We thank the Cytometry and Antibody Core Facility at the University of Chicago for support on flow cytometry instrumentation. The Animal Research Center at the University of Chicago provided animal housing.

<ol>
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The authors have no conflicts of interest to declare for this study.

Over the last decade or so, flow cytometry has become a more common assay platform in cancer research due to the emerging popularity of tumor immunology, the development of lower-cost flow cytometers, and the improvement of shared instrumentation facilities at academic institutions. Multicolor assays are now standard, as most newer instruments are equipped with violet, blue-green, and red to far-red optical arrays. Thus, this DDAOG protocol is likely to be compatible with a wide array of flow cytometers. Of course, any f...

Senescent cells normally accumulate in organisms over years during normal biological aging but may also develop rapidly in tumor cells as a response to damage induced by various cancer treatments, including radiation and chemotherapy. Though no longer proliferating, therapy-induced senescent (TIS) tumor cells may contribute to treatment resistance and drive recurrence1,2,3. Factors secreted by TIS cells can exacerbate tumor malignancy by promoting immune evasion or metastasis4,5. TIS cells develop complex, context-specific phenotypes, altered metabolic profiles, and unique immune responses6,7,8. Therefore, the identification and characterization of TIS tumor cells induced by various cancer treatment approaches is a topic of ongoing interest to the cancer research community.
To detect TIS tumor cells, conventional senescence assays are widely used, primarily based on detecting increased activity of the senescence marker enzyme, the lysosomal beta-galactosidase GLB19. Detection at a near-neutral (rather than acidic) lysosomal pH allows for specific detection of senescence-associated beta-galactosidase (SA-&#946;-Gal)10. A standard SA-&#946;-Gal assay that has been used for several decades uses X-Gal (5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside), a blue chromogenic beta-galactosidase substrate, to detect SA-&#946;-Gal in fixed cells by light microscopy11. The X-Gal assay allows the qualitative visual confirmation of TIS utilizing commonly available reagents and laboratory equipment. A basic transmitted light microscope is the only instrumentation required to evaluate the presence of the blue chromogen. However, the X-Gal staining procedure can lack sensitivity, sometimes requiring more than 24 h for color to develop. Staining is followed by low-throughput, subjective scoring of individual senescent cells based on counting the cells exhibiting some level of intensity of the blue chromogen under a light microscope. As X-Gal is cell-impermeable, this assay requires solvent-fixed cells, which cannot be recovered for downstream analysis. When working with limited samples from animals or patients, this can be a major drawback.
Improved SA-&#946;-Gal assays using cell-permeant, fluorescent enzyme substrates, including C12-FDG (5-dodecanoylaminofluorescein Di-&#946;-D-Galactopyranoside, green) and&#160;DDAOG (9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) &#946;-D-Galactopyranoside, far-red)&#160;have previously appeared in the literature12,13,14,15. The chemical probe structure and optical characteristics of DDAOG are shown in Supplementary Figure S1. These cell-permeant probes&#160;permit the analysis of living (rather than fixed) cells, and fluorescent rather than chromogenic probes facilitate the use of rapid high-throughput fluorescent analysis platforms, including high-content screening instruments and flow cytometers. Sorting flow cytometers enable the recovery of enriched populations of living senescent cells from cell cultures or tumors for downstream analysis (e.g., western blotting, ELISA, or &#39;omics). Fluorescence analysis also provides a quantitative signal, allowing for more accurate determination of the percentage of senescent cells within a given sample. Additional fluorescent probes, including viability probes and fluorophore-labeled antibodies, can readily be added for multiplexed analysis of targets beyond SA-&#946;-Gal.
Similar to DDAOG, C12-FDG is a fluorescent probe for SA-&#946;-Gal, but its green fluorescent emission overlaps with intrinsic cellular AF, which arises during senescence due to the accumulation of lipofuscin aggregates in cells16. By utilizing the far-red DDAOG probe, green cellular AF can be used as a secondary parameter to confirm senescence17. This improves assay reliability by using a second marker in addition to SA-&#946;-Gal, which can often be unreliable as a single marker for senescence18. As the detection of endogenous AF in senescent cells is a label-free approach, it is a rapid and simple way to expand the specificity of our DDAOG-based assay.
In this protocol, we demonstrate the use of DDAOG and AF as a rapid, dual-parameter flow cytometry assay for the identification of viable TIS tumor cells from in vitro cultures or isolated from drug-treated tumors established in mice (Figure 1). The protocol uses fluorophores compatible with a wide range of standard commercial flow cytometry analyzers and sorters (Table 1). Quantitation of the percentage of viable senescent cells using standard flow cytometry analysis is enabled. If desired, an optional immunolabeling step may be performed to evaluate cell surface antigens of interest concurrently with senescence. Identified senescent cells can also be enriched using standard fluorescence-activated cell sorting (FACS) methodology.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64176/64176fig01.jpg" /> 
Figure 1: Experimental workflow. A schematic summarizing key points of the DDAOG assay. (A) A TIS-inducing drug is added to mammalian cultured cells or administered to tumor-bearing mice. Time is then allowed for the onset of TIS: for cells, 4 days following treatment; for mice, 22 days total, with three treatments every 5 days plus 7 days recovery. Cells are harvested or tumors are dissociated into suspension.&#160;(B)&#160;Samples are treated with Baf to adjust lysosomal pH for detection of SA-&#946;-Gal for 30 min; then, DDAOG probe is added for 60 min to detect SA-&#946;-Gal. Samples are washed 2x in PBS, and a viability stain is briefly added (15 min). Optionally, samples can be stained with fluorescent antibodies in open fluorescence channels and/or fixed for later analysis. (C) Samples are analyzed using a standard flow cytometer. Viable cells are visualized in dot plots showing red DDAOG (indicating SA-&#946;-Gal) versus green autofluorescence (lipofuscin). A gate to determine the percentage of TIS cells is established based on untreated control samples (not shown). If a sorting cytometer (FACS) is used, TIS cells can be collected and placed back into culture for further in vitro assays or lysed and processed for molecular biology assays. Abbreviations: DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside; TIS = therapy-induced senescence; FL-Ab = fluorophore-conjugated antibody; Baf = Bafilomycin A1; SA-&#946;-Gal = senescence-associated beta-galactosidase; PBS = phosphate-buffered saline; FACS = fluorescence-activated cell sorting. <a href="https://www.jove.com/files/ftp_upload/64176/64176fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fluorophore</td>
			<td>Detects</td>
			<td>Ex/Em (nm)</td>
			<td>Cytometer laser (nm)</td>
			<td>Cytometer detector / bandpass filter (nm)</td>
		</tr>
		<tr>
			<td>DDAOG</td>
			<td>SA-&#946;-Gal</td>
			<td>645/6601</td>
			<td>640</td>
			<td>670 / 30</td>
		</tr>
		<tr>
			<td>AF</td>
			<td>Lipofuscin</td>
			<td>&#60; 600</td>
			<td>488</td>
			<td>525 / 50</td>
		</tr>
		<tr>
			<td>CV450</td>
			<td>Viability</td>
			<td>408/450</td>
			<td>405</td>
			<td>450 / 50</td>
		</tr>
		<tr>
			<td>PE</td>
			<td>Antibody/surface marker</td>
			<td>565/578</td>
			<td>561</td>
			<td>582 / 15</td>
		</tr>
	</tbody>
</table>
Table 1: Fluorophores and cytometer optical specifications. Cytometer specifications used in this protocol are listed for an instrument with a total of 4 lasers and 15 emission detectors.&#160;DDAOG detected at 645/660 nm is the form of the probe cleaved by SA-&#946;-Gal1. Uncleaved DDAOG can exhibit low level fluorescence at 460/610 nm but is removed by wash steps in the protocol. Abbreviations: DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside; AF = autofluorescence; PE = phycoerythrin; SA-&#946;-Gal = senescence-associated beta-galactosidase.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bafilomycin A1</td>
			<td>Research Products International</td>
			<td>B40500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleomycin sulfate&#160;</td>
			<td>Cayman</td>
			<td>13877</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>US Biological</td>
			<td>A1380</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein Violet 450 AM viability dye</td>
			<td>ThermoFisher Scientific</td>
			<td>65-0854-39</td>
			<td>eBioscience</td>
		</tr>
		<tr>
			<td>DPP4 antibody, PE conjugate</td>
			<td>Biolegend</td>
			<td>137803</td>
			<td>Clone H194-112</td>
		</tr>
		<tr>
			<td>Cell line: A549 human lung adenocarcinoma</td>
			<td>American Type Culture Collection</td>
			<td>CCL-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell line: B16-F10 mouse melanoma</td>
			<td>American Type Culture Collection</td>
			<td>CRL-6475</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Corning</td>
			<td>3008</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainers, 100 &#181;m</td>
			<td>Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>DDAO-Galactoside</td>
			<td>Life Technologies</td>
			<td>D6488</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM medium 1x</td>
			<td>Life Technologies</td>
			<td>11960-069</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Sigma</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin, hydrochloride injection (USP)</td>
			<td>Pfizer</td>
			<td>NDC 0069-3032-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin, PEGylated liposomal (USP)</td>
			<td>Sun Pharmaceutical</td>
			<td>NDC 47335-049-40</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA 0.5 M</td>
			<td>Life Technologies</td>
			<td>15575-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Etoposide&#160;</td>
			<td>Cayman</td>
			<td>12092</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Omega&#160;</td>
			<td>FB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc receptor blocking reagent</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td>Anti-mouse CD16/32</td>
		</tr>
		<tr>
			<td>Flow cytometer (cell analyzer)</td>
			<td>Becton Dickinson (BD)</td>
			<td>Various</td>
			<td>LSRFortessa</td>
		</tr>
		<tr>
			<td>Flow cytometer (cell sorter)</td>
			<td>Becton Dickinson (BD)</td>
			<td>Various</td>
			<td>FACSAria</td>
		</tr>
		<tr>
			<td>GlutaMax 100x</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1 M</td>
			<td>Lonza</td>
			<td>BW17737</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase TL</td>
			<td>Sigma</td>
			<td>5401020001</td>
			<td>Roche</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 100x</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) 1x</td>
			<td>Corning</td>
			<td>MT21031CV</td>
			<td>Dulbecco&#39;s PBS (without calcium and magnesium)</td>
		</tr>
		<tr>
			<td>Rainbow calibration particles, ultra kit</td>
			<td>SpheroTech</td>
			<td>UCRP-38-2K</td>
			<td>3.5-3.9 &#181;m, 2E6/mL</td>
		</tr>
		<tr>
			<td>RPMI-1640 medium 1x</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride 0.9% (USP)</td>
			<td>Baxter Healthcare Corporation</td>
			<td>2B1324</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for cytometer data acquisition, &#34;FACSDiva&#34;</td>
			<td>Becton Dickinson (BD)</td>
			<td>n/a</td>
			<td>Contact BD for license</td>
		</tr>
		<tr>
			<td>Software for cytometer data analysis, &#34;FlowJo&#34;</td>
			<td>TreeStar</td>
			<td>n/a</td>
			<td>Contact TreeStar for license</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25%</td>
			<td>Life Technologies</td>
			<td>25200-114</td>
			<td></td>
		</tr>
	</tbody>
</table>

far-red fluorescent senescence-associated &#946;-galactosidase probe for identification and enrichment of senescent tumor cells by flow cytometry

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge rapidly in tumor cells as a response to damage induced by various cancer treatments. Tumor cell senescence is generally considered undesirable, as senescent cells become resistant to death and block tumor remission while exacerbating tumor malignancy and treatment resistance. Therefore, the identification of senescent tumor cells is of ongoing interest to the cancer research community. Various senescence assays exist, many based on the activity of the well-known senescence marker, senescence-associated beta-galactosidase (SA-&#946;-Gal). 
Typically, the SA-&#946;-Gal assay is performed using a chromogenic substrate (X-Gal) on fixed cells, with the slow and subjective enumeration of "blue" senescent cells by light microscopy. Improved assays using cell-permeant, fluorescent SA-&#946;-Gal substrates, including C12-FDG (green) and DDAO-Galactoside (DDAOG; far-red), have enabled the analysis of living cells and allowed the use of high-throughput fluorescent analysis platforms, including flow cytometers. C12-FDG is a well-documented probe for SA-&#946;-Gal, but its green fluorescent emission overlaps with intrinsic cellular autofluorescence (AF) that arises during senescence due to the accumulation of lipofuscin aggregates. By utilizing the far-red SA-&#946;-Gal probe DDAOG, green cellular autofluorescence can be used as a secondary parameter to confirm senescence, adding reliability to the assay. The remaining fluorescence channels can be used for cell viability staining or optional fluorescent immunolabeling.
Using flow cytometry, we demonstrate the use of DDAOG and lipofuscin autofluorescence as a dual-parameter assay for the identification of senescent tumor cells. Quantitation of the percentage of viable senescent cells is performed. If desired, an optional immunolabeling step may be included to evaluate cell surface antigens of interest. Identified senescent cells can also be flow cytometrically sorted and collected for downstream analysis. Collected senescent cells can be immediately lysed (e.g., for immunoassays or 'omics analysis) or further cultured.

