Clinical Significance of Disseminated Pluripotent Tumor Cell Signature Expression in the Bone Marrow from Patients with Colorectal Cancer.

PURPOSE: Disseminated tumor cells (DTCs) are critically involved in tumor relapse and survival in several invasive tumors. We previously showed that the ATP-binding cassette (ABC) transporter, ABCB5, is a chemoresistance mediator expressed on specific cell subsets in colorectal cancer (CRC) and other malignancies. This study evaluated the molecular signature expression and its clinical relevance of DTCs in bone marrow from patients with colon cancer. METHODS: This study included 49 consecutive patients (UICC stage I-IV) that underwent curatively intended or palliative surgery for CRC. We analyzed cells from bone marrow aspirates obtained before surgery and derived from patients that had completed minimally a 5-year follow-up. The gene expression of ABCB5 in comparison to CD133 (molecule for identifying cancer initiating cells), Lgr5 (an intestinal stem cell marker) as well as Cytokeratin (CK) 20 (terminally differentiated tumor cells of epithelial origin) in these cells was evaluated. RESULTS: Bone marrow analysis showed differential expression between the analyzed genes. ABCB5 and Lgr5 and to lesser extent CD133 and CK20 genes were significantly expressed in the analyzed cells from bone marrow aspirates while only ABCB5 and Lgr5 were significantly negative associated with tumor progress and overall survival. CONCLUSION: Overexpression of ABCB5 and Lgr5 in bone marrow negatively influenced patient survival pointing to a specific chemo resistant and pluripotent cell subgroup of DTCs in the bone marrow. ABCB5 like Lgr5 positive cells seem to be involved in limited tumor related patient survival, suggesting that ABCB5- and Lgr5-positive cells may be relevant for specific clinical intervention strategies.

Ten-year follow-up of a prospective trial for the targeted therapy of gastric cancer with the human monoclonal antibody PAT-SC1.

The fully human monoclonal antibody PAT-SC1 is specific for an isoform of CD55 (decay-accelerating factor) designated CD55PAT-SC1. This antigen is expressed in the majority (80%) of gastric cancers (GCs), and the antibody induces tumour cell-specific apoptosis in vitro as well as in vivo. PAT-SC1, therefore, has been deemed promising as a therapeutic agent. Here, we describe the results of an academic clinical study performed in a neoadjuvant setting with resectable GC patients. Patients undergoing treatment for GC between 1997 and 2001 were tested for CD55PAT-SC1 expression. Fifty-one resectable patients that tested positively received a single administration of 20 mg PAT-SC1 48 h prior to surgery. They underwent standard surgery with either subtotal or total gastrectomy with bursectomy, omentectomy and a modified D2-lymphadenectomy, aimed at R0 resection. Primary endpoints of the present study were to evaluate side-effects of the PAT-SC1 antibody treatment and to evaluate histopathological effects such as tumour regression and induction of apoptosis. Long-term survival was a secondary endpoint. Administration of PAT-SC1 appeared safe with only reversible side-effects according to WHO grade I and II. Despite the low­dose of the antibody, 81.6% of the patients showed signs of increased apoptosis within the primary tumour and 60% showed signs of tumour cell regression. Comparison of the 10-year survival rates of the R0-resected CD55PAT-SC1-positive patients treated with the PAT-SC1 antibody with a historical collective of R0-resected CD55PAT-SC1-positive patients not treated with PAT-SC1 indicated a survival benefit in the treated patients. Furthermore, comparison of the patient survival of CD55PAT­SC1-positive vs. CD55PAT-SC1-negative groups suggested that CD55PAT-SC1 antigen expression is an independent predictor of poor survival in a Cox regression analysis. Antibody PAT-SC1 may be a useful additive therapeutic agent in the treatment of patients with CD55PAT-SC1-expressing GCs. In combination with radical standard surgery, PAT-SC1 given as an adjuvant or neoadjuvant immunotherapeutic agent induces apoptosis in tumour cells which may improve survival of these patients. Because of the human origin and its specific binding to the CD55PAT-SC1 antigen, PAT-SC1 was well tolerated in this trial.

Differential effects of PPARgamma activation by the oral antidiabetic agent pioglitazone in Barrett's carcinoma in vitro and in vivo.

BACKGROUND AND PURPOSE: The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is a key transcription factor regulating genes involved in adipogenesis, glucose homeostasis and cell differentiation. Moreover, PPARgamma has been demonstrated to control proliferation and apoptosis in various cancer cells. We investigated the biological effects of PPARgamma activation by the oral antidiabetic agent pioglitazone in Barrett's adenocarcinoma cells in vitro and in vivo. RESULTS: PPARgamma mRNA and protein were overexpressed in endoscopic biopsies of Barrett's epithelium and the human Barrett's adenocarcinoma cancer cell line OE33 as compared to normal esophagus and stomach and the esophageal squamous epithelium cancer cell line Kyse-180. PPARgamma activation by pioglitazone in OE33 cells in vitro led to reduced cell growth by induction of apoptosis. Effects of systemic PPARgamma activation by the thiazolidinedione pioglitazone on tumor cell proliferation and apoptosis were then assessed in vivo in nude mice bearing transplantable Barrett's adenocarcinomas derived from OE33 cells. Unexpectedly, enhanced growth of OE33 derived transplantable adenocarcinomas was observed in Balb/c nu/nu mice upon systemic pioglitazone treatment due to increased cell proliferation. CONCLUSION: These results indicate that PPARgamma is involved in the molecular pathogenesis of Barrett's adenocarcinoma formation and growth. However, activation of PPARgamma exerts differential effects on growth of Barrett's adenocarcinoma cells in vitro and in vivo emphasizing the importance of additional cell context specific factors and systemic metabolic status for the modulation of PPARgamma action in vivo.

The intraportal injection model: a practical animal model for hepatic metastases and tumor cell dissemination in human colon cancer.

BACKGROUND: The development of new therapeutic strategies for treatment of metastasized colorectal carcinoma requires biologically relevant and adequate animal models that generate both reproducible metastasis and the dissemination of tumor cells in the form of so-called minimal residual disease (MRD), an expression of the systemic character of neoplastic disease. METHODS: We injected immunoincompetent nude mice intraportally with different numbers (1 x 10(5), 1 x 10(6) and 5 x 10(6) cells) of the human colon carcinoma cell lines HT-29 and SW-620 and investigated by histological studies and CK-20 RT-PCR the occurrence of hematogenous metastases and the dissemination of human tumor cells in bone marrow. RESULTS: Only the injection of 1 x 10(6) cells of each colon carcinoma cell line produced acceptable perioperative mortality with reproducible induction of hepatic metastases in up to 89% of all animals. The injection of 1 x 10(6) cells also generated tumor cell dissemination in the bone marrow in up to 63% of animals with hepatic metastases. CONCLUSION: The present intraportal injection model in immunoincompetent nude mice represents a biologically relevant and adequate animal model for the induction of both reproducible hepatic metastasis and tumor cell dissemination in the bone marrow as a sign of MRD.

Growth of human gastric cancer cells in nude mice is delayed by a ketogenic diet supplemented with omega-3 fatty acids and medium-chain triglycerides.

BACKGROUND: Among the most prominent metabolic alterations in cancer cells are the increase in glucose consumption and the conversion of glucose to lactic acid via the reduction of pyruvate even in the presence of oxygen. This phenomenon, known as aerobic glycolysis or the Warburg effect, may provide a rationale for therapeutic strategies that inhibit tumour growth by administration of a ketogenic diet with average protein but low in carbohydrates and high in fat enriched with omega-3 fatty acids and medium-chain triglycerides (MCT). METHODS: Twenty-four female NMRI nude mice were injected subcutaneously with tumour cells of the gastric adenocarcinoma cell line 23132/87. The animals were then randomly split into two feeding groups and fed either a ketogenic diet (KD group; n = 12) or a standard diet (SD group; n = 12) ad libitum. Experiments were ended upon attainment of the target tumor volume of 600 mm3 to 700 mm3. The two diets were compared based on tumour growth and survival time (interval between tumour cell injection and attainment of target tumour volume). RESULTS: The ketogenic diet was well accepted by the KD mice. The tumour growth in the KD group was significantly delayed compared to that in the SD group. Tumours in the KD group reached the target tumour volume at 34.2 +/- 8.5 days versus only 23.3 +/- 3.9 days in the SD group. After day 20, tumours in the KD group grew faster although the differences in mean tumour growth continued significantly. Importantly, they revealed significantly larger necrotic areas than tumours of the SD group and the areas with vital tumour cells appear to have had fewer vessels than tumours of the SD group. Viable tumour cells in the border zone surrounding the necrotic areas of tumours of both groups exhibited a glycolytic phenotype with expression of glucose transporter-1 and transketolase-like 1 enzyme. CONCLUSION: Application of an unrestricted ketogenic diet enriched with omega-3 fatty acids and MCT delayed tumour growth in a mouse xenograft model. Further studies are needed to address the impact of this diet on other tumour-relevant functions such as invasive growth and metastasis.

Neoadjuvant therapy of gastric cancer with the human monoclonal IgM antibody SC-1: impact on the immune system.

Adjuvant therapies for minimal residual disease are a promising approach to improve the poor survival rates after surgery of gastric tumors. A pilot study of a neoadjuvant therapy was performed using a human monoclonal IgM antibody (SC-1) specifically inducing apoptosis in signet ring cell stomach carcinomas. However, scarce information exists on how such a treatment affects the immune system, in particular what are the effects of apoptosis induction and infusion of large amounts of IgM. Thus, the leukocyte composition (CD3, CD4, CD8, CD19, CD16+56, CD14) and several cytokines (TNF-alpha, IL6, IL12, IFN-gamma, GM-CSF, Neopterin) before and after SC-1 application were measured and compared to results of patients that underwent surgical removal of gastric carcinoma without antibody treatment. After SC-1 application, an increase in TNF-alpha and a decrease of lymphocytes and CD3+ T-cells but in the range obtained in healthy individuals was observed before surgery. After surgery, the IL6 levels increased and the TNF-alpha levels remained at the elevated level. Furthermore, there was a significant drop in lymphocytes and CD3+ T-cells. These effects were due to the surgical treatment. Other parameters did not show significant changes. It seems that the application of an apoptosis-inducing antibody prior to surgery of gastric tumors has mild if any effect on the immune system. Therefore, from an immunological point of view, the treatment with this monoclonal antibody is extremely safe.

Who benefits from gastric banding?

BACKGROUND: In the present study, criteria were investigated to predict major benefit after laparoscopic adjustable gastric banding (LAGB). MATERIALS AND METHODS: 85 morbidly obese patients were operated with LAGB between 1999 and 2005. Seventy-one of these patients were analyzed according to several possible predictive characteristics for success as the primary endpoint. Success was defined as excess body weight loss (EBWL) >50% and no band removal. Median follow-up was 27 months (range 8-90 months). RESULTS: In total, median EBWL was 43% (-41 to 171.5%) with a decrease in BMI of 8.0 kg/m(2) (-9 to 35 kg/m(2)). Success rate was 37% (n = 26). These patients were compared to all other patients (n = 45). Significant success predictors were baseline absolute BW, EBW, BMI (p < 0.01), BMI with a threshold value of 50 kg/m(2) (p = 0.02), and female sex (p = 0.02) as well as postoperative vomiting (p = 0.02), eating behavior and physical activity after LAGB (p < 0.01). Baseline EBW and change in eating behavior after surgery were identified as independent predictors in multivariate analysis. CONCLUSION: Patients with a lower excess body weight who improve especially their eating behavior after surgery have the highest chance of success after LAGB.

Feasibility and limits of an orthotopic human colon cancer model in nude mice.

We sought to develop an accurate colorectal cancer model in nude mice with stable local growth, tumor cell dissemination, and reproducible metastatic capacity. To this end, we orthotopically transplanted histologically intact human colorectal cancer tissue from 10 human patients into nude mice. After successful local tumor growth, tumor tissues were retransplanted as many as 9 times in serial passage. All specimens were transplanted using microsurgical techniques. Histologic, immunohistochemical, and polymerase chain reaction techniques were used to determine tumor growth rates and kinetics, development of regional lymph node and distant hepatic metastases, and the induction of minimal residual disease (MRD). Stable local tumor growth rates with variable growth kinetics were detected in 73.4% of all mice. The lymph node and hepatic metastasis rates were low, at 18.4% and 4.9%, respectively. MRD, as reflected by CK20 positivity of the bone marrow in animals with lymph node and hepatic metastases, was present in 22.2%. The orthotopic colorectal cancer model described here is feasible for the induction of reproducible local tumor growth but is limited by variable growth kinetics and the low rate of lymph node and hepatic metastases. Cytokeratin-positive cells indicative of MRD could be detected in the bone marrow of approximately 25% of the nude mice with metastases. The observed induction of MRD after orthotopic implantation of intact human colon cancer in animals with lymph node and hepatic metastases might be improved if established colon cancer cell lines were used.

