Colorectal Cancer Cell Line SW480 and SW620 Released Extravascular Vesicles: Focus on Hypoxia-induced Surface Proteome Changes.

BACKGROUND/AIM: Extravascular vesicle (EV) proteome closely reflects the proteome of the cell of origin. Therefore, cancer cell-derived EV proteomic analysis could help in identifying cancer biomarkers. This study's goal was to investigate hypoxia-induced proteomic changes in EV released from hypoxic human isogenic non-metastatic colorectal cancer cells SW480 and metastatic colorectal cancer cells SW620. MATERIALS AND METHODS: EV were characterized by western blot, transmission electron microscopy, proteomic analysis using liquid chromatography time-of-flight-mass spectrometry and quantified by an label-free intensity-based absolute quantitation (iBAQ) approach. RESULTS: A total of 16 proteins in hypoxic EV exceeded normoxic EV protein levels in SW480 EV. Of them, 15 were also found in EV of hypoxic SW620 cells. The expression levels of proteins differed quantitatively: iBAQ (log 10) scores of the levels of five proteins in SW620 EV exceeded those in hypoxic SW480 EV and levels of 11 proteins in SW480 EV exceeded those of SW620 EV. CONCLUSION: Under hypoxia, colorectal cancer cells release EV that qualitatively and quantitatively change the surface proteome. In the future, the specific hypoxia-induced proteins could be developed as new biomarkers for non-invasive assessment of tumour hypoxia.

Extracellular Vesicles Derived from Hypoxic Colorectal Cancer Cells Confer Metastatic Phenotype to Non-metastatic Cancer Cells.

BACKGROUND/AIM: Tumor-secreted extracellular vesicles (EVs) play an important role as mediators of intercellular communication. Hypoxia is a common feature of solid tumors frequently associated with an aggressive clinical behavior. This study aimed to gain a deeper understanding into the functions of EVs in intercellular communication between primary and metastatic cancer cells under hypoxic conditions. MATERIALS AND METHODS: EVs were isolated from two isogenic colorectal cancer (CRC) cell lines SW480 and SW620 cultured under normoxic and hypoxic conditions. Their uptake and effects in SW480 and SW620 cells were studied using EV uptake, proliferation, spheroid-formation, wound healing and invasion assays. RESULTS: Our data showed that hypoxia enhanced the release of EVs from CRC cells in a Hypoxia Induced Factor (HIF)-1-dependent manner. Hypoxic EVs were taken up by CRC cells more efficiently than normoxic EVs. Hypoxic EVs stimulated motility, invasiveness and stemness of primary tumour-derived SW480 cells, whereas they had a little effect on metastasis-derived SW620 cells. CONCLUSION: Hypoxic colorectal cancer-derived EVs confer aggressiveness and invasiveness to hypoxia-naïve cancer cells.

Miniature diamond-anvil cells for FTIR-microspectroscopy of small quantities of biosamples.

Fourier transform infrared (FTIR) spectroscopy techniques and data analyses have become widely available, are easy to use, and are convenient for studies of various biosamples, especially in biomedical science. Yet, cultivation of cells and purification of cell components are costly, often methodically challenging, and time and labor consuming. Therefore, reduction of the sample amount is of high value. Here we propose a novel method for the analysis of small quantities of biosamples by FTIR-microscopy of dry films using a diamond-anvil cell (DAC). This approach allows us to decrease the sample volume at least a hundred times compared to that for a high-throughput screening device (HTS-XT, Bruker, Germany), while still obtaining homogeneous films, acquiring qualitative spectra, and using a conventional 15× objective instead of an ATR-objective. Both FTIR methods were applied for analyses of human colorectal cancer cell lines SW480 and SW620 cultured under hypoxic conditions to estimate the strengths and weaknesses of each approach. FTIR absorption spectra acquired by both methods were compared and no significant spectral differences were detected. It was shown that FTIR-microscopy of films on the DAC can be used for evaluation, screening, discrimination and identification of biochemical markers in biosamples like cells. We conclude that the DAC can be transferred to other biosamples like tissues, biofluids, their components and extracellular matrix, and is especially valuable when the available quantities of biosamples are limited.

Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages.

