Graft versus host disease (GVHD), mediated by donor T cells, is a significant source of morbidity and mortality following allogeneic stem cell transplantation. Mesenchymal stem cells (MSC) can successfully treat ongoing graft versus host disease, presumably due to their ability to suppress donor T cell proliferation. Little is known about the potential of MSC to prevent GVHD. Here we show that bone marrow-isolated MSC can suppress the development of GVHD if given after donor T cell recognition of antigen. IFN-c was required to initiate MSC efficacy. Recipients of IFN-c -/-T cells did not respond to MSC treatment and succumbed to GVHD. MSC, pre-treated with IFN-c, became immediately active and could suppress GVHD more efficiently than a fivefold-greater number of MSC that were not activated. When given at the time of bone marrow transplantation, activated MSC could prevent GVHD mortality (100% survival, p=0.006). MSC activation was dependent on the magnitude of IFN-c exposure, with increased IFN-c exposure leading to increased MSC suppression of GVHD. Activated MSC present a new strategy for preventing GVHD using fewer MSC. Key words: Mesenchymal stem cell Á GVH disease Á IFN-c See accompanying commentary by Dazzi and Marelli-Berg IntroductionAllogeneic hematopoietic stem cell transplants have the potential to play a significant curative role in the treatment of malignant and non-malignant hematopoietic disorders, autoimmune diseases, and immunological deficiencies, and in the induction of transplantation tolerance [1][2][3][4][5][6][7][8][9][10]. Widespread application of this therapeutic modality is limited due to the morbidity and mortality of graft versus host disease (GVHD), which affects 50% of stem cell transplant recipients [11][12][13][14][15][16]. While grafts highly matched to the recipient, young donors, donor/recipient sex match, and posttransplant immunosuppression are strategies used to reduce the risk of GVHD [17], thus far, the greatest preventative measure has been intentional underutilization of stem cell transplantation. Theoretically, strategies aimed at preventing GVHD would target early initiating factors either during the inflammatory milieu created in the wake of tissue damage from conditioning regimens [18,19] or during T cell antigen recognition and proliferation [20,21]. Once the efferent effector phase occurs, donor T cell-mediated destruction of host tissues occurs and preventive strategies are replaced with treatment regimens [19].Mesenchymal stem cells (MSC) have been used in the efferent phase of GVHD to successfully treat ongoing, acute, steroidresistant GVHD [22,23]. In contrast, when given at the time of BM transplant, for the prevention of GVHD, the incidence of grade III/ IV GVHD was not significantly improved [24], suggesting the [26,27,29,30]. In addition, MSC do not suppress the modest T cell proliferative response to recall antigens [31]. These findings suggest MSC may exert their optimal effects during the events surrounding larger scale T cell activation and proliferat...

There is an accumulation of evidence in the literature demonstrating the integral role of vimentin intermediate filaments (IFs) in the progression of lung cancers. Vimentin IF proteins have been implicated in many aspects of cancer initiation and progression, including tumorigenesis, epithelial-to-mesenchymal transition (EMT), and the metastatic spread of cancer. Specifically, vimentin IFs have been recognized as an essential component regulating EMT, major signal transduction pathways involved in EMT and tumor progression, cell migration and invasion, the positioning and anchorage of organelles, such as mitochondria, and cell-cell and cell-substrate adhesion. In tumorgenesis, vimentin forms a complex with 14-3-3 and beclin 1 to inhibit autophagy via an AKT-dependent mechanism. Vimentin is a canonical marker of EMT, and recent evidence has shown it to be an important regulator of cellular motility. Transcriptional regulation of vimentin through hypoxia-inducible factor-1 may be a potential driver of EMT. Finally, vimentin regulates 14-3-3 complexes and controls various intracellular signaling and cell cycle control pathways by depleting the availability of free 14-3-3. There are many exciting advances in our understanding of the complex role of vimentin IFs in cancer, pointing to the key role vimentin IFs may play in tumor progression.

Gene delivery from hydrogels represents a versatile approach for localized expression of tissue inductive factors than can promote cellular processes that lead to regeneration. Lentiviral gene therapy vectors were entrapped within fibrin hydrogels, either alone or complexes with hydroxylapatite (HA) nanoparticles. The inclusion of HA into the hydrogel led to the formation of small aggregates distributed throughout the hydrogel, with no obvious alteration of the pore structure outside the aggregates. The presence of HA slowed hydrogel degradation by collagenase and plasmin relative to fibrin alone, and also decreased the rate of cell migration. Lentivirus had similar release from the fibrin hydrogels formed with or without HA. The altered hydrogel properties suggest an interaction between the nanoparticle and fibrin, which may displace the virus from the particle leading to similar release profiles. Transgene expression by cells migrating into the hydrogel in vitro was reduced in the presence of HA, consistent with the role of cell migration on transgene expression. In vivo, lentivirus loaded fibrin hydrogels promoted localized transgene expression that increased through day 9 and decreased through day 14. For the fibrin only hydrogels, expression continued to decline after day 14. However, hydrogels with HA maintained this transgene expression level for an additional two weeks before declining. Immunostaining identified transgene primarily outside the fibrin-HA gel at day 9; however, at day 21, transgene expression was observed primarily within the fibrin-HA gel. The localized delivery of lentivirus provides an opportunity to enhance the bioactivity of fibrin hydrogels for a wide range of applications in regenerative medicine.

1Vimentin, a type III intermediate filament, is highly expressed in aggressive epithelial 2 cancers and is associated with increased rates of metastasis. We show that vimentin is causally 3 required for lung cancer metastasis using a genetic mouse model of lung adenocarcinoma (LSL-4 Kras G12D ;Tp53 fl/fl , termed KPV +/+ ) crossed with vimentin-null mice (thereby creating KPV −/− mice). 5Both KPV +/+ and KPV −/− mice developed lung tumors, yet KPV −/− mice had delayed tumorigenesis 6 and prolonged survival. KPV +/+ cells implanted in the flank metastasized to the lung while KPV −/− 7 cells did not, providing additional evidence that vimentin is required for metastasis. Differential 8 expression analysis of RNA-seq data demonstrated that KPV −/− cells had suppressed expression 9 of genes that drive epithelial-to-mesenchymal transition, migration, and invasion, processes that 10 are critical to the metastatic cascade. Integrative metabolomic and transcriptomic analysis 11 revealed altered glutaminolysis, with KPV −/− cells accumulating glutathione, leading to impaired 12 cell motility in response to oxidative stress. Together, these results show that loss of vimentin 13 impairs epithelial-to-mesenchymal transition and regulation of the oxidative stress response, 14 resulting in decreased metastasis in murine lung adenocarcinoma. 15 16 17 18Non-small-cell lung cancers (NSCLCs) represent 80% of all lung cancers and are often 20 diagnosed at more advanced stages of the disease resulting in high rates of mortality (1). 21Adenocarcinoma is the most common subtype of NSCLC and is characterized by activating 22 mutations in the Kras proto-oncogene in up to 30% of diagnoses and by inactivating mutations in 23 the tumor suppressor gene Tp53 in up to 60% of diagnoses (2)(3)(4)(5). Despite the prevalence of lung 24 adenocarcinoma, the metastatic mechanisms that drive lung cancer progression are incompletely 25 understood. 26The type III intermediate filament vimentin is associated with increased metastatic spread 27 and lower rates of survival in patients with NSCLC (6-8). Vimentin is a canonical marker of 28 epithelial-to-mesenchymal transition (EMT), an initiating event of the metastatic cascade (9). EMT 29 is the process by which epithelial cells remodel cell-cell and cell-extracellular-matrix (ECM) 30 contacts, lose their apical-basal polarity, and adopt the spindle-shaped morphology associated 31 with a mesenchymal cell phenotype (10). During EMT, cells undergo a downregulation of 32 epithelial cell associated genes, including E-cadherin and cytokeratins, and an upregulation of 33 mesenchymal cell associated genes, including N-cadherin and vimentin. In addition to acting as 34 a marker of EMT, vimentin is functionally involved in EMT. Structurally, vimentin intermediate 35 filaments control cell shape, and thus facilitate the transition toward a mesenchymal phenotype 36(11). Twist1, a transcription factor critical to EMT, upregulates vimentin expression (12). Vimentin 37 also serves as a scaffold for Slug, another transc...

Vimentin is highly expressed in metastatic cancers, and its expression correlates with poor patient prognoses. However, no causal in vivo studies linking vimentin and non–small cell lung cancer (NSCLC) progression existed until now. We use three complementary in vivo models to show that vimentin is required for the progression of NSCLC. First, we crossed LSL-KrasG12D; Tp53fl/fl mice (KPV+/+) with vimentin knockout mice (KPV−/−) to demonstrate that KPV−/− mice have attenuated tumor growth and improved survival compared with KPV+/+ mice. Next, we therapeutically treated KPV+/+ mice with withaferin A (WFA), an agent that disrupts vimentin intermediate filaments (IFs). We show that WFA suppresses tumor growth and reduces tumor burden in the lung. Finally, luciferase-expressing KPV+/+, KPV−/−, or KPVY117L cells were implanted into the flanks of athymic mice to track cancer metastasis to the lung. In KPVY117L cells, vimentin forms oligomers called unit-length filaments but cannot assemble into mature vimentin IFs. KPV–/– and KPVY117L cells fail to metastasize, suggesting that cell-autonomous metastasis requires mature vimentin IFs. Integrative metabolomic and transcriptomic analysis reveals that KPV–/– cells upregulate genes associated with ferroptosis, an iron-dependent form of regulated cell death. KPV–/– cells have reduced glutathione peroxidase 4 (GPX4) levels, resulting in the accumulation of toxic lipid peroxides and increased ferroptosis. Together, our results demonstrate that vimentin is required for rapid tumor growth, metastasis, and protection from ferroptosis in NSCLC.