Aronia Berry Extract Inhibits Cancer Stemness and Overcomes 5-Fluorouracil Resistance by Targeting TLR3/NF-κB Signaling in Colorectal Cancer

TL;DR

Aronia berry extract helps overcome chemotherapy resistance in colorectal cancer by targeting specific signaling pathways.

Contribution

Aronia berry extract is shown to target TLR3/NF-κB signaling to reverse 5-FU resistance in colorectal cancer.

Findings

ABE combined with 5-FU significantly reduced the effective concentration needed to inhibit resistant CRC cells.

ABE reduced cancer stemness markers like CD44, Nanog, and Oct4 in resistant CRC cells.

ABE treatment decreased NF-κB expression and organoid growth in patient-derived models.

Abstract

Background: Colorectal cancer (CRC) remains a major clinical challenge, in part due to the limited efficacy of 5-fluorouracil (5-FU)-based chemotherapy, which is often compromised by the emergence of acquired resistance. Aronia berry extract (ABE), a phenolic-rich natural compound, has gained increasing attention for its anticancer and chemosensitizing properties. This study aimed to investigate whether ABE can overcome 5-FU resistance (5-FU-R) in CRC and to elucidate the molecular mechanisms underlying its therapeutic effects. Methods: We conducted a series of in vitro experiments using 5-FU-R CRC cell lines to evaluate the synergistic effects of combined ABE and 5-FU treatment. Genome-wide transcriptomic profiling was performed to identify key regulatory pathways associated with chemoresistance and to determine potential ABE-responsive targets. Findings were further validated using patient-derived 3D organoids (PDOs). Results: Co-treatment with ABE and 5-FU significantly reduced the effective concentration of 5-FU required to inhibit 5-FU-R CRC cells, yielding a Bliss synergy score greater than 10. The combination markedly suppressed cell viability, clonogenic potential, migration, and invasion. ABE also reduced cancer stemness, as evidenced by reduced CD44, Nanog, and Oct4 expression. Functional inhibition of Toll-like receptor 3 (TLR3) impaired spheroid growth, and PDO experiments corroborated these findings, demonstrating reduced organoid growth, diminished survival, and decreased NF-κB expression following ABE treatment. Conclusions: Our findings reveal that ABE effectively overcomes 5-FU resistance in CRC by targeting the TLR3/NF-κB signaling axis. This study highlights ABE as a safe, accessible, and promising adjunctive strategy to enhance therapeutic responses in 5-FU-resistant CRC.

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1. Introduction

Colorectal cancer (CRC) remains one of the most commonly diagnosed malignancies and a leading cause of cancer-related mortality worldwide, with an estimated more than a million new cases and approximately 903,859 deaths annually [1]. Despite improvements in early detection and therapeutic interventions, CRC continues to pose a significant clinical burden, particularly in advanced stages. Current standard-of-care treatment strategies involve surgery followed by adjuvant chemotherapy for high-risk stage II and stage III patients, as well as systemic therapy for those with stage IV disease, aimed at eradicating minimal residual disease and improving overall survival [2,3,4].

Among available chemotherapeutic agents, 5-fluorouracil (5-FU) remains the standard of care for CRC treatment and serves as the backbone of most combination regimens. However, its long-term efficacy is severely limited by the development of intrinsic or acquired chemoresistance. Alarmingly, more than 90% of patients with metastatic CRC ultimately fail to respond to 5-FU-based therapies, resulting in disease progression and poor clinical outcomes [5]. Resistance to 5-FU is a multifactorial process involving alterations in drug metabolism, dysregulation of apoptotic signaling, enhanced DNA repair capacity, and enrichment of therapy-resistant tumor cell subpopulations. These challenges underscore the urgent need to identify novel, mechanism-based approaches to overcome chemoresistance and restore therapeutic sensitivity in CRC.

Natural products and dietary phytochemicals have emerged as promising candidates for cancer prevention and therapy due to their pleiotropic biological activities and favorable safety profiles [6,7,8]. Aronia berry (Aronia melanocarpa), a polyphenol-rich fruit native to North America, has gained increasing attention for its potent antioxidant, anti-inflammatory, and anticancer properties. Aronia berries contain exceptionally high concentrations of phenolic compounds, including anthocyanins, flavonoids, and phenolic acids, which collectively contribute to their bioactivity. Among these, cyanidin-3-O-galactoside, quercetin, and chlorogenic acid have been identified as key bioactive constituents with demonstrated antitumor effects [9,10,11].

