Therapeutic potential of isochlorogenic acid A from Taraxacum officinale in improving immune response and enhancing the efficacy of PD-1/PD-L1 blockade in triple-negative breast cancer

Taraxacum officinale (Product Standard Number: GHT1091) was purchased from Liaoning Senkangyuan Ecological Agriculture Co., Ltd., and authenticated by Wang Kun. The dried whole plant was subjected to reflux extraction with 75% ethanol at 60°C for three cycles, each lasting 3 hours. The combined extracts were filtered and concentrated under reduced pressure to obtain the crude extract. The crude extract was then further purified using D-101 macroporous resin column chromatography. Initially, the column was washed with water to remove hydrophilic impurities such as sugars, followed by elution with 100% ethanol to collect the active compounds. The ethanol fraction was subsequently evaporated under vacuum and spray-dried to obtain the final TOE. For in vitro studies, TOE was dissolved in 50% DMSO to prepare a stock solution, which was then diluted with cell culture medium. The final DMSO concentration was maintained at 0.5% to minimize solvent-induced cytotoxicity.

0.50 g of TOE was taken and mixed with 10 ml of solvent (methanol, acetonitrile, and water = 2:2:1), centrifuged at 13000 rpm for 5 min, and filtered through a 0.22 µm membrane filter.

A heat electro-spray ionization (HESI) was paired with a Q-Orbitrap MS (Thermo Fisher Scientific) in the MS analysis. The main parameters included the scan range, 100.0 to 1300.0 m/z; spray voltage, 2.80 KV (negative mode); capillary temperature, 300°C; aux gas heater temperature, 300°C; sheath gas flow rate, 40 arbitrary units; aux gas flow rate, 10 arbitrary units; maximum injection time, 50 ms; automatic gain control target, 1.0 e6. Molecular weights [M-H]- and [M-H]+ were used for qualitative analysis. UPLC separation was carried out on an AccucoreaQ C18 column (150 mm×2.1 mm, 2.6 μm, Thermo Fisher Scientific) with a flow rate of 0.3 μL/min. The optimized mobile phase was a mixture of water with 0.9% acetic acid (A) and methanol (B). For identification samples, the gradient was operated as follows: 0-9 min, 20%-100% B; 9-10 min, held at 100% B; 10-11 min, 100%-20% B; and 11-14 min, held at 20% B. The injection volume was 3 μL, and the temperature of the column was 30°C.

The 4T1 and MDA-MB-231 cell lines were obtained from the Procell Life Science & Technology Co., Ltd. MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Procell) and 1% penicillin/streptomycin (Procell). 4T1 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) supplemented with 10% fetal bovine serum (Procell) and 1% penicillin/streptomycin (Procell). Cells were cultured at 37°C with a 5% CO2 atmosphere.

CCK-8 assays were performed using the CCK-8 kit (Elabscience, China) according to the manufacturer’s instructions. Briefly, MDA-MB-231 or 4T1 cells were seeded at a density of 2.0×10^5 cells/mL in 96-well plates and incubated with culture medium at 37°C and 5% CO2 for 24 hours. MDA-MB-231 and 4T1 cells were then treated with a series of concentrations of TOE for 24 hours (24, 48, 72 hours). CCK-8 assay results indicated that TOE exhibited the most significant inhibitory effect on the viability of TNBC cells at 24 hours (). To maintain consistency, the 24-hour time point was also chosen for assessing the effects of ICGA-A and CRA on cell viability. At the end of the treatment, the culture medium was removed, and 20 µL of CCK-8 reagent was added to each well. The plates were incubated for an additional 1 hour, and the absorbance of each well was measured at 450 nm. 1

Cell migration and invasion were assessed using Transwell assays. For the migration assay, approximately 2×10^4 cells were seeded into the upper chamber of the Transwell, which had been treated with TOE, ICGA-A, and CRA, using serum-free medium, while 750 μL of medium was added to the lower chamber. For the invasion assay, 30 µg of Matrigel Matrix (Corning) was added to the upper chamber and incubated for 1 hour at 37°C before seeding the cells. After 20 hours of incubation at 37°C with 5% CO₂, the medium was removed and the cells were washed twice with PBS. The cells were fixed in paraformaldehyde for 15 minutes and then washed twice with PBS. The chambers were stained with 0.1% crystal violet for 10 minutes, and five fields of view were selected, photographed, and counted under a microscope to evaluate migration and invasion.

