Cancer-associated stromal fibroblasts (CAFs) are the main cellular constituents of reactive stroma in primary and metastatic cancer. We analyzed phenotypical characteristics of CAFs from human ...colorectal liver metastases (CLMs) and their role in inflammation and cancer progression. CAFs displayed a vimentin+ , α-smooth-muscle actin+ , and Thy-1+ phenotype similar to resident portal-located liver fibroblasts (LFs). We demonstrated that CLMs are inflammatory sites showing stromal expression of interleukin-8 (IL-8), a chemokine related to invasion and angiogenesis. In vitro analyses revealed a striking induction of IL-8 expression in CAFs and LFs by tumor necrosis factor-α (TNF-α). The effect of TNF-α on CAFs is inhibited by the nuclear factor-κB inhibitor parthenolide. Conditioned medium of CAFs and LFs similarly stimulated the migration of DLD-1, Colo-678, HuH7 carcinoma cells, and human umbilical vein endothelial cells in vitro . Pretreatment of CAFs with TNF-α increased the chemotaxis of Colo-678 colon carcinoma cells by conditioned medium of CAFs; however, blockage of IL-8 activity showed no inhibitory effect. In conclusion, these data raise the possibility that the majority of CAFs in CLM originate from resident LFs. TNF-α-induced up-regulation of IL-8 via nuclear factor-κB in CAFs is an inflammatory pathway, potentially permissive for cancer invasion that may represent a novel therapeutic target.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cells release heterogeneous nano-sized vesicles either as exosomes, being derived from endosomal compartments, or through budding from the plasma membrane as so-called microvesicles, commonly ...referred to as extracellular vesicles (EVs). EVs are known for their important roles in mammalian physiology and disease pathogenesis and provide a potential biomarker source in cancer patients. EVs are generally often analysed in bulk using Western blotting or by bead-based flow-cytometry or, with limited parameters, through nanoparticle tracking analysis. Due to their small size, single EV analysis is technically highly challenging. Here we demonstrate imaging flow cytometry (IFCM) to be a robust, multiparametric technique that allows analysis of single EVs and the discrimination of distinct EV subpopulations. We used IFCM to analyse the tetraspanin (CD9, CD63, CD81) surface profiles on EVs from human and murine cell cultures as well as plasma samples. The presence of EV subpopulations with specific tetraspanin profiles suggests that EV-mediated cellular responses are tightly regulated and dependent on cell environment. We further demonstrate that EVs with double positive tetraspanin expression (CD63
+
/CD81
+
) are enriched in cancer cell lines and patient plasma samples. In addition, we used IFCM to detect tumour-specific GFP-labelled EVs in the blood of mice bearing syngeneic intracerebral gliomas, indicating that this technique allows unprecedented disease modelling. In summary, our study highlights the heterogeneous and adaptable nature of EVs according to their marker profile and demonstrates that IFCM facilitates multiparametric phenotyping of EVs not only in vitro but also in patient plasma at a single EV level, with the potential for future functional studies and clinically relevant applications.
Abbreviation: EDTA = ethylenediamine tetraacetic acid
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Forty percent of non-small cell lung cancer (NSCLC) patients develop brain metastases, resulting in a dismal prognosis. However, patients in an oligo-metastatic brain disease setting seem to have ...better outcomes. Here, we investigate the possibility of using circulating tumor cells (CTCs) as biomarkers to differentiate oligo-metastatic patients for better risk assessment. Using the CellSearch
system, few CTCs were detected among NSCLC patients with brain metastases (
= 52, 12.5% ≥ two and 8.9% ≥ five CTC/7.5 mL blood) and especially oligo-metastatic brain patients (
= 34, 5.9%, and 2.9%). Still, thresholds of both ≥ two and ≥ five CTCs were independent prognostic indicators for shorter overall survival time among all of the NSCLC patients (
= 90, two CTC ≥ HR: 1.629,
= 0.024, 95% CI: 1.137⁻6.465 and five CTC ≥ HR: 2.846,
= 0.0304, CI: 1.104⁻7.339), as well as among patients with brain metastases (two CTC ≥ HR: 4.694,
= 0.004, CI: 1.650⁻13.354, and five CTC ≥ HR: 4.963,
= 0.003, CI: 1.752⁻14.061). Also, oligo-brain NSCLC metastatic patients with CTCs had a very poor prognosis (
= 0.019). Similarly, in other tumor entities, only 9.6% of patients with brain metastases (
= 52) had detectable CTCs. Our data indicate that although patients with brain metastases more seldom harbor CTCs, they are still predictive for overall survival, and CTCs might be a useful biomarker to identify oligo-metastatic NSCLC patients who might benefit from a more intense therapy.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Abstract Cancer stem cells are an attractive target for immunotherapeutic approaches to glioblastoma. However, an immune inhibitory phenotype of cells currently classified as “glioma-initiating ...cells” (GIC) might counteract recognition by immune effector cells. Here, we investigate the contribution of the non-classical MHC molecule HLA-E to the immunosuppressive phenotype of GIC. HLA-E is expressed in GIC lines and its expression is reduced upon differentiation of GIC in serum-containing culture conditions. Constitutive HLA-E inhibits natural killer (NK) cell-mediated lysis of GIC since small-interfering RNA-mediated HLA-E gene silencing enhances the immunogenicity of GIC. Increased GIC lysis was observed both in the CD133+ and in the CD133− compartment. Furthermore, the use of interferon-γ as a possible agent to boost an immune response against glioblastoma cells might be limited by the concurrent upregulation of HLA-E.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
DNA methylation analysis has become a powerful tool in neuropathology. Although DNA methylation-based classification usually shows high accuracy, certain samples cannot be classified and remain ...clinically challenging. We aimed to gain insight into these cases from a clinical perspective. To address, central nervous system (CNS) tumors were subjected to DNA methylation profiling and classified according to their calibrated score using the DKFZ brain tumor classifier (V11.4) as "≥ 0.84" (score ≥ 0.84), "0.3-0.84" (score 0.3-0.84), or "< 0.3" (score < 0.3). Histopathology, patient characteristics, DNA input amount, and tumor purity were correlated. Clinical outcome parameters were time to treatment decision, progression-free, and overall survival. In 1481 patients, the classifier identified 69 (4.6%) tumors with an unreliable score as "< 0.3". Younger age (P < 0.01) and lower tumor purity (P < 0.01) compromised accurate classification. A clinical impact was demonstrated as unclassifiable cases ("< 0.3") had a longer time to treatment decision (P < 0.0001). In a subset of glioblastomas, these cases experienced an increased time to adjuvant treatment start (P < 0.001) and unfavorable survival (P < 0.025). Although DNA methylation profiling adds an important contribution to CNS tumor diagnostics, clinicians should be aware of a potentially longer time to treatment initiation, especially in malignant brain tumors.
Non-small cell lung cancer (NSCLC) is currently the leading cause of cancer-related death worldwide, and the incidence of brain metastases (BM) in NSCLC patients is continuously increasing. The ...recent improvements of systemic treatment in NSCLC necessitate continuous updates on prognostic subgroups and factors determining overall survival (OS). In order to improve clinical decision-making in tumor boards, we investigated the clinical determinants affecting survival in patients with resectable NSCLC BM. A retrospective analysis was conducted of NSCLC patients with surgically resectable BM treated in our institution between 01/2015 and 12/2020. The relevant clinical factors affecting survival identified by univariate analysis were included in a multivariate logistic regression model. Overall, 264 patients were identified, with a mean age of 62.39 ± 9.98 years at the initial diagnosis of NSCLC BM and OS of 23.22 ± 1.71 months. The factors that significantly affected OS from the time of primary tumor diagnosis included the systemic metastatic load (median: 28.40 ± 4.82 vs. 40.93 ± 11.18 months, p = 0.021) as well as a number of BM <2 (median: 17.20 ± 2.52 vs. 32.53 ± 3.35 months, p = 0.014). When adjusted for survival time after neurosurgical intervention, a significant survival benefit was found in patients <60 years (median 16.13 ± 3.85 vs. 9.20 ± 1.39 months, p = 0.011) and, among others, patients without any concurrent systemic metastases at time of NSCLC BM diagnosis. Our data shows that the number of BM (singular/solitary), the Karnofsky Performance Status, gender, and age but not localization (infra-/supratentorial), mass-edema index or time to BM occurrence impact OS, and postsurgical survival in NSCLC BM patients. Additionally, our study shows that patients in prognostically favorable clinical subgroups an OS, which differs significantly from current statements in literature. The described clinically relevant factors may improve the understanding of the risks and the course of this disease and Faid future clinical decision making in tumor boards.
Glioblastomas are highly aggressive brain tumors of adults with poor clinical outcome. Despite a broad range of new and more specific treatment strategies, therapy of glioblastomas remains ...challenging and tumors relapse in all cases. Recent work demonstrated that the posttranslational hypusine modification of the eukaryotic initiation factor 5A (eIF-5A) is a crucial regulator of cell proliferation, differentiation and an important factor in tumor formation, progression and maintenance. Here we report that eIF-5A as well as the hypusine-forming enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) are highly overexpressed in glioblastoma patient samples. Importantly, targeting eIF-5A and its hypusine modification with GC7, a specific DHS-inhibitor, showed a strong antiproliferative effect in glioblastoma cell lines in vitro, while normal human astrocytes were not affected. Furthermore, we identified p53 dependent premature senescence, a permanent cell cycle arrest, as the primary outcome in U87-MG cells after treatment with GC7. Strikingly, combined treatment with clinically relevant alkylating agents and GC7 had an additive antiproliferative effect in glioblastoma cell lines. In addition, stable knockdown of eIF-5A and DHS by short hairpin RNA (shRNA) could mimic the antiproliferative effects of GC7. These findings suggest that pharmacological inhibition of eIF-5A may represent a novel concept to treat glioblastomas and may help to substantially improve the clinical course of this tumor entity.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cement augmentation via percutaneous vertebroplasty or kyphoplasty for treatment of spinal metastasis is a well-established treatment option. We assessed whether elevated intrametastatic pressure ...during cement augmentation results in an increased dissemination of tumour cells into the vascular circulation. We prospectively collected blood from patients with osteolytic spinal column metastases and analysed the prevalence of circulating tumour cells (CTCs) at three time-points: preoperatively, 20 minutes after cement augmentation, and 3-5 days postoperatively. Enrolling 21 patients, including 13 breast- (61.9%), 5 lung- (23.8%), and one (4.8%) colorectal-, renal-, and prostate-carcinoma patient each, we demonstrate a significant 1.8-fold increase of EpCAM+/K+ CTCs in samples taken 20 minutes post-cement augmentation (P < 0.0001). Despite increased mechanical CTC dissemination due to cement augmentation, follow-up blood draws demonstrated that no long-term increase of CTCs was present. Array-CGH analysis revealed a specific profile of the CTC collected 20 minutes after cement augmentation. This is the first study to report that peripheral CTCs are temporarily increased due to vertebral cement augmentation procedures. Our findings provide a rationale for the development of new prophylactic strategies to reduce the increased release of CTC after cement augmentation of osteolytic spinal metastases.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Kinesins play an important role in many physiological functions including intracellular vesicle transport and mitosis. The emerging role of kinesins in different cancers led us to investigate the ...expression and functional role of kinesins in meningioma. Therefore, we re-analyzed our previous microarray dataset of benign, atypical, and anaplastic meningiomas (
= 62) and got evidence for differential expression of five kinesins (KIFC1, KIF4A, KIF11, KIF14 and KIF20A). Further validation in an extended study sample (
= 208) revealed a significant upregulation of these genes in WHO°I to °III meningiomas (WHO°I
= 61, WHO°II
= 88, and WHO°III
= 59), which was most pronounced in clinically more aggressive tumors of the same WHO grade. Immunohistochemical staining confirmed a WHO grade-associated upregulated protein expression in meningioma tissues. Furthermore, high mRNA expression levels of KIFC1, KIF11, KIF14 and KIF20A were associated with shorter progression-free survival. On a functional level, knockdown of kinesins in Ben-Men-1 cells and in the newly established anaplastic meningioma cell line NCH93 resulted in a significantly inhibited tumor cell proliferation upon siRNA-mediated downregulation of KIF11 in both cell lines by up to 95% and 71%, respectively. Taken together, in this study we were able to identify the prognostic and functional role of several kinesin family members of which KIF11 exhibits the most promising properties as a novel prognostic marker and therapeutic target, which may offer new treatment options for aggressive meningiomas.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Glioblastoma (GBM) is the most frequent and lethal primary brain tumor in adults. Despite multimodal therapy combining resection, radio- and alkylating chemotherapy, disease recurrence is universal ...and prognosis of patients is poor. Glioblastoma stem-like cells (GSC), which can be grown as neurospheres from primary tumors in vitro, appear to be resistant to the established therapies and are suspected to be the driving force for disease recurrence. Thus, efficacy of emerging therapies may depend on targeting GSC. 5-aminolaevulinic acid-mediated photodynamic therapy (5-ALA/PDT) is a promising therapeutic approach in GBM. It utilizes the selective accumulation of the photosensitizer protoporphyrin IX (PPIX) in GBM cells after application of 5-ALA. When exposed to laser light of 635nm wavelength, PPIX initiates a photochemical reaction resulting in the generation of reactive oxygen species, which kill the tumor cells. Whether GSC accumulate PPIX and are sensitive to 5-ALA/PDT is currently unknown. Therefore, human GSC were derived from primary tumors and grown as neurospheres under serum free conditions. When subjected to exogenous 5-ALA, a dose- and time-dependent accumulation of PPIX in GSC was observed by flow cytometry, which varied between individual GSC preparations. Subsequent exposure to laser light of 635nm wavelength substantially killed GSC, whereas treatment with 5-ALA or exposure to laser light only had no effect. LD50 values differed between GSC preparations, but were negatively correlated with PPIX accumulation in GSC. In summary, we report for the first time that glioblastoma stem-like cells accumulate PPIX when subjected to 5-aminolaevulinic acid and are sensitive to 5-aminolaevulinc acid based photodynamic therapy.
•GSC accumulate PPIX upon application of 5-ALA.•GSC are sensitive to 5-ALA/PDT.•Sensitivity of GSC to ALA/PDT correlates with PPIX accumulation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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