Abstract 5651: Achieving simultaneous improvements in workflow and multiplexing in whole-slide tissue imaging

With very high spatial and biological complexity, tumor micro-environments present a significant characterization challenge, which is only heightened by the need to image entire sections of cancer tissue on many different samples. A variety of techniques offer very high levels of multiplexing, but always fail on either speed requirements (incredibly long times required to image a whole slide) or on compatibility issues (expensive, hard-to-operate CapEx not suitable for high-throughput CLIA operation). Ultivue has developed a platform reagent technology (InSituPlex™) that simultaneously enables whole- slide higher multiplexing by immunofluorescence AND complete compatibility with existing instrumentation. The InSituPlex technology comprises five key foundational elements. First, short unique DNA barcodes are covalently attached to each primary antibody (Ab). These conjugates are purified and functionally characterized in tissue for optimal staining specificity. Importantly, all of this work is done in advance by Ultivue. Second, tissue samples are stained in a single step. Third, all the DNA sequences attached to targets (via primary Ab binding) are simultaneously amplified, allowing for detection of less abundant markers e.g. PD-1, PDL-1 etc. Fourth, complementary DNA sequences each with different, covalently- attached fluorescent labels (“imager strands”) are simultaneously hybridized to the amplified DNA on the targets. All these wet chemistry steps are carried out in walkaway mode using commercially available auto-stainers. The samples are then imaged using a standard, whole-slide multi-color fluorescence imaging system. Finally, in an optional fifth step, the imager strands are gently removed via an enzymatic dehybridzation- with complete retention of antigenicity- to enable higher multiplexing via additional rounds of hybridization/imaging. The InSituPlex reagent technology offers many advantages for cancer research over current approaches: • the ability to detect 5, 10, or more targets simultaneously via whole-slide imaging on an each and every sample, with no loss in antigenicity; • the ability to leverage existing auto-stainers and existing fluorescent microscopes with Ultivue9s reagent kits to carry out high-throughput analyses of multiple cancer biomarkers; and • the ability to design custom biomarker panels that leverage Ultivue9s unique barcoding capability and infrastructure. This poster will elaborate on each step of the technology, and provide multiple examples of data acquired using different biomarker panels and different cancer tissue types. Citation Format: Michael Natan. Achieving simultaneous improvements in workflow and multiplexing in whole-slide tissue imaging [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 5651.