Abstract 2876: Early Retention and Subsequent Engraftment of Transplanted Progenitor Cells is Improved by Delivery within Collagen Matrix

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Yan Zhang ◽  
Marc Lamoureux ◽  
Stephanie Thorn ◽  
Vincent Chan ◽  
Joel Price ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Progenitor Cells ◽  
Pet Imaging ◽  
Ex Vivo ◽  
Collagen Matrix ◽  
Cell Labeling ◽  
Type I ◽  
The Matrix ◽  
Early Retention

Background: To investigate the mechanisms involved in the potentiation of cell therapy by delivery matrices, we evaluated the retention and engraftment of transplanted human circulating progenitor cells (CPCs) injected in a collagen matrix by using in vivo positron emission tomography (PET) imaging, ex vivo biodistribution, and immunohistochemistry. Methods: CPCs were labeled with 18 F-FDG and injected with or without a collagen type I-based matrix in the ischemic hindlimb muscle (IM) of rats (2x10 6 cells; n=15/group). Localization of cells was acquired by PET imaging (15 min) at 150 min post-injection. In addition, radionuclide biodistribution, immunofluorescence, and immunohistochemical examination of transplanted CPCs were performed at up to 14 days. Results: Cell labeling efficiency was CPC-concentration dependent (r=0.61, p <0.001), but not 18 F-FDG-dose dependent. Labeled CPCs exhibited excellent short-term stability and viability. Persistence of 18 F-FDG radioactivity in cells was markedly greater than non-specific retention in the matrix. Wholebody (WB) PET images revealed better CPC retention in the IM and less non-specific leakage to other tissues when CPCs were delivered within the matrix (IM/WB retention ratio of 43.9±8.2%), compared to cells injected alone (22.3±10.4%; p =0.040) and to 18 F-FDG injected with or without the matrix (9.7±5.5% and 11.0±5.5%, respectively; p <0.005). Radioactivity biodistribution confirmed that accumulation was increased (by 92.5%; p =0.024) in the IM and reduced (by 1.1 to 23.8%; p <0.05) in non-specific tissues when cells were injected within the matrix, compared to cells injected alone. Anti-human mitochondria staining showed increased cell retention in the IM with use of matrices (3.0±2.1%) versus cells only (1.9±0.8%; p =0.048). At 14 days the number of CD31 + transplanted human cells was greater (1.6±0.1%) when injected within the matrix than injected alone (0.7±0.1%; p =0.004). Conclusions: Collagen-based delivery matrices improve the early retention of transplanted CPCs, which in turn favors subsequent cell engraftment in the ischemic tissue. This mechanism conferred by the matrix has potential implications for the optimization of cell therapy at the early stages after cell delivery.

scholarly journals Efficient Ex Vivo Expansion of Highly Purified Human Umbilical Cord Blood-Derived Very Small CD34+lin-CD45- Stem Cells into Functional Endothelial Cells in Vitro in Chemically Identified, Feeder Layer-Free Medium Supplemented with Nicotinamide

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4882-4882
Author(s):  
Alison Domingues ◽  
Kamila Bujko ◽  
Magdalena Kucia ◽  
Janina Ratajczak ◽  
Mariusz Z Ratajczak
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Cell Therapy ◽  
Progenitor Cells ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Germ Layer ◽  
Differentiation Factors

