Disruption of TGF-β signaling pathway is required to mediate effective killing of hepatocellular carcinoma by human iPSC-derived NK cells

Hepatobiliary System Biochemistry Genetics Bioengineering Life Sciences Immunology Oncology Medicine Cell Biology
TGF-β signaling pathway NK cells hepatocellular carcinoma iPSC anti-tumor immunotherapy

Background Introduction

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, with a five-year survival rate of less than 20%, and treatment options are extremely limited. Traditional targeted drug therapies, such as Sorafenib and other kinase inhibitors, have been used to treat HCC, but their efficacy is limited and difficult to achieve a cure. In recent years, immunotherapy has gained attention in HCC treatment; however, immunotherapies targeting solid tumors (such as chimeric antigen receptor T cells and natural killer cells) face challenges from inhibitory factors in the tumor microenvironment. In the HCC microenvironment, a high concentration of Transforming Growth Factor Beta (TGF-β) has been confirmed to inhibit the activity of immune cells, creating an obstacle to the effectiveness of anti-tumor immunity. Therefore, inhibiting TGF-β signaling might become an important path to enhancing the efficacy of immunotherapy.

This research was completed by Jaya Lakshmi Thangaraj’s team at the University of California, San Diego, and published in the journal “Cell Stem Cell” on September 5, 2024. The study used genetically engineered natural killer cells derived from human induced pluripotent stem cells (iPSCs) to maintain high-efficiency anti-tumor effects in high TGF-β environments of HCC, providing a new research perspective for future NK cell therapy in HCC.

Research Process

Overview of Experimental Process

The study utilized iPSCs as the starting cell source, first via CRISPR-Cas9 gene editing technology to delete TGF-β receptor 2 (TGFBR2), generating TGFBR2 knockout (KO) and TGFBR2 dominant negative (DN) forms of NK cells. Subsequently, the chimeric antigen receptor (CAR) targeting HCC-specific antigens Glypican-3 (GPC3) and Alpha-fetoprotein (AFP) was introduced into iPSC-derived NK cells to achieve specific killing. The study proceeded with two main strategies: one being TGF-β signaling inhibition (TGFBR2-KO and TGFBR2-DN) and the other combining CAR technology to enhance the anti-tumor effectiveness of NK cells.

Detailed Experimental Process

  1. Construction of TGFBR2 Knockout and Dominant Negative Receptor Expression: Using CRISPR-Cas9 editing, the research team designed guide RNA targeting exon 2 of TGFBR2 to edit iPSCs, forming TGFBR2-KO clones. The PiggyBac transposon system was used to introduce the dominant negative form of the TGFBR2 gene (TGFBR2-DN) into iPSCs, yielding functionally complete TGFBR2-DN iPSC-NK cells.

  2. Differentiation and Expansion of iPSC-NK Cells: After six days of differentiation culture, these edited iPSCs successfully differentiated into early hematopoietic progenitor cells. After further culturing under NK cell conditions for five weeks, the cells expressed typical NK cell markers (CD45 and CD56), demonstrating typical NK cell activity and phenotype, proving that TGFBR2 editing does not affect NK cell differentiation.

  3. Functional Validation and Anti-tumor Activity Testing: The study tested the anti-tumor activity of TGFBR2-KO and TGFBR2-DN iPSC-NK cells under high concentrations of TGF-β. Results showed that the edited NK cells effectively killed HCC cell lines (HepG2 and SNU-449) at TGF-β concentrations ranging from 0 to 100 ng/ml. In contrast, unedited NK cells showed significantly reduced anti-tumor effects under high TGF-β concentrations. Further testing investigated the degranulation function (CD107a expression) and cytokine secretion capacity (IFN-γ and TNF-α expression) of these NK cells, confirming that TGFBR2-KO and TGFBR2-DN iPSC-NK cells maintained high-efficiency anti-tumor ability in the presence of TGF-β.

  4. CAR Expression Anti-tumor Activity: The introduction of CAR targeting GPC3 and AFP into TGFBR2-KO and wild type (WT) iPSC-NK cells validated the tumor cell killing activity of CAR-specific NK cells. Although CAR expression enhanced the specific killing ability of NK cells, the TGF-β environment had a significant inhibitory effect on WT iPSC-NK cells, preventing effective anti-tumor effects under high TGF-β concentrations. Conversely, TGFBR2-KO CAR-NK cells maintained high anti-tumor activity within the TGF-β environment, proving that inhibiting TGF-β signaling is crucial for achieving CAR-NK cells’ anti-tumor activity.

