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. 2024 Feb 20;19(2):e0298292.
doi: 10.1371/journal.pone.0298292. eCollection 2024.

Fluorescence-guided assessment of bone and soft-tissue sarcomas for predicting the efficacy of telomerase-specific oncolytic adenovirus

Koji Uotani  1 , Hiroshi Tazawa  2   3 , Joe Hasei  1 , Tomohiro Fujiwara  1 , Aki Yoshida  1 , Yasuaki Yamakawa  1 , Toshinori Omori  1 , Kazuhisa Sugiu  1 , Tadashi Komatsubara  1 , Hiroya Kondo  1 , Takuya Morita  1 , Masahiro Kiyono  1 , Suguru Yokoo  1 , Toshiaki Hata  1 , Toshiyuki Kunisada  1   4 , Ken Takeda  1 , Yasuo Urata  5 , Toshiyoshi Fujiwara  3 , Toshifumi Ozaki  1
Affiliations

Fluorescence-guided assessment of bone and soft-tissue sarcomas for predicting the efficacy of telomerase-specific oncolytic adenovirus

Koji Uotani et al. PLoS One. .

Abstract

Bone and soft-tissue sarcomas are rare malignancies with histological diversity and tumor heterogeneity, leading to the lack of a common molecular target. Telomerase is a key enzyme for keeping the telomere length and human telomerase reverse transcriptase (hTERT) expression is often activated in most human cancers, including bone and soft-tissue sarcomas. For targeting of telomerase-positive tumor cells, we developed OBP-301, a telomerase-specific replication-competent oncolytic adenovirus, in which the hTERT promoter regulates adenoviral E1 gene for tumor-specific viral replication. In this study, we present the diagnostic potential of green fluorescent protein (GFP)-expressing oncolytic adenovirus OBP-401 for assessing virotherapy sensitivity using bone and soft-tissue sarcomas. OBP-401-mediated GFP expression was significantly associated with the therapeutic efficacy of OBP-401 in human bone and soft-tissue sarcomas. In the tumor specimens from 68 patients, malignant and intermediate tumors demonstrated significantly higher expression levels of coxsackie and adenovirus receptor (CAR) and hTERT than benign tumors. OBP-401-mediated GFP expression was significantly increased in malignant and intermediate tumors with high expression levels of CAR and hTERT between 24 and 48 h after infection. Our results suggest that the OBP-401-based GFP expression system is a useful tool for predicting the therapeutic efficacy of oncolytic virotherapy on bone and soft-tissue sarcomas.

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Conflict of interest statement

Y.U. is the President and CEO of Oncolys BioPharma, Inc. H.T. and Tos.F. are consultants of Oncolys BioPharma, Inc. The other authors have no potential conflicts of interest to disclose.

Figures

Fig 1
Fig 1. CAR and hTERT expression levels in human bone and soft-tissue sarcoma cells.
(A,B) The CAR expression on the surface of bone and soft-tissue sarcoma cells and fibroblasts was analyzed by flow cytometric analysis. Representative histograms in each sample were shown (A). The mean fluorescence intensity (MFI) for each cell was determined by calculating the differences between the MFI in antibody-incubated and isotype IgG-incubated cells (B). (C) The hTERT mRNA expression in bone and soft-tissue sarcoma cells and fibroblasts was analyzed by RT-PCR. Data are expressed as mean values ± SD of independent experiments (n = 3). The statistical significance of the differences was calculated using an unpaired t-test. N.S., not significant.
Fig 2
Fig 2. OBP-401-mediated GFP expression and cell viability in human bone and soft-tissue sarcoma cells.
(A) Representative photographs of bone and soft-tissue sarcoma cells 72 h after infection with OBP-401 at the indicated doses. Scale bars: 100 μm. (B,D) The GFP intensity was analyzed using FlexScan in bone and soft-tissue sarcoma cells (B) and normal fibroblasts (D) 24, 48, and 72 h after infection with OBP-401 at the indicated doses. Data are expressed as mean values ± SD of independent experiments (n = 3). (C,E) Cell viability was analyzed using the WST-1 assay in bone and soft-tissue sarcoma cells (C) and normal fibroblasts (E) at 24, 48, and 72 h after infection with OBP-401 at the indicated doses. Data are expressed as mean values ± SD of independent experiments (n = 5). (F) Relationship between the GFP intensity and cell viability in bone and soft-tissue sarcoma cells and fibroblasts infected with OBP-401.
Fig 3
Fig 3. CAR and hTERT expressions in clinical specimens of bone and soft-tissue tumors.
(A) Representative photographs of immunohistochemical staining for CAR and hTERT using clinical tumor specimens. The top panel shows hematoxylin and eosin (HE) staining, the middle panel shows CAR expression, and the bottom panel shows hTERT expression. The ATM score is shown in each photograph. Scale bars: 50 μm. (B,C) Comparison of ATM score for CAR (B) or hTERT (C) between the malignant (n = 48) and intermediate (n = 6) group and the benign (n = 14) group. The statistical significance was determined using the Mann−Whitney test.
Fig 4
Fig 4. OBP-401-mediated GFP expression in clinical specimens of bone and soft-tissue tumors.
(A) Representative photographs of tumor specimens at 24 and 48 h after OBP-401 infection. The top panel shows phase-contrast images, and the bottom panel shows fluorescent images for GFP expression. The scale bars represent 1 mm and 500 μm in the top and bottom panels, respectively. (B) The GFP intensity was compared between 24 and 48 h after OBP-401 infection in the malignant (n = 48) and intermediate (n = 6) group and the benign (n = 14) group. The statistical significance of the differences was calculated using an unpaired t-test. N.S., not significant.
Fig 5
Fig 5. OBP-401-mediated GFP expression in clinical specimens of bone and soft-tissue tumors with different expressions of CAR and hTERT.
(A,B) Comparison of ATM score for CAR (A) or hTERT (B) between the high and low group in the malignant (n = 48) and intermediate (n = 6) group and the benign (n = 14) group. (C,D) The GFP intensity was compared between 24 and 48 h after OBP-401 infection in the malignant and intermediate group and the benign group with different expressions of CAR (C) and hTERT (D). The statistical significance of the differences was calculated using an unpaired t-test. N.S., not significant.
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