Genes of anti Nectin-4 antibody were generated (General Biosystems, Anhui, China), and subcloned into a pCDNA 3.4 vector (Invitrogen). HEK 293F cells were transiently transfected with plasmids loading antibody genes and cultured. The supernatant containing anti Nectin-4 antibody was collected for antibody extraction and purification as previously described .
Cell lines (TNBC, MDA-MB-468; non-TNBC, MCF-7) were provided by the Shanghai Type Culture Collection of the Chinese Academy of Sciences. MDA-MB-468 cells were cultivated with modified L-15 medium (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin–streptomycin at 37 °C under a humidified atmosphere with 0% CO2. MCF-7 cells were cultivated with DMEM medium (10% FBS + 1% penicillin–streptomycin; 37 °C, 5% CO2).
When the growth density of MDA-MB-468 and MCF-7 cells reached about 80%, cells were dispersed and collected for protein extraction. The total protein concentration was measured and the protein (20 µg each lane) was added to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After separation, trarsmembran was performed using polyvinylidene fluoride (PVDF) film. The film was incubated with the primary antibody (Nectin-4 antibody; Abcam, ab192033; 4 °C, overnight) and secondary antibody (goat anti-rabbit IgG, 1:10,000; 4 °C, 2 h) orderly. The films were visualized on a Visionwork system. Glyceraldehyde 3‑phosphate dehydrogenase (GAPDH) was applied as internal reference.
The cells were collected and suspended in phosphate buffer saline (PBS) (concentration: 1 × 106 cells/100 μL). After dispersing, cells were incubated with Cy5 labeled mAbNectin-4 on ice for 30 min. For blocking assay, pre-treat the MDA-MB-468 cells using excess unlabeled mAbNectin-4 1 h prior to mAbNectin-4-Cy5 incubation. After flushing with PBS for three times, cell were tested by FACSCalibur flow cytometer (Becton,Dickinson and Company, USA).
Confocal laser scanning microscopy (CLSM) imaging
Seed the cells in 35 mm plates (density: 1 × 104) for incubation overnight. Replace 1 mL serum-free medium containing Cy5 labeled mAbNectin-4 (20 µg/mL) to each plate and incubation was performed for 1 h. Rinse the Cy5-treated cells with pre-cooling PBS for three times, 200 μL 4% paraformaldehyde solution was added to fix cells. Then DAPI and FITC-Phalloidin was used for nuclear and cytoskeleton staining respectively. Images with different channels (Excitation/Emission of the Cy5, DAPI, and FITC were 650/670 nm, 360/450 nm, and 490/520 nm, respectively) were acquired by fluorescence optics of a confocal microscope (Zeiss, NOL-LSM 710). For blocking studies, MDA-MB-468 cells were incubated with excess mAbNectin-4 for 1 h previously before experiments.
Radiolabeling of mAbNectin-4 with 99mTc
Nectin-4-specific mAb (mAbNectin-4) was purified by Zeba™ desalting column (7 K, 0.5 mL) first. Fourty µg bifunctional chelator succinimidyl 6-hydraziniumnicotinate hydrochloridem (SHNH), (Solulink, Inc., USA) was added to the purified mAbNectin-4 (150 µg, 1 nmol) and reacted overnight at 4 °C for the synthesis of Hynic-conjugated mAbNectin-4. After filtering with Zeba™ desalting column to remove the excess SHNH, tricine solution (100 μL,100 mg/mL), SnCl2 solution (4 μL, 7 mg/mL) and 99mTcO4− eluate (500 μL, 1110 MBq; eluted from 99mTc/99Mo generator) were added orderly into the Hynic-conjugated mAbNectin-4 solution. Thhe mixture was incubated away from light at 25 °C for 30 min, then purified with PD-10 desalting column (GE, USA). The labeling efficiency (prior to PD-10 purification) and radiochemical purity (post to PD-10 purification) were examined with instant thin layer chromatography (ITLC; mobile phase: PBS).
Cell uptake assays
Seed the cells were in 24-well plates with a cell concentration of 1.0 × 105 cells/well. After complete attachment, replace 1 mL serum-free medium containing 99mTc-Hynic-mAbNectin-4 (37 kBq) to each well and incubation was performed at 37 °C for multiple time points (1, 2, 3, 4,6, and 8 h). For each time point, rinse the radiotracer-treated cells with 800 μL pre-cooling PBS twice. Collect the rinsed PBS as supernatants. Then 800 µL NaOH (1 N) was added for cell lysis and collected as lysates. Radioactivity of the supernatants and lysates was detected using automatic gamma-counter (PerkinElmer, USA). After attenuation correction, the cell uptake rate is determined as: Alysate/(A supernatant + Alysate) × 100%. For blocking studies, experimental cells were incubated with excess unradiolabeled mAbNectin-4 for 1 h before experiments.
Animals and tumor modeling
All experimental animal examinations were conducted following Institutional Animal Care and Use Committee of Huazhong University of Science and Technology-approved protocol. 150 µL cell suspension (containing 1 × 107 MDA-MB-468 or MCF-7 cells, Matrigel and sterile PBS mixed in a ratio of 1:1) was implanted into the right axilla to each mouse (Balb/c-nude, female, aged between 4 and 6 weeks,) for subcutaneous xenograft inoculation. Mice with a tumor volumes of 200–300 mm3 were applied for in vivo imaging and that of 100 mm3 for PTT.
SPECT/CT imaging and ex vivo biodistribution
All SPECT/CT images were scanned by microPET/SPECT/CT multimodal imaging system (InliView-3000B, Novel Medical™, Yongxin Medical Equipment Co., Ltd., Beijing, China). Thirty-seven MBq 99mTc-Hynic-mAbNectin-4 (about 10 µg mAb, within 150 µL PBS) was administrated via the tail vein. Maximum intensity projection (MIP) and transaxial images were acquired at 3, 6, 12, 24, and 36 h post injection (p.i.). Non-contrast-enhanced CT (50 keV, 0.5 mA) was performed for the attenuation-correction of SPECT data. Anaesthetization was perfomed with isofurane amid the scanning duration. For the blocking study, MDA-MB-468 xenograft tumor-bearing mice received excess cool mAbNectin-4 (1 mg) 2 h prior to the radiotracer injection. For semi-quantitative analysis, the regions of interest (ROIs) of the tumor and the contralateral normal muscle were delineated on selected transaxial images and the radioactivity count was obtained. The T/M ratio was calculated as counttumor/countmuscle.
The ex vivo biodistribution studies were performed to quantify the 99mTc-Hynic-mAbNectin-4 uptake in relevant organs. After the last time-point of SPECT/CT imaging, sacrifice the mice and collect the chosen organs/tissues (the blood, brain, heart, lung, liver, spleen, kidney, pancreas, stomach, small intestine, large intestine, muscle, bone, and the tumor). Following washing with PBS, weights and radioactivity of each organ were examined. After attenuation correction, the uptake of each tissue was computed and described as %ID/g. For semi-quantitative analysis, the tumor to blood (T/B) and tumor to muscle (T/M) radioactivity ratio were also computed.
Synthesis and characterization of mAbNectin-4-ICG
ICG-NHS-ester (4 mg, dissolved in 50 μL DMSO; Xi’an Ruixi Biological Technology, China) was added to purified mAbNectin-4 (150 μg, 1 nmol), the mixture was reacted with continuous oscillation for 12 h (4 °C, away form light) to synthesize mAbNectin-4-ICG mixture. The mAbNectin-4-ICG mixture was further purified with PD-10 columns (mobile phase: PBS). The absorption spectrum of samples were detected by UV−vis–NIR spectrometer. The storage stability of mAbNectin-4-ICG was also tested away light till 48 h.
Prepare ICG solution with graded concentrations within PBS (1, 5, 10, 20, 50, 100, 250, 500, and 1000 μg/mL), and detect the optical density (OD) values with an microplate reader. Take the ICG concentrations as the abscissas and the OD value as the ordinates to built the ICG concentration-absorbance standard curve and obtain a regression equation. The standard curve is applied to calculate the ICG concentration in the in mAbNectin-4-ICG solution in further studies.
Photothermal property of mAbNectin-4-ICG
To explore the photothermal performance of the mAbNectin-4-ICG, 100 μL of mAbNectin-4-ICG aqueous solution containing different ICG concentrations [0 (water), 1, 5, 10, 20, and 30 μg/mL] placed in separate plate (Corning™, Stripwell Plates, 1 × 8 strips, 0.36 mL) was irradiated with 808 nm laser (Honglan Electronic Technology Co., Ltd. Beijing, China) with multiple power densities (0.1, 0.3, 0.5, 0.8, 1.0, and 1.5 W/cm2). The solution temperature was detected by a handheld infrared thermal detector (FLIR®, E8xt, FLIR Systems, Inc. USA). Photothermal stability was explored following 4 laser on–off cycles: mAbNectin-4-ICG solution (20 μg/mL) was treated with laser irradiation (1 W/cm2, 3 min), then the laser was turned off for cooling solution to room temperature.
Biological safety and in vitro targeting of mAbNectin-4-ICG
Seed MDA-MB-468 cells in a 96-well plate (density: 1.0 × 104) in 100 μL complete L-15 medium. After overnight cultivation, switch to serum-free medium containing mAbNectin-4-ICG/free ICG with different ICG doses (1, 5, 10, 20, 30, 40, 50, 100, and 200 μg/mL) and continue incubating for 4 h. Rinsed the cells by PBS twice, and co-incubate the cells with 1 × Cell counting kit-8 (CCK-8) solution for another 4 h. Utilizing a multi-functional microplate reader to detect the absorbance at 450 nm and calculate the cell viability (n = 5).
Following same seeding conditions, after complete attachment, co-incubate MDA-MB-468 cells with mAbNectin-4-ICG or free ICG (20 μg/mL, 100 μL/well; within serum-free medium) for 4 h. The treated cells were flushed using PBS twice, then imaged with an IVIS Spectrum imaging system (Bruker, Germany; excitation/emission of 750/790 nm fliters). For blocking studies, cells were incubated previously with excess mAbNectin-4 for 1 h.
In vitro PTT
MDA-MB-468 cells (seeded in 96-well plate; 1.0 × 104 cells/well; 100 μL medium; incubated overnight) was applied for in vitro PTT studies. Employ a standard CCK-8 assay to test the cell viability: (1) For ICG concentration-dependent study, cells were treated with serum-free medium containing graded ICG doses (0, 1, 5, 10, 20, and 30 μg/mL) in either mAbNectin-4-ICG or free ICG and continue incubated for 4 h. Serum-free L-15 medium was switched after washing cells with pre-cooling PBS twice, and then the cells were exposed to the laser irradiation (1.0 W/cm2, 10 min); (2) For power density-dependent study, all cells were incubated with mAbNectin-4-ICG/free ICG (containing 20 μg/mL ICG) and the irradiation was performed with graded power densities (0.1, 0.3, 0.5, 0.8, 1.0, and 1.5 W/cm2; 10 min); and (3) For irradiated time-dependent study, cells were incubated with mAbNectin-4-ICG/free ICG (20 μg/mL ICG), following by laser irradiation with 1.0 W/cm2 power for different durations (0, 2, 4, 6, 8, and 10 min). In vitro PTT on MCF-7 cells was also performed (ICG concentration: 20 μg/mL; laser intensity: 1 W/cm2; and irradiated time: 10 min).
Calcein AM/PI staining was also performed for verifying the cellular viability. Seeded MDA-MB-468 cells (1.0 × 104 cells/well in 96-well plates) were co-incubated with mAbNectin-4-ICG or free ICG (20 μg/mL ICG) for 4 h. Then irradiate the cells using 808 nm laser (1.0 W/cm2, 10 min). Calcein AM/PI doulbe staining was taken and the FL imaging was performed by microscope.
In vivo and ex vivo fluorescence imaging
MDA-MB-468 xenograft tumor-bearing mice were administrated intravenously with 200 μL mAbNectin-4-ICG, free ICG (1 mg/kg ICG dose), or saline. Mice were anesthetized by 1% pentobarbital sodium during imaing process. The in vivo fluorescence (FL) imaging at multiple time points (3, 6, 12, 24, 36, and 48 h) was achieved with IVIS Spectrum imaging system with Excitation/Emission of 750/790 nm) and analyzed using Bruker MI software. The ROIs of the tumor area (tumor) and the lower limb muscle (muscle) were delineated and the corresponding FL intensity was obtained. The T/M ratio was calculated as Intensitytumor/Intensitymuscle. After the last scanning time point, all mice were killed to collect the tumors and major organs to conduct the ex vivo FL imaging. The intensity of each organ and tumors were measured for quantification and comparison.
In vivo PTT for TNBC tumor
The tumor-bearing mice were indiscriminately assigned to 4 groups (① mAbNectin-4-ICG + laser; ② free ICG + laser; ③ saline + laser; and ④ saline; n = 6) and received different treatments. All groups were administrated with 200 μL mAbNectin-4-ICG, free ICG solution (1 mg ICG/kg) or saline via tail veins, respectively. Twenty-four h post injection, ①, ②, and ③ groups were irradiated with 808 nm laser (1.0 W/cm2, 5 min) whereas ④ group did not underwent laser therapy. During the irradiation, anesthesia was maintained with isoflurane, and the tumor temperature and the corresponding thermal images were detected. The tumor volume (length and width) and body weight were measured during the 30-day follow-up period. The tumor volume and relative tumor volume were computed as V = D × d2/2, and VR = VX/V0, respectively (D refer the maximum diameter of tumor, and d refer the minor diameter; VX refer the volume on day X, and V0 refer the initial tumor volume prior to treatment). On the 30th day, sacrifice all mice and weight the tumor tissues. Moreover, harvest the blood and the selected organs. The hematology tests [white blood cells (WBC), platelets (PLT), red blood cells (RBC), and hemoglobin (HGB)], blood biochemistry tests [alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), and creatinine (CRE)].Hematoxylin and eosin (H&E) staining of selected organs were processed to evaluate the systematic toxicity.
Statistical analysis and charting were conducted employing GraphPad Prism software (version 8.0, USA). The data are described as mean ± standard deviation. p < 0.05 was regarded as statistically significant.