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Fibrosis is a progressive biological condition, leading to organ dysfunction in various clinical settings. Although fibroblasts and macrophages are known as key cellular players for fibrosis development, a comprehensive functional model that considers their interaction in the metabolic/immunologic context of fibrotic tissue has not been set up. Here we show, by transcriptome-based mathematical modeling in an in vitro system that represents macrophage-fibroblast interplay and reflects the functional effects of inflammation, hypoxia and the adaptive immune context, that irreversible fibrosis development is associated with specific combinations of metabolic and inflammatory cues. The in vitro signatures are in good alignment with transcriptomic profiles generated on laser captured glomeruli and cortical tubule-interstitial area, isolated from human transplanted kidneys with advanced stages of glomerulosclerosis and interstitial fibrosis/tubular atrophy, two clinically relevant conditions associated with organ failure in renal allografts. The model we describe here is validated on tissue based quantitative immune-phenotyping of biopsies from transplanted kidneys, demonstrating its feasibility. We conclude that the combination of in vitro and in silico modeling represents a powerful systems medicine approach to dissect fibrosis pathogenesis, applicable to specific pathological conditions, and develop coordinated targeted approaches., (© 2022. The Author(s).)

Background: More than 50% of all patients with colorectal cancer (CRC) develop liver metastases (CLM), a clinical condition characterized by poor prognosis and lack of reliable prognostic markers. Vδ1 cells are a subset of tissue-resident gamma delta (γδ) T lymphocytes endowed with a broad array of antitumor functions and showing a natural high tropism for the liver. However, little is known about their impact in the clinical outcomes of CLM., Methods: We isolated human γδ T cells from peripheral blood (PB) and peritumoral (PT) tissue of 93 patients undergone surgical procedures to remove CLM. The phenotype of freshly purified γδ T cells was assessed by multiparametric flow cytometry, the transcriptional profiles by single cell RNA-sequencing, the functional annotations by Gene Ontology enrichment analyses and the clonotype by γδ T cell receptor (TCR)-sequencing., Results: The microenvironment of CLM is characterized by a heterogeneous immune infiltrate comprising different subsets of γδ tumor-infiltrating lymphocytes (TILs) able to egress the liver and re-circulate in PB. Vδ1 T cells represent the largest population of γδ TILs within the PT compartment of CLM that is greatly enriched in Vδ1 T effector (T EF ) cells expressing constitutive high levels of CD69. These Vδ1 CD69 + TILs express a distinct phenotype and transcriptional signature, show high antitumor potential and correlate with better patient clinical outcomes in terms of lower numbers of liver metastatic lesions and longer overall survival (OS). Moreover, intrahepatic CD69 + Vδ1 TILs can egress CLM tissue to re-circulate in PB, where they retain a phenotype, transcriptional signature and TCR clonal repertoires resembling their liver origin. Importantly, even the increased frequencies of the CD69 + terminally differentiated (T EMRA ) Vδ1 cells in PB of patients with CLM significantly correlate with longer OS. The positive prognostic score of high frequencies of CD69 + T EMRA Vδ1 cells in PB is independent from the neoadjuvant chemotherapy and immunotherapy regimens administered to patients with CLM prior surgery., Conclusions: The enrichment of tissue-resident CD69 + Vδ1 T EMRA cells re-circulating at high frequencies in PB of patients with CLM limits tumor progression and represents a new important clinical tool to either predict the natural history of CLM or develop alternative therapeutic protocols of cellular therapies., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Tumor-associated macrophages (TAMs) are correlated with the progression of prostatic adenocarcinoma (PCa). The mechanistic basis of this correlation and therapeutic strategies to target TAMs in PCa remain poorly defined. Here, single-cell RNA sequencing was used to profile the transcriptional landscape of TAMs in human PCa, leading to identification of a subset of macrophages characterized by dysregulation in transcriptional pathways associated with lipid metabolism. This subset of TAMs correlates positively with PCa progression and shorter disease-free survival and is characterized by an accumulation of lipids that is dependent on Marco. Mechanistically, cancer cell-derived IL-1β enhances Marco expression on macrophages, and reciprocally, cancer cell migration is promoted by CCL6 released by lipid-loaded TAMs. Moreover, administration of a high-fat diet to tumor-bearing mice raises the abundance of lipid-loaded TAMs. Finally, targeting lipid accumulation by Marco blockade hinders tumor growth and invasiveness and improves the efficacy of chemotherapy in models of PCa, pointing to combinatorial strategies that may influence patient outcomes., Competing Interests: Disclosures: R.A. DePinho reported being a Founder and Advisor for Tvardi Therapeutics, Asylia Therapeutics, Nirogy Therapeutics, Stellanova Therapeutics, and Sporos Bioventures. The focus of these companies is not directly related to the content of this manuscript. S. Gordon reported personal fees from Verseau, Myeloid Therapeutics, and Alnylam outside the submitted work. No other disclosures were reported., (© 2021 Masetti et al.)

The mammalian circadian clock, expressed throughout the brain and body, controls daily metabolic homeostasis. Clock function in peripheral tissues is required, but not sufficient, for this task. Because of the lack of specialized animal models, it is unclear how tissue clocks interact with extrinsic signals to drive molecular oscillations. Here, we isolated the interaction between feeding and the liver clock by reconstituting Bmal1 exclusively in hepatocytes (Liver-RE), in otherwise clock-less mice, and controlling timing of food intake. We found that the cooperative action of BMAL1 and the transcription factor CEBPB regulates daily liver metabolic transcriptional programs. Functionally, the liver clock and feeding rhythm are sufficient to drive temporal carbohydrate homeostasis. By contrast, liver rhythms tied to redox and lipid metabolism required communication with the skeletal muscle clock, demonstrating peripheral clock cross-talk. Our results highlight how the inner workings of the clock system rely on communicating signals to maintain daily metabolism.

The cancer-free photosensitive trichothiodystrophy (PS-TTD) and the cancer-prone xeroderma pigmentosum (XP) are rare monogenic disorders that can arise from mutations in the same genes, namely ERCC2/XPD or ERCC3/XPB Both XPD and XPB proteins belong to the 10-subunit complex transcription factor IIH (TFIIH) that plays a key role in transcription and nucleotide excision repair, the DNA repair pathway devoted to the removal of ultraviolet-induced DNA lesions. Compelling evidence suggests that mutations affecting the DNA repair activity of TFIIH are responsible for the pathological features of XP, whereas those also impairing transcription give rise to TTD. By adopting a relatives-based whole transcriptome sequencing approach followed by specific gene expression profiling in primary fibroblasts from a large cohort of TTD or XP cases with mutations in ERCC2/XPD gene, we identify the expression alterations specific for TTD primary dermal fibroblasts. While most of these transcription deregulations do not impact on the protein level, very low amounts of prostaglandin I 2 synthase (PTGIS) are found in TTD cells. PTGIS catalyzes the last step of prostaglandin I 2 synthesis, a potent vasodilator and inhibitor of platelet aggregation. Its reduction characterizes all TTD cases so far investigated, both the PS-TTD with mutations in TFIIH coding genes as well as the nonphotosensitive (NPS)-TTD. A severe impairment of TFIIH and RNA polymerase II recruitment on the PTGIS promoter is found in TTD but not in XP cells. Thus, PTGIS represents a biomarker that combines all PS- and NPS-TTD cases and distinguishes them from XP., Competing Interests: The authors declare no competing interest.

It has long been known that in vitro polarized macrophages differ in morphology. Stemming from a conventional immunohistology observation, we set out to test the hypothesis that morphology of tumor-associated macrophages (TAMs) in colorectal liver metastasis (CLM) represents a correlate of functional diversity with prognostic significance. Density and morphological metrics of TAMs were measured and correlated with clinicopathological variables. While density of TAMs did not correlate with survival of CLM patients, the cell area identified small (S-TAM) and large (L-TAM) macrophages that were associated with 5-yr disease-free survival rates of 27.8% and 0.2%, respectively (P < 0.0001). RNA sequencing of morphologically distinct macrophages identified LXR/RXR as the most enriched pathway in large macrophages, with up-regulation of genes involved in cholesterol metabolism, scavenger receptors, MERTK, and complement. In single-cell analysis of mononuclear phagocytes from CLM tissues, S-TAM and L-TAM signatures were differentially enriched in individual clusters. These results suggest that morphometric characterization can serve as a simple readout of TAM diversity with strong prognostic significance., Competing Interests: Disclosures: A. Mantovani reported personal fees from Ventana, Pierre Fabre, Verily, AbbVie, Astra Zeneca, Verseau Therapeutics, Compugen, Myeloid Therapeutics, Third Rock Venture, Imcheck Therapeutics, Ellipses, Novartis, Roche, Macrophage Pharma, Biovelocita, Merck, and Principia; grants from Novartis; and "other" from Cedarlane Laboratories Ltd, Hycult Biotechnology, eBioscience, BioLegend, ABCAM Plc, Novus Biologicals, Enzo Life (ex-Alexis Corp.), and Affymetrix outside the submitted work. He is the inventor of patents related to PTX3 and other innate immunity molecules and receives royalties for reagents related to innate immunity. No other disclosures were reported., (© 2020 Donadon et al.)

Deciphering the molecular alterations leading to disease initiation and progression is currently crucial to identify the most relevant targets for precision therapy in cancer patients. Cancers express a complex chemokine network influencing leucocyte infiltration and angiogenesis. Moreover, malignant cells also express a selective repertoire of chemokine receptors that sustain their growth and spread. At present, different cancer types have been shown to overexpress C-X-C chemokine receptor type 4 (CXCR4) and to respond to its ligand C-X-C motif chemokine 12 (CXCL12). The CXCL12/CXCR4 axis influences cancer biology, promoting survival, proliferation, and angiogenesis, and plays a pivotal role in directing migration of cancer cells to sites of metastases, making it a prognostic marker and a therapeutic target. More recently, mutations in the C-terminus of CXCR4 have been identified in the genomic landscape of patients affected by Waldenstrom's macroglobulinemia, a rare B cell neoplasm. These mutations closely resemble those occurring in Warts, Hypogammaglobulinemia, Immunodeficiency, and Myelokathexis (WHIM) syndrome, an immunodeficiency associated with CXCR4 aberrant expression and activity and with chemotherapy resistance in clinical trials. In this review, we summarize the current knowledge on the relevance of CXCR4 mutations in cancer biology, focusing on its importance as predictors of clinical presentation and response to therapy.

Background: Inflammation is a key component of cardiac disease, with macrophages and T lymphocytes mediating essential roles in the progression to heart failure. Nonetheless, little insight exists on other immune subsets involved in the cardiotoxic response., Methods: Here, we used single-cell RNA sequencing to map the cardiac immune composition in the standard murine nonischemic, pressure-overload heart failure model. By focusing our analysis on CD45 + cells, we obtained a higher resolution identification of the immune cell subsets in the heart, at early and late stages of disease and in controls. We then integrated our findings using multiparameter flow cytometry, immunohistochemistry, and tissue clarification immunofluorescence in mouse and human., Results: We found that most major immune cell subpopulations, including macrophages, B cells, T cells and regulatory T cells, dendritic cells, Natural Killer cells, neutrophils, and mast cells are present in both healthy and diseased hearts. Most cell subsets are found within the myocardium, whereas mast cells are found also in the epicardium. Upon induction of pressure overload, immune activation occurs across the entire range of immune cell types. Activation led to upregulation of key subset-specific molecules, such as oncostatin M in proinflammatory macrophages and PD-1 in regulatory T cells, that may help explain clinical findings such as the refractivity of patients with heart failure to anti-tumor necrosis factor therapy and cardiac toxicity during anti-PD-1 cancer immunotherapy, respectively., Conclusions: Despite the absence of infectious agents or an autoimmune trigger, induction of disease leads to immune activation that involves far more cell types than previously thought, including neutrophils, B cells, Natural Killer cells, and mast cells. This opens up the field of cardioimmunology to further investigation by using toolkits that have already been developed to study the aforementioned immune subsets. The subset-specific molecules that mediate their activation may thus become useful targets for the diagnostics or therapy of heart failure.

CRISPR/Cas9 has become a powerful method to engineer genomes and to activate or to repress genes expression. As such, in cancer research CRISPR/Cas9 technology represents an efficient tool to dissect mechanisms of tumorigenesis and to discover novel targets for drug development. Here, we employed the CRISPR/Cas9 technology for studying the role of prostaglandin-endoperoxide synthase 2 (PTGS2) in melanoma development and progression. Melanoma is the most aggressive form of skin cancer with a median survival of less than 1 year. Although oncogene-targeted drugs and immune checkpoint inhibitors have demonstrated a significant success in improving overall survival in patients, related toxicity and emerging resistance are ongoing challenges. Gene therapy appears to be an appealing option to enhance the efficacy of currently available melanoma therapeutics leading to better patient prognosis. Several gene therapy targets have been identified and have proven to be effective against melanoma cells. Particularly, PTGS2 is frequently expressed in malignant melanomas and its expression significantly correlates with poor survival in patients. In this study we investigated on the effect of ptgs2 knockdown in B16F10 murine melanoma cells. Our results show that reduced expression of ptgs2 in melanoma cells: i ) inhibits cell proliferation, migration, and invasiveness; ii ) modulates immune response by impairing myeloid derived suppressor cell differentiation; iii ) reduces tumor development and metastasis in vivo . Collectively, these findings indicate that ptgs2 could represent an ideal gene to be targeted to improve success rates in the development of new and highly selective drugs for melanoma treatment., (Copyright © 2019 Ercolano, De Cicco, Rubino, Terrazzano, Ruggiero, Carriero, Kunderfranco and Ianaro.)

