Dennis Otali

Combined effects of formalin fixation and tissue processing on immunorecognition.

Abstract It is accepted that aldehyde-based fixation of cells can affect immunodetection of antigens; however, the effects of tissue processing on immunodetection have not been analyzed systematically. We investigated the effects of aldehyde-based fixation and the various cumulative steps of tissue processing on immunohistochemical detection of specific antigens. DU145 (prostate) and SKOV3 (ovarian) cancer cell lines were cultured as monolayers on microscope slides. Immunohistochemical detection of Ki67/MIB-1 and proliferating cell nuclear antigen (PCNA) was evaluated after various fixation times in 10% neutral buffered formalin and after each of the several cumulative steps of tissue processing. The effect of antigen retrieval (AR) was evaluated concomitantly as an additional variable. Our results indicate that in addition to fixation, each of the tissue processing steps has effects on immunorecognition of the epitopes recognized by these antibodies. Extensive dehydration through ethanols to absolute ethanol had only modest effects, except for the detection of Ki67/MIB-1 in SKOV-3 cells where the effect was stronger. In general, however, establishment of a hydrophobic environment by xylene resulted in the greatest decrease in immunorecognition. AR compensated for most, but not all, of the losses in staining following fixation and exposure to xylene; however, AR gave consistent results for most steps of tissue processing, which suggests that AR also should be used for staining PCNA. The cellular variations that were observed indicate that the effects of fixation and other steps of tissue processing may depend on how antigens are packaged by specific cells.

Preservation of immunorecognition by transferring cells from 10% neutral buffered formalin to 70% ethanol

Abstract Prolonged fixation of cells and tissues in 10% neutral buffered formalin (NBF) may decrease immunorecognition in some antigen-antibody pairs. Short fixation in 10% NBF followed by transfer to 70% ethanol has been used to overcome these effects, but the effects of this transfer on immunorecognition have not been explored adequately. We used two cell lines, DU145 (prostate cancer) and SKOV3 (ovarian cancer), grew them on coverslips and fixed them with 10% NBF at room temperature for 5 min and 12, 15, 18, 36, 108 and 180 h. Aliquots of the same cells were fixed in 10% NBF for 12 h, then transferred to 70% ethanol for 3, 6, 24, 96 and 168 h. Immunostaining with PCNA, Ki67-MIB-1, cytokeratins AE1/AE3 and EGFr was done concomitantly. In both cell lines, immunorecognition decreased between 18 and 36 h of fixation in 10% NBF for PCNA, Ki67-MIB-1 and cytokeratins AE1/AE3. By 108 to 180 h of 10% NBF exposure, there was complete loss of immunorecognition of PCNA and extensive loss of Ki67-MIB-1 and cytokeratins AE1/AE3. The effects on EGFr immunorecognition were less. Transfer to 70% ethanol after fixation for 12 h in 10% NBF preserved immunorecognition of the antibodies.

A standard tissue as a control for histochemical and immunohistochemical staining.

The variable quality of histochemical and immunohistochemical staining of tissues may be attributed to pre-analytical and analytical variables. Both categories of variables frequently are undefined or inadequately controlled during specimen collection and preparation. Pre-analytical variables may alter the molecular composition of tissues, which results in variable staining; such variations may cause problems when different tissues are used as staining controls. We developed a standard tissue for use as a staining control. Our standard tissue contains five components: 1) nine combined human cell lines mixed with stroma from human spleen; 2) a squamous cancer cell line, A431; 3) fungus; 4) transverse sections of the mosquitofish and 5) normal human spleen. The first three components were embedded in HistoGel™ and all components were processed to paraffin and used to construct a single standard paraffin block. The muscles of mosquitofish and arteries of the spleen are positive controls for eosin staining, while other tissues are useful for assessing hematoxylin staining. The mosquitofish tissues also are excellent controls for the Masson trichrome stain and all mucin-related histochemical stains that we tested. The goblet cells of the intestine and skin stained strongly with Alcian blue, pH 2.5 (AB-2.5), mucicarmine, colloidal iron, periodic acid Schiff (PAS) or PAS-hematoxylin (PASH) and combination stains such as colloidal iron-PASH. Cell lines were not useful for evaluating histochemical stains except for PASH. The splenic stroma was a useful control for AB-2.5; however, eosin and mucin stains stained cell lines poorly, probably due to their rapid growth and associated loss of some differentiated characteristics such as production of mucins. Nevertheless, the cell lines were a critical control for immunohistochemical stains. Immunostaining of specific cell lines was consistent with the presence of markers, e.g., EGFr in DU145 cells. The cell lines expressed a wide range of markers, so they were useful controls for immunohistochemical staining including EGFr, HER2, E-cadherin, cytokeratins, Ki67, PCNA, estrogen receptor, progesterone receptor, CD3, CD20 and CD45, activated (cleaved) caspase 3 and Bcl-2. The cell lines also were a control for the TUNEL stain.

Optoacoustic imaging identifies ovarian cancer using a microenvironment targeted theranostic wormhole mesoporous silica nanoparticle

At the intersection of the newly emerging fields of optoacoustic imaging and theranostic nanomedicine, promising clinical progress can be made in dismal prognosis of ovarian cancer. An acidic pH targeted wormhole mesoporous silica nanoparticle (V7-RUBY) was developed to serve as a novel tumor specific theranostic nanoparticle detectable using multispectral optoacoustic tomographic (MSOT) imaging. We report the synthesis of a small, < 40 nm, biocompatible asymmetric wormhole pore mesoporous silica core particle that has both large loading capacity and favorable release kinetics combined with tumor-specific targeting and gatekeeping. V7-RUBY exploits the acidic tumor microenvironment for tumor-specific targeting and tumor-specific release. In vitro, treatment with V7-RUBY containing either paclitaxel or carboplatin resulted in increased cell death at pH 6.6 in comparison to drug alone (p < 0.0001). In orthotopic ovarian xenograft mouse models, V7-RUBY containing IR780 was specifically detected within the tumor 7X and 4X higher than the liver and >10X higher than in the kidney using both multispectral optoacoustic tomography (MSOT) imaging with secondary confirmation using near infrared fluorescence imaging (p < 0.0004). The V7-RUBY system carrying a cargo of either contrast agent or an anti-neoplastic drug has the potential to become a theranostic nanoparticle which can improve both diagnosis and treatment of ovarian cancer.

Factors Affecting the Use of Human Tissues in Biomedical Research: Implications in the Design and Operation of a Biorepository

The availability of high-quality human tissues is necessary to advance medical research. Although there are inherent and induced limitations on the use of human tissues in research, biorepositories play critical roles in minimizing the effects of such limitations. Specifically, the optimal utilization of tissues in research requires tissues to be diagnosed accurately, and the actual specimens provided to investigators must be carefully described (i.e., there must be quality control of each aliquot of the tissue provided for research, including a description of any damage to tissues). Tissues also should be collected, processed, stored, and distributed (i.e., handled) uniformly under a rigorous quality management system (QMS). Frequently, tissues are distributed to investigators by tissue banks which have collected, processed, and stored them by standard operating procedures (SOPs). Alternatively, tissues for research may be handled via SOPs that are modified to the specific requirements of investigators (i.e., using a prospective biorepository model). The primary goal of any type of biorepository should be to ensure its specimens are of high quality and are utilized appropriately in research; however, approaches may vary based on the tissues available and requested. For example, extraction of specific molecules (e.g., microRNA) to study molecular characteristics of a tissue may require less clinical annotation than tissues that are utilized to identify how the molecular expression might be used to clarify a clinical outcome of a disease or the response to a specific therapy. This review focuses on the limitations of the use of tissues in research and how the design and operations of a tissue biorepository can minimize some of these limitations.

MP29-15 MAKE IT FROM SCRATCH OR HAVE IT DELIVERED? SATISFYING THE FATTY ACID DEMAND OF PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the transforming growth factor-b family. We previously reported that high-fat diet (HFD) induced prostate cancer progression through the upregulation of MIC1 in cancer cells. Here, we investigated the role of MIC-1 in controlling tumor microenvironment in HFD-induced prostate cancer progression. METHODS: PC-3M-luc-C6 cells were intraperitoneally injected into BALB/c-nu/nu mice, and equal numbers of mice were categorized into HFD and controlled diet (CD) groups. The extent of tumor burden was detected using the IVIS imaging system, and expression of MIC, proportion of stromal cells in tumors, serum level of MIC-1, and cytokine profile were examined. In vitro, functional analyses in PC-3 cells cultured with prostate stromal cell (PrSC) condition medium, recombinant MIC-1, and cytokine-blocking antibodies were performed. In humans, the relationship between serum level of MIC-1, MIC-1 expression in surgically resected prostate tissues, and clinicopathological parameters were assessed. RESULTS: The mean luciferase activity in mice at 4 weeks was significantly higher in the HFD group than in the CD group (p < 0.01). In the HFD group, MIC-1 expression and alpha smooth muscle actin (aSMA)-positive cells significantly increased in tumors, and serum levels of MIC-1 and IL-8 were significantly elevated. In vitro, recombinant MIC-1 (rMIC-1) induced PrSCs to secrete proinflammatory cytokines, including IL-8 and IL-6, and activate ERK in addition to the directly stimulate cancer cell proliferation and invasion. PrSCs and rMIC-1 increased the invasive capacity of PC-3 cells; however, the effects were impaired by IL-6 and IL-8 capture antibody. Patients with high staining scores of aSMA had significantly higher stage tumors (1⁄4pT2a) than those with low staining scores of aSMA (p 1⁄4 0.031), and the serum level of MIC-1 tended to be higher in patients with high staining scores of aSMA (p 1⁄4 0.056). CONCLUSIONS: MIC-1 stimulates stromal cytokine production in HFD-induced prostate cancer progression. MIC-1 and tumor stromal microenvironment may have implications in the prevention and treatment of HFD-induced prostate cancer progression.

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 2209: Characterization of FABP5 antibodies in prostate

FABP5, a member of a family of small proteins (~15 kDa) that transport lipids, is emerging as an important biomarker because it is differentially expressed in prostate cancers (PrCa) compared to uninvolved prostate glands and lipids are important to the progression of prostate cancer. This study was to select an antibody (Ab) for use in immunostaining (IHC) FABP5 in formalin fixed paraffin embedded (FFPE) tissue and also an Ab for use in Western blotting (WB). A mouse monoclonal antibody (mAb) clone A9 to FABP5 from Santa Cruz (SC) Biotechnology, Inc. and a rabbit polyclonal antibody (pAb) to FABP5 from Abcam (AB) (cat. # ab37267) were tested. Six prostate cell lines (PC3, DU145, 22Rv1, MDA PCa 2b, LNCaP, and normal primary prostate epithelial cells (NH Pri Pro) were used to study protein and mRNA levels of FABP5. Protein expression of FABP5 was determined by WB of whole cell lysates as well as cytoplasmic and nuclear lysates. GAPDH, a “housekeeping” gene expressed at similar levels in most types of cells, was used as a loading control for WB. Cells from the six prostate cells lines also were embedded in HistoGel, processed to FFPE and immunostained. The prostate cells were transfected with FABP5 siRNA for WB with aliquots of the transfected prostate cells processed to FFPE. qrtPCR was performed to measure mRNA levels. The expression of whole cell lysates on WB probed with the AB pAb to FABP5 was NH Pri Pro > PC3 > DU145 > 22Rv1 >> MDA PCa 2b > LNCaP compared to NH-Pri Pro > PC3 > DU145 > 22Rv1 when probed with the SC mAb. WB of the cytoplasmic fractions from each cell line has a similar pattern. In HistoGel sections immunostained with AB pAb, the FABP5 cytoplasmic expression was PC3 > DU145 > 22Rv1 > MDA PCa 2b > NH Pri Pro >> LNCaP. These levels of expression among the cell lines followed a similar trend in evaluation of immunostaining of cell membranes, nuclear, and the perinuclear area. In HistoGel sections immunostained with SC mAb, the FABP5 expression was PC3 > DU145 with weak signals in 22Rv1 > MDA PCa 2b > NH Pri Pro >> LNCaP. A recombinant blocking peptide (RBP) to FABP5 strongly inhibited the immunostaining with the antibodies to FABP5 in FFPE sections from archival radical prostatectomy specimens. This RBP is the same sequence as the peptide used to produce the AB antibody. No RBP to FABP5 was available from SC. Incubation with the RBP strongly inhibited the extent of immunostaining with FABP5 SC mAb. Similarly, immunostaining of the 6 cell lines with the AB pAb following incubation with RBP strongly inhibited staining. In analysis of mRNA levels, the expression of FABP5 was PC3 > 22Rv1 > > DU145 > MDA PCa 2b >> LNCaP. We demonstrated by siRNA and the use of a RBP that both these antibodies are specific and sensitive for detecting FABP5 by immunostaining but the AB Ab is more useful for WB. These results are presented with the IHC of FABP5 in normal and uninvolved prostate glands in PrCa. Citation Format: Dennis A. Otali, Denise K. Oelschlager, James Kearns, Sandra M. Gaston, William E. Grizzle. Characterization of FABP5 antibodies in prostate [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 2209. doi:10.1158/1538-7445.AM2017-2209