Cells were seeded at a density of 2.5 × 10^5 cells per well in six-well plates and allowed to adhere overnight. The following day, the cells were treated with ICGA-A and CRA for 24 hours. After treatment, both adherent and floating cells were collected by trypsinization (using trypsin without EDTA, Procell) and centrifuged at 300 × g for 5 minutes at 4°C. The cell pellets were then washed twice with cold PBS and resuspended in 100 µL of 1× Annexin V binding buffer. Subsequently, 5 µL of Annexin V-FITC and 5 µL of propidium iodide (PI) were added to each sample, followed by incubation in the dark at room temperature for 15 minutes. After staining, 400 µL of 1× binding buffer was added to each sample. Apoptosis was analyzed using a flow cytometer (Beckman Coulter, China) within 1 hour. Apoptotic populations were identified based on fluorescence signals, with Annexin V⁺/PI^– indicating early apoptosis and Annexin V⁺/PI^– indicating late apoptosis. All antibodies and buffers were provided by Elabscience Biotechnology Co., Ltd., with antibodies diluted at a 1:2 ratio. Data analysis was performed using FlowJo software.

To establish the tumor model, 10 g of tribromoethanol (Sigma) was dissolved in 10 mL of tert-pentanol (Sigma) at room temperature to prepare a clear stock solution. The solution was filtered through a 0.22 μm Millex-GP SLGPR33RB filter (Millipore) and then diluted with physiological saline (National Medicine Standard H11021190, 0.9 g/100 mL) to achieve a final concentration of 19.2 mg/mL (2.5% tribromoethanol). The working solution was incubated at 37°C for 4 hours, shaken, and stored at 4°C in the dark. For tumor induction, 160 μL (307 mg/kg) of 2.5% tribromoethanol was administered intraperitoneally to induce anesthesia. Once the mice were fully anesthetized, they were positioned in a sterile work area in a supine position, and the area was disinfected with povidone-iodine. A small incision (approximately 0.5 cm) was made above the right fourth mammary gland using ophthalmic scissors. The skin was gently lifted with a cotton swab to expose the mammary fat pad, and 40 μL of a serum-free suspension containing 5 × 10^5 4T1 cells was injected into the fat pad. Tumor volume was measured using calipers, and the volume was calculated using the formula: Volume (mm^3) = (Width^2 × Length)/2.

When the tumor volume reached 100 mm^3, treatment commenced. In the in vivo experiments using TOE, the low-dose (100 mg/kg) and high-dose (200 mg/kg) monotherapy groups were administered TOE daily by oral gavage. The combination therapy group received 5 mg/kg PD-1/PD-L1 inhibitor 2 via intraperitoneal injection every two days for a total of 14 days. In the experiments using commercial compounds, the monotherapy groups received daily intraperitoneal injections of ICGA-A (10 mg/kg) or every two days of CRA (20 mg/kg). The combination therapy group received 5 mg/kg PD-1/PD-L1 inhibitor 2 via intraperitoneal injection every two days for 14 days.

On day 15 of treatment, mice were euthanized using 2.5% isoflurane anesthetic. Tumors were carefully excised and transferred into enzyme digestion solution containing collagenase IV (2 mg/mL, Thermo Fisher Scientific), DNase I (0.1 mg/mL, Thermo Fisher Scientific), and RPMI-1640 medium (Procell) to degrade extracellular matrix tissue and remove impurities. Red blood cells were then eliminated using red blood cell lysis buffer (Biyuntian Biotechnology). Lymphocytes were isolated using Percoll (Solarbio) density gradient centrifugation. The resulting cell pellet was resuspended in PBS (Procell), and a single-cell suspension suitable for flow cytometry analysis was prepared.

