Cancer Drug Screening Research Articles

Development of two layer fibrous scaffolds for 3D in vitro modelling: Effects of morphology and surface properties on cell proliferation, adhesion and drug sensitivity

Three-dimensional (3D) in vitro cell models are gaining popularity in cancer research compared to two-dimensional (2D) cell culture models because of their ability to more accurately represent the tissue microenvironment. This study introduces a novel approach for the fabrication of 3D cell culture models by combining modified melt-electrospinning technology and solution electrospinning to create 3D two-layer fibrous scaffolds. By manipulating electrospinning parameters (voltage 11–12 kV and feed rate 0.85–1.55 mg/min), we engineered poly (ε-caprolactone) (PCL) scaffolds with varying fiber diameters (8.9 ± 2.2 to 23.1 ± 3.4 μm) and pore sizes (32.7 ± 12.8 to 122.5 ± 21.1 μm). Non-thermal plasma (NTP) treatment at energy density of 0.23 J/cm2 enhanced surface hydrophilicity, evident in a significant reduction in water contact angles from 113 ± 0° to 64 ± 0°. A comprehensive set of characterization techniques, including X-ray diffraction (XRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA) confirmed that plasma treatment altered the surface wettability without compromising the scaffold's chemical stability or crystalline structure. Biological assays involving human breast cancer MDA-MB-231 cells and human glioblastoma U-87 MG cells demonstrated high cell viability, indicating biocompatibility of the scaffolds. A pronounced difference in proliferation patterns was noted between MDA-MB-231 and U-87 MG, with the latter fully utilizing voids for the formation of cell aggregates, especially in the “fine” scaffold, indicating the dependence of cell behavior on scaffold architecture, which must be adjusted according to the modelled system and duration. Furthermore, investigation of the therapeutic efficacy of doxorubicin revealed a distinct drug response in 3D cultures, EC50 values of doxorubicin in 3D culture was approximately twice higher compared to 2D. These findings underscore the potential of tailored PCL scaffolds to create more physiologically relevant models for cancer research, tissue engineering, and drug screening applications, highlighting the necessity for careful consideration of scaffold design to achieve the desired in vitro model.

Unveiling Cellular Microenvironments with a Near-Infrared Fluorescent Sensor: A Dual-Edge Tool for Cancer Detection and Drug Screening.

Mitochondria and lysosomes are pivotal intracellular organelles, and the injury or dysfunction of these organelles can trigger a range of pathological processes. Early diagnosis and treatment of cancer are of paramount importance due to cancer's status as a leading health threat. This study introduces a novel fluorescent probe, BDHV, for detecting mitochondrial and lysosomal viscosity and pH abnormalities in tumors, facilitating early cancer detection and screening of anticancer drugs. Under acidic conditions, the red fluorescence of the probe gradually increases with increasing viscosity. Conversely, in alkaline environments, an increase in viscosity leads to a decrease in green fluorescence and an increase in red fluorescence. The inclusion of a benzothiazole group endows BDHV with strong dual-targeting capability for mitochondria and lysosomes and without being affected by the mitochondrial membrane potential. Most notably, BDHV has potential applications for early cancer diagnosis and in effectively assessing the efficacy of various anticancer drugs.

An innovative electrochemical platform based on graphene for rapid screening of cancer drugs in blood plasma

This study introduces a new electrochemical platform for the rapid and sensitive detection of the potent anticancer drug epirubicin (EPB) in blood plasma. The platform utilizes a graphene/Zinc Oxide (ZnO) nanoparticle nanocomposite modified glassy carbon electrode (GCE), which was synthesized through a simple one-pot method where ZnO nanoparticles were dispersed onto graphene sheets. The graphene/ZnO/GCE nanocomposite was characterized using advanced techniques such as FE-SEM, EDS, XRD, FT-IR, and XPS in order to analyze its structural properties. The graphene/ZnO/GCE electrode exhibited minimal interference and demonstrated high selectivity and sensitivity in detecting EPB. The sensor's selectivity was validated by a recovery rate exceeding 98.40 % and a relative standard deviation below 4.65 %. The limits of detection and quantification were determined to be 6.0 nM and 9.0 nM, respectively, indicating the sensor's high sensitivity within a practical concentration range. With a sensitivity of 1.09509 µA/µM, the sensor exhibited a linear response to EPB concentrations ranging from 4.0 to 730.0 μM. Furthermore, successful detection of EPB in serum samples with a recovery rate exceeding 98.40 % underscores the practical utility of the graphene/ZnO/GCE sensor. The detailed characterization of the graphene/ZnO/GCE nanocomposite provides insights into its structural and chemical attributes, furthering its potential for biomedical applications.

MODL-04. ZEBRAFISH: THE FUTURE OF PEDIATRIC BRAIN CANCER RESEARCH

Abstract Pediatric brain cancers cause the highest rate of mortality amongst all childhood cancers. Most high-grade gliomas are difficult to treat mainly due to impermeability across the blood-brain barrier, or systemic toxicity. Here, we explore the benefits of zebrafish to assess drug toxicity, orthotopic pediatric brain cancer model development and drug screening. We assessed the toxicity of eight anti-cancer drugs with diverse mechanisms of action. In addition to the survival assay (LC50), we analyzed other teratogenic characteristics to demonstrate the detrimental developmental toxicity, which was critical in determining drug safety for future human usage. To develop fast and reproducible orthotopically xenografted pediatric brain cancer models, we injected approximately 80-100 mCherry expressing JHH-NF1-PA1 and TM-31 cells into the midbrain of 2 days post-fertilization (dpf) zebrafish embryos and imaged daily for next 4 days to monitor the survival, progression, and invasion of the injected cells. Our results show that both cell types were engrafted successfully and multiplied inside the developing zebrafish brain. As compared to JHH-NF1-PA1 cells, which mostly migrated caudally within the developing spinal cord, TM-31 migrated in multiple directions and metastasized five times more rapidly. To check the efficiency of VAL-083 and AZD-1775 in high-grade glioma, 2 dpf zebrafish embryos were injected with mCherry expressing HGG cells, SF8628. At one day post-injection (dpi), the xenografted embryos were exposed to different combinations of Val-083 and AZD-1775 for 72 hours. After 4 dpi, tumor cell invasion and migration in zebrafish were quantified. We found that the combination group had the lowest tumor burden with significantly lower fluorescence intensity, total area of migration, and reduced number of metastatic cells (p<0.001) compared to single or no drug treatment groups. Our study showed that zebrafish is a valuable, rapid, and cost-effective in-vivo pediatric brain cancer pre-clinical model to assess tumor proliferation, metastasis, and rapid drug screening.

Azoxymethane-induced carcinogenesis-like model of mouse intestine and mouse embryonic stem cell-derived intestinal organoids.

Tumor modeling using organoids holds potential in studies of cancer development, enlightening both the intracellular and extracellular molecular mechanisms behind different cancer types, biobanking, and drug screening. Intestinal organoids can be generated in vitro using a unique type of adult stem cells which are found at the base of crypts and are characterized by their high Lgr5 expression levels. In this study, we successfully established intestinal cancer organoid models by using both the BALB/c derived and mouse embryonic stem cells (mESCs)-derived intestinal organoids. In both cases, carcinogenesis-like model was developed by using azoxymethane (AOM) treatment. Carcinogenesis-like model was verified by H&E staining, immunostaining, relative mRNA expression analysis, and LC/MS analysis. The morphologic analysis demonstrated that the number of generated organoids, the number of crypts, and the intensity of the organoids were significantly augmented in AOM-treated intestinal organoids compared to non-AOM-treated ones. Relative mRNA expression data revealed that there was a significant increase in both Wnt signaling pathway-related genes and pluripotency transcription factors in the AOM-induced intestinal organoids. We successfully developed simple carcinogenesis-like models using mESC-based and Lgr5 + stem cell-based intestinal organoids. Intestinal organoid based carcinogenesi models might be used for personalized cancer therapy in the future.

Abstract 6773: Tissue engineered colorectal cancer microspheres for drug screening

