Enhancer-targeted CRISPR-Activation Rescues Haploinsufficient Autism Susceptibility Genes.

Autism Spectrum Disorder (ASD) is a highly heritable condition with diverse clinical presentations. Approximately 20% of ASD's genetic susceptibility is imparted by de novo mutations of major effect, most of which cause haploinsufficiency. We mapped enhancers of two high confidence autism genes - CHD8 and SCN2A and used CRISPR-based gene activation (CRISPR-A) in hPSC-derived excitatory neurons and cerebral forebrain organoids to correct the effects of haploinsufficiency, taking advantage of the presence of a wildtype allele of each gene and endogenous gene regulation. We found that CRISPR-A induced a sustained increase in CHD8 and SCN2A expression in treated neurons and organoids, with rescue of gene expression levels and mutation-associated phenotypes, including gene expression and physiology. These data support gene activation via targeting enhancers of haploinsufficient genes, as a therapeutic intervention in ASD and other neurodevelopmental disorders.

Broad transcriptomic dysregulation occurs across the cerebral cortex in ASD.

Neuropsychiatric disorders classically lack defining brain pathologies, but recent work has demonstrated dysregulation at the molecular level, characterized by transcriptomic and epigenetic alterations1-3. In autism spectrum disorder (ASD), this molecular pathology involves the upregulation of microglial, astrocyte and neural-immune genes, the downregulation of synaptic genes, and attenuation of gene-expression gradients in cortex1,2,4-6. However, whether these changes are limited to cortical association regions or are more widespread remains unknown. To address this issue, we performed RNA-sequencing analysis of 725 brain samples spanning 11 cortical areas from 112 post-mortem samples from individuals with ASD and neurotypical controls. We find widespread transcriptomic changes across the cortex in ASD, exhibiting an anterior-to-posterior gradient, with the greatest differences in primary visual cortex, coincident with an attenuation of the typical transcriptomic differences between cortical regions. Single-nucleus RNA-sequencing and methylation profiling demonstrate that this robust molecular signature reflects changes in cell-type-specific gene expression, particularly affecting excitatory neurons and glia. Both rare and common ASD-associated genetic variation converge within a downregulated co-expression module involving synaptic signalling, and common variation alone is enriched within a module of upregulated protein chaperone genes. These results highlight widespread molecular changes across the cerebral cortex in ASD, extending beyond association cortex to broadly involve primary sensory regions.

Challenges and opportunities for precision medicine in neurodevelopmental disorders.

Neurodevelopmental Disorders (NDDs) encompass a broad spectrum of disorders, linked because of their origins in brain developmental processes, including diverse conditions across the age span, including autism spectrum disorders (ASD) and schizophrenia (SCZ). Clinical treatment of these disorders has traditionally focused on symptom management, as the severity of developmental disruption varies widely and the precise molecular mechanisms, timing, and progression of these disorders is usually not known. Several hundred genes have been identified as major risk factors for ASD and SCZ, which creates new potential therapeutic avenues, and there is strong evidence that these genes converge upon key molecular pathways, pointing to opportunities for precision medicine. In this review, we focus on forms of ASD and SCZ with known genetic etiologies and discuss advances in research technologies that enable a more systemic understanding of disease progression. We highlight recent advances in targeted clinical treatment and discuss ongoing preclinical efforts as well as new initiatives aimed at developing scalable platforms for NDD precision medicine.

Disruption of ß-Catenin-Dependent Wnt Signaling in Colon Cancer Cells Remodels the Microenvironment to Promote Tumor Invasion.

The recent classification of colon cancer into molecular subtypes revealed that patients with the poorest prognosis harbor tumors with the lowest levels of Wnt signaling. This is contrary to the general understanding that overactive Wnt signaling promotes tumor progression from early initiation stages through to the later stages including invasion and metastasis. Here, we directly test this assumption by reducing the activity of ß-catenin-dependent Wnt signaling in colon cancer cell lines at either an upstream or downstream step in the pathway. We determine that Wnt-reduced cancer cells exhibit a more aggressive disease phenotype, including increased mobility in vitro and disruptive invasion into mucosa and smooth muscle in an orthotopic mouse model. RNA sequencing reveals that interference with Wnt signaling leads to an upregulation of gene programs that favor cell migration and invasion and a downregulation of inflammation signatures in the tumor microenvironment. We identify a set of upregulated genes common among the Wnt perturbations that are predictive of poor patient outcomes in early-invasive colon cancer. Our findings suggest that while targeting Wnt signaling may reduce tumor burden, an inadvertent side effect is the emergence of invasive cancer. IMPLICATIONS: Decreased Wnt signaling in colon tumors leads to a more aggressive disease phenotype due to an upregulation of gene programs favoring cell migration in the tumor and downregulation of inflammation programs in the tumor microenvironment; these impacts must be carefully considered in developing Wnt-targeting therapies.

An in vitro vascularized micro-tumor model of human colorectal cancer recapitulates in vivo responses to standard-of-care therapy.

Around 95% of anti-cancer drugs that show promise during preclinical study fail to gain FDA-approval for clinical use. This failure of the preclinical pipeline highlights the need for improved, physiologically-relevant in vitro models that can better serve as reliable drug-screening and disease modeling tools. The vascularized micro-tumor (VMT) is a novel three-dimensional model system (tumor-on-a-chip) that recapitulates the complex human tumor microenvironment, including perfused vasculature, within a transparent microfluidic device, allowing real-time study of drug responses and tumor-stromal interactions. Here we have validated this microphysiological system (MPS) platform for the study of colorectal cancer (CRC), the second leading cause of cancer-related deaths, by showing that gene expression, tumor heterogeneity, and treatment responses in the VMT more closely model CRC tumor clinicopathology than current standard drug screening modalities, including 2-dimensional monolayer culture and 3-dimensional spheroids.

Inhibition of nuclear Wnt signalling: challenges of an elusive target for cancer therapy.

The highly conserved Wnt signalling pathway plays an important role in embryonic development and disease pathogenesis, most notably cancer. The 'canonical' or ß-catenin-dependent Wnt signal initiates at the cell plasma membrane with the binding of Wnt proteins to Frizzled:LRP5/LRP6 receptor complexes and is mediated by the translocation of the transcription co-activator protein, ß-catenin, into the nucleus. ß-Catenin then forms a complex with T-cell factor (TCF)/lymphoid enhancer binding factor (LEF) transcription factors to regulate multiple gene programmes. These programmes play roles in cell proliferation, migration, vasculogenesis, survival and metabolism. Mutations in Wnt signalling pathway components lead to constitutively active Wnt signalling that drives aberrant expression of these programmes and development of cancer. It has been a longstanding and challenging goal to develop therapies that can interfere with the TCF/LEF-ß-catenin transcriptional complex. This review will focus on the (i) structural considerations for targeting the TCF/LEF-ß-catenin and co-regulatory complexes in the nucleus, (ii) current molecules that directly target TCF/LEF-ß-catenin activity and (iii) ideas for targeting newly discovered components of the TCF/LEF-ß-catenin complex and/or downstream gene programmes regulated by these complexes. LINKED ARTICLES: This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc.

Mathematical modeling links Wnt signaling to emergent patterns of metabolism in colon cancer.

Cell-intrinsic metabolic reprogramming is a hallmark of cancer that provides anabolic support to cell proliferation. How reprogramming influences tumor heterogeneity or drug sensitivities is not well understood. Here, we report a self-organizing spatial pattern of glycolysis in xenograft colon tumors where pyruvate dehydrogenase kinase (PDK1), a negative regulator of oxidative phosphorylation, is highly active in clusters of cells arranged in a spotted array. To understand this pattern, we developed a reaction-diffusion model that incorporates Wnt signaling, a pathway known to upregulate PDK1 and Warburg metabolism. Partial interference with Wnt alters the size and intensity of the spotted pattern in tumors and in the model. The model predicts that Wnt inhibition should trigger an increase in proteins that enhance the range of Wnt ligand diffusion. Not only was this prediction validated in xenograft tumors but similar patterns also emerge in radiochemotherapy-treated colorectal cancer. The model also predicts that inhibitors that target glycolysis or Wnt signaling in combination should synergize and be more effective than each treatment individually. We validated this prediction in 3D colon tumor spheroids.

A serial 4DCT study to quantify range variations in charged particle radiotherapy of thoracic cancers.

Weekly serial 4DCT scans were acquired under free breathing conditions to assess water-equivalent path length (WEL) variations due to both intrafractional and interfractional changes in tissue thickness and density and to calculate proton dose distributions resulting from anatomical variations observed in serial 4DCT. A template of region of interests (ROIs) was defined on the anterior-posterior (AP) beam's eye view, and WEL measurements were made over these ROIs to quantify chest wall thickness variations. Interfractional proton dose distributions were calculated to assess changes in the expected dose distributions caused by range variations. Mean intrafractional chest wall WEL changes during respiration varied by: -4.1 mm (<-10.2 mm), -3.6 mm (<-7.1 mm), -3.2 mm (<-5.6 mm) and -2.5 mm (<-5.1 mm) during respiration in the ITV, upper, middle and lower lung regions, respectively. The mean interfractional chest wall WEL variation at Week 6 decreased by -4.0 mm (<-8.6 mm), -9.1 mm (<-17.9 mm), -9.4 mm (<-25.3 mm) and -4.5 mm (<-15.6 mm) in the ITV, upper, middle and lower lung regions, respectively. The variations were decomposed into anterior and posterior chest wall thickness changes. Dose overshoot beyond the target was observed when the initial boli was applied throughout the treatment course. This overshoot is due to chest wall thickness variations and target positional variations. The radiological path length can vary significantly during respiration as well as over the course of several weeks of charged particle therapy. Intrafractional/interfractional chest wall thickness changes can be a significant source of range variation in treatment of lung tumors with charged particle beams, resulting in dose distribution perturbations from the initial plan. Consideration of these range variations should be made in choosing the therapeutic charged particle beam range.

A four-dimensional computed tomography analysis of multiorgan abdominal motion.

PURPOSE: To characterize and quantify multiorgan respiration-induced motion in the abdomen in liver and pancreatic cancer patients. METHODS AND MATERIALS: Four-dimensional computed tomography scans were acquired for 18 patients treated for abdominal tumors. Contours of multiple abdominal organs were drawn by the radiation oncologist at one respiratory phase; these contours were propagated to other respiratory phases by deformable registration. Three-dimensional organ models were generated from the resulting contours at each phase. Motions of the bounding box and center of mass were extracted and analyzed for the clinical target volume and organs at risk. RESULTS: On average, the center of mass motion for liver clinical target volumes was 9.7 mm (SD 5 mm) in the superior-inferior direction, with a range of 3 to 18 mm; for pancreatic tumors, the average was 5 mm (SD 1 mm) m with a range of 3 to 7 mm. Abdominal organs move in unison, but with varying amplitudes. Gating near exhale (T40-T60) reduces the range of motion by a factor of ∼10. CONCLUSION: We have used deformable registration to calculate the trajectories of abdominal organs in four dimensions, based on center of mass and bounding box motion metrics. Our results are compared with previously reported studies. Possible reasons for differences are discussed.

Proton radiography and fluoroscopy of lung tumors: a Monte Carlo study using patient-specific 4DCT phantoms.

PURPOSE: Monte Carlo methods are used to simulate and optimize a time-resolved proton range telescope (TRRT) in localization of intrafractional and interfractional motions of lung tumor and in quantification of proton range variations. METHODS: The Monte Carlo N-Particle eXtended (MCNPX) code with a particle tracking feature was employed to evaluate the TRRT performance, especially in visualizing and quantifying proton range variations during respiration. Protons of 230 MeV were tracked one by one as they pass through position detectors, patient 4DCT phantom, and finally scintillator detectors that measured residual ranges. The energy response of the scintillator telescope was investigated. Mass density and elemental composition of tissues were defined for 4DCT data. RESULTS: Proton water equivalent length (WEL) was deduced by a reconstruction algorithm that incorporates linear proton track and lateral spatial discrimination to improve the image quality. 4DCT data for three patients were used to visualize and measure tumor motion and WEL variations. The tumor trajectories extracted from the WEL map were found to be within 1 mm agreement with direct 4DCT measurement. Quantitative WEL variation studies showed that the proton radiograph is a good representation of WEL changes from entrance to distal of the target. CONCLUSIONS: MCNPX simulation results showed that TRRT can accurately track the motion of the tumor and detect the WEL variations. Image quality was optimized by choosing proton energy, testing parameters of image reconstruction algorithm, and comparing to ground truth 4DCT. The future study will demonstrate the feasibility of using the time resolved proton radiography as an imaging tool for proton treatments of lung tumors.

