Heterogeneous Cell Populations Research Articles

IMMU-46. CROSS-TALK OF INNATE IMMUNE CELLS IN GLIOBLASTOMA PATHOPHYSIOLOGY: UNDERSTANDING NLRP3 AND NLRP12

Abstract Glioblastoma (GBM) are malignant gliomas with an average survival of less than 14 months after diagnosis, despite surgery, chemotherapy, and radiotherapy. Tumor survival is aided by the heterogeneous cell populations in the tumor microenvironment. Two crucial innate immune cells of the brain, namely microglia and macrophages, favor a tumor-promoting microenvironment IN GBM. The nucleotide-binding domain leucine-rich repeat-containing receptor (NLR) family in astrocytes and microglia cells recognizes pathogen- and damage-associated molecular patterns that induce inflammation. NLRP3, upon activation, leads to cleavage and activation of Caspase-1, causing the proteolytic cleavage-mediated activation and conversion of pro-IL-1ß and pro-IL-18 to IL-1ß and IL-18, leading to pyroptosis. NLRP12 downregulates inflammatory responses in microglia and macrophages by regulating the NF-kB pathway. NLRP3 and NLRP12 have tumor-promoting and anti-tumour functions in various cancers. NLRP12 is a prognostic marker for glioblastoma, and its increased expression correlates with poor survival in GBM patients. However, the cellular and molecular mechanisms of NLRP3 and NLRP12 in GBM pathophysiology are largely unknown. This study aims to understand the effect of NLRP3 and NLRP12 expression in GBM, to interpret the pathophysiology, and to help develop future targeted therapeutic interventions. To understand the baseline expression of NLRP3 and NLRP12 in cell lines and patient-derived GBM cells, PCR, western blotting, and Immunocytochemistry were performed comparing WT and nlrp3-/- and nlrp12-/- cells. Differential expression of NLRP3 and NLRP12 in patient-derived cells can be compared to GBM, astrocyte, and microglia cell lines. This may be the underlying cause for intra and inter-tumor heterogeneity and resultant differential therapy outcomes. To understand cell-to-cell communication in GBM-like tumor microenvironments, heterocellular 3D organoids, and patient-derived tumor organoids were developed. Cell viability in 3D patient-derived tumor organoids decreased by up to 10% with Doxorubicin and 40% with Carboplatin treatment decreases compared to untreated controls. This suggests the scope of developing drugs targeting inflammation regulatory genes.

Exploring heterogeneous cell population dynamics in different microenvironments by novel analytical strategy based on images.

Understanding the dynamic states and transitions of heterogeneous cell populations is crucial for addressing fundamental biological questions. High-content imaging provides rich datasets, but it remains increasingly difficult to integrate and annotate high-dimensional and time-resolved datasets to profile heterogeneous cell population dynamics in different microenvironments. Using hepatic stellate cells (HSCs) LX-2 as model, we proposed a novel analytical strategy for image-based integration and annotation to profile dynamics of heterogeneous cell populations in 2D/3D microenvironments. High-dimensional features were extracted from extensive image datasets, and cellular states were identified based on feature profiles. Time-series clustering revealed distinct temporal patterns of cell shape and actin cytoskeleton reorganization. We found LX-2 showed more complex membrane dynamics and contractile systems with an M-shaped actin compactness trend in 3D culture, while they displayed rapid spreading in early 2D culture. This image-based integration and annotation strategy enhances our understanding of HSCs heterogeneity and dynamics in complex extracellular microenvironments.

Functional analysis of antigen presentation by enteric glial cells during intestinal inflammation.

