Patient-derived Spheroids Research Articles

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.

3D keloid spheroid model: Development and application for personalized drug response prediction

Research on keloid is limited by the lack of proper in vitro and animal model reflecting in vivo status. Based on heterogeneity of keloid and important role of endothelial cells in its pathogenesis, a novel 3D in vitro keloid spheroid prepared with keloid fibroblasts and endothelial cells was evaluated in this study. Commercial cell lines of keloid fibroblasts and endothelial cells were used at various cellular ratios to generate keloid spheroids to determine the optimal condition. Keloid spheroids from three keloid patients were also made and their usefulness as in vitro models, including their responses to drugs, were assessed. Spheroids with higher endothelial cell proportions exhibited increased viability and propagation ability. Patient-derived keloid spheroids showed heterogeneity which might reflect individual clinical conditions. The optimal ratio of fibroblasts to endothelial cells was determined to be 4:1 for keloid spheroids based on gene expression and viability analyses. Patient-derived keloid spheroid showed better keloidal changes in genetic expressions than 2D monolayer culture. Spheroids exhibited varied responses and resistance to each drug used for keloids, depending on the cell type used. 3D keloid spheroids might provide an effective in vitro model for investigating disease pathogenesis and appropriate treatment modalities for future precision medicine.

Differential Effects of Extracellular Vesicles from Two Different Glioblastomas on Normal Human Brain Cells.

Background/Objectives: Glioblastomas (GBMs) are dreadful brain tumors with abysmal survival outcomes. GBM extracellular vesicles (EVs) dramatically affect normal brain cells (largely astrocytes) constituting the tumor microenvironment (TME). We asked if EVs from different GBM patient-derived spheroid lines would differentially alter recipient brain cell phenotypes. This turned out to be the case, with the net outcome of treatment with GBM EVs nonetheless converging on increased tumorigenicity. Methods: GBM spheroids and brain slices were derived from neurosurgical patient tissues following informed consent. Astrocytes were commercially obtained. EVs were isolated from conditioned culture media by ultrafiltration, concentration, and ultracentrifugation. EVs were characterized by nanoparticle tracking analysis, electron microscopy, biochemical markers, and proteomics. Astrocytes/brain tissues were treated with GBM EVs before downstream analyses. Results: EVs from different GBMs induced brain cells to alter secretomes with pro-inflammatory or TME-modifying (proteolytic) effects. Astrocyte responses ranged from anti-viral gene/protein expression and cytokine release to altered extracellular signal-regulated protein kinase (ERK1/2) signaling pathways, and conditioned media from EV-treated cells increased GBM cell proliferation. Conclusions: Astrocytes/brain slices treated with different GBM EVs underwent non-identical changes in various omics readouts and other assays, indicating "personalized" tumor-specific GBM EV effects on the TME. This raises concern regarding reliance on "model" systems as a sole basis for translational direction. Nonetheless, net downstream impacts from differential cellular and TME effects still led to increased tumorigenic capacities for the different GBMs.

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.

