Selective and effective killing of angiogenic vascular endothelial cells and cancer cells by targeting tissue factor usinga factor VII-targeted photodynamic therapy for breast cancer
Zhiwei Hu, Benqiang Rao, Shimin Chen and Jinzhong Duanmu
Supplemental figures
Fig. S1. Incubation time of fVII-SnCe6 affects the efficacy of fVII-tPDT in vitro. Murine (EMT6) and human (MDA-MB-231 and MCF-7) breast cancer cells and a normal non-TF-expressing 293 cell line were incubated with the fVII-SnCe6 conjugate (2 μM SnCe6) for various amounts of time (x-axis) and then irradiated with 36 J/cm2 635 nm laser light. The efficacy of SnCe6 was determined by crystal violet staining.
Fig. S2. Comparison of the crystal violet staining assay to clonogenic assaysfor determining the therapeutic efficacy of fVII-tPDT in vitro. MDA-MB-231 cells were treated with fVII-tPDT (2 μM, 18 J/cm2), and then the crystal violet staining assay and the clonogenic assays (counting viable cells using trypan blue exclusion and colony formation) were carried out separately. Procedures are described in Methods. The difference of percent of surviving cells was not statistically significant using unpaired t test (P=0.1813).
Fig. S3. SnCe6 is stable at least for one year at 4C. MDA-MB-231 cells were seeded and treated with ntPDT (36 J/cm2) as described in Methods using various concentration of SnCe6 diluted from a 10 mg/ml freshly dissolved SnCe6 solution or an SnCe6 solution that had been stored at 4C for more than one year. The efficacy of each solution was determined by crystal violet staining. Both SnCe6 solutions had similar abilities to kill MDA-MB-231 cells (P=0.3767 by paired t-test), indicating that one year of storage at 4C did not change the photoactivity of SnCe6.
Fig. S4. TF is expressed on 293 cells from ATCC and 293FT cells from Invitrogen. TF expression on these 293 cells were determined by the same flow cytometry procedure using goat anti-HTF antibody (American Diagnostica) (solid line) followed by secondary anti-goat FITC as described in Fig. 4. The control was incubated only with secondary anti-goat FITC (dashed line).
Supplemental methods and procedures
Cell lines. Chinese Hamster Ovary (CHO-K1, ATCC) cells were grown in F-12 medium, the mouse mammary cancer cell line EMT6, the human breast cancer MCF-7, and the human embryonic kidney 293 cell line (kind gift from Dr. Albert Deisseroth during his tenure at Yale University) were grown in DMEM and the human breast cancer MDA-MB-231 were grown in L-15 supplemented with 10% FBS and 1:100 penicillin/streptomycin (Sigma) at 37C and 5% CO2. Human umbilical vein endothelial cells (HUVECs) at passages 2-5 were used and grown in M199 (Invitrogen) supplemented with 20% heat-inactivated FBS (Gemini) and endothelial cell growth supplement (ECGS, 1:100 dilution of 5 mg/ml stock solution). The HUVECs and ECGS were purchased from the Vascular Biology & Transplantation Group at the Yale Medical School.
Endocytosis of fVII by TF-expressing VECs and breast cancer cells. TF expression was induced on HUVECs using VEGF as previously described (28). Briefly, HUVECs were starved in human endothelial SFM (Invitrogen) overnight then incubated with 1.1 nM VEGF (BD Biosciences) diluted in human endothelial SFM for 4 hrs to induce TF expression. Control HUVECs were starved but not stimulated.
To study the endocytosis of fVII, HUVECs (pre-stimulated for 4 hrs with VEGF) and human breast cancer MDA-MB-231 cells were grown on glass coverslips overnight and then blocked with HBSS buffer/1% BSA/5 mM CaCl2 (blocking buffer) followed by incubation with 10 g/ml mouse Icon (mfVII/hIgG1 Fc) diluted in blocking buffer for 15, 30, 60, or 120 min. Binding of fVII to TF was detected by using an anti-human IgG Fc FITC antibody to determine the localization of fVII (cell membrane-bound or cytoplasmic). After incubation with fVII, the cells were washed once with HBSS-T (HBSS, 0.1% Triton X-100, 10 mM CaCl2), fixed with 4% paraformaldehyde for 20 min, washed again, permeabilized with HBSS, 1% Triton X-100, and 10 mM CaCl2 at RT for 10 min, blocked with HBSS/BSA at 37C for 30 min, and incubated with a 1:40 dilution of Alexa Fluor 633-phalloidin (Molecular Probes) in HBSS/1% BSA/0.1% Triton X-100 at RT for 20 min to stain intracellular F-actin, allowing assessment of its co-localization with the TF/fVII complex. The cells on the coverslips were washed three times, briefly air-dried, mounted onto glass slides using anti-fade reagent (Molecular Probes), and photographed with a confocal microscope (Zeiss LM 100).
In vitro PDT in tissue culture plates. The in vitro PDT tests were done in 96-well plates containing 1-2 104 cells in 100 l growth medium per well or in 48-well plates containing 9 104 cancer cells in 200 l per well. In the experimental wells, fVII-tPDT or ntPDT was carried out by incubating the cells with fVII-SnCe6 conjugate or unconjugated SnCe6 diluted in HBSS/5 mM CaCl2/1% BSA at 37C for 90 min, then discarding the drug solution, washing the cells once with pre-warmed complete growth medium and adding 100 l or 200 l growth medium to the 96- or 48-well plates, respectively. The cells were irradiated with a 635 nm fiber-coupled diode laser (BWF2-635-0.1-100-0.22, B&W Tek, Inc.) for 0, 1, 3, 6, or 12 min at 100 mW/cm2 (corresponding to a laser fluence of 0, 6, 18, 36 or 72 J/cm2). The fluence rate of the laser unit was measured by using a laser power meter (LaserCheck, Coherent, Inc.) prior to carrying out PDT. After treatment with PDT, the cells were incubated at 37C and 5% CO2 until they reached 95% confluence and were ready for the crystal violet staining assay or clonogenic assay described below. Controls included cells treated only with the drug (mfVII-SnCe6 or free SnCe6 without laser irradiation), cells treated with the laser alone, untreated cells, medium only, and a maximal killing control. All of the controls included the same number of cancer cells except for the medium only control, which included growth medium that did not contain cells to ensure that medium alone would not be stained by crystal violet during the staining assays. In the maximal killing control wells, cells were lysed by addition of 1/10 volume of 9% Triton X-100 45 min prior to crystal violet staining. The efficacy of PDT was determined by crystal violet staining and/or the clonogenic assay described below.
Crystal violet staining and clonogenic assays for determining the in vitro efficacy of SnCe6 PDT.
Non-clonogenic crystal violet staining was performed as described previously[1] with a minor modification to the more convenient reading of absorbance at 595 nm (A595nm). The percent of surviving cells was calculated based on A595nm readings using the following formula: Percent of surviving cells(%)=(Average A595nm of PDT-treated cells - Average A595nm of maximal killing controls)/(Average A595nm of untreated controls - Average A595nm of maximal killing controls) x 100%.
For conventional clonogenic assays, the fVII-tPDT treated and untreated control cells were dissociated and, based on the original seeding cell numbers, 100, 200 or 500 cells were seeded per 100 mm dish in duplicate dishes. After formation of colonies, the colonies were fixed and stained with crystal violet, and the number of colonies with at least 50 cells per colony was counted. The percent of surviving cells was calculated using the following formula: Percent surviving cells = (number of colonies fVII-tPDT treated cells number of colonies of untreated control cells with the same seeding cell number) 100%.
Assessing apoptosis and necrosis as mechanism of action of SnCe6 PDT
Human MDA-MB-231 breast cancer cells were treated in 96-well plates with fVII-tPDT or ntPDT (2 M, 72 J/cm2) as described above. At various time points after PDT treatment, the plates were separately assayed for necrosis (cytotoxicity) and apoptosis. Briefly, the fluorescence-based Apo-ONE Homogeneous Caspase-3/7 assay (Promega) was used to measure caspase-3/7 activity (excitation 485 20 nm and emission 530 25 nm) using a fluorescence microplate reader (Bio-Tek). The percent of apoptosis was calculated using the following formula: Percent of apoptosis (%) =(experimental fluorescent units – average fluorescent units of untreated controls)/(average fluorescent units of maximal killing controls - average fluorescent units of untreated controls) 100%. The CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) was used to measure lactate dehydrogenase (LDH, a stable cytosolic enzyme that is released upon cell lysis and can be used as an indicator of necrosis) in the culture supernatants as evidence of cytotoxicity in the PDT-treated cancer cells following the manufacturer’s instructions.The percent of cytotoxicity (%)was calculated based on the A490nm using the following formula: Percent of cytotoxicity (%)=(A490nm of PDT treated cells – average A490nm of untreated controls)/(average A490nm of maximal killing controls – average A490nm of untreated controls) 100%.
Supplemental results/discussion
Incubation time of fVII-SnCe6 conjugate with cancer cells affects the efficacy of fVII-tPDT in vitro.
We showed in Fig. S1 that after 90 min of treatment, fVII-tPDT achieved its strongest killing effect on TF-expressing human and murine breast cancer cells, including breast cancer cell lines (MDA-MB-231, MCF7, and EMT6; See Fig. 4a-c). fVII-tPDT did not kill TF-negative 293 cells (Fig. 4c), a human embryonic kidney cell line used here as a model of normal tissue cells. Surprisingly, flow cytometry using monoclonal anti-HTF antibody showed that two other commonly used 293 lines, 293 (ATCC, CRL-1573) and 293FT (Invitrogen), did express TF (Fig. S4). The reason for different TF expression on these 293 cell lines remains to be investigated.
Based on the results of endocytosis assays, however, 90 min was not the time point at which the maximal endocytosis of fVII/Fc protein occurred. One obvious difference was the molecular weight of fVII proteins in these two experiments; in the endocytosis studies, the dimeric fVII/Fc protein had larger MW (210 kDa) than that of the monomeric fVII protein in fVII-tPDT (52.3 kDa), which could potentially contribute to a different rate/destination of internalized fVII/TF. Nevertheless, 90 min was chosen as the interval between in vitro incubation with or in vivo injection of fVII-SnCe6 and 635 nm laser light irradiation.
Crystal violet staining assay for measuring the loss of monolayer adherence has comparable results to clonogenic assays for determining the efficacy of fVII-tPDT in vitro.
The choice of crystal violet staining was based on previous work(reference 27 in the published paper) in which Mickuviene et al. (2004) found that crystal violet staining was the most sensitive non-clonogenic assay for determining the in vitro efficacy of PDT. We show in S2 that the percent of surviving cells determined by crystal violet staining was slightly higher than that obtained by counting colony numbers, but the difference between these two methods was not statistically significant (P= 0.1813 vs. colony formation) (Fig. S2). The slightly higher percent of surviving cells observed by crystal violet staining probably resulted from the staining of dead cell debris membranes. Although the cancer cells were killed by fVII-tPDT, the debris membranes remained in the assay wells. Because it is less time consuming, the non-clonogenic crystal violet staining assay was used to determine the efficacy of PDT in vitro.
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