Cancer Stem Cells Research Articles

STEM-18. A HIGH THROUGHPUT NEUROSPHERE BASED COLONY FORMATION ASSAY TO VALIDATE DRUG AND RADIATION EFFECTIVENESS IN PATIENT DERIVED XENOLINES

Abstract Glioblastoma (GBM) is a primary malignant brain tumor developed from normal astrocytes. GBM patients’ median survival is less than 2 years despite safe surgical resection, fractionated conventional radiation, and temozolomide (TMZ) therapy. Few currently used drugs are successful in GBM although there are many FDA approved blood-brain barrier (BBB) penetrant drugs, for other uses that have not been screened in GBM. We utilized a panel of 734 of these BBB penetrant drugs in different GBM patient derived xenolines (PDX) along with acquired radio-resistant derivatives (-RT) including X1441, JX14P, JX14P-RT, JX39P, JX39P-RT and normal human astrocytes (NHA) as control and screened for viability with Cell Titer-Glo. We identified multiple candidate drugs that preferentially killed GBM PDX cells at 10 µM relative to NHA control. As we used stem cell media (serum free) in our drug screening study, it indicates that the potent drugs are also effective in killing cancer stem cells or brain tumor initiating cells. These drugs had targets ranging from PDGFR, DNA/RNA synthesis, as well as other kinase inhibitors. We developed a high throughput (HT), neurosphere based colony formation assay (CFA) in 96-well plate to screen multiple drugs and doses in combination with radiation. For visualizing the colonies, we use a Cytation 5 imager at 4X resolution and count the colonies based on parameters including object size, width, intensity and others. We plan on validating our drug screen and HT-CFA assay with high throughput PamChip kinase activity assays and with in vivo orthotopic tumor PDX models.

TMET-36. INCREASED METHIONINE CYCLE DRIVES PYRIMIDINE METABOLISM IN LETHAL PEDIATRIC EPENDYMOMAS

Abstract Supratentorial ependymomas (ST-EPN) are pediatric brain tumors found in the cerebral hemisphere, driven by the oncogenic fusion of ZFTA with RELA. Other than surgery and radiotherapy, these tumors do not have effective therapeutic options. Recurrent ST-EPN tumors are highly aggressive and hard to treat, which makes these tumors lethal. To understand the disease mechanism and potentially identify or develop therapeutic targets, we established patient-derived models and interrogated the genetic and metabolic dependencies using cutting-edge, near-genome-wide genetic screening and metabolomic tools. Our integrative transcriptomics and metabolic analysis identify that the ZFTA-RELA fusion drives methionine metabolic reprogramming in the cancer stem cells to regulate permissive epigenetic status and pyrimidine nucleotide synthesis to fuel tumor growth. Limiting methionine or inhibiting MAT2A, a rate-limiting enzyme of methionine metabolism in ST-EPN cells, results in profound cell cycle arrest and make the cells vulnerable to pyrimidine inhibitors compared to other pediatric brain tumors. Thus, for treatment, along with radiotherapy, blocking the methionine cycle by using the methionine limited diet or in combination with pyrimidine metabolism inhibitors can be a novel therapeutic strategy against the aggressive paediatric ST-EPN tumors.

Insight into the mechanisms regulating liver cancer stem cells by hepatitis B virus X protein.

Hepatocellular carcinoma (HCC) is a heterogeneous disease with high recurrence and mortality. It is well known that a large proportion of HCCs are caused by hepatitis B virus (HBV) infection. In particular, the HBV X protein (HBX), a multifunctional molecule produced by the virus, plays a leading role in hepatocarcinogenesis. However, the molecular mechanisms underlying HBX-mediated HCC remain not fully elucidated. Recently, liver cancer stem cells (LCSCs), a unique heterogeneous subpopulation of the malignancy, have received particular attention owing to their close association with tumorigenesis. Especially, the modulation of LCSCs by HBX by upregulating CD133, CD44, EpCAM, and CD90 plays a significant role in HBV-related HCC development. More importantly, not only multiple signaling pathways, including Wnt/β-catenin signaling, transforming growth factor-β (TGF-β) signaling, phosphatidylinositol-3-kinase (PI-3K)/AKT signaling, and STAT3 signaling pathways, but also epigenetic regulation, such as DNA and histone methylation, and noncoding RNAs, including lncRNA and microRNA, are discovered to participate in regulating LCSCs mediated by HBX. Here, we summarized the mechanisms underlying different signaling pathways and epigenetic alterations that contribute to the modulation of HBX-induced LCSCs to facilitate hepatocarcinogenesis. Because LCSCs are important in hepatic carcinogenesis, understanding the regulatory factors controlled by HBX might open new avenues for HBV-associated liver cancer treatment.