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

Background & AimsHepatocellular carcinoma (HCC) is the third leading and fastest rising cause of cancer-related death worldwide. The discovery and preclinical development of compounds targeting HCC is hampered by the absence of authentic tractable systems recapitulating the heterogeneity of patient HCC tumors and the tumor microenvironment (TME). MethodsWe established a novel and simple patient-derived multicellular tumorspheroid model based on clinical HCC tumor tissues, processed using enzymatic and mechanical dissociation. After quality controls, 22 HCC tissues and 17 HCC serum were selected for tumorspheroid generation and perturbation studies. Cells were grown in 3D in optimized medium in presence of patient serum. Characterization of the tumorspheroid cell populations was performed by flow cytometry, immunohistochemistry and functional assays. As a proof of concept, we treated patient-derived spheroids with FDA approved anti-HCC compounds. ResultsThe model was successfully established independently from cancer etiology and grade from 22 HCC tissues. We show that the use of HCC patient serum was essential for tumorspheroid generation, the TME function and to maintain cell viability. The tumorspheroids comprised the main cell compartments including epithelial cancer cells as well as all major cell populations of the TME (cancer-associated fibroblasts, macrophages and T cells, endothelial cells). Tumorspheroids reflect HCC heterogeneity, including variability in cell type proportions and TME, and mimic the original tumor features. Moreover, we observed differential responses to FDA HCC approved drugs between donors as observed in patients. ConclusionThis patient HCC serum-tumorspheroid model provides novel opportunities for drug discovery and development as well as mechanism of action studies including compounds targeting the TME. This model will likely contribute to improve the dismal outcome of patients with HCC. Impact and implicationsHepatocellular carcinoma (HCC) is a leading and fast rising cause of cancer-related death worldwide. Despite approval of novel therapies, the outcome of advanced HCC remains unsatisfactory. By developing a novel patient-derived tumorspheroid model recapitulating tumor heterogeneity and microenvironment, we provide new opportunities for HCC drug development and analysis of mechanism of action in authentic patient tissues. The application of the patient-derived tumorspheroids combined with other HCC models will likely contribute to drug development and to improve the dismal outcome of patients with HCC.

ZCCHC3 and Efp coordinately contribute to the pathophysiology of triple-negative breast cancer by modulating NCAPH

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited targeted therapies and high rates of recurrence. We previously showed that Efp promotes TNBC cell proliferation by regulating cell cycle-related gene expression. Recent studies showed that ZCCHC3 interacts with Efp, promoting Efp signaling in innate immune responses. We here characterize whether ZCCHC3 plays a pathophysiological role in TNBC tumorigenesis. We showed that ZCCHC3 silencing significantly repressed the proliferation of TNBC conventional cultured cells and three-dimensional patient-derived spheroid culture, which we established from a clinical TNBC tissue. RNA-sequencing in TNBC cells defined that “cell division” was a major pathway commonly downregulated by ZCCHC3 and Efp silencing, and NCAPH was a cell division-related gene highly downregulated by ZCCHC3 silencing. In a TNBC cell-derived xenograft model, ZCCHC3-specific siRNA injection successfully reduced in vivo TNBC tumor growth and downregulated NCAPH expression. Overall, our findings demonstrate that ZCCHC3 and Efp coordinately promote TNBC progression by regulating NCAPH expression and that ZCCHC3/Efp/NCAPH pathway can be applied to clinical TNBC management.

Anti-Cancer Properties of Two Intravenously Administrable Curcumin Formulations as Evaluated in the 3D Patient-Derived Cancer Spheroid Model.

Curcumin (Cur) is a heavily used complementary derived drug from cancer patients. Spheroid samples derived from 82 patients were prepared and treated after 48 h with two Cur formulations (CurA, CurB) in mono- and combination therapy. After 72 h, cell viability and morphology were assessed. The Cur formulations had significant inhibitory effects of -8.47% (p < 0.001), CurA of -10.01% (-50.14-23.11%, p = 0.001) and CurB of -6.30% (-33.50-19.30%, p = 0.006), compared to their solvent controls Polyethylene-glycol, β-Cyclodextrin (CurA) and Kolliphor-ELP, Citrate (CurB). Cur formulations were more effective in prostate cancer (-19.54%) and less effective in gynecological non-breast cancers (0.30%). CurA showed better responses in samples of patients <40 (-13.81%) and >70 years of age (-17.74%). CurB had stronger effects in metastasized and heavily pretreated tumors. Combinations of Cur formulations and standard therapies were superior in 20/47 samples (42.55%) and inferior in 7/47 (14.89%). CurB stimulated chemo-doublets more strongly than monotherapies (-0.53% vs. -6.51%, p = 0.022) and more effectively than CurA (-6.51% vs. 3.33%, p = 0.005). Combinations of Cur formulations with Artesunate, Resveratrol and vitamin C were superior in 35/70 (50.00%) and inferior in 16/70 (22.86%) of samples. Cur formulations were significantly enhanced by combination with Artesunate (p = 0.020). Cur formulations showed a high variance in their anti-cancer effects, suggesting a need for individual testing before administration.

Targeting the liver clock improves fibrosis by restoring TGF-β signaling

Liver fibrosis is the major driver of hepatocellular carcinoma and liver disease-related death. Approved antifibrotic therapies are absent and compounds in development have limited efficacy. Increased TGF-β signaling drives collagen deposition by hepatic stellate cells (HSCs)/myofibroblasts. Here, we aimed to dissect the role of the circadian clock (CC) in controlling TGF-β signaling and liver fibrosis. Using CC-mutant mice, enriched HSCs and myofibroblasts obtained from healthy and fibrotic mice in different CC phases and loss-of-function studies in human hepatocytes and myofibroblasts, we investigated the relationship between CC and TGF-β signaling. We explored hepatocyte-myofibroblast communication through bioinformatic analyses of single-nuclei transcriptomes and performed validation in cell-based models. Using mouse models for MASH (metabolic dysfunction-associated steatohepatitis)-related fibrosis and spheroids from patients with liver disease, we performed proof-of-concept studies to validate pharmacological targetability and clinical translatability. We discovered that the CC oscillator temporally gates TGF-β signaling and this regulation is broken in fibrosis. We demonstrate that HSCs and myofibroblasts contain a functional CC with rhythmic expression of numerous genes, including fibrogenic genes. Perturbation studies in hepatocytes and myofibroblasts revealed a reciprocal relationship between TGF-β activation and CC perturbation, which was confirmed in patient-derived exvivo and invivo models. Pharmacological modulation of CC-TGF-β signaling inhibited fibrosis in mouse models invivo as well as in patient-derived liver spheroids. The CC regulates TGF-β signaling, and the breakdown of this control is associated with liver fibrosis in patients. Pharmacological proof-of-concept studies across different models have uncovered the CC as a novel therapeutic target for liver fibrosis - a growing unmet medical need. Liver fibrosis due to metabolic diseases is a global health challenge. Many liver functions are rhythmic throughout the day, being controlled by the circadian clock (CC). Here we demonstrate that regulation of the CC is perturbed upon chronic liver injury and this perturbation contributes to fibrotic disease. By showing that a compound targeting the CC improves liver fibrosis in patient-derived models, this study provides a novel therapeutic candidate strategy to treat fibrosis in patients. Additional studies will be needed for clinical translation. Since the findings uncover a previously undiscovered profibrotic mechanism and therapeutic target, the study is of interest for scientists investigating liver disease, clinical hepatologists and drug developers.

GDNF/GFRA1 signaling contributes to chemo- and radioresistance in glioblastoma

Glioblastoma is the most common primary brain tumor in adults, characterized by an inherent aggressivity and resistance to treatment leading to poor prognoses. While some resistance mechanisms have been elucidated, a deeper understanding of these mechanisms is needed to increase therapeutic efficacy. In this study we first discovered glial-cell derived neurotrophic factor (GDNF) to be upregulated in patient-derived glioblastoma spheroid cultures after chemotherapeutic temozolomide treatment, through RNA-Seq experiments. Therefore, we investigated the role of the GDNF/GDNF receptor alpha 1 (GFRA1) signaling pathway as a resistance mechanism to chemotherapy with temozolomide and lomustine, as well as irradiation using patient-derived glioblastoma spheroid cultures. With qPCR experiments we showed a consistent upregulation of GDNF and its primary receptor GFRA1 following all three lines of treatment. Moreover, CRISPR/Cas9 knock-outs of GDNF in two patient-derived models sensitized these cells to chemotherapy treatment, but not radiotherapy. The increased sensitivity was completely reversed by the addition of exogeneous GDNF, confirming the key role of this factor in chemoresistance. Finally, a CRISPR KO of GFRA1 demonstrated a similar increased sensitivity to temozolomide and lomustine treatment, as well as radiotherapy. Together, our findings support the role of the GDNF/GFRA1 signaling pathway in glioblastoma chemo and radioresistance.

Investigating the Radiobiological Response to Peptide Receptor Radionuclide Therapy Using Patient-Derived Meningioma Spheroids.

