The complex interplay between tumor cells and the multiple microenvironments with which they interact during metastasis provides numerous potential targets for therapeutic intervention. Since metastasis is the primary cause of cancer morbidity and mortality, critical evaluation and prioritization of those targets represents the first step in developing treatments specifically targeting metastases. I will focus on the four features of metastatic cells that distinguish them from malignant cancer cells: motility & invasion, modulating local or secondary microenvironments, plasticity/adaptability, and colonizing secondary tissues. By defining the hallmarks of metastasis, my hope is that new therapies will be developed that will improve survival and quality of life.

For >4000 years, medical practitioners have recognized that tumor cell dissociation, dissemination and colonization of discontiguous secondary sites is the most lethal attribute of neoplastic cells. Cure rates are high for most cancers when diagnosis occurs before spread; but, once metastases develop, cancer is frequently considered incurable. I prefer to think of it as currently incurable, but potentially curable. Otherwise, what's the point? The premise is that the hallmarks when coupled with deeper understanding of the molecular mechanisms responsible will accelerate development of therapies that prevent and/or treat metastasis, reduce cancer deaths and improve patients' quality of life.

The context requires recognizing that metastasis is a distinct (but interrelated) phenotype to cancer. Development of cancer involves several hallmarks, as defined by Hanahan and Weinberg - immortality, genomic instability, resisting cell death, altered metabolism and invasion/metastasis, including how cancer cells interact with the stroma - sustained angiogenesis, promote inflammation, immune evasion, resistance to growth inhibition, and relative autonomy. Acquisition of malignant characteristics, in general, follow a progression through precancerous, transformed, benign and malignant (invasive) states. Superimposed upon the hallmarks of cancer development are the hallmarks of metastasis. Critically, the definition of metastasis is the process of spreading to a nearby or distant, discontiguous secondary site and the establishment of macroscopic secondary foci.

Every metastatic cell must accomplish an entire cascade series of sequential steps. To spread, a subset of heterogenous primary tumor cells enter a circulatory compartment (hematogenous, lymphatic, or body cavity) and disseminate (note: cells can move between circulatory compartments). This early step is surprisingly common with >106 cells/gm tumor entering the vasculature. Indeed, many cells disseminate long prior to tumor detection.

Before tumors are detectable, cells begin to manipulate local stroma and distant sites. Establishment of a pre-metastatic niche occurs via tumor cell-derived soluble factors and extracellular vesicles communicating with HSC or MSC. Induced bone marrow-derived stem cells mobilize to seed and manipulate secondary microenvironments, making each niche favorable for colonization.

Cells must migrate, restructure or degrade the ECM, often co-opting normal physiologic activities and responding to motility-inducing and chemotactic factors. Migration can occur as cooperating cell clusters (collective migration) or as rogue individual cells. Some cells usurp a reversible embryologic process, the epithelial-mesenchymal transition (EMT), or entosis, or use amoeboid characteristics to squeeze through small spaces without proteolytic ECM degradation, or a combinations of these mechanisms to invade. Invasion through a basement membrane is the defining feature of malignancy. But the ability to invade is insufficient to metastasize. Since millions to billions of cancer cells begin the process but the vast majority fail to complete it, mobility is also necessary, but not sufficient. Importantly, tumor cells can manipulate stromal cells and immune cells into assisting their motile and invasive phenotypes.

Upon entry into a transport compartments, tumor cells distribute to wherever that compartment leads using both active and passive mechanisms. Circulating tumor cells (CTC) can provide very useful information regarding probabilities of metastatic spread as well as therapeutic efficacy. But, as noted above, many cells disseminate, but few successfully form metastases. Therefore, it is critical to distinguish between mere dispersal of cells and actual metastasis formation.

To successfully form a metastatic focus, a disseminated cell must eventually arrest at a compatible ectopic site and extravasate. Arrest can either be active or passive. However, the distribution of metastases is non-random.

The last step of metastasis, colonization, utilizes the same processes that lead to primary tumor growth - response to pro-growth factors, resistance to growth inhibitors, adequate oxygenation, nutrients and angiogenesis. Successful colonization often requires landing at a pre-metastatic niche. Despite extensive study the molecular mechanisms of colonization are still largely unknown. Some cells undergo prolonged (months to years) quiescence between seeding and colonization. What happens during that time is unknown except that some cells temporarily mitotically arrest, while others await angiogenesis.

Lineage tracing shows that metastases utilized both parallel and sequential evolution processes. Single cell analyses confirm that >90% of metastases are clonally derived and possess the same driver mutations that led to tumorigenesis. Individual metastases arise from different subpopulations from the primary tumor, many sharing overlapping gene changes. All steps of metastasis involve coordinated expression of multiple genes. Cells in the process of metastasizing sometimes exhibit an incomplete cohort of those gene cassettes. Intervention will require distinguishing between cells partially capable versus those destined to become metastases. This distinction is most critical when cells that have seeded, but not yet colonized, a secondary site.

So, what constitutes the ‘hallmarks' of metastasis? The challenge when defining any hallmarks in cancer is that cancer cells utilize the same molecules and processes that normal cells use. It is the combination of hallmarks that distinguish metastatic cells, not just the presence of a subset of hallmarks. All metastatic cells are motile and penetrate basement membranes. All metastatic cells somehow manipulate myriad microenvironments prior to and during transit. All cells must continuously adapt to the conditions in which they find themselves (i.e., sheer forces in the blood stream, immune attack, deformability, etc.). And finally, all metastatic cells must successfully colonize secondary tissues. This last hallmark distinguishes mere dissemination from metastasis.

Is defining the hallmarks of metastasis helpful in clinical oncology? Distinguishing cells with the potential from those which actually metastasize is key to identifying targets for new treatments. Some insights have already led to improved 5-year survival and quality-of-life. But unfortunately, improvements have not yet been realized for all cancers. Yet, I believe we are poised to make significant inroads by applying the principles outlined here.

Besides the biology, there are other aspects of metastasis which are just as important. While not the focus of this presentation, they deserve attention. They include better management of the emotional toll treatments exert on patients (and families). Every scan is anticipated with ‘scanxiety,' a fear that the tumor has grown or recurred. Metastatic patients constantly fear of running out of options. And financial toxicities cannot be overlooked and are not sustainable. By proposing these hallmarks of metastasis, I hope a framework is provided which will accelerate translating the growing knowledge of molecular circuitry into treatments that prevent and eliminate metastases.

Acknowledgments: I thank colleagues, friends, trainees and metastatic patients who have taught me much and inspired me with your strength and courage. My lab has been supported by grants from the NIH, NCI, DoD, Komen, NFCR, METAvivor, Theresa's Foundation and Hall Family Professorship.

For additional details and references, see Cancer Res (2019) 79(12):3011-3027.

Citation Format: Danny R. Welch. Using the hallmarks of metastasis to develop antimetastatic treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr SY02-01.