Directing the migration of serum-free, ex vivo-expanded Vγ9Vδ2 T cells.

Vγ9Vδ2 T cells represent a promising cancer therapy platform because the implementation of allogenic, off-the-shelf product candidates is possible. However, intravenous administration of human Vγ9Vδ2 T cells manufactured under good manufacturing practice (GMP)-compliant, serum-free conditions are not tested easily in most mouse models, mainly because they lack the ability to migrate from the blood to tissues or tumors. We demonstrate that these T cells do not migrate from the circulation to the mouse bone marrow (BM), the site of many malignancies. Thus, there is a need to better characterize human γδ T-cell migration in vivo and develop strategies to direct these cells to in vivo sites of therapeutic interest. To better understand the migration of these cells and possibly influence their migration, NSG mice were conditioned with agents to clear BM cellular compartments, i.e., busulfan or total body irradiation (TBI), or promote T-cell migration to inflamed BM, i.e., incomplete Freund's adjuvant (IFA), prior to administering γδ T cells. Conditioning with TBI, unlike busulfan or IFA, increases the percentage and number of γδ T cells accumulating in the mouse BM, and cells in the peripheral blood (PB) and BM display identical surface protein profiles. To better understand the mechanism by which cells migrate to the BM, mice were conditioned with TBI and administered γδ T cells or tracker-stained red blood cells. The mechanism by which γδ T cells enter the BM after radiation is passive migration from the circulation, not homing. We tested if these ex vivo-expanded cells can migrate based on chemokine expression patterns and showed that it is possible to initiate homing by utilizing highly expressed chemokine receptors on the expanded γδ T cells. γδ T cells highly express CCR2, which provides chemokine attraction to C-C motif chemokine ligand 2 (CCL2)-expressing cells. IFNγ-primed mesenchymal stromal cells (MSCs) (γMSCs) express CCL2, and we developed in vitro and in vivo models to test γδ T-cell homing to CCL2-expressing cells. Using an established neuroblastoma NSG mouse model, we show that intratumorally-injected γMSCs increase the homing of γδ T cells to this tumor. These studies provide insight into the migration of serum-free, ex vivo-expanded Vγ9Vδ2 T cells in NSG mice, which is critical to understanding the fundamental properties of these cells.

Microfragmented adipose tissue is associated with improved ex vivo performance linked to HOXB7 and b-FGF expression.

INTRODUCTION: Adipose tissue (AT) has become a source of mesenchymal stromal/stem cells (MSC) for regenerative medicine applications, in particular skeletal disorders. Several enzymatic or mechanical procedures have been proposed to process AT with the aim to isolate cells that can be locally implanted. How AT is processed may impact its properties. Thus, we compared AT processed by centrifugation (C-AT) to microfragmentation (MF-AT). Focusing on MF-AT, we subsequently assessed the impact of synovial fluid (SF) alone on both MF-AT and isolated AT-MSC to better understand their cartilage repair mechanisms. MATERIALS AND METHODS: MF-AT and C-AT from the same donors were compared by histology and qRT-PCR immediately after isolation or as ex vivo cultures using a micro-tissue pellet system. The in vitro impact of SF on MF-AT and AT-MSC was assessed by histological staining and molecular analysis. RESULTS: The main AT histological features (i.e., increased extracellular matrix and cellularity) of the freshly isolated or ex vivo-cultured MF-AT persisted compared to C-AT, which rapidly deteriorated during culture. Based on our previous studies of HOX genes in MSC, we investigated the involvement of Homeobox Protein HOX-B7 (HOXB7) and its target basic Fibroblast Growth Factor (bFGF) in the molecular mechanism underlying the improved performance of MF-AT. Indeed, both these biomarkers were more prominent in freshly isolated MF-AT compared to C-AT. SF alone preserved the AT histological features of MF-AT, together with HOXB7 and bFGF expression. Increased cell performance was also observed in isolated AT-MSC after SF treatment concomitant with enhanced HOXB7 expression, although there was no apparent association with bFGF. CONCLUSIONS: Our findings show that MF has a positive effect on the maintenance of AT histology and may trigger the expression of trophic factors that improve tissue repair by processed AT.

Key Metabolic Pathways in MSC-Mediated Immunomodulation: Implications for the Prophylaxis and Treatment of Graft Versus Host Disease.

Mesenchymal stromal cells (MSCs) are spindle-shaped, plastic-adherent cells in vitro with potent immunosuppressive activity both in vitro and in vivo. MSCs have been employed as a cellular immunotherapy in diverse preclinical models and clinical trials, but most commonly as agents for the prophylaxis or therapy of graft versus host disease after hematopoietic cell transplantation. In addition to the oft studied secreted cytokines, several metabolic pathways intrinsic to MSCs, notably indoleamine 2,3-dioxygenase, prostaglandin E2, hypoxia-inducible factor 1 α, heme oxygenase-1, as well as energy-generating metabolism, have been shown to play roles in the immunomodulatory activity of MSCs. In this review, we discuss these key metabolic pathways in MSCs which have been reported to contribute to MSC therapeutic effects in the setting of hematopoietic cell transplantation and graft versus host disease. Understanding the contribution of MSC metabolism to immunomodulatory activity may substantially inform the development of future clinical applications of MSCs.

Author Correction: A Novel 3D In Vitro Platform for Pre-Clinical Investigations in Drug Testing, Gene Therapy, and Immuno-oncology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Challenges in Clinical Development of Mesenchymal Stromal/Stem Cells: Concise Review.

