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Fluorescent microscopy images of PANC1 A. and PANC1 GemR D. cells stained with biotinylated MAL-II and SNA on ice followed with avidin-fluorescein and fixed. Background control has only the avidin-fluorescein. Stained cells were visualized by epifluorescence microscopy using a x20 objective. B, C, E, F, G. Quantitative analysis and flow cytometry analyses are similarly described in Figure 3 except for the flow data, where the median fluorescence for each histogram is indicated for 200000 acquired cells (100% gated).
Source publication
Multicellular tumor spheroids (MTS) have been at the forefront of cancer research, designed to mimic tumor-like developmental patterns in vitro. Tumor growth in vivo is highly influenced by aberrant cell surface-specific sialoglycan structures on glycoproteins. Aberrant sialoglycan patterns that facilitate MTS formation are not well defined. Matrix...
Context in source publication
Context 1
... viability is expressed as cell viability (% of control) ± S.E. of two independent experiments. the spheroid cells [32], we examined prior to spheroid formation the cell surface sialylation of monolayer MCF- 7, MCF-7 TMX cells (Figure 3), and PANC1 and PANC1- GemR cells (Figure 4). This analysis was conducted using lectin histochemistry staining and flow cytometry with α-2,3-sialic acid (SA) specific Maackia amurensis (MAL- II) and α-2,6-SA specific Sambucus nigra (SNA). ...
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Background
Prostaspheres-based three dimensional (3D) culture models have provided insight into prostate cancer (PCa) biology, highlighting the importance of cell–cell interactions and the extracellular matrix (EMC) in the tumor microenvironment. Although these 3D classical spheroid platforms provide a significant advance over 2D models mimicking i...
Citations
... Cells in spheroid culture grow, resulting in a dense cell structure in multilayers and the extracellular matrix development [74,75]. Within this context, the remarkably increased viability in spheroids, compared to the 2D system when applying DNA-PFs-AlClPc to PDT, can be attributed to the diffusion difficulty of these compounds or even to the MTT and FA in the inner layers of the spheroid [74]. ...
The present study examines the designer of DNA polymeric films (DNA-PFs) associated with aluminum chloride phthalocyanine (AlClPc) (DNA-PFs-AlClPc), as a promising drug delivery system (DDS), applicable for breast cancer treatment and early-stage diagnosis using photodynamic therapy (PDT). This study starts evaluating (MCF7) as a model for breast cancer cell behavior associated with DNA-PFs. Analyses of the morphological behaviors, biochemical reaction, and MCF7 cell adhesion profile on DNA-PFs were evaluated. SEM and AFM analysis allowed the morphological characterization of the DNA-PFs. Cell viability and cell cycle kinetics studies indicate highly biocompatible material capable of anchoring MCF7 cells, allowing the attachment and support of cell in the same structure where the insertion of AlClPc (DNA-PFs-AlClPc). The application of visible light photoactivation based on classical PDT protocol over the DNA-PFs-AlClPc showed a reduction in cell viability with increased cell death proportional to the fluency energy range from 600, 900, and 1800 mJ cm-2. The 3D organoid system mimics the tumor microenvironment which was precisely observed in human breast cancer in early-stage progression in the body. The results observed indicate that the viability was reduced by more than 80% in monolayer culture and around 50% in the 3D organoid cell culture at the highest energy fluency (1800 mJ cm-2). We could also point out that with low energy fluency (100 mJ cm-2,), the DNA-PFs-AlClPc did not show a cytotoxic effect on MCF7 cells, enabling this user dose for the photodiagnosis of early-stage human breast cancer detection in the initial stage of progression.
... It has been demonstrated that they have a capability to form in vivo xenograft tumors particularly in the cancer cell lines. It is considered that the obtainable data will provide a supportive information infrastructure in selection of the appropriate cells, administration of the oriented treatment and establishment of the early diagnosis (Akasov et al., 2016). ...
... It has been demonstrated that they have a capability to form in vivo xenograft tumors particularly in the cancer cell lines. In the light of these data; it may be concluded that the ability to specifically label these xenograft tumor formations to differentiate cancer cells from the normal cells is highly important (Akasov et al., 2016). ) that can specifically bind to some sialic acid types (GlcNAc, Neu5Ac) and particularly ß-type N-Acetyl Glucosamine (ß-GlcNAc) sialic acid glycan. ...
