Background and aim: Melanoma is a fatal form of skin cancer that carries a grave prognosis if the cancer cells spread and form metastases. The Kynurenine (Kyn) pathway is activated by the enzyme indoleamine 2,3-dioxygenase 1 (IDO-1) and has been shown to have a role in tumour progression. We have previously shown that interferon-γ (IFN-γ) acts as an inducer of tryptophan (Trp) degradation to Kyn in keratinocytes of the basal layer in a 3D epidermis model. Before extending our reconstructed human epidermis model to not only contain keratinocytes but also fibroblasts and melanocytes/melanoma cells, we have in this study set out to investigate possible differences between primary adult melanocytes and six melanoma cell lines regarding the expression of the immune checkpoint inhibitors IDO-1 and programmed death ligand 1 (PD-L1) together with Kyn production.

Methods: The melanocytes and melanoma cells were stimulated with 1–20 ng/ml of IFN-γ and the levels of Trp to Kyn degradation were monitored with high-performance liquid chromatography (HPLC). To analyze the viability of the cell types after IFN-γ treatment, an MTT assay was performed. mRNA quantity of IDO-1, PD-L1 and IFN-γ receptor (IFN-GR1) was analyzed with qPCR.

Results: After 24 h, only the metastatic cell line WM-266-4 was affected by all concentrations of IFN-γ, whereas at 48 h, the higher IFN-γ concentrations gave a more pronounced effect on the viability in all cell types. Trp was detected at various levels in the culture medium from all cell types before and after IFN-γ treatment. The degradation to Kyn was detected in primary melanocytes, Mel Juso, and Mel Ho cell lines after 24 h of treatment and low levels of IFN-γ. However, the higher concentration of IFN-γ, 20 ng/ml, induced Kyn to various degrees in all cell types after 24 h. The change in mRNA quantity of IDO-1 and PD-L1 was similar in all cell types.

Conclusion: To conclude, no significant difference in upregulation of the immune checkpoint inhibitors PD-L1 and IDO-1 was seen between primary tumour and metastatic melanoma. IFN-γ stimulation of melanocytes and different stages of melanoma cell lines resulted in an increased Kyn/Trp ratio in the more aggressive melanoma cells when a high concentration was used (20 ng/ml) but when a lower concentration of IFN-γ (5 ng/ml) was used an increased Kyn/Trp ratio were detected in media from primary melanocytes and early-stage melanoma.

Spherical, multicellular aggregates of tumor cells, or three-dimensional (3D) tumor models, can be grown from established cell lines or dissociated cells from tissues in a serum-free medium containing appropriate growth factors. Air–liquid interfaces (ALIs) represent a 3D approach that mimics and supports the differentiation of respiratory tract and skin 3D models in vitro. Many 3D tumor cell models are cultured in conjunction with supporting cell types, such as fibroblasts, endothelial cells, or immune cells. To further mimic the in vivo situation, several extracellular matrix models are utilized to support tumor cell growth. Scaffolds used for 3D tumor cell culture growth include both natural and synthetic hydrogels. Three-dimensional cell culture experiments in vitro provide more accurate data on cell-to-cell interactions, tumor characteristics, drug discovery, metabolic profiling, stem cell research, and diseases. Moreover, 3D models are important for obtaining reliable precision data on therapeutic candidates in human clinical trials before predicting drug cytotoxicity. This review focuses on the recent literature on three different tissue types of 3D tumor models, i.e., tumors from a colorectal site, prostate, and skin. We will discuss the establishment of 3D tumor cell cultures in vitro and the requirement for additional growth support.

