clinical cancer research - role of the microenvironment in liver metastasis: from … · 2016. 10....

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Role of the microenvironment in liver metastasis: from pre- to pro-metastatic

niches

Pnina Brodt*

Departments of Surgery, Medicine, and Oncology McGill University and the McGill University Health

Centre, Montreal, Quebec, Canada

Running Title: The microenvironment in liver metastasis

Key Words: liver metastasis, tumor microenvironment, colon cancer, immunosuppression

This work was made possible by support from the Canadian Institute for Health Research and by a

PSR-SIIRI-843 grant from the Québec Ministère de l'Économie, de l'Innovation et des Exportations.

The author has no conflict of interest to disclose.

* All correspondence should be addressed to:

Dr. Pnina Brodt

The Research Institute of the McGill University Health Centre

The Glen Site, 1001 Décarie Blvd, Room E.02.6220

Montréal, QC, H4A 3J1, Canada

Telephone: (514) 934-1934, Ext. 36692, Fax: (514) 938-7033

Email: [email protected]

Research. on June 29, 2021. © 2016 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 19, 2016; DOI: 10.1158/1078-0432.CCR-16-0460

http://clincancerres.aacrjournals.org/

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Summary

Liver metastases remain a major barrier to successful management of malignant disease, particularly

for cancers of the gastrointestinal (GI) tract but also for other malignancies such as breast carcinoma

and melanoma. The ability of metastatic cells to survive and proliferate in the liver is determined by

the outcome of complex, reciprocal interactions between the tumor cells and different local resident

subpopulations including the sinusoidal endothelium, stellate, Kupffer and inflammatory cells that are

mediated through cell-cell and cell-extracellular matrix adhesion and the release of soluble factors.

Cross communication between different hepatic resident cells in response to local tissue damage and

inflammation and the recruitment of bone marrow cells further enhance this inter-cellular

communication network. Both resident and recruited cells can play opposing roles in the progression

of metastasis and the balance of these divergent effects determines whether the tumor cells will die,

proliferate and colonize the new site or enter a state of dormancy. Moreover, this delicate balance

can be tilted in favor of metastasis, if factors produced by the primary tumor pre-condition the

microenvironment to form niches of activated resident cells that promote tumor expansion. This

review aims to summarize current knowledge on these diverse interactions and the impact they can

have on the clinical management of hepatic metastases.




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Introduction

Cancer metastasis remains the major challenge to successful management of malignant disease.

The liver is the main site of metastatic disease and a major cause of death from gastrointestinal (GI)

malignancies such as colon, gastric and pancreatic carcinomas, as well as melanoma, breast cancer

and sarcomas (1).

Circulating metastatic cells that enter the liver encounter unique cellular populations. These include

the parenchymal hepatocytes and the non–parenchymal, liver sinusoidal endothelial cells (LSEC),

hepatic stellate cells (HSC), Kupffer cells (KC), dendritic cells, liver associated lymphocytes and

portal fibroblasts. Circulating and bone marrow (BM)-derived immune cells are also recruited to the

liver in response to, and possibly in preparation for invading tumor cells and can affect the final

outcome (reviewed in (2, 3), (see Table 1).

This review summarizes our current understanding of the role of the liver microenvironment in the

growth of hepatic metastases and the reciprocal interactions between metastatic tumor cells and

different liver cell populations that regulate the process.

Pre- and pro metastatic niches drive the progression of liver metastasis.

The process of liver metastasis (LM) has previously been divided into four major phases (detailed in

Table 1) that follow tumor cell entry into the liver (4, 5). Emerging evidence suggests that in addition,

a pre-metastatic phase could set the stage for liver colonization by disseminating tumor cells.

Evidence for pre-metastatic niche formation in the liver:

The term “pre-metastatic niche” was coined to describe a microenvironment in a secondary organ site

that has been rendered permissive to metastatic outgrowth in advance of cancer cell entry through

the activity of circulating factors released by the primary tumor (6) (7, 8). While the dependency of

metastasis on pre-metastatic niches remains controversial and difficult to verify in the clinical setting

(9, 10), it appears that a consensus is emerging on two scores 1. That their existence may have

important implications for the clinical management of metastatic disease and 2. That much can be




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learned from their interrogation about the cellular/molecular changes required to facilitate tumor cell

growth in a distant site, such as the liver. Two recent studies have confirmed their potential relevance

to LM. In a mouse model of metastatic pancreatic ductal adenocarcinoma (PDAC), Costa-Silva et al

showed that PDAC-derived exosomes taken up by hepatic KC, upregulate TGFβ production leading

to increased fibronectin production by HSC and recruitment of BM-derived macrophages. They

identified macrophage-derived MIF as essential for pre-metastatic niche formation and metastasis.