The importance of early cancer diagnosis has led to development of many different diagnostic methods. In this context, the studies investigating the presence and amount of sugar residues to use as indicators in the identification of cancer cell type have become prominent. In the present study, sialic acids found on the membrane surfaces of ER (+) MCF-7 and ER (-) MDA-MB-231 breast cancer cell lines were labeled using three-dimensional (3D) cell culture (Spheroid) model as the closest method to the patient sample, thus its natural environment, among the cell culture methods. These sugar units that play a role in regulation of important immune characteristics such as recognition, binding and metastasis were made visualizable by applying fluorescent-labeled lectins such as FITC-(Wheat Germ Agglutinin) specifically binding to sialic acid units (GlcNAc, Neu5Ac) including particularly ß-GlcNAc and FITC-(Maackia Amurensis-Lectin-1) specifically binding to Galß4GlcNAc type sialic acids. These glycan units were specifically labeled with FITC-(Maackia Amurensis-Lectin-1) and FITC- (Wheat Germ Agglutinin) and radiation intensities were analyzed relatively. The two different breast cancer cell cultures were compared with respect to change in the amounts of sialic acid residues containing α-2,3- and α-2,6 bonds using fluorescent-labeled lectins. In the present study, we have performed a precise, accurate and rapid determination of the sugar content in the different breast cancer cell surface lines by means of fluorescent-labeled lectins and carried out a relative comparison between the micrographs.
... The results showed that CA19-9 was hardly detected on positive population of PANC-1 and MIA PaCa-2 but was detected on AsPC-1 and Capan-2 (Fig. 3a). The analysis also showed that PANC-1 and MIA PaCa-2 had an affinity for SNA and AAL, which is consistent with the results of previous work (Akasov et al. 2016), whereas both AsPC-1 and Capan-2 had an affinity for AAL and the CA19-9 antibody. H6C7 presented weak affinities for AAL and CA19-9 antibody but a relatively strong affinity for SNA (Fig. 3a). ...
The unique profile of upregulated glycosylation in metastatic cancer cells may form the basis for the development of new biomarkers for the targeting and diagnosis of specific cancers. This study introduces a pancreatic cancer cell-derived exosome detection technology, which is based on the specific binding of lectins to distinctive glycan profiles on the surface of exosomes. Lectins with a high and specific affinity for sialic acid or fucose were attached to bifunctional Janus nanoparticles (JNPs), which facilitated interactions with pancreatic cancer cell-derived exosomes in a microfluidic device. Here, we show that pancreatic cancer cell-derived exosomes from two cell lines and plasma samples collected from patients diagnosed with pancreatic cancer were successfully captured on the lectin-conjugated JNPs with affinities that were comparable to those of CA19-9, a conventional antibody. In addition, exosome detection using our platform could differentiate between metastatic and nonmetastatic pancreatic cancer cells. This study opens the possibility to achieve a new early diagnosis marker based on the glycan properties of pancreatic cancer cell-derived exosomes.
... Recently, a simple universal technique based on RGD-induced cell self-assembly after peptide adding directly to monolayer cell culture was reported by us.19 Recently, we have used this RGD-peptide based platform to generate tumor spheroids from different tumor cells, namely pancreatic PANC1 cancer cells.20 Using this technique we also demonstrated that sialylation facilitates generation of the tumor spheroids (prostospheres) from human prostate carcinoma cells.21 ...