Peptide receptor radionuclide therapy (PRRT) using 177Lu-DOTA-TATE has recently been evaluated for the treatment of meningioma patients. However, current knowledge of the underlying radiation biology is limited, in part due to the lack of appropriate in vitro models. Here, we demonstrate proof-of-concept of a meningioma patient-derived 3D culture model to assess the short-term response to radiation therapies such as PRRT and external beam radiotherapy (EBRT). We established short-term cultures (1 week) for 16 meningiomas with high efficiency and yield. In general, meningioma spheroids retained characteristics of the parental tumor during the initial days of culturing. For a subset of tumors, clear changes towards a more aggressive phenotype were visible over time, indicating that the culture method induced dedifferentiation of meningioma cells. To assess PRRT efficacy, we demonstrated specific uptake of 177Lu-DOTA-TATE via somatostatin receptor subtype 2 (SSTR2), which was highly overexpressed in the majority of tumor samples. PRRT induced DNA damage which was detectable for an extended timeframe as compared to EBRT. Interestingly, levels of DNA damage in spheroids after PRRT correlated with SSTR2-expression levels of parental tumors. Our patient-derived meningioma culture model can be used to assess the short-term response to PRRT and EBRT in radiobiological studies. Further improvement of this model should pave the way towards the development of a relevant culture model for assessment of the long-term response to radiation and, potentially, individual patient responses to PRRT and EBRT.

DIPG-69. IN-VITRO DETERMINATION OF 5-AMINOLEVULINIC-ACID-TREATMENT EFFICACY IN PATIENT-DERIVED DIPG MODELS

Abstract BACKGROUND Diffuse Intrinsic Pontine Glioma (DIPG) presents a significant clinical challenge due to its inoperable nature and poor response to current treatments. Sonodynamic Therapy (SDT) is a non-invasive therapy that combines low-intensity ultrasound stimulation with a sonosensitizing agent. 5-Aminolevulinic Acid (5-ALA) as a sonosensitizer has yielded positive outcomes in in-vivo glioblastoma models, and a clinical trial (NCT05123534) for DIPG patients is currently ongoing. 5-ALA is a precursor of Protoporphyrin IX (PpIX), which preferentially accumulates in cancer cells and generates cytotoxic reactive oxygen species upon activation by ultrasound or light. Our work is aimed at investigating the accumulation of PpIX in in-vitro DIPG patient-derived cell models to predict the efficacy of 5-ALA SDT treatment. METHODS Patient derived DIPG cell lines (H3K27M, P53 mutant and wild type) were used to create three-dimensional spheroids. The spheroids were dosed with 5-ALA for 4 hours, and PpIX accumulation was quantified by measuring its fluorescence intensity within the cells. The spheroids were then exposed to controlled light stimulation as an in-vitro proxy for SDT. RESULTS We confirmed the uptake of 5-ALA and subsequent PpIX accumulation in all patient-derived DIPG spheroids. Light exposure of 5-ALA treated DIPG spheroids resulted in reduced viability of the tumor models, while 5-ALA itself did not show cytotoxic effects. The extent of PpIX accumulation and cytotoxicity upon PpIX light activation varied among DIPG models with higher sensitivity of p53 wild type cells. Further studies incorporating a larger number of DMG cells with p53 alterations are underway. CONCLUSIONS These findings indicate that SDT using 5-ALA as sonosensitizer may offer clinical benefits for treatment of DIPG, however treatment efficacy could vary considerably between patients. In vitro evaluation of PpIX accumulation and sensitivity to activation could potentially provide a means to stratify patients and to identify those most likely to benefit from 5-ALA-mediated SDT.

