The underappreciated role of resident epithelial cell populations in metastatic progression: contributions of the lung alveolar epithelium

Abstract

Metastatic cancer is difficult to treat and is responsible for the majority of cancer-related deaths. After cancer cells initiate metastasis and successfully seed a distant site, resident cells in the tissue play a key role in determining how metastatic progression develops. The lung is the second most frequent site of metastatic spread, and the primary site of metastasis within the lung is alveoli. The most abundant cell type in the alveolar niche is the epithelium. This review will examine the potential contributions of the alveolar epithelium to metastatic progression. It will also provide insight into other ways in which alveolar epithelial cells, acting as immune sentinels within the lung, may influence metastatic progression through their various interactions with cells in the surrounding microenvironment.

INTRODUCTION

Metastasis is the spread of cancer cells from the original tumor to distant organs. Metastases are routinely difficult to treat and are ultimately responsible for the majority of cancer-related deaths (1–3). The cascade of events that occur during metastasis is well defined and includes the sequential stages of invasion, intravasation, circulation, extravasation, and colonization (reviewed in Ref. 4). Dissemination of cancer cells occurs early on in tumor development (5), but only a very limited number of cancer cells effectively metastasize (6, 7). As described by Welch and Hurst (8) in their review defining the hallmarks of metastasis, cancer cells must possess four key features to successfully metastasize: 1) motility and invasion, 2) the ability to modulate the secondary site, 3) plasticity, and 4) the ability to colonize the secondary tissue. Metastatic progression, however, involves more than dissemination of cancer cells from the primary site and seeding of a distant organ. Several distinct stages in progression occur after cancer cells reach the metastatic site, such as quiescence, colonization, outgrowth, and even secondary dissemination from the metastatic site (Fig. 1). Bearing that in mind, resident cells at the metastatic site undoubtedly play a key role in determining how metastatic progression develops and distinctions such as whether a metastatic cell undergoes quiescence/dormancy or colonizes the lung can have a dramatic impact on patient survival.

Figure 1.

Metastatic progression. The well-known steps of the metastatic cascade define the early stages of metastatic progression involving dissemination and seeding of a distant site. Later stages of progression, including quiescence, colonization, and outgrowth, are distinct processes that occur within the metastatic site. The final stages of metastatic progression, which lead to terminal disease, include the outgrowth of macrometastases and/or secondary dissemination to other organs. It is critical that every stage of metastatic progression be considered when attempting to block or treat metastatic cancer. [Figure created with BioRender.com and published with permission.]

The lung is the second most frequent site of metastatic spread (9), and up to 54% of all cancer types, including breast and prostate, metastasize to the lung (10). Even when cancers metastasize to other organs initially, the lung is a common secondary metastatic site (11). Patients with lung metastases generally have poor overall survival. For instance, the median overall survival for patients with metastatic breast cancer is 2–5 years (12), but patients with lung metastases have substantially shortened survival with median survival dropping to just 13 mo (13). To effectively treat patients with lung metastases, it is critical that we understand the many and varied cellular interactions that occur in the metastatic microenvironment. Since the most abundant cell type in the lung is the epithelium, this review will examine the potential contributions of the alveolar epithelium to metastatic progression.

THE LUNG ALVEOLAR AIRSPACE

The primary site of metastasis within the lung is the parenchyma, the portion of the lung involved in gas exchange (14, 15). The parenchyma in the adult human lung comprises upward of 500 million alveoli, designed to maximize gas exchange by expanding the total surface area of the lung (16, 17). Alveoli are the terminal structures of the branched lung architecture and consist of four main components: 1) type I alveolar epithelial (AT1) cells, 2) type II alveolar epithelial (AT2) cells, 3) pulmonary surfactant, and 4) alveolar macrophages (AM) (Fig. 2).

Figure 2.

The alveolar airspace. Alveoli are the terminal structures of the branched lung architecture where gas exchange occurs through the close association with pulmonary vasculature. There are four main components within the lumen of the alveolar airspace: type I alveolar epithelial cells, type II alveolar epithelial cells, pulmonary surfactant, and alveolar macrophages. RBCs, red blood cells. [Figure created with BioRender.com and published with permission.]

