Targeting stem-property and vasculogenic mimicry for sensitizing paclitaxel therapy of triple-negative breast cancer by biomimetic codelivery

Targeting stem-property and vasculogenic mimicry for sensitizing paclitaxel therapy of triple-negative breast cancer by biomimetic codelivery

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Siqi Wu^a^,^b, Qing Tang^c, Weifeng Fang^d, Zhe Sun^a, Meng Zhang^e, Ergang Liu^b, Yang Cao^a^,^*, Yongzhuo Huang^a^,^b^,^d^,^f^,^*

Acta Pharmaceutica Sinica B | 2025, 15(6) : 3226 - 3242

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Acta Pharmaceutica Sinica B | 2025, 15(6): 3226-3242

• ORIGINAL ARTICLE •

Targeting stem-property and vasculogenic mimicry for sensitizing paclitaxel therapy of triple-negative breast cancer by biomimetic codelivery

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Siqi Wu^a^,^b, Qing Tang^c, Weifeng Fang^d, Zhe Sun^a, Meng Zhang^e, Ergang Liu^b, Yang Cao^a^,^*, Yongzhuo Huang^a^,^b^,^d^,^f^,^*

Affiliations

^aDepartment of Oncology, the First Affiliated Hospital and the First Clinical School of Guangzhou University of Chinese Medicine, Guangzhou 510000, China

^bZhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan 528400, China

^cState Key Laboratory of Traditional Chinese Medicine Syndrome, Clinical and Basic Research Team of TCM Prevention and Treatment of NSCLC, Guangdong Provincial Hospital of Chinese Medicine, the Second Clinical College of Guangzhou University of Chinese Medicine, Guangzhou 510000, China

^dSchool of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China

^eDepartment of Pharmacy, Women's Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China

^fState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

doi: 10.1016/j.apsb.2025.04.006

Outline

Abstract

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Triple-negative breast cancer (TNBC) is aggressive, with high recurrence rates and poor prognosis. Paclitaxel (PTX) remains a key chemotherapeutic agent for TNBC, but its efficacy diminishes due to the emergence of drug resistance, largely driven by cancer stem-like cells (CSCs), vasculogenic mimicry (VM) formation and tumor immunosuppressive microenvironment (TIME). Pyruvate kinase M2 (PKM2) is highly expressed in TNBC, and is a potential target for TNBC treatment. In this study, we developed a biomimetic codelivery system using albumin nanoparticles (termed S/P NP) to co-encapsulate PTX and shikonin (SHK), a natural inhibitor of PKM2. By inhibiting PKM2, SHK suppressed β-Catenin signaling, thereby reversing CSC stemness and preventing VM formation. The S/P NP system exhibited tumor-targeting delivery effect and significantly inhibited TNBC growth and lung metastasis. Mechanistically, the treatment reversed epithelial–mesenchymal transition (EMT) and stem-like properties of TNBC cells, suppressed VM formation, and remodeled the TIME. It reduced immunosuppressive cells (M2 macrophages, MDSCs) while promoting anti-tumor immunity (M1 macrophages, dendritic cells, cytotoxic T cells, and memory T cells). This dual-action strategy holds promise for improving TNBC therapy by targeting CSCs, VM, and the immune microenvironment, and for overcoming PTX resistance and reducing metastasis.

Key words

Shikonin / Paclitaxel / Triple-negative breast cancer / Pyruvate kinase M2 (PKM2) / Cancer stem-like cells / Vasculogenic mimicry / Tumor microenvironment / Albumin

Cite this Article

Siqi Wu, Qing Tang, Weifeng Fang, Zhe Sun, Meng Zhang, Ergang Liu, Yang Cao, Yongzhuo Huang. Targeting stem-property and vasculogenic mimicry for sensitizing paclitaxel therapy of triple-negative breast cancer by biomimetic codelivery[J]. Acta Pharmaceutica Sinica B, 2025 , 15 (6) : 3226 -3242 . DOI: 10.1016/j.apsb.2025.04.006

Full Text

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1.　Introduction

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Triple-negative breast cancer (TNBC) is characterized by high invasiveness, proneness to relapse, and poor prognosis^1,2. Paclitaxel (PTX) is a mainstay of TNBC treatment. However, a wealth of clinical and experimental evidence indicates that PTX alone is insufficiently effective against TNBC, with significantly decreased efficacy after repeated administration^3,4. Consequently, the conventional and classic TNBC treatment usually combines PTX with other chemotherapeutic drugs⁵, immune checkpoint blockade⁶, or anti-angiogenic therapies⁷. To sensitize PTX treatment and to seek an efficient and affordable combination therapy are important for improving TNBC treatment.

Cancer stem-like cells (CSCs) have been identified as a major factor in the poor response to PTX therapy and cancer recurrence in breast cancer (BC)⁸, which is attributed to their capacity for self-renewal and production of heterogeneous cell lineages^9,10. CSCs, characterized as a typical undifferentiated cancer cell phenotype, are rich in poorly differentiated tumors, which are linked to high-grade malignancy and poor prognosis compared to well-differentiated tumors^11,12. Vasculogenic mimicry (VM) is a specific type of vasculature formation characterized by endothelial-like tumor cells themselves constituting the tubular structures that acts as pseudo blood vessels; VM is an important mechanism for poor response to PTX therapy^13,14. VM is an alternative means to transport oxygen and nutrients to tumor cells¹⁵, which is linked to poor overall survival (OS) in patients, thus being a viable therapy target^16-18. Notably, CSCs and VM are closely associated with each other. CSCs lose some epithelial properties and gain endothelial-like cell characteristics that contribute to VM formation¹⁹. Specifically, CSCs inside TNBC can form VM, serving as its source²⁰. On the other hand, the process of VM can maintain stemness characteristics of cancer cells²¹. Importantly, factors such as hypoxia can upregulate the genes associated with both CSC properties and VM, creating a feedback loop to promote tumor aggressiveness²². It is a widely-accepted paradigm that there is a close link between CSCs, VM and tumor immunosuppressive microenvironment (TIME).

Pyruvate kinase M2 (PKM2) is a crucial target involved in the stem-property of cancer cells²³, VM formation²⁴ and TIME²⁵. The tetramer of PKM2 is a crucial enzyme that controls glycolysis rate²⁶. By contrast, the dimeric PKM2 performs its nonmetabolic functions via translocation into nucleus, where PKM2 binds with β-Catenin, leading to the activation of β-Catenin signaling pathway²⁷. Notably, this pathway plays a vital role in maintaining the stem property of CSCs by promoting their renewal, proliferation, and differentiation²⁸. It, in turn, makes the cancer cell resistant to PTX and other chemotherapeutic drugs²⁹. Therefore, the inhibition of PKM2 is expected to decrease the stem-property of cancer cells and the formation of VM, and remodel TIME, leading to re-sensitization of PTX therapy.

