Multiplexed Tissue Tomography.

Multiplexed tissue tomography enables comprehensive spatial analysis of markers within a whole tissue or thick tissue section. Clearing agents are often used to make tissue transparent and facilitate deep tissue imaging. Many methods of clearing and tissue tomography are currently used in a variety of tissue types. Here we detail a workflow known as transparent tissue tomography (T3), which builds upon previous methods and can be applied to difficult to clear tissues such as tumors.

UltraPlex Hapten-Based Multiplexed Fluorescent Immunohistochemistry.

The UltraPlex method for multiplexed two-dimensional fluorescent immunohistochemistry is described, in which hapten tags conjugated to primary antibodies facilitate multiplexed imaging of four or more antigens per tissue section at once. Anti-hapten secondary antibodies labeled with fluorophores provide amplified signal for detection, which is accomplished using a standard fluorescent microscope or digital slide scanner. The protocol is rapid and straightforward and utilizes conventionally prepared tissue samples. The resulting staining is highly sensitive and specific, enabling high-resolution imaging of multiple cellular subtypes within tissue samples. Tumor cells and tumor-infiltrating lymphocytes are presented as examples. Multiple 4-plex-stained tissue samples can be digitally overlaid to create 8-plex (or more) high-content images, enabling visualization of distribution of complex cellular subtypes across tissues.

O-GlcNAcylation Enhances Double-Strand Break Repair, Promotes Cancer Cell Proliferation, and Prevents Therapy-Induced Senescence in Irradiated Tumors.

The metabolic reprogramming associated with characteristic increases in glucose and glutamine metabolism in advanced cancer is often ascribed to answering a higher demand for metabolic intermediates required for rapid tumor cell growth. Instead, recent discoveries have pointed to an alternative role for glucose and glutamine metabolites as cofactors for chromatin modifiers and other protein posttranslational modification enzymes in cancer cells. Beyond epigenetic mechanisms regulating gene expression, many chromatin modifiers also modulate DNA repair, raising the question whether cancer metabolic reprogramming may mediate resistance to genotoxic therapy and genomic instability. Our prior work had implicated N-acetyl-glucosamine (GlcNAc) formation by the hexosamine biosynthetic pathway (HBP) and resulting protein O-GlcNAcylation as a common means by which increased glucose and glutamine metabolism can drive double-strand break (DSB) repair and resistance to therapy-induced senescence in cancer cells. We have examined the effects of modulating O-GlcNAcylation on the DNA damage response (DDR) in MCF7 human mammary carcinoma in vitro and in xenograft tumors. Proteomic profiling revealed deregulated DDR pathways in cells with altered O-GlcNAcylation. Promoting protein O-GlcNAc modification by targeting O-GlcNAcase or simply treating animals with GlcNAc protected tumor xenografts against radiation. In turn, suppressing protein O-GlcNAcylation by blocking O-GlcNAc transferase activity led to delayed DSB repair, reduced cell proliferation, and increased cell senescence in vivo. Taken together, these findings confirm critical connections between cancer metabolic reprogramming, DDR, and senescence and provide a rationale to evaluate agents targeting O-GlcNAcylation in patients as a means to restore tumor sensitivity to radiotherapy. IMPLICATIONS: The finding that the HBP, via its impact on protein O-GlcNAcylation, is a key determinant of the DDR in cancer provides a mechanistic link between metabolic reprogramming, genomic instability, and therapeutic response and suggests novel therapeutic approaches for tumor radiosensitization.

HMG-CoA Reductase Inhibition Delays DNA Repair and Promotes Senescence After Tumor Irradiation.

Despite significant advances in combinations of radiotherapy and chemotherapy, altered fractionation schedules and image-guided radiotherapy, many cancer patients fail to benefit from radiation. A prevailing hypothesis is that targeting repair of DNA double strand breaks (DSB) can enhance radiation effects in the tumor and overcome therapeutic resistance without incurring off-target toxicities. Unrepaired DSBs can block cancer cell proliferation, promote cancer cell death, and induce cellular senescence. Given the slow progress to date translating novel DSB repair inhibitors as radiosensitizers, we have explored drug repurposing, a proven route to improving speed, costs, and success rates of drug development. In a prior screen where we tracked resolution of ionizing radiation-induced foci (IRIF) as a proxy for DSB repair, we had identified pitavastatin (Livalo), an HMG-CoA reductase inhibitor commonly used for lipid lowering, as a candidate radiosensitizer. Here, we report that pitavastatin and other lipophilic statins are potent inhibitors of DSB repair in breast and melanoma models both in vitro and in vivo When combined with ionizing radiation, pitavastatin increased persistent DSBs, induced senescence, and enhanced acute effects of radiation on radioresistant melanoma tumors. shRNA knockdown implicated HMG-CoA reductase, farnesyl diphosphate synthase, and protein farnesyl transferase in IRIF resolution, DSB repair, and senescence. These data confirm on-target activity of statins, although via inhibition of protein prenylation rather than cholesterol biosynthesis. In light of prior studies demonstrating enhanced efficacy of radiotherapy in patients taking statins, this work argues for clinical evaluation of lipophilic statins as nontoxic radiosensitizers to enhance the benefits of image-guided radiotherapy. Mol Cancer Ther; 17(2); 407-18. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

A signature of enhanced lipid metabolism, lipid peroxidation and aldehyde stress in therapy-induced senescence.

At their proliferative limit, normal cells arrest and undergo replicative senescence, displaying large cell size, flat morphology, and senescence-associated beta-galactosidase (SA-ß-Gal) activity. Normal or tumor cells exposed to genotoxic stress undergo therapy-induced senescence (TIS), displaying a similar phenotype. Senescence is considered a DNA damage response, but cellular heterogeneity has frustrated identification of senescence-specific markers and targets. To explore the senescent cell proteome, we treated tumor cells with etoposide and enriched SA-ß-GalHI cells by fluorescence-activated cell sorting (FACS). The enriched TIS cells were compared to proliferating or quiescent cells by label-free quantitative LC-MS/MS proteomics and systems analysis, revealing activation of multiple lipid metabolism pathways. Senescent cells accumulated lipid droplets and imported lipid tracers, while treating proliferating cells with specific lipids induced senescence. Senescent cells also displayed increased lipid aldehydes and upregulation of aldehyde detoxifying enzymes. These results place deregulation of lipid metabolism alongside genotoxic stress as factors regulating cellular senescence.

Repurposing cephalosporin antibiotics as pro-senescent radiosensitizers.

Radiation therapy remains a significant therapeutic modality in the treatment of cancer. An attractive strategy would be to enhance the benefits of ionizing radiation (IR)with radiosensitizers. A high-content drug repurposing screen of approved and investigational agents, natural products and other small molecules has identified multiple candidates that blocked repair of IR damage in vitro. Here, we validated a subset of these hits in vitro and then examined effects on tumor growth after IR in a murine tumor model. Based on robust radiosensitization in vivo and other favorable properties of cephalexin, we conducted additional studies with other beta-lactam antibiotics. When combined with IR, each cephalosporin tested increased DNA damage and slowed tumor growth without affecting normal tissue toxicity. Our data implicate reactive oxygen species in the mechanism by which cephalosporins augment the effects of IR. This work provides a rationale for using commonly prescribed beta-lactam antibiotics as non-toxic radiosensitizers to enhance the therapeutic ratio of radiotherapy.

DNA-directed assembly of antibody-fluorophore conjugates for quantitative multiparametric flow cytometry.

Multiparametric flow cytometry offers a powerful approach to single-cell analysis with broad applications in research and diagnostics. Despite advances in instrumentation, progress in methodology has lagged. Currently there is no simple and efficient method for antibody labeling or quantifying the number of antibodies bound per cell. Herein, we describe a DNA-directed assembly approach to fluorescent labeling that overcomes these barriers. Oligonucleotide-tagged antibodies and microparticles can be annealed to complementary oligonucleotides bearing fluorophores to create assay-specific labeling probes and controls, respectively. The ratio of the fluorescence intensity of labeled cells to the control particles allows direct conversion of qualitative data to quantitative units of antibody binding per cell. Importantly, a single antibody can be labeled with any fluorophore by using a simple mix-and-match labeling strategy. Thus, any antibody can provide a quantitative probe in any fluorescent channel, thus overcoming major barriers to the use of flow cytometry as a technique for systems biology and clinical diagnostics.