2.10 Primary tumor growth and spontaneous metastasis
Forty BALB/c mice (female, 6–8 weeks old) were randomly divided into five groups. At day 0, 4T1 cells re-suspended in 0.1 mL PBS were injected into the right mammary gland of BALB/c mice at 1 × 106/mouse. At 14, 17, 20 and 23 d, mice in the five groups were administered via tail vein injection with saline, DOX, DHA, DOX + DHA solution, and DOX/DHA-LNs, respectively, at the equivalent doses of 5.00 mg kg-1 DOX and 2.83 mg kg-1 DHA. The animal weight and tumor volume were measured twice a week until the end of the experiment. Tumor volume was estimated by measuring the minimum and maximum tumor diameters using the formula: (minimum diameter)2 × (maximum diameter)/2. At day 42, the mice were sacrificed. Part of primary tumors were digested for flow cytometry analysis (n = 3). The residual primary tumors, lungs, and spleens were collected and washed with cold saline (n = 5). The final tumor weights were measured and the tumor inhibiting rates (TIR) were calculated by the following equation:
TIR = (1 - Wtest/Wsaline) × 100%
Wsaline represented the average tumor weight of saline group and Wtest referred to that of the tested groups. Then, primary tumors, lungs, and spleens were fixed with 4% paraformaldehyde and imaged with a digital camera. The primary tumor sections were stained by H&E, Ki67, TUNEL, IL-6, and CD34. The spleen sections were stained by H&E, CD4, and CD8. Moreover, the metastatic nodules on the pulmonary tissues were counted, and the lung sections were stained by H&E, F4/80, neutrophil and IL-6. The H&E stained lung sections were subjected to scanning using Pathological section scanner (Pannoramic MIDI, Hungary).
2.11 Experimental metastasis assay
Seventy-five BALB/c mice (female, 6–8 weeks old) were randomly divided into five groups. At day 0, all of them were given 2 × 105 4T1 tumor cells suspended in 0.1 mL PBS by tail vein injection to generate lung metastasis of breast cancer bearing mouse model. At day 4, 7, 10 and 13, mice were systemically given saline, DOX (5.00 mg kg-1), DHA (2.83 mg kg-1), DOX + DHA and DOX/DHA-LNs. The body weight was recorded. At day 16, five mice were sacrificed in each group (n = 5). The lungs fixed with 4% paraformaldehyde were photographed with a digital camera and the number of lung metastasis nodules was counted. Microscopic metastatic lesions in lungs were revealed after H&E staining of lung tissue sections, where were further subjected to scanning as previously described.
2.12 Postsurgial breast cancer recurrence murine model
BALB/c mice (female, 6–8 weeks) were randomly divided into five groups. First, 1 × 106 4T1-luc cells were inoculated into the right mammary gland of each mouse. When tumors reached ~ 300 mm3 after approximately 10 days, tumors were surgically removed. Specifically, animals were anaesthetized via intraperitoneal injection of 4% chloral hydrate, and the majority of the tumor mass was removed but left with a layer of surrounding skin with tumor residues to allow tumor relapse. The surgical wound was then closed with sutures. The five groups were subsequently administered via tail vein injection with saline, DOX (5.00 mg/kg), DHA (2.83 mg/kg), DOX + DHA solution and DOX/DHA-LNs at equivalent doses, respectively. Formulations were injected every 3 days for four consecutive times. Meanwhile, the animal weight and tumor volume were measured every 3 days until the end of the experiment. Tumor volume was also estimated by measuring the minimum and maximum tumor diameters using the formula: (minimum diameter)2 × (maximum diameter)/2. At day 12, three mice from each group were sacrificed. The remaining mice were left for survival study. The tumors, lungs, and spleens were collected and fixed with 4% paraformaldehyde. The tumor sections were stained by H&E, TLR4, Ki67, TUNEL, IL-6, TNF-α and CD34. The spleen sections were stained by H&E, CD4, and CD8.
2.13 In vivo bioluminescence and imaging
At day − 1, 0, 4, and 8, mice were anesthetized with 4% chloral hydrate after intraperitoneal injection of D-luciferin potassium salt (150 mg/kg), and imaged using the IVIS® Spectrum in vivo imaging system (Perkin Elmer, USA).
2.14 Patient samples
Human samples were obtained from the Sichuan Provincial People’s Hospital. Immunostaining of TLR4 was performed on sections from formalin-fixed paraffin-embedded tissue biopsies obtained from breast cancer patients prior to receiving any chemotherapy. Analysis of human samples was in line with the Institutional Review Board of Sichuan Provincial People’s Hospital. Collection of patient samples for scientific purposes was approved by the local ethics committee of Sichuan Provincial People’s Hospital (2016-16-1), and written informed consents were obtained.
2.15 Pharmacokinetics and biodistributions in vivo
4T1 tumor-bearing mice with tumor size of about 200 mm3 were fasted for 12 h before experiment and were intravenously given DOX, DOX + DHA, and DOX/DHA-LNs at an equivalent dose of 5.00 mg kg-1 DOX. At each predetermined time point (0.5, 1, 3, 6, 12, 24, and 48 h), three mice were sacrificed, and the blood and tissues (heart, liver, spleen, lung, kidney, and tumor) were collected. Plasma samples were obtained following centrifugation at 6000 rpm for 8 min. All samples were stored at -20°C until analysis. Every tissue sample was accurately weighed, homogenized, and extracted with 0.9% NaCl solution. Homogenized tissue and plasma samples were mixed with four volumes of acetonitrile, vortexed for 10 min and centrifuged at 10000 rpm for 5 min. The supernatants were subjected to LC-MS/MS analysis as previously described. The pharmacokinetic data were generated and analyzed by DAS 3.2.5 (Drug and Statistics, Anhui, China).
2.16 Safety evaluation
Female Sprague Dawley rats (200 ± 20 g) were randomized into 6 groups with 5 rats in each group and fasted for 12 h before administration. Saline, DOX solution (6.00 µg mL-1), DHA solution (3.40 µg mL-1), DOX + DHA, DOX/DHA-LNs and blank LNs were administered intravenously every three days for three consecutive times. The animal weight was recorded every three days. About 0.4 mL of blood samples were collected in centrifuge tubes containing ethylenediamine tetra acetic acid dipotassium salt (EDTA-2K) on either the day before administration or day 3 after administration. White blood cells (WBC) were counted by MEK-6318K automated hematology analyzer (Nijon-kohden, Shinjuku-ku, Japan) as an index of bone marrow suppression. To assess the cardiac and gastrointestinal toxicity, all rats were sacrificed on day 3 after administration, and heart, stomach, duodenum, jejunum, ileum and colon samples were collected. The obtained tissues were fixed with 4% paraformaldehyde for at least 48 h for paraffin sectioning and hematoxylin and eosin (H&E) staining. Degrees of injury such as bleeding, necrosis, hyperaemia, glandular expansion, and decrease in glandular tubes were examined and photographed under light microscope (Axiovert 40CFL, Zeiss, Germany).
2.17 Flow cytometry
Flow cytometry was performed on tumor-associated macrophage (TAM), myeloid-derived suppressor cells (MDSCs), neutrophil and T cells obtained from tumors. Minced mice tumors were digested with 0.5% (w/v) collagenase II solution for 8 h (n = 3). Cells were incubated with anti-CD11b-FITC (BD Pharmagin; 557396) and anti-F4/80-PE (BD Pharmagin; 565410) for TAM. For neutrophil and MDSC staining, anti-CD11b-FITC (BD Pharmagin; 557396), anti-Ly6C-APC (BD Pharmagin; 560595), and anti-Ly6G-PE (BD Pharmagin; 561104) were used. For CD4/CD8 staining, anti-CD4-FITC (BD Pharmagin; 561835) and anti-CD8a-APC (BD Pharmagin; 561093) were used. All stained samples were analyzed by flow cytometry (BD FACS CelestaTM, USA).
Routine histological analysis was performed on 4 µm paraformaldehyde-fixed, paraffin-embedded sections. To stain CD8, CD4, IL-6, TLR4, TNF-α, Ki67, TAM, neutrophil and blood vessels, the sections were incubated with anti-mouse CD8 (Abcam, ab203035), CD4 (Abcam, ab221775), IL-6 (Abcam; ab7737), TLR4 (Abcam; ab13556), TNF-α (Abcam, ab6671), Ki67 (Servicebio, GB13030-2), F4/80 (Abcam; ab1000790), neutrophil (Abcam; ab2557) and CD34 (Abcam; ab187282) at 4 ºC overnight, followed by biotinylated anti-IgG secondary antibodies (ZSGB-BIO). Signal detection was performed using DAB kit (ZSGB-BIO, K135925C) for 2 min at room temperature. Sections were visualized under light microscope (Axiovert 40CFL, Zeiss, Germany).
2.19 TUNEL assay
TUNEL staining was performed according to the manufacturer’s instructions (Roche; 11684817910). TUNEL-stained images of were captured using confocal laser scanning microscope (LSM 800, Zeiss, Germany) at 20 × magnification.
Cells deposited on glass bottom dishes were rinsed with 1× PBS twice, fixed in 4% (w/v) paraformaldehyde for 20 min, permeabilized with 0.1% (w/v) Triton X in PBS, washed and blocked with 1% (w/v) bovine serum albumin (Sigma-Aldrich, USA) in PBS for 60 min. TLR4 antibody (1:100) (Abcam; ab22048) or NF-κB p65 antibody (1:400) (Cell Signaling, D14E12) served as primary reagents. Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L) (ZSGB-BIO, ZF-0511) and Alexa Flour 488-conjugated goat anti-mouse IgG (H + L) (ZSGB-BIO, ZF-0512) served as secondary reagents. Cells were incubated with primary antibodies in 1% bovine serum albumin in a moist chamber at 4°C overnight and the secondary antibodies (1:400) for 1 h at room temperature (RT) in dark. After DAPI staining, cells were observed with a confocal laser scanning microscope (Leica, Wetzlar, Germany).
Paraffin sections of recurrent tumors were rinsed with 1× PBS three times, permeabilized, blocked, and incubated with antibodies according to the above procedure. TLR4 antibody (1:100) (Abcam; ab22048) served as primary reagents, Cy3-conjugated goat anti-mouse IgG (H + L) (Servicebio; GB21301) served as secondary reagents. After DAPI staining. TLR4 expression of tumor sections were observed with a confocal laser scanning microscope (LSM 800, Zeiss, Germany).
2.21 Statistics analysis
Statistical analyses were performed with Graphpad Prism 6 (GraphPad Software, La Jolla, CA). All data represent mean ± standard deviation (SD). Pairwise comparison testing in experiments with more than two groups was performed using one-way analysis of variance (ANOVA) followed with post hoc Tukey test. P values less than 0.05 were considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.