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 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 "tumor hotspot" 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.
1. Preparation of SG imaginal ring tumor
2. Preparation of adult wild type Drosophila for allotransplantation
3. Assembly of the autoinjector apparatus
4. Dissection of the SG imaginal ring tumor
5. Allograft of primary SG imaginal ring tumor
6. Re-allograft of transplanted tumors
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 5A features a live imaging capture of a 1st generation (G1) SG imaginal ring tumor growing in an adult host abdomen on day 10 post-allotransplantation. This level of imaging can be used to track the process of tumor division. Figure 5B depicts a 6th generation (G6) SG imaginal ring tumor occupying a large portion of the host abdomen on day 10 post-allotransplantation. Imaging at this stage may help reveal tumor growth patterns, as well as its migration and invasion behaviors. It is important to note that even though this image was captured using a confocal laser scanning microscope, a stereomicroscope with a GFP fluorescence adapter could also be used at 2x to 5x magnification, depending on the tumor size.
Figure 1: Host flies taped and secured for allotransplantation. The host flies are taped down by their wings and oriented neatly to prepare for the subsequent transplantation procedure. Please click here to view a larger version of this figure.
Figure 2: A well-clipped injection capillary. The red arrow points to a sharp edge needed to effectively pierce the abdominal cuticle of adult Drosophila hosts. Please click here to view a larger version of this figure.
Figure 3: Dissection of the primary salivary gland ImR tumor. The process of dissecting and isolating two primary salivary gland ImR tumors is demonstrated chronologically from Panel (A) to Panel (B), using two separate incisions. Panel (A) shows the salivary gland before tumor dissection. The red arrowheads indicate the first incision points. The blue arrowheads indicate the second incision points. The tumor lies between the red and blue arrowheads. Panel (B) shows the isolated ImR tumor after the two incisions are made to separate it from normal salivary gland tissue. Please click here to view a larger version of this figure.
Figure 4: Appropriate tumor location within the capillary and injection of tumor into Drosophila host abdomen. Panel (A) shows the most appropriate tumor location within the capillary. The tumor expresses eGFP (488 nm). Panel (B) shows the injection process. The red arrow indicates the tumor injection site. The blue arrow shows the placement of forceps to help hold down the terminalia of the fly for easier injection. Please click here to view a larger version of this figure.
Figure 5: A G1 and G6 ImR tumor seen in WT Drosophila host abdomen on day 10 post-allotransplantation. These are ventral views of the fly abdomen with the transplanted tumors in green. Panel (A) shows a G1 tumor on day 10 post-allotransplantation expressing eGFP (488 nm). Panel (A) is captured using a confocal microscope using a 20x lens with 0.8 NA, and 3x zoom. Panel (B) shows a G6 tumor on day 10 post-allotransplantation expressing eGFP (488 nm). Panel (B) is captured using a confocal microscope using a 5x lens with 0.25 NA, and 1x scan zoom. Please click here to view a larger version of this figure.
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 aspects of host-tumor interactions7,13. Host genotypes can be manipulated prior to tumor allograft to allow for evaluation of the host effect on tumor growth and migration, and on tumor-induced cachexia14,15. Fly hosts with different genotypes may exhibit distinct manifestations of cachexia-like wasting in response to the same tumor. Post-allotransplantation, the tumor hosts can be mounted in preparation for in vivo imaging using a protocol adapted from Koyama et al. and Ji et al.11,16.
The application of the autoinjector apparatus toward Drosophila tumor allotransplantation provides a convenient and straightforward protocol that possesses enhanced efficiency. As compared to the manual injector1, this method allows for reproducible and large-scale allotransplantations, which can expedite and standardize tumor behavior studies and drug screening procedures. This improved method produces impressive host survival and tumor yield rates. A trained researcher can achieve post-allograft host survival rates of >90%. Tumor yield rates can differ depending on whether the tumor is primary or re-allografted. Researchers can expect to achieve tumor yield rates of >50% for primary tumors and >70% for re-allografted tumors. In addition, this method reduces injection time per host fly by nearly 50% compared to the manual injector method.
This procedure has its limitations, however, mainly due to inconsistency of tumor incision, injection location and wound size. If the primary tumors are not cut into uniformly sized fragments prior to allotransplantation, certain fly hosts can receive larger fragments than others. This is a confounding factor affecting studies that aim to track the rate of tumor growth. This can potentially be mitigated by measuring the differential rate of tumor growth at two-day intervals post-allograft. In addition, during the injection, the operator should choose a consistent site in the abdominal cuticle across all host flies. This helps mitigate another confounding variable that may affect fly host survival and the final location of tumor attachment.
The authors have nothing to disclose.
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 analysis, decision to publish, or preparation of the manuscript.
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 |