3D stem-like spheroids-on-a-chip for personalized combinatorial drug testing in oral cancer

BackgroundFunctional drug testing (FDT) with patient-derived tumor cells in microfluidic devices is gaining popularity. However, the majority of previously reported microfluidic devices for FDT were limited by at least one of these factors: lengthy fabrication procedures, absence of tumor progenitor cells, lack of clinical correlation, and mono-drug therapy testing. Furthermore, personalized microfluidic models based on spheroids derived from oral cancer patients remain to be thoroughly validated. Overcoming the limitations, we develop 3D printed mold-based, dynamic, and personalized oral stem-like spheroids-on-a-chip, featuring unique serpentine loops and flat-bottom microwells arrangement.ResultsThis unique arrangement enables the screening of seven combinations of three drugs on chemoresistive cancer stem-like cells. Oral cancer patients-derived stem-like spheroids (CD 44+) remains highly viable (> 90%) for 5 days. Treatment with a well-known oral cancer chemotherapy regimen (paclitaxel, 5 fluorouracil, and cisplatin) at clinically relevant dosages results in heterogeneous drug responses in spheroids. These spheroids are derived from three oral cancer patients, each diagnosed with either well-differentiated or moderately-differentiated squamous cell carcinoma. Oral spheroids exhibit dissimilar morphology, size, and oral tumor-relevant oxygen levels (< 5% O2). These features correlate with the drug responses and clinical diagnosis from each patient’s histopathological report.ConclusionsOverall, we demonstrate the influence of tumor differentiation status on treatment responses, which has been rarely carried out in the previous reports. To the best of our knowledge, this is the first report demonstrating extensive work on development of microfluidic based oral cancer spheroid model for personalized combinatorial drug screening. Furthermore, the obtained clinical correlation of drug screening data represents a significant advancement over previously reported personalized spheroid-based microfluidic devices. Finally, the maintenance of patient-derived spheroids with high viability under oral cancer relevant oxygen levels of less than 5% O2 is a more realistic representation of solid tumor microenvironment in our developed device.Graphical

Human-derived Tumor-On-Chip model to study the heterogeneity of breast cancer tissue

One of the leading causes that complicate the treatment of some malignancies, including breast cancer, is tumor heterogeneity. In addition to inter-heterogeneity and intra-heterogeneity of tumors that reflect the differences between cancer cell characteristics, heterogeneity in the tumor microenvironment plays a critical role in tumor progression and could be considered an overlooked and a proper target for the effective selection of therapeutic approaches. Due to the difficulty of completely capturing tumor heterogeneity in conventional detection methods, Tumor-on-Chip (TOC) devices with culturing patient-derived spheroids could be an appropriate alternative. In this research, human-derived spheroids from breast cancer individuals were cultured for 6 days in microfluidic devices. To compare TOC data with conventional detection methods, immunohistochemistry (IHC) and ITRAQ data were employed, and various protein expressions were validated using the transcriptomic databases. The behavior of the spheroids in the collagen matrix and the cell viability were monitored over 6 days of culture. IHC and immunocytochemistry (ICC) results revealed that inter and intra-heterogeneity of tumor spheroids are associated with HER2/ER expression. HER2 expression levels revealed a more important biomarker associated with invasion in the 3D culturing of spheroids. The expression levels of CD163 (as a marker for Ma2 macrophages) and CD44 (a marker for cancer stem cells (CSCs)) were also evaluated. Interestingly, the levels of M2a macrophages and CSCs were higher in triple-negative specimens and samples that showed higher migration and invasion. Cell density and extracellular matrix (ECM) stiffness were also important factors affecting the migration and invasion of the spheroids through the matrix. Among these, rigid ECM revealed a more crucial role than cell density. To sum up, these research findings demonstrated that human-derived spheroids from breast cancer specimens in microfluidic devices provide a dynamic condition for predicting tumor heterogeneity in patients, which can help move the field forward for better and more accurate therapeutic strategies.

Ethyl Pyruvate Decreases Collagen Synthesis and Upregulates MMP Activity in Keloid Fibroblasts and Keloid Spheroids.

