HIF-1α and MIF enhance neutrophil-driven type 3 immunity and chondrogenesis in a murine spondyloarthritis model.

The hallmarks of spondyloarthritis (SpA) are type 3 immunity-driven inflammation and new bone formation (NBF). Macrophage migration inhibitory factor (MIF) was found to be a key driver of the pathogenesis of SpA by amplifying type 3 immunity, yet MIF-interacting molecules and networks remain elusive. Herein, we identified hypoxia-inducible factor-1 alpha (HIF1A) as an interacting partner molecule of MIF that drives SpA pathologies, including inflammation and NBF. HIF1A expression was increased in the joint tissues and synovial fluid of SpA patients and curdlan-injected SKG (curdlan-SKG) mice compared to the respective controls. Under hypoxic conditions in which HIF1A was stabilized, human and mouse neutrophils exhibited substantially increased expression of MIF and IL-23, an upstream type 3 immunity-related cytokine. Similar to MIF, systemic overexpression of IL-23 induced SpA pathology in SKG mice, while the injection of a HIF1A-selective inhibitor (PX-478) into curdlan-SKG mice prevented or attenuated SpA pathology, as indicated by a marked reduction in the expression of MIF and IL-23. Furthermore, genetic deletion of MIF or HIF1A inhibition with PX-478 in IL-23-overexpressing SKG mice did not induce evident arthritis or NBF, despite the presence of psoriasis-like dermatitis and blepharitis. We also found that MIF- and IL-23-expressing neutrophils infiltrated areas of the NBF in curdlan-SKG mice. These neutrophils potentially increased chondrogenesis and cell proliferation via the upregulation of STAT3 in periosteal cells and ligamental cells during endochondral ossification. Together, these results provide supporting evidence for an MIF/HIF1A regulatory network, and inhibition of HIF1A may be a novel therapeutic approach for SpA by suppressing type 3 immunity-mediated inflammation and NBF.

Cell-free DNA methylation-defined prognostic subgroups in small-cell lung cancer identified by leukocyte methylation subtraction.

Small-cell lung cancer (SCLC) methylome is understudied. Here, we comprehensively profile SCLC using cell-free methylated DNA immunoprecipitation followed by sequencing (cfMeDIP-seq). Cell-free DNA (cfDNA) from plasma of 74 patients with SCLC pre-treatment and from 20 non-cancer participants, genomic DNA (gDNA) from peripheral blood leukocytes from the same 74 patients, and 7 accompanying circulating tumor cell-derived xenografts (CDXs) underwent cfMeDIP-seq. Peripheral blood leukocyte methylation (PRIME) subtraction to improve tumor specificity. SCLC cfDNA methylation is distinct from non-cancer but correlates with CDX tumor methylation. PRIME and k-means consensus identified two methylome clusters with prognostic associations that related to axon guidance, neuroactive ligand-receptor interaction, pluripotency of stem cells, and differentially methylated at long noncoding RNA and other repeats features. We comprehensively profiled the SCLC methylome in a large patient cohort and identified methylome clusters with prognostic associations. Our work demonstrates the potential of liquid biopsies in examining SCLC biology encoded in the methylome.

Targeting the Ubiquitin-Proteasome System Using the UBA1 Inhibitor TAK-243 is a Potential Therapeutic Strategy for Small-Cell Lung Cancer.

