Subpopulation Of Stem-like Cells Research Articles

Current relation between Durante-Conheim theory and radiation resistance. Commentary on “Embryonic stem cell-like subpopulations are present within Schwannoma”

Current relation between Durante-Conheim theory and radiation resistance. Commentary on “Embryonic stem cell-like subpopulations are present within Schwannoma”

Oncolytic herpes simplex virus expressing IL-2 controls glioblastoma growth and improves survival

BackgroundGlioblastoma (GBM), a highly immunosuppressive and often fatal primary brain tumor, lacks effective treatment options. GBMs contain a subpopulation of GBM stem-like cells (GSCs) that play a central role in...

Retraction: Long Non-Coding RNA HOTAIR Regulates the Proliferation, Self-Renewal Capacity, Tumor Formation and Migration of the Cancer Stem-Like Cell (CSC) Subpopulation Enriched from Breast Cancer Cells.

Retraction: Long Non-Coding RNA HOTAIR Regulates the Proliferation, Self-Renewal Capacity, Tumor Formation and Migration of the Cancer Stem-Like Cell (CSC) Subpopulation Enriched from Breast Cancer Cells.

Abstract 4264: The TWEAK-Fn14-NF-kB signaling axis enhances expression of integrin AVB3 in a post-chemotherapy tumor microenvironment

Abstract High grade serous ovarian cancer (HGSOC) patients initially respond to chemotherapy but a majority relapse with chemoresistant disease. Research supports the notion that tumor-initiating cells (TICs), a subpopulation of drug resistant stem-like tumor cells, are responsible for facilitating relapse and therapies targeting these fecund cells may prolong remission. The tumor microenvironment (TME) plays a crucial role in therapy resistance, and tumor relapse. Cytotoxic chemotherapies have been shown to modify the stroma of the TME which may favor the survival of TICs displaying specific adhesion proteins. We and others have shown that NF-kB transcription factors RelA and RelB respond to signals from the ovarian TME to promote proliferation, chemoresistance, and survival. Our results further show that RelA and RelB regulate expression of specific integrins. Recently we found that TNF-like inducer of apoptosis (TWEAK), a cytokine involved in tissue repair, and its only known receptor Fn14, are enriched following chemotherapy, leading to enhanced chemoresistance and survival of TICs. TWEAK also induces expression of specific integrins in ovarian TICs. Therefore, we hypothesize that NF-kB mediated integrin expression enables adhesion and survival of TICs to a chemotherapy modified extracellular matrix (ECM) to facilitate tumor repopulation and relapse. Using magnetic activated cell sorting (MACS) for CD117+ TICs, we found that a significant majority are enriched for integrin AVB3. Supporting a role for TWEAK-Fn14-NF-kB in mediating integrin expression we further show that integrin AVB3, but not AVB5 or A5B1, was significantly increased in CD117+ TICs in the presence of TWEAK and was dependent on Fn14 as demonstrated through knockdown experiments. RNA-sequencing analysis of tumor cells grown in spheroid conditions with RelA or RelB shRNA knockdown relative to control, show ITGAV is dependent on RelA while ITGB3 expression is dependent on RelB. Taken together, our findings suggest CD117+ TICs have enhanced expression of AVB3 and is further induced by TWEAK mediated NF-kB activity for adaptation to a stressful post-chemotherapy environment. Ongoing in vitro and in vivo studies are investigating the therapeutic benefit of targeting AVB3 or TWEAK in CD117+ TICs for prevention of adhesion and tumor repopulation following chemotherapy. These studies will uncover interactions between TICs and ECM through their unique expression of integrins and clarifies the role of NF-kB transcription factors RelA and RelB in regulating TIC survival in the post-chemotherapy TME. Citation Format: Omar Lujano Olazaba, Mikella Robinson, Cecilia Gallo, Mena Shammas, Katelyn Shelby, Luisjesus Cruz, Samuel F. Gilbert, Carrie D. House. The TWEAK-Fn14-NF-kB signaling axis enhances expression of integrin AVB3 in a post-chemotherapy tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4264.

Comprehensive transcriptional atlas of human adenomyosis deciphered by the integration of single-cell RNA-sequencing and spatial transcriptomics.

