Positron emission tomography imaging of CD105 expression during tumor angiogenesis

Abstract

Purpose: Overexpression of CD105 (endoglin) correlates with poor prognosis in many solid tumor types. Tumor microvessel density (MVD) assessed by CD105 staining is the current gold standard for evaluating tumor angiogenesis in the clinic. The goal of this study was to develop a positron emission tomography (PET) tracer for imaging CD105 expression.

Methods: TRC105, a chimeric anti-CD105 monoclonal antibody, was conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and labeled with (64)Cu. FACS analysis and microscopy studies were performed to compare the CD105 binding affinity of TRC105 and DOTA-TRC105. PET imaging, biodistribution, blocking, and ex vivo histology studies were performed on 4T1 murine breast tumor-bearing mice to evaluate the ability of (64)Cu-DOTA-TRC105 to target tumor angiogenesis. Another chimeric antibody, cetuximab, was used as an isotype-matched control.

Results: FACS analysis of human umbilical vein endothelial cells (HUVECs) revealed no difference in CD105 binding affinity between TRC105 and DOTA-TRC105, which was further validated by fluorescence microscopy. (64)Cu labeling was achieved with high yield and specific activity. Serial PET imaging revealed that the 4T1 tumor uptake of the tracer was 8.0 ± 0.5, 10.4 ± 2.8, and 9.7 ± 1.8%ID/g at 4, 24, and 48 h post-injection, respectively (n = 3), higher than most organs at late time points which provided excellent tumor contrast. Biodistribution data as measured by gamma counting were consistent with the PET findings. Blocking experiments, control studies with (64)Cu-DOTA-cetuximab, as well as ex vivo histology all confirmed the in vivo target specificity of (64)Cu-DOTA-TRC105.

Conclusion: This is the first successful PET imaging study of CD105 expression. Fast, prominent, persistent, and CD105-specific uptake of the tracer in the 4T1 tumor was observed. Further studies are warranted and currently underway.

Fig. 1

In vitro investigation of DOTA-TRC105. a Flow cytometry analysis of TRC105 and DOTA-TRC105 in HUVECs (CD105-positive) and MCF-7 (CD105-negative) cells at different concentrations. b Fluorescence microscopy images of HUVECs using either TRC105 or DOTA-TRC105 (2 μg/mL) as the primary antibody. Various control images are also shown.

Fig. 2

In vivo investigation of ⁶⁴Cu-DOTA-TRC105 in 4T1 tumour-bearing mice. a Serial coronal PET images of 4T1 tumour-bearing mice at 4, 24, and 48 h post-injection of ⁶⁴Cu-DOTA-TRC105, TRC105 before ⁶⁴Cu-DOTA-TRC105 (i.e. blocking), and ⁶⁴Cu-DOTA-cetuximab. Tumors are indicated by arrowheads. b Representative PET/CT images of ⁶⁴Cu-DOTA-TRC105 in 4T1 tumour-bearing mice at 24 h post-injection. c Time-activity curves of the tumour, liver, blood, and muscle upon intravenous injection of ⁶⁴Cu-DOTA-TRC105 into 4T1 tumour-bearing mice (n = 3). d Comparison of 4T1 tumour uptake of ⁶⁴Cu-DOTA-TRC105, ⁶⁴Cu-DOTA-TRC105 with a blocking does of TRC105, and ⁶⁴Cu-DOTA-cetuximab. *: P < 0.05 (n = 3).

Fig. 3

Biodistribution studies after non-invasive PET scans. a Biodistribution of ⁶⁴Cu-DOTA-TRC105 in 4T1 tumour-bearing mice at 24 and 48 h post-injection (n = 3). b Biodistribution of ⁶⁴Cu-DOTA-TRC105 and ⁶⁴Cu-DOTA-cetuximab at 48 h post-injection (n = 3). *: P < 0.05.

Fig. 4

Immunofluorescence CD105/CD31 double-staining of the 4T1 tumour, liver, and spleen tissue sections. a TRC105 and AlexaFluor488-labeled goat anti-human IgG was used for CD105 staining (green). Afterwards, the tissue slices were stained with rat anti-mouse CD31 antibody and Cy3-labeled donkey anti-rat IgG (red). b A higher magnification merged image revealed that CD105 expression in the 4T1 tumour was almost exclusively on the vessels, evidenced by almost perfect overlay of the CD105 and CD31 staining.