Type I Alveolar Epithelial Cells

Epithelial cells within the alveolar niche are called pneumocytes. AT1 pneumocytes cover ∼95% of the interior of each alveolus (17). These thin cells, with few organelles, share a basement membrane with capillary endothelium and form tight junctions with surrounding cells to form a barrier to limit fluid infiltration into the alveolus (17). Although the primary role of AT1 cells is to perform gas exchange, which is enabled by their close proximity to pulmonary capillaries, they also assist with ion and fluid homeostasis and communicate directly with AT2 cells in response to pulmonary stretch and injury (17, 18).

Type II Alveolar Epithelial Cells

AT2 pneumocytes are large, cuboidal cells with microvilli on their apical surface (17). They are distinguished by large granular secretory bodies, called lamellar bodies, that occupy up to 25% of their cytoplasmic space (17). AT2 cells are commonly called the “defenders of the alveolus” (19, 20) and have three primary functions: 1) to facilitate AT1 gas exchange by secreting pulmonary surfactant, 2) to serve as the stem cells of the alveolar epithelium, a necessary component of wound resolution (20), and 3) to serve as the master regulators of host defense and repair within the lung by initiating and coordinating the repair process. This critical immunological coordination is achieved through direct and indirect interactions with cells in the microenvironment (20–22).

Pulmonary Surfactant

AT2 cells produce and secrete pulmonary surfactant, which is stored intracellularly in lamellar bodies (17). Surfactant is a lipid and protein conglomerate that reduces surface tension within the alveolus and prevents airway collapse during exhalation (23). Surfactant evenly covers the inner surface of alveoli and is composed of a lower aqueous hypophase and upper phospholipid phase. Once released into the hypophase, the contents of AT2 lamellar bodies transform into lattice-like tubular myelin to form the upper phospholipid layer (reviewed in Ref. 24). Surfactant is composed of 90% lipids, 80%–90% of which are phospholipids, and 10% proteins (25), including surfactant proteins (SP-) A, B, C, and D (26). Hydrophobic SP-B and SP-C are required for regulation of surface tension and are located within the upper phase of phospholipids. In contrast, hydrophilic SP-A and SP-D are present in the hypophase and are involved in modulating innate host immunity, another major function of pulmonary surfactant (26). SP-A and SP-D bind to the surfaces of various pathogens to segregate them from epithelial cell membranes and facilitate their elimination by AM (20).

Alveolar Macrophages

AM are located within the lumen of alveoli, directly exposed to air (27). AM are a resident cell population distinct from recruited macrophages, which arise from circulating monocytes, and those that reside between the airway epithelium, termed interstitial macrophages (reviewed in Refs. 27 and 28). AM populate the alveoli soon after birth and originate from yoke sac-derived fetal monocytes, a process completely independent of bone marrow-derived monocytes. AM are long-lived and replenished throughout life by self-renewal (29, 30). In fact, ∼40% of the AM population within the lung is replaced each year (31). AM phagocytose and destroy inhaled particles and pathogens as well as dead cells and irregularly formed surfactant (17, 27). AM activity increases during infection (17), and AM secrete proinflammatory cytokines to enhance pulmonary immune responses (20, 28).

THE ROLE OF ALVEOLAR EPITHELIAL CELLS IN METASTASIS

AT1 and AT2 cells are the most prominent cells within the alveolus and are, thus, closely associated with cancer cells during metastasis. Although further research is needed and much remains unknown, several initial studies indicate that alveolar epithelial cells may have a considerable impact on metastatic progression. The following discussion will enumerate our current understanding of how the alveolar epithelium contributes to different stages of metastatic progression (Fig. 3).

Figure 3.

The role of the lung alveolar epithelium in metastatic progression. Alveoli are the primary location of metastasis within the lung, and alveolar epithelial cells are the most prominent resident cells within the alveolar airspace. Recent studies indicate that interactions between metastatic and epithelial cells in the alveoli promote metastatic progression at several stages throughout this process (32–39). [Figure created with BioRender.com and published with permission.]

