In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish

Summary

Described here is a method for utilizing zebrafish embryos to study the ability of functionalized nanoparticles to target human cancer cells in vivo. This method allows for the evaluation and selection of optimal nanoparticles for future testing in large animals and in clinical trials.

Abstract

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal models are required for bridging the nanotechnology to its biomedical application. The mouse represents the traditional animal model for preclinical testing; however, mice are relatively expensive to keep and have long experimental cycles due to the limited progeny from each mother. The zebrafish has emerged as a powerful model system for developmental and biomedical research, including cancer research. In particular, due to its optical transparency and rapid development, zebrafish embryos are well suited for real-time in vivo monitoring of the behavior of cancer cells and their interactions with their microenvironment. This method was developed to sequentially introduce human cancer cells and functionalized nanoparticles in transparent Casper zebrafish embryos and monitor in vivo recognition and targeting of the cancer cells by nanoparticles in real time. This optimized protocol shows that fluorescently labeled nanoparticles, which are functionalized with folate groups, can specifically recognize and target metastatic human cervical epithelial cancer cells labeled with a different fluorochrome. The recognition and targeting process can occur as early as 30 min postinjection of the nanoparticles tested. The whole experiment only requires the breeding of a few pairs of adult fish and takes less than 4 days to complete. Moreover, zebrafish embryos lack a functional adaptive immune system, allowing the engraftment of a wide range of human cancer cells. Hence, the utility of the protocol described here enables the testing of nanoparticles on various types of human cancer cells, facilitating the selection of optimal nanoparticles in each specific cancer context for future testing in mammals and the clinic.

Introduction

The development of nanoparticles that are capable of detecting, targeting, and destroying cancer cells is of great interest to both physicists and biomedical researchers. The emergence of nanomedicine led to the development of several nanoparticles, such as those conjugated with targeting ligands and/or chemotherapeutic drugs^1,2,3. The added properties of nanoparticles enable their interaction with the biological system, sensing and monitoring biological events with high efficiency and accuracy along with therapeutic applications. Gold and iron oxide nanoparticles are primarily used in computed tomography and magnetic resonance imaging applications, respectively. While the enzymatic activities of gold and iron oxide nanoparticles allow the detection of cancer cells through colorimetric assays, fluorescent nanoparticles are well suited for in vivo imaging applications⁴. Among them, ultrabright fluorescent nanoparticles are particularly beneficial, due to their ability to detect cancers early with fewer particles and reduced toxicities⁵.

Despite these advantages, nanoparticles require experimentation using in vivo animal models for the selection of suitable nanomaterials and optimization of the synthesis process. Additionally, just like drugs, nanoparticles rely on animal models for preclinical testing to determine their efficacy and toxicities. The most widely used preclinical model is the mouse, which is a mammal whose upkeep comes at a relatively high cost. For cancer studies, either genetically engineered mice or xenografted mice are typically used^6,7. The length of these experiments often spans from weeks to months. In particular, for cancer metastasis studies, cancer cells are directly injected into the circulatory system of the mice at locations such as tail veins and spleens^8,9,10. These models only represent the end stages of metastasis when tumor cells extravasate and colonize distant organs. Moreover, due to visibility issues, it is particularly challenging to monitor tumor cell migration and nanoparticle targeting of tumor cells in mice.

The zebrafish (Danio rerio) has become a powerful vertebrate system for cancer research due to its high fecundity, low cost, rapid development, optical transparency, and genetic conservations^11,12. Another advantage of the zebrafish over the mouse model is the fertilization of the fish eggs ex utero, which allows the embryos to be monitored throughout their development. Embryonic development is rapid in zebrafish, and within 24 hours postfertilization (hpf), the vertebrate body plane has already formed¹³. By 72 hpf, eggs are hatched from the chorion, transitioning from the embryonic to the fry stage. The transparency of the zebrafish, the Casper strain in particular¹⁴, provides a unique opportunity to visualize the migration of cancer cells and their recognition and targeting by nanoparticles in a living animal. Finally, zebrafish develop their innate immune system by 48 hpf, with the adaptive immune system lagging behind and only becoming functional at 28 days postfertilization¹⁵. This time gap is ideal for the transplantation of various types of human cancer cells into zebrafish embryos without experiencing immune rejections.