Adenomyosis is a poorly understood gynecological disorder lacking effective treatments. Controversy persists regarding "invagination" and "metaplasia" theories. The endometrial-myometrial junction (EMJ) connects the endometrium and myometrium and is important for diagnosing and classifying adenomyosis, but its in-depth study is just beginning. Using single-cell RNA sequencing and spatial profiling, we mapped transcriptional alterations across eutopic endometrium, lesions, and EMJ. Within lesions, we identified unique epithelial (LGR5+) and invasive stromal (PKIB+) subpopulations, along with WFDC1+ progenitor cells, supporting a complex interplay between "invagination" and "metaplasia" theories of pathogenesis. Further, we observed endothelial cell heterogeneity and abnormal angiogenic signaling involving VEGF and ANGPT pathways. Cell-cell communication differed markedly between ectopic and eutopic endometrium, with aberrant signaling in lesions involving PTN, TWEAK, and WNT cascades. This study reveals unique stem cell-like and invasive cell subpopulations within adenomyosis lesions identified, dysfunctional signaling, and EMJ abnormalities critical to developing precise diagnostic and therapeutic strategies.

P02.16.B COMPENSATORY CROSS-TALK BETWEEN AUTOPHAGY AND GLYCOLYSIS REGULATES SENESCENCE AND STEMNESS IN HETEROGENEOUS GLIOBLASTOMA TUMOR SUBPOPULATIONS

Abstract BACKGROUND Despite tremendous research efforts, the successful targeting of aberrant tumor metabolism in clinical practice has remained elusive. Tumor heterogeneity and metabolic plasticity may play a role in the clinical failure of metabolism-targeting interventions for treating cancer patients. Moreover, compensatory growth-related processes and adaptive responses exhibited by heterogeneous tumor subpopulations to metabolic inhibitors are poorly understood. Hence, a deeper understanding of the cellular adaptations in response to metabolic interventions is urgently required to develop more effective therapeutic options against deadly glioblastoma (GBM) brain tumors. MATERIAL AND METHODS Using clinically-relevant patient-derived GBM models, we discovered a cross-talk between glycolysis, autophagy, and senescence. Protein and mRNA expression of glycolytic enzymes and stemness markers were measured using western blot and bioinformatic analysis, respectively. We were able to measure senescence with the use of β-galactosidase staining and upregulation of cell cycle regulators, such as p21/CDKN1A and p16/CDKN2A. Autophagy flux and EGFP-MAP1LC3B+ puncta formation analysis was used to determine autophagy induction. RESULTS We found that stem cell-like GBM tumor subpopulations possessed higher basal levels of glycolytic activity and increased expression of several glycolysis-related enzymes including, GLUT1/SLC2A1, PFKP, ALDOA, GAPDH, ENO1, PKM2, and LDH, compared to their non-stem-like counterparts. Importantly, the mRNA expression of glycolytic enzymes positively correlates with stemness markers (CD133/PROM1 and SOX2) in patient GBM tumors. While treatment with glycolysis inhibitors induced senescence in stem cell-like GBM tumor subpopulations, these cells maintained their aggressive stemness features and failed to undergo apoptotic cell death. Moreover, we determined that inhibition of glycolysis led to the induction of autophagy in stem cell-like GBM tumor subpopulations, but not in their non-stem-like counterparts. Similarly, blocking autophagy in stem cell-like GBM tumor subpopulations induced senescence-associated growth arrest without hampering stemness capacity or triggering apoptosis while reciprocally upregulating glycolytic activity. Combinatorial treatment of stem cell-like GBM tumor subpopulations with autophagy and glycolysis inhibitors blocked the induction of senescence while drastically impairing their stemness capacity which drove cells towards apoptotic cell death. CONCLUSION These findings identify a novel and complex compensatory interplay between glycolysis, autophagy, and senescence that helps maintain stemness in heterogeneous GBM tumor subpopulations and provides a survival advantage during metabolic stress. This work was supported by the Cancer Research Society (CRS).

Phenotype-Directed DNA Nanomachine for in Situ Analysis of Stem Cell-like Subpopulation in Breast Cancer

Phenotype-Directed DNA Nanomachine for in Situ Analysis of Stem Cell-like Subpopulation in Breast Cancer

Single-cell sequencing analysis reveals development and differentiation trajectory of Schwann cells manipulated by M. leprae.

