See in 3D
Scientists at Stanford University have developed a 3D model that was more accurately able to model the effects of putative cancer drivers than traditional monolayer cell cultures. With new bioinformatics methods, the list of potential cancer driver genes has been growing rapidly. Methods for wet lab validation, however, have lagged. The team created 3D cancer spheroids and used CRISPR gene editing to compare spheroids to 2D cell culture sheets. They found that “CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumors, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D… Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.” They reported their results in the March 12, 2020, issue of Nature.
GOF, LOF mutations take different paths to same result
Scientists at the Baylor College of Medicine have shown that loss of function and gain of function mutations in the ACOX1 (acyl-CoA oxidase 1) gene both led to glial loss, but did so via distinct mechanisms. ACOX1 encodes the rate-limiting gene in a pathway that degrades very long chain fatty acids, and mutations lead to loss of glial cells. However, the authors showed that mutations that caused a gain of function (GOF) were toxic because of the accumulation of reactive oxygen species, but loss of function (LOF) mutations were toxic because of the accumulation of very long chain fatty acids themselves. They concluded that “both loss and gain of ACOX1 lead to glial and neuronal loss, but different mechanisms are at play and require different treatments.” The team also noted that while flies have no myelin sheath, which has led to some skepticism about their value as a model organism for glial diseases, “humans and flies share similar phenotypes in the absence of ACOX1 that have not yet been observed in mice. We propose this may be due to redundancy with murine Acox2 and Acox3.” The team reported its results in the March 13, 2020, online issue of Neuron.
Tolerizing DCs are made, not born
Investigators at the University of Marseille have discovered that in order to tolerize CD8 T cells to self-antigens, class 1 dendritic cells (cDC1s) first needed to interact with regulatory T cells in the draining lymph nodes. The induction of tolerance to self-antigens by dendritic cells including cDC1s is key to preventing massive autoimmunity. The authors set out to understand the mechanisms which maintain cDC1's ability to tolerize CD8 T cells. They showed that in the absence of MHC class II, the cDC1s were not able to transfer information from regulatory to CD8 T cells, and as a result, cDC1/CD8 T-cell interactions led to autoimmune reactions. “Our results revealed that the cross-tolerizing capacity of cDC1s is not a property fully acquired at the time they undergo homeostatic maturation but needs to be enforced via MHCII-restricted, suppressive interactions with regulatory T cells,” the authors wrote. They published their work in the March 13, 2020, issue of Science Immunology.
NEFA’s nefarious role in pancreatitis
Scientists at Mayo Clinic have gained new insights into the role of visceral fat tissue on organ inflammation. Abdominal fat contributes to multiple inflammation-mediated diseases, but the severity of those diseases varies greatly. Diverticulitis is on the mild end of the spectrum and usually self-resolving, while pancreatitis can be severe and progress to even more severe organ failure. The authors compared the molecular events of diverticulitis with those of pancreatitis, and found that the enzyme pancreatic triglyceride lipase (PNLIP) increased in adipose tissue during pancreatitis and entered fat cells, metabolizing triglyceride and generating excess nonesterified fatty acids (NEFAs). Knocking out PNLIP protected obese mice from organ failure and improved survival during acute pancreatitis. Triglycerides are usually broken down by adipocyte triglyceride lipase (ATGL), but knocking out AGTL was not protective, leading the authors to propose a role for PNLIP in the progression from pancreatitis to organ failure. Their work appeared in the March 9, 2020, print issue of the Journal of Clinical Investigation after earlier publication online.
Comprehensive look identifies insulin autoantigens
Researchers at Washington University in St. Louis have biochemically identified the insulin peptides that generated autoimmune responses in a mouse model of autoimmune diabetes. “The nature of autoantigens that trigger autoimmune diseases has been much discussed, but direct biochemical identification is lacking for most,” the authors wrote. By testing the entire proinsulin peptidome that could be presented by MHC II, which is the major histocompatibility complex whose presentation can lead to autoimmune responses, the authors “generated conventional epitopes but also resulted in the presentation of post-translationally modified peptides, including deamidated sequences. These analyses reveal the key features of a restricted component in the self-MHC-II peptidome that caused autoreactivity.” Their work appeared in the March 9, 2020, issue of Nature Immunology.