Peptides
CKS1 (NGRKACLNPASPIVKKIIEKMLNS), FITC-CKS1 (CKS1 with FITC tagged at the N-terminus), the helical region of CKS1 (IVKKIIEKMLNS), the loop region of CKS1 (NGRKACLNPASP), NC1 (NGRKACLNPASPIVEEIIEEMLNS), and NC2 (NGRKACLNPASPIVKKIIPEKMLNS) were synthesized by Genscript (Piscataway, NJ) using a solid-phase peptide synthesis method. There is a free amine at the N-termini of all these peptides while they were all amidated at the C-terminus. The purity was > 90% as verified by HPLC and MS analyses. CKS1, the fragments of CKS1, and FITC-CKS1 were solubilized in water. NC1 and NC2 were solubilized in DMSO.
Cell culture
4T1 murine mammary carcinoma cells (CRL-2539), CT26 murine colon carcinoma cells (CRL-2638), U2OS human osteosarcoma cells (HTB-96), and NIH/3T3 normal murine fibroblast cells (CRL-1658) were purchased from the American Type Culture Collection (Manassas, VA). MCA205 murine fibrosarcoma cells (SCC173) were purchased from MilliporeSigma (Burlington, MA). Cell lines were mycoplasma-tested before being used. 4T1 cells and MCA205 cells were propagated in RPMI 1640 (Corning, Corning, NY) supplemented with 10% FBS (MilliporeSigma). NIH/3T3 cells and U2OS cells were propagated in DMEM (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS. HUVECs were propagated in EGM-2 bulletkit (LONZA, Basel, Switzerland). All cell lines were grown in T75 tissue culture flasks (Sarstedt, Nümbrecht, Germany) under standard culture conditions of 37°C and 5% CO2.
Prediction of the peptide structures
The structures of the peptides were predicted by the protein structure prediction software AlphaFold 2 [31]. The prediction of the protein structure was done using the ColabFold platform [32] and was visualized by Pymol [32]. The electrostatic property of the peptides was calculated using the APBS plugin in Pymol.
Animal models
The protocols used in this study were approved by the Institutional Care and Use Committee at Johns Hopkins Medical Institutions. Four- to eight-week-old female Balb/c mice were obtained from Charles River (Wilmington, MA). For the 4T1 triple breast cancer model, 2.5 × 104 4T1 cells were injected into the first mammary fat pad of each mouse. For the CT26 colon carcinoma model, 1.0 x 106 CT26 cells were injected subcutaneously in the left flank. After 1 week, we started daily intratumoral treatment with 40 mg/kg CKS1. The tumor size was measured by using a caliper, and the volume was calculated by using the formula 0.52 × (length) × (width)2.
Real-time cell viability assay
3,000 cells were seeded in the wells of an xCELLigence RTCA E-plate 16 (Agilent, Santa Clara, CA). After the cells adhered to the plate, the media was replaced with serum free media, and the cells were treated with CKS1 at the indicated concentrations. The viability of the cells was monitored using the xCELLigence RTCA analyzer (Agilent).
LDH assay
1.0 x 104 cells were seeded in a 96-well plate and incubated overnight. The cells were washed once, and the medium was replaced with serum free media. Cells were treated with CKS1 for the indicated time and concentration. CyQUANT LDH cytotoxicity assay (Thermo Fisher Scientific) was used following the manufacturer's instructions to measure the release of LDH. LDH release was normalized to the amount of LDH released from cells treated with lysis buffer.
BrdU assay
1,000 cells were seeded in wells of a 96-well plate. The cells were treated with the indicated concentration of CKS1 immediately after seeding. 48 hours later, 5-bromo-2'-deoxyuridine (BrdU) was added to each well. 24 hours later, BrdU incorporation was quantified using the BrdU cell proliferation assay kit (Cell Signaling Technology, Danvers, MA) following the manufacturer's instructions.
Electron microscopy
Samples were fixed in 2.5% glutaraldehyde, 3 mM MgCl2 in 0.1 M sodium cacodylate buffer, pH 7.2 overnight at 4°C. After buffer rinse, samples were postfixed in 2% osmium tetroxide in 0.1 M sodium cacodylate for at least one hour (no more than two) on ice in the dark. After osmium, samples were rinsed in 0.1 M Maleate buffer (pH 6.2), followed by uranyl acetate in 0.1 M Maleate (0.22 µm filtered, 1 h, dark), dehydrated in a graded series of ethanol and embedded in Epon (PolySci) resin. Samples were polymerized at 60°C overnight. Thin sections, 60 to 90 nm, were cut with a diamond knife on a Leica UCT ultramicrotome and picked up with 2x1 mm Formvar copper slot grids. Grids were stained with 2% uranyl acetate (aq.) followed by lead citrate and observed with a Hitachi 7600 TEM at 80 kV. Images were captured with an AMT CCD XR80 (8-megapixel camera - side mount AMT XR80 – high-resolution high-speed camera).
Fluorescence labeling
Fluorescence labeling of cells was performed by seeding 5.0 x 104 4T1 cells in a 35mm dish on a collagen-coated No. 1.5 coverslip with a 14 mm glass diameter (MatTek, Ashland, MA). After overnight incubation, the cells were washed once with serum free media and replaced with fresh serum free media. For cell membrane labeling, NR12S (R&D Systems, Minneapolis, MN, #7509) was added at a final concentration of 0.3 µM and incubated for 30 minutes. For cell cytosol labeling, Calcein-AM (Thermo Fisher Scientific, C1430) was added at a final concentration of 5 µg/mL and incubated for 10 minutes. For mitochondrial membrane labeling, JC-1 (Thermo Fisher Scientific, T3168) was added at a final concentration of 10 µg/mL and incubated for 10 minutes. For nuclear staining, Hoechst 33342 (Cell Signaling Technology, #4082) was added at a final concentration of 5 µg/mL and incubated for 10 minutes.
Fluorescence microscopy
Cells were imaged on either a Zeiss LSM 700 or a 3i spinning disk confocal microscope. Samples were imaged with optimized microscope settings. Z-stack, time-lapse, or multi-channel acquisition settings were adjusted based on the experimental requirements. Images were then acquired with the appropriate excitation and emission wavelengths for each fluorophore.
Image analysis
Image analysis was performed using Fiji software. For each image/video, the brightness and the contrast were adjusted. The time stamp and the scale bar were added.
Caspase-3 activity assay
1.0 x 104 4T1 cells were seeded in 96-well plates and incubated overnight. After CKS1 treatment, the cells were lysed using lysis buffer (Cell Signaling Technology, #7018). The activity of caspase-3 in the cell lysate was quantified using the caspase-3 activity assay kit (Cell Signaling Technology, #5723) following the manufacturer’s instructions. Briefly, the cell lysate was incubated with a substrate of caspase-3 which was designed to emit fluorescence when cleaved. After incubation, the fluorescence was measured using a plate reader. The protein concentration of the cell lysate was quantified using the DC protein assay kit (Bio-Rad, Hercules, CA). The caspase-3 activity was divided by the protein concentration to obtain the caspase-3 activity per µg/mL protein in the cell lysate.
Quantification of ATP release
1.5 x 104 cells of 4T1 cells or CT26 cells in growth media were seeded in wells of a 96-well plate. After 12 hours, the media was replaced with serum free media, and the cells were treated with the corresponding concentration of CKS1. The cells were treated with CKS1 for 6 hours and the supernatant was collected from each well. After filtering the supernatant with a 0.2 µm pore filter (Corning, 431229), the amount of ATP in the supernatant was quantified using the ENLITEN ATP assay system (Promega Corporation, Madison, WI, FF2000). We followed the instructions from the manufacturer when using the ENLITEN ATP assay system.
Quantification of HMGB-1 release
1.5 x 104 cells of 4T1 cells or CT26 cells in growth media were seeded in wells of a 96-well plate. After 12 hours, the media was replaced with serum free media, and the cells were treated with the corresponding concentration of CKS1. The cells were treated with CKS1 for 6 hours and the supernatant was collected from each well. After filtering the supernatant with a 0.2 µm pore filter (Corning, 431229), the amount of HMGB-1 in the supernatant was quantified using the Mouse/Rat HMGB1 ELISA kit (arigo biolaboratories, ARG81310).
H&E staining
Tumors were fixed by immersion in 10% neutral buffered formalin (Millipore Sigma) for 48 hours and subsequently dehydrated by immersion in 70%, 90%, and 100% ethanol followed by xylene for two changes of 30 minutes each. Tissues were embedded in paraffin blocks at 58°C and sectioned at 4 µm using a microtome. Sections were floated in a water bath at 56°C, embedded onto glass slides, and dried overnight. Next, sections were rehydrated by immersion in xylene, followed by 100%, 95%, 70% ethanol, and finally water for two changes of 5 minutes each. For staining, slides were immersed in Gill’s Hematoxylin #2 (Millipore Sigma) for 1 minute, immediately rinsed in tap water to prevent overstaining, and immersed in Eosin Y (Millipore Sigma) for 1 minute and rinsed. Finally, slides were again dehydrated, mounted with Citramount, coverslipped, and scanned at 40X with a NanoZoomer slide scanner.
Statistics
All statistics analyses were performed using R. Differences between two groups were determined using unpaired two-tailed Welch’s t-test and were considered statistically significant when p < 0.05. To determine the difference between the control group and the treatment groups, we used Dunnett’s test. We used two-way repeated-measures ANOVA to determine the significance of the treatment in the in vivo experiment monitoring tumor growth. All data were replicated in at least three independent experiments and the results are expressed as mean ± SEM.