Identification of lysyl oxidase as an adipocyte-secreted mediator that promotes a partial mesenchymal-to-epithelial transition in MDA-MB-231 cells.

Aim: Breast cancer (BC) is the most common cancer in women worldwide, where adiposity has been linked to BC morbidity. In general, obese premenopausal women diagnosed with triple-negative BC (TNBC) tend to have larger tumours with more metastases, particularly to the bone marrow, and worse prognosis. Previous work using a 3-dimensional (3D) co-culture system consisting of TNBC cells, adipocytes and the laminin-rich extracellular matrix (ECM) trademarked as Matrigel, demonstrated that adipocytes and adipocyte-derived conditioned media (CM) caused a partial mesenchymal-to-epithelial transition (MET). Given that MET has been associated with secondary tumour formation, this study sought to identify molecular mediators responsible for this phenotypic change. Methods: Adipocytes were cultured with and without Matrigel, where semi-quantitative proteomics was used to identify proteins whose presence in the CM was induced or enhanced by Matrigel, which were referred to as adipocyte-secreted ECM-induced proteins (AEPs). The AEPs identified were assessed for association with prognosis in published proteomic datasets and prior literature. Of these, 4 were evaluated by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), followed by a functional and MET marker analysis of 1 AEP on MDA-MB-231 cells grown on Matrigel or as monolayers. Results: The 4 AEPs showed a positive correlation between protein expression and poor prognosis. RT-qPCR analysis reported no significant change in AEPs mRNA expression. However, lysyl oxidase (LOX) was increased in CM of ECM-exposed adipocytes. Recombinant LOX (rLOX) caused the mesenchymal MDA-MB-231 TNBC cells to form less branched 3D structures and reduced the expression of vimentin. Conclusions: The data suggest that adipocyte-secreted LOX changes the mesenchymal phenotype of BC cells in a manner that could promote secondary tumour formation, particularly at sites high in adipocytes such as the bone marrow. Future efforts should focus on determining whether targeting LOX could reduce BC metastasis in obese individuals.

Extracellular vesicle small RNA cargo discriminates non-cancer donors from pediatric B-lymphoblastic leukemia patients.

Pediatric B-acute lymphoblastic leukemia (B-ALL) is a disease of abnormally growing B lymphoblasts. Here we hypothesized that extracellular vesicles (EVs), which are nanosized particles released by all cells (including cancer cells), could be used to monitor B-ALL severity and progression by sampling plasma instead of bone marrow. EVs are especially attractive as they are present throughout the circulation regardless of the location of the originating cell. First, we used nanoparticle tracking analysis to compare EVs between non-cancer donor (NCD) and B-ALL blood plasma; we found that B-ALL plasma contains more EVs than NCD plasma. We then isolated EVs from NCD and pediatric B-ALL peripheral blood plasma using a synthetic peptide-based isolation technique (Vn96), which is clinically amenable and isolates a broad spectrum of EVs. RNA-seq analysis of small RNAs contained within the isolated EVs revealed a signature of differentially packaged and exclusively packaged RNAs that distinguish NCD from B-ALL. The plasma EVs contain a heterogenous mixture of miRNAs and fragments of long non-coding RNA (lncRNA) and messenger RNA (mRNA). Transcripts packaged in B-ALL EVs include those involved in negative cell cycle regulation, potentially suggesting that B-ALL cells may use EVs to discard gene sequences that control growth. In contrast, NCD EVs carry sequences representative of multiple organs, including brain, muscle, and epithelial cells. This signature could potentially be used to monitor B-ALL disease burden in pediatric B-ALL patients via blood draws instead of invasive bone marrow aspirates.

The breast cancer microenvironment and lipoprotein lipase: Another negative notch for a beneficial enzyme?

The energy demand of breast cancers is in part met through the ß-oxidation of exogenous fatty acids. Fatty acids may also be used to aid in cell signaling and toward the construction of new membranes for rapidly proliferating tumor cells. A significant quantity of fatty acids comes from the hydrolysis of lipoprotein triacylglycerols and phospholipids by lipoprotein lipase (LPL). The lipid obtained via LPL in the breast tumor microenvironment may thus promote breast tumor growth and development. In this hypothesis article, we introduce LPL, provide a meta-analysis of RNAseq data showing that LPL is associated with poor prognosis, and explain how LPL might play a role in breast cancer prognosis over time.

CD24 as a Potential Therapeutic Target in Patients with B-Cell Leukemia and Lymphoma: Current Insights.