The Enteric Nervous System is composed of a vastly interconnected network of neurons and glial cells that coordinate to regulate homeostatic gut function including intestinal motility, nutrient sensing, and mucosal barrier immunity. Enteric Glial Cells (EGCs) are a heterogeneous cell population located throughout the gastrointestinal tract and have well described roles in regulating intestinal immune responses. Enteric Glial Cells have been suggested to act as nonconventional antigen presenting cells via the Major Histocompatibility Complex II (MHC II), though this has not been confirmed functionally. Here, we investigate the capability of EGCs to present antigen on MHC I and MHC II using in vitro antigen presentation assays performed with primary murine EGC cultures. We found that EGCs are capable of functional antigen presentation on MHC I, including antigen cross-presentation, but are not capable of functional antigen presentation on MHC II. We also determined EGC cell surface MHC I and MHC II expression levels by flow cytometry during intestinal inflammation during Dextran Sodium Sulfate-induced colitis or acute Toxoplasma gondii infection. We found that EGCs upregulate MHC I during acute T. gondii infection and induce low-level MHC II expression. These findings suggest that EGCs may be important in the regulation of CD8+ T cell responses via MHC I mediated antigen (cross) presentation but may not be relevant for MHC II-mediated antigen presentation.

Microwell-assembled aluminum substrates for enhanced single-cell analysis: A novel approach for cancer cell profiling by Raman spectroscopy

Single-cell analysis is critical for advancing personalized medicine, as it reveals cell population heterogeneity that influences disease outcomes. We present a microwell-assembled aluminum substrate platform that enhances single-cell Raman spectroscopy in liquid suspension by isolating individual cells and preventing stacking and movement, which significantly improves signal stability and the signal-to-noise ratio (SNR).We applied this novel platform to analyze PC-9 lung cancer cells and BEAS-2B normal bronchial epithelial cells, identifying distinct biochemical differences. Notably, cancer cells showed higher levels of adenine, cytochromes, DNA/RNA, and unsaturated lipids, along with an increased unsaturation ratio and protein content. These findings were further validated using machine learning models. An eXtreme Gradient Boosting (XGBoost) model achieved perfect classification accuracy of 100 %, underscoring the robustness of the spectral features identified by our platform.Our platform not only enhances single-cell Raman signal detection but also holds promise for biomedical applications, including early cancer detection, treatment monitoring, and drug development. The high-throughput capacity of this platform featuring over 120,000 wells, along with its compatibility with techniques such as Raman-activated cell sorting (RACS) further extends its potential for clinical diagnostics and personalized medicine.

Design, performance, processing, and validation of a pooled CRISPR perturbation screen for bacterial toxins.

Unbiased forward genetic screens have been extensively employed in biological research to elucidate functional genomics. In pooled clustered regularly interspaced short palindromic repeats (CRISPR) perturbation screens, various genetically encoded gain-of-function or loss-of-function mutations are introduced into a heterogeneous population of cells. Subsequently, these cells are screened for phenotypes, perturbation-associated genotypes are analyzed and a connection between genotype and phenotype is determined. CRISPR screening techniques enable the investigation of important biological questions, such as how bacterial toxins kill cells and cause disease. However, the broad spectrum of effects caused by diverse toxins presents a challenge when selecting appropriate screening strategies. Here, we provide a step-by-step protocol for a genome-wide pooled CRISPR perturbation screen to study bacterial toxins. We describe technical considerations, pilot experiments, library construction, screen execution, result analysis and validation of the top enriched hits. These screens are applicable for many different types of toxins and are anticipated to reveal a repertoire of host factors crucial in the intoxication pathway, such as receptors, trafficking/translocation factors and substrates. The entire protocol takes 21-27 weeks and does not require specialized knowledge beyond basic biology.

Rapid manufacture of low-seed CAR-T cells in a GMP-grade hollow-fiber bioreactor platform

Both quality of product and rapidity of manufacture are critical parameters if ex vivo manufacturing of autologous chimeric antigen receptor T cell (CAR-T) therapies is to reach its full potential. The Quantum Flex™ Cell Expansion System from Terumo Blood and Cell Technologies (Terumo BCT), a hollow-fiber bioreactor platform, is one of several cell expansion systems available to cell and gene therapy manufacturers to generate such cells in a GMP-compliant manner. In this study, the dynamic range of the Quantum Flex platform to expand CD19 CAR-T cells from variable quantities of starting material was investigated. Reflecting the industry's utilization of contract development manufacturing organizations (CDMOs) for accelerating clinical timelines, Terumo Blood and Cell Technologies performed a technology transfer of application protocols for study execution. Four different amounts starting material (1, 3, 6, and 15 million cells) were expanded on Quantum Flex, using a unique donor's cells for each run. In this study, CAR-T cells were created using commercially obtained T cells and an anti-CD19 CAR-T lentiviral construct. The resultant heterogenous cell populations were expanded for 7 days in the functionally closed bioreactor platform. Expansion kinetics for all 4 starting material amounts were remarkedly similar, resulting in a 150- to 200-fold increase in cell numbers. This allowed for a study maximum of 2.6 billion cells from loading 15 million cells. Viability remained high throughout the expansion process with > 93% for all 4 donors at harvest. To complete the manufacturing cycle, the automated and functionally closed Finia™ Fill and Finish System (Terumo Blood and Cell Technologies, Lakewood, CO) was used to formulate the cells for cryopreservation. Post-procedure analysis for potency and cytotoxicity demonstrated the production of efficacious cells. With this range of starting numbers, the platform is relevant to adult, pediatric, and compassionate CAR-T expansion dosing. Today, several platforms are available to achieve sufficient cell yields for therapeutic applications of CAR-T, and awareness of the capabilities, pros, and cons of each platform is critical to drive progress.

Origin of dedifferentiated adipocyte-derived cells (DFAT) during ceiling culture in an Adiponectin Cre-Recombinase mouse model.

Dedifferentiated adipose tissue-derived (DFAT) cells represent an attractive source of stem cells for tissue engineering and the potential treatment of several clinical conditions. Our objective was to determine whether DFAT cells originate from mature adipocytes and address whether contamination from the stromal vascular fraction (SVF) could be as a source for these cells. A murine adiponectin-creERT; mT/mG model was used with the excision of the cassette induced by tamoxifen injection for the cells expressing adiponectin (adipoq). This model allows distinguishing mature adipocytes (green fluorescence) from other SVF cell types (red fluorescence) based on the fluorescent protein expressed. Mature adipocytes and SVF cells were isolated from adipose tissues by collagenase digestion. Ceiling cultures were imaged by time-lapse microscopy. Confocal microscopy was used to follow cells over 21 days. Time-lapse microscopy experiments showed liposecretion occurring in mature adipocytes displaying green fluorescence. Confocal imaging allowed the identification of a heterogeneous cell population expressing green but also red fluorescence after 21 days of culture. Asymmetrical division of mature adipocytes was not observed. In conclusion, liposecretion of mature adipocytes is a phenomenon that can be observed in vitro and DFAT cells do originate from mature adipocytes. However, the population of DFAT cells is heterogenous.

Clinically Relevant and Precisely Printable Live Adipose Tissue-Based Bio-Ink for Volumetric Soft Tissue Reconstruction.

Autologous fat is widely used in soft tissue reconstruction; however, significant volume reduction owing to necrosis and degradation of the transplanted adipose tissue (AT) remains a major challenge. To address this issue, a novel live AT micro-fragment-based bio-ink (ATmf bio-ink) compatible with precision 3D printing, is developed. Live AT micro-fragments of ≈280µm in size are prepared using a custom tissue micronizer and they are incorporated into a fibrinogen/gelatin mixture to create the ATmf bio-ink. AT micro-fragments exhibit high viability and preserve the heterogeneous cell population and extracellular matrix of the native AT. The developed bio-ink enables precise micropatterning and provides an excellent adipo-inductive microenvironment. AT grafts produced by co-printing the bio-ink with polycaprolactone demonstrate a 500% improvement in volume retention and a 300% increase in blood vessel infiltration in vivo compared with conventional microfat grafts. In vivo engraftment of AT grafts is further enhanced by using a stem cell-laden ATmf bio-ink. Last, it is successfully demonstrated that the bio-ink is enabled for the creation of clinically relevant and patient-specific AT grafts for patients undergoing partial mastectomy. This novel ATmf bio-ink for volumetric soft tissue reconstruction offers a pioneering solution for addressing the limitations of existing clinical techniques.

Stemness in solid malignancies: coping with immune attack.

Immunotherapy has become a key new pillar of cancer treatment, and this has sparked interest in understanding mechanisms of cancer immune evasion. It has long been appreciated that cancers are constituted by heterogeneous populations of tumour cells. This feature is often fuelled by specialized cells that have molecular programs resembling tissue stem cells. Although these cancer stem cells (CSCs) have capacity for unlimited self-renewal and differentiation, it is increasingly evident that some CSCs are capable of achieving remarkable immune resistance. Given that most immunotherapy regiments have overlooked CSC-specific immune-evasive mechanisms, many current treatment strategies often lead to cancer relapse. This Review focuses on advancements in understanding how CSCs in solid tumours achieve their unique immune-evasive properties, enabling them to drive tumour regrowth. Moreover, as cancers often arise from tissue stem cells that acquired oncogenic mutations, we discuss how tissue stem cells undergoing malignant transformation activate intrinsic immune-evasive mechanisms and establish close interactions with suppressive immune cells to escape immune surveillance. In addition, we summarize how in advanced disease stages, CSCs often hijack features of normal stem cells to resist antitumour immunity. Finally, we provide insights in how to design a new generation of cancer immunotherapies to ensure elimination of CSCs.

Proteogenomic characterization of highly enriched viable leukemic blasts in acute myeloid leukemia: A SWOG report

AbstractIntroductionAcute myeloid leukemia (AML) remains one of the deadliest hematopoietic malignancies. A better understanding of the molecular biology governing AML may lead to improved risk stratification and facilitate the development of novel therapies. Proteins are responsible for much of the biology of cells. Several studies have examined the global proteome in bulk mononuclear cells (MNCs) from AML specimens, which are comprised a heterogenous population of cells at various stages of differentiation.MethodsGiven the potential impact of the nonleukemic cells on protein expression profiles, we applied an integrative proteogenomic approach utilizing next‐generation sequencing and mass spectrometry‐based proteomics to identify novel protein biomarkers in unsorted MNCs and viable leukemic blasts (VLBs) isolated from blood and bone marrow specimens obtained at the time of AML diagnosis.ResultsWe identified significant differences in protein expression between VLBs and MNCs. Subsequent studies (N = 27) focused on proteomic profiling of VLBs that identified novel candidate biomarkers associated with mutational genotypes and clinical outcome, some of which were recapitulated in an independent cohort of patients. Using mass spectrometry, we also detected mutated protein products, some of which were predicted via in silico analyses to be potential neoantigens amenable to adoptive immunotherapy. As previously described, analyses comparing transcript and protein expression showed an overall modest correlation between mRNA and protein dataset, but enriching for genes associated with mutations significantly improved the protein–RNA correlation.ConclusionTogether, the results provide insight into the biology of VLBs and demonstrate the gains derived from examining the proteome in addition to genome and transcriptome.

Computational identification of surface markers for isolating distinct subpopulations from heterogeneous cancer cell populations

Intratumor heterogeneity reduces treatment efficacy and complicates our understanding of tumor progression and there is a pressing need to understand the functions of heterogeneous tumor cell subpopulations within a tumor, yet systems to study these processes in vitro are limited. Single-cell RNA sequencing (scRNA-seq) has revealed that some cancer cell lines include distinct subpopulations. Here, we present clusterCleaver, a computational package that uses metrics of statistical distance to identify candidate surface markers maximally unique to transcriptomic subpopulations in scRNA-seq which may be used for FACS isolation. With clusterCleaver, ESAM and BST2/tetherin were experimentally validated as surface markers which identify and separate major transcriptomic subpopulations within MDA-MB-231 and MDA-MB-436 cells, respectively. clusterCleaver is a computationally efficient and experimentally validated workflow for identification of surface markers for tracking and isolating transcriptomically distinct subpopulations within cell lines. This tool paves the way for studies on coexisting cancer cell subpopulations in well-defined in vitro systems.

Tolerance of Oncogene-Induced Replication Stress: A Fuel for Genomic Instability.

