2.1 Cell culture and animal feeding
BT474 breast cancer cells (Cell bank of Chinese Academy of Sciences, China) were cultured in RPMI-1640 medium (Gibco, 31870074, USA) containing 10% fetal bovine serum (FBS) (Gibco,12483020, USA) and 1% penicillin (Beijing Solarbio Technology Co., Ltd, 10378016, China) mixture at incubator (Thermo Fisher Scientific, USA) with 37 ℃ and 5% CO2.
FVB-MMTV-PyMT mice (Gempharmatech Co., Ltd, China) and FVB mice (Beijing Vital River Laboratory Animal Technology Co., Ltd, China) were fed in SPF laboratory animal rooms which the temperature was 23 ± 3℃, the day and night cycle was alternated for 12 hours. This animal research was reviewed and approved by the Ethics Committee of Ningxia Medical University (license number: SYXK (Ning) 2020-0001).
2.2 Establishment of orthotopic transplantation model of HER2-positive breast cancer in mice
According to Doornebal's research method [21], using FVB-MMTV-PyMT (FVB/N background) mice as donors and FVB/N mice with the same background as recipients, "breast orthotopic tumor transplantation" was performed under Sevoflurane inhalation anesthesia to establish the orthotopic transplantation model of HER2-positive breast cancer in mice. When the growth of orthotopic breast tumor in recipient mice reached about 2 × 2 mm3 (defined as "diagnosis time"), the mice were randomly grouped and intervened according to the method of "2.3 Grouping and processing of in vivo experiments". When the growth of orthotopic breast tumor in recipient mice reached about 15×15 mm3 (1687.5 mm3), the "breast orthotopic transplantation tumor resection" was performed, and the transplanted breast tumor tissues were collected for other experiments (immunohistochemistry, Western blot). Suture the incision, keep warm and supply oxygen during operation. After the operation, the "2.3 Grouping and processing of in vivo experiments" method was given to complete postoperative analgesia.
During the post-operation period, disease monitoring was carried out on the recipient mice. When the recipient mice showed painful clinical symptoms caused by metastatic diseases (respiratory distress, abdominal distension, ascites, rapid weight gain, severe anemia, obvious metastatic lesions in lymphatic organs, or local in-situ recurrence of tumor, about 15×15 mm3), the tumors tissues and organs were collected.
2.3 Experimental grouping and processing in vivo and in vitro
Cells were randomly divided into control group (C group, RPMI 1640 complete medium only), parecoxib sodium (Pfizer Inc, USA) group (P group, 300 mmol/L), sufentanil (Yichang Humanwell Pharmaceutical Co., Ltd, China) group (SF group, 1 nmol/L [22,23]) and sufentanil + parecoxib sodium combination group (SF+P group, 1 nmol/L + 300 mmol/L). The processing time was 24h.
When the orthotopic breast tumor growth of FVB/N mice reached about 2 × 2 mm3, the mice were randomly divided into control group (C group, normal saline), parecoxib sodium group (P group, 5 [24]mg/kg), sufentanil group (SF group, 1 μg/kg [25]) and sufentanil + parecoxib sodium combination group (SF+P group, 1 μg/kg +5 mg/kg). The processing was administered by intraperitoneal injection every 12h for 3 consecutive days.
2.4 Substrate adhesion assay
Matrigel matrix gel (Becton, Dickinson and Company, USA) was diluted to 0.04 mg/mL with serum-free RPMI 1640 medium, and 50 mL was added to each well of a 96-well plate, and hydrated in PBS for 60-90 min the next day. BT474 breast cancer cells at logarithmic growth stage were treated according to "2.3 Grouping and processing of in vitro experiments". The cells were collected and adjusted to density of 5×104 /well, with 5 replicate wells in each group, and incubated in the incubator for 2 h. The cells were washed with PBS, incubated with 20 mL/well of MTT for 4 h, and then incubated with DMSO 150 mL/well, shaken for 10 min, and the OD value was measured on an enzyme marker at 570 nm. The cell adhesion rate was calculated after 24 h of drug treatment. Cell adhesion rate (%) = (OD experimental group/OD control group) × 100%.
2.5 Wound healing assay
BT474 breast cancer cells in logarithmic growth phase were digested by trypsin (Gibco, 12563011, USA), centrifuged, blown and mixed to make a single cell suspension, inoculated with 2×105 cells/well in a 6-well plate. When the cell growth density reached about 70%, intervention was performed according to "2.3 Grouping and processing of in vitro experiments". After treatment, the cells in each well were scratched with a 200 mL pipette tip, and the cells were gently washed with PBS solution 3 times after scratching. The marked photographic sites were observed and photographed at 0h and 24h and the wound healing rate was calculated. Wound healing rate (100%) = (0 h area - 24 h area)/0 h area × 100%. The results were analyzed and processed using ImageJ (version 1.52) image software.
