Patient-derived Spheroids Research Articles

Unlocking the Potential of Spheroids in Personalized Medicine: A Systematic Review of Seeding Methodologies.

Three-dimensional (3D) spheroid models have revolutionized in vitro cancer research by offering more physiologically relevant alternatives to traditional two-dimensional (2D) cultures. A systematic search identifies English-language studies on patient-derived cancer spheroids for drug screening, using defined inclusion and exclusion criteria, with data extracted on cancer type, culture methods, spheroid characteristics, and therapeutic responses. This manuscript evaluates the methods for spheroid formation and the cellular sources used, highlighting the diverse applications and preferences in this field. The five most investigated cancer origins for spheroid seeding are breast, colon, lung, ovary, and brain cancers, reflecting their clinical importance and research focus. Among seeding methodologies, forced-floating and scaffold-based methods predominate, demonstrating reliability and versatility in spheroid generation. Other techniques, including microfluidics, bioprinting, hanging drop, and suspension culture also play significant roles, each with distinct advantages and limitations. This review underscores the increasing use of spheroid models and the need for standardization in methodologies to enhance the reproducibility and translational potential in cancer research.

Recent advances on modeling retinal disease: Towards efficient gene/drug therapy.

Recent advances on modeling retinal disease: Towards efficient gene/drug therapy.

Strong Hsp90α/β Protein Expression in Advanced Primary CRC Indicates Short Survival and Predicts Response to the Hsp90α/β-Specific Inhibitor Pimitespib

The prognosis of advanced (UICC IIb-IV) primary colorectal cancer (pCRC) remains poor. More effective targeted therapies are needed. Heat shock protein 90 alpha/beta (Hsp90α/β) expression was immunohistologically quantified in 89 pCRCs and multivariately correlated with survival. Pimitespib (Pim, TAS-116), a Hsp90α/β-specific inhibitor, was tested in pCRC cell lines and patient-derived cancer spheroids (PDCS) and referenced to the pan-Hsp90 inhibitor ganetespib (Gan, STA-9090) and standard-of-care therapies. A total of 26.97% pCRCs showed strong tumoral Hsp90α/β expression (Hsp90α/β > 40%), which correlated with reduced PFS (HR: 3.785, 95%CI: 1.578–9.078, p = 0.003) and OS (HR: 3.502, 95%CI: 1.292–9.494, p = 0.014). Co-expression of Hsp90α/β > 40% with its clients BRAF-V600E and Her2/neu aggravated the prognosis (BRAF-V600E mutated: PFS, p = 0.002; OS, p = 0.012; Her2/neu score3: PFS, p = 0.029). The prognostic cut-off Hsp90α/β > 40% was also a predictor for response to Pim-based therapy. Pim efficacy was increased in combination with 5-FU, 5-FU + oxaliplatin, and 5-FU + irinotecan (all p < 0.001). Pim induced sensitization to all chemotherapies in HT-29 (p < 0.001), Caco-2 (p < 0.01), and HCT116 (p < 0.05) cells. Pim combined with encorafenib in HT-29 and with trastuzumab in Caco-2 cells was most effective in dual-target inhibition approaches (HT-29: p < 0.005; Caco-2: p < 0.05). The anti-cancer effect and chemosensitization of Pim-based therapy were prospectively confirmed in PDCS directly generated from Hsp90α/β > 40% pCRCs. Protein profiling combined with functional drug testing stratifies Hsp90α/β > 40% pCRC patients diagnosed with UICC IIb-IV for effective Pim-based therapy.

A 3D vascularized tumor spheroid microfluidic platform for head and neck cancer research: new insights.

