Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

Summary

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

Abstract

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Introduction

Overt cancer metastasis in the clinic comprises a series of complex and multi-step events known as the "metastatic cascade". The cascade has been extensively reviewed and can be dissected into successive steps: local invasion, intravasation, dissemination, arrest, extravasation, and colonization^1,2. A better understanding of the pathogenesis of cancer metastasis and the development of potential treatment strategies in vivo require robust host models of cancer cell spread. Rodent models are well established and are widely used to evaluate metastasis³, but these approaches have low efficiency and ethical limitations and are costly as a forefront model to determine whether a particular manipulation could affect the metastatic phenotype. Other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. Due to their high genetic homology to humans and the transparency of their embryos zebrafish (Danio rerio) have emerged as an important vertebrate model and are being increasingly applied to the study of developmental processes, microbe-host interactions, human diseases, drug screening, etc.⁴. The cancer metastasis models established in zebrafish may provide an answer to the shortcomings of rodent models^5,6.

Although spontaneous neoplasia is scarcely seen in wild zebrafish⁷, there are several longstanding techniques to induce the desired cancer in zebrafish. Carcinogen-induced gene mutations or signaling pathway activation can histologically and molecularly model carcinogenesis, mimicking human disease in zebrafish^7,8,9. By taking advantage of diverse forward and reverse genetic manipulations of oncogenes or tumor suppressors, (transgenic) zebrafish have also enabled potential studies of cancer formation and maintenance^6,10. The induced cancer models in zebrafish cover a broad spectrum, including digestive, reproductive, blood, nervous system, and epithelial⁶.

The utilization of zebrafish in cancer research has expanded recently due to the establishment of human tumor cell xenograft models in this organism. This was first reported with human metastatic melanoma cells that were successfully engrafted in zebrafish embryos at the blastula stage in 2005¹¹. Several independent laboratories have validated the feasibility of this pioneering work by introducing a diverse range of mammalian cancer cells lines into zebrafish at various sites and developmental stages⁵. For example, injections near the blastodisc and blastocyst of the blastula stage; injections into the yolk sac, perivitelline space, duct of Cuvier (Doc), and posterior cardinal vein of 6-h- to 5-day-old embryos; and injections into the peritoneal cavity of 30-day-old immunosuppressed larvae have been performed^5,12. Additionally, allogeneic tumor transplantations were also reported in zebrafish^12,13. One of the great advantages of using xenografts is that the engrafted cancer cells can be easily fluorescently labeled and distinguished from normal cells. Hence, investigations into the dynamic behaviors of microtumor formation¹⁴, cell invasion and metastasis^15,16,17, tumor-induced angiogenesis^15,18, and the interactions between cancer cells and host factors¹⁷ can be clearly visualized in the live fish body, especially when transgenic zebrafish lines are applied⁵.

Inspired by the high potential of zebrafish xenograft models to evaluate metastasis, we demonstrated the transvascular extravasation properties of different breast cancer cell lines in the tailfin area of Tg (fli:EGFP) zebrafish embryos through Doc injections¹⁶. The role of transforming growth factor-β (TGF-β)¹⁶ and bone morphogenetic protein (BMP)¹⁹ signaling pathways in pro-/anti-breast cancer cell invasion and metastasis were also investigated in this model. Moreover, we also recapitulated the intravasation ability of various breast cancer cell lines into circulation using xenograft zebrafish models with perivitelline space injections.

This article presents detailed protocols for zebrafish xenograft models based upon the injection of human breast cancer cells into the perivitelline space or Doc. Using high-resolution fluorescence imaging, we show the representative process of intravasation into blood vessels and the invasive behavior of different human breast cancer cells, which move from the blood vessels into the avascular tailfin area.

Protocol

All research using the transgenic fluorescent zebrafish Tg (fli:EGFP) strain, which has enhanced green fluorescent protein (EGFP)-labeled vasculature²⁰, including housing and experiments, was carried out according to the international guidelines and was approved by the local Institutional Committee for Animal Welfare (Dier Ethische Commissie (DEC) of the Leiden University Medical Center.

