Diversification and progression of malignant tumors

Tumor-cell diversification mechanisms insure that malignant neoplasms contain diversified tumor-cell subpopulations. Because of the instability of tumor cell phenotypes, some malignant cells will evolve with the most favorable properties for their progression to highly metastatic cells. The rates of cellular phenotypic diversification vary greatly among different tumors, and they are probably modulated, in part, by genetic and chromosome defects and by epigenetic events that may vary widely depending upon the nature of the tumor cells and their microenvironments. As tumor diversification and selection proceed, the most malignant cell subpopulations may eventually become dominant and gradually lose their microenvironmental responsiveness. Tumor-cell diversification mechanisms may be similar or identical to normal, developmentally regulated diversification mechanisms that are used during embryonic cell diversification and differentiation.     

Reduction of TGF-beta activity abrogates growth promoting tumor cell-cell interactions in vivo.

We have shown in previous studies that metastatically-competent variant subpopulations (B5, C1) derived from a non-metastatic murine mammary adenocarcinoma (SP1) have a pronounced growth advantage over their non-metastatic tumor cell counterparts in primary tumors. As a result, primary tumors can be progressively overgrown by cells having the competence to spread elsewhere in the body. This occurs despite any evidence to indicate an intrinsic in vivo growth rate advantage of the metastatic cells when grown as isolated populations. This suggested that cell-cell interactions between metastatic and non-metastatic tumor populations may be involved in the metastatic cell growth dominance process. Evidence was therefore sought for growth factors released by SP1 cells which could preferentially stimulate the B5 or C1 variants and thereby mediate this cell-cell interaction process. We found that cocultures of SP1 and C1 or B5 cells with irradiated C1, B5, or SP1 "feeder" cells showed significant stimulation of C1 and B5 by SP1 "feeder" cells. Cell growth stimulation in response to EGF, TGF-alpha, TGF-beta 1, bFGF, PDGF, NGF, IGF-1, or IGF-2 demonstrated that only TGF-beta 1 could duplicate this effect. A repeat of the coculture experiment in the presence of specific neutralizing anti-TGF-beta antibodies was therefore undertaken and this was found to markedly reduce the stimulation of C1 or B5 cells by irradiated SP1 cells. Conditioned media from the SP1 and C1 cell lines was quantitated for TGF-beta activity and contained 4.5 ng/ml and 2.0 ng/ml, respectively. However, the majority of the TGF-beta released by SP1 cells was found to be spontaneously active, whereas 70% of the TGF-beta released by C1 cells was in its latent form. Scatchard analysis revealed approximately four times the number of TGF-beta receptors, of similar type and affinity, present on C1 as compared with SP1 cells. The in vitro results support the hypothesis that active TGF-beta released by SP1 cells may stimulate the proliferation of metastatic variant cells in a paracrine like fashion. In vivo evidence for this was obtained by showing that coinjection of irradiated SP1 cells could selectively stimulate tumor growth of viable C1 cells and this effect was markedly diminished by neutralizing polyclonal anti-TGF-beta antibodies. Taken together, the results suggest a novel role for TGF-beta in clonal evolution of malignant tumor growth and as a molecular mediator of tumor cell-tumor cell interactions involved in facilitating tumor progression.     

Alpha IIb beta 3 integrin expression and function in subpopulations of murine tumors.

