Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

There is currently no gold standard for analyzing tail-vein injection induced metastatic lung tumor burdens. We utilized digital image analysis to accurately and reproducibly quantify lung metastases in a highly efficient manner. This method is time and cost effective.

And, when coupled with digital image analysis, allows a highly accurate and comprehensive analysis of the resulting lung metastatic tumor burden. To prepare human breast cancer cells for injection, first, place an appropriate number of cells in a suitable cell culture medium, based on the number of mice and cell concentration to be used. When the cells reach an 80 to 90%confluency, wash the plates with PBS, and detach the cells in a minimal volume of trypsin, according to standard protocols.

When the cells have detached, re-suspend the cells in fresh culture medium for counting, and collect the cells by centrifugation. Then, re-suspend the cells at the appropriate concentration in 100 microliters of PBS per mouse, on ice. Just before the injection, thoroughly re-suspend cells on ice to avoid clumping, and load 100 microliters of the cells into a 28-gauge insulin syringe.

Keeping the syringe vertical, tap the shaft while slowly adjusting the plunger to remove any bubbles. If permitted, carefully recap the needle and bend the needle tip to a 20 to 30-degree angle. Restrain a greater than six-week-old female mouse in a plastic mouse restrainer and position the mouse on its side such that its lateral tail vein is easily viewed.

Locate the ventral artery in line with the genitalia, a dorsal vein, and two lateral caudle veins, and use an aseptic wipe to clean the tail surface. Grasping the tail between the index finger and thumb of the non-dominant hand, apply slight tension, and beginning at the distal portion of the tail, insert the needle, bevel side up, parallel to the vein. Slowly deliver the entire volume of cells into the vein.

A small volume of blood will likely be displaced after the injection. Apply gentle pressure with a sterile gauze to the injection site. Then discard the syringe in an appropriate sharps container, and place the mouse in a clean, ventilated cage with monitoring.

Within no more than one hour of the tail-vein injection, perform live animal in vivo imaging to confirm a successful cell injection, and to obtain time zero data. To maintain the structural format of the lungs for histopathology, secure the mouse to a dissecting board and wet the fur with 70%ethanol. Open the thorax with a midline incision, extending the incision cranially and caudally through the peritoneum, and grasp the xiphoid process to allow removal of the diaphragm.

Using a second pair of scissors, cut the ribs along each side of the sternum and carefully remove the ribcage to leave room for the lungs to expand. To isolate the trachea, remove the submandibular salivary glands and infrahyoid musculature, and place pins on either side of the trachea to prevent unwanted movement during the needle insertion. Load a three milliliter syringe, equipped with a 26-gauge needle, with two to three milliliters of 10%neutral buffered Formelin, and insert the needle into the trachea.

Slowly inject the Formelin, while watching for the lungs to expand. Once the Formelin begins leaking out of the lungs, use forceps to pinch off the trachea and remove the needle. Then detach the entire respiratory apparatus and place the organs directly into a container of fresh Formelin.

For analysis of the metastatic lung tumor burden, scan H&E stained lung tissue sections on a high resolution slide scanner at a 40 times magnification, and import the images into an appropriate image analysis software program. Adjust the parameters defining the shape and sparseness to best fit the images. To display the segmented areas of tumor metastases and normal lung tissue, using different colored labels for each tissue type, open the Decision Forest program, and follow the prompted series of yes or no questions to appropriately train each class for an image.

To adjust the features for each class, apply filters to sharpen, blur, or sort by shape to enhance the accuracy of the algorithm. In this analysis, the tumor metastases appear in blue. The normal tissue is green, and the bronchiolar epithelium is yellow.

Red blood cells can be observed in red, and air spaces appear in pink. When all of the features have been set, save the modified settings and apply the algorithm to the entire set or series of H&E stained tissues. Then export all of the output variables to allow the area in micron squared to be quantified for each tissue type, and to allow calculation of the percentages from the specimen total net tissue area.

The presence of a bioluminescence signal within the thoracic space less than two hours after the tail-vein injection of luciferase-tagged human epithelial breast cancer cells can be considered confirmation of an accurate injection. In this experiment, the photon counts in the thoracic region increased over time and a strong bioluminescence signal was still present at day 24 post-injection. At the time of necropsy, many macroscopic lung lesions were observed in these mice.

Using image analysis software and a custom algorithm as demonstrated, whole lung tissue can be segmented by different tissue features. By segmenting the lung tissue in this manner, the software can quantify various parameters. Here, raw data from an analysis performed on lung tissue from mice injected with human epithelial breast cancer cells followed by treatment with a drug designed to block metastatic colonization are shown.

As demonstrated by these images, a difference in metastatic tumor burden between these treatment groups may have easily been overlooked, as the total number of lung nodules is no different. A comprehensive analysis of all the parameters, however, indicated a significant difference in the percent net lung metastasis area, underscoring the need for a thorough approach to metastatic lung tumor burden analysis A proper cell re-suspension without air bubbles is critical for preventing pulmonary embolisms, and a successful lung inflation helps to maintain the tissue structure integrity for an accurate image analysis. Immunostaining of the lung tissue can be performed to determine the presence of cells or proteins of interest within the lung lesions or the lung micro environment.