Abstract Three-dimensional (3D) disease models have garnered widespread interest for use in later stages of the drug discovery process, such as preclinical efficacy and toxicology studies, due to their pathophysiologically relevant properties. However, there is a need and opportunity for 3D cancer models to be used earlier in the drug screening process. To meet this need, the 3D models must strike a balance between throughput, which includes scalability and uniformity, and physiological relevance, such as the ability to modulate key attribute of the tumor microenvironment. Here we report the creation of 3D colorectal cancer (CRC) tissue models, referred to as VivoSpheres, and demonstrated their relevance to cancer drug screening. The VivoSphere production platform couples tissue engineering toolkits with microfluidics, enabling the scalable production of engineered cancer microspheres. The model supports the long-term maintenance of the cancer cell phenotype. In a preliminary study, we were able to generate more physiologically relevant drug responses. We formed CRC VivoSpheres by encapsulating HT-29 CRC cells within poly(ethylene glycol)-fibrinogen hydrogel microspheres using our previously developed microfluidic platform. CRC VivoSpheres were rapidly produced with high cell densities (20 × 106 cells/ml) and high uniformity on day 0 with a coefficient of variation (COV) < 7%. This high uniformity was maintained for 15 days (COV ≤ 10%), which is critical for long-term dose studies. The cells maintained high viability and showed high proliferative capability with a significant increase in colony size and expression of Ki67 up to day 29. The encapsulated cells maintained the CRC phenotype over time with the expression of CD44 (cancer stem cell marker) and CK20 (CRC marker). After establishing shipping conditions that maintained cell viability for remote use, the HT-29 VivoSpheres were shipped to the oncology team at Southern Research for drug testing. The CRC VivoSpheres were treated with DMSO, GANT61, and SRI-38832, the latter two of which are GLI1 inhibitors. Phase contrast images and western blot were used to assess the response of CRC VivoSpheres to the treatments. Oncogenic GLI1 transcription activity and NBS1 overexpression have been found to contribute to chemotherapeutic resistance, negating the anti-tumor effects of 5-fluorouracil. While 2D cultured HT-29s responded to treatment with GANT61, HT-29 VivoSpheres continued to express NBS1 following GANT1 treatment, but downregulated NBS1 in response to the GLI1 inhibitor SRI-38832, which is the same response Southern Research has seen in in vivo tumor models. In conclusion, we have developed tissue-engineered 3D CRC models that hold promise for use in drug screening. These models have demonstrated an initial capability to reproduce the CRC phenotype and mimic in vivo drug response. Citation Format: Yuan Tian, Mohammadjafar Hashemi, Benjamin Anbiah, Ruowen Zhang, Iman Hassani, Rebecca Boohaker, Elizabeth Lipke. Tissue engineered colorectal cancer microspheres for drug screening [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 6773.

Abstract 221: Novel organotypic patient derived primary tumor tissues for oncology drug safety and efficacy studies

Abstract Organotypic, three-dimensional (3D) cancer tissue models (OCTM) have enabled investigators to develop predictive markers that can be used in preclinical cancer drug development. However, a drawback of the existing models is that they are mostly based on cell lines based are not physiological. In vitro development of 3D tumoroids using patient derived primary tumors (PDPT) in tumor microenvironment (TME) will have clinical significant and can predict human responses to cancer therapeutics. Here, we describe the generation of OCTM from patient-derived metastatic colorectal cancer, the second most deadly cancer. PDPT were grown in Matrigel for 5 passages and for biobanking purposes tumoroids were further expanded in faCellitate 384 well plates or Sun Bioscience plates to harvest large amounts of well-defined tumoroids cultured for over 21 days. Expanded tumoroids were collected and cryopreserved or gently mixed several times using a pipet tip to break up tumoroids into smaller fragments. These tumor fragments were mixed with endothelial cells, dendritic cells, fibroblasts and embedded in a collagen matrix to simulate TME. The collagen-cell mixture was then added to Transwells and induced to gel to form a collagen gel matrix that simulates TME. The tumoroids were cultured with a specialized medium that supports the growth of complex cell types. The tumor development was monitored daily using bright field microscopy and image analysis was used to monitor tumor size. To evaluate the utility of the OCTM, treatment was initiated at day 10 of the culture period by exposing tissues with the chemotherapeutic drug Cisplatin on days 0, 2, 4, 7, 9, and 11. After each treatment, OCTM tissues (N=2) were fixed for H&E and live/dead staining. Overall, the results of the study showed that: 1) PDPT can be expanded for 5 passages (this low passage number is not expected to change the phenotype of the tumor) and further expanded in high throughput format, 384 well plates, 2) the PDPT could be cultured with multiple cell types in a collagen-gel matrix to recreate the TME, 3) histological evaluation of the OCTM showed a glandular-like tumoroid, 4) immunohistochemical staining revealed that the PDPT in the in vivo-like microenvironment were positive for CK20, and 5) the PDPT shrank following 3 treatment cycles with Cisplatin (day 4). Conclusion: The data shows metastatic colorectal PDPT can be expanded in vitro and cultured in a complex tumor microenvironment on tissue culture inserts. These OCTMs can be used to screen drug safety and efficacy. This study opens new avenues for high-throughput cancer drug screening format using PDPT embedded in a in vivo like TME. This model can be used as a tool for personalized oncology drug testing and for translational assays tailored to other cancer types from our patient derived primary tumor pool. Citation Format: Seyoum Ayehunie, Megan Groves, Bryda Bryda, Alex Armento, Anthony Tolcher. Novel organotypic patient derived primary tumor tissues for oncology drug safety and efficacy studies [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 221.

Using 3-Dimensional Cultures to Propagate Genetically Modified Lung Organoids.

Transformed lung organoids have extensive applications in lung cancer modeling and drug screening. Traditional two-dimensional (2D) cultures fail to propagate a large subpopulation of murine primary tumors in vitro. However, three-dimensional (3D) air-liquid interface (ALI) cultures, which are employed to grow normal lung organoids, can be used to efficiently culture cancerous lung tumor cells. Here, we detail a procedure for cultivating genetically modified lung organoids in 3D-ALI cultures. This protocol contains two parts. The first part describes how to transduce lung epithelial cells, which are either freshly sorted from lungs or from actively growing murine organoids, with virus in order to modify gene expression. The target lung cells are incubated with virus for 1-2h for transduction. Then, the transduced cells are thoroughly washed and mixed with stromal support cells and Matrigel and are loaded into transwell inserts for culture and validated for genetic modifications through downstream assays. The second part describes how to isolate tumor cells growing orthotopically in genetically engineered mouse models to produce organoid cell lines that can be used for ex vivo drug discovery assays. For this protocol, tumors are isolated from lungs of mice, finely chopped and washed. Then, tumor chunks are mixed with Matrigel for 3D-ALI culture. Finally, organoids budding from tumor chunks are trypsinized and passaged to establish an organoid line. Together these two protocols provide a promising platform to study the genesis, progression, and treatment of lung cancer.

Suspension Culture System for Isolating Cancer Spheroids using Enzymatically Synthesized Cellulose Oligomers.

Isolating cancer cells from tissues and providing an appropriate culture environment are important for a better understanding of cancer behavior. Although various three-dimensional (3D) cell culture systems have been developed, techniques for collecting high-purity spheroids without strong stimulation are required. Herein, we report a 3D cell culture system for the isolation of cancer spheroids using enzymatically synthesized cellulose oligomers (COs) and demonstrate that this system isolates only cancer spheroids under coculture conditions with normal cells. CO suspensions in a serum-containing cell culture medium were prepared to suspend cells without settling. High-purity cancer spheroids could be separated by filtration without strong stimulation because the COs exhibited antibiofouling properties and a viscosity comparable to that of the culture medium. When human hepatocellular carcinoma (HepG2) cells, a model for cancer cells, were cultured in the CO suspensions, they proliferated clonally and efficiently with time. In addition, only developed cancer spheroids from HepG2 cells were collected in the presence of normal cells by using a mesh filter with an appropriate pore size. These results indicate that this approach has potential applications in basic cancer research and cancer drug screening.

Multi-level characteristics recognition of cancer core therapeutic targets and drug screening for a broader patient population

Multi-level characteristics recognition of cancer core therapeutic targets and drug screening for a broader patient population

A one-pot transcriptional assay method that detects the tumor biomarker FEN1 based on its flap cleavage activity

BackgroundDetection of tumor biomarkers in body fluids is a significant advancement in cancer treatment because it allows diagnosis without invasive tissue biopsies. Nucleases have long been regarded as a potential class of biomarkers that can indicate the occurrence and progression of cancers. Among these, flap endonuclease 1 (FEN1) plays an important role in DNA replication and repair, and also overexpressed in abnormally proliferating cells such as cancer cells. FEN1 is thus considered to be a potential biomarker as well as a target for cancer therapy. ResultsWe developed a novel method for detecting FEN1 based on its specific endonuclease activity which incises bifurcated nucleic acids (flaps), in combination with in vitro transcription. Developed method uses a simple DNA structure (substrate DNA) carrying a short 5′-flap sequence, and a single-stranded sensor DNA encoding the Broccoli light-up aptamer. When the assay mixture was supplied with a FEN1-containing sample, the flap sequence encoding the sense sequence of T7 promoter was cleaved and released from the substrate DNA. Because the sensor DNA was designed to carry the Broccoli RNA aptamer under the antisense sequence of T7 promoter, hybridization of the excised flap onto the sensor DNA initiated the transcription of the Broccoli RNA aptamer, enabling determination of the FEN1 titer based on the fluorescence of transcribed Broccoli aptamer. By using a combination of FEN1-mediated generation of a short oligonucleotide and subsequent oligonucleotide-dependent in vitro transcription, this method could detect FEN1 in biological samples within 1 h. Significance and noveltyDeveloped method enables the detection of FEN1 by a simple one-pot reaction. It can detect sub-nanomolar concentrations of FEN1 within an hour, and has the potential to be used for cancer diagnosis, prognosis, and drug screening. It also enables easy identification of compounds that inhibit FEN1 activity and is thus a versatile platform for screening anti-cancer drugs. We anticipate that the basic principles of this assay can be applied to detect other biomolecules, such as nucleic acids.

Automation, live-cell imaging, and endpoint cell viability for prostate cancer drug screens.

Androgen deprivation therapy (ADT) is the standard of care for high risk and advanced prostate cancer; however, disease progression from androgen-dependent prostate cancer (ADPC) to lethal and incurable castration-resistant prostate cancer (CRPC) and (in a substantial minority of cases) neuroendocrine prostate cancer (NEPC) is common. Identifying effective targeted therapies is challenging because of acquired resistance to established treatments and the vast heterogeneity of advanced prostate cancer (PC). To streamline the identification of potentially active prostate cancer therapeutics, we have developed an adaptable semi-automated protocol which optimizes cell growth and leverages automation to enhance robustness, reproducibility, and throughput while integrating live-cell imaging and endpoint viability assays to assess drug efficacy in vitro. In this study, culture conditions for 72-hr drug screens in 96-well plates were established for a large, representative panel of human prostate cell lines including: BPH-1 and RWPE-1 (non-tumorigenic), LNCaP and VCaP (ADPC), C4-2B and 22Rv1 (CRPC), DU 145 and PC3 (androgen receptor-null CRPC), and NCI-H660 (NEPC). The cell growth and 72-hr confluence for each cell line was optimized for real-time imaging and endpoint viability assays prior to screening for novel or repurposed drugs as proof of protocol validity. We demonstrated effectiveness and reliability of this pipeline through validation of the established finding that the first-in-class BET and CBP/p300 dual inhibitor EP-31670 is an effective compound in reducing ADPC and CRPC cell growth. In addition, we found that insulin-like growth factor-1 receptor (IGF-1R) inhibitor linsitinib is a potential pharmacological agent against highly lethal and drug-resistant NEPC NCI-H660 cells. This protocol can be employed across other cancer types and represents an adaptable strategy to optimize assay-specific cell growth conditions and simultaneously assess drug efficacy across multiple cell lines.

Engineered bone marrow as a clinically relevant ex vivo model for primary bone cancer research and drug screening

Osteosarcoma (OS) is the most common primary malignant bone cancer in children and adolescents. While numerous other cancers now have promising therapeutic advances, treatment options for OS have remained unchanged since the advent of standard chemotherapeutics and offer less than a 25% 5-y survival rate for those with metastatic disease. This dearth of clinical progress underscores a lack of understanding of OS progression and necessitates the study of this disease in an innovative system. Here, we adapt a previously described engineered bone marrow (eBM) construct for use as a three-dimensional platform to study how microenvironmental and immune factors affect OS tumor progression. We form eBM by implanting acellular bone-forming materials in mice and explanting the cellularized constructs after 8 wk for study. We interrogate the influence of the anatomical implantation site on eBM tissue quality, test ex vivo stability under normoxic (5% O2) and standard (21% O2) culture conditions, culture OS cells within these constructs, and compare them to human OS samples. We show that eBM stably recapitulates the composition of native bone marrow. OS cells exhibit differential behavior dependent on metastatic potential when cultured in eBM, thus mimicking in vivo conditions. Furthermore, we highlight the clinical applicability of eBM as a drug-screening platform through doxorubicin treatment and show that eBM confers a protective effect on OS cells that parallel clinical responses. Combined, this work presents eBM as a cellular construct that mimics the complex bone marrow environment that is useful for mechanistic bone cancer research and drug screening.

Extracellular matrix remodelling and stiffening contributes to tumorigenesis of salivary carcinoma ex pleomorphic adenoma——A study based on patient-derived organoids

BackgroundSalivary carcinoma ex pleomorphic adenoma (CXPA) is defined as a carcinoma that develops from benign pleomorphic adenoma (PA). Abnormally activated Androgen signaling pathway and amplification of HER-2/neu(ERBB-2) gene are known to be involved in CXPA tumorigenesis. Recent progress in tumour microenvironment research has led to identification that extracellular matrix (ECM) remodelling and increased stiffness act as critical contributing role in tumour carcinogenesis. This study examined ECM modifications to elucidate the mechanism underlying CXPA tumorigenesis.ResultsPA and CXPA organoids were successfully established. Histological observation, immunohistochemistry (IHC), and whole-exome sequencing demonstrated that organoids recapitulated phenotypic and molecular characteristics of their parental tumours. RNA-sequencing and bioinformatic analysis of organoids showed that differentially expressed genes are highly enriched in ECM-associated terms, implying that ECM alternations may be involved in carcinogenesis. Microscopical examination for surgical samples revealed that excessive hyalinized tissues were deposited in tumour during CXPA tumorigenesis. Transmission electron microscopy confirmed that these hyalinized tissues were tumour ECM in nature. Subsequently, examination by picrosirius red staining, liquid chromatography with tandem mass spectrometry, and cross-linking analysis indicated that tumour ECM was predominantly composed of type I collagen fibers, with dense collagen alignment and an increased level of collagen cross-linking. IHC revealed the overexpression of COL1A1 protein and collagen-synthesis-related genes, DCN and IGFBP5 (p < 0.05). Higher stiffness of CXPA than PA was demonstrated by atomic force microscopy and elastic imaging analysis. We utilized hydrogels to mimic ECM with varying stiffness degrees in vitro. Compared with softer matrices (5Kpa), CXPA cell line and PA primary cells exhibited more proliferative and invasive phenotypes in stiffer matrices (50Kpa, p < 0.01). Protein-protein interaction (PPI) analysis of RNA-sequencing data revealed that AR and ERBB-2 expression was associated with TWIST1. Moreover, surgical specimens demonstrated a higher TWIST1 expression in CXPA over PA. After knocking down TWIST1 in CXPA cells, cell proliferation, migration, and invasiveness were significantly inhibited (p < 0.01).ConclusionDeveloping CXPA organoids provides a useful model for cancer biology research and drug screening. ECM remodelling, attributed to overproduction of collagen, alternation of collagen alignment, and increased cross-linking, leads to increased ECM stiffness. ECM modification is an important contributor in CXPA tumorigenesis.

3D Porous Scaffold-Based High-Throughput Platform for Cancer Drug Screening.

Natural polymer-based porous scaffolds have been investigated to serve as three-dimensional (3D) tumor models for drug screening owing to their structural properties with better resemblance to human tumor microenvironments than two-dimensional (2D) cell cultures. In this study, a 3D chitosan-hyaluronic acid (CHA) composite porous scaffold with tunable pore size (60, 120 and 180 µm) was produced by freeze-drying and fabricated into a 96-array platform for high-throughput screening (HTS) of cancer therapeutics. We adopted a self-designed rapid dispensing system to handle the highly viscous CHA polymer mixture and achieved a fast and cost-effective large-batch production of the 3D HTS platform. In addition, the adjustable pore size of the scaffold can accommodate cancer cells from different sources to better mimic the in vivo malignancy. Three human glioblastoma multiforme (GBM) cell lines were tested on the scaffolds to reveal the influence of pore size on cell growth kinetics, tumor spheroid morphology, gene expression and dose-dependent drug response. Our results showed that the three GBM cell lines showed different trends of drug resistance on CHA scaffolds of varying pore size, which reflects the intertumoral heterogeneity across patients in clinical practice. Our results also demonstrated the necessity to have a tunable 3D porous scaffold for adapting the heterogeneous tumor to generate the optimal HTS outcomes. It was also found that CHA scaffolds can produce a uniform cellular response (CV < 0.15) and a wide drug screening window (Z' > 0.5) on par with commercialized tissue culture plates, and therefore, can serve as a qualified HTS platform. This CHA scaffold-based HTS platform may provide an improved alternative to traditional 2D-cell-based HTS for future cancer study and novel drug discovery.

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models recapitulating bone- and bone marrow-specific functions, including vascularization, that are compatible with drug screening are highly desirable. Such models can bridge the gap between simplistic, structurally irrelevant two-dimensional (2D) in vitro models and the more expensive, ethically challenging in vivo models. This article describes a controllable three-dimensional (3D) co-culture assay based on engineered poly(ethylene glycol) (PEG) matrices for the generation of vascularized, osteogenic bone-marrow niches. The PEG matrix design allows the development of 3D cell cultures through a simple cell seeding step requiring no encapsulation, thus enabling the development of complex co-culture systems. Furthermore, the matrices are transparent and pre-cast onto glass-bottom 96-well imaging plates, rendering the system suitable for microscopy. For the assay described here, human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are cultured first until a sufficiently developed 3D cell network is formed. Subsequently, GFP-expressing human umbilical vein endothelial cells (HUVECs) are added. The culture development is followed by bright-field and fluorescence microscopy. The presence of the hBM-MSC network supports the formation of vascular-like structures that otherwise would not form and that remain stable for at least 7 days. The extent of vascular-like network formation can easily be quantified. This model can be tuned toward an osteogenic bone-marrow niche by supplementing the culture medium with bone morphogenetic protein 2 (BMP-2), which promotes the osteogenic differentiation of the hBM-MSCs, as assessed by increased alkaline phosphatase (ALP) activity at day 4 and day 7 of co-culture. This cellular model can be used as a platform for culturing various cancer cells and studying how they interact with bone- and bone marrow-specific vascular niches. Moreover, it is suitable for automation and high-content analyses, meaning it would enable cancer drug screening under highly reproducible culture conditions.

Synthetic lethality prediction in DNA damage repair, chromatin remodeling and the cell cycle using multi-omics data from cell lines and patients.

