An array of phenotypically diverse myeloid cells and macrophages (MC&M) resides in the tumor microenvironment, requiring multiplexed detection systems for visualization. Here we report an automated, multiplexed staining approach, named PLEXODY, that consists of five MC&M-related fluorescently-tagged antibodies (anti - CD68, - CD163, - CD206, - CD11b, and - CD11c), and three chromogenic antibodies, reactive with high- and low-molecular weight cytokeratins and CD3, highlighting tumor regions, benign glands and T cells. The staining prototype and image analysis methods which include a pixel/area-based quantification were developed using tissues from inflamed colon and tonsil and revealed a unique tissue-specific composition of 14 MC&M-associated pixel classes. As a proof-of-principle, PLEXODY was applied to three cases of pancreatic, prostate and renal cancers. Across digital images from these cancer types we observed 10 MC&M-associated pixel classes at frequencies greater than 3%. Cases revealed higher frequencies of single positive compared to multi-color pixels and a high abundance of CD68+/CD163+ and CD68+/CD163+/CD206+ pixels. Significantly more CD68+ and CD163+ vs. CD11b+ and CD11c+ pixels were in direct contact with tumor cells and T cells. While the greatest percentage (~70%) of CD68+ and CD163+ pixels was 0–20 microns away from tumor and T cell borders, CD11b+ and CD11c+ pixels were detected up to 240 microns away from tumor/T cell masks. Together, these data demonstrate significant differences in densities and spatial organization of MC&M-associated pixel classes, but surprising similarities between the three cancer types.
Human umbilical tissue-derived cells (hUTC or palucorcel) are currently under clinical investigation for the treatment of geographic atrophy, a late stage of macular degeneration, but how hUTC transplantation mediates vision recovery is not fully elucidated. Subretinal administration of hUTC preserves visual function in the Royal College of Surgeons (RCS) rat, a genetic model of retinal degeneration caused by loss of function. hUTC secrete synaptogenic and neurotrophic factors that improve the health and connectivity of the neural retina. Therefore, we investigated the progression of synapse and photoreceptor loss and whether hUTC treatment preserves photoreceptors and synaptic connectivity in the RCS rats of both sexes. We found that RCS retinas display significant deficits in synaptic development already by postnatal day 21 (P21), before the onset of photoreceptor degeneration. Subretinal transplantation of hUTC at P21 is necessary to rescue visual function in RCS rats, and the therapeutic effect is enhanced with repeated injections. Synaptic development defects occurred concurrently with morphological changes in Müller glia, the major perisynaptic glia in the retina. hUTC transplantation strongly diminished Müller glia reactivity and specifically protected the α2δ-1-containing retinal synapses, which are responsive to thrombospondin family synaptogenic proteins secreted by Müller glia. Müller glial reactivity and reduced synaptogenesis observed in RCS retinas could be recapitulated by CRISPR/Cas9-mediated loss-of- in Müller glia in wild-type rats. Together, our results show that hUTC transplantation supports the health of retina at least in part by preserving the functions of Müller glial cells, revealing a previously unknown aspect of hUTC transplantation-based therapy. Despite the promising effects observed in clinical trials and preclinical studies, how subretinal human umbilical tissue-derived cell (hUTC) transplantation mediates vision improvements is not fully known. Using a rat model of retinal degeneration, the RCS rat (lacking ), here we provide evidence that hUTC transplantation protects visual function and health by protecting photoreceptors and preserving retinal synaptic connectivity. Furthermore, we find that loss of function only in Müller glia is sufficient to impair synaptic development and cause activation of Müller glia. hUTC transplantation strongly attenuates the reactivity of Müller glia in RCS rats. These findings highlight novel cellular and molecular mechanisms within the neural retina, which underlie disease mechanisms and pinpoint Müller glia as a novel cellular target for hUTC transplantation.
