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Seminars in Cancer Biology 21 (2011) 99–106

Contents lists available at ScienceDirect

Seminars in Cancer Biology

journa l homepage: www.e lsev ier .com/ locate /semcancer

eview

etastatic colonization: Settlement, adaptation and propagation of tumor cellsn a foreign tissue environment

sukasa Shibuea,b, Robert A. Weinberga,b,c,∗

Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USAMIT Ludwig Center for Molecular Oncology, 77 Massachusetts Ave., Cambridge, MA 02139, USADepartment of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, USA

r t i c l e i n f o

eywords:etastasis

xtravasationolonizationormancyumor–host interactions

a b s t r a c t

Disseminated tumor cells must negotiate multiple situations that challenge their viability and/or prolif-erative capacity before they can successfully colonize distant organ sites. Thus, the shear stress causedby the blood flow may physically damage tumor cells during their translocation from primary tumors todistant organs via the circulation. In addition, the tissue microenvironment of distant organs is gener-ally unfamiliar to tumor cells, limiting their proliferation within the parenchyma of these organs. Eachof these situations involves various types of interactions between tumor cells and host components,which either support or inhibit the establishment and subsequent progression of metastases. The initialformation of micrometastases, as well as their subsequent growth – often termed colonization – there-fore require complex adaptations by tumor cells to various host components, most of which are never

encountered by these cells during their growth within primary tumor sites. These difficulties explain whythe colonization of distant organs by disseminated tumor cells is an extraordinarily demanding task andthus inefficient, and suggests a number of potential targets that might be used in the future to interferetherapeutically with this process. Studying the details of tumor–host interactions at each of the stepsleading up to successful metastatic colonization may therefore pave the way for designing therapeutic

he m

strategies to counteract t

Hematogenous metastasis is the primary cause of cancer-ssociated mortality. This process, often referred to as theinvasion-metastasis cascade”, proceeds in a stepwise manner thategins with the invasion of surrounding host tissues by the tumorells. This is followed by the penetration of the blood vessel walls byhe tumor cells and the entrance of these cells into the circulation“intravasation”). After being disseminated via the blood stream toites anatomically distant from the primary tumor (“transport”),irculating tumor cells are arrested in the capillary beds (“arrest”),nvade through the microvascular walls, and enter the parenchymaf the target organs (“extravasation”), in which they may survive,roliferate and thereby establish metastatic colonies (“coloniza-

ion”) [1].

Various lines of evidence support the notion that systemic dis-emination of tumor cells can already occur at relatively earlytages of the progression of primary tumors in certain types of can-

∗ Corresponding author at: Whitehead Institute for Biomedical Research, 9 Cam-ridge Center, Cambridge, MA 02142, USA. Tel.: +1 617 258 5159;ax: +1 617 258 5213.

E-mail addresses: shibue@wi.mit.edu (T. Shibue), weinberg@wi.mit.eduR.A. Weinberg).

044-579X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcancer.2010.12.003

etastatic spread of malignant tumors.© 2010 Elsevier Ltd. All rights reserved.

cers, including those in the breast and prostate [2]. In addition, in anepidemiological study of breast cancer, tumor cells are estimated todisseminate from the primary site 5–7 years prior to the diagnosisof the primary tumor [3]. This phenomenon indicates the need tounderstand the steps of metastases that occur after disseminationof tumor cells from the primary site and to develop strategies toblock these later steps of the invasion-metastasis cascade.

In the present review, we will provide a brief summary of thecurrent understanding, controversies and future prospects in thestudies of tumor–host interactions, specifically those associatedwith the last steps of the invasion-metastasis cascade that fol-low the dissemination of tumor cells from the primary site, withspecial emphasis on the direct interactions between tumor cellsand the insoluble components of the host microenvironment. Forunderstanding the details of the individual steps of this cascade,readers are encouraged to refer to more specific review articles[4–9].

1. Transport and arrest in the capillary beds

Tumor cells that have entered the circulation are likely to betransported quickly to distant organ sites via the blood stream andto become lodged in the capillary beds of these organs. Thus, in mul-

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iple experimental models of metastasis in mice, the great majorityf the tumor cells that are injected directly into the venous circula-ion are arrested within minutes in the microvasculature of the firstrgan that they encounter [10]. In accord with these experimentalbservations, a clinical study of breast cancer demonstrated thathe half-life of the tumor cells within the circulation was in rangef 1–2.4 h, a number that was calculated by taking blood samplesrom metastasis-free breast cancer patients at short intervals start-ng immediately after the surgical removal of the primary tumornd tracing the changes in the numbers of circulating tumor cellsithin these blood samples [11].

