Phenotypic Heterogeneity Of Cells Research Articles

Imaging Mass Cytometry Reveals a Highly Heterogenous Cell Makeup of the Follicular Lymphoma Tumour Microenvironment

The highly variable clinical course and response to treatment of follicular lymphoma (FL) is strongly influenced by the tumor microenvironment (TME). While both pro- and anti-tumor effects depend on cell-cell interactions within the TME, these mechanisms remain incompletely understood. Bulk (Dave et al. NEJM 2004) and single-cell RNA-seq studies have identified signatures associated with prognosis, e.g., ‘T cell depleted’ microenvironments that are linked with inferior clinical outcomes (Han et al. Blood Cancer Discov. 2022). While such research has shed light on the TME, single-cell studies often lack spatial context and conventional immunohistochemistry is limited by a low number of parameters. Imaging mass cytometry (IMC) allows multiplexed analysis of up to 40 markers in tissue simultaneously, offering a unique opportunity to study the spatial features of single cells within the TME, informing both disease biology and predictors of clinical outcome. To our knowledge, IMC has more commonly been applied to solid malignancies. FL presents unique challenges in this remit, such as its morphologic variability and phenotyping cells within highly structured, dense lymph-node tissue composed of inherent tight-knit microenvironments. To analyse FL tissue by IMC, we first optimized a 31-plex panel to identify key immune and non-immune lineages including tumor cells, T and NK cells, macrophages, fibroblasts, dendritic, endothelial and reticular cells. Additionally, we wished to identify functional states such as memory, effector or regulatory phenotypes in addition to other parameters including activation, exhaustion and proliferation. Formalin-fixed paraffin embedded (FFPE) biopsies from 100 untreated FL patients enrolled on the Gallium trial (NCT01332968) of frontline chemo-immunotherapy, were subjected to IMC. In total, 381 highly multiplexed images were acquired; 13 were excluded due to suboptimal image quality. Following image pre-processing and deep-learning based cell segmentation, we analysed >3.5 million cells across 368 high-dimensional images (mean=9659 cells, SD=4600). To effectively phenotype large numbers of cells, we constructed a workflow incorporating ‘ASTIR’ (Geuenich et al. Cell Syst. 2021) - a publicly available marker-based algorithm - and cell clustering to identify population subsets. We identified 95 cell populations including Lymphoma subsets (variable CD10, CD11c, CD80 expression), CD4+ and CD8+ T cell subsets (memory, naïve, exhausted etc) and a range of non-lymphoid cells including myeloid and stromal cells. Of particular interest, we found a CD4+ CD3+ PD1+ ICOS+ population which are likely T follicular helper cells (Tfh). On average, Tfh-like cells constitute 1.9% of cells per image (SD=3.1%), although we a found considerable range in their presence. Given previous associations with Tfh cells and FL prognosis we are investigating this variability in further detail. In addition, we have identified ~30 samples enriched for CD57+ T cells, a phenotype suggestive of functional senescence. Amongst substantial heterogeneity of identified cell phenotypes (Fig A), we have identified variable sample sets characterised by enrichment or depletion of i) Lymphoma vs. T-cells (Fig B) and ii) Macrophage vs. T-cell subsets, consistent with previous single cell studies. Here, we report the use of IMC to examine the TME of untreated FL, observing marked heterogeneity of cell phenotypes across FL. Future work seeks to examine prognostic associations between the frequency of cell subsets and their spatial interactions, with this clinically annotated dataset derived from the front-line GALLIUM study. This work will provide a detailed characterization of how the spatial context of FL TME influences disease behaviour, response to treatment and survival. Given the high-throughput and high-plex nature of this study, ultimately, we aim to distil this multitude of information into condensed biomarker(s), to provide potential insights into enhancing diagnostic and/or prognostic capabilities. Therefore, we aim to derive meaningful clinical correlates associated to outcome that can be ultimately translated into practice.

