Protein kinase C delta involvement in mammary tumor cell metastasis.

Metastasis requires cytoskeletal remodeling for migration, adhesion, and extravasation of metastatic cells. Although protein kinase C (PKC) is involved in tumor promotion/progression and cytoskeletal remodeling, its role in metastasis has not been defined. PKCdelta levels are increased in highly metastatic 13762NF mammary tumor cells (MTLn3) compared with less metastatic, parental cell lines. To determine whether the increase in endogenous PKCdelta is functionally related to their increased metastatic potential, we prepared MTLn3 cells that express the inhibitory regulatory domain fragment of PKCdelta (RDdelta) under the control of a tetracycline-inducible promoter. RDdelta expression attenuated endogenous PKCdelta activity, as demonstrated by decreased phosphorylation of the PKCdelta substrate adducin in migrating cells. Thus, in MT cells, RDdelta appears to primarily influence cytoskeleton-dependent processes rather than cell cycle progression. To determine whether RDdelta expression influenced metastatic potential in vivo, MTLn3/RDdelta cells were either grown in the mammary fat pad or injected into the tail vein of syngeneic rats, and effects of doxycycline-induced RDdelta expression on pulmonary metastases were studied. Consistent with the in vitro data, induction of RDdelta significantly reduced the number of lung metastases without affecting growth of the primary tumor. These results suggest that interfering with endogenous PKCdelta activity by expressing the inhibitory RDdelta fragment inhibits cytoskeleton-regulated processes important for MTLn3 cell metastasis.     

Differential organ tissue adhesion,invasion, and growth properties of metastatic rat mammary adenocarcinoma cells

Metastatic lines and clones of the rat 13762NF mammary adenocarcinoma have been established that show reproducible spontaneous metastasis from the mammary fat pad to regional lymph node and lung. Poorly (MTC) and highly (MTLn3) metastatic cloned lines derived from tumor growing in the mammary fat pad (MTC) and its spontaneous lung metastasis (MTLn3) were tested in vitro for their abilities to attach to and invade into syngeneic organ tissue and to survive and grow in medium conditioned by target and nontarget syngeneic organ tissues. The highly metastatic MTLn3 cells adhered to and invaded target lung tissue at significantly higher rates than the MTC cells, and bound to and invaded other organ tissues although at lower rates than lung tissue. Similarly, the MTLn3 cells showed significantly higher growth stimulation by lung-conditioned medium than medium conditioned by other tissues. Poorly metastatic MTC cells were not significantly stimulated by any of the organ-conditioned media. The results are consistent with previous proposals that explain preferential organ metastasis in terms of 'seed and soil', and further suggest that metastasis of mammary tumors to specific organ secondary sites is mediated by specific properties, such as those involved in tumor-cell organ-cell adhesion, invasion, and growth.     

Epidermal growth factor receptor overexpression results in increased tumor cell motility in vivo coordinately with enhanced intravasation and metastasis

Although overexpression of the epidermal growth factor receptor (EGFR; ErbB1) has been correlated with poor prognosis in breast and other cancers, clinical trials of ErbB1 inhibitors have shown limited efficacy in inhibiting tumor proliferation. To evaluate other possible roles of ErbB1 in tumor malignancy besides proliferation, we have developed a series of tools for analysis of intravasation. Overexpression of ErbB1 in MTLn3 mammary adenocarcinoma cells results in increased intravasation and lung metastasis from tumors formed by injection of cells in the mammary fat pad. However, increased ErbB1 expression has no effect on primary tumor growth and lung seeding efficiency of cells injected i.v. Chemotactic responses to low concentrations of EGF in vitro and cell motility in vivo in the primary tumor measured using intravital imaging are significantly increased by ErbB1 overexpression. The increased cell motility is restricted to ErbB1-overexpressing cells in tumors containing mixtures of cells expressing different ErbB1 levels, arguing for a cell-autonomous effect of increased ErbB1 expression rather than alteration of the tumor microenvironment. In summary, we propose that ErbB1 overexpression makes more significant contributions to intravasation than growth in some tumors and present a novel model for studying ErbB1 contributions to tumor metastasis via chemotaxis and intravasation.     

