This work was supported by grants R01-CA169354&#160;and R01-047822&#160;from the National Institutes of Health and a Merit Award from the Department of Veterans Affairs (ECB). RKP was supported by NIH T32 CA009287-30A1, an ASCO Young Investigator Award, California Breast Cancer Research Project Fellowship, and an American Cancer Society Mentored Research Scholar Grant; BAZ was supported by NIH grant AI079320. JM was supported by fellowships from NIH T-32 training grant T32-AI07290- 25, T32-AI07290-24 and American Cancer Society post-doctoral fellowship PF-12-052-01-CSM.

NOTE: All animal experiments were conducted in accordance with approved Stanford, Palo Alto VA HCS, and National Institutes of Health Institutional AnimalCare and Use Committee guidelines.
1. Preparing for Sample Collection and Processing (Time Required: ~10-15 min)
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	<li>Inoculate murine B16F0 melanoma cells (0.5-1 x 106) subcutaneously at or near the midline of the abdomen in female C57BL/6 mice as described previously6. Alternatively, inoculate tumor cells at add...

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Performing detailed analysis of the tumor microenvironment is critical in determining the mechanisms and effects of immunomodulation. With the increasing presence of immunotherapeutics in the human clinical realm, understanding the impact of these agents on the tumor infiltrating leukocytes is becoming requisite in defining their mechanism of action. In humans, there often exist clinical and/or logistic difficulties in obtaining and analyzing tumor tissue for leukocyte subset analysis, and thus analysis of the peripheral...

The balance between tumor growth and promotion and regression is, in part, dependent on the balance of pro- and anti- tumor infiltrating leukocytes present in the microenvironment1,2. In order to study the tumor microenvironment (TME) and specifically identify the infiltrating leukocyte subpopulations, we developed a method for evaluation of subcutaneous tumors in a murine tumor model. The importance of studying the tumor microenvironment is well known and supported in the literature. Numerous studies have shown that the balance of pro- and anti-tumor infiltrating immune cells can impact the outcomes of tumor growth, not only in mouse but also human studies (reviewed in3,4). For example, Curiel et al. showed that worsened clinical outcomes in ovarian cancer patients were correlated with the presence of increasing percentages of tumor-infiltrating regulatory CD4+ T cells (Tregs)5. Our own work also showed the effect of a novel leukocyte chemoattractant on the ratio of leukocyte subsets in a mouse melanoma model6, which also correlated with decreased tumor growth. Thus, the detailed analyses of the leukocyte subsets within a tumor is now more broadly recognized and increasingly important.
There are many ways to evaluate the tumor microenvironment for infiltrating leukocytes; for example groups have engineered transgenic mice to express various fluorescent proteins in order to image the TME7, classical immunohistochemistry and immunofluorescence of preserved sections8, including various imaging modalities such as MRI, PET, confocal microscopy9-11 - some with the ability to monitor intravitally10,12. These can be used with various molecular imaging agents, such as nanoparticles13 or novel contrast agents14 that label immune cells. Our method is a flow cytometry-based approach and has several advantages. First, the entire tumor microenvironment can be sampled; at the time of analysis, the entire subcutaneous tumor and surrounding periphery is surgically resected for processing. This eliminates any potentially sampling bias within a single tumor and gives a more &#8220;global&#8221; analysis of the tumor as a whole. Secondly, using multicolor flow cytometry to analyze the leukocyte subsets allows us to more specifically gauge the phenotype of infiltrating leukocytes. Depending on the number of colors used, very specific subsets can be identified. This is important as there are several leukocyte subsets within a particular cell type - or even in a general subtype classification &#8211; that have disparate functions that are potentially significant in determining the fate of the tumor. For example, plasmacytoid dendritic cells (pDC) have been implicated in anti-tumor immunity15. However, the CCR9+ subset of pDCs has been shown to be tolerogenic16, and shifting the balance of such a subset may have an impact on tumor growth.
Our method is appropriate for subcutaneous or other tumors that can be resected en bloc. In our hands, tumors were uniformly resected at the time of euthanasia. However, it is conceivable, as has been done in some studies, that a subcutaneous tumor could be fully resected with closure of the surrounding skin in a survival surgery17, thus allowing additional evaluation of the animal. The analysis is then performed on the resected tumor. Thus, the results represent a single timepoint in the tumor&#8217;s development. While this allows a detailed look into the microenvironment, it is also a static picture of what is no doubt a dynamic process. However, isolated leukocytes (e.g. via magnetic separation or density gradient) can then be analyzed separately from the tumor epithelia and stroma, or used in other, functional assays to further define their phenotype, as has been previously described18. This method, then, would be useful for any investigators interested in understanding the composition of the leukocytes within the tumor microenvironment at a given time point, whether in the setting of the natural disease course, or after a specific therapeutic perturbation. Although not done by us, variations of this procedure could also potentially be used to analyze specific portions of a tumor in isolation. For example, given the size of the tumor, the peripheral zone(s) could be dissected away from the central, possibly necrotic core of the tumor to give the researcher a more spatially segregated view of the tumor microenvironment.
In the burgeoning field of tumor immunology, there will no doubt be an exponentially growing number of studies evaluating novel immunomodulatory agents in murine tumor models. Several reports have highlighted the differences in specific leukocyte function within the tumor versus the peripheral environment. For example, Shafer-Weaver et al. showed in a mouse model that antigen-specific T effector CD8+ cells, while active in the periphery, were transformed into CD8+ T suppressor cells once they trafficked into the tumor microenvironment19. This was in part due to TGF&#946;, but other factors are likely involved as well. Thus, evaluating leukocyte subsets &#8211; numbers and ratios, as well as functional status &#8211; within the tumor itself will give a more accurate representation of the effect of a particular immunomodulation on tumor fate.
Our technique allows detailed analysis of the tumor and provides the researcher an opportunity to more closely identify changes in leukocyte populations than previous approaches.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Name of the Material/Equipment</td>
			<td style="width:229px;">Company</td>
			<td style="width:229px;">Catalog Number</td>
			<td style="width:229px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI</td>
			<td>Cellgro</td>
			<td>10-040</td>
			<td>http://cellgro.com; keep on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Cellgro</td>
			<td>35-011-CV</td>
			<td>http://cellgro.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml conical tubes</td>
			<td>Falcon</td>
			<td>14-432-22</td>
			<td>fischersci.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40 micron filter</td>
			<td>Falcon</td>
			<td>08-771-1</td>
			<td>fischersci.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 ml syringe</td>
			<td>BD</td>
			<td>14-823-35</td>
			<td>fischersci.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">surgical scissors/forceps</td>
			<td>Roboz</td>
			<td>RS-5910</td>
			<td>roboz.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Cellgro</td>
			<td>MT-21-030-CM</td>
			<td>http://cellgro.com; keep on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trypan blue</td>
			<td>Cellgro</td>
			<td>MT-25-900-CI</td>
			<td>fischersci.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hemacytometer</td>
			<td>Hausser Scientifice&#160;</td>
			<td>02-671-54&#160;</td>
			<td>fischersci.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Live/Dead stain</td>
			<td>Life Technologies</td>
			<td>L34957</td>
			<td>lieftechnologies.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowJo software</td>
			<td>TreeStar, Inc</td>
			<td></td>
			<td>flowjo.com</td>
		</tr>
	</tbody>
</table>

