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Various aspects of cancer cells have been studied, explored, and researched for generations. One such feature is the study of cancer biomarkers.
A biomarker is a biological molecule found in the blood, bodily fluids, or tissues that indicates normality or abnormality of processes, conditions, or diseases (1). Specifically, a cancer biomarker will be unique to a cancerous state. This provides insight in risk assessment, screening, diagnosis, prognosis, progression of disease, and response to treatment (2).
Cancer biomarkers have a wide variety of forms including proteins, nucleic acids, antibodies, and peptides, as well as a collection of alterations. The importance of studying biomarkers is accentuated by the fact that many can be detected in circulation or by excretions or secretions allowing for non-invasive assessment. An alternative cancer biomarker evaluation comes from tissue sections via biopsy.
Once the sample is collected, cancer biomarkers can be studied by various laboratory techniques, including immunohistochemistry (IHC) and immunofluorescence (IF). These two methods provide color and contrast to microscopic images for the labeling of biological structures. Next, we will explore each of these techniques and how they have been used for identifying cancer biomarkers.
IHC assays offer a unique advantage compared to other laboratory tests because they are performed with intact histological architecture. This gives an assessment of expression patterns in the context of the microenvironment (3). IHC protocols have also been widely developed and established, giving a strong platform to use in novel research. It is important to note that even though an assay may have been standardized, individual optimization may still be necessary.
The following is an excellent example of harnessing the power of IHC for cancer biomarker analysis. A study was conducted to address the relationship between a biomarker, nuclear factor kappa B p65 (NF-κB p65), and prostate cancer. IHC staining for NF-κB p65 was conducted on tissue microarray samples from the Canadian Prostate Cancer Biomarker Network.
The study found a strong association between NF-κB p65 nuclear frequency and more aggressive prostate cancer. This can help better identify patients with a higher risk of disease progression and influence patient treatment (4).
The previous example implements IHC as the primary research tool, but IHC can also be used as an important reference to confirm the validity of a study. For example, a study was done to highlight the advantages of a novel assay in breast cancer diagnostics (5). The researchers used IHC as the benchmark for comparison because it is well established as a practice in protein identification.
In addition to the advantages of IHC, IF offers some unique advantages. Primarily, the use of fluorescent detection allows for more successful multiplexing. This is especially pertinent when multiple target antigens are co-localized in the same cellular compartment of the same cell type.
Multiplexing gives the advantage of comparing the spatial relation and expression levels of multiple targets in a single tissue. For example, two cancer biomarkers could be evaluated in a single sample, or a single target biomarker could be assessed against a ubiquitous internal control.
A notable example of cancer biomarkers detected by an IF assay comes from the following study of triple negative breast cancer (TNBC). Multiplex IF was used on tissue microarrays to assess a variety of proteins including CD3, CD8, CD20, CD68, PD-1, PD-L1, and FOXP3. The study found high CD68 PD-L1 cell counts were associated with improved overall survival and breast cancer-specific survival (6). This information is clinically valuable for identifying a prognosis and guiding treatment decisions for TNBC.
Often, an assay can be carried out using either IHC or IF, but there are times when one is preferred over the other.
As mentioned above, IF is the preferred method when working with co-localized targets. IHC has the advantage of a more permanent stain compared to IF, which can experience fading of the fluorophore over time and exposure.
Additionally, tissue architecture and morphology are typically more easily observed in IHC stained tissue. Another principal factor is the available equipment in the laboratory. IHC involves visualization under a brightfield microscope whereas IF visualization requires a fluorescent microscope.
Some of the reagents necessary for the identification of cancer biomarkers will vary depending on whether IHC or IF is used. In either case, the primary antibody, antigen retrieval reagents, and some blocking reagents will likely be the same. The main difference will be in selecting a secondary antibody, tertiary reagent, and mounting media.
For example, an IHC protocol may include a biotinylated secondary antibody used in conjugation with a streptavidin conjugated peroxidase. This would be followed by a peroxidase reactive substrate such as DAB for visualization.
Secondary antibodies come in a variety of different formats including bound to a detection enzyme, a fluorophore, unconjugated, or biotinylated like in the previous example. For an in-depth explanation of these differences, check out previous blog posts on the selection of secondary antibodies for IHC and secondary antibodies for IF applications.
Regardless of the method chosen, immunostainings play a pivotal role in the detection of cancer biomarkers. This is seen in novel research, diagnostics, prognosis, responsiveness to treatment, and more. Significant strides have been made in these areas, but there is still massive potential for the future study of biomarkers and their implication in the cancer field.
Check out the blog for other insights on how IHC and IF applications can push your research forward, and stay tuned for more tips and tricks.