Disseminated tumor cells in the blood of patients with gastric cancer are an independent predictive marker of poor prognosis.

OBJECTIVE: Gastric cancer carries a poor prognosis even after curative resection (R0). Tumor progression in gastric cancer patients has been attributed to the persistence of disseminated tumor cells (DTC) in various body compartments as a sign of minimal residual disease, although the prognostic relevance of DTC is still unclear. In this study the prognostic relevance of DTC in the blood of gastric cancer patients was investigated. MATERIALS AND METHODS: Venous blood samples of 70 cancer patients were taken intraoperatively before surgical manipulation and examined by reverse transcription-polymerase chain reaction (RT-PCR) for expression of cytokeratin 20 (CK20) as a marker for DTC. Tumor-related survival was analyzed using univariate and multivariate models assessing occurrence of DTC, residual tumor classification, and tumor stage. Median follow-up was 20 months (range 1-57 months). RESULTS: Twenty-eight of the 70 patients (40%) were CK20 positive. The prevalence of DTC in patients following R0 resection (15/41, 37%) was similar to that in patients with residual tumor (13/29, 45%, NS). Furthermore, expression of CK20 was independent of TNM stage. Univariate analysis of R0-resected patients revealed CK20 to be a marker for significantly shorter tumor-related survival (p = 0.0363). In a multivariate analysis, CK20 was an independent prognostic marker. Detection of CK20 had greatest impact for early tumor stages (T1 and T2, N0; p < 0.0032). CONCLUSIONS: Detection of DTC in venous blood of gastric cancer patients is an independent predictive marker of poor prognosis and thus could help to define patients for adjuvant therapy with this tumor entity.

Human antibody SC-1 reduces disseminated tumor cells in nude mice with human gastric cancer.

Advanced gastric cancer is a systemic disease that requires adjuvant therapy targeted at eliminating disseminated tumor cells (DTCs). We investigated whether the apoptosis-inducing human monoclonal IgM antibody SC-1 was able to reduce the number of disseminated gastric cancer cells in blood and bone marrow. Human gastric tumor specimens with positive expression of the SC-1 receptor were transplanted in nude mice with metastasizing gastric cancer. After tumor growth (4-6 weeks) animals were randomly allocated to intraperitoneal 100 microg SC-1 (n=23) or 100 microg human IgM (n=23). One week later, animals were sacrificed and blood and bone marrow specimens were obtained. A nested RT-PCR for cytokeratin 20 (CK-20) from blood and bone marrow of mice was performed for detection of disseminated tumor cells. Animals receiving SC-1 had significantly fewer DTCs than did control animals (p=0.0011). None of the SC-1 mice had DTCs simultaneously in both blood and bone marrow versus four of the control animals (p=0.0363). The reduction of DTCs in SC-1 animals was due to reduction in bone marrow (p=0.032 compared to controls), but not in blood (p=0.1158). Treatment with SC-1 significantly reduced the number of DTCs in bone marrow in this animal model.

Tumor induction by activated JNK occurs through deregulation of cellular growth.

Activation of the cytoplasmic (Ras-Raf-MEK-ERK) signaling cascade was shown to be both, necessary and sufficient for transformation in vitro as well as in vivo. However, over the last years the involvement of stress-activated protein kinases (SAPKs)/Jun N-terminal kinases (JNKs), and their substrate c-Jun in the process of cellular transformation has been suggested. To dissect the mechanisms through which JNK signaling contributes to the transformation process we employed a recently generated constitutively active version of this kinase, SAPKbeta-MKK7, which behaves like a weakly transforming oncogene in vitro. Dissection of the transforming potential of oncogenic JNK demonstrates that it is sufficient for tumor induction in nude mice. In vitro studies and analysis of tumor material support the conclusion that oncogenic JNK primarily transforms through its effects on cell proliferation and tumor vascularization but does not affect cell survival.

Expression profiling and genetic alterations of the selenoproteins GI-GPx and SePP in colorectal carcinogenesis.