Abstract 4461: Optimized human immunophenotyping panels enhance the flexibility for high-dimensional flow cytometry analysis with CyTOF

Abstract The immune system plays a pivotal role in the pathogenesis of cancer. Beyond its importance in leukemia and lymphoma, scientists now understand that the immune system is involved in virtually all malignancies. Heterogeneity of cell phenotypes within a tumor microenvironment, including blood cell phenotypes, is a hallmark of cancer that is poorly understood. Resolution of these phenotypes is essential to direct therapeutic development. CyTOF® technology has pioneered the field of high-dimensional flow cytometry through the use of isotopically pure metal-tagged antibodies and a highly sensitive mass cytometer to enable 50-plus-parameter analysis. Easy panel design without the need for compensation controls or issues of autofluorescence allows for comprehensive single-cell analysis in complex biological samples and is particularly well suited for revealing the intricacies of oncogenesis. Maxpar® OnDemand reagents were recently introduced for CyTOF to increase flexibility and facilitate larger panel design in a short period of time. To this end, lineage markers that have strong, reliable expression were reassigned to optimal metal isotopes to reserve the high-sensitivity lanthanide channels for more difficult-to-detect markers with low endogenous expression, common in immuno-oncology studies. In this study, we aimed to test the newly released Maxpar OnDemand™ Antibodies to create an optimized immune phenotyping panel and maximize further customization for CyTOF. Individual antibody performance was compared between the new and existing antibodies with different metal tags. Human peripheral blood mononuclear cells (PBMC) and fresh whole blood (WB) were stained to confirm equivalent capabilities for population gating. Importantly, we demonstrated the power of these antibodies to generate reproducible, impactful data by creating functional human immunophenotyping panels. A 20-marker human phenotyping panel was tested in PBMC and WB. We identified major immune cell populations including T cells, B cells, granulocytes, natural killer (NK) cells, and monocytes. Furthermore, the addition of CD45RO to the panel enhanced the delineation of naive, memory and effector T cell subsets. Additional myeloid markers were included to resolve the complexity within this compartment. With the updated placement of key lineage markers, the optimized phenotyping panel is ideal for future immuno-oncology studies as it can be easily expanded to interrogate cell cycle proteins, cytokines, cell signaling proteins, and oncogenic transcription factors. This work demonstrates the capability of CyTOF for robust, high-parameter immunophenotyping and a high degree of flexibility to expand our understanding of the complex processes underlying carcinogenesis and response to therapy. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Lauren J. Tracey, Michael Cohen, Christina Loh. Optimized human immunophenotyping panels enhance the flexibility for high-dimensional flow cytometry analysis with CyTOF. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4461.

Deep learning-based phenotyping of breast cancer cells using lens-free digital in-line holography.

Lens-free digital in-line holography (LDIH) produces cellular diffraction patterns (holograms) with a large field of view that lens-based microscopes cannot offer. It is a promising diagnostic tool allowing high-throughput cellular analysis. Holograms are, however, too complicated to discern by the human eye, and time-consuming computation is required to recover object images. To directly analyze holograms from LDIH, we developed a novel deep learning architecture called a holographical deep learning network (HoloNet) for cellular phenotyping. The HoloNet extracts large features from diffraction patterns, integrates them with small features from convolutional layers, and outperforms other state-of-the-art deep learning methods for the classification of breast cancer cells as well as the prediction of the intensities of breast cancer markers, ER/PR and HER2 from raw holograms. Moreover, to identify previously unknown subclusters of breast cancer cells, we developed the feature-fusion HoloNet model to integrate diffraction features related to breast cancer cell types and marker intensities of ER/PR and HER2. This hologram embedding allowed us to identify the subclusters within the known breast cancer cells. Some of the subclusters identified in our study have phenotypes shared by multiple breast cancer cell types since they are located near the class boundaries in the feature space. Identifying such rare and subtle phenotypes of breast cancer cells will enable us to perform detailed analyses of the heterogeneity of cell phenotypes for precise breast cancer diagnosis.

Mechanical transmission enables EMT cancer cells to drive epithelial cancer cell migration to guide tumor spheroid disaggregation.