Keloids, marked by abnormal cellular proliferation and excessive extracellular matrix (ECM) accumulation, pose significant therapeutic challenges. Ethyl pyruvate (EP), an inhibitor of the high-mobility group box 1 (HMGB1) and TGF-β1 pathways, has emerged as a potential anti-fibrotic agent. Our research evaluated EP's effects on keloid fibroblast (KF) proliferation and ECM production, employing both in vitro cell cultures and ex vivo patient-derived keloid spheroids. We also analyzed the expression levels of ECM components in keloid tissue spheroids treated with EP through immunohistochemistry. Findings revealed that EP treatment impedes the nuclear translocation of HMGB1 and diminishes KF proliferation. Additionally, EP significantly lowered mRNA and protein levels of collagen I and III by attenuating TGF-β1 and pSmad2/3 complex expression in both human dermal fibroblasts and KFs. Moreover, metalloproteinase I (MMP-1) and MMP-3 mRNA levels saw a notable increase following EP administration. In keloid spheroids, EP induced a dose-dependent reduction in ECM component expression. Immunohistochemical and western blot analyses confirmed significant declines in collagen I, collagen III, fibronectin, elastin, TGF-β, AKT, and ERK 1/2 expression levels. These outcomes underscore EP's antifibrotic potential, suggesting its viability as a therapeutic approach for keloids.

Memory-like differentiation enhances NK cell responses against colorectal cancer

ABSTRACT Metastatic (m) colorectal cancer (CRC) is an incurable disease with a poor prognosis and thus remains an unmet clinical need. Immune checkpoint blockade (ICB)-based immunotherapy is effective for mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) mCRC patients, but it does not benefit the majority of mCRC patients. NK cells are innate lymphoid cells with potent effector responses against a variety of tumor cells but are frequently dysfunctional in cancer patients. Memory-like (ML) NK cells differentiated after IL-12/IL-15/IL-18 activation overcome many challenges to effective NK cell anti-tumor responses, exhibiting enhanced recognition, function, and in vivo persistence. We hypothesized that ML differentiation enhances the NK cell responses to CRC. Compared to conventional (c) NK cells, ML NK cells displayed increased IFN-γ production against both CRC cell lines and primary patient-derived CRC spheroids. ML NK cells also exhibited improved killing of CRC target cells in vitro in short-term and sustained cytotoxicity assays, as well as in vivo in NSG mice. Mechanistically, enhanced ML NK cell responses were dependent on the activating receptor NKG2D as its blockade significantly decreased ML NK cell functions. Compared to cNK cells, ML NK cells exhibited greater antibody-dependent cytotoxicity when targeted against CRC by cetuximab. ML NK cells from healthy donors and mCRC patients exhibited increased anti-CRC responses. Collectively, our findings demonstrate that ML NK cells exhibit enhanced responses against CRC targets, warranting further investigation in clinical trials for mCRC patients, including those who have failed ICB.

Abstract P32: Cell Type-specific Genetic Therapy of Cancer, Virus Infection and Mitochondrial Disease

Abstract Cell type-specific gene delivery which restricts gene transfer to therapy-relevant cells, hence reducing unwanted side effects caused by the ectopic expression of transgenes, remains one of the biggest challenges. To mitigate potential off-target effects, we developed a programmable cell type-specific gene therapy using an RNA trans-splicing-based approach for gene replacement therapy or suicide gene therapy that can selectively express therapeutic proteins or suicide signals based on the cell’s endogenous pre-mRNA expression profile. Using the liver as a medical target organ, we developed a hepatocellular carcinoma (HCC)-specific trans-splicing based Herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) suicide gene therapy approach. We rationally designed suicide RNAs that can target multiple HCC-related pre-mRNA biomarkers thereby increasing the overall concentration of targetable pre-mRNA. For delivery, non-viral redosable dumbbell-shaped DNA vectors were designed, manufactured, and tested in tissue culture cells, in 3D patient-derived xenograft spheroids and in mouse model. For cell-type specific delivery, the conjugation of dumbbell vectors with GalNAc3 residues enabled targeted delivery into hepatoblastoma-derived human tissue culture cells. To deliver nucleic acids into the mitochondria of human cells, we studied the long non-coding β2.7 RNA of the human cytomegalovirus which was previously reported to colocalise with mitochondrial complex I. Using thermodynamic profiling, we identified four thermodynamically conserved structural subdomains within the β2.7 RNA that are responsible for its mitochondrial targeting activity. A vector comprising eight mitochondrial targeting domains exhibited a three times higher mitochondrial targeting activity as compared with the parental β2.7 RNA. The amalgamation of mitochondrial targeting RNA and trans-splicing allows cell type-specific transcription of mRNA which can be delivered into mitochondria for mitochondrial gene therapy. Trans-splicing based gene therapy represent a technology that is highly translational offering promising perspectives towards suicide gene therapy of any cancer types or incurable infections with integrating viruses and may enable cell type-specific mitochondrial gene therapy. Citation Format: Pei She Loh (Rachel), Sushmita Poddar, Kaustav Chatterjee, Chiu Jing Wen, Derrick Wee Zan Liang, Fritz Lai, Chen Qingfeng, Volker Patzel. Cell Type-specific Genetic Therapy of Cancer, Virus Infection and Mitochondrial Disease [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P32.

