4.1 Synthesis and Characterization of PAC-SABIs: The intermediates including Pep and Pep-PEG were acquired by solid phase peptide synthesis, and PAC-SABIs were obtained by liquid phase synthesis. The detailed synthesis procedures and mass spectrum analysis are presented in the Supporting Information.
4.2 Fluorescence Assays: The CACs of Pep and Pep-PEG were determined using the ANS fluorescence assay. ANS was dissolved in dimethylformamide (DMF) to a concentration of 1 mM. Subsequently, 1 µL of ANS was added to 100 µL of varying concentrations of Pep and Pep-PEG. The resulting solution was transferred to a quartz cuvette and analyzed using a Perkin Elmer LS-55 fluorescence spectrophotometer (Perkin Elmer, USA). The CAC was determined from the matched curve, which was obtained by the ANS fluorescent intensities at 475 nm plotted against different concentrations of Pep and Pep-PEG.
The self-assembly process of Pep and Pep-PEG was monitored using NBD fluorescence. The fluorescence intensities of NBD-labeled Pep and Pep-PEG were measured at CAC and half of the CAC concentrations via a fluorescence spectrometer (excitation wavelength: 467 nm). To evaluate the ALP-triggered dephosphorylation and in situ self-assembly process, the NBD fluorescence intensity was detected after incubating Pep and Pep-PEG at CAC concentration with ALP (1 U / mL) at 37 °C for 1 h.
4.3 CD Spectroscopy: All the CD spectra analyses were recorded on a CD spectropolarimeter (Applied Photophysics Ltd, UK) in quartz cuvettes with an optical path length of 0.1 cm. Data were collected from 190 to 260 nm with a scanning speed of 100 nm/min at room temperature.
4.4 FTIR Spectroscopy: All the FTIR analyses were carried out by a Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific, USA). A 100 µL solution of different formulations was spun down at 10000 g for 1 h, and the pellet was blown dry with nitrogen. The dry samples were mixed with KBr and pressed into pellets for further analysis.
4.5 TEM Characterization: Pep and PAC-SABI solutions (100 μM) with or without ALP (1 U / mL) incubation were prepared and placed at room temperature for morphology observation at different time points. Copper grids with a porous carbon mesh were used for sample preparation. 10 µL of the Pep and PAC-SABI solution was placed onto a copper grid with a carbon membrane and left for 3 min, followed by removal of excess solutions with filter papers. Then, a small drop of uranyl acetate solution (2% weight/volume in water) was added to the copper grid for negative staining of samples, and the grid was blotted with filter paper after 1 minute. Finally, the samples were left on filter paper overnight to facilitate further analysis.
4.6 Cell Culture, Animal studies and human specimens: Murine BC 4T1 cell line, murine PC PAN02 cell line, human BC MDA-MB-231 cell line, murine macrophage cell line RAW264.7, and human macrophage cell line THP-1 were purchased from the American Type Culture Collection (ATCC) and cultured according to the supplier’s recommendations, supplemented with 10% fetal bovine serum (FBS) and antibiotics. The cell culture supernatant of BC cell lines 4T1 and MDA-M-231 was collected as TCM, and was used to induce the transformation of RAW264.7 macrophages and phorbol myristate acetate (PMA; 100 ng/ml)-treated THP-1 macrophages into TAMs, respectively.
C57BL/6 mice (male, 6 weeks old) were purchased from BesTest Bio-Tech Co., Ltd (Zhuhai, China), and BALB/c mice (female, 6 weeks old) were purchased from Guangdong Nanmo Biotechnology Co., Ltd (Zhongshan, China). All animal experiments were carried out in compliance with NIH guidelines and all animal-experimental protocol were approved by the Animal Experimentation Ethics Committee of Zhujiang Hospital of Southern Medical University (LAEC-2021-040 and LAEC-2022-073).
After obtaining approval from the institutional review board of Nanfang Hospital and Guangdong Provincial People’s Hospital of Southern Medical University (NFEC-2023-309, KY2023-179-01), human peripheral blood, BC and PC specimens were obtained, and the appropriate informed consent was obtained for all sample donors.
