Cousins at work: How combining medical with optical imaging enhances in vivo cell tracking

Abstract

Microscopy and medical imaging are related in their exploitation of electromagnetic waves, but were developed to satisfy differing needs, namely to observe small objects or to look inside subjects/objects, respectively. Together, these techniques can help elucidate complex biological processes and better understand health and disease. A current major challenge is to delineate mechanisms governing cell migration and tissue invasion in organismal development, the immune system and in human diseases such as cancer where the spatiotemporal tracking of small cell numbers in live animal models is extremely challenging. Multi-modal multi-scale in vivo cell tracking integrates medical and optical imaging. Fuelled by basic research in cancer biology and cell-based therapeutics, it has been enabled by technological advances providing enhanced resolution, sensitivity and multiplexing capabilities. Here, we review which imaging modalities have been successfully used for in vivo cell tracking and how this challenging task has benefitted from combining macroscopic with microscopic techniques.

Keywords: Cancer metastasis; Cell therapy; Microscopy; Reporter genes; Whole-body imaging.

Fig. 1

Macroscopic and microscopic imaging modalities. Imaging modalities are ordered according to the electromagnetic spectrum they exploit for imaging (top: high energy; bottom: low energy). Routinely achievable spatial resolution (left end) and fields of view (right end) are shown in red. Where bars are blue they overlap red bars and indicate the same parameters but achievable with instruments used routinely in the clinic. Imaging depth is shown in green alongside sensitivity ranges. Instrument cost estimations are classified as ($) <125,000 $, ($$) 125–300,000 $ and ($$$) >300,000 $. * Fluorophore detection can suffer from photobleaching by excitation light. ** Generated by positron annihilation (511 keV). *** Contrast agents sometimes used to obtain different anatomical/functional information. **** In ‘emission mode’ comparable to other fluorescence modalities (∼nM). ***** Highly dependent on contrast agent. ^& Multichannel MRI imaging has been shown to be feasible (Zabow et al., 2008). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 2

Dual-mode radionuclide-fluorescence metastasis tracking is quantitative and provides data across multiple length scales. Representative results of metastasis tracking in a murine model of inflammatory breast cancer using the radionuclide-fluorescence fusion reporter NIS-GFP are shown. NIS served as an in vivo reporter and was imaged by PET/CT using the NIS tracer [¹⁸F]BF₄⁻. (A/left) On day 19 post tumour inoculation, the primary tumour (yellow dashed line) was clearly identified but no metastasis. It is noteworthy that endogenous NIS signals (white descriptors) were also recorded, i.e. the thyroid and salivary glands (Th + SG), the stomach (S), and, at very low levels, some parts of the mammary and lachrymal glands. Neither of these endogenous signals interfered with sites of expected metastasis in this tumour model. The bladder (B) signal stems from tracer excretion. (A/right) On day 29 post tumour inoculation, metastases were clearly identified in the lung (yellow dotted line; numbered individual metastases) and in some lymph nodes (inguinal (ILN), axillary (AxLN); yellow arrowheads). The primary tumour (yellow dashed line) had also invaded into the peritoneal wall. Images presented are maximum intensity projections (MIP). (B) A 3D implementation of the Otsu thresholding technique enabled 3D surface rendering of cancerous tissues; these are superimposed onto a PET MIP. Lung metastases are shown in white, metastatic axillary lymph nodes in red, the metastatic inguinal lymph node in yellow, and the primary tumour that invaded into the peritoneal wall in turquoise. (C) Radiotracer uptake into cancerous tissues was quantified from 3D images (%injected dose (ID)) and normalized by the corresponding volumes (%ID/mL). Individual lung metastases correspond to the numbers in (A). (D) NIS-GFP’s fluorescence properties guided animal dissection. As exemplars birghtfield and fluorescence images of the lung with several metastatic lesions and two positive lymph nodes are shown. (E) Immunofluorescence histology of the primary tumour. NIS-GFP expressing cancer cells were directly identified without the need for antibody staining. Blood vessels were stained with a rabbit antibody against mouse PECAM-1/CD31 and for nuclei (DAPI) before being imaged by confocal fluorescence microscopy. Data demonstrated vascularization heterogeneity of the primary tumour. The image also shows that the NIS-GFP reporter predominantly resides in the plasma membranes of the tumour cells demonstrating its correct localization to be functional in vivo and enabling tumour cell segmentation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 3

Tracking a nanomedicine to primary and secondary cancer lesions. Liposomal alendronate was radiolabelled with the PET isotope ⁸⁹Zr (⁸⁹Zr-PLA) and administered to animals bearing primary breast tumours that had already spontaneously metastasized (as determined by ^99mTcO4⁻-afforded NIS-SPECT/CT). (A) Coronal and sagittal SPECT-CT (top; cancer cells) and PET-CT (bottom; nanomedicine) images centred at the tumours of the same animal are shown at indicated time points after intravenous administration of ⁸⁹Zr-PLA. SPECT-CT images show identical biodistribution over time with high uptake in endogenous NIS-expressing organs (stomach, thyroid) and NIS-FP-expressing cancer cells in the primary tumour (T) and metastases (LN_met and Lu_met). PET-CT images show the increasing uptake of ⁸⁹Zr-PLA over time in the primary tumour (T), spleen (Sp), liver (L), and bone (B) and decreasing amounts in the blood pool/heart (H). For corresponding time–activity curves refer to (Edmonds et al., 2016). (B) Co-registered SPECT/PET/CT images of the primary tumour (from left to right: sagittal, coronal, transverse) showing a high degree of colocalization but also intra-tumoral heterogeneity of ⁸⁹Zr-PLA (purple scale); ^99mTcO₄⁻NIS signals (green scale) show live cancer cells. (C) Autoradiography images (left, ^99mTc; right, ⁸⁹Zr) of a coronal slice from the same tumour as in (B) showing a high degree of colocalization and heterogeneity. (D) Fluorescence microscopy of an adjacent slice of the same tumour as in (B/C) showing areas of high and low microvascular density (determined by anti-CD31 staining). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).