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Advanced Drug Delivery Reviews xxx (2011) xxx–xxx

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Advanced Drug Delivery Reviews

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Cancer Cell Invasion: Treatment and Monitoring Opportunities in Nanomedicine

Omid Veiseh a, Forrest M. Kievit a, Richard G. Ellenbogen b, Miqin Zhang a,b,⁎a Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USAb Department of Neurological Surgery, University of Washington, Seattle, WA 98195, USA

⁎ Corresponding author at: Department of MaterialsRoberts Hall, University of Washington, Seattle, WA 99356; fax: +1 206 543 3100.

E-mail address: [email protected] (M. Zha

0169-409X/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.addr.2011.01.010

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a b s t r a c t
a r t i c l e i n f o
Article history:Received 4 November 2010Accepted 25 January 2011Available online xxxx

Keywords:NanoparticlesNanotechnologyMolecular targetsAngiogenesisMetastasisContrast agentsGene therapyDrug deliveryImaging

Cell invasion is an intrinsic cellular pathway whereby cells respond to extracellular stimuli to migrate throughand modulate the structure of their extracellular matrix (ECM) in order to develop, repair, and protect thebody's tissues. In cancer cells this process can become aberrantly regulated and lead to cancer metastasis. Thiscellular pathway contributes to the vast majority of cancer related fatalities, and therefore has been identifiedas a critical therapeutic target. Researchers have identified numerous potential molecular therapeutic targetsof cancer cell invasion, yet delivery of therapies remains a major hurdle. Nanomedicine is a rapidly emergingtechnology which may offer a potential solution for tackling cancer metastasis by improving the specificityand potency of therapeutics delivered to invasive cancer cells. In this reviewwe examine the biology of cancercell invasion, its role in cancer progression and metastasis, molecular targets of cell invasion, and therapeuticinhibitors of cell invasion. We then discuss how the field of nanomedicine can be applied to monitor and treatcancer cell invasion. We aim to provide a perspective on how the advances in cancer biology and the field ofnanomedicine can be combined to offer new solutions for treating cancer metastasis.

Science & Engineering, 302L8195–2120. Tel.: +1 206 616

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ancer Cell Invasion: Treatment and Monitorin0

© 2011 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. Cell invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.1. Immune response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.2. Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.3. Lymphangiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.4. Cancer metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3. Molecular therapeutic targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.1. Cell adhesion proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.2. Proteinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.3. Ion/water channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.4. Transcription factors and signal transducers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4. Nanomedicine in treatment of cancer cell invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.1. Fundamentals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.2. Directing nanoparticles to cancer cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.3. Nanomedicine in imaging cancer cell invasion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.4. Nanomedicine in treating cancer cell invasion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

1. Introduction

Cell invasion is the migration of cells within a tissue and a criticalmechanism in tissue development, repair, and immune surveillance.

g Opportunities in Nanomedicine, Adv. Drug Deliv.

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However, this pathway can become aberrantly regulated in cancercells and lead to malignant invasion within local tissue, blood vesselformation, and lymphatic vessel formation [1]. Combined, theseevents lead to spread of cancer from its tissue of origin and itssubsequent growth in other organs, a process known as cancermetastasis [2]. Cancer metastasis attributes to the most life-threat-ening aspect of the disease, accounting for approximately 90% ofhuman cancer related deaths [3,4]. The clinical importance of thisprocess has garnered significant attention from oncologists and, todate, numerous molecular therapeutic targets have been identified[2,5]. However, inefficient delivery of therapies and development ofcancer cell resistance both remain major hurdles towards treatmentof invasion and metastasis. To circumvent these limitations research-ers are turning to the rapidly advancing field of nanotechnology todevelop nanomedicine-based solutions.

Nanomedicine is an emerging field that holds great potential tointervene with cancer at the molecular scale and deliver potent dosesof therapeutic agents to cancer cells with improved specificity andreduced toxicities [6,7]. At the core of nanomedicine is thedevelopment of nanoparticles (NPs; e.g., liposomes, dendrimers,magnetic NPs, quantum dots, and carbon nanotubes) that functionas carriers for therapeutics and molecular beacons for detection. NPsare materials assembled at the nanoscale (1–100 nm) in at least onedimension, and can be engineered to have multifunctional propertiesthrough the incorporation of multiple therapeutic, sensing, andtargeting agents [8–10]. Since Richard Feynman's prediction of theopportunities associated with nano-sized materials in 1959, numer-ous nanomaterial formulations have been introduced and evaluatedas tools for detection, prevention, and treatment in oncology [11]. Therecent advances in our understanding of cancer cell invasion havecreated new opportunities to develop NPs engineered to monitor andtreat cancer invasion and metastasis.

Nanoparticles developed for imaging and treatment of cancer cellinvasion offer numerous advantages over conventional medicine inthat they have the potential to enable preferential delivery of drugs totumors and delivery of more than one therapeutic agent forcombinatorial therapy. Other advantages of NPs include specificbinding of drugs to targets in cancer cells or the tumor microenvi-ronment, simultaneous visualization of tumors using innovativeimaging techniques, prolonged drug-circulation times, controlleddrug-release kinetics, and superior dose scheduling for improvedpatient compliance [6,12–15].

In this review we first examine the cancer cell invasion pathwayand identify a set of potential therapeutic targets that could beexploited in conjunction with nanomedicine to monitor and treatcancer cell invasion and metastasis. Next, we evaluate the currentstate of nanomedicine and present some examples used for treatmentand imaging of cancer cell invasion and metastasis. Finally, we discussthe direction of the field and opportunities available to further expandthe application of nanomedicine in tracking and treating cancer cellinvasion. We hope that our review will raise more interest forresearchers and oncologists to drive this emerging technology innanomedicine towards improved outcome of cancer treatment.

2. Cell invasion

Cell invasion is a complex and integrated process, whichorchestrates natural pathological processes in the body such asembryonic development, tissue repair, wound healing, and immuneresponse [16]. Cell invasion can be defined as the migration of cellswithin a tissue in response to chemical signals (e.g. hormones, growthfactors, or metabolites), physical cues (e.g. tissue stiffness, cell density,or cellular pattern and organization), and physicochemical processes(e.g. diffusion, or cell activation and deactivation). Deleteriousmutations in the cell invasion pathway can lead to disorders such asarthritis, atherosclerosis, aneurism, multiple sclerosis, and chronic

Please cite this article as: O. Veiseh, et al., Cancer Cell Invasion: TreatmeRev. (2011), doi:10.1016/j.addr.2011.01.010

obstructive pulmonary disease (COPD) [17–19]. In cancer, cellinvasion can lead to metastasis (i.e. the development of tumors insecondary locations away from the primary tumor) which accountsfor 90% of cancer related deaths [3,4].

Depending on the cell type and the host tissuematrix, cell invasioncan occur both as a single cell or as a collections of cells in clusters orsheets [20,21]. Single cell invasion facilitates the repositioning of a cellwithin tissues or secondary growths. Depending on the process, thecell movement can occur at a constant pace, or intermittently. Forexample, during morphogenesis cell movement occurs persistently ina highly orchestrated fashion. Conversely, during immune response,cells of the immune system transiently infiltrate intermittently,surveying the host tissue cells for infection or damage.

Collective cell invasion is the second principal mode by which cellrepositioning occurs within tissue [21,22]. This mode differs fromsingle cell invasion in that cells remain connected through cell-celljunctions and move as 2 or 3 dimensional sheets or clusters of cells.Collective cell invasion is prevalently observed during embryogenesis,tissue repair, angiogenesis, lymphangiogenesis, and drives theformation of many complex tissues and organs.

