Invasion and Metastasis

Andy J. Minn

Joan Massagué

Many of the gains in our understanding of the genetics and molecular mechanisms of cancer have been driven by the quest to understand characteristic anatomic and cellular traits of the disease. Pathologists have long observed that cancer seemingly can evolve from hyperplasia through a series of increasingly disorganized and invasiveappearing tumors that can then colonize distant organs in a nonrandom fashion. This spread of cancer from the organ of origin (primary site) to distant tissues is called metastasis. Much of the complex knowledge that has been acquired about cancer biology has been from a reductionistic approach that has focused on the inner workings of cancer cells with limited regard to interactions with the microenvironment and host biology. Although our understanding about cell proliferation, cell death, genomic instability, and signal transduction pathways has rapidly progressed, detailed understanding about the molecular mechanisms of metastasis has lagged considerably behind.

Inherent difficulties in studying metastasis have been due to technological limitations in analyzing a complex in vivo process rich with heterotypic interactions. Invasion, survival in the circulation, and growth in distant organs are not amenable to methods that primarily use in vitro models. Despite technical challenges, elegant experiments that started in the 1950s were done with mouse xenograft models and resulted in an important descriptive understanding of the biology of metastasis. With the accumulation of knowledge from studying cancer cells in isolation, subsequent advances in metastasis built on the classic studies. Unfortunately, metastasis remains responsible for the vast majority of cancer-related morbidity and mortality. Therefore, advancing our scientific and clinical understanding of metastasis is a high priority. In this chapter, we will first review the classic paradigm of cancer metastasis and then describe recent advances that are starting to better characterize metastasis on the molecular, cellular, and organismal level.

THE EVOLUTION AND PATHOGENESIS OF METASTASIS

Somatic Evolution of Cancer

Hyperplastic and dysplastic lesions need not always progress to cancer, but when they do, it can take years if not decades for this to occur. This protracted course to malignancy is consistent with epidemiologic studies that show an age-dependent increase in the incidence of cancer.¹ Mathematically, this precipitous rise can be explained by the accumulation of many stochastic events. These ideas have contributed to the widely accepted view that cancer requires several genetic alterations during a course of somatic evolution. However, the mutation frequency of human cells is thought to be too low to explain the high prevalence of the disease if so many stochastic genetic alterations are needed. To account for this disparity, cancer cells are widely believed to have a “mutator phenotype,” a concept with much experimental support.²

Driven by increased mutability of the genome, the dynamics of tumor progression depend on mutation, selection, and tissue organization.³ Mutations can result in activation of oncogenes or loss of tumor suppressor genes that increase fitness and cell autonomy. To oppose the accumulation of cells with tumorigenic mutations, tissue architecture often limits the spread of mutant cells that have reached fixation. For example, large compartments containing many cells accumulate advantageous mutations more rapidly than smaller compartments. Similarly, if there are only a limited number of precursor cells that have self-renewal capabilities (stem cells), this also has the effect of reducing the risk of enriching for tumorigenic mutations. However, despite the sequential mutations and steps predicted by the somatic evolution of cancer, the nature and/or sequence of genes that are altered during this evolution are mostly unknown.

Clinical, Pathologic, and Anatomic Correlations

Metastasis is often associated with several clinical and pathologic characteristics. Among these, tumor size and regional lymph node involvement are consistently associated with distant relapse. For tumor size, no clear threshold exists but trends are clear. For example, metastatic risk for breast cancer rises sharply after 2 cm,⁴ while distant metastasis in sarcoma is more common for tumor sizes larger than 5 cm.⁵ The involvement of regional lymph nodes is often, but not always, a harbinger for increased risk of distant metastasis. For head and neck cancer, the association between lymph node involvement and metastasis is predictable. Metastasis rarely occurs without prior involvement of cervical neck lymph nodes, and the lower down in the neck nodal involvement occurs, the more likely distant metastasis becomes.⁶ For breast cancer, the presence of positive lymph nodes is the strongest clinicopathologic prognostic marker for distant relapse. Like head and neck cancer, the extent of nodal involvement is telling, as a precipitous rise in metastatic risk is observed for patients with more than four axillary lymph nodes.⁴ However, lymph node metastasis does not always precede distant relapse. In sarcomas, for example, metastasis is often seen in the absence of nodal disease.⁵

