Deep learning-enabled detection of rare circulating tumor cell clusters in whole blood using label-free, flow cytometry.

Metastatic tumors have poor prognoses for progression-free and overall survival for all cancer patients. Rare circulating tumor cells (CTCs) and rarer circulating tumor cell clusters (CTCCs) are potential biomarkers of metastatic growth, with CTCCs representing an increased risk factor for metastasis. Current detection platforms are optimized for ex vivo detection of CTCs only. Microfluidic chips and size exclusion methods have been proposed for CTCC detection; however, they lack in vivo utility and real-time monitoring capability. Confocal backscatter and fluorescence flow cytometry (BSFC) has been used for label-free detection of CTCCs in whole blood based on machine learning (ML) enabled peak classification. Here, we expand to a deep-learning (DL)-based, peak detection and classification model to detect CTCCs in whole blood data. We demonstrate that DL-based BSFC has a low false alarm rate of 0.78 events per min with a high Pearson correlation coefficient of 0.943 between detected events and expected events. DL-based BSFC of whole blood maintains a detection purity of 72% and a sensitivity of 35.3% for both homotypic and heterotypic CTCCs starting at a minimum size of two cells. We also demonstrate through artificial spiking studies that DL-based BSFC is sensitive to changes in the number of CTCCs present in the samples and does not add variability in detection beyond the expected variability from Poisson statistics. The performance established by DL-based BSFC motivates its use for in vivo detection of CTCCs. Using transfer learning, we additionally validate DL-based BSFC on blood samples from different species and cancer cell types. Further developments of label-free BSFC to enhance throughput could lead to critical applications in the clinical detection of CTCCs and ex vivo isolation of CTCC from whole blood with minimal disruption and processing steps.

A mitochondrial NADPH-cholesterol axis regulates extracellular vesicle biogenesis to support hematopoietic stem cell fate.

Mitochondrial fatty acid oxidation (FAO) is essential for hematopoietic stem cell (HSC) self-renewal; however, the mechanism by which mitochondrial metabolism controls HSC fate remains unknown. Here, we show that within the hematopoietic lineage, HSCs have the largest mitochondrial NADPH pools, which are required for proper HSC cell fate and homeostasis. Bioinformatic analysis of the HSC transcriptome, biochemical assays, and genetic inactivation of FAO all indicate that FAO-generated NADPH fuels cholesterol synthesis in HSCs. Interference with FAO disturbs the segregation of mitochondrial NADPH toward corresponding daughter cells upon single HSC division. Importantly, we have found that the FAO-NADPH-cholesterol axis drives extracellular vesicle (EV) biogenesis and release in HSCs, while inhibition of EV signaling impairs HSC self-renewal. These data reveal the existence of a mitochondrial NADPH-cholesterol axis for EV biogenesis that is required for hematopoietic homeostasis and highlight the non-stochastic nature of HSC fate determination.

Restoration of metabolic functional metrics from label-free, two-photon human tissue images using multiscale deep-learning-based denoising algorithms.

Significance: Label-free, two-photon excited fluorescence (TPEF) imaging captures morphological and functional metabolic tissue changes and enables enhanced understanding of numerous diseases. However, noise and other artifacts present in these images severely complicate the extraction of biologically useful information. Aim: We aim to employ deep neural architectures in the synthesis of a multiscale denoising algorithm optimized for restoring metrics of metabolic activity from low-signal-to-noise ratio (SNR), TPEF images. Approach: TPEF images of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavoproteins (FAD) from freshly excised human cervical tissues are used to assess the impact of various denoising models, preprocessing methods, and data on metrics of image quality and the recovery of six metrics of metabolic function from the images relative to ground truth images. Results: Optimized recovery of the redox ratio and mitochondrial organization is achieved using a novel algorithm based on deep denoising in the wavelet transform domain. This algorithm also leads to significant improvements in peak-SNR (PSNR) and structural similarity index measure (SSIM) for all images. Interestingly, other models yield even higher PSNR and SSIM improvements, but they are not optimal for recovery of metabolic function metrics. Conclusions: Denoising algorithms can recover diagnostically useful information from low SNR label-free TPEF images and will be useful for the clinical translation of such imaging.

Extracellular Hsp90 Binds to and Aligns Collagen-1 to Enhance Breast Cancer Cell Invasiveness.