In cancer, both types of cell invasion have been observed andfound with different degrees and combinations [21,23,24]. In generalcancer invasion occurs with less uniformity in organization and pacein comparison to cell invasion associated with normal pathologicalprocesses. In many types of tumors, both single cells and collectivesare simultaneously present [24]. However, at early stages of tumordevelopment one mode of invasion may be observed to be moreprevalent in certain types of cancer. For example in leukemias,lymphomas, and most solid stromal tumors such as sarcomas andgliomas, cancer cells are observed invading in heterogeneous patternsof individual single cells. Conversely, in epithelial tumors, patterns ofcollective cells can be observed infiltrating as poorly organizedclusters or sheets. As epithelial tumors expand, de-differentiationoccurs (Epithelial-mesenchymal transformation (EMT)) and thecancer cells become more prone to disseminate as single cells,resulting in increased metastasis, and poor prognosis [25,26].

Here we focus on single cell invasion as it is the principal mode ofinvasion in cancer and is the most well studied pathway. There are anumber of complex molecular pathways involved in modulating theprocess of cancer cell invasion. We provide a synopsis of theinvolvement of cell invasion in immune response, vessel formation(angiogenesis and lymphangiogenesis), and cancer metastasis.

2.1. Immune response

The normal immune response due to infection or wound healingrequires immune cells to infiltrate the disrupted site to perform theirtherapeutic function. Immune cell invasion is a major componentnecessary for this infiltration. For example, upon injury to a tissuethere is the release of various growth factors and cytokines along withthe formation of a blood clot composed of cross-linked fibrin and ECMproteins which serves as a matrix reservoir of growth factors forinvading cells [27]. Neutrophils are the first cells to invade the injurysite followed by monocytes and lymphocytes, which must invadethroughout the wound site to deposit ECM. Fibroblasts then invadeand provide a contractile force for wound closure. While the invasionpathway of immune cell migration is critical for tissue repair, it alsocan be correlated with disease progression.

In cancer, the infiltration of immune cells has been associated withits progression [28]. Furthermore, the types of immune cells found inthe tumor microenvironment have been proposed as a prognosticfactor [29]. Macrophages and mast cells are thought to maintaintumor inflammation and promote tumor growth while lymphocytesare thought to manage tumor growth [29]. The ability to selectivelyinhibit the invasive potential of macrophages and mast cells could bean effective concomitant anticancer therapy.

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2.2. Angiogenesis

In both morphogenesis and regeneration new vasculature sproutsto provide nutrients to the tissue (angiogenesis). In this process,strands of endothelial cells penetrate the tissue matrix to form avessel [21]. In cancer, angiogenesis occurs when a tumor becomes toolarge to rely on diffusion for nutrient and oxygen exchange [30]. Thisangiogenic switch relies on the effect of pro-angiogenic molecules tooutweigh the effect of anti-angiogenic molecules expressed by thecancer cells [31]. This erratic signaling causes the newly formed bloodvessels to display altered structure as compared to neovasculature inhealthy tissue. The endothelial cells are poorly aligned with irregularshape which leads to large fenestrations and leaky vasculature.Furthermore, many tumors lack sufficient lymphatic drainage. Theseabnormalities lead to the enhanced permeability and retention (EPR)effect which has been exploited in delivery of macromolecular drugs[32,33].

The idea of targeting angiogenesis as an anticancer therapy,proposed by Professor Folkman 40 years ago, has led to thedevelopment of many effective therapies [34]. However, recentevidence indicates that some of these anti-angiogenesis therapiescan actually lead to a more malignant tumor and promote cancer cellinvasion andmetastases [35–38]. Furthermore, the lack of vasculaturewithin, and peripheral to, a tumor prohibits drugs from reachingtarget cells. Many anti-angiogenic therapies that target endothelialcell invasion are being evaluated in the clinic, and on the horizon arecombinational approaches that focus on inhibiting both endothelialcell and cancer cell invasion [39].

2.3. Lymphangiogenesis

Just as with angiogenesis, both morphogenesis and regenerationrely on the sprouting of new lymph vasculature to drain waste(lymphangiogenesis). While lymphangiogenesis has received littleattention in comparison to angiogenesis, recent findings indicate itplays a large role in cancer progression and metastasis. It has beengenerally accepted that tumors lack sufficient lymphatic vessels,which in part causes the EPR effect. However, the lymphaticvasculature serves as the primary route for lymph node metastasis,especially in cancers such as breast, colon, and prostate [40,41].Furthermore, some tumors have even been found to express pro-lymphangiogenesis factors, promoting lymph node metastasis [42].Therefore, anti-lymphangiogenetic drugs could provide an effectivetherapy against tumor metastasis [40].

Fig. 1. Cell invasion process. a) Invasive cells from a primary tumor intravasate into surrounform a metastatic tumor. Invasive cells from the primary tumor can also invade the surrouinvading cell, a process that involves: (I) protrusion of the leading edge of the cell into the straction; (III) proteolysis of ECM to provide room for infiltration; (IV) cell contraction to pullthe cell from the ECM and surrounding cells to move forward. Additionally, throughout thisinward and outward flux of ions regulate cell volume and protein function, and (c) water e


2.4. Cancer metastasis

Cancer metastasis involves the invasion of a tumor cell to a bloodor lymph vessel, intravasation into the vessel, extravasation from theblood vessel in another location, and invasion into the tissue to form asecondary tumor [2]. In some tumors such as in the brain, the cancercells do not typically metastasize to other organs, but rather theyinfiltrate extensively within the organ of origin through the cellinvasion pathway [43]. As brain tumors progress, individual cancercells infiltrate distant sites away from the primary tumor and sproutnumerous new micro-tumors throughout the brain. The extent ofdistant metastasis or brain infiltration extending from the primarysite typically correlates with poor survival outcome, therefore theability to inhibit the invasive potential of cancer cells woulddramatically improve outcome of therapy [44–46]. Likewise, themetastasis of breast cancer correlates with poor survival outcome.However, unlike in brain cancer, breast cancer cells metastasize toother organs such as the lungs and bone marrow.

Fig. 1 provides a generalized illustration of the two pathways ofcancer cell invasion to form secondary infiltrative or metastatictumors. Cell invasion is a 5-step process that involves: (I) theprotrusion of the leading edge of the cell into the surrounding ECM;(II) the formation of focal contacts between the cell and ECM toprovide forward traction; (III) proteolysis of ECM to provide room forinfiltration; (IV) cell contraction to pull itself forward toward theinvasion front; and (V) detachment of the trailing edge of the cell toprovide forward movement [24]. In addition, throughout this process,transcription factors promote the expression of pro-invasion mole-cules, inward and outward flux of ions helps regulate cell volume andprotein function, and water efflux modulates cell volume. The steps ofcancer cell invasion are a highly dependent on the expression of manydifferent interacting biomolecules, each of which provide an oppor-tunity for therapy.

3. Molecular therapeutic targets

There are many different molecules involved in cell invasion thatperform a specific yet critical role. Table 1 provides the mostnoteworthy biomolecules involved in cell invasion classified by theirgeneral function in cell adhesion, proteolysis, ion and water transport,and signal transduction. Table 2 lists some of the most prominentinhibitors of these pathways implicated in cell invasion. Here, webriefly describe the specific function of these molecules in cellinvasion and various therapies developed to inhibit them. For morecomprehensive reviews of the biology of these molecules see [47–49].

ding vasculature to enter the circulation, and extravasate into a secondary location tonding tissue to form a micro-tumor within the same organ. b) Expanded view of anurrounding ECM; (II) formation of focal contacts between the cell and ECM to provideitself forward towards the invasive direction; and (V) detachment of the trailing edge ofprocess, (a) transcription factors promote the expression of pro-invasion molecules, (b)fflux modulates cell volume.


image of Fig.�1


Table 1Molecular targets for invasion.

