Tumor Cell Attachment Research Articles

GRP-induced up-regulation of Hsp72 promotes CD16+/94+ natural killer cell binding to colon cancer cells causing tumor cell cytolysis

Gastrin-releasing peptide (GRP) and its receptor (GRPR) are not normally expressed by epithelial cells lining the adult human colon. However post malignant transformation both GRP and its receptor are aberrantly expressed in the colon where we have previously shown they act to retard metastasis by enhancing tumor cell attachment to the extracellular matrix. In the present study, we show that GRP signaling via its cognate receptor when both are aberrantly expressed in human colon cancer cells causes heat shock protein 72 (Hsp72) to be expressed. We show that GRP/GRPR induces expression of Hsp72 by signaling via focal adhesion kinase. When expressed, Hsp72 promotes the binding of CD16+ and CD94+ natural killer cells, resulting in tumor cell cytolysis. These findings demonstrate the presence of a novel mechanism whereby aberrantly expressed GRP/GRPR in human colorectal cancer attenuates tumor progression and may promote a favorable outcome.

Stepping out of the flow: capillary extravasation in cancer metastasis

In order for cancer cells to successfully colonize a metastatic site, they must detach from the primary tumor using extracellular matrix-degrading proteases, intravasate and survive in the circulation, evade the immune response, and extravasate the vasculature to invade the target tissue parenchyma, where metastatic foci are established. Though many of the steps of metastasis are widely studied, the precise cellular interactions and molecular alterations associated with extravasation are unknown, and further study is needed to elucidate the mechanisms inherent to this process. Studies of leukocytes localized to inflamed tissue during the immune response may be used to elucidate the process of cancer extravasation, since leukocyte diapedesis through the vasculature involves critical adhesive interactions with endothelial cells, and both leukocytes and cancer cells express similar surface receptors capable of binding endothelial adhesion molecules. Thus, leukocyte extravasation during the inflammatory response has provided a model for transendothelial migration (TEM) of cancer cells. Leukocyte extravasation is characterized by a process whereby rolling mediated by cytokine-activated endothelial selectins is followed by firmer adhesions with beta1 and beta2 integrin subunits to an activated endothelium and subsequent diapedesis, which most likely involves activation of Rho GTPases, regulators of cytoskeletal rearrangements and motility. It is controversial whether such selectin-mediated rolling is necessary for TEM of cancer cells. However, it has been established that similar stable adhesions between tumor and endothelial cells precede cancer cell transmigration through the endothelium. Additionally, there is support for the preferential attachment of tumor cells to the endothelium and, accordingly, site-specific metastasis of cancer cells. Rho GTPases are critical to TEM of cancer cells as well, and some progress has been made in understanding the specific roles of the Rho GTPase family, though much is still unknown. As the mechanisms of cancer TEM are elucidated, new approaches to study and target metastasis may be utilized and developed.

Binding of endostatin to human ovarian cancer cells inhibits cell attachment

Endostatin, a C-terminal fragment of collagen type XVIII, is one of the well-characterized endogenous inhibitors of angiogenesis. Endostatin is known to bind integrin alpha(5)beta(1), which is upregulated on tumor endothelium. Most of the ovarian cancer cells express significant amounts of alpha(5)beta(1) integrin, which is important for ovarian cancer cells to attach to the peritoneal wall. Therefore we investigated whether endostatin could directly bind ovarian cancer cells and inhibit tumor cell attachment to extracellular matrix. Binding of endostatin to ovarian cancer cells was characterized by preincubation with function blocking antibodies to integrin subunits. These studies showed that ovarian cancer cell attachment to fibronectin-coated wells can be inhibited by alpha(5)beta(1) integrin specific antibodies as well as endostatin. Downregulation of integrin alpha(5) and beta(1) by siRNA abrogated the binding of OVCAR5 and human umbilical vein endothelial cell to endostatin. Although endostatin treatment did not affect ovarian cancer cell migration, treated cells failed to attach mouse peritoneal wall preparations. These studies suggest an extra-antiangiogenic role for endostatin, which can be used prevent peritoneal attachment and dissemination of ovarian cancer cells.

