Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

Summary

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

Abstract

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

Introduction

The development of patient-derived tumor xenografts (PDXs), where surgically resected tumor samples are engrafted directly into immune-compromised mice, offers several advantages over standard cell-line xenograft models and represents a major advance in cancer research^1,2. PDXs can be maintained and expanded by successive passages with minimal alteration of the genetic and biological characteristics of the tumor grown at the first passage; and more accurately reflect tumor heterogeneity than xenografts derived from human cancer cell lines^3-8. These models are now extensively used as a platform for personalizing cancer therapeutics^9,10,....

Protocol

All steps requiring the use of animals in this protocol follows the guidelines of University of Colorado animal research ethics committee (IACUC).

1. Preparation of Instruments, Culture Media and Other Reagents

1. Prepare 100 ml mammosphere media containing Dulbecco's Modified Eagle Medium and Han's F-12 medium (DMEM/F12) (1:1), basic Fibroblast Growth Factor (bFGF, 20 ng/ml), epidermal growth factor (EGF, 10 ng/ml), Heparin (4 µg/ml), 1x B27, Penicillin (100 U/ml), streptomycin (100 µg/ml). Make media in sterile conditions and store at 4 °C for up to 3 months.
2. Prepare epithelial enrichment buffer (EEB) containing PBS p....

Results

This method describes the transduction of PDX-dissociated breast cancer cells using high titer lentiviral vectors pSIH1-H1-copGFP-T2A-puro and pHAGE-EF1aL-luciferase-UBC-GFP-W. These vectors express a fluorescent marker that allows estimating the efficiency of transduction in vitro, as early as 24 hr after infection (Figure 1a). For most PDXs, expression of GFP will be delayed up to 72 hr after infection (Figure 1b), at this time the formation of.......

Discussion

Critical steps within the protocol:

The use of high titer lentiviral particles (>10⁸ TU/ml) is a critical step in the success of this protocol, as allows careful control of the media composition during in vitro transduction. While multiple methods for production of high-titer viral particles have been well described^18,19; this protocol uses lentiviral particles produced as described in detail at www.kottonla.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM/F12 (1:1)	Hyclone	SH30023.01
bFGF	BD Biosciences	354060
EGF	BD Biosciences	354001
Heparin	Sigma	H4784
B27	Gibco/Thermo Fisher	17504-44
Anti-fungi-antibiotics	Hyclone	SV30010
Accumax	Innovative Cell Technologies	AM-105-500	Digestion Buffer
FBS	Atlanta Biologicals	S11550
HBSS Red Ca2+/Mg2+ free	Hyclone	SH30031.02
Hepes
10x PBS	Hyclone	SH30258.01
Cultrex	Cultrex	3433-005-01	Basement Matrix Extract (BME)
30 °C shaker	NewBrunswick Scientific CO. INC	Series 25 Incubator Shaker
70 μm filters	Falcon	7352350
scalpels	Fisher	22079690
Clorhexidine disinfectant	Durvet	NDC# 30798-624-35
Red blood  cell lysis reagent	Sigma	R7757
Neuraminidase	Sigma	N7885-1UN
EpCAM (CD326+) microbeads*	Miltenyil Biotec	130-061-101
Lineage cell depletion Kit, mouse*	Miltenyil Biotec	130-090-858
MiniMACS Separator	Miltenyil Biotec	130-042-102
Mini MACS Magnetic Stand	Miltenyil Biotec	130-042-303
MS Columns	Miltenyil Biotec	130-042-201	MS or LS columns can be used, adjust to number of cells.
Illumatool Tunable light system	Lightools research	Various	For in vivo fluorescence imaging
Xenogen IVIS200 imaging device	Xenogen	Various	For in vivo luminiscence imaging
Human Cytokeratin Clone MNF116 Monoclonal antibody	DAKO	M0821	Pan-cytokeratin
Epidermal Growth factor receptor antibody	Cell signaling	4267S	EGFR