Target Function in invasion References

Cell AdhesionProteins

Integrins Cell surface receptors that bindto ECM to promote cell motility.

[47]

Cadherins Cell surface receptors that bind toadjacent cells decreasing cell motility

[23]

Proteinases MMP-1 Activates PAR-1 through proteolyticcleavage. Cleaves ECM to provideroom for invasion.

[48,61]

MMP-2 Cleaves ECM to provide roomfor invasion.

[48]

MMP-3 Cleaves ECM to provide room forinvasion. Cleaves cell surfaceadhesion molecule E-cadherin toinhibit cell-cell adhesions.

[48]

MMP-7 Cleaves cell surface adhesionmolecule E-cadherin to inhibit cell-cell adhesions.

[48]

proMMP-9 Hemopexin domain promotes cellinvasion through an unclearsignaling pathway.

[62]

MMP-13 Cleaves ECM to provide roomfor invasion.

[48]

MMP-14/MT1-MMP

Cleaves ECM to provide roomfor invasion.Cleaves cell surfaceadhesion molecule CD-44 to inhibitcell adhesion to ECM and cell-cell adhesions.Membrane bound MMPactive in ECMremodeling.

[48,60]

ADAM-10 Cleaves cell surface adhesionmolecule E-cadherin to inhibitcell-cell adhesions.

[48]

ADAM-17 Cleaves cell surface adhesionmolecule E-cadherin to inhibitcell-cell adhesions.

[48]

Thrombin Activates PAR-1 throughproteolytic cleavage.

[89]

ADF/Cofilin Actin depolymerization forincreased cell motility.

[67]

ROCK Crosslink myosin to promotecontraction for cell motility

[68,90]

Ion/waterChannels

Aquaporins Redistribution of water forcell volume regulation

[69]

NKCC1 Major pathway for Cl- accumulationin glioma for cell volume regulation.

[71]

ClC-3 Cl- channel for cell volume regulation. [72]TRPC6 Increases intracellular Ca2+

concentration resulting inNFAT activation.

[75]

IKCa

channelCell volume regulation. [73]

TranscriptionFactors/SignalTransducers

NFATs Transcription factors that promotethe expression of genes involvedin invasion.

[76]

NF-κB Transcription factor involved inpromoting the expression ofpro-invasion genes.

[82,83]

STAT3 Transcription factor involved inpromoting the expression ofpro-invasion genes.

[82,83]

CCR7 Promotes actin polymerizationand pseudopodia formationto increase cell motility.

[86]

CXCR4 Promotes actin polymerizationand pseudopodia formationto increase cell motility.

[86]

PAR-1 G-protein coupled receptorimplicated in the activation ofinvasion upon proteolytic cleavage.

[61,79–81]

Twist Decreases cell surface expressionof E-cadherin to reduce cell-celladhesions and increase cell motility.

[84]

Table 2Therapies affecting the invasion pathway.

Therapeutic Molecular target References

Cilengitide Integrin inhibitor [52–55]Etaracizumab Integrin inhibitor [91–93]CNTO 95 Integrin inhibitor [94–97]Marimastat(BB2516)

MMP inhibitor [98]

TIMP-1 MMP-9 inhibition [62,99]LY294002 MMP-9 inhibition [62]PD98059 MMP-9 inhibition [62]Wortmannin MMP-9 inhibition [62]RhoE ROCK inactivation [100]Fasudil(HA-1077)

ROCK inhibitor [90,101]

Chlorotoxin Inhibits MMP-2 activity, Reducesmembrane ion channel concentrationinhibiting cell volume regulation.

[74,102]

DAPT TRPC6 inhibition [75]Bumex NKCC1 inhibition [71]Charybdotoxin Potassium ion channel blocker [73]Dopamine Aquaporins through increased protein

kinase C phosphorylation.[103]

CsA Inhibit nuclear transport of NFATs [104]FK506 Inhibit nuclear transport of NFATs [104]VIVIT NFAT inhibitor [78]L-732531 NFAT inhibitor [105]ISATX47 NFAT inhibitor [106]P1pal-7 PAR1 inhibitor [61]AMD3100(Plerixafor)

CXCR4 inhibitor [85,88]



3.1. Cell adhesion proteins

In order to start the process of invasion, a cell must sever itsinteraction with surrounding cells and strengthen its hold on the ECMfor motility. In the tumor microenvironment cells have manyadhesion sites with adjacent cells and with the ECM through specificadhesion proteins expressed on the cell surface. Cadherins, a class oftype-1 transmembrane proteins involved in cell-cell interactions, aregenerally found with reduced function on the surface of invading cells[23]. Indeed, metastasis is higher when E-cadherin (a member of thecadherin family found in epithelial tissue) expression is reduced orlost [50]. On the other hand, integrins, receptors that mediate cell-ECM adhesions, are found at higher concentrations on the surface ofinvading cancer cells, particularly on their leading edges. Endothelialcells migrating into the tumor microenvironment for angiogenesisand lymphangiogenesis also rely on integrins for motility [51]. Inparticular, the integrins αvβ3, αvβ5, α5β1, α6β4, α4β1 and αvβ6have been implicated in disease progression and are thus most widelystudied [47].

In order to move forward, an invading cell requires attachment tothe surrounding ECM through integrin-tissue interactions; therefore,integrin inhibitors have been extensively studied as anti-cancer drugs.These anti-cancer agents function by inhibiting both the invasion oftumor cells out of the tumor site and into metastatic sites, and theinvasion of endothelial cells into the tumor site. This has theadvantage over strictly angiogenesis inhibitors in that integrininhibitors also reduce the risk of metastasis, a current challengewith anti-angiogenesis drugs. For example, cilengitide, a cyclic RGDpeptide that inhibits αvβ3 and αvβ5 integrins binding to ECM, hasshown promise in lung and prostate cancer patients and notably inglioblastoma patients [52–55]. Likewise, therapeutically increasingexpression of cadherins could inhibit cell invasion by strengtheningcell-cell adhesions, preventing the cell from escaping the bulk tumor.Thus, forcing overexpression of cadherins has been the focus of someanti-invasion therapy studies [56–58].




3.2. Proteinases

The restructuring of the ECM is a critical step in the process of cellinvasion. The ECM is a dense network of fibrous proteins such ascollagen and fibronectin that an invading cell must break down toprovide room to migrate. Furthermore, the invading cell must cleavecell-cell and cell-ECM adhesions which is mainly achieved throughthe secretion of matrix metalloproteinases (MMPs) [59].

Many of the MMPs (MMP-1, MMP-2, MMP-3, MMP-13, and MMP-14) are involved in the breakdown of ECM, but also function in otheraspects of cell invasion. For example, along with the breakdown ofECM, MMP-3 breaks down E-cadherin to cleave cell-cell adhesions. E-cadherin is also broken down by MMP-7, but has no function in ECMdegradation. MMP-14 cleaves CD44, a cell-surface glycoprotein whichprovides both cell-ECM and cell-cell adhesion sites [60]. MMP-1functions in cleaving the cell membrane bound receptor PAR1,activating it for subsequent intracellular signaling [61]. AlthoughMMP-9 does not have a direct function in ECM remodeling, its non-enzymatic form, proMMP-9, promotes cell invasion through anintracellular signaling pathway [62]. In fact, down regulation ofMMP-9 has been shown to inhibit the invasion and tumor growth inhuman models of gastric adenocarcinoma [63], prostate cancer [64],and laryngeal cancer [65].