Preparation of the Premetastatic Niche and Seeding

A groundbreaking study in 2015 found that neutrophils promote breast cancer lung metastasis by preparing the metastatic niche for seeding. This study showed that neutrophils traffic to the lung before metastasis. Metastasis was significantly enhanced by neutrophil trafficking to the premetastatic niche and the recruitment of neutrophils to the lung was dependent on both the presence of a primary tumor as well as granulocyte colony-stimulating factor (G-CSF) signaling from the lung. These data suggested that primary cancer cells induce cells within the lung to recruit neutrophils (40). It was later discovered that primary tumor-derived exosomal RNA activates toll-like receptor 3 (TLR3) on AT2 cells, subsequently inducing secretion of chemokines that recruit neutrophils to the lung (32, 33). This process is reminiscent of what happens in the lung during inflammation and infection. TLRs recognize bacterial products, and TLR activation in AT2 cells induces chemokine secretion and recruitment of neutrophil to the lung (41–43). Similarly, primary tumor-derived exosomal microRNA can induce C-C motif chemokine ligand 2 (CCL2) expression and secretion in AT2 cells. Lung CCL2 serves as a chemoattractant for myeloid derived suppressor cells (MDSC), which then promote metastasis (34). Incidentally, these MDSC effects may be dependent on diminished levels of lysosomal acid lipase (LAL) in AT2 cells. LAL expression in AT2 cells downregulates the synthesis and secretion of tumor-promoting cytokines and decreases the recruitment and expansion of MDSC. Essentially, LAL^Hi AT2 cells, which are responsible for regulation of lung homeostasis and immune responses (44), prevent metastasis by limiting the number of MDSC present within the lung. What these data suggest is that primary tumor signaling to lung AT2 cells plays a significant role in determining whether disseminating tumor cells can effectively seed the lung.

Disseminated Tumor Cell Quiescence and Dormancy

When disseminated tumor cells enter the alveolus, they frequently come into direct contact with AT1 cells. A recent study found that the direct cell-cell interactions between AT1 and disseminated tumor cells stimulate tumor cell dormancy (35). Interestingly, this effect is predicated on the ability of cancer cells to form cellular protrusions that maximized their direct contact with AT1 cells. AT1 cells induce prosurvival and growth-restrictive gene expression in cancer cells that subsequently lead to quiescence. Mechanistically, AT1 cells suppress apoptosis and induced dormancy in cancer cells by activating signaling pathways associated with cancer dormancy, including epidermal growth factor receptor (EGFR) and Src family kinase (SFK) signaling. These effects were only observed in more indolent cancer cells, as opposed to more aggressive, highly proliferative cancer cells, which suggests that cancer cells themselves play a critical role in this reciprocal interaction (35). Another more recent study showed similar results, indicating that coculture with AT1 cells induces a transcriptional program in tumor cells that is associated with disseminated dormant cancer cells (36).

Metastatic Niche Formation and Colonization

Metastatic cells can alter the metastatic niche early in metastatic progression in ways that allow for subsequent metastatic colonization. In an effort to better understand and characterize changes to the early metastatic niche, Ombrato et al. (37) developed a unique labeling system that could assist in isolating cells directly adjacent to metastatic cells. In this system, mammary carcinoma cells secrete a soluble red fluorescent protein (RFP) that is taken up by surrounding cells in the lung. These RFP-positive cells could then be sorted out and compared with nonadjacent RFP-negative cells (37). Using this system, they found that early metastases induced a more stem-like phenotype in adjacent AT2 cells. These epithelial cells began to express progenitor markers and exhibit higher proliferative activity (37). Similar results were observed in a model of ovarian cancer pulmonary metastasis. In that study, alveolar epithelial cells adopted a stem-like lineage and began to express the pulmonary progenitor marker CD90 (38, 45). Together, these data indicate that tumor cells modify the surrounding metastatic niche by inducing stemness in adjacent AT2 cells to create a more tumor-permissive microenvironment. The metastasis-associated AT2 phenotype described in these studies is remarkably similar to what happens to AT2 cells during wound repair. To restore normal alveolar structure following injury, AT2 cells proliferate, enhance stemness, and transdifferentiate into AT1 cells to finalize wound resolution (46, 47). Thus, cancer cells may co-opt this AT2 wound repair process to facilitate metastatic progression.

