Progression and metastasis of lung cancer

When tumor cells start to form nodules within the stroma, they need to communicate with the surrounding microenvironment, which is composed mainly by macrophages, fibroblasts/myofibroblasts, neutrophils, lymphocytes, and dendritic cells. To facilitate angiogenesis, tumor cells can either directly release angiogenic factors such as vascular endothelial growth factors (VEGFs) to directly stimulate the formation of new blood vessels, or tumor cells cooperate with macrophages, which can release angiogenic growth factors [6‐8]. A good example for angiogenesis induced by tumor cells is the vascular variant of squamous cell dysplasia, whereas in well-differentiated adenocarcinomas, angiogenesis seems to relay on cooperating macrophages [9‐12] (Figs. 1a, b and 2a). To understand the function of macrophages, it is necessary to briefly discuss the two different populations of macrophages, the M1 and M2 types. M1 macrophages are acting against tumor cell invasion by secreting interleukin 12 (IL-12), which function tumoricidal by an interaction with cytotoxic lymphocytes and NK cells. M2 macrophages produce IL-10, which promote tumor progression. The differentiation of naïve macrophages into either M1 or M2 types is facilitated by NOTCH, where low Notch via SOCS3 drives macrophages into M2 types [13]. M1 macrophages act proinflammatory, inactivate autophagy by production of radical oxygen species, and can also induce apoptosis of tumor cells [14‐16]. Notably, mutation and inactivation of Notch are found in neuroendocrine carcinomas, whereas activation in other non-small-cell carcinomas, which questions the function of this gene as either oncogene or tumor suppressor [17‐20]. Most probably different members of the Notch family proteins function differently in squamous cell, small cell, and adenocarcinomas and in addition, act differently during tumor development [21‐23].

Usually, tumor cells produce many modified proteins, which are recognized as foreign by dendritic cells and lymphocytes. Tumor cells are therefore attacked and destroyed by cytotoxic lymphocytes (CD8+). However, pulmonary carcinoma cells have developed different escape mechanisms to prevent this cytotoxic attack. By modulating the innate immune system, macrophages are preferentially forced to differentiate into M2 types as already explained. Another mechanism to protect tumor cell is to modify the pool of antigen presenting dendritic cells (DCs). Within dendritic cells, several functional quite opposite acting cell populations are discerned; conventional DC will present tumor antigens to T lymphocytes and force the production of cytotoxic T cells, whereas plasmocytoid and monocytoid DCs act as tumor protective cells. For example, Bombesin derived from SCLC inhibits IL-12 production by DC and their ability to activate T cells [48]. Tumor cells by secretion of TGF-β and prostaglandin E2 induce DCs to differentiate into regulatory DCs with a CD11c(low)CD11b(high) phenotype (also named plasmocytoid DC) and high expressions of IL-10, VEGF, and arginase I. These regulatory plasmocytoid DCs inhibited CD4+ T cell proliferation [49] and thus act protumorigenic. Another action to prevent cytotoxic lymphocyte attack is to induce an influx of regulatory T cells (Treg). Treg downregulate the production and influx of cytotoxic T cells and NK cells and promote immune tolerance [50‐52]. Finally, also bone marrow-derived myeloid precursor cells (MDSCs) may downregulate a T cell-based immune reaction towards growing tumor cells by secreting arginase I [53]. Indoleamine 2,3-dioxygenase (IDO) and IL-6 seem to play a regulatory role for these MDSCs, as downregulation of IDO resulted in reduced lung tumor burden and improved survival in experimental settings. Loss of IDO resulted in an impairment of protumorigenic MDSC, whereas IL-6 recovered both MDSC suppressor function and metastasis susceptibility. In addition, vascular density was significantly reduced in Ido1-nullizygous mice [54].

In some carcinomas, preferentially in pulmonary squamous cell carcinomas, eosinophils are found in abundance. The role eosinophilic granulocytes play in NSCLC is not fully understood. It may be that variants of IL-17 (IL-17E) induce a helper 2 type of immune response, which in turn by the release of IL-4 and IL-5 causes tissue eosinophilia. In one study, it was shown that IL-17E has antitumor activity. Injections of recombinant IL-17E resulted in significant antitumor activity. Combining IL-17E with chemotherapy increased the antitumor efficacy in a xenograft model [55]. If eosinophils are directly acting cytotoxic against the tumor cells, for example, by releasing cytotoxic basic proteins was not explored in this study.

