This work was funded by a grant from the National Medical Research Council of Singapore (NMRC/CIRG/1335/2012) awarded to Professor Kesavan Esuvaranathan.

All animal work adhered to the Institutional Animal Care and Use Committee (IACUC) guidelines on animal use and handling (Protocol number 084/12) at the National University of Singapore.
1. Growing MB49-PSA Cells In Vitro and Measuring PSA Secretion
<ol>
	<li>Maintain murine bladder cancer cells MB49-PSA15 in complete Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, and 0.05 mg/mL Penicillin-Strepto...

<ol>
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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=Histopathological+characterization+of+a+syngeneic+orthotopic+murine+bladder+cancer+model.">Histopathological characterization of a syngeneic orthotopic murine bladder cancer model.</a> Int Braz J Urol. 34, 220-226 (2008).</li><li>Chan, E. 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The most critical steps in the protocol are 1) successfully maintaining the tumorigenicity of the cell line; 2) ensuring measurable PSA secretion before tumor cell implantation in mice; 3) generating a single-cell suspension for implantation so as to reduce variation in tumor size; and 4) instilling cells at a slow rate to prevent vesico-ureteral reflux, resulting in tumor cell implantation in the kidney.
After prolonged passage in vitro, MB49/MB49-PSA cells can lose their tumorigenic...

The goal of this method is to generate murine orthotopic bladder tumors and to monitor the implanted tumors as accurately as possible, so that mice without tumor implantation are not thought to have been cured at end-point analysis. Overall, the method shown will reduce the need for large numbers of mice for experimental analysis and ensure greater accuracy in determining therapeutic outcomes.
The development of an orthotopic model for cancer is important, as implanting tumor cells subcutaneously does not recapitulate the environment of the clinical disease or enable the development of therapeutic strategies. The architecture of the bladder permits the instillation of bladder cancer therapies directly into the bladder with minimal systemic effects. Thus, animal models that recapitulate this environment, such as an orthotopic model, are important to evaluate new therapies. The conclusions drawn from any experimental set-up are dependent upon the limitations of the model.
Several techniques have been developed for the production of orthotopic bladder tumors in mice. These rely on damaging the glycosaminoglycan layer of the bladder, enabling tumor cells to be implanted. The techniques used include electrocautery, which results in a single point of damage in the bladder wall, leading to tumor development at one site in the bladder1,2. However, the success rate of tumor implantation using electrocautery is operator-dependent varying from 10 - 90%, and it includes the danger that the bladder wall will be punctured, leading to tumors developing in the peritoneal cavity. Chemical cautery is performed using silver nitrate, which damages the bladder wall3. Similarly, acid has been used to damage the bladder wall4. Trypsin has also been used to damage the bladder as well5. These methods may result in the development of more than one tumor in the bladder. Furthermore, there is a danger of severe damage to the bladder if the chemicals are left in contact with the bladder wall for too long. The method developed by Ninalga et al. uses the positively charged poly-L-lysine (PLL)6 molecules to coat the bladder wall; this enables the negatively charged tumor cells to stick to the glycosaminoglycan layer of the bladder. This method generally results in more than one tumor developing in the bladder, but tumor implantation is at 80 - 100%4,7. Technically, it is also the easiest procedure to perform. To ensure that the tumors that develop are fairly even in size, it is important that the tumor cells are not grouped in large clumps before implantation.
In order to evaluate therapeutic efficacy, it is best to perform this study on mice with fairly similar-sized tumors. Thus, a good detection system that can quantify tumor size soon after implantation is important. Several strategies have been used to evaluate tumors. These include magnetic resonance imaging (MRI)8-10, fluorescence11, bioluminescence12,13, ultrasound14, and enzyme-linked immunosorbent assay (ELISA)15,16. While MRI and ultrasound do not require modifications of the tumor cells, there is a need for sensitive equipment and contrast agents for MRI. The fluorescence-, luminescence-, and ELISA-based assays require modification of the tumor cells to express marker proteins that can be detected by these methods. For luminescence, a substrate is required for the detection of the luciferase activity; thus, there is an added step and increased cost. Both luminescence and fluorescence require specialized equipment. To produce fluorescence, green fluorescent protein (GFP) cyclization, which is catalyzed by molecular oxygen, is required. Thus, GFP expression may be variable within a tumor mass depending on access to oxygen, making this a rather unreliable marker17. Modification of the murine bladder cancer cell line MB49 to secrete human Prostate Specific Antigen (PSA)15,16 as a surrogate marker is another strategy. These markers also provide an alternative means of confirming tumor presence at the termination of the experiment, making them an alternative to immunohistochemistry. This study reports the PLL method of orthotopic tumor implantation and presents a comparison of tumor detection systems, namely ELISA, fluorescence, and ultrasound imaging.

<table border="0" cellpadding="0" cellspacing="0" width="893">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:404px;">MB49-PSA cells</td>
			<td style="width:188px;">N/A</td>
			<td style="width:188px;">N/A</td>
			<td style="width:113px;">ref (Wu QH, 2004)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RPMI 1640 media</td>
			<td>HyClone</td>
			<td>SH30027.01&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) media</td>
			<td>Biowest</td>
			<td>L0102</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS) South American</td>
			<td>Biowest</td>
			<td>S1810</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS) South American, Premium</td>
			<td>Biowest</td>
			<td>S181B</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS)</td>
			<td>HyClone</td>
			<td>SH30088.03&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">L-glutamine</td>
			<td>Biowest</td>
			<td>X0550</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Penicillin-Streptomycin</td>
			<td>Biowest</td>
			<td>L0022</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hygromycin B</td>
			<td>Invitrogen</td>
			<td>10687-010</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">free PSA (Human) ELISA kit</td>
			<td>Abnova</td>
			<td>KA0209</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TRIzol Reagent for RNA extraction&#160;</td>
			<td>Ambion</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor</td>
			<td>Applied Biosystems</td>
			<td>4374967</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Universal PCR Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Gene Expression Assay &#8211; Mouse Actb</td>
			<td>Applied Biosystems</td>
			<td>4331182</td>
			<td>Mm00607939_s1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Gene Expression Assay &#8211; Human KLK3</td>
			<td>Applied Biosystems</td>
			<td>4331182</td>
			<td>Hs00426859_g1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">C57BL/6J female mice</td>
			<td>In Vivos</td>
			<td></td>
			<td>4-6 wk old</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anesthesia (75mg/kg Ketamine and 1mg/kg Medetomidine)</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Reversal drug (1mg/kg Atipamezole)</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ear punch</td>
			<td>Electron Microscopy Sciences</td>
			<td>72893-01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hartmann&#39;s solution or Compound sodium lactate</td>
			<td>B Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ophthalmic ointment - Duratears sterile ocular lubricant ointment</td>
			<td>Alcon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heat pack - HotHands handwarmers</td>
			<td>Heatmax Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Introcan Certo IV catheter</td>
			<td>B Braun</td>
			<td>4251300</td>
			<td>24G x 3/4&#8243;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Aquagel Lubricating jelly</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#160;Poly-L-lysine solution, 0.01%,</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>4693159001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Quantichrom Creatinine Assay Kit</td>
			<td>BioAssay Systems</td>
			<td>DICT-50&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fluorescent dye - VivoTrack 680</td>
			<td>Perkin Elmer</td>
			<td>NEV12000&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RNAlater-ICE Frozen Tissue Transition Solution</td>
			<td>Ambion</td>
			<td>4427575</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Equipment and Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">7500 Realtime PCR System</td>
			<td>Applied Biosystems</td>
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			<td height="17" style="height:17px;">7500 Software v2.3</td>
			<td>Applied Biosystems</td>
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			<td height="17" style="height:17px;">Metabolic Cage</td>
			<td>Tecniplast&#160;</td>
			<td>Vertical type rack for 12 cages</td>
			<td></td>
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			<td height="17" style="height:17px;">BD FACSCanto I system&#160;</td>
			<td>BD Biosciences</td>
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			<td height="17" style="height:17px;">BD FACSDiva software v7</td>
			<td>BD Biosciences</td>
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			<td height="17" style="height:17px;">IVIS SpectrumCT in vivo imaging system&#160;</td>
			<td>Caliper Life Sciences&#160;</td>
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			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">Living Image Software v3.1</td>
			<td>Caliper Life Sciences&#160;</td>
			<td></td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">Vevo 2100 imaging system&#160;</td>
			<td>VisualSonics&#160;</td>
			<td></td>
			<td></td>
		</tr>
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</table>

a murine orthotopic bladder tumor model and tumor detection system

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

PSA secretion from MB49 cells was found to vary with the growth media. MB49-PSA is grown in DMEM media because this results in increased PSA secretion (Figure 1A). In order to determine the sensitivity of the PSA ELISA and real-time PCR, different numbers of MB49-PSA-secreting cells were mixed with MB49 parental cells. PSA ELISA detects a minimum of 1 x 105 PSA-secreting cells/1 x 106 cells (Figure 1B), while real-time PCR analysis d...

