Geotemporal Fluorophore Biodistribution Mapping of Colorectal Cancer: Micro and Macroscopic Insights

Abstract

Fluorescence-guided oncology promises to improve both the detection and treatment of malignancy. We sought to investigate the temporal distribution of indocyanine green (ICG), an exogenous fluorophore in human colorectal cancer. This analysis aims to enhance our understanding of ICG's effectiveness in current tumour detection and inform potential future diagnostic and therapeutic enhancements.

Methods: Fifty consenting patients undergoing treatment for suspected/confirmed colorectal neoplasia provided near infrared (NIR) video and imagery of transanally recorded and ex vivo resected rectal lesions following intravenous ICG administration (0.25 mg/kg), with a subgroup providing tissue samples for microscopic (including near infrared) analysis. Computer vision techniques detailed macroscopic 'early' (<15 min post ICG administration) and 'late' (>2 h) tissue fluorescence appearances from surgical imagery with digital NIR scanning (Licor, Lincoln, NE, USA) and from microscopic analysis (Nikon, Tokyo, Japan) undertaken by a consultant pathologist detailing tissue-level fluorescence distribution over the same time.

Results: Significant intra-tumoural fluorescence heterogeneity was seen 'early' in malignant versus benign lesions. In all 'early' samples, fluorescence was predominantly within the tissue stroma, with uptake within plasma cells, blood vessels and lymphatics, but not within malignant or healthy glands. At 'late' stage observation, fluorescence was visualised non-uniformly within the intracellular cytoplasm of malignant tissue but not retained in benign glands. Fluorescence also accumulated within any present peritumoural inflammatory tissue.

Conclusion: This study demonstrates the time course diffusion patterns of ICG through both benign and malignant tumours in vivo in human patients at both macroscopic and microscopic levels, demonstrating important cellular drivers and features of geolocalisation and how they differ longitudinally after exposure to ICG.

Keywords: colorectal cancer; fluorescence microscopy; fluorescence-guided surgery; fluorophore biodistribution.

Figure 1

Images showing ‘early’ timepoint malignant and benign rectal lesions with white light imagery and 2D dynamic perfusion profile mapping. The outer grey borders around images represent regions lost during tracking. X and Y co-ordinates are constant across all images. (a1): White light view of a malignant rectal polyp. (a2): Pixel-by-pixel dynamic perfusion curves of malignant rectal polyp represented by a centre of mass heat map demonstrating intra-lesional heterogeneity. (a3): Graph representations of the two clusters (red and blue) created from assessment of time–fluorescence curves. (a4): Resulting heatmap following image creation using unsupervised clustering. Intralesional heterogeneity (red and blue) consistent with malignancy. (b1): White light view of benign rectal polyp. (b2): Pixel-by-pixel dynamic perfusion curves of benign rectal polyp represented by a centre of mass heat map demonstrating intra-lesional homogeneity. (b3): Three cluster centres created from all tracked pixel data. (b4): Clustering was applied to create an image showing intra-lesional homogeneity.

Figure 2

Images depicting macroscopic appearances of ‘late’ timepoint malignant lesions. White light images of malignancy are shown in (a1,b1). Fire colourmaps of fluorescence intensity show a relatively homogenous lesion demarcated from surrounding healthy tissue in (a2) with a less clearly demarcated lesion shown in (b2). (Fire colourmaps were created by converting the NIR images to 8-bit greyscale followed by application of a “fire” heatmap using a lookup table (LUT) in ImageJ).

Figure 3

Compound white light and NIR fluorescence microscopy images of ‘early’ phase tissue samples: (A): 10× magnification image of relative stromal fluorescence concentration with minimal uptake seen within the glandular tissue at early timepoints. Concentration within plasma cells marked with white circle (top right). (B): Images taken from a low grade, poorly differentiated adenocarcinoma demonstrating a small volume of tumour with a surrounding predominantly inflammatory component in which ICG is seen to accumulate. (C): Images from a poorly differentiated adenocarcinoma demonstrating early strong uptake within lymphatics and vascular channels (black and white circles) but no relative uptake within abnormal glands.

Figure 4

‘Late’ obtained sample of colorectal adenocarcinoma demonstrating a reversal of appearances seen at the ‘early’ timepoints, with intracellular cytoplasmic uptake of ICG within malignant glands and a relative lack of ICG within the neoplastic stroma.

Figure 5

Microscopic images of a ‘late’ sample, poorly differentiated mucinous tumour with a large surrounding inflammatory component. NIR fluorescence image taken from the region marked by the black rectangle, with malignant regions marked by white outline, and with a concentration of fluorescence within surrounding inflammatory cells.

Figure 6

Narrower field of view from patient in Figure 5 demonstrating a concentration of ICG within the inflammatory interphase between malignant and healthy glands.

Figure 7

‘Late’ phase fluorescence appearances with relative concentration within malignant cells (right and bottom) and with washout from adjacent normal tissue (top left).