Imaging and biodistribution of Her2/neu expression in non-small cell lung cancer xenografts with Cu-labeled trastuzumab PET

Abstract

Non-small cell lung carcinomas (NSCLC) overexpress the Her2/neu gene in approximately 59% of cases. Trastuzumab, a humanized monoclonal antibody, interferes with Her2 signaling and is approved for the treatment of Her2/neu overexpressing breast cancer. However, its therapeutic use in Her2/neu overexpressing NSCLC remains obscure. The present study aimed to determine the role of (64)Cu-labeled trastuzumab positron emission tomography (PET) for non-invasive imaging of Her2/neu expression in NSCLC. Trastuzumab was conjugated with the bifunctional chelator 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) and radiolabeled with (64)Cu. The molecular specificity of DOTA-trastuzumab was determined in NSCLC cell lines with Her2/neu overexpression (NCI-H2170) and negative expression (NCI-H520). Imaging of Her2/neu expression was performed in NCI-H2170 tumor-bearing mice with (64)Cu-DOTA-trastuzumab PET and (64)Cu-DOTA-IgG. In vitro studies revealed specific binding of DOTA-trastuzumab in the Her2/neu positive NCI-H2170 cells, while no binding was seen in the Her2/neu negative NCI-H520 cell line. Biodistribution and PET studies revealed a significantly high accumulation of (64)Cu-DOTA-trastuzumab in the Her2/neu overexpressing NCI-H2170 tumor at 24 and 48 h post-injection (21.4 +/- 1.4% and 23.2 +/- 5.1% injection dose/gram (% ID/g), respectively). PET imaging of Her2/neu negative NCI-H520 tumors showed much less uptake of (64)Cu-DOTA-trastuzumab (4.0% ID/g). The NCI-H2170 tumor uptake of (64)Cu-DOTA-trastuzumab was significantly higher than that of (64)Cu-DOTA-IgG (P < 0.0001). (64)Cu-DOTA-trastuzumab showed a very clear image of a Her2/neu positive tumor and appeared to be effective as a PET tracer for imaging of Her2/neu gene expression in NSCLC, suggesting its potential clinical use for identifying patients that might benefit from trastuzumab-based therapy.

Figure 1

Fluorescence microscopy imaging of NSCLC cell lines. (A) Significant binding of DOTA‐trastuzumab is demonstrated in the membrane of Her2/neu positive NCI‐H2170 cells (magnification ×1200). (B) Her2/neu negative NCI‐H520 cells show no binding of DOTA‐trastuzumab (magnification ×800). (C) DOTA‐IgG did not show any binding to Her2/neu positive NCI‐H2170 cells (magnification ×800).

Figure 2

Radioactivity profiles of ⁶⁴Cu‐DOTA‐tras‐tuzumab after incubation in murine serum (in vitro) for 1 and 48 h. The radioactivity of the sample was also analyzed. The right hand graph shows the radioactivity profiles in the blood of mice after administration of ⁶⁴Cu‐DOTA‐trastuzumab at 1 and 48 h. The radioactivity was analyzed by SE‐HPLC.

Figure 3

In vivo and ex vivo PET images of mice bearing Her2/neu negative NCI‐H520 and Her2/neu positive NCI‐H2170 tumors after injection of ⁶⁴Cu‐DOTA‐trastuzumab or ⁶⁴Cu‐DOTA‐IgG. In vivo and ex vivo studies showed no uptake of ⁶⁴Cu‐DOTA‐trastuzumab in the Her2/neu positive NCI‐H2170 tumor after 1 h of administration. After 24 h of injection, significant accumulation was observed in the tumor and liver. At 48 h after the injection, the accumulation increased in the tumor but it decreased in the normal organs, including the liver. Negative tumor model NCI‐H520 xenografts injected with ⁶⁴Cu‐DOTA‐trastuzumab showed no prominent accumulation. Similarly, Her2/neu positive NCI‐H2170 xenografts also did not show specific accumulation in the tumor when injected with ⁶⁴Cu‐DOTA‐IgG. The arrow shows the tumor. Bl, blood; Li, liver; Ki, kidney; I, intestine; St, stomach; Sp, spleen; P, pancreas; H, heart; M, muscle; B, bone; L, lung; Br, brain; T, tumor.

Figure 4

Biodistribution of the Her2/neu overexpressing NCI‐H2170 xenografts at different time points. (A) % ID/g of the organs after injection with ⁶⁴Cu‐DOTA‐trastuzumab. (B) % ID/g in the organs after injection with ⁶⁴Cu‐DOTA‐IgG. Tumor uptake was significantly higher in the mice injected with ⁶⁴Cu‐DOTA‐trastuzumab (P < 0.0001).

Figure 5

Tumor‐to‐blood ratio at various time points. The ratio was 1.52 ± 0.20 at 24 h post‐injection and it was 2.45 ± 1.29 at 48 h post‐injection. Each value represents the mean ± SD of three or four mice.