Cell Suspension Research Articles

A case of hemolytic-uremic syndrome that developed in a 5-year-old child due to acute intestinal infection with hemocolitis

Purpose: to demonstrate a case of hemolytic-uremic syndrome (HUS) in a 5-year-old child associated with an acute intestinal infection of unknown etiology, occurring with hemocolitis. Results. During clinical and laboratory observation, edematous syndrome, oligoanuria, severe microangiopathic hemolytic anemia (decrease in hemoglobin, haptoglobin, increase in Lactate dehydrogenase), thrombocytopenia, consumption coagulopathy, hyperazotemia, acute kidney injury (AKI) according to KDIGO grade 2 (increase in serum creatinine more than 2 times) developed. As part of complex therapy with balanced crystalloid solutions with stimulation of diuresis, it was possible to achieve rapid restoration of diuresis on the 2nd day of hospital stay without the use of renal replacement therapy; he also received antibiotics, anticoagulants, transfusion of fresh frozen plasma and red blood cell suspension. Hyperazotemia was relieved within 5 days, as was thrombocytopenia. The follow-up recovery of renal function showed.

Growth promotion and biological control of fungal diseases in tomato by a versatile rhizobacterium, Pseudomonas chlororaphis subsp. aureofaciens SPS-41

Pseudomonas chlororaphis subsp. aureofaciens SPS-41, a beneficial bacterium isolated from rhizosphere of sweet potato, has been explored as a biological control agent for the management of soil-borne diseases. In this study, its plant growth-promoting and antifungal properties against two major tomato fungal pathogens (Fusarium oxysporum and Botrytis cinerea) were evaluated using in vitro and in vivo experiments. Strain SPS-41 produced indole-3-acetic acid (IAA), siderophore, biofilm, protease, amylase, chitinase, and could solubilize phosphate. It also inhibited fungal mycelial growth, with an inhibition rate of more than 80% in dual culture assays. Volatile organic compounds (VOCs) produced by SPS-41 also exhibited strong antifungal activity. In addition, SPS-41 promoted the growth of tomato seedlings in greenhouse conditions, and the severity of wilt disease and grey mold decreased significantly with rhizospheric inoculation of cell suspension and VOC fumigation comparing to untreated controls. The composition of VOCs emitted by SPS-41 was analyzed, and 57 volatiles were identified by SPME/GC-MS. Of these, 2-tridecanone, 2-undecanone, 2-nonanone, 2-acetylthiazole, 9-decenyl acetate, 2-hexadecanol, α-farnesene, and nonanal were the major volatiles. Nonanal, 2-nonanone and 2-acetylthiazole were confirmed to inhibit the growth of all three pathogens. The complete genome of strain SPS-41 was sequenced and annotated. We identified a series of gene clusters responsible for the synthesis of active secondary metabolites with antimicrobial and biocontrol functions. Moreover, several genes related to plant growth promotion, such as nitrogen fixation and indole-3-acetic acid and siderophore biosynthesis were also identified. This research indicates that strain SPS-41 is a plant growth promoting bacterium with the potential for development as a new biocontrol agent for tomato fungal pathogens.

Synthetic auxin herbicide 2,4-D and its influence on a model BY-2 suspension.

2,4-D is a broadly used auxin herbicide. The presence of the 2,4-D synthetic auxin in the medium is imperative for long-term BY-2 tobacco suspension viability. The precise mechanism of this symbiosis of the suspension and the synthetic auxin remains unclear. Our goal was to study the hormonal regulation of the growth of the cell suspension; and to describe the experiments clarifying the interaction between the chosen growth regulators and phytohormones on the cellular level, specifically between the 2,4-D synthetic auxin and the native stress phytohormone - ethylene. This study examined the influence of low 2,4-D concentrations stimulating cell growth in vitro as well as the influence of high herbicide concentrations on the model tobacco BY-2 suspension. The culture took 6 days. Different parameters were evaluated, including the influence of different 2,4-D concentrations on the production of the phytohormone ethylene and its precursor 1-Aminocyclopropane-1-carboxylic acid (ACC) in the tobacco cells. The content of 2,4-D in the cells and the medium was established. The observations of the morphological changes showed that a heavy impregnation of the cell walls taking place depending on the concentration of 2,4-D. A dramatic increase in protective polysaccharides and a remodulation of the cell walls by the formation of a pectin shield in artificial conditions were expected and observed. At the same time, massive production of the stress phytohormone ethylene took place, and, because of that, plant mutagenicity, anomalous tumour-type proliferation growth, and the production of supercells were observed. The hypothesis of the protective shield is discussed.

Abstract 1489: The role of EPAS1 and TEAD1 in gastric cancer liver metastasis

Abstract Gastric cancer ranks fifth in incidence and fourth for cancer-related mortality worldwide. While the liver is widely recognized as the leading site of metastasis in gastric cancer, the intricate mechanisms underpinning its tropism toward the liver and the hematogenous metastasis process remain poorly understood and demand further investigation. Hematogenous metastasis encompasses the generation of circulating tumor cells from the primary tumor site and their subsequent attachment to the metastatic niche. This process involves the transition between adherent and suspension states. In our investigation of anchorage dependency, we identified 20 transcription factors upregulated in suspension cells compared to adherent cells and 18 transcription factors upregulated in adherent cells relative to suspension cells, respectively. To investigate factors involved in gastric cancer metastasis, RNA sequencing of paired primary and metastatic tissue samples obtained from 15 patients diagnosed with advanced gastric cancer was conducted, which disclosed the selective upregulation of EPAS1 and TEAD1 in metastatic liver tissue compared to the matched primary tumor tissue and the metastatic tissue originating from other sites. Through in vitro analysis, we observed a portion of adherent cells transitioning into suspension cells when subjected to a hyperdense environment. We also observed cell reattachment upon harvesting and replating these suspension-state transited cells. Comparing EPAS1 and TEAD1 expression in these states unveiled increased expression of EPAS1 and TEAD1 in reattached cells relative to suspension-state cells. We established a gastric cancer metastasis mouse model introducing gastric cancer cells into the mouse circulatory system via intracardiac injection to validate our findings. In vivo imaging was utilized to confirm systemic metastasis. After fluorescence-based cell sorting, EPAS1 and TEAD1 expression in cancer cells collected from the blood, liver, and ovary were assessed. We detected higher expression of EPAS1 and TEAD1 in liver metastasis compared to the circulating tumor cells. Furthermore, EPAS1 and TEAD1 were upregulated in liver metastatic cancer cells compared to ovarian metastatic cancer cells. Our findings underscore EPAS1 and TEAD1 as pivotal factors governing gastric cancer liver tropism and metastasis, offering insights that may contribute to targeted interventions in the clinical landscape. Citation Format: DONG KI Lee, Hyun Woo Park, Jae-Ho Cheong, Heon Yung Gee. The role of EPAS1 and TEAD1 in gastric cancer liver metastasis [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 1489.

Abstract 6786: Assessment of immune cell infiltration and cancer metastatic potential in Akura™ Immune Flow Chip - A microfluidic 3D spheroid system

Abstract Evaluation of novel therapeutics often fails to reliably predict severe complications in patients, especially when circulating cells are involved. The reasons may include inter-species differences upon using animal models or the lack of relevant in vitro systems. Moreover, existing in vitro systems mostly rely on static investigations of different cell types leading to overestimation of effects due to non-representative cell-tissue interactions. The incorporation of medium flow may facilitate to mimic physiological conditions more closely. To tackle current limitations of perfused organ-on-chip approaches, we developed a microfluidic chip and operation concept, which prevents undesired sedimentation and accumulation of cells in suspensions. The Akura™ Immune Flow platform was designed to study flow-dependent recruitment of circulating cells to different tissue spheroids via gravity-driven perfusion over several days. The Akura™ Immune Flow platform features standardized formats for co-culturing up to seven spheroids with suspended cells in each microfluidic channel. On-chip staining protocols enabled monitoring of the co-culture. The system allowed for application and extractions of cellular components and liquid for further off-chip analysis. Primary tissue and cell line spheroids (liver and tumor) could be retained in the respective chambers and remained viable over six to 14 days. Circulating cells were kept viable and in suspension for up to six days (PBMCs) and14 days (Prostate Cancer (PCa) cells) respectively. PBMCs were not activated by flow-induced shear forces. Unspecific stimulation of T-cells led to physiological expression of early and late activation markers (CD69 and CD25), which were detected by flow cytometry in periodically collected cells. Multi-photon microscopy imaging confirmed that PBMCs interacted with and infiltrated into 3D tumor spheroids. Application of PCa cells to primary liver spheroids, cultured in Akura™ Immune Flow chips allowed for assessment of their invasive potential. Confocal imaging revealed adhesion of cancer cells to the liver spheroids and offers the potential to study the ability of tissue invasion at later time points. The simple design of the chip facilitates user-friendly operation and high-throughput implementation in cancer research and drug testing. The selected applications support the relevance of using flow solutions for physiologically more complex processes. This work constitutes an important step towards device application as a tool for basic research as well as for efficacy studies of immunotherapeutic drug candidates Citation Format: Tamara Häfeli, Silas Riss, Christian Lohasz, Dzhansu Hasanova, Svenja Lützow, Lisa Hölting, Michal Rudnik, Franziska Linke, Wytske M van Weerden, Laure-Anne Ligeon, Andreas Hierlemann, Mario Modena, Olivier Frey. Assessment of immune cell infiltration and cancer metastatic potential in Akura™ Immune Flow Chip - A microfluidic 3D spheroid system [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 6786.

Abstract 2912: SFRP2 and CD38 colocalize in triple negative breast cancer microenvironment

Abstract Introduction: A promising target for breast cancer is secreted frizzled-related protein 2 (SFRP2), and a humanized monoclonal antibody to SFRP2 (hSFRP2 mAb) inhibits primary triple negative breast cancer (TNBC) in vivo. CD38 is a known cause of PD-1 inhibitor resistance in some tumors, and in metastatic osteosarcoma hSFRP2 mAb overcomes PD-1 mAb resistance by reducing CD38 in tumor infiltrating lymphocytes (TILs). Could this phenomenon be replicated in TNBC? This is unknown as the expression of CD38 in TNBC has not been studied. This study will establish the presence of SFRP2 and CD38 in the TNBC microenvironment. Methods: Immunohistochemistry (IHC) was performed on an 88 core human TNBC tissue microarray (TMA) with antibodies to SFRP2 and CD38. Spatial analysis software was used to determine percent positivity. Each core was categorized using the scoring system for the estrogen receptor in breast cancer (<1% negative, 1-10% low positive, and >10% positive). Multiplex IHC was performed on 4 human triple TNBCs sections. Tissues were stained with antibodies to CD38, CD68 (macrophage), CD3 (T-cell), CD19 (B-cell), CytoKeratin (tumor), and SFRP2. Percent positivity of each marker was analyzed with a software package. For flow cytometry of tumor associated macrophages (TAMs), 1 million EO771 cells were injected into the mammary fat pad of female C57/BL6 mice. After 4 weeks tumors were extracted and a cell suspension was incubated in a mastermix with antibodies to SFRP2, CD38, PD1, CD11B and F480. Cells were fixed and conjugated to a fluorochrome secondary antibody. TAM gated cells were analyzed for the presence of SFRP2 and CD38. Results: For the TNBC TMA, 83 cores were positive and 4 cores were low positive for SFRP2, 1 not evaluable; 87 cores were positive and 1 low positive for CD38. For multiplex IHC there was a total of 1,753,544 individual cells in all 4 human TNBC tumors that were stained. 94% of cells stained positive for SFRP2, and 65% stained positive for CD38. Of the cells that were positive for SFRP2, 70% were also positive for CD38, 15% were positive for CD68, 1% were positive for CD19, 77% were positive for CytoKeratin, and 10% were positive for CD3. Of the cells that were CD38 positive, 99% were also positive for SFRP2, 15% were positive for CD68, 1% were positive for CD19, 81% were positive for CytoKeratin, and 9% were positive for CD3. Flow cytometry analysis of TAMs confirmed the presence of SFRP2, CD38 on TAMs. TAMs that were negative for PD1 and CD38 (pro-inflammatory) had a 56% reduction in SFRP2 compared to TAMs that were double positive for PD1 and CD38 (anti-inflammatory). Conclusion: SFRP2 and CD38 are prevalent in human TNBC and colocalize in the TNBC microenvironment in tumor cells, TAMs and TILs, but not B-cells. The presence and co-localization of SFRP2 and CD38 in TAMs was confirmed with flow cytometry. This confirms that SFRP2 is a therapeutic target in TNBC and provides a mechanistic rational for combination therapy with PD-1 inhibitor. Citation Format: Lillian Hsu, Patrick Nasarre, Rupak Mukherjee, Eleanor Hilliard, Martin Romeo, Elizabeth O'Quinn, Whitney Christians, Nancy Klauber DeMore. SFRP2 and CD38 colocalize in triple negative breast cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2912.