Moreover, in exosomes derived from stage I PDAC patients who later developed LM, MIF levels were

found to be higher than in patients whose tumors did not progress, identifying MIF as a potential

predictor of PDAC LM (11). This group also showed that exosomes from human breast and

pancreatic cancer cell lines that metastasize selectively to the lung (MDA-MB-231) or liver (BxPC-3

and HPAF-II) fused preferentially with fibroblasts and epithelial cells in the lung and KC in the liver,

and this was mediated by the exosomal integrin laminin receptors α6β1and α6β4 and the fibronectin

receptor αvβ5, respectively. In exosome-fused KC, the pro-inflammatory factors S100P and S100A8

were upregulated (Table 1, Fig 1). Significantly, in PDAC patients, a correlation was documented

between the levels of circulating, αv-bearing exosomes and disease stage, suggesting that exosomes

may play a role clinically and their levels and integrin cargo may predict metastases in specific organs

(12). Kowanetz et al reported that tumor-derived GM-CSF could mobilize Ly6C+Ly6G+ myeloid cells

into metastases - enabling niches in the lung and liver and also identified S100A8 as a driving factor

(13). Tumor-derived TIMP-1 was identified as another potential inducer of increased liver

susceptibility to metastasis, acting via hepatic SDF-1 and neutrophil recruitment (14). Interestingly,

S100A8 was identified as a driver of liver pre-metastatic niche formation in several studies (12-15),

suggesting that it may have clinical utility as a predictor of LM.

The stage of primary tumor development at which pre-metastatic niches can be formed is difficult to

assess in the clinical setting. Costa-Silva et al found exosomal MIF upregulation in mice with pre-

tumoral pancreatic lesions and high plasma exosomal MIF levels were also detected in patients with




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stage I PDAC (11), suggesting that they could be formed at very early stages of cancer development.

The potential contribution of circulating cancer cells (CTC) that may be present early in tumor

development and even after resection of the primary tumor (16, 17) is also unknown.

Resident and immune cells can play diverse and opposing roles in the development of liver

metastases.

Host innate resistance mechanism can destroy tumor cells prior to extravasation. Blood-born

cancer cells entering the liver first encounter the LSEC, KC and hepatic NK cells (pit cells) that

together mount the first line of defence (18). Cancer cells entrapped in the sinusoids may undergo

destruction due to mechanical stress and deformation-associated trauma or die due to phagocytosis

by KC or cytolysis by NK cell-released perforin/granzyme (18), (reviewed in (4)). NO, ROS and toxic

radicals released by LSEC in response to local ischemia/reperfusion can contribute to tumor cell

death (19, 20). Release of NO and INFγ by LSEC and NK cells can result in upregulation of tumor

cell Fas and apoptosis (18) that can be augmented by NK and LSEC-derived TNFα (21). TNFα is one

of numerous cytokines and chemokines unleashed by KC in response to inflammation (Table 1) (22).

In addition to causing cell death directly, some of these factors can mobilize and activate additional

innate immune cells such as neutrophils, thereby adding to the local tumoricidal capacity (reviewed in

(23)). In several animal tumor models, including colon cancer, loss of NK cells increased cancer cell

growth in the liver, whereas enhanced NK activity reduced LM (24-26). Indirect evidence also

suggests that susceptibility to NK-mediated immune attack can affect the ability of human cancer

cells to generate LM (27). Circulating neutrophils can also release various factors abundant in their

granules to kill tumor cells (see Table 1) and their cytokines and chemokines can activate the

tumoricidal potential of resident KC and recruit host immune T cells with anti-tumorigenic activities

(reviewed in (28))

Cancer cells can escape these cytotoxic effects by forming clusters with blood or other cancer cells

that shield them from the lethal effects of shear stress or NK-mediated cytotoxicity (reviewed in (29)).




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The release of inflammatory mediators such as IL-1β, TNFα and IL-18 can also initiate a cascade that

facilitates their rapid exit from the vasculature to a less “toxic” microenvironment (see below and Fig

2).

The sinusoidal endothelium actively participates in different phases of metastasis.

Although the rapid inflammatory response initiated by cancer cell entry can lead to cell death, it may

also have a tumor-protective effect by upregulating the expression of LSEC CAM and in this way,

enhance cancer cell adhesion and trans-endothelial migration into the space of Disse, where they can

escape the cytotoxic effects of KC and NK cells (30). Cancer cells can adhere to inflammation -

induced E-selectin, VCAM-1 and ICAM-1, either as single cells or in association with host cells such

as KC or neutrophils (reviewed in (31)). Blockade of this inflammatory cascade was shown to reduce

LM (32-34) and TNFR1 signaling was identified as a major driver of this process (35).

Ultimately, the outcome of these opposing effects on tumor cell survival and progression to the next

phase depends on multiple factors including the expression on the cancer cells of the respective

counter receptors namely, E- selectin ligands sLewa and sLewx and CD44 isoforms (36), (reviewed in

(37)(29)), and VCAM-1 and ICAM-1 counter receptors integrins α4β1 and LFA-1 and Mac-1 (38),

respectively. E-selectin binding triggers a signaling cascade in both tumor cells and LSEC, leading to

diapedesis and trans-endothelial migration (39) and altering gene expression in a tumor-type specific

manner (40). Clinical studies have documented increased expression of E-selectin in and around

CRC liver metastases and elevated soluble E-selectin, ICAM-1 and VCAM-1 levels that correlated

with disease outcome in CRC patients (reviewed in (23)).

