A risk-reward examination of sample multiplexing reagents for single cell RNA-Seq.

Single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for understanding cellular heterogeneity and function. However the choice of sample multiplexing reagents can impact data quality and experimental outcomes. In this study, we compared various multiplexing reagents, including MULTI-Seq, Hashtag antibody, and CellPlex, across diverse sample types such as human peripheral blood mononuclear cells (PBMCs), mouse embryonic brain and patient-derived xenografts (PDXs). We found that all multiplexing reagents worked well in cell types robust to ex vivo manipulation but suffered from signal-to-noise issues in more delicate sample types. We compared multiple demultiplexing algorithms which differed in performance depending on data quality. We find that minor improvements to laboratory workflows such as titration and rapid processing are critical to optimal performance. We also compared the performance of fixed scRNA-Seq kits and highlight the advantages of the Parse Biosciences kit for fragile samples. Highly multiplexed scRNA-Seq experiments require more sequencing resources, therefore we evaluated CRISPR-based destruction of non-informative genes to enhance sequencing value. Our comprehensive analysis provides insights into the selection of appropriate sample multiplexing reagents and protocols for scRNA-Seq experiments, facilitating more accurate and cost-effective studies.

The antidiabetic drug metformin acts on the bone microenvironment to promote myeloma cell adhesion to preosteoblasts and increase myeloma tumour burden in vivo.

Multiple myeloma is a haematological malignancy that is dependent upon interactions within the bone microenvironment to drive tumour growth and osteolytic bone disease. Metformin is an anti-diabetic drug that has attracted attention due to its direct antitumor effects, including anti-myeloma properties. However, the impact of the bone microenvironment on the response to metformin in myeloma is unknown. We have employed in vitro and in vivo models to dissect out the direct effects of metformin in bone and the subsequent indirect myeloma response. We demonstrate how metformin treatment of preosteoblasts increases myeloma cell attachment. Metformin-treated preosteoblasts increased osteopontin (OPN) expression that upon silencing, reduced subsequent myeloma cell adherence. Proliferation markers were reduced in myeloma cells cocultured with metformin-treated preosteoblasts. In vivo, mice were treated with metformin for 4 weeks prior to inoculation of 5TGM1 myeloma cells. Metformin-pretreated mice had an increase in tumour burden, associated with an increase in osteolytic bone lesions and elevated OPN expression in the bone marrow. Collectively, we show that metformin increases OPN expression in preosteoblasts, increasing myeloma cell adherence. In vivo, this translates to an unexpected indirect pro-tumourigenic effect of metformin, highlighting the importance of the interdependence between myeloma cells and cells of the bone microenvironment.

Animal Models of Multiple Myeloma.

Multiple myeloma (MM) is a plasma cell neoplasm which is defined by strong interactions with the bone marrow microenvironment, a compartment with high cellular heterogeneity and unique structural and extracellular components. This necessitates the use of in vivo models for research to fully recapitulate MM growth conditions. The selection of appropriate model system is crucial, as each has advantages and shortcomings. Here, we describe the murine models available for studying MM, and focus on the methods for inoculating mice with MM cells via intravenous, intratibial or subcutaneous delivery, as well as monitoring of disease and organ processing for further analysis. The interaction and destruction of bone is a hallmark symptom of MM, and therefore many other complementary techniques used in calcified tissue research can be used, such as microCT, histomorphometry, and biomechanical testing.

Small Animal Video Tracking for Activity and Path Analysis Using a Novel Open-Source Multi-Platform Application (AnimApp).

Experimental biological model system outcomes such as altered animal movement capability or behaviour are difficult to quantify manually. Existing automatic movement tracking devices can be expensive and imposing upon the typical environment of the animal model. We have developed a novel multiplatform, free-to-use open-source application based on OpenCV, called AnimApp. Our results show that AnimApp can reliably and reproducibly track movement of small animals such as rodents or insects, and quantify parameters of action including distance and speed in order to detect activity changes arising from handling, environment enrichment, or temperature alteration. This system offers an accurate and reproducible experimental approach with potential for simple, fast and flexible analysis of movement and behaviour in a wide range of model systems.

Multiple myeloma increases nerve growth factor and other pain-related markers through interactions with the bone microenvironment.