When tumor cells appear to have aggressive traits on microscopic analysis, this often translates into increased risk for distant disease. Although many histopathologic traits for different cancer types have been reported to associate with poor prognosis, there are several that consistently appear to track with metastatic risk across various tumor types. These traits include: (1) Tumor grade. Tumors that are poorly differentiated, or retain few features of their normal tissue counterparts, are generally considered to be high grade. High-grade tumors often exhibit infiltrative rather than pushing borders and show signs of rapid cell division. Breast cancer and sarcomas are well recognized for displaying a markedly elevated risk of metastasis with higher tumor grade. (2) Depth of invasion beyond normal tissue compartmental boundaries. Some cancers like melanoma and gastrointestinal malignancies are staged by how deeply they extend beyond the basement membrane. Violation of deeper layers of the dermis, or invasion through the lamina propria, muscularis mucosa, and serosa, represent progressively more extensive invasion and higher risk of metastasis. (3) Lymphovascular invasion. Tumor emboli seen in the blood or lymphatic vessels generally carry a poorer prognosis than cancer without these features. Breast cancer and squamous cell cancers of the head and neck or female cervix are examples.

Tissue Tropism and the Seed and Soil Hypothesis

Despite apparent similarities in clinical and/or histologic features, different cancer types do not exhibit the same proclivity to metastasize to the same organs, and the same cancer type can preferentially metastasize to different organs (Table 10.1). This tissue tropism has long been recognized and has intrigued clinicians and pathologists to seek an explanation. In 1889, Stephen Paget proposed his “seed and soil” hypothesis (reviewed in ref. 7). This stated that the propensity of different cancers to form metastases in specific organs was due to the dependence of the seed (the cancer) on the soil (the distant organ). In contrast, James Ewing and others argued that tissue tropism could be accounted for based on mechanical factors and circulatory patterns of the primary tumor. For example, colorectal cancer can enter the hepaticportal system, explaining its propensity for liver metastasis, and prostate cancer can traverse a presacral plexus that connects the periprostatic and vertebral veins, explaining its propensity for metastases to the lower spine and pelvis. Supporting the arguments for both views, current understanding would suggest that both seed and soil factors and anatomic (“plumbing”) considerations contribute to metastatic tropism. A modern interpretation of the seed and soil hypothesis is an active area of investigation, with molecular definitions accumulating for both the cancer and the microenvironment.

Basic Steps in the Metastatic Cascade

From clinical, anatomic, and pathologic observations of metastasis, a picture of the steps involved in a metastatic cascade emerges. Numerous prerequisites and steps can be envisioned.

Invasion and motility. Normal tissue requires proper adhesions with basement membrane and/or neighboring cells to signal to each other that proper tissue compartment size and homeostasis is being maintained. Tumor cells display diminished cellular adhesion, allowing them to become motile, a fundamental property of metastatic cells. Tumor cells use their migratory and invasive properties in order to burrow through surrounding extracellular stroma and to gain entry into blood vessels and lymphatics.

Intravasation and survival in the circulation. Once tumor cells enter the circulation, or intravasate, they must be able to withstand the physical shear forces and the hostility of sentinel immune cells. Solid tumors are not accustomed to surviving as single cells without attachments
and often interact with each other or blood elements to form intravascular tumor emboli.

TABLE 10.1 STEREOTYPIC PATTERNS OF METASTASIS TO DISTANT ORGANS BY CANCER TYPE

Cancer Type	Site of Metastasis
Breast carcinomas	Primarily bone, lung, pleura and liver; less frequently brain and adrenal. ER-positive tumors preferentially spread to bone; ER-negative tumors metastasize more aggressively to visceral organs.
Lung cancers	The two most common types of lung cancer have different etiologies. SCLC disseminates rapidly to many organs including the liver, brain, adrenals, pancreas, contralateral lung, and bone. NSCLC often spreads to the contralateral lung and the brain, and also to adrenal glands, liver, and bones.
Prostate carcinoma	Almost exclusively to bone; forms osteoblastic lesions filling the marrow cavity with mineralized osseous matrix, unlike the osteolytic metastasis caused by breast cancer.
Pancreatic cancer	Aggressive spread to the liver, lungs, and surrounding viscera.
Colon cancer	The portal circulation pattern favors dissemination to the liver and peritoneal cavity, but metastasis also occurs in the lungs.
Ovarian carcinoma	Local spread in the peritoneal cavity.
Sarcomas	Various types of sarcoma; mesenchymal origin; mainly metastasize to the lungs.
Myeloma	Hematologic malignancy of the bone marrow that causes osteolytic bone lesions, sometimes spreading to other organs.
Glioma	These brain tumors display little propensity for distance organ metastasis, despite aggressively invading the central nervous system.
Neuroblastoma	Pediatric tumors arising from nervous tissue of the adrenal gland. Forms bone, liver, and lung metastases, which spontaneously regress in some cases.
ER, estrogen receptor; SCLC, small cell lung cancer; NSCLC, non-small cell lung carcinoma.

Arrest and extravasation. Once arrested in the capillary system of distant organs, tumor cells must extravasate, or exit the circulation, into foreign parenchyma.
Growth in distant organs. Successful adaptation to the new microenvironment results in sustained growth. Of all the steps in the metastatic cascade, the ability to grow in distant organs has the greatest clinical impact and lies at the core of the seed and soil hypothesis. Accomplishing this step may be rate-limiting and may determine whether distant relapse occurs rapidly or dormancy ensues.

Heterogeneity in Cancer Metastasis and Rarity of Metastatic Cells

Because numerous sequential steps are needed for metastasis, multiple genetic changes are envisioned. A failure in any step would prevent metastasis altogether. Accordingly, tumor cells that can accumulate a full complement of needed alterations to endow them with metastatic ability should be rare. These ideas are supported by early experiments. Work by Fidler and colleagues⁷ showed that subpopulations of tumor cells exist that display significant variation in their metastatic ability and metastatic lesions likely arose from single progenitor cells. Early cell fate studies revealed that less than 0.01% of tumor cells gave rise to metastases. More recent studies using in vivo video microscopy to visualize and quantitate cell fate confirmed that metastasis is an inefficient process (reviewed in ref. 8). Thus, important early studies helped to establish the idea that primary tumors are heterogeneous in their metastatic ability and that tumor cells that can successfully metastasize are exceedingly rare.

The Traditional Progression Model for Metastasis and Its Implications

A synthesis of clinical observation, deduced steps in the metastatic cascade, and early studies of experimental metastasis in mice led to a traditional model for metastatic progression.⁷ In this view, primary tumor cells undergo somatic evolution and accumulate genetic changes. Because numerous steps are required for metastasis, the number of genetic changes that are needed for full metastatic competency is large; hence, tumor cells that have acquired these changes are rare. Many clinicopathologic traits such as lymphovascular invasion and regional lymph node involvement represent successful completion of some of the steps in the metastatic cascade but not necessarily all. The clinical observation that metastatic risk increases with tumor size is explained
by mathematical considerations predicting that genetic changes accumulate faster with increased population size. Larger tumors are more likely to contain rare cells that are metastatically competent, making metastasis a late event in tumorigenesis.

One of the primary objectives in the clinical management of cancer is to prevent or decrease the risk of metastasis. How this objective is approached is shaped by empiricism and perceptions about how metastasis proceeds. The idea that metastasis occurs as a late event in tumorigenesis argues that early detection and early eradication of the primary tumor will prevent metastasis and be sufficient for cure. Screening programs, radical versus more limited surgical excisions, and the use of adjuvant radiation to the surgical bed can be justified based on the idea that cancers caught early have not likely spread. Metastatic heterogeneity within the primary tumor and the rarity of tumor cells that can complete all the sequential steps in the metastatic cascade suggest that the detection of tumor cells caught in the act of undergoing an early step in the cascade may still represent an opportunity to stop metastasis in its tracks. This is a rationale for oncologic surgeries that include regional lymph node dissections and the use of regional radiation therapy. The likely emergence of rare metastatic cells late during tumorigenesis provides reason to add adjuvant systemic chemotherapy after local treatment of larger and more advanced primary tumors rather than smaller tumors with less aggressive features.

Alternative Models

Although the traditional model for metastasis has enjoyed favor, alternative models have been proposed. The clinical data for breast cancer has inspired a long-standing debate on whether metastasis follows a traditional progression model or a predetermination paradigm—also known as the Halsted model versus the Fisher model (discussed in ref. 9) for metastasis.⁹ Both models seek to justify and explain clinical data looking at the benefit of aggressive local treatment of the primary tumor and draining lymph nodes versus the early use of adjuvant systemic chemotherapy. Although more anatomic than cellular in nature, the Halsted model looked at breast cancer from a traditional vantage point and imposed on it an orderly anatomic spread pattern from primary site, to regional lymph nodes, to distant organs. This orderly progression would make complete eradication of the primary and regional tumor burden sufficient to stop metastasis. In contrast, Fisher hypothesized that whether distant relapse occurs in breast cancer is predetermined from the onset of tumorigenesis (discussed in ref. 9). This view emphasizes breast cancer as a systemic disease for those tumors so fated and the importance of adjuvant systemic chemotherapy. The data from randomized trials for adjuvant treatment and from breast cancer screening programs do not clearly rule-out one model or the other.¹⁰ To reconcile the clinical data, Hellman⁹ proposed that breast cancer is best considered a spectrum of diseases bound by predetermination models and traditional progression models. Other models that conceptually differ from the traditional progression model include the clonal dominance model¹¹ and the dynamic heterogeneity model.¹²

Compatibility of Metastasis Models with Somatic Evolution

Both alternative and traditional progression models alike need to be compatible with the paradigm of somatic evolution, which presents a potential problem. Because it is not obvious why metastasis genes that promote growth at a distant site should have a fitness advantage for a primary tumor, the likelihood that multiple metastasis-specific genes will become fixed in a primary tumor would seem unlikely. To reconcile this, it has been suggested that the genes selected to drive primary tumor formation and progression are also the genes that mediate metastasis.¹³ This notion would imply that metastasis is a predetermined property of primary tumors that principally depends on the history of oncogenes and tumor suppressor genes that the primary tumor acquires. Such early onset of metastatic ability could explain phenomenon like cancers of unknown primary and support earlier predetermination metastasis models for breast cancer. However, as previously mentioned, predetermination models are not always consistent with clinical data, in particular the ability of screening and early detection to decrease cancer mortality. Furthermore, the phenomenon of metastatic dormancy, whereby metastasis remains inactive and undetectable for years if not decades after treatment of the primary tumor, is difficult to explain unless further metastasis-promoting changes occur after the primary tumor has been removed.

AN INTEGRATED MODEL FOR METASTASIS

Different concepts on how metastasis progresses have individual merits and limitations. A clearer understanding of metastasis requires sophisticated insight on a molecular level. Recent advances in the field of metastasis research are beginning to bring together an integrated and more complex paradigm (Fig. 10.1) whereby elements from different models may be interconnected.¹⁴ At the heart of this integrated paradigm are the principles of somatic evolution. Somatic evolution selects for functions and not directly for specific genes. Therefore, during primary tumor growth, the principal functions that are selected are tumorigenic functions that can be met by a large repertoire of oncogenic mutations. Examples of these tumorigenic functions include proliferative and metabolic autonomy, self-renewal ability, resistance to cell death, resistance to inhibitory signals, immune evasion, motility, invasion, and angiogenesis. Most of these traits were enumerated by Hanahan and Weinberg¹⁵ as being hallmarks of cancer. Many of these tumorigenic functions allow transformed cells to attract supporting stroma and migrate and invade surrounding tissue, regardless of whether or not cells reside in the primary tumor. This subset of tumorigenic functions is a prerequisite for metastasis because such functions are needed for cells to invade, penetrate blood vessels, and give rise to circulating tumor cells. These functions are shared by primary tumors and metastasis and are defined as metastasis initiation functions. A prominent example includes epithelial-to-mesenchymal transition (EMT).

FIGURE 10.1 Selective pressures and steps from primary tumor growth to metastasis. Selective pressures at the primary tumor (labeled in magenta) can determine metastatic potential. Cancer is initiated by oncogenic changes. Of particular relevance to metastatic potential may be self-renewal pathways and the need to overcome apoptosis and senescence. Hypoxia and inflammation have important roles and lead to tumor cells co-opting bone marrow-derived cells (BMDCs), myeloid-derived suppressor cells (MDSCs), and mesenchymal stem cells (MSCs), to name a few. These cells and the cytokines that they produce enhance the ability of the tumor to migrate, invade, overcome hypoxia, and maintain an immunosuppressed environment. The ability of primary tumor cells to undergo an epithelial-to-mesenchymal transition (EMT) is also influenced by the selective pressures faced during primary tumor growth. EMT results in migration, invasion, and intravasation. Such functions (labeled in red) are examples of metastasis initiation functions. Although non-EMT-related migration and invasion can also lead to intravasation, EMT is likely a principle means by which circulating tumor cells (CTCs) are promoted. Further steps in the metastatic cascade are shown by green arrows, with the size of the arrow representing the likelihood that the step is successfully completed for many cancer types (e.g., breast cancer). Selective pressures encountered from the local microenvironment shape metastatic proclivity by selecting for tumorigenic functions that secondarily help cells navigate the metastatic cascade (metastasis-progression functions). Hypoxia and inflammation-related events contribute to metastasis-progression functions (labeled in purple) by enhancing the ability of CTCs to survive in the circulation, extravasate, and form a premetastatic niche. CTCs can either self-seed the primary tumor, which results in augmentation of tumor mass and further selection of metastatic traits, or extravasate into distant organs. Although a premetastatic niche facilitates adaptation to the foreign microenvironment of distant organs, further selection is needed for full colonization. This results in a period of dormancy whereby micrometastases remain quiescent or growth is counterbalanced by apoptosis and the lack of angiogenesis. Further somatic evolution within the distant organ can eventually result in selection of macrometastatic-colonization functions (labeled in blue) and organ-specific growth. On the bottom of the figure are examples of specific genes that play a role in metastasis initiation, metastasis progression, and macrometastatic colonization.

