Taewook Kang
Apr 7, 2020; 0:RA119.001882v1-mcp.RA119.001882
Research

Xing-Huang Gao
May 1, 2020; 19:852-870
Research

Xiao-Kang Lun
May 1, 2020; 19:744-756
Review

Chia-Feng Tsai
May 1, 2020; 19:828-838
Research

The purpose of this study was to assess the predictive and prognostic value of interim FDG PET (iPET) in evaluating early response to immuno-chemotherapy after two cycles (PET-2) in diffuse large B-cell lymphoma (DLBCL) by applying two different methods of interpretation: the Deauville visual five-point scale (5-PS) and a change in standardised uptake value by semi-quantitative evaluation. Methods: 145 patients with newly diagnosed DLBCL underwent pre-treatment PET (PET-0) and PET-2 assessment. PET-2 was classified according to both the visual 5-PS and percentage SUV changes (SUV). Receiver operating characteristic (ROC) analysis was performed to compare the accuracy of the two methods for predicting progression-free survival (PFS). Survival estimates, based on each method separately and combined, were calculated for iPET-positive (iPET+) and iPET-negative (iPET–) groups and compared. Results: Both with visual and SUV-based evaluations significant differences were found between the PFS of iPET– and iPET+ patient groups (p<0.001). Visually the best negative (NPV) and positive predictive value (PPV) occurred when iPET was defined as positive if Deauville score 4-5 (89% and 59%, respectively). Using the 66% SUV cut-off value, reported previously, NPV and PPV were 80 and 76%, respectively. SUV at 48.9% cut-off point, reported for the first time here, produced 100% specificity along with the highest sensitivity (24%). Visual and semi-quantitative SUV<48.9% assessment of each PET-2 gave the same PET-2 classification (positive or negative) in 70% (102/145) of all patients. This combined classification delivered NPV and PPV of 89% and 100% respectively, and all iPET+ patients failed to achieve or remain in remission. Conclusion: In this large consistently treated and assessed series of DLBCL, iPET had good prognostic value interpreted either visually or semi-quantitatively. We determined that the most effective SUV cut-off was at 48.9%, and that when combined with visual 5-PS assessment, a positive PET-2 was highly predictive of treatment failure.

Purpose: The aim of this retrospective multicentric study was to develop and evaluate a prognostic FDG PET/CT radiomics signature in early-stage non-small cell lung cancer (NSCLC) patients treated with stereotactic radiotherapy (SBRT). Material and Methods: Patients from 3 different centers (n = 27, 29 and 8) were pooled to constitute the training set, whereas the patients from a fourth center (n = 23) were used as the testing set. The primary endpoint was local control (LC). The primary tumour was semi-automatically delineated in the PET images using the Fuzzy locally adaptive Bayesian algorithm, and manually in the low-dose CT images. A total of 184 IBSI-compliant radiomic features were extracted. Seven clinical and treatment parameters were included. We used ComBat to harmonize radiomic features extracted from the four institutions relying on different PET/CT scanners. In the training set, variables found significant in the univariate analysis were fed into a multivariate regression model and models were built by combining independent prognostic factors. Results: Median follow-up was 21.1 (1.7 – 63.4) and 25.5 (7.7 – 57.8) months in training and testing sets respectively. In univariate analysis, none of the clinical variables, 2 PET and 2 CT features were significantly predictive of LC. The best predictive models in the training set were obtained by combining one feature from PET, namely information correlation 2 (IC2) and one from CT (Flatness), reaching a sensitivity of 100% and a specificity of 96%. Another model combining 2 PET features (IC2 and Strength), reached sensitivity of 100% and specificity of 88%, both with an undefined hazard ratio (HR) (p<0.001). The latter model obtained an accuracy of 0.91 (sensitivity 100%, specificity 81%), with a HR undefined (P = 0.023) in the testing set, however other models relying on CT radiomics features only or the combination of PET and CT features failed to validate in the testing set. Conclusion: We showed that two radiomic features derived from FDG PET were independently associated with LC in patients with NSCLC undergoing SBRT and could be combined in an accurate predictive model. This model could provide local relapse-related information and could be helpful in clinical decision-making.

Due to their peculiar mechanism of action, the evaluation of radiological response to immune checkpoint inhibitors (ICI) presents many challenges in solid tumors. We aimed to compare the evaluation of first response to Nivolumab by means of CT-based criteria with respect to fluorodeoxyglucose positron emission tomography (FDG-PET) response criteria in non-small-cell lung cancer (NSCLC) patients. Methods: 72 patients with advanced NSCLC were recruited in a mono-institutional ancillary trial within the expanded access program (EAP; NCT02475382) for Nivolumab. Patients underwent CT scan and FDG-PET at baseline and after 4 cycles (first evaluation). In case of progressive disease (PD), an additional evaluation was performed after two further cycles in order to confirm progression. We evaluated the response to treatment with CT scan by means of response evaluation criteria in solid tumors (RECIST) 1.1 and Immuno-related Response Criteria (IrRC) and with FDG-PET by means of PERCIST and immunotherapy-modified-PERCIST (imPERCIST) criteria. The concordance between CT- and PET-based criteria and the capability of each method to predict overall survival (OS) were evaluated. Results: 48/72 patients were evaluable for first response assessment with both PET- and CT-based criteria. We observed low concordance between CT- and PET-based criteria (Kappa value of 0.346 and 0.355 and Kappa value of 0.128 and 0.198 between PERCIST and imPERCIST versus RECIST and irRC respectively). Looking at OS, IrRC were more reliable to distinguish responders from non-responders. However thanks to the prognostic value of partial metabolic response assessed by both PERCIST and Immuno-PERCIST, PET-based response maintained prognostic significant in patients classified as progressive disease on the basis of irRC. Conclusion: Even though the present study did not support the routine use of FDG-PET in the general population of NSCLC patients treated with ICI, it suggests the added prognostic value of the metabolic response assessment, potentially improving the therapeutic decision-making.

Introduction: A new pattern of response, so-called hyper-progressive disease (HPD), is emerging during treatment with immune checkpoint inhibitors (ICI). Our aim was to investigate the prevalence of such phenomenon and to assess its association with clinical variables and metabolic parameters by ¹⁸F-fludeoxyglucose positron emission tomography/computed tomography (¹⁸F-FDG PET/CT). Methods: Data from 50 patients (34 male, 16 female, median age 73) with non-small cell lung carcinoma (NSCLC) and treated with ICI were prospectively collected. All patients underwent contrast-enhanced CT, ¹⁸F-FDG PET/CT, and complete peripheral blood sample at baseline before ICI. HPD was defined according to clinical and radiologic criteria. Because of the rapid disease progression or worsening of clinic conditions, radiologic response assessment was available for 46 patients. OS were analyzed using the Kaplan–Meier method and the log-rank test. A Cox proportional hazards regression analysis was used to evaluate factors independently associated with OS. Median follow-up was 12.4 months (9.7-15.2 months). Results: We identified the following response categories: 10 cases as complete/partial response (CR/PR), 17 cases with stable disease (SD), 5 patients with progressive disease (PD), and 14 with HPD. Among metabolic parameters we observed a statistically significant association between HPD status and tumor burden, expressed by both MTV (756.1ml for HPD vs 475.6ml for non-HPD, P = 0.011) and TLG (287.3 for HPD vs 62.1 for non-HPD, P = 0.042). Among clinical variables, 12/14 patients (85.7%) within the HPD group compared with 8/32 patients (25%) in the non-HDP group had more than two metastatic sites (p<0.001). In addition, the derived neutrophil-to-lymphocyte ratio (dNLR) and platelet counts was significantly associated with HPD status (P = 0.038, P = 0.025, respectively). Survival analysis showed a median OS of 4 months for HPD group compared with 15 months within non-HPD patients (P = 0.003). Likewise, median OS was significantly different when we considered all the response categories: CR/PR, SD, PD, and HPD (P = 0.001). Finally, Multivariate analysis identified MTV and dNLR as independent predictors for OS. Conclusion: Our results suggest that the use of ICI might represent a concern in patients with high metabolic tumor burden and inflammatory indexes at baseline. However Additional studies are needed.

Objective: To develop a novel approach to generate individual maps of white matter (WM) innate immune cell activation using ¹⁸F-DPA-714 translocator protein (TSPO) positron emission tomography (PET), and to explore the relationship between these maps and individual trajectories of disability worsening in patients with multiple sclerosis (MS). Methods: Patients with MS (n = 37), whose trajectories of disability worsening over the 2 years preceding study entry were calculated, and healthy controls (n = 19) underwent magnetic resonance magnetic and ¹⁸F-DPA-714 PET. A threshold of significant activation of ¹⁸F-DPA-714 binding was calculated with a voxel-wise randomized permutation-based comparison between patients and controls, and used to classify each WM voxel in patients as characterized by a significant activation of innate immune cells (DPA+) or not. Individual maps of innate immune cell activation in the WM were employed to calculate the extent of activation in WM regions-of-interests and to classify each WM lesion as "DPA-active", "DPA-inactive" or "unclassified". Results: Compared with the WM of healthy controls, patients with MS had a significantly higher percentage of DPA+ voxels in the normal-appearing WM, (NAWM in patients=24.9±9.7%; WM in controls=14.0±7.8%, p<0.001). In patients with MS, the percentage of DPA+ voxels showed a significant increase from NAWM, to perilesional areas, T2 hyperintense lesions and T1 hypointense lesions (38.1±13.5%, 45.0±17.9%, and 51.9±22.9%, respectively, p<0.001). Among the 1379 T2 lesions identified, 512 were defined as DPA-active and 258 as DPA-inactive. A higher number of lesions classified as DPA-active (OR=1.13, P = 0.009), a higher percentage of DPA+ voxels in the NAWM (OR=1.16, P = 0.009) and in T1-spin-echo lesions (OR=1.06, P = 0.036), were significantly associated with a retrospective more severe clinical trajectory in patients with MS. Conclusion: A more severe trajectory of disability worsening in MS is associated with an innate immune cells activation inside and around WM lesions. ¹⁸F-DPA-714 PET may provide a promising biomarker to identify patients at risk of severe clinical trajectory.

The aim of this work was to quantify the uptake of [¹⁸F]BMS-986192, a PD-L1 adnectin PET tracer, in patients with non-small-cell lung cancer (NSCLC). To this end, plasma input kinetic modeling of dynamic tumor uptake data with online arterial blood sampling was performed. In addition, the accuracy of simplified uptake metrics such as standardized uptake value (SUV) was investigated. Methods: Data from a study with [¹⁸F]BMS-986192 in patients with advanced stage NSCLC eligible for nivolumab treatment were used if a dynamic scan was available and lesions were present in the field of view of the dynamic scan. After injection of [¹⁸F]BMS-986192, a 60-minutes dynamic PET-CT scan was started, followed by a 30-min whole body PET-CT scan. Continuous arterial and discrete arterial and venous blood sampling were performed to determine a plasma input function. Tumor time activity curves were fitted by several plasma input kinetic models. Simplified uptake parameters included tumor to blood ratio as well as several SUV measures. Results: Twenty two tumors in nine patients were analyzed. The arterial plasma input single-tissue reversible compartment model with fitted blood volume fraction seems to be the most preferred model as it best fitted 11 out of 18 tumor time activity curves. The distribution volume V_T ranged from 0.4 to 4.8 mL·cm-3. Similar values were obtained with an image derived input function. From the simplified measures, SUV normalized for body weight (SUV_BW) at 50 and 67 minutes post injection correlated best with V_T, with an R² > 0.9. Conclusion: A single tissue reversible model can be used for the quantification of tumor uptake of the PD-L1 PET tracer [¹⁸F]BMS-986192. SUV_BW at 60 minutes post injection, normalized for body weight, is an accurate simplified parameter for uptake assessment of baseline studies. In order to assess its predictive value for response evaluation during PD-(L)1 immune checkpoint inhibition further validation of SUV against V_T based on an image derived input function is recommended.

Recently, N-(4-¹⁸F-fluorobenzoyl)-interleukin-2 (¹⁸F-FB-IL2) was introduced as PET tracer for T-cell imaging. However, production is complex and time-consuming. Therefore, we developed two radiolabeled interleukin-2 (IL-2) variants, namely aluminum ¹⁸F-fluoride-(restrained complexing agent)-IL-2 (¹⁸F-AlF-RESCA-IL2) and ⁶⁸Ga-gallium-(1,4,7-triazacyclononane-4,7-diacetic acid-1-glutaric acid)-IL-2 (⁶⁸Ga-Ga-NODAGA-IL2) and compared their in-vitro and in-vivo characteristics with ¹⁸F-FB-IL2. Methods: Radiolabeling of ¹⁸F-AlF-RESCA-IL2 and ⁶⁸Ga-Ga-NODAGA-IL2 was optimized and stability was evaluated in human serum. Receptor binding was studied with activated human peripheral blood mononuclear cells (hPBMCs). Ex-vivo tracer biodistribution in immunocompetent BALB/cOlaHsd (BALB/c) mice was performed at 15, 60 and 90 min after tracer injection. In-vivo binding characteristics were studied in severe combined immune-deficient (SCID) mice inoculated with activated hPBMCs in Matrigel. Tracer was injected 15 min after hPBMCs inoculation and a 60-min dynamic PET scan was acquired, followed by ex-vivo biodistribution studies. Specific uptake was determined by co-injection of tracer with unlabeled IL2 and by evaluating uptake in a control group inoculated with Matrigel only. Results: ⁶⁸Ga-Ga-NODAGA-IL2 and ¹⁸F-AlF-RESCA-IL2 were produced with radiochemical purity >95% and radiochemical yield of 13.1±4.7% and 2.4±1.6% within 60 and 90 min, respectively. Both tracers were stable in serum, with >90% being intact tracer after 1h. In-vitro, both tracers displayed preferential binding to activated hPBMCs. Ex-vivo biodistribution studies in BALB/c mice showed higher uptake of ¹⁸F-AlF-RESCA-IL2 than ¹⁸F-FB-IL2 in liver, kidney, spleen, bone and bone marrow. ⁶⁸Ga-Ga-NODAGA-IL2 uptake in liver and kidney was higher than ¹⁸F-FB-IL2 uptake. In-vivo, all tracers revealed uptake in activated hPBMCs in SCID mice. Low uptake was seen after a blocking dose of IL2 or in the Matrigel control group. In addition, ¹⁸F-AlF-RESCA-IL2 yielded highest contrast PET images of target lymph nodes. Conclusion: Production of ¹⁸F-AlF-RESCA-IL2 and ⁶⁸Ga-Ga-NODAGA-IL2 is simpler and faster than ¹⁸F-FB-IL2. Both tracers showed good in-vitro and in-vivo characteristics with high uptake in lymphoid tissue and hPBMC xenografts.

