Research

The main focus of research in the CGR are two diseases caused by ectopic growth of the endometrium to form lesions: endometriosis (endometrium growing outside the uterus) and adenomyosis (endometrium growing in the uterine muscle, or myometrium). Whether these two conditions are twins, or just siblings, is a matter of great debate.

CGR’s “Systems Biology” Approach

The observations expressed in the questions above – and others regarding the huge disparity in patients’ ages are when they first discover symptoms, their differences in responses to therapies, and the wide range of co-morbidities – led us to bring a “systems biology” approach to the study of endometriosis, adenomyosis and related diseases.

Endometriosis is currently staged by the numbers, size, and location of lesions and adhesions.  Why isn’t endometriosis also characterized by molecular markers related to disease mechanism, prognosis, and treatment, like breast cancer? One reason is that cancers have “somatic mutations”—tumor cells have mutated genes that drive malignancy. The mutations in cancer patients can be identified, and in many cases, linked to prognosis and therapies. Endometriosis, like rheumatoid arthritis, Type 2 diabetes, Alzheimer’s, Crohn’s disease, and other chronic inflammatory diseases, is not known involve somatic mutations as a disease driver.

We borrowed from systems biology research in these other diseases to propose that irregularities in cell-cell communication networks – which might arise to many different kinds of gene-environment interactions – might provide insight into general mechanisms and even discern different patient groups. Although many researchers for decades have discovered how individual molecules or pathways are aberrant in endometriosis patients, in the systems approach, we examine how pathways operate in networks, and networks of different kinds intersect to control phenotype. This approach has led to identification of possible molecular subclasses of patients implicating new disease targets.

Explaining CGR’s Systems Biology Approach

Complex diseases like endometriosis and adenomyosis are challenging to dissect with genetic studies alone, as they often involve many different genes interacting with many different environmental cues. Protein activity states – for example, the activities of intracellular kinases and extracellular cytokines, chemokines, growth factors, and proteases – integrate information from genes in a dynamic fashion and are therapeutic targets.

A major challenge is that these proteins and related metabolic signaling molecules operate in large networks that usually contain recursive features, such that thwarting an individual pathway can accentuate malignant activity of the network as a whole. (Fig. 1)

Our research focuses on understanding how cell communication networks within and between cells and between organs are disrupted in disease, using a compendium of computational and experimental approaches that often involve highly multiplexed measurements from patient samples including peritoneal fluid, endometrial biopsies, and lesions. These approaches derive from extensive research by CGR investigator Doug Lauffenburger in cancer and other diseases. (For more information about Lauffenburger’s research, click here.)

For example, by measuring the concentration of 50 different cytokines in the peritoneal fluid of endometriosis patients and analyzing the data in a multivariate way, we found a macrophage-driven immune network (Fig. 2) in a subset of patients and identified Jun kinase as a driver of cytokine release (1).  (Fig. 3)

We are now applying these approaches to parse immune networks in infertility (2, 3, 4, 5) and in adolescent endometriosis (3).

Endometriosis and adenomyosis are also invasive diseases driven in part by growth factors shed proteolytically from the cell surface. We developed a new combined experimental and computational approach to analyze a compendium of protease activities in the context of endometrial cell migration, and found that that ADAM-10 and -17 dynamically integrate numerous signaling pathways to direct endometrial cell motility (Fig. 4) and that growth-factor-driven ADAM-10 activity and MET shedding are jointly dysregulated in the peritoneal fluid of endometriosis patients (6).

To study protease network activity, we developed two new approaches to measure the concentrations of active proteases in fluid and tissue samples and applied these to measurement of proteases in menstrual blood and myometrium in adenomyosis patients (7, 8).

Beyond endometriosis, CGR affiliate Doug Lauffenburger is applying systems biology to analyze how pathogen-induced antibodies and associated immune cell functions, such as against SARS-CoV-2, are transferred from mother to fetus during pregnancy.

Tissue Engineering and Physiomimetics

Systems biology approaches enable identification of potential disease mechanisms, however animal and standard cell culture models do not capture the full spectrum of human disease. We are therefore using advanced tissue engineering approaches to model the eutopic endometrium and ectopic lesions using samples of tissues derived from patients, to create patient avatars for testing personalized medicine approaches. We use results and insights from the systems biology analysis of patients to design these in vitro models, and term this integrated approach “Physiomimetics”(9). (Fig. 5)

We first create cell banks by isolating, expanding, and cryopreserving individual cell types from tissues and blood, so that many different experiments can be done on individual patient samples at different times. (Fig. 6) We then reconstruct the important features of tissues combining different cell types with synthetic biomaterials that foster 3D tissue organization. Standard protocols for expanding and culturing endometrial epithelial cells involve organoid culture in basement membrane Matrigel, a complex mix of extracellular matrix (ECM) proteins derived from a tumor. (Fig. 7)

Matrigel is not a preferred matrix for stromal cells and immune cells, and it is broken down quickly by cells. We therefore developed a “one size fits all” synthetic hydrogel that can be used for expansion and culture of human organoids from intestine (Fig. 8, Fig. 9) and endometrium (Fig. 10) while allowing integration of stromal, endothelial, and immune cells and creation of 3D mucosal barrier structures (10-14). (Fig. 11)

A particular advantage to this synthetic ECM is that it can be dissolved with the microbial enzyme Sortase A, which does not cleave human proteins, so that cells and local pericellular proteins can be analyzed. Another advantage is that the biochemical and biophysical features can be tailored, for example, to create a stiff microenvironment that mimics the environment in a fibrotic lesion or to induce morphogenesis of gland-lumen structures in endometrial co-cultures.

Organs-on-Chips

“Organs-on-Chips” or “Tissue Chips” refers to complex mimics of human tissues maintained in microreactors, such that fluid flow provides oxygen and nutrients, as well as mechanical stimulation, to 3D tissues. Most of the work to date has been on other organ systems that are important systemically for endometriosis and adenomyosis, and approaches are now being applied to the endometrium and to lesions. The Griffith lab pioneered a microreactor for long term 3D microperfused culture of liver (15), and together with collaborator David Trumper, translated the design to a multi-well plate format (16) by designing a novel microfluidic on-board pumping technology (17); this technology has been commercialized by CN BioInnovations.

The liver chip technology has been used continuously in the Griffith lab since its introduction (18-22), and is currently used to study Type 2 diabetes, in a project sponsored by NovoNordisk. More recently, the lab has developed interconnected organs-on-chips to study systemic diseases involving organ crosstalk, developing a proof-of-principle 10-organ interactome incorporating the endometrium as part of a demonstration of drug pharmacokinetics (23).

The lab has recently applied version of this platform to illuminate how short chain fatty acids produced in the gut influence gut-liver inflammatory interactions, possibly potentiating inflammation in ulcerative colitis (24).  A more sophisticated model to co-culture a healthy colon mucosal barrier with the colon microbiome is being used to study how the gut microbiome regulates immune function (25), and can potentially be applied to co-culture of the endometrial or cervical mucosal barrier with a microbiome.

With this advanced microfluidic device (Fig. 12), a healthy human colon mucosal barrier can be co-cultured with the most oxygen-sensitive gut microbe, Fecalibacterium prausnitzii. (Fig. 13)

An exciting frontier is the adaptation to endometriosis of microfluidic device technologies used to create microvascular structures in tumor tissues (26). Conceptually, these microfluidic devices can be used to model the birth and early life of lesions and how they interact with blood vessels to recruit immune cells and stem cells, and how they respond to current therapies (e.g. progestins, combined oral contraceptives, GNRH agonists/antagonists) and to proposed new therapies). (Fig. 14)

Surgical Tools and Instrumentation for Diagnostics and Treatment