It is evident how genes with tumorigenic functions and genes with metastasis initiation functions can be selected for during primary tumor growth. However, how are metastasis-specific functions (i.e. functions that are not characteristic of general tumorigenesis) selected during growth at the primary site? Metastasis-specific functions include survival in the circulation, extravasation, survival in the microenvironment of distant organs, and organ-specific colonization. Recent experimental evidence reveals that some genes can mediate tumorigenic functions and secondarily serve metastasis-specific functions either in a general way or with particular organ selectivity.¹⁶,¹⁷ These types of functions are called metastasis-progression functions and genes with this duality are defined as metastasis-progression genes. Metastasis-progression genes form the basis for predetermination models for metastasis. When metastasis-progression genes are selected for, their expression by the primary tumor will track with increased risk of metastasis. These genes will also mechanistically couple certain traits of primary tumor progression (e.g., rapid growth, invasiveness, resistance to hypoxia) with distant spread.

Cancer cells that have acquired metastasis-progression genes can undergo additional selective pressure during life away from the primary tumor. Functionally, genes selected by the pressures of a distant site are similar to metastasis-progression genes but they are not coupled to tumorigenic genes and so confer no advantage to a primary tumor. Therefore, altered expression of these genes would be rare or absent in the primary tumor and discernible only in the metastatic lesion. These genes are called macrometastatic-colonization genes and provide macrometastatic-colonization functions. Macrometastatic-colonization genes form the basis of traditional progression models for metastasis.

In this integrated view that stratifies genes into tumorigenic, metastasis initiation, metastasis progression, and macrometastatic colonization, the selection for tumorigenic functions during primary tumor growth provides essential prerequisites for future metastasis. Certain biases in the genes that are selected to fulfill particular tumorigenic functions may result in genes that can also fulfill specific metastatic functions, leading to an early proclivity toward distant spread. The further selection of metastasis-specific functions after infiltration of distant organs can continue to modify metastatic behavior through the acquisition of macrometastatic-colonization genes. Although the emerging evidence does not always allow clear delineation between genes that serve tumorigenic versus metastasis initiation versus metastasis-progression functions, recent molecular understanding and insight offer the underpinnings of this integrated view.

SELECTIVE PRESSURES AT THE PRIMARY TUMOR DRIVING ACQUISITION OF METASTASIS FUNCTIONS

Of all the tumorigenic functions required by aggressive primary cancers, several may additionally select for metastasis initiation or metastasis-progression genes (Fig. 10.1). Experimental and clinical evidence point toward the following factors.

Hypoxia

In order to disrupt tissue homeostasis during primary tumorigenesis, many barriers that can limit growth must be overcome. A near-universal need is for tumors to respond to hypoxia (reviewed in refs. 18 through 20). Normal tissue such as epithelium is separated from blood vessels by a basement membrane. When preinvasive tumor growth occurs, hypoxia can ensue because oxygen and glucose typically can only diffuse 100 to 150 microns, resulting in portions of the expanding mass becoming hypoxic. This can be seen in come-do-type ductal carcinoma in situ (DCIS) of the breast, whereby a necrotic center characterizes these preinvasive breast tumors. The fact that DCIS can take years to progress to invasive cancer, or never progresses to cancer, suggests that hypoxia can be a significant barrier.