Two distinct mechanisms are thought to underlie the arrest ofirculating tumor cells in the microvasculature: mechanical entrap-ent due to size restriction and adhesion of the tumor cells to the

ascular endothelium. Indeed, B16F1 mouse melanoma cells thatre injected into mice via the portal vein are arrested in the liverear the ends of terminal portal venules due to size restriction [12].imilarly, CT-26 mouse colon carcinoma cells injected through theecal vein do not adhere to the larger portal vessels but are insteadrapped mechanically at sinusoids [13]. In contrast, HT-29 humanolon carcinoma cells can easily pass through the microvessels ofhe liver when injected into rats via the left ventricle; however, araction of these cells adhere to the walls of microvessels whoseuminal diameters are larger than those of these cells [14]. Hence,oth the mechanical entrapment and adhesion can contribute func-ionally to the initial arrest of tumor cells in the capillary beds; theelative contributions of these two mechanisms is likely to varyepending on the conditions such as the types of tumor cells andarget organs.

The adhesion of circulating tumor cells to the capillary wallsf target organs is analogous, at least superficially, to the processf leukocyte adhesion to the capillary endothelium [15]. Thus, innflammatory responses, leukocytes activated by proinflammatoryytokines – such as interleukin (IL) -1, -6, -8 and tumor necrosisactor (TNF)-� – first become arrested on the luminal surfaces ofndothelial cells in inflammatory tissues, which also is activatedy the same set of cytokines; this arrest is mediated by low-affinity

nteractions between the glycoproteins, such as P-selectin glyco-rotein ligand-1 (PSGL-1), on the surface of these leukocytes andelectins – the transmembrane receptors for these glycoproteins –hat are expressed on endothelial cell surface. These weak interac-ions, together with the propulsion provided by the blood flow,esult in the rolling of leukocytes on the luminal surface of thendothelium. This rolling behavior is soon followed by the forma-ion of more stable adhesions between the cell adhesion moleculesCAMs) of the immunoglobulin (Ig) superfamily, such as ICAM-1Inter-Cellular Adhesion Molecule-1) and VCAM-1 (Vascular Celldhesion Molecule-1), on the luminal surfaces of endothelial cellsnd their ligands – mainly integrins – on the surface of the leuko-ytes [15].

Certain types of tumor cells exhibit similar rolling behav-or on the endothelial surface, which is also mediated by theelectin–glycoprotein interactions (Fig. 1). For example, MDA PCab human prostate carcinoma cells exhibit rolling behavior inonolayer culture of bone marrow endothelial cells (BMECs) under

onditions of shear flow. This behavior is dependent on the associ-tions between sialylated glycoproteins on MDA PCa 2b cells and-selectin on BMECs [16]. However, there also are other situationsn which tumor cells do not exhibit rolling behavior prior to theirrrest in the capillary beds [17,18]. For example, lung metastasisormation by cells of the DU-145 human prostate carcinoma cell

ine and the MDA-MB-435 human breast cancer cell line is notnhibited by the functional blocking of selectins with neutraliz-ng antibodies [19]. Hence, the general importance of the selectin-nd glycoprotein-dependent rolling behavior for the subsequentormation of metastases remains unclear.

ancer Biology 21 (2011) 99–106

Regardless of the relative importance of the rolling behavior, thesubsequent stable adhesions between tumor cells and the endothe-lium are likely to be mediated by the binding of integrins onthe surface of tumor cells to CAMs of the Ig superfamily on theendothelial cell surface. These stable interactions have been foundto be important for the subsequent extravasation of tumor cellsinto the parenchyma of target organs and thus for the formationof metastatic colonies. For example, studies using A375M humanmelanoma cells and B16BL6 mouse melanoma cells have revealedthe essential role of interactions between tumor cell integrin �4�1and endothelial cell VCAM-1 in the development of lung metastases[20,21].

