Ultrasound-Mediated Mesenchymal Stem Cells Transfection as a Targeted Cancer Therapy Platform

Abstract

Mesenchymal stem cells (MSCs) hold tremendous potential as a targeted cell-based delivery platform for inflammatory and cancer therapy. Genetic manipulation of MSCs, however, is challenging, and therefore, most studies using MSCs as therapeutic cell carriers have utilized viral vectors to transduce the cells. Here, we demonstrate, for the first time, an alternative approach for the efficient transfection of MSCs; therapeutic ultrasound (TUS). Using TUS with low intensities and moderate frequencies, MSCs were transfected with a pDNA encoding for PEX, a protein that inhibits tumor angiogenesis, and studied as a cell vehicle for in vivo tumor therapy. TUS application did not alter the MSCs' stemness or their homing capabilities, and the transfected MSCs transcribed biologically active PEX. Additionally, in a mouse model, 70% inhibition of prostate tumor growth was achieved following a single I.V. administration of MSCs that were TUS-transfected with pPEX. Further, the repeated I.V. administration of TUS-pPEX transfected-MSCs enhanced tumor inhibition up to 84%. Altogether, these results provide a proof of concept that TUS-transfected MSCs can be effectively used as a cell-based delivery approach for the prospective treatment of cancer.

Figure 1. Effect of TUS transfection on PEX expression level in MSCs.

PEX secreted to culture media of TUS-transfected MSCs with or without USCA (Optison) was quantified using ELISA and presented as ng PEX/10⁵ cells ± SD, **p < 0.001, n = 5.

Figure 2. Effect of PEX from TUS-transfected MSCs with and without USCA (Optison) on the migration, proliferation and apoptosis of endothelial cells.

(A,B) Migration assay of HUVECs after incubation in conditioned media of TUS-transfected MSCs at different time points. Percentage of migrated cells is compared to control. n = 9 (C) Apoptosis (n = 4) and (D) Proliferation (n = 6) of HUVECs after incubation with conditioned media of TUS-transfected MSCs, expressed as % of control ± SD. *p < 0.01, **p < 0.001.

Figure 3. Effect of MSC-secreting PEX post TUS transfection with and without USCA (Optison) on the viability and proliferation of cancer cells.

(A) Proliferation of PC3 cells after incubation with conditioned media of MSCs transfected using TUS (n = 7), (B) Viability of PC3 cells co-cultured with TUS-transfected MSCs along 5 days (n = 4). Results are presented as mean ± SD (C) Representative micrographs of PC3 cells (green) after being co-cultured with TUS-transfected MSCs. *p < 0.01, **p < 0.001.

Figure 4. Effect of TUS on the morphology and stemness of the transfected MSCs.

(A) Effect of TUS + USCA on cell morphology. Representative fluorescent images of actin (green), membrane (red) and nucleus (blue) of TUS treated and untreated MSCs. (B–E) FACS analyses of MSC cell surface markers (CD31, CD34, CD90 and CD44) 2 (B) and 7 (D) days post TUS transfection. All markers were compared to their isotype control.

Figure 5. Bio-distribution of TUS-transfected MSCs.

(A) Representative micrographs of TUS-PEX-transfected MSCs (red) in tumor (green) bearing mice. Cells were administered I.V. and visualized using a whole-body Maestro in vivo imaging system^TM. (B) % of MSCs in the tumor and other organs of tumor-bearing mice after I.V. administration of TUS-PEX-transfected MSCs. Cells were analyzed by flow-cytometry using PE anti-CD90 and results are presented as mean ± SD *p < 0.01, n = 5.

Figure 6

In vivo efficacy studies using single (A,B) and multiple (C,D) treatments with TUS-transfected MSCs. (A,C) Average tumor volume at different time points following treatment, and (B,C) tumor weight 21 days following treatment. Results are presented as mean ± SE *p < 0.05, **p < 0.001, n = 8.

Figure 7. Histology and immunohistochemistry (IHC) of treated tumors.

(A) Representative micrographs of histology (scale bar: 50 mm) and IHC – vascularization (CD31, scale bar: 100 μm), proliferation (Ki67, scale bar: 100 μm) and apoptosis (caspase 3, scale bar: 50 μm) of tumors from mice after single (DNA I) and multiple treatments (DNA III) compared to control. Quantification of IHC (B) % vascularization, (C) Proliferation and (D) Apoptosis. Results are presented as mean ± SD ***p < 0.001, **p < 0.01, *p < 0.05, n = 10.