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>

<ol>
	<li>Rossi, F., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+tumor+growth+in+Drosophila+using+the+tissue+allograft+method.">Studying tumor growth in Drosophila using the tissue allograft method.</a> Nature Protocols. 10 (10), 1525-1534 (2015).</li><li>Magadi, S. 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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><tbody><tr><td>Confocal Laser Scanning Microscope</td><td>Zeiss</td><td>LSM 980</td><td>Also known as &quot;Zeiss LSM 980&quot;</td></tr><tr><td>Cornmeal Fly Food</td><td>Bloomington Drosophila Stock Center</td><td>N/A</td><td>Also known as &quot;BDSC Standard Cornmeal Food&quot;</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 &quot;NightSea Fluorescence Adapter&quot;</td></tr><tr><td>Fluoresence Microscope</td><td>Zeiss</td><td>495015-0001-000</td><td>Also known as &quot;Zeiss Stereo Discovery.V8&quot;</td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11251-10</td><td>Also known as &quot;Dumont #5 Forceps&quot;&amp;nbsp;</td></tr><tr><td>Glass Capillary (3.5&#039;&#039;)</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 &quot;Elmer Washabale Clear Glue&quot;</td></tr><tr><td>Light Microscope</td><td>Zeiss</td><td>435063-9010-100</td><td>Also known as &quot;Zeiss Stemi 305&quot;</td></tr><tr><td>Micropipette Puller</td><td>World Precision Instruments</td><td>PUL-1000</td><td>Also known as &quot;Four Step Micropipette Puller&quot;</td></tr><tr><td>Nanoject Apparatus</td><td>Drummond</td><td>3-000-204</td><td>Also known as &quot;Nanoject II Auto-Nanoliter Injector&quot;</td></tr><tr><td>Schneider&#039;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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JoVE Journal

Cancer Research

2.3K Views.  Tulane University School of Medicine. Dit protocol is een van de eerste om het gebruik van een auto-injectorapparaat bij Drosophila tumor allotransplantatie te beschrijven. Een onderwerp dat de laatste jaren in populariteit is gegroeid. Het belangrijkste voordeel van deze methode is dat het de efficiëntie van kankeronderzoek in Drosophila verhoogt door de snelheid, consistentie en opbrengst van tumor allotransplantatie te verbeteren.Tumor allotransplantatie in Drosophila is een delicate techniek die moeilijk onder de knie...

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 ...

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the tumor milieu can possess pro- or anti-tumor capacity. One of the important cytokines regulating neutrophil functions in a tumor context are type I interferons. In the presence of interferons, neutrophils gain anti-tumor properties, including cytotoxicity or stimulation of the immune system. Conversely, the absence of an interferon signaling results in prominent pro-tumor activity, characterized with strong stimulation of tumor angiogenesis. Recently, we could demonstrate that pro-angiogenic properties of neutrophils depend on the activation of nicotinamide phosphoribosyltransferase (NAMPT) signaling pathway in these cells. Inhibition of this pathway in tumor-associated neutrophils leads to their potent anti-angiogenic phenotype. Here, we demonstrate our newly established model allowing in vivo evaluation of tumorigenic potential of manipulated tumor-associated neutrophils (TANs). Shortly, pro-angiogenic tumor-associated neutrophils can be isolated from tumor-bearing interferon-deficient mice and repolarized into anti-angiogenic phenotype by blocking of NAMPT signaling. The angiogenic activity of these cells can be subsequently evaluated using an aortic ring assay. Anti-angiogenic TANs can be transferred into tumor-bearing wild type recipients and tumor growth should be monitored for 14 days. At day 14 mice are sacrificed, tumors removed and cut with their vascularization assessed. Overall, our protocol provides a novel tool to in vivo evaluate angiogenic capacity of primary cells, such as tumor-associated neutrophils, without a need to use artificial neutrophil cell line models. vc

Here, we show therapeutic potential of anti-angiogenic tumor-associated neutrophils after their transfer into tumor-bearing mice. This protocol can be used to manipulate neutrophil activity ex vivo and to subsequently evaluate their functionality in vivo&#160;in developing tumors. It is an appropriate model for studying potential neutrophil-based immunotherapies.

