Cell Fate Reprogramming in the Era of Cancer Immunotherapy
Advances in understanding how cancer cells interact with the immune system allowed the development of immunotherapeutic strategies, harnessing patients’ immune system to fight cancer. Dendritic cell-based vaccines are being explored to reactivate anti-tumor adaptive immunity. Immune checkpoint inhibitors and chimeric antigen receptor T-cells (CAR T) were however the main approaches that catapulted the therapeutic success of immunotherapy. Despite their success across a broad range of human cancers, many challenges remain for basic understanding and clinical progress as only a minority of patients benefit from immunotherapy. In addition, cellular immunotherapies face important limitations imposed by the availability and quality of immune cells isolated from donors. Cell fate reprogramming is offering interesting alternatives to meet these challenges. Induced pluripotent stem cell (iPSC) technology not only enables studying immune cell specification but also serves as a platform for the differentiation of a myriad of clinically useful immune cells including T-cells, NK cells, or monocytes at scale. Moreover, the utilization of iPSCs allows introduction of genetic modifications and generation of T/NK cells with enhanced anti-tumor properties. Immune cells, such as macrophages and dendritic cells, can also be generated by direct cellular reprogramming employing lineage-specific master regulators bypassing the pluripotent stage. Thus, the cellular reprogramming toolbox is now providing the means to address the potential of patient-tailored immune cell types for cancer immunotherapy. In parallel, development of viral vectors for gene delivery has opened the door for in vivo reprogramming in regenerative medicine, an elegant strategy circumventing the current limitations of in vitro cell manipulation. An analogous paradigm has been recently developed in cancer immunotherapy by the generation of CAR T-cells in vivo. These new ideas on endogenous reprogramming, cross-fertilized from the fields of regenerative medicine and gene therapy, are opening exciting avenues for direct modulation of immune or tumor cells in situ, widening our strategies to remove cancer immunotherapy roadblocks.
Here, we review current strategies for cancer immunotherapy, summarize technologies for generation of immune cells by cell fate reprogramming as well as highlight the future potential of inducing these unique cell identities in vivo, providing new and exciting tools for the fast-paced field of cancer immunotherapy.
Reprogramming, The Journal
Cellular Reprogramming | Vol. 23, No. 3 | Editorial
© Mary Ann Liebert, Inc
Cellular reprogramming is a diverse and growing discipline that studies the reversal or modification of
cellular identity. The field aims to understand how cell fate is acquired, maintained, and inherited in homeostatic conditions and what happens when cell identity is hijacked in disease. Owing to the vast therapeutic potential of cellular reprogramming, efforts have also been placed to harness cell fate engineering for clinical applications. Cellular reprogramming history began addressing a fundamental question in biology: how are the myriad of cell types that compose an adult organism generated?
Ontogenic Shifts in Cellular Fate Are Linked to Proteotype Changes in Lineage-biased Hematopoietic Progenitor Cells
- >4,000 proteins quantified in fetal and adult hematopoietic progenitor cells (HPCs)
- Protein expression in HPCs separates cells based on ontogenic stage and lineage potential
- Generic fetal features are suppressed in myeloid-restricted progenitors
- Low Irf8 expression partially drives an impairment in monopoiesis in fetal HPCs
The process of hematopoiesis is subject to substantial ontogenic remodeling that is accompanied by alterations in cellular fate during both development and disease. We combine state-of-the-art mass spectrometry with extensive functional assays to gain insight into ontogeny-specific proteomic mechanisms regulating hematopoiesis. Through deep coverage of the cellular proteome of fetal and adult lympho-myeloid multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs), and granulocyte-monocyte progenitors (GMPs), we establish that features traditionally attributed to adult hematopoiesis are conserved across lymphoid and myeloid lineages, whereas generic fetal features are suppressed in GMPs. We reveal molecular and functional evidence for a diminished granulocyte differentiation capacity in fetal LMPPs and GMPs relative to their adult counterparts. Our data indicate an ontogeny-specific requirement of myosin activity for myelopoiesis in LMPPs. Finally, we uncover an ontogenic shift in the monocytic differentiation capacity of GMPs, partially driven by a differential expression of Irf8 during fetal and adult life.
Mononuclear Phagocyte Regulation by the Transcription Factor Blimp-1 in Health and Disease
Blimp‐1, the transcription factor encoded by the gene Prdm1, plays a number of crucial roles in the adaptive immune system, which result in the maintenance of key effector functions of B and T cells. Emerging clinical data, as well as mechanistic evidence from mouse studies, has additionally identified critical functions of Blimp‐1 in the maintenance of immune homeostasis by the mononuclear phagocyte system. Blimp‐1 regulation of gene expression affects various aspects of mononuclear phagocyte biology, including developmental programs such as fate decisions of monocytes entering peripheral tissue, and functional programs such as activation, antigen presentation, and secretion of soluble inflammatory mediators. The highly tissue‐, subset‐, and state‐specific regulation of Blimp‐1 expression in mononuclear phagocytes suggests that Blimp‐1 is a dynamic regulator of immune activation, integrating environmental cues to fine‐tune the function of innate cells. In this review, we will discuss the current knowledge regarding Blimp‐1 regulation and function in macrophages and dendritic cells.