Reversal of Disseminated Tumor Cell Dormancy

When disseminated tumor cells invade a distant metastatic site, the cells either undergo cell death or establish themselves within the tissue, termed seeding. At this stage, metastatic tumor cells can actively proliferate to colonize the site or they enter a dormant state of quiescence where they can survive for long periods of time (Fig. 1 and reviewed in Ref. 48). This state of dormancy is what accounts for patients presenting with metastatic disease years after their initial diagnosis. The reversal of tumor cell dormancy is an active area of research for cancer biologists and AT2 transdifferentiation may support this process. One study found that coculturing AT2 cells with cancer cells enhance transdifferentiation to AT1 cells, which subsequently promotes the proliferation of indolent breast cancer cells in a three-dimensional (3-D) culture model of dormancy. This interaction caused an upregulation of AT2 transforming growth factor β (TGF-β) secretion and snail family transcriptional repressor 2 (SNAI2) expression in cancer cells (39). Although it is unclear how to reconcile these results with the other AT1 cell data described earlier, future research may clarify the link between these disparate studies. In fact, the “reversal of dormancy” described in this study was recapitulated by culturing indolent cancer cells in conditioned media from AT2 cells stimulated with proinflammatory lipopolysaccharide (LPS), which suggests that it may be AT2 activation, rather than transdifferentiation, that induces cancer cell proliferation. It is also possible that additional characterization could discover whether the transdifferentiated phenotype described in this study is similar to the stem-like phenotype described earlier (37, 38). Ultimately, these disparate AT1 cell functions may not be mutually exclusive, but only context-dependent.

Metastatic Outgrowth

AT2 cells have also been shown to promote cancer cell proliferation. Ombrato et al. (37) used the niche labeling system described above to isolate AT2 cells and coculture them with cancer cells. Metastasis-adjacent AT2 cells promoted the growth of cancer cells in 3-D culture (37). Moreover, while looking at the growth-suppressive effects of AT1 cells, Montagner et al. (35) postulate that the AT2 cells emanate proliferative cues that AT1 cells block to induce dormancy. Although these studies are limited, they strongly suggest that AT2 cells may play a role in metastatic outgrowth as well.

ALVEOLAR EPITHELIAL CELL INTERACTIONS

Although the number of studies examining how alveolar epithelial cells contribute to metastasis is limited, numerous studies examine the interactions between alveolar epithelial cells and cells within the microenvironment. Every day we inhale more than 2,500 gallons of nonsterile air (49), which causes some distinct challenges for maintenance of homoeostasis within the lung. Mason (49) described the situation succinctly in his review on lung epithelial cells in which he states that “The alveolus must deal with low levels of inhaled organisms without inducing much of an inflammatory response [to avoid injury] but then must switch and produce a significant immune response once an overt infection is established.” The lung epithelium preserves homeostasis and is the first line of host defense against pathogens and inhaled particles (50). AT2 cells, in particular, are considered master regulators of immune responses within the alveolus (51), integrating signals from a variety of cells to coordinate responses to injury and infection (Fig. 4). Listed below are some of the primary ways in which AT2 cells influence adjacent cells in the lung microenvironment. This list includes cellular interactions compiled from studies investigating various lung conditions such as chronic disease, allergies, bacterial infection, inflammation, viral infection, and injury. Many of these studies were conducted using mouse models, and while the mouse lung is incredibly similar to the human lung, there are some subtle differences that may influence the translation of some of these results to humans (reviewed in Refs. 52 and 53). The extent to which AT2 cells interact with surrounding cells strongly suggests that similar interactions may occur during metastasis, especially considering that injury and inflammation often occur in the lung during metastasis.

Figure 4.

Type II alveolar epithelial (AT2) cells are the master regulators of immune responses within the alveolus. AT2 cells act as sentinels within the lung, integrating signals from surrounding immune and stromal cells in response to injury and disease. Through these interactions AT2 cells influence the behavior of these cells to coordinate immune responses. B, B cells; DCs, dendritic cells; EC, endothelial cells; FB, fibroblasts; Gran, granulocytes; ILC, innate lymphoid cells; Mφ, macrophages; NK, natural killer cells; T, T cells. [Figure created with BioRender.com and published with permission.]

Granulocytes

Granulocytes (Gran) are the most common type of immune cells in circulation (54). These relatively short-lived cells are distinguished by the presence of numerous cytoplasmic granules that release cytokines in response to immunogenic stimuli (55). Gran rapidly migrate from the blood into tissues following chemoattractant signals where they play important roles in clearing pathogens and regulating immune responses. There are three types of Gran (neutrophils, eosinophils, and basophils) with specialized biological functions (reviewed in Ref. 56). Neutrophils are the most abundant Gran, and they play a significant role in metastasis. Neutrophil plasticity means they can be both pro- or antimetastatic depending on the context and microenvironmental signals (reviewed in Ref. 57). For instance, the most established antitumor effects of neutrophils is direct killing through the production of reactive oxygen species (58), whereas secretion of matrix metalloproteinases (MMPs) by mature neutrophils is known to promote tumor progression (59). The body of literature characterizing the contributions of neutrophils and basophils to metastatic progression is expansive (reviewed in Refs. 60–62). Eosinophils, on the other hand, seem to be primarily antitumor (63). Interestingly, the lung has resident populations of both basophils and eosinophils with functions distinct from their circulating counterparts (64, 65), but more research is needed to fully characterize their functions in normal lung homeostasis and disease.

