MDA-MB-231 / 40.2 µM±0.6 / 28.6 µM±1.2 / 5.3 µM±0.3
TMD231 / 19.4 µM±3.3 / 6.3 µM±1.7 / 0.5 µM±0.5
MDA-MB-468 / 23.8 µM±7 / 5 µM±1.8 / 1.3 µM±0.3
MCF-7 / 0.8 µM±0.04 / 23 µM±3.3 / 1.2 µM±0.02
IC50 values for Nultin-3a, Carboplatin, and 1:1 Combination
Supplemental Table S1
Supplemental Information
Supplemental Materials and Methods
p73siRNA Constructs
p73siRNA constructs included: GAGACGAGGACACGUACUA; GCAAUAAUCUCUCGCAGUA; GAACUUUGAGAUCCUGAUG; CCACCAUCCUGUACAACUU.
Analysis of H2AXFoci
For image acquisition and processing, all slides were examined at 600X magnification using a Confocal/two-photon Olympus Fluoview FV-1000 MPE system (Olympus America). Fields were randomly selected on the basis of DAPI-counter stained nuclei. After acquisition of the DAPI image, the amplification of the FITC signal, z-step and magnification parameters were kept constant during the study. The number of foci per nucleus was determined from 5 different fields per sample using ImageJ software.
Cell Cycle
TMD231 cells were treated with Nutlin-3a, carboplatin, 1:1 combination, or appropriate vehicle controls for 72-hours in three independent experiments. Cells were collected and stained with propidium iodide solution containing 0.1% (v/v) Triton X-100 (Sigma), 10 µg/mL propidium iodide (BD Biosciences), and 100 µg/mL DNase-free RNaseA (Invitrogen), and analyzed by flow cytometry. Cell cycle subpopulations were quantitated using ModFit LT for Mac version 3.2 software with the diploid-aneuploid model option (Verity Software House).
Cycloheximide Protein Stability
TMD231 cells were treated with Vehicle or Nutlin-3a in combination with carboplatin as well as 10 or 80 µg/mL cycloheximide (Sigma-Aldrich) for 2, 8, or 16 hours. Protein was collected and run on SDS-PAGE. Blots were probed for Mdm2, p53, p21, PUMA and GAPDH. The stability of Mdm2, p21, and PUMA served as controls for CHX activity.
Invasion Assay
Cell invasion was determined using CytoSelect Cell Invasion Assay Kit (Cell BioLabs) as per manufacturer’s specifications. TMD231 cells were seeded on membranes and serum starved overnight. Cells were treated with Nutlin-3a, carboplatin, 1:1 combination, or vehicle for 24 hours. Concurrent cell survival assays were conducted with TMD231 cells using trypan blue to enumerate total viable cells.
In Vitro Cell Number Imaging
TMD231-CR and TMD231 cells were counted and 0.03125, 0.0625, 0.125, 0.5, 1, 2, and 4x106 cells in triplicate were placed into 96-well plates. The TMD231 parental cells were used as a control for any auto-fluorescence. Imaging analysis was completed and fluorescent intensity was calculated.
In Vivo Cell Number Imaging
Each of three NSG mice was implanted with 0.125, 0.25, 0.5, and 1x106 TMD231 or TMD231-CR cells in the mammary fat pad, and fluorescence measured using an Optix MX3 within 1 hour after implantation.
Aperio Automatic Image Analysis
Computer-assistedmorphometric analysis of digital images was carried out using the Aperio software of the Aperio Imaging system (Leica Biosystems). A specific positive pixel algorithm was used for imaging the H&E lung tumor metastases. The positive pixel algorithm was modified to distinguish between the red and blue colors. Alterations from the normal “hue value” (0.1 to 0.78) and “hue width” (0.5 to 0.1) were made for the H&E evaluation. For the Ki67 immunostain, the AperioImageScope standard positive pixel algorithm was used. The Image Analysis software was used and the software package (positive pixel algorithm) calculated the percent of positive pixels (brown staining) in one large cross section from tumors derived from the vehicle and treated mice.
In VivoPharmacodynamic Study
NSG mice were implanted with 1x106 TMD231-CR cells. When tumors reached ~700mm3 by caliper measurement, mice were randomized based on tumor volume into treatment groups. Mice were administered vehicle (Veh), 20 mg/kg carboplatin i.p. (Carb) (in AM), 200 mg/kg Nutlin-3a p.o. (Nut) (in PM), or 20 mg/kg carboplatin+200 mg/kg Nutlin-3a (Combo) for 3 consecutive days. Two hours after the last drug dose, the mice were sacrificed and primary tumors were weighed, snap frozen, and maintained at -80C until processed. Tumors were disrupted using an Omni Tissue Homogenizer (Omni International). In PD study 1, tumors were lysed in buffer containing 1% SDS, 1 Complete-EDTA free mini tablet (Roche) and 1% phosphatase buffer (Sigma) and sample boiled. In PD study 2, tumors were gently lysed in RIPA buffer. Protein concentration was determined as described previously.
In Vivo Measurement of Activated Caspase-3 and Cleaved PARP
Tumors were processed and the levels of activated Caspase-3 and cleaved PARPwere measured using MILLIPLEX MAP Caspase-3/GAPDH and PARP/GAPDH Magnetic Beads (EMD Millipore) as per manufacturer’s instructions.
Supplemental Figure Legends
Supplemental Table S1.IC50 values for Nutlin-3a, carboplatin, and 1:1 combination
Supplemental Figure S1. Combination treatment decreases combination index (CI) values in a panel of breast cancer cell lines. MDA-MB-231, TMD231, MDA-MB-468, and wt-p53 MCF-7 breast cancer cells were treated with the indicated dose-ratios of Nutlin-3a:carboplatin for 5-days. CI values less than 1 indicate a synergistic effect.
Supplemental Figure S2. Total apoptosis/necrosis in normal fibroblast cells.Total apoptosis/necrosis in normal fibroblast cells treated with Nutlin-3a (N), carboplatin (C), or the combination (NC) at the indicated dose-ratios.
Supplemental Figure S3.Carboplatin and combination treatment induces accumulation in S and G2/M. (A-B) Cell cycle analysis was evaluated following a 72- hour drug treatment with vehicle or varying concentrations of Nutlin-3a, carboplatin, or 1:1 combinations of Nutlin-3a and carboplatin. (C) Representative histograms of Vehicle, 15 µM Nutlin-3a (Nut), 15 µM carboplatin (Carb), or 1:1 combination (Combo) showing ModFit data analysis for diploid (Dip) (Red) and aneuploid (An) (Yellow) cells.
Supplemental Figure S4. Nutlin-3a does not affect mutant p53 stability in TMD231 cells. TMD231 cells were treated with Vehicle or 15 µM Nutlin-3a in combination with 15 µM carboplatin (Combo) as well as 10 or 80 µg/mL cycloheximide (CHX) for the indicated times. Cells treated with 15 µM Nutlin-3a in combination with 15 µM carboplatin without CHX (Combo) were used as a control. Cells were lysed and the protein levels of Mdm2, p53, p21, PUMA, and GAPDH were evaluated by Western blot.
Supplemental Figure S5.Validation of TMD231-Crimson cells in vitro and in vivo for sensitive detection of primary tumor burden.(A) The E2-Crimson fluorescent protein was expressed from the p2CL7CR2wo lentiviral vector under control of a modified spleen focus-forming virus (SFFV) promoter. TMD231 cells were transduced with the E2-Crimson lentiviral vector (TMD231-CR). (B) After three days, the transduction efficiency was ~80% E2-Crimson-positive cells as determined by flow cytometry. (C) Representative images of implantation of varying number of TMD231 and TMD231-CR cells in vivo (n=3 per cell line). (D) Fluorescence intensity was linearly correlated with the number of cells implanted (n=3 per cell line). (E) Dose-response curves and IC50 values of Nutlin-3a, carboplatin, and the 1:1 combination in TMD231-CR cells. (F) Representative images on day 7 of NSG mice implanted with 1x106 TMD231 or TMD231-CR cells in the mammary fat pad on day 0. (G) After randomization, treatment groups were well balanced with respect to tumor size and standard deviation as assessed by fluorescence intensity of primary tumors on day 7.
Supplemental Figure S6. Single-agent or combination treatment does not affect cell invasion. (A) Cell invasion following 7.5 µM of Nutlin-3a, carboplatin, or the combination for 24 hours expressed as % of control. Cytochalasin D was used as a positive control and inhibited invasion by ~50% (n=3). (B) Concurrent cell survival assays were performed to assess cell viability in each group following treatment (n=3).
Supplemental Figure S7.Evaluation of pharmacodynamic markers in vivo.NSG mice were implanted in the mammary fat pad with 1x106 TMD231-CR cells and randomized into treatment groups once tumor volumes reached based ~700 mm3. Mice were administered Vehicle (Veh), 200 mg/kg Nutlin-3a (Nut), 20 mg/kg carboplatin (Carb), or combination (Combo) for 3 consecutive days. Lysates from primary tumors were evaluated for target protein levels by Western blot in 2 independent experiments (PD study 1 and PD study 2). (A) Lysates were prepared in 1% SDS extraction buffer. Densitometry values were normalized to GAPDH relative to vehicle-treated mice (right panel, # p<0.05 vs Carb, *p<0.05 vsVeh, Student’s t-test, n=4 per group, ±SEM). Each lane represents a different mouse sample. (B) Lysates were prepared in RIPA extraction buffer. Tumors were randomly selected from each treatment group for Western Analysis. Densitometry values were normalized to -tubulin relative to vehicle treated mice (right panel, p > 0.05 for all comparisons; n=3 per group ±SEM). (A-B) Each lane represents a different mouse tumor sample. (C-D) Tumors from each group were extracted in MILLIPLEX buffer (PD study 2, n=6 per group) and evaluated by MILLILPLEX for (C) activated Caspase-3 or (D) cleaved PARP. Mean fluorescence intensities (MFI) per µg protein were normalized to GAPDH MFI. Each point represents a tumor sample from a different mouse.