Figure 1.
Bioluminescence imaging detection and quantification of 4T1-GL cancer cells spiked in mouse whole blood samples.
(A) Quantification of BLI imaging of high numbers of 4T1-GL cells spiked into 100 µL culture medium or 100 µL mouse whole blood samples, shown as mean ± SEM on a log-log scale. There was a significant linear correlation (R2 = 0.95 for culture medium; R2 = 0.92 for whole blood). (B) Quantification of BLI imaging of low numbers (0–150) of 4T1-GL cells cells spiked into 100 µL culture medium or 100 µL mouse whole blood samples. There was a significant linear correlation (R2 = 0.90 for culture medium; R2 = 0.92 for whole blood). (C) Zoom on very low number of cells (0–50) in (B). The shaded regions represent background BLI radiance.
Figure 2.
Bioluminescence imaging of primary tumor and metastases in the 4T1-GL orthotopic mammary tumor model.
(A) Primary tumor growth in the mammary fat pad (m.f.p) as monitored using BLI, following implantation of 2×107 4T1-GL cells in the fourth left mammary fat pad (image scales in p/s/cm2/sr, n = 7 mice). (B) Corresponding quantification of BLI signal in the m.f.p area, shown as mean ± SEM and fit of the mean values to a Gompertzian tumor growth equation (dashed line, R2 = 0.86). (C) Metastases in the upper body (lung area) as monitored using BLI in the same animals (image scales in p/s/cm2/sr). (D) Corresponding quantification of the signal in the lung area, shown as mean ± SEM. (E) Metastases in excised organs on day 23, as monitored using BLI in the same animals: a. tumor (scale: 2×108–3×109 p/s/cm2/sr), b. lungs (scale: 2×108–3×109 p/s/cm2/sr), c. liver (scale: 2×105–7×106 p/s/cm2/sr), d. brain (scale: 1×104–1×105 p/s/cm2/sr), e. heart (scale: 1×107–6×107 p/s/cm2/sr), f. kidneys (scale: 2×105–7×106 p/s/cm2/sr), g. spleen (scale: 2×105–7×106 p/s/cm2/sr), h. bone (scale: 2×105–7×106 p/s/cm2/sr). (F) Corresponding quantification of BLI signal in these organs, shown as mean ± SD.
Figure 3.
Bioluminescence imaging of CTCs dynamics in the 4T1-GL orthotopic mammary tumor model.
(A) CTCs in 100 µL blood samples from n = 7 mice, as monitored using BLI (image scale in p/s/cm2/sr). (B) Corresponding quantification of BLI signal in (A), shown as mean ± SEM. The pink shaded region corresponds to average background signal. (C) Correlation of blood BLI signal and primary tumor signal in the MFP area for all timepoints measured and all animals studied. The line indicates a linear regression (R2 = 0.43) and the red dotted line shows the 95% confidence interval. The black dotted line represents the average BLI background signal of whole blood from healthy mice. (D) Correlation of CTC detection BLI signal and lung metastasis signal in the torso area for all timepoints measured and all animals studied. There was no significant linear correlation in this case. The black dotted line represents the average BLI background signal of whole blood from healthy mice. (E) CTCs and CTC microemboli in terminal blood samples from animals bearing day 23 tumors, as imaged by bioluminescence microscopy. (F) Statistical analysis showing the onset of lung metastasis and of CTCs detected in blood over time in the n = 7 animals.
Figure 4.
Optimization of BLI detection and quantification of 4T1-GL and 4T1-F3 cancer cells spiked in mouse blood samples.
(A) Quantification of BLI imaging of low numbers (0–50) of 4T1-GL cells spiked into 100 µL blood with or without RBC lysis There was a significant linear correlation (R2 = 0.64 for RBC lysis; R2 = 0.78 for unprocessed). (B) Zoom on very low number of cells (0–50) in (A). The shaded regions represent background BLI photon counts. (C) Quantification of BLI signal per µg protein for the following cell lines: 4T1-GL, 4T1-GL-TS, monoclonal populations sorted from 4T1-GL-TS: 4T1-A4, 4T1-C3, 4T1-D3 and 4T1-F3, shown as mean ± SD (D) Quantification of BLI imaging of low numbers (0–25) of 4T1-F3 cells spiked into 100 µL blood with or without RBC lysis, shown as mean ± SEM. The label numbers for each point indicate number of replicates (n = 3–12). There was a significant linear correlation (R2 = 0.68) for RBC-lysed samples.
Figure 5.
Bioluminescence imaging of primary tumor, metastatic growth and CTC dynamics in the 4T1-F3 orthotopic mammary tumor model.
(A) Primary tumor growth in the mammary fat pad (m.f.p) as monitored using Bioluminescence (BLI) imaging, following implantation of 5×106 4T1-F3 cells in the fourth left mammary fat pad (image scales in p/s/cm2/sr), and (B) Corresponding quantification of BLI signal in the m.f.p area, shown as mean ± SEM and fit of the mean values to an exponential tumor growth equation (dashed line, R2 = 0.99). (C) Metastases in the upper body (lung area) as monitored using BLI in the same animals (image scales in p/s/cm2/sr) and (D) Corresponding quantification of the signal in the lung area, shown as mean ± SEM. (E) Corresponding quantification of blood BLI signal in 1-mL terminal blood samples from n = 3–4 animals/day (image scales in p/s/cm2/sr), shown as mean ± SEM. The image on the right shows BLI signal from CTCs in n = 3 terminal blood samples on day 12, (image scales in p/s/cm2/sr) (I) Statistical analysis showing the onset of lung metastasis and blood CTCs over time in the n = 40 animals.