NOTE: As summarized in Figure 1, the protocol is roughly broken down into four steps: embryo collection (Figure 1A), microinjection (Figure 1B), screening (Figure 1C), and analysis (Figure 1D).

1. Prepare the Injection Needles

1. Prepare injection needles with borosilicate glass microcapillaries. Put a microcapillary into a micropipette puller device with the following settings: air pressure, 500; heat, 650; pull, 100; velocity, 200; time, 40. Keep the injection needles in a needle holder plate until they are used for injection.

2. Prepare the Fluorescent, Genetically Labeled Breast Cancer Cells for Injection

1. Culture human breast cancer MDA-MB-231 cells at 37 °C in DMEM-high glucose medium containing L-glutamine, 10% fetal bovine serum, and 1:100 penicillin-streptomycin (pen-strep).
2. Culture the breast epithelial cell lines, MCF10A (M1) and MCF10A-Ras (M2), at 37 °C in DMEM/F12 media containing L-glutamine with 5% horse serum, 20 ng/mL epidermal growth factor, 10 mg/mL insulin, 100 ng/mL cholera enterotoxin, 0.5 mg/mL hydrocortisone, and 1:100 pen-strep.
3. Produce mCherry lentivirus by co-transfecting PLV-mCherry, pCMV-VSVG²¹, pMDLg-RRE (gag/pol)²², and pRSV-REV²² plasmid into HEK293T cells. Harvest cell supernatants 48 h after transfection and store at -80 °C.
4. Infect MDA-MB-231, M1, and M2 cells at 30% confluence for 24 h with lentiviral supernatants diluted 1:1 with normal culture medium in the presence of 5 ng/mL polybrene.
5. Select single-cell clones by diluting cells in a 96-well plate, which allows the outgrowth of isolated cell clones, until obtaining the stable mCherry-expressing cell lines.
6. Culture one T75 flask of cells for injection. Harvest the cells at 80% confluence with a 0.5% trypsin-EDTA treatment. Wash the cells with 1x PBS 2-3 times.
7. Re-suspend the cells in about 200 µL of PBS. Store at them 4 °C for less than 5 h before injection.

3. Prepare Zebrafish Embryos for Injection

1. Set up zebrafish breeding pairs and collect embryos, as shown in a previous JoVE article by Rosen et al.²³.
2. Select the embryos that are at 0-4 hpf by removing the unfertilized and abnormal embryos. Keep the embryos in a Petri dish full of egg water (60 µg/mL sea salts; ~60 embryos/dish) and incubate at 28 °C.
3. Dechorionate the embryos with fine tweezers at 48 hpf.
4. Anesthetize the embryos by transferring them to 40 µg/mL tricaine (3-aminobenzoic acid) containing egg water approximately 2 min prior to injection, but no longer than 2 h prior to injection.
   NOTE: Tricaine stock solution (4 mg/mL, 100x) is prepared as 400 mg of tricaine powder in 97.9 mL of double-distilled water and 2.1 mL of 1 M Tris-base (pH 9), with the pH adjusted to 7.4. Store in the -20 °C freezer.

4. Inject Human Breast Cancer Cells into the Perivitelline Space

1. Load 15 µL of the cell suspension into an injection needle. Mount the needle onto the micromanipulator and break off the needle tip with fine tweezers to obtain a tip opening diameter of 5-10 µm.
2. Use a pneumatic picopump and a manipulator to perform the microinjection. Adjust the picopump to inject 400 cells each time. Prior to injection, count the cell numbers manually by injecting the cells on the top of a Petri dish containing 1% agarose.
3. Line up anesthetized embryos (2-3 days post fertilization (dpf)) on a flat, 1% agarose injecting plate, around 10 embryos each time.
4. Orient the injection plate by hand during the injections to place the embryos in the preferred position for inserting the needle (i.e., diagonally).
5. Point the needle tip at the injection site and gently insert the needle tip into the perivitelline space between the yolk sac and the periderm of the zebrafish embryo (Figure 2A).
6. Inject approximately 400 mCherry-labeled tumor cells. Make sure that the yolk sac is not ruptured to avoid implantation into the yolk sac.

5. Inject Human Breast Cancer Cells into the Doc

1. Prepare the injection needle and zebrafish embryos as described in protocol steps 1, 2, and 3.
2. Use a 45° needle angle so that the Doc can be approached from the dorsal side of the embryo.
3. Insert the needle into the starting point of the Doc (Figure 3A), just dorsal to where the duct starts broadening over the yolk sac, and inject approximately 400 cells; the injection is correct if the volume within the duct expands directly after the pulse and the yolk sac.
   NOTE: Several consecutive injections can be performed without extracting the needle.
4. Transfer the injected zebrafish embryos to egg water.
   ​NOTE: As considerable variation exists among individual zebrafish embryos, and as the death of embryos after injection can occur, a relatively large number of zebrafish embryos (around 100) should be injected with cancer cells.
5. Maintain the zebrafish embryos at 33 °C to accommodate the optimal temperature requirements for fish and mammalian cells.

6. Screen the Injected Embryos

1. Screen each fish under a fluorescence stereomicroscope at 2 h post-injection (hpi) for the perivitelline space injection (Figure 2) or at 2-24 hpi for the Doc injection (Figure 2) to ensure that all of the embryos are injected with a similar number of tumor cells. Remove the embryos with injection errors, such as ruptures (Figure 2B) or injections (Figure 3B) of the yolk sac, and pick out embryos with injected cells below (Figures 2C and 3B) or above (Figures 2D and 3B) threshold. Keep only the embryos with approximately 400 cells in culture.
2. Rule out the possibility that the cells are introduced directly into circulation during the injection process by removing the embryos with cells already in circulation. Also, remove any embryo with a cell mass close to the Doc (Figure 2D).

7. Image and Analyze the Metastatic Process

1. Collect several anesthetized embryos with a wide-tip Pasteur pipette and transfer them to the glass bottom of a polystyrene dish.
2. Remove excess water and keep a limited amount of egg water. Manipulate the embryo into position with a hair loop tool and place a cover on top of the glass.
3. Use an inverted confocal microscope in combination with water-immersion or long-distance dry objectives. Position the embryo such that the region of interest is as close to the objective as possible.
4. Perform imaging immediately after anesthesia to reduce the risk of death due to liquid evaporation.
   1. Capture signals from EGFP-labeled vasculature and mCherry-labeled tumor cells at the same position on the embryos to co-register injected cells with blood vessels by merging the two imaging channels.
   2. For each zebrafish embryo, collect two different sets of images from the head region and tail region.
5. Quantify the number of disseminated cells.
   1. For perivitelline space injections, count the number of cells in each fish that have disseminated from the cell mass towards the embryonic fish body within the head and tail regions^4,15; the regions are beyond the boundaries of the heart cavity frontally, on top of the swim bladder dorsally, and beyond the urogenital opening caudally.
   2. For the Doc injection, count the number of individual cells that have invaded the collagen fibers of the tailfin from circulation (MDA-MB-231) or the number of clusters formed by cells collectively (M2) in the caudal hematopoietic tissue (CHT) of each zebrafish¹⁹.
6. Study invasion and metastasis in more detail by using confocal microscopy (highly recommended).
   1. Use low magnification (4X objective) to image the whole body and to obtain an overview of the tumor cell dissemination pattern.
      ​NOTE: Higher magnification (20X and 40X objectives) is suitable for studying intra- and peri-tumoral angiogenesis and the precise localization of disseminated cells in the embryo body.
   2. Use a 488 nm laser to scan the zebrafish embryo vasculature and a 543 nm laser to scan implanted tumor cells labeled with red fluorescence. Obtain a high-quality image by scanning each embryo in eight to ten steps. Scan and average each step six times.
7. Carefully place the embryo back into the egg water if it is required for further experiments.