BiTE® (Bispecific T-cell engager) molecules are designed to engage and activate cytotoxic T-cells to kill tumor cells. Little is known about their biodistribution in immunocompetent settings. To explore their pharmacokinetics and the role of the immune cells, BiTE molecules were radiolabeled with positron emission tomography (PET) isotope zirconium-89 (89Zr) and studied in immunocompetent and immunodeficient mouse models. PET images and ex-vivo biodistribution in immunocompetent mice with 89Zr-muS110, targeting mouse CD3 (Kd = 2.9 nM) and mouse EpCAM (Kd = 21 nM), and 89Zr-hyS110, targeting only mouse CD3 (Kd = 2.9 nM), showed uptake in tumor, spleen and other lymphoid organs, while the human-specific control BiTE 89Zr-AMG 110 showed similar tumor uptake but lacked spleen uptake. 89Zr-muS110 spleen uptake was lower in immunodeficient than in immunocompetent mice. After repeated administration of non-radiolabeled muS110 to immunocompetent mice 89Zr-muS110 uptake in spleen, and other lymphoid tissues, decreased and was comparable to uptake in immunodeficient mice, indicating saturation of CD3 binding sites. Autoradiography and immunohistochemistry demonstrated colocalization of 89Zr-muS110 and 89Zr-hyS110 with CD3-positive T-cells in the tumor and spleen but not with EpCAM expression. Also, uptake in the duodenum correlated with a high incidence of T-cells. This study shows that in immunocompetent mice the BiTE 89Zr-muS110 distribution is predominantly based on its high affinity CD3 binding arm. Significance: 89Zr-muS110 biodistribution is mainly dependent on the T-cell targeting arm with limited contribution of its second arm, targeting EpCAM. These findings highlight the need for extensive biodistribution studies of novel bispecific constructs as results might have implications for their respective drug development and clinical translation.

C-X-C chemokine receptor 4 is a transmembrane chemokine receptor involved in growth, survival, and dissemination of cancer, including aggressive B-cell lymphoma. Magnetic resonance imaging (MRI) is the standard imaging technology for central nervous system involvement of B-cell lymphoma and provides high sensitivity but moderate specificity. Therefore, novel molecular and functional imaging strategies are urgently required. Methods: In this proof-of-concept study, 11 patients with lymphoma of the CNS (CNSL, n = 8 primary and n = 3 secondary involvement) were imaged with the CXCR4-directed positron emission tomography (PET) tracer ⁶⁸Ga-Pentixafor. To evaluate the predictive value of this imaging modality, treatment response, as determined by MRI, was correlated with quantification of CXCR4 expression by ⁶⁸Ga-Pentixafor PET in vivo before initiation of treatment in 7 of 11 patients. Results: ⁶⁸Ga-Pentixafor-PET showed excellent contrast characteristics to the surrounding brain parenchyma in all patients with active disease. Furthermore, initial CXCR4 uptake determined by PET correlated with subsequent treatment response as assessed by MRI. Conclusion: ⁶⁸Ga-Pentixafor-PET represents a novel diagnostic tool for central nervous system lymphoma with potential implications for theranostic approaches as well as response and risk assessment.

In randomized clinical trials (RCTs), no survival benefit has been observed for selective internal radiotherapy (SIRT) over sorafenib in patients with advanced hepatocellular carcinoma (aHCC). This study aimed to assess by means of a meta-analysis whether overall survival (OS) with SIRT, as monotherapy or followed by sorafenib, is non-inferior to sorafenib, and compare safety profiles for patients with aHCC. Methods: We searched MEDLINE, EMBASE, and the Cochrane Library up to February 2019 to identify RCTs comparing SIRT as monotherapy, or followed by sorafenib, to sorafenib monotherapy among patients with aHCC. The main outcomes were OS and frequency of treatment-related severe adverse events (AEs grade ≥3). The per-protocol population was the primary analysis population. A non-inferiority margin of 1.08 in terms of hazard ratio (HR) was pre-specified for the upper boundary of 95% confidence interval (CI) for OS. Pre-specified subgroup analyses were performed. Results: Three RCTs, involving 1,243 patients, comparing sorafenib with SIRT (SIRveNIB and SARAH) or SIRT followed by sorafenib (SORAMIC), were included. After randomization, 411/635 (64.7%) patients allocated to SIRT and 522/608 (85.8%) allocated to sorafenib completed the studies without major protocol deviations. Median OS with SIRT, whether or not followed by sorafenib, was non-inferior to sorafenib (10.2 and 9.2 months, [HR 0.91, 95% CI 0.78–1.05]). Treatment-related severe adverse events were reported in 149/515 patients (28.9%) who received SIRT and 249/575 (43.3%) who received sorafenib only (p<0.01). Conclusion: SIRT as initial therapy for aHCC is non-inferior to sorafenib in terms of OS, and offers a better safety profile.

