Name: generation of prostate cancer patient derived xenograft models from circulating tumor cells
Uploaded: 2015-10-20T15:00:00.000+00:00
Duration: 8 min 3 s
Description: Watch this Scientific Journal Video about Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells at JoVE.com

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>

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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><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 &amp;micro;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>2 x 2 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 G 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, ...

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14.1K 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

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

Cancer Research

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer&#160;the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.

Resistance to cancer therapies contributes to disease progression and death. Determining the mechanistic underpinnings of resistance is crucial for improving therapeutic response. This manuscript details the protocol to generate taxane-resistant cell models of prostate cancer (PC) to help dissecting the pathways involved in progression to Docetaxel resistance in PC patients.

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

Genetics

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

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

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