Positive Control Cell Line Research Articles

Abstract 5157: Development and evaluation of a high-throughput method for rapid detection of surface antigen expression in fixed cells

Abstract Cell surface proteins are increasingly considered in the development of novel anticancer agents, as demonstrated by recent clinical successes in the field of immuno-oncology and antibody-drug conjugates (ADCs). These conjugates specifically target antigens present on the surface of tumor cells, enabling the delivery of cytotoxic agents to specific tumor sites. During the preclinical development of such compounds, for example, when they are tested on a wide panel of cell lines, it is essential to have a rapid and reliable method for monitoring target expression and distribution. Complementing our 160 Cell line panel ProLiFiler™, we report here the development of a novel method named oncoFLOW-Profiler™ which includes steps of cell fixation, micronic storage, staining, and measurement by flow cytometry. This method is applied in a 96-well format, enabling screening of all cell lines in parallel and analysis of the expression of different targets in just one day. In this proof-of-concept study, we investigated the level of ERBB2 expression across the ProLiFiler panel of 160-cell lines in order to validate this methodology. We first compared the impact of fixation methods and sample storage on knowingly positive and negative control cell lines over a period of several months. ERBB2 measurements from the oncoFLOW-Profiler™ will be analyzed and validated by comparison with various OMICs datasets from the Cancer DataMiner platform (4HF Biotec), including RNA expression, mutational status, and protein expression levels. To determine the expression cutoff of ERBB2 amplified models, results will be analyzed according to gene copy number variation. Finally, the flow cytometry data generated will be investigated for use as a predictor of response to clinically approved ADCs targeting ERBB2: Kadcyla and Enhertu. Here we present a method that we have demonstrated is feasible for investigating cell surface receptor/antigens during preclinical phase of drug development. Our method has enabled us to successfully assess ERBB2 in biological samples and to identify models sensitive to ADCs targeting ERBB2. We will also discuss the results of ongoing experiments analyzing the applicability of this screen to intracellular targets and carbohydrate-based surface antigens, which cannot be analyzed either by transcriptomics or proteomics. Citation Format: Kaja Holtorf, Anne-Lise Peille, Daniel Feger, Vincent Vuaroqueaux, Jan Erik Ehlert, Nadine Obier. Development and evaluation of a high-throughput method for rapid detection of surface antigen expression in fixed 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 5157.

Abstract 7486: Development and analytic validation of an ARv7/PSMA dual biomarker circulating tumor cell assay

Abstract Background Prostate cancer drug targets and biomarkers may be investigated non-invasively on circulating tumor cells (CTCs) obtained via liquid biopsies. PSMA is an important drug target in prostate cancer, and ARv7 is a splice variant that confers resistance to second-generation androgen receptor inhibitors. In this study, we describe the development and analytic validation of a novel assay to quantify ARv7 and PSMA protein expression on CTCs. The assay was validated with blood samples from patients with Stage IV prostate cancer to demonstrate clinical feasibility. To our knowledge, a PSMA/ARv7 dual protein assay for CTCs has not previously been reported. Methods LNCaP (PSMA positive), PC3 (PSMA negative), 22Rv1 (ARv7 positive), and BT474 (ARv7 negative) cells were spiked into healthy donor blood to generate model CTCs. Nucleated cells were collected from these spike-in samples using the AccuCyte® sample preparation kit and spread onto microscope slides. Slides were fixed and stained with antibodies to cytokeratin, EpCAM, and CD45 to detect CTCs and exclude white blood cells (WBCs), and with PSMA and ARv7 to evaluate protein expression on identified CTCs. The slides were imaged with the CyteFinder® automated immunofluorescence scanning microscope that identifies CTCs for confirmation by trained reviewers based on machine learning algorithms and quantitatively measures biomarker signal mean fluorescence intensity (MFI). Results For ARv7/PSMA expression classification of the model CTCs, the dual biomarker assay showed analytic sensitivity of 83%/97% and specificity 98%/99%, yielding accuracy of 90.2%/97.9%, respectively. The dynamic range of MFI for both biomarkers was approximately three orders of magnitude. Inter-run reproducibility CV was 13.6% (ARv7) and 6.9% (PSMA). Slide repeatability mean CV was 11.1% (ARv7) and 12.0% (PSMA). Clinical feasibility of the assay was investigated in 9 progressing Stage IV prostate cancer patient blood samples. CTCs were identified in all clinical samples (median 4, range 1 - 384). Thresholds for biomarker positivity were established based on MFI measurements of positive and negative control cell lines. ARv7 positivity in clinical samples ranged from 33% to 100%, and PSMA positivity ranged from 0% to 75%. Conclusions We have developed a dual ARv7/PSMA assay that simultaneously measures protein expression of both biomarkers on individual CTCs. The assay is accurate and reproducible for the assessment of ARv7 and PSMA expression by liquid biopsy in clinical studies. Citation Format: Jon Ladd, Erin Bayer, Brock Bartels, Eric Kaldjian, Arturo Ramirez. Development and analytic validation of an ARv7/PSMA dual biomarker circulating tumor cell assay [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 7486.

