*2.6. In Vivo Experiments*

The A549 tumor cell line was inoculated, by subcutaneous injection, in the right shoulder of homozygous female BALB/c nude mice. The tumor was allowed to grow under observation for five weeks, and imaging procedures were started when 80% of the animals developed tumors of sufficient size (i.e., >0.5 cm). A first group of mice (*n* = 5) was intravenously injected with [111]In-IP-001 (2.06 nmol, 7.4 MBq), and tomographic single photon emission tomography (SPECT)/CT images were acquired at 2, 4, 8, and 24 h post injection to monitor the pharmacokinetics of the radiotracer. For blocking experiments, a second group of mice (*n* = 5) was injected with approximately the same amount of [ <sup>111</sup>]In-IP-001 of the first group with the addition of a 50-fold molar excess of unlabeled IP-001 ligand and imaged at 4 and 24 h. In a parallel independent experiment, three mice belonging to both groups were sacrificed at 4 and 24 h post injection, and their main organs were explanted for measuring the biodistribution of the radiotracer. As per SPECT/CT imaging analysis, the tumor could already be visualized after 4 h, even though [111]In-IP-001 showed a high accumulation in all the main organs. Blood radioactivity was high, suggesting that a significant amount of the radiotracer was in circulation or bound to blood constituents, such as serum albumin, as found in the in vitro experiments. The kinetics of clearance appeared to be slow with a tumor to background ratio improving over time up to 24 h, when radioactivity accumulation mainly persisted in the tumor, the gastro-intestinal tract, and the kidneys. Paradigmatic SPECT/CT images are reported in Figure 3. In the biodistribution studies, the tumor achieved the maximal uptake (2.36 ± 0.26% IA/g; *n* = 3) at 4 h post injection despite the greater (blood: 4.21 ± 0.40% IA/g; liver: 8.25 ± 2.21%; kidneys: 6.99 ± 0.97%; spleen: 3.88± 0.36%; *n* = 3) or at least comparable (intestines: 2.39 ± 0.28%; lungs: 2.82 ± 0.21%; pancreas: 2.03 ± 0.16%; *n* = 3) accumulation in other organs and tissues, suggesting a low specific biodistribution of the radiotracer at this time. At 24 h post injection, tumor uptake decreased to 1.52 ± 0.71% IA/g (*n* = 3), but the tumor to organ ratios highly improved with respect to 4 h, as shown in Figure 4. Actually, only the liver and kidneys still exhibited higher uptake as compared to the tumor tissue, with ratios of 0.47 and 0.48, respectively. Conversely, the tumor to muscle (T/M) and tumor to blood (T/B) ratios were highly favorable (15.72 and 10.19, respectively), thus confirming

the rapid accumulation and persistence in the tumor visualized by SPECT/CT images, as well as a slow kinetics of clearance that occurred via both renal and hepatobiliary excretion. Residual radioactivity in all the other main organs and, in particular, the intestines and pancreas was low (0.55 ± 0.29% IA/g and 0.26 ± 0.04% IA/g, respectively).

**Figure 3.** Representative single photon emission tomography (SPECT)/CT scans as maximum intensity projection (MIP) views of an A549 tumor-bearing mouse at 4 (**A**) and 24 h (**B**) post injection of about 7.4 MBq of [111In]In-IP-001. Tumor position in the shoulder is indicated by the arrow.

At 4 h post injection, a blocking experiment reflected a low specific distribution of the radiotracer, as no significant differences were found in the radioactivity measured in the organs and tumors of the normal and blocked groups. After 24 h, a more pronounced gap could be appreciated when, for instance, the tumor uptake was 1.52 ± 0.71% IA/g (*n* = 3) in the experimental group versus 0.84 ± 0.33% IA/g (*n* = 3) in the blocked group. However, the differences were still not statistically significant (p = 0.08). Complete comparisons between the two groups at 4 and 24 h are shown in Figure 5. On the other hand, a clear effect of the blocking could be visualized in the SPECT/CT images at 24 h post injection. In Figure 6, for instance, it is shown that a negligible uptake in the tumor was found in the blocked mouse in comparison with the unblocked one.

**Figure 4.** Biodistribution in multiple organs of [111In]In-IP-001 in A549 cancer-bearing nude mice (*<sup>n</sup>* = 3; mean <sup>±</sup> SD) at 4 and 24 h post injection (**A**); ratios of the tumor uptake to major organs (**B**).

**Figure 5.** Comparison among the biodistributions in multiple organs of [111In]In-IP-001 and [111In]In-IP-001, as well as unlabeled-IP-001 (blocking) in A549 cancer-bearing nude mice (*n* = 3; mean ± SD) at 4 (**A**) and 24 h (**B**) post injection.

**Figure 6.** Representative SPECT/CT scans as MIP views of two A549 tumor-bearing mice injected with [111In]-IP-001 (**A**), as well as [111In]-IP-001 and 50-fold molar excess of unlabeled IP-001 (blocking) (**B**) at 24 h post injection. Mice developing tumors approximately of the same size (about 112 mm<sup>3</sup> ) are shown. Tumor position in the shoulder is indicated by the arrow.