Reduction of peritoneal carcinomatosis by intraperitoneal administration of phospholipids in rats

BackgroundIntraperitoneal tumor cell attachment after resection of gastrointestinal cancer may lead to a developing of peritoneal carcinosis. Intraabdominal application of phospholipids shows a significant decrease of adhesion formation even in case of rising tumor cell concentration.MethodsIn experiment A 2*106 colonic tumor cells (DHD/K12/Trb) were injected intraperitonely in female BD-IX-rats. A total of 30 rats were divided into three groups with treatments of phospholipids at 6% or 9% and the control group. In experiment B a total of 100 rats were divided into ten groups with treatments of phospholipids at 9% and the control group. A rising concentration of tumor cells (10,000, 50,000, 100,000, 250,000 and 500,000) were injected intraperitonely in female BD-IX-rats of the different groups. After 30 days, the extent of peritoneal carcinosis was determined by measuring the tumor volume, the area of attachment and the Peritoneal Cancer Index (PCI).ResultsIn experiment A, we found a significant reduction (control group: tumor volume: 12.0 ± 4.9 ml; area of tumor adhesion: 2434.4 ± 766 mm2; PCI 28.5 ± 10.0) of peritoneal dissemination according to all evaluation methods after treatment with phospholipids 6% (tumor volume: 5.2 ± 2.2 ml; area of tumor adhesion: 1106.8 ± 689 mm2; PCI 19.0 ± 5.0) and phospholipids 9% (tumor volume: 4.0 ± 3.5 ml; area of tumor adhesion: 362.7 ± 339 mm2; PCI 13.8 ± 5.1). In experiment B we found a significant reduction of tumor volume in all different groups of rising tumor cell concentration compared to the control. As detected by the area of attachment we found a significant reduction in the subgroups 1*104, 25*104 and 50*104. The reduction in the other subgroups shows no significance. The PCI could be reduced significantly in all subgroups apart from 5*104.ConclusionIn this animal study intraperitoneal application of phospholipids resulted in reduction of the extent of peritoneal carcinomatosis after intraperitoneal administration of free tumor cells. This effect was exceptionally noticed when the amount of intraperitoneal tumor cells was limited. Consequently, intraperitoneal administration of phospholipids might be effective in reducing peritoneal carcinomatosis after surgery of gastrointestinal tumors in humans.

Arsenic trioxide (As 2O 3) reduces the invasive and metastatic properties of cervical cancer cells in vitro and in vivo

Objectives Arsenic trioxide (As 2O 3) was found to induce apoptosis in certain types of cancer cells including acute promyelocytic leukemia, and recently in solid tumors. We have previously demonstrated that As 2O 3 has a therapeutic effect on cervical cancer by apoptosis promotion in vitro and in vivo. Here we further our study on the role of arsenic trioxide in regulating invasive activity of cervical cancer cells in vitro and in vivo. Methods The effects of As 2O 3 on human cervical cancer cell lines (HeLa, SiHa, Caski) adhesion, migration and invasion were observed by means of cell adhesion test, cell migration test and cell invasion test. The effects of As 2O 3 on p-IκB, MMP-2, E-cadherin, caveolin-1 and β-catenin protein expressions of tumor cells were determined by Western blot. In addition, the effects of As 2O 3 on NF-κB activity of tumor cells were analyzed by immunoblot in whole lysates, cytosol and nucleus, respectively. In animal experiments, cervical cancer cells TC-1 were injected into tail veins of C57BL/6 mice and then the mice were treated by intraperitoneal injection of different doses As 2O 3. Lung weights and the foci on the surface of lungs were measured. Results As 2O 3 inhibited attachment of tumor cells to Fibronectin and Matrigel, reduced cell motility and inhibited tumor invasion potential. As 2O 3 treatment also resulted in a positive regulation of caveolin-1, upregulation of E-cadherin and decreased activity of β-catenin, NF-κB and NF-κB-regulated gene MMP-2. In animal experiments, lung weights in PBS group (0.31 ± 0.07 g) were significantly elevated compared with those in As 2O 3-treated groups (0.21 ± 0.03 g and 0.17 ± 0.03 g) also As 2O 3 reduced number of metastatic lesions of lungs (15.4 ± 3.5 vs. 8.3 ± 2.0 and 6.3 ± 2.3) in a dose-dependent manner. Conclusions This study is the first to report the effectiveness of As 2O 3 as an inhibitor of cervical cancer invasion both in vitro and in vivo, suggesting a potential clinical application of As 2O 3 in cervical cancer therapies combining apoptosis induction and metastasis inhibition.