Survival of rat mammary tumor cell clones and DNA strand damage following adriamycin treatment

Summary Tumor cell subpopulations have been shown to be heterogenous in a number of phenotypic characteristics, including responses to cytotoxic drugs. This phenotypic heterogeneity has been used here to study mechanisms associated with Adriamycin (doxorubicin HCl)-induced cytotoxicity. Clonogenic survival and alkaline elution methods were employed to examine the response of two tumor cell subpopulations to Adriamycin. The cells were derived from a primary 13762NF rat mammary adenocarcinoma (clone MTC) and a lung metastasis in the same animal (clone MTLn3). The MTC cells were significantly more resistant to Adriamycin than were the MTLn3 cells; the dose effective in reducing cell survival by 50% was 10-fold higher. Protein-associated DNA strand breakage assayed by alkaline elution was dose-dependent in both clones, and MTC cells were again more resistant to break induction than were MTLn3. These results showed that clonal tumor subpopulations isolated from a primary tumor and its metastases possessed different intrinsic survival responses to Adriamycin treatment in vitro and that this survival response correlated with Adriamycin-induced production of protein-associated DNA single-strand breaks.The investigation reported in this paper was supported in part by USPHS grants CA-32745 (SPT) and CA-23270 (REM) and NIH training grant CA-09299-06 (DPE)     

Expression of epidermal growth-factor receptor correlates with metastatic potential of 13762nf rat mammary adenocarcinoma cells

Increased expression of EGFR in metastases of human mammary carcinoma as compared to cells of the primary cancer suggests a contribution of EGFR to mammary carcinoma metastasis. To test for a positive correlation, we investigated 13762NF rat mammary adenocarcinoma cloned tumor cell lines of high (MTLn3) or low (MTC) metastatic potential. While MTC cells expressed barely detectable amounts of EGFR, MTLn3 cells expressed readily detectable levels of receptor. This was demonstrated in Northern blot analysis, in immunoprecipitation studies using metabolically labeled whole cell lysates and in Western blot analysis of membrane fractions. Cross-linking of radiolabeled ligand to intact cells identified on both cell types specific binding to a 170 kd protein, however, at much lower levels on low-metastatic MTC cells and not in sufficient amounts to estimate receptor numbers by Scatchard analysis. In contrast, Scatchard plot analysis of I-125-EGF binding to MTLn3 cells revealed the expression of about 10,000 high and 46,000 low affinity sites. Both cell lines expressed the ligand in comparable amounts as was demonstrated by using a specific rat TGFalpha cDNA probe in Northern blot and an antibody recognising membrane bound TGF in FACS analysis. Adhesion of MTC cells to immobilized collagen or fibronectin was rapid reaching 50% after 30 min while control MTLn3 cells demonstrated lower adhesion to collagen. Addition of 10 ng/ml EGF increased the rate and the maximal adhesion of MTLn3 cells to collagen G, while the adhesion kinetics of MTC cells to collagen G or fibronectin were unaffected.     

