Distinct Subpopulations Of Tumor Cells Research Articles

P52-ZER6/IGF1R axis maintains cancer stem cell population to promote cancer progression by enhancing pro-survival mitophagy.

Cancer stem cells (CSCs), which are distinct subpopulations of tumor cells, have a substantially higher tumor-initiating capacity and are closely related to poor clinical outcomes. Damage to organelles can trigger CSC pool exhaustion; however, the underlying mechanisms are poorly understood. ZER6 is a zinc-finger protein with two isoforms possessing different amino termini: p52-ZER6 and p71-ZER6. Since their discovery, almost no study reported on their biological and pathological functions. Herein, we found that p52-ZER6 was crucial for CSC population maintenance; p52-ZER6-knocking down almost abolished the tumor initiation capability. Through transcriptomic analyses together with in vitro and in vivo studies, we identified insulin like growth factor 1 receptor (IGF1R) as the transcriptional target of p52-ZER6 that mediated p52-ZER6 regulation of CSC by promoting pro-survival mitophagy. Moreover, this regulation of mitophagy-mediated CSC population maintenance is specific to p52-ZER6, as p71-ZER6 failed to exert the same effect, most possibly due to the presence of the HUB1 domain at its N-terminus. These results provide a new perspective on the regulatory pathway of pro-survival mitophagy in tumor cells and the molecular mechanism underlying p52-ZER6 oncogenic activity, suggesting that targeting p52-ZER6/IGF1R axis to induce CSC pool exhaustion may be a promising anti-tumor therapeutic strategy.

Abstract PR-006: Brain metastases roadmap reveals conserved regulatory dynamics rooted in dormant tumor cells

Abstract Capturing comprehensive brain metastasis landscape is critical to the establishment of effective anti-tumor strategies. This study took advantage of single cell multiomics sequencing to profile the molecular and cellular dynamics in tumor cells and associated microenvironment during brain metastasis. Among different primary cancer types, our data suggest that there are conserved yet distinct tumor cell subpopulations, governed by specific changes in gene expression, chromatin accessibility and tumor-stroma interactions. We characterized the conserved tumor subpopulations of brain metastasis with features of: (1) dormancy, (2) enhanced translation and migration, (3) epithelial to mesenchymal transition and (4) high proliferation rate. In line with previous observations, dormant cells exhibit distinct stimulation for interferon and oxidative phosphorylation. Furthermore, our data suggest that dormant tumor cells increase amino acids metabolism and antigen presentation by upregulating HLA class molecules. Stromal and tumor cells interaction network analysis predicted most active ligands that might shape intratumoral heterogeneity. For example, TGFβ signaling induced by immune cells and astrocytes is associated with tumor cell epithelial to mesenchymal transition. Additionally, results suggest the vascular endothelial niche supports brain metastasis proliferation. Interestingly, dormant tumor cells appear to limit cell-cell interactions with the surrounding microenvironment when compared to other tumor cells subpopulations. Despite pronounced cell plasticity, trajectory analysis uncovered a cell progression pattern and its major sequential states and driving genes. Notably, the trajectory embarked with dormant tumor cells at its root and ended with a prevalence of rapidly proliferating tumor cells. As cycling cancer cells arise, we observed a disruption in circadian rhythm regulators activities, indicating that altered circadian regulation may contribute to brain metastasis progression. This work demonstrated that despite a pronounced intratumoral heterogeneity, brain metastases present conserved tumor subpopulations regardless of the patient and primary type. Importantly, the tumor trajectory provides new insights into the regulatory landscape controlling the transition from dormant to proliferating tumor cells. A novel and key finding in this study is the multiomics characterization of the regulatory landscape shaping human brain metastases. Thus, we believe further understanding of these key regulatory elements could lead to novel tumor targets with broad translational potential. Citation Format: Remi Klotz, Yiru Jess Wang, Alexis Zukowski, Frank Attenelo, Srinivas Ramachandran, Min Yu. Brain metastases roadmap reveals conserved regulatory dynamics rooted in dormant tumor cells [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr PR-006.