TMET-18. MANNOSE INHIBITS GLIOMA CELL PROLIFERATION AND POTENTIATES TEMOZOLOMIDE EFFICACY

Abstract Gliomas are among the most aggressive types of brain tumors, often displaying resistance to standard therapies such as temozolomide (TMZ). Recent research suggests that metabolic modulation may influence tumor growth and therapeutic response. This study explores the effects of mannose, a monosaccharide, on glioma cell growth and its potential to enhance the efficacy of TMZ. In vitro experiments were conducted to evaluate the impact of mannose and its combination with TMZ on glioma cells. Cell viability assays, ATP measurements, and flow cytometry (FACS) analysis were performed to assess cell cycle and apoptosis post-treatment with various monosaccharides, including mannose. The synergistic effects of the combined treatment with mannose and TMZ on cell viability were evaluated using Bliss score analysis and FACS. The impact on cancer stem cell characteristics was investigated through neurosphere formation assays, while tumor invasiveness was assessed using 3D invasion assays. Expression of stemness and invasion markers was analyzed by western blot. Differentially expressed gene (DEG) analysis was conducted to assess changes in gene expression related to stemness and invasiveness. Mannose treatment significantly reduced glioma cell viability and ATP levels, indicating decreased metabolic activity. Combined treatment with mannose and TMZ demonstrated a synergistic effect, enhancing cell death compared to either treatment alone, as evidenced by Bliss score and FACS analysis. Co-treatment reduced neurosphere formation and 3D invasion capabilities, with western blot results showing downregulation of stemness and invasion markers. DEG analysis revealed significant changes in gene expression related to stemness and invasiveness with the combined treatment. Mannose significantly impairs glioma cell growth and enhances the therapeutic efficacy of TMZ in vitro. Combining mannose with standard chemotherapy offers a promising strategy for improving glioma treatment outcomes. Further investigation in clinical settings is warranted to validate these findings.

Biomimetic Nano-delivery of Small-Molecule Piceatannol Modulates Tumor Stemness and Suppresses Colorectal Cancer Metastasis via Hippo/YAP1/SOX9 Signaling.

Suppressing tumor metastasis is a crucial strategy for improving survival rates in patients with colorectal cancer (CRC), with cancer stem cells (CSCs) being the primary drivers of metastasis. Current therapeutic approaches targeting CSCs are limited, and their molecular mechanisms remain unclear. To address this challenge, a biomimetic nanoparticle delivery system, CMD-BHQ3-PTL/DOX@RBCM is developed, to deliver the stem cell regulator, piceatannol (PTL). This system used carboxymethyl dextran (CMD) and Black Hole Quencher 3 (BHQ3) to encapsulate PTL and the cytotoxic drug doxorubicin (DOX) within a red blood cell membrane (RBCm), enhancing stability and biocompatibility while allowing gradual drug release under hypoxic conditions. The effects of PTL are investigated on CSCs using molecular biology experiments, plasmid construction, and high-throughput sequencing and elucidated the molecular mechanisms underlying this biomimetic nanoparticle delivery system. The therapeutic efficacy of PTL is validated at the tissue level using subcutaneous and metastatic tumor models in human and murine systems. The results demonstrated that CMD-BHQ3-PTL/DOX@RBCM effectively addressed the challenges of specificity and biocompatibility in vivo, significantly inhibiting CSC-related tumor metastasis. This inhibitory effect is closely associated with the Hippo/YAP1/SOX9 pathway. This study highlights the effectiveness of the pH-responsive biomimetic nanoparticle system CMD-BHQ3-PTL/DOX@RBCm in delivering PTL to tumor sites, with SOX9 and its upstream Hippo/YAP1 pathway playing a critical role in the underlying mechanism.

Membrane-Cloaked Nanodrug for Homologous Targeting and Treatment of Therapeutic Stress Escaped Cancer Stem Cells

Membrane-Cloaked Nanodrug for Homologous Targeting and Treatment of Therapeutic Stress Escaped Cancer Stem Cells

Retraction Note: LINC00115 promotes stemness and inhibits apoptosis of ovarian cancer stem cells by upregulating SOX9 and inhibiting the Wnt/β-catenin pathway through competitively binding to microRNA-30a

Retraction Note: LINC00115 promotes stemness and inhibits apoptosis of ovarian cancer stem cells by upregulating SOX9 and inhibiting the Wnt/β-catenin pathway through competitively binding to microRNA-30a

Breast cancer stem cells origins and the memory stem cells: clinical significance of biomarkers and the active therapeutic approaches

Despite recent improvements in diagnosis and treatment, breast cancer (BC) remains one of the leading causes of cancer-related deaths among women. In this complex disease, breast cancer stem cells (BCSCs) are a small but significant subset of different cancer cells with the ability to proliferate and self-renew. According to an increasing amount of studies, BCSCs are essential for breast cancer metastasis, drug resistance, and recurrence. Due to its diverse nature, BC includes numerous subtypes, each of which displays unique BCSC types and concentrations that are connected to different therapy outcomes and outcomes. Despite significant advancements in the treatment of early-stage breast cancer, there are still few effective therapy approaches for metastatic BC. The development, progression, and dissemination of BC are largely attributed to cancer stem-like cells (CSCs), which are characterized by their exceptional adaptability and self-renewal ability. An overview of the development of BCSCs, their biomarkers, clinical significance, and the mechanisms behind their behavior is the goal of the current study. The active therapy strategies being employed to address BCSCs will also be examined.

NSDHL contributes to breast cancer stem-like cell maintenance and tumor-initiating capacity through TGF-β/Smad signaling pathway in MCF-7 tumor spheroid

BackgroundNAD(P)-dependent steroid dehydrogenase-like protein (NSDHL), which is involved in breast tumor growth and metastasis, has been implicated in the maintenance of cancer stem cells. However, its role in regulating breast cancer stem-like cells (BCSCs) remains unclear. We have previously reported the clinical significance of NSDHL in patients with estrogen receptor-positive (ER +) breast cancer. This study aimed to elucidate the molecular mechanisms by which NSDHL regulates the capacity of BCSCs in the ER + human breast cancer cell line, MCF-7.MethodsNSDHL knockdown suppressed tumor spheroid formation in MCF-7 human breast cancer cells grown on ultralow-attachment plates. RNA sequencing revealed that NSDHL knockdown induced widespread transcriptional changes in the MCF-7 spheroids. TGF-β signaling pathway was the most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (fold change ≥ 2, P ≤ 0.05) identified in NSDHL-knockdown MCF-7 spheroids compared with the control. In orthotopic tumor models injected with NSDHL-knockdown MCF-7 spheroids, tumor initiation and growth were strongly suppressed compared with those in the control.ResultsBCSC populations with CD44+/CD24- and CD49f+/EpCAM + phenotypes and high ALDH activity were decreased in NSDHL-knockdown MCF-7 spheroids and xenograft tumors relative to controls, along with decreased secretion of TGF-β1 and 3, phosphorylation of Smad2/3, and expression of SOX2. In RNA-sequencing data from The (TCGA) database, a positive correlation between the expression of NSDHL and SOX2 was found in luminal-type breast cancer specimens (n = 998). Our findings revealed that NSDHL plays an important role in maintaining the BCSC population and tumor-initiating capacity of ER-positive MCF-7 spheroids, suggesting that NSDHL is an attractive therapeutic target for eliminating BCSCs, thus preventing breast cancer initiation and progression.ConclusionsOur findings suggest that NSDHL regulates the BCSC/tumor-initiating cell population in MCF-7 spheroids and xenograft tumors.

Identification of Crucial Cancer Stem Cell Genes Linked to Immune Cell Infiltration and Survival in Hepatocellular Carcinoma

Identification of Crucial Cancer Stem Cell Genes Linked to Immune Cell Infiltration and Survival in Hepatocellular Carcinoma

Targeting ketone body metabolism in mitigating gemcitabine resistance.

Chemotherapy is often combined with surgery for muscle invasive and non-muscle invasive bladder cancer. However, 70% of the patients recur within 5 years. Metabolic reprogramming is an emerging hallmark in cancer chemoresistance. Here, we report a gemcitabine resistance mechanism which promotes cancer reprogramming via the metabolic enzyme, OXCT1. This mitochondrial enzyme, responsible for the rate-limiting step in β-hydroxybutyrate (βHB) catabolism, was elevated in muscle invasive disease and in chemo-resistant bladder cancer patients. Resistant orthotopic tumors presented an OXCT1-dependent rise in mitochondrial oxygen consumption rate, ATP, and nucleotide biosynthesis. In resistant bladder cancer, knocking out OXCT1 restored gemcitabine sensitivity, and administering the non-metabolizable βHB, enantiomer (S-βHB) only partially restored gemcitabine sensitivity. Suggesting an extra-metabolic role for OXCT1, multi-omics analysis of gemcitabine sensitive and resistant cells revealed an OXCT1-dependent signature with the transcriptional repressor, OVOL1, as a master regulator of epithelial differentiation. The elevation of OVOL1 target genes was associated with its cytoplasmic translocation and poor prognosis in a chemotherapy-treated BCa patient cohort. The knockout of OXCT1 restored OVOL1 transcriptional repressive activity by its nuclear translocation. Orthotopic mouse models of bladder cancer supported OXCT1 as a mediator of gemcitabine sensitivity through ketone metabolism and regulating cancer stem cell differentiation.

DLK1 Is Associated with Stemness Phenotype in Medullary Thyroid Carcinoma Cell Lines

Medullary thyroid carcinoma (MTC) is a rare and aggressive tumor, often requiring systemic treatment in advanced or metastatic stages, where drug resistance presents a significant challenge. Given the role of cancer stem cells (CSCs) in cancer recurrence and drug resistance, we aimed to identify CSC subpopulations within two MTC cell lines harboring pathogenic variants in the two most common MEN2-associated codons. We analyzed 15 stemness-associated markers, along with well-established thyroid stem cell markers (CD133, CD44, and ALDH1), a novel candidate (DLK1), and multidrug resistance proteins (MRP1 and MRP3). The ability to efflux the fluorescent dye Hoechst 3342 and form spheroids, representing CSC behavior, was also assessed. MZ-CRC-1 cells (p.M918T) displayed higher expressions of canonical markers, DLK1, and MRP proteins than TT cells (p.C634W). MZ-CRC-1 cells also formed more spheroids and showed less dye accumulation (p < 0.0001). Finally, we observed that DLK1+ cells (those expressing DLK1) in both cell lines exhibited significantly higher levels of stemness markers compared to DLK1− cells (those lacking DLK1 expression). These findings underscore DLK1’s role in enhancing the stemness phenotype, providing valuable insights into MTC progression and resistance and suggesting potential therapeutic implications.

Histological transformation in lung adenocarcinoma: Insights of mechanisms and therapeutic windows.

Histological transformation from lung adenocarcinoma (ADC) to small cell lung carcinoma (SCLC), large cell neuroendocrine carcinoma (LCNEC), squamous cell carcinoma (SCC), and sarcomatoid carcinoma (PSC) after targeted therapies is recognized as a mechanism of resistance in ADC treatments. Patients with transformed lung cancer typically experience a poor prognosis and short survival time. However, effective treatment options for these patients are currently lacking. Therefore, understanding the mechanisms underlying histological transformation is crucial for the development of effective therapies. Hypotheses including intratumoral heterogeneity, cancer stem cells, and alteration of suppressor genes have been proposed to explain the mechanism of histological transformation. In this review, we provide a comprehensive overview of the known molecular features and signaling pathways of transformed tumors, and summarized potential therapies based on previous findings.

Editorial: Reciprocal crosstalk between the tumor microenvironment and cancer stem cells

Editorial: Reciprocal crosstalk between the tumor microenvironment and cancer stem cells

Deciphering normal and cancer stem cell niches by spatial transcriptomics: opportunities and challenges.

Cancer stem cells (CSCs) often exhibit stem-like attributes that depend on an intricate stemness-promoting cellular ecosystem within their niche. The interplay between CSCs and their niche has been implicated in tumor heterogeneity and therapeutic resistance. Normal stem cells (NSCs) and CSCs share stemness features and common microenvironmental components, displaying significant phenotypic and functional plasticity. Investigating these properties across diverse organs during normal development and tumorigenesis is of paramount research interest and translational potential. Advancements in next-generation sequencing (NGS), single-cell transcriptomics, and spatial transcriptomics have ushered in a new era in cancer research, providing high-resolution and comprehensive molecular maps of diseased tissues. Various spatial technologies, with their unique ability to measure the location and molecular profile of a cell within tissue, have enabled studies on intratumoral architecture and cellular cross-talk within the specific niches. Moreover, delineation of spatial patterns for niche-specific properties such as hypoxia, glucose deprivation, and other microenvironmental remodeling are revealed through multilevel spatial sequencing. This tremendous progress in technology has also been paired with the advent of computational tools to mitigate technology-specific bottlenecks. Here we discuss how different spatial technologies are used to identify NSCs and CSCs, as well as their associated niches. Additionally, by exploring related public data sets, we review the current challenges in characterizing such niches, which are often hindered by technological limitations, and the computational solutions used to address them.

Comprehensive Analysis and Verification of the Prognostic Significance of Cuproptosis-Related Genes in Colon Adenocarcinoma.

Colon adenocarcinoma (COAD) is a frequently occurring and lethal cancer. Cuproptosis is an emerging type of cell death, and the underlying pathways involved in this process in COAD remain poorly understood. Transcriptomic and clinical data for COAD patients were collected from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. We investigated alterations in DNA and chromatin of cuproptosis-related genes (CRGs) in COAD. In order to identify predictive differentially expressed genes (DEGs) and various molecular subtypes, we used consensus cluster analysis. Through univariate, multivariate, and Lasso Cox regression analyses, four CRGs were identified. A risk prognostic model for cuproptosis characteristics was constructed based on four CRGs. This study also examined the association between the risk score and the tumor microenvironment (TME), the immune landscape, and drug sensitivity. We distinguished two unique molecular subtypes using consensus clustering analysis. We discovered that the clinical characteristics, prognosis, and TME cell infiltration characteristics of patients with multilayer CRG subtypes were all connected. The internal and external evaluations of the predicted accuracy of the prognostic model built using data derived from a cuproptosis risk score were completed at the same time. A nomogram and a clinical pathological analysis make it more useful in the field of medicine. A significant rise in immunosuppressive cells was observed in the high cuproptosis risk score group, with a correlation identified between the cuproptosis risk score and immune cell infiltration. Despite generally poor prognoses, the patients with a high cuproptosis risk but low tumor mutation burden (TMB), cancer stem cell (CSC) index, or microsatellite instability (MSI) may still benefit from immunotherapy. Furthermore, the cuproptosis risk score positively correlated with immune checkpoint gene expression. Analyzing the potential sensitivity to medications could aid in the development of clinical chemotherapy regimens and decision-making. CRGs are the subject of our in-depth study, which exposed an array of regulatory mechanisms impacting TME. In addition, we performed additional data mining into clinical features, prognosis effectiveness, and possible treatment medications. COAD's molecular pathways will be better understood, leading to more precise treatment options.

Computational investigations of flavonoids as ALDH isoform inhibitors for treatment of cancer

ABSTRACT Human aldehyde dehydrogenases (ALDHs) are a group of 19 isoforms often overexpressed in cancer stem cells (CSCs). These enzymes play critical roles in CSC protection, maintenance, cancer progression, therapeutic resistance, and poor prognosis. Thus, targeting ALDH isoforms offers potential for innovative cancer treatments. Flavonoids, known for their ability to affect multiple cancer-related pathways, have shown anticancer activity by downregulating specific ALDH isoforms. This study aimed to evaluate 830 flavonoids from the PubChem database against five ALDH isoforms (ALDH1A1, ALDH1A2, ALDH1A3, ALDH2, ALDH3A1) using computational methods to identify potent inhibitors. Extra precision (XP) Glide docking and MM-GBSA free binding energy calculations identified several flavonoids with high binding affinities. MD simulation highlighted flavonoids 1, 2, 18, 27, and 42 as potential specific inhibitors for each isoform, respectively. Flavonoid 10 showed high binding affinities for ALDH1A2, ALDH1A3, and ALDH3A1, emerging as a potential multi-ALDH inhibitor. ADMET property evaluation indicated that the promising hits have acceptable drug-like profiles, but further optimization is needed to enhance their therapeutic efficacy and reduce toxicity, making them more effective ALDH inhibitors for future cancer treatment.

Bisphenol S exposure promotes stemness of triple-negative breast cancer cells via regulating Gli1-mediated Sonic hedgehog pathway

Bisphenol S (BPS), one of the most common alternatives for bisphenol A (BPA), has been implied to increase the risk of breast cancer. Triple-negative breast cancer (TNBC) is a highly aggressive type of breast cancer with a poor prognosis. However, the association between BPS and TNBC remains unclear. Cancer stem cells (CSCs) have a crucial role in breast cancer initiation, metastasis, and recurrence. Here, we proposed that BPS, equivalent to the human internal exposure and the environmental concentrations, enhanced CSC-like properties by upregulating sphere formation, self-renewal, the percentage of CD44+/CD24- cells, and the expression of CSC markers. Moreover, BPS promoted the migration, invasion, and epithelial-mesenchymal transition (EMT) in TNBC cells. Mechanistically, BPS activated the Sonic Hedgehog (SHH) signaling pathway in TNBC cells. Molecular docking analysis further showed that BPS upregulated SHH signaling pathway via directly binding Gli1 protein. Furthermore, inhibitor of SHH pathway or Gli1 siRNA attenuated the promoting effects of BPS on stemness, invasion, and migration of TNBC cells. In summary, our data firstly provide evidence that environmentally relevant BPS concentration treatment significantly enhanced TNBC malignant phenotype by activating the Sonic Hedgehog/Gli1 signaling pathway, raising high concerns about the potential population biology hazards of BPS.

IMPA1-derived inositol maintains stemness in castration-resistant prostate cancer via IMPDH2 activation.

Acquisition of prostate cancer stem cells (PCSCs) manifested during androgen ablation therapy (ABT) contributes to castration-resistant prostate cancer (CRPC). However, little is known about the specific metabolites critically orchestrating this process. Here, we show that IMPA1-derived inositol enriched in PCSCs is a key metabolite crucially maintaining PCSCs for CRPC progression and ABT resistance. Notably, conditional Impa1 knockout in the prostate abrogates the pool and properties of PCSCs to orchestrate CRPC progression and prolong the survival of TRAMP mice. IMPA1-derived inositol serves as a cofactor that directly binds to and activates IMPDH2, which synthesizes guanylate nucleotides for maintaining PCSCs with ARlow/- features leading to CRPC progression and ABT resistance. IMPA1/inositol/IMPDH2 axis is upregulated in human prostate cancer, and its overexpression predicts poor survival outcomes. Genetically and pharmacologically targeting the IMPA1/inositol/IMPDH2 axis abrogates CRPC and overcomes ABT resistance in various CRPC xenografts, patient-derived xenograft (PDX) tumor models, and TRAMP mouse models. Our study identifies IMPDH2 as an inositol sensor whose activation by inositol represents a key mechanism for maintaining PCSCs for CRPC and ABT resistance.

Ex-vivo models of post-surgical residual disease in human glioblastoma

Background Glioblastoma is a highly infiltrative, currently incurable brain cancer. To date, translation of novel therapies for glioblastoma from the laboratory into clinical trials has relied heavily on in vitro cell culture and murine (subcutaneous and orthotopic) xenograft models using cells derived from the main bulk of patient tumours. However, it is the residual cells left-behind after surgery that are responsible for disease progression and death in the clinic. A lack of substantial improvements in patient survival for decades suggests commonly used murine xenograft models, a key step before clinical trials, do not reflect the biology of residual disease in patients. Methods To address this, we have developed the ‘Sheffield Protocol’ to generate ex vivo models that reflect both resected, and post-surgical residual disease from the same patient. The protocol leverages parallel derivation of inherently treatment-resistant glioblastoma stem cells (GSCs) from ‘core’ and distant ‘edge’ regions through careful macrodissection of a large en bloc specimen, such as from a partial lobectomy for tumour, followed by tissue dissociation and propagation in serum-free media. Opportunistic en bloc specimen use can liberate the most distant infiltrative cells feasibly accessible from living patients. Results We provide an example illustrating that resected and residual disease models represent spatially divergent tumour subpopulations harbouring distinct transcriptomic and cancer stem cell marker expression profiles. We also introduce the ‘Sheffield Living Biobank’ of glioma models (SLB) that incorporates over 150 GSC lines from 60+ patients, including 44+ resected and residual models, which are available for academic use via MTA. Conclusions These models provide a novel tool to reduce animal xenograft usage by improving candidate drug triage in early preclinical studies and directly replacing animal studies for some therapies that are post-Phase 1+ clinical trial for other cancers/conditions to, ultimately, deliver more effective treatments for post-surgical residual disease in glioblastoma.