This work was supported by the philanthropic contributions of the John G. and Marie Stella Kenedy Memorial Foundation (Grant #0727033) and the Melanoma Research Alliance. The authors wish to thank the University of Texas MD Anderson Cancer Center Institutional Tissue Bank (ITB) for coordinating tissue distribution and collection and facilitating translational research, as well as Dr. Ignacio Wistuba, Dr. Victor Prieto, the MD Anderson Cancer Center Department of Pathology and the Melanoma Moon Shot Program. Finally, the authors wish to acknowledge all patients and families affected by melanoma.

Ethics statement: This protocol is approved by the University of Texas MD Anderson Cancer Center.
1. Specimen Procurement
<ol>
	<li>Collect research tissue samples from surplus tumor and related normal tissue10-12. Store specimens on wet ice to avoid autolysis.</li>
	<li>Send all tissues, medical devices and foreign bodies removed during a surgical procedure to the Department of Pathology for necessary examination in order to facilitate a pathologic diagnosis.</li>
	<li>Handle tissue allocated for research in accordance with applicable research and tissue banking policies and procedures. 
	NOTE: Biospecimen collection involves the coordination of pathologists, pathologists&#39; assistants, histotechnologists and tissue procurement specialists.</li>
</ol>
2. Pathology Quality Assurance (QA)&#160;
<ol>
	<li>Complete and verify patient records as an early QA measure and consult a pathologist to review diagnostic patient records (pathology reports, tissue slides, etc.) and tissue samples. Judge tissue samples on morphological characteristics of interest such as percentage of tumor, necrosis, tissue-tumor interface, etc. This is in accordance with biorepository best practices10-12.</li>
	<li>Following initial review, conduct periodic reviews of biorepository data and research samples during the entire length of stay of the biospecimen.</li>
	<li>Link results and annotations from histopathological and other investigations to the samples and provide this data to the researchers through a proprietary biorepository software system. &#160; 
	NOTE: QA processes ensure the integrity of both the clinical and technical aspects of biorepository operations.</li>
	<li>Perform genetic and molecular tests to verify and characterize the samples for clinical use.</li>
	<li>Collect and analyze data based on feedback from researchers who have been using the samples for laboratory experiments. Track samples using barcode technology. This electronic tracking system is also advocated in biorespository best practices10-12.</li>
</ol>
3. Specimen/Tissue Processing &#160;
<ol>
	<li>Always store fresh tissue specimens on wet ice, and process as quickly as possible, in order to limit changes induced by extraction of the tissue sample from its natural environment. Process normal tumor-matched and tumor tissue samples separately to avoid cross-contamination. 
	NOTE: Based on research protocols, biospecimen processing can involve collection of the specimens in one or more types of media.</li>
	<li>Before beginning the process, prepare a sterile Petri culture dish, a scalpel, as well as a needle or forceps for tissue processing. Ensure that tubes have been prepared, labeled, and laid out in order to limit time of sample processing (Figure 2).</li>
	<li>All relevant tissue information, such as patient name, number, treatment, date of surgery, and other relevant information as may be appropriate for the study, should be made readily accessible in an institutional tissue database. Limited patient information should be held in the lab in paper or spreadsheet form. 
	NOTE: Only collect biospecimen information relevant to the nature of the research viz., basic biomedical research, clinical research, translational research, etc. For disease-specific information, the biorepository staff should collect data deemed necessary by the current literature and governing organizations to ensure proper diagnosis and meaningful research. This includes but is not limited to tumor grades and cancer staging information.</li>
	<li>Plan all desired subsequent experiments in advance, to decide on the size of tissue sections required for each type of analysis, as well as the location of excisions. Ideally, although impossible, all analyses should be representative of the entire tissue section in order to account for tissue heterogeneity and limit bias of downstream analyses. Lay out all required items in advance as depicted in Figure 2.</li>
	<li>Place the tissue specimen in the open Petri culture dish, flat against the surface. 
	NOTE: Allow only authorized biorepository staff to handle identified samples and to review identified health data. Investigators should access only those patient identifiers as allowed by the signed informed consents and as regulated by the IRB. All specimens must be adequately labeled and should include at minimum, a unique tissue identification number and tissue type. Do not include any PHI in the labels, in accordance with HIPAA regulations.</li>
	<li>Inspect the sample from all angles, to gain a better understanding of its structure and dimensions. Pin the sample to the bottom of the dish with the needle, and proceed to bisect the tissue along its largest and best representative axis.</li>
	<li>Cut out a small piece of tissue and place it in a sterile 1.5 ml microcentrifuge tube containing 1ml of RNA-stabilizing solution. This sample can be placed on ice for the remainder of the process and stored at 4 &#176;C until needed. 
	NOTE: When working with RNA, gloves should always be worn, and RNAse-free filter tips should be used to limit chances of degradation and RNA contamination.</li>
	<li>Lay out an open and pre-labeled biopsy cassette in the lid of the Petri culture dish. Cut out a slice of tissue no thicker than 3 to 5 mm and transfer it to the cassette. Place the cassette in 10% Neutral Buffered Formalin (NBF) for a minimum of 72 hr. Transfer the cassette in 70% ethanol for long-term storage before paraffin-embedding. 
	NOTE: Labeling of cassettes should be carried out in pencil or with recommended markers as use of xylene during processing will otherwise erase labels.</li>
	<li>Cut out a piece of tissue and place it in a tissue mold. Cover the tissue section in optimal cutting temperature (OCT) compound, and freeze it immediately at -20 &#176;C for cryosectioning. This tissue section may be stored at -20 &#176;C until ready for cryosectioning.</li>
	<li>Cut out one or many pieces of tissue and transfer them to a 50 ml tube containing MACS buffer (1x phosphate buffered saline (PBS), 0.5% fetal bovine serum (FBS), 2 mM Ethylenediaminetetraacetic acid (EDTA)) for subsequent flow cytometry analysis. For flow cytometry, larger samples may be preferable as cells may be lost during tumor disassociation, cell fixation and permeabilization when required. Generally, this section may be taken at the end, once all other pieces have been processed. Leftover representative tumor sample may be used for flow cytometry. Optionally, this tube may be stored at 4 &#176;C O/N or used for flow cytometric staining immediately following digestion into a cell suspension (see Step 4 - Flow Cytometric Staining).</li>
	<li>Cut remainder of tissue into pieces no larger than a maximum size of 0.5 mm x 0.5 mm and transfer them to cryogenic vials for immediate freezing in liquid nitrogen. These snap-frozen samples may be used for future analyses and collaborative purposes. 
	NOTE: Extra care should be taken when working with liquid nitrogen as direct contact can cause severe injury. Eyes should be protected with safety glasses with side shields or with a face shield. Cryogenic gloves should also be worn to protect from liquid nitrogen burns caused by splashing and direct contact. Gloves should fit properly but be loose enough to be removed quickly should liquid nitrogen splash into them.</li>
</ol>
4. Flow Cytometric Staining
<ol>
	<li>Tissue Homogenization
	<ol>
		<li>Prepare digestion medium with DMEM (40 ml), DNase I (50 U/ml) and Collagenase I (2 mg/ml). Place fresh or O/N-stored tumor in MACS buffer in the lid of a Petri dish, and submerge with digestion medium. 
		NOTE: Different digestion methods exist, each with advantages (i.e., cell surface protein expression, viability). Ensure you have the proper method for your desired downstream application.</li>
		<li>Pin tumor to the bottom of the dish with a needle, and cut from the outside to center with a scalpel, until the tumor has a paste-like texture. Transfer tumor mix to a 50 ml tube, seal and place in a plastic bag to avoid leaks. Transfer tube to an incubator, and rotate at 225 RPM, 37 &#176;C, 1 hr.</li>
		<li>Filter tumor cell suspension through a 70&#181;m cell strainer. Centrifuge cell suspension at 250 x g, 4 &#176;C, 5 min. Resuspend cell pellet in 1 ml FACS buffer (500 ml PBS, 1 ml 0.5 M stock EDTA, 10 ml FCS or FBS).</li>
		<li>Count cells using a hemocytometer and re-suspend at a concentration of 1x107 cells/ml. Transfer 100 &#181;l per well (106 cells) in a 96 well round bottom plate for staining.</li>
	</ol>
	</li>
	<li>Viability and Antibody Staining
	<ol>
		<li>Add 200 &#181;l of viability stain solution to each well and mix. Incubate for 30 minutes, at 4 &#176;C, in the dark. Spin down cells at 250 x g, 4 &#176;C, 5 min, and aspirate supernatant.</li>
		<li>Add antibody mixture at recommended/titrated concentration (as per manufacturer) for surface antigens in 200 &#181;l of FACS buffer. Incubate cells at 4 &#176;C, 30 min, in the dark. Spin down cells at 250 x g, 4 &#176;C, 5 min, and aspirate supernatant.</li>
		<li>Wash with 200 &#181;l of FACS buffer, spin down cells at 250 x g, 4 &#176;C, 5 min, and aspirate supernatant. Resuspend cells in 200 &#181;l of FACS buffer and proceed to flow cytometer for acquisition and analysis. 
		NOTE : If performing intracellular flow cytometry staining, perform fixation and permeabilization steps and intracellular staining between steps 4.2.2 and 4.2.3. If performing cytokine staining, cells should be pre-activated preceding staining as recommended, and an inhibitor of intracellular protein transport and secretion such as monensin should be used during activation to ensure retention of soluble factors. Fixed cells may be stored short-term (one week) at 4 &#176;C in the dark prior to acquisition on a flow cytometer.</li>
	</ol>
	</li>
</ol>

