The concept of organotropism is by no means novel; for over a century it has been observed that certain primary tumor types have a tendency to metastasize to specific secondary sites. Yet it is only since the dawn of the 21st century that genomics has emerged as a valuable tool for probing the mysteries behind what drives organotropism. Metastasis is a complicated process involving many steps: intravasation, survival in circulation, extravasation, and colonization and growth at a distant site. The multi-step nature of metastasis and the inherent complications associated with cancer cells make finding and studying the genes associated with organotropism difficult. However, in recent years, great strides have been made in the field of organotropism and metastasis. Here we examine some specific examples of genes associated with organotropism, along with the complicated processes involved with the regulation of metastatic tumor growth.

Introduction

The hypothesis that metastasis is organotropic has influenced medical research since the publication of Dr. Pagets "Seed and Soil" paper.¹ Throughout the first half of the 20th century, researchers attempted to find a rationale for the seemingly random process of metastasis. Dr. Pagets idea was simple: if a primary tumor sends out "seeds" (i.e., cells) throughout the body, they can only grow in good "soil" (i.e., a new organ site). Indeed, as techniques for locating metastatic tumors were refined, it became clearer that there is a distinct patterns of metastasis to distant organs: breast cancer tends to metastasise to the bone, liver, lymph nodes, and lung, and later to the brain; gastrointestinal, ovarian, and colorectal cancers to the liver and lung; prostate cancer to the bones, liver, and lung; lung cancer to the liver, brain, bone, and lymph nodes; neuroblastoma to the lung, liver, bone, and skin; and melanoma cells to lung, liver, brain and bone^2-6 (Fig. 1). Although correlations could be found between primary tumors and their preferred metastatic destination, there was no clear rationale for the organ preference.

Figure 1.

Common sites of metastases from breast, colon, lung, and prostate cancers, neuroblastomas and melanomas. A few genes shown to be involved in metastasis of the primary tumor to a specific organ are listed next to some of these organs. These lists are by (more...)

Logic would dictate that primary tumors in certain organs would spread spatially through the body, affecting the nearest neighboring organs.⁷ In the early 1950s, it was shown that intravascular injection of tumor cells into rabbits led to visible metastases in many different visceral organs, e.g., the adrenal glands, diaphragm, iris, kidneys, masseter muscle, pituitary gland, spleen, testis, and thyroid.⁸ In the organs where metastases were found, tumor cells were lodged in the capillaries. These results illustrate the importance of the circulatory system and mechanical factors in the delivery of metastatic cancer "seeds" to new locations. Indeed, others have shown by videomicroscopy that circulating tumors cells get lodged in capillaries and begin to grow in the organs where they arrest.^9,10 Additionally, autopsy studies provide evidence that many metastases are in secondary sites that would be predicted based on blood circulation pattern, e.g, liver metastases in colon cancer patients. At the same time, however, there is an absence of metastases in organs where metastases would be expected based on circulation patterns, e.g., no metastases to the thyroid in colon cancer patients.^11,12 Established correlative studies have proved that metastases have a tendency to consistently form in known secondary locations, distant from the primary tumor.¹³

Technology has now advanced to allow for the examination of gene regulation in regards to organotropism. One of the earliest tools applied to genetic studies was the bacterialLacZ gene, which allowed researchers to stain and quantify micrometastases.¹⁴ More recently, the use of firefly andrenillaluciferase in mouse model systems has allowed for a more refined picture of metastatic progression. Studies show that human tumor cells injected via different routes into immunocompromised mice lead to high metastases burdens in different locations.⁴ The tumor cells can be tracked over a period of time using bioluminescent imaging to see what organ the cells preferentially colonize. For example, immediately after injection of breast cancer cells into the left ventricle of the mouse heart, the cells are spread across the circulatory system, but 412 weeks later the cells have high rate of colonization almost exclusively in the brain and bone.¹⁵ One would expect that since the cells make contact with many different organs via the circulatory system there would be metastases all over the body; yet instead there are an increased number of metastases in the specific organs seen in human patients, e.g., bone and brain. Circulation has an obvious role in metastasis, but it is not the key regulator of organtropism in cancer metastasis. A more thorough review of the concept of seed and soil is covered in the chapter entitled "Metastatic cascade: seed and soil revisited."

