This research was funded by grant number PID2019-105128RB-I00 to RSA, JMB, and AA, and PGC2018-095795-B-I00 to PFS and RML, both funded by MCIN/AEI/10.13039/501100011033 and grant numbers B31_20R (RSA, JMA, and AA) and E35_17R (PFS and RML) and funded by Gobierno de Arag&#243;n. The work of RSA was supported by a grant from the Asociaci&#243;n Espa&#241;ola Contra el C&#225;ncer (AECC) PRDAR21487SOLE. The authors would like to acknowledge the use of Servicio General de Apoyo a la Investigaci&#243;n-SAI, Universidad de Zaragoza.

NOTE: All culture media and buffer compositions are specified in Table 1. Prior to cybrid generation, both mitochondrial and nuclear DNA profiles from the donor and recipient cells must be typed to confirm the presence of genetic differences in both genomes between cell lines. In this study, a commercially available L929 cell line and its derived cell line, L929dt, which was spontaneously generated in our laboratory (see13 for more information) were used. These cell lines present two differences in the sequence of their mt-Nd2 gene which can be used to confirm the purity of mtDNA once the cybridization process has been finished13. In this case, the purity of the nuclear background was confirmed by antibiotic sensitivity, since, contrary to L929dt cells, L929 were resistant to geneticin.
1. Mitochondrial depletion by rhodamine 6G treatment (recipient cells)
NOTE: The first step for successful cybrid generation is to completely and irreversibly abolish mitochondrial functions in the recipient cells. For this purpose, it is necessary to previously determine, for each cell line, the appropriate concentration and treatment duration with rhodamine 6G. This adequate concentration should be just below drug-induced cell death (the highest that does not kill the cells during the treatment). Perform the following once the optimal conditions are defined.
<ol>
	<li>Seed 106 cells in a 6-well plate using complete culture medium (see Table 1 for details) and treat them daily with the optimal concentration of rhodamine 6G for 3-10&#160;days, depending on the cell line. Traditional concentrations range from 2 to 5 &#181;g/mL for 3-10 days20,21. In this study, for L929-derived cells, 2.5 &#181;g/mL rhodamine 6G for 7 days was the selected treatment13,22.</li>
	<li>To keep the cells alive, supplement the cell culture medium with 50 &#181;g/mL of uridine and 100 &#181;g/mL of pyruvate and renew it every 24 h.</li>
	<li>After treatment and prior to fusion, change the medium of rhodamine 6G-treated cells to complete cell culture medium without rhodamine 6G, and leave them in this medium for 3-4 h in an incubator at 37 &#176;C with 5% CO2. 
	NOTE: The rhodamine 6G toxic effect is irreversible, and cells with non-functional mitochondria should not recover20. Thus, after treatment, cells that do not receive functional mitochondria should die even in uridine-supplemented culture medium. Therefore, no nuclear selection should be necessary. However, a control fusion of rhodamine 6G-treated cells without mitochondria is recommended.</li>
</ol>
2. Expansion and mitochondrial isolation (donor cells)
<ol>
	<li>Expand the mitochondria donor cells during the time lapse of rhodamine 6G treatment to obtain around 25 x 106 cells.</li>
	<li>On day 7, harvest exponentially growing cells in a 50 mL tube and collect them by centrifugation at 520 x g for 5 min at room temperature (RT). Wash the cells 3x with cold phosphate-buffered saline (PBS) and sediment them by centrifugation at 520 x g for 5 min at RT. From now on, perform all the steps of mitochondrial extraction at 4 &#176;C, using cold reagents and keeping the tubes with cells or mitochondria on ice.</li>
	<li>After the third centrifugation, discard the supernatant by aspiration using a glass pipette coupled to a vacuum pump and resuspend the packed cells in a volume of hypotonic buffer equal to 7x the cell pellet volume. Then, transfer the cell suspension into a homogenizer tube and let the cells swell by incubating them on ice for 2 min.</li>
	<li>Break the cell membranes by performing 8 to 10 strokes in the homogenizer coupled to a motor-driven pestle rotating at 600 rpm. 
	NOTE: The step of cell membrane disruption can vary between cell types; thus, it has to be optimized for each cell type.</li>
	<li>Add the same volume of hypertonic buffer to the cell suspension (7x the cell pellet volume) to generate an isotonic environment.</li>
	<li>Transfer the homogenate into a 15 mL tube and centrifuge it in a fixed rotor at 1,000 x g for 5 min at 4 &#176;C. Then, collect only 3/4 of the supernatant leaving a large margin from the pellet, to avoid contamination with nuclei or intact cells, and transfer it to another tube. Repeat the same process twice. Note that the supernatant must be kept and the pellet discarded.</li>
	<li>Save the mitochondrial fraction (supernatant). Transfer it into 1.5 mL tubes and centrifuge at maximum speed (18,000 x g) for 2 min at 4 &#176;C.</li>
	<li>Discard the supernatant and wash the mitochondria-enriched pellet with buffer A, combining the content of the two tubes into one and centrifuging at the same conditions as described in step 2.7. Repeat the same process until all the material is in only one tube.</li>
	<li>Make an additional wash with 300 &#181;L of buffer A and quantify the mitochondrial protein concentration using the Bradford assay23. For each cybridization assay, the optimal amount of mitochondria for the transfer procedure has to be determined (in our case, a concentration between 10-40 &#181;g of mitochondrial protein per 106 cells).</li>
	<li>Simultaneously, prepare the rhodamine 6G-pretreated cells for the fusion by collecting them in a 15 mL tube and centrifuging at 520 x g for 5 min at RT. Note that the pellet acquires a neon pink color due to rhodamine 6G treatment.</li>
	<li>To ensure that both mitochondrial function abolishment in receptor cells as well as organelle purification from donors have been properly performed, seed a small number of rhodamine 6G-treated cells and isolated mitochondria using the complete culture medium in a 6-well plate and culture them for a month to check that no surviving cells remain in any of the wells (Figure 2).</li>
	<li>In parallel, evaluate the absence of nuclei contaminants in the mitochondrial fraction by immunodetection of nuclear proteins (i.e., lamin beta, histone H3, etc.) or by quantitative polymerase chain reaction (qPCR) amplification of a nuclear gene (i.e., SDH, 18S rRNA, etc.). 
	NOTE: To avoid contaminations, it is recommended to perform all the steps in aseptic conditions working under a laminar flow hood.</li>
</ol>
3. Fusion and cybrid generation
<ol>
	<li>To proceed with the fusion, carefully add 106&#160;of the rhodamine 6G-treated cells to the isolated mitochondria pellet (10-40 &#181;g of mitochondrial protein) and centrifuge at 520 x g for 5 min to allow the cells to mix with the mitochondria.</li>
	<li>Add 100 &#181;L of polyethylene glycol (PEG, 50%) and gently resuspend the pellet for 30 s. Then, allow to rest untouched for another 30 s.</li>
	<li>Finally, transfer the mix into a 6-well plate with fresh complete cell culture medium and place in the incubator at 37 &#176;C with 5% CO2. After a few days (usually 1 week), transmitochondrial cybrids should start growing (Figure 2), giving rise to clones that can be individually selected or mixed in a pool prior to their analysis.</li>
</ol>
4. Verification of both mitochondrial and nuclear background
NOTE: Once the new cell line has been established and cells begin to grow exponentially, the purity of their mitochondrial and nuclear DNAs must be verified. Thus, the original cell lines should harbor different mutations or polymorphisms within their genomes to make them recognizable.
<ol>
	<li>Total DNA isolation
	<ol>
		<li>Isolate the genomic DNA of all cell lines used for cybrid generation by employing a commercial genomic DNA extraction kit (see Table of Materials) or by performing a standard protocol using phenol-chloroform-isoamyl alcohol extraction and alcohol precipitation24.</li>
	</ol>
	</li>
	<li>mtDNA evaluation (Figure 3) 
	NOTE: Several techniques, such as sequencing, restriction fragment length polymorphism (RFLP) analysis, or allele-specific qPCR, can be performed to analyze the purity of mtDNA. To confirm the presence of mtDNA sequence variations by RFLP, follow the next protocol steps.
	<ol>
		<li>Amplify an mtDNA fragment containing the nucleotide change by PCR.
		<ol>
			<li>Here, L929dt cells present an mtDNA mutation at position 4206, within an mt-Nd2 gene (m.4206C&#62;T) that is absent in L929 cells. To confirm the presence of this substitution in the transmitochondrial cybrids, amplify a 397 bp fragment by PCR using a standard protocol and the following primers: 1) 5&#39;-AAGCTATCGGG 
			CCCATACCCCG-3&#39;&#160;(positions 3862-3884) and 2) 5&#39;-TAATCAGAAGTGGAATGGGGCG -3&#39; (positions 4236-4258).</li>
		</ol>
		</li>
		<li>Analyze the presence of the desired nucleotide substitution by RFLP, using a specific endonuclease that recognizes the sequence change and generates a different cut pattern for both cell lines. 
		NOTE: When the L929dt mtDNA variant is present (4206T), the amplicon obtained in step 4.2.1 contains two restriction sites for SspI (see Table of Materials) that produces three DNA fragments of 306 bp, 52 bp, and 39 bp. The restriction site that generates the 52 and 39 bands is disrupted when the wild-type (WT) version C4206 is present, and a new band of 91 bp appears. Therefore, an internal control for full digestion for SspI is included in the analysis. The digestion reaction is performed at 37 &#176;C, following the manufacturer&#39;s instructions.</li>
		<li>Separate the restriction fragments by electrophoresis and compare the band pattern.
		<ol>
			<li>Once the digestion is performed, analyze the obtained restriction fragments by electrophoresis in 10% polyacrylamide-Tris-borate-EDTA (TBE) gels (see Table 1 for 1x TBE composition). Run the electrophoresis at 80 V for 1 h at RT. Visualize the DNA fragments after gel staining in a solution of ethidium bromide in 1x TBE for 15 min at RT (see Table 1 for gel staining solution composition).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Nuclear DNA genotyping 
	NOTE: Nuclear DNA genotyping must be performed using the previous DNA extraction sample employed for RFLP analysis.
	<ol>
		<li>Amplify 16 loci (D21S11, CSF1PO, vWA, D8S1179, TH01, D18S51, D5S818, D16S539, D3S1358, D2S1338, TPOX, FGA, D7S820, D13S317, D19S433, and AMEL) by using a pool of commercial-specific oligonucleotides (see Table of Materials).</li>
		<li>Perform electrophoresis by using a genetic analyzer in order to separate the fragments previously obtained. 
		NOTE: Once amplified, the different loci are analyzed by electrophoresis using a capilar system (see Table of Materials). The fragments are separated in a 50 cm long capilar filled with a commercial polymer. Cathode and anode buffers are also commercially available, as shown in the Table of Materials. Electrophoresis is performed at 19.5 kV for 20 min at 60 &#176;C.</li>
		<li>Use bioinformatical tools to determine the alleles corresponding to each amplified locus (see Table of Materials).</li>
		<li>Compare the data obtained in step 4.3.3 with the nuclear DNA profile database (Table 1), to check if the nuclear DNA profile matches with the mitochondria receptor cell line profile (Figure 4).</li>
	</ol>
	</li>
</ol>

Transmitochondrial Cybrid Generation From Suspension-Growing Cancer Cells

The authors declare no conflicts of interest.

Since Otto Warburg reported that cancer cells shift their metabolism and potentiate &#34;aerobic glycolysis&#34;3,4 while reducing mitochondrial respiration, the interest in the role of mitochondria in cancer transformation and progression has grown exponentially. In recent years, mutations in the mtDNA and mitochondrial dysfunction have been postulated as hallmarks of many cancer types25. To date, numerous studies have analyzed the mtDNA ...

Reprogramming energy metabolism is a hallmark of cancer1 that was observed for the first time by Otto Warburg in the 1930s2. Under aerobic conditions, normal cells convert glucose into pyruvate, that then generates acetyl-coA, fuelling the mitochondrial machinery and promoting cellular respiration. Nevertheless, Warburg demonstrated that, even under normoxic conditions, most cancer cells convert pyruvate obtained from the glycolysis process into lactate, shifting their way to obtain energy. This metabolic adjustment is known as the &#34;Warburg effect&#34; and enables some cancer cells to supply their energetic demands for rapid growth and division, despite generating ATP less efficiently than the aerobic process3,4,5. In recent decades, numerous works have supported the implication of metabolism reprogramming in cancer progression. Hence, tumor energetics is considered an interesting target against cancer1. As a central hub in energetic metabolism and in the supply of essential precursors, mitochondria play a key role in these cell adaptations that, to date, we only partially understand.
In line with the above, mitochondrial DNA (mtDNA) mutations have been proposed as one of the possible causes of this metabolic reprogramming, which could lead to an impaired electron transport chain (ETC) performance6 and would explain why some cancer cells enhance their glycolytic metabolism to survive. Indeed, it has been reported that mtDNA accumulates mutations within cancer cells, being present in at least 50% of tumors7. For example, a recent study carried out by Yuan et al. reported the presence of hypermutated and truncated mtDNA molecules in kidney, colorectal, and thyroid cancers8.&#160;Moreover, many works have demonstrated that certain mtDNA mutations are associated with a more aggressive tumor phenotype and with an increase in the metastatic potential of cancer cells9,10,11,12,13,14,15,16.
Despite the apparent relevance of the mitochondrial genome in cancer progression, the study of these mutations and their contribution to the disease have been challenging due to limitations in the experimental models and technologies currently available17. Thus, new techniques to understand the real impact of mitochondria DNA in cancer disease development and progression are needed. In this work, we introduce a protocol for transmitochondrial cybrid generation from suspension-growing cancer cells, based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria, that overcomes the main challenges of traditional cybridization methods18,19. This methodology allows the use of any nuclei donor regardless of the availability of their corresponding &#961;0 cell line and the transfer of mitochondria from cells that, following the traditional techniques, would be difficult to enucleate (i.e., non-adherent cell lines).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3500XL Genetic Analyzer&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>4406016</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Corning</td>
			<td>08-772-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>AmpFlSTR Identifiler Plus PCR Amplification Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>4427368</td>
			<td></td>
		</tr>
		<tr>
			<td>Anode Buffer Container 3500 Series</td>
			<td>Applied Biosystems</td>
			<td>4393927</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>PanReac</td>
			<td>131015</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford assay</td>
			<td>Biorad</td>
			<td>5000002</td>
			<td></td>
		</tr>
		<tr>
			<td>Cathode Buffer Container 3500 Series</td>
			<td>Applied Biosystems</td>
			<td>4408256</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks</td>
			<td>TPP</td>
			<td>90076</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>PanReac</td>
			<td>131026</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Sigma-Aldrich</td>
			<td>E8751</td>
			<td></td>
		</tr>
		<tr>
			<td>Geneticin</td>
			<td>Gibco</td>
			<td>10131027</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer Teflon pestle</td>
			<td>Deltalab</td>
			<td>196102</td>
			<td></td>
		</tr>
		<tr>
			<td>L929 cell line</td>
			<td>ATCC</td>
			<td>CCL-1</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniProtean Tetra4 Gel System</td>
			<td>BioRad</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma-Aldrich</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Sigma-Aldrich</td>
			<td>Custom products</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyacrylamide Solution 30%</td>
			<td>PanReac</td>
			<td>A3626</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol</td>
			<td>Sigma-Aldrich</td>
			<td>P7181</td>
			<td></td>
		</tr>
		<tr>
			<td>POP-7</td>
			<td>Applied Biosystems</td>
			<td>4393714</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAmp DNA Mini Kit</td>
			<td>Qiagen</td>
			<td>51306</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine-6G</td>
			<td>Sigma-Aldrich</td>
			<td>R4127</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum Fetal Bovine</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>SspI</td>
			<td>New England Biolabs</td>
			<td>R3132</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin/penicillin</td>
			<td>PAN biotech</td>
			<td>P06-07100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S3089</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Sigma-Aldrich</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>PanReac</td>
			<td>P14030b</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma-Aldrich</td>
			<td>U3750</td>
			<td></td>
		</tr>
	</tbody>
</table>

