role of the microenvironment in liver metastasis: from pre ......review role of the microenvironment...

Review

Role of the Microenvironment in Liver Metastasis:From Pre- to Prometastatic NichesPnina Brodt

Abstract

Liver metastases remain a major barrier to successful manage-ment of malignant disease, particularly for cancers of the gastro-intestinal tract but also for other malignancies, such as breastcarcinoma and melanoma. The ability of metastatic cells tosurvive and proliferate in the liver is determined by the outcomeof complex, reciprocal interactions between tumor cells anddifferent local resident subpopulations, including the sinusoidalendothelium, stellate, Kupffer, and inflammatory cells that aremediated through cell–cell and cell–extracellularmatrix adhesionand the release of soluble factors. Cross-communication betweendifferent hepatic resident cells in response to local tissue damageand inflammation and the recruitment of bone marrow cells

further enhance this intercellular communication network. Bothresident and recruited cells can play opposing roles in the pro-gression of metastasis, and the balance of these divergent effectsdetermines whether the tumor cells will die, proliferate, andcolonize the new site or enter a state of dormancy. Moreover,this delicate balance can be tilted in favor of metastasis, if factorsproduced by the primary tumor precondition the microenviron-ment to formniches of activated resident cells that promote tumorexpansion. This review aims to summarize current knowledge onthese diverse interactions and the impact they can have on theclinical management of hepatic metastases. Clin Cancer Res; 22(24);5971–82. �2016 AACR.

IntroductionCancer metastasis remains the major challenge to successful

management of malignant disease. The liver is the main site ofmetastatic disease and a major cause of death from gastrointes-tinal malignancies, such as colon, gastric, and pancreatic carci-nomas as well as melanoma, breast cancer, and sarcomas (1).

Circulating metastatic cells that enter the liver encounterunique cellular populations. These include the parenchymalhepatocytes and the nonparenchymal hepatocytes, including liversinusoidal endothelial cells (LSEC), hepatic stellate cells (HSC),Kupffer cells (KC), dendritic cells, liver-associated lymphocytes,and portal fibroblasts. Circulating and bone marrow–derivedimmune cells are also recruited to the liver in response to, andpossibly in preparation for, invading tumor cells and can affect thefinal outcome (reviewed in refs. 2, 3; see Table 1).

This review summarizes our current understanding of the roleof the livermicroenvironment in the growth of hepaticmetastasesand the reciprocal interactions betweenmetastatic tumor cells anddifferent liver cell populations that regulate the process.

Pre- and Prometastatic Niches Drive theProgression of Liver Metastasis

The process of livermetastasis has previously been divided intofour major phases (detailed in Table 1) that follow tumor cell

entry into the liver (4, 5). Emerging evidence suggests that inaddition, a premetastatic phase could set the stage for livercolonization by disseminating tumor cells.

Evidence for premetastatic niche formation in the liverThe term "premetastatic niche" was coined to describe a

microenvironment in a secondary organ site that has beenrendered permissive to metastatic outgrowth in advance ofcancer cell entry through the activity of circulating factorsreleased by the primary tumor (6–8). Although the dependencyof metastasis on premetastatic niches remains controversial anddifficult to verify in the clinical setting (9, 10), it appears that aconsensus is emerging on two scores: (i) that their existence mayhave important implications for the clinical management ofmetastatic disease, and (ii) that much can be learned from theirinterrogation about the cellular/molecular changes required tofacilitate tumor cell growth in a distant site, such as the liver.Two recent studies have confirmed their potential relevance toliver metastasis. In a mouse model of metastatic pancreaticductal adenocarcinoma (PDAC), Costa-Silva and colleaguesshowed that PDAC-derived exosomes taken up by hepatic KCs,upregulate TGFb production, leading to increased fibronectinproduction by HSCs and recruitment of bone marrow–derivedmacrophages. They identified macrophage migration inhibitoryfactor (MIF) as essential for premetastatic niche formation andmetastasis. Moreover, in exosomes derived from patients withstage I PDAC who later developed liver metastasis, MIF levelswere found to be higher than in patients whose tumors did notprogress, thus identifying MIF as a potential predictor of PDACliver metastasis (11). This group also showed that exosomesfrom human breast and pancreatic cancer cell lines that metas-tasize selectively to the lung (MDA-MB-231) or liver (BxPC-3and HPAF-II) fused preferentially with fibroblasts and epithelialcells in the lung and KCs in the liver, and this was mediated by

Departments of Surgery, Medicine, and Oncology, McGill University and theMcGill University Health Centre, Montreal, Quebec, Canada.

Corresponding Author: Pnina Brodt, McGill University Health Centre, 1001Boulevard D�ecarie, Montr�eal, Quebec H4A 3J1, Canada. Phone: 514-934-1934,ext. 36692; Fax: 514-938-7033; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-0460

�2016 American Association for Cancer Research.

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the exosomal integrin laminin receptors a6b1 and a6b4 and thefibronectin receptor avb5, respectively. In exosome-fused KCs,the proinflammatory factors S100P and S100A8 were upregu-lated (Table 1; Fig. 1). Significantly, in PDAC patients, a corre-lation was documented between the levels of circulating, av-bearing exosomes and disease stage, suggesting that exosomesmay play a role clinically and their levels and integrin cargo maypredict metastases in specific organs (12). Kowanetz and col-leagues reported that tumor-derived GM-CSF could mobilizeLy6CþLy6Gþ myeloid cells into metastases, enabling niches inthe lung and liver, and also identified S100A8 as a driving factor(13). Tumor-derived TIMP-1 was identified as another potentialinducer of increased liver susceptibility to metastasis, acting viahepatic SDF-1 and neutrophil recruitment (14). Interestingly,S100A8 was identified as a driver of liver premetastatic nicheformation in several studies (12–15), suggesting that it mayhave clinical utility as a predictor of liver metastasis.

The stage of primary tumor development at which premeta-static niches can be formed is difficult to assess in the clinicalsetting. Costa-Silva and colleagues found exosomal MIF upre-gulation in mice with pretumoral pancreatic lesions, and highplasma exosomal MIF levels were also detected in patientswith stage I PDAC (11), suggesting that they could be formedat very early stages of cancer development. The potentialcontribution of circulating cancer cells (CTC) that may bepresent early in tumor development and even after resectionof the primary tumor (16, 17) is also unknown.

Resident and Immune Cells Can PlayDiverse and Opposing Roles in theDevelopment of Liver MetastasesHost innate resistance mechanism can destroy tumor cellsprior to extravasation

Blood-borne cancer cells entering the liver first encounter theLSECs, KCs, and hepatic natural killer (NK) cells (pit cells) thattogether mount the first line of defense (18). Cancer cellsentrapped in the sinusoids may undergo destruction due tomechanical stress and deformation-associated trauma or may diedue to phagocytosis by KCs or cytolysis by NK cell–releasedperforin/granzyme (18; reviewed in ref. 4). NO, ROS, and toxicradicals released by LSECs in response to local ischemia/reperfu-sion can contribute to tumor cell death (19, 20). Release of NOand IFNg by LSECs and NK cells can result in upregulation oftumor cell Fas and apoptosis (18) that can be augmented by NKand LSEC-derived TNFa (21). TNFa is one of the numerouscytokines and chemokines unleashed by KCs in response toinflammation (Table 1; ref. 22). In addition to causing cell deathdirectly, some of these factors can mobilize and activate addi-tional innate immune cells, such as neutrophils, thereby adding tothe local tumoricidal capacity (reviewed in ref. 23). In severalanimal tumor models, including colon cancer, loss of NK cellsincreased cancer cell growth in the liver, whereas enhanced NKactivity reduced liver metastasis (24–26). Indirect evidence alsosuggests that susceptibility to NK-mediated immune attack canaffect the ability of human cancer cells to generate livermetastases(27). Circulating neutrophils can also release various factorsabundant in their granules to kill tumor cells (see Table 1), andtheir cytokines and chemokines can activate the tumoricidalpotential of resident KCs and recruit host immune T cells withantitumorigenic activities (reviewed in ref. 28).

Cancer cells can escape these cytotoxic effects by formingclusters with blood or other cancer cells that shield them fromthe lethal effects of shear stress or NK-mediated cytotoxicity(reviewed in ref. 29). The release of inflammatory mediators,such as IL1b, TNFa, and IL18, can also initiate a cascade thatfacilitates their rapid exit from the vasculature to a less "toxic"microenvironment (see below and Fig. 2).

The sinusoidal endothelium actively participates in differentphases of metastasis

Although the rapid inflammatory response initiated by cancercell entry can lead to cell death, it may also have a tumor-protective effect by upregulating the expression of LSEC celladhesion molecules (CAM) and in this way, enhance cancercell adhesion and transendothelial migration into the space ofDisse, where they can escape the cytotoxic effects of KCs andNK cells (30). Cancer cells can adhere to inflammation-inducedE-selectin, VCAM-1, and ICAM-1, either as single cells or inassociation with host cells, such as KCs or neutrophils (reviewedin ref. 31). Blockade of this inflammatory cascade was shownto reduce liver metastasis (32–34), and TNFR1 signaling wasidentified as a major driver of this process (35).

Ultimately, the outcomeof these opposing effects on tumor cellsurvival and progression to the next phase depends on multiplefactors, including the expression on the cancer cells of the respec-tive counter receptors, namely the E-selectin ligands sLewa andsLewx and CD44 isoforms (36; reviewed in refs. 29, 37), andVCAM-1 and ICAM-1 counter receptors integrins a4b1 and LFA-1and Mac-1 (38), respectively. E-selectin binding triggers a signal-ing cascade in both tumor cells and LSECs, leading to diapedesisand transendothelial migration (39) and altering gene expressionin a tumor type–specific manner (40). Clinical studies havedocumented increased expression of E-selectin in and aroundcolorectal cancer liver metastases and elevated soluble E-selectin,ICAM-1, and VCAM-1 levels that correlated with disease outcomein patients with colorectal cancer (reviewed in ref. 23).

LSECs may have other metastasis-promoting functions. LSEC-secreted fibronectin can induce epithelial–mesenchymal transi-tion (EMT) in colorectal cancer cells via integrin a9b1, enhancingERK signaling and tumor invasion (41). Human LSECs wereshown to induce colorectal cancer migration and EMT via MIF(42), thereby increasing their metastatic potential.

