Open-ST: High-resolution spatial transcriptomics in 3D.

Spatial transcriptomics (ST) methods unlock molecular mechanisms underlying tissue development, homeostasis, or disease. However, there is a need for easy-to-use, high-resolution, cost-efficient, and 3D-scalable methods. Here, we report Open-ST, a sequencing-based, open-source experimental and computational resource to address these challenges and to study the molecular organization of tissues in 2D and 3D. In mouse brain, Open-ST captured transcripts at subcellular resolution and reconstructed cell types. In primary head-and-neck tumors and patient-matched healthy/metastatic lymph nodes, Open-ST captured the diversity of immune, stromal, and tumor populations in space, validated by imaging-based ST. Distinct cell states were organized around cell-cell communication hotspots in the tumor but not the metastasis. Strikingly, the 3D reconstruction and multimodal analysis of the metastatic lymph node revealed spatially contiguous structures not visible in 2D and potential biomarkers precisely at the 3D tumor/lymph node boundary. All protocols and software are available at https://rajewsky-lab.github.io/openst.

Mutant huntingtin impairs neurodevelopment in human brain organoids through CHCHD2-mediated neurometabolic failure.

Expansion of the glutamine tract (poly-Q) in the protein huntingtin (HTT) causes the neurodegenerative disorder Huntington's disease (HD). Emerging evidence suggests that mutant HTT (mHTT) disrupts brain development. To gain mechanistic insights into the neurodevelopmental impact of human mHTT, we engineered male induced pluripotent stem cells to introduce a biallelic or monoallelic mutant 70Q expansion or to remove the poly-Q tract of HTT. The introduction of a 70Q mutation caused aberrant development of cerebral organoids with loss of neural progenitor organization. The early neurodevelopmental signature of mHTT highlighted the dysregulation of the protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a transcription factor involved in mitochondrial integrated stress response. CHCHD2 repression was associated with abnormal mitochondrial morpho-dynamics that was reverted upon overexpression of CHCHD2. Removing the poly-Q tract from HTT normalized CHCHD2 levels and corrected key mitochondrial defects. Hence, mHTT-mediated disruption of human neurodevelopment is paralleled by aberrant neurometabolic programming mediated by dysregulation of CHCHD2, which could then serve as an early interventional target for HD.

Modelling viral encephalitis caused by herpes simplex virus 1 infection in cerebral organoids.

Herpes simplex encephalitis is a life-threatening disease of the central nervous system caused by herpes simplex viruses (HSVs). Following standard of care with antiviral acyclovir treatment, most patients still experience various neurological sequelae. Here we characterize HSV-1 infection of human brain organoids by combining single-cell RNA sequencing, electrophysiology and immunostaining. We observed strong perturbations of tissue integrity, neuronal function and cellular transcriptomes. Under acyclovir treatment viral replication was stopped, but did not prevent HSV-1-driven defects such as damage of neuronal processes and neuroepithelium. Unbiased analysis of pathways deregulated upon infection revealed tumour necrosis factor activation as a potential causal factor. Combination of anti-inflammatory drugs such as necrostatin-1 or bardoxolone methyl with antiviral treatment prevented the damages caused by infection, indicating that tuning the inflammatory response in acute infection may improve current therapeutic strategies.

Ibrutinib-based therapy impaired neutrophils microbicidal activity in patients with chronic lymphocytic leukemia during the early phases of treatment.

Ibrutinib is a tyrosine kinase inhibitor used in the treatment of a variety of lymphoid malignancies, including chronic lymphocytic leukemia (CLL). Drugs inhibiting B-cell-receptor (BCR)-associated kinases, including BTK inhibitors, act on B cells and on a wide spectrum of tissues and cells, including innate immunity cells. Thus, alterations in the Bruton's tyrosine kinase (BTK) kinase function could lead to an impairment of innate immune cells functions and to an increased infectious risk in patients receiving BTK inhibitors. We analyzed in vivo neutrophils oxidative burst, neutrophils granules release and cytokine production in relapsed/refractory CLL patients treated over time with ibrutinib as single-agent. We observed a dramatic reduction of neutrophils oxidative burst, Fc gamma receptors (FcγRs)-mediated degranulation and IL-8 plasma levels already after the first forty-eight hours of therapy with ibrutinib. However, ibrutinib treatment did not alter the surface expression of CD11b nor cytokine and proteinases release not mediated by FcγRs engagement. After three weeks, oxidative burst was still impaired, while degranulation and IL-8 levels were restored. In a group of CLL patients who survived for more than three years, all processes triggered by FcγRs completely recovered except the release of neutrophil elastase (NE) and IL-8. In conclusion, during the initial phases of ibrutinib therapy, the reduction of IL-8, NE, myeloperoxidase (MPO) levels and oxidative burst negatively impacted on mechanisms involved in neutrophils microbicidal activity.