The interactions between tumor cells and endothelial cells aresupposed to account, in part, for the organ tropisms of metastases.Thus, endothelial cells from different anatomical locations exhibitdistinct adhesive properties on their luminal surfaces and certaintumor cells may selectively bind to the endothelium of organs thatare preferentially colonized by these tumor cells. Indeed, in vitroexamination of the adhesion of various human prostate cancer celllines, including PC-3, TSU, LNCaP and DU-145, to the endothelialcells of various origins has revealed that these prostate cancer cellspreferentially adhere to the endothelial cells derived from the bonemarrow relative to those derived from other origins such as thelungs or umbilical vein [22,23]. This is consistent with the patternsof metastasis in prostate cancer, a cancer type that metastasizesalmost exclusively to the bone.

Another example of the mechanisms accounting for the organ-specific metastasis formation comes from a study of Metadherin,a transmembrane protein that was identified in a phage displayscreening for proteins mediating breast cancer cell adhesion to thelung endothelium [24]. After intracardiac injection into mice, phageexpressing Metadherin accumulated in the lungs, but not in liver,brain or bone; this argued that Metadherin mediates selective bind-ing of cells to the lung endothelium. Indeed, blocking the expressionor function of Metadherin by small interfering RNA (siRNA) or bya neutralizing antibody, respectively, resulted in impaired lungmetastasis formation by 4T1 mouse mammary carcinoma cells,indicating that Metadherin-dependent homing of tumor cells to thelungs enables the efficient development of lung metastases [24].

Yet other mechanisms that have been proposed to contribute tothe organ tropisms of metastases include the combination of locallyproduced chemokines and chemokine receptors expressed on thesurface of tumor cells. The role of chemokines and their receptorsin the chemotaxis of leukocytes during inflammatory responsesis well established [15]; tumor cells may exploit these interac-tions, explaining their preferential metastasis to certain organs. Forexample, the CXCR4 receptor expressed on the surface of MDA-MB-231 human breast cancer cells has been shown to facilitate themetastasis of these cells to the lungs, an organ rich in the CXCR4ligand CXCL12/SDF-1 [25]. Similarly, the CCR10-CCL27 pair is impli-cated in melanoma metastasis to skin [26]. However, the patterns ofcirculation between the primary site and the secondary site, as wellas other properties of the target organs, such as tissue architectureand the local availability of growth factors, might also exert strongeffects on the organ specificity of metastases. Consequently, theimportance of tumor cell–endothelial cell adhesion and chemokinereceptor signaling to the organ tropisms of metastases remainsunclear.

Tumor cells also interact with various components of the bloodwhile passing through the general circulation and becoming lodgedin the lumina of capillaries (Fig. 1). These interactions involve

platelets, polymorphonuclear leukocytes (PMNs), monocytes, lym-phocytes as well as the multiple plasma proteins. Among these, themetastasis-promoting role of the interactions between tumor cellsand components of the blood-clotting machinery, including fib-rin clots and aggregated platelets, is well established [27]. Indeed,

T. Shibue, R.A. Weinberg / Seminars in Cancer Biology 21 (2011) 99–106 101

Fig. 1. Tumor–host interactions during the transport, arrest and extravasation steps of metastasis. During transport via the blood stream, tumor cells interact with multiplec y, sucb or cellh ch of tr for th

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onstituents of the blood; these include components of the blood clotting machinereds of target organs and extravasation into the parenchyma of these organs, tumost-derived factors (green) involved in the tumor–host interactions occurring in eaetraction (VEGF, 12(S)-HETE) or death (ROS) of the endothelial cells. See main text

icroscopic observations of B16F10 mouse melanoma cells lodgedithin the lung microvasculature have revealed their close associ-

tions with fibrin clots and platelets [28]. The deposition of fibrinnd platelets around the tumor cells appears to serve as a barrierhat protects tumor cells from the mechanical stress of blood flows well as attack by immunocytes, such as natural killer (NK) cells27]. Moreover, fibrin and platelets might also facilitate the arrestf tumor cells in small-diameter capillaries, doing so via a mech-nism involving bridge formation between tumor cells that havereviously adhered to the endothelium and those flowing freely inhe blood [29]. In support of these notions, pharmacological inhibi-ions of blood clotting by various agents, including heparin, vitamin

antagonists and prostacyclin, all result in impaired metastasisormation in various experimental models in mice and rats [27].imilarly, a genetic depletion of fibrinogen, the precursor of fib-in, as well as genetic defects of platelet production, both result inemarkable reductions in the efficiency of lung metastasis forma-ion by B16F10 mouse melanoma cells [30].

While residing in the blood, tumor cells can trigger coagu-ation and platelet activation through mechanisms that includehe elevated expression of tissue factor (TF) on their surface. TFs a transmembrane glycoprotein that produces thrombin, a keynzyme in the coagulation machinery, from its precursor pro-hrombin; thrombin, in turn, induces fibrin clot formation andlatelet activation [31]. The importance of TF-dependent clottingachinery on the progression of metastases is supported by the

ight correlation between elevated TF expression and poor prog-osis in multiple tumor types, including colorectal, breast andon-small cell lung carcinomas [31].

The interactions between tumor cells and platelets are alsoediated, in part, via the associations between mucin-like glyco-

roteins on the surface of tumor cells and P-selectin expressedy platelets [29]. The importance of these specific associations

s supported by a study in which LS180 human colon carcinomaells were injected intravenously into wild-type and P-selectin-

eficient mice [32]. Within the circulation of P-selectin-deficientice, LS180 cells did not become associated with platelets, whereas

hose cells injected into wild-type mice acquired a thick coat oflatelets. In the P-selectin-deficient mice, the failure by LS810 cellso acquire a platelet coat was associated with a reduced arrest in the

h as fibrin clots and platelets. In the subsequent processes of arrest in the capillarys closely interact with the endothelial cells. Tumor cell-derived factors (blue) andhese steps are listed. During extravasation, tumor cell-derived factors induce eithere details.

lung microvasculature and an impaired lung metastasis formation[32]. These various pieces of experimental and clinical evidencecollectively point to the importance to metastasis formation oftumor–host interactions occurring intraluminally within the bloodvessels.

In addition to the heterotypic interactions between tumorcells and the host components, homotypic interactions withinthe tumor cell population (i.e., cell clumping) might also con-tribute to the progression of metastases. Thus, in a study of T241mouse fibrosarcoma model, when cells were administered intra-venously as clumps of 10–12 cells, lung metastases developedfar more efficiently than when the same number of cells wereinjected as a single cell suspension [33]. Indeed, the formation ofintravascular tumor clumps in lung arteries has long been recog-nized clinically [34]. Further studies will be required to assess thegeneral importance of intravascular tumor cell clumping in metas-tases. For example, it will be interesting to determine whethermetastasis formation by cell clumps can be reconciled with thelargely monoclonal nature of subsequently arising metastaticcolonies [35].

2. Extravasation/intravascular growth

A fraction of tumor cells that are arrested in the capillarybeds of target organs may subsequently extravasate through theendothelial walls and enter the parenchyma of these organs, whichis usually considered to be essential for the eventual establish-ment of metastatic colonies [1] (Fig. 1). The precise mechanismsunderlying tumor cell extravasation still remain elusive. Often, thisprocess is analogized to the better understood process of leukocyteextravasation into inflammatory tissues—the process of diapedesis[6,36]. Indeed, the extravasation of both tumor cells and leuko-cytes is preceded by, and probably requires, the formation of stableinteractions between the extravasating cells (i.e., tumor cells andleukocytes) and endothelial cells.

Nonetheless, the differences between extravasating cancer cellsand leukocytes undergoing diapedesis are fundamental: leuko-cytes are biologically adapted to execute diapedesis, resulting inits completion within minutes, while tumor cells do so only ineffi-ciently and often require a day or two to successfully extravasate

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37,38]. Moreover, while leukocytes usually transmigrate throughhe endothelium without significantly disrupting the endothelial

onolayer, the tumor cell extravasation is frequently associatedith retraction of endothelial cells from one another or their death

6,36]. In sinusoidal capillaries, such as those observed in liver andone marrow [39], tumor cells may pass through preexisting gapsetween endothelial cells without damaging these cells.

Several distinct mechanisms have been reported by whichumor cells induce endothelial cells to retract from one another orndothelial death during extravasation. The involvement of tumorell-derived bioactive lipids in endothelial retraction has been sug-ested; some tumor cells can induce this process via production of2(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], a major lipoxy-enase metabolite of arachidonic acid [40–42]. Indeed, the additionf 12(S)-HETE to a monolayer culture of endothelial cells induces aeversible retraction of these cells through a mechanism involvinghe redistribution of integrin �v�3 on the endothelial cell surface41]. In a series of B16a mouse melanoma cell lines, the produc-ion of 12(S)-HETE was correlated with the activity of these cells tonduce endothelial cell retraction as well as with the ability of theseells to form metastases [42]. More recently, 12(S)-HETE was alsodentified as a factor responsible for the endothelial cell-retractingbility within the culture medium of MCF7 human breast cancerells [43].

As an alternative to endothelial retraction, tumor cell extravasa-ion might also be facilitated by mechanisms involving endothelialnjury; these include the oxidative stress arising from the interac-ions between tumor cells and endothelial cells. Indeed, multipleines of malignant melanoma cells release reactive oxygen speciesROS) upon adherence to the confluent monolayer of endothe-ial cells in vitro, which subsequently cause irreversible damageo the endothelial cells [44]. Moreover, in an experimental lung

etastasis model of rats, Walker 256 rat carcinosarcoma cells thatre arrested at the lung microvasculature exhibit H2O2 productionlong the contact sites with the lung tissue [45], suggesting the con-ribution of ROS to the in vivo interactions between tumor cells andndothelial cells.

Accumulating evidence also implicates regulators of angiogene-is/vascular remodeling in tumor cell extravasation. Thus, vascularndothelial growth factor (VEGF), a central regulator of angiogen-sis, appears to play essential roles in the tumor cell extravasationnder several experimental conditions. The extravasation of CT-6 colon carcinoma cells through the lung endothelium dependsn the VEGF-induced dissolution of endothelial cell–cell junctions;his response is mediated, in turn, by the VEGF-dependent activa-ion of Src family kinases in the endothelial cells and the resultingissociation of �-catenin from VE-cadherin, which is responsibleor forming the adherens junctions between adjacent endothelialells [46]. VEGF was also found to be essential for the migra-ion of MDA-MB-231 human breast cancer cells through a layerf human brain microvascular endothelial cells (HBMECs) in vitro47].

More recently, in a search for factors whose expression is corre-ated with the capability of breast cancer cells to metastasize to theungs, Massagué and colleagues identified multiple factors that arelso known to be involved in vascular remodeling; these includepiregulin, cyclooxygenase 2, as well as matrix metalloproteinaseand 2 [48]. Knocking-down the expression of these factors in com-ination impairs the extravasation of MDA-MB-231 human breastancer cells into the lung parenchyma. Accordingly, these agentsromote breast cancer metastasis at least in part by facilitating

he tumor cell extravasation in this tissue. Interestingly, expres-ion profiling of clinical samples has revealed that the expression ofhese factors within the primary tumor correlated with the devel-pment of metastases to the lungs but not to the bone [49]; thiss consistent with the notion that the mechanism of tumor cell

ancer Biology 21 (2011) 99–106

extravasation may differ depending on the identity of the targetorgan.

There are also cases in which tumor cells initially proliferateintraluminally within the vasculature and begin to form metas-tases without undergoing extravasation; as these metastases growin size, they will inevitably rupture the microvascular wall andthereby invade into the tissue parenchyma. Several cancer celllines, including HT1080 human fibrosarcoma cells, 2.10.10 ratembryo fibroblast-derived cells [50] and PC-3 human prostate can-cer cells [51] do not effectively extravasate into the parenchymaof the lungs and liver when injected into mice. Instead, thesecells start proliferating while still attached to the luminal surfacesof the endothelium. Analyses of metastases from the sponta-neous mammary tumor formed in C3H/He mice have also revealedthe intravascular origin of lung metastases [52]. Accordingly,metastatic colonization may result either from the proliferation oftumor cells within the organ parenchyma following extravasationor from their initial proliferation within the vascular lumen prior toextravasation. The relative contributions of these alternative pro-cesses may well vary depending on the types of tumor cells andtarget organs.

3. Colonization

Independent on the mode of extravasation, a rapidly growingbody of evidence indicates the key role of tumor–host interactionswithin the parenchyma of target organs as a primary determinantof the subsequent success of metastatic colonization—the processinvolving the growth of micrometastases into macroscopic tumors.Using the technique of intravital imaging and various mouse mam-mary carcinoma and melanoma cell lines, Chambers and colleagueshave demonstrated that the inability to actively proliferate follow-ing extravasation into the parenchyma of a target organ is a majorobstacle in metastatic colonization [12,53,54]. In fact, untrans-formed mammary epithelial cells can survive in the parenchyma ofthe lungs for up to 4 months after intravenous injection, indicatingthat even these untransformed cells can pass through the process ofhematogenous transport, arrest in microvasculature and extrava-sation, albeit in the absence of vigorous proliferation within thelung parenchyma [55]. In the sections that follow, we will focus onthe interactions between extravasated tumor cells and componentswithin the host parenchyma that either foster or hinder subsequenttumor cell proliferation.

In general, extravasated tumor cells will follow one of the threealternative courses—cell death, dormancy (i.e., survival withoutapparent increase in cell number), or colony formation by contin-uous proliferation in the absence of counterbalancing cell death,permitting net increases in cell number [56]. The determinantsof these alternative fates include the interactions of tumor cellswith the constituents of the target organ parenchyma, such as ECMcomponents and various host stromal cells (Fig. 2). In addition, vas-cularization and immune surveillance appear to have significanteffects on the fate of extravasated tumor cells (Fig. 2).

3.1. Interactions with the ECM components of the target organparenchyma

The important role of cell–ECM interactions in the prolifera-tion and survival of tumor cells has been well established [9,57].Thus, binding of tumor cell integrins to ECM ligands activates

a form of intracellular signaling that promotes cell proliferationand/or survival; this type of integrin-dependent intracellular signaltransduction is generally referred to as “outside-in signaling” [58].Integrins can directly activate signaling proteins, such as focal adhe-sion kinase (FAK). In addition, they can also crosstalk with other

T. Shibue, R.A. Weinberg / Seminars in Cancer Biology 21 (2011) 99–106 103

Fig. 2. Tumor–host interactions in the post-extravasation process of metastasis. The interactions between tumor cells and host components also play key roles in determiningthe fate of tumor cells after extravasating into the parenchyma of target organs. The host components interacting with tumor cells in this post-extravasation process ofmetastasis include extracellular matrix (ECM), immunocytes (such as macrophages, CD8+ T cells, and NK cells), blood vessels (i.e., endothelial cells), and other organ-specificc er). Asi r cellc sts anp prost

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ells (such as osteoclasts and osteoblasts in the bone and hepatocytes in the livnteractions are listed in blue and green text, respectively. (*) Different types of tumoancer metastases to the bone often result in the preferential activation of osteoclaredominantly osteoblastic because of the selective activation of osteoblasts by the

ignaling pathways, such as those governed by growth factor recep-ors, to augment the effects of ligand-activated signaling [57]. Givenhe differences that are likely to distinguish the organization of ECMn the primary tumor site from that present in the ECM of targetrgans, it is likely that certain integrin-dependent signals that per-itted tumor cell proliferation and/or survival within the primary

ite are no longer available for tumor cells after their extravasationnto the parenchyma of target organs. This thinking points to theotential importance of integrin-mediated cell–ECM interactions

n determining the fate of extravasated tumor cells (Fig. 2).For example, recent studies by others and ourselves have

evealed the essential contribution of integrin �1-mediatedell–ECM interactions and the resulting activation of FAK, a centralediator of integrin-dependent signaling, in enabling the pro-

iferation of tumor cells following their extravasation into theung parenchyma [59,60]. Thus, post-extravasation proliferationn the lung parenchyma of colonization-competent D2A1 mouse

ammary carcinoma cells is diminished by knocking down thexpression of either integrin �1 or FAK in these cells [59].

Our subsequent analyses show that the related, D2.0R and D2.1olonization-deficient mouse mammary carcinoma cells exhibitower levels of FAK activation after entering the lung parenchyma,

lthough these cells display levels of integrin �1 and FAK expressionomparable to those observed in the colonization-competent D2A1ells. Moreover, the patterns of integrin �1 distribution are dif-erent between colonization-competent D2A1 cells and the otherwo colonization-deficient cells; thus, only D2A1 cells, but not the

in Fig. 1, tumor cell-derived factors and host-derived factors involved in theses interact with the organ-specific cell types in distinct manners. For example, breastd are therefore osteolytic. In contrast, prostate cancer metastases to the bone areate cancer cells. See main text for the details.

other two cell types, developed abundant integrin �1-containingadhesion plaques of elongated morphology within the parenchymaof the lungs (T.S. and R.A.W., manuscript in preparation). This,together with the functional connection between elongated adhe-sion plaque formation and FAK activation, suggested that themechanism enabling the assembly of these adhesion plaques is alsolikely to govern the proliferation of cancer cells that have alreadyextravasated into the parenchyma of target organs, doing so byregulating the activation of FAK.

Cell-surface receptors of ECM other than integrins, such as CD44– a receptor of the ECM component hyaluronan – also play impor-tant roles in determining the fate of extravasated tumor cells. Thus,the overexpression of a dominant-inhibitory, soluble isoform ofCD44 (sCD44) in extravasated TA3/St mouse mammary carcinomacells resulted in a remarkable increase in their rate of apoptosiswithin the lung parenchyma [61]. Interestingly, the overexpressionof sCD44 does not inhibit the adhesion of these cells to the lungendothelium or their penetration to the parenchyma of the lungs;this indicated the specific role of the CD44–hyaluronan interactionsin promoting the post-extravasation survival of these tumor cells.

The ECM also functions as a reservoir of multiple growthfactors, such as transforming growth factor beta (TGF-�), bone mor-

phogenic proteins (BMPs) and vascular endothelial growth factor(VEGF) [62]. These growth factors, some of which become avail-able to tumor cells upon ECM processing by proteases like matrixmetalloproteinases (MMPs), may also contribute to the prolifer-ation and/or survival of extravasated tumor cells. These diverse

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ines of evidence illustrate the critical role of the ECM within thearenchyma of target organs in controlling the fate of extravasatedumor cells.

.2. Interactions with the host cells residing in the target organarenchyma

In addition complex ECM interactions, as detailed above, dis-eminated tumor cells must also cope with a variety of cells thateside within these sites (Fig. 2). In particular, cells of the immuneystem have both supportive and inhibitory effects on the processf colonization [63]. Early clinical experience with organ transplan-ation revealed cases of the outgrowth of donor-derived tumor cellsithin the bodies of recipients shortly after the transplantation

64]. In most of these cases, the donors had a history of potentiallyetastatic cancer in other organs; the formation of donor-derived

umor was therefore attributable to the minimal metastatic dis-ase within the donated organs whose outgrowth was permitted byhe immune suppression of the transplant recipients. These caseseveal the role of immune surveillance in suppressing the forma-ion of macroscopic metastases after tumor cells have establishedesidence within distant organ sites.

In fact, several distinct types of immune cells exert inhibitoryffects on metastatic colonization in experimental models. Forxample, Killion and Fidler demonstrated that macroscopic metas-asis formation by B16BL6 mouse melanoma cells in the lungs wasfficiently blocked by systemically activating macrophages via thentravenous administration of liposome-encapsulated macrophagectivators; in these experiments, the liposomes were introducedfter the melanoma cells had taken up residence within the lungarenchyma [65], indicating that activated macrophages blockedhe post-extravasation metastatic progression. In the RET.AAD

odel of mouse melanoma, the depletion of CD8+ cytotoxic T cellsesulted in the more rapid outgrowth of metastases in multiple vis-eral organs, including lungs, liver and bladder, without affectinghe efficiency of initial melanoma cell seeding to these organs [66].n the liver metastasis model of mouse fibrosarcoma L929 cells,he cytotoxic effect of NK cells eliminated the formation of macro-copic metastases via a mechanism involving TRAIL (tumor necrosisactor-related apoptosis-inducing ligand) expressed on the surfacef NK cells [67]. In contrast to these, certain types of immune cells,uch as macrophages, may also have supportive effects on the pro-ess of colonization [68], possibly by remodeling the environmentf the target organ parenchyma, as discussed below.

Endothelial cells represent yet another important cellular com-onent of the host tissue that can influence metastatic colonization.hus, access to sufficient supplies of oxygen and nutrients is essen-ial for tumors to grow continuously. Since oxygen can only diffusedistance of 150–200 �m from the capillaries, the formation of

ew blood vessels (i.e., angiogenesis) is critical for tumors to groweyond a certain size (generally 1–2 mm3); this applies to both pri-ary tumors and secondary tumors (metastases). A study using

uman liposarcoma cells has revealed that cancer cells within arimary tumor exhibit heterogeneity in their angiogenic activities69]. This, taken together with the largely monoclonal origin of

etastases [35], indicates that tumor angiogenesis, a process thats mediated by direct and indirect interactions between tumor cellsnd endothelial cells or their precursors [70], can be rate-limitingor the outgrowth of metastases.

