Pericytes in the pre-metastatic niche

Abstract

The pre-metastatic niche formed by primary tumor-derived molecules contributes to fixation of cancer metastasis. The design of efficient therapies is limited by the current lack of knowledge about the details of cellular and molecular mechanisms involved in the pre-metastatic niche formation. Recently, the role of pericytes in the pre-metastatic niche formation and lung metastatic tropism was explored by using state-of-the-art techniques, including in vivo lineage-tracing and mice with pericyte-specific KLF4 deletion. Strikingly, genetic inactivation of KLF4 in pericytes inhibits pulmonary pericyte expansion, and decreases metastasis in the lung. Here, we summarize and evaluate recent advances in the understanding of pericyte contribution to pre-metastatic niche formation.

INTRODUCTION

Tumor malignancy is characterized by its capacity to disseminate to distant organs in the human body, and nearly all related deaths happen due to metastasis rather than to primary tumor growth (1). The molecular basis of tumor dissemination is still poorly understood. Understanding how metastatic tumors form and spread is key for successful cancer treatment. The relationship between the disseminating cancer cells and the microenvironment of the organs which they colonize is key to determining whether these malignant cells grow to form secondary tumors (2). Non-withstanding the recent improvement in the mechanistic understanding of essential events that constitute the metastatic process, the ability to limit, prevent, or reverse metastatic tumor growth in cancer patients unfortunately has not progressed significantly, due to the complexity of the mechanisms underlying this process (1). The chase to identify metastatic cellular and molecular processes is driven by the hope that some of them may be therapeutically targetable.

The exact properties of specific tissues that regulate their involvement with metastatic tumor cells are still poorly understood. Currently, it is believed that the primary tumors may produce specific microenvironments in distant organs that assist metastatic cell survival and growth. These microenvironments are termed pre-metastatic niches, whose appearance is believed to be coordinated by messages released from primary tumors before the arrival of metastatic cells (3–5). This communication may pre-instruct local cells to form a supportive niche for metastasis survival and growth (6). Tumors have a tendency to metastasize in specific organs. Lungs are a common site for the metastatic spread of cancer cells, and a number of pre-metastatic modifications have been described there (7). These pre-formed lung niches include both resident cells, and cells recruited from other tissues (8). The original findings about the pre-metastatic niche, by Lynden’s group, showed that cancer-derived molecules induce the recruitment of bone marrow-derived cells to the secondary organs, where later metastases will reside (3). Investigation and characterization of lung cells that play a dominant role in setting pre-metastatic niche are important to develop targeted therapies that revert alterations of local microenvironment, inhibiting metastatic establishment (8).

The architecture of the lung is represented by an extensive external surface area with a large vascular bed for adequate gas exchange (9). Lung blood vessels are composed of endothelial cells associated with perivascular cells, such as pericytes (10). Pericytes are identified by specific molecular markers in combination with their anatomical location, with thin cytoplasmic processes surrounding endothelial cells (11). Classically, the main function of pericytes was considered physical stabilization of blood vessels (12). Recent discoveries made by lineage tracing, targeted gene deletion, and conditional genetic ablation of pericytes in mice have expanded our knowledge of pericyte role in health and disease (13,14). Pericytes function as stem cells (15–19), forming several cell types in a variety of pathophysiologic conditions (20). Also, they may regulate the behavior of other stem cells, such as hematopoietic stem cells (21,22). Additionally, pericytes play several immune functions (23), and are important cellular constituents of the tumor microenvironment (24). Due to their role in tumor angiogenesis, strategies targeting pericytes have been proposed as anti-angiogenic therapies for cancer (25). Nevertheless, higher pericytes’ coverage was associated with better prognosis in some patients (26), and, so far, clinical trials blocking pericytes have failed to improve patients’ outcome (27,28). Interestingly, pericyte targeting may even increase metastasis in certain conditions (29–32). For instance, Semb’s group have shown, by using PDGFB^ret/ret mouse model, that pericyte deficiency leads to spreading of metastatic insulinoma-derived cells (29), while Kalluri’s group have shown, by using NG2-TK mouse model, that genetic depletion of tumoral pericytes in breast cancer increases pulmonary metastasis (32). This is possibly due to the lack of specificity of these strategies which target pericytes as a whole. Therefore, a better understanding of the molecular mechanisms in which pericytes are involved during tumor progression may reveal specific targets within pericyte subpopulations, beneficial for anti-cancer treatments. Importantly, little was known about pericytic regulation of lung tumor metastasis. Now, in a recent article in Nature Medicine, Murgai and colleagues show that pericytes form a pre-metastatic niche in the lung (33). Following implantation of primary tumor with metastatic ability, pericytes detach and migrate away from the lung vasculature, expand in the pre-metastatic lung parenchyma, and become matrix-producing cells. Murgai and colleagues investigated the role of lung pericytes as pre-metastatic niche components by using state-of-the-art technologies, including confocal microscopy, in vivo lineage-tracing, and sophisticated Cre/loxP techniques to delete specific genes exclusively in pericytes. These experiments revealed that tumor-derived factors induce the expression of the transcription Kruppel-like factor 4 (KLF4) in pericytes (33). Genetic deactivation of KLF4 in pericytes decreased fibronectin formation by those cells. Strikingly, pericyte-specific deletion of KLF4 inhibited pericyte expansion in the lung, decreasing pulmonary tumor cell colonization (metastasis), while not affecting primary tumor growth (33) (Figure 1). Additionally, disruption of metastatic tumor cells binding to fibronectin by β1-integrin-neutralizing antibody decreased lung metastasis. This work provides a novel role for pericytes in the lung. Here, we discuss the findings from this study, and evaluate recent advances in our understanding of pericyte role in pre-metastatic niche formation.

