3.2.2. 3D Cell Culture Using a Stirred-Tank Culture System

CRC spheroids (3D cells model) were generated as previously described [19] with some modifications. Briefly, HT29 single cells were inoculated into a 100 mL spinner flask (Corning, Tewksbury, MA, USA) in a culture medium with 10% (*v/v*) FBS, accounting for a cell density of 2.5 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL. The spinner vessel was placed on a magnetic stirrer under 40 rpm, and cell culture was carried out in a humidified atmosphere with 5% CO<sup>2</sup> at 37 ◦C, with an increasing stirring speed to 50 rpm and 60 rpm at the time-point of 8 h and 28 h post-inoculation, respectively. After the 4th day post-inoculation, half of the spinner flask volume was renewed daily. Experiments were performed using spheroids collected at day eight of culture with an average diameter of 500 µm.

### 3.2.3. Cytotoxicity Assay in Caco-2 Cells

The cytotoxicity of FJPP was assessed using confluent and undifferentiated Caco-2 cells as a model of the human intestinal epithelium, as previously described [29]. Briefly, Caco-2 cells were seeded into 96-well plates at a density of 2 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/well and allowed to grow for seven days, with medium renewal every 48 h. At day seven, cells were incubated with FJPP that was diluted in a culture medium at concentrations ranging from 125 µg to 10,000 µg of JPP-IN equivalents mL−<sup>1</sup> Cells incubated with culture medium and cells incubated with control fermentation medium (fecal suspension without JPP-IN) were used as controls. Another batch of control cells were incubated with Milli-Q water (FJPP vehicle) to account for culture medium dilution effects during FJPP addition. After 72 h of incubation, cells were washed with Phosphate Buffered Saline (PBS, Sigma-Aldrich, St. Louis, MO, USA) and cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay [4]. Cell viability was calculated relative to the respective feces control (no JPP-IN) and relative to medium control. Six independent experiments were performed in triplicate.

### 3.2.4. Antiproliferative Assay in HT29 Cell Monolayers

HT29 cells were seeded in 96-well plates at a density of 1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells per well. After incubation for 24 h, cells were treated with several concentrations (0-control, 125, 250, 500, 1000 and 2000 µg of JPP-IN equivalents mL−<sup>1</sup> ) of JPP previously fermented for 0, 2, 8, 24 and 48 h (FJPP). Cells incubated with culture medium and cells incubated with control fermentation medium (fecal suspension without JPP-IN) were used as controls. After 72 h of incubation, cells were washed with PBS and cell viability was assessed using the MTT assay, as described by [4]. Cell viability was calculated relative to the respective feces control (no JPP-IN) and relative to the medium control. Six independent experiments were performed in triplicate.

### 3.2.5. Antiproliferative Assay in HT29 Cell Spheroids (3D Cells)

HT 29 spheroids were seeded at a density of approximately five spheroids/well, in 96 well plates and incubated with PrestoBlue Viability Reagent (Molecular Probes, Invitrogen, CA, USA) to determine the basal viability. Then, spheroids were treated with FJPP at 10,000 µg of JPP-IN equivalents.mL−<sup>1</sup> (five times the highest concentration used in the 2D cells antiproliferative assay, but still non-toxic). After 72 h of incubation, the spheroids were washed with PBS and cell viability was assessed using the MTT assay, as described above. Cell viability was calculated relative to the respective feces control (no JPP-IN) as previously described [8]. Six independent experiments were performed in triplicate.

### *3.3. HPLC-Q-TOF-MS/MS Analysis of PC during Colonic Fermentation*

The extraction, identification and quantification of individual PC and metabolites from the FJPP of this assay were described in our previous study [12]. Briefly, anthocyanins were separated in a C-18 Core-Shell Kinetex column (2.6 µm particle size, 100 mm, 4.6 mm; Phenomenex, Torrance, CA, USA) at 38 ◦C using a gradient of 3% formic acid in water and 100% acetonitrile at a flow rate of 0.9 mL·min−<sup>1</sup> . Non-anthocyanin PC and metabolites were separated in a C-18 Hypersil Gold column (5 µm particle size, 150 mm, 4.6 mm; Thermo Fisher Scientific, Waltham, MA, USA) at 38 ◦C using a gradient of 5% methanol in acidified water (0.1%, *v/v*, of formic acid) and 0.1% acetonitrile at a flow rate of 1.0 mL·min−<sup>1</sup> . The identification of PC and metabolites was performed in a HPLC system connected to a diode array detector (DAD) and a Q-TOF mass spectrometer analyzer and electrospray ionization (ESI) source (micrOTOF-QIII, Bruker Daltonics, Bremen, Germany). Compounds were identified based on their elution order and the comparison of their UV to visible spectra and mass spectrometry fragmentation patterns with authentic standards and literature data. The quantification of PC and metabolites was conducted using DAD peak area data using the method previously validated [10]. Hydroxybenzoates were quantified at 280 nm as equivalents of gallic acid or protocatechuic acid, tannins were quantified at 280 nm as equivalents of gallic acid, anthocyanins were quantified at 520 nm as equivalents of cyanidin 3-glucoside, and flavonols and urolithins were quantified at 360 nm as equivalents of quercetin or myricetin. The limits of detection (LOD) and quantification (LOQ) for gallic acid, protocatechuic acid, cyanidin 3-glucoside, quercetin and myricetin were 0.012 and 0.037, 0.027 and 0.083, 0.020 and 0.068, 0.562 and 1.363, 0.166 and 0.503 mg·L −1 .

## *3.4. Statistical Analysis*

Antiproliferative activity data were expressed as mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Data were submitted to one-way analysis of variance (ANOVA) and the means were compared by Tukey's test at a 5% significance level. Additionally, antiproliferative activity data was also submitted to regression analysis. For antiproliferative assay in monolayer HT29 cells, the EC<sup>50</sup> values were calculated using the former software.

The bioactivity of PC metabolites formed during colonic fermentation of JPP was investigated using chemometric analyses. Principal Component Analysis and Cluster Analysis (CA) were used to investigate the association between HPLC-MS-fingerprinting assessment of PC (parent compounds and metabolites) and the antiproliferative activity during the colonic fermentation of JPP. Data were processed using the software SAS® OnDemand for Academics (SAS Institute Inc., Cary, NC, USA).

### **4. Conclusions**

This study demonstrated the antiproliferative effect of JPP fermented by colonic microbiota against CRC using a complex 3D cell model. The potential effects of JPP against CRC were increased in the intermediate times of fermentation, and were associated to HHDP-digalloylglucose isomer and dihydroxyphenyl-γ-valerolactone rather than to other colonic PC metabolites or to the PC found at highest concentrations in the undigested fruit. Studies regarding the antiproliferative effect of these isolated compounds in CRC 3D models should be carried out in the near future.

**Supplementary Materials:** The following are available online, Figure S1: Effect of FJPP (10,000 µg mL−<sup>1</sup> ) on viability of Caco-2 cells. Cell viability was evaluated after exposure of Caco-2 cells to FJPP at 10,000 µg mL−<sup>1</sup> for 72 h. Results are means of at least six independent experiments performed in triplicate ± SEM., Table S1: The loadings of the first three principal components.

**Author Contributions:** Conceptualization, P.R.A., T.E. and A.T.S.; formal analysis, P.R.A., A.Q., V.C.B., E.R., I.D.P., A.C.M., S.C.O.-A.; resources, T.E., A.T.S. and M.R.B.; data curation, P.R.A., A.T.S., T.E. and R.M.; writing—original draft preparation, P.R.A., T.E. and A.T.S.; funding acquisition, T.E., A.T.S. and M.R.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Brazilian National Council for Scientific and Technological Development (CNPq) [grant number 303654/2017-1 and postdoctoral scholarship 205295/2018-5] and Coordination for the Improvement of Higher Education Personnel (CAPES) [finance code 001]. This research was also funded by the Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior [Grant CEECIND/04801/2017] and iNOVA4Health–UIDB/04462/2020 and UIDP/04462/2020, a program financially supported by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior.

**Institutional Review Board Statement:** This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of Federal University of Santa Maria (protocol of study CAAE 50151015.6.0000.5346). Informed consent was obtained from all subjects involved in the study.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data supporting the findings of this study are available on request from the corresponding author. Supporting information is provided in the supplementary material.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

**Sample Availability:** Samples used in this study are not available from the authors.

### **References**