Tumor imaging with multicolor fluorescent protein expression

Imaging with fluorescent proteins has been revolutionary and has led to the new field of in vivo cell biology. Many new applications of this technology have been developed. Green fluorescent protein (GFP)-labeled or red fluorescent protein (RFP)-labeled HT-1080 human fibrosarcoma cells were used to determine clonality of metastasis by imaging of metastatic colonies after mixed implantation of the red and green fluorescent cells. Resulting pure red or pure green colonies were scored as clonal, whereas mixed yellow colonies were scored as nonclonal. Dual-color fluorescent cancer cells expressing GFP in the nucleus and RFP in the cytoplasm were engineered. The dual-color cancer cells enable real-time nuclear–cytoplasmic dynamics to be visualized in living cells in vivo, including mitosis and apoptosis. The nuclear and cytoplasmic behavior of dual-color cancer cells in real time in blood vessels was observed as they trafficked by various means or extravasated in an abdominal skin flap. Dual-color cancer cells were also visualized trafficking through lymphatic vessels where they were imaged via a skin flap. Seeding and arresting of single dual-color cancer cells in the lung, accumulation of cancer-cell emboli, cancer-cell viability, and metastatic colony formation were imaged in real time in an open-chest nude mouse model using assisted ventilation. Novel treatment was evaluated in these imageable models. UVC irradiation killed approximately 70% of the dual-color cancer cells in a nude mouse model. An RFP-expressing glioma was transplanted to the spinal cord of transgenic nude mice expressing nestin-driven green fluorescent protein (ND-GFP). In ND-GFP mice, GFP is expressed in nascent blood vessels and neural stem cells. ND-GFP cells staining positively for neuronal class III-β-tubulin or CD31 surrounded the tumor, suggesting that the tumor stimulated both neurogenesis and angiogenesis. The tumor caused paralysis and also metastasized to the brain. The Salmonella typhimurium A1-R tumor-targeting bacterial strain was administered in the orthotopic spinal cord glioma model. The treated animals had a significant increase in survival and decrease in paralysis. S. typhimurium A1-R was effective against primary bone tumor and lung metastasis expressing RFP in a nude mouse model. S. typhimurium A1-R was effective against both axillary lymph and popliteal lymph node metastases of human dual-color pancreatic cancer and fibrosarcoma cells, respectively, as well as lung metastasis of the fibrosarcoma in nude mice. Imaging with fluorescent proteins will reveal mechanisms of cancer progression and provide visual targets for novel therapeutics.     

Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling

We have developed animal models of breast cancer that allow the direct examination of the behavior of individual green fluorescent protein-expressing carcinoma cells in live nonmetastatic and metastatic primary tumors in situ. We have combined this model with multiphoton microscopy to image differences in cell behavior within the primary tumor. Differences in cell behavior between nonmetastatic and metastatic cells in culture and within live primary tumors were correlated with results from cDNA microarray analyses to identify potentially important genetic determinants for breast cancer invasion and metastasis. Using multiphoton microscopy, we found five major differences in carcinoma cell behavior between the nonmetastatic and metastatic primary breast tumors involving extracellular matrix, cell motility, and chemotaxis. Behavioral differences were correlated with seven categories of molecules that were differentially expressed and related to these behaviors. We have found that extracellular matrix composition, actin nucleation factors, molecules involved in mechanical stability and survival, and cell polarity and chemotaxis showed large and consistent differences in gene expression. We conclude that aligning cell behavior in vivo with patterns of gene expression can lead to new insights into the microenvironment of carcinoma cells in the primary tumor and the molecular mechanisms behind cell behavior.     

The role of polymorphonuclear leukocytes (PMN) on the growth and metastatic potential of 13762NF mammary adenocarcinoma cells

Circulating neutrophil (PMN) levels can increase in rats bearing subcutaneously growing clones of the 13762NF mammary adenocarcinoma and the level of increase correlates with the metastatic potential of the clone. In rats with poorly metastatic MTC tumors, numbers of circulating PMN did not rise, whereas PMN levels rose 50-fold in rats bearing highly metastatic MTLn3, 12-fold in rats with weakly metastatic MTLn2, and 14-fold in those with moderately metastatic MTF7 tumors. Neutrophilia was caused partly by tumor size, but metastatic potential was a stronger determinant, suggesting that PMNs may play a role in the metastatic process. To determine whether circulating PMNs indeed contribute to cellular metastatic potential, we examined effects of PMN on various aspects of the metastatic process. Experimental metastasis assays involving i.v. co-injections of PMNs yielded a dose-dependent increase in extrapulmonary metastases for MTLn3, but no change in lung colonization potential for any of the clones examined. The change in the metastatic profile was not due to any modification in in vivo distribution of i.v. injected tumor cells or in adhesion to endothelial monolayers in vitro. PMNs also had no effect on in vitro DNA, RNA or protein synthesis and were not cytolytic (E:T 100:1). However, PMNs collected from high-passage MTLn3 tumor-bearing rats had a 50% increase in heparanase and type-IV collagenolytic activity as compared to unstimulated PMNs isolated from normal rats. These results indicate that polymorphonuclear cells may contribute to the metastatic potential of highly metastatic clones from the 13762NF mammary adenocarcinoma cells by assisting in the degradation of basement membrane during extravasation.     

Preferential growth stimulation of metastatic rat mammary adenocarcinoma cells by organ-derived syngeneic fibroblasts in vitro.

To determine whether organ-derived fibroblasts differentially affect the growth of cells from tumors that preferentially metastasize to specific organs, we investigated the effect of medium conditioned with primary cultured rat fibroblasts from various organs on the in vitro growth of metastatic cell lines and clones of the rat 13762NF mammary adenocarcinoma. The conditioned medium from fibroblasts derived from rat mammary fat pad differentially stimulated tumor cell growth in monolayer culture and clonogenic growth in soft agarose of the highly metastatic clone MTLn3 in a dose-dependent manner. Conditioned medium from fibroblasts derived from the lung and liver also stimulated the growth of clone MTLn3 cells but to a lesser extent than did mammary fat pad fibroblasts. In contrast, poorly metastatic cell clones (MTC, MTPa) did not respond to the growth stimulatory factor(s) from the fibroblast-conditioned medium. The factor(s) responsible for the growth stimulation were inactivated by heat and trypsin treatment and inhibited by low pH and cycloheximide. The result suggest that fibroblasts in different organs have different effects on tumor cell growth, and they may determine, in part, the organ specificity of tumor development and metastasis.     

Decreased expression of nucleoside diphosphate kinase α isoform,an nm23-H2 gene homolog,is associated with metastatic potential of rat mammary-adenocarcinoma cells

The nm23 gene [encoding nucleoside diphosphate kinase (NDPK)] may act as a metastasis suppressor in certain tumor cells. We investigated the role of NDPK isoforms (α and β) in the metastatic processes, using rat mammary-adenocarcinoma cell lines of poor (MTC) and high (MTLn3) spontaneous metastatic potential respectively. In these cell lines, as in most rat tissues, the α isoform (nm23-H2 homolog) was more highly expressed than the β isoform (nm23-H1 homolog) at the mRNA and protein levels. When examined by Northern- and Western-blot analyses, expression of the 2 isoforms was reduced in highly metastatic MTLn3 cells compared with poorly metastatic MTC cells. The reduced expression was also associated with diminished NDPK-enzyme activity in the cell extracts. Southern-blot and RT-PCR-SSCP analyses suggested that the 2 genes were not grossly altered or mutated in their translation regions. MTLn3 cell clones transfected with NDPKα or NDPKβ cDNA were all tumorigenic when implanted into the mammary fat pad of syngeneic rats. Among those, only clones transfected with the NDPKα gene exhibited reduced lung metastasis in a spontaneous metastasis assay. © 1996 Wiley-Liss, Inc.     

Effect of prostaglandin E2-producing nonmetastatic Lewis lung carcinoma cells on the migration of prostaglandin E2-responsive metastatic Lewis lung carcinoma cells

The role of prostaglandin E2 (PGE2) in directly stimulating metastatic spread by Lewis lung carcinoma (LLC) cells was examined with the use of an in vitro migration model for tumor dissemination. The extent to which cloned metastatic and nonmetastatic LLC cells migrated out of glass capillary tubes in vitro reflected their capacity to form pulmonary metastases in vivo. The addition of PGE2 to metastatic LLC cells further stimulated their migration. Other cyclooxygenase products, besides PGE2, did not stimulate the migration of metastatic LLC cells. Nonmetastatic LLC cells did not migrate out of capillary tubes, even in the presence of exogenous PGE2. The amount of PGE2 secreted by cloned LLC cells was quantitated by a radioimmunoassay. Nonmetastatic LLC cells secreted more PGE2 than did the metastatic LLC cells. When the nonmetastatic LLC cells were either mixed with or placed adjacent to cloned metastatic LLC cells, the migration by the metastatic LLC cells was stimulated. The migration-stimulatory capacity of the nonmetastatic LLC cells was minimized in the presence of indomethacin, a prostaglandin synthesis inhibitor. Studies were conducted to relate these in vitro results to tumor metastasis in vivo. Injection of a mixture of metastatic and nonmetastatic LLC cells into mice s.c. resulted in a greater number of lung metastases than did injection of metastatic cells alone. This increase in metastasis formation was prevented by indomethacin. Formation of pulmonary metastases was also augmented when irradiated nonmetastatic LLC cells were injected into metastatic LLC-bearing mice. The results of our studies suggest that nonmetastatic LLC cells, by producing PGE2, can augment in vitro migration and in vivo dissemination of metastatic LLC cells. Thus, the response of tumor cells to PGE2, rather than simply their production of PGE2, appears to be important in regulating tumor dissemination.     