LSECs contribute to tumor-induced angiogenesis. A recentstudy identified Notch1 as a negative regulator of sinusoidalendothelial cells sprouting into micrometastases and thereby ofangiogenesis and liver metastasis (43), raising questions aboutthe advisability of Notch targeting for cancer therapy. Moreover,liver metastasis of different carcinomas, including colorectalcancer, can co-opt the sinusoidal endothelium at the tumor–liverinterface, giving rise to a histologic growth pattern termed the"replacement" or "sinusoidal" growth pattern that seems to resultfrom tumor cell invasion between LSECs and the matrix in thespace of Disse and is characterized by the formation of intrameta-static vessels that appear continuous with the sinusoidal vessels(reviewed in ref. 23). The factors that drive this co-opting mech-anism remain to be identified, but a recent study by Im andcolleagues suggests that Ang2 may play a regulatory role (44).

Collectively, the evidence shows that LSECs are not a passivebarrier to tumor cell extravasation but participate actively in themetastatic process. Cancer cell interaction with LSECs can recip-rocally alter thephenotypes of both cell types, and thismay lead to

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Clin Cancer Res; 22(24) December 15, 2016 Clinical Cancer Research5972




intravascular tumor cell destruction but can also promote metas-tasis through enhanced tumor cell migration and increasedangiogenesis (Table 1; Figs. 2 and 3).

The role of macrophages is context dependentKCs constitute approximately 10% of all liver cells. Recent

studies suggest that they may be established prenatally andmaintained into adulthood, independently of replenishmentfrom bloodmonocytes but in anM-CSF andGM-CSF–dependentmanner (45). They reside mainly in the hepatic sinusoids andare anchored to the endothelium by long cytoplasmic processes(46; reviewed in ref. 47). In their interaction with invading can-cer cells, KCs can play diverse and opposing roles, dependingon factors such as the stage of the metastatic process, tumorload, and interactions with other immune cells. As discussed,KCs can promote metastasis by orchestrating the premetastaticniche. However, tumor–KC interactions can have opposingeffects. Several studies have documented a rapid adhesion oftumor cells to KCs in the sinusoidal lumen, resulting mainly in

tumor cell phagocytosis or apoptosis, within hours of tumor cellentry (47). This tumoricidal effect may require cooperation withlocal or recruited NK cells (48), and its efficiency in removingcancer cells likely depends on the tumor load (49). An intravitalmicroscopy study (50) recently revealed that 74% of intra-arte-rially injected rat colon cancer cells adhered to the KCs within6 hours, and this was associated with increased liver TNFa andIL1b levels, consistent with our own findings (51, 52) andindicative of KC activation. Extensive phagocytosis of tumorcells was observed within 2 hours postinjection. Interestingly,although elimination of KCs 2 days prior to tumor injectionincreased liver metastasis, it had no significant effect up to 1 weekafter tumor injection, suggesting that KCs exerted their mostpotent antitumor effect within the first 24 hours of tumor cellentry into the liver (50). A bimodal effect of KCdepletionwas alsodocumented in another study of murine colorectal cancer, and itwas attributed to decreased VEGF and increased iNOS levels inlivers subjected to late-stage KC depletion (53). Indeed, tumorcells that survive the initial tumoricidal assault by KCs can benefit

Table 1. Cells and mediators involved in the different phases of liver metastasis

Soluble factors involvedPhase of the metastaticprocess Cell type involved Cell surface markers Recruiting or activating Produced References

Premetastatic nicheKCs CD11b, F4/80 M-CSF, GM-CSF, MIF TGFb, S100P, S100A8 (11–14)HSCs a-SMA, desmin, GFAP KC-derived TGFb and TNFa Fibronectin (11, 13)MDSCs CD11b, Ly6C, Ly6G Tumor-derived GM-CSF S100A8 (12, 15)Neutrophils CD11b, Ly6G TIMP-1, SDF-1 S100A8 (7, 14, 67)

Post-tumor invasion(1) Microvascular phase:

Tumor cells trapped inthe vasculature

LSECs CD31, TNFR1, TNFR2, avb3,E-selectin, VCAM-1, ICAM-1

TNFa, IL1b, IL18 ROS, NO, IFNg , TNFa (5, 18–20, 23, 31)

KCs CD11b, F4/80 M-CSF, GM-CSF, IFNg TNFa, IL1, IL6, IL8, IFNg ,MIP-1a, MIP-2, IP-10,MCP-1, CCL5, VEGF

(5, 23, 31, 45–47, 51)

Neutrophils CD11b, Ly6G S100A8, S100A9, IL8, CCL2 TNFa, ROS, defensins,perforins, elastase

(28, 31, 64)

Hepatic NK cells NK1.1, NK1.2, FcgRIII CXCL9, CXCL10, IL1, IL12,IL15, IL18

Grz, Perforin, IFNg ,TNFa

(5, 31, 48)

(2) Extravascular,preangiogenic phase:Tumor cells transmigrateinto the space of Disse,activating a local stromalresponse

HSCs a-SMA, desmin, GFAP KC-derived TGFb, PDGF,bFGF, SDF-1, IGF-I, CCL2

ECM deposition, MCP-1,CCL5, CCL21, TGFb

(74–77, 84)

Neutrophils CD11b, Ly6G; N1: ICAM-1;N2: CXCR4

S100A8, S100A9, CXCL1,CXCL2, CXCL5, TNFa,IL8, G-CSF, IFNg , CCL2,CCL5, TGFb

TNFa, ROS, NOS,MMP-8, MMP-9,elastase, cathepsin G,VEGF

(28, 60, 64, 71)

KCs CD11b, F4/80 M-CSF, GM-CSF, IFNg IL6, VEGF, HGF,MMP-9, MMP-14

(5, 50, 53)

(3) Angiogenic phase:Micrometastases arevascularized

Blood-derivedmonocytes

CD11b, F4/80, CD68, CD163,CD206

TNFa, MCP-1 VEGF, MMPs, bFGF (57, 60)

HSCs a-SMA, desmin, GFAP KC-derived TGFb, PDGF,bFGF, SDF-1, IGF-I, CCL2

VEGF, angiopoietin-1,MMP-2, -9, and -13

(74, 79–82, 88)

LSECs CD31, TNFR1, ICAM-1 VEGF, Ang1, inhibited byNotch1 and regulated byAng2

Fibronectin, MIF (23, 41–44)

Macrophages (M2) CD11b, F4/80, Ly6C, CD163 TGFb, IL10 NO, TNFa, IFN, VEGF,EGF, bFGF, TGFb,IL10

(57–61)

(4) Growth phase:Metastases expansion

Hepatocytes Albumin, tyrosineaminotransferase

Adhesion to tight junctionintegral proteins

AREG, EGF, HB-EGF,ErB2, IGF-I, HGFL,ErB3, bFGF

(92–98, 101–105,107)

Tregs CD4, CD25, Foxp3 TGFb, IL10, CCL5, CCL22 VEGF, TGFb, IL10 (35, 111, 124)

NOTE: Shown are the four phases of the metastatic process that follow tumor cell invasion into the liver. Listed are the hepatic cells known to be involvedat each phase, the mediators regulating their recruitment and activation, and the factors they produce to block or promote the metastatic process. Theprocess has been simplified and divided into distinct phases in the interest of clarity. However, it should be viewed as dynamic with overlapping cellular andmolecular drivers at different phases. Moreover, as the metastatic process advances, the cancer and hepatic cells evolve and their phenotypes change as aconsequence of their interactions. These newly acquired properties help propel the process forward (see also Figs. 1–3).Abbreviations: ECM, extracellular matrix; MDSC, myeloid-derived suppressor cell; MMP, matrix metalloproteinase, NK, natural killer; Treg, regulatory T cell.

The Microenvironment in Liver Metastasis

www.aacrjournals.org Clin Cancer Res; 22(24) December 15, 2016 5973




from their protumorigenic functions. Adhesion to KC can facil-itate tumor cell extravasation, among others, by sequential acti-vation of endothelial CAMs (29, 54). In addition, KCs canproduce cytokines and growth factors, including IL6, hepatocytegrowth factor (HGF), and VEGF, and matrix metalloproteinases(MMP), such as MMP-9 and MMP-14, that can accelerate tumorcell invasion into and within the parenchymal space as well aspromote tumor cell proliferation and angiogenesis, therebyenhancing liver metastasis (see Table 1; Figs. 2 and 3).

Taken together, the evidence suggests that KC targeting couldbecome an effective antimetastatic strategy only within a verynarrow window. Limiting KC activity may be beneficial if it couldprevent premetastatic niche formation,whereas enhancing theKCtumoricidal potential using agents such as IFNg , GM-CSF, or

muramyl dipeptide may be most effective if achieved beforetumor cells enter the liver in large numbers (47, 55). The contri-bution of CTCs that may be present before liver micrometastasesare established or after surgical resection of the primary tumor(16, 17) to the tumor load and KC activation is unknown. Thismultifaceted role of KCs in liver metastasis may explain thelimited success to date of KC-targeting interventions (47).

Importantly, in addition to KCs, blood-derived monocytescan also be recruited to the tumor site in response to localinjury and inflammation and differentiate locally into matureCD11bloF4/80hi macrophages in a CCR2-dependent manner(56). These macrophages can also trigger HSC activation andfibrogenesis (57), a process important in the early stages ofextravascular tumor expansion (see below).

© 2016 American Association for Cancer Research

A B

ExosomesG-CSF

PDAC

LSEC

BM-derivedmyeloid cells

Vessel lumen

Space of Disse

KC

HSC

Hepatocyte

Fibronectin

S100A8 S100A8

Potential therapeutic strategies:1. Enhancing KC tumoricidal activities2. Inducing HSC apoptosis3. Inhibition of inflammatory mediators

Ly6C+Ly6G+

myeloid cells

Neutrophil32

1

aHSC

MIF

?

TGFβ

TGFβ

TGFβ

Tumor-derivedexosomes

Tumor-derivedexosomes

S100A8

Tumor-derived G-CSF

aKC

αβ

Figure 1.

The premetastatic niche in the liver. Shown is a diagrammatic representation of the multistep process involved in the formation of premetastatic niches inthe liver based on data described in refs. 11, 12. The symbols used for depiction of different hepatic cells are listed in Fig. 3. A and B, A diagrammaticrepresentation of a PDAC is shown in inset A, and an enlarged image of a Kupffer cell activated by PDAC-derived exosomes is shown in inset B.Encircled numbers denote cellular and molecular interactions that can potentially be targeted for liver metastasis prevention, as detailed in the right-hand corner box. BM, bone marrow.

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Macrophages have inherent plasticity, and their phenotypescan change within a spectrum of activation states between thepolar M1 and M2 phenotypes (58, 59). M1 macrophages aretumoricidal due to high NO and TNFa production levels andproduce Th1, Th17, and the NK-attracting chemokines CXCL9and CXCL10 (60). In contrast, M2 macrophages can promotetumor growth through production of growth factors, such asVEGF, EGF, bFGF, and TGFb (61). In addition, M1 macrophagesactivate Th1-type immune responses that can further amplifyM1/killer-type activity through the production of IFNg , whereasM2 macrophages induce regulatory T cells (Treg) and thereby animmunotolerant microenvironment through release of TGFb

and IL10 (Table 1; ref. 62). Evidence regarding the role ofmacrophage polarization in liver metastasis is scant, becausemacrophages within or around hepatic metastases have, with fewexceptions, not been subtyped. A recent clinical study identifiedthe M2/M1 ratio in hepatic resections as a correlate of colorectalcancer metastasis (63), implicating M2 macrophages in the clin-ical disease. Furthermore, F4/80, the cell surface marker frequent-ly used to identify KCs, is also expressed on recruited monocytes,and strategies used to eliminate macrophages in vivo are not KCspecific. The source and identities of macrophages in manypublished studies remain therefore to be verified. As immuneediting of the tumor microenvironment and the conversion of

III. Angiogenic phase

Fas/FasL

Fas/FasL

VEGF

VEGF-R

MMP-9

MMP-9VEGF

LSEC

aKC

I. Microvascular phase

II. Preangiogenic phase

Potential therapeutic strategies:3. TGFbRII blockade4. MDSC depletion5. TNFR2 blockade6. PD-1/PD-L1 blockade

Potential therapeutic strategies:10. IGF-I neutralization11. Claudin-2 blockade

IV. Growth phase

Potential therapeutic strategies:1. TNF inhibition2. E-selectin blockade

Potential therapeutic strategies:7. Blocking M2 polarization8. VEGF inhibition9. N1 induction

sLew×/E-Sel.

TNFα

TNFα

TNFα

TGFβ

TGFβ

TGFβ

IL10TGFβ

NOTNFα

TNFR1

HSC

aHSC

aHSC

M1

N1

N2

IGF-I Hepatocyte

M2

CoIIVMDSC

ArgROS

Treg MMPsVEGFTGFβIGF

CD8+

T cell

Tumor cell

Vessel lumen

Space of Disse

Parenchyma

12

3

4

56

7

8

9

11

10


Figure 2.

The multistep process of liver colonization by disseminated cancer cells. Shown is a diagrammatic representation of the major cell types, soluble factors, andextracellular matrix (ECM) components in the liver microenvironment that impact the progression of liver metastasis. The four major phases in the process aredelineated by dashed lines. Top, major intercellular interactions that occur upon entry of the tumor cells into the sinusoidal vessels (the microvascular phase)and precede tumor extravasation; middle, events following tumor cell extravasation into the space of Disse, divided into the preangiogenic phase (middle left)that is orchestrated by stellate cell activation, ECM deposition, cytokine production, and the recruitment of innate and adaptive immune cells and culminatesin neovascularization (the vascular phase; middle right); bottom, these interactions enable tumor expansion (the growth phase). The symbols used fordepiction of different hepatic cells are listed in Fig. 3 or indicated in the diagram. The encircled numbers denote potential targets for therapeuticintervention, as detailed in the boxes inserted within each of the depicted phases. MDSC, myeloid-derived suppressor cell; MMP, matrix metalloproteinase;Treg, regulatory T cell.






Produce ROS, NO, IFNγ,TNFα

Express CAM, protect tumorcells from apoptosis, facilitatetransmigration, participate inangiogenesis, induce EMT

Participate in premetastaticniche formation, drive fibrogenicresponse, ECM production, mediate angiogenesis,recruit inflammatory cells,produce MMPs

Mediate tumor adhesion, producegrowth factors (e.g., IGF-I, HGFL),alter tumor transcriptome, undergoEMT to provide stromal support,enhance angiogenesis via bFGF

(5, 11, 12, 47–53)

(59–63)

(113, 117, 123, 124)

Produce perforin and TNFα,induce Fas/FasL interaction,kill tumor cells

Induce Tregs, block T-cellexpansion, suppress cytotoxicT-cell function, produce ROS,produce arginase

M2 macrophages produceVEGF, EGF, and bFGF; induce Tregs via IL10 and TGFβ

M1 macrophages produce NO,TNFα, and T-cell– and NK-recruiting chemokines

Participate in premetastaticniche formation; activate LSECCAM; produce VEGF, IL6,HGF, and MMP-9; activate HSC

Phagocytose tumor cells, causeapoptosis (early in the process),produce TNFα, mobilizeneutrophils and NK cells

(5, 18–20, 31, 33, 41)

Hepatocytes

Antimetastatic effects ReferencesPrometastatic effectsCell type

Liver sinusoidendothelial cells(LSEC)

Hepatic stellate cells (HSC)

Kupffer cells(KC)

Macrophages

Myeloid-derivedsuppressor cells(MDSC)

Natural killer (NK) cells

T lymphocytes

Neutrophils

(11, 23, 77, 82–86)

Resting Activated

(93–96, 101, 103, 105)

(4)

(28, 64, 77)

(109, 111)Tregs inhibit effector T-cellexpansion, eliminate cytotoxicT cells, subvert immuneactivation, produce VEGF

Cytotoxic T cells kill tumorcells, release perforin andTNFα, recruit macrophages

Participate in premetastaticniche formation, enhancetransendothelial migration,entrap tumor cells; N2 neutrophilsproduce VEGF, MMP-9, and CCL5

N1 neutrophils produce ROS,NO, TNFα, and defensins; recruitCD8+ T cells


Figure 3.

Parenchymal, nonparenchymal, and immune cells of the liver and their role in metastasis. Listed are the parenchymal and nonparenchymal cells of the liver andtheir anti- and prometastatic functions. Symbols shown for each cell type were used to depict intercellular communications in Figs. 1 and 2. EMT, epithelial-mesenchymal transition; Treg, regulatory T cell.

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tumor-associated (TAM) M2 macrophages to M1 killer macro-phages are emerging as potentially relevant anticancer strategies(62), further characterization of both the source and the statusof macrophage populations involved in liver metastasis willbecome critical.

The neutrophils: A double-edged swordNeutrophils are part of the innate immune response to patho-

gens and are also rapidly activated in response to invading cancercells (28). Bone marrow–derived neutrophils are mobilized tosites of inflammation or cancer via their cell-surface receptorCXCR2 and in response to chemokines and cytokines (Table 1)secreted by activatedmacrophages, endothelial cells, or the tumorcells (reviewed in ref. 28). At the tumor site, the neutrophils canexert opposing effects that depend on the inflammatory/immunecontext (reviewed in ref. 64). They can release cytolytic factors(detailed in Table 1) or inhibit tumor growth indirectly viarecruitment of CD8þ cytotoxic T cells and macrophages. Thismay require direct contact with the tumor cells (64), such as canoccur within the liver sinusoidal lumen (65, 66).

On the other hand, neutrophils can be mobilized into pre-metastatic niches in the liver in response to S100A8 and S100A9(7) and were shown to contribute to niche formation in anorthotropic colon carcinoma model (67). Moreover, in the vas-cular lumen, the physical interaction with tumor cells thatcould lead to tumor cell kill may, alternatively, anchor circulatingtumor cells to the vascular endothelium and enhance their migra-tion into the extravascular space. Tumor-derived cytokines, suchas IL8, induce expression of integrins, such as CD11b/CD18, onthe neutrophils, increasing their adhesion to tumor cells viathe counter receptor ICAM-1 and facilitating transendothelialmigration, as shown in murine melanoma and lung carcinomamodels (66, 68). Tumor cell entrapment in the sinusoids canalso be mediated by neutrophil-produced extracellular (DNA)traps, resulting in increased tumor retention in the sinusoids,enhanced tumor cell adhesion, proliferation, migration and inva-sion, and increased livermetastasis (69, 70). Neutrophils can alsorelease extracellular matrix (ECM)–degrading proteinases, suchas MMP-8, MMP-9, elastase, and cathepsin G, thereby increasingtumor invasion (64), and can promote angiogenesis through therelease of VEGF (Table 1; Figs. 2 and 3).

Moreover, a TGFb-mediated neutrophil polarization thatalters the phenotype of tumor-associated neutrophils fromtumor inhibitory (N1) to tumor promoting (N2) was recentlydescribed (71). TGFb produced by M2 macrophages may con-tribute to this process (reviewed in ref. 59). However, the contri-bution of polarized neutrophils to liver metastasis remains tobe confirmed.

Clinical studies implicate neutrophils in tumor promotion.With few exceptions, elevated circulating neutrophil counts orneutrophil-to-lymphocyte ratios were associated with pooreroutcomes and distant metastases in patients with variousepithelial malignancies (72), including carcinomas of the gas-trointestinal tract. High neutrophil counts may also be associ-ated with increased resistance to therapy (64, 72). The 5-yearsurvival for patients with colorectal cancer undergoing hepaticresections that had neutrophil-to-lymphocyte ratios greaterthan 5 was worse than for those with normal ratios (73).However, high neutrophil infiltration into the tumor site maybe a consequence of advanced tumor stage (and, therefore,poorer prognosis) rather than its cause.

HSCs orchestrate a prometastatic microenvironment that isrequired for transition from the avascular to the vascularstage of metastasis

HSCs orchestrate the characteristic fibrogenic response of theliver to injury. Normally quiescent in the space of Disse (74),they are activated (aHSC) in response to liver damage andinflammatory stimuli, acquire a myofibroblast-like phenotype(a-SMAþ), and produce ECM rich in collagen I and IV (74, 75).Chemokines and cytokines released by aHSCs (Table 1) alsorecruit inflammatory/immune cells (76, 77), thereby shapingthe immune microenvironment.

In addition to their role in premetastatic niche formation(above), HSCs can orchestrate a prometastatic niche followingtumor extravasation. In response to growth factors and inflam-matory mediators (detailed in Table 1), HSCs are activated (74)and can trigger a process akin to the early events in liver repair.This was observed in animal models (4) and is also evidencedby the increased production of collagen IV in and aroundhepatic metastases in clinical specimens (78). Macrophages,hepatocytes, and LSECs contribute to this process by releasingTGFb and/or TNFa (57). In turn, aHSC-derived proangiogenicfactors, such as VEGF and angiopoietin-1 (79, 80), initiateangiogenesis (3), and this is enhanced by HSC-derived MMPsthat facilitate endothelial cell migration and tumor invasion(23, 74; reviewed in refs. 5, 81; Table 1; Fig. 2).

Several lines of evidence confirm the essential role of HSCs inliver metastasis. Olaso and colleagues showed that myofibro-blasts that infiltrated avascular micrometastases of B16 mela-noma cells induced a stromal response favorable to angio-genesis, which preceded endothelial cell recruitment and wasfollowed by colocalization of myofibroblasts and endothelialcells within angiogenic structures (82, 83). HSC and macro-phage recruitment into metastatic sites and subsequent angio-genesis were shown to be CCR2/CCL2 dependent (84). Morerecently, Eveno and colleagues (85), using immunofluores-cence, showed that 9 days postinjection of colorectal cancerLS174 cells into SCID mice, the hepatic micrometastases con-sisted of proliferating cancer cells, a well-organized network ofaHSC and laminin deposits, but no vascular network. As theliver metastases grew, an organized vascular network appearedand laminin colocalized with CD31þ endothelial cells. Co-injection of tumor cells and aHSCs enhanced metastasis. More-over, analysis of liver metastasis from patients with colorectalcancer revealed a surface-marker expression pattern similar tothat observed in the coinjection studies.

Recently, suppression of TGFb receptor II signaling byQGAP1 was shown to prevent HSC activation, and IQGAP1downregulation was observed in myofibroblasts associatedwith human colorectal cancer liver metastases, consistent withtheir activation in this context (86). Furthermore, a-SMAþ cells,whose presence correlated with the degree of fibrous stroma,were identified in liver metastases of colon, gastric, and pan-creatic adenocarcinomas (87).

The importance of HSCs to metastatic niche formation andtheir role in primary liver cancer (88) have raised interest inHSC targeting as an anticancer/antimetastatic strategy. There islittle direct evidence that such an approach can succeed, duepartially to the present lack of agents that specifically targetHSCs without toxicity. Indirect evidence from a rat model ofcholangiocarcinoma (89) and pancreatic stellate cell targetingin a PDAC model (81) suggests a potential therapeutic benefit,






but this remains to be verified. Of note, portal fibroblasts andbone marrow–derived fibrocytes (90) can also contribute to thestromal response. Liver-invading cancer cells located in portaltracts and unable to activate HSCs may engage portal tractfibroblasts that produce IL8, a chemokine involved in invasionand angiogenesis (91). Specific targeting of HSCs may, there-fore, not be sufficient to deprive metastatic cells of a growth-promoting stroma.

Hepatocytes can promote metastasis directly and indirectlyThe role of the parenchymal hepatocytes in liver metastasis is

not well understood. Tumor cell adhesion to hepatocytes wasidentified as one of the earliest events in liver metastasis for-mation and a correlate of the metastatic potential (92, 93).Desmosomes (94), integrins av, a6, and b1 that bind tohepatocyte ECM (93), osteopontin binding via counter recep-tors CD44 and integrin av (95), and claudins (96, 97) were allimplicated. Adhesion of human colorectal cancer cells to hepa-tocyte-derived ECM was shown to upregulate the expression ofgenes involved in tumor cell survival, motility, and prolifera-tion, in particular EGF family members implicated in livermetastasis (98) such as AREG, EGFR, HBEGF, and erbB2 andstem cell markers CD133 (PROM1) and LGR5 (99), suggestingthat colorectal cancer adhesion to hepatocyte ECM inducesautocrine growth-promoting mechanisms for tumor expansion.Tumor cell adhesion to hepatocytes can have other conse-quences. The FasL/FLICE-like inhibitory protein on murineMC38 cells was shown to induce apoptosis in hepatocytes byactivating Fas signaling, and this destructive process created aniche for tumor expansion (100). Furthermore, hepatocytesproduce several growth factors, including IGF-I (101). As wehave shown, blockade of IGF-I signaling in metastatic tumorcells could inhibit liver metastasis (102–104). Other factorsinclude the HGF-like protein/macrophage-stimulating protein(HGFL) that can enhance tumor growth, motility, and inva-sion via the Ron receptor (105); heregulin, a ligand of ErbB3shown to enhance integrin avb5-mediated cell migration anderbB3/erbB2 signaling in human colorectal cancer cells (106);and the proangiogenic factor bFGF (107).

The clinical relevance of tumor–hepatocyte interactions isnot clear. Three different histologic growth patterns have beenidentified in liver metastases specimens, namely the desmo-plastic, pushing, and replacement growth patterns. The lattertwo are characterized by close tumor–hepatocyte proximity(23). Whether or not direct adhesion between metastaticcancer cells and hepatocytes influences the eventual growthpattern of colorectal cancer liver metastases is not known.Blocking tumor–hepatocyte interactions could have beneficialantimetastatic effects, as recently demonstrated by Tabariesand colleagues (108).

An immunosuppressive microenvironment facilitatesmetastatic expansion

Tumor cells can activate specific T-cell–mediated immuneresponses that curtail tumor expansion through various mechan-isms (reviewed in ref. 109). However, tumor cells can evadeT-cell–mediated killing, among others, by giving rise to and/orrecruiting immunosuppressive cells, such as myeloid-derivedsuppressor cells (MDSC) and regulatory T cells (Treg) that alterthe immune landscape, inducing a state of immune tolerance thatis permissive to tumor expansion (reviewed in ref. 110).

The role of Tregs. Treg cells may be thymically derived (natural,nTregs) or induced from na€�ve CD4þ T-cell precursors (iTreg)in response to IL10 and TGFb. They exert an immunosup-pressive effect by different mechanisms that result in theinhibition of an effective, antitumorigenic T-cell response(reviewed in ref. 111). Although high Treg levels were docu-mented in the blood and local tumor sites of patients withvarious epithelial cancers (e.g., ref. 112), relatively little isknown about their role in liver metastasis. Connolly andcolleagues, using models of early, preinvasive pancreaticneoplasia and advanced colorectal cancer showed that Tregsaccelerated the development of liver metastases (113). Werecently documented the accumulation of Tregs around coloncarcinoma MC38 liver micrometastases and showed that thiswas TNFR2 dependent (35).

Intriguingly, in 57 colon cancer specimens frompatients under-going chemotherapy or chemoimmunotherapy, higher Treg infil-tration scores were associated with a better prognosis and a betteroutcome (114). However, the relationship between Treg accu-mulation in the primary tumors and in the corresponding livermetastases has not been examined. Although Treg-targetingstrategies are in development (115, 116; reviewed in ref. 110),their potential therapeutic benefit for liver metastases has notyet been examined.

The role of MDSCs. MDSCs are a heterogeneous population ofmyeloid cells that can be of the monocytic (Mo-MDSC,CD11bþLy6Cþ) or granulocytic (G-MDSC, CD11bþLy6Gþ)lineages (117). In humans, MDSCs are CD33þ and/or CD11bþ

andHLA-DR� (118). Under normal physiologic conditions, bonemarrow–derived immature myeloid cells differentiate intomature granulocytes or monocytes, able to mediate host innateimmune responses in the target tissue. However, in the tumormicroenvironment, these precursors do not mature and exertimmunosuppressive and tumor-promoting effects instead(119). Although they express granulocyte and monocyte surfacemarkers, these cells have been characterized on the basis offunctional criteria, namely their immunosuppressive capabilities(117, 120). An increase in circulating MDSCs in cancer patientsand their recruitment into tumor sites have been documented(117, 121, 122).

In the liver, MDSCs can be recruited to the metastasesthrough chemokines, such as CXCL1 and CXCL2, producedby LSECs and KCs or by activated HSCs (77). There, they canpromote tumor growth by inducing immunosuppression(123), producing arginase (117), and increasing Treg numbers(Table 1; Fig. 2; ref. 124). Clinical and experimental evidenceconfirms their role in metastasis (125). Recruitment of tumor-promoting myeloid cells into colorectal cancer liver metastaseshas recently been documented, and their depletion was shownto result in a marked reduction in liver metastasis (126). Werecently reported that MDSCs accumulate in hepatic microme-tastases of MC38 cells within days of tumor cell injectionand documented a relative preponderance of Ly6Chigh cells(the more suppressive, arginase-high subtype; ref. 127). Wealso identified CD33þHLA-DR�TNFR2þ myeloid cells in theperiphery of hepatic metastases from patients with colorectalcancer, implicating MDSCs in the clinical disease (35).

Several strategies are being developed to selectively elim-inate MDSCs, including pharmacologic induction of MDSC dif-ferentiation, inhibition of MDSC expansion from bone marrow

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precursors, or blockade of their function (128). However, some ofthe drugs developed are not MDSC specific, or their long-termadministration is not possible due to toxicity (128). Their trans-lational potential remains therefore to be verified.

ConclusionsCancer cells entering the liver encounter a unique and complex

microenvironment. Parenchymal and nonparenchymal livercells, as well as recruited inflammatory and immune cells, par-ticipate in the response to invading tumor cells andmay inhibit orfavor the progression ofmetastasis. The factors that determine theoutcomeof these opposing effects and the pattern of growth of themetastases are not yet well understood. This duality of functionand the shifts in the types of cells and mediators involved atdifferent stages of the metastatic process render the attempts totarget specific cellular interactions in the microenvironmentextremely challenging and may explain the slow progress to datein identifying agents that can successfully and specificallyinhibit metastatic disease. With the advent of genomic profilingfor personalized cancer treatment and the increased availabilityof surgically resected liver metastases for cellular/molecular

interrogation, our understanding of the relationship betweenthe genetic background and clinical history of the patient, thegenomic/transcriptomic profile of the cancer cells, and the typeof response mounted by the microenvironment may improve,opening new avenues for prevention and/or treatment of livermetastatic disease.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsThe author is indebted to Dr. Janusz Rak (McGill University Health Centre)

for insightful editorial comments and to Simon Milette for the superb artisticrendering of the metastatic niches of the liver and for his help with the table.

Grant SupportThis work was made possible by support from the Canadian Institute for

Health Research and by a PSR-SIIRI-843grant from the Qu�ebec Minist�ere de

l'�Economie, de l'Innovation et des Exportations.

Received February 21, 2016; revised September 19, 2016; accepted September22, 2016; published OnlineFirst October 19, 2016.

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