Proof of concept of a multimodal intravital molecular imaging system for tumour transpathology investigation

Background Transpathology highlights the interpretation of the underlying physiology behind molecular imaging. However, it remains challenging due to the discrepancies between in vivo and in vitro measurements and difficulties of precise co-registration between trans-scaled images. This study aims to develop a multimodal intravital molecular imaging (MIMI) system as a tool for in vivo tumour transpathology investigation. Methods The proposed MIMI system integrates high-resolution positron imaging, magnetic resonance imaging (MRI) and microscopic imaging on a dorsal skin window chamber on an athymic nude rat. The window chamber frame was designed to be compatible with multimodal imaging and its fiducial markers were customized for precise physical alignment among modalities. The co-registration accuracy was evaluated based on phantoms with thin catheters. For proof of concept, tumour models of the human colorectal adenocarcinoma cell line HT-29 were imaged. The tissue within the window chamber was sectioned, fixed and haematoxylin–eosin (HE) stained for comparison with multimodal in vivo imaging. Results The final MIMI system had a maximum field of view (FOV) of 18 mm × 18 mm. Using the fiducial markers and the tubing phantom, the co-registration errors are 0.18 ± 0.27 mm between MRI and positron imaging, 0.19 ± 0.22 mm between positron imaging and microscopic imaging and 0.15 ± 0.27 mm between MRI and microscopic imaging. A pilot test demonstrated that the MIMI system provides an integrative visualization of the tumour anatomy, vasculatures and metabolism of the in vivo tumour microenvironment, which was consistent with ex vivo pathology. Conclusions The established multimodal intravital imaging system provided a co-registered in vivo platform for trans-scale and transparent investigation of the underlying pathology behind imaging, which has the potential to enhance the translation of molecular imaging.

Proof of Concept of a Multimodal Intravital Molecular Imaging System for Tumour Transpathology Investigation

Background: Transpathology highlights the interpretation of underlying physiology behind molecular imaging. However, it remains challenging due to the discrepancies between in vivo and in vitro measurements and difficulties of precise co-registration between multi-scaled images. This study aims to develop a multimodal intravital molecular imaging (MIMI) system as a tool for in vivo tumour transpathology investigation.Methods: The proposed MIMI system integrates high-resolution positron imaging, magnetic resonance imaging (MRI) and microscopic imaging on a dorsal skin window chamber on a nude rat. The window chamber frame was designed to be compatible with multimodal imaging and its fiducial markers were customized for precise physical alignment among modalities. The co-registration accuracy was evaluated based on phantoms with thin catheters. For proof of concept, the human colorectal adenocarcinoma cell line HT-29 was imaged. The tissue within the window chamber was sectioned, fixed, hematoxylin-eosin (HE) stained for comparison with multimodal in vivo imaging.Results: With the physical alignment via fiducial markers, the co-registration errors of the MIMI system were 0.18 ± 0.27 mm between MRI and positron imaging, 0.19 ± 0.22 mm between positron imaging and microscopic imaging, and 0.15 ± 0.27 mm between MRI and microscopic imaging. A pilot test demonstrated that the MIMI system provides an integrative visualization of the tumour anatomy, perfusion and metabolism of the in vivo tumour microenvironment.Conclusions: The established MIMI system provided a co-registered in vivo platform for trans-scale and transparent investigation of the underlying pathology behind imaging, which has the potential to enhance the translation of molecular imaging.

Multi-Modal Multi-Spectral Intravital Microscopic Imaging of Signaling Dynamics in Real-Time during Tumor–Immune Interactions

Intravital microscopic imaging (IVM) allows for the study of interactions between immune cells and tumor cells in a dynamic, physiologically relevant system in vivo. Current IVM strategies primarily use fluorescence imaging; however, with the advances in bioluminescence imaging and the development of new bioluminescent reporters with expanded emission spectra, the applications for bioluminescence are extending to single cell imaging. Herein, we describe a molecular imaging window chamber platform that uniquely combines both bioluminescent and fluorescent genetically encoded reporters, as well as exogenous reporters, providing a powerful multi-plex strategy to study molecular and cellular processes in real-time in intact living systems at single cell resolution all in one system. We demonstrate that our molecular imaging window chamber platform is capable of imaging signaling dynamics in real-time at cellular resolution during tumor progression. Importantly, we expand the utility of IVM by modifying an off-the-shelf commercial system with the addition of bioluminescence imaging achieved by the addition of a CCD camera and demonstrate high quality imaging within the reaches of any biology laboratory.

Harnessing lipid signaling pathways to target specialized pro-angiogenic neutrophil subsets for regenerative immunotherapy

To gain insights into neutrophil heterogeneity dynamics in the context of sterile inflammation and wound healing, we performed a pseudotime analysis of single-cell flow cytometry data using the spanning-tree progression analysis of density-normalized events algorithm. This enables us to view neutrophil transitional subsets along a pseudotime trajectory and identify distinct VEGFR1, VEGFR2, and CXCR4 high-expressing pro-angiogenic neutrophils. While the proresolving lipid mediator aspirin-triggered resolvin D1 (AT-RvD1) has a known ability to limit neutrophil infiltration, our analysis uncovers a mode of action in which AT-RvD1 leads to inflammation resolution through the selective reprogramming toward a therapeutic neutrophil subset. This accumulation leads to enhanced vascular remodeling in the skinfold window chamber and a proregenerative shift in macrophage and dendritic cell phenotype, resulting in improved wound closure after skin transplantation. As the targeting of functional immune subsets becomes the key to regenerative immunotherapies, single-cell pseudotime analysis tools will be vital in this field.