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.
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.
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 discovery3,4. 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 spread6,7. 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 research8,9. The technique is reliable, reproducible, efficient, and safe for the establishment of relevant metastatic tumor xenografts10,11.
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.
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
2. Preparation of Primary Patient-Derived Tumor Cells
3. Luciferase Tagging of Cancer Cells
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.
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 (Figure 1B). The needle was then advanced into the adrenal gland under direct visualization (Figure 1C). During initial utilization of this technique, a mixture of methylene blue dye and matrigel was used to confirm correct localization of the adrenal gland. The animal was sacrificed, and the success of the procedure was confirmed on necropsy by visualizing methylene blue dye beneath the adrenal gland capsule (Figure 1D).
Weekly ultrasound and bioluminescence imaging were the modalities used to monitor tumor engraftment and growth rate. Bioluminescence imaging of the region of interest was measured as photons/s/cm2/steridian (p/s/cm2/sr) with a minimal count of 106 to 108 indicative of adequate tumor size for preclinical experiments9 (Figure 2). Analysis using t-test showed statistically significant increase in bioluminescence was noted over the eight-week period (*p<0.05; **p<0.001; ***p<0.0001). Ultrasound imaging allowed noninvasive measurements of tumor area and volume. This along with bioluminescence measurement monitored both tumor engraftment and progression (Figure 3). Similar procedures were utilized for ES cells injected into the renal sub capsule (Figure 4).
Gross and histological analysis of resultant tumors characterized the primary tumors and metastases at necropsy. Tissue samples were examined for morphologic cell patterns and stained for tumor-specific protein markers (Figure 5, Figure 6). Primary tumor engraftment for NB cell lines (IMR32, SH-SY-5Y, SK-N-BE2, UMNBL001, UMNBL002) ranged from 62-100%, with metastases seen in 0-100% of injected mice. The most robust metastases were seen in the SK-N-BE2 (33%) and UMNBL002 (100%). Metastatic sites for NB were lymph nodes, liver, lung and cortical bone. Renal sub capsular engraftment for ES cell lines (A4573, A673, CHLA-25, TC-32) ranged from 60-100%, with metastases seen in 0-40% of injected mice. Metastatic sites examined for ES included lymph nodes, cortical bones and lungs. The most robust engraftment (100%) and metastases (40%) were seen in TC-32 xenograft mice.
Figure 1. Murine adrenal gland localization and ultrasound-guided NB cell implantation. (A) High resolution ultrasound imaging of murine abdominal organs allowed adrenal gland implantation of tumor cells. (B) Left kidney was identified. (C) The left adrenal gland is located adjacent to the left kidney as a hypoechoic (dark) round structure. The needle was advanced into the adrenal gland followed by cell injection. (D) A mixture of matrigel and methylene blue dye was used to assess the accuracy of injection procedures. Representative ex vivo image of the left kidney and adrenal gland with matrigel and methylene blue dye at the adrenal gland capsule and peri-adrenal space. This figure has been modified with permission from Van Noord, R.A. et al.11. Please click here to view a larger version of this figure.
Figure 2. In vivo bioluminescence imaging of tumor engraftment and growth rate. (A) Human NB adrenal xenografts were monitored for engraftment and tumor growth over eight weeks. (B) Maximal bioluminescence signal was determined within the region of interest. NB cell lines (SH-SY5Y, IMR 32, SK-N-BE2) and patient-derived cells (UMNBL001, UMNBL002) had increased radiance and growth patterns over time. Increase of bioluminescence over the eight-week period was statistically significant (*p<0.05; **p<0.001; ***p<0.0001). This figure has been modified with permission from Van Noord, R.A. et al.11. Please click here to view a larger version of this figure.
Figure 3. In vivo ultrasound imaging of tumor engraftment and growth. Ultrasound imaging monitored in vivo tumor progression. Ultrasound images and corresponding bioluminescence images at one, two and eight weeks are shown. At one week, engraftment was visualized in both imaging modalities. Tumor area and volume were calculated using ultrasound imaging. 3D ultrasound images mirrored excised tumors at the completion of the study. This figure has been modified with permission from Van Noord, R.A. et al.11. Please click here to view a larger version of this figure.
Figure 4. Murine kidney localization and ultrasound-guided implantation of ES cells. (A) Bioluminescence was used to monitor tumor radiance and growth. (B-C) Animals developed locally invasive tumors that distorted surrounding abdominal structures and invaded into local structures such as muscle. This figure has been modified with permission from Van Noord, R.A. et al.11. Please click here to view a larger version of this figure.
