[Construction and identification of a stable CT26 cell line expressing CD19-FLUC-GFP].

Solid tumors lack well-defined targets for chimeric antigen receptor T-cell (CAR-T) therapy. Therefore, introducing a known target molecule, CD19, into solid tumor cell lines via lentiviral transduction to investigate the cytotoxicity of CD19 CAR-T cells can potentially support CAR-T cell therapy against solid tumors. In this study, a stable colon cancer CT26 cell line, CT26-CD19-FLUC-GFP, expressing CD19, firefly luciferase (FLUC), and green fluorescent protein (GFP), was constructed using a triple-plasmid lentiviral system. The growth characteristics of this cell line were consistent with those of the CT26 cell line. Subsequent flow cytometry analysis confirmed stable expression of CD19 and GFP in CT26-CD19-FLUC-GFP cells after serial passaging up to the 5th, 10th, and 22nd generations. Further validation revealed significantly higher levels of CD19 mRNA and FLUC expression in CT26-CD19-FLUC-GFP cells continuously passaged up to the 22nd generation compared to the control CT26 cells. In comparison to T cells, CD19 CAR-T cells demonstrated substantial cytotoxicity against CT26-CD19-FLUC-GFP cells and MC38-CD19 cells. One week after intraperitoneal implantation of CT26-CD19-FLUC-GFP cells into mice, FLUC expression in the peritoneal region could be detected. These results indicate the successful establishment of a stable CT26 cell line expressing CD19-FLUC-GFP, which can be specifically targeted by CD19 CAR-T cells.

A novel cocktail therapy based on quintuplet combination of oncolytic herpes simplex virus-2 vectors armed with interleukin-12, interleukin-15, GM-CSF, PD1v, and IL-7 × CCL19 results in enhanced antitumor efficacy.

BACKGROUND: Selectively replicating herpes simplex virus-2 (HSV-2) vector is a promising treatment for cancer therapy. The insertion of multiple transgenes into the viral genome has been performed to improve its oncolytic activity. METHODS: Herein, we simultaneously constructed five "armed" oncolytic viruses (OVs), designated oHSV2-IL12, -IL15, GM-CSF, -PD1v, and IL7 × CCL19. These OVs delete the ICP34.5 and ICP47 genes with the insertion of transgenes into the deleted ICP34.5 locus. The anti-tumor efficacy in vivo was tested in the syngeneic 4T1 and CT26 tumor-bearing mice model. RESULTS: The OVs showed comparable oncolytic capability in vitro. The combination therapy of oHSV2-IL12, -IL15, GM-CSF, -PD1v, and IL7 × CCL19 exhibited the highest tumor inhibition efficacy compared with the treatment of single OV or two OVs combination. CONCLUSIONS: The OVs armed with different transgenes combination therapy also named 5-valent oHSV2 (also called cocktail therapy) might be an effective therapeutic strategy for solid tumors.

BGC823 Cell Line with the Stable Expression of iRFP720 Retains Its Primary Properties with Promising Fluorescence Imaging Ability.

Reliable animal models are required for understanding the molecular events of gastric tumor growth and metastasis. Tracing techniques based on iRFP720 may optimize the noninvasive monitoring of tumors in vivo. The present study established a human gastric adenocarcinoma cell line BGC823-iRFP720-GFP (abbreviated as BGC823-iRFP) that stably expressed iRFP720 and green fluorescent protein (GFP) by piggyBac transposon system. The monoclonal cell line BGC823-iRFP was isolated under puromycin selection. The cell morphology and proliferation ability of BGC823-iRFP cells in vitro were similar to that of the BGC823 cells. The iRFP720 and GFP expressions were confirmed by laser confocal microscopy and Cytation™ 5. Hematoxylin and eosin staining, immunohistochemical analysis, and animal experiments also revealed that BGC823-iRFP exhibited no significant changes in morphology, growth kinetics, and tumorigenicity in vivo. IVIS Lumina III imaging indicated that the iRFP720 signals of the BGC823-iRFP cells could be used to evaluate the antitumor efficacy of oncolytic viruses and chemotherapy drugs. Therefore, the BGC823-iRFP cells would be a useful tool for gastric cancer research and antitumor drug evaluation.

The construction of a new oncolytic herpes simplex virus expressing murine interleukin-15 with gene-editing technology.

The treatment of tumors with oncolytic viruses is an important cancer immunotherapy strategy. Interleukin-15 (IL-15) can enhance the antitumor effect of natural killer cells and T cells. An oncolytic herpes simplex type II virus (oHSV2-mIL-15CherryFP) expressing mouse IL-15 was constructed using the CRISPR/Cas9 system, and its antitumor activity in vitro and in vivo was evaluated. In vitro, the mouse interleukin-15 (mIL-15) present in the culture supernatant expressed by oHSV2-mIL-15CherryFP was able to enhance the killing of CT26-GFP tumor cells by T cells. In addition, the intratumoral injection of oHSV2-mIL-15CherryFP inhibited tumor growth in the CT26-iRFP and BGC823-iRFP model. These results indicate that the use of oncolytic herpes simplex virus expressing IL-15 may be a potential therapeutic strategy in tumor immunotherapy.

PiggyBac-modified CD19-expressing 4T1 cell line for the evaluation of CAR construct.

Reliable and stable target cell lines are required for evaluating the efficiency and studying the mechanism of chimeric antigen receptor T (CAR-T) immunotherapy both in vitro and in vivo. Jurkat cells can be used as an alternative for human primary lymphocytes to evaluate the constructs and function of the "CAR". This study established a murine 4T1-CD19 cell line that stably expressed a cd19 gene. The 4T1-CD19 cells had similar growth kinetics to its parent cell 4T1. The protein CD19 expression of the 4T1-CD19 was detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. The second-generation CAR was constructed and transfected into Jurkat cells. The expression of CAR protein was analyzed by flow cytometry and western blot. Finally, the interaction between the CAR and CD19 was confirmed by the upregulation of the IL-2 mRNA level of Jurkat-CAR stimulated by 4T1-CD19. Therefore, the 4T1-CD19 cell line and Jurkat-CAR have been successfully established, and may be used to access the function of various CAR constructs both in vitro and in vivo.