Therapeutic Targeting Cancer-Initiating Cell Markers by Exosome miRNA: Efficacy and Functional Consequences Exemplified for claudin7 and EpCAM

Abstract

Aim: Transfer of exosomes (Exo) miRNA was described interfering with tumor progression. We here explored for claudin7 (cld7) and EpCAM (EpC), cancer-initiating-cell markers in colorectal and pancreatic cancer, the efficacy of Exo loading with miRNA and miRNA transfer.

Methods: Exo were collected from nontransformed mouse (NIH3T3) and rat lung fibroblasts (rFb), which were transfected with Tspan8 cDNA (NIH3T3-Tspan8, rFb-Tspan8). Exo were loaded by electroporation with miRNA. The transfer of Exo-miRNA was evaluated in vitro and in vivo in a rat pancreatic (ASML) and a human colon (SW948) cancer line.

Results: NIH3T3-Tspan8- or rFb-Tspan8-Exo were efficiently loaded with cld7- or EpC-miRNA. Exo targeting in vivo was strongly improved by tailoring with Tspan8. Exo-miRNA transfer into tumor targets promoted cld7, respectively, EpC downregulation by 33%-60%. Cld7 silencing was accompanied by reduced expression of additional cancer-initiating cell markers and NOTCH. EpC silencing reduced vimentin, N-cadherin, and Nanog expression. The Exo-miRNA transfer affected anchorage-independent growth, motility, and invasion.

Conclusions: Exo are efficiently loaded with miRNA, miRNA-delivery being supported by Exo tailoring. Partial cld7 and EpC silencing by Exo miRNA affects metastasis-promoting tumor cell activities. The findings suggest miRNA loading of tailored Exo as an easy approachable and efficient adjuvant therapy.

Figure 1

Fibroblast exosome uptake in vitro and in vivo. (A) Dio-labeled Exo from nontransfected and Tspan8-transfected rFb and NIH3T3 were cocultured for 3 and 6 hours with ASML or SW948 cells. Exo uptake was evaluated by flow cytometry after acid wash to remove bound Exo; mean percent ± SD (triplicates) of stained tumor cells; significant differences between Exo from nontransfected versus transfected Fb: *; (B-D) Dio-labeled Exo and Tspan8-Exo were iv injected into tumor-free and ASML-bearing (ifp application) rats and tumor-free or SW948-bearing (sc injection) nude mice. Animals were sacrificed after 48 hours; organs were excised and meshed. Dio-labeled Exo uptake was evaluated by flow cytometry or confocal microscopy. (B) Flow cytometry analysis of recovery of Dio-labeled Exo and Tspan8-Exo in tumor tissue and confocal microscopy of tumor cells seeded on glass slides (3 examples, 1 counterstained with DAPI (scale bar: 10 μm) are shown). (C) Tumor-free and TB nude mice and rats received a single iv injection of Dio-labeled Tspan8-Exo, flow-cytometry analysis of the percent±SD stained cells (3 animals / group) in the indicated organs; significant differences in Exo uptake between tumor-free and TB animals: *; (D) Tumor-free and TB mice and rats received a single iv injection of Dio-labeled Tspan8-Exo; dispersed cells of the indicated organs were counterstained with the indicated antibodies; the percent of marker+/Exo−, marker+/Exo+ and marker−/Exo + cells (3 animals / group); (B-D) mean (±SD) of triplicates; significant uptake by tumor cells: s, significantly uptake by host cells: σ. Tspan8-overexpressing Exo have an advantage in uptake by tumor cells. High in vivo uptake by SW948 tumor tissue and by draining LN and lung of ASML bearing rats relies exclusively on the uptake by tumor cells; comparably high uptake by liver cells and low / medium uptake by SC and BMC largely depends on Mϕ and/or MDSC.