CD24 is a highly glycosylated glycophosphatidylinositol (GPI)-anchored protein that is expressed in many types of differentiating cells and some mature cells of the immune system as well as the central nervous system. CD24 has been extensively used as a biomarker for developing B cells as its expression levels change over the course of B cell development. Functionally, engagement of CD24 induces apoptosis in developing B cells and restricts cell growth in more mature cell types. Interestingly, CD24 is also expressed on many hematological and solid tumors. As such, it has been investigated as a therapeutic target in many solid tumors including ovarian, colorectal, pancreatic, lung and others. Most of the B-cell leukemias and lymphomas studied to date express CD24 but its role as a therapeutic target in these malignancies has, thus far, been understudied. Here, I review what is known about CD24 biology with a focus on B cell development and activation followed by a brief overview of how CD24 is being targeted in solid tumors. This is followed by an assessment of the value of CD24 as a therapeutic target in B cell leukemia and lymphoma in humans, including an evaluation of the challenges in using CD24 as a target considering its pattern of expression on normal cells.

Overview of the Maintenance of Authentic Cancer Cell Lines.

The proper maintenance of cancer cell lines is critical to maintain the integrity of any experiment. Proper maintenance includes handling of cell lines and maintaining records on usage and confirmation of identity. Cross-contamination of cancer cell lines has been well documented by the ATCC and must be avoided at all costs. In addition, contamination by Mycoplasma sp. is a well-known issue, which, if left unchecked, can affect all data collected as this contamination can affect cell line metabolism and has uncharacterized effects. Considerations and specific recommendations for the proper maintenance of cancer cell lines are presented here.

Culturing Suspension Cancer Cell Lines.

Suspension cell lines grow free-floating in the cell culture media without any attachment to the culture plate/vessel. Suspension cells typically mimic cells that exist in the circulation of multicellular animals such as mouse and humans. Generally, cell lines derived from the blood such as lymphocytes, megakaryocyte, and neutrophils grow in suspension. These cell lines can be used for experimental studies to understand the biology/biochemistry of cancer cells. In this chapter, procedures for working with suspension cell lines are provided, including protocols for thawing, culturing, and cryopreserving cancer cell lines. Importantly, this chapter demonstrates the best practices required to work with suspension cell lines, to minimize the risk of contaminations from adventitious microorganisms or from other cell lines.

Three-Dimensional Co-Culture Method for Studying Interactions Between Adipocytes, Extracellular Matrix, and Cancer Cells.

Three-dimensional (D) culture models are increasingly becoming the model of choice for studying different biological phenomena such as cell-cell interaction, drug resistance, and gene expression. These models include extracellular matrix (ECM) proteins that better model the in vivo conditions as it allows cells to have both cell-cell and cell-ECM contacts. In the context of the tumor microenvironment, there are additional types of cells present in addition to the ECM. Thus, an intermediate between 2D cell culture and in vivo mouse models can be desired to interrogate the interactions between multiple cell types under the influence of the ECM. Here we describe a 3D co-culture technique for studying breast cancer-adipocyte interactions. This technique could easily be modified to analyze interactions between other cancer cell types and different fibroblast-like cells.

Characterizing Extracellular Vesicles Using Nanoparticle-Tracking Analysis.

Extracellular vesicles (EVs) are nanosized lipid bound particles secreted by cells. EVs transfer biologic material and can be found in all body fluids. Accurately characterizing the size and concentration of EVs is difficult because of the nanoscale size of EVs. An evolving solution to this problem is tracking the Brownian motion of EVs in suspension, a technique known as nanoparticle-tracking analysis (NTA). This technique is used by many researchers in the EV field. The ability to accurately replicate data between studies and laboratories is critical to advancing the knowledge surrounding EVs for use in liquid biopsy and cancer studies, in general. Thus, this chapter provides a step-by-step guide on isolating EVs using a variety of methods, for characterizing EVs using the NS300 NTA instrument, troubleshooting tips, and a robust guide to reporting key parameters that will help improve cross-lab reproducibility of NTA data.

Meta-analysis of microRNA profiling data does not reveal a consensus signature for B cell acute lymphoblastic leukemia.