Herein, we proposed a PKM2 inhibition strategy for simultaneously blocking the cytoplasmic tetramer PKM2 to inhibit lactate production, as well as the nuclear ectopic of dimeric PKM2 to deactivate β-Catenin. By this way, it is expected that the stem-property of cancer cells and the formation of VM could be reversed, and TIME remodeled, leading to re-sensitization of PTX therapy. Shikonin (SHK), a naturally sourced naphthoquinone compound, is a classic PKM2 inhibitor with potent anticancer activity³⁰. In our study, an albumin-based nanoparticulate system was developed for codelivery of SHK and PTX (S/P NP) for synergistic therapy. Albumin is a main nutritional source for tumors, which primarily targets the albumin-binding proteins (such as SPARC) that are overexpressed in tumor cells and tumor vasculature, thus serving as an ideal drug carrier^31-34.

2.　Materials and methods

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2.1.　 Materials

Shikonin (SHK) was purchased from Woge Dongfang Biotechnology Co., Ltd. (Beijing, China). Paclitaxel (PTX) was purchased from Shaoyuan Biotechnology Co., Ltd. (Shanghai, China). Lecithin was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) was purchased from Aladdin Bio-chem technology Co., Ltd. (Shanghai, China). RPMI-1640 medium, fetal bovine serum (FBS) were purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). Cell Counting Kit-8 (CCK8) was purchased from GLPBIO (Montclair, USA). Cy5.5 was purchased from MedChemExpress LLC (NJ, USA). Collagenase IV and hyaluronidase were purchased from Biosharp (Beijing, China). The Total RNA Isolation Kit was purchased from FOREGENE (Chengdu, China). PKM2 antibody, SOX2 antibody, OCT4 antibody, vimentin antibody, E-cadherin antibody, β-tubulin antibody, CDH5 antibody were purchased from Abmart (Shanghai, China). β-Catenin antibody, GAPDH antibody were purchased from Abcam (Cambridge, UK). VEGFA antibody was purchased from Abclonal (Wuhan, China). Calreticulin monoclonal antibody (Alexa Fluor 488 conjugate), Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody, HRP-conjugated Affinipure goat anti-mouse IgG, HRP-conjugated Affinipure goat anti-rabbit IgG were purchased from Proteintech (Wuhan, China). Recombinant murine macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-γ), and interleukin-4 (IL-4) were obtained from Peprotech (NJ, USA). cDNA synthesis SuperMix kit, qPCR SYBR® Green Master Mix, Matrigel Basement Membrane Matrix were purchased from Yeasen Biotechnology Co., Ltd. (Shanghai, China). Mouse HMGB-1 ELISA Kit was purchased from Chenglin Huihuang Biotechnology Co., Ltd. (Beijing, China). ATP Assay Kit was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). CD45 antibody, CD11b antibody, Ly6G antibody, Ly6C antibody, CD3e antibody, CD8a antibody, CD62L antibody, CD86 antibody, CD11c antibody, F4/80 antibody, CD80 antibody, CD4 antibody, CD25 antibody, Granzyme B antibody, IFN-γ antibody were purchased from Biolegend (San Diego, CA, USA). MHC II antibody, CD206 antibody, CD44 antibody, CD24 antibody were purchased from Elabscience (Wuhan, China).

2.2.　 Cell lines

The murine breast cancer cells (4T1) and the human breast cancer cells (MDA-MB-231) were obtained from The Cell Bank of the Chinese Academy of Sciences. 4T1 cells and MDA-MB-231 cells were cultured in RPMI-1640 medium with 10% FBS and 1% penicillin-streptomycin (Dalian Meilun Biotechnology, Dalian, China). All cells were cultured at 37 ℃ in a humidified incubator (Thermo Scientific, 3111, Waltham, USA) with 5% CO₂.

2.3.　 Animals

All the animal experimental procedures were complied with the institutional ethical guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of Zhongshan Institute for Drug Discovery. Female BALB/c mice (6–8 weeks old) were purchased from the Zhuhai BesTest Bio-Tech Co., Ltd. (Zhuhai, China). Animals were kept in a facility that was pathogen-free, had free access to food and water, and maintained a 12-h light/dark cycle.

2.4.　 Orthotopic breast cancer model

The abdomens of the mice were shaved. The mice were anesthetized by isoflurane. Approximately 1 × 10⁶ 4T1 cells in 100 μL of PBS were injected orthotopically into BALB/c mouse mammary fat pad through insulin needle, and a cotton swab was then applied to the injection site to prevent leakage of tumor cells.

2.5.　 Orthotopic breast cancer lung metastasis model

When the orthotopic tumor volume reached about 50 mm³, approximately 5 × 10⁴ 4T1 cells in 100 μL of PBS were intravenously injected into each mouse to develop a lung metastasis model.

2.6.　 Database

Based on various databases, the bioinformatics analysis was carried out. Differential expression of PKM and β-Catenin in normal tissues and breast cancer tumor samples were analyzed based on Breast Cancer Gene-Expression Miner (https://bcgenex.ico.unicancer.fr/BC-GEM/GEM-Chronologie.php), TIMER2.0 database (http://timer.cistrome.org/) and GEPIA (http://gepia.cancer-pku.cn/) database. Based on BEST database (https://rookieutopia.com/app_direct/BEST/) and Kaplan–Meier Plotter database (https://kmplot.com/analysis/), the relationship between PKM and β-Catenin expression and prognosis in breast cancer (BRCA) was analyzed. PKM and β-Catenin expression in various cell types of tumor tissues was analyzed based on the TISCH database (http://tisch.comp-genomics.org/), specifically in EMTAB8107, GSE110686, GSE114727, GSE148673, GSE161529, and GSE138536 subsets. The correlation of PKM and β-Catenin expression in BRCA with macrophages was analyzed based on the TIMER2.0 database. Analysis of PKM and β-Catenin expression and immune infiltration in the immune microenvironment of BRCA was performed based on the TCGA database (https://www.cancer.gov/ccg/research/genome-sequencing/tcga).

