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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Cancer Research

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

Published: February 2nd, 2021

DOI:

10.3791/62229

1Department of Biochemistry and Molecular Biology, Tulane University School of Medicine
* These authors contributed equally

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.

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.

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 allotransp....

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1. Preparation of SG imaginal ring tumor

  1. 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 °C. The selected adult flies should be 5-9 days old to ensure high fertility.
  2. Allow the adult flies to lay eggs in the fly food contained in vials for 24 h at 18 ˚C, then remove the adult flies.
    NOTE: Fly food is prepared using the standard cor.......

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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.......

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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.......

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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'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/) and funding (IOS-155790) from the National Science Foundation (htps://nsf.gov/). The funders had no role in study design, data collection and a....

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

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