The Secretome of Parental and Bone Metastatic Breast Cancer Elicits Distinct Effects in Human Osteoclast Activity after Activation of ß2 Adrenergic Signaling.

The sympathetic nervous system (SNS), particularly through the ß2 adrenergic receptor (ß2-AR), has been linked with breast cancer (BC) and the development of metastatic BC, specifically in the bone. Nevertheless, the potential clinical benefits of exploiting ß2-AR antagonists as a treatment for BC and bone loss-associated symptoms remain controversial. In this work, we show that, when compared to control individuals, the epinephrine levels in a cohort of BC patients are augmented in both earlier and late stages of the disease. Furthermore, through a combination of proteomic profiling and functional in vitro studies with human osteoclasts and osteoblasts, we demonstrate that paracrine signaling from parental BC under ß2-AR activation causes a robust decrease in human osteoclast differentiation and resorption activity, which is rescued in the presence of human osteoblasts. Conversely, metastatic bone tropic BC does not display this anti-osteoclastogenic effect. In conclusion, the observed changes in the proteomic profile of BC cells under ß-AR activation that take place after metastatic dissemination, together with clinical data on epinephrine levels in BC patients, provided new insights on the sympathetic control of breast cancer and its implications on osteoclastic bone resorption.

Profiling the Adrenergic System in Breast Cancer and the Development of Metastasis.

Epidemiological studies and preclinical models suggest that chronic stress might accelerate breast cancer (BC) growth and the development of metastasis via sympathetic neural mechanisms. Nevertheless, the role of each adrenergic pathway (α1, α2, and ß) in human samples remains poorly depicted. Herein, we propose to characterize the profile of the sympathetic system (e.g., release of catecholamines, expression of catecholamine metabolic enzymes and adrenoreceptors) in BC patients, and ascertain its relevance in the development of distant metastasis. Our results demonstrated that BC patients exhibited increased plasma levels of catecholamines when compared with healthy donors, and this increase was more evident in BC patients with distant metastasis. Our analysis using the BC-TCGA database revealed that the genes coding the most expressed adrenoreceptors in breast tissues (ADRA2A, ADRA2C, and ADRB2, by order of expression) as well as the catecholamine synthesizing (PNMT) and degrading enzyme (MAO-A and MAO-B) genes were downregulated in BC tissues. Importantly, the expression of ADRA2A, ADRA2C, and ADRB2 was correlated with metastatic BC and BC subtypes, and thus the prognosis of the disease. Overall, we gathered evidence that under stressful conditions, both the α2- and ß2-signaling pathways might work on a synergetic matter, thus paving the way for the development of new therapeutic approaches.

Stress in Metastatic Breast Cancer: To the Bone and Beyond.

Breast cancer (BRCA) remains as one the most prevalent cancers diagnosed in industrialised countries. Although the overall survival rate is high, the dissemination of BRCA cells to distant organs correlates with a significantly poor prognosis. This is due to the fact that there are no efficient therapeutic strategies designed to overcome the progression of the metastasis. Over the past decade, critical associations between stress and the prevalence of BRCA metastases were uncovered. Chronic stress and the concomitant sympathetic hyperactivation have been shown to accelerate the progression of the disease and the metastases incidence, specifically to the bone. In this review, we provide a summary of the sympathetic profile on BRCA. Additionally, the current knowledge regarding the sympathetic hyperactivity, and the underlying adrenergic signalling pathways, involved on the development of BRCA metastasis to distant organs (i.e., bone, lung, liver and brain) will be revealed. Since bone is a preferential target site for BRCA metastases, greater emphasis will be given to the contribution of α2- and ß-adrenergic signalling in BRCA bone tropism and the occurrence of osteolytic lesions.

A metastasis-on-a-chip approach to explore the sympathetic modulation of breast cancer bone metastasis.

Organ-on-a-chip models have emerged as a powerful tool to model cancer metastasis and to decipher specific crosstalk between cancer cells and relevant regulators of this particular niche. Recently, the sympathetic nervous system (SNS) was proposed as an important modulator of breast cancer bone metastasis. However, epidemiological studies concerning the benefits of the SNS targeting drugs on breast cancer survival and recurrence remain controversial. Thus, the role of SNS signaling over bone metastatic cancer cellular processes still requires further clarification. Herein, we present a novel humanized organ-on-a-chip model recapitulating neuro-breast cancer crosstalk in a bone metastatic context. We developed and validated an innovative three-dimensional printing based multi-compartment microfluidic platform, allowing both selective and dynamic multicellular paracrine signaling between sympathetic neurons, bone tropic breast cancer cells and osteoclasts. The selective multicellular crosstalk in combination with biochemical, microscopic and proteomic profiling show that synergistic paracrine signaling from sympathetic neurons and osteoclasts increase breast cancer aggressiveness demonstrated by augmented levels of pro-inflammatory cytokines (e.g. interleukin-6 and macrophage inflammatory protein 1α). Overall, this work introduced a novel and versatile platform that could potentially be used to unravel new mechanisms involved in intracellular communication at the bone metastatic niche.

Sympathetic activity in breast cancer and metastasis: partners in crime.

