![]() ( D) Flow cytometry charts showing expression of Dendra2-green (X-axis) and Dendra2-red (Y-axis) in converted tumor cells and non-converted controls. Dissociated tissue from each group are pooled and sorted for subsequent applications. In different fragments, microcolonies (♜ol) or macrometastases are photoconverted. Orthotopic tumors derived from Dendra2-expressing cells produce liver metastasis. ( C) Experimental scheme utilized for photoconversion-based isolation of metastatic cells from liver fragments. The 40× region with a full FOV demonstrating specificity of photoconversion to field-stop confined area. Field-stop confined FOV of an 40× objective following photoconversion (‘Postconversion’). Field-stop image of a photoconversion session (violet light exposure, ‘Photoconversion’). In boxes, images representing individual green and red channels for the field-stop FOV. Field-stop-confined FOV within the 40× objective region, encircling the targeted three cell focus prior to photoconversion (‘Preconversion’). Focus on the marked area with 40× objective (scale bar = 25 µm). 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). ( B) Photoconversion of Dendra2 in microcolonies spontaneously arising in the liver of a pancreatic cancer tumor model. Here, we report ‘PIC-IT’ (‘Photomark and Isolate Cells If Tiny’), which enables unbiased and efficient isolation of size-defined metastatic colonies from live tissues through photoconversion-based marking ( Figure 1A). However, existing systems require specialized equipment and extended handling times, thus limiting scalability and precluding effective acquisition of rare or sporadic cells from multiple compartments such as microcolonies. Photoactivable and photoconvertible proteins provide a promising alternative for targeted cell isolation ( Medaglia et al., 2017 Nicenboim et al., 2015). Spatial transcriptomics methods continue to improve and can distinguish local patterns across large tumor regions ( Moncada et al., 2020), but resolution limits and low representation of small metastatic colonies in the tissue hinders their isolation. ![]() However, the use of these methods is restricted to compartments of particular size and only for specific applications due to several limitations, including (1) contamination by undesired cells within the capture field (2) low throughput (3) a need for expensive and temperamental hardware (4) loss of cell viability and (5) lack of control over population composition. Several methods have been developed to isolate cells from specific compartments in vivo, laser-capture microdissection representing the most widely used platform ( Basnet et al., 2019 Espina et al., 2006 Lovatt et al., 2014 Tang et al., 2009). While fluorescent reporters allow detection of metastatic colonies in pre-clinical models ( Aiello et al., 2016 Fluegen et al., 2017), an inability to recover pure micron-scale colonies has precluded systematic phenotypic analysis of early-stage metastases. A considerable fraction of metastasis is occult ( Haeno et al., 2012 Vanharanta and Massagué, 2013) and thus serves a potential source for residual disease and recurrence ( Sosa et al., 2014 Tohme et al., 2017). Metastasis is the primary cause of cancer-associated mortality and remains a significant therapeutic challenge. Moreover, the technique can be applied to other biological systems in which isolation and characterization of spatially distinct cell populations is not currently feasible. PIC-IT thus enables systematic investigation of metastatic heterogeneity. Pharmacological inhibition of NF-κB depleted microcolonies but had no effect on macrometastases, suggesting microcolonies are particularly dependent on this pathway. In a murine pancreatic cancer model, transcriptional profiling of spontaneously arising microcolonies revealed phenotypic heterogeneity, functionally reduced propensity to proliferate and enrichment for an inflammatory-response phenotype associated with NF-κB/AP-1 signaling. We developed PIC-IT, a photoconversion-based isolation technique allowing efficient recovery of cell clusters of any size – including single-metastatic cells – which are largely inaccessible otherwise. Studies of this occult fraction have been limited by a lack of tools with which to isolate discrete cells on spatial grounds. Cancer patients often harbor occult metastases, a potential source of relapse that is targetable only through systemic therapy.
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