Protocol for generating high-fidelity proteomic profiles using DROPPS.

Deep mass spectrometry-based proteomic profiling of rare cell populations has been constrained by sample input requirements. Here, we present a protocol for droplet-based one-pot preparation for proteomic samples (DROPPS), an accessible low-input platform that generates high-fidelity proteomic profiles of 100-2,500 cells. We describe steps for depositing cellular material, cell lysis, and digesting proteins in the same microliter-droplet well. We anticipate DROPPS will accelerate biology-driven proteomic research for a multitude of rare cell populations. For complete details on the use and execution of this protocol, please refer to Waas et al.1.

Prostate cancer reshapes the secreted and extracellular vesicle urinary proteomes.

Urine is a complex biofluid that reflects both overall physiologic state and the state of the genitourinary tissues through which it passes. It contains both secreted proteins and proteins encapsulated in tissue-derived extracellular vesicles (EVs). To understand the population variability and clinical utility of urine, we quantified the secreted and EV proteomes from 190 men, including a subset with prostate cancer. We demonstrate that a simple protocol enriches prostatic proteins in urine. Secreted and EV proteins arise from different subcellular compartments. Urinary EVs are faithful surrogates of tissue proteomes, but secreted proteins in urine or cell line EVs are not. The urinary proteome is longitudinally stable over several years. It can accurately and non-invasively distinguish malignant from benign prostatic lesions and can risk-stratify prostate tumors. This resource quantifies the complexity of the urinary proteome and reveals the synergistic value of secreted and EV proteomes for translational and biomarker studies.

Droplet-based proteomics reveals CD36 as a marker for progenitors in mammary basal epithelium.

Deep proteomic profiling of rare cell populations has been constrained by sample input requirements. Here, we present DROPPS (droplet-based one-pot preparation for proteomic samples), an accessible low-input platform that generates high-fidelity proteomic profiles of 100-2,500 cells. By applying DROPPS within the mammary epithelium, we elucidated the connection between mitochondrial activity and clonogenicity, identifying CD36 as a marker of progenitor capacity in the basal cell compartment. We anticipate that DROPPS will accelerate biology-driven proteomic research for a multitude of rare cell populations.

Organelle resolved proteomics uncovers PLA2R1 as a novel cell surface marker required for chordoma growth.

Chordomas are clinically aggressive tumors with a high rate of disease progression despite maximal therapy. Given the limited therapeutic options available, there remains an urgent need for the development of novel therapies to improve clinical outcomes. Cell surface proteins are attractive therapeutic targets yet are challenging to profile with common methods. Four chordoma cell lines were analyzed by quantitative proteomics using a differential ultracentrifugation organellar fractionation approach. A subtractive proteomics strategy was applied to select proteins that are plasma membrane enriched. Systematic data integration prioritized PLA2R1 (secretory phospholipase A2 receptor-PLA2R1) as a chordoma-enriched surface protein. The expression profile of PLA2R1 was validated across chordoma cell lines, patient surgical tissue samples, and normal tissue lysates via immunoblotting. PLA2R1 expression was further validated by immunohistochemical analysis in a richly annotated cohort of 25-patient tissues. Immunohistochemistry analysis revealed that elevated expression of PLA2R1 is correlated with poor prognosis. Using siRNA- and CRISPR/Cas9-mediated knockdown of PLA2R1, we demonstrated significant inhibition of 2D, 3D and in vivo chordoma growth. PLA2R1 depletion resulted in cell cycle defects and metabolic rewiring via the MAPK signaling pathway, suggesting that PLA2R1 plays an essential role in chordoma biology. We have characterized the proteome of four chordoma cell lines and uncovered PLA2R1 as a novel cell-surface protein required for chordoma cell survival and association with patient outcome.

Prostate Cancer Reshapes the Secreted and Extracellular Vesicle Urinary Proteomes.

Urine is a complex biofluid that reflects both overall physiologic state and the state of the genitourinary tissues through which it passes. It contains both secreted proteins and proteins encapsulated in tissue-derived extracellular vesicles (EVs). To understand the population variability and clinical utility of urine, we quantified the secreted and EV proteomes from 190 men, including a subset with prostate cancer. We demonstrate that a simple protocol enriches prostatic proteins in urine. Secreted and EV proteins arise from different subcellular compartments. Urinary EVs are faithful surrogates of tissue proteomes, but secreted proteins in urine or cell line EVs are not. The urinary proteome is longitudinally stable over several years. It can accurately and non-invasively distinguish malignant from benign prostatic lesions, and can risk-stratify prostate tumors. This resource quantifies the complexity of the urinary proteome, and reveals the synergistic value of secreted and EV proteomes for translational and biomarker studies.