Abstract 4161: Identification of pancreatic adenocarcinoma using urokinase plasminogen activator probe detected using optoacoustic imaging

Abstract Purpose: Diagnosing and treating pancreatic cancer remains a challenge. The hypovascularized nature of pancreatic cancer renders modern chemotherapeutics and cancer detection contrast agents ineffective. A promising approach to the challenges is the adoption of a novel imaging technique known as Multispectral Tomography (MSOT). We hypothesize that a urokinase plasminogen activator (UPA) probe will enhance the detection of orthotopic pancreatic cancer xenografts. Methods: Pancreatic tumor cell lines (Panc1, MiaPaca-2, S2VP10, and S2C9) were assessed for UPA-receptor expression using western blot analysis. UPA peptide was synthesized and to the HiLyte-750 NIR dye. Flow cytometry and immunofluorescence were utilized to examine the specificity and activity of this UPA-750. The UPA-750 binding efficiency was assessed through a competitive inhibition assay with a UPAR-blocking antibody using fluorescence microscopy. Then SCID mice (5 mice/group) received orthotopic implantation of S2C9 (1.5 x 105) or MiaPaCa2 (2.0 × 106) pancreatic tumor cells. UPA-750 or 750 dye alone was IV injected into mice after the tumors reached 3mm. Biodistribution and accumulation of UPA-750 were observed through MSOT images taken at 2-hour intervals over 8 hours and at 24 hours. UPA-750 in the tumor, liver, and kidney was confirmed through ex vivo fluorescent imaging. Results: UPAR expression was observed in every pancreatic cancer cell line, with the greatest relative abundance in S2P9 cells (3.0X), followed by S2VP10 (2.5X), Panc1 (1.77X), and MiaPaca-2 (1.3X). Application of the UPAR blocking antibody on S2CP9, S2VP10, MiaPaCa2, and SKOV3p1 (positive control) cell lines resulted in the inhibition of cellular binding of the UPA-750, as evidenced by fluorescence microscopy. UPA-probe was successfully observed in pancreas tumor slices with peak intensity at 4-hrs of imaging by MSOT. Ex vivo NIR fluorescence imaging confirmed UPA-750 signal in pancreatic tumors but not in liver or kidney. Conclusion: UPA-probe exhibits potential as a marker for pancreatic cancer. Citation Format: Vung Kim, Phillip Chuong, Ryan Bynum, William Grizzle, Lacey R. McNally. Identification of pancreatic adenocarcinoma using urokinase plasminogen activator probe detected using optoacoustic imaging [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 4161.

Smartphone-assisted lab-in-a-tube device using gold nanocluster-based aptasensor for detection of MUC1-overexpressed tumor cells

Developing smartphone technology for point-of-care diagnosis is one of the current favorable trends in the field of biosensors. In fact, using smartphones can provide better accessibility and facility for rapid diagnosis of diseases. On the other hand, the detection of circulating tumor cells (CTCs) is one of the recent methods for the early diagnosis of cancer. Here, a new smartphone-assisted lab-in-a-tube device is introduced for the detection of Mucin 1 (MUC1) overexpressed tumor-derived cell lines using gold nanoclusters (GNCs)-based aptasensor. Accordingly, commercial polyurethane (PU) foam was first coated with graphene oxide (GO) to increase its surface area (8.45-fold), and improve its wettability. The surface of the resulting three-dimensional PU-GO (3DPU-GO) platform was then modified by MUC1 aptamer-GNCs to provide the required sensitivity and specificity through a turn “on/off” detection system. The proposed biosensor was first optimized with a spectrophotometer method. Afterward, findings were evaluated based on the red color intensity of the lab-in-a-tube system; and indicated the high ability of the biosensor for detection of MUC1-overexpressed tumor cell lines in the range of 250–20,000 cells mL−1 with a limit of detection of 221 cells mL−1. In addition, the developed biosensor showed a decent selectivity against positive-control cell lines (MCF-7, and HT-29) in comparison to negative-control cell lines (HEK293, and L929). Notably, the results represented good accordance with reference methods including spectroscopy devices. Ultimately, the results of this work bring a new perspective to the field of point-of-care detection and can be considered in future biosensors.

Effect of aspirin on the TNF-α-mediated cell survival and death pathways in breast cancer.

Aspirin is an anti-inflammatory drug commonly used as an analgesic and in cardiovascular disorders. However, many studies have highlighted its anti-cancer properties, especially in colorectal, lung, head and neck, and breast cancers. In this work, we tried to study the effect of aspirin on the TNF-α-mediated cell survival and death pathways in two cell lines representing two different subtypes of breast cancer. TNF-α-mediated stimulation of a cell can result in its proliferation via theNF-κB pathway or its death via either apoptosis or a programmed form of necrosis called necroptosis. The latter is believed to come into the picture only when apoptosis is inhibited. In this work, we studied the effect of aspirin on the TNF-α-mediated cell survival pathway and observed a decrease in expression of the NF-κB pathway regulators, itsnuclear translocation, and phosphorylation in a dose-dependent manner. The effect of aspirin on the TNF-α-mediated cell death showed significant cytotoxicity at the higher doses (5-20mM) of aspirin in both the breast cancer cell lines. The effect of aspirin on necroptosis was investigated after stimulating the cells with TNF-α and inhibiting apoptosis using Z-VAD-FMK. Though no significant effect was noted in breast cancer cell lines, the above protocol successfully induced necroptosis in L929, i.e.,a positive control cell line for necroptosis having an intact necroptosis machinery. Even when combined with the chemotherapeutic drugs, the above regime failed to induce any significant necroptosis in breast cancer cells but was found effective in L929. Overall, the findings show that while aspirin has the potential to inhibit the TNF-α-mediated cell survival pathway, it does not help sensitize breast cancer cells to necroptotic cell death induction.

Relative refractoriness of breast cancer cells to tumour necrosis factor-α induced necroptosis.

Necroptosis, a recently identified programmed cell death pathway, has attracted attention as an alternative route to target apoptosis-resistant cancer cells. The status of the necroptosis pathway in different subtypes of breast cancer has not been well explored. Stimulating the cells by TNF-α can trigger cell survival or death depending on the combination of downstream players involved. In this work, we attempted to induce necroptosis in them using a combination of TNF-α and Z-VAD-FMK with and without chemotherapy. Cell viability, apoptosis, and necroptosis were assessed using MTT and Annexin-V/PI assays, respectively. Gene and protein expression was analysed by qPCR and immunophenotyping. Both the cell lines were resistant to induction of cell death by necroptosis. There was no enhancement in cell death when chemotherapeutic drugs were combined with necroptosis induction. Expression studies showed reduced translational expression of key necroptosis molecules like RIP kinases and MLKL in breast cancer cells compared to positive control cell line L929. Also, cell survival molecules were expressed more in MDA-MB-231 in contrast to death pathway molecules which were expressed more in T47D cells. In this work, the two breast cancer cell lines were observed to be resistant to TNF-α induced necroptosis with or without chemotherapy. Expression of key necroptosis players revealed relative insufficiency of the molecular machinery involved in the above pathway. In our opinion this may be the cause for resistance to necroptosis and novel strategies to upregulate these molecules need to be developed to sensitize the breast cancer cells towards cell death by necroptosis.