Cell phenotype heterogeneity within tumor tissue, especially which due to the emergence of epithelial-mesenchymal transition (EMT) in cancer cells, is associated with cancer invasion and metastasis. However, our understanding of the cellular mechanism(s) underlying the cooperation between EMT cell and epithelial cancer cell migration remains incomplete. Herein, heterotypic tumor spheroids containing both epithelial and EMT cancer cells were generated in vitro. We observed that EMT cells dominated the peripheral region of the self-organized heterotypic tumor spheroid. Furthermore, our results demonstrated that EMT cells could serve as leader cells to improve the collective migration efficiency of epithelial cancer cells and promote dispersion and invasion of the tumor spheroids, which was regulated by the force transition between EMT cells and epithelial cancer cells. Mechanistically, our data further suggest that force transmission is mediated by heterophilic N-cadherin/E-cadherin adhesion complexes between EMT and epithelial cancer cells. Impairment of N-cadherin/E-cadherin adhesion complex formation abrogated the ability of EMT cells to guide epithelial cancer cell migration and blocked the dispersion of tumor spheroids. Together, our data provide new insight into the mechanical interaction between epithelial and EMT cancer cells through heterophilic cadherin adhesion, which enables cooperative tumor cell migration, highlighting the role of EMT cells in tumor invasion.

Spontaneous formation and spatial self-organization of mechanically induced mesenchymal-like cells within geometrically confined cancer cell monolayers

There is spatiotemporal heterogeneity in cell phenotypes and mechanical properties in tumor tissues, which is associated with cancer invasion and metastasis. It is well-known that exogenous growth factors like transforming growth factor (TGF)-β, can induce epithelial-mesenchymal transition (EMT)-based phenotypic transformation and the formation of EMT patterning on geometrically confined monolayers with mechanics heterogeneity. In the absence of exogenous TGF-β stimulation, however, whether geometric confinement-caused mechanics heterogeneity of cancer cell monolayers alone can trigger the EMT-based phenotypic heterogeneity still remains mysterious. Here, we develop a micropattern-based cell monolayer model to investigate the regulation of mechanics heterogeneity on the cell phenotypic switch. We reveal that mechanics heterogeneity itself is enough to spontaneously induce the emergence of mesenchymal-like phenotype and asymmetrical activation of TGF-β-SMAD signaling. Spatiotemporal dynamics of patterned cell monolayers with mesenchymal-like phenotypes is essentially regulated by tissue-scale cell behaviors like proliferation, migration as well as heterogeneous cytoskeletal contraction. The inhibition of cell contraction abrogates the asymmetrical TGF-β-SMAD signaling activation level and the emergence of mesenchymal-like phenotype. Our work not only sheds light on the key regulation of mechanics heterogeneity caused by spatially geometric confinement on regional mesenchymal-like phenotype of cancer cell monolayers, but highlights the key role of biophysical/mechanical cues in triggering phenotypic switch.

Decomposition of cell activities revealing the role of the cell cycle in driving biofunctional heterogeneity

Heterogeneity of cell phenotypes remains a barrier in progressing cell research and a challenge in conquering cancer-related drug resistance. Cell morphology, the most direct property of cell phenotype, evolves along the progression of the cell cycle; meanwhile, cell motility, the dynamic property of cell phenotype, also alters over the cell cycle. However, a quantifiable research understanding the relationship between the cell cycle and cell migration is missing. Herein, we coordinate the migratory behaviours of NIH 3T3 fibroblasts to their corresponding phases of the cell cycle, the G1, the S, and the G2 phases, and explain the relationship through the spatiotemporal arrangements between the Rho GTPases’ signals and cyclin-dependent kinase inhibitors, p21Cip1, and p27Kip1. Taken together, we demonstrate that both cell morphology and the dynamic subcellular behaviour are homogenous within each stage of the cell cycle phases but heterogenous between phases through quantitative cell analyses and an interactive molecular mechanism between the cell cycle and cell migration, posing potential implications in countering drug resistance.

Extracellular Vesicles Protect the Neonatal Lung from Hyperoxic Injury through the Epigenetic and Transcriptomic Reprogramming of Myeloid Cells.

