EB1089 Increases the Antiproliferative Response of Lapatinib in Combination with Antiestrogens in HER2-Positive Breast Cancer Cells.

HER2-positive breast cancer is associated with aggressive behavior and reduced survival rates. Calcitriol restores the antiproliferative activity of antiestrogens in estrogen receptor (ER)-negative breast cancer cells by re-expressing ERα. Furthermore, calcitriol and its analog, EB1089, enhance responses to standard anti-cancer drugs. Therefore, we aimed to investigate EB1089 effects when added to the combined treatment of lapatinib and antiestrogens on the proliferation of HER2-positive breast cancer cells. BT-474 (ER-positive/HER2-positive) and SK-BR-3 (ER-negative/HER2-positive) cells were pre-treated with EB1089 to modulate ER expression. Then, cells were treated with EB1089 in the presence of lapatinib with or without the antiestrogens, and proliferation, phosphorylation array assays, and Western blot analysis were performed. The results showed that EB1089 restored the antiproliferative response to antiestrogens in SK-BR-3 cells and improved the inhibitory effects of the combination of lapatinib with antiestrogens in the two cell lines. Moreover, EB1089, alone or combined, modulated ERα protein expression and reduced Akt phosphorylation in HER2-positive cells. EB1089 significantly enhanced the cell growth inhibitory effect of lapatinib combined with antiestrogens in HER2-positive breast cancer cells by modulating ERα expression and Akt phosphorylation suppression. These results highlight the potential of this therapeutic approach as a promising strategy for managing HER2-positive breast cancer.

In Vitro Culturing of Human Term Placental Explants.

The study of the human placenta has always been appealing, given the importance of this temporal organ capable of sustaining the beginning of life and development of a new human being within the womb. Culturing placental explants has been an easy and reliable method to study some placental morphological, biochemical, and physiological features for a very long time. Besides low time consumption, requirement of few resources, and wide versatility, the placental explant in vitro culture retains cell-cell interaction in a 3D structure resembling the in vivo setting, which is why it is the option of choice for many researchers in the field. This chapter will describe a simplified method for culturing explants from human term placentas.

In Vitro Culturing of Human Trophoblasts from Term Placenta.

Since the early 1960s, researchers began culturing placental cells to establish an in vitro model to study the biology of human trophoblasts, including their ability to differentiate into syncytiotrophoblasts and secrete steroid and peptide hormones that help sustain a viable pregnancy. This task was addressed by testing different serum concentrations, cell culture media, digestive enzymes, growth factors, substrate coating with diverse proteins from the extracellular matrix, and so on. Among the many methodological challenges, the contamination of trophoblasts with other cell types, such as immune and stromal cells, was a matter of concern. However, introducing the Percoll gradient to isolate cytotrophoblasts was an excellent contribution, and later, the depletion of contaminating cells by using magnetic bead-conjugated antibodies also helped increase the purity of cytotrophoblasts. Herein, with some modifications, we describe a rapid and easy method for cytotrophoblast isolation from the term human placenta based on the previously reported method by Harvey Kliman et al. (Endocrinology 118:1567-1582, 1986). This method yields about 40-90 million cells from a single placenta, with a purity of around 85-90%.

Evaluation of Leukocyte Chemotaxis Induced by Human Fetal Membranes in an In Vitro Model.

Leukocyte infiltration into the maternal-fetal interface is a consequence of the robust inflammation in the gestational tissues during term labor and preterm labor with or without infection. During pregnancy, the fetal membranes act as a physical barrier that isolates the fetus into the amniotic cavity, keeping it in an optimal environment for its development. In addition, the fetal membranes possess immunological competencies such as the secretion of cytokines and chemokines in response to different stimuli. Clinical and experimental evidence indicates that these tissues are involved in the extensive chemotaxis of immune cells in normal or pathological conditions.Few studies have evaluated the chemotactic capacities of the fetal membranes considering that this tissue is composed of two adjacent tissues, the amnion and the chorion, which have different characteristics. Although these tissues function as a unit, their response is complex since there is an interaction between them, where each tissue contributes differently. The protocol described here allows us to evaluate the in vitro chemotactic capacities of fetal membranes in response to various applied stimuli, considering the contribution of each of their components (amnion and choriodecidua) using a Boyden chamber assay and phenotyping the chemo-attracted leukocytes by flow cytometry.

Culture of Human Fetal Membranes in a Two Independent Compartment Model: An Ex Vivo Approach.

