We thank J. Vaughen for critical reading of the manuscript. This work was supported by grants from JSPS KAKENHI Grant Numbers 26891025, 15H01500 and The Takeda Science Foundation Research Grant to Y.T.

1. Fly Crosses and Clone Induction
<ol>
	<li>Remove all flies in the vial 12 h before collecting virgin flies.</li>
	<li>Anesthetize the flies in the vial by injecting CO2 gas and place flies onto a CO2 fly pad.</li>
	<li>Transfer 10 - 20 virgin females and 10 males from the CO2 fly pad into a fresh vial and incubate for 1 day at 25 &#176;C.</li>
	<li>Transfer these flies into a fresh vial and incubate for 12 h at 25 &#176;C. 
	NOTE: Discard the first vial as virgin females do not lay enough eggs in the first day.</li>
	<li>For enhancer-GAL4 lines, remove the adult flies and incubate the vial at 25 &#176;C until dissection.</li>
	<li>For induction of mosaic clones by flip-out-GAL4, remove the adult flies and incubate the vial for 2 - 4 days at 25 &#176;C. The incubation time before heat shock is dependent on the experimental purpose. A heat shock 2 days after egg deposition (AED) generates mosaic clones in immature second instar imaginal epithelia. A heat shock after 3 or 4 days AED generates mosaic clones in differentiated early- to mid-third instar imaginal epithelia. 
	NOTE: It is important to examine the effect of altered heat-shock timing on tumorigenic phenotypes.</li>
	<li>For induction of mosaic clones by flip-out-GAL4, heat shock the vial by immersion in a 37 &#176;C water bath for 10 - 45 min followed by incubation for 2 - 3 days at 25 &#176;C. 
	NOTE: Information of heat-shock time, timing, and incubation time in each experiment is shown in the figure legends.</li>
</ol>
2. Dissection of Larvae
<ol>
	<li>Collect wandering third instar larvae with a wooden stick or blunt forceps, genotype them by appropriate fluorescent markers (e.g., EGFP) under a fluorescence stereoscopic microscope and place them in a dissection dish with PBS (phosphate buffered saline).</li>
	<li>Wash the larvae in PBS by pipetting with 2 mL plastic transfer pipettes.</li>
	<li>Pinch the center of the larva with one forceps and tear the body in half with the other forceps.</li>
	<li>Pinch the mouth hook of the anterior half with one forceps and push the mouth towards the body with the other forceps to turn the body inside out.</li>
	<li>Remove unnecessary materials such as salivary glands or fat bodies with forceps.</li>
</ol>
3. Fixation and Antibody Staining
<ol>
	<li>Transfer the dissected anterior half of the larval body (including imaginal wing discs) to a 1.5 mL plastic tube and fix in 1 mL of Fix solution (4% Formaldehyde in PBS) for 10 min at room temperature in the dark with gentle rotation. 
	CAUTION: Formaldehyde is toxic and has carcinogenic potential. Wear protective gloves and clothing to prevent skin contact. 
	NOTE: In this part, all steps take place on a nutator at room temperature in the dark unless otherwise noted.</li>
	<li>Remove the Fix solution and discard. Wash the tissues with 1 mL of PBT (0.3% Triton X-100 in PBS) three times for 15 min each.</li>
	<li>Remove the PBT and add 1 mL of PBTG (0.2% bovine serum albumin and 5% normal goat serum in PBT) for blocking and nutate 1 h at room temperature or overnight at 4 &#176;C.</li>
	<li>Remove PBTG and add primary antibody solution appropriately diluted with PBTG (see Materials Table) and nutate overnight at 4 &#176;C.</li>
	<li>Remove the primary antibody solution and wash the tissues with 1 mL PBT three times for 15 min each.</li>
	<li>Remove PBT and add secondary antibody solution appropriately diluted with PBTG (1:400). Nutate for 2 h at room temperature or overnight at 4 &#176;C.</li>