Conventional in vitro cancer models often fail to replicate the complexity of the tumor microenvironment. We have developed a 3D micro-engineered vascularized organoid chip (VOC) platform to enhance the physiological relevance of in vivo tumor models. This platform incorporates patient-derived tumor spheroids from head and neck cancer patients, providing a more accurate simulation of the native tumor microenvironment. We evaluated the efficacy of 5-fluorouracil (5-FU) and sunitinib on angiogenic sprouting and cell viability of red fluorescent protein-expressing human umbilical vein endothelial cells (RFP-HUVECs) and head and neck cancer patient-derived tumor spheroids cultured in the VOC platform. A 3D micro-engineered VOC platform was developed to provide a physiologically relevant environment for RFP-HUVECs and head and neck cancer patient-derived tumor spheroids. Cellular responses to 5-FU and sunitinib were examined over 14 days, focusing on interactions and behavior in the VOC setup. 5-FU and sunitinib significantly inhibited angiogenic sprouting and reduced cell viability. Notably, these drugs induced changes in cellular network formation and disrupted the structural integrity of patient-derived spheroids, emphasizing the effectiveness of these drugs in a model that closely simulates the tumor microenvironment of head and neck cancer. Our study demonstrates the potential of the 3D vascularized tumor spheroid microfluidic chip as a valuable tool for personalized treatment and investigation of head and neck squamous cell carcinoma. This platform simulates the tumor microenvironment and offers exceptional precision in evaluating drug efficacy.

Abstract 5756: Quantification of SHetA2 in patient-derived ovarian cancer spheroids to study drug uptake and resistance in a physiologically relevant model

Abstract Ovarian cancer presents therapeutic challenges, particularly in advanced stages, where malignant ascites—a buildup of fluid in the abdominal cavity—complicates treatment. Within this ascitic environment, clusters of cancer cells, known as spheroids, form, proliferate, and implant upon various organs, contributing to the metastatic nature of ovarian cancer. As they limit drug penetration and have a hypoxic environment that fosters angiogenesis, spheroids exhibit significant chemoresistance, amplified by the inflammatory and tumor-promoting microenvironment (Peng). Therefore, to reveal the potential mechanisms of chemoresistance in ovarian cancer, directly targeting these spheroids is crucial to develop effective therapies that could help reduce the high mortality rate associated with ovarian cancer. ShetA2 (Sulfur Heteroarotinoid A2) is a novel, promising anticancer drug that exhibits unique potential by inducing apoptosis in cancer cells and disrupting mitochondrial pathways without harming healthy cells (Benbrook). Currently in phase 1 clinical trials, it is vital to understand its efficacy and optimize its therapeutic potential. However, conventional quantification methods for SHetA2 primarily rely on plasma analysis in animal models or human samples, providing only indirect measures and failing to replicate microenvironmental factors in the tumor’s 3D structure that inhibit drug access to spheroids (Sharma et al.). Consequently, a direct approach to measure drug concentration in single spheroids is necessary to understand the pharmacodynamics and pharmacokinetics of anticancer drugs (Peng). To address this gap, we developed a highly sensitive nano-flow liquid chromatography-mass spectrometry (nanoLC-MS) method for quantification of SHetA2 in patient-derived spheroids treated ex vivo with cell free-ascites. First, spheroids are washed and lysed. Second, an isotopically labeled compound d4-SHetA2 with known concentration is added to the lysate as the internal standard. Third, a C18 ZipTip is used to clean and desalt the sample to remove competing ions that could reduce detection sensitivity. Lastly, the prepared samples are analyzed using flow ultra-high-performance liquid chromatography (UHPLC) coupled with a high-resolution Thermo Orbitrap mass spectrometer. This integrated approach will provide highly sensitive and accurate quantification of SHetA2 at low concentrations, offering insights into its distribution, penetration, and activity within single patient-derived spheroids. Furthermore, by enabling a more precise and clinically relevant analysis of SHetA2’s efficacy in targeting spheroids, this approach will help establish robust criteria for dose selection and scheduling intervals in clinical applications. Citation Format: Annelise Huynh, Amit Singh, Zongkai Peng, Doris M. Benbrook, Anthony W. Burgett, Zhibo Yang. Quantification of SHetA2 in patient-derived ovarian cancer spheroids to study drug uptake and resistance in a physiologically relevant model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5756.