B cell acute lymphoblastic leukemia (B-ALL) is the most prevalent pediatric cancer. MicroRNAs (miRNAs) are 18-22nt non-coding transcripts shown to be essential for the development of many cancers. While some miRNAs are reportedly expressed differentially between healthy and B-ALL, no studies have reported a consensus miRNA signature. Therefore, we performed a reanalysis of five miRNA datasets to identify differentially expressed miRNAs (DEmiRs) and a meta-analysis of previously identified DEmiRs from 25 studies. Overall, the re-analysis showed that the DEmiR data clustered by platform and not by disease state. The meta-analysis also did not reveal a consensus miRNA signature as there were many miRNAs upregulated in some studies and downregulated in others. However, eight promising miRNAs (miR-181b, miR-128b, miR-181a, miR-128, miR-128a, miR-181c, miR-155, miR-142-3p, and miR-451) were identified from the meta-analysis, which could be the basis of future investigations. These analyses reveal that standardization of miRNA isolation and analysis is needed in B-ALL to enable cross-study comparisons and identify a consensus signature.

CD24 and IgM Stimulation of B Cells Triggers Transfer of Functional B Cell Receptor to B Cell Recipients Via Extracellular Vesicles.

Extracellular vesicles (EVs) are membrane-encapsulated nanoparticles that carry bioactive cargo, including proteins, lipids, and nucleic acids. Once taken up by target cells, EVs can modify the physiology of the recipient cells. In past studies, we reported that engagement of the glycophosphatidylinositol-anchored receptor CD24 on B lymphocytes (B cells) causes the release of EVs. However, a potential function for these EVs was not clear. Thus, we investigated whether EVs derived from CD24 or IgM-stimulated donor WEHI-231 murine B cells can transfer functional cargo to recipient cells. We employed a model system where donor cells expressing palmitoylated GFP (WEHI-231-GFP) were cocultured, after stimulation, with recipient cells lacking either IgM (WEHI-303 murine B cells) or CD24 (CD24 knockout mouse bone marrow B cells). Uptake of lipid-associated GFP, IgM, or CD24 by labeled recipient cells was analyzed by flow cytometry. We found that stimulation of either CD24 or IgM on the donor cells caused the transfer of lipids, CD24, and IgM to recipient cells. Importantly, we found that the transferred receptors are functional in recipient cells, thus endowing recipient cells with a second BCR or sensitivity to anti-CD24-induced apoptosis. In the case of the BCR, we found that EVs were conclusively involved in this transfer, whereas in the case in the CD24 the involvement of EVs is suggested. Overall, these data show that extracellular signals received by one cell can change the sensitivity of neighboring cells to the same or different stimuli, which may impact B cell development or activation.

Transcriptome Profiling of Atlantic Salmon Adherent Head Kidney Leukocytes Reveals That Macrophages Are Selectively Enriched During Culture.

The Atlantic salmon (Salmo salar) is an economically important fish, both in aquaculture and in the wild. In vertebrates, macrophages are some of the first cell types to respond to pathogen infection and disease. While macrophage biology has been characterized in mammals, less is known in fish. Our previous work identified changes in the morphology, phagocytic ability, and miRNA profile of Atlantic salmon adherent head kidney leukocytes (HKLs) from predominantly "monocyte-like" at Day 1 of in vitro culture to predominantly "macrophage-like" at Day 5 of culture. Therefore, to further characterize these two cell populations, we examined the mRNA transcriptome profile in Day 1 and Day 5 HKLs using a 44K oligonucleotide microarray. Large changes in the transcriptome were revealed, including changes in the expression of macrophage and immune-related transcripts (e.g. csf1r, arg1, tnfa, mx2), lipid-related transcripts (e.g. fasn, dhcr7, fabp6), and transcription factors involved in macrophage differentiation and function (e.g. klf2, klf9, irf7, irf8, stat1). The in silico target prediction analysis of differentially expressed genes (DEGs) using miRNAs known to change expression in Day 5 HKLs, followed by gene pathway enrichment analysis, supported that these miRNAs may be involved in macrophage maturation by targeting specific DEGs. Elucidating how immune cells, such as macrophages, develop and function is a key step in understanding the Atlantic salmon immune system. Overall, the results indicate that, without the addition of exogenous factors, the adherent HKL cell population differentiates in vitro to become macrophage-like.

Lipoprotein lipase hydrolysis products induce pro-inflammatory cytokine expression in triple-negative breast cancer cells.

