The progression and metastatic capacity of solid tumors are strongly influenced by immune cells in the tumor microenvironment. In non–small cell lung cancer (NSCLC), accumulation of anti-inflammatory tumor-associated macrophages (TAM) is associated with worse clinical outcome and resistance to therapy. Here we investigated the immune landscape of NSCLC in the presence of protumoral TAMs expressing the macrophage receptor with collagenous structure (MARCO). MARCO-expressing TAM numbers correlated with increased occurrence of regulatory T cells and effector T cells and decreased natural killer (NK) cells in these tumors. Furthermore, transcriptomic data from the tumors uncovered a correlation between MARCO expression and the anti-inflammatory cytokine IL37. In vitro studies subsequently showed that lung cancer cells polarized macrophages to express MARCO and gain an immune-suppressive phenotype through the release of IL37. MARCO-expressing TAMs blocked cytotoxic T-cell and NK-cell activation, inhibiting their proliferation, cytokine production, and tumor killing capacity. Mechanistically, MARCO+ macrophages enhanced regulatory T (Treg) cell proliferation and IL10 production and diminished CD8 T-cell activities. Targeting MARCO or IL37 receptor (IL37R) by antibody or CRISPR knockout of IL37 in lung cancer cell lines repolarized TAMs, resulting in recovered cytolytic activity and antitumoral capacity of NK cells and T cells and downmodulated Treg cell activities. In summary, our data demonstrate a novel immune therapeutic approach targeting human TAMs immune suppression of NK- and T-cell antitumor activities. Significance: This study defines tumor-derived IL37 and the macrophage scavenger receptor MARCO as potential therapeutic targets to remodel the immune-suppressive microenvironment in patients with lung cancer.

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Direct in vivo reprogramming of cardiac fibroblasts into myocytes is an attractive therapeutic intervention in resolving myogenic deterioration. Current transgene‐dependent approaches can restore cardiac function, but dependence on retroviral delivery and persistent retention of transgenic sequences are significant therapeutic hurdles. Chemical reprogramming has been established as a legitimate method to generate functional cell types, including those of the cardiac lineage. Here, we have extended this approach to generate progenitor cells that can differentiate into endothelial cells and cardiomyocytes using a single inhibitor protocol. Depletion of terminally differentiated cells and enrichment for proliferative cells result in a second expandable progenitor population that can robustly give rise to myofibroblasts and smooth muscle. Deployment of a genome‐wide knockout screen with clustered regularly interspaced short palindromic repeats‐guide RNA library to identify novel mediators that regulate the reprogramming revealed the involvement of DNA methyltransferase 1‐associated protein 1 (Dmap1). Loss of Dmap1 reduced promoter methylation, increased the expression of Nkx2‐5, and enhanced the retention of self‐renewal, although further differentiation is inhibited because of the sustained expression of Cdh1. Our results hence establish Dmap1 as a modulator of cardiac reprogramming and myocytic induction. Stem Cells 2019;37:958–972

p53 is an important modulator of stem cell fate, but its role in cardiac progenitor cells (CPCs) is unknown. Here, we tested the effects of a single extra-copy of p53 on the function of CPCs in the presence of oxidative stress mediated by doxorubicin in vitro and type-1 diabetes in vivo. CPCs were obtained from super-p53 transgenic mice (p53-tg), in which the additional allele is regulated in a manner similar to the endogenous protein. Old CPCs with increased p53 dosage showed a superior ability to sustain oxidative stress, repair DNA damage and restore cell division. With doxorubicin, a larger fraction of CPCs carrying an extra-copy of the p53 allele recruited γH2A.X reestablishing DNA integrity. Enhanced p53 expression resulted in a superior tolerance to oxidative stress in vivo by providing CPCs with defense mechanisms necessary to survive in the milieu of the diabetic heart; they engrafted in regions of tissue injury and in three days acquired the cardiomyocyte phenotype. The biological advantage provided by the increased dosage of p53 in CPCs suggests that this genetic strategy may be translated to humans to increase cellular engraftment and growth, critical determinants of successful cell therapy for the failing heart.

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As a result of stress, injury, or aging, cardiac fibrosis is characterized by excessive deposition of extracellular matrix (ECM) components resulting in pathological remodeling, tissue stiffening, ventricular dilatation, and cardiac dysfunction that contribute to heart failure (HF) and eventually death. Currently, there are no effective therapies specifically targeting cardiac fibrosis, partially due to limited understanding of the pathological mechanisms and the lack of predictive in vitro models for high-throughput screening of antifibrotic compounds. The use of more relevant cell models, three-dimensional (3D) models, and coculture systems, together with high-content imaging (HCI) and machine learning (ML)-based image analysis, is expected to improve predictivity and throughput of in vitro models for cardiac fibrosis. In this review, we present an overview of available in vitro assays for cardiac fibrosis. We highlight the potential of more physiological 3D cardiac organoids and coculture systems and discuss HCI and automated artificial intelligence (AI)-based image analysis as key methods able to capture the complexity of cardiac fibrosis in vitro. As 3D and coculture models will soon be sufficiently mature for application in large-scale preclinical drug discovery, we expect the combination of more relevant models and high-content analysis to greatly increase translation from in vitro to in vivo models and facilitate the discovery of novel targets and drugs against cardiac fibrosis.

Cover: Confocal image of primary human cardiac fibroblasts in culture. Focus on Fibroblasts: Development, plasticity, and therapeutic challenges in the cardiac fibroblast lineage THESIS FOR DOCTORAL DEGREE (Ph.D.

BackgroundThe progression and metastatic capacity of solid tumors are strongly influenced by immune cells in the tumor microenvironment (TME). In non-small cell lung cancer (NSCLC) accumulation of anti-inflammatory tumor-associated macrophages (TAMs) is associated with worse clinical outcome and resistance to therapy. Numerous clinical trials aiming to recover T cell anti-tumor activity have been failing due to the persistence immune suppression in TME. Thus, there is a clinical need for alternative treatments targeting the suppressive function of the TME. We have previously shown that antibodies targeting the scavenger receptor MARCO reprograms the pro-tumoral TAMs in murine cancer models. Here, we investigated the immune landscape of NSCLC in the presence of MARCO expressing TAMs. We tested targeting MARCO or the tumor mechanisms inducing MARCO on human TAMs and hypothesized that targeting these mechanisms will remodel the suppressive environment and relive the anti-tumor responses to increase the efficacy of immunotherapy.MethodsTo test our hypothesis, we first investigated the immune landscape of NSCLC in the presence of pro-tumoral MARCO+TAMs compared with tumors infiltrated by MARCO-TAMs. We next used RNAseq to analyze differential gene expression in NSCLC tumors infiltrated by MARCO positive or negative macrophages. In vitro, cytokine differentiated macrophages alternatively cultured with lung cancer cell lines were co-cultured with Natural Killer (NK) cells and T cells to mimic their interaction in the TME. Later, macrophages were treated with targeting antibodies and their phenotype and function were examined prior and following interaction with other immune cells.ResultsWe found that MARCO expressing TAM numbers correlated with increased occurrence of regulatory T cell and effector T cells and decreased NK cells in NSCLC infiltrated by MARCO+TAMs. Furthermore, transcriptomic data from the tumors uncovered a correlation between MARCO expression and the anti-inflammatory cytokine IL-37. Studies in vitro subsequently showed that lung cancer cells polarized macrophages to express MARCO and gain an anti-inflammatory phenotype through the release of IL-37. These human MARCO expressing TAMs blocked cytotoxic T cell and NK cell activation, inhibiting their proliferation, cytokine production and tumor killing capacity. Mechanistically, MARCO+ macrophages enhanced regulatory T (Treg) cell proliferation and IL-10 production and diminished CD8 T cell activities. Targeting MARCO or IL-37 receptor (IL-37R) repolarized TAMs resulted in recovered cytolytic activity and anti-tumoral capacity of NK cells and T cells.ConclusionsIn summary, our data demonstrate a novel immune therapeutic approach targeting human TAM immune suppression of NK and T cell anti-tumor activities and remodel immune suppression.Ethics ApprovalThe study was approved by Institutional Ethics Board, approval number Dnr 2013.977-31.1.