Rationale: Mesenchymal stem/stromal cell (MSC)-small extracellular vesicle (MEx) treatment has shown promise in experimental models of neonatal lung injury. The molecular mechanisms by which MEx afford beneficial effects remain incompletely understood. Objectives: To investigate the therapeutic mechanism of action through assessment of MEx biodistribution and impact on immune cell phenotypic heterogeneity. Methods: MEx were isolated from the conditioned medium of human umbilical cord Wharton's jelly-derived MSCs. Newborn mice were exposed to hyperoxia (HYRX, 75% O2) from birth and returned to room air at Postnatal Day 14 (PN14). Mice received either a bolus intravenous MEx dose at PN4 or bone marrow-derived myeloid cells (BMDMy) pretreated with MEx. Animals were killed at PN4, PN7, PN14, or PN28 to characterize MEx biodistribution or for assessment of pulmonary parameters. The therapeutic role of MEx-educated BMDMy was determined invitro and invivo. Measurements and Main Results: MEx therapy ameliorated core histological features of HYRX-induced neonatal lung injury. Biodistribution and mass cytometry studies demonstrated that MEx localize in the lung and interact with myeloid cells. MEx restored the apportion of alveolar macrophages in the HYRX-injured lung and concomitantly suppressed inflammatory cytokine production. In vitro and ex vivo studies revealed that MEx promoted an immunosuppressive BMDMy phenotype. Functional assays demonstrated that the immunosuppressive actions of BMDMy are driven by phenotypically and epigenetically reprogrammed monocytes. Adoptive transfer of MEx-educated BMDMy, but not naive BMDMy, restored alveolar architecture, blunted fibrosis and pulmonary vascular remodeling, and improved exercise capacity. Conclusions: MEx ameliorate hyperoxia-induced neonatal lung injury though epigenetic and phenotypic reprogramming of myeloid cells.

Analysis of the phenotypic heterogeneity of CD123-positive cells in Kikuchi-Fujimoto disease using a sequential immunoperoxidase labeling and erasing method

To identify the phenotypic heterogeneity of CD123-expressing cells in the affected lymph nodes in patients with KFD by a sequential immunoperoxidase labeling and erasing (SIMPLE) method. Excision biopsies of lymph nodes were examined in 3 patients with KFD. After an immunohistochemical reaction using a single antibody, the tissue specimen was digitized with a Pannoramic 250 Flash III scanner (3DHISTECH, Hungary), then the cover glass was removed from the section, the specimen was hydrated and placed in a specialized buffer. Then the following primary antibody was applied to the washed tissue specimen and further immunohistochemical reaction and scanning were performed. As a result, each tissue specimen was sequentially stained in reactions with 4 antibodies. The microphotographs of specimens stained in a reaction with anti-CD123 antibody showed positive cells for their identification in the Pannoramic Viewer program (3DHISTECH, Hungary) on the remaining microphotographs displaying the expression of the other 3 markers. The selected fields of view were exported to a JPG format. Assessing the co-expression of the antigens CD123, MNDA, CD68, and TCL1A detected 4 CD123+ cell subpopulations: No. 1. CD68+/ MNDA+/ TCL1A+; No. 2. CD68+/ MNDA+/ TCL1A-; No. 3. CD68+/ MNDA-/ TCL1A+; No. 4. CD68-/ MNDA-/ TCL1A+. SIMPLE has shown the phenotypic heterogeneity of CD123-positive cells (some of them may be PDCs) and could identify 4 immunophenotypically distinct subpopulations in the affected lymph nodes in patients with KFD. Further investigations are needed to define the role of subpopulations in the pathogenesis of KFD and other diseases.

Phenotypically supervised single-cell sequencing parses within-cell-type heterogeneity.

SummaryTo better understand cellular communication driving diverse behaviors, we need to uncover the molecular mechanisms of within-cell-type functional heterogeneity. While single-cell RNA sequencing (scRNAseq) has advanced our understanding of cell heterogeneity, linking individual cell phenotypes to transcriptomic data remains challenging. Here, we used a phenotypic cell sorting technique to ask whether phenotypically supervised scRNAseq analysis (pheno-scRNAseq) can provide more insight into heterogeneous cell behaviors than unsupervised scRNAseq. Using a simple 3D in vitro breast cancer (BRCA) model, we conducted pheno-scRNAseq on invasive and non-invasive cells and compared the results to phenotype-agnostic scRNAseq analysis. Pheno-scRNAseq identified unique and more selective differentially expressed genes than unsupervised scRNAseq analysis. Functional studies validated the utility of pheno-scRNAseq in understanding within-cell-type functional heterogeneity and revealed that migration phenotypes were coordinated with specific metabolic, proliferation, stress, and immune phenotypes. This approach lends new insight into the molecular systems underlying BRCA cell phenotypic heterogeneity.

Senescence-Associated Pro-inflammatory Cytokines and Tumor Cell Plasticity.

