A Productive Scholar's Guide to Respectful, Responsible Inquiry During the COVID-19 Pandemic: Moving Forward.

As a result of the COVID-19 pandemic of 2020, schools across the country have pivoted to providing a range of instructional opportunities including remote, hybrid, and modified in-person options with a commitment to keeping our society safe and supporting our nations' youth in continuing their education. One byproduct of this most challenging situation has been the interruption of scientific inquiry in many fields, including educational research. Using a framework of self-leadership, in this feature article, we provide reflections to guide discussion among scholars involved in school-based research during and after the COVID-19 pandemic. Based on lessons learned in our own mentee-mentor relationships and from our collective experiences, the purpose of this article is to offer considerations primarily for early-career scholars, but also to engage dialogue among mid- and late-career scholars to promote school-based inquiry in this defining moment in history, at a time when educators are concerned with mitigating learning loss. We focus on (a) revisiting and refining programmatic lines of inquiry, (b) crafting articles to disseminate lessons learned from research conducted to date-including those with lost time points, (c) retooling in a focused area of inquiry (e.g., methodology), (d) conceptualizing future studies featuring procedures that do-and do not-allow for interpersonal contact, and (e) appreciating and acknowledging individuals who have facilitated inquiry. We conclude with a note of encouragement and call to action for future educational research.

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging.

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Significantly higher expression levels of androgen receptor are associated with erythroblastosis virus E26 oncogene related gene positive prostate cancer.

Erythroblastosis virus E26 related gene (ERG) overexpression is correlated with the TMPRSS2-ERG fusion gene, a rearrangement known to be present in about 50% of cases of prostate cancer. Androgen receptor (AR) is a known regulator of the TMPRSS2 gene. Despite knowledge of this relationship, limited data is available on the specific relationship of AR expression to TMPRSS2-ERG fusion (ERG) status in prostate cancer (PCa). We used multiplexed immunohistochemistry, multispectral imaging technology and tissue microarray (TMA) to elucidate this relationship. Two prostate tissue microarrays were created from two cohorts of hormonal naïve patients' prostatectomy specimens: progression TMA (pTMA, from 95 PCa patients) and outcome TMA (oTMA, from 183 PCa patients with at least 5-year follow-up information). Each of the two TMAs were triple-stained with ERG, AR and E-cadherin antibodies and visualized with a different chromogen. We found marked difference in AR expression levels between ERG positive (ERG(+)) and ERG negative (ERG(-)) prostate cancer. The difference was significant in localized (pT2) prostate cancer. We also found that AR expression levels were significantly higher in PCa tissue compared to benign prostate tissue, with the highest expression levels in ERG(+) metastatic cancer. Neither AR nor ERG expression was associated with clinical outcome. Our findings confirm that TMPRSS2-ERG fusion is AR-dependent and is associated with increased AR expression. Our data suggest that the AR pathway may play an important role in the development of ERG(+) PCa and ERG status may be useful in stratifying PCa patients for hormonal therapy.

Sex steroid receptor expression and localization in benign prostatic hyperplasia varies with tissue compartment.

Androgens and estrogens, acting via their respective receptors, are important in benign prostatic hyperplasia (BPH). The goals of this study were to quantitatively characterize the tissue distribution and staining intensity of androgen receptor (AR) and estrogen receptor-alpha (ERα), and assess cells expressing both AR and ERα, in human BPH compared to normal prostate. A tissue microarray composed of normal prostate and BPH tissue was used and multiplexed immunohistochemistry was performed to detect AR and ERα. We used a multispectral imaging platform for automated scanning, tissue and cell segmentation and marker quantification. BPH specimens had an increased number of epithelial and stromal cells and increased percentage of epithelium. In both stroma and epithelium, the mean nuclear area was decreased in BPH relative to normal prostate. AR expression and staining intensity in epithelial and stromal cells was significantly increased in BPH compared to normal prostate. ERα expression was increased in BPH epithelium. However, stromal ERα expression and staining intensity was decreased in BPH compared to normal prostate. Double positive (AR and ERα) epithelial cells were more prevalent in BPH, and fewer double negative (AR and ERα) stromal and epithelial negative cells were observed in BPH. These data underscore the importance of tissue layer localization and expression of steroid hormone receptors in the prostate. Understanding the tissue-specific hormone action of androgens and estrogens will lead to a better understanding of mechanisms of pathogenesis in the prostate and may lead to better treatment for BPH.

A colorful future of quantitative pathology: validation of Vectra technology using chromogenic multiplexed immunohistochemistry and prostate tissue microarrays.

The Vectra platform (Caliper Life Sciences, Hopkinton, MA) is an advanced multispectral imaging system for biomarker quantitation in tissue microarray or intact tissue sections. This is the first study to validate its reliability for quantitating spatially overlapping biomarkers using chromogenic multiplexed immunohistochemistry on prostate tissue microarrays. Two tissue microarray cohorts (an outcome tissue microarray and a progression tissue microarray) were used. The outcome tissue microarray cohort consists of 462 duplicate cores with more than 5-year outcome information. The progression tissue microarray cohort consists of 384 duplicate cores from different disease (stage) groups. The tissue microarray slides were stained with different combinations of antibodies (anti-androgen receptor, anti-E-cadherin, anti-erythroblastosis virus E26 oncogene-related gene product, and anti-α-methylacyl-CoA racemase). Three outcome tissue microarrays were stained with androgen receptor + erythroblastosis virus E26 oncogene-related gene + E-cadherin (outcome tissue microarray 1), androgen receptor + E-cadherin (outcome tissue microarray 2), and erythroblastosis virus E26 oncogene-related gene + E-cadherin (outcome tissue microarray 3), respectively. One progression tissue microarray section was stained with E-cadherin and α-methylacyl-CoA racemase; tissue microarray slides were then scanned with the Vectra platform. Biomarker expression analysis was performed with Vectra software-Nuance 3.0.0, and inForm 1.2. IBM SPSS Statistics 19 was used for statistical and correlation analysis (SPSS, Chicago, IL). Close concordance was found between the triple- and double-immunostaining assays used for quantitating spatially overlapping biomarkers androgen receptor and erythroblastosis virus E26 oncogene-related gene using outcome tissue microarrays (r = 0.897 for androgen receptor and 0.613 for erythroblastosis virus E26 oncogene-related gene, respectively). α-Methylacyl-CoA racemase and E-cadherin expression levels measured in progression tissue microarray were consistent with previously published data by other groups. In conclusion, Vectra technology is reliable for objective and high-throughput biomarker quantitation and colocalization study using chromogenic multiplexed immunohistochemistry.

Oncocytoma can be differentiated from its renal cell carcinoma mimics by a panel of markers: an automated tissue microarray study.

BACKGROUND: Differentiating oncocytoma from its renal cell carcinoma (RCC) mimics, particularly chromophobe RCC, can be difficult, especially when limited tissue is available for evaluation. This study presents a panel of markers that are readily available, easy to use, and useful for differential diagnoses of renal tumors. DESIGN: A renal cell neoplasm tissue microarray was constructed including oncocytoma (n=30), chromophobe RCC (n=18), conventional RCC (n=64), papillary RCC (n=50), and benign renal tissues (n=31). CK7, CD10, epithelial membrane antigen, renal cell carcinoma marker (RCCma), vimentin, and endogenous avidin-binding activity (EABA) were studied. An Automated Cellular Imaging System, was used to quantify the staining intensity. RESULT: EABA was positive in 97% of oncocytoma, 26% of conventional RCC and 35% of papillary RCC with granular/eosinophilic (G/E) features and 6% of chromophobe RCC. EABA was negative in RCC without G/E features. Vimentin and RCCma were positive in most RCC with G/E features (conventional, 78% and 71%; and papillary, 85% and 76%, respectively), and negative in oncocytoma. Vimentin was also negative in chromophobe RCC. CK7 was positive in up to 81% of papillary RCC and 63% of chromophobe RCC, and essentially negative in conventional RCC and oncocytoma. CONCLUSIONS: EABA is an excellent marker for oncocytoma, which can be useful in differentiating oncocytoma from chromophobe RCC. A panel of EABA, vimentin, and RCCma markers can be useful in discerning oncocytoma from RCC with G/E features. Vimentin can be useful in discriminating chromophobe RCC from papillary or conventional RCCs.

Heparan sulfate proteoglycans as regulators of fibroblast growth factor-2 signaling in brain endothelial cells. Specific role for glypican-1 in glioma angiogenesis.

Fibroblast growth factor-2 (FGF2) is a potent angiogenic factor in gliomas. Heparan sulfate promotes ligand binding to receptor tyrosine kinase and regulates signaling. The goal of this study was to examine the contribution of heparan sulfate proteoglycans (HSPGs) to glioma angiogenesis. Here we show that all brain endothelial cell HSPGs carry heparan sulfate chains similarly capable of forming a ternary complex with FGF2 and fibroblast growth factor receptor-1c and of promoting a mitogenic signal. Immunohistochemical analysis revealed that glypican-1 was overexpressed in glioma vessel endothelial cells, whereas this cell-surface HSPG was consistently undetectable in normal brain vessels. To determine the effect of increased glypican-1 expression on FGF2 signaling, we transfected normal brain endothelial cells, which express low base-line levels of glypican-1, with this proteoglycan. Glypican-1 expression enhanced growth of brain endothelial cells and sensitized them to FGF2-induced mitogenesis despite the fact that glypican-1 remained a minor proteoglycan. In contrast, overexpression of syndecan-1 had no effect on growth or FGF2 sensitivity. We conclude that the glypican-1 core protein has a specific role in FGF2 signaling. Glypican-1 overexpression may contribute to angiogenesis and the radiation resistance characteristic of this malignancy.

Heparan sulfate proteoglycans as regulators of fibroblast growth factor-2 receptor binding in breast carcinomas.

Binding of fibroblast growth factors (FGFs) to their tyrosine kinase-signaling receptors (FGFRs) requires heparan sulfate (HS). HS proteoglycans (HSPGs) determine mitogenic responses of breast carcinoma cells to FGF-2 in vitro. For this study, we examined the role of HSPGs as modulators of FGF-2 binding to FGFR-1 in situ and in vitro. During stepwise reconstitution of the FGF-2/HSPG/FGFR-1 complex in situ, we identified an elevated ability of breast carcinoma cell HSPGs to promote receptor complex formation compared to normal breast epithelium. HSPGs isolated from the MCF-7 breast-carcinoma cell line were then fractionated according to their ability to assemble the FGF-2 receptor complex. All MCF-7 HSPGs are decorated with HS chains similarly capable of promoting FGF-2 receptor complex formation. In this in vitro model, syndecan-1 and syndecan-4 are the cell surface HSPGs contributing most to the complex formation. Relative expression levels of these syndecans in human breast carcinoma tissues correlate well with receptor complex formation in situ, indicating that in breast carcinomas, core protein levels determine FGF-2 receptor complex formation. However, variances in syndecan expression levels do not explain the difference in FGF-2 receptor complex formation between normal and malignant epithelial cells, suggesting that alterations in HS structure occur during malignant transformation.