Subpopulations of B16 amelanotic melanoma (B16a) cells, isolated by centrifugal elutriation from enzymatically dispersed solid tumors, demonstrated different abilities to form lung colonies when injected intravenously. In contrast, no differences in experimental metastasis were observed among subpopulations obtained from Lewis lung (3LL) tumors. Lung colonization by B16a and 3LL subpopulations correlated positively with observed differences (B16a) or lack of differences (3LL) in tumor cell ability to induce aggregation of homologous platelets, to adhere to subendothelial matrix or fibronectin, and with the percentage of cells in the G2/M phase of the cell cycle. Both B16a and 3LL cells express alpha IIb beta 3 integrin receptors; however, differences in the receptor expression level were found only among B16a subpopulations. Comparison of the amount of alpha IIb beta 3 receptor expressed on cell surface with tumor cell ability to induce platelet aggregation (TCIPA) and to adhere to fibronectin or subendothelial matrix revealed a positive correlation. Pretreatment of tumor cells with alpha IIb beta 3-specific antibodies inhibited tumor cell matrix adhesion, TCIPA, and lung colony formation. We propose that alpha IIb beta 3 integrin receptor expression, tumor cell matrix adhesion, and tumor cell-induced platelet aggregation can be important parameters to indicate the metastatic potential of some tumor cells and that the alpha IIb beta 3 is a multifunctional receptor involved in both tumor cell-matrix and tumor cell-platelet interactions. Further, the correlation among cell cycle phase, metastatic ability, and receptor expression suggests that metastatic propensity may be transiently expressed and/or increased in some tumor cell subpopulations.     

TrAp: a tree approach for fingerprinting subclonal tumor composition

Revealing the clonal composition of a single tumor is essential for identifying cell subpopulations with metastatic potential in primary tumors or with resistance to therapies in metastatic tumors. Sequencing technologies provide only an overview of the aggregate of numerous cells. Computational approaches to de-mix a collective signal composed of the aberrations of a mixed cell population of a tumor sample into its individual components are not available. We propose an evolutionary framework for deconvolving data from a single genome-wide experiment to infer the composition, abundance and evolutionary paths of the underlying cell subpopulations of a tumor. We have developed an algorithm (TrAp) for solving this mixture problem. In silico analyses show that TrAp correctly deconvolves mixed subpopulations when the number of subpopulations and the measurement errors are moderate. We demonstrate the applicability of the method using tumor karyotypes and somatic hypermutation data sets. We applied TrAp to Exome-Seq experiment of a renal cell carcinoma tumor sample and compared the mutational profile of the inferred subpopulations to the mutational profiles of single cells of the same tumor. Finally, we deconvolve sequencing data from eight acute myeloid leukemia patients and three distinct metastases of one melanoma patient to exhibit the evolutionary relationships of their subpopulations.     

Exposure of human primary colon carcinoma cells to anti-Fas interactions influences the emergence of pre-existing Fas-resistant metastatic subpopulations

Fas, an important death receptor-mediated signaling pathway, has been shown to be down-regulated during human colon tumorigenesis; however, how alterations in Fas expression influence the metastatic process remains unresolved. In mouse models, loss of Fas function was found to be both necessary and sufficient for tumor progression. In this study, we investigated the link between functional Fas status and malignant phenotype using a matched pair of naturally occurring primary (Fas-sensitive) and metastatic (Fas-resistant) human colon carcinoma cell lines in both in vitro and in vivo (xenograft) settings. Metastatic sublines were produced in vitro from the primary tumor cell line by functional elimination of Fas-responsive cells. Conversely, sublines derived from the primary tumor in vivo at distal metastatic sites were Fas-resistant. In contrast, simply disrupting the Fas pathway by molecular-based strategies in the Fas-sensitive primary tumor failed to achieve the same metastatic outcome. Interestingly, both in vitro- and in vivo-produced sublines resembled the naturally occurring metastatic population, based on functional and morphologic studies and genome-scale gene expression profiling. Overall, using this human colon carcinoma model, we: 1) showed that loss of Fas function was linked to, but alone was insufficient for, acquisition of a detectable metastatic phenotype; 2) demonstrated that metastatic subpopulations pre-existed within the heterogeneous primary tumor, and that anti-Fas interactions served as a selective pressure for their outgrowth; and 3) identified a large set of differentially expressed genes distinguishing the primary from metastatic malignant phenotypes. Thus, Fas-based interactions may represent a novel mechanism for the biologic or immunologic selection of certain types of Fas-resistant neoplastic clones with enhanced metastatic ability.     

Heterogeneity of fibroblast response in host-tumor cell-cell interactions in metastatic tumors

The spread and invasion of tumor cells into host tissues are associated with the release of elevated levels of collagenolytic activity of both host and tumor cell origins. However, the mechanisms of regulation of the enzyme activity is still unresolved. Histological examination of human and animal tumors revealed morphological changes in stromal fibroblasts and mast cells at the tumor periphery. Numerous mast cells appeared at microfoci along the tumor: host tissue junction and mast cell degranulation were associated with collagenolysis. In vitro studies, using rat mammary adenocarcinoma and human lung adenocarcinoma cells, showed that both tumor cells and host fibroblasts participate in matrix degradation. Tumor-associated stromal fibroblasts released higher levels of enzyme activity than normal fibroblasts and were more responsive to stimulation by tumor-conditioned media and soluble mast cell products. Host fibroblasts appear to be heterogeneous populations of responsive and nonresponsive subpopulations based on their response to tumor- or mast-cell-mediated stimulation of collagenase release. Fibroblast subpopulations were obtained by density fractionation of serum-deprived, synchronized confluent fibroblasts on discontinuous Percoll gradient. Density-fractionated fibroblast subpopulations differed in their response to stimulation by mast cell products and tumor-cell-conditioned media. The stimulatory activity of tumor-cell-conditioned media also varied as a function of the metastatic potential of the tumor cells. The data suggest that cellular interactions between tumor cells and select subpopulations of host fibroblasts at the tumor periphery play a key role in host tissue degradation. However, heterogeneity of stromal fibroblasts may determine the site and extent of the tissue damage at foci of tumor invasion.     

ABCB1 identifies a subpopulation of uveal melanoma cells with high metastatic propensity

Metastasis of tumor cells to distant organs is the leading cause of death in melanoma. Yet, the mechanisms of metastasis remain poorly understood. One key question is whether all cells in a primary tumor are equally likely to metastasize or whether subpopulations of cells preferentially give rise to metastases. Here, we identified a subpopulation of uveal melanoma cells expressing the multidrug resistance transporter ABCB1 that are highly metastatic compared to ABCB1(-) bulk tumor cells. ABCB1(+) cells also exhibited enhanced clonogenicity, anchorage-independent growth, tumorigenicity and mitochondrial activity compared to ABCB1(-) cells. A375 cutaneous melanoma cells contained a similar subpopulation of highly metastatic ABCB1(+) cells. These findings suggest that some uveal melanoma cells have greater potential for metastasis than others and that a better understanding of such cells may be necessary for more successful therapies for metastatic melanoma.     

The immunobiology of metastatic processes: analysis of NK sensitivity and the metastatic potential of H-2 gene transfected fibrosarcoma cells

The transformation of a potentially neoplastic cell into an autonomous highly malignant and metastatic tumor cell involves a multifactorial cascade of events. This will eventually lead not only to the emergence of a tumor cell with an unlimited potential of replication, but more important will contribute to its ability to ignore and evade homeostatic immune and nonimmune regulatory mechanisms. Specifically, those mechanisms which may restrict and direct its growth, dissemination, patterns of differentiation and interaction with the cellular and humoral factors comprising its environment. In the present studies we have investigated the contribution of three major factors which may be the cause or result of alterations at the level of the cell membrane: MHC encoded antigen expression, susceptibility to the cytolytic activity of NK cells and enhanced expression of the c-K-ras proto-oncogene, as to their development of the metastatic capacity of a malignant cell. To address these questions we used metastatic (IE7) and nonmetastatic (IC9) variants of the murine 3-methylcholanthrene-induced T-10 fibrosarcoma. Using this system, the following major conceptually important observations were made: (A) The restoration by transfection of the expression of membrane associated H-2K encoded glycoproteins abrogates the metastatic capacity of the highly metastatic tumor cell clone, IE7, irrespective of the degree of susceptibility to NK or c-K-ras oncogene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Why do cancer cells metastasize into particular organs?