Tc-99m-tamoxifen: A novel diagnostic imaging agent for estrogen receptor-expressing breast cancer patients

PURPOSE The aim of the study was to radiolabel, characterize, and perform in vitro and in vivo assessment of Technetium-99m (Tc-99m) tamoxifen for screening ER expressing lesions in breast cancer patients. METHODS In this study, tamoxifen has been radiolabeled with Tc-99m via Tc-99m-tricarbonyl core. The characterization and quality control tests of Tc-99m-tamoxifen were performed. In vitro recep- tor binding and blocking studies were performed in both positive control (MCF-7) and negative control cell lines (MDA-MB-231). Normal biodistribution studies were performed in female Wistar albino rats. The pilot clinical studies were performed in 4 ER-expressing breast cancer patients. Of the 4 patients, 1 was on tamoxifen therapy. All 4 patients had also undergone Fluorine-18 fluorodeoxyglucose (F-18-FDG) positron emission tomography/computed tomography. RESULTS Tamoxifen was radiolabeled with Tc-99m via Tc-99m-tricarbonyl core with more than 95% radio- chemical yield. Mass spectra showed a peak corresponding to the molecular weight of Tc-99m- tricarbonyl and Tc-99m-tamoxifen. The site of binding of Tc-99m-tricarbonyl with tamoxifen was determined by proton nuclear magnetic resonance. The Tc-99m-tamoxifen showed 30% binding with MCF-7 and only 1%-2% receptor binding with MDA-MB-231 cell lines. Also, the percentage of receptor binding was drastically reduced (up to 72%) when ER was saturated with 50 times the excess molar ratio of unlabeled tamoxifen. In a pilot patient study, Tc-99m-tamoxifen uptake was observed in primary and metastatic lesions. However, no uptake was observed in a patient who was on tamoxifen therapy. The uptake of F-18-FDG was noted in all the patients. CONCLUSION Tamoxifen was radiolabeled with an in-house-synthesized Tc-99m-tricarbonyl core. The radio- labeled complex has been characterized and evaluated for receptor specificity in in vitro and in vivo studies. Also, this is the first clinical study using Tc-99m-tamoxifen for imaging ER. More patients need to be evaluated to further explore the role of Tc-99m-tamoxifen in ER-expressing lesions.

DDEL-01. The role of key pharmacodynamic and pharmacokinetic parameters in drug response prediction of pediatric tumors in the precision oncology study INFORM

Abstract INTRODUCTION: The INFORM (INdividualized Therapy FOr Relapsed Malignancies in Childhood) study is a European pediatric precision oncology program. Using state of the art molecular assays, INFORM aims for identification of targetable genetic alterations, matching drugs and clinical trials. High evidence targets were associated with doubling of progression free survival when patients received a matching drug. However, the fraction of tumors with high evidence drug targets remains low requiring functional layers of information such as drug sensitivity profiling. The aim of this project is to identify and investigate the role of key pharmacodynamic and pharmacokinetic parameters to improve the predictivity of ex vivo drug response of pediatric tumors. METHODS: Positive control cell lines harboring specific mutations (n=7) and primary tumors (n=121) from INFORM, including 10% ependymomas, 7% high grade gliomas, 5% neuroblastomas and 4% medulloblastomas, were profiled ex vivo using a library of n=76 clinically relevant oncology drugs in a 384 well plate format. Metabolic activity was measured after 72h of treatment. Quality control (QC) was done using the robust z-factor, correlation of replicates and mean negative control. Hit selection was based on maximum percentage inhibition, normalized AUC metric (DSSasym) and maximum serum concentration (Cmax) of the drug. Clinical follow-up was collected using a questionnaire. RESULTS: A linear mixed model revealed the DSSasym to be the strongest pharmacodynamic parameter in drug prediction in cell lines. Drug screens of n=105 INFORM cases passed QC. Application of the filtering parameters resulted in prediction of n=1-16 drugs/case (min-max). A data base of published pediatric pharmacokinetic parameters of the drug library was generated. Analysis of predictive parameters and clinical follow-up of clinical samples is ongoing. CONCLUSION: Including pharmacodynamic as well as clinical pharmacokinetic parameters is paramount to identify potentially clinically active compounds from ex vivo drug screen data. Further algorithm development is warranted.

Rarity of fetal cells in exocervical samples for noninvasive prenatal diagnosis.

The possibility to isolate fetal cells from pregnant women cervical samples has been discussed for five decades but is not currently applied in clinical practice. This study aimed at offering prenatal genetic diagnosis from fetal cells obtained through noninvasive exocervical sampling and immuno-sorted based on expression of HLA-G. We first developed and validated robust protocols for cell detection and isolation on control cell lines expressing (JEG-3) or not (JAR) the HLA-G antigen, a specific marker for extravillous trophoblasts. We then applied these protocols to noninvasive exocervical samples collected from pregnant women between 6 and 14weeks of gestational age.Sampling was performed through insertion and rotation of abrush at the ectocervix close to the external os of the endocervical canal. Finally, we attempted to detect and quantify trophoblasts in exocervical samples from pregnant women by ddPCR targeting the male SRY locus. For immunohistochemistry, a strong specific signal for HLA-G was observed in the positive control cell line and for rare cells in exocervical samples, but only in non-fixative conditions. HLA-G positive cells diluted in HLA-G negative cells were isolated by flow cytometry or magnetic cell sorting. However, no HLA-G positive cells could be recovered from exocervical samples. SRY gene was detected by ddPCR in exocervical samples from male (50%) but also female (27%) pregnancies. Our data suggest that trophoblasts are too rarely and inconstantly present in noninvasive exocervical samples to be reliably retrieved by standard immunoisolation techniques and therefore cannot replace the current practice for prenatal screening and diagnosis.

Design, Synthesis, In silico and In Vitro Anticancer Activity of Novel Bis‐Furanyl‐Chalcone Derivatives Linked through Alkyl Spacers

AbstractA novel series of bis(furan‐based chalcone) derivatives linked to aliphatic linkers, with the furan units at the A‐ or B‐rings, were synthesized and evaluated as anticancer agents. Chalcones 5 a–c in which the furan ring designed to be a B‐ring were obtained from the Knoevenagel condensation reactions of the appropriate bis(acetyl) compounds 3 a–c with two equivalents of furan‐2‐aldehyde. Likewise, the condensation of bis(aldehydes) 7 a–c with two equivalents of 2‐acetylfuran afforded the corresponding chalcones 9 a–c, in which the furan rings represent the A‐rings of the chalcones, in excellent yields. The synthesized compounds have been fully characterized by 1H‐NMR, 13C‐NMR, and elemental analysis. The in vitro anticancer activity of the prepared compounds was tested against A549, HCT116, HePG2, PC3, A431 and BJ1 cell lines using MTT assay, gene expression analysis of skin and lung cancer related genes, comet assay, and DNA fragmentation assy. Compounds 9 a and 9 c were found to be the most promising, with IC50 (24.9 and 13.7 μg/ml, respectively) against A549 compared with doxorubicin (IC50, 28.3 μg/ml) and IC50 (26.1 and 14.4 μg/ml, respectively) against A431 in comparison to doxorubicin (IC50, 24.9 μg/ml). Comapred with neagtive control cell lines, the gene expression levels of hBD‐2 and hBD‐3 genes were decreased singnificantly (P<0.05) in positive control and skin cancer cell lines (A431) treated with compounds 9 a and 9 c. The expression levels of FOSB and IL‐8 genes were increased singnificantly (P<0.05) in positive control and lung cancer cell lines (A549 and A541) treated with compounds 9 a and 9 c, respectively, comapred with neagtive control cell lines. The DNA damage was increased significantly (P<0.001) in treated skin and lung cell lines samples compared with negative control. The negative control skin and lung cancer cell lines showed a significant decrease (P< 0.05) in DNA fragmentation rate compared with positive control. On the other hand, the DNA fragmentation rates were increased significantly (P<0.001) in treated skin and lung cell lines samples compared with negative control. In summary, the synthesized compounds 9 a and 9 c showed a significant anticancer activity against lung and skin cancer cell lines compared with known chemotherapeutic drug (doxorubicin).

Fit-for-purpose quantitative liquid biopsy based droplet digital PCR assay development for detection of programmed cell death ligand-1 (PD-L1) RNA expression in PAXgene blood samples.

Development of a clinically applicable liquid biopsy-based test for PD-L1 mRNA expression would be beneficial in providing complementary evidence to current immunohistochemistry assays. Hence, we report the development of a fit-for-purpose assay for detection of blood PD-L1 mRNA expression using droplet digital polymerase chain reaction (ddPCR). TaqMan® assays were selected based on coverage of the PD-L1 gene and were tested for linearity and efficiency using real-time quantitative PCR. Four reference genes were analyzed in positive control cell line (A549 treated with interferon gamma, [IFN γ]) genomic DNA. The PD-L1 primer/probe sets were also evaluated in ddPCR for limit of blank, limit of detection, and precision. Finally, thirty-five healthy volunteer samples were evaluated to establish a baseline level of PD-L1 expression. In ddPCR, the limit of blank was determined to be 0 copies and the limit of detection was determined to be less than or equal to 19 copies of PD-L1. The average intra-run coefficient of variation in the ddPCR assay was 7.44% and average inter-run CV was 7.70%. Treatment of A549 cells with IFN γ resulted in a 6.7-fold increase in PD-L1 expression (21,580 copies in untreated cDNA versus 145,000 copies in treated cDNA). Analysis of healthy human samples yielded a median value of 1659 PD-L1 copies/μL with a range of 768-7510 copies/μL. The assay was transferred to an external service provider and results from our in-house experiments and those conducted externally shows a correlation of 0.994. In conclusion, a fit-for-purpose liquid biopsy-based, purely quantitative ddPCR assay for the detection of PD-L1 mRNA expression was developed and validated using PAXgene RNA blood samples. Linearity, reproducibility, limit of blank and limit of detection were measured and deemed suitable for clinical application. This ultra-sensitive liquid biopsy ddPCR assay has promising clinical potential in screening, longitudinal monitoring and disease progression detection.

Tissue-based SARS-CoV-2 detection in fatal COVID-19 infections: Sustained direct viral-induced damage is not necessary to drive disease progression

Coronavirus disease 2019 (COVID-19) is an ongoing pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although viral infection is known to trigger inflammatory processes contributing to tissue injury and organ failure, it is unclear whether direct viral damage is needed to sustain cellular injury. An understanding of pathogenic mechanisms has been handicapped by the absence of optimized methods to visualize the presence and distribution of SARS-CoV-2 in damaged tissues. We first developed a positive control cell line (Vero E6) to validate SARS-CoV-2 detection assays. We then evaluated multiple organs (lungs, kidneys, heart, liver, brain, intestines, lymph nodes, and spleen) from fourteen COVID-19 autopsy cases using immunohistochemistry (IHC) for the spike and the nucleoprotein proteins, and RNA in situ hybridization (RNA ISH) for the spike protein mRNA. Tissue detection assays were compared with quantitative polymerase chain reaction (qPCR)-based detection. SARS-CoV-2 was histologically detected in the Vero E6 positive cell line control, 1 of 14 (7%) lungs, and none (0%) of the other 59 organs. There was perfect concordance between the IHC and RNA ISH results. qPCR confirmed high viral load in the SARS-CoV-2 ISH-positive lung tissue, and absent or low viral load in all ISH-negative tissues. In patients who die of COVID-19-related organ failure, SARS-CoV-2 is largely not detectable using tissue-based assays. Even in lungs showing widespread injury, SARS-CoV-2 viral RNA or proteins were detected in only a small minority of cases. This observation supports the concept that viral infection is primarily a trigger for multiple-organ pathogenic proinflammatory responses. Direct viral tissue damage is a transient phenomenon that is generally not sustained throughout disease progression.

Abstract 3232: In vitro cell based cytotoxicity and T cell activation assays to assess safety and efficacy of engineered T cell therapies

Abstract Chimeric antigen receptor (CAR) and T cell receptor (TCR) engineered T cells are part of a big wave of immunotherapies showing great promise in cancer clinical trials. With the first T cell based therapies targeting hematological malignances now approved, their next challenge is solid tumors. Solid tumors create an additional challenge due to the lack of tumor specific target antigens, posing significant safety risks, i.e. on-target on-tumor, on-target off-tumor and off-target toxicities. Toxic effects previously reported vary from mild-severe cytokine release syndrome (CRS) to neurotoxicity and death. We have developed in vitro assays utilizing primary human cells from healthy tissue and/or differentiated iPCS-derived cells to assess on-target off-tumor and/or off-target cytotoxicity for engineered T cell therapies. The presence of CAR-T cell mediated cytotoxicity was measured through co-culture with healthy human primary cells to assess unwanted CAR-T reactivity as well as a high target antigen expressing control cancer cell line to confirm CAR-T functionality. The human primary cell type was selected based on its potential safety risk by establishing low level protein expression of the target antigen. Readouts included IFNγ production determined by MesoScale Discovery platform as a measure of T cell activation and Hoechst/PI staining of target cells by flow cytometry. Our study generated high quality data of the CAR-T cell, confirming functionality by showing consistent T cell activation and killing against a positive control cell line. Moreover, we were able establish a clear absence of activity against the primary human cells thus providing insight into the safety of the CAR-T therapy. The developed safety assays provide a robust and rapid platform to assess on-target off-tumor and off-target effects within immuno-oncology therapies, either TCR or CAR-T cells, in both early stage development or late stage testing of the therapeutic product. Through inclusion of a wide range of human primary cells, both high risk tissues and major organs at risk of off-target toxicity, a clear safety profile can be generate in vitro for these novel T cell therapies. Safety risks associated with cell based IO-therapies is the biggest challenge for the success of these therapies, performing a thorough safety screen on healthy primary human tissues is therefore crucial. Citation Format: Sanne Holt, Sophie Vermond, Monique Hazenoot Hazenoot, Jeroen Overman, Jamil Aarbiou Aarbiou, Jeroen DeGroot DeGroot. In vitro cell based cytotoxicity and T cell activation assays to assess safety and efficacy of engineered T cell therapies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3232.

Flow cytometry and live cell imaging for characterization of cell surface markers on human tissue-derived progenitors

Background & Aim Podoplanin (PDPN) was recently reported as a valuable potential marker for identifying human skeletal stem cells (SSCs). These are a subset of connective tissue progenitors (CTPs), a highly variable, and tissue specific group of cells capable of clonal propagation, proliferation and differentiation in vitro. Fluorescence-activated cell sorting (FACS) is a power tool for parsing cell markers, but FACS has a negative effect on colony forming efficiency. Development of better methods for isolating SSCs from human tissues using cell markers is a key step toward more viable methods of cell expansion. Our team has developed methods of live-cell large field of view (LFOV) imaging techniques that enable the identification and characterization of colony founding CTPs in culture soon after isolation and prior to cell division. The goal of this project is to define and validate the sensitivity and specificity of two LFOV imaging systems to detect the surface marker PDPN in comparison to flow cytometry. Methods, Results & Conclusion Bone marrow was aspirated from the iliac crest of consented donors. Nucleated cells were isolated. Live staining methods using anti-PDPN antibody, tested on positive control cell line U87 and negative control Jurkat, were defined for bone marrow-derived cells in suspension (Figure 1). The Cell X™ automation platform was used (Cell X Technologies) and the EVOS FL Auto 2 microscope (ThermoFisher Scientific) were used to capture LFOV images at 2 and 24 hours after plating. Subsequently, LFOV phase contrast images were collected every hour for 10 days, until colonies formed. Videos were reversed to identify individual colony founding CTPs. Quantitative image analysis was used to overlay areas of anti-PDPN and anti-CD45 staining with colony founding cells to determine the prevalence of PDPN+ and PDPN- cells among CTPs as well as the overall prevalence of PDPN+ cells in the entire adherent cell population as assessed using these methods. These data were then used to calculate the apparent prevalence of PDPN+ cells and the range of intensity of staining among CTPs, non-CTP adherent cells and total cells using each methods, with a parallel analysis using flow cytometry (LSR II). EVOS FL Auto 2 and Cell X devices are both able to detect PDPN+ and PDPN- cells in control cell lines (Figure 2) and among bone-marrow derived cells, enabling their use in further characterization of colony founding cell surface markers (CD146, CD271, HA).

Abstract LB-011: Fit-for-purpose quantitative programmed cell death ligand -1 (PD-L1) droplet digital PCR assay development using PAXgene RNA blood samples

Abstract Purpose Programmed death ligand receptor 1, (PD-L1) is a key immune checkpoint inhibitory receptor expressed on activated tumor specific immune cells. While immunohistochemistry assays have been developed for the detection of PD-L1 expression in several cancer types for patient treatment decision, a blood-based test for PD-L1 expression evaluation would be beneficial in providing complimentary evidence for potential clinical application. This study developed a fit-for-purpose clinical biomarker assay for the detection of PD-L1 mRNA expression in blood using droplet digital polymerase chain reaction (ddPCR) methods. Study Procedure Taqman primer/probe assays were selected based on coverage of PD-L1 gene and were tested for linearity and efficiency using a cDNA construct. Four housekeeping genes were analyzed in positive control cell line (A549 treated with interferon gamma), negative control cell line (A549 untreated) and PAXgene blood from healthy volunteers. Real-time quantitative PCR method was tested first, following initial experiments there were concerns with the performance of our qPCR assay prompting the switch to ddPCR which offers absolute quantification and a lower limit of detection. Assay performance characteristics of the ddPCR assay were evaluated including linearity, limit of blank (LOB), limit of detection (LOD), and precision. Thirty-five healthy volunteer samples were evaluated to establish a baseline level of PD-L1 expression. Data Summary Conducting linearity comparison between Taqman primer/probe kits using cDNA titrations showed no significant difference in linearity (r2=0.99) or efficiency (~95%) among the three primer/probe kits. Housekeeping genes B2M, GUSB, RPLP0, and TBP were compared and GUSB was most consistently expressed between IFN gamma treated and untreated cells; it was selected for use thereafter. In ddPCR, the LOB was determined to be 0 copies and the LOD was determined to be 19 copies of PD-L1. The average intra-run coefficient of variation (CV) was 7.44% and average inter-run CV was 7.70%. Analysis of healthy control samples yielded an average of 2415 PD-L1 copies with a range of 768-7510 copies. Treatment of A549 cells with IFN gamma resulted in a 6.7-fold increase in PD-L1 expression (21,580 copies in untreated cDNA versus 145,000 copies in treated cDNA). Conclusion Our fit-for purpose clinical biomarker assay development efforts have demonstrated that the ddPCR assay is capable of reliably and accurately detecting PD-L1 in PAXgene blood with low limit of detection and minimal inter-and-intra run variability. Compared to real-time PCR, ddPCR is a purely quantitative and offers a much broader dynamic range and provides more sensitive detection of PD-L1 in blood. Further evaluation and comparison of cancer to healthy volunteer samples is underway for more information on assay performance and disease biology. Citation Format: Dennis O'Rourke, Danyi Wang, Maria Cusano, Jorge F. Sanchez-Garcia, Ti Cai, Juergen Scheuenpflug, Zheng Feng. Fit-for-purpose quantitative programmed cell death ligand -1 (PD-L1) droplet digital PCR assay development using PAXgene RNA blood samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-011.

Abstract 2322: ICAM-1 targeting CAR T cell therapy for triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is an aggressive disease with a poor prognosis. TNBC patients do not respond to hormone receptor or HER2-targeted therapies owing to low expression of these cell surface biomarkers. The lacking of targeted therapy leaves conventional combination of surgery, chemotherapy and radiation therapy as the standard-of-care treatment options for TNBC. But even this combination usually fails to prevent disease recurrence. Intercellular adhesion molecule-1 (ICAM-1) was recently discovered to be upregulated in TNBC and could serve as an attractive molecular target (Guo et al., PNAS 2014). Chimeric antigen receptor (CAR) T cell therapy has shown remarkable success against hematological malignancies, however there has been little success in the treatment of solid tumors. Here, we developed an ICAM-1 targeting CAR T cell-based immunotherapeutic strategy to redirect T cell to kill solid TNBC. Methods: Patient-derived xenograft (PDX) models of TNBC were utilized to determine ICAM-1 expression by immunohistochemistry. ICAM-1 surface protein expression in TNBC cell line MDA-MB-231 was measured by flow cytometry. Primary T cells (n = 4 donors) were isolated and transduced with ICAM-1 CAR lentivirus twice at 24 and 48 hours after activation with anti-CD3/CD28 Dynabeads. In vitro killing ability was determined by co-incubation of GFP-Firefly Luciferase (GFP/Fluc) transduced target cells (HeLa, MDA-MB-231, and 293T) with transduced and non-transduced T cells. NSG mice bearing MDA-MB-231 xenografts were treated with CAR T cells to test the in vivo efficacy. Bioluminescence was used to quantify cell lysis in vitro and to track tumor growth in vivo. Results: 4 out of 8 TNBC PDX models (JAX, tumor markers: TER−/PR−/HER2−) showed strong IHC ICAM-1 staining. We validated by flow cytometry that ICAM-1 is highly expressed on TNBC cell line MDA-MB-231. These results provide further evidence supporting ICAM-1 as a molecular target for TNBC by CAR T cell therapy. Primary T cells were transduced to express ICAM-1 targeting CAR at approximately 50%. Co-incubation of CAR T cells or non-transduced T cells with ICAM-1 positive cell lines (HeLa and MDA-MB-231) or negative control cell line (293T) showed that CAR T cell-mediated killing was ICAM-1 expression dependent. After 48 hours, 70% of HeLa and 85% of MDA-MB-231 cells were specifically lysed at effector to target ratio of 5:1, while no obvious killing of 293T cells was observed. Additionally, non-transduced T cells produced little killing with less than 20% of target cell lysis. Ongoing in vivo studies will determine the efficacy of this ICAM-1 targeting CAR T cell against TNBC. Conclusions: We developed an ICAM-1 targeting CAR T cell with the ability to induce potent and specific killing of TNBC cells. Preclinical studies are being conducted to evaluate the feasibility of ICAM-1 specific CAR T cells as a potential therapy for patients with ICAM-1 positive TNBC. Citation Format: Yanping Yang, Yogindra Vedvyas, Jaclyn E. McCloskey, Irene M. Min, Moonsoo M. Jin. ICAM-1 targeting CAR T cell therapy for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2322.