Abstract 1140: Characterization of a novel PDE10 inhibitor in lung tumor cells and an orthotopic mouse model of lung cancer

BACKGROUND: Screening a focused library of indene derivatives for PDE10 inhibitory activity identified novel leads with potent and selective tumor cell growth inhibitory activity. ADT-030 emerged from lead optimization chemistry with excellent drug-like properties and oral bioavailability. Here we characterize the anti-tumor activity of ADT-030 in human lung tumor cells and an orthotopic mouse model of lung cancer. METHODS: Growth inhibitory activity of ADT-030 was measured in a panel of human lung tumor cell lines by ATP quantification following 72 h of treatment. The effect of ADT-030 on intracellular cGMP/cAMP was measured in whole cell lysates using a competitive ELISA assay. PDE inhibitory activity of ADT-030 was evaluated in lysates of human lung tumor cells and by recombinant PDE isozymes using the IMAP fluorescence polarization PDE assay. Activation of PKG signaling and suppression of β-catenin levels in response to ADT-030 treatment was evaluated by Western blot using whole cell lysates of human lung tumor cells. ADT-030 was orally administrated to C57BL/6 mice and free levels quantified in plasma and tissues by LC-MS. Anti-tumor activity of ADT-030 was evaluated in athymic nude-Foxn1nu mice after inoculating the left lung with 1x106 A549 lung tumor cells and treating once daily by oral administration at dosages ranging from 25 - 125 mg/kg. Tumor growth was monitored by in situ bioluminescence using IVIS as well as necropsy and pathological grading after 4 weeks of treatment. RESULTS: ADT-030 inhibited the growth of human lung tumor cell lines with IC50 values in the low micromolar range by inducing apoptosis, while appreciably higher concentrations were required to affect the growth of normal human airway epithelial cells. ADT-030 treatment of human lung tumor cells increased both intracellular cGMP and cAMP levels, activated PKG and suppressed β-catenin within the same concentration range as required for tumor cell growth inhibition. Pharmacokinetic studies in mice demonstrated a half-life suitable for once a day dosing. Tissue distribution studies revealed appreciably higher concentrations of ADT-030 in lungs relative to plasma and other tissues, with the highest accumulation measured in the parenchyma. ADT-030 was well tolerated in mice implanted with A549 tumor cells and displayed strong anti-tumor activity as evident by reduced luminescence, tumor grading, and double-blinded pathological evaluation. CONCLUSIONS: ADT-030 represents a prospective drug development candidate with favorable drug-like properties that concentrates in lung after oral administration exhibiting a strong anti-tumor activity in a pre-clinical mouse model. The mechanism of lung tumor cell growth inhibition involves PDE10 inhibition, elevation of cGMP, activation of PKG, and attenuation of β-catenin. Citation Format: Veronica Ramirez-Alcantara, Bing Zhu, Xi Chen, Rajkumar Savai, Prema Subbarayal, Michele A. Schuler, Kevin J. Lee, Ashley S. Lindsey, Kristy L. Berry, Dennis Otali, Joshua Canzoneri, Jacob Valiyaveettil, Adam Keeton, Lori Coward, Gregory Gorman, William Grizzle, Michael Boyd, Gary A. Piazza. Characterization of a novel PDE10 inhibitor in lung tumor cells and an orthotopic mouse model of lung cancer [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 1140. doi:10.1158/1538-7445.AM2017-1140

Abstract C40: A biopsy-focused approach to molecular studies of prostate cancer in African Americans: the Birmingham Alabama Prostate Cancer Consortium (BAPrCa)

Cancer health disparities can arise from research studies in which the study subjects are not representative of the populations affected by the disease. In prostate cancer (PrCa) biorepositories, most of the frozen samples collected for molecular analyses are obtained from radical prostatectomy (RP) specimens. However, tissue collections based on RP specimens do not include patients who are diagnosed with relatively advanced disease and treated with first-line hormonal and/or radiation therapy rather than surgery; African Americans (AA) are more likely than European Americans (EA) to be diagnosed with this type of advanced prostate cancer. With support from the DOD Prostate Cancer Research Program, our research team has established the Birmingham Alabama Prostate Cancer Consortium (BAPrCa) with a major focus on studies that use prostate biopsies for the molecular analysis of PrCa in AAs. To ensure that there is no compromise of biopsy diagnosis, we use innovative tissue print technologies to obtain snap frozen nitrocellulose blots from each of the prostate biopsy cores. These biopsy tissue prints provide high quality RNA and DNA for molecular and genetic studies. Our biopsy-based analysis of high risk cancers has revealed PrCa subtypes that were either unrecognized or significantly underestimated in previous prostatectomy-based studies. As of June 2016 more than 250 prostate biopsy patients have been prospectively enrolled in the BAPrCa prostate biopsy tissue print study; just under half (48%) of these study subjects self-identify as AA. Each biopsy from each study subject was tissue printed to generate more than 3000 unique pairs of high quality RNA and DNA samples. AA men were enrolled from two study sites in Birmingham Alabama, an academic medical center (University of Alabama at Birmingham, UAB) and a large urology private practice (Urology Centers of Alabama, UCA). The prevalence of prostate cancer diagnosed in our AA study subjects undergoing standard biopsies was 51%, with 30% showing cancer of Gleason 7 or more. A higher proportion of AA subjects were diagnosed with PrCa at UAB (61% cancer positive; 45% with Gleason 7 or more) compared to UCA (44% cancer positive; 20% with Gleason 7 or more). The difference between AA cancer diagnoses observed between our two study sites may in part result from differences in the criteria for biopsy; pre-biopsy PSA was less than 4 ng/ml for 25% of UCA subjects but for only 2% of UAB subjects. PSA criteria for prostate biopsy may have a significant and unrecognized impact on the detection of prostate cancer in contemporary AA populations. The BAPrCa study cohort provides a unique opportunity to evaluate the PrCa subtypes diagnosed in AAs using different PSA criteria for biopsy, especially in a deep south AA population. Citation Format: Sandra M. Gaston, Dennis Otali, James Kearns, Kerry Dehimer, George W. Adams, Soroush Rais-Bahrami, Jeffrey Nix, James E. Bryant, Peter Kolettis, William E. Grizzle. A biopsy-focused approach to molecular studies of prostate cancer in African Americans: the Birmingham Alabama Prostate Cancer Consortium (BAPrCa). [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr C40.