Background . There is an ongoing search for multipotent stem cells from umbilical cord blood (UCB) with trans-germ layer differentiation potential that can be employed in repairing damaged organs and also expanded into transplantable hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs). The existence of such cells in postnatal life could also revive the concept of hemangioblasts or hemangioblast-like cells in adult hematopoietic organs. Our group was the first to isolate a population of small CD34+CD133+lin-CD45- early-development stem cells from human hematopoietic tissues, including UCB. Based on the validated expression of early-development markers, these cells were named "very small embryonic-like stem cells" (VSELs, Circulation Res 2019; 124:208-210). Currently, more than 25 independent groups worldwide who have carefully followed the multicolor-staining cell-sorting strategy described by us (Current Protocols in Cytometry 2010, 9.29.1-9.29.15) have successfully isolated these cells and demonstrated their in vivo contribution to all three germ layer lineages. Thus, VSELs could be very useful in regenerative medicine in the field of angiogenesis, and UCB is an attractive source, with easy accessibility and tolerance to allogenic grafts. However, the low number of these cells in UCB and their quiescence are limiting factors. Therefore, in vitro differentiation of VSELs into endothelial progenitor cells (EPCs) would allow improvement in the ability to expand endothelial cells and could represent a clinically relevant alternative to embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS) for cell therapy without ethical problems and undesirable side effects. Hypothesis. We hypothesized that UCB-purified, very small, early-developmentCD34+lin-CD45-stem cells can be ex vivo expanded into functional EPCs. Materials and Methods. VSELs highly purified by FACS were expanded into EPCs in pro-angiogenic medium supplemented with mesodermic differentiation factors and then endothelial differentiation factors in the presence of nicotinamide and UM171. In parallel, we expanded EPCs from MNCs isolated from the same UCB units by employing a classical protocol (Methods in Enzymology 2008, 445:303-29). The EPC nature of the expanded VSEL-derived cells was confirmed by the expression of typical EPC markers as well as by in vitro angiogenic assays. Results. Our differentiation cocktail allowed us to differentiate and expand VSELs into EPCs. In our expansion medium (Figure 1), the very small, round VSELs smaller than 6 mm in diameter proliferated and differentaited over time into larger and extended cells with a cobblestone morphology similar to the EPC control cells, and we confirmed their endothelial characteristics by cytometry analysis. Like EPCs, VSEL-derived EPCs were positive for CD31, CD144, KDR, and CD105 and negative for mesenchymal surface markers, such as CD90. They also performed similarly to EPCs in classical vasculogenic tests, including adhesion, proliferation, migration, and tubulogenesis assays. Conclusions. This work shows, for the first time, efficient VSEL differentiation into functional endothelial cells with vasculogenic properties without the help of co-culture over feeder-layers or viral vectors in medium supplemented with nicotinamide and UM171. These findings allow us to propose these cells as an interesting cell therapy product. These results also reopen the question of the existence of hemangioblast-like cells in postnatal tissues. We are currently testing these cells in vivo in model of hind limb ischemia. Figure 1 Disclosures No relevant conflicts of interest to declare.

Abstract 491: Injury-Activated TGFβ Recruits MSCs for Vascular Remodeling

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Mei Wan ◽  
Changjun Li ◽  
Gehua Zhen ◽  
Wenying He ◽  
Kai Jiao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Migration ◽  
Progenitor Cells ◽  
Vascular Remodeling ◽  
Ex Vivo ◽  
Vascular Lesions ◽  
Type I ◽  
Post Injury ◽  
Local Resident

Stem/progenitor cells can be recruited to participate in tissue remodeling, regeneration, or repair following injury. In particular, bone marrow-derived or local resident stem /progenitor cells are implicated in vascular repair and remodeling. It is believed that migratory factor(s) released by injured tissue creates a gradient to mediate the migration of stem cells to the injury sites. The key mediator(s) inducing the recruitment of the cells to the vascular lesions remains to be defined. Here we show that nestin + cells, which represent similar population as Sca1 + CD29 + CD11b - CD45 - mesenchymal/stromal stem cells (MSCs), were recruited and incorporated into the neointimal tissue in both rat model of balloon injury of carotid artery and mouse model of wire injury of femoral artery. Importantly, elevated active TGFβ1 level was observed in the injured vessels as early as 8 hr post injury, and the elevation lasted for 2 wks. To investigate whether injury-induced activation of TGFβ1 in vascular matrix stimulates migration of MSCs, we developed an aorta-conditioned medium (Aorta-CM)-based cell migration assay in which MSCs were placed in the upper chamber and the Aorta-CM prepared by culturing ex vivo injured aorta was placed in the lower chamber of a transwell chamber. Aorta-CM prepared from ex vivo injured aorta significantly enhanced cell migration compare to CM prepared from uninjured aorta. When neutralizing antibody specific for TGFβ1 or the inhibitor of TGFβ type I receptor (TβRI) was added to the CM, the migration of MSCs was almost abolished, indicating that active TGFβ1 released by the injured arteries mediates MSCs migration. To examine the role of TGFβ in MSCs recruitment following vascular injury in vivo , TβRI inhibitor was injected into mice with injury of femoral arteries. Both the development of neointima and the recruitment of nestin + cells were blocked with the inhibitor treatment compare to those with vehicle treatment. Collectively, the results suggest that TGFβ is an injury-activated messenger essential for the recruitment of MSCs to participate in vascular remodeling.

