Adenoviral targeting of malignant melanoma for fluorescence-guided surgery prevents recurrence in orthotopic nude-mouse models

Abstract

Malignant melanoma requires precise resection in order to avoid metastatic recurrence. We report here that the telomerase-dependent, green fluorescent protein (GFP)-containing adenovirus OBP-401 could label malignant melanoma with GFP in situ in orthotopic mouse models. OBP-401-based fluorescence-guided surgery (FGS) resulted in the complete resection of malignant melanoma in the orthotopic models, where conventional bright-light surgery (BLS) could not. High-dose administration of OBP-401 enabled FGS without residual cancer cells or recurrence, due to its dual effect of cancer-cell labeling with GFP and killing.

Keywords: OBP-401; adenovirus; fluorescence-guided surgery (FGS); melanoma; nude mice.

Figure 1. OBP-401 targets human malignant melanoma cell lines with GFP in vitro

RFP-expressing human malignant melanoma cell lines, Mel526, FEMX1, LOMVIXI, MV3 were seeded on 6 well plates (1×10⁵ cells well). OBP-401 was added at the indicated multiple of infection (MOI) 24 hours after cell seeding. Images were acquired with an FV1000 confocal laser scanning microscope (Olympus). A. Time-course images of Mel526, FEMX1, LOMVIXI, MV3 cells 2, 3, and 4 days after infection with OBP-401, at indicated MOIs. B. Histograms show the frequency of GFP-expressing Mel526, FEMX1, LOMVIXI, MV3 cells at indicated days after infection with OBP-401. Data are shown as average ± SD. N = 5. Scale bars = 500 μm.

Figure 2. Comparison of OBP-401-targeted fluorescence-guided surgery with bright-light surgery for orthotopic malignant melanoma using a stationary imaging system

RFP-expressing FEMX1 cells (5×10⁶) in 20 μl Matrigel (BD) were inoculated in the femoral skin of nude mice (5 weeks old). OBP-401 (1×10⁸ PFU) was injected intratumorally when tumors reached approximately 100 mm³ (6 mm diameter). A. Representative whole-tumor images of mock-infected REP-expressing orthotopic melanoma before and after bright-light surgery (BLS). B. Representative whole-tumor images of OBP-401-GFP labeled, RFP-expressing orthotopic malignant melanoma before and after OBP-401-based fluorescence-guided surgery (OBP-401 FGS). C. Representative whole-tumor images of a tumor resected by BLS and a tumor resected by OBP-401 FGS using the OV100 whole body imaging system. D. Bar graph shows the comparison of fluorescent area in the surgical bed after BLS or OBP-401 FGS (left). Bar graph shows the comparison of fluorescence intensity in the surgical bed after BLS or OBP-401 FGS (right). Fluorescent area and fluorescence intensity are calculated with ImageJ software. Data are shown as average ± SD. N = 12. Scale bars = 1 cm.

Figure 3. OBP-401 based FGS of melanoma using the Dino-Lite hand-held portable fluorescence scope

A. Representative whole tumor images of OBP-401-GFP labeled, RFP-expressing orthotopic malignant melanoma before and after OBP-401-FGS using the Dino-Lite hand-held fluorescence scope. B. Step-by-step procedure for OBP-401 based FGS of malignant melanoma using the Dino-Lite hand-held-fluorescence scope.

Figure 4. OBP-401 targeting visualizes residual malignant melanoma cells after BLS

A. Representative high-magnification images of the surgical bed of orthotopic malignant melanoma after BLS. B. OBP-401 targeting enabled visualization of residual tumor after BLS and results in complete resection. Representative high-magnification images of surgical bed after FGS of malignant melanoma. C. Step-by-step procedure of OBP-401 based FGS of residual malignant melanoma after BLS using the Dino-Lite hand-held fluorescence scope. D. Representative images of resected malignant melanoma nodule by FGS. Images were acquired with the OV100. E. Representative single-cell level images of resected malignant melanoma nodule after FGS. Images were acquired with the FV1000 confocal microscope.

Figure 5. OBP-401 kills human malignant melanoma cell lines in vitro

A. Representative images of RFP-expressing human malignant melanoma cell lines, Mel526-RFP, FEMX1-RFP, LOMVIXI-RFP, and MV3-RFP, 7 days after targeting with OBP-401 at a MOI of 5, 15, and 30. B. Bar graphs show the surviving fraction of Mel526-RFP, FEMX1-RFP, LOMVIXI-RFP, and MV3-RFP cells 7 days after targeting with OBP-401 in vitro. The number of live cells was counted. Data are shown as average ± SD. N = 5. Scale bars = 250 μm.

Figure 6. OBP-401 enables minimal precise surgery for orthotopic melanoma

A. Representative whole-tumor images of orthotopic malignant melanoma before and after mock-infection or before and after i.t. administration of low-dose OBP-401 (1×10⁸ PFU) or high-dose OBP-401 (at 3×10⁸ PFU). B. Bar graphs show the fluorescence intensity of a control tumor and low-dose or high-dose OBP-401-targeted tumors (left). Fluorescent area is calculated with ImageJ software. Bar graphs show the fluorescence area of control tumor and low-dose or high-dose OBP-401-targeted tumors (right). Fluorescence intensity is calculated with ImageJ software. Data are shown as average ± SD. N = 10. C. Representative images of surgical area after BLS (upper) and low-dose (middle) or high-dose OBP-401-FGS (lower). Bright field images (left panels) and fluorescence images (right panels). D. Bar graphs show the comparison of surgical area after BLS, low-dose FGS, or high doe FGS. E. Bar graphs show the comparison of tumor weight after BLS, low-dose FGS, or high-dose FGS. Data are shown as average ± SD. N=10, *p<0.05.

Figure 7. OBP-401-based FGS prevents local recurrence

A. Representative whole-body images of orthotopic malignant melanoma 90 days after BLS. B. Representative whole body images of orthotopic malignant melanoma 90 days after low-dose OBP-401-FGS. C. Representative whole body images of orthotopic malignant melanoma 90 days after precise minimal FGS with high-dose administration of OBP-401. Upper panels in A, B, C: brightfield; lower panels: fluorescence. D. Comparison of fluorescent areas of recurrent tumors after BLS, low-dose OBP-401-FGS, or high-dose OBP-401-FGS (left panel). Fluorescent area is calculated with ImageJ software. Comparison of fluorescence intensity of recurrent tumors after BLS, low-dose OBP-401-FGS, or high-dose OBP-401-FGS (right panel). Fluorescence intensity is calculated with ImageJ software. Data are shown as average ± SD. N = 10. E. Kaplan-Meyer curves show the over-all survival after BLS or OBP-401-FGS.