Name: A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster (Video) | JoVE Journal
Uploaded: 2013-09-11T15:15:00.000+00:00
Duration: 7 min 23 s
Description: Learn to induce and visualize benign and invasive tumors in Drosophila melanogaster using larval cephalic complex dissection. This protocol enables study of oncogene and cell polarity interactions in cancer biology.

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5 minutes

Research in my laboratory is supported by the WKU Department of Biology startup funds, WKU Research Foundation RCAP-I grant # 11-8032 and by a KBRIN-AREA grant funded through a parent grant from the National Institute of General Medical Sciences of the National Institutes of Health under award number 5P20GM103436-13. I would also like to acknowledge Dr. Tian Xu in whose laboratory I was introduced to this technique and Dr. Raymond Pagliarini who first established this technique in the Xu laboratory.

1. Induction of Benign Non-invasive Tumors
<ol>
	<li>Use stocks listed in Table 2 for the induction of benign tumors.</li>
	<li>Prepare a starter culture for the "tester" stock of the following genotype: y,w, ey-FLP1; Tub-Gal80,FRT40A; Act5C&gt;y+&gt;Gal4, UAS-GFP</li>
	<li>Prepare a starter culture for the "tested" stock of the following genotype: w; FRT40A, UAS-RasV12/CyO</li>
	<li>Collect ten female virgins from the "tester" stock and ten males from the "tested" stock.</li>
	<li>Cross the males and females from the "tester" and the "tested" stock by placing them both in a vial with fly food.</li>
	<li>Place the cross in a 25 &deg;C incubator and allow the males and females to mate and lay eggs for 24-48 hr.</li>
	<li>Check the vials for sufficient egg deposition and for the presence of first instar larvae making sure that the culture is not drying out.
	<ol>
		<li>Add a few drops of autoclaved distilled water to the culture to keep it moist (if the culture is drying out).</li>
		<li>Continue to monitor the culture and repeat step 1.7.1 as needed.</li>
	</ol>
	</li>
	<li>Observe wandering third instar larvae under a fluorescence stereomicroscope as outlined in section 3.</li>
</ol>
2. Induction of Invasive Tumors
<ol>
	<li>Use stocks listed in Table 2 for the induction of invasive tumors.</li>
	<li>Use the starter culture from step 1.2 above of the following genotype: y,w, ey-FLP1; Tub-Gal80,FRT40A; Act5C&gt;y+&gt;Gal4, UAS-GFP</li>
	<li>Prepare a starter culture for the "tested" stock of the following genotype: w; lgl4, FRT40A, UAS-RasV12/CyO</li>
	<li>Collect ten female virgins from the "tester" stock and ten males from the "tested" stock.</li>
	<li>Cross the males and females from the "tester" and the "tested" stock by placing them both in a vial with fly food.</li>
	<li>Place the cross in a 25 &deg;C incubator and allow the males and females to mate and lay eggs for 24-48 hr.</li>
	<li>Check the vials for sufficient egg deposition and for the presence of first instar larvae making sure that the culture is not drying out.
	<ol>
		<li>Add a few drops of autoclaved distilled water to the culture to keep it moist (if the culture is drying out).</li>
		<li>Continue to monitor the culture for dryness and repeat step 2.7.1 as needed.</li>
	</ol>
	</li>
	<li>Observe third instar larvae under a fluorescence stereomicroscope as outlined in section 3.</li>
</ol>
3. Observation of Benign and Invasive Tumors
<ol>
	<li>Use third instar larvae for benign tumor isolation and visualization.
	<ol>
		<li>Wet a paint brush in distilled water, and using this paint brush scrape a few third instar larvae from the wall of the culture vial.</li>
		<li>Place larvae in a depression slide with 1X PBS and then using a wet paint brush scrape off any food material from the larval epidermis.</li>
		<li>Place the larvae in a Petri plate and leave at -20 &deg;C freezer for 30 min to immobilize the larva.
		<ol>
			<li>Alternative method to immobilize larvae using FLYNAP.</li>
			<li>Place a FLYNAP wand dipped in FLYNAP to the vial from 3.1.3.2 and after plugging the vial let plugged vial with larvae stand for 30 to 40 min.</li>
		</ol>
