Compared to endogenous biomolecules, small-molecule drugs are intrinsically challenging to image in vivo by fluorescence owing to their small size. Capturing drug distribution with high spatial resolution can help pinpoint direct drug actions onto specific cellular populations. Recently, we developed volumetric clearing-assisted tissue click chemistry (vCATCH), a pipeline that maps covalent drug binding with cellular resolution across an adult mouse body; this allowed us to systematically characterize and compare the distribution of two clinical cancer drugs in wild-type mice: the epidermal growth factor receptor (EGFR) inhibitor afatinib and the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib.

To do this, we built on our earlier work, in which we utilized CATCH to image alkyne-functionalized drugs in situ via a copper(I)-catalysed azide alkyne cycloaddition click reaction in thin tissue sections. However, such 2D works could inevitably omit important anatomical information. We therefore thoroughly optimized clearing, click labelling and lightsheet imaging workflows to achieve drug mapping in the intact adult mouse body. Comparisons across the whole mouse revealed that afatinib showed the strongest enrichment in the lung, which is consistent with high local EGFR expression. Interestingly, whereas both compounds were highly enriched in the liver, ibrutinib featured sparse yet strong cellular signals scattered around the liver. Although BTK expression is crucial for B cell function, detailed characterizations identified that nearly half of the cells with ibrutinib bound were T cells. However, because BTK is minimally expressed in T cells, the findings suggest that ibrutinib is associated with off-target engagement in T cells. Furthermore, we found that ibrutinib showed clear vascular and heart enrichment, which may explain the known bleeding and cardiac side effects.