Selection of internalizing RNA aptamers into human breast cancer cells derived from primary sites.

Breast cancer is the most common cancer in women. Although chemotherapy is still broadly used in its treatment, adverse effects remain a challenge. In this scenario, aptamers emerge as a promising alternative for theranostic applications. Studies using breast cancer cell lines provide useful information in laboratory and preclinical investigations, most of which use cell lines established from metastatic sites. However, these cell lines correspond to cell populations of the late stage of tumor progression. On the other hand, studies using breast cancer cells established from primary sites make it possible to search for new theranostic approaches in the early stages of the disease. Therefore, this work aimed to select RNA aptamers internalized by MGSO-3 cells, a human breast cancer cell line, derived from a primary site previously established in our laboratory. Using the Cell-Internalization SELEX method, we have selected two candidate aptamers (ApBC1 and ApBC2). We evaluated their internalization efficiencies, specificities, cellular localization by Reverse Transcription-qPCR (RT-qPCR) and confocal microscopy assays. The results suggest that both aptamers were efficiently internalized by human breast cancer cells, MACL-1, MDA-MB-231, and especially by MGSO-3 cells. Furthermore, both aptamers could effectively distinguish human breast cancer cells derived from normal human mammary cell (MCF 10A) and prostate cancer cell (PC3) lines. Therefore, ApBC1 and ApBC2 could be promising candidate molecules for theranostic applications, even in the early stages of tumor progression.

Translocation of Epidermal Growth Factor (EGF) to the nucleus has distinct kinetics between adipose tissue-derived mesenchymal stem cells and a mesenchymal cancer cell lineage.

Nuclear Epidermal Growth Factor Receptor (EGFR) has been associated with worse prognosis and treatment resistance for several cancer types. After Epidermal Growth Factor (EGF) binding, the ligand-receptor complex can translocate to the nucleus where it functions in oncological processes. By three-dimensional quantification analysis of super-resolution microscopy images, we verified the translocation kinetics of fluorescent conjugated EGF to the nucleus in two mesenchymal cell types: human adipose tissue-derived stem cells (hASC) and SK-HEP-1 tumor cells. The number of EGF clusters in the nucleus does not change after 10 min of stimulation with EGF in both cells. The total volume occupied by EGF clusters in the nucleus of hASC also does not change after 10 min of stimulation with EGF. However, the total volume of EGF clusters increases only after 20 min in SK-HEP-1 cells nuclei. In these cells the nuclear volume occupied by EGF is 3.2 times higher than in hASC after 20 min of stimulation, indicating that translocation kinetics of EGF differs between these two cell types. After stimulation, EGF clusters assemble in larger clusters in the cell nucleus in both cell types, which suggests specific sub-nuclear localizations of the receptor. Super-resolution microscopy images show that EGF clusters are widespread in the nucleoplasm, and can be localized in nuclear envelope invaginations, and in the nucleoli. The quantitative study of EGF-EGFR complex translocation to the nucleus may help to unravel its roles in health and pathological conditions, such as cancer.

Inhibition of the osteogenic differentiation of mesenchymal stem cells derived from the offspring of rats treated with caffeine during pregnancy and lactation.

Caffeine is an alkaloid that is widely consumed due to its presence in drugs, coffee, tea, and chocolate. This compound passes to offspring through the placenta and milk; can cause teratogenic mutations; and reduces the formation, growth, and mass of bone. Because mesenchymal stem cells (MSCs) are responsible for generating the entire skeleton, we hypothesized that these cells are targets of caffeine. This study evaluated the osteogenic differentiation of MSCs derived from the offspring of rats treated with caffeine during pregnancy and lactation. Twenty-four adult Wistar rats were randomly divided into four groups, including one control group and three experimental groups treated with 25, 50, or 100 mg/kg of caffeine. At weaning, three 21-day-old pups from each dam in each group were euthanized for extraction of bone marrow cells for in vitro tests. Caffeine doses of 50 and 100 mg/kg significantly reduced the activity of alkaline phosphatase at 7, 14, and 21 days and the expression of collagen I at 21 days. However, the expression of gene transcripts for alkaline phosphatase, Runx-2, and bone sialoprotein, as well as the synthesis of mineralization nodules, decreased significantly in all groups treated with caffeine. The expression of osteocalcin was significantly reduced only in the group treated with 50 mg/kg caffeine. The caffeine that passes from the mother to the offspring during pregnancy and lactation reduces the osteogenic differentiation of MSCs. We propose that this reduction in the osteogenic potential of MSCs may be involved in the pathogenesis of osteopenia resulting from caffeine consumption.

Adipose-derived stem/stromal cell secretome modulates breast cancer cell proliferation and differentiation state towards aggressiveness.

It is becoming increasingly evident that mesenchymal stem/stromal cells are recruited by cancer cells from nearby endogenous host stroma and promote events such as tumor proliferation, angiogenesis, invasion, and metastasis, as well as mediate therapeutic resistance. Consequently, understanding the regulatory mechanisms of ASCs that influence the tumor microenvironment may provide an avenue for further treatment. To understand the role of the ASC secretome in breast cancer cell proliferation, death, and phenotype alteration, adipose-derived stem cell-conditioned medium (mASC) was used to cultivate MCF-7 and MDA-MB-231 cells. These breast cancer cells in mASC showed a shorter doubling time, higher frequency of EdU positivity, and higher levels of phosphorylated histone 3. In addition, increased expression of cyclin B1 was observed, suggesting that proliferation was induced. The mASC was also able to increase apoptosis in MCF-7 cells, which was confirmed by caspase-7 activation. The number of tumor-initiating cells (CD44+ CD24-/low) and migration capacity were increased in cells cultivated in mASC. These data collectively suggest that ASC-conditioned medium can induce selective pressure by increasing cell proliferation, giving rise to a more aggressive phenotype in MCF-7 and MDA-MB-231 cells. Our study provides a foundation for further elucidation of the precise mechanism underlying ASCs in breast cancer cells and the modulation of ASCs in potential therapeutic uses.

Stem Cell Extracellular Vesicles in Skin Repair.

Stem cell extracellular vesicles (EVs) have been widely studied because of their excellent therapeutic potential. EVs from different types of stem cell can improve vascularization as well as aid in the treatment of cancer and neurodegenerative diseases. The skin is a complex organ that is susceptible to various types of injury. Strategies designed to restore epithelial tissues' integrity with stem cell EVs have shown promising results. Different populations of stem cell EVs are able to control inflammation, accelerate skin cell migration and proliferation, control wound scarring, improve angiogenesis, and even ameliorate signs of skin aging. However, large-scale production of such stem cell EVs for human therapy is still a challenge. This review focuses on recent studies that explore the potential of stem cell EVs in skin wound healing and skin rejuvenation, as well as challenges of their use in therapy.

Phospholipase C delta 4 (PLCδ4) is a nuclear protein involved in cell proliferation and senescence in mesenchymal stromal stem cells.

Ca2+ is an important second messenger, and it is involved in many cellular processes such as cell death and proliferation. The rise in intracellular Ca2+ levels can be due to the generation of inositol 1,4,5-trisphosphate (InsP3), which is a product of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by phospholipases C (PLCs), that leads to Ca2+ release from endoplasmic reticulum by InsP3 receptors (InsP3R). Ca2+ signaling patterns can vary in different regions of the cell and increases in nuclear Ca2+ levels have specific biological effects that differ from those of Ca2+ increase in the cytoplasm. There are PLCs in the cytoplasm and nucleus, but little is known about the functions of nuclear PLCs. This work aimed to characterize phenotypically the human PLCδ4 (hPLCδ4) in mesenchymal stem cells. This nuclear isoform of PLC is present in different cell types and has a possible role in proliferative processes. In this work, hPLCδ4 was found to be mainly nuclear in human adipose-derived mesenchymal stem cells (hASC). PLCδ4 knockdown demonstrated that it is essential for hASC proliferation, without inducing cell death. An increase of cells in G1, and a reduction of cells on interphase and G2/M in knockdown cells were seen. Furthermore, PLCδ4 knockdown increased the percentage of senescent cells, p16INK4A+ and p21Cip1 mRNAs expression, which could explain the impaired cell proliferation. The results show that hPLCδ4 is in involved in cellular proliferation and senescence in hASC.

Extracellular Vesicles from Adipose-Derived Mesenchymal Stem/Stromal Cells Accelerate Migration and Activate AKT Pathway in Human Keratinocytes and Fibroblasts Independently of miR-205 Activity.

Mesenchymal stem/stromal cells (MSCs) are promising tools in cell therapy. They secrete extracellular vesicles (EVs) that carry different classes of molecules that can promote skin repair, but the mechanisms are poorly understood. Skin wound healing is a complex process that requires the activity of several signaling pathways and cell types, including keratinocytes and fibroblasts. In this study, we explored whether adipose tissue MSC-derived EVs could accelerate migration and proliferation of keratinocytes and fibroblasts, activate the AKT pathway, and promote wound healing in vivo. Furthermore, we evaluated if EV effects are miR-205 dependent. We found that MSC EVs had an average diameter of 135 nm. Keratinocytes and fibroblasts exposed to EVs exhibited higher levels of proliferation, migration, and AKT activation. Topical administration of EVs accelerated skin wound closure. Knockdown of miR-205 decreased AKT phosphorylation in fibroblasts and keratinocytes, whereas migration was decreased only in keratinocytes. Moreover, knockdown of miR-205 failed to inhibit AKT phosphorylation in fibroblasts and keratinocytes exposed to EVs. About the mechanism of EV effects, we found that incubation with EVs prevented inhibition of AKT activation by miR-205 knockdown, suggesting that EVs activate AKT independently of miR-205. In conclusion, we demonstrated that EVs are a promising tool for wound healing.