https://orcid.org/0000-0002-7949-3471
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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.
Acknowledgments
We thank Dr. Jolanta Topczewska (Director of the Imaging Facility) for help with confocal microscopy. We also thank the Developmental Therapeutics Core for assistance with in vivo experiments. Additionally, we are grateful to Dr. Ntziachristos for providing Jurkat cells and to Dr. Rintaro Hashizume for providing DIPG cells.
We also thank the Northwestern University Robert H. Lurie Comprehensive Cancer Center Developmental Therapeutics Core and Flow Cytometry Core Facility.
Additionally, we thank the NUSeq Core: Center for Genetic Medicine (Northwestern University) and the University of Chicago Genomics Facility for their services.
Disclosure Statement
No conflict of interest was reported by the author(s).
Compliance with Ethical Standards
All animal procedures were approved by the Institutional Animal Care and Use Committee at Northwestern University (Approved Animal Use Protocol #IS00009762) and performed in accordance with the Guide for the Care and Use of Laboratory Animals.
Author Contribution
Study Conception and Design: V.G, I.B, O.B, Y.G, Y.D, X-N.L, M.P, and P.M.I
Mouse Engraftment, Flow Cytometry: Y.D and X-N.L
HPC Derivation, Flow Cytometry: Y.G, V.G, and W.T
NK Differentiation, NK Functional Assays: Y.G and V.G
RNA Isolation, Bioinformatic Sequencing Analyses: M.P, Y.G, and V.G
NK Characterization: S.D, Y.G, S.S, M.P, and V.G
Material Contribution: I.B, O.B, D.G, and V.G
Manuscript Editing: Y.G, M.P, S.D, O.B, V.G, P.M.I, Y.D, X-N.L, I.B, S.S, and D.G
All authors read and approved the final manuscript.
Data Availability Statement
The datasets that support this study are available from the corresponding authors (Y.G and M.P) upon reasonable request.
Abbreviations
| cAMP | = | cyclic adenosine monophosphate |
| BBB | = | blood-brain barrier |
| CIS | = | cytokine-inducible Src homology 2-containing protein |
| CML | = | chronic myelogenous leukemia |
| CREB | = | cyclic adenosine monophosphate response element-binding protein |
| EBs | = | embryonic bodies |
| EP4 | = | prostaglandin E2 receptor 4 |
| DIPG | = | diffuse intrinsic pontine glioma |
| FBS | = | fetal bovine serum |
| FC | = | fold change |
| GBM | = | glioblastoma multiforme |
| GSCs | = | GBM cells with stem cell properties |
| HE | = | hemogenic endothelium |
| HPCs | = | hematopoietic progenitor cells |
| HSCs | = | hematopoietic stem cells |
| hPSCs | = | human pluripotent stem cells |
| NKs | = | natural killer cells |
| PB | = | peripheral blood |
| PBS | = | phosphate-buffered saline |
| PI | = | propidium iodide |
| PKA | = | protein kinase A |
| pV | = | p-value |
| TME | = | tumor microenvironment |
| TNF | = | tumor necrosis factor |
Statements and Declarations
The Robert H. Lurie Cancer Center is partly supported by the NCI Cancer Center Support Grant P30CA060553.
The IL13Rα2 hAb was developed with the help of the Specialized Program of Research Excellence for Translational Approaches to Brain Cancer, Developmental Research Project (I.V.B.) [Grant P50CA221747].
Supplementary Material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/2162402X.2023.2240670