Name: modeling osteosarcoma using li-fraumeni syndrome patient-derived induced pluripotent stem cells
Uploaded: 2018-06-13T14:00:00
Description: Watch this Scientific Journal Video about Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells at JoVE.com

R. Z. is supported by UTHealth Innovation for Cancer Prevention Research Training Program Pre-Doctoral Fellowship (Cancer Prevention and Research Institute of Texas grant RP160015). J.T. is supported by the Ke Lin Program of the First Affiliated Hospital of Sun Yat-sen University. D.-F.L. is the CPRIT scholar in Cancer Research and supported by NIH Pathway to Independence Award R00 CA181496 and CPRIT Award RR160019.

This work was approved by The University of Texas Health Science Center at Houston (UTHealth) Animal Welfare Committee. The experiments are performed in strict accordance with the standards established by the UTHealth Center for Laboratory Animal Medicine &#38; Care (CLAMC) which is accredited by American Association for Laboratory Animal Care (AAALAC International).The human subjects in this study fall under Scenario A (&#34;No Human Subjects Research&#34;) as defined by the NIH SF424 documentation. Therefore, it does n...

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To achieve higher MSC differentiation efficiency, several aspects are critical. One is the culture condition of iPSCs before initiating MSC differentiation. The protocol presented in the manuscript is based on previous studies 9,17. iPSCs need to be cultured on MEFs for at least 2 weeks. Maintaining iPSCs in good conditions on MEFs are critical for cells to attach on the gelatin-coated plate for MSC differentiation. Another important aspect is the density of iPSC...

Between 2006 and 2007, several breakthrough findings from the laboratories of Drs. Shinya Yamanaka and James A. Thomson led to the development of induced pluripotent stem cells (iPSCs)1,2,3. By reprogramming somatic cells with defined transcriptional factors to form iPSCs, researchers were able to generate cells with key characteristics namely, pluripotency, and self-renewal, which was previously thought to only exist in human embryonic stem cells (hESCs). iPSCs could be generated from any individual or patient and did not have to be derived from embryos, vastly expanding the repertoire of available diseases and backgrounds for the study. Since then, patient-derived iPSCs have been used to recapitulate the phenotype of various human diseases, from Alzheimer&#39;s disease4 and amyotrophic lateral sclerosis5 to long QT syndrome6,7,8.
These advances in iPSC research also have opened new avenues for the cancer research. Several groups recently have used patient iPSCs to model cancer development under a susceptible genetic background9,10,11, with successful application demonstrated to date in osteosarcoma9, leukemia10,11,12, and colorectal cancer13. Although iPSC-derived cancer models are still in their infancy, they have demonstrated great potential in phenocopying disease-associated malignancies, elucidating pathological mechanisms, and identifying therapeutic compounds14.
Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder caused by TP53 germline mutation15. Patients with LFS are predisposed to a various type of malignancies including osteosarcoma, making LFS iPSCs and their derived cells particularly well-suited to studying this malignancy16. An iPSC-based osteosarcoma model was first established in 2015 using LFS patient-derived iPSCs9 subsequently differentiated into mesenchymal stem cells (MSCs) and then to osteoblasts, the originating cells of osteosarcoma. These LFS osteoblasts recapitulate osteosarcoma-associated osteogenic differentiation defects and oncogenic properties, demonstrating the model potential as a &#34;bone tumor in a dish&#34; platform. Interestingly, genome-wide transcriptome analyses reveal aspects of an osteosarcoma gene signature in LFS osteoblasts and that features of this LFS gene expression profile are correlated with poor prognosis in osteosarcoma9, indicating the potential of LFS iPSCs disease models to reveal features of clinical relevance.
This manuscript provides a detailed description of how to use LFS patient-derived iPSCs to model osteosarcoma. It details the generation of LFS iPSCs, differentiation of iPSCs to MSCs and then to osteoblasts, and use of an in vivo xenograft model using LFS osteoblasts. The LFS disease model comprises several advantages, most notably the ability to generate unlimited cells at all stages of osteosarcoma development for mechanistic studies, biomarker identification, and drug screening9,14,16.
In summary, the LFS iPSC-based osteosarcoma model offers an attractive complementary system for advancing osteosarcoma research. This platform also provides a proof-of-concept for cancer modeling using patient-derived iPSCs. This strategy described below can be readily extended to model malignancies associated with other genetic disorders with cancer predispositions.