The well-recognized cell phenotypic heterogeneity in tumors is a great challenge for cancer treatment. Dynamic interconversion and movement within a spectrum of different cell phenotypes (cellular plasticity) with the acquisition of specific cell functions is a fascinating biological puzzle, that represent an additional difficulty for cancer treatment and novel therapies development. The understanding of the molecular mechanisms responsible for moving or stabilizing tumor cells within this spectrum of variable states constitutes a valuable tool to overcome these challenges. In particular, cell transitions between epithelial and mesenchymal phenotypes (EMT-MET) and de-and trans-differentiation processes are relevant, since it has been shown that they confer invasiveness, drug resistance, and metastatic ability, due to the simultaneous acquisition of stem-like cell properties. Multiple drivers participate in these cell conversions events. In particular, cellular senescence and senescence-associated soluble factors have been shown to unveil stem-like cell properties and cell plasticity. By modulating gradually the composition of their secretome and the time of exposure, senescent cells may have differential effect not only on tumor cells but also on surrounding cells. Intriguingly, tumor cells that scape from senescence acquire stem-like cell properties and aggressiveness. The reinforcement of senescence and inflammation by soluble factors and the participation of immune cells may provide a dynamic milieu having varied effects on cell transitions, reprogramming, plasticity, stemness and therefore heterogeneity. This will confer different epithelial/mesenchymal traits (hybrid phenotype) and stem-like cell properties, combinations of which, in a particular cell context, could be responsible for different cellular functions during cancer progression (survival, migration, invasion, colonization or proliferation). Additionally, cooperative behavior between cell subpopulations with different phenotypes/stemness functions could also modulate their cellular plasticity. Here, we will discuss the role of senescence and senescence-associated pro-inflammatory cytokines on the induction of cellular plasticity, their effect role in establishing particular states within this spectrum of cell phenotypes and how this is accompanied by stem-like cell properties that, as the epithelial transitions, may also have a continuum of characteristics providing tumor cells with functional adaptability specifically useful in the different stages of carcinogenesis.

Single-Cell RNA Sequencing Reveals Renal Endothelium Heterogeneity and Metabolic Adaptation to Water Deprivation.

Renal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown. We inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model in vitro and in dehydrated mice in vivo. We identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and-surprisingly-oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration. This study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation.

Influence of Phenotypic Dissociation in Bacillus subtilis Strain ET-1 on Iturin A Production.

Iturin A is a very important cyclic lipopeptide produced by several B. subtilis strains and has large commercial and therapeutic application potentials but its production on industrial scale has not been realized yet. In the present study, we have observed that the strain ET-1 of Bacillus subtilis, a producer of Iturin A, can present at least three different colony morphologies, which we arbitrarily called Rough, Smooth, and Mucoid morphotypes (R-, S-, and M-form). Performing HPLC analysis, a significant difference between the amounts of Iturin A produced by the three morphotypes was found. The morphotype R-form showed the highest productivity with yields about 10 and 100 times higher than morphotypes S and M, respectively. The results show that the production of Iturin A by B. subtilis could be strongly influenced by the phenotypic heterogeneity of cells within the inoculum. Indeed, we have observed that, pasteurizing the inoculum before seeding in order to improve the homogeneity removing the phenotypes less able to synthesize the Iturin A, its yields in a bench-scale production could be significantly improved. This can represent an important control factor also at industrial scale to improve the Iturin A yields, the robustness, the replicability, and consequently the cost-effectiveness of fermentation processes.

Distinctive association of peripheral immune cell phenotypes with capillaroscopic microvascular patterns in systemic sclerosis.

The pathological changes in SSc include immune system dysregulation and microvascular damage. However, the association of immune cell phenotype heterogeneity and microvascular abnormalities is unclear. The aim of this study is to elucidate this association in SSc. Peripheral blood mononuclear cells obtained from 150 SSc patients were used for comprehensive flow cytometric analysis based on the Human Immunology Project. Hierarchical cluster analysis was used to classify SSc patients into subgroups and their association with microvascular abnormalities, as assessed by nailfold videocapillaroscopy (i.e. 'early', 'active' and 'late' patterns), was analysed. The proportions of activated CD4+ T cells, T cells re-expressing CD45RA, activated Th1 and Th17 cells and IgD-CD27- B cells were higher in SSc patients than in healthy individuals. Hierarchical cluster analysis stratified SSc patients into three groups: patients with few immune abnormalities (fewer abnormalities group), patients with high proportions of activated T and Treg cells (Treg-dominant group) and patients with high proportions of Tfh and plasmablasts (Tfh-dominant group). Age and disease duration were comparable among the groups. On the other hand, microvascular abnormalities, especially the 'late' nailfold videocapillaroscopy pattern, correlated with internal organ involvement. Among the groups stratified according to immune cell phenotype, the progression to the 'late' nailfold videocapillaroscopy pattern was more frequent in the Tfh-dominant group. Our study confirmed the presence of immunophenotypic abnormalities in SSc. Immunological abnormalities were not uniform but rather limited to subpopulations, particularly the Tfh-dominant group, where they were highly associated with microvascular abnormalities and organ involvement.

Diversity of collective migration patterns of invasive breast cancer cells emerging during microtrack invasion.

Understanding the mechanisms underlying the diversity of tumor invasion dynamics, including single-cell migration, multicellular streaming, and the emergence of various collective migration patterns, is a long-standing problem in cancer research. Here we have designed and fabricated a series of microchips containing high-throughput microscale tracks using protein repelling coating technology, which were then covered with a thin Matrigel layer. By varying the geometrical confinement (track width) and microenvironment factors (Matrigel concentration), we have reproduced a diversity of collective migration patterns in the chips, which were also observed in vivo. We have further classified the collective patterns and quantified the emergence probability of each class of patterns as a function of microtrack width and Matrigel concentration to devise a quantitive "collective pattern diagram." To elucidate the mechanisms behind the emergence of various collective patterns, we employed cellular automaton simulations, incorporating the effects of both direct cell-cell interactions and microenvironment factors (e.g., chemical gradient and extracellular matrix degradation). Our simulations suggest that tumor cell phenotype heterogeneity, and the associated dynamic selection of a favorable phenotype via cell-microenivronment interactions, are key to the emergence of the observed collective patterns in vitro.

Meet the First Authors.

Meet the First Authors.

Estimating intratumoral heterogeneity from spatiotemporal data.

Glioblastoma multiforme (GBM) is a malignant brain cancer with a tendency to both migrate and proliferate. We propose modeling GBM with heterogeneity in cell phenotypes using a random differential equation version of the reaction-diffusion equation, where the parameters describing diffusion (D) and proliferation ([Formula: see text]) are random variables. We investigate the ability to perform the inverse problem to recover the probability distributions of D and [Formula: see text] using the Prohorov metric, for a variety of probability distribution functions. We test the ability to perform the inverse problem for noisy synthetic data. We then examine the predicted effect of treatment, specifically, chemotherapy, when assuming such a heterogeneous population and compare with predictions from a homogeneous cell population model.

Regional differences in endothelial cell cytoskeleton, junctional proteins and phosphorylated tyrosine labeling in the porcine vortex vein system

We previously demonstrated endothelial phenotype heterogeneity in the vortex vein system. This study is to further determine whether regional differences are present in the cytoskeleton, junctional proteins and phosphorylated tyrosine labeling within the system.The vortex vein system of twenty porcine eyes was perfused with labels for f-actin, claudin-5, VE-Cadherin, phosphorylated tyrosine and nucleic acid. The endothelial cells of eight different regions (choroidal veins, pre-ampulla, anterior ampulla, mid-ampulla, posterior ampulla, post-ampulla, intra-scleral canal and the extra-ocular vortex vein) were studied using confocal microscopy.There were regional differences in the endothelial cell structures. Cytoskeleton labeling was relatively even in intensity throughout Regions 1 to 6. Overall VE-Cadherin had a non-uniform distribution and thicker width endothelial cell border staining than claudin-5. Progressing downstream there was an increased variation in thickness of VE-cadherin labeling. There was an overlap in phosphorylated tyrosine and VE-Cadherin labeling in the post-ampulla, intra-scleral canal and extra-ocular vortex vein. Intramural cells were observed that were immune-positive for VE-Cadherin and phosphorylated tyrosine. There were significant differences in the number of intramural cells in different regions.Significant regional differences with endothelial cell labeling of cytoskeleton, junction proteins, and phosphorylated tyrosine were found within the vortex vein system. These findings support existing data on endothelial cell phenotype heterogeneity, and may aid in the knowledge of venous pathologies by understanding regions of vulnerability to endothelial damage within the vortex vein system. It could be valuable to further investigate and characterize the VE-cadherin and phosphotyrosine immune-positive intramural cells.