Evaluation of tumor response to immune checkpoint inhibitors by a 3D immunotumoroid model.

Only 20 percent of renal and bladder cancer patients will show a significant response to immune checkpoint inhibitor (ICI) therapy, and no test currently available accurately predicts ICI response. We developed an "immunotumoroid" cell model system that recapitulates the tumor, its microenvironment, and necessary immune system components in patient-derived spheroids to enable ex vivo assessment of tumor response to ICI therapy. Immunotumoroids were developed from surgically resected renal cell carcinomas and bladder carcinomas selected for high tumor-infiltrating lymphocytes (TILs) and survived more than a month without media exchange. Immunohistochemistry was used to detect immune and non-immune cells in cryopreserved source tumors and the resulting immunotumoroids. Immunotumoroid response to ICIs (nivolumab, pembrolizumab, and durvalumab) and chemotherapy (cisplatin, gemcitabine, and paclitaxel) was monitored in real-time with Cytotox Red staining in an Incucyte device, and the immunotumoroid response was compared to retrospective clinical drug responses. Six of the 13 cases tested grew viable immunotumoroid models, with failed cases attributed to extensive tumor tissue necrosis or excess lymphocytes preventing spheroid formation. One successfully cultured case was excluded from the study due to low TIL infiltration (<5%) in the primary tumor sample. The five remaining models contained immune cells (CD4+ and CD8+ T cells, and macrophages), non-immune cells (fibroblasts), and tumor cells. Chemotherapy and ICI drugs were tested in immunotumoroids from 5 cases and compared to clinical outcomes where data was available. Four/five models showed cell killing in response to chemotherapy and two/five showed sensitivity to ICI. In three cases, the immunotumoroid model accurately predicted the patient's clinical response or non-response to ICIs or chemotherapy. Our immunotumoroid model replicated the multicellular nature of the tumor microenvironment sufficiently for preclinical ICI screening. This model could enable valuable insights into the complex interactions between cancer cells, the immune system, and the microenvironment. This is a feasibility study on a small number of cases, and additional studies with larger case numbers are required including correlation with clinical response.

Monolayer culture alters EGFR inhibitor response through abrogation of microRNA-mediated feedback regulation

