https://openworks.mdanderson.org/sumexp21/1201/thumbnail.jp

Daniele, Joseph R.

Draetta, Giulio

Han, Jing

Hu, Jian

Machado, Annette

Marisetty, Sujay P.

Marszalek, Joe

Zamler, Daniel B.

Zorrilla-Veloz, Rocio Isabel

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OpenWorks @ MD Anderson

• TMAs mediate rapid validation of antibodies against immune markers, eliminating the need for single-tissue IHC staining and analysis.• mIHC paired with TMA utilization could further increase the efficiency of validating antibodies. Fig 6. Image of Anti-MBP stained WT eye tissue (specific). RET –retina. ON – optic nerve.Utilizing Tissue Microarrays for Medium-Throughput Validation of Antibodies Against Immune MarkersSujay Marisetty1, Joseph R. Daniele, PhD2, Annette Machado, PhD2, Jing Han, PhD2, Rocio Isabel Zorrilla-Veloz3, Daniel B. Zamler*1,3, Joe Marszalek*, PhD2, Jian Hu*, PhD3, and Giulio Draetta*, MD, PhD1,21Department of Genomic Medicine, The University of Texas MD Anderson Cancer, Houston, TX; 2The Translational Research to AdvanCe Therapeutics and Innovation in ONcology (TRACTION), Houston, TX;  3Department of Cancer Biology, The University of Texas MD Anderson Cancer, Houston, TX • Novel immune biomarkers are constantly discovered, sometimes replacing old markers for identification.• Antibodies – including those targeting immune markers – should be rigorously validated via methods such as immunohistochemistry (IHC) to ensure specificity. • Single-tissue IHC staining and analysis is resource and time-consuming.• Tissue Microarrays (TMAs) are an efficient platform to implement validation using multiple tissues, which allows technical replicates and on-slide controls.1,2Fig 6. Map of TMA design with 60-1mm cores. Tissues from WT mice and NSG mice are labeled blue and white, respectively. Spln – spleen. Liv –liver. Lng – lung. Panc – pancreas. Thy - thymus. LN – lmph node. Ret – retina. Brn – brain. Kid –kidney. Hrt – heart. Methods:Methods-cont.:Fig 7. Panel of Anti-CD20 stained tissues (moderately specific). WT Spleen scanned at 20x (Top left). NSG spleen scanned at 20x (Top right). WT kidney scanned at 20x (Bottom left). WT Brain scanned at 20x (Bottom right).Results:We classified anti-CD20 as moderately specific because while the NSG spleen showed positive signal, the NSG kidney showed no signal as expected despite the positive signal in the WT kidney. We classified anti-CSF1R as moderately specific because the WT spleen showed positive signal, as expected, but the NSG spleen showed considerable background staining. We classified anti-LAG3 as nonspecific because the WT spleen shows positive signal, but the NSG spleen showed more positive signal in comparison to the WT spleen. Similarly, we classified anti-NCR1 as nonspecific because the WT spleen shows positive signal, but the NSG spleen showed more positive signal in comparison to the WT spleen. Lastly, we classified anti-FOXP3 as nonspecific because both the WT and NSG spleen show similar staining intensity. Fig 7. Panel of Anti-LAG3 stained tissues (nonspecific). WT Spleen at 20x – specific (Top). NSG spleen at 20x - nonspecific (Bottom). Methods-cont.:23Fig 1. Sample Multiplex Stain of brain tumor border. Rigorous antibody validation has significant implications for many procedures, such as mIHC. Fig 2. (Step 1-3). (1) Image of C57BL/6J (wild-type) mouse. Organs were harvested from 3 wild-type (WT) and 3 NSG mice. (2) Paraffin-embedded WT mouse liver. Paraffin-embedded single-organ blocks were sectioned. (3) Sample QuPath annotation of WT mouse brain. H&E-stained 5μm slices are scanned and resulting scans are annotated with QuPath.Background:Conclusions:1• 1. Zhang X, Su D, Cheng Q. Advantages and applications of tissue microarray technology on cancer research. Chin J Cancer Res. 2003;15(1):74-78. doi:10.1007/s11670-003-0017-z• 2. Jawhar NM. Tissue Microarray: A rapidly evolving diagnostic and research tool. Ann Saudi Med. 2009;29(2):123-127. doi:10.4103/0256-4947.51806References:Galileo semiautomatic Tissue Microarrayer• The authors would like to thank Brittney Edwards, Kara Lewis, Khandan Keyomarsi, and Stephanie Watowichfor facilitating this research through the CPRIT-CURE program and the NIH R25 Training Grant. Acknowledgements:Caliper-Measuring OffsetFig 3. (Steps 4 -5). (4) Caliper used to measure donor block offset. Offsets of the donor blocks relative to the TMA recipient block were measured prior to tissue core extraction. (5) TMA generation using the Galileo semiautomatic tissue microarrayer. Cylindrical tissue cores were extracted from single-organ blocks (donor blocks) and inserted into the recipient TMA block.47Fig 6. (Step 7). Image of Vectra Polaris. Slides are scanned on H&E, Brightfield preset. 5Fig 5. (Step 6). Image of H&E stained TMA section.5 μm sections underwent IHC staining with respective antibodies.6Fig 7. (Step 8). Sample portion of GeneCards protein expression profile of CSF1R. IHC-stained sections are analyzed using existing literature, as well as protein and gene expression databases. Adapted from GeneCards. (https://www.genecards.org/cgi-bin/carddisp.pl?gene=CSF1R).8LivLngPancThyLNBrnKidHrt RetSplnDonor Block TMA Recipient BlockONRET

Utilizing Tissue Microarrays for Medium-Throughput Validation of Antibodies Against Immune Markers

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1201&context=sumexp21

Utilizing Tissue Microarrays for Medium-Throughput Validation of Antibodies Against Immune Markers

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