Abstract 1867: Development and validation of fluorescent lipid nanoparticles for RNA delivery applicable to personalized in vitro oncology models

Abstract Tebubio offers contract services in RNA-based therapeutic discovery, in vitro modeling, biomarker mapping, and data analysis. Leveraging these platforms, we have developed innovative lipid nanoparticles (LNPs) designed to encapsulate and deliver RNA. This advancement features a fluorescent lipid, created by Echelon Inc., which enables imaging-based tracking of the LNPs. Using this fluorescent lipid, we formulated LNPs encapsulating a mRNA Coding for a fluorescent protein. We validated the distribution of these LNPs within 2D mammalian cell line model and confirmed RNA expression in these cells through imaging techniques. This technology is applicable to in vitro oncology models, including patient-derived spheroids and tumoroids, which allow for a personalized approach to therapeutic testing. Ultimately, Tebubio’s platform provides end-to-end support, from RNA optimization and custom LNP formulation to efficacy validation in vitro, enabling personalized medicine models for RNA-based therapeutics. Citation Format: Xavier Warnet, Célia Bosso-Lefèvre, Erica Cirri, Eric Mennesson, Marie Morin, Mélanie Sapateiro, Maxime Rochet, Laetitia Edmond, Alexandra Foucher, Armelle Vindrios, Alzbeta Komarkova, Flavien Carpenier, Romain Cordonnier-Goulay. Development and validation of fluorescent lipid nanoparticles for RNA delivery applicable to personalized in vitro oncology models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1867.

Abstract 4000: The significance of GLP-1R and its agonist in neuroendocrine neoplasms

Abstract Neuroendocrine neoplasms (NENs) are rare cancers originating from neuroendocrine cells that are found throughout the body. NENs are subdivided into 2 categories: Well-differentiated neuroendocrine tumors (NET) and poorly differentiated neuroendocrine carcinomas (NEC). Both NET and NEC are highly metastatic and difficult to treat. NEN cases are on the rise and yet the etiology remains unclear. Commonly used drugs such as proton pump inhibitors can promote NET growth and result in poor prognosis for NET patients. Nowadays, the diabetes and weight loss drug semaglutide, a glucagon-like peptide 1 receptor (GLP-1R) agonist, has gain extensive popularity and are currently being taken by over 15 million people in the USA. Semaglutide is contraindicated for NET patients with medullary thyroid carcinoma (MTC) or multiple endocrine neoplasia syndrome type 2 (MEN2) since some of these cancers express the GLP-1R. However, this area of research remains understudied. Little is known about the expression levels of GLP-1R in NEN from different anatomical locations and their response to semaglutide since these are rare cancers and few NET models are available for drug testing. We recently identified 2 human NET cell lines (GOT1 and NT-3) that express the GLP-1R and showed that semaglutide promotes tumor cell growth both in vitro and in vivo. In this study, we aim to investigate the levels of GLP-1R expression in a large collection of NEN tissue microarrays and determine the effects of semaglutide in novel GLP-1R positive NET patient-derived spheroid cultures. We stained for GLP-1R expression by immunohistochemistry in 357 NET tissue microarrays covering 7 classes of NEN: 78 pancreas NET, 62 duodenum NET, 33 ileal NET, 42 lung NET, 10 small cell lung NEC, 12 extrapulmonary visceral NEC, 29 thyroid NET, 12 gastric NET, 6 appendix NET, 6 rectum NET, 22 pheochromocytoma, 22 paraganglioma, and 23 Merkel cell carcinoma. Furthermore, we generated GLP-1R positive pancreas, ileal, and duodenal NET spheroids for drug testing. Our data showed 45% of duodenum NET, 17% of gastric NET, and 14% of pancreas NET stained positive for GLP-1R expression. Less than 2% of other classes NET or NEC stained positive for GLP-1R expression. Using newly established NET patient-derived spheroid models, we demonstrated that 100 nM of semaglutide accelerates tumor cell growth by 1.4 to 2-fold. Surprisingly, duodenum NET is the class of NET that frequently express GLP-1R and should be highly cautioned for the semaglutide usage. Citation Format: Sophia A. Hueser, Reese E. Townsend, Casandro J. Chan, Leona Rupp, Leopaul J. Chan, Dawn E. Quelle, Joseph S. Dillon, Andrew M. Bellizzi, James R. Howe, Po Hien H. Ear. The significance of GLP-1R and its agonist in neuroendocrine neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4000.

Abstract 1460: Clinical relevance of a super-enhancer-associated actin polymerization promoting-factor in endometrial cancer

Abstract Super-enhancers (SEs) are defined as large clusters of enhancers regulating transcription of genes that exhibit cell-type specific functions. SEs in cancers are often different from those in healthy states and responsible for the development and progression of the disease. We here study the clinical role of a super-enhancer-associated actin polymerization promoting-factor, cordon-bleu WH2 repeat protein-like 1 (COBLL1), in endometrial cancer, since we previously demonstrated its tumor-promoting role in castration-resistant prostate cancer [Proc Natl Acad Sci USA 115:4975, 2018]. In the present study, using multiple patient-derived spheroid cultures established by our group [Endocrinology 160:1895, 2029], endometrial cancer-specific SEs were identified by chromatin immunoprecipitation (ChIP) sequencing for H3K27ac binding sites. Among top-ranked SE-associated genes, we focused on the relevance of COBLL1 in endometrial cancer. Then, we showed that COBLL1 silencing in endometrial cancer cells substantially repressed cell proliferation and reduced the populations of cells in S phase of the cell cycle. Of note, immunohistochemical study of clinical endometrial cancer tissues revealed that COBLL1 immunoreactivity was well correlated with poor prognosis of the patients. Our findings suggest that COBLL1 plays a relevant role in the pathophysiology of endometrial cancer and can be applied as a promising diagnostic and therapeutic target in endometrial cancer management. Citation Format: Kuniko Horie, Erina Iwabuchi, Kazuhiro Ikeda, Takashi Suzuki, Satoshi Inoue. Clinical relevance of a super-enhancer-associated actin polymerization promoting-factor in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1460.

Abstract 2805: A lncRNA-specific CRISPRi screen identifies an essential role of PDCD4-AS1 in colorectal cancer growth and survival

Colorectal cancer (CRC) ranks as the third most commonly diagnosed cancer worldwide and is the second leading cause of cancer-related mortality. Recent studies have highlighted the critical role of long non-coding RNAs (lncRNAs) in CRC development, influencing processes such as proliferation, invasion, and therapy resistance. However, the functional roles of most annotated lncRNAs in CRC remain poorly understood, underscoring the need to uncover novel lncRNAs that may impact CRC initiation, progression, and treatment response. To identify lncRNAs involved in CRC cell growth, we conducted a comprehensive lncRNA-specific CRISPR interference (CRISPRi) dropout screen targeting 675 lncRNAs highly expressed across various cancer entities. We utilized four CRC models with diverse molecular backgrounds, including two patient-derived 3D models. As a control, we included HEK293T cells to filter out common essential lncRNAs. This approach revealed 11 lncRNAs significantly depleted across all CRC models, indicating a shared regulatory role in CRC. To validate these findings, we inhibited expression of selected top-hit lncRNA candidates using CRISPRi and antisense oligonucleotide-mediated knockdown. In both approaches, we observed reduced cell growth compared to control conditions, closely aligning with the dropout screen findings. Among the candidates, PDCD4-AS1 was prioritized for further characterization, as it is not located near an oncogenic or common essential protein-coding gene, minimizing the likelihood of off-target effects. By generating loss-of-function patient-derived spheroid models, we showed that PDCD4-AS1 silencing significantly reduced cell growth, induced G2/M cell cycle arrest, and promoted apoptosis. To evaluate the therapeutic potential of targeting PDCD4-AS1, we performed knockdown experiments in a panel of patient-derived organoids (PDOs) both in vitro and in vivo. In a PDO xenograft mouse model, PDCD4-AS1 knockdown significantly suppressed tumor growth and prolonged overall survival. To explore the molecular mechanisms underlying the PDCD4-AS1 knockdown phenotype, we performed transcriptomic analysis by RNA sequencing. Here, we identified significant alterations in amino acid metabolism, suggesting a trans-regulatory role of PDCD4-AS1 in CRC. However, further analyses are required to identify the interaction partners of PDCD4-AS1 and gain deeper insights into its mechanism of action. Collectively, our findings highlight the potential of CRISPRi screens in uncovering novel molecular vulnerabilities in patient-derived CRC models. We present the previously uncharacterized lncRNA PDCD4-AS1 as a mediator of CRC progression and provide strong evidence for lncRNAs as promising therapeutic targets and/or biomarkers for personalized oncology. Citation Format: Vida Kufrin, Silvia Schäfer, Silke Brilloff, Maria Gabriela Pereira dos Santos, Elahe Rahimian, Sandra Küchler, Denis M. Schewe, Claudia R. Ball, Hanno Glimm, Marius Bill, Alexander A. Wurm. A lncRNA-specific CRISPRi screen identifies an essential role of PDCD4-AS1 in colorectal cancer growth and survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2805.

Integrin targeted photodynamic therapy in patient-derived glioblastoma spheroids.

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor, with a median overall survival of 14.6 months. GBM is incurable because of its invasive growth. These local invasive cells, most significantly glioblastoma stem cells (GSCs), when left behind, resist standard treatment, and cause almost all recurrences. However, the treatment of these infiltrative margins remains a significant challenge, as there are currently no options to reach these margins safely. Photodynamic therapy (PDT) shows promise as localized treatment option using light-activated compounds that target tumor cells and that generate reactive oxygen species (ROS) to destroy them. Far red light, combined with silicon phthalocyanines, could penetrate deeper making it more effective for reaching cancer cells in the tumor margin without compromise of healthy brain. In this study, we used patient-derived GBM spheroids invitro as a preclinical model to evaluate a new dual-cRGDfK-silicon phthalocyanine conjugate targeting integrin αvβ3, a protein expressed by GBM cells and vasculature. Targeted PDT was efficient in killing GSC spheroids, showing that the combination of far-red light with more precise targeting can reach the type of cells found in the invasive margin, using silicon phthalocyanine as the photosensitizer.

The histone modifier KAT2A presents a selective target in a subset of well-differentiated microsatellite-stable colorectal cancers.

Lysine acetyltransferase 2 A (KAT2A) plays a pivotal role in epigenetic gene regulation across various types of cancer. In colorectal cancer (CRC), increased KAT2A expression is associated with a more aggressive phenotype. Our study aims to elucidate the molecular underpinnings of KAT2A dependency in CRC and assess the consequences of KAT2A depletion. We conducted a comprehensive analysis by integrating CRISPR-Cas9 screening data with genomics, transcriptomics, and global acetylation patterns in CRC cell lines to pinpoint molecular markers indicative of KAT2A dependency. Additionally, we characterized the phenotypic effect of a CRISPR-interference-mediated KAT2A knockdown in CRC cell lines and patient-derived 3D spheroid cultures. Moreover, we assessed the effect of KAT2A depletion within a patient-derived xenograft mouse model in vivo. Our findings reveal that KAT2A dependency is closely associated with microsatellite stability, lower mutational burden, and increased molecular differentiation signatures in CRC, independent of the KAT2A expression levels. KAT2A-dependent CRC cells display higher gene expression levels and enriched H3K27ac marks at gene loci linked to enterocytic differentiation. Furthermore, loss of KAT2A leads to decreased cell growth and viability in vitro and in vivo, downregulation of proliferation- and stem cell-associated genes, and induction of differentiation markers. Altogether, our data show that a specific subset of CRCs with a more differentiated phenotype relies on KAT2A. For these CRC cases, KAT2A might represent a promising novel therapeutic target.

Epigenetic modulation drives inflammatory responses in sarcoma

Compared with adult tumors, childhood malignancies are typically driven by a relatively small number of mutations, frequently in genes encoding epigenetic regulators. High-grade osteosarcoma (OS) is an aggressive bone malignancy characterized by massive genomic rearrangements and one of the most prevalent cancers in young adults. However, the role of epigenetic reprogramming in OS remains unclear. In this study, we sought to investigate the potential of histone modifications as prognostic and therapeutic biomarkers in OS. An in silico predictive algorithm of histone modification landscape from RNA sequencing data revealed that genome-wide levels of acetylation of the lysin 27 in histone 3 (H3K27ac) inversely correlated with survival. Notably, H3K27ac levels positively correlated with immune-related signatures indicating an inflamed tumor microenvironment. To investigate if H3K27ac expression influences inflammatory responses, the histone deacetylase 1-3 (HDAC) inhibitor Entinostat was used to induce H3K27ac expression in sarcoma tumors. Compared with untreated sarcoma tumors, Entinostat treatment resulted in higher infiltration of tissue resident T cells in ex vivo patient-derived sarcoma spheroids and in an in vivo model of OS. Mechanistically, our preliminary results highlight the involvement of the Hippo pathway mediators YAP1 and VGLL3 in Entinostat-mediated tumor infiltration of T cells. Together, these findings show that H3K27ac drives T cells infiltration into sarcoma. Furthermore, genome wide H3K27ac levels may be used to predict survival in OS. In addition, H3K27ac levels are associated with immune-related signatures and could be explored to promote inflammatory responses in sarcoma.

A Novel 3D High-Throughput Phenotypic Drug Screening Pipeline to Identify Drugs with Repurposing Potential for the Treatment of Ovarian Cancer.

Ovarian cancer (OC) poses a significant clinical challenge due to its high recurrence rates and resistance to standard therapies, particularly in advanced stages where recurrence is common, and treatment is predominantly palliative. Personalized treatments, while effective in other cancers, remain underutilized in OC due to a lack of reliable biomarkers predicting clinical outcomes. Accordingly, precision medicine approaches are limited, with PARP inhibitors showing efficacy only in specific genetic contexts. Drug repurposing offers a promising, rapidly translatable strategy by leveraging existing pharmacological data to identify new treatments for OC. Patient-derived polyclonal spheroids, isolated from ascites fluid closely mimic the clinical behavior of OC, providing a valuable model for drug testing. Using these spheroids, a high-throughput drug screening pipeline capable of evaluating both cytotoxicity and anti-migratory properties of a diverse drug library, including FDA-approved, investigational, and newly approved compounds is developed. The findings highlight the importance of 3D culture systems, revealing a poor correlation between drug efficacy in traditional 2D models and more clinically relevant 3D spheroids. This approach has expedited the identification of promising candidates, such as rapamycin, which demonstrated limited activity as a monotherapy but synergized effectively with standard treatments like cisplatin and paclitaxel in vitro. In combination with platinum-based therapy, Rapamycin led to significant in vitro cytotoxicity and a marked reduction in tumor burden in a syngeneic in vivo model. This proof-of-concept study underscores the potential of drug repurposing to rapidly advance new treatments into clinical trials for OC, offering renewed hope for patients with advanced disease.

Hypoxia-Responsive Polymersomes for Stemness Reduction in Patient-Derived Solid Tumor Spheroids.

