ABSTRACT
The pathogenesis of many rare tumor types is poorly understood, preventing the design of effective treatments. Solitary fibrous tumors (SFTs) are neoplasms of mesenchymal origin that affect 1/1,000,000 individuals every year and are clinically assimilated to soft tissue sarcomas. SFTs can arise throughout the body and are usually managed surgically. However, 30-40% of SFTs will relapse local-regionally or metastasize. There are no systemic therapies with durable activity for malignant SFTs to date. The molecular hallmark of SFTs is a gene fusion between the NAB2 and STAT6 loci on chromosome 12, resulting in a chimeric protein of poorly characterized function called NAB2-STAT6. We use primary samples and an inducible cell model to discover that NAB2-STAT6 operates as a transcriptional coactivator for a specific set of enhancers and promoters that are normally targeted by the EGR1 transcription factor. In physiological conditions, NAB2 is primarily localized to the cytoplasm and only a small nuclear fraction is available to operate as a co-activator of EGR1 targets. NAB2-STAT6 redirects NAB1, NAB2, and additional EGR1 to the nucleus and bolster the expression of neuronal EGR1 targets. The STAT6 moiety of the fusion protein is a major driver of its nuclear localization and further contributes to NAB2's co-activating abilities. In primary tumors, NAB2-STAT6 activates a neuroendocrine gene signature that sets it apart from most sarcomas. These discoveries provide new insight into the pathogenesis of SFTs and reveal new targets with therapeutic potential.
ABSTRACT
The human colon is inhabited by a complex community of microbes. These microbes are integral to host health and physiology. Understanding how and when the microbiome causally influences host health will require microbiome models that can be tightly controlled and manipulated. While in vivo models are unrivalled in their ability to study host-microbial interplay, in vitro models are gaining in popularity as methods to study the ecology and function of the gut microbiota, and benefit from tight controllability and reproducibility, as well as reduced ethical constraints. In this set of protocols, we describe the Robogut, a single-stage bioreactor system designed to replicate the conditions of the distal human colon, to culture whole microbial communities derived from stool and/or colonic biopsy samples, with consideration of methods to create culture medium formulations and to build, run, and sample the bioreactor apparatus. Cleaning and maintenance of the bioreactor system are also described. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Growth medium preparation Support Protocol 1: Preparing medium supplements Basic Protocol 2: Preparing the bioreactor vessels Support Protocol 2: Making acid and base bottles Support Protocol 3: Preparing the effluent bottles Support Protocol 4: Making acid solution Support Protocol 5: Making base solution Basic Protocol 3: Preparing inoculum and inoculating bioreactors Alternate Protocol 1: Preparing inoculum less than 0.5% (w/v) of vessel volume Alternate Protocol 2: Preparing synthetic community aliquots and inoculation via the septum Alternate Protocol 3: Preparing inoculum from a tissue sample Basic Protocol 4: Sampling the bioreactor vessel Basic Protocol 5: Harvesting bioreactor vessel contents at end of experiment Support Protocol 6: Cleaning and sterilizing sampling needles Basic Protocol 6: Cleaning the bioreactor vessel Support Protocol 7: Cleaning bioreactor support bottles.
