## 4 Discussion  
BSGI with [99 mTc]Tc-Sestamibi can represent a remarkable opportunity to detect both primary and metastatic lesions characterized by the presence of breast cancer cells with high amounts of mitochondria. In fact, the current data about the chemical-physical characteristics of setsamibi, as well as its pharmacokinetics, seem to indicate its tropism for the accumulation in the cell organelles with a negative membrane potential, such as mitochondria. In support of this, it is known that once in the cytoplasm, setsamibi may translocate into the mitochondria according to its cationic nature [15; 16]. The positive charge on setsamibi may drive this molecule into the mitochondria during cell metabolic activities that increase the negative plasma membrane potential. Despite this evidence, few studies have been performed about the possible effect of setsamibi uptake in breast cancer cells. The accumulation of setsamibi in the cell allows it to hypothesize the alteration of normal mitochondria functions and consequently of the cancer cell's homeostasis, mainly at the level of cell proliferation and cell death. Ex vivo data reported here strengthened the known association between setsamibi uptake and Ki67 expression, thus confirming the capability of BSGI with [99 mTc]Tc-Sestamibi to identify breast cancer lesions characterized by high proliferation index [17]. In line with this, [PERSON] et al. also demonstrated the association between setsamibi uptake and proliferation index in parathyroid lesions [18]. Surprisingly, linear regression analysis also  
Figure 3: Comparison between apoptosis and proliferation of BT474 breast cancer cells treated with [99 mTc]Tc-Sestamibi and ultrastructural analysis. (**A**) Graph shows the number of both caspase-3- and Ki67-positive BT474 breast cancer cells after 24, 48, 72, 96, and 120 h of [99 mTc]Tc-Sestamibi incubation (10 ug/mL). (**B**) Graph displays the number of both caspase-3- and Ki67-positive BT474 breast cancer cells after 24, 48, 72, 96, and 120 h of [99 mTc]Tc-Sestamibi incubation (10 ug/mL). (**C**) Graph shows the number of both caspase-3- and Ki67-positive BT474 breast cancer cells after 24, 48, 72, 96, and 120 h of [99 mTc]Tc-Sestamibi incubation (10 ug/mL). (**D**) Electron micrograph displays ultrastructure of BT474 breast cancer cells (CTRL T0). (**E**) Image shows an apoptotic breast cancer cell (asterisk) close to a non-apoptotic cell (10 ug/mL at 48 h). (**F,G**) EDX spectrum revealed the presence of [99 mTc]Technetium into the cytoplasm of apoptotic cells (10 ug/mL at 120 h). showed a significant association between the secstanibi uptake and the number of apoptotic cells evaluated as caspase-3-positive cells. Caspase-3 is a protein effector involved in apoptosis [19]. Activated caspase-3, as well as caspase-6 and caspase-7, may cleave multiple structural and regulatory proteins that are critical for cell survival and maintenance [18]. However, caspase-3 is considered the most important executioner caspase and it is involved in both intrinsic and extrinsic apoptotic pathways [20]. Alteration in both mitochondrial function and structure induce the release of mediators such as BCL-2 family proteins that amplify caspase-3 activity, inducing the intrinsic pathway of apoptosis [21]. Thus, the expression of caspase-3 in breast cells can be related to the occurrence of intrinsic apoptosis. Data on both Ki67 and caspase-3, despite being in disagreement, can be related to the same processes involved in breast cancer progression. Indeed, as demonstrated by [PERSON] and [PERSON] [22], the caspase-3 related apoptotic phenomenon may be related to cancer progression due to the communication between apoptotic cells and surrounding ones. This biological process occurs physiologically during embryogenesis, where proapoptotic proteins--mostly caspases--can induce the proliferation of neighboring surviving cells to replace dying cells. It is demonstrated that the deregulation of this process in cancer tissues could sustain tumor proliferation and progress.