Ashley S. Lindsey

Thin-film tunable filters for hyperspectral fluorescence microscopy

Abstract. Hyperspectral imaging is a powerful tool that acquires data from many spectral bands, forming a contiguous spectrum. Hyperspectral imaging was originally developed for remote sensing applications; however, hyperspectral techniques have since been applied to biological fluorescence imaging applications, such as fluorescence microscopy and small animal fluorescence imaging. The spectral filtering method largely determines the sensitivity and specificity of any hyperspectral imaging system. There are several types of spectral filtering hardware available for microscopy systems, most commonly acousto-optic tunable filters (AOTFs) and liquid crystal tunable filters (LCTFs). These filtering technologies have advantages and disadvantages. Here, we present a novel tunable filter for hyperspectral imaging—the thin-film tunable filter (TFTF). The TFTF presents several advantages over AOTFs and LCTFs, most notably, a high percentage transmission and a high out-of-band optical density (OD). We present a comparison of a TFTF-based hyperspectral microscopy system and a commercially available AOTF-based system. We have characterized the light transmission, wavelength calibration, and OD of both systems, and have then evaluated the capability of each system for discriminating between green fluorescent protein and highly autofluorescent lung tissue. Our results suggest that TFTFs are an alternative approach for hyperspectral filtering that offers improved transmission and out-of-band blocking. These characteristics make TFTFs well suited for other biomedical imaging devices, such as ophthalmoscopes or endoscopes.

An approach for characterizing and comparing hyperspectral microscopy systems.

Hyperspectral imaging and analysis approaches offer accurate detection and quantification of fluorescently-labeled proteins and cells in highly autofluorescent tissues. However, selecting optimum acquisition settings for hyperspectral imaging is often a daunting task. In this study, we compared two hyperspectral systems—a widefield system with acoustic optical tunable filter (AOTF) and charge coupled device (CCD) camera, and a confocal system with diffraction gratings and photomultiplier tube (PMT) array. We measured the effects of system parameters on hyperspectral image quality and linear unmixing results. Parameters that were assessed for the confocal system included pinhole diameter, laser power, PMT gain and for the widefield system included arc lamp intensity, and camera gain. The signal-to-noise ratio (SNR) and the root-mean-square error (RMS error) were measured to assess system performance. Photobleaching dynamics were studied. Finally, theoretical sensitivity studies were performed to estimate the incremental response (sensitivity) and false-positive detection rates (specificity). Results indicate that hyperspectral imaging assays are highly dependent on system parameters and experimental conditions. For detection of green fluorescent protein (GFP)-expressing cells in fixed lung tissues, a confocal pinhole of five airy disk units, high excitation intensity and low detector gain were optimal. The theoretical sensitivity studies revealed that widefield hyperspectral microscopy was able to detect GFP with fewer false positive occurrences than confocal microscopy, even though confocal microscopy offered improved signal and noise characteristics. These studies provide a framework for optimization that can be applied to a variety of hyperspectral imaging systems.

In the absence of effector proteins, the Pseudomonas aeruginosa type three secretion system needle tip complex contributes to lung injury and systemic inflammatory responses.

Herein we describe a pathogenic role for the Pseudomonas aeruginosa type three secretion system (T3SS) needle tip complex protein, PcrV, in causing lung endothelial injury. We first established a model in which P. aeruginosa wild type strain PA103 caused pneumonia-induced sepsis and distal organ dysfunction. Interestingly, a PA103 derivative strain lacking its two known secreted effectors, ExoU and ExoT [denoted PA103 (ΔU/ΔT)], also caused sepsis and modest distal organ injury whereas an isogenic PA103 strain lacking the T3SS needle tip complex assembly protein [denoted PA103 (ΔPcrV)] did not. PA103 (ΔU/ΔT) infection caused neutrophil influx into the lung parenchyma, lung endothelial injury, and distal organ injury (reminiscent of sepsis). In contrast, PA103 (ΔPcrV) infection caused nominal neutrophil infiltration and lung endothelial injury, but no distal organ injury. We further examined pathogenic mechanisms of the T3SS needle tip complex using cultured rat pulmonary microvascular endothelial cells (PMVECs) and revealed a two-phase, temporal nature of infection. At 5-hours post-inoculation (early phase infection), PA103 (ΔU/ΔT) elicited PMVEC barrier disruption via perturbation of the actin cytoskeleton and did so in a cell death-independent manner. Conversely, PA103 (ΔPcrV) infection did not elicit early phase PMVEC barrier disruption. At 24-hours post-inoculation (late phase infection), PA103 (ΔU/ΔT) induced PMVEC damage and death that displayed an apoptotic component. Although PA103 (ΔPcrV) infection induced late phase PMVEC damage and death, it did so to an attenuated extent. The PA103 (ΔU/ΔT) and PA103 (ΔPcrV) mutants grew at similar rates and were able to adhere equally to PMVECs post-inoculation indicating that the observed differences in damage and barrier disruption are likely attributable to T3SS needle tip complex-mediated pathogenic differences post host cell attachment. Together, these infection data suggest that the T3SS needle tip complex and/or another undefined secreted effector(s) are important determinants of P. aeruginosa pneumonia-induced lung endothelial barrier disruption.

