Preclinical Molecular Imaging using Multi-Isotope Digital Autoradiography - Techniques and Applications

Molecular imaging, both in vivo and ex vivo, is playing an increasingly important role in preclinical medical research. When using radionuclide-labeled tracers, e.g. in the development of radiopharmaceuticals for diagnostic imaging or for radionuclide therapy, quantitative in vivo imaging can be performed using emission tomography. Quantitative autoradiography of thin tissue sections is employed to obtain high-resolution images of the radioactivity distribution ex vivo, commonly using photo¬graphic film or a storage phosphor screen. The aim of the work presented in this thesis was to evaluate the potential of digital auto-radiography with multi-radionuclide imaging capabilities to contribute to pre¬clinical, small-animal, research studies using radiolabeled targeting molecules for diagnosis or therapy. The characteristics of a digital autoradiography system employing a double-sided silicon strip detector were investigated with regard to spatial resolution, detection sensitivity, background and noise, system dead time, and the ability to resolve energy spectra. This system, although it has a smaller field of view, was found to perform favorably compared to a storage phosphor system. Methods of separating the contributions from several radionuclides imaged simultaneously were developed and evaluated. The intratumoral distribution of 177Lu-labeled monoclonal antibodies (mAbs) over time was studied in a syngeneic rat model of colon carcinoma at both therapeutic and lower activity levels. The activity was initially found in the tumor periphery, then in areas of viable, antigen-expressing cells, and at 24-48 h and later in areas of granulation tissue and low antigen expression. A point-dose kernel was used to calculate the absorbed-dose rate distribution in tumor sections, which was found to be twice the section mean in areas with high activity accumulation. The distributions of 111In- and 177Lu-labeled mAbs targeting intercellular adhesion molecule 1 were studied in a xenograft model of human prostate cancer, and compared with a control mAb as well as clinically used 18F-labelled tracers using small animal SPECT/CT and PET/CT, as well as multi-radionuclide digital auto¬radio¬graphy. Results from this study, and those from an 125I-labelled mAb compared with a co-injected smaller antibody fragment labeled with 131I, both targeting carcino¬embryonic antigen, confirmed that barriers to tumor penetration by macro-molecules are a major problem, especially in radioimmunotherapy. A mAb against free prostate-specific antigen was also studied. Using multi-radionuclide digital autoradiography, the plaque-to-aorta contrast of a mAb targeting oxidized low-density lipoprotein was observed to be higher than that of 18FDG in slide mounted aortas from atherosclerotic mice. These studies show that digital autoradiography, including multi-radionuclide imaging, has the potential to provide novel data for preclinical imaging studies, but that further development and optimiz¬ation of the method are needed

Preclinical efficacy of hK2 targeted [177Lu]hu11B6 for prostate cancer theranostics

Androgen ablating drugs increase life expectancy in men with metastatic prostate cancer, but resistance inevitably develops. In a majority of these recurrent tumors, the androgen axis is reactivated in the form of increased androgen receptor (AR) expression. Targeting proteins that are expressed as a down-stream effect of AR activity is a promising rationale for management of this disease. The humanized IgG1 antibody hu11B6 internalizes into prostate and prostate cancer (PCa) cells by binding to the catalytic cleft of human kallikrein 2 (hK2), a prostate specific enzyme governed by the AR-pathway. In a previous study, hu11B6 conjugated with Actinium-225 (225Ac), a high linear energy transfer (LET) radionuclide, was shown to generate an AR-upregulation driven feed-forward mechanism that is believed to enhance therapeutic efficacy. We assessed the efficacy of hu11B6 labeled with a low LET beta-emitter, Lutetium-177 (177Lu) and investigated whether similar tumor killing and AR-enhancement is produced. Moreover, single-photon emission computed tomography (SPECT) imaging of 177Lu is quantitatively accurate and can be used to perform treatment planning. [177Lu]hu11B6 therefore has significant potential as a theranostic agent. Materials and Methods: Subcutaneous PCa xenografts (LNCaP s.c.) were grown in male mice. Biokinetics at 4-336 h post injection and uptake as a function of the amount of hu11B6 injected at 72 h were studied. Over a 30 to 120-day treatment period the therapeutic efficacy of different activities of [177Lu]hu11B6 were assessed by volumetric tumor measurements, blood cell counts, molecular analysis of the tumor as well as SPECT/CT imaging. Organ specific mean absorbed doses were calculated, using a MIRD-scheme, based on biokinetic data and rodent specific S-factors from a modified MOBY phantom. Tumor tissues of treated xenografts were immunohistochemically (IHC) stained for Ki-67 (proliferation) and AR, SA-β-gal activity (senescence) and analyzed by digital autoradiography (DAR). Results: Organ-to-blood and tumor-to-blood ratios were independent of hu11B6 specific activity except for the highest amount of antibody (150 µg). Tumor accumulation of [177Lu]hu11B6 peaked at 168 h with a specific uptake of 29 ± 9.1 percent injected activity per gram (%IA/g) and low accumulation in normal organs except in the submandibular gland (15 ± 4.5 %IA/g), attributed to a cross-reaction with mice kallikreins in this organ, was seen. However, SPECT imaging with therapeutic amounts of [177Lu]hu11B6 revealed no peak in tumor accumulation at 7 d, probably due to cellular retention of 177Lu and decreasing tumor volumes. For [177Lu]hu11B6 treated mice, tumor decrements of up to 4/5 of the initial tumor volume and reversible myelotoxicity with a nadir at 12 d were observed after a single injection. Tumor volume reduction correlated with injected activity and the absorbed dose. IHC revealed retained expression of AR throughout treatment and that Ki-67 staining reached a nadir at 9-14 d which coincided with high SA- β-gal activity (14 d). Quantification of nuclei staining showed that Ki-67 expression correlated negatively with activity uptake. AR expression levels in cells surviving therapy compared to previous timepoints and to controls at 30 d were significantly increased (p = 0.017). Conclusions: This study shows that hu11B6 labeled with the low LET beta-emitting radionuclide 177Lu can deliver therapeutic absorbed doses to prostate cancer xenografts with transient hematological side-effects. The tumor response correlated with the absorbed dose both on a macro and a small scale dosimetric level. Analysis of AR staining showed that AR protein levels increased late in the study suggesting a therapeutic mechanism, a feed forward mechanism coupled to AR driven response to DNA damage or clonal lineage selection, similar to that reported in high LET alpha-particle therapy using 225Ac labeled hu11B6, however emerging at a later timepoint

The Intratumoral Distribution of Radiolabeled 177Lu-BR96 Monoclonal Antibodies Changes in Relation to Tumor Histology over Time in a Syngeneic Rat Colon Carcinoma Model.

The therapeutic effect of radioimmunotherapy depends on the distribution of the absorbed dose in relation to viable cancer cells within the tumor, which in turn is a function of the activity distribution. The aim of this study was to investigate the distribution of (177)Lu-DOTA-BR96 monoclonal antibodies targeting the Lewis Y antigen over 7 d using a syngeneic rat model of colon carcinoma

Eye tracking: empirical foundations for a minimal reporting guideline

In this paper, we present a review of how the various aspects of any study using an eye tracker (such as the instrument, methodology, environment, participant, etc.) affect the quality of the recorded eye-tracking data and the obtained eye-movement and gaze measures. We take this review to represent the empirical foundation for reporting guidelines of any study involving an eye tracker. We compare this empirical foundation to five existing reporting guidelines and to a database of 207 published eye-tracking studies. We find that reporting guidelines vary substantially and do not match with actual reporting practices. We end by deriving a minimal, flexible reporting guideline based on empirical research (Section "empirically based minimal reporting guideline")