The single-cell suspension was aliquoted into two tubes for different fluorescent antibody labeling. One tube was labeled with F4/80, CD11b, and CD45.2 antibodies, while the other tube was labeled with CD3e, CD4, CD8a, CD279 (PD-1), CD223 (LAG-3), and CD366 (TIM3) antibodies, all obtained from Thermo Fisher Scientific. Antibodies used here are listed in. Flow cytometry (FACS) analysis was performed to assess the antitumor immune response, with 500,000 cells collected for evaluation. Flow cytometry data were analyzed using FlowJo software. Initial gating was performed based on forward scatter (FSC) and side scatter (SSC) plots. Immunophenotyping analysis was conducted using multiple fluorescence channels. The gating strategy is outlined in. 1 1

The relevant targets of nine compounds identified in TOE were obtained using SwissADME and SwissTargetPrediction. Interaction standardization between compounds and targets was achieved by processing target information from the Uniprot protein database. Using “triple-negative breast cancer” as the keyword, potential therapeutic targets were identified in the GeneCards, OMIM, and DisGeNET databases, with targets scoring above the median selected as potential “disease targets” for TNBC.

Cells were seeded in 10 cm culture dishes and treated with DMSO, ICGA-A, and CRA for 24 hours in a 5% CO₂ environment at 37°C. Following treatment, cells were collected using Trizol (Vazyme). RNA quality control, library construction, and sequencing were performed by GeneWiz Inc. GO and KEGG analyses were conducted using the DAVID v6.8 database, with results visualized through bar and bubble charts generated on the Bioinformatics platform.

The compound structure files were obtained from the PubChem database. The three-dimensional structure of the FAK protein (PDB code: 6dj6) was retrieved from the Protein Data Bank (PDB). Molecular docking and virtual screening were performed using AutoDock Vina, and the visualizations were generated with PyMOL.

PD-1/PD-L1 inhibitor 2 (BMS202) were purchased from Selleck. Isochlorogenic acid A and Chicoric acid were purchased from TOPSCIENCE. All chemicals were dissolved in DMSO for the in vivo and in vitro studies.

After the cells were treated with ICGA-A and CRA for 48 hours, they were lysed on ice using RIPA buffer (Solarbio) containing 1% PMSF and 1% phosphatase inhibitor (Proteintech). The protein concentration was measured using the Pierce BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein (20 µg each) were loaded onto 10% SDS-PAGE (Vazyme) and FuturePAGE™ 4%–12% 12 Wells (Ace Biotechnology) and then transferred to a PVDF membrane (Millipore). The membrane was blocked at room temperature with 5% nonfat milk and then incubated overnight with the primary antibody at 4˚C. The membrane was washed with TBST, followed by incubation with the secondary antibody at room temperature. The protein signals in the BG-gdsAUTO730 were detected using a high-sensitivity ECL chemiluminescent detection kit (Proteintech). Original images of the Western blot analysis from three independent experiments are shown in. Antibodies used here are listed in. 1 1

Data were collected from independent experiments and expressed as the mean ± SEM. All graphs and analyses were generated using GraphPad Prism software. The significance among multiple (three or more) groups was compared using one-way ANOVA analysis, and differences between the different groups were analyzed by a Student’s t-test.

