This work was funded by the Academy of Finland (grants 25013080481 and 25013142041 (I.J.), 286377 and 295814 (M.P.), 287907 (T.J.)), P&#228;ivikki and Sakari Sohlberg Foundation (M.P., T.J.), Finnish Medical Foundation (T.P.), The Competitive State Research Financing of the Expert Responsibility Area of Tampere University Hospital (grant 9V049 and 9X044 (M.P.), 9X011 and 9V010 (T.J.)), The Competitive State Research Financing of the Expert Responsibility Area of Fimlab Laboratories (grant X51409 (I.J.)), Tays Support Foundation (I.J., M.P., T.J.), Tampere Tuberculosis Foundation (I.J., M.P., T.J.), the Finnish Cultural Foundation (M.V.), the Paulo Foundation (T.P.), Cancer Society of Finland (M.P.), and the Emil Aaltonen Foundation (T.P.).

The protocol described here has been approved by the National Animal Ethics Committee of Finland (protocol number ESAVI/23659/2018).
1. Experimental Animals, Reagents, and Equipment
<ol>
	<li>Use age and sex-matched mice. Start the study at 7&#8211;9 weeks of age, because the skin in most mice is in telogen (the resting phase) around that age2.</li>
	<li>Observe the behavior of the animals during the study and if they fight, which often happens with males, house them ...

DMBA-TPA-induced skin cancer is one of the most commonly used cancer models because it is highly reproducible and provides information on tumor progression from initiation to malignancy. The key outcome measure, papilloma formation, is easily and reliably quantitative. The model addresses both tumor initiation (tumor-free survival) and progression (tumor numbers and sizes) simultaneously. The model is suitable for studying different compounds, such as potential therapeutics, and the effects of individual genes on tumor p...

Cancer is one of the leading causes of death in the world. Therefore, there is a demand to develop reliable experimental disease models to obtain a better understanding of the disease as well as to explore potential therapeutic approaches. One of the most commonly used experimental in vivo models to study skin cancer development is the chemically induced two-stage skin carcinogenesis model1,2. The model provides a tool to study tumor initiation, promotion, and progression in addition to specific events such as immune cell infiltration and angiogenesis.
To use the two-stage skin carcinogenesis model, the back skin of mice is treated with two different chemicals that together induce tumor formation. The model is initiated with a low dose of the mutagen, DMBA, followed by prolonged exposure to the tumor promoter, TPA3 (Figure 1). DMBA mutates DNA randomly by forming covalent adducts with the DNA of epidermal cells and primary keratinocyte stem cells4,5,6,7. Some of these random mutations take place in a proto-oncogene, such as Hras1 (mutations in Kras and Nras are also detected) and the conversion of proto-oncogenes to oncogenes drives the tumor formation under proper stimuli. TPA, in turn, is the most commonly used tumor growth-promoting agent. Its molecular target is protein kinase C (PKC)8. TPA also activates Wnt/&#946;-catenin signaling that is crucial for tumor formation in the model9. Repeated and prolonged exposure to the promoting agent leads to enhanced cell signaling, increased production of growth factors, and a local inflammatory reaction, which are evident due to increased DNA synthesis and inflammatory cell infiltration in the treated skin.
The key inflammatory mediators in the DMBA-TPA model have been identified10. Interleukin-17A (IL-17A) is known to be particularly tumorigenic in the DMBA-TPA model11,12. It works in synergy with interleukin 6 (IL-6) and participates in macrophage and neutrophil recruitment13,14. In addition, CD4+ T cells and neutrophils have been shown to be tumorigenic in the DMBA-TPA model. Finally, macrophages can also promote tumorigenesis in the model15,16,17.
During the promotion phase, the cell proliferation of the mutated cells is enhanced and a sustained hyperplasia of the epidermis is maintained1. This leads to papilloma development in the skin in 10&#8211;20 weeks, after which the papillomas start to convert to malignant tumors, squamous cell carcinomas (SCCs)2. However, less than 10% of the papillomas progress to malignancy, although this percentage also depends on the genetic background of the mice2,18. For decades it was not known what type of cells were initially mutated in the tumors leading to malignancy, even though some studies had reported clearly distinct features in the malignant tumors when compared to benign papillomas19,20. However, recent studies have greatly increased our understanding on the clonal origin of tumor formation in the DMBA-TPA model21.22.23. It was demonstrated that both bone marrow-derived epithelial cells and hair follicle stem cells contribute to the tumor formation22. Stage-specific lineage tracing studies have unveiled that benign papillomas are of monoclonal origin, but they recruit new epithelial cell populations21,23. However, only one of the cell clones functions as a driver for carcinogenesis; it contains an Hras mutation23. The progression to carcinoma formation is associated with a clonal sweep23.
The carcinogen DMBA initiates the papilloma formation and TPA promotes tumor growth. Hence, the tumor initiation can be studied separately from the promotion by interrupting the experiment before the TPA treatment period. As the tumor progression is studied weekly it offers a great opportunity for detailed tumor growth analysis throughout the study. Because the tumors are generated by external chemicals, an oncogenic mutation in the germline is unnecessary. Thus, studying the effects of a genetic background (e.g., knockout/transgene vs. wild type) on tumorigenesis is straightforward2. In sum, the DMBA/TPA skin cancer model is a particularly useful approach for studying the role of the immune system in tumor progression as well as for the evaluation of tumor initiation and promotion steps independently or interdependently.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60445/60445fig1a.jpg" /> 
Figure 1: DMBA-TPA-induced skin carcinogenesis model outline. The carcinogen DMBA is topically applied to induce DNA mutations in the initiation phase of the model. The growth-promoting agent TPA is administered 2x a week to enhance cell proliferation during the promotion phase, leading to the development of papillomas in the skin. Animals are sacrificed after the papilloma response reaches a plateau, usually within weeks 15&#8211;20, depending on the genetic background of the mice. A small proportion of the papillomas can further develop into SCCs within 20&#8211;50 weeks. To study early events in the initiation and early promotion phase, samples can be collected (e.g., shortly after the second TPA application). A representative photograph and hematoxylin and eosin stained cross section of papillomas on a C57BL/6 mouse skin after 19 weeks of treatment are shown. Scale bar = 0.1 mm. <a href="https://www.jove.com/files/ftp_upload/60445/60445fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1000 ul RPT XL Graduated Filter Tip (Sterile), Refill</td>
			<td>Starlab</td>
			<td>S1182-1730-C</td>
			<td></td>
		</tr>
		<tr>
			<td>300 ul RTP Graduated Filter Tip (sterile), Refill</td>
			<td>Starlab</td>
			<td>S1180-9710-C</td>
			<td></td>
		</tr>
		<tr>
			<td>7,12-Dimethylbenz[a]anthracene (DMBA)</td>
			<td>Sigma</td>
			<td>D3254-100MG</td>
			<td>Harmful if swallowed and may cause cancer. Store protected from light.</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma</td>
			<td>1000141011</td>
			<td>Evaporates rapidly and is inflammable.</td>
		</tr>
		<tr>
			<td>Attane vet 1000 mg/g</td>
			<td>Piramal Critical Care Limited</td>
			<td></td>
			<td>Liquid isoflurane for inhalation</td>
		</tr>
		<tr>
			<td>Battery-Operated Clipper Isis</td>
			<td>Albert Kerlb GmbH</td>
			<td>GT421</td>
			<td>For shaving the fur</td>
		</tr>
		<tr>
			<td>CONTRAfluran-Restgasfilter</td>
			<td>ZeoSys GmbH</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Linex Nature N1030 Ruler 30 cm</td>
			<td>Staples Business Advantage</td>
			<td>60383</td>
			<td>For measuring papillomas</td>
		</tr>
		<tr>
			<td>Medium CO2 Chamber 300 x 200 x 200mm - Red</td>
			<td>VetTech Solutions Ltd</td>
			<td>AN045AR</td>
			<td>For sacrifice</td>
		</tr>
		<tr>
			<td>Mekasoft</td>
			<td>Mekalasi</td>
			<td>23008</td>
			<td>Table cover</td>
		</tr>
		<tr>
			<td>Mice (Balb/c JRj)</td>
			<td>Janvier labs</td>
			<td></td>
			<td>Other strains also possible</td>
		</tr>
		<tr>
			<td>Mice (C57BL/6JRj)</td>
			<td>Janvier labs</td>
			<td></td>
			<td>Other strains also possible</td>
		</tr>
		<tr>
			<td>Panasonic Lumix DMC-FS5 Digital Camera</td>
			<td>Panasonic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck</td>
			<td>30525-89-4</td>
			<td>For histology samples</td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate aka 12-Otetradecanoylphorbol-13-acetate (TPA)</td>
			<td>Enzo</td>
			<td>BML-PE160-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision balance PLJ-C/PLJ-G</td>
			<td>KERN &#38; SOHN GmbH</td>
			<td>PLJ 600-3CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-Set CO2 System-2 Chamber-S/S Housing</td>
			<td>VetTech Solutions Ltd</td>
			<td>AN044BX</td>
			<td>For sacrifice</td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>Qiagen</td>
			<td>76104</td>
			<td>For nucleic acid samples</td>
		</tr>
		<tr>
			<td>Tacta pipette 100-1000 ul</td>
			<td>Sartorius</td>
			<td>LH-729070</td>
			<td></td>
		</tr>
		<tr>
			<td>Tacta pipette 20-200 ul</td>
			<td>Sartorius</td>
			<td>LH-729060</td>
			<td></td>
		</tr>
		<tr>
			<td>UNO Anaesthetic Key Filler</td>
			<td>Scintica instrumentation inc.</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>UNO Face Mask for Mouse</td>
			<td>Scintica instrumentation inc.</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>UNO FM2200 Flowmeter</td>
			<td>Scintica instrumentation inc.</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>UNO Gas Exhaust Unit</td>
			<td>Scintica instrumentation inc.</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>UNO Induction Box</td>
			<td>Scintica instrumentation inc.</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>UNO200VAP Vaporizer</td>
			<td>Scintica instrumentation inc.</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
	</tbody>
</table>

chemical-induced skin carcinogenesis model using dimethylbenz[a]anthracene and 12-o-tetradecanoyl phorbol-13-acetate (dmba-tpa)

Cancer is one of the most devastating human diseases. Experimental cancer models are important to gain insight into the complex interplay of different cell types and genes in promoting tumor progression and to provide a platform for testing the efficacy of different therapeutic approaches. One of the most commonly used experimental inflammatory cancer models is the DMBA-TPA two-stage skin carcinogenesis model. Tumor formation is induced in this model by the topical application of two different chemicals, 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoyl phorbol-13-acetate (TPA), that together cause papilloma formation in the skin. As the primary outcome is papilloma formation in the skin, the model is an ideal, reliable, and reproducible way to address both tumor initiation (tumor-free survival) and tumor progression (number and size of visible tumors). The effects of the DMBA-TPA treatment are transmitted via an inflammatory mechanism, which makes this model especially suitable for studying the role of the immune system in tumor formation. However, this model is restricted to the skin and other surfaces where the chemicals can be applied on. A detailed protocol is provided in this article to use the model successfully.

The main outcome is the survival (i.e., papilloma free) time between the treatment or genotype groups. The secondary outcome is the number of papillomas per week in each group (Figure 2). The expected results are a statistically significant difference in the papilloma free time and in the number of papillomas between the experimental (two or more) groups. It is recommended to count the number of papillomas and draw a curve during the promotion (TPA) phase to get an idea of the differences be...

Watch this Scientific Journal Video about Chemical-Induced Skin Carcinogenesis Model Using Dimethylbenz[a]Anthracene and 12-O-Tetradecanoyl Phorbol-13-Acetate DMBA-TPA at JoVE.com

Chemical-Induced Skin Carcinogenesis Model Using Dimethylbenz[a]Anthracene and 12-O-Tetradecanoyl Phorbol-13-Acetate DMBA-TPA

Two-stage skin carcinogenesis is induced by two topically applied chemicals. A mutagen 7,12-dimethylbenz[a]anthracene) causes mutations in the epidermal cells and a continuous application of general growth stimulator 12-O-tetradecanoyl phorbol-13-acetate accelerates skin papilloma formation.

Chemical-Induced Skin Carcinogenesis Model Using Dimethylbenz[a]Anthracene and 12-O-Tetradecanoyl Phorbol-13-Acetate (DMBA-TPA)

Cancer is one of the most devastating human diseases. Experimental cancer models are important to gain insight into the complex interplay of different ...

This two step skin cancer model enables the study of the role of inflammation in skin cancer and allows the study the tumor initiation as a separate process from tumor progression. The tumor formation in this model is chemically induced and allows the use of knockout strains to study the effects of environmental factors or therapeutic candidates. The model is well suited for studying the role of immune system and angiogenesis in cancer.It demonstrates a similar disease pathology to squamous cell carcinoma in human patients. Although this method is relatively easy to conduct, accurate timing is essential. It's also important to take appropriate safety measures when handling DMBA, which is a carcinogen.Before beginning the experiment, house any aggressive seven to nine week old mice in separate cages to avoid fighting and skin lesions. For skin papilloma induction, first shave the back skins of the experimental animals and weigh the mice individually. 48 hours after shaving, use a pipette to apply 50 micrograms of DMBA in 200 microliters of acetone onto the shaved area of each anesthetized mouse.For skin papilloma promotion, seven days after DMBA application treat the skin with five micrograms of TPA in 200 microliters of acetone two times a week through the end of the experiment. Examine the animals for papillomas and photograph and record the size of each individual papilloma on a map every week. A palpable mass greater than one millimeter in diameter is considered a papilloma if it remains longer than one week.When the tumor response reaches a plateau, harvest the appropriate sample material from the animals 24 hours after the last TPA application and collection full thickness pieces of skin for immunohistochemical analysis. Use a biopsy punch to collect skin pieces from papilloma and non-papilloma skin samples for gene expression and or protein anaylses and use a scalpel to collect pieces from papilloma and non-papilloma skin for immunohistochemical analysis. Then collect the skin draining lymph nodes and a piece of the spleen for flow cytometric analysis as desired.A statistically significant difference in the papilloma free time and in the number of papillomas between the experimental groups is typically observed in the DMBA TPA tumor model. Histological analyses are suitable for studying the structure of the papillomas, the morphology of the skin, or the infiltration of immune cells of interest. It is essential to apply the DMBA and TPA evenly on the skin and to apply TPA in consistent intervals.It also important to count the papillomas regularly. The mechanisms that affect the number and time of tumor formation can be studied using different methods, including immunohistochemistry, flow cytometry, quantitative PCR, and protein analysis. This conventional technique can be easily adapted and applied to study the effects of different genes, environmental factors, or therapeutics of interest on tumor initiation and progression.Caution is required when performing the technique since DMBA is carcinogenic and acetone evaporates quickly. A suitable breathing mask and the use of flow cabinet are recommended.

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JoVE Journal

Cancer Research

13.6K Views.  Tampere University and Tampere University Hospital. This two step skin cancer model enables the study of the role of inflammation in skin cancer and allows the study the tumor initiation as a separate process from tumor progression. The tumor formation in this model is chemically induced and allows the use of knockout strains to study the effects of environmental factors or therapeutic candidates. The model is well suited for studying the role of immune system and angiogenesis in cancer.It demonstrates a similar disease pathology to squ...

Video: Chemical-Induced Skin Carcinogenesis Model Using Dimethylbenz[a]Anthracene and 12-O-Tetradecanoyl Phorbol-13-Acetate DMBA-TPA

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 ...

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without the ability to target a specific or well-delineated tumor volume. The delivery of radiation was achieved using fixed radiation sources or linear accelerators producing megavoltage (MV) X-rays. These devices are unable to achieve sub-millimeter precision required for small animals. Furthermore, the high doses delivered to healthy surrounding tissue hamper response assessment. To increase the translation between small animal studies and humans, our goal was to mimic the treatment of human glioblastoma in a rat model. To enable a more accurate irradiation in a preclinical setting, recently, precision image-guided small animal radiation research platforms were developed. Similar to human planning systems, treatment planning on these micro-irradiators is based on computed tomography (CT). However, low soft-tissue contrast on CT makes it very challenging to localize targets in certain tissues, such as the brain. Therefore, incorporating magnetic resonance imaging (MRI), which has excellent soft-tissue contrast compared to CT, would enable a more precise delineation of the target for irradiation. In the last decade also biological imaging techniques, such as positron emission tomography (PET) gained interest for radiation therapy treatment guidance. PET enables the visualization of e.g., glucose consumption, amino-acid transport, or hypoxia, present in the tumor. Targeting those highly proliferative or radio-resistant parts of the tumor with a higher dose could give a survival benefit. This hypothesis led to the introduction of the biological tumor volume (BTV), besides the conventional gross target volume (GTV), clinical target volume (CTV), and planned target volume (PTV).
At the preclinical imaging lab of Ghent University, a micro-irradiator, a small animal PET, and a 7 T small animal MRI are available. The goal was to incorporate MRI-guided irradiation and PET-guided sub-volume boosting in a glioblastoma rat model.

In the past, small animal irradiation was usually performed without the ability to target a well-delineated tumor volume. The goal was to mimic the treatment of human glioblastoma in rats. Using a small animal irradiation platform, we performed MRI-guided 3D conformal irradiation with PET-based sub-volume boosting in a preclinical setting.

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell ...

Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

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

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

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

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

Three Differential Expression Analysis Methods for RNA Sequencing: limma, EdgeR, DESeq2

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration and complex biological processes and has great value in diagnostics, prognostics, and therapeutics of tumors. Differential analysis of RNA-seq data is crucial to identify aberrant transcriptions, and limma, EdgeR and DESeq2 are efficient tools for differential analysis. However, RNA-seq differential analysis requires certain skills with R language and the ability to choose an appropriate method, which is lacking in the curriculum of medical education.
Herein, we provide the detailed protocol to identify differentially expressed genes (DEGs) between cholangiocarcinoma (CHOL) and normal tissues through limma, DESeq2 and EdgeR, respectively, and the results are shown in volcano plots and Venn diagrams. The three protocols of limma, DESeq2 and EdgeR are similar but have different steps among the processes of the analysis. For example, a linear model is used for statistics in limma, while the negative binomial distribution is used in edgeR and DESeq2. Additionally, the normalized RNA-seq count data is necessary for EdgeR and limma but is not necessary for DESeq2.
Here, we provide a detailed protocol for three differential analysis methods: limma, EdgeR and DESeq2. The results of the three methods are partly overlapping. All three methods have their own advantages, and the choice of method only depends on the data.

A detailed protocol of differential expression analysis methods for RNA sequencing was provided: limma, EdgeR, DESeq2.

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration ...