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BACKGROUND Brain metastases are most common in adults with lung cancer,predicting uniformly poor patient outcome,with a median survival of only months. Despite their frequency and severity,very little is known about tumorigenesis in brain metastases. METHODS We applied previously developed primary solid tumor-initiating cell models to the study of brain metastases from the lung to evaluate the presence of a cancer stem cell population. Patient-derived brain metastases (n = 20) and the NCI-H1915 cell line were cultured as stem-enriching tumorspheres. We used in vitro limiting-dilution and sphere-forming assays,as well as intracranial human-mouse xenograft models. To determine genes overexpressed in brain metastasis tumorspheres,we performed comparative transcriptome analysis. All statistical analyses were two-sided. RESULTS Patient-derived brain metastasis tumorspheres had a mean sphere-forming capacity of 33 spheres/2000 cells (SD = 33.40) and median stem-cell frequency of 1/60 (range = 0-1/141),comparable to that of primary brain tumorspheres (P = .53 and P = .20,respectively). Brain metastases also expressed CD15 and CD133,markers suggestive of a stemlike population. Through intracranial xenotransplantation,brain metastasis tumorspheres were found to recapitulate the original patient tumor heterogeneity. We also identified several genes overexpressed in brain metastasis tumorspheres as statistically significant predictors of poor survival in primary lung cancer. CONCLUSIONS For the first time,we demonstrate the presence of a stemlike population in brain metastases from the lung. We also show that NCI-H1915 tumorspheres could be useful in studying self-renewal and tumor initiation in brain metastases. Our candidate genes may be essential to metastatic stem cell populations,where pathway interference may be able to transform a uniformly fatal disease into a more localized and treatable one. View Publication

Patients with triple-negative breast cancer display the highest rates of early relapse of all patients with breast cancer. The basal-like subtype,a subgroup of triple-negative breast cancer,exhibits high levels of constitutively active NF-κB signalling. Here we show that NF-κB activation,induced by inflammatory cytokines or by epigenetically dysregulated NIK expression,cell-autonomously upregulates JAG1 expression in non-cancer stem cells. This upregulation stimulates NOTCH signalling in cancer stem cells in trans,leading to an expansion of cancer stem cell populations. Among breast cancers,the NF-κB-dependent induction of JAG1 and the NOTCH-dependent expansion of the cancer stem cell population occur only in the basal-like subtype. Collectively,our results indicate that NF-κB has a non-cell-autonomous role in regulating cancer stem cell populations by forming intratumoural microenvironments composed of JAG1-expressing non-cancer stem cells with a basal-like subtype. View Publication

During pancreatic development,proliferating pancreatic progenitors activate the proendocrine transcription factor neurogenin 3 (NEUROG3),exit the cell cycle,and differentiate into islet cells. The mechanisms that direct robust NEUROG3 expression within a subset of progenitor cells control the size of the endocrine population. Here we demonstrate that NEUROG3 is phosphorylated within the nucleus on serine 183,which catalyzes its hyperphosphorylation and proteosomal degradation. During progression through the progenitor cell cycle,NEUROG3 phosphorylation is driven by the actions of cyclin-dependent kinases 2 and 4/6 at G1/S cell-cycle checkpoint. Using models of mouse and human pancreas development,we show that lengthening of the G1 phase of the pancreatic progenitor cell cycle is essential for proper induction of NEUROG3 and initiation of endocrine cell differentiation. In sum,these studies demonstrate that progenitor cell-cycle G1 lengthening,through its actions on stabilization of NEUROG3,is an essential variable in normal endocrine cell genesis. View Publication

Any epithelial portion of a normal mouse mammary gland can reproduce an entire functional gland when transplanted into an epithelium-free mammary fat pad. Mouse mammary hyperplasias and tumors are clonal dominant populations and probably represent the progeny of a single transformed cell. Our study provides evidence that single multipotent stem cells positioned throughout the mature fully developed mammary gland have the capacity to produce sufficient differentiated progeny to recapitulate an entire functional gland. Our evidence also demonstrates that these stem cells are self-renewing and are found with undiminished capacities in the newly regenerated gland. We have taken advantage of an experimental model where mouse mammary tumor virus infects mammary epithelial cells and inserts a deoxyribonucleic acid copy(ies) of its genome during replication. The insertions occur randomly within the somatic genome. CzechII mice have no endogenous nucleic acid sequence homology with mouse mammary tumor virus; therefore all viral insertions may be detected by Southern analysis provided a sufficient number of cells contain a specific insertional event. Transplantation of random fragments of infected CzechII mammary gland produced clonal-dominant epithelial populations in epithelium-free mammary fat pads. Serial transplantation of pieces of the clonally derived outgrowths produced second generation glands possessing the same viral insertion sites providing evidence for self-renewal of the original stem cell. Limiting dilution studies with cell cultures derived from third generation clonal outgrowths demonstrated that three multipotent but distinct mammary epithelial progenitors were present in clonally derived mammary epithelial populations. Estimation of the potential number of multipotent epithelial cells that may be evolved from an individual mammary-specific stem cell by self-renewal is in the order of 10(12)-10(13). Therefore,one stem cell might easily account for the renewal of mammary epithelium over several transplant generations. View Publication

Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently,reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture,apoptosis assays,protein expression,limiting dilution clonal frequency assay,genetic affymetrix analysis,and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin,P=0.9) were similar as well. Likewise,markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue,DiIC,caspase-3,and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition,genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally,glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional,protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence,both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture. View Publication

Hematopoietic stem/progenitor cells (HSPC) are characterized by their unique capacities of self-renewal and multi-differentiation potential. This second property makes them able to adapt their differentiation profile depending on the local environment they reach. Taking advantage of an animal model of peritonitis,induced by injection of the TLR-2 ligand,zymosan,we sought to study the relationship between bone marrow-derived hematopoietic stem/progenitor cells (BM-HSPCs) and innate lymphoid cells (ILCs) regarding their emergence and differentiation at the site of inflammation. Our results demonstrate that the strength of the inflammatory signals affects the capacity of BM-derived HSPCs to migrate and give rise in situ to ILCs. Both low- and high-dose of zymosan injections trigger the appearance of mature ILCs in the peritoneal cavity where the inflammation occurs. Herein,we show that only in low-dose injected mice,the recovered ILCs are dependent on an in situ differentiation of BM-derived HSPCs and/or ILC2 precursors (ILC2P) wherein high-dose,the stronger inflammatory environment seems to be able to induce the emergence of ILCs independently of BM-derived HSPCs. We suggest that a relationship between HSPCs and ILCs seems to be affected by the strength of the inflammatory stimuli opening new perspectives in the manipulation of these early hematopoietic cells. View Publication

To facilitate the characterization of unlabeled induced pluripotent stem cells (iPSCs) during culture and expansion,we developed an AI pipeline for nuclear segmentation and mitosis detection from phase contrast images of individual cells within iPSC colonies. The analysis uses a 2D convolutional neural network (U-Net) plus a 3D U-Net applied on time lapse images to detect and segment nuclei,mitotic events,and daughter nuclei to enable tracking of large numbers of individual cells over long times in culture. The analysis uses fluorescence data to train models for segmenting nuclei in phase contrast images. The use of classical image processing routines to segment fluorescent nuclei precludes the need for manual annotation. We optimize and evaluate the accuracy of automated annotation to assure the reliability of the training. The model is generalizable in that it performs well on different datasets with an average F1 score of 0.94,on cells at different densities,and on cells from different pluripotent cell lines. The method allows us to assess,in a non-invasive manner,rates of mitosis and cell division which serve as indicators of cell state and cell health. We assess these parameters in up to hundreds of thousands of cells in culture for more than 36 hours,at different locations in the colonies,and as a function of excitation light exposure. View Publication

BackgroundMacrophage-based cell therapies have shown modest success in clinical trials,which can be attributed to their phenotypic plasticity,where transplanted macrophages get reprogrammed towards a pro-tumor phenotype. In most tumor types,including melanoma,the balance between antitumor M1-like and tumor-promoting M2-like macrophages is critical in defining the local immune response with a higher M1/M2 ratio favoring antitumor immunity. Therefore,designing novel strategies to increase the M1/M2 ratio in the TME has high clinical significance and benefits macrophage-based cell therapies.MethodsIn this study,we reprogrammed antitumor and proinflammatory macrophages ex-vivo with HDAC6 inhibitors (HDAC6i). We administered the reprogrammed macrophages intratumorally as an adoptive cell therapy (ACT) in the syngeneic SM1 murine melanoma model and patient-derived xenograft bearing NSG-SGM3 humanized mouse models. We phenotyped the tumor-infiltrated immune cells by flow cytometry and histological analysis of tumor sections for macrophage markers. We performed bulk RNA-seq profiling of murine bone marrow-derived macrophages treated with vehicle or HDAC6i and single-cell RNA-seq profiling of SM1 tumor-infiltrated immune cells to determine the effect of intratumor macrophage ACT on the tumor microenvironment (TME). We further analyzed the single-cell data to identify key cell-cell interactions and trajectory analysis to determine the fate of tumor-associated macrophages post-ACT.ResultsMacrophage ACT resulted in diminished tumor growth in both mouse models. We also demonstrated that HDAC6 inhibition in macrophages suppressed the polarization toward tumor-promoting phenotype by attenuating STAT3-mediated M2 reprogramming. Two weeks post-transplantation,ACT macrophages were viable,and inhibition of HDAC6 rendered intratumor transplanted M1 macrophages resistant to repolarization towards protumor M2 phenotype in-vivo. Further characterization of tumors by flow cytometry,single-cell transcriptomics,and single-cell secretome analyses revealed a significant enrichment of antitumor M1-like macrophages,resulting in increased M1/M2 ratio and infiltration of CD8 effector T-cells. Computational analysis of single-cell RNA-seq data for cell-cell interactions and trajectory analyses indicated activation of monocytes and T-cells in the TME.ConclusionsIn summary,for the first time,we demonstrated the potential of reprogramming macrophages ex-vivo with HDAC6 inhibitors as a viable macrophage cell therapy to treat solid tumors.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13046-024-03182-w. View Publication

