(A) Schematic of the PIC-IT protocol. (B) Photoconversion of Dendra2 in microcolonies spontaneously arising in the liver of a pancreatic cancer tumor model. Left to right: Microscopic views acquired through a 4× objective with region of interest containing three cell clusters destined for photoconversion located in the marked area (arrows denote the three cells of interest, scale bar = 250 µm). Focus on the marked area with 40× objective (scale bar = 25 µm). Field-stop-confined FOV within the 40× objective region, encircling the targeted three cell focus prior to photoconversion (‘Preconversion’). In boxes, images representing individual green and red channels for the field-stop FOV. Field-stop image of a photoconversion session (violet light exposure, ‘Photoconversion’). Field-stop confined FOV of an 40× objective following photoconversion (‘Postconversion’). The 40× region with a full FOV demonstrating specificity of photoconversion to field-stop confined area. (C) Experimental scheme utilized for photoconversion-based isolation of metastatic cells from liver fragments. Orthotopic tumors derived from Dendra2-expressing cells produce liver metastasis. In different fragments, microcolonies (µCol) or macrometastases are photoconverted. Dissociated tissue from each group are pooled and sorted for subsequent applications. (D) Flow cytometry charts showing expression of Dendra2-green (X-axis) and Dendra2-red (Y-axis) in converted tumor cells and non-converted controls. (E) Seeding efficiency of photoconverted tumor cells, calculated as the fraction of cells counted in tissue culture plates 24 hr post-isolation relative to the number sorted by FACS (n = 4). (F) UMI profiles indicate expression of the markers Krt19 and Dendra2 in the sorted cells but no expression of other lineage markers (N = 361). (G, H) High prevalence of RNA-seq transcripts of CLCA3A1 (G, NµCol = 94, Nmacro = 111) and the corresponding CLCA1 protein expression (H) determined by flow cytometry on macrometastasis-derived vs. microcolonies (n = 4). (I, J) Concordance between elevated transcription (UMI per million) of BNIP3 in RNA-seq (I) and (J) BNIP3 MFIs in (left) immunostained microcolonies and macrometastases populating the livers of 6419c5-YFP tumor-bearing mice (NµCol = 53, Nmacro = 60). On the right, representative images of microcolonies and macrometastases immunostained for BNIP3 (red), YFP (green), and DAPI (blue). Scale bar = 50 µm. Bars represent mean ± SEM in all graphs. p-values were calculated by unpaired two-tailed Student’s t-test.