Figure 1.
Schematic diagram depicting the interaction of multiple myeloma (MM) and stromal cells.
A. Very late antigen-4 (VLA-4, also known as integrin α4β1) is over expressed on MM cells. VLA-4 mediates myeloma cell adhesion to the bone marrow (BM) stroma through interactions with fibronectin, a glycoprotein of extracellular matrix (ECM) protein and vascular cell adhesion molecule-1 (VCAM-1) protein expressed on bone marrow (BM) stromal cells. B. Structure of CB-TE1A1P-LLP2A.
Figure 2.
Flow cytometry, cell uptake and saturation binding data.
A. Greater than 85% of α4 (VLA-4)-positive cells in total 5TGM1 tumor cell population as determined by flow cytometry (Anti-Mouse CD49d (integrin α-4). B. Cell uptake of 64Cu-CB-TE1A1P-LLP2A (0.1 nM), in 5TGM1 cells at 37°C (p<0.0001). C. Saturation binding curve for 64Cu-CB-TE1A1P-LLP2A gave a Kd of 2.2 nM (±1.0) and Bmax of 136 pmol/mg (±19). N = 3 (Inset: Scatchard transformation of saturation binding data).
Figure 3.
Tissue biodistribution of 64Cu-CB-TE1A1P-LLP2A in 5TGM1 s.c. tumor mice.
Biodistribution of 64Cu-CB-TE1A1P-LLP2A in 5TGM1 s.c. tumor mice (black bars). The open bars represent biodistribution in the presence of non-radioactive blocking agent (∼ 200 fold excess LLP2A). Mice were injected with 64Cu-CB-TE1A1P-LLP2A (0.01 µg, 0.2 MBq, SA: 37 MBq/µg) and sacrificed at 2 h post injection. N = 4 mice/group.
Figure 4.
Representative maximum intensity projection (MIP) small animal PET/CT images.
A. non-tumor KaLwRij control mouse. B. a small sized, non-palpable, early stage subcutaneous (s.c.) 5TGM1 murine tumor in the nape of the neck inoculated without the use of matrigel (tumor SUV 2.24). White arrows point to suspected tumor cells and associated tumor supporting cells in the BM of the long bones and spine. C. matrigel assisted s.c. 5TGM1 tumor in the nape of the neck (tumor SUV 6.2). D. mouse injected intraperitoneally (i.p.) with 5TGM1 murine myeloma cells. All the mice were injected with 64Cu-CB-TE1A1P-LLP2A (0.9 MBq, 0.05 µg, 27 pmol) and were imaged by small animal PET/CT at 2 h post-injection. *All tumor bearing animals were SPEP (Serum Protein Electrophoresis) positive. T = Tumor; S = Spleen. N = 4/group.
Figure 5.
Graph representing tumor to muscle and blood respectively at early and late time-points.
The Tumor/Muscle and Tumor/Blood ratios at 2 h and 24 h respectively calculated from the MIP images (SUVs). The ratios were higher at 24 h indicating improved contrast after clearance of the radioactive probe from the background tissues over time.
Figure 6.
Graph representing in vivo blocking of 64Cu-CB-TE1A1P-LLP2A.
Averaged tumor MIP SUV’s (N = 4) calculated from small animal PET images in the presence and absence of the blocking agent, LLP2A. Inset-Representative MIP image showing mice with similar tumors in the nape of the neck imaged in the absence (L) and presence (R) of the blocking agent (LLP2A). Mice were imaged with small animal PET and 64Cu-CB-TE1A1P-LLP2A at 2 h post-injection (0.9 MBq, 0.05 µg, 27 pmol, (SA: 37 MBq/µg). Blocking dose: ∼200 fold excess than the tracer amount. P<0.05.
Figure 7.
Tumor histology and SPEP (Serum Protein Electrophoresis) analysis on the serum of KaLwRij mice.
A. Hematoxylin and eosin (H&E) stained slide of a representative 5TGM1 s.c. tumor tissue. The tumor cells show irregularly shaped nuclei and increased mitosis consistent with myeloma pathogenic features. B. SPEP gel showing qualitatively the γ-globulin (M protein) in tumor bearing (lanes 5, 6 & 7) and non-tumor bearing (lanes 1, 2, 3 & 4) mice. The 5TGM1 tumor bearing mice (lanes 5, 6 & 7) were analyzed two weeks post tumor cell inoculation. The top arrow represents the M Protein band and the lanes represent serum SPEP for each mouse. C) Quantitative representation of the total γ-globulin g/dl in the mice (1–7).
Figure 8.
Cellular uptake of 64Cu-CB-TE1A1P-LLP2A in human myeloma RPMI-8226 cells.
Cell uptake of 64Cu-CB-TE1A1P-LLP2A (0.1 nM), in human RPMI-8226 cells at 37°C in the absence (red bar) and presence (blue bar) of excess LLP2A (P<0.0001).