After having established the primary tumor and organized nutrition as well as protection for immune cell attacks, the tumor cells have to acquire changes to migrate to distant sites and establish metastasis. There are two different forms how tumor cells migrate, single-cell or small-cell cluster movement as it is seen in small-cell carcinoma as well as undifferentiated NSCLC and movement by large clusters of organized cells such as in acinar adenocarcinoma or some cases of squamous cell carcinoma (Figs. 6a, b, 7a, b, and 8a, b). For single cell and small clusters, migration seems to be much easier since single cells can more easily adapt, for example, a spindle cell morphology, which enables better movement. Tumor cells during migration reduce or even abolish cytokeratin filaments and increase/or de novo express α-actin and vimentin; this is commonly seen in pleomorphic carcinomas (Fig. 5b), carcinosarcomas, high-grade squamous cell and adenocarcinomas, and SCLC. Lung adenocarcinomas with high smooth muscle actin gene ACTA2 expression showed significantly enhanced distant metastasis and unfavorable prognosis. ACTA2 downregulation remarkably impaired in vitro migration, invasion, clonogenicity, and transendothelial penetration of adenocarcinoma cells without affecting proliferation. ACTA2 upregulation in lung adenocarcinoma cells was also connected to expression of c-MET and focal adhesion kinase (FAK), whereas ACTA2 targeting by small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) resulted in loss of mesenchymal characteristics [56]. Migration within the stroma requires several changes in tumor cells, one is formation of invadopodia. Tyrosine kinase substrate 5 (Tks5) is a scaffolding protein necessary for the formation of invadopodia. There are different isoforms, some of them (short isoforms) associated with reduced other (long isoforms) increased metastasis [57‐59]. Expression of Tks5 together with the expression of α-actin is further regulated by cortactin and neural Wiskott-Aldrich syndrome protein (N-WASP), which also regulate the expression of metalloprotease membrane type 1 matrix metalloprotease (MMP14) [58]. However, migration of tumor cells seems to be regulated by different genes, so probably, there is not a single mechanism for each tumor type, but more likely that tumor cells individually have adapted different mechanisms of migration protocols and used it during carcinogenesis. As an example, myosin heavy chain 9 (MYH9) and Copine III (CPNE3) positively correlate with the migration and invasion properties of lung cancer cell lines. If CPNE3 was knocked down, the metastatic abilities were inhibited in a mouse model. Also, CPNE3 protein expression levels were positively correlated with the clinical stage in NSCLC [60]. In another study, nestin protein expression significantly correlated with tumor size and lymph node metastasis in NSCLC and also poor survival in patients with adenocarcinoma. Nestin inhibition by shRNA decreased proliferation, migration, invasion, and sphere formation in adenocarcinoma cells [61]. One of the major studied mechanisms of tumor cell migration is EMT, which again is seen in tumors with single-cell or small-cluster migration type (Fig. 9a–c).

When looking up studies on EMT, a huge amount of published article can be found in databases. The major surprise is that different genes are associated with EMT. And even when focusing on lung cancer studies, there are still different genes found to trigger EMT. Some of the most often found EMT-associated genes are Twist, Snail, and TGFβ1.

Suppression of Twist expression in metastatic mammary carcinoma cells inhibits their ability to metastasize from the mammary gland to the lung. Ectopic expression of Twist resulted in loss of E-cadherin-mediated adhesion, activation of mesenchymal markers, and induction of cell motility, suggesting that Twist promotes EMT [62]. In another study, Twist was selectively associated with EGFR-mutated adenocarcinomas. Twist expressed in lung adenocarcinoma cell lines with EGFR mutation showed increased cell mobility. A decrease of EGFR pathway through EGF retrieval or inhibition of Twist expression by small RNA reversed the phenomenon. These findings supported that Twist promotes EMT in EGFR-mutated lung adenocarcinoma [63]. In the study by Pirozzi, the focus was on TGFβ1. They used two epithelial cell lines which acquired a fibroblast-like appearance when treated by TGFβ1. By inhibiting TGFβ1, vimentin and CD90 were downregulated and cytokeratin, E-cadherin, and CD326 were upregulated. TGFβ1 also upregulated Slug, Twist, and β-catenin, thus confirming EMT. Interestingly, also some stem cell markers as Oct4, Nanog, Sox2, and CD133 were overexpressed too, linking EMT to tumor stem cells [64]. Adhesion plays a major role in EMT; therefore, not surprisingly, studies have focused on Wnt, catenin, and GSK3β pathway. Loss of SARI (suppressor of AP-1, also called BATF2) expression initiates EMT, causing repression of E-cadherin and upregulation of vimentin in lung adenocarcinoma cell lines and in human lung adenocarcinomas. By knockdown endogenous SARI in a human lung xenograft-mouse model, multiple lymph node metastases developed. SARI has been shown to regulate EMT by modulating the (GSK)-3beta-beta-catenin signaling pathway [65]. In the study by Blaukovitsch, another pathway for EMT was shown; Snail and Twist were not involved in pulmonary sarcomatoid carcinomas but instead upregulation of c-Jun and consecutive overexpressions of Vimentin and Fascin were seen [66].

