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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Abstract

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Introduction

Most breast cancer deaths can be ascribed to recurrent disease that is resistant to conventional therapies1,2. The inter- and intra-tumor heterogeneity of breast cancers contribute to therapy resistance. Moreover, tumor heterogeneity can impinge on accurate prognosis and challenge disease management3,4. Identification of predictive biomarkers of response will significantly improve clinical outcomes of patients with breast cancer. Even though most breast cancer types are immunologically 'cold' tumors that are likely unresponsive to immunotherapy....

Protocol

All protocols using animals have been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The tumor fragments, around 1−2 mm3 in size, are from viably frozen stock obtained from the Patient-Derived Xenograft and Advanced In Vivo Models Core at Baylor College of Medicine.

1. Preparation of cryopreserved mammary tumor fragments for transplantation

  1. Transfer the cryovial with tumor fragments from liquid nitrogen to a 37 °C water bath........

Representative Results

Figure 1 shows the equipment (Figure 1A) and key procedures (Figure 1B) of orthotopic transplantation. Figure 2 shows characterization of a transplanted PDX tumor (MC1). Tumor fragments (1 mm3) of MC1 model were transplanted into the #4 fat pad of SCID/Beige mice. One month later, the average tumor size reached around 350 mm

Discussion

To reduce variations in tumor growth across animals, it is critical to cut the tumor tissue into 1 mm3 fragments for transplantation. Models that grow soft tissue are harder to work with and the tumor fragments need to be cut slightly larger (1−2 mm3). When placing the tissue into the mammary fat pad pocket take care not to split the tissue into multiple pieces as this will result in multiple small tumors or oddly shaped tumors.

In addition, use fresh tumor for tran.......

Acknowledgements

This work was supported by the National Institutes of Health (R37CA228304 and R01HL146642 to Xi Chen, CA148761 to Jeffrey M. Rosen), US Department of Defense (W81XWH-19-1-0524 to Xi Chen, W81XWH-19-1-0035 to Xiangdong Lv), American Cancer Society (RSG-18-181-01-TBE to Xi Chen) and Cancer Prevention and Research Institute of Texas (RR150009 CPRIT Scholar in Cancer Research Award to Xi Chen), the Patient-Derived Xenograft and Advanced In Vivo Models Core at Baylor College of Medicine (funding from RP170691 CPRIT Core Facility Award and NCI-CA125123 P30 Cancer Center Support Grant).

....

Materials

NameCompanyCatalog NumberComments
1 mg/mL Buprenorphine-SRZooPharm (via BCM veterinarians)Sterile
26G syringeBD148232ESterile
Betadine ScrubFisher19-027132
Cotton SwabsVWR International Laboratory89031-272Sterile
DMEMFisherMT 10-013-CMSterile
Electric shaverOster78005-050
Glass beads sterilizer (Germinator)Roboz Surgical StoreDS-401
Lubricant ophthalmic ointmentAkorn Animal Health17478-062-35
Micro Dissecting Forceps; Serrated, Angular (regular forceps)Roboz Surgical StoreRS-5139Sterile
Micro Dissecting Spring Scissors (fat pad cutter)Roboz Surgical StoreRS-5658BTSterile
Micro Forceps (tissue placing forceps)Roboz Surgical StoreRS-5069Sterile
Petri DishFisher08-757- 100DSterile
Sterile drapeSai Infusion TechnologyPSS-SD1Sterile
Surgery scissorsRoboz Surgical StoreRS-5960Sterile
Tissue Forceps (claw forceps)Roboz Surgical StoreRS-5158Sterile
Wound clip applierBD Autoclip Wound System01-804Sterile
Wound clip removerBD Autoclip Wound System01-804-15Sterile
Wound clipsBD Autoclip Wound System01-804-5Sterile

References

  1. Waks, A. G., Winer, E. P. Breast Cancer Treatment: A Review. JAMA. 321 (3), 288-300 (2019).
  2. Harbeck, N., et al. Breast cancer. Nature Reviews Disease Primers. 5 (1), 66 (2019).
  3. Harbeck, N., Salem, M., Nitz, U., Gluz, O., Liedtke, C.

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Orthotopic TransplantationBreast TumorPreclinical ModelBreast CancerTumor BiologyDrug ResponseDrug ResistanceMammary Fat PadPatient derived XenograftSubcutaneous ImplantationCryopreservationThawingTumor DissectionDMEM

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