Use of Precision-Cut Lung Slices as an Ex Vivo Tool for Evaluating Viruses and Viral Vectors for Gene and Oncolytic Therapy

Abstract

Organotypic slice cultures recapitulate many features of an intact organ, including cellular architecture, microenvironment, and polarity, making them an ideal tool for the ex vivo study of viruses and viral vectors. Here, we describe a procedure for generating precision-cut ovine and murine tissue slices from agarose-perfused normal and murine melanoma tumor-bearing lungs. Furthermore, we demonstrate that these precision-cut lung slices can be maintained up to 1 month and can be used for a range of applications, which include characterizing the tissue tropism of viruses that cannot be propagated in cell monolayers, evaluating the transducing properties of gene therapy vectors, and, finally, investigating the tumor specificity of oncolytic viruses. Our results suggest that ex vivo lung slices are an ideal platform for studying the tissue specificity and cancer cell selectivity of gene therapy vectors and oncolytic viruses prior to in vivo studies, providing justification for pre-clinical work.

Graphical abstract

Figure 1

Setup for the Perfusion of Sheep Lungs (A) Two styrofoam box lids are lined with paper towels and enclosed with plastic wrap prior to being positioned 90° to each other with the use of wooden skewers. Clamps (hemostats) are then attached to the trachea and used to position the lungs upright. Another set of hemostats is put through the clamps holding the lungs and adhered to the vertical styrofoam cover. The initial state of the sheep lungs is characterized by the lobes appearing deflated and folded onto themselves (A); however, upon perfusion (B), they no longer fold and appear engorged. (C) A section approximately 2 mm thick is generated using a scalpel and then (D) an 8 mm puncher is used to core the slice to generate 8 mm-wide 2 mm-thick cores. The cores may vary in perfusion level, as the lungs will be differentially perfused throughout. The colored bars under the lung cores denotes the difference in perfusion level that can be observed in one section, with the orange bar being the highest, leaving the core spongy, and the light blue bar showing a flat section, lacking perfusion. The appropriate lung cores (pink bar or second from the left) can then be moved onto a Petri dish and embedded within LMPa (E). (F) The use of antibiotics extends culturing of sheep lung slices. Greater survival was observed when lung slices were cultured over a period of 1 to 4 weeks throughout 6 months with or without antibiotics. Those maintained in media with antibiotics were also subjected to 5 washes prior to culturing. Error bars represent SEM.

Figure 2

Experimental Overview and Timeline for Generating Precision-Cut Lung Tissue Slices for Viral Transduction or Infection

Figure 3

Equipment Setup for Use of the Vibratome The vibratome contains two setting panels: (A) a blade settings panel on the left and (B) a tissue cutting settings panel on the right. The blade settings panel can adjust the frequency and speed of the setting, while the tissue cutting settings accounts for the thickness of the slice and the blade travel range along the buffer tray platform. The reservoir should be filled with ice throughout the procedure. (C) and (D) show representative tissue blocks for ovine and murine lung tissues, respectively.

Figure 4

Sheep Lung Slices Remain Viable for a Month (A) Sheep lung slices were cultured in lung slice maintenance medium-C (Table 1) and harvested after 1 and 4 weeks. At both time points, the lung slices were tested for viability using a live-dead viability stain, and the results were visualized using a Zeiss Leica inverted fluorescence microscope using the FITC (for live cells) and Texas Red (for dead cells) channels. Lung slices treated with 10% Triton X-100 and left in the fridge for 24 hr were used as positive controls for cell death. After 4 weeks, slices where there was more observable red than green throughout the entire tissue slice were deemed as dead. (B) Mouse lung slice viability was assessed by a metabolic assay. Resazurin was added to mouse lung slices in LSMM-B for 2 hours and then the fluorescent intensity of each sample was read by a plate reader (530/25 nm; 595/5 nm). Lung slices 2 more SD below the mean were excluded from experimentation (indicated by red arrows). Error bars represent SEM.

Figure 5

Ovine and Murine Lung Slices Can Be Transduced with Gene Therapy Vectors and Murine Tumor-Bearing Lung Tissue Slices Can Be Infected with Oncolytic Viruses VSV-G-LV-transduced ovine lung cells (A) and Ad-GFP-transduced mouse lung cells (B) can be seen as green fluorescent foci in the center and edges of the lung slices. Transduction was possible in standard cell culture medium supplemented with additional antibiotics (LSMM-A; Table 1). Migrant cells from the lung slices are also susceptible to transduction (A). Lung slices were imaged by fluorescence microscopy to detect infection with NDV-GFP or VACV-GFP 72 hr post-infection (C). B16-F10 tumors are outlined in white dotted lines in the BF images. GFP expression can be observed as punctate spots in and around the tumors. Uninfected lung slices show dark B16-F10 tumors (arrows). GFP puncta localized in and/or around the tumors can be identified in NDV-GFP- and VACV-GFP-treated slices.

Figure 6

Sheep and Murine Lung Slices Can Be Infected and Virus Production Can Be Detected Lung slices grown in LSMM-C were infected with JSRV and incubated for 4 weeks at 37°C. (A) Robust immunohistochemical staining of a lung slice infected with JSRV using a monoclonal antibody against the JSRV Env oncoprotein. An uninfected lung slice was used as a negative control and lacked staining. Tissue from the lung of an adult sheep suffering from ovine pulmonary adenocarcinoma was used as a positive control. (B) Reverse transcriptase (RT) activity in the supernatant of mock and JSRV-infected lung slices. Murine B16-F10 melanoma tumor-bearing lung slices grown in LSMM-B were infected with oncolytic Maraba virus (MG1-eGFP). (C) Top: uninfected lung slices show dark B16-F10 melanoma tumors (arrows). Bottom: GFP puncta localized around and within the tumor (arrows) indicates infection by MG1-eGFP. (D) MG1-eGFP virus is detectable in supernatant from infected lung slices. Viral titers (1 × 10⁵ PFU/mL) from infected lung slice supernatants (N = 6) were determined by TCID50 48 hr post-infection. Error bars represent SEM.