Molecular Digital CytoImaging for Leukemia Drug Screening

目前的细胞术技术查询细胞表型和蛋白质表达，但不能可靠地测量 由于其基础水平较低，在单细胞中检测具有挑战性的关键临床相关蛋白质 和/或药物治疗后水平降低（例如磷蛋白、免疫调节蛋白）。作为 因此，许多药物靶点没有得到评估，无法可靠地解释检测的缺失 作为负面结果。为了克服敏感蛋白检测的主要瓶颈，OHSU和Becton 迪金森（BD）将商业翻译成像技术，使研究人员能够执行 在原始患者样本中以前所未有的分子水平灵敏度进行药物筛选。我们已经建立了一个 DigiCount平台，它实现了一种新的策略，在细胞中计数离散的免疫标记蛋白质。 在单细胞中存在扩散背景噪声（例如细胞自发荧光）（Jacob等，Nat Sci 2016年报告; OHSU：1项专利已提交，2项正在申请中）。专有的数字化分子定位算法 辨别用超亮抗体-荧光团探针标记的离散蛋白质复合物， 1-2对数倍增加的信噪比超过常规流式细胞术/荧光显微术。我们将 扩大DigiCount的吞吐量，以实现高效的临床筛选。我们还将生成额外的探测器 包括临床相关/低丰度蛋白（干细胞，免疫调节， 磷蛋白）以进一步增加靶筛选信息。探头面板利用了一类新的 具有高效光捕获放大化学和超亮发射的导电聚合物染料 （2000年诺贝尔奖）。这些探针选自BD的化合物库，并针对以下方面进行了专门优化： 数字化分子检测在急性髓细胞白血病的试点筛查中，1例患者入选Beat AML研究，一项正在进行的900例患者研究，以确定改进的AML靶向治疗（OHSU Knight 癌症研究所），我们已经确定了协同小分子/免疫检查点抑制剂组合 其比单分子更有效地产生AML细胞杀伤并诱导免疫细胞增殖 剂.我们将应用DigiCount筛选每位患者的数百种组合，以确定是否存在 是小分子/免疫检查点抑制剂组合的主要类别， 单一代理我们还将分析有希望的组合的效果，以表征它们对 免疫和骨髓细胞靶点。研究团队由单细胞成像的创新者 技术（PI：Vu，OHSU），一位在串联聚合物荧光团方面具有专业知识的首席科学家， 来自商业部门（PI：Martin，Becton Dickinson）和临床医生的新型/HCA检测试剂盒开发- 致力于鉴定用于临床部署的AML靶向治疗剂的科学家（co-PI：泰纳，Lind， OHSU）。来自关键意见领袖的支持信证明了 DigiCount技术和BD和OHSU的商业翻译关系支持。

Current cytometry technologies query cell phenotype and protein expression but do not reliably measure key, clinically-relevant proteins that are challenging to detect in single cells due to their low basal levels and/or diminished levels following drug treatment (e.g. phosphoproteins, immune regulatory proteins). As a result, many drug targets are not evaluated and the absence of detection cannot be reliably interpreted as a negative result. To overcome major bottlenecks in sensitive protein detection, OHSU and Becton Dickinson (BD) will commercially translate an imaging technology that will enable investigators to perform drug screening with unprecedented molecular-level sensitivity in primary patient samples. We have built a platform, the DigiCount, that implements a new strategy of counting discrete immunolabeled proteins in the presence of diffuse background noise (e.g. cell autofluorescence) in single cells (Jacob et al, Nat Sci Reports 2016; OHSU: 1 patent filed, 2 pending). Proprietary digitized molecular-localization algorithms discriminate discrete protein complexes tagged with ultrabright antibody-fluorophore probes, offering up to 1-2 log times increased signal to noise over conventional flow cytometry/fluorescence microscopy. We will scale up the DigiCount throughput for efficient clinical screening. We will also generate additional probe panels that include markers of clinically-relevant/low abundance proteins (stem cell, immune regulatory, phosphoproteins) to further increase target screening information. Probe panels leverage a new class of conductive polymer dyes with efficient light harvesting amplification chemistry and ultrabright emission (Nobel Prize, 2000). The probes are selected from BD's compound library, and specifically optimized for digitized molecular detection. In pilot screening of acute myeloid leukemia 1 patients enrolled in the Beat AML study, an ongoing 900 patient study to identify improved AML targeted therapies (OHSU Knight Cancer Institute), we have identified synergistic small molecule/immune checkpoint inhibitor combinations that produce AML cell kill and induce immune cell proliferation more effectively than single molecule agents. We will apply the DigiCount to screen hundreds of combinations per patient to determine if there are main classes of small-molecule/immune checkpoint inhibitor combinations that are more effective than single agents. We will also profile the effects of promising combinations to characterize their effect on immune and myeloid cell targets. The research team consists of the innovator of single cell imaging technologies (PI: Vu, OHSU), a lead scientist with expertise in tandem polymer fluorophores and novel/HCA assay development from the commercial sector (PI: Martin, Becton Dickinson), and clinician- scientists leading efforts to identify AML targeted therapeutics for clinical deployment (co-PIs: Tyner, Lind, OHSU). Letters of support from key opinion leaders attest to the innovation and clinical significance of the DigiCount technology and support of relationships at BD and OHSU for commercial translation.