Activation of oncogenes disturbs a wide variety of cellular processes and induces physiological dysregulation of DNA replication, widely referred to as replication stress (RS). Oncogene-induced RS can cause replication forks to stall or collapse, thereby leading to DNA damage. While the DNA damage response (DDR) can provoke an anti-tumor barrier to prevent the development of cancer, a small subset of cells triggers replication stress tolerance (RST), allowing precancerous cells to survive, thereby promoting clonal expansion and genomic instability (GIN). Genomic instability (GIN) is a hallmark of cancer, driving genetic alterations ranging from nucleotide changes to aneuploidy. These alterations increase the probability of oncogenic events and create a heterogeneous cell population with an enhanced ability to evolve. This review explores how major oncogenes such as RAS, cyclin E, and MYC induce RS through diverse mechanisms. Additionally, we delve into the strategies employed by normal and cancer cells to tolerate RS and promote GIN. Understanding the intricate relationship between oncogene activation, RS, and GIN is crucial to better understand how cancer cells emerge and to develop potential cancer therapies that target these vulnerabilities.

Limited Adipogenic Differentiation Potential of Human Dental Pulp Stem Cells Compared to Human Bone Marrow Stem Cells.

Bone marrow and teeth contain mesenchymal stem cells (MSCs) that could be used for cell-based regenerative therapies. MSCs from these two tissues represent heterogeneous cell populations with varying degrees of lineage commitment. Although human bone marrow stem cells (hBMSCs) and human dental pulp stem cells (hDPSCs) have been extensively studied, it is not yet fully defined if their adipogenic potential differs. Therefore, in this study, we compared the in vitro adipogenic differentiation potential of hDPSCs and hBMSCs. Both cell populations were cultured in adipogenic differentiation media, followed by specific lipid droplet staining to visualise cytodifferentiation. The in vitro differentiation assays were complemented with the expression of specific genes for adipogenesis and osteogenesis-dentinogenesis, as well as for genes involved in the Wnt and Notch signalling pathways. Our findings showed that hBMSCs formed adipocytes containing numerous and large lipid vesicles. In contrast to hBMSCs, hDPSCs did not acquire the typical adipocyte morphology and formed fewer lipid droplets of small size. Regarding the gene expression, cultured hBMSCs upregulated the expression of adipogenic-specific genes (e.g., PPARγ2, LPL, ADIPONECTIN). Furthermore, in these cells most Wnt pathway genes were downregulated, while the expression of NOTCH pathway genes (e.g., NOTCH1, NOTCH3, JAGGED1, HES5, HEY2) was upregulated. hDPSCs retained their osteogenic/dentinogenic molecular profile (e.g., RUNX2, ALP, COLIA1) and upregulated the WNT-specific genes but not the NOTCH pathway genes. Taken together, our in vitro findings demonstrate that hDPSCs are not entirely committed to the adipogenic fate, in contrast to the hBMSCs, which are more effective to fully differentiate into adipocytes.

CD81-guided heterologous EVs present heterogeneous interactions with breast cancer cells

BackgroundExtracellular vesicles (EVs) are cell-secreted particles conceived as natural vehicles for intercellular communication. The capacity to entrap heterogeneous molecular cargoes and target specific cell populations through EV functionalization promises advancements in biomedical applications. However, the efficiency of the obtained EVs, the contribution of cell-exposed receptors to EV interactions, and the predictability of functional cargo release with potential sharing of high molecular weight recombinant mRNAs are crucial for advancing heterologous EVs in targeted therapy applications.MethodsIn this work, we selected the popular EV marker CD81 as a transmembrane guide for fusion proteins with a C-terminal GFP reporter encompassing or not Trastuzumab light chains targeting the HER2 receptor. We performed high-content imaging analyses to track EV-cell interactions, including isogenic breast cancer cells with manipulated HER2 expression. We validated the functional cargo delivery of recombinant EVs carrying doxorubicin upon EV-donor cell treatment. Then, we performed an in vivo study using JIMT-1 cells commonly used as HER2-refractory, trastuzumab-resistant model to detect a more than 2000 nt length recombinant mRNA in engrafted tumors.ResultsFusion proteins participated in vesicular trafficking dynamics and accumulated on secreted EVs according to their expression levels in HEK293T cells. Despite the presence of GFP, secreted EV populations retained a HER2 receptor-binding capacity and were used to track EV-cell interactions. In time-frames where the global EV distribution did not change between HER2-positive (SK-BR-3) or -negative (MDA-MB-231) breast cancer cell lines, the HER2 exposure in isogenic cells remarkably affected the tropism of heterologous EVs, demonstrating the specificity of antiHER2 EVs representing about 20% of secreted bulk vesicles. The specific interaction strongly correlated with improved cell-killing activity of doxorubicin-EVs in MDA-MB-231 ectopically expressing HER2 and reduced toxicity in SK-BR-3 with a knocked-out HER2 receptor, overcoming the effects of the free drug. Interestingly, the fusion protein-corresponding transcripts present as full-length mRNAs in recombinant EVs could reach orthotopic breast tumors in JIMT-1-xenografted mice, improving our sensitivity in detecting penetrant cargoes in tissue biopsies.ConclusionsThis study highlights the quantitative aspects underlying the creation of a platform for secreted heterologous EVs and shows the limits of single receptor-ligand interactions behind EV-cell engagement mechanisms, which now become the pivotal step to predict functional tropism and design new generations of EV-based nanovehicles.

In-Depth Proteomic Analysis Reveals Phenotypic Diversity of Macrophages in Liver Fibrosis.

Macrophages make up a heterogeneous population of immune cells that exhibit diverse phenotypes and functions in health and disease. Although macrophage epigenomic and transcriptomic profiles have been reported, the proteomes of distinct macrophage populations under various pathological conditions remain largely elusive. Here, we employed a label-free proteomic approach to characterize the diversity of the hepatic macrophage pool in an experimental model of CCl4-induced liver fibrosis. We found a decrease in the proportion of liver resident embryo-derived KCs (EmKCs), and a drastic increase in the proportion of monocyte-derived KCs (MoKCs) and CLEC2-Macs. Proteomic profiling revealed that MoKCs largely resembled EmKCs, whereas CLEC2-Macs exhibited greater proteomic alternations compared with EmKCs, suggesting two distinct destinations for monocyte differentiation during liver fibrosis. Furthermore, CLEC2-Macs were characterized by increased expression of proteins associated with inflammatory response, antigen processing and presentation processes, which may be involved in the pathogenesis of liver fibrosis. Collectively, our study provides insights into the considerable heterogeneity within the hepatic macrophage pool during liver fibrosis.

Substrate stiffness-dependent activation of Hippo pathway in cancer associated fibroblasts

The tumor microenvironment (TME) comprises a heterogenous cell population within a complex three-dimensional (3D) extracellular matrix (ECM). Stromal cells within this TME are altered by signaling cues from cancer cells to support uncontrolled tumor growth and invasion events. Moreover, the ECM also plays a fundamental role in tumor development through pathological remodeling, stiffening and interaction with TME cells. In healthy tissues, Hippo signaling pathway actively contributes to tissue growth, cell proliferation and apoptosis. However, in cancer, the Hippo signaling pathway is highly dysregulated, leading to nuclear translocation of the YAP/TAZ complex, which directly contributes to uncontrolled cell proliferation and tissue growth, and ECM remodeling and stiffening processes. Here, we compare the effect of increasing cell culture substrate stiffness, derived from tumor progression, upon the dysregulation of the Hippo signaling pathway in colorectal cancer-associated fibroblasts (CAFs) and normal colorectal fibroblasts (NFs). We correlate the dysregulation of Hippo pathway with the magnitude of the traction forces exerted by healthy and malignant stromal cells. We found that ECM stiffening is crucial in Hippo pathway dysregulation in CAFs, but not in normal fibroblasts.

BIT: Bayesian Identification of Transcriptional Regulators from Epigenomics-Based Query Region Sets.

