Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.
Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.
1. Tissue processing
2. Assessment of tissue viability
3. Visualization of GFP transgene expression by florescence microscopy
4. Visualization of luciferase expression using an In Vivo imaging System (IVIS)
5. Assessing viral titers by plaque assay
6. Representative Results:
In order to accurately determine whether a surgically obtained normal/tumor tissue sample is or is not infectable with virus, one must first ensure that the tissue sample is at least viable. Fig 1A shows that viability can be assessed using a metabolic dye (Alamar blue) and that both normal and tumor tissue can remain metabolically active over a period of at least 72h. This suggests that tissues cultured ex vivo can support viral replication. A significant advantage of oncolytic viruses is that they can be engineered to express therapeutic or imaging transgenes. Fig 2D-E shows that GFP or Luciferase can be detected from tissues infected ex vivo, further supporting that these tissues are viable and also infectable. Although increased transgene expression is generally associated with viral replication, it does not necessarily equate to a productive viral life cycle that leads to self-amplification and spread, which is thought to be important for therapeutic activity. For this reason, it is necessary to determine whether more virus is produced than what was used to initially infect the tissue. Fig 2B shows that upon viral quantification by plaque assay, more virus is obtained after 72 hours infection then tissue collected immediately after infection. Overall, these data show that surgically excised tumor tissue can remain viable in cell culture for a period of at least 72 hours, during which time viral replication can be supported.
Figure 1. Overview of the tissue sampling / sectioning protocol. Tissue sample is processed by removing several 2 mm cores which are subsequently divided into four quarters which are randomly distributed into wells A1-A4. The color codes indicate the reagents used in each well. The grey-shaded squares within the wells represent each quarter from the 6 individual cores depicted. The tissue in well A1 is transferred to a new well (C1) with media after the initial alamar blue reading. A second reading is done at the end of the 72 hour incubation with viruses. Wells A4 and B4 are supplemented with Luciferin after the 72 hour incubation post-infection
Figure 2. Viability and infectability of patient tissue samples. A) Tissue viability as measured by alamar blue signal at 2 hours and 72 hours post collection. Y-axis represents the blank corrected alamar blue signal. B) Titer of vaccinia virus collected per gram of tissue samples 2 and 72 hours post-infection. C) Example of vaccinia virus plaques on U2OS cells following coomassie staining. D) Luminescence signal obtained from patient VV-luciferase-infected tissue imaged using IVIS. E) Fluorescence microscopy pictures of patient tissue sample infected with VV-GFP.
One of the critical steps in this protocol is obtaining fresh tissue sample. If the sample is put into cell culture following a long wait at the operating room in an inappropriate media (eg. PBS), this may compromise tissue viability and prevent infectability. Of note, normal tissue is inherently more prone to these effects than tumor tissues. Another critical point is how many cores are used to sample the tissue and the consistency of their size. Inconsistencies in size will lead to variability across patient samples since factors such as tissue hypoxia and infectectable surface will fluctuate depending on the size of the cores and core quarters. While this can be partially resolved by using tissue slicers, one advantage of the method presented here is that it is relatively easy, less prone to contamination, and widely applicable to a variety of tissue types, including soft or viscous tissues which are not easily amenable to tissue slicing. Notably, the number of cores can be increased in order to get better tissue representation. Also, the cores can be cut into more pieces (eg. 5-8) to accommodate other potential assays to be done in parallel such as DNA, RNA, or protein extraction. However, the number of pieces in which the cores can be cut to reproducible sizes will depend on the actual size of the cores, which can be modified by using different sized corers. Optionally, one way to help obtain reproducibly sized tissue pieces is to even out the length of the cores using a ruler prior to further subdividing them into smaller pieces. The protocol can naturally be modified to accommodate other viruses and we have found that tissues can be viable and support replication for up to 6 days. The protocol can further be extended to measurements of other virally-expressed transgenes including cytrokines. Following infection, tissues can also be embedded in paraffin for further sectioning and staining by immunohistochemical methods, which allows for further refinement of the tissue histology and how it relates to viral infection10-12.
The authors have nothing to disclose.
We would like to thank Dr. Hesham Abdelbary for providing human surgical specimens for the data presented in Fig 2.
Name of the reagent | Company | Catalogue number | Comments (optional) |
---|---|---|---|
High glucose DMEM | Hyclone | SH30243.01 | |
Fetal Bovine Serum | NorthBio Inc. | NBSF-701 | |
Amphothericin B solution | Sigma Aldrich | A2942 | Use at 0.1% |
Pennicilin-Streptomycin | Sigma Aldrich | P4333 | Use at 1% |
Alamar Blue | Invitrogen | DAL1025 | |
D-Luciferin potassium salt | Molecular Imaging Products | D-Luciferin potassium salt 1g | Resuspend in PBS at 10 mg/ml and filter on 0.22 μm filter |
MEM powder | Gibco | #41500018 | Make in half the suggested volume to make 2X MEM and filter on 0.22 μm filter prior to use |
Carboxymethyl cellulose (CMC) | Sigma Aldrich | C9481 | Make a 3% solution in deionized water and autoclave. Note that powder can take some time to resuspend |
Coomassie Brilliant Blue R | Sigma Aldrich | B7920 | |
2 mm Biopsy punch | Miltex | MX-33-31 | |
Double Edge Prep Blades | Personna Medical Care | 74-0002 | |
Fluorescence disection Microscope | Leica | model M205 FA | |
In vivo imaging system (IVIS) | Caliper Life Sciences | IVIS® 200 series | |
Tissue Tearor | Biospec products | model 985370-395 |