Although there are multiple paths that cancer cells can take to adapt to hypoxia, the hypoxia-inducible factor (HIF) transcription factors have a central role. Under hypoxic conditions, HIF-1α and HIF-2α become stabilized, resulting in the transcription of over 100 HIF-α regulated genes. These target genes are involved in angiogenesis, glycolysis, and invasion, which together help
hypoxic cells adapt. Up-regulated angiogenesis genes include vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). These factors cause quiescent blood vessels to undergo remodeling, including the laying down of a matrix that activated endothelial cells use to form newly vascularized areas. Various glycolysis genes are expressed and their metabolic by-products lead to acidification of the extracellular space. This is normally toxic to cells and requires further adaptation either by up-regulation of H⁺ transporters or acquired resistance to apoptosis. To assist in invasion toward newly vascularized areas, HIF-α upregulates matrix metalloproteinase 1 and 2 (MMP1, MMP2), lysyl oxidase (LOX), and the chemokine receptor CXCR4. Degradation of the basement membrane by MMP2 and alteration of the extracellular matrix (ECM) by MMP1 and LOX clears away a barrier to migration. The activation of CXCR4 then stimulates cancer cells to migrate to regions of angiogenesis. Thus, if these series of events can be successfully completed, not only will preinvasive tumors successfully deal with hypoxia, but they will also likely invade through the basement membrane in the process. Invasion through the basement membrane defines invasive carcinomas.

Inflammation

When normal tissue homeostasis and architecture are disrupted, this can lead to vessel injury, hypoxic zones, extravasation of blood proteins, and the entry of foreign pathogens (reviewed in refs. 21 and 22). A rapid response is mounted by a front line composed of immune and bone marrow-derived cells (BMDCs) such as lymphocytes, neutrophils, macrophages, dendritic cells, eosinophils, and natural killer (NK) cells. The purpose is to restore homeostasis through several phases: inflammation, tissue formation, and tissue remodeling. In the initial phase, tissue breakdown attracts neutrophils to infiltrate the wounded area and release various proinflammatory cytokines such as interleukin (IL)-8), IL-1β, and tumor necrosis factor-α (TNF-α). In addition, reactive oxygen species and proteases such as urokinasetype plasminogen activator (uPA) are produced by neutrophils to fight pathogens and debride devitalized tissue. After a few days, neutrophils begin to undergo cell death and are replaced by macrophages that are either resident or recruited from circulating monocytes in response to proinflammatory cytokines and chemotactic gradients. Activated macrophages are thought to play an integral part in coordinating the wound response by providing matrix remodeling capabilities (uPA, MMP9), synthesis of growth factors (fibroblast growth factor [FGF], PDGF, transforming growth factor beta [TGF-β]), and production of angiogenesis factors (VEGF). These factors activate fibroblasts to synthesize new ECM and promote neovascularization in the formation of granulation tissue. Other cells that are important in wound healing include mesenchymal stem cells.²³ These fibroblastoidlike cells can be mobilized from the bone marrow or from niches within various tissues in order to aid wound healing by differentiating into different connective tissue cell types.

Cancer cells are often surrounded by activated fibroblasts and BMDCs. Because of the resemblances between primary tumors and normal tissue wound response, cancer has been described as a “wound that does not heal.” Although Virchow hypothesized in the 1850s that inflammation was the cause of cancer, the presence of an inflammatory response has generally been interpreted as evidence that the immune system actively fights the cancer as it does with invading bacterial or viral pathogens. Under this scenario, the inflammatory response would apply significant selective pressure on the tumor to evade immune-mediated attack, and the nonhealing nature of the response suggests a back-and-forth struggle. Tumors that progress do so by orchestrating an immunosuppressive environment, a process known as immunoediting.²⁴

To facilitate an immunosuppressive environment, the tumor microenvironment selects for cells that favor production of immunomodulatory factors like TGF-β, cycoloxygenase-2 (COX2), CSF-1 (macrophage growth factor, colony-stimulating factor-1), IL-10, and IL-6. These cytokines inhibit maturation of dendritic cells and promote tumor-associated macrophages (TAMs) that are immunosupressed.²⁵ Tumors also recruit BMDCs that have immunosuppressive properties such as myeloid-derived suppressor cells (MDSCs).²⁶ These cells are recruited through signaling events that involve stromal cell-derived factor-1 (SDF-1, also known as CXCL12) and its ligand CXCR4 and CXCL5/CXCR2, another chemokine/receptor pair. On arrival, the MDSCs increase local production of TGF-β, block T-lymphocyte function, and inhibit the activation of NK cells.²⁷ Thus, although the inflammatory response undoubtedly can help to limit cancer growth, cancers seem to select for cells that create immunosuppressive surroundings.