evaluation of tumor-infiltrating leukocyte subsets in a subcutaneous tumor model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

Our results showed that forced over-expression of chemerin in murine B16 tumors augmented the percentage of tumor infiltrating leukocytes (TILs). Additionally, changes to the relative ratio of leukocyte subsets represented within the tumor microenvironment associated with chemerin expression were identified. Re-print with permission from Pachynski et al.6.
Figure 1 shows that there was a significant increase in total CD45+ cells (TILs) within in the tumors th...

Watch this Scientific Journal Video about Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model at JoVE.com

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

evaluation-tumor-infiltrating-leukocyte-subsets-subcutaneous-tumor

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19.9K ビュー.  Washington University School of Medicine. This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses. 

ビデオ: Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

enrichment and characterization of the tumor immune and non-immune microenvironments in established subcutaneous murine tumors

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune...

flow cytometry-based isolation and therapeutic evaluation of tumor-infiltrating lymphocytes in a mouse model of pancreatic cancer

Author Spotlight: Advancing Adoptive Cell Therapy 

We describe a method to utilize tumor-infiltrating lymphocytes (TILs) from mice through flow cytometry for adoptive cell transfer. This protocol aims to verify the specific cytotoxicity of TILs against tumors in a syngeneic pancreatic cancer mouse model, providing insights into the development of adoptive cell therapies for pancreatic...

Flow Cytometry-Based Isolation and Therapeutic Evaluation of Tumor-Infiltrating Lymphocytes in a Mouse Model of Pancreatic Cancer

tumor transplantation for assessing the dynamics of tumor-infiltrating cd8+ t cells in mice

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

a three-dimensional co-culture model of infiltrating t cells in tumor spheroids

Use a light microscope to check whether any spheroids have been aspirated, and holding the P200 loaded pipette about 1/2 a centimeter above each cast, carefully seed the T cells onto the casts in a dropwise fashion, without dislodging the spheroids. When all of the casts have been seeded, carefully return the plate to the cell culture incubator for 15 minutes, before adding fresh cell culture medium supplemented with fetal bovine serum to the outside of each cast for another 48-hour...

A Three-Dimensional Co-culture Model of Infiltrating T Cells in Tumor Spheroids

an adjuvant therapy in a mouse model with an incompletely resected subcutaneous tumor

This video illustrates testing of adjuvant or additional therapy in a mouse model with incompletely resected subcutaneous tumors. The partially tumor-resected mouse is treated with an antibody targeting the anti-cytotoxic protein of T regulatory cells, potentially reducing tumor regrowth and suggesting effective adjuvant...

An Adjuvant Therapy in a Mouse Model with an Incompletely Resected Subcutaneous Tumor

This video demonstrates a method to establish a three-dimensional tumor spheroid and T cell co-culture. An agarose cast with microwells containing tumor spheroids is prepared, followed by the addition of T cells. After incubation, this setup allows studying the interaction of T cells with tumor spheroids in a controlled three-dimensional...

Do not cut the skin directly covering the tumor, as it will not heal well. Avoid using a scalpel to cut the tumor, and use tweezers to scoop the...

functional analysis of tumor-infiltrating myeloid cells by flow cytometry and adoptive transfer

Functional Analysis of Tumor-Infiltrating Myeloid Cells by Flow Cytometry and Adoptive Transfer

This protocol provides reliable methods of solid tumor dissociation and myeloid cell isolation in murine intradermal or subcutaneous tumor models. Flow cytometry allows for phenotypic characterization of heterogeneous myeloid populations within the tumor microenvironment and sorting will demonstrate their functionality in the context of adoptive...

tumor implantation and injection of a tumor vaccine in a mouse model

This video demonstrates a cell-based tumor vaccination assay. A mouse, intradermally injected with B16-F10 melanoma cells for tumor formation, is vaccinated with B16-F10 cells secreting Flt3L cytokine near the developed tumor. The secreted Flt3L from the vaccine promotes the maturation and proliferation of dendritic cells that then present tumor antigens to T cells in lymph nodes, triggering a targeted anti-tumor immune...

Tumor Implantation and Injection of a Tumor Vaccine in a Mouse Model

a murine orthotopic bladder tumor model and tumor detection system

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...

isolating tumor-infiltrating immune cells from a murine brain

The video demonstrates the isolation of tumor-infiltrating peripheral blood mononuclear cells from a mouse brain using mechanical dissociation and enzymatic digestion. This is followed by density gradient centrifugation to collect the immune cells for further...

Isolating Tumor-Infiltrating Immune Cells from a Murine Brain

Next, place the target tissue into the labeled glass-down tissue grinder, containing 1 milliliter of DPBS. Push the plunger all the way down, and twist seven times to disrupt the tissue. After that, lift the plunger to allow the liquid to settle back to the bottom of the tissue grinder. Repeat this step thrice. However, only twist the plunger four times during the next two repeats and three times during the last repeat to avoid over-triturating the brain...

generation of induced pluripotent stem cells from human melanoma tumor-infiltrating lymphocytes

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem...

an orthotopic bladder tumor model and the evaluation of intravesical sarna treatment

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent...

real time detection of in vitro tumor cell apoptosis induced by cd8+ t cells to study immune suppressive functions of tumor-infiltrating myeloid cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

generation of orthotopic pancreatic tumors and ex vivo characterization of tumor-infiltrating t cell cytotoxicity

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...

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

After implementation, measure the tumor length and width three times a week using an electronic digital caliper. Calculate the tumor volume, and treat the mice with tumor vaccine when tumors have reached a size of 2 millimeters as described in the text...

generation of 3d tumor spheroids for drug evaluation studies

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

tumor engraftment in a xenograft mouse model of human mantle cell lymphoma

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

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...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

Osmotic pumps continuously deliver compounds at a constant rate into small animals. This article introduces a standard protocol used to induce aortic aneurysms via subcutaneous infusion of angiotensin II (AngII) from implanted osmotic pumps. This protocol includes calculation of AngII amount and dissolution, osmotic pump filling, implantation of osmotic pumps subcutaneously, observation after pump implantation, and harvest of aortas to visualize aortic aneurysms in mice. Subcutaneous infusion of AngII through osmotic pumps following this protocol is a reliable and reproducible technique to induce both abdominal and thoracic aortic aneurysms in mice. Infusion durations range from a few days to several months based on the purpose of the study. AngII 1,000 ng/kg/min is sufficient to provide maximal effects on abdominal aortic aneurysmal formation in male hypercholesterolemic mouse models such as apolipoprotein E deficient or low-density lipoprotein receptor deficient mice. Incidence of abdominal aortic aneurysms induced by AngII infusion via osmotic pumps is 5 - 10 times lower in female hypercholesterolemic mice and also lower in both genders of normocholesterolemic mice. In contrast, AngII-induced thoracic aortic aneurysms in mice are not hypercholesterolemia or gender-dependent. Importantly, multiple features of this mouse model recapitulate those of human aortic aneurysms.

Subcutaneous implantation of osmotic pumps provides a convenient approach for prolonged and consistent delivery of compounds. This approach has been used extensively to study both abdominal and thoracic aortic aneurysms in mice.

Osmotic pumps continuously deliver compounds at a constant rate into small animals.  This article introduces a standard protocol used to induce aortic ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...