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

Neuroscience

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases 1-7. Progression of tumors from a benign to a metastatic state is a complex event 8 and has been modeled in Drosophila to help us better understand the genetic basis of this disease 9. Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system 10 and exploits the cooperation between an activated oncogene, RasV12 and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors 9. I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Cooperation between an activated oncogene like RASV12 and mutations in cell polarity genes like scribbled, result in tumor growth in Drosophila where tumor cells also display invasive behaviors. Here a simple protocol for the induction and observation of the benign and invasive tumors is presented.

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

Medicine

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

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

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

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

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

phiC31 Integrase-Mediated Embryo Transgenesis in Drosophila

Embryo Microinjection for Transgenesis in Drosophila

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope.&#160;Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and&#160;presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

Author Spotlight: Optimizing Embryo Microinjection for Transgenesis in Drosophila

This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents an optimized protocol for embryo microinjection,&#160;a crucial step for creating transgenic flies.

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the ...

Genetics

Utilizing Larval Zebrafish for Efficient In Vivo Tumor Studies

A Simple, Rapid, and Effective Method for Tumor Xenotransplantation Analysis in Transparent Zebrafish Embryos

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we present larval zebrafish as a transplant model that has numerous advantages over murine models, including ease of handling, low expense, and short experimental duration. Moreover, the absence of an adaptive immune system during larval stages obviates the need to generate and use immunodeficient strains. While established protocols for xenotransplantation in zebrafish embryos exist, we present here an improved method involving embryo staging for faster transfer, survival analysis, and the use of flow cytometry to assess disease burden. Embryos are staged to facilitate rapid cell injection into the yolk of the larvae and cell marking to monitor the consistency of the injected cell bolus. After injection, embryo survival analysis is assessed up to 7 days post injection (dpi). Finally, disease burden is also assessed by marking transferred cells with a fluorescent protein and analysis by flow cytometry. Flow cytometry is enabled by a standardized method of preparing cell suspensions from zebrafish embryos, which could also be used in establishing the primary culture of zebrafish cells. In summary, the procedure described here allows a more rapid assessment of the behavior of tumor cells in vivo with larger numbers of animals per study arm and in a more cost-effective manner.

Author Spotlight: Advancing Cancer Therapeutics Using Tumor Xenotransplantation in Zebrafish

We describe a protocol for xenotransplantation into the yolk of transparent zebrafish embryos that is optimized by a simple, rapid staging method. Post-injection analyses include survival and assessing the disease burden of xenotransplanted cells by flow cytometry.

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we ...

Iron Oxide Nanoparticles for Image-Guided Therapy Delivery in Murine Glioblastoma

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

Author Spotlight: Innovative Cancer Therapies with Iron Oxide Nanoparticles for Glioblastoma Treatment

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

Drosophila melanogaster Larva Injection Protocol

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly and raise ethical issues. Unconventional models are notoriously cheap, easy to handle and culture, and do not take much space. They are genetically amenable and possess complete genome sequences, and their use presents no ethical considerations. The fruit fly Drosophila melanogaster, for instance, has provided great insights into a variety of behavior, development, metabolism, and immunity research. More specifically, D. melanogaster adult flies and larvae possess several innate defense reactions that are shared with vertebrate animals. The mechanisms regulating immune responses have been mostly revealed through genetic and molecular studies in the D. melanogaster model. Here a novel larval injection technique is provided, which will further promote investigations of innate immune processes in D. melanogaster larvae and explore the pathogenesis of a wide range of microbial infections.

Drosophila melanogaster Larva Injection Protocol

Drosophila melanogaster adult flies have been extensively utilized as model organisms to investigate the molecular mechanisms underlying host antimicrobial innate immune responses and microbial infection strategies. To promote the D. melanogaster larva stage as an additional or alternative model system, a larval injection technique is described.

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly ...

Immunology and Infection

Subcutaneous Transplantation Assay: A Method to Transplant Melanoma Cells from a Donor to a Recipient Zebrafish Model

Source: Iyengar, S. et al. <a href="https://www.jove.com/v/50086"> Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model</a>. J. Vis. Exp.(2012)
This video describes the method for transplantation of tumor cells from the melanoma-bearing donor zebrafish model to an immunocompromised recipient Casper zebrafish. It helps to visualize tumor engraftment and proliferation in the recipient fish.

Source: Iyengar, S. et al.  Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model. J. Vis. Exp.(2012)
This video describes the ...

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

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