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 Assay  

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. 

IF Assay  

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. 

IHC vs. IF Assays 

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.

References 

1. National Cancer Institute. 2023. NCI Dictionary of Cancer Terms, Biomarker. 
2. Henry NL, et al. 2012. Cancer Biomarkers. Molecular Oncology. 
3. Schacht V, et al. 2015. Basics of Immunohistochemistry. Journal of Investigative Dermatology. 
4. Grosset AA, et al. 2019. Validation of the Prognostic Value of NF-κB p65 in Prostate Cancer: A Retrospective Study Using a Large Multi-Institutional Cohort of the Canadian Prostate Cancer Biomarker Network. Journal of the Public Library of Science. 
5. Erfani P, et al. 2023. Breast Cancer Molecular Diagnostics in Rwanda: A Cost-Minimization Study of Immunohistochemistry Versus a Novel GeneXpert® mRNA Expression Assay. Bulletin of the World Health Organization. 
6. Wang J, et al. 2021. Multiplexed immunofluorescence identifies high stromal CD68+PD-L1+ Macrophages as a Predictor of Improved Survival in Triple Negative Breast Cancer. Scientific Reports. 

Heart Health Awareness Month

Heart diseases comprise many different conditions, and together, they are responsible for about 20% of all deaths in the United States (1). Coronary heart diseases—blockage or narrowing of the heart’s main blood vessels—are the most common type of heart disease and can lead to myocardial infarction (MI), a serious and life-threatening condition (1). MI happens when blood flow to the heart completely stops. As a result, lack of oxygen and nutrients results causes damage to the heart muscle and contribute to heart failure (1).  

Current treatments for MI focus on preventing reoccurrence. Patients often receive medications to prevent blood clots, improve blood flow, slow heart rate, and reduce blood pressure (2). Clinical management of MI still lacks interventions focused on counteracting pathophysiological changes and preventing damage to the heart muscle. The development of such treatments can have a major impact on public health, as it has the potential to improve health outcomes in patients who have had MI.  

Researchers from the Oregon Health and Science University, Sepe et al., characterized pathophysiological changes in the heart in response to novel therapeutics that disrupt protein tyrosine phosphatase-s signaling (3). They observed that the treatment restores sympathetic innervation and shifts the immune response after MI to promote tissue repair. As a result, heart function improved and susceptibility to arrhythmia decreased (3). 

Keep reading to learn more details about the research study, and check out the blog for other publication highlights. 

Myocardial Infarction Pathophysiology  

After MI, 2 waves of immunological response recruit and activate innate and adaptative immune cells to clear cellular debris and remodel the infarcted heart (4). A pro-inflammatory response characterizes the initial phase: presence of M1 macrophages, release of pro-inflammatory cytokines, proteolysis, and phagocytosis (4). In the second phase, the pro-inflammatory response tapers off (but doesn’t fully resolve), and the healing and remodeling processes begin (4). The number of reparative immune cells, such as M2 macrophages and regulatory T cells (Tregs), increases, contributing to the development of a mature scar (4). Persistence of the inflammatory response beyond the initial phase often results in pathological remodeling of the myocardium and contributes to heart failure (4). 

MI pathophysiology also includes reperfusion-induced sympathetic nerve damage (5,6). And chondroitin sulfate proteoglycans in the cardiac scar activate protein tyrosine kinase phosphatase receptor-s on sympathetic nerves and prevent nerve regeneration even in the presence of nerve growth factor (5). In the end, the extent of nerve damage predicts the development of ventricular arrhythmias (7–9).  

Study Rationale and Hypothesis 

Previous studies have identified 3 novel therapeutics that can promote sympathetic reinnervation in the heart after MI: Intracellular sigma peptide (ISP), HJ-01, and HJ-02 (6,10,11). ISP prevents the binding of chondroitin sulfate proteoglycans to protein tyrosine phosphatase-s, and HJ-01 and HJ-02 disrupt protein tyrosine phosphatase-s–tropomyosin kinase A interactions (6,10,11). As a result, sympathetic nerves can regrow even in the presence of chondroitin sulfate proteoglycan. In addition, disrupting protein tyrosine phosphatase receptor-s normalizes heart function and confers resistance to isoproterenol-induced arrhythmias (6). 

