We thank Dr. Jordi Ochando from the Flow Cytometry Shared Resources at the Mount Sinai Medical Center for their assistance in flow cytometry analysis. We thank Dr. Rumana Huq from the Microscopy Shared Resource Facility at the Mount Sinai Medical Center for their imaging assistance. The authors thank the TJ Martell Foundation for its support in this project.

This protocol has been conducted at our institution with approval from the institutional research ethics board and is in compliance with all institutional, national, and international guidelines for human welfare.
1. Collection of Peripheral Blood from Patients with Advanced Prostate Cancer
Note: Select patients with metastatic prostate cancer. Obtain written patient consent and record clinical characteristics of patients, including age at isolation and previous chemotherapy and hormonal treatments. Patients with metastatic disease will potentially have highest concentration of CTCs&#160;in peripheral blood.
<ol>
	<li>Collect blood samples after informed consent and under an appropriate Institutional Review Board approved protocol. Wear latex gloves and a laboratory coat throughout the procedure.</li>
	<li>Connect 25 G&#160;butterfly needle and tubing to needle holder. Use an alcohol swab to clean area of antecubital vein between bicep and forearm, then apply tourniquet to patient&#8217;s upper arm.</li>
	<li>Insert butterfly needle into antecubital vein and push blood collection tube into needle holder to start collection. Collect approximately 10 ml&#160;of blood in collection tubes that contain ethylenediaminetetraacetic acid (EDTA) to prevent clotting.</li>
	<li>Remove butterfly needle from patient and apply pressure with sterile gauze pad over site of vein puncture for 1 min. Cover site with a sterile bandage.</li>
</ol>
2. Isolation of Mononuclear Cells
Note: The blood collected from step 1 will contain peripheral blood mononuclear cells (PBMCs) (e.g.,&#160;lymphocytes and monocytes) in addition to the mononuclear CTCs.
<ol>
	<li>Pipette whole blood into 50 ml polystyrene conical tube with a 5 ml serological pipette and add Hanks Balanced Salt Solution (HBSS) in a 1:1 ratio. Gently pipette mixture to homogenize.</li>
	<li>Add 15 ml of a commercially prepared solution (Ficoll-Paque) to separate blood components by low speed density gradient centrifugation to an empty 50 ml polystyrene conical tube.</li>
	<li>Gently pipette whole blood and HBSS mixture, from step 2.1, into the polystyrene tube from step 2.2, to form distinct top layer. Set pipette to lowest setting to minimize mixing of solutions, this will ensure efficient separation.</li>
	<li>Centrifuge at 400 x g for 30 min at RT&#160;(25 &#176;C). Set deceleration to lowest setting to prevent mixing of solutions after separation. Acceleration may be set to any speed.</li>
	<li>After centrifugation, identify the thin white-grey stripe of PBMCs and CTCs sandwiched layer between plasma on top and separation solution at the bottom of the tube.</li>
	<li>Collect the cells from the white-grey stripe in step 2.5 into a 50 ml polystyrene tube using a plastic transfer pipette.</li>
	<li>Add HBSS to 50 ml polystyrene conical tube with PBMCs and CTCs and centrifuge again at 400 x g for 10 min RT&#160;to wash.</li>
	<li>Decant liquid and resuspend pellet in 50 ml of HBSS and centrifuge again at 400 x g for 3 min at RT to wash.</li>
	<li>Discard the supernatant, resuspend the remaining pellet with 5 ml of Red Blood Cell Lysis buffer and incubate the solution for 5 min at RT.</li>
	<li>Remove the red blood cell lysis buffer by centrifugation at 400 x g for 3 min at RT.</li>
	<li>Discard the supernatant and resuspend the pellet in 1 ml PBS 1x&#160;supplemented with 10% fetal bovine serum (FBS) to block prior to antibody staining.</li>
</ol>
3. Staining Mononuclear Cells with CD45-FITC Antibody for Fluorescence Activated Cell Sorting (FACS)
<ol>
	<li>Quantify the number of viable (trypan blue-negative) cells (from step 2.11) using a hemocytometer or automated cell counter. Prepare a 1 x 106 cells/ml suspension (in PBS 1x&#160;with 10% FBS) and place it on ice for 1 hr.</li>
	<li>Distribute the quantified cell suspension from the previous step into two tubes. Label tubes as control and CD45 staining.</li>
	<li>In the control tube, add IgG1&#954;-FITC (2.5 &#181;g/ml) at a dilution of 1:250. In the CD45 staining tube, add CD45 FITC (2.5 &#181;g/ml) conjugated primary antibody at a dilution of 1:250 and incubate the cell suspensions on ice for 30 min. CD45 antigen labeling is used to exclude hematopoietic cells.</li>
	<li>Centrifuge the cells at 400 x g for 3 min at 4 &#176;C, and discard the supernatant.</li>
	<li>Wash the cells twice, by suspending each pellet with sterile 1 x PBS supplemented with 10% FBS followed by centrifugation at 400 x g for 3 min at 4 &#176;C.</li>
	<li>Resuspend the cells in a 1 ml solution of PBS 1x&#160;containing 4&#39;,6-diamidino-2-phenylindole (DAPI) at a concentration of 10 &#181;g/ml.</li>
	<li>Filter the final solution through 35 &#181;m strainer caps into 12 mm x 75 mm polystyrene tubes.</li>
</ol>
4. Isolation of Prostate CTCs by FACS
Note: Utilize a flow cytometer to collect live CD45 negative CTCs.
<ol>
	<li>Set compensation controls using cells from the tube labeled, &#8220;Control.&#8221;</li>
	<li>Create gates that exclude debris and clusters of cells using appropriate forward-scatter and side-scatter parameters.</li>
	<li>Establish the FITC gates using the cell suspensions from the tube labeled, &#8220;CD45-FITC.&#8221;</li>
	<li>Gate viable (DAPI-negative) cells using the pacific blue-A channel.</li>
	<li>Collect CD45 negative population into a sterile 15 ml polystyrene conical tube containing 2 ml of Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% FBS.</li>
	<li>Centrifuge the sorted prostate tumor cell suspension at 400 x g for 3 min, then resuspend the pellets in 200 - 500 &#181;l&#160;of RPMI supplemented with 10% FBS.</li>
</ol>
5. Injection of CTCs into Mice and Monitoring of Xenograft Growth
Note: Conduct all animal procedures in compliance with protocols approved by the institutional animal care committee. This protocol has been conducted at our institution under a specific Animal Use Protocol approved by our Animal Care Committee in accordance and compliance with all relevant regulatory and institutional agencies, regulations and guidelines.
<ol>
	<li>Mix the sorted prostate tumor cell suspension with extracellular matrix at a 1:1 ratio and place on ice.</li>
	<li>Anesthetize mouse prior to subcutaneous implantation in a chamber supplying 5% (v/v) inhaled isoflurane in 1 L/min of oxygen. Ensure appropriate anesthesia by checking for loss of corneal and toe reflex in the mouse.</li>
	<li>Using a 25 G needle and a 1 ml syringe, inject 250 &#181;l of prostate tumor cell and extracellular matrix cell suspension subcutaneously into both upper flanks of a 8-10 week old male immunodeficient mouse. Inject 250 &#181;l regardless of CTC concentration as the injection volume is the important value.&#160;Maximum SQ administration in mouse is no more than 1 ml. &#160;</li>
	<li>Monitor mice by performing weekly palpation of mouse injection sites for growth of subcutaneous nodular densities and by measuring mice weight twice a week.</li>
	<li>Euthanize mice by carbon dioxide asphyxiation followed by cervical dislocation and harvest subcutaneous human prostate cancer xenografts when tumor size is more than 0.5 cm and less than 1 cm in greatest diameter to ensure enough tumor tissue for molecular analyses and serial passage. Euthanize mice with signs of distress or weight loss of 15% despite no tumor is palpable.&#160;</li>
</ol>

