Name: in vivo model for testing effect of hypoxia on tumor metastasis
Uploaded: 2016-12-09T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis at JoVE.com

This work was supported by National Institutes of Health (NIH) grants: UL1TR000101 (previously UL1RR031975) through the Clinical and Translational Science Awards Program, 1RO1CA123211, 1R03CA178809, R01CA197964 and 1R21CA198698 to JK. MRI was performed in the Georgetown-Lombardi Comprehensive Cancer Center's Preclinical Imaging Research Laboratory (PIRL) and tissue processing in the Georgetown-Lombardi Comprehensive Cancer Center's Histopathology &amp; Tissue Shared Resource, both supported by NIH/NCI grant P30-CA051008. The authors thank Dan Chalothorn and James E. Faber, Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, for their assistance with postmortem x-ray angiography, and providing insight and expertise on collaterogenesis.

All procedures were approved by the Georgetown University Institutional Animal Care and Use Committee.
1. Cell Preparation for Orthotopic Injections
<ol>
	<li>Culture human ES cells under standard conditions. Use approximately one 15-cm cell culture plate not exceeding 70% of confluency for injection of 5 mice. 
	NOTE: For this study, SK-ES1 cells were cultured in McCoy&#39;s 5A medium with 15% fetal bovine serum (FBS) on collagen-coated plates and TC71 cells were cultured in RPMI with ...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropeptide+Y+induces+ischemic+angiogenesis+and+restores+function+of+ischemic+skeletal+muscles.">Neuropeptide Y induces ischemic angiogenesis and restores function of ischemic skeletal muscles.</a> J Clin Invest. 111, 1853-1862 (2003).</li><li>Tilan, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+neuropeptide+Y+is+critical+for+ischemic+revascularization+in+mice.">Platelet neuropeptide Y is critical for ischemic revascularization in mice.</a> FASEB J. , (2013).</li><li>Toffoli, S., Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittent+hypoxia+is+a+key+regulator+of+cancer+cell+and+endothelial+cell+interplay+in+tumours.">Intermittent hypoxia is a key regulator of cancer cell and endothelial cell interplay in tumours.</a> The FEBS journal. 275, 2991-3002 (2008).</li><li>Das, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+enhances+tumor+stemness+by+increasing+the+invasive+and+tumorigenic+side+population+fraction.">Hypoxia enhances tumor stemness by increasing the invasive and tumorigenic side population fraction.</a> Stem cells (Dayton, Ohio). 26, 1818-1830 (2008).</li><li>Arndt, C. 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Our model involves the comparison of metastasis in two experimental groups &#8212; a control group, where tumors are allowed to develop in the hindlimb followed by amputation upon reaching a calf volume of 250 mm3, and a hypoxia-exposed group, in which the tumor-bearing hindlimb is subjected to FAL at the same volume, followed by amputation 3 days later. Even though in these experiments the FAL-treated tumors are amputated with a slight delay, as compared to the control tumors, their size does not increase dur...

