A Cancer Initiation Study Using Immunofluorescence

Notes from the KTB Lab

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In the search for new preventive strategies, determining the molecular mechanism of breast cancer initiation is critical. However, studying the early stages of breast cancer development is challenging for the scientific community mostly because of the lack of tissue specimens at an initial phase of the process, when the tumor mass is not yet clinically detectable.


In our lab, we are able to address this challenge because of the availability of normal breast tissue samples donated to the KTB by women who were “healthy” at the time of donation and few years later were diagnosed with breast cancer.

BCRF Investigator in Breast Cancer Research Natascia Marino, PhD, is leading the Cancer Initiation study, where breast tissue samples from women who at the time of donation were clinically healthy, but after 2-5 years were diagnosed with sporadic breast cancer. In order to reduce the biological differences between individuals as much as we can, we match the susceptible and healthy women by age, racial background, BMI and menstrual phase. The specimens donated by these women provide a window into the early stage of breast cancer development and evaluating how their breast tissue differs from those of unaffected women.


Dr. Marino and KTB lab tech Rana German


We are investigating changes in the expression of genes in very early stages of breast cancer. Because each breast compartment has its own critical role in maintaining tissue homeostasis, as well as in cancer progression, we have performed a comprehensive study of the three breast tissue compartments: milk-producing cells or epithelium, surrounding stroma and adipose tissue (the cells that store fat). We detected a significant increase in metabolic pathways in the breast epithelium of the susceptible samples as well as increase in genes involved in cell movement, suggesting early cell dissemination. Interestingly, the data from the stroma compartment showed a significant reduction in immune cells homing in the susceptible as compared with healthy samples, as indicated by the downregulation of genes coding for several immune cells markers and receptors. The adipose tissue of the susceptible breast also showed dysregulation of genes associated with metabolic activation and immune response inhibition.

Fatty acids are known to be utilized by the cancer cells as a source of energy for cell growth. In this study, we are also interested in evaluating whether an increase in free fatty acids in the breast tissue may cause DNA damage in the breast epithelial cells.


One of the technical methodologies commonly utilized in our lab is immunofluorescence. This technique uses fluorescent dyes that identify a specific target. The process allows the target(s) in the tissue to be localized and quantified.


In this image, obtained using a Keyence BZ-X800E fluorescence microscope, you can see three different targets that were detected in one frozen normal breast tissue:

Blue: A dye called DAPI, which is a fluorescent stain that binds the DNA, is used to detect the nuclei or an area of the cell that contains the nucleic acids, or DNA.

Green: A green labeled antibody recognizes and binds to the γ-H2AX protein, a key protein that helps to protect the DNA from damage.

Red: BODIPY dye detects free fatty acids.