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Supplementary Figure from B7-H3 Specific CAR T Cells for the Naturally Occurring, Spontaneous Canine Sarcoma Model

One obstacle for human solid tumor immunotherapy research is the lack of clinically relevant animal models. In this study, we sought to establish a chimeric antigen receptor (CAR) T-cell treatment model for naturally occurring canine sarcomas as a model for human CAR T-cell therapy.Canine CARs specific for B7-H3 were constructed using a single-chain variable fragment derived from the human B7-H3–specific antibody MGA271, which we confirmed to be cross-reactive with canine B7-H3. After refining activation, transduction, and expansion methods, we confirmed target killing in a tumor spheroid three-dimensional assay. We designed a B7-H3 canine CAR T-cell and achieved consistently high levels of transduction efficacy, expansion, and in vitro tumor killing. Safety of the CAR T cells were confirmed in two purposely bred healthy canine subjects following lymphodepletion by cyclophosphamide and fludarabine. Immune response, clinical parameters, and manifestation were closely monitored after treatments and were shown to resemble that of humans. No severe adverse events were observed.In summary, we demonstrated that similar to human cancers, B7-H3 can serve as a target for canine solid tumors. We successfully generated highly functional canine B7-H3–specific CAR T-cell products using a production protocol that closely models human CAR T-cell production procedure. The treatment regimen that we designed was confirmed to be safe in vivo. Our research provides a promising direction to establish in vitro and in vivo models for immunotherapy for canine and human solid tumor treatment.

One obstacle for human solid tumor immunotherapy research is the lack of clinically relevant animal models. In this study, we sought to establish a chimeric antigen receptor (CAR) T-cell treatment model for naturally occurring canine sarcomas as a model for human CAR T-cell therapy. Canine CARs specific for B7-H3 were constructed using a single-chain variable fragment derived from the human B7-H3–specific antibody MGA271, which we confirmed to be cross-reactive with canine B7-H3. After refining activation, transduction, and expansion methods, we confirmed target killing in a tumor spheroid three-dimensional assay. We designed a B7-H3 canine CAR T-cell and achieved consistently high levels of transduction efficacy, expansion, and in vitro tumor killing. Safety of the CAR T cells were confirmed in two purposely bred healthy canine subjects following lymphodepletion by cyclophosphamide and fludarabine. Immune response, clinical parameters, and manifestation were closely monitored after treatments and were shown to resemble that of humans. No severe adverse events were observed. In summary, we demonstrated that similar to human cancers, B7-H3 can serve as a target for canine solid tumors. We successfully generated highly functional canine B7-H3–specific CAR T-cell products using a production protocol that closely models human CAR T-cell production procedure. The treatment regimen that we designed was confirmed to be safe in vivo. Our research provides a promising direction to establish in vitro and in vivo models for immunotherapy for canine and human solid tumor treatment.

Additively manufactured materials are gaining wide attention owing to the manufacturing benefits as it results in near net shape components. It is well known that the manufacturing processes affects the performance of the components via microstructural features and the mechanical properties. There is an urgent need to understand the processing-structure-property-performance relationship for the materials manufactures via such innovative techniques. Strategies are needed to quantify and modify the mechanical properties. This study assists to design and tailor the process parameters based on the final properties required. Current work predicts the yield strength of additively manufactured Ti-6Al-4V with different post heat treatments. A thermal model predicted by ABAQUS is fed into an implementation of Langmuir equation that predicts the chemistry which is then used in a phenomenological equation predicting the yield strength. The model is confirmed via experiments showing less than 2% deviation from the predicated properties. A statistical model gives design allowables that have an uncertainty of less than 1 ksi.

Advanced manufacturing approaches, including additive manufacturing (i.e., “3D printing”) of metallic structures requires a change to qualification strategies. One approach, informed qualification, integrates modeling strategies to make predictions of material characteristics, including the prediction of tensile properties for given chemistries and microstructures. In this work, constitutive equations are developed and presented that can predict the yield strength of additively manufactured Ti-6Al-4V subjected to one of three different heat-treatments: a stress relief anneal in the α+β phase field; a hot isostatic press treatment in the α+β phase field; and a β-anneal. The equations are nominally identical, though different strengthening mechanisms are active according to subtle microstructural differences. To achieve an equation that can predict the yield strength of the material, it is also necessary to include an assessment of dramatic reduction in the tensile strength due to texture (i.e., a “knock-down” effect). This has been experimentally measured, and included in this paper. The resulting predictions of yield strength are generally within 5% of their experimentally measured values.

A constituent-based phenomenological equation to predict yield strength values from quantified measurements of the microstructure and composition of β processed Ti-6Al-4V alloy was developed via the integration of artificial neural networks and genetic algorithms. It is shown that the solid solution strengthening contributes the most to the yield strength (~80% of the value), while the intrinsic yield strength of the two phases and microstructure have lower effects (~10% for both terms). Similarities and differences between the proposed equation and the previously established phenomenological equation for the yield strength prediction of the α+β processed Ti-6Al-4V alloys are discussed. While the two equations are very similar in terms of the intrinsic yield strength of the two constituent phases, the solid solution strengthening terms and the ‘Hall-Petch’-like effect from the alpha lath, there is a pronounced difference in the role of the basketweave factor in strengthening. Finally, Monte Carlo simulations were applied to the proposed phenomenological equation to determine the effect of measurement uncertainties on the estimated yield strength values.

Chronic liver injury leads to fibrosis and cirrhosis. Cirrhosis, the end stage of chronic liver disease, is a leading cause of death worldwide and increases the risk of developing hepatocellular carcinoma. Currently, there is a lack of effective antifibrotic therapies to treat fibrosis and cirrhosis. Development of antifibrotic therapies requires an in-depth understanding of the cellular and molecular mechanisms involved in inflammation and fibrosis after hepatic injury. Two growth factor signaling pathways that regulate liver fibrosis are transforming growth factor-β (TGFβ) and platelet-derived growth factor (PDGF). However, their specific contributions to fibrogenesis are not well understood. Using a genetic model of liver fibrosis, we investigated whether the canonical TGFβ signaling pathway was necessary for fibrogenesis. PDGF-C transgenic ( PDGF-C Tg) mice were intercrossed with mice that lack Smad3, and molecular and histological fibrosis was analyzed. PDGF-C Tg mice that also lacked Smad3 had less fibrosis and improved liver lobule architecture. Loss of Smad3 also reduced expression of collagen genes, which were induced by PDGF-C, but not the expression of genes frequently associated with hepatic stellate cell (HSC) activation. In vitro HSCs isolated from Smad3-null mice proliferated more slowly than cells from wild-type mice. Taken together, these findings indicate that PDGF-C activates TGFβ/Smad3 signaling pathways to regulate HSC proliferation, collagen production and ultimately fibrosis. In summary, these results suggest that inhibition of both PDGF and TGFβ signaling pathways may be required to effectively attenuate fibrogenesis in patients with chronic liver disease.

During World War II, 2710 Liberty ships were built in the United States across 18 ship yards. The rate of production of these ships was at a scale not previously witnessed, reflecting a strategic marshaling of national assets critical to the war effort. For the metallurgist, metallurgical engineer, or materials scientist, these ships also struck commanding images regarding their catastrophic failures. The study of these failures led to increased understanding of brittle fracture, fracture mechanics, and ductile-to-brittle transition temperatures. The post-mortem studies of Liberty ships highlighted the importance of composition and microstructure in controlling the properties of steel in fracture-critical applications. This study examines a rivet from the SS “John W. Brown”, which was assembled in Baltimore, Maryland, and launched in September 1942, The “John W. Brown” was restored between 1988 and 1991. Classical metallurgical analysis of a rivet from the original 1942 vessel is compared with modern rivets used during its restoration. The rivets provide an analogue to the plate material used in these ships. A comparison of these materials is presented along with a discussion of the importance of composition–microstructure–property relationships that concomitantly evolved.

Electron beam direct manufacturing, synonymously known as electron beam additive manufacturing, along with other additive “3-D printing” manufacturing processes, are receiving widespread attention as a means of producing net-shape (or near-net-shape) components, owing to potential manufacturing benefits. Yet, materials scientists know that differences in manufacturing processes often significantly influence the microstructure of even widely accepted materials and, thus, impact the properties and performance of a material in service. It is important to accelerate the understanding of the processing–structure–property relationship of materials being produced via these novel approaches in a framework that considers the performance in a statistically rigorous way. This article describes the development of a process model, the assessment of key microstructural features to be incorporated into a microstructure simulation model, a novel approach to extract a constitutive equation to predict tensile properties in Ti-6Al-4V (Ti-64), and a probabilistic approach to measure the fidelity of the property model against real data. This integrated approach will provide designers a tool to vary process parameters and understand the influence on performance, enabling design and optimization for these highly visible manufacturing approaches.

Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet-derived growth factor C (PDGF-C) induces progressive fibrosis, chronic inflammation, neoangiogenesis and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF-CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast-like cells that deposit extracellular matrix and lead to production of growth factors and cytokines. We demonstrate induction of cytokine genes at 2 months, and stromal cell-derived hepatocyte growth factors that coincide with the onset of dysplasia at 4 months. Our results support a paracrine signaling model wherein hepatocyte-derived PDGF-C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF-CC levels were observed in livers of patients with nonalcoholic fatty steatohepatitis and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF-C transgenic mice provide a unique model for the in vivo study of tumor–stromal interactions in the liver.

Liver fibrosis is mediated by hepatic stellate cells (HSCs), which respond to a variety of cytokine and growth factors to moderate the response to injury and create extracellular matrix at the site of injury. G-protein coupled receptor (GPCR)-mediated signaling, via endothelin-1 (ET-1) and angiotensin II (AngII), increases HSC contraction, migration and fibrogenesis. Regulator of G-protein signaling-5 (RGS5), an inhibitor of vasoactive GPCR agonists, functions to control GPCR-mediated contraction and hypertrophy in pericytes and smooth muscle cells (SMCs). Therefore we hypothesized that RGS5 controls GPCR signaling in activated HSCs in the context of liver injury. In this study, we localize RGS5 to the HSCs and demonstrate that Rgs5 expression is regulated during carbon tetrachloride (CCl4)-induced acute and chronic liver injury in Rgs5LacZ/LacZ reporter mice. Furthermore, CCl4 treated RGS5-null mice develop increased hepatocyte damage and fibrosis in response to CCl4 and have increased expression of markers of HSC activation. Knockdown of Rgs5 enhances ET-1-mediated signaling in HSCs in vitro. Taken together, we demonstrate that RGS5 is a critical regulator of GPCR signaling in HSCs and regulates HSC activation and fibrogenesis in liver injury.

Platelet-derived growth factors (PDGFs) are critical for development; their over-expression is associated with fibrogenesis. Full-length PDGF-C is secreted as an inactive dimer, requiring cleavage to allow receptor binding. Previous studies indicate that tissue-type plasminogen activator (tPA) is the specific protease that performs this cleavage; in vivo confirmation is lacking. We demonstrate that primary hepatocytes from tpa KO mice produce less cleaved active PDGF-CC than do wild type hepatocytes, suggesting that tPA is critical for in vitro activation of this growth factor. We developed mice that over-express full-length human PDGF-C in the liver; these mice develop progressive liver fibrosis. To test whether tPA is important for cleavage and activation of PDGF-C in vivo, we intercrossed PDGF-C transgenic (Tg) and tpa knock-out (KO) mice, anticipating that lack of tPA would result in decreased fibrosis due to lack of hPDGF-C cleavage. To measure levels of cleaved, dimerized PDGF-CC in sera, we developed an ELISA that specifically detects cleaved PDGF-CC. We report that the absence of tpa does not affect the phenotype of `PDGF-C Tg mice. PDGF-C Tg mice lacking tPA have high serum levels of cleaved growth factor, significant liver fibrosis, and gene expression alterations similar to those of PDGF-C Tg mice with intact tPA. Furthermore, urokinase plasminogen activator and plasminogen activator inhibitor-1 expression are increased in PDGF-C Tg; tpa KO mice. Our ELISA data suggest a difference between in vitro and in vivo activation of this growth factor, and our mouse model confirms that multiple proteases cleave and activate PDGF-C in vivo.

Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet-derived growth factor-c (PDGF-C) induces progressive fibrosis, chronic inflammation, neoangiogenesis, and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF-CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast-like cells that deposit extracellular matrix (ECM) and lead to production of growth factors and cytokines. We demonstrate induction of cytokines genes at two months, and stromal cell-derived hepatocyte growth factors that coincide with the onset of dysplasia at four months. Our results support a paracrine signaling model wherein hepatocyte-derived PDGF-C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury, and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF-CC levels were observed in livers of patients with non-alcoholic fatty steatohepatitis (NASH) and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF-C transgenic mice provide a unique model for the in vivo study of tumor-stromal interactions in the liver.

There is increasing evidence that p38 MAPK, which is classified as a stress-activated kinase, also participates in cell cycle regulation, functioning as a suppressor of cell proliferation and tumorigenesis. We conducted a study of p38 MAPK phosphorylation during liver regeneration in mice to determine whether p38 MAPK activation or inactivation may correlate with events that lead to DNA replication after partial hepatectomy (PH), and whether p38 MAPK activation may be required for hepatocyte DNA replication in vivo and in culture. We report that active p38 (Pi-p38 MAPK) is present in normal liver, is rapidly inactivated starting 30 min after PH, and is re-activated by 12h. Although levels of Pi-MKK 3/6, the upstream kinases that activate p38 MAPK increase after PH, the expression of the dual protein phosphatase 1 is also elevated, and may be responsible for Pi-p38 MAPK dephosphorylation after PH. Inactivation and re-activation of p38 MAPK inversely correlates with the stimulation of protein synthesis and translation pathways, as indicated by activation of p70S6 kinase, increases in the phosphorylation of initiation factor elF-4E and translational repressor, 4E-BP. The activity of a p38 MAPK downstream substrate, MAPKAPK2 (MK2), did not reflect the changing levels of Pi-p38 MAPK during liver regeneration. Pi-p38 MAPK may be involved in TNF-stimulated DNA replication of murine hepatocytes in culture, but is not necessary for hepatocyte DNA replication after PH. Our results suggest that p38 MAPK inactivation plays a permissible role in DNA replication during liver regeneration and is consistent with a role for p38 MAPK in the maintenance of hepatocyte cell cycle arrest in adult liver.

Chronic liver injury leads to fibrosis, cirrhosis, and loss of liver function. Liver cirrhosis is the 12th leading cause of death in the United States, and it is the primary risk factor for developing liver cancer. Fibrosis and cirrhosis result from activation of hepatic stellate cells (HSCs), which are the primary collagen producing cell type in the liver. Here, we show that platelet-derived growth factor receptor α (PDGFRα) is expressed by human HSCs, and PDGFRα expression is elevated in human liver disease. Using a green fluorescent protein (GFP) reporter mouse strain, we evaluated the role of PDGFRα in liver disease in mice and found that mouse HSCs express PDGFRα and expression is upregulated during carbon tetrachloride (CCl4) induced liver injury and fibrosis injection. This fibrotic response is reduced in Pdgfrα heterozygous mice, consistent with the hypothesis that liver fibrosis requires upregulation and activation of PDGFRα. These results indicate that Pdgfrα expression is important in the fibrotic response to liver injury in humans and mice, and suggest that blocking PDGFRα–specific signaling pathways in HSCs may provide therapeutic benefit for patients with chronic liver disease.