8. Perform Statistical Analysis Using One-way Analysis of Variance (ANOVA) Followed by Post Hoc Analysis

Results

In the embryonic xenograft zebrafish model with a perivitelline space injection, the hematogenous dissemination of labeled cancer cells in the fish body is considered as active migration. This process can be detected and quantified under a fluorescent microscope, as described in the methods above. To illustrate this xenograft model, we followed the dissemination process of different breast cancer cell lines with known (or without) invasion/metastasis potential according to in vitro

Discussion

Here, we described two methods to investigate the invasive behavior of breast cancer cells in Tg (fli1:EGFP) zebrafish embryos, with perivitelline space and Doc injections. By injecting cancer cells labeled with chemical dye or fluorescent protein into transgenic zebrafish embryos, the dynamic and spatial characteristics of invasion and metastasis can be clearly tracked in real-time at the single-cell or cluster level under a fluorescence microscope. In most cases, the rapid progression of metastasis in zebrafis...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose	MP Biomedicals	AGAF0500
Borosilicate glass capillary	Harvard Apparatus	300038
Cholera enterotoxin	Calbiochem	227035
Confocal microscope	Leica	SP5 STED
DMEM-high glucose media containing L-glutamine	ThermoFisher Scientific	11965092
DMEM/F-12 media containing L-glutamine	ThermoFisher Scientific	21041025
Dumont #5 forceps	Fine Science Tools Inc	11252-20
Epidermal growth factor	Merck Millipore	01-107
Fetal bovine serum	ThermoFisher Scientific	16140071
Fluorescent stereo microscope	Leica	M165 FC
HEK293T cell line	American Type Culture Collection	CRL-1573
Hydrocortisone	SigmaAldrich	227035
Horse serum	ThermoFisher Scientific	26050088
Insulin	SigmaAldrich	I-6634
MCF10A (M1) cell line			Kindly provided by Dr. Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA)
MCF10Aras (M2) cell line			Kindly provided by Dr. Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA)
MDA-MB-231 cell line	American Type Culture Collection	CRM-HTB-26
Manual micromanipulator	World Precision Instruments	M3301R
Micropipette puller	Sutter Instruments	P-97
Wide-tip Pasteur pipette (0.5-20 µL)	Eppendorf	F276456I
pCMV-VSVG plasmid			Kindly provided by Prof. Dr. Rob Hoeben (Leiden University Medical Center, Leiden, The Netherlands)
Penicillin-Streptomycin (10,000 U/mL)	ThermoFisher Scientific	15140122
PLV-mCherry plasmid	Addgene	36084
pMDLg-RRE (gag/pol) plasmid			Kindly provided by Prof. Dr. Rob Houben (Leiden University Medical Center, Leiden, The Netherlands)
Pneumatic picoPump	World Precision Instruments	SYS-PV820
Polybrene	SigmaAldrich	107689
Prism 4 software	GraphPad Software
pRSV-REV plasmid			Kindly provided by Prof. Dr. Rob Hoeben (Leiden University Medical Center, Leiden, The Netherlands)
Stereo microscope	Leica	MZ16FA
Tg (fli:EGFP) zebrafish strain			Kindly provided by Dr. Ewa Snaar-Jagalska (Institute of Biology, Leiden University, Leiden, The Netherlands)
Tris-base	SigmaAldrich	11814273001
Tricaine (3-aminobenzoic acid)	SigmaAldrich	A-5040
Trypsin-EDTA (0.5%)	ThermoFisher Scientific	15400054
Petri dishes, polystyrene (60 × 15 mm)	SigmaAldrich	P5481-500EA
Polystyrene dish with glass bottom	WillCo	GWST-5040