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	<li>Lorsch, J. R., Collins, F. S., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Biology.+Fixing+problems+with+cell+lines.">Cell Biology. Fixing problems with cell lines.</a> Science. 346, 1452-1453 (2014).</li><li>van der Worp, H. B., 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=Can+animal+models+of+disease+reliably+inform+human+studies.">Can animal models of disease reliably inform human studies.</a> , (2010).</li><li>McCabe, P. M., 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=Animal+models+of+disease.">Animal models of disease.</a> Physiol. Behav. 68, 501-507 (2000).</li><li>Woolf, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+meaning+of+translational+research+and+why+it+matters.">The meaning of translational research and why it matters.</a> JAMA. 299, 211-213 (2008).</li><li>Brockbank, K. G., Taylor, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Preservation.">Tissue Preservation.</a> Advances in Biopreservation. , 157-159 (2006).</li><li>Bulmer, M., Gilbert, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ethics+of+social+research.">The ethics of social research.</a> Researching social life. , 45-57 (2001).</li><li>Penslar, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> IRB guidebook. , (1993).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> MD Anderson Cancer Center Institutional Tissue Bank Standard Operating Procedures. 3, (2009).</li><li>Ruffell, B., 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=Leukocyte+composition+of+human+breast+cancer.">Leukocyte composition of human breast cancer.</a> Proc. of the Ntnl. Aca. of Sci. 109, 2796-2801 (2012).</li><li>Shapiro, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical flow cytometry. , (2005).</li><li>Baker, A. F., 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=Stability+of+phosphoprotein+as+a+biological+marker+of+tumor+signaling.">Stability of phosphoprotein as a biological marker of tumor signaling.</a> Clin. Cancer Res. 11, 4338-4340 (2005).</li><li>Jacobs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+processing+considerations+for+detecting+copy+number+changes+in+formalin-fixed,+paraffin-embedded+tissues.">Sample processing considerations for detecting copy number changes in formalin-fixed, paraffin-embedded tissues.</a> Cold Spring Harbor protocols. 2012, 1195-1202 (2012).</li><li>Chung, J. -. Y., 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=Factors+in+tissue+handling+and+processing+that+impact+RNA+obtained+from+formalin-fixed,+paraffin-embedded+tissue.">Factors in tissue handling and processing that impact RNA obtained from formalin-fixed, paraffin-embedded tissue.</a> J. Histochem. Cytochem. 56, 1033-1042 (2008).</li><li>Guo, H., 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=An+efficient+procedure+for+protein+extraction+from+formalin-fixed,+paraffin-embedded+tissues+for+reverse+phase+protein+arrays.">An efficient procedure for protein extraction from formalin-fixed, paraffin-embedded tissues for reverse phase protein arrays.</a> Proteome Sci. 10, 1477-5956 (2012).</li><li>Feldman, A. L., 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=Advantages+of+mRNA+amplification+for+microarray+analysis.">Advantages of mRNA amplification for microarray analysis.</a> Biotechniques. 33, 906-914 (2002).</li><li>Francino, O., 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=Advantages+of+real-time+PCR+assay+for+diagnosis+and+monitoring+of+canine+leishmaniosis.">Advantages of real-time PCR assay for diagnosis and monitoring of canine leishmaniosis.</a> Vet Parasitol. 137, 214-221 (2006).</li><li>Grunberg-Manago, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Messenger+RNA+stability+and+its+role+in+control+of+gene+expression+in+bacteria+and+phages.">Messenger RNA stability and its role in control of gene expression in bacteria and phages.</a> Annu. Rev. Genet. 33, 193-227 (1999).</li><li>Lesnik, E. A., Freier, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+thermodynamic+stability+of+DNA,+RNA,+and+DNA:RNA+hybrid+duplexes:+relationship+with+base+composition+and+structure.">Relative thermodynamic stability of DNA, RNA, and DNA:RNA hybrid duplexes: relationship with base composition and structure.</a> Biochemistry. 34, 10807-10815 (1995).</li><li>MacBeath, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+microarrays+and+proteomics.">Protein microarrays and proteomics.</a> Nat Genet. 32, 526-532 (2002).</li></ol>

The authors have no conflicts of interest to disclose.

Ultimately, the methods of processing must be dictated by the desired hypothesis and anticipated technical assays to be performed (Figure 1). The following section serves as an overview to describe potential assays that may be performed on such tissue sections. It is by no means an exhaustive overview, but is intended to help guide sample processing methods and choice of downstream assays by describing techniques and listing their strengths and weaknesses.
Flow cytometry stain...

Medical research has benefited immensely from the study of both human cell lines and animal models. Cell lines allow for better control of components in a system, as well as use of cells from the proper organism. Working with human cells, while more representative, presents a drastically simplified snapshot of mechanisms which may be responsible for health and disease, because cell lines may not recapitulate the impact of other cells, stromal components, and organs which may affect responses1. On the other hand, animal models, although not entirely accurate in reproducing human disease and genetic heterogeneity, offer further insight by allowing for input of whole animal systems in most models2,3.
Shortcomings aside, both methods have their benefits and are complementary. However, the main goal remains to study human cells and organs, in a full body, multi-organ system. Accordingly, translational research has taken great strides in facilitating the study of human organs and tissues extracted from patients, in order to better grasp the mechanisms underlying disease. Translational research offers the advantage of studying the result of treatments and diseases in the entire body and in appropriate cells, thereby providing the best of both worlds between cell and animal work4.
Translational research is also not without flaws. Harvested specimens from human patients, upon removal from the host, are immediately subjected to ischemia and other external factors from which they would be otherwise protected. This may result in stress-induced molecular and genetic changes, and cause bias in subsequent studies. Therefore, much effort has been put into extracting and processing tissues through strict methods to best preserve organ and cell integrity for downstream studies5. Importantly, future applications must be considered closely when selecting methods of processing human samples, as certain methods may be detrimental to cellular components and signaling pathways while sparing others.
For any research involving human tissues, regulatory issues must be addressed. After the Tuskegee and Belmont reports, there was a growing acceptance of the fact that biomedical research was associated with inherent risks often resulting in unavoidable ethical dilemmas6. The need for national standards was recognized and the federal government created Institutional Review Boards (IRBs) as a means to uphold the principles of the Belmont Report (Respect for persons, Beneficence, Justice).
Any institution&#39;s IRB is a committee of doctors, researchers and community members who are responsible for protecting research participants. It requires that voluntary consent be obtained from all participants of biomedical research studies, and that this consent be documented in an informed consent form. The informed consent process aims to provide adequate information to a participant, so that an informed decision may be made on whether or not to enroll in a study, or to continue participation. In this regard, the informed consent document must have good readability and should be written in language that can be easily comprehended (6th to 8th grade reading level) by the target population. The possibility of coercion or undue influence must be minimized, and the subject must be given ample time to consider participation. The participant must also be allowed to exercise their right to withdraw consent at any stage during the course of the study without penalty. Voluntary informed consent is a mandatory pre-requisite for a subject&#39;s participation in research and it is also legally effective7.
As per the regulations for the protection of human subjects 21 CFR 50.25 (<a href="http://www.hhs.gov/ohrp/humansubjects/guidance/45cfr46.html">http://www.hhs.gov/ohrp/humansubjects/guidance/45cfr46.html</a>8), study participants must be informed of the purpose of the research, procedures involved in the research, alternatives to participation, all foreseeable risks and discomforts (including not only physical injury but also possible psychological, social, or economic harm, discomfort, or inconvenience), benefits of the research to society and possibly to the individual, length of time the subject is expected to participate, person to contact for answers to questions or in the event of a research-related injury or emergency, statement indicating that participation is voluntary and that refusal to participate will not result in any consequences or any loss of benefits such as a compromise in regular clinical care that the participant is otherwise entitled to receive as a result of his/her diagnoses, and finally a statement regarding the subject&#39;s right to confidentiality and right to withdraw from the study at any time without any consequences. Tissue specimens from participants who withdraw consent during the course of the study should not be banked and must be immediately exhausted. Waiver of one or more elements of informed consent may be obtained from the IRB for some research projects that could not practically be done without an alteration to the required elements or for studies where required elements are not applicable. Great care must be taken to ensure data confidentiality. The Health Insurance Portability and Accountability Act (HIPAA) is a federal law that prevents using or disclosing &#8220;Protected Health Information&#8221; (PHI) without written consent from the participants. PHI includes but is not limited to health information transmitted or maintained in any form or medium and includes fields that could be used to identify an individual9.
Through this work, we aim to outline the protocol that must be followed during tissue processing - including procurement, and storage and emphasize the importance of following these strict guidelines so as to minimize the biases that may result due to the stresses a tissue is subjected to upon extraction. We also endeavor to provide a brief overview of the downstream assays or analyses (Figure 1) that could be performed on such specimens so that readers may make a qualified judgment on what assay(s) best suits their needs.