Cyanidin-3-O-galactoside, the predominant anthocyanin in aronia berry, exhibits strong antiproliferative, antioxidant, and anti-inflammatory activities and has been shown to suppress CRC cell growth in multiple preclinical models [12,13,14,15,16]. Quercetin, another abundant flavonoid, exerts broad anticancer effects by modulating apoptosis, oxidative stress, cell cycle progression, and oncogenic signaling pathways, including the PI3K/AKT and MAPK cascades [17,18]. Chlorogenic acid further contributes to aronia berries’ anticancer potential by inhibiting tumor cell proliferation, migration, and invasion through the regulation of key signaling mediators, including p53, p38 MAPK, JNK, c-Myc, and reactive oxygen species (ROS) [16]. Collectively, these findings suggest that aronia-derived polyphenols act on multiple molecular targets relevant to tumor growth and survival.

Importantly, accumulating evidence indicates that aronia berry constituents may also impact cancer stem cells (CSCs), a subpopulation of tumor cells characterized by self-renewal capacity, tumor-initiating potential, and intrinsic resistance to chemotherapy. CSCs are increasingly recognized as key drivers of CRC recurrence, metastasis, and treatment failure. Previous studies have demonstrated that aronia juice selectively inhibits P19 mouse embryonal carcinoma stem cells by upregulating the tumor suppressors p53 and p73, highlighting a potential role for aronia-derived compounds in CSC-targeted therapy [19]. Moreover, aronia polyphenols have been shown to enhance the cytotoxic efficacy of gemcitabine in AsPC-1 pancreatic cancer cells, further supporting their ability to function as chemosensitizing agents that potentiate conventional anticancer therapies [20].

Despite substantial evidence supporting the antitumor and chemosensitizing properties of aronia berry components, their role in modulating 5-FU resistance (5-FU-R) in CRC has not yet been investigated. Addressing this gap is particularly important given the central role of 5-FU in CRC management and the pressing need for strategies capable of reversing resistance. In the present study, we conducted a comprehensive evaluation of the effects of aronia berry extract (ABE) on 5-FU-R CRC. Specifically, we sought to determine whether ABE can restore sensitivity to 5-FU and to elucidate the underlying molecular mechanisms. By integrating in vitro functional assays, genome-wide transcriptomic profiling, and validation using patient-derived three-dimensional organoid models, this study aims to define the therapeutic potential of ABE and to provide mechanistic insights that may inform the development of novel adjuvant strategies to overcome chemoresistance in CRC.

2. Results

2.1. The Treatment of ABE and 5-FU Shows Synergistic Inhibition in 5-FU-R CRC Cells

We initially utilized the two 5-FU-R cell lines as described in our prior study [21]. Notably, the half maximal inhibitory concentration (IC50) of 5-FU was substantially higher in 5-FU-R HCT116 and 5-FU-R SW480 cells compared to their respective parental cell lines (Figure S1). Specifically, IC_50_ values in parental HCT116 and SW480 cells were 15.38 μM and 11.96 μM, respectively, whereas both 5-FU-R cell lines exhibited IC_50_ values exceeding 160 μM. We next evaluated whether ABE could restore 5-FU sensitivity in 5-FU-R CRC cells. Cells were treated with increasing concentrations of ABE (0–120 μg/mL) and 5-FU (0–60 μM), either alone or in combination. At identical 5-FU concentrations, the addition of ABE consistently enhanced growth inhibition compared with 5-FU monotherapy, indicating increased chemosensitivity. Combination treatment resulted in a synergistic reduction in cell viability. Bliss synergy analysis identified an optimal ABE:5-FU ratio of 60:40, yielding synergy scores > 10 in both cell lines. Notably, the presence of ABE markedly reduced the effective 5-FU concentration required to achieve comparable inhibitory effects. At this ratio, inhibitory rates reached 45.37% in 5-FU-R HCT116 cells (Figure 1A–C) and 49.28% in 5-FU-R SW480 cells (Figure 1D–F). Therefore, subsequent experiments were performed using 60 μg/mL ABE and 40 μM 5-FU.