In vitro cultured 3D models of CRC have been demonstrated to hold considerable worth in drug discovery, drug resistance analysis, and in studying cell-cell and cell-matrix interactions that occur in the tumor microenvironment. The 3D models resemble the in vivo physiological microenvironment by replicating the cell type composition and tissue architecture. Molecularly imprinted polymers (MIPs) have been investigated for use instead of antibodies against small non-immunogenic structures, such as sialic acid (SA). Glyco-conjugates including SA are present on all cells, and often deregulated on cancer cells. Here, we present a novel approach for targeting and detecting colorectal cancer cells (CRC) by using in vitro cultured HT29 3D spheroids co-cultured in vitro with either fluorescent MIPs targeting SA, SA-MIPs, or the two lectins targeting SA, MAL I, and SNA. Both formaldehyde-fixed and viable HT29 3D spheroids with or without SA-MIPs were imaged in 3D by confocal microscopy. The results revealed a preserved cell morphology and viability of the HT29 3D spheroids co-cultured in vitro with SA-MIPs. However, the lectins MAL I and SNA targeting the a-2,3 or a-2,6 SA glycosidic linkages, respectively, affected the cell viability when co-cultured with the viable HT29 3D spheroids, and no living cells could be detected. Here, we have shown that the SA-MIPs could be used as a safe and low-cost diagnostic tool for targeting and detecting cancer cells in a physiologically relevant 3D cancer model in vitro.

BACKGROUND AND OBJECTIVES: L was investigated both in normal and malignant skin cells.

METHODS: Antioxidant activity of the extracts was analyzed by using the Trolox Equivalent Antioxidant Capacity (TEAC) assay. High-Performance Thin-Layer Chromatography (HPTLC) was performed to demonstrate the phytochemical profile, and the total flavonoid content was analyzed with an aluminum chloride colorimetric method. The anti-inflammatory effect was investigated by cell treatments using the plant extracts. Thereafter, the possible suppression of induced IL-6 response was measured from the cultured skin cancer cell lines A2058 and A431, and normal primary keratinocytes with Enzyme-Linked Immunosorbent Assay (ELISA).

RESULTS: also had the highest flavonoid content and showed the highest antioxidant activity of the three extracts tested.

CONCLUSION: possess properties such as antioxidant and anti-inflammatory activities in both normal and malignant keratinocytes, and thus could be a promising agent controlling the pro-inflammatory IL-6 production.

Molecularly imprinted polymers (MIPs) and the imprinting technique provide polymeric material with recognition elements similar to natural antibodies. The template of choice (i.e., the antigen) can be almost any type of smaller or larger molecule, protein, or even tissue. There are various formats of MIPs developed for different medical purposes, such as targeting, imaging, assay diagnostics, and biomarker detection. Biologically applied MIPs are widely used and currently developed for medical applications, and targeting the antigen with MIPs can also help in personalized medicine. The synthetic recognition sites of the MIPs can be tailor-made to function as analytics, diagnostics, and drug delivery systems. This review will cover the promising clinical applications of different MIP systems recently developed for disease diagnosis and treatment.

Colorectal cancer (CRC) is the second most metastatic disease with the majority of cases detected in Western countries. Metastases are formed by circulating altered phenotype tumor cells causing 20% of CRC related deaths. Metastatic cells may show higher expression of surface molecules such as CD44, and changes in morphological properties are associated with increased invasiveness and poor prognosis. In this study, we intended to mimic the environment for metastasizing cells. Here, we used digital holographic cytometry (DHC) analysis to determine cellular morphological properties of three metastatic and two non-metastatic colorectal cancer cell lines to show differences in morphology between the CRC cells and peripheral blood mononuclear cells (PBMCs). By establishing differences in cell area, cell thickness, cell volume, and cell irregularity even when the CRC cells were in minority (5% out of PBMCs), DHC does discriminate between CRC cells and the PBMCs in vitro. We also analyzed the epithelial marker EpCAM and migration marker CD44 using flow cytometry and demonstrate that the CRC cell lines and PBMC cells differ in EpCAM and CD44 expression. Here, we present DHC as a new powerful tool in discriminating cells of different sizes in suspension together with a combination of biomarkers.