The Host Inflammatory Response Promotes Liver Metastasis by Increasing Tumor Cell Arrest and Extravasation

Inflammation can play a regulatory role in cancer progression and metastasis. Previously, we have shown that metastatic tumor cells entering the liver trigger a proinflammatory response involving Kupffer cell-mediated release of tumor necrosis factor-alpha and the up-regulation of vascular endothelial cell adhesion receptors, such as E-selectin. Here, we analyzed spatio-temporal aspects of the ensuing tumor-endothelial cell interaction using human colorectal carcinoma CX-1 and murine carcinoma H-59 cells and a combination of immunohistochemistry, confocal microscopy, and three-dimensional reconstruction. E-selectin expression was evident mainly on sinusoidal vessels by 6 and 10 hours, respectively, following H-59 and CX-1 inoculation, and this corresponded to a stabilization of the number of tumor cells within the sinuses. Tumor cells arrested in E-selectin(+) vessels and appeared to flatten and traverse the vessel lining, away from sites of intense E-selectin staining. This process was evident by 8 (H-59) and 12 (CX-1) hours after inoculation, coincided with increased endothelial vascular cell adhesion molecule-1 expression, and involved tumor cell attachment in areas of intense vascular cell adhesion molecule-1 and platelet endothelial cell adhesion molecule-1 expression. Nonmetastatic (human) MIP-101 and (murine) M-27 cells induced a weaker response and could not be seen to extravasate. The results show that metastatic tumor cells can alter the hepatic microvasculature and use newly expressed endothelial cell receptors to arrest and extravasate.

Carcinoembryonic antigen-stimulated THP-1 macrophages activate endothelial cells and increase cell–cell adhesion of colorectal cancer cells

The liver is the most common site for metastasis by colorectal cancer, and numerous studies have shown a relationship between serum carcinoembryonic antigen (CEA) levels and metastasis to this site. CEA activates hepatic macrophages or Kupffer cells via binding to the CEA receptor (CEA-R), which results in the production of cytokines and the up-regulation of endothelial adhesion molecules, both of which are implicated in hepatic metastasis. Since tissue macrophages implicated in the metastatic process can often be difficult to isolate, the aim of this study was to develop an in vitro model system to study the complex mechanisms of CEA-induced macrophage activation and metastasis. Undifferentiated, human monocytic THP-1 (U-THP) cells were differentiated (D-THP) to macrophages by exposure to 200 ng/ml phorbol myristate acetate (PMA) for 18 h. Immunohistochemistry showed two CEA-R isoforms present in both U- and D-THP cells. The receptors were localized primarily to the nucleus in U-THP cells, while a significant cell-surface presence was observed following PMA-differentiation. Incubation of D-THP-1 cells with CEA resulted in a significant increase in tumor necrosis factor-alpha (TNF-alpha) release over 24 h compared to untreated D-THP-1 or U-THP controls confirming the functionality of these cell surface receptors. U-THP cells were unresponsive to CEA. Attachment of HT-29 cells to human umbilical vein endothelial cells significantly increased at 1 h after incubation with both recombinant TNF-alpha and conditioned media from CEA stimulated D-THP cells by six and eightfold, respectively. This study establishes an in vitro system utilizing a human macrophage cell line expressing functional CEA-Rs to study activation and signaling mechanisms of CEA that facilitate tumor cell attachment to activated endothelial cells. Utilization of this in vitro system may lead to a more complete understanding of the expression and function of CEA-R and facilitate the design of anti-CEA-R therapeutic modalities that may significantly diminish the metastatic potential of CEA overexpressing colorectal tumors.