β-catenin nuclear translocation in colorectal cancer cells is suppressed by PDE10A inhibition, cGMP elevation, and activation of PKG

Phosphodiesterase 10A (PDE10) is a cGMP and cAMP degrading PDE isozyme that is highly expressed in the brain striatum where it appears to play an important role in cognition and psychomotor activity. PDE10 inhibitors are being developed for the treatment of schizophrenia and Huntingtons disease and are generally well tolerated, possibly because of low expression levels in most peripheral tissues. We recently reported high levels of PDE10 in colon tumors and that genetic silencing of PDE10 by siRNA or inhibition with small molecule inhibitors can suppress colon tumor cell growth with a high degree of selectivity over normal colonocytes (Li et al., Oncogene 2015). These observations suggest PDE10 may have an unrecognized role in tumorigenesis. Here we report that the concentration range by which the highly specific PDE10 inhibitor, Pf-2545920 (MP-10), inhibits colon tumor cell growth parallels the concentration range required to increase cGMP and cAMP levels, and activates PKG and PKA, respectively. Moreover, PDE10 knockdown by shRNA reduces the sensitivity of colon tumor cells to the growth inhibitory activity of Pf-2545920. Pf-2545920 also inhibits the translocation of β-catenin to the nucleus, thereby reducing β-catenin mediated transcription of survivin, resulting in caspase activation and apoptosis. PDE10 mRNA was also found to be elevated in colon tumors compared with normal tissues. These findings suggest that PDE10 can be targeted for cancer therapy or prevention whereby inhibition results in cGMP elevation and PKG activation to reduce β-catenin-mediated transcription of survival proteins leading to the selective apoptosis of cancer cells.

Abstract 331: Beta-catenin nuclear translocation in colorectal cancer cells is suppressed by PDE10A inhibition, cGMP elevation, and activation of PKG

Phosphodiesterase 10A (PDE10A) is a cGMP and cAMP degrading PDE isozyme that is highly expressed in the brain striatum where it appears to play an important role in cognition and psychomotor activity. PDE10 inhibitors are being developed for the treatment of schizophrenia and Huntington9s disease and are generally well tolerated, possibly because of low expression levels in most peripheral tissues. We recently reported high levels of PDE10 in colon tumors and that genetic silencing of PDE10 by siRNA or inhibition with small molecule inhibitors can suppress colon tumor cell growth with a high degree of selectivity over normal colonocytes (Li et al., Oncogene 2015). These observations suggest PDE10 may have an unrecognized role in tumorigenesis. Here we report that the concentration range by which the highly specific PDE10 inhibitor, Pf-2545920 (MP-10), inhibited colon tumor cell growth parallels the concentration range required to increase cGMP and cAMP levels, and activate PKG and PKA, respectively. Moreover, PDE10 knockdown by shRNA reduced the sensitivity of colon tumor cells to the growth inhibitory activity of Pf-2545920. Pf-2545920 also inhibited the translocation of β-catenin to the nucleus, thereby reducing β-catenin mediated transcription of survivin, which resulted in caspase activation and apoptosis. This was determined to be through a PKG mediated pathway through the use of small molecule inhibitors of PKG and PKA. PDE10 mRNA was also found to be elevated in colon tumors compared with normal tissues in a cDNA array. We also report the increase in PDE10 mRNA in 50% of a small collection of human clinical specimens collected at the Mitchell Cancer Institute (n = 13). In addition, novel PDE10 inhibitor, MCI-030, reduced tumor size and activated PDE10 signaling mechanisms in vivo. These findings suggest that PDE10 can be targeted for cancer therapy or prevention whereby inhibition results in cGMP elevation and PKG activation to reduce β-catenin-mediated transcription of survival proteins leading to the selective apoptosis of cancer cells. Citation Format: Kevin J. Lee, Ashley S. Lindsey, Luciana Madeira da Silva, Alisa Trinh, Bernard Gary, Joel Andrews, Veronica Ramirez-Alcantara, Adam B. Keeton, Wen-Chi Chang, Margie Clapper, Gary A. Piazza. Beta-catenin nuclear translocation in colorectal cancer cells is suppressed by PDE10A inhibition, cGMP elevation, and activation of PKG. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 331.

Phosphodiesterase 10A is overexpressed in lung tumor cells and inhibitors selectively suppress growth by blocking β-catenin and MAPK signaling

Phosphodiesterase 10A (PDE10) is a cyclic nucleotide (e.g. cGMP) degrading enzyme highly expressed in the brain striatum where it plays an important role in dopaminergic neurotransmission, but has limited expression and no known physiological function outside the central nervous system. Here we report that PDE10 mRNA and protein levels are strongly elevated in human non-small cell lung cancer cells and lung tumors compared with normal human airway epithelial cells and lung tissue, respectively. Genetic silencing of PDE10 or inhibition by small molecules such as PQ10 was found to selectively inhibit the growth and colony formation of lung tumor cells. PQ10 treatment of lung tumor cells rapidly increased intracellular cGMP levels and activated cGMP-dependent protein kinase (PKG) at concentrations that inhibit lung tumor cell growth. PQ10 also increased the phosphorylation of β-catenin and reduced its levels, which paralleled the suppression of cyclin D1 and survivin but preceded the activation of PARP and caspase cleavage. PQ10 also suppressed RAS-activated RAF/MAPK signaling within the same concentration range and treatment period as required for cGMP elevation and PKG activation. These results show that PDE10 is overexpressed during lung cancer development and essential for lung tumor cell growth in which inhibitors can selectively induce apoptosis by increasing intracellular cGMP levels and activating PKG to suppress oncogenic β-catenin and MAPK signaling.

Abstract A30: Novel non-COX inhibitory sulindac derivative with PDE10 inhibitory activity reduces incidence and multiplicity of colorectal adenomas in the APC+/min-FCCC mouse model

Sulindac has been reported to reduce the number and size of precancerous colonic adenomas in patients with familial adenomatous polyposis (FAP), but is not recommended for long-term use as a cancer chemopreventive drug because of potentially fatal toxicities associated with cyclooxygenase (COX) inhibition. We report here a novel sulindac derivative, ADT-061, that lacks COX-1 and COX-2 inhibitory activity, yet potently and selectively inhibits colon tumor cells with IC 50 values of 0.3-0.5 μM with no significant effect on the growth of normal colonocytes. By comparison, sulindac sulfide inhibits colon tumor cell growth with IC 50 values of 40-60 μM, and has only modest tumor cell selectivity. ADT-061 inhibits phosphodiesterase 10A (PDE10), which increases intracellular cGMP levels, thus activates protein kinase G, suppresses nuclear levels of β-catenin, and induces cell cycle arrest and apoptosis, all within the same concentration range that inhibits tumor cell growth. ADT-061was evaluated for chemopreventive activity in the APC +/min-FCCC mouse model of colon cancer, which harbors colorectal adenomas that express PDE10. Colonoscopy exams were initially performed on all mice to ensure the absence of colorectal adenomas prior to treatment. At 7-8 weeks of age, male mice were treated with ADT-061 (1000ppm and 1500ppm) in the diet for 14 weeks. ADT-061 reduced the incidence of colorectal adenomas from 95.8% in the control group to 73.9% in the 1000ppm group (p=0.048), and 59.1% (p=0.004) in the 1500ppm group. Control untreated mice had an average of 2.98 colorectal adenomas per mouse, while mice treated with 1000ppm and 1500ppm ADT-061 had an average of 2.04 (p=0.102) and 1.91 (p=0.039) adenomas per mouse, respectively. These observations suggest that ADT-061, or analogs thereof, merit further drug development efforts for colorectal cancer chemoprevention. Citation Format: Kevin J. Lee, Xi Chen, Jacob Valiyaveettil, Ashley S. Lindsey, Joel Andrews, Veronica Ramirez-Alcantara, Adam B. Keeton, Gary A. Piazza, Margie L. Clapper, Wen-Chi L. Chang. Novel non-COX inhibitory sulindac derivative with PDE10 inhibitory activity reduces incidence and multiplicity of colorectal adenomas in the APC+/min-FCCC mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr A30.