Watch this Scientific Journal Video about A Murine Orthotopic Bladder Tumor Model and Tumor Detection System at JoVE.com

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

The overall goal of this non-surgical method is to consistently produce orthotopic non-muscle-invasive bladder tumors in mice that can be easily monitored for the evaluation of intravesical therapies. This method can help answer key questions in the field of bladder cancer. Such as the evaluation of novel intravesical therapies.The main advantage of this technique is that it is easy to implant tumors in mice and monitor their growth, which helps to accurately determine responses to therapies. One day before the implantation, when the cells are about 80%confluent passage to the MB49 PSA tumor cells in complete DMEM media with a one to two split. On the day of the procedure, collect and count the cells, making sure they are sufficiently healthy.Mix in equal volume of cell suspension and 4%Trypan Blue, and count the blue-stained live cells using a hemocytometer. The cells should have at least 80%viability. Next, resuspend the cells at two million per milliliter in a 15 milliliter centrifuge tube, and wash them three times in DMEM.Then, keep them chilled until they are to be injected. Once a female mouse has been anesthetized, provide it hydration by an intraperitoneal injection of Hartmanns Solution at 1 milliliters per 10 grams of body weight. Repeat this every one or two hours during the anesthesia.Next, apply sterile ophthalmic ointment to both eyes, reapply as needed. Then, position the mouse supine on paper towels over a heat pad to maintain body tempurature. Next, secure the hind legs with tape.Now apply gentle pressure to the lower abdominal region, and collect urine into a 1.5 milliliter tube to measure the basal level of urinary PSA. Next, load a one-milliliter syringe with sterile PLL and attach a 24-gauge IV catheter with the needle stylet removed. Apply lubricant to the tip of the catheter.Then, insert the catheter into the urethra, and using forceps, guide it to the bladder. Stop when resistance is felt. Then, slowly eject 50 microliters of PLL at a rate of 10 microliters every 20 seconds to avoid vesicoureteral reflux.Now leave the catheter in the bladder for 20 minutes, with a stopper to prevent outflow. After 20 minutes, remove the catheter and vacate the bladder of any contents, by gently pressing on the lower abdomen. Then, using a one-milliliter syringe, flush any remaining contents out of the catheter.Now mix the MB49 PSA cells thoroughly by pipetting, and load them into a 1-mL syringe. Attach the catheter and lubricate it as before. Insert the catheter into the bladder as before.And commence with injecting the cells, using a gradual ejection of 10 microliters per 20 seconds. Then wait one hour. After an hour, remove the catheter and vacate the bladder of any contents.Then, release the mouse, positioning on its ventral side and revive it with an injection of atipamezole. Now, monitor the mouse until it regains sternal recumbency, and then return it to its home cage. PSA secretion of MB49 cells was found to vary with the growth media.DMEM media is used because it resulted in the most PSA secretion. In order to determine the sensitivity of the PSA ELISA, different numbers of MB49 PSA secreting cells were mixed with MB49 parent cells. The assay detected at least 100, 000 PSA-secreting cells per million cells.The PLL method of tumor implantation results in multiple tumors developing in the bladder. Both overnight collections of urine using metabolic cages and spot urine samples could be used to measure PSA by ELISA. For all animal studies, mouse weight was recorded as a measure of health.And urinary PSA is monitored on a weekly basis as a measure of tumor growth. Different therapies were used on each mouse, resulting in different tumor sizes. Two weeks after implantation, the bladders were harvested and processed.Tumor sizes varied, and the PSA measurements predicted this. After watching this video, you should have a good understanding of how to produce orthotropic bladder tumors in mice, and monitor their growth. Once mastered, this technique can be done in two hours per mouse.While attempting this procedure, it's important to remember to ensure cells are mixed before implantation, and to perform the implantation at a slow rate. Following this procedure, other methods, like flow cytometric analysis of Uvm or ELISA can be performed in order to investigate immune activation post-therapy.

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JoVE Journal

Cancer Research

14.4K ビュー.  National University of Singapore, National University Health System. The overall goal of this non-surgical method is to consistently produce orthotopic non-muscle-invasive bladder tumors in mice that can be easily monitored for the evaluation of intravesical therapies. This method can help answer key questions in the field of bladder cancer. Such as the evaluation of novel intravesical therapies.The main advantage of this technique is that it is easy to implant tumors in mice and monitor their growth, which helps to accurately determine responses to the...

ビデオ: A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

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

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

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

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

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

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 ...