LSEC may have other metastasis-promoting functions. LSEC-secreted fibronectin can induce

epithelial-mesenchymal transition (EMT) in CRC cells via integrin α9β1, enhancing ERK signaling and

tumor invasion (41). Human LSEC were shown to induce CRC migration and EMT via MIF (42),

thereby increasing their metastatic potential.




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The LSEC contribute to tumor-induced angiogenesis. A recent study identified Notch1 as a negative

regulator of sinusoidal endothelial cell sprouting into micrometastases and thereby of angiogenesis

and LM (43), raising questions about the advisability of Notch targeting for cancer therapy. Moreover,

LM of different carcinomas, including CRC, can co-opt the sinusoidal endothelium at the tumor-liver

interface, giving rise to a histological growth pattern (GP) termed the “replacement” or “sinusoidal ”GP

that appears to result from tumor cell invasion between LSEC and the matrix in the space of Disse

and is characterized by formation of intra-metastatic vessels that appear continuous with the

sinusoidal vessels (reviewed in (23)). The factors that drive this co-opting mechanism remain to be

identified, but a recent study by Im et al suggests that Ang2 may play a regulatory role (44).

Collectively, the evidence shows that LSEC are not a passive barrier to tumor cell extravasation but

participate actively in the metastatic process. Cancer cell interaction with LSEC can reciprocally alter

the phenotypes of both cell types and this may lead to intravascular tumor cell destruction but can

also promote metastasis through enhanced tumor cell migration and increased angiogenesis (Table

1, Fig 2 and 3).

The role of macrophages is context dependent. KC constitute approximately 10% of all liver cells.

Recent studies suggest that they may be established prenatally and maintained into adulthood,

independently of replenishment from blood monocytes, but in an M-CSF and GM-CSF-dependent

manner (45). They reside mainly in the hepatic sinusoids and are anchored to the endothelium by

long cytoplasmic processes (46) (reviewed in (47)). In their interaction with invading cancer cells, KC

can play diverse and opposing roles, depending on factors such as the stage of the metastatic

process, tumor load and interactions with other immune cells. As discussed, KC can promote

metastasis by orchestrating the pre-metastatic niche. However, tumor-KC interactions can have

opposing effects. Several studies documented a rapid adhesion of tumor cells to KC in the sinusoidal

lumen resulting mainly in tumor cell phagocytosis or apoptosis, within hours of tumor cell entry (47).

This tumoricidal effect may require cooperation with local or recruited NK cells (48) and its efficiency




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in removing cancer cells likely depends on the tumor load (49). An intravital microscopy study (50)

recently revealed that 74% of intra-arterially injected rat colon cancer cells adhered to the KC within 6

hrs., and this was associated with increased liver TNFα and IL-1β levels, consistent with our own

findings (51, 52), and indicative of KC activation. Extensive phagocytosis of tumor cells was observed

within 2 hrs. post injection. Interestingly, while elimination of KC two days prior to tumor injection

increased LM, it had no significant effect up to one week post tumor injection, suggesting that KC

exerted their most potent anti-tumor effect within the first 24 hrs. of tumor cell entry into the liver (50).

A bi-modal effect of KC depletion was also documented in another study of murine CRC and it was

attributed to decreased vascular endothelial growth factor (VEGF) and increased iNOS levels in livers

subjected to late stage KC depletion (53). Indeed, tumor cells that survive the initial tumoricidal

assault by KC can benefit from their pro-tumorigenic functions. Adhesion to KC can facilitate tumor

cell extravasation, among others by sequential activation of endothelial CAM (29, 54). In addition, KC

can produce cytokines and growth factors including IL-6, HGF and VEGF and matrix

metalloproteinases such as MMP-9 and MMP-14 that can accelerate tumor cell invasion into, and

within the parenchymal space, as well as promote tumor cell proliferation and angiogenesis, thereby

enhancing LM (see Table 1 and Figures 2 and 3).

Taken together, the evidence suggests that KC targeting could become an effective anti-metastatic

strategy only within a very narrow window. Limiting KC activity may be beneficial if it could prevent

pre-metastatic niche formation, while enhancing the KC tumoricidal potential using agents such as

INFγ, GM-CSF or muramyl dipeptide may be most effective if achieved before tumor cells enter the

liver in large numbers (47, 55). The contribution of CTC that may be present before liver

micrometastases are established or after surgical resection of the primary tumor (16, 17) to the tumor

load and KC activation is unknown. This multi-faceted role of KC in LM may explain the limited

success to date of KC-targeting interventions (47).




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Importantly, in addition to KC, blood-derived monocytes can also be recruited to the tumor site in

response to local injury and inflammation and differentiate locally into mature CD11bloF4/80hi

macrophages in a CCR2-dependent manner (56). These macrophages can also trigger HSC

activation and fibrogenesis (57), a process important in the early stages of extravascular tumor

expansion (see below).

Macrophages have inherent plasticity and their phenotypes can change within a spectrum of

activation states between the polar M1 and M2 phenotypes (58, 59). M1 macrophages are tumoricidal

due to high NO and TNFα production levels and produce Th1, Th17 and the NK attracting

chemokines CXCL9 and CXCL10 (60). In contrast, M2 macrophages can promote tumor growth

through production of growth factors such as VEGF, EGF, bFGF and TGFβ (61). In addition, M1

macrophages activate Th1-type immune responses that can further amplify M1/killer-type activity

through the production of IFNγ, while M2 macrophages induce regulatory T cells (Treg) and thereby

an immunotolerant microenvironment, through release of TGFβ and IL-10 (62) (Table 1). Evidence

regarding the role of macrophage polarization in LM is scant because macrophages within or around

hepatic metastases have, with few exceptions, not been subtyped. A recent clinical study identified

the M2/M1 ratio in hepatic resections as a correlate of CRC metastasis (63), implicating M2

macrophages in the clinical disease. Furthermore, F4/80 – the cell surface marker frequently used to

identify KC is also expressed on recruited monocytes, and strategies used to eliminate macrophages

in vivo are not KC-specific. The source and identities of macrophages in many published studies

remain therefore to be verified. As immune-editing of the tumor microenvironment and the conversion

of tumor associated (TAM) M2 macrophages to M1 killer macrophages are emerging as potentially

relevant anti-cancer strategies (62), further characterization of both the source and the status of

macrophage populations involved in LM will become critical.

The Neutrophils: a double-edged sword. Neutrophils are part of the innate immune response to

pathogens and are also rapidly activated in response to invading cancer cells (28). BM-derived




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neutrophils are mobilized to sites of inflammation or cancer via their cell surface receptor CXCR2 and

in response to chemokines and cytokines (Table 1) secreted by activated macrophges, endothelial

cells or the tumor cells (reviewed in (28)). At the tumor site, the neutrophils can exert opposing effects

that depend on the inflammatory/immune context (reviewed in (64)). They can release cytolytic

factors (Detailed in Table 1) or inhibit tumor growth indirectly, via recruitment of CD8+ cytotoxic T cells

and macrophages. This may require direct contact with the tumor cells (65) such as can occur within

the liver sinusoidal lumen (66, 67).

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 an orthotropic colon

carcinoma model (68). Moreover, in the vascular lumen, the physical interaction with tumor cells that

could lead to tumor cell kill may, alternatively, anchor circulating tumor cells to the vascular

endothelium and enhance their migration into the extravascular space. Tumor-derived cytokines such

as IL-8 induce expression of integrins such as CD11b/CD18 on the neutrophils, increasing their

adhesion to tumor cells via the counter receptor ICAM-1 and facilitating trans-endothelial migration,

as shown in murine melanoma and lung carcinoma models (67, 69). Tumor cell entrapment in the

sinusoids can also be mediated by neutrophil-produced extracellular (DNA) traps (NETs), resulting in

increased tumor retention in the sinusoids, enhanced tumor cell adhesion, proliferation, migration and

invasion and increased LM (70, 71). Neutrophils can also release ECM-degrading proteinases such

as MMP-8, MMP-9, elastase and cathepsin G, thereby increasing tumor invasion (65) and can

promote angiogenesis through the release of VEGF (Table 1 and Figures 2 and 3).

Moreover, a TGFβ-mediated neutrophil polarization that alters the phenotype of tumor-associated

neutrophils from tumor inhibitory (N1) to tumor promoting (N2) was recently described (72). TGFβ

produced by M2 macrophages may contribute to this process (reviewed in (59)). The contribution of

polarized neutrophils to LM remains, however, to be confirmed.




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Clinical studies implicate neutrophils in tumor promotion. With few exceptions, elevated circulating

neutrophil counts or neutrophil-to-lymphocyte ratios were associated with poorer outcomes and

distant metastases in patients with various epithelial malignancies (73), including carcinomas of the

GI tract. High neutrophil counts may also be associated with increased resistance to therapy (64,

73). The 5-year survival for CRC patients undergoing hepatic resections that had neutrophil-to-

lymphocyte ratios greater than 5 was worse than for those with normal ratios (74). However, high

neutrophil infiltration into the tumor site may be a consequence of advanced tumor stage (and

therefore poorer prognosis) rather than its cause.

Hepatic stellate cells orchestrate a pro-metastatic microenvironment that is required for

transition from the avascular to the vascular stage of metastasis.

The HSC orchestrate the characteristic fibrogenic response of the liver to injury. Normally quiescent

in the space of Disse (75), they are activated (aHSC), in response to liver damage and inflammatory

stimuli, acquire a myofibroblast-like phenotype (α-SMA+) and produce ECM rich in collagens I and IV

(75, 76). Chemokines and cytokines released by aHSC (Table 1) also recruit inflammatory/immune

cells (77, 78), thereby shaping the immune microenvironment.

In addition to their role in pre-metastatic niche formation (above), HSC can orchestrate a pro-

metastatic niche following tumor extravasation. In response to growth factors and inflammatory

mediators (detailed in Table 1), HSC are activated (75) and can trigger a process akin to the early

events in liver repair. This was observed in animal models (4) and is also evidenced by the increased

production of collagen IV in and around hepatic metastases in clinical specimens (79). Macrophages,

hepatocytes and LSEC contribute to this process by releasing TGFβ and/or TNFα (57). In turn, aHSC

–derived pro-angiogenic factors such as VEGF and angiopoietin-1 (80, 81) initiate angiogenesis (3)

and this is enhanced by HSC-derived MMPs that facilitate endothelial cell migration and tumor

invasion (23, 75) (reviewed in (5, 82)) (Table 1, Fig 2).




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Several lines of evidence confirm the essential role of HSC in LM. Olaso et al showed that

myofibroblasts infiltrating into avascular micrometastases of B16 melanoma cells induced a stromal

response favorable to angiogenesis that preceded endothelial cell recruitment and was followed by

co-localization of myofibroblasts and endothelial cells within angiogenic structures (83),(84). HSC and

macrophage recruitment into metastatic sites and subsequent angiogenesis were shown to be

CCR2/CCL2-dependent (85). More recently, Eveno et al (86) using immunofluorescence, showed

that 9 days post injection of CRC LS174 cells into SCID mice, the hepatic micrometastases consisted

of proliferating cancer cells, a well-organized network of aHSC and laminin deposits but no vascular

network. As the LM grew, an organized vascular network appeared and laminin co-localized with

CD31+ endothelial cells. Co-injection of tumor cells and aHSC enhanced metastasis. Moreover,

analysis of LM from CRC patients revealed a surface marker expression pattern similar to that

observed in the co-injection studies.

Recently, suppression of TGFβ receptor II signalling by QGAP1 was shown to prevent HSC activation

and IQGAP1 downregulation was observed in myofibroblasts associated with human CRC LM,

consistent with their activation in this context (87). Furthermore, α-SMA+ cells whose presence

correlated with the degree of fibrous stroma were identified in LM of colon, gastric and pancreatic

adenocarcinomas (88).

The importance of HSC to metastatic niche formation and their role in primary liver cancer (89) have

raised interest in HSC-targeting as an anti-cancer/anti-metastatic strategy. There is little direct

evidence that such an approach can succeed, due partially to the present lack of agents that

specifically target HSC, without toxicity. Indirect evidence from a rat model of cholangiocarcinoma

(90) and pancreatic stellate cell targeting in a PDAC model (82) suggest a potential therapeutic

benefit, but this remains to be verified. Of note, portal fibroblasts and BM derived fibrocytes (91) can

also contribute to the stromal response. Liver-invading cancer cells located in portal tracts and unable

to activate HSC may engage portal tract fibroblasts that produce IL-8, a chemokine involved in




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invasion and angiogenesis (92). Specific targeting of HSC may therefore not be sufficient to deprive

metastatic cells of a growth-promoting stroma.

Hepatocytes can promote metastasis directly and indirectly.

The role of the parenchymal hepatocytes in LM is not well understood. Tumor cell adhesion to

hepatocytes was identified as one of the earliest events in LM formation and a correlate of the

metastatic potential (93, 94). Desmosomes (95), integrins αv, α6 and β1 that bind to hepatocyte ECM

(94), osteopontin binding via counter receptors CD44 and integrin αv (96) and claudins (97, 98) were

all implicated. Adhesion of human CRC cells to hepatocyte-derived ECM was shown to upregulate

the expression of genes involved in tumor cell survival, motility and proliferation, in particular EGF

family members implicated in LM (99) such as AREG, EGFR, HBEGF and erbB2 and stem cell

markers CD133 (PROM1) and LGR5 (100), suggesting that CRC adhesion to hepatocytes ECM

induces autocrine growth-promoting mechanisms for tumor expansion. Tumor cell adhesion to

hepatocytes can have other consequences. The FasL/FLICE-like inhibitory protein on murine MC38

cells was shown to induce apoptosis in hepatocytes by activating Fas signaling and this destructive

process created a niche for tumor expansion (101). Furthermore, hepatocytes produce several

growth factors including IGF-I (102). As we have shown, blockade of IGF-I signaling in metastatic

tumor cells could inhibit LM (103-105). Other factors include the hepatocyte growth factor-like

protein/macrophage stimulating protein (HGFL) that can enhance tumor growth, motility and invasion

via the Ron receptor (106), heregulin - a ligand of ErbB3 shown to enhance integrin αvβ5-mediated

cell migration and erbB3/erbB2 signaling in human CRC cells (107) and the pro-angiogenic factor

bFGF (108) .