Metastatic spread of tumor cells is one of the most common causes of death in cancer patients. Therefore, elucidation of the molecular mechanisms that underlie the formation of metastatic colonies has been one of the major objectives of cancer research during the last two decades. In this review we will mainly discuss the mechanisms that cause a malignant cell to grow at a given site rather than at other possible sites, taking into account experimental and clinical evidence published on the subject. As a whole this evidence tends to confirm the hypothesis that organ-specific colonization by malignant cells often follows very specific and close interactions between the cancer cell and the target organ, either in terms of specific cellular adhesion or growth promotion. In this paper we would like to underscore the fact that cellular adhesion, either specific or unspecific, is a necessary but, by itself, insufficient condition for the development of metastases. It is the ability of the tumor cells to grow at the site where they arrested that ultimately determines whether a metastatic colony develops or fails to develop at that site.     

Epithelial plasticity, cancer stem cells, and the tumor-supportive stroma in bladder carcinoma

High recurrence rates and poor survival rates of metastatic bladder cancer emphasize the need for a drug that can prevent and/or treat bladder cancer progression and metastasis formation. Accumulating evidence suggests that cancer stem/progenitor cells are involved in tumor relapse and therapy resistance in urothelial carcinoma. These cells seem less affected by the antiproliferative therapies, as they are largely quiescent, have an increased DNA damage response, reside in difficult-to-reach, protective cancer stem cell niches and express ABC transporters that can efflux drugs from the cells. Recent studies have shown that epithelial-to-mesenchymal transition (EMT), a process in which sessile, epithelial cells switch to a motile, mesenchymal phenotype may render cancer cells with cancer stem cells properties and/or stimulate the expansion of this malignant cellular subpopulation. As cancer cells undergo EMT, invasiveness, drug resistance, angiogenesis, and metastatic ability seem to increase in parallel, thus giving rise to a more aggressive tumor type. Furthermore, the tumor microenvironment (tumor-associated stromal cells, extracellular matrix) plays a key role in tumorigenesis, tumor progression, and metastasis formation. Taken together, the secret for more effective cancer therapies might lie in developing and combining therapeutic strategies that also target cancer stem/progenitor cells and create an inhospitable microenvironment for highly malignant bladder cancer cells. This review will focus on the current concepts about the role of cancer stem cells, epithelial plasticity, and the supportive stroma in bladder carcinoma. The potential implications for the development of novel bladder cancer therapy will be discussed. Mol Cancer Res; 10(8); 995-1009. ?2012 AACR.     

Identification of the angiogenesis signaling domain in pleiotrophin defines a mechanism of the angiogenic switch

Neoplasms progress through genetic and epigenetic mutations that deregulate pathways in the malignant cell that stimulate more aggressive growth of the malignant cell itself and/or remodel the tumor microenvironment to support the developing tumor mass. The appearance of new blood vessels in malignant tumors is known as the "angiogenic switch." The angiogenic switch triggers a stage of rapid tumor growth supported by extensive tumor angiogenesis and a more aggressive tumor phenotype and its onset is a poor prognostic indicator for host survival. Identification of the factors that stimulate the angiogenic switch thus is of high importance. Pleiotrophin (PTN the protein, Ptn the gene) is an angiogenic factor and the Ptn gene has been found to be constitutively expressed in many human tumors of different cell types. These studies use a nude mouse model to test if Ptn constitutively expressed in premalignant cells is sufficient to trigger an angiogenic switch in vivo. We introduced an ectopic Ptn gene into "premalignant" SW-13 cells and analyzed the phenotype of SW-13 Ptn cell tumor implants in the flanks of nude mice. SW-13 Ptn cell subcutaneous tumor implants grew very rapidly and had a striking increase in the density of new blood vessels compared to the SW-13 cell tumor implants, suggesting that constitutive PTN signaling in the premalignant SW-13 cell implants in the nude mouse recapitulates fully the angiogenic switch. It was found also that ectopic expression of the C-terminal domain of PTN in SW-13 cell implants was equally effective in initiating an angiogenic switch as the full-length PTN whereas implants of SW-13 cells in nude mice that express the N-terminal domain of PTN grew rapidly but failed to develop tumor angiogenesis. The data suggest the possibility that mutations that activate Ptn in premalignant cells are sufficient to stimulate an angiogenic switch in vivo and, since these mutations are frequently found in human malignancies, that constitutive PTN signaling may be an important contributor to progression of human tumors. The data also suggest that the C-terminal and the N-terminal domains of PTN equally initiate switches in premalignant cells to cells of a more aggressive tumor phenotype but the separate domains of PTN signal different mechanisms and perhaps signal through activation of a separate receptor-like protein.     

Transglutaminases: key regulators of cancer metastasis

The ability to metastasize represents the most important characteristic of malignant tumors. The biological details of the metastatic process remain somewhat unknown, due to difficulties in studying tumor cell behaviour with high spatial and temporal resolution in vivo. Several lines of evidence involve transglutaminases (TGs) in the key stages of tumor progression cascade, even though the molecular mechanisms remain controversial. TG expression and activity display a different role in the primary tumor or in metastatic cells. In fact, TG expression is low in the primary tumor mass, but augmented when cells acquire the metastatic phenotype. Nevertheless, in other cases, the use of inducers of TG transamidating activity seems to contrast tumor cell plasticity, migration and invasion. In the following review, the function of TGs in cancer cell migration into the extracellular matrix, adhesion to the capillary endothelium and its basement membrane, invasion and angiogenesis is discussed.     

Development of a metastatic fluorescent Lewis Lung carcinoma mouse model: Identification of mRNAs and microRNAs involved in tumor invasion

Cancer metastasis is the foremost cause of death in cancer patients. A series of observable pathological changes takes place during progression and metastasis of cancer, but the underlying genetic changes remain unclear. Therefore, new approaches are required, including insights from cancer mouse models. To examine the mechanisms involved in tumor metastasis, we first generated a stably transfected Lewis Lung carcinoma cell line expressing a far-red fluorescent protein, called Katushka. After in vivo growth in syngeneic mice, two fluorescent Lewis Lung cancer subpopulations were isolated from primary tumors and lung metastases. The metastasis-derived cells exhibited a significant improvement in in vitro invasive activity compared to the primary tumor-derived cells, using a quantitative invasion chamber assay. Moreover, expression levels of 84 tumor metastasis-related mRNAs, 88 cancer-related microRNAs as well as Dicer and Drosha were determined using RT-qPCR. Compared to the primary Lewis Lung carcinoma subculture, the metastasis-derived cells exhibited statistically significantly increased mRNA levels for several matrix metalloproteinases as well as hepatocyte growth factor (HGF) and spleen tyrosine kinase (SYK). A modest decrease in Drosha and Dicer mRNA levels was accompanied by significant downregulation of ten microRNAs, including miR-9 and miR-203, in the lung metastatic Lewis Lung carcinoma cell culture. Thus, a tool for cancer metastasis studies has been established and the model is well suited for the identification of novel microRNAs and mRNAs involved in malignant progression. Our results suggest that increases in metalloproteinase expression and impairment of microRNA processing are involved in the acquirement of metastatic ability.     