The vast majority of patients with advanced breast cancer present skeletal complications that severely compromise their quality of life. Breast cancer cells are characterized by a strong tropism to the bone niche. After engraftment and colonization of bone, breast cancer cells interact with native bone cells to hinder the normal bone remodeling process and establish an osteolytic "metastatic vicious cycle". The sympathetic nervous system has emerged in recent years as an important modulator of breast cancer progression and metastasis, potentiating and accelerating the onset of the vicious cycle and leading to extensive bone degradation. Furthermore, sympathetic neurotransmitters and their cognate receptors have been shown to promote several hallmarks of breast cancer, such as proliferation, angiogenesis, immune escape, and invasion of the extracellular matrix. In this review, we assembled the current knowledge concerning the complex interactions that take place in the tumor microenvironment, with a special emphasis on sympathetic modulation of breast cancer cells and stromal cells. Notably, the differential action of epinephrine and norepinephrine, through either α- or ß-adrenergic receptors, on breast cancer progression prompts careful consideration when designing new therapeutic options. In addition, the contribution of sympathetic innervation to the formation of bone metastatic foci is highlighted. In particular, we address the remarkable ability of adrenergic signaling to condition the native bone remodeling process and modulate the bone vasculature, driving breast cancer cell engraftment in the bone niche. Finally, clinical perspectives and developments on the use of ß-adrenergic receptor inhibitors for breast cancer management and treatment are discussed.

The alliance between nerve fibers and stem cell populations in bone marrow: life partners in sickness and health.

The bone marrow (BM) is the central hematopoietic organ in adult mammals, with great potential to be used as a tool to improve the efficacy of the body's response to a number of malignancies and stressful conditions. The nervous system emerges as a critical regulatory player of the BM both under homeostatic and pathologic settings, with essential roles in cellular anchorage and egress, stem cell differentiation, and endothelial cell permeability. This review collects the current knowledge on the interplay between the nervous system and the BM cell populations, with a focus on how the nervous system modulates hematopoietic stem and progenitor cell, mesenchymal stromal cell, and endothelial progenitor cell activity in BM. We have also highlighted the pathologies that have been associated with disturbances in the neuronal signaling in BM and discussed if targeting the nervous system, either by modulating the activity of specific neuronal circuits or by pharmacologically leveling the activity of sympathetic and sensorial signaling-responsive cells in BM, is a promising therapeutic approach to tackling pathologies from BM origin.-Leitão, L., Alves, C. J., Sousa, D. M., Neto, E., Conceição, F., Lamghari, M. The alliance between nerve fibers and stem cell populations in bone marrow: life partners in sickness and health.

Bone marrow cell response after injury and during early stage of regeneration is independent of the tissue-of-injury in 2 injury models.

Selectively recruiting bone marrow (BM)-derived stem and progenitor cells to injury sites is a promising therapeutic approach. The coordinated action of soluble factors is thought to trigger the mobilization of stem cells from the BM and recruit them to lesions to contribute to tissue regeneration. Nevertheless, the temporal response profile of the major cellular players and soluble factors involved in priming the BM and recruiting BM-derived cells to promote regeneration is unknown. We show that injury alters the BM cellular composition, introducing population-specific fluctuations during tissue regeneration. We demonstrate that injury causes an immediate, transient response of mesenchymal stromal cells and endothelial cells followed by a nonoverlapping increase in hematopoietic stem and progenitor cells. Moreover, BM reaction is identical whether the injury is inflicted on skin and muscle or also involves a bone defect, but these 2 injury paradigms trigger distinct systemic cytokine responses. Together, our results indicate that the BM response to injury in the early stages of regeneration is independent of the tissue-of-injury based on the 2 models used, but the injured tissue dictates the systemic cytokine response.-Leitão, L., Alves, C. J., Alencastre, I. S., Sousa, D. M., Neto, E., Conceição, F., Leitão, C., Aguiar, P., Almeida-Porada, G., Lamghari, M. Bone marrow cell response after injury and during early stage of regeneration is independent of the tissue-of-injury in 2 injury models.

Axonal outgrowth, neuropeptides expression and receptors tyrosine kinase phosphorylation in 3D organotypic cultures of adult dorsal root ganglia.

Limited knowledge from mechanistic studies on adult sensory neuronal activity was generated, to some extent, in recapitulated adult in vivo 3D microenvironment. To fill this gap there is a real need to better characterize the adult dorsal root ganglia (aDRG) organotypic cultures to make these in vitro systems exploitable for different approaches, ranging from basic neurobiology to regenerative therapies, to address the sensory nervous system in adult stage. We conducted a direct head-to-head comparison of aDRG and embryonic DRG (eDRG) organotypic culture focusing on axonal growth, neuropeptides expression and receptors tyrosine kinase (RTK) activation associated with neuronal survival, proliferation and differentiation. To identify alterations related to culture conditions, these parameters were also addressed in retrieved aDRG and eDRG and compared with organotypic cultures. Under similar neurotrophic stimulation, aDRG organotypic cultures displayed lower axonal outgrowth rate supported by reduced expression of growth associated protein-43 and high levels of RhoA and glycogen synthase kinase 3 beta mRNA transcripts. In addition, differential alteration in sensory neuropeptides expression, namely calcitonin gene-related peptide and substance P, was detected and was mainly pronounced at gene expression levels. Among 39 different RTK, five receptors from three RTK families were emphasized: tropomyosin receptor kinase A (TrkA), epidermal growth factor receptors (EGFR, ErbB2 and ErbB3) and platelet-derived growth factor receptor (PDGFR). Of note, except for EGFR, the phosphorylation of these receptors was dependent on DRG developmental stage and/or culture condition. In addition, EGFR and PDGFR displayed alterations in their cellular expression pattern in cultured DRG. Overall we provided valuable information particularly important when addressing in vitro the molecular mechanisms associated with development, maturation and regeneration of the sensory nervous system.