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.

Filamin A is involved in human intrahepatic cholangiocarcinoma aggressiveness and progression.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (iCCA) is a primary liver tumour, characterized by poor prognosis and lack of effective therapy. The cytoskeleton protein Filamin A (FLNA) is involved in cancer progression and metastasis, including primary liver cancer. FLNA is cleaved by calpain, producing a 90 kDa fragment (FLNACT ) that can translocate to the nucleus and inhibit gene transcription. We herein aim to define the role of FLNA and its cleavage in iCCA carcinogenesis. METHODS & RESULTS: We evaluated the expression and localization of FLNA and FLNACT in liver samples from iCCA patients (n = 82) revealing that FLNA expression was independently correlated with disease-free survival. Primary tumour cells isolated from resected iCCA patients expressed both FLNA and FLNACT , and bulk RNA sequencing revealed a significant enrichment of cell proliferation and cell motility pathways in iCCAs with high FLNA expression. Further, we defined the impact of FLNA and FLNACT on the proliferation and migration of primary iCCA cells (n = 3) and HuCCT1 cell line using silencing and Calpeptin, a calpain inhibitor. We observed that FLNA silencing decreased cell proliferation and migration and Calpeptin was able to reduce FLNACT expression in both the HuCCT1 and iCCA cells (p < .05 vs. control). Moreover, Calpeptin 100 µM decreased HuCCT1 and primary iCCA cell proliferation (p <.00001 vs. control) and migration (p < .05 vs. control). CONCLUSIONS: These findings demonstrate that FLNA is involved in human iCCA progression and calpeptin strongly decreased FLNACT expression, reducing cell proliferation and migration.

Multimodal single-cell profiling of intrahepatic cholangiocarcinoma defines hyperactivated Tregs as a potential therapeutic target.

BACKGROUND & AIMS: The landscape and function of the immune infiltrate of intrahepatic cholangiocarcinoma (iCCA), a rare, yet aggressive tumor of the biliary tract, remains poorly characterized, limiting development of successful immunotherapies. Herein, we aimed to define the molecular characteristics of tumor-infiltrating leukocytes with a special focus on CD4+ regulatory T cells (Tregs). METHODS: We used high-dimensional single-cell technologies to characterize the T-cell and myeloid compartments of iCCA tissues, comparing these with their tumor-free peritumoral and circulating counterparts. We further used genomics and cellular assays to define the iCCA-specific role of a novel transcription factor, mesenchyme homeobox 1 (MEOX1), in Treg biology. RESULTS: We found poor infiltration of putative tumor-specific CD39+ CD8+ T cells accompanied by abundant infiltration of hyperactivated CD4+ Tregs. Single-cell RNA-sequencing identified an altered network of transcription factors in iCCA-infiltrating compared to peritumoral T cells, suggesting reduced effector functions by tumor-infiltrating CD8+ T cells and enhanced immunosuppression by CD4+ Tregs. Specifically, we found that expression of MEOX1 was highly enriched in tumor-infiltrating Tregs, and demonstrated that MEOX1 overexpression is sufficient to reprogram circulating Tregs to acquire the transcriptional and epigenetic landscape of tumor-infiltrating Tregs. Accordingly, enrichment of the MEOX1-dependent gene program in Tregs was strongly associated with poor prognosis in a large cohort of patients with iCCA. CONCLUSIONS: We observed abundant infiltration of hyperactivated CD4+ Tregs in iCCA tumors along with reduced CD8+ T-cell effector functions. Interfering with hyperactivated Tregs should be explored as an approach to enhance antitumor immunity in iCCA. LAY SUMMARY: Immune cells have the potential to slow or halt the progression of tumors. However, some tumors, such as intrahepatic cholangiocarcinoma, are associated with very limited immune responses (and infiltration of cancer-targeting immune cells). Herein, we show that a specific population of regulatory T cells (a type of immune cell that actually suppresses the immune response) are hyperactivated in intrahepatic cholangiocarcinoma. Targeting these cells could enable cancer-targeting immune cells to act more effectively and should be looked at as a potential therapeutic approach to this aggressive cancer type.