The clinical relevance of tumor-hepatocyte interactions is not clear. Three different histological GPs

have been identified in LM specimens namely, the desmoplastic, pushing, and replacement GPs. The

latter two are characterized by close tumor-hepatocyte proximity (23). Whether or not direct adhesion

between metastatic cancer cells and hepatocytes influences the eventual GP of CRC LM is not




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known. Blocking tumor-hepatocyte interactions could have beneficial anti-metastatic effects, as

recently demonstrated by Tabaries et al (109).

An immunosuppressive microenvironment facilitates metastatic expansion.

Tumor cells can activate specific T-cell mediated immune responses that curtail tumor expansion

through various mechanisms (reviewed in (110)). However, tumor cells can evade T-cell mediated kill

among others, by giving rise to and/or recruiting immunosuppressive cells, such as myeloid derived

suppressor cells (MDSC) and Treg that alter the immune landscape, inducing a state of immune-

tolerance, permissive to tumor expansion (reviewed in (111)).

Role of Treg: Treg cells may be thymically derived (natural, nTregs) or induced from naïve CD4+ T

cell precursors (iTreg) in response to IL-10 and TGFβ. They exert an immunosuppressive effect by

different mechanisms that result in the inhibition of an effective, anti-tumorigenic T cell response

(reviewed in (112)). Although high Treg levels were documented in the blood and local tumor sites of

patients with various epithelial cancers (e.g. (113)), relatively little is known about their role in LM.

Connolly et al, using models of early, pre-invasive pancreatic neoplasia and advanced colorectal

cancer showed that Tregs accelerated the development of LM (114). We recently documented the

accumulation of Treg around colon carcinoma MC38 liver micrometastases and showed that this was

TNFR2 dependent (35).

Intriguingly, in fifty seven colon cancer specimens from patients undergoing chemo- or

chemoimmunotherapy, higher Treg infiltration scores were associated with a better prognosis and a

better outcome (115). However, the relationship between Treg accumulation in the primary tumors

and in the corresponding liver metastases has not been examined. While Treg-targeting strategies

are in development (116, 117) (reviewed in (111)), their potential therapeutic benefit for LM has not

yet been examined.

The Role of MDSC. The MDSC are a heterogeneous population of myeloid cells that can be of

monocytic (Mo-MDSC, CD11b+Ly6C+) or granulocytic (G-MDSC, CD11b+Ly6G+) lineages (118). In




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humans, MDSCs are CD33+ and/or CD11b+ and HLA-DR- (119). Under normal physiological

conditions, BM-derived, immature myeloid cells differentiate into mature granulocytes or monocytes,

able to mediate host innate immune responses in the target tissue. However, in the tumor

microenvironment, these precursors do not mature and exert immunosuppressive and tumor-

promoting effects (120). Although they express granulocyte and monocyte surface markers, these

cells have been characterized based on functional criteria namely, their immunosuppressive

capability (118, 121). An increase in circulating MDSC in cancer patients and their recruitment into

tumor sites has been documented (118, 122, 123).

In the liver, MDSCs can be recruited to the metastases through chemokines such as CXCL1 and

CXCL2 produced by LSEC and KC or by activated HSC (78). There, they can promote tumor growth

by inducing immunosuppression (124), producing arginase (118) and increasing Treg numbers (125)

(Table 1 and Fig 2). Clinical and experimental evidence confirm their role in metastasis (126).

Recruitment of tumor-promoting myeloid cells into CRC LM has recently been documented and their

depletion was shown to result in a marked reduction in LM (127). We recently reported that MDSC

accumulate in hepatic micrometastases of MC38 cells within days of tumor cell injection and

documented a relative preponderance of Ly6Chigh cells (the more suppressive, arginase-high subtype

(128)). We also identified CD33+HLA-DR-TNFR2+ myeloid cells in the periphery of hepatic

metastases from CRC patients, implicating MDSC in the clinical disease (35).

Several strategies are being developed to selectively eliminate MDSC, including pharmacological

induction of MDSC differentiation, inhibition of MDSC expansion from BM precursors or blockade of

their function (129). However, some of the drugs developed are not MDSC-specific or their long term

administration is not possible due to toxicity (129). Their translational potential remains therefore to

be verified.

Conclusion




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Cancer cells entering the liver encounter a unique and complex microenvironment. Parenchymal and

non-parenchymal liver cells, as well as recruited inflammatory and immune cells participate in the

response to invading tumor cells and may inhibit or favor the progression of metastasis. The factors

that determine the outcome of these opposing effects and the pattern of growth of the metastases are

not yet well understood. This duality of function and the shifts in the types of cells and mediators

involved at different stages of the metastatic process render the attempts to target specific cellular

interactions in the microenvironment extremely challenging and may explain the slow progress, to

date, in identifying agents that can successfully and specifically inhibit metastatic disease. With the

advent of genomic profiling for personalized cancer treatment and the increased availability of

surgically resected LM for cellular/molecular interrogation, our understanding of the relationship

between the genetic background and clinical history of the patient, the genomic/transcriptomic profile

of the cancer cells and the type of response mounted by the microenvironment may improve, opening

new avenues for prevention and/or treatment of liver metastatic disease.




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Acknowledgements

The author is indebted to Dr. Janusz Rak (McGill University Health Center) for insightful editorial

comments and to Simon Milette for the superb artistic rendering of the metastatic niches of the liver

and for his help with the Table. Studies by the author’s group cited in this manuscript were supported

by grants from the Canadian Institute for Health Research and by a PSR-SIIRI-843 grant from the

Québec Ministère de l'Économie, de l'Innovation et des Exportations.




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Table 1. Cells and mediators involved in the different phases of liver metastasis. 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 involved at each phase, the mediators regulating their recruitment and

activation and the factors they produce to block or promote the metastatic process. Note that the

process has been simplified and divided into distinct phases in the interest of clarity. However, it

should be viewed as dynamic with overlapping cellular and molecular drivers at different phases.

Moreover, as the metastatic process advances, the cancer and hepatic cells evolve and their

phenotypes change, as a consequence of their interactions. These newly acquired properties help

propel the process forward (see also Fig 1-3).

Figure legends

Figure 1. The pre-metastatic niche in the liver. Shown is a diagrammatic representation of the

multi-step process involved in the formation of pre-metastatic niches in the liver based on data

described in (11, 12). The symbols used for depiction of different hepatic cells are listed in Figure 3. A

diagrammatic representation of a pancreatic ductal adenocarcinoma (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.

Fig 2. The multi-step process of liver colonization by disseminated cancer cells. Shown is a

diagrammatic representation of the major cell types and ECM components in the liver

microenvironment that impact the progression of liver metastasis. The four major phases in the

process are delineated by dashed lines. Depicted in the upper panel are the major inter-cellular

interactions that occur upon entry of the tumor cells into the sinusoidal vessels (the Microvascular

phase) and precede tumor extravasation. Shown in the center two panels are the events following

tumor cell extravasation into the space of Disse divided into the Pre-angiogenic phase (center-left)




29

that is orchestrated by stellate cell activation, ECM deposition, cytokine production and the

recruitment of innate and adaptive immune cells and culminates in neovascularization (the vascular

phase)(center-right). These interactions enable tumor expansion (the Growth phase) (bottom panel).

The symbols used for depiction of different hepatic cells are listed in Figure 3 or indicated in the

diagram. The encircled numbers denote potential targets for therapeutic intervention, as detailed in

the boxes inserted within each of the depicted phases.

Fig 3. Parenchymal, non-parenchymal and immune cells of the liver and their role in

metastasis. Listed are the parenchymal and non-parenchymal cells of the liver and their anti and pro-

metastatic functions. Symbols shown for each cell type were used to depict inter-cellular

communications in Figures 1 and 2.




Phase of the

metastatic process Cell type involved Cell surface markers Soluble factors involved References

Recruiting or activating Produced Pre-metastatic niche Kupffer cell (KC) CD11b, F4/80 M-CSF, GM-CSF, MIF TGF-β, S100P, S100A8 (11-14)

Hepatic stellate cell (HSC) α-SMA, desmin, GFAP KC-derived TGF-β and TNF-α Fibronectin (11, 13)

Myeloid-derived

suppressor cells (MDSC) CD11b, Ly6C, Ly6G Tumor-derived GM-CSF S100A8 (12, 15)

Neutrophils CD11b, Ly6G TIMP-1, SDF-1 S100A8 (7, 14, 68) Post-tumor invasion

(1) Microvascular phase: Tumor cells trapped in the vasculature

Liver sinusoid endothelial

cell (LSEC) CD31, TNFR1, TNFR2, αvβ3, E-

selectin, VCAM-1, ICAM-1 TNF-α, IL-1β, IL-18 ROS, NO, INF, TNF-α (5, 18-20, 23, 31)

Kupffer cell (KC) CD11b, F4/80 M-CSF, GM-CSF, IFNγ TNF-α, IL-1, IL-6, IL-8, IFNγ, MIP-1α, MIP-2, IP-10, MCP-1, CCL5, VEGF

(5, 23, 31, 45-47,

51) Neutrophils CD11b, Ly6G S100A8, S100A9, IL-8, CCL2 TNF-α, ROS, defensins, perforins, elastase (28, 31, 64)

Hepatic natural killer cells

(NK) NK1.1, NK1.2, FcγRIII CXCL9, CXCL10, IL-1, IL-12, IL-15, IL-

18 Grz, Perforin, INFγ, TNF-α (5, 31, 48)

(2) Extravascular, pre-angiogenic: Tumor cells transmigrate into the space of Disse activating a local stromal response

Hepatic stellate cell (HSC) α-SMA, desmin, GFAP KC-derived TGF-β, PDGF, bFGF, SDF-1,IGF-I, CCL2

ECM deposition, MCP-1, CCL5, CCL21,

TGF-β (75-78, 85)