The trace element selenium is discussed as a chemopreventive agent in colorectal carcinogenesis. Selenocysteine-containing proteins, so-called selenoproteins, represent potential molecular targets for nutritive selenium supplementation. Due to their antioxidative potential, the selenoproteins gastrointestinal glutathione peroxidase (GI-GPx) and selenoprotein P (SePP) are considered to provide protection against reactive oxygen species (ROS), thereby reducing DNA damage and preventing development of colon cancer. GI-GPx and SePP are abundantly expressed in normal colon mucosa. Recently, we demonstrated both reduced SePP expression and increased GI-GPx expression in colorectal adenomas. In this study, we investigated the expression of SePP and GI-GPx in colorectal cancers compared with corresponding normal mucosa. Further, the occurrence of genetic alterations within the SePP and GI-GPx genes was analyzed. We observed a significant reduction or loss of SePP mRNA expression in colon cancers, whereas GI-GPx mRNA and protein expression varied between different tumor samples. In addition, we identified novel polymorphisms within the SePP and GI-GPx genes with so far unknown relevance for protein function. Our results argue against a general decrease of selenoprotein expression in colorectal carcinogenesis but imply specific differential regulation of expression of individual selenoproteins.

Detection of disseminated tumor cells in nude mice with human gastric cancer.

Disseminated tumor cells (DTC) are a potential contributor to relapse of cancer. In the present study we developed a model for induction of disseminated tumor cells in nude mice, which can aid in the search for therapeutic approaches as well as improve our understanding of metastasizing gastric cancer. To detect DTC in blood and bone marrow we established a modified animal model of orthotopic transplantation. Two groups of nude mice were used for xenotransplantation of gastric cancer specimens. In group I tumor specimens originating from a gastric adenocarcinoma cell line were transplanted onto the stomach; in group II they were transplanted subcutaneously into both axillaries. Tumor growth, metastases and presence of DTC were compared in both groups. For detection of DTC a nested reverse transcriptase polymerase chain reaction (RT-PCR) for human cytokeratin (CK)-20 was performed on blood and bone marrow of all mice. Tumor growth occurred in both groups (9/10 animals in group I, 10/10 in group II) within 14 weeks. Only animals in group I developed local invasive tumor growth, stenosis of the stomach and distant metastases. Tumors in animals of group II grew with local displacement only and developed no metastases. There were no signals of CK-20 detected in the blood in both groups. In group I, 5 of 9 animals had positive signals of human CK-20 in their bone marrow as a sign of DTC. In group II no DTC were detected in bone marrow. We conclude that orthotopic transplantation is a prerequisite for the development of DTC and metastasizing tumor growth in this modified gastric cancer model.

Short-term immunosuppression after rat parathyroid allotransplantation.

The aim of this study was to evaluate whether short-term postoperative immunosuppression is able to sufficiently prolong graft survival after experimental allogeneic parathyroid transplantation. Heterotopic parathyroid transplantation was performed in 6 groups: 1) syngeneic control Lewis (LEW) to LEW; 2) allogeneic control Wistar-Furth (WF) to LEW; 3-5) WF to LEW plus short-term immunosuppression, postoperative days 1-13 (cyclosporine 5/10/20 mg/kg); and 6) WF to LEW plus 10 mg/kg CyA from preoperative day 7 to postoperative day 7. Graft function was examined up to 60 days; histological and immunohistological examination was performed on all grafts with impaired function. Graft function after syngeneic transplantation was indefinite, while recipients of allogeneic grafts turned hypocalcemic after 13 +/- 2 days. With immunosuppression, graft function was 21 +/- 2 days (groups 5 and 6) and 28 +/- 3 days (groups 3 and 4). Histologically, a cellular infiltrate responsible for graft destruction was found. The results show that indefinite parathyroid allograft survival cannot be achieved by short-term immunosuppression alone. Whether the combination of an additional graft pretreatment and immunosuppression has an impact on graft function will be further examined.

Optimization of a metastasizing human gastric cancer model in nude mice.