Abstract 5243: Novel non-COX inhibitory sulindac derivative with β-catenin suppressing activity reduces the formation of colorectal adenomas and adenocarcinomas in theAPC+/min-FCCCmouse model

Sulindac can reduce the number and size of precancerous colonic adenomas in patients with familial adenomatous polyposis (FAP), but is not recommended for long-term use as a cancer chemopreventive drug because of potentially fatal toxicities associated with cyclooxygenase (COX) inhibition. Here we characterize the antineoplastic properties of a novel sulindac derivative, ADT-061, that lacks COX-1 and COX-2 inhibitory activity. ADT-061 potently and selectively inhibited colon tumor cell growth in vitro with IC50 values of 0.3-0.5 μM by inducing apoptosis without affecting the growth of normal colonocytes at concentrations 100x higher. By comparison, sulindac sulfide (SS) inhibited colon tumor cell growth with IC50 values of 40-60 μM, and had only modest tumor cell selectivity. While SS non-selectively inhibits multiple cyclic GMP phosphodiesteraes (PDE) isozymes, previous studies have suggested an important role for PDE10 in colon cancer (Li et al, Oncogene, 2014; Lee et al, Oncotarget, 2015). At concentrations comparable to its IC50 values, ADT-061 selectively inhibits PDE10 to induce an increase in intracellular cGMP levels and activate protein kinase G (PKG). ADT-061 also suppressed nuclear levels of β-catenin and induced cell cycle arrest and apoptosis, within the same concentration range that inhibits tumor cell growth. With attractive drug-like properties, the chemopreventive activity ADT-061was assessed using the APC+/min-FCCC mouse model of spontaneous colorectal tumorigenesis. Prior to treatment, colonoscopy exams were performed on all APC+/min-FCCC mice (males, 6-7 weeks of age) to ensure the absence of colorectal adenomas. At 7-8 weeks of age, mice were assigned (19 per group) to receive either unsupplemented chow (control) or chow supplemented with ADT-061 (1500ppm) for 14 weeks. No discernible toxicity was observed during the experimental period. Double-blinded pathological analysis of fixed colon tissues revealed that ADT-061 not only reduced the multiplicity of colonic adenomas by 50% (Mean ± SEM, control vs. ADT-061, 3.95 ± 0.81 vs. 1.95 ± 0.58, P Citation Format: Kevin J. Lee, Xi Chen, Jacob Valiyaveettil, Ashley S. Lindsey, Joel Andrews, Veronica Ramirez-Alcantara, Adam B. Keeton, Gary A. Piazza, Harry Cooper, Margie Clapper, Wen-Chi L. Chang. Novel non-COX inhibitory sulindac derivative with β-catenin suppressing activity reduces the formation of colorectal adenomas and adenocarcinomas in the APC+/min-FCCC mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5243. doi:10.1158/1538-7445.AM2017-5243

Abstract B21: PDE10A overexpression in colon cancer cells and tumors relative to normal colonocytes and colon mucosa