  5. Gene Expression and Transcriptome Analysis: RNA sequencing analysis comparing WT, TGFBR2-KO, and TGFBR2-DN iPSC-NK cells revealed significant changes in the expression of multiple tumor-related genes (such as CD226, IFNG, GZMB, CXCR4, etc.) after TGFBR2 editing. The analysis results showed significantly increased expression of genes related to NK cell activation, tumor infiltration, and chemokine receptors in TGFBR2-KO and TGFBR2-DN NK cells, forming an enhanced anti-tumor function basis.

  6. In Vivo Experiment Validation: In xenograft mouse models, different treatments were applied to HepG2 transplanted tumors. Results showed that TGFBR2-KO iPSC-NK cells significantly outperformed the WT iPSC-NK cell group in inhibiting tumor growth in vivo, and their anti-tumor effect was not affected by CAR expression. Furthermore, TGFBR2-KO iPSC-NK cells demonstrated better persistence and survival in mice, further confirming the importance of TGF-β signaling inhibition in NK cell therapy.

Research Results

  1. Application of TGFBR2 Knockout or Dominant Negative Receptors in iPSC-NK Cells Effectively Prevented TGF-β Inhibition: TGFBR2-KO and TGFBR2-DN iPSC-NK cells maintained high-efficiency anti-tumor activity in the presence of TGF-β, indicating that inhibiting TGF-β signaling is crucial for NK cell survival and anti-tumor activity in the tumor microenvironment.

  2. Combining CAR Expression for Specific Targeting of HCC: Although CAR-NK cells can recognize specific antigens GPC3 and AFP of HCC cells, their anti-tumor effect is still limited under the TGF-β environment. TGFBR2-KO iPSC-NK cells still maintained high-efficiency anti-tumor activity under these conditions, affirming the importance of TGF-β signal inhibition.

  3. Significant Changes in Gene Expression Profiles Support Enhanced Anti-tumor Activity: RNA sequencing showed significant upregulation of genes related to anti-tumor activity and NK cell activation in TGFBR2-KO and TGFBR2-DN iPSC-NK cells, providing a molecular basis for their enhanced anti-tumor function.

  4. In Vivo Xenograft Mouse Experiment Further Validates Anti-tumor Effects and Persistence: In a xenograft mouse model with HepG2 tumors, TGFBR2-KO iPSC-NK cells significantly slowed tumor growth and increased NK cell survival rate in vivo, indicating great potential for TGFBR2-KO and DN designs in NK cell applications for solid tumors.

Research Value and Significance

This study provides a new perspective for the application of NK cells in high TGF-β tumor microenvironments. Although CAR technology improves the targeting capability of NK cells towards specific tumors, the presence of TGF-β in the HCC microenvironment remains a major obstacle to effective anti-tumor activity. By gene-editing to inhibit TGF-β signaling, this study successfully enhanced the survival and anti-tumor effect of NK cells in TGF-β-rich environments, opening new pathways for future NK cell therapy.

Additionally, the standardized advantage of iPSCs as a source of NK cells supports the scalability and multi-gene editing of this therapy, giving it the potential for “off-the-shelf” cell therapy. This study lays the foundation for the application of NK cells in HCC and other high TGF-β tumors, providing critical experimental evidence for further developments in immunotherapy.

Highlights and Innovations

  • Efficient TGF-β Inhibition: Utilization of TGFBR2 gene knockout or dominant negative receptors ensures the anti-tumor activity of NK cells in a TGF-β inhibitory environment.
  • Standardization of iPSC-NK Cell Platform: iPSC-based NK cell generation achieves the convenience of multiple gene editing and the feasibility of mass production.
  • Validation of Efficacy in Xenograft Models: In vivo mouse experiments validated the inhibitory effects of TGFBR2-KO and CAR expression against HCC.

This study demonstrates the potential enhancement of NK cell therapy for solid tumors through TGF-β signal inhibition and provides new gene editing strategies and cell therapy solutions for future clinical trials.