During pregnancy, the fetal membranes composed of the amnion and chorodecidua constitute a selective barrier separating two distinct environments, maternal and fetal. These tissues have the function of delimiting the amniotic cavity. Their histological complexity gives them physical, mechanical, and immunological properties to protect the fetus. Although the study of the amnion, chorion, and decidua separately provides knowledge about the functions of the fetal membranes, the protocol we describe in this chapter has the advantage of maintaining the biological and functional complexity of these tissues. In addition, this experimental model allows the researcher to recreate various pathological scenarios because this model allows for differential stimulation of the amnion or choriodecidua.

Enhancing Tamoxifen Therapy with α-Mangostin: Synergistic Antiproliferative Effects on Breast Cancer Cells and Potential Reduced Endometrial Impact.

Breast cancer is the most prevalent neoplasia among women worldwide. For the estrogen receptor-positive (ER+) phenotype, tamoxifen is the standard hormonal therapy; however, it carries the risk of promoting endometrial carcinoma. Hence, we aimed to evaluate the antiproliferative effect of the phytochemical α-mangostin (AM) as a co-adjuvant alongside tamoxifen on breast cancer cells to improve its efficacy while reducing its adverse effects on endometrium. For this, ER+ breast cancer cells (MCF-7 and T-47D) and endometrial cells (N30) were treated with AM, 4-hydroxytamoxifen (4-OH-TMX), and their combination. Cell proliferation was evaluated using sulforhodamine B assay, and the pharmacological interaction was determined through the combination index and the dose reduction index calculation. The genes KCNH1, CCDN1, MKI67, and BIRC5 were amplified by real-time PCR as indicators of oncogenesis, cell cycle progression, cell proliferation, and apoptosis, respectively. Additionally, genes involved in ER signaling were analyzed. In breast cancer cells, the combination of AM with 4-OH-TMX showed a synergistic antiproliferative effect and favorable dose reduction. AM and 4-OH-TMX decreased KCNH1, CCND1, and BIRC5 gene expression. In endometrial cells, AM decreased MKI-67 gene expression, while it reverted the 4-OH-TMX-dependent CCND1 upregulation. This study establishes the benefits of incorporating AM as a co-adjuvant for first-line ER+ breast cancer therapy.

The Inhibition of the FGFR/PI3K/Akt Axis by AZD4547 Disrupts the Proangiogenic Microenvironment and Vasculogenic Mimicry Arising from the Interplay between Endothelial and Triple-Negative Breast Cancer Cells.

Vasculogenic mimicry (VM), a process in which aggressive cancer cells form tube-like structures, plays a crucial role in providing nutrients and escape routes. Highly plastic tumor cells, such as those with the triple-negative breast cancer (TNBC) phenotype, can develop VM. However, little is known about the interplay between the cellular components of the tumor microenvironment and TNBC cells' VM capacity. In this study, we analyzed the ability of endothelial and stromal cells to induce VM when interacting with TNBC cells and analyzed the involvement of the FGFR/PI3K/Akt pathway in this process. VM was corroborated using fluorescently labeled TNBC cells. Only endothelial cells triggered VM formation, suggesting a predominant role of paracrine/juxtacrine factors from an endothelial origin in VM development. Via immunocytochemistry, qPCR, and secretome analyses, we determined an increased expression of proangiogenic factors as well as stemness markers in VM-forming cancer cells. Similarly, endothelial cells primed by TNBC cells showed an upregulation of proangiogenic molecules, including FGF, VEGFA, and several inflammatory cytokines. Endothelium-dependent TNBC-VM formation was prevented by AZD4547 or LY294002, strongly suggesting the involvement of the FGFR/PI3K/Akt axis in this process. Given that VM is associated with poor clinical prognosis, targeting FGFR/PI3K/Akt pharmacologically may hold promise for treating and preventing VM in TNBC tumors.

The Preventive Role of the Vitamin D Endocrine System in Cervical Cancer.

Vitamin D along with its active metabolite calcitriol and its metabolic and signaling system, known as the vitamin D endocrine system, have been widely recognized as a pivotal regulator of calcium homeostasis in addition to non-calcemic antitumoral effects in a variety of human cancers, including cervical cancer. Several studies have found an inverse relationship between the incidence of cervical neoplasia and vitamin D levels. This narrative review updates the current evidence supporting the notion that the vitamin D endocrine system has a preventive role on cervical cancer, mainly in the early phases of the disease, acting at the level of suppressing cell proliferation, promoting apoptosis, modulating inflammatory responses, and probably favoring the clearance of human papillomavirus-dependent cervical lesions. Although an optimal vitamin D status helps in the prevention and regression of low-grade squamous intraepithelial lesions of the cervix, it appears that vitamin D alone or combined with chemotherapeutic agents has little effectivity once advanced cervical cancer is established. These observations suggest that an optimal vitamin D status might exert beneficial actions in the early phases of cervical cancer by preventing its onset and progression.

