Bone and muscle have been recognized as endocrine organs since they produce and secrete “hormone-like factors” that can mutually influence each other and other tissues, giving rise to a “bone–muscle crosstalk”. In our study, we made use of myogenic (C2C12 cells) and osteogenic (2T3 cells) cell lines to investigate the effects of muscle cell-produced factors on the maturation process of osteoblasts. We found that the myogenic medium has inhibitory effects on bone cell differentiation and we identified sclerostin as one of the myokines produced by muscle cells. Sclerostin is a secreted glycoprotein reportedly expressed by bone/cartilage cells and is considered a negative regulator of bone growth due to its role as an antagonist of the Wnt/β-catenin pathway. Given the inhibitory role of sclerostin in bone, we analyzed its expression by muscle cells and how it affects bone formation and homeostasis. Firstly, we characterized and quantified sclerostin synthesis by a myoblast cell line (C2C12) and by murine primary muscle cells by Western blotting, real-time PCR, immunofluorescence, and ELISA assay. Next, we investigated in vivo production of sclerostin in distinct muscle groups with different metabolic and mechanical loading characteristics. This analysis was done in mice of different ages (6 weeks, 5 and 18 months after birth) and revealed that sclerostin expression is dynamically modulated in a muscle-specific way during the lifespan. Finally, we transiently expressed sclerostin in the hind limb muscles of young mice (2 weeks of age) via in vivo electro-transfer of a plasmid containing the SOST gene in order to investigate the effects of muscle-specific overproduction of the protein. Our data disclosed an inhibitory role of the muscular sclerostin on the bones adjacent to the electroporated muscles. This observation suggests that sclerostin released by skeletal muscle might synergistically interact with osseous sclerostin and potentiate negative regulation of osteogenesis possibly by acting in a paracrine/local fashion. Our data point out a role for muscle as a new source of sclerostin.

Subcellular spatial location is an essential descriptor of molecules biological function. Presently, super-resolution microscopy techniques enable quantification of subcellular objects distribution in fluorescence images, but they rely on instrumentation, tools and expertise not constituting a default for most of laboratories. We propose a method that allows resolving subcellular structures location by reinforcing each single pixel position with the information from surroundings. Although designed for entry-level laboratory equipment with common resolution powers, our method is independent from imaging device resolution, and thus can benefit also super-resolution microscopy. The approach permits to generate density distribution maps (DDMs) informative of both objects’ absolute location and self-relative displacement, thus practically reducing location uncertainty and increasing the accuracy of signal mapping. This work proves the capability of the DDMs to: (a) improve the informativeness of spatial distributions; (b) empower subcellular molecules distributions analysis; (c) extend their applicability beyond mere spatial object mapping. Finally, the possibility of enhancing or even disclosing latent distributions can concretely speed-up routine, large-scale and follow-up experiments, besides representing a benefit for all spatial distribution studies, independently of the image acquisition resolution. DDMaker, a Software endowed with a user-friendly Graphical User Interface (GUI), is also provided to support users in DDMs creation.

The volume is one of the most relevant features that define the treatment of an in vivo tumour. When using cancer 3D in vitro models in pre-clinical studies, it becomes important to evaluate the macroscopic effects of drugs and radiotherapy treatments. Depending on the nature of the 3D in vitro model used, different open-source solutions can be used for measuring the volume by starting from microscope-acquired images. In this work, we introduced several open-source tools today available for estimating the volume of 3D multicellular aggregates (e.g., spheroids, organoids), also giving hints for defining the “best software” by analysing characteristics of 3D in vitro models and limits of the tools. Finally, using several cancer organoids imaged by a fluorescent microscope, we compared volume estimations obtained with different tools, besides presenting a new version of the Reconstruction and Visualization from Multiple Sections (ReViMS version 2.0) tool. This work aims to be the reference for researchers interested in estimating the volume of 3D multicellular aggregates through an open-source tool.

Cellular and subcellular spatial colocalization of structures and molecules in biological specimens is an important indicator of their co-compartmentalization and interaction. Presently, colocalization in biomedical images is addressed with visual inspection and quantified by co-occurrence and correlation coefficients. However, such measures alone cannot capture the complexity of the interactions, which does not limit itself to signal intensity. On top of the previously developed density distribution maps (DDMs), here, we present a method for advancing current colocalization analysis by introducing co-density distribution maps (cDDMs), which, uniquely, provide information about molecules absolute and relative position and local abundance. We exemplify the benefits of our method by developing cDDMs-integrated pipelines for the analysis of molecules pairs co-distribution in three different real-case image datasets. First, cDDMs are shown to be indicators of colocalization and degree, able to increase the reliability of correlation coefficients currently used to detect the presence of colocalization. In addition, they provide a simultaneously visual and quantitative support, which opens for new investigation paths and biomedical considerations. Finally, thanks to the coDDMaker software we developed, cDDMs become an enabling tool for the quasi real time monitoring of experiments and a potential improvement for a large number of biomedical studies.