Functional in vivo optical imaging of tumor angiogenesis, growth, and metastasis prevented by administration of anti-human VEGF antibody in xenograft model of human fibrosarcoma HT1080 cells

Angiogenesis plays a crucial role in cancer progression and metastasis. Thus, blocking tumor angiogenesis is potentially a universal approach to prevent tumor establishment and metastasis. In this study, we used in vivo and ex vivo fluorescence imaging to show that an antihuman vascular endothelial growth factor (VEGF) antibody represses angiogenesis and the growth of primary tumors of human fibrosarcoma HT1080 cells in implanted nude mice. Interestingly, administering the antihuman VEGF antibody reduced the development of new blood vessels and normalized pre-existing tumor vasculature in HT1080 cell tumors. In addition, antihuman VEGF antibody treatment decreased lung metastasis from the primary tumor, whereas it failed to block lung metastasis in a lung colonization experiment in which tumor cells were injected into the tail vein. These results suggest that VEGF produced by primary HT1080 cell tumors has a crucial effect on lung metastasis. The present study indicates that the in vivo fluorescent microscopy system will be useful to investigate the biology of angiogenesis and test the effectiveness of angiogenesis inhibitors. ( Cancer Sci 2009)     

Morpho-functional study of vascular fluorochrome delivery to lung and liver metastases of Lewis lung carcinoma (3LL).

The growth of 3LL liver and lung metastases related to its vascular organization was studied by morphological and functional methods, using the Hoechst 33342 fluorescent DNA staining technique. Experimental liver and lung metastases were produced in syngeneic C57BL/6 mice by injection of 3LL tumor cells into a lateral tail vein or into the spleen, respectively. The resulting neoplasms were composed of large cells arranged in sheets with a thin irregularly distributed stroma. Scattered blood vessels with an open or closed lumen were observed within the tumor. Functional study of H33342 diffusion showed a single and reticular fluorescent pattern in liver metastases. In contrast, in lung metastases the fluorochrome diffusion revealed two different fluorescent patterns related to the location of the metastasis. Thus, parenchymal and subpleural metastasis presented a fluorescent pattern similar to that observed in the liver whereas metastases located around blood vessels and conducting airways never displayed fluorescence. In summary, our results suggest that the target metastatic organ and/or intra-organ location modulates the characteristics of metabolic exchange of the tumor cells in relation to the vascular organization of the metastatic focus.     

Correlation of beta-actin messenger RNA localization with metastatic potential in rat adenocarcinoma cell lines

The actin cytoskeleton is involved in the motility of tumor cells. It has been shown in several cell types that beta-actin mRNA is localized in the protrusions of cells in which actin is actively polymerized, and the ability to localize mRNA is correlated with the efficiency of motility. In this context, we studied the distribution of beta-actin mRNA in two different tumor cell lines and correlated it with their metastatic potential. The two cell lines used were the highly metastatic MTLn3 cells and nonmetastatic MTC cells. Nonmetastatic MTC cells have two different pools of beta-actin mRNA (perinuclear and at the leading edge), whereas highly metastatic MTLn3 cells have only a perinuclear distribution of beta-actin mRNA. These differences in mRNA localization are correlated with profound differences in the polarity and plasticity of cell motility of these cells in culture and the histopathology of primary breast tumors derived from these cells. In particular, MTLn3 cells are unpolarized by all cell shape and motility criteria in culture and in their histopathological organization in primary tumors. By comparison, MTC cells are polarized in all identical measurements. These results suggest that the increased plasticity of cell locomotion and the invasiveness of MTLn3 cells result from the failure of metastatic cells to localize beta-actin mRNA properly, causing them to be less polarized and therefore more flexible in their direction of motility. Thus, differences in the polarized organization of cells in the primary tumor that are correlated with beta-actin mRNA localization may have prognostic value in predicting metastatic potential.     