MMPs are not the only molecules involved in modifying theinteraction of the invading cell with its surrounding microenviron-ment. A disintegrin and metalloproteinase (ADAM), a family ofpeptidase proteins, cleaves cell-cell adhesions and regulates integrinfunction to promote cell invasion [66]. Both ADAM-10 and ADAM-17cleave E-cadherin, severing cell-cell interactions to promote single-cell invasion [48].

The invading cell pulls itself forward through cell-ECMadhesion siteson the leading edge of the cell. This also requires polymerization andcontraction of the cytoskeleton of the cell to provide a force topropel thecell forward. Actin filaments, which are the main constituent of thecytoskeleton,arerapidlycleavedandpolymerizedinaninvadingcell. Theactindepolymerizingfactor(ADF)/cofilin familyofproteins is involvedinthe depolymerization of actin allowing the cell to change shape [67].Specifically, cofilin 1 depolymerizes F-actin (the polymer form of actin),replenishing the G-actin (monomer form of actin) of the cell for re-polymerization and restructuring. Inhibiting this pathway is expected tohaveprofoundanti-tumoreffects,but to thisdatenosuch inhibitorsexist[67]. Rhoassociatedproteinkinase (ROCK), on theotherhand, crosslinksmyosin to promote contraction of the cytoskeleton to squeeze throughtightspacesandpull itself forward[68]. Inordertofullyregulate itsshape,the invadingcellmust alsobeable to control its volume.

3.3. Ion/water channels

An invading cancer cell must be able to squeeze through the densenetwork of extracellular matrix and cell-cell junctions that make upthe tumor microenvironment. This movement is achieved byregulating the cell volume (both total volume and local volume).The leading edge of the cell must be able to become very thin andelongated to fit through narrow spaces, and if the narrow space islonger than the cell, the entire cell must be able to greatly reducevolume. This is generally achieved by altering the osmotic balancebetween the cell and extracellular space causing water to flow in andout of the cell through aquaporins, transmembrane proteins thatregulate water flow [69].

The osmolarity of the cell is controlled by ion channels on the cellmembrane that can actively pump in or out specific ions. Chloride ionsplay an important role in cell invasion by providing the osmoticdriving force for cell shrinkage through their electrochemical gradientacross the cell membrane [70]. One of the major Cl− ion transportersis the sodium-potassium-chloride co-transporter isoform-1 (NKCC1)transporter which helps maintain this electrochemical gradient [71].


The ClC-3 also plays a role in Cl− ion transport to drive cell volumeregulation for invasion [72]. The activity of the NKCC1 transporter isdependent on intracellular potassium concentrations. Therefore, inorder for the cells to transport chloride ions, potassium ions must alsobe present for the co-transport. Thus, potassium pumps are also vitalion channels involved in cell invasion [73].

Chloride channels are attractive therapeutic targets because theyare critical for cell invasion through the tight spaces imposed by thetumor microenvironment. Bumex® has been found to inhibit NKCC1which reduced cell invasion by over 50%, simply by inhibiting theability of the cell to regulate volume [71]. Interestingly, indirectinhibition of chloride channels can inhibit cell invasion. Chlorotoxin(CTX), a peptide derived from scorpion venom, binds MMP-2 on thesurface of cancer cells which causes the MMP-2 complex and lipid raftwhich contains chloride channels to be internalized [74]. This, in turn,reduces the number of chloride channels on the surface of the cancercell, mediating its ability to regulate cell volume.

Calcium ion transport is also important for invasion, but is notdirectly involved in the regulation of cell volume. The transientreceptor potential channel 6 (TRPC6) maintains a high intracellularcalcium ion concentration which is correlated with increased invasion[75]. Rather than disrupting the osmotic balance, the TRPC6 inducedintracellular calcium ion concentration leads to activation of otherpathways involved in invasion, as discussed below. The same is true inT lymphocytes where calcium ion influx activates the NFAT promoterleading to T cell activation.While calcium ion channel inhibition couldreduce the invasive potential of the cancer, it could also diminish anyimmune activity of anticancer T cells within the tumor. Therefore,effective calcium ion transport channel inhibition should be selectiveto those active on the tumor cell but not lymphocytes.

3.4. Transcription factors and signal transducers

The activation of transcription factors by direct interaction orindirect signal transduction plays the initial role in cell invasion byturning on the invasive phenotype. The TRPC6 calcium ion channeldiscussed above leads to the activation of nuclear factor of activated Tcells (NFAT) transcription factors [75]. The NFATs promote theexpression of a wide variety of pro-invasion molecules [76].Furthermore, NFATs have been implicated in promoting angiogenesisand lymphangiogenesis indicating the crucial role they play in cellmigration and invasion. This makes NFATs favorable targets for cancertherapy. Both Cyclosporin A (CsA) and FK506 inhibit the function ofNFATs by preventing their import into the nucleus [77]. These drugsare very effective for immune suppression in organ transplantpatients, but their use in cancer therapy is limited due to severetoxic side effects. L-732531 and ISATX47 are less toxic analogues ofFK506 and CsA, but are very early in development for cancer therapy[76]. The lack of successful NFAT inhibitors has lead to thedevelopment of an NFAT inhibitory peptide, namely VIVIT, whichshows minimal side effects [78]. However, its activity is limited tospecific NFATs indicating it is not as potent or robust as the CsA andFK506 drugs [77].

Protease-activated receptor 1 (PAR-1) is another moleculeactivated by a pro-invasion molecule. MMP-1 cleaves PAR-1,activating it for downstream signaling that promotes invasionthrough various pathways [61,79–81]. In addition, NF-κB and STAT3are transcription factors involved in promoting the expression ofproteins that help in invasion [82,83]. Twist is another transcriptionfactor implicated in tumor metastasis. Twist expression reduces thecell surface expression of E-cadherin which results in decreased cell-cell adhesion and increased cancer cell motility [84]. Inhibiting thesetranscription factors should have a direct and potent effect on cellinvasion.

Chemokine receptors also play a critical role in cancer cell invasion[85]. The CXCR4 and CCR7 chemokine receptors are highly expressed



Fig. 2. General architecture and assembly of a multifunctional NP. Generally, a solid NPcore is coated with a biocompatible polymer coating which can then be derivatizedwith targeting agents, fluorophores, radionuclides, gene therapeutics, and chemother-apy drugs.


on the surface of breast cancer cells and promote the polymerizationof actin and formation of pseudopodia to increase cell motility [86].The bicyclam AMD3100 is used to inhibit CXCR4 and has been shownto reduce breast cancer and melanoma metastasis [87,88].

4. Nanomedicine in treatment of cancer cell invasion

4.1. Fundamentals

Nanomedicine is an emerging technology that combines the fieldsof biology, chemistry, engineering, and medicine to develop newsolutions for major clinical problems. Cancer is one disease where theapplication of nanomedicine has potential to provide clinicians theability to overcome many existing shortcomings in screening andtreatment. At the heart of nanomedicine is the development ofprecisely engineered nanomaterials (e.g., NPs) with desired proper-ties. Typically, NPs in nanomedicine have dimensions of tens tohundreds of nanometers across, putting them on the same size scaleas biomolecules. For example, proteins are typically in the size rangeof 1–20 nm, DNA has a diameter of 2 nm, and cell surface receptors areapproximately 10 nm [107,108]. Therefore, the size of NPs affordsthem the opportunity to interact with biomolecules on a scale that canmodulate biological pathways elusive in medicine, such as the cellinvasion pathway.

