Focal adhesion pathway inhibition is the central axis of macrophage phenotypic responses to monoclonal antibody therapy in aggressive lymphoma via high-throughput screening and high-content imaging

Blakemore, S. J.; Zentis, P.; Mueller, L.; Brinker, R.; Michalik, M.; Juengst, C.; Beielstein, A.; Zhang, J.; Neuhaus, R.; Florin, A.; Hallek, M.; Meinel, J.; Buettner, R.; Schauss, A.; Pallasch, C.

doi:10.1101/2025.09.23.677924

bioRxiv · 10.1101/2025.09.23.677924

Focal adhesion pathway inhibition is the central axis of macrophage phenotypic responses to monoclonal antibody therapy in aggressive lymphoma via high-throughput screening and high-content imaging

Blakemore, S. J.·Zentis, P.·Mueller, L.·Brinker, R.·Michalik, M.·Juengst, C.·Beielstein, A.·Zhang, J.·Neuhaus, R.·Florin, A.·Hallek, M.·Meinel, J.·Buettner, R.·Schauss, A.·Pallasch, C.

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Abstract

High-grade B-cell lymphoma (HGBCL) frequently arises as a refractory or relapsed state of diffuse large B-cell lymphoma (DLBCL) and is associated with poor outcomes due to multi-drug resistance and hallmark oncogenic translocations. To identify novel therapeutic strategies, we developed a dual high-throughput screening (HTS) and high-content imaging (HCI) macrophage-tumour co-culture platform that quantifies antibody-dependent and antibody-independent cellular phagocytosis (ADCP/AICP) across a 1,241-compound library. Using GFP+ HGBCL cells and mCherry+ macrophages, we validated our methodology through time-resolved phenotypic profiling, Euclidean distance-based analysis, and hit compound prioritisation. Pathway interrogation revealed focal adhesion as a central hub of macrophage phenotypic modulation, highlighting focal adhesion kinase (FAK/PTK2) as a candidate therapeutic target. Pharmacological inhibition with PF-562271 enhanced phagocytic activity, altered macrophage morphology, and synergised with anti-CD20 monoclonal antibodies in vitro and ex vivo. In vivo, Rituximab plus PF-562271 significantly reduced lymphoma burden and prolonged survival in xenograft models. Collectively, our work demonstrates that HTS/HCI-driven phenotypic profiling of tumour-associated macrophages can uncover actionable therapeutic combinations and nominates FAK inhibition as a promising strategy to potentiate antibody immunotherapy in HGBCL.

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Blakemore, S. J., Zentis, P., Mueller, L., Brinker, R., Michalik, M., Juengst, C., Beielstein, A., Zhang, J., Neuhaus, R., Florin, A., Hallek, M., Meinel, J., Buettner, R., Schauss, A., Pallasch, C.. 2025-09-23. Focal adhesion pathway inhibition is the central axis of macrophage phenotypic responses to monoclonal antibody therapy in aggressive lymphoma via high-throughput screening and high-content imaging. https://doi.org/10.1101/2025.09.23.677924

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