Molecular imaging of very late antigen-4 (α4β1 integrin) in the premetastatic niche

Despite advances in cancer treatment over the past few decades, metastatic disease remains the primary cause of morbidity and mortality. Recent reports suggest the formation of a "premetastatic niche" before the metastatic cascade, where niche is defined as the microenvironment for tumor cells to be able to engraft and proliferate at secondary sites. Bone marrow-derived (BMD) cells that express vascular endothelial growth factor receptor-1 and very late antigen-4 (VLA-4) have been shown to arrive at sites of metastasis to form a receptive environment for tumor cells. Here we describe experiments toward imaging of VLA-4-positive BMD cells using a high-affinity PET probe, ⁶⁴Cu-labeled 11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[ 6.6.2] hexadecane (CB-TE2A)-LLP2A. Methods: VLA-4-negative MDA-MB-231/firefly luciferase (fluc) human breast tumor cells were injected intraarterially in the left ventricle in nude mice. Tumor metastasis in mice was monitored for 30 d by bioluminescence imaging and small-animal PET/CT. Small-animal PET images were collected 2 h after mice were injected in the tail vein with ⁶⁴Cu-CB-TE2A-LLP2A (5.6-11.1 MBq [150-300 μCi; specific activity, 400 μCi/mg]). Cellular uptake of ⁶⁴Cu-CB-TE2A-LLP2A was determined in VLA-4- positive B16F10 mouse melanoma cells and VLA-4-negative MDA-MB-231/fluc human breast cancer tumor cells. Biodistribution experiments in nude mice bearing VLA-4-positive B16F10 subcutaneous tumors in the flank were conducted to validate targeting of VLA-4-positive cells in vivo. Results: Uptake of ⁶⁴Cu-CB-TE2A-LLP2A was higher in VLA-4-positive human melanoma B16F10 cells than in VLA-4-negative MDAMB- 231 cells (P <0.05). In B16F10 tumor-bearing mice, ⁶⁴Cu- CB-TE2A-LLP2A had high uptake in the VLA-4-rich organs marrow, spleen, and tumor (11.26% ± 2.59%, 8.36% ± 2.15%, and 3.09% ± 0.58% injected dose/g, respectively). Cumulative standardized uptake value data from 2 independent studies (n 5 7 and 8 mice) on nude mice implanted with VLA- 4-negative MDA-MB-231/fluc human breast tumor cells suggested an influx of VLA-4-positive BMD cells that corresponded to metastasis (P <0.05). Immunohistochemical analysis and flow cytometry also showed upregulation of VLA-4-positive cell clusters and BMD cells at the metastatic sites, providing evidence for noninvasive imaging of BMD cells in the premetastatic niche. Conclusion: The results of the study demonstrated the potential of PET with VLA-4-targeted ⁶⁴Cu-CB-TE2A-LLP2A to visualize BMD cell reorganization and expansion noninvasively in vivo.