Name: dried blood and serum spots as a useful tool for sample storage to evaluate cancer biomarkers
Uploaded: 2018-06-11T13:00:00
Description: Watch this Scientific Journal Video about Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers at JoVE.com

We would like to thank Gr&#225;inne Tierney for editorial assistance.

Peripheral blood and serum were collected and stored from healthy donors and patients who gave written informed consent to take part in the study. The study protocol was approved by the local Ethics Committee in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.
1. Blood Storage in DBS
<ol>
	<li>Blood sample collection
	<ol>
		<li>Collect 3 mL of peripheral blood sample in a 3 mL tube with 5.4 mg of ethylenediaminetetraacetic acid (EDTA).<br...

<ol>
	<li>Bertagnolio, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+drug+resistance+surveillance+using+dried+whole+blood+spots.">HIV-1 drug resistance surveillance using dried whole blood spots.</a> Antivir Ther. 12 (1), 107-113 (2007).</li><li>Mei, J. V., Alexander, J. R., Adam, B. W., Hannon, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+filter+paper+for+the+collection+and+analysis+of+human+whole+blood+specimens.">Use of filter paper for the collection and analysis of human whole blood specimens.</a> J Nutr. 131 (5), 1631S-1636S (2001).</li><li>Garrido, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtype+variability,+virological+response+and+drug+resistance+assessed+on+dried+blood+spots+collected+from+HIV+patients+on+antiretroviral+therapy+in+Angola.">Subtype variability, virological response and drug resistance assessed on dried blood spots collected from HIV patients on antiretroviral therapy in Angola.</a> J Antimicrob Chemoth. 61 (3), 694-698 (2008).</li><li>Steegen, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+of+detecting+human+immunodeficiency+virus+type+1+drug+resistance+in+DNA+extracted+from+whole+blood+or+dried+blood+spots.">Feasibility of detecting human immunodeficiency virus type 1 drug resistance in DNA extracted from whole blood or dried blood spots.</a> J Clin Microbiol. 45 (10), 3342-3351 (2007).</li><li>Costenaro, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+load+detection+using+dried+blood+spots+in+a+cohort+of+HIV-1-infected+children+in+Uganda:+correlations+with+clinical+and+immunological+criteria+for+treatment+failure.">Viral load detection using dried blood spots in a cohort of HIV-1-infected children in Uganda: correlations with clinical and immunological criteria for treatment failure.</a> J Clin Microbiol. 52 (7), 2665-2667 (2014).</li><li>Gong, Z. H., Tian, G. L., Huang, Q. W., Wang, Y. M., Xu, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+glutathione+and+glutathione+disulfide+in+the+blood+of+glucose-6-phosphate+dehydrogenase-deficient+newborns.">Reduced glutathione and glutathione disulfide in the blood of glucose-6-phosphate dehydrogenase-deficient newborns.</a> BMC Pediatr. 20 (17), 172 (2017).</li><li>Lukacs, Z., Barr, M., Hamilton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Best+practice+in+the+measurement+and+interpretation+of+lysosomal+acid+lipase+in+dried+blood+spots+using+the+inhibitor+Lalistat+2.">Best practice in the measurement and interpretation of lysosomal acid lipase in dried blood spots using the inhibitor Lalistat 2.</a> Clin Chim Acta. 471, 201-205 (2017).</li><li>Skogstrand, P. T., Bent, N. P., Schendel, D. E., S&#248;rensen, L. C., Hougaard, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+measurement+of+25+inflammatory+markers+and+neurotrophins+in+neonatal+dried+blood+spots+by+immunoassay+with+xMAP+technology.">Simultaneous measurement of 25 inflammatory markers and neurotrophins in neonatal dried blood spots by immunoassay with xMAP technology.</a> Clin Chem. 51 (10), 1854-1866 (2005).</li><li>Amadori, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+D+receptor+polymorphisms+or+serum+levels+as+key+drivers+of+breast+cancer+development?+The+question+of+the+vitamin+D+pathway.">Vitamin D receptor polymorphisms or serum levels as key drivers of breast cancer development? The question of the vitamin D pathway.</a> Oncotarget. 8 (8), 13142-13156 (2017).</li><li>Gupta, B. P., Jayasuryan, N., Jameel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+detection+of+hepatitis+B+virus+from+dried+blood+spots+by+polymerase+chain+reaction+amplification.">Direct detection of hepatitis B virus from dried blood spots by polymerase chain reaction amplification.</a> J Clin Microbiol. 30 (8), 1913-1916 (1992).</li><li>Colson, K. E., Potter, A., Conde-Glez, C., Hernandez, B., R&#237;osZertuche, D., Z&#250;&#241;iga-Brenes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+commercial+ELISA+for+the+detection+of+measles-specific+immunoglobulin+G+(IgG)+in+dried+blood+spots+collected+from+children+living+in+low-resource+settings.">Use of a commercial ELISA for the detection of measles-specific immunoglobulin G (IgG) in dried blood spots collected from children living in low-resource settings.</a> J Med Virol. 87 (9), 1491-1499 (2015).</li><li>Drabe, C. H., Blauenfeldt, T., Ruhwald, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ELISA-based+assay+for+IP-10+detection+from+filter+paper+samples.">ELISA-based assay for IP-10 detection from filter paper samples.</a> Methods Mol Biol. 1172, 27-37 (2014).</li><li>St Julien, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+quality+genome-wide+genotyping+from+archived+dried+blood+spots+without+DNA+amplification.">High quality genome-wide genotyping from archived dried blood spots without DNA amplification.</a> PLoS One. 8 (5), e64710 (2013).</li><li>Shaner, R. L., Schulze, N. D., Seymour, C., Hamelin, E. I., Thomas, J. D., Johnson, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+fentanyl+analogs+in+dried+blood+spots+by+flow-through+desorption+coupled+to+online+solid+phase+extraction+tandem+mass+spectrometry.">Quantitation of fentanyl analogs in dried blood spots by flow-through desorption coupled to online solid phase extraction tandem mass spectrometry.</a> Anal Methods. 9, 3876-3883 (2017).</li></ol>

This protocol investigates the potential for storing blood and serum on filter paper when the labs do not have the staff or infrastructures needed for the correct handling of blood samples. In particular, whole blood or serum collected in standard tubes or by finger-prick can be stored using this method and there is no need to freeze samples at -20 &#176;C or -80 &#176;C immediately after the blood collection. DBS/DSS can remain at room temperature for up to 14 days without any change in blood/serum integrity. A critical...

The main aim of the research into cancer biomarkers is the identification of new biological parameters that can be used for diagnosis, for predicting patient prognosis, and for determining whether a patient will respond to a specific treatment. This research area is fundamental for the discovery of innovative cancer treatments and plays a key role in tailored treatment.
The procedures carried out during each step of biomarker identification and validation must be reliable and reproducible. A cornerstone for the success of translational research is the correct storage of biological samples such as blood and serum. This is the first step towards obtaining high quality biological material that can be used to perform molecular biology experiments or protein assays.
Multicenter studies are often needed to recruit enough patients to obtain robust data. Not all institutes are able to store samples at -80 &#176;C or to send samples to other international centers in dry ice. The use of filter paper for blood collection is a simple method for storing blood and serum and does not require the immediate freezing of samples1,2. A drop of blood or serum can be spotted onto the paper, left to dry overnight and then stored for up to 14 days at room temperature1,2. This gives researchers time to send the samples to other laboratories. The use of dried blood spots (DBS) and dried serum spots (DSS) could thus simplify the collaboration between institutes in developed and developing countries.
Given its ease of use, DBS sampling is widely used in several types of assays for serological or genetic downstream analyses. For example, in the past, DBS were frequently used for HIV screening in developing countries1,2,3,4,5. Another advantage of this storage method is that blood samples can be collected from finger-pricks, thus enabling its use for newborn screening tests 6,7,8. Easy sample handling and transport are further advantages of DBS, especially for samples collected in remote sites where there is no laboratory equipment. In a previous publication, we used DBS and DSS to test vitamin D and vitamin D binding protein (DBP) in a series of Caucasian and African patients9. Our African colleagues were not able to obtain dry ice. To compare the biological markers to understand the differences in the vitamin D pathway between the two ethnic populations, we further improved the procedure using matched samples stored under standard conditions and on filter paper. After optimizing the DBS/DSS procedure, we were able to analyze DBP and vitamin D in patient serum in both cohorts. We also evaluated a number of single nucleotide polymorphisms (SNPs) after DNA extraction from whole blood for Caucasians and from DBS for Africans9. The present protocol permits high quality blood samples to be stored at room temperature without affecting different types of downstream analyses ranging from molecular biology to ELISA assays. It is recommended for use to manage biological materials in multicenter studies or for centers that do not have facilities for standard storage conditions. The following protocol represents the culmination of these optimized procedures.

<table>
	<tbody>
		<tr>
			<td>Whatman protein saver cards 903 Protein saver card, 100/pk&#8206;</td>
			<td>Sigma</td>
			<td>Z761575</td>
			<td>Useful to store samples at room temperature for downstream analyses</td>
		</tr>
		<tr>
			<td>Falcon Serological Pipettes, 5 mL</td>
			<td>Stem cell</td>
			<td>#38003</td>
			<td></td>
		</tr>
		<tr>
			<td>50-200 &#181;L tips</td>
			<td>Star-Lab</td>
			<td>S1120-8810</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL centrifuge tube</td>
			<td>Eppendorf</td>
			<td>4036-3204</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp DNA microkit</td>
			<td>Qiagen</td>
			<td>56304</td>
			<td>This is a DNA extraction kit designed to isolate small quantities of DNA&#160;&#160;</td>
		</tr>
		<tr>
			<td>Buffer ATL (included in QIAamp DNA microkit)</td>
			<td>Qiagen</td>
			<td></td>
			<td>This is reported as Lysis Buffer 1 in the text</td>
		</tr>
		<tr>
			<td>Buffer AL (included in QIAamp DNA microkit)</td>
			<td>Qiagen</td>
			<td></td>
			<td>This is reported as Lysis Buffer 2 in the text</td>
		</tr>
		<tr>
			<td>QIAamp mini elute column</td>
			<td>Qiagen</td>
			<td></td>
			<td>This is reported as column in the text</td>
		</tr>
		<tr>
			<td>AW1&#160; (included in QIAamp DNA microkit)</td>
			<td>Qiagen</td>
			<td></td>
			<td>This is reported as Wash Buffer 1 IN THE TEXT</td>
		</tr>
		<tr>
			<td>AW2&#160; (included in QIAamp DNA microkit)</td>
			<td>Qiagen</td>
			<td></td>
			<td>This is reported as Wash Buffer 2 in the text</td>
		</tr>
		<tr>
			<td>Human Vitamin D BP Quantikine ELISA Kit</td>
			<td>R&#38;D systems</td>
			<td>DVDBP0</td>
			<td></td>
		</tr>
	</tbody>
</table>

dried blood and serum spots as a useful tool for sample storage to evaluate cancer biomarkers

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the starting point to achieve reproducible and reliable results. All samples should be treated in the same way from blood collection to storage. Depending on the analyses to be performed, whole blood and serum samples should be stored at -20 &#176;C or -80 &#176;C until use. Blood/serum samples should also be aliquoted to avoid multiple freeze-thawing. Another important issue is the sample conditions during shipment from one laboratory to another. If dry ice is not available or the shipment takes longer than a few days, alternative approaches are needed. One option is to use filter paper for blood collection. Here, we propose a method for blood and serum sample collection that takes advantage of dried blood spots (DBS) and dried serum spots (DSS). We developed the procedure to extract DNA from DBS for the downstream evaluation of some single nucleotide polymorphisms (SNPs) by real time PCR. We also optimized an ELISA assay starting from proteins eluted from DSS. This method can be used with other ELISA assays or procedures evaluating proteins.

We took advantage of the DBS and DSS procedures to store blood and serum at room temperature without affecting the quality of the biological material. Figure 1 shows an example of the protein card saver without blood and after blood collection. In order to confirm that the storage on filter paper and the procedure to elute blood do not interfere with sample quality, we performed a comparison with standard storage methods. We extracted DNA from 3 matched sampl...

Watch this Scientific Journal Video about Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers at JoVE.com

Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers

This protocol describes a simple and useful method to store peripheral blood and serum/plasma for downstream analyses such as single nucleotide polymorphism (SNP) evaluation and ELISA assay.

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the ...

This method can help answer key questions in translational research such as the chance to study biomarkers in patients from developing countries. The main advantage of this technique is the possibility to store blood and serum samples for at least two weeks without freezing and provide high quality material for downstream analysis such as qPCR and ELISA. The implications of this technique extend toward diagnosis of diseases such as cancer and infectious diseases.This method can also be applied toward the diagnostic or research fields like the study of prenatal diseases or the analysis of infectious pathology such as HIV. We first had the idea for this method when we started comparing biomarkers between a Caucasian and an African case series for which frozen samples were not available. Visual demonstration of this method is critical as the blood spotting steps are difficult to learn.In this procedure, three milliliters of a blood sample is collected in a three milliliter tube with 5.4 milligrams of EDTA. Write the patient code number on the bottom right-hand corner of the saver card. Carefully re-suspend the blood with a five milliliter pipette.Next, pipette and transfer one spot of blood onto the saver card. Using a new tip for each blood aliquot, repeat the spotting to obtain a total of three to five blood spots. Leave the dried blood spots, or DBS, to dry overnight at room temperature with the cover of the saver card open in a horizontal position over an open, non-absorbent surface and avoiding direct sunlight.On the following day, store the card at room temperature in a plastic bag with desiccant until use. DNA will be extracted from three dried blood spots from the saver card. Begin by cutting each of the DBS from the card and separating them from the card.Cut the separated DBS into small pieces of about one millimeter in diameter. Place the small pieces from one DBS into a 1.5 milliliter centrifuge tube. To digest the samples, add to the tube 180 microliters of lysis buffer one from a commercially available DNA extraction kit, then add 20 microliters of proteinase K.Mix by vortexing.Place the tube in a thermo-incubator and incubate with shaking at 56 degrees celsius for 60 minutes. At the end of the incubation, briefly centrifuge the tube to remove drops from the lid. Start the wash by adding 200 microliters of lysis buffer two and vortexing for 10 seconds.Place the tube in a thermo-mixer and incubate at 70 degrees celsius for 10 minutes. Place the elusion column in a new two milliliter collection tube and discard the old one. Add 500 microliters of wash buffer and centrifuge at 6000 times gravity for one minute.Place the column in a new two milliliter collection tube and discard the old one. Add 500 microliters of wash buffer two and centrifuge at 6000 times gravity for one minute. Place the column in a new two milliliter collection tube and discard the old one.Centrifuge at 20, 000 times gravity for three minutes to dry the membrane. To elute the DNA, place the column in a new 1.5 milliliter centrifuge tube and discard the collection tube. Place 50 microliters of distilled water onto the center of the membrane and incubate at room temperature for 10 minutes.Centrifuge the tube at 20, 000 times gravity for one minute. Collection the elute and place again on the center of the membrane. Centrifuge the tube again at 20, 000 times gravity for one minute.Discard the membrane. After measuring the DNA concentration using a spectrophotometer, store the DNA at minus 20 degrees celsius until use. For this procedure, seven milliliters of peripheral blood sample is first collected in a seven milliliter tube without EDTA.Leave the blood sample to coagulate at room temperature for 30 minutes. Write the patient code number on the bottom right-hand corner of the saver card. Centrifuge the blood sample at 1100 times gravity at room temperature for 15 minutes.Remove the supernatant from the tube and transfer it to a new 15 milliliter tube. Carefully re-suspend the blood with a five milliliter pipette. Pipette and transfer one spot of serum onto the saver card.Repeat the spotting to have a total of three to five blood spots. Leave the dried sero spots to dry overnight at room temperature with the cover of the saver card open. On the following day, store the card at room temperature in a plastic bag with desiccant until use.If storage is longer than 14 days, store at minus 20 degrees celsius. Prepare for the ELISA assay by thawing the DSS for each sample if necessary. To elute the protein from the DSS, cut one DSS into small pieces of about one millimeter in diameter and put the pieces in a 1.5 milliliter tube.Then, add four microliters of PBS. Shake the samples at low speed at four degrees celsius overnight. On the following day, use an appropriate dilution of the protein elute as a serum sample for secreted protein or growth factor analysis using a commercially available kit.In this protocol, blood and serum samples were stored as dried spots on filter paper at room temperature. A representative protein saver card is shown before and after blood sampling. In order to confirm that the storage on filter paper and the procedure to elute blood do not interfere with the sample quality, ELISA assays for vitamin D binding protein, or DBP levels, were performed on eight samples of serum collected in a two milliliter tube and stored immediately at minus 80 degrees celsius or collected as dried serum spots.The coefficient of variation, or CV, between matched samples ranged from 2%to 24%and the mean CV was 9.6%Once mastered, this technique can be done in 10, 15 minutes if it is performed properly. Following this procedure, downstream methods of molecular biology or protein analysis can be performed. After its development, this technique enables research in the field of translational research and biomarker analysis to collaborate with institutes from all over the world that do not have the lab equipment to store biological samples in standard conditions.After watching this video, you should have a good understanding of how to correctly store blood samples in dried blood and serum spots. Don't forget that working with blood and chemical reagent can be extremely hazardous and precautions such as wearing gloves and a lab coat should always be taken while performing this procedure.

