IGF1R Expression in Canine Immune Cells


Imagine a great dane standing next to a Chihuahua. With the Great Dane weighing almost twenty times as much as the Chihuahua, it’s impressive that these two animals are members of the same species, Canis familiaris. In addition to size differences, there is an inverse relationship between breed size and longevity.1 The genetic basis behind the wide variation in sizes has been a prominent subject of research, and in recent years, several specific regions in the canine genome have been found to influence size.2 In 2008, a genetic analysis of large and small breed dogs discovered a difference in the promoter region of the gene for a small protein, insulin-like growth factor 1 (IGF1), which is produced in the liver and circulates throughout the body. The promoter region of a gene helps regulate when the gene is transcribed into a protein. The change in the promoter region of IGF1 means less of the protein is produced and consequently, smaller breeds have lower levels of IGF1 in their blood.3 Cells with a receptor protein, insulin-like growth factor 1 receptor (IGF1R), in their membranes can respond to IGF1. When IGF1 binds to IGF1R, a cascade of events occurs within the cell leading to cell growth, proliferation, and differentiation. The interaction of IGF1 and IGF1R is especially important for the growth and development of an organism.4 As IGF1 has been studied more, it has become apparent that IGF1 plays a role beyond growth, specifically in anti-inflammatory immune pathways.5 The goal of my summer research is to investigate if the canine immune system responds to IGF1 with the hypothesis that IGF1 could play a role in health differences between breeds.


This summer, I have been working in the lab of Dr. Jennifer Punt and Dr. John Wagner at the University of Pennsylvania. Jenni is currently Dean of One Health at the University of Pennsylvania School of Veterinary Medicine, as well as Haverford’s Pre-Vet advisor. Additionally, Jenni was previously a biology professor at Haverford. As an aspiring student of veterinary medicine, I’ve been in contact with Jenni since I arrived on Haverford’s campus. Interested in gaining an experience with a veterinarian different from those I’ve had with general clinical practice, I asked Jenni if I could work in her lab for the summer, and she said yes! I then applied for, and received, the KINSC Summer Scholar’s Scholarship to fund my research. In the lab, I’m working with three Penn undergrads, a second year Penn Vet student, and two high school students. It’s been a great experience involving teamwork, communication, and research.


The majority of my work has been focused on using a technique called flow cytometry to investigate our hypothesis that there are canine immune cells that can respond to IGF1. The easiest way to tell if a cell can respond to IGF1 is to see if the cell has the receptor, IGF1R. Luckily, there is a well-established technique for identifying proteins, like IGF1R, on cells. It involves capitalizing on another type of protein that is naturally produced as part of the immune system – an antibody. Antibodies have two regions to them – an Fc region and a Fab region. The Fc region is constant between many different antibodies, whereas the Fab region is variable and binds to a specific target called an antigen. This is a property of antibodies taken advantage of by scientists. Essentially, we can manufacture antibodies that bind specifically to any protein of interest. Since cells and antibodies are miniscule and cannot be readily visualized, antibodies of interest can be made such that they are conjugated with a small molecule that fluoresces: a fluorophore.

This is where flow cytometry comes in. A flow cytometer uses fluidics and optics to analyze cells one-by-one. Through a system of lasers, the machine can determine the volume of the cell (forward scatter), the internal complexity of the cell (side scatter), and whatever fluorophores are attached to its surface. By incubating immune cells with various fluorophore-conjugated antibodies, we can begin to characterize the cells that can respond to IGF1.

So where do we find these immune cells? The immune system is partly composed of white blood cells, found, as expected, in the blood (among other places). The diagram below illustrates the basic pathway for how blood cells, including white blood cells, are made.

Modified from: opentextbc.ca/anatomyandphysiology/wp-content/uploads/sites/142/2016/03/2204_The_Hematopoietic_System_of_the_Bone_Marrow_new.jpg%5B/caption%5D

For our project, we received canine blood samples from Penn Vet’s Veterinary Clinical Investigations Center (VCIC). Since the blood contains more than just white blood cells, we had to get rid of some of the extra material. One way to do this is to destroy, or lyse, the red blood cells with ammonium chloride. We can also spin the blood through a density gradient. The hypodense layer (above the gradient) is called the peripheral blood mononuclear cell (PBMC) fraction and contains mostly monocytes and lymphocytes (T and B cells).

As I mentioned previously, existing research indicates a connection between IGF1 and anti-inflammatory pathways, specifically pathways that suppress the immune system. Within many types of immune cells, there have been subsets identified whose main functions appear to be suppressive. The ones my project has focused on are polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs), a subset of neutrophils; monocytic myeloid derived suppressor cells (M-MDSCs), a subset of monocytes; and T regulatory cells (T regs), a subset of T cells. Luckily, all three of these cell types are found in the PBMC fraction of blood.

To determine which, if any, of these might respond to IGF1, we stained them for characteristic surface markers in addition to IGF1R. The list of antibodies we used is below. Due to the limitations of our flow machine, we were only able to stain with four at a time, but we were innovative in the combinations we used.

Work done in the lab last summer yielded some promising results that suggested the vast majority of  PMN-MDSCs might express IGF1R. PMN-MDSCs are generally defined as being low density (PBMC) fraction and CADO48A+ (an antigen present on canine neutrophils.)

This figure, made by Trevor Esilu ‘21 who worked in the lab last summer, looks at the CADO48A+ population, labeled as neutrophils, and highlights their expression of IGF1R (gray) versus the whole sample (black). In whole blood, only about 2-3% of neutrophils express IGF1R. In contrast, 87% of the CADO48A+ cells (which are PMN-MDSCs) in the low density fraction express IGF1R. This result was very exciting!

At the beginning of this summer, we conducted an experiment that is a basic control for any antibody staining. Many cell types, especially neutrophils, have receptors on their surface that can bind to the Fc region of an antibody. If this happens, the cell will still be “positive” according to the flow cytometry, even though the antibody has not actually bound to the correct target.

In order to prevent this from happening, the sample has to be flooded with generic antibodies that do not fluoresce. This can be done by adding serum to the sample. Serum is blood without any cells, but it contains a wide variety of particles including hormones, lipids, cholesterol, sugars, proteins, and most important, antibodies. Generally, whole blood samples already contain serum, but PBMCs, due to the nature of their isolation, do not. One of the first things I did was conduct an experiment to see if the addition of serum changes the expression profile of IGF1R. The results from that experiment are below.

In both samples, whole blood and PBMC, without serum there is significant expression of IGF1R. Once the serum is added, however, this positive reading almost disappears. This was a disappointing way to start out my project, but we recognized that there was still some expression of IGF1R, we just still had to determine exactly where.

We still had some more suppressor populations to investigate. We decided to move onto T regs. To investigate if T regs expressed IGF1R, we stained with for IGF1R along with three characteristics of T regs: CD4, CD5, and Foxp3. Some of those results are below.

These graphs show the IGF1R expression in CD5+ CD4+ cells that are either Foxp3+ (left) or Foxp3– (right). As you can see, there is a population of the T regs (CD5+ CD4+ Foxp3+) cells that do express IGF1R. Additionally, the proportion of IGF1R+ cells is higher in the Foxp3+ cells than the Foxp3– cells. While these results clearly show expression of IGF1R in some T regs, some of our other samples indicated no expression of IGF1R in T regs. While the lack of reproducibility is disappointing, these results are very interesting and provide direction for further investigation.

While my part in the project will likely end this summer, our work will be continued by a Superlab class at Penn in the fall. Hopefully they can use our results to continue investigating IGF1R expression in T regs by testing more samples and varying ages, sizes, and disease states. Other future directions of the project include investigating potential IGF1R expression in M-MDSCs and possible stimuli that can induce IGF1R expression. I look forward to seeing which directions the project takes!

– Johanna ’21


(1) Galis, F., Sluijs, I. V. D., Dooren, T. J. M. V., Metz, J. A. J., and Nussbaumer, M. (2007, March 15) Do large dogs die young? J. Exp. Zoolog. B Mol. Dev. Evol.

