Blood-borne Cancer Cells Research Articles

Platelets, Constant and Cooperative Companions of Sessile and Disseminating Tumor Cells, Crucially Contribute to the Tumor Microenvironment.

Although platelets and the coagulation factors are components of the blood system, they become part of and contribute to the tumor microenvironment (TME) not only within a solid tumor mass, but also within a hematogenous micrometastasis on its way through the blood stream to the metastatic niche. The latter basically consists of blood-borne cancer cells which are in close association with platelets. At the site of the primary tumor, the blood components reach the TME via leaky blood vessels, whose permeability is increased by tumor-secreted growth factors, by incomplete angiogenic sprouts or by vasculogenic mimicry (VM) vessels. As a consequence, platelets reach the primary tumor via several cell adhesion molecules (CAMs). Moreover, clotting factor VII from the blood associates with tissue factor (TF) that is abundantly expressed on cancer cells. This extrinsic tenase complex turns on the coagulation cascade, which encompasses the activation of thrombin and conversion of soluble fibrinogen into insoluble fibrin. The presence of platelets and their release of growth factors, as well as fibrin deposition changes the TME of a solid tumor mass substantially, thereby promoting tumor progression. Disseminating cancer cells that circulate in the blood stream also recruit platelets, primarily by direct cell-cell interactions via different receptor-counterreceptor pairs and indirectly by fibrin, which bridges the two cell types via different integrin receptors. These tumor cell-platelet aggregates are hematogenous micrometastases, in which platelets and fibrin constitute a particular TME in favor of the cancer cells. Even at the distant site of settlement, the accompanying platelets help the tumor cell to attach and to grow into metastases. Understanding the close liaison of cancer cells with platelets and coagulation factors that change the TME during tumor progression and spreading will help to curb different steps of the metastatic cascade and may help to reduce tumor-induced thrombosis.

Complement Inhibitors Block Complement C3 Opsonization and Improve Targeting Selectivity of Nanoparticles in Blood.

Complement is one of the critical branches of innate immunity that determines the recognition of engineered nanoparticles by immune cells. Antibody-targeted iron oxide nanoparticles are a popular platform for magnetic separations, in vitro diagnostics, and molecular imaging. We used 60 nm cross-linked iron oxide nanoworms (CLIO NWs) modified with antibodies against Her2/neu and EpCAM, which are common markers of blood-borne cancer cells, to understand the role of complement in the selectivity of targeting of tumor cells in whole blood. CLIO NWs showed highly efficient targeting and magnetic isolation of tumor cells spiked in lepirudin-anticoagulated blood, but specificity was low due to high uptake by neutrophils, monocytes, and lymphocytes. Complement C3 opsonization in plasma was predominantly via the alternative pathway regardless of the presence of antibody, PEG, or fluorescent tag, but was higher for antibody-conjugated CLIO NWs. Addition of various soluble inhibitors of complement convertase (compstatin, soluble CD35, and soluble CD55) to whole human blood blocked up to 99% of the uptake of targeted CLIO NWs by leukocytes, which resulted in a more selective magnetic isolation of tumor cells. Using well-characterized nanomaterials, we demonstrate here that complement therapeutics can be used to improve targeting selectivity.

Fluorometric Quantification of Single-Cell Velocities to Investigate Cancer Metastasis.

Hematogenous metastasis is a multistep, selectin-regulated process whose mechanisms remain poorly understood. To investigate this biological pathway of cancer dissemination and better understand circulating cancer cells, we developed a high-throughput methodology that integrates organ-on-chip-like microfluidic and photoconvertible protein technologies. Our approach can ascribe single-cell velocity as a traceable cell property for off-chip analysis of the direct relationships between cell molecular profiles and adhesive phenotypes in the context of physiologically relevant fluid flow. We interrogate how natively expressed selectin ligands relate to colon cancer cell rolling frequencies and velocities and provide context for previously reported disparities in invitro and invivo models of selectin-mediated adhesion and metastasis. This integrated methodology represents a versatile approach for the development of anti-metastatic therapeutics as well as to generate and test mechanistic hypotheses regarding spatiotemporal processes that occur over timescales of seconds to hours with single-cell resolution.