		</li>
		<li>Place the immobilized larva on a glass slide, add a drop of light halocarbon oil, cover with a cover glass and observe under a fluorescence stereomicroscope with the capability to visualize Green Fluorescent Protein (GFP).</li>
		<li>The benign tumor bearing larvae will fluoresce green in the anterior region where the cephalic complex is present.</li>
	</ol>
	</li>
	<li>Select day 10 larvae (after induction) from the culture set in step #2 above for observation of invasive tumors. Day 10 larvae are selected because invasive tumor bearing larvae have an extended larval stage and generally fail to pupariate.</li>
	<li>Follow steps 3.1.1 to 3.1.4 except instead of third instar larvae use day 10 larvae.
	<ol>
		<li>The day 10 invasive tumor bearing larvae will fluoresce green in the anterior region where the cephalic complex is present.</li>
	</ol>
	</li>
	<li>If the fluorescent microscope is equipped with a digital camera then take pictures of the fluorescing benign and invasive tumor bearing larvae.</li>
</ol>
4. Dissection of the Cephalic Complex and Further Observation of Benign and Invasive Tumors
The translucent epidermis of the larva makes it difficult to observe the extent of invasion of tumors. Thus the extent of invasion is better visualized by dissecting the cephalic complex out of the larva. The following steps should be utilized to dissect the cephalic complex.
<ol>
	<li>Using a wet paint brush select third instar larvae (for benign tumors) and day 10 larvae (for invasive tumors) from the respective culture vials.</li>
	<li>Place larvae in a well of a dissection dish containing cold 1X PBS. Use paint brush to scrape off any food particles from the larval epidermis.
	<ol>
		<li>Transfer clean larvae to a fresh well of the dissection dish containing cold 1X PBS.</li>
		<li>Check for presence of GFP fluorescence using a stereomicroscope capable of detecting fluorescence. Discard non-GFP bearing larvae.</li>
	</ol>
	</li>
	<li>Add 1.0ml of cold 1X PBS to a fresh well on the dissection dish and using a pair of forceps transfer a larva bearing either the benign or invasive tumors (the dissection protocol remains the same for these two types of tumors).</li>
	<li>Hold the larva down using a pair of forceps about 2/3rds from the anterior end.
	<ol>
		<li>Use the other pair of forceps and smartly separate the posterior 1/3rd of the larva and discard it.</li>
		<li>Let go of the anterior 2/3rds of the larva. As the pressure inside the larva is released the contents of the larva will ooze out of the body cavity.</li>
		<li>Use a pair of forceps to remove the gut, fat body and other inner contents of the larva that have oozed out of the body cavity.</li>
		<li>Use a pair of forceps to hold the mouth hook of the larva and push it into the larval epidermis and using the other pair of forceps invert the larva completely.</li>
		<li>Use the forceps to gently and carefully remove the fat body, salivary glands, gut, the wing disc complex and any tracheal tubes. The cephalic complex should now be visible attached to the mouth hook of the inverted larva. The brain hemispheres and the ventral nerve cord (VNC) would still be connected to the larval epidermis through the nerves emanating from the VNC.</li>
		<li>Use a pair of forceps to gently break the connections between the cephalic nerves and the larval epidermis.</li>
		<li>As the nerve connections between the VNC and the larval epidermis are broken and excess fat body and other tissue removed, the cephalic complex would become clearly visible and will be attached to the larval epidermis only at the mouth hooks.</li>
		<li>The cephalic complex can be left attached to the larval epidermis if the complex needs to be fixed for downstream applications like antibody staining or for later visualization.</li>
		<li>If no further downstream applications would be performed then the cephalic complex can be detached from the larval epidermis and placed in a drop of a glycerol based mounting media for further observation and analysis. Use VECTASHIELD as the mounting media.</li>
	</ol>
	</li>
</ol>

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I have nothing to disclose.

Cancer is a complex disease with a much better understanding today than in the past. However, much still needs to be learnt and explained before we have a complete picture of the underlying mechanisms. The simple protocol presented here makes it possible to genetically induce benign and invasive tumors in a whole organism and then study the biology associated with the progression of tumors in this model. Most of the existing techniques in Drosophila and other organisms either utilize a cell culture based system ...

Progression of tumors from a benign to a metastatic state is a step wise process that is characterized by evasion of protective mechanisms present in the body 8. For example tumor cells in the body must be able to evade apoptosis and the immune system, breakthrough the specialized extracellular matrix (ECM) called Basement Membrane, and overcome any social controls imposed by the surrounding cells 8. It is through a step wise progression that the cancer cells acquire the ability to migrate and colonize distant sites in a process called metastasis. Our understanding of how the tumor cell overcomes the barriers imposed by the body is still in its infancy, however, the emerging picture from research done thus far points to a repeated use of normal developmental processes and signaling pathways by the cancer cells 11-13.
The fruit fly Drosophila melanogaster has contributed tremendously to our understanding of normal development and disease through use of sophisticated genetic techniques developed over the past several decades 14-17. Using mutagenesis and overexpression tools we have arrived at a better understanding of various oncogenes and tumor suppressor genes 18-22. However, tumor metastasis is a result of cooperation between several genetic lesions that has been studied primarily in cell culture models 23,24 as well as various xenograft models 25-27. These models though powerful have their limitations as they do not mimic entirely the conditions found in a living organism. Furthermore, transgenic models available in mice are cumbersome and not conducive to genetic analysis of invasive behavior 28,29. Several studies have attempted to understand invasion of tumor cells in Drosophila 30,31. These techniques primarily utilize transplantation of primary tumors to hosts and then rely on tracking the transplanted tumors for invasion of neighboring tissues 32,33. A powerful technique called MARCM 10 was adapted by Pagliarini and Xu to model tumor invasion in Drosophila 9. This elegant genetic modeling of tumor invasion exploited the cooperation between an activated oncogene and the loss of cell polarity. The power of this modeling lies in the fact that the invasive tumors are created in an intact organism thus circumventing the need for transplantation of tissues. To bring about the oncogenic cooperation, an activated oncogene like RasV12 is expressed in clones of cells in the larval eye-antennal disc. As a result of the MARCM technique these clones are also marked with green fluorescent protein (GFP) for easy visualization and are made homozygous for cell polarity mutants like lethal giant larvae, scribbled, and discs large. The result is GFP tagged invasive tumors in the cephalic complex. In this report I demonstrate how to induce, and visualize these invasive tumors both in the context of an intact larvae and in dissected out cephalic complex. The tumor induction presented here utilizes reagents on the second chromosome of Drosophila. In Table 2, I provide a listing of stocks on X and 3rd chromosomes that can be utilized for the same purpose. I believe that this simplified protocol will make this technique readily accessible to researchers interested in understanding the molecular basis of tumor progression.

<table><tbody><tr><td>10X PBS (phosphate buffered saline) pH 7.2 stock solution</td><td>Invitrogen, Sigma Aldrich</td><td></td><td></td></tr><tr><td>Chilled 1X PBS pH7.2 working solution</td><td>Invitrogen, Sigma Aldrich</td><td></td><td>Make fresh and refrigerate, can be used up to a week</td></tr><tr><td>Flynap</td><td>Carolina Biologicals</td><td></td><td>Fly anesthesia needed to anesthetize larvae</td></tr><tr><td>Fixative</td><td>0.1M PIPES, pH 7.2, 4% Paraformaldehyde</td><td></td><td>Needed to fix the dissected cephalic complex</td></tr><tr><td>Ice Bucket</td><td>Several</td><td></td><td>Maintain solutions on ice. Also, dissect cephalic complex in chilled 1X PBS and then place on ice in an Eppendorf tube</td></tr><tr><td>1.7ml Eppendorf tube</td><td>Various</td><td></td><td></td></tr><tr><td>Glass slides, cover glass</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Vectashield Mounting Media or any other mounting media</td><td>Vector Laboratories</td><td></td><td></td></tr><tr><td>Halocarbon 200 or 700 Oil</td><td>Polysciences Inc. or Halocarbon.com</td><td></td><td>Halocarbon 200 is used to mount the larvae for visualization on a fluorescence stereoscope</td></tr><tr><td>Sally Hansen &quot;Hard as Nails&quot; nail polish</td><td>Can be found at any general merchandise store</td><td></td><td>Needed to seal the edges of Coverslip</td></tr><tr><td>A Leica MZ16.5 fluorescence stereomicroscope or any other fluorescence stereomicroscope</td><td>Leica and others</td><td></td><td>Needed to observe the GFP fluorescence in larvae</td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Pyrex 9 well spot plate or any other dissection dish</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Paint Brush</td><td>Can be found at any general merchandise store</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Table 1.&lt;/strong&gt; Materials needed to perform the experimental protocol presented in this article.</td></tr></tbody></table>

a protocol for genetic induction and visualization of benign and invasive tumors in cephalic complexes of drosophila melanogaster

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.

As a result of the protocol presented here the user will be able to induce benign tumors by overexpressing an activated oncogene in the larval eye antennal imaginal disc. The user will also be able to induce invasive tumors in the eye antennal disc by overexpressing an activated oncogene in clones of cells also mutant for a cell polarity gene. The tumors could be easily visualized with the help of a fluorescent stereomicroscope as "green fluorescent" tissue in the whole larvae or in cephalic complexes that have been diss...

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 ...

The overall goal of this procedure is to induce benign and invasive tumors in drosophila, and then visualize them by dissecting the larval cephalic complex. This begins with setting up across between the desired strains and isolating the larval progeny, bearing either the invasive or benign tumors. The next step is to dissect the selected larvae.To expose the cephalic complex by first cutting them two thirds of the way down, securing them, and then turning them inside out. Next fat body and gut tissues are cleaned off, and then any nerves still attached to the ventral nerve cord and the larval epidermis are also broken. The final step is to image the cephalic complex or fix it for downstream applications.Ultimately, results can show GFP tagged invasive or benign tumors through the use of fluorescence microscopy. This method can help answer some of the key questions in cancer biology. For example, you may want to understand which genes could inhibit progression of tumors in an intact organism.Once the required genetic stocks have been obtained, prepare a starter culture of the tester strain. Also prepare a starter culture for the tested stock. Cross 10 female virgins from the tester stock with 10 males from the tested stock.Allow the flies to mate and lay eggs for 24 to 48 hours at 25 degrees Celsius, and then transfer them to a fresh vial to produce more progeny. Monitor the cross vials for eggs and first instar larvae. Set up a new cross.If the collected vials show poor egg deposition. If the collected vials start drying out, add just three to four drops of autoclave distilled water to keep the culture moist. Continue monitoring the vials until wandering.Third instar larvae are present. Then proceed with monitoring them under a fluorescence stereo microscope. Using the same procedure, collect flies bearing invasive tumors using the listed stocks by culture day five or six, wandering third instar larvae will be available from the benign tumor cultures.However, they will not be available until day 10 from the invasive tumor cultures. To collect the third instar larvae, wet a fine paintbrush with distilled water and scrape them off the wall of the vial. Place the larvae into a depression slide with cold one XPBS, and then using a wet paint brush.Scrape off any food material from their epidermis. For a few minutes of immobilization, place the larva on the plate at minus 20 degrees Celsius for 30 minutes. For a longer immobilization time, place the larvae in a culture vial with only fly nap on a wand.Plug the culture vial and let it stand for 40 minutes. Next, transfer the immobilized larvae to a glass slide. Add a drop of light halo carb oil and cover the larvae with a cover glass.Now, observe the larvae under a fluorescent stereo microscope. For GFP expression, select the green fluorescent larvae and proceed with the dissection. Benign tumor bearing larvae will fluoresce green in the anterior region where the cephalic complex is present.Invasive tumors will also fluoresce green in the anterior region of the larvae at the cephalic complex. If possible, be sure to photo document the fluorescing tumors. So the translucent epidermis of the larva makes it difficult to observe the extent of migration of the tumors, and that is why we must dissect the tumors.Begin with using forceps to transfer a fresh larva with either tumor. Type to a well of a dissection dish containing one milliliter of cold one XPBS. Hold the larvae down using a pair of forceps at about two thirds the distance from the anterior end.Using another pair of forceps smartly separate the posterior third of the larvae and discard it. It then let go of the anterior two thirds. As the pressure inside the larvae is released, its contents will ooze out of the body cavity.Using forceps, remove the tissues that have oozed out of the body cavity. Next, hold the mouth hook of the larva and push it into the larva epidermis. Using the other pair of forceps completely invert the larvae.Now with the forceps, gently and carefully, remove the fat body salivary glands, gut, the wing disc complex and any tracheal tubes. The cephalic complex may now be visible attached to the mouth hook. The brain hemispheres and the VNC are still connected to the larval epidermis via nerves emanating from the VNC.With forceps, gently break these connections. Then remove any excess fat body or other tissues so that the cephalic complex becomes clearly visible. Attached to the larval epidermis at the mouth hooks.Leave the cephalic complex as it is for further processing prior to visualization. Otherwise, to directly visualize the cephalic complex, detach it from the epidermis and place it in a drop of glycerol based mounting media such as vector shield. Using the described protocol, tumors were induced in the eye.Anin disc, benign tumors remained localized to the eye anin disc where they are induced. Notice that the GFP labeled tissue has not migrated to contiguous organs like the ventral nerve cord in the cephalic complex bearing invasive tumors from a day 10 larva overgrowth of tumor tissue was noted. Also, as pointed out by the red arrow, the tumors invaded contiguous organs like the VNC and mouth hooks.After watching this video, you should have a good understanding of how to genetically induce benign and invasive tumors, and then visualize them by dissecting the cephalic complex intra ano gastro.

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A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster (Video) | JoVE Journal

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

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