Thread by @SizunJ

Looking to do multiplexed tissue imaging on your archival clinical samples? We describe a ready blueprint to study tumor immunology and enable insights into immunotherapy Together with Darci Philips, @ChrisSchuerch, Mike Khodadoust, Youn Kim, @GarryPNolan frontiersin.org/articles/10.33…

First off, thank you @FrontImmunol, our Editor Pedro Berraondo and Reviewers for a first-class experience in getting this manuscript out the door! The reviews were reasonable, well-meaning and timely, and they certainly helped strengthen our manuscript.

Using CODEX @AkoyaBio, we wanted to enable >50 markers to be robustly enabled on FFPE tissue sections. To this end, we assembled, validated and combined 56 antibodies into a final panel applied to selected cutaneous T cell lymphoma (CTCL) patient samples. All clones available:

Mega thanks to @ChrisSchuerch for his amazing patience and work in pathological confirmation of these markers. Aka if something is wrong, blame him 😉

Testing and validating antibodies for highly multiplexed platforms like @AkoyaBio and @IONpathOfficial is a very expensive process, since antibodies need to be stained concurrently (1 condition for >50 Abs!) and we buy them purified (even more $$$) for conjugation to oligos/metal

Even if an Ab worked superbly in standard IHC, it doesn't mean they survive the conjugation process (which is done under mildly reducing conditions). ~ 20% of Abs don't survive this

We then have to make sure these Abs play nice with each other. >50 Abs in a pot!

A GREAT thing for new-gen pathologists and immunologists: When you have >3 markers, you can use them to validate each other. Eg CD3+ CD4+ CD8- cells are CD4 T cells!

Eyeballing is great, but to really scale these up we need computational methods to stratify and annotate these cells. And then go back and confirm they are correctly assigned!

Finally, we wanted to have a strong basis to start clinical studies into immuno-modulatory markers, such as Lag3 (recent good stuff from @bmsnews), OX40, PD1/PD-L1, TIM3 and VISTA. Here we show the expression pattern on both immune (T/Macrophage) and tumor cells

We hope this work will save considerable time, effort, and resources for researchers interested in studying the TME and immunotherapy responsiveness with multiplexed tissue imaging approaches. Thanks for coming to my TED talk 😉