A Microfluidic Liquid Biopsy Platform to Monitor Protein Biomarker Heterogeneity in Single Circulating Therapy‐Resistance Cancer Cell

AbstractTumor cells display heterogenous molecular signatures during the course of cancer and create distinct tumor cell subpopulations which challenge effective therapeutic decisions. Detection and monitoring of these heterogenous molecular events at single cell level are imperative to identify tumor cell subpopulations and to engage the best therapeutic options for the individual patient. Herein, a microfluidic liquid biopsy platform to analyze circulating tumor cells (CTCs) at single cell level is reported. The individual CTCs are captured in an alternating current‐induced microfluidic platform and analyzed by using surface‐enhanced Raman scattering spectroscopy. This platform selectively captures single CTCs from the patient's peripheral blood mononuclear cells. Using cell line models and patient samples, it is shown that the assay can simultaneously detect multiple protein biomarkers on a single CTC. The platform can stratify the CTCs into different subpopulations based on their cancer‐associated protein signature changes in response to drug treatment. This enables the identification of CTC subpopulations that are probably not responding to treatment and may assist clinicians in specifically monitoring and eliminating therapy‐resistant cancer cells within a lesion. This single CTC monitoring chip will likely have high clinical importance in disease diagnosis and treatment monitoring, and advance the knowledge of cancer heterogeneity.

Resolving the Pathogenesis of Anaplastic Wilms Tumors through Spatial Mapping of Cancer Cell Evolution.

While patients with intermediate-risk (IR) Wilms tumors now have an overall survival (OS) rate of almost 90%, those affected by high-stage tumors with diffuse anaplasia have an OS of only around 50%. We here identify key events in the pathogenesis of diffuse anaplasia by mapping cancer cell evolution over anatomic space in Wilms tumors. We spatially mapped subclonal landscapes in a retrospective cohort of 20 Wilms tumors using high-resolution copy-number profiling and TP53 mutation analysis followed by clonal deconvolution and phylogenetic reconstruction. Tumor whole-mount sections (WMS) were utilized to characterize the distribution of subclones across anatomically distinct tumor compartments. Compared with non-diffuse anaplasia Wilms tumors, tumors with diffuse anaplasia showed a significantly higher number of genetically distinct tumor cell subpopulations and more complex phylogenetic trees, including high levels of phylogenetic species richness, divergence, and irregularity. All regions with classical anaplasia showed TP53 alterations. TP53 mutations were frequently followed by saltatory evolution and parallel loss of the remaining wild-type (WT) allele in different regions. Morphologic features of anaplasia increased with copy-number aberration (CNA) burden and regressive features. Compartments demarcated by fibrous septae or necrosis/regression were frequently (73%) associated with the emergence of new clonal CNAs, although clonal sweeps were rare within these compartments. Wilms tumors with diffuse anaplasia display significantly more complex phylogenies compared with non-diffuse anaplasia Wilms tumors, including features of saltatory and parallel evolution. The subclonal landscape of individual tumors was constrained by anatomic compartments, which should be considered when sampling tissue for precision diagnostics.

Abstract 1242: Single cell multiomic map reveals regulatory landscape of human brain metastases

Abstract Capturing comprehensive brain metastasis landscape is critical to the establishment of effective anti-tumor strategies. This study took advantage of single-cell multiomics sequencing to profile the molecular and cellular dynamics in tumor cells and associated microenvironment during brain metastasis. Among different primary cancer types, our data suggest that there are conserved yet distinct tumor cell subpopulations, governed by specific changes in gene expression, chromatin accessibility and tumor-stroma interactions. We characterized the conserved tumor subpopulations of brain metastasis with features of: (1) dormancy, (2) enhanced translation and migration, (3) cell death and (4) high proliferation rate. Dormant tumor cells in brain metastasis appear to limit cell-cell interactions with the surrounding microenvironment. In line with previous observations, dormant cells exhibit distinct stimulation for interferon, oxidative phosphorylation and HIPPO signaling. Despite a pronounced cell plasticity, trajectory analysis uncovered a cell progression pattern and its major sequential states and driving genes. Importantly, the tumor trajectory provides new insights into the regulatory landscape controlling the transition from dormant to proliferating tumor cells. Our findings also point to a valuable use of matched isolated circulating tumor cells and cell-free DNA to map the molecular and cellular dynamics of brain metastases. This work demonstrated that despite a pronounced intratumoral heterogeneity, brain metastases present conserved tumor subpopulations regardless of the patient and primary type. A novel and key finding in this study is the multiomics characterization of the regulatory landscape shaping human brain metatastases. Thus, we believe further understanding of these key regulatory elements could lead to novel tumor targets with broad translational potential. Citation Format: Remi Klotz, Alexis Zukowski, Mohamed Kamal, Frank Attenelo, Srinivas Ramachandran, Min Yu. Single cell multiomic map reveals regulatory landscape of human brain metastases [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 1242.

