*3.9. Cell Viability Assays and Determination of the OOPs' EC50 Values*

For the determination of the EC50 values, cell viability was assayed after 72 h treatment using the ViaLightTM Cell Proliferation and Cytotoxicity BioAssay Kit according to the supplier's protocol with slight modifications. Briefly, after the treatment with OOPs, the adapted medium was removed from the dish wells and the cells were washed twice with medium without FBS. Then, the C-lysis (LT27-076) was diluted in PBS (1:2) and a volume of 50 μL was added to each well. Cells were incubated with C-lysis for at least 10 min. Finally, an equal volume of lysed cells from each well and ATP monitoring reagent (AMR; LT27-212) was transferred to the wells of a white-walled illuminometer plate (Greiner bio-one; 655074). Cell viability was quantified by measuring luminescence using a multi-mode microplate reader Safire2 Tecan (software Magellan V6.00 STD.2PC WIN.20000/XP). EC50 values were calculated after 72 h treatment using the GraphPad Prism 6 software.

For the assessment of cell viability after treatment with OOPs the 3-(4, 5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay was also used. MTT solution was added in cells at a final concentration of 0.5 mg/mL and the cells were incubated for 3 h. The MTT solution was subsequently discarded, and a 100 μL volume of DMSO was added into each well to dissolve the generated formazan crystals. Each sample's optical density was measured at 570 nm on a microplate reader (Dynatech Laboratories MRX Microplate Reader, Chantilly, VA, USA). Results from two or three independent experiments performed in triplicate were presented either in tables or in bar graphs as mean cell count ± SE for each treatment group normalized to the control group (cells treated with 0.2% (*v*/*v*) DMSO).

### *3.10. Cell Proliferation Assay—Cell Preparation and Staining*

Human cancer cells were plated onto sterile glass coverslips (10-mm diameter, 5161063, ThermoFisher Scientific, Waltham, MA, USA) in 24-well tissue culture plates, at a density 5 times the density of cells seeded in 96-well plate and at a final medium volume of 500 μL. In order to obtain homogeneous plating, 250 μL of the medium was added directly to the wells already containing coverslips. Using a tip perpendicularly, coverslips were pressed in order to ensure their attachment to the bottom of the well. The cells were then seeded by adding 250 μL of single-cell suspension drop by drop following a cross path. Immediately after seeding, the plate was shaken back and forth at least ten times to achieve homogeneous plating. The cells were allowed to adhere overnight (~16 h) at 37 ◦C and 5% (*v*/*v*) CO2. The next day, a volume of 500 μL fresh medium was added to each well and the treatment with OOPs was initiated by adding directly to the wells 2 μL of OOPs from the 500× stock in DMSO and immediately mixed afterward by pipetting. The concentration of each OOP used was its EC50 value and the treatment lasted 24 h. Control cells were prepared under the same experimental conditions using DMSO instead of the OOPs' solutions in DMSO at the same final concentration (0.2% (*v*/*v*) DMSO). Only the compounds with EC50 ≤ 50 μM were tested for antiproliferative effect. Each condition was performed in duplicate. After treatment with the OOPs, live proliferating mammalian cells were labeled with 5-ethynyl-2'-deoxyuridine (EdU), a nucleoside analog of thymidine, using the Cell proliferation kit III (EdU-FM, PK-CA724-488FM, Promokine, Heidelberg, Germany). More specifically, a 20 μM EdU solution from a 10 mM stock in DMSO was prepared in a fresh culture medium. Culture supernatant was removed from treated cells to leave only 250 μL. An equal volume of 20 μM EdU solution was added and mixed with the medium to obtain a 10 μM EdU final concentration. The treated cells were incubated for the desired time of pulse length (2–4 h) under conditions optimal for each cell type depending on each cell line's doubling time. Following incubation, cells were washed

twice with PBS, then fixed with 4% (*w*/*v*) paraformaldehyde in PBS for 15 min at room temperature (RT), and subsequently washed twice with 3% (*w*/*v*) BSA for 5 min each time. Cells were permeabilized by incubation (20 min, RT, in the dark) with 0.5% (*v*/*v*) Triton-X 100 in PBS. Following permeabilization the cells were washed twice with 3% (*w*/*v*) BSA in PBS and incubated for 30 min with the reaction cocktail, according to the instructions of the Cell proliferation kit III (EdU-488; FM). After staining for replicating DNA, the cells were washed three times with 3% (*w*/*v*) BSA in PBS, and were then incubated (10 min, at RT) with Hoechst 33342 in PBS (1:10.000) to visualize all the nuclei. The treated and stained cells on coverslips were washed twice with PBS, mounted on glass coverslips with Mowiol at RT and stored protected from light at 4 ◦C until analyzed by confocal microscopy imaging. Before imaging, glass slides with mounted coverslips were allowed to warm up at RT for proper emission of fluorophores. Additionally, the coverslips were carefully cleaned with 70% (*v*/*v*) EtOH in order to eliminate remaining mounting medium that could damage the objective lenses upon contact.

