In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

To begin this procedure, luciferase expressing mammary tumor cell line is thawed from frozen stalks and then expanded in culture to 90%COF fluency. Serial dilution of suspended cells are then seeded in 96. Well plates exposed to Luciferian and then the optimal cell number generating fluorescent emissions above 500 photons is determined using a plate Reader cells are then injected, subcutaneously into the flank of an animal, and then luciferase is injected.

Intraperitoneal cells can be imaged as early as 10 minutes following administration of Lucifer, and the tumor mass can be visualized up to a month in vivo until the onset of necrosis. Hi, I'm Ed Lim from the Imaging Laboratory at Caliper Life Sciences in Alameda, California. Today we'll show you a procedure for monitoring tumor growth in mice non-invasively using bioluminescence.

This procedure is used in our laboratory and in ivus lab, the worldwide to study tumor growth and development, especially after treatment with drug compounds. So let's get started. This procedure begins by preparing the tumor cell line for injection into the mouse.

A wide range of luciferase expressing cancer cell lines can be used for bioluminescent preclinical experiments. These cells are provided as a pathogen free frozen culture, which will readily grow in standard media with no need for selection markers. For our experiment, we'll use the 41 Luke two murine mammary tumor cell line, which expresses the luciferase gene that serves as an optical indicator of gene expression or tumorgenesis.

In vivo, we will use luciferase expression to track growth of the primary tumor non-invasively, but it can also be used to locate and monitor metastatic lesions. Luciferase activity must be verified before injection, and in order to do this, a 90%confluent flask is harvested by trypsin and then counted. 50, 000 cells are then dispensed in a single well of a microtiter plate and serial dilution are performed.

Lucifer is added to the wells at 150 micrograms per milliliter and incubated for two minutes. The microplate can be imaged in ivus or a luminescent plate reader to determine expression levels. This cell line expresses up to 6, 500 photons per second per cell, but any expression level above 30 photons per second per cell can be imaged successfully in vivo.

Now that we have cells of optimal activity, we can proceed to the subcutaneous injection step. In order to facilitate optimal detection of the tumor, we are using an A thymic immunocompromised nude mouse strain. Prior to injection, animals are anesthetized using 3%ISO fluorine for five minutes for deep anesthesia.

Next, we will inject 250, 000 cells in a hundred microliter PBS subcutaneously into the flank. Load the cells in a one milliliter syringe and attach a 26 gauge needle. Lift the skin gently with forceps to make a tent and inject the cells at the base.

The newly injected cells can be imaged immediately. A total of 150 milligrams of Lucifer per kilogram body weight is then administered via two injections into the peritoneal cavity. In this study, animals are imaged 10 minutes after luciferian injection to ensure consistent photon flux.

We'll show you this in the next step. For our experiment, we'll use the IVUS spectrum in vivo imaging system, which uses a back thinned charge coupled device cooled to negative 90 degrees Celsius to achieve maximum sensitivity. To support absolute quantitation, the system measures dark charge during downtime and runs a self calibration during initialization.

To start imaging initialize the IVUS system and set the imaging parameters for the experiment. Select field of view. For the number of animals being imaged, up to five animals can be maintained in the instrument using the integral anesthetic manifold.

The stage is at a constant 37 degrees Celsius to maintain body temperature in the animals. An EKG port is provided to monitor animal health during stressful procedures in living image software exposure, time F-stop and pix binding can be optimized based on the expression level of the cell line. These settings can be changed at any time during an experiment without impacting the quantitative result.

Ivus acquires a photographic image of the animal under white light in a quantitative bioluminescent or fluorescent signal, which is overlaid on the image, the bioluminescent signal is expressed in photons per second and displayed as an intensity map. The image display is adjusted to provide optimal contrast and resolution in the image without affecting quantitation. Luminescence from the cells can be measured at the site of injection using a region of interest.Tool.

Measurement data are displayed in the table together with all experimental parameters relating to the image capture, which can be saved or exported. For analysis, multiple images can be acquired and compared and longitudinal studies covering seconds or months depending on the nature of the experiment. We will measure photon flux from the tumor at time zero and monitor for four weeks.

With imaging at biweekly intervals, photon flux from the tumor is proportional to the number of live cells expressing luciferase. So bioluminescence correlates directly with tumor size at five days post implantation. The tumor is not yet palpable, but the cells can be quantified through bioluminescence and the tumor is seen to be actively growing.

At this stage, the bioluminescence signal is much stronger. The exposure time f-stop and pixel spinning can be adjusted so that the image is clear and the camera does not saturate. IVUS automatically compensates for the changes in light collection.

So these measurements could be compared to those collected earlier and later. In the experiment. At 15 days post implantation tumors are palpable and bioluminescence measurement has already generated 15 days of data.

At 28 days, post implantation tumors are becoming necrotic and cells begin to die. Tumor size estimated by caliper measurement does not change appreciably, but luminescence from the tumor will decrease. Indicating cell death caliber and bioluminescence measurement can be continued until a humane endpoint is reached.

Tumor necrosis due to hypoxia or treatment regimens will be indicated by reduced bioluminescence even if they do not reduce the tumor mass. We've just shown you how to monitor subcutaneous tumor development, a simple xenograft mouse model using bioluminescent imaging. When doing this procedure, it's important to remember to check the expression level of your cells before implantation.

Secondly, for maximum reproducibility image the animals at the optimum time post luciferian injection. The sensitivity of the method enables one, to monitor the tumor development at early stages because tumors can be measured by bioluminescence long before they can be reliably measured by calipers. Remember to optimize your imaging parameters at each stage That luminescence signal does not saturate, but by luminescence, the animals can be monitored quantitatively for months to study remission and relapse effects.

So that's it. Thanks for watching and good luck with your experiments.