Formation of Hyaluronan- and Versican-rich Pericellular Matrix by Prostate Cancer Cells Promotes Cell Motility

Previous studies have demonstrated that high levels of hyaluronan (HA) and the chondroitin sulfate proteoglycan, versican in the peritumoral stroma are associated with metastatic spread of clinical prostate cancer. In vitro integration of HA and versican into a pericellular sheath is a prerequisite for proliferation and migration of vascular smooth muscle cells. In this study, a particle exclusion assay was used to determine whether human prostate cancer cell lines are capable of assembling a pericellular sheath following treatment with versican-containing medium and whether formation of a pericellular sheath modulated cell motility. PC3 and DU145, but not LNCaP cells formed prominent polarized pericellular sheaths following treatment with prostate fibroblast-conditioned medium. The capacity to assemble a pericellular sheath correlated with the ability to express membranous HA receptor, CD44. HA and versican histochemical staining were observed surrounding PC3 and DU145 cells following treatment with prostatic fibroblast-conditioned medium. The dependence on HA for integrity of the pericellular sheath was demonstrated by its removal following treatment with hyaluronidase. Purified versican or conditioned medium from Chinese hamster ovary K1 cells overexpressing versican V1, but not conditioned medium from parental cells, promoted pericellular sheath formation and motility of PC3 cells. Using time lapse microscopy, motile PC3 cells treated with versican but not non-motile cells exhibited a polar pericellular sheath. Polar pericellular sheath was particularly evident at the trailing edge but was excluded from the leading edge of PC3 cells. These studies indicate that prostate cancer cells recruit stromal components to remodel their pericellular environment and promote their motility.

Tenascin-C Stimulates Glioma Cell Invasion through Matrix Metalloproteinase-12

The capacity of glioma cells to invade extensively within the central nervous system is a major cause of the high morbidity rate of primary malignant brain tumors. Glioma cell invasion involves the attachment of tumor cells to extracellular matrix (ECM), degradation of ECM components, and subsequent penetration into adjacent brain structures. These processes are accomplished in part by matrix metalloproteinases (MMP) within a three-dimensional milieu of the brain parenchyma. As the majority of studies have used a two-dimensional monolayer culture system, we have used a three-dimensional matrix of collagen type I gel to address glioma-secreted proteases, ECM, and invasiveness of glioma cells. We show that in a three-dimensional collagen type I matrix, the presence of tenascin-C, commonly elevated in high-grade gliomas, increased the invasiveness of glioma cells. The tenascin-C-mediated invasiveness was blocked by metalloproteinase inhibitors, but this did not involve the gelatinases (MMP-2 and MMP-9) commonly implicated in two-dimensional glioma growth. A thorough analysis of 21 MMPs and six members of a disintegrin and metalloproteinase domain showed that MMP-12 was increased in gliomas by tenascin-C in three-dimensional matrix. Furthermore, examinations of resected specimens revealed high MMP-12 levels in the high-grade glioblastoma multiforme tumors. Finally, a function-blocking antibody as well as small interfering RNA to MMP-12 attenuated the tenascin-C-stimulated glioma invasion. These results identify a new factor, MMP-12, in regulating glioma invasiveness through interaction with tenascin-C.

Gastrin-releasing peptide mediates its morphogenic properties in human colon cancer by upregulating intracellular adhesion protein-1 (ICAM-1) via focal adhesion kinase