To evaluate the effect of TOE on PD-1/PD-L1 inhibitor 2-mediated suppression of tumor growth in the 4T1 mouse model and its impact on TNBC immunotherapy, we established a 4T1 mouse model. Mice were randomly assigned to six different treatment groups: PD-1/PD-L1 inhibitor 2 alone, TOE (100 mg/kg), TOE (200 mg/kg), TOE (100 mg/kg) combined with PD-1/PD-L1 inhibitor 2, TOE (200 mg/kg) combined with PD-1/PD-L1 inhibitor 2, and saline control. Treatment was initiated on Day 0 when the tumor volume reached 100 mm^3. In the TOE (100 mg/kg) monotherapy group, 100 mg/kg of TOE was administered orally once daily from Day 0 to Day 14. In the TOE (200 mg/kg) monotherapy group, 200 mg/kg of TOE was administered orally once daily from Day 0 to Day 14. In the combination therapy groups, TOE was administered orally once daily from Day 0 to Day 14, while PD-1/PD-L1 inhibitor 2 (5 mg/kg) was administered intraperitoneally every 48 hours on Days 0, 2, 4, 6, 8, 10, 12, and 14 throughout the treatment period. For consistency, the PD-1/PD-L1 inhibitor 2 monotherapy group followed the same dosing schedule, receiving a single intraperitoneal injection of 5 mg/kg every 48 hours, ensuring a comparable treatment frequency across groups (Figure 1A). TOE (200 mg/kg) exhibited a more pronounced antitumor effect compared to the low-dose TOE (100 mg/kg), indicating a dose-dependent antitumor activity. Therefore, in subsequent analyses, TOE refers to the high-dose TOE (200 mg/kg) (Figure 1B). The combined use of PD-1/PD-L1 inhibitor 2 and TOE significantly inhibited tumor progression compared to TOE or PD-1/PD-L1 inhibitor 2 alone (Figures 1C, D). Compared with the control group, the combination of TOE and PD-1/PD-L1 inhibitor 2 did not cause significant changes in body weight or the visceral indices of the spleen, liver, or lungs, indicating that the combination therapy did not induce systemic toxicity (Supplementary Figure S4). Taken together, these findings suggest that TOE can enhance PD-1/PD-L1 inhibitor 2-mediated suppression of tumor growth in TNBC in vivo.

In vitro experiments were conducted using TNBC cell lines MDA-MB-231 and 4T1. Cells were treated with different concentrations of TOE, and cell viability was assessed using CCK-8 analysis. Results showed a dose-dependent decrease in cell viability, indicating that TOE has cytotoxic effects (Figure 1E). The 24-hour IC50 values for MDA-MB-231 and 4T1 cells were 135.9µg/mL and 150.2µg/mL, respectively. Furthermore, Transwell migration and Matrigel invasion assays indicated that TOE significantly reduced the migration and invasion capabilities of TNBC cells (Figure 1F).

To assess the ability of combination therapy to enhance tumor lymphocyte infiltration and modulate the immune-suppressive tumor microenvironment in 4T1 tumors, flow cytometry results revealed that PD-1/PD-L1 inhibitor 2 monotherapy had a negligible effect on macrophage infiltration. In contrast, combination therapy with PD-1/PD-L1 inhibitor 2 and TOE significantly increased macrophage recruitment into the tumor microenvironment (Figures 2A, B). Both TOE monotherapy and its combination with PD-1/PD-L1 inhibitor 2 significantly elevated the frequency of M1-type macrophages within tumor-infiltrating lymphocytes (Figures 2C, D). Moreover, TOE treatment notably reduced the frequency of M2-type macrophages (Figure 2F). These findings suggest that TOE promotes macrophage polarization toward the M1 phenotype in PD-1/PD-L1 inhibitor 2 treatment, although its underlying mechanism requires further elucidation. Evaluation of effector T cells revealed that, compared to the saline control group, PD-1/PD-L1 inhibitor 2 monotherapy and TOE monotherapy had a limited impact on CD8+ T cell infiltration. However, combination therapy with PD-1/PD-L1 inhibitor 2 and TOE significantly increased the proportion of CD8+ T cells in the tumor microenvironment, while the proportion of CD4+ T cells remained unchanged (Figures 2E, G, H).

Reversing T cell exhaustion is crucial for revitalizing the antitumor immune response, aiding the immune system in recognizing and eliminating tumor cells. Monotherapy with PD-1/PD-L1 inhibitor 2 did not significantly affect the antitumor activity of LAG-3+, TIM-3+, or PD-1+ T cell subsets. However, monotherapy with TOE and combination therapy with PD-1/PD-L1 inhibitor 2 and TOE significantly reduced the frequency of exhausted LAG-3+, TIM-3+, and PD-1+ T cells. Notably, the combination therapy more effectively reversed the exhaustion of LAG-3+ and TIM-3+ T cells, thereby enhancing the antitumor activity of T cells (Figures 2I-N). These results indicate that the combination of TOE and PD-1/PD-L1 inhibitor 2 not only enhances the antitumor effects of PD-1/PD-L1 inhibitor 2 therapy but also limits immune escape by tumors and improves the tumor microenvironment, thereby increasing the overall efficacy of cancer treatment. This combination therapy can reprogram the tumor microenvironment from a “cold” to a “hot” state, thereby activating immune cells to eliminate tumor cells and significantly improving the efficacy of immunotherapy.