Ewing sarcoma (ES) is an aggressive malignancy affecting children and adolescents.1 The tumors develop in soft tissues and bones, commonly in limbs. While the presence of metastases is the single most powerful adverse prognostic factor for ES patients, the mechanisms underlying their development remain unclear.2 Tumor hypoxia is one of the few factors implicated in ES progression. In ES patients, the presence of non-perfused areas within the tumor tissue is associated with poor prognosis.3 In vitro, hypoxia increases invasiveness of ES cells and triggers expression of pro-metastatic genes.4-6 However, despite these lines of evidence, no direct proof for hypoxia-induced ES progression and spread exists. Moreover, the mechanisms by which hypoxia exerts such effects are, at present, unknown. Hence, we have created an in vivo model to fill the gap between existing in vitro data and clinical observations. This model system enables direct testing of the effects of hypoxia on tumors occurring in their natural environment, using magnetic resonance imaging (MRI) to follow tumor progression and metastasis in vivo in combination with ex vivo pathological and molecular analyses (Figure 1).
Since no established transgenic model of ES is currently available, the in vivo studies on metastatic properties of these tumors rely on injections of human cells into immunocompromised mice. While the use of immunologically impaired animals may underestimate the impact of the immune system on the disease progression, the ability to use human cells increases translatability of such studies. Among different xenograft models, systemic injections into tail vein are the easiest to perform, yet they omit the initial steps of tumor cell intravasation and escape from the primary site of growth.7-12 On the other hand, orthotopic xenografting, which involves injections of tumor cells into bones (femur, rib) or muscles, is more technically challenging, but also more biologically relevant to human cancer.13-16 However, in the past, the high morbidity associated with rapid growth of primary tumors has often necessitated animal euthanasia before metastasis development. In this study, we employed a previously established model of cell injections into the gastrocnemius muscle followed by excision of the resulting primary tumor combined with longitudinal monitoring of metastatic progression by MRI.17,18 Such injections into gastrocnemius muscle in close proximity to the tibia allow for tumor growth in two natural ES environments &#8212; muscles and bones &#8212; and result in distant metastases to locations typically affected in humans.18 Thereby, this model accurately recapitulates the metastatic processes occurring in ES patients during disease progression.
The localization of primary tumors in the lower hindlimb also facilitates the precise control of the blood supply to the tumor tissue. Femoral artery ligation (FAL) is a well-established technique utilized in angiogenesis research to block blood flow to distal regions of the leg and investigate tissue vascularization in response to ischemia.19,20 Importantly, the initial drop in blood flow is followed by collateral vessel opening and tissue reperfusion observed approximately 3 days after FAL.20 Thus, when performed in a tumor-bearing limb, this model recreates hypoxia/reperfusion events that occur naturally in rapidly growing tumors and enables the escape of metastatic tumor cells due to restoration of perfusion to the lower hindlimb via newly opened collateral vessels.21 Importantly, this procedure must be performed when the tumor size is small enough to prevent excessive hypoxia in control tumors (typically at the tumor-bearing calf volume of 150 - 250 mm3), ensuring significant differences in levels of tumor hypoxia between control and FAL-treated groups.
In addition to longitudinal monitoring of the effect of hypoxia on ES latency and the frequency of metastases, this model also allows for the collection of tissues and the development of new cell lines from both primary tumors and metastases. Importantly, previous work established that metastases-derived cell lines exhibit enhanced metastatic potential upon reintroduction to animals, indicating that tumor dissemination is associated with permanent changes in the tumor cell phenotype, and thereby validating the use of these cell lines to decipher the metastatic processes.18 Collectively, these models can now be used for the genetic and molecular analyses required for identifying hypoxia-induced metastatic pathways.
As hypoxia is a pro-metastatic factor enhancing the malignancy of various tumors, our model can be used as a platform to investigate the role of hypoxia in other tumor types that naturally develop in limbs, such as osteosarcoma and rhabdomyosarcoma.21-23 Moreover, a similar approach can be applied to malignancies growing in other anatomical locations with a well-defined route of blood supply. Ultimately, the model can be modified and its utility further extended, depending on individual research needs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>SK-ES1 Human Ewing sarcoma (ES) cells</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TC71 Human ES cells</td>
			<td></td>
			<td></td>
			<td>Kindly provided from Dr. Toretsky</td>
		</tr>
		<tr>
			<td>McCoy&#39;s 5A (modified) Medium</td>
			<td>Gibco by Life Technologies</td>
			<td>12330-031</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>ATCC</td>
			<td>30-2001</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning Cellgro</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F2442-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA (1X)</td>
			<td>Gibco by Life Technologies</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco by Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fungizone&#174; Antimycotic</td>
			<td>Gibco by Life Technologies</td>
			<td>15290-018</td>
			<td></td>
		</tr>
		<tr>
			<td>MycoZap&#8482; Prophylactic</td>
			<td>Lonza</td>
			<td>VZA-2032</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type I Rat tail high concetration</td>
			<td>BD Biosciences</td>
			<td>354249</td>
			<td></td>
		</tr>
		<tr>
			<td>SCID/beige mice</td>
			<td>Harlan or Charles River</td>
			<td>250 (Charles River) or 18602F (Harlan)</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Insulin syringes with permanently attached 28G&#189; needle&#160;</td>
			<td>BD</td>
			<td>329424</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline (0.9% Sodium Chloride Injection, USP)</td>
			<td>Hospira, INC</td>
			<td>NDC 0409-7984-37</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital calipers</td>
			<td>World Precision Instruments, Inc</td>
			<td>501601</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Tools</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rimadyl (Carprofen) Injectable&#160;</td>
			<td>Zoetis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoxyprobe-1 (Pimonidazole Hydrochloride solid)</td>
			<td>HPI, Inc</td>
			<td>HP-100mg</td>
			<td></td>
		</tr>
		<tr>
			<td>hypoxyprobe-2 (CCI-103F-250mg)</td>
			<td>HPI, Inc</td>
			<td>CCI-103F-250mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine Swabstick</td>
			<td>PDI</td>
			<td>S41350</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile alcohol prep pad</td>
			<td>Fisher HealthCare</td>
			<td>22-363-750</td>
			<td></td>
		</tr>
		<tr>
			<td>LubriFresh P.M. (eye lubricant ointment)&#160;</td>
			<td>Major Pharaceuticals</td>
			<td>NDC 0904-5168-38</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Absorbent Underpads with Waterproof Moisture Barrier</td>
			<td>VWR</td>
			<td>56617-014&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Oster Golden A5 Single Speed Vet Clipper with size 50 blade</td>
			<td>Oster</td>
			<td>078005-050-002 (clipper), 078919-006-005 (blade)</td>
			<td></td>
		</tr>
		<tr>
			<td>Nair Lotion with baby oil</td>
			<td>Church &#38; Dwight Co., Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silk 6-0</td>
			<td>Surgical Specialties Corp</td>
			<td>752B</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolene (polypropylene) suture 6-0</td>
			<td>Ethicon</td>
			<td>8680G</td>
			<td></td>
		</tr>
		<tr>
			<td>Vicryl (Polyglactin 910) suture 4-0&#160;</td>
			<td>Ethicon</td>
			<td>J386H</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Applicators (Purified cotton)</td>
			<td>Fisher Scientific</td>
			<td>23-400-115</td>
			<td></td>
		</tr>
		<tr>
			<td>GelFoam Absorbable Dental Sponges - Size 4</td>
			<td>Pfizer Pharmaceutical</td>
			<td>9039605</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclip Wound Clip Applier</td>
			<td>BD</td>
			<td>427630</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclip remover</td>
			<td>BD</td>
			<td>427637</td>
			<td></td>
		</tr>
		<tr>
			<td>Aound clip</td>
			<td>BD</td>
			<td>427631</td>
			<td></td>
		</tr>
		<tr>
			<td>MRI 7 Tesla</td>
			<td>Bruker Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paravision 5.0 software</td>
			<td>Bruker Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Euthanasia system</td>
			<td>VetEquip</td>
			<td></td>
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			<td>25G 5/8 Needle (for heart-puncture)</td>
			<td>BD</td>
			<td>305122</td>
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			<td>0.1 mL syringe (for heart-puncture)</td>
			<td>Terumo</td>
			<td>SS-01T</td>
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			<td>&#160;K3 EDTA Micro tube 1.3ml</td>
			<td>Sarstedt</td>
			<td>41.1395.105</td>
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			<td>10% Neutral Buttered Formalin</td>
			<td>Fisher Scientific</td>
			<td>SF100-4</td>
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in vivo model for testing effect of hypoxia on tumor metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