The value of interim ¹⁸F-fluorodeoxyglucose positron emission tomography (iPET) guided treatment decisions in patients with diffuse large B-cell lymphoma (DLBCL) has been the subject of much debate. This investigation focuses on a comparison of the Deauville score and the deltaSUV_max (SUV_max) approach – two methods to assess early metabolic response to standard chemotherapy in DLBCL. Methods: Of 609 DLBCL patients participating in the Positron Emission Tomography-guided Therapy of Aggressive non-Hodgkin Lymphomas (PETAL) trial, iPET scans of 596 patients originally evaluated using the SUV_max method were available for post-hoc assessment of the Deauville score. A commonly used definition of an unfavorable iPET result according to the Deauville score is an uptake greater than that of the liver, whereas an unfavorable iPET scan with regard to the SUV_max approach is characterized as a relative reduction of the maximum standardized uptake value between baseline and iPET staging of less than or equal to 66%. We investigated the two methods’ correlation and concordance by Spearman’s rank correlation coefficient and the agreement in classification, respectively. We further used Kaplan-Meier curves and Cox regression to assess differences in survival between patient subgroups defined by the pre-specified cut-offs. Time-dependent receiver operating curve analysis provided information on the methods’ respective discrimination performance. Results: Deauville score and SUV_max approach differed in their iPET-based prognosis. The SUV_max approach outperformed the Deauville score in terms of discrimination performance – most likely due to a high number of false-positive decisions by the Deauville score. Cut-off-independent discrimination performance remained low for both methods, but cut-off-related analyses showed promising results. Both favored the SUV_max approach, e.g. for the segregation by iPET response, where the event-free survival hazard ratio was 3.14 (95% confidence interval (CI): 2.22 – 4.46) for SUV_max and 1.70 (95% CI: 1.29 – 2.24) for the Deauville score. Conclusion: When considering treatment intensification, the currently used Deauville score cut-off of an uptake above that of the liver seems to be inappropriate and associated with potential harm for DLBCL patients. The SUV_max criterion of a relative reduction of the maximum standardized uptake value of less than or equal to 66% should be considered as an alternative.

The molecular mechanisms underlying exceptional radioresistance in pancreatic cancer remain elusive. In the present study, we established a stable radioresistant pancreatic cancer cell line MIA PaCa-2-R by exposing the parental MIA PaCa-2 cells to fractionated ionizing radiation (IR). Systematic proteomics and bioinformatics analysis of protein expression in MIA PaCa-2 and MIA PaCa-2-R cells revealed that several growth factor-/cytokine-mediated pathways, including the OSM/STAT3, PI3K/AKT, and MAPK/ERK pathways, were activated in the radioresistant cells, leading to inhibition of apoptosis and increased epithelial-mesenchymal plasticity. In addition, the radioresistant cells exhibited enhanced capabilities of DNA repair and antioxidant defense compared with the parental cells. We focused functional analysis on one of the most up-regulated proteins in the radioresistant cells, ecto-5'-nucleotidase (CD73), which is a cell surface protein that is overexpressed in different types of cancer. Ectopic overexpression of CD73 in the parental cells resulted in radioresistance and conferred resistance to IR-induced apoptosis. Knockdown of CD73 re-sensitized the radioresistant cells to IR and IR-induced apoptosis. The effect of CD73 on radioresistance and apoptosis is independent of the enzymatic activity of CD73. Further studies demonstrate that CD73 up-regulation promotes Ser-136 phosphorylation of the proapoptotic protein BAD and is required for maintaining the radioresistant cells in a mesenchymal state. Our findings suggest that expression alterations in the IR-selected pancreatic cancer cells result in hyperactivation of the growth factor/cytokine signaling that promotes epithelial-mesenchymal plasticity and enhancement of DNA repair. Our results also suggest that CD73, potentially a novel downstream factor of the enhanced growth factor/cytokine signaling, confers acquired radioresistance by inactivating proapoptotic protein BAD via phosphorylation of BAD at Ser-136 and by maintaining the radioresistant pancreatic cancer cells in a mesenchymal state.

Aberrantly high mTORC1 signaling is a known driver of many cancers and human disorders, yet pharmacological inhibition of mTORC1 rarely confers durable clinical responses. To explore alternative therapeutic strategies, herein we conducted a proteomics survey to identify cell surface proteins upregulated by mTORC1. A comparison of the surfaceome from Tsc1^–/– versus Tsc1^+/+ mouse embryonic fibroblasts revealed 59 proteins predicted to be significantly overexpressed in Tsc1^–/– cells. Further validation of the data in multiple mouse and human cell lines showed that mTORC1 signaling most dramatically induced the expression of the proteases neprilysin (NEP/CD10) and aminopeptidase N (APN/CD13). Functional studies showed that constitutive mTORC1 signaling sensitized cells to genetic ablation of NEP and APN, as well as the biochemical inhibition of APN. In summary, these data show that mTORC1 signaling plays a significant role in the constitution of the surfaceome, which in turn may present novel therapeutic strategies.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with very limited therapeutic options. We have recently shown that the combined inhibition of EGFR and ROCK in TNBC cells results in cell death, however, the underlying mechanisms remain unclear. To investigate this, here we applied a mass spectrometry-based proteomic approach to identify proteins altered on single and combination treatments. Our proteomic data revealed autophagy as the major molecular mechanism implicated in the cells' response to combinatorial treatment. We here show that EGFR inhibition by gefitinib treatment alone induces autophagy, a cellular recycling process that acts as a cytoprotective response for TNBC cells. However, combined inhibition of EGFR and ROCK leads to autophagy blockade and accumulation of autophagic vacuoles. Our data show impaired autophagosome clearance as a likely cause of antitumor activity. We propose that the inhibition of the autophagic flux on combinatorial treatment is attributed to the major cytoskeletal changes induced on ROCK inhibition, given the essential role the cytoskeleton plays throughout the various steps of the autophagy process.

The prediction of metastatic properties from molecular analyses still poses a major challenge. Here we aimed at the classification of metastasis-related cell properties by proteome profiling making use of cutaneous and brain-metastasizing variants from single melanomas sharing the same genetic ancestry. Previous experiments demonstrated that cultured cells derived from these xenografted variants maintain a stable phenotype associated with a differential metastatic behavior: The brain metastasizing variants produce more spontaneous micro-metastases than the corresponding cutaneous variants. Four corresponding pairs of cutaneous and metastatic cells were obtained from four individual patients, resulting in eight cell-lines presently investigated. Label free proteome profiling revealed significant differences between corresponding pairs of cutaneous and cerebellar metastases from the same patient. Indeed, each brain metastasizing variant expressed several apparently metastasis-associated proteomic alterations as compared with the corresponding cutaneous variant. Among the differentially expressed proteins we identified cell adhesion molecules, immune regulators, epithelial to mesenchymal transition markers, stem cell markers, redox regulators and cytokines. Similar results were observed regarding eicosanoids, considered relevant for metastasis, such as PGE2 and 12-HETE. Multiparametric morphological analysis of cells also revealed no characteristic alterations associated with the cutaneous and brain metastasis variants. However, no correct classification regarding metastatic potential was yet possible with the present data. We thus concluded that molecular profiling is able to classify cells according to known functional categories but is not yet able to predict relevant cell properties emerging from networks consisting of many interconnected molecules. The presently observed broad diversity of molecular patterns, irrespective of restricting to one tumor type and two main classes of metastasis, highlights the important need to develop meta-analysis strategies to predict cell properties from molecular profiling data. Such base knowledge will greatly support future individualized precision medicine approaches.