The kinase PKCα selectively upregulates interleukin-17A during Th17 cell immune responses.

Transforming growth-factor ß (TGFß) has been implicated in T helper 17 (Th17) cell biology and in triggering expression of interleukin-17A (IL-17A), which is a key Th17 cell cytokine. Deregulated TGFß receptor (TGFßR) signaling has been implicated in Th17-cell-mediated autoimmune pathogenesis. Nevertheless, the full molecular mechanisms involved in the activation of the TGFßR pathway in driving IL-17A expression remain unknown. Here, we identified protein kinase C α (PKCα) as a signaling intermediate specific to the Th17 cell subset in the activation of TGFßRI. We have shown that PKCα physically interacts and functionally cooperates with TGFßRI to promote robust SMAD2-3 activation. Furthermore, PKCα-deficient (Prkca(-/-)) cells demonstrated a defect in SMAD-dependent IL-2 suppression, as well as decreased STAT3 DNA binding within the Il17a promoter. Consistently, Prkca(-/-) cells failed to mount appropriate IL-17A, but not IL-17F, responses in vitro and were resistant to induction of Th17-cell-dependent experimental autoimmune encephalomyelitis in vivo.

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.

Relationship between Epithelial-to-Mesenchymal Transition and Tumor-Associated Macrophages in Colorectal Liver Metastases.

The liver is the most common metastatic site in colorectal cancer (CRC) patients. Indeed, 25-30% of the cases develop colorectal liver metastasis (CLM), showing an extremely poor 5-year survival rate and resistance to conventional anticancer therapies. Tumor-associated macrophages (TAMs) provide a nurturing microenvironment for CRC metastasis, promoting epithelial-to-mesenchymal transition (EMT) through the TGF-ß signaling pathway, thus driving tumor cells to acquire mesenchymal properties that allow them to migrate from the primary tumor and invade the new metastatic site. EMT is known to contribute to the disruption of blood vessel integrity and the generation of circulating tumor cells (CTCs), thus being closely related to high metastatic potential in numerous solid cancers. Despite the fact that it is well-recognized that the crosstalk between tumor cells and the inflammatory microenvironment is crucial in the EMT process, the association between the EMT and the role of TAMs is still poorly understood. In this review, we elaborated on the role that TAMs exert in the induction of EMT during CLM development. Since TAMs are the major source of TGF-ß in the liver, we also focused on novel insights into their role in TGF-ß-induced EMT.

Deficient Natural Killer Cell NKp30-Mediated Function and Altered NCR3 Splice Variants in Hepatocellular Carcinoma.

The activating natural cytotoxicity receptor NKp30 is critical for natural killer (NK) cell function and tumor immune surveillance. The natural cytotoxicity receptor-3 (NCR3) gene is transcribed into several splice variants whose physiological relevance is still incompletely understood. In this study, we investigated the role of NKp30 and its major ligand B7 homolog 6 (B7-H6) in patients with hepatocellular carcinoma (HCC). Peripheral blood NK cell phenotype was skewed toward a defective/exhausted immune profile with decreased frequencies of cells expressing NKp30 and natural killer group 2, member D and an increased proportion of cells expressing T-cell immunoglobulin and mucin-domain containing-3. Moreover, NKp30-positive NK cells had a reduced expression of NCR3 immunostimulatory splice variants and an increased expression of the inhibitory variant in patients with advanced tumor, resulting in deficient NKp30-mediated functionality. Tumor-infiltrating lymphocytes showed a prevalent inhibitory NKp30 isoform profile, consistent with decreased NKp30-mediated function. Of note, there were significant differences in the cytokine milieu between the neoplastic and the surrounding non-neoplastic tissue, which may have further influenced NKp30 function. Exposure of NK cells to B7-H6-expressing HCC cells significantly down-modulated NKp30, that was prevented by small interfering RNA-mediated knockdown, suggesting a role for this ligand in inhibiting NKp30-mediated responses. Interestingly, B7-H6 expression was reduced in HCC tissue and simultaneously augmented as a soluble form in HCC patients, particularly those with advanced staging or larger nodule size. Conclusion: These findings provide evidence in support of a role of NKp30 and its major ligand in HCC development and evolution.

Rapid automated diagnosis of primary hepatic tumour by mass spectrometry and artificial intelligence.

BACKGROUND AND AIMS: Complete surgical resection with negative margin is one of the pillars in treatment of liver tumours. However, current techniques for intra-operative assessment of tumour resection margins are time-consuming and empirical. Mass spectrometry (MS) combined with artificial intelligence (AI) is useful for classifying tissues and provides valuable prognostic information. The aim of this study was to develop a MS-based system for rapid and objective liver cancer identification and classification. METHODS: A large dataset derived from 222 patients with hepatocellular carcinoma (HCC, 117 tumours and 105 non-tumours) and 96 patients with mass-forming cholangiocarcinoma (MFCCC, 50 tumours and 46 non-tumours) were analysed by Probe Electrospray Ionization (PESI) MS. AI by means of support vector machine (SVM) and random forest (RF) algorithms was employed. For each classifier, sensitivity, specificity and accuracy were calculated. RESULTS: The overall diagnostic accuracy exceeded 94% in both the AI algorithms. For identification of HCC vs non-tumour tissue, RF was the best, with 98.2% accuracy, 97.4% sensitivity and 99% specificity. For MFCCC vs non-tumour tissue, both algorithms gave 99.0% accuracy, 98% sensitivity and 100% specificity. CONCLUSIONS: The herein reported MS-based system, combined with AI, permits liver cancer identification with high accuracy. Its bench-top size, minimal sample preparation and short working time are the main advantages. From diagnostics to therapeutics, it has the potential to influence the decision-making process in real-time with the ultimate aim of improving cancer patient cure.

Constitutively active Lck kinase in T cells drives antigen receptor signal transduction.

T cell antigen receptor (TCR) and coreceptor ligation is thought to initiate signal transduction by inducing activation of the kinase Lck. Here we showed that catalytically active Lck was present in unstimulated naive T cells and thymocytes and was readily detectable in these cells in lymphoid organs. In naive T cells up to approximately 40% of total Lck was constitutively activated, part of which was also phosphorylated on the C-terminal inhibitory site. Formation of activated Lck was independent of TCR and coreceptors but required Lck catalytic activity and its maintenance relied on monitoring by the HSP90-CDC37 chaperone complex to avoid degradation. The amount of activated Lck did not change after TCR and coreceptor engagement; however it determined the extent of TCR-zeta phosphorylation. Our findings suggest a dynamic regulation of Lck activity that can be promptly utilized to initiate T cell activation and have implications for signaling by other immune receptors.

Filamin A expression predicts early recurrence of hepatocellular carcinoma after hepatectomy.

BACKGROUND & AIMS: Recurrence of hepatocellular carcinoma (HCC) after hepatectomy is very high. A predictive marker of early recurrence (ER) capable of personalizing follow-up and developing a new target therapy would be beneficial. The overexpression of Filamin-A (FLNA), a cytoskeleton protein with scaffolding properties, has recently been associated with progression in tumours. The aim of this study was to test the expression of FLNA in a cohort of patients operated for HCC. METHODS: A retrospective cohort of patients who underwent hepatic resection at Humanitas Clinical and Research Center between January 2004 and December 2014 was analysed. FLNA was tested, using a tissue microarray, in the HCC and in the surrounding tissues. The endpoint was the role of FLNA expression in predicting ER of HCC after hepatectomy. Analyses were performed following the REMARK guidelines. RESULTS: A total of 113 patients were considered. FLNA was expressed only in the tumoral tissue. Several variables, including T stage, tumour number, tumour size, type of viral hepatitis, type of hepatectomy and intra and peritumoral immune-reactivity to FLNA were significantly associated with ER by univariate analysis. With multivariate analysis, only T stage (HR=2.108; P=.002), tumour number (HR=1.586; P=.023), intra-tumoral (HR=2.672; P<.001) and peritumoral immune-reactivity to FLNA (HR=2.569; P<.001), significantly correlated with ER. The logistic regression analysis revealed that advanced T stage (OR=2.985; P=.001), HCV-infection (OR=1.219; P=.008) and advanced tumour grading (OR=2.781; P=.002) were associated with intratumoral FLNA immune-reactivity. CONCLUSIONS: FLNA expression predicts recurrence of HCC after hepatectomy. This finding provides important insights that would help physicians to personalize follow-up strategies.