Brittle and "tiger-tail" hair is the diagnostic hallmark of trichothiodystrophy (TTD), a rare recessive disease associated with a wide spectrum of clinical features including ichthyosis, intellectual disability, decreased fertility, and short stature. As a result of premature abrogation of terminal differentiation, the hair is brittle and fragile and contains reduced cysteine content. Hypersensitivity to UV light is found in about half of individuals with TTD; all of these individuals harbor bi-allelic mutations in components of the basal transcription factor TFIIH, and these mutations lead to impaired nucleotide excision repair and basal transcription. Different genes have been found to be associated with non-photosensitive TTD (NPS-TTD); these include MPLKIP (also called TTDN1), GTF2E2 (also called TFIIEβ), and RNF113A. However, a relatively large group of these individuals with NPS-TTD have remained genetically uncharacterized. Here we present the identification of an NPS-TTD-associated gene, threonyl-tRNA synthetase (TARS), found by next-generation sequencing of a group of uncharacterized individuals with NPS-TTD. One individual has compound heterozygous TARS variants, c.826A>G (p.Lys276Glu) and c.1912C>T (p.Arg638 ∗ ), whereas a second individual is homozygous for the TARS variant: c.680T>C (p.Leu227Pro). We showed that these variants have a profound effect on TARS protein stability and enzymatic function. Our results expand the spectrum of genes involved in TTD to include genes implicated in amino acid charging of tRNA, which is required for the last step in gene expression, namely protein translation. We previously proposed that some of the TTD-specific features derive from subtle transcription defects as a consequence of unstable transcription factors. We now extend the definition of TTD from a transcription syndrome to a "gene-expression" syndrome., (Copyright © 2019 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Neutrophils are a component of the tumor microenvironment and have been predominantly associated with cancer progression. Using a genetic approach complemented by adoptive transfer, we found that neutrophils are essential for resistance against primary 3-methylcholantrene-induced carcinogenesis. Neutrophils were essential for the activation of an interferon-γ-dependent pathway of immune resistance, associated with polarization of a subset of CD4 - CD8 - unconventional αβ T cells (UTC αβ ). Bulk and single-cell RNA sequencing (scRNA-seq) analyses unveiled the innate-like features and diversity of UTC αβ associated with neutrophil-dependent anti-sarcoma immunity. In selected human tumors, including undifferentiated pleomorphic sarcoma, CSF3R expression, a neutrophil signature and neutrophil infiltration were associated with a type 1 immune response and better clinical outcome. Thus, neutrophils driving UTC αβ polarization and type 1 immunity are essential for resistance against murine sarcomas and selected human tumors., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Moderate DNA damage resulting from metabolic activities or sub-lethal doses of exogenous insults may eventually lead to cancer onset. Human 46BR.1G1 cells bear a mutation in replicative DNA ligase I (LigI) which results in low levels of replication-dependent DNA damage. This replication stress elicits a constitutive phosphorylation of the ataxia telangiectasia mutated (ATM) checkpoint kinase that fails to arrest cell cycle progression or to activate apoptosis or cell senescence. Stable transfection of wild type LigI, as in 7A3 cells, prevents DNA damage and ATM activation. Here we show that parental 46BR.1G1 and 7A3 cells differ in important features such as cell morphology, adhesion and migration. Comparison of gene expression profiles in the two cell lines detects Bio-Functional categories consistent with the morphological and migration properties of LigI deficient cells. Interestingly, ATM inhibition makes 46BR.1G1 more similar to 7A3 cells for what concerns morphology, adhesion and expression of cell-cell adhesion receptors. These observations extend the influence of the DNA damage response checkpoint pathways and unveil a role for ATM kinase activity in modulating cell biology parameters relevant to cancer progression.

In the past few years, the role of long noncoding RNAs (lncRNAs) in tumor development and progression has been disclosed although their mechanisms of action remain to be elucidated. An important contribution to the comprehension of lncRNAs biology in cancer could be obtained through the integrated analysis of multiple expression datasets. However, the growing availability of public datasets requires new data mining techniques to integrate and describe relationship among data. In this perspective, we explored the powerness of the Association Rule Mining (ARM) approach in gene expression data analysis. By the ARM method, we performed a meta-analysis of cancer-related microarray data which allowed us to identify and characterize a set of ten lncRNAs simultaneously altered in different brain tumor datasets. The expression profiles of the ten lncRNAs appeared to be sufficient to distinguish between cancer and normal tissues. A further characterization of this lncRNAs signature through a comodulation expression analysis suggested that biological processes specific of the nervous system could be compromised.