Purpose Higher education institutions (HEI) play a critical role in developing student leaders equipped with the skills and knowledge needed to mobilize societal changes that the United Nations Sustainable Development Goals (SDGs) call for. To broaden this understanding, this study aimed to engage with student leaders of a grassroots, student-led initiative at the University of Calgary, the Sustainable Development Goals Alliance (SDGA), to better understand the experience of students who took on leadership roles in organizing SDG engagement activities. Design/methodology/approach A qualitative thematic analysis was used to understand the experiences of 12 student leaders involved in SDG programming. Semi-structured interviews asked participants to reflect on their key learnings, skills development and overall student’s experiences of leaders involved in SDG programming. Thematic analysis was applied to determine emerging themes. Findings Analyses showed that taking a leadership role in the SDGA empowered students to deepen their engagement with the SDGs and overcome barriers such as lack of knowledge and feelings of powerlessness. Secondary findings showed that community-building, flexibility and a sense of ownership were key strengths of the program and contributed toward student leaders’ feelings of hopefulness, self-confidence and inspiration. Originality/value This work offers a window into the experiences of student leaders who have worked to advance SDG engagement within their institution. Our findings suggest that student-led initiatives represent untapped potential for HEIs to prioritize and support to help deliver on their SDG implementation and engagement efforts. As HEIs offer a vital space for innovation, policy and capacity building towards implementation of the SDGs, this work demonstrates how student leadership can yield grassroots influence on HEI commitments and responses to the needs of students.

Microglia and macrophages are the largest component of the inflammatory infiltrate in glioblastoma (GBM). However, whether there are differences in their representation and activity in the prognostically-favorable isocitrate dehydrogenase (IDH)-mutated compared to -wild type GBMs is unknown. Studies on human specimens of untreated IDH-mutant GBMs are rare given they comprise 10% of all GBMs and often present at lower grades, receiving treatments prior to dedifferentiation that can drastically alter microglia and macrophage phenotypes. We were able to obtain large samples of four previously untreated IDH-mutant GBM. Using flow cytometry, immunofluorescence techniques with automated segmentation protocols that quantify at the individual-cell level, and comparison between single-cell RNA-sequencing (scRNA-seq) databases of human GBM, we discerned dissimilarities between GBM-associated microglia and macrophages (GAMMs) in IDH-mutant and -wild type GBMs. We found there are significantly fewer GAMM in IDH-mutant GBMs, but they are more pro-inflammatory, suggesting this contributes to the better prognosis of these tumors. Our pro-inflammatory score which combines the expression of inflammatory markers (CD68/HLA-A, -B, -C/TNF/CD163/IL10/TGFB2), Iba1 intensity, and GAMM surface area also indicates that more pro-inflammatory GAMMs are associated with longer overall survival independent of IDH status. Interrogation of scRNA-seq databases demonstrates microglia in IDH-mutants are mainly pro-inflammatory, while anti-inflammatory macrophages that upregulate genes such as FCER1G and TYROBP predominate in IDH-wild type GBM. Taken together, these observations are the first head-to-head comparison of GAMMs in treatment-naïve IDH-mutant versus -wild type GBMs. Our findings highlight biological disparities in the innate immune microenvironment related to IDH prognosis that can be exploited for therapeutic purposes.

Most immune cells in the glioblastoma (GBM) microenvironment are microglia and macrophages (MMs), but they are poorly understood. We sought to characterize these innate immune cells in human untreated IDH-WT and rare IDH-MUT GBM tissue to elucidate differences underlying their disparate prognoses relevant to immunotherapy design. An in-house automated segmentation protocol that quantifies at the single-cell level was used to analyze newly diagnosed human GBM (9 IDH-WT, 4 IDH-MUT). Three large sections (3-8mm in diameter) were quantitated to capture potential spatial heterogeneity. Expression of CD68, HLA-A/B/C, TNFa, CD163, IL10, TGFB2, Iba1 intensity, and surface area were enumerated and combined into an activation profile in Iba1+ cells (MMs). Results were validated with flow cytometry. Human IDH-MUT (GSE89567) and –WT (GSE84465) single-cell RNA-seq databases were then compared using novel bioinformatics techniques to affirm results. MM content is drastically reduced in IDH-MUT compared to –WT GBMs (4.9 ± 1.4% vs. 37.2 ± 7.3% of all GBM cells, respectively; p=0.0154). Surprisingly, a large range of MM content was found in IDH-WT GBMs, from 1.6 ± 0.6% to 71.9 ± 13.4%. Positive correlation with flow cytometry corroborated these results (Pearson r=0.7296; p=0.026). Inflammatory phenotypic variability was again seen in MMs in both IDH-MUT and –WT GBMs, but IDH-MUT GBM-associated MMs were more activated/pro-inflammatory (124.5 ± 21.6 pro-inflammatory units vs. 54.0 ± 15.6 pro-inflammatory units; p=0.0265). Comparison of single-cell RNA-seq databases after normalization and dynamic pruning of hierarchical clustering dendrograms verified MMs in IDH-MUT GBMs were more pro-inflammatory, but that this was driven by anti-inflammatory macrophages in IDH-WT GBMs as opposed to microglia which were pro-inflammatory in all tumors (p < 0.01). This is one of the first studies to characterize MMs in untreated human IDH-MUT GBMs and identify dissimilarities to the IDH-WT innate immune microenvironment that can be targeted by immunotherapies. Also, considerable MM phenotypic heterogeneity suggests precision immunotherapy approaches are crucial.

CD47 is a cell surface glycoprotein that interacts with signal regulatory protein alpha (SIRPα) on macrophages and dendritic cells triggering a “don9t eat me” signal that inhibits phagocytosis. Many tumors evade immune surveillance by overexpressing CD47, thereby preventing their recognition by phagocytes. Blocking the interaction of SIRPα/CD47 promotes phagocytosis and tumor cell destruction leading to a reduction in tumor burden. We have developed a humanized anti-CD47 antibody, AO-176, that blocks the interaction between CD47 and SIRPα and exhibits several additional novel functional characteristics. These characteristics include the induction of cell death in multiple human tumor cell lines in a cell autonomous manner (not ADCC), assessed by an increase in phosphatidylserine/7AAD positive staining. A second novel characteristic is enhanced binding to tumor cells at acidic pH. AO-176 binds to human tumor cell lines in the high pM to low nM range at physiologic pH, however, binding is enhanced up to 20-fold at an acidic pH of 6.5. The acidic pH of the tumor microenvironment which ranges from 6.4-7.2 is characteristic of solid tumors and correlates with tumor progression and metastasis. As a result of this enhanced binding at acidic pH, AO-176 has the potential added advantage of tumor-specific targeting. A third novel characteristic exhibited by AO-176 is its selective binding to tumor cells while exhibiting reduced binding to normal cells including red blood cells (cynomolgus monkey and human), endothelial, epithelial and skeletal muscle cells. In addition to these novel characteristics, AO-176 also exhibits dose-dependent efficacy in multiple mouse tumor models. Taken together, the unique combination of functional characteristics of AO-176, including induction of cell-autonomous killing, enhanced binding to tumor cells at acidic pH, significantly reduced binding to normal cells and potent in vivo efficacy provides the preclinical rationale for further development. Citation Format: Robyn Puro, Katherine Liu, Benjamin Capoccia, Michael Donio, Ronald Hiebsch, Myriam Bouchlaka, Alun Carter, Pamela Manning, Kathleen Crowley, Robert Karr. A humanized anti-CD47 monoclonal antibody that directly kills human tumor cells and has additional unique functional characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1765.

Background: CNS innate immune cells, microglia and macrophages (MMs), are the largest component of the inflammatory infiltrate in glioblastoma (GBM). They initially participate in tumor surveillance, but are co-opted by GBM to further angiogenesis and invasion. There are no effective immunotherapies against GBM in part because GBM-associated MMs are not well understood. We hypothesized that the extent and inflammatory phenotype of MM infiltration into GBM is variable between patients. This variability could have important implications on immunotherapy selection and treatment outcomes. Methods: Using automated quantitation of fluorescently labeled human GBMs, flow cytometry/live cell sorting, collection of conditioned GBM-associated MM media, and corroboration with TCGA and previously published scRNA-seq data, we have uncovered there is surprisingly marked variation in the amount of MM infiltration between tumors. Results: MM infiltration can range from almost non-existent, to comprising ~70% of GBM cells. By detecting cell surface markers and secreted cytokines, we determined that a mixture of pro- and anti-inflammatory MMs are found in each tumor. The overall inflammatory phenotype did not depend on the amount of infiltration. Interestingly, IDH-mutant GBM-associated MMs are more pro-inflammatory and less heterogeneous than IDH-wildtype GBMs. Conclusions: Taken together, the highly variable immunologic status of GBMs suggests the success of immunotherapies hinges on selecting appropriately vulnerable tumors.

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections.Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments.In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software.Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software.In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible.Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run.This technique will save researchers time and effort and create an automated protocol for slide analysis.

Background: CNS innate immune cells, microglia and macrophages (MMs), are the largest component of the inflammatory infiltrate in glioblastoma (GBM). They initially participate in tumor surveillance, but are subverted by GBM. Immunotherapies have proven incredibly successful in cancers such as melanoma, but not against GBM in part because GBM-associated MMs are not well understood. We hypothesized the content and inflammatory phenotype of MMs in GBM is variable between patients. We suspect MMs in IDH-wildtype and –mutant GBMs display divergent inflammatory phenotypes that helps explain the latter's better prognosis. Understanding GBM-associated MM heterogeneity will allow for better immunotherapy development and selection. Methods: MMs were isolated from untreated human IDH-wildtype and -mutant GBMs using flow cytometry and cultured for collection of conditioned media and analysis of secretory products. Automated segmentation with a high-content analysis system was used to quantitate MM content and inflammatory phenotype in frozen sections. New bioinformatics techniques allowed the comparison of MM profiles in publicly available single-cell RNA-sequencing databases with IDH-wildtype and -mutant GBMs. Results: Surprisingly marked variation in MM content exists between GBMs ranging from ~0-70%. A mixture of pro- and anti-inflammatory MMs are found in each GBM. Interestingly, IDH-mutant GBM-associated MMs were more activated than MMs in IDH-wildtype GBMs. Conclusions: Taken together, the highly variable MM content and phenotype of GBMs suggests the success of immunotherapies hinges on taking a precision medicine approach. MM-rich GBMs would benefit more from therapies that target them. MM activation in IDH-mutant GBMs may contribute to better patient prognoses.

Innate immune cells in the CNS, microglia and macrophages (MMs), are the largest component of the inflammatory infiltrate in glioblastoma (GBM). They initially participate in tumor surveillance, but are co-opted by GBM to adopt anti-inflammatory, immunosuppressive phenotypes and aid neoplastic progression. The bulk of immunotherapy research in GBM has been directed at T cells, which are part of the adaptive immune system, but make up a much smaller part of the inflammatory infiltrate. An effective immunotherapy against GBM has still not been found, in part because of a lack in understanding of GBM-associated MMs and the way they affect the immune microenvironment in which T cell therapies are expected to work. Our studies on human GBM tissue have uncovered there is surprisingly marked variation in the amount of MM infiltration between tumors, and this has bearing on clincopathologic parameters. Using automated quantitation methods, immunohistofluorescence, and validation with flow cytometry, we found that MM infiltration can range from almost non-existent, to comprising approximately 70% of GBM cells. With canonical markers and conditioned media, we determined that a mixture of pro-inflammatory and anti-inflammatory MMs were found in each tumor. Despite having a similar level of infiltration, GBM-associated MMs could still have drastically different gross inflammatory profiles. Age at diagnosis, time to progression, overall survival, comorbidities, and tumor volume were not associated with extent of MM infiltration. However, volumetric MRI analysis revealed heavier MM infiltration correlated with decreased peritumoral edema, contrary to previous reports. Taken together, we have found the inflammatory nature of the immune infiltrate can be drastically different between GBMs, and can have clinically significant effects on parameters such as peritumoral edema. These findings also demonstrate the importance of tailoring immunotherapies to individual patients given the considerable variability in magnitude and immunosuppression of the innate immune cells in the GBM microenvironment.

Abstract Antibodies vs CD47 (CD47mAbs) that block the CD47-SIRPalpha interaction promote the phagocytosis of cancer cells and have efficacy in several tumor models. A select few CD47mAbs also directly kill cancer cells by lowering cellular cAMP levels leading to mitochondrial damage and cell death. These CD47mAbs are thus referred to as “dual-function” mAbs. Activation of protein kinase A prevents CD47mAb-mediated death suggesting that phosphorylation of one or more target proteins in the cancer cell can block the death mechanism. BNIP3, a member of the BH3-only family, is induced by hypoxia and oncogenes and is necessary for induction of cell death by dual-function CD47mAbs. BNIP3 can activate autophagy, a pro-survival function, and can also induce cell death by damaging mitochondria. The role of BNIP3 in cancer is controversial and context-dependent with some cancers over-expressing BNIP3 compared to low levels of expression in normal tissue, while other cancers cannot tolerate BNIP3 expression and silence the gene, often by methylation of the BNIP3 promoter. This suggests that cancers that tolerate BNIP3 expression employ an as yet unknown mechanism to protect themselves from its toxic effects. The C-terminal transmembrane (TM) domain of BNIP3 (residues 164-184) penetrates the outer mitochondrial membrane allowing the extreme C-terminal ten residue tail of BNIP3 (RRLTTSTSTF, residues 185-194) to extend across the intermembrane space to bind OPA1 on the inner mitochondrial membrane, a key step in the death mechanism. We used a phosphosite-specific antibody to detect phosphorylation of BNIP3 immunoprecipitated from Jurkat leukemia cells at residue T188 which resides in a canonical protein kinase A site (RRLT, amino acids 185-188). Using mass spectrometry of 6His-tagged BNIP3 isolated from HEK293 cells treated with 8BrcAMP, we identified as many as 4 additional phosphorylated sites in the C-terminal tail sequence (residues 189-194, TSTSTF). We generated phosphomimetic (S/T to D) and unphosphorylated (S/T to A or N) mutations at these residues and expressed the mutant BNIP3 proteins in 293 cells. All of the BNIP3 mutants associated with mitochondria but only the phosphomimetic mutants prevented BNIP3-induced mitochondrial damage and cell death. In contrast, mutation of the phosphorylated S/T residues to unphosphorylated residues resulted in rapid and extensive cell death. Importantly, phosphomimetic C-terminal BNIP3 residues blocked cell death without preventing autophagy, providing evidence that the two roles of BNIP3 can be regulated independently. We replicated these results in one lung cancer and three breast cancer cell lines. These findings suggest that phosphorylation at the C-terminus of BNIP3 is a switch that determines the pro-survival vs pro-death effects of BNIP3. Dual-function CD47mAbs may act by dephosphorylating BNIP3 expressed in tumor cells thus unleashing its killing potential. Citation Format: William Frazier, Katherine Liu, Julie Dimitry, Benjamin Capoccia, Pamela Manning, Robert Karr. Phosphorylation of BNIP3 is a switch between life and death in cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2. doi:10.1158/1538-7445.AM2015-2

The Evidence-based Practice Center (EPC) program within the Agency for Healthcare Research and Quality (AHRQ) provides detailed evidence reports for partner organizations that they can translate into activities that improve patient care. A review of these dissemination activities provides a rich opportunity to understand how to create more successful linkages between best evidence and best practice. On the basis of interviews with EPC directors, AHRQ staff, and representatives of public and private users of EPC reports, we summarize the variety of efforts to disseminate the work of the EPCs. We also identify a case example of a successful dissemination of an EPC report. Experience to date reinforces the importance of creating close ties between researchers and the policymakers, clinicians, and other decision makers who use EPC evidence reports; developing a conceptual framework to guide the process; and establishing the resource foundation for the entire effort.