Cancer cell-secreted eHsp90 binds and activates proteins in the tumor microenvironment crucial in cancer invasion. Therefore, targeting eHsp90 could inhibit invasion, preventing metastasis-the leading cause of cancer-related mortality. Previous eHsp90 studies have solely focused on its role in cancer invasion through the 2D basement membrane (BM), a form of extracellular matrix (ECM) that lines the epithelial compartment. However, its role in cancer invasion through the 3D Interstitial Matrix (IM), an ECM beyond the BM, remains unexplored. Using a Collagen-1 binding assay and second harmonic generation (SHG) imaging, we demonstrate that eHsp90 directly binds and aligns Collagen-1 fibers, the primary component of IM. Furthermore, we show that eHsp90 enhances Collagen-1 invasion of breast cancer cells in the Transwell assay. Using Hsp90 conformation mutants and inhibitors, we established that the Hsp90 dimer binds to Collagen-1 via its N-domain. We also demonstrated that while Collagen-1 binding and alignment are not influenced by Hsp90's ATPase activity attributed to the N-domain, its open conformation is crucial for increasing Collagen-1 alignment and promoting breast cancer cell invasion. These findings unveil a novel role for eHsp90 in invasion through the IM and offer valuable mechanistic insights into potential therapeutic approaches for inhibiting Hsp90 to suppress invasion and metastasis.

Formalin fixation and paraffin embedding interfere with the preservation of optical metabolic assessments based on endogenous NAD(P)H and FAD two-photon excited fluorescence.

Endogenous NAD(P)H and FAD two-photon excited fluorescence (TPEF) images provide functional metabolic information with high spatial resolution for a wide range of living specimens. Preservation of metabolic function optical metrics upon fixation would facilitate studies which assess the impact of metabolic changes in the context of numerous diseases. However, robust assessments of the impact of formalin fixation, paraffin embedding, and sectioning on the preservation of optical metabolic readouts are lacking. Here, we evaluate intensity and lifetime images at excitation/emission settings optimized for NAD(P)H and FAD TPEF detection from freshly excised murine oral epithelia and corresponding bulk and sectioned fixed tissues. We find that fixation impacts the overall intensity as well as the intensity fluctuations of the images acquired. Accordingly, the depth-dependent variations of the optical redox ratio (defined as FAD/(NAD(P)H + FAD)) across squamous epithelia are not preserved following fixation. This is consistent with significant changes in the 755 nm excited spectra, which reveal broadening upon fixation and additional distortions upon paraffin embedding and sectioning. Analysis of fluorescence lifetime images acquired for excitation/emission settings optimized for NAD(P)H TPEF detection indicate that fixation alters the long lifetime of the observed fluorescence and the long lifetime intensity fraction. These parameters as well as the short TPEF lifetime are significantly modified upon embedding and sectioning. Thus, our studies highlight that the autofluorescence products formed during formalin fixation, paraffin embedding and sectioning overlap highly with NAD(P)H and FAD emission and limit the potential to utilize such tissues to assess metabolic activity.

Evaluation of a polarization-enhanced laparoscopy prototype for improved intra-operative visualization of peritoneal metastases.

Despite careful staging, the accuracy for preoperative detection of small distant metastases remains poor, creating a clinical need for enhanced operative staging to detect occult peritoneal metastases. This study evaluates a polarization-enhanced laparoscopy (PEL) prototype and assesses its potential for label-free contrast enhancement of peritoneal metastases. This is a first-in-human feasibility study, including 10 adult patients who underwent standard staging laparoscopy (SSL) for gastrointestinal malignancy along with PEL. Image frames of all detectable peritoneal lesions underwent analysis. Using Monte Carlo simulations, contrast enhancement based on the color dependence of PEL (mPEL) was assessed. The prototype performed safely, yet with limitations in illumination, fogging of the distal window, and image co-registration. Sixty-five lesions (56 presumed benign and 9 presumed malignant) from 3 patients represented the study sample. While most lesions were visible under human examination of both SSL and PEL videos, more lesions were apparent using SSL. However, this was likely due to reduced illumination under PEL. When controlling for such effects through direct comparisons of integrated (WLL) vs differential (PEL) polarization laparoscopy images, we found that PEL imaging yielded an over twofold Weber contrast enhancement over WLL. Further, enhancements in the discrimination between malignant and benign lesions were achieved by exploiting the PEL color contrast to enhance sensitivity to tissue scattering, influenced primarily by collagen. In conclusion, PEL appears safe and easy to integrate into the operating room. When controlling for the degree of illumination, image analysis suggested a potential for mPEL to provide improved visualization of metastases.

