Longitudinal imaging of T cell-based immunotherapy with multi-spectral, multi-scale optoacoustic tomography

Abstract

Most imaging studies of immunotherapy have focused on tracking labeled T cell biodistribution in vivo for understanding trafficking and homing parameters and predicting therapeutic efficacy by the presence of transferred T cells at or in the tumour mass. Conversely, we investigate here a novel concept for longitudinally elucidating anatomical and pathophysiological changes of solid tumours after adoptive T cell transfer in a preclinical set up, using previously unexplored in-tandem macroscopic and mesoscopic optoacoustic (photoacoustic) imaging. We show non-invasive in vivo observations of vessel collapse during tumour rejection across entire tumours and observe for the first time longitudinal tumour rejection in a label-free manner based on optical absorption changes in the tumour mass due to cellular decline. We complement these observations with high resolution episcopic fluorescence imaging of T cell biodistribution using optimized T cell labeling based on two near-infrared dyes targeting the cell membrane and the cytoplasm. We discuss how optoacoustic macroscopy and mesoscopy offer unique contrast and immunotherapy insights, allowing label-free and longitudinal observations of tumour therapy. The results demonstrate optoacoustic imaging as an invaluable tool in understanding and optimizing T cell therapy.

Figure 1

Longitudinal multi-scale imaging of anatomical tumour characteristics during immunotherapy. (a) Anatomical whole-body optoacoustic tomography (left) and corresponding cryoslice image (right) with indication of prominent tissue markers. MSOT (b,c upper row) and RSOM images (b,c lower row) of one representative animal corresponding to the therapy (b) and the control group (c). The tumour area is marked by a rectangle. In the RSOM image large vessels are colour-coded in red, small vessels in green. Mice of both groups were imaged at day 3, day 5 and day 7 after T cell transfer. (d,e) Merged CD31 (red signal) and DAPI (blue signal) immunofluorescence of the tumours presented in (B) and (C). A lack of the vasculature in the core of the tumour due to cellular decline is visible in the therapy group (d). The vasculature in the control tumour is unaffected both in the core and the periphery (e). Scale bar (RSOM) = 1 mm, scale bar (MSOT) = 2.5 mm, scale bar (IF) = 0.5 mm.

Figure 2

MSOT imaging of tumour behaviour during T cell therapy. (a) Optoacoustic images at different excitation wavelengths (700 nm, left and 750 nm, right) reveal a gradient signal in animals of the therapy group. The spectrum of the gradient signal is shown in the graph. (b,c) Spectral detection of the gradient signal within tumours of 3 representative animals of the therapy (b) and the control group (c) at the last imaging time-point (up to day 9 after T cell transfer). The gradient signal (red colour) is consistently detected in the tumours (labeled) of all animals of the therapy (b) but not in the case of the control group (c). Scale bar (MSOT) = 2.5 mm.

Figure 3

Longitudinal assessment of tumour rejection using MSOT. (a,b) Merged MSOT images of the gradient signal indicating cellular (red colour-coding) overlaid onto the anatomical optoacoustic images. One representative animal of the therapy (a) and one of the control group (b) is shown. Animals were imaged every second day starting at day 3 after T cell transfer. (c) 3D maximum intensity projection rendering of the merged MSOT images for the whole tumour volume of the animal shown in (a). Scale bar (MSOT) = 2.5 mm. (d) Tumour volumes (in mm³, blue colour-coding) from animals of the therapy (left image) and the control group (right image). The gradient signal per tumour volume (in %) is illustrated in red. Values are given as boxplots (mean; whiskers: min to max). Day 3 was used as baseline. Statistically significant differences of the gradient signal per tumour volume within the therapy are depicted above the relative day (A,B,C). The statistically significant difference of the tumour volume within the control group is marked for day 9 (D). Statistically significant differences of the gradient signal per tumour volume between the two groups (therapy and control) are depicted in the therapy group for day 7 and day 9 (1,2) and for the tumour volume in the control group at day 9 (3). p ≤ 0.05.

Figure 4

Multimodal analysis of T cell-based immunotherapy. (a–d) MSOT images of animals from the therapy (a,b) and control group (c, d) at indicated time points. Red colour-coding corresponds to the gradient signal. (e-h) Episcopic fluorescence imaging of the mice presented in (a–d). Accumulation of double labeled T cells was found in tumours of both the therapy (e,f) and the control group (g,h). Within the therapy group, T cells are concentrated around the gradient signal. In the control group, T cells can also be found at the tumour edge and within the tumours. (i,j) Cryo RGB image correlating to the episcopic fluorescence images. In tumours of the therapy group (i,j), cellular decline is visible in central parts of the tumour. Tumours of control animals (k,l) do not present large areas of cell death. Scale bar (MSOT) = 2.5 mm.