DOI: 10.6084/m9.figshare.7906313.v1
title: MOESM2 of Delivery of oncolytic vaccinia virus by matched allogeneic stem cells overcomes critical innate and adaptive immune barriers

description: Additional file 2: Figure S2. ADSCs promote the oncolysis of resistant tumor cell lines through a combination of virus amplification, tumor cell recruitment and secretion of factors sensitizing the resistant tumor cells to virus infection. (A) Human ADSC promote the oncolysis of resistant B16 melanoma cells through augmented amplification of the TurboFP635-engineered L14 vaccinia virus. The figure shows fluorescence image analysis of 1 × 106 B16 cells cocultured with 2 × 105 eGFP-labelled RM20 adipose-derived stem cells (4× magnification) in a 12-well plate. B16 and stem cells were infected together with 1 × 105 pfu virus (MOI = 0.1 to B16) and incubated for up to 72 h (data party shown in Fig. 2a). (B) Human RM35 ADSC can also promote the oncolysis of the resistant murine B16 melanoma cells in vitro. Fluorescence imaging analysis of 1 × 106 B16 cells cocultured with 200,000 ADSC and infected with 100,000 pfu L14 VV for up to 4 days. (C) IFNγ pretreatment protects stem cells only in the presence of relatively resistant B16 but not the highly permissive ADSC and A549 cells. 200,000 RM20-eGFP cells (0.2 M) were pretreated with 20 ng/ml IFNγ for 24 h, cocultured with 200,000 (0.2 M) RM20 ADSC, A549 or B16 cells, and infected with the L14 virus as described in (Fig. 2a). Note that IFNγ pretreatment of the stem cells compromised the oncolysis of the B16 monolayer. (D) Insufficient number of stem cells (2% or lower) results in incomplete oncolysis of the B16 monolayer. B16 cells and RM20-eGFP cells were cocultured and infected with L14 as described in (Fig. 2A). To evaluate the role of stem cell number/dose, we compared the oncolysis of the B16 monolayer in the presence of 200,000 (0.2 M) and 20,000 (0.02 M) stem cells. (E) Fluorescence imaging analysis of B16 (10,000) and K562 (100,000) cells infected with L14 virus at MOI of 0.1 for 96 h in 96-well flat-bottom plates in the presence of ADSC supernatants from different stem cell donors as indicated. (F) Plaque assay analysis of L14 (top) and WT1 (medium) vaccinia virus amplification in B16 cells as in (E) and MTT assay showing the absence of significant impact of ADSC supernatants alone on the survival of the infected B16 cells (Bottom). (G) Flow cytometry analysis of ADSC supernatant-potentiated infection of K562 cells as evidenced by slight increases in the frequency of infected cells, TurboFP635 + MFI, and viral titers, but lack of a significant effect on the overall survival of the highly resistant K562 cells, as measured by the MTT assay. (H) K562 cells were infected with L14 VV at MOI of 0.1 as in (E) but instead of supernatants K562 cells were cocultured with 5000 or 20,000 RM20-eGFP ADSCs in triplicates. Fluorescence imaging and flow cytometry analysis were used to show that the green fluorescent stem cells attract the unlabeled/grey K562 cells and dramatically increase the percentage of infected eGFP-negative TurboFP635 + K562 cells. Despite the potentiated infectivity of the highly resistant K562 cells, the stem cells ultimately fail to eradicate or significantly impact their overall survival, consistent with the minimal ability of these cells to amplify vaccinia virus, as shown in the NCI-60 human cell line screen previously. Statistically significant differences (Student T-test, p