Sepe et al. conducted experiments to validate previous findings and determine the impact of treatments with ISP, HJ-01, and HJ-02 on the profile of the immune cells after MI. They hypothesized that therapeutics would shift the immune response into a reparative phenotype and used quantitative multiplex immunohistochemistry (mIHC) to test their hypothesis. 

Modulation of Immune Response 

Mice underwent 2 surgeries: The first one to implant telemetry electrodes in the heart, and the second one, 5 days later, to induce ischemia-reperfusion. For the latter, researchers occluded the left coronary artery for 45 minutes and then released the ligature to induce reperfusion. Sham mice underwent surgery but had no occlusion. Between 3–10 days after surgery, mice received daily intraperitoneal injections of ISP, HJ-01, HJ-02, or vehicle. Data collection took place 14–15 days after surgery. 

As previous studies have shown, treatment with ISP, HJ-01, and HJ-02 promoted sympathetic reinnervation of the heart, as assessed by immunohistochemistry (IHC) for tyrosine hydroxylase (5,6). Mice that received treatment had sympathetic nerve density similar to that of sham animals. In addition, sympathetic reinnervation of the heart resulted in functional improvement. Mice that received treatment had fewer isoproterenol-induced arrhythmia than mice that received vehicle.  

Next, researchers used mIHC to phenotype immune cells in the heart after MI. They performed 15 rounds of IHC to target a total of 23 cell markers. Each round of the mIHC process consisted of antigen retrieval, incubation with primary and secondary antibodies, staining with ImmPACT® AEC Substrate Kit, Peroxidase (HRP), slide visualization and image acquisition, and stripping (Figure 1). The subsequent image analysis process took place in 3 steps: Image processing, cell classification, and quantification of cell types (Figure 1). 

I could see things that my eyes alone can’t. And so, I was passionate about biology from the first time that I started to study it. I was also fascinated with history and archaeology. I saw being a biologist or an archaeologist as my ideal job because both required the curiosity that I had to make a discovery. Both had the potential for excitement and the ability to discover something new, but in the end, I decided that biology was right for me.  

How did you decide to leave Italy and come to San Diego? 

I’ve been in San Diego for 4 years now. I love this place. Honestly, it was the dream of my life, to come from Italy and live in California. As part of my PhD program, I had the opportunity to spend 6 months outside Italy so I was looking at several labs that I could join. I first thought about a lab in San Francisco, but in the end, I decided to join a cancer research lab at the Institute of Genetics and Molecular Medicine at the University of Edinburgh in Scotland. The Institute of Genetics and Cancer is a strategic partnership of the Medical Research Council (MRC) Human Genetics Unit (HGU), Cancer Research UK Edinburgh Center, and the Centre for Genomic and Experimental Medicine. I had a fantastic experience in Edinburgh but when I went back to Italy after discussing my PhD, I was still thinking about needing to go to California to work. I believed that I could have a well-balanced lifestyle and access to job opportunities within a strong research environment. They say that California and San Diego are perfect because they also have beaches, which help you achieve a nice balance. My other alternative was in Boston. And while I loved the research they were doing, I was not very excited about their much colder climate. In some ways, I think destiny was involved because whenever I looked at the location of the affiliation of the people authoring a paper, it was always La Jolla, La Jolla, La Jolla. And then after I checked what this area looked like, I said, “Wow, this is wonderful, I want to go there.” 

How was your science education different in Italy? 

When I went to middle school and high school in Italy, we didn’t have laboratory science classes where you could do practical experiments. We studied a lot, but only with books and both oral and written tests. I never had the opportunity to work in a lab until I went to university. My initial response for each college lab was that it was super exciting! Unfortunately, our lab sections were not very long, and we didn’t have many during the year. But to get a PhD in Italy, we must first earn both a Bachelor’s and Master’s degree. And for each, we need to write a research thesis, requiring us to join a lab, follow a project, and write about those experiments.  

How did you become interested in cancer research? 

For my Bachelor’s research, I did three months of laboratory research with a team focused on cancer research. Initially, I was excited about the genetics and molecular biology focus of the work. But, for personal reasons associated with the events that were happening in my life, I grew to understand that I wanted to do science to help people. In my family, a lot of people die of cancer. I get a little bit emotional when I think about that because I know first-hand what happens to a person with cancer and how it affects their family. We were working on a tumor suppressor protein, and I had a nice experience—you can tell because I worked there not only through my Bachelor’s and Master’s programs but also for my PhD, which allowed me to see this project grow. And so, I’m (still) attached to that project, the team, and the place. Looking back, I think it was the best school for me to learn how to work in science. 

How did you learn to adapt to funding difficulties? 

In our labs in Italy, we didn’t have enough money to do the work—there were funding issues all the time. But there’s a lesson to be learned in that: when you don’t have many resources, you develop the skills you need to adapt and learn to accept the things that you can do. But to me, it made me question why we should do these experiments or figure out whom we could ask for a reagent when we didn’t have it. It was complicated. But it also gave me a push to want to do more and find a better situation when possible. You need to be able to adapt when something goes wrong. And, unfortunately, one of the main problems in science is the availability of funding: even in the best lab, there are periods when you know that the funding is going to expire, and you need to adapt to a stricter budget for a short time. And to me and where I come from, you give value to each thing, you don’t throw a thing away, and if there is an issue, you find a solution.  

What is most interesting about your current research?  

I joined this lab to work on tumor metastasis. While they have strong expertise in breast cancer, they also have subgroups that are each looking at a different aspect of tumor metastasis. My subgroup is looking at the actin-based structure that carcinoma cells form to degrade the extracellular matrix, invade, and metastasize. My project’s goals are to understand the molecular mechanisms, identify new proteins that affect this subcellular structure or the metastatic cascade, and find a new therapeutic target in the long term. What excites me about it is that this could translate into a positive impact on a cancer patient. We are still assessing which kind of cancer to focus on so that I can analyze the right kind of target, and we have resources available for that. And the project itself is interesting from a molecular biology standpoint. Also, the mechanisms of these small and highly dynamic structures are difficult to study, so having good microscopy skills is essential. We are developing good resources for live imaging and confocal microscopy in cancer cells. 

Why do you believe so strongly in achieving a strong work-life balance? 

It’s something that I’m learning here, to have this good balance between my work and life. As I see with my colleagues who do research in academia, a lot lose focus on themselves. They are too involved with only doing science. But having a 100% science focus can result in a loss of enjoyment for both your life and your science.  

What’s next for you after you complete your postdoc?  

I don’t know. I haven’t decided yet because everything depends on how all this research right now will go. I’m still evaluating the possibility of a career in academia or sometimes I also think about the job of an editor, reviewing papers, or maybe a job in a biotech company to develop a product that can be helpful to treat patients. I need to think more about my future direction in science. I’ll be a postdoc at least until next October. Unlike in Italy, where careers in science are limited to academia and that’s it, in the US, there are a lot of different kinds of jobs that you can do with a PhD in biology: you can be what you want and change your career path anytime along the way just by a little bit or by a lot. 

How important is it to be passionate about your work? 

I can tell you that my best memories of my previous lab are related to passion. While the people were going to work and coming to the lab day after day, year after year, everyone was motivated by their passion. When this happens, it feels like there is a mental interaction, a strong connection. When that happens, you feel comfortable messaging each other in the evening to share ideas. And honestly, I would be happy if everyone in life could have this kind of experience. When you can find someone that is on the same wavelength in your job, it’s the best feeling that you can have. You know that that person thinks similarly to you. And when you interact with her or with him, the conversation can go on for hours. And while it’s job-related, you don’t think of it as work because you are talking about your passion.  

What is the most rewarding facet of working in the sciences? 

Reward is the feeling that I have when I am discovering something, and that is one of the best feelings. You can especially see it at the end of a project when you pull the story together in a publication. I also love the rewards of communicating with people and talking about science:  sharing ideas, hearing what others think, seeing how people react, and taking suggestions. I see communication as a moment of interaction with others and not as a moment where they are judging you. Because I think that science is all about teamwork, one of the best parts of this job is interacting with others that you work with day-to-day and side-by-side. Being open to suggestions from others who might be able to see something that you don’t notice can provide a new way to look at things. And that is even more rewarding.  

What is the biggest life lesson you’ve learned from your work? 

I learned that you need to be persistent, but not fixed. A lot of people in this job can get fixed on some concept and then they spend a month or year or more on it rather than accepting that it just doesn’t work. It can be difficult to separate the failure of an experiment or a negative result from who you are and how well you understand the science. I couldn’t do that during my PhD, but I’ve gotten better since then. Now I try to focus on the main goal and find alternative ways to solve issues. This fosters discovery, and that’s what I do.