<table>
	<tbody>
		<tr>
			<td>Needles</td>
			<td>BD</td>
			<td>305167</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel disposable scalpels</td>
			<td>Miltex</td>
			<td>4-410</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Cassettes</td>
			<td>Thermo Fisher</td>
			<td>58931</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA-stabilizing solution</td>
			<td>Ambion</td>
			<td>AM7021</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml Eppendorf tubes</td>
			<td>Phenix</td>
			<td>MAX-815</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml tubes</td>
			<td>Corning</td>
			<td>430290</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>StatLab Medical Products</td>
			<td>28600-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin Containers</td>
			<td>Fisher Scientific</td>
			<td>23-032-059</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal cutting temperature solution (OCT)</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Cryomold</td>
			<td>Sakura Finetek</td>
			<td>4557</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Roche</td>
			<td>11088793001</td>
			<td></td>
		</tr>
		<tr>
			<td>70&#956;m cell strainers</td>
			<td>BD Bioscience</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well round bottom plates</td>
			<td>Corning</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead stain solution</td>
			<td>Life Technologies</td>
			<td>L34957</td>
			<td></td>
		</tr>
	</tbody>
</table>

working with human tissues for translational cancer research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

The results displayed below depict flow cytometry gating strategy for broad immune phenotyping on a processed melanoma tumor. The day of staining, tissue sample was digested and homogenized with collagenase I and DNase I incubation for 60 min, at 37 &#176;C under 225 RPM rotation. Following digestion, cell suspension was filtered through a 70 &#181;m cell strainer to eliminate debris and cells were stained with fluorescence-labeled flow cytometry antibodies for immune cell phenotyping. Shown below (Figure 3</stro...

Watch this Scientific Journal Video about Working with Human Tissues for Translational Cancer Research at JoVE.com

Working with Human Tissues for Translational Cancer Research

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

working-with-human-tissues-for-translational-cancer-research

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9.8K Views.  University of Texas MD Anderson Cancer Center. Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

Video: Working with Human Tissues for Translational Cancer Research

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer specimens and clinical data to be available to researchers within the Thoracic Oncology Research Program. This database also captures a large volume of genomic and proteomic data obtained from various tumor tissue studies. A team of clinical and basic science researchers, a biostatistician, and a bioinformatics expert was convened to design the database. Variables of interest were clearly defined and their descriptions were written within a standard operating manual to ensure consistency of data annotation. Using a protocol for prospective tissue banking and another protocol for retrospective banking, tumor and normal tissue samples from patients consented to these protocols were collected. Clinical information such as demographics, cancer characterization, and treatment plans for these patients were abstracted and entered into an Access database. Proteomic and genomic data have been included in the database and have been linked to clinical information for patients described within the database. The data from each table were linked using the relationships function in Microsoft Access to allow the database manager to connect clinical and laboratory information during a query. The queried data can then be exported for statistical analysis and hypothesis generation.

A thoracic oncology database was developed to serve as a comprehensive repository for clinical and laboratory data for the purposes of translational research. The database will serve translational cancer researchers within the Thoracic Oncology Research Program. This database is adaptable to other cancer models, as well as other human diseases.

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer ...

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.
This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.
Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...