An integrated microfluidic chip for immunomagnetic detection and isolation of rare prostate cancer cells from blood.

The quantitative and qualitative analysis of circulating tumor cells (CTCs) has the potential to improve the clinical management of several cancers, including prostate cancer. As such, there is much interest in the isolation of CTCs from the peripheral blood of cancer patients. We report the design, fabrication, and proof-of-principle testing of an integrated permalloy-based microfluidic chip for immunomagnetic isolation of blood-borne prostate cancer cells using an antibody targeting prostate surface membrane antigen (PSMA). The preliminary results using spiked blood samples indicate that the proposed device is consistently capable of isolating prostate cancer cells with high sensitivity (up to 98%) at clinically relevant low concentrations (down to 20 cells/mL) and an acceptable throughput (100μL/min).

Targeted drug delivery to circulating tumor cells via platelet membrane-functionalized particles

Circulating tumor cells (CTCs) are responsible for metastases in distant organs via hematogenous dissemination. Fundamental studies in the past decade have suggested that neutralization of CTCs in circulation could represent an effective strategy to prevent metastasis. Current paradigms of targeted drug delivery into a solid tumor largely fall into two main categories: unique cancer markers (e.g. overexpression of surface receptors) and tumor-specific microenvironment (e.g. low pH, hypoxia, etc.). While relying on a surface receptor to target CTCs can be greatly challenged by cancer heterogeneity, targeting of tumor microenvironments has the advantage of recognizing a broader spectrum of cancer cells regardless of genetic differences or tumor types. The blood circulation, however, where CTCs transit through, lacks the same tumor microenvironment as that found in a solid tumor. In this study, a unique “microenvironment” was confirmed upon introduction of cancer cells of different types into circulation where activated platelets and fibrin were physically associated with blood-borne cancer cells. Inspired by this observation, synthetic silica particles were functionalized with activated platelet membrane along with surface conjugation of tumor-specific apoptosis-inducing ligand cytokine, TRAIL. Biomimetic synthetic particles incorporated into CTC-associated micro-thrombi in lung vasculature and dramatically decreased lung metastases in a mouse breast cancer metastasis model. Our results demonstrate a “Trojan Horse” strategy of neutralizing CTCs to attenuate metastasis.

Abstract 2846: Molecular platforms for mutation analysis from whole blood derived clinical samples by nextgen sequencing

Abstract Both cell free DNA (cfDNA) and circulating tumor cells (CTC) represent important possible templates for mutation analysis of clinical samples with different theoretical advantages for a clinical test. cfDNA is easy to access and isolate, while CTC can provide both DNA as well as RNA for clinical testing. We have tested matched cfDNA and CTC DNA in a Next Generation sequencing test of clinical samples. Both cfDNA and CTC samples provided sufficient quantity for a direct sequencing clinical test. No whole genome amplification was required. The test consisted of coupling these purification technologies to an amplicon re-sequencing panel of 50 cancer-associated genes using a CLIA validated sequencing pipeline for single nucleotide variant (SNV) mutations with a sensitivity of 1%. Typically using this pipeline, blood borne cancer cells are isolated, extracted, sequenced and analyzed in 7 days. In the CLIA setting, the validated DNA sequencing process applied to breast cancer patient samples, has demonstrated useful and actionable clinical data. Results of SNV detection from known clinical positive control samples were quite definitive. We observed perfect concordance in True Positive (TP) SNV detection between the cfDNA and CTC templates. The only discordant results observed were in the detection of False Positives (FP). Using the identical software for SNV detection and variant calls, CTC templates exhibited ∼5x greater sensitivity in our sample set. cfDNA samples showed a much greater noise spectrum of FP. As blood samples are routinely fixed at collection, we evaluated whether fixation might contribute to the cfDNA observed high FP frequency. Our experimental results conclusively show that fixation did not contribute to increased FP observed with cfDNA. Whereas CTC samples showed zero FP over the reportable range, the cfDNA samples exhibited variable FP output ranging from 0 to >10 FP per sample. To extend the capabilities of sequencing rare cells, several RNA sequencing panels have been developed that allow for both SNV, expression, and fusion transcript detection. Although these panels have not been successfully used with cell free templates, they have been successfully applied to rare cell templates and promise to be an important capability for clinical use of CTC in the future. Looking at both lung and breast cancer derived samples we have measured both SNV, CNV (expression based), and fusion transcripts. We describe simultaneous detection of SNV mutations (KRAS: G12S, STK11:Q37*, PIK3CA:E545K, EGFR:L858R, EGFR:T790M, TP53:R273H) as well as fusion transcripts events EML4-ALK.E6aA20.AB374361, and EML4-ALK.E13A20.AB462411. In summary, both cfDNA and CTC derived DNA provided concordant results in SNV mutation detection. cfDNA exhibited a much higher FP rate as compared to CTC DNA. We further demonstrate that rare cells can provide useful template for RNA sequencing. Citation Format: William M. Strauss, Erich Klem, Maria Cuellar, Jill Simmons, Jessamine Winer-Jones, Paul W. Dempsey. Molecular platforms for mutation analysis from whole blood derived clinical samples by nextgen sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2846. doi:10.1158/1538-7445.AM2014-2846