Described here is a method that takes advantage of the transparency and rapid development of zebrafish to demonstrate the recognition and targeting of human cancer cells by fluorescent nanoparticles in vivo. In this assay, human cervical cancer cells (HeLa cells) genetically engineered to express a red fluorescent protein were injected into the vascularized area in the perivitelline cavity of 48 hpf embryos. After 20-24 h, HeLa cells had already spread throughout the embryos through the fish circulatory system. Embryos with apparent metastasis were microinjected with ~0.5 nL of a nanoparticle solution directly behind the eye, where the rich capillary bed is located. Using this technique, the ultrabright fluorescent silica nanoparticles can target HeLa cells as quickly as 20-30 min postinjection. Due to its simplicity and effectiveness, the zebrafish represents a robust in vivo model to test a variety of nanoparticles for their ability to target specific cancer cells.

Protocol

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Boston University School of Medicine under the protocol #: PROTO201800543.

1. Generation of Casper zebrafish embryos

1. Choose adult Casper fish that are at least 3 months of age for natural breeding to generate transparent Casper zebrafish embryos.
2. Fill two-chamber mating tanks with fish water in the evening, separate the upper tanks using dividers, place one male fish into one side of the chamber and one or two female fish into the other side of the chamber, and leave the fish separated overnight by dividers.
3. Pull out the dividers the next morning at 8:00 AM when the lights are on. Add artificial enrichment plants and tilt the top chamber slightly to create a shallow area of water. Allow the fish to breed for 3-4 h.
4. Lift the top chambers of the mating tanks that contain the fish and return them to their original tanks.
5. Collect eggs located in the bottom chambers by pouring the water through a mesh net. Transfer eggs to a sterile Petri dish at a density no greater than 200 eggs per dish. Remove any dead or unfertilized eggs and fill the dish 2/3 full with fresh fish water.
   NOTE: Fertilized and healthy eggs should be translucent and round. Any eggs that are cloudy, white, or disfigured should be removed. Fish water is obtained from fish tanks in the fish facility.
6. Incubate embryos in the incubator at 28.5 °C overnight.
7. Bleach the embryos the next morning using the standard protocol as described in The Zebrafish Book¹⁶, and put the embryos back to the incubator (optional step).
8. Take the 24 hpf embryos out of the incubator in the afternoon and dechorionate the embryos using pronase.
   1. Remove as much fish water as possible from the embryos in the Petri dish and add a few drops of the pronase solution (1 mg/mL in fish water) to the dish. Gently swirl the Petri dish. Once the chorions show signs of disintegration, pipette the embryos up and down a few times to break down the chorions to release the embryos.
   2. Add fresh fish water immediately into the Petri dish to terminate the process once a majority of the embryos are out of the chorions. Rinse the embryos 3x more using fish water to remove the floating chorions. Return the embryos to the incubator.

2. Preparation of human cancer cells for transplantation

1. Set the incubator temperature to exactly 35.5 °C. Monitor the incubator to ensure a consistent and stable temperature using a thermometer inside the incubator.
2. Autoclave 1 L of fish water in a glass bottle. Make a 3% agarose solution by adding 3 g of electrophoresis-grade agarose to 100 mL of autoclaved fish water and microwaving until the agarose is completely dissolved.
   1. Pour hot agarose solution into a Petri dish until it is 3/4 full. Place the microinjection mold on the agarose. Ensure that the mold is not in contact with the bottom of the Petri dish and no bubbles form underneath.
   2. Allow the solution to solidify and carefully remove the mold from the plate. Fill the plate with autoclaved fish water and store the plate at 4 °C. Prewarm the agarose plate and fish water in the 35.5 °C incubator before harvesting HeLa cells.
3. Pull 1.0 mm O.D. x 0.78 mm borosilicate glass capillaries on a pipette puller using the following settings: pressure at 500, heat at 560, pull at 100, velocity at 100, and time/delay at 200. Store needles on putty in a large Petri dish that has been wiped with an ethanol towel.
   CAUTION: Pulled needles are very sharp and fragile. Use caution when handling.
4. Prepare a stock solution of tricaine methanesulfonate (MS222, 4 mg/mL) by dissolving MS222 into autoclaved fish water. Vortex well before use. Dilute MS222 stock solution 1:100 in fish water (i.e., add 200 µL of MS222 stock solution to 20 mL of fish water to a final concentration of 40 µg/mL) to anesthetize embryos for the following procedures.
5. Culture hLabel HeLa cells by transducing them with PLenti6.2_miRFP670 lentivirus using the protocol described¹⁷. Harvest RFP+ HeLa cells 30 min-1 h before the transplantation.
   NOTE: Human HeLa cells have been cultured in a tissue culture incubator up to 70% confluency in complete growth medium (DMEM medium with 10% FBS) at 37 °C supplemented with 5% CO₂.
   1. Remove the HeLa cell medium by aspiration in a tissue culture hood. Briefly rinse the cell layer with sterile PBS to remove all traces of the serum.
   2. Add 3.0 mL of sterile Trypsin-EDTA solution to a T-75 flask and place the cells back into the 37 °C tissue culture incubator for 3-5 min to facilitate enzymatic digestion. Observe the flask under the microscope until ~80% of the cells become suspended.
   3. Add 6-8 mL of complete growth medium into the flask. Collect the cells into a 15 mL sterile tube by gently pipetting. Centrifuge at 135 x g for 5 min.
   4. Aspirate the supernatant and resuspend HeLa cells in 3 mL of complete growth medium. Repeat the above washing step 2x. Resuspend the cells in 1 mL of complete growth medium and count the cells under the microscope using a hemocytometer.
   5. Spin the cells down again, remove the supernatant, and resuspend the cells in a 1.5 mL microcentrifuge tube at a concentration of 5 x 10⁷ cells/mL. Keep the cells warm by holding the tube in one hand when transporting to the fish facility.
      NOTE: Always keep the cells warm by storing the cells inside the 35.5 °C incubator before or during injecting the embryos.

3. Transplantation of human cancer cells

1. Clean up the work area using ethanol towels before transplantation (e.g., scissors, tweezers, plastic pipettes, razor blades).
2. Align embryos within the grooves of the agarose plate using a plastic pipette. Lay embryos on the side with the anterior facing forward.
   NOTE: Make sure that the fish water covers the embryos. Set some embryos aside to not inject cancer cells and use as controls.
3. Turn on the air source and microinjector. Take HeLa cells out of the incubator and pipette the cells up and down 20-30x using a P200 tip. Load 3 µL of cell mixture immediately into a needle using a gel loading tip with a cut end. Carefully insert the tip toward the sharp bottom end of the needle. If needed, shake the needle to ensure the cell mixture moves down the needle to fill up the sharp end.
   1. Insert the needle into the needle holder. Use a pair of tweezers to carefully break open the tip of the needle. Adjust the pressure and duration of time on the microinjector to push out all of the air bubbles inside the needle tip. Reduce the pressure and injection duration time until the size of the injection droplets is ~1 nL.
   2. Place the injection plate under the microscope in an appropriate position to have the yolk side of embryos facing the needle. Anesthetize the embryos by adding five drops of the diluted MS222 solution (40 µg/mL).
   3. Position the injector and allow the needle to touch the perivitelline cavity of each embryo.
4. Inject the cell mixture into the embryos at the vascularized area under the perivitelline cavity by pressing the foot pedal.
5. Use the nondominant hand to move the injection plate to the next embryo. Use the dominant hand to extend and retract the injector while pressing the foot pedal simultaneously to continue the injection. Pipette a few drops of sterile fish water onto embryos that have been injected. Once the injection of all embryos on the plate is completed, wash the embryos off with sterile fish water, put them on a sterile Petri dish and immediately move them to the 35.5 °C incubator.
6. After 3 h, examine the injected embryos and remove the dead ones.
7. Return the live embryos to the 35.5 °C incubator and incubate them for 20-24 h to allow the HeLa cells to spread from the injection site to other parts of the body.
   NOTE: Embryos are kept in 35.5 °C incubator to allow the survival and migration of human cancer cells because the cells do not do well at 28.5 °C, the temperature fish embryos are normally incubated.

4. Injection of nanoparticles or vehicle

1. Anesthetize the transplanted embryos the next morning with five drops of diluted MS222 solution. Do not to add too much MS222, because this will kill the embryos. Under a fluorescent microscope, carefully pick up embryos with tail metastasis of RFP+ HeLa cells and place them into a new Petri dish with sterile fish water.
2. Make the injection needles as previously described in section 2 using the following settings: pressure at 500, heat at 645, pull at 60, velocity at 50, and time/delay at 100.
3. Follow the procedures in steps 3.1-3.3 to align embryos and load vehicle (e.g., H₂O) or the nanoparticle solution into the needle.
4. Inject 0.5 nL of 1 mg/mL nanoparticle solution behind the eye and continue the injection as described in steps 3.5-3.6 (Figure 1B). This location behind the eyes is enriched with capillaries, allowing the nanoparticles to enter circulation.
5. Following a similar procedure, inject the vehicle (e.g., H₂O) that was used to suspend the nanoparticles into embryos with HeLa cells transplanted and those without (i.e., controls) (Figure 1A, C).
6. Incubate all injected embryos at 35.5 °C.

5. Imaging and tracking of nanoparticles and cancer cells

1. Examine injected embryos under a fluorescent microscope at 0, 30, 60, 90, 120, 180, and 210 min postinjection of nanoparticles to monitor their distribution in circulation and the degree of cancer cell targeting. The targeting of cancer cells by nanoparticles can be observed as early as 30 min postinjection depending on the type of nanoparticle tested.
2. Pipette 2-3 embryos into a Petri dish and immobilize them by adding five drops of diluted MS222 solution (40 µg/mL). Once the embryos stop swimming, remove most of the water to allow the embryos to lie on their sides.
3. Use a pipette with a thin, soft brush attached to its end to align the embryos so they lie on their sides with the anterior facing forward and only one eye visible.
4. Image the embryos in red, blue, and brightfield channels at low magnification (2x) to capture the whole embryo and repeat at higher magnification (6.4x) to capture the tail area. Focus the embryo under the red channel to avoid the bleeding of nanoparticles.
   NOTE: The embryos must not move during imaging. Any movement will lead to blurry images and the inability to overlap images from different channels.
5. Add fresh fish water to the embryos immediately after imaging and return them to the incubator. Repeat steps 5.2-5.4 to image the embryos at different time points.

Results

The protocol schematic in Figure 1 illustrates the overall procedures for this study. Transparent Casper male and female adult fish were bred to generate embryos (section 1). RFP+ HeLa cells were injected into the vascularized area under the perivitelline cavity of the zebrafish embryos at 48 hpf, with uninjected embryos as controls (section 3). For individuals experienced in microinjection, the survival rate of embryos is often high, with at le...

Discussion

The protocol described here utilizes the zebrafish as an in vivo system to test the ability of nanoparticles to recognize and target metastatic human cancer cells. Several factors can impact the successful execution of the experiments. First, embryos need to be fully developed at 48 hpf. The correct developmental stage of the embryos enables them to endure and survive the transplantation of human cancer cells. Embryos younger than 48 hpf have a significantly lower survival rate compared to older and more developed embryo...

Materials

Name	Company	Catalog Number	Comments
Agarose	KSE scientific	BMK-A1705
Borosilicate glass capillaries	World Precision Instruments	1.0 mm O.D. x 0,78 mm
Computer and monitor	ThinkCentre	X000335
DMEM (Dulbecco's Modified Eagle's Medium)	Corning	10-013-CV	sold by Fisher
Fetal Bovine Serum	Sigma-Aldrich	F0926
Fish incubator	VWR	35960-056
Hemocytometer	Fishersci brand	02-671-51B
Magnetic stand	World Precision Instruments	M10
Microloader tip	Eppendorf	E5242956003	sold by Fisher
Micromanipulator	Applied Scientific Instrumentation	MMPI-3
Needle Puller	Sutter instruments	P-97
Olympus MVX-10 fluorescent microscope	Olympus	MVX-10
P200 tip	Fishersci brand	07-200-293
PBS (Dulbecco's Phosphate-Buffered Salt Solution 1X)	Corning	21-030-CV	sold by Fisher
Petri dish	Corning	SB93102	sold by Fisher
Plastic pipette	Fishersci brand	50-998-100
pLenti6.2_miRFP670	Addgene	13726
Pneumatic pico pump	World Precision Instruments	SYSPV820
Pronase	Roche-Sigma-Fisher	50-100-3275	Roche product made by Sigma- sold by Fisher
Razor blade	Fishersci brand	12-640
SZ51 dissection microscope	Olympus	SZ51
Tricaine methanesulfonate	Western Chemicals	NC0872873	sold by Fisher
Trypsin-EDTA	Corning	MT25053CI	sold by Fisher
Tweezer	Fishersci brand	12-000-122