Deep Learning-Enabled, Detection of Rare Circulating Tumor Cell Clusters in Whole Blood Using Label-free, Flow Cytometry.

Metastatic tumors have poor prognoses for progression-free and overall survival for all cancer patients. Rare circulating tumor cells (CTCs) and rarer circulating tumor cell clusters (CTCCs) are potential biomarkers of metastatic growth, with CTCCs representing an increased risk factor for metastasis. Current detection platforms are optimized for ex vivo detection of CTCs only. Microfluidic chips and size exclusion methods have been proposed for CTCC detection; however, they lack in vivo utility and real-time monitoring capability. Confocal backscatter and fluorescence flow cytometry (BSFC) has been used for label-free detection of CTCCs in whole blood based on machine learning (ML) enabled peak classification. Here, we expand to a deep-learning (DL) -based, peak detection and classification model to detect CTCCs in whole blood data. We demonstrate that DL-based BSFC has a low false alarm rate of 0.78 events/min with a high Pearson correlation coefficient of 0.943 between detected events and expected events. DL-based BSFC of whole blood maintains a detection purity of 72% and a sensitivity of 35.3% for both homotypic and heterotypic CTCCs starting at a minimum size of two cells. We also demonstrate through artificial spiking studies that DL-based BSFC is sensitive to changes in the number of CTCCs present in the samples and does not add variability in detection beyond the expected variability from Poisson statistics. The performance established by DL-based BSFC motivates its use for in vivo detection of CTCCs. Further developments of label-free BSFC to enhance throughput could lead to critical applications in the clinical detection of CTCCs and ex vivo isolation of CTCC from whole blood with minimal disruption and processing steps.

Mitochondria dysregulation contributes to secondary neurodegeneration progression post-contusion injury in human 3D in vitro triculture brain tissue model.

Traumatic Brain injury-induced disturbances in mitochondrial fission-and-fusion dynamics have been linked to the onset and propagation of neuroinflammation and neurodegeneration. However, cell-type-specific contributions and crosstalk between neurons, microglia, and astrocytes in mitochondria-driven neurodegeneration after brain injury remain undefined. We developed a human three-dimensional in vitro triculture tissue model of a contusion injury composed of neurons, microglia, and astrocytes and examined the contributions of mitochondrial dysregulation to neuroinflammation and progression of injury-induced neurodegeneration. Pharmacological studies presented here suggest that fragmented mitochondria released by microglia are a key contributor to secondary neuronal damage progression after contusion injury, a pathway that requires astrocyte-microglia crosstalk. Controlling mitochondrial dysfunction thus offers an exciting option for developing therapies for TBI patients.

Non-invasive Imaging Techniques for Monitoring Cellular Response to Treatment in Stable Vitiligo.

Punch grafting procedures, where small pieces of normal skin are transplanted into stable vitiligo patches, results in repigmentation in only half of patients treated, yet the factors that determine whether a patient responds to treatment or not are still unknown. Reflectance confocal microscopy (RCM) is adept at visualizing melanocyte migration and epidermal changes over large areas while multiphoton microscopy (MPM) can capture metabolic changes in keratinocytes. With the overall goal of identifying optical biomarkers for early treatment response, we followed 12 vitiligo lesions undergoing punch grafting. Dendritic melanocytes adjacent to the graft site were observed before clinical evidence of repigmentation in treatment responsive patients but not in treatment non-responsive patients, suggesting that the early visualization of melanocytes is indicative of a therapeutic response. Keratinocyte metabolic changes in vitiligo skin adjacent to the graft site also correlated with treatment response, indicating that a keratinocyte microenvironment that more closely resembles normal skin is more hospitable for migrating melanocytes. Taken together, these studies suggest that successful melanocyte transplantation requires both the introduction of new melanocytes and modulation of the local tissue microenvironment.