Abstract 3531: Discrete extracellular matrix-secreting tumor cell subpopulations remodel the Ewing sarcoma tumor microenvironment to promote invasion

Abstract Ewing sarcoma (EwS) cells exist along a neuro-mesenchymal transcriptional continuum that is largely determined by transcriptional activity of the EWS::FLI1 fusion oncoprotein. Although EWS::FLI1-low state cells are more metastatic in experimental models, the role of these cells in established EwS tumor ecosystems is unknown. Here we have leveraged multimodal single-cell sequencing, spatial transcriptomics, and immunohistochemical profiling to characterize EwS cell subpopulations in cell lines, PDX models, and patient tumors. We identify CD73 as a marker of mesenchymal-high state EwS cells and show that CD73+ tumor cells share properties of experimentally-induced EWS::FLI1-low cells including altered cytoskeletons, enhanced migration and invasion, and increased expression of EWS::FLI1-repressed genes. However, CD73+ cells retain proliferative capacity and do not down-regulate the fusion or the EWS::FLI1-activated signature. Instead, CD73+ EwS cells selectively express a gene signature that is enriched in extracellular matrix (ECM) genes. Examination of xenograft and EwS patient biopsies confirms the existence of subpopulations of spatially and transcriptionally distinct ECM-producing tumor cells and shows them to be more prevalent in infiltrating regions. Importantly, the local tumor microenvironment (TME) surrounding these cells is rich in tumor-derived TNC, SPARC, and biglycan, ECM proteins that have been widely implicated in cancer metastatic progression. Significantly, CD73+ EwS cells also upregulate expression of ECM-sensing genes, including ITGA5 and ITGB1, suggesting potential to activate autocrine feedback loops. In support of this, interrupting outside-in ECM:integrin signaling cascades, through pharmacologic inhibition of FAK or SRC pathways, strongly reduces the invasive properties of CD73+ tumor spheroids in 3D collagen. Together these studies confirm the presence and functional importance of transcriptionally distinct tumor cell subpopulations in established EwS tumor ecosystems. In particular, our data reveal that highly mesenchymal tumor cells both generate and respond to pro-metastatic ECM proteins to support invasion. Studies are ongoing to determine the role of tumor cell-derived ECM on EwS progression and treatment resistance. Citation Format: Emma D. Wrenn, April A. Apfelbaum, Shireen Ganapathi, Erin R. Rudzinski, Xuemei Deng, Nicolas Garcia, Katherine Braun, Trisha Lipson, Shruti Bhise, Sami B. Kanaan, Olivia Waltner, Erika Newman, Scott Furlan, Elizabeth R. Lawlor. Discrete extracellular matrix-secreting tumor cell subpopulations remodel the Ewing sarcoma tumor microenvironment to promote invasion. [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 3531.

Single-cell transcriptomics uncovers cellular architecture and developmental trajectories in hepatoblastoma.

Hepatoblastoma (HB) is the predominant type of childhood liver cancer. Treatment options for the clinically advanced HB remain limited. We aimed to dissect the cellular and molecular basis underlying HB oncogenesis and heterogeneity at the single-cell level, which could facilitate a better understanding of HB at both the biological and clinical levels. Single-cell transcriptome profiling of tumor and paired distal liver tissue samples from five patients with HB was performed. Deconvolution analysis was used for integrating the single-cell transcriptomic profiles with the bulk transcriptomes of our HB cohort of post-neoadjuvant chemotherapy tumor samples. A single-cell transcriptomic landscape of early human liver parenchymal development was established for exploring the cellular root and hierarchy of HB oncogenesis. As a result, seven distinct tumor cell subpopulations were annotated, and an effective HB subtyping method was established based on their compositions. A HB tumor cell hierarchy was further revealed to not only fit with the classical cancer stem cell (CSC) model but also mirror the early human liver parenchymal development. Moreover, FACT inhibition, which could disrupt the oncogenic positive feedback loop between MYC and SSRP1 in HB, was identified as a promising epigenetic-targeted therapeutic strategy against the CSC-like HB1-Pro-like1 subpopulation and its related high-risk "Pro-like1" subtype of HB. Our findings illustrate the cellular architecture and developmental trajectories of HB via integrative bulk and single-cell transcriptome analyses, thus establishing a resourceful framework for the development of targeted diagnostics and therapeutics in the future.

Computational quantification and characterization of independently evolving cellular subpopulations within tumors is critical to inhibit anti-cancer therapy resistance

