Evolving concepts in margin strategies and adaptive radiotherapy for glioblastoma: A new future is on the horizon.

Chemoradiotherapy is the standard treatment after maximal safe resection for glioblastoma (GBM). Despite advances in molecular profiling, surgical techniques, and neuro-imaging, there have been no major breakthroughs in radiotherapy (RT) volumes in decades. Although the majority of recurrences occur within the original gross tumor volume (GTV), treatment of a clinical target volume (CTV) ranging from 1.5 to 3.0 cm beyond the GTV remains the standard of care. Over the past 15 years, the incorporation of standard and functional MRI sequences into the treatment workflow has become a routine practice with increasing adoption of MR simulators, and new integrated MR-Linac technologies allowing for daily pre-, intra- and post-treatment MR imaging. There is now unprecedented ability to understand the tumor dynamics and biology of GBM during RT, and safe CTV margin reduction is being investigated with the goal of improving the therapeutic ratio. The purpose of this review is to discuss margin strategies and the potential for adaptive RT for GBM, with a focus on the challenges and opportunities associated with both online and offline adaptive workflows. Lastly, opportunities to biologically guide adaptive RT using non-invasive imaging biomarkers and the potential to define appropriate volumes for dose modification will be discussed.

Endometrial mesenchymal stromal/stem cells improve regeneration of injured endometrium in mice.

BACKGROUND: The monthly regeneration of human endometrial tissue is maintained by the presence of human endometrial mesenchymal stromal/stem cells (eMSC), a cell population co-expressing the perivascular markers CD140b and CD146. Endometrial regeneration is impaired in the presence of intrauterine adhesions, leading to infertility, recurrent pregnancy loss and placental abnormalities. Several types of somatic stem cells have been used to repair the damaged endometrium in animal models, reporting successful pregnancy. However, the ability of endometrial stem cells to repair the damaged endometrium remains unknown. METHODS: Electrocoagulation was applied to the left uterine horn of NOD/SCID mice causing endometrial injury. Human eMSC or PBS was then injected into the left injured horn while the right normal horn served as controls. Mice were sacrificed at different timepoints (Day 3, 7 and 14) and the endometrial morphological changes as well as the degree of endometrial injury and repair were observed by histological staining. Gene expression of various inflammatory markers was assessed using qPCR. The functionality of the repaired endometrium was evaluated by fertility test. RESULTS: Human eMSC successfully incorporated into the injured uterine horn, which displayed significant morphological restoration. Also, endometrium in the eMSC group showed better cell proliferation and glands formation than the PBS group. Although the number of blood vessels were similar between the two groups, gene expression of VEGF-α significantly increased in the eMSC group. Moreover, eMSC had a positive impact on the regeneration of both stromal and epithelial components of the mouse endometrium, indicated by significantly higher vimentin and CK19 protein expression. Reduced endometrial fibrosis and down-regulation of fibrosis markers were also observed in the eMSC group. The eMSC group had a significantly higher gene expression of anti-inflammatory factor Il-10 and lower mRNA level of pro-inflammatory factors Ifng and Il-2, indicating the role of eMSC in regulation of inflammatory reactions. The eMSC group showed higher implantation sites than the PBS group, suggesting better endometrial receptivity with the presence of newly emerged endometrial lining. CONCLUSIONS: Our findings suggest eMSC improves regeneration of injured endometrium in mice.

Endometrial mesenchymal stromal/stem cells improve regeneration of injured endometrium in mice

