In vitro vascular differentiation system efficiently produces natural killer cells for cancer immunotherapies

Abstract

Background Immunotherapeutic innovation is crucial for limited operability tumors. CAR T-cell therapy displayed reduced efficiency against glioblastoma (GBM), likely due to mutations underlying disease progression. Natural Killer cells (NKs) detect cancer cells despite said mutations – demonstrating increased tumor elimination potential. We developed an NK differentiation system using human pluripotent stem cells (hPSCs). Via this system, genetic modifications targeting cancer treatment challenges can be introduced during pluripotency – enabling unlimited production of modified “off-the-shelf” hPSC-NKs. Methods hPSCs were differentiated into hematopoietic progenitor cells (HPCs) and NKs using our novel organoid system. These cells were characterized using flow cytometric and bioinformatic analyses. HPC engraftment potential was assessed using NSG mice. NK cytotoxicity was validated using in vitro and in vitro K562 assays and further corroborated on lymphoma, diffuse intrinsic pontine glioma (DIPG), and GBM cell lines in vitro . Results HPCs demonstrated engraftment in peripheral blood samples, and hPSC-NKs showcased morphology and functionality akin to same donor peripheral blood NKs (PB-NKs). The hPSC-NKs also displayed potential advantages regarding checkpoint inhibitor and metabolic gene expression, and demonstrated in vitro and in vivo cytotoxicity against various cancers. Conclusions Our organoid system, designed to replicate in vivo cellular organization (including signaling gradients and shear stress conditions), offers a suitable environment for HPC and NK generation. The engraftable nature of HPCs and potent NK cytotoxicity against leukemia, lymphoma, DIPG, and GBM highlight the potential of this innovative system to serve as a valuable tool that will benefit cancer treatment and research – improving patient survival and quality of life.

Image‐based assessment of natural killer cell activity against glioblastoma stem cells

“…Therefore, utilizing 3D GSC neurospheres could better emulate the in vivo environment. Galat et al recently employed GSC neurospheres to assess NK cell function [25], though ensuring uniform sphere sizes at the outset, especially for high-throughput screening, is crucial yet challenging for enhancing assay accuracy. In addition, the influence of other immune cell players, such as T cells or macrophages, on the effects exerted by NK cells against GSCs in GBM microenvironment remains a pertinent question.…”

Current immunotherapeutic approaches to diffuse intrinsic pontine glioma

“…There has also been interest in natural killer (NK) cells for cell transfers other than CAR-T cells. NK cells have increased anti-tumour activity compared to T cells in glioblastoma due to T cell targeting mutations in the tumour ( Lieberman et al, 2019 ; Galat et al, 2023 ). Galat et al, 2023 differentiated human pluripotent stem cells into NK cells and assessed their cytotoxicity in vitro using DIPG cell lines.…”

“…NK cells have increased anti-tumour activity compared to T cells in glioblastoma due to T cell targeting mutations in the tumour ( Lieberman et al, 2019 ; Galat et al, 2023 ). Galat et al, 2023 differentiated human pluripotent stem cells into NK cells and assessed their cytotoxicity in vitro using DIPG cell lines. They found that the cells could successfully engraft in peripheral blood samples and are cytotoxic against DIPG cells.…”