A multimodal imaging pipeline to decipher cell-specific metabolic functions and tissue microenvironment dynamics

Abstract

Tissue microenvironments are extremely complex and heterogeneous. It is challenging to study metabolic interaction between the different cell types in a tissue with the techniques that are currently available. Here we describe a multimodal imaging pipeline that allows cell type identification and nanoscale tracing of stable isotope-labeled compounds. This pipeline extends upon the principles of correlative light, electron and ion microscopy, by combining confocal microscopy reporter or probe-based fluorescence, electron microscopy, stable isotope labeling and nanoscale secondary ion mass spectrometry. We apply this method to murine models of hepatocellular and mammary gland carcinomas to study uptake of glucose derived carbon (¹³C) and glutamine derived nitrogen (¹⁵N) by tumor-associated immune cells. In vivo labeling with fluorescent-tagged antibodies (B220, CD3, CD8a, CD68) in tandem with confocal microscopy allows for the identification of specific cell types (B cells, T cells and macrophages) in the tumor microenvironment. Subsequent image correlation with electron microscopy offers the contrast and resolution to image membranes and organelles. Nanoscale secondary ion mass spectrometry tracks the enrichment of stable isotopes within these intracellular compartments. The whole protocol described here would take ~6 weeks to perform from start to finish. Our pipeline caters to a broad spectrum of applications as it can easily be adapted to trace the uptake and utilization of any stable isotope-labeled nutrient, drug or a probe by defined cellular populations in any tissue in situ.

Figure 1. Using correlative fluorescence microscopy, EM and NanoSIMS analysis to evaluate glucose and glutamine catabolism in specific cells of bi-clonal mammary gland tumours.

(A) Schematic of inducible and traceable mouse model of heterogeneity in breast cancer and experimental workflow used by Kreuzaler et al. (B) Representative panel of region of interest in the bi-clonal tumour with red clones, green clones and macrophages (CD68) identified with confocal fluorescence image analysis (IF). (C) SEM and correlative NanoSIMS nitrogen/carbon isotope ratio images of region of interest with tumour cells and a macrophage (yellow ROI). Isotope ratio images are displayed as a hue saturation intensity (HSI) transformation. The blue hue represents the natural abundance ratios which are 0.37% and 1.1% respectively, while pink hue represents a value of twice the natural ratio in the ¹²C¹⁵N/¹²C¹⁴N and ¹³C¹⁴N/¹²C¹⁴N HSI image. Figure adapted from Kreuzaler et al.

Figure 2. Using correlative fluorescence microscopy, EM and NanoSIMS analysis to evaluate glucose and glutamine catabolism in specific cells of MYC-induced liver tumours.

(A) SEM images with cells of interest (B cells – blue ROI, T cells – red ROI, tumour cells – green ROI) – top panel. B and T cells are identified by confocal fluorescence analysis using fluorescently labled anti-B220 and anti-CD8a antibodies, respectively – bottom panel. (B) Correlative NanoSIMS isotope ratio images (¹²C¹⁵N/¹²C¹⁴N, middle panel, and ¹³C¹⁴N/¹²C¹⁴N, right panel) of the B (blue ROI) and T (red ROI) cells from A. Isotope ratio images are displayed as a hue saturation intensity (HSI) transformation. The blue hue represents the natural abundance ratios which are 0.37% and 1.1% respectively.

Figure 3. Flow diagram showing the steps of the multimodal imaging pipeline (Created with BioRender.com).

Figure 4. Correlating SEM images for NanoSIMS acquisition.

(A) SEM image acquired at high pixel resolution enabling easy digital zooming for fine detail of tissue structure. (B) Corresponding optical image obtained using NanoSIMS CCD camera showing microtomed thin sections and structure within.

Figure 5. Example of ROI selection for quantitative analysis.

(A) SEM image showing a B-cell (blue ROI). (B) ¹²C¹⁴N NanoSIMS image showing ROIs defined in nucleus of the B-cell (green ROIs) and cytoplasm (red ROIs). Similar ROIs are shown for a cancer cell in the top right of the image. (C) Corresponding nitrogen isotope ratio image. ¹²C¹⁵N/¹²C¹⁴N Isotope ratio images displayed as an HIS transformation. The blue hue represents the natural abundance ratio which is 0.37%.

Figure 6. Enrichment of stable isotopes within the intracellular compartments of B cells and CD8 T cells.

(A) Comparison of ¹²C¹⁵N/¹²C¹⁴N isotope ratio of ROIs from the nucleus of B cells and CD8 T cells (n=15 ROIs). (B) ¹³C¹⁴N/¹²C¹⁴N isotope ratio of ROIs from the nucleus of B cells and CD8 T cells (n=15 ROIs). (C) ¹²C¹⁵N/¹²C¹⁴N isotope ratio of ROIs from the cytoplasm and (D) ¹³C¹⁴N/¹²C¹⁴N isotope ratio of ROIs from the cytoplasm of B cells and CD8 T cells (n=20 ROIs). Statistical significance was assessed by unpaired t-test (mean +/- SD,**p<0.01, ****p<0.0001).