Radio-photothermal therapy mediated by a single compartment nanoplatform depletes tumor initiating cells and reduces lung metastasis in the orthotopic 4T1 breast tumor model.

Preparation and characterization of CuS NPs CuS

NPs were prepared following our previously published method. 12 In brief, 1.0 mg of methoxy-PEG-thiol (mPEG-SH; 5000 Da, Sigma-Aldrichwas added into 10.0 mL aqueous solution of CuCl 2 (4 mM) upon stirring at room temperature. Ten minutes later, 40 μL sodium sulfide solution (Na 2 S, 1M) was added to the solution. Then, the reaction solution was heated to 90 °C until the color changed to dark green. The mixture solution was moved to ice water. Finally, the as-prepared CuS NPs were purified by a Centrifugal Filter Units (Amicon Ultra 15a, Millipore, Billerica, MA) and stored at 4°C till use. The morphology and size of the CuS NPs particle were measured by transmission electron microscopy (JEOL JEM-2010) and dynamic light scattering analysis (ZetaPLUS, Brookhaven Instruments Corp., Holtsville, NY). The optical property of the CuS NPs was monitored by a a UV–vis spectrometer (Beckman Coulter DU-800, Brea, CA). TGA was performed using a thermogravimetric analyzer (TGA-50, Shimadzu Inc., Columbia, MD, USA). Samples were heated from 25 °C to 600 °C at an increasing rate of 20 °C per minute. Radiolabelling Radiolabeled [ 64 Cu]CuS NPs were prepared following previously published protocol. 10 Briefly, a trace amount of radioactive 64 CuCl 2 (10 μL, 148 MBq) was mixed with non-radioactiveCuCl 2 solution (190 μL, 4 mM) containing 0.2 g/L of PEG-SH. After 10 minutes, sodium sulfide (10 μL, 100 mM) was added to the 64 Cu-CuCl 2 mixture. Then, the mixture was heated to 90 °C for 15 min. The solution was put on ice-water to obtain radioactive [ 64 Cu]CuS NPs. The labeling efficiency of the [ 64 Cu]CuS NPs were tested using instant thin-layer chromatography (ITLC, IAR-2000, Bioscan). Aliquot of [ 64 Cu]CuS NPs was dropped on a ITLC strip. The strip was then developed with a phosphate-buffered saline (PBS) containing ethylenediaminetetraacetic acid (4 mM). Radioactivity on teh strip was read and quantified by a radio-TLC imaging scanner (model IAR-2000, Bioscan, Washington, DC). To investigate the radiolabeling stability, [ 64 Cu]CuS NPs were incubated in PBS or mouse serum at 37 °C for 24 h. The solution was then re-analyzed using ITLC. Photothermal Effect in CuS NPs solution A 808-nm NIR laser (continuous-wave, 15PLUS Laser, Diomed) was used to record temperature change in the presence of CuS NPs. The laser light (1.5 W/cm 2 ) was passed through a quartz cuvette containing either an aqueous solution of CuS NPs (100 μg/mL, 400 μL) or pure water (control). The temperature of the samples were monitored by a thermocouple over a period of 4 min. Micro-PET/CT imaging, biodistribution, and autoradiography All experiments involving animals were approved by the MD Anderson Institutional Animal Care and Use Committee (IACUC). All mice were supplied by Charles River Laboratories (Wilmington, MA). The BT-474 tumor models were generated by subcutaneous injection of 2 × 10 6 BT-474 (American Type Culture Collection, Manassas, VA) into the right front arms of female nude mice. Each mice were supplemented with 0.72 mg of 17β-estradiol pellets (Innovative Research of America, Sarasota, FL) 3 days prior to tumor cells inoculation. The tumor-bearing mice were randomly divided into two groups (n = 3 mice/group) when the tumor size reached 50-150 mm 3 . Mice in Group 1 were intratumorally injected with [ 64 Cu]CuS NPs (2 mM, 0.2 mg/mL, 4 OD, 10 μL, 7.4 MBq/mouse). Mice in group 2 were intratumorally injected with 64 CuCl 2 (2 mM, 10 μL, 7.4 MBq/mouse). Twenty-four hours later, the mice were anesthetized, and PET/CT images were obtained by a μPET/CT scanner (Inveon, Siemens). All the mice were sacrificed by CO 2 exposure at the end of the imaging session. Major organs including blood, heart, liver, spleen, kidney, lung, stomach, intestine, muscle, bone, brain, bladder, and tumor were removed and weighed. The radioactivities of the organs were counted by a gamma counter (Packard Cobra). Uptake of [ 64 Cu]CuS NPs or 64 CuCl 2 in different organs was defined as a percentage of the injected dose per gram of tissue (%ID/g). For autoradiography, tumor tissues were resected and cryosectioned into 20-μm slices. Then, the slices were exposed on a Fuji film (BAS-SR 2025) for 24 h. The film was scanned by a Multifunctional Imaging System (Fuji Film FLA5100 Life Science, Valhalla, NY). Tumor growth delay in subcutaneous BT-474 breast tumor model Twenty-four BT-474 mice were randomly arranged into 4 groups (n = 6 mice/group) when the tumor volume reached 50 to 150 mm 3 . Mice in the PTT group were intratumorally injected with nonradioactive CuS NPs (2 mM, 0.2 mg/mL, 4 OD; 10 μL) followed by NIR laser light exposure. Mice in the RT group were intratumorally injected with [ 64 Cu]CuS NPs (2 mM, 0.2 mg/mL, 4 OD; 10 μL, 7.4 MBq/mouse). Mice in the RT/PTT group were intratumorally injected with [ 64 Cu]CuS NPs (2 mM, 0.2 mg/mL, 4 OD; 10 μL, 7.4 MBq/mouse) followed by NIR laser treatment. Laser treatment was instituted 24 h after NP injection with a 808-nm NIR laser beam at 1.5 W/cm 2 for 3 min. Mice in the control group were not treated. Thermographic photos were acquired by a Flir i7 thermal camera (Flir Systems Inc., Portland, OR). The tumor sizes were monitored 2 or 3 times a week using a caliper and were calculated as follows: (tumor length) × (tumor width) 2 /2. All mice were killed on day 30. The tumor tissues were removed, snapfrozen, and sectioned into 5-μm slices. The slices were stained with hematoxylin & eosin (H&E) for histology analysis. Antitumor activity against orthotopic 4T1 breast tumors in Balb/c mice To established orthotopic breast tumor, 4T1 cancer cells (5 × 10 6 , American Type Culture Collection) were inoculated into mammary fat pads of female Balb/c mice. When the tumor volume reached 50 to 150 mm 3 , 4T1 tumor–bearing mice were randomly divided into 4 groups (n = 10 mice/group). Mice in the control group were not treated. Mice in the RT and RT/PTT groups were injected intratumorally with [ 64 Cu]CuS NPs (2 mM, 0.2 mg/mL, 4 OD; 10 μL, 7.4 MBq/mouse). Mice in the PTT group were injected intratumorally with nonradioactive CuS NPs (2 mM, 0.2 mg/mL, 4 OD; 10 μL). Mice in PTT and RT/PTT treatment groups were also treated with NIR laser (808 nm, 1.5 W/cm 2 for 3 min) 24 h after NP injection. The animals were monitored daily for survival. The mice were sacrificed when one dimension of tumor reached 15 mm.

