In Vitro Characterization
illustrates binding of (Cu-DOTA)n-trastuzumab-(IRDye800)m in SKBr3 (HER-2-overexpressing) and MDA-MB-231 (low expressors of HER-2) cells as visualized through fluorescence microscopy. The imaging agent binds with high affinity to SKBr3 cells, whereas there is negligible binding in MDA-MB-231 cells. Also, the binding seems to be extracellular, which is consistent with the location of the transmembrane HER-2 receptors. The dual-labeled imaging agent bound to greater than 90% of SKBr3 cells, as determined by NIR FACS.
Fluorescence microscopy imaging shows significant binding of (Cu-DOTA)n-trastuzumab-(IRDye800)m to HER-2-overexpressing SKBr3 cells in comparison to low HER-2 expressing MDA-MB-231 cells.
represents an in-cell Western blot to quantify the difference in HER-2 levels between 4T1.2 and 4T1.2neu cells. The top panel in shows the fluorescence signal from HER-2/neu staining in cells, the middle panel shows the DRAQ5 nuclear staining for normalization with cell number, whereas the bottom panel shows the combined fluorescence signal. The quantitative results are depicted graphically in . compares the binding of (Cu-DOTA)n-trastuzumab-(IRDye800)m in DsRed-transfected 4T1.2R and 4T1.2neu/R cells. Both cell lines express DsRed as illustrated, but 4T1.2neu/R has a significantly higher fluorescence because of binding of the dual-labeled imaging agent compared with 4T1.2R.
Figure 2 4T1.2neu cells have an elevated level of HER-2/neu receptor density compared with 4T1.2 breast cancer cells. (A) In-cell Western blot shows fluorescence signal intensity after HER-2/neu staining (top panel). Cells were normalized using DRAQ5 nuclear staining (more ...)
In Vivo Characterization
Because DsRed expression was lost with tumor progression, DsRed fluorescence imaging was conducted to assess inoculation and initial growth. DsRed fluorescence imaging of a shaved Balb/c mouse with a subcutaneous 4T1.2neu/R tumor confirms the location of the tumor (). The corresponding NIR fluorescence image acquired 24 hours after (64Cu-DOTA)n-trastuzumab-(IRDye800)m administration is shown in . PET signal fused with CT after (64Cu-DOTA)n-trastuzumab-(IRDye800)m and 18FDG administration revealed significant uptake within the tumor compared with the muscle region. Representative coronal sections are represented in , C and D, with crosshairs indicating tumor location. In addition to tumor uptake, the dual-labeled imaging agent accumulates within the liver, as seen in both PET and NIR fluorescence images. The nonspecific liver uptake may be attributed to the interaction of the Fc portion of the antibody with hepatocytes and is consistent with the site of antibody degradation and clearance.
Figure 3 DsRed fluorescence imaging (A) shows the location of 4T1.2neu/R primary tumor in Balb/c mice, 2 weeks after subcutaneous inoculation into the mammary pad. NIR fluorescence (B) and PET/CT (C) imaging performed 24 hours after intravenous administration (more ...)
Tumor uptake after 18FDG and (64Cu-DOTA)n-trastuzumab-(IRDye800)m administration shows comparable results as observed by PET imaging, but differences were observed in the in vivo distribution between the two imaging agents. In vivo TMRs between 18FDG-PET, 64Cu-PET, and NIR imaging ranged from 2.09 ± 0.37, 2.21 ± 0.40, and 2.81 ± 0.70, respectively (). No statistically significant differences (P > .1) between the TMR were observed between the modalities, indicating that the diagnostic capabilities of the molecularly specific dual-labeled imaging agent—(64Cu-DOTA)n-trastuzumab-(IRDye800)m—are similar to those of clinically approved but nonspecific 18FDG for detecting primary tumors.
Metastases in the 4T1.2 animal model are known to occur by 5 to 6 weeks [23
]. To assess the imaging capabilities of (64
in detecting HER-2-overexpressing metastatic lesions arising from the primary tumor, we acquired PET/CT and NIR fluorescence images after administration of the dual-labeled imaging agent in 4T1.2neu/R tumor-bearing Balb/c mice and compared the results with 18
FDG. We also compared uptake between nontumor control, 4T1.2R, and 4T1.2neu/R tumor-bearing mice injected with (64
FDG. shows NIR fluorescence, 64
Cu-PET, and 18
FDG-PET images from a representative mouse from each group. All mice showed nonspecific uptake of the dual-labeled imaging agent in the liver. The primary tumor region of the 4T1.2R tumor-bearing mice showed some uptake of the imaging agent, but there was significantly higher binding within the 4T1.2neu/R tumors as confirmed ex vivo
. Nevertheless, because 18
FDG is molecularly nonspecific, it did not differentiate between the two tumor types, and we were able to detect both 4T1.2R and 4T1.2neu/R tumors. Although we were able to visualize the primary tumor with all modalities, 18
FDG-PET was not effective in detecting metastases. In contrast, we were able to detect multiple sites of 4T1.2neu/R metastases with the molecularly specific dual-labeled (64
. Owing to intrinsic differences between PET and NIR imaging capabilities, the metastatic lesions identified through both modalities complemented each other. Lung metastases were clearly visualized noninvasively in vivo
Cu-PET (, B
) in 4T1.2neu/R tumor-bearing mice, whereas limitations associated with scatter in deep tissue prevented detection with planar in-vivo
NIR fluorescence imaging. , D
and E, I
, and N
, shows PET/CT sections acquired after (64
FDG administration in representative 4T1.2neu/R, 4T1.2/R, and non-tumor-bearing mice, respectively. The crosshairs indicate lung location. 18
FDG-PET from both the tumor- and non-tumor-bearing mouse shows no accumulation within the lung. In contrast, 64
Cu-PET shows significant accumulation of the dual-labeled imaging agent within the lung region of a 4T1.2neu/R tumor-bearing mouse compared with the 4T1.2R and nontumor control.
Figure 4 Comparison of (64Cu-DOTA)n-trastuzumab-(IRDye800)m and 18FDG distribution on Balb/c mice subcutaneously inoculated with 4T1.2neu/R (A–E), 4T1.2/R (F–J), and non-tumor-bearing (K–O) mice. NIR fluorescence (A) and PET imaging with (more ...)
The high spatial resolution achieved using NIR fluorescence imaging enabled us to detect metastatic lesions (~2 mm diameter) close to the skin (, A
), whereas PET imaging could not identify them. Optical imaging enabled us to visualize trafficking of the imaging agent from the primary tumor to proximal and distant lymph nodes when we removed the skin to reveal the mammary pad (, C
). This correlates with previous reports of lymphatic involvement in tumor growth and metastatic process of this animal model [26
]. When we performed whole skin dissection of the tumor-bearing mouse, in addition to visualizing the primary tumor, we observed accumulation of the imaging agent in lymph nodes and sections of the mammary pad. A representative photograph and NIR fluorescence image of a whole skin dissection is represented in , F
, respectively. In addition to the primary tumor, the tumor-draining lymph node (inguinal) is fluorescent and confirmed to be cancer-positive from H&E histologic staining. The skin metastatic lesions are also indicated.
Figure 5 (64Cu-DOTA)n-trastuzumab-(IRDye800)m detected superficial skin metastases in Balb/c mice implanted with 4T1.2neu/R tumor as visualized by NIR fluorescence imaging (A, B). Ex vivo NIR fluorescence imaging revealed trafficking of the dual-labeled (64Cu-DOTA) (more ...)
We identified a total of 15 fluorescent lymph nodes from six tumor-bearing mice used in this study and performed further investigation using histologic staining. Only seven nodes were infiltrated by cancer cells. However, extensive inflammation and presence of granulopoieisis was observed in all the lymph nodes in addition to spleen and liver, consistent with previous reports [26–29
]. Although in vivo
whole-body NIR fluorescence imaging of (64
did not show accumulation within the lung, we were able to confirm the presence of the imaging agent ex vivo
on harvesting the organs after sacrifice (). Representative histologic H&E staining of the 4T1.2neu/R primary tumor and lung, lymph nodes, muscle, and skin metastases are shown in , B
Figure 6 (A) Ex vivo NIR fluorescence imaging of organs harvested 24 hours after administration of (64Cu-DOTA)n-trastuzumab-(IRDye800)m. Compared with muscle, there is high uptake in tumor, lung, and organs involved in antibody degradation, namely liver and kidneys. (more ...)
We removed the skin of mice to reveal the mammary pad and compared whole skin dissections (, A–F) within the three groups of mice to assess the specificity of 64Cu-DOTA)n-trastuzumab-(IRDye800)m binding in lymph nodes. The nontumor control mice and the mice inoculated with 4T1.2/R did not show accumulation of the imaging agent within the mammary pad or lymph nodes. In contrast, mice with 4T1.2neu/R tumors showed an increased background fluorescence signal within the entire skin in addition to sections of the mammary pad. shows another representative image where trafficking of the imaging agent from the primary tumor and its associated lymph node to the axillary region can be visualized.
Figure 7 Comparison of ex vivo NIR fluorescence imaging of whole skin dissections between non-tumor-bearing (A, D), 4T1.2/R (B, E), and 4T1.2neu/R (C, F) tumor-bearing Balb/c mice 6 weeks after subcutaneous inoculation. TMR comparisons showed that there was significantly (more ...)
We hypothesize that the NIR fluorescence signal from within lymph nodes and the mammary pad may be due to the interaction of the antibody-based imaging agent with elevated levels of HER-2/neu antigen expression as a result of the 4T1.2neu/R tumor.
The ex vivo NIR fluorescence LMR in 4T1.2neu/R tumor-bearing mice (2.85 ± 0.67) was found to be statistically higher in comparison with non-tumor-bearing mice (1.23 ± 0.15) with a P < .05 (). The large error bar found in the LMR in tumor-bearing mice can be due to the variability in the extent of lung metastases within each mouse.
Ex vivo NIR fluorescence TMR estimated from 4T1.2neu/R tumors (4.03 ± 0.57) was also found to be statistically higher than 4T1.2R tumors (1.83 ± 0.33) (P < .05) as shown in . LMR of 4T1.2neu/R tumor-bearing mice (2.85 ± 0.67) was found to be statistically higher in comparison with 4T1.2/R (1.45 ± 0.19) and non-tumor-bearing mice (1.23 ± 0.15) with a P < .05 ().