Protein Kinase Cϵ Mediates Polymeric Fibronectin Assembly on the Surface of Blood-borne Rat Breast Cancer Cells to Promote Pulmonary Metastasis

Malignant breast cancer cells that have entered the blood circulation from primary mammary fat pad tumors or are grown in end-over-end suspension culture assemble a characteristic, multi-globular polymeric fibronectin (polyFn) coat on their surfaces. Surface polyFn is critical for pulmonary metastasis, presumably by facilitating lung vascular arrest via endothelial dipeptidylpeptidase IV (CD26). Here, we show that cell-surface polyFn assembly is initiated by the state of suspension, is dependent upon the synthesis and secretion of cellular Fn, and is augmented in a dose- and time-dependent manner by plasma Fn. PolyFn assembly is regulated by protein kinase Cepsilon (PKCepsilon), which translocates rapidly and in increasing amounts from the cytosol to the plasma membrane and is phosphorylated. PolyFn assembly is impeded by select inhibitors of this kinase, i.e. bisindolylmaleimide I, Ro-32-0432, Gö6983, and Rottlerin, by the phorbol 12-myristate 13-acetate-mediated and time-dependent loss of PKCepsilon protein and decreased plasma membrane translocation, and more specifically, by stable transfection of lung-metastatic MTF7L breast cancer cells with small interfering RNA-PKCepsilon and dominant-negative PKCepsilon constructs (e.g. RD-PKCepsilon). The inability to assemble a cell surface-associated polyFn coat by knockdown of endogenous Fn or PKCepsilon impedes cancer cells from metastasis to the lungs. The present studies identify a novel regulatory mechanism for polyFn assembly on blood-borne breast cancer cells and depict its effect on pulmonary metastasis.

Frequent detection and immunophenotyping of prostate-derived cell clusters in the peripheral blood of prostate cancer patients.

Recent scientific studies have failed to determine parameters for the assessment of prostate cancer aggressiveness. The present study deals with the detection of blood-borne cancer cells based on polymerase chain reaction (PCR) and cell enrichment methods. The contradictory results reported in the literature have called into question the clinical usefulness of this diagnostic method in the preoperative staging of clinically localized prostate cancer. We established a combined method of density gradient centrifugation and immunomagnetic separation using epithelium-specific antibodies, i.e. cytokeratins, to isolate prostate-derived circulating cells from the peripheral blood of patients with prostate cancer. Isolated cells were characterized by DNA staining and immunocytochemistry using antibodies for the detection of prostate-specific antigen (PSA), proliferation-associated proteins (MIB-1, H1 and H3) and apoptosis-associated proteins (M30, c-FasR). We applied these methods to 68 prostate cancer patients and were able to isolate cell clusters in 98%. Immunophenotypic and morphological characterization of PSA-positive prostate-derived cell clusters found in the peripheral blood of prostate cancer patients showed two main populations: 1) in 35% of the investigated prostate cancer patients we detected rounded cell aggregates of probable cancer cells expressing proliferation-associated proteins and lacking apoptosis-associated protein expression; 2) in all cases there was a high frequency of circulating dysmorphic cell clusters positive for apoptosis-associated protein expression. Our results demonstrate the existence of at least two different types of blood-borne prostate-derived circulating cell clusters. Of these, only the less frequent, round, small cell clusters harbor features that are probably necessary for the cells to survive for metastatic spread.

