Cell surface patching via CXCR4-targeted nanothreads for cancer metastasis inhibition – Nature Communications

Animals and ethics statement

Female BALB/c mice (6–8 weeks, 20 ± 2 g) were provided by SPF Biotechnology Co., Ltd (Beijing, China). Mice were housed in a specific pathogen-free environment at 21 ± 1 °C and 60 ± 5% humidity, with a 12 h light-dark cycle. Mice were access to food and water free. All animal experiments were approved by the Institutional Animal Care and Ethics Committee of Sichuan University. All animal experiments were conducted in the Animal Laboratory of West China School of Pharmacy in Sichuan University (accreditation number: SYXK (Chuan) 2018-113). Female mice were chosen because the majority of metastatic breast cancers is seen in female patients. According to the guidelines of ethics committee, the maximal tumor size permitted was 1500 mm³. Mice were euthanized when the tumor burden exceeded this threshold.

Materials

CXCR4 binding sequence (LGASWHRPDKCCLGYQKRPLPA) was synthesized by Apeptide Co. Ltd. (Shanghai, China). Coiled motif 1 (CM1, CYGGKVSALKEKVSALKEEVSANKEKVSALKEKVSALKE), and coiled motif 2 (CM2, CYGGEVSALEKEVSALEKKNSALEKEVSALEKEVSALEK) were synthesized by Chinapeptides Co. Ltd. (Shanghai, China). Chlorin e6 (Ce6) was purchased from Meilunbio Co., Ltd. (Dalian, China). N-(2-hydroxypropyl) methacrylamide (HPMA) and N-(3-aminopropyl) methacrylamide (APMA) was purchased from Bide Pharmatech Ltd. (Shanghai, China). SulfoCy3 SE and SulfoCy5 SE were provided by Beijing Fluorescence Biotechnology Co. Ltd. ADM3100 (Plerixafor, Cat No.: M1898) and CCK-8 (Cell Counting Kit, Cat No.: M4839) were purchased from AbMole (USA). D-fluorescein potassium salt (Cat No.: E011306) was provided by Energy Chemical. Annexin V-FITC/PI Apoptosis detection Kit (Cat No.: 40302ES60) and Fluo-4 AM (Cat No.: 40704ES50) were provided by Yeasen (Shanghai, China). Enhanced BCA Protein Assay Kit (Cat No.: P0009) and Enhanced ATP Assay Kit (Cat No.: S0027) were provided by Beyotime Biotechnology (Shanghai, China). 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI, Cat No.: D8200), Red Blood Cell Lysis Buffer (Cat No.: R1010), Modified Hematoxylin-Eosin Stain Kit (Cat No.: G1121), Modified Masson’s Trichrome Stain Kit (Cat No.: G1346), and Modified Sirius Red Stain Kit (No Picric Acid) (Cat No.: GG1472) were provided by Beijing Solarbio Science & Technology Co., Ltd. Recombinant Murine SDF-1α (CXCL12) (Cat No.: 250-20 A) was purchased from PEPROTECH inc. Goat anti-mouse IgG H&L Alexa Fluor® 488 (Cat No.: ab150113) and goat anti-rabbit IgG H&L Alexa Fluor® 647 (Cat No.: ab150079) were provided by Abcam. Anti-CD8α-APC (Cat No.: 100711; clone: 53-6.7), anti-IFN-γ-PE (Cat No.: 505808; clone: XMG1.2), anti-CD45-PerCP/Cy5.5 (Cat No.: 103132; clone: 30-F11), anti-CD44-PE (Cat No.: 163610; clone: QA19A43), anti-CD62L-Percp/Cy5.5 (Cat No.: 161210; clone: 30-F11), anti-CXCR2-PE (Cat No.: 149304; clone: SA004G4), anti-CXCR5-PE (Cat No.: 145504; clone: L138D7), anti-CXCR7-PE (Cat No.: 331104; clone: 8F11-M16) were purchased from Biolegend. Anti-CD16/32 (Cat No.: 553142; clone: 2.4G2), anti-Foxp3-PE (Cat No.: 560408; clone: MF23), transcription factor buffer set (Cat No.: 562574) were provided by BD Biosciences. Anti-Vimentin Recombinant Rabbit Monoclonal Antibody (Cat No.: ET1610-39; clone: SC60-05), Anti-Vimentin Mouse Monoclonal Antibody (Cat No.: EM0401; clone: D4-B11), Anti-PI 3 Kinase p85 alpha Recombinant Rabbit Monoclonal Antibody (Cat No.: ET1608-70; clone: SU04-07), Anti-Fibronectin Mouse Monoclonal Antibody (Cat No.: RT1224; clone: 3G4), Anti-alpha smooth muscle Actin Recombinant Rabbit Monoclonal Antibody (Cat No.: ET1607-53; clone: SY04-07), Anti-Calreticulin Recombinant Rabbit Monoclonal Antibody (Cat No.: ET1608-60; clone: SU37-03), Anti-LOX Recombinant Rabbit Monoclonal Antibody (Cat No.: ET1706-31; clone: JU30-23) Anti-TGF beta 1 Recombinant Rabbit Monoclonal Antibody (Cat No.: HA721143; clone: PD00-17), Anti-HIF1 alpha Rabbit Polyclonal Antibody (Cat No.: ER1802-41), and Anti-HIF-1 alpha Recombinant Rabbit Monoclonal Antibody (Cat No.: HA721997 clone: JE75-33) were provided by Hangzhou HuaAn Biotechnology Co., Ltd. (HUABIO). S100-A8 / MRP8 Rabbit pAb (Cat No.: bs-2696R) and MMP9 Rabbit pAb (Cat No.: bs-0397R) were provided by Bioss Co.,Ltd. FITC Anti-Mouse CD3 Antibody (Cat No.: E-AB-F1013C clone: 17A2), PerCP/Cyanine5.5 Anti-Mouse CD4 Antibody (Cat No.: E-AB-F1097J; clone: GK1.5), PE Anti-Mouse/Human CD11b Antibody (Cat No.: E-AB-F1081D; clone: M1/70), FITC Anti-Mouse Ly-6G/Ly-6C (Gr-1) Antibody (Cat No.: E-AB-F1120C; clone: RB6-8C5) PE Anti-Human CD184/CXCR4 Antibody (Cat No.: E-AB-F1157D; clone: 12G5), and PE Anti-Human CD194/CCR4 Antibody (E-AB-F1366D; clone: L291H4) were provided by Elabscience® Biotechnology Co., Ltd. CoraLite® Plus 647 Anti-Mouse CD324 (E-cadherin; Cat No.: CL647-65241; clone: DECMA-1) was provided by Proteintech Co., Ltd. InvivoMab anti-mouse CD8α (Cat No.: BE0061; clone: 2.43) was provided by BioXCell. QuantiCyto® Human/Mouse/Rat HMGB1 ELISA Kit (Cat No.: EHRC01.96) was provided by Neobioscience Technology Co,Ltd. Mouse Lysyl oxidase homolog 2 (LOXL2) ELISA kit (Cat No.: CSB-EL013041MO) was provided by CUSABIO. Mouse CXCL12 ELISA Kit (Cat No.: RX201119M) was provided by Quanzhou Ruixin Biological Technology Co., LTD. Mouse TGF-β ELISA Kit (Cat No.: HB1328-Mu) was provided by Shanghai hengyuan biological technology co., LTD. Hydroxyproline Assay Kit (Cat No.: A030-2-1), Alanine aminotransferase Assay Kit (Cat No.: C009-2-1), and Aspartate aminotransferase Assay Kit (Cat No.: C010-2-1) were provided by Nanjing Jiancheng Bioengineer Institute (NJJCBIO). All other reagents were of analytical grade.

Cell lines

4T1 (Cat No.: CL-0007) murine breast cancer cells were provided by Pricella Life Science&Technology Co., Ltd. Luciferase-expressing 4T1 (4T1-luc, Cat No.: YC-B004-Luc-P) were provided by Guangzhou Ubigene Biosciences Co., Ltd. 4T1 cells and 4T1-luc cells were cultured in RPMI-1640 medium supplemented with 10% v/v fetal bovine serum, 1% antibiotics (penicillin and streptomycin), and incubated at 37 °C humidified environment with 5% CO₂ supply.

Synthesis and characterizations of Nanothread-1 and Nanothread-2

HPMA copolymer containing pendant amino groups (P-NH₂) was synthesized by reversible addition-fragmentation chain transfer (RAFT) copolymerization as previously reported^21,22,23,24. Briefly, HPMA (1.40 mmol) and N-(3-aminopropyl) methacrylamide (APMA, 0.16 mmol) were dissolved with 1.1 mL deionized water. 4-cyanopentanoic acid dithiobenzoate as a chain transfer agent (0.57 mg in 10 μL methanol) and 2,2‘-azobis[2-(2-dimidazolin-2-yl)propane] dihydrochloride as an initiator (0.19 mg in 10 μL methanol) were added. The solution was bubbled with argon in ice bath for 20 min, sealed and then reacted at 50 °C for 24 h. The copolymer was precipitated three times in acetone and diethyl ether to ensure removal of excess monomers. The dithiobenzoate end group was then removed using excess of 2,2′-Azobis(2,4 dimethyl) valeronitrile at 50 °C for 3 h. After dialysis and lyophilization, P-NH₂ was obtained. Afterwards, the pendent amino groups of P-NH₂ were converted to maleimides (P-Mal) by reacting with a heterobifunctional amino-thiol coupling agent, succinimidyl-4-(N-maleimidomethyl) cyclohexane-1- carboxylate (SMCC). P-NH₂ and SMCC were dissolved in N, N-dimethylformamide (DMF) adding moderate amount of N, N-diisopropylethylamine (DIPEA) (P-NH2: SMCC: DIPEA = 1:1:3 mol%). After reaction at room temperature for 6 h, the solution was added into the mixture of acetone and diethyl ether. The HPMA copolymer containing pendant maleimide groups (P-Mal) was obtained, filtered, and dried under vacuum. The remaining amino group were subsequently reacted with SulfoCy5 SE or SulfoCy3 SE to obtain fluorescent-labeled copolymers.

Next, P-Mal and CXCR4 binding sequence (BS) (P-Mal: BS = 1: 4 mol%) were reacted in phosphate buffer saline solution (PBS, pH 7.0) under an argon atmosphere for 24 h. After dialysis and lyophilization, HPMA copolymer-BS conjugates (P-BS) were obtained. Then, P-BS and coil motif 1 (CM1) were dissolved in PBS (pH 7.0) under an argon atmosphere. After 24 h, the product (P-BS-CM1) was obtained after dialysis and lyophilization. Similarly, HPMA copolymer-CM2 conjugates (P-CM2) were synthesized using the same method as P-BS, except that the BS was replaced with CM2.

To synthesize HPMA copolymer conjugates containing the photosensitizer Ce6, Ce6, DCC, and NHS (Ce6: DCC: NHS = 1:1.2:1.2 mol%) were stirred in DMSO for 3 h. P-Mal was added to the solution (P-Mal: Ce6 = 1:4 mol%) and reacted at room temperature. After 24 h, the reaction solution was filtered to remove insoluble byproducts, and the filtrate was dialyzed against deionized water for 2 days.

To synthesize the HPMA copolymer conjugates containing photosensitizer Ce6, Ce6, DCC, and NHS (Ce6: DCC: NHS = 1: 1.2: 1.2 mol%) were stirred in DMSO for 3 h. P-Mal was added into the solution (P-Mal: Ce6 = 1:4 mol%) and reacted at room temperature. After 24 h, the reaction solution was filtered to remove insoluble byproducts, and the filtrate was dialyzed against deionized water for 2 days. Subsequently, P-PS and CM2 or P-PS and BS were separately dissolved in PBS (pH 7.4) under an argon atmosphere. After 24 h, the products (P-PS-CM2 and P-BS-PS) were purified by dialysis and obtained after lyophilization.

The average molecular weight (Mw) and polydispersity (PDI) of the HPMA copolymer conjugates were determined by gel permeation chromatography (GPC) on an AKTA purifier equipment with a Superose 6 10/300 GL analytical column (GE Healthcare, USA) and a differential refraction detector (KNAUER, 2300, Germany). Hydrodynamic sizes and zeta potentials were determined using a Malvern Zetasizer Nano ZS90 equipment (Malvern Instruments Ltd, Malvern, UK). The contents of BS, CM1, and CM2 were determined using a bicinchoninic acid (BCA) protein assay. The PS concentration was determined by ultraviolet-visible (UV-Vis) spectroscopy at its characteristic absorbance peak (660 nm).

Investigation of coiled-coil assembly between Nanothread-1 and Nanothread-2

To investigate the coiled-coil assembly, varying CM1-to-CM2 ratios (3:1, 1:1, and 1:3) of P-BS-CM1 and P-CM2 were mixed in 10 mM PBS (pH 7.4) at 25 °C for 10 min. The total polymer concentration was kept constant at 10 mg/mL. Circular dichroism spectra, size distribution, and rheological properties of P-BS-CM1, P-CM2, and their mixture were measured. The surface morphology of P-BS-CM1 and the mixture (P-BS-CM1 + P-CM2) was observed using scanning electron microscopy. Additionally, for the FRET assay, CM1-to-CM2 equimolar of P-BS-CM1-Cy5 and P-CM2-Cy3 were mixed in 10 mM PBS (pH 7.4) at 25 °C for 10 min. Fluorescent spectra of P-BS-CM1-Cy5, P-CM2-Cy3, and the mixture (P-BS-CM1-Cy5+P-CM2-Cy3) were recorded at the Cy3 excitation wavelength of 550 nm.

Investigation of the specificity of BS to CXCR4

To validate the specificity of the BS (CXCR4-binding sequence) to CXCR4, a dose-dependent chemokine receptors occupation assay and a chemokine receptors competitive binding assay were conducted. In the dose-dependent chemokine receptors occupation assay, 4T1 cells were exposed to AMD3100 or free BS, with concentrations ranging from 0.1 nM to 1 mM equivalent, for 1 h. Unoccupied chemokine receptors (CXCR4, CXCR7, CXCR2, CXCR5, and CCR4) on cell surface were stained with PE anti-human CD184/CXCR4 Antibody (1:300 dilution), anti-CXCR7-PE (1:300 dilution), anti-CXCR2-PE (1:300 dilution), anti-CXCR5-PE (1:300 dilution), and PE anti-human CD194/CCR4 Antibody (1:300 dilution) at 4 °C for 1 h, prior to flow cytometry analysis. In the chemokine receptors competitive binding assay, 4T1 cells (5 × 10⁵ cells) were pre-blocked with PE anti-human CD184/CXCR4 Antibody (1:300 dilution), anti-CXCR7-PE (1:300 dilution), anti-CXCR2-PE (1:300 dilution), anti-CXCR5-PE (1:300 dilution), and PE anti-human CD194/CCR4 Antibody (1:300 dilution) at 4 °C for 1 h. Subsequently, these cells were treated with P-Cy5-BS-CM1-Cy5 (5 nM Cy5 equivalence) for 1 h. Following the treatments, cells were washed three times with PBS and subjected to flow cytometry analysis.

Investigation of Nanothreads crosslinking on 4T1 tumor cell surface

For in vitro studies, 4T1 cells (2 × 10⁵ cells) were seeded on coverslips (NEST Biotechnology) for attachment. Subsequently, the cells were treated with i) Cy5-labeled P-CM1 (P-CM1-Cy5) for 1 h, followed by incubation in cell culture medium for an additional 1 h, ii) Cy5-labeled P-BS-CM1 (P-BS-CM1-Cy5) for 1 h, followed by incubation in cell culture medium for an additional 1 h, or iii) P-BS-CM1-Cy5 for 1 h, followed by Cy3-labeled P-CM2 (P-CM2-Cy3) for an additional 1 h (5 nM Cy5 and 5 nM Cy3 equivalence), respectively. Following the treatments, cells were washed three times with PBS, stained with DAPI, and observed using a confocal microscope. For in vivo studies, 4T1 cells (1 × 10⁶) were injected into the third mammary fat pad of female BALB/c mice (6–8 weeks, 20 ± 2 g) (n = 3) on day 0 to establish orthotopic breast tumor models. For investigation of CXCR4 binding, mice on day 14 (tumor volume, ~200 mm³) were intravenously injected with P-CM1-Cy5 or P-BS-CM1-Cy5 (equivalent Cy5 dose, 5 nmol/mouse, n = 3). Whole-body living imaging at 24, 48, and 72 h post-administration as well as excised tumors at the end point, were captured using the IVIS optical imaging system (IVIS Lumina Series III, PerkinElmer, USA). For the analysis of CM1/CM2 biorecognition in the second step, 4T1 tumor-bearing mice (tumor volume, ~200 mm³) were intravenously injected with Cy5 labeled P-CM2 (P-CM2-Cy5) alone on day 15, or pre-injected P-BS-CM1 on day 14 followed by intravenous injection of P-CM2-Cy5 on day 15 (equivalent Cy5 dose, 5 nmol/mouse, n = 3). Whole-body living imaging at 24 and 48 h post-P-CM2-Cy5 administration, as well as excised tumors at the end point, were captured using the IVIS optical imaging system. For further investigation, mice (tumor volume, ~200 mm³) were intravenously injected with P-BS-CM1-Cy5 on day 14 and then P-CM2-Cy3 on day 15 (5 nmol of Cy5 and 5 nmol of Cy3 per mouse). One day later, tumor tissues were collected for frozen sectioning and further observation by confocal microscope.

Cell transfection and investigation of CXCR4 clustering

Lipofectamine™ 3000 (Cat No.: L3000008, Thermo Fisher Scientific) was employed to transfect EGFP-tagged Mus musculus C-X-C motif chemokine receptor 4 (EGFP-CXCR4) plasmids (GENEWIZ) into 4T1 cells. Prior to transfection, 4T1 cells (2 × 10⁵ cells) were seeded on coverslips (NEST Biotechnology) and cultured until reaching 50 − 70% confluence. For the transfection procedure, 5 μg of EGFP-CXCR4 plasmid was mixed with 7.5 μL of Lipofectamine 3000 reagent and 10 μL of P3000 reagent in 250 μL Opti-MEM™ (Cat No.: 31985062, Thermo Fisher Scientific), followed by a 15-minute incubation. Subsequently, the DNA−lipid complex was added to the cells cultured in Opti-MEM™ and incubated for 1 day. The transfected cells were then either left untreated or treated with: i) free BS for 1 h followed by culture in fresh medium for 13 h, ii) P-BS for 1 h followed by culture in fresh medium for 13 h, or iii) P-BS-CM1 for 1 h followed by P-CM2 for an additional 1 h, then cultured in fresh medium for 12 h (1 mg/mL BS equivalence, CM1:CM2, 1:1 mol%). Following the respective treatments, cells were observed using a confocal microscope. To investigate the inhibition of calcium influx following CXCR4 clustering, 4T1 cells (5 × 10⁵ cells) were seeded in 12-well plates (NEST Biotechnology). For free BS and P-BS groups, 4T1 cells were treated with concentrations ranging from 0 to 1 mM BS equivalence for 1 h, followed by cultivation in fresh medium for 25 h. In the P-BS-CM1 → P-CM2 group, 4T1 cells underwent consecutive treatment with P-BS-CM1 (concentrations ranging from 0 to 1 mg/mL BS equivalence) for 1 h and P-CM2 (CM1:CM2, 1:1 mol%) for 1 h, followed by culture in fresh medium for 24 h. Subsequent to the treatments, intracellular calcium levels were assayed.

Investigation of the influence of delivery sequences on tumor accumulation and pharmacokinetics

Three distinct delivery approaches were implemented in 4T1 tumor-bearing female BALB/c mice (6–8-week, tumor volume~200 mm³, n = 5 per group). i) Consecutive delivery: Nanothread-1 and nanothread-2 were sequentially administered via intravenously injection with a 24 h time interval. ii) Simultaneous delivery: Nanothread-1 and Nanothread-2 were concurrently administered through two tail veins of one mouse. iii) Post-assembly delivery: Nanothread-1 and Nanothread-2 were pre-mixed before intravenous injection (5 nmol of Cy5 and 5 nmol of Cy3 per mouse). For the tumor accumulation study, whole-body living imaging was conducted at 24, 48, and 72 h post-administration of Cy5-labeled Nanothread. Additionally, excised tumors were imaged at the end point using the IVIS optical imaging system. For the pharmacokinetics study, blood samples were extracted at predetermined time points post-administration of fluorescence-labeled Nanothread, n = 5 per group. The fluorescent intensity in blood samples was measured with a microplate reader at the wavelengths of Cy5 (Ex: 630 nm, Em: 670 nm) and Cy3 (Ex: 530 nm, Em: 570 nm). Pharmacokinetic parameters, including half-life (T_1/2z), area under curve (AUC), and mean residence time (MRT), were calculated using DAS 2.0 software.

Investigation of Nanothread-1 binding, Nanothread-2 crosslinking, and downstream calcium influx interference

For in vitro studies, 4T1 cells (2 × 10⁵ cells/well) were seeded into 24-well plates (NEST Biotechnology) and subjected to the following treatments: i) P-CM1-Cy5 for 1 h followed by P-CM2-Cy3 for another 1 h, ii) P-BS-Cy5 for 1 h followed by P-CM2-Cy3 for another 1 h, or iii) P-BS-CM1-Cy5 for 1 h followed by P-CM2-Cy3 for another 1 h, respectively (5 nM Cy5 and 5 nM Cy3 equivalence). For Nanothread-1 binding investigation, cells were washed three times with PBS, and the fluorescent intensity was measured in the APC channel of flow cytometry (Ex: 538 nm, Em: 660 ± 20 nm), representing Cy5 labeled Nanothread-1. For Nanothread-2 crosslinking investigation, cells were washed, and the fluorescent intensity was measured on the PC5.5 channel (Ex: 488 nm, Em: 690 ± 50 nm), representing the FRET signal. For calcium influx interference investigation, cells were cultured in fresh medium for an additional 24 h and then subjected to intracellular calcium assay. For in vivo studies, 4T1 tumor-bearing female BALB/c mice (6–8-week, tumor volume~200 mm³, n = 3 per group) were intravenously injected with: i) P-CM1-Cy5→P-CM2-Cy3, ii) P-BS-Cy5→P-CM2-Cy3, iii) P-BS-CM1-Cy5→P-CM2-Cy3 with a time lag of 24 h (5 nmol of Cy5 and 5 nmol of Cy3 per mouse, n = 5). 24 h post-P-CM2-Cy3 administration, tumor cells were isolated from excised tumors for flow cytometry analysis, as describe above in in vitro studies.

Investigation of Nanothreads biodistribution and biosafety

For biodistribution study, 4T1 tumor-bearing female BALB/c mice (6–8-week, tumor volume~200 mm³, n = 3 per group) were intravenously injected with either i) P-BS-CM1-Cy5 → P-CM2, or ii) P-BS-CM1 → P-CM2-Cy5 with a time interval of 24 h (equivalent Cy5 dose, 5 nmol/mouse). After 72 h post-injection of Cy5-labeled Nanothreads, tumors, hearts, livers, spleens, lungs, and kidneys were harvested and imaged using the IVIS optical imaging system. For biosafety study, female BALB/c mice (6–8-week, tumor volume ~200 mm³, n = 4 per group) were intravenously injected with P-BS-CM1 on day 7, 14, 21, and 28 post-tumor inoculation, followed by P-PS-CM2 on day 8, 15, 22, and 29 post-tumor inoculation (5 mg/kg BS equivalence, CM1:CM2 1:1 mol%, 2.5 mg/kg PS equivalence). On day 30, blood samples, hearts, livers, spleens, lungs, and kidneys were harvested. Serum biochemistry and hematological cell status in bloods were analyzed. Organ histological morphologies were assessed by hematoxylin-eosin staining.

Investigation of metastasis inhibition

Wound healing assay and transwell migration and invasion assay were conducted to investigate in vitro metastasis inhibition. For the wound healing assay, 4T1 cells (5 × 10⁵ cells) were cultured in 12-well plates (NEST Biotechnology) as monolayer with more than 90% coverage. Sterile 200 μL pipette tips were used to scrape off the cells, generating a linear cell-free area in the middle of the well. Subsequently, cells were washed by PBS to remove debris. In the stimulated group, 4T1 cells were stimulated with CXCL12 (100 ng/mL) for 1 h. For the free BS and P-BS groups, 4T1 cells received the treatments for 1 h prior to CXCL12 stimulation. In the P-BS-CM1 → P-CM2 group, 4T1 cells underwent consecutive treatment with P-BS-CM1 for 1 h and P-CM2 for 1 h before CXCL12 stimulation (1 mg/mL BS equivalence, CM1:CM2, 1:1 mol%). Images of the wound were recorded by a microscope at 0 h and 24 h after CXCL12 stimulation, and the wound healing rate was calculated using Image J software. For the transwell migration and invasion assay, 4T1 cells (5 × 10⁵ cells) were seeded in the non-coated upper chamber (Millipore, 8 μm) or GelNest™ matrigel (NEST Biotechnology) pre-coated upper chamber. The cells underwent the same treatment conditions as wound healing assay. After 24 h of treatment, migrated or invaded cells across the membrane were stained with 0.1% crystal violet, imaged by microscope, and quantified by absorbance at 570 nm after dissolution.

For proteomics analysis, 4T1 cells (5 × 10⁵ cells) were seeded in 12-well plates (NEST Biotechnology). Subsequently, cells were either left untreated or treated as follows: i) with free BS for 1 h followed by culture in fresh medium for 25 h, ii) with P-BS for 1 h followed by culture in fresh medium for 25 h, or iii) with P-BS-CM1 for 1 h followed by P-CM2 for another 1 h, and then cultured in fresh medium for 24 h (1 mg/mL BS equivalence, CM1:CM2, 1:1 mol%). Cell samples were submitted for label-free proteomics analysis (Shanghai Omics-space Biotech Co., Ltd., Shanghai, China). The analysis included protein extraction, protein digestion, LC − MS/MS detection (Thermo Scientific Orbitrap-Fusion Lumos), protein quantitation and identification (MaxQuant 1.5.5.1), and bioinformatics analysis.

For the in vivo investigation of pulmonary metastasis, 4T1 cells (1 × 10⁶ cells) were injected into the third mammary fat pad of female BALB/c mice (6–8 weeks). When tumor volumes reached nearly 100 cm³ on day 7 after tumor inoculation, mice were randomly divided into 4 groups (n = 5 per group) and intravenously injected with the following samples: (1) saline on day 7, 14, and 21, (2) free BS on day 7, 14, and 21, (3) P-BS on day 7, 14, and 21, or (4) P-BS-CM1 on day 7, 14, and 21, followed by P-CM2 on day 8, 15, and 22 (5 mg/kg BS equivalence, CM1:CM2 1:1 mol%), respectively. Tumor volumes were recorded every other day. On day 28, lungs were harvested and fixed with Bouin’s solutions. The pulmonary metastatic nodules were counted and analyzed by hematoxylin-eosin staining. In another experiment, the anti-metastasis effect of P-BS-CM1 → P-CM2 therapy was compared with daily treatment of AMD3100 at 2 mg/kg and 5 mg/kg via i.p. injection from day 7 to day 28 (n = 5).

Investigation of CXCR4 associated metastasis cascade

4T1 cells (1 × 10⁶ cells) were injected into the third mammary fat pad of female BALB/c mice (6–8-week). Upon reaching tumor volumes of nearly 100 cm³ on day 7 post-tumor inoculation, mice were randomly divided into saline, free BS, P-BS, and P-BS-CM1 → P-CM2 groups (n = 5 per group), with the treatment regimens as described above. On day 28, blood samples, lungs and tumors were harvested.

To assess fibrosis in tumor tissues, Masson staining, Sirius staining, immunofluorescent staining of α-SMA and fibronectin, and a quantitative hydroxyproline assay were employed. For immunofluorescent staining of α-SMA and fibronectin, tumor slices were stained with anti-alpha smooth muscle actin recombinant rabbit monoclonal antibody (1:500 dilution) and anti-fibronectin mouse monoclonal antibody (1:500 dilution) at 4 °C overnight, followed by staining with goat anti-mouse IgG H&L Alexa Fluor® 488 (1:1000 dilution) and goat anti-rabbit IgG H&L Alexa Fluor® 647 (1:1000 dilution).

For evaluating the hypoxic conditions in tumor tissues, immunofluorescent staining and flow cytometry analysis of HIF-1α were conducted. For immunofluorescent staining of HIF-1α, tumor slices were sequentially stained with anti-HIF-1 alpha recombinant rabbit monoclonal antibody (1:500 dilution) and goat anti-rabbit IgG H&L Alexa Fluor® 647 (1:1000 dilution). For flow cytometry analysis of HIF-1α, single-cell suspensions of tumor tissues were sequentially stained with anti-HIF1 alpha rabbit polyclonal antibody (1:500 dilution) and goat anti-rabbit IgG H&L Alexa Fluor® 647 (1:1000 dilution) before analysis.

To investigate the epithelial-mesenchymal transition process in tumor tissues, immunofluorescent staining of E-cadherin, vimentin, MMP-9, and TGF-β, along with flow cytometry analysis of E-cadherin and vimentin, were applied. For immunofluorescent staining of E-cadherin and vimentin, tumor slices were stained with CoraLite® Plus 647 anti-mouse CD324 (E-cadherin, 1:300 dilution) and anti-vimentin mouse monoclonal antibody (1:500 dilution) at 4 °C overnight, followed by staining with goat anti-mouse IgG H&L Alexa Fluor® 488 (1:1000 dilution). For flow cytometry analysis of E-cadherin and vimentin, single-cell suspensions of tumor tissues were stained with CoraLite® Plus 647 anti-mouse CD324 (E-cadherin, 1:300 dilution) or sequentially stained with anti-vimentin recombinant rabbit monoclonal antibody and goat anti-rabbit IgG H&L Alexa Fluor® 647 (1:1000 dilution), respectively. For immunofluorescent staining of MMP-9 and TGF-β, tumor slices were separately stained with MMP9 Rabbit pAb (1:500 dilution) or anti-TGF beta 1 recombinant rabbit monoclonal antibody (1:500 dilution), followed by staining with goat anti-rabbit IgG H&L Alexa Fluor® 647 (1:1000 dilution), respectively.

To explore the “seed-soil” crosstalk, the serum, intra-tumoral, and pulmonary concentration of LOX was determined using enzyme-linked immunosorbent assay kits (CSB-EL013041MO, CUSABIO, https://www.cusabio.com/) according to the manufacturer’s instructions. The serum, intra-tumoral, and pulmonary concentration of TGF-β was determined using mouse TGF-β ELISA Kit (Cat No.: HB1328-Mu). The formation of PMN was investigated through lung slices subjected to immunofluorescent staining of LOX, E-cadherin, and S100A8. For immunofluorescent staining of E-cadherin, Lung slices were stained with CoraLite® Plus 647 anti-mouse CD324 (E-cadherin, 1:300 dilution). For immunofluorescent staining of LOX and S100A8, Lung slices were separately stained with anti-LOX recombinant rabbit monoclonal antibody (1:500 dilution) or S100-A8 / MRP8 rabbit pAb (1:500 dilution), followed by staining with goat anti-rabbit IgG H&L Alexa Fluor® 647 (1:1000 dilution), respectively. Single-cell suspensions of lungs were analyzed using flow cytometry to assess BMDC (CD11b⁺Gr1⁺) with PE anti-mouse/human CD11b antibody (1:300 dilution) and FITC anti-mouse Ly-6G/Ly-6C (Gr-1) antibody (1:300 dilution). Chemokine concentrations in lungs were determined using a mouse CXCL12 ELISA kit.

Investigation of PDT potentiation

For cell binding investigation, 4T1 cells (4 × 10⁵ cells/well) were seeded onto 12-well plates (NEST Biotechnology), and treated with i) P-PS for 1 h, ii) P-BS-PS for 1 h, or iii) P-BS-CM1 for 1 h followed by P-PS-CM2 for another 1 h (50 μg/mL BS equivalence, CM1:CM2 1:1 mol%, 25 μg/mL PS equivalence), respectively. Subsequently, cells were washed three times with PBS and observed using flow cytometry and confocal microscopy.

Cytotoxicity of PDT was investigated by CCK-8 cell viability assay and Annexin V-FITC/PI double staining cell apoptosis assay. For the CCK-8 assay, 4T1 cells (5 × 10³ cells/well) were seeded in the 96-well plate (NEST Biotechnology), and treated with: i) P-PS for 1 h, ii) P-BS-PS for 1 h, or iii) P-BS-CM1 for 1 h followed by P-PS-CM2 for another 1 h (concentrations ranging from 0–50 μg/mL PS equivalence, CM1:CM2 1:1 mol%), respectively. After treatment, cells were irradiated with a 660 nm laser for 5 min (280 mW/cm²) and then incubated for an additional 24 h. Subsequently, CCK-8 solution (1x) was added, and cell viability was calculated based on the absorbance at 450 nm.

For the Annexin V-FITC/PI double staining cell apoptosis assay, 4T1 cells (2 × 10⁵ cells/well) were seeded into 24-well plates (NEST Biotechnology) and treated with: i) P-PS for 1 h, ii) P-BS-PS for 1 h, or iii) P-BS-CM1 for 1 h followed by P-PS-CM2 for another 1 h (25 μg/mL BS equivalence, CM1:CM2 1:1 mol%, 12.5 μg/mL PS equivalence), respectively. After treatment, cells were irradiated with a 660 nm laser for 5 min (280 mW/cm²) and then incubated for an additional 24 h. Cells were stained with FITC Annexin-V and PI, and analyzed using flow cytometry.

For the investigation of CXCR4 downstream PI3K signals, cells were fixed, permeabilized, and stained with anti-PI 3 kinase p85 alpha recombinant rabbit monoclonal antibody (1:500 dilution, 4 °C, 1 h) and goat anti-rabbit IgG H&L Alexa Fluor® 647 (1:1000 dilution, 4 °C, 30 min), followed by flow cytometry analysis. For the investigation of ICD hallmarks, cells were stained with anti-calreticulin recombinant rabbit monoclonal antibody (1:500 dilution, 4 °C, 1 h) and goat anti-rabbit IgG H&L Alexa Fluor® 647 (1:1000 dilution, 4 °C, 40 min), followed by flow cytometry analysis. ATP levels in the cell culture supernatant were measured following the protocol of luciferase-based ATP assay kit.

Investigation of local immune response against primary tumor growth and metastasis

4T1 cells (1 × 10⁶ cells) were injected into the third mammary fat pad of female BALB/c mice (6–8-week). When tumor volumes reached nearly 100 cm³ on day 7 after tumor inoculation, mice were randomly divided into 8 groups (n = 8 per group). For four groups without laser irradiation (−), mice were intravenously injected with the following samples: (1) saline on day 7, 14, 21, and 28, (2) P-PS on day 7, 14, 21, and 28, (3) P-BS-PS on day 7, 14, 21, and 28, or (4) P-BS-CM1 on day 7, 14, 21, and 28, followed by P-PS-CM2 on day 8, 15, 22, and 29 (5 mg/kg BS equivalence, CM1:CM2 1:1 mol%, 2.5 mg/kg PS equivalence), respectively. For four groups with laser irradiation (+), mice were treated with the same regimens and irradiated at 650 nm laser (580 mW/cm², 5 min) on day 9, 16, 23 and 30. Tumor volumes and mice survivals were recorded every other day. For the investigation of immune response and lung metastasis, 4T1 tumor-bearing female BALB/c mice (6–8-week, tumor volume~100 mm³) were randomly divided into 8 groups (n = 5 per group) and given three rounds of above treatments. On day 28, lungs and tumors were harvested. lungs were fixed with Bouin’s solution. The pulmonary metastatic nodules were counted and analyzed by hematoxylin-eosin staining. Single-cell suspensions of tumor tissues were stained with anti-CD45-PerCP/Cy5.5 (1:300 dilution) and anti-calreticulin recombinant rabbit monoclonal antibody (1:500 dilution) at 4 °C for 1 h, and then stained with goat anti-rabbit IgG H&L Alexa Fluor® 647 (1:1000 dilution, 4 °C, 45 min), followed by flow cytometry analysis. ATP and HMGB1 concentrations in the supernatant of cell suspension were measured using a luciferase-based ATP assay kit and a mouse HMGB1 ELISA kit, respectively. Single-cell suspensions of tumors were stained with FITC anti-mouse CD3 antibody (1:300 dilution), PerCP/Cyanine5.5 anti-mouse CD4 antibody (1:300 dilution), anti-CD8α-APC (1:300 dilution), and anti-Foxp3-PE (1:300 dilution) for flow cytometry analysis of T cell subtypes (CD8⁺ T cells, CD4⁺ effector T cells, and Foxp3⁺ regulatory T cells), and stained with PE anti-mouse/human CD11b antibody (1:300 dilution) and FITC anti-mouse Ly-6G/Ly-6C (Gr-1) antibody (1:300 dilution) for flow cytometry analysis of MDSCs (CD11b⁺Gr1⁺). Cytotoxic T cells in tumor tissues were investigated with immunofluorescent staining of IFN-γ and flow cytometry analysis using anti-IFN-γ-PE (1:300 dilution). For CD8-depletion assay, 4T1 tumor-bearing female BALB/c mice (6–8-week, tumor volume~100 mm³) were randomly divided into 3 groups (n = 8 per group). Mice were intravenously given four doses of P-BS-CM1 on day 7, 14, 21 and 28, combined with P-PS-CM2 on day 8, 15, 22 and 29 (5 mg/kg BS equivalence, CM1:CM2 1:1 mol%, 2.5 mg/kg PS equivalence) in the presence or absence of InvivoMab anti-mouse CD8α (100 μg/mice). Mice were irradiated at 650 nm laser (580 mW/cm², 5 min) on day 9, 16, 23 and 30. Tumor volumes and mice survivals were recorded every other day.

Investigation of abscopal immune memory against disseminated tumor metastasis

4T1 tumor-bearing female BALB/c mice (6–8-week, tumor volume~100 mm³) were randomly divided into 3 groups (n = 5 per group) and treated with: (1) saline on day 7 and 14, (2) P-BS-CM1 + P-PS-CM2 without laser irradiation (−) on day 7-8 and 14-15, or (3) P-BS-CM1 + P-PS-CM2 with laser irradiation (+) on day 7–9 and 14–16, as indicated above. On day 21, mice were intravenously injected with 4T1−luc cells (4 × 10⁵ cells). Tumor-free mice were also intravenously injected with 4T1-luc cells (4 × 10⁵ cells) to function as controls. Disseminated tumor metastatic niches were captured on day 28 using the IVIS optical imaging system. Lungs, spleens, and bloods of these mice were collected on day 28. Lung slices underwent immunofluorescent staining of CD11b and Gr1. Single-cell suspensions of spleen tissues were subjected to flow cytometry analysis of CD8⁺ Tems (CD8⁺CD44⁺CD62L⁻) using anti-CD8α-APC (1:300 dilution), anti-CD44-PE (1:300 dilution), and anti-CD62L-Percp/Cy5.5 (1:300 dilution). Peripheral blood mononuclear cells (PBMCs) were isolated from bloods of these mice and cultured in T cell medium. A total of 5 × 10⁵ PBMCs were incubated with 1 × 10⁵ living 4T1 cells for 16 h in the presence of brefeldin A. Then, IFN-γ expression in PBMCs was analyzed with flow cytometry using anti-CD8α-APC (1:300 dilution) and anti-IFN-γ-PE (1:300 dilution).

Statistical analysis

Statistical data was analyzed using Graphpad Prism 8 software and presented as mean ± SD. For two-group comparison, statistical significance was determined using Student’s two-sided t-test. For multiple comparison, statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons without adjustments. A significant difference was considered when the P value was less than 0.05.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41467-024-47111-z