Low affinity immunoglobulin gamma Fc region receptor III-A (FcγRIIIa) is a cell surface protein that belongs to a family of Fc receptors that facilitate the protective function of the immune system against pathogens. However, the role of FcγRIIIa in prostate cancer (PCa) progression remained unknown. In this study, we found that FcγRIIIa expression was present in PCa cells and its level was significantly higher in metastatic lesions than in primary tumors from the PCa cohort (p=0.006). PCa patients with an elevated level of FcγRIIIa expression had poorer biochemical recurrence (BCR)-free survival compared with those with lower FcγRIIIa expression, suggesting that FcγRIIIa is of clinical importance in PCa. We demonstrated that overexpression of FcγRIIIa increased the proliferative ability of PCa cell line C4-2 cells, which was accompanied by the upregulation of androgen receptor (AR) and phosphatidylinositol-4-phosphate 5-kinase alpha (PIP5Kα), which are the key players in controlling PCa progression. Conversely, targeted inhibition of FcγRIIIa via siRNA-mediated knockdown or using its inhibitory antibody suppressed growth of xenograft PC-3 and PC-3M prostate tumors and reduced distant metastasis in xenograft mouse models. We further showed that elevated expression of AR enhanced FcγRIIIa expression, whereas inhibition of AR activity using enzalutamide led to a significant downregulation of FcγRIIIa protein expression. Similarly, inhibition of PIP5K1α decreased FcγRIIIa expression in PCa cells. FcγRIIIa physically interacted with PIP5K1α and AR via formation of protein-protein complexes, suggesting that FcγRIIIa is functionally associated with AR and PIP5K1α in PCa cells. Our study identified FcγRIIIa as an important factor in promoting PCa growth and invasion. Further, the elevated activation of FcγRIII and AR and PIP5K1α pathways may cooperatively promote PCa growth and invasion. Thus, FcγRIIIa may serve as a potential new target for improved treatment of metastatic and castration-resistant PCa.

Sialic acid (SA) is a monosaccharide usually linked to the terminus of glycan chains on the cell surface. It plays a crucial role in many biological processes, and hypersialylation is a common feature in cancer. Lectins are widely used to analyze the cell surface expression of SA. However, these protein molecules are usually expensive and easily denatured, which calls for the development of alternative glycan-specific receptors and cell imaging technologies. In this study, SA-imprinted fluorescent core-shell molecularly imprinted polymer particles (SA-MIPs) were employed to recognize SA on the cell surface of cancer cell lines. The SA-MIPs improved suspensibility and scattering properties compared with previously used core-shell SA-MIPs. Although SA-imprinting was performed using SA without preference for the α2,3- and α2,6-SA forms, we screened the cancer cell lines analyzed using the lectins Maackia Amurensis Lectin I (MAL I, α2,3-SA) and Sambucus Nigra Lectin (SNA, α2,6-SA). Our results show that the selected cancer cell lines in this study presented a varied binding behavior with the SA-MIPs. The binding pattern of the lectins was also demonstrated. Moreover, two different pentavalent SA conjugates were used to inhibit the binding of the SA-MIPs to breast, skin, and lung cancer cell lines, demonstrating the specificity of the SA-MIPs in both flow cytometry and confocal fluorescence microscopy. We concluded that the synthesized SA-MIPs might be a powerful future tool in the diagnostic analysis of various cancer cells.

Purpose: Despite the high mortality of metastatic colorectal cancer (mCRC), no new biomarker tools are available for predicting treatment response. We developed gene-mutation-based algorithms as a biomarker classifier to predict treatment response with better precision than the current predictive factors.

Methods: Random forest machine learning (ML) was applied to identify the candidate algorithms using the MSK Cohort (n = 471) as a training set and validated in the TCGA Cohort (n = 221). Logistic regression, progression-free survival (PFS), and univariate/multivariate Cox proportional hazard analyses were performed and the performance of the candidate algorithms was compared with the established risk parameters.

Results: A novel 7-Gene Algorithm based on mutation profiles of seven KRAS-associated genes was identified. The algorithm was able to distinguish non-progressed (responder) vs. progressed (non-responder) patients with AUC of 0.97 and had predictive power for PFS with a hazard ratio (HR) of 16.9 (p < 0.001) in the MSK cohort. The predictive power of this algorithm for PFS was more pronounced in mCRC (HR = 16.9, p < 0.001, n = 388). Similarly, in the TCGA validation cohort, the algorithm had AUC of 0.98 and a significant predictive power for PFS (p < 0.001).

Conclusion: The novel 7-Gene Algorithm can be further developed as a biomarker model for prediction of treatment response in mCRC patients to improve personalized therapies.Keywords: KRAS; algorithm; colorectal cancer biomarkers; colorectal cancer metastasis; colorectal cancer progression; gene mutations.

PURPOSE: There is an urgent need for developing new biomarker tools to accurately predict treatment response of breast cancer, especially the deadly triple-negative breast cancer. We aimed to develop gene-mutation-based machine learning (ML) algorithms as biomarker classifiers to predict treatment response of first-line chemotherapy with high precision.

METHODS: = 807) with up to 220 months follow-up. Subtypes of breast cancer including triple-negative and luminal A (ER+, PR+ and HER2-) were also assessed. The predictive performance of the candidate algorithms as classifiers was further assessed using logistic regression, Kaplan-Meier progression-free survival (PFS) plot, and univariate/multivariate Cox proportional hazard regression analyses.

RESULTS: < 0.0001).

CONCLUSIONS: The novel 12-Gene algorithm based on multitude gene-mutation profiles identified through ML has a potential to predict breast cancer treatment response to therapies, especially in triple-negative subgroups patients, which may assist personalized therapies and reduce mortality.

Molecularly imprinted polymers (MIPs) against sialic acid (SA) have been developed as a detection tool to target cancer cells. Before proceeding to in vivo studies, a better knowledge of the overall effects of MIPs on the innate immune system is needed. The aim of this study thus was to exemplarily assess whether SA-MIPs lead to inflammatory and/or cytotoxic responses when administered to phagocytosing cells in the innate immune system. The response of monocytic/macrophage cell lines to two different reference particles, Alhydrogel and PLGA, was compared to their response to SA-MIPs. In vitro culture showed a cellular association of SA-MIPs and Alhydrogel, as analyzed by flow cytometry. The reference particle Alhydrogel induced secretion of IL-1β from the monocytic cell line THP-1, whereas almost no secretion was provoked for SA-MIPs. A reduced number of both THP-1 and RAW 264.7 cells were observed after incubation with SA-MIPs and this was not caused by cytotoxicity. Digital holographic cytometry showed that SA-MIP treatment affected cell division, with much fewer cells dividing. Thus, the reduced number of cells after SA-MIP treatment was not linked to SA-MIPs cytotoxicity. In conclusion, SA-MIPs have a low degree of inflammatory properties, are not cytotoxic, and can be applicable for future in vivo studies.

PIP5K1α has emerged as a promising drug target for the treatment of castration-resistant prostate cancer (CRPC), as it acts upstream of the PI3K/AKT signaling pathway to promote prostate cancer (PCa) growth, survival and invasion. However, little is known of the molecular actions of PIP5K1α in this process. Here, we show that siRNA-mediated knockdown of PIP5K1α and blockade of PIP5K1α action using its small molecule inhibitor ISA-2011B suppress growth and invasion of CRPC cells. We demonstrate that targeted deletion of the N-terminal domain of PIP5K1α in CRPC cells results in reduced growth and migratory ability of cancer cells. Further, the xenograft tumors lacking the N-terminal domain of PIP5K1α exhibited reduced tumor growth and aggressiveness in xenograft mice as compared to that of controls. The N-terminal domain of PIP5K1α is required for regulation of mRNA expression and protein stability of PIP5K1α. This suggests that the expression and oncogenic activity of PIP5K1α are in part dependent on its N-terminal domain. We further show that PIP5K1α acts as an upstream regulator of the androgen receptor (AR) and AR target genes including CDK1 and MMP9 that are key factors promoting growth, survival and invasion of PCa cells. ISA-2011B exhibited a significant inhibitory effect on AR target genes including CDK1 and MMP9 in CRPC cells with wild-type PIP5K1α and in CRPC cells lacking the N-terminal domain of PIP5K1α. These results indicate that the growth of PIP5K1α-dependent tumors is in part dependent on the integrity of the N-terminal sequence of this kinase. Our study identifies a novel functional mechanism involving PIP5K1α, confirming that PIP5K1α is an intriguing target for cancer treatment, especially for treatment of CRPC.

Cells adhering onto surfaces sense and respond to chemical and physical surface features. The control over cell adhesion behavior influences cell migration, proliferation, and differentiation, which are important considerations in biomaterial design for cell culture, tissue engineering, and regenerative medicine. Here, we report on a supramolecular-based approach to prepare reversible self-assembled monolayers (rSAMs) with tunable lateral mobility and dynamic control over surface composition to regulate cell adhesion behavior. These layers were prepared by incubating oxoacid-terminated thiol SAMs on gold in a pH 8 HEPES buffer solution containing different mole fractions of ω-(ethylene glycol)2-4- and ω-(GRGDS)-, α-benzamidino bolaamphiphiles. Cell shape and morphology were influenced by the strength of the interactions between the amidine-functionalized amphiphiles and the oxoacid of the underlying SAMs. Dynamic control over surface composition, achieved by the addition of inert filler amphiphiles to the RGD-functionalized rSAMs, reversed the cell adhesion process. In summary, rSAMs featuring mobile bioactive ligands offer unique capabilities to influence and control cell adhesion behavior, suggesting a broad use in biomaterial design, tissue engineering, and regenerative medicine.

Glycoproteins are abundant on the cell surface of mammals, providing structural support, modulating cell membrane properties, and acting as signaling agents. Variation of glycosylation patterns has been found to indicate various disease states, including cell malignancy. Sialic acid (SA) is present as a terminating group on cell-surface glycans, and its overexpression has been linked to several types of cancer. Detection of SA on the cell surface is therefore critical for detection of cancer in its early stages. In this work, a fluorescent molecularly imprinted polymer layer targeting SA was synthesized on the surface of silica-coated polystyrene (PS) particles. Compared to previous works, a PS core supplies a lighter, lower-density support for improved suspension stability and scattering properties. Moreover, their smaller size provides a higher surface-area-to-volume ratio for binding. The incorporation of a fluorescent monomer in the MIP shell allowed for simple and rapid determination of binding specificity in polar media due to a deprotonation-reprotonation interaction mechanism between the fluorescent monomer and SA, which led to spectral changes. Upon titration of the MIP particles with SA in suspension, an increase in fluorescence emission of the particles was observed, with the MIP particles binding SA more selectively compared to the nonimprinted polymer (NIP) control particles. In cell staining experiments performed by flow cytometry, the binding behavior of the MIP particles compared favorably with that of SA-binding lectins. NIPs prepared with a "dummy" template served as a better negative control in cell binding assays due to the favorable inward orientation of template-binding functional groups in the polymer shell, which reduced nonspecific binding. The results show that fluorescent MIPs targeting SA are a promising tool for in vitro fluorescence staining of cancerous cells and for future diagnosis of cancer at early stages.

Sialylations are post-translational modifications of proteins and lipids that play important roles in many cellular events, including cell-cell interactions, proliferation, and migration. Tumor cells express high levels of sialic acid (SA), which are often associated with the increased invasive potential in clinical tumors, correlating with poor prognosis. To overcome the lack of natural SA-receptors, such as antibodies and lectins with high enough specificity and sensitivity, we have used molecularly imprinted polymers (MIPs), or "plastic antibodies", as nanoprobes. Because high expression of epithelial cell adhesion molecule (EpCAM) in primary tumors is often associated with proliferation and a more aggressive phenotype, the expression of EpCAM and CD44 was initially analyzed. The SA-MIPs were used for the detection of SA on the cell surface of breast cancer cells. Lectins that specifically bind to the a-2,3 SA and a-2,6 SA variants were used for analysis of SA expression, with both flow cytometry and confocal microscopy. Here we show a correlation of EpCAM and SA expression when using the SA-MIPs for detection of SA. We also demonstrate the binding pattern of the SA-MIPs on the breast cancer cell lines using confocal microscopy. Pre-incubation of the SA-MIPs with SA-derivatives as inhibitors could reduce the binding of the SA-MIPs to the tumor cells, indicating the specificity of the SA-MIPs. In conclusion, the SA-MIPs may be a new powerful tool in the diagnostic analysis of breast cancer cells.