Gastrin-releasing peptide (GRP) and its receptor (GRPR) act as morphogens when expressed in colorectal cancer (CRC), promoting the assumption of a better differentiated phenotype by regulating cell motility in the context of remodeling and retarding tumor cell metastasis by enhancing cell-matrix attachment. Although we have shown that these processes are mediated by focal adhesion kinase (FAK), the downstream target(s) of GRP-induced FAK activation are not known. Since osteoblast differentiation is mediated by FAK-initiated upregulation of ICAM-1 (Nakayamada S, Okada Y, Saito K, Tamura M, Tanaka Y. J Biol Chem 278: 45368-45374, 2003), we determined whether GRP-induced activation of FAK alters ICAM-1 expression in CRC and, if so, determined the contribution of ICAM-1 to mediating GRP's morphogenic properties. Caco-2 and HT-29 cells variably express GRP/GRPR. These cells only express ICAM-1 when GRPR are present. In human CRC, GRPR and ICAM-1 are only expressed by better differentiated tumor cells, with ICAM-1 located at the basolateral membrane. ICAM-1 expression was only observed subsequent to GRPR signaling via FAK. To study the effect of ICAM-1 expression on tumor cell motility, CRC cells expressing GRP, GRPR, and ICAM-1 were cultured in the presence and absence of GRPR antagonist or monoclonal antibody to ICAM-1. CRC cells engaged in directed motility in the context of remodeling and were highly adherent to the extracellular matrix, only in the absence of antagonist or ICAM-1 antibody. These data indicate that GRP upregulation of ICAM-1 via FAK promotes tumor cell motility and attachment to the extracellular matrix.

Arsenic trioxide (As 2O 3) inhibits peritoneal invasion of ovarian carcinoma cells in vitro and in vivo

Objectives. To study the role of arsenic trioxide (As 2O 3) in regulating peritoneal invasive activity of ovarian carcinoma cells in vitro and in vivo. Methods. The effects of As 2O 3 on human ovarian cancer cell lines (3AO, SW626 and HO-8910PM) migration, invasion and adhesion with tumor cells and human peritoneal mesothelial cells (HPMC) were observed by means of cell migration test, cell invasion test and cell adhesion test. The effects of As 2O 3 on MMP-2, MMP-9, TIMP-1 and TIMP-2 gene expressions and protein expressions of tumor cells were determined by RT-PCR and ELISA, respectively. In animal experiments, ovarian tumor cells were implanted into abdominal cavity of nude mice and then the nude mice were treated by intraperitoneal injection of different doses As 2O 3. The foci on the surface of peritoneum were counted. Results. As 2O 3 inhibited tumor cells migration, invasion and adhesion with HPMC in a dose-dependent manner, while the same treatment enhanced tumor cell–tumor cell interactions. As 2O 3 inhibited mRNA and protein expressions of MMP-2, MMP-9 and TIMP-2 of tumor cells. In contrast, As 2O 3 increased mRNA and protein expressions of TIMP-1. As 2O 3 could reduce tumor cells peritoneal metastasis in nude mice. Conclusion. As 2O 3 inhibits in vitro and in vivo peritoneal invasive activity of ovarian carcinoma cells in a dose-dependent manner. Its anti-invasive activity may be the results of reduced cell motility, inhibited attachment of tumor cells to HPMC and enhanced tumor cell–tumor cell interaction, as well as down-regulation of MMP-2 and MMP-9 levels and up-regulation of TIMP-1 level.

Melanoma cell extravasation under flow conditions is modulated by leukocytes and endogenously produced interleukin 8.

Attachment of tumor cells to the endothelium (EC) under flow conditions is critical for the migration of tumor cells out of the vascular system to establish metastases. Innate immune system processes can potentially promote tumor progression through inflammation dependant mechanisms. White blood cells, neutrophils (PMN) in particular, are being studied to better understand how the host immune system affects cancer cell adhesion and subsequent migration and metastasis. Melanoma cell interaction with the EC is distinct from PMN-EC adhesion in the circulation. We found PMN increased melanoma cell extravasation, which involved initial PMN tethering on the EC, subsequent PMN capture of melanoma cells and maintaining close proximity to the EC. LFA-1 (CD11a/CD18 integrin) influenced the capture phase of PMN binding to both melanoma cells and the endothelium, while Mac-1 (CD11b/CD18 integrin) affected prolonged PMN-melanoma aggregation. Blocking E-selectin or ICAM-1 (intercellular adhesion molecule) on the endothelium or ICAM-1 on the melanoma surface reduced PMN-facilitated melanoma extravasation. Results indicated a novel finding that PMN-facilitated melanoma cell arrest on the EC could be modulated by endogenously produced interleukin-8 (IL-8). Functional blocking of the IL-8 receptors (CXCR1 and CXCR2) on PMN, or neutralizing soluble IL-8 in cell suspensions, significantly decreased the level of Mac-1 up-regulation on PMN while communicating with melanoma cells and reduced melanoma extravasation. These results provide new evidence for the complex role of hemodynamic forces, secreted chemokines, and PMN-melanoma adhesion in the recruitment of metastatic cancer cells to the endothelium in the microcirculation, which are significant in fostering new approaches to cancer treatment through anti-inflammatory therapeutics.