Abstract 4628: Validation of an automated, scalable comprehensive genomic profiling assay for hematologic malignancies

Abstract Optimal diagnosis, prognosis, and treatment selection for hematologic malignancies requires assessment of somatic mutations across a subset of clinically relevant genes. Here we present the analytical validation results of an expanded targeted next generation sequencing (NGS) panel of 141 clinically relevant genes for myeloid and lymphoid malignancies. Based on review of clinical guidelines, 141 genes including 79 myeloid and 84 lymphoid associated genes were selected. The expanded hematological panel interrogates all coding exons of the 141 genes to detect single nucleotide variants (SNVs) and insertions/deletions (indels). The panel also identifies copy number aberrations (CNAs) in 16 genes, FLT3 internal tandem duplicates (ITDs), select non-coding pathogenic variants, and patient sex. The custom hybrid capture-based assay utilizes genomic libraries created from 250 ng gDNA extracted from peripheral blood, bone marrow, or cell suspensions, followed by sequencing on Illumina® instruments. Concordance studies were performed on clinical samples previously assessed using an orthogonal NGS-based laboratory developed test for SNVs/indels/FLT3-ITDs or digital multiplexed ligation-dependent probe amplification for CNAs. In total, 308 patient samples were evaluated and included 72 (23.4%) acute myeloid leukemia and 58 (18.8%) myelodysplastic syndromes samples among other indications, of which 240 (77.9%) were bone marrow specimens. Analysis of concordance demonstrated a positive percent agreement (PPA) of 99.7% for SNVs (750/752), 99.5% for indels <25bp (206/207), and 95.7% for indels ≥ 25bp (22/23). Variants were from 45 genes including TP53, NPM1, and IDH1/2. PPA was 100% for FLT3-ITDs (38/38; size range 18-300 bp) and sex (294/294). For CNAs, PPA was 96.4% (132/137) and negative percent agreement (NPA) was 98.3% (931/947). Assay precision was determined using three replicates of 15 clinical samples for both intra and inter-assay precision using multiple operators, instruments, and reagent lots. 100% concordance was observed for SNVs (36/36), indels (11/11), a 24 bp FLT3-ITD (1/1), CNAs (24/24), and sex (15/15). Analytical specificity was assessed using 5 replicates of NA12878 showing specificity >99.99% for SNVs/indels with variant allele frequency ≥ 3%, and >99.99% for CNAs with copy number ≤0.85 or ≥1.15. Dilution series to determine analytical sensitivity are in progress. This study highlights the analytical validation of the expanded NGS panel for the detection of clinically informative genomic alterations in hematologic malignancies. Results from this validation study, when complete, will include at least 576 samples plus orthogonal testing. These data describe the performance of the assay to enable a comprehensive evaluation of genomic alterations using a single sample, further facilitating the use of broad NGS assays in patients with hematologic malignancies. Citation Format: Grant Hogg, Tong Liu, Helen Cao, Adib Shafi, Ashraf Shabaneh, Jon Williams, John Howitt, Amanda Williamson, Rachel Dango, Xiaojun Guan, Heidi Hoffmann, Michael Mooney, John Pruitt, Scott Parker, Henry Dong, Stan Letovsky, Li Cai, Eric A. Severson, Shakti H. Ramkissoon, Anjen Chenn, Marcia Eisenberg, Brian Caveney, Eyad Almasri, Taylor J. Jensen. Validation of an automated, scalable comprehensive genomic profiling assay for hematologic malignancies [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 4628.

Abstract 4120: Adherent-to suspension transition by hematopoietic factors in metastatic dissemination of circulating tumor cells

Abstract Although metastasis is the foremost cause of cancer-related death, a specialized mechanism that reprograms anchorage dependency of solid tumor cells into circulating tumor cells (CTCs) during the metastatic cascade remains elusive. We discovered a biological phenomenon referred to as Adherent-to-Suspension Transition (AST) that reprograms adherent cells into suspension cells via aberrant induction of hematopoietic transcriptional regulators, which are hijacked by solid tumor cells to disseminate into CTCs. During dissemination, tumor microenvironment triggers epigenetic alteration of AST factors in the invasive front of solid tumors to evoke spontaneous cell-matrix dissociation, acquire anoikis resistance, and bypass immune surveillance of CTCs, however, in the absence of lineage differentiation and EMT. Using mouse models and de novo metastatic patient specimens, we uncovered how Adherent-Suspension Plasticity (ASP) dictates anchorage plasticity during the dissemination and colonization process within the metastatic cascade. Finally, we demonstrate therapeutic strategies that target AST factors to specifically abrogate CTC formation and suppress distant metastases. Together, these findings underscore an important early mechanism of how cancer cells spread and highlight novel targets for the development of anti-metastatic therapy. Furthermore, AST opens unexplored scenarios how reprogramming anchorage dependency orchestrates physiological events such as development, organ morphogenesis and immune cell activation. Citation Format: Hyun Woo Park. Adherent-to suspension transition by hematopoietic factors in metastatic dissemination of circulating tumor cells [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 4120.