Glycoproteomics Identifies Plexin-B3 as a Targetable Cell Surface Protein Required for the Growth and Invasion of Triple-Negative Breast Cancer Cells.

Driven by the lack of targeted therapies, triple-negative breast cancers (TNBCs) have the worst overall survival of all breast cancer subtypes. Considering that cell surface proteins are favorable drug targets and are predominantly glycosylated, glycoproteome profiling has significant potential to facilitate the identification of much-needed drug targets for TNBCs. Here, we performed N-glycoproteomics on six TNBCs and five normal control (NC) cell lines using hydrazide-based enrichment. Quantitative proteomics and integrative data mining led to the discovery of Plexin-B3 (PLXNB3), a previously undescribed TNBC-enriched cell surface protein. Furthermore, siRNA knockdown and CRISPR-Cas9 editing of in vitro and in vivo models show that PLXNB3 is required for TNBC cell line growth, invasion, and migration. Altogether, we provide insights into N-glycoproteome remodeling associated with TNBCs and functional evaluation of an extracted target, which indicate the surface protein PLXNB3 as a potential therapeutic target for TNBCs.

Proteomics of High-Grade Serous Ovarian Cancer Models Identifies Cancer-Associated Fibroblast Markers Associated with Clinical Outcomes.

The tumor microenvironment has recently emerged as a critical component of high-grade serous ovarian cancer (HGSC) disease progression. Specifically, cancer-associated fibroblasts (CAFs) have been recognized as key players in various pro-oncogenic processes. Here, we use mass-spectrometry (MS) to characterize the proteomes of HGSC patient-derived CAFs and compare them to those of the epithelial component of HGSC to gain a deeper understanding into their tumor-promoting phenotype. We integrate our data with primary tissue data to define a proteomic signature of HGSC CAFs and uncover multiple novel CAF proteins that are prognostic in an independent HGSC patient cohort. Our data represent the first MS-based global proteomic characterization of CAFs in HGSC and further highlights the clinical significance of HGSC CAFs.

High-throughput approaches for precision medicine in high-grade serous ovarian cancer.

High-grade serous carcinoma (HGSC) is the most prevalent and aggressive subtype of ovarian cancer. The large degree of clinical heterogeneity within HGSC has justified deviations from the traditional one-size-fits-all clinical management approach. However, the majority of HGSC patients still relapse with chemo-resistant cancer and eventually succumb to their disease, evidence that further work is needed to improve patient outcomes. Advancements in high-throughput technologies have enabled novel insights into biological complexity, offering a large potential for informing precision medicine efforts. Here, we review the current landscape of clinical management for HGSC and highlight applications of high-throughput biological approaches for molecular subtyping and the discovery of putative blood-based biomarkers and novel therapeutic targets. Additionally, we present recent improvements in model systems and discuss how their intersection with high-throughput platforms and technological advancements is positioned to accelerate the realization of precision medicine in HGSC.

Surgical Approach and Reaming Depth Influence the Direction and Magnitude of Acetabular Center of Rotation Changes During Total Hip Arthroplasty.

BACKGROUND: Changes in acetabular or hip center of rotation (HCOR) commonly occur during acetabular component preparation during total hip arthroplasty (THA). HCOR displacement in mediolateral or superoinferior directions is known to influence offset and leg length, but the incidence and range of HCOR change in the anteroposterior direction is less understood as the sagittal plane cannot be measured on standard anteroposterior radiographs. This study assessed the 3-dimensional displacement of HCOR after cup implantation and evaluated for potential factors associated with increased acetabular component translations. METHODS: A total of 894 THAs were performed using a posterior, lateral, or direct anterior approach. Only intraoperative data from the navigation device were included in the analysis. All THAs performed between September 2015 and October 2017 were included. Paired t -tests were used to compare native HCOR and new HCOR values. RESULTS: The mean HCOR displacement in 3 directions was 4.97mm medially (P < .001), 0.83mm superiorly (P < .001), and 0.64mm posteriorly (P < .001). Subgroup analysis revealed greater posterior HCOR displacement with the anterior approach than the lateral/posterior approach (2.32mm vs 0.44mm; P < .001). Increasing medial HCOR displacement also resulted in increased superior and posterior HCOR displacement across surgical cases (P < .001). CONCLUSIONS: HCOR displacement is commonly observed in medial, superior, and posterior directions. HCOR changes are influenced by surgical approach, potentially secondary to patient positioning, with greater posterior HCOR displacement observed in anterior cases. Surgeons should be aware of these factors, particularly in cases with deficient or reduced posterior column bone stock.

A Novel Method for Correcting Pelvic Tilt on Anteroposterior Pelvic Radiographs.

Background Anteroposterior (AP) pelvic radiographs remain the standard for pre- and postoperative imaging during total hip arthroplasty (THA), despite the known limitation of plain films, including the inability to adequately account for distortion caused by variations in pelvic orientation such as pelvic tilt. The purpose of this study was to develop a reliable method for correcting pelvic tilt on AP pelvic radiographs in patients undergoing THA. Methods CT scans from 20 patients/cadaver specimens (10 male, 10 female) were used to create 3D renderings, from which synthetic radiographs of each pelvis were generated. For each pelvis, 13 synthetic radiographs were generated, showing the pelvis at between -30° and 30° of pelvic tilt, in 5° increments. On each image, eight unique parameters/distances were measured to determine the most appropriate parameters for the calculation of pelvic tilt. The most reliable and accurate of these parameters was determined via regression analysis and used to create gender-specific nomograms from which pelvic tilt measurements could be calculated. The accuracy and reliability of the nomograms and correction method were subsequently validated using both synthetic radiographs (n=50) and stereoradiographic images (n=58). Results Of the eight parameters measured, the vertical distance between the superior margin of the pubic symphysis and the transischial line (PSTI) was determined to be the most reliable (r=-0.96, ICC=0.94). Using that parameter and applying the associated nomograms to 50 synthetic radiographs of random pelvic tilt, the mean difference between the actual pelvic tilt and that calculated using the correction method was 0.1°±5.1° (p=0.98, r=0.96). In 58 stereoradiographic images, the mean difference between actual and measured pelvic tilt was -0.2°±6.4° (p=0.74, r=0.77). The pooled results indicate no significant difference between actual (2.2°±13.9°) and measured pelvic tilt (2.1°±14.3°, p=0.93, r=0.91). No significant differences were noted based on gender. Conclusions Our method of correcting for pelvic tilt using the vertical distance from the pubic symphysis to the transischial line provides a reliable method for correcting for pelvic tilt on AP pelvic radiographs.

Evaluation of Tilt-correction of Anteversion on Anteroposterior Pelvic Radiographs in Total Hip Arthroplasty.

Despite inaccuracies due to artifact and variations in patient positioning, anteroposterior (AP) radiographs remain the clinical standard for post-operative evaluation of component placement following total hip arthroplasty (THA). However, cup position, specifically anteversion, can be significantly affected by variations in patient positioning on an X-ray. A major cause of such artifact is unaccounted for pelvic tilt. Several methods for correcting the effects of pelvic tilt on radiographic anteversion have been proposed, with varying degrees of accuracy. The purpose of this study was to evaluate the accuracy and reliability of a commonly referenced method for correcting acetabular cup anteversion in a cohort undergoing total hip arthroplasty and determine its appropriateness for use in this population of patients. Radiographs from patients who underwent primary or revision hip arthroplasty between February 2016 and February 2017 were retrospectively reviewed. Corrected anteversion was calculated by measuring the vertical distance between the symphysis pubis and the sacrococcygeal joint, per the method outlined by Tannast et al. This symphococcygeal distance was then applied to Tannast's nomograms to calculate the magnitude of pelvic tilt. Corrected and uncorrected anteversion values were compared to anteversion values collected intraoperatively using an imageless computer-assisted navigation device. A total of 71 cases were initially eligible for inclusion in the study. The correction method could not be applied in 44% (31/71) of the cases, chiefly due to difficulties in visualizing the required landmarks. In cases where it could be applied, corrected values correlated very poorly with navigation measurements (r = -0.07). Mean corrected anteversion (36.9°, SD: 7.4°) differed from uncorrected anteversion (25.2°, SD: 7.6°) by an average of 13.5° (p<0.001). Mean navigated anteversion (27.4°, SD: 5.7°) differed from corrected values by an average of 10.8° (p=0.16). The evaluated correction method could not be consistently applied to radiographs and did not reliably correct anteversion due to pelvic tilt in this population of patients undergoing hip arthroplasty. This correction method does not appear to be appropriate for use in this patient population.