Identified 50 years ago, mesenchymal stromal/stem cells (MSCs) immediately generated a substantial interest among the scientific community because of their differentiation plasticity and hematopoietic supportive function. Early investigations provided evidence of a relatively low engraftment rate and a transient benefit for challenging congenital and acquired diseases. The reasons for these poor therapeutic benefits forced the entire field to reconsider MSC mechanisms of action together with their ex vivo manipulation procedures. This phase resulted in advances in MSCs processing and the hypothesis that MSC-tissue supportive functions may be prevailing their differentiation plasticity, broadening the spectrum of MSCs therapeutic potential far beyond their lineage-restricted commitments. Consequently, an increasing number of studies have been conducted for a variety of clinical indications, revealing additional challenges and suggesting that MSCs are still lagging behind for a solid clinical translation. For this reason, our aim was to dissect the current challenges in the development of still promising cell types that, after more than half a century, still need to reach their maturity. Stem Cells Translational Medicine 2019;8:1135-1148.

A Novel 3D In Vitro Platform for Pre-Clinical Investigations in Drug Testing, Gene Therapy, and Immuno-oncology.

Tumors develop within complex cell-to-cell interactions, with accessory cells playing a relevant role starting in the early phases of cancer progression. This event occurs in a three-dimensional (3D) environment, which to date, has been difficult to reproduce in vitro due to its complexity. While bi-dimensional cultures have generated substantial data, there is a progressive awareness that 3D culture strategies may rapidly increase the understanding of tumor development and be used in anti-cancer compound screening and for predicting response to new drugs utilizing personalized approaches. However, simple systems capable of rapidly rebuilding cancer tissues ex-vivo in 3D are needed and could be used for a variety of applications. Therefore, we developed a flat, handheld and versatile 3D cell culture bioreactor that can be loaded with tumor and/or normal cells in combination which can be monitored using a variety of read-outs. This biocompatible device sustained 3D growth of tumor cell lines representative of various cancers, such as pancreatic and breast adenocarcinoma, sarcoma, and glioblastoma. The cells repopulated the thin matrix which was completely separated from the outer space by two gas-permeable membranes and was monitored in real-time using both microscopy and luminometry, even after transportation. The device was tested in 3D cytotoxicity assays to investigate the anti-cancer potential of chemotherapy, biologic agents, and cell-based therapy in co-cultures. The addition of luciferase in target cancer cells is suitable for comparative studies that may also involve parallel in vivo investigations. Notably, the system was challenged using primary tumor cells harvested from lung cancer patients as an innovative predictive functional assay for cancer responsiveness to checkpoint inhibitors, such as nivolumab. This bioreactor has several novel features in the 3D-culture field of research, representing a valid tool useful for cancer investigations, drug screenings, and other toxicology approaches.

Impact of HOXB7 overexpression on human adipose-derived mesenchymal progenitors.

BACKGROUND: The ex vivo expansion potential of mesenchymal stromal/stem cells (MSC) together with their differentiation and secretion properties makes these cells an attractive tool for transplantation and tissue engineering. Although the use of MSC is currently being tested in a growing number of clinical trials, it is still desirable to identify molecular markers that may help improve their performance both in vitro and after transplantation. METHODS: Recently, HOXB7 was identified as a master player driving the proliferation and differentiation of bone marrow mesenchymal progenitors. In this study, we investigated the effect of HOXB7 overexpression on the ex vivo features of adipose mesenchymal progenitors (AD-MSC). RESULTS: HOXB7 increased AD-MSC proliferation potential, reduced senescence, and improved chondrogenesis together with a significant increase of basic fibroblast growth factor (bFGF) secretion. CONCLUSION: While further investigations and in vivo models shall be applied for better understanding, these data suggest that modulation of HOXB7 may be a strategy for innovative tissue regeneration applications.

MSC-Delivered Soluble TRAIL and Paclitaxel as Novel Combinatory Treatment for Pancreatic Adenocarcinoma.

Pancreatic cancer is the fourth leading cause of cancer death in western countries with more than 100,000 new cases per year in Europe and a mortality rate higher than 90%. In this scenario, advanced therapies based on gene therapies are emerging, thanks to a better understanding of tumour architecture and cancer cell alterations. We have demonstrated the efficacy of an innovative approach for pancreatic cancer based on mesenchymal stromal cells (MSC) genetically engineered to produce TNF-related Apoptosis Inducing Ligand (TRAIL). Here we investigated the combination of this MSC-based approach with the administration of a paclitaxel (PTX)-based chemotherapy to improve the potential of the treatment, also accounting for a possible resistance onset. Methods: Starting from the BXPC3 cell line, we generated and profiled a TRAIL-resistant model of pancreatic cancer, testing the impact of the combined treatment in vitro with specific cytotoxicity and metabolic assays. We then challenged the rationale in a subcutaneous mouse model of pancreatic cancer, assessing its effect on tumour size accounting stromal and parenchymal organization. Results: PTX was able to restore pancreatic cancer sensitivity to MSC-delivered TRAIL by reverting its pro-survival gene expression profile. The two compounds cooperate both in vitro and in vivo and the combined treatment resulted in an improved cytotoxicity on tumour cells. Conclusion: In summary, this study uncovers the potential of a combinatory approach between MSC-delivered TRAIL and PTX, supporting the combination of cell-based products and conventional chemotherapeutics as a tool to improve the efficacy of the treatments, also addressing possible mechanisms of resistance.