High-Throughput Dissociation and Orthotopic Implantation of Breast Cancer Patient-Derived Xenografts

Summary

This protocol describes an efficient, non-surgical method for the orthotopic implantation of breast cancer patient-derived xenografts in mice. The technique involves enzymatic tumor dissociation followed by direct injection into the mammary fat pads, enabling high-throughput implantation. Comprehensive validation ensures model fidelity, facilitating rigorous studies across various breast cancer subtypes.

Abstract

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which is essential for advancing knowledge of breast cancer (BC). Additionally, PDX models enable the study of BC systemic effects, which is not possible using in vitro models. Traditional methods for implanting BC xenografts typically involve anesthesia and sterile surgical procedures, which are time-consuming, invasive, and limit the scalability of PDX models in BC research. This protocol describes a simple and scalable method for the orthotopic implantation of BC PDXs in mice. The immunodeficient mouse strain NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) was used for PDX engraftment. Human BC samples obtained from IRB-consented patients were mechanically and enzymatically dissociated, then resuspended in a solution of basement membrane extract (BME) and RPMI 1640. Animals were restrained by scruffing, and depilatory cream was applied to remove hair from the fat pads at the fourth inguinal nipple, followed by injection. Approximately 2 million cells in a 100 µL suspension were bilaterally injected orthotopically into the mammary fat pads using a 26 G needle. Notably, no anesthetic was required, and the total procedure time was under 5 min, from cell preparation to injection. After a growth period of several months, tumors were excised and processed for authentication. Validation included receptor status assessment using immunohistochemistry with specific antibodies for traditional BC receptors (i.e., ER, PR, HER2). Tumor morphology was confirmed with hematoxylin and eosin (H&E) staining, which was interpreted by a pathologist. Genetic similarity to the patient sample was verified through bulk RNA sequencing and short tandem repeat (STR) analysis. This approach to PDX engraftment and validation supports the rigorous development of models and high-throughput tumor implantation, enabling well-powered studies across various BC subtypes.

Introduction

Breast cancer (BC) is diagnosed in over 2 million women worldwide each year¹. In order to advance the basic biological understanding of BC and successfully translate novel therapeutics to approved treatments, preclinical animal models that retain the defining features of patient tumors are necessary. Patient-derived xenografts (PDXs) have been demonstrated to recapitulate tumor heterogeneity with high fidelity when implanted orthotopically, including histopathology, receptor expression, and genetic aberrations^2,3,4. Importantly, they also retain stroma....

Protocol

This protocol adheres to the guidelines established by the West Virginia University (WVU) Institutional Animal Care and Use Committee. Female NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice, aged 8 weeks or older and weighing approximately 25 g, were used for tumor cell injections. Breast cancer tumor tissue was procured at West Virginia University Cancer Institute (Morgantown, WV) and by the NCI Cooperative Human Tissue Network under approved WVU Institutional Review Board protocol and WVU Canc.......

Discussion

This article presents an efficient, high-throughput method for orthotopically implanting BC PDXs into the inguinal mammary fat pads of immunodeficient mice. This optimized workflow enables the single researcher to implant PDXs into dozens of mice per day, supporting large-scale and adequately powered studies. The capacity to simultaneously dissociate and implant multiple unique PDXs also enables precision oncology approaches. Beyond time efficiency, the dissociation of the tumors creates a homogenous suspension of cell t.......

Materials

Name	Company	Catalog Number	Comments
1 mL tuberculin syringe	BD	305945	sterile
1x phosphate buffered saline, pH 7.4	Gibco	10010023	sterile
2.0 mL round-bottom microcentrifuge tube	Eppendorf	0030123620	sterile
26 G needle, ½ in.	BD	305111	sterile
5.0 mL microtube	Eppendorf	0030119401	sterile
8+ week old NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice	The Jackson Laboratory	#005557
Alcohol prep pads	Fisher Scientific	22-363-750	sterile
Basement membrane extract (Matrigel)	Cultrex	3632-005-02
Cotton-tipped applicators, 6 in.	Fisher Scientific	22-029-553	sterile
Curved Forceps with Medium Non-serrated Tips, 152 mm	Electron Microscopy Sciences	50-365-845	sterile
Depilatory cream	Nair
gentleMACS C Tubes	Miltenyi Biotec	130-093-237
gentleMACS Octo dissociator with heaters	Miltenyi Biotec	130-096-427
No. 10 scalpel blades	Fisher Scientific	12-000-162	sterile
Non-woven gauze sponges, 4x4 inch	Fisher Scientific	22-028-558	sterile
RPMI 1640 1x + L-Glutamine	Gibco	11875093	sterile
Tumor dissociation kit, human	Miltenyi Biotec	130-095-929