Phosphodiesterase 10A (PDE10A) is a cGMP and cAMP degrading PDE isozyme expressed in areas of the brain controlling motor function and cognition. PDE10A has been strongly linked to diseases such as schizophrenia and Huntington9s disease, for which PDE10A inhibitors are currently in clinical trials and appear to be well tolerated. Although, PDE10A has limited expression and no known function in peripheral tissues, high levels were measured in colon tumor cells in vitro and in vivo compared with cells derived from normal colon and colonic mucosa (Li et al., Oncogene 2015). PDE10A inhibition by small molecules or genetic silencing attenuated colon tumor cell growth by a mechanism involving cGMP/PKG activation, the suppression of oncogenic β-catenin, and a decrease in TCF transcriptional activity (Li et al., Oncogene 2015). These observations suggest that PDE10A may play an unrecognized role in tumorigenesis and provide a novel therapeutic target for colorectal cancer chemoprevention or therapy. The human colon tumor cell lines, HCT116, HT29, Caco2, and SW480, show elevated expression of PDE10A as compared to the normal colon mucosal epithelial cell line, NCM460, and are more sensitive to PDE10 inhibitors relative to NCM460 cells. Subcutaneous tumors established from HT29 colon tumor cells in athymic mice expressed elevated levels of PDE10A as compared to normal mouse colon tissue. Colon tumors collected from heterozygous APCmin/+FCCC mice displayed an elevation in PDE10A levels as compared to uninvolved mucosa from the APCmin/+FCCC mice and wild type colon mucosa. Overexpression of PDE10A mRNA and protein levels was also observed in colon adenocarcinomas relative to uninvolved colon mucosa in specimens obtained from colorectal cancer patients. These observations, along with recent findings that a novel sulindac derivative (ADT-061) with PDE10A inhibitory activity suppresses tumor formation in the APCmin/+FCCC mouse model of colon cancer without apparent toxicity, suggests that PDE10A provides a novel therapeutic target for cancer cell specific inhibition of tumor growth. Citation Format: Ashley S. Lindsey, Kevin Lee, Joel Andrews, Wen-Chi L. Chang, Veronica Ramirez-Alcantara, Marcus Tan, William Grizzle, Margie L. Clapper, Gary Piazza. PDE10A overexpression in colon cancer cells and tumors relative to normal colonocytes and colon mucosa. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B21.

Abstract 5159: Characterization of a novel class of RAS inhibitory compounds with potent anti-tumor activity

Introduction: Over 30% of all human cancers harbor activating RAS mutations which induce deregulation of the cell cycle, uncontrolled proliferation, and decreased apoptosis. Through a phenotypic screening strategy, we have identified a series of indene derivatives which potently and selectively inhibit growth of tumor cells harboring activated RAS. A development candidate from this series, DC070-547, disrupts RAS-RAF binding, inhibits RAS signaling, causes cell cycle arrest and induces apoptosis, and exhibits strong anti-tumor activity in a mouse KRAS mutant tumor model. Methods: Viable cell number was measured using a luminescent indicator of ATP. RAS activation was measured by GST-RAF1-RBD pull-down and western blotting using an anti-RAS antibody. Disruption of RAS-RAF binding was determined by pre-incubation of GST-RAF1-RBD beads with cell lysates or recombinant RAS in the presence of test compounds for 30 min. Cell cycle distribution was measured by DNA content and immunofluorescent detection of cell cycle proteins. Antitumor activity was determined in a subcutaneous mouse tumor model involving KRAS mutant colon tumors. Results: Low nanomolar concentrations of DC070-547 selectively inhibited growth of a diverse panel of tumor cell lines harboring activated RAS relative to tumor cell lines lacking activated RAS. Transfection of HT-29 cells lacking activated RAS with mutant RAS conferred sensitivity to DC070-547. The compounds blocked binding of RAF1-RBD to recombinant RAS, RAS from cell lysates, as well as RAS in intact cells. Sustained and potent growth inhibitory effects of DC070-547 were demonstrated by colony formation assays. Immunoblotting showed that DC070-547 inhibited EGF-induced signaling in HCT-116 colon tumor cells with activated RAS at concentrations that inhibit growth. DC070-547 also induced apoptosis as evident by Annexin V labeling and M-phase arrest in HCT116 cells as shown by DNA content and immunostaining of phospho-Histone H3B and Cdc25C, an important downstream mediator of RAS signaling. DC070-547 and three analogs from this series were evaluated for antitumor activity in an athymic mouse model using subcutaneously implanted KRAS mutant colon tumor cells. Treatments either completely suppressed tumor growth or caused tumor regression with no discernible toxicity. Conclusion: While RAS is widely considered to be non-druggable, a novel compound series was identified that potently and selectivity inhibit in vitro and in vivo the growth of tumor cells harboring activated RAS by inhibiting RAS-effector binding. Together, these findings support further preclinical development of this compound class for Phase I/II clinical evaluation for the treatment of RAS-driven cancers. Citation Format: Antonio Ward, Xi Chen, Jacob Valiyaveettil, Bing Zhu, Veronica Ramirez-Alcantara, Kevin J. Lee, Ashley Lindsey, Kristy Berry, Tyler E. Mattox, Kate McConnell, Michael R. Boyd, Gary A. Piazza, Adam B. Keeton. Characterization of a novel class of RAS inhibitory compounds with potent anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5159. doi:10.1158/1538-7445.AM2017-5159