Precision nephrology identified tumor necrosis factor activation variability in minimal change disease and focal segmental glomerulosclerosis.

The diagnosis of nephrotic syndrome relies on clinical presentation and descriptive patterns of injury on kidney biopsies, but not specific to underlying pathobiology. Consequently, there are variable rates of progression and response to therapy within diagnoses. Here, an unbiased transcriptomic-driven approach was used to identify molecular pathways which are shared by subgroups of patients with either minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS). Kidney tissue transcriptomic profile-based clustering identified three patient subgroups with shared molecular signatures across independent, North American, European, and African cohorts. One subgroup had significantly greater disease progression (Hazard Ratio 5.2) which persisted after adjusting for diagnosis and clinical measures (Hazard Ratio 3.8). Inclusion in this subgroup was retained even when clustering was limited to those with less than 25% interstitial fibrosis. The molecular profile of this subgroup was largely consistent with tumor necrosis factor (TNF) pathway activation. Two TNF pathway urine markers were identified, tissue inhibitor of metalloproteinases-1 (TIMP-1) and monocyte chemoattractant protein-1 (MCP-1), that could be used to predict an individual's TNF pathway activation score. Kidney organoids and single-nucleus RNA-sequencing of participant kidney biopsies, validated TNF-dependent increases in pathway activation score, transcript and protein levels of TIMP-1 and MCP-1, in resident kidney cells. Thus, molecular profiling identified a subgroup of patients with either MCD or FSGS who shared kidney TNF pathway activation and poor outcomes. A clinical trial testing targeted therapies in patients selected using urinary markers of TNF pathway activation is ongoing.

ATL-derived exosomes modulate mesenchymal stem cells: potential role in leukemia progression.

BACKGROUND: Exosomes are membrane nano-vesicles secreted by a multitude of cells that harbor biological constituents such as proteins, lipids, mRNA and microRNA. Exosomes can potentially transfer their cargo to other cells, implicating them in many patho-physiological processes. Mesenchymal stem cells (MSCs), residents of the bone marrow and metastatic niches, potentially interact with cancer cells and/or their derived exosomes. In this study, we investigated whether exosomes derived from adult T-cell leukemia/lymphoma (ATL) cells act as intercellular messengers delivering leukemia-related genes that modulate the properties of human MSCs in favor of leukemia. We hypothesized that the cargo of ATL-derived exosomes is transferred to MSCs and alter their functional behavior to support the establishment of the appropriate microenvironment for leukemia. RESULTS: We showed that both ATL cells (C81 and HuT-102) and patient-derived cells released Tax-containing exosomes. The cargo of HuT-102-derived exosomes consisted of miR-21, miR-155 and vascular endothelial growth factor. We demonstrated that HuT-102-derived exosomes not only deliver Tax to recipient MSCs, but also induce NF-κB activation leading to a change in cellular morphology, increase in proliferation and the induction of gene expression of migration and angiogenic markers. CONCLUSIONS: This study demonstrates that ATL-derived exosomes deliver Tax and other leukemia-related genes to MSCs and alter their properties to presumably create a more conducive milieu for leukemia. These findings highlight the contribution of leukemia-derived exosomes in cellular transformation and their potential value as biomarkers and targets in therapeutic strategies.

Interferon-γ induces combined pyroptotic angiopathy and APOL1 expression in human kidney disease.

Elevated interferon (IFN) signaling is associated with kidney diseases including COVID-19, HIV, and apolipoprotein-L1 (APOL1) nephropathy, but whether IFNs directly contribute to nephrotoxicity remains unclear. Using human kidney organoids, primary endothelial cells, and patient samples, we demonstrate that IFN-γ induces pyroptotic angiopathy in combination with APOL1 expression. Single-cell RNA sequencing, immunoblotting, and quantitative fluorescence-based assays reveal that IFN-γ-mediated expression of APOL1 is accompanied by pyroptotic endothelial network degradation in organoids. Pharmacological blockade of IFN-γ signaling inhibits APOL1 expression, prevents upregulation of pyroptosis-associated genes, and rescues vascular networks. Multiomic analyses in patients with COVID-19, proteinuric kidney disease, and collapsing glomerulopathy similarly demonstrate increased IFN signaling and pyroptosis-associated gene expression correlating with accelerated renal disease progression. Our results reveal that IFN-γ signaling simultaneously induces endothelial injury and primes renal cells for pyroptosis, suggesting a combinatorial mechanism for APOL1-mediated collapsing glomerulopathy, which can be targeted therapeutically.

An integrated organoid omics map extends modeling potential of kidney disease.

Kidney organoids are a promising model to study kidney disease, but their use is constrained by limited knowledge of their functional protein expression profile. Here, we define the organoid proteome and transcriptome trajectories over culture duration and upon exposure to TNFα, a cytokine stressor. Older organoids increase deposition of extracellular matrix but decrease expression of glomerular proteins. Single cell transcriptome integration reveals that most proteome changes localize to podocytes, tubular and stromal cells. TNFα treatment of organoids results in 322 differentially expressed proteins, including cytokines and complement components. Transcript expression of these 322 proteins is significantly higher in individuals with poorer clinical outcomes in proteinuric kidney disease. Key TNFα-associated protein (C3 and VCAM1) expression is increased in both human tubular and organoid kidney cell populations, highlighting the potential for organoids to advance biomarker development. By integrating kidney organoid omic layers, incorporating a disease-relevant cytokine stressor and comparing with human data, we provide crucial evidence for the functional relevance of the kidney organoid model to human kidney disease.

Data on migration of the non-invasive breast cancer cell line, MCF-7 treated with Bevacizumab using Real Time Cell Analyzer (RTCA).

Bevacizumab or Avastin® (Av), the recombinant antibody targeting VEGF, improves progression-free but not overall survival of metastatic breast cancer patients due to development of Av resistance. We showed that Av-therapy-induced inflammatory microenvironment contributes to the refractoriness to Av treatment. Here we present data regarding the effect of Av treatment on migration of a non-invasive breast cancer cell line, MCF-7. The data presented hereis related to the research article "Bevacizumab induces inflammation in MDA-MB-231 breast cancer cell line and in a mouse model" (Hajjar et al., 2018).

Bevacizumab induces inflammation in MDA-MB-231 breast cancer cell line and in a mouse model.

BACKGROUND: Bevacizumab or Avastin® (Av) is an anti-vascular endothelial growth factor agent. It does not improve survival of breast cancer patients due to development of refractoriness. Av treatment was shown to increase inflammation in a diabetic mouse model, and also to induce epithelial-to-mesenchymal transition of non-transformed breast epithelia. This study aimed to understand if the Av-induced inflammatory microenvironment could be a mechanism of Av refractoriness. Expression profiles of inflammatory mediators, in vitro in MDA-MB-231 cells, in vivo in a mouse model xenografted with MDA-MB-231 cells and from archived cases of human breast carcinoma tissues were evaluated. Gap junctions are also crucial for angiogenesis and tumor cell extravasation. The effect of connexin 43 (Cx43) overexpression on the expression of inflammatory markers in MDA-MB-231 cells treated with Av was assessed. METHODS: MDA-MB-231 cells, control or overexpressing Cx43, were used in this study. Proliferation and invasion assays were performed. Quantitative PCR, ELISA and western blotting were performed to assess the regulation of inflammatory mediators and other factors upon Av treatment. Immunofluorescence was performed to document the translocation of Nuclear Factor-kappa B p65. RESULTS: Breast cancer tissues had elevated transcriptional levels of inflammatory mediators. Av treatment increased expression levels of inflammatory mediators and metastatic factors in vitro and in vivo. Interestingly, overexpressing Cx43 in MDA-MB-231 cells alleviated the inflammatory effects induced by Av treatment. CONCLUSION: This study attributes Av refractoriness to the Av therapy-induced inflammatory microenvironment.

Cx43 Expression Correlates with Breast Cancer Metastasis in MDA-MB-231 Cells In Vitro, In a Mouse Xenograft Model and in Human Breast Cancer Tissues.

Connexins regulate multiple cellular functions and are considered tumor suppressors. Connexin43 (Cx43) is frequently down-regulated in breast tumors. However, Cx43 regulation during cancer onset and metastasis is complex and context-dependent. We investigated the effect of Cx43 over-expression or knock-down on the metastatic potential of MDA-MB-231 breast cancer cells in vitro and in vivo and in human breast cancer tissues. MDA-MB-231 cells over-expressing (Cx43D) or down-regulating Cx43 (shCx43) were generated and used in proliferation, migration, and invasion assays. The regulation of genes/proteins implicated in progression, invasion and metastasis was assessed in vitro and in immune-compromized mice injected with MDA-MB-231, Cx43D or shCx43 cells. Primary tumor onset/growth, metastasis and overall survival of these animals was monitored and evaluated. In addition, Cx43 expression in human breast carcinoma samples was assessed by qPCR. Cx43 over-expression increased protein levels of epithelial markers E-cadherin and zonula occludens 1 expression and resulted in the sequestration of ß-catenin at the cell membrane, while Cx43 knock-down induced protein expression of the mesenchymal marker N-cadherin and an increased invasive potential of shCx43 cells. In vivo, in mice xenografted with breast cancer cells, Cx43 over-expression decreased tumor volume, attenuated cell metastasis to lungs and liver and increased overall mice survival. Importantly, the expression of Cx43 in triple negative human breast cancer tissues is also down-regulated. Collectively, Cx43 over-expression induced an epithelial-like phenotype in MDA-MB-231 cells and suppressed tumor growth and metastasis to secondary organs in vivo. In contrast, Cx43 knock-down in MDA-MB-231 cells induced a mesenchymal phenotype with increased cell invasion leading to an enhanced metastatic phenotype. These data provide evidence for a pivotal role of Cx43 in breast cancer metastasis and support the potential targeting of connexins in breast cancer therapy.

