全瘤、单纯K-ras突变体多肽和K-ras突变体表位肽阳离子纳米颗粒分别致敏DC。致敏DC刺激T 淋巴细胞得到肿瘤抗原特异的细胞毒性T 淋巴细胞（CTL）。 Patu 8988、SW1990细胞系制备荷瘤裸鼠模型评价CTL 体内抗肿瘤活性。结果:负载全瘤抗原的DC其诱导产生的CTL 对胰腺癌有较好的抑制,负载单纯K-ras（12-Val ）突变体多肽、K-ras（12-Val ）突变体表位肽阳离子纳米颗粒的DC其诱导产生的CTL 对表达K-ras（12-Val ）突变体阳性（Patu 8988）的胰腺癌有较特异的抑制作用,而对K-ras（12-Val ）突变体阴性（SW1990）的胰腺癌的抑制作用与对照组比较无显著性差异。结论:负载肿瘤抗原的DC诱导的CTL 可显著提高对荷瘤裸鼠的生存时间,抑制肿瘤的生长速度,并显示其可增加抗肿瘤特异性。      

High expression of tumor-associated antigen RCAS1 in pancreatic ductal adenocarcinoma is an unfavorable prognostic marker

RCAS1 (receptor-binding cancer antigen expressed on SiSo cells) is a recently identified human tumor-associated antigen expressed on various cancer cells. It is thought that tumor cells evade immune surveillance by expression of RCAS1, which induces apoptotic cell death in receptor-positive immune cells. The purpose of our study was to investigate the relation between RCAS1 expression and the clinicopathological variables and clinical outcome in patients with pancreatic adenocarcinoma. Immunohistochemical analysis for RCAS1 was performed on paraffin-embedded specimens of 80 patients (mean age, 62 years) who underwent surgical resection for pancreatic adenocarcinoma. Of the 80 specimens, 77 (96%) were positive for RCAS1. No significant correlation was found between RCAS1 expression and age, gender, depth of invasion, tumor diameter, surgical margin, lymphatic invasion, venous invasion or histopathological grading. Borderline correlations between RCAS1 expression were noted for lymph node metastasis and stage (p = 0.0608 and 0.0934, respectively). RCAS1 expression was very frequently observed and the survival of patients with high RCAS1 expression was significantly shorter than that of those with low expression (p = 0.0012). Multivariate analysis using the Cox regression model indicated that high RCAS1 expression was an independent prognostic factor (risk ratio, 3.090; p = 0.0090). These results suggested that RCAS1 might be a significant tumor marker for pancreatic adenocarcinoma and an unfavorable predictor for prognosis of patients who have undergone surgical resection.     

Forskolin increases the expression of the pancreatic tumor antigen, Nd2, and uptake of Nd2 antibody

Nd2 antibody recognizes an antigen that is tumor specific in pancreas. It has been used successfully in clinical radioimmunodetection studies to identify exocrine pancreatic tumors. In the present study we show that the uptake of radiolabeled Nd2 antibody by SW1990 pancreatic carcinoma cells was increased by the adenyl cyclase activator, forskolin. Dibutyryl cyclic AMP and forskolin were both effective in increasing the level of Nd2 antigen in SW1990 cells. Immunoprecipitation studies showed that the Nd2 epitope is associated with MUC1 mucin. Forskolin increased Nd2/MUC1 antigen in both a membrane fraction and a high buoyant density mucin-like fraction. Nd2 immunoreactivity was reduced by treatment of mucins with proteases and beta-mercaptoethanol. Immunohistochemical studies showed that periodate catalyzed beta-elimination greatly reduced Nd2 immunoreactivity. These results suggest that the Nd2 epitope is unusual in having characteristics of both a peptide and a carbohydrate, protease and conformation sensitivities and involvement of O-linked oligosaccharides. Nd2 antibody does not react with several known pancreatic cancer antigens. In summary, activation of the cyclic AMP pathway increased cellular uptake of Nd2 antibody and the cellular expression of the tumor-specific, mucin-associated Nd2 antigen. These results suggest a means of improving the effectiveness of monoclonal antibodies in targeting tumor antigens for the diagnosis and treatment of malignancy.     