Compounds of the TOE were identified using UPLC-Q-Orbitrap MS, and nine major components were obtained. The UPLC-Q-Orbitrap MS total ion chromatograms of the TOE are shown in Figure 3A. The mass spectra of these nine components were given in Figures 3B-I. The detailed information of the identified constituents in TOE was summarized in Table 1.

SwissADME and SwissTargetPrediction were used to identify 327 potential targets for nine compounds meeting pharmacokinetic standards. The GeneCards database identified 7,257 TNBC-related gene targets. A cross-analysis between the 327 TOE-related complementary targets and the 7,257 TNBC-related targets revealed 255 overlapping genes (Figure 4A). Cytoscape 3.9.1 was employed to construct a network linking compounds, targets, and disease, displaying the names of 255 genes involved in drug-disease interactions (Figure 4B).

GO functional analysis and KEGG pathway enrichment were used to predict potential regulatory pathways through which TOE may exert anti-TNBC effects via these 255 targets. Figure 4C presents several enriched terms, including phosphorylation, extracellular matrix disassembly, cell surface, and protein tyrosine kinase activity, suggesting that TOE may exert anti-TNBC effects through these biological processes. Additionally, as shown in Figure 4D, several signaling pathways involved in tumorigenesis and tumor progression were enriched, including “receptor activation”, “cancer pathways”, “focal adhesion”, “apoptosis”, and the “PI3K/AKT signaling pathway”. These findings are closely related to signal transduction via cell surface receptors, suggesting that TOE may exert antitumor effects through these critical pathways and targets, including the PI3K/AKT signaling pathway. Further molecular docking studies and experimental validations will be conducted to confirm these predictions. FAK serves as an upstream regulatory factor of the PI3K/AKT signaling pathway, directly affecting the function of this pathway. To assess whether these compounds in TOE can directly interact with FAK, molecular docking simulations were performed (Table 2). Results indicate that the nine compounds exhibit varying binding affinities with FAK, with ICGA-A and CRA demonstrating the strongest affinities (Figures 4E, F).

The highest-scoring bioactive compounds in TOE, ICGA-A and CRA, exhibited binding energies of -8.2 kcal/mol and -7.8 kcal/mol, respectively. These two compounds were subsequently selected for in vitro validation. CCK-8 assays demonstrated that treatment with ICGA-A and CRA significantly reduced cell viability, inhibited cell proliferation, and slowed cell growth (Figure 5A). The 24-hour IC50 values of ICGA-A for MDA-MB-231 and 4T1 cells were 135.8µM and 154.9µM, and the 24-hour IC50 values of CRA for MDA-MB-231 and 4T1 cells were 193.8µM and 198.6µM, respectively. Transwell assays indicated that ICGA-A and CRA significantly decreased the migration and invasion capabilities of MDA-MB-231 and 4T1 cells (Figure 5B). Flow cytometry was utilized to assess whether ICGA-A and CRA induced apoptosis. The results revealed that treatment with ICGA-A and CRA led to a substantial increase in the frequency of Annexin V-positive cells, effectively promoting apoptosis (Figure 5C).

To evaluate the potential of ICGA-A and CRA to inhibit TNBC tumor proliferation in vivo, a 4T1 mouse tumor model was established. Mice were randomly assigned to six groups, each receiving a different treatment regimen: PD-1/PD-L1 inhibitor 2 monotherapy, ICGA-A (10 mg/kg), CRA (20 mg/kg), combined ICGA-A with PD-1/PD-L1 inhibitor 2, combined CRA with PD-1/PD-L1 inhibitor 2, and a saline control group. Compared to the control group, both ICGA-A and CRA significantly inhibited the growth of 4T1 TNBC tumors. Furthermore, the combination of ICGA-A and PD-1/PD-L1 inhibitor 2 exhibited superior tumor-suppressive effects compared to PD-1/PD-L1 inhibitor 2 alone, suggesting the potential of this combination as a therapeutic strategy for TNBC (Figures 5D-F). In the combination treatment group, body weight and the organ indices of the spleen, liver, and lung remained unchanged, indicating the safety of this therapeutic approach (Supplementary Figure S5).