A ferutinin analogue with enhanced potency and selectivity against ER-positive breast cancer cells in vitro.

Estrogen is considered a risk factor for breast cancer since it promotes breast-cell proliferation. The jaesckeanadiol-3-p-hydroxyphenylpropanoate, a hemi-synthetic analogue of the natural phytoestrogen ferutinin (jaesckeanadiol-p-hydroxybenzoate), is designed to be devoid of estrogenic activity. This analogue induces a cytotoxic effect 30 times higher than that of ferutinin towards MCF-7 breast cancer cell line. We compared these two compounds with respect to their effect on proliferation, cell cycle distribution and cancer stem-like cells in the MCF-7 cell line. Treatment with ferutinin (30 µM) and its analogue (1 µM) produced significant accumulation of cells at the pre G0/G1 cell cycle phase and triggered apoptosis. Importantly, this compound retains its anti-proliferative activity against breast cancer stem/progenitor cells that are naturally insensitive to ferutinin at the same dose. These results position ferutinin analogue as an effective compound inhibiting the proliferation of estrogen-dependent breast cancer cells and consistently targeting their stem-like cells.

Anti-angiogenesis therapy and gap junction inhibition reduce MDA-MB-231 breast cancer cell invasion and metastasis in vitro and in vivo.

Cancer cells secrete VEGF, which plays a key role in their growth, invasion, extravasation and metastasis. Direct cancer cell-endothelial cell interaction, mediated by gap junctions, is of critical importance in the extravasation process. In this study, we evaluated avastin (Av), an anti-VEGF antibody; and oleamide (OL), a gap junction inhibitor, using MDA-MB-231 human breast cancer cells in vitro and a xenograft murine model in vivo. Results showed that Av/OL significantly decreased proliferation, induced cell cycle arrest and decreased migration and invasion of MDA-MB-231 cells in vitro. In addition, Av/OL significantly decreased homo and hetero-cellular communication interaction between MDA-MDA and MDA-endothelial cells, respectively. The expression levels of several factors including VEGF, HIF1α, CXCR4, Cx26, Cx43, and MMP9 were attenuated upon Av/OL treatment in vitro. On the other hand, avastin, but not oleamide, reduced tumor size of NSG mice injected subdermally (s.d.) with MDA-MB-231 cells, which was also associated with increased survival. Furthermore, Av but also OL, separately, significantly increased the survival rate, and reduced pulmonary and hepatic metastatic foci, of intravenously (i.v.) injected mice. Finally, OL reduced MMP9 protein expression levels, better than Av and in comparisons to control, in the lungs of MDA-MB-231 i.v. injected NSG mice. In conclusion, while avastin has anti-angiogenic, anti-tumor and anti-metastatic activities, oleamide has anti-metastatic activity, presumably at the extravasation level, providing further evidence for the role of gap junction intercellular communication (GJIC) in cancer cell extravasation.

Connexins: a junctional crossroad to breast cancer.

The mammary gland presents a valuable model for developmental studies, spanning the embryonic stage through menarche to menopause. The dynamic remodeling of this gland is orchestrated by cellular heterogeneity, integrating mammogenic, systemic and local cues. Gap junctional intercellular communication provides pivotal cross talk of mammary epithelial cells with the surrounding cells and their local microenvironment. Connexins are involved in regulating normal and pathological mammary gland development, through channel-dependent and channel-independent roles. Modulation of the isoforms of connexins expressed, as well as their differential assembly into connexons and recruitment of a variety of associated partners, contributes to the complexity of signaling relayed at the membrane. This confers context-dependent functions of connexins at different stages of development and carcinogenesis. This review will summarize available knowledge about the functional dynamics of connexins and gap junctions in regulating normal mammary gland development and its pathophysiology.