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

Summary

Cooperation between an activated oncogene like RAS^V12 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.

Abstract

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, Ras^V12 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.

Introduction

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 Ras^V12 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 3^rd 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.

Protocol

1. Induction of Benign Non-invasive Tumors

1. Use stocks listed in Table 2 for the induction of benign tumors.
2. Prepare a starter culture for the "tester" stock of the following genotype: y,w, ey-FLP1; Tub-Gal80,FRT40A; Act5C>y+>Gal4, UAS-GFP
3. Prepare a starter culture for the "tested" stock of the following genotype: w; FRT40A, UAS-Ras^V12/CyO
4. Collect ten female virgins from the "tester" stock and ten males from the "tested" stock.
5. Cross the males and females from the "tester" and the "tested" stock by placing them both in a vial with fly food.
6. Place the cross in a 25 °C incubator and allow the males and females to mate and lay eggs for 24-48 hr.
7. Check the vials for sufficient egg deposition and for the presence of first instar larvae making sure that the culture is not drying out.
   1. Add a few drops of autoclaved distilled water to the culture to keep it moist (if the culture is drying out).
   2. Continue to monitor the culture and repeat step 1.7.1 as needed.
8. Observe wandering third instar larvae under a fluorescence stereomicroscope as outlined in section 3.

2. Induction of Invasive Tumors

1. Use stocks listed in Table 2 for the induction of invasive tumors.
2. Use the starter culture from step 1.2 above of the following genotype: y,w, ey-FLP1; Tub-Gal80,FRT40A; Act5C>y+>Gal4, UAS-GFP
3. Prepare a starter culture for the "tested" stock of the following genotype: w; lgl⁴, FRT40A, UAS-Ras^V12/CyO
4. Collect ten female virgins from the "tester" stock and ten males from the "tested" stock.
5. Cross the males and females from the "tester" and the "tested" stock by placing them both in a vial with fly food.
6. Place the cross in a 25 °C incubator and allow the males and females to mate and lay eggs for 24-48 hr.
7. Check the vials for sufficient egg deposition and for the presence of first instar larvae making sure that the culture is not drying out.
   1. Add a few drops of autoclaved distilled water to the culture to keep it moist (if the culture is drying out).
   2. Continue to monitor the culture for dryness and repeat step 2.7.1 as needed.
8. Observe third instar larvae under a fluorescence stereomicroscope as outlined in section 3.

3. Observation of Benign and Invasive Tumors

1. Use third instar larvae for benign tumor isolation and visualization.
   1. 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.
   2. 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.
   3. Place the larvae in a Petri plate and leave at -20 °C freezer for 30 min to immobilize the larva.
      1. Alternative method to immobilize larvae using FLYNAP.
      2. 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.
   4. 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).
   5. The benign tumor bearing larvae will fluoresce green in the anterior region where the cephalic complex is present.
2. 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.
3. Follow steps 3.1.1 to 3.1.4 except instead of third instar larvae use day 10 larvae.
   1. The day 10 invasive tumor bearing larvae will fluoresce green in the anterior region where the cephalic complex is present.
4. If the fluorescent microscope is equipped with a digital camera then take pictures of the fluorescing benign and invasive tumor bearing larvae.

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.

1. Using a wet paint brush select third instar larvae (for benign tumors) and day 10 larvae (for invasive tumors) from the respective culture vials.
2. 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.
   1. Transfer clean larvae to a fresh well of the dissection dish containing cold 1X PBS.
   2. Check for presence of GFP fluorescence using a stereomicroscope capable of detecting fluorescence. Discard non-GFP bearing larvae.
3. 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).
4. Hold the larva down using a pair of forceps about 2/3rds from the anterior end.
   1. Use the other pair of forceps and smartly separate the posterior 1/3rd of the larva and discard it.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
   6. Use a pair of forceps to gently break the connections between the cephalic nerves and the larval epidermis.
   7. 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.
   8. 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.
   9. 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.

Results

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...

Discussion

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 ...

Materials

List of materials used in this article

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