Several experiments were performed to demonstrate the comparability of DDAOG to X-Gal and C12-FDG for the detection of senescence by SA-&#946;-Gal. First, X-Gal was used to stain senescent B16-F10 melanoma cells induced by ETO (Figure 2A). An intense blue color developed in a subset of ETO-treated cells, while other cells exhibited less intense blue staining. Morphology was enlarged in most ETO-treated cells. Staining ETO-treated cells with fluorescent SA-&#946;-Gal substrate C<su...

Watch this Scientific Journal Video about Far-Red Fluorescent Senescence-Associated ÃŽÂ²-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry at JoVE.com

Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

A protocol for fluorescent, flow cytometric quantification of senescent cancer cells induced by chemotherapy drugs in cell culture or in murine tumor models is presented. Optional procedures include co-immunostaining, sample fixation to facilitate large batch or time point analysis, and the enrichment of viable senescent cells by flow cytometric sorting.

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge ...

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4.3K Views.  University of Chicago. A protocol for fluorescent, flow cytometric quantification of senescent cancer cells induced by chemotherapy drugs in cell culture or in murine tumor models is presented. Optional procedures include co-immunostaining, sample fixation to facilitate large batch or time point analysis, and the enrichment of viable senescent cells by flow cytometric sorting.

Video: Far-Red Fluorescent Senescence-Associated β-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median survival following diagnosis is less than 15 months with maximal surgical resection, radiation, and temozolomide chemotherapy. The challenges inherent in developing more effective GBM treatments have become increasingly clear, and include its unyielding invasiveness, its resistance to standard treatments, its genetic complexity and molecular adaptability, and subpopulations of GBM cells with phenotypic similarities to normal stem cells, herein referred to as glioblastoma stem cells (GSCs). Because GSCs are required for tumor growth and progression, differentiation-based therapy represents a viable treatment modality for these incurable neoplasms. 
The following protocol describes a collection of procedures to establish a high throughput screening platform aimed at the identification of small molecules that promote GSC astroglial differentiation. At the core of the system is a glial fibrillary acidic protein (GFAP) differentiation reporter-construct. The protocol contains the following general procedures: (1) establishing GSC differentiation reporter lines; (2) testing/validating the relevance of the reporter to GSC self-renewal/clonogenic capacity; and (3) high-capacity flow-cytometry based drug screening. 
The screening platform provides a straightforward and inexpensive approach to identify small molecules that promote GSCs differentiation. Furthermore, utilization of libraries of FDA-approved drugs holds the potential for the identification of agents that can be repurposed more rapidly. Also, therapies that promote cancer stem cell differentiation are expected to work synergistically with current "standard of care" therapies that have been shown to target and eliminate primarily more differentiated cancer cells.

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and protein functions, and cell signaling aberrations may therefore serve as biomarkers to indicate personalized treatment options. As opposed to DNA and RNA analyses, changes in protein activity can more efficiently evaluate the mechanisms underlying drug sensitivity and resistance. Phospho flow cytometry is a powerful technique that measures protein phosphorylation events at the cellular level, an important feature that distinguishes this method from other antibody-based approaches. The method allows for simultaneous analysis of multiple signaling proteins. In combination with fluorescent cell barcoding, larger medium- to high-throughput data-sets can be acquired by standard cytometer hardware in short time. Phospho flow cytometry has applications both in studies of basic biology and in clinical research, including signaling analysis, biomarker discovery and assessment of pharmacodynamics. Here, a detailed experimental protocol is provided for phospho flow analysis of purified peripheral blood mononuclear cells, using chronic lymphocytic leukemia cells as an example.

Here, a protocol for medium- to high-throughput analysis of protein phosphorylation events at the cellular level is presented. Phospho flow cytometry is a powerful approach to characterize signaling aberrations, identify and validate biomarkers, and assess pharmacodynamics.

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&#38;E) annotations are used to target small tumor areas using a blade that is 250 &#181;m2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 &#181;m in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

Digital annotation with automated tissue dissection provides an innovative approach to enriching tumor in low tumor content cases and is adaptable to both paraffin and frozen tissue types. The described workflow improves accuracy, reproducibility and throughput and could be applied to both research and clinical settings.

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly ...

Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as drug resistance, epithelial-mesenchymal transition (EMT), and metastasis. Intratumoral eATP is 103 to 104 times higher in concentration than in normal tissues. While eATP functions as a messenger to activate purinergic signaling for EMT induction, it is also internalized by cancer cells through upregulated macropinocytosis, a specific type of endocytosis, to perform a wide variety of biological functions. These functions include providing energy to ATP-requiring biochemical reactions, donating phosphate groups during signal transduction, and facilitating or accelerating gene expression as a transcriptional cofactor. ATP is readily available, and its study in cancer and other fields will undoubtedly increase. However, eATP study remains at an early stage, and unresolved questions remain unanswered before the important and versatile activities played by eATP and internalized intracellular ATP can be fully unraveled.
These authors&#39; laboratories&#39; contributions to these early eATP studies include microscopic imaging of non-hydrolysable fluorescent ATP, coupled with high- and low-molecular weight fluorescent dextrans, which serve as macropinocytosis and endocytosis tracers, as well as various endocytosis inhibitors, to monitor and characterize the eATP internalization process. This imaging modality was applied to tumor cell lines and to immunodeficient mice, xenografted with human cancer tumors, to study eATP internalization in vitro and in vivo. This paper describes these in vitro and in vivo protocols, with an emphasis on modifying and finetuning assay conditions so that the macropinocytosis-/endocytosis-mediated eATP internalization assays can be successfully performed in different systems.

We developed a reproducible method to visualize the internalization of nonhydrolyzable fluorescent adenosine triphosphate (ATP), an ATP surrogate, with high cellular resolution. We validated our method using independent in vitro and in vivo assays-human tumor cell lines and immunodeficient mice xenografted with human tumor tissue.

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Flow Cytometry-Based Monitoring of Cancer-Immunity Cycle 

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between cancer and immune cells, the so-called cancer-immunity cycle. In vitro co-culture systems combined with multiparametric flow cytometry (mFC) and tumor-on-a-chip microfluidic devices (ToCs) enable simple, fast, and reliable monitoring and characterization of each step of the cancer-immunity cycle and lead to the identification of the mechanisms responsible for tipping the balance between cancer immunosurveillance and immunoevasion. A thorough understanding of the dynamic interplays between cancer and immune cells provides critical insights to outsmart tumors and will accelerate the pace of therapeutic personalization and optimization in patients. Specifically, here we detail a straightforward mFC- and ToC-assisted protocol for unraveling the dynamic complexities of each step of the cancer-immunity cycle in murine cancer cell lines and mouse-derived immune cells and focus on immunosurveillance. Considering the time- and cost-related features of this protocol, it is certainly feasible on a large scale. Moreover, with minor variations, this protocol can be both adapted to human cancer cell lines and human peripheral-blood-derived immune cells and combined with genetic and/or pharmacologic inhibition of specific pathways in order to identify biomarkers of immune response.

Monitoring the Cancer-Immunity Cycle and Exploring Tumor Microenvironment Dynamics

Here, we detail a simple, fast, and reliable multiparametric flow cytometry- and tumor-on-a-chip-assisted protocol to monitor and characterize the steps constituting the cancer-immunity cycle. Indeed, a thorough understanding of the interplays between cancer and immune cells provides critical insights to outsmart tumors and guide clinical care.

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between ...

Utilizing Larval Zebrafish for Efficient In Vivo Tumor Studies

A Simple, Rapid, and Effective Method for Tumor Xenotransplantation Analysis in Transparent Zebrafish Embryos

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we present larval zebrafish as a transplant model that has numerous advantages over murine models, including ease of handling, low expense, and short experimental duration. Moreover, the absence of an adaptive immune system during larval stages obviates the need to generate and use immunodeficient strains. While established protocols for xenotransplantation in zebrafish embryos exist, we present here an improved method involving embryo staging for faster transfer, survival analysis, and the use of flow cytometry to assess disease burden. Embryos are staged to facilitate rapid cell injection into the yolk of the larvae and cell marking to monitor the consistency of the injected cell bolus. After injection, embryo survival analysis is assessed up to 7 days post injection (dpi). Finally, disease burden is also assessed by marking transferred cells with a fluorescent protein and analysis by flow cytometry. Flow cytometry is enabled by a standardized method of preparing cell suspensions from zebrafish embryos, which could also be used in establishing the primary culture of zebrafish cells. In summary, the procedure described here allows a more rapid assessment of the behavior of tumor cells in vivo with larger numbers of animals per study arm and in a more cost-effective manner.

Author Spotlight: Advancing Cancer Therapeutics Using Tumor Xenotransplantation in Zebrafish

We describe a protocol for xenotransplantation into the yolk of transparent zebrafish embryos that is optimized by a simple, rapid staging method. Post-injection analyses include survival and assessing the disease burden of xenotransplanted cells by flow cytometry.

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we ...