VEGF significance in peritoneal recurrence from gastric cancer

In gastric cancer, the management of peritoneal dissemination in the Peritoneal cavity is extremely important; however, peritoneal dissemination in the final stage of gastric cancer remains untreatable. Peritoneal dissemination involves several steps, including tumor-cell attachment, invasion, and growth in the peritoneum. Many cytokines, growth factors, matrix metalloproteinases (MMPs), and angiogenic factors play important roles in these steps. So far, few studies have investigated the correlation, if any, between peritoneal dissemination and the angiogenic factor, vascular endothelial growth factor (VEGF). Immunohistochemical staining, using the avidin-biotin peroxidase complex method, was performed on slides of surgical specimens from 40 patients with stage II gastric cancer with serosal invasion, who underwent surgery at our hospital between 1990 and 2000. Anti-human VEGF rabbit polyclonal IgG was used as the primary antibody. VEGF expression was classified in one of four categories depending on the percentage of tumor-cell staining (P). VEGF expression was also classified in one of three categories depending on the staining intensity (I). The VEGF expression score was calculated as P x I. There were ten patients with peritoneal recurrence. Of these, seven had macroscopic type-4 scirrhous-type gastric carcinoma. In the immunohistochemical study, the VEGF score of patients with peritoneal recurrence was 9.40 +/- 2.46; on the other hand, that of patients without peritoneal recurrence was 3.47 +/- 2.36. The VEGF score of patients with peritoneal recurrence was significantly higher than that of patients without peritoneal recurrence. In patients with macroscopic type 4, the VEGF score of those with peritoneal recurrence was 9.14 +/- 2.19, while on the other hand, that of the patients without peritoneal recurrence was 3.80 +/- 3.03. The VEGF score of these patients with peritoneal recurrence was significantly higher than that of those without peritoneal recurrence. The survival rate in the VEGF low-expression group was significantly higher than that in the VEGF high-expression group. Multivariate analysis showed that the VEGF score was a significant parameter of peritoneal recurrence. These results suggested that VEGF was correlated with peritoneal metastasis from gastric cancer, and that VEGF was a useful indicator of peritoneal recurrence.

Hierarchy of ADAM12 binding to integrins in tumor cells

ADAMs (a disintegrin and metalloprotease) comprise a family of cell surface proteins with protease and cell-binding activities. Using different forms and fragments of ADAM12 as substrates in cell adhesion and spreading assays, we demonstrated that alpha9beta1 integrin is the main receptor for ADAM12. However, when alpha9beta1 integrin is not expressed--as in many carcinoma cells--other members of the beta1 integrin family can replace its ligand binding activity. In attachment assays, the recombinant disintegrin domain of ADAM12 only supported alpha9 integrin-dependent tumor cell attachment, whereas full-length ADAM12 supported attachment via alpha9 integrin and other integrin receptors. Cells that attached to full-length ADAM12 in an alpha9 integrin-dependent manner also attached to ADAM12 in which the putative alpha9beta1 integrin-binding motif in the disintegrin domain had been mutated. This attachment was mediated through use of an alternate beta1 integrin. We also found that cell spreading in response to ADAM12 is dependent on the apparent level of integrin activation. Binding of cells to ADAM12 via the alpha9beta1 integrin was Mn(2+)-independent and resulted in attachment of cells with a rounded morphology; attachment of cells with a spread morphology required further activation of the alpha9beta1 integrin. We demonstrated that phosphoinositide-3-kinase appears to be central in regulating alpha9beta1 integrin cell spreading activity in response to ADAM12.

