MR was funded by a fellowship from the Danish cancer society (R146-A9394-16-S2). MFB and MKT were funded by AUFF NOVA (AUFF-E-2015-FLS-9-8). MR and MFB were co-funded by Graduate School, HEALTH, AU. The E.F.W. laboratory is supported by grants from the Spanish Ministry of Economy (SAF2015-70857, co-funded by the ERDF-EU) and an ERC advanced grant (741888 - CSI-Fun).
We want to thank Liliana Fajardo Mellor (Genes, Development and Disease; National Cancer Research Center) for critical reading of the manuscript.

This protocol involves a surgical procedure in laboratory mice. All animal experiments must be individually reviewed and approved by an Institutional Animal Care and Use Committee (IACUC). As the approach is based on animal recovery and survival, ensure appropriate anesthesia, pain management, and an aseptic surgical environment at all time. Use a heating pad to prevent hypothermia during surgery and until recovery from anesthesia.
1. Starting Considerations
<ol>
	<li>Depending on the virus used, different titer requirements may apply; dilute the virus accordingly. 
	NOTE: In this example, an Adeno-associated virus diluted in PBS is injected at a titer of 1 x 1012 IU/mL. The virus has serotype 9 and contains guide RNA to Pten and expression of Cre protein under a ubiquitin promotor (Figure 1). Loss of Pten increased pAKT and proliferation of the prostatic epithelium13.</li>
	<li>Use male mice at an age of 8&#160;weeks at the time of surgery to ensure adequate prostate development. 
	NOTE: Mice shown in this experiment are Rosa26-LSL-Cas9-EGFP knockin mice on C57BL/6J background12 (Figure 1).</li>
	<li>Freshly prepare a general anesthesia mix and if possible keep it sterile. 
	NOTE: For better animal recovery after surgery, an anesthesia antidote is highly recommended.
	<ol>
		<li>To prepare a total volume of 5 mL anesthetic, mix 0.15 mL medetomidine hydrochloride (1 mg/mL), 0.4 mL midazolam (5 mg/mL), and 0.25 mL butorphanol (10 mg/mL) with 4.2 mL sterile saline water. 
		NOTE: Alternative anesthesia methods, such as inhalable isoflurane, can be used according to the institutional animal care guidelines.</li>
		<li>To reverse the effect of medetomidine hydrochloride post-surgery, mix 0.1 mL atipamezole (5 mg/mL) with 4.9 mL sterile saline water.</li>
	</ol>
	</li>
	<li>Prepare an aseptic surgical environment by proper disinfection and sterilization. Autoclave surgical instruments before use.</li>
</ol>
2. Virus-delivery to the Murine Prostate
<ol>
	<li>Anesthetize the animal by intraperitoneal injection with a sterile 1 mL syringe and a 27G x &#189;&#34; needle. Use an anesthesia dose of 0.01 mL/g body weight. If the procedure lasts longer than 30 min, maintain anesthesia by injecting 1/3 of the starting dose every 30 min.</li>
	<li>Examine the anesthetic depth by assessing muscle relaxation, pedal withdrawal, and palpebral reflexes. When loss of reflexes is observed, shave the lower abdomen of the animal.</li>
	<li>Carefully cover the animal&#39;s eyes with veterinary ophthalmic ointment using a sterile cotton swab to prevent blindness due to lack of corneal reflex.</li>
	<li>Wipe the shaved abdomen with 70% ethanol and 10% povidone iodine to disinfect the surgical area.&#160;Scrub the surgical area from the center of the surgical area toward the periphery.&#160;</li>
	<li>Perform a vertical skin incision at the low-abdominal midline of approximately 1 cm using a sterile surgical scissor.</li>
	<li>Lift the peritoneum using a fine point forceps to prevent damaging the organs that are laying underneath. Carefully make an &#60; 8 mm incision using surgical scissors through the peritoneum. 
	NOTE: In contrast to human, a rodent&#39;s prostate gland comprises four separate lobes. The anterior lobe is attached to the seminal vesicle.</li>
	<li>Gently move the fat tissue aside to uncover the seminal vesicle. Using a ring forceps, carefully lift up the seminal vesicle until the anterior prostate can be identified (Figure 1).</li>
	<li>Inject a total volume of 30 &#181;L virus (3 x 1010 viral particles) solution in the anterior prostate epithelium using a 0.5 mL insulin syringe with a 30G x 8 mm needle. Minimize leakage and ensure the fluid is absorbed within the tissue, forming a small bubble. Place the seminal vesicle back in the abdominal cavity.</li>
	<li>Suture the peritoneum with 2-3 simple interrupted stitches using 6-0 absorbable sutures with a 13 mm, 3/8 circle, and a taper point needle.</li>
	<li>Staple the skin with 3 sterile 4.8 x 6.5 mm clips lifting the skin with forceps to avoid damaging the peritoneum.</li>
	<li>For better recovery, apply the anesthesia antidote in a dose of 0.01 mL/g body weight by intraperitoneal injection using a sterile 1 mL syringe and a 27G x &#189;&#34; needle. Carefully place the animal back in its cage.</li>
</ol>
3. Post-surgery Procedures
<ol>
	<li>Keep the cage with mice that underwent surgery on a heating pad for 1 h after the procedure, to prevent hypothermia until fully recovered. If the antidote is applied, the animal should be awake a few minutes after the injection.</li>
	<li>Monitor the animals daily for appropriate wound healing and pain indications. If necessary, administer additional pain killers according to institutional animal care guidelines.</li>
	<li>Remove skin clips after 4-5 days, once the wound is closed.</li>
</ol>