Background: The repair of double-stranded breaks in DNA occurs through homologous recombination (HR) or nonhomologous enjoining (NHEJ) pathways. Germline mutations in DNA damage repair enzymes reduce efficiency of DNA damage repair (DDR) and increase cancer risk, but also the sensitivity to DNA damaging agents, such as cisplatinum and PARP1 inhibitors. Because the regulation of DDR occurs through protein-protein interactions and post-translational modifications, genomic methods are insufficient to identify cancers with defective DDR pathways. The formation of DDR protein complexes underlies strict spatial regulation. Therefore, the measurement of DDR efficiency requires in situ detection of DDR foci by visualizing the location of multiple proteins within the nucleus. This can be accomplished by multiantibody immunofluorescent staining combined with digital image analysis. The main goal of the project is to develop tissue staining assays for protein complexes involved in DDR using 5 antibodies per slide and quantitative digital image analysis pipelines to integrate data from consecutive slides in order to establish a DDR index of a patient’s prostate cancer. Methods: We developed 2 fully automated 5-plex fluorescent (RPA32, RAD51, Ku80, XRCC4, pH2AX) (53BP1, pDNA-PK, PARP1, PAR, AR) antibody protocols on the Ventana Discovery autostainer. The fluorescent assays were combined with an automated cytokeratin (CK), Ki-67, and Geminin chromogenic staining protocol on the exact same tissue. The panels were applied to a TMA of high-grade prostate cancer and for comparison to a bladder and a rectal cancer TMA slide. Slides were imaged after the fluorescent portion of the assay was completed and subsequently processed for staining with CK (yellow) and Ki-67 (teal) and Geminin (brown). Chromogenic and fluorescent images were coregistered. To integrate images from adjacent slides, a transformation matrix was applied to distort the images in order to maximize the overlap of nuclear DAPI stains. The nuclear staining of each antibody was quantified through intensity, area, and texture measurements and compared in nuclei positive and negative for pH2AX. After histogram normalization and subtraction of background and autofluorescence, the amount of colocalized pixels between antibody pairs was determined. Results: We have demonstrated feasibility of automated multiplex staining protocols that increase the reproducibility and future clinical utility of DDR assays. Specific staining of individual antibodies was identified as a punctate nuclear staining pattern in a fraction of cancer cells, and multiple parameters of the staining pattern (intensity, stained area, subnuclear distribution) were quantified using digital image analysis. DDR occurred in high-grade and low-grade prostate cancer as determined by the percentage of cancer cells that stained positive for pH2AX. pH2AX staining was also noticed in normal epithelium (weakly proliferative), stroma (nonproliferative), and inflammatory cells (proliferative). At least 5 nuclei from each compartment (tumor, normal, stroma, and inflammation) per TMA core were selected for analyses based on pH2AX positivity. Using a consistent image analysis pipeline to set staining intensity thresholds, overlapping pixels between RPA32-RAD51, Ku80-XRCC4, 53BP1-pDNAPK, and the overlap of pixels of each protein complex with AR were determined. The intracase and intercase variance of DDR complexes was compared for each cell type (cancer, normal epithelium, stroma, and inflammation) and correlated with the amount of cell proliferation. Conclusion: We demonstrate feasibility of measuring DDR complexes in single nuclei. The assay has the potential to be used in a CLIA-certified laboratory and the quantitative measurements from the multiplex DDR assays can be compared to those from other OMICS platforms, such as mutation burden and copy number alterations. Funding: Precision Medicine initiative and Translational Research Core at Cedars-Sinai Medical Center. Citation Format: Joshua Saylor, Eric Weterings, Nathan Ellis, James Hinton, Esteban Roberts, Anne Cress, Beatrice Knudsen. An automated tissue staining and quantitative digital image analysis pipeline for quantification of DNA damage repair at the single-cell level [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B095.
Background: Stem cell products are increasingly entering early stage clinical trials for treating retinal degeneration. The field is learning from experience about comparability of cells proposed for preclinical and clinical use. Without this, preclinical data supporting translation to a clinical study might not adequately reflect the performance of subsequent clinical-grade cells in patients. Methods: Research- grade human neural progenitor cells (hNPC) and clinical-grade hNPC (termed CNS10-NPC) were injected into the subretinal space of the Royal College of Surgeons (RCS) rats, a rodent model for retinitis pigmentosa (RP); An IND-enabling study with CNS10-NPC was perform in the same rodent model; Finally, surgical methodology for subretinal cell delivery in the clinic was optimized in large animal model-Yucatan minipig. Results: Both research grade hNPC and clinical-grade hNPC (termed CNS10-NPC) can survive and provide functional and morphological protection in a dose-dependent fashion in the RCS rats and defined the optimal cell dose used for an investigational new drug (IND) enabling study. Grafted CNS10-NPC migrated from the injection site without differentiation into retinal cell phenotypes. Additionally, CNS10-NPC showed long-term survival, safety and efficacy in a toxicity and tumorigenicity study, with no observed cell overgrowth even at the maximum deliverable dose. Finally, using a large animal model-Yucatan minipig, which has eye size comparable to the human, we optimized the surgical methodology for subretinal cell delivery in the clinic. Conclusions: These extensive studies supported an approved IND and the translation of CNS10-NPC to an ongoing Phase 1/2a clinical trial (NCT04284293) for the treatment of retinitis pigmentosa.
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