Endothelial adhesion molecules in breast cancer invasion into the vascular and lymphatic systems

Aims It is well recognised that intravasation of tumour cells into the vasculature and/or lymphatics is a key stage in the metastatic process. It is also clear that very little is known about the mechanisms underlying this event. In this review, we will focus on cell surface molecules that may be instrumental in mediating the attachment of tumour cells, and in particular breast carcinoma cells, to the lymphatic and microvascular endothelia and discuss the therapeutic and prognostic value in targeting these receptors in metastatic disease. Methods A literature search was carried out from PubMed for indexed articles and reviews. Websites containing information on gene expression profiles were located using standard web browser search functions. For articles containing gene expression data, relevant information was frequently located in supplementary tables or in associated websites. Findings The search yielded a very large number of indexed published articles and websites. Important major reports and studies were reviewed, screened and tracked for other relevant publications. The most important articles were analysed and discussed. Conclusions The lack of knowledge as to the mechanism by which tumour cells intravasate into the vasculature and/or lymphatics is perhaps not surprising given the lack of suitable models with which to investigate tumour cell intravasation. However, recent advances in the identification of molecular markers of angiogenic and lymphangiogenic endothelium, the development of techniques to image tumour cells in vivo and a better understanding of the architecture of these vessels is beginning to offer hope that this least well understood event in the metastatic process is becoming more amenable to study.

Novel in vitro perfusion model to study the interaction between coagulation and blood‐borne metastasis

The association between cancer and hemostasis has long been studied in cell culture, animal models, and cancer patients developing thrombosis. The variety of biologic mechanisms involved in malignancy and metastasis makes the understanding of the relative importance of each mechanism difficult. We have developed a novel in vitro perfusion model that allows for the isolated study of the interactions between tumor cells and components of the hemostatic system under normal physiologic conditions. Segments of denuded umbilical cord or saphenous vein are cut longitudinally and mounted in a perfusion chamber under sterile conditions. Human breast cancer cells are perfused for 24 h under venous flow conditions with either whole blood (WB), platelet-rich plasma (PRP), platelet-poor plasma (PPP), or serum. Tissue samples are fixed and stained with hematoxylin and eosin as well as with pan-cytokeratin. Morphometric analysis is performed to quantify cancer cell adhesion. With PRP, this model maintains normal human physiologic conditions for the duration of the experiment. It differentiates between previously characterized high and low metastatic breast cancer cell lines. In addition, different vein tissue types do not alter tumor cell attachment. This model appears to be an accurate representation of the pathophysiology of in vivo metastasis. This model may serve as a useful bridge between cell culture studies and animal models. It may be a useful tool to elucidate the role of selected hemostatic systems in blood-borne metastasis and may potentially serve as a screening tool for the development of antimetastatic pharmaceutical agents.

Micromechanics of tumor cell adhesion and migration under dynamic flow conditions

Attachment of tumor cells to endothelial cells is critical for migration of tumor cells out of the vascular system to establish metastases. We have studied human melanoma cell (C8161) adhesion and migration in response to stimulation by soluble collagen IV (CIV) using a modified Boyden chamber. In this modified chamber, shear flow can be introduced over the cell-substrate interface affecting tumor cell chemotactic migration through a microporous filter. Our results suggest that transmigration of C8161 cells under flow conditions can be influenced by PMNs, mediated by Mac-1 and ICAM-1 adhesive interactions and enhanced by altered cytokine production.

Phospholipids reduce the intraperitoneal adhesion of colonic tumor cells in rats and adhesion on extracellular matrix in vitro.

