Reprogramming the spleen into a functioning ‘liver’ in vivo

ObjectiveLiver regeneration remains one of the biggest clinical challenges. Here, we aim to transform the spleen into a liver-like organ via directly reprogramming the splenic fibroblasts into hepatocytes in vivo.DesignIn the mouse spleen, the number of fibroblasts was through silica particles (SiO2) stimulation, the expanded fibroblasts were converted to hepatocytes (iHeps) by lentiviral transfection of three key transcriptional factors (Foxa3, Gata4 and Hnf1a), and the iHeps were further expanded with tumour necrosis factor-α (TNF-α) and lentivirus-mediated expression of epidermal growth factor (EGF) and hepatocyte growth factor (HGF).ResultsSiO2 stimulation tripled the number of activated fibroblasts. Foxa3, Gata4 and Hnf1a converted SiO2-remodelled spleen fibroblasts into 2×106 functional iHeps in one spleen. TNF-α protein and lentivirus-mediated expression of EGF and HGF further enabled the total hepatocytes to expand to 8×106 per spleen. iHeps possessed hepatic functions—such as glycogen storage, lipid accumulation and drug metabolism—and performed fundamental liver functions to improve the survival rate of mice with 90% hepatectomy.ConclusionDirect conversion of the spleen into a liver-like organ, without cell or tissue transplantation, establishes fundamental hepatic functions in mice, suggesting its potential value for the treatment of end-stage liver diseases.

Mutant KRAS-associated proteome is mainly controlled by exogenous factors

KRAS signaling has been extensively studied, yet the clarification between KRAS-autonomous and non-autonomous mechanisms are still less explored. Understanding how KRAS signaling and effects are affected by exogenous stimuli can provide valuable insights not only to understand resistance mechanisms that justify pathway inhibition failure, but also to uncover novel therapeutic targets for mutant KRAS patients. Hence, aiming at understanding KRAS-autonomous versus non autonomous mechanisms, we studied the response of two mutant KRAS colorectal cancer cell lines (HCT116 and LS174T) - control and KRAS silenced- to TGFβ1-activated fibroblasts secretome. By performing a total proteome analysis, we observed that TGFβ1-activated fibroblast-secreted factors triggered cell line-specific proteome alterations and that mutant KRAS governs approximately 1/3 of those alterations. Moreover, the analysis of the impact of exogenous factors on the modulation of KRAS proteome revealed that, in both cell lines, more than 2/3 of the KRAS-associated proteome is controlled in a KRAS-non-autonomous manner and dependent on the exogenous factors. This work highlights the context-dependency of KRAS-associated signaling and reinforces the importance of establishing more integrative models resembling the complexity of the tumor microenvironment to study KRAS-associated signals.

CXCL12 in Pancreatic Cancer: Its Function and Potential as a Therapeutic Drug Target

Pancreatic ductal adenocarcinoma (PDAC) is a disease with limited therapeutic options and dismal long-term survival. The unique tumor environment of PDAC, consisting of desmoplastic stroma, immune suppressive cells, and activated fibroblasts, contributes to its resistance to therapy. Activated fibroblasts (cancer-associated fibroblasts and pancreatic stellate cells) secrete chemokines and growth factors that support PDAC growth, spread, chemoresistance, and immune evasion. In this review, we focus on one such chemokine, CXCL12, secreted by the cancer-associated fibroblasts and discuss its contribution to several of the classical hallmarks of PDAC and other tumors. We review the various therapeutic approaches in development to target CXCL12 signaling in PDAC. Finally, we propose an unconventional use of tipifarnib, a farnesyl transferase inhibitor, to inhibit CXCL12 production in PDAC.

Fibroblast to Myofibroblast Transition is Enhanced by Increased Cell Density

Idiopathic pulmonary fibrosis (IPF) is a chronic disease of the lung caused by a rampant inflammatory response that results in the deposition of excessive extracellular matrix (ECM). IPF patient lungs also develop fibroblastic foci that consist of activated fibroblasts and myofibroblasts. In concert with ECM deposition, the increased cell density within fibroblastic foci imposes confining forces on lung fibroblasts. In this work, we observed that increased cell density increases the incidence of fibroblast to myofibroblast transition (FMT), but mechanical confinement imposed by micropillars has no effect on FMT incidence. We found that human lung fibroblasts (HLFs) express more α-SMA and deposit more collagen matrix, which are both characteristics of myofibroblasts, in response to TGF-β1 when cells were seeded at a high density compared to a medium or a low density. These results support the hypothesis that HLFs undergo FMT more readily in response to TGF-β1 when cells are densely packed, and this effect could be dependent on increased OB-cadherin expression. This work demonstrates that cell density is an important factor to consider when modelling IPF in vitro, and it may suggest decreasing cell density within fibroblastic foci as a strategy to reduce IPF burden.

IL-1-driven stromal–neutrophil interactions define a subset of patients with inflammatory bowel disease that does not respond to therapies

Current inflammatory bowel disease (IBD) therapies are ineffective in a high proportion of patients. Combining bulk and single-cell transcriptomics, quantitative histopathology and in situ localization across three cohorts of patients with IBD (total n = 376), we identify coexpressed gene modules within the heterogeneous tissular inflammatory response in IBD that map to distinct histopathological and cellular features (pathotypes). One of these pathotypes is defined by high neutrophil infiltration, activation of fibroblasts and vascular remodeling at sites of deep ulceration. Activated fibroblasts in the ulcer bed display neutrophil-chemoattractant properties that are IL-1R, but not TNF, dependent. Pathotype-associated neutrophil and fibroblast signatures are increased in nonresponders to several therapies across four independent cohorts (total n = 343). The identification of distinct, localized, tissular pathotypes will aid precision targeting of current therapeutics and provides a biological rationale for IL-1 signaling blockade in ulcerating disease.

Serological assessment of collagen fragments and tumor fibrosis may guide immune checkpoint inhibitor therapy

Despite the overall clinical success of immune checkpoint inhibitors (ICIs) for treating patients with solid tumors, a large number of patients do not benefit from this approach. Consequently, there is a need for predictive biomarkers. The most prevalent biomarkers such as PD-L1 expression and tumor mutational burden (TMB) do not reliably predict response to ICIs across different solid tumor types suggesting that a broader view of regulating factors in the tumor microenvironment is needed. Emerging evidence indicates that one central common denominator of resistance to ICIs may be fibrotic activity characterized by extracellular matrix (ECM) and collagen production by cancer-associated fibroblasts (CAFs). A fibroblast-and collagen-rich stroma attenuates immunotherapy response by contributing to inhibition and exclusion of T cells. Here we review opportunities and limitations in the utilization of the most prevalent biomarkers for ICIs and elaborate on the unique opportunities with biomarkers originating from the activated fibroblasts producing an impermeable ECM. We propose that ECM and collagen biomarkers measured non-invasively may be a novel and practical approach to optimize treatment strategies and improve patient selection for ICI therapy.

The Role of Hippo/YAP Signaling in Alveolar Repair and Pulmonary Fibrosis

Pulmonary fibrosis is characterized by loss of normal alveoli, accumulation of pathologic activated fibroblasts, and exuberant extracellular matrix deposition that over time can lead to progressive loss of respiratory function and death. This loss of respiratory function is associated with the loss of alveolar type 1 cells (AT1) that play a crucial role in gas exchange and the depletion of the alveolar type 2 cells (AT2) that act as progenitor cells to regenerate the AT1 and AT2 cell populations during repair. Understanding the mechanisms that regulate normal alveolar repair and those associated with pathologic repair is essential to identify potential therapeutic targets to treat or delay progression of fibrotic diseases. The Hippo/YAP developmental signaling pathway has been implicated as a regulator of normal alveolar development and repair. In idiopathic pulmonary fibrosis, aberrant activation of YAP/TAZ has been demonstrated in both the alveolar epithelium and activated fibroblasts associated with increased fibrotic remodeling, and there is emerging interest in this pathway as a target for antifibrotic therapies. In this review, we summarize current evidence as to the role of the Hippo-YAP/TAZ pathway in alveolar development, homeostasis, and repair, and highlight key questions that must be resolved to determine effective strategies to modulate YAP/TAZ signaling to prevent progressive pulmonary fibrosis and enhance adaptive alveolar repair.

Low Ozone Concentrations Differentially Affect the Structural and Functional Features of Non-Activated and Activated Fibroblasts In Vitro

Oxygen–ozone (O2–O3) therapy is increasingly applied as a complementary/adjuvant treatment for several diseases; however, the biological mechanisms accounting for the efficacy of low O3 concentrations need further investigations to understand the possibly multiple effects on the different cell types. In this work, we focused our attention on fibroblasts as ubiquitous connective cells playing roles in the body architecture, in the homeostasis of tissue-resident cells, and in many physiological and pathological processes. Using an established human fibroblast cell line as an in vitro model, we adopted a multimodal approach to explore a panel of cell structural and functional features, combining light and electron microscopy, Western blot analysis, real-time quantitative polymerase chain reaction, and multiplex assays for cytokines. The administration of O2–O3 gas mixtures induced multiple effects on fibroblasts, depending on their activation state: in non-activated fibroblasts, O3 stimulated proliferation, formation of cell surface protrusions, antioxidant response, and IL-6 and TGF-β1 secretion, while in LPS-activated fibroblasts, O3 stimulated only antioxidant response and cytokines secretion. Therefore, the low O3 concentrations used in this study induced activation-like responses in non-activated fibroblasts, whereas in already activated fibroblasts, the cell protective capability was potentiated.

WNT/β-Catenin Signaling Promotes TGF-β-Mediated Activation of Human Cardiac Fibroblasts by Enhancing IL-11 Production

Cardiac fibrosis is a pathological process associated with the development of heart failure. TGF-β and WNT signaling have been implicated in pathogenesis of cardiac fibrosis, however, little is known about molecular cross-talk between these two pathways. The aim of this study was to examine the effect of exogenous canonical WNT3a and non-canonical WNT5a in TGF-β-activated human cardiac fibroblasts. We found that WNT3a and TGF-β induced a b-catenin-dependent response, whereas WNT5a prompted AP-1 activity. TGF-β triggered profibrotic signatures in cardiac fibroblasts, and co-stimulation with WNT3a or co-activation of the β-catenin pathway with the GSK3β inhibitor CHIR99021 enhanced collagen I and fibronectin production and development of active contractile stress fibers. In the absence of TGF-β, neither WNT3a nor CHIR99021 exerted profibrotic responses. On a molecular level, in TGF-β-activated fibroblasts, WNT3a enhanced phosphorylation of TAK1 and production and secretion of IL-11 but showed no effect on the Smad pathway. Neutralization of IL-11 activity with the blocking anti-IL-11 antibody effectively reduced the profibrotic response of cardiac fibroblasts activated with TGF-β and WNT3a. In contrast to canonical WNT3a, co-activation with non-canonical WNT5a suppressed TGF-β-induced production of collagen I. In conclusion, WNT/β-catenin signaling promotes TGF-β-mediated fibroblast-to-myofibroblast transition by enhancing IL-11 production. Thus, the uncovered mechanism broadens our knowledge on a molecular basis of cardiac fibrogenesis and defines novel therapeutic targets for fibrotic heart diseases.