Figure 1. Pericytes activate pre-metastatic niche formation and metastasis in the lung.

Pericytes are associated to pulmonary blood vessels. The study of Murgai and colleagues now reveals a novel very important function for pericytes in the pulmonary pre-metastatic niche formation (33). Pericytes undergo phenotypic switching, and migrate away from the blood vessels in response to primary tumor factors. These cells also contribute to the increase of extracellular matrix deposition, and the formation of a niche prone for the colonization of the lung by cancer cells. Murgai and colleagues by using NG2-CreER/KLF4 floxed mice demonstrate that KLF4 expression in pericytes is necessary for these processes to occur.

THE COMPLEXITY OF THE PRE-METASTATIC MICROENVIRONMENT

The pre-metastatic niche involves an intricate microenvironment which contains, in addition to pericytes, several other types of stromal cells, immune cells (34), innervations (35,36), extracellular matrix proteins, and tumor-produced molecules. Deciphering the individual and combinatorial signals that influence the development of the pre-metastatic niche will help develop effective therapeutic interventions. Although the exact nature of the pre-metastatic niche remains poorly understood, significant progress has been made in the last decade. Various immune cells were identified as essential constituents of the pre-metastatic pulmonary niche (37), especially from the myeloid lineage: immature myeloid CD11b-expressing cells (3,38), neutrophils (39,40), macrophages (41,42), and monocytes (43). These cells are recruited to pre-metastatic lung, enhancing pulmonary metastasis by production of cancer growth-stimulating factors. In contrast, the role of the non-myeloid cells in the establishment of the pre-metastatic niche is still not completely determined. Although CD4-expressing T cells were shown to contribute to the pre-metastatic niche in the bone (44), the role of CD4+ T cells in the pre-metastatic lung remains to be elucidated. Interestingly, regulatory T cells (T_reg) increase in the pre-metastatic lung (45).

Recently it was shown that pericytes interact actively with immune cells, and also display multiple immune properties (46). For instance, pericytes overexpress adhesion molecules involved in the control of immune cells trafficking through the vasculature, such as ICAM-1 and VCAM-1 (47), and produce a repertoire of chemokines important for immune cells roles (21,48). Overall, pericyte functions are complex, and our knowledge about the crosstalk between pericytes and immune cells remains limited. Thus, what is the cross-talk between distinct immune cell subsets involved in pre-metastatic niche formation and pericytes remains to be examined. Further studies are required to evaluate the importance of pericytes’ interactions with immune cells in pre-metastatic niche formation.

The cells from pre-metastatic niche communicate via complex molecular pathways, promoting metastasis. Exosomes are especially crucial in this inter-cellular exchange of information for pre-metastatic niche formation, affecting several cell types, including immune cells (5). Cancer-derived exosomes, liberated into the blood stream, originate pre-metastatic microenvironment in secondary organs by affecting several cells (49), including macrophages (50), neutrophils (51), myeloid-derived suppressor cells (52), natural killer cells (53) cytotoxic T cells, and antigen-presenting cells (54). Exosomes are involved in several types of cell-to-cell communications, not necessarily involving cancer cells (55), such as bidirectional communication between endothelial cells and pericytes (56). It remains to be explored whether pericytes in the pulmonary pre-metastatic niche communicate via exosomes present in the lung microenvironment.

THE VASCULAR PRE-METASTATIC NICHE

The vasculature is essential in multiple stages of the metastatic process, including at the entrance of cancer cells from the primary tumor into the blood stream, as well as the entry into the metastatic organ from the blood stream (57,58). In contrast, more studies are needed to understand the role of the vasculature in the pre-metastatic niche formation. The vascular niche is heterogeneous and contains, besides pericytes, several other cells, such as endothelial cells (59,60), fibroblasts (61), adventitial cells (62), smooth muscle cells (21), and macrophages (63,64). The main difference between pericytes and other perivascular cells is the vascular basal lamina (basement membrane) on the endothelial cell abluminal surface covering pericytes (65–70), composed of several macromolecules, such as proteoglycans, collagen, and glycoproteins (71). Pericytic markers are not specific, and could be expressed by other cells (12), for example, perivascular macrophages could express NG2 proteoglycan (72). The combination of pericytic molecular markers with immunolabeling of the vascular basal lamina and genetic cell fate mapping analysis will confirm the nature of perivascular cells in future studies. Endothelial cells have been shown to be important cells for the pre-metastatic niche formation in the bone (73). Cancer-produced factors induce the expression of chemoattractants by pulmonary endothelial cells, which may be important for this interaction (74,75). In the lung, cancer cells may recruit molecular players that disrupt the vasculature by a mechanism of recruitment of pre-existing blood vessels from the surrounding tissue, called vessel co-option (76,77). This phenomenon could be the reason for anti-angiogenic treatments failure in the lung (78). Its relevance for the pre-metastatic niche formation was not yet explored in deep. Even though the metastatic tropism to lung probably requires interactions of malignant cells with endothelial cells (79), how the endothelial cells and other perivascular cells interact with pericytes in the lung’s pre-metastatic microenvironment remains to be elucidated. Further studies will reveal the exact roles of endothelial cells in lung pre-metastatic niche formation.

Importantly, in addition to the vascular endothelium, lymphatic endothelial cells have also been suggested to play important roles in metastatic spread (80). Lymphatic vessels form the initial routes for metastatic cancer cells (81). Moreover, the formation of new lymphatic vessels is essential for metastatic success (82), and these vessels in the pre-metastatic organs may affect immune cell behavior (83). Thus, future studies should further explore the role of lymphatic endothelial cells in the pre-metastatic niche formation, and the interactions between these cells and the other cellular constituents of this complex microenvironment.

PERICYTE HETEROGENEITY IN THE PRE-METASTATIC NICHE

Pericytes have been shown to be heterogeneous regarding their phenotype, distribution, origin, marker expression, and function (84). Nevertheless, in their work, Murgai and colleagues consider pericytes as a homogeneous cell population. Pericyte heterogeneity was first described almost a century ago. Zimmermann distinguished pericytes into three types according to their vascular location: in the pre-capillary, capillary, or post-capillary (85). Pre-capillary pericytes have multiple round branches which cover the blood vessel (86). Capillary pericytes are spindle-shaped, highly elongated, have multiple short secondary processes, and extend mainly in the long axis of the blood vessels (86). Post-capillary pericytes are shorter stellate-shaped cells, and wrap themselves around the endothelial bed in post-capillaries. Pericytes also differ in their embryonic origin between organs, and even within the same tissue (84,87). Pericytes in the cephalic region and thymus derive from the neuroectoderm (88), while in most organs, they derive from the mesoderm; specifically, the sclerotomal compartment (89). In the lung, heart, liver and gut, the mesothelium is the main source of pericytes (22). Interestingly, recent studies show that cardiac endothelial cells are a source of a subset of pericytes in the murine embryonic heart (90), and myeloid cells give rise to a subpopulation of pericytes in the embryonic skin (84). Thus, the developmental sources of pericytes could be heterogeneous even within the same organ. These unexpected findings raise the possibility that distinct pericytes’ subpopulations, depending on their developmental origin, could vary in their contribution to different pathological conditions. Several molecular markers to identify pericytes have been discovered and characterized, such as platelet derived growth factor receptor β (PDGFRβ), aminopeptidase N (CD13), nerve/glial antigen 2 (NG2) proteoglycan (CSPG4), α smooth muscle actin (αSMA), desmin, regulator of G protein signaling 5 (RGS5), ATP-binding cassette, subfamily C (CFTR/MRP), member 9 (SUR2), alkaline phosphatase (ALP), vimentin, CD133, CD146, potassium inwardly rectifying channel, subfamily J, member 8 (Kir6.1), endosialin, Tbx18, and others (91). Pericyte heterogeneity was also confirmed based on their marker expression profiles. For instance, capillary pericytes express desmin but usually are negative for αSMA, while venular pericytes express both desmin and αSMA proteins (92). Also, Kir6.1 is highly expressed in pericytes in the brain, but undetectable in pericytes in the heart and skin (93). In the bone marrow, arterioles-associated leptin receptor (LEPR)-negative pericytes are distinct from sinusoid-associated LEPR-expressing ones (22,94). In the skin, NG2+ and NG2− pericytes have been described (95). Also, pericytes that express YFP differ from the ones that express desmin and αSMA surrounding blood vessels in the spinal cord of glutamate aspartate transporter (Glast)-CreER/R26R-YFP double transgenic mice (96).

In the lung, pericytes are also not homogeneous, and there are at least two different subsets, which were distinguished based on the presence or absence of Nestin-GFP expression: Nestin+ (type-1) and Nestin-negative (type-2) pericytes were reported surrounding pulmonary blood vessels (97). Whether these pericyte subpopulations vary in their function, and if only a fraction of pericytes promote pre-metastatic niche formation still need to be clarified. Murgai and colleagues reveal that extracellular matrix deposition by pericytes is essential to support tumor metastatic behavior (33). Interestingly, type-1, but not type-2, pericytes accumulate at the injury site in response to lung damage, and produce collagen, contributing to pulmonary fibrosis (97). The other pericyte subpopulation, type-2 pericytes, lack fibrogenic capacity, and do not form collagen (13). It will be interesting to test in the future whether, in the formation of the pre-metastatic niche, it is also only type-1 pericytes that produce extracellular matrix proteins, which dictate tumor cell fate and metastasis in the lung. Additionally, although both described pulmonary pericyte subsets express the well-established pericytic marker NG2 proteoglycan, it was not explored yet whether NG2− pericytes are also present in the lung, as reported in the skin (95). The presence of pericytes not expressing NG2 in the lung, as well as their role in pre-metastatic niche formation, should be explored in future works.

As mentioned above, the complex architecture of the pulmonary vasculature ensures an extensive surface area for gas exchange (98). Pulmonary vascular beds are composed of distinct blood vessel types, which, considering anatomical position, size, and morphology, are divided into the following types: pre-acinar and intra-acinar; extra-alveolar and those within the alveolar compartment; as well as pre-capillary, capillary, and post-capillary vessels (99). Pericytes associated with particular blood vessel types may differ in their behavior from the ones associated with others (21). For instance, in the bone marrow, pericytes from arterioles, sinusoids, and transition zone capillaries control hematopoietic stem cells maintenance and activation differently (21). Thus, future studies should distinguish lung pericytes attached to varying kinds of blood vessels in their roles as metastatic niche-forming cells.

CLINICAL INTERVENTIONS

Murgai and colleagues propose KLF4-targeting to prevent metastasis (33). Although in several conditions KLF4 is pro-tumorigenic (100,101), it may also act to inhibit tumor growth in some contexts (102), therefore clinical trials will be needed to test the efficacy of KLF4-targeted strategies. Importantly, targeted activation of KLF4 in cancer patients has been approved for clinical trial (NCT01281592) (103). This clinical trial will need to consider the negative effect of blocking KLF4 in pericytes on metastasis formation. Future clinical trials will benefit from the development of drugs that function in a cell type-specific fashion; for instance, by tumor targeted nanoparticles carrying drugs. This will allow to specifically activate KLF4 only in malignant cells, or that block it specifically in pericytes. β1 integrin signaling is also suggested to play important role in metastatic progression (33). A blocking peptide for β1 integrin has been developed (ATN-161), and it is currently being used in phase II clinical trials in combination with chemotherapy (104). Notably, a monoclonal anti-β1 integrin antibody for use in clinics is currently under development (104). Murgai et al. (2017) results are promising. Unfortunately, experimental data does not always predict success at the clinical stage. However, the efforts in the field to identify specific inhibitors that influence uniquely the tumor niche will lead to more efficacious combined therapies.

Other potential molecular targets in pericytes have been proposed as anti-cancer therapies, such as PDGFRβ, expressed by these cells. Receptor tyrosine kinase inhibitors that target PDGFRβ, upregulated in pericytes, such as sunitinib and imatinib, have been tested (105). Nevertheless, these treatments result in lung-enhanced metastasis (32). Interestingly, combination of imatinib with anti-angiopoietin 2 treatment control lung metastasis (25). Clinical trials using drugs targeting pericytes are underway (106). Nonetheless, in future studies the gene expression analysis of human pre-metastatic pulmonary pericytes in distinct cancer subtypes may reveal new potential molecular targets.

