Construction of self-driving anti-αFR CAR-engineered NK cells based on IFN-γ and TNF-α synergistically induced high expression of CXCL10

Neoplasia. 2024 Dec:58:101065. doi: 10.1016/j.neo.2024.101065. Epub 2024 Oct 3.

Abstract

Introduction: Ovarian cancer is the most malignant gynecological tumor. Previous studies have demonstrated that chimeric antigen receptor (CAR)-engineered NK-92 cells targeting folate receptor α (αFR) (NK-92-αFR-CAR) can specifically kill αFR-positive ovarian cancer cells. However, the migration barrier restricts antitumor effects of CAR-engineered cells.

Objectives: To elucidate the mechanism by which NK-92-αFR-CAR cells induce the secretion of chemokine CXCL10 during killing ovarian cancer cells. It is speculated that NK-92-αFR-CAR-CXCR3A can target αFR and have chemotaxis of CXCL10, and they may have stronger killing effect of ovarian cancer.

Methods: Study the mechanism of CXCL10 expression strongly induced by TNF-α and IFN-γ combined stimulation in ovarian cancer cells. Construct the fourth generation of NK-92-αFR-CAR-CXCR3A cells, which were co-expressed CXCR3A and αFR-CAR. Evaluate the killing and migration effects of NK-92-αFR-CAR-CXCR3A in vitro and in vivo.

Results: RNA sequencing (RNA-seq) first revealed that the expression level of the chemokine CXCL10 was most significantly increased in ovarian cancer cells co-cultured with NK-92-αFR-CAR. Secondly, cytokine stimulation experiments confirmed that IFN-γ and TNF-α secreted by NK-92-αFR-CAR synergistically induced high CXCL10 expression in ovarian cancer cells. Further signaling pathway experiments showed that IFN-γ and TNF-α enhanced the activation level of the IFN-γ-IFNGR-JAK1/2-STAT1-CXCL10 signaling axis. Cytotoxicity experiments showed that NK-92-αFR-CAR-CXCR3A cells could not only efficiently kill αFR-positive ovarian cancer cells in vitro but also secrete IFN-γ and TNF-α. Higher migration than that of NK-92-αFR-CAR was detected in NK-92-αFR-CAR-CXCR3A using transwell assay. NK-92-αFR-CAR-CXCR3A effectively killed tumor cells in different mouse xenograft models of ovarian cancer and increased infiltration into tumor tissue.

Conclusion: This study confirmed that IFN-γ and TNF-α secreted by αFR-CAR-engineered NK cells can synergistically induce high expression of CXCL10 in ovarian cancer cells and constructed self-driving αFR-CAR-engineered NK cells that can break through migration barriers based on CXCL10, which may provide a new therapeutic weapon for ovarian cancer.

Keywords: CAR-NK; CXCL10; CXCR3; Immunotherapy; Ovarian cancer.

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Chemokine CXCL10* / genetics
  • Chemokine CXCL10* / metabolism
  • Cytotoxicity, Immunologic
  • Female
  • Folate Receptor 1 / genetics
  • Folate Receptor 1 / metabolism
  • Humans
  • Immunotherapy, Adoptive / methods
  • Interferon-gamma* / metabolism
  • Killer Cells, Natural* / immunology
  • Killer Cells, Natural* / metabolism
  • Mice
  • Ovarian Neoplasms* / genetics
  • Ovarian Neoplasms* / immunology
  • Ovarian Neoplasms* / metabolism
  • Ovarian Neoplasms* / pathology
  • Receptors, CXCR3 / genetics
  • Receptors, CXCR3 / metabolism
  • Receptors, Chimeric Antigen* / genetics
  • Receptors, Chimeric Antigen* / immunology
  • Receptors, Chimeric Antigen* / metabolism
  • Tumor Necrosis Factor-alpha* / metabolism
  • Xenograft Model Antitumor Assays*

Substances

  • Chemokine CXCL10
  • Tumor Necrosis Factor-alpha
  • Interferon-gamma
  • CXCL10 protein, human
  • Receptors, Chimeric Antigen
  • Folate Receptor 1
  • Receptors, CXCR3