Formalin fixation and paraffin embedding interfere with preservation of optical metabolic assessments based on endogenous NAD(P)H and FAD two photon excited fluorescence.

Endogenous NAD(P)H and FAD two-photon excited fluorescence (TPEF) images provide functional metabolic information with high spatial resolution for a wide range of living specimens. Preservation of metabolic function optical metrics upon fixation would facilitate studies which assess the impact of metabolic changes in the context of numerous diseases. However, robust assessments of the impact of formalin fixation, paraffin embedding, and sectioning on the preservation of optical metabolic readouts are lacking. Here, we evaluate intensity and lifetime images at excitation/emission settings optimized for NAD(P)H and FAD TPEF detection from freshly excised murine oral epithelia and corresponding bulk and sectioned fixed tissues. We find that fixation impacts the overall intensity as well as the intensity fluctuations of the images acquired. Accordingly, the depth-dependent variations of the optical redox ratio (defined as FAD/(NAD(P)H + FAD)) across squamous epithelia are not preserved following fixation. This is consistent with significant changes in the 755 nm excited spectra, which reveal broadening upon fixation and additional distortions upon paraffin embedding and sectioning. Analysis of fluorescence lifetime images acquired for excitation/emission settings optimized for NAD(P)H TPEF detection indicate that fixation alters the long lifetime of the observed fluorescence and the long lifetime intensity fraction. These parameters as well as the short TPEF lifetime are significantly modified upon embedding and sectioning. Thus, our studies highlight that the autofluorescence products formed during formalin fixation, paraffin embedding and sectioning overlap highly with NAD(P)H and FAD emission and limit the potential to utilize such tissues to assess metabolic activity.

Restoration of metabolic functional metrics from label-free, two-photon cervical tissue images using multiscale deep-learning-based denoising algorithms.

Label-free, two-photon imaging captures morphological and functional metabolic tissue changes and enables enhanced understanding of numerous diseases. However, this modality suffers from low signal arising from limitations imposed by the maximum permissible dose of illumination and the need for rapid image acquisition to avoid motion artifacts. Recently, deep learning methods have been developed to facilitate the extraction of quantitative information from such images. Here, we employ deep neural architectures in the synthesis of a multiscale denoising algorithm optimized for restoring metrics of metabolic activity from low-SNR, two-photon images. Two-photon excited fluorescence (TPEF) images of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavoproteins (FAD) from freshly excised human cervical tissues are used. We assess the impact of the specific denoising model, loss function, data transformation, and training dataset on established metrics of image restoration when comparing denoised single frame images with corresponding six frame averages, considered as the ground truth. We further assess the restoration accuracy of six metrics of metabolic function from the denoised images relative to ground truth images. Using a novel algorithm based on deep denoising in the wavelet transform domain, we demonstrate optimal recovery of metabolic function metrics. Our results highlight the promise of denoising algorithms to recover diagnostically useful information from low SNR label-free two-photon images and their potential importance in the clinical translation of such imaging.

Label-Free Optical Metabolic Imaging in Cells and Tissues.

Over the last half century, the autofluorescence of the metabolic cofactors NADH (reduced nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) has been quantified in a variety of cell types and disease states. With the spread of nonlinear optical microscopy techniques in biomedical research, NADH and FAD imaging has offered an attractive solution to noninvasively monitor cell and tissue status and elucidate dynamic changes in cell or tissue metabolism. Various tools and methods to measure the temporal, spectral, and spatial properties of NADH and FAD autofluorescence have been developed. Specifically, an optical redox ratio of cofactor fluorescence intensities and NADH fluorescence lifetime parameters have been used in numerous applications, but significant work remains to mature this technology for understanding dynamic changes in metabolism. This article describes the current understanding of our optical sensitivity to different metabolic pathways and highlights current challenges in the field. Recent progress in addressing these challenges and acquiring more quantitative information in faster and more metabolically relevant formats is also discussed.

Label-free, multi-parametric assessments of cell metabolism and matrix remodeling within human and early-stage murine osteoarthritic articular cartilage.

Osteoarthritis (OA) is characterized by the progressive deterioration of articular cartilage, involving complicated cell-matrix interactions. Systematic investigations of dynamic cellular and matrix changes during OA progression are lacking. In this study, we use label-free two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging to assess cellular and extracellular matrix features of murine articular cartilage during several time points at early stages of OA development following destabilization of medial meniscus surgery. We detect significant changes in the organization of collagen fibers and crosslink-associated fluorescence of the superficial zone as early as one week following surgery. Such changes become significant within the deeper transitional and radial zones at later time-points, highlighting the importance of high spatial resolution. Cellular metabolic changes exhibit a highly dynamic behavior, and indicate metabolic reprogramming from enhanced oxidative phosphorylation to enhanced glycolysis or fatty acid oxidation over the ten-week observation period. The optical metabolic and matrix changes detected within this mouse model are consistent with differences identified in excised human cartilage specimens from OA and healthy cartilage specimens. Thus, our studies reveal important cell-matrix interactions at the onset of OA that may enable improved understanding of OA development and identification of new potential treatment targets.

Development of a novel bioengineered 3D brain-like tissue for studying primary blast-induced traumatic brain injury.

Primary blast injury is caused by the direct impact of an overpressurization wave on the body. Due to limitations of current models, we have developed a novel approach to study primary blast-induced traumatic brain injury. Specifically, we employ a bioengineered 3D brain-like human tissue culture system composed of collagen-infused silk protein donut-like hydrogels embedded with human IPSC-derived neurons, human astrocytes, and a human microglial cell line. We have utilized this system within an advanced blast simulator (ABS) to expose the 3D brain cultures to a blast wave that can be precisely controlled. These 3D cultures are enclosed in a 3D-printed surrogate skull-like material containing media which are then placed in a holder apparatus inside the ABS. This allows for exposure to the blast wave alone without any secondary injury occurring. We show that blast induces an increase in lactate dehydrogenase activity and glutamate release from the cultures, indicating cellular injury. Additionally, we observe a significant increase in axonal varicosities after blast. These varicosities can be stained with antibodies recognizing amyloid precursor protein. The presence of amyloid precursor protein deposits may indicate a blast-induced axonal transport deficit. After blast injury, we find a transient release of the known TBI biomarkers, UCHL1 and NF-H at 6 h and a delayed increase in S100B at 24 and 48 h. This in vitro model will enable us to gain a better understanding of clinically relevant pathological changes that occur following primary blast and can also be utilized for discovery and characterization of biomarkers.

Riboflavin depletion promotes longevity and metabolic hormesis in Caenorhabditis elegans.

Riboflavin is an essential cofactor in many enzymatic processes and in the production of flavin adenine dinucleotide (FAD). Here, we report that the partial depletion of riboflavin through knockdown of the C. elegans riboflavin transporter 1 (rft-1) promotes metabolic health by reducing intracellular flavin concentrations. Knockdown of rft-1 significantly increases lifespan in a manner dependent upon AMP-activated protein kinase (AMPK)/aak-2, the mitochondrial unfolded protein response, and FOXO/daf-16. Riboflavin depletion promotes altered energetic and redox states and increases adiposity, independent of lifespan genetic dependencies. Riboflavin-depleted animals also exhibit the activation of caloric restriction reporters without any reduction in caloric intake. Our findings indicate that riboflavin depletion activates an integrated hormetic response that promotes lifespan and healthspan in C. elegans.

Multimodal analyses of vitiligo skin identify tissue characteristics of stable disease.

Vitiligo is an autoimmune skin disease characterized by the destruction of melanocytes by autoreactive CD8+ T cells. Melanocyte destruction in active vitiligo is mediated by CD8+ T cells, but the persistence of white patches in stable disease is poorly understood. The interaction between immune cells, melanocytes, and keratinocytes in situ in human skin has been difficult to study due to the lack of proper tools. We combine noninvasive multiphoton microscopy (MPM) imaging and single-cell RNA-Seq (scRNA-Seq) to identify subpopulations of keratinocytes in stable vitiligo patients. We show that, compared with nonlesional skin, some keratinocyte subpopulations are enriched in lesional vitiligo skin and shift their energy utilization toward oxidative phosphorylation. Systematic investigation of cell-to-cell communication networks show that this small population of keratinocyte secrete CXCL9 and CXCL10 to potentially drive vitiligo persistence. Pseudotemporal dynamics analyses predict an alternative differentiation trajectory that generates this new population of keratinocytes in vitiligo skin. Further MPM imaging of patients undergoing punch grafting treatment showed that keratinocytes favoring oxidative phosphorylation persist in nonresponders but normalize in responders. In summary, we couple advanced imaging with transcriptomics and bioinformatics to discover cell-to-cell communication networks and keratinocyte cell states that can perpetuate inflammation and prevent repigmentation.

