Name: establishment of a primary culture of patient-derived soft tissue sarcoma
Uploaded: 2018-04-11T13:00:00
Description: Watch this Scientific Journal Video about Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma at JoVE.com

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:
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	<li>Collect tumor ...

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	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.">Cancer statistics.</a> CA Cancer J Clin. 66 (1), 7-30 (2016).</li><li>Linch, M., Miah, A. B., Thway, K., Judson, I. R., Benson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+treatment+of+soft-tissue+sarcoma+gold+standard+and+novel+therapies.">Systemic treatment of soft-tissue sarcoma gold standard and novel therapies.</a> Nat Rev Clin Oncol. 11 (4), 187-202 (2014).</li><li>Fletcher, C. 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Biol. 946, 363-381 (2013).</li><li>Daigeler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+in+vitro+effects+of+doxorubicin+on+gene+expression+in+primary+human+liposarcoma+cultures.">Heterogeneous in vitro effects of doxorubicin on gene expression in primary human liposarcoma cultures.</a> BMC Cancer. 29, 313 (2008).</li><li>Pan, X., Yoshida, A., Kawai, A., Kondo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+publicly+available+sarcoma+cell+lines+for+use+in+proteomic+studies.">Current status of publicly available sarcoma cell lines for use in proteomic studies.</a> Expert Rev Proteomics. 13 (2), 227-240 (2016).</li><li>Liverani, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innovative+approaches+to+establish+and+characterize+primary+cultures:+an+ex+vivo.+3D+system+and+the+zebrafish+model.">Innovative approaches to establish and characterize primary cultures: an ex vivo. 3D system and the zebrafish model.</a> Biol Open. 15 (2), 309 (2017).</li><li>De Vita, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myxofibrosarcoma+primary+cultures:+molecular+and+pharmacological+profile.">Myxofibrosarcoma primary cultures: molecular and pharmacological profile.</a> Ther Adv Med Oncol. 9 (12), 755-767 (2017).</li><li>Blay, J. Y., Ray-Coquard, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcoma+in+2016:+Evolving+biological+understanding+and+treatment+of+sarcomas.">Sarcoma in 2016: Evolving biological understanding and treatment of sarcomas.</a> Nat Rev Clin Oncol. 14 (2), 78-80 (2017).</li><li>Dairkee, S. H., Deng, G., Stampfer, M. R., Waldman, F. M., Smith, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+cell+culture+of+primary+breast+carcinoma.">Selective cell culture of primary breast carcinoma.</a> Cancer Res. 55 (12), 2516-2519 (1995).</li><li>Kar, R., Sharma, C., Sen, S., Jain, S. K., Gupta, S. 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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...

Watch this Scientific Journal Video about Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma at JoVE.com

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 ...

The overall goal of this human preclinical model derived from surgically resected tumor tissue is to obtain the establishment of a primary culture of patient-derived soft tissue sarcoma. This method can help answer key questions about the pathophysiology of soft tissue sarcoma and it represents an important preclinical tool for predicting response to therapy. The main advantage of this technique is that it is a fully human preclinical model.The implications of this technique extend towards therapy of soft tissue sarcoma because this preclinical model can be tested with conventional innovative drugs to investigate their activity and mechanism of action. Although this method can provide insight into soft tissue sarcoma, it can also be applied to the study of cancer cell soft tissue cell interaction. Individuals new to this method will struggle because of the heterogeneity of the tumor tissues and in isolating tumor cells.Visual demonstration of this method is critical as the initial soft tissue sarcoma cell selection and CD steps requires an amount of experience before proficiency can be achieved. Tumor specimens should be received in a 100 milliliter sterile urine container with 50 milliliters of DMEM low glucose medium sealed with a paraffin film. The samples may be kept at four degrees Celsius for a maximum of three hours after tumor resection.Sterilize the tissue culture hood with 70%ethanol and sterilize steel INOX tweezers with 70%ethanol. Remove the paraffin film from the urine container and discard. Clean the exterior of the urine container with 70%ethanol.Open a square Petri dish, then open the urine container and use the sterilized steel INOX tweezers to transfer the tumor specimens into the dish. Add PBS and wash the tumor specimen twice. After washing, finely shred the tumor specimen into pieces of one to two millimeters in size.Transfer one-quarter of the total sample to a two milliliter tube. Then add one milliliter of TRIzol reagent to the tube and immediately freeze at minus 80 degrees Celsius. Collect the rest of the finely shredded tumor material in a 15 milliliter tube and add four milliliters of collagenase type one solution.Then dilute the collagenase type one solution with four milliliters of culture medium, close the tube, and seal with paraffin film. Incubate at 37 degrees Celsius on a rolling shaker for 15 minutes to activate the digestion. After 15 minutes, place the tube with the rolling shaker at room temperature and leave it overnight.The following day, gently filter the cell suspension through a 100 micron sterile filter into a 50 milliliter tube. Then wash the filter with culture medium and centrifuge the filtrate at 225 times g for five minutes at room temperature. Discard the supernatant by inverting the tube and resuspend the cells with one to two milliliters of culture medium.After performing a cell count and calculating the cell number, plate the cells in a standard monolayer culture at a density of 80, 000 cells per square centimeter. To prepare a cytological sample, begin by collecting 100, 000 cells in 200 microliters of culture medium. Pipette the solution into a disposable funnel equipped with a filter and mounted with a glass slide.Open the cytocentrifuge and insert the samples. Following cytocentrifugation, discard the funnel equipped with the filter. Then fix the slide in acetone for 10 minutes, followed by chloroform for five minutes.Passage the STS cells when confluence is around 90%After washing the cell sheet with PBS, add two to three milliliters of 1X Trypsin in PBS and incubate for three to five minutes at 37 degrees Celsius. After the incubation, use a 200 to 1, 000 microliter pipette to collect the detached cells and transfer to a 15 milliliter tube. Add four milliliters of medium to stop the enzymatic reaction.Then centrifuge at 225 times g for five minutes at room temperature. After discarding the supernatant, resuspend the cell pellet by adding one milliliter of medium and seed the cells into a new culture plate or a flask. Tumor cells were isolated from a liposarcoma surgically removed from a 54-year-old patient.Fluorescent in situ hybridization was performed to detect MDM2 amplification in the patient tissue specimen. Cytological analysis of MDM2 amplification in isolated tumor cells confirm the establishment of a patient-derived primary culture. Morphological changes from base were induced when cultures were treated with Ifosfamide, Epirubicin, Ifosfamide and Epirubicin, Trabectedin and Eribulin.Three independent replicates were performed for each viability experiment. The results confirmed the sensitivity of the cultures to chemotherapy. The most active treatment was combined Ifosfamide and Epirubicin, a first-line therapy used in advanced or metastatic ALT-DDLPS.Once mastered, this technique can be done in 15-16 hours if it is performed properly. While attempting this procedure, it's important to remember to finely shred the tumor specimen. Following this procedure, a variety of downstream analysis including gene expression profiling, immunohistochemical analysis, or cytotoxicity assay can be performed in order to answer additional questions about soft tissue sarcoma biology.After its development, this technique paved the way for researchers in the field of soft tissue sarcoma to study the natural history of these poorly explored malignancies. After watching this video, you should have a good understanding of how to establish a primary culture of patient-derived soft tissue sarcoma. Don't forget that working with biological samples and drugs can be extremely hazardous and precautions such as wearing gloves and working with a fume hood should always be taken while performing this procedure.

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Research

JoVE Journal

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 ...

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 ...

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 ...

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 ...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Author Spotlight: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

Establishment and Characterization of Patient-Derived Xenograft Models of Anaplastic Thyroid Carcinoma and Head and Neck Squamous Cell Carcinoma

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its heterogeneity. Pharmacodynamic results based on PDX models are highly correlated with clinical practice. Anaplastic thyroid carcinoma (ATC) is the most malignant subtype of thyroid cancer, with strong invasiveness, poor prognosis, and limited treatment. Although the incidence rate of ATC accounts for only 2%-5% of thyroid cancer, its mortality rate is as high as 15%-50%. Head and neck squamous cell carcinoma (HNSCC) is one of the most common head and neck malignancies, with over 600,000 new cases worldwide each year. Herein, detailed protocols are presented to establish PDX models of ATC and HNSCC. In this work, the key factors influencing the success rate of model construction were analyzed, and the histopathological features were compared between the PDX model and the primary tumor. Furthermore, the clinical relevance of the model was validated by evaluating the in vivo therapeutic efficacy of representative clinically used drugs in the successfully constructed PDX models.

The present protocol establishes and characterizes a patient-derived xenograft (PDX) model of anaplastic thyroid carcinoma (ATC) and head and neck squamous cell carcinoma (HNSCC), as PDX models are rapidly becoming the standard in the field of translational oncology.