SlyD Accelerates trans-to-cis Prolyl Isomerization in a Mechanosignaling Protein under Load.

Prolyl isomerization is recognized as one of the key regulatory mechanisms, which plays a crucial role in cell signaling, ion channel gating, phage virus infection, and molecular timing. This isomerization is usually slow but often accelerated by an enzyme, called peptidyl-prolyl isomerase (PPIase). In the current project, we investigate using single-molecule force spectroscopy (SMFS) the impact of a bacterial PPIase, SlyD, on the cis-trans isomerization of the proline 2225 (P2225) in an isolated 20th domain of a cytoskeletal mechanosensing protein filamin-A (FlnA20). To explore the FlnA20-PPIase interaction, we have used multiple SMFS modes, like constant velocity, constant distance, and jumping trap experiments. In our previous study, we reported the unique nature of the P2225, which is conserved in all naturally occurring filamins and can slowly (minutes) interconvert between cis-trans isomers, in absence of any PPIase. Our current results show a staggering 25-fold acceleration of the trans-to-cis isomerization rate in the presence of saturating SlyD concentration (7.25 µM) compared to the unenzymatic condition. A SlyD concentration-dependent depletion of the trans isomeric lifetime was also observed. Additionally, we observed that SlyD stabilizes the cis-isomer in the native state of FlnA20 by ∼2 kBT. This is the first single-molecule observation of the cis-trans isomerization catalysis by a PPIase in a mechanosensing protein.

Characterization of the immune microenvironment of NSCLC by multispectral analysis of multiplex immunofluorescence images.

Multiplex immunofluorescence (MIF) staining of tumor sections combined with computational pathology quantifies phenotypic variants of tumor and immune cells and assesses their spatial relationships. Here, we discuss a MIF panel composed of cytokeratin, PD-L1, PD1, CD8, CD68, and Ki67 applied to non-small cell lung cancer (NSCLC) to demonstrate key components of the immune response to this cancer. We also describe a method of whole-slide multiplex imaging and digital multispectral image analysis. Key aspects of marker labeling and digital tissue and cellular classification are highlighted. We then illustrate how digital analysis can measure the spatial relationships among important cell types. This approach is presented in the context of a multidisciplinary team of scientists who together can optimize the combined methods to increase the impact of the study findings. Recommendations are provided to assist others to apply similar methods to further understand the immune response to NSCLC.

Automated image analysis of NSCLC biopsies to predict response to anti-PD-L1 therapy.

BACKGROUND: Immune checkpoint therapies (ICTs) targeting the programmed cell death-1 (PD1)/programmed cell death ligand-1 (PD-L1) pathway have improved outcomes for patients with non-small cell lung cancer (NSCLC), particularly those with high PD-L1 expression. However, the predictive value of manual PD-L1 scoring is imperfect and alternative measures are needed. We report an automated image analysis solution to determine the predictive and prognostic values of the product of PD-L1+ cell and CD8+ tumor infiltrating lymphocyte (TIL) densities (CD8xPD-L1 signature) in baseline tumor biopsies. METHODS: Archival or fresh tumor biopsies were analyzed for PD-L1 and CD8 expression by immunohistochemistry. Samples were collected from 163 patients in Study 1108/NCT01693562, a Phase 1/2 trial to evaluate durvalumab across multiple tumor types, including NSCLC, and a separate cohort of 199 non-ICT- patients. Digital images were automatically scored for PD-L1+ and CD8+ cell densities using customized algorithms applied with Developer XD™ 2.7 software. RESULTS: For patients who received durvalumab, median overall survival (OS) was 21.0 months for CD8xPD-L1 signature-positive patients and 7.8 months for signature-negative patients (p = 0.00002). The CD8xPD-L1 signature provided greater stratification of OS than high densities of CD8+ cells, high densities of PD-L1+ cells, or manually assessed tumor cell PD-L1 expression ≥25%. The CD8xPD-L1 signature did not stratify OS in non-ICT patients, although a high density of CD8+ cells was associated with higher median OS (high: 67 months; low: 39.5 months, p = 0.0009) in this group. CONCLUSIONS: An automated CD8xPD-L1 signature may help to identify NSCLC patients with improved response to durvalumab therapy. Our data also support the prognostic value of CD8+ TILS in NSCLC patients who do not receive ICT. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01693562 . Study code: CD-ON-MEDI4736-1108. Interventional study (ongoing but not currently recruiting). Actual study start date: August 29, 2012. Primary completion date: June 23, 2017 (final data collection date for primary outcome measure).