BackgroundDrug resistance continues to be a major limiting factor across diverse anti-cancer therapies. Contributing to the complexity of this challenge is cancer plasticity, in which one cancer subtype switches to another in response to treatment, for example, triple-negative breast cancer (TNBC) to Her2-positive breast cancer. For optimal treatment outcomes, accurate tumor diagnosis and subsequent therapeutic decisions are vital. This study assessed a novel approach to characterize treatment-induced evolutionary changes of distinct tumor cell subpopulations to identify and therapeutically exploit anticancer drug resistance.MethodsIn this research, an information-theoretic single-cell quantification strategy was developed to provide a high-resolution and individualized assessment of tumor composition for a customized treatment approach.Briefly, this single-cell quantification strategy computes cell barcodes based on at least 100,000 tumor cells from each experiment and reveals a cell-specific signaling signature (CSSS) composed of a set of ongoing processes in each cell.ResultsUsing these CSSS-based barcodes, distinct subpopulations evolving within the tumor in response to an outside influence, like anticancer treatments, were revealed and mapped. Barcodes were further applied to assign targeted drug combinations to each individual tumor to optimize tumor response to therapy.The strategy was validated using TNBC models and patient-derived tumors known to switch phenotypes in response to radiotherapy (RT).ConclusionsWe show that a barcode-guided targeted drug cocktail significantly enhances tumor response to RT and prevents regrowth of once-resistant tumors. The strategy presented herein shows promise in preventing cancer treatment resistance, with significant applicability in clinical use.

DIPG-39. New preclinical models for Diffuse Midline Glioma

Malignant brain tumors are the leading cause of childhood death in Germany, with Diffuse Midline Glioma (DMG) being the most lethal of all paediatric brain tumors. Current treatment strategies are limited to irradiation which prolongs survival only by a few months. Preclinical studies have identified effective drug candidates, but translation into the clinic remains a major obstacle. It is known that interactions between tumor cells and components of the TME (tumor microenvironment), such as cell to cell contacts between malignant and non-malignant cells or secreted factors, can increase therapy resistance and progression of brain tumors. However, these important factors are not present in most conventional cell culture models for drug testing. Consequently, there is a need for more realistic DMG models to improve the relevance and translational potential of current drug screening. Therefore, the goal of this study was to develop a new DMG model for drug testing, consisting of induced pluripotent stem cell (iPSC) derived human brain cells and patient derived DMG cells to better mimic the complex tumor microenvironment. We co-cultured three-dimensional cerebral organoids with DMG tumor spheres resulting in the formation of DMG-Brain-Organoids (DBO). Preliminary results show that co-culture induces distinct tumor cell subpopulations corresponding to those detected in DMG tumors by single cell RNA sequencing (Filbin et al., 2018). These subpopulations mainly differ in their proliferative capacity and their differential response to clinical interventions may be critical for therapeutic success. DBOs subjected to drug treatments (single or combination) were sectioned and individual therapy effects on tumor cell subpopulations and proliferative capacity were monitored using multiplexed immunofluorescence imaging. By observing drug effects in a realistic setup, we hope to improve the predictive power of our preclinical drug screens and to find new combination therapies for DMG.

MEDB-44. Transcriptomic resolution of subgroup-specific medulloblastoma architecture

Despite a growing understanding and stratification of medulloblastoma, it remains an aggressive childhood brain tumor with high morbidity and mortality. Multimodal genomic and epigenomic analysis has permitted the classification of medulloblastoma into four subgroups with varying biology and clinical behavior: WNT, Sonic-Hedgehog (SHH), Group 3, and Group 4. In our previously published work, Single-cell RNA sequencing (scRNAseq) identified distinct tumor cell subpopulations in specific medulloblastoma groups. However, this technology is limited by its lack of architectural information. Spatial transcriptomics is a relatively new technology that permits the analysis of gene expression as it occurs within organized tissue. In our ongoing study, we utilized Visium spatial transcriptomics, integrated with scRNAseq data and immunohistochemistry, to analyze frozen samples of medulloblastomas (SHH, Group 4, and Group 3 with and without MYC amplification). In SHH in particular, we were able to identify scRNAseq populations within the geographically constricted Visium data, including SHH-C2, a population located in histologic nodules, the predominant neuronal-differentiated population SHH-C1, and progenitor populations (SHH-B1 and B2). In addition, we were able to visualize clusters not detectable by scRNAseq – a cluster lining nodules with expression of vascular endothelium marker, reticulin and M2-macrophage genes, and a novel DNA-repair cluster. In addition, Visium data permits the spatial constraint of proliferating cells, which is frequently problematic in scRNAseq, as dividing cells cluster independently. The proliferation is highest in the SHH-B2 minor progenitor population, absent in the SHH-C1 major differentiated population, and is moderate in other population including the SHH-C2 nodules. Group 3 and 4 medulloblastoma are more complex but show preliminary corroboration with scRNAseq data. In summary, Visium allows us to map subpopulations identified by scRNAseq to tumor architecture more definitively and rapidly than IHC. These novel insights advance our understanding of medulloblastoma, a critical step in improving treatment options for children with this disease.

Single-Cell Transcriptomic Analysis Revealed a Critical Role of SPP1/CD44-Mediated Crosstalk Between Macrophages and Cancer Cells in Glioma.

High-grade glioma is one of the most lethal human cancers characterized by extensive tumor heterogeneity. In order to identify cellular and molecular mechanisms that drive tumor heterogeneity of this lethal disease, we performed single-cell RNA sequencing analysis of one high-grade glioma. Accordingly, we analyzed the individual cellular components in the ecosystem of this tumor. We found that tumor-associated macrophages are predominant in the immune microenvironment. Furthermore, we identified five distinct subpopulations of tumor cells, including one cycling, two OPC/NPC-like and two MES-like cell subpopulations. Moreover, we revealed the evolutionary transition from the cycling to OPC/NPC-like and MES-like cells by trajectory analysis. Importantly, we found that SPP1/CD44 interaction plays a critical role in macrophage-mediated activation of MES-like cells by exploring the cell-cell communication among all cellular components in the tumor ecosystem. Finally, we showed that high expression levels of both SPP1 and CD44 correlate with an increased infiltration of macrophages and poor prognosis of glioma patients. Taken together, this study provided a single-cell atlas of one high-grade glioma and revealed a critical role of macrophage-mediated SPP1/CD44 signaling in glioma progression, indicating that the SPP1/CD44 axis is a potential target for glioma treatment.

Ferroptosis: Cancer Stem Cells Rely on Iron until "to Die for" It.

Cancer stem cells (CSCs) are a distinct subpopulation of tumor cells with stem cell-like features. Able to initiate and sustain tumor growth and mostly resistant to anti-cancer therapies, they are thought responsible for tumor recurrence and metastasis. Recent accumulated evidence supports that iron metabolism with the recent discovery of ferroptosis constitutes a promising new lead in the field of anti-CSC therapeutic strategies. Indeed, iron uptake, efflux, storage and regulation pathways are all over-engaged in the tumor microenvironment suggesting that the reprogramming of iron metabolism is a crucial occurrence in tumor cell survival. In particular, recent studies have highlighted the importance of iron metabolism in the maintenance of CSCs. Furthermore, the high concentration of iron found in CSCs, as compared to non-CSCs, underlines their iron addiction. In line with this, if iron is an essential macronutrient that is nevertheless highly reactive, it represents their Achilles’ heel by inducing ferroptosis cell death and therefore providing opportunities to target CSCs. In this review, we first summarize our current understanding of iron metabolism and its regulation in CSCs. Then, we provide an overview of the current knowledge of ferroptosis and discuss the role of autophagy in the (regulation of) ferroptotic pathways. Finally, we discuss the potential therapeutic strategies that could be used for inducing ferroptosis in CSCs to treat cancer.

Targeting CDK4 overcomes EMT-mediated tumor heterogeneity and therapeutic resistance in KRAS-mutant lung cancer.

Lack of sustained response to therapeutic agents in patients with KRAS-mutant lung cancer poses a major challenge and arises partly due to intratumor heterogeneity that defines phenotypically distinct tumor subpopulations. To attain better therapeutic outcomes, it is important to understand the differential therapeutic sensitivities of tumor cell subsets. Epithelial-mesenchymal transition is a biological phenomenon that can alter the state of cells along a phenotypic spectrum and cause transcriptional rewiring to produce distinct tumor cell subpopulations. We utilized functional shRNA screens, in in vitro and in vivo models, to identify and validate an increased dependence of mesenchymal tumor cells on cyclin-dependent kinase 4 (CDK4) for survival, as well as a mechanism of resistance to MEK inhibitors. High zinc finger E-box binding homeobox 1 levels in mesenchymal tumor cells repressed p21, leading to perturbed CDK4 pathway activity. Increased dependence on CDK4 rendered mesenchymal cancer cells particularly vulnerable to selective CDK4 inhibitors. Coadministration of CDK4 and MEK inhibitors in heterogeneous tumors effectively targeted different tumor subpopulations, subverting the resistance to either single-agent treatment.

Therapeutic and prognostic implications of NOTCH and MAPK signaling in bladder cancer.

Signaling pathways that drive bladder cancer (BC) progression may be promising and specific targets for systemic therapy. Here, we investigated the clinical significance and targetability of NOTCH and mitogen‐activated protein kinase (MAPK) signaling for this aggressive malignancy. We assessed NOTCH1 and MAPK activity in 222 stage III and IV BC specimens of patients that had undergone radical cystectomy, and tested for clinical associations including cancer‐specific and overall survival. We examined therapeutic effects of NOTCH and MAPK repression in a murine xenograft model of human bladder cancer cells and evaluated tumor growth and tumor cell plasticity. In BC, NOTCH1 and MAPK signaling marked two distinct tumor cell subpopulations. The combination of high NOTCH1 and high MAPK activity indicated poor cancer‐specific and overall survival in univariate and multivariate analyses. Inhibition of NOTCH and MAPK in BC xenografts in vivo depleted targeted tumor cell subpopulations and revealed strong plasticity in signaling pathway activity. Combinatorial inhibition of NOTCH and MAPK signaling most strongly suppressed tumor growth. Our findings indicate that tumor cell subpopulations with high NOTCH and MAPK activity both contribute to tumor progression. Furthermore, we propose a new concept for BC therapy, which advocates specific and simultaneous targeting of these different tumor cell subpopulations through combined NOTCH and MAPK inhibition.

