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Osteosarcoma is the most frequent primary bone tumor with poor prognosis. Through RNA-sequencing of 100,987 individual cells from 7 primary, 2 recurrent, and 2 lung metastatic osteosarcoma lesions, 11 major cell clusters are identified based on unbiased clustering of gene expression profiles and canonical markers. The transcriptomic properties, regulators and dynamics of osteosarcoma malignant cells together with their tumor microenvironment particularly stromal and immune cells are characterized. The transdifferentiation of malignant osteoblastic cells from malignant chondroblastic cells is revealed by analyses of inferred copy-number variation and trajectory. A proinflammatory FABP4 + macrophages infiltration is noticed in lung metastatic osteosarcoma lesions. Lower osteoclasts infiltration is observed in chondroblastic, recurrent and lung metastatic osteosarcoma lesions compared to primary osteoblastic osteosarcoma lesions. Importantly, TIGIT blockade enhances the cytotoxicity effects of the primary CD3 + T cells with high proportion of TIGIT + cells against osteosarcoma. These results present a single-cell atlas, explore intratumor heterogeneity, and provide potential therapeutic targets for osteosarcoma. Osteosarcomas are difficult to treat and have a limited response to immunotherapy. Here, the authors analyse osteosarcomas at the single-cell level, and identify both the transdifferentiation of malignant cells and an array of immune cells that could have implications for metastasis and immunotherapy.

Yeast flocculation is a community-building cell aggregation trait that is an important mechanism of stress resistance and a useful phenotype for brewers; however, it is also a nuisance in many industrial processes, in clinical settings, and in the laboratory. Chemostat-based evolution experiments are impaired by inadvertent selection for aggregation, which we observe in 35% of populations. These populations provide a testing ground for understanding the breadth of genetic mechanisms Saccharomyces cerevisiae uses to flocculate, and which of those mechanisms provide the biggest adaptive advantages. In this study, we employed experimental evolution as a tool to ask whether one or many routes to flocculation are favored, and to engineer a strain with reduced flocculation potential. Using a combination of whole genome sequencing and bulk segregant analysis, we identified causal mutations in 23 independent clones that had evolved cell aggregation during hundreds of generations of chemostat growth. In 12 of those clones, we identified a transposable element insertion in the promoter region of known flocculation gene FLO1, and, in an additional five clones, we recovered loss-of-function mutations in transcriptional repressor TUP1, which regulates FLO1 and other related genes. Other causal mutations were found in genes that have not been previously connected to flocculation. Evolving a flo1 deletion strain revealed that this single deletion reduces flocculation occurrences to 3%, and demonstrated the efficacy of using experimental evolution as a tool to identify and eliminate the primary adaptive routes for undesirable traits.

Human induced pluripotent stem cells (hiPSCs) represent an almost limitless source of cells for disease modelling and drug screening applications. Here we established an efficient and robust 3D platform for cardiomyocyte (CMs) production from hiPSCs, solely through small-molecule-based temporal modulation of the Wnt signalling, which generates more than 90% cTNT + cells. The impact of performing the differentiation process in 3D conditions as compared to a 2D culture system, was characterized by transcriptomic analysis by using data collected from sequential stages of 2D and 3D culture. We highlight that performing an initial period of hiPSC aggregation before cardiac differentiation primed hiPSCs towards an earlier mesendoderm lineage differentiation, via TGF-β/Nodal signaling stabilization. Importantly, it was also found that CMs in the 3D microenvironment mature earlier and show an improved communication system, which we suggested to be responsible for a higher structural and functional maturation of 3D cardiac aggregates.

Background Maturation of complex N-glycans involves the action of Golgi mannosidases and plays a major role in cancer progression. We recently showed a favourable prognostic role of α-mannosidase MAN1A1 in breast cancer mainly caused by alteration of certain adhesion molecules. Methods We analysed the protein expression of MAN1A1 in ovarian cancer ( n  = 204) using western blot and studied the impact of MAN1A1 itself and of MAN1A1-related glycosylation on the prognostic relevance of two adhesion molecules. Functional consequences of mannosidase inhibition using kifunensine and MAN1A1 knock out were investigated in ovarian cancer cells in vitro. Results Patients with high MAN1A1 expression in tumours showed significantly shorter RFS than those with low-MAN1A1 levels. Moreover, high MAN1A1 expression correlated significantly with advanced stage, lymph node involvement and distant metastasis. Further, the glycosylated adhesion molecule ALCAM reveals a significant adverse prognostic effect only in the presence of high MAN1A1 expression. In spheroid-formation assays, mannosidase inhibition and especially MAN1A1 knock out led to strong reduction of tumour cell aggregation. Conclusions Our study demonstrates the unfavourable prognostic role of MAN1A1 in ovarian cancer, probably caused by an altered ability of spheroid formation, and the strong influence of this glycosylation enzyme on the prognostic impact of ALCAM.