Abstract 6840: Automated preparation of single nuclei from FFPE samples for snRNA-Seq

Abstract S2 Genomics developed the Singulator™ 100 and 200 Systems to automate the processing of fresh, frozen, or OCT tissues into single cell or single nuclei suspensions using disposable cartridges, reagents, and customizable protocols. The systems have been used to prepare cells or nuclei from a wide range of tissues and organisms, including human, mouse, chicken, insects, snails, zebrafish, and plants. Formalin-fixed, paraffin-embedded (FFPE) tissues are the preferred format for pathologists and as many as one billion FFPE slides are in repositories. FFPE tissue samples have traditionally been difficult to study with molecular techniques due to the chemical modifications caused by the formalin fixation, which can lead to degradation and sequestration of RNA and DNA. Recent advances in single cell/nuclei RNA-Seq library preparation technology have made it possible to overcome these challenges and generate high-quality single cell or nuclei genomic data from FFPE samples. Protocols have been released for processing previously deparaffinized and rehydrated FFPE samples on the Singulator 100 or 200 into single nuclei. We now report the development of a fully automated workflow for the deparaffinization and rehydration of FFPE tissues in about 40 min, with an optional enzymatic treatment of 45 min, and processing of FFPE tissues into singulated cells or nuclei on prototype Singulator 200 systems. Data will be presented for the optimization of process parameters for cell or nuclei recovery and RNA quality for six tissues. Finally, single nuclei sequencing data will be presented for snRNA-Seq libraries from healthy and cancerous human FFPE samples including glioblastomas and colorectal cancer. These data demonstrate the successful recovery of genomic materials from FFPE samples using an automated sample preparation system for single cell/nuclei transcriptomics. Citation Format: Stevan Jovanovich, Nate Pereira, Danielle Meyer, Catherine Snopkowski, Ignas Masilionis, Brigita Meškauskaitė, Meril Takizawa, Zaki Abou-Mrad, Kenny Yu, Viviane Tabar, Kathleen Luckett, Karuna Ganesh, Nabiha Khan, Ronan Chaligne. Automated preparation of single nuclei from FFPE samples for snRNA-Seq [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 6840.

Abstract 3200: Assessing the utility of cell membrane lipid-extracted nanoliposomes (CLENs) as a targeted drug delivery system for pancreatic cancer

Abstract Background: Pancreatic cancer is a challenging type of cancer to treat as evidenced by a 9% 5-year survival and a dismal rate of mortality. Nanoparticles represent a revolutionary treatment method, primarily due to characteristics including cellular absorption, boosting therapeutic effectiveness, and mitigating side effects through localized actions. For nanomedicine, optimizing liposome formulations is crucial for achieving the highest level of specificity in drug targeting. This study aims to evaluate cellular interaction of pancreatic cancer lipid-extract modified nanoliposomes when compared to unmodified varieties in vitro. Method: Cell membrane lipid-extracted nanoliposomes (CLENs) were prepared via the thin film evaporation technique. The composition included various compositions and ratios of DOPC, DOTAP, Cholesterol, DPPE-PEG-5000, and lipid extracts derived from MS1-VEGF target cells. MS1-VEGF was the cellular model of pancreatic cancer used for this study. Cellular binding studies were performed using cellular suspension and adherent cell populations. Studies were performed in 48-well plates under different experimental conditions including alterations in time of incubation, and periods of cellular exposure to dual humidified and oscillation environments in the presence of lipid-extracted (LE) nanoliposomal preparations and conventional controls. Results: The particle size for nanoliposomes employed for this study was approximately 115 ± 0.7 nm for control and 128 ± 2.7 nm for experimental (lipid extract-modified) nanoliposomes. Following cellular exposure to different lipid extract concentrations (typically 0 to 100 nmoles per ml of growth medium), the fluorescence values demonstrated increased cellular uptake with greater LE content for floating and adhered MS1-VEGF cell populations. With the floating cells, LE-modified nanoliposomes demonstrated significantly higher fluorescence intensity values compared to conventional nanoliposomes. Similarly, with MS1-VEGF adherent cells, LE-modified nanoliposomes targeted cells to a greater extent compared to unmodified controls. Conclusions: The increased cellular uptake of CLENs designed for targeting pancreatic tumor cells (both floating and adherent cells) demonstrated an improvement over the use of conventional nanoliposomes under similar experimental conditions. Further research is needed to fully explore the potential use of CLENs for targeting and treatment of pancreatic cancer. Citation Format: Khaznah Alshammari, Sergio Verdery, Sujin George, Robert B Campbell. Assessing the utility of cell membrane lipid-extracted nanoliposomes (CLENs) as a targeted drug delivery system for pancreatic cancer [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 3200.

Abstract 1631: A homogeneous assay system for discernment of proliferative, antiproliferative, and cytotoxic effects in culture

