Cytocidal Effect of Irradiation on Gastric Cancer Cells Infected with a Recombinant Mammalian Orthoreovirus Expressing a Membrane-Targeted KillerRed.

The outcomes of unresectable gastric cancer (GC) are unfavorable even with chemotherapy; therefore, a new treatment modality is required. The combination of an oncolytic virus and photodynamic therapy can be one of the promising modalities to overcome this. Mammalian orthoreovirus (MRV) is an oncolytic virus that has been used in clinical trials for several cancers. In this study, we developed and evaluated a recombinant MRV strain type 3 Dearing (T3D) that expresses membrane-targeting KillerRed (KRmem), a phototoxic fluorescent protein that produces cytotoxic reactive oxygen species upon light irradiation. KRmem was fused in-frame to the 3' end of the σ2 viral gene in the S2 segment using a 2A peptide linker, enabling the expression of multiple proteins from a single transcript. RNA electrophoresis, Western blotting, and immunofluorescence analyses confirmed functional insertion of KRmem into the recombinant virus. The growth activity of the recombinant virus was comparable to that of the wild-type MRV in a cultured cell line. The recombinant virus infected two GC cell lines (MKN45P and MKN7), and a significant cytocidal effect was observed in MKN45P cells infected with the recombinant virus after light irradiation. Thus, recombinant MRV-expressing KRmem has the potential to serve as a novel treatment tool for GC.

Development of an oncolytic mammalian orthoreovirus expressing the near-infrared fluorescent protein iRFP720.

Fluorescence-guided surgery (FGS) is a useful method for removing invasive tumor tissues. For this, near-infrared (NIR) fluorescence probes are suitable for visualizing cancer cells due to their low autofluorescence, and an oncolytic mammalian orthoreovirus (MRV) expressing an NIR fluorescent protein is expected to be a novel tool for FGS. In this study, we identified the optimal insertion site of the NIR fluorescent protein gene iRFP720 (915 nt) in the MRV genome. We constructed genome plasmids for the L1, M1, and S2 segments, where a gene cassette comprising iRFP720 and T2A self-cleaving peptide was inserted in the 5' or 3' region of each segment. Through virus recovery, the recombinant MRV with the gene cassette at the M1 segment's 3' end, T3D-L(M1/3'iRFP720), was capable of replication and passaging with bright NIR fluorescence. However, the replication of T3D-L(M1/3'iRFP720) was approximately 1,000-fold lower than that of the wild-type virus. T3D-L(M1/3'iRFP720) production improved due to the transfection of a fusion-associated small transmembrane protein gene of fusogenic reovirus. Further, fluorescence signals were detected in T3D-L(M1/3'iRFP720)-infected human gastric and pancreatic cancer cells. Thus, the M1 segment's 3' end tolerates the expression of the long iRFP720 gene, which may propel the development of recombinant MRV vectors for FGS.

Clinical significance of sarcoid-like reaction in lymph nodes of gastric cancer patients.

BACKGROUND: Although granulomatous formation (sarcoid-like reaction [SR]) similar to sarcoidosis occasionally occurs in regional lymph nodes of malignant tumors, the pathological significance of SR is unknown. We aimed to elucidate the clinicopathological significance and prognostic relevance of SR in gastric cancer. METHODS: We evaluated 391 patients who underwent curative gastrectomy for gastric cancer between 2007 and 2016 at our hospital. The patients were divided into two groups according to presence of SR in lymph nodes, and clinicopathological factors and prognosis were compared between the two groups. RESULTS: SR was found in 67 (17.1%) of the 391 patients, and 332 (3.3%) of 10,149 lymph nodes examined. Regarding clinicopathological factors, SR-positive group showed significantly higher average age (p < 0.01) and numbers of differentiated tumors than SR-negative group (p < 0.05). Three-year overall survival rate in elderly patients aged ≥75 years was significantly more favorable in SR-positive group (n = 27, 85.2%) versus SR-negative group (n = 97, 63.4%) (p < 0.05). Immunohistochemical studies showed the predominant presence of CD68-positive macrophages for SR, with CD4/CD8-positive T cells and interleukin-10 also positive. CONCLUSION: The presence of SR in gastric cancer is frequent in elderly patients and might be a favorable indicator of prognosis. SR is suggested to reflect immune activation in the host.

Asymmetric cooperativity in tandem hybridization of enantiomeric metal complex-tethered short fluorescent DNA probes.

The complex [Ru(phen)(2)(dppz)](2+)(phen = 1,10-phenanthroline, dppz = dipyrido[3,2-aratio2',3'-c]phenazine) was attached to the 5' end of a short oligonucleotide to form conjugates, the Delta-isomer of which showed a high cooperativity during the recognition of the repetitive sequence, while the Lambda-isomer did not.

Highly enhanced duplex stability of dipyrido [3,2-a:2',3'-c] phenazine-modified oligonucleotide conjugate.

Dipyrido [3,2-a:2',3'-c] phenazine (DPPZ) or 1,10-phenanthroline (Phen) was tethered to the 5'-end of a short oligonucleotide (ODN) to generate two ODN conjugates. The conjugates formed stable duplexes with complementary 6 mer (d(TTAGGG)), which is one unit of telomeric repeats of human. The melting temperature of the duplex with DPPZ conjugate was higher than that of the corresponding duplex with unmodified 6 mer by 19.6 degrees C. This stabilization is enormous compared with those observed in other ODN conjugates reported previously. It would be attributed to the effective interaction of tethered heteroaromatic groups with DNA base stack of the duplex.