Agrin is required for survival and function of monocytic cells.

Agrin, an extracellular matrix protein belonging to the heterogeneous family of heparan sulfate proteoglycans (HSPGs), is expressed by cells of the hematopoietic system but its role in leukocyte biology is not yet clear. Here we demonstrate that agrin has a crucial, nonredundant role in myeloid cell development and functions. We have identified lineage-specific alterations that affect maturation, survival and properties of agrin-deficient monocytic cells, and occur at stages later than stem cell precursors. Our data indicate that the cell-autonomous signals delivered by agrin are sensed by macrophages through the α-DC (DG) receptor and lead to the activation of signaling pathways resulting in rearrangements of the actin cytoskeleton during the phagocytic synapse formation and phosphorylation of extracellular signal-regulated kinases (Erk 1/2). Altogether, these data identify agrin as a novel player of innate immunity.

Unveiling the prognostic role of blood inflammatory indexes in a retrospective cohort of patients undergoing liver resection for intrahepatic cholangiocarcinoma.

BACKGROUND: Systemic inflammation is relevant in intrahepatic cholangiocarcinoma (iCCA), but controversial results exist on the prognostic role of inflammatory indexes and their correlation with tumor microenvironment (TME). We aimed to explore the biological and prognostic values of these indexes. MATERIALS AND METHODS: A retrospective cohort study involving iCCA patients who underwent hepatic resection between 2010-2021 was conducted. The neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), and clinic-pathological factors were recorded. Immune-cell subpopulations, isolated from surgical specimens, were analyzed by flow cytometry. NLR and LMR cut-offs were calculated by X-Tile software. Linear regression, Kaplan-Meier, and Cox regression analyses were conducted. RESULTS: A total of 101 iCCA patients were considered. NLR ≥3.83 and LMR <2.28 correlated with worse survival. Patients were divided into groups: 67 (66.3%) in the low-risk and 34 (33.7%) in the high-risk (having at least one worse prognostic ratio). The 5-year overall survival was 49.8% and 18.9% for low- and high-risk groups, respectively (P=0.003). An elevated CA19.9 in the high-risk group gives 2.148 HR (95%CI:1.060-4.349) of mortality and 2.182 HR (95%CI:1.206-3.948) of disease recurrence. Flow cytometry analysis of 20 surgical specimens highlighted that NLR was associated with tumor-derived NLR (P=0.026) and LMR with tumor-infiltrating lymphocytes (P=0.002). In a subset of five high-risk vs five low-risk patients, T-cell evaluation showed a higher prevalence of CD4+ compared to CD8+ cells in the high-risk group (78.5% vs. 21.5%, P<0.0001). Conversely, low-risk patients demonstrated a noteworthy infiltration of CD8+ cells compared to the high-risk group (21.5% vs. 48.7%, P=0.037). CONCLUSIONS: The combination of blood inflammatory indexes determined two survival-risk profiles. The correlation between the blood scores and the iCCA microenvironment suggests a link between immune-cell infiltration and the risk group. These findings open the possibility of patient stratification with the chance to identify subgroups suitable for dedicated follow-up and targeted immuno-chemotherapy protocols.

The tumor microenvironment of VETC+ hepatocellular carcinoma is enriched of immunosuppressive TAMs spatially close to endothelial cells.

BACKGROUND AND AIM: VETC (vessel that encapsulate tumor cluster) is a peculiar vascular phenotype observed in hepatocellular carcinoma (HCC), associated with distant metastases and poor outcome. VETC has been linked to the Tie2/Ang2 axis and is characterized by lymphocytes poor (cold) tumor microenvironment (TME). In this setting the role of Tumor Associated Macrophages (TAMs) has never been explored. Aim of the study is to investigate the presence and features of TAMs in VETC+ HCC and the possible interplay between TAMs and endothelial cells (ECs). METHODS: The series under study included 42 HCC. Once separated according to the VETC phenotype (21 VETC+; 21 VETC-) we stained consecutive slides with immunohistochemistry for CD68, CD163 and Tie2. Slides were then scanned and QuPath used to quantify morphological features. RESULTS: VETC+ cases were significantly (p < 0.001) enriched with large, lipid rich CD163+ TAMs (M2 oriented) that were spatially close to ECs; HCC cells significantly (p: 0.002) overexpressed Tie2 with a polarization toward ECs. CONCLUSIONS: The pro-metastatic attitude of VETC is sustained by a strict morphological relationship between immunosuppressive M2-TAMs, ECs and Tie2-expressing HCC cells.

Cholangiocarcinoma-on-a-chip: A human 3D platform for personalised medicine.

Background & Aims: Cholangiocarcinoma (CCA) is a primary liver tumour characterised by a poor prognosis and limited therapeutic options. Available 3D human CCA models fail to faithfully recapitulate the tumour niche. We aimed to develop an innovative patient-specific CCA-on-chip platform. Methods: A CCA tumour microenvironment was recapitulated on a microfluidic three-channel chip using primary CCA cells, cancer-associated fibroblasts (CAFs), endothelial cells, and T cells isolated from CCA specimens (n = 6). CAF and CCA cells were co-cultured in the central channel, flanked by endothelial cells in one lateral channel, recreating a tubular structure. An extensive characterisation of this platform was carried out to investigate its diffusion ability, hydrogel properties, and changes in matrix composition. Cell phenotype and functional properties were assessed. Results: Primary cells seeded on the microfluidic device were shown to reproduce the architectural structure and maintain the original phenotype and functional properties. The tumour niche underwent a deep remodelling in the 3D device, with an increase in hydrogel stiffness and extracellular matrix deposition, mimicking in vivo CCA characteristics. T cells were incorporated into the device to assess its reliability for immune cell interaction studies. Higher T cell migration was observed using cells from patients with highly infiltrated tumours. Finally, the drug trial showed the ability of the device to recapitulate different drug responses based on patient characteristics. Conclusions: We presented a 3D CCA platform that integrates the major non-immune components of the tumour microenvironment and the T cell infiltrate, reflecting the CCA niche. This CCA-on-chip represents a reliable patient-specific 3D platform that will be of help to further elucidate the biological mechanisms involved in CCA and provide an efficient tool for personalised drug testing. Impact and implications: An innovative patient-specific cholangiocarcinoma (CCA)-on-chip platform was successfully developed, integrating the major components of the tumour microenvironment (tumour cells, cancer-associated fibroblasts, endothelial cells, and immune infiltrate) and faithfully mimicking the CCA niche. This CCA-on-chip represents a powerful tool for unravelling disease-associated cellular mechanisms in CCA and provides an efficient tool for personalised drug testing.

Self-antigen presentation by mouse B cells results in regulatory T-cell induction rather than anergy or clonal deletion.

Multiple mechanisms operate to ensure T-cell tolerance toward self-antigens. Three main processes have been described: clonal deletion, anergy, and deviation to CD4(+) regulatory T cells (Tregs) that suppress autoreactive T cells that have escaped the first 2 mechanisms. Although it is accepted that dendritic cells (DCs) and B cells contribute in maintaining T-cell tolerance to self-antigens, their relative contribution and the processes involved under physiologic conditions remain only partially characterized. In this study, we used different transgenic mouse models to obtain chimeras where a neo self-antigen is expressed by thymic epithelium and/or by DCs or B cells. We found that expression of cognate ligand in the thymus enhances antigen-specific FoxP3(+) cells independently of whether the self-antigen is expressed on thymic epithelium or only on DCs, but not on B cells. On the contrary, self-antigen expression by B cells was very efficient in inducing FoxP3(+) cells in the periphery, whereas self-antigen expression by DC led mainly to deletion and anergy of antigen-specific FoxP3(-) cells. The results presented in this study underline the role of B cells in Treg induction and may have important implications in clinical protocols aimed at the peripheral expansion of Tregs in patients.

The critical role of agrin in the hematopoietic stem cell niche.

Hematopoiesis is the process leading to the sustained production of blood cells by hematopoietic stem cells (HSCs). Growth, survival, and differentiation of HSCs occur in specialized microenvironments called "hematopoietic niches," through molecular cues that are only partially understood. Here we show that agrin, a proteoglycan involved in the neuromuscular junction, is a critical niche-derived signal that controls survival and proliferation of HSCs. Agrin is expressed by multipotent nonhematopoietic mesenchymal stem cells (MSCs) and by differentiated osteoblasts lining the endosteal bone surface, whereas Lin(-)Sca1(+)c-Kit(+) (LSK) cells express the α-dystroglycan receptor for agrin. In vitro, agrin-deficient MSCs were less efficient in supporting proliferation of mouse Lin(-)c-Kit(+) cells, suggesting that agrin plays a role in the hematopoietic cell development. These results were indeed confirmed in vivo through the analysis of agrin knockout mice (Musk-L;Agrn(-/-)). Agrin-deficient mice displayed in vivo apoptosis of CD34(+)CD135(-) LSK cells and impaired hematopoiesis, both of which were reverted by an agrin-sufficient stroma. These data unveil a crucial role of agrin in the hematopoietic niches and in the cross-talk between stromal and hematopoietic stem cells.

Regulatory T cells target chemokine secretion by dendritic cells independently of their capacity to regulate T cell proliferation.

The clinical manipulation of regulatory T cells (Tregs) represents a promising strategy for the regulation of unwanted immune responses. It is now becoming clear that Tregs exert multiple effects on different cell targets under particular conditions; however, the interplay between these different factors remains unclear. Using mouse Tregs of known Ag specificity, we report in this study two different levels of Treg-mediated suppression: one that targets T cell proliferation and one that targets dendritic cell-mediated proinflammatory chemokine (CCL3 and CCL4) production. These two effects can be dissociated, and whereas modulation of T cell proliferation depends on the strength of the antigenic stimulus, modulation of chemokine production by dendritic cells does not. We also provide evidence that the bystander effect of Tregs on immune responses observed in vivo may be in great part explained by a decrease in the recruitment of target T cells, and therefore in the magnitude of the response, rather than by a direct effect on their priming or proliferation. Overall, our results shed some light on the different aspects that need to be considered when attempting to modulate Tregs for clinical purposes.

Implication of metabolism in the polarization of tumor-associated-macrophages: the mass spectrometry-based point of view.

Tumor-associated macrophages (TAMs) represent one of the main tumor-infiltrating immune cell types and are generally categorized into either of two functionally contrasting subtypes, namely classical activated M1 macrophages and alternatively activated M2 macrophages. TAMs showed different activation states that can be represent by the two extremes of the complex profile of macrophages biology, the M1-like phenotype (pro-inflammatory activity) and the M2-like phenotype (anti-inflammatory activity). Based on the tumor type, and grades, TAMs can acquire different functions and properties; usually, the M1-like phenotype is typical of early tumor stages and is associated to an anti-tumor activity, while M2-like phenotype has a pro-inflammatory activity and is related to a poor patients' prognosis. The classification of macrophages into M1/M2 groups based on well-defined stimuli does not model the infinitely more complex tissue milieu where macrophages (potentially of different origin) would be exposed to multiple signals in different sequential order. This review aims to summarize the recent mass spectrometry-based (MS-based) metabolomics findings about the modifications of metabolism in TAMs polarization in different tumors. The published data shows that MS-based metabolomics is a promising tool to help better understanding TAMs metabolic phenotypes, although it is still poorly applied for TAMs metabolism. The knowledge of key metabolic alterations in TAMs is an essential step for discovering TAMs polarization novel biomarkers and developing novel therapeutic approaches targeting TAM metabolism to repolarize TAMs towards their anti-tumor phenotype.

Cellular Senescence in Liver Cancer: How Dying Cells Become "Zombie" Enemies.

Liver cancer represents the fourth leading cause of cancer-associated death worldwide. The heterogeneity of its tumor microenvironment (TME) is a major contributing factor of metastasis, relapse, and drug resistance. Regrettably, late diagnosis makes most liver cancer patients ineligible for surgery, and the frequent failure of non-surgical therapeutic options orientates clinical research to the investigation of new drugs. In this context, cellular senescence has been recently shown to play a pivotal role in the progression of chronic inflammatory liver diseases, ultimately leading to cancer. Moreover, the stem-like state triggered by senescence has been associated with the emergence of drug-resistant, aggressive tumor clones. In recent years, an increasing number of studies have emerged to investigate senescence-associated hepatocarcinogenesis and its derived therapies, leading to promising results. In this review, we intend to provide an overview of the recent evidence that unveils the role of cellular senescence in the most frequent forms of primary and metastatic liver cancer, focusing on the involvement of this mechanism in therapy resistance.

Macrophage morphology and distribution are strong predictors of prognosis in resected colorectal liver metastases: results from an external retrospective observational study.

INTRODUCTION: Tumor-associated macrophages (TAMs) are key components of a tumoral microenvironment and have been shown to impact prognosis in different cancers. Previously reported data showed that TAM morphology correlates with prognosis in colorectal liver metastases (CLMs) after hepatectomy, with smaller TAMs (S-TAMs) conferring a more favorable prognosis than larger ones (L-TAMs). This study aims to externally validate this finding. MATERIAL AND METHODS: The external cohort consisted of 84 formalin-fixed and paraffin-embedded surgical samples of CLMs and peritumoral tissue. Two-micrometer-section slides were obtained; the area and perimeter of 21 macrophages in each slide were recorded. The endpoints were TAMs morphometrics and their prognostic significance in relation to disease-free survival (DFS). RESULTS: The average macrophage perimeter was 71.5±14.1 µm whilst the average area was 217.7±67.8 µm 2 . At univariate analysis, the TAM area demonstrated a statistically significant association with DFS ( P =0.0006). Optimal area cutoff value was obtained, showing a sensitivity and specificity of 92 and 56%, respectively. S-TAMs and L-TAMs were associated with 3-year DFS rates of 60 and 8.5%, respectively ( P <0.001). Multivariate analysis confirmed the predictive role of TAM area for DFS [hazard ratio (HR)=5.03; 95% CI=1.70-14.94; P =0.003]. Moreover, in a subset of patients ( n =12) characterized by unfavorable ( n =6, recurrence within 3 months) or favorable ( n =6, no recurrence after 48 months) prognosis, TAMs showed a different distribution: L-TAMs were more abundant and closer to the tumor invasive margin in patients that encountered early recurrence and tended to cluster in foci significantly larger ( P =0.02). CONCLUSIONS: This external validation confirms that morphometric characterization of TAMs can serve as a simple readout of their diversity and allows to reliably stratify patient outcomes and predict disease recurrence after hepatectomy for CLMs.