<ol>
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A., De Gasperi, R., Elder, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+transgenesis:+an+overview.">Animal transgenesis: an overview.</a> Brain Struct Funct. 214, 91-109 (2010).</li><li>Wu, X., 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=Generation+of+a+prostate+epithelial+cell-specific+Cre+transgenic+mouse+model+for+tissue-specific+gene+ablation.">Generation of a prostate epithelial cell-specific Cre transgenic mouse model for tissue-specific gene ablation.</a> Mech Dev. 101, 61-69 (2001).</li><li>Ma, X., 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=Targeted+biallelic+inactivation+of+Pten+in+the+mouse+prostate+leads+to+prostate+cancer+accompanied+by+increased+epithelial+cell+proliferation+but+not+by+reduced+apoptosis.">Targeted biallelic inactivation of Pten in the mouse prostate leads to prostate cancer accompanied by increased epithelial cell proliferation but not by reduced apoptosis.</a> Cancer Res. 65, 5730-5739 (2005).</li><li>Chen, Z., 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=Crucial+role+of+p53-dependent+cellular+senescence+in+suppression+of+Pten-deficient+tumorigenesis.">Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis.</a> Nature. 436, 725-730 (2005).</li><li>Leow, C. C., Wang, X. D., Gao, W. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+method+of+generating+prostate-specific+Cre-LoxP+gene+switching+via+intraductal+delivery+of+adenovirus.">Novel method of generating prostate-specific Cre-LoxP gene switching via intraductal delivery of adenovirus.</a> Prostate. 65, 1-9 (2005).</li><li>Thomsen, M. K., 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=Loss+of+JUNB/AP-1+promotes+invasive+prostate+cancer.">Loss of JUNB/AP-1 promotes invasive prostate cancer.</a> Cell Death Differ. 22, 574-582 (2015).</li><li>Cho, H., 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=a+novel+GEM+model+for+metastatic+prostate+cancer+analysis+and+therapy,+reveals+myc+as+a+driver+of+Pten-mutant+metastasis.">a novel GEM model for metastatic prostate cancer analysis and therapy, reveals myc as a driver of Pten-mutant metastasis.</a> Cancer Discov. 4, 318-333 (2014).</li><li>Cho, H., 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=Rapid+in+vivo+validation+of+candidate+drivers+derived+from+the+PTEN-mutant+prostate+metastasis+genome.">Rapid in vivo validation of candidate drivers derived from the PTEN-mutant prostate metastasis genome.</a> Methods. 77-78, 197-204 (2015).</li><li>Platt, R. J., 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=CRISPR-Cas9+knockin+mice+for+genome+editing+and+cancer+modeling.">CRISPR-Cas9 knockin mice for genome editing and cancer modeling.</a> Cell. 159, 440-455 (2014).</li><li>Wang, S., 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=Prostate-specific+deletion+of+the+murine+Pten+tumor+suppressor+gene+leads+to+metastatic+prostate+cancer.">Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer.</a> Cancer Cell. 4, 209-221 (2003).</li><li>Liu, W., 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=Copy+number+analysis+indicates+monoclonal+origin+of+lethal+metastatic+prostate+cancer.">Copy number analysis indicates monoclonal origin of lethal metastatic prostate cancer.</a> Nat Med. 15, 559-565 (2009).</li><li>Haffner, M. 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=Tracking+the+clonal+origin+of+lethal+prostate+cancer.">Tracking the clonal origin of lethal prostate cancer.</a> J Clin Invest. 123, 4918-4922 (2013).</li><li>Daya, S., Berns, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+using+adeno-associated+virus+vectors.">Gene therapy using adeno-associated virus vectors.</a> Clin Microbiol Rev. 21, 583-593 (2008).</li><li>Price, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Aspects+of+Development+and+Structure+in+the+Prostate.">Comparative Aspects of Development and Structure in the Prostate.</a> Natl Cancer Inst Monogr. 12, 1-27 (1963).</li><li>McNeal, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+prostate:+an+historical+survey+of+divergent+views.">Anatomy of the prostate: an historical survey of divergent views.</a> Prostate. 1, 3-13 (1980).</li><li>Thomsen, M. K., 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=SOX9+elevation+in+the+prostate+promotes+proliferation+and+cooperates+with+PTEN+loss+to+drive+tumor+formation.">SOX9 elevation in the prostate promotes proliferation and cooperates with PTEN loss to drive tumor formation.</a> Cancer Res. 70, 979-987 (2010).</li></ol>