We present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on a small but diverse set of PPIs (eleven protein families from six organisms) with different affinities; the dissociation constants range from 117 pm to 17 μm. After only two hours of reaction, expression of the reporter can be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical expression levels of the reaction partners. As a result of this standardization of expression levels, the mean reporter levels directly reflect the affinities of the studied PPIs. With a set of PPIs with known affinities, it is straightforward to construct an affinity ladder that permits rapid classification of PPIs with thus far unknown affinities. Conventional software can be used for this analysis. To permit automated analysis, we provide a graphical user interface for the Python-based FlowCytometryTools package.

Anal squamous cell carcinoma is a rare tumor. Chemo-radiotherapy yields a 50% 3-year relapse-free survival rate in advanced anal cancer, so improved predictive markers and therapeutic options are needed. High-throughput proteomics and whole-exome sequencing were performed in 46 paraffin samples from anal squamous cell carcinoma patients. Hierarchical clustering was used to establish groups de novo. Then, probabilistic graphical models were used to study the differences between groups of patients at the biological process level. A molecular classification into two groups of patients was established, one group with increased expression of proteins related to adhesion, T lymphocytes and glycolysis; and the other group with increased expression of proteins related to translation and ribosomes. The functional analysis by the probabilistic graphical model showed that these two groups presented differences in metabolism, mitochondria, translation, splicing and adhesion processes. Additionally, these groups showed different frequencies of genetic variants in some genes, such as ATM, SLFN11 and DST. Finally, genetic and proteomic characteristics of these groups suggested the use of some possible targeted therapies, such as PARP inhibitors or immunotherapy.

The cell cycle is a highly conserved process involving the coordinated separation of a single cell into two daughter cells. To relate transcriptional regulation across the cell cycle with oscillatory changes in protein abundance and activity, we carried out a proteome- and phospho-proteome-wide mass spectrometry profiling. We compared protein dynamics with gene transcription, revealing many transcriptionally regulated G2 mRNAs that only produce a protein shift after mitosis. Integration of CRISPR/Cas9 survivability studies further highlighted proteins essential for cell viability. Analyzing the dynamics of phosphorylation events and protein solubility dynamics over the cell cycle, we characterize predicted phospho-peptide motif distributions and predict cell cycle-dependent translocating proteins, as exemplified by the S-adenosylmethionine synthase MAT2A. Our study implicates this enzyme in translocating to the nucleus after the G1/S-checkpoint, which enables epigenetic histone methylation maintenance during DNA replication. Taken together, this data set provides a unique integrated resource with novel insights on cell cycle dynamics.

Acute myeloid leukemia (AML) is a clonal disorder arising from hematopoietic myeloid progenitors. Aberrantly activated tyrosine kinases (TK) are involved in leukemogenesis and are associated with poor treatment outcome. Kinase inhibitor (KI) treatment has shown promise in improving patient outcome in AML. However, inhibitor selection for patients is suboptimal.

In a preclinical effort to address KI selection, we analyzed a panel of 16 AML cell lines using phosphotyrosine (pY) enrichment-based, label-free phosphoproteomics. The Integrative Inferred Kinase Activity (INKA) algorithm was used to identify hyperphosphorylated, active kinases as candidates for KI treatment, and efficacy of selected KIs was tested.

Heterogeneous signaling was observed with between 241 and 2764 phosphopeptides detected per cell line. Of 4853 identified phosphopeptides with 4229 phosphosites, 4459 phosphopeptides (4430 pY) were linked to 3605 class I sites (3525 pY). INKA analysis in single cell lines successfully pinpointed driver kinases (PDGFRA, JAK2, KIT and FLT3) corresponding with activating mutations present in these cell lines. Furthermore, potential receptor tyrosine kinase (RTK) drivers, undetected by standard molecular analyses, were identified in four cell lines (FGFR1 in KG-1 and KG-1a, PDGFRA in Kasumi-3, and FLT3 in MM6). These cell lines proved highly sensitive to specific KIs. Six AML cell lines without a clear RTK driver showed evidence of MAPK1/3 activation, indicative of the presence of activating upstream RAS mutations. Importantly, FLT3 phosphorylation was demonstrated in two clinical AML samples with a FLT3 internal tandem duplication (ITD) mutation.

Our data show the potential of pY-phosphoproteomics and INKA analysis to provide insight in AML TK signaling and identify hyperactive kinases as potential targets for treatment in AML cell lines. These results warrant future investigation of clinical samples to further our understanding of TK phosphorylation in relation to clinical response in the individual patient.

The redox-based modifications of cysteine residues in proteins regulate their function in many biological processes. The gas molecule H₂S has been shown to persulfidate redox sensitive cysteine residues resulting in an H₂S-modified proteome known as the sulfhydrome. Tandem Mass Tags (TMT) multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent in detecting cysteine modifications. Here we developed a TMT-based proteomics approach for selectively trapping and tagging cysteine persulfides in the cellular proteomes. We revealed the natural protein sulfhydrome of two human cell lines, and identified insulin as a novel substrate in pancreatic beta cells. Moreover, we showed that under oxidative stress conditions, increased H₂S can target enzymes involved in energy metabolism by switching specific cysteine modifications to persulfides. Specifically, we discovered a Redox Thiol Switch, from protein S-glutathioinylation to S-persulfidation (RTS^GS). We propose that the RTS^GS from S-glutathioinylation to S-persulfidation is a potential mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.

Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in isobaric labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with isobaric labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g. in general <100x) and optimizing MS automatic gain control (AGC) and ion injection time settings in MS/MS analysis (e.g. 5E5 and 300 ms, respectively, which is significantly higher than that used in typical bulk analysis). By coupling with a nanodroplet-based single cell preparation (nanoPOTS) platform, iBASIL enabled identification of ~2500 proteins and precise quantification of ~1500 proteins in the analysis of 104 FACS-isolated single cells, with the resulting protein profiles robustly clustering the cells from three different acute myeloid leukemia cell lines. This study highlights the importance of carefully evaluating and optimizing the boosting ratios and MS data acquisition conditions for achieving robust, comprehensive proteomic analysis of single cells.

Autoimmune thyroid diseases (AITD) are the most common group of autoimmune diseases, associated with lymphocyte infiltration and the production of thyroid autoantibodies, like thyroid peroxidase antibodies (TPOAb), in the thyroid gland. Immunoglobulins and cell-surface receptors are glycoproteins with distinctive glycosylation patterns that play a structural role in maintaining and modulating their functions. We investigated associations of total circulating IgG and peripheral blood mononuclear cells glycosylation with AITD and the influence of genetic background in a case-control study with several independent cohorts and over 3,000 individuals in total. The study revealed an inverse association of IgG core fucosylation with TPOAb and AITD, as well as decreased peripheral blood mononuclear cells antennary α1,2 fucosylation in AITD, but no shared genetic variance between AITD and glycosylation. These data suggest that the decreased level of IgG core fucosylation is a risk factor for AITD that promotes antibody-dependent cell-mediated cytotoxicity previously associated with TPOAb levels.

Signaling networks process intra- and extracellular information to modulate the functions of a cell. Deregulation of signaling networks results in abnormal cellular physiological states and often drives diseases. Network responses to a stimulus or a drug treatment can be highly heterogeneous across cells in a tissue because of many sources of cellular genetic and non-genetic variance. Signaling network heterogeneity is the key to many biological processes, such as cell differentiation and drug resistance. Only recently, the emergence of multiplexed single-cell measurement technologies has made it possible to evaluate this heterogeneity. In this review, we categorize currently established single-cell signaling network profiling approaches by their methodology, coverage, and application, and we discuss the advantages and limitations of each type of technology. We also describe the available computational tools for network characterization using single-cell data and discuss potential confounding factors that need to be considered in single-cell signaling network analyses.

Phosphoglycerate kinase 1 (PGK1) plays important roles in glycolysis, yet its forward reaction kinetics are unknown, and its role especially in regulating cancer cell glycolysis is unclear. Here, we developed an enzyme assay to measure the kinetic parameters of the PGK1-catalyzed forward reaction. The Km values for 1,3-bisphosphoglyceric acid (1,3-BPG, the forward reaction substrate) were 4.36 μm (yeast PGK1) and 6.86 μm (human PKG1). The Km values for 3-phosphoglycerate (3-PG, the reverse reaction substrate and a serine precursor) were 146 μm (yeast PGK1) and 186 μm (human PGK1). The Vmax of the forward reaction was about 3.5- and 5.8-fold higher than that of the reverse reaction for the human and yeast enzymes, respectively. Consistently, the intracellular steady-state concentrations of 3-PG were between 180 and 550 μm in cancer cells, providing a basis for glycolysis to shuttle 3-PG to the serine synthesis pathway. Using siRNA-mediated PGK1-specific knockdown in five cancer cell lines derived from different tissues, along with titration of PGK1 in a cell-free glycolysis system, we found that the perturbation of PGK1 had no effect or only marginal effects on the glucose consumption and lactate generation. The PGK1 knockdown increased the concentrations of fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and 1,3-BPG in nearly equal proportions, controlled by the kinetic and thermodynamic states of glycolysis. We conclude that perturbation of PGK1 in cancer cells insignificantly affects the conversion of glucose to lactate in glycolysis.

Triple-negative breast cancer (TNBC) is characterized by its aggressive biology, early metastatic spread, and poor survival outcomes. TNBC lacks expression of the targetable receptors found in other breast cancer subtypes, mandating use of cytotoxic chemotherapy. However, resistance to chemotherapy is a significant problem, encountered in about two-thirds of TNBC patients, and new strategies are needed to mitigate resistance. In this issue of the Journal of Biological Chemistry, Geck et al. report that TNBC cells are highly sensitive to inhibition of the de novo polyamine synthesis pathway and that inhibition of this pathway sensitizes cells to TNBC-relevant chemotherapy, uncovering new opportunities for addressing chemoresistance.

Treatment of patients with triple-negative breast cancer (TNBC) is limited by a lack of effective molecular therapies targeting this disease. Recent studies have identified metabolic alterations in cancer cells that can be targeted to improve responses to standard-of-care chemotherapy regimens. Using MDA-MB-468 and SUM-159PT TNBC cells, along with LC-MS/MS and HPLC metabolomics profiling, we found here that exposure of TNBC cells to the cytotoxic chemotherapy drugs cisplatin and doxorubicin alter arginine and polyamine metabolites. This alteration was because of a reduction in the levels and activity of a rate-limiting polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). Using gene silencing and inhibitor treatments, we determined that the reduction in ODC was mediated by its negative regulator antizyme, targeting ODC to the proteasome for degradation. Treatment with the ODC inhibitor difluoromethylornithine (DFMO) sensitized TNBC cells to chemotherapy, but this was not observed in receptor-positive breast cancer cells. Moreover, TNBC cell lines had greater sensitivity to single-agent DFMO, and ODC levels were elevated in TNBC patient samples. The alterations in polyamine metabolism in response to chemotherapy, as well as DFMO-induced preferential sensitization of TNBC cells to chemotherapy, reported here suggest that ODC may be a targetable metabolic vulnerability in TNBC.

Liam R. Brunham
Apr 1, 2020; 61:468-469
Commentary

Mustafa Yalcinkaya
Apr 1, 2020; 61:492-504
Research Articles

Kotaro Hama
Apr 1, 2020; 61:523-536
Patient-Oriented and Epidemiological Research

Henry J. Pownall
May 1, 2020; 61:595-597
Commentary

Kristen A. Johnson
Apr 23, 2020; 0:jlr.ILR120000836v1-jlr.ILR120000836
Images in Lipid Research

Xia Meng
May 1, 2020; 61:591-591
Images in Lipid Research

Protein tyrosine kinases (PTKs) play key roles in cellular signal transduction, cell cycle regulation, cell division, and cell differentiation. Dysregulation of PTK-activated pathways, often by receptor overexpression, gene amplification, or genetic mutation, is a causal factor underlying numerous cancers. In this study, we have developed a parallel reaction monitoring (PRM)-based assay for quantitative profiling of 83 PTKs. The assay detects 308 proteotypic peptides from 54 receptor tyrosine kinases and 29 nonreceptor tyrosine kinases in a single run. Quantitative comparisons were based on the labeled reference peptide method. We implemented the assay in four cell models: 1) a comparison of proliferating versus epidermal growth factor (EGF)-stimulated A431 cells, 2) a comparison of SW480Null (mutant APC) and SW480APC (APC restored) colon tumor cell lines, and 3) a comparison of 10 colorectal cancer cell lines with different genomic abnormalities, and 4) lung cancer cell lines with either susceptibility (11-18) or acquired resistance (11-18R) to the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib. We observed distinct PTK expression changes that were induced by stimuli, genomic features or drug resistance, which were consistent with previous reports. However, most of the measured expression differences were novel observations. For example, acquired resistance to erlotinib in the 11-18 cell model was associated not only with previously reported upregulation of MET, but also with upregulation of FLK2 and downregulation of LYN and PTK7. Immunoblot analyses and shotgun proteomics data were highly consistent with PRM data. Multiplexed PRM assays provide a targeted, systems-level profiling approach to evaluate cancer-related proteotypes and adaptations. Data are available through Proteome eXchange Accession PXD002706.

