Tom J. L. Schreurs’s research while affiliated with Eindhoven University of Technology and other places

Targeted photodynamic therapy (PDT) has the potential to selectively damage tumor tissue and to increase tumor vessel permeability. Here we characterize the tissue biodistribution of two EGFR-targeted nanobody-photosensitizer conjugates (NB-PS), the monovalent 7D12-PS and the biparatopic 7D12-9G8-PS. In addition, we report on the local and acute phototoxic effects triggered by illumination of these NB-PS which have previously shown to lead to extensive tumor damage. Methods: Intravital microscopy and the skin-fold chamber model, containing OSC-19-luc2-cGFP tumors, were used to investigate: a) the fluorescence kinetics and distribution, b) the vascular response and c) the induction of necrosis after illumination at 1 or 24 h post administration of 7D12-PS and 7D12-9G8-PS. In addition, dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) of a solid tumor model was used to investigate the microvascular status 2 h after 7D12-PS mediated PDT. Results: Image analysis showed significant tumor colocalization for both NB-PS which was higher for 7D12-9G8-PS. Intravital imaging showed clear tumor cell membrane localization 1 and 2 h after administration of 7D12-9G8-PS, and fluorescence in or close to endothelial cells in normal tissue for both NB-PS. PDT lead to vasoconstriction and leakage of tumor and normal tissue vessels in the skin-fold chamber model. DCE-MRI confirmed the reduction of tumor perfusion after 7D12-PS mediated PDT. PDT induced extensive tumor necrosis and moderate normal tissue damage, which was similar for both NB-PS conjugates. This was significantly reduced when illumination was performed at 24 h compared to 1 h after administration. Discussion: Although differences were observed in distribution of the two NB-PS conjugates, both led to similar necrosis. Clearly, the response to PDT using NB-PS conjugates is the result of a complex mixture of tumor cell responses and vascular effects, which is likely to be necessary for a maximally effective treatment.

Early evaluation of response to therapy is crucial for selecting the optimal therapeutic follow-up strategy for cancer patients. PDT is a photochemistry-based treatment modality that induces tumor tissue damage by cytotoxic oxygen radicals, generated by a pre-injected photosensitive drug upon light irradiation of tumor tissue. Vascular shutdown is an important mechanism of tumor destruction for most PDT protocols. In this study, we assessed the suitability of Dynamic Contrast-Enhanced Magnetic Resonance Imaging (DCE-MRI) to evaluate treatment efficacy within a day after photodynamic therapy (PDT), using the tumor vascular response as a biomarker for treatment success. Methods: DCE-MRI at 7 T was used to measure the micro-vascular status of subcutaneous colon carcinoma tumors before, right after, and 24 h after PDT in mice. Maps of the area under the curve (AUC) of the contrast agent concentration were calculated from the DCE-MRI data. Besides, tracer kinetic parameters including Ktrans were calculated using the standard Tofts-Kermode model. Viability of tumor tissue at 24 h after PDT was assessed by histological analysis. Results: PDT led to drastic decreases in AUC and Ktrans or complete loss of enhancement immediately after treatment, indicating a vascular shutdown in treated tumor regions. Histological analysis demonstrated that the treatment induced extensive necrosis in the tumors. For PDT-treated tumors, the viable tumor fraction showed a strong correlation (ρ ≥ 0.85) with the tumor fraction with Ktrans > 0.05 min⁻¹ right after PDT. The viable tumor fraction also correlated strongly with the enhanced fraction, the average Ktrans, and the fraction with Ktrans > 0.05 min⁻¹ at 24 h after PDT. Images of the viability stained tumor sections were registered to the DCE-MRI data, demonstrating a good spatial agreement between regions with Ktrans > 0.05 min⁻¹ and viable tissue regions. Finally, 3D post-treatment viability detection maps were constructed for the tumors of three mice by applying a threshold (0.05 min⁻¹) to Ktrans at 24 h after PDT. As a proof of principle, these maps were compared to actual tumor progression after one week. Complete tumor response was correctly assessed in one animal, while residual viable tumor tissue was detected in the other two at the locations where residual tumor tissue was observed after one week. Conclusion: This study demonstrates that DCE-MRI is an effective tool for early evaluation of PDT tumor treatment.

