RESUMEN
BACKGROUND & AIMS: Surgical resection of the cancerous tissue represents one of the few curative treatment options for neoplastic liver disease. Such partial hepatectomy (PHx) induces hepatocyte hyperplasia, which restores liver function. PHx is associated with bacterial translocation, leading to an immediate immune response involving neutrophils and macrophages, which are indispensable for the priming phase of liver regeneration. Additionally, PHx induces longer-lasting intrahepatic apoptosis. Herein, we investigated the effect of apoptotic extracellular vesicles (aEVs) on neutrophil function and their role in this later phase of liver regeneration. METHODS: A total of 124 patients undergoing PHx were included in this study. Blood levels of the apoptosis marker caspase-cleaved cytokeratin-18 (M30) and circulating aEVs were analyzed preoperatively and on the first and fifth postoperative days. Additionally, the in vitro effects of aEVs on the secretome, phenotype and functions of neutrophils were investigated. RESULTS: Circulating aEVs increased at the first postoperative day and were associated with higher concentrations of M30, which was only observed in patients with complete liver recovery. Efferocytosis of aEVs by neutrophils induced an activated phenotype (CD11bhighCD16highCD66bhighCD62Llow); however, classical inflammatory responses such as NETosis, respiratory burst, degranulation, or secretion of pro-inflammatory cytokines were not observed. Instead, efferocytosing neutrophils released various growth factors including fibroblast growth factor-2 and hepatocyte growth factor (HGF). Accordingly, we observed an increase of HGF-positive neutrophils after PHx and a correlation of plasma HGF with M30 levels. CONCLUSIONS: These data suggest that the clearance of PHx-induced aEVs leads to a population of non-inflammatory but regenerative neutrophils, which may support human liver regeneration. LAY SUMMARY: In this study, we show that the surgical removal of a diseased part of the liver triggers a specific type of programmed cell death in the residual liver tissue. This results in the release of vesicles from dying cells into the blood, where they are cleared by circulating immune cells. These respond by secreting hepatocyte growth factors that could potentially support the regeneration of the liver remnant.
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Vesículas Extracelulares , Hiperplasia Nodular Focal , Humanos , Hepatectomía , Neutrófilos , Transporte Biológico , Regeneración HepáticaRESUMEN
OBJECTIVES: Chemotherapy-induced hematological toxicities are potentially life-threatening adverse drug reactions that vary between individuals. Recently, JMJD1C has been associated with gemcitabine/carboplatin-induced thrombocytopenia in non-small-cell lung cancer patients, making it a candidate marker for predicting the risk of toxicity. This study investigates if JMJD1C knockdown affects gemcitabine/carboplatin-sensitivity in cell lines. METHODS: Lentiviral transduction-mediated shRNA knockdown of JMJD1C in the cell lines K562 and MEG-01 were performed using shRNA#32 and shRNA#33. The knockdown was evaluated using qPCR. Cell proliferation, viability, and gemcitabine/carboplatin-sensitivity were subsequently determined using cell counts, trypan blue, and the MTT assay. RESULTS: ShRNA#33 resulted in JMJD1C downregulation by 56.24% in K562 and 68.10% in MEG-01. Despite incomplete knockdown, proliferation (reduction of cell numbers by 61-68%, day 7 post-transduction) and viability (reduction by 21-53%, day 7 post-transduction) were impaired in K562 and MEG-01 cells. Moreover, JMJD1C knockdown reduced the gemcitabine IC50-value for K562 cells (P < 0.01) and MEG-01 cells (P < 0.05) compared to scrambled shRNA control transduced cells. CONCLUSIONS: Our results suggest that JMJD1C is essential for proliferation, survival, and viability of K562 and MEG-01 cells. Further, JMJD1C also potentially affects the cells gemcitabine/carboplatin-sensitivity. Although further research is required, the findings show that JMJD1C could have an influential role for gemcitabine/carboplatin-sensitivity.
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Proliferación Celular/efectos de los fármacos , Histona Demetilasas con Dominio de Jumonji/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Oxidorreductasas N-Desmetilantes/genética , Carboplatino/efectos adversos , Carboplatino/farmacología , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/efectos adversos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Técnicas de Silenciamiento del Gen , Humanos , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Células Mieloides/efectos de los fármacos , Células Mieloides/patología , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , GemcitabinaRESUMEN
BACKGROUND AND AIMS: Systemic chemotherapy followed by hepatic resection is the treatment of choice for patients with colorectal cancer liver metastasis (CRCLM) but reliable biomarkers predicting response to therapy are needed. Spontaneous apoptosis of single tumour cells is common in CRCLM. We explored the potential of circulating apoptosis markers to predict treatment response. MATERIALS AND METHODS: Fifty-eight patients with CRCLM or hepatocellular carcinoma (HCC) were included in this study. Tumour tissue and blood samples were obtained before and after initiation of chemotherapy. Immunohistochemistry and ELISA assays were utilized to quantify the apoptosis marker caspase-cleaved cytokeratin 18 (M30) in tissue and circulation. RESULTS: CRCLM tissues showed more apoptotic tumour cells than HCC, or healthy liver. This was associated with elevated levels of circulating M30 (median = 244 U/l vs. 37 U/l in healthy controls, p = 0.009) which correlated with tumour volume (r2 = 0.92). Patients with progressive disease during chemotherapy showed higher M30 levels before therapy than responders (745 U/l vs. 136 U/l, p = 0.016). The predictive potential of M30 was higher than that of the tumour markers CA19-9 or CEA (AUC: 0.93, 0.63, and 0.78, respectively). CONCLUSIONS: Apoptotic tumour cells release cellular debris into the circulation, which provides information about tumour size and vitality.
