## 3 Results
### Effect of Secstamibi on Breast Cancer Cells: In Vitro Study  
At the end of each experimental point, BT-474 cells were fixed with 4% paraformaldehyde-hyde and used to evaluate the number of both Ki67- and caspase-3-positive cells. One-way ANOVA showed significant data distribution for both the number of caspase-3- (\(p\) < 0.0001) and Ki67- (\(p\) = 0.0038) positive cells. Notably, a high concentration of [99 mTc]Tc-Sestamibi (10 ug/mL) induced a significant increase in the number of apoptotic cells (caspase-3-positive cells) when compared with all other experimental conditions, including the control  
Figure 1: Breast-specific \(\gamma\) imaging with [99 mTc]Tc-Sestamibi and both caspase-3 and Ki67 expression. (**A**) Maximum-intensity projection of breast-specific \(\gamma\) imaging with [99 mTc]Tc-Sestamibi in a 69-year-old breast cancer patient. (**B**) The graph shows the positive and significant association between the number of caspase-3-positive breast cancer cells and the secstamibi uptake (L/M ratio). (**C**) The graph displays significant association between the number of Ki67-positive breast cancer cells and the secstamibi uptake (L/M ratio). (**D**) T hematoxylin and eosin section shows G3 infiltrating breast carcinomas (scale bar represents 50 μm). (**E**) The image displays numerous caspase-3-positive breast cancer cells (scale bar represents 50 μm). (**F**) High percentage of Ki67-positive breast cancer cells (scale bar represents 50 μm). (Figure 2A-I). Specifically, a great increase in the number of caspase-3-positive cells was observed after 48h (Figure 2A,C). This datum suggests that [99 mTc]Tc-Sestamibi could be able to induce the apoptotic process by caspase-3 signal only at high concentrations. In other experimental conditions, no significant effects were detected (Figure 2A). Concerning the cell proliferation (Ki67), a significant decrease in the number of Ki67-positive cells was observed in cell cultures incubated with both 10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL and 1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of [99 mTc]Tc-Sestamibi when compared with cells treated with a concentration of 1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL (Figure 2M-P). Indeed, the data reported here showed that a low concentration of [99 mTc]Tc-Sestamibi (1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) does not influence the proliferation index (Figure 2M). Cell cultures treated with both 1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL and 0.1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL [99 mTc]Tc-Sestamibi displayed a proliferation index similar to that observed in the control (Figure 2M). This datum can explain the association observed in vivo between secantibi uptake and the percentage of Ki67-positive cells. It is important to note that high concentration of [99 mTc]Tc-Sestamibi (10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) induced a significant reduction in the proliferation index already after 24 h of treatment (Figure 2M-L). By comparing the value of Ki67 and caspase-3 at every time point it became clear that by only using 10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of [99 mTc]Tc-Sestamibi concentration, a complete reversion between proliferation and apoptosis was obtained (Figure 3A-C). In particular, after 72h, the number of caspase-3-positive cells was higher than those positive for Ki67, suggesting an imbalance capable of arresting the tumor's proliferation (Figure 3A). Morphological  
Figure 2: In vitro evaluation of the effect of [99 mTc]Tc-Sestamibi on BT474 breast cancer cells. (**A**) The graph shows the number of caspase-3-positive BT474 breast cancer cells after secantibi treatment. (**B**) No/rare caspase-3-positive cells at T0. (**C**) The image shows caspase-3-positive cells after 48 h of [99 mTc]Tc-Sestamibi incubation (10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL). (**D**) Numerous caspase-3-positive cells after 72 h of [99 mTc]Tc-Sestamibi incubation (10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL). (**E**) High number of caspase-3-positive cells after 120 h of [99 mTc]Tc-Sestamibi incubation (10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL).