We thank former lab members Dr. Sheng-An Yang and Mr. Juan-Martin Portilla for their contribution in developing this protocol. We are grateful for Dr. Yan Song&#39;s lab at Peking University School of Life Sciences for sharing their protocol on manual allotransplantation. We also thank Mr. Calder Ellsworth and Mr. Everest Shapiro for critical reading of the manuscript.
WMD received funding (GM072562, CA224381, CA227789) for this work from National Institute of Health (https://www.nih.gov/)&#160;and funding (IOS-155790) from the National Science Foundation (htps://nsf.gov/).&#160;The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1. Preparation of SG imaginal ring tumor
<ol>
	<li>Cross adult flies with genotypes of UAS-NICD (Male: 10-15 flies) and Act-Gal4, UAS-GFP/CyO; tub-Gal80ts (Virgin female: 10-15 flies) and allow them to breed for 1 day at 18 &#176;C. The selected adult flies should be 5-9 days old to ensure high fertility.</li>
	<li>Allow the adult flies to lay eggs in the fly food contained in vials for 24 h at 18 &#730;C, then remove the adult flies. 
	NOTE: Fly food is prepared using the standard cornmeal food recipe from Drosophila Stock Center8. Each vial should contain around 10mL of fly food.</li>
	<li>Allow the eggs to incubate for 6 days at 18 &#176;C. During this period, the larvae will hatch.</li>
	<li>Transfer the vials containing larvae to a 29 &#176;C incubator and incubate for another 7 days. 
	&#8203;NOTE: This incubation step is optional depending on the specific experimental design.</li>
</ol>
2. Preparation of adult wild type Drosophila for allotransplantation
<ol>
	<li>Anesthetize wild type or appropriate mutant adult flies&#160;with 100% CO2&#160;and sort flies based on sexes. Both male and female flies can be used as tumor hosts.</li>
	<li>Secure a 5 cm long piece of fly tape to a microscope slide with the sticky side up by fixing it with two smaller pieces of tape, one at each end.</li>
	<li>Immobilize the flies by adhering their wings to the tape. Use forceps while maneuvering the flies.
	<ol>
		<li>Repeat the above step for 60-80 adult flies used as allograft acceptors. 
		NOTE: It is best to organize flies into neat rows, with their body axis aligned parallel to each other for a more time-efficient injection process later. Figure 1 shows rows of host flies taped down in this manner.</li>
	</ol>
	</li>
</ol>
3. Assembly of the autoinjector apparatus
<ol>
	<li>Connect both the autoinjector apparatus and power cord to the controller box.
	<ol>
		<li>Set the injection volume to 59.8 nL. This will help maintain the appropriate amount of suction and injection forces during allotransplantation.</li>
	</ol>
	</li>
	<li>Place the&#160;controller box and the autoinjector on opposite sides of the light microscope. 
	NOTE: For right-handed operators, the control box should be placed on the left side of the microscope with the injector on the right side. Vice versa for left-handed operators.</li>
	<li>Prepare the 3.5&#39;&#39; glass capillary for use by clipping off the closed end with forceps.
	<ol>
		<li>Process one end of the glass capillary using a four-step micropipette puller and heat to a narrow, closed end using the following specifications in the instrument: Heat = 650, Force = 200, and Distance = 8. Neatly place the capillaries into the puller apparatus and run the program after inputting the above settings.</li>
		<li>Use forceps to clip the capillary at an approximately 60&#176; angle to make a sharper end for easy entry into the adult fly abdomen1. See Figure 2 for an example of a well-clipped capillary.</li>
	</ol>
	</li>
	<li>Lightly unscrew the cap of the injector. Hold down the Empty button to advance the injector needle until 70%-80% of its total length is showing. To accelerate capillary advancement, press the Fill button once while simultaneously holding down the Empty button.</li>
	<li>Use a syringe to fill the glass capillary with mineral oil. Then, carefully insert the glass capillary onto the injector needle until the former is firmly attached to the rubber stopper of the injector. Now screw the injector cap tight.
	<ol>
		<li>Wipe off the mineral oil residue on the external surface of the glass capillary cover to avoid contaminating the medium during allotransplantation.</li>