Following injection of ES cells into gastrocnemius muscle, the primary tumors are allowed to grow to a calf size of 250 mm3 (Figure 1, 2). The time necessary for the tumors to reach this volume typically ranges from 10 - 15 days for TC71 to 20-25 days for SK-ES1 xenografts, respectively. Tumors at a calf volume of 250 mm3 exhibit a relatively low level of endogenous hypoxia (approximately 3% of tumor tissue), based on hypoxybrobe-1 (pimonidazole) sta...

Watch this Scientific Journal Video about In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis at JoVE.com

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

The overall goal of this procedure is to directly test the effect of tumor hypoxia on metastasis using an animal model. This method can help to identify the mechanism underlying the prometastatic effects of hypoxia and potential novel therapeutic targets for preventing tumor dissemination. Two advantages of this technique are the ability to test the effects of tumor hypoxia in vivo, and to recapitulate the steps of metastasis and cell interactions that occur in cancer patients.In our model, tumor hypoxia is created by ligation of the femoral artery supplying blood to the tumor-bearing hilum of the mouse. Subsequent hind limb amputation prevents the mobility associated with repeat primary tumor growth, allowing for metastasis to form. Demonstrating the procedure will be Jason Tilan, Sung-Kyeok Hung, and Dr.Olga Rodriguez, co-director of the Preclinical Imaging Research Laboratory here at Georgetown.To inject the Ewing sarcoma cells, gently restrain a four to six week old female SCID Beige mouse, stabilizing the left leg between the fourth and fifth fingers to expose the medial side of the lower hind limb. Insert a 28 gauge 1/2"inch needle at an approximately 30-45 degree angle anteriorly into the gastrocnemius muscle toward the tibial crest. When the needle tip touches the crest, withdraw the needle slightly and slowly inject 0.1 milliliters of the Ewing sarcoma cells while continuing to withdraw the needle.Then use digital calipers to measure the calf size daily via the medial/lateral and anterior/posterior lengths until the tumor size reaches the desired volume. Before beginning the ligation, subcutaneously inject an analgesic agent followed by the administration of Hypoxyprobe-1 for hypoxia confirmation. Next, place the anesthetized mouse on a warming pad on the operating surface and apply ointment to the animal's eyes.Then, extend and secure the hind limb with a piece of tape approximately 45 degrees from the midline. Depilate the surgical area with hair-removal cream removing the cream with an ethanol prep pad after 10 seconds and disinfect the exposed skin with three consecutive 10%povidone-iodine and ethanol wipes. Transfer the mouse under a stereo microscope and make a 1 centimeter incision in the skin from the mid-thigh towards the inguinal region.Using saline-moistened fine cotton swabs, gently brush away the subcutaneous fat tissue surrounding the thigh muscle. Then carefully blunt dissect the subcutaneous fat tissue to reveal the underlying femoral artery. Stabilize the wound and surgical field to expose the vasculature of the mid-upper adductor muscle and use fine forceps to gently pierce through the membranous femoral sheath to expose the neurovascular bundle.Using a clean set of fine forceps, dissect and separate the femoral artery from the femoral vein and nerve at the proximal location near the groin, distal to the inguinal ligament. Then, pass a 6-0 silk suture under the femoral artery distal to the brach of the lateral circumflex femoral artery, and occlude the femoral artery with double knots. Close the incision with 6-0 polypropylene sutures and subcutaneously inject 0.5 milliliters of warm saline for fluid balance therapy.Then place the animal on top of a draped warm pad in the recovery cage with monitoring until fully recovered. At the appropriate experimental time point, gently restrain the animal and use clippers to shave the hair from the tumor-bearing limb from the distal tibia to the pelvic region. Inject the analgesic agent and place the anesthetized animal in the right lateral recumbent position on a sterile drape placed on a warming pad.Apply ointment to the eyes and disinfect the surgical site as just demonstrated. Then, place a sterile drape over the animal and make a middle femoral circumferential skin incision followed by blunt dissection and proximal retraction of the skin. Expose the medial femoral neurovascular pedicle on the median side of the leg.Then use a 4-0 polyglactin 910 coated absorbable suture to ligate near the inguinal ligament. Using scissors, perform a mid-femoral transection of the muscle followed by blunt dissection of the soft tissue to the coxofemoral joint. Using a