Fig 1.
Sorafenib, a tyrosine kinase inhibitor, effectively suppresses HCT116-luc2 tumor growth.
Nude mice bearing subcutaneous tumors on the flanks were treated with vehicle or with 120 mg/kg of sorafenib on days 0–4 and days 7–9 via oral gavage. A: Longitudinal tumor volume measurements of subcutaneous HCT116-luc2 tumors (***p < 0.0001, n = 5 per group, two-way ANOVA, mean and s.e.m., representative of three studies) B: H&E staining of tumor tissue sections harvested from the vehicle and sorafenib-treated (120 mg/kg) tumors on day 9. The drug effectively induced necrosis in treated tumors. Bar: 50 μm.
Fig 2.
Sorafenib dose-titration study on HCT116-luc tumors in nude mice.
Tumor progression was assessed by BLI and tumor volume from the start of sorafenib treatments (mice enrolled with ~50–75 mm3 tumor volumes) though day 11. A: Representative mouse images of longitudinal BLI of HCT116-luc2 tumors in response to daily treatments of 120, 80 or 40 mg/kg. Treatments were given daily on day 0–4 and day 7–11. B: Relative tumor BLI of firefly luciferase activity was determined for each treatment group and represented using each group’s day 0 signal as 100%. The lower panel shows relative BLI signal of each dose group on day 2. C: Relative tumor volumes of each treatment group calculated using the day 0 volume as 100%. The lower panel shows relative tumor volume of each dose group on day 2. Statistics for longitudinal readouts were done by two-way ANOVA (***p < 0.0001, n = 5 per group, representative of three studies) with data represented as mean and s.e.m. The bar charts in the lower panels show the mean relative tumor readouts on day 2 with statistics assessed by Student’s t-test (#p<0.05, n = 5 per group).
Fig 3.
Tumor biology changes in progression and during anti-cancer treatment.
There are several tumor biological changes during disease progression and in response to a targeted anti-cancer drug. Without treatment, the cancer cells are metabolic active and can produce sufficient energy for cancer cell proliferation and overall tumor growth. Active cancer cell proliferation also triggers new vasculature formation in tumor (angiogenesis) that is critical for tumor growth. On the other hand, a treatment using targeted anti-cancer drugs, such as sorafenib, can suppress metabolic activity in tumors during the immediate, early phase of treatment. Such metabolic suppression could reduce the growth of not only the cancer cells, but also the vascular endothelial cells that are responsible for angiogenesis. At the late state of treatment, the drug induces extensive tumor death which triggers inflammatory responses and recruitment of granulocytes and macrophages. We hypothesize that the transition from early to late responses can be modulated by the dose level of the targeted drug.
Fig 4.
Using 18F-FDG PET to visualize early treatment responsiveness.
A: Tumor volume data of mice receiving vehicle or daily sorafenib treatment at 40 mg/kg. The treatment was initiated on day 0, with mice enrolled into the study with tumor volumes ~ 50–75 mm3. B: Relative tumor 18F-FDG uptake levels using the day 0 data as 100%. Each animal was injected IV with 100 μCi of 18F-FDG and were acquired using the G8 PET/CT system after 1 h incubation period for tracer uptake (n = 5 per group; *p < 0.01,**p < 0.001; two-way ANOVA, mean and s.e.m., representative of two experiments). C: Representative microPET images of animals treated with vehicle or 40 mg/kg of sorafenib (unit: SUVtotal).
Fig 5.
Molecular imaging of tumor viability and metabolism in response to sorafenib treatment.
A: Images for representative mice bearing HCT116-luc2 tumors are shown that were imaged on day 9 by BLI to assess tumor burden/viability. Compared with vehicle control, HCT116-luc2 tumors treated with 120 mg/kg of sorafenib show significant quantitative reduction of BLI signal on day 9, indicating loss of tumor viability. (*p < 0.01, n = 15, student’s t-test, bar: s.e.m., representative of three independent studies) B: Images of tumor-bearing mice imaged using fluorescent BRS-680 and TfV-750. Both probes were administered IV on day 8 and the animals were imaged on day 9, allowing sufficient time to generate a tumor-specific fluorescent contrast. Compared with control tumors, the sorafenib treatment significantly reduced tumor metabolic activity on day 9. (#p < 0.05, n = 5, Student’s t-test, bar: s.e.m., representative of three independent studies).
Fig 6.
Monitor changes of HCT116-luc2 tumor viability in response to low dose sorafenib.
Viability of HCT116-luc2 tumors was visualized using BLI imaging of luciferase light production. Tumor-bearing mice treated with vehicle or 40 mg/kg sorafenib were imaged on day 1, 2, 3, and 9. A: BLI images of representative mice bearing HCT116-luc2 tumors on day 1, 2, 3 and 9. B: Quantitative presentation of the tumor BLI signals (n = 5 per group, bar: s.e.m., representative of three independent studies). C: Corresponding tumor volume measurements during the imaging study represented as mean and s.e.m. (#p < 0.05, n = 15 per group, Student’s t-test). Results are representative of three independent studies.
Fig 7.
Monitoring subtle tumor metabolic changes in response to low dose sorafenib.
Metabolic changes in HCT116-luc2 tumors from the study represented in Fig 6 were visualized using BRS-680 and TfV-750. The probes were injected IV the day before FLI imaging on the IVIS Spectrum CT imaging system. A: Representative mouse images are shown for 2D FLI of BRS-680 tumor uptake, with quantitation of tumor fluorescent signal graphed below. Tumor regions of interest (ROI) are represented by solid line circles, and background control ROI are represented by dotted line circles. Average background signal was determined for each probe, normalized for ROI size, and subtracted from each tumor FLI value to more accurately determine levels of inhibition. B: Representative mice are shown for TfV-750 uptake in tumors, with quantitation of tumor fluorescent signal graphed below. Results are representative of three independent studies and are shown as mean and s.e.m. (#p < 0.05, *p < 0.01, day 1–2: n = 10, day 3–10: n = 5, Student’s t-test).
Fig 8.
Summary analysis of tumor volume, BLI, PET, and FLI results normalized for comparison.
The quantitative imaging data obtained in Figs 4, 6 and 7 were normalized for the mean of each day’s vehicle control group. A: Tumor volume (n = 15). B: BLI signal (n = 5). C: PET signal (n = 5). D and E: BRS-680 and TfV-750 FLI signals respectively (day 1–2: n = 10, day 3–10: n = 5). All data is represented as mean and s.e.m. (#p < 0.05, *p < 0.01, Student’s t-test). Normalized vehicle group data (means all at 100%) were included to allow representation of error bars for comparison to treatment groups.