Understanding the mechanism of metastasis organotropism is of interest to cancer biologists and oncologists since oftentimes the metastasis proves fatal, rather than the primary malignancy. Metastasis is a complicated process, and most likely there is not a single gene in any type of cancer cell that determines the destination of its metastases. Over the past decade, more refined tools such as qPCR, fluorescent and bioluminescent imaging, and microarray analysis have allowed a more complete understanding of both the genetic profile and the phenotypic expression of specific cancer cell types. Gene signatures have been identified which indicate which genes play a role in the metastasis of the primary tumor to a specific organ.^16-19 Understanding the link between the regulation of these genes and the destination of certain metastases will impact the treatment of patients and the prevention of cancer fatalities. Presently, breast cancer is the most well characterized in terms of its genetic profile in relation to organotropism, and therefore, much of this review will discuss genes that mediate organotropism of breast cancer. A few examples of other cancers will also be examined. This review is by no means exhaustive; many genes in a variety of cancers have been implicated in the tropism of metastases, and it is not feasible to address all of them.

Genes That Mediate Breast Cancer Organotropism

Metastasis occurring from primary breast tumors has been studied exhaustively for many years. Breast cancer is known to most often metastasize initially to the lymph nodes, bone, and lungs, and later to the brain and liver; how and why these organs are preferred is not fully elucidated.^4,20 Studies regarding the genetic forces driving organotropism in breast cancer have advanced dramatically in the past decade as a result of more refined molecular probing. As these phenomena become better understood, they open up possibilities for new biomarkers, drug targets, or other molecular therapies to prevent potentially fatal metastases.

Early studies in organotropism sought to establish a correlation between key prognostic markers in the primary tumor and the site of relapse. Breast cancer has long been classified based on the positive or negative presence of the estrogen receptor (ER), progesterone receptor (PR), and the human epidermal growth factor receptor 2 (HER2). The tropism of breast cancer to the bone and brain was first linked with the presence or absence of ER/PR in the primary tumor, respectively²¹; however, the presence or absence of ER, PR, or HER2 alone could not account for the phenotypic diversity seen in breast cancer. Five molecular subtypes have now been mapped to specific gene expression patterns describing this phenotypic diversity and predicting patient outcome.^22-24 Follow up studies utilizing immunohistochemical (IHC) staining of key markers from each subtype found that frequencies of the site of relapse of breast cancer patients are related to the molecular subtype of the patients tumor, with brain relapses in patients with basal tumors and HER2+ tumors, bone relapses in patients with luminal type tumors, and lung relapses associated with patients with basal tumors.²⁵ Though there are some clinicopathologic analyses that suggest these subtypes have little correlation with any specific tropisms,²⁶ many other studies utilizing both IHC staining and gene array analysis suggest the contrary.^27,28 Harrell et al.²⁷ recently analyzed 1,319 genes in an expression microarray from patients primary tumors, metastases, and cancer cell lines. They found that patients with breast tumors that were ER/PR negative subtypes generally presented with higher metastases, except those found in the bones, which had both ER+ and ER- tumors; HER2+ tumors were associated with tropism to the liver; and the basal-like and claudin-low subtypes were associated with brain and lung tropisms.²⁷ These findings were similar to those of earlier studies.^25,28 Further analysis showed that the brain-seeking metastases had basal-like, low claudin markers, with a gene signature similar to that of lung metastases and the primary tumors from which they originated. Overall there were very similar gene signatures shared between the primary tumor and metastatic tumor. These results suggest that the primary tumor molecular subtype can predict the potential metastatic site. Analysis with multiple organotropic-metastatic gene signatures (described below) further predict the site of relapse and indicate the importance of identifying genes and gene signatures that mediate organotropism.