It is difficult to achieve minimally invasive injectable cell delivery while maintaining high cell retention and animal survival for in vivo stem cell therapy of myocardial infarction. Here we show that pluripotent stem cell aggregates pre-differentiated into the early cardiac lineage and encapsulated in a biocompatible and biodegradable micromatrix, are suitable for injectable delivery. This method significantly improves the survival of the injected cells by more than six-fold compared with the conventional practice of injecting single cells, and effectively prevents teratoma formation. Moreover, this method significantly enhances cardiac function and survival of animals after myocardial infarction, as a result of a localized immunosuppression effect of the micromatrix and the in situ cardiac regeneration by the injected cells. Stem cell therapy of myocardial infarction is hampered by poor survival of injected cells. Here the authors develop injectable aggregates of stem cells differentiated to an early cardiac stage and encapsulated in a biodegradable micromatrix, and show their enhanced therapeutic efficacy in a heart infarction mouse model.

Ascitic multicellular aggregates (MCAs) promote peritoneal metastasis of ovarian cancer. The aim of the present study was to elucidate the role of cancer-associated fibroblasts (CAFs) in MCA formation and metastasis in patients with high-grade serous ovarian cancer (HGSOC). Immunohistochemistry was used to identify the cell phenotypes and the presence of CAFs in ascitic MCAs. The role of CAFs in tumor-cell MCA formation was assessed by co-culture in suspension. Primary ascitic tumor cells and omental CAFs were used to generate ex vivo MCAs in hanging drops, and the invasiveness of MCAs was evaluated by mesothelial clearance and adhesion assays in vitro and in vivo. MCAs containing CAFs and tumor cells were identified in the ascitic fluid. CAFs facilitated tumor cell aggregation and compaction to form MCAs, and enhanced the mesothelial clearance and adhesion abilities of tumor-cell MCAs. These findings suggest that ascitic CAFs promote peritoneal metastasis by forming heterotypic aggregates with tumor cells, and that they may serve as potential targets for the treatment of HGSOC.

Telomerase, particularly its main subunit, the reverse transcriptase, TERT, prevents DNA erosion during eukaryotic chromosomal replication, but also has poorly understood non-canonical functions. Here, in the model social amoeba Dictyostelium discoideum, we show that the protein encoded by tert has telomerase-like motifs, and regulates, non-canonically, important developmental processes. Expression levels of wild-type (WT) tert were biphasic, peaking at 8 and 12 h post-starvation, aligning with developmental events, such as the initiation of streaming (~7 h) and mound formation (~10 h). In tert KO mutants, however, aggregation was delayed until 16 h. Large, irregular streams formed, then broke up, forming small mounds. The mound-size defect was not induced when a KO mutant of countin (a master size-regulating gene) was treated with TERT inhibitors, but anti-countin antibodies did rescue size in the tert KO. Although, conditioned medium (CM) from countin mutants failed to rescue size in the tert KO, tert KO CM rescued the countin KO phenotype. These and additional observations indicate that TERT acts upstream of smlA/countin: (i) the observed expression levels of smlA and countin, being respectively lower and higher (than WT) in the tert KO; (ii) the levels of known size-regulation intermediates, glucose (low) and adenosine (high), in the tert mutant, and the size defect's rescue by supplemented glucose or the adenosine-antagonist, caffeine; (iii) the induction of the size defect in the WT by tert KO CM and TERT inhibitors. The tert KO's other defects (delayed aggregation, irregular streaming) were associated with changes to cAMP-regulated processes (e.g. chemotaxis, cAMP pulsing) and their regulatory factors (e.g. cAMP; acaA, carA expression). Overexpression of WT tert in the tert KO rescued these defects (and size), and restored a single cAMP signaling centre. Our results indicate that TERT acts in novel, non-canonical and upstream ways, regulating key developmental events in Dictyostelium.

