Rueda Lab

Overview

The Rueda lab is involved in basic, translational and clinical research focused on women’s reproductive health. The lab specializes in reproductive tract and cancer biology, with an emphasis on the investigation of exogenous or endogenous factors that contribute to infertility, benign gynecologic diseases, malignant transformation of gynecologic tissues, and mechanisms driving drug resistance in recurrent gynecologic cancers.

A secondary initiative of the Rueda laboratory has been to increase the number of samples collected by our fertility and gynecologic biorepository. The samples are needed for developing pre-clinical models for our translational investigations. In addition, it serves as a valuable resource for developing diagnostics for early detection of disease and or biomarkers to inform options for targeted therapy or treatment response.

Research Projects

PETAL (Plastics Exposure & Tubal-associated Lesions)

Environmental contamination by plastics and their eventual breakdown into micro- and nanoplastics (MNPs) presents a global climate and health crisis. MNPs can enter the human body through ingestion, inhalation, or physical contact and are found in circulation and in various human organs, including the lungs, brain, gut, and reproductive tract. MNPs may negatively impact human health through physical interactions and/or cellular uptake, leading to inflammatory signaling, DNA damage, and potential carcinogenic exposure. MNPs have been found in follicular fluid (FF), which is rich in hormones, cytokines, reactive oxygen species, and more. FF normally supports the oocyte, signals follicular rupture for ovulation, and promotes subsequent wound healing. Incessant ovulation, and thus the repeated exposure to FF, has been linked to high-grade serous ovarian cancer (HGSOC), the most common form of ovarian cancer, by promoting inflammation and inducing DNA damage that may lead to eventual chromosomal instability. HGSOC has recognized precursor lesions originating from fallopian tube epithelial (FTE) cells, including p53 signatures and Serous Tubal Intraepithelial Carcinomas, that are more prevalent in patients harboring BRCA mutations. We hypothesized that MNPs found in FF can cause nucleotide or organelle damage in FTE, potentially leading to the generation of HGSOC precursor lesions. Thus, our objective is to interrogate the presence of micro- and nanoplastics in FF and determine their potential to promote aberrant change in fallopian tube epithelial cells that might lead to precursor lesions.

Determining the role of the retrotransposon LINE-1 (L1) in Ovarian Carcinogenesis

High-grade serous ovarian cancer (HGSOC) is one of the most lethal gynecologic malignancies, largely due to its silent progression and late clinical presentation. Studies have shown that a majority of HGSOC initiates in the secretory epithelium of the distal fallopian tube rather than on the ovarian surface. Despite these advances, the earliest events that trigger malignant transformation in the tube remain poorly understood. Repeated ovulation has been strongly associated with increased risk of HGSOC, suggesting that physiological processes linked to ovulation may inadvertently damage the fallopian tube epithelium over time. During ovulation, human follicular fluid (hFF), enriched with inflammatory mediators, reactive oxygen species, and proteolytic enzymes, is released near the fimbriae of the tube. Exposure to these factors can induce DNA damage and replication stress, which are alterations characteristic of early precursor lesions. Our preliminary studies have suggested that this stress environment may activate L1 retrotransposons, a mobile genetic element normally silenced in healthy adult tissues. Aberrant L1 expression is associated with the formation of DNA double-strand breaks, chromosomal instability, and impairment of p53 tumor-suppressor function, all of which are recognized hallmarks of early serous tubal carcinogenesis. These observations have led to the hypothesis that ovulation-associated derepression of L1 contributes causally to the initiation of or progression of tubal precursor lesions linked to malignant transformation. To investigate this hypothesis, advanced experimental model systems, including fallopian tube epithelial patient-derived organoids, immortalized tubal epithelial cell lines, and genetic engineering to control L1 expression, are being used. These models and modifications allow controlled evaluation of genome integrity, retrotransposon activity, and cellular phenotypic shifts following exposure to human follicular fluid. High-resolution genomic analyses and ultrasensitive protein-detection technologies are being employed to define the timing and consequences of L1 activation at the earliest stages of transformation. This aspect is being conducted in collaboration with Dr. Kathy Burns at the Dana-Farber Cancer Institute and Dr. Martin Taylor at Brown University. By elucidating how physiologic reproductive processes influence genomic instability in the fallopian tube, this research aims to identify molecular events that precede cancer development. The discovery of L1-driven genomic alterations may ultimately support new strategies for prevention and early detection, enabling clinical interception of ovarian cancer before lethality becomes inevitable.

Identifying and impeding inherent and acquired mechanisms of BCL-XL-mediated drug resistance in endometrial cancer

Uterine cancer has recently surpassed ovarian cancer in lethality, becoming the deadliest gynecologic cancer in the United States. This is due, in part, to an increase in uterine cancer deaths associated with the more aggressive subtypes. Although uterine serous cancer (USC) is considered rare compared to the more common endometrioid subtype, it accounts for 40% of all uterine cancer deaths due to its metastatic potential and high likelihood of developing drug-resistant recurrent disease. Inhibitors of apoptosis, a programmed cell death process, have long been suspected in the genesis, progression, and drug resistance of many solid tumors. While the antiapoptotic Bcl-2 protein family members are postulated to contribute to drug resistance in USC, their role is not yet known. Solid tumors reportedly depend more on BCL-X Long (BCL-XL) than on other antiapoptotic BCL-2 proteins. Unfortunately, drugs targeting BCL-XL are known to have severe toxicities. Consequently, a PROteolysis TArgeting Chimera (PROTAC) against BCL-XL, currently being tested in ovarian cancer, is being tested in in vitro and in vivo preclinical models of USC. Using established USC cell lines and USC patient-derived organoids (PDOs), we are testing the effect of a PROTAC targeting BCL-XL as monotherapy and/or in combination with standard-of-care cytotoxic agents, biologics, and antibody-drug conjugates (ADCs). The endpoint analyses included MTT, Cell Titer Glo (CTG), flow cytometry, and BCL-XL by immunoblotting. Drug synergy was determined using a zero-interaction potency model. To validate our in vitro findings, we are conducting in vivo experiments using a mouse xenograft model. We aim to demonstrate that combining a BCL-XL targeting PROTAC with current treatment strategies has the potential to overcome recurrent, inherent, or acquired drug-resistant USC.

Defining mechanisms of ADC resistance in high-grade endometrial cancer

High-grade uterine cancer, unlike many other solid tumors, is on the rise. Moreover, the incidence of mortality rate is increasing, which is attributed, in part, to limited options for a durable treatment of recurrent drug-resistant disease. Antibody-drug conjugates (ADCs) are a rapidly emerging treatment strategy for advanced recurrent endometrial cancer. ADCs are designed to exploit antibody specificity to bind an antigen on a tumor cell and deliver a cytotoxic or other bioactive payload that would be intolerable if delivered systemically. ADCs are known to be highly effective in other solid tumor types; mounting evidence suggests that tumors can develop resistance. Our objective is to be proactive and investigate how high-grade endometrial cancers might develop resistance to ADCs, with a focus on therapies targeting the HER2 and FLRα receptors. Although ADCs, such as trastuzumab deruxtecan (TDXd), show promising activity in HER2-positive endometrial cancers, the mechanisms of resistance in endometrial cancer remain undefined. While no ADC has been explicitly approved for this disease, several new ADCs are in the pipeline. Consequently, we plan to use our growing number of patient-derived organoids with varying HER2 or FLRa expression to elucidate the cellular mechanisms underlying ADC resistance in high-grade endometrial cancer, encompassing alterations in antigen presentation, receptor density, signaling, ADC uptake/internalization, extracellular vesicle export, drug efflux pumps, and impaired apoptosis.

Defining how extracellular vesicles facilitate the cell-to-cell transfer of stem-like properties driving drug resistance

It is well accepted that ovarian cancer stem cells (CSCs) can seed recurrent drug-resistant disease. Similarly, it has been shown that non-CSCs can acquire CSC-like phenotypes in response to treatment. Understanding how this process is mediated is critical for informing how it might be prevented. We are testing the hypothesis that ovarian CSC and/or drug-resistant tumor cells confer stem-like properties via extracellular vesicles (EVs). Specifically, we are investigating how EVs might mediate Enhancer of Zeste Homolog 2 (EZH2) signaling to promote a phenotypic change in drug-sensitive, non-CSCs. To accomplish this, we utilized paired PARP inhibitor-sensitive and -resistant ovarian cancer cell lines, EZH2 knockdown lines, and patient-derived organoids (PDOs) originating from recurrent high-grade serous ovarian cancer. Small EVs isolated from drug-sensitive, CSC and/or drug-resistant enriched cultures, PARP inhibitor (olaparib) resistant lines, or drug-treated (olaparib or carboplatin) lines were cultured with treatment naïve or sensitive lines for defined time points. We are assessing the impact of small EV exposure by assessing cell number, metabolic activity, viability, sphere and colony-forming capacity, ALDH activity, DNA damage, and changes in associated signaling pathways. To date, we have found that EVs from CSC-enriched or drug-resistant cell fractions communicate CSC-like phenotypes to more sensitive tumor cells via EZH2 canonical and non-canonical signaling pathways, promoting stemness. Our data suggest that EV-mediated activation of EZH2 signaling represents a targetable mechanism contributing to stemness-associated drug resistance in ovarian cancer. We are currently exploring targeted strategies to disrupt this EV-mediated communication.

Identification of Liquid Biomarker and Diagnostics for early detection and monitoring of gynecologic malignancies

The Rueda Lab, along with their collaborators, Drs. Castro, Im, and Lee from the Center for Systems Biology at MGH, and Dr. Oladapo, a member of the VCRB and MGB Cancer Cener are are heavily invested in using the blood samples in our biorepository to develop diagnostic platforms for the detection of early onset and recurrence of gynecologic cancers, as well as identifying novel companion biomarkers to inform which patients might benefit most from specific treatment strategies. The team's focus has been on circulating extracellular vesicles (EVs), small lipid-bound nanoparticles, shuttling cargo into and out of cells, and mediating cell-to-cell signaling. The EVs contain proteins, lipids, and nucleic acids (i.e., lncRNA, miRNA, mRNA, and DNA). The team is developing strategies to detect, isolate, and enrich for tumor-derived EVs, aiming to improve the detection of gynecologic cancers and serve as companion biomarkers to inform treatment.

VCRB/MGB Gynecologic – Fertility Biorepository

The Rueda Lab and its collaborators were responsible for the establishment of the VCRB/MGB GYN – Fertility Biorepository. The repository was established to develop a collection of clinically applicable tissue and correlate biologic samples (ex. blood, ascites) collected from women diagnosed with gynecologic or fertility conditions and or gynecologic cancers along with their annotated medical information. From the inception one of our objectives was to establish an infrastructure whereby all biologic samples were collected under a single carefully maintained IRB approved repository protocol. Moreover, the presence of an established well-maintained repository that has appropriate management would be the best way to ensure a quality product, equitable distribution, and oversight. At present the repository has collected over 3000 samples. The VCRB/MGB Gynecologic – Fertility BioRepository is led by Dr. Bo Rueda who serves as the Executive Director. Other leaders include members of the MGB Community from the applicable clinical divisions.

This rich resource serves to support our investigators whose research is focused on developing or improving early diagnostics, defining molecular pathways contributing to the genesis of infertility, disease, and or malignancies. Importantly, these samples enable the development of pre-clinical models for testing of novel therapies and or combination drug strategies including patient derived organoids and xenografts, and discerning mechanisms contributing to inherent and acquired treatment resistance.

The overall success of our banking efforts is the byproduct of collegial interactions between the fertility, service, gyn surgeons, medical oncologists, pathology staff, clinical fellows, post-doctoral fellows, clinical research coordinators, clinical support staff and research technologists. Importantly, we recognize the unselfish generosity of the patients themselves for providing informed consent which allows us to collect the discarded samples and the annotated clinical information which we hope will benefit others suffering from similar diagnoses and conditions. Similarly, we appreciate that this resource was initiated with the support of the Nile Albright Research Foundation (A.K.A Advanced Medical Research Foundation) and the Vincent Memorial Hospital Foundation.

The benefits of this effort are evidenced by the increase in clinically meaningful publications in high quality peer reviewed publications, increased extramural funding from federal funding agencies and foundations. Also of importance is that this resource serves to promote active collaborations with pharmaceutical companies to test novel agents while recognizing the potential for opportunities to move forward into phase I trials should they be successful.

The MGH Fertility and Gynecology Biorepository actively collects fluids in addition to cells or tissues and correlate data from informed patients who have provided written consent for the collection. These fluids are important for diagnostics for early detection or for biomarkers to assess outcomes of treatment or onset of recurrent disease.

To learn more about our Biorepository, please contact Bo Rueda at brueda@mgh.harvard.edu.

Maryam Azimi, PhD – Postdoctoral Fellow

Maryam Azimi, PhD, received her PhD in Medical Immunology from Tehran University of Medical Science, Tehran, Iran. Her PhD was focused on the immunomodulatory effects of Regulatory T cells derived Exosomes (EVs) on Helper T cells (CD4+ T cells) in immunologic disorders. One main objective of her study was to find diagnostic and therapeutic biomarkers, especially MicroRNAs encapsulated in EVs.

After her graduation, Dr. Azimi continued her research field as a researcher at the Immunology Research Center at the Iran University of Medical Sciences. In 2020, she resumed her work as an Assistant Professor. During this time, she worked on the immunomodulatory effects of mesenchymal stem cell-derived exosomes in suppressing immune responses in tumors. Due to her interest in obtaining international experience, she joined Dr. Bo Rueda's Laboratory at Massachusetts General Hospital, Harvard Medical School, as a postdoctoral research fellow in August 2022.

Dr. Azimi's current research focuses on understanding the role of retrotransposons, specifically Long Interspersed Element-1 (LINE-1), in promoting DNA damage and chromosomal instability leading to tumorigenesis in gynecological tissue. Specifically, Dr. Azimi is delineating how follicular fluid might influence the LINE-1 activity in the epithelial cells of the fallopian tube using pre-clinical models, including tubal patient derived organoids.

Eugene Kim – Research Technician II

Eugene Kim grew up in Edison, NJ before receiving his bachelor’s degree in mathematics at Amherst College. He currently works as a research technician in the Rueda Lab and supports the tissue banking efforts as well as other post-docs/fellows with their projects. Eugene hopes to attend medical school in the future and attributes a lot of his motivation to his experiences at this job.

Anna Schnieder – Senior Research Lab Technician

Anna Schneider earned her bachelor’s degree in biology from Saint Michael’s College and her master’s in medical sciences from Boston University’s Chobanian & Avedisian School of Medicine. Her graduate research in Boston Medical Center’s Vascular Surgery Department investigated sex disparities and hemodialysis access outcomes. She concurrently led a Surgery Department project focused on the association of social determinants of health and safety outcomes in various device and combination product clinical trials. Now at the VCRB, she values the integration of her scientific interests, wet lab research, and patient-centered care.

Lissah Johnson - Student

Lissah Johnson is a PhD candidate in the Biological Sciences in Public Health program at Harvard T.H. Chan School of Public Health and works jointly in the laboratories of Dr. Bo Rueda and Dr. Kristopher Sarosiek. In the Rueda Lab, her project focuses on understanding how ovulation-related exposures can trigger pro-cancerous changes in fallopian tube epithelial cells — where high grade serous ovarian cancer originates. Prior to starting graduate school, she received a BS in Chemistry with Honors from the University of Redlands in Southern California and then worked as a chemist in an environmental health laboratory at the California Department of Public Health.

Esther Rodman, PhD – Postdoctoral Fellow

Esther Rodman completed her bachelor's degree in biology at Macalester College in St. Paul, MN and earned her PhD in Biochemistry and Molecular Biology at the Mayo Clinic Graduate School of Biomedical Sciences in Rochester, MN. Her thesis work focused on mechanisms of action of a JAK/STAT inhibitor for the treatment therapy resistant ovarian cancer, the molecular mechanisms by which STAT1 and STAT3 function to support ovarian cancer cell viability and growth, and the establishment of a 3D bioprinted ovarian cancer cell model to study the role extracellular matrix composition on drug treatments. Now, as a postdoctoral fellow, Dr. Rodman is interested in identifying early detection strategies and understanding the biology of early transformation events of ovarian cancer. She is particularly interested in how the environment, or the ovarian/gynecologic microenvironment, influences carcinogenesis, particularly the role of epigenetic dysregulation in driving these changes and how the underlying genetic background of each patient may contribute to this dysregulation. Dr. Rodman's current research is investigating the potential impact of environmental micro- and nanoplastics found in the follicular fluid on fallopian tube epithelial cell pathology and the development of ovarian cancer precursor lesions.