Thesis Defense

*Why did you use a commercial olive leaf extract rather than purified oleuropein?

  • Standardized to 20% oleuropein — consistent dosing across experiments

  • More translationally relevant — reflects what would realistically be used clinically

  • Limitation: extract contains other polyphenols so effects cannot be attributed exclusively to oleuropein

*Why did you use glycerol as a vehicle control?

  • Glycerol is the excipient in the olive leaf extract formulation

  • Prepared at 0.3% v/v to match glycerol concentration in the highest OLE dilution

  • Isolates treatment effect from solvent effect

*What are the limitations of using an immortalized cell line versus primary patient-derived cells?

  • May not reflect patient heterogeneity across individuals, lesion sites, and disease stages

  • Immortalization alters baseline signaling, stress responses, and treatment sensitivity

  • Findings are preclinical — validation in primary cells is the most important next step

*Why does scratch assay closure not exclusively reflect migration?

  • Closure reflects both migration and proliferation

  • Without mitomycin C you cannot separate these two contributions

  • Results are therefore described as impaired wound closure not exclusively impaired migration

*What would mitomycin C have told you?

  • DNA crosslinking agent that blocks cell division without impairing motility

  • Would have allowed attribution of wound closure changes to migration rather than proliferation

  • Standard control in scratch assay studies — priority for future work

*Why these timepoints (24h and 48h)?

  • Standard in scratch assays

  • Capture both early and sustained effects on wound closure

  • Allow observation of initial migration and longer-term dynamics

*Why these concentrations/dilution ranges?

  • Selected to span a physiologically relevant and literature-supported concentration window

  • Designed to avoid overt cytotoxicity

  • Possible the active concentration range was not fully captured — may explain lack of dose-dependence

*What are the black dots on the assay image on the discussion slide?

  • Sharpie marks added to the bottom of the plate

  • Ensured imaging of the exact same wound region at each time point

  • Maintained consistency in scratch assay imaging across timepoints

*Why was OLE+CAL less effective than OLE alone at 48 hours?

  • Calcitriol may partially attenuate oleuropein's anti-migratory effects over time through competing pathway effects

  • Calcitriol alters cellular metabolism which could allow partial recovery of migration by 48h

  • Interaction is not simply additive — warrants formal synergy analysis

*Could calcitriol be interfering with oleuropein's mechanism?

  • Cannot be ruled out

  • Both converge on NF-kB through distinct upstream mechanisms

  • VDR-mediated transcriptional changes from calcitriol may alter cellular context and modify response to oleuropein

  • Bliss independence or Loewe additivity analysis in future work would clarify this

*What is the single most important takeaway from your study?

  • Endometriotic cell behavior is more meaningfully altered at the level of migration than cytotoxicity

  • Targeting inflammatory pathways to impair migration may be more relevant than inducing cytotoxicity for endometriosis

  • Oleuropein shows particular promise as a functional modulator of this process

*What is the difference between stromal and epithelial cells, and why does it matter?

  • Epithelial cells form structured layers involved in barrier function and glandular activity

  • Stromal cells are connective tissue that provide structural support, secrete extracellular matrix, and regulate the local microenvironment

  • In endometriosis: epithelial cells drive lesion formation and migration, stromal cells drive inflammation, fibrosis, and hormone responsiveness

  • This study used 12Z epithelial cells — findings reflect epithelial behavior only, not stromal-driven processes

*How is the cell line immortalized?

  • SV40 large T antigen inactivates key tumor suppressor proteins

  • Allows cells to bypass normal cell cycle regulation and divide indefinitely

  • Limitation: behavior may differ from primary endometriotic cells

*What is the chemistry of OLE and how does it act?

  • Polyphenol with aromatic rings and hydroxyl groups

  • Hydroxyl groups donate a hydrogen atom to reactive oxygen species

  • Stabilizes free radicals by converting them into less reactive molecules

  • ROS plus phenolic hydroxyl group produces reduced ROS and stabilized phenoxyl radical

  • Aromatic ring stabilizes the remaining radical via resonance — does not become damaging itself

ADDITIONAL QUESTIONS

What is the difference between Vitamin D3 and calcitriol, and why does that matter for your study?

  • Vitamin D3 is the inactive prohormone synthesized in the skin following UV exposure

  • Requires magnesium-dependent enzymatic activity throughout the conversion process

  • Undergoes two hydroxylation steps: first in the liver to form 25-hydroxyvitamin D, then in the kidneys to form calcitriol

  • Calcitriol is 1,25-dihydroxyvitamin D3 — the biologically active form that binds the vitamin D receptor

  • Used calcitriol directly to assess the active form on endometriotic cells without relying on metabolic conversion

How do you know the concentration of oleuropein in your extract is accurate?

  • Relied on manufacturer's standardization — 20% oleuropein content

  • Did not independently verify through HPLC or other analytical method — acknowledged limitation

  • Future studies should use purified oleuropein for more precise dosing

Why did you use DMSO as the solvent for calcitriol?

  • Calcitriol is fat-soluble with very low water solubility — requires organic solvent

  • DMSO is standard for calcitriol in cell culture studies

  • Final concentration kept below cytotoxic levels, typically under 0.1%

Why did you use 12Z cells specifically?

  • Immortalized human endometriotic epithelial cell line derived from peritoneal endometriotic tissue

  • Maintains stable endometriotic epithelial phenotype

  • Reproducible across experiments and well characterized in literature

  • Provided by Dr. Fazleabas who was central to their original development

Why are PBMCs not an adequate comparator for epithelial selectivity?

  • PBMCs are immune cells — differ substantially in metabolism, adhesion biology, migratory programming, and baseline signaling

  • Absence of cytotoxicity in PBMCs indicates compounds do not broadly kill immune cells — useful for safety but not selectivity

  • A healthy endometrial epithelial cell line would be a more appropriate comparator — acknowledged as a limitation

Why can't you conclude that viability changes equal changes in cell number?

  • Assay measures metabolic activity not cell number

  • A metabolically more or less active cell produces a different signal even if cell number is unchanged

  • OLE+CAL values exceeding 100% likely reflect calcitriol-induced mitochondrial metabolism changes not increased proliferation

  • Complementary assay such as cell count, proliferation assay, or flow cytometry would be needed to confirm

Why didn't you see dose-dependence?

  • Concentrations tested may all be below the threshold for graded cytotoxic effects in this cell type

  • Inhibitory effect may be a threshold or binary response rather than linear within this range

  • Dilution series may not have captured the active concentration window

  • Important limitation for translational interpretation

What does the treatment by time interaction actually mean statistically?

  • Effect of treatment on wound area was not consistent across time points

  • Difference between OLE-treated and control wells changed between 24 and 48 hours

  • Shows OLE altered the dynamics of closure over time, not just shifted it uniformly

  • Captures the time-dependent divergence between treatment groups — why OLE sustained inhibition while OLE+CAL attenuated

Why did OLE+CAL show viability above 100%?

  • Calcitriol modulates mitochondrial function and cellular metabolism — increases resazurin reduction independent of cell number

  • Combination may have altered cells' redox state enhancing metabolic activity without increasing proliferation

  • Interaction between calcitriol and oleuropein may partially counteract oleuropein's modest cytostatic effects

  • Cannot definitively distinguish between these without complementary assays — interpreted as altered metabolism not increased cell number

You say OLE inhibits NF-kB — did you measure NF-kB in this study?

  • No — NF-kB was not directly measured

  • Mechanistic interpretation is grounded in existing literature from inflammatory models and cancer cell lines

  • Cannot confirm a causal pathway from this data alone

  • Mechanistic claims are explicitly described as inferential

How can you claim a mechanistic link if you didn't measure any signaling pathways?

  • No confirmed mechanistic link is claimed — a plausible interpretation based on convergent published evidence is proposed

  • Study establishes functional outcomes: migration impaired, cytotoxicity limited

  • Mechanistic explanation informed by prior work on these compounds

  • Direct mechanistic validation is identified as the most important future direction

What is the difference between migration and invasion?

  • Migration: cell movement across a two-dimensional surface — what the scratch assay captures

  • Invasion: movement through a three-dimensional extracellular matrix requiring matrix metalloproteinase activity

  • In endometriosis invasion is critical for lesion establishment — ectopic cells must penetrate the peritoneum

  • A compound can inhibit migration without inhibiting invasion

  • Study addresses migration only — invasion would need to be tested separately with transwell or Matrigel system

Why did you not test a calcitriol-only arm?

  • Study was designed to evaluate oleuropein as the primary compound and the combination as secondary

  • Calcitriol-only arm would have allowed direct comparison of its independent effects on 12Z cells

  • Would have strengthened interpretation of the combination results

  • Identified as a priority for future work

How does your study advance the field beyond what Park et al. 2022 already showed?

  • Park et al. used a murine in vivo model — this is the first study in human endometriotic epithelial cells

  • First to test oleuropein in combination with calcitriol

  • First to use quantitative statistical modeling for treatment, time, and concentration effects simultaneously in this cell type

  • Demonstrates migration rather than cytotoxicity is the more sensitive functional endpoint

How does your in vitro dilution series relate to what would actually reach endometriotic tissue in a patient?

  • Cannot be fully answered from this data — designed to test a range not mirror physiologic exposure

  • Oleuropein is rapidly metabolized following ingestion producing hydroxytyrosol and other phenolic metabolites

  • Tissue concentrations in peritoneal cavity depend on route of administration, absorption, distribution, and metabolic conversion

  • Bridging in vitro concentrations to in vivo exposure requires pharmacokinetic modeling — identified as future direction

Oleuropein is metabolized rapidly in vivo — how does that affect the relevance of your findings?

  • Real translational limitation

  • First-pass hepatic metabolism primarily produces hydroxytyrosol and other phenolic metabolites

  • Metabolites retain antioxidant and anti-inflammatory activity but whether they replicate parent compound effects is not confirmed

  • Findings are most directly relevant to the in vitro context — preclinical functional evidence not a direct prediction of in vivo efficacy

Would your results be different with primary cells?

  • Possibly yes — primary cells reflect actual biological heterogeneity of the disease

  • Vary in receptor expression, signaling pathway activation, hormone responsiveness, and migratory behavior

  • 12Z cells have stable uniform phenotype that may not capture this variability

  • Validating in primary cells is the most important next step before translational conclusions