Presently, most of anticancer drugs are high toxic for normal cells and, and as a result, they have severe side effects. Moreover, most of the formulations are lipophilic and have poor selectivity. To overcome these limitations, various drug delivery systems could be proposed. The aim of the current study was to fabricate novel polysaccharide nanocontainers (NC) by one-step ultrasonication technique and to evaluate their accumulation efficacy and cytotoxicity in 2D (monolayer culture) and 3D (tumor spheroids) in vitro models. NC with mean sizes in a range of 340-420 nm with the core-shell structure are synthetized and characterized. The NC shell is composed from diethylaminoethyl dextran/xanthan gum polyelectrolyte complex, while the hydrophobic core was loaded with the lipophilic anticancer drug thymoquinone. To enhance NC accumulation in human breast adenocarcinoma MCF-7 cells, the NC surface was modified with poly-L-lysine (PLL) or polyethylene glycol. Cell uptake of the NC loaded with Nile Red into the tumor cells was investigated by laser scanning confocal microscopy, fluorescent flow cytometry and fluorimetry. Modification of the NC with PLL allowed to obtain the optimal drug delivery system with maximal cytotoxicity, which was tested by MTT-test. The developed NC are promising for lipophilic anticancer drug delivery.
... The two measured diameters were then averaged and divided to calculate the average radius. The following formulae were used to determine MCTS/cell aggregate volume, as previously described in detail: [37][38][39][40] 10× objective images: Volume = (4/3) πr 3 where r = average radius (μm); 4× objective images: Volume = (2.5)(4/3) πr 3 where r = average radius (μm) The formula for the 4× objective images includes 2.5 factor to normalize values to the 10× objective images. ...
Introduction
Targeted multimodal approaches need to be strategically developed to control tumour growth and prevent metastatic burden successfully. Breast cancer presents a unique clinical problem because of the variety of cellular subtypes that arise. The tumour stage and cellular subtypes often dictate the appropriate clinical treatment regimen. Also, the development of chemoresistance is a common clinical challenge with breast cancer. Higher doses and additional drug agents can produce additional adverse effects leading to a more aggressive malignancy. Acetylsalicylic acid (ASA), metformin (Met), and oseltamivir phosphate (OP) were investigated for their efficacy to sensitize MDA-MB-231 triple-negative breast cancer and its tamoxifen (Tmx) resistant variant (MDA-MB-231-TmxR) together in combination with Tmx treatment.
Methods
Microscopic imaging, the formation of 3D multicellular tumour spheroids, immunocytochemistry, flow cytometry, Annexin V Assay, Caspase 3/7 Apoptosis Assay, tube formation assay and analysis, and WST-1 cell viability assay evaluated the formation of MCTS, morphologic changes, cell viability, apoptosis activity and the expression levels of ALDH1A1, CD44 and CD24 on the cell surface, MDA-MB231 triple-negative breast cancer, tamoxifen (Tmx) resistant variant (MDA-MB-231-TmxR).
Results
The results using a triple combination of ASA, Met and OP on MDA-MB-231 and MDA-MB-231-TmxR cells and their matrix-free 3D multicellular tumour spheroids (MCTS) formed by using the cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK(TPP)) peptide method demonstrate a consistent and significant decrease in cell and tumour spheroid viability and volume with increased apoptotic activity, and increased sensitivity to Tmx therapy. Tmx treatment of MDA-MB-231 cells in combination with ASA, Met and OP markedly reduced the CD44/CD24 ratio by 6.5-fold compared to the untreated control group. Tmx treatment of MDA-MB-231-TmxR cells in combination with ASA, Met and OP markedly reduced the ALDH1A1 by 134-fold compared to the same treatment for the parental cell line. Also, the triple combination treatment of ASA, Met, and OP inhibited vasculogenic endothelial cell tube formation and induced endothelial cell apoptosis.
Conclusion
For the first time, the findings demonstrate that repurposing ASA, Met, and OP provides a novel and promising targeted multimodal approach in the treatment of triple-negative breast cancer and its chemoresistant variant.
... These limitations of monolayer cultures can be partially overcome by using 3D cultures. A 3D culture presented by spheroids, i.e. spherical conglomerates of tumor cells, is closer by its structure to natural tumor [4][5][6]8,20,24,27]. Unlike monolayer culture, spheroids are multicellular self-organized spherical clustered colonies with tissue-like architecture similar to avascular solid tumors formed at the initial stages of tumorigenesis [5,27]. ...
... A 3D culture presented by spheroids, i.e. spherical conglomerates of tumor cells, is closer by its structure to natural tumor [4][5][6]8,20,24,27]. Unlike monolayer culture, spheroids are multicellular self-organized spherical clustered colonies with tissue-like architecture similar to avascular solid tumors formed at the initial stages of tumorigenesis [5,27]. Dense aggregates are formed due strong cell-cell contacts because of enhanced expression of cell adhesion proteins and stromal markers [4,20,22]. In a spheroid, like in a tumor in vivo, cells are presented by a heterogeneous population with different gene expression in diverse parts of the spheroid, and this state can be maintained for a long time [22,23,27]. ...
... Cell growth in 3D cultures affects their response to cytotoxic factors, and spheroids are often more resistant to anticancer drugs than monolayer cultures [4,8,21,22,24]. To study the response of spheroid culture to cytotoxic agents, we examined a number of platinum complexes with previously established physicochemical properties and toxicity to monolayer cultured cells [1][2][3]. ...
We performed a comparative study of the cytotoxicity of cisplatin, JM216 complex, and aminonitroxyl platinum(IV) complexes for HeLa cells grown in monolayer and 3D culture. The growth dynamics of spheroids was studied and optimal conditions for evaluation of cytotoxicity were determined. Spheroids were less sensitive to the test compounds than cells in a monolayer. The resistance index (RI) of spheroids was determined as the ratio of IC50 for spheroids to IC50 for monolayer culture. Resistance index was 5.0±1.5 for cisplatin and ranged from 1.8 to 2.3 for platinum(IV) complexes. The observed differences are related to different physicochemical properties of the complexes and different mechanisms of their penetration into cells.
... To this end, Akasov et al have demonstrated that sialylation facilitates MCTS formation in parental and chemoresistant breast MCF7 and pancreatic PANC1 cell lines using the cyclo-RGDfK(TPP) peptide method. 23 The specific sialoglycan structures expressed on the cell surface also correlated with the ability of prostate cancer cells to form avascular multicellular prostaspheres. 24 Until now, the effect of fucosylation on the formation of avascular MCTS is unknown. ...
... MCTS were established from prostate cells using previously reported protocols. 6,7,23 Cells were first grown in T25 or T75 tissue culture flasks until at least 90% confluent, then plated in flat-bottom Microwell 96-well plates at a density of 10,000 cells/well or 100µL/well. They settled at the bottom of the wells in the 2 to the 3 hr incubation period. ...
... A concentration of 50µM of cyclo-RGDfK(TPP) was used as per previously established protocols. 6,23 The cells were incubated for six days in a 37ºC humidified incubator and monitored daily. Spheroid measurements were taken on days four, five and six. ...
Introduction
Core fucosylation of N-glycans on the integrin β1 subunit is essential for the functional activity of the integrin. The binding of α5β1 integrin with the tripeptide Arg-Gly-Asp (RGD) motif within the extracellular matrix protein fibronectin may be influenced by the α-1,6-fucose core or α-1,2-fucose and α-1,3/4-fucose peripheral N-glycan profiles. Here, we investigated whether fucosylation impacts the formation of matrix-free 3D multicellular tumor spheroids (MCTS) from human triple negative breast MDA-MB231 cell line, prostate PC3 and DU145 cell lines and DU145 gemcitabine resistant (GemR) variant by using the cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK(TPP)) peptide method.
Methods
Microscopic imaging, lectin histochemistry, flow cytometry, WST-1 cell viability assay and You Only Look Once version 2 (YOLOv2) training object detection using cyclic learning rates were used to evaluate the formation of MCTS, morphologic changes, and the expression levels of α-1,6-fucose and α-1,2-fucose linkages on the cell surface.
Results
DU145 prostate cancer cells expressed higher α-1,6-fucose than α-1,2-fucose linkages on their cell surface, as determined by lectin cytochemistry and flow cytometry. Blockage of the α-1,6- and α-1,2-fucose linkages with Aspergillus oryzae lectin (AOL) and Ulex Europaeus agglutinin I (UEA I) one hour before the addition of cyclic-RGDfK(TPP) peptide to the monolayer of the cancer cells resulted in a statistically significant dose-dependent reduction in spheroid volumes using threshold diameters of 40 and 60 µm. Application of a 40 µm threshold diameter measurements of spheroids resulted in fewer false-positive ones compared to the 60 µm diameter threshold previously used in our studies. A state-of-the-art, image object detection system YOLOv2 was used to automate the analysis of spheroid measurements and volumes. The results showed that YOLOv2 corroborated manual spheroid detection and volume measurements with high precision and accuracy.
Conclusion
For the first time, the findings demonstrate that α-1,6- and α-1,2-fucose linkages of N-glycans on the cell surface receptors facilitate cyclo-RGDfK(TPP)-mediated self-assembly of cancer cells to form 3D multicellular tumor spheroids.
... However, it is not uncommon for research laboratories to employ their own definitions of a spheroid, which contributes to the lack of standardization in spheroid analysis. For the purposes of this study, the following definition was used, as per the specifications of the laboratory of Dr. Myron R. Szewczuk: "compact rounded spheroids with a distinct border that has a diameter of at least 60 microns, and contain cells which are indistinguishable from one another" [14,15]. Spheroids are capable of reproducing growth kinetics, oxygen, gases, nutrient and waste gradients, proliferate distribution, and gene expression profiles among other properties of small, solid avascular tumors [16]. ...
... For instance, the system could be implemented for the detection and volume estimation of cell aggregates (Fig. 1c), defined as "loose packages of cells" that lack a spherical geometry and may lack cell-cell or cellextracellular matrix (ECM) interactions [38]. In the context of 3D spheroids generated using cyclo-RGDfK (TPP), PANC-1 pancreatic cancer cells form cellular aggregates as opposed to true spheroids [14]. Unlike measuring spheroids, measuring the size of cell aggregates using a manual approach is challenging due to their non-uniform shape, thereby highlighting the importance of approaches such as YOLOv2 to automate this process. ...
... Sialic binding lectins, such as Maackia amurensis lectin-II (MAL-II, α2,3-sialylated glycan binding lectin) and Sambucus nigra agglutinin (SNA, α2,6-sialylated glycan binding lectin), have been used for detecting and studying biological roles of sialylated-glycans in human diseases [9,11,12,14,15,17,[22][23][24][25]. ...
Background and objectives:
Sialylation plays important roles in tumor progression. Our present study aimed to demonstrate the alteration of sialylation and its role in cholangiocarcinoma (CCA).
Materials and methods:
The α2,3- and α2,6-sialylation in CCA tissue was analyzed by lectin-histochemistry using Maackia amurensis lectin-II (MAL-II) and Sambucus nigra agglutinin (SNA). CCA cell lines were treated with the pan-sialylation inhibitor 3Fax-peracetyl-Neu5Ac (3F-Sia) followed by proliferation and chemosensitivity assays.
Results:
MAL-II binding α2,3-Sialylated Glycan (MAL-SG) and SNA binding α2,6-Sialylated Glycan (SNA-SG) were both elevated in CCA compared with hyperplastic/dysplastic (HP/DP) and normal bile ducts (NBD). The positive staining for MAL-SG or SNA-SG were found in 82% (61/74) of the CCA cases. Higher expression of MAL-SG in CCA was associated with shorter survival of the patients. The median survival of patients with high and low MAL-SG were 167 and 308 days, respectively, with overall survival of 233 days, suggesting the involvement of MAL-SG in CCA progression. MAL-SG expression of CCA cell lines was markedly decreased after treatment with 3F-Sia for 48 to 72 h. While proliferation of CCA cells were not affected by 3F-Sia treatment, their susceptibility to 5-fluorouracil (5-FU) was significantly enhanced. These results suggest that sialylation is involved in the development of 5-FU resistance and the sialylation inhibitor 3F-Sia can be used as a chemosensitizer for CCA.
Conclusions:
Sialylation is critically involved in the development of chemoresistance of CCA, and sialylation inhibitors may be used as a chemosensitizer in CCA treatment.
... Recently, we have developed a simple universal approach based on RGD-induced cell self-assembly technique. This approach has been already successfully used by us to generate tumor spheroids from different tumor cells, in particular pancreatic PANC1 cancer cells [28]. Recently, this RGD-peptide based platform was also used to demonstrate that sialylation facilitates formation of tumor spheroids from human prostate carcinoma cells, so called prostospheres [29]. ...