HGG-53. PROJECT HOPE: “PEDIATRIC AND AYA HIGH-GRADE GLIOMA OMICS PROJECT”- A LONGITUDINAL MOLECULAR LANDSCAPE OF HIGH-GRADE GLIOMAS RESOLVED AT SINGLE-CELL LEVEL

High-grade gliomas (HGG) are among the most prevalent and fatal cancers in pediatric, adolescent, and young adult (AYA) patients. Especially understudied are older children and young adults, aged 16–39 years. Previously, we profiled primary pediatric HGGs through single-cell transcriptomics and identified the genetic, epigenetic and developmental programs that drive their malignant progression. However, the questions of how these programs compare to those in older HGG patients, what the mechanisms are by which these tumors ultimately evolve to drive recurrence and treatment resistance, and how distinct tumor cell subpopulations bidirectionally communicate with their microenvironment remain to be elucidated. Here, we use single-nucleus RNA sequencing to compare 11 paired, matched high-grade gliomas at diagnosis and recurrence and 15 additional H3K27M primary and recurrent DMG samples in pediatric and AYA patients. In all tumors, we find both undifferentiated and differentiated tumor cells recapitulating distinct glial lineages, as well as diverse microenvironmental cell populations. When longitudinally comparing this tumor architecture within matched pairs, we find substantial differences in transcriptional program expressions. Diagnostic samples include more differentiated, astrocyte-like tumor cells, while cells from recurrent samples more highly express ribosomal and heat-shock protein genes, suggesting tumor progression- and treatment-related shifts. Ongoing sequencing and analysis will allow for unprecedented insight into the evolutionary dynamics of pediatric and AYA high-grade gliomas as well as delineate differences in the biology of DMGs occurring in different age groups. This multi-institutional project was funded by the National Institute of Health.

EPCO-26. PROJECT HOPE: “PEDIATRIC AND AYA HIGH-GRADE GLIOMA OMICS PROJECT”- A LONGITUDINAL MOLECULAR LANDSCAPE OF HIGH-GRADE GLIOMAS RESOLVED AT SINGLE-CELL LEVEL

Abstract High-grade gliomas (HGG) are among the most prevalent and fatal cancers in pediatric, adolescent, and young adult (AYA) patients. Especially understudied are older children and young adults, aged 16–39 years. Previously, we profiled primary pediatric HGGs through single-cell transcriptomics and identified the genetic, epigenetic and developmental programs that drive their malignant progression. However, the questions of how these programs compare to those in older HGG patients, what the mechanisms are by which these tumors ultimately evolve to drive recurrence and treatment resistance, and how distinct tumor cell subpopulations bidirectionally communicate with their microenvironment remain to be elucidated. In order to investigate these questions, we use single-nucleus RNA sequencing to compare 11 paired, matched high-grade gliomas at diagnosis and recurrence and 15 additional H3K27M primary and recurrent DMG samples in pediatric and AYA patients. In all tumors, we find both undifferentiated and differentiated tumor cells recapitulating distinct glial lineages, as well as diverse microenvironmental cell populations. When longitudinally comparing this tumor architecture within matched pairs, we find substantial differences in transcriptional program expressions. In particular, recurrent samples showed a higher proportion of cells expressing heat- shock proteins (HSPs) and a novel cancer cell program characterized by synaptic formation and neurotransmitter secretory processes, suggesting tumor progression- and treatment-related shifts. Ongoing sequencing and analysis will allow for unprecedented insight into the evolutionary dynamics of pediatric and AYA high-grade gliomas as well as delineate differences in the biology of DMGs occurring in different age groups. This multi-institutional project was funded by the National Institute of Health.