Influence of MUC18/MCAM/CD146 expression on human melanoma growth and metastasis in SCID mice.

The cell surface glycoprotein MUC18MCAM/CD146 was originally defined as a marker of melanoma progression and has been suspected to be directly linked to the metastatic process of this malignancy. In order to address this question, 2 MCAM negative human melanoma cell lines, SK-2 and XP44RO(Mel), were transfected with MCAM-encoding cDNA. Surface MCAM expression on SK-2 and XP44RO(Mel) transfectants was similar to that observed in naturally occurring MCAM positive human melanoma cells and transfectants demonstrated MCAM-dependent increase in homotypic adhesion in vitro. The growth behavior of 7 MCAM transfectants and their respective vector controls was evaluated in SCID mice. Tumor size at 4-5 weeks after s.c. implantation was highly variable, but did not correlate with MCAM expression. Despite massive primary tumor formation at the injection site, no spontaneous metastasis was observed with any of the investigated MCAM transfectants. The influence of MCAM expression on lung metastases formation in an experimental metastasis assay was system dependent, converting only XP44RO(Mel) transfectants into metastatic cells, although increased homotypic adhesion, leading to formation of tumor cell clusters, was observed with transfectants of both cell lines in vitro. Our findings indicate that MCAM expression of human melanoma cells has an influence on later stages of the metastatic process only, namely, extravasation and establishment of new foci of growth, but is per se not sufficient for this process.     

Tissue distribution, immunochemical characterization, and biosynthesis of 47D10, a tumor-associated surface glycoprotein

This report describes a new monoclonal antibody (MAb) designated 47D10 which was produced by immunizing mice against a human lung adenocarcinoma line, A549. The MAb 47D10 reacts with a surface antigen found in 95% of adenocarcinomas of the pancreas as well as on high percentages of adenocarcinomas from colon, breast, lung, and bile duct. The antigen was not detected in normal pancreas, in pancreatitis, or in a variety of normal tissues with the exception of colon and mature granulocytes. Lymphocytes and erythrocytes were also negative. The binding of 47D10 to tumor cells was unaffected by treatment of cells with neuraminidase. Immunoprecipitation followed by polyacrylamide gel electrophoresis showed that 47D10 MAb recognized a group of glycoproteins ranging in molecular weight from 67,000-98,000 on A549 lung carcinoma cells. Pulse-chase labeling showed two precursor proteins with molecular weights of 69,000 and 67,000 which were processed to the larger polypeptides in 1.5 h. At least part of the carbohydrates associated with the 47D10 antigen was asparagine linked because the antigen was sensitive to endoglycosidases, and tunicamycin inhibited the biosynthesis of 47D10 antigen. The 47D10 antigen was expressed on the cell surface because it could be detected on live A549 cells by enzyme-linked immunosorbant assays as well as by immunofluorescent staining. Furthermore, 47D10 antigens on tumor cell lines and granulocytes were vectorially labeled with 125I. The antigen found on granulocytes showed a higher molecular weight of 150,000-180,000, which was digested by endoglycosidase F to polypeptides with molecular weights ranging from 23,000-27,000. In contrast, the degradation product of the A549 antigen was a Mr 39,000 polypeptide after treatment with endoglycosidase F. The immunochemical characteristics of 47D10 antigen suggest that it is distinct from other antigens associated with pancreatic tumors, such as carcinoembryonic antigen, 19-9, and Du-PAN-2. By virtue of its broad range of tumor cell reactivity and low activity on normal cells, the 47D10 MAb may represent an important immunological reagent for differential diagnosis, especially of pancreatic carcinoma.     