M. leprae preferentially infects Schwann cells (SCs) in the peripheral nerves leading to nerve damage and irreversible disability. Knowledge of how M. leprae infects and interacts with host SCs is essential for understanding mechanisms of nerve damage and revealing potential new therapeutic strategies. We performed a time-course single-cell sequencing analysis of SCs infected with M. leprae at different time points, further analyzed the heterogeneity of SCs, subpopulations associated with M. leprae infection, developmental trajectory of SCs and validated by Western blot or flow cytometry. Different subpopulations of SCs exhibiting distinct genetic features and functional enrichments were present. We observed two subpopulations associated with M. leprae infection, a stem cell-like cell subpopulation increased significantly at 24 h but declined by 72 h after M. leprae infection, and an adipocyte-like cell subpopulation, emerged at 72 h post-infection. The results were validated and confirmed that a stem cell-like cell subpopulation was in the early stage of differentiation and could differentiate into an adipocyte-like cell subpopulation. Our results present a systematic time-course analysis of SC heterogeneity after infection by M. leprae at single-cell resolution, provide valuable information to understand the critical biological processes underlying reprogramming and lipid metabolism during M. leprae infection of SCs, and increase understanding of the disease-causing mechanisms at play in leprosy patients as well as revealing potential new therapeutic strategies.

Abstract 5009: Inhibition of TGFBR2 signaling re-sensitizes TMZ-resistant glioblastoma cells to therapy

Abstract Glioblastoma (GBM), the most aggressive and lethal form of brain tumors, recurs in 90% of cases and the standard protocol of care—temozolomide (TMZ)—is an ineffective method to combat chemo-resistant GBM. It is therefore imperative that we identify, develop, and design novel therapeutics to effectively combat recurrent GBM (rGBM). Multi-potent stem-like cell subpopulations within GBM (also referred to as glioma stem cells or GSCs) play important roles in tumor propagation, therapeutic resistance, and recurrence following treatment. Understanding the molecular mechanisms that drive resistance in GSCs can lead to novel therapeutic approaches for rGBM. Bioinformatics analyses of transcriptome data sets from rGBM clinical specimens identified transforming growth factor-beta receptor type 2 (TGFBR2) as a putative driver of resistance and recurrence in GBM. We show that TMZ-resistant cells become more stem-like concurrent with higher expression of TGFBR2 protein and mRNA. Importantly, shRNA-mediated inhibition of TGFBR2 decreases proliferation of TMZ-resistant cells and enhances the effects of TMZ. We hypothesize that by developing targeted therapeutics to reduce TGFBR2 expression we will inhibit the growth capacity and/or re-sensitize rGBM cells to ionizing radiation (IR) or TMZ treatment by simultaneously targeting multiple putative oncogenes implicated in the induction of stem-like resistant GBM cell subsets. Future directions include inhibiting TGFBR2 signaling in clinical specimens derived from rGBM patients using two translatable approaches: (1) ITD1, a small molecule TGFBR2 inhibitor predicted to cross the blood-brain-barrier and (2) microRNAs to target multiple TGFBR2 target genes up-regulated in rGBM. Citation Format: Maya K. Johnson, Jack Korleski, Amanda Johnson, Sophie Sall, John Laterra, Hernando Lopez-Bertoni. Inhibition of TGFBR2 signaling re-sensitizes TMZ-resistant glioblastoma cells to therapy. [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 5009.

A nanotherapeutic approach to selectively eliminate metastatic breast cancer cells by targeting cell surface GRP78.

Here, rational engineering of doxorubicin prodrug loaded peptide-targeted liposomal nanoparticles to selectively target metastatic breast cancer cells in vivo is described. Glucose-regulated protein 78 (GRP78), a heat shock protein typically localized in the endoplasmic reticulum in healthy cells, has been identified to home to the cell surface in certain cancers, and thus has emerged as a promising therapeutic target. Recent reports indicated GRP78 to be expressed on the cell surface of an aggressive subpopulation of stem-like breast cancer cells that exhibit metastatic potential. In this study, a targeted nanoparticle formulation with a GRP78-binding peptide (Kd of 7.4 ± 1.0 μM) was optimized to selectively target this subpopulation. In vitro studies with breast cancer cell lines showed the targeted nanoparticle formulation (TNPGRP78pep) achieved enhanced cellular uptake, while maintaining selectivity over the control groups. In vivo, TNPGRP78pep loaded with doxorubicin prodrug was evaluated using a lung metastatic mouse model and demonstrated inhibition of breast cancer cell seeding to lungs down at the level of negative control groups. Combined, this study established that specific-targeting of surface GRP78 expressing a subpopulation of aggressive breast cancer cells was able to inhibit breast cancer metastasis to lungs, and underpinned the significance of GRP78 in breast cancer metastasis.

Single-cell analysis reveals the chemotherapy-induced cellular reprogramming and novel therapeutic targets in relapsed/refractory acute myeloid leukemia

Chemoresistance and relapse are the leading cause of AML-related deaths. Utilizing single-cell RNA sequencing (scRNA-seq), we dissected the cellular states of bone marrow samples from primary refractory or short-term relapsed AML patients and defined the transcriptional intratumoral heterogeneity. We found that compared to proliferating stem/progenitor-like cells (PSPs), a subpopulation of quiescent stem-like cells (QSCs) were involved in the chemoresistance and poor outcomes of AML. By performing longitudinal scRNA-seq analyses, we demonstrated that PSPs were reprogrammed to obtain a QSC-like expression pattern during chemotherapy in refractory AML patients, characterized by the upregulation of CD52 and LGALS1 expression. Flow cytometric analysis further confirmed that the preexisting CD99+CD49d+CD52+Galectin-1+ (QSCs) cells at diagnosis were associated with chemoresistance, and these cells were further enriched in the residual AML cells of refractory patients. Interaction of CD52-SIGLEC10 between QSCs and monocytes may contribute to immune evading and poor outcomes. Furthermore, we identified that LGALS1 was a promising target for chemoresistant AML, and LGALS1 inhibitor could help eliminate QSCs and enhance the chemotherapy in patient-derived primary AML cells, cell lines, and AML xenograft models. Our results will facilitate a better understanding of the AML chemoresistance mechanism and the development of novel therapeutic strategies for relapsed/refractory AML patients.

Implications of Possible HBV-Driven Regulation of Gene Expression in Stem Cell-like Subpopulation of Huh-7 Hepatocellular Carcinoma Cell Line.

Elevated levels of STIM1, an endoplasmic reticulum Ca2+ sensor/buffering protein, appear to be correlated with poor cancer prognosis in which microRNAs are also known to play critical roles. The purpose of this study is to investigate possible HBV origins of specific microRNAs we identified in a stem cell-like subpopulation of Huh-7 hepatocellular carcinoma (HCC) cell lines with enhanced STIM1 and/or Orai1 expression that mimicked poor cancer prognosis. Computational strategies including phylogenetic analyses were performed on miRNome data we obtained from an EpCAM- and CD133-expressing Huh-7 HCC stem cell-like subpopulation with enhanced STIM1 and/or Orai1 expression originally cultured in the present work. Results revealed two putative regions in the HBV genome based on the apparent clustering pattern of stem loop sequences of microRNAs, including miR3653. Reciprocal analysis of these regions identified critical human genes, of which their transcripts are among the predicted targets of miR3653, which was increased significantly by STIM1 or Orai1 enhancement. Briefly, this study provides phylogenetic evidence for a possible HBV-driven epigenetic remodeling that alters the expression pattern of Ca2+ homeostasis-associated genes in STIM1- or Orai1 overexpressing liver cancer stem-like cells for a possible mutual survival outcome. A novel region on HBV-X protein may affect liver carcinogenesis in a genotype-dependent manner. Therefore, detection of the viral genotype would have a clinical impact on prognosis of HBV-induced liver cancers.

