Invasion and Metastasis of Malignant Neoplasms

Neoplasm refers to an abnormal and excessive growth of new cells. This growth is uncontrolled and uncoordinated with the surrounding normal tissues. The newly formed tissue is disorganized and non-functional. These cells continue to replicate, completely ignoring the regulatory mechanisms that control normal cell growth. In general, neoplasm is also referred to as a tumor. A tumor is a swelling or mass, but not all swellings are tumors. In oncology, the term "tumor" specifically refers to a neoplastic mass that causes a visible or palpable lump on the body's surface. Neoplastic cells are those that have undergone transformation, usually due to genetic mutations, with DNA damage considered the primary cause.

Carcinogenesis, also known as oncogenesis, is the multi-step process through which cancer develops. For a cell to start dividing uncontrollably, the genes that regulate cell growth must become dysregulated. Two main classes of genes are involved in carcinogenesis: proto-oncogenes and tumor suppressor genes. When proto-oncogenes are activated by mutations, they send positive proliferative signals to the tumor. On the other hand, tumor suppressor genes act to inhibit neoplastic growth, and must be inactivated or lost in tumor cells. Generally, multiple mutations in these genes are required for a normal cell to become cancerous. These mutations provide the signals that trigger uncontrolled division in tumor cells.

In oncology, neoplasms are classified as either benign or malignant. A tumor is considered benign if it remains localized, does not spread, can be removed surgically, and does not cause death—unless its location interferes with vital bodily functions. Malignant tumors, however, invade and destroy surrounding structures, can spread to distant sites (metastasize), often recur after removal or radiation, and may lead to death. Malignant tumors are commonly referred to as cancer. The key differences between benign and malignant tumors include differentiation and anaplasia, growth rate, local invasion, and metastasis.

Metastasis is defined as the spread of cancer from one organ to another or to a different part of the same organ. Cancer cells can spread through one or more of the following metastatic routes:

1. 1. Lymphatic spread: via lymphatic vessels, primarily through lymph nodes.
2. 2. Hematogenous spread: through blood vessels to distant organs.
3. 3. Transcoelomic spread: via direct contact between serosal surfaces in the body cavity.

Lymphatic spread involves invasion of lymphatic vessels and transport through the lymphatic system to regional lymph nodes and distant organs. This is often combined with hematogenous spread. In hematogenous spread, tumor cells invade nearby blood vessels, enter the circulation, and are transported to distant sites. A few tumors, such as canine osteosarcoma, metastasize almost exclusively through the bloodstream and rarely involve lymph nodes. Cancers like those of the pancreas, ovaries, and mesothelioma commonly spread via transcoelomic metastasis, sometimes along with the other two routes. Transcoelomic spread involves the direct implantation of tumor cells onto the serosal surfaces of adjacent organs.

Figure 1. Metastasis cascade

Distant metastasis, primarily via hematogenous and often prior lymphatic spread, can be described as a metastatic cascade, consisting of five main steps:

1. 1. Loss of adhesion between neighboring cells.
2. 2. Invasion into the surrounding extracellular matrix and blood/lymphatic vessels—often involving epithelial-to-mesenchymal transition (EMT).
3. 3. Survival in the bloodstream as circulating tumor cells (CTCs).
4. 4. Extravasation and formation of micrometastases.
5. 5. Development into macrometastases.

The first step in the cascade is the separation of potentially metastatic cells from their neighbors, often associated with reduced expression of cell adhesion proteins such as E-cadherin, leading to loss of cell-cell contact.

Conversely, the expression of proteins involved in invasion and migration through the extracellular matrix increases. These include CD44, focal adhesion kinase (FAK), and matrix metalloproteinases (MMPs) that help degrade the extracellular matrix. Detachment of neoplastic epithelial cells and invasion into the surrounding matrix are associated with changes in cell shape from a polygonal epithelial form to a spindle-shaped mesenchymal form—this is known as epithelial-mesenchymal transition (EMT). It's unclear whether mesenchymal tumors like sarcomas undergo EMT. This invasive process is also influenced by surrounding stromal stem cells and macrophages, which contribute to MMP production.

After invading the vessels, tumor cells are carried through the bloodstream either individually or in small clusters, known as circulating tumor cells (CTCs). Many efforts are underway to develop liquid biopsy methods to detect CTCs in patient blood samples, which are less invasive and more informative about disease status than traditional tumor biopsies. Early studies show that CTCs from canine mammary tumors can be detected in peripheral blood using markers such as CLDN7, CRYAB, ATP8B1, and EGFR. Their presence is significantly and sensitively associated with metastatic disease progression in dogs. Despite tumors shedding thousands to millions of cells into circulation, the number of detectable CTCs is usually low—fewer than 10 CTCs per milliliter of blood and per million peripheral leukocytes. Moreover, not all CTCs are clinically relevant. It’s estimated that fewer than 0.1% of CTCs can actually lead to macrometastatic disease, and the total number in the blood does not necessarily correlate with metastasis development.

The question of why and where CTCs extravasate and form metastases remains largely unanswered. However, it is clear that metastases from different tumor types tend to follow specific patterns and affect certain organs preferentially. For instance, canine mammary tumors and osteosarcomas most commonly metastasize to the lungs, while feline lung carcinomas often metastasize to the distal phalanges. In contrast, organs like the heart or skin are rarely affected by metastasis. This pattern is explained by the “seed and soil” theory: cancer cells (the seeds) can only grow into metastases in suitable environments (the soil). A seed can only grow where the soil supports it. The factors that make certain tissues or organs more hospitable to metastasis are still under investigation. Identifying these factors is of great interest because they could lead to the development of targeted therapies to prevent metastatic disease. Among all organs, the bone has been studied most extensively due to its relevance in human medicine. Various mechanisms and factors are involved in tumor spread and bone colonization, including secretion of chemokines (e.g., CXCL12, CXCL13) and RANKL by osteoblasts and bone marrow stromal cells, which may attract cancer cells to the bone marrow.

Micrometastasis formation is the next stage following CTC extravasation. Micrometastases are more commonly observed clinically than macrometastases. They consist of small clusters of tumor cells that grow slowly or are dormant, and are often undetectable with standard imaging techniques. In the next stage, micrometastatic tumors must adapt to the new tissue microenvironment and grow into macrometastases—a process called colonization. Colonization appears to be a major hurdle for tumor cells. It’s believed that out of millions of tumor cells circulating in the blood, only a few successfully form micrometastases. Most of them remain dormant.

Dormancy is defined as a state in which cancer cells do not divide or proliferate. During dormancy, cells stay in the G0 or G1 phase of the cell cycle, waiting for signals to start proliferating again.