2.2. The Combination of ABE and 5-FU Inhibits Cell Proliferation, Migration, and Invasion in 5-FU-R CRC Cell Lines

To assess the potential of ABE to enhance chemosensitivity to 5-FU in 5-FU-R CRC cells, we conducted a series of functional assays, including colony formation, wound-healing, and invasion assays. To evaluate the impact of the combination of ABE and 5-FU on CRC cell proliferation, we treated 5-FU-R HCT116 and 5-FU-R SW480 cells with 5-FU (40 µM) and ABE (60 µg/mL), either separately or in combination, for 48 h. We performed a colony formation assay, which demonstrated a significant reduction in clonogenicity with the combination of 5-FU and ABE compared to individual treatments in 5-FU-R HCT116 cells (Combination vs. 5-FU: Fold change = 2.26, p < 0.01; Combination vs. ABE: Fold change = 1.64, p < 0.01) and 5-FU-R SW480 cells (Combination vs. 5-FU: Fold change = 2.24, p < 0.01; Combination vs. ABE: Fold change = 1.63, p < 0.01, Figure 2A). To determine whether the combination of 5-FU and ABE had a greater impact on the motility and invasive potential of CRC cells, we conducted wound-healing and transwell assays. The wound-healing assay revealed that the combination of 5-FU and ABE significantly inhibited cell migration to a greater extent than individual treatments in both 5-FU-R HCT116 (Combination vs. 5-FU: Fold change = 2.74, p < 0.01; Combination vs. ABE: Fold change = 2.13, p < 0.01) and 5-FU-R SW480 cells (Combination vs. 5-FU: Fold change = 2.00, p < 0.01; Combination vs. ABE: Fold change = 1.83, p < 0.01, Figure 2B). The trans well assay showed that a combination of 5-FU and ABE shows a significantly more significant reduction in invasion than individual treatment in both 5-FU-R HCT116 (Combination vs. 5-FU: Fold change = 8.63, p < 0.01; Combination vs. ABE: Fold change = 3.00, p < 0.01) and 5-FU-R SW480 cells (Combination vs. 5-FU: Fold change = 1.80, p < 0.01; Combination vs. ABE: Fold change = 1.49, p < 0.01, Figure 2C). Consistent with the previous results, our findings demonstrated that the combination of 5-FU and ABE significantly reduced the proliferation, migration, and invasion abilities of CRC cells.

2.3. ABE, in Combination with 5-FU, Prevents Cell Stemness

CSCs within the tumor bulk possess the unique capacity to self-renew, differentiate, and initiate tumor formation. Therefore, CSCs drive tumor recurrence, metastasis, and drug resistance by generating new tumor cells [22,23]. Spheroids have emerged as a helpful tool that more closely simulates the in vivo organization of solid tumors than the standard in vitro monolayer culture approach, and provides a better in vitro platform to study the enhanced properties of tumor cells [24]. Using a sphere formation assay, we evaluated the effects of combined 5-FU and ABE treatment on spheroid morphology and viability in CRC cells. Compared with untreated cells or cells treated with either agent alone, co-treatment with 5-FU and ABE markedly disrupted spheroid formation and significantly reduced overall spheroid size in both 5-FU-R HCT116 and 5-FU-R SW480 cells (Figure 3A,B, p < 0.05). To determine whether alterations in CSC-associated gene expression accompanied these phenotypic changes, we next examined key stemness markers. CD44 is a well-established surface marker of CRC CSCs, whereas Oct4 and Nanog are core transcription factors essential for maintaining pluripotency and self-renewal [25,26]. Quantitative gene expression analysis revealed that combined treatment significantly reduced transcript levels of CD44, OCT4, and NANOG compared with untreated controls or single-agent treatments (Figure 3C). Consistent with these findings, Western blot analysis of spheroid lysates showed marked downregulation of CD44, Oct4, and Nanog in the combination group compared with the untreated and 5-FU-treated groups (Figure 3D).

Collectively, these results indicate that ABE enhances the anti-tumor efficacy of 5-FU by suppressing CSC-associated stemness properties at both the transcriptional and protein levels in 5-FU-resistant CRC cells. Collectively, these results indicate that ABE potentiates the anti-tumor effects of 5-FU by suppressing CSC-associated stemness programs at both transcriptional and protein levels, providing mechanistic support for the observed chemosensitization in 5-FU-resistant CRC cells.

2.4. The Toll-like Receptor 3 (TLR3) Expression Is Positively Correlated with 5-FU Resistance in CRC Cells

To investigate the mechanism associated with 5-FU-R in CRC cells, we performed a genome-wide transcriptomic analysis using RNA-seq in parental and 5-FU-R HCT116 and SW480 cell lines (GSE196900). In this analysis, we identified 657 upregulated genes in the 5-FU-R HCT116 cell line vs. the HCT116 cell line and 3060 upregulated genes in the 5-FU-R SW480 cell line vs. the SW480 cell line. 117 genes were upregulated in both 5-FU-R HCT116 and 5-FU-R SW480 cells compared with their parental cells. Furthermore, we performed a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially up-regulated genes in 5-FU-R versus parental CRC cells (Figure 4A). We selected the top 7 significant signaling pathways based on their p-value. It was observed that several important cancer-associated signaling pathways, including the TLR3 signaling pathway and cytokine pathway, were significantly suppressed in 5-FU-R CRC cells compared with parental cells (Figure 4B). In our study, we observed that the TLR3 signaling pathway is enriched and activated in 5-FU-R cells; the relative mRNA expression of TLR3 in 5-FU-R HCT116 (fold change = 1.46, p < 0.01, Figure 4C) and 5-FU-R SW480 cells (fold change = 3.24, p < 0.01, Figure 4D) was significantly higher compared with parental CRC cells.