Background The monthly regeneration of human endometrial tissue is maintained by the presence of human endometrial mesenchymal stromal/stem cells (eMSC), a cell population co-expressing the perivascular markers CD140b and CD146. Endometrial regeneration is impaired in the presence of intrauterine adhesions, leading to infertility, recurrent pregnancy loss and placental abnormalities. Several types of somatic stem cells have been used to repair the damaged endometrium in animal models, reporting successful pregnancy. However, the ability of endometrial stem cells to repair the damaged endometrium remains unknown. Methods Electrocoagulation was applied to the left uterine horn of NOD/SCID mice causing endometrial injury. Human eMSC or PBS was then injected into the left injured horn while the right normal horn served as controls. Mice were sacrificed at different timepoints (Day 3, 7 and 14) and the endometrial morphological changes as well as the degree of endometrial injury and repair were observed by histological staining. Gene expression of various inflammatory markers was assessed using qPCR. The functionality of the repaired endometrium was evaluated by fertility test. Results Human eMSC successfully incorporated into the injured uterine horn, which displayed significant morphological restoration. Also, endometrium in the eMSC group showed better cell proliferation and glands formation than the PBS group. Although the number of blood vessels were similar between the two groups, gene expression of VEGF-α significantly increased in the eMSC group. Moreover, eMSC had a positive impact on the regeneration of both stromal and epithelial components of the mouse endometrium, indicated by significantly higher vimentin and CK19 protein expression. Reduced endometrial fibrosis and down-regulation of fibrosis markers were also observed in the eMSC group. The eMSC group had a significantly higher gene expression of anti-inflammatory factor Il-10 and lower mRNA level of pro-inflammatory factors Ifng and Il-2, indicating the role of eMSC in regulation of inflammatory reactions. The eMSC group showed higher implantation sites than the PBS group, suggesting better endometrial receptivity with the presence of newly emerged endometrial lining. Conclusions Our findings suggest eMSC improves regeneration of injured endometrium in mice.

Chemical Exchange Saturation Transfer MRI: What Neuro-Oncology Clinicians Need To Know.

Chemical exchange saturation transfer (CEST) is a relatively novel magnetic resonance imaging (MRI) technique with an image contrast designed for in vivo measurement of certain endogenous molecules with protons that are exchangeable with water protons, such as amide proton transfer commonly used for neuro-oncology applications. Recent technological advances have made it feasible to implement CEST on clinical grade scanners within practical acquisition times, creating new opportunities to integrate CEST in clinical workflow. In addition, the majority of CEST applications used in neuro-oncology are performed without the use gadolinium-based contrast agents which are another appealing feature of this technique. This review is written for clinicians involved in neuro-oncologic care (nonphysicists) as the target audience explaining what they need to know as CEST makes its way into practice. The purpose of this article is to (1) review the basic physics and technical principles of CEST MRI, and (2) review the practical applications of CEST in neuro-oncology.

Diffusion-weighted imaging on an MRI-linear accelerator to identify adversely prognostic tumour regions in glioblastoma during chemoradiation.

BACKGROUND AND PURPOSE: Survival in glioblastoma might be extended by escalating the radiotherapy dose to treatment-resistant tumour and adapting to tumour changes. Diffusion-weighted imaging (DWI) on MRI-linear accelerators (MR-Linacs) could be used to identify a dose escalation target, but its prognostic value must be demonstrated. The purpose of this study was to determine whether MR-Linac DWI can assess treatment response in glioblastoma and whether changes in DWI show greater prognostic value than changes in the contrast-enhancing gross tumour volume (GTV). MATERIALS AND METHODS: Seventy-five patients with glioblastoma were treated with chemoradiotherapy, of which 32 were treated on a 1.5 T MRI-linear accelerator (MR-Linac). Patients were imaged with simulation MRI scanners (MR-sim) at treatment planning and weeks 2, 4, and 10 after treatment start. Twenty-eight patients had additional MR-Linac DWI sequences. Cox modelling was used to evaluate the correlation of overall and progression-free survival (OS and PFS) with clinical variables and volumetric changes in the GTV and low-ADC regions (ADC < 1.25 µm2/ms within GTV). RESULTS: In total, 479 MR-Linac DWI and 289 MR-sim DWI datasets were analyzed. MR-Linac low-ADC changes between weeks 2 and 5 inclusive were prognostic for OS (hazard ratio lower limits ≥ 1.2, p-values ≤ 0.02). MR-sim low-ADC changes showed greater correlation with OS and PFS than GTV changes (e.g., OS hazard ratio at week 2 was 3.4 (p <0.001) for low-ADC versus 2.0 (p = 0.022) for GTV). CONCLUSION: MR-Linac DWI can measure low-ADC tumour volumes that correlate with OS and PFS better than contrast-enhancing GTV. Low-ADC regions could serve as dose escalation targets.

Chemical Exchange Saturation Transfer MRI for Differentiating Radiation Necrosis From Tumor Progression in Brain Metastasis-Application in a Clinical Setting.