Improved Relapse Prediction in Pediatric Acute Myeloid Leukemia By Deconvolving Lineage-Specific and Cancer-Specific Features in Single-Cell Data

Introduction While most children with acute myeloid leukemia (AML) achieve first remission, nearly 40% will relapse. Of these children, few survive to a second remission even with highly-escalated treatment protocols. Recent studies have shown that many AML patients harbor rare, stem cell-like subpopulations that resist chemotherapy and drive relapse. However, the exact characteristics of these relapse-associated cells are a matter of contention, with reported phenotypes spanning the hematopoietic developmental continuum. In some patients, treatment-resistant cells can be detected as minimal residual disease (MRD), which is often used to predict relapse, albeit with limited accuracy and only after induction chemotherapy. Thus, the identity of treatment-resistant cells as well as their relationship to normal progenitors remain mysterious, thereby limiting the development of targeted therapies for pediatric AML. Here, we present a computational approach for decomposing high-dimensional single-cell measurements into two components: a lineage-specific component that can be used to align cancer cells with specific stages of myeloid development and a cancer-specific component that can be used to identify aberrant phenotypes unique to AML cells. We show that, together, these components can be used at the time of diagnosis to predict relapse more accurately than clinical information alone. Methods and Results Using mass cytometry, we analyzed paired diagnostic and post-induction samples collected from 19 (8 relapse, 11 non-relapse) pediatric patients who enrolled on the Children's Oncology Group trial AAML1031. All patients were treated on the control arm and consented to banking of tissue for research. We also included 5 bone marrow samples from healthy donors. After thawing, samples were divided in half and stimulated with conditioned medium from the human bone marrow stromal cell line HS-5 to activate relevant signaling pathways, or left unstimulated. An average of 5 x 105 cells per patient were analyzed for each condition. The mass cytometry panel included 31 antibodies to surface markers, 6 antibodies to intracellular signaling mediators, and 4 antibodies to intracellular proteins and transcription factors. Following data collection, the singular value decomposition was applied to the data matrix of healthy single-cell measurements to construct a linear subspace representing the predominant protein expression programs ("eigencells") within the healthy myeloid developmental continuum. By projecting AML single-cell measurements onto this subspace, we derived a healthy feature vector aligned with the healthy subspace and a cancer-specific feature vector orthogonal to the healthy subspace for each AML cell. These feature vectors-along with clinical metadata about each patient including age, blast percentage at diagnosis, and cytogenetic status-were used as the input to regularized Cox proportional hazards models predicting time-to-relapse for each patient. Using the relative risk scores from the proportional hazards model, patients were assigned to high-risk or low-risk groups according to the optimal log-rank test threshold. The baseline clinical model used only age, blast percentage at diagnosis, and cytogenetic status as predictors and predicted relapse status with an accuracy of 13/19 (68%). This baseline model was outperformed by the model constructed using the average value of the single-cell cancer-specific feature vectors for each patient, which predicted relapse status with an accuracy of 16/19 (84%). Interestingly, despite using only information available at diagnosis, the single-cell model also outperformed a clinical model incorporating patients' MRD status after induction chemotherapy, which predicted relapse with an accuracy of 15/19 (79%). Interrogation of the coefficients of the single-cell feature model revealed specific cellular signaling programs associated with relapse, including enhanced pCreb and pSTAT1 signaling as well as depleted pSTAT5 signaling relative to healthy lineage cells (Figure 1). Conclusions These results support the feasibility of predicting relapse in AML as early as diagnosis by leveraging a computational approach that compares cancer cells to the native lineage from which they arise. Validation of this approach in an independent cohort is ongoing and will be presented. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

STEM-03. MOLECULAR PROFILING OF PRIMARY VERSUS RECURRENT GLIOBLASTOMA BRAIN TUMOR STEM CELLS UNCOVERS SIGNALING MECHANISMS THAT PROMOTE THE AGGRESSIVENESS OF RECURRENT GLIOBLASTOMA

Abstract Glioblastoma (GBM) is a devastating brain cancer with a median overall survival of a mere 12-15 months. Patients receive the standard of care treatment, comprised of maximum surgical resection, ionizing radiation, and temozolomide chemotherapy, however the tumor inevitably recurs. Recurrent GBM is more invasive, difficult to resect, and treatment resistant. GBM brain tumor stem cells (BTSCs), a sub-population of stem-like tumor cells with the ability to self-renew and differentiate into a heterogeneous tumor, are thought to be at the root of recurrent disease. BTSCs isolated from primary and recurrent tumors from the same patient are rare due to fewer resections of recurrent tumors and reduced quality of recurrent tumor samples used to initiate cell lines. As a result, the process of tumor recurrence is vastly understudied in BTSCs. To further understand the process of recurrence and elucidate potential mechanisms of invasion and treatment resistance, we explored global transcriptomics in 40 primary versus 17 recurrent BTSC cultures. Additionally, we profiled changes at the chromatin level in a subset of primary versus recurrent BTSCs using ATAC sequencing. Several pathways were found to be upregulated in recurrent GBM BTSCs, including innate immune signaling at the mRNA and chromatin level, which may mediate the aggressive nature of recurrent BTSCs. Moreover, these signaling changes may be unique to the stem cell population within the tumor, as they are not observed when profiling primary versus recurrent bulk tumors. We are currently targeting these pathways genetically and pharmacologically in primary and recurrent GBM BTSCs established from the same patient and have uncovered mechanisms for treatment resistance, invasion, and recurrence. Our work investigating global signaling changes in primary versus recurrent BTSCs holds promise for uncovering new therapeutic targets or biomarkers for treatment of recurrent GBM.

Glioblastoma CD105+ cells define a SOX2− cancer stem cell-like subpopulation in the pre-invasive niche

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. Glioma stem like cells (GSC) represent the highest cellular hierarchy in GBM and have a determining role in tumor growth, recurrence and patient prognosis. However, a better definition of GSC subpopulations, especially at the surgical resection margin, is warranted for improved oncological treatment options. The present study interrogated cells expressing CD105 (CD105+) specifically within the tumor front and the pre-invasive niche as a potential GSC subpopulation. GBM primary cell lines were generated from patients (n = 18) and CD105+ cells were isolated and assessed for stem-like characteristics. In vitro, CD105+ cells proliferated and enriched in serum-containing medium but not in serum-free conditions. CD105+ cells were characterized by Nestin+, Vimentin+ and SOX2−, clearly distinguishing them from SOX2+ GCS. GBM CD105+ cells differentiated into osteocytes and adipocytes but not chondrocytes. Exome sequencing revealed that GBM CD105+ cells matched 83% of somatic mutations in the Cancer cell line encyclopedia, indicating a malignant phenotype and in vivo xenotransplantation assays verified their tumorigenic potential. Cytokine assays showed that immunosuppressive and protumorigenic cytokines such as IL6, IL8, CCL2, CXCL-1 were produced by CD105+ cells. Finally, screening for 88 clinical drugs revealed that GBM CD105+ cells are resistant to most chemotherapeutics except Doxorubicin, Idarubicin, Fludarabine and ABT-751. Our study provides a rationale for targeting tumoral CD105+ cells in order to reshape the tumor microenvironment and block GBM progression.

Glioblastoma Stem-like Cell Detection Using Perfusion and Diffusion MRI.

Simple SummaryGlioblastoma stem-like cells (GSCs) are known to be aggressive and radio-resistant and proliferate heterogeneously in preferred environments. Additionally, quantitative diffusion and perfusion MRI biomarkers provide insight into the tissue micro-environment. This study assessed the sensitivity of these imaging biomarkers to GSCs in the hyperintensities-FLAIR region, where relapses may occur. A total of 16 patients underwent an MRI session and biopsies were extracted to study the GSCs. In vivo and in vitro biomarkers were compared and both Apparent Diffusion Coefficient (ADC) and relative Cerebral Blood Volume (rCBV) MRI metrics were found to be good predictors of GSCs presence and aggressiveness.Purpose: With current gold standard treatment, which associates maximum safe surgery and chemo-radiation, the large majority of glioblastoma patients relapse within a year in the peritumoral non contrast-enhanced region (NCE). A subpopulation of glioblastoma stem-like cells (GSC) are known to be particularly radio-resistant and aggressive, and are thus suspected to be the cause of these relapses. Previous studies have shown that their distribution is heterogeneous in the NCE compartment, but no study exists on the sensitivity of medical imaging for localizing these cells. In this work, we propose to study the magnetic resonance (MR) signature of these infiltrative cells. Methods: In the context of a clinical trial on 16 glioblastoma patients, relative Cerebral Blood Volume (rCBV) and Apparent Diffusion Coefficient (ADC) were measured in a preoperative diffusion and perfusion MRI examination. During surgery, two biopsies were extracted using image-guidance in the hyperintensities-FLAIR region. GSC subpopulation was quantified within the biopsies and then cultivated in selective conditions to determine their density and aggressiveness. Results: Low ADC was found to be a good predictor of the time to GSC neurospheres formation in vitro. In addition, GSCs were found in higher concentrations in areas with high rCBV. Conclusions: This study confirms that GSCs have a critical role for glioblastoma aggressiveness and supports the idea that peritumoral sites with low ADC or high rCBV should be preferably removed when possible during surgery and targeted by radiotherapy.

Activation of Hedgehog signaling by the oncogenic RELA fusion reveals a primary cilia-dependent vulnerability in supratentorial ependymoma.

Supratentorial RELA fusion (ST-RELA) ependymomas (EPNs) are resistant tumors without an approved chemotherapeutic treatment. Unfortunately, the molecular mechanisms that lead to chemoresistance traits of ST-RELA remain elusive. The aim of this study was to assess RELA fusion-dependent signaling modules, specifically the role of the Hedgehog (Hh) pathway as a novel targetable vulnerability in ST-RELA. Gene expression was analyzed in EPN from patient cohorts, by microarray, RNA-seq, qRT-PCR, and scRNA-seq. Inhibitors against Smoothened (SMO) (Sonidegib) and Aurora kinase A (AURKA) (Alisertib) were evaluated. Protein expression, primary cilia formation, and drug effects were assessed by immunoblot, immunofluorescence, and immunohistochemistry. Hh components were selectively overexpressed in EPNs induced by the RELA fusion. Single-cell analysis showed that the Hh signature was primarily confined to undifferentiated, stem-like cell subpopulations. Sonidegib exhibited potent growth-inhibitory effects on ST-RELA cells, suggesting a key role in active Hh signaling; importantly, the effect of Sonidegib was reversed by primary cilia loss. We, thus, tested the effect of AURKA inhibition by Alisertib, to induce cilia stabilization/reassembly. Strikingly, Alisertib rescued ciliogenesis and synergized with Sonidegib in killing ST-RELA cells. Using a xenograft model, we show that cilia loss is a mechanism for acquiring resistance to the inhibitory effect of Sonidegib. However, Alisertib fails to rescue cilia and highlights the need for other strategies to promote cilia reassembly, for treating ST-RELA tumors. Our study reveals a crucial role for the Hh pathway in ST-RELA tumor growth, and suggests that rescue of primary cilia represents a vulnerability of the ST-RELA EPNs.

Lung Cancer Stem-like Cells and Drug Resistance

肺癌是全球致死率最高的肿瘤，耐药和复发是肺癌致死率居高不下的关键原因之一。大量研究证据表明，在肺癌组织和细胞中存在一小群具有干性样特征的细胞，这群细胞被证实能够自我更新、多向分化和无限增殖，在体内可以高效成瘤，能在放疗和化疗中存活，最终引起肺癌的耐药和复发。本文着重针对肺癌干性样细胞的来源、分子生物学特征和鉴定方法以及广谱耐药机制进行综述，旨在为进一步研究其临床诊断和靶向策略提供依据。

Abstract P5-11-01: Identification of novel ER and ER-NFκB driven stem-like cell populations in ER+ breast cancer