Our purpose was to optimize the surgical orthotopic implantation (SOI) technique to create a reproducible gastric cancer model in nude mice with stable tumor growth and metastasizing course. We performed xenotransplantation of primary human tumor specimens from patients with gastric cancer (series 1) and orthotopic transplantation of tumor specimens originating from the gastric cancer cell line 23132/87 (series 2). All specimens were transplanted using microsurgical techniques. The two series were compared with regard to tumor growth rates and kinetics, development of metastases, and induction of minimal residual disease (MRD), as determined by histology and PCR techniques. In series 1 mice, the tumor growth rate was slow; in series 2 mice, it was both fast and reproducible. Unlike animals in series 1, animals in series 2 developed metastases and MRD. In conclusion, the optimized SOI technique presented here represents a reproducible and reliably metastasizing gastric cancer model.

Ultrastructural alterations of primary human liver sinusoidal cells in patients treated for peritonitis.

Acute peritonitis is still associated with a high mortality rate. Bacterial toxins are rapidly cleared from the peritoneal cavity and may induce general sepsis. The hepatic sinusoidal cells are part of the primary defence against these toxins. The object of this study was to examine ultrastructural alterations of human sinusoidal liver cells from patients undergoing surgical treatment for peritonitis. Liver specimens, obtained from five patients treated with programmed interval peritoneal lavages for peritonitis, were analyzed by electron microscopy. Despite interindividual differences in etiology of peritonitis, the detected ultrastructural alterations displayed a high degree of similarity. Kupffer cells displayed enhanced phagocytotic activity. Numerous Kupffer cell-lymphocyte contacts were observed. Notably, the morphological appearance of the endothelial cells resembled that of an activated phagocytotic cell. The ultrastructural alterations peaked on day 7, and regressed during the course of treatment. Our findings demonstrate that major changes occur in the ultrastructural appearance of both Kupffer cells and sinusoidal endothelial cells in patients with acute peritonitis treated successfully with programmed interval peritoneal lavages. Our data suggest that in peritonitis, a septic spread of toxins and antigens may be modulated by sinusoidal liver cells.

Constitutive JNK activation in NIH 3T3 fibroblasts induces a partially transformed phenotype.

The c-Jun N-terminal kinases (JNKs) (also known as stress-activated protein kinases or SAPKs), members of the mitogen-activated protein kinase (MAPK) family, regulate gene expression in response to a variety of physiological and unphysiological stimuli. Gene knockout experiments and the use of dominant interfering mutants have pointed to a role for JNKs in the processes of cell differentiation and survival as well as oncogenic transformation. Direct analysis of the transforming potential of JNKs has been hampered so far by the lack of constitutively active forms of these kinases. Recently, such mutants have become available by fusion of the MAPK with its direct upstream activator kinase. We have generated a constitutively active SAPK beta-MKK7 hybrid protein and, using this constitutively active kinase, we are able to demonstrate the transforming potential of activated JNK, which is weaker than that of classical oncogenes such as Ras or Raf. The inducible expression of SAPK beta-MKK7 caused morphological transformation of NIH 3T3 fibroblasts. Additionally, these cells formed small foci of transformed cells and grew anchorage-independent in soft agar. Furthermore, similar to oncogenic Ras and Raf, the expression of activated SAPK beta resulted in the disassembly of F-actin stress fibers. Our data suggest that constitutive JNK activation elicits major aspects of cellular transformation but is unable to induce the complete set of changes which are required to establish the fully transformed phenotype.

Human monoclonal IgM antibodies with apoptotic activity isolated from cancer patients.

Monoclonal antibodies are accepted as ideal adjuvant therapeutic reagents for all kinds of diseases. Polyvalent (cross-linking) and low-mutated IgM antibodies (less immunogenic) are believed to be the most effective weapons against cancer. The best sources for these types of antibodies are the cancer patients themselves. Using conventional hybridoma technology, not only are fully human monoclonal IgM antibodies isolated, but also new tumor-related targets can be identified using the same experimental approach. The resulting antibodies can be used directly for therapeutic purposes without further modulation and manipulation. This report describes five newly established human monoclonal IgM antibodies; antibody LM-1 that was isolated from a patient with lung cancer, antibodies PM-1 und PM-2 that were isolated from a patient with pancreatic cancer, and antibodies CM-1 and CM-2 which were isolated from a patient with colon carcinoma. The mainly germ-line encoded antibodies are specific for malignant tissues and show only restricted reactivity with healthy cells. When tested for in vitro functional activity, all five antibodies inhibit tumor cell proliferation of carcinoma cells by inducing apoptosis.