 Hi, I'm David Clinky, associate professor at West Virginia University, and what I'd like to do is introduce a new paper that's going to come out in an upcoming issue of biotechnology and bioengineering entitled Inferring Alterations in Cell-to-Cell Communication in Her Two Positive Breast Cancer Using Secretome Profiling of Three Cell Models. Now before we get into the summary of the paper, I'd like to introduce two concepts. The first concept is related to how we think of tissues as dynamic systems, and the second one is related to thinking of cancer as an evolutionary process. Using the mammary gland as an example, tissues are typically made up of multiple cell types. As shown in this picture, the milk duct includes luminal epithelial cells, which respond to female hormones, and myoepithelial cells that express smooth muscle contractile proteins. During lactation, the luminal epithelial cells produce milk, and the myoepithelial cells aid in milk release. While this figure presents a static picture, the mammary gland, like other reproductive organs, is also a dynamic structure. During the ovarian cycle, endocrine hormones coordinate the remodeling of the organs in preparation for reproduction, as shown in the top two graphs. The bottom two graphs illustrate how these hormone changes correspond to ovulation and preparation of the uterus for implantation of a fertilized egg. The middle graph illustrates the remodeling of the mammary gland, which undergoes a cycle of cell proliferation, followed by cell differentiation, and finally, an involution phase that involves programmed cell death at the end of the cycle. To summarize these dynamics, we can think of this process controlled by global triggers in the form of endocrine hormones and local control mechanisms for cell-to-cell communication that help maintain the overall structure and function as new cells join this system. Understanding how these local cues help maintain the integrity of this dynamic system remains a central challenge, and we're going to use proteomics to sort of eavesdrop on basal levels of communication between different cell types that arise in these different tissues. So when we think about this dynamic tissue, it may be helpful to have maybe an appropriate mental model for which we're more familiar, and that mental model may be one of team sports. When we think of teams are made up of a bunch of individuals, you know, we're all kind of similar, but on a team we play different roles, and ultimately its communication among members of the team determines the success of the team in a competition. The second concept is related to evolution, and when we talk about evolution, we can't help but think about Charles Darwin and his trip to the Galapagos Islands. So this trip and subsequent studies really laid the foundation with what we understand now about our views of evolutionary biology. In the intervening years, we've established a couple of key traits associated with Darwinian evolution, specifically we know that genetic alterations, which are created through sexual reproduction, can create variants that dominate a population based on their ability to survive and reproduce. The spatial influence of the environment on survival is thought of in terms of its apology, and we express this in the terms that we use are a fitness landscape. So we also know that the majority of genetic alterations have a neutral effect on the survival of the individual while detrimental variants are lost. Similarly, a mental model for Darwinian evolution may be the survival across generations of humans in a hostile environment like the Alaskan wilderness. In the field of cancer biology, there's really been a resurgence in thinking about cancer as an evolutionary process. In part, this is driven by some interesting results that have come about by sequencing cancer genomes. In a commentary published in conjunction with five studies that sequenced breast cancer genomes, Joe Gray and Brian Drucker noted that these studies show that individual breast cancers typically have a few consistent and functionally characterized abnormalities, along with tens to thousands of other changes that are rare or unique to the individual tumor about which little is known. This quote highlights a couple of points. First, the prevalence of advantageous versus neutral mutations in breast cancer is consistent with an evolutionary process. Second, there are a few genes that we understand why they happen, but for many genes we don't. So if we think about cancer from a Darwinian evolution perspective, the emphasis really is on the individual, which in this case is the cell. And most of these well-characterized alterations impart some sort of intrinsic advantage to the cell, like the ability to survive in a nutrient-poor environment, a reluctance to respond to cues that initiate programmed cell death, or a cell proliferation program that is stuck on. We collectively know a lot about how these intrinsic properties change in cancer cells as embodied in the hallmarks of cancer shown here. Though given the importance of cell communication within a tissue, we know much less about how cells' cell communication is changed during oncogenesis. To address this last point, let's review some of the key differences between Darwinian evolution and somatic evolution. As summarized here, Darwinian and somatic evolution differ in terms of the source of genetic variation and the dynamics of evolutionary change. This dynamics of change are really related to the time to reproduce, which for humans is on the order of decades and for cells is the order of days. But one of the biggest differences is the fitness landscape. In Darwinian evolution, the environment is much larger than the individual, and the individual's actions have a limited effect on the shape of this fitness landscape. In contrast, the fitness landscape in somatic evolution includes an intracellular component that helps maintain the genomic integrity of the cell, and also the local environment in terms of the interactions among components of that system. And these interactions can ultimately influence this fitness landscape. So there's a bi-directional interaction between components. In short, the objective of this paper is to test whether cell-to-cell communication is an important component of the fitness landscape that gets manipulated during oncogenesis. And we can sort of represent two alternative hypotheses graphically, as sort of shown here. So the first hypothesis we can think of as a null hypothesis is that cell-to-cell communication is not part of the fitness landscape. So the fitness landscape is essentially flat. This is shown on the left. At the center of this figure, the red dot represents a normal tissue, and the wiggly red lines represent the evolution of malignant clones. Given the random nature of mutation and the flat fitness landscape, we would expect the secretome of the malignant clones to diverge from a normal cell. On the right, the fitness landscape suggests that there is a particular way in which cell-to-cell communication should be altered for a malignant cell to survive in that system. Therefore, the different malignant clones should exhibit the same secretome and be different from the normal cell. In summary, the results of the paper suggests that cell-to-cell communication is an important component of the fitness landscape that becomes altered in breast cancer. Specifically in this paper, we used a proteomics workflow to compare the secretomes of two HER2 positive cell lines against the normal human mammary epithelial cell line. A subset of the identified proteins were validated by Western blot. Then, in comparing the inferred protein-protein interaction networks and enriched pathways, the secretomes from the two HER2 positive cell lines were similar but distinct from the normal mammary epithelial cell line. The protein-protein interaction networks suggested that the secretome contained exosomes, which we confirmed by scanning electron microscopy. Pathway enrichment results suggest that the exosomes play a role in antigen presentation and may transfer metabolic enzymes that increase glycolysis. These pathways are related to emerging hallmarks of cancer. We encourage you to delve into the details presented in the paper and wish you happy reading. Thank you.