Interactions between AT2 cells and granulocyte populations have been relatively well studied. AT2 cells are a major source of neutrophil-stimulating cytokines within the lung. AT2 secretion of chemoattractants like C-X-C motif chemokine ligand 5 (CXCL5), granulocyte-macrophage colony-stimulating factor (GM-CSF), and G-CSF accelerate neutrophil recruitment to the lung (66–68). Direct interactions between AT2 cells and neutrophils also alter expression of surface receptor levels on neutrophils, including toll-like receptor 4 (TLR4) which is a principal regulator of neutrophil survival (69, 70). Similarly, secretion of macrophage inflammatory protein-1α (MIP-1α/CCL3) by infected AT2 cells serves as a strong inducer of eosinophil recruitment (71). There is even evidence that AT2 cells engulf apoptotic eosinophils, which is believed to contribute to resolution of inflammation (72, 73). Neutrophils have reciprocal effects on AT2 cells as well. Depending on the circumstances, neutrophils can promote AT2 proliferation (73), induce cytokine secretion (74), and trigger oxidant-independent cell death (75). How the extensive cross talk between these two cell types may contribute to metastatic progression needs to be explored in more detail and given the importance of neutrophils in seeding the lung, future studies exploring these interactions could lead to the discovery of effective antimetastatic treatment strategies.

Monocytes/Macrophages

Macrophages (Mφ) are innate immune cells that engulf dead cells and pathogens and secrete immune modulatory cytokines. There are many tissue resident Mφ populations that maintain homeostasis (reviewed in Ref. 76). Alternatively, during inflammation monocytes are recruited from the bone marrow to sites of injury or infection where they differentiate into Mφ (reviewed in Ref. 77). A key feature of Mφ is their incredible plasticity (78). Depending on the microenvironmental signals, Mφ can alter their phenotypes to appropriately respond to localized situations (reviewed in Ref. 79). In cancer, both tissue-resident and recruited Mφ can be anti- or protumor, and in their protumor state, they contribute to immune suppression and tumor progression. For example, antitumor Mφ secrete classic inflammatory cytokines like tumor necrosis factor α (TNF-α) and interleukin (IL)-12, whereas protumor Mφ secrete immune suppressive cytokines like IL-8 and IL-10 (reviewed in Ref. 80). The role of Mφ in metastasis has been extensively studied, and they facilitate metastasis at multiple stages throughout progression (reviewed in Ref. 81).

In the lung, AT2 cells interact with both resident AM and recruited monocytes/Mφ during injury and disease. To maintain homeostasis, AM and AT2 cells engage in a significant amount of cross talk (reviewed in Ref. 82). Secreted factors from bacterial-activated AT2 cells, tested using conditioned media, modulate AM activation and promote migration, phagocytosis, and polarization toward a protumor phenotype (83). Alternatively, incubation with AT2-derived surfactant proteins (SP-A and SP-D) suppresses Mφ phagocytic activity (84). AT2 cells also influence monocyte behavior. They produce the inflammatory modulator LL-37 that acts as a chemoattractant for monocytes (85, 86). Proinflammatory stimulated AT2 cells promote monocyte migration across the alveolar epithelial barrier in a baso-apical direction by secreting monocyte chemoattractant protein-1 (MCP-1) and CCL5 apically and upregulating intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1) integrin expression (87). These complex interactions are intricately regulated and given that Mφ plays such a critical role in metastasis, it seems probable that interactions between Mφ and alveolar epithelial cells contribute to metastatic progression. Future studies will determine to what extent.