To evaluate the influence of intraperitoneal treatment with phospholipids on the formation of peritoneal carcinosis after inoculation of colonic tumor cells in rats. The presence of tumor cells in the peritoneal cavity serves as a prognostic marker for postoperative survival after resection of gastrointestinal cancer. Intraperitoneal tumor cell attachment is a pivotal step in developing peritoneal carcinosis. Intraabdominal application of phospholipids resulted in a significant decrease of adhesion formation, especially at sites of peritoneal lesions. 2x10(6) colonic tumor cells (DHD/K12/TRb) were injected intraperitonely in female BD-IX rats. A total of 90 rats were divided into three groups with treatments of phospholipids at 75 mg/kg or 150 mg/kg bodyweight or sodium chloride at 0.9% in the control group. The treatment groups were subdivided into animals with defined peritoneal lesions and animals without lesions. After 30 days, the extent of peritoneal carcinosis was determined by measuring the tumor volume, the area of tumor attachment and the Peritoneal Cancer Index. Over a 90-day observation period, the survival rate was analyzed. In vitro, we examined the reduction of tumor cell adhesion on extracellular matrix components after treatment with phospholipids. Microtiter plates were coated with laminin, fibronectin or collagen IV for adhesion experiments. In our study, we found a significant reduction of peritoneal dissemination with respect to all evaluation methods after treatment with phospholipids at 150 mg/kg in animals without peritoneal lesions. This could not be achieved using the lower concentration of phospholipids (75 mg/kg). In vitro, the maximum reductions of tumor cell adhesion by phospholipids compared with the control values for laminin and fibronectin were 46% and 37%, respectively, whereas for collagen IV the reduction was only 24% ( p<0.0001). A new method of prevention of intraperitoneal tumor cell adhesion, possibly leading to a reduced incidence of peritoneal carcinosis after surgery of gastrointestinal tumors, is introduced.

Effects of tumor-enhancing IgG 2 on macrophage function

Macrophages (Mϕ) play a significant role in allograft rejection. We investigated whether tumor-enhancing (te) IgG 2 acting as a cytophilic opsonin affects allograft destruction. Our results demonstrate that immune Tennessee Swiss (TS) (H-2 s) mouse Mϕ destroyed greater numbers of target C3H f/He (H-2 k) tumor cells than did nonimmune Mϕ. The percentage of 51Cr release from labeled tumor cells induced by immune Mϕ was 39.10 + 3.24% compared to nonimmune Mϕ 28.0 + 3.87%, while te IgG suppressed cytotoxicity toward C3H f/He tumor cells of normal TS Mϕ as manifested by less isotope release (19.60 + 3.13%) than that produced by normal TS Mϕ with non-enhancing IgG 2 (39.90 + 5.8%). Rather than facilitating survival of allogeneic fibrosarcoma cells, te IgG 2 alloantibody potentiated their destruction by immune TS Mϕ (isotope release of 52.90 + 3.46%) compared with that produced by immune Mϕ alone (39.10 + 3.24%). Cytophilic te IgG 2 on TS Mϕ was demonstrated with either C3H f/He red cells or tumor cells. Electron micrographs of Mϕ revealed tumor cell ingestion and attachment of ferritin-labeled IgG 2 alloantibody on Mϕ and target tumor cells. In contrast to protection, te IgG 2 alloantibody facilitates microphage-mediated allograft destruction.

Effects of tumor-enhancing IgG2 on macrophage function

Macrophages (Mϕ) play a significant role in allograft rejection. We investigated whether tumor-enhancing (te) IgG2 acting as a cytophilic opsonin affects allograft destruction. Our results demonstrate that immune Tennessee Swiss (TS) (H-2s) mouse Mϕ destroyed greater numbers of target C3Hf/He (H-2k) tumor cells than did nonimmune Mϕ. The percentage of 51Cr release from labeled tumor cells induced by immune Mϕ was 39.10 + 3.24% compared to nonimmune Mϕ 28.0 + 3.87%, while te IgG suppressed cytotoxicity toward C3Hf/He tumor cells of normal TS Mϕ as manifested by less isotope release (19.60 + 3.13%) than that produced by normal TS Mϕ with non-enhancing IgG2 (39.90 + 5.8%). Rather than facilitating survival of allogeneic fibrosarcoma cells, te IgG2 alloantibody potentiated their destruction by immune TS Mϕ (isotope release of 52.90 + 3.46%) compared with that produced by immune Mϕ alone (39.10 + 3.24%). Cytophilic te IgG2 on TS Mϕ was demonstrated with either C3Hf/He red cells or tumor cells. Electron micrographs of Mϕ revealed tumor cell ingestion and attachment of ferritin-labeled IgG2 alloantibody on Mϕ and target tumor cells. In contrast to protection, te IgG2 alloantibody facilitates microphage-mediated allograft destruction.