Discovering synthetic lethal (SL) gene partners of cancer genes is an important step in developing cancer therapies. However, identification of SL interactions is challenging, due to a large number of possible gene pairs, inherent noise and confounding factors in the observed signal. To discover robust SL interactions, we devised SLIDE-VIP, a novel framework combining eight statistical tests, including a new patient data-based test iSurvLRT. SLIDE-VIP leverages multi-omics data from four different sources: gene inactivation cell line screens, cancer patient data, drug screens and gene pathways. We applied SLIDE-VIP to discover SL interactions between genes involved in DNA damage repair, chromatin remodeling and cell cycle, and their potentially druggable partners. The top 883 ranking SL candidates had strong evidence in cell line and patient data, 250-fold reducing the initial space of 200K pairs. Drug screen and pathway tests provided additional corroboration and insights into these interactions. We rediscovered well-known SL pairs such as RB1 and E2F3 or PRKDC and ATM, and in addition, proposed strong novel SL candidates such as PTEN and PIK3CB. In summary, SLIDE-VIP opens the door to the discovery of SL interactions with clinical potential. All analysis and visualizations are available via the online SLIDE-VIP WebApp.

Ultrasensitive PEC cytosensor for breast cancer cells detection and inhibitor screening based on plum-branched CdS/Bi2S3 heterostructures

Breast cancer is the most common malignant tumor in women, which seriously threatens the life and health of patients. Therefore, facile and sensitive detection of human breast cancer cells is crucial for cancer diagnosis. In this work, plum-branched CdS/Bi2S3 heterostructures (CdS/Bi2S3 HSs) were synthesized under hydrothermal condition, whose photoelectrochemical (PEC) property and biocompatibility were scrutinously investigated. In parallel, a signal amplification strategy was designed based on immune recognition between epidermal growth factor receptor (EGFR) overexpressed on membrane of breast cancer cells MDA-MB-231 and its aptamer. Integration of the above together, a highly sensitive PEC cytosensor was developed for analysis of target MDA-MB-231 cells, exhibiting a wider linear range of 1 × 102 ∼ 3 × 105 cells mL−1 with a limit of detection (LOD) down to 6 cells mL−1 (S/N = 3). Further, the biosensor was explored for anticancer drug (e.g., dacomitinib) screening by monitoring the variations in the PEC signals of the expressed EGFR upon drug stimulation. The obtained CdS/Bi2S3 HSs are identified as promising and feasible photoactive material for determination of cancer cells and drug screening in clinic and related research.

Abstract 3118: Predicting response to PARP inhibitors in pediatric cancer via synthetic lethal networks

Abstract Introduction: PARP inhibitors (PARPi) are known to have a synthetic lethal (SL) relationship with BRCA1/2 mutated cancers (ie: BRCA loss confers sensitivity to PARPi), but heterogeneity in response exists. PARPi have also found utility in BRCA-non mutated cancers, suggesting other factors may contribute to response. Recently, PARPi trials in pediatric solid tumors have had mixed results. SL interactions have been shown to exist in a network relationship, as such the aim of this study was to identify a clinically relevant SL signature (including synthetic dosage lethality and synthetic rescue) that would predict response to PARPi in pediatric cancers. Methods: Pre-treatment genomic copy number alteration, RNA sequencing, and survival data from 519 pediatric cancer patients (INFORM Consortium, 91% solid tumor) were obtained. SL partner genes for PARPi were selected if co-alteration of the drug target (PARP1/2/3) and candidate gene was 1) found to be under-represented in the patient transcriptome, 2) associated with improved survival, and 3) the genes were phylogenetically similar to PARP1/2/3. The 100 gene signature was validated on two independent pediatric cancer drug screens: in-vivo orthotopic patient-derived xenograft (OPDX) mouse models (n=24) and in-vitro PRISM cell lines (n=54). It was also validated on a cisplatin (PARP surrogate) treated pediatric osteosarcoma clinical trial (n=37). Comparisons were made to 1) a signature generated with the same pipeline using adult TCGA data, 2) a validated adult synthetic lethality pipeline (SELECT), and 3) a published 60-gene PARPi response signature (BRCAness score). Results: The pediatric signature predicted response in all drug screens evaluated. Four cell line screens: Talazoparib (AUC 0.72, p = 0.003), Rucaparib (AUC 0.69, p = 0.01), Niraparib (AUC 0.67, p = 0.03), Olaparib (AUC 0.64, p = 0.06), and two OPDX screens: Talazoparib (AUC 0.74, p = 0.03), Olaparib (AUC 0.74, p = 0.03). The pediatric signature outperformed both adult signatures (AUCs 0.34-0.61, all p’s &amp;gt;0.05), and the BRCAness score (AUCs 0.41-0.53, p’s &amp;gt;0.05) except in the Olaparib cell line (BRCAness: AUC 0.68, p = 0.01). In the pediatric osteosarcoma dataset, the pediatric signature predicted death (AUC 0.68) and two-year recurrence (AUC 0.72), outperforming the other measures (death: AUC 0.49-0.66; two-year recurrence: AUCs 0.36-0.44). Discussion: We developed a pediatric derived, clinically relevant, synthetic lethal signature that predicts response to PARPi in pan-pediatric cancer drug screens and in a cisplatin treated osteosarcoma clinical trial. Our findings support the goal of identifying precision oncology frameworks to improve the care of children with cancer. Citation Format: Matthew Nagy, Fiorella Schischlik, Kun Wang, Nishanth Ulhas Nair, E. Michael Gertz, Lipika R. Pal, Natalie Jaeger, Christopher Previti, Dina ElHarouni, Stefan M. Pfister, Eytan Ruppin. Predicting response to PARP inhibitors in pediatric cancer via synthetic lethal networks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3118.

Abstract 5272: 3D cell-culture strategy for screening novel agents in Fanconi anemia chemoprevention

Abstract Fanconi anemia (FA) patients have DNA repair mutations predisposing them to oral cancers so treatment with non-DNA damaging agents is highly desired. As cancer experimental therapeutics advance towards study paradigms that more carefully mimic physiology, e.g. organoids and spheroids. Presently, we are using a 3D cell culture method emphasizing layers of extracellular matrix (ECM) as FA premalignant lesions retain epithelial architecture. We conducted 3D screening studies using 2 layered geometries: 1) nanofibers coated with 1 ng/mL 2:1 Collagen III/IV (C3C4) then 100 pg/mL 2.75:1 Tenascin C/Fibronectin (TNFN) and 2) polystyrene layered with 50 ug/mL C3C4 then 3.5 ug/mL TNFN. We developed these coatings to maximize lipid raft recovery, a drug transport feature lost in 2D culture. The ECM impact on FA cell drug transport was characterized by comparing effective nuclear delivery of a 20nm nanoencapsulated FITC-labeled RNAi compound (s50-TBG-RNAi3UTR), known to require lipid rafts, to the same oligo formulated with Dotap in a FA HNSCC cell line (FA1). Using confocal microscopy and IMARS software to segment delivery at maximal nuclear intensity into nuclear or endolysosomal compartments, we found, for FA1 cells on ECM, nuclear oligo signal colocalization with nuclear compartment ± coefficient of variation was 62 ± 2.2% for the capsule vs 9.8 ± 8.5% for Dotap oligo. On glass, nuclear delivery was 10 ± 52% for s50 capsule vs 25 ± 32% for Dotap. Endolysosomal compartment sequestration was respectively 28 ± 12, 62 ± 10, 44 ± 19 and 51 ± 15%. This illustrates a relevant decrease in variation in FA cell biology from ECM addition. Next, we studied metformin (Met) and pioglitazone (Pio)(agents in current oral cancer prevention studies) combined with G2/M blockade inhibitors, MK1775 (Wee1 kinase inhibitor) and GSK461364 (PLK inhibitor) in FA1s. Inhibiting G2/M blockade, already induced by FA-derived DNA damage, could promote inappropriate cell division, mitotic catastrophe (MOC) and death. In initial 2D FA1 growth studies, increased death was observed with Met plus MK1775. 3D studies, in contrast, showed increased cell death for FA1s treated with Pio plus either MK1775 or s50-TBG-RNAiCK2 (RNAiCK2). Confocal mechanistic studies in cells plated on ECM and treated for 18 hours, indicated Pio + MK1775 inhibited Survivin and ß-catenin upregulation and FA1 cells did proceed into MOC and death. For Pio (PPARg agonist) + RNAiCK2, RXRa was dephosphorylated at S260, enabling its escape from cytosolic sequestration to bind nuclear PPARg, initiating differentiation. Differentiation was indicated by upregulation panKeratin and Transglutaminase-3. We conclude combination therapies with high interest agents in FA-associated oral cancer can be performed in 3D culture systems and might confirm drug mechanisms of action, thus augmenting other standard methods of cancer drug evaluation and screening (e.g. cell proliferation and clonogenicity). Citation Format: Gretchen M. Unger, Beverly R. Wuertz, Mary E. Brown, Sanjana Arji, Janeen H. Trembley, Frank G. Ondrey. 3D cell-culture strategy for screening novel agents in Fanconi anemia chemoprevention. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5272.