Abstract 5190: Development of a multi-parameter immunofluorescence assay for identification of circulating tumor cells with epithelial-mesenchymal phenotype

Abstract Background. The epithelial-mesenchymal transition (EMT) is understood to be an important step in invasion and metastasis of cancer. It is of increasing investigational interest to identify circulating tumor cells (CTCs) that express mesenchymal markers that indicate entrance into EMT. Such cells may not express surface epithelial markers (such as EpCAM) that are often used to capture CTCs. RareCyte has developed a platform for automated visual identification and retrieval of rare cells in blood by immunofluorescence (IF) that does not rely on surface marker capture. We developed a 5-parameter assay to identify epithelial CTCs with or without mesenchymal differentiation. Methods. Buffy coats isolated from blood by AccuCyte® separation were spread onto slides. A 5-parameter IF assay was developed with the following markers: Sytox orange (nuclear dye) / cytokeratin and EpCAM (epithelial) / vimentin (mesenchymal) / CD45, CD11b and CD105 (exclusion) / EGFR (investigational biomarker). Slides were stained with the assay on an auto-stainer, and were scanned and imaged with CyteFinder®. Specificity of the assay was validated on positive and negative control cell lines spiked into normal donor blood. It was applied to a pilot set of blood samples from 3 prostate and 3 breast cancer patients that were collected under an IRB-approved protocol. Nucleated cells that were epithelial marker-positive and exclusion marker-negative were identified as CTCs. CTCs were categorized as epithelial (epiCTC) if they were vimentin-negative and epithelial-mesenchymal (E-M) if they were vimentin-positive. In a sample from a patient with known mutation in TP53 (G244S) from tissue tumor analysis, individual epiCTCs, E-M CTCs and WBC control cells were mechanically retrieved by CytePicker® and whole genome amplification was performed. The resulting DNA was PCR amplified at the TP53 mutation site and sequenced. Results. Between 15 and 193 CTCs were counted in the patient samples. The average percentage of epiCTCs was 74% (range 33 - 100%) and of E-M CTCs was 26% (range 0 - 67%). E-M CTC fraction was higher in breast than in prostate cancer samples. EGFR-positive epiCTCs and E-M CTCs were identified. In the patient with the tumor-associated TP53 G244S mutation, 2 of 2 epiCTC, 6 of 7 E-M CTC and 0 of 6 WBC contained the mutation. Conclusions. We have developed a multi-parameter immunofluorescence assay for identification of circulating tumor cells with epithelial-mesenchymal phenotype and applied it to cancer patient samples. Confirmation of malignancy of epiCTCs and E-M CTCs by single cell mutational analysis was demonstrated. The expression of EGFR on CTCs in prostate and breast cancer confirms literature reports. Citation Format: Arturo Ramirez, Nolan Ericson, Daniel Campton, Melinda Duplessis, Tanisha Mojica, Alisa Clein, Celestia Higano, VK Gadi, Daniel E. Sabath, Eric Kaldjian. Development of a multi-parameter immunofluorescence assay for identification of circulating tumor cells with epithelial-mesenchymal phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5190.

Abstract 5656: Developing metal-labeled antibody panels for multiplex proteomic characterization of rare circulating tumor cells by imaging mass cytometry

Abstract Imaging mass cytometry (IMC) is a novel technology with the potential of multiplex proteomic profiling of rare circulating and disseminated tumor cells (CTCs and DTCs) and immune cells from liquid biopsies. The high-definition single-cell analysis (HD-SCA) identifies CTCs and DTCs by the virtue of morphometric and fluorescent staining with exact locations of all the cells, which could be further characterized using downstream genomics and proteomics platform. This study aims to develop and employ metal-labeled antibody panels for multiplex proteomic characterization of CTCs, DTCs and immune cells obtained from longitudinal liquid biopsies from cancer patients on the basis of their epithelial-to-mesenchymal transition (EMT) status, cancer stem cell (CSC), dormancy, proliferation and drug resistance markers. For every antibody panel, 6-10 biomarkers were chosen, and metal-labeled antibodies were processed in a three-stage validation program using known positive and negative control cell lines. In the first stage, basal level marker expression was evaluated in positive control cells seeded on a glass slide. In the second stage, cell lines were spiked in blood and slides were prepared as per the HD-SCA protocol, stored at -800 C, and expression of biomarkers was evaluated in IMC staining. In the third stage, cell line-spiked slides were first stained in three colors immunofluorescent staining in HD-SCA, mimicking the clinical setup followed by IMC staining with metal-labeled antibodies. Antibodies showing expressions in all three stages were included to build a panel targeting EMT, CSC, vasculature mimicry, dormancy, proliferation and drug resistance biomarkers. Out of 69 metal-labeled antibodies, 24 antibodies targeting cancer and immune cell-specific biomarkers have been validated for multiplex proteomic profiling in HD-SCA platform. This includes epithelial markers EpCAM and E-cadherin; mesenchymal markers vimentin and fibronectin; CSC markers CD44, CD24, ALDH and beta-catenin; prostate-specific markers PSA, PSMA, AR-C terminal, AR-N terminal; and immune cell markers CD45, CD66a, CD3, HLA-DR, CD45RA, CD8a, CD4, CD38, CD14 and CD31. Building and validating metal-labeled antibody panels for EMT, CSC, dormancy and drug resistance biomarkers could achieve multiplex proteomic characterization of the rare cell IMC. It would be further used in monitoring treatment efficacy, mutational switchovers, and immune surveillance from longitudinal liquid biopsies collected from cancer patients to better understand the biology of a disease. Citation Format: Milind Pore, Erik Gerdtsson, Christopher Boldt, Sophia Wix, Preeya Katti, Mohan Singh, Akil Merchant, James Hicks, Peter Kuhn. Developing metal-labeled antibody panels for multiplex proteomic characterization of rare circulating tumor cells by imaging mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5656.

Comparison of HLA DQA1 and DQB1 Genes by Multiplex Ligation-Dependent Probe Amplification and Next Generation Sequencing Technologies for Celiac Disease Risk Determination

Introduction: Celiac disease is a systemic autoimmune disease with chronic gastrointestinal disorder caused by intolerance to gluten proteins. The most dominant genetic risk factors are the specific genotypes encoding the Human Leukocyte Antigen (HLA) class II molecule genes, DQA1 and DQB1. The DQ2.5 variant (DQA1*05/DQB1*02 alleles) is present in 90-95% of patients with celiac disease. The DQ8 variant (DQA1*03/DQB1*0302) and DQ2.2 variant (DQA1*02/DQB1*02) are found in most of the remaining patients. A positive test is indicative of genetic susceptibility but does not necessarily mean disease development. A negative test has a more significant value because gluten intolerance rarely occurs in the absence of specific HLA predisposing alleles. In the current study we compared Multiplex Ligation-dependent Probe Amplification (MLPA) and Next Generation Sequencing (NGS) technologies for identifying Celiac disease risk HLA alleles. Methods: DNA was extracted from either saliva or whole-blood archival samples obtained from commercial vendors using Qiagen DNA mini extraction kits. MLPA assay probe mix designed to detect the HLA alleles implicated in Celiac disease consists of 29 specific fluorescent labeled amplification products. The PCR products are separated by capillary gel electrophoresis on the ABI 3500 Genetic Analyzer yielding a specific electropherogram. Based on the presence and relative height of individual probe, as compared to the relative probe peak height in various reference samples, the HLA allele in the patient sample was determined. For Next Generation sequencing assay, long range PCR products of HLA DQA1 and DQB1 genes were pooled to prepare bar coded NGS libraries. Equimolar pools of the barcoded libraries were sequenced on the Illumina MiSeq platform by paired end 151 bp sequencing. Demultiplexed FASTQ files were aligned to HLA variant database and genotypes were identified. Results: A total of 26 samples with different HLA variants were analyzed by both MLPA and NGS assay panel. The samples included DQ2.5, DQ8, DQ2.2, both heterozygous and homozygous variants, negatives and positive control cell line DNA samples. Data is summarized in Table 1. There was 100% concordance between the two technologies in determining the Celiac disease risk alleles. DNA dilution studies showed that both technologies were optimal with a minimum of 40 ng of DNA input. The workflow for MLPA was simple with sample to data generation completed within 24 hours. NGS assay was laborious with highly technical library preparation, sequencing and data analysis with sample to data in 48 hours. However, NGS technology results in a high resolution genotyping whereas MLPA assay is limited to 5 alleles of DQA1 and DQB1 genes.Table: Table. HLA DQA1 and DQB1 genotyping data summary- MLPA and NGS dataConclusion: Both methods are reliable and efficient to provide necessary information for celiac disease risk evaluation. In the future if high resolution HLA genotyping is needed, NGS may be more applicable.

Abstract 1950: Introducing c-Myc into transformed human mesenchymal stem cells and osteoblasts to recapitulate the osteosarcoma phenotype

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults. It remains unclear at what point in the pathway of differentiation between human mesenchymal stem cells (hMSCs) and osteoblasts (OBs), OS originates. Since high grade OS frequently demonstrates histologic variability, as well as the potential for multi-lineage differentiation, some consider the hMSC as the cell of origin in OS, whereas others believe the osteoblast to be the most likely cell of origin. Identifying the primary cell of origin is crucial in understanding the molecular pathogenesis of OS. To address the potential for hMSCs or OBs to transform into OS, we have performed insertional mutagenesis in hMSCs and OBs with defined genetic elements which have the potential to lead to tumorigenesis and whose pathways have been altered in OS patient specimens. Serial introduction of the viral constructs hTERT (T), SV40Tag (S), and H-RAS (R), led to spindle cell tumor formation in mice. However, MSC-TSR tumors did not form osteoid, whereas OB-TSR tumors showed only scant production of osteoid. Also the addition of β-catenin to MSC-TS cells failed to lead to tumor formation in mice. Based on these findings, MSC-TS and OB-TS cell lines may be ideal platforms for further analysis of the impact of the genetic transformation of hMSCs and OBs into OS. c-Myc has been shown to be overexpressed in OS cells and hence has been suggested as an oncogene. It has been also suggested as a novel target of RUNX2 through rescue from the apoptosis. MSC-TS and OB-TS cells were transformed with a retrovirus containing human c-Myc. Drug resistant colonies were picked up 21 days after selection to obtain stably transformed cell lines. Quantitative PCR and western blots were carried out to detect both gene and protein expression patterns in transformed cell lines, respectively. Furthermore, in order to determine the expression pattern of c-Myc in OS, quantitative PCR and western blots were also performed on human osteosarcoma primary samples, xenografts and cell lines. All functional assays outlined below will be performed to determine the tumorigenic properties of transformed cell lines. Six out of seven (86%) osteosarcoma primary samples showed a significantly greater c-Myc gene expression level compared to the positive control cell lines. Selection of stably transformed clones and comparison between cell lines and characterizations are underway. Further ongoing characterization includes: soft agar assays, in-vivo tumorigenic assays, histological examination for osteoid production and other OS specific immunohistochemical markers, proliferation, invasion and migration, and differentiation assays. It is our hope that this study will highlight the model that closely recapitulates the human osteosarcoma phenotype by forming a malignant spindle cell tumor that produces aberrant osteoid, and has the potential for multi-lineage differentiation. Citation Format: Sajida Piperdi (Thein), Wendong Zhang, Daria Ivenitsky, Yidan Zhang, Yunjia Zhang, David S. Geller, Bang Hoang, Rui Yang, Jonathan B. Gill, Michael Roth, Richard Gorlick. Introducing c-Myc into transformed human mesenchymal stem cells and osteoblasts to recapitulate the osteosarcoma phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1950. doi:10.1158/1538-7445.AM2017-1950