Uncertainties in lung motion prediction relying on external surrogate: a 4DCT study in regular vs. irregular breathers.

This paper examines the uncertainty in estimating lung motion from external surrogates for lung cancer patients with regular and irregular breathing. 4DCT data sets were analyzed using a template matching algorithm to track the spatial movement of vessel bifurcations in 12 patients. The detected internal movement of features in 3D was retrospectively synchronized with the RPM surrogate signal, and the correlation index R(2) and the prediction error were computed. Patients were classified into two groups depending on the presence or not of irregularities in their breathing pattern. Peak-to-peak values of feature motion in the SI direction ranged from 0.8 mm (upper lung) to 25.3 mm (lower lung). Some patients exhibited large motion also in the latero-lateral (10.6 mm) and anterior-posterior (12.2 mm) directions. The median +/- quartile of R(2) in SI direction was 0.89 +/- 0.09. Prediction error values were up to 4.2 mm (95th percentile) with a maximum value of 4.9 mm. Statistical differences between regular and irregular breathers were found for R(2), while prediction error depended only on the range of motion. This study is relevant for image guided radiotherapy methods that rely on external surrogates to monitor motion.

Variations in tumor size and position due to irregular breathing in 4D-CT: a simulation study.

PURPOSE: To estimate the position and volume errors in 4D-CT caused by irregular breathing. METHODS: A virtual 4D-CT scanner was designed to reproduce axial mode scans with retrospective resorting. This virtual scanner creates an artificial spherical tumor based on the specifications of the user, and recreates images that might be produced by a 4D-CT scanner using a patient breathing waveform. 155 respiratory waveforms of patients were used to test the variability of 4D-CT scans. Each breathing waveform was normalized and scaled to 1, 2, and 3 cm peak-to-peak motion, and artificial tumors with 2 and 4 cm radius were simulated for each scaled waveform. The center of mass and volume of resorted 4D-CT images were calculated and compared to the expected values of center of mass and volume for the artificial tumor. Intrasubject variability was investigated by running the virtual scanner over different subintervals of each patient's breathing waveform. RESULTS: The average error in the center of mass location of an artificial tumor was less than 2 mm standard deviation for 2 cm motion. The corresponding average error in volume was less than 4%. In the worst-case scenarios, a center of mass error of 1.0 cm standard deviation and volume errors of 30%-60% at inhale were found. Systematic errors were observed in a subset of patients due to irregular breathing, and these errors were more pronounced when the tumor volume is smaller. CONCLUSIONS: Irregular breathing during 4D-CT simulation causes systematic errors in volume and center of mass measurements. These errors are small but depend on the tumor size, motion amplitude, and degree of breathing irregularity.

Evaluation and commissioning of a surface based system for respiratory sensing in 4D CT.

The purpose of this study is to assess the temporal and reconstruction accuracy of a surface imaging system, the GateCT under ideal conditions, and compare the device with a commonly used respiratory surrogate: the Varian RPM. A clinical CT scanner, run in cine mode, was used with two optical devices, GateCT and RPM, to detect respiratory motion. A radiation detector, GM-10, triggers the X-ray on/off to GateCT system, while the RPM is directly synchronized with the CT scanner through an electronic connection. Two phantoms were imaged: the first phantom translated on a rigid plate along the anterior-posterior (AP) direction, and was used to assess the temporal synchronization of each optical system with the CT scanner. The second phantom, consisting of five spheres translating 3 cm peak-to-peak in the superior-inferior direction, was used to assess the quality of rebinned images created by GateCT and RPM. Calibration assessment showed a nearly perfect synchronization with the scanner for both the RPM and GateCT systems, thus demonstrating the good performance of the radiation detector. Results for the volume rebinning test showed discrepancies in volumes for the 3D reconstruction (compared to ground truth) of up to 36% for GateCT and up to 40% for RPM. No statistical difference was proven between the two systems in volume sorting. Errors are mainly due to phase detection inaccuracies and to the large motion of the phantom. This feasibility study assessed the consistency of two optical systems in synchronizing the respiratory signal with the image acquisition. A new patient protocol based on both RPM and GateCT will be soon started.

Effects of interfractional anatomical changes on water-equivalent pathlength in charged-particle radiotherapy of lung cancer.

