Anti-microRNA-21 Therapy on Top of ACE Inhibition Delays Renal Failure in Alport Syndrome Mouse Models.

Col4a3-/- Alport mice serve as an animal model for renal fibrosis. MicroRNA-21 (miR-21) expression has been shown to be increased in the kidneys of Alport syndrome patients. Here, we investigated the nephroprotective effects of Lademirsen anti-miR-21 therapy. We used a fast-progressing Col4a3-/- mouse model with a 129/SvJ background and an intermediate-progressing F1 hybrid mouse model with a mixed genetic background, with angiotensin-converting enzyme inhibitor (ACEi) monotherapy in combination with anti-miR-21 therapy. In the fast-progressing model, the anti miR-21 and ACEi therapies showed an additive effect in the reduction in fibrosis, the decline of proteinuria, the preservation of kidney function and increased survival. In the intermediate-progressing F1 model, the anti-miR-21 and ACEi therapies individually improved kidney pathology. Both also improved kidney function and survival; however, the combination showed a significant additive effect, particularly for survival. RNA sequencing (RNA-seq) gene expression profiling revealed that the anti-miR-21 and ACEi therapies modulate several common pathways. However, anti-miR-21 was particularly effective at normalizing the expression profiles of the genes involved in renal tubulointerstitial injury pathways. In conclusion, significant additive effects were detected for the combination of anti-miR-21 and ACEi therapies on kidney function, pathology and survival in Alport mouse models, as well as a strong differential effect of anti-miR-21 on the renal expression of fibrotic factors. These results support the addition of anti-miR-21 to the current standard of care (ACEi) in ongoing clinical trials in patients with Alport syndrome.

Regulators of Metastasis Modulate the Migratory Response to Cell Contact under Spatial Confinement.

The breast tumor microenvironment (TMEN) is a unique niche where protein fibers help to promote invasion and metastasis. Cells migrating along these fibers are constantly interacting with each other. How cells respond to these interactions has important implications. Cancer cells that circumnavigate or slide around other cells on protein fibers take a less tortuous path out of the primary tumor; conversely, cells that turn back upon encountering other cells invade less efficiently. The contact response of migrating cancer cells in a fibrillar TMEN is poorly understood. Here, using high-aspect ratio micropatterns as a model fibrillar platform, we show that metastatic cells overcome spatial constraints to slide effectively on narrow fiber-like dimensions, whereas nontransformed MCF-10A mammary epithelial cells require much wider micropatterns to achieve moderate levels of sliding. Downregulating the cell-cell adhesion protein, E-cadherin, enables MCF-10A cells to slide on narrower micropatterns; meanwhile, introducing exogenous E-cadherin in metastatic MDA-MB-231 cells increases the micropattern dimension at which they slide. We propose the characteristic fibrillar dimension (CFD) at which effective sliding is achieved as a metric of sliding ability under spatial confinement. Using this metric, we show that metastasis-promoting genetic perturbations enhance cell sliding and reduce CFD. Activation of ErbB2 combined with downregulation of the tumor suppressor and cell polarity regulator, PARD3, reduced the CFD, in agreement with their cooperative role in inducing metastasis in vivo. The CFD was further reduced by a combination of ErbB2 activation and transforming growth factor ß stimulation, which is known to enhance invasive behavior. These findings demonstrate that sliding is a quantitative property and a decrease in CFD is an effective metric to understand how multiple genetic hits interact to change cell behavior in fibrillar environments. This quantitative framework sheds insights into how genetic perturbations conspire with fibrillar maturation in the TMEN to drive the invasive behavior of cancer cells.

Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell- and patient-derived tumor organoids.

There are few in vitro models of exocrine pancreas development and primary human pancreatic adenocarcinoma (PDAC). We establish three-dimensional culture conditions to induce the differentiation of human pluripotent stem cells into exocrine progenitor organoids that form ductal and acinar structures in culture and in vivo. Expression of mutant KRAS or TP53 in progenitor organoids induces mutation-specific phenotypes in culture and in vivo. Expression of TP53(R175H) induces cytosolic SOX9 localization. In patient tumors bearing TP53 mutations, SOX9 was cytoplasmic and associated with mortality. We also define culture conditions for clonal generation of tumor organoids from freshly resected PDAC. Tumor organoids maintain the differentiation status, histoarchitecture and phenotypic heterogeneity of the primary tumor and retain patient-specific physiological changes, including hypoxia, oxygen consumption, epigenetic marks and differences in sensitivity to inhibition of the histone methyltransferase EZH2. Thus, pancreatic progenitor organoids and tumor organoids can be used to model PDAC and for drug screening to identify precision therapy strategies.

The adjuvancy of OX40 ligand (CD252) on an HIV-1 canarypox vaccine.

The immunogenicity of current human immunodeficiency virus-1 (HIV-1) canarypox vaccines is weak and needs to be improved. Ligation of OX40 (CD134), a member of tumor necrosis factor receptor superfamily (TNFRSF), by its ligand OX40L (CD252), a tumor necrosis factor superfamily (TNFSF) molecule, has been demonstrated to provide a pivotal costimulatory signal to enhance CD4(+) T cell help of humoral and cytotoxic T cell immune responses. The present study examined whether an OX40L-expressing vector could boost the immunogenicity of the HIV-1 canarypox vaccine, vCP1452, in mice. Co-immunization of mice with OX40L-expressing canarypox and vCP1452 augmented HIV-1 specific CD8(+) T cell responses in terms of frequency and cytokine expression. OX40L-expressing canarypox enhanced the frequency of antigen specific CD8(+) T cells with an effector (CD127(-)CD62L(-)) phenotype, which was associated with an ex vivo expansion of HIV-1 specific CD4(+) T cells. This was in contrast to our previous work in which a CD40L-expressing construct preferentially enhanced antigen specific memory responses [Liu J, Yu Q, Stone GW, Yue FY, Ngai N, Jones RB, et al. CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals. Vaccine 2008;26(32):4062-72]. Surprisingly, OX40L did not enhance antibody responses elicited by the HIV-1 canarypox vaccine. We saw no added benefit by combining OX40L and CD40L vectors as an adjuvant strategy for vCP1452. Our results indicate that, similar to CD40L, canarypox vectors expressing OX40L can enhance the cellular but not humoral immunogenicity of HIV-1 canarypox vaccines. In summary, our findings show that OX40L can be used as a molecular adjuvant to enhance T cell immune responses.

CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals.

Human immunodeficiency virus type 1 (HIV-1) canarypox vaccines are safe but poorly immunogenic. CD40 ligand (CD40L), a member of the tumor necrosis factor superfamily (TNFSF), is a pivotal costimulatory molecule for immune responses. To explore whether CD40L can be used as an adjuvant for HIV-1 canarypox vaccine, we constructed recombinant canarypox viruses expressing CD40L. Co-immunization of mice with CD40L expressing canarypox and the canarypox vaccine expressing HIV-1 proteins, vCP1452, augmented HIV-1 specific cytotoxic T lymphocyte (CTL) responses in terms of frequency, polyfunctionality and interleukin (IL)-7 receptor alpha chain (IL-7Ralpha, CD127) expression. In addition, CD40L expressed from canarypox virus could significantly augment CD4+ T cell responses against HIV-1 in mice. CD40L expressed from canarypox virus matured human monocyte-derived dendritic cells (MDDCs) in a tumor necrosis factor-alpha (TNF-alpha) independent manner, which underwent less apoptosis, and could expand ex vivo Epstein-Barr virus (EBV)-specific CTL responses from healthy human individuals and ex vivo HIV-1-specific CTL responses from HIV-1-infected individuals in the presence or absence of CD4+ T cells. Taken together, our results suggest that CD40L incorporation into poxvirus vectors could be used as a strategy to enhance their immunogenicity.