Aggressive solid tumors are associated with rapid growth, early hypoxia, a lack of targeted therapies, and a poor prognosis. The hypoxic niches within the rapidly growing solid tumors give rise to a stem-cell-like phenotype with higher metastasis and drug resistance. To overcome the drug resistance of these regions, we used hypoxia-responsive polymersomes with an encapsulated anticancer drug (doxorubicin, Dox) and a stemness modulator (all-trans retinoic acid, ATRA). Reductase enzymes overexpressed in hypoxia reduce the azobenzene linker of the polymers, disrupt the bilayer structure of the polymersomes, and release the encapsulated drugs. We used triple-negative breast cancer (TNBC) as a representative of aggressive and hypoxic solid tumors. We observed that ATRA synergistically enhanced the efficacy of Dox in killing cancer cells. A synergistic combination of the two drug-encapsulated polymersomes reduced the volumes of patient-derived TNBC spheroids by 90%. In contrast, Dox alone decreased the spheroid volumes by 70% and encapsulated ATRA by 19%. Mechanistic studies revealed that ATRA inhibited efflux pumps, leading to a higher concentration of doxorubicin within TNBC cells. In addition, the combination of encapsulated Dox and ATRA significantly decreased stemness expression of the TNBC cells in hypoxia compared to that of Dox alone.

A patient-derived HCC spheroid system to model the tumor microenvironment and treatment response

A patient-derived HCC spheroid system to model the tumor microenvironment and treatment response

Preliminary study of utilizing a patient derived tumor spheroid model to augment precision therapy in metastatic brain tumors

Treating metastatic brain tumors remains a significant challenge. This study introduces and applies the Patient-Derived Tumor Spheroid (PDTS) system, an ex vivo model for precision drug testing on metastatic brain tumor. The PDTS system utilizes a decellularized extracellular matrix (dECM) derived from adipose tissue, combined with the tumor cells, to form tumor spheroids. These spheroids were subsequently used to test anticancer drugs, with results compared to the clinical outcomes observed after administering these treatments to patients. To assess the validity of the data, the correlation between the drug responses observed in the PDTS model and actual patient outcomes was analyzed. Chi-square tests evaluated the significance of associations between lab predictions and clinical outcomes, using a significance threshold of p < 0.05. In preliminary data, 17 patients met the criteria for final analysis, which showed an overall 57% accuracy (p-value = 0.463), with improvements to 73% accuracy (p-value = 0.072) when patients receiving certain treatments were excluded. This PDTS offers real-time results within three weeks, simultaneous testing of multiple drugs, and the ability to culture and store tumor cells for reproducibility. Despite some limitations, further development of this model could enhance its clinical application and improve patient outcomes.

A Novel Microfluidic Platform for Personalized Anticancer Drug Screening Through Image Analysis.

The advancement of personalized treatments in oncology has garnered increasing attention, particularly for rare and aggressive cancer with low survival rates like the bone tumors osteosarcoma and chondrosarcoma. This study introduces a novel PDMS-agarose microfluidic device tailored for generating patient-derived tumor spheroids and serving as a reliable tool for personalized drug screening. Using this platform in tandem with a custom imaging index, we evaluated the impact of the anticancer agent doxorubicin on spheroids from both tumor types. The device produces 20 spheroids, each around 300 µm in diameter, within a 24 h timeframe, facilitating assessments of characteristics and reproducibility. Following spheroid generation, we measured patient-derived spheroid diameters in bright-field images, calcein AM-positive areas/volume, and the binary fraction area, a metric analyzing fluorescence intensity. By employing a specially developed equation that combines viability signal extension and intensity, we observed a substantial decrease in spheroid viability of around 75% for both sarcomas at the highest dosage (10 µM). Osteosarcoma spheroids exhibited greater sensitivity to doxorubicin than chondrosarcoma spheroids within 48 h. This approach provides a reliable in vitro model for aggressive sarcomas, representing a personalized approach for drug screening that could lead to more effective cancer treatments tailored to individual patients, despite some implementation challenges.

Microglia induce an interferon-stimulated gene expression profile in glioblastoma and increase glioblastoma resistance to temozolomide.