bone-cutter, perform a mid-femoral osteotomy followed by gentle pressure at the site of osteotomy with a absorbable gelatin sponge to minimize the bleeding.Then use surgical wound clips to close the overlying skin and subcutaneously inject 0.5 milliliters of warm saline for fluid balance therapy. At least twice a week, carefully palpate the head, neck, and axillary regions and contralateral hind limb of the tumor-injected animals. Check for internal organ metastases via abdominal distension and for the presence of lung metastases via gentle xiphoid process pressure with the index finger.To evaluate the internal organ metastasis by MRI at the appropriate experimental time points, turn on the pump connected to a warm water circulating system to maintain the mouse at a physiological body temperature. Then, place the respiratory sensor in contact with the upper abdomen of the animal and turn on the BIOPAC Systems respiratory monitoring equipment. Place the anesthetized mouse onto a custom-designed mouse stereotaxic holder and apply ophthalmic ointment to the animal's eyes.To reduce the motion artifacts and noise, select the gating option in the MRI protocol to synchronize the data collection with the respiratory cycle. Then, run a two dimensional T2-weighted anatomical imaging sequence to image the metastases. For primary tumor cell culture after amputation, incision, or necropsy, harvest two to three 2-3 millimeter segments of wet lustrous pink-red viable tumor tissue, from each tissue sample of interest and place the isolated segments in 6 centimeter cell culture plates containing fresh primary culture medium.Then, transfer the tissues to a cell culture incubator for the appropriate cellular outgrowth period. Following injection of the Ewing sarcoma cells into the gastrocnemius muscle, the primary tumors are allowed to grow to a calf size of 250 cubic millimeters. Control tumors at this volume exhibit a relatively low level of endogenous hypoxia solely in the cells distant from the vasculature as determined by Hypoxyprobe-1 staining.In contrast, femoral artery ligation induces profound hypoxia in the vast majority of the tumor cells, even those proximal to the blood vessels. The effectiveness of the femoral artery ligation is supported by the complete inhibition of the primary tumor growth over the three-day period between the femoral artery ligation and the amputation. Histopathological analysis reveals extensive areas of necrosis in femoral artery ligation treated primary tumors harvested three days post-surgery.Nevertheless, there are groups of viable tumor cells present within the tumor tissue located at the edges of the tumor close to the vasculature. These remaining viable tumor cells are highly invasive as evidenced by their massive intravasation. Many cells within the vessel lumen are Hypoxyprobe-1 positive, indicating that cells that are hypoxic post femoral artery ligation may have initiated angioinvasion.In these experiments, hypoxia significantly increased the frequency and multiplicity of the metastases, often observed in locations not affected in mice bearing control tumors. Once mastered, each surgical procedure, including preparation, anesthesia, and recovery can be completed in 30 minutes per animal. The length of the entire experiment depends on the cell line used and its rates of growth and metastasis.The MRI and euthanasia timelines vary according to the latency of the metastases, and must be established for each individual cell line in a pilot experiment. While attempting this procedure, it's important to remember to maintain the appropriate pain management and animal monitoring post-procedure. If the procedure is performed perfectly, no severe adverse effect or death related to the surgery should be observed.Following this procedure, tissues and cells from control and hypoxic primary tumors as well as from their corresponding metastasis can be subjected to further analysis, including global gene expression profiling for identifying the mechanisms of hypoxia-induced metastasis. This technique can be used for the researchers in the field of cancer metastasis for directly testing the effects of tumor hypoxia on disease dissemination. Although here we focused on Ewing sarcoma, a similar approach can be applied to other tumors.After watching this video, you should have a very good understanding of how to create an animal model of tumor hypoxia and how to monitor the resulting metastases both in vivo and ex vivo.

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Research

JoVE Journal

Cancer Research

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 ...

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

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 ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...