The reprogramming effects of active compound monomers on the polarization of tumor-associated macrophages (TAMs) and their potential to modulate the immunosuppressive microenvironment were assessed by quantifying the frequencies of total macrophages, M1-type TAMs, and M2-type TAMs using flow cytometry. The results indicated that combination treatment with ICGA-A and PD-1/PD-L1 inhibitor 2 significantly increased the frequency of anti-tumor M1-type TAMs, while having no significant effect on immunosuppressive M2-type TAMs (Figures 6B, E). Moreover, compared to the control group, the frequency of total tumor-associated macrophages was also significantly increased (Figures 6A, D). Analysis of effector T cells revealed that, compared to other groups, combination therapy with ICGA-A and PD-1/PD-L1 inhibitor 2 significantly increased the frequency of effector CD8+ T cells, surpassing the PD-1/PD-L1 inhibitor 2 monotherapy group. Additionally, the frequency of effector T cells in the CRA monotherapy group was also higher than that in the PD-1/PD-L1 inhibitor 2 monotherapy group (Figures 6C, F). Notably, none of the drug treatments had a significant impact on Treg levels (Supplementary Figure S6). These findings suggest that the ICGA-A combination therapy strategy can effectively enhance the immune response to immune checkpoint blockade (ICB) therapy.

This study also explored the effects of ICGA-A and CRA as monotherapy or in combination with PD-1/PD-L1 inhibitor 2 on T cell exhaustion. The results indicated that both compounds, whether used alone or in combination with PD-1/PD-L1 inhibitor 2, effectively reduced the frequency of exhausted T cells expressing LAG-3 (Figures 6G, J). Combination treatment with both compounds exhibited a similar trend in reducing TIM-3 expression levels (Figures 6H, K). Both the ICGA-A monotherapy and its combination with PD-1/PD-L1 inhibitor 2 effectively reduced the frequency of PD-1-positive exhausted T cells (Figures 6I, L). These results not only highlight the potential immunomodulatory roles of ICGA-A and CRA in regulating T cell exhaustion but also suggest that their combination with PD-1/PD-L1 inhibitor 2 may enhance T cell-mediated tumor immune responses. These findings provide robust experimental evidence for the development of novel tumor immunotherapy strategies, particularly those that utilize natural products to modulate the tumor microenvironment and enhance existing immunotherapies.

To further elucidate the molecular mechanisms by which ICGA-A and CRA synergize with PD-1/PD-L1 inhibitor 2 to exert antitumor immune functions, RNA sequencing (RNA-seq) was conducted. Cross-analysis of MDA-MB-231 cells treated with ICGA-A and CRA revealed 1,347 overlapping DEGs (p < 0.05) (Figure 7A). Cells treated with ICGA-A and CRA showed significant enrichment in the “Cancer pathway”, “VEGF pathway”, “PI3K-Akt signaling pathway”, “Focal adhesion pathway”, and “ECM-receptor interaction pathway” (Figures 7B, C). In compound library screening conducted using HTS², ICGA-A showed strong specificity for the PI3K signaling pathway, ranking in the top 8 out of 20,000 compounds and targeting the PI3K/Akt/mTOR signaling pathway (Figure 7D).

By overlapping the 255 targets identified through network pharmacology analysis with the differentially expressed genes (DEGs) (p < 0.05) from ICGA-A and CRA-treated MDA-MB-231 cells, RNA-seq analysis demonstrated a similar trend for these compounds in suppressing antitumor immunity-related genes (e.g., IDO1, MMP9, ADORA1, and AURKB) (Figure 7E). ICGA-A and CRA significantly inhibited the expression of various antitumor immunity-related genes, thereby modulating the immune microenvironment. Western blot results indicated that ICGA-A and CRA reduced the phosphorylation levels of FAK, PI3K, AKT, and mTOR, suggesting that these compounds may be key bioactive components within TOE (Figure 7F).

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/↗, PRJNA1215589.