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 ...

<ol>
	<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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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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