The pre-metastatic niche: finding common ground.

TL;DR: This review provides an overview of the current understanding of underlying mechanisms of pre-metastatic niche formation and highlights the common links as well as discrepancies between independent studies.

Abstract: It is rapidly becoming evident that the formation of tumor-promoting pre-metastatic niches in secondary organs adds a previously unrecognized degree of complexity to the challenge of curing metastatic disease. Primary tumor cells orchestrate pre-metastatic niche formation through secretion of a variety of cytokines and growth factors that promote mobilization and recruitment of bone marrow-derived cells to future metastatic sites. Hypoxia within the primary tumor, and secretion of specific microvesicles termed exosomes, are emerging as important processes and vehicles for tumor-derived factors to modulate pre-metastatic sites. It has also come to light that reduced immune surveillance is a novel mechanism through which primary tumors create favorable niches in secondary organs. This review provides an overview of our current understanding of underlying mechanisms of pre-metastatic niche formation and highlights the common links as well as discrepancies between independent studies. Furthermore, the possible clinical implications, links to metastatic persistence and dormancy, and novel approaches for treatment of metastatic disease through reversal of pre-metastatic niche formation are identified and explored.

Summary

Metastasis: new thoughts

• Metastasis remains the cause of over 90% of cancer-related deaths from solid tumors [1] .
• The aggressive nature and widespread distribution of metastatic tumors limits the effectiveness of cancer therapeutics, and as such a cure for metastatic disease remains elusive.
• The process of metastasis is defined by distinct steps involving local invasion, intravasation into adjacent blood and lymphatic vessels, transit through circulation and evasion of host immune systems, extravasation into the parenchyma of distant organs, colonization and formation of micrometastases, followed by proliferation and progression to macrometastases.
• This process is largely inefficient due to the many obstacles tumor cells must overcome to successfully metastasize, and has until recently been regarded as a late event in tumorigenesis [2] [3] .
• Emerging evidence suggests distinct forms of invasion and metastasis may occur in different cancer types, and that how, when and where tumor cells metastasize needs to be explored in greater detail [1] [2].

Learning from the past

• Steven Paget's 'seed and soil' hypothesis, proposed over a century ago, still forms the basis of their understanding of the metastatic process.
• In a study of 735 breast cancer autopsies, Paget noted that metastatic tumors were not randomly distributed in patients [5] .
• Instead he proposed the 'seed' (tumor cells) selectively colonized the 'soil' of distant organs with an environment favorable for survival and proliferation [5] .
• It is now well established that specific organs are predisposed to metastases in certain cancers, and that signaling between cytokines, chemokines and their receptors regulates tumor cell-homing to secondary organs [3] .
• The microenvironment at secondary sites of metastasis, while equally as important to allow metastatic tumor cell colonization and growth, is poorly understood in comparison.

The pre-metastatic niche: a new era in metastasis research

• The components crucial to pre-metastatic niche formation include tumorderived secreted factors and BMDCs.
• Tumor-derived VEGF and PlGF were demonstrated to promote the recruitment of VEGFR1 + hematopoietic progenitor cells (HPCs) that formed distinct clusters of cells in secondary organs.
• HPCs expressing the fibronectin receptor integrin VLA-4 interact with resident fibroblasts to stimulate fibronectin production and secrete MMP9 to create pre-metastatic niches for disseminating CXCR4 + tumor cells.
• While it is likely that pre-metastatic niches are not essential for metastases to form, various studies suggest that they greatly enhance the likelihood of metastatic progression [9] .
• This review will collate and explore the similarities and differences in the TDSFs and BMDC components implicated in pre-metastatic niche formation, highlight the roles of hypoxia, myeloid cells and immunosuppression in regulating microenvironments at distant organs, discuss potential links to tumor dormancy and investigate how this knowledge may help in the treatment of metastatic disease.

The primary tumor drives pre-metastatic niche formation: a role for hypoxia?

• A variety of TDSFs including VEGF, PlGF, TNF-α, TGF-β, Lysyl oxidase (LOX), versican and G-CSF have been shown to drive pre-metastatic niche formation in various tumor models (Table 1 & Figure 1 ).
• Pre-metastatic niches may simply arise as a consequence of systemic disturbances caused by the presence of the primary tumor.
• The hypoxia inducible factors (HIFs) are the main downstream regulators of the hypoxic response-signaling pathway.
• Recently, hypoxic tumor cells were demonstrated to be one of the main sources of pre-metastatic niche promoting-TDSFs [24] .

Myeloid cell diversity in the pre-metastatic niche is controlled by the local environment and tumor-derived factors

• As discussed above, the disparity in TDSFs and their apparently different roles in pre-metastatic niche formation have generally been attributed to the different tumor models used.
• This suggests that the organotropism observed in different tumor types is largely determined and driven by the TDSFs secreted from the primary tumor.
• A common theme amongst different models of the pre-metastatic niche (Table 1 ) is the mobilization of myeloid cell lineages from the bone marrow and recruitment to specific pre-metastatic sites.
• A common myeloid progenitor derives from haematopoietic stem cells, and can give rise to a variety of monocytic and granulocytic cell subtypes including macrophages, dendritic cells (DCs), neutrophils and myeloid-derived suppressor cells [25] .
• MDSCs were originally defined by the co-expression of CD11b and Gr-1 antigens in tumor-bearing mice [36] .

Immunosuppression as a mechanism of tumor promotion in

• Recently, CD11b + /Ly6C med /Ly6G + granulocytic myeloid cells were identified as the main myeloid cell constituent of the pre-metastatic niche, mobilized by hypoxic TDSFs [24] .
• Contact-independent mechanisms of NK cell suppression by MDSCs have also been reported.
• MDSCs affect the viability, proliferation, effector functions and migration of T cells (reviewed in [36] ).
• T cells, capable of suppressing cells of both the innate and adaptive immune response.
• Hence, while very little is known regarding immune surveillance in pre-metastatic organs, its importance as a potential mechanism of pre-metastatic niche formation should not be underestimated.

Does the pre-metastatic niche control tumor cell dormancy?

• Exactly when pre-metastatic niche formation is initiated during tumor progression has not yet been clearly defined.
• The microenvironment of the secondary organs then becomes extremely important in controlling the fate of these DTCs.
• Creation of pre-metastatic niches means DTCs may not need to acquire all of the mutations necessary to complete the metastatic cascade, and can instead rely on the pre-metastatic niche environment to make up for anything the tumor cell alone may lack in order to successfully metastasize (Figure 2 ).
• The ECM, angiogenesis, immune suppression and hypoxia are all associated with either the initiation of tumor cells into, or the escape from, dormancy [60, [62] [63] [64] .
• Depending on the nature of the primary cancer, this could occur months or decades after successful treatment.

Exosomes as emerging coordinators of the pre-metastatic niche

• Interactions with and between cells in the pre-metastatic niche have generally been assumed to occur through cell-cell contact or the release of soluble tumorderived factors.
• Exosomes are small membrane-bound vesicles, 50 to 100 nm in size, capable of mediating communication with surrounding cells or ECM components through cell-surface receptor interactions, or the horizontal transfer of their contents into recipient cells.
• Tumor-derived exosomes also have other functions that further implicate them as drivers of pre-metastatic niche formation.
• Secondly, two recent studies have demonstrated a role for tumor-derived exosomes in the differentiation and mobilization of MDSCs.
• Additionally, the MyD88-Toll-like receptor (TLR) signaling pathway has been demonstrated to be critical for tumor exosome-mediated expansion of these CD11b + /Gr-1 + MDSCs and induction of the proinflammatory cytokines that promote metastasis [77] .

Is the pre-metastatic niche reversible?

• Metastatic disease is the major cause of cancer-related morbidity and mortality.
• Therefore, targeting pre-metastatic niches to reduce or prevent metastatic disease in these patients, and not relying on complete removal of the primary tumor alone, would be highly desirable.
• Exosomes also show potentially more promise as pre-metastatic niche biomarkers.
• As discussed earlier, the immunosuppressive function of MDSCs in the context of the pre-metastatic niche has not been explored in great detail, but could be a key factor limiting the overall success of cancer immunotherapy in the treatment of metastatic disease.
• Treatment regimes, such as adoptive transfer of NK cells early during disease, may prove beneficial in preventing metastatic relapse through recovery of NK cell activity in distant organs.

Conclusions and perspective

• Formation of pre-metastatic niches in ectopic organs driven by the primary tumor is now a well-established process promoting metastatic progression (Figure 1 ), yet many unanswered questions still remain regarding the exact mechanisms of its formation.
• Better targets for treatment of metastatic disease need to be found.

Figures

Table 2 Correlation between TDSFs implicated in pre-metastatic niche formation and their reported effects on MDSC populations in cancer

Table 1 Current published mechanisms of pre-metastatic niche formation

Full text

1
The pre-metastatic niche: finding common ground
Jaclyn Sceneay
1,2,3
, Mark J. Smyth
2,4,5,6,*
, Andreas Möller
3,*
1
Cancer Genomics and Genetics Laboratory,
4
Cancer Immunology Program,
Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, Victoria
3002, Australia.
2
Department of Pathology,
5
Sir Peter MacCallum Department of Oncology,
The University of Melbourne, Parkville, Victoria 3010, Australia.
3
Tumour Microenvironment Laboratory and
6
Immunology in Cancer and
Infection Laboratory, Queensland Institute of Medical Research, 300 Herston
Road, Herston, Queensland 4006, Australia.
*
Contributed equally to this work.
Corresponding Authors: Andreas Möller (email: andreas.moller@qimr.edu.au,
Telephone: +61 7 3845 3950) and Mark Smyth (email: mark.smyth@qimr.edu.au).
Running title: The pre-metastatic niche: finding common ground
Keywords: Pre-metastatic niche, Hypoxia, Immunosuppression, Myeloid-derived
suppressor cells, Exosomes, Tumor dormancy
Potential conflict of interest: The authors declare no conflict of interest.
Word count: Abstract: 166 words; Text: 6,577 words (w/o Acknowledgment);
Number of Figures/Tables: 2 Figure, 2 Tables.

2
Abstract
It is rapidly becoming evident that the formation of tumor-promoting pre-metastatic
niches in secondary organs adds a previously unrecognized degree of complexity to
the challenge of curing metastatic disease. Primary tumor cells orchestrate pre-
metastatic niche formation through secretion of a variety of cytokines and growth
factors that promote mobilization and recruitment of bone marrow-derived cells to
future metastatic sites. Hypoxia within the primary tumor, and secretion of specific
microvesicles termed exosomes, are emerging as important processes and vehicles for
tumor-derived factors to modulate pre-metastatic sites. It has also come to light that
reduced immune surveillance is a novel mechanism through which primary tumors
create favorable niches in secondary organs. This review provides an overview of our
current understanding of underlying mechanisms of pre-metastatic niche formation,
and highlights the common links as well as discrepancies between independent
studies. Furthermore the possible clinical implications, links to metastatic persistence
and dormancy, and novel approaches for treatment of metastatic disease through
reversal of pre-metastatic niche formation, are identified and explored.

3
Metastasis: new thoughts
Metastasis remains the cause of over 90% of cancer-related deaths from solid
tumors [1]. The aggressive nature and widespread distribution of metastatic tumors
limits the effectiveness of cancer therapeutics, and as such a cure for metastatic
disease remains elusive. The high mortality rate associated with metastatic disease
emphasizes the need to move away from the current limiting paradigms regarding
metastatic progression.
The process of metastasis is defined by distinct steps involving local invasion,
intravasation into adjacent blood and lymphatic vessels, transit through circulation
and evasion of host immune systems, extravasation into the parenchyma of distant
organs, colonization and formation of micrometastases, followed by proliferation and
progression to macrometastases. This process is largely inefficient due to the many
obstacles tumor cells must overcome to successfully metastasize, and has until
recently been regarded as a late event in tumorigenesis [2-3]. Emerging evidence
suggests distinct forms of invasion and metastasis may occur in different cancer types,
and that how, when and where tumor cells metastasize needs to be explored in greater
detail [1-2, 4].
Learning from the past
Steven Paget’s ‘seed and soil’ hypothesis, proposed over a century ago, still
forms the basis of our understanding of the metastatic process. In a study of 735
breast cancer autopsies, Paget noted that metastatic tumors were not randomly
distributed in patients [5]. Instead he proposed the ‘seed’ (tumor cells) selectively
colonized the ‘soil’ of distant organs with an environment favorable for survival and
proliferation [5]. It is now well established that specific organs are predisposed to
metastases in certain cancers, and that signaling between cytokines, chemokines and
their receptors regulates tumor cell-homing to secondary organs [3]. An example is
breast cancer, where tissues such as lungs, bone, liver, brain and regional lymph
nodes, which express high levels of stromal cell derived factor-1 (SDF-1α/CXCL12),
a ligand of the CXCR4 receptor expressed on breast tumor cells, are the most
common sites of metastases [6-7]. Yet, while chemokine signaling directs tumor cells
to particular organs, the crosstalk between metastatic tumor cells, stromal and bone
marrow-derived cell (BMDC) lineages once at the metastatic site is crucial in creating
a supportive microenvironment. Creation of a metastatic microenvironment through

4
the recruitment of BMDCs determines whether a disseminated tumor cell (DTC)
survives and proliferates, becomes quiescent or dies at metastatic sites [3]. The
importance of the tumor microenvironment to primary tumor growth and progression
is well-established. The microenvironment at secondary sites of metastasis, while
equally as important to allow metastatic tumor cell colonization and growth, is poorly
understood in comparison. Recent evidence suggests the primary tumor itself is able
to influence and alter the environment of secondary organs by promoting the
formation of supportive metastatic microenvironments, termed pre-metastatic niches,
prior to tumor cell dissemination.
The pre-metastatic niche: a new era in metastasis research
The components crucial to pre-metastatic niche formation include tumor-
derived secreted factors (TDSFs) and BMDCs. TDSFs from the primary tumor
promote the mobilization and recruitment of BMDCs that interact with the local
stroma and extracellular matrix (ECM) at secondary organs, to help create
microenvironments suitable for colonization by metastasizing tumor cells (Figure 1).
The pre-metastatic niche was first described by Kaplan and colleagues in 2005
[8]. Tumor-derived VEGF and PlGF were demonstrated to promote the recruitment of
VEGFR1
+
hematopoietic progenitor cells (HPCs) that formed distinct clusters of cells
in secondary organs. Once at the secondary organ, clusters of VEGFR1
+
HPCs
expressing the fibronectin receptor integrin VLA-4 interact with resident fibroblasts to
stimulate fibronectin production and secrete MMP9 to create pre-metastatic niches for
disseminating CXCR4
+
tumor cells. Subsequent research has identified various
TDSFs and BMDCs important in pre-metastatic niche formation in different tumor
models [9]. Although pre-metastatic niches are now widely accepted to be a true
biological process promoting metastatic growth, speculation still exists as to whether
their formation is necessary and required for metastases formation [10-11]. While it is
likely that pre-metastatic niches are not essential for metastases to form, various
studies suggest that they greatly enhance the likelihood of metastatic progression [9].
This review will collate and explore the similarities and differences in the TDSFs and
BMDC components implicated in pre-metastatic niche formation, highlight the roles
of hypoxia, myeloid cells and immunosuppression in regulating microenvironments at
distant organs, discuss potential links to tumor dormancy and investigate how this
knowledge may help in the treatment of metastatic disease.

5
The primary tumor drives pre-metastatic niche formation: a role for hypoxia?
A variety of TDSFs including VEGF, PlGF, TNF-α, TGF-β, Lysyl oxidase
(LOX), versican and G-CSF have been shown to drive pre-metastatic niche formation
in various tumor models (Table 1 & Figure 1). While the role of individual TDSFs in
promoting pre-metastatic niche formation is many and varied (Table 1), little has been
done in the way of investigating the processes occurring at the primary tumor site to
stimulate their initial production. Pre-metastatic niches may simply arise as a
consequence of systemic disturbances caused by the presence of the primary tumor.
The induction of angiogenesis for example, is crucial to the development and growth
of solid tumors and results in the production of many pro-angiogenic TDSFs
including VEGF and PlGF from tumor cells and surrounding stromal cells such as
bone marrow-derived macrophages [12], neutrophils [13] and mast cells [14]. Pre-
metastatic niche formation could therefore simply be a bystander effect caused by the
induction of angiogenesis at the primary tumor. Yet while the influence of the primary
tumor may be systemic, pre-metastatic niche formation appears not be, with niches
generally observed in organs predisposed to metastases in certain cancer types as
discussed later.
Defining the state or processes occurring in the primary tumor that result in the
production of the pre-metastatic niche-promoting secreted factors is crucial to prevent
niche formation, as many studies have demonstrated that silencing or neutralizing
certain TDSFs can directly or indirectly, decrease metastasis. Tumor-bearing mouse
serum deprived of TNF-α, TGF-β and VEGF-A by means of a neutralizing antibody
for each protein, reduced the expression of proinflammatory proteins S100A8 and
S100A9 in pre-metastatic lungs and reduced metastatic burden in a Lewis Lung
Carcinoma (LLC) model [15]. Furthermore, administration of anti–G-CSF antibody to
mice bearing 4T1, 66c14, or MMTV-PyMT tumors was shown to decrease pre-
metastatic niche promoting-Ly6G
+
Ly6C
+
myeloid cells in the peripheral blood and
lungs, and thus decrease metastatic burden [16].
One process known to be associated with tumor progression, and more recently, pre-
metastatic niche formation, is hypoxia. Hypoxia is a reduction in tissue oxygen
tension and occurs in all solid tumors larger than 1 cm
3
due to an inadequate blood
supply resulting from the aberrant vasculature present in most solid tumors [17].
Cancer cells undergo genetic and adaptive changes to allow them to survive in
hypoxic conditions, resulting in the promotion of an aggressive tumor phenotype

Frequently asked questions

Q1. What have the authors contributed in "It is rapidly becoming evident that the formation of tumor-promoting pre-metastatic niches in secondary organs adds a previously unrecognized degree of complexity to the challenge of curing metastatic disease. primary tumor cells orchestrate pre- metastatic niche formation through secretion of a variety of cytokines and growth factors that promote mobilization and recruitment of bone marrow-derived cells to future metastatic sites. hypoxia within the primary tumor, and secretion of specific" ?

In this paper, the authors presented a study of the relationship between cancer genomics and genetics at the University of Melbourne 's Cancer Genomics and Genetics Laboratory. 

Q2. What other potential therapeutic approaches have been used in the treatment of multiple myeloma?

Other potential therapeutic approaches include redistributing cytotoxic NK cells [92], boosting NK cell activity by upregulating activating ligands (such as NKG2D) on tumor cells using chemotherapeutics as demonstrated in multiple myeloma [92], or through the use of monoclonal antibodies targeting tumor cells to enhance antibody-dependent cell-mediated cytotoxicity [86, 93]. 

Q3. What is the secretome of various tumor cell lines?

The secretome of various tumor cell lines contains both soluble factors and exosome-associated proteins [79], suggesting that exosomes were most likely present in tumor cell conditioned media in previous models of the pre-metastatic niche. 

Q4. What is the role of TDSFs in the pre-metastatic niche?

In addition to influencing myeloid cells mobilized directly from the bonemarrow to the pre-metastatic niche, TDSFs also influence the differentiation of myeloid cells in the primary tumor microenvironment, which in turn influences the myeloid cell subpopulations found in pre-metastatic organs. 

Q5. What is the role of myeloid cells in the pre-metastatic niche?

whether myeloid cells are first mobilized from the bone marrow to the primary tumor microenvironment before migrating to pre-metastatic sites, or from the bone marrow to pre-metastatic niches directly is an important determinant of their differentiation and function. 

Q6. How have NK cells been used in the treatment of different forms of leukaemia?

Adoptive transfer of human NK cells cultured and activated in vitro have been used, with varying degrees of success, in the treatment of different forms of leukaemia (reviewed in [91]). 

Q7. What is the need to move away from the current limiting paradigms regarding metastatic disease?

The high mortality rate associated with metastatic disease emphasizes the need to move away from the current limiting paradigms regarding metastatic progression. 

Q8. What is the role of stromal components in the formation of premetastatic niches?

However in spontaneous cancer, both the tumor cells and the stromal components that make up the primary tumor microenvironment could release factors capable of changing the composition of premetastatic organs. 

Q9. What could be the role of stromal components in the development of pre-metastatic?

Factors secreted by hypoxic endothelial and immune cells, fibroblasts and other stromal components of the tumor microenvironment could potentially have a substantial impact on the development of pre-metastatic niches. 

Q10. What is the common theme amongst different models of the pre-metastatic niche?

A common theme amongst different models of the pre-metastatic niche (Table 1) is the mobilization of myeloid cell lineages from the bone marrow and recruitment to specific pre-metastatic sites. 

Q11. What are the components that are important in the formation of pre-metastatic niches?

A variety of TDSFs including VEGF, PlGF, TNF-α, TGF-β, Lysyl oxidase(LOX), versican and G-CSF have been shown to drive pre-metastatic niche formation in various tumor models (Table 1 & Figure 1).