Our preprint on cellular behavior analysis in TCR T cells & cancer cell live-cell imaging data is out! This 3-year collaboration led by pd Archit Verma w/ Alex Marson & Julia Carnevale, with segmentation & tracking by @davidvv & team!
biorxiv.org/content/10.1101/…
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Incucyte live cell imaging is ubiquitous, but from this complex data cancer immunologists typically plot one thing: the number of red pixels in the well, which is a proxy for the cancer cell coverage (RFP marks cancer cell nuclei). From [Carnevale et al. 2022]:
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A lot of interesting signal is left on the cutting room floor. For example, proliferation (rates) in cancer cells...
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We teamed up with Alex Marson and Julia Carnevale’s Labs, who imaged TCR T cells co-cultured with RFP+ A375 tumor cells 3 ways: Safe harbor knockout (SH KO; control), RASA2 KO (Carnevale et al., 2022), CUL5 KO (Liao et al., 2024).
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Each well was imaged every 4 minutes at 10X magnification on Sartorius Incucyte for 72 hrs. Images include brightfield, RFP (cancer cell nuclei) channels.
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Then, the Van Valen Lab developed Caliban to segment and track each cell (green are T cells, red are cancer cells, white are detritus).
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With the masked, tracked cells, we went to work to develop Occident. We were curious how well the RFP markers captured cancer cell number; we found that RFP lags as a proxy for cancer cell numbers.
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We found that T cell proliferation increased in the two beneficial KO T cells, in the CUL5 KO T cells in particular.
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We can study differences in cancer cell division events (lower in beneficial KO T cells) and average T cell speed (faster in beneficial KO T cells).
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We found that the number of T cells attached to cancer cells reduces the likelihood that the cancer cell will proliferate, with the beneficial KO T cells having greater effects on proliferation reduction.
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Cancer cell and T cell morphology changes dramatically depending on state. These changes are visible in the brightfield imaging – active interacting T cells are larger and change to less circular shapes. Cancer cell begin to aggregate together when interacting with T cells.
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While the # of T cell--cancer cell interactions increased similarly, their effects were modulated by the CRISPR KOs. E.g., the time a T cell remained attached to a cancer cell (as estimated by a negative binomial and Markov model separately) was highest in RASA2 KO T cells.
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Even more exciting, the speed of cancer cells decreased after interactions with T cells, as did their overall size (indicating stress).
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With a Markov model, we deconvolved when, in frame t-1, there is 1 cancer cell and 1 T cell in a window, and in frame t there is 1 cancer cell and 2 T cells. We quantified how often this doubling of T cells attacking a cancer cell was due to proliferation or due to recruitment.
Nov 26, 2024 · 8:12 PM UTC
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In sum, compared to the SH KO control condition, TCR T cells with the RASA2 KO have a longer dwell time and cripple cancer cells more effectively this way, whereas TCR T cells with the CUL5 KO proliferated more frequently upon activation, adding more T cells to the fight.
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With five new collaborations in the works, and a paper characterizing the differences using explainable AI already accepted as an oral presentation at
#PSB2025 (lead by high school senior Marcus Blennemann), look for future work in this space! biorxiv.org/content/10.1101/…
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Please play with these data! There is a lot more signal there. Thank you to @bioimagearchive.bsky.social
for hosting these Incucyte image data -- this is a new thing for them, and they have been so kind in working through the details of submission (link coming soon!)! 🎉
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Feedback welcome! And join us over in the Good Place: @thebeehive.bsky.social
Try out Caliban and Occident on your own Incucyte data! More phenotypes and analyses added regularly.
github.com/vanvalenlab/Calib…
github.com/bee-hive/occident
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