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

S100A8, S100A9, CXCL1, CXCL2,

CXCL5, TNFα, IL-8, G-CSF, IFNγ, CCL2,

CCL5, TGF-β

TNF-α, ROS, NOS, MMP-8, MMP-9,

elastase, cathepsin G, VEGF (28, 60, 65, 72)

Kupffer cell (KC) CD11b, F4/80 M-CSF, GM-CSF, IFNγ IL-6, VEGF, HGF, MMP-9, MMP-14 (5, 50, 53)

(3) Angiogenic phase: Micrometastases are vascularized

Blood-derived monocytes CD11b, F4/80 TNF-α, MCP-1 VEGF, MMPs, bFGF (57, 60)

Hepatic stellate cell (HSC) α-SMA, desmin, GFAP KC-derived TGF-β, PDGF, bFGF, SDF-1,IGF-I, CCL2

VEGF, angiopoietin-1, MMP-2, -9 and-13 (75, 80-83, 89)

Liver sinusoid endothelial

cell (LSEC) CD31, TNFR1, ICAM-1 VEGF, Ang1, inhibited by Notch1 and

regulated by Ang2 Fibronectin, MIF (23, 41-44)

Macrophages (M2) CD11b, F4/80, Ly6C TGF-β, IL-10 NO, TNFα, IFN, VEGF, EGF, bFGF, TGF-β, IL-10

(57-61)

(4) Growth phase: Metastases expansion

Hepatocytes Albumin, tyrosine aminotransferase

Adhesion to tight junction integral

proteins AREG, EGF, HB-EGF, erB2, IGF-I, HGFL,

ErB3, bFGF (93-99, 102-106,

108)

Tregs CD4, CD25, Foxp3 TGF-β, IL-10, CCL5, CCL22 VEGF, TGF-β, IL-10 (35, 112,125)

Research.

on June 29, 2021. © 2016 A

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anuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Author M

anuscript Published O

nlineFirst on O

ctober 19, 2016; DO

I: 10.1158/1078-0432.CC

R-16-0460


© 2016 American Association for Cancer Research

Figure 1:

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

?

TGF-

TGF-

TGF-

Tumor-derivedexosomes

Tumor-derivedexosomes

S100A8

Tumor-derived G-CSF

aKC





III. Angiogenic phase

Figure 2:

Fas/FasL

Fas/FasL

VEGF

VEGF-R

MMP-9

MMP-9VEGF

LSEC

aKC

I. Microvascular phase

II. Preangiogenic phase

Potential therapeutic strategies3. TGF-bRII blockade4. MDSC depletion5. TNFR2 blockade6. PD-1/PD-L1 blockade

Potential therapeutic strategies10. IGF-I neutralization11. Claudin-2 blockade

IV. Growth phase

Potential therapeutic strategies1. TNF inhibition2. E-selectin blockade

Potential therapeutic strategies7. Blocking M2 polarization8. VEGF inhibition9. N1 induction

sLew×/E-Sel.

TNF-α

TNF-α

TNF-α

TGF-β

TGF-β

TGF-β

IL-10TGF-β

NOTNF-α

TNFR1

HSC

aHSC

aHSC

M1

N1

N2

IGF-I Hepatocyte

M2

CoIIVMDSC

ArgROS

Treg MMPsVEGFTGF-βIGF

CD8+T cell

Tumor cell

Vessel lumen

Space of Disse

Parenchyma

12

3

4

56

7

8

9

11

10





Figure 3:

Produce ROS, NO, IFN ,TNF-

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

Premetastatic niche formationdrive fibrogenic response, ECMproduction, mediate angiogenesis,recruit inflammatory cells,produce MMPs

Mediate tumor adhesion, producegrowth factors (e.g., IGF-1, HGFL),alter tumor transcriptome, undergoEMT to provide stromal support,enhance angiogenesis via bFGF

(5, 11, 12, 47–53)

(59–63)

(114, 118, 124, 125)

Produce perforin, TNF- ,induce Fas/FasL interaction,kill tumor cells

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

M2 macrophages produceVEGF, EGF, bFGF, induce Tregvia IL-10 and TGF-

M1 macrophages produce NO,TNF- , and T-cell and NKrecruiting chemokines

Participate in premetastaticniche formation, activate LSECCAM, produce VEGF, IL-6,HGF, and MMP-9, activate HSC

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

(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 cells (NK)

T lymphocytes

Neutrophils

(11, 23, 78, 83-87)

Resting Activated

(94–97, 102, 104, 106)

(4)

(28, 64, 78)

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

Cytotoxic T cells kill tumorcells, release perforin andTNF- , recruit macrophages

Participate in premetastaticniche, enhance transendothelialmigration, entrap tumor cells;N2 neutrophils produce VEGF,MMP-9, and CCL5

N1 neutrophils produce ROS,NO, TNF- , defensins, recruitCD8+ T cells




Published OnlineFirst October 19, 2016.Clin Cancer Res Pnina Brodt pro-metastatic nichesRole of the microenvironment in liver metastasis: from pre- to

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