The authors have nothing to disclose.

In this protocol, we describe a method to alter gene expression in the anterior lobe of the murine prostate by virus injection, creating a powerful new mouse model for prostate cancer (Figure 2). The successful administration of an Adenovirus was first described by Leow et al. in 20058. We have previously shown how an Adenovirus coding for a Cre recombinase protein can replace time-consuming cross-breeding of a Cre allele for tissue-specific deletion <sup cla...

Detection and treatment of prostate cancer have significantly improved over the last decade. Still, the incidence of prostate cancer is increasing, following life expectancy. With an estimated 1.1 million new cases worldwide, it is among the most common causes of cancer-related death in men 1. Prostate cancer is slow in its development, but when the cancer has progressed to an advanced metastatic state, prognosis is poor due to limited treatment options. So far, only a few genes have been identified as common drivers in this cancer, and its heterogeneity and multifocality impedes detection of biomarkers and targetable disease drivers2,3.
Classical techniques of generating GEMMs are often impaired by their complexity, timely expenses, and costs. Conditional knockout models have been widely used to study prostate cancer candidate genes, that result in embryonic lethality when inactivated in the germline4. Most common models involve a prostate-specific Cre recombinase driven by either a modified Probasin5 or a PSA6 promoter integrated in the GEMM by additional cross-breeding. In these models, the gene of interest will be targeted in the majority of prostate epithelial cells, generating hyperplasia in the entire organ, which may impair the animal&#39;s urinary tract function7.
Viral delivery of the Cre protein by injection into the anterior lobe of the murine prostate can resolve this problem by only targeting a few cells8. Taking laboratories technical prerequisites, expertise, and objectives into account, the method benefits from a broad range of possible variations. Successful approaches utilizing Adenovirus targeting JunB and Pten9 or Lentivirus targeting Pten and Trp5310 have been shown amongst others. Adding transgenes, such as luciferase, to the viral construct or to the GEMM will furthermore enable non-invasive monitoring of disease progression via bioluminescence imaging11.
Genome editing based on the CRISPR/Cas9 technology reveals a new and rapid opportunity to study cancer through rapid generation of somatic knockouts12. Viral delivery of single guide RNAs (sgRNAs) directed to the anterior lobe of the murine prostate establishes a physiologically more relevant model of prostate cancer. By this means, single cells carrying chosen mutations can form clones that are capable of expansion and invasion. Furthermore, use of guide RNAs for multiple target genes will generate cell clones with alterations in different genes. This will allow tumor heterogeneity and a natural selection pressure on cancer progression, which can reveal the importance of each gene alteration or epistatic mechanisms.
Here we present a method to deliver viral particles to the murine prostate for alteration of gene expression. By a small abdominal incision, the murine anterior prostate lobe is exposed and viral particles are injected into the lobe. Five days post-surgery, surgical clips can be removed from the skin and the prostatic cancer can be analyzed from 8 weeks after. Overall, this is a rapid and cost-efficient procedure, which has little impact on the mouse and allows larger tumor to develop without compromising the mouse.

<table>
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B6J.129(B6N)-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J mice</td>
			<td>The Jackson Laboratory</td>
			<td>26175</td>
			<td></td>
		</tr>
		<tr>
			<td>0,5 ml U-100 insulin syringe</td>
			<td>BD</td>
			<td>324825</td>
			<td>for virus injection</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>BD</td>
			<td>300013</td>
			<td>for anesthesia injection</td>
		</tr>
		<tr>
			<td>30 G 1/2&#39;&#39; needle</td>
			<td>BD</td>
			<td>304000</td>
			<td>for anesthesia injection</td>
		</tr>
		<tr>
			<td>6-0 Polysorb Suture</td>
			<td>Medtronic</td>
			<td>GL889</td>
			<td>to close the peritoneum</td>
		</tr>
		<tr>
			<td>Disposable sterilized surgery drape</td>
			<td>multiple suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>multiple suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-Iodine Prep Pad</td>
			<td>Fisher Scientific</td>
			<td>06-669-70</td>
			<td></td>
		</tr>
		<tr>
			<td>Trimming machine</td>
			<td>Aesculap</td>
			<td>GT415</td>
			<td>to shave the abdomen</td>
		</tr>
		<tr>
			<td>Dumont Forceps</td>
			<td>F.S.T</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey Micro Needle Holder</td>
			<td>F.S.T</td>
			<td>12500-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>F.S.T</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow Pattern Forceps</td>
			<td>F.S.T</td>
			<td>11002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring Forceps</td>
			<td>F.S.T</td>
			<td>11106-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Wound Clip System Handle incl. Clips</td>
			<td>F.S.T</td>
			<td>12030-01</td>
			<td>to close the skin</td>
		</tr>
		<tr>
			<td>Wound Clip System Remover</td>
			<td>F.S.T</td>
			<td>12030-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td></td>
			<td>different models have been used</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Antisedan</td>
			<td></td>
			<td></td>
			<td>obtained from the animal facility</td>
		</tr>
		<tr>
			<td>Butorphanol</td>
			<td></td>
			<td></td>
			<td>obtained from the animal facility</td>
		</tr>
		<tr>
			<td>Medetomidinhydrochlorid</td>
			<td></td>
			<td></td>
			<td>obtained from the animal facility</td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td></td>
			<td></td>
			<td>obtained from the animal facility</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Fisher Scientific</td>
			<td>22-032-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye ointment</td>
			<td>Takeda</td>
			<td>7242</td>
			<td></td>
		</tr>
		<tr>
			<td>Virus (AAV, AV)</td>
			<td>multiple suppliers</td>
			<td></td>
			<td>virus used in this protocol is an in-house production</td>
		</tr>
		<tr>
			<td>pAKT antibody</td>
			<td>CST</td>
			<td>4060</td>
			<td>used 1:200 dillution</td>
		</tr>
		<tr>
			<td>GFP anitbody</td>
			<td>CST</td>
			<td>2956</td>
			<td>used 1:100 dillution</td>
		</tr>
	</tbody>
</table>