OBJECTIVES: Breast cancer cell growth and proliferation requires lipids for energy production, cell membrane synthesis, or as signaling molecules. Lipids can be delivered to cells by lipoprotein lipase (LPL), an extracellular lipase that hydrolyzes triacylglycerols and phospholipids from lipoproteins, that is expressed by adipose tissue and some breast cancer cell lines. Studies have shown that lipoprotein hydrolysis products induce pro-inflammatory cytokine secretion by endothelial cells. Thus, our objective was to determine if hydrolysis products generated by LPL from total lipoproteins can also promote pro-inflammatory cytokine secretion from breast cancer cells. RESULTS: Using cytokine arrays, we found that MDA-MB-231 cells increased secretion of seven cytokines in response to treatment with lipoprotein hydrolysis products. In contrast, MCF-7 cells showed decreased secretion of two cytokines. Expanding the analysis to additional cell lines by ELISA, we found increased secretion of TNF-α and IL-6 by MDA-MB-468 cells, and increased secretion of IL-4 by MDA-MB-468 and SKBR3 cells. The changes to cytokine secretion profiles of the breast cancer cell types examined, including the non-cancerous MCF-10a breast cells, were independent of increased cell metabolic activity. These results provide information on how lipoprotein hydrolysis products within the tumor microenvironment might affect breast cancer cell viability and progression.

Deciphering the messages carried by extracellular vesicles in hematological malignancies.

Extracellular vesicles (EVs) are nanosized membrane-bound particles released from all living cells examined thus far. EVs can transfer information in the form of proteins, nucleic acids, and lipids from donor cells to recipient cells. Here we summarize recent advances in understanding the role(s) EVs play in hematological malignancies (HM) and outline potential prognostic and diagnostic strategies using EVs. EVs have been shown to promote proliferation and angiogenesis, and alter the bone marrow microenvironment to favour the growth and survival of diverse HM. They also promote evasion of anti-cancer immune responses and increase multi-drug resistance. Using knowledge of EV biology, including HM-specific packaging of cargo, EV based diagnostics and therapeutic approaches show substantial clinical promise. However, while EVs may represent a new paradigm to solve many of the challenges in treating and/or diagnosing HM, much work is needed before they can be used clinically to improve patient outcomes.

Characterization of miRNAs in Extracellular Vesicles Released From Atlantic Salmon Monocyte-Like and Macrophage-Like Cells.

Cell-derived extracellular vesicles (EVs) participate in cell-cell communication via transfer of molecular cargo including genetic material like miRNAs. In mammals, it has previously been established that EV-mediated transfer of miRNAs can alter the development or function of immune cells, such as macrophages. Our previous research revealed that Atlantic salmon head kidney leukocytes (HKLs) change their morphology, phagocytic ability and miRNA profile from primarily "monocyte-like" at Day 1 to primarily "macrophage-like" at Day 5 of culture. Therefore, we aimed to characterize the miRNA cargo packaged in EVs released from these two cell populations. We successfully isolated EVs from Atlantic salmon HKL culture supernatants using the established Vn96 peptide-based pull-down. Isolation was validated using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. RNA-sequencing identified 19 differentially enriched (DE) miRNAs packaged in Day 1 versus Day 5 EVs. Several of the highly abundant miRNAs, including those that were DE (e.g. ssa-miR-146a, ssa-miR-155 and ssa-miR-731), were previously identified as DE in HKLs and are associated with macrophage differentiation and immune response in other species. Interestingly, the abundance relative of the miRNAs in EVs, including the most abundant miRNA (ssa-miR-125b), was different than the miRNA abundance in HKLs, indicating selective packaging of miRNAs in EVs. Further study of the miRNA cargo in EVs derived from fish immune cells will be an important next step in identifying EV biomarkers useful for evaluating immune cell function, fish health, or response to disease.

Adipose Tissue from Lean and Obese Mice Induces a Mesenchymal to Epithelial Transition-Like Effect in Triple Negative Breast Cancers Cells Grown in 3-Dimensional Culture.

Breast cancer is the second leading cause of cancer-related mortality among women globally with obesity being one risk factor. Obese breast cancer patients have at least a 30% increased risk of death from breast cancer compared to non-obese breast cancer patients because they present with larger tumors and generally have increased rates of metastasis. Moreover, obese breast cancer patients respond more poorly to treatment compared to non-obese patients, particularly pre-menopausal women diagnosed with triple negative breast cancer (TNBC). To help understand the molecular mechanisms underlying the increased metastasis associated with obesity, we previously established a three-dimensional culture system that permits the co-culture of adipocytes and TNBC cells in a manner that mimics an in vivo milieu. Using this system, we demonstrate that white adipose tissue from both lean and obese mice can induce a partial mesenchymal-to-epithelial transition (MET). Triple negative breast cancer cells adopt an epithelial morphology and have an increased expression of some epithelial markers, but they maintain the expression of mesenchymal markers, furnishing the breast cancer cells with hybrid properties that are associated with more aggressive tumors. Thus, these data suggest that adipose tissue has the potential to promote secondary tumor formation in lean and obese women. Further work is needed to determine if targeting the partial MET induced by adipose tissue could reduce metastasis.

Characterization of miRNAs in Cultured Atlantic Salmon Head Kidney Monocyte-Like and Macrophage-Like Cells.

Macrophages are among the first cells to respond to infection and disease. While microRNAs (miRNAs) are involved in the process of monocyte-to-macrophage differentiation in mammals, less is known in teleost fish. Here, Atlantic salmon head kidney leukocytes (HKLs) were used to study the expression of miRNAs in response to in vitro culture. The morphological analysis of cultures showed predominantly monocyte-like cells on Day 1 and macrophage-like cells on Day 5, suggesting that the HKLs had differentiated from monocytes to macrophages. Day 5 HKLs also contained a higher percentage of phagocytic cells. Small RNA sequencing and qPCR analysis were applied to examine the miRNA diversity and expression. There were 370 known mature Atlantic salmon miRNAs in HKLs. Twenty-two miRNAs (15 families) were downregulated while 44 miRNAs (25 families) were upregulated on Day 5 vs. Day 1. Mammalian orthologs of many of the differentially expressed (DE) miRNAs are known to regulate macrophage activation and differentiation, while the teleost-specific miR-2188, miR-462 and miR-731 were also DE and are associated with immune responses in fish. In silico predictions identified several putative target genes of qPCR-validated miRNAs associated with vertebrate macrophage differentiation. This study identified Atlantic salmon miRNAs likely to influence macrophage differentiation, providing important knowledge for future functional studies.

Adipocytes in the Tumour Microenvironment.

Adipose tissue contribution to body mass ranges from 6% in male athletes to over 25% in obese men and over 30% in obese women. Crosstalk between adipocytes and cancer cells that exist in close proximity can lead to changes in the function and phenotype of both cell types. These interactions actively alter the tumour microenvironment (TME). Obesity is one of the major risk factors for multiple types of cancer, including breast cancer. In obesity, the increase in both size and number of adipocytes leads to instability of the TME, as well as increased hypoxia within the TME, which further enhances tumour invasion and metastasis. In this chapter, we will discuss the diverse aspects of adipocytes and adipocyte-derived factors that affect the TME as well as tumour progression and metastasis. In addition, we discuss how obesity affects the TME. We focus primarily on breast cancer but discuss what is known in other cancer types when relevant. We finish by discussing the studies needed to further understand these complex interactions.

A Comparison of the Innate and Adaptive Immune Systems in Cartilaginous Fish, Ray-Finned Fish, and Lobe-Finned Fish.

The immune system is composed of two subsystems-the innate immune system and the adaptive immune system. The innate immune system is the first to respond to pathogens and does not retain memory of previous responses. Innate immune responses are evolutionarily older than adaptive responses and elements of innate immunity can be found in all multicellular organisms. If a pathogen persists, the adaptive immune system will engage the pathogen with specificity and memory. Several components of the adaptive system including immunoglobulins (Igs), T cell receptors (TCR), and major histocompatibility complex (MHC), are assumed to have arisen in the first jawed vertebrates-the Gnathostomata. This review will discuss and compare components of both the innate and adaptive immune systems in Gnathostomes, particularly in Chondrichthyes (cartilaginous fish) and in Osteichthyes [bony fish: the Actinopterygii (ray-finned fish) and the Sarcopterygii (lobe-finned fish)]. While many elements of both the innate and adaptive immune systems are conserved within these species and with higher level vertebrates, some elements have marked differences. Components of the innate immune system covered here include physical barriers, such as the skin and gastrointestinal tract, cellular components, such as pattern recognition receptors and immune cells including macrophages and neutrophils, and humoral components, such as the complement system. Components of the adaptive system covered include the fundamental cells and molecules of adaptive immunity: B lymphocytes (B cells), T lymphocytes (T cells), immunoglobulins (Igs), and major histocompatibility complex (MHC). Comparative studies in fish such as those discussed here are essential for developing a comprehensive understanding of the evolution of the immune system.