Dendritic Cells

Dendritic cells (DCs) are myeloid-derived professional antigen-presenting immune cells that facilitate communication between the innate and adaptive immune systems (88). These phagocytic cells process antigens and present them to T and B lymphocytes to establish long-lasting adaptive immune responses (89, 90). In cancer, DCs promote metastasis through immune evasion (91). This process, termed “DC tolerization,” involves the reprogramming of DCs to block antitumor immune recognition (92). Reduced DC maturation and recruitment leads to dysfunctional lymphocyte activation (reviewed in Refs. 93 and 94). Although there are limited numbers of DCs present in the lung, they are highly sensitive to changes within the microenvironment (95). They reside beneath the epithelium in the airways and sample antigens on the luminal surface using their finger-like protrusions (96). Overall, DCs maintain homeostasis within the lung by fostering adaptive immunity in response to pathogenesis and tissue damage (reviewed in Ref. 89). Due to their close proximity, lung epithelial cells can profoundly affect DC localization and function. AT2-derived CCL20 promotes DC recruitment to the lung (97, 98). Similarly, AT2-derived GM-CSF induces DC recruitment to the lung in addition to promoting DC activation and migration to draining lymph nodes to present antigen to lymphocytes (99). Ultimately, AT2 cells modulate T cell proliferation and cytokine release through contact-dependent interactions with DCs (100). Given the wide-ranging effects that DCs have on downstream adaptive immunity, AT2-DC interactions certainly have the potential to affect metastatic progression.

Innate Lymphoid Cells

Innate lymphoid cells (ILCs) exist functionally at the intersection between innate and adaptive immunity (101). These lymphocytes, which lack antigen-specific receptors, sense changes in the microenvironment through cytokine receptors like innate immune cells. They then secrete a group of cytokines and inflammatory mediators similar to those secreted by T cells (101, 102). The primary role of ILCs is to protect epithelial barriers and maintain tissue homeostasis (103, 104). There are three types of ILC (ILC1, ILC2, and ILC3) that are distinguished by the signaling molecules they respond to and secrete (102). ILC2s are the most prevalent ILC population in the lung and are regarded to be a tissue-resident cell type (105, 106). ILC2s are responsive to lung injury and play a significant role in inflammation and tissue repair (reviewed in Ref. 107). ILCs have both pro- and antimetastatic functions, depending on the tissue and cancer type (reviewed in Ref. 108). One prominent study found that activated ILC2 in the lung promotes metastasis by suppressing the function of immune-mediated antitumor activity (109). ILCs in the lung are located in the alveolar airspace, in close proximity to epithelial cells (110). Several studies show that AT2 cells secrete stimulatory factors, such as IL-33, IL-25, and thymic stromal lymphopoietin (TSLP) (110, 111), which can activate ILC secretion of cytokines that support airway remodeling and motility (111–113). Future studies are needed to determine the direct link between AT2 cells, ILCs, and metastasis.

Natural Killer Cells

Natural killer (NK) cells are large granular lymphocytic innate immune cells with cytotoxic and cytokine-producing effector functions. They recognize cells in distress, with problems such as microbial infection or cancer, and induce autologous cell death. Receptor recognition ensures a balance between activating and inhibitory signals that make certain that healthy cells are tolerated, whereas stressed cells are destroyed (114, 115). NK cells play a critical role in lung pathogenesis (reviewed in Refs. 116 and 117). They also have a well-documented role in immunosurveillance during metastasis by targeting and directly killing disseminated tumor cells (reviewed in Ref. 118). Interestingly, recent data indicate that interactions with tumor cells may shift NK cells toward a more prometastatic phenotype (reviewed in Ref. 119). The lung has one of the highest frequencies of NK cells of any tissue in the body (120, 121), and NK cells are exclusively found within the parenchyma of the lung (122). During pathogenesis, lung epithelial cells begin to express stress signals, such as upregulation of natural killer group 2 member D (NKG2D) receptor ligand, that promote recruitment and activation of NK cells (123, 124). It remains unclear whether the effects of lung epithelial cells on NK cells could lead to a more pro- or antimetastatic NK phenotype within the metastatic microenvironment. Perhaps future studies will help elucidate the details of these interactions and fill this intriguing gap in knowledge.

T Cells

T lymphocytes have multiple, sometimes contradictory, roles in immunity, depending on the type of T cells being considered. There are three main T cell types that are activated upon antigen recognition: helper (Th), cytotoxic (Tc), and regulatory (Treg) T cell. CD4+ Th cells secrete cytokines that enhance immune responses and CD8+ Tc kill target cells by inducing cell death, whereas CD4+ Tregs suppress other T cell functions to dampen the immune system. T cells recognize antigens presented by cells on major histocompatibility complex (MHC) molecules. Many cells within the body present intracellular antigens on MHC class I molecules (MHC-I) to CD8+ Tc cells. However, in general, only professional antigen-presenting cells (Mφ, DCs, and B cells) express MHC class II molecules (MHC-II) to CD4+ cells (125, 126). Similarly, based on their alternative functions, CD4+ Th and CD8+ Tc are considered antimetastatic, whereas CD4+ Treg are prometastatic (127). There is currently a significant amount of research engaged in manipulating T cell populations toward antitumor phenotypes to facilitate patient survival (128).