High Glucose Promotes Inflammation and Weakens Placental Defenses against E. coli and S. agalactiae Infection: Protective Role of Insulin and Metformin.

Placentas from gestational diabetes mellitus (GDM) patients undergo significant metabolic and immunologic adaptations due to hyperglycemia, which results in an exacerbated synthesis of proinflammatory cytokines and an increased risk for infections. Insulin or metformin are clinically indicated for the treatment of GDM; however, there is limited information about the immunomodulatory activity of these drugs in the human placenta, especially in the context of maternal infections. Our objective was to study the role of insulin and metformin in the placental inflammatory response and innate defense against common etiopathological agents of pregnancy bacterial infections, such as E. coli and S. agalactiae, in a hyperglycemic environment. Term placental explants were cultivated with glucose (10 and 50 mM), insulin (50-500 nM) or metformin (125-500 µM) for 48 h, and then they were challenged with live bacteria (1 × 105 CFU/mL). We evaluated the inflammatory cytokine secretion, beta defensins production, bacterial count and bacterial tissue invasiveness after 4-8 h of infection. Our results showed that a GDM-associated hyperglycemic environment induced an inflammatory response and a decreased beta defensins synthesis unable to restrain bacterial infection. Notably, both insulin and metformin exerted anti-inflammatory effects under hyperglycemic infectious and non-infectious scenarios. Moreover, both drugs fortified placental barrier defenses, resulting in reduced E. coli counts, as well as decreased S. agalactiae and E. coli invasiveness of placental villous trees. Remarkably, the double challenge of high glucose and infection provoked a pathogen-specific attenuated placental inflammatory response in the hyperglycemic condition, mainly denoted by reduced TNF-α and IL-6 secretion after S. agalactiae infection and by IL-1ß after E. coli infection. Altogether, these results suggest that metabolically uncontrolled GDM mothers develop diverse immune placental alterations, which may help to explain their increased vulnerability to bacterial pathogens.

The Phytochemical α-Mangostin Inhibits Cervical Cancer Cell Proliferation and Tumor Growth by Downregulating E6/E7-HPV Oncogenes and KCNH1 Gene Expression.

Cervical cancer is the fourth most common cancer among women worldwide. The main factor associated with the onset and progression of this neoplasia is the human papillomavirus (HPV) infection. The HPV-oncogenes E6 and E7 are critical drivers of cellular transformation, promoting the expression of oncogenes such as KCNH1. The phytochemical α-mangostin (AM) is a potent antineoplastic and antiviral compound. However, its effects on HPV oncogenes and KCNH1 gene expression remain unknown. This study evaluated the effects of AM on cell proliferation, cell cycle distribution and gene expression, including its effects on tumor growth in xenografted mice. AM inhibited cell proliferation in a concentration-dependent manner, being the most sensitive cell lines those with the highest number of HPV16 copies. In addition, AM promoted G1-cell cycle arrest in CaSki cells, while led to cell death in SiHa and HeLa cells. Of interest was the finding of an AM-dependent decreased gene expression of E6, E7 and KCNH1 both in vitro and in vivo, as well as the modulation of cytokine expression, Ki-67, and tumor growth inhibition. On these bases, we suggest that AM represents a good option as an adjuvant for the treatment and prevention of cervical cancer.

The Interaction of Human Papillomavirus Infection and Prostaglandin E2 Signaling in Carcinogenesis: A Focus on Cervical Cancer Therapeutics.

Chronic infection by high-risk human papillomaviruses (HPV) and chronic inflammation are factors associated with the onset and progression of several neoplasias, including cervical cancer. Oncogenic proteins E5, E6, and E7 from HPV are the main drivers of cervical carcinogenesis. In the present article, we review the general mechanisms of HPV-driven cervical carcinogenesis, as well as the involvement of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) and downstream effectors in this pathology. We also review the evidence on the crosstalk between chronic HPV infection and PGE2 signaling, leading to immune response weakening and cervical cancer development. Finally, the last section updates the current therapeutic and preventive options targeting PGE2-derived inflammation and HPV infection in cervical cancer. These treatments include nonsteroidal anti-inflammatory drugs, prophylactic and therapeutical vaccines, immunomodulators, antivirals, and nanotechnology. Inflammatory signaling pathways are closely related to the carcinogenic nature of the virus, highlighting inflammation as a co-factor for HPV-dependent carcinogenesis. Therefore, blocking inflammatory signaling pathways, modulating immune response against HPV, and targeting the virus represent excellent options for anti-tumoral therapies in cervical cancer.

Endothelium-Dependent Induction of Vasculogenic Mimicry in Human Triple-Negative Breast Cancer Cells Is Inhibited by Calcitriol and Curcumin.

In highly aggressive tumors, cancer cells may form channel-like structures through a process known as vasculogenic mimicry (VM). VM is generally associated with metastasis, mesenchymal phenotype, and treatment resistance. VM can be driven by antiangiogenic treatments and/or tumor microenvironment-derived factors, including those from the endothelium. Curcumin, a turmeric product, inhibits VM in some tumors, while calcitriol, the most active vitamin D metabolite, exerts potent antineoplastic effects. However, the effect of these natural products on VM in breast cancer remains unknown. Herein, we studied the effect of both compounds on triple-negative breast cancer (TNBC) VM-capacity in a co-culture model. The process of endothelial cell-induced VM in two human TNBC cell lines was robustly inhibited by calcitriol and partially by curcumin. Calcitriol promoted TNBC cells' morphological change from spindle-like to cobblestone-shape, while curcumin diminished VM 3D-structure. Notably, the treatments dephosphorylated several active kinases, especially those involved in the PI3K/Akt pathway. In summary, calcitriol and curcumin disrupted endothelium-induced VM in TNBC cells partially by PI3K/Akt inactivation and mesenchymal phenotype inhibition. Our results support the possible use of these natural compounds as adjuvants for VM inactivation in patients with malignant tumors inherently capable of forming VM, or those with antiangiogenic therapy, warranting further in vivo studies.

AZD4547 and calcitriol synergistically inhibited BT-474 cell proliferation while modified stemness and tumorsphere formation.

Fibroblast growth factor receptor (FGFR) overamplification/activation in cancer leads to increased cell proliferation. AZD4547, a FGFR selective inhibitor, hinders breast cancer cells growth. Although luminal B breast tumors may respond to chemotherapy and endocrine therapy, this subtype is associated with poor prognosis, inadequate response and/or acquired drug resistance. Calcitriol, the vitamin D most active metabolite, exerts anti-neoplastic effects and enhances chemotherapeutic drugs activity. In this study, we sought to decrease the concentration of AZD4547 needed to inhibit the luminal-B breast cancer cell line BT-474 proliferation by its combination with calcitriol. Anti-proliferative inhibitory concentrations, combination index and dose-reduction index were analyzed from Sulforhodamine B assays. Western blot and qPCR were used to study FGFR molecular targets. The compound's ability to inhibit BT-474 cells tumorigenic capacity was assessed by tumorspheres formation. Results: BT-474 cells were dose-dependently growth-inhibited by calcitriol and AZD4547 (IC50 = 2.9 nM and 3.08 µM, respectively). Calcitriol at 1 nM synergistically improved AZD4547 antiproliferative effects, allowing a 2-fold AZD4547 dose-reduction. Mechanistically, AZD4547 downregulated p-FGFR1, p-Akt and tumorsphere formation. Calcitriol also decreased tumorspheres, while induced cell differentiation. Both compounds inhibited MYC and CCND1 expression, as well as ALDH, a stemness marker that positively correlated with FGFR1 and negatively with VDR expression in breast cancer transcriptomic data. In conclusion, the drugs impaired self-aggregation capacity, reduced stemness features, induced cell-differentiation and when combined, synergistically inhibited cell proliferation. Overall, our results suggest that calcitriol, at low pharmacological doses, may be a suitable candidate to synergize AZD4547 effects in luminal B breast tumors, allowing to reduce dose and adverse effects.

Compartmentalized Innate Immune Response of Human Fetal Membranes against Escherichia coli Choriodecidual Infection.

An infectious process into the uterine cavity represents a major endangered condition that compromises the immune privilege of the maternal-fetal unit and increases the risk for preterm birth (PTB) and premature rupture of membranes (PROM). Fetal membranes are active secretors of antimicrobial peptides (AMP), which limit bacterial growth, such as Escherichia coli. Nevertheless, the antibacterial responses displayed by chorioamniotic membranes against a choriodecidual E. coli infection have been briefly studied. The objective of this research was to characterize the profile of synthesis, activity, and spatial distribution of a broad panel of AMPs produced by fetal membranes in response to E. coli choriodecidual infection. Term human chorioamniotic membranes were mounted in a two independent compartment model in which the choriodecidual region was infected with live E. coli (1 × 105 CFU/mL). Amnion and choriodecidual AMP tissue levels and TNF-α and IL-1ß secretion were measured by the enzyme-linked immunosorbent assay. The passage of bacterium through fetal membranes and their effect on structural continuity was followed for 24 h. Our results showed that E. coli infection caused a progressive mechanical disruption of the chorioamniotic membranes and an activated inflammatory environment. After the challenge, the amnion quickly (2-4 h) induced production of human beta defensins (HBD)-1, HBD-2, and LL-37. Afterwards (8-24 h), the amnion significantly produced HBD-1, HBD-2, HNP-1-3, S100A7, sPLA2, and elafin, whereas the choriodecidua induced LL-37 synthesis. Therefore, we noticed a temporal- and tissue-specific pattern regulation of the synthesis of AMPs by infected fetal membranes. However, fetal membranes were not able to contain the collagen degradation or the bacterial growth and migration despite the battery of produced AMPs, which deeply increases the risk for PTB and PROM. The mixture of recombinant HBDs at low concentrations resulted in increased bactericidal activity compared to each HBD alone in vitro, encouraging further research to study AMP combinations that may offer synergy to control drug-resistant infections in the perinatal period.

Combinations of Calcitriol with Anticancer Treatments for Breast Cancer: An Update.

Preclinical, clinical, and epidemiological studies indicate that vitamin D3 (VD) deficiency is a risk factor for the development of breast cancer. Underlying mechanisms include the ability of calcitriol to induce cell differentiation, inhibit oncogenes expression, and modify different signaling pathways involved in the control of cell proliferation. In addition, calcitriol combined with different kinds of antineoplastic drugs has been demonstrated to enhance their beneficial effects in an additive or synergistic fashion. However, a recognized adjuvant regimen based on calcitriol for treating patients with breast cancer has not yet been fully established. Accordingly, in the present work, we review and discuss the preclinical and clinical studies about the combination of calcitriol with different oncological drugs, aiming to emphasize its main therapeutic benefits and opportunities for the treatment of this pathology.

Cord Serum Calcitriol Inversely Correlates with Maternal Blood Pressure in Urinary Tract Infection-Affected Pregnancies: Sex-Dependent Immune Implications.

Urinary tract infections (UTI) during pregnancy are frequently associated with hypertensive disorders, increasing the risk of perinatal morbidity. Calcitriol, vitamin D3's most active metabolite, has been involved in blood pressure regulation and prevention of UTIs, partially through modulating vasoactive peptides and antimicrobial peptides, like cathelicidin. However, nothing is known regarding the interplay between placental calcitriol, cathelicidin, and maternal blood pressure in UTI-complicated pregnancies. Here, we analyzed the correlation between these parameters in pregnant women with UTI and with normal pregnancy (NP). Umbilical venous serum calcitriol and its precursor calcidiol were significantly elevated in UTI. Regardless of newborn's sex, we found strong negative correlations between calcitriol and maternal systolic and diastolic blood pressure in the UTI cohort (p < 0.002). In NP, this relationship was observed only in female-carrying mothers. UTI-female placentas showed higher expression of cathelicidin and CYP27B1, the calcitriol activating-enzyme, compared to male and NP samples. Accordingly, cord-serum calcitriol from UTI-female neonates negatively correlated with maternal bacteriuria. Cathelicidin gene expression positively correlated with gestational age in UTI and with newborn anthropometric parameters. Our results suggest that vitamin D deficiency might predispose to maternal cardiovascular risk and perinatal infections especially in male-carrying pregnancies, probably due to lower placental CYP27B1 and cathelicidin expression.

Immunoendocrine Dysregulation during Gestational Diabetes Mellitus: The Central Role of the Placenta.

Gestational Diabetes Mellitus (GDM) is a transitory metabolic condition caused by dysregulation triggered by intolerance to carbohydrates, dysfunction of beta-pancreatic and endothelial cells, and insulin resistance during pregnancy. However, this disease includes not only changes related to metabolic distress but also placental immunoendocrine adaptations, resulting in harmful effects to the mother and fetus. In this review, we focus on the placenta as an immuno-endocrine organ that can recognize and respond to the hyperglycemic environment. It synthesizes diverse chemicals that play a role in inflammation, innate defense, endocrine response, oxidative stress, and angiogenesis, all associated with different perinatal outcomes.

Antitumoral effects of dovitinib in triple-negative breast cancer are synergized by calcitriol in vivo and in vitro.

Chemotherapy is a standard therapeutic option for triple-negative breast cancer (TNBC); however, its effectiveness is often compromised by drug-related toxicity and resistance development. Herein, we aimed to evaluate whether an improved antineoplastic effect could be achieved in vitro and in vivo in TNBC by combining dovitinib, a multi-kinase inhibitor, with calcitriol, a natural anticancer hormone. In vitro, cell proliferation and cell-cycle distribution were studied by sulforhodamine B-assays and flow cytometry. In vivo, dovitinib/calcitriol effects on tumor growth, angiogenesis, and endothelium activation were evaluated in xenografted mice by caliper measures, Itgb3/VEGFR2-immunohistochemistry and 99mTc-Ethylenediamine-N,N-diacetic acid/hydrazinonicotinamyl-Glu[cyclo(Arg-Gly-Asp-D-Phe-Lys)]2 (99mTc-RGD2)-tumor uptake. The drug combination elicited a synergistically improved antiproliferative effect in TNBC-derived cells, which allowed a 7-fold and a 3.3-fold dovitinib dose-reduction in MBCDF-Tum and HCC-1806 cells, respectively. Mechanistically, the co-treatment induced a cell cycle profile suggestive of cell death and DNA damage (accumulation of cells in SubG1, S, and G2/M phases), increased the number of multinucleated cells and inhibited tumor growth to a greater extent than each compound alone. Tumor uptake of 99mTc-RGD2 was reduced by dovitinib, suggesting angiogenesis inhibition, which was corroborated by decreased endothelial cell growth, tumor-vessel density and VEGFR2 expression. In summary, calcitriol synergized dovitinib anticancer effects in vitro and in vivo, allowing for a significant dose-reduction of dovitinib while maintaining its antiproliferative potency. Our results suggest the beneficial convergence of independent antitumor mechanisms of dovitinib and calcitriol to inhibit TNBC-tumor growth.

Vasculogenic Mimicry in Breast Cancer: Clinical Relevance and Drivers.

In solid tumors, vasculogenic mimicry (VM) is the formation of vascular structures by cancer cells, allowing to generate a channel-network able to transport blood and tumor cells. While angiogenesis is undertaken by endothelial cells, VM is assumed by cancer cells. Besides the participation of VM in tumor neovascularization, the clinical relevance of this process resides in its ability to favor metastasis and to drive resistance to antiangiogenic therapy. VM occurs in many tumor types, including breast cancer, where it has been associated with a more malignant phenotype, such as triple-negative and HER2-positive tumors. The latter may be explained by known drivers of VM, like hypoxia, TGFB, TWIST1, EPHA2, VEGF, matrix metalloproteinases, and other tumor microenvironment-derived factors, which altogether induce the transformation of tumor cells to a mesenchymal phenotype with a high expression rate of stemness markers. This review analyzes the current literature in the field, including the participation of some microRNAs and long noncoding RNAs in VM-regulation and tumorigenesis of breast cancer. Considering the clinical relevance of VM and its association with the tumor phenotype and clinicopathological parameters, further studies are granted to target VM in the clinic.

Ethical considerations in animal research: the principle of 3r's

In the last century, progress in the knowledge of human diseases, their diagnosis and treatment have grown exponentially, due in large part to the introduction and use of laboratory animals. Along with this important progress, the need to provide training and guidance to the scientific community in all aspects related to the proper use of experimental animals has been indispensable. Animal research committees play a primary role in evaluating experimental research protocols, from their feasibility to the rational use of animals, but above all in seeking animal welfare. The Institutional Committee for the Care and Use of Animals (IACUC) has endeavored to share several relevant aspects in conducting research with laboratory animals. Here, we present and discuss the topics that we consider of utmost importance to take in the account during the design of any experimental research protocol, so we invite researchers, technicians, and undergraduate and graduate students to dive into the fascinating subject of proper animal care and use for experimentation. The main intention of these contributions is to sensitize users of laboratory animals for the proper and rational use of them in experimental research, as well as to disseminate the permitted and unpermitted procedures in laboratory animals. In the first part, the significance of experimental research, the main functions of IACUC, and the principle of the three R's (replacement, reduction, and refinement) are addressed.