Feed‐forward loops between metastatic cancer cells and their microenvironment—the stage of escalation

Review4 May 2022Open Access Feed-forward loops between metastatic cancer cells and their microenvironment—the stage of escalation Zora Baumann Zora Baumann orcid.org/0000-0002-0796-5011 Tumor Heterogeneity Metastasis and Resistance, Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland Contribution: Conceptualization, ​Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Priska Auf der Maur Priska Auf der Maur orcid.org/0000-0002-9059-8979 Tumor Heterogeneity Metastasis and Resistance, Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland Contribution: Conceptualization, ​Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Mohamed Bentires-Alj Corresponding Author Mohamed Bentires-Alj [email protected] orcid.org/0000-0001-6344-1127 Tumor Heterogeneity Metastasis and Resistance, Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland Contribution: Supervision, Funding acquisition, Writing - original draft, Writing - review & editing Search for more papers by this author Zora Baumann Zora Baumann orcid.org/0000-0002-0796-5011 Tumor Heterogeneity Metastasis and Resistance, Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland Contribution: Conceptualization, ​Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Priska Auf der Maur Priska Auf der Maur orcid.org/0000-0002-9059-8979 Tumor Heterogeneity Metastasis and Resistance, Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland Contribution: Conceptualization, ​Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Mohamed Bentires-Alj Corresponding Author Mohamed Bentires-Alj [email protected] orcid.org/0000-0001-6344-1127 Tumor Heterogeneity Metastasis and Resistance, Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland Contribution: Supervision, Funding acquisition, Writing - original draft, Writing - review & editing Search for more papers by this author Author Information Zora Baumann1,†, Priska Auf der Maur1,† and Mohamed Bentires-Alj *,1 1Tumor Heterogeneity Metastasis and Resistance, Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland † These authors contributed equally to this work *Corresponding author. Tel: +41 61 26 53 313; E-mail: [email protected] EMBO Mol Med (2022)14:e14283https://doi.org/10.15252/emmm.202114283 See the Glossary for abbreviations used in this article. Correction added on 16 September 2022, after first online publication: The reference had been improperly cited. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Breast cancer is the most frequent cancer among women, and metastases in distant organs are the leading cause of the cancer-related deaths. While survival of early-stage breast cancer patients has increased dramatically, the 5-year survival rate of metastatic patients has barely improved in the last 20 years. Metastases can arise up to decades after primary tumor resection, hinting at microenvironmental factors influencing the sudden outgrowth of disseminated tumor cells (DTCs). This review summarizes how the environment of the most common metastatic sites (lung, liver, bone, brain) is influenced by the primary tumor and by the varying dormancy of DTCs, with a special focus on how established metastases persist and grow in distant organs due to feed-forward loops (FFLs). We discuss in detail the importance of FFL of cancer cells with their microenvironment including the secretome, interaction with specialized tissue-specific cells, nutrients/metabolites, and that novel therapies should target not only the cancer cells but also the tumor microenvironment, which are thick as thieves. Glossary Cancer dormancy Two forms of dormancy have been observed: Cellular dormancy describes a reversible non-proliferative state of a cancer cell that can last for several years. Tumor mass dormancy represents the offset of cancer cell proliferation by cell death, resulting in net-constant cell numbers. Colonization Growth of micrometastases into macrometastases. Disseminated tumor cells (DTCs) Cancer cells that infiltrate and survive in distant sites. DTCs may succumb, remain dormant, or colonize the tissue. Extracellular Matrix (ECM) Three-dimensional network surrounding cells, consisting of macromolecules and minerals, including collagens, glycoproteins, and cell adhesion proteins. The ECM provides essential structural support and serves diverse biochemical activities. Components of the ECM are produced intracellularly by resident cells and subsequently secreted into the extracellular space. The composition thus varies widely between organs and can be transiently remodeled upon physiological injury or chronic stimuli, including cancer/metastases. Extracellular Vesicles (EV) EV is a collective term covering a variety of cell-derived membranous structures that encapsulate and transport