The role of the intravascular microenvironment in spontaneous metastasis development

Metastasis is primarily responsible for the morbidity and mortality of cancer. Improved therapeutic outcomes and prognosis depend on improved understanding of mechanisms regulating the establishment of early metastasis. In this study, use of green fluorescent protein (GFP)‐expressing PC‐3 orthotopic model of human prostate cancer and two complementary fluorescence in vivo imaging systems (Olympus OV100 and VisEn FMT) allowed for the first time real‐time characterization of cancer cell–endothelium interactions during spontaneous metastatic colonization of the liver and lung in live mice. We observed that prior to the detection of extra‐vascular metastases, GFP‐expressing PC‐3 cancer cells resided initially inside the blood vessels of the liver and the lung, where they proliferated and expressed Ki‐67 and exhibited matrix metalloprotenases (MMP) activity. Thus, the intravascular cancer cells produced their own microenvironment, where they could continue to proliferate. Extravasation occurred earlier in the lung than in the liver. Our results demonstrate that the intravascular microenvironment is a critical staging area for the development of metastasis that later can invade the parenchyma. Intravascular tumor cells may represent a therapeutic target to inhibit the development of extravascular metastases. Therefore, this imageable model of intravascular metastasis may be used for evaluation of novel anti‐metastatic agents.     

Candidate metastasis-associated genes of the rat 13762NF mammary adenocarcinoma

Summary Differential hybridization was used to isolate genes potentially involved in the process of metastasis. Ten complementary DNAs (cDNAs) that were differentially expressed between a highly metastatic (MTLn3) and a nonmetastatic (MTC.4) line of the rat 13762NF mammary adenocarcinoma were isolated and sequenced. Examination of the EMBL/GenBank database revealed that one of the genes had a high degree of homology (98.8%) to annexin I (also known as calpactin II). Quantitative analysis of Northern blot hybridizations showed that the annexin I-like sequence was expressed 4- to 7-fold higher in MTLn3 than in MTC.4 cells. Steady state mRNA levels were also low in MTLn2, a cell line of low metastatic potential closely related to MTLn3, but were not related to metastatic potential in colon adenocarcinoma or melanoma cells. Two of the cDNAs (designated 8.11 and 10.14) were found to be novel. The expression of 10.14 mRNA (3.2 kb) was 4-fold higher in MTLn3 than in MTC.4 cells. Sequencing of the 10.14 cDNA (2.2 kb) revealed a putative open reading frame of 583 amino acids that was also novel. Expression of 8.11 mRNA (>7 kb) inversely correlated with metastatic potential. Another differentially transcribed gene was highly homologous to ERK2 (extracellular signal related kinase 2), a mitogen-activated protein kinase (MAPK). Northern analysis of ERK2 expression revealed 3-fold higher amounts of a 1.3 kb mRNA in MTLn3 than in MTC.4 cells. Higher levels of ERK2 mRNA were generally seen in the more metastatic human colon but not in melanoma cell lines. We also corroborated the work of Taniguchi (Nucl Acids Res 19:6949, 1991) by independently identifying EF-1 as a putative metastasis-associated gene.Supported by an educational grant from CIBA-GEIGY Corp., at the San Antonio Breast Cancer Symposium, Dec. 8, 1992.     