Cell heterogeneity and subpopulations in solid tumors characterized by simultaneous immunophenotyping and DNA content analysis

BACKGROUND: Heterogeneity in human malignant tumors is a well-described phenomenon and of interest with regard to subpopulations with differences in clonality, metastatic potential, and response to therapy under different treatment regimes. The aim of this study was the simultaneous characterization of surface markers and DNA content of solid tumors to identify tumor cell subpopulations and to study the association between the expression of antigens and DNA content. METHODS: In the present study, six different malignant tumors grown as xenografts in nude mice were characterized by five-parameter flow cytometry. Immunophenotyping was performed using a variety of direct fluorescence-conjugated antibodies. In all cases, simultaneous detection of DNA content was done after staining with 7-aminoactinomycin D. RESULTS: Tumor cells were characterized by light scatter properties, antigen expression, and DNA content. Tumor cell heterogeneity, subpopulations, and DNA content-dependent antigen expression were identified. CONCLUSIONS: This method offers the possibility of characterizing solid tumors according to their immunophenotype and DNA content. The results obtained can be used to identify changes in immunophenotypic and DNA profiles of tumor cell populations before and after therapy and might be useful to define parameters predictive for response to therapy.     

Chemotactic response of rat mammary adenocarcinoma cell clones to tumor-derived cytokines

A cytokine with an apparent molecular weight of 53,000 daltons was isolated from serum-free medium conditioned by MTLn3 cells or from homogenates of MTLn3 cells, a highly metastatic variant of the rat 13762NF mammary adenocarcinoma. The chemotactic responses of MTLn3 and the low metastatic variant MTLn2 cells to this cytokine were tested in vitro using modified Boyden chambers. Both the chemotactic and chemokinetic movements of MTLn3 cells were stimulated by the MTLn3-derived cytokine. In addition, the MTLn3-derived cytokine stimulated a relatively small, but significant chemotactic migration of MTLn2 tumor cells, while these cells did not respond to medium conditioned by MTLn2 cells. MTLn3 cells themselves did not respond chemotactically to type I collagen or medium conditioned by MTLn2 cells. These results suggest that the chemotactic response may be a function of metastatic potential of the invading tumor cells. The production of tumor cytokines that enhance tumor cell motility may thus represent a phenotypic difference between 13762NF tumor cell subpopulations of high and low metastatic potential.     

Involvement of highly sulfated chondroitin sulfate in the metastasis of the Lewis lung carcinoma cells

The altered expression of cell surface chondroitin sulfate (CS) and dermatan sulfate (DS) in cancer cells has been demonstrated to play a key role in malignant transformation and tumor metastasis. However, the functional highly sulfated structures in CS/DS chains and their involvement in the process have not been well documented. In the present study, a structural analysis of CS/DS from two mouse Lewis lung carcinoma (3LL)-derived cell lines with different metastatic potentials revealed a higher proportion of Delta(4,5)HexUA-GalNAc(4,6-O-disulfate) generated from E-units (GlcUA-GalNAc(4, 6-O-disulfate)) in highly metastatic LM66-H11 cells than in low metastatic P29 cells, although much less CS/DS is expressed by LM66-H11 than P29 cells. This key finding prompted us to study the role of CS-E-like structures in experimental lung metastasis. The metastasis of LM66-H11 cells to lungs was effectively inhibited by enzymatic removal of tumor cell surface CS or by preadministration of CS-E rich in E-units in a dose-dependent manner. In addition, immunocytochemical analysis showed that LM66-H11 rather than P29 cells expressed more strongly the CS-E epitope, which was specifically recognized by the phage display antibody GD3G7. More importantly, this antibody and a CS-E decasaccharide fraction, the minimal structure recognized by GD3G7, strongly inhibited the metastasis of LM66-H11 cells probably by modifying the proliferative and invading behavior of the metastatic tumor cells. These results suggest that the E-unit-containing epitopes are involved in the metastatic process and a potential target for the diagnosis and treatment of malignant tumors.     

Quantitative variations in gene expression: possible role in cellular diversification and tumor progression.