cellular materials, and nearly, all cell types can produce them. EV cargo include proteins, lipids, microRNAs (miRs), mRNA, and noncoding RNAs. One example of EVs are exosomes. In the context of cancer, EVs released from primary tumors can establish a pre-metastatic niche in distant organs. Inter-site heterogeneity Heterogeneity between lesions in different sites. Inter-site heterogeneity is generally used to describe cancer cells; however, it also applies to other cell types that are part of the tumor microenvironment. Intra-site heterogeneity Heterogeneity within the same primary tumor or the same metastasis. Commonly, intra-site heterogeneity is used for cancer cells only; however, it also applies to other cell types that are part of the tumor microenvironment. Metastasis Metastasis is a multi-step process in which cancer cells invade surrounding tissues at the primary site, intravasate, and survive in the circulation as circulating tumor cells (CTCs) that extravasate at distant organs (referred to as disseminated tumor cells; DTCs). Metastases are responsible for the majority of breast cancer-related deaths. Minimal residual disease (MRD) This disease stage is when a patient is in remission, and only a small number of cancer cells have persisted therapy. Minimal residual disease can endure for several months or, in some cases, up to decades and represents a significant challenge for long-term remission because it is a reservoir of cancer cells that can regrow anytime. Additionally, these cells are often of a more aggressive type because they are treatment-resistant and will therefore be more challenging to eradicate. Organotropism Process of cancer cells spreading to and surviving in distant organs in a non-arbitrary way. Broadly categorized, it depends on cancer cell intrinsic factors (i.e., clonal fitness, genetic alterations, or metabolism) and non-cancer cell autonomous features (i.e., endothelial structure, or immune cells). These factors allow seeding to specific distant organs and enable survival in this foreign microenvironment. Pre-metastatic Niche (PMN) Primary tumors secrete soluble factors and EVs that reprogram distant sites and facilitate future cancer cell homing and survival. Numerous factors influence PMN formation, including vascular changes, stromal cell activation, immune cell recruitment, ECM remodeling, and metabolic reprogramming. Tumor Microenvironment (TME) The TME can include endothelial, immune, tissue-resident cells, nerve cells, adipocytes, a stroma composed of extracellular matrix, cancer-associated fibroblasts, mesenchymal cells, and numerous soluble factors. Breast cancer cells and the TME co-evolve dynamically through reciprocal interactions that corrupt homeostatic networks and contribute actively to disease progression. Introduction Metastases are the leading cause of cancer-related deaths in breast cancer patients (Siegel et al, 2020). This spread to distant organs is a multi-step process in which cancer cells invade surrounding tissues at the primary site, intravasate, and survive in the circulation as circulating tumor cells (CTCs) that extravasate at distant organs (referred to as disseminated tumor cells; DTCs), and possibly form metastasis (Chambers et al, 2002; Alix-Panabières & Pantel, 2014; Lambert et al, 2017; Esposito et al, 2021; Ganesh & Massagué, 2021). Especially, CTC clusters are rare but are more metastatic than single CTCs (Aceto et al, 2014). Despite significant advances in our understanding of the metastatic cascade, therapeutic targeting of metastasis remains poor and is a significant impediment to the clinical management of patients. Much of the reduction in cancer-related mortality achieved by therapeutic interventions has been based on early detection and improved surgery, combined with adjuvant treatments that eliminate DTCs. However, for breast cancer patients that reach the metastatic stage, the 5-year survival rate has barely improved in the last 20 years (Esposito et al, 2021). Therefore, a mechanistic understanding of the metastatic odyssey that suggests specific effective therapies is of the utmost clinical importance. One of the main challenges for successful treatment is tumor heterogeneity, which is found at several levels within a single patient. First, there is the heterogeneity within the same primary tumor or the same metastases, known as intra-site heterogeneity. Second, there is heterogeneity between different lesions, so-called inter-site heterogeneity (Koren & Bentires-Alj, 2015; Lüönd et al, 2021). Over the years, several studies have shed light on the diverse origins of genetic heterogeneity during cancer evolution (Burrell et al, 2013; Yates, 2017). While metastases of breast cancer patients harbor more mutations than primary tumors (Angus et al, 2019; Priestley et al, 2019), the gain of actionable driver mutations seems to play a subordinate role in the metastatic process (Vanharanta & Massagué, 2013; van de Haar et al, 2021; Reiter et al, 2018). Therefore, phenotypic alterations of DTCs, including changes in epigenome, metabolism, and interactions with immune and stromal cells, seem to be key for metastatic progression. The combination of genetic heterogeneity, phenotypic plasticity, and various selection pressures at different stages of the metastatic progression is a major hurdle to successful therapy. Cancers can arise from a single “mutated” cell; however, disease progression is often a consequence of sequential alterations that enrich for aggressive subpopulations within the tumor (Swanton, 2012). To a certain degree, cancer progression resembles the Darwinian “survival of the fittest” principle and tumors can be viewed as constantly evolving ecosystems (Tabassum & Polyak, 2015; Vendramin et al, 2021). One of the most important non-genetic drivers of cancer development is the tumor microenvironment (McGranahan & Swanton, 2017; Black & McGranahan, 2021). Tumor development is heavily shaped by physical and architectural constraints of the tissue, competition for space, the enduring effects of the immune system, and the changing nutritional environment (Altea-Manzano et al, 2020). In particular, the reactions of cancer cells to the potentially harsh environment outside of primary tumors is a driver in the selection of metastatic cancer clones (Massagué & Obenauf, 2016; Vendramin et al, 2021). This review summarizes how the environment of metastatic sites is influenced by the phenotype of the primary tumor and by the varying dormancy of DTCs, with a special focus on how established metastases persist and grow in distant organs due to feed-forward loops (FFLs). Finally, we discuss the concept that novel therapies should target not only the cancer cells but also the tumor microenvironment. The (co-)evolution of metastasis and the microenvironment Properties of the pre-metastatic niche An elaborate interplay of the primary tumor’s secretome with immune and tissue-resident cells results in a microenvironment in secondary organs (the pre-metastatic niche—PMN) that favors subsequent cancer cell homing. PMN alterations can be broadly categorized into (i) vascular changes including vascular leakiness, expression of adhesion molecules, clot formation, (ii) activation of stromal components and extracellular matrix (ECM) reorganization, for example, by distant secretion of matrix metalloproteinases, (iii) immune cell recruitment, (iv) changes in resident cells, including metabolic adaptions (Peinado et al, 2017; Wang et al, 2019). The concept that metastases do not seed arbitrarily was proposed in 1889 by Stephen Paget, who suggested that cancer cells (“seeds”) preferentially home to specific secondary organs (“soil”) (Paget, 1889). It is nowadays evident that the “seeds” can prime the “soil” on multiple levels. The “seed and soil” theory was amended with the concept of the PMN. Systemic effects of tumor-secreted factors and vesicles result in changes in secondary organs devoid of cancer cells (the PMN) that favor subsequent CTC homing (Peinado et al, 2017). Organ-specific metastases (organotropism) are a common occurrence in multiple solid cancers. Clinically, breast cancer often spreads to several distant organs and different subtypes have been associated with differential patterns of metastasis. The most common site of metastasis shared between all subtypes is the bone, with the highest percentage in the estrogen receptor (ER)-positive subtype. In ER-positive/HER2-negative patients, there is a pattern of bone only disease (Leone et al, 2017). Patients with triple-negative breast cancer have a higher risk to develop brain and lung metastases than patients with other subtypes. On the other hand, HER2-positive tumors more often spread to the liver (Chen et al, 2017; Wu et al, 2017). Moreover, there is a 25-50% incidence of brain metastasis in advanced HER2-positive disease (Zimmer et al, 2020). Multiple factors contribute to organotropism; for example, secretion of lysyl oxidase by the hypoxic primary tumor induces Wnt signaling in pre-metastatic bone lesions (Cox et al, 2015); periostin expression in resident cells modifies the ECM and enhances lung metastases (Malanchi et al, 2012); and lactate secretion by cancer cells limits NK cell cytotoxicity and increases brain metastases (Parida et al, 2022). The metastatic niche After extravasation into the blood stream (Fig 1), only approximately 0.01% of CTCs infiltrate and eventually colonize distant organs (Massagué & Obenauf, 2016). The initial arrest of CTCs at distant sites is regulated primarily by blood circulation and flow patterns, the vascular architecture, and whether they adhere to the endothelium (Chambers et al, 2002). CTC