Pathogenic inflammation and immune suppression are the cardinal features that underlie the pathogenesis of severe systemic inflammatory syndrome and sepsis. Neutrophil exhaustion may play a key role during the establishment of pathogenic inflammation and immune suppression through elevated expression of inflammatory adhesion molecules such as ICAM1 and CD11b as well as immune-suppressors such as PD-L1. However, the mechanism of neutrophil exhaustion is not well understood. We demonstrated that murine primary neutrophils cultured in vitro with the prolonged lipopolysaccharides (LPS) stimulation can effectively develop an exhaustive phenotype resembling human septic neutrophils with elevated expression of ICAM1, CD11b, PD-L1 as well as enhanced swarming and aggregation. Mechanistically, we observed that TICAM2 is involved in the generation of neutrophil exhaustion, as TICAM2 deficient neutrophils have the decreased expression of ICAM1, CD11b, PD-L1, and the reduced aggregation following the prolonged LPS challenge as compared to wild type (WT) neutrophils. LPS drives neutrophil exhaustion through TICAM2 mediated activation of Src family kinases (SFK) and STAT1, as the application of SFK inhibitor Dasatinib blocks neutrophil exhaustion triggered by the prolonged LPS challenge. Functionally, TICAM2 deficient mice were protected from developing severe systemic inflammation and multi-organ injury following the chemical-induced mucosal damage. Together, our data defined a key role of TICAM2 in facilitating neutrophil exhaustion and that targeting TICAM2 may be a potential approach to treating the severe systemic inflammation.

Kin selection theory predicts that costly cooperative behaviors evolve most readily when directed toward kin. Dispersal plays a controversial role in the evolution of cooperation: dispersal decreases local population relatedness and thus opposes the evolution of cooperation, but limited dispersal increases kin competition and can negate the benefits of cooperation. Theoretical work has suggested that plasticity of dispersal, where individuals can adjust their dispersal decisions according to the social context might help resolve this paradox and promote the evolution of cooperation. Here, we experimentally tested the hypothesis that conditional dispersal decisions are mediated by a cooperative strategy: we quantified the density-dependent dispersal decisions and subsequent colonization efficiency from single cells or groups of cells among six genetic strains of the unicellular Tetrahymena thermophila that differ in their aggregation level (high, medium, and low), a behavior associated with cooperation strategy. We found that the plastic reaction norms of dispersal rate relative to density differed according to aggregation level: highly aggregative genotypes showed negative density-dependent dispersal, whereas low-aggregation genotypes showed maximum dispersal rates at intermediate density, and medium-aggregation genotypes showed density-independent dispersal with intermediate dispersal rate. Dispersers from highly aggregative genotypes had specialized long-distance dispersal phenotypes, contrary to low-aggregation genotypes; medium-aggregation genotypes showing intermediate dispersal phenotype. Moreover, highly aggregation genotypes showed evidence for beneficial kin-cooperation during dispersal. Our experimental results should help to resolve the evolutionary conflict between cooperation and dispersal: cooperative individuals are expected to avoid kin-competition by dispersing long distances, but maintain the benefits of cooperation by dispersing in small groups.

Here we describe a novel method in which embryonic kidneys are dissociated into single-cell suspensions and then reaggregated to form organotypic renal structures. Kidney cell reaggregates were transiently cultured with small-molecule Rho kinase inhibitors, which caused ureteric bud structures to form and induced formation of nephrons. These structures displayed normal morphology, expressed appropriate differentiation markers, and were connected at their distal ends to the ureteric buds, thus forming artificial tissues very similar to those found in normal embryonic kidneys. Using this culture method, it was straightforward to make fine-grained chimeras by mixing different cell types or by mixing cells transfected with different constructs before reaggregation. Chimeric renal cultures were formed using mixtures of unmarked normal host embryonic kidney cells and CellTracker-marked WT1 siRNA-carrying cells to test the hypothesis that WT1 is important to a cell's ability to contribute to nephron formation. We found a significant reduction in the ability of WT1 knockdown cells to contribute to nephron formation. This dissociation and reaggregation procedure can also be applied to embryonic lungs and to form coarse-grained hybrid tissues from mixtures of lung and kidney cells. Overall, our protocol allows very simple mixing of cells from different sources or cells subjected to different pretreatments to make fine-grained, highly dispersed chimera tissues.