Abstract Approximately 70% of breast cancers are estrogen receptor (ER) positive and can be treated with endocrine therapy. Unfortunately, 30-40% of tumors will recur, often as therapy-resistant metastatic disease. One contributor to disease recurrence is a population of cells with stem-like features (i.e. breast cancer stem cells, BCSCs). Previously, we have shown that positive crosstalk between two critical transcription factors, ER and NFκB, can contribute to the expansion of ER+ BCSCs, as indicated by increased mammosphere (MS) forming efficiency and BCSC markers. NFκB has been previously described as a major player in BCSC biology. However, the role of ER, as well as the interaction between ER and NFκB, in driving BCSCs remains unclear. We found in MS that both ER and NFκB are endogenously active and required for MS formation. To investigate whether ER and NFκB work together or independently, we made dual ERE and NFκB-RE reporter cell lines. We confirmed activation of both ER and NFκB in MS, but with a high degree of heterogeneity as indicated by 4 different cell populations (ERE+/NFκB-RE+, ERE+/NFκB-RE-, ERE-/NFκB-RE+ and ERE-/NFκB-RE-). Interestingly, we found >90% of MS arose from ERE+ cells while NFκB-RE+ cells failed to from MS, suggesting that ER but not NFκB activity was sufficient to initiate MS development. Moreover, ERE+ cells, with or without NFκB-RE activity, formed more secondary MS and expressed higher levels of stem cell-associated genes than ERE- cells, indicating ERE+ cells are more stem-like. To study ER and NFκB function in more detail, we utilized single cell RNA sequencing of MS. We identified 4 clusters with clusters 1 and 2 enriched for stem cell gene signatures. Bioinformatic analysis revealed Cluster 2 to be enriched for ER, proliferation, and DREAM complex signatures, and we confirmed these genes were enriched in ERE+ cells. However, we also found that ER activity is associated with activation of the DREAM complex in standard cell culture. This suggests that ER regulation of proliferation via the DREAM complex may not be specific for BCSCs. In contrast to Cluster 2, Cluster 1 was enriched with ER and NFκB signatures, as well as an ER-NFκB crosstalk signature. In addition, Cluster 1 was enriched with pathways related to adaptive stress response such as: oxidative phosphorylation, unfolded protein response, and protein secretion. As some studies have reported activation of stress pathways is associated with maintenance of stemness, we hypothesize that ER and NFκB may work together to promote stem features through activation of multiple stress pathways to increase survival and growth in an anchorage-independent environment. To determine whether either Cluster 1 or Cluster 2 derived signatures are associated with clinical outcome, we first interrogated single cell RNA sequencing data from a metastatic ER+ PDX model. The Cluster 2 signature was enriched in proliferative cell populations found in both primary and metastatic tumors. In contrast, the Cluster 1 signature was enriched only in a metastatic tumor cell population. We also showed that expression of signatures derived from Cluster 1 or 2 in ER+ breast cancer patient tumors predict poor relapse free survival in the METABRIC dataset. However, only co-expression of both signatures predicted poor disease-free survival. Taken together, our findings suggest there are two unique subpopulations of stem-like cells, one of which is dependent on ER driven proliferation, whereas the other population relies on an ER-NFkB mediated stress response for survival and self-renewal. Furthermore, both of these population signatures in patients’ tumors are associated with more aggressive disease and poor outcome. Citation Format: Svetlana E. Semina, Mark Maienschein-Cline, Elaine T. Alarid, Carol A. Sartorius, Jonna Frasor. Identification of novel ER and ER-NFκB driven stem-like cell populations in ER+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-11-01.

The protein kinase LKB1 promotes self-renewal and blocks invasiveness in glioblastoma.

The role of liver kinase B1 (LKB1) in glioblastoma (GBM) development remains poorly understood. LKB1 may regulate GBM cell metabolism and has been suggested to promote glioma invasiveness. After analyzing LKB1 expression in GBM patient mRNA databases and in tumor tissue via multiparametric immunohistochemistry, we observed that LKB1 was localized and enriched in GBM tumor cells that co-expressed SOX2 and NESTIN stemness markers. Thus, LKB1-specific immunohistochemistry can potentially reveal subpopulations of stem-like cells, advancing GBM patient molecular pathology. We further analyzed the functions of LKB1 in patient-derived GBM cultures under defined serum-free conditions. Silencing of endogenous LKB1 impaired 3D-gliomasphere frequency and promoted GBM cell invasion in vitro and in the zebrafish collagenous tail after extravasation of circulating GBM cells. Moreover, loss of LKB1 function revealed mitochondrial dysfunction resulting in decreased ATP levels. Treatment with the clinically used drug metformin impaired 3D-gliomasphere formation and enhanced cytotoxicity induced by temozolomide, the primary chemotherapeutic drug against GBM. The IC50 of temozolomide in the GBM cultures was significantly decreased in the presence of metformin. This combinatorial effect was further enhanced after LKB1 silencing, which at least partially, was due to increased apoptosis. The expression of genes involved in the maintenance of tumor stemness, such as growth factors and their receptors, including members of the platelet-derived growth factor (PDGF) family, was suppressed after LKB1 silencing. The defect in gliomasphere growth caused by LKB1 silencing was bypassed after supplementing the cells with exogenous PFDGF-BB. Our data support the parallel roles of LKB1 in maintaining mitochondrial homeostasis, 3D-gliomasphere survival, and hindering migration in GBM. Thus, the natural loss of, or pharmacological interference with LKB1 function, may be associated with benefits in patient survival but could result in tumor spread.