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Cancer Research
Generazione e verifica funzionale di cellule T del recettore dell'antigene chimerico sensibili al...
Generazione e verifica funzionale di cellule T del recettore dell'antigene chimerico sensibili al...
JoVE Journal
Cancer Research
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JoVE Journal Cancer Research
Generation and Functional Verification of Hypoxia-Sensitive Chimeric Antigen Receptor-T Cells

Generazione e verifica funzionale di cellule T del recettore dell'antigene chimerico sensibili all'ipossia

Full Text
1,503 Views
09:12 min
June 14, 2024

DOI: 10.3791/66697-v

Ying Xue*1, Yunyu Mao*2, Qibin Liao3, Chen Zhao4, Xiaoyan Zhang1,2,4, Jianqing Xu1,2,4

1Institutes of Biomedical Sciences,Fudan University, 2Clinical Center of Biotherapy at Zhongshan Hospital,Fudan University, 3Department of Oncology and Bio-therapeutic Center, Shenzhen Third People's Hospital,Southern University of Science and Technology, 4Shanghai Public Health Clinical Center,Fudan University

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Overview

This article presents a protocol for generating hypoxia-sensitive chimeric antigen receptor (CAR)-T cells and verifying their functionality. The focus is on the lentivirus-based generation of these CAR-T cells, emphasizing their hypoxia-dependent expression and selective cytotoxicity against tumors.

Key Study Components

Area of Science

  • Cancer Immunotherapy
  • Chimeric Antigen Receptor (CAR) Technology
  • Hypoxia in Tumor Microenvironment

Background

  • CAR T-cell therapy is a promising approach in cancer treatment.
  • Hypoxia is a common feature of the tumor microenvironment that can affect therapy outcomes.
  • Improving the safety and efficacy of CAR T-cells is crucial for better patient outcomes.
  • This research explores hypoxia-sensitive CAR T-cells as a novel strategy.

Purpose of Study

  • To develop a protocol for creating hypoxia-sensitive CAR-T cells.
  • To validate the functionality of these CAR-T cells in hypoxic conditions.
  • To enhance understanding of CAR T-cell behavior in the tumor microenvironment.

Methods Used

  • Lentivirus-based generation of CAR-T cells.
  • Characterization of CAR expression under hypoxic conditions.
  • Assessment of selective cytotoxicity against tumor cells.
  • Establishment of hypoxia models for evaluation.

Main Results

  • Successful generation of hypoxia-sensitive CAR-T cells.
  • Validation of hypoxia-dependent CAR expression.
  • Demonstration of selective cytotoxicity in hypoxic environments.
  • Improved safety profile and efficacy in solid tumors.

Conclusions

  • Hypoxia-sensitive CAR-T cells represent a promising advancement in cancer immunotherapy.
  • This protocol can aid researchers in developing safer and more effective CAR-T therapies.
  • Understanding hypoxia's role in CAR T-cell function is essential for future research.

Frequently Asked Questions

What are hypoxia-sensitive CAR-T cells?
Hypoxia-sensitive CAR-T cells are engineered to respond to low oxygen levels in the tumor microenvironment, enhancing their targeting ability.
How does hypoxia affect CAR-T cell therapy?
Hypoxia can influence CAR-T cell functionality and survival, making hypoxia-sensitive designs crucial for effective therapy.
What is the significance of using lentivirus in CAR-T cell generation?
Lentivirus allows for stable integration of CAR genes into T-cells, ensuring long-term expression and functionality.
What are the potential benefits of hypoxia-sensitive CAR-T cells?
They may improve safety profiles and reduce exhaustion, leading to enhanced efficacy in treating solid tumors.
How can researchers evaluate hypoxia-sensitive CAR-T cells?
Researchers can establish hypoxia models to assess CAR expression and cytotoxicity in controlled environments.
What challenges exist in CAR-T cell therapy?
Challenges include managing side effects and ensuring effective targeting of tumor cells without harming healthy tissues.

Qui presentiamo un protocollo per la generazione e la verifica funzionale di cellule T sensibili al recettore chimerico dell'antigene (CAR)-T sensibili all'ipossia. Questo protocollo presenta la generazione di cellule CAR-T sensibili all'ipossia basate su lentivirus e la loro caratterizzazione, inclusa la convalida dell'espressione CAR ipossia-dipendente e della citotossicità selettiva.

La nostra ricerca si concentra principalmente sull'immunoterapia del cancro, in particolare sull'immunoterapia basata su cellule T CAR. Negli ultimi 10 anni abbiamo esplorato nuove strategie per consentire alle cellule T CAR di colpire specificamente un tumore riducendo al minimo l'effetto collaterale o addirittura nessun effetto collaterale. Nuove strategie sono state costantemente esplorate per migliorare la sicurezza e l'efficacia dell'immunoterapia basata su cellule T CAR, in particolare mirando al microambiente tumorale, come il CAR condizionato da ipossia, come esemplificato in questo protocollo.

È interessante notare che studi recenti hanno dimostrato che la condizione di ipossia CAR T non solo ha notevolmente migliorato il profilo di sicurezza, ma ha anche ridotto l'esaurimento delle CAR T e ha migliorato l'efficacia nei tumori solidi. L'ipossia è una caratteristica del microambiente tumorale. La nostra ricerca mira ad aiutare i ricercatori nel campo dell'immunoterapia del cancro a comprendere il concetto di cellule T CAR sensibili all'ipossia, la loro costruzione e come stabilire modelli di ipossia per la valutazione.

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Parole chiave: Cellule T chimeriche del recettore dell'antigene Cellule CAR-T Ipossia-sensibile Microambiente tumorale Dominio di degradazione ossigeno-dipendente Verifica funzionale Citotossicità Tumori solidi

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