Numeric definition of the clinical performance of the nested reverse transcription-PCR for detection of hematogenous epithelial cells and correction for specific mRNA of non-target cell origin as evaluated for prostate cancer cells.

Inappropriate quality management of reverse transcription-PCR (RT-PCR) assays for the detection of blood-borne prostate cancer (PCa) cells hampers clinical conclusions. Improvement of the RT-PCR methodology for prostate-specific antigen (PSA) mRNA should focus on an appropriate numeric definition of the performance of the assay and correction for PSA mRNA that is not associated with PCa cells. Repeated (RT-)PCR tests for PSA mRNA in single blood specimens from PCa patients and PCa-free controls, performed by four international institutions, showed a large percentage (approximately equal to 50%) of divergent test results. The best estimates of the mean, lambda (SD), of the expected Poisson frequency distributions of the number of positive tests among five replicate assays of samples from PCa-free individuals were 1.0 (0.2) for 2 x 35 PCR cycles and 0.2 (0.1) for 2 x 25 PCR cycles. Assessment of the numeric value of the mean can be considered as a new indicator of the performance of a RT-PCR assay for PSA mRNA under clinical conditions. Moreover, it determines the required number of positive test repetitions to differentiate between true and false positives for circulating prostate cells. At a predefined diagnostic specificity of > or = 98%, repeated PCRs with lambda of either 1.0 or 0.2 require, respectively, more than three or more than one positive tests to support the conclusion that PSA mRNA-containing cells are present. Repeated nested PCR tests for PSA and appropriate handling of the data allow numeric quantification of the performance of the assay and differentiation between analytical false and true positives at a predefined accuracy. This new approach may contribute to introduction of PSA RT-PCR assays in clinical practice.

Regulation of the metastatic process by E-selectin and stress-activated protein kinase-2/p38.

The formation of metastasis is a dreadful complication of cancer that is associated with a poor prognosis. Several clinical observations and experimental findings indicate that the metastatic process is nonrandom and involves a sequence of multistep events that may all be targeted for therapy. This includes angiogenesis of the primary neoplasm, release of malignant cells from this neoplasm, entry of cancer cells into the blood circulation, interaction of cancer cells with vascular endothelial cells in distant organs, and growth of blood-borne cancer cells locally in the vessels or distally following extravasation. Our working hypothesis is that metastatic cancer cells exploit the mechanisms of the inflammation process to successfully migrate into distant organs. This implies a pivotal role for specific adhesive interactions between cancer cells and vascular endothelial cells and activation of migratory pathways in the cancer cells. We review here the roles played by the endothelial adhesive molecule E-selectin and by the motogenic stress-activated protein kinase-2 (SAPK2/p38) pathway of cancer cells in modulating transendothelial migration of cancer cells.

Focal Adhesion Kinase Activated by β4 Integrin Ligation to mCLCA1 Mediates Early Metastatic Growth

Early metastatic growth occurs at sites of vascular arrest of blood-borne cancer cells and is entirely intravascular. Here we show that lung colonization by B16-F10 cells is licensed by beta(4) integrin adhesion to the mouse lung endothelial Ca(2+)-activated chloride channel protein mCLCA1. In a manner independent of Met, beta(4) integrin-mCLCA1-ligation leads to complexing with and activation of focal adhesion kinase (FAK) and downstream signaling to extracellular signal-regulated kinase (ERK). FAK/ERK signaling is Src-dependent and is interrupted by adhesion blocking antibodies and by dominant-negative (dn)-FAK mutants. Levels of ERK activation in B16-F10 cells transfected with wild-type or mutant FAK are closely associated with rates of proliferation and bromodeoxyuridine (BrdUrd) incorporation of tumor cells grown in mCLCA1-coated dishes, the ability to form tumor cell colonies on CLCA-expressing endothelial cell monolayers, and the extent of pulmonary metastatic growth. Parallel with the transfection rates, B16-F10 cells transfected with dn-FAK mutants and injected intravenously into syngeneic mice generate approximately half the number and size of lung colonies that vector-transfected B16-F10 cells produce. For the first time, beta(4) integrin ligation to its novel CLCA-adhesion partner is shown to be associated with FAK complexing, activation, and signaling to promote early, intravascular, metastatic growth.