Abstract B02: Dissecting tumor cell heterogeneity to identify therapeutic vulnerabilities in Kras-mutant lung cancer

Abstract Lung cancer is the foremost cause of cancer-related deaths in the U.S. KRAS mutations are one of the most frequent oncogenic aberrations in lung adenocarcinoma patients. Despite harboring identical mutations, the response of distinct tumor cells to therapeutic agents varies due to epigenetic reprogramming of the tumor cells. Epithelial-mesenchymal transition (EMT) contributes to phenotypic heterogeneity and determines the response of tumor cells to therapeutic agents. Kras-mutant NSCLC cells have been shown to acquire EMT as a mechanism to evade therapeutic targeting. We have previously observed that murine and human epithelial lung cancer cells are highly sensitive to MEK inhibition whereas mesenchymal tumor cells remain largely resistant. We aim to identify the mechanisms conferring survival advantage and an intrinsic resistance to MEK inhibition to mesenchymal cells. Using a loss-of-function shRNA dropout screen for targets with FDA-approved drugs, we identified cyclin dependent kinase 4 (CDK4) as an important driver of growth in mesenchymal cells both in vitro and in vivo. Murine NSCLC cells are resistant to cell cycle arrest induced by serum starvation, which corresponds to higher protein levels of CDK4 and cyclin D1 allowing cells to transition from G1 to S phase. After release of cells from arrested state, the mesenchymal cells transition into S phase more rapidly than the epithelial cells, suggesting a higher dependency on CDK4-cyclin D1 complex of mesenchymal tumor cells. An Ex Vivo Tumor (EVT) system to culture lung tumors in a 3D matrix environment that retains tumor cell heterogeneity was utilized to test the therapeutic sensitivity of distinct tumor cell subpopulations. In response to treatment with a CDK4 inhibitor, we observed a depletion of mesenchymal tumor cells within heterogeneous EVTs. In combination with MEK inhibitor treatment, there was a net decrease in size and viability of EVTs. Single-agent treatment with CDK4 inhibitor upregulated pErk in mesenchymal tumor cells, increasing their sensitivity to MEK inhibition and achieving an additive response with combination treatment. Mechanistically, the effects are mediated by FOXM1, a transcriptional regulator of cell cycle. FOXM1 positively regulates CDK4-cyclin D1 activity and is negatively regulated by the miR-200 family, which is preferentially expressed in epithelial cells. Upon induction of miR-200 expression, there was a decrease in FOXM1. CDK4, cyclin D1 and pRB levels follow a similar trend, suggesting the phenotype observed in mesenchymal cells is mediated by FOXM1. Since EMT is modulated by the miR-200/Zeb1 axis, higher levels of the transcriptional repressor Zeb1 and consequently FOXM1 in mesenchymal tumor cells confer a survival advantage and an intrinsic resistance to MEK inhibition. Combinatorial treatment approaches with MEK and CDK4 inhibitors targeting different subpopulations within a tumor are an effective strategy to combat resistant outgrowth of epigenetic subsets in a heterogeneous Kras mutant tumor. Citation Format: Aparna Padhye, B. Leticia Rodriguez, Jared J. Fradette, Christin Ungewiss, Don L. Gibbons. Dissecting tumor cell heterogeneity to identify therapeutic vulnerabilities in Kras-mutant lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B02.

Cancer stem cells: A comprehensive review on identification and therapeutic implications.

Cancer stem cells (CSCs) are distinct subpopulations of tumor cells that possess the ability for perpetual self-renewal and proliferation. They produce downstream progenitor cells and cancer cells that drive tumor growth. Studies of many cancer types including oral squamous cell carcinoma (OSCC) have identified CSCs using specific markers, but it is still unclear as to where in the stem cell hierarchy these markers fall. This is compounded further by the presence of multiple CSC subtypes within OSCC, making investigation reliant on the use of multiple markers. This review paper focuses on the current knowledge in CSC markers including OCT4, SOX2, NANOG, aldehyde dehydrogenase 1, CD44, CD24, CD133 and Musashi-1, highlighting their use and validity in OSCC CSC research.

Abstract A132: Characterization of intratumoral heterogeneity in drug sensitivity and non-cell autonomous mechanisms of drug resistance using subclonal cell populations derived from a single breast cancer cell line

Abstract Intratumoral heterogeneity impacts both tumor progression and drug sensitivity. While intratumoral genetic heterogeneity has been extensively described, there is still little understanding of the extent of heterogeneity in drug sensitivity within a single tumor, the intra tumor dynamics of different cell populations during anti-cancer compound treatment, and the role of non-cell autonomous mechanism in drug resistance. The objective of this study is to characterize heterogeneity in drug sensitivity within a model for human breast cancer, identify interactions between clonal population influencing drug sensitivity, and use this to model the dynamics of sub populations in a tumor during anti-cancer compound treatment. We have characterized 31 subclonal cell populations (SCPs) that were generated by single cell cloning of the triple-negative breast cancer cell line MDA-MB-468. These SCPs display considerable phenotypic and molecular heterogeneity with respect to morphology, proliferation rate, RNA expression and tumorigenicity in mice. To examine the extent of heterogeneity in drug sensitivity, we designed and screened a 200-small molecule compound library in 24 SCPs and the parental cell line. We observed considerable heterogeneity in drug sensitivity and selected 36 compounds for validation in the SCPs that expressed the greatest range of sensitivity (determined by IC50). To investigate whether interactions between clonal population can influence drug sensitivity heterogeneously responsive subclonal cell lines were transduced with lentiviruses encoding for the H2B-GFP or H2B-RFP fusion protein and then cocultured in the absence or presence of different compounds. We observed shifts in drug sensitivity for SCPs that were cocultured with differentially responsive clones. Examples include SCPs 19 and 27 two highly sensitive populations that were protected by the presence of less sensitive SCPs when under gemcitabine treatment, resulting in IC50-values that were 20-fold higher then when cultured alone. RNA-sequencing and RPPA analysis will be used to characterize this non-cell autonomous mechanism of resistance and the relationship with identified transcriptional programs or genes which expression correlates with drug sensitivity. We are also generating and will evaluate predictive models for drug combination using the SCP drug sensitivity profiles. The results of this study show that there is considerable heterogeneity in drug sensitivity among clonal subpopulations derived from a single cancer cell line. Ongoing experiments will provide insight into the effect of drug combinations and unique treatment schedules on distinct tumor cell subpopulations and drug sensitivity within heterogenous mixtures. Moreover, we will be able to test for and investigate non-cell autonomous modes of drug resistance, which may guide the development of new drug combination treatments or schedules. Citation Format: Chandler M Friend, Joan S Brugge, Hendrik J Kuiken, Steven M Corsello, Christopher C Mader. Characterization of intratumoral heterogeneity in drug sensitivity and non-cell autonomous mechanisms of drug resistance using subclonal cell populations derived from a single breast cancer cell line [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A132. doi:10.1158/1535-7163.TARG-19-A132

Abstract 3775: Characterization of intratumoral heterogeneity in drug sensitivity and modeling of drug combination effects using subclonal cell populations derived from a single breast cancer cell line

Abstract Intratumoral heterogeneity can have profound effects on tumor progression and drug resistance. While tumor cell heterogeneity has been described at many levels, there is a poor understanding of the extent of heterogeneity in drug sensitivity within a single tumor, the dynamics of tumor cell subpopulations during treatment with anti-cancer agents and non-cell autonomous mechanisms of drug resistance. The overall objective of this study is to characterize heterogeneity in drug sensitivity within a model for human breast cancer and to model and evaluate the effect of drug combinations on the dynamics of subpopulations within heterogeneous mixtures. We have characterized 31 subclonal cell populations (SCPs) that were generated through single cell cloning of the triple-negative breast cancer cell line MDA-MB-468. These SCPs display considerable heterogeneity with respect to morphology, proliferation rate, colony formation in soft agar and tumorigenicity in mice. To examine the extent of heterogeneity in drug sensitivity, we designed and screened a 200-small molecule compound library in 24 SCPs and the parental cell line. We observed considerable heterogeneity in drug sensitivity and selected 36 compounds for validation, including GPX4 inhibitor ML-210 and gemcitabine, to which the SCPs displayed 12-fold and 150-fold differences in the EC50, respectively. The results of these validation experiments were used to identify transcriptional programs and individual genes which expression correlates with drug sensitivity. We have generated and will evaluate predictive models for drug combination using the SCP drug sensitivity profiles. To test the effect of individual drugs and combinations, as well as different treatment schedules on heterogeneous populations, we will examine the dynamics of individual SCPs within mixed cultures. To this end, we have transduced 22 SCPs with CloneTracer lentiviruses, each encoding for a unique DNA barcode. The distribution of these unique barcodes can be determined by next generation sequencing. Previously, we have found that propagation of a 22 SCP mixture results in reproducible changes in the composition of mixed monolayer cultures with approximately half of the SCPs having a relative abundance of 1% each in the mixed population following propagation for four months. The results of this study show that there is considerable heterogeneity in drug sensitivity among clonal subpopulations derived from a single cancer cell line. Ongoing experiments will provide insight into the effect of drug combinations and unique treatment schedules on distinct tumor cell subpopulations within heterogenous mixtures. Moreover, using this panel of SCPs, we will be able to test for and investigate non-cell autonomous modes of drug resistance, which may guide the development of new drug combinations. Citation Format: Hendrik J. Kuiken, Chandler M. Friend, Steven M. Corsello, Christopher C. Mader, Joan S. Brugge. Characterization of intratumoral heterogeneity in drug sensitivity and modeling of drug combination effects using subclonal cell populations derived from a single breast cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3775.