When dissecting sites of metastasis, the way EMT is regulated get more diverse; PREP1 accumulation was found in a large number of human brain metastases of various solid tumors, including NSCLC. PREP1 induces the expression of multiple activator protein 1 components including Fos-related antigen 1 (FRA-1). FRA-1 and PBX1 are required for EMT triggered by PREP1 in lung tumor cells [67]. The study by Shen showed that increased levels of long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) promotes lung cancer brain metastasis by EMT, whereas silencing of MALAT1 inhibits lung cancer cell migration and metastasis in the brain [68]. So far, nothing similar was investigated for bone metastasis by pulmonary carcinomas.

So far, we have focused on single-cell and small-cluster migration. However, in surgical pathology routine, most well-differentiated carcinomas including lung carcinomas move in large cell clusters; for example, acinar adenocarcinomas will show nice structured acini deep within the stroma and even within blood vessels (Fig. 10a, b). The mechanisms how these tumor cells manage their coordinated movement by retaining their epithelial structure is almost unknown. These carcinomas do not undergo EMT. Recently, in an investigation using drosophila border cells as a model, the processes of migration of large-cell complexes were elucidated. By RNAi silencing, 360 conserved signaling transduction genes were knocked down to identify essential pathways for border cell migration. The following four genes associated with TGF-beta signaling were identified: Rack1 (receptor of activated C kinase), brk (brinker), mad (mother against dpp), and sax (saxophone). Inhibition of Src activity by Rack1 may be important for border cell migration and cluster cohesion maintenance. Although this study focused on signaling pathways involved in collective migration during embryogenesis and organogenesis, these data could be the first step in understanding migration of carcinoma complex cancer metastasis [69].

Tumor cells orient themselves along adhesion molecules as expressed by matrix proteins, but in addition, they also sense for oxygen and most probably also orient themselves for higher oxygen tension [70]. Invasion into blood vessels is very similar to invasion into the stroma. Tumor cells have already learned to degrade proteins of the basal lamina at the epithelial border, and similar proteins form the matrix of small blood vessels. Forming holes into the basal lamina of these blood vessels is therefore easily done, and tumor cells migrate into the intima. However, a new problem arises for tumor cells within the circulation, shear stress due to tumor cell deformation in small blood vessels and the problem with coagulation.

Shear stress is usually well tolerated by those tumor cells which underwent EMT. Tumor cells expressing Vimentin and α-actin can adapt to the capillary diameters, but cells still expressing cytokeratins might burst. This is one of the reasons why a majority of tumor cells do not survive within the circulation [71]. With respect to coagulation, tumor cells on one hand have to avoid being trapped within a blood clot but on the other hand, will need a clot to slow down the speed of the blood stream, attach to the clot, and use it for extravasation [72]. Clot formation might be induced by tissue factor being produced and released by macrophages. Impairment of macrophage function decreased tumor cell survival without altering clot formation, demonstrating that the recruitment of functional macrophages was essential for tumor cell survival [73]. Another way how tumor cells might trigger clot formation has been demonstrated in mucinous adenocarcinomas. Mucins secreted by the tumor cells induced platelet aggregation and furthermore interacted with L-selectin and platelet-derived P-selectin without thrombin generation [74]. This interaction already points to the next step, adherence to vascular walls for extravasation. Coming back to tumor cell trapping by blood clots, it seems that carcinoma cells require the assistance of macrophages and granulocytes for fibrinolysis. In a study of lung carcinomas, fibrinolytic components as tissue plasminogen activators (t-PA) and the inhibitors PAI-1 and PAI-2 were all negative in tumor cells, whereas urokinase-specific antibodies stained loosely packed tumor cells and macrophages. Both PAI-1 and PAI-2 were most prominently expressed within interstitial and alveolar macrophages [75]. In another study analyzing pulmonary adenocarcinomas, a positive correlation was found between Ets-1 and urokinase-type plasminogen activator (u-PA) expression [76].

Invasion into lymph vessels is easier than into blood vessels due to the tiny wall of the former. In addition, carcinoma cells might already enter the lymphatic stream by the interstitial channels of the lymph draining system. On the contrary, lymph vessels can easily be congested by tumor cells. This can reverse the lymph flow, which might explain unusual sites of lymph node metastasis and so-called skip lesions. In contrast to the situation within blood vessels, carcinoma cells in lymphatics have to deal with the immune system. So, survival is dependent on induction of immune cell escape mechanisms (see above).

Whereas carcinoma cells entering the blood stream might early onset distant metastasis and thus shorten overall survival of the patient [1], propagation of carcinoma cells along the lymphatics will set distant metastasis later. These tumor cells will set primarily metastasis within regional lymph nodes.