Abstract We describe a novel homogeneous cell-based assay developed for the quantitative detection of Ki-67 protein, a recognized marker of cell proliferation, utilizing Lumit™ immunoassay technology. This luminescent assay has been effectively multiplexed with a fluorescent measure of cell death, employing a cell-impermeant fluorogenic DNA dye. This multiplex allows for the clear distinction between antiproliferative activity and overt cytotoxicity in cell culture models. Through varying lytic conditions, we demonstrated effective cell lysis and detection of nuclear Ki-67 protein. Specifically, Lumit™ Lysis Buffer II provided efficient recovery of Ki-67 protein without compromising assay performance, including the requisite binding of labeled antibodies to the target protein. This assay exhibited excellent signal-to-background (up to 40-fold) and signal linearity (R^2 = 0.99) in wildtype HeLa cells (~1000 to 80,000/well), while the signal in Ki-67 knockout HeLa cells was comparable to the no-cell background, indicating excellent specificity for Ki-67 protein. Due to the unavailability of recombinant full-length Ki-67 protein, two different recombinant Ki-67 protein fragments were used to corroborate assay sensitivity of <1 pM Ki-67 protein. The assay's suitability for human Ki-67 protein detection, not mouse, was observed through further cell-based testing. The proliferation/cytotoxicity assay multiplex was validated in both suspension and adherent cell lines, as well as primary cell culture, detecting antiproliferative and proliferative responses. CDK4/6 inhibitors are a class of antiproliferative agents of significant interest for their potential in treating cancer. In Jurkat cells, treatment with the CDK4/6 inhibitor palbociclib led to a dose-dependent reduction in Ki-67 protein expression with an EC50 of 88 nM, and total Ki-67 elimination at 2 μM compound concentration. Notably, there was no cytotoxicity detected at the tested doses and treatment duration (up to 48 h), but a significant reduction in viable cell number at maximal palbociclib concentrations, aligning with an antiproliferative mechanism. This work establishes a rapid, simple, luminescent assay for determining Ki-67 levels that can be paired with a fluorogenic readout of cell death, facilitating efficient and quantitative discernment of altered proliferation in mammalian cell culture using standard microplate readers. Citation Format: Dan F. Lazar, Kevin R. Kupcho, Andrew L. Niles, James J. Cali. A homogeneous assay system for discernment of proliferative, antiproliferative, and cytotoxic effects in culture [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 1631.

Retrospective analysis of hand and foot function after application of cellular technologies in patients with post–traumatic ischemic and denervation–reinervation injuries of limb muscles

Objective. To determine the effect of cell therapy on the function of the hand and foot in patients with ischemic and denervation–reinervation injuries of the limb muscles.
 Materials and methods. Over 9 years (2014 – early 2023), 47 patients with posttraumatic ischaemia and/or peripheral nerve damage were treated at the Institute of Traumatology and Orthopedics. Cell therapy, namely the injection of bone marrow aspirate and adipose tissue suspension (cell substrates) into the target muscles, was used in 32 (68.1%) patients (main group), while 15 (31.9%) patients did not undergo cell therapy (control group). In 16 (50%) patients, cell therapy was applied to the upper limb, and in 16 (50%) – to the lower limb. Patients in the control and intervention groups were divided into three subgroups: with ischaemic contracture, peripheral nerve damage, and a combination of these pathologies.
 Results. The treatment with the use of cellular technology in patients with ischaemic and denervation–reinervation injuries of the muscles of the hand and foot had a statistically significant positive effect. In patients with ischaemic contracture of the hand 12 months after the introduction of cell substrates, the loss of its function decreased to less than 30%.
 Conclusions. Cell therapy helps to restore hand and foot function in patients with ischemic and/or denervation–reinervation injury of the limb muscles.

Abstract 1604: Cancer-associated fibroblasts modulate T cell killing activity & phenotype in PDAC

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is highly aggressive malignancy characterised by its abundant extracellular matrix and diverse stromal cell components such as cancer associated fibroblasts (CAFs). CAFs play a pivotal role in shaping the tumour microenvironment and influencing the behaviour of immune cells, particularly T cells, through various mechanisms. The impact of CAFs on T cells is multifaceted as they can exert both pro- and anti-tumoral effects, influencing the delicate balance between immunosurveillance and immune evasion. In this study, we examined the phenotype and function of T cells when co-cultured with CAFs and Adjacent-normal fibroblasts (ANF) isolated from primary PDAC tissue following surgical resection. Methods: Tumour and juxta-tumoral tissue from patients underwent digestion into a single cell suspension. Subsequently, the isolated fibroblasts were cultured and stained with a 22-plex antibody panel designed for flow cytometry. Additional functional assays involved co-culture of CAFs and ANFs with unstimulated or anti-CD3/CD28 stimulated allogeneic T cells. Cell proliferation rates were quantified through the CFSE assay, while the T cells were simultaneously stained to distinguish CD4+ and CD8+ T cells subpopulations. Additionally, the expression of key co-stimulatory molecules: CD69, NKG2D and DNAM-1 and the co-inhibitory molecule: PD-1 were assessed, providing a thorough examination of the immune dynamics within the tumour microenvironment. Results: Flow Cytometry analysis revealed a distinct expression profile in CAFs compared to ANFs, with CAFs expressing higher levels of FAP and CD29. The CFSE assay further revealed that CAFs induced T cell proliferation of resting T cells with no significant increase in stimulated T cells. Notably, this co-culture led to a highly differentiated phenotype in both unstimulated and stimulated CD4+ and CD8+ T cells, characterised by elevated expression of activating markers (CD69 and NKG2D) and the inhibitory molecule PD-1. These findings suggest a dynamic interplay between fibroblasts and T cells in the tumour microenvironment, influencing both the proliferation and phenotypic aspects of T cell behaviour. Citation Format: Fouzia Zayou, Hayden Pearce, Samantha Nicol, Sandra Margielewska-Davies, Sarah Powell-Brett, Rachel Brown, Jianmin Zuo, Keith Roberts, Helen M. McGettrick, Paul Moss. Cancer-associated fibroblasts modulate T cell killing activity & phenotype in PDAC [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 1604.