Will differential culturing of circulating human lung cancer cells aid in understanding metastasis?

It is thought that there is need to resolve the incompatibilities present in current concepts of cancer metastasis. In presenting as many as 10 such anomalies in lung cancer dissemination in this Journal, my explanatory hypothesis rested on the contradistinctive status of the blood-borne and lymph-borne lung cancer cell (LCC). Perhaps, researches slanted towards differential culturing of these two circulating cells will open avenues for advancing the bioscience of cell transportation in human cancer metastasis.

Predictive laboratory diagnostics in oncology utilizing blood-borne cancer cells – current best practice and unmet needs

The aim of laboratory diagnostics in oncology is to improve the clinical outcome of cancer by allowing earlier detection. Molecular knowledge of cancer should increase the number of risk and prognostic factors and will allow development of methods for detection and elimination of even very small tumors. Thus, the race for the specific tumor antigen in peripheral blood and the race for the blood-borne cancer cell happened simultaneously. The direct detection of the cells which have the highest probability to harbor all the properties mandatory to be life-threatening, conceivably metastatic, would be the most promising way to find the target structure of malignancy. Methods applying enrichment techniques based on density, morphology, tissue specific protein and tumor-associated protein detection enabled multi-parametric analysis of those blood-borne cancer cells. In exemplary studies it was demonstrated that the count of cell clusters positive for the tissue-specific proteins cytokeratin and prostate-specific antigen (PSA) from the peripheral blood of prostate cancer patients and a combination of a tissue-specific protein, a oncogenic receptor protein cytokeratin and p185 c- erbB-2 from the peripheral blood of breast cancer patients is related to the stage of the diseases. Breast cancer patients who presented with cytokeratin/p185 c- erbB-2 positive cell clusters showed a decrease of those cells under adriamycin adjuvant therapy. Nevertheless, additional molecular markers are required to characterize the functional properties of blood-borne cancer cells. Therefore, the genome of the cells can be investigated using a procedure for indirectly detecting aberrations of defined gene locations, i.e. multiplex microsatellite polymerase chain reaction. Up to now, the methods applied to the separation of blood-borne cancer cells are time-consuming and rather expensive. They consist of an initial enrichment step of density gradient centrifugation or buffy coat preparation followed by a specific isolation step using superparamagnetic microbeads coupled to antibodies, filter techniques or multi-parametric flow cytometry. Novel technologies have to be applied using miniaturization, integration and parallel-processing techniques based on those used in the computer industry to overcome the drawbacks.

The Breast Cancer β4 Integrin and Endothelial Human CLCA2 Mediate Lung Metastasis

Adhesion of blood-borne cancer cells to the endothelium is a critical determinant of organ-specific metastasis. Here we show that colonization of the lungs by human breast cancer cells is correlated with cell surface expression of the alpha(6)beta(4) integrin and adhesion to human CLCA2 (hCLCA2), a Ca(2+)-sensitive chloride channel protein that is expressed on the endothelial cell luminal surface of pulmonary arteries, arterioles, and venules. Tumor cell adhesion to endothelial hCLCA2 is mediated by the beta(4) integrin, establishing for the first time a cell-cell adhesion property for this integrin that involves an entirely new adhesion partner. This adhesion is augmented by an increased surface expression of the alpha(6)beta(4) integrin in breast cancer cells selected in vivo for enhanced lung colonization but abolished by the specific cleavage of the beta(4) integrin with matrilysin. beta(4) integrin/hCLCA2 adhesion-blocking antibodies directed against either of the two interacting adhesion molecules inhibit lung colonization, while overexpression of the beta(4) integrin in a model murine tumor cell line of modest lung colonization potential significantly increases the lung metastatic performance. Our data clearly show that the beta(4)/hCLCA2 adhesion is critical for lung metastasis, yet expression of the beta(4) integrin in many benign breast tumors shows that this integrin is insufficient to bestow metastatic competence on cells that lack invasiveness and other established properties of metastatic cells.

The role of blood group antigens in malignant progression, apoptosis resistance, and metastatic behavior

T HE METASTATIC phenotype is an extremely complex biologic phenomenon comprising multiple biologic features such as matrix-independent growth and survival, motility, invasion, aberrant adhesive behavior, induction of neoangiogenesis, and apoptosis resistance. Correlation of metastatic human cancer with the expression of certain blood group carbohydrates or corresponding cell surface lectins is well established for several major types of human malignancies, such as colon, breast, and prostate cancers. Similar data are beginning to emerge for many other types of human cancer. It is well documented that bloocl group carbohydrates expressed on the cell surface of metastatic cancer cells function as cell adhesion molecules. The homotypic and heterotypic cell-cell adhesion mediated by interactions of certain blood group carbohydrates with corresponding lectins is a critically important event at the extravasation step of the metastatic cascade when metastatic cancer cells escape from the circulation into distant sites of secondary tumor growth. These adhesive interactions are believed to be essential for matrixindependent survival of blood-borne cancer cells. We describe initial experimental evidence that supports the hypothesis that carbohydrate-lectin interactions trigger an intracellular signaling cascade culminating in alterations of transcriptional control of gene expression. Furthermore, modification of gene expression triggered by carbohydratelectin interactions is manifested in differential gene expression profiles that distinguish highly and poorly metastatic cancer cells and may be responsible for matrix-independent survival and apoptosis resistance of metastatic cancer cells. Thus, the

Blood-borne cancer cells--quo vadis?

The detection of blood-borne cancer cells may help in clinical staging and further understanding of cancer metastasis. We developed a cytokeratin-based immunomagnetic method to isolate epithelium-derived cells from the circulating blood of patients. The number of cell clusters positive for cytokeratin/prostate-specific antigen (PSA) from the peripheral blood of prostate cancer patients and cytokeratin/p185c-erbB-2 from the peripheral blood of breast cancer patients has been related to stage of the disease. Breast cancer patients who presented cytokeratin/p185c-erbB-2-positive cell clusters showed a decrease in such cells under adriamycin adjuvant therapy with Further molecular characterization by a highly sensitive microsatellite multiplex-PCR enabled reproducible detection of microsatellite alterations. The impact of these individually targeted results may contribute to an individual diagnostic and therapeutic strategy.

Cell adhesion molecules in the formation of liver metastasis.

Metastasis is the most life-threatening event in patients with cancer, with the liver being one of the most frequently affected organs. In the development and establishment of metastasis, blood-borne cancer cells utilise various cell adhesion mechanisms (cell-cell, cell matrix, tumour-endothelial and hepatocyte). Thus, cell adhesion molecules have a pivotal role in this process of metastasis formation. This article discusses recent progress in the biology of cell adhesion molecules in the formation of liver metastasis and the clinical implications of the findings.

Endothelial Cell Adhesion Molecules and Their Role in Organ Preference of Metastasis.

Endothelial cell adhesion molecules expressed in distinct vascular branches of select organs are implicated in organ preference of metastasis. Two constitutively expressed adhesion molecules of the lung vasculature are described here. The first is the murine B 16 melanoma cell-binding endothelial cell adhesion molecule Lu-ECAM-1. The 90kDa Lu-ECAM-1 selectively promotes adhesion and lung metastasis of B 16 melanoma cells. Melanoma cell attachment to immobilized Lu-ECAM-1 is inhibited in a dose dependant manner by anti-Lu-ECAM-1 mAb 6D3, soluble Lu-ECAM-1, and lacto-N-fucopentose. In vivo blocking of Lu-ECAM-1 by its mAb 6D3 prevents lung colonization of B16-F10. However, Lu-ECAM-1 either binds nor affects metastasis of other lung colonizing tumor cells (e.g., KLN205 carcinoma cells), or tumor cell lines that metastasize to other organs than the lung (e.g., liver-metastatic B16-L8-F10, RAW117-H10), and does not bind lymphocytes and neutrophils. The second adhesion molecule is rat dipeptidyl peptidase IV(DPP IV). This sialoglycoprotein binds lung-metastatic rat prostate and breast carcinoma cells via fibronectin which is expressed in significantly higher amounts on the surface of metastatic than non-metastatic tumor cells. Increased fibronectin expression is associated with increases in tumor cell β1 and β3 integrins. DPP IV binding is mediated by a fibronectin sequence other than RGD and is independent of its peptidase domain. These constitutively expressed adhesion molecules are believed to be responsible for site-specific vascular arrest of blood-borne cancer cells and, through as yet unknown signaling events, may cause upregulation of other (transiently expressed) adhesion molecules to enhance tumor cell binding to endothelium and/or initiate gap junctional communication to promote extravasation and secondary tumor growth.

Mediation of lung metastasis of murine melanomas by a lung-specific endothelial cell adhesion molecule.

Organ-specific adhesion molecules expressed by vascular endothelial cells have been implicated in the arrest of blood-borne cancer cells in selective, secondary sites. A lung-specific endothelial cell adhesion molecule (Lu-ECAM-1) localized on endothelia of distinct branches of lung blood vessels has been purified by immunoaffinity chromatography from detergent extracts of lung matrix-modulated endothelial cells using monoclonal antibody (mAb) 6D3. It has a molecular mass of 90 kDa and promotes the selective attachment of lung-metastatic B16 melanoma cells. Corresponding with their metastatic performance, B16-F10 tumor cells selected for higher lung colonization bind to Lu-ECAM-1 in significantly higher numbers than their low lung metastatic counterpart B16-F0. Binding of B16-F0 and B16-F10 is reduced with mAb 6D3 to slightly lower levels than B16-F0 bound to Lu-ECAM-1. mAb 6D3 injected into C57BL/6 mice 1 hr prior to an i.v. challenge with B16-F10 causes a 90% reduction in the number of lung colonies compared with animals injected with control mAb (6D8 or 3C6). Lu-ECAM-1 neither binds nor effects metastasis of other lung-colonizing tumor cells (e.g., KLN205). Thus, site-specific metastasis of tumor cells is regulated by similar mechanisms as the homing of lymphocytes--namely, by the ability of blood-borne cancer cells to recognize and adhere to distinct endothelial cell adhesion molecules.

Organ-preference of metastasis

The initial, site-specific colonization of secondary organs by blood-borne cancer cells appears to be mediated by endothelial cell adhesion molecules. These molecules are part of the organ-specific microvascular phenotype and are regulated through complex interactions of the endothelium with the extracellular matrix (e.g., distinct matrix macromolecules and growth factors). They are induced in vitro by growing 'unspecific' (large vessel) endothelial cells on extracts of organ-specific biomatrices. In many respects, these molecules are similar to the various classes of chemically different adhesion molecules that regulate lymphocyte traffic, but are believed to be distinct from the inducible adhesion molecules that govern leukocyte adhesion during acute episodes of inflammation. Biochemical and biophysical data indicate that preference of tumor cell adhesion to organ-specific microvascular endothelium may not require qualitative differences of such homing receptors between endothelia, but may be explained on the basis of quantitative receptor differences as well as differences of receptor avidity. Following adhesion, the metastatic cascade proceeds by the establishment of metabolic conduits between the endothelium and adherent tumor cells. This heterotypic coupling represents an early step in the extravasation of cancer cells from the microvasculature, initiating endothelial cell retraction from its basement membrane and recanalization around the arrested tumor cell. These events, together with local growth promoting effects exerted by the metastasized organ, are believed to provide the basis for Paget's 'seed and soil' hypothesis of metastasis.