Cluster of differentiation (CD81) is a type of protein, which is encoded by CD81 gene. Beside that CD81 is also known under other names such as Target of the Antiproliferative Antibody 1 (TAPA-1) and Tetraspanin-28 (TSPAN28). Location of CD81 is known to be on chromosome 11 (11p15.5), where it contains 15-20 bases in length. It is expressed mostly in cells of testis, ovary, endometrium, placenta, bone marrow, smooth muscles and others. The main function of the CD81 protein is to mediate signal transduction events, which are important for cells' development, activation, growth and motility. The CD81 gene is also known as a candidate for many malignancies because of its location. The characteristic feature of CD81 is that it is highly hydrophobic and contains a short N- and C-terminal cytoplasmic domains together with cytoplasmic cysteines, potential sites of palmitoylation as well as four transmembrane domains where they together hold the protein in a cell membrane. There are two CD81 isoforms, isoform 1 and isoform 2. Isoforms of CD81 are usually found in a tumor-suppressor region where they have a great impact on tumor development. There has always been a high interest in research on CD81 function in viral disease development. In fact, it is known that CD81 contributes in the development of diseases such as hepatitis C, malaria and various types of cancer. Since the complete effect of CD81 is unknown, further research and scientific methodology could potentially discover all possible functions and mechanisms regulated by the CD81 protein in human body.

Breast cancer is the second most common cancer worldwide. Metastasis is the main reason for death in breast cancer, and today, there is a lack of methods to detect and isolate circulating tumor cells (CTCs), mainly due to their heterogeneity and rarity. There are some systems that are designed to detect rare epithelial cancer cells in whole blood based on the most common marker used today, the epithelial cell adhesion molecule (EpCAM). It has been shown that aggressive breast cancer metastases are of non-epithelial origin and are therefore not always detected using EpCAM as a marker. In the present study, we used an in vitro-based circulating tumor cell model comprising a collection of six breast cancer cell lines and white blood cell lines. We used digital holographic cytometry (DHC) to characterize and distinguish between the different cell types by area, volume and thickness. Here, we present significant differences in cell size-related parameters observed when comparing white blood cells and breast cancer cells by using DHC. In conclusion, DHC can be a powerful diagnostic tool for the characterization of CTCs in the blood.

BACKGROUND: Interferon-gamma (IFN-γ) represents a potent inducer for keratinocyte inflammatory and immune activation in vitro. Since tryptophan (trp) conversion to kynurenine (kyn) is involved in inflammation, the topical kyn/trp ratio may serve as a biomarker of skin inflammation. However, the trp metabolism in keratinocytes exposed to IFN-γ is not yet fully understood.

OBJECTIVE: The aim of this study was to establish a human epidermis model in order to quantify cytokine and kyn/trp secretion from IFN-γ stimulated cells and tissues. Moreover, to compare the cell response of 2D-cultured keratinocytes and the 3D epidermis model.

METHODS: Polycarbonate filters were used on which primary keratinocytes could attach and stratify in order to form the typical layers of reconstructed human epidermis (RHE). After IFN-γ treatment, secretion of kyn/trp was measured by high performance liquid chromatography. Gene and protein expression of indoleamine 2,3-dioxygenase 1 (IDO) was analyzed with real-time PCR and immunohistochemistry. The secretion of cytokines was quantified with ELISA.

RESULTS: Trp catabolism to kyn was significantly increased (P < 0.01) in the 2D culture in response to IFN-γ treatment. Before kyn secretion, IDO was strongly upregulated (P < 0.001). IFN-γ treatment also increased the secretion of IL-6 and IL-8 from the keratinocytes. In the RHE, IDO was upregulated by IFN-γ, and kyn secretion could be detected. Interestingly, IDO expression was only present in the basal cells of the RHE.

CONCLUSION: Our results suggest that IFN-γ acts as an inducer of trp degradation preferentially in undifferentiated keratinocytes, indicated by the IDO expression in the basal layer of the RHE.

Molecularly imprinted polymers (MIPs) are currently widely used and further developed for biological applications. The MIP synthesis procedure is a key process, and a wide variety of protocols exist. The templates that are used for imprinting vary from the smallest glycosylated glycan structures or even amino acids to whole proteins or bacteria. The low cost, quick preparation, stability and reproