Riboflavin-LSD1 axis participates in the in vivo tumor-associated macrophage morphology in human colorectal liver metastases.

Tumor-associated macrophages (TAMs) are key components of the tumor microenvironment (TME). In colorectal liver metastasis (CLM), TAM morphology correlates with prognosis, with smaller TAMs (S-TAMs) conferring a more favorable prognosis than larger TAMs (L-TAMs). However, the metabolic profile of in vivo human TAM populations remains unknown. Multiparametric flow cytometry was used to freshly isolate S- and L-TAMs from surgically resected CLM patients (n = 14S-, 14L-TAMs). Mass spectrometry-based metabolomics analyses were implemented for the metabolic characterization of TAM populations. Gene expression analysis and protein activity were used to support the biochemical effects of the enzyme-substrate link between riboflavin and (lysine-specific demethylase 1A, LSD1) with TAM morphologies. L-TAMs were characterized by a positive correlation and a strong association between riboflavin and TAM morphologies. Riboflavin in both L-TAMs and in-vitro M2 polarized macrophages modulates LSD1 protein expression and activity. The inflammatory stimuli promoted by TNFα induced the increased expression of riboflavin transporter SLC52A3 and LSD1 in M2 macrophages. The modulation of the riboflavin-LSD1 axis represents a potential target for reprogramming TAM subtypes, paving the way for promising anti-tumor therapeutic strategies.

Flow Cytometry Analysis of IL-1 Receptors and Toll-Like Receptors on Platelets and Platelet-Derived Extracellular Vesicles.

Flow cytometry is largely used for the immunophenotyping and quantification of several cell types or related components including platelets and extracellular vesicles. Platelets and platelet-derived extracellular vesicles (PEVs) are receiving increased interest in inflammatory diseases including sepsis. Thus, in this chapter, we will describe protocols for the flow cytometry analysis of platelets, platelet/neutrophils hetero aggregates, and PEVs mainly focusing on the evaluation of the surface expression of some IL-1 receptor (ILR) and Toll-like receptor (TLR) family members.

Size-advantage of monovalent nanobodies against the macrophage mannose receptor for deep tumor penetration and tumor-associated macrophage targeting.

Rationale: Nanobodies (Nbs) have emerged as an elegant alternative to the use of conventional monoclonal antibodies in cancer therapy, but a detailed microscopic insight into the in vivo pharmacokinetics of different Nb formats in tumor-bearers is lacking. This is especially relevant for the recognition and targeting of pro-tumoral tumor-associated macrophages (TAMs), which may be located in less penetrable tumor regions. Methods: We employed anti-Macrophage Mannose Receptor (MMR) Nbs, in a monovalent (m) or bivalent (biv) format, to assess in vivo TAM targeting. Intravital and confocal microscopy were used to analyse the blood clearance rate and targeting kinetics of anti-MMR Nbs in tumor tissue, healthy muscle tissue and liver. Fluorescence Molecular Tomography was applied to confirm anti-MMR Nb accumulation in the primary tumor and in metastatic lesions. Results: Intravital microscopy demonstrated significant differences in the blood clearance rate and macrophage targeting kinetics of (m) and (biv)anti-MMR Nbs, both in tumoral and extra-tumoral tissue. Importantly, (m)anti-MMR Nbs are superior in reaching tissue macrophages, an advantage that is especially prominent in tumor tissue. The administration of a molar excess of unlabelled (biv)anti-MMR Nbs increased the (m)anti-MMR Nb bioavailability and impacted on its macrophage targeting kinetics, preventing their accumulation in extra-tumoral tissue (especially in the liver) but only partially influencing their interaction with TAMs. Finally, anti-MMR Nb administration not only allowed the visualization of TAMs in primary tumors, but also at a distant metastatic site. Conclusions: These data describe, for the first time, a microscopic analysis of (m) and (biv)anti-MMR Nb pharmacokinetics in tumor and healthy tissues. The concepts proposed in this study provide important knowledge for the future use of Nbs as diagnostic and therapeutic agents, especially for the targeting of tumor-infiltrating immune cells.

Workflow for high-dimensional flow cytometry analysis of T cells from tumor metastases.

We describe a multi-step high-dimensional (HD) flow cytometry workflow for the deep phenotypic characterization of T cells infiltrating metastatic tumor lesions in the liver, particularly derived from colorectal cancer (CRC-LM). First, we applied a novel flow cytometer setting approach based on single positive cells rather than fluorescent beads, resulting in optimal sensitivity when compared with previously published protocols. Second, we set up a 26-color based antibody panel designed to assess the functional state of both conventional T-cell subsets and unconventional invariant natural killer T, mucosal associated invariant T, and gamma delta T (γδT)-cell populations, which are abundant in the liver. Third, the dissociation of the CRC-LM samples was accurately tuned to preserve both the viability and antigenic integrity of the stained cells. This combined procedure permitted the optimal capturing of the phenotypic complexity of T cells infiltrating CRC-LM. Hence, this study provides a robust tool for high-dimensional flow cytometry analysis of complex T-cell populations, which could be adapted to characterize other relevant pathological tissues.

Intratumoral combination therapy with poly(I:C) and resiquimod synergistically triggers tumor-associated macrophages for effective systemic antitumoral immunity.

BACKGROUND: Tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer and hinder the antitumoral efficacy of most treatments currently applied in the clinic. Previous studies have evaluated the antitumoral immune response triggered by (TLR) agonists, such as poly(I:C), imiquimod (R837) or resiquimod (R848) as monotherapies; however, their combination for the treatment of cancer has not been explored. This study investigates the antitumoral efficacy and the macrophage reprogramming triggered by poly(I:C) combined with R848 or with R837, versus single treatments. METHODS: TLR agonist treatments were evaluated in vitro for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry using primary human and murine M-CSF-differentiated macrophages. Cytotoxic activity of TLR-treated macrophages toward cancer cells was evaluated with an in vitro functional assay by flow cytometry. For in vivo experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry, RT-qPCR, multispectral immunophenotyping, quantitative proteomic experiments, and protein-protein interaction analysis. RESULTS: Results demonstrated the higher efficacy of poly(I:C) combined with R848 versus single treatments or combined with R837 to polarize macrophages toward M1-like antitumor effectors in vitro. In vivo, the intratumoral synergistic combination of poly(I:C)+R848 significantly prevented tumor growth and metastasis in lung cancer and fibrosarcoma immunocompetent murine models. Regressing tumors showed increased infiltration of macrophages with a higher M1:M2 ratio, recruitment of CD4+ and CD8+ T cells, accompanied by a reduction of immunosuppressive CD206+ TAMs and FOXP3+/CD4+ T cells. The depletion of both CD4+ and CD8+ T cells resulted in complete loss of treatment efficacy. Treated mice acquired systemic antitumoral response and resistance to tumor rechallenge mediated by boosted macrophage cytotoxic activity and T-cell proliferation. Proteomic experiments validate the superior activation of innate immunity by poly(I:C)+R848 combination versus single treatments or poly(I:C)+R837, and protein-protein-interaction network analysis reveal the key activation of the STAT1 pathway. DISCUSSION: These findings demonstrate the antitumor immune responses mediated by macrophage activation on local administration of poly(I:C)+R848 combination and support the intratumoral application of this therapy to patients with solid tumors in the clinic.

Myocardial hypoxic stress mediates functional cardiac extracellular vesicle release.

AIMS: Increased shedding of extracellular vesicles (EVs)-small, lipid bilayer-delimited particles with a role in paracrine signalling-has been associated with human pathologies, e.g. atherosclerosis, but whether this is true for cardiac diseases is unknown. METHODS AND RESULTS: Here, we used the surface antigen CD172a as a specific marker of cardiomyocyte (CM)-derived EVs; the CM origin of CD172a+ EVs was supported by their content of cardiac-specific proteins and heart-enriched microRNAs. We found that patients with aortic stenosis, ischaemic heart disease, or cardiomyopathy had higher circulating CD172a+ cardiac EV counts than did healthy subjects. Cellular stress was a major determinant of EV release from CMs, with hypoxia increasing shedding in in vitro and in vivo experiments. At the functional level, EVs isolated from the supernatant of CMs derived from human-induced pluripotent stem cells and cultured in a hypoxic atmosphere elicited a positive inotropic response in unstressed CMs, an effect we found to be dependent on an increase in the number of EVs expressing ceramide on their surface. Of potential clinical relevance, aortic stenosis patients with the highest counts of circulating cardiac CD172a+ EVs had a more favourable prognosis for transcatheter aortic valve replacement than those with lower counts. CONCLUSION: We identified circulating CD172a+ EVs as cardiac derived, showing their release and function and providing evidence for their prognostic potential in aortic stenosis patients.

Flow Cytometry Instrument Setting as a Crucial Checkpoint for Optimal T-Cell Analysis and Sorting.

Although flow cytometry and cell sorting are widely used by immunologists both for basic and translation research, many aspects of these techniques should be optimized to obtain reproducible and meaningful data. In this chapter we provide some protocols and tips on instrument setting, multicolor panel design and T-cell immunophenotyping and proliferation assay.

Gut vascular barrier impairment leads to intestinal bacteria dissemination and colorectal cancer metastasis to liver.

Metastasis is facilitated by the formation of a "premetastatic niche," which is fostered by primary tumor-derived factors. Colorectal cancer (CRC) metastasizes mainly to the liver. We show that the premetastatic niche in the liver is induced by bacteria dissemination from primary CRC. We report that tumor-resident bacteria Escherichia coli disrupt the gut vascular barrier (GVB), an anatomical structure controlling bacterial dissemination along the gut-liver axis, depending on the virulence regulator VirF. Upon GVB impairment, bacteria disseminate to the liver, boost the formation of a premetastatic niche, and favor the recruitment of metastatic cells. In training and validation cohorts of CRC patients, we find that the increased levels of PV-1, a marker of impaired GVB, is associated with liver bacteria dissemination and metachronous distant metastases. Thus, PV-1 is a prognostic marker for CRC distant recurrence and vascular impairment, leading to liver metastases.

Unveiling role of sphingosine-1-phosphate receptor 2 as a brake of epithelial stem cell proliferation and a tumor suppressor in colorectal cancer.

BACKGROUND: Sphingosine-1-phosphate receptor 2 (S1PR2) mediates pleiotropic functions encompassing cell proliferation, survival, and migration, which become collectively de-regulated in cancer. Information on whether S1PR2 participates in colorectal carcinogenesis/cancer is scanty, and we set out to fill the gap. METHODS: We screened expression changes of S1PR2 in human CRC and matched normal mucosa specimens [N = 76]. We compared CRC arising in inflammation-driven and genetically engineered models in wild-type (S1PR2+/+) and S1PR2 deficient (S1PR2-/-) mice. We reconstituted S1PR2 expression in RKO cells and assessed their growth in xenografts. Functionally, we mimicked the ablation of S1PR2 in normal mucosa by treating S1PR2+/+ organoids with JTE013 and characterized intestinal epithelial stem cells isolated from S1PR2-/-Lgr5-EGFP- mice. RESULTS: S1PR2 expression was lost in 33% of CRC; in 55%, it was significantly decreased, only 12% retaining expression comparable to normal mucosa. Both colitis-induced and genetic Apc+/min mouse models of CRC showed a higher incidence in size and number of carcinomas and/or high-grade adenomas, with increased cell proliferation in S1PR2-/- mice compared to S1PR2+/+ controls. Loss of S1PR2 impaired mucosal regeneration, ultimately promoting the expansion of intestinal stem cells. Whereas its overexpression attenuated cell cycle progression, it reduced the phosphorylation of AKT and augmented the levels of PTEN. CONCLUSIONS: In normal colonic crypts, S1PR2 gains expression along with intestinal epithelial cells differentiation, but not in intestinal stem cells, and contrasts intestinal tumorigenesis by promoting epithelial differentiation, preventing the expansion of stem cells and braking their malignant transformation. Targeting of S1PR2 may be of therapeutic benefit for CRC expressing high Lgr5.

Two subsets of stem-like CD8+ memory T cell progenitors with distinct fate commitments in humans.

T cell memory relies on the generation of antigen-specific progenitors with stem-like properties. However, the identity of these progenitors has remained unclear, precluding a full understanding of the differentiation trajectories that underpin the heterogeneity of antigen-experienced T cells. We used a systematic approach guided by single-cell RNA-sequencing data to map the organizational structure of the human CD8+ memory T cell pool under physiological conditions. We identified two previously unrecognized subsets of clonally, epigenetically, functionally, phenotypically and transcriptionally distinct stem-like CD8+ memory T cells. Progenitors lacking the inhibitory receptors programmed death-1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) were committed to a functional lineage, whereas progenitors expressing PD-1 and TIGIT were committed to a dysfunctional, exhausted-like lineage. Collectively, these data reveal the existence of parallel differentiation programs in the human CD8+ memory T cell pool, with potentially broad implications for the development of immunotherapies and vaccines.

Macrophage morphology correlates with single-cell diversity and prognosis in colorectal liver metastasis.

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.

Impact of RAS mutations on the immune infiltrate of colorectal liver metastases: A preliminary study.

Kirsten rat sarcoma viral oncogene homolog KRAS proto-oncogene is the most common altered gene in colorectal cancer (CRC). Determining its mutational status, which is associated with worse prognosis and resistance to anti-epidermal growth factor receptor (EGFR) inhibitors, is essential for managing patients with CRC and colon liver metastases (CLM). Emerging studies highlighted the relationship of KRAS-mutated cancers and tumor microenvironment components, mainly with T cells. The aim of this study was to analyze the relationship of CLM immune cell infiltrate with KRAS mutational status. We performed a retrospective study on paraffin-embedded CLM tissue sections from patients surgically resected at the Department of Hepatobiliary and General Surgery of Humanitas Clinical and Cancer Center. We studied the distribution of lymphocytes (CD3+ cells), macrophages (CD163+), and neutrophils (CD66b+) in CLM tumoral and peritumoral area. Percentage of positive cells was correlated with tumor macroscopic characteristic, clinical aspects, and KRAS mutation. We observed a significant increase in CD66b+ cells in the peritumoral area in patients KRAS-mutated compared to KRAS wild-type patients. Percentages of lymphocytes and macrophages did not show significant differences. Further, neutrophils were found to be significantly increased also in the bloodstream of KRAS-mutated patients, indicating increased mobilization of neutrophils and recruitment in the CLM site. In conclusion, this study reveals a new intriguing aspect of the peritumoral microenvironment, which could pave the way for new prognostic and predictive markers for patient stratification.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Intracerebral Injection of Extracellular Vesicles from Mesenchymal Stem Cells Exerts Reduced Aß Plaque Burden in Early Stages of a Preclinical Model of Alzheimer's Disease.

Bone marrow Mesenchymal Stem Cells (BM-MSCs), due to their strong protective and anti-inflammatory abilities, have been widely investigated in the context of several diseases for their possible therapeutic role, based on the release of a highly proactive secretome composed of soluble factors and Extracellular Vesicles (EVs). BM-MSC-EVs, in particular, convey many of the beneficial features of parental cells, including direct and indirect ß-amyloid degrading-activities, immunoregulatory and neurotrophic abilities. Therefore, EVs represent an extremely attractive tool for therapeutic purposes in neurodegenerative diseases, including Alzheimer's disease (AD). We examined the therapeutic potential of BM-MSC-EVs injected intracerebrally into the neocortex of APPswe/PS1dE9 AD mice at 3 and 5 months of age, a time window in which the cognitive behavioral phenotype is not yet detectable or has just started to appear. We demonstrate that BM-MSC-EVs are effective at reducing the Aß plaque burden and the amount of dystrophic neurites in both the cortex and hippocampus. The presence of Neprilysin on BM-MSC-EVs, opens the possibility of a direct ß-amyloid degrading action. Our results indicate a potential role for BM-MSC-EVs already in the early stages of AD, suggesting the possibility of intervening before overt clinical manifestations.

TNF-Stimulated Gene-6 Is a Key Regulator in Switching Stemness and Biological Properties of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are well established to have promising therapeutic properties. TNF-stimulated gene-6 (TSG-6), a potent tissue-protective and anti-inflammatory factor, has been demonstrated to be responsible for a significant part of the tissue-protecting properties mediated by MSCs. Nevertheless, current knowledge about the biological function of TSG-6 in MSCs is limited. Here, we demonstrated that TSG-6 is a crucial factor that influences many functional properties of MSCs. The transcriptomic sequencing analysis of wild-type (WT) and TSG-6-/- -MSCs shows that the loss of TSG-6 expression leads to the perturbation of several transcription factors, cytokines, and other key biological pathways. TSG-6-/- -MSCs appeared morphologically different with dissimilar cytoskeleton organization, significantly reduced size of extracellular vesicles, decreased cell proliferative rate, and loss of differentiation abilities compared with the WT cells. These cellular effects may be due to TSG-6-mediated changes in the extracellular matrix (ECM) environment. The supplementation of ECM with exogenous TSG-6, in fact, rescued cell proliferation and changes in morphology. Importantly, TSG-6-deficient MSCs displayed an increased capacity to release interleukin-6 conferring pro-inflammatory and pro-tumorigenic properties to the MSCs. Overall, our data provide strong evidence that TSG-6 is crucial for the maintenance of stemness and other biological properties of murine MSCs.

IL1R8 Deficiency Drives Autoimmunity-Associated Lymphoma Development.

Chronic inflammation, including that driven by autoimmunity, is associated with the development of B-cell lymphomas. IL1R8 is a regulatory receptor belonging to the IL1R family, which negatively regulates NF-κB activation following stimulation of IL1R or Toll-like receptor family members. IL1R8 deficiency is associated with the development of severe autoimmune lupus-like disease in lpr mice. We herein investigated whether concomitant exacerbated inflammation and autoimmunity caused by the deficiency of IL1R8 could recapitulate autoimmunity-associated lymphomagenesis. We thus monitored B-cell lymphoma development during the aging of IL1R8-deficient lpr mice, observing an increased lymphoid cell expansion that evolved to diffuse large B-cell lymphoma (DLBCL). Molecular and gene-expression analyses showed that the NF-κB pathway was constitutively activated in Il1r8 -/-/lpr B splenocytes. In human DLBCL, IL1R8 had reduced expression compared with normal B cells, and higher IL1R8 expression was associated with a better outcome. Thus, IL1R8 silencing is associated with increased lymphoproliferation and transformation in the pathogenesis of B-cell lymphomas associated with autoimmunity.

MicroRNA-127-3p controls murine hematopoietic stem cell maintenance by limiting differentiation.

The balance between self-renewal and differentiation is crucial to ensure the homeostasis of the hematopoietic system, and is a hallmark of hematopoietic stem cells. However, the underlying molecular pathways, including the role of micro-RNA, are not completely understood. To assess the contribution of micro-RNA, we performed micro-RNA profiling of hematopoietic stem cells and their immediate downstream progeny multi-potent progenitors from wild-type control and Pbx1-conditional knockout mice, whose stem cells display a profound self-renewal defect. Unsupervised hierarchical cluster analysis separated stem cells from multi-potent progenitors, suggesting that micro-RNA might regulate the first transition step in the adult hematopoietic development. Notably, Pbx1-deficient and wild-type cells clustered separately, linking micro-RNAs to self-renewal impairment. Differential expression analysis of micro-RNA in the physiological stem cell-to-multi-potent progenitor transition and in Pbx1-deficient stem cells compared to control stem cells revealed miR-127-3p as the most differentially expressed. Furthermore, miR-127-3p was strongly stem cell-specific, being quickly down-regulated upon differentiation and not re-expressed further downstream in the bone marrow hematopoietic hierarchy. Inhibition of miR-127-3p function in Lineage-negative cells, achieved through a lentiviral-sponge vector, led to severe stem cell depletion, as assessed with serial transplantation assays. miR-127-3p-sponged stem cells displayed accelerated differentiation, which was uncoupled from proliferation, accounting for the observed stem cell reduction. miR-127-3p overexpression in Lineage-negative cells did not alter stem cell pool size, but gave rise to lymphopenia, likely due to lack of miR-127-3p physiological downregulation beyond the stem cell stage. Thus, tight regulation of miR-127-3p is crucial to preserve the self-renewing stem cell pool and homeostasis of the hematopoietic system.

Macrophage ferroportin is essential for stromal cell proliferation in wound healing.

Iron recycling by macrophages is essential for erythropoiesis, but may also be relevant for iron redistribution to neighboring cells at the local tissue level. Using mice with iron retention in macrophages due to targeted inactivation of the iron exporter ferroportin, we investigated the role of macrophage iron release in hair follicle cycling and wound healing, a complex process leading to major clinical problems, if impaired. Genetic deletion of ferroportin in macrophages resulted in iron deficiency and decreased proliferation in epithelial cells, which consequently impaired hair follicle growth and caused transient alopecia. Hair loss was not related to systemic iron deficiency or anemia, thus indicating the necessity of local iron release from macrophages. Inactivation of macrophage ferroportin also led to delayed skin wound healing with defective granulation tissue formation and diminished fibroplasia. Iron retention in macrophages had no impact on the inflammatory processes accompanying wound healing, but affected stromal cell proliferation, blood and lymphatic vessel formation, and fibrogenesis. Our findings reveal that iron/ferroportin plays a largely underestimated role in macrophage trophic function in skin homeostasis and repair.