Multiplex immunofluorescence was performed using
OPAL reagents (Akoya Biosciences) on 4-mm sections of FFPE tumor biopsies. In brief, stainings were performed in multiple cycles of the following: antigen retrieval (15-minute boiling in antigen retrieval buffer, pH 6 or pH 9 depending on primary antibodies) followed by cooling, blocking, and consecutive staining with primary antibodies, HRP-polymer, and Opal fluorophores. Cycles were repeated until all markers were stained. Finally, nuclei were stained with DAPI. The sequence of antibody stainings was as follows: (i)
CD4 (FP1600/EP204, Akoya Biosciences, 1:100) -OPAL540; (ii)
CD8 (FP1601/144B, Akoya Biosciences, 1:200) -OPAL690; (iii) CXCR3 (HPA045942, Sigma, 1:100) -OPAL620; (iv)
T-bet (4B10, eBioscience, 1:50) -OPAL570; (v) PD-1 (NAT105, ImmunoLogic, 1:50) -OPAL520; (vi)
CD11b (EP1345Y, Abcam, 1:200) -OPAL650; (vii)
Cytokeratin-Pan (AE1/ AE3, Invitrogen, 1:200) -Coumarin (tyramide signal amplification coumarin system, Akoya Biosciences); and (viii) DAPI.
To assess immune cell clusters for the presence of B cells, we additionally stained consecutive tissue sections of responders with the following antibodies: (i)
CD3 (SP7, Sigma Aldrich, 1:250) -OPAL520; (ii) CD20 (L26, Cell Marque, 1:1,000) -OPAL620; (iii) CD8 (144B, Cell Marque, 1:400) -OPAL570; (iv) Cytokeratin-Pan (AE1/AE3, Invitrogen, 1:200) -OPAL690; and (v) DAPI.
Rijnders M., Balcioglu H.E., Robbrecht D.G.J., Oostvogels A.A.M., Wijers R., Aarts M.J.B., Hamberg P., van Leenders G.J.L.H., Nakauma-González J.A., Voortman J., Westgeest H.M., Boormans J.L., de Wit R., Lolkema M.P., van der Veldt A.A.M, & Debets R. (2022). Anti-PD-1 Efficacy in Patients with Metastatic Urothelial Cancer Associates with Intratumoral Juxtaposition of T Helper-Type 1 and CD8+ T cells. Clinical cancer research : an official journal of the American Association for Cancer Research, 28(1).
