Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Summary

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

Abstract

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Introduction

Animal xenograft models are essential tools for preclinical studies of novel anticancer therapies. Standard murine xenografts rely on subcutaneous flank implantation of cells, providing an efficient and easily accessible site for monitoring tumor growth. The disadvantage of subcutaneous models is their lack of tumor-specific biologic characteristics, which may limit their potential to metastasize¹. Such limitations are overcome by the use of orthotopic xenografts in which tumor cells are engrafted at native tissue sites, providing a relevant microenvironment with metastatic potential². Orthotopic xenograft models maintain original biological features and provide reliable models for preclinical drug discovery^3,4. The cancer cells utilized for tissue-directed implantation are either established cell lines or patient-derived cells from patient tumors. Xenografts established from cancer cell lines may exhibit high genetic divergence from the primary tumor compared to patient derived xenografts⁵. Given this, the establishment of patient-derived orthotopic xenografts has become the preferred standard for testing novel therapeutics in cancer drug discovery.

In the pediatric cancer neuroblastoma (NB), orthotopic xenograft models recapitulate primary tumor biology and develop metastasis to typical sites of NB spread^6,7. NB develops in the adrenal gland or along the paravertebral sympathetic chain. The most common methods of orthotopic implantation require open trans-abdominal surgical procedures. Such methods are often tedious, have high animal morbidity, and complex recovery periods. High-resolution ultrasound has been recently utilized for tissue-directed implantation of tumor cells in development of several murine models for cancer research^8,9. The technique is reliable, reproducible, efficient, and safe for the establishment of relevant metastatic tumor xenografts^10,11.

The establishment of pediatric cancer xenografts by ultrasound-guided target organ localization and needle implantation of cell lines and patient-derived tumor cells is demonstrated¹¹. The technique was utilized for NB targeted to the murine adrenal gland. Ewing's sarcoma (ES) is predominantly an osseous cancer, commonly seen in the long bones such as femur and pelvic bones¹². Case reports have shown that to determine whether growth of a predominantly osseous cancer is feasible in renal tissue, a renal sub capsular location was chosen for orthotopic implantation¹³. Renal sub capsular cell implantation of tumor cells has been utilized as a promising model to study spontaneous metastases for ES¹⁴.

Protocol

All work was done in accordance with The University of Michigan Institutional Review Board (HUM 00052430) and conforms to procedures approved by the University Committee on Use and Care of Animals (UCUCA). The Unit for Laboratory of Animal Medicine (ULAM) oversaw animal care.

All work was done with approval from The University of Michigan Institutional Review Board (HUM 00052430) and conforms to all human research ethics committee regulations. Human cells are considered to be a potentially biohazardous material, so follow all special precautions and appropriate biosafety practices that are required by your institution. NSG mice are severely immunocompromised and susceptible to disease caused by bacteria commonly found in the environment.
Always use strict sterile technique when preparing materials for implantation and performing the injections.

1. Cell Culture

1. Grow established human NB cell lines (SH-SY5Y, SK-N-BE2, and IMR32) in minimal essential medium, supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. Supplement IMR32 cells further with 1 mM pyruvate and 0.075% NaHCO₃. Maintain all cells at 37 °C, 5% CO₂ and 70-80% confluence_.
2. Grow human ES cell lines (TC32, A673, CHLA-25, and A4573) in RPMI medium supplemented with 10% fetal bovine serum and 6 mM L-glutamine. Maintain all cells at 37 °C, 5% CO₂ at 70-80% confluence_.
3. Send all cell lines for authentication.
   NOTE: Cell authentication is done at an outside facility, please refer to acknowledgements.

2. Preparation of Primary Patient-Derived Tumor Cells

1. With patient consent and assent, harvest discarded human NB tumor tissue from patients undergoing surgical resection for local control and transport to the laboratory in tissue storage solution for preservation.
   NOTE: All patient samples are de-identified and handled in accordance with Institutional Review Board guidelines.
2. Generate a single patient-derived cancer cell suspension (UMNBL001, UMNBL002) from tumor tissue using a tumor dissociation kit. Transfer approximately 0.5 g of tumor to a 100 mm cell culture dish containing 5 mL of RPMI buffer, 200 µL of enzyme H, 100 µL of enzyme R and 25 µL of enzyme A from the human tumor dissociation kit.
3. Mince the tumor into small pieces (2-4 mm each) using scissors and tissue forceps. Mix these fragments with the solutions in step 2.2.
4. Pipette the tumor mixture into a dissociator tube and close the tube. Invert the tube and attach onto the sleeve of the tissue dissociator. Run on program h_tumor_01.
5. Incubate the tumor cell suspension obtained above at 37 °C for 1 h on a rotating rack with intermittent trituration every 15 min.
6. Transfer the cell suspension to a new 50 mL conical tube and add 10 mL of RPMI. Centrifuge the cell suspension at 314 x g for 5 min.
7. Remove the supernatant and suspend the pellet in 5 mL of RPMI. Pass this solution through a 40 µm cell strainer and collect the strained solution into a fresh 50 mL conical tube. Wash this strainer once with 5 mL of RPMI media and centrifuge the mixture at 314 x g for 5 min to collect a pellet.
8. Measure the cell density using a hemacytometer¹⁵. Suspend the pellet obtained from above in RPMI to obtain a final concentration of 4 × 10⁵ cells/10 µL. Take 5 µL of this cell suspension and 5 µL of matrigel to make 10 µL of cell solution for each injection.
   NOTE: Amount of RPMI used depends on the cell density measurement. For example, if the measured cell density of the obtained pellet is 4 x 10⁵ cells, suspend the pellet in 10 µL of RPMI.

3. Luciferase Tagging of Cancer Cells

1. Make 10 mL of transduction media with 5 mL of viral supernatant EF1a-Luc2-IRES-mCherry and 5 mL of stem cell media. Add 7.5 µL of 1x polybrene.
2. Mix 5 × 10⁶ cancer cells in transduction media and plate in 100 mm ultra-low attachment plate. Incubate at 37 °C and 5% CO₂ overnight.
   NOTE: As low attachment plates are used, cells will not adhere to plate.
3. After 24 h, bring the 100 mm plate containing cells under tissue culture hood and transfer the mixture into a 15 mL conical tube. Centrifuge the cell suspension at 314 x g for 5 min to get the cell pellet. Wash the cell pellet once with 1 mL of 1x phosphate-buffered saline (PBS) and suspend the cell pellet in 1 mL of HBSS containing 1% fetal bovine serum (FBS).
   1. Sort luciferase cells based on GFP-positive signal on fluorescence-activated cell sorting (FACS) analysis. Plate the sorted cells on 100 mm tissue culture treated dish and maintain at 70-80% confluence, 37 °C and 5% CO₂ until required.
4. Confirm luciferase signal with luciferase assay kit. Plate 10 x 10⁴ sorted cells per well in an illuminometer compatible 96 well plate and incubate cells overnight at 37 °C and 5% CO₂. After 24 h, bring the 96 well plate to room temperature and add Steady-Glo reagent to cultured media in wells in 1:1 ratio. Allow cells to lyse for 5 min and read luminescence in spectrophotometer.
5. Bring the 100 mm dish under a tissue culture hood. Discard the supernatant media. Wash the cells once with 2 mL of 1x PBS.
6. Add 2 mL of 0.25% trypsin and return the 100 mm dish to the incubator for 2-3 min. After 2-3 min, check for dislodged cells under microscope and add 8 mL of RPMI to deactivate trypsin.
7. Collect the cell suspension in a 15 mL conical tube and centrifuge at 314 x g for 5 min to get a pellet. Discard the supernatant. Wash the cell pellet with 1 mL of 1x PBS, suspend the pellet in 5 mL of RPMI and determine the cell density using a hemacytometer¹⁵. Centrifuge yhe remaining cell solution at 314 x g for 5 min to obtain a pellet.
8. Suspend the pellet in RPMI to obtain a concentration of 4 x 10⁵ cells/10 µL. Take 5 µL of the cell suspension and 5 µL of matrigel to make 10 µL of cell solution for each injection.

4. Ultrasound Guided Adrenal Gland (NB) or Renal Sub Capsule (ES) Implantation

NOTE: All ultrasound procedures are performed using a high resolution in vivo imaging system. For the described procedures, the transducer, which has a center frequency of 40 MHz and a bandwidth of 22-55 MHz was used.

1. Utilize 6-8-week-old immune-deficient NSG mice for all injection procedures.
2. Chill the matrigel and autoclaved Hamilton syringes fitted with a 27G needle (1.25"), and 22G catheters (0.9 mm external diameter, 25 mm length) overnight at 4 °C.
3. Place the cell solution prepared in step 3 on ice.
4. Anesthetize the mice in an induction chamber using 2% isoflurane in O₂ delivered at 2 L/min.
   NOTE: Adequate anesthesia is determined with the lack of active movement and the maintenance of spontaneous respiration.
5. Once anesthetized, depilate the back and flank of the animals using hair removal lotion (such as Nair) and a shaver.
6. Transfer the animal to the imaging platform with abdominal side down, where the nose of the animal is placed into a nosecone and secured while inhaling isoflurane.
7. Place optical ointment in the animal's eyes to prevent drying. Tape the mouse in place to prevent any inadvertent movement.
8. Identify the murine liver, vena cava, spleen, left kidney, and adjacent left adrenal gland using ultrasound visualization.
9. Use sterile gloves, mask and cap for personal protection and maintenance of sterile conditions. Under ultrasound-guidance, gently insert a 22G catheter through the skin and back muscle directly into the left adrenal gland to provide a channel for cellular injection. Remove the needle and leave the catheter in place.
10. Load a chilled Hamilton syringe fitted with a small-bore needle with 10 µL of cell solution, then guide the syringe stereotactically through the catheter positioned into the center of the adrenal gland.
11. Inject the cells into the targeted adrenal tissue. Leave the needle in place for 1-2 min to allow the matrigel to set. Slowly remove the needle, followed by the removal of the catheter.
12. Place the mice back into their cage and ensure that the mouse regains sufficient consciousness to maintain sternal recumbency.
    NOTE: Due to the minimally invasive nature of this technique, post procedural pain is minimal, but still monitored on a daily basis with mice health checks." should be followed with a sentence: "If unexpected signs of pain or distress are identified during the course of an experiment, consult with your institutional veterinary support unit for information regarding analgesia or other medical care.
    

Results

Using the procedures presented, ultrasound-guided implantation of NB cells into the adrenal gland was done in a dedicated procedure room equipped with a heated surgical table. Arm and foot pads were placed for monitoring murine heart activity (Figure 1A). The animal remained anesthetized under isoflurane using nose cone inhalation. Using a high-resolution ultrasound probe, the left kidney was identified with the adrenal gland just cranial to the kidney (

Discussion

Ultrasound-guided implantation of NB and ES cells is an efficient and safe method to establish reliable murine xenografts for preclinical studies in cancer biology. Critical to the success of ultrasound-guided tissue-targeted implantation is the presence and availability of trained personnel with expertise in anatomically localizing the organ of interest and in stereotactic injection of tumor cells.

The dissociation of tumor tissue proved to be a crucial step in developing the described patien...

Materials

Name	Company	Catalog Number	Comments
Mice
NOD-SCID	Charles River	394
NSG	The Jackson Laboratory	5557
Cell Line
NB
IMR-32	ATCC	CCL-127	Established human neuroblastoma cell line
SH-SY5Y	ATCC	CRL-2266	Established human neuroblastoma cell line
SK-N-Be2	ATCC	CRL-2271	Established human neuroblastoma cell line
ES
TC32	COGcell.ORG		Established human Ewing's Sarcoma cell line
A673	COGcell.ORG		Established human Ewing's Sarcoma cell line
CHLA-25	COGcell.ORG		Established human Ewing's Sarcoma cell line
A4573	COGcell.ORG		Established human Ewing's Sarcoma cell line
Cell Line media
RPMI	Life Technologies	11875-093
Matrigel	BD BioSciences	354234
Dissociation
Dissection Tools	KentScientific	INSMOUSEKIT
Human Tumor Dissociation Kit	MACS Miltenyi Biotec	130-095-929
gentleMACS dissociator	MACS Miltenyi Biotec	130-093-235
gentleMACS C tubes	MACS Miltenyi Biotec	130-096-334
Cell Strainer	Corning	431751
Luciferase Tagging
Lenti-GFP1 virus	University of Michigan, Vector Core		Luciferase Virus
Steady Glo-Luciferase Assay Kit	Promega	E2510
Bioluminescence Imaging
Ivis Spectrum Imaging System	PerkinElmer	124262
D-Luciferin	Promega	E160X
Anesthetic
Inhaled Isoflurane	Piramal Critical Care Inc	66794-0017-25
Ultrasound Guided Injection
Vevo 2100 High Resolution Imaging	Vevo	2100
Hamilton Syringes (27 gauge needle)	Hamilton	80000
22 Gauge Angiocatheter	BD Biosciences	381423
Optical ointment	Major Pharmaceuticals	301909
Nair	Church & Dwight Co		Hair Removal agent
Aquasonic 100 Ultrasound Transmission gel	Parker		Ultrasound gel
Histology
CD99	DAKO	M3601	Primary Antibody
Tyrosine Hydroxylase	Sigma-Aldrich	T2928	Primary Antibody
Secondary HRP-Polymer antibody	Biocare	BRR4056KG
Miscelleneous
10 mL Pipettes	Fisher Scientific	13-676-10J
5 mL Pipettes	Fisher Scientific	13-676-10H
1.5 mL Microcentrifuge tubes	Fisher Scientific	05-408-129
P1000 pipette	Eppendorf	3120000062
P200 pipette	Eppendorf	3120000054
P1000 pipette tips	Fisher Scientific	21-375E
P200 pipette tips	Fisher Scientific	21-375D
Portable pipette aid	Drummond	4-000-101
digital animal Weighing Scale	KentScientific	SCL-1015
Calipers	Fisher Scientific	06-664-16
6well low attachment plates	Corning	07-200-601
10 cm Tissue Culture Treated Dishes	Fisher Scientific	FB012924
Polybrene	Sigma-Aldrich	TR-1003-G