PURPOSE: Small cell lung cancer (SCLC) is an aggressive disease with an overall 5-year survival rate of less than 10%. Treatment for SCLC with cisplatin/etoposide chemotherapy (C/E) ± radiotherapy has changed modestly over several decades. The ubiquitin-proteasome system is an underexplored therapeutic target for SCLC. We preclinically evaluated TAK-243, a first-in-class small molecule E1 inhibitor against UBA1. EXPERIMENTAL DESIGN: We assessed TAK-243 in 26 SCLC cell-lines as monotherapy and combined with C/E, the PARP-inhibitor, olaparib, and with radiation using cell viability assays. We interrogated TAK-243 response with gene expression to identify candidate biomarkers. We evaluated TAK-243 alone and in combination with olaparib or radiotherapy with SCLC patient-derived xenografts (PDX). RESULTS: Most SCLC cell lines were sensitive to TAK-243 monotherapy (EC50 median 15.8 nmol/L; range 10.2 nmol/L-367.3 nmol/L). TAK-243 sensitivity was associated with gene-sets involving the cell cycle, DNA and chromatin organization, and DNA damage repair, while resistance associated with cellular respiration, translation, and neurodevelopment. These associations were also observed in SCLC PDXs. TAK-243 synergized with C/E and olaparib in vitro across sensitive and resistant SCLC cell lines. Considerable TAK-243-olaparib synergy was observed in an SCLC PDX resistant to both drugs individually. TAK-243 radiosensitization was also observed in an SCLC PDX. CONCLUSIONS: TAK-243 displays efficacy in SCLC preclinical models. Enrichment of gene sets is associated with TAK-243 sensitivity and resistance. TAK-243 exhibits synergy when combined with genotoxic therapies in cell lines and PDXs. TAK-243 is a potential therapeutic strategy to improve SCLC patient outcomes, both as a single agent and in combination with existing therapies.

The Utility of Liquid Biopsies in Radiation Oncology.

The use of therapeutic radiation is primarily guided by clinicopathologic factors and medical imaging, whereas molecular biomarkers currently play a comparatively minor role in most settings. Liquid biopsies provide a rich source of noninvasive tumor-specific biomarkers and are amenable to repeated and noninvasive assessment. Here, we review the current status of liquid biopsies and their potential impact on the field of radiation oncology. We focus on established and emerging approaches to analyze circulating tumor DNA and circulating tumor cells from peripheral blood. These promising classes of biomarkers could have an outsized impact on cancer management by meaningfully stratifying patients into risk groups, tracking radiation therapy efficacy during and after treatment, and identifying patients with radiosensitive or radioresistant disease. Finally, we highlight opportunities for future investigation including the need for prospective interventional studies employing liquid biopsies to guide the management of radiation therapy-treated patients.

Engineering Photocrosslinkable Bicomponent Hydrogel Constructs for Creating 3D Vascularized Bone.

Engineering bone tissue requires the generation of a highly organized vasculature. Cellular behavior is affected by the respective niche. Directing cellular behavior and differentiation for creating mineralized regions surrounded by vasculature can be achieved by controlling the pattern of osteogenic and angiogenic niches. This manuscript reports on engineering vascularized bone tissues by incorporating osteogenic and angiogenic cell-laden niches in a photocrosslinkable hydrogel construct. Two-step photolithography process is used to control the stiffness of the hydrogel and distribution of cells in the patterned hydrogel. In addittion, osteoinductive nanoparticles are utilized to induce osteogenesis. The size of microfabricated constructs has a pronounced effect on cellular organization and function. It is shown that the simultaneous presence of both osteogenic and angiogenic niches in one construct results in formation of mineralized regions surrounded by organized vasculature. In addition, the presence of angiogenic niche improves bone formation. This approach can be used for engineered constructs that can be used for treatment of bone defects.

Biodegradable Gelatin Methacrylate Gel as a Potential Scaffold for Bone Tissue Engineering of Canine Adipose-Derived Stem Cells.

Therapeutic potential of adipose derived stem cells (ADSCs) has widely been explored for treatment of orthopedic ailments. Transplantation of cells encapsulated in a scaffold facilitates 3 dimensional modelling of the tissue for the cases where well-defined spatial distribution of cells is desired for implantation. Present study aims to encapsulate canine ADSCs (cADSCs) in biodegradable methacrylated gelatin gel (GelMA) scaffold followed by their osteogenic differentiation for fabrication of a three dimensional bone tissue construct. Different percentages (5, 10 and 20%) and different methacrylation levels of gel (GelMAhigh and GelMAlow) were tested for degradation. Porosity of 10% GelMA was compared by SEM imaging. Gels with the fastest degradation rate (5% GelMAhigh and GelMAlow) were chosen for cell encapsulation since degradation of scaffold is of prime importance when the gel is intended to be used for implantation. Finally, cADSCs encapsulated in 5% GelMAlow demonstrated best morphology and were differentiated osteogenically. We developed a modified protocol for isolation of RNA from cells encapsulated in GelMA. Osteogenic differentiation was affirmed by the presence of osteo-specific gene expression by reverse transcriptase PCR in addition to von Kossa staining of the construct. GelMA is an excellent biodegradable scaffold for encapsulation of cADSCs without altering their osteogenic potential. This osteo-induced cellular scaffold implant opens a new therapeutic horizon in the area of tissue engineering in orthopedics.

In vitro silencing of myostatin gene by shRNAs in chicken embryonic myoblast cells.

RNA interference represents one of the potential mechanisms of regulation of gene expression. Selective downregulation of myostatin (MSTN), a member of transforming growth factor-ß (TGF-ß) superfamily and a negative regulator of myogenesis, has been demonstrated to enhance skeletal muscle growth. In this study, we studied short hairpin RNA (shRNA)-induced myostatin gene silencing in chicken embryonic myoblast cells using seven different shRNA-expressing constructs by reverse transcription-quantitative real time PCR (RT-qPCR). Myostatin-silencing efficiency of all shRNA constructs were first evaluated in human embryonic kidney cell line 293T (HEK293T) cells, where we observed 30-75.6% reduction in myostatin expression, followed by chicken embryo myoblast cells that revealed up to 55% reduction in myostatin expression along with upregulation of MyoD by 4.65-folds. Consistent with the earlier observations, the transfection of cells with plasmids led to significant increase in interferon responsive genes OAS1 and IFN ß (2-112-folds), independent of myostatin silencing in both HEK293T and chicken embryonic myoblast cells. Our study suggests that apart from shRNA sequences, cell type-specific factors may play a significant role in determining the knockdown efficiency of shRNAs.

Myostatin gene silencing by RNA interference in chicken embryo fibroblast cells.

Myostatin (MSTN), a member of transforming growth factor-ß (TGF-ß) superfamily, is a negative regulator of the skeletal muscle growth, and suppresses the proliferation and differentiation of myoblast cells. Dysfunction of MSTN gene either by natural mutation or genetic manipulation (knockout or knockdown) has been reported to interrupt its proper function and to increase the muscle mass in many mammalian species. RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful tool for gene knockdown studies. In the present study transient silencing of MSTN gene in chicken embryo fibroblast cells was evaluated using five different shRNA expression constructs. We report here up to 68% silencing of myostatin mRNA using these shRNA constructs in transiently transfected fibroblasts (p<0.05). This was, however, associated with induction of interferon responsive genes (OAS1, IFN-ß) (3.7-64 folds; p<0.05). Further work on stable expression of antimyostatin shRNA with minimum interferon induction will be of immense value to increase the muscle mass in the transgenic animals.

In vitro osteogenic potential of canine adipose derived stem cells.

Stem cells based tissue engineering is a promising approach for the regenerative treatment of various tissue disorders. Adipose tissue is an abundant source of cells which are competent of multipotential differentiation, called adipose-derived stem cells (ADSCs). The present study was contemplated with the objective of assessing the osteogenic differentiation potential of the canine ADSCs in vitro. The canine ADSCs were isolated from adipose tissue around falciform ligament and abdominal subcutaneous fat. Yield of viable ADSCs from both the tissue sources was found to be nearly equivalent. Tissue subjected to trypsinization yielded more viable, but lesser number of cells as compared to collagenase treatment. The stemness of ADSCs was affirmed by reverse transcriptase PCR which exhibited the expression of stem cell specific genes, OCT4 and NANOG.The monolayer of ADSCs was subjected to differentiation into adipogenic and osteogenic lineages. Assessment of the osteogenic potential of ADSCs in vitro opens a new therapeutic horizon for development of in vivo strategies employing autologous stem cell based tissue regeneration in orthopedics.