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		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Plastic ware</td>
			<td style="width:157px;"></td>
			<td style="width:136px;"></td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">100 mm Dish</td>
			<td style="width:157px;">Corning</td>
			<td style="width:136px;">430107</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:327px;">60 mm Dish</td>
			<td style="width:157px;">Corning</td>
			<td style="width:136px;">430166</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">6-well Plate</td>
			<td style="width:157px;">Falcon</td>
			<td style="width:136px;">353046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">12-well Plate</td>
			<td style="width:157px;">Falcon</td>
			<td style="width:136px;">353043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">48-well Plate</td>
			<td style="width:157px;">Falcon</td>
			<td style="width:136px;">353078</td>
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			<td height="21" style="height:21px;width:327px;">1 mL Pipet Tip</td>
			<td style="width:157px;">USA Scientific</td>
			<td style="width:136px;">1111-2721</td>
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			<td height="21" style="height:21px;width:327px;">10 &#181;L Pipet Tip</td>
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			<td style="width:136px;">1111-3700</td>
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			<td height="21" style="height:21px;width:327px;">5 mL Serological Pipette</td>
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			<td height="21" style="height:21px;width:327px;">10 mL Serological Pipette</td>
			<td style="width:157px;">SARSTEDT</td>
			<td style="width:136px;">86.1254.001</td>
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			<td height="21" style="height:21px;width:327px;">25 mL Serological Pipette</td>
			<td style="width:157px;">SARSTEDT</td>
			<td style="width:136px;">86.1685.001</td>
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			<td height="21" style="height:21px;width:327px;">50 mL Tube, PP</td>
			<td style="width:157px;">SARSTEDT</td>
			<td style="width:136px;">62.547.100</td>
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			<td height="21" style="height:21px;width:327px;">15 mL Tube, PP</td>
			<td style="width:157px;">SARSTEDT</td>
			<td style="width:136px;">62.554.100</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Culture materials and Reagents</td>
			<td style="width:157px;"></td>
			<td style="width:136px;"></td>
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			<td height="42" style="height:42px;width:327px;">CytoTune- iPS 2.0 Sendai Reprogramming Kit</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:136px;">A16517</td>
			<td style="width:208px;">Commercial Sendai virus reprogramming kit</td>
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			<td height="21" style="height:21px;width:327px;">Corning hESC-Qualified Matrix</td>
			<td style="width:157px;">Corning</td>
			<td style="width:136px;">354277</td>
			<td style="width:208px;">Basement membrane matrix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">CF1 MEFs, irradiated</td>
			<td style="width:157px;">ThermoFisher</td>
			<td style="width:136px;">A34180</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:327px;">DMEM</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:136px;">D5671</td>
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			<td height="21" style="height:21px;width:327px;">DMEM/F12</td>
			<td style="width:157px;">Corning</td>
			<td style="width:136px;">10-090-CV</td>
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			<td height="21" style="height:21px;">&#945;MEM</td>
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			<td height="21" style="height:21px;width:327px;">StemMACS iPS-Brew XF</td>
			<td style="width:157px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-104-368</td>
			<td>Commercial iPSC medium</td>
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			<td height="21" style="height:21px;width:327px;">KnockOut DMEM/F-12</td>
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			<td height="21" style="height:21px;width:327px;">FBS Opti-Gold</td>
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			<td style="width:136px;">A3181502</td>
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			<td style="width:157px;">Corning</td>
			<td style="width:136px;">21-031-CV</td>
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			<td height="21" style="height:21px;width:327px;">StemMACS Passaging Solution XF</td>
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			<td style="width:136px;">130-104-688</td>
			<td>Commercial passaging solution</td>
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			<td height="21" style="height:21px;width:327px;">Collagenase, Type II&#160;&#160;</td>
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			<td style="width:136px;">17101015</td>
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			<td height="21" style="height:21px;width:327px;">Human NANOG Antibody</td>
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			<td height="42" style="height:42px;width:327px;">Alexa Fluor 555 Mouse Anti-Human TRA-1-81 Antigen</td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">FITC Mouse Anti-Human CD44</td>
			<td style="width:157px;">DB Biosciences</td>
			<td style="width:136px;">555478</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">PE Mouse Anti-Human CD73</td>
			<td style="width:157px;">DB Biosciences</td>
			<td style="width:136px;">550257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">PE Mouse Anti-Human CD166</td>
			<td style="width:157px;">DB Biosciences</td>
			<td style="width:136px;">560903</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">FITC Mouse Anti-Human CD24</td>
			<td style="width:157px;">DB Biosciences</td>
			<td style="width:136px;">555427</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Donkey Serum</td>
			<td style="width:157px;">Jackson ImmunoResearch</td>
			<td style="width:136px;">017-000-121</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Goat Serum</td>
			<td style="width:157px;">Jackson ImmunoResearch</td>
			<td style="width:136px;">005-000-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Alkaline Phosphatase Staining Kit II</td>
			<td style="width:157px;">Stemgent</td>
			<td style="width:136px;">00-0055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Alizarin Red S</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:136px;">A5533</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">TRIzol Reagent</td>
			<td style="width:157px;">ThermoFisher</td>
			<td style="width:136px;">15596018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Chloroform</td>
			<td style="width:157px;">ThermoFisher</td>
			<td style="width:136px;">C298-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">2-Propanol</td>
			<td style="width:157px;">ThermoFisher</td>
			<td style="width:136px;">A416-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Ethanol, Absolute, Molecular Biology Grade</td>
			<td style="width:157px;">ThermoFisher</td>
			<td style="width:136px;">BP28184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">DNase I, RNase-free (1 U/&#181;L)</td>
			<td style="width:157px;">ThermoFisher</td>
			<td style="width:136px;">EN0521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">iScript cDNA Synthesis Kit</td>
			<td style="width:157px;">BioRad</td>
			<td style="width:136px;">1708891BUN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">iQ SYBR Green Supermix</td>
			<td style="width:157px;">BioRad</td>
			<td style="width:136px;">1708884</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Matrigel Matrix High Concentration (HC), Phenol-Red Free</td>
			<td style="width:157px;">Corning</td>
			<td style="width:136px;">354262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">1 mL Slip Tip Syringe, 26 Gauge x 5/8 Inch</td>
			<td style="width:157px;">DB Biosciences</td>
			<td style="width:136px;">309597</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling osteosarcoma using li-fraumeni syndrome patient-derived induced pluripotent stem cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

This protocol presents the procedures including LFS iPSC generation, MSC differentiation, osteoblast differentiation, and in vivo tumorigenesis assay using LFS MSC-derived osteoblasts.
Scheme for the generation of LFS iPSCs from fibroblasts by using a commercially available Sendai virus reprogramming kit is shown in Figure 1A. Sendai virus-based delivery of the Yamanaka four factors is a no...

Watch this Scientific Journal Video about Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells at JoVE.com

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

This Li-Fraumeni syndrome iPSC-based osteosarcoma model can help answer key questions in the osteosarcoma research field about using patient iP cells in cancer modeling. The main advantage of this model is that it allows the generation unlimited cells at all stages of osteosarcoma development for mechanistic studies, biomarker identification, and drug screening. Ruoji will be demonstrating the cell culture procedure and I will be demonstrating the animal procedure.After two weeks of mouse embryonic fibroblast, or MEF, co-culture wash the 80 to 90%confluent iPS cell culture with five milliliters of DPBS followed by the addition of one milliliter of cell detachment solution to the cells for a three minute incubation at room temperature. It is critical to maintain iPS cells in good conditions on MEFs for at least 14 days for the cells to be able to attach to a gelatin-coated plate for their differentiation into mesenchymal stem cells. When the cells have detached from the plate bottom add nine milliliters of MEF/MSC culture medium to the cells to neutralize the cell detachment solution and transfer the cell suspension into an uncoated 100 millimeter tissue culture plate.Incubate the cells at room temperature for 30 minutes to allow the MEFs to attach to the plate and carefully collect the unattached iPS cell-containing supernatant. Centrifuge the iPS cell solution and resuspend the pellet in six milliliters of MSE differentiation medium. Then seed two milliliters of cells into each of three 0.1%gelatin-coated wells of a six-well culture plate and place the plate in the cell culture incubator for 28 days feeding the cells with fresh medium every two days to remove any unattached or dead cells.On day 28 large masses of differentiated cells will be visible with fibroblast-like MSCs observed at the edges of the clusters. Wash the cell cultures with one milliliter of DPBS per well followed by their detachment with 0.5 milliliters of 0.25%Trypsin-EDTA per well at room temperature. After three minutes neutralize the Tyrypsin with 1.5 milliliters of MEF/MSE culture medium per well and pool the detached MSEs into a single 15 milliliter conical tube for their centrifugation.Resuspend the pellet in three milliliters of MEF/MSE culture medium and seed the cells onto a 0.1%gelatin-coated 60 millimeter plate, changing the medium every two days until the cells reach 100%confluence. After splitting the 100%confluent culture at a one to three ratio the MSCs will proliferate quickly demonstrating a fibroblast-like morphology and formi&ng a swirl-like culture pattern when the culture reaches a high density. The iPS cell-derived MSCs can also be examined by a immunofluorescent staining for mesenchymal stem cell markers at any point during their differentiation.To differentiate the MSE into osteoblasts plate the appropriate number of MSE from a 95%confluent cell culture onto a cell culture plate in the appropriate volume of MEF/MSC culture medium for 24 hours. Then begin replacing the supernatant with the appropriate volume of osteoblast differentiation medium the next day and every two days after until the end of the culture period. To detect the bone-associated alkaline phosphatase produced by preosteoblasts and the mineral deposition produced by mature osteoblasts perform alkaline phosphatase and Alizarin Red S staining, respectively, according to the manufacturer's protocol at the appropriate experimental time points.To set up an in vivo tumorigenesis model with the differentiated osteoblasts seed the appropriate number of MSC into five 100 millimeter dishes to start osteoblastic differentiation. On day 14 of osteoblastic differentiation wash five culture plates of osteoblasts with five milliliters of DPBS and treat the cells with 1.5 milliliters of osteoblast detachment solution per dish. After a 30 minute incubation at 37 degrees celsius confirm detachment of the cells by light microscopy and neutralize the detachment solution with fresh osteoblast differentiation medium.Pool the cells in a 50 milliliter conical tube for their centrifugation and resuspend the pellet in 25 milliliters of fresh osteoblast differentiation medium. Filter the cell solution through 70 micron strainer to remove any aggregated cells prior to counting and set aside 1 X 10 to the 7 cells per subcutaneous injection. Pellet the cells by centrifugation and reconstitute the cells at a 1 X 10 to the 7 osteoblasts per 50 microliters of ice-cold DPBS concentration.Mix the osteoblasts with an equal volume of phenol-red free basement membrane matrix and place the cells on ice until their injection. After confirming a lack of response to toe pinch in an eight-week old female immuno-compromised nude mouse load the cells into a one milliliter syringe equipped with the 27 gauge needle and subcutaneously inject 100 microliters of the cells into the disinfected flank of the animal. Then monitor the mice with weekly palpitations at the injection site for the growth of subcutaneous nodular densities.Tumor formation should be observed between six to 10 weeks after injection. Established LFS iPS cell clones should exhibit a typical human embryonic stem cell morphology and demonstrate a positive alkaline phosphatase activity. LFS iPS cells also highly express pluripotency factor mRNAs comparable to human embryonic stem cell lines and much higher than levels observed in parental fibroblasts.Successfully differentiated LFS MSCs also express typical MSC markers including CD44, CD73, CD105 and CD166 as assessed by immunofluoresent staining. When the MSCs are subjected to osteogenic differentiation signals the differentiated cells begin to exhibit positive alkaline phosphatase activity by day nine and mineral deposition by day 21. The differentiating osteoblasts also demonstrate an upregulated expression of pre-osteoblast and mature osteoblast genes throughout their differentiation process.Six to 10 weeks after osteoblast injection LFS osteoblast derived tumors demonstrate immature osteoblast characteristics including positive alkaline phosphatase activity, positive collagen matrix deposition, and negative mineralization. After watching this video you should have a good understanding of how to differentiate Li-Fraumeni Syndrome iPS Cells into osteoblasts and to generate an in vivo osteosarcoma model using patient-derived osteoblasts. In addition to osteosarcoma this Li-Fraumeni Syndrome iPS cell-based disease model can be also applied to other Li-Fraumeni Syndrome related malignancies such as soft tissue sarcoma, breast cancer, and brain tumors.

modeling-osteosarcoma-using-li-fraumeni-syndrome-patient-derived

Research

JoVE Journal

Cancer Research

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many studies in the last decades. Therefore, it is important to develop methods to investigate the role of key genes involved in cancer cell stemness. Gastric cancer (GC) is one of the most common and mortal types of cancers. Gastric cancer stem cells (GCSCs) are thought to be the root of gastric cancer relapse, metastasis and drug resistance. Understanding GCSCs&#160;biology is needed to advance the development of targeted therapies and eventually to reduce mortality among patients. In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...