Angiogenesis at the metastatic site in one part follows the same principles as in the primary focus; however, there is one major problem. Whereas at the primary focus lung carcinoma cells cross-talk with stroma cells by mechanisms and transmitters which have been developed during the process of developing from the precursor lesion to in situ carcinoma to invasive carcinoma, this cross-talk is different in the new metastatic site. Brain glial cells or bone marrow stroma cells might response to other signals than those stroma cells within the lung. So, the major developmental step to establish a metastatic focus is communication with the stroma, and further, more communication might be different depending on the location. In one investigation, a bridge was built between angiogenesis at the primary and metastatic sites. CXCL12 was expressed in tumor cells and in tumor vessels; CXCR7 was expressed by tumor and endothelial cells in the primary tumor and in the brain metastasis. CXCR4 showed a nuclear positivity in all samples, but only CXCL12 expression in tumor endothelial cells was significantly correlated with shorter survival [92].

In interaction with stroma, there are no published data which could highlight general mechanisms by which lung carcinoma cells communicate with their stromal counterparts; however, communication at different organ sites have been studied and therefore will be discussed in the following paragraph.

When discussing metastasis, many questions arise, which are still incompletely answered. When does metastasis occur? Is there a need for a certain size of the primary tumor that cells leave and start migrating? Are tumor cells randomly moving out from the tumor or are these selective clones, and are these genetically different from the dominant clone? Do carcinoma cells move collectively or as single cells? These questions have been discussed extensively in the literature, but especially when comparing metastasis in lung cancer, several good examples are there to answer at least some of these questions.

Small-cell neuroendocrine carcinoma has some unique features. When looking at the invasion front, it is evident that this carcinoma prefers migration of single-cell and small-cell clusters composed of three to five cells (Figs. 6a, and 7a). In blood and lymphatic vessels, SCLC usually presents with single cells or small clusters of cells. Quite common is the finding of several large brain metastases and a very small primary tumor, which might even escape the detection by HRCT. This raises the question of early migration of carcinoma cells from the initial focus and setting metastasis early in the tumor development. In contrast, squamous cell carcinoma can form a large primary tumor, and when surgically removed has not formed metastasis; even some cases have not set regional lymph node metastasis. Migrating squamous cell carcinoma (SCC) often form large complexes of cells and when seen intravascular again present with large cell complexes. So, both extremes do occur in lung carcinomas. In adenocarcinomas, both types of migration and metastasis do occur, usually large migrating complexes of well-differentiated acinar or papillary adenocarcinomas (Fig. 11a), and small-cell clusters of solid or mucinous adenocarcinomas. The aspect of genetic heterogeneity in primary and metastatic tumor clones has been investigated in studies comparing primary and metastatic carcinomas. More importantly, it seems that not only primary carcinomas are different from metastasis but also metastases are different among each other. To be clear, driver mutations or general genetic aberrations in primary and secondary tumors are still identical, but additional genetic modifications arose within the metastases.

When looking up the frequency of metastasis of lung carcinomas, there are some preferential sites, as bone 34.3 %, lung 32.1 %, brain 28.4 %, adrenals 16.7 %, and liver 13.4 % [3]. We will discuss some of these, depending on the availability of research data.

Although lymph nodes are among the first metastatic foci of lung carcinomas, much less is known about specific molecular events in facilitating this colonization. Peng et al. studied the effect of hypoxia and chemokines. CCR7 expression correlated positively with HIF-1α and HIF-2α, and all together correlated with lymph node metastasis. It was shown that hypoxia induced HIF-1α and HIF-2α expression, which upregulated CCR7; inhibiting HIF-1α or HIF-2α resulted in decreased CCR7 expression and furthermore in inhibition of tumor cell migration and invasion [126]. However, it seems obvious that more than these three molecules are involved, which has been shown by a study on genetic aberrations. Gains at 7q36, 8p12, 10q22, and 12p12; loss at 4p14; and the homozygous deletions at 4q occurred significantly more frequent in SCC from patients with lymph node metastases only. Gains at 7q, 8p, and 10q were restricted to SCC with lymph node metastasis, and gain at 8q was restricted to patients with distant metastasis [127].

In summarizing our knowledge in the metastatic process in lung carcinomas, it can be stated that many mechanisms and involved genes/proteins have been identified, but the major breakthrough is still not achieved. The major problem is that the process of metastasis has so many steps that we still do not overlook the interactions of hypoxia, migration, EMT and MET, homing, interaction with stoma cells, and preparation of the metastatic niche, which probably occur not as a time sequence but more likely in parallel.

Finally, we will not discuss metastasis to the lung, although there are complementing factors hidden, which might help in understanding homing mechanisms, such as the exclusive metastasis of glioblastomas and meningosarcomas to the lung. But, this would too much expand this review.