Activation of TLR3 signaling in 5-FU–R CRC cells by poly (I:C) stimulation resulted in increased spheroid formation, as evidenced by enlarged adherent spherical clusters (Figure 4E). Furthermore, pharmacological inhibition of TLR3 significantly enhanced sensitivity to 5-FU in both 5-FU–R HCT116 and SW480 cells, as demonstrated by reduced cell viability compared with control-treated cells (p < 0.01; Figure 4F,G). Collectively, these results indicate that TLR3 activation is associated with enhanced cancer stem-like phenotypes and contributes to chemoresistance in 5-FU-resistant CRC cells.

2.5. Combined Treatment with 5-FU and ABE Down-Regulates Cell Stemness Through the TLR3/NF-κB Axis

As mentioned above, the TLR3 activation may promote the capacity of 5-FU-R CRC cells to form spheroids. The cellular responses to TLR3 activation are widely acknowledged to be primarily regulated by the NF-κB signaling pathway [27]. Given that NF-κB activation drives pro-survival and stemness-associated pathways, we hypothesized that NF-κB signaling plays a pivotal role in TLR3-mediated CSC phenotypes. Consistent with this concept, Western blot analyses demonstrated that combination treatment with 5-FU and ABE resulted in the most pronounced reduction in both TLR3 and NF-κB expression compared with untreated cells or cells treated with either agent alone (Figure 5A). Although 5-FU monotherapy partially reduced TLR3 levels, combination treatment achieved markedly greater suppression. Because nuclear translocation of NF-κB is a critical step for transcriptional activation, we further examined NF-κB localization in cytoplasmic and nuclear fractions. Notably, combined treatment significantly decreased NF-κB levels in both compartments in both CRC cell lines (Figure 5B), indicating effective inhibition of NF-κB nuclear translocation and signaling activity.

Together, these findings suggest that ABE enhances 5-FU efficacy by suppressing CSC-associated stemness via inhibition of the TLR3/NF-κB signaling axis.

2.6. The Combination of 5-FU and ABE Suppressed the Growth of PDOs

Three-dimensional organoid culture systems have become indispensable tools for modeling different facets of cancer biology, especially for investigating drug responses and evaluating treatment effectiveness [28,29,30]. In this study, patient-derived organoid (PDO) models from two CRC patients were used to validate the anti-tumor effects of combined ABE and 5-FU treatment. The combination significantly reduced organoid size and viability compared with single-agent treatments (Figure 6A). In addition, TLR3 mRNA expression was significantly suppressed in PDOs following combination treatment (p < 0.01, Figure 6B). Immunofluorescence analysis further demonstrated reduced NF-κB staining in the combination-treated organoids (Figure 6C), corroborating the mechanistic findings observed in CRC cell lines.

3. Discussion

CRC is the third most common cancer and a significant cause of morbidity and mortality worldwide [31]. Despite advances in early detection and therapeutic strategies, CRC remains a major cause of cancer-related mortality, largely due to disease recurrence and the emergence of chemoresistance. Among currently available chemotherapeutic agents, 5-FU continues to serve as the backbone of CRC treatment; however, intrinsic and acquired resistance severely limit its long-term efficacy [32,33,34]. Multiple signaling pathways, including PI3K/AKT, FOXO1, FZD6, and NF-κB, have been implicated in the molecular mechanisms underlying chemoresistance [35,36,37,38,39,40,41]. Accumulating evidence indicates that tumor spheroids and cancer stem-like cells play central roles in therapeutic resistance, tumor heterogeneity, and disease relapse [42,43]. These high-risk cell populations exhibit enhanced self-renewal capacity and pronounced resistance to cytotoxic agents, highlighting the importance of targeting stemness-associated pathways. Accordingly, targeting these resistant stem-like tumor cells may represent a promising strategy to improve treatment efficacy in high-risk CRC patients, including those with metastatic disease.

Natural products have shown promise in cancer therapy by minimizing toxicity to healthy tissues and overcoming drug resistance [44,45,46]. In this study, we investigated whether ABE could enhance 5-FU sensitivity in 5-FU–resistant CRC cells. Combined treatment with ABE significantly reduced the effective concentration of 5-FU, yielding a Bliss synergy score exceeding 10. Moreover, the combination of ABE and 5-FU effectively suppressed cell viability, clonogenic capacity, migration, and invasion in CRC cells. This dual treatment also reduced stemness-associated markers, including CD44, Nanog, and Oct4, indicating attenuation of cancer stem-like properties. The results from the PDO model support the dual-drug efficacy. 5-FU and ABE combination treatment of PDOs resulted in a significant reduction in organoid growth and the number of alive organoids. The decreased survival rate is associated with reduced NF-kB levels.

To elucidate the molecular mechanisms underlying ABE-mediated chemosensitization, we analyzed RNA-seq profiles from parental and 5-FU–R CRC cells. Pathway enrichment analysis revealed significant activation of Toll-like receptor signaling, with marked upregulation of TLR3 in resistant cells. TLR3 has been reported to activate NF-κB through both MYD88-dependent and MYD88-independent pathways [47,48]. Consistent with this concept, our Western blot analyses demonstrated that ABE, particularly in combination with 5-FU, markedly reduced TLR3 and NF-κB expression. Importantly, subcellular fractionation revealed decreased NF-κB levels in both the cytoplasm and the nucleus, indicating effective suppression of NF-κB nuclear translocation and transcriptional activity. These findings support a functional link between TLR3 inhibition and attenuation of downstream NF-κB signaling. KEGG pathway enrichment analysis additionally suggested involvement of the JAK/STAT signaling cascade. STAT3 is known to stabilize nuclear NF-κB activity [48,49] and regulate cancer malignancy [50,51]. Although phosphorylated STAT3 was not directly evaluated in the present study, TLR signaling showed more substantial fold enrichment in our pathway analysis, guiding our mechanistic focus toward TLR3. Nevertheless, these findings suggest potential crosstalk between the TLR3/NF-κB and STAT3 pathways, warranting further investigation in future studies. NF-κB signaling has been widely implicated as a central regulator of chemoresistance through its ability to promote cancer cell survival, inflammatory signaling, epithelial–mesenchymal transition, and maintenance of cancer stem-like properties [52,53]. Persistent NF-κB activation has been reported to confer resistance to 5-FU by upregulating anti-apoptotic genes and stemness-associated transcriptional programs [54]. Therefore, NF-κB suppression represents an attractive therapeutic strategy to overcome drug resistance.

Notably, natural products such as ABE are unlikely to target a single signaling pathway exclusively; instead, they may exert broader regulatory effects across interconnected oncogenic networks. Consistent with this concept, recent reviews have demonstrated that plant-derived natural products and their analogs can modulate NF-κB signaling at multiple regulatory levels, including membrane receptors, upstream kinases, and NF-κB subunits [55,56], and can also regulate Toll-like receptor signaling via suppression of MyD88/NF-κB cascades [57]. Together, these reports support the notion that natural products can target NF-κB and TLR pathways, reinforcing our observation that ABE suppresses TLR3/NF-κB signaling in 5-FU-resistant CRC cells. In this context, the pronounced reduction in NF-κB observed following combined ABE and 5-FU treatment provides a mechanistic basis for the enhanced chemosensitivity and CSC suppression identified in our study. Several limitations of this study should be acknowledged. First, a well-recognized challenge of natural product–based therapies is the difficulty in standardizing complex extracts for clinical application. Aronia berry extract contains diverse bioactive compounds, including anthocyanins, flavonols, and phenolic acids, which have been reported to inhibit topoisomerase I/II activity [58,59,60,61]. While our findings demonstrate the therapeutic potential of ABE, future studies should focus on isolating active components and developing standardized formulations. Another limitation is that, although patient-derived organoids were used to enhance clinical relevance, they were not derived from 5-FU–resistant tumors. While our PDO data support synergistic efficacy of ABE and 5-FU, further validation using resistant organoid models and in vivo studies will be essential to confirm translational applicability.

In summary, our study documented that the combination of 5-FU and ABE inhibits CRC stem cell proliferation. The mechanism involves inhibition of stemness and NF-κB signaling and is supported by in vitro data. This article provides valuable insights into ABE’s potential to overcome 5-FU resistance in CRC. These findings add to the growing evidence for ABE’s potential as a novel therapy for CRC, especially for patients with 5-FU resistance.

4. Materials and Methods

4.1. Cell Culture

The human CRC cell lines, HCT116 and SW480, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin (Gibco), and 1% streptomycin (Sigma-Aldrich, St. Louis, MO, USA). The cells were grown at 37 °C in a humidified atmosphere containing 5% CO_2_ and harvested using 0.05% trypsin-0.03% EDTA (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). 5-FU-R cell lines, 5-FU-R HCT116 and 5-FU-R SW480, were established by continuously culturing the cells with increasing doses of 5-FU, as described previously [21,62].

4.2. Herbal Preparations

Aronia Berry extract (ABE; Aronia Berry Complex, EuroPharma USA, Green Bay, WI, USA) was employed in this study. This extract is derived from the fruit of Aronia melanocarpa and prepared in 70% ethanol, standardized to contain 40% polyphenols. This powdered extract, derived from the fruit of black chokeberry (native to North America), offers a characteristic sensory profile ranging from purple to dark red. The extract is dissolved in dimethyl sulfoxide to obtain stock solutions, which are then diluted to appropriate levels in the culture media.

4.3. Reagents

Poly (I:C) was obtained from InvivoGen (San Diego, CA, USA), and CU-CPT 4a was purchased from MedChemExpress (MCE; Monmouth Junction, NJ, USA). 5-fluorouracil (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) for use in this study. The stock solution of 5-fluorouracil and ABE was stored at −20 °C in opaque containers and diluted with complete culture medium to achieve the desired experimental concentrations before each application.

4.4. Cell Counting MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide) Assays

To confirm the 5-FU resistance, we compared the proliferation rates of 5-FU-R and parental cell lines by treating them with increasing doses of 5-FU. Cells were seeded at a density of 5 × 10^3^ cells per well in 96-well flat-bottom plates and incubated for 24 h. Subsequently, they were exposed to escalating concentrations of 5-FU (0–160 μM). After 48 h of treatment, the supernatant was removed, and each well was supplemented with 90 μL of DMEM and 10 μL of MTT solutionas previously described [63,64]. The plates were then incubated for an additional 4 h, and the absorbance of the final product was measured at 490 nm (OD490) using a microplate reader (Tecan Trading AG, Männedorf, Switzerland). To evaluate the combined impact of ABE and 5-FU on CRC cell proliferation, cells were seeded at 3 × 10^3^ cells/well in 100 µL of complete medium in 96-well plates and grown for 24 h. Following this, the cells were exposed to appropriate concentrations of 5-FU (40 μM), ABE (60 μg/mL), and their combination. Proliferation was assessed at different time points using the MTT assay.

4.5. TLR3 Activation and Inhibition Assays

TLR3 activation was achieved by treating 5-FU–resistant CRC cell lines with poly (I:C) (InvivoGen) as a specific TLR3 ligand. Cells were exposed to poly (I:C) for 2–6 days, and spheroid formation was monitored under adherent culture conditions. To inhibit TLR3 signaling, cells were treated with the TLR3 inhibitor CU-CPT 4a (MCE). Following inhibitor treatment, cells were subsequently exposed to 5-FU, and cell viability was assessed using the MTT assay.

4.6. Drug Response Testing

The CRC cell lines were cultured in 96-well plates at a density of 5 × 10^3^ cells per well and incubated for 24 h prior to drug introduction. Subsequent treatment involved exposing the cells to varying concentrations of 5-FU (0, 20, 40, and 60 μM) and ABE (0, 30, 60, 90, 120, and 150 μg/mL), individually and in various combinations, for 48 h. Cell viability was evaluated using the MTT assay as mentioned above. To analyze the combined effects of the drugs, synergy scores were computed utilizing SynergyFinder 3.0 (https://synergyfinder.fimm.fi, accessed on 28 January 2026), a user-friendly tool tailored for interactive assessment and visualization of combination treatment outcomes.

4.7. Colony Formation Assay

Colony formation assays were performed as previously described [9,11,65]. 5 × 10^2^ cells per well were seeded in 6-well flat-bottom plates, followed by treatment with 5-FU (40 μM), ABE (60 μg/mL), or their combination for 48 h. Colony formation was allowed to proceed for 10 to 14 days, with the culture medium refreshed every 3 days. Upon completion of the incubation period, the cell colonies were fixed with methanol for 30 min and subsequently stained with 1% crystal violet (Thermo Fisher Scientific, Waltham, MA, USA). Quantification of colony numbers was performed using ImageJ software (version 1.53; National Institutes of Health, Bethesda, MD, USA).

4.8. The Wound Healing Assay

The wound healing assay was performed as previously described [9,11,65]. 5 × 10^5^ cells per well were seeded in 6-well flat-bottom plates after 48 h of treatment with 5-FU, ABE, or their combination. Upon reaching 90% to 100% confluence, a controlled scratch was created in the monolayer using a sterile 200 μL micropipette tip, followed by a wash with serum-free medium to remove detached cells. Subsequently, the cells were cultured in medium devoid of FBS. Photographs of cellular migration were captured 24 h post-wounding, and the percentage of wound closure was quantified using ImageJ.

4.9. Invasion Assays

Invasion assays were conducted as previously described [9,11,65]. BioCoat Matrigel Invasion Chambers with 8.0 μm Pore Polyester Membrane (BD Biosciences, Franklin Lakes, NJ, USA) were used. Following treatment with 5-FU, ABE, or their combination for 48 h, 5 × 10^4^ cells per well were seeded in 24-well flat-bottom plates for the invasion assay. Subsequently, the cells were placed onto inserts in serum-free medium and transferred to wells containing culture medium supplemented with 10% FBS. After 72 h of incubation, the cells that had invaded the bottom surface of the membrane were fixed and stained using a Diff-Quick staining kit (Thermo Fisher Scientific). Quantification of the stained cells was performed under a microscope.

4.10. Spheroid Formation Assay

Spheroid formation assays were performed as previously described [64]. After 48 h of treatment with 5-FU, ABE, and their combination, 5-FU-R HCT116 and 5-FU-R SW480 cells were seeded in ultra-low attachment plates at a density of 500–1000 cells per well in serum-free DMEM-F12 medium (STEMCELL Technologies, Vancouver, BC, Canada) containing epidermal growth factor (EGF; STEMCELL Technologies), basic fibroblast growth factor (bFGF; Gibco), and B27 supplement (Gibco). After 7–10 days, the spheroids were observed under a bright-field microscope at 40× magnification and counted using ImageJ.

4.11. Genome-Wide Transcriptomic Profiling and Pathway Enrichment Analysis

Genome-wide mRNA expression profiling was conducted to compare parental CRC cells with their 5-FU-R counterparts [66]. Total RNA was isolated and used for next-generation sequencing library preparation with the SureSelect XT HS2 mRNA Library Preparation Kit (Agilent Technologies, Santa Clara, CA, USA), using up to 1 µg of input RNA per sample in accordance with the manufacturer’s instructions. The raw Fastq files and the processed, filtered count matrix for mRNA sequencing were deposited in the NCBI GEO database under accession GSE196900. RNA sequencing was performed on an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA). Initial base calling was carried out using Illumina Real-Time Analysis software (RTA3, version 3.4.4), followed by conversion to FASTQ format using bcl2fastq (version 2.20.0.422). Low-quality bases at the 5′ and 3′ ends of reads were removed based on Phred quality scores prior to downstream processing. The filtered reads were aligned to the human reference genome (GRCh38) using the STAR aligner (version 2.7.8a) with default alignment settings [67]. After alignment, the final BAM files were quantified using the Partek E/M algorithm [68], and gene-level annotations were assigned based on Ensembl transcript release 102. Normalization and differential expression analysis were subsequently performed using the DESeq2 package to identify transcriptional differences between 5-FU-R and parental CRC cells [69]. Genes exhibiting an absolute log_2_ fold change greater than 1 with a nominal p-value < 0.05 were considered differentially expressed. To explore the biological significance of the identified differentially expressed genes (DEGs), pathway enrichment analysis was performed using KEGG pathways through DAVID Bioinformatics Resources (https://davidbioinformatics.nih.gov/summary.jsp, accessed on 22 Janually 2025).

4.12. Protein Isolation and Western Blot

Total protein was extracted from CRC cell lines treated with 5-FU, ABE, or their combination for 48 h. The cells were collected using a plastic scraper and rinsed three times with cold phosphate buffered solution (PBS; Gibco). Subsequently, the cells were lysed using ice-cold RIPA protein extraction solution supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific). Protein concentration was determined using the BCA method (Thermo Fisher Scientific). Equal amounts of protein samples (50 μg) were resolved via SDS-PAGE using 10% Mini-PROTEAN TGXTM Precast Gels (BIO-RAD, Hercules, CA, USA) and then transferred onto a 0.45 μm PVDF membrane (Cytiva, Marlborough, MA, USA). The membrane was blocked with 5% bovine serum albumin (BSA; Sigma-Aldrich) in Tris-buffered saline (BIO-RAD) containing 0.1% Tween-20 (Sigma-Aldrich) for 1 h at room temperature. Primary antibodies, including anti-CD44 (1:1000, 5023S; Cell Signaling Technology [CST], Danvers, MA, USA), anti-Nanog (1:1000, 9505; CST), anti-Oct4 (1:1000, 9532S; CST), anti-TLR3 (1:1000, PA5-20183; Thermo Fisher Scientific), anti-P65 (1:1000, 3034; CST), anti-GAPDH (1:2000, 5174T; CST), anti-β-actin (1:5000, 58169S; CST), and anti-Lamin A/C (1:1000, 10298-1-AP; Proteintech, Rosemont, IL, USA) were incubated with the membranes at 4 °C overnight. After three washes with TBST, the membrane was incubated with the appropriate anti-rabbit (1:2000, 7074; CST) or anti-mouse (1:2000, 7076; CST) secondary antibodies for 1 h at room temperature. Blots were visualized using an HRP-based chemiluminescence kit (Thermo Fisher Scientific) and Gel Imaging Systems (BIO-RAD). GAPDH, β-actin, and Lamin A/C protein were used as internal controls, and protein band intensities were quantified using ImageJ. The original Western blot images are provided in Figure S2.

4.13. Quantitative Reverse Transcription PCR (qRT-PCR)

Total RNA extraction was performed using the Qiagen miRNeasy Kit (Qiagen, Hilden, Germany). cDNA synthesis was performed by reverse transcription of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). RT-PCR assays were performed using the QuantStudio 6 Flex RT-PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocols and the SensiFAST SYBR Lo-ROX Kit (Bioline, London, UK). Relative mRNA expression levels were calculated using the 2^−ΔΔCt^ method and normalized to β-actin, the internal control. The primers used in the study are detailed in Table S1.

4.14. Nuclear Protein Extraction

CRC cells were harvested after treatment and washed twice with cold PBS. Nuclear protein extraction was performed using a standard hypotonic lysis protocol. Briefly, cell pellets were resuspended in 500 μL hypotonic buffer [20 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl_2_, 0.5 mM DTT] supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich, cat. #P8340; Thermo Fisher Scientific, cat. #78420) and incubated on ice for 15 min. Subsequently, 25 μL of 10% Nonidet P-40 (Sigma-Aldrich) was added, followed by centrifugation at 1000× g for 10 min at 4 °C. The resulting nuclear pellet was washed once, then lysed in 180 μL of complete lysis buffer provided in the transcription factor extraction kit, according to the manufacturer’s instructions, as previously described [10]. Nuclear protein concentrations were determined prior to downstream analyses.

4.15. Patient-Derived CRC Tumor Organoids

Patient-derived CRC organoids were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured according to previously published procedures [65]. For the culture of human organoids, the Human Complete Feeding Medium (hCPLT) comprised IntestiCult Organoid Growth Medium (STEMCELL Technologies, Vancouver, BC, Canada) supplemented with Y-27632 (1:1000; STEMCELL Technologies). The Human Wash Medium with BSA consisted of Advanced DMEM/F-12 (STEMCELL Technologies), 10 mM HEPES, 1× GlutaMAX Supplement (Gibco), 100 μg/mL Primocin (InvivoGen), and 0.1% BSA (Sigma-Aldrich). CRC organoids were seeded in 24-well plates, forming domes within 40 μL of Matrigel (Corning, Tehama County, CA, USA), and cultured in 700 μL of the hCPLT. Subsequently, the organoids were divided into four treatment groups, and appropriate concentrations of ABE, 5-FU, and their combination were administered. After a 3-day treatment period, the number and size of the organoids were assessed by microscopy (magnification ×40), with measurements obtained using ImageJ software. Harvested organoids were also aliquoted for total RNA extraction.

4.16. Immunofluorescence Assays

For the immunofluorescence assay, the organoids were treated with 5-FU, ABE, or a combination of both for 48 h. The organoids were subsequently fixed with 4% paraformaldehyde for 10 min at room temperature, then permeabilized with 0.5% Triton X-100 (Thermo Fisher Scientific) for 10 min at room temperature. To remove endogenous peroxides, the slides were treated and subsequently blocked in TBST (Tris-buffered saline (Thermo Fisher Scientific) containing Tween-20 (Sigma-Aldrich)) containing 3% BSA (Sigma-Aldrich) for 1 h at room temperature. The slides were then incubated overnight at 4 °C with an anti-NF-kB Polyclonal antibody, followed by incubation with a FITC-labeled secondary antibody (1:1000, A-21202; Thermo Fisher Scientific) for 1 h at 37 °C. After the incubation periods, the organoids were subjected to three additional washes with PBS (Gibco), each lasting 5 min. Finally, the organoid nucleus was stained with DAPI (Thermo Fisher Scientific), and all images were captured using a Carl Zeiss fluorescent microscope (Zeiss, Oberkochen, Germany).

4.17. Statistical Analysis

Statistical analyses were conducted using GraphPad Prism v. 6.0 software (GraphPad Software, San Diego, CA, USA). Student’s t-test was employed to assess the significance of differences between any two groups, while one-way analysis of variance (ANOVA) was utilized to evaluate differences across multiple comparisons. Each experiment was independently performed in triplicate using biological replicates, and data were expressed as mean ± standard deviation. A significance level of p < 0.05 was adopted to determine statistical significance.

5. Conclusions

We have introduced a novel perspective on ABE’s potential to address 5-FU resistance in CRC cells, through a comprehensive series of experiments using 5-FU-resistant cell cultures and patient-derived tumor organoids. Furthermore, our observations indicate a pivotal role for the TLR3/NF-κB axis in mediating stemness and 5-FU resistance in CRC, and ABE mitigates this resistance by downregulating TLR3 and its associated signaling pathways in CRC cells. These findings underscore a promising, economically feasible strategy to enhance therapeutic efficacy in CRC.