	<li>Remove secondary antibody solution and wash the tissues with 1 mL of PBT two times for 15 min each.</li>
	<li>To stain F-actin, remove PBT and add Phalloidin solution appropriately diluted in PBS (1:40). Then nutate for 20 min. Remove the Phalloidin solution and wash the tissues with 1 mL of PBT two times for 15 min each.</li>
	<li>To counterstain nuclear DNA, remove PBT and add DAPI (4&#39;, 6-diamidino-2-phenylindole) solution (0.5 &#181;g/mL of DAPI in PBS). Then nutate 10 min. 
	CAUTION: DAPI has carcinogenic potential. Wear protective gloves and clothing to prevent skin contact.</li>
	<li>Remove DAPI solution and wash the tissues with 1 mL of PBT two times for 15 min each.</li>
	<li>Rinse once in 1 mL of PBS for 10 min at room temperature.</li>
	<li>Remove PBS and add 500 &#181;L of 100 % glycerol as the pre-mounting medium.</li>
</ol>
4. Mounting onto Microscope Slides
<ol>
	<li>Place the stained tissues on a microscope slide using a 2 mL plastic transfer pipette.</li>
	<li>Transfer the tissues to drops of mounting medium on another microscope slide with forceps.</li>
	<li>Hold down the end of dissected tissue with one forceps and pull away brain and eye antennal discs with the other forceps. 
	NOTE: Keep the dissected brains to place them near the wing imaginal discs. The brains act as a platform preventing the coverslip from crushing the wing imaginal discs.</li>
	<li>To isolate the wing imaginal discs hold down the end of dissected tissue with one forceps and gently scratch the body wall and tear off the discs with the other forceps. 
	NOTE: If it is difficult to find wing imaginal discs, peel the trachea from the posterior to the anterior side. Wing imaginal discs stick to the trachea.</li>
	<li>Gently cover the imaginal discs with a coverslip and seal with nail polish; store at 4 &#176;C.</li>
</ol>
5. Confocal Microscopy
<ol>
	<li>To acquire confocal images using a confocal microscope set image acquisition parameters including range of emission wavelength, laser intensity, gain, offset, scanning speed and image size 24. 
	NOTE: For detailed procedure of image acquisition settings, refer to the instruction manual supplied by each microscope manufacturer.</li>
	<li>To capture single confocal sections of an entire wing imaginal disc, use a 20X objective lens.</li>
	<li>Acquire 3-dimensional images by z-stack scanning at step-size of 0.5 - 1.0 &#181;m using 40X or 60X lenses. 
	NOTE: To analyze cellular phenotypes in high resolution, an image size should be larger than 512 x 512 pixels.</li>
</ol>
6. Image Analysis Using ImageJ
<ol>
	<li>Use Fiji, an open-source ImageJ software focused on biological-image analysis (https://fiji.sc/) 25, to acquire and analyze confocal z-stack images.</li>
	<li>To acquire vertical sections, open the z-stack images in ImageJ and select the menu item &#34;Image/Stacks/Reslice.&#34; 
	NOTE: Either X-Z axis or Y-Z axis are selectable in the Reslice menu. Vertical section can be obtained also in an arbitrary direction by drawing a straight line or rectangle onto the z-stack image.</li>
</ol>
7. Diagnosis of Neoplastic Phenotypes
<ol>
	<li>Open the vertical sections (X-Z or Y-Z axis) obtained from one set of z-stack images and analyze morphological phenotypes and antibody staining.</li>
	<li>For the diagnosis of tumor phenotypes, mainly focus on the following three points: (1) if a cell mass, including knockdown clones, deviates from the main epithelial layer, (2) if the subcellular localization of junctional proteins is altered in this cell mass, and (3) if the diameter of this cell mass is larger than 4 cells.</li>
	<li>Categorize tumor phenotypes according to the flowchart described in Figure 1.</li>
</ol>