Phenotypic drift of metastatic and cell-surface properties of mammary adenocarcinoma cell clones during growth in vitro

We have examined cell clones obtained from a 13762 mammary adenocarcinoma tumor and its spontaneous lung metastasis for phenotypic stability during serial culture passage in vitro. Two clones that varied markedly in their metastatic properties were chosen for further examination. One of these clones (MTC) obtained from the parental transplanted tumor initially failed to metastasize within 23 days post-injection s.c. but gained the ability to form spontaneous pulmonary metastases after several serial passages in vitro. Another clone (MTLn₃) derived from a spontaneous lung metastasis was initially highly metastatic after short-term culture, but lost the potential to form large numbers of spontaneous lung metastases with long-term culture. In contrast to MTA, clone MTLn₃ displayed lymph-node metastasis, and the frequency of lymph-node involvement increased when late-passage cultures of MTLn₃ cells were assayed in vivo. Both clones from late-passage cultures produced larger tumor sizes at the primary (mammary fat pad) injection sites compared to early passage cells. The morphologies of MTC cells changed with serial tissue culture passage, while the morphologies of MTLn₃ cells did not change. The display of fibronectin on MTC cells by immunofluorescence did not change with culture passage; fibronectin was not detected in cultures of clone MTLn₃. Fibronectin was also found on MTC cells by cell surface labelling using lactoperoxidase-catalyzed iodination-sodium dodecylsulfate polyacrylamide gel electrophoresis-autoradio-graphy. lodination of fibronectin on MTC cells did not vary with culture passage, and as in immunofluorescence experiments it was not detected on MTLn₃ cells. There was a decrease in exposure of certain cell surface proteins on MTC cells with culture passage, but we did not detect modifications with this procedure that correlated with culture passage of MTLn₃ cells. We conclude that prolonged culture in vitro can result in modifications of metastatic and cell-surface properties of tumor cell clones.     

The inhibition of tumor cell intravasation and subsequent metastasis via regulation of in vivo tumor cell motility by the tetraspanin CD151

In vivo tumor cell migration through integrin-dependent pathways is key to the metastatic behavior of malignant cells. Using quantitative in vivo assays and intravital imaging, we assessed the impact of cell migration, regulated by the integrin-associated tetraspanin CD151, on spontaneous human tumor cell metastasis. We demonstrate that promoting immobility through a CD151-specific metastasis blocking mAb prevents tumor cell dissemination by inhibiting intravasation without affecting primary tumor growth, tumor cell arrest, extravasation, or growth at the secondary site. In vivo, this loss of migration is the result of enhanced tumor cell-matrix interactions, promoted by CD151, which prevent dissociation by individual cells and leads to a subsequent inhibition of invasion and intravasation at the site of the primary tumor.     

The collection of the motile population of cells from a living tumor

In this study, we report that needles containing chemoattractants can be used to collect the subpopulation of motile and chemotactic tumor cells from a primary tumor in a live rat as a pure population suitable for further analysis. The most efficient cell collection requires the presence of chemotactic cytokines, such as epidermal growth factor and serum components, and occurs with 15-fold higher efficiency in metastatic tumors compared with nonmetastatic tumors. Although tumor cells of the nonmetastatic tumors show a motility response to serum, they were not collected with high efficiency into needles in vivo in response to serum, indicating that additional factors besides motility are required to explain differences in cell collection efficiencies between metastatic and nonmetastatic tumors. The results reported here indicate that needles filled with growth factors and matrigel, when inserted into the primary tumor, can faithfully mimic the environment that supports invasion and intravasation in vivo. Furthermore, the results indicate that the same cell behaviors that contribute to chemotaxis in vitro also contribute to invasion in vivo.     

Imaging of cancer invasion and metastasis using green fluorescent protein

The use of green fluorescent protein to fluorescently tag tumour cells has allowed investigators to open the “black box” of metastasis in order to visualise the behaviour of tumour cells in living tissues. Analysis of cells leaving the primary tumour indicates that highly metastatic cells are able to polarise more effectively towards blood vessels while poorly metastatic cells fragment more often when interacting with blood. In addition, there appear to be greater numbers of host immune system cells interacting with metastatic tumours. After arresting in target organs such as the lungs or liver, most tumour cells become dormant or apoptose. A small fraction of the arrested cells form metastases. In some target organs, migration of tumour cells may enhance the ability to form metastases.