Additional Examples of Organotropic Genes in Other Primary Tumor Types

While breast cancer remains the best categorized in terms of organotropic metastases, a few other genes have been found to regulate specific tropisms of other cancers. In primary lung cancer bone-, liver-, and kidney-tropic genes have been identified. Lung cancer is often diagnosed late in progression, after metastases have already been established in different secondary locations. The preferred destinations of lung metastases are the liver, lymph nodes, brain, and bone.⁶⁵ SBC-5 cells have been established as a lung cancer cell line with a proclivity to metastasise to the bone, liver, kidney and lung. For this experiment, researchers used gene array analysis to examine the genetic profiles of the SBC-5 cells after they had reached their metastatic terminus. Genes specific to each metastatic location were identified from each subpopulation. Liver-tropic genes include IGFBP7, CDH2, CTSL2, ADAM17 and P4HA1; bone tropic genes include FGFR, FST, PTHrP, COL3A1, MST1, FAP and POV1; kidney tropic genes include LGALS9, COL1A1, BMP6, INHBA, and C1S.⁶⁵ Notably, the bone metastases from the primary lung tumor-derived cells had a similar genetic profile to bone-tropic metastases derived from breast cancer; upregulated genes included RANKL, PTHrP, and CXCR4. RANKL and CXCR4 are also involved in neuroblastoma and prostate cancer tropism to bone.^66-69 These results indicate that the same genes present in different primary tumor types can guide metastases to the bone. Similarly, genes correlated with breast cancer metastasis to the brain are linked to lung and melanoma tropism to the brain. In lung cancer, 10 miRNAs were found to correlate with metastasis to the brain, including miR-450b-3p, miR-29c, miR-145, miR-148a, miR-1, miR-30d, miR-187, miR-218, miR-708, and miR-375.⁷⁰ miR-145 has been shown to suppress invasion and metastasis through inhibition of mucin-1 expression.⁷¹ Interestingly, expression of mucin-1 has also been correlated with breast cancer metastasis to the brain.⁷² In melanoma cells, which were selected for their ability to metastasize to the brain, expression of ANGPTL4, PTGS2/ COX2, and MMP1, were upregulated.⁷³ These genes are also upregulated in the breast cancer BrMS.¹⁶ Additionally, another 35 genes were up or downregulated in the brain metastasis and micrometastasis cells. Higher levels of preferentially expressed antigen in melanoma (PRAME) and lower levels of claudin-1 and cysteine-rich protein 61 were found in the brain metastasizing cells versus their corresponding parental cell lines.⁷³ Claudin-1 functions in tight junction formation suggesting that loss of cell-cell adhesion is important in tropism of melanoma cells to the brain. The molecular subtype claudin-low also correlates with breast cancer metastasis to the brain.²⁷ These results suggest that gene clusters or similar gene families may play a role in organotropism of primary tumors from different organs to the same destination.

The current understanding the mechanism of colon cancer growth in the liver and lung is very limited, and there is very little known about which genes or gene signatures regulate the tropism of colon cancer cells to the liver or lung. Genes that are believed to increase colon cancer cell survival in the lungs include E2F and thymidylate synthase. High levels of E2F expression are associated with lung metastases of colon cancer patients. This transcription factor upregulates thymidylate synthase and is believed to increase the cells' survival and chemo resistance of colon cancer lung metastases.⁷⁴ Recently, expression of miR-493 was shown to inhibit colon cancer metastasis to the liver.⁷⁵ IGF1R is a direct target of miR-493 and inhibition of IGF1R leads to decreased metastasis to the liver and induced cancer cell death in the liver after the cells arrive. High levels of miR-493 in colon cancer tumors were associated with decreased liver metastases in these patients.⁷⁵ Further understanding of miR-493 function in colon cancer metastasis to the liver will help elucidate additional genes involved in liver tropism.

In neuorblastoma, the suppression of caspase-8 and integrin α4 may confer tropism to the bone. Caspase-8 is a known tumor suppressor gene.⁷⁶ When caspase-8 deficient neuroblastomas were injected into chick embryos, they showed increased metastatic capabilities compared with their wild-type counterparts.⁷⁷ In addition, caspase-8 is not found in murine neuroblastomas that spontaneously metastasize. If integrins on a neuroblastoma cell are not activated through interactions with another cells surface receptor or extracellular matrix, they trigger integrin mediated death (IMD) via caspase-8, which recruits other apoptotic factors leading to cell death. Thus, downregulation of caspase-8 expression allows the neuroblastoma cells to survive better after they enter circulation. Loss of caspase-8 would confer a general increase in metastasis. Interestingly, some metastatic tumor cells still retain caspase-8 expression. In order for these cells to metastasize, IMD must be bypassed. Integrin α4 is expressed in neuroblastomas, and bone marrow contains the ligands for this integrin⁷⁷; thus, the presence of ligated integrin-α4 on neuroblastoma cells in the bone could allow for the bypass of IMD and the survival of neuroblastoma cells in the bone. These results suggest that, as with breast cancer, coordinated gene expression is necessary for bone-tropism of neuroblastomas.

This is by no means a comprehensive list of all primary tumors and genetic profiles and their metastatic propensities. While breast, lung, bone, lymph, and brain cancers, as well as neuroblastomas and melanomas, exhibit an overlap in their metastases, the liver, gallbladder, bile ducts, and intestines have completely different primary profiles and metastatic tendencies. The genes which regulate specific organotropisms of these tumors remain poorly defined.⁷⁸ Furthermore, much of this genetic profiling has been done only in the past ten years; while many genes have been identified, they have not all been closely examined for their regulatory roles in organ-specific metastasis. Additionally, as the technology for genetic profiling and in vivo studies is refined, there is no doubt that more genes, in both the cancer cells and their microenvironment, will be elucidated.

