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.

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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><tbody><tr><td>Bafilomycin A1</td><td>Research Products International</td><td>B40500</td><td></td></tr><tr><td>Bleomycin sulfate&amp;nbsp;</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 &amp;micro;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&amp;nbsp;</td><td>Cayman</td><td>12092</td><td></td></tr><tr><td>FBS</td><td>Omega&amp;nbsp;</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&#039;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 &amp;micro;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, &quot;FACSDiva&quot;</td><td>Becton Dickinson (BD)</td><td>n/a</td><td>Contact BD for license</td></tr><tr><td>Software for cytometer data analysis, &quot;FlowJo&quot;</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 Î²-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 ...

far-red-fluorescent-senescence-associated-galactosidase-probe-for

Research

JoVE Journal

Cancer Research

4.5K Views.  University of Chicago. Ons multi-parameter flowcytometrieprotocol maakt snelle en verbeterde detectie van therapie-geïnduceerde senescentie in tumorcellen mogelijk, wat een voorwaarde is voor lopende studie in kankerbiologie en -therapie. Onze flowcytometrie senescentietest is sneller dan bestaande op microscopie gebaseerde assays. We gebruiken twee markers, in plaats van slechts één, om senescente cellen te identificeren, waardoor de betrouwbaarheid van de test wordt verbeterd.Cellen kunnen worden sameng...

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 ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

A Sensitive Method to Quantify Senescent Cancer Cells

Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce.
	Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required.
	To this end, the standard &beta;-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.

Whether senescence prevents or promotes tumorigenesis remains controversial. Since chemotherapeutical drugs can induce cancer cells to senesce, studying senescence is essential for proposing new therapies. However, the standard and broadly used &beta;-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

Human cells do not indefinitely proliferate. Upon external and/or intrinsic  cues, cells might die or enter a stable cell cycle arrest called senescence. ...

Medicine

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

Developmental Biology

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, ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Biology

Flow Cytometry-Based Quantification of Therapy-Induced Senescent Cancer Cells

Source: Flor, A., et al. <a href="https://app.jove.com/v/64176">Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry.</a> J. Vis. Exp. (2022)
This video illustrates a flow cytometry-based assay for quantifying therapy-induced senescent cancer cells with dual senescence markers. The detection and quantification of senescent cells involve using a &#946;-galactosidase substrate. The substrate is cleaved by active lysosomal &#946;-galactosidase within the senescent cells, producing a fluorescent product that, in conjunction with autofluorescent lipofuscin granules, marks the senescent cancer cells.

1. Induction of senescence by chemotherapy drugs in cultured cancer cells
NOTE: All cell manipulation steps in this section should be performed in a ...

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

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