Live imaging in zebrafish reveals neu(trophil) insight into the metastatic niche

Abstract

Non-cancerous immune cells can significantly contribute to tumour progression and metastases. Neutrophils associated with tumours can both promote and inhibit tumour progression, but less is known about how non-associated immune cells contribute to cancer biology. In a recent issue of the Journal of Pathology, He and colleagues use non-invasive, high-resolution imaging of the whole living animal to provide a compelling glimpse at how physiological migration of neutrophils can prepare a metastatic niche and how their activities can be altered by the unintended consequences of targeted therapeutics. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

How cancer cells establish metastases and respond to therapy are fundamental questions in cancer research 1. Metastases are cancer cells that have disseminated from the primary tumour, established themselves at a distant site and formed small nodules (micrometastases) or grown into macroscopic tumours (macrometastases). Most cancer deaths are due to metastatic spread and survival rates for patients with metastatic disease are bleak 2. There are few effective treatments for metastatic cancers and most show acquired or inherent therapeutic resistance. The series of events that permit metastases are complex and involve genetic changes within the tumour, access to circulation, proinvasive inflammatory cells and a permissive microenvironment 1–3. Permissive microenvironments are critical for metastases formation and are thought to evolve via active reciprocal signalling between tumour and stromal cells 1, 4, 5. However, some microenvironments, called pre-metastatic niches, are already primed for metastases, such as by tumour-secreted factors or by pre-existing physiological characteristics 5, 6.

One of the important research tools to address the biology of metastasis is vital imaging. In the mouse, intravital imaging—including imaging windows, fluorescent reporters, labelled small molecules and photoswitchable proteins—has allowed remarkable visualization of cancer cells in the physiological setting 7. More recently, imaging in zebrafish has provided a complementary approach. Small, vertebrate aquarium fish, zebrafish have become an important model system for cancer, immune and stem cell biology. In addition to the benefits of the relative ease and low cost of animal husbandry, zebrafish (∼2–5 cm as adults; 1–4 mm as early embryos) can be studied in the adult or embryonic/larval forms. Adult zebrafish develop cancers that share histological and molecular pathways similar to those of human cancers, and cancers can develop following carcinogenic treatments and/or through genetic modification of cancer genes 8. Cancers can be serially transplanted and, while the pigmented stripes (for which the zebrafish is named) obscure intravital imaging in the adult, transparent zebrafish platforms have been developed that permit the imaging of cancer within the living adult fish 9, 10. Cancer cells can also be transplanted into the embryonic and larval stages 11–14. This approach has a variety of advantages, including transparency, a small size that permits imaging of the entire animal system and treatment with small molecules 15–17. Cancer cells injected into the fish embryo, or cancer cells developing in situ, can be visualized undergoing the fundamental processes of cancer biology and, when coupled with fluorescent transgenic reporter lines, can provide unprecedented detail of the dynamic interactions between host and cancer cells 12.

In their study reported in the Journal of Pathology, He and colleagues use the zebrafish system to visualize human cancer cells undergoing invasion and metastasis in living animals, and report an important role for neutrophils in the development of the metastatic niche 18. Key to their success is the use of fluorescent transgenic lines that permit the visualization of the vasculature (fli1–GFP), transplanted cancer cells (dsRed or mCherry) and myeloid cells (mpx–GFP). Using these tools, they show the development and vascularization of small tumours after transplantation of human cancer cells, and also that cancer cells can disperse throughout the animal via the vascular system. Critically, the authors observe that only a subset of circulating cancer cells form micrometastases and always in the same tissue, indicative of a metastatic niche. Close examination reveals that neutrophils actively migrate at this site and leaving tracks in the collagen matrix, inadvertently leaving a path for tumour cell invasion and micrometastases. As discussed below, the authors then go onto show that the pharmacological alteration of neutrophil migration can have important consequences for the establishment of micrometastases.

He and colleagues 18 present three advances in the field of cancer biology, all of which build on imaging. First, they present a reproducible and refined protocol for embryonic zebrafish xenograftment (Figure 1). He and colleagues inject fluorescently labelled cancer cells directly into a large channel called the duct of Cuvier that directs blood flow toward the embryonic heart. From here, the cancer cells either form a small, vascularized tumour or are pumped into the perivasculature. Both normal and tumour vasculature are clearly visible because the experiments are done in transgenic zebrafish expressing the endothelial marker fli1–GFP. Cancer cell transplantation into the zebrafish embryo is a powerful technique that has led to the identification of critical cancer signalling pathways and tumour cell reprogramming and the visualization of host–cancer cell interactions 5, 13, 19–21. The xenografting protocol described by He and colleagues provides an approach that bypasses the early proliferation stages of tumour formation (although localized tumours do grow) and focuses on the biology of the metastatic processes once cancer cells have entered the blood stream.

Figure 1. Zebrafish as a model system to study micrometastasis. Human cancer cells (red) are injected into the blood supply of zebrafish embryos and form tumours at that site, as well as micrometastases at a distant site in the tail fin. Microscopic analysis indicates that cancer cell invasion (red cells) into the tail fin tissue is facilitated by the collagen matrix conditioning (light gray) of migrating neutrophils (beige cell)