Changes in the quantitative expression of certain genes or in the amounts of their products can quickly stimulate progression to the metastatic phenotype. This has been done experimentally by transferring dominantly acting oncogenes such as c-H-rasEJ into susceptible cells or more recently by interfering with metastasis suppressor genes. In vivo such rapid qualitative changes in dominantly acting oncogenes or suppressor genes occur only rarely, and progression to highly metastatic phenotypes is thought to occur through a process involving the slow stepwise progression of a subpopulation of neoplastic cells to more malignant states. Such slow changes can be reversible and need not involve known dominantly acting oncogenes or metastatic suppressor genes, consistent with clinical and experimental observations on naturally occurring, highly advanced metastatic tumors. An important element in the natural progression of tumors to more malignant states may be their ability to circumvent host environmental controls that regulate growth and cellular diversity. They also evolve into heterogeneous cellular phenotypes, a process that appears to mainly involve quantitative changes in gene expression but can be rapidly stimulated in cell culture by the introduction of a dominantly acting oncogene or inhibited by the introduction of a suppressor gene. The oncogenes and suppressor genes that affect malignancy may control important steps in the quantitative regulation of sets of genes that are ultimately responsible for the cellular alterations seen in adhesion receptors, cell motility responses, cell-cell communication components, degradative enzymes and their inhibitors, growth factor receptors, components that aid in escape from host surveillance mechanisms and others that are important in malignancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human Breast Tumor Cells Are More Resistant to Cardiac Glycoside Toxicity Than Non-Tumorigenic Breast Cells

Cardiotonic steroids (CTS), specific inhibitors of Na,K-ATPase activity, have been widely used for treating cardiac insufficiency. Recent studies suggest that low levels of endogenous CTS do not inhibit Na,K-ATPase activity but play a role in regulating blood pressure, inducing cellular kinase activity, and promoting cell viability. Higher CTS concentrations inhibit Na,K-ATPase activity and can induce reactive oxygen species, growth arrest, and cell death. CTS are being considered as potential novel therapies in cancer treatment, as they have been shown to limit tumor cell growth. However, there is a lack of information on the relative toxicity of tumor cells and comparable non-tumor cells. We have investigated the effects of CTS compounds, ouabain, digitoxin, and bufalin, on cell growth and survival in cell lines exhibiting the full spectrum of non-cancerous to malignant phenotypes. We show that CTS inhibit membrane Na,K-ATPase activity equally well in all cell lines tested regardless of metastatic potential. In contrast, the cellular responses to the drugs are different in non-tumor and tumor cells. Ouabain causes greater inhibition of proliferation and more extensive apoptosis in non-tumor breast cells compared to malignant or oncogene-transfected cells. In tumor cells, the effects of ouabain are accompanied by activation of anti-apoptotic ERK1/2. However, ERK1/2 or Src inhibition does not sensitize tumor cells to CTS cytotoxicity, suggesting that other mechanisms provide protection to the tumor cells. Reduced CTS-sensitivity in breast tumor cells compared to non-tumor cells indicates that CTS are not good candidates as cancer therapies.     

Role of platelets and platelet receptors in cancer metastasis

The interaction of tumor cells with platelets is a prerequisite for successful hematogenous metastatic dissemination. Upon tumor cell arrival in the blood, tumor cells immediately activate platelets to form a permissive microenvironment. Platelets protect tumor cells from shear forces and assault of NK cells, recruit myeloid cells by secretion of chemokines, and mediate an arrest of the tumor cell platelet embolus at the vascular wall. Subsequently, platelet-derived growth factors confer a mesenchymal-like phenotype to tumor cells and open the capillary endothelium to expedite extravasation in distant organs. Finally, platelet-secreted growth factors stimulate tumor cell proliferation to micrometastatic foci. This review provides a synopsis on the current literature on platelet-mediated effects in cancer metastasis and particularly focuses on platelet adhesion receptors and their role in metastasis. Immunoreceptor tyrosine-based activation motif (ITAM) and hemi ITAM (hemITAM) comprising receptors, especially, glycoprotein VI (GPVI), FcγRIIa, and C-type lectin-like-2 receptor (CLEC-2) are turned in the spotlight since several new mechanisms and contributions to metastasis have been attributed to this family of platelet receptors in the last years.