Genes that Mediate the Pre-Metastatic Niche in Organotropism

Many of the studies mentioned above demonstrate that the "soil," or the metastatic microenviroment, is important for organotropism. Studies have examined the role a pre-metastatic niche plays in attracting certain tropisms. Haematopoietic progenitor cells (HPCs) in the bone marrow that are positive for vascular endothelial growth factor receptor 1 (VEGFR1) are stimulated by conditioned media from either B16 melanoma or Lewis Lung carcinoma cells to increase fibronectin in the nearby fibroblasts, which primes the local environment into a niche for incoming metastases.⁷⁹ Bone marrow-derived cells (BMDCs) tagged with GFP or b-galactosidase were found to inhabit the lungs several days before the arrival of either the B16 melanoma or Lewis Lung carcinoma cells (LLCs). Intravenously injecting B16 melanoma cell conditioned media into mice was enough to stimulate the mobilization of BMDCs into the pre-metastatic niche. Additionally, LLCs are known to colonize the lungs and liver preferentially, and it was shown that when intradermally injected into mice, a BMDC pre-metastatic niche in the lung and liver was established prior to actual tumor metastasis. Likewise, the B16 melanoma cells, which have a broader metastatic tropism potential, stimulated BMDC cluster formation in the lung, liver, testis, spleen, and kidney. Functionally, it is believed the VEGFR1 receptor is the key regulator of this clustering of BMDCs and the driving force behind the pre-metastatic niche; when the receptor was silenced using monoclonal antibodies, incidence of lung metastases from the LLCs dropped to nearly zero. It is speculated that VEGFR1+ cells express the integrin VLA-4, which allows for the interaction of VEGFR1+ cells with VCAM1 on endothelial cells in the pre-metastatic niche in the new location.⁷⁹ These results suggest that soluble factors secreted from the primary tumor mediate the formation of the pre-metastatic niche in certain organs and thus organotropism. The chemokines VEGF and PIGF are believed to be two of these soluble factors.⁸⁰ Further discovery of genes that mediate the interaction of the tumor cells with the BMDCs and how this interaction generates the pre-metastatic niche in an organ-specific manner will provide a better understanding of what mediates organotropism. The factors regulating the pre-metastatic niche are described in more detail in the chapter entitled "Pre-metastatic hematopoietic niche."

Conclusion

Metastasis is an elaborate process in the progression of cancer, and understanding the mechanism of how it is regulated is crucial for the prevention and treatment of patients with advanced disease. The tendency of primary tumors to have preferred orthotopic destinations for their metastases is known as organotropism, and understanding the genes that control a specific tropism can help researchers develop more targeted treatments for the disease. With new technologies, the concept of a "seed" and "soil" has advanced since over a century ago. Though the molecular basis for organotropism is still largely unclear, mounting evidence shows there are unique genes and gene signatures that modulate specific tropisms of a primary tumor. To date, researchers have found genes that mediate: (a) the attraction of cancer cells to specific organs, e.g., CXCR4 in tumor cells binding the chemokine CXCL12 in bone, brain and lung,⁴¹ (b) adhesion/"homing" of cells to the endothelium in specific organs, e.g., expression of integrin β4 on breast cancer cells and its ligand, hCLCA2,⁵¹ on lung endothelium, (c) growth or survival in the metastatic site, e.g., the expression of PTHrP in prostate, lung, and breast cancer cells³² mediates tumor growth in the bone, (d) angiogenesis at the metastatic site, e.g., integrin αvβ3 activations leads to increased VEGF production and angiogenesis in brain metastases but not the primary tumor,⁵⁸ (e) the premetastatic niche, e.g., VEGF and PIGF mediate formation of the premetastatic niche⁸⁰ and, (f) the ability of cells to extravasate into a specific organ, e.g., expression of ST6GALNAC5 in breast cancer cells increases extravasation through the BBB.¹⁶ The evidence that genes involved in the cyclical interaction of primary tumors, their metastases, and the cellular microenvironment involved in organotropism indicates this process requires a coordinated effort. Indeed many of the genes identified functioned in coordination to increase metastasis to a specific organ.^16-18 When multiple gene signatures and/or clinical variables are combined, the ability to predict the organ of metastasis is greatly increased.²⁷ Individual genes found to play a role in organotropism can allow for the development of targeted therapy for specific metastases; however, since a coordinated effort is required for metastasis to a specific organ, a multi-therapeutic approach is needed to prevent and inhibit metastatic growth. Several recent studies indicate that loss of specific miRNAs play a role in organotropism and provide a selection advantage for cells in specific organs,^43,70,75 suggesting that expression of certain miRNAs may be responsible for some of the coordinated gene expression required for organotropism. We are just now beginning to understand the mechanisms involved in the arrival and growth of cancer cells to specific organs. Future research will work toward defining the organotropic genes more extensively in cancers, determining when or if these genes are activated in the primary tumor cells or in the tumor cells at the metastatic site, and discovering new therapies and targets to prevent the occurrence of potentially fatal metastases.

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