T cells play a significant role in lung immunity (reviewed in Ref. 129), and AT2 cells activated by pathogens secrete factors that recruit T lymphocytes to the lung (130). T cells also interact directly with the lung epithelium. Although epithelial cell types are not traditionally considered immunogenic, AT2 cells, unlike AT1 cells, constitutively express MHC-II (131, 132), and several studies have established that AT2 cells present antigens via MHC-II to CD4+ T cells (133, 134). What remains unclear is how exactly AT2 cell antigen presentation affects CD4+ function given the somewhat inconsistent data. AT2 cells do not express the classic CD4+ T cell costimulatory molecules CD80 and CD86 (131), which has caused some researchers to postulate that AT2-CD4+ T cell interactions cause T cell tolerance (135). However, other studies identified alternative T cell costimulatory molecules, like CD54, CD58, and TGF-β, that are highly expressed in AT2 cells (131, 136). What is clear is that AT2 antigen presentation is limited and causes relatively poor CD4+ T cell stimulation, when compared with professional antigen-presenting cells (135, 137). Physiologically this means that AT2 cells are able to activate a constrained immune response without causing additional damage to the lung (137). Most relevant to metastasis are several studies that found that AT2 antigen presentation stimulated T cell activation. AT2-T cell activation subsequently caused altered T cell localization, accompanied by loss of barrier immunity (138), and induction of Tregs (134), which could promote metastatic seeding and outgrowth, respectively. Based on these data, there is a high likelihood that AT2 activation during metastasis could subsequently, in a reciprocal manner, promote metastatic outgrowth through acquisition of suppressive CD4+ T cell phenotypes.

B Cells

B cells are probably best known for their roles in antibody production and long-term immunity. They are the principal regulators of the antibody-mediated adaptive immune response (139, 140). In cancer, regulatory B cells (Breg) are known to promote tumor growth and metastasis by suppressing antitumor immune responses (reviewed in Refs. 141 and 142). In the tumor microenvironment, these cells suppress effector T and NK cell function, promote the generation of Treg, and educate MDSCs (143, 144). B cells are also the most common lymphocytic cell population in the lung (145), and activation of airway epithelial cells leads to epithelial secretion of BAFF (B cell activating factor of the TNF family, TNFSF13B) (146). BAFF is known to stimulate B cell maturation, proliferation, and promote immunoglobulin A (IgA) secretion, a known B cell mechanism of immune suppression (147–149). Although more research is needed, B cell stimulation may be another way in which alveolar epithelial cells facilitate metastatic progression by dampening immune responses within the lung.

Fibroblasts

Fibroblasts (FBs) are mesenchymal cells known for their plasticity. They are the most common cells in connective tissue and are responsible for synthesizing and maintaining the extracellular matrix (ECM) (150). FBs, within both the tumor and metastatic microenvironments, promote cancer development and metastatic progression in several ways, such as establishing premetastatic niches, promoting angiogenesis, and modulating immune responses (reviewed in Refs. 151 and 152). Interstitial FBs are the resident population within the lung (153, 154). AT2 cells and FB communicate extensively during lung development (reviewed in Ref. 155). In alveoli, fibroblasts directly link AT2 cells to capillary endothelial cells through gaps in the basement membrane, forming a bridge from the vasculature to the airway lumen for immune cells (156). In coculture and conditioned media experiments, AT2 cells induce FB collagen type I secretion (157), reduce adhesion, increase aggregation (158), inhibit proliferation (159), and block differentiation (160). These cell-cell interactions also have effects on AT2 cells. For instance, FBs, depending on the circumstances, can promote AT2 proliferation and activate SP-A gene expression (157, 159), both of which could have considerable repercussions for metastatic progression.

Endothelial Cells

Endothelial cells (ECs) form the lining of blood vessels throughout the body and control the passage of materials from circulation into tissues. Mature endothelial cells are long-lived but also retain the ability to proliferate and migrate allowing for rapid repair of damage to vessels and the creation of new vasculature (161). A major step in metastasis is the intravasation into and extravasation out of blood vessels, which allows tumor cells to disseminate to distant organs. Although endothelial cells can act as a barrier to metastasis, they can also promote metastatic progression. Signaling from tumor cells dramatically alters the phenotypes of adjacent endothelial cells (reviewed in Ref. 162). One example of this involves secretion of cancer-derived exosomes that induce full body vascular permeability, which ultimately facilitates disseminating tumor cell transendothelial migration during metastasis (163). Pulmonary ECs line the vasculature of the alveolus and interact directly with AT1 cells to form a thin, gas-diffusible surface to facilitate gas exchange and cell trafficking (reviewed in Ref. 164). Based on their close proximity, the cross talk between alveolar epithelial and pulmonary ECs is extensive (reviewed in Refs. 165 and 166). Certain outcomes of these interactions may have relevance to cancer metastasis. For instance, AT2 cells can modulate vascular permeability to maintain the alveolar-capillary/air-blood barrier (167). During sepsis, when vasculature becomes leaky, AT2 cells secrete lipid-related factors to decrease permeability (168). Similarly, alveolar epithelial cells upregulate endothelial growth factor expression during injury (20, 169). In a reciprocal manner, pulmonary EC dysfunction can cause abnormal epithelial differentiation (170). Although more research is needed to determine how these interactions may influence metastatic progression, the field is ripe for investigation. Several cell culture models have been created to mimic the alveolar-capillary barrier (171, 172), and a recently developed imaging technique, using 3-D optical imaging/computed tomography (OI/CT), can image alveolar-capillary barrier permeability in real-time in live mice (173).

AT1 Cell Interactions

Although AT2 cells are the primary alveolar epithelial cells that interact with stromal cells in the microenvironment, AT1 cells can influence stromal cell behavior as well. A primary way that AT1 cells affect the microenvironment is by impacting AT2 cells directly. AT1 cells can inhibit AT2 proliferation in a contact-dependent manner (174). Beyond that AT1 cells can directly mediate innate immune responses during infection by secreting CXCL5 (175), which plays a major role in immune cell chemotaxis (176). More specifically, AT1 cells contribute to neutrophil transepithelial migration (177), inhibit proliferation of fibroblasts (178), and promote alveolar angiogenesis through vascular endothelial growth factor A (VEGFA) expression (179). These studies, while limited, suggest that AT1 cells may have a greater impact on alveolar homeostasis than previously believed. Likewise, they may have a greater-than-expected role in metastatic progression.

DISCUSSION

Research on AT2 cells has escalated dramatically in the past few years due to the fact that AT2 cells are the main targets of SAR-CoV-2 (COVID-19) viral infection. Given this, it is likely that interest in the role of AT2 cells in other contexts, such as cancer, will increase in coming years. Having a better understanding of how alveolar epithelial cells respond to and affect metastases could dramatically impact patient survival by addressing some of the current challenges in managing metastatic disease, such as developing more effective treatment strategies (180). Based on some of the similarities observed in alveolar epithelial cell responses to disease/injury and metastasis, therapies currently approved for common lung conditions may be viable therapies in the treatment of patients with lung metastases. Furthermore, more research is needed to determine how currently approved therapies for the treatment of lung metastasis, such as chemotherapy, affect resident cells within metastatic tissues. It is certainly possible that while current treatments have antitumor effects on metastatic cells, these drugs could have dramatically different effects on resident cells that could subsequently lead to treatment failure or recurrence. Another topic that should be considered is how aging may affect how AT2 cells interact with different cell populations within the alveolar niche, especially considering that the majority of cancers occur in aged individuals. It is increasingly accepted that aging causes tissue dysfunction that is significantly relevant to cancer development (reviewed in Ref. 181). Less is known regarding how aging affects metastasis, but since aging impairs AT2 cell function (182), lung metastatic progression could be significantly affected by patient age as well. Ultimately, published data strongly suggest that alveolar epithelial cells play a role in metastatic progression. Future research will help to determine the mechanisms underlying these effects, and targeting the interactions between resident lung cells, immune cells, and tumor metastases could have dramatic effects on metastatic progression and patient survival.

GRANTS

This work was supported by an ACS IRG No. 16-184-56 from the American Cancer Society to the University of Colorado Cancer Center, a METAvivor Early Career Investigator Award No. 25B0307 (to J.L.C.), and National Cancer Institute Grants T32 CA190216-01A1 (to J.L.C.) and F32 CA239436 (to M.M.W.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.L.C. prepared figures; J.L.C. drafted manuscript; J.L.C., M.M.W., and J.K.R. edited and revised manuscript; J.L.C., M.M.W., and J.K.R. approved final version of manuscript.