2.6 Transwell invasion assay
Matrigel matrix gel was diluted with serum-free RPMI 1640 medium in the ratio of 1:8, and 80 mL was added to each well and incubated at 37 °C for 2 h to solidify into a gel. Mix and suspend BT474 breast cancer cells at logarithmic growth stage with RPMI 1640 medium containing 0.1% FBS, adjust the cell inoculation density to 5×105 /well, add 150 mL of cell suspension in the upper chamber of each well and 600 mL of RPMI 1640 complete medium in the lower chamber of each well. The interventions were performed according to "2.3 Grouping and processing of in vitro experiments". After treatment, the chambers were fixed with 4% paraformaldehyde for 30 min, washed 3 times with PBS, air-dried upside down, stained with 0.1% crystal violet for 5 min, washed 3 times with PBS, gently wiped off the Matrigel matrix gel and cells on the gel with cotton swabs, dried and observed under an inverted microscope, and the number of membranes piercing cells was counted in 3 randomly selected fields. The results were analyzed and processed by ImageJ (version 1.52) image software.
2.7 Western blot
The total protein was extracted from BT474 breast cancer cells and mice breast cancer tissues, and the concentration of total protein was detected by BCA method (KeyGen Biotech, KGP2100, China). The proteins were separated by SDS-PAGE with a loading volume of 30 µg/well, and the separated proteins on the gel were transferred to PVDF membrane at 270 mA for 2 hours and closed for 2 hours. Wash 3 times with TBST. Add primary antibody Cyclin D1 (1:1000, CST, 55506t, USA), MMP-9 (1:500, Abcam, ab58803, USA), COX-2 (1:3000, Abcam, ab179800, USA), VEGFA (1:200, Abcam, ab1316, USA), E-cadherin (1:1000, Abcam, ab231303, USA), N-cadherin (1:1000, Abcam, ab98952, USA), Vimentin (1:3000, Abcam, ab92547, USA), Snail (1:1000, Abcam, ab180714, USA), and β-actin (1:1000, Proteintech, 20536-1-AP, USA), incubated overnight at 4°C in a shaker. The next day, TBST was washed 3 times and the secondary antibodies of the corresponding species were added and incubated under the chamber for 2 h. After washing 3 times with TBST, the images were developed in a dark room environment using ECL chromogenic solution exposed on a gel imaging system. The results were analyzed and processed using Image Lab (version 6.0.1) image software.
2.8 Growth of mice
FVB/N recipient mice were observed daily from the day of "breast orthotopic tumor transplantation", and the growth of transplanted tumors, diet and feces of the mice were observed. The body weight of the mice was recorded every 3 days, and the longest diameter (A) and the shortest diameter (B) of the transplanted tumor were measured with vernier calipers, and the volume was calculated as V=1/2 × A × B2. The survival time of recipient mice was recorded and the survival curve was plotted.
2.9 HE staining
The lung tissues of the mice were fixed with 4% paraformaldehyde, dehydrated by ethanol and embedded in paraffin, then the slices with a thickness of 5 μm were prepared conventionally, dewaxed in xylene Ⅰ and Ⅱ for about 10 ~ 15 min, hydrated by absolute ethanol, 95% ethanol and 80% ethanol in turn for 2 min, dyed with tap water. Hematoxylin nuclear staining for about 5 min, tap water washing, hydrochloric acid ethanol differentiation for 10 s and tap water washing for 30 min. Re-dyeing with 0.5% Eosin for about 2 min, washing with tap water, soaking with 95% ethanol Ⅰ and Ⅱ for 90 s, soaking with absolute ethanol Ⅰ and Ⅱ for 90 s, making xylene Ⅰ and Ⅱ transparent for 10 min, and sealing with neutral gum to dry. Motic digital section scanning and application system (VM1000, China) was used to analyze the images.
2.10 Immunohistochemistry
Paraffin sections of breast cancer tissues were dewaxed and hydrated, and antigen repair was performed with sodium citrate antigen repair solution, goat serum was added and closed at room temperature for 30 min, primary antibody CD31 (1:500, Proteintech, 28083-1-AP, USA), was added and incubated overnight at 4 ℃; the next day, the sections were rewarmed and incubated dropwise with secondary antibody of the corresponding species for 30 min at 37 ℃, washed twice with PBS, DAB staining solution was added dropwise and incubated for 10 min at room temperature, and hematoxylin staining solution was used for restaining. The stains were re-stained with hematoxylin staining solution, dehydrated with ethanol and xylene, sealed with neutral resin, and then photographed under an orthomorphic fluorescence microscope, and the results were analyzed by ImageJ (version 1.52) image software.
2.11 Statistics
Statistical analyses were performed with SPSS 26.0 and GraphPad Prism 9.0 software. The data was expressed in the form of mean ± standard deviation (`𝑥±𝑠). Between-group data comparison is conducted by normality test (P>0.05) and homogeneity test of variance (P>0.05). If the data meet the requirements at the same time, one-way ANOVA is used, and LSD-t back testing is used for inter-group comparison. If the data does not meet the normal distribution or variance homogeneity test, the nonparametric rank sum test is selected. The changes of body weight and tumor volume of mice were examined by univariate analysis of variance. Survival analysis was tested by Log-Rank. P<0.05, the difference was statistically significant.