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Research

JoVE Journal

Cancer Research

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

Using the E1A Minigene Tool to Study mRNA Splicing Changes

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic treatment.

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides ...

Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties make it an excellent in vivo experimental model to study angiogenesis, tumor growth, drug delivery systems, or photodynamic diagnosis (PDD) and photodynamic therapy (PDT). At the same time, this model addresses the requirement for the replacement of experimental animals with a suitable alternative. Ex ovo cultivated embryo allows easy substance application, access, monitoring, and documentation. The most frequently used is chick CAM; however, this article describes the advantages of the Japanese quail CAM as a low-cost and high-throughput model. Another advantage is the shorter embryonic development, which allows higher experimental turnover. The suitability of quail CAM for PDD and PDT of cancer and microbial infections is explored here. As an example, the use of the photosensitizer hypericin in combination with lipoproteins or nanoparticles as a delivery system is described. The damage score from images in white light and changes in fluorescence intensity of the CAM tissue under violet light (405 nm) was determined, together with analysis of histological sections. The quail CAM clearly showed the effect of PDT on the vasculature and tissue. Moreover, changes like capillary hemorrhage, thrombosis, lysis of small vessels, and bleeding of larger vessels could be observed. Japanese quail CAM is a promising in vivo model for photodynamic diagnosis and therapy research, with applications in studies of tumor angiogenesis, as well as antivascular and antimicrobial therapy.

The chorioallantoic membrane (CAM) of the avian embryo is a very useful and applicable tool for various areas of research. A special ex ovo model of Japanese quail CAM is suitable for photodynamic treatment investigation.

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties ...