Spatial heterogeneity of nanomedicine investigated by multiscale imaging of the drug, the nanoparticle and the tumour environment

Abstract

Genetic and phenotypic tumour heterogeneity is an important cause of therapy resistance. Moreover, non-uniform spatial drug distribution in cancer treatment may cause pseudo-resistance, meaning that a treatment is ineffective because the drug does not reach its target at sufficient concentrations. Together with tumour heterogeneity, non-uniform drug distribution causes "therapy heterogeneity": a spatially heterogeneous treatment effect. Spatial heterogeneity in drug distribution occurs on all scales ranging from interpatient differences to intratumour differences on tissue or cellular scale. Nanomedicine aims to improve the balance between efficacy and safety of drugs by targeting drug-loaded nanoparticles specifically to tumours. Spatial heterogeneity in nanoparticle and payload distribution could be an important factor that limits their efficacy in patients. Therefore, imaging spatial nanoparticle distribution and imaging the tumour environment giving rise to this distribution could help understand (lack of) clinical success of nanomedicine. Imaging the nanoparticle, drug and tumour environment can lead to improvements of new nanotherapies, increase understanding of underlying mechanisms of heterogeneous distribution, facilitate patient selection for nanotherapies and help assess the effect of treatments that aim to reduce heterogeneity in nanoparticle distribution. In this review, we discuss three groups of imaging modalities applied in nanomedicine research: non-invasive clinical imaging methods (nuclear imaging, MRI, CT, ultrasound), optical imaging and mass spectrometry imaging. Because each imaging modality provides information at a different scale and has its own strengths and weaknesses, choosing wisely and combining modalities will lead to a wealth of information that will help bring nanomedicine forward.

Keywords: Clinical Imaging; Drug distribution; Mass Spectrometry Imaging.; Nanomedicine; Optical imaging.

Figure 1

Non-invasive clinical methods to image spatial heterogeneity of nanomedicine. (A) Patterns of HER2-PET/CT confronted with FDG-PET/CT, Maximum intensity projection. Lesion uptake was considered pertinent when visually higher than blood pool. Top: dominant part of tumour load showed tracer uptake. Lung, liver and bone involvement seen of FDG-PET: not all lung lesions are seen on HER2-PET. Bottom: entire tumour load lacked tracer uptake. Liver and bone involvement seen on FDG-PET are not seen on HER2PET. (Adapted with permission from , copyright 2016 Oxford University Press on behalf of the European Society for Medical Oncology). (B) In vivo computed tomography (CT), positron emission tomography (PET)/CT, and SPECT/CT images of a nude mouse injected with 14 MBq of [¹⁸F]-FCP encapsulated [¹¹¹In]-Liposome through tail vein injection 1 h post-administration. Coronal images. Both PET/CT and SPECT/CT images show the uptake of [¹⁸F]-FCP encapsulated in [¹¹¹In]-Liposome in the liver and spleen. Both images correspond to each other in the uptake profile, demonstrating the feasibility of dual-tracer imaging from a single nano-construct. (Adapted with permission from , copyright 2017 MDPI). (C)MR T2* images of CL1-5-F4/NF-κB-luc2-xenograft-bearing mice treated with erlotinib-conjugated iron oxide nanoparticles. Voxelwise estimates of the intratumoural iron concentration derived from changes in the ΔR2* signal (P < 0.0001), which correlates to the amount of intratumoural erlotinib content. Top: T2* weighted MR image. Bottom: T2*-weighted MR image with color-coded overlay of voxelwise estimates of intratumoural iron concentration (Adapted with permission from , copyright 2018 Elsevier). (D) A panel of images showing point-based measurements of IFP overlaid on the intratumoural distribution of CT-liposomes in an orthotopic tumour. Images from left to right represent: interstitial Fluid Pressure (IFP); permeability; perfusion; interstitial volume fraction; plasma volume fraction. The coloured circles and corresponding numbers represent the region of interest (ROI) locations, ROI size used for point-based analysis, and measured IFP. Predominantly peripheral CT-liposome enhancement was observed, with some heterogeneous accumulation within the central tumour region. Metrics of perfusion were spatially heterogeneous, but tended to increase towards the tumour periphery. (Adapted with permission from , copyright 2015 Elsevier). (E)Motion model ultrasound localization microscopy (mULM). Super-resolution ultrasound images of an A431 tumour provide detailed information on the microvascular architecture including insights into vascular connectivity and the number of vascular branching points (see arrows in magnifications). Functional information such as MB velocities (left image) and MB flow directions (right image; color-coding illustrating the direction of flow according to the coloured circle) can be determined for each individual vessel and evaluated together with the morphological characteristics. Scale bar = 1 mm. (Adapted with permission from , copyright 2018 Nature Research).

Figure 2

Optical technologies to image spatial heterogeneity of nanomedicine. (A) Combination of bioluminescence imaging (BLI) of luciferase expressing glioblastoma and photoluminescence (PL) imaging of theranostic photonic nanoparticles to verify nanoparticle tumour targeting efficacy. (Adapted with permission from , copyright 2016 Wiley). (B) 3D fluorescence-enhanced diffuse optical tomography (fDOT) image after injection of NIR-decorated nanoparticles in tumour-bearing mouse. (Adapted with permission from copyright 2012 SPIE Digital Library). (C) Representative examples of real-time intravital microscopy (IVM) used to visualizing tumour microenvironment and track nanoparticles. The combination of bright-field illumination, non-linear optical imaging effects, endogenous fluorescence, and i.v. administration of fluorescent dyes contribute to a high quality tumour microenvironment characterization. Left: Green fluorescent protein (GFP) expressing endothelium (green) in a TIE2GFP mouse, 70 KDa TMR-dextran positive TAM (red), collagen (blue). Middle: Rhodamine-labelled nanoemulsions, passive diffusion on inflamed tissue over 30 min. Right: Atto633-labelled Doxil-like liposomes in circulation (red blur within vessel) and phagocytosed by a slow-moving circulating immune cell (red blob), GFP expressing endothelium (green) on TIE2GFP mouse (green). Scale bars 100 μm (right: 20 μm). (A.M. Sofias and S. Hak, unpublished data). (D) Multispectral Optoacoustic Tomography (MSOT) images of nude mouse with A2780 tumour Left: gold nanorod accumulation (overlaid in red) 24 hours after injection Right: MSOT images of oxyhemoglobin (red) and deoxyhemoglobin (blue) distribution visualizes vasculature. (Adapted with permission from , copyright 2012 Radiological Society of North America (United States)). (E) Heterogeneity of transport and structural properties of 4T1 breast cancer metastases in mouse liver. Several magnified metastases with different sizes and the red fluorescence of extravasated doxorubicin delivered by PEGylated liposomal doxorubicin (PLD) and colocalizing (yellow arrows) with Kupffer cells (green) outside tumours; stars denote doxorubicin fluorescence in tumours, the white-dashed line indicates the tumour boundary (Adapted with permission from , copyright 2018 Elsevier).

Figure 3

Mass Spectrometry Imaging to image spatial heterogeneity of nanomedicine. (A) Paclitaxel distribution by MALDI MSI. Necrotic areas, highlighted with dashed lines, are those were there is the lower drug signal. (Adapted with permission from , copyright 2016 Nature Research). (B) DESI image overlay representing the spatial distribution of the drug compound (m/z 378) and its most abundant metabolites (m/z 380 and 394) in a tissue section of a formalin fixed frozen rabbit kidney. (Adapted with permission from , copyright 2016 Springer US). (C) ToF SIMS 2D images of 3D data acquired in higher spatial resolution mode from HeLa cells completely consumed by the argon cluster source during analysis. The cells were incubated for 2 h with 9.7 nmol/mL amiodarone hydrochloride. Composite image where red represents ribose m/z 81, blue shows the signal from the phosphatidylcholine lipid fragment (m/z 184), and green shows the amiodarone signal, [M + H]+ (m/z 646). (Adapted with permission from , copyright 2017 American Chemical Society). (D) Cisplatin effects on tumour proliferation, DNA damage and cisplatin distribution in the tumour. Representative Pan-Keratin, EF5, Collagen I, 195Pt, and Histone H3 images of cisplatin-treated (40 mg/kg for 24 h) mice with OCIP28 patient derived xenografts. Scale bar = 100 μm. (Adapted with permission from , copyright 2016 Nature Research). (E) MALDI MSI images performed on brain slices of mice that were dosed with liposomes. Four images on the left: Half of the mice were perfused before being sacrificed (right panels) to reduce the remaining blood in the tissue. MALDI images of liposomal marker 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and indocyanine green (ICG) were acquired in reflector negative ion mode, of PEG36-DSPE in reflector positive mode and of Hb α chain in linear positive mode. DPPG, ICG and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated with monodisperse polyethylene glycol (PEG36-DSPE) were measured with 4-Phenyl-α-cyanocinnamic acid amide (PhCCAA) MALDI matrix. Hb was detected after delipidation and 2,5-dihydroxybenzoic acid (sDHB) deposition on the same tissue region. Magnifications: MALDI-MS images of the boxed parts marked in perfused brain in pixels indicated by an arrow shows the co-localisation of the liposomal components and hemoglobin at pixel X442 Y071 and the absence of HB at pixel X449 Y074. (Adapted with permission from , copyright 2016 Nature Research)