#### *3.11. Image Acquisition by Confocal Microscopy and Digital Image Analysis*

Cells were imaged with "sequential z scan" and "tile scan" modes of an SP8 confocal microscope using a 20× objective and a 512 × 512 pixel resolution format. The solid-state laser lines 405 and 488 nm were used in order to image the fluorescence of Hoechst and Alexa 488 emission signals respectively. Fluorescence signals for each fluorophore were collected separately. The same laser intensity and detector acquisition parameters of gain and offset were used in the OOP-treated and untreated samples. The Gain [V] was adjusted so that the brightest areas fall just below the limit for signal saturation. Each field size imaged consisted of 45 (9 × 5) tiles "stitched" with "seams smoothed" using the "Merge images" application after completion of the image acquisition. The z-stack was acquired 'Between Stacks' with a z-step size of 1 μm.

For further quantitative analysis of the digital images, a series of data from z-stacks were processed as follows: Eleven out of forty-five acquired series (25%) were analyzed using the open source image analysis software Icy Version 2.4.2.0 [100]. A Maximum Z Projection was applied to all of them and the HK-Means segmentation plugin [101] was used to extract objects corresponding to nuclei labeled with Hoechst (total cell population) and to proliferating cells' nuclei labeled with EdU488. Segmentation was performed simultaneously for both channels, or separately depending on the set-up for the channels. The number of Hoechst- and EdU-labelled nuclei was used to calculate the % of proliferating cells as the % of EdU positive/total number of nuclei labelled with Hoechst (% EdU +ve). At least 180 cells (181–2500) from two or three independent experiments were observed for each experimental group in most cases.

#### *3.12. Annexin V/PI Staining and Analysis by Flow Cytometry*

Apoptosis and necrosis were assessed by double staining with annexin V-FITC and propidium iodide (PI) and were analyzed using flow cytometry (FACS). Human cancer cells were plated in 96-well culture plates in 100 μL of complete medium and were allowed to adhere overnight (~16 h). The next day, 150 μL of fresh complete medium was added to each well and the cells were treated with OOPs at their EC50 concentration for 48 h (compounds with EC50 ≤ 50 μM were tested). In detail, 0.5 μL of a 500× stock of each tested compound in DMSO was added directly into each well in a final volume of 250 μL culture medium and was gently mixed by pipetting. Control wells were prepared under the same experimental conditions by adding only DMSO at a final concentration of 0.2% (*v*/*v*). At the end of the treatment, the cells were detached by trypsinization, media with serum was added to deactivate trypsin, and the cells from three wells were pooled together by centrifugation (1000 rpm, 5 min, 24 ◦C), washed with cold PBS, centrifuged (1000 rpm, 5 min, 24 ◦C) and finally resuspended in cold 1× annexin V binding buffer (10 mM HEPES pH = 7.4, 150 mM NaCl, 2.5 mM CaCl2) at a density of 10<sup>4</sup> cells/mL. Staining was performed by incubating with annexin V- FITC and propidium iodide (PI; BioLegend) for 15 min at room temperature in the dark, according to the manufacturer's instructions. Negative control samples consisted of cells treated only with 0.2% (*v*/*v*) DMSO for the same incubation time length (i.e., 48 h). Cells treated with Triton X-100 (0.25% (*v*/*v*)) for 5 min at 4 ◦C were used as controls for 100% permeabilization of the plasma membrane and maximum fluorescence staining with PI (positive control for cells in necrosis). The presence of live, apoptotic or necrotic cells was assessed with the FACS Calibur (Becton– Dickinson, San Jose, CA, USA). In total, 10,000 cells were analyzed per measurement and the acquired data were analyzed using the FlowJo V.10.0.8 software (Tree Star Inc., Ashland, OR, USA). Each condition was analyzed in duplicate, and the results presented are from 2 or 3 independent experiments.

#### *3.13. Statistical Analysis*

All data were derived from multiple experiments conducted at least in triplicate. Statistical analysis was performed using the GraphPad Prism v8 (GraphPad Software Inc., San Diego, CA, USA) and Office Excel 365 (Microsoft, Redmond, WA, USA). For the cell-viability assays, the data obtained from cells treated with OOPs were normalized to the average luminescence of the control group treated with the vehicle compound (i.e., DMSO), which was considered 100% viability, and the EC50s were calculated using the GraphPad Prism algorithm.

Data showing OOPs' antiproliferative effect as the percentage of S-phase cells were derived from two or three independent experiments and were presented as mean values ± SE (Excel Formula applied for three experiments: SE = STDEV (A1, A2, A3)/SQRT(COUNT(A1, A2, A3)). Differences in proliferation levels between treated and untreated control cells were analyzed for significance using the unpaired two tailed Student's *t*-test, and *p* values were estimated using the GraphPad algorithm. Values were considered significant at a 0.05 level of confidence. The levels of antiproliferative effect shown as % inhibition of cell proliferation are presented as means ± SE after normalization, with the average proliferation levels of the control cells.

Data showing OOPs' apoptotic/necrotic effect were derived from two or three independent experiments and were presented as the means ± SE of the percentage (%) of both annexin V-positive cells (early apoptotic events) and % of annexin V/PI positive cells (late apoptotic events) over the whole cell population normalized to the corresponding events of the control cells, as determined using the FlowJo software.

#### **4. Conclusions**

The present work is the first systematic comparative ex vivo study evaluating the anti-cancer potential of extra virgin olive oil phenols. It was performed with secoiridoid phenols isolated in pure form (i.e., oleocanthal (**1**), oleacein (**2**), oleuropein aglycone (**3a,b**), ligstroside aglycone (**4a,b**), oleomissional (**6a,b,c**) and oleocanthalic acid (**7**)) using new methods for large-scale selective extraction from different olive plant parts. EC50 values of these OOPs' bioactivity on multiple cancer and non-cancer cell lines from different tissue origins were calculated. For this, the same experimental protocols were followed enabling thereby valid comparisons between the effects of all tested OOPs in either the same cell line or amongst different cell lines. The variability in the activity of the analyzed OOPs in different cell lines and different cancer types was clearly highlighted. The antiproliferative and pro-apoptotic bioactivity was confirmed for OOPs studied before, i.e., oleocanthal (**1**), [28–33] oleuropein aglycone (**3a,b**) [17,27,53], ligstroside aglycone (**4a,b**) [53] and oleacein (**2**) [19], and for the first time for oleomissional (**6a,b,c**). Important information was generated, encouraging further in vivo investigations of the OOPs presenting strong bioactivity in several cancer cell lines. Moreover, the important antiproliferative cytostatic effect of oleuropein and ligstroside aglycones ((**3a,b**) and (**4a,b**)) in the H1437 lung cancer and Caco-2 colon carcinoma cells, stronger than that of oleocanthal (**1**), which has been the most effective and well-studied OOP until now, highlights the selectivity in the action of the different OOPs on different cancer types.

To conclude, this study, besides the new methodologies for the isolation of olive oil secoiridoids, provides important information about the methodology of handling them for in vitro analysis of their bioactivity in cell culture models. The major messages from already performed studies converge to a conclusion that the analyzed secoiridoid phenols hold significant potential for further analysis in in vivo studies evaluating their anti-tumor properties [7]. However, a large variability exists in results already generated by others with respect to the EC50 values of each phenol as well as its activity in different cancer cell models. This is perhaps due to the fact that each compound was tested in one or very few cell lines, or in cell lines of only one cancer type. Moreover, the bioactivity of new phenols recently isolated (i.e., oleomissional (**6a,b,c**) and oleocanthalic acid (**7**)) had not been studied until now [34,40]. The present study aspires to fill gaps in knowledge such as the above-mentioned, and to become a reference report for the EC50 values of oleocanthal (**1**), oleacein (**2**), oleuropein aglycone (**3a,b**), ligstroside aglycone (**4a,b**), oleomissional (**6a,b,c**) and oleocanthalic acid (**7**) in the large array of cell lines analyzed herein, forming thereby a base for further in vivo studies in animal cancer models investigating their potential anti-tumoral effects.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms24010003/s1. Refs [99,102] are cited in supplementary files.

**Author Contributions:** Conceived and designed the experiments: H.B., P.M., E.M., A.P., P.K. and N.V.; performed the experiments: A.P., P.K., A.R., P.D., E.F. and E.C.; analyzed the data: A.P., P.K., A.R., P.M. and H.B.; contributed reagents/materials/analysis tools: H.B., P.M. and E.M.; wrote the paper: A.P., P.K., P.M. and H.B.; edited the paper: E.C. and N.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was co-financed by Greece and the European Union (European Social Fund-ESF) through the Operational Program «Human Resources Development, Education and Lifelong Learning 2014-2020» in the context of the project "In vitro study of anticancer properties of olive oil polyphenols" (MIS 5048983).

**Acknowledgments:** The authors acknowledge the help of the World Olive Center for Health (https: //worldolivecenter.com) and Leventis Foundation for the scholarship provided to A.P. and A.R. The authors also thank Evangelia Xingi at the Light Microscopy facility of the Hellenic Pasteur Institute for her assistance with the use of the SP8 confocal microscope, as well as all the scientists listed in the Experimental Section who donated cell lines used in this study, and Panagiotis Georgiadis at the National Research Foundation in Greece for the permission to use the multi-mode microplate reader Safire2/Tecan in his laboratory.

**Conflicts of Interest:** The authors P.M., E.M., A.R. and P.D. are inventors of the patents: "Method For Obtaining Oleocanthal Type Secoiridoids And For Producing Respective Pharmaceutical Preparations" WO/2020/165614 and "Method for obtaining of oleacein and oleomissional secoiridoids and method of producing pharmaceutical preparations thereof" WO/2020/165613. The remaining authors declare no conflicts of interest.
