Non-invasive intravital imaging of head and neck squamous cell carcinomas in live mice

Abstract

Head and neck squamous cell carcinoma is one of the most common cancers with a 50% 5-year survival rate. Understanding the mechanisms that control development, progression, and spreading of the tumor to distal sites is of paramount importance to develop effective therapies. Here, we describe a minimally invasive procedure, which enables performing intravital microscopy of the mouse tongue in models for oral cancer and provides structural and dynamic information of the tumors at cellular and subcellular level.

Figure 1. Schematics of the two main experimental models used to image tumor development in the mouse tongue

A. Xenograft model in immunocompromised mice. Human HNSCC cell lines are transfected with fluorescent probes and/or shRNA using lentiviral-based vectors and FACS sorting. Engineered cells are injected in the ventral anterior side of the tongue of immunocompromised mice (nude or SCID). Animals develop tumors within 2–3 weeks from the injection. Tumors are allowed to grow for a maximum period of 15 weeks. Metastatic cell lines invade the locoregional lymph-nodes in 5–10 weeks. B. Carcinogen model. 4NQO is administered to immunocompetent mice (e.g. C57B6 background) in the drinking water for 16 weeks. Cancerous lesions become visible on the surface of the tongue starting from 16–18 weeks. Tumors are allowed to grow for a maximum of 25–30 weeks.

Figure 2. Diagram of the tongue holder

A. The holder is composed of two plastic flaps connected by a flexible rubber hinge. The flaps have one hole each and the lower one has a grove that can accommodate a thin squared coverslip (18×30 mm). B. The tongue is pulled out of the mouth with toothless forceps and carefully sandwiched in between the two flaps, which are kept in place by a screw. C. The holder is secured to a pre-warmed metallic platform by a metallic pin, which is tightened by a screw. Finally, the platform is placed on the pre-warmed stage of an inverted microscope with the hole coinciding with the location of the objective. D. Overall view of the setup.

Figure 3. Low and high magnification imaging of the tongue using transgenic mice

K5-GFP/mTomato mice were anesthetized and the tongue placed in the holder, as described in the legend to Fig. 2. The tongue was imaged by MPM (excitation wavelength 930 nm, emission filters 505–560-nm for the GFP and the 590–650 nm for the td-Tomato) with a 10X (A) or a 40X (B) objective. A. Low magnification imaging of the whole tongue using 3D stitching. Scanning was initiated from one of the anterior corners of the tongue. 200 nm z-stack were automatically acquired following the path described in the left diagram. Left images show the maximal projection of an individual field of view acquired on the dorsal (upper panel) or the ventral (lower panel) side of the tongue. In the dorsal side filiform (arrowheads) and fungiform (arrows) are shown. In the ventral side and area with blood vessels are shown (arrows). Bar 300 μm. Right images show the overall reconstruction of the tongue highlighting the basal layer (green). Bar 1 mm. B. High magnification imaging of selected areas of the tongue. Left images show volume rendering of the dorsal (upper panel) or the ventral (lower panel) side of the tongue. Bar 100 μm. Right panels show side view of the volume rendering of the filiform (upper) and fungiform (lower) papillae. The GFP expressing cells are primarily localized in the basal layer (BL) and to a lesser extent to the body of the papillae (asterisks). Bars 100 μm.

Figure 4. Volume rendering of Xenograft in situ

A- OSCC3 cells were engineered to express GFP-Rab25 (green) or mCherry (red). Cells were mixed in a 1:1 ratio and injected in the tongue of a nude mouse. After 17 days (D17) the margin of the tumor was identified by epifluorescence and a z-scan (150 μm) was performed by using MPM (same parameters as legend to Fig. 2 and using a 25x water immersion objective). SHG signal was collected using a 400–480 nm emission filter. The two cell populations and the edge of the tumor are clearly distinguished in both the xy and xz view. After 2 days (D19), the tumor was imaged in the same location using the SHG signal as a reference point. As previously shown (4), a reduction in the number of cells lacking Rab25 was observed, indicating that this cell population has a higher metastatic potential. Bars 150 μm. B- Maximal projections of z-stacks imaged over 4 consecutive days (D23–D26). Upper panels show the SHG signal and lower panels show a comparison of the behavior of cells lacking Rab25 (red) or cells expressing GFP-Rab25 (green). Cell lacking Rab25 migrate away from the primary tumor (arrows), whereas cell expressing GFP-Rab25 do not. Bar 100 μm (adapted from [3])

Figure 5. Time-lapse analysis of the tumor

OSCC3 cells were engineered to express either GFP-Rab25 or GFP and injected into the tongue of nude mice. After 5 weeks, the edge of the tumor was imaged by MPM (930 nm excitation wavelength) in time-lapse modality using a 60X water immersion objective. The upper panels show a close-up of a single cell expressing GFP (broken line) and a series of still images extracted from the time-lapse series showing the formation of membrane protrusions from the plasma membrane (insets). The lower panels show that Rab25 is localized in a series of dynamic vesicles that undergo fusion over time (insets). Bars 5μm. Time min:sec.

Figure 6. Long term imaging of the carcinogen-induced tumors

A. K5-GFP/mTomato mice were treated for 16 weeks with 4NQO as described in the Methods. The dorsal sides of the tongues were imaged by MPM once a week for 7 weeks (D12 to D19), as described in legend to Fig. 3, and volume rendering were generated. B. Cells expressing the GFP are organized in large clusters, which represent an expansion of the basal layer (compare to untreated animals in Fig. 3) Bar 1 mm. Inset 1 and 2 show the slow changes in the size of two main clusters. Bars 500 μm. C. An area with high GFP intensity was image by using a 25X objective. A z-scan was performed in the enlarged area showing a significant enrichment in GFP expressing cells in one fungiform papillae as a consequence of the expansion of the basal layer. The right panel shows the maximal projection of the Z-scan. Bar 150 μm.