https://openworks.mdanderson.org/sumexp22/1015/thumbnail.jp

Chien, Derek L

Hu, Jian

Yasar, Fatma Betul Aksoy

Zaman, Mohammad F

Zamler, Daniel B.

Zeng, Ailiang

English

OpenWorks @ MD Anderson

Figure 2. A. Representative 10x IF staining of implanted QPP with Iba1 (green). B. Classification of Iba1+ myeloid cells based on morphology. C. Proportion of myeloid cell morphotypes by brain region.Results• Microglia change morphology based on location. Morphotypes have obvious but unexplained functional differences.Introduction• Glioblastoma, the most common type of malignant brain tumor, has evaded conventional adaptive immunotherapeutic efforts.1• Little is understood about the myeloid composition in the glioma microenvironment. Modulating glioma-associated macrophage (GAM) activity presents an alternative immunotherapeutic strategy.• QkL/L;PtenL/L;Trp53L/L (QPP) mice develop glioma with immune environments resembling that of human glioma.2-3 They are thus ideal in determining myeloid composition across a tumorigenic brain.• We sought to probe the following:1. Myeloid cell morphology in non-tumor and tumor regions2. Distinction between resident microglia and circulation-derived macrophages (CDMs)3. Presence of pro- or anti-phagocytic markersMethodsMouse models:• QPP7 (genetic tumors), injected with tamoxifen at P7, and harvested when moribund• Cx3cr1-CreERT2 adult mice injected with QPP7 tumor cells (implanted tumors), harvested when moribundSlide preparation: Following euthanasia, brains were removed, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm via microtome.Immunofluorescence (IF) staining or co-staining: Sections were stained with 1:250 primary antibody dilution and 1:1000 secondary antibody dilution (488 nm or 594 nm). The following antibodies were used: Iba1, TMEM119, CD45, CD47, Arg1, and GFP. Images were taken via widefield microscopy.References1. Garg A. D. et. Al. Oncoimmunology. 2017; 6(4); e1295903.2. Shingu T. et. al. Nat. Genet. 2017; 49(1); 75-86.3. Zamler D. B. et. al. JCI Insight. 2022; 7(12); e148990.4. Kenkhuis B. et. al. Neurobiol Dis. 2022; 167; 105684.Title of the Poster Presentation Goes HereAuthors of the Poster Presentation Goes HereInstitutional and/or Graduate School of Biomedical Sciences Affiliation Goes Here Probing Spatial Myeloid Heterogeneity in GlioblastomaDerek L. Chien1, Mohammad F. Zaman2,3, Fatma B. A. Yasar2, Daniel B. Zamler2, Ailiang Zeng2, Jian Hu2School of Arts and Sciences, University of Rochester, Rochester, NY, USA.1Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.2Cancer Biology, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA.3Conclusions• Microglia play some significant role in defending against or promoting gliomagenesis, with gene signatures likely differing based on brain location.• There are phagocytic suppression and high CDM trafficking into the proliferating tumor.• Future experiments to probe myeloid heterogeneity in glioma might include multiplex immunofluorescence, confocal microscopy, FACS, scRNAseq, secretomics, lineage tracing, in vivotracking, and time course studies.AcknowledgmentsThe authors would like to acknowledge the American Brain Tumor Association (ABTA) for funding this project, as well as Marites Melancon, Chandra Bartholomeusz, and Nancy Strange for hosting and managing the MD Anderson Summer Undergraduate Research Program.• TMEM119 is downregulated in glioma conditions, supporting its identity as a marker of homeostatic conditions.4• Iba1+ CD45high cells are increasingly found towards tumor core, suggesting higher likely CDM infiltration.• Tumor cells are largely CD47+, indicating phagocytic suppression.• Arg1+ expression increases towards tumor core, suggesting increasing myeloid polarization to “M2” subtype.• GFP signal was found only in some Iba1+ cells, suggesting high phagocytic heterogeneity. It remains unclear whether there is downregulation of uptake and/or endolysosomal activity, and at what point of development.Figure 1. A. Design of Nes-CreERT2;QkL/L;PtenL/L;Trp53L/L (QPP) mouse model. B. Representative image of QPP murine brain after harvesting.2A BADB CA B CA BCD EFigure 3. A. Representative 10x IF co-staining of implanted QPP with Iba1 (green) and TMEM119 (red), showing little co-localization. B. Positive control of 40x IF co-staining using genetic QPP brain harvested at 7 weeks with Iba1 (green) and TMEM119 (red). C. Representative 20x multiplex IF staining using Vectra Polaris slide scanner (more powerful imaging) of implanted QPP with 1:2000 CD11b (red) + TMEM119 solution (yellow), showing co-localization. D. Representative 10x (top) and 20x (bottom) IF co-staining of implanted QPP with Iba1 (green) and CD45 (red). E. Proportion of CD45high cells by brain region. Figure 4. A. Representative 10x/40x IF staining of implanted QPP with CD47 (red). B. Representative 10x/40x IF co-staining of implanted QPP with Iba1 (green) and Arg1 (red). C. Proportion of Arg1+ cells by brain region. D. Representative 10x and 40x IF co-stainings of genetic QPP with Iba1 (red) and GFP (green).Ramified Hyper-RamifiedRod Activated or CDM

Probing Spatial Myeloid Heterogeneity in Glioblastoma

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1015&context=sumexp22

Probing Spatial Myeloid Heterogeneity in Glioblastoma

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