Article Figures & Data
Figures
- Figure 1.
In vitro analysis of fusion protein binding and enzyme prodrug system cytotoxicity. A, The dissociation constants for the fusion proteins were determined from the specific binding curves (available in Supplementary Data) on breast cancer and nonconfluent endothelial cells representative of tumor vasculature endothelium. B, Live cell imaging indicates membrane binding of Dylight 680 conjugated fusion protein the cell membrane of GFP expressing MDA-MB-231 cells. C and D, The cytotoxic effects of the mCTH-ANXA1, mCTH-ANXA5, and MGL-ANXA5 enzyme prodrug systems were compared for MDA-MB-231 (C) and 4T1 (D) breast cancer cells. Groups that received fusion protein were treated on day 0. Selenomethionine was administered daily. Viability was determined by the Alamar Blue assay on day 3, and each sample was represented as a percentage of the vehicle-treated control. Statistical analysis was performed with a one-way ANOVA test with data presented as mean ± SEM (n = 3). Statistical significance versus vehicle-treated control is denoted by * (P < 0.001).
- Figure 2.
Preliminary in vivo analysis in immune competent 4T1 model. A, The pharmacokinetic profile of mCTH-ANXA5 was determined via ELISA assay of serum samples collected at the time points indicated after fusion protein administration (10 mg/kg i.p.). B, The efficacy of ANXA1 and ANXA5-targeted mCTH enzyme prodrug therapies with selenomethionine on 4T1 tumors in BALB/cJ mice were compared (arrow, treatment period). C, The mammalian (mCTH-ANXA5) and bacterial (MGL-ANXA5) enzyme prodrug systems were evaluated in BALB/cJ mice (arrow, treatment period). B and C, Fusion protein was administered daily (10 mg/kg i.p.), and selenomethionine (5 mg/kg i.p.) was administered 10 hours after fusion protein administration. Statistical significance versus vehicle is indicated by * (P < 0.001). B, No significant difference was observed between mCTH-ANXA1 and mCTH-ANXA5 groups. C, mCTH-ANXA5 and Sel versus MGL-ANXA5 and Sel is indicated by # (P < 0.001). No side effects were observed in any group. Data, mean volume ± SEM (n = 6–10). D, Blood samples were collected weekly during the treatment period and analyzed for the presence of antibodies specific to the administered protein. Antibody detection was performed via ELISA of serum samples, with a positive detection reported as a maximum serum dilution factor (antibody titer).
- Figure 3.
In vivo tests in the immunodeficient MDA-MB-231 model. A, The enzyme prodrug therapy in combination with rapamycin (Rap) was evaluated in SCID mice bearing orthotopic MDA-MB-231 tumors (bottom arrow, enzyme prodrug treatment period). MGL-ANXA5 was administered daily (10 mg/kg i.p.). Selenomethionine (5 mg/kg i.p.) was administered 10 hours after fusion protein administration. Rapamycin (5 mg/kg i.p.) was administered daily indicated by top arrow. Data, mean volume ± SEM (n = 5–10 at the start of the study). Statistical significance versus vehicle-treated group is indicated by * (P < 0.001). Statistical significance of enzyme prodrug treatment versus enzyme prodrug treatment with rapamycin is indicated by # (P < 0.05). B, Kaplan–Meier survival curves are shown for groups indicated from A. C, Negligible change in mouse mass observed throughout the duration of study is shown (arrow, treatment period). In addition, no side effects were observed for any treatment.
- Figure 4.
Combination therapy in the immunocompetent 4T1 model. A, The enzyme prodrug therapy in combination with rapamycin (Rap) and cyclophosphamide (Cyc) was evaluated in BALB/cJ mice bearing orthotopic 4T1 tumors (arrow, treatment period). mCTH-ANXA5 was administered daily (10 mg/kg i.p.). Selenomethionine (Sel; 5 mg/kg i.p.) was administered 10 hours after fusion protein administration. Rapamycin (5 mg/kg i.p.) and cyclophosphamide (10 mg/kg i.p.) were administered daily. Statistical significance versus vehicle treated on same day or last available point is indicated by * (P < 0.001). Data, mean volume ± SEM (n = 5–10 at the start of the study). B, Kaplan–Meier survival curves are shown for groups indicated from A. C, Negligible change in mouse mass observed throughout the duration of study is shown (arrow, treatment period). In addition, no side effects were observed for any treatment.
- Figure 5.
IHC and cellular analyses of combination therapy in immunocompetent model. BALB/cJ mice bearing orthotopic 4T1 tumors treated for 3 weeks with the enzyme prodrug treatment result in increased staining of the apoptosis marker, activated casapse-3, and decreased staining of the proliferation marker, Ki-67. Rapamycin (Rap) reduced staining of HIF1A in the same mice. A Nikon Eclipse E800 microscope was used to capture 15 fields of view of tumor sections from 3 mice per group (necrotic tumor cores were excluded) for activated caspase-3 and Ki-67 staining. Immunostaining for activated caspase-3 (A) and Ki-67 (B) was quantified as percent of cells (hematoxylin counterstain) with DAB and is presented as mean ± SEM. C, IHC staining of HIF1A with DAB development was quantified from whole section images counterstained with hematoxylin. Data, mean ± SEM (n = 3 mice). Statistical significance between groups is indicated by * (P < 0.01). Cyclophosphamide (Cyc) reduces the number of pulmonary metastasis in the orthotopic 4T1-TdTomato mouse model despite poor correlation with Treg numbers in the spleen of BALB/cJ mice. D, A Leica stereomicroscope with an automated ImageJ macro was used to quantify fluorescent nodules in the lung. Data are shown as individual nodules from the lungs of 3 mice per group after 3 weeks of treatment on a log-normal scale, as the nodule sizes were logarithmically distributed. Median nodule size on the log scale is marked. Total nodules per group are summed and shown. Statistical significance between groups is indicated by * (P < 0.05). E, CD4+ CD25+ FoxP3+ Treg levels were quantified with flow cytometry and are presented as a percentage of spleen lymphocytes in BALB/cJ mice with 4T1 grafts after 3 weeks of treatment or healthy BALB/cJ mice with no tumor. Data, mean ± SEM (n = 3 mice). No statistical significance was found in the difference of the level of Tregs for mice with tumors in the vehicle-treated group compared with any of the treated groups.
Tables
- Table 1.
Kaplan–Meier survival curves for all groups of mice with 4T1 tumors were analyzed for significance against vehicle-treated mice, and the combination therapies were additionally analyzed against the enzyme prodrug therapy
Synergism assessment factors Median P vs. P vs. Day of treatment survival vehicle mCTH-ANXA5 & Sel 0 2 6 9 13 Vehicle 14 days Cyc 23 days 0.2739 Rap 14 days 0.7830 Sel 18 days 0.8813 mCTH-ANXA5 18 days 0.5215 Cyc & Rap 24 days 0.1315 0.00 −0.03 0.06 −0.10 −0.02 mCTH-ANXA5 & Sel 26 days 0.1005 0.00 0.12 0.07 0.06 0.04 mCTH-ANXA5 & Sel + Rap 27 days 0.0015a 0.0303 0.00 −0.23 0.10 0.06 0.20 mCTH-ANXA5 & Sel + Cyc 28 days 0.0079 0.0560 0.00 0.09 −0.01 0.00 0.12 mCTH-ANXA5 & Sel + Cyc & Rap 36 days 0.0002a 0.0014a 0.00 −0.03 0.07 0.15 0.19 NOTE: The quantification of the synergism assessment based on primary tumor growth inhibition using the Bliss independence model is summarized for percent tumor inhibition for the combination therapies. Values presented as a function of the day of treatment are a “synergism assessment factor” with positive values indicative of synergism (bold), calculated from experimental values for the individual constituents of the combination therapies, and compared with the experimentally determined values of the combination therapies (see Supplementary Data for additional information about the assessment of synergism).
Abbreviations: Cyc, cyclophosphamide; Rap, rapamycin; Sel, selenomethionine.
↵aLog-rank–determined P values are presented and significance is indicated (P < 0.005).
Supplementary Data
- Supplementary Data - This file contains includes 9 figures of supplementary data (S1-S9) and a description of live cell microscopy and the assessment of synergism for the combination therapies.
Volume 16, Issue 9