Direct Reprogramming of Mouse Embryonic Fibroblasts to Conventional Type 1 Dendritic Cells by Enforced Expression of Transcription Factors
Ectopic expression of transcription factor combinations has been recently demonstrated to reprogram differentiated somatic cells towards the dendritic cell (DC) lineage without reversion to a multipotent state. DCs have the ability to induce potent and long-lasting adaptive immune responses. In particular, conventional type 1 DCs (cDC1s) excel on antigen cross-presentation, a critical step for inducing CD8+ T cell cytotoxic responses. The rarity of naturally occurring cDC1s and lack of in vitro methodologies for the generation of pure cDC1 populations strongly hinders the study of cDC1 lineage specification and function. Here, we describe a protocol for the generation of induced DCs (iDCs) by lentiviral-mediated expression of the transcription factors PU.1, IRF8 and BATF3 in mouse embryonic fibroblasts. iDCs acquire DC morphology, cDC1 phenotype and transcriptional signatures within 9 days. iDCs generated with this protocol acquire functional ability to respond to inflammatory stimuli, engulf dead cells, process and cross-present antigens to CD8+ T cells. DC reprogramming provides a simple and tractable system to generate high numbers of cDC1-like cells for high content screening, opening new avenues to better nderstand cDC1 specification and function. In the future, faithful induction of cDC1 fate in fibroblasts may lead to the generation of patient-specific DCs for vaccination
A SOX2 Reporter System Identifies Gastric Cancer Stem-Like Cells
Gastric cancer remains a serious health burden with few therapeutic options. Therefore, the recognition of cancer stem cells (CSCs) as seeds of the tumorigenic process makes them a prime therapeutic target. Knowing that the transcription factors SOX2 and OCT4 promote stemness, our approach was to isolate stem-like cells in human gastric cancer cell lines using a traceable reporter system based on SOX2/OCT4 activity (SORE6-GFP). Cells transduced with the SORE6-GFP reporter system were sorted into SORE6+ and SORE6– cell populations, and their biological behavior characterized. SORE6+ cells were enriched for SOX2 and exhibited CSC features, including a greater ability to proliferate and form gastrospheres in non-adherent conditions, a larger in vivo tumor initiating capability, and increased resistance to 5-fluorouracil (5-FU) treatment. The overexpression and knockdown of SOX2 revealed a crucial role of SOX2 in cell proliferation and drug resistance. By combining the reporter system with a high-throughput screening of pharmacologically active small molecules we identified monensin, an ionophore antibiotic, displaying selective toxicity to SORE6+ cells. The ability of SORE6-GFP reporter system to recognize cancer stem-like cells facilitates our understanding of gastric CSC biology and serves as a platform for the identification of powerful therapeutics for targeting gastric CSCs.
Understanding and Modulating Immunity With Cell Reprogramming
Cell reprogramming concepts have been classically developed in the fields of developmental and stem cell biology and are currently being explored for regenerative medicine, given its potential to generate desired cell types for replacement therapy. Cell fate can be experimentally reversed or modified by enforced expression of lineage specific transcription factors leading to pluripotency or attainment of another somatic cell type identity. The possibility to reprogram fibroblasts into induced dendritic cells (DC) competent for antigen presentation creates a paradigm shift for understanding and modulating the immune system with direct cell reprogramming. PU.1, IRF8, and BATF3 were identified as sufficient and necessary to impose DC fate in unrelated cell types, taking advantage of Clec9a, a C-type lectin receptor with restricted expression in conventional DC type 1. The identification of such minimal gene regulatory networks helps to elucidate the molecular mechanisms governing development and lineage heterogeneity along the hematopoietic hierarchy. Furthermore, the generation of patient-tailored reprogrammed immune cells provides new and exciting tools for the expanding field of cancer immunotherapy. Here, we summarize cell reprogramming concepts and experimental approaches, review current knowledge at the intersection of cell reprogramming with hematopoiesis, and propose how cell fate engineering can be merged to immunology, opening new opportunities to understand the immune system in health and disease.
Is Immunotherapy the Holy Grail for Pancreatic Cancer?
Novel approaches to trigger the immune system against cancer have recently gained much attention. The pioneers within the field, James Allison (MD Anderson Cancer Center, USA) and Tasuku Honjo (Kyoto University, Japan), were awarded the Nobel Prize in Physiology or Medicine 2018 for their breakthrough research on CTLA-4 and PD-1/PD-L1, respectively. These contributions have been fundamental for the development of immune checkpoint blockade drugs and have transformed the treatment of patients with advanced melanoma and several other tumors [1–3]. Given the success of immunotherapy in several solid tumors, the question remains whether immunotherapy is also an option in a recalcitrant tumor such as pancreatic cancer?
Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors
This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.
The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.
Induction of Human Hemogenesis in Adult Fibroblasts by Defined Factors and Hematopoietic Coculture
Transcription factor (TF)-based reprogramming of somatic tissues holds great promise for regenerative medicine. Previously, we demonstrated that the TFs GATA2, GFI1B, and FOS convert mouse and human fibroblasts to hemogenic endothelial-like precursors that generate hematopoietic stem progenitor (HSPC)-like cells over time. This conversion is lacking in robustness both in yield and biological function. Herein, we show that inclusion of GFI1 to the reprogramming cocktail significantly expands the HSPC-like population. AFT024 coculture imparts functional potential to these cells and allows quantification of stem cell frequency. Altogether, we demonstrate an improved human hemogenic induction protocol that could provide a valuable human in vitro model of hematopoiesis for disease modeling and a platform for cell-based therapeutics