Figure 2

The impact of cld7- and EpC-targeting miRNA on tumor cells. (A) ASML and SW948 cells were transfected with cld7- or EpC-miRNA. After 48 hours, the relative quantity of cld7 and EpC mRNA was evaluated by qRT-PCR and of the proteins by WB. (B) Flow cytometry analysis of CIC markers (cld7, EpC, Tspan8, CD44v6) and EMT markers in miR-3541 and miR-615-transfected ASML and miR-498-and miR-342 transfected SW948 cells; mean percent±SD (triplicates) of stained cells; significant differences between nontransfected and transfected tumor cells: *; (C) representative WB examples of control and miR-transfected ASML and SW948 cells. From eight miRNAs suggested to bind with high confidence to cld7 or EpC mRNA, only two rat cld7-miR and one human cl7- and human-EpC-miR promoted a reduction in cld7, respectively, EpC mRNA and protein expression. Only cld7 downregulation was accompanied by reduced expression of additional CIC markers. Reduced expression of both cld7 and EpC was accompanied by a reduction in vimentin expression. A reduction in the EMT transcription factors Nanog and Notch was restricted to reduced cld7 expression.

Figure 3

Exosome transfection with miRNA and transfer into tumor cells. (A) Exo were transfected with miRNA by electroporation, and recovery was evaluated by qRT-PCR; mean ± SD (triplicates) are shown; (B-D) SW948 and ASML cells were cocultured for 48 hours with miRNA transfected Fb. (B) Recovery of the miRNA and (C) recovery of cld7, EpCAM, and CD71 mRNA were evaluated by qRT-PCR (mean ± SD of triplicates, significant differences: *) and (D) confirmed for cld7, EpC and CD71 at the protein level by flow cytometry (mean ± SD of triplicates, significant differences by Exo-miR treatment: *). miRNA is efficiently transferred into Exo by electroporation and is recovered in tumor cells after coculture, where it promotes a reduction in cld7, EpC, or CD71, respectively, at the mRNA and the protein level.

Figure 4

The impact of exosome miRNA transfer into tumor cells on CIC and EMT marker expression. (A-D) miRNA-loaded Exo were cocultured for 48 hours with SW948, SW480, ASML, and AS cells. (A) Flow cytometry analysis of CIC marker and (C) EMT marker expression; mean ± SD of the percent stained cells (three assays); significant differences to cells cocultured with nontransfected Exo: *; (B-D) WB of selected CIC- and EMT-markers after coculture with miR-loaded Exo. The transfer of cld7-miRNA affects expression of additional CIC-markers, particularly CD44v6 and Tspan8. The EMT-markers vimentin N-cadh and E-cadh are more strongly affected by EpC than cld7 silencing. Only cld7 silencing affects NOTCH expression. Nanog expression becomes weakly downregulated by EpC silencing. CD71-miRNA transfer did not affect EMT marker expression.

Figure 5

The impact of exosome miRNA transferred into tumor cells on tumor progression-associated activities. (A-F) ASML and SW948 cells were cocultured for 48 hours with 30 μg/ml miRNA transfected Exo, where indicated Exo also were included during the assay period. (A) Exo-treated tumor cells were seeded in 0.3% agar containing 30 μg/ml Exo on a 1% agar monolayer. Cells were cultured for 3 weeks. Thereafter, the number of soft agar colonies was counted; representative examples and mean ± SD of 10 wells are shown; significant differences between cocultures with nontransfected versus transfected Exo are indicated; (B) Morphology of SW948 cells after 48-hour culture in the presence of nontransfected and miRNA-transfected Exo; (C) SW948 and ASML cells were seeded in 24-well plates and cultured in the presence of unloaded or miR-loaded Exo. Reaching subconfluence, medium was exchanged, and the monolayer was scratched with a yellow pipette tip. Wound healing was followed for 72 hours by light microscopy. Representative examples and the mean (five wells) of the wound area as % of the starting wound area are shown; significant differences between treatment with unloaded and miRNA-loaded Exo: *; (D) Exo-treated ASML and SW948 cells were seeded on Matrigel in 48-Transwell plates; the Matrigel contained 30 μg/ml Exo, and the lower chamber contained medium with Exo-depleted FCS. After 48 hours, invading cells (OD595 after lysis and crystal violet staining) and penetrating cells (counting after crystal violet staining) were evaluated. Representative examples and mean ± SD (five wells) as % of the seeded number of cells are shown; significant differences between treatment with unloaded versus miRNA-loaded Exo: *. The transfer of cld7- or EpC-miRNA affects anchorage-independent growth, migration, and invasion.