BACKGROUND: High radiation doses of stereotactic radiosurgery (SRS) for brain metastases (BM) can increase the likelihood of radiation necrosis (RN). Advanced MRI sequences can improve the differentiation between RN and tumor progression (TP). PURPOSE: To use saturation transfer MRI methods including chemical exchange saturation transfer (CEST) and magnetization transfer (MT) to distinguish RN from TP. STUDY TYPE: Prospective cohort study. SUBJECTS: Seventy patients (median age 60; 73% females) with BM (75 lesions) post-SRS. FIELD STRENGTH/SEQUENCE: 3-T, CEST imaging using low/high-power (saturation B1  = 0.52 and 2.0 µT), quantitative MT imaging using B1  = 1.5, 3.0, and 5.0 µT, WAter Saturation Shift Referencing (WASSR), WAter Shift And B1 (WASABI), T1 , and T2 mapping. All used gradient echoes except T2 mapping (gradient and spin echo). ASSESSMENT: Voxel-wise metrics included: magnetization transfer ratio (MTR); apparent exchange-dependent relaxation (AREX); MTR asymmetry; normalized MT exchange rate and pool size product; direct water saturation peak width; and the observed T1 and T2 . Regions of interests (ROIs) were manually contoured on the post-Gd T1 w. The mean (of median ROI values) was compared between groups. Clinical outcomes were determined by clinical and radiologic follow-up or histopathology. STATISTICAL TESTS: t-Test, univariable and multivariable logistic regression, receiver operating characteristic, and area under the curve (AUC) with sensitivity/specificity values with the optimal cut point using the Youden index, Akaike information criterion (AIC), Cohen's d. P < 0.05 with Bonferroni correction was considered significant. RESULTS: Seven metrics showed significant differences between RN and TP. The high-power MTR showed the highest AUC of 0.88, followed by low-power MTR (AUC = 0.87). The combination of low-power CEST scans improved the separation compared to individual parameters (with an AIC of 70.3 for low-power MTR/AREX). Cohen's d effect size showed that the MTR provided the largest effect sizes among all metrics. DATA CONCLUSION: Significant differences between RN and TP were observed based on saturation transfer MRI. EVIDENCE LEVEL: 3 Technical Efficacy: Stage 2.

Resolving the gene expression maps of human first-trimester chorionic villi with spatial transcriptome.

The placenta is important for fetal development in mammals, and spatial transcriptomic profiling of placenta helps to resolve its structure and function. In this study, we described the landscape of spatial transcriptome of human placental villi obtained from two pregnant women at the first trimester using the modified Stereo-seq method applied for paraformaldehyde (PFA) fixation samples. The PFA fixation of human placenta villi was better than fresh villi embedded in optimum cutting temperature (OCT) compound, since it greatly improved tissue morphology and the specificity of RNA signals. The main cell types in chorionic villi such as syncytiotrophoblasts (SCT), villous cytotrophoblasts (VCT), fibroblasts (FB), and extravillous trophoblasts (EVT) were identified with the spatial transcriptome data, whereas the minor cell types of Hofbauer cells (HB) and endothelial cells (Endo) were spatially located by deconvolution of scRNA-seq data. We demonstrated that the Stereo-seq data of human villi could be used for sophisticated analyses such as spatial cell-communication and regulatory activity. We found that the SCT and VCT exhibited the most ligand-receptor pairs that could increase differentiation of the SCT, and that the spatial localization of specific regulons in different cell types was associated with the pathways related to hormones transport and secretion, regulation of mitotic cell cycle, and nutrient transport pathway in SCT. In EVT, regulatory pathways such as the epithelial to mesenchyme transition, epithelial development and differentiation, and extracellular matrix organization were identified. Finally, viral receptors and drug transporters were identified in villi according to the pathway analysis, which could help to explain the vertical transmission of several infectious diseases and drug metabolism efficacy. Our study provides a valuable resource for further investigation of the placenta development, physiology and pathology in a spatial context.

High grade glioma radiation therapy on a high field 1.5 Tesla MR-Linac - workflow and initial experience with daily adapt-to-position (ATP) MR guidance: A first report.