Develoapmet of a Tissue Analog for Cartilage Repair

1991 ◽  
Vol 252 ◽  
Author(s):  
J. M. Pachence ◽  
S. R. Frenkel ◽  
H. Lin
Keyword(s):  
Type I Collagen ◽  
Collagen Matrix ◽  
In Vitro Studies ◽  
In Vivo Studies ◽  
Type I ◽  
Collagen Matrices ◽  
Pore Sizes ◽  
The Matrix

ABSTRACTPurified type I collagen was formed into matrices whose pore sizes were defined on the basis of previous results. The first series of in vitro studies measured the metabolism of chondrocytes grown in matrices with various pore sizes; results revealed that the growth rate was independent of the average matrix pore size, but that ckmdrocyte infiltration throughout the matrix was optimal for pore sizes of 100 to 150 un. In a second series of studies, type I collagen was combined with hyaluranic acid; the HyA/collagen matrices had little effect on chcrdrocyte cell growth versus the collagen matrices. A third set of in vitro studies used collagen matrices incorporating varying cornentrations of insulin-like growth factor. It was found that the IGF-1/collagen matrices can significantly effect the growth and metabolism of the clxrihrocytes. These experiments were vital in establishing the collagen matrix parameters which will be used in subsequent in vivo studies.

scholarly journals Characterization of Intrinsically Radiolabeled Poly(lactic-co-glycolic acid) Nanoparticles for ex Vivo Autologous Cell Labeling and in Vivo Tracking

2021 ◽  
Author(s):  
Massis Krekorian ◽  
Gerwin G. W. Sandker ◽  
Kimberley R. G. Cortenbach ◽  
Oya Tagit ◽  
N. Koen van Riessen ◽  
...  
Keyword(s):  
Glycolic Acid ◽  
Ex Vivo ◽  
Cell Labeling ◽  
Autologous Cell

Abstract 18845: Induced Cardiac Progenitor Cells Differentiate Into Cardiac Lineage Cells in vivo and Improve Survival in Mice post Myocardial Infarction

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Pratik A Lalit ◽  
Max R Salick ◽  
Daryl O Nelson ◽  
Jayne M Squirrell ◽  
Christina M Shafer ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Disease Modeling ◽  
Ex Vivo ◽  
Cardiac Progenitor Cells ◽  
Heart Tube ◽  
Whole Embryo Culture ◽  
Cardiac Actin ◽  
Cardiac Lineage

Several studies have reported reprogramming of fibroblasts (Fibs) to induced cardiomyocytes, and we have recently reprogrammed mouse Fibs to induced cardiac progenitor cells (iCPCs), which may be more favorable for cardiac repair because of their expandability and multipotency. Adult cardiac (AC), lung and tail-tip Fibs from an Nkx2.5-EYFP reporter mouse were reprogrammed using a combination of five defined factors into iCPCs. Transcriptome and immunocytochemistry analysis revealed that iCPCs were cardiac mesoderm-restricted progenitors that expressed CPC markers including Nkx2.5, Gata4, Irx4, Tbx5, Cxcr4, Flk1 etc. iCPCs could be extensively expanded (over 30 passages) while maintaining multipotency to differentiate in vitro into cardiac lineage cells including cardiomyocytes (CMs), smooth muscle cells and endothelial cells. iCPC derived CMs upon co-culture with mESC-derived CMs formed intercellular gap junctions, exhibited calcium transients, and contractions. The purpose of this study was to determine the in vivo potency of iCPCs. Given that the Nkx2.5-EYFP reporter identifies embryonic CPCs, we first tested the embryonic potency of iCPCs using an ex vivo whole embryo culture model injecting cells into the cardiac crescent (CC) of E8.5 mouse embryos and culturing for 24 to 48 hours. GFP labeled AC Fibs were first tested and live imaging revealed that after 24 hours these cells were rejected from the embryo proper and localized to the ecto-placental cone. In contrast, iCPCs reprogrammed from AC Fibs when injected into the CC localized to the developing heart tube and differentiated into MLC2v, αMHC and cardiac actin expressing CMs. Further we injected iCPCs into infarcted adult mouse hearts and determined their regenerative potential after 1-4 wks. The iCPCs significantly improved survival (p<0.01 Mantel-Cox test) in treated animals (75%) as compared to control (11%). Immunohistochemistry revealed that injected iCPCs localized to the scar area and differentiated into cardiac lineage cells including CMs (cardiac actin). These results indicate that lineage reprogramming of adult somatic cells into iCPCs provides a scalable cell source for cardiac regenerative therapy as well as drug discovery and disease modeling.

Cells in regenerating deer antler cartilage provide a microenvironment that supports osteoclast differentiation

2001 ◽  
Vol 204 (3) ◽  
pp. 443-455
Author(s):  
C. Faucheux ◽  
S. Nesbitt ◽  
M. Horton ◽  
J. Price
Keyword(s):  
Type I Collagen ◽  
Mononuclear Cells ◽  
Osteoclast Differentiation ◽  
Cathepsin K ◽  
Collagen Matrix ◽  
Tartrate Resistant Acid Phosphatase ◽  
Type I ◽  
Deer Antler

Deer antlers are a rare example of mammalian epimorphic regeneration. Each year, the antlers re-grow by a modified endochondral ossification process that involves extensive remodelling of cartilage by osteoclasts. This study identified regenerating antler cartilage as a site of osteoclastogenesis in vivo. An in vitro model was then developed to study antler osteoclast differentiation. Cultured as a high-density micromass, cells from non-mineralised cartilage supported the differentiation of large numbers of osteoclast-like multinucleated cells (MNCs) in the absence of factors normally required for osteoclastogenesis. After 48 h of culture, tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells (osteoclast precursors) were visible, and by day 14 a large number of TRAP-positive MNCs had formed (783+/−200 per well, mean +/− s.e.m., N=4). Reverse transcriptase/polymerase chain reaction (RT-PCR) showed that receptor activator of NF κ B ligand (RANKL) and macrophage colony stimulating factor (M-CSF) mRNAs were expressed in micromass cultures. Antler MNCs have the phenotype of osteoclasts from mammalian bone; they expressed TRAP, vitronectin and calcitonin receptors and, when cultured on dentine, formed F-actin rings and large resorption pits. When cultured on glass, antler MNCs appeared to digest the matrix of the micromass and endocytose type I collagen. Matrix metalloproteinase-9 (MMP-9) may play a role in the resorption of this non-mineralised matrix since it is highly expressed in 100 % of MNCs. In contrast, cathepsin K, another enzyme expressed in osteoclasts from bone, is only highly expressed in resorbing MNCs cultured on dentine. This study identifies the deer antler as a valuable model that can be used to study the differentiation and function of osteoclasts in adult regenerating mineralised tissues.

A novel mouse model based on intersectional genetics enables unambiguous in vivo discrimination between plasmacytoid and other dendritic cells and their comparative characterization

2022 ◽  
Author(s):  
Michael Valente ◽  
Nils Collinet ◽  
Thien-Phong Vu Manh ◽  
Karima Naciri ◽  
Gilles Bessou ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Steady State ◽  
Mouse Model ◽  
Single Cell ◽  
Ex Vivo ◽  
High Specificity ◽  
Type I ◽  
Specific Expression ◽  
Physiological Functions

Plasmacytoid dendritic cells (pDC) were identified about 20 years ago, based on their unique ability to rapidly produce copious amounts of all subsets of type I and type III interferon (IFN-I/III) upon virus sensing, while being refractory to infection. Yet, the identity and physiological functions of pDC are still a matter of debate, in a large part due to their lack of specific expression of any single cell surface marker or gene that would allow to track them in tissues and to target them in vivo with high specificity and penetrance. Indeed, recent studies showed that previous methods that were used to identify or deplete pDC also targeted other cell types, including pDC-like cells and transitional DC (tDC) that were proposed to be responsible for all the antigen presentation ability previously attributed to steady state pDC. Hence, improving our understanding of the nature and in vivo choreography of pDC physiological functions requires the development of novel tools to unambiguously identify and track these cells, including in comparison to pDC-like cells and tDC. Here, we report successful generation of a pDC-reporter mouse model, by using an intersectional genetic strategy based on the unique co-expression of Siglech and Pacsin1 in pDC. This pDC-Tomato mouse strain allows specific ex vivo and in situ detection of pDC. Breeding them with Zbtb46GFP mice allowed side-by-side purification and transcriptional profiling by single cell RNA sequencing of bona fide pDC, pDC-like cells and tDC, in comparison to type 1 and 2 conventional DC (cDC1 and cDC2), both at steady state and during a viral infection, revealing diverging activation patterns of pDC-like cells and tDC. Finally, by breeding pDC-Tomato mice with Ifnb1EYFP mice, we determined the choreography of pDC recruitment to the micro-anatomical sites of viral replication in the spleen, with initially similar but later divergent behaviors of the pDC that engaged or not into IFN-I production. Our novel pDC-Tomato mouse model, and newly identified gene modules specific to combinations of DC types and activations states, will constitute valuable resources for a deeper understanding of the functional division of labor between DC types and its molecular regulation at homeostasis and during viral infections.

scholarly journals Oncofetal Protein CRIPTO Is Involved in Wound Healing and Fibrogenesis in the Regenerating Liver and Is Associated with the Initial Stages of Cardiac Fibrosis

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3325
Author(s):  
Sofia Karkampouna ◽  
Danny van der Helm ◽  
Mario Scarpa ◽  
Bart van Hoek ◽  
Hein W. Verspaget ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Mouse Models ◽  
Cardiac Fibrosis ◽  
Ex Vivo ◽  
Pressure Overload ◽  
Collagen Type I ◽  
Type I ◽  
Liver Fibrogenesis ◽  
End Stage

Oncofetal protein, CRIPTO, is silenced during homeostatic postnatal life and often re-expressed in different neoplastic processes, such as hepatocellular carcinoma. Given the reactivation of CRIPTO in pathological conditions reported in various adult tissues, the aim of this study was to explore whether CRIPTO is expressed during liver fibrogenesis and whether this is related to the disease severity and pathogenesis of fibrogenesis. Furthermore, we aimed to identify the impact of CRIPTO expression on fibrogenesis in organs with high versus low regenerative capacity, represented by murine liver fibrogenesis and adult murine heart fibrogenesis. Circulating CRIPTO levels were measured in plasma samples of patients with cirrhosis registered at the waitlist for liver transplantation (LT) and 1 year after LT. The expression of CRIPTO and fibrotic markers (αSMA, collagen type I) was determined in human liver tissues of patients with cirrhosis (on a basis of viral hepatitis or alcoholic disease), in cardiac tissue samples of patients with end-stage heart failure, and in mice with experimental liver and heart fibrosis using immuno-histochemical stainings and qPCR. Mouse models with experimental chronic liver fibrosis, induced with multiple shots of carbon tetrachloride (CCl4) and acute liver fibrosis (one shot of CCl4), were evaluated for CRIPTO expression and fibrotic markers. CRIPTO was overexpressed in vivo (Adenoviral delivery) or functionally sequestered by ALK4Fc ligand trap in the acute liver fibrosis mouse model. Murine heart tissues were evaluated for CRIPTO and fibrotic markers in three models of heart injury following myocardial infarction, pressure overload, and ex vivo induced fibrosis. Patients with end-stage liver cirrhosis showed elevated CRIPTO levels in plasma, which decreased 1 year after LT. Cripto expression was observed in fibrotic tissues of patients with end-stage liver cirrhosis and in patients with heart failure. The expression of CRIPTO in the liver was found specifically in the hepatocytes and was positively correlated with the Model for End-stage Liver Disease (MELD) score for end-stage liver disease. CRIPTO expression in the samples of cardiac fibrosis was limited and mostly observed in the interstitial cells. In the chronic and acute mouse models of liver fibrosis, CRIPTO-positive cells were observed in damaged liver areas around the central vein, which preceded the expression of αSMA-positive stellate cells, i.e., mediators of fibrosis. In the chronic mouse models, the fibrosis and CRIPTO expression were still present after 11 weeks, whereas in the acute model the liver regenerated and the fibrosis and CRIPTO expression resolved. In vivo overexpression of CRIPTO in this model led to an increase in fibrotic markers, while blockage of CRIPTO secreted function inhibited the extent of fibrotic areas and marker expression (αSMA, Collagen type I and III) and induced higher proliferation of residual healthy hepatocytes. CRIPTO expression was also upregulated in several mouse models of cardiac fibrosis. During myocardial infarction CRIPTO is upregulated initially in cardiac interstitial cells, followed by expression in αSMA-positive myofibroblasts throughout the infarct area. After the scar formation, CRIPTO expression decreased concomitantly with the αSMA expression. Temporal expression of CRIPTO in αSMA-positive myofibroblasts was also observed surrounding the coronary arteries in the pressure overload model of cardiac fibrosis. Furthermore, CRIPTO expression was upregulated in interstitial myofibroblasts in hearts cultured in an ex vivo model for cardiac fibrosis. Our results are indicative for a functional role of CRIPTO in the induction of fibrogenesis as well as a potential target in the antifibrotic treatments and stimulation of tissue regeneration.

scholarly journals Small Molecule Screening Identifies Rho-Associate Protein Kinase (ROCK) As a Regulator of NK Cell Cytotoxicity Against Cancer

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3607-3607
Author(s):  
Grace Lee ◽  
Sheela Karunanithi ◽  
Zachary Jackson ◽  
David Wald
Keyword(s):  
Cell Therapy ◽  
Cytotoxic Activity ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Akt Activation ◽  
Nk Cell Cytotoxicity ◽  
Rock Inhibition ◽  
Nk Cell Therapy

NK cells are a subset of lymphocytes that directly recognize and lyse tumor cells without the limitation of antigen specific receptor recognition. In addition to behaving as cytotoxic effector cells, NK cells unlike T cells are not thought to elicit graft versus host disease. The combination of these characteristics makes NK cells a powerful tool for adoptive cell therapy. Despite the promise of NK cell therapy, key hurdles in achieving significant clinical efficacy include both generating sufficient numbers of highly tumoricidal NK cells and maintaining the cytotoxic activity of these cells in vivo despite the immunosuppressive tumor microenvironment. Our lab and others have developed several feeder cell line-based expansion modules that robustly stimulate the ex vivo proliferation of NK cells. However, strategies to enhance and sustain the activity of NK cells once administered in vivo are still limited. In order to identify strategies to enhance the cytotoxic activity of NK cells, we developed a high-throughput small molecule screen (Figure 1A) that involved a calcein-based cytotoxicity assay of ex vivo expanded and treated NK cells against ovarian cancer cells (OVCAR-3). 20,000 compounds were screened and the screen was found to be highly robust (Z'&gt;0.59). We identified 29 hits that led to at least a 25% increase in cytotoxicity as compared to DMSO control-treated NK cells. One of the most promising hits was the pan-ROCK inhibitor, Y-27632 that led to an 30% increase in NK killing of the OVCAR-3 cells. We validated that ROCK inhibition leads to enhanced NK cell cytotoxic activity using Y-27632 (Figure 1B) as well as other well-established ROCK inhibitors such as Fasudil using a flow cytometry based killing assay. Y-27632 increased NK cell cytotoxicity in a dose- and time- dependent manner. ROCK inhibition consistently led to ~10-25% increase in NK cell cytotoxic activity directed against a variety of ovarian (Figure 1C) and other solid tumor cell lines (Figure 1D). Interestingly, we found that the NK hyperactivation persists for up to 48hrs after washing off the drug that may enable ex vivo stimulation before NK cell infusion. Our preliminary results showed that ROCK inhibition activates PI3K-dependent Akt activation (Figure 1E). We hypothesize that ROCK inhibition restores Akt activation which may be critical for NK cell activating receptor pathways and our current investigations will test these hypotheses. ROCK inhibitors, such as Y-27632 and Fasudil have been utilized in both preclinical and clinical studies for a variety of diseases such as atherosclerosis, neurodegenerative disorders, and ocular diseases. However, the consequences of ROCK inhibition in NK cells has not been thoroughly investigated. Our work shows a promising novel strategy to significantly enhance NK cell therapy against cancer that has high translational potential. Disclosures No relevant conflicts of interest to declare.

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