4.7 BMDMs and HDDMs Generation and Stimulation: BMDM isolation was performed according to a previously published protocol with minor modification. Briefly, after sacrificing C57BL/6 mice and disinfecting the skin with 75% alcohol, the hind legs were cut off and placed in a sterile petri dish containing sterile and ice-cold PBS. The bone marrow was flushed with PBS using a syringe with a 25-gauge needle. The supernatant bone marrow cells were collected and then washed with PBS and resuspended with complete conditioned media for BMDM differentiation (100 mL complete medium consisted of 74 mL Dulbecco’s modified Eagle’s medium (DMEM) + 15 mL macrophage colony-stimulating factor (M-CSF, 25 ng/mL) + 10 mL FBS + 1 mL penicillin-streptomycin solution (PS)), seeded on tissue culture plates, and incubated at 37 °C with 5% CO2. Primary HDDMs were generated from venous blood of healthy volunteers, diluted with 2 × PBS (pH 7.4) and separated with Ficoll density gradient as described previously. Monocytes were then differentiated into macrophages by culture in Iscove’s modified Dulbecco’s medium (IMDM) + 10% AB human serum (Life Technologies) for 7 to 10 days. To stimulate macrophages with TCM, we cultured BMDMs and HDDMs with complete medium containing 50% TCM from 4T1 and MDA-MB-231 cells, respectively.
4.8 Characterization of the Self-Assembly Process of PAC-SABI in 2D and 3D Cell Culture Environments: 4T1 or PAN02 cells were inoculated and incubated overnight at a density of 1 × 105 cells / dish in laser confocal petri dishes. NBD-labeled PAC-SABIs (100 μM) were co-incubated with 4T1 or PAN02 cells for 30, 60, and 120 min, followed by washing with PBS three times. Then, the NBD fluorescence at different time points was observed by CLSM imaging. To reveal the self-assembly process of PAC-SABI on cancer cell membranes, 4T1 (1 × 105 cells/dish) cells were cultured with NBD-labeled PAC-SABI (100 μM) for 120 min. After 3 rounds of washing with PBS, the membrane dye CellTracker CM-Dil (1 mM; Invitrogen, USA) was added and incubated for 5 min at 37 °C, and then for an additional 15 min at 4 °C. CLSM imaging was used to detect the spatial distribution of NBD and Dil fluorescence.
3D spheroids of 4T1 cells were constructed according to the previously reported method. Cy5.5-labeled PAC-SABI (100 μM) was co-incubated with 4T1 3D spheroids for 90 min, followed by washing with PBS three times. Then, the outermost cells and nuclei of the spheroids were stained with Calcein-AM and Hoechst 33342, respectively. Finally, CLSM imaging was used to detect spatial distributions of fluorescence signals with Cy5.5, FITC and DAPI channels in spheroids and different focal plane along the z axis.
4.9 Detection of Cell Surface Distribution Using Bio-TEM: The 4T1 and PAN02 cells were seeded on cell culture dishes with a diameter of 10 mm at a density of 1 × 105 cells / dish and cultured overnight, followed by replacement with fresh culture medium containing PAC-SABIs at a concentration of 100 μM for 120 min. Then, the PAC-SABI-containing medium was removed and the cells were washed three times with PBS. The cells attached to the bottom of the dishes were gently harvested using a cell scraper and collected in a centrifuge tube. After centrifugation at 1500–3000 rpm for 5 min, the supernatant was carefully discarded, and then a 4 °C precooled fixative was slowly added along the tube wall and then placed in a 4 °C refrigerator overnight. The samples were then fixed with a 1% osmium acid solution for 1 h followed by dehydration using a gradient concentration of ethanol. The samples were embedded and sliced with a LEICA EM UC7 ultra-thin microtome to obtain 70–90 nm slices. The sections were stained with lead citrate solution, uranyl acetate, and 50% ethanol saturated solution for 5-10 min, respectively, and then observed under TEM imaging.
4.10 Detection of Cell Surface Distribution Using SEM: 4T1 and PAN02 cells were seeded into 12-well plates containing cover glass slips at a density of 1 × 105 cells / well and cultured overnight. PAC-SABIs were added into each well at the final concentration of 100 μM and cultured for 120 min. Then, PAC-SABI-treated 4T1 and PAN02 cells were dehydrated for the time course of 24 h, processing from 30% to 100% ethanol. The samples were then sputter coated with gold (CRC-150 Sputter Coater, USA) and imaged using a Zeiss Ultra 55 SEM (Carl Zeiss, Germany).
4.11 Phagocytosis Assay Using Flow Cytometry: The in vitro phagocytosis assays described in this study were performed by co-culture GFP+ 4T1 cells and macrophages at a ratio of 100,000 target cells to 50,000 macrophages for 120 min in a humidified, 5% CO2 incubator at 37 °C in ultra-low-attachment 96-well U-bottom plates (Corning, USA) in serum-free IMDM. 4T1 cells with endogenous fluorescence were harvested from plates using TrypLE Express (Life Technologies, Poland) and treated with PAC-SABIs for 120 min prior to co-culture. After co-culture, phagocytosis assays were stopped by placing plates on ice, centrifuged at 400 g for 5 min at 4 °C and stained with anti-CD11b (Biolegend, cat. no. 101209, clone M1/70) to identify macrophages. Assays were analyzed by flow cytometry on a Sony SA3800 Flow Cytometer (Sony Biotechnology, Japan) or a CytoFLEX (Beckman, USA). Phagocytosis was measured as the number of CD11b+, GFP+ macrophages, quantified as a percentage of the total CD11b+ macrophages.
4.12 Phagocytosis Assay Using Live-cell Microscopy: Non-fluorescently labeled 4T1, PAN02, or MDA-MB-231 cells were harvested and labeled with pHrodo Red, SE (Thermo Fisher Scientific, USA) as per manufacturer instructions at a concentration of 1:30,000 in PBS for 1 h at 37°C, followed by two washes with DMEM + 10% FBS + 100 U/mL PS. 50,000 macrophages were added to a transparent 96 well plate and allowed to adhere at 37°C. After macrophage adherence, 100,000 pHrodo-Red-labeled 4T1, PAN02, or MDA-MB-231 cells pretreated with PAC-SABIs for 120 minutes were added in serum-free IMDM. The plate was centrifuged gently at 50 g for 2 min in order to promote the timely settlement of 4T1, PAN02, or MDA-MB-231 cells into the same plane as adherent macrophages. Phagocytosis events were calculated as the number of pHrodo red+ events per visual field and the fluorescent signals were captured by a Nikon AX confocal laser microscope (Nikon, Japan).
4.13 In Vivo and Ex Vivo Fluorescence Imaging: To obtain breast and pancreatic subcutaneous xenografts, 1 × 106 4T1 or PAN02 cells were implanted into the right hind limb of BALB/c and C57BL/6 mice, respectively. When the tumor size reached ≈ 50 mm3 mice were randomly sorted into free Cy5.5, Cy5.5SAMI, and Cy5.5PAC-SABI groups. The 4T1 and PAN02 tumor-bearing mice were correspondingly intravenously injected with free Cy5.5, Cy5.5-labled SAMI, and Cy5.5-labled PAC-SABI (the dose of Cy5.5 was 1 mg / kg) for in vivo fluorescence imaging with IVIS system (Perkin Elmer, USA). Then, the mice were sacrificed to collect the major organs (heart, liver, spleen, lung, and kidney) and tumors for ex vivo fluorescence imaging. Finally, the 4T1 and PAN02 tumors were embedded in Tissue-Tek OCT compound (Sakura, Japan), and cryosections of 8 µm thickness were prepared. The Cy5.5 signal was detected using a fluorescence microscope (Nikon, Japan).
4.14 Blood Examination and Histology: To record the complete blood count data, healthy BALB/c mice were intravenously injected with PAC-SABIs at a dose of 15 mg / kg every other day for a total of 4 administrations. The mice were sacrificed before blood collection (0.5 mL), and complete blood count evaluations at 1, 7, 14 and 21 d postinjection of PAC-SABIs were carried out at the Nanfang Hospital of Southern Medical University. After about 1 month following treatment with PAC-SABIs, the mice were sacrificed and the major organs (heart, liver, spleen, lung and kidney) were collected. The organs were immersed in a 4% paraformaldehyde solution for an overnight fixation period, followed by dehydration in a 25% sucrose solution. Subsequently, the fixed tissues were sliced into sections with a thickness of 8 μm, and the sections were stained with H&E (Beyotime Biotech, China) as per the manufacturer’s instructions. Finally, a microscope was employed to examine the samples for any histological alterations.
4.15 Anticancer Treatment Studies: The anticancer efficacy of PAC-SABIs was evaluated in the breast and pancreatic orthotopic xenograft tumor models. 4T1-luc (1 × 106) suspended in a 25 µL PBS and Matrigel (Corning, USA) mixture (1:1, v/v) was injected into the right third mammary fat pad of BALB/c mice. PAN02-luc (1 × 106) suspended in a 25 µL PBS and Matrigel (Corning, USA) mixture (1:1, v/v) were injected into the tail region of the pancreatic parenchyma of C57BL/6 mice. The PAC-SABI treatment began 5 days after the tumor implantation, and the BLIs of orthotopic 4T1-luc and PAN02-luc tumor-bearing mice were detected every 7 days to monitor the tumor growth in each treatment group. For BLI, mice were given D-luciferin potassium salt (150 mg/kg) intraperitoneally and imaged 10 min later in an IVIS system (Perkin Elmer, USA). Survival (in days) of mice in the different treatment groups were monitored throughout the period of study.
In order to study the amplified anticancer immune therapy, 4T1-luc (1 × 106) suspended in a 25 µL PBS and Matrigel (Corning, USA) mixture (1:1, v/v) was injected into the right third mammary fat pad of BALB/c mice. On the 5th day, 4T1 tumor-bearing mice were randomly divided into 4 groups, and were immunized with PAC-SABIs (15 mg / kg) or PBS on day 5, 7, 9, and 11 through intravenous injection. Then, intraperitoneal injection with or without anti-PD-1 antibody (10 mg / kg) was performed on days 12, 15, 18, and 21. Mouse body weights and tumor sizes (length and width measured by calipers) were measured every other day. The tumor volume was calculated with following the formula: V = (L × W2)/2, where V is the volume (mm3), L is the biggest diameter (mm), and W is the smallest diameter (mm). To study the role of PVA-CD40 in prevention of lung metastasis, the histological examination of representative lung sections was conducted using H&E staining at the end points of different treatment procedures. The mice were monitored regularly for death throughout the whole 60-day survival period.
4.16 Analysis of Macrophages and T Cells in Tumor: The 4T1 tumor-bearing mice were euthanized, and tumor tissues were collected and frozen in optimal cutting temperature medium on dry ice. Tumor sections were cut using a cryotome, mounted on slides and stained with different primary antibodies: Anti-F4/80 antibody (Abcam, cat. no. ab6640), Anti-CK19 antibody (Abcam, cat. no. ab52625), Anti-CD8 antibody (Invitrogen, cat. no. MA5-29682) overnight at 4 °C according to the manufacturer’s instructions. Following the addition of fluorescently labelled goat anti-rat IgG H&L (Abcam, cat. no. ab150167) and goat anti-rabbit IgG H&L (Abcam, cat. no. ab150077), the slides were analyzed with a confocal microscope (Nikon, Japan)
For flow cytometry analysis, 4T1 tumors were cut into small pieces and homogenized in cold staining buffer to form single cell suspensions in the presence of digestive enzyme. Cells were stained with fluorescence-labelled antibodies: anti-mouse CD45 antibody (Elabscience, cat. no. E-AB-F1136J, clone 30-F11), anti-mouse CD11b antibody (Biolegend, cat. no. 101209, clone M1/70), anti-mouse F4/80 antibody (Biolegend, cat. no. 123116, clone BM8), anti-mouse CD206 antibody (Biolegend, cat. no. 141703, clone C068C2), anti-mouse CD80 antibody (Biolegend, cat. no. 104705, clone 16-10A1), anti-mouse CD3 antibody (BDbioscience, cat. no. 555274, clone 17A2), anti-mouse CD4 antibody (BDbioscience, cat. no. 553051, clone RM4-5), anti-mouse CD8 antibody (BDbioscience, cat. no. 553033, clone 53-6.7), and anti-mouse Foxp3 antibody (BDbioscience, cat. no. 563101, clone R16-715) following the manufacturer’s instructions. The stained cells were measured on a Sony SA3800 Flow Cytometer (Sony Biotechnology, Japan) or a CytoFLEX (Beckman, USA). The numbers presented in the flow cytometry analysis images are percentage based.
4.17 Statistical analysis: Data are presented as mean ± standard error of the mean (S.E.M.). Student’s t-test was used for two-group comparisons, and Kaplan–Meier curves were used to analyze the survival study. Statistical significance was defined as *p < 0.05, **p < 0.01 and ***p < 0.001. Statistical analysis was performed using GraphPad Prism 5 and SPSS version 22.0.