Another advantage of NPs is the unique properties of the materialthat arise only at the nanoscale. Themost well studied phenomenon isthat nanoscaled materials have a high surface area to volume ratio[109]. This implies that the percentage of atoms on the surface of anNP is high compared to a macroscaled or evenmicroscaled particles ofthe same material. This physical property renders NP surfaces highlyreactive and amendable. Using nanoengineering strategies research-ers can tailor the unique physical properties (e.g., size, charge,biocompatibility, solubility, hydrophilicity/hydrophobicity) of NPs tomodulate their behavior in biological systems [110]. Through theseapproaches, critical pharmacokinetic properties such as circulationhalf-life, biodistribution, non-specific adsorption, premature degra-dation, and toxicity can be dictated [6,111–115]. A number of otherphysical phenomena can occur in nanoscaled versions of materialssuch as the development of unique optical, electronic, and magneticproperties depending on their core material and size [10,11]. Theseproperties are highly desirable for sensing, tracking, and activationapplications [116].

NPs can be synthesized from myriad different material formula-tions to create numerous nanoarchitectures. Examples from thevarious common classes of NP formulations developed to date can besummarized into the following categories: liposomes, albumin-basedparticles, nanocrystals, polymeric micelles, polymer-based NPs,dendrimers, inorganic NPs, nanotubes, and/or other solid NPs[113,117–123].

Another desirable property of NPs is that they are amenable tochemical modification, and through organic chemistries, can beengineered as multifunctional devices that carry multiple detectionsignals, tumor cell recognizing targeting ligands, and therapeuticcargos [7,9,10,124]. Multifunctional devices are capable of deliveringprecisely targeted treatments to tumor cells, avoiding healthy tissues,and being tracked non-invasively through incorporated detectionsignals (contrast agents). Fig. 2 shows a cartoon diagram depicting thegeneral architecture of amultifunctional NP device and its assembly. Atypical multifunctional NP device comprises a NP core, a biocompat-ible coating, surface bound or encapsulated targeting and therapeuticpayloads, and/or additional detection signals.

Many NP formulations have been examined for clinical use andsome formulations have already been approved for use in humans [7].Less complex formulations, such as liposomes loaded with chemo-therapeutic drugs, have been approved for cancer therapy for morethan a decade [125,126]. In these early NP formulations, the liposome


enhanced the solubility of the chemotherapeutic for improvedbiodistributions and extended blood circulation time, which ulti-mately led to a higher therapeutic index for the delivered drug.

These liposomal formulations have also been used to overcomecancer cell drug resistance. This drug resistance generally occurs dueto the overexpression of ATP-binding cassette (ABC) transporterswhich increase the efflux of a broad class of hydrophobic drugs fromcancer cells [127]. Nanotechnology provides an alternative strategy tocircumvent drug resistance by encapsulating or attaching drugs tonanomaterials that are resistant to drug efflux [128,129]. Indeed,several NP-based chemotherapies (e.g. Doxil, Caelyx, DaunoXome)have been approved for clinical use or are in clinical trials [130,131].

Formulations of crystalline NPs have also been examined forclinical applications. For example, a number of iron oxide NPs are inearly-stage clinical trials or experimental study stages [132–134].Several formulations have already been approved for widespreadclinical use in medical imaging and therapy. Some examples include:Lumiren® for bowel imaging [135], Feridex IV® for liver and spleenimaging [136], and Combidex® for lymph node metastases imaging[137]. Iron oxide NPs are desirable because of their magneticproperties that can be exploited for non-invasive tracking throughmagnetic resonance imaging (MRI) [113]. Furthermore, in contrast tomany other inorganic NP formulations, iron oxide NPs are biocom-patible, and iron from degraded NPs are used in the body's naturaliron stores such as hemoglobin in red blood cells. In fact, a formulationof iron oxide NPs (Ferumoxytol®) was recently approved for ironreplacement therapy [138].

Recently, more complex formulations of NPs, such as multifunc-tional devices that incorporate both cancer specific targeting andtherapeutic delivery functionalities, have emerged in the clinicalsetting. One example is the multifunctional polymeric NP formulationCALAA-01 (Calando Pharmaceuticals, Inc.) [139]. This formulationconsists of: (1) a linear, cyclodextrin-based polymer, (2) a humantransferrin protein (TF) targeting ligand displayed on the exterior ofthe NP to engage TF receptors on the surface of the cancer cells, (3) ahydrophilic polymer (polyethylene glycol) used to promote NPstability in biological fluids, and (4) siRNA designed to reduce theexpression of ribonucleotide reductase M2 (RRM2), a criticalbiomolecule in DNA synthesis. In a recently completed phase I clinicaltrial, this NP formulation showed favorable tolerability and thera-peutic efficacy in patients with solid cancers [140]. Most notably, thetrial revealed that incorporating a targeting ligand could drasticallyimprove the amount of NPs internalized by cancer cells and lead tohigher therapeutic efficacy.

These advancements highlight the promise of nanomedicine beingtranslated into clinical practice. Further, this emergence is opening up


image of Fig.�2



new avenues in nanomedicine for targeting more specialized cancer-specific pathways, such as cancer cell invasion, for more effectivetherapy with reduced side effects. Cancer cell invasion is highlycomplex and involves numerous environmentally and temporallyregulated processes. This makes the multifunctional nature ofnanomedicine well suited to tackle this phenomenon. By simulta-neously targeting various molecular targets in the progression of cellinvasion, we can produce a much more effective therapy that is lessprone to development of resistance. Furthermore, the sensing andtracking capabilities that can be developed through nanomedicineprovide opportunities to monitor and study the progression of cancermetastasis.

The development of NPs for the treatment and monitoring ofcancer cell invasion is a new and emerging application in the field ofnanomedicine. Traditionally, cancer nanomedicine strategies focus ondelivering therapies and imaging contrast agents to the solid tumormass. Tackling cell invasion will require novel NP formulations andnew strategies for targeting NPs specifically to the invasive cells thathave become segregated from the bulk tumor. In the followingsections we will review examples of currently developed andemerging nanoparticle directing strategies, and evaluate theirapplicability for targeting cancer cell invasion. Furthermore, we willdescribe several examples of nanoparticle formulations which showpromise for monitoring and treatment of cancer cell invasion.

4.2. Directing nanoparticles to cancer cells

Nanoparticles developed for cancer applications are typicallyadministered systemically through intravenous injection. If properlyengineered, the NPs travel as discrete, individual entities through theblood, bypass biological barriers (e.g., vascular tumor barriers,extracellular matrices, cell membranes), and reach their moleculartarget for biorecognition and activation [141]. Directing NPs in vivohas been the focus of tremendous amounts of research and manyinnovative strategies have been introduced and investigated. Variousreviews have specifically focused on this engineerable feature of NPs[142–145]. Here we will examine several of the generalized strategiesutilized and discuss their utility for targeting cancer cell invasion.

Many earlier NP-directing strategies focused on modifying theNP's physiochemical properties to promote uptake by tumor cells. Onewell-studied approach is to enhance the circulation time of NPsthrough surface modification strategies. Long-circulating NPs canpassively target tumors through a phenomenon known as theenhance permeability and retention (EPR) effect [146]. This effectarises due to the poorly functioning blood and lymphatic vessels intumor tissue that enable macromolecules of 1–500 nm in size to leakinto tumor tissue over time [147]. Due to inefficient lymphaticdrainage, there is poor clearance of NPs leading to their prolongedaccumulation [148,149]. The most widely known method to impartthe long-circulating property onto NPs is through surface modifica-tion with polymers such as polyethylene glycol (PEG) that possessnon-fouling properties. These polymers help limit protein absorptiononto the NP and the recognition of NPs by the body's immune system[150]. This strategy has been exploited in numerous studies topassively direct numerous NP formulations including liposomes [151–153], polymers [154,155], and crystalline NPs [156–158] to tumorcells.