Ex vivo drug screening is a potentially powerful tool for the future of cancer care, but the accuracy of results is contingent on the culture model. Both monolayer (2D) and spheroid (3D) culture systems offer advantages, but given the differences in mechanical environment, we hypothesized that that the suitability of one system over another would be critical for screening drugs with mechanical targets in mechanical tissues. HCC827 lung adenocarcinoma cells were challenged with EGFR tyrosine kinase inhibitors in monolayer and spheroid culture. RNA sequencing was performed on cells in both conditions to assess culture-induced transcriptional changes that could account for differences in drug response and differences in EGFR expression detected by immunostain. A microRNA microarray was performed to assess culture-induced differences in regulation of microRNA, and the impact of miR-146a-5p on drug response was verified by inhibition. Results were confirmed in human lung adenocarcinoma tissue. HCC827 spheroids were resistant to erlotinib and gefitinib, but significantly more sensitive in 2D culture. RNA-seq and immunostaining show a discrepancy in EGFR transcript and protein expression between the two conditions, which we attribute to miR-146a-5p. This microRNA targets EGFR and is differentially expressed between 2D and 3D culture. Inhibition of miR-146a-5p significantly increased erlotinib cytotoxicity, but validation in patient-derived spheroids suggests that the effect may be mutation-specific. Analysis of RNA-seq data suggests that cells in 2D culture become highly dependent on EGFR signaling to drive proliferation and cell spreading, resulting in a misleading level of sensitivity to EGFR TKIs, while the same cells in spheroid culture retain microRNA-driven EGFR feedback regulation that leaves them less vulnerable to EGFR inhibition. These findings underscore the need for close scrutiny of culture-induced effects on drug target regulation in model design for ex vivo drug screening.

Abstract 3578: A systematic NEN spheroid drug screen reveals a novel drug resistance mechanism in small bowel NETs

Abstract Neuroendocrine neoplasms (NENs) are rare cancers that arise from neuroendocrine cells. NENs are classified as well-differentiated neuroendocrine tumors (NETs) and poorly differentiated neuroendocrine carcinomas (NECs). Small bowel NETs (SBNETs) and pancreatic NETs (PNETs) are generally slow growing but they commonly metastasize to the liver and can become aggressive cancers. NECs are rapidly growing, and patients have poor prognosis. Little is known about the drug sensitivity profile of SBNETs, PNETs and NECs due to a paucity of cellular and animal models of these malignancies. We have successfully cultured NEN cells from clinical samples as patient-derived spheroids (PDS) and showed that they express appropriate tumor markers. We systematically screened 20 NEN (12 SBNET, 5 PNET, and 3 NEC) spheroid cultures against a library of 175 compounds (147 FDA-approved anti-cancer drugs, 8 lab selected compounds, and 20 structurally diverse molecules) and compared their drug sensitivity profiles to identify the most effective drug classes and to better understand the biology of each NEN subtype. Top drug hits were validated for their anti-tumor properties in NEN PDS and patient-derived xenograft (PDX) mouse models. Our NEN PDS cultures identified common and unique drug sensitivity profiles for each type of NEN. SBNET spheroids were more resistant to many classes of anti-cancer drugs, which was due to overexpression of cytochrome P450 genes. Consistent with clinical findings, PNET spheroids showed increased sensitivity to tyrosine kinase and mTOR/PI3K inhibitors compared to SBNET &amp; NEC spheroids. NEC spheroids showed the broadest sensitivity to many anti-neoplastic compounds. The top candidate drug identified from our screen was romidepsin, a histone deacetylase inhibitor. Romidepsin displayed anti-tumor properties in vitro and in vivo for all 3 NEN models and was highly synergistic with rapamycin, an mTOR inhibitor similar to the SBNET approved drug everolimus. Excitingly, low-dose romidepsin effectively inhibited tumor growth when combined with low-dose rapamycin in an SBNET PDX mouse model. These NEN PDS drug screens enabled direct drug testing in primary tumor cultures to identify promising drugs that could be used alone or in combination with currently approved-NEN therapies. Histone deacetylase inhibitors, such as romidepsin, may be effective against SBNETs. NEN PDS models also serve as a valuable resource for understanding the unique biology and mechanisms of drug resistance for specific NEN subtypes. Citation Format: Catherine G. Tran, Luis C. Borbon, Dane H. Tow, Jonathan Shilyansky, Guiying Li, James Egan, Scott K. Sherman, Ellen Abusada, Jing Tang, Ramaswamy govindan, Ryan C. Fields, Terry A. Braun, Carlos HF Chan, Chandrikha Chandrasekharan, Douglas Spitz, Dawn E. Quelle, Andrew M. Bellizzi, James R. Howe, Po Hien Ear. A systematic NEN spheroid drug screen reveals a novel drug resistance mechanism in small bowel NETs [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 3578.