Abstract 7041: Tumor-immune interactions and cisplatin resistance in localized muscle-invasive bladder cancer

Abstract Background: Cisplatin-based chemotherapy followed by radical cystectomy remains the standard-of-care treatment for patient with localized muscle-invasive bladder cancer (MIBC). Neoadjuvant clinical trials studying the combination of cisplatin-based chemotherapy and immune checkpoint blockade (ICB) in MIBC reported encouraging pathologic complete response rates. However, the molecular mechanisms and cellular programs involved in response and resistance to cisplatin and ICB in MIBC remain incompletely characterized. Our goal is to define cellular transcriptional programs and chromatin accessible regions in cancer cells and infiltrating immune cells associated with response and resistance to neoadjuvant systemic therapy. Methods: We performed single-cell Multiome ATAC and Gene expression sequencing (simultaneous identification of gene expression and open chromatin regions within the same cell) of tumors that had a complete response – “responders” – and tumors that had no response or progressed – “non-responders” – through neoadjuvant cisplatin-based chemotherapy (n=15). Single cell suspensions were prepared following the 10x Genomics protocol. Cell Ranger ARC was used for sequence alignment, peak calling, and generation of count matrices. Downstream single-cell gene expression analysis was performed with Scanpy and ATAC-seq with Seurat. Results: We identified two distinct urothelial cell populations in "responders" vs "non-responders" based on single cell gene expression and genome wide chromatin accessibility regions. Cancer cells in “non-responders” preferentially express basal cell gene expression signatures. “Non-responders” were significantly enriched in suppressive tumor-associated macrophages (TAMs). Interestingly, SPP1+ TAMs known to have a pro-tumoral role and promote resistance to ICB were exclusively identified in “non-responders”. Conclusion: Single cell and genome wide chromatin accessibility regions identify heterogenous urothelial cell populations expressing basal cell markers in tumors resistant to neoadjuvant cisplatin-based chemotherapy. “Non-responders” are significantly enriched with TAMs implicated in resistance to ICB. Future studies will better define the implications of these findings in cancer therapeutics for patients with MIBC. Citation Format: Filipe La Fuente De Carvalho, Jihyun Lee, Kevin Bi, Breanna Titchen, Konrad Stawiski, Jihye Park, Amanda Garza, Daniel Michaud, Jennifer Guerriero, Elizabeth Mittendorf, Kent Mouw, Eliezer Van Allen. Tumor-immune interactions and cisplatin resistance in localized muscle-invasive bladder cancer [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 7041.

Abstract 5526: Activation of platelet-activating factor (PAF) in high-risk melanoma patients with nodal metastasis

Abstract Background: Platelet-activating factor (PAF) is one of the lipid mediators in ultraviolet-induced immunosuppression signaling. Sentinel lymph nodes (SLNs) are the first organs to receive lymphatic drainage via afferent lymphatic channels leading directly from the primary melanoma. In patients with nodal metastasis, it has been shown that the SLN has a profoundly immunotolerant microenvironment. We sought to determine whether lymphatic fluid signals, including PAF, may be involved in modulating the SLN immune microenvironment to favor melanoma metastasis. Hypothesis: Upregulation of PAF can bind to its receptor on monocytes and activate downstream immunosuppressive chemokines, resulting in an immunosuppressed environment in SLNs that permits nodal metastasis and melanoma progression. Methods: Lymphatic fluids, plasma samples, and SLN samples were collected from patients undergoing surgical excision of melanoma with SLN biopsy under an IRB-approved protocol. Metabolic analyses were performed on lymphatic fluids from high- versus low-risk patients by two-dimensional liquid chromatography system coupled with mass spectrometry (2D LC-MS). Single cell sequencing was done on single cell suspensions from SLN samples. Protein array was used to compare the cytokine and chemokine levels in plasma samples from high- versus low-risk melanoma patients. We compared two groups: low-risk (no SLN metastasis) and high-risk (with SLN metastasis). Results: Lymphatic fluid PAF levels were 3-fold greater in high-risk melanoma patients compared with low-risk patients (p=0.0039). Macrophages were identified as the cell type with greatest PTAFR (platelet-activating factor receptor) expression by single cell sequencing. CCL17 levels in plasma samples from high-risk patients were significantly lower than in low-risk patients (p<0.05). These results suggest that increased PAF in lymphatic fluid may bind to PTAFR in SLN macrophages, inhibit chemokines (such as CCL17), and result in an immunosuppressive SLN environment to favor nodal metastasis and melanoma progression. Conclusion: Lymphatic fluid PAF signaling may contribute to an immunotolerant microenvironment in the SLN that allows nodal metastasis and melanoma progression through downregulation of CCL17. Further studies will investigate whether this pathway can be exploited to therapeutic advantage. Citation Format: Austin McMasters, Julia Chariker, Jae Yeon Hwang, Xinmin Yin, Xipeng Ma, Raobo Xu, Xiang Zhang, Juw Won Park, Kelly M. McMasters, Hongying Hao. Activation of platelet-activating factor (PAF) in high-risk melanoma patients with nodal metastasis [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 5526.

Abstract 2500: Technical feasibility of CRISPR-based functional profiling for solid tumor precision medicine

Abstract Introduction: Next generation sequencing (NGS) has revolutionized precision oncology by enabling identification of cancer patients harboring actionable mutations who may benefit from corresponding targeted therapies. However, sequencing-based biomarkers are not detected for most patients and imperfectly predict response to therapy even when present. To address this unmet need, we have developed a CRISPR-based diagnostics platform for ‘personalized functional genomics’, allowing identification of patient-specific therapeutic vulnerabilities irrespective of mutation status. We have previously applied this approach to primary hematologic malignancies, and here report application to solid tumor-derived samples. Methods: Patient-derived solid tumor samples from several tumor types including breast, ovarian, colon and lung were obtained from 3 sources: fresh tissue (obtained from patients via core biopsy, surgical resection, or malignant fluid sampling), primary Patient-Derived Cell Lines (PDCLs, generated from surgical material and adapted for ex vivo culture), and commercially obtained viably frozen dissociated tumor cells (DTCs). Samples were dissociated to single cell suspensions and transduced with a lentivirus encoding Cas9 and an sgRNA library covering a custom targeted oncology gene panel. The panel consists of genes encoding targets of FDA-approved oncology drugs, as well as reference genes. After a short culture period, transduced cells were harvested and relative sgRNA distribution was measured by amplicon NGS. Changes in sgRNA abundance were collapsed into gene-level log2-fold changes, and significantly depleted genes scored as functional dependencies. For select PDCLs, cells were treated with cognate small molecule inhibitors over the course of 4 days. Cell viability was read out using CTG assay and a calculated IC50 lower than 500nM inferred as inhibitor sensitive. Results: In a double-blind study using primary PDCLs, functional dependencies identified by CRISPR perfectly discriminated alpelisib sensitive (2/2) from resistant samples (6/6). Using fresh primary samples from varying cancer types and specimen sources, we achieve a technical success rate >70%. Among gene dependencies identified, targets of FDA-approved oncology drugs and those under late-stage clinical investigation were both observed including BRAF, CDK4/6, VEGFR1/2 and PARP1/2. Conclusion: We demonstrate technical proof of concept of the FX platform for CRISPR-based personalized functional genomics. This approach is feasible when applied to primary patient samples from a variety of tumor types and tissue sources, and results are validated by comparison with orthogonal methods. CRISPR-based functional profiling represents a powerful adjunct to traditional genomic profiling, for the first time allowing drug target dependency mapping in a patient-specific and mutation-agnostic manner. Citation Format: Karl Hodel, Andreas Varkaris, Nicholas Chevalier, Maci Meyers, Allison Kehlmann, Stephane Corbel, Anastasia Stavridi, Peter Kilfeather, Leonardo Hagmann, Kathleen Tran, Yali Li, Christian Schmedt, Ioannis Sanidas, Srinath Sampath, Srihari Sampath, Dejan Juric. Technical feasibility of CRISPR-based functional profiling for solid tumor precision medicine [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 2500.

Abstract 780: Normal breast tissues harbor rare populations of aneuploid epithelial cells

Abstract Although aneuploidy is a hallmark of invasive breast cancers, the presence of aneuploid cells in breast tissues of disease-free women is poorly understood. Furthermore, the expansion of aneuploid cells and the specific copy number events that they harbor, as well as their relationship to invasive breast cancers has not been characterized in large cohorts of women. In this study, we obtained viable cell suspensions from 31 disease-free women that underwent reduction mammoplasty surgeries and fresh tissue collection in the HBCA project along with the collection of clinical metadata. The viable cell suspensions from 31 women were enriched for epithelial cells and used to perform high-thoughput nanowell scDNA-seq. In total, 55,321 cells were analyzed. The normal breast tissue samples were also embedded into OCT and frozen to perform Spatial Transcriptomics (Visium 10X Genomics) profiling and embedded into FFPE to perform histopathological analysis. Our data shows that all women harbored rare populations of aneuploid epithelial cells (mean 4.3%) that increased in frequency and their number of copy number aberrations with age. Many aneuploid epithelial cells (mean 79%) in normal breast tissues underwent clonal expansions of their genomes and harbored chromosomal aberrations detected in invasive breast cancers (gain of 1q and loss of 7q, 10q, 16q). Spatial mapping showed that aneuploid cells are localized in focal regions of ductal and lobular epithelial structures with normal histopathological features. Collectively, these data show that aneuploid epithelial cells exist in the normal breast tissues of disease-free women and increase with age. In many women, the aneuploid cells expand and harbor CNA events associated with invasive breast cancer. Citation Format: Yiyun Lin, Junke Wang, Kaile Wang, Shanshan Bai, Aatish Thennavan, Runmin Wei, Yun Yan, Jianzhuo Li, Heba Elgamal, Emi Sei, Anna Casasent, Mitchell Rao, Chenling Tang, Jessica Montalvan, Chandandeep Nagi, Sebastrian Winocour, Bora Lim, Alastair Thompson, Nicholas Navin. Normal breast tissues harbor rare populations of aneuploid epithelial cells [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 780.

Abstract 5263: The impact of MRTX1133 on anti-tumor immunity in lung cancer with KRASG12D mutation

Abstract Background: To date, there have been no clinical molecules that are selective for KRASG12D mutant tumors. Although immune checkpoint inhibitors (ICIs) have improved the survival for patients with lung cancer, inferior efficacy of ICIs has been reported in KRASG12D mutant NSCLC. MRTX1133 is a selective KRASG12D inhibitor, which has been investigated in early clinical studies. In this study, we aimed to reveal the immune status using preclinical murine model of KRASG12D mutated lung cancer. In addition, we investigated whether MRTX1133 effects the anti-tumor immunity in mice bearing lung cancer with KRASG12D. Method: CCSP-rtTA/TetO-Cre mice were crossed with LSL-KRASG12D knock-in mice to obtain CCSP-rtTA/TetO-Cre/LSL-KRASG12D conditional mutant mice (KRASG12D mice). These mice were fed an autoclaved rodent diet supplemented with 0.0625% doxycycline when the mice were about 8~12 weeks old. Mice were treated with 10 mg/kg MRTX1133 in vehicle once daily by intraperitoneal injection for 4 or 8 days. After administration of MRTX1133, the lungs and spleens were harvested, and single cell suspensions were labeled with fluorophore-conjugated antibodies for FACS analyses. Results: More regulatory T cells (Tregs) infiltrated the tumors of lung of mice treated with doxycycline for a longer period of time. The expression of immune checkpoint molecules (ICMs), including PD-L1, PD-1, TIGIT, TIM-3, LAG-3, were also upregulated on CD4+ and CD8+ T cells in the KRASG12D mice. Treatment with MRTX1133 strongly inhibited KRASG12D tumor progression, reduced the percentage of Tregs and downregulated the expression of PD-1 on CD4+ and CD8+ T cells infiltrating in lung cancers. The expression of the other ICMs remained high after MRTX1133 administration. In addition, MRTX1133 tended to increase the percentage of tumor-specific CD8+ effector T cells infiltration in lung tumors. Conclusion: T cells were exhausted in mice with KRASG12D mutant lung cancer. MRTX1133 demonstrates the therapeutic efficacy in preclinical murine KRASG12D mutated lung cancer model. This efficacy is more likely to relate not only signal inhibition but also enhances the anti- tumor immune responses. Our findings could aid in developing an alternative immunotherapy strategy in patients with KRASG12D mutant lung cancer. Citation Format: Kunihiro Shono, Satoshi Watanabe, Takaaki Masuda, Ryo Suzuki, Tomoya Wakabayashi, Tomoki Sekiya, Susumu Tanaka, Aya Ohtsubo, Tomohiro Tanaka, Koichiro Nozaki, Rie Kondo, Yu Saida, Satoshi Hokari, Naohiro Yanagimura, Masashi Arita, Riuko Ohashi, Kenjiro Shima, Yosuke Kimura, Nobumasa Aoki, Yasuyoshi Ohshima, Toshiyuki Koya, Toshiaki Kikuchi. The impact of MRTX1133 on anti-tumor immunity in lung cancer with KRASG12D mutation [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 5263.