Measuring multiple parameters of CD8+ tumor-infiltrating lymphocytes in human cancers by image analysis.

BACKGROUND: Immuno-oncology and cancer immunotherapies are areas of intense research. The numbers and locations of CD8+ tumor-infiltrating lymphocytes (TILs) are important measures of the immune response to cancer with prognostic, pharmacodynamic, and predictive potential. We describe the development, validation, and application of advanced image analysis methods to characterize multiple immunohistochemistry-derived CD8 parameters in clinical and nonclinical tumor tissues. METHODS: Commercial resection tumors from nine cancer types, and paired screening/on-drug biopsies of non-small-cell lung carcinoma (NSCLC) patients enrolled in a phase 1/2 clinical trial investigating the PD-L1 antibody therapy durvalumab (NCT01693562), were immunostained for CD8. Additional NCT01693562 samples were immunostained with a CD8/PD-L1 dual immunohistochemistry assay. Whole-slide scanning was performed, tumor regions were annotated by a pathologist, and images were analyzed with customized algorithms using Definiens Developer XD software. Validation of image analysis data used cell-by-cell comparison to pathologist scoring across a range of CD8+ TIL densities of all nine cancers, relying primarily on 95% confidence in having at least moderate agreement regarding Lin concordance correlation coefficient (CCC = 0.88-0.99, CCC_lower = 0.65-0.96). RESULTS: We found substantial variability in CD8+ TILs between individual patients and across the nine types of human cancer. Diffuse large B-cell lymphoma had several-fold more CD8+ TILs than some other cancers. TIL densities were significantly higher in the invasive margin versus tumor center for carcinomas of head and neck, kidney and pancreas, and NSCLC; the reverse was true only for prostate cancer. In paired patient biopsies, there were significantly increased CD8+ TILs 6 weeks after onset of durvalumab therapy (mean of 365 cells/mm2 over baseline; P = 0.009), consistent with immune activation. Image analysis accurately enumerated CD8+ TILs in PD-L1+ regions of lung tumors using the dual assay and also measured elongate CD8+ lymphocytes which constituted a fraction of overall TILs. CONCLUSIONS: Validated image analysis accurately enumerates CD8+ TILs, permitting comparisons of CD8 parameters among tumor regions, individual patients, and cancer types. It also enables the more complex digital solutions needed to better understand cancer immunity, like analysis of multiplex immunohistochemistry and spatial evaluation of the various components comprising the tumor microenvironment. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01693562 . Study code: CD-ON-MEDI4736-1108. Interventional study (ongoing but not currently recruiting). Actual study start date: August 29, 2012. Primary completion date: June 23, 2017 (final data collection date for primary outcome measure).

Force-dependent isomerization kinetics of a highly conserved proline switch modulates the mechanosensing region of filamin.

Proline switches, controlled by cis-trans isomerization, have emerged as a particularly effective regulatory mechanism in a wide range of biological processes. In this study, we use single-molecule mechanical measurements to develop a full kinetic and energetic description of a highly conserved proline switch in the force-sensing domain 20 of human filamin and how prolyl isomerization modulates the force-sensing mechanism. Proline isomerization toggles domain 20 between two conformations. A stable cis conformation with slow unfolding, favoring the autoinhibited closed conformation of filamin's force-sensing domain pair 20-21, and a less stable, uninhibited conformation promoted by the trans form. The data provide detailed insight into the folding mechanisms that underpin the functionality of this binary switch and elucidate its remarkable efficiency in modulating force-sensing, thus combining two previously unconnected regulatory mechanisms, proline switches and mechanosensing.

Dynamic force sensing of filamin revealed in single-molecule experiments.

Mechanical forces are important signals for cell response and development, but detailed molecular mechanisms of force sensing are largely unexplored. The cytoskeletal protein filamin is a key connecting element between the cytoskeleton and transmembrane complexes such as integrins or the von Willebrand receptor glycoprotein Ib. Here, we show using single-molecule mechanical measurements that the recently reported Ig domain pair 20-21 of human filamin A acts as an autoinhibited force-activatable mechanosensor. We developed a mechanical single-molecule competition assay that allows online observation of binding events of target peptides in solution to the strained domain pair. We find that filamin force sensing is a highly dynamic process occurring in rapid equilibrium that increases the affinity to the target peptides by up to a factor of 17 between 2 and 5 pN. The equilibrium mechanism we find here can offer a general scheme for cellular force sensing.