2.7.　 Bone marrow-derived MΦ (BMDM) and dendritic cell (BMDC) induction

BALB/c female mice were euthanized and the femur and tibia were then collected, with the muscle removed from the bones. The bones were subsequently immersed in 75% alcohol solution for 10 min and then washed twice with pre-cooled PBS for 5 min each time. The epiphyses were cut at both ends, followed by aspiration of PBS with a syringe to flush the marrow from the bone cavity. The bone marrow cell suspension was transferred to a 15-mL tube and then centrifuged at 2000 rpm for 5 min using a centrifuge (Xiangyi centrifuge Co., Ltd., Hunan, China). Two mililiters of erythrocyte lysate were added to the bone marrow cell precipitate, followed by incubation at room temperature for 5 min, and then centrifuged at 1000 rpm for 5 min using a centrifuge (Xiangyi Centrifuge Co., Ltd.). The bone marrow cells were resuspended in DMEM medium (20% FBS) and cultured for 5 days, with the addition of 20 ng/mL M-CSF to stimulate BMDM differentiation. After that, the medium was changed to 40 ng/mL IL-4-containing DMEM complete media for the induction of BMDM polarization towards M2 by 24-h incubation. To induce BMDM polarization towards M1, 100 ng/mL LPS and 20 ng/mL IFN-γ were added to DMEM complete media and incubated for 24 h. To induce BMDC differentiation, the bone marrow cells were resuspended in DMEM medium (20% FBS) for 4 days, with the addition of 20 ng/mL GM-CSF and 10 ng/mL IL-4. During this period, the semi-adherent cell population was retained by half-exchanging the medium every 48 h.

2.8.　 The optimum ratio of drugs combination

The optimal drug combination ratio was established by performing CCK8 assay. 4T1 cells were seeded to a 96-well plate (5 × 10³ cells/well) and placed in incubator for incubation for 12 h. Then treated with SHK and PTX free or in a series of molar ratios for 24 h. Following the addition of 100 μL of serum-free media containing 10% CCK8 at 37 ℃ for another 2 h, each well's absorbance (450 nm wavelength) was measured using a microplate reader. Graphpad Prism 8.0.1 (San Diego, CA, USA) was used to determine the IC₅₀ values. The combination index (CI) of SHK&PTX was calculated by CompuSyn (CA, USA) software.

Proportions were screened for CI values < 1 at each combined dose. The screened ratios were subsequently administered to mice with orthotopic breast cancer. Briefly, the established orthotopic breast cancer mice were randomly divided into five groups (3 mice per group), and received saline (control), SHK (5 mg/kg), PTX (10 mg/kg), SHK + PTX (5 mg/kg+ 1 mg/kg, equivalent to at a molar ratio of 1:0.2), SHK + PTX (5 mg/kg + 10 mg/kg, equivalent to at a molar ratio of 1:2), via intratumoral injection (q.o.d., 5 times). Tumor volume was closely monitored to evaluate therapeutic efficacy. Mice were deemed dead in accordance with animal welfare regulations, and euthanasia occurred in one of the following situations. Animals were extremely lethargic, with a weight loss of more than 20% compared to the control, or with a tumor size reached 2000 mm³, or with severe ulceration.

2.9.　 Preparation and characterization of the albumin nanoparticles (NPs)

The hybrid NPs were created using the high-speed dispersion emulsification-homogenization technique. Briefly, PTX and lecithin were dissolved in absolute ethanol, and SHK was dissolved in DMSO to form the oil phase. Albumin was dissolved in water to constitute the aqueous phase. Once the two phases were combined, the high-speed dispersion machine (IKA, T 25 Digital, Staufen, Germany) was used to generate the primary emulsion. Subsequently, after homogenizing the primary emulsion for ten cycles at a pressure of 12,000 psi, the BSA nanoparticle suspension was prepared using a high-pressure homogenizer (Avestin Inc., EmulsiFlex-B15, Ottawa, Canada). The NPs were purified by a Sephadex™ G-50 (Beijing Solarbio Science & Technology Co., Ltd., S8151, Beijing, China) column.

The dynamic light scattering device (Malvern, Zetasizer Pro, Malvern City, UK) was used to detect the particle size and zeta potential. The particle size change in PBS (pH 7.4) containing 10% neonatal bovine serum and in water at 4 ℃ was used to gauge the stability of the NPs. HPLC (Agilent, 1260 Infinity II, Santa Clara, USA) was used to assess the drug-loading capacity and encapsulation efficiency, and calculated by the formulas as shown in Eqs. (1) and (2):

(1)

(2)

The HPLC mobile phase of SHK was composed of methanol (65%) and ultra-pure water (35%), and the HPLC mobile phase of PTX was composed of acetonitrile (40%), methanol (25%), and ultra-pure water (35%). Both of them were at a flow rate of 1.0 mL/min. SHK and PTX were isocratic elution by a C18 column (250 mm × 4.6 mm, FLM Scientific Instrument Co. Ltd., Guangzhou, China) at room temperature. The wavelength was 227 nm for PTX and 216 nm for SHK. A traditional dialysis membrane (MWCO 14 kDa) method was used to assess the in vitro drug release in PBS (pH 7.4) after 0.5% (w/v) SDS was added and the mixture was shaken at 37 ℃ by a thermostatic shaker (Shanghai HerryTech Co., Ltd., Shanghai, China). Released drugs were also measured employing HPLC (Agilent), as previously mentioned, and the cumulative release was computed.

2.10.　 Cellular uptake efficiency

4T1 cells were seeded to a 12-well plate (1 × 10⁵ cells/well) and placed in incubator to continue incubation for 12 h. The cells were then co-cultured with coumarin 6-labeled nanoparticles. After 2-h co-incubation the fluorescent images were obtained by a fluorescence microscope (Keyence Co., Ltd. BZ-X800LE, Osaka, Japan) and the intracellular fluorescence was detected by a flow cytometer (NovoCyte Quanteon VBYR, Agilent, California, USA).

2.11.　 In vitro cytotoxicity test

The cytotoxicity of the free drugs and S/P NP was conducted by the CCK8 method according to the previous description (section 2.8). SHK, PTX, SHK&PTX, and S/P NP were applied to the 4T1 cells in a series of concentration gradients, respectively. Graphpad Prism 8.0.1 was employed to estimate the half-maximal inhibitory concentration (IC₅₀).