Glioblastoma is the most malignant primary brain tumour. Even with standard treatment comprising surgery followed by radiation and concomitant temozolomide (TMZ) chemotherapy, glioblastoma remains incurable. Almost all patients with glioblastoma relapse owing to various intrinsic and extrinsic resistance mechanisms of the tumour cells. Glioblastomas are densely infiltrated with tumour-associated microglia and macrophages (TAMs). These immune cells affect the tumour cells in experimental studies and are associated with poor patient survival in clinical studies. The aim of the study was to investigate the impact of microglia on glioblastoma chemo-resistance. We co-cultured patient-derived glioblastoma spheroids with microglia at different TMZ concentrations and analysed cell death. In addition, we used RNA sequencing to explore differentially expressed genes after co-culture. Immunostaining was used for validation. Co-culture experiments showed that microglia significantly increased TMZ resistance in glioblastoma cells. RNA sequencing revealed upregulation of a clear interferon-stimulated gene (ISG) expression signature in the glioblastoma cells after co-culture with microglia, including genes such as IFI6, IFI27, BST2, MX1 and STAT1. This ISG expression signature is linked to STAT1 signalling, which was confirmed by immunostaining. The ISG expression profile observed in glioblastoma cells with enhanced TMZ resistance corresponded to the interferon-related DNA damage resistance signature (IRDS) described in different solid cancers. Here, we show that the IRDS signature, linked to chemo-resistance in other cancers, can be induced in glioblastoma by microglia. ISG genes and the microglia inducing the ISG expression could be promising novel therapeutic targets in glioblastoma.

DDDR-27. BMP SIGNALING MODULATES APOPTOTIC AND NON-APOPTOTIC CELL DEATH IN GLIOBLASTOMA

Abstract Recurrence occurs in approximately 90% of glioblastoma (GBM) patients, in part due to the tumors becoming resistant to cell death in response to existing therapies. These therapies often trigger apoptosis; however, apoptosis is only one of several different forms of cell death. We hypothesize that activating non-canonical death mechanisms could provide a back door to killing treatment-resistant GBM cells. To test this hypothesis, we first developed a new imaging-based high-throughput method to directly quantify cell death in 3D models. Using this method, we find patient-derived GBM spheroids to be resistant to the induction of cell death in response to various standard-of-care and experimental therapeutic compounds. To further investigate mechanisms underlying this resistance, we screened a panel of extracellular protein ligands for the modulation of cell death response. We find that bone morphogenic protein (BMP) signaling increases resistance to several apoptosis-inducing compounds, including temozolomide and tyrosine kinase inhibitors. Intriguingly, BMP signaling simultaneously enhanced sensitivity to a novel non-apoptotic cell death pathway triggered by the small molecule CIL56. Efforts are ongoing to further understand how BMP signaling governs apoptotic and non-apoptotic cell death mechanisms. Nevertheless, our results suggest a novel approach to treating GBM, centered upon the modulation of BMP signaling combined with the use of agents that induce non-apoptotic cell death.

DDDR-17. A FACILE HIGH THROUGHPUT DRUG SCREENING PLATFORM ENABLES THE CONSTRUCTION OF A PHARMACO-PROTEOGENOMIC INTERACTION LANDSCAPE IN GLIOBLASTOMA

Abstract Despite decades of intensive research, the number of treatment options for glioblastoma (GBM), a devastating disease, remains very limited. The vast tumor heterogeneity of GBM and the blood-brain barrier (BBB) represent major hurdles to developing effective therapeutics. Here, we present CARPOOL, a high-throughput therapeutic screening approach designed to facilitate accurate and parallel interrogation of numerous patient-derived GBM spheroid cultures (GSCs), capturing the disease’s heterogeneity. We validated CARPOOL in vitro and in vivo and then applied it for drug screening using a library of over 700 BBB-penetrating compounds across 23 omics-profiled GSCs. This screening identified numerous potential anti-GBM compounds, including previously non-oncology drugs. Clustering analysis provided insights into the mechanisms of action of some of the compounds. Coupling these results with omics data of the GSCs enabled the identification of genetic, subtype, and gene expression-based predictors of drug sensitivity. Our study presents a new approach for advancing drug screening and in vivo preclinical therapy evaluation for precision oncology. Furthermore, our construction of a pharmaco-proteogenomic interaction landscape offers a valuable resource for both basic and translational glioblastoma research.