transmitochondrial cybrid generation using cancer cell lines

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more efforts are still needed to fully understand the link involving alterations in mitochondria and tumorigenesis, as well as to identify tumor-associated mitochondrial phenotypes. For instance, to evaluate the contribution of mitochondria in tumorigenesis and metastasis processes, it is essential to understand the influence of mitochondria from tumor cells in different nuclear environments. For this purpose, one possible approach consists of transferring mitochondria into a different nuclear background to obtain the so-called cybrid cells. In the traditional cybridization techniques, a cell line lacking mtDNA (&#961;0, nuclear donor cell) is repopulated with mitochondria derived from either enucleated cells or platelets. However, the enucleation process requires good cell adhesion to the culture plate, a feature that is partially or completely lost in many cases in invasive cells. In addition, another difficulty found in the traditional methods is achieving complete removal of the endogenous mtDNA from the mitochondrial-recipient cell line to obtain pure nuclear and mitochondrial DNA backgrounds, avoiding the presence of two different mtDNA species in the generated cybrid. In this work, we present a mitochondrial exchange protocol applied to suspension-growing cancer cells based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria. This methodology allows us to overcome the limitations of the traditional approaches, and thus can be used as a tool to expand the comprehension of the mitochondrial role in cancer progression and metastasis.

After following the above-presented protocol, a homoplasmic cybrid cell line with a conserved nuclear background but with a new mitochondria genotype should be obtained, as represented in the schematics in Figure 1 and Figure 2. The purity of the mitochondrial and nuclear DNA present in the cybrids can be confirmed by RFLP, as shown in Figure 3, and by nuclear DNA genotyping analysis, as shown in Figure 4</stron...

Watch this Scientific Journal Video about Transmitochondrial Cybrid Generation Using Cancer Cell Lines at JoVE.com

Author Spotlight: Transmitochondrial Cybrid Generation Using Cancer Cell Lines  

This protocol describes a technique for cybrid generation from suspension-growing cancer cells as a tool to study the role of mitochondria in the tumorigenic process.

Transmitochondrial Cybrid Generation Using Cancer Cell Lines

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more ...

transmitochondrial-cybrid-generation-using-cancer-cell-lines

Research

JoVE Journal

Cancer Research

2.2K Views.  University of Zaragoza. This protocol describes a technique for cybrid generation from suspension-growing cancer cells as a tool to study the role of mitochondria in the tumorigenic process. 

Video: Author Spotlight: Transmitochondrial Cybrid Generation Using Cancer Cell Lines  

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

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

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

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

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

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. However, the application of this method is challenging due to its low sensitivity in analyzing the trace amount of ctDNA in the blood. Furthermore, the method may generate false positive and negative results from this sequencing and subsequent analysis. To improve the feasibility and reliability of ctDNA detection in the clinic, here we present a technique which enriches rare mutations for sequencing, Enrich Rare Mutation Sequencing (ER-Seq). ER-Seq can distinguish a single mutation out of 1 x 107 wild-type nucleotides, which makes it a promising tool to detect extremely low frequency genetic alterations and thus will be very useful in studying disease heterogenicity. By virtue of the unique sequencing adapter&#39;s ligation, this method enables an efficient recovery of ctDNA molecules, while at the same time correcting for errors bidirectionally (sense and antisense). Our selection of 1021 kb probes enriches the measurement of target regions that cover over 95% of the tumor-related driver mutations in 12 tumors. This cost-effective and universal method enables a uniquely successful accumulation of genetic data. After efficiently filtering out background error, ER-seq can precisely detect rare mutations. Using a case study, we present a detailed protocol demonstrating probe design, library construction, and target DNA capture methodologies, while also including the data analysis workflow. The process to carry out this method typically takes 1-2 days.

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. ...

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell lines or increasing its expression with overexpression cell lines. Transient and stable transfection are two methods that are often used for exogenous gene expression. Transient transfection is only useful for short-term expression, whereas stable transfection allows exogenous genes to be integrated into the host cell genome where it will be continuously expressed. As a result, stable transfection is usually employed for research into long-term genetic regulation. Here we describe a simple protocol to generate a stable cell line overexpressing tagged death receptor 3 (DR3) to explore DR3 function. We picked single clones after a retroviral infection in order to maintain the homogeneity and purity of the stable cell lines. The stable cell lines generated using this protocol render DR3-deficient HT29 cells sensitive to antimitotic drugs, thus reconstituting the apoptotic response in HT29 cells. Moreover, the FLAG tag on DR3 compensates for the unavailability of good DR3 antibody and facilitates the biochemical study of the molecular mechanism by which antimitotic agents induce apoptosis.

Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell ...

Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in most countries. The discovery of "oncogenic driver mutations," such as epidermal growth factor receptor (EGFR)-activating mutations, and subsequent development of molecular targeted agents of EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib) have dramatically altered lung cancer treatment in recent decades. However, these drugs are still not effective in patients with non-small cell lung cancer (NSCLC) carrying EGFR-activating mutations. Following acquired resistance, the systemic progression of NSCLC remains a significant obstacle in treating patients with EGFR mutation-positive NSCLC. Here, we present a stepwise dose escalation method for establishing three independent acquired afatinib-resistant cell lines from NSCLC PC-9 cells harboring EGFR-activating mutations of 15-base pair deletions in EGFR exon 19. Methods for characterizing the three independent afatinib-resistance cell lines are briefly presented. The acquired resistance mechanisms to EGFR TKIs are heterogeneous. Therefore, multiple cell lines with acquired resistance to EGFR-TKIs must be examined. Ten to twelve months are required to obtain cell lines with acquired resistance using this stepwise dose escalation approach. The discovery of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies.

A method for establishing afatinib-resistance cell lines from lung adenocarcinoma PC-9 cells was developed, and resistant cells were characterized. The resistant cells can be used to investigate epidermal growth factor receptor tyrosine kinase inhibitor-resistance mechanisms, applicable for patients with non-small cell lung cancer.

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in ...

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. ...

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

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

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

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

Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide clues to overcoming radioresistance. Voltage-gated calcium channel &#945;2&#948;1 subunit isoform 5 has been reported as a marker for radioresistant CSCs in non-small cell lung cancer (NSCLC) cell lines. Using calcium channel &#945;2&#948;1 subunit as an example of a CSC marker, methods to study the radiosensitivity of CSCs in NSCLC cell lines are presented. CSCs are sorted with putative markers by flow cytometry, and the self-renewal capacity of sorted cells is evaluated by sphere formation assay. Colony formation assay, which determines how many cells lose the ability to generate descendants forming the colony after a certain dose of radiation, is then performed to assess the radiosensitivity of sorted cells. This manuscript provides initial steps for studying the radiosensitivity of CSCs, which establishes the basis for further understanding of the underlying mechanisms.

The presence of cancer stem cells have been associated with relapse or poor outcomes after radiotherapy. This manuscript describes the methods to study the radiosensitivity of cancer stem cells in lung cancer cell lines.

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...