Exosomes are transportation and logistics services of the body that carry biomolecules to specific cellular targets and have capability to heal the defective cells and tissues. We harness the targeting capability of exosomes to improve the efficacy of current drugs and healing capability of exosomes to usher a new era of therapeutics for the brain disorders.

 

Menstrual-Effluent Exosomes as a Non-Invasive Biopsy for Gynecological Health

 

Group 40: Angela Neighbors

Mentor: Dr. Pranav Sharma, Xosomix

 

Project Objective

 

To develop a noninvasive and reliable diagnostic of endometriosis that tests the in vitro angiogenic activity of exosomes found in menstrual blood.

Background

 

Endometriosis: An invisible pandemic of silent pain

 

 

Endometriosis is a chronic condition that occurs when uterine tissue grows outside the uterus, on or around other reproductive organs in the pelvic cavity. Approximately 11% of reproductive age women, more than 4 million in United States alone, are affected by endometriosis. This is an underestimate as invasive surgery is the only FDA approved diagnosis of endometriosis. Endometriosis is seen in 12-32% of women undergoing surgery for pelvic pain and up to 50% of women undergoing surgery for infertility. About 60% of cases are undiagnosed and it's common to see a delay in diagnosis by 8-10 years. Economic impact from endometriosis is estimated to be ~$121B per year. In spite of tremendous physical, psychological, and economic costs, a non-invasive FDA-approved diagnosis of endometriosis remains elusive.1 

 

 

 

Exosomes are nanoscopic sacs secreted by all cells and are key mediators of communication and transport in the body. In a disease state, the message in exosomes is changed, relaying the earliest 911 distress call to the body asking for help. Exosomes carry nucleic acids, proteins, lipids, and metabolites.3 Since exosome signaling is altered in disease, we aim to investigate how increased angiogenesis due to exosome signaling is involved in the pathogenesis and development of endometriosis.

 

Hypothesis

 

Signaling via exosomes is altered in disease. Increased angiogenesis is known to be involved in pathogenesis and development of endometriosis. We aim to test if menstrual effluent (ME) exosomes can be used to report increased angiogenic activity in endometriosis.

 

Methods

 

Comparison groups:

 

  • Endometriosis (confirmed through laparoscopy)
  • Asymptomatic Control

 

Sample collection:

 

Participants for the study were recruited under the guidance and approval from UCSD Institutional Review Board (IRB). Volunteers were asked to collect menstrual sample during the day 1 of the patient’s menstrual cycle through the use of a silicone menstrual cup and bring to Xosomix lab in a sterile centrifuge tube for further processing as described below. Typical sample collection volumes are in the range of 3mL to 7mL.

 

Schematic describing procedure for purification of exosomes from menstrual effluent 

 

Exosome purification:

 

Menstrual samples were first diluted with DPBS in a 1:2 ratio and 10 μL EDTA is added per 1mL of diluted blood. After mixing thoroughly, the blood was carefully layered on top of Lymphocyte Separation Medium (LSM, Ficoll-Paque), where the volume of LSM is ¾ of the total volume of blood.

 

Blood/menstrual effluent layered on top of Ficol density gradient medium

 

The layered blood and LSM was centrifuged without the brake applied at 800g for 30 minutes at 18-24°C. Upon centrifugation in Ficoll density gradient, the blood separates into a pellet of granulocytes and RBCs, buffy coat of larger PBMCs and uterine cells, and a topmost layer of plasma. Once separated, the topmost plasma layer was taken from the tube, without contaminating the plasma with any of the cells from buffy coat directly below.

 

Blood/menstrual effluent separated into plasma (topmost layer), buffy coat (middle), and pellet.

 

The plasma was placed in a separate tube and centrifuged at 2500g for 10 minutes to remove any remaining cells. After centrifugation, the plasma was decanted into a new tube, and the 2500g spin was repeated for another 10 minutes to ensure that plasma is completely cell free.

 

The plasma was decanted into a ultracentrifuge tube and spun at 12000 g for 40 minutes at 4°C, which pellets the larger microvesicles (MV) out of the plasma. The MV pellet was resuspended in RIPA with protein inhibitors and nuclease, then stored at -80°C.

 

The plasma was decanted into a new centrifuge tube, and then spun at 120000g at 4°C for 2 hours to pellet exosomes.  After the initial 120k spin, the supernatant was decanted, and the pellet was washed with DPBS and spun twice as much at 120000g for additional washes. The exosome pellet was then resuspended in ~0.5mL of DPBS until completely dissolved, then stored at -80°C until use.  

 

Exosomes retrieved from menstrual samples were quantified using a microBCA protein quantification kit (ThermoFisher Scientific).

 

Angiogenesis assay:

TERT immortalized HUVECs (Human Umbilical Vein Endothelial Cells; ATCC CRL-4053) seeded on a gel basement membrane (Matrigel, Corning Inc) respond to angiogenic signals. They form tubular mesh like structures after exposure to angiogenic signals. The tubular meshes can be measured to provide a quantitative measure of angiogenic activity.

 

 

Corning® Matrigel® Matrix was used, with 10μL of Matrigel plated onto the bottom of the well within a well of specialized imaging 96-well plates (ibidi USA, Inc.), to provide a flat substrate for growing HUVECs. 50 μL of DPBS was added to the perimeter wells of the 96-well plate to prevent drying of matrigel and evaporation of media. 

 

Phase contrast image of HUVECs (ATCC CRL-4053)

 

HUVECs were passaged 2 days prior to the assay at a concentration of 2.25 X 105 in T-25 flasks with aim to reach an optimal confluency of ~80% at assay. After washing the adherent cells with DPBS, they were detatched from the flask with 0.5% Trypsin and neutralized before being transferred to a centrifuge tube to be pelleted down at 150g for seven minutes.

 

HUVECs were mixed with treatment agents of the following groups and seeded onto an Ibidi 96 well µ-Plate at a concentration of ~15,000 cells per well.

 

  • Negative Control (DPBS)
  • Positive Control (Serum free HUVEC growth media or VEGF)
  • 0.2 μG exosomes (as measured by protein)/well (AC & Endometriosis)
  • 2.0 μG exosomes (as measured by protein)/well (AC & Endometriosis)
  • 5.0 μG exosomes (as measured by protein)/well (AC & Endometriosis)

 

These concentrations were determined following initial dose response experiments using a 1000-fold range, where it was determined that significant response was found in 2 μG exosomes (as measured by protein)/well and 5 μG exosomes (as measured by protein)/well. Past exosome concentrations tested ranged from 0.002 μG to 20 μG of protein, and protein concentrations were tested using venous blood exosomes to minimize use of volunteer menstrual effluent samples.

 

Schematic showing steps of angiogenesis assay

 

Each well was imaged at 2-hour and 4-hour timepoints, and the 4-hour timepoint images were used for quantification due to the higher amounts of differentiation and best representative activity. The 4-hour images were quantified using MetaMorph® software (Molecular Devices, Inc.) based on the type of structure formed.

 

Data analysis and statistics:

 

Criterion for identification of tubular structures in images:

  • Meshes (closed structures)
  • Segments (5+ cells which have differentiated)
  • Branches (3+ differentiated cells branching off a segment)

 

Structures were quantified by manually drawing regions of interest (ROIs) representing meshes, segments and branches. Region measurements data for types of structures and length was recorded for each ROI in the image and logged into Excel worksheet. Once images were quantified in MetaMorph®, each well was normalized to negative control (DPBS). The average for each group was calculated from data measured from 5 independent wells (biological replicates), with standard error. After this normalization, the following categories are calculated for each group:

 

  • Mesh number
  • Mesh Area
  • Mesh length
  • Segment number
  • Segment length
  • Average Segment length
  • Branch number
  • Branch length
  • Total segment length (segments + branches)
  • Total length (meshes + segments + branches)

 

The p value was obtained using two independent samples, unequal variance (Welch's) T-test. 

 

Angiogenesis assay standardization:

Positive Control: In initial experiments, positive control cells were treated with Vascular Endothelial Growth Factor (VEGF) as it is a potent angiogenic factor and its contribution to tumor angiogenesis is well defined.4

However, VEGF performance showed varying results across experiments, with low activity in all concentrations from 10 ng/mL up to 300 ng/mL. FGF, that is known to have angiogenic activity as well was also tested and showed less activity than VEGF in all groups. B-27, which is a serum-free supplement, was also tested. As an alternative, Serum-Free Media (SFM) was substituted in, and eventually determined as a better positive control due to consistent and high angiogenic activity from experiment to experiment.

 

Phase contrast image of a well treated with serum free growth media (SFM, right) showed consistent and robust angiogenesis compared to VEGF (left) 

 

Quantification across patient samples (n = 4) showed significant (p < 0.05) angiogenic increases in a dose dependent manner across the following groups:

 

  • Mesh quantity
  • Segment quantity
  • Segment length
  • Branch quantity
  • Branch length

 

Smaller mesh areas with high quantities of meshes denote more complexity and advancement of angiogenesis, as shown in the 4-hour Endometriosis treatment images.

 

High resolution poster data

 

Phase contrast image of a well treated with asymptomatic control exosomes

 

 

Zoom of phase contrast image of a well treated with asymptomatic control exosomes

 

Phase contrast image of a well treated with endometriosis exosomes

 

 

Zoom of phase contrast image of a well treated with endometriosis exosomes

 

Exosomes from ME of endometriosis patients lead to significant increase in mesh formation

Exosomes from ME of endometriosis patients lead to significant increase in branch formation

 
References

 

  1. Ellis, K., Munro, D., & Clarke, J. (2022). Endometriosis Is Undervalued: A Call to Action. Frontiers in global women's health, 3, 902371.
  2. World Health Organization: WHO & World Health Organization: WHO. (2023, March 24). Endometriosis.
  3. Kalluri, R., & LeBleu, V. S. (2020). The biology, function, and biomedical applications of exosomes. Science (New York, N.Y.), 367(6478), eaau6977. Darbà J, Marsà A. Economic Implications of Endometriosis: A Review. Pharmacoeconomics. 2022 Dec;40(12):1143-1158. doi: 10.1007/s40273-022-01211-0. Epub 2022 Nov 8. PMID: 36344867.
  4. Duffy AM, Bouchier-Hayes DJ, Harmey JH. Vascular Endothelial Growth Factor (VEGF) and Its Role in Non-Endothelial Cells: Autocrine Signalling by VEGF. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013. Available from: https://www.ncbi.nlm.nih.gov/books/NBK6482/

 

Acknowledgements

 

This project was performed at Xosomix under supervision of Dr. Pranav Sharma.

Special Thanks: Catherine Novick from Xosomix; Dr. Sanjay Agarwal, Dr. Omar Mesina, Ava Gordon, and Amy Lee from Center for Endometriosis Research (CERT) at UCSD Medical Center; Aayush Somani, & Dr. Alyssa Taylor Amos from UCSD Bioengineering.

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