Label-free flow cytometry of rare circulating tumor cell clusters in whole blood.

Circulating tumor cell clusters (CTCCs) are rare cellular events found in the blood stream of metastatic tumor patients. Despite their scarcity, they represent an increased risk for metastasis. Label-free detection methods of these events remain primarily limited to in vitro microfluidic platforms. Here, we expand on the use of confocal backscatter and fluorescence flow cytometry (BSFC) for label-free detection of CTCCs in whole blood using machine learning for peak detection/classification. BSFC uses a custom-built flow cytometer with three excitation wavelengths (405 nm, 488 nm, and 633 nm) and five detectors to detect CTCCs in whole blood based on corresponding scattering and fluorescence signals. In this study, detection of CTCC-associated GFP fluorescence is used as the ground truth to assess the accuracy of endogenous back-scattered light-based CTCC detection in whole blood. Using a machine learning model for peak detection/classification, we demonstrated that the combined use of backscattered signals at the three wavelengths enable detection of ~ 93% of all CTCCs larger than two cells with a purity of > 82% and an overall accuracy of > 95%. The high level of performance established through BSFC and machine learning demonstrates the potential for label-free detection and monitoring of CTCCs in whole blood. Further developments of label-free BSFC to enhance throughput could lead to important applications in the isolation of CTCCs in whole blood with minimal disruption and ultimately their detection in vivo.

Specialized Pro-Resolving Mediators Reduce Scarring After Cleft Lip Repair.

Objective: Residual scarring after cleft lip repair surgery remains a challenge for both surgeons and patients and novel therapeutics are critically needed. The objective of this preclinical experimental study was to evaluate the impact of the methyl-ester of pro-resolving lipid mediator lipoxin A4 (LXA4-ME) on scarring in a novel rabbit model of cleft lip repair. Methods: A defect of the lip was surgically created and repaired in eight six-week old New Zealand white rabbits to simulate human cleft lip scars. Rabbits were randomly assigned to topical application of PBS (control) or 1 ug of LXA4-ME (treatment). 42 days post surgery all animals were euthanized. Photographs of the cleft lip area defect and histologic specimens were evaluated. Multiple scar assessment scales were used to compare scarring. Results: Animals treated with LXA4-ME exhibited lower Visual Scar Assessment scores compared to animals treated with PBS. Treatment with LXA4-ME resulted in a significant reduction of inflammatory cell infiltrate and density of collagen fibers. Control animals showed reduced 2D directional variance (orientation) of collagen fibers compared to animals treated with LXA4-ME demonstrating thicker and more parallel collagen fibers, consistent with scar tissue. Conclusions: These data suggest that LXA4-ME limits scarring after cleft lip repair and improves wound healing outcomes in rabbits favoring the resolution of inflammation. Further studies are needed to explore the mechanisms that underlie the positive therapeutic impact of LXA4-ME on scarring to set the stage for future human clinical trials of LXA4-ME for scar prevention or treatment after cleft lip repair.

Polarization enhanced laparoscope for improved visualization of tissue structural changes associated with peritoneal cancer metastasis.

A polarization enhanced laparoscopy (PEL) imaging system was developed to examine the feasibility of utilizing PEL to augment conventional white light laparoscopy (WLL) in the visualization of peritoneal cancer metastases. The system includes a modified tip to illuminate tissue with linearly polarized light and elements in the detection path enabling recording of corresponding images linearly co- and cross-polarized relative to the incident light. WLL and PEL images from optical tissue phantoms with features of distinct scattering cross-section confirm the enhanced sensitivity of PEL to such characteristics. Additional comparisons based on images acquired from collagen gels with different levels of fiber alignment highlight another source of PEL contrast. Finally, PEL and WLL images of ex vivo human tissue illustrate the potential of PEL to improve visualization of cancerous tissue surrounded by healthy peritoneum. Given the simplicity of the approach and its potential for seamless integration with current clinical practice, our results provide motivation for clinical translation.