There are a number of disadvantages in solely relying on the EPReffect to direct NPs to tumor cells for treatment of cancer cell invasion.First, tumors are heterogeneous in vascularization, blood flow, andlymphatic drainage rate, which make delivering drugs to the entiretumor difficult. Second, not all tumors will develop an EPR effect, andin fact, certain types of solid cancers, including those of the brain, areprotected by more restrictive vasculature that prohibits passivelytargeted NPs from reaching tumor cells [159]. Lastly, the EPR effect islimited to the bulk tumor which means NPs cannot interact with


metastasized/invasive cancer cells that have migrated away from thetumor bulk [2]. Therefore, more specific and active targetingapproaches are necessary to improve the NP uptake by invadingcells dissociated from the bulk tumor.

Active targeting relies on the use of specific targeting ligandswhich can recognize and bind to receptors that are upregulated oncancer cells or associated stromal cells. Incorporated onto the surfaceof NPs, these targeting ligands can direct NPs to specific cells [160].Numerous targeting ligands have been evaluated to actively deliverNPs specifically to cancer cells [6,113,142,143,161]. Of note, manycrystalline systems have implemented active tumor targeting strat-egies with varying success, including ligands such as small organicmolecules [162–164], peptides [102,165–179], proteins [180], anti-bodies [181–183], and aptamers [184–186]. Some of these examplesinclude ligands which recognize molecular receptors involved incancer cell invasion, such as CTX which binds to MMP-2 upregulatedon the surface of cancer cells during invasion [102,166,171–178], andthe peptide RGD, which binds integrin receptors upregulated onendothelial cells associated with tumor neovasculature [114,187–189]. Other examples include antibodies directed against ion channelsupregulated on the surface of invading cells which inhibit channelfunction [190]. Attaching one of these ion channel-targeting anti-bodies to the surface of a NP could provide the dual function of ionchannel inhibition and NP mediated imaging and/or therapy.

In addition to enhancing specificity of NPs to cancer cells, targetingagents can help initiate endocytosis of the NPs to which they areattached [191]. Therefore, targeting ligands can improve the deliveryof drugs into cancer cells and the therapeutic index of thenanotherapeutic formulation. A notable study by Bartlett et al.evaluated this phenomenon by comparing the in vivo efficacy ofdelivering RNAi-based therapeutics using actively targeted versuspassively targeted NPs. They found that active targeting can enhancethe therapeutic efficacy by 50% [192]. Interestingly, this studyrevealed that although similar amounts of both NP formulationswere delivered to the tumor tissue, the therapeutic efficacy wasenhanced for the actively targeted formulation due to improvedcancer cell uptake and tumor distribution.

Active targeting strategies also improve the percentage of cancercells that are exposed to NPs. Our group demonstrated this concept intwo recent studies utilizing magnetic NPs prepared with and withoutthe active targeting ligand CTX to compare their efficacy in deliveringnucleic acids (siRNA and plasmid DNA) to brain cancer cells [171,176].These in vitro and in vivo studies revealed that the percentage ofcancer cells that received therapies was two-fold higher with theactively targeted CTX modified nanovector in comparison to thepassively targeted nanovector. In a recent landmark study, Sugaharaet al. demonstrated that co-administration of the tumor penetratingpeptide iRGD with NPs can improve their therapeutic index in tumorbearingmice [193]. Here the peptide iRGD has the capacity to increasetumor vascular permeability. This peptide functions by first associat-ing with integrins that are specifically expressed on the endotheliumof tumor vessels, and then the peptide is proteolytically cleaved in thetumor to produce a truncated sequence that has no integrin-bindingactivity, but gains affinity for neuropilin-1 (NRP-1), and thus enhancestissue permeability. Notably the iRGD peptide was just as effectivewhen co-administered with NPs as when chemically bound. Thisstrategy opens up new opportunities for multistage therapy wherebynumerous levels of targeting are included.

Other tumor directing strategies for nanomedicine includesystems that can recognize tumor specific microenvironmental cuesfor activation of the NPs [171,192,194,195]. In a series of recent ofstudies by, Nguyen et al. and Olson et al., protease activatable cellpenetrating peptides (ACPPs) which respond to the activity of MMPsin tumors were incorporated onto the surface of dendrimeric NPs[194,196]. In the presence of proteinases, a 4- to 15-fold higher cellinternalization of ACPP modified NPs was observed in comparison to



Fig. 3. Summary of data obtained through this original study that demonstrated the applicability of NPCP-CTX for delineating tumor boundaries through in vivo MRI, and in vivofluorescence imaging. a) In vivo MR images of autochthonous medulloblastoma tumors in genetically engineered ND2:SmoA1 acquired before and 48 hrs after administration ofeither NPCP-CTX or NPCP NPs. b) In vivo NIRF imaging of ND2:SmoA1mice injected with either NPCP-CTX or NPCP-Cy5.5, or receiving no injection (from left to right). Post-injectionex vivo fluorescence images of mice brains from the same mice following necropsy are shown in the inset of b. The spectrum gradient bar at right corresponds to fluorescenceintensity (p/s/cm2/sr) of images. Reprinted by permission from the American Association for Cancer Research [178], copyright 2009.


the passively targeted version of the same NP. Their studies revealedthe ability to use the invasive tumor environment to activatenanotherapeutics.

Ultimately, it is likely that successful formulations designed totarget invasive cancer cells will exploit a combination of strategies todirect NPs to cancer cells. In the next two sections we will evaluatecurrent strategies that have been utilized for delivering nanoparticlesto cancer cells for imaging cancer cell invasion and for therapy.

4.3. Nanomedicine in imaging cancer cell invasion

Non-invasive monitoring of cancer cell progression andmetastasisis of great interest to clinicians. Until recently, most studies ofmetastasis only measured the end point of the process: macroscopicmetastases. Although these studies have provided much usefulinformation, the details of the metastatic process remain somewhatmysterious owing to difficulties in studying cell behavior with highspatial and temporal resolution in vivo. Nanomedicine provides anavenue for monitoring cancer cell invasion and metastasis in situthrough various imaging platforms and can aid clinicians in visualrepresentation, characterization, and quantification of this biological

Fig. 4. Dual-Labeled ACPPD. (A–D) Example of HT1080 xenograft treated with ACPPD duallyuptake in tumor (a, black arrow). Following skin incision and retraction, the tumor (blacksurgery, repeat MRI (c) showed a small area of tissue with increased gadolinium uptake (d insecond surgery. Histological analysis of this tissue confirmed the presence of cancer cells (Ddually labeled with gadolinium and Cy5. Preoperative MR image of mouse showing contra(black arrow) on the left chest wall was visible with Cy5 fluorescence (f). Tumor was reseremoved (g). Repeat MR imaging following surgery showed complete removal of all tumcopyright 2010.


process at the cellular and molecular levels [7,8,149]. NPs have beendeveloped for imaging application across different platforms includ-ing MR, optical, and nuclear imaging systems [195]. In some casesthese platforms can be combined to offer clinicians the ability toobtain a variety of pathologic information using the unique imagingcapabilities of each system with a common NP formulation [116].

Visualizing cancer cell invasion is especially critical in tumorsarising at anatomical sites where surgery is complex (e.g., head andneck tumors, brain tumors, and others). Here, having the ability tovisualize the extent of cancer cell infiltration into the brain couldprovide improved guidance to neurosurgeons in planning andexecuting surgical resection. In many brain tumors the extent ofresection is predicative of outcome, with more complete resectionscorrelating to improved progression free survival. This addedinformation could drastically aid in improving the outcome of surgeryas a result of a more radical resection, and thus numerousmultifunctional NP formulations have been developed [166–168,178,197,198]. Our group recently demonstrated that multifunc-tional magnetic/optical detectable NPsmodifiedwith CTX could safelypermeate across the blood brain barrier (BBB) and highlight theextent of tumor cell infiltration into normal brain tissue under both

labeled with gadolinium and Cy5. Preoperative MR image of mouse showing contrastarrow) on the left chest wall was visible with Cy5 fluorescence (b). Following initialset, white arrowhead). This area of tissue was identified using fluorescence imaging at a). (Scale bar: 100 μm). (E–H) Example of MDA-MB 435 xenograft treated with ACPPDst uptake in tumor (e, black arrow). Following skin incision and retraction, the tumorcted using ACPPD-Cy5 imaging guidance until all visible fluorescence was completelyor (h). Reproduced with permission from National Academy of Sciences, USA [194],


image of Fig.�3

image of Fig.�4



MRI and fluorescence optical imaging [178]. In this formulation, thecombination of using CTX to actively target MMP-2 on brain tumorcells and engineering NPs to have extended blood circulation timefacilitated access of the NP across the BBB to brain tumor cells [199].Fig. 3 shows the imaging data obtained through this study inmedulloblastoma brain tumor bearing mice with intact BBBs.

As described in the preceding section, NP formulations have beendeveloped to sense biochemical changes and molecular activity ofcancer cells. This approachwas recently demonstrated in imaging theextent of tumor infiltration through a series of studies performed byNguyen et al. andOlson et al. [194,196]. Fig. 4 shows a series of imagesfrom this study depicting how this NP formulation can be applied toimprove the outcome of tumor resection by highlighting tumormargins under pre-operative MRI and intra-operative opticalimaging. This nanomedicine based diagnostic tool was evaluated inits ability to improve surgical outcome by aiding surgeons inidentifying and resecting residual metastatic cancer cells both pre-and intra-operatively. Their formulation consisted of protease-activatable cell penetrating peptides linked to dendrimers duallylabeledwith a fluorophore for optical imaging and gadolinium forMRimaging. Thus, MMPs in the tumor microenvironment cleave andactivate the cell penetrating peptide which promotes uptake intocancer cells. Once internalized, the optical and MR signaturesassociated with the nanoprobes provide navigation to aid in complex

Fig. 5. Membrane dynamics in metastatic cancer cells in vessels. a, imaging of cells in the blocells. Red dotted lines show outlines of vessels determined by superimposed images of autoflof the barycentric position of the cell in A at every 2 s (green line). c, fluorescent image of a ceshows an outline of the cancer cell. d, trajectory of the barycentric position of the cell in C aton the inner vascular surface. The yellow line represents an outline of the cancer cell. Whitbarycentric position of the cell in E at every second. g, cells in E superimposed for 16–17lamellipodia-like structures. h, traces of blue, purple, and orange squares, as shown withmembranes of cells in the bloodstream (blue), on the inner vascular surface without direcD=diffusion constant. Error bars indicate±S.E. Blue data, n=88 (22 trajectories/cell×4trajectories/cell×3 cells). Squares in A, C, and E show typical QDs on the edge of cells. Expermission from the Journal of Biological Chemistry [202], copyright 2009.


surgical resection of large and invasive tumors. This approachdemonstrated a 90% reduction in residual cancer cells left aftersurgery.

Outside of clinical screening and staging applications, nanomedi-cine approaches can be used to further understand cell invasion andmetastasis processes in vivo in animal models. For example, cancercells loaded with magnetic NPs have been implanted in rat brains andmonitored through MRI which has provided insights into brain tumorcell invasion [200]. Furthermore, advances in microscopic imagingtechniques now provide opportunities to monitor single cells in vivo.These emerging techniques include spatiotemporally resolved imag-ing, fluorescent reporter reagents, and multiparametric imageanalysis, which can contribute to a better insight into single cellmigration and invasion [201]. Gonda et al. recently illustrated theseconcepts in a study where cancer cells labeled with semiconductorquantum dots (QDs) were temporally tracked in vivo through theprocess of invasion and metastasis [202]. Fig. 5 exemplifies how thisapproach can characterize individual cell migration over an extendedperiod of time. In this study, QDs were labeled with an anti-PAR1antibody and used to target and track metastatic breast cancer cells ina mouse model. Imaging was performed with a spatial precision of 7–9 nm under a confocal microscope, which provided information onmembrane dynamics of invading and metastasizing cells. Forexample, the membrane fluidity of metastasizing cells in the blood

odstream. Cells are shown after 1 s, 17 s, and 41 s. Yellow lines show outlines of canceruorescent blood cells. White dotted lines indicate outlines of red blood cells. b, trajectoryll adhering to the inner vascular surface without directional movement. The yellow lineevery second. Numbers show the tracking order. e, imaging of directional cell migratione arrowheads show red blood cells with a comet-like configuration. f, trajectory of thes and 27–28 s. Yellow lines show outlines of cancer cells. Red arrowheads representarrowheads in A, C, and E. Numbers show the tracking order. i, MSD plots of QDs ontional migration (purple), and on the inner vascular surface with migration (orange),cells). Purple data, n=115 (23 trajectories/cell×5 cells). Orange data, n=78 (26

citation, 532 nm; emission, 580 nm; exposure time, 0.2 s. Bars, 10 μm. Reprinted with


image of Fig.�5



was 1100-fold greater than that of cells in the bulk tumor, whichindicates a lack of cytoskeletal actin structure near the cell membrane.This bit of information can direct therapeutic strategies towardsinhibiting actin polymerization in these metastasizing cells to preventtheir invasion into secondary locations.

4.4. Nanomedicine in treating cancer cell invasion

There are several immediate benefits of using NPs as drug carriers.Most nanotechnology-based drug formulations aim to increase thetherapeutic index for established chemotherapeutic drugs via im-proving pharmacokinetics, biodistribution, and selectivity in deliveryto cancerous tissue. Combined, these formulations have utilizednanotechnology-based strategies for tumor targeting, imaging, anddelivery of therapeutics [7,11,114,116,145,203,204]. In most of thesecases, well-established chemotherapeutic drug molecules (e.g.,paclitaxel, doxorubicin, docetaxel, and methotrexate) have beencombined with liposomal or polymeric NP platforms. More recently,biotherapeutic agents (e.g., therapeutic peptides, antibodies, genes,and siRNAs) have been combined with nanomedicine to treat cellinvasion more specifically.

Fig. 6. Schematic representations of CTX-enabled nanoparticles (NPCs) inhibiting tumor cellchemistry of NPC conjugate. b) NPC binding to lipid rafts of glioma cells containingMMP-2 anpresence of gelatin. Comparable inhibition by CTX and NPC indicates retention of catalytisubsequent to NPC binding. Scale bars represent 5 μm and 200 nm for whole cell (first rowidentify NPC and endosomes, respectively. e) Quantitative assessment matrigel cell invasfluorescence imaging, showing the morphological changes of C6 cells exposed to NPC (scalSmall [102] Copyright 2009.