The second advance by He and colleagues 18 is that they shed light on the biology of the metastatic niche. Because the authors can visualize the entire animal for each experiment, He and colleagues can carefully examine where micrometastases form. They find that while cancer cells disseminate and undergo extravasation in a non-biased fashion throughout the embryo, micrometastases only form at the caudal hematopoietic tissue (CHT; a site of haematopoiesis and leukocyte differentiation) and associated tail fin tissue (Figure 1). At this stage in zebrafish development, neutrophils and macrophages are the only functional leukocytes, and neutrophils can be clearly visualized in live animals using a myeloid-specific peroxidase (mpx)–GFP transgenic line 22. Using time-lapse imaging, the authors show that neutrophils randomly migrate between the CHT and the tail fin. As the neutrophils do so, they make tracks in the collagen fibres that are closely associated with invading cancer cells. Cancer cell invasion and micrometastasis formation are dependent on neutrophil migration, because when neutrophils are genetically knocked down, or when the embryos are treated with beclomethasone, a known inhibitor of neutrophil migration, there are reduced metastases. The take-home message from these observations is that neutrophil movement is important for establishing the metastatic niche, presumably by preconditioning the collagen matrix. Interestingly, the development of this metastatic niche in zebrafish is not a pathological process and is not enhanced by activating neutrophils through wounding. Rather, the neutrophils appear to be ‘neutral’ to the presence of cancer cells, and it is their normal migration that inadvertently contributes to the development of the metastatic niche. While the authors use multiple metastatic cell lines in their work, determining whether all cancer cells have the potential to migrate on neutrophil tracks or whether only subpopulations of cells have this ability will be important future question to explore.

The third advance by He and colleagues 18 addresses how targeted therapies affect tumour growth and metastases. As in human tumours, the tumours that develop in the xenografted embryo are vascularized with a tortuous matrix of blood vessels. Therapies that target the vascular endothelial growth factor (VEGF) pathways would ideally lead to a reduction in tumour burden 23, 24. Indeed, for some patients, VEGF pathway inhibitors have led to a meaningful improvement in outcome 25, 26. However, for many patients resistance inevitably occurs, and this can be associated with increased spreading in some tumour types 25–27. Evidence from preclinical mouse models indicate that while tumours are sensitive to VEGF pathway treatment, they can become more invasive and metastatic in the process 23, 25, 28–30. He and colleagues present a similar intriguing observation using their zebrafish xenograft model. They find that VEGFR inhibitors successfully reduce vascularization of tumours but concomitantly increase micrometastases formation. Careful tracking of neutrophil movement reveals that VEGFR inhibitor treatment enhances the movement of neutrophils, thereby indirectly developing the metastatic niche.

Studies in animal models raise important questions about how the results in a model system can be applied to our understanding of human disease. Zebrafish are a powerful system for imaging cancer biology within the whole animal, but also have significant biological differences from mammals. A future question to address from this work is the role ‘neutral’ neutrophils play in preparing the metastatic niche in mammals. Both tumour-promoting and tumour-antagonizing immune cells are often associated with tumours, including macrophages and neutrophils, and can contribute to tumour progression 1, 4, 31. These cells can also play a role in preparing metastatic microenvironments, creating an inflammation-like state that recruits tumour cells 5. Indeed, in another zebrafish cancer model, it is the H₂O₂-activated leukocytes that favour tumour cell growth 19, rather than the neutral immune cells seen in this study. Certainly, the immune system of a zebrafish embryo at the stages reported by He and colleagues is less complex than that of humans, mice or even an adult zebrafish, and we do not yet know whether this type of collagen remodelling occurs during zebrafish adult tissue homeostasis or is a feature of development. We do know, however, that there are at least some differences in the injection of cancer cells into the zebrafish compared with cancer cell development in situ 19. Nonetheless, it is becoming increasingly recognized that the zebrafish immune system shares many features with other vertebrates 22, suggesting that the immune–tumour cell interactions observed here and in other studies 19 may be relevant to mammals.

Another major question that emerges from this study is the identity of the molecular targets of the VEGF pathway inhibitors that lead to enhanced neutrophil movement. The authors argue that the VEGFR inhibitors are not targeting the collagen matrix directly, and it is the neutrophils themselves that are directly responding to the VEGFR inhibitor. While it is too early to know how VEGF pathway inhibitors cause increased micrometastases, it seems unlikely that the changes in this zebrafish model are due to disrupted vasculature 29. It seems more likely that similar activity may be at work a similar to that reported in preclinical mouse cancer models, where VEGF-pathway inhibitors can condition the metastatic niche even before tumour cell inoculation 32, 33. The mechanism of the preconditioning is unknown, but may include the up-regulation of circulating pro-angiogenic cytokines and growth factors 33. An important consideration for this study is that VEGFR inhibitors are known to target additional kinases and may have additional targets in vivo 34, and it is still not clear how the final intracellular concentrations in preclinical animal models relate to final concentrations in patients. Of course, once the target has been identified in the zebrafish system, it will be important to test whether the compound's target is also conserved in mammalian systems. While it is possible that the target is zebrafish-specific, it does appear that many of the responses induced by small molecules in zebrafish are conserved, and new uses for clinically active drugs identified in zebrafish are effective in mice and primates and are being tested in clinical trials [ 35, 36; LI Zon, personal communication].

Clinical trials are inherently different from laboratory model systems, involving individuals with diverse genetic and environmental backgrounds and different cancer types, often involving additional chemotherapeutic treatments. In the context of anti-VEGF therapy, it is still not clear how the increased metastases observed in mouse models and this zebrafish model relates to the responses seen in the clinic 23, 25. There is clearly much work to do to understand the role of anti-VEGF therapies in cellular homeostasis and cancer biology. Having multiple, tractable model systems to address these problems will surely enhance our understanding of how therapies function at the whole-animal systems level.

Acknowledgements

I am grateful to Professor Ian Jackson, Dr James Amatruda, Dr Nick Trede, Dr Jennifer Richardson, Dr Zhiqiang Zeng and Professor Paul Martin for critical reading of the manuscript. EEP is supported by Medical Research Scotland and the Medical Research Council.