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The authors have nothing to disclose.

The GAL4-UAS system is one of the most powerful genetic tools for targeted gene expression in Drosophila 26 and greatly facilitates tumor cell induction and analysis in vivo 4. This system enables the generation of clones bearing knockdown of tumor-suppressor genes or overexpression of oncogenes within wild-type epithelial tissue, a situation highly similar to the initial stages of human cancer where transformed pro-tumor cells are surrounded by normal epi...

Epithelial tissues are highly organized systems that have the remarkable homeostatic ability to maintain their organization through development and cell turnover. This robust self-organizing system, however, is progressively disrupted during tumor development. At the beginning of tumor development, individual mutant cells arising from oncogene activation or tumor-suppressor gene inactivation emerge within an epithelial layer. When this transformed &#34;pro-tumor cell&#34; evades a suppressive environment, disrupts epithelial organization, and begins uncontrolled proliferation, tumorigenesis occurs 1. During the past few decades, outstanding technological advances in genetics and molecular biology have made remarkable progresses on cancer research. In particular, recent studies using the genetically mosaic analysis tools in Drosophila melanogaster, such as FLP-FRT (flippase recombinase/flippase recombinase target) mitotic recombination 2 and flip-out-GAL4-UAS (upstream activating sequence) systems 3, have greatly contributed to better understanding the genetic mechanisms involved in the formation and metastasis of tumors 4,5,6.
Studies of a group of conserved Drosophila tumor-suppressor genes, lethal giant larvae (lgl), discs large (dlg), and scribble (scrib), highlighted the critical relationship between loss of epithelial organization and tumor development, as these genes play key roles in regulation of apical-basal cell polarity and cell proliferation in epithelial tissues 7. While Drosophila imaginal discs are normally monolayered epithelia, homozygous mutations in any of these three genes cause cells to lose structure and polarity, fail to differentiate, overproliferate, and ultimately form multilayered amorphous masses that fuse with adjacent tissues 7. Similarly, disruption of these genes in mammals is involved in the development of malignant tumors 8,9. The neoplastic phenotypes exhibited by the mutant tissues have led to the classification of these three genes as conserved, neoplastic tumor-suppressor genes (nTSGs) 7,8. However, when homozygous nTSG mutant cells are sporadically generated in developing wild-type imaginal discs using FLP-FRT-mediated mitotic recombination, mutant cells are eliminated from the tissue through c-Jun N-terminal kinase (JNK)-dependent apoptosis 10,11,12,13,14, extrusion 15,16, or engulfment and phagocytosis by neighbors 17. In this genetically mosaic epithelia, apoptosis is mostly detected in nTSG mutant cells located at the clone boundary, suggesting that adjacent normal cells trigger the apoptosis of nTSG mutant cells 10,11,12,18. Recent studies in mammalian cells have confirmed that this cell competition-dependent elimination of pro-tumor cells is an evolutionarily conserved epithelial self-defense mechanism against cancer 19,20,21,22,23.
A recent study in Drosophila imaginal discs, however, showed that mosaic nTSG-knockdown clones induces neoplastic tumors in specific regions of wing imaginal discs 16. Initial tumor formation was always found in the peripheral &#34;hinge&#34; region and never observed in the central &#34;pouch&#34; region of the wing disc epithelium, suggesting that the tumorigenic potential of nTSG-knockdown cells depends on the local environment. The central pouch region functions as a &#34;tumor coldspot&#34; where pro-tumor cells do not show dysplastic overgrowth, whereas the peripheral hinge region behaves as a &#34;tumor hotspot&#34; 16. In &#34;coldspot&#34; pouch regions, nTSG-knockdown cells delaminate from the basal side and undergo apoptosis. In contrast, as &#34;hotspot&#34; hinge cells possess a network of robust cytoskeletal structures on their basal sides, nTSG-knockdown cells delaminate from the apical side of the epithelium and initiate tumorigenic overgrowth 16. Therefore, analysis of tumor phenotypes in imaginal discs requires careful consideration of the region-specific susceptibility to tumorigenic stimuli.
Here, we describe a protocol to induce neoplastic tumor formation in the Drosophila wing imaginal discs utilizing the GAL4-UAS-RNAi system by which nTSG-knockdown cells are generated in normal wing disc epithelia. Although these experimental systems are useful to study the early stages of cancer, a clear classification method to evaluate the stages of tumor progression in imaginal disc epithelia has not been clearly described before. Therefore, we also propose a diagnosis method to classify pro-tumor clonal phenotypes induced in the wing disc epithelia into three categories: hyperplasia (accumulation of an excessive number of normal-appearing cells with increased proliferation), dysplasia (premalignant tissue composed of abnormally appearing cells), and neoplasia (benign or malignant tumor composed of cells having an abnormal appearance and abnormal proliferation pattern).

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Wako</td>
			<td>162-19321</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Wako</td>
			<td>168-11805</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Wako</td>
			<td>064-00406</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>normal goat serum</td>
			<td>Sigma</td>
			<td>G6767</td>
			<td></td>
		</tr>
		<tr>
			<td>mounting medium, Vectashield</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse-anti-Dlg 4F3</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>4F3 anti-discs large, RRID:AB_528203</td>
			<td>dilute in PBTG, 1:40</td>
		</tr>
		<tr>
			<td>mouse-anti-MMP1</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>3A6B4, RRID:AB_579780</td>
			<td>3 mixed 1:1:1 and dilute in PBTG, 1:40</td>
		</tr>
		<tr>
			<td>mouse-anti-MMP1</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>3B8D12, RRID:AB_579781</td>
			<td>3 mixed 1:1:1 and dilute in PBTG, 1:40</td>
		</tr>
		<tr>
			<td>mouse-anti-MMP1</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>5H7B11, RRID:AB_579779</td>
			<td>3 mixed 1:1:1 and dilute in PBTG, 1:40</td>
		</tr>
		<tr>
			<td>mouse-anti-atubulin</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>AA4.3, RRID:AB_579793</td>
			<td>dilute in PBTG, 1:100</td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 Phalloidin</td>
			<td>Molecular probes</td>
			<td>A22283</td>
			<td>dilute in PBS, 1:40</td>
		</tr>
		<tr>
			<td>goat anti-mouse IgG antibody, Alexa Fluor 546</td>
			<td>Molecular probes</td>
			<td>A11030</td>
			<td>dilute in PBTG, 1:400</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Fly strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sd-Gal4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#8609</td>
			<td>recombined with UAS-EGFP</td>
		</tr>
		<tr>
			<td>upd-Gal4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#26796</td>
			<td>recombined with UAS-EGFP</td>
		</tr>
		<tr>
			<td>UAS-lgl-RNAi</td>
			<td>Vienna Drosophila RNAi center</td>
			<td>#51247</td>
			<td></td>
		</tr>
		<tr>
			<td>UAS-scrib-RNAi</td>
			<td>Vienna Drosophila RNAi center</td>
			<td>#105412</td>
			<td></td>
		</tr>
		<tr>
			<td>UAS-RasV12</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#64196</td>
			<td></td>
		</tr>
		<tr>
			<td>UAS-Yki3SA</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#28817</td>
			<td></td>
		</tr>
		<tr>
			<td>hsFLP</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#6</td>
			<td></td>
		</tr>
		<tr>
			<td>Act&#62;CD2&#62;GAL4 (flip-out GAL4)</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#4780</td>
			<td>recombined with UAS-EGFP</td>
		</tr>
		<tr>
			<td>UAS-EGFP</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#5428</td>
			<td>X chromosome</td>
		</tr>
		<tr>
			<td>UAS-EGFP</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#6658</td>
			<td>third chromosome</td>
		</tr>
		<tr>
			<td>UAS-Dicer2</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#24650</td>
			<td>second chromosome</td>
		</tr>
		<tr>
			<td>UAS-Dicer2</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#24651</td>
			<td>third chromosome</td>
		</tr>
		<tr>
			<td>vkg-GFP</td>
			<td>Morin et al. 2001</td>
			<td></td>
			<td>GFP protein trap</td>
		</tr>
	</tbody>
</table>

induction and diagnosis of tumors in drosophila imaginal disc epithelia

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue organization. Drosophila melanogaster has emerged as a popular experimental model system in cancer biology to study the genetic and cellular mechanisms of tumorigenesis. In particular, genetic tools for Drosophila imaginal discs (developing epithelia in larvae) enable the creation of transformed pro-tumor cells within a normal epithelial tissue, a situation similar to the initial stages of human cancer. A recent study of tumorigenesis in Drosophila wing imaginal discs, however, showed that tumor initiation depends on the tissue-intrinsic cytoarchitecture and the local microenvironment, suggesting that it is important to consider the region-specific susceptibility to tumorigenic stimuli in evaluating tumor phenotypes in imaginal discs. To facilitate phenotypic analysis of tumor progression in imaginal discs, here we describe a protocol for genetic experiments using the GAL4-UAS system to induce neoplastic tumors in wing imaginal discs. We further introduce a diagnosis method to classify the phenotypes of clonal lesions induced in imaginal epithelia, as a clear classification method to discriminate various stages of tumor progression (such as hyperplasia, dysplasia, or neoplasia) had not been described before. These methods might be broadly applicable to the clonal analysis of tumor phenotypes in various organs in Drosophila.

To demonstrate neoplastic tumor formation experimentally induced by RNAi-mediated nTSG-knockdown in Drosophila wing imaginal discs, three different GAL4 drivers were used to express UAS-RNAi for lgl or scrib: (1) sd-GAL4, which drives strong UAS expression in the wing pouch and mild expression in the hinge regions (Figure 2 and Figure 3A); (2) upd-GAL4, which drives i...

Watch this Scientific Journal Video about Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia at JoVE.com

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

Mosaic clone analysis in Drosophila imaginal disc epithelia is a powerful model system to study the genetic and cellular mechanisms of tumorigenesis. Here we describe a protocol to induce tumors in Drosophila wing imaginal discs using the GAL4-UAS system, and introduce a diagnosis method to classify the tumor phenotypes.

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue ...

induction-diagnosis-tumors-drosophila-imaginal-disc

Research

JoVE Journal

Cancer Research

14.8K Views.  SOKENDAI. Mosaic clone analysis in Drosophila imaginal disc epithelia is a powerful model system to study the genetic and cellular mechanisms of tumorigenesis. Here we describe a protocol to induce tumors in Drosophila wing imaginal discs using the GAL4-UAS system, and introduce a diagnosis method to classify the tumor phenotypes.

Video: Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

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 ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

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

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

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 microenvironment.

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties make it an excellent in vivo experimental model to study angiogenesis, tumor growth, drug delivery systems, or photodynamic diagnosis (PDD) and photodynamic therapy (PDT). At the same time, this model addresses the requirement for the replacement of experimental animals with a suitable alternative. Ex ovo cultivated embryo allows easy substance application, access, monitoring, and documentation. The most frequently used is chick CAM; however, this article describes the advantages of the Japanese quail CAM as a low-cost and high-throughput model. Another advantage is the shorter embryonic development, which allows higher experimental turnover. The suitability of quail CAM for PDD and PDT of cancer and microbial infections is explored here. As an example, the use of the photosensitizer hypericin in combination with lipoproteins or nanoparticles as a delivery system is described. The damage score from images in white light and changes in fluorescence intensity of the CAM tissue under violet light (405 nm) was determined, together with analysis of histological sections. The quail CAM clearly showed the effect of PDT on the vasculature and tissue. Moreover, changes like capillary hemorrhage, thrombosis, lysis of small vessels, and bleeding of larger vessels could be observed. Japanese quail CAM is a promising in vivo model for photodynamic diagnosis and therapy research, with applications in studies of tumor angiogenesis, as well as antivascular and antimicrobial therapy.

The chorioallantoic membrane (CAM) of the avian embryo is a very useful and applicable tool for various areas of research. A special ex ovo model of Japanese quail CAM is suitable for photodynamic treatment investigation.

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties ...

High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes significantly. Genetically engineered murine models that mimic ATC&#39;s progression may help researchers to study treatments for this disease. Crossing three different genotypes of mice, a TPO-cre/ERT2; BrafCA/wt; Trp53&#916;ex2-10/&#916;ex2-10&#160;transgenic ATC model was developed. The ATC murine model was induced by an intraperitoneal injection of tamoxifen with overexpression of BrafV600E and deletion of Trp53, and the tumors were generated within about 1 month. High-resolution ultrasound was applied to investigate the tumor initiation and progression, and the dynamic growth curve was obtained by measuring the tumor sizes. Compared to magnetic resonance imaging (MRI) and computed tomography scanning, ultrasound has advantages in observing the ATC murine model, such as being noninvasive, portable, in real-time, and without radiation exposure. High-resolution ultrasound is suitable for dynamic and multiple measurements. However, ultrasonographic examination of the thyroid in mice requires relevant anatomical knowledge and experience. This article provides a detailed procedure for utilizing high-resolution ultrasound to scan tumors in the transgenic ATC model. Meanwhile, ultrasonic parameter adjustment, ultrasound scanning skills, anesthesia and recovery of the animals, and other elements that need attention during the process are listed.

The present protocol describes high-frequency ultrasonography for visualizing the entire mouse thyroid gland and monitoring the growth of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes ...