Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

This protocol quantifies lung metastasis, which is an aggressive and common cause of breast cancer related death, with increased precision and efficiency in a preclinical breast cancer model. Compared to the original technique, this protocol gives researchers quicker, more consistent results. It alleviates human counting error through easy-to-use computer technology.

This technique can absolutely be extended to preclinical research studying the effects of breast cancer therapies on lung metastasis by allowing researchers to demonstrate decreased metastatic burden following successful treatment. Begin by labeling a 15 milliliter conical tube for the tissue sample. Then add 2.5 milliliters of type four collagenase mixture and 30 units of elastase to the tube.

Move the lung to a clean 1X HBSS well and swirl it with forceps to remove any remaining blood, then transfer it to an empty 3.5 centimeter tissue culture plate. Mince the lung with scissors, then rinse the plate with 2.5 milliliters of HBSS and transfer the HBSS with the lung pieces into a prepared 15 milliliter conical tube with the collagenase and elastase cocktail. Incubate the tube for 75 minutes at four degrees Celsius on a rocker or rotating wheel.

Meanwhile, label one 50 milliliter centrifuge tube and one 10 centimeter tissue culture plate for each mouse. If doing a dilution, label enough tissue culture plates for the dilutions. Bring the volume of the tube up to 10 milliliters with 1X HBSS, then pour the contents over a 70 micrometer cell strainer into the 50 milliliter conical tube.

Use the plunger of a one milliliter syringe to gently grind the sample through the strainer. Centrifuge the tube for five minutes at 350 times G and discard the supernatant, then wash the pellet twice with 10 milliliters of 1X HBSS. Resuspend the pellet in 10 milliliters of 60 micromolar 6-thioguanine complete culture media, and plate the samples in the 10 centimeter cell culture plates using a dilution scheme if desired.

Incubate the plates at 37 degrees Celsius and 5%carbon dioxide for five days. Pour culture media off of the plates into the appropriate waste container. To fix the cells, add five milliliters of undiluted methanol to each plate and incubate for five minutes at room temperature, making sure to swirl the methanol so that it covers the entire plate.

Pour the methanol off the plates into the appropriate waste container, then rinse each plate with five milliliters of distilled water. Add five milliliters of 0.03%methylene blue per plate and incubate it for five minutes at room temperature, making sure to swirl the methylene blue solution so that it covers the entire plate. Pour the methylene blue into the appropriate waste container and rinse each plate again with five milliliters of distilled water.

Turn the plate upside down and blot it against a paper towel to remove excess liquid. Place the plate on its lid and let it air dry overnight. Metastatic colonies will be blue.

Once the plates have dried, they can be stored at room temperature indefinitely. Remove the labeled lids from the plates, taking care to ensure clear identification of the samples. Line up all stained lung plates on a clean, light surface.

Take a picture of the collection of plates in a well-lit area, making sure to minimize reflections. Reflections in the plates will influence image analysis and should be avoided. Pay close attention to the photographs taken, and take several pictures from several angles.

After photographing the plates, crop the image to exclude the lids or anything in the background. Open the image in Fiji ImageJ and change it to black and white by clicking image, adjust, and color threshold. Then select default for thresholding method, black and white for threshold color, and lab for color space.

Make sure that the dark background box is not selected. The image should now be black and white. Black represents the light background, and white represents the blue metastatic colonies.

Use the circle tool to select the area to be analyzed. Draw one circle to use for all of the plates to ensure that each plate is analyzed for the same sized area. Choose a size that maximizes the analyzed area on the plates while minimizing the background noise that appears on the edge of the plates.

The size appears in the toolbar as it is drawn. Analyze the selected circle to determine the percentage of area that is white. Click on analyze and analyze particles, then select zero to infinity for size, zero to one for circularity, and nothing for show.

Check the summarized box and hit okay. Move the circle to the next plate in the picture by grabbing it center and repeat the analysis. Copy and paste the result into a spreadsheet.

The percent area, which is the percentage of the selected area that is white, represents the metastatic burden. Once all plates and images have been analyzed, average the percent area results between different images for each plate to mitigate any inconsistencies between pictures. Fiji ImageJ analysis was compared to manual counting and histopathological analysis.

When three separate researchers manually counted metastatic colonies, the results were inconsistent between counters. Fiji ImageJ results were consistent between counters for each of the three images. The results from the three images and the three counters were combined for each lung plate.

The results were averaged for each plate to account for the variations between the images, which provided consistent results between counters. When ranking the plates from most to least metastatic, manual counting agreed on the most confluent plate but all following ranks were inconsistent. The ranks from the average Fiji ImageJ results were much more consistent between counters.

To demonstrate the importance of avoiding reflections in the images, an image with the reflection of a hand and its subsequent Fiji ImageJ analysis is shown along with an image of the same plate without a reflection. Dark blemishes from a dirty background surface or a blood sample residue on the plates can also negatively impact Fiji ImageJ analysis. This blood plate only has two metastatic colonies but the dark residue caused Fiji ImageJ to consider it as 31.6%metastatic.

When attempting this protocol, remember to carefully examine pictures for reflection, use the same size circle for each plate in the image, and average the results for each plate between at least three separate images.