Three-Dimensional Magnetic Levitation Culture System Simulating White Adipose Tissue.

White adipose tissue (WAT) has attracted interest for tissue engineering and cell-based therapies as an abundant source of adipose stem/stromal cells (ASC). However, technical challenges in WAT cell culture have limited its applications in regenerative medicine. Traditional two-dimensional (2D) cell culture models, which are essentially monolayers of cells on glass or plastic substrates, inadequately represent tissue architecture, biochemical concentration gradients, substrate stiffness, and most importantly for WAT research, cell phenotypic heterogeneity. Physiological cell culture platforms for WAT modeling must recapitulate the native diversity of cell types and their coordination within the organ. For this purpose, we developed a three-dimensional (3D) model using magnetic levitation. Here, we describe our protocol that we successfully employed to build adipose tissue organoids (adipospheres) that preserve the heterogeneity of the constituent cell types in vitro. We demonstrate the capacity of assembling adipospheres from multiple cell types, including ASCs, endohtelial cells, and leukocytes that recreate tissue organization. These adipospheres mimicked WAT organogenesis in that they enabled the formation of vessel-like endothelial structures with lumens and differentiation of unilocular adipocytes. Altogether, magnetic levitation is a cell culture platform that recreates tissue structure, function, and heterogeneity in vitro, and serves as a foundation for high-throughput WAT tissue culture and analysis.

Meis2 as a critical player in MN1-induced leukemia

Meningioma 1 (MN1) is an independent prognostic marker for normal karyotype acute myeloid leukemia (AML), with high expression linked to all-trans retinoic acid resistance and poor survival. MN1 is also a potent and sufficient oncogene in murine leukemia models, strongly dependent on the MEIS1/AbdB-like HOX protein complex to transform common myeloid progenitors, block myeloid differentiation, and promote leukemic stem cell self-renewal. To identify key genes and pathways underlying leukemic activity, we functionally assessed MN1 cell phenotypic heterogeneity, revealing leukemic and non-leukemic subsets. Using gene expression profiling of these subsets combined with previously published comparisons of full-length MN1 and mutants with varying leukemogenic activity, we identified candidate genes critical to leukemia. Functional analysis identified Hlf and Hoxa9 as critical to MN1 in vitro proliferation, self-renewal and impaired myeloid differentiation. Although critical to transformation, Meis1 knockdown had little impact on these properties in vitro. However, we identified Meis2 as critical to MN1-induced leukemia, with essential roles in proliferation, self-renewal, impairment of differentiation and disease progression in vitro and in vivo. Here, we provide evidence of phenotypic and functional hierarchy in MN1-induced leukemic cells, characterise contributions of Hlf, Hoxa9 and Meis1 to in vitro leukemic properties, and reveal Meis2 as a novel player in MN1-induced leukemogenesis.

From autonomy to community; new perspectives on tumorigenicity and therapy resistance.

Subclones of cancer cells evading treatment represent the major challenge in oncology. Despite recent advances, tumors not responding to treatments are still a severe risk to cancer patients, and oncologists have, as of now, little effective therapy to offer patients with systemic cancer disease. The widely discussed cancer stem cell (CSC) paradigm was originally launched as an explanation to the existence of small cell populations resistant to therapy within the heterogeneous tumor, but has so far unfortunately, offered little concrete improvement in cancer treatment regimes. The launch of the CSC hypothesis did, however, highlight the significance of therapy targeting specific tumor-driving processes, and even more importantly, an increased awareness of a phenomenon well known to stem cell researchers; non-genetic phenotypic heterogeneity of cells with common origin. Here, the scientific background of the CSC theory is revisited and the evidence for CSCs is discussed, along with the importance of considering CSC's dependency of their habitat for survival and growth. Furthermore, recent advances in cancer cell heterogeneity and new possibilities for studying therapy responses in cell clones within the natural tumor environment using patient derived xenograft (PDX) models, are reviewed.