Intrafractional motion and interfractional changes affect the accuracy of the delivered dose in radiotherapy, particularly in charged-particle radiotherapy. Most recent studies are focused on intrafractional motion (respiratory motion). Here, we report a quantitative simulation analysis of the effects of interfractional changes on water-equivalent pathlength (WEL) in charged-particle lung therapy. Serial four-dimensional (4D) CT scans were performed under free breathing conditions; the time span between the first and second 4DCT scans was five weeks. We quantified WEL changes between the first and second CT scans due to interfractional changes (tumor shrinkage and tissue density changes) and compared the particle-beam-stopping point between the serial 4DCT scans with use of the same initial bolus. Both tumor-shrinkage and lung-density changes were observed in a single patient over the course of therapy. The lung density decreased by approximately 0.1 g/cm(3) between the first and second-CT scans, resulting in a 1.5 cm WEL changes. Tumor shrinkage resulted in approximately 3 cm WEL changes. If the same initial bolus and plan were used through the treatment course, an unexpected significant beam overshoot would occur by interfractional changes due to tumor shrinkage and lung density variation.

Exploration of the potential of liquid scintillators for real-time 3D dosimetry of intensity modulated proton beams.

In this study, the authors investigated the feasibility of using a 3D liquid scintillator (LS) detector system for the verification and characterization of proton beams in real time for intensity and energy-modulated proton therapy. A plastic tank filled with liquid scintillator was irradiated with pristine proton Bragg peaks. Scintillation light produced during the irradiation was measured with a CCD camera. Acquisition rates of 20 and 10 frames per second (fps) were used to image consecutive frame sequences. These measurements were then compared to ion chamber measurements and Monte Carlo simulations. The light distribution measured from the images acquired at rates of 20 and 10 fps have standard deviations of 1.1% and 0.7%, respectively, in the plateau region of the Bragg curve. Differences were seen between the raw LS signal and the ion chamber due to the quenching effects of the LS and due to the optical properties of the imaging system. The authors showed that this effect can be accounted for and corrected by Monte Carlo simulations. The liquid scintillator detector system has a good potential for performing fast proton beam verification and characterization.

Accuracy in breast shape alignment with 3D surface fitting algorithms.

Surface imaging is in use in radiotherapy clinical practice for patient setup optimization and monitoring. Breast alignment is accomplished by searching for a tentative spatial correspondence between the reference and daily surface shape models. In this study, the authors quantify whole breast shape alignment by relying on texture features digitized on 3D surface models. Texture feature localization was validated through repeated measurements in a silicone breast phantom, mounted on a high precision mechanical stage. Clinical investigations on breast shape alignment included 133 fractions in 18 patients treated with accelerated partial breast irradiation. The breast shape was detected with a 3D video based surface imaging system so that breathing was compensated. An in-house algorithm for breast alignment, based on surface fitting constrained by nipple matching (constrained surface fitting), was applied. Results were compared with a commercial software where no constraints are utilized (unconstrained surface fitting). Texture feature localization was validated within 2 mm in each anatomical direction. Clinical data show that unconstrained surface fitting achieves adequate accuracy in most cases, though nipple mismatch is considerably higher than residual surface distances (3.9 mm vs 0.6 mm on average). Outliers beyond 1 cm can be experienced as the result of a degenerate surface fit, where unconstrained surface fitting is not sufficient to establish spatial correspondence. In the constrained surface fitting algorithm, average surface mismatch within 1 mm was obtained when nipple position was forced to match in the [1.5; 5] mm range. In conclusion, optimal results can be obtained by trading off the desired overall surface congruence vs matching of selected landmarks (constraint). Constrained surface fitting is put forward to represent an improvement in setup accuracy for those applications where whole breast positional reproducibility is an issue.

A review of image-guided radiotherapy.

Image-guided radiotherapy (IGRT) is in the midst of a strong development and implementation cycle, stimulated by pioneering work performed in Japan. We present a review of the rationale, technology, and methodology of image guidance, as well as an overview of current work in IGRT at the Massachusetts General Hospital. The technology is rapidly evolving, and synergisms between the various acquisition approaches are converging to provide unparalleled information on target and normal tissue location and motion. With these new approaches to patient localization, we expect improved clinical results to be forthcoming.