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Evaluating human cancer cell metastasis in zebrafish
© Teng et al.; licensee BioMed Central Ltd. 2013
Received: 12 June 2013
Accepted: 24 September 2013
Published: 4 October 2013
In vivo metastasis assays have traditionally been performed in mice, but the process is inefficient and costly. However, since zebrafish do not develop an adaptive immune system until 14 days post-fertilization, human cancer cells can survive and metastasize when transplanted into zebrafish larvae. Despite isolated reports, there has been no systematic evaluation of the robustness of this system to date.
Individual cell lines were stained with CM-Dil and injected into the perivitelline space of 2-day old zebrafish larvae. After 2-4 days fish were imaged using confocal microscopy and the number of metastatic cells was determined using Fiji software.
To determine whether zebrafish can faithfully report metastatic potential in human cancer cells, we injected a series of cells with different metastatic potential into the perivitelline space of 2 day old embryos. Using cells from breast, prostate, colon and pancreas we demonstrated that the degree of cell metastasis in fish is proportional to their invasion potential in vitro. Highly metastatic cells such as MDA231, DU145, SW620 and ASPC-1 are seen in the vasculature and throughout the body of the fish after only 24–48 hours. Importantly, cells that are not invasive in vitro such as T47D, LNCaP and HT29 do not metastasize in fish. Inactivation of JAK1/2 in fibrosarcoma cells leads to loss of invasion in vitro and metastasis in vivo, and in zebrafish these cells show limited spread throughout the zebrafish body compared with the highly metastatic parental cells. Further, knockdown of WASF3 in DU145 cells which leads to loss of invasion in vitro and metastasis in vivo also results in suppression of metastasis in zebrafish. In a cancer progression model involving normal MCF10A breast epithelial cells, the degree of invasion/metastasis in vitro and in mice is mirrored in zebrafish. Using a modified version of Fiji software, it is possible to quantify individual metastatic cells in the transparent larvae to correlate with invasion potential. We also demonstrate, using lung cancers, that the zebrafish model can evaluate the metastatic ability of cancer cells isolated from primary tumors.
The zebrafish model described here offers a rapid, robust, and inexpensive means of evaluating the metastatic potential of human cancer cells. Using this model it is possible to critically evaluate whether genetic manipulation of signaling pathways affects metastasis and whether primary tumors contain metastatic cells.
Metastasis is the primary cause of human cancer mortality, accounting for >90% of deaths due to cancer . There is now abundant evidence that, independent of the process of cellular transformation, the metastasis phenotype is genetically controlled . Metastasis is a multistep process that involves local tumor invasion followed by dissemination to, and re-establishment at, distant sites. Families of genes have been described which have no effect on cell proliferation but which can suppress or promote metastasis [3, 4]. Thus, targeting metastasis may prove to be effective in reducing cancer mortality if specific targets can be identified that suppress this phenotype. Here, we present a robust in vivo system for rapidly and accurately evaluating the effectiveness of candidate suppressor molecules.
Much of the analysis of metastasis pathways is conducted in tightly controlled in vitro cell systems, usually involving overexpression or ablation of a particular gene. Assays such as wound healing, transwell motility, invasion assays and hanging drop assays have been developed which provide readouts of cellular phenotypes related to metastasis [5–7]. These assays, however, do not address the issue of intravasation of tumor cells into blood vessels and extravasation into distant organs, a process requiring an in vivo assay system. Typically, such assays are performed in mice using experimental or spontaneous metastasis models [8, 9]. While it is ultimately necessary to demonstrate that a pathway identified in vitro also affects invasion and metastasis in vivo, mouse models have significant drawbacks: 1) it is difficult to study early stages of the process where it is necessary to rapidly evaluate whether a particular drug or genetic manipulation has affected the metastasis phenotype, 2) evaluating the complete process in mice can require up to 6 months (depending on the cell system), 3) these experiments are expensive, immunosuppressed mice are required to study human cells and per diem charges in barrier facilities are costly, 4) in vivo imaging of small metastatic lesions is not possible in the deep tissues of the mouse, thus typically requiring termination and autopsy, thus extrapolation across experimental populations to realize the result, 5) popular immunosuppressed mice such as, nude (nu/nu), the severe combined immunodeficiency (SCID), or mice null for the recombination activating gene (Rag), have residual immune competence, which can actually prevent metastasis and, 6) the cohort size in these experiments is often pragmatically limited by high costs, thus statistical verification of metastasis modulation cannot be adequately assessed when the effect is mild.
Zebrafish provide an experimentally and genetically tractable animal model of a wide variety of human diseases . Recent studies have demonstrated that zebrafish form spontaneous tumors with similar histopathological and gene expression profiles as human tumors [11–13]. The zebrafish-cancer model overcomes the drawbacks of murine xenograft models and offers alternative options for studying human tumor angiogenesis and metastasis [14–21]. Following early reports of the application of zebrafish to evaluate metastasis , we now tested whether metastasis in fish faithfully reports the metastatic potential of a broad range of cancer cells. To do so, we correlated in vitro invasion efficacy to in vivo metastasis metrics following manipulation of the metastatic phenotype. Without exception, we show that gene manipulations that affect in vitro invasion, alter metastasis in fish in a corresponding manner, demonstrating that the zebrafish is a tractable model to assay metastatic potential of human cancer cells. We also show that primary human cancer cells can metastasize in fish and that this ability can be used to predict metastatic potential in a clinical setting.
The endogenous metastasis phenotype of human cancer cells is maintained in zebrafish
We have also shown previously that DU145 prostate cancer cells invade in vitro and metastasize in vivo in mouse models  compared with LNCaP cells which do not. As shown in Figure 1b, DU145 cells metastasize throughout the body of the fish at 30 hpi but LNCaP cells do not. The same correlation was seen for invasive SW620 and non-invasive HT29 colon cancer cells (Figure 1c), where again the correlation between invasion and metastasis in the fish was observed. Finally we studied the pancreatic ductal adenocarcinoma cells AsPC-1 and BxPC3 (Figure 1d). In this case, the AsPC-1 cells showed higher invasion potential than BxPC3 in the fish model which was consistent with in vitro invasion assays (Figure 1d). The extent of metastasis in fish for the highly invasive cells lines was readily apparent with large numbers of cells throughout the body of the fish (>25) after 24–48 hours. In contrast, the cells with low metastatic potential rarely showed cells in the body of the fish. These correlations were consistent within the cohort of fish used for each experiment. To evaluate the metastatic potential, therefore, we determined the number of fish that showed metastasis in the four different cancer cell systems, compared with the number that did not. In the highly invasive cell lines, MDA231 (n = 58), DU145 (n = 72), SW620 (n = 63) and ASPC-1 (n = 57), metastasis was seen in 97%, 89%, 92% and 84% of fish respectively (Figure 1e). In contrast, in the poorly invasive cells, T47D (n = 64), LNCaP (n = 53), HT29 (n = 84) and BxPC3 (n = 42) metastasis was seen in only 9%, 15%, 14% and 26% of fish respectively (Figure 1e). We then performed quantitative invasion assays (see Methods) for all of these cell lines (Figure 1e) where the relative proportion of invading cells mirrored the distribution of metastasis in the in vivo fish model.
To demonstrate that cells from the perivitelline cavity could intravasate into the circulation and extravasate into the fish body, we used the Tg(kdrl:EGFP) transgenic zebrafish which highlights the vasculature (Figure 1f). In this study, using invasive MDA231 cells, for example, we could clearly see CM-Dil-labeled human cancer cells both in the vasculature and in the body of the fish adjacent to the vasculature (Figure 1f). This analysis demonstrated that the human cancer cells showed the range of phenotypes associated with metastasis. In contrast, the non-invasive T47D cells, were never seen in the vasculature of the host fish, nor in tissues distant from the injection site (Figure 1a). Thus, these data suggest that zebrafish can robustly report metastasis potential of different types of human cancer cells.
Assessment of the metastatic potential of primary human cancer cells in zebrafish
The genetic regulation of tumor metastasis is maintained in zebrafish
Quantitation of metastasis in zebrafish using Fiji algorithms
Zebrafish show variation in metastasis potential related to tumor cell progression
Suppression of oncogenic kinases affects metastasis in zebrafish
Dissection of the functional aspects of genes that impact the metastasis phenotype requires a robust assay for tumor spread. While it is accepted that, in the final analysis, rodent models should be used to evaluate metastasis, this approach is costly and inefficient as an up-front assay to determine whether a particular genetic manipulation affects the metastasis phenotype. The spontaneous metastasis assay [8, 9] has shortcomings, since it cannot be used to evaluate intravasation into the blood vessels. The zebrafish model, on the other hand provides a solution to the time-consuming and costly mouse experiments, since in many cases the assay can be performed within 24–36 hours of xenotransplantation, in large cohorts of fish, providing statistical power to the results. Although we have only studied cancer cells from breast, pancreas, colon and sarcomas thus far, in all cases in vitro invasion ability correlated with the metastatic potential of tumor cells to spread in vivo. Importantly, we have shown that genetic manipulations of human cancer cells which affect invasion, also affect metastasis in fish. Although the technical dexterity needed to inject 48 hpf zebrafish can be demanding, the absence of an adaptive immune response for the first 14 days post fertilization (dpf)  avoids side effects and dosing issues related to using immunosuppressants [34, 35]. Early studies targeting the yolk sac as a site of injection truly challenged cancer cells to enter the blood stream. The blood supply to the yolk sac is extensive since this sustains the fish for the first 5 dpf and maximizes the opportunity for intravasation. It has been shown using the cloche mutant fish, which do not develop a vasculature or circulation, that metastatic human cells injected into the yolk sac cannot metastasize in these fish, demonstrating the requirement for a functional circulatory system in this process . Injection into the yolk sac, however, has complications apparently associated with poor resealing of the yolk sac membrane which leads to spillage of the cancer cells or yolk sac contents. The perivitelline space between the body of the fish and the yolk sac provides an alternative, which does not suffer from these associated problems. The technical challenge is successfully targeting the perivitelline space, and avoiding injection directly into the circulatory system. For this reason we examined fish after 12 hours for the presence of cells in the vasculature and excluded these fish from the analysis. Since the injection involves large numbers of fish, excluding those that have been compromised during the injection process does not have any impact on the final analysis.
Although zebrafish have been used as a model for metastasis previously, protocols between different groups were not consistent in terms of the number of cells injected, the site of injection, the age of the fish used and the method of quantitation of metastasis [18, 22]. To evaluate this metastasis model more robustly, we have used a standardized protocol with a variety of different cancer cells and cell systems. In this report we clearly demonstrate that metastasis in the zebrafish correlates with in vitro invasion assays and in one case (DU145 cells, Figure 3) with metastasis potential in murine models of metastasis. We observed that metastatic spread in the fish was achieved as early as 24 hpi, and possibly sooner, and that the maximum tumor spread was achieved within 48 hours in most cases, without a significant increase over subsequent days. A reduction in the numbers of cancer cells, however, occurred when the analysis was extended to 5 dpi. The metastasis assay, however, if initiated at 2 dpi, can be completed before it is necessary to feed the fish (4 dpi) and, at least in the cells we tested, do not need to be followed for more that 2–3 days to evaluate metastasis. In prior studies, the number of cells injected into the fish varied between 50–2000 [14–17, 19–22]. We observed that injecting too many cells can lead to mortality in our system and, following preliminary evaluation of the optimal number to demonstrate metastasis, we consistently injected cells ~300 cells per fish.
In many of our studies, the difference in the number of disseminated cells between the metastatic and non-metastatic tumors was usually striking. The same was observed in experimental cell systems where inactivation of a particular gene led to almost complete loss of invasion or metastasis, e.g. the JAK1 deficient 2C4 cells or the DU145 WASF3 knockdown cells. In these experiments, however, cells were seen outside the yolk sac region, which raised the issue of how metastasis is defined? In cell lines such as T47D, LNCAP and HT29, which are generally considered non-metastatic, our analysis showed that, even if there were disseminated cells, in the majority there were usually only between 1–5 cells in the body of the fish, where 5 cells was the exception. There are several reasons why small numbers of cells may appear in the fish body. Firstly, on rare occasions, the injection procedure could have inadvertently penetrated the vasculature and cells were introduced directly, although we screened all fish 12 hours after injection and excluded any that already showed cells outside the yolk sac region to overcome this being a major factor in the analysis. In practice this was only a very small number (<2%) for each cohort. It is also possible that cells defined as non-metastatic, are in fact weakly metastatic, and so occasional cells will disseminate into the fish. This is particularly true in experimental systems when, for example, shRNA knockdown of a particular gene is not complete, leaving some cells with gene expression levels above the threshold that will allow metastasis. It is important to note, however, that in many of our experiments involving apparently non metastatic cells, the presence of >5 disseminated cells was only seen in a minority of fish in the cohort. The main criterion for metastasis, therefore, is the presence of >5 cells in the majority of fish. In practice, however, the numbers of metastatic cells throughout the various cohorts for metastatic cells was far greater than 5 as shown in Figure 4 (35–55 cells after 48 hours). The number of disseminated cells becomes particularly important when defining relative metastatic potential, as seen in the MCF10A continuum. In this case the overall number of cells found outside the yolk sac area correlated with the invasiveness of the cells in vitro. We expect, however, most metastasis assays will want to determine whether ablation or overexpression of a gene leads to changes in metastatic potential, and following the protocol described here will facilitate this determination.
Although we were limited by the number of clinical samples available to us, the demonstration that primary human cancer cells can survive, grow, and metastasize in zebrafish provides a very encouraging proof-of-principle and opens opportunities to evaluate the metastatic potential in primary cells from biopsies or following surgery, which can have important advantages for clinical management of the patient. Even though it may take 2–3 weeks to establish the primary cell cultures, the presence of metastatic cells in a tumor for which there is a well-differentiated histopathology, could affect the future screening and management protocol. In addition, the well- established systems [36–38] for drug screening in zebrafish, opens up the possibility of identifying therapeutics that can target metastasis on a tumor-by-tumor basis, so providing a personalized approach to individual tumors.
In summary, we provide a side-by-side critical evaluation showing that zebrafish can evaluate the metastatic potential of human cancer cell lines and primary tumors. The ability to perform these analyses in large cohorts of fish allow for robust statistical analysis in a short time frame (24–120 hours) and provides a rapid means of evaluating whether genetic manipulation of cells, or cell origin, affects metastasis in vivo. Where it has been investigated, the metastasis phenotype in fish is identical to that in rodents with the advantages that individual cells can be imaged during the metastasis process.
Cell culture and primary lung cancer cell isolation
Human breast cancer cells (MDA231 and T47D), prostate cancer cells (DU145 and LNCaP), pancreatic ductal adenocarcinoma cells (AsPC-1 and BxPC3) and colon cancer cells (SW620 and HT29) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The parental human fibrosarcoma cell line 2C4, the JAK1-deficient U4C and JAK2-deficient γ2A derivatives were a gift from Dr. Ivo P. Touw (Erasmus University Medical Center, The Netherlands) and were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS). The normal MCF10A breast epithelial cell line (M-I) and its derivatives M-II, M-III and M-IV [27, 39], were a gift from Dr. Shuang Huang (Georgia Regents University, USA). The M-III and M-IV cells were maintained in DMEM/F-12 supplemented with 5% horse serum. The culture medium for the M-I and M-II was similar but also included 20 ng/ml recombinant human EGF (R&D Systems, Minneapolis, MN, USA), 0.5 μg/ml hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 10 μg/ml insulin (Sigma-Aldrich) and 100 ng/ml cholera toxin (Sigma-Aldrich). Primary lung cancer cells were obtained immediately after resection, disaggregated with trypsin and cultured in DMEM for 5–10 days.
In vitroinvasion assay and experimental metastasis mouse model
To measure cell invasion potential, matrigel invasion assays were performed as described previously [7, 25] using Transwells (BD biosciences, San Diego, CA, USA) with 8-μm pore size filters. The invading cells were fixed in 3.7% paraformaldehyde and stained with 0.5% crystal violet in 2% ethanol. The lower surface of the filter was photographed and the invading cells were counted from six fields at 200× magnification. Each experiment was performed in triplicate on at least three occasions. For experimental metastasis, 6-week-old male SCID mice were injected with 1 × 106 WASF3 knockdown DU145 cells or the knockdown control cells through the lateral tail vein . Mice were sacrificed 3 months after injection and the lung tissues were processed for hematoxylin and eosin (HE) staining. All experimental procedures were approved by the Animal Care and Use Committee of Georgia Regents University.
Cell preparation and transplantation
For cell labeling, cells were incubated with cell tracker CM-Dil (Invitrogen, Carlsbad, CA, USA) at a final concentration of 2.5 μg/ml for 4 min at 37°C followed by 15 min at 4°C. To remove unincorporated dye, cells were rinsed twice with phosphate-buffered saline (PBS), and then resuspended at a higher concentration (5 × 106 cells/ml). Before injection, CM-Dil labeled cells were assessed for viability using trypan blue exclusion and only samples in which there was >90% viability were used. We also evaluated the cells for uniform red staining and membrane integrity using a Zeiss Axiovert microscope (Zeiss, Thornwood, NY, USA) before being transplanted into the fish.
Zebrafish husbandry and the metastasis model
Zebrafish were maintained using established temperature and light cycle conditions as previously described [40, 41]. All experimental procedures were approved by the Animal Care and Use Committee of Georgia Regents University. The Tg(kdrl:EGFP) transgenic fish line was a gift from Dr. Daniel Wagner (Department of Biochemistry and Cell Biology, Rice University, USA). For zebrafish xenotrasplantation, 48 hpf wild-type AB or Tg(kdrl:EGFP) strains of transgenic zebrafish embryos were dechorionated and anaesthetized in 0.3× Danieau's solution containing phenythiourea (PTU, Sigma-Aldrich) and 0.04 mg/ml tricaine (Sigma-Aldrich) before human cell injection. Approximately 300 CM-Dil labeled human cells were injected into the perivitelline cavity of each embryo, and zebrafish were maintained in 0.3× Danieau's solution containing PTU for 1 h at 28°C. After confirmation of a visible cell mass at the injection site, zebrafish were transferred to an incubator and maintained at 34°C.
Living zebrafish embryos were anesthetized using 0.04 mg/ml tricaine and were then embedded in a lateral orientation in 0.5% agarose. Serial sections were captured using an Olympus FLUOVIEW™ FV1000 laser scanning confocal microscope (Olympus, Tokyo, Japan) and 2.5 μm z-step intervals. Low magnification (×4 objective) was used to provide an overview of the tumor cell metastasis pattern throughout the fish, and higher magnification (×20 objective) was used to define the precise localization of metastatic cells and foci within the zebrafish. Z-stack images were processed using ImageJ/Fiji as previously described [40, 42].
Automated cell counting and statistical analysis
The custom Fiji (ImageJ2) software package  was used for automated cell counting. Briefly, a 190–255 intensity threshold was set to select cells and the ‘analyze’ particle tool was used with default selection of cell size and cell shape during counting. A Fiji macro was generated using the ‘record’ function to streamline analyses and remove bias. To count the metastatic cell number in the tail, the fish tails were selected for cell counts using the polygon selection tool. A P-value of 0.05 or less was considered to be statistically significant and determined by the Student’s t-test. Values of three or more experiments were given as mean ± SEM.
This work was supported in part by grant CA120510 from the National Institutes of Health. Dr. JK Cowell is A Georgia Cancer Coalition Scholar. We are grateful for Dr. Z. Hao for providing the primary lung cancer cells.
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