Abstract 3778: Comprehensive analysis of single disseminated cancer cells identifies a stem-like phenotype with strong prognostic impact

Abstract Introduction: Recent studies on disseminated cancer cells (DCCs) in Non-Small-Cell Lung Cancer (NSCLC) patients demonstrated their clinical relevance even in early tumor stages. However, their molecular and phylogenetic link to the primary tumor is currently unclear. Certain genetic alterations, such as TP53 or KRAS mutations, have been proposed to be clonal events and drivers of dissemination and metastasis. In this study, we analyzed the genome and transcriptome of single DCCs to identify the characteristics of early DCCs and their translational clinical value. Materials and methods: From 2011 to 2019 we prospectively collected and analyzed bone marrow aspirates and lymph node samples from 296 newly diagnosed NSCLC patients undergoing tumor resection with curative intention. Presence of DCC was examined by i) immunocytology (IC) for Cytokeratin (clone A45-B/B3, bone marrow, BM-DCCs) or EpCAM (clone BerEp4, lymph node, LN-DCCs) and ii) immunofluorescent (IF) staining for EpCAM in a live cell suspension (clone HEA-125) from BM or LN. Single cells were isolated using micromanipulation and subjected to Whole Genome Amplification or combined Whole Genome/Transcriptome Amplification for IC and IF, respectively. Sanger sequencing for known hotspot mutations in TP53 and KRAS was performed on single DCCs. Gene expression profiles of DCCs were analyzed by single cell RNA sequencing and the results were compared with clinical data with a mean follow-up of 2.93 years (range 0.03-7.22). Results: LN-DCCs were found in 44.8% and 59.6% by IC and IF, respectively. CKIC pos BM-DCCs were found in 45.2%, while EpCAMIF pos BM-DCCs were present in 52.8%. LN-DCCs correlated with reduced tumor-specific survival (TSS, p=0.009) and progression-free survival (PFS, p=0.025). EpCAMIF pos, but not CKIC pos BM-DCCs correlated with reduced TSS (p=0.003) and PFS (p=0.083). Genetic analysis of single DCCs revealed that TP53 or KRAS mutations could be found in 3.6% and 2.8% of analyzed BM-DCCs and 5.0% and 29.3% of LN-DCCs for IF and IC, respectively. Single cell RNA-seq showed distinct transcriptomic profiles for LN- and BM-DCCs, and identified a DCC phenotype with high stemness score in the bone marrow. Further analysis of potential transcriptome-based biomarkers identified a novel candidate marker for activated EpCAMpos BM-DCCs that strongly correlates with PFS and TSS (p<0.001). Conclusions: DCCs in early-stage NSCLC have high prognostic value and predict reduced PFS and TSS. While genetic alterations in the TP53 and KRAS gene were not detected in the majority of analyzed DCCs, transcriptomic phenotypes provided relevant information and identified a subset of activated DCCs that correlate strongly with relapse and poor outcome. Citation Format: Tobias Mederer, Daniel Spitzl, Felix Elsner, Bernhard Polzer, Michael Ried, Reiner Neu, Tobias Robold, Hans-Stefan Hofmann, Christoph A. Klein. Comprehensive analysis of single disseminated cancer cells identifies a stem-like phenotype with strong prognostic impact [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 3778.

Abstract 2060: Automation of 3D cancer spheroid assay for compound screening

Abstract Finding efficient drug combinations to treat cancer patients is critical for therapy success. Accordingly, there is a critical need to develop methods for efficient testing drug efficacy to discover new therapeutic targets. 3D cancer models are highly valuable tools for cancer research and drug development. However, the complexity of performing 3D assays remains a hurdle for adopting these methods for compound screening. To alleviate the bottlenecks that come with labor-intensive manual protocols we developed cell culture automation methods using CellXpress.ai instrument. The instrument enables automation of 2D or 3D culture for prolonged complex workflows. The CellXpress.ai provides automated plating, passaging, media exchanges, monitoring organoids, alone with compound treatment and the end point assays with high content image analysis. In the present study we describe full automation of imaging, analysis, and cell culture process which enables scaling up complex 3D cell-based assays and compound screening. As an example of 3D protocol, we automated culture and imaging of colorectal cancer 3D spheroids formed from HCT116 cell line in U-shape low attachment plates. HCT116 cells were expanded in 2D, then spheroids were formed after automated dispensing of cell suspension into U-shape 96 or 384 plates. After 48h spheroids were treated with 12 anti-cancer compounds, at multiple concentrations for 3-5 days, then stained and imaged. Cell plating, compound additions, media exchange, and staining were performed automatically by CellXpressAI instrument. During the culture spheroids were monitored using transmitted light, with immediate analysis of phenotypic changes, including inhibition of growth or spheroid disintegration. For endpoint assay spheroids were stained with combination of Hoechst nuclear stain and viability dyes Calcein AM and EtHD, then imaged, and analyzed for multiple read-outs including spheroid size, marker intensities, and live-dead cell scoring. We observed concentration-dependent inhibition of spheroid growth and cell death in response to anti-cancer compounds and evaluated effective compound concentrations for cytotoxic or cytostatic effects. Cell culture process automation for 3D assays, powered by imaging and image analysis has a potential to bring 3D biology into another level, allowing to increase throughput and reproducibility, and enabling variety of high throughput drug discovery and precision medicine applications. Citation Format: Oksana Sirenko, Angeline Lim, Astrid Michlmayr, Zhisong Tong, Emilie Keidel, Felix Spira. Automation of 3D cancer spheroid assay for compound 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 2060.