Other organ-specific cell types can also exert considerablenfluence on the fate of extravasated tumor cells. This is well doc-

mented in the case of breast cancer metastasis to the bone. Thus,hese metastases to bone are usually osteolytic, a response that is

ediated by the production of osteoclast-activating factors, suchs parathyroid hormone-related protein (PTHrP), interleukin-1, 6nd 11, and granulocyte-macrophage colony stimulating factor

ancer Biology 21 (2011) 99–106

(GM-CSF), by the tumor cells [71–73]. Activated osteoclasts, inturn, facilitate the outgrowth of metastases by releasing bone-derived growth factors, such as transforming growth factor beta(TGF-�) and insulin-like growth factor 1 (IGF-1) [74,75]. In con-trast, prostate cancer metastases to the bone usually stimulatebone formation, which is mediated by the production by the tumorcells of osteoblast-activating factors, such as endothelin-1, bonemorphogenic proteins (BMPs), and platelet-derived growth factor(PDGF). Activated osteoblasts may, in turn, support the prolifera-tion and survival of tumor cells by releasing growth factors suchas IGF-1 [76]. Hence, multiple distinct host cell types have a majorimpact on the fate of extravasated tumor cells.

3.3. Processing the microenvironment of target organparenchyma by tumor cells

In addition to interacting with pre-existing components of thetarget organ parenchyma, active remodeling by tumor cells of themicroenvironment within the target organs appears to play a keyrole in their post-extravasation behavior. In some cases, tumorcells growing in the primary site secrete factors that can directlyor indirectly alter the microenvironments of distant sites prior tothe dissemination of tumor cells, thereby preparing these sitesfor the subsequent arrival of tumor cells. Such altered microen-vironments of the distant sites are refereed to as “pre-metastaticniches”. Thus, mice bearing subcutaneous Lewis lung carcinoma(LLC) tumors or B16 melanoma tumors exhibit accumulations ofbone-marrow derived cells in the parenchyma of distant organs,notably the lungs, prior to the appearance of disseminated tumorcells in these sites [77,78]. The types of cells reported to accumu-late to these distant sites include VEGFR1+ hematopoietic precursorcells and Mac-1+ myeloid cells, and experiments have shown thatthese cells facilitate the subsequent development of metastases atthese sites [77,78].

Another mechanism reported to enable the microenvironmen-tal remodeling of target organs includes the secretion of lysyloxidase (LOX) into the circulation by orthotopoically implantedMDA-MB-231 breast cancer cells; this LOX supports metastasisdevelopment by crosslinking collagen IV in the basement mem-brane of the lungs and subsequently recruiting Mac-1+ myeloidcells to the lung tissue prior to the dissemination of tumor cells[79]. Moreover, a recent study in our group showed that a secretedglycoprotein osteopontin released from certain primary tumorscan contribute to the outgrowth of metastases through a mech-anism involving the enhanced mobilization of bone marrow cells;in this case, the mobilized bone marrow cells appear to instigatethe growth of already-established but otherwise-indolent tumorsat distant sites rather than priming the microenvironment prior tothe arrival of disseminated tumor cells [80].

4. Concluding remarks

Since the proposal of “seed and soil” hypothesis by StephenPaget in 1889 [81], it has been recognized that interactions betweentumor cells and host components have a major impact on the pro-gression of metastases. Similarly, the idea of “invasion-metastasiscascade”, i.e., the stepwise progression of metastases, was proposedmore than 30 years ago [82]. However, the molecular mechanismsunderlying the tumor–host interactions associated with each stepsof the invasion-metastasis cascade still remain largely elusive.

Attempts to understand tumor cell dissemination and metasta-sis formation at the molecular level have been thwarted, in part,by the difficulties in experimentally accessing and manipulatingthe in vivo conditions in which metastasis occur as well as thelack of in vitro model systems that closely recapitulate the com-

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lex conditions within the microenvironments of living tissues. Inight of recent technical progresses in both in vitro culture, such ashe development of three-dimensional tissue culture systems [83],nd in vivo experimental systems, such as advances in intravitalicroscopy [84], we anticipate that our understanding of metasta-

is will progress rapidly in the near future. With such informationn hand, we will finally begin to understand in detail the pathogenic

echanisms that are responsible for 90% of cancer-associated mor-ality.

unding

Breast Cancer Research Foundation (R.A.W.).

onflict of interests

The authors declare that there is no conflict of interest.

cknowledgements

We thank present and former members of Weinberg Labora-ory for the productive discussions. T.S. is a recipient of Long-Termellowship from Human Frontier Science Program, Postdoctoralellowship from Japan Society for the Promotion of Science, andostdoctoral Fellowship from the Ludwig Fund for Cancer Research..A.W. is an American Cancer Society research professor and aaniel K. Ludwig Foundation cancer research professor. This workas funded by Breast Cancer Research Foundation.

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