Transcriptional regulators (TRs) are master controllers of gene expression and play a critical role in both normal tissue development and disease progression. However, existing computational methods for identification of TRs regulating specific biological processes have significant limitations, such as relying on distance on a linear chromosome or binding motifs that have low specificity. Many also use statistical tests in ways that lack interpretability and rigorous confidence measures. We introduce BIT, a novel Bayesian hierarchical model for in-silico TR identification. Leveraging a comprehensive library of TR ChIP-seq data, BIT offers a fully integrated Bayesian approach to assess genome-wide consistency between user-provided epigenomic profiling data and the TR binding library, enabling the identification of critical TRs while quantifying uncertainty. It avoids estimation and inference in a sequential manner or numerous isolated statistical tests, thereby enhancing accuracy and interpretability. BIT successfully identified critical TRs in perturbation experiments, functionally essential TRs in various cancer types, and cell-type-specific TRs within heterogeneous cell populations, offering deeper biological insights into transcriptional regulation.

Spatiotemporal spread of oncolytic virus in a heterogeneous cell population

Oncolytic (cancer-killing) virus treatment is a promising new therapy for cancer, with many viruses currently being tested for their ability to eradicate tumors. One of the major stumbling blocks to the development of this treatment modality has been preventing spread of the virus to non-cancerous cells. Our recent ability to manipulate RNA and DNA now allows for the possibility of creating designer viruses specifically targeted to cancer cells, thereby significantly reducing unwanted side effects in patients. In this study, we use a partial differential equation model to determine the characteristics of a virus needed to contain spread of an oncolytic virus within a spherical tumor and prevent it from spreading to non-cancerous cells outside the tumor. We find that oncolytic viruses that have different infection rates or different cell death rates in cancer and non-cancerous cells can be made to stay within the tumor. We find that there is a minimum difference in infection rates or cell death rates that will contain the virus and that this threshold value depends on the growth rate of the cancer. Identification of these types of thresholds can help researchers develop safer strains of oncolytic viruses allowing further development of this promising treatment.

Mechanisms of Germline Stem Cell Competition across Species.

In this review, we introduce the concept of cell competition, which occurs between heterogeneous neighboring cell populations. Cells with higher relative fitness become "winners" that outcompete cells of lower relative fitness ("losers"). We discuss the idea of super-competitors, mutant cells that expand at the expense of wild-type cells. Work on adult stem cells (ASCs) has revealed principles of neutral competition, wherein ASCs can be stochastically lost and replaced, and of biased competition, in which a winning ASC with a competitive advantage replaces its neighbors. Germline stem cells (GSCs) are ASCs that are uniquely endowed with the ability to produce gametes and, therefore, impact the next generation. Mechanisms of GSC competition have been elucidated by studies in Drosophila gonads, tunicates, and the mammalian testis. Competition between ASCs is thought to underlie various forms of cancer, including spermatocytic tumors in the human testis. Paternal age effect (PAE) disorders are caused by de novo mutations in human GSCs that increase their competitive ability and make them more likely to be inherited, leading to skeletal and craniofacial abnormalities in offspring. Given its widespread effects on human health, it is important to study GSC competition to elucidate how cells can become winners or losers.

Microglia and monocyte-derived macrophages drive progression of pediatric high-grade gliomas and are transcriptionally shaped by histone mutations

Pediatric high-grade gliomas (pHGGs), including hemispheric pHGGs and diffuse midline gliomas (DMGs), harbor mutually exclusive tumor location-specific histone mutations. Using immunocompetent de novo mouse models of pHGGs, we demonstrated that myeloid cells were the predominant infiltrating non-neoplastic cell population. Single-cell RNA sequencing (scRNA-seq), flow cytometry, and immunohistochemistry illustrated the presence of heterogeneous myeloid cell populations shaped by histone mutations and tumor location. Disease-associated myeloid (DAM) cell phenotypes demonstrating immune permissive characteristics were identified in murine and human pHGG samples. H3.3K27M DMGs, the most aggressive DMG, demonstrated enrichment of DAMs. Genetic ablation of chemokines Ccl8 and Ccl12 resulted in a reduction of DAMs and an increase in lymphocyte infiltration, leading to increased survival of tumor-bearing mice. Pharmacologic inhibition of chemokine receptors CCR1 and CCR5 resulted in extended survival and decreased myeloid cell infiltration. This work establishes the tumor-promoting role of myeloid cells in DMG and the potential therapeutic opportunities for targeting them.