<ol>
	<li>Hidalgo, 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=Patient-derived+xenograft+models:+an+emerging+platform+for+translational+cancer+research.">Patient-derived xenograft models: an emerging platform for translational cancer research.</a> Cancer discovery. 4, 998-1013 (2014).</li><li>Siolas, D., Hannon, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+tumor+xenografts:+transforming+clinical+samples+into+mouse+models.">Patient-derived tumor xenografts: transforming clinical samples into mouse models.</a> Cancer research. 73, 5315-5319 (2013).</li><li>Joosse, S. A., Gorges, T. M., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology,+detection,+and+clinical+implications+of+circulating+tumor+cells.">Biology, detection, and clinical implications of circulating tumor cells.</a> EMBO molecular medicine. 1, 1-11 (2014).</li><li>Yu, M., Stott, S., Toner, M., Maheswaran, S., Haber, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells:+approaches+to+isolation+and+characterization.">Circulating tumor cells: approaches to isolation and characterization.</a> The Journal of cell biology. 192, 373-382 (2011).</li><li>Liu, Z., 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=Negative+enrichment+by+immunomagnetic+nanobeads+for+unbiased+characterization+of+circulating+tumor+cells+from+peripheral+blood+of+cancer+patients.">Negative enrichment by immunomagnetic nanobeads for unbiased characterization of circulating tumor cells from peripheral blood of cancer patients.</a> Journal of translational medicine. 9, 1-9 (2011).</li><li>Siegel, R., Naishadham, D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics+,+2013.">Cancer statistics , 2013.</a> CA: a cancer journal for clinicians. 63, 11-30 (2013).</li><li>Domingo-Domenech, J., 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=Suppression+of+acquired+docetaxel+resistance+in+prostate+cancer+through+depletion+of+notch-+and+hedgehog-dependent+tumor-initiating+cells.">Suppression of acquired docetaxel resistance in prostate cancer through depletion of notch- and hedgehog-dependent tumor-initiating cells.</a> Cancer cell. 22, 373-388 (2012).</li><li>Klein, K. A., 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=Progression+of+metastatic+human+prostate+cancer+to+androgen+independence+in+immunodeficient+SCID+mice.">Progression of metastatic human prostate cancer to androgen independence in immunodeficient SCID mice.</a> Nature Medicine. 3, 402-408 (1997).</li><li>Yu, 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=Cancer+therapy.+Ex+vivo+culture+of+circulating+breast+tumor+cells+for+individualized+testing+of+drug+susceptibility.">Cancer therapy. Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility.</a> Science. 345, 216-220 (2014).</li><li>Hodgkinson, C. 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=Tumorigenicity+and+genetic+profiling+of+circulating+tumor+cells+in+small-cell+lung+cancer.">Tumorigenicity and genetic profiling of circulating tumor cells in small-cell lung cancer.</a> Nature medicine. 20, 897-903 (2014).</li><li>Vidal, 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=A+Targetable+GATA2-IGF2+Axis+Confers+Aggressiveness+in+Lethal+Prostate+Cancer.">A Targetable GATA2-IGF2 Axis Confers Aggressiveness in Lethal Prostate Cancer.</a> Cancer cell. 27, 223-239 (2015).</li><li>Quintana, E., 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=Efficient+tumour+formation+by+single+human+melanoma+cells.">Efficient tumour formation by single human melanoma cells.</a> Nature. 456, 593-598 (2008).</li><li>DeRose, Y. 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=Tumor+grafts+derived+from+women+with+breast+cancer+authentically+reflect+tumor+pathology,+growth,+metastasis+and+disease+outcomes.">Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes.</a> Nature. 17, 1514-1520 (2011).</li></ol>

The authors declare that they have no competing financial interests.

This manuscript describes a method for the generation of prostate cancer PDX models from CTCs. The use of CTCs for the generation of PDX models has several potential important advantages when compared to existing methods. First, accessible collection of CTCs from peripheral blood enables the generation of experimental models from the same patient at different disease stages. Second, blood collection represents a safer and inexpensive method to isolate tumor cells when compared to existing methodologies t...

A correction was made to <a href="http://www.jove.com/video/53182">Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells</a>. There was a spelling error in one of the authors' surname. The author's name was corrected from:
Veronica Rodriquez-Bravo
to:
Veronica Rodriguez-Bravo

A correction was made to Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells.

Erratum: Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenografts are increasingly popular experimental models used for cancer research. They can be used for characterization of biomarkers and biological pathways, pre-clinical evaluation of drug efficacy, and creation of avatars for personalized cancer therapies 1,2. Previously, other research groups have developed PDX models either by implanting or injecting single tumor cell suspensions or whole tumor explants into immunocompromised mice 1. These PDX models require surgical collection of fresh solid tumor, malignant ascites or pleural effusions from a patient undergoing a surgical procedure which is both costly and exposes the patient to increased risk of iatrogenic morbidity.
A significant recent development in cancer research has been the detection, isolation and characterization of circulating tumor cells. These tumor cells escape from the primary tumor mass and enter circulation where they play a critical role in metastasis and relapse, the most common cause of cancer related mortality 3. The evaluation and characterization of CTCs from several solid tumor types have provided clinical information for diagnosis, prognosis, and monitoring residual disease 3. A variety of currently used approaches relying on either the physical properties, expression of biomarkers, or functional characteristics of CTCs can be used to efficiently isolate CTCs 4. Existing macroscale CTC isolation methods include density gradient centrifugation, physical filtration with filter pores and separation against surface molecules. The most widely used CTC isolation methodologies are based on antibody-based capture of CTCs. Both positive and negative selection of cell surface markers can be employed to isolate CTCs from peripheral blood. Positive selection for CTCs in the peripheral circulation commonly uses epithelial markers (e.g., EpCAM) which are expressed on CTCs but not hematopoietic cells. The disadvantage of this method is that CTCs with metastatic potential have often undergone epithelial-to-mesenchymal transition (EMT), which downregulates epithelial surface markers 3. In order to isolate CTCs with metastatic potential, a negative selection methodology which employs the hematopoietic surface marker, CD45, to deplete the normal cell population of leukocytes can be used 5.
Prostate cancer is the most commonly diagnosed cancer and a major cause of cancer-related deaths in men 6. The mechanisms of tumor progression and aggressiveness are not completely understood and therefore the generation and characterization of experimental models that recapitulate the molecular heterogeneity of prostate cancer are of significant interest. PDX models of prostate cancer have been generated previously by engraftment of human prostate cancer cells into immunocompromised mice 7,8. However the generation of such models has been hampered by the low engraftment rate of prostate cancer into immunocompromised mice, which is primarily attributed to the indolent nature of the disease. Recently, CTCs have been used to generate breast cancer 9, lung cancer 10 and prostate cancer 11 PDX models. These proof-of-concept studies introduced the possibility of generating PDX models independent of the need for surgical sample collection. In this article we describe in detail a method for the generation of this novel experimental model.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640</td>
			<td>Gibco Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Corning Cell Gro</td>
			<td>21-031-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>35 &#181;m Cell Strainer</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml polystyrene conical tube</td>
			<td>Crystalgen</td>
			<td>23-2263</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysing buffer</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>d3571</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm x 75 mm Polystyrene tubes with cell strainer cap</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Lavender Blood Collection Tubes with EDTA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Winged Blood Collection Set with Push Button Retract Needle 23 gauge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer One Use Needle Holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Latex Tourniquet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Latex or non-latex gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>alcohol swabs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2x2 cotton gauze pads</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive bandage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 gauge needle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of prostate cancer patient derived xenograft models from circulating tumor cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This protocol will lead to the generation of PDX models from isolated CD45 negative prostate cancer CTCs. Based on the negative selection method used in our protocol it is necessary to exclude dead cells using DAPI stain. The percentage of CD45 negative cells detected by flow cytometry is variable and depends on the tumor burden of the patient&#160;(Figure 1A). Immunofluorescent staining of unsorted cells using CD45 and DAPI (to identify cell nuclei) reveals CD45 negative cells, as indicated by the white...

Watch this Scientific Journal Video about Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells at JoVE.com

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

generation-prostate-cancer-patient-derived-xenograft-models-from

Research

JoVE Journal

Medicine

14.0K Views.  Icahn School of Medicine at Mount Sinai. This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Video: Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche.&#160;The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines.&#160;Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions.&#160;Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs.&#160;Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions.&#160;However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly.&#160;CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs.&#160;Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques.&#160;In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system.&#160;This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.