Interactions between multiple myeloma (MM) and bone marrow (BM) are well documented to support tumour growth, yet the cellular mechanisms underlying pain in MM are poorly understood. We have used in vivo murine models of MM to show significant induction of nerve growth factor (NGF) by the tumour-bearing bone microenvironment, alongside other known pain-related characteristics such as spinal glial cell activation and reduced locomotion. NGF was not expressed by MM cells, yet bone stromal cells such as osteoblasts expressed and upregulated NGF when cultured with MM cells, or MM-related factors such as TNF-α. Adiponectin is a known MM-suppressive BM-derived factor, and we show that TNF-α-mediated NGF induction is suppressed by adiponectin-directed therapeutics such as AdipoRON and L-4F, as well as NF-κB signalling inhibitor BMS-345541. Our study reveals a further mechanism by which cellular interactions within the tumour-bone microenvironment contribute to disease, by promoting pain-related properties, and suggests a novel direction for analgesic development.

Transcriptomic profiling of the myeloma bone-lining niche reveals BMP signalling inhibition to improve bone disease.

Multiple myeloma is an incurable, bone marrow-dwelling malignancy that disrupts bone homeostasis causing skeletal damage and pain. Mechanisms underlying myeloma-induced bone destruction are poorly understood and current therapies do not restore lost bone mass. Using transcriptomic profiling of isolated bone lining cell subtypes from a murine myeloma model, we find that bone morphogenetic protein (BMP) signalling is upregulated in stromal progenitor cells. BMP signalling has not previously been reported to be dysregulated in myeloma bone disease. Inhibition of BMP signalling in vivo using either a small molecule BMP receptor antagonist or a solubilized BMPR1a-FC receptor ligand trap prevents trabecular and cortical bone volume loss caused by myeloma, without increasing tumour burden. BMP inhibition directly reduces osteoclastogenesis, increases osteoblasts and bone formation, and suppresses bone marrow sclerostin levels. In summary we describe a novel role for the BMP pathway in myeloma-induced bone disease that can be therapeutically targeted.

Contributions of the host microenvironment to cancer-induced bone disease.

The bone marrow provides a specialized and highly supportive microenvironment for tumor growth and development of the associated bone disease. It is a preferred site for breast and prostate cancer bone metastasis and the hematologic malignancy, multiple myeloma. For many years, researchers have focused upon the interactions between tumor cells and the cells directly responsible for bone remodeling, namely osteoclasts and osteoblasts. However, there is ever-increasing evidence for a multitude of ways in which the bone marrow microenvironment can promote disease pathogenesis, including via cancer-associated fibroblasts, the hematopoietic stem cell niche, myeloid-derived suppressor cells, and the sympathetic nervous system. This review discusses the recent advances in our understanding of the contribution of the host microenvironment to the development of cancer-induced bone disease.

Hypoxic induction of the regulator of G-protein signalling 4 gene is mediated by the hypoxia-inducible factor pathway.

The transcriptional response to hypoxia is largely dependent on the Hypoxia Inducible Factors (HIF-1 and HIF-2) in mammalian cells. Many target genes have been characterised for these heterodimeric transcription factors, yet there is evidence that the full range of HIF-regulated genes has not yet been described. We constructed a TetON overexpression system in the rat pheochromocytoma PC-12 cell line to search for novel HIF and hypoxia responsive genes. The Rgs4 gene encodes the Regulator of G-Protein Signalling 4 (RGS4) protein, an inhibitor of signalling from G-protein coupled receptors, and dysregulation of Rgs4 is linked to disease states such as schizophrenia and cardiomyopathy. Rgs4 was found to be responsive to HIF-2α overexpression, hypoxic treatment, and hypoxia mimetic drugs in PC-12 cells. Similar responses were observed in human neuroblastoma cell lines SK-N-SH and SK-N-BE(2)C, but not in endothelial cells, where Rgs4 transcript is readily detected but does not respond to hypoxia. Furthermore, this regulation was found to be dependent on transcription, and occurs in a manner consistent with direct HIF transactivation of Rgs4 transcription. However, no HIF binding site was detectable within 32 kb of the human Rgs4 gene locus, leading to the possibility of regulation by long-distance genomic interactions. Further research into Rgs4 regulation by hypoxia and HIF may result in better understanding of disease states such as schizophrenia, and also shed light on the other roles of HIF yet to be discovered.