A noninvasive method is introduced for quantification and visualization of fluence rate in light-irradiated biological tissues. The method is based on magnetic resonance thermometry (MRT) measurements of tissue temperature changes resulting from absorption of light. From the spatial-temporal temperature data, the generated heat is calculated. Finally, fluence rate maps are reconstructed by dividing the heat data by the tissue absorption coefficient. Simulations were performed using virtual MRT datasets based on analytically described fluence rate distributions, which could be accurately reconstructed by the method. Next, the approach was tested in gel phantoms. Resulting fluence rate maps matched well with theoretical predictions in a nonscattering phantom (R²=0.93). Experimental validation was further obtained in a scattering phantom, by comparing fluence rates to invasive fluence rate probe measurements along and perpendicular to the optical axis (R²≥0.71 for both cases). Finally, our technique was applied in vivo in a mouse tumor model. The resulting fluence rates matched invasive probe measurements (Pearson's ρ=0.90, p=0.0026). The method may be applied to investigate the relation between light dose and biological response in light-based treatments, such as photodynamic therapy. It may also be useful for experimental validation of light transport models. © 2017 Society of Photo-Optical Instrumentation Engineers (SPIE).

MRE pre and post indentation movie. Movie of a 16 offsets 900 Hz SE-EPI-MRE (A) before and (B) after indentation. (AVI)

Detail of indentation and MRE actuator part of setup. Following parts are labeled: u-shaped profile (a), cutout for the rat’s groin (b), indentor (c), movable indentor holder (d), rotatable half arch (e), dovetail profile (f), spacer plates (g), MRE piston (h), drive rod (i), cantilever (j). (TIF)

Deformation of skeletal muscle in the proximity of bony structures may lead to deep tissue injury category of pressure ulcers. Changes in mechanical properties have been proposed as a risk factor in the development of deep tissue injury and may be useful as a diagnostic tool for early detection. MRE allows for the estimation of mechanical properties of soft tissue through analysis of shear wave data. The shear waves originate from vibrations induced by an external actuator placed on the tissue surface. In this study a combined Magnetic Resonance (MR) compatible indentation and MR Elastography (MRE) setup is presented to study mechanical properties associated with deep tissue injury in rats. The proposed setup allows for MRE investigations combined with damage-inducing large strain indentation of the Tibialis Anterior muscle in the rat hind leg inside a small animal MR scanner. An alginate cast allowed proper fixation of the animal leg with anatomical perfect fit, provided boundary condition information for FEA and provided good susceptibility matching. MR Elastography data could be recorded for the Tibialis Anterior muscle prior to, during, and after indentation. A decaying shear wave with an average amplitude of approximately 2 μm propagated in the whole muscle. MRE elastograms representing local tissue shear storage modulus Gd showed significant increased mean values due to damage-inducing indentation (from 4.2 ± 0.1 kPa before to 5.1 ± 0.6 kPa after, p<0.05). The proposed setup enables controlled deformation under MRI-guidance, monitoring of the wound development by MRI, and quantification of tissue mechanical properties by MRE. We expect that improved knowledge of changes in soft tissue mechanical properties due to deep tissue injury, will provide new insights in the etiology of deep tissue injuries, skeletal muscle damage and other related muscle pathologies.

Photograph of a rat positioned in the setup. Rat, Indentation and MRE component are indicated with arrows. In A, the indentor, put through the surface RF coil, positioned on top of TA muscle in rats hindleg, and the MRE piston attached at distal side of TA muscle are shown. Indentation component is removed in B, revealing the MRE component and surface coil. Pre-amplifier block of the surface coil, the respiratory sensor and the rectal temperature probe are also visible. Anesthesia mask and rat’s head are underneath the pre-amplifier block. (TIF)

Overview of experiment groups and their timelines. Numbers of animals are indicated per group. Grey blocks represent different measurement days. The following abbreviations are used: CT26: subcutaneous inoculation of CT26.WT tumor cells. MRI: Multi-parametric MRI scan session (approximate duration: 2.5 h). PS: photosensitizer injection, 6 h before PDT. PDT: photodynamic therapy (10 min tumor irradiation). †: kill animal and excise tumor for storage at -80°C. (TIF)

Ktrans and AUC maps of all slices of the entire tumor of three animals. The first example is a mouse with complete tumor response at 2 weeks after PDT, while the other two had recurring growth, both in the distal part of the tumor. Ktrans right after PDT and AUC at 72 h after PDT are shown. For Ktrans, only the contrast-enhanced pixels in the tumor are color coded. Both for example 2 and 3, high Ktrans values and significant enhancement were seen in the distal part of the tumor (red arrows). In the entire tumor of example 1, and in the central and proximal tumor parts of examples 2 and 3, few pixels with high Ktrans and AUC were observed. (TIF)