	</ol>
	</li>
</ol>
4. Dissection of the SG imaginal ring tumor
<ol>
	<li>Select one of the larvae and transfer it to a dissection plate filled with 100 &#181;L of Schneider&#39;s Medium to prepare for SG imaginal ring tumor dissection. 
	&#8203;NOTE: Only the specimens harboring the tumor will remain as larvae. This is because tumor growth delays larval development and progression9. Two thirds of the specimens will not harbor the tumor and will thus have progressed to pupae/adults.
	<ol>
		<li>For dissection and allotransplantation purposes, use a stereomicroscope with a 10x-20x magnification range.</li>
	</ol>
	</li>
	<li>Using one pair of forceps to hold the mid-section of the larval body, pinch the larval head using another pair of forceps and apply a stretching force lengthwise.</li>
	<li>Locate the Y-shaped SG of the larvae and isolate it from the rest of the larval tissue10.</li>
	<li>Dissect and isolate the SG imaginal ring tumor by removing the adjacent tissue. See Figure 3 for a depiction of this dissection process.</li>
	<li>Repeat steps 4.1-4.4 for an additional 10 to 20 SG imaginal ring tumors based on research needs.</li>
</ol>
5. Allograft of primary SG imaginal ring tumor
<ol>
	<li>Submerge the capillary into the Schneider&#39;s Medium containing the primary SG imaginal ring tumors. Hold the Fill button to fill the glass capillary with Schneider&#39;s Medium all the way to the top 0.5 cm segment. This top segment should remain filled with mineral oil. 
	NOTE: Accelerate this process by pressing the Empty button once while simultaneously holding down the Fill button.</li>
	<li>Locate a primary tumor and press the Fill button until the tumor is suctioned into the capillary.
	<ol>
		<li>Ensure that the tumor sits at the tip of the capillary or several millimeters away from the tip of the capillary. This helps avoid the tumor drifting and becoming lost in the solution contained within the capillary. See Figure 4A for a demonstration of the appropriate tumor location as it sits in the capillary.</li>
	</ol>
	</li>
	<li>Locate an adult fly immobilized to the tape on the microscope slide. Using forceps, gently hold down the lower abdomen. Then, pierce the lower lateral cuticle of the abdomen with the capillary. Press the Empty button until the tumor enters the new host abdomen. See Figure 4B for a demonstration of this technique.</li>
	<li>Using forceps, gently pinch the wings of the host up to remove it from the tape. Place the host into a new vial with fresh food. It is best to place the vial sideways for the initial 24 h after injection. Each vial should only contain up to a maximum of 20 flies. 
	NOTE: Some host flies will have missing wings and other wounds on their bodies after injection and may stick to the fly food if the vial is placed upright.</li>
	<li>Repeat steps 5.2 through 5.4 to transplant the remaining primary tumors into their new adulthosts.</li>
	<li>Dispose capillaries into sharps&#39; container and clean the mineral oil residue from the exterior of the autoinjector before replacing the apparatus back into its box.</li>
	<li>Store the vial of hosts at room temperature for 1 day, then transfer the vial to an incubation chamber at 29 &#730;C. Transfer fly hosts to new vials every 2-3 days.</li>
	<li>Monitor the fly hosts daily and calculate survival rates. After a week, tumors should be visible under a stereo microscope with fluorescence adapter and can be continually monitored for their size and progression.</li>
</ol>
6. Re-allograft of transplanted tumors
<ol>
	<li>Around 10-14 days post-allograft, screen for tumors that have grown in the host abdomens using a fluorescence microscope.</li>
	<li>Anesthetize a host with CO2 and place it in a dissection plate filled with 100 &#956;L Schneider&#39;s Medium. Dissect the grown allografted tumor out of the host using two pairs of forceps.
	<ol>
		<li>Use one pair of forceps to hold down the abdomen and the other pair to incise open the abdominal cuticle, exposing the allografted tumor1.</li>
		<li>Carefully isolate the tumor from the attached host tissues as much as possible using fluorescence markers as guidance.</li>
	</ol>
	</li>
	<li>Repeat step 6.2 to prepare for two to three additional allografted tumors.</li>
	<li>Transfer the dissection plate containing the harvested tumors onto the stage of a light microscope.</li>
	<li>Use sterile needles and dissect the tumors into smaller pieces that are appropriate for the capillary size.</li>
	<li>Repeat steps 4.1-4.5 to prepare the new generation of adult hosts and repeat steps 5.1-5.7 to complete the allotransplantation of the tumors. 
	NOTE: Success rates are generally higher for non-primary tumors compared with those of primary tumors.</li>
	<li>Repeat steps 6.1-6.6 for every subsequent generation of flies used in the study. 
	NOTE: Choose Drosophila host lines appropriate for the experimental needs.</li>
</ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+Hes-centred+transcriptional+networks+in+neural+stem+cell+maintenance+and+tumorigenesis+in+Drosophilia.">Dissecting Hes-centred transcriptional networks in neural stem cell maintenance and tumorigenesis in Drosophilia.</a> Development. 147 (22), (2020).</li><li>Haller, S., Limmer, S., Ferrandon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pseudomonas Methods and Protocols. , 723-740 (2014).</li><li>Letini&#263;, B., Kemp, A., Christian, R., Koekemoer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inoculation+protocol+for+the+African+malaria+vector,+Anopheles+arabiensis,+by+means+of+nano-injection.">Inoculation protocol for the African malaria vector, Anopheles arabiensis, by means of nano-injection.</a> African Entomology. 26 (2), 422-428 (2018).</li><li>Mejia, M., Heghinian, M. D., Busch, A., Mar&#237;, F., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired+nanoinjection+and+electrophysiology+assay+to+screen+for+bioactivity+of+compounds+using+the+Drosophila+melanogaster+giant+fiber+system.">Paired nanoinjection and electrophysiology assay to screen for bioactivity of compounds using the Drosophila melanogaster giant fiber system.</a> Journal of Visualized Experiments: JoVE. (62), e3597 (2012).</li><li>Miles, W. O., Dyson, N. J., Walker, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tumor+invasion+and+metastasis+in+Drosophila.">Modeling tumor invasion and metastasis in Drosophila.</a> Disease Models &amp; Mechanisms. 4 (6), 753 (2011).</li><li>Yang, S. A., Portilla, J. M., Mihailovic, S., Huang, Y. C., Deng, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogenic+notch+triggers+neoplastic+tumorigenesis+in+a+transition-zone-like+tissue+microenvironment.">Oncogenic notch triggers neoplastic tumorigenesis in a transition-zone-like tissue microenvironment.</a> Developmental Cell. 49 (3), 461-472 (2019).</li><li>Bloomington Drosophila Stock Center. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> BDSC Cornmeal Food. , (2020).</li><li>Garelli, A., Gontijo, A. M., Miguela, V., Caparros, E., Dominguez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaginal+discs+secrete+insulin-like+peptide+8+to+mediate+plasticity+of+growth+and+maturation.">Imaginal discs secrete insulin-like peptide 8 to mediate plasticity of growth and maturation.</a> Science. 336 (6081), 579-582 (2012).</li><li>Kennison, J. 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(156), (2020).</li><li>Mirzoyan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster:+a+model+organism+to+study+cancer.">Drosophila melanogaster: a model organism to study cancer.</a> Frontiers in Genetics. 10, 51 (2019).</li><li>Bangi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+at+the+intersection+of+infection,+inflammation,+and+cancer.">Drosophila at the intersection of infection, inflammation, and cancer.</a> Frontiers in Cellular and Infection Microbiology. 3, 103 (2013).</li><li>Saavedra, P., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+tumor-induced+organ+wasting.">Drosophila as a model for tumor-induced organ wasting.</a> Advances in Experimental Medicine and Biology. 1167, 191-205 (2019).</li><li>Figueroa-Clarevega, A., Bilder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+drosophila+tumors+interrupt+insulin+signaling+to+induce+cachexia-like+wasting.">Malignant drosophila tumors interrupt insulin signaling to induce cachexia-like wasting.</a> Developmental Cell. 33 (1), 47-55 (2015).</li><li>Koyama, L. 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There are no conflicts of interest to declare among the authors.

Tumor allotransplantation can help researchers address certain problems that arise during Drosophila tumor growth and progression. One such challenge is the circumvention of premature deaths of tumor-bearing larvae or adults during primary tumor culture12. In this context, continued tumor allotransplantation allows tumors to grow indefinitely, which facilitates longitudinal studies of tumor growth, metastasis, and evolution. Tumor allotransplantation is also useful for assessing various a...

This protocol provides a step-by-step guidance for allotransplantation of Drosophila larval salivary gland (SG) imaginal ring tumors into abdomens of adult hosts using an auto-nanoliter injection apparatus (e.g., Nanoject). This protocol also provides directions for the subsequent re-allografting of tumors into new generations of adult hosts, which provides opportunities for continued longitudinal study of tumor characteristics, such as tumor evolution and tumor-host interactions. The protocol can also be applied toward drug screening experiments.
This method was developed to improve upon the efficacy of performing tumor allotransplantation in Drosophila using manual injectors1, which are often inconsistent in their suction and injection forces, leading to suboptimal results for tumor allotransplantation. An autoinjector apparatus provides better control and can result in lower rates of fly mortality post-allograft. A trained operator could achieve a host-survival rate of over 90% with the autoinjector, compared to around 80% when the manual injector was used1. The overall tumor acquisition rate is 60%-80% at day 8-12 post-allograft. The average injection time has also been improved from 30-40 s per fly using a manual injector to 20-25 s per fly using the autoinjector.
This protocol is among the first few protocols to use the autoinjector apparatus in Drosophila tumor allotransplantation. A recent study also used the autoinjector for allotransplantation of tumorous neural stem cells2. Previously, the autoinjector apparatus was used in Drosophila to study bacterial virulence3, parasitic infections and host defense4, as well as screening for bioactivity of different compounds5. Our protocol adapts the autoinjector apparatus for tumor injection use and seeks to provide Drosophila researchers with higher quality and more consistent results while saving them considerable time. This protocol can not only be used for the allotransplantation of tumors, but can also be tailored to the allotransplantation of wildtype and mutant tissues of similar caliber6.
The Drosophila NICD tumor used in this protocol was first introduced by Yang et al.7 in the SG imaginal ring transitional zone, a &#34;tumor hotspot&#34; that exhibits high levels of endogenous Janus Kinase/Signal Transducer and Activators of Transcription (JAK-STAT), and c-Jun N-terminal Kinase (JNK) activity. Additionally, the transition zone has high levels of matrix metalloproteinase-1 (MMP1)7, which makes this region particularly conducive to tumorigenesis. Notch pathway activation through NICD overexpression alone is sufficient to consistently initiate tumor formation. These tumors can be subsequently allotransplanted to allow investigation of a broad range of topics, including tumor cell division, invasion, and tumor-host interactions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Confocal Laser Scanning Microscope</td>
			<td>Zeiss</td>
			<td>LSM 980</td>
			<td>Also known as &#34;Zeiss LSM 980&#34;</td>
		</tr>
		<tr>
			<td>Cornmeal Fly Food</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>N/A</td>
			<td>Also known as &#34;BDSC Standard Cornmeal Food&#34;</td>
		</tr>
		<tr>
			<td>Dissection Needle (30Gx1/2)</td>
			<td>BD PrecisionGlide</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Plate</td>
			<td>Fisher Scientific</td>
			<td>12-565B</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly Tape</td>
			<td>Fisherbrand</td>
			<td>159015A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresence Adapter for Stero Microscope</td>
			<td>Electron Microscopy Sciences</td>
			<td>SFA-UV</td>
			<td>Also known as &#34;NightSea Fluorescence Adapter&#34;</td>
		</tr>
		<tr>
			<td>Fluoresence Microscope</td>
			<td>Zeiss</td>
			<td>495015-0001-000</td>
			<td>Also known as &#34;Zeiss Stereo Discovery.V8&#34;</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td>Also known as &#34;Dumont #5 Forceps&#34;&#160;</td>
		</tr>
		<tr>
			<td>Glass Capillary (3.5&#39;&#39;)</td>
			<td>Drummond</td>
			<td>3-000-203-G/X</td>
			<td></td>
		</tr>
		<tr>
			<td>Glue</td>
			<td>Elmer</td>
			<td>E305</td>
			<td>Also known as &#34;Elmer Washabale Clear Glue&#34;</td>
		</tr>
		<tr>
			<td>Light Microscope</td>
			<td>Zeiss</td>
			<td>435063-9010-100</td>
			<td>Also known as &#34;Zeiss Stemi 305&#34;</td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>World Precision Instruments</td>
			<td>PUL-1000</td>
			<td>Also known as &#34;Four Step Micropipette Puller&#34;</td>
		</tr>
		<tr>
			<td>Nanoject Apparatus</td>
			<td>Drummond</td>
			<td>3-000-204</td>
			<td>Also known as &#34;Nanoject II Auto-Nanoliter Injector&#34;</td>
		</tr>
		<tr>
			<td>Schneider&#39;s Medium</td>
			<td>ThermoFisher</td>
			<td>21720001</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (27G x1/2)</td>
			<td>BD PrecisionGlide</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>Vial</td>
			<td>Fisherbrand</td>
			<td>AS507</td>
			<td></td>
		</tr>
	</tbody>
</table>

tumor allotransplantation in drosophila melanogaster with a programmable auto-nanoliter injector

This protocol describes the allotransplantation of tumors in Drosophila melanogaster using an auto-nanoliter injection apparatus. With the use of an autoinjector apparatus, trained operators can achieve more efficient and consistent transplantation results compared to those obtained using a manual injector. Here, we cover topics in a chronological fashion: from the crossing of Drosophila lines, to the induction and dissection of the primary tumor, transplantation of the primary tumor into a new adult host and continued generational transplantation of the tumor for extended studies. As a demonstration, here we use Notch intracellular domain (NICD) overexpression induced salivary gland imaginal ring tumors for generational transplantation. These tumors can first be reliably induced in a transition-zone microenvironment within larval salivary gland imaginal rings, then allografted and cultured in vivo to study continued tumor growth, evolution, and metastasis. This allotransplantation method can be useful in potential drug screening programs, as well as for studying tumor-host interactions.

Here, we carried out generational allotransplantation of SG imaginal ring tumors using the nanoliter injection autoinjector apparatus and conducted subsequent tumor live-imaging with a confocal laser scanning microscope, which allowed for a deeper dive into topics of tumor growth, tumor cell migration, and tumor-host interactions. When mounting flies, glue them to a microscope slide and restrain them via a polydimethylsiloxane (PDMS) block11.
Figure...

Watch this Scientific Journal Video about Tumor Allotransplantation in Drosophila melanogaster with a Programmable Auto-Nanoliter Injector at JoVE.com

Tumor Allotransplantation in Drosophila melanogaster with a Programmable Auto-Nanoliter Injector

This protocol provides detailed guidance for the initial and continued generational allotransplantation of Drosophila tumors into the abdomen of adult hosts for studying various aspects of neoplasia. Using an autoinjector apparatus, researchers can achieve improved efficiency and tumor yields compared to those achieved by traditional, manual methods.

Tumor Allotransplantation in Drosophila melanogaster with a Programmable Auto-Nanoliter Injector

This protocol describes the allotransplantation of tumors in Drosophila melanogaster using an auto-nanoliter injection apparatus. With the use of an ...

tumor-allotransplantation-drosophila-melanogaster-with-programmable

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Cancer Research

2.3K Views.  Tulane University School of Medicine. This protocol provides detailed guidance for the initial and continued generational allotransplantation of Drosophila tumors into the abdomen of adult hosts for studying various aspects of neoplasia. Using an autoinjector apparatus, researchers can achieve improved efficiency and tumor yields compared to those achieved by traditional, manual methods.

Video: Tumor Allotransplantation in Drosophila melanogaster with a Programmable Auto-Nanoliter Injector

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 ...

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

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

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

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

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

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

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

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 biology.

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

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

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

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

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

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

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of brain tumors, the molecular selectivity of BBB also shields the neoplastic cells by blocking the delivery of peripherally administered chemotherapies. The development of novel drugs (including nanoparticles) targeting malignant brain tumors ideally requires the use of preclinical animal models to study the drug&#8217;s transcytosis and antitumor efficacy. In order to comply with the 3R principle (refine, reduce, and replace) to reduce the number of laboratory animals in experimental setup and perform the high-throughput screening of a large library of antitumor agents, we developed a reproducible in vitro human and murine mimic of the blood-brain tumor-barrier (BBTB) using three-layered cultures of endothelial cells, astrocytes, and patient-derived glioblastoma spheres. For higher scalability and reproducibility, commercial cell lines or immortalized cells have been used in tailored conditions to allow the formation of a barrier resembling the actual BBB. Here we describe a protocol to obtain a BBTB mimic by culturing endothelial cells in contact with astrocytes at specific cell densities on inserts. This BBTB mimic can be used, for instance, for the quantification and confocal imaging of the nanoparticle passage through the endothelial and astrocytic barriers, in addition to the evaluation of the tumor cell targeting within the same assay. Moreover, we show that the obtained data can be used to predict the behavior of nanoparticles in preclinical animal models. In a broader perspective, this in vitro model could be adapted to other neurodegenerative diseases for the determination of the passage of new therapeutic molecules through the BBB and/or be supplemented with brain organoids to directly evaluate the efficacy of drugs.

Drug targeting to central nervous system tumors is a major challenge. Here we describe a protocol to produce an in vitro mimic of the blood-brain tumor-barrier using murine and/or human cells and discuss their relevance for the predictability of central nervous system tumor targeting in vivo.

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Tumor Allotransplantation in Drosophila melanogaster with a Programmable Auto-Nanoliter Injector

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Chapters in this video

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

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

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

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

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

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish

In Vitro Assay to Study Tumor-macrophage Interaction