PERSPECTIVES/FUTURE DIRECTIONS

Pericytes’ potential to differentiate into various cell populations is well known; and the general consensus holds that pericytes are cells with high plasticity (107,108). A recent study even showed that pericytes expressing NG2 proteoglycan can be the cell of origin for mesenchymal tumors, such as bone and soft tissue sarcomas (109). It will be interesting to explore whether some of those pericytes activated by tumor-derived factors in the lung pre-metastatic niche can transform into metastatic tumor cells.

One question that remains open in the study of Murgai and colleagues is the exact origin of pericytes in the lung’s pre-metastatic niche. Is it possible that those pericytes are derived from another organ? For example, it has been shown by cell-lineage tracing that, in the brain, glioblastoma stem cells can give rise to pericytes that support blood vessel function and tumor growth (110–113). It still remains to be investigated whether metastatic cancer cells can also originate lung pericytes that form the pre-metastatic microenvironment.

Kaplan and colleagues showed that the removal of VEGFR1-expressing hematopoietic cells from the bone marrow abrogated the formation of the pre-metastatic niche (3). It will be interesting to examine whether or not pre-formation of the pre-metastatic niche by pericytes and bone marrow-derived cells is reversible upon removal of the primary tumor. Niche cells-intrinsic changes may be reversible or not but, are continuous reinforcing signals from the primary tumors necessary for these cells’ support of tumor growth in the lung? Thus, analyses of the lung pre-metastatic microenvironment, after removal of the primary tumor, should be done in future experimental settings. The possible presence of tumor dormant cells needs to be taken into account, as they may remain at low numbers after primary tumor removal (114).

The permissiveness of the different tissues to tumor growth varies between organs. Different tumor types have a propensity to form metastases in distinct sites. Murgai and colleagues suggested that the enhanced proliferation and migration of pericytes was specific to the pre-metastatic lung tissue, as a similar expansion of pericytes was not seen in the liver (33). However, the tumor cell lines tested (B16-F10, melanoma, and M3-9M, rhabdomyosarcoma) rarely metastasize to the liver. It will be interesting, in future studies, to explore other organs/tumors where metastatic cells colonize other tissues, besides the lung. This will reveal whether pericytes from other organs are also activated after primary tumor implantation, or whether this is an only-lung pericyte specific role.

Interestingly, a recent study showed that the lung is an organ that supports hematopoiesis. Hematopoietic stem cells reside within the lung and migrate out repopulating the bone marrow, and contributing to multiple hematopoietic lineages (115–118). Future studies should reveal whether there are common cellular and molecular mechanisms to form stem cell and metastatic niches in the lung.

It still remains poorly explored which factors produced by pericytes are important for the support of metastatic growth in the lung. The study by Murgai et al. (2017) shows that the transcription factor KLF4 in pericytes is essential for early tumor cell colonization and metastatic burden (33). Pericytes release a plethora of molecules, including growth factors and cytokines (119,120). Which molecules produced by lung pericytes are important in the pre-metastatic niche to attract tumor cells remains to be discovered. Also, it is known that primary tumor secretes inflammatory chemoattractants, such as transforming growth factor β (TGFβ), tumor necrosis factor α (TNFα), and vascular endothelial growth factor A (VEGFA), which activate pulmonary endothelial cells (75). Nevertheless, it is still unclear which molecules released by primary tumor cells act directly on pericytes, inducing the pre-metastatic niche formation. In addition to transcriptomic and single cell analysis, genetic mouse models will help to address this. For instance, by using pericyte-specific inducible CreER drivers crossed to cytokines-specific floxed mice, specific cytokines could be deleted genetically at different time points specifically in pericytes in the lung, and pulmonary metastatic colonization can be analysed. By similar methods, it was recently demonstrated that CXCL12 from bone marrow arteriolar pericytes is essential for hematopoietic stem cell maintance in the bone marrow (21).

In conclusion, the study by Murgai and colleagues reveals a new important role of pericytes in the lung, complementing other existing studies in the area. However, our understanding of pericytes biology in the lung still remains limited, and future studies should shed light on the complexity and interactions of different cellular components of the lung microenvironment during metastatic progression. A great challenge for the future will be to translate the research from experimental models into humans. Whether tumor cells at an early stage of human cancer development promote the same pre-metastatic niche formation in the lung remains to be determined. Improving the availability of human tissue samples will be essential to reach this aim.