Development of an ectopic huLiver model for Plasmodium liver stage infection.

Early Plasmodium falciparum and P. vivax infection requires parasite replication within host hepatocytes, referred to as liver stage (LS). However, limited understanding of infection dynamics in human LS exists due to species-specificity challenges. Reported here is a reproducible, easy-to-manipulate, and moderate-cost in vivo model to study human Plasmodium LS in mice; the ectopic huLiver model. Ectopic huLiver tumors were generated through subcutaneous injection of the HC-04 cell line and shown to be infectible by both freshly dissected sporozoites and through the bite of infected mosquitoes. Evidence for complete LS development was supported by the transition to blood-stage infection in mice engrafted with human erythrocytes. Additionally, this model was successfully evaluated for its utility in testing antimalarial therapeutics, as supported by primaquine acting as a causal prophylactic against P. falciparum. Presented here is a new platform for the study of human Plasmodium infection with the potential to aid in drug discovery.

Comparison of automated full-body bone metastases delineation methods and their corresponding prognostic power.

Objective.Manual disease delineation in full-body imaging of patients with multiple metastases is often impractical due to high disease burden. However, this is a clinically relevant task as quantitative image techniques assessing individual metastases, while limited, have been shown to be predictive of treatment outcome. The goal of this work was to evaluate the efficacy of deep learning-based methods for full-body delineation of skeletal metastases and to compare their performance to existing methods in terms of disease delineation accuracy and prognostic power.Approach.1833 suspicious lesions on 3718F-NaF PET/CT scans of patients with metastatic castration-resistant prostate cancer (mCRPC) were contoured and classified as malignant, equivocal, or benign by a nuclear medicine physician. Two convolutional neural network (CNN) architectures (DeepMedic and nnUNet)were trained to delineate malignant disease regions with and without three-model ensembling. Malignant disease contours using previously established methods were obtained. The performance of each method was assessed in terms of four different tasks: (1) detection, (2) segmentation, (3) PET SUV metric correlations with physician-based data, and (4) prognostic power of progression-free survival.Main Results.The nnUnet three-model ensemble achieved superior detection performance with a mean (+/- standard deviation) sensitivity of 82.9±ccc 0.1% at the selected operating point. The nnUnet single and three-model ensemble achieved comparable segmentation performance with a mean Dice coefficient of 0.80±0.12 and 0.79±0.12, respectively, both outperforming other methods. The nnUNet ensemble achieved comparable or superior SUV metric correlation performance to gold-standard data. Despite superior disease delineation performance, the nnUNet methods did not display superior prognostic power over other methods.Significance.This work showed that CNN-based (nnUNet) methods are superior to the non-CNN methods for mCRPC disease delineation in full-body18F-NaF PET/CT. The CNN-based methods, however, do not hold greater prognostic power for predicting clinical outcome. This merits more investigation on the optimal selection of delineation methods for specific clinical tasks.

Exploring Spatial-Temporal Changes in 18F-Sodium Fluoride PET/CT and Circulating Tumor Cells in Metastatic Castration-Resistant Prostate Cancer Treated With Enzalutamide.

PURPOSE: Intrapatient treatment response heterogeneity is under-recognized. Quantitative total bone imaging (QTBI) using 18F-NaF positron emission tomography/computed tomography (PET/CT) scans is a tool that allows characterization of interlesional treatment response heterogeneity in bone. Understanding spatial-temporal response is important to identify individuals who may benefit from treatment beyond progression. PATIENTS AND METHODS: Men with progressive metastatic castration-resistant prostate cancer (mCRPC) with at least two lesions on bone scintigraphy were enrolled and treated with enzalutamide 160 mg daily (ClinicalTrials.gov identifier: NCT02384382). 18F-NaF PET/CT scans were obtained at baseline (PET1), week 13 (PET2), and at the time of prostate-specific antigen (PSA) progression, standard radiographic or clinical progression, or at 2 years without progression (PET3). QTBI was used to determine lesion-level response. The primary end point was the proportion of men with at least one responding bone lesion on PET3 using QTBI. RESULTS: Twenty-three men were enrolled. Duration on treatment ranged from 1.4 to 34.1 months. In general, global standardized uptake value (SUV) metrics decreased while on enzalutamide (PET2) and increased at the time of progression (PET3). The most robust predictor of PSA progression was change in SUVhetero (PET1 to PET3; hazard ratio, 3.88; 95% CI, 1.24 to 12.1). Although overall functional disease burden improved during enzalutamide treatment, an increase in total burden (SUVtotal) was seen at the time of progression, as measured by 18F-NaF PET/CT. All (22/22) evaluable men had at least one responding bone lesion at PET3 using QTBI. CONCLUSION: We found that the proportion of progressing lesions was low, indicating that a substantial number of lesions appear to continue to benefit from enzalutamide beyond progression. Selective targeting of nonresponding lesions may be a reasonable approach to extend benefit.

Phase II Trial of a DNA Vaccine Encoding Prostatic Acid Phosphatase (pTVG-HP [MVI-816]) in Patients With Progressive, Nonmetastatic, Castration-Sensitive Prostate Cancer.

PURPOSE: We previously reported the safety and immunologic effects of a DNA vaccine (pTVG-HP [MVI-816]) encoding prostatic acid phosphatase (PAP) in patients with recurrent, nonmetastatic prostate cancer. The current trial evaluated the effects of this vaccine on metastatic progression. PATIENTS AND METHODS: Ninety-nine patients with castration-sensitive prostate cancer and prostate-specific antigen (PSA) doubling time (DT) of less than 12 months were randomly assigned to treatment with either pTVG-HP co-administered intradermally with 200 µg granulocyte-macrophage colony-stimulating factor (GM-CSF) adjuvant or 200 µg GM-CSF alone six times at 14-day intervals and then quarterly for 2 years. The primary end point was 2-year metastasis-free survival (MFS). Secondary and exploratory end points were median MFS, changes in PSA DT, immunologic effects, and changes in quantitative 18F-sodium fluoride (NaF) positron emission tomography/computed tomography (PET/CT) imaging. RESULTS: Two-year MFS was not different between study arms (41.8% vaccine v 42.3%; P = .97). Changes in PSA DT and median MFS were not different between study arms (18.9 v 18.3 months; hazard ratio [HR], 1.6; P = .13). Preplanned subset analysis identified longer MFS in vaccine-treated patients with rapid (< 3 months) pretreatment PSA DT (12.0 v 6.1 months; n = 21; HR, 4.4; P = .03). PAP-specific T cells were detected in both cohorts, including multifunctional PAP-specific T-helper 1-biased T cells. Changes in total activity (total standardized uptake value) on 18F-NaF PET/CT from months 3 to 6 increased 50% in patients treated with GM-CSF alone and decreased 23% in patients treated with pTVG-HP (n = 31; P = .07). CONCLUSION: pTVG-HP treatment did not demonstrate an overall increase in 2-year MFS in patients with castration-sensitive prostate cancer, with the possible exception of a subgroup with rapidly progressive disease. Prespecified 18F-NaF PET/CT imaging conducted in a subset of patients suggests that vaccination had detectable effects on micrometastatic bone disease. Additional trials using pTVG-HP in combination with PD-1 blockade are under way.

Friomaramide, a Highly Modified Linear Hexapeptide from an Antarctic Sponge, Inhibits Plasmodium falciparum Liver-Stage Development.

The cold waters of Antarctica are known to harbor a rich biodiversity. Our continuing interest in the chemical analysis of Antarctic invertebrates has resulted in the isolation of friomaramide (1), a new, highly modified hexapeptide, from the Antarctic sponge Inflatella coelosphaeroides. The structure of friomaramide was determined using spectroscopic methods and its configuration established by Marfey's method. Friomaramide, which bears the unusual permethylation of the amino acid backbone and is the longest polypeptide bearing a tryptenamine C-terminus, blocks >90% of Plasmodium falciparum liver-stage parasite development at 6.1 µM.

Impact of Anatomic Location of Bone Metastases on Prognosis in Metastatic Castration-Resistant Prostate Cancer.

BACKGROUND: Whole-body assessments of 18F-NaF positron emission tomography (PET)/computed tomography (CT) provide promising quantitative imaging biomarkers of metastatic castration-resistant prostate cancer (mCRPC). This study investigated whether the distribution of metastases across anatomic regions is prognostic of progression-free survival. PATIENTS AND METHODS: Fifty-four mCRPC patients with osseous metastases received baseline NaF PET/CT. Patients received chemotherapy (n = 16) or androgen receptor pathway inhibitors (n = 38). Semiautomated analysis using Quantitative Total Bone Imaging software extracted imaging metrics for the whole, axial, and appendicular skeleton as well as 11 skeletal regions. Five PET metrics were extracted for each region: number of lesions (NL), standardized maximum uptake value (SUVmax), average uptake (SUVmean), sum of uptake (SUVtotal), and diseased fraction of the skeleton (volume fraction). Progression included that discovered by clinical, biochemical, or radiographic means. Univariate and multivariate Cox proportional hazard regression analyses were performed between imaging metrics and progression-free survival, and were assessed according to their hazard ratios (HR) and concordance (C)-indices. RESULTS: The strongest univariate models of progression-free survival were pelvic NL and SUVmax with HR = 1.80 (NL: false discovery rate adjusted P = .001, SUVmax: adjusted P = .001). Three other region-specific metrics (axial NL: HR = 1.59, adjusted P = .02, axial SUVmax: HR = 1.61, adjusted P = .02, and skull SUVmax: HR = 1.58, adjusted P = .04) were found to be stronger prognosticators relative to their whole-body counterparts. Multivariate model including region-specific metrics (C-index = 0.727) outperformed that of whole-body metrics (C-index = 0.705). The best performance was obtained when region-specific and whole-body metrics were included (C-index = 0.742). CONCLUSION: Quantitative characterization of metastatic spread by anatomic location on NaF PET/CT enhances potential prognostication. Further study is warranted to optimize the prognostic and predictive value of NaF PET/CT in mCRPC patients.

Computational modelling of resistance and associated treatment response heterogeneity in metastatic cancers.

Metastatic cancer patients invariably develop treatment resistance. Different levels of resistance lead to observed heterogeneity in treatment response. The main goal was to evaluate treatment response heterogeneity with a computation model simulating the dynamics of drug-sensitive and drug-resistant cells. Model parameters included proliferation, drug-induced death, transition and proportion of intrinsically resistant cells. The model was benchmarked with imaging metrics extracted from 39 metastatic prostate cancer patients who had 18F-NaF-PET/CT scans performed at baseline and at three cycles into chemotherapy or hormonal therapy. Two initial model assumptions were evaluated: considering only inter-patient heterogeneity and both inter-patient and intra-patient heterogeneity in the proportion of intrinsically resistant cells. The correlation between the median proportion of intrinsically resistant cells and baseline patient-level imaging metrics was assessed with Spearman's rank correlation coefficient. The impact of model parameters on simulated treatment response was evaluated with a sensitivity study. Treatment response after periods of six, nine, and 12 months was predicted with the model. The median predicted range of response for patients treated with both therapies was compared with a Wilcoxon rank sum test. For each patient, the time was calculated when the proportion of disease with a non-favourable response outperformed a favourable response. By taking into account inter-patient and intra-patient heterogeneity in the proportion of intrinsically resistant cells, the model performed significantly better ([Formula: see text]) than by taking into account only inter-patient heterogeneity ([Formula: see text]). The median proportion of intrinsically resistant cells showed a moderate correlation (ρ = 0.55) with mean patient-level uptake, and a low correlation (ρ = 0.36) with the dispersion of mean metastasis-level uptake in a patient. The sensitivity study showed a strong impact of the proportion of intrinsically resistant cells on model behaviour after three cycles of therapy. The difference in the median range of response (MRR) was not significant between cohorts at any time point (p  > 0.15). The median time when the proportion of disease with a non-favourable response outperformed the favourable response was eight months, for both cohorts. The model provides an insight into inter-patient and intra-patient heterogeneity in the evolution of treatment resistance.

Immunization efficacy of cryopreserved genetically attenuated Plasmodium berghei sporozoites.

Malaria is transmitted through the injection of Plasmodium sporozoites into the skin by Anopheles mosquitoes. The parasites first replicate within the liver before infecting red blood cells, which leads to the symptoms of the disease. Experimental immunization with attenuated sporozoites that arrest their development in the liver has been extensively investigated in rodent models and humans. Recent technological advances have included the capacity to cryopreserve sporozoites for injection, which has enabled a series of controlled studies on human infection with sporozoites. Here, we used a cryopreservation protocol to test the efficiency of genetically attenuated cryopreserved sporozoites for immunization of mice in comparison with freshly isolated controls. This showed that cryopreserved sporozoites are highly viable as judged by their capacity to migrate in vitro but show only 20% efficiency in liver infection, which impacts their capacity to generate protection of animals in immunization experiments.

Subcellular and in-vivo Nano-Endoscopy.

Analysis of individual cells at the subcellular level is important for understanding diseases and accelerating drug discovery. Nanoscale endoscopes allow minimally invasive probing of individual cell interiors. Several such instruments have been presented previously, but they are either too complex to fabricate or require sophisticated external detectors because of low signal collection efficiency. Here we present a nanoendoscope that can locally excite fluorescence in labelled cell organelles and collect the emitted signal for spectral analysis. Finite Difference Time Domain (FDTD) simulations have shown that with an optimized nanoendoscope taper profile, the light emission and collection was localized within ~100 nm. This allows signal detection to be used for nano-photonic sensing of the proximity of fluorophores. Upon insertion into the individual organelles of living cells, the nanoendoscope was fabricated and resultant fluorescent signals collected. This included the signal collection from the nucleus of Acridine orange labelled human fibroblast cells, the nucleus of Hoechst stained live liver cells and the mitochondria of MitoTracker Red labelled MDA-MB-231 cells. The endoscope was also inserted into a live organism, the yellow fluorescent protein producing nematode Caenorhabditis elegans, and a fluorescent signal was collected. To our knowledge this is the first demonstration of in vivo, local fluorescence signal collection on the sub-organelle level.