TRPM8 Activation via 3-Iodothyronamine Blunts VEGF-Induced Transactivation of TRPV1 in Human Uveal Melanoma Cells

In human uveal melanoma (UM), tumor enlargement is associated with increases in aqueous humor vascular endothelial growth factor-A (VEGF-A) content that induce neovascularization. 3-Iodothyronamine (3-T1AM), an endogenous thyroid hormone metabolite, activates TRP melastatin 8 (TRPM8), which blunts TRP vanilloid 1 (TRPV1) activation by capsaicin (CAP) in human corneal, conjunctival epithelial cells, and stromal cells. We compare here the effects of TRPM8 activation on VEGF-induced transactivation of TRPV1 in an UM cell line (92.1) with those in normal primary porcine melanocytes (PM) since TRPM8 is upregulated in melanoma. Fluorescence Ca2+-imaging and planar patch-clamping characterized functional channel activities. CAP (20 μM) induced Ca2+ transients and increased whole-cell currents in both the UM cell line and PM whereas TRPM8 agonists, 100 μM menthol and 20 μM icilin, blunted such responses in the UM cells. VEGF (10 ng/ml) elicited Ca2+ transients and augmented whole-cell currents, which were blocked by capsazepine (CPZ; 20 μM) but not by a highly selective TRPM8 blocker, AMTB (20 μM). The VEGF-induced current increases were not augmented by CAP. Both 3-T1AM (1 μM) and menthol (100 μM) increased the whole-cell currents, whereas 20 μM AMTB blocked them. 3-T1AM exposure suppressed both VEGF-induced Ca2+ transients and increases in underlying whole-cell currents. Taken together, functional TRPM8 upregulation in UM 92.1 cells suggests that TRPM8 is a potential drug target for suppressing VEGF induced increases in neovascularization and UM tumor growth since TRPM8 activation blocked VEGF transactivation of TRPV1

Low Energy Electron Irradiation Is a Potent Alternative to Gamma Irradiation for the Inactivation of (CAR-)NK-92 Cells in ATMP Manufacturing

Background: With increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these “off-the-shelf” therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function. Methods: Effectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others. Results: Our results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NKmediated specific lysis of tumor cells was maintained at stable levels for three days postirradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30. Conclusions: The presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application

Humanized mouse model: Hematopoietic stemcell transplantation and tracking using short tandem repeat technology

Introduction Models of mice carrying a human immune system, so-called humanized mice, are used increasingly as preclinical models to bridge the gap between model organisms and human beings. Challenges of the humanized mouse model include finding suitable sources for human hematopoietic stem cells (HSC) and reaching sufficient engraftment of these cells in immunocompromised mice. Methods In this study, we compared the use of CD34(+)HSC from cord blood (CB) vs HSC from adult mobilized peripheral blood. Furthermore, we developed a simple and highly specific test for donor identification in humanized mice by applying the detection method of short tandem repeats (STR). Results It was found that, in vitro, CB-derived and adult HSC show comparable purity, viability, and differentiation potential in colony-forming unit assays. However, in vivo, CB-derived HSC engrafted to a significantly higher extent in NOD.Cg-Prkdc(scid)IL2r gamma(tm1Wjl)/SzJ (NSG) mice than adult HSC. Increasing the cell dose of adult HSC or using fresh cells without cryopreservation did not improve the engraftment rate. Interestingly, when using adult HSC, the percentage of human cells in the bone marrow was significantly higher than that in the peripheral blood. Using the STR-based test, we were able to identify and distinguish human cells from different donors in humanized mice and in a humanized allogeneic transplantation model. Conclusion From these findings, we conclude that adult mobilized HSC are less suitable for generating a humanized immune system in mice than CB-derived cells

Humanized mouse model: Hematopoietic stemcell transplantation and tracking using short tandem repeat technology

Introduction Models of mice carrying a human immune system, so-called humanized mice, are used increasingly as preclinical models to bridge the gap between model organisms and human beings. Challenges of the humanized mouse model include finding suitable sources for human hematopoietic stem cells (HSC) and reaching sufficient engraftment of these cells in immunocompromised mice. Methods In this study, we compared the use of CD34(+)HSC from cord blood (CB) vs HSC from adult mobilized peripheral blood. Furthermore, we developed a simple and highly specific test for donor identification in humanized mice by applying the detection method of short tandem repeats (STR). Results It was found that, in vitro, CB-derived and adult HSC show comparable purity, viability, and differentiation potential in colony-forming unit assays. However, in vivo, CB-derived HSC engrafted to a significantly higher extent in NOD.Cg-Prkdc(scid)IL2r gamma(tm1Wjl)/SzJ (NSG) mice than adult HSC. Increasing the cell dose of adult HSC or using fresh cells without cryopreservation did not improve the engraftment rate. Interestingly, when using adult HSC, the percentage of human cells in the bone marrow was significantly higher than that in the peripheral blood. Using the STR-based test, we were able to identify and distinguish human cells from different donors in humanized mice and in a humanized allogeneic transplantation model. Conclusion From these findings, we conclude that adult mobilized HSC are less suitable for generating a humanized immune system in mice than CB-derived cells

Effect of combined sublethal X-ray irradiation and cyclosporine A treatment in NOD scid gamma (NSG) mice

Cyclosporine A (CsA) is used in hematopoietic stem cell transplantations (HSCT) to prevent graft-versus-host disease (GvHD). GvHD is the most severe side effect of allogeneic HSCT and efficient therapies are lacking. Mouse models are an essential tool for assessing potential new therapeutic strategies. Our aim is to mimic a clinical setting as close as possible using CsA treatment after sublethal irradiation in NSG mice and thereby evaluate the feasibility of this mouse model for GvHD studies. The effect of CsA (7.5 mg/kg body weight) on sublethally X-ray irradiated (2 Gy) and non-irradiated NSG mice was tested. CsA was administered orally every twelve hours for nine days. Animals irradiated and treated with CsA showed a shorter survival (n=3/10) than irradiated animals treated with NaCl (n=10/10). Furthermore, combined therapy resulted in severe weight loss (82 ± 6% of initial weight, n=7, day 8), with weight recovery after the CsA application was ceased. A high number of apoptotic events in the liver was observed in these mice (0.431 ± 0.371 apoptotic cells/cm2, n=2, compared to 0.027 ± 0.034 apoptotic cells/cm2, n=5, in the non-irradiated group). Other adverse effects, including a decrease in white blood cell counts were non-CsA-specific manifestations of irradiation. The combination of CsA treatment with irradiation has a hepatotoxic and lethal effect on NSG mice, whereas the treatment without irradiation is tolerated. Therefore, when using in vivo models of GvHD in NSG mice, a combined treatment with CsA and X-ray irradiation should be avoided or carefully evaluated

Publisher Correction: Automated application of low energy electron irradiation enables inactivation of pathogen- and cell-containing liquids in biomedical research and production facilities

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

Low Energy Electron Irradiation Is a Potent Alternative to Gamma Irradiation for the Inactivation of (CAR-)NK-92 Cells in ATMP Manufacturing

Background: With increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these “off-the-shelf” therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function. Methods: Effectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others. Results: Our results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NKmediated specific lysis of tumor cells was maintained at stable levels for three days postirradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30. Conclusions: The presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application

Low Energy Electron Irradiation Is a Potent Alternative to Gamma Irradiation for the Inactivation of (CAR-)NK-92 Cells in ATMP Manufacturing

Background: With increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these “off-the-shelf” therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function. Methods: Effectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others. Results: Our results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NKmediated specific lysis of tumor cells was maintained at stable levels for three days postirradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30. Conclusions: The presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application