survival is promoted by neutrophils in the circulation and the PMN (Szczerba et al, 2019). Additionally, CTC homing to distant sites is enhanced by neutrophils and macrophages facilitate anchorage and escape from immune surveillance and provide survival signals and metabolites (Kitamura et al, 2015; Celià-Terrassa & Kang, 2018). Once DTCs intravasate into distant organs, whether they succumb, remain dormant, or colonize the tissue depends on their surroundings (Fig 1) (Lukanidin & Sleeman, 2012; Liu & Cao, 2016). In patients, metastasis cannot be fully distinguished from therapy resistance as most metastases of breast cancer are recurrences after systemic therapy (Weiss et al, 2022). Thus, the metastatic niche is not only important for metastatic growth but also for resistance to therapy. Figure 1. The path of cancer escalation Breast cancer disease progresses over several stages. After local invasion and dissemination from the primary tumor, CTCs enter the blood circulation, traveling as single cells or in clusters combined with neutrophils or T cells. Several factors secreted from primary tumor cells, including exosomes, S100 proteins, VEGF-A, TGFβ, TNFα, SAAs, and CCL2, influence the properties of pre-metastatic niches in particular organs). Additionally, these same factors can facilitate the extravasation of DTCs at distant sites. Metastatic cancer cells adhere to and multiply at distant sites (pre-metastatic niches) with surface and immunological affinities. Cancer cell dormancy can, at this stage, persist for several years or even decades. Disseminated cancer cells are in constant interaction with tissue-resident cells and with the resident homeostatic programs that foster metastatic niche formation and survival. This results in self-enhancing loops and disease escalation that is in many cases fatal. CTCs, circulating tumor cells; CCL2, C-C motif chemokine ligand 2; DTCs, disseminated tumor cells; SAAs, serum amyloid A proteins; TGFβ, transforming growth factor-beta; TNFα, tumor necrosis factor-alpha; VEGF-A, vascular endothelial growth factor-A. Download figure Download PowerPoint Dormancy Following primary tumor removal and therapy, minimal residual disease (MRD) that persists several years to decades without clinical detection is referred to as “dormancy”. Two forms of dormancy have been proposed: cellular dormancy and tumor mass dormancy. Cellular dormancy is characterized by three traits: dormant DTCs persist in foreign organs (“soil”), they are—for the time being—arrested in G0 and are frequently resistant to treatments (Ghajar, 2015). In tumor mass dormancy, cancer cell proliferation is offset by cell death due to immune surveillance and/or insufficient vascularization, resulting in insignificant net change in cell number (Chambers et al, 2002; Kang & Pantel, 2013). Notably, the risk of breast cancer relapse is subtype-dependent. While this risk remains constant in the ER-positive subtype, it decreases over time in ER-negative disease (Lee & Djamgoz, 2018; Rueda et al, 2019). Cancer cells may enter or exit cellular dormancy through cancer cell-intrinsic mechanisms (Vera-Ramirez et al, 2018; La Belle Flynn et al, 2019) or extrinsic stimuli (Ghajar et al, 2013; Senft & Jeremias, 2019; Perego et al, 2020; Correia et al, 2021). We have shown recently that IFNγ secretion from NK cells maintains breast cancer cell dormancy in the liver (Correia et al, 2021). The reawakening of DTCs and subsequent colonization is mediated by CXCL12 secretion from activated hepatic stellate cells. This suppresses immune surveillance by inducing NK cell quiescence and results in metastatic outgrowth (Correia et al, 2021; Lopes & Vivier, 2021). Additionally, it was reported that tissue-resident type I innate lymphoid cells and conventional NK cells impede liver colonization (Ducimetière et al, 2021).§ Other mechanisms have been reported to tip the balance from dormancy to overt metastasis. Neutrophil activity can wake dormant DTCs upon exposure to environmental inflammatory cues (Albrengues et al, 2018). Depending on the host’s NK cell status, neutrophils can be facilitators or inhibitors of metastatic colonization (Li et al, 2020). Additionally, sprouting neovasculature can lead to breast cancer DTC outgrowth (Ghajar et al, 2013). Moreover, surgical resection of primary breast tumors can also trigger outgrowth of previously immune-controlled metastases by macrophage engagement (Krall et al, 2018). Unfortunately, the triggers leading to clinically detectable metastases are probably the least characterized—yet potentially most important components—in the metastatic cascade (Esposito et al, 2018). Admittedly, monitoring and studying the switch from dormancy to metastatic outgrowth in clinical specimens is challenging and most of our current understanding of these processes is derived from preclinical studies. Colonization and feed-forward loops (FFLs)—the stage of escalation Colonization (the transition from a micro to macro metastasis) is the last and most fatal stage of breast cancer and the most difficult to treat due to tumor heterogeneity, metabolic flexibility, and complex interactions of cancer cells with the tumor microenvironment. It is important to note that no single therapeutic agent has been approved that specifically targets breast cancer metastases. That clinically detectable metastases can develop years after resection of the primary tumor highlights the fact that colonization is the most complex and rate-limiting phase of the metastatic cascade (Fig 1) (Massagué & Obenauf, 2016). The signals governing the development of nascent metastatic lesions are still being defined, but it is already known that stimuli range from stress hormones to chemokines (Obradović et al, 2019; Ozga et al, 2021). One example is CCL2 secretion from mammary tumors, which recruits C-C motif receptor 2 (CCR2)-expressing inflammatory monocytes to the metastatic site (Qian et al, 2011; Bonapace et al, 2014; Kitamura et al, 2015). We have shown previously that anti-CCL2 therapy efficiently retains monocytes in the bone marrow. However, interruption of the treatment results in rapid dissemination and direct colonization of metastatic cells fueled by elevated monocyte release from the bone marrow due to vascular endothelial growth factor A (VEGF-A) and interleukin 6 (IL6) signaling (Bonapace et al, 2014). This highlights that targeting colonization is complicated by interactions between tumor cells, their microenvironment, and remote sites such as the bone marrow (Keklikoglou & De Palma, 2014; Hitchcock & Watson, 2015). The outgrowth of metastases can be influenced by factors from resident immune and stromal cells. However, crosstalk between cancer cells and the tumor microenvironment (TME) can also result in detrimental self-enhancing loops that increase the survival and proliferation of metastatic tumor cells. One example is that mammary tumor-initiating cells exhibit elevated G-CSF production due to increased mTOR signaling, which then leads to accumulation of myeloid-derived suppressor cells (MDSC). Notch activation by MDSCs subsequently increase tumor-cell initiating frequency (Welte et al, 2016). Such vicious cycles vary between different tissues and are therefore discussed separately for specific organs later in this review. These organ-specific self-enhancing loops are challenging but potentially offer important therapeutic opportunities for treatment of metastases that will be discussed at the end of this review. Factors influencing the TME The secretome of cancer cells and their microenvironment A key aspect of metastasis initiation is the cancer secretome, that is, cytokines, metabolites, and extracellular vesicles that transmit reprogramming signals to cells in the vicinity of the cancer as well as in distant organs. The secretome is relevant from the onset of tumorigenesis and embodies crucial stimuli throughout the metastatic cascade. In the following paragraph, we focus on initial FFLs that prime and establish the metastatic niche (Fig 2). Figure 2. The intricate soluble network that creates a (pre-)metastatic niche The vast secretome of primary tumors includes chemokines, growth factors, and a range of tumor vesicles, including exosomes. These enter the blood system and reach secondary sites before cancer cells disseminate. This results in a microenvironment that is compatible for tumor cells and immune-suppressive. Examples include TNFα, TGFβ, and VEGF-A, which induce serum amyloid A (SAA) proteins that recruit MDSCs to the organ. Additionally, S100 proteins are induced and secreted by various mechanisms, including chemokines and exosomes, from the primary tumor that act on tissue-resident cells, including fibroblasts, Kupffer cells, and lung epithelial cells. S100 proteins are pro-inflammatory proteins diversely involved in metastatic niche properties. Furthermore, S100 proteins secreted from immune-suppressive immune cells including MDSCs are additionally able to initiate their proliferation in an S100-autocrine manner. The proteins are also involved in a vicious FFL with neutrophils that are attracted to the niche, where they in turn secrete even more S100 proteins. Exosomes induce higher lactate secretion from tumor-associated macrophages (TAM), which in turn upregulates PD-L1, a crucial immune checkpoint inhibitor. Moreover, CCL2 from primary tumors attracts TAMs and regulatory T cells (Tregs) to pre-metastatic niches, which deters anti-tumorigenic NK cells and T cells. In addition, reactive oxygen species (ROS) secreted from neutrophils further impact cytotoxic NK cells and T cells. All of these listed factors promote metastatic signaling and thus enhance exacerbating disease progression. CCL2, C-C motif chemokine ligand 2; MDSC, myeloid-derived suppressor cells; PD-L1, programmed death-ligand 1; ROS, reactive oxygen species; SAA, serum amyloid A proteins; TAM, tumor-associated macrophages; TGFβ, transforming growth factor β; TNFα, tumor necrosis factor α; Treg, regulatory T cells; VEGF-A, vascular endothelial growth factor-A. Download figure Download PowerPoint The PMN is composed of several soluble factors secreted by cancer cells in the primary tumor, by bone marrow-derived cells, suppressive immune cells, and host tissue stromal cells (Paolillo & Schinelli, 2019). The main pro-inflammatory signal proteins secreted from the cancer cells include VEGF-A, transforming growth factor β (TGFβ), and tumor necrosis factor (TNF) (Paolillo & Schinelli, 2019). In turn, all these factors further induce expression of S100 chemoattractants, a family of Ca2+-binding proteins that are implicated in many aspects of cancer progression (Lukanidin & Sleeman, 2012; Rinaldi et al, 2021). S100 proteins lie at the center of many vicious FFLs that foster metastasis. Examples include S100A8 and S100A9 secreted from breast cancer cells, as well as MDSCs that promote metastases in xenograft models in an auto- and paracrine fashion (Bresnick et al, 2015). The cancer secretome is critical for recruitment of immune cells. Tumor-derived cytokines and chemokines such as CCL2, mentioned above, recruit regulatory and immunosuppressive immune cells, including tumor-associated macrophages (TAMs), to secondary organs, where they are potent orchestrators of PMN formation through immune-modulation and immune-suppression (Qian et al, 2011; Bonapace et al, 2014; Ozga et al, 2021). Recently, β-catenin-mediated CCL2 secretion has been implicated in enhancing TAM recruitment and promoting metastasis (Zhang et al, 2021a). For an extensive overview of chemokines and the immune response to cancer, we refer to the recent review by Ozga et al (2021). Importantly, the same cytokines are often involved in recruiting both pro- and anti-tumorigenic immune cells. For example, the CCR5/CCL5 axis attracts both Tregs and cytotoxic CD8+ T cells (Ozga et al, 2021). Ultimately, cytokines and chemokines are double-edged swords as they are indispensable for homeostatic tissue functions but may become delinquent in metastases formation. Heterogeneous immunosuppressive bone marrow-derived cells recruited by the cancer secretome are also important contributors to the PMN. Examples include the ability of VEGF to mobilize VEGFR1+ hematopoietic bone marrow progenitor (HPCs) cells or VEGF-A, TGFβ, and TNFα-evoked induction of serum amyloid A proteins that recruit MDSCs (Kaplan et al, 2005; Hiratsuka et al, 2006, 2008). MDSCs are a heterogenous population of immunosuppressive cells that can be broadly categorized into CD11b+CD68+F4/80+ myeloid cells, CD11b+Ly6C+ monocytes, and CD11b+Ly6G+Ly6C+ granulocytes (Liu & Cao, 2016). MDSCs expression of integrins, and secretion of chemokines, inflammatory mediators, and growth and angiogenic factors promote the PMN (Liu & Cao, 2016). Intriguingly, MDSCs can reduce NK cell cytotoxicity in the pre-metastatic lung in breast cancer models (Sceneay et al, 2012). For further reading on MDSCs, we refer to a review by Wang et al (2019). Neutrophils are another important immune cell type that is attracted by tumor-derived factors, including G-CSF and S100 proteins, as well as by the CXCL12/CXCR4 axis (Dumitru et al, 2013; Leach et al, 2019; Gonzalez et al, 2020; Wang et al, 2020). Leukotriene signaling from neutrophils can support metastasis-initiating cells in pre-metastatic lung of mammary cancer mouse models (Wculek & Malanchi, 2015). This also contributes to the reawakening of dormant tumor cells by neutrophil extracellular traps (NETs) formation (Albrengues et al, 2018) and to metastatic colonization, as they are recruited by γδ-T cell-released G-CSF that leads to expansion and polarization of neutrophils in the lung metastatic niche (Coffelt et al, 2015). Moreover primary tumor angiopoietin-like protein 2 (ANGPTL2) secretion increases recruitment of neutrophils to the lung, thus contributing to PMN formation (Charan et al, 2020). Extracellular vesicles (EVs), including exosomes released from the primary tumor, can dictate organotropic behavior of metastatic tumor cells (Hoshino et al, 2015). Breast cancer-derived exosomes containing several integrins travel in the bloodstream and are preferentially endocytosed into organ-specific cells such as lung fibroblasts or liver Kupffer cells (Hoshino et al, 2015). Various S100 family proteins in these cells are then highly upregulated and secreted, leading to a pro-inflammatory supportive niche (Sakaguchi, 2017). Additionally, exosome-packaged RNA can activate Toll-like receptor 3 (TLR3) signaling in lung epithelial cells