virus delivery of crispr guides to the murine prostate for gene alteration

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) for prostate cancer are hampered by tumor heterogeneity and its complex microevolution dynamics. Traditional prostate cancer mouse models include, amongst others, germline and conditional knockouts, transgenic expression of oncogenes, and xenograft models. Generation of de novo mutations in these models is complex, time-consuming, and costly. In addition, most of traditional models target the majority of the prostate epithelium, whereas human prostate cancer is well known to evolve as an isolated event in only a small subset of cells. Valuable models need to simulate not only prostate cancer initiation, but also progression to advanced disease.
Here we describe a method to target a few cells in the prostate epithelium by transducing cells by viral particles. The delivery of an engineered virus to the murine prostate allows alteration of gene expression in the prostate epithelia. Virus type and quantity will hereby define the number of targeted cells for gene alteration by transducing a few cells for cancer initiation and many cells for gene therapy. Through surgery-based injection in the anterior lobe, distal from the urinary track, the tumor in this model can expand without impairing the urinary function of the animal. Furthermore, by targeting only a subset of prostate epithelial cells the technique enables clonal expansion of the tumor, and therefore mimics human tumor initiation, progression, as well as invasion through the basal membrane.
This novel technique provides a powerful prostate cancer model with improved physiological relevance. Animal suffering is limited, and since no additional breeding is required, overall animal count is reduced. At the same time, analysis of new candidate genes and pathways is accelerated, which in turn is more cost efficient.

To assess virus delivery to the murine prostate, samples were analyzed three months after the surgery. The Rosa26-LSL-Cas9-EGFP mice12 express GFP in cells that have been exposed to Cre protein expressed by the virus. The prostate samples were examined with a fluorescence microscopy to identify areas with GFP signal (Figure 2A). The GFP signal indicates Cre activity in the prostate epithelium but not whether gene editing has been induc...

Watch this Scientific Journal Video about Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration at JoVE.com

Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

This protocol describes a newly established method for virus delivery to the murine prostate. Using either CRISPR/Cas9 technology, gene overexpression, or Cre recombinase delivery, the technique allows orthotopic alteration of gene expression and implements a novel mouse model for prostate cancer.

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

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8.6K Views.  Aarhus University. This protocol describes a newly established method for virus delivery to the murine prostate. Using either CRISPR/Cas9 technology, gene overexpression, or Cre recombinase delivery, the technique allows orthotopic alteration of gene expression and implements a novel mouse model for prostate cancer.

Video: Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis and to identify novel therapeutic targets. To investigate molecular events that drive the progression from primary cancer to metastasis, we have developed a bitransgenic mouse model, RIP-Tag; RIP-tva. In this mouse model, the rat insulin promoter (RIP) drives the expression of the SV40 T antigen (Tag) and the receptor for subgroup A avian leukosis virus (tva) in pancreatic &#946; cells. The mice develop pancreatic neuroendocrine tumors with 100% penetrance through well-defined stages that are similar to human tumorigenesis, with stages including hyperplasia, angiogenesis, adenoma, and invasive carcinoma. Because RIP-Tag; RIP-tva mice do not develop metastatic disease, genetic alterations that promote metastasis can be identified easily. Somatic gene transfer into tva-expressing, proliferating pancreatic &#946; premalignant lesions is achieved through intracardiac injection of avian retroviruses harboring the desired genetic alteration. A titer of &#62;1 x 108 infectious units per ml is considered appropriate for in vivo infection. In addition, avian retroviruses can infect cell lines derived from tumors in RIP-Tag; RIP-tva mice with high efficiency. The cell lines can also be used to characterize the metastatic factors. Here we demonstrate how to utilize this mouse model and cell lines to assess the functions of candidate genes in tumor metastasis.

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

We present a protocol to demonstrate a novel somatic gene transfer system utilizing RIP-Tag; RIP-tva mouse model to study the function of genes in metastasis. The avian retroviruses are delivered intracardiacally to ensure gene transfer into pre-malignant, noninvasive lesions of pancreatic &#946; cells in adult mice.

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis ...

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 ...

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...