Assessment of lung metastasis

For the assessment of lung metastasis, female Balb/c mice bearing 4T1 tumors were treated as described in the previous section (n = 3 mice/group). Mice were killed when tumor size at the primary sites had reached 15 mm. Lungs were removed, fixed in formalin, sectioned to 5-μm slices, and stained with H&E for histology analysis.

Tumorsphere formation assay

A fraction of harvested 4T1 tumor tissues (~0.5 g) was washed with PBS, and minced in digestion solution (RPMI 1640 medium with 0.2% collagenase-II and 1.5% bovine serum albumin faction V) into 1 mm 3 slices. The minced tumor was then added to 10 mL digestion solution and shaken vigorously at 37 °C for 45 min. After digestion, the mixture was filtered through 100-μm strainer to remove acellular debris. The resultant cell suspension was then centrifuged at 900 RPM for 5 min and washed again with RPMI 1640 with 1% bovine serum albumin. Red blood cells were removed by pipetting the cell pellet in RBC lysis buffer. The resultant cells were washed again, resuspended in RPMI 1640 (1% BSA), and counted. Tumorspheres were grown in 24-well ultralow attachment plates (sigma-aldrich). Each well was added with 5000 tumor cells and 0.5 mL sphere-forming medium (RPMI-1640, 10 ng/mL β-FGF, 10 ng/mL EGF, and 4 μg/mL heparin). Cells were cultured for 7 days at 37 °C. The suspension was collected into Eppendorf tubes and pipetted to disperse cell aggregates. Tumorspheres were collected by centrifugation at 200 g for 5 min, and fixed with 4% formalin in PBS. The number of tumorspheres was counted under a microscope.

Statistical analysis

Quantitative data analysis were defined as mean ± standard deviation (SD). Student's t- test was used to compare mean value. A logrank test was used to determine significance of survival.. p < 0.05 was considered statistically significant.