Concentration and localization of carbohydrate antigen 19-9 in tissues of pancreatic cancer

Carbohydrate antigen (CA) 19-9 concentration in the tissue-extracts of cancerous and noncancerous pancreatic tissues were determined by radioimmunoassay. Pancreatic cancer tissues revealed significantly elevated CA 19-9 concentrations, when compared with chronic pancreatitis, normal adult pancreas, or fetal pancreas tissues. Metastatic liver tumors from pancreatic cancer also showed extremely high CA 19-9 concentrations, and there was no significant correlation between tissue CA 19-9 concentration and serum CA 19-9 level in patients with pancreatic cancer. In addition, positive localization of CA 19-9 was clearly observed in cancer cells of pancreatic cancerous tissues by immunohistochemical study, confirming the remarkable increase of CA 19-9 concentration in tissues of pancreatic cancer, although CA 19-9 was also partially found in non-malignant pancreatic tissues. The results indicated that CA 19-9 would be produced in great quantities by cancer cells in tissues of pancreatic cancer, and thus could be a valuable tumor associated antigen suggesting its clinical use as a tumor marker for cancer of the pancreas.     

Pancreatic stellate cells: partners in crime with pancreatic cancer cells

Pancreatic stellate cells (PSC) produce the stromal reaction in pancreatic cancer, but their role in cancer progression is not fully elucidated. We examined the influence of PSCs on pancreatic cancer growth using (a) an orthotopic model of pancreatic cancer and (b) cultured human PSCs (hPSC) and human pancreatic cancer cell lines MiaPaCa-2 and Panc-1. Athymic mice received an intrapancreatic injection of saline, hPSCs, MiaPaCa-2 cells, or hPSCs + MiaPaCa-2. After 7 weeks, tumor size, metastases, and tumor histology were assessed. In vitro studies assessed the effect of cancer cell secretions on PSC migration and the effect of hPSC secretions on cancer cell proliferation, apoptosis, and migration. Possible mediators of the effects of hPSC secretions on cancer cell proliferation were examined using neutralizing antibodies. Compared with mice receiving MiaPaCa-2 cells alone, mice injected with hPSCs + MiaPaCa-2 exhibited (a) increased tumor size and regional and distant metastasis, (b) fibrotic bands (desmoplasia) containing activated PSCs within tumors, and (c) increased tumor cell numbers. In vitro studies showed that, in the presence of pancreatic cancer cells, PSC migration was significantly increased. Furthermore, hPSC secretions induced the proliferation and migration, but inhibited the apoptosis, of MiaPaCa-2 and Panc-1 cells. The proliferative effect of hPSC secretions on pancreatic cancer cells was inhibited in the presence of neutralizing antibody to platelet-derived growth factor. Our studies indicate a significant interaction between pancreatic cancer cells and stromal cells (PSCs) and imply that pancreatic cancer cells recruit stromal cells to establish an environment that promotes cancer progression.     

In vivo Adenovirus-mediated Prodrug Gene Therapy for Carcinoembryonic Antigen-producing Pancreatic Cancer

In gene therapy for malignancy, the herpes simplex virus thymidine kinase (HSVtk)-ganciclovir (GCV) system has been widely used. For pancreatic cancer targeting, we estimated the therapeutic efficacy of gene transduction by an adenovirus-carrying HSVtk gene under the control of a carci-noembryonic antigen (CEA) promoter (AdCEAtk) followed by systemic administration of GCV. Four cell lines, CEA-producing Su.86.86, BxPC-3 (pancreatic cancer cells), MKN45 (gastric cancer cells) and CEA-nonproducing HeLa, were used for analysis of GCV sensitivity induced by adeno-viral gene transduction. To evaluate the therapeutic efficacy of AdCEAtk and GCV administration in human CEA-positive pancreatic cancer in vivo , a subcutaneously implanted tumor-bearing nude mouse model was used. When the HSVtk gene was transduced with a ubiquitous promoter into these cells, increase of the GCV sensitivity was independent of CEA-production. In contrast, when the cells were transduced with a CEA promoter, the cell-killing effect of GCV was increased in only CEA-producing cells. For in vivo analysis, AdCEAtk was delivered into subcutaneously established tumors of Su.86.86 cells. Immunohistochemical staining of the tumor showed that HSVtk protein was expressed only in tumor cells, and tumor growth was markedly suppressed by administration of GCV. These results suggest that the adenovirus-mediated transfer of HSVtk gene with CEA promoter specifically increases the GCV sensitivity of CEA-producing pancreatic cancer cells in vitro and in vivo . This strategy may provide a useful tool for treating pancreatic cancer, especially CEA-producing tumor cells.