As neovascularization is essential for tumor growth and metastasis, controlling angiogenesis is a promising tactic in limiting cancer progression. Melatonin has been studied for their inhibitory properties on angiogenesis in cancer. We performed an in vivo study to evaluate the effects of melatonin treatment on angiogenesis in breast cancer. Cell viability was measured by MTT assay after melatonin treatment in triple-negative breast cancer cells (MDA-MB-231). After, cells were implanted in athymic nude mice and treated with melatonin or vehicle daily, administered intraperitoneally 1 hour before turning the room light off. Volume of the tumors was measured weekly with a digital caliper and at the end of treatments animals underwent single photon emission computed tomography (SPECT) with Technetium-99m tagged vascular endothelial growth factor (VEGF) C to detect in vivo angiogenesis. In addition, expression of pro-angiogenic/growth factors in the tumor extracts was evaluated by membrane antibody array and collected tumor tissues were analyzed with histochemical staining. Melatonin in vitro treatment (1 mM) decreased cell viability (p<0.05). The breast cancer xenografts nude mice treated with melatonin showed reduced tumor size and cell proliferation (Ki-67) compared to control animals after 21 days of treatment (p<0.05). Expression of VEGF receptor 2 decreased significantly in the treated animals compared to that of control when determined by immunohistochemistry (p0.05) images. In addition, there was a decrease of micro-vessel density (Von Willebrand Factor) in melatonin treated mice (p<0.05). However, semiquantitative densitometry analysis of membrane array indicated increased expression of epidermal growth factor receptor and insulin-like growth factor 1 in treated tumors compared to vehicle treated tumors (p<0.05). In conclusion, melatonin treatment showed effectiveness in reducing tumor growth and cell proliferation, as well as in the inhibition of angiogenesis.

It is well-recognized that solid tumors are genomically, anatomically, and physiologically heterogeneous. In general, more heterogeneous tumors have poorer outcomes, likely due to the increased probability of harboring therapy-resistant cells and regions. It is hypothesized that the genomic and physiologic heterogeneity are related, because physiologically distinct regions will exert variable selection pressures leading to the outgrowth of clones with variable genomic/proteomic profiles. To investigate this, methods must be in place to interrogate and define, at the microscopic scale, the cytotypes that exist within physiologically distinct subregions (“habitats”) that are present at mesoscopic scales. MRI provides a noninvasive approach to interrogate physiologically distinct local environments, due to the biophysical principles that govern MRI signal generation. Here, we interrogate different physiologic parameters, such as perfusion, cell density, and edema, using multiparametric MRI (mpMRI). Signals from six different acquisition schema were combined voxel-by-voxel into four clusters identified using a Gaussian mixture model. These were compared with histologic and IHC characterizations of sections that were coregistered using MRI-guided 3D printed tumor molds. Specifically, we identified a specific set of MRI parameters to classify viable-normoxic, viable-hypoxic, nonviable-hypoxic, and nonviable-normoxic tissue types within orthotopic 4T1 and MDA-MB-231 breast tumors. This is the first coregistered study to show that mpMRI can be used to define physiologically distinct tumor habitats within breast tumor models. Significance: This study demonstrates that noninvasive imaging metrics can be used to distinguish subregions within heterogeneous tumors with histopathologic correlation.

Cancer stem cells (CSCs) have been associated with metastasis and therapeutic resistance and can be generated via epithelial mesenchymal transition (EMT). Some studies suggest that the hormone melatonin acts in CSCs and may participate in the inhibition of the EMT. The objectives of this study were to evaluate the formation of mammospheres from the canine and human breast cancer cell lines, CMT-U229 and MCF-7, and the effects of melatonin treatment on the modulation of stem cell and EMT molecular markers: OCT4, E-cadherin, N-cadherin and vimentin, as well as on cell viability and invasiveness of the cells from mammospheres. The CMT-U229 and MCF-7 cell lines were subjected to three-dimensional culture in special medium for stem cells. The phenotype of mammospheres was first evaluated by flow cytometry (CD44⁺/CD24^low/- marking). Cell viability was measured by MTT colorimetric assay and the expression of the proteins OCT4, E-cadherin, N-cadherin and vimentin was evaluated by immunofluorescence and quantified by optical densitometry. The analysis of cell migration and invasion was performed in Boyden Chamber. Flow cytometry proved the stem cell phenotype with CD44⁺/CD24^low/- positive marking for both cell lines. Cell viability of CMT-U229 and MCF-7 cells was reduced after treatment with 1mM melatonin for 24 h (P<0.05). Immunofluorescence staining showed increased E-cadherin expression (P<0.05) and decreased expression of OCT4, N-cadherin and vimentin (P<0.05) in both cell lines after treatment with 1 mM melatonin for 24 hours. Moreover, treatment with melatonin was able to reduce cell migration and invasion in both cell lines when compared to control group (P<0.05). Our results demonstrate that melatonin shows an inhibitory role in the viability and invasiveness of breast cancer mammospheres as well as in modulating the expression of proteins related to EMT in breast CSCs, suggesting its potential anti-metastatic role in canine and human breast cancer cell lines.

The use of nanosized particles has emerged to facilitate selective applications in medicine. Drug-delivery systems represent novel opportunities to provide stricter, focused, and fine-tuned therapy, enhancing the therapeutic efficacy of chemical agents at the molecular level while reducing their toxic effects. Melatonin (N-acetyl-5-methoxytriptamine) is a small indoleamine secreted essentially by the pineal gland during darkness, but also produced by most cells in a non-circadian manner from which it is not released into the blood. Although the therapeutic promise of melatonin is indisputable, aspects regarding optimal dosage, biotransformation and metabolism, route and time of administration, and targeted therapy remain to be examined for proper treatment results. Recently, prolonged release of melatonin has shown greater efficacy and safety when combined with a nanostructured formulation. This review summarizes the role of melatonin incorporated into different nanocarriers (e.g., lipid-based vesicles, polymeric vesicles, non-ionic surfactant-based vesicles, charge carriers in graphene, electro spun nanofibers, silica-based carriers, metallic and non-metallic nanocomposites) as drug delivery system platforms or multilevel determinations in various in vivo and in vitro experimental conditions. Melatonin incorporated into nanosized materials exhibits superior effectiveness in multiple diseases and pathological processes than does free melatonin; thus, such information has functional significance for clinical intervention.