Bioluminescent Imaging of Breast Tumor Progression in a Female Mouse Model

Abstract

Source:
Marina Arantes Radicchi¹, Victor Carlos Mello¹,
Victória Paz Machado¹, Márcia Cristina Oliveira da Rocha¹, Jaqueline Vaz de Oliveira¹, Luana Cristina Camargo^1,2, Luís Alexandre Muehlmann¹, Ricardo Bentes Azevedo¹, Sônia Nair Bao¹, João Paulo Figueiró Longo<sup>1

1</sup>Institute of Biological Sciences, University of Brasília, ²Department of Basic Psychological Processes, Institute of Psychology, University of Brasília

This video demonstrates the procedure for tracking tumor progression in vivo by injecting luciferase-expressing breast adenocarcinoma cells into a female mouse and monitoring bioluminescent signals to evaluate primary and metastatic tumor growth over time

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Cell culture

1. Maintain 4T1 cell line (ATCC-CRL-2539), a murine breast adenocarcinoma cell, in culture with DMEM medium (Dulbecco's modified eagle medium) supplemented with 10% fetal bovine serum (v:v), penicillin (100 U/mL), and streptomycin (100 U/mL), at 37 °C and in a humidified atmosphere containing 5% CO₂.
2. Transfect the original cell line with the Luc2 gene, which expresses luciferase. This bioluminescent enzyme is constitutively produced.

2. Animal model - Tumor cell transplantation

1. Subcutaneously inoculate bioluminescent, luciferase-expressing 4T1 cells (2 × 10⁵ cells per mouse) into the left dorsum of Balb/c female mice under anesthesia (ketamine and xylazine at 80 mg/kg and 10 mg/kg, respectively).
   1. For this procedure, anesthetize the mouse prior to the experiment using intraperitoneal injection of the specified anesthetic agents (ketamine and xylazine at 80 and 10 mg/kg, respectively).
   2. After 5 min of anesthesia induction, shave the mouse and inject the cells subcutaneously using 1 mL insulin syringes.
      NOTE: A total of five 8-week-old mice, weighing approximately 25 g each, were utilized for tumor induction and imaging.
2. To quantify tumor growth in vivo, evaluate the tumor-bearing mouse by bioluminescent imaging, by monitoring tumor growth for 4 weeks.
   1. For bioimaging experiments, anesthetize the mouse with isoflurane (2.5% diluted in purified compressed air). To evaluate tumor progression, image the mouse once a week for 4 weeks using equipment that measures bioluminescent signals.
      NOTE: The 4T1-luc luciferase bioluminescent signal was utilized as a parameter for assessing growth and indicating tumor progression.
3. During the imaging process, position the mouse in a dorsal recumbent position within the designated area of the equipment to capture the bioluminescent images.
4. After anesthesia, inject the mouse with D-luciferin (100 µL; 15 mg/mL) intraperitoneally.
   NOTE: Since D-luciferin is a substrate for the luciferase enzyme, which produces light photons when cleaved, and the 4T1-bioluminescent luciferase cells express this enzyme constitutively, quantify and detect the tumor in live animals.
5. To detect signals, capture 20 bioluminescent images over 20 min for each animal at each endpoint. Each of these images represents the bioluminescent signal emitted over a period of 60 s.
6. Construct a bioluminescent kinetics curve representing the signal emitted each minute. To ensure optimal results for each animal, record the maximum bioluminescent emission for each endpoint.
7. For bioluminescence image capture, use the following parameters: emission filter open, without excitation, binning 8, f/stop 2, and field of view type-D. To generate a growth curve, image the animals once a week for a total of 4 weeks.
8. Bioluminescence imaging - Primary Tumor and Lung Metastasis.
   1. To distinguish between the primary tumor and lung metastasis signals, define specific regions of interest using the equipment's software.
   2. Identify bioluminescent signals in the injection site and the thoracic region where the cells metastasized. Based on these images, calculate the progression of the bioluminescent signal, which correlates strongly with tumor progression.
   3. With images collected over several weeks, create a growth kinetics profile for the different tumor sites, including the primary tumor and metastatic lesions.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
D-luciferin	Perkin Elmer	122799	Chemical Reagent
Dulbecos’s modified Eagle’s médium	Gibko	A7030	Cell Culture Media
Fetal Bovine Serum	Merck	F2442	Cell Culture Media Supplement
Isoflurane	Cristália	NA	Anesthetics
IVIS Lumina	XR	Series III	Equipment – USA
Penicillin	Merck	P4333	Cell Culture Antibiotic
Phalloidin-Alexa Fluor 488	Thermo Fischer	A12379	Chemical Reagent
Phosphate buffer saline	Merck	4437568	Chemical Reagent
Streptomycin	Merck	P4333	Cell Culture Antibiotic