Rather than simply suppress the inflammatory response, cancer cells actually develop mechanisms to both co-opt and perpetuate it. For example, at the same time MDSCs are contributing to immunosuppression, these cells also facilitate tumor invasion by residing at the invasive front and secreting MMPs. The comingling of various stromal cells and other BMDCs with the cancer also actively contributes to tumor growth in a similar fashion. For example, TAMs are often found at points of basement membrane breakdown and, like MDSCs, end up at the invasive front to help tumors degrade extracellular proteins using uPA and MMPs or stimulate tumor growth and motility through EGF receptor ligands and PDGF. As in normal wound healing, growth factors secreted by the TAMs
activate fibroblasts. These activated fibroblasts become carcinoma-associated fibroblasts (CAFs) and promote primary tumor growth by secreting CXCL12 to stimulate CXCR4 on tumor cells. Angiogenesis is also aided by the action of CAFs through recruitment of endothelial progenitor cells by CXCL12 and by the action of TAMs that are recruited to areas of hypoxia to produce VEGF. Figure 10.2 presents a summary of this interaction. Thus, although the question of whether the immune system is a friend or foe of malignancies is not a new one, it would seem that recent answers suggest that cancers actually find ways to turn an enemy into an accomplice.

FIGURE 10.2 Interactions between cancer and stroma that promote invasion and metastasis. Cancerized stroma consists of fibroblasts, inflammatory cells, and other bone marrow-derived cells that have been conscripted to aid the tumor in overcoming hypoxia and in invasion and migration. Tissue breakdown, hypoxia, and inflammatory cytokines and chemokines secreted by the tumor cells result in recruitment of tumor-associated macrophages (TAMs), carcinoma-associated fibroblasts (CAFs), mesenchymal stem cells (MSCs), and myeloid-derived suppressor cells (MDSCs). TAMs and MDSCs can be found at points of basement membrane breakdown and at the invasive front of the tumor. These cells produce angiogenic factors to promote vascularization, proteases to degrade the extracellular matrix, and growth factors that stimulate tumor invasion and motility. CAFs also produce similar angiogenic factors, protease, and tumor growth factors. In addition, CAFs recruit bone marrow-derived endothelial precursors for angiogenesis. The cytokines and growth factors that TAMs and CAFs secrete are mutually beneficial to each other as part of an inflammatory/woundlike response. Cancers have been described as “wounds that do not heal.” This chronic state is maintained by immunomodulatory cytokines that suppress immune functions to ensure a protumorigenic environment.

Escaping Apoptosis and Senescence

A major mechanism to safeguard against a breakdown in tissue homeostasis due to cells that stray, become damaged, or spent, is to have these cells commit programmed cell death, or apoptosis.28 This form of cell suicide is genetically regulated and can be triggered by a variety of signal transduction pathways linked to proteins that monitor environmental cues or act as damage sensors. Common cell intrinsic triggers for apoptosis include oncogene activation or tumor suppressor gene loss. For example, the inappropriate activation of c-MYC or the loss of Rb results in programmed cell death that must be countered by overexpression of antiapoptosis genes such as Bcl-2 or loss of proapoptotic regulators like p53.²⁹ Extrinsic triggers for apoptosis include hypoxia, low pH, reactive oxygen species, loss of cell contact, and immune-mediated killing. Members of the TNF-receptor family can act as death receptors that mediate activation of proapoptotic proteases called caspases in response to loss of ECM adhesion³⁰ or after being engaged
by immune cells for elimination. Cancer cells invariably ignore these cues, and their ability to resist cell death likely contributes to successful establishment of tumors.

In addition to apoptosis, senescence is another important barrier to cancer. This exit from the pool of proliferating cells results from telomere erosion, oncogene induction, and DNA damage. Similar to some forms of apoptosis, senescence is p53-dependent. Thus, pressure to escape senescence can result in the loss of p53 or mutations in p53.

