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 cor...

<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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+larval+salivary+glands+and+polytene+chromosome+preparation.">Dissection of larval salivary glands and polytene chromosome preparation.</a> CSH Protocols. 2008, (2008).</li><li>Ji, H., Han, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LarvaSPA,+a+method+for+mounting+drosophila+larva+for+long-term+time-lapse+imaging.">LarvaSPA, a method for mounting drosophila larva for long-term time-lapse imaging.</a> Journal of Visualized Experiments: JoVE. (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 ...

This protocol is among the first to describe the use of an autoinjector apparatus in Drosophila tumor allotransplantation. A topic that has grown in popularity in recent years. The main advantage of this method is that it increases the efficiency of cancer research in Drosophila by improving the speed, consistency and yield of tumor allotransplantation.Tumor allotransplantation in Drosophila is a delicate technique that can be difficult to master. Be patient and trust that practice makes perfect. After crossing the flies and incubating the eggs for six days at 18 degrees Celsius, transfer the vials containing the hatched larvae to an incubator set at 29 degrees Celsius.Sort the anesthetized wild type or mutant adult flies based on their sexes. Secure a five centimeter long piece of fly tape to a microscope slide with the sticky side up. Immobilize the flies by adhering their wings to the tape with forceps.Place the controller box and the autoinjector on opposite sides of the light microscope. Process one end of the glass capillary using a four-step micro pipette puller, heating it to a narrow closed end. Use forceps to clip the capillary at an approximately 60 degree angle to prepare a 3.5 inch glass capillary.Lightly unscrew the cap of the injector. Hold down the empty button to advance the injector needle until 70 to 80%of it's total length is showing. Use a syringe to fill the glass capillary with mineral oil.Select one of the larvae and transfer to a dissection plate filled with 100 microliters of Schneider's Medium to prepare for SG imaginal ring tumor dissection. Using one pair of forceps to hold the midsection of the larval body, pinch the larval head using another pair of forceps and apply a stretching force lengthwise. Locate and isolate the Y shaped SG of the larvae, then dissect the SG imaginal ring tumor by removing the adjacent tissue.Fill the glass capillary with Schneider's Medium all the way to the top 0.5 centimeter segment by holding the fill button. Locate a primary tumor and press the fill button until the tumor is suctioned into the capillary. Ensure that the tumor sits at the tip of the capillary or several millimeters away from the tip of the capillary.Locate an adult fly immobilized to the tape. Then use forceps to gently hold down the lower abdomen and pierce the lower lateral cuticle of the abdomen with the capillary. Press the empty button until the tumor enters the new host abdomen.Using forceps, gently pinch the wings of the host up to remove it from the tape. Then place it into a new vial with fresh food. Screen for tumors that have grown in the host abdomens using a fluorescence microscope around 10 to 14 days post allograph and dissect the grown allografted tumor out of the host using two pairs of forceps.Use one pair of forceps to hold down the abdomen and the other pair to incise open the abdominal cuticle, exposing the allografted tumor. Carefully isolate the tumor from the attached host tissues. Use sterile needles and dissect the tumors into smaller pieces that are appropriate for the capillary size.Repeat this process for a new generation of adult hosts to complete the allotransplantation of the tumor. The process of tumor development was tracked by live imaging of a first generation SG imaginal ring tumor growing in an adult host abdomen on day 10, post-allotransplantation. A sixth generation SG imaginal ring tumor occupying a large portion of the host abdomen on day 10, post-allotransplantation is shown here.Imaging at this stage may help reveal tumor growth patterns as well as its migration and invasion behaviors. Tumor transplantation is a great technique for longitudinal studies for tumor progression, evolution and a metastasis. Results of great importance, for studying tumor host interactions.This proto has great potential in cancer drug screens using the drugs Temodal. Using this technique, researchers will be able to save significant time and effort in the investigation.

tumor-allotransplantation-drosophila-melanogaster-with-programmable

Research

JoVE Journal

Cancer Research

2.2K visualizações.  Tulane University School of Medicine. Este protocolo está entre os primeiros a descrever o uso de um aparelho autoinjetor na aotransplantação do tumor Drosophila. Um tema que cresceu em popularidade nos últimos anos. A principal vantagem desse método é que ele aumenta a eficiência da pesquisa sobre câncer em Drosophila, melhorando a velocidade, consistência e rendimento da aotransplantação tumoral.A aotransplantação tumoral em Drosophila é uma técnica delicada que pode ser difícil de dominar. Seja paciente ...