Figure 5. Tumor histopathology of NB and ES xenograft tumors. (A) NB xenograft mice had locally infiltrative primary tumors at the adrenal and peri-adrenal gland sites, which distorted adjacent abdominal organs. (B) Microscopically (20X, bar = 50 µm), the xenograft adrenal tumors showed morphologic features consistent with human NB (clusters of poorly differentiated angular to round cells), including occasional pseudo rosette formation (arrow). (C) Tumor tissue showed strong tyrosine hydroxylase (NB tumor marker) immunoreactivity (40X, bar = 40 µm; positive control staining shown in the left panel; 1:100). (D) ES xenograft tumor had histologic features consistent with infiltrative ES (40X, bar = 40 µm), clusters of angular to round epithelioid cells separated by a fine fibro vascular stroma, effacing and compressing normal adjacent kidney (arrow; compressed remnants of kidney parenchyma). (E) ES tumor tissue showed strong membrane immunoreactivity for the ES tumor marker CD99 (40X, bar = 40 µm; positive control staining shown in the left panel; 1:100). This figure has been modified with permission from Van Noord, R.A. et al.11. Please click here to view a larger version of this figure.
Figure 6. Distant metastases developed in xenografts established by ultrasound-guided injection technique. (A) Bioluminescence imaging showing the primary NB tumor at the adrenal gland space and metastatic foci detected at cortical bone. (B) Microscopic analysis demonstrated a metastatic tumor (arrow) effacing the marrow cavity and cortical bone of the proximal femur (4X, bar = 400 µm). (C) Tyrosine hydroxylase cytoplasmic immunoreactivity of the cortical bone NB metastatic site (40X, bar = 40 µm). (D) Microscopy of patient-derived orthotopic xenograft (UMNBL002) with a hepatic metastasis (arrow) within a hepatic vein (arrowhead demonstrates endothelial cells), and infiltrating the adjacent liver (L) parenchyma (20X, bar = 50 µm). (E) Neuroblastoma micro metastasis (arrow) within the lung parenchyma (40X, bar = 40 µm). This figure has been modified with permission from Van Noord, R.A. et al.11. Please click here to view a larger version of this figure.
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 patient-derived xenograft model. Native tumor tissue includes a cellular microenvironment and surrounding stroma that may affect the tumor uptake or engraftment. Cell counts, after the dissociation process was complete, provided a gauge of the cellular density in the sample, with 2 x 105 cells/10 μL considered an optimal cell count for implantation and successful tumor engraftment.
Luciferase tagging of cells allowed for the validation of tumor engraftment and monitoring of tumor growth by measuring bioluminescence signal, with 106 to 108 being considered indicative of tumor uptake9. To ensure successful transduction, luciferase activity was confirmed in vitro prior to injection of cells. This was done by immunofluorescence, luciferase assay kits, and also by examining the cells for bioluminescence signal. Settings used to measure bioluminescence were kept constant, providing consistent data during weekly measurements.
Ex vivo histopathological analysis of tumors confirmed the cell type and morphology of resultant tumors with specific staining for protein markers. Histopathological analysis confirmed tumor engraftment and metastasis demonstrated by ultrasound and bioluminescent imaging.
Advantages of ultrasound-guided cellular implantation include its non-invasive nature, minimal animal recovery time, and growing success in the establishment of several successful xenograft models. Ultrasound imaging also provides a reliable method to monitor in vivo tumor progression and tumor volumes. In these preclinical models, tumors progressed to locally invasive disease with metastasis within 5 weeks. Limitations include the availability of high resolution imaging technology and the ability to consistently localize the target organ. Availability of personnel with expertise in ultrasound imaging of the organ of interest are critical components of this technique.
The availability of high-resolution ultrasound imaging has increased substantially within the last decade and its use continues to expand not only in the clinical practice but also in laboratory research. The utilization of ultrasound for the development of modern murine xenografts is a promising application, with potential for advancing preclinical research models for cancer drug discovery.
The authors have nothing to disclose.
This work received support from the Robert Wood Johnson Foundation/Amos Medical Faculty Development Program, Taubman Research Institute, and the Section of Pediatric Surgery, The University of Michigan. The authors wish to thank Kimber Converso-Baran and Dr. Marcus Jarboe for the assistance with ultrasound injection procedures and the imaging platform. We thank Paul Trombley for his assistance with figure graphics. We also thank the Department of Radiology at The University of Michigan for the use of The Center for Molecular Imaging and the Tumor Imaging Core, which are supported in part by Comprehensive Cancer Center NIH, grant P30 CA046592. The University of Michigan Physiology Phenotyping Core that is supported in part by grant funding from the NIH (OD016502) and the Frankel Cardiovascular Center. Cell line authentication was done at IDEXX RADIL Bioresearch Facilities, Columbia, MO. We thank Tammy Stoll, Dr. Rajen Mody and the Mott Solid Tumor Oncology Program. Our patients and families are gratefully acknowledged for their inspiration, courage, and ongoing support of our research.
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 |