Ultrasensitive determination of ß-conglutin food allergen by means an aptamer assay based on inductively coupled plasma mass spectrometry detection.

The overall objective of this work is the evaluation of different competitive aptamer assays based on inductively coupled plasma mass spectrometry (ICP-MS) detection for the determination of ß-conglutin (food protein allergen from lupin) in flour samples. To this end, two competitive aptamer assay schemes were developed using either thiolated aptamers chemisorbed onto gold nanoparticles (AuNPs) or biotinylated aptamers linked to streptavidin-AuNPs. The influence of ICP-MS detection mode (i.e., conventional vs single particle) on assay performance was explored. In the case of the thiolated aptamer, the limit of detection (LoD) obtained using the single particle mode was improved 2-fold as compared to the LoD provided by the conventional mode. With regards to the biotinylated aptamer, the use of the conventional mode provided a 5-fold improvement of LoD as compared to that obtained for the single particle one. Using the optimized conditions, the best LoD of 2 pM was obtained with the biotinylated aptamer operating with conventional ICP-MS detection. When compared to previous reports using the same aptamer in a competitive assay, the developed method significantly improved the LoD by at least an order of magnitude. Different flour samples containing lupin were successfully analyzed according to European Conformity guidelines for the analysis of food contaminants.

Novel nandrolone aptamer for rapid colorimetric detection of anabolic steroids.

The illicit use of anabolic androgenic steroids (AAS) as performance-enhancing drugs remains a global issue threatening not only the credibility of competitive sports but also public health due to the well-documented adverse effects they elicit. AAS abuse is not restricted only to professional sports, but also extends to recreational athletes and adolescents as well as in livestock production as growth-promoting agents. Testosterone and nandrolone are among the AAS most frequently exploited. Gas chromatography-mass spectrometry is the reference method for AAS detection, but it is strictly laboratory-based and cannot be performed on-site. The great potential of aptamers in bioanalytical applications and specifically for the development of simple analytical tools suitable for on-site analysis has been extensively documented. In this report, we describe the selection and identification of aptamers binding nandrolone, exhibiting affinity dissociation constants in the low nanomolar range. A label-free colorimetric assay based on gold nanoparticles was developed using one of these novel aptamers for the detection of nandrolone and/or its metabolites. The assay could be deployed for the rapid, on-site, facile and cost-effective screening of samples and provide qualitative visual results with a red to purple/blue color change being indicative of a positive result.

Synthesis and Characterization of New Boron Compounds Using Reaction of Diazonium Tetraphenylborate with Enaminoamides.

A family of oxazaborines, diazaborinones, triazaborines, and triazaborinones was prepared by reaction of polarized ethylenes, such as ß-enaminoamides, with 4-methylbenzenediazonium tetraphenylborates. The reaction conditions (stirring in CH2Cl2 at room temperature (Method A) or stirring with CH3COONa in CH2Cl2 at room temperature (Method B) or refluxing in the CH2Cl2/toluene mixture (Method C)) controlled the formation and relative content of these compounds in the reaction mixtures from one to three products. Substituted oxazaborines gradually rearranged into diazaborinones at 250 °C. The prepared compounds were characterized by 1H NMR, 13C NMR, IR, and UV-Vis spectroscopy, HRMS, or microanalysis. The structure of individual compounds was confirmed by 11B NMR, 15N NMR, 1D NOESY, and X-ray analysis. The mechanism of reaction of enaminoamides with 4-methylbenzenediazonium tetraphenylborate was proposed.

Aptasensors for mycotoxin detection: A review.

Mycotoxins are toxic compounds produced by fungi, which represent a risk to the food and feed supply chain, having an impact on health and economies. A high percentage of feed samples have been reported to be contaminated with more than one type of mycotoxin. Systematic, cost-effective and simple tools for testing are critical to achieve a rapid and accurate screening of food and feed quality. In this review, we describe the various aptamers that have been selected against mycotoxins and their incorporation into optical and electrochemical aptasensors, outlining the strategies exploited, highlighting the advantages and disadvantages of each approach. The review also discusses the different materials used and the immobilization methods employed, with the aim of achieving the highest sensitivity and selectivity.

Aptamer Sandwich Assay for the Detection of SARS-CoV-2 Spike Protein Antigen.

The novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) emerged at the end of 2019, resulting in the ongoing COVID-19 pandemic. The high transmissibility of the virus and the substantial number of asymptomatic individuals have led to an exponential rise in infections worldwide, urgently requiring global containment strategies. Reverse transcription-polymerase chain reaction is the gold standard for the detection of SARS-CoV-2 infections. Antigen tests, targeting the spike (S) or nucleocapsid (N) viral proteins, are considered as complementary tools. Despite their shortcomings in terms of sensitivity and specificity, antigen tests could be deployed for the detection of potentially contagious individuals with high viral loads. In this work, we sought to develop a sandwich aptamer-based assay for the detection of the S protein of SARS-CoV-2. A detailed study on the binding properties of aptamers to the receptor-binding domain of the S protein in search of aptamer pairs forming a sandwich is presented. Screening of aptamer pairs and optimization of assay conditions led to the development of a laboratory-based sandwich assay able to detect 21 ng/mL (270 pM) of the protein with negligible cross-reactivity with the other known human coronaviruses. The detection of 375 pg of the protein in viral transport medium demonstrates the compatibility of the assay with clinical specimens. Finally, successful detection of the S antigen in nasopharyngeal swab samples collected from suspected patients further establishes the suitability of the assay for screening purposes as a complementary tool to assist in the control of the pandemic.

Hybrid Antibody-Aptamer Assay for Detection of Tetrodotoxin in Pufferfish.

The marine toxin tetrodotoxin (TTX) poses a great risk to public health safety due to its severe paralytic effects after ingestion. Seafood poisoning caused by the consumption of contaminated marine species like pufferfish due to its expansion to nonendemic areas has increased the need for fast and reliable detection of the toxin to effectively implement prevention strategies. Liquid chromatography-mass spectrometry is considered the most accurate method, although competitive immunoassays have also been reported. In this work, we sought to develop an aptamer-based assay for the rapid, sensitive, and cost-effective detection of TTX in pufferfish. Using capture-SELEX combined with next-generation sequencing, aptamers were identified, and their binding properties were evaluated. Finally, a highly sensitive and user-friendly hybrid antibody-aptamer sandwich assay was developed with superior performance compared to several assays reported in the literature and commercial immunoassay kits. The assay was successfully applied to the quantification of TTX in pufferfish extracts, and the results obtained correlated very well with a competitive magnetic bead-based immunoassay performed in parallel for comparison. This is one of the very few works reported in the literature of such hybrid assays for small-molecule analytes whose compatibility with field samples is also demonstrated.

Toxicokinetics of hydrophobic organic compounds in oligochaeta: A critical review.

Toxicokinetic studies appertain to the fundamental research of soil bioavailability. However, the research outcomes of aspects influencing uptake and elimination of hydrophobic organic compounds have not been summarized so far. In our review, a recapitulation of available toxicokinetic data (i.e. experimental conditions, if the steady state was reached, uptake and elimination rate constants, and bioaccumulation factors) is presented in well-arranged tables. Further, toxicokinetic models are overviewed in the schematic form. In the review, the required information could be quickly found and/or the experimental gaps easily identified. Generally a little is known about the effects of soil properties other than soil organic matter. Limited or no data are available about soil treatment, food supply during laboratory exposure, and metabolization in oligochaeta. The impact of these factors might be important especially for arable soils with typically low organic matter content but high consequences on humans. Besides these circumstances, other uncertainties between published studies have been found. Firstly, the scientific results are provided in heterogenous units: bioaccumulation factors as well as the rate constants are reported in dry or wet weight of soil and earthworms. The steady state is another critical factor because the time to reach the equilibrium is influenced not only by soil and compound characteristics but for example also by aging. Nevertheless, toxicokinetic studies bring irreplaceable information about the real situation in soil and our review help to define missing knowledge and estimate the scientific priorities.

Aptamers against the ß-Conglutin Allergen: Insights into the Behavior of the Shortest Multimeric (Intra)Molecular DNA G-Quadruplex.

In previous work, a 93-mer aptamer was selected against the anaphylactic allergen, ß-conglutin and truncated to an 11-mer, improving the affinity by two orders of magnitude, whilst maintaining the specificity. This 11-mer was observed to fold in a G-quadruplex, and preliminary results indicated the existence of a combination of monomeric and higher-order structures. Building on this previous work, in the current study, we aimed to elucidate a deeper understanding of the structural forms of this 11-mer and the effect of the structure on its binding ability. A battery of techniques including polyacrylamide gel electrophoresis, high-performance liquid chromatography in combination with electrospray ionization time-of-flight mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight, thermal binding analysis, circular dichroism and nuclear magnetic resonance were used to probe the structure of both the 11-mer and the 11-mer flanked with TT- at either the 5' or 3' end or at both ends. The TT-tail at the 5' end hinders stacking effects and effectively enforces the 11-mer to maintain a monomeric form. The 11-mer and the TT- derivatives of the 11-mer were also evaluated for their ability to bind its cognate target using microscale thermophoresis and surface plasmon resonance, and biolayer interferometry confirmed the nanomolar affinity of the 11-mer. All the techniques utilized confirmed that the 11-mer was found to exist in a combination of monomeric and higher-order structures, and that independent of the structural form present, nanomolar affinity was observed.

The Importance of Cancer-Associated Fibroblasts in the Pathogenesis of Head and Neck Cancers.

BACKGROUND: Despite progress in anticancer therapies, head and neck squamous cell carcinoma (HNSCC) has still a low survival rate. Recent studies have shown that tumour stroma may play an important role in the pathogenesis of this malignant disease. Fibroblasts are a major component of the tumour microenvironment and may significantly influence HNSCC progression as indicated by the contribution they make to important hallmarks of cancer, such as inflammation, non-restricted growth, angiogenesis, invasion, metastasis, and therapy resistance. It is well known that tumour cells can confer a cancer-associated fibroblast (CAF) phenotype that supports the growth and dissemination of cancer cells. CAFs can stimulate cancer progression through cell-cell contacts and communication, remodelling of extracellular matrix, and production of many signal molecules and matrix metalloproteinases. Consequently, genetic changes in epithelial cells are probably not the only factor that drives HNSCC carcinogenesis. Non-genetic changes in the tumour stroma can also be significantly involved. Stress-induced signals can induce a multicellular program, creating a field of tissue that is predisposed to malignant transformation. The “field cancerization” concept represents a process of active evolution of intercellular interactions and feedback loops between tumour and stromal cells. This model paves the way to study cancer from a new perspective and identify new therapeutic targets. PURPOSE: In this review, we discuss current knowledge about CAFs, such as their cellular origin, phenotypical plasticity and functional heterogeneity, and stress their contribution to HNSCC progression. This article was supported by the project AZV 16-29835A. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 18. 6. 2019 Accepted: 9. 9. 2019.

Uptake kinetics of four hydrophobic organic pollutants in the earthworm Eisenia andrei in aged laboratory-contaminated natural soils.

Laboratory studies of pollutant uptake kinetics commonly start shortly after experimental soil contamination when it is not clear if the processes between soil and chemicals are equilibrated and stabilized. For instance, when the concentration in soil quickly decreases due to initial biodegradation, bioaccumulation may show a peak-shape accumulation curve instead of conventional first order kinetics with a plateau at the end. The results of such experiments with soil freshly contaminated in the laboratory are then hardly comparable to bioaccumulation observed in soils from historically contaminated sites. Therefore, our study focused on the uptake kinetics of four hydrophobic organic compounds (pyrene, lindane, p,p'-DDT and PCB 153) in two laboratory-contaminated natural soils with different soil properties (e.g. total organic carbon content of 1.6 and 9.3%) aged for 203 days to mimic long-term contamination. For pyrene, the results surprisingly showed peak-shape accumulation curves despite long aging. It seems compound biodegradation might be significant in aged soils when the conditions change (e.g. by distribution to the experimental vessels) and this should be also considered when testing historically contaminated soils. For lindane, longer aging seems to guarantee stability of the soil-compound-earthworm system and the steady state was reached after 5 days of exposure. Furthermore, although concentrations of p,p'-DDT and PCB 153 in earthworms after 11-15-day exposure did not statistically differ, which is a commonly-used indicator that a steady state was reached, they continuously increased until the end of the exposure. Therefore, despite the aging, longer exposure was probably needed to reach the true equilibrium between concentrations in earthworms and soil. In summary, aging does not warranty the conventional first order kinetic curve with the equilibrium at the end of the exposure but may have diverse effects for compounds with different environmental properties and should be taken into account in the bioaccumulation factor calculation and the risk assessment.

One-Pot SELEX: Identification of Specific Aptamers against Diverse Steroid Targets in One Selection.

Aptamers are well-established biorecognition molecules used in a wide variety of applications for the detection of their respective targets. However, individual SELEX processes typically performed for the identification of aptamers for each target can be quite time-consuming, labor-intensive, and costly. An alternative strategy is proposed herein for the simultaneous identification of different aptamers binding distinct but structurally similar targets in one single selection. This one-pot SELEX approach, using the steroids estradiol, progesterone, and testosterone as model targets, was achieved by combining the benefits of counter-SELEX with the power of next-generation sequencing and bioinformatics analysis. The pools from the last stage of the selection were compared in order to discover sequences with preferential abundance in only one of the pools. This led to the identification of aptamer candidates with potential specificity to a single steroid target. Binding studies demonstrated the high affinity of each selected aptamer for its respective target, and low nanomolar range dissociation constants calculated were similar to those previously reported for steroid-binding aptamers selected using traditional SELEX approaches. Finally, the selected aptamers were exploited in microtiter plate assays, achieving nanomolar limits of detection, while the specificity of these aptamers was also demonstrated. Overall, the one-pot SELEX strategy led to the discovery of aptamers for three different steroid targets in one single selection without compromising their affinity or specificity, demonstrating the power of this approach of aptamer discovery for the simultaneous selection of aptamers against multiple targets.

Comparison of different label-free imaging high-throughput biosensing systems for aptamer binding measurements using thrombin aptamers.

To enable the analysis of several hundreds to thousands of interactions in parallel, high-throughput systems were developed. We used established thrombin aptamer assays to compare three such high-throughput imaging systems as well as analysis software and user influence. In addition to our own iRIf-system, we applied bscreen and IBIS-MX96. As non-imaging reference systems we used Octet-RED96, Biacore3000, and Monolith-NT.115. In this study we measured 1378 data points. Our results show that all systems are suitable for analyzing binding kinetics, but the kinetic constants as well as the ranking of the selected aptamers depend significantly on the applied system and user. We provide an insight into the signal generation principles, the systems and the results generated for thrombin aptamers. It should contribute to the awareness that binding constants cannot be determined as easily as other constants. Since many parameters like surface chemistry, biosensor type and buffer composition may change binding behavior, the experimenter should be aware that a system and assay dependent KD is determined. Frequently, certain conditions that are best suited for a given biosensing system cannot be transferred to other systems. Therefore, we strongly recommend using at least two different systems in parallel to achieve meaningful results.

Fate and bioavailability of four conazole fungicides in twelve different arable soils - Effects of soil and pesticide properties.

The objective of this study is to characterize changes in the fate, behaviour and bioavailability of four conazole fungicides - CFs (prochloraz - PRO, tebuconazole - TEB, epoxiconazole - EPO, flusilazole - FLU) in 12 diverse agricultural soils in complex microcosm systems consisting of agriculturally-used fluvisols, plants (Lactuca sativa), earthworms (Eisenia fetida) and passive samplers (SPME, Empore™ discs, silicone rubber). Due to great variability of the data and other methodological problems, the in-matrix passive samplers failed to be indicators of pore-water concentration and (bio)availability/(bio)accessibility of CFs. A dissipation of all CFs followed the first order kinetics (usually after initial lag phase) with large span of resulting half-lives (7-670 d) depending on soils and compounds. In many soils, the model revealed the ending plateau, which indicates the non-degradable or slowly-degradable residues. The half-lives and the residues were generally higher for EPO and FLU, than for PRO and TEB. Greater but slower total dissipation of CFs was observed in soils with higher percentage of organic matter. Earthworm concentrations were highest at first sampling time (14 days) and considerably decreased afterwards often resulting in PRO concentration below LOQ. Earthworm uptake was influenced by amount of organic matter and soil texture. Accumulation to lettuce roots was generally higher than to leaves and differed greatly among CFs. Concentration shoot to root ratios were generally the lowest for FLU (0.04) and highest for TEB (0.37). PRO was not detected in lettuce leaves during experiment. The study brings new results on fate and bioavailability of CFs in soils.

Prognostic Significance of Serum Free Amino Acids in Head and Neck Cancers.

Despite distinctive advances in the field of head and neck squamous cell cancer (HNSCC) biomarker discovery, the spectrum of clinically useful prognostic serum biomarkers is limited. As metabolic activities in highly proliferative transformed cells are fundamentally different from those in non-transformed cells, specific shifts in concentration of different metabolites may serve as diagnostic or prognostic markers. Blood amino acids have been identified as promising biomarkers in different cancers before, but little is known about this field in HNSCC. Blood amino acid profiles of 140 HNSCC patients were examined using high-performance liquid chromatography. Cox proportional hazards regression model was used to assess the prognostic value of amino acid concentrations in serum. Colony forming assay was used to identify the effect of amino acids that were significant in Cox proportional hazards regression models on colony forming ability of FaDu and Detroit 562 cell lines. In the multivariable Cox regression model for overall survival (OS), palliative treatment was associated with an unfavourable prognosis while high serum levels of methionine have had a positive prognostic impact. In the relapse-free survival (RFS) multivariable model, methionine was similarly identified as a positive prognostic factor, along with tumor localization in the oropharynx. Oral cavity localization and primary radio(chemo)therapy treatment strategy have been linked to poorer RFS. 1mM serine was shown to support the forming of colonies in both tested HNSCC cell lines. Effect of methionine was exactly the opposite.

Aptamer Selection against a Trichomonas vaginalis Adhesion Protein for Diagnostic Applications.

Trichomoniasis, caused by Trichomonas vaginalis, is the leading nonviral sexually transmitted infection worldwide. We report the selection of a DNA aptamer against a T. vaginalis adhesion protein, AP65, using a microtiter plate-based in vitro combinatorial chemistry process termed systematic evolution of ligands by exponential enrichment. The enriched library pool was sequenced by next-generation sequencing, and several aptamer candidates with high affinity and specificity were identified. The aptamer with the highest affinity and specificity had a KD in the low nanomolar range, as confirmed by three different techniques: surface plasmon resonance, enzyme-linked aptamer assay, and biolayer interferometry. The selected aptamer was demonstrated to have a high specificity to the AP65 protein and to T. vaginalis cells with no cross-reactivity to other enteric and urogenital microorganisms. Current work is focused on the development of inexpensive and easy-to-use aptamer-based diagnostic assays for the reliable and rapid detection of T. vaginalis in vaginal swabs.

Prostate-Specific Membrane Antigen-Targeted Site-Directed Antibody-Conjugated Apoferritin Nanovehicle Favorably Influences In Vivo Side Effects of Doxorubicin.

Herein, we describe the in vivo effects of doxorubicin (DOX) encapsulated in ubiquitous protein apoferritin (APO) and its efficiency and safety in anti-tumor treatment. APODOX is both passively (through Enhanced Permeability and Retention effect) and actively targeted to tumors through prostate-specific membrane antigen (PSMA) via mouse antibodies conjugated to the surface of horse spleen APO. To achieve site-directed conjugation of the antibodies, a HWRGWVC heptapeptide linker was used. The prostate cancer-targeted and non-targeted nanocarriers were tested using subcutaneously implanted LNCaP cells in athymic mice models, and compared to free DOX. Prostate cancer-targeted APODOX retained the high potency of DOX in attenuation of tumors (with 55% decrease in tumor volume after 3 weeks of treatment). DOX and non-targeted APODOX treatment caused damage to liver, kidney and heart tissues. In contrast, no elevation in liver or kidney enzymes and negligible changes were revealed by histological assessment in prostate cancer-targeted APODOX-treated mice. Overall, we show that the APO nanocarrier provides an easy encapsulation protocol, reliable targeting, high therapeutic efficiency and very low off-target toxicity, and is thus a promising delivery system for translation into clinical use.

Uptake kinetics of pesticides chlorpyrifos and tebuconazole in the earthworm Eisenia andrei in two different soils.

Agriculture is today indispensably connected with enormous use of pesticides. Despite tough regulation, their entrance into soil cannot be excluded and they might enter soil organisms and plants and continue further to terrestrial food chains. This study was conducted to investigate the bioaccumulation of two pesticides currently used in large amounts, the insecticide chlorpyrifos (CLP) and the fungicide tebuconazole (TBZ). Their detailed uptake kinetics in the model earthworm species Eisenia andrei were measured in two arable soils differing in organic carbon content (1.02 and 1.93% respectively). According to our results, a steady state was reached after 3-5 days for both pesticides and soils. The values of bioaccumulation factors calculated at the steady state ranged from 4.5 to 6.3 for CLP and 2.2-13.1 for TBZ. Bioaccumulation factors were also calculated as the ratio of uptake and elimination rate constants with results comparable with steady-state bioaccumulation factors. The results suggested that the degradation and bioaccumulation of tested compounds might be influenced by other factors than only total organic carbon (e.g. clay content). The lower Koc and hydrophobicity of TBZ relative to CLP probably led to higher availability of TBZ through pore water exposure. On the other hand, CLP's higher hydrophobicity probably caused an increase in availability by its additional uptake via ingestion. To enable a proper ecological risk assessment of current pesticides in soils, it is necessary to accurately determine their bioaccumulation in soil invertebrates. We believe that our study not only brings such information for two specific pesticides but also addresses key methodological issues in this area.

Currently and recently used pesticides in Central European arable soils.

Although large amounts of pesticides are used annually and a majority enters the soil to form short- or long-term residues, extensive soil surveys for currently used pesticides (CUPs) are scarce. To determine the status of CUPs' occurrence in arable land in Central Europe, 51 CUPs and 9 transformation products (TPs) were analysed in 75 arable soils in the Czech Republic (CR) several months after the last pesticide application. Moreover, two banned triazines (simazine and atrazine) and their TPs were analysed because of their frequent detection in CR waters. Multi-residue pesticide analysis on LC-MS/MS after soil QuEChERS extraction was used. The soils contained multiple pesticide residues frequently (e.g. 51% soils with ≥5 pesticides). The levels were also noticeable (e.g. 36% soils with ≥3 pesticides exceeding the threshold of 0.01mg/kg). After triazine herbicides (89% soils), conazole fungicides showed the second most frequent occurrence (73% soils) and also high levels (53% soils with total conazoles above 0.01mg/kg). Frequent occurrence was found also for chloroacetanilide TPs (25% of soils), fenpropidin (20%) and diflufenican (17%). With the exception of triazines' negative correlation to soil pH, no clear relationships were found between pesticide occurrence and soil properties. Association of simazine TPs with terbuthylazine and its target crops proved the frequent residues of this banned compound originate from terbuthylazine impurities. In contrast, frequent atrazine-2-hydroxy residue is probably a legacy of high atrazine usage in the past. The occurrence and levels of compounds were closely associated with their solubility, hydrophobicity and half-life. The results showed links to CR water-monitoring findings. This study represents the first extensive survey of multiple pesticide residues in Central European arable soils, including an insight into their relationships to site and pesticide properties.

Establishment of oral squamous cell carcinoma cell line and magnetic bead-based isolation and characterization of its CD90/CD44 subpopulations.

In this study, we describe the establishment of the human papillomavirus 18-positive, stage II, grade 1, T2N0M0 head and neck tumor primary cell line derived from oral squamous cell carcinoma of a non-smoking patient by using two different protocols. Furthermore, a preparation of subpopulations derived from this primary cell line according to the cluster of differentiation molecules CD44/CD90 status using magnetic bead-based separation and their characterization was performed. Impedance-based real-time cell analysis, enzyme-linked immunsorbant assay (ELISA), wound-healing assay, flow-cytometry, gene expression analysis, and MTT assay were used to characterize these four subpopulations (CD44+/CD90-, CD44-/CD90-, CD44+/CD90+, CD44-/CD90-). We optimised methodics for establishement of primary cell lines derived from oral squamous cell carcinoma tissue samples and subsequent separation of mesenchymal (CD90+) and epithelial (CD90-) types of tumorous cells. Primary cell line prepared by using trypsin proteolysis was more viable than the one prepared by using collagenase. According to our results, CD90 separation is a necessary step in preparation of permanent tumor-tissue derived cell lines. Based on the wound-healing assay, CD44+ cells exhibited stronger migratory capacity than CD44- subpopulations. CD44+ subpopulations had also significantly higher expression of BIRC5 and SOX2, lower expression of FLT1 and IL6, and higher levels of basal autophagy compared to CD44- subpopulations. Furthermore, co-cultivation experiments revealed that CD44-/CD90+ cells supported growth of epithelial tumor cells (CD44+/CD90-). On the contrary, factors released by CD44+/CD90+ type of cells seem to have rather inhibiting effect. The most cisplatin-resistant subpopulation with the shortest doubling time was CD44-/CD90+, but this subpopulation had a low migratory capacity.