Transduction to Label PDX Tumor Cells: Introducing Lentivirus Expressing Fluorescent Marker into the Tumor Cell In Vitro

Overview

The following video describes a technique of transduction to label (patient-derived xenografts) PDX tumor cells, which is  used for introducing lentivirus expressing green-fluorescent protein and luciferase reporters into the tumor cell in vitro.

Protocol

1. Transduction of PDX-derived Tumor Cells

1. Centrifuge dissociated tumor cells at 300 g x 5 min and resuspend in 2 ml mammosphere media. Count viable cells using trypan blue exclusion (Resuspend digested cells in 5 ml wash buffer and count both viable and non-viable cells using trypan blue exclusion. Briefly, mix 50 µl of cells and 50 µl of trypan blue and count trypan-blue excluding cells using a hemocytometer).
2. Prepare 10 ml of mammosphere media containing 8 µg/ml polybrene (2 µl of stock polybrene per ml of media).
3. Determine the volume needed for 2 x 10⁵ viable tumor cells. Centrifuge cells at 300 x g for 5 min, and resuspend pellet in 2 ml of polybrene containing media. Plate 2 x 10⁵ viable tumor cells per well in a 6-well ultra-low adherence tissue culture plate.
   NOTE: This is an optimal number of cells required for transduction with one lentiviral vector.
   CAUTION! Lentiviral particles and all consumables used with lentiviral particles should be handled following institutional procedures for recombinant DNA biohazards.
   NOTE: Lentiviral transduction of primary breast cells strongly favors myoepithelial cells which can result in poor labelling of luminal cells and selection of subpopulations of tumor cells during labelling.
4. Incubate the virus with 200 mU/ml neuraminidase at 37 °C for 1 hr prior to transduction to increase the binding of viral particles to different primary cell subpopulations and correct for this potential bias.
5. Add lentiviral particles at 10 MOI (2 x 10⁶ TU for 2 x 10⁵ viable cells) if PDX-dissociated cells are depleted from Lin+ mouse cells or enriched in EpCam+ cells. Add lentiviral particles at 30 MOI (6 x 10⁶ TU for 2 x 10⁵ viable cells) if using non-enriched PDX-dissociated cells.
   NOTE: The increased MOI allows for efficient transduction even in the presence of large amounts of debris and dead cells in crude extracts. Keep one well with unlabeled cells to serve as a control for viability and transduction efficiency.
6. Swirl to mix virus with cells. Incubate at 37 °C, 5% CO₂ for up to 96 hr. Add 500 µl of fresh mammosphere media 24 hr after transfection. Cells will not attach to plates, do not aspirate media.

2. Evaluation of Transduction Efficiency and Re-implantation of Labeled Cells in Immunocompromised Mice

1. Monitor expression of the traceable marker (GFP) every 24 hr using a fluorescent microscope at 10X magnification.
   NOTE: GFP expression can be observed as early as 24 hr after infection but in most PDXs GFP expression is clearly visible after 72 hr (Figure 1).
2. Estimate the efficiency of transduction by evaluating the percentage of GFP+ cells within the total well.
   NOTE: Since cultures contain tumor cells, residual stromal cells, and dead cells at various degrees, wells with as low as 10% GFP+ cells can be implanted in a host mouse.
3. Transfer transduced cells from 6-well plates into a 15 ml conical tube. Add 1 ml of mammosphere media to the well to collect all the cells left behind. Place on ice.
4. Add 10 ml of HBSS/hepes to transduced cells and centrifuge at 300 x g for 5 min, 4 °C. Carefully aspirate supernatant and resuspend in 50 µl basement matrix extract (BME) on ice.
   NOTE: BME aliquots must be thawed on ice, 1 - 2 hr prior to use. BME will solidify at room temperature; keep on ice at all times.
5. Load BME-embedded cells into a 0.5 ml insulin syringe, keep on ice, and bring to the animal facility for reimplantation into NSG mice.

Representative Results

Figure 1: Tracking Transduction Efficiency in Dissociated PDX Cells. A) PDX-dissociated cells enriched for human epithelial cells (Lin+ depletion) 24 hr after transduction with pSIH1-H1-copGFP-T2A-puro lentiviral particles. B) PDX-dissociated cells from a separate experiment, without epithelial cell enrichment, 72 hr after transduction with pHAGE-EF1aL-luciferase-UBC-GFP-W. Both panels show live cells. BF: Bright field images, bar represents 50 µm

Materials

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
HBSS Red Ca2+/Mg2+ free	Hyclone	SH30031.02
Hepes
Cultrex	Cultrex	3433-005-01	Basement Matrix Extract (BME)
30 °C shaker	NewBrunswick Scientific CO. INC	Series 25 Incubator Shaker