The onset of obesity-linked type 2 diabetes (T2D) is marked by an eventual failure in pancreatic β-cell function and mass that is no longer able to compensate for the inherent insulin resistance and increased metabolic load intrinsic to obesity. However, in a commonly used model of T2D, the db/db mouse, β-cells have an inbuilt adaptive flexibility enabling them to effectively adjust insulin production rates relative to the metabolic demand. Pancreatic β-cells from these animals have markedly reduced intracellular insulin stores, yet high rates of (pro)insulin secretion, together with a substantial increase in proinsulin biosynthesis highlighted by expanded rough endoplasmic reticulum and Golgi apparatus. However, when the metabolic overload and/or hyperglycemia is normalized, β-cells from db/db mice quickly restore their insulin stores and normalize secretory function. This demonstrates the β-cell’s adaptive flexibility and indicates that therapeutic approaches applied to encourage β-cell rest are capable of restoring endogenous β-cell function. However, mechanisms that regulate β-cell adaptive flexibility are essentially unknown. To gain deeper mechanistic insight into the molecular events underlying β-cell adaptive flexibility in db/db β-cells, we conducted a combined proteomic and post-translational modification specific proteomic (PTMomics) approach on islets from db/db mice and wild-type controls (WT) with or without prior exposure to normal glucose levels. We identified differential modifications of proteins involved in redox homeostasis, protein refolding, K48-linked deubiquitination, mRNA/protein export, focal adhesion, ERK1/2 signaling, and renin-angiotensin-aldosterone signaling, as well as sialyltransferase activity, associated with β-cell adaptive flexibility. These proteins are all related to proinsulin biosynthesis and processing, maturation of insulin secretory granules, and vesicular trafficking—core pathways involved in the adaptation of insulin production to meet metabolic demand. Collectively, this study outlines a novel and comprehensive global PTMome signaling map that highlights important molecular mechanisms related to the adaptive flexibility of β-cell function, providing improved insight into disease pathogenesis of T2D.

Accumulation and propagation of hyperphosphorylated tau (p-tau) is a common neuropathological hallmark associated with neurodegeneration of Alzheimer's disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and related tauopathies. Extracellular vesicles, specifically exosomes, have recently been demonstrated to participate in mediating tau propagation in brain. Exosomes produced by human induced pluripotent stem cell (iPSC)-derived neurons expressing mutant Tau (mTau), containing the P301L and V337M Tau mutations of FTDP-17, possess the ability to propagate p-tau pathology after injection into mouse brain.  To gain an understanding of the mTau exosome cargo involved in tau pathogenesis, these pathogenic exosomes were analyzed by proteomics and bioinformatics. The data showed that mTau expression dysregulates the exosome proteome to result in (1) proteins uniquely present only in mTau, and not control exosomes, (2) the absence of proteins in mTau exosomes, uniquely present in control exosomes, and (3) shared proteins which were significantly up-regulated or down-regulated in mTau compared to control exosomes. Notably, mTau exosomes (not control exosomes) contain ANP32A (also known as I1PP2A), an endogenous inhibitor of the PP2A phosphatase which regulates the phosphorylation state of p-tau.  Several of the mTau exosome-specific proteins have been shown to participate in AD mechanisms involving lysosomes, inflammation, secretases, and related processes.  Furthermore, the mTau exosomes lacked a substantial portion of proteins present in control exosomes involved in pathways of localization, vesicle transport, and protein binding functions. The shared proteins present in both mTau and control exosomes represented exosome functions of vesicle-mediated transport, exocytosis, and secretion processes. These data illustrate mTau as a dynamic regulator of the biogenesis of exosomes to result in acquisition, deletion, and up- or down-regulation of protein cargo to result in pathogenic mTau exosomes capable of in vivo propagation of p-tau neuropathology in mouse brain. 

The degradation of intra- and extracellular proteins is essential in all cell types and mediated by two systems, the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. This study investigates the changes in autophagosomal and lysosomal proteomes upon inhibition of proteasomes by bortezomib (BTZ) or MG132. We find an increased abundance of more than 50 proteins in lysosomes of cells in which the proteasome is inhibited. Among those are dihydrofolate reductase (DHFR), ß-Catenin and 3-hydroxy-3-methylglutaryl-coenzym-A (HMGCoA)-reductase. Since these proteins are known to be degraded by the proteasome they seem to be compensatorily delivered to the autophagosomal pathway when the proteasome is inactivated. Surprisingly, most of the proteins which show increased amounts in the lysosomes of BTZ or MG132 treated cells are proteasomal subunits. Thus an inactivated, non-functional proteasome is delivered to the autophagic pathway. Native gel electrophoresis shows that the proteasome reaches the lysosome intact and not disassembled. Adaptor proteins, which target proteasomes to autophagy, have been described in Arabidopsis, Saccharomyces and upon starvation in mammalians. However, in cell lines deficient of these proteins or their mammalian orthologues, respectively, the transfer of proteasomes to the lysosome is not impaired. Obviously, these proteins do not play a role as autophagy adaptor proteins in mammalian cells. We can also show that chaperone-mediated autophagy (CMA) does not participate in the proteasome delivery to the lysosomes. In autophagy-related (ATG)-5 and ATG7 deficient cells the delivery of inactivated proteasomes to the autophagic pathway was only partially blocked, indicating the existence of at least two different pathways by which inactivated proteasomes can be delivered to the lysosome in mammalian cells.

Perlecan is a critical proteoglycan found in the extracellular matrix (ECM) of cartilage. In healthy cartilage, perlecan regulates cartilage biomechanics and we previously demonstrated perlecan deficiency leads to reduced cellular and ECM stiffness in vivo. This change in mechanics may lead to the early onset osteoarthritis seen in disorders resulting from perlecan knockdown such as Schwartz-Jampel syndrome (SJS). To identify how perlecan knockdown affects the material properties of developing cartilage, we used imaging and liquid chromatography–tandem mass spectrometry (LC-MS/MS) to study the ECM in a murine model of SJS, Hspg2C1532Y-Neo. Perlecan knockdown led to defective pericellular matrix formation, whereas the abundance of bulk ECM proteins, including many collagens, increased. Post-translational modifications and ultrastructure of collagens were not significantly different; however, LC-MS/MS analysis showed more protein was secreted by Hspg2C1532Y-Neo cartilage in vitro, suggesting that the incorporation of newly synthesized ECM was impaired. In addition, glycosaminoglycan deposition was atypical, which may explain the previously observed decrease in mechanics. Overall, these findings provide insight into the influence of perlecan on functional cartilage assembly and the progression of osteoarthritis in SJS.

We previously described the expression of CD36 and LPL by breast cancer (BC) cells and tissues and the growth-promoting effect of VLDL observed only in the presence of LPL. We now report a model in which LPL is bound to a heparan sulfate proteoglycan motif on the BC cell surface and acts in concert with the VLDL receptor to internalize VLDLs via receptor-mediated endocytosis. We also demonstrate that gene-expression programs for lipid synthesis versus uptake respond robustly to triglyceride-rich lipoprotein availability. The literature emphasizes de novo FA synthesis and exogenous free FA uptake using CD36 as paramount mechanisms for lipid acquisition by cancer cells. We find that the uptake of intact lipoproteins is also an important mechanism for lipid acquisition and that the relative reliance on lipid synthesis versus uptake varies among BC cell lines and in response to VLDL availability. This metabolic plasticity has important implications for the development of therapies aimed at the lipid dependence of many types of cancer, in that the inhibition of FA synthesis may elicit compensatory upregulation of lipid uptake. Moreover, the mechanism that we have elucidated provides a direct connection between dietary fat and tumor biology.­.

Phospholipases A₂ (PLA₂s) catalyze hydrolysis of the sn-2 substituent from glycerophospholipids to yield a free fatty acid (i.e., arachidonic acid), which can be metabolized to pro- or anti-inflammatory eicosanoids. Macrophages modulate inflammatory responses and are affected by Ca²⁺-independent phospholipase A₂ (PLA₂)β (iPLA₂β). Here, we assessed the link between iPLA₂β-derived lipids (iDLs) and macrophage polarization. Macrophages from WT and KO (iPLA₂β–/–) mice were classically M1 pro-inflammatory phenotype activated or alternatively M2 anti-inflammatory phenotype activated, and eicosanoid production was determined by ultra-performance LC ESI-MS/MS. As a genotypic control, we performed similar analyses on macrophages from RIP.iPLA₂β.Tg mice with selective iPLA₂β overexpression in β-cells. Compared with WT, generation of select pro-inflammatory prostaglandins (PGs) was lower in iPLA₂β^–/–, and that of a specialized pro-resolving lipid mediator (SPM), resolvin D2, was higher; both changes are consistent with the M2 phenotype. Conversely, macrophages from RIP.iPLA₂β.Tg mice exhibited an opposite landscape, one associated with the M1 phenotype: namely, increased production of pro-inflammatory eicosanoids (6-keto PGF₁α, PGE₂, leukotriene B₄) and decreased ability to generate resolvin D2. These changes were not linked with secretory PLA₂ or cytosolic PLA₂α or with leakage of the transgene. Thus, we report previously unidentified links between select iPLA₂β-derived eicosanoids, an SPM, and macrophage polarization. Importantly, our findings reveal for the first time that β-cell iPLA₂β-derived signaling can predispose macrophage responses. These findings suggest that iDLs play critical roles in macrophage polarization, and we posit that they could be targeted therapeutically to counter inflammation-based disorders.

Cholesteryl ester transfer protein (CETP) exists as full-length (FL) and exon 9 (E9)-deleted isoforms. The function of E9-deleted CETP is poorly understood. Here, we investigated the role of E9-deleted CETP in regulating the secretion of FL-CETP by cells and explored its possible role in intracellular lipid metabolism. CETP overexpression in cells that naturally express CETP confirmed that E9-deleted CETP is not secreted, and showed that cellular FL- and E9-deleted CETP form an isolatable complex. Coexpression of CETP isoforms lowered cellular levels of both proteins and impaired FL-CETP secretion. These effects were due to reduced synthesis of both isoforms; however, the predominate consequence of FL- and E9-deleted CETP coexpression is impaired FL-CETP synthesis. We reported previously that reducing both CETP isoforms or overexpressing FL-CETP impairs cellular triglyceride (TG) storage. To investigate this further, E9-deleted CETP was expressed in SW872 cells that naturally synthesize CETP and in mouse 3T3-L1 cells that do not. E9-deleted CETP overexpression stimulated SW872 triglyceride synthesis and increased stored TG 2-fold. Expression of E9-deleted CETP in mouse 3T3-L1 cells produced a similar lipid phenotype. In vitro, FL-CETP promotes the transfer of TG from ER-enriched membranes to lipid droplets. E9-deleted CETP also promoted this transfer, although less effectively, and it inhibited the transfer driven by FL-CETP. We conclude that FL- and E9-deleted CETP isoforms interact to mutually decrease their intracellular levels and impair FL-CETP secretion by reducing CETP biosynthesis. E9-deleted CETP, like FL-CETP, alters cellular TG metabolism and storage but in a contrary manner.

Niemann-Pick disease type C (NPC) disease is a lipid-storage disorder that is caused by mutations in the genes encoding NPC proteins and results in lysosomal cholesterol accumulation. 2-Hydroxypropyl-β-cyclodextrin (CD) has been shown to reduce lysosomal cholesterol levels and enhance sterol homeostatic responses, but CD’s mechanism of action remains unknown. Recent work provides evidence that CD stimulates lysosomal exocytosis, raising the possibility that lysosomal cholesterol is released in exosomes. However, therapeutic concentrations of CD do not alter total cellular cholesterol, and cholesterol homeostatic responses at the ER are most consistent with increased ER membrane cholesterol. To address these disparate findings, here we used stable isotope labeling to track the movement of lipoprotein cholesterol cargo in response to CD in NPC1-deficient U2OS cells. Although released cholesterol was detectable, it was not associated with extracellular vesicles. Rather, we demonstrate that lysosomal cholesterol trafficks to the plasma membrane (PM), where it exchanges with lipoprotein-bound cholesterol in a CD-dependent manner. We found that in the absence of suitable extracellular cholesterol acceptors, cholesterol exchange is abrogated, cholesterol accumulates in the PM, and reesterification at the ER is increased. These results support a model in which CD promotes intracellular redistribution of lysosomal cholesterol, but not cholesterol exocytosis or efflux, during the restoration of cholesterol homeostatic responses.

Pyruvate kinase M2 (PKM2) links metabolic and inflammatory dysfunction in atherosclerotic coronary artery disease; however, its role in oxidized LDL (Ox-LDL)-induced macrophage foam cell formation and inflammation is unknown and therefore was studied. In recombinant mouse granulocyte-macrophage colony-stimulating factor-differentiated murine bone marrow-derived macrophages, early (1–6 h) Ox-LDL treatment induced PKM2 tyrosine 105 phosphorylation and promotes its nuclear localization. PKM2 regulates aerobic glycolysis and inflammation because PKM2 shRNA or Shikonin abrogated Ox-LDL-induced hypoxia-inducible factor-1α target genes lactate dehydrogenase, glucose transporter member 1, interleukin 1β (IL-1β) mRNA expression, lactate, and secretory IL-1β production. PKM2 inhibition significantly increased Ox-LDL-induced ABCA1 and ABCG1 protein expression and NBD-cholesterol efflux to apoA1 and HDL. PKM2 shRNA significantly inhibited Ox-LDL-induced CD36, FASN protein expression, DiI-Ox-LDL binding and uptake, and cellular total cholesterol, free cholesterol, and cholesteryl ester content. Therefore, PKM2 regulates lipid uptake and efflux. DASA-58, a PKM2 activator, downregulated LXR-α, ABCA1, and ABCG1, and augmented FASN and CD36 protein expression. Peritoneal macrophages showed similar results. Ox-LDL induced PKM2- SREBP-1 interaction and FASN expression in a PKM2-dependent manner. Therefore, this study suggests a role for PKM2 in Ox-LDL-induced aerobic glycolysis, inflammation, and macrophage foam cell formation.