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Colorectal cancer progression is intrinsically linked to stepwise deregulation of the intestinal differentiation trajectory. In this process, sequential mutations of APC, KRAS, TP53, and SMAD4 enable oncogenic signaling and establish the hallmarks of cancer. Here, we use mass cytometry of isogenic human colon organoids and patient-derived cancer organoids to capture oncogenic signaling, cell phenotypes, and differentiation states in a high-dimensional single-cell map. We define a differentiation axis in all tumor progression states from normal to cancer. Our data show that colorectal cancer driver mutations shape the distribution of cells along the differentiation axis. In this regard, subsequent mutations can have stem cell promoting or restricting effects. Individual nodes of the cancer cell signaling network remain coupled to the differentiation state, regardless of the presence of driver mutations. We use single-cell RNA sequencing to link the (phospho-)protein signaling network to transcriptomic states with biological and clinical relevance. Our work highlights how oncogenes gradually shape signaling and transcriptomes during tumor progression.

Background: The choroid plexus (CP), which constitutes the blood-cerebrospinal fluid barrier, was recently identified as an important component of the circadian clock system. Objective: The fact that circadian rhythm disruption is closely associated to Alzheimer’s disease (AD) led us to investigate whether AD pathology can contribute to disturbances of the circadian clock in the CP. Methods: For this purpose, we evaluated the expression of core-clock genes at different time points, in 6- and 12-month-old female and male APP/PS1 mouse models of AD. In addition, we also assessed the effect of melatonin pre-treatment in vitro before amyloid-β stimulus in the daily pattern of brain and muscle Arnt-like protein 1 (Bmal1) expression. Results: Our results showed a dysregulation of circadian rhythmicity of Bmal1 expression in female and male APP/PS1 transgenic 12-month-old mice and of Period 2 (Per2) expression in male mice. In addition, a significant circadian pattern of Bmal1 was measured the intermittent melatonin pre-treatment group, showing that melatonin can reset the CP circadian clock. Conclusion: These results demonstrated a connection between AD and the disruption of circadian rhythm in the CP, representing an attractive target for disease prevention and/or treatment.

Colorectal cancer progression is intrinsically linked to stepwise deregulation of the intestinal differentiation trajectory. In this process, sequential mutations of APC/Wnt, KRAS, TP53 and SMAD4 stepwisely enable an oncogenic signalling network. Here, we developed a novel mass cytometry antibody panel to analyse colorectal cancer cell differentiation and signalling in human isogenic colorectal cancer organoids and in patient-derived cultures. We define a differentiation axis following EphrinB2 abundance in all tumour progression states from normal to cancer. We show that during colorectal cancer progression, oncogenes decrease dependence on external factors and shape distribution of cells along the differentiation axis. In this regard, subsequent mutations can have stem cell-promoting or restricting effects. Individual nodes of the signalling network remain coupled to the differentiation state, regardless of the presence of oncogenic signals. Our work underscores the key role of cell plasticity as a hallmark of cancer that is gradually unlocked during colorectal cancer progression.

SummarySevere COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathogenesis, and it remains unclear if T cells also contribute to disease pathology. Here, we combined single-cell transcriptomics and proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated, CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Age-dependent generation of C3a in severe COVID-19 induced activated CD16+ cytotoxic T cells. The proportion of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a correlated with clinical outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.

Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathogenesis, and it remains unclear if T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and proteomics with mechanistic studies assessing pathogenic T cell functions and inducing signals. We identified activated, CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Age-dependent generation of C3a in severe COVID-19 induced activated CD16 + cytotoxic T cells. The proportion of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a correlated with clinical outcome, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19. Funding: This work was supported by the German Research Foundation (DFG): SA1383/3-1 to B.S.; SFB-TR84 114933180 to L.E.S., S.B., P.G., S.H. and W.M.K. INST 37/1049-1, INST 216/981- 1, INST 257/605-1, INST 269/768-1, INST 217/988-1, INST 217/577-1, and EXC2151- 390873048 to J.L.S.; GRK 2168 – 272482170, ERA CVD (00160389 to J.L.S.; SFB 1454 – 432325352 to A.C.A. and J.L.S.; SFB TR57 and SPP1937 to J.N.; GRK2157 to A.-E.S.; and ME 3644/5-1 to H.E.M.; RTG2424 to N.B.; SFB-TRR219 322900939, BO3755/13-1 Project- ID 454024652 to P.B.; the Berlin University Alliance (BUA) (PreEP-Corona grant to L.E.S. and V.M.C.); the Berlin Institute of Health (BIH) (to L.E.S., V.M.C.,B.S. and W.M.K.); Helmholtz- Gemeinschaft Deutscher Forschungszentren, Germany (sparse2big to J.L.S.), EU projects SYSCID (733100 to J.L.S.); European Research Council Horizon 2020 (grant agreement No 101001791 to P.B.); the DZIF, Germany (TTU 04.816 and 04.817 to J.N.); the Hector Foundation (M89 to J.N.); the EU projects ONE STUDY (260687), BIO-DrIM (305147) and INsTRuCT (860003) to B.S.); German Registry of COVID-19 Autopsies through Federal Ministry of Health (ZMVI1-2520COR201 to P.B.); Federal Ministry of Education and Research (DEFEAT PANDEMICs, 01KX2021 and STOP-FSGS-01GM1901A to P.B.); the Berlin Senate to German Rheumatism Research Centre (DRFZ); the Berlin Brandenburg School for regenerative Therapies (BSRT) to C.B.; the German Federal Ministry of Education and Research (BMBF) projects RECAST (01KI20337) to B.S., V.M.C., L.E.S and M.R.; VARIPath (01KI2021) to V.M.C.; NUM COVIM (01KX2021) to L.E.S., V.M.C., F.K., J.L.S., J.N. and B.S.; RAPID to and S.H.,; SYMPATH to N.S. and W.M.K.; PROVID to S.H. and W.M.K.; ZissTrans (02NUK047E) to N.B; National Research Node ‘Mass spectrometry in Systems Medicine (MSCoresys) (031L0220A) to M.R. and N.B.; Diet–Body–Brain (DietBB) (01EA1809A) to J.L.S.; the UKRI/NIHR through the UK Coronavirus Immunology Consortium (UK-CIC), the Francis Crick Institute through the Cancer Research UK (FC001134), the UK Medical Research Council (FC001134), the Wellcome Trust (FC001134 and IA 200829/Z/16/Z) to M.R.; a Charite 3R project (to B.S., S.H., W.M.K.); and an intramural grant from the Department of Genomics & Immunoregulation at the LIMES Institute to A.C.A. We are grateful to the patients and donors volunteering to participate in this study making this research possible in the first place and wish for a speedy and full recovery. Conflict of Interest: V.M.C. is named together with Euroimmun GmbH on a patent application filed recently regarding SARS-CoV-2 diagnostics via antibody testing. A.R.S. and H.E.M. are listed as inventors on a patent application by the DRFZ Berlin in the field of mass cytometry. Ethical Approval: The study was approved by the Institutional Review board of Charite (EA2/066/20).

Opportunistic modification of the tumour microenvironment by cancer cells enhances tumour expansion and consequently eliminates tumour suppressor components. We studied the effect of fibroblasts on the circadian rhythm of growth and protein expression in colon cancer HCT116 cells and found diminished oscillation in the proliferation of HCT116 cells co-cultured with naive fibroblasts, compared with those co-cultured with tumour-associated fibroblasts (TAFs) or those cultured alone, suggesting that TAFs may have lost or gained factors that regulate circadian phenotypes. Based on the fibroblast paracrine factor analysis, we tested IL6, which diminished HCT116 cell growth oscillation, inhibited early phase cell proliferation, increased early phase expression of the differentiation markers CEA and CDX2, and decreased early phase ERK5 phosphorylation. In conclusion, our data demonstrate how the cancer education of naive fibroblasts influences the circadian parameters of neighbouring cancer cells and highlights a putative role for IL6 as a novel candidate for preoperative treatments.

Malfunctions of circadian clock trigger abnormal cellular processes and influence tumorigenesis. Using an in vitro and in vivo xenograft model, we show that circadian clock disruption via the downregulation of the core-clock genes BMAL1, PER2, and NR1D1 impacts the circadian phenotype of MYC, WEE1, and TP53, and affects proliferation, apoptosis, and cell migration. In particular, both our in vitro and in vivo results suggest an impairment of cell motility and a reduction in micrometastasis formation upon knockdown of NR1D1, accompanied by altered expression levels of SNAI1 and CD44. Interestingly we show that differential proliferation and reduced tumour growth in vivo may be due to the additional influence of the host-clock and/or to the 3D tumour architecture. Our results raise new questions concerning host–tumour interaction and show that core-clock genes are involved in key cancer properties, including the regulation of cell migration and invasion by NR1D1 in zebrafish xenografts.

The circadian clock is an endogenous time-generating system accountable for the synchrony between the internal and the geophysical time. In recent years, chronobiology research has demonstrated that the circadian regulation of numerous molecular and cellular processes leads to a temporal control of physiology and behaviour. These findings alert to the negative impact on health caused by a disrupted internal timing. In this review we address the relation between atypical external factors in long-term space flights (or other extreme environments) and circadian clock misalignment, stressing the need of establishing preventive measures to minimize the effect on human health and performance. For this purpose, daily activities of astronauts (or humans living in extreme environments) could be planned according to the individual’s internal biological time, which can be achieved through the synergy between the molecular characterization of the circadian clock and computational predictive mathematical models.