Purpose: This study reports the workflow and initial clinical experience of high grade glioma (HGG) radiotherapy on the 1.5 T MR-Linac (MRL), with a focus on the temporal variations of the tumor and feasibility of multi-parametric image (mpMRI) acquisition during routine treatment workflow. Materials and methods: Ten HGG patients treated with radiation within the first year of the MRL's clinical operation, between October 2019 and August 2020, were identified from a prospective database. Workflow timings were recorded and online adaptive plans were generated using the Adapt-To-Position (ATP) workflow. Temporal variation within the FLAIR hyperintense region (FHR) was assessed by the relative FHR volumes (n = 281 contours) and migration distances (maximum linear displacement of the volume). Research mpMRIs were acquired on the MRL during radiation and changes in selected functional parameters were investigated within the FHR. Results: All patients completed radiotherapy to a median dose of 60 Gy (range, 54-60 Gy) in 30 fractions (range, 30-33), receiving a total of 287 fractions on the MRL. The mean in-room time per fraction with or without post-beam research imaging was 42.9 minutes (range, 25.0-69.0 minutes) and 37.3 minutes (range, 24.0-51.0 minutes), respectively. Three patients (30%) required re-planning between fractions 9 to 12 due to progression of tumor and/or edema identified on daily MRL imaging. At the 10, 20, and 30-day post-first fraction time points 3, 3, and 4 patients, respectively, had a FHR volume that changed by at least 20% relative to the first fraction. Research mpMRIs were successfully acquired on the MRL. The median apparent diffusion coefficient (ADC) within the FHR and the volumes of FLAIR were significantly correlated when data from all patients and time points were pooled (R=0.68, p<.001). Conclusion: We report the first clinical series of HGG patients treated with radiotherapy on the MRL. The ATP workflow and treatment times were clinically acceptable, and daily online MRL imaging triggered adaptive re-planning for selected patients. Acquisition of mpMRIs was feasible on the MRL during routine treatment workflow. Prospective clinical outcomes data is anticipated from the ongoing UNITED phase 2 trial to further refine the role of MR-guided adaptive radiotherapy.

The role of Notch signaling in endometrial mesenchymal stromal/stem-like cells maintenance.

Human endometrium undergoes cycles of regeneration in women of reproductive age. The endometrial mesenchymal stromal/stem cells (eMSC) contribute to this process. Notch signaling is essential for homeostasis of somatic stem cells. However, its role in eMSC remains unclear. We show with gain- and loss-of-function experiments that activation of Notch signaling promotes eMSC maintenance, while inhibition induces opposite effect. The activation of Notch signaling better maintains eMSC in a quiescent state. However, these quiescent eMSC can re-enter the cell cycle depending on the Notch and Wnt activities in the microenvironment, suggesting a crosstalk between the two signaling pathways. We further show that the Notch signaling is involved in endometrial remodeling event in a mouse menstrual-like model. Suppression of Notch signaling reduces the proliferation of Notch1+ label-retaining stromal cells and delays endometrial repair. Our data demonstrate the importance of Notch signaling in regulating the endometrial stem/progenitor cells in vitro and in vivo.

Three-dimensional culture models of human endometrium for studying trophoblast-endometrium interaction during implantation.

During implantation, a symphony of interaction between the trophoblast originated from the trophectoderm of the implanting blastocyst and the endometrium leads to a successful pregnancy. Defective interaction between the trophoblast and endometrium often results in implantation failure, pregnancy loss, and a number of pregnancy complications. Owing to ethical concerns of using in vivo approaches to study human embryo implantation, various in vitro culture models of endometrium were established in the past decade ranging from two-dimensional cell-based to three-dimensional extracellular matrix (ECM)/tissue-based culture systems. Advanced organoid systems have also been established for recapitulation of different cellular components of the maternal-fetal interface, including the endometrial glandular organoids, trophoblast organoids and blastoids. However, there is no single ideal model to study the whole implantation process leaving more research to be done pursuing the establishment of a comprehensive in vitro model that can recapitulate the biology of trophoblast-endometrium interaction during early pregnancy. This would allow us to have better understanding of the physiological and pathological process of trophoblast-endometrium interaction during implantation.

Hypoxia Regulates the Self-Renewal of Endometrial Mesenchymal Stromal/Stem-like Cells via Notch Signaling.

Human endometrium is an incredibly dynamic tissue undergoing cyclic regeneration and shedding during a woman's reproductive life. Endometrial mesenchymal stromal/stem-like cells (eMSC) contribute to this process. A hypoxic niche with low oxygen levels has been reported in multiple somatic stem cell types. However, the knowledge of hypoxia on eMSC remains limited. In mice, stromal stem/progenitor cells can be identified by the label-retaining technique. We examined the relationship between the label-retaining stromal cells (LRSC) and hypoxia during tissue breakdown in a mouse model of simulated menses. Our results demonstrated that LRSC resided in a hypoxic microenvironment during endometrial breakdown and early repair. Immunofluorescence staining revealed that the hypoxic-located LRSC underwent proliferation and was highly colocalized with Notch1. In vitro studies illustrated that hypoxia activated Notch signaling in eMSC, leading to enhanced self-renewal, clonogenicity and proliferation of cells. More importantly, HIF-1α played an essential role in the hypoxia-mediated maintenance of eMSC through the activation of Notch signaling. In conclusion, our findings show that some endometrial stem/progenitor cells reside in a hypoxic niche during menstruation, and hypoxia can regulate the self-renewal activity of eMSC via Notch signaling.

WNT5A Interacts With FZD5 and LRP5 to Regulate Proliferation and Self-Renewal of Endometrial Mesenchymal Stem-Like Cells.

Endometrial mesenchymal stem-like cells (eMSC) reside in the basal layer of the endometrium and are responsible for cyclic regeneration during the reproductive lives of women. Myometrial cells act as a component of the niche and regulate the stem cell fate through the activation of WNT/ß-catenin signaling via WNT5A. Since WNT5A-responsive mechanisms on eMSC are still uncertain, we hypothesize that the WNT ligand-WNT5A works to activate WNT/ß-catenin signaling through binding to Frizzled receptors (FZDs) and co-receptor low-density lipoprotein receptor-related protein 5 (LRP5). Among the various receptors that have been reported to interact with WNT5A, we found FZD5 abundantly expressed by eMSC when compared to unfractionated stromal cells. Neutralizing the protein expression by using anti-FZD5 antibody suppressed the stimulatory effects on phenotypic expression and the clonogenicity of eMSC in a myometrial cell-eMSC co-culture system as well as in an L-Wnt5a conditioned medium. Gene silencing of FZD5 not only reduced the binding of WNT5A to eMSC but also decreased the TCF/LEF transcriptional activities and expression of active ß-catenin. Inhibition of LRP coreceptors with recombinant Dickkopf-1 protein significantly reduced the binding affinity of eMSC to WNT5A as well as the proliferation and self-renewal activity. During postpartum remodeling in mouse endometrium, active ß-catenin (ABC) was detected in label-retaining stromal cells (LRSCs), and these ABC+ LRSCs express FZD5 and LRP5, suggesting the activation of WNT/ß-catenin signaling. In conclusion, our findings demonstrate the interaction of WNT5A, FZD5, and LRP5 in regulating the proliferation and self-renewal of eMSC through WNT/ß-catenin signaling.

Accuracy and precision of apparent diffusion coefficient measurements on a 1.5 T MR-Linac in central nervous system tumour patients.

BACKGROUND AND PURPOSE: MRI linear accelerators (MR-Linacs) may allow treatment adaptation to be guided by quantitative MRI including diffusion-weighted imaging (DWI). The aim of this study was to evaluate the accuracy and precision of apparent diffusion coefficient (ADC) measurements from DWI on a 1.5 T MR-Linac in patients with central nervous system (CNS) tumours through comparison with a diagnostic scanner. MATERIALS AND METHODS: CNS patients were treated using a 1.5 T Elekta Unity MR-Linac. DWI was acquired during MR-Linac treatment and on a Philips Ingenia 1.5 T. The agreement between the two scanners on median ADC over the gross tumour/clinical target volumes (GTV/CTV) and in brain regions (white/grey matter, cerebrospinal fluid (CSF)) was computed. Repeated scans were used to estimate ADC repeatability. Daily changes in ADC over the GTV of high-grade gliomas were characterized from MR-Linac scans. RESULTS: DWI from 59 patients was analyzed. MR-Linac ADC measurements showed a small bias relative to Ingenia measurements in white matter, grey matter, GTV, and CTV (bias: -0.05 ± 0.03, -0.08 ± 0.05, -0.1 ± 0.1, -0.08 ± 0.07 µm2/ms). ADC differed substantially in CSF (bias: -0.5 ± 0.3 µm2/ms). The repeatability of MR-Linac ADC over white/grey matter was similar to previous reports (coefficients of variation for median ADC: 1.4%/1.8%). MR-Linac ADC changes in the GTV were detectable. CONCLUSIONS: It is possible to obtain ADC measurements in the brain on a 1.5 T MR-Linac that are comparable to those of diagnostic-quality scanners. This technical validation study adds to the foundation for future studies that will correlate brain tumour ADC with clinical outcomes.

Deuterium MRS of early treatment-induced changes in tumour lactate in vitro.

Elevated production of lactate is a key characteristic of aberrant tumour cell metabolism and can be non-invasively measured as an early marker of tumour response using deuterium (2 H) MRS. Following treatment, changes in the 2 H-labelled lactate signal could identify tumour cell death or impaired metabolic function, which precede morphological changes conventionally used to assess tumour response. In this work, the association between apoptotic cell death, extracellular lactate concentration, and early treatment-induced changes in the 2 H-labelled lactate signal was established in an in vitro tumour model. Experiments were conducted at 7 T on acute myeloid leukaemia (AML) cells, which had been treated with 10 µg/mL of the chemotherapeutic agent cisplatin. At 24 and 48 h after cisplatin treatment the cells were supplied with 20 mM of [6,6'-2 H2 ]glucose and scanned over 2 h using a two-dimensional 2 H MR spectroscopic imaging sequence. The resulting signals from 2 H-labelled glucose, lactate, and water were quantified using a spectral fitting algorithm implemented on the Oxford Spectroscopy Analysis MATLAB toolbox. After scanning, the cells were processed for histological stains (terminal deoxynucleotidyl transferase UTP nick end labelling and haematoxylin and eosin) to assess apoptotic area fraction and cell morphology respectively, while a colorimetric assay was used to measure extracellular lactate concentrations in the supernatant. Significantly lower levels of 2 H-labelled lactate were observed in the 48 h treated cells compared with the untreated and 24 h treated cells, and these changes were significantly correlated with an increase in apoptotic fraction and a decrease in extracellular lactate. By establishing the biological processes associated with treatment-induced changes in the 2 H-labelled lactate signal, these findings suggest that 2 H MRS of lactate may be valuable in evaluating early tumour response.

Chemical exchange saturation transfer MRI in central nervous system tumours on a 1.5 T MR-Linac.

PURPOSE: To describe the implementation and initial results of using Chemical Exchange Saturation Transfer (CEST) for monitoring patients with central nervous system (CNS) tumours treated using a 1.5 tesla MR-guided radiotherapy system. METHODS: CNS patients were treated with up to 30 fractions (total dose up to 60 Gy) using a 1.5 T Elekta Unity MR-Linac. CEST scans were obtained in 54 subjects at one or more time points during treatment. CEST metrics, including the amide magnetization transfer ratio (MTRAmide), nuclear Overhauser effect (NOE) MTR (MTRNOE) and asymmetry, were quantified in phantoms and CNS patients. The signal was investigated between tumour and white matter, across time, and across disease categories including high- and low-grade tumours. RESULTS: The gross tumour volume (GTV) exhibited lower MTRAmide and MTRNOE and higher asymmetry compared to contralateral normal appearing white matter. Signal changes in the GTV during fractionated radiotherapy were observed. There were differences between high- and low-grade tumours, with higher CEST asymmetry associated with higher grade disease. CONCLUSION: CEST MRI using a 1.5 T MR-Linac was demonstrated to be feasible for in vivo imaging of CNS tumours. CEST images showed tumour/white-matter contrast, temporal CEST signal changes, and associations with tumour grade. These results show promise for the eventual goal of using metabolic imaging to inform the design of adaptive radiotherapy protocols.

Single-cell RNA sequencing of cultured human endometrial CD140b+CD146+ perivascular cells highlights the importance of in vivo microenvironment.

BACKGROUND: Endometrial mesenchymal-like stromal/stem cells (eMSCs) have been proposed as adult stem cells contributing to endometrial regeneration. One set of perivascular markers (CD140b&CD146) has been widely used to enrich eMSCs. Although eMSCs are easily accessible for regenerative medicine and have long been studied, their cellular heterogeneity, relationship to primary counterpart, remains largely unclear. METHODS: In this study, we applied 10X genomics single-cell RNA sequencing (scRNA-seq) to cultured human CD140b+CD146+ endometrial perivascular cells (ePCs) from menstrual and secretory endometrium. We also analyzed publicly available scRNA-seq data of primary endometrium and performed transcriptome comparison between cultured ePCs and primary ePCs at single-cell level. RESULTS: Transcriptomic expression-based clustering revealed limited heterogeneity within cultured menstrual and secretory ePCs. A main subpopulation and a small stress-induced subpopulation were identified in secretory and menstrual ePCs. Cell identity analysis demonstrated the similar cellular composition in secretory and menstrual ePCs. Marker gene expression analysis showed that the main subpopulations identified from cultured secretory and menstrual ePCs simultaneously expressed genes marking mesenchymal stem cell (MSC), perivascular cell, smooth muscle cell, and stromal fibroblast. GO enrichment analysis revealed that genes upregulated in the main subpopulation enriched in actin filament organization, cellular division, etc., while genes upregulated in the small subpopulation enriched in extracellular matrix disassembly, stress response, etc. By comparing subpopulations of cultured ePCs to the publicly available primary endometrial cells, it was found that the main subpopulation identified from cultured ePCs was culture-unique which was unlike primary ePCs or primary endometrial stromal fibroblast cells. CONCLUSION: In summary, these data for the first time provides a single-cell atlas of the cultured human CD140b+CD146+ ePCs. The identification of culture-unique relatively homogenous cell population of CD140b+CD146+ ePCs underscores the importance of in vivo microenvironment in maintaining cellular identity.

Method of computing direction-dependent margins for the development of consensus contouring guidelines.

BACKGROUND: Clinical target volume (CTV) contouring guidelines are frequently developed through studies in which experts contour the CTV for a representative set of cases for a given treatment site and the consensus CTVs are analyzed to generate margin recommendations. Measures of interobserver variability are used to quantify agreement between experts. In cases where an isotropic margin is not appropriate, however, there is no standard method to compute margins in specified directions that represent possible routes of tumor spread. Moreover, interobserver variability metrics are often measures of volume overlap that do not account for the dependence of disagreement on direction. To aid in the development of consensus contouring guidelines, this study demonstrates a novel method of quantifying CTV margins and interobserver variability in clinician-specified directions. METHODS: The proposed algorithm was applied to 11 cases of non-spine bone metastases to compute the consensus CTV margin in each direction of intraosseous and extraosseous disease. The median over all cases for each route of spread yielded the recommended margins. The disagreement between experts on the CTV margin was quantified by computing the median of the coefficients of variation for intraosseous and extraosseous margins. RESULTS: The recommended intraosseous and extraosseous margins were 7.0 mm and 8.0 mm, respectively. The median coefficient of variation quantifying the margin disagreement between experts was 0.59 and 0.48 for intraosseous and extraosseous disease. CONCLUSIONS: The proposed algorithm permits the generation of margin recommendations in relation to adjacent anatomy and quantifies interobserver variability in specified directions. This method can be applied to future consensus CTV contouring studies.

Quantitative CEST and MT at 1.5T for monitoring treatment response in glioblastoma: early and late tumor progression during chemoradiation.

PURPOSE: Quantitative MRI (qMRI) was performed using a 1.5T protocol that includes a novel chemical exchange saturation transfer/magnetization transfer (CEST/MT) approach. The purpose of this prospective study was to determine if qMRI metrics at baseline, at the 10th and 20th fraction during a 30 fraction/6 week standard chemoradiation (CRT) schedule, and at 1 month following treatment could be an early indicator of response for glioblastoma (GBM). METHODS: The study included 51 newly diagnosed GBM patients. Four regions-of-interest (ROI) were analyzed: (i) the radiation defined clinical target volume (CTV), (ii) radiation defined gross tumor volume (GTV), (iii) enhancing-tumor regions, and (iv) FLAIR-hyperintense regions. Quantitative CEST, MT, T1 and T2 parameters were compared between those patients progressing within 6.9 months (early), and those progressing after CRT (late), using mixed modelling. Exploratory predictive modelling was performed to identify significant predictors of early progression using a multivariable LASSO model. RESULTS: Results were dependent on the specific tumor ROI analyzed and the imaging time point. The baseline CEST asymmetry within the CTV was significantly higher in the early progression cohort. Other significant predictors included the T2 of the MT pools (for semi-solid at fraction 20 and water at 1 month after CRT), the exchange rate (at fraction 20) and the MGMT methylation status. CONCLUSIONS: We observe the potential for multiparametric qMRI, including a novel pulsed CEST/MT approach, to show potential in distinguishing early from late progression GBM cohorts. Ultimately, the goal is to personalize therapeutic decisions and treatment adaptation based on non-invasive imaging-based biomarkers.