2.12.　 ICD, lactate assay and activate immune-related cells

The 4T1 cells were seeded in a 6-well plate for 12 h and then treated with PBS, SHK (1.0 μmol/L), PTX (0.2 μmol/L), SHK&PTX (0.5 μmol/L+0.1 μmol/L), or S/P NP (equal dose with the combination therapy) for 10 h. The supernatant was collected by ELISA kit for HMGB1 detection according to the manufacturer instruction (Chenglin, Beijing, China). The 4T1 cells were collected and co-incubated with calreticulin (CRT) antibody-Alexa Fluor 488 conjugate and corallite 488-conjugated goat anti-rabbit IgG (H + L) for flow cytometry analysis, and the ICD-cells were characterized by FITC positive cells. To quantify ATP level in 4T1 cells, the cells were processed in accordance with the manufacturer's instructions (Beyotime, Shanghai, China). A lactic acid assay kit was utilized to determine the concentrations of lactate in the 4T1 cell culture medium (Jiancheng Bioengineering Co., Ltd., Nanjing, China).

The BMDC cells were induced differentiation as described above. 4T1 cells were seeded in a 12-well plate for 12 h and then treated with PBS, SHK (1.0 μmol/L), PTX (0.2 μmol/L), SHK&PTX (0.5 μmol/L+0.1 μmol/L), or S/P NP (equal dose with the combination therapy) for 10 h. The BMDC cells were co-cultured with the drug-treated 4T1 cells (also its supernatant) in a Transwell device (an aperture of 0.4 μm, Corning, USA) for 24 h. LPS (200 ng/mL) treatment was added as the positive control. CD11c, MHC II, CD80, and CD86 were applied to characterize the mature BMDC through flow cytometry analysis.

2.13.　 Combination therapy regulation of macrophages

The bone marrow cells were collected, cultured, and induced as mentioned above. After 24-h stimulation, the M2-type BMDM cells were treated with PBS, SHK (1.0 μmol/L), PTX (0.2 μmol/L), SHK&PTX (0.5 μmol/L+0.1 μmol/L), or S/P NP (equal dose with the combination therapy) for 24 h. M1-type BMDM cells were used as a control group. F4/80, CD86, and CD206 were applied to characterize the phenotype of BMDM through flow cytometry analysis.

2.14.　 Scratch assay

The 4T1 cells were seeded in a 12-well plate with 8 × 10⁵ cells/well for 12 h and scratched with a 200 μL pipette tip. The cells were then washed with sterile PBS and treated with PBS, SHK (1.0 μmol/L), PTX (0.2 μmol/L), SHK&PTX (0.5 μmol/L+0.1 μmol/L), or S/P NP (equal dose with the combination therapy) for 24 h in the serum-free 1640 medium. The same site was selected and photographed using a microscope (Olympus, IX73P1F, Tokyo, Japan) at 0 and 24 h after drug administration, respectively.

2.15.　 Matrigel invasion assay

The matrigel invasion assay was evaluated using a Transwell device (an aperture of 8.0 μm, Corning, USA). Firstly, the upper chamber in a Transwell plate was coated with 100 μL matrigel (matrigel:serum-free 1640 medium 1:8), and then the plate was incubated in a 37 ℃ incubator for 1 h. After that, 75 μL 4T1 cells (4 × 10⁵ cells/well) with 75 μL drugs (PBS, terminal concentration SHK 1.0 μmol/L, PTX 0.2 μmol/L, SHK&PTX 0.5 μmol/L+0.1 μmol/L, or S/P NP equal dose with the combination therapy) and with 5% FBS were seeded into the upper chamber. The lower chamber was filled with 600 µL 1640 medium containing 20% FBS. After 24 h, the upper chamber was washed twice with PBS, followed by formaldehyde fixation for 30 min. The invasion cells in the bottom of the upper chamber were dyed with 0.5% purple crystal and washed with PBS. The image figures were obtained by an inverted microscope.

2.16.　 Anti-angiogenesis assay

The anti-angiogenesis was evaluated with lactate addition. Briefly, 100 μL of matrigel per well was added to a pre-cooled 96-well plate, and then the plate was incubated in a 37 ℃ incubator for 1 h. After that, 40 μL 4T1 cells (3 × 10⁴ cells/well) were seeded into the plate containing 10 μL lactate (final concentration of 10 mmol/L), and 50 μL drug (PBS, terminal concentration SHK 1.0 μmol/L, PTX 0.2 μmol/L, SHK&PTX 0.5 μmol/L+0.1 μmol/L, or S/P NP equal dose with the combination therapy). The 4T1 cell tube formation was observed by a microscope in the bright field.

2.17.　 Tumorsphere destruction

The 4T1 cells were seeded in a 6-well plate for 12 h and then treated with PBS, SHK (1.0 μmol/L), PTX (0.2 μmol/L), SHK&PTX (0.5 μmol/L+0.1 μmol/L), or S/P NP (equal dose with the combination therapy) for 24 h. The serum-free suspension culture was utilized to foster the tumorsphere of 4T1 cells. After trypsinization, the drug-treated adherent cells were cultivated in DMEM/F12 media with the addition of B27 supplement (1 ×), insulin (5 μg/mL), epidermal growth factor (20 ng/mL), basic fibroblast growth factor (20 ng/mL) and then seeded in the 6-well ultra-low attachment plate at 37 ℃ in a humidified incubator with 5% CO₂, respectively. After 8–12 days, the growth of tumorspheres was observed under a microscope and photographed, and the diameter larger than 50 μm was counted and the average diameter was calculated.

2.18.　 Flow cytometric detection of cell stemness

The cells were washed twice with phosphate-buffered saline (PBS) and then harvested using 0.05% trypsin/0.025% EDTA. The detached cells were resuspended in 1% BSA at a concentration of 10⁶ cells/100 μL. Combinations of CD44 and CD24 antibodies were added to the cell suspension at the concentrations recommended by the manufacturer's instructions and incubated at 4 ℃ in the dark for 40 min. The labeled cells were then washed with PBS and analyzed using a flow cytometer.

2.19.　 In vivo distribution

The free Cy5.5 and Cy5.5-labeled S/P NP were intravenously administered into the established mice bearing orthotopic breast tumors, and the IVIS imaging system (PerkinElmer Instruments Co., Ltd., Spectrum, MA, USA) was utilized to monitor the biodistribution of free Cy5.5 and Cy5.5-labeled S/P NP. The mice were humanely euthanized 24 h after injection, and the tumor, heart, liver, spleen, lung, and kidney were collected for ex vivo imaging. The distribution of Cy5.5 in tumor tissues was measured by a flow cytometer.

2.20.　 Establishment of orthotopic model of breast cancer

(3)

All the mice were humanely euthanized to collect the tumor tissues and were weighted to calculate the tumor inhibition ratio (%) according to the formula as shown in Eq. (4):

(4)

W is the tumor weight of the free drug and S/P NP-treated group. W₀ is the tumor weight of the saline-treated group. In order to prepare the single-cell solution, the tumor tissues were physically cut and processed using collagenase IV and hyaluronidase. After incubation with antibodies (CD45 antibody, CD11b antibody, Ly6G antibody, Ly6C antibody, CD3e antibody, CD8a antibody, CD62L antibody, CD86 antibody, CD11c antibody, F4/80 antibody, CD80 antibody, CD4 antibody, CD25 antibody, Granzyme B antibody, IFN-γ antibody, MHC II antibody, CD206 antibody, CD44 antibody), the immune cell subsets in the tumor tissues were analyzed by a flow cytometer.

2.21.　 Establishment of lung metastasis model of orthotopic breast cancer

The 4T1 orthotopic tumor mice were randomly assigned into three groups (6 mice per group). The mice were treated with saline (control), SHK&PTX (2 mg/kg+1.25 mg/kg, equal to SHK:PTX = 5:1, mol/mol), or S/P NP (equivalent to a dose of free drug) via tail vein injection (a treatment every 4 days, 6 times). The body weight and the tumor growth were monitored throughout the experiment. The tumor volume was calculated as described above. The blood samples were collected from the animals and plasma was isolated via centrifugation for biochemical analysis of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine (CRE). All the mice were euthanized to collect the tumor tissues and the major organs (heart, liver, spleen, lung, and kidney). In order to further evaluate the system toxicities and visualize the number of pulmonary metastatic nodules, the organs were preserved with 4% paraformaldehyde for histological examination. The tumor tissues were homogenized. The expression levels of the target proteins were determined by Western blot. The PAS/CD31 staining was conducted to estimate the VM.

2.22.　 Statistical analysis

All data are represented as mean ± standard deviation (SD, n ≥ 3) with statistical analysis. The semi-quantitative analysis of Western blot bands was conducted by ImageJ software (NIH, USA). Statistical significance was performed by Student's t-test or one-way ANOVA (∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001).

3.　Results

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3.1.　 The optimum ratio of drug combination

The anti-proliferative effects on the 4T1 cells and the MDA-MB-231 cells were measured to optimize the combination ratio of SHK and PTX. The combination index (CI) of SHK and PTX was utilized to evaluate the combination therapy efficacy, and CI < 1 indicates the synergistic effect³⁵. The synergistic ratio of SHK and PTX in the 4T1 cells was 1:0.2 and 1:2 (mol/mol), with CIs <1 at all doses and the lowest CI was up to 0.21 (SHK:PTX = 1:0.2) and 0.22 (SHK:PTX = 1:2), respectively (Fig. 1A, Supporting Information Fig. S1). Moreover, the most effective synergistic ratio of SHK and PTX in the MDA-MB-231 cells was 1:0.2 (mol/mol) with the lowest CI was up to 0.18 (Supporting Information Fig. S2). Furthermore, in an orthotopic 4T1 BC model, both synergistic ratio treatments achieved a significantly higher tumor inhibition ability compared to the SHK or PTX alone groups (Fig. 1B and C). However, the body weight declined remarkably in the group (SHK:PTX = 1:2) (Supporting Information Fig. S3), suggesting the potential side toxicity. By contrast, the animals maintained their body weight during the treatment of SHK:PTX = 1:0.2, indicating the biosafety. Therefore, a synergistic ratio of 1:0.2 (SHK:PTX) was selected for further studies.

3.2.　 Bioinformatics analysis of PKM2 and β-Catenin

PKM2 is highly expressed in various cancers and closely associated with reduced efficacy of chemotherapeutics³⁶. Therefore, via suppression of PKM2, we hypothesized that SHK could enhance PTX efficacy. First of all, we performed a bioinformatics analysis, which demonstrated that PKM2 expression is higher in the breast tumors and TNBC than in the normal areas (Fig. 1D, Supporting Information Figs. S4 and S5) and overall survival (OS) is much longer in cases of low PKM2 than in patients with high PKM2 both in BC and TNBC patients (Fig. 1E, Supporting Information Fig. S6). Recurrence free survival (RFS) is negatively correlative to PKM2 expression in BC patients, too (Supporting Information Fig. S7). Furthermore, by examining the results of scRNA-seq in multiple datasets, PKM2 was found to be expressed in several immune cells, with macrophages exhibiting the highest levels of PKM2 in BC patients (Supporting Information Fig. S8). Moreover, intratumoral PKM2 expression was positively connected with the increased infiltration of macrophages in both BC and TNBC tumor microenvironment (TME) and it also increased with the purity of the tumor cells in the tumors (Fig. 1F and G, Supporting Information Fig. S9). In addition, PKM2 expression was positively related to the increased infiltration of Tregs in BC patients (Fig. 1G).

Notably, PKM2 plays a dual role, serving as a glycolytic enzyme in cytoplasm in a tetramer form and as a non-metabolizing enzyme via entering the nucleus in a dimer form and triggering the transcription factors²⁷. As a case in point, the dimer PKM2 triggers β-Catenin transactivation (Fig. 1H), which subsequently promotes the stemness of cancer cells³⁷, VM formation³⁸, and poor chemotherapy efficacy³⁹. The qPCR and WB results verified that SHK effectively inhibited the mRNA and protein expression of PKM2 in a dose-dependent manner (Fig. 1I‒K). Similarly, we also conducted a bioinformatics analysis of β-Catenin, which was higher both in the BC and TNBC tissues than that in normal tissues (Supporting Information Figs. S10 and S11) and negatively correlative to OS (Supporting Information Figs. S12 and S13). Moreover, β-Catenin was also expressed on the intratumoral macrophages, endothelial cells, and fibroblasts (Supporting Information Fig. S14). Furthermore, β-Catenin high expression was positively correlated with infiltration of naïve B cells, M2 macrophages, and resting DCs and memory CD4⁺ T cells in TME (Supporting Information Fig. S15).

More importantly, the wet experiment also demonstrated that SHK significantly suppressed β-Catenin in a dose-dependent manner (Fig. 1J, L and M). Taken together, PKM2/β-Catenin might be a crucial signaling pathway for SHK in enhancing the anticancer effect of PTX in BC and TNBC.

3.3.　 Characterization, cellular studies and in vivo imaging performance of the nanoparticles

SHK and PTX were successfully encapsulated in albumin nanoparticles (S/P NP). The S/P NP showed a spherical morphology and the particle size was about 133 nm (PDI<0.20) (Fig. 2A, Supporting Information Table S1). The S/P NP displayed a negative zeta potential about −26 mV (Fig. 2B and Table S1). The SHK- or PTX-loading capacity was 1.39% and 0.89%, respectively, at a drug loading ratio was about 5:1 (SHK:PTX, mol/mol, equal to SHK:PTX 1.6:1, mg/mg) (Table S1). The S/P NP remained stable at 4 ℃ (Supporting Information Fig. S16) with only a slight increase in size when placed in serum-containing medium, likely due to interactions with blood proteins that form protein coronas (Fig. 2C). The S/P NP showed a drug release pattern at a ratio around 5:1 (SHK:PTX, mol/mol) (Fig. 2D and E).

The combination of SHK/PTX (5:1, mol/mol) displayed a synergistic effect with an IC₅₀ of 0.70 μmol/L (indicated by SHK), compared to 2.18 μmol/L for SHK, while the 4T1 cells showed substantial resistance to PTX (Fig. 1A). The S/P NP showed the strongest inhibitory effect with an IC₅₀ of 0.11 μmol/L (Fig. 2F).

The Coumarin-6-labeled S/P NP exhibited an efficient internalization in 4T1 cells with a 1.8-fold higher efficiency than the free dye (Fig. 2G‒I). Following the approach used in other studies, we chose the Cy5.5 fluorescent dye to assess the targeting ability of the S/P NP^32,40-42. In vivo imaging results showed that the Cy5.5-labeled NPs efficiently accumulated in the tumor after 6 h and reached a maximum at 8 h (Fig. 2J, Supporting Information Fig. S17). The ex vivo imaging showed that intratumor accumulation of the NP group was 3-fold higher than the free dye group (Fig. 2K and L), while these two groups showed a similar distribution in the normal organs (Fig. 2L, Supporting Information Fig. S18). Flow cytometric assay also revealed that the intracellular fluorescence intensity was also higher in the NP group (Fig. 2M and N).

3.4.　 Regulation of the stemness and vasculogenic mimicry (VM) in 4T1 cells

The stemness of tumor cells is an important indicator for assessing the characteristics of self-renewal, differentiation, and proliferation of CSCs⁴³. Our results showed that the S/P NP had the highest inhibition rates against invasion and migration of 4T1 cells (Fig. 3A‒C). The S/P NP efficiently suppressed the formation of tumor spheroids (Fig. 3D‒F). The inhibitory effect of S/P NP on tumor stemness is due to the effects of SHK (Fig. 3G). Moreover, the S/P NP-treated 4T1 cells showed downregulation in the expression of the typical stem-property associated transcription factors including OCT4, NANOG, and SOX2 (Fig. 3H‒J). The flow cytometry revealed that after S/P NP treatment, the proportion of high stem-property cell (CD44⁺/CD24^‒) decreased (Fig. 3K and L). Epithelial–mesenchymal transition (EMT) is an index to evaluate the stem-property of cancer cells⁴⁴. Our results showed that the S/P NP treatment led to upregulation of E-Cadherin and downregulation of Vimentin in 4T1 cells, demonstrating the strong ability to reverse EMT (Fig. 3H‒J). These results suggested that the S/P NP significantly reduced the stem-property of 4T1 cells. Furthermore, we also detected the protein and mRNA expression of PKM2 and β-Catenin, the S/P NP exhibited the remarkable down-regulation of PKM2 and β-Catenin (Fig. 3H‒J).

Distinct from tumor angiogenesis, VM refers to a vasculature network formed by aggressive, plastic tumor cells independent of endothelial cells, and provides a blood supply for tumor cells¹⁶. CSCs have a higher potential for developing into VM because of their propensity for self-renewal and multidirectional differentiation²². Our results revealed that the S/P NP treatment significantly inhibited the tube formation of 4T1 cells (Supporting Information Fig. S19). Lactate is a primary end product of glycolysis, contributing to the acidic TME⁴⁵. Lactate can facilitate the survival of tumor cells by acting as an energy source, while also promoting the formation of vasculature network by tumor cells⁴⁶. After adding lactate, tube formation rate was effectively upregulated (up to 1.3-fold of non-lactate added group), because lactate is a potent angiogenesis inducer. The S/P NP significantly weakened the ability of tube formation in 4T1 cells (Fig. 4A‒C). It was attributed to the suppression of lactate production (Fig. 4D and E). Notably, the S/P NP downregulated the mRNA and protein of VM-associated transcription factors including CDH5 and VEGFA (Fig. 4F‒J). Taken together, these results indicated that the S/P NP efficiently inhibited the formation of vasculature network of the tumor cells via blocking the CDH5/VEGFA pathway and reducing lactate production, thus yielding anti-VM effect.

3.5.　 Immunogenic cell death (ICD) induction and immunoactivation

The ICD effect triggers DAMPs that are characterized by calreticulin (CRT) eversion, the release of high mobility group box 1 (HMGB-1) and adenosine triphosphate (ATP)⁴⁷. Our results demonstrated the ICD effect induced by the S/P NP, showing CRT eversion by flow cytometry (Fig. 5A and B), HMGB-1 and ATP release to supernatant in the 4T1 cells (Fig. 5C and D).

Tumor-associated macrophages (TAMs) are a promising target for BC therapy⁴⁸. The S/P NP dramatically reduced the proportion of CD206⁺ MΦ (protumor M2-like subset) (Fig. 5E and F). DCs connect innate immunity and adaptive immunity by presenting antigens to naïve T cells and thereby triggering T cell immunity⁴⁹. In a co-culture system (Fig. 5G), Our results showed that the S/P NP-treated 4T1 cells promoted the maturation of BMDCs, with the upregulation of co-stimulatory molecules (CD80, CD86), indicating the induction of ICD effect in 4T1 cells (Fig. 5H and I).

3.6.　 Treatment efficacy in a mouse model with orthotopic BC

In an orthotopic 4T1 mouse model (Fig. 6A), the S/P NP effectively arrested tumor growth with an inhibition rate of 80%, compared to 45% of free SHK/PTX (Fig. 6B‒D, Supporting Information Fig. S20). The body weight change of the S/P NP group was no significantly different from the control group (Fig. 6E and F).

To profile the remodeled TME, the intratumoral immune cells were detected after treatment. The S/P NP treatment significantly increased the proportion of anti-cancer M1Φ but reducing protumor M2Φ (Fig. 6G and H, Supporting Information Fig. S21). Besides, the percentage of matured DCs was highest in the S/P NP group (Fig. 6I, Supporting Information Fig. S22), while the population of protumoral PMN-MDSCs in the S/P NP group decreased to about 47% of the saline group (Fig. 6J, Supporting Information Fig. S23). These results indicated that the S/P NP treatment effectively relieved the immunosuppression of TME by reprogramming macrophages, promoting DCs maturation, and suppressing PMN-MDSCs. Moreover, the S/P NP treatment significantly promoted memory T cells (CD62L⁺ CD44⁺) (Fig. 6K, Supporting Information Fig. S24), suggesting the induction of long-term immune responses. The infiltration of CD8⁺ T cells was increased (Fig. 6L, Supporting Information Figs. S25A and S25B), and the IFN-γ⁺/Granzyme B⁺ CD8⁺ T cell subsets were also significantly expanded (Fig. 6M and N, Fig. S25C and S25D). In addition, we detected the percentage of Treg cells, however, there was no statistical difference among the groups (Supporting Information Fig. S26). The discrepancy could be due to the difference between animal model and human, but further investigation should be carried out for better understanding this. These results confirmed that the S/P NP treatment effectively relieved the immunosuppression of TME and activated T cells immunity against breast tumor.

3.7.　 Therapeutic efficacy in a mouse model with lung metastasis of orthotopic BC

There are common cases of lung metastasis of advanced BC. The therapeutic efficacy of the S/P NP was evaluated in a lung metastasis mouse model bearing orthotopic 4T1 (Fig. 7A). The S/P NP not only successfully inhibited the progression of primary breast tumor (Fig. 7B and C), but also effectively minimized the lung metastasis nodules with an inhibition rate of 86%, compared to 50% of the free SHK/PTX group (Fig. 7D‒F, Supporting Information Fig. S27). The S/P NP significantly downregulated the expression levels of PKM2, β-Catenin, the stem-property markers SOX2, OCT4, NANOG, VM formation related biotargets CDH5, VEGFA and EMT associated marker Vimentin, while upregulated EMT associated marker E-cadherin (Fig. 7G). Crucially, the PAS/CD31 staining results showed that the S/P NP exhibited a potent inhibitory capacity against VM formation, with the remarkably reduced proportion of CD31^‒/PAS⁺ (Fig. 7H). These results suggested the regulation of the crosstalk among stem-property and VM contributed to the remission of lung metastasis.

The S/P NP treatment showed a good biosafety in terms of body weight change (Supporting Information Fig. S28). There were also no obvious lesions observed in the H&E staining sections of the organs (Supporting Information Fig. S29). Moreover, no statistically significant difference in ALT, AST, and CRE levels was found among the groups (Supporting Information Fig. S30).

4.　Discussion

Less

TNBC patients suffer from poor prognosis, with a median survival time of approximately 4.2 years after diagnosis⁵⁰. PTX is ready to develop drug resistance in TNBC treatment. Hence its combination with other drugs has emerged as a recommended option^6,7. SHK, one of the main components of Lithospermum. erythrorhizon, has been widely investigated in cancer therapy as a PKM2 inhibitor⁵¹. In our previous report, the glycolysis inhibition effect of SHK was demonstrated, including the down-regulation of PKM2, as well as reduction of ATP and lactate^42,52,53. Co-delivery systems are commonly employed to overcome the poor efficacy of chemotherapy and improve the in vivo delivery of hydrophobic drugs. For example, a co-delivery system of quercetin and doxorubicin, modified with the KC26 peptide, facilitates deep penetration into tumors and strongly inhibits breast tumor growth⁵⁴. In this work, we developed an albumin delivery system (S/P NP) to co-load SHK and PTX in an optimized ratio of 5:1, aiming to enhance the anti-tumor effect of PTX in TNBC.

PKM2 is associated with poor prognosis for patients with cancers and is highly expressed in various malignancies⁵⁵, which is in agreement with our results of bioinformatics analysis. PKM2 expression levels were significantly higher in PTX-resistant cancer cells. The upregulated PKM2 contributed to drug resistance by regulating cellular energy metabolism and influencing the stress response of tumor cells⁵⁶. Moreover, PKM2 can affect cellular sensitivity to PTX by modulating mitochondrial function and reactive oxygen species (ROS) production⁵⁷. Therefore, this SHK-based therapeutic strategy can take a dual action for simultaneously inhibiting both glycolysis and β-Catenin signaling pathway, thus synergizing PTX treatment against TNBC by decreasing the stem-property and VM formation, remodeling the TME.

One of the major culprits for unsatisfied outcomes of PTX accounts for the stem-property of TNBC cells which is regulated by the activation of β-Catenin⁸. Meanwhile, the stemness-associated EMT not only causes drug resistance but also promotes tumor metastasis^58,59. Another mechanism for the ineffectiveness of PTX therapy is VM. There is a correlation between VM and CSCs, and some tumor vessels may originate from the tumor cells through vasculogenesis⁶⁰, in which CSCs may act as progenitors of tumor stromal components like tumor vasculogenic stem cells⁶¹. Furthermore, in the perivascular environment, CSCs primordially grow in VM niches⁶², where they transdifferentiate into endothelial-like cells in VM formation. Our results demonstrated that the S/P NP successfully reversed the stem-property and EMT, as well as suppressed VM formation. As a result, the S/P NP significantly enhanced the treatment efficacy of PTX against TNBC and lung metastasis.

TNBC is a typical “cold tumor” with a strong immunosuppressive microenvironment⁶³. CSCs are preferentially dispersed in niches with typical TME characteristics⁶⁴. The interaction between CSCs and immune cells in TME often leads to unproductive therapy outcomes⁶⁵. Therefore, targeting CSCs and VM can remodel the tumor immune microenvironment. Our results showed that the S/P NP treatment decreased M2Φ and MDSCs, but promoted M1Φ, matured DCs, CTLs, and memory T cells.

5.　Conclusions

Less

In this work, we developed a therapeutic strategy using the S/P NP for targeting the stem-property and VM formation by inhibiting PKM2. This codelivery system enhanced the tumor drug accumulation. Such treatment efficiently arrested the primary tumor growth of TNBC and suppressed lung metastasis.

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Appendix

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Year 2025 volume 15 Issue 6

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doi: 10.1016/j.apsb.2025.04.006

Receive Date：2024-12-15
Online Date：2026-04-03

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History

Received：2024-12-15
Revised：2025-02-20
Accepted：2025-02-22

Affiliations

^aDepartment of Oncology, the First Affiliated Hospital and the First Clinical School of Guangzhou University of Chinese Medicine, Guangzhou 510000, China

^bZhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan 528400, China

^dSchool of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China

^eDepartment of Pharmacy, Women's Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China

^fState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

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^* Corresponding authors.

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表12种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Figure 1 Optimization of drug combination and the high expression of PKM2 in BC. (A) The viability of 4T1 cells under the different combination ratios (SHK: PTX). The value in parentheses is IC₅₀ (μmol/L); (B) The dissected tumors at the endpoint under the different combination ratios (SHK: PTX); (C) The tumor volume curves during the treatment; (D) BC tissues overexpressed PKM2 mRNA compared to normal tissues in the GEPIA database; (E) Correlation analysis between the differential expression of PKM2 and the overall survival of patients with BC (Data collected from BEST database GSE58812 array); (F) The correlations between PKM2 gene expression and macrophage infiltration in BC in TIMER2.0 database; (G) The correlations between PKM2 gene expression and immune cell infiltration in BC in TCGA database; (H) Schematic mechanism of SHK enhanced the anti-breast cancer effect of PTX; (I, L) The regulatory effect of SHK on the expression of Pkm2 mRNA and β-catenin mRNA in 4T1 cells; The regulatory effect (J, K, M) of SHK on the expression of PKM2 and β-catenin proteins in 4T1 cells. Data are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 2 Characterization, cellular uptake and in vivo distribution of the nanoparticles. (A) The particle size distribution and the morphological observation with TEM, scale bar = 100 μm; (B) The zeta potential of S/P NP; (C) The stability of S/P NP in serum-containing PBS; (D) In vitro release profiles of SHK and PTX from the NP; (E) Cumulative drug release ratios; (F) The cytotoxicity test in 4T1 cells; (G) The inverted microscope image of uptake of S/P NP in 4T1 cells, scale bar = 50 μm; (H, I) The cellular uptake was detected by flow cytometry and statistical analysis of mean fluorescence intensity; (J) The tumors radiant efficiency in vivo at different time points; (K) The radiant efficiency semi-quantitative analysis in tumor tissue; (L) Ex vivo images of the tumor tissues and major organs (heart, liver, spleen, lung and kidney); (M, N) The Cy5.5⁺ proportion of the tumors was analyzed by flow cytometry and statistical analysis. NP: Nanoparticles. Data are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 3 Regulation of the stem-property in 4T1 cells. (A) Transwell analysis in 4T1 cells, scale bars = 200 μm; (B, C) The representative images of scratch assay in 4T1 cells and the statistical analysis of wound area, scale bar = 100 μm; (D) Representative images of the 4T1 tumorspheres on Day 12 after different treatments, scale bar = 50 μm; (E, F) The statistical analysis of number and diameter of tumorspheres; (G) Schematic mechanism of SHK inhibiting stem-property of 4T1 cells; (H) Western blot analysis of PKM2, β-Catenin, epithelial or mesenchymal markers, stem-property markers in 4T1 cells; Quantization of Western blot analysis (I) and the mRNA levels (J) of β-catenin, Pkm2, Vimentin, Oct4, Sox2, Nanog and E-cadherin in 4T1 cells; (K, L) The population of CD44⁺/CD24^‒ subtype in drug treated 4T1 cells and statistical analysis. Data are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns, not significant.

Figure 4 Regulation of the vasculogenic mimicry in 4T1 cells. (A, B) The representative images of tube formation after lactate treatment in 4T1 cells and the statistical analysis of total segments length, scale bar = 100 μm; (C) The statistical analysis of number of meshes; (D) Schematic mechanism of tumor cell vasculogenic mimicry; (E) Quantitative of exocellular lactate of 4T1 cells treated with nanoparticles; The mRNA levels of Cdh5 (F), Vegfa (G) in 4T1 cells; Western blot analysis (H) and quantization (I, J) for CDH5 and VEGFA proteins in 4T1 cells. Data are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns, not significant.

Figure 5 Remodeling of immune microenvironment. (A, B) Analysis of calreticulin (CRT) eversion in 4T1 cells by flow cytometry and statistical analysis; (C) Analysis of the released HMGB-1 of 4T1 cells by ELISA; (D) Quantitative of intracellular ATP of 4T1 cells treated with nanoparticles; (E, F) The population of M2 macrophages after treatment and statistical analysis; (G) Schematic of the ICD stimulated maturation of BMDC; (H, I) The population of matured DC after co-culturing with drug treated 4T1 cells and statistical analysis. Data are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns, not significant.

Figure 6 In vivo therapeutic efficacy of nanoparticles in a mouse model with orthotopic breast cancer. (A) Therapeutic schedule; (B) The tumor image, (C) tumor volume curves and (D) tumor weight, (E) Body weight curves; (F) Statistical analysis of body weight on the last day during treatment; (G) The ratio of the M1-like TAM (F4/80⁺ CD86⁺) subset; (H) The ratio of the M2-like TAM (F4/80⁺CD206⁺) subset; (I) The ratio of the matured DC (CD80⁺CD86⁺) subset; (J) The ratio of the PMN-MDSC (Ly6G⁺Ly6C⁻) subset; (K) The ratio of the memory T cells (CD62L⁺CD44⁺) subset; (L) The ratio of the CD8⁺ T cells (CD3⁺CD8⁺) subset; (M, N) The ratio of the cytotoxicity CD8⁺ T cells (IFN γ⁺/Granzyme B⁺) subset. Data are presented as mean ± SD (n = 3–6). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns, not significant.

Figure 7 In vivo therapeutic efficacy of nanoparticles in a mouse model with lung metastasis of orthotopic BC. (A) Therapeutic schedule; (B) The tumor volume variations and (C) tumor weight; (D, E) The image of lung nodules of groups and the statistical analysis; (F) Representative H&E staining images of the lungs after different treatments (The area circled by the red line represents breast cancer lung metastasis nodules), scale bar = 50 μm; (G) Western blot analysis and quantization of β-Catenin, PKM2, OCT4, SOX2, NANOG, CDH5, VEGFA, Vimentin and E-Cadherin in tumor tissues; (H) Representative CD31/PAS staining of tumor tissue after different treatments. CD31⁺ represents vascular endothelial cell tube formation. CD31^‒/PAS⁺ represents vasculogenic mimicry, scale bar = 50 μm. Data are presented as mean ± SD (n = 3–6). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

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