While there is a wealth of studies focused on developing NPs forcancer therapy, there are only a limited number of nanoformulationsreported in treating cancer cell invasion [102,179,205]. However,there is tremendous potential to combine NP formulations withknown inhibitors of cancer cell invasion to curb tumormetastasis. Oneexample of this approach is a recently published study by our groupwhere the therapeutic effect of CTX bound to NPs was compared tofree CTX in its ability to inhibit glioma tumor cell invasion [102]. CTX isan inhibitor of MMP-2 (Section 3 above) and also plays a role ininhibiting volume regulating ion channels. In this in vitro study wedemonstrated that when bound to NPs, CTX provided enhancedtherapeutic potency compared to free CTX. Fig. 6 summarizes the dataobtained through this study describing comparative effect of free CTXvs. NP bound CTX. Notably, NP-CTX can simultaneously interact withnumerous MMP-2 receptors expressed on glioma cell surfaces. Thismultivalent binding promotes cellular internalization of a largerportion of lipid rafts which contain MMP-2 receptors and volumeregulating ion channels. Combined, these interactions and processeslead to inhibition of MMP-2 and ion channel activity in targetedglioma cells. Thus, an enhanced ability of NP formulation to inhibitglioma cell invasion is observed.

invasion and summary of MMP-2 and cell invasion inhibition data from [102]. a) Surfaced select ion channels. c) Functional inhibition of NPC, free CTX, and NP onMMP-2 in thec activity of CTX bound to NPC. d) TEM images showing increased membrane uptake) and high magnification imaging (second row), respectively. White and black arrowsion post-treatment. f) Confocal differential interference contrast (DIC) and confocale bar: 20 μm). Reproduced with permission from Wiley-VCH Verlag GmbH & Co. KGa:


image of Fig.�6



The inhibition of ion channels is an exciting strategy to treatinvasive tumors. The use of CTX and other ion channel inhibitors inclinical trials have shown promising results [206–209]. As shownabove, nanotechnology can enhance the inhibition of ion channelssolely through the multivalent effect wherein a larger portion of thecell membrane is internalized. Recent studies have also utilized thesmall scale of NPs to directly interact with ion channels to diminishcells' ability to regulate cell volume. For example, Park et al. showedthat single-walled carbon nanotubes (SWCNTs) are able to inhibit K+

ion channels in Chinese hamster ovary cells if engineered properly[210]. They found that the SWCNTs with an inner diameter of 0.9 nmhad the highest K+ ion channel blocking ability, but in a reversiblemanner indicating the effect was highly concentration-dependent.This work was followed up by the same group in a paper byChhowalla et al. who developed functionalized SWCNTs for irrevers-ible inhibition of K+ ion channels [211]. By attaching the chemical 2-trimethylammoniumethylmethane thiosulfonate (MTSET) to theSWCNTs, they showed this functionalized nanotube was able tospecifically interact with the cysteine groups of amino acids withinthe ion channel for higher binding affinity and irreversible channelinhibition. K+ channel inhibition has also been established with multi-walled carbon nanotubes (MWCNTs) [212]. Likewise, Kraszewski et al.modeled the interaction of fullerenes (C60) with K+ ion channels andproposed that this carbon based nanomaterial has an affinity towardsthe transmembrane domain of K+ ion channels, and that the K+ ioncurrent could be greatly inhibited through the attachment of hydro-phobic drugs [213]. Since invading cells rely on the intracellularconcentration of K+ ions to regulate cell volume, inhibiting thesechannels could provide significant treatment efficacy.

Actively targeted nanoparticles have also been evaluated ascarriers of conventional drug therapies designed to treat cancermetastasis. For example, Murphy et al. evaluated the use of polymericnanoparticels loaded with the chemotherapeutic agent doxorubicinand modified with the targeting ligand RGD peptide which bindsαvß3 integrins expressed on neovascular endothelial cells [214]. Inthis system, RGD was integrated to direct the doxorubicin loaded NPsto a subset of tumor blood vessels associated with angiogenesis. In thestudy, the NP formulation was shown to produce a therapeutic indexthat was 15-fold more superior to the free drug for treating cancermetastasis, and furthermore, contrary to the free drug, no toxicity wasobserved in mice treated with the NP formulation. This studydemonstrates the potential of NP formulations for improving thetherapeutic index of conventional drugs while minimizing theirrelated toxicity.

NPs have also proven to be effective vehicles in delivering DNA orsiRNAs for gene therapy, a powerful tool that could simultaneouslyaffect multiple pathways leading to invasion. For example, Alshamsanet al. delivered anti-STAT3 siRNA using PLGA NPs to melanomatumors and showed this knockdown of STAT3 diminished tumorgrowth [215].While they did not directly correlate this to inhibition oftumor cell invasion, this study shows the utility of nanotechnology indisrupting pathways involved in cancer cell invasion as an anticancertherapy.

A study by Han et al. actually showed the correlation of NPmediated gene therapy with reduced cell invasion [216]. They usedmagnetic NPs coated with polyamidoamine dendrimers to carry anti-epidermal growth factor receptor (EGFR) siRNA to brain tumor cells.Knockdown of EGFR led to the downstream reduction in expression ofpro-invasion biomolecules, namely MMP-2 and MMP-9, and reducedtumor cell invasion in a transwell migration assay. Gao et al. alsoshowed reduced cell invasion through siRNA treatment using NPs[217]. In this study they used PEGylated liposomes to deliver anti-RhoA siRNA to breast cancer cells and showed that knockdown ofRhoA led to reduced cell invasion through a migration assay. Thesestudies highlight the potential of nanotechnology to treat specificcellular functions that lead to invasion.


In a study that showed the knockdown of a pro-invasion genedoes, in fact, lead to reduced metastases, Villares et al. employedliposomal NPs as their gene delivery vehicle [218]. In this study theydelivered anti-PAR-1 siRNA loaded into DOPC liposomes tomelanomacells and monitored lung metastases. Mice receiving intravenouslyinjected melanoma cells treated with anti-PAR-1 siRNA showed adramatically reduced number of lung metastases indicating thistreatment prevented these cells from invading into potentiallymetastatic lung sites. This exciting finding demonstrates the abilityof nanotechnology to inhibit cell invasion, and thus reduce cancermetastasis.

5. Conclusions

Cancer cell invasion is an aberrantly regulated pathway leading tohallmark events in tumor progression including angiogenesis,lymphangiogenesis, and ultimately metastasis to distant tissues andorgans. Recent advancements in the biology of cancer cell invasionhave crystallized into new opportunities to combat cancer cellmetastasis. In this review we examined a number of moleculartargets that have been identified as key regulators of the variouspathways that are involved in cancer cell invasion, and the emergingtechnology of nanomedicine as a potential solution to remedy thechallenge of metastasis. We described several examples of NPformulations developed for drug delivery and imaging applications,and highlighted the emergence of multifunctional devices thatincorporate targeting, therapeutic, and detecting capabilities fortheranostic applications. We discussed the few formulations thathave been developed to recognize specific biomolecular and bio-chemical signatures associated with cancer cell invasion.

As we move forward, it is expected that nanomedicine willimprove the specificity and potency of existing therapies, and newsolutions will emerge through development of multifunctional NPdevices. These nanoformulations could combine sensing, imaging,molecular targeting, and enhanced therapeutic delivery to further aidin themonitoring and treatment of cancer cell metastasis. Approachesthat focus on improving the therapeutic index of current therapeuticsare likely to make the quickest impact towards improving the clinicaloutcome. However, successful application of nanomedicine willrequire advancements in fabrication strategies and characterizationtechniques to properly evaluate the uniformity, reproducibility, andsafety of nanomedicine formulations.

Many NP formulations developed have focused on deliveringapoptosis inducing therapies to the bulk tumor. Tackling cell invasionwill require development of novel platforms which are more specificin targeting residual cancer cells that have migrated away from thebulk tumor, rather than only debulking the main tumor mass.Although there are a number of different NP directing strategiescurrently being evaluated, it will likely be necessary to develop NPformulations that are directed to cancer cells through multipletargeting strategies. Once these concerns are addressed, this combi-natorial targeting strategy could produce NP formulations with higheraffinity and specificity to invading cancer cells, and lead to thedevelopment of more effective nanomedicine based tools.

Acknowledgments

Thiswork is supported byNIH grants (R01CA119408, R01EB006043,and R01CA134213). O. Veiseh and F. Kievit acknowledge supportthrough the NIH/NCI training grant (T32CA138312).

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