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Carcinoma Hepatocelular , Neoplasias Colorrectales , Neoplasias Hepáticas , Humanos , Apoptosis , Biomarcadores de Tumor , Caspasas , Neoplasias Colorrectales/tratamiento farmacológico , Queratina-18 , Neoplasias Hepáticas/tratamiento farmacológico , Carga TumoralRESUMEN
Sporadic apoptosis of tumour cells is a commonly observed feature of colorectal cancer (CRC) and strongly correlates with adverse patient prognosis. The uptake of apoptotic cell debris by neutrophils induces a non-inflammatory, pro-regenerative, and hence potentially pro-tumorigenic phenotype. In this study, we therefore sought to investigate the impact of apoptotic CRC cells on neutrophils and its consequence on other immune cells of the tumour microenvironment. Apoptosis induced by combined TNFα-treatment and UV-C irradiation, as well as various chemotherapeutic agents, led to a substantial release of neutrophil-attracting chemokines, most importantly interleukin-8 (IL-8), in both primary patient-derived and established CRC cells. Accordingly, conditioned media of apoptotic tumour cells selectively stimulated chemotaxis of neutrophils, but not T cells or monocytes. Notably, caspase-inhibition partially reduced IL-8 secretion, suggesting that caspase activity might be required for apoptosis-induced IL-8 release. Moreover, apoptotic tumour cell-conditioned media considerably prolonged neutrophil lifespan and induced an activated CD66bhighCD11bhighCD62Llow phenotype, comparable to that of tumour-associated neutrophils in CRC patients, as assessed by flow cytometry of dissociated CRC tissues. Immunohistochemical analyses of 35 CRC patients further revealed a preferential accumulation of neutrophils at sites of apoptotic tumour cells defined by the expression of epithelial cell-specific caspase-cleaved cytokeratin-18. The same areas were also highly infiltrated by macrophages, while T cells were virtually absent. Notably, neutrophils induced an M2-like CD86lowCD163+CD206+ phenotype in co-cultured monocyte-derived macrophages and suppressed LPS-induced pro-inflammatory cytokine release. In an in vitro transwell model, IL-8 blockade efficiently prevented neutrophil-induced anti-inflammatory macrophage polarisation by inhibiting neutrophil migration towards IL-8 gradients generated by apoptotic CRC cells. To conclude, our data suggest that apoptotic cancer cells release chemotactic factors that attract neutrophils into the tumour, where their interaction with neighbouring macrophages might promote an immunologically unfavourable tumour microenvironment. This effect may contribute to tumour recurrence after chemotherapy-induced apoptosis.
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Neoplasias Colorrectales , Interleucina-8 , Apoptosis , Caspasas/metabolismo , Neoplasias Colorrectales/patología , Medios de Cultivo Condicionados/metabolismo , Medios de Cultivo Condicionados/farmacología , Humanos , Interleucina-8/metabolismo , Infiltración Neutrófila , Neutrófilos/metabolismo , Microambiente TumoralRESUMEN
Fibroblasts are the most abundant stromal constituents of the tumour microenvironment in primary as well as metastatic colorectal cancer (CRC). Their supportive effect on tumour cells is well established. There is growing evidence that stromal fibroblasts also modulate the immune microenvironment in tumours. Here, we demonstrate a difference in fibroblast-mediated immune modulation between primary CRC and peritoneal metastasis. Cancer-associated fibroblasts (CAFs) were isolated from primary cancer and from peritoneal metastases (MAFs) from a total of 17 patients. The ectoenzyme CD38 was consistently expressed on the surface of all MAFs, while it was absent from CAFs. Furthermore, MAFs secreted higher levels of IGFBP2, CXCL2, CXCL6, CXCL12, PDGF-AA, FGFb, and IL-6. This was associated with a decreased activation of macrophages and a suppression of CD25 expression and proliferation of co-cultivated T-cells. Downregulation of IGFBP2 abolished these immunosuppressive effects of MAFs. Taken together, these results show that MAFs contribute to an immunosuppressive tumour microenvironment in CRC metastases by modulating the phenotype of immune cells through an IGFBP2-dependent mechanism.