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

STS cells are isolated from a tumor mass surgically excised from patients with STS. The protocol has been approved by the Local Ethics Committee and is performed according to Good Clinical Practice guidelines and the Declaration of Helsinki. All patients gave written informed consent to take part in the study. The surgical specimens are analyzed by an experienced sarcoma pathologist and processed within 3 hours of surgery.
1. Tumor Specimen Collection and Processing:
<ol>
	<li>Collect tumor specimens with the help of a sarcoma pathologist in a 100-mL sterile urine container with 50 mL of DMEM low glucose medium sealed with a paraffin film.</li>
	<li>Store the samples at 4 &#176;C for a maximum of 3 h after tumor resection.</li>
	<li>Perform all the following steps under a sterile tissue culture hood.
	<ol>
		<li>Sterilize the hood with 70% ethanol and wear sterile gloves.</li>
		<li>Sterilize steel inox tweezers with 70% ethanol (use a sterile gauze).</li>
		<li>Remove the paraffin film from the urine container and discard.</li>
		<li>Wash exterior of the urine container with 70% ethanol.</li>
		<li>Open a square petri dish.</li>
		<li>Open the urine container and transfer the tumor specimens into the square petri dish using the sterilized steel inox tweezers.</li>
	</ol>
	</li>
	<li>Wash the tumor specimens with PBS twice.</li>
	<li>Open a sterile surgical scalpel.
	<ol>
		<li>Finely shred the tumor specimens into 1 - 2 mm3 pieces.</li>
		<li>Collect one quarter of the total sample in a 2-mL tube.</li>
		<li>Add 1 mL of trizol reagent (purchased) to the tube and immediately freeze at -80 &#176;C (the sample will be used in the downstream analysis for the STS gene expression evaluation).</li>
	</ol>
	</li>
	<li>Collect the rest of the finely shredded tumor material in a 15-mL tube.
	<ol>
		<li>Add 4 mL of collagenase type I solution (collagenase type I is dissolved in PBS, at a concentration of 4 &#181;g/mL).</li>
		<li>Dilute the collagenase type I solution with 4 mL of DMEM high culture media (final concentration of collagenase type I is 2 &#181;g/mL).</li>
		<li>Close the 15-mL tube and seal with paraffin film.</li>
		<li>Put the 15-mL tube in a rolling shaker in an incubator at 37 &#176;C in stirring conditions for 15 min to activate the digestion.</li>
		<li>After 15 min store the tube in the rolling shaker under stirring conditions at room temperature overnight.</li>
	</ol>
	</li>
	<li>The following day put the 15-mL tube under the sterile hood.</li>
	<li>Gently filter the cell suspension through a 100 &#181;m sterile filter attached to the top of a 50-mL tube.</li>
	<li>Wash the filter with DMEM high culture media supplemented with 10% fetal bovine serum, 1% glutamine, and 1% penicillin/streptomycin.</li>
	<li>Discard the 100 &#181;m sterile filter, close the 50-mL tube and centrifuge at 225 x g for 5 minutes at room temperature.</li>
	<li>Discard the supernatant by inverting the tube.</li>
	<li>Re-suspend the cells with 1 - 2 mL DMEM high culture media supplemented with 10% fetal bovine serum, 1% glutamine,&#160;and 1% penicillin/streptomycin. 
	Note: The volume depends on the amount of tumor sample processed.</li>
	<li>Count the cells with a Neubauer chamber.
	<ol>
		<li>Dilute the sample 1:2 in trypan blue and use 10 &#181;L of the dilution to count cells.</li>
		<li>Count at least twice and calculate the mean of the replicates to know the total mean number of cells collected.</li>
	</ol>
	</li>
	<li>Plate cells in a standard monolayer culture at a density of 80,000 cells/cm2. 
	Note: Always include controls in experiments. Perform at least 3 technical replicates for each condition. Perform at least 2 biological replicates. For preclinical pharmacology, gene expression and protein expression analyses, experiments should be carried out with a low number of culture passages within 30 - 45 days of cell seeding to avoid losing cancer cell phenotypes.</li>
	<li>Cytological sample preparation.
	<ol>
		<li>Collect 100,000 cells in 200 &#181;L of DMEM high culture media supplemented with 10% fetal bovine serum, 1% glutamine,&#160;and 1% penicillin/streptomycin.</li>
		<li>Pipette the solution (200 &#181;L pipette) into a disposable funnel equipped with a filter and mounted with a glass slide.</li>
		<li>Open the cytocentrifuge and insert the samples.</li>
		<li>Run the cytocentrifuge at 18.6 x g for 20 min.</li>
		<li>Discard the funnel equipped with the filter.</li>
		<li>Fix the slides in acetone for 10 min followed by chloroform for 5 min.</li>
		<li>Store the slides at -20 &#176;C. 
		Note: Thaw and hydrate the samples before staining and perform the staining following the manufacturer&#39;s instructions. Prepare at least 3 technical replicates.</li>
	</ol>
	</li>
</ol>
2. STS Cell Cultures:
<ol>
	<li>Change media once a week (DMEM high culture media supplemented with 10% fetal bovine serum, 1% glutamine and 1% penicillin/streptomycin).
	<ol>
		<li>Discard the culture medium using a 200 - 1,000 &#181;L pipette.</li>
		<li>Add new fresh medium using a 200 - 1,000 &#181;L pipette.</li>
		<li>Cell passage:
		<ol>
			<li>Detach cells when the confluence is around 90%: 
			Note: STS cell culture amplification should be performed once a week on the basis of the culture conditions.</li>
			<li>Discard medium, wash with PBS and add 2-3 mL of trypsin 1x in PBS 
			Note: The volume of trypsin used depends on the amount of cultured cells obtained.</li>
			<li>Incubate cells for 3 - 5 min at 37 &#176;C.</li>
			<li>Collect detached cells from the plate using a 200 - 1,000 &#181;L pipette.</li>
			<li>Pipette the detached cells into a 15-mL tube and add 4 mL of DMEM high culture media supplemented with 10% fetal bovine serum, 1% glutamine,&#160;and 1% penicillin/streptomycin to stop the enzymatic reaction.</li>
			<li>Centrifuge at 225 x g for 5 min at room temperature.</li>
			<li>Discard the supernatant.</li>
			<li>Re-suspend the cell pellet by adding 1 mL of DMEM high culture media supplemented with 10% fetal bovine serum, 1% glutamine, and 1% penicillin/streptomycin.</li>
			<li>Seed the cells in a new culture plate or a flask with a final volume according to the manufacturer&#39;s instructions. 
			Note: Do not split cells more than 1:3.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Downstream Analyses:
Note: A variety of different experiments can be carried out using this model (see below). It is, therefore, crucial to decide during the design phase of the study which analyses will be performed so that an adequate number of samples and replicates can be prepared.
<ol>
	<li>Proliferation assays such as MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide</li>
	<li>Apoptotic assays such as terminal deoxynucleotidyl transferase (TdT) nick end labeling (TUNEL) assay</li>
	<li>Immunohistochemical analysis</li>
	<li>Gene expression analysis</li>
	<li>Western blot analysis</li>
	<li>ELISA assay</li>
	<li>Fluorescence-activated cell sorting (FACS) analysis</li>
	<li>In vivo xenograft model</li>
</ol>

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The authors have no conflicts of interest to disclose.

Well defined preclinical models are needed to elucidate the molecular background of tumors, predict poor prognosis, and develop new therapeutic strategies for cancer patients. This is especially important for rare tumors such as STS whose high heterogeneity in terms of morphology, aggressive potential, and clinical behavior challenges our understanding of STS biology and patient management21. Moreover, the few commercial sarcoma cell lines available limits preclinical investigations into this grou...

Soft tissue sarcomas (STS) include a spectrum of heterogeneous lesions of mesenchymal origin accounting for 1% of all solid tumors1,2, and the new World Health Organization classification recognizes the presence of more than 50 different subtypes3. Among these, the most common histotypes are adipocyte sarcoma or liposarcoma and leiomyosarcoma, accounting for 15% and 11% of all adult STS, respectively4,5. Although STS can develop in different parts of the body, the extremities and retroperitoneum are the most common sites, occurring in 60% and 20% of cases, respectively6.
The cornerstone of therapy for localized disease is wide surgical resection, whereas the benefits of adjuvant and neoadjuvant chemotherapy are still unclear and are only considered in selected cases7. In the metastatic setting, chemotherapy is the standard of care but shows limited results8. A better understanding of the molecular biology of STS would help to improve the current differential diagnosis, optimize the available treatments, and identify new targets for therapy.
Within this context, research into cancer has been performed for many years using immortalized cell lines9. These experimental models are normally cultured in standard monolayer supports or implanted into immunodeficient rodents to establish in vivo xenograft models10. Immortalized cell lines represent a manageable and easy-to-store material with which an ample number of research experiments can be performed. They are, in fact, the least expensive and most widely used tool in preclinical studies11.
However, cell-line based cancer models also have some limitations, e.g. prolonged culture in a monolayer system together with an increasing number of passages induces phenotypic and genetic drift, making cells less likely to recapitulate key tumor features. Furthermore, cell line cultures fail to reproduce the cell-to-cell interaction and signaling molecule crosstalk that mimic the biological behavior of tumors and characterize the tumor microenvironment. These issues form the basis of the gap existing between preclinical and clinical results12.
Given the above, the interest in patient-derived primary cultures has increased13. Indeed, the excision of malignant and normal tissue reduces the risk of losing cancer cell phenotype and heterogeneity, thus offering starting material that is more representative of the tumor microenvironment. Moreover, the use of fresh tumor specimens harvested from patients undergoing surgical resection enables us to investigate single tumors and to compare different lesions from the same part of a patient&#39;s body14. For the above reasons, tissue-derived cell cultures provide a valuable material to study tumor pathophysiology and pharmacology15,16,17, especially in STS in which commercially available sarcoma cell lines are limited18. We thus optimized a method to establish a patient-derived STS primary cell culture starting from surgically excised tumor tissue13,19,20. Our method consists of disaggregating the tumor specimen into small tissue fragments followed by overnight enzymatic tissue dissociation to obtain a single cell suspension. The following day, the enzymatic digestion is stopped by adding fresh culture media and the obtained suspension is filtered to remove tissue fragments, cell-aggregates, and excess matrix material or debris. Finally, a fraction of isolated tumor cells is cytospinned onto glass slides, fixed and stored for downstream analysis aimed at confirming the establishment of STS primary culture by cytomorphological, immunohistochemical and molecular cytogenetic evaluation (e.g. FISH analysis of MDM2 amplification represents the standard differential diagnosis for a well-differentiated liposarcoma and the dedifferentiated liposarcoma)4. The remaining cells are seeded into a standard monolayer culture for gene expression profiling and pharmacological studies. All the experiments are performed using low passage primary cultures to avoid the selection of specific cancer subclones, and analyzed by an experienced sarcoma pathologist. We have thus created a fully human STS ex-vivo model13,19,20.This highly versatile, easy to handle model could represent a useful tool for different types of preclinical research, e.g. to identify new molecular markers for diagnosis and prognosis or to gain a deeper understanding of the activity and mechanisms of action of standard and new drugs used in the management of STS.

<table border="0" cellpadding="0" cellspacing="0" style="width:599px;" width="597">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="53">
			<td height="53" style="height:53px;width:163px;">DMEM High Glucose</td>
			<td style="width:180px;">EuroClone</td>
			<td style="width:111px;">ECB7501L</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:163px;">Fetal bovine serum</td>
			<td style="width:180px;">EuroClone</td>
			<td style="width:111px;">ECS0180D</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:163px;">Glutamine</td>
			<td style="width:180px;">Gibco</td>
			<td style="width:111px;">25030-024</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="112">
			<td height="112" style="height:112px;width:163px;">Paraformaldehyde 4% aqueous solution, EM grade</td>
			<td style="width:180px;">Electron Microscopy Sciences</td>
			<td style="width:111px;">157-4-100</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Penicillin streptomycin</td>
			<td style="width:180px;">Gibco</td>
			<td style="width:111px;">15140122</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:163px;">Triazol reagent</td>
			<td style="width:180px;">Ambion Life-Technologies</td>
			<td style="width:111px;">15596018</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;">Trypsin</td>
			<td>EuroClone</td>
			<td style="width:111px;">ECB3052D</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a primary culture of patient-derived soft tissue sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

We designed a simple method to obtain the establishment of a primary culture of patient-derived soft tissue sarcoma and report here an example of results obtained on one specific STS histotype13. The protocol was used for the establishment of primary cultures of different STS histotypes including well-differentiated liposarcoma, dedifferentiated liposarcoma, myxoid liposarcoma, pleomorphic liposarcoma, myxofibrosarcoma, undifferentiated pleomorphic sarcoma, GIST, a...

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Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

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Cancer Research

14.0K Views.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Video: Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

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 ...

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 ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

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 ...

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. ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies.

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.

Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics.

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...

Establishment and Culture of Patient-Derived Breast Organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms of screening, prevention, and treatment; however, preclinical models that predict the chemosensitivity profile of cancer patients are still lacking. To fill this gap, an in vivo patient-derived xenograft model was developed and validated. The model was based on zebrafish (Danio rerio) embryos at 2 days post fertilization, which were used as recipients of xenograft fragments of tumor tissue taken from a patient's surgical specimen. 
It is also worth noting that bioptic samples were not digested or disaggregated, in order to maintain the tumor microenvironment, which is crucial in terms of analyzing tumor behavior and the response to therapy. The protocol details a method for establishing zebrafish-based patient-derived xenografts (zPDXs) from primary solid tumor surgical resection. After screening by an anatomopathologist, the specimen is dissected using a scalpel blade. Necrotic tissue, vessels, or fatty tissue are removed and then chopped into 0.3 mm x 0.3 mm x 0.3 mm pieces. 
The pieces are then fluorescently labeled and xenotransplanted into the perivitelline space of zebrafish embryos. A large number of embryos can be processed at a low cost, enabling high-throughput in vivo analyses of the chemosensitivity of zPDXs to multiple anticancer drugs. Confocal images are routinely acquired to detect and quantify the apoptotic levels induced by chemotherapy treatment compared to the control group. The xenograft procedure has a significant time advantage, since it can be completed in a single day, providing a reasonable time window to carry out a therapeutic screening for co-clinical trials.

Preclinical models aim to advance the knowledge of cancer biology and predict treatment efficacy. This paper describes the generation of zebrafish-based patient-derived xenografts (zPDXs) with tumor tissue fragments. The zPDXs were treated with chemotherapy, the therapeutic effect of which was assessed in terms of cell apoptosis of the transplanted tissue.

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...