BACKGROUND: Macrophages are one of the most important players in the tumor microenvironment. The polarization status of tumor associated macrophages into a pro-inflammatory type M1 or anti-inflammatory type M2 may influence cancer progression and patient survival. Extracellular vesicles (EVs) are membrane-bound vesicles containing different biomolecules that are involved in cell to cell signal transfer. Accumulating evidence suggests that cancer-derived EVs are taken up by macrophages and modulate their phenotype and cytokine profile. However, the interactions of cancer-derived EVs with monocytes and macrophages at various differentiation and polarization states are poorly understood. In the current study, we have analyzed the uptake and functional effects of primary (SW480) and metastatic (SW620) isogenic colorectal cancer (CRC) cell line-derived EVs on monocytes (M), inactive macrophages (M0) and M1 and M2 polarized macrophages. METHODS: THP-1 monocytes were differentiated into M0 macrophages by addition of phorbol-12-myristate-13-acetate. Then M0 macrophages were further polarized into M1 and M2 macrophages in the presence of LPS, IFN- γ, IL-4, and IL-13 respectively. Internalization of SW480 and SW620-derived EVs was analyzed by flow cytometry and fluorescence microscopy. Changes in monocyte and macrophage immunophenotype and secretory profile upon EV exposure were analyzed by flow cytometry, quantitative PCR and Luminex assays. RESULTS: THP-1 monocytes and M0 macrophages efficiently take up SW480 and SW620-derived EVs, and our results indicate that dynamin-dependent endocytic pathways may be implicated. Interestingly, SW480 and SW620-derived EVs increased CD14 expression in M0 macrophages whereas SW480-derived EVs decreased HLA-DR expression in M1 and M2 polarized macrophages. Moreover, SW480-derived EVs significantly increased CXCL10 expression in monocytes and M0 macrophages. In contrast, SW620-derived EVs induced secretion of IL-6, CXCL10, IL-23 and IL-10 in M0 macrophages. However, addition of CRC cell line-derived EVs together with LPS, IFN- γ (M1) and IL-4, IL-13 (M2) stimuli during macrophage polarization had no additional effect on cytokine expression in M1 and M2 macrophages. CONCLUSION: Our results suggest that CRC cell line-derived EVs are internalized and reprogram the immunophenotype and secretory profile in monocytes and inactive macrophages inducing mixed M1 and M2 cytokine response. Although CRC EVs decreased HLA-DR expression in M1, M2 polarized macrophages, their effect on the secretory profile of M1 and M2 polarized macrophages was negligible.

A novel 3D heterotypic spheroid model for studying extracellular vesicle-mediated tumour and immune cell communication.

Cancer-derived extracellular vesicles (EVs) have emerged as important mediators of tumour-host interactions, and they have been shown to exert various functional effects in immune cells. In most of the studies on human immune cells, EVs have been isolated from cancer cell culture medium or patients' body fluids and added to the immune cell cultures. In such a setting, the physiological relevance of the chosen EV concentration is unknown and the EV isolation method and the timing of EV administration may bias the results. In the current study we aimed to develop an experimental cell culture model to study EV-mediated effects in human T and B cells at conditions mimicking the tumour microenvironment. We constructed a human prostate cancer cell line PC3 producing GFP-tagged EVs (PC3-CD63-GFP cells) and developed a 3D heterotypic spheroid model composed of PC3-CD63-GFP cells and human peripheral blood mononuclear cells (PBMCs). The transfer of GFP-tagged EVs from PC3-CD63-GFP cells to the lymphocytes was analysed by flow cytometry and fluorescence imaging. The endocytic pathway was investigated using three endocytosis inhibitors. Our results showed that GFP-tagged EVs interacted with a large fraction of B cells, however, the majority of EVs were not internalised by B cells but rather remained bound at the cell surface. T cell subsets differed in their ability to interact with the EVs - 15.7-24.1% of the total CD3+ T cell population interacted with GFP-tagged EVs, while only 0.3-5.8% of CD8+ T were GFP positive. Furthermore, a fraction of EVs were internalised in CD3+ T cells via macropinocytosis. Taken together, the heterotypic PC3-CD63-GFP and PBMC spheroid model provides the opportunity to study the interactions and functional effects of cancer-derived EVs in human immune cells at conditions mimicking the tumour microenvironment.

Depletion of carbonic anhydrase IX abrogates hypoxia-induced overexpression of stanniocalcin-1 in triple negative breast cancer cells.

Carbonic anhydrase IX (CAIX) is a pH-regulating enzyme that plays a key role in maintaining an alkaline intracellular pH under hypoxic conditions. It is overexpressed in a variety of solid cancers, including breast cancer (BC), and has been implicated in the migration, invasion and stemness of breast cancer cells. Therefore, CAIX recently emerged as a novel therapeutic target for the treatment of BC. To gain an insight into the mechanism of action of CAIX inhibitors, we investigated the impact of CAIX knock-down on the transcriptional response to hypoxia in 2 BC cell lines - MCF7 and MDA-MB-231, by performing a global gene expression analysis. This showed that CAIX knock-down had a relatively minor effect on the global transcriptional response to hypoxia, however it blocked hypoxia-induced upregulation of stanniocalcin-1 (STC1), a secreted glycoprotein that has been shown to promote tumor progression and metastasis in BC. Kaplan-Meier survival analysis showed that high STC1 expression is significantly associated with poor survival in patients with basal-type breast cancer but not luminal A and HER2+ subtypes. Moreover, the association was particularly high in a subgroup of basal-type BC patients with TP53 mutations thus revealing a putative cooperation of STC1 with mutated TP53 in generating highly aggressive BC subgroup. Taken together, these findings show that CAIX inhibitors at least partially act through blocking STC1 induction in BC cells and reveal a subgroup of BC patients, who potentially would benefit most from the treatment with CAIX inhibitors.

Prognostic relevance of carbonic anhydrase IX expression is distinct in various subtypes of breast cancer and its silencing suppresses self-renewal capacity of breast cancer cells.

PURPOSE: Carbonic anhydrase IX (CAIX) is a hypoxia-inducible enzyme with extracellular catalytic domain that is overexpressed in a variety of cancers including breast cancer and plays a crucial role in maintaining favourable intracellular pH and reducing extracellular pH. The purpose of the current study was to elucidate the prognostic significance of CAIX in the intrinsic subtypes of breast cancer and to characterise CAIX as a drug target in breast cancer. METHODS: The prognostic significance of CAIX mRNA expression was interrogated in a cohort of 3,455 breast tumours by using an online tool, Kaplan-Meier plotter. The functional effects of stable CAIX depletion by shRNA in three breast cancer cell lines­MDA-MB-231, MCF7 and SKBR-3, representing basal-like, luminal A and HER2+ subtypes, respectively­were studied by proliferation, invasion, clonal spheroid formation and chemosensitivity assays under normoxia and hypoxia. Finally, the effect of pharmacological CA inhibition alone or in the combination with doxorubicin on self-renewal was assessed by spheroid-forming assay. RESULTS: High CAIX mRNA expression was significantly associated with poor survival in patients with basal-like, luminal B and triple-negative breast cancer, but not luminal A and HER+ subtypes. Silencing of CAIX expression had no significant effect on the cell proliferation or viability upon treatment with doxorubicin in any of the cell lines studied, while it inhibited spheroid formation in hypoxic conditions. Furthermore, pharmacological inhibition of CAs using acetazolamide had a synergistic effect with doxorubicin on decreasing the spheroid-forming efficiency in MDA-MB-231 cells. CONCLUSIONS: Inhibition of CAIX reduces the self-renewal capacity of breast cancer cells, and the combination of doxorubicin and CAIX inhibition is an attractive therapeutic strategy in basal-like and triple-negative breast cancer, which warrants further investigations.

Cell-free microRNAs as diagnostic, prognostic, and predictive biomarkers for lung cancer.

Lung cancer is the most common cancer worldwide, accounting for over 1.37 million deaths annually. The clinical outcome and management of lung cancer patients could be substantially improved by the implementation of non-invasive biomarker assays for the early detection, prognosis as well as prediction and monitoring of treatment response. MicroRNAs (miRNAs) have been implicated in the regulation of virtually all signaling circuits within a cell and their dysregulation has been shown to play an essential role in the development and progression of cancer. Recently, miRNAs were found to be released into the circulation and to exist there in a remarkably stable form. Furthermore, various cancers were shown to leave specific miRNA fingerprints in the blood of patients suggesting that cell-free miRNAs could serve as non-invasive biomarkers for the detection or monitoring of cancer and putative therapeutic targets. Since that, a considerable effort has been devoted to decode the information carried by circulating miRNAs. In the current review, we give an insight into the mechanisms of miRNA release into the bloodstream, their putative functional significance and systematically review the studies focused on the identification of cell-free miRNAs with the diagnostic, prognostic, and predictive significance in lung cancer and discuss their potential clinical utility.