Fig. 3: Design and characterization of the CtC platform.

a Schematic showing the proof-of-principle assay of the CtC platform. Two expression vectors were used here. First, there were two independent cMSC systems expressing GFP^DR (cMSC-GFP^DR) and mCherry (cMSC-mCherry). The cMSC-GFP^DR and cMSC-mCherry strains were constructed with the same expression vector (Reporter vector). Second, a cMSC was used to drive the expression of the 5’ CD63-trCas13d expression cassette and the T2A and 3’ MUC1 scFv-Lamp2b expression cassettes (CtC expression vector). In the cells transfected with the reporter vector, both GFP and mCherry were expressed in the MYC^high cells simultaneously. In the cells cotransfected with the reporter vector and the CtC expression vector, GFP was expressed in the MYC^high cells, and its mRNA could be packaged into the CtC exosomes for shuttling to nearby cells, especially those with high MUC1 expression. mCherry was expressed in only the MYC^high cells, which were used to label the transfected cells. b–d Representative images (b) of cells transfected with the reporter vector alone (mock group, top) and cotransfected with the reporter vector and the CtC expression vector (CtC group, bottom) are shown. FACS analysis of GFP and mCherry expression (c). FACS was used to quantify the proportion of GFP-positive and mCherry-positive cells among the 293T cells transfected as described above (d). These data indicated that the presence of CtC events resulted in more cells in the group expressing GFP. The data were presented above as the mean ± SD, n = 3 individual experiments, and significance was determined via two-tailed Student’s t-tests, NS not significant. Scale bar: 300 μm (the line in white) and 50 μm (the line in red). e Quantification of the expression levels of GFP and mCherry in a qPCR assay. These data indicated that the presence of CtC events could not increase the total amount of GFP expressed in a cell population. The data were presented above as the mean ± SD, n = 3 individual experiments, and significance was determined via two-tailed Student’s t-tests; NS not significant. f, g Illustration of CtC events in a cancer xenograft model. GFP and mCherry expression in T24 tumor tissue infected with AAV (cMSC-GFP^DR-cMSC-mCherry) alone or with both AAV (cMSC-CD63-dCas13d) and AAV (MSC-GFP^DR-cMSC-mCherry) (f). Areas marked by white arrows represent spatial segregation of GFP and mCherry fluorescence, indicating nonoverlapping cellular expression domains. FACS quantification confirmed the differential distribution of GFP-positive versus RFP-positive cells (g). The data were presented above as the mean ± SD, n = 3 individual experiments, and significance was determined via two-tailed Student’s t-tests. Scale bar: 300 μm (the line in white) and 50 μm (the line in red). h, i Illustration of CtC events in bladder cancer organoids. Quantification of GFP-positive and mCherry-positive cells in bladder cancer organoids (h). The tissue indicated by the white arrow is the tissue where GFP and mCherry expression are not colocalized. Bladder cancer organoids were infected with the above treatments (i). The data were presented above as the mean ± SD, n = 3 individual experiments, and significance was determined via two-tailed Student’s t-tests. Scale bar: 300 μm (the line in white) and 50 μm (the line in red).