Crystalline silica (CS) particle exposure leads to silicosis which is characterized as progressive fibrosis. Fibroblasts are vital effector cells in fibrogenesis. Emerging studies have identified immune sentinel roles for fibroblasts in chronic disease,while their immune-modulatory roles in silicosis remain unclear. Herein,we show that group 2 innate lymphoid cell (ILC2) conversion to ILC1s is closely involved in silicosis progression,which is mediated by activated fibroblasts via interleukin (IL)−18. Mechanistically,Notch3 signaling in mechanics-activated fibroblasts modulates IL-18 production via caspase 1 activity. The mouse-specific Notch3 knockout in fibroblasts retards pulmonary fibrosis progression that is linked to attenuated ILC conversion. Our results indicate that activated fibroblasts in silicotic lungs are regulators of ILC2–ILC1 conversion,associated with silicosis progression via the Notch3–IL-18 signaling axis. This finding broadens our understanding of immune-modulatory mechanisms in silicosis,and indicates potential therapeutic targets for lung fibrotic diseases. Crystalline silica particle exposure in the airways can lead to lung silicosis and progressive fibrosis. Here the authors use mouse silicosis models to show mechanics activated fibroblasts promote conversion of ILC2 to ILC1-like cells pulmonary fibrosis and that this is associated with a Notch3-IL-18 signalling pathway. View Publication

The generation of the post-cranial embryonic body relies on the coordinated production of spinal cord neurectoderm and presomitic mesoderm cells from neuromesodermal progenitors (NMPs). This process is orchestrated by pro-neural and pro-mesodermal transcription factors that are co-expressed in NMPs together with Hox genes,which are essential for axial allocation of NMP derivatives. NMPs reside in a posterior growth region,which is marked by the expression of Wnt,FGF and Notch signalling components. Although the importance of Wnt and FGF in influencing the induction and differentiation of NMPs is well established,the precise role of Notch remains unclear. Here,we show that the Wnt/FGF-driven induction of NMPs from human embryonic stem cells (hESCs) relies on Notch signalling. Using hESC-derived NMPs and chick embryo grafting,we demonstrate that Notch directs a pro-mesodermal character at the expense of neural fate. We show that Notch also contributes to activation of HOX gene expression in human NMPs,partly in a non-cell-autonomous manner. Finally,we provide evidence that Notch exerts its effects via the establishment of a negative-feedback loop with FGF signalling. View Publication

Functional cellular heterogeneity in tumours often underlies incomplete response to therapy and relapse. Previously,we demonstrated that the growth of the paediatric brain malignancy,sonic hedgehog subgroup medulloblastoma,is rooted in a dysregulated developmental hierarchy,the apex of which is defined by characteristically quiescent SOX2 + stem-like cells. Integrating gene expression and chromatin accessibility patterns in distinct cellular compartments,we identify the transcription factor Olig2 as regulating the stem cell fate transition from quiescence to activation,driving the generation of downstream neoplastic progenitors. Inactivation of Olig2 blocks stem cell activation and tumour output. Targeting this rare OLIG2-driven proliferative programme with a small molecule inhibitor,CT-179,dramatically attenuates early tumour formation and tumour regrowth post-therapy,and significantly increases median survival in vivo. We demonstrate that targeting transition from quiescence to proliferation at the level of the tumorigenic cell could be a pivotal medulloblastoma treatment strategy. Subject terms: Cancer stem cells,Mechanisms of disease,Cancer therapy View Publication

Autoantibodies to transglutaminase 2 (TG2) are hallmarks of celiac disease. To address B cell tolerance and autoantibody formation to TG2,we generated immunoglobulin knock-in (Ig KI) mice that express a prototypical celiac patient-derived anti-TG2 B cell receptor equally reactive to human and mouse TG2. We studied B cell development in the presence/absence of autoantigen by crossing the Ig KI mice to Tgm2-/- mice. Autoreactive B cells in Tgm2+/+ mice were indistinguishable from their naive counterparts in Tgm2-/- mice with no signs of clonal deletion,receptor editing,or B cell anergy. The autoreactive B cells appeared ignorant to their antigen,and they produced autoantibodies when provided T cell help. The findings lend credence to a model of celiac disease where gluten-reactive T cells provide help to autoreactive TG2-specific B cells by involvement of gluten-TG2 complexes,and they outline a general mechanism of autoimmunity with autoantibodies being produced by ignorant B cells on provision of T cell help. View Publication