(2) Boyko, A. R., Quignon, P., Li, L., Schoenebeck, J. J., Degenhardt, J. D., Lohmueller, K. E., Zhao, K., Brisbin, A., Parker, H. G., vonHoldt, B. M., Cargill, M., Auton, A., Reynolds, A., Elkahloun, A. G., Castelhano, M., Mosher, D. S., Sutter, N. B., Johnson, G. S., Novembre, J., Hubisz, M. J., Siepel, A., Wayne, R. K., Bustamante, C. D., and Ostrander, E. A. (2010) A Simple Genetic Architecture Underlies Morphological Variation in Dogs. PLOS Biol. 8, e1000451.

(3) Sutter, N. B., Bustamante, C. D., Chase, K., Gray, M. M., Zhao, K., Zhu, L., Padhukasahasram, B., Karlins, E., Davis, S., Jones, P. G., Quignon, P., Johnson, G. S., Parker, H. G., Fretwell, N., Mosher, D. S., Lawler, D. F., Satyaraj, E., Nordborg, M., Lark, K. G., Wayne, R. K., and Ostrander, E. A. (2007) A Single IGF1 Allele Is a Major Determinant of Small Size in Dogs. Science 316, 112–115.

(4) Laron, Z. (2001) Insulin-like growth factor 1 (IGF-1): a growth hormone. Mol. Pathol. 54, 311–316.

(5) Smith, T. J. (2010) Insulin-Like Growth Factor-I Regulation of Immune Function: A Potential Therapeutic Target in Autoimmune Diseases? Pharmacol. Rev. 62, 199–236.

The Manifestation of Colorectal Cancers and How to Proactively Protect Yourself from the Third Most Common Form of Cancer.

Colorectal cancers have been widely studied due to their massive impact on Americans. In 2019, estimates from the American Cancer Society expect slightly over 100,000 new cases of colon cancer and 44,000 new cases of rectal cancer to be diagnosed. Additionally, colorectal cancer is expected to cause around 51,000 deaths in the U.S. in 2019 alone. Although these statistics are quite powerful, the number of cases and deaths have been decreasing as further scientific understanding of the mechanisms of these cancers is elucidated.

This summer, I am working in the Kalady Lab at the Lerner Research Institute of the Cleveland Clinic. My lab specializes in providing insight into the genetic underpinnings of colorectal cancers and applying this knowledge to the clinical realm in order to treat patients. I will be reporting on my project further along in the summer, but I first wanted to start with a blog post explaining how colon cancer manifests and how the worst aspects of this illness are often preventable.

Understanding tumor formation requires understanding the cellular mechanisms that tumor cells hijack. Tumors cells are abnormal cells that have somehow beaten the system and have acquired the capacity to uncontrollably divide. The methods in which tumor cells diversify or transform often confer a selective advantage in comparison to regular cells, indicating why tumors can grow at such a fast and uncontrollable pace. For colorectal cancers, there have been two major pathways established for how tumors can develop. These separate pathways include modifications to genetic, epigenetic, and DNA mismatch-repair (MMR) systems that are associated with cell growth, differentiation, motility, and survival. For the purpose of this blog post, I am going to define the genetic and epigenetic roots of tumor formation in addition to a shorter explanation of MMR.

The underlying genetics involved in cancer have been studied for decades, leading to an understanding that certain mutations that can lead to benign and malignant tumors. The Chromosomal Instability Pathway, described by Bert Vogelstein in 1988-1990, establishes that a certain number of genetic mutations in specific genes are correlated to different stages of tumor development.

As seen in the figure, three genes are often referred to within the Vogelstein pathway as genes or processes that must be mutated or upset in order to undergo tumor formation. All of the mutations in the Vogelstein Pathway are additive; there isn’t a necessary order to the mutation process. Because tumor formation requires all of these mutations but not in any order, tumors are much more likely to win out as random mutation is a byproduct of the imperfection of nature.

The APC gene, or adenomatous polyposis coli gene, codes for a regulatory protein in the Wnt pathway. When a mutation occurs in APC, its protein’s ability to interact with and bind to β-catenin is ceased. β-catenin is a signaling molecule that can call for the upregulation of genes associated with proliferation. Therefore, if APC is mutated and loses functionality, proliferative genes can be more highly expressed, leading to one of the hallmarks of cancer. The next gene mentioned in this figure is Kirsten-ras or KRAS, which codes for a cell-signaling protein that works within the RAS/ERK pathway of signaling. When RAS proteins are phosphorylated, they can pass their phosphate to the next protein in the pathway: BRAF. Eventually, the pathway involves a protein called ERK that has the potential to upregulate more genes associated with proliferation and survival. Mutations in either KRAS or BRAF have been clinically observed in early adenoma formation, a benign growth that needs only one more mutation to become cancerous. The final gene that provides a barrier against tumor formation is the p53 gene. p53 is a transcription factor that assists in the control of the cell cycle. This transcription factor will bind to DNA and can downregulate genes associated with survival and proliferation; however, if p53 is mutated, this function is lost. p53 is somewhat of a last resort in tumor prevention, although the exact reasoning as to why has not yet been fully elucidated.

The second pathway to colon cancer development was established in a study by Toyota et al. in 1999. This pathway arises from the CpG island methylator phenotype (CIMP), which is an observed phenotype of epigenetic silencing of certain DNA repair and cell maintenance genes through promoter methylation. The physical process that occurs includes the sequestering of a protein complex to certain promoter locations of DNA. At the promoter, methyl groups are added by the complex to cytosines that share phosphodiester bonds to guanine nucleotides. When CpG’s form in groups, this clustering of methylation is deemed an island and can lead to the inability of other transcription factors successfully interacting with the promoter. Ultimately, methylation leads to downregulation of transcription of the following gene, thus cutting off any cellular outcome attached to the target gene.

The identification of CIMP has been made only in relation to genes associated with regulatory processes within the cell, which, when hijacked, can lead to tumor formation. Most of the genome is actually methylated at any given time; proteins must often demethylate DNA via nucleotide excision repair or mismatch repair in order to express the following gene. Therefore, when studying CIMP, it is critical to have a thorough and accurate process in how to delineate between CIMP+, CIMP-, and CIMP0. This delineation has been established differently between studies, but the most common identification technique of CIMP-status is a five-panel marker established by Weisenberger et al. in 2006. This panel includes genes that are all somehow associated with CIMP and that when methylated, resulting tumors show symptoms correlated to CIMP+.

The distinction of why CIMP-status becomes important is in the type of tumor formed by CIMP+ versus CIMP- status. CIMP+ tumors are often less differentiated and are more aggressive tumors. Prognostically, data differ between whether CIMP+ or CIMP- possess more clinically favorable outcomes, though. These differences arise from the multiple panels used to analyze CIMP status. Regardless of which panel is used, a certain gene correlated to MMR is always addressed: MLH1.

MLH1, mutL homolog 1, is a protein that assists in fixing errors in DNA replication prior to cell division. MLH1 complexes with PMS2 to cut out erroneous nucleotides and properly replace the necessary nucleotide as replication occurs. MLH1 mutation causes the gene to lose its ability to regulate DNA replication through mismatch repair. Without MLH1 functionality, mutation rates drastically increase causing DNA hypermutability, designated as microsatellite instable, or MSI-H. The outcome of microsatellite instability is that if a tumor is formed, the tumor genome is unstable as MLH1 cannot spell-check its replication. Conversely, without an MLH1 mutation, a tumor is designated as microsatellite stable, or MSS. This distinction provides a more controlled environment for tumor growth and is disadvantageous prognostically.

Throughout this background on the formation of cancer, it is still crucial to acknowledge that although it may be interesting to study the causes of this disease and where the body is prone to failure, individual people still suffer from the outcomes of these failures. The only reason that we have access to such a great deal of information about colorectal cancers is because thousands of patients have been willing to undergo additional testing or provide samples of their tumors during surgery. Ideally, more effective screening should reduce the number of patients who have to experience the physical and mental ramifications of colorectal cancer. Currently, the American Cancer Society recommends that people with average risk for colorectal cancer should begin screening at age 45.

Major issues exist in making this screening accessible to all peoples, but if the option exists to receive the screen, there is no reason to not immediately schedule testing. Colorectal cancer is one of the only cancers that can be controlled or prevented simply by adequate screening, and for most, there is no reason why this illness should be a risk. Therefore, please advocate for screening or sign up for your own screening soon! This can easily be a life-changing or even life-saving decision.