Self-Renewal Ability

Normal tissues result from the differentiation of precursor cells called stem cells, which are multipotent cells with self-renewal ability. In the adult, mature differentiated cells serve specialized tasks and have limited proliferative potential. However, adult tissue still undergoes turnover and is maintained through the self-renewal and multilineage differentiation of adult stem cells. Examples of this include skin, mucosa, and hematopoietic cells whereby a limited and spatially restricted pool of adult stem cells asymmetrically divides. One daughter cell maintains the stem cell pool by self-renewal, and the other daughter cell starts the process of terminal differentiation for tissue maintenance. The majority of cancers maintain some resemblance to their tissue of origin by virtue of persistent differentiation, albeit in an abnormal way. Thus, many cells in a tumor population may have limited proliferative potential and be incapable of sustained self-renewal, similar to their normal counterparts. The idea that only a limited subset of cells in a cancer is capable of self-renewal is called the cancer stem cell hypothesis.

The existence of cancer stem cells was first demonstrated in acute myeloid leukemia and recently shown in breast cancer, glioblastoma, and other cancer types.³¹ These studies use cell surface markers to enrich for putative stem cell populations. In the case of breast cancer, CD44^high/CD24^low cells were found to form tumors when injected into immunocompromised mice in low numbers and give rise to a diverse population that contained additional CD44^high/CD24^low cells. In contrast, the injection of thousands of cells from other populations was nontumorigenic. Thus, the need for cancer cells to acquire self-renewal ability is paramount to the productive growth of the tumor. In principal, however, there is no advantage for cancer to keep this subpopulation of tumor-initiating cells small or fixed. Thus, tumors may contain a large proportion of cells with a tumor-perpetuating stem phenotype,³² and this phenotype may be subject to back-and-forth reprogramming.

COUPLING TUMORIGENESIS WITH METASTASIS INITIATION

The selective pressures previously described that are encountered during primary tumor growth—hypoxia, inflammation, apoptosis, senescence, and need for proliferative, metabolic, and self-renewal sufficiency—drive primary tumors to acquire tumorigenic alterations that support aggressive growth. These same pressures collaterally support the initial stems of metastasis, and remain important throughout the subsequent malignant steps. One of the most striking types of metastasis initiation functions is EMT.

Selecting for Epithelial-to-Mesenchymal Transition

During development, the generation of many adult tissues and organs results from a series of EMT events and the reverse process, a mesenchymal-to-epithelial transition (MET).³³ For example, gastrulation and delamination of the neural crest are early developmental processes that layout the three germ layers (ectoderm, mesoderm, and endoderm) and give rise to diverse cell lineages (craniofacial cartilage and bone, smooth muscles, neurons, melanocytes), respectively. Both of these developmental events are characterized by epithelial cells loosening their cell-cell adhesion, losing cell polarity, and gaining the ability to invade and migrate under controlled cues. Important regulators include Notch and Wnt/β-catenin pathways, TGF-β family members, and FGF proteins that serve to set up regulatory networks involving EMT transcription factors such as Snail and Twist. These networks do not necessarily regulate cell fate, but rather drive morphogenetic movements by repression of the cell-cell adhesion protein E-cadherin, promoting cytoskeletal rearrangement, and increasing MMP activity. After cells complete EMT-mediated morphogenetic migration, they can then transiently differentiate into epithelial structures by repressing Snail and undergoing a MET. An example of cells that undergo a primary EMT that is followed by a MET includes neural crest cells giving rise to somites. These epithelial somites that have already experienced a primary EMT that is followed by a MET can then initiate a secondary EMT, which eventually results in development of muscle cells.

Growing evidence points toward EMT as an important characteristic of metastasis-prone cancers. EMT in cancer is not a concrete and tidy single process but rather a collection of cell reprogramming phenomena that share the property of down-regulating epithelial cell markers
and, for convenience, the collective denomination of EMT. Hypoxia can induce Snail and Twist, a direct target of HIF-1α.³⁴ Low oxygen enhances β-catenin activity by inhibiting the activity of glycogen synthase kinase-3β, which normally induces the destruction of β-catenin. Accordingly, the presence of enhanced β-catenin signaling promotes Snail expression and subsequent EMT. Interestingly, the ability of hypoxia to liberate active β-catenin may set in place a feed-forward loop to help maintain EMT. Activation of Snail represses E-cadherin, which can then further enhance β-catenin and reinforce Snail expression.

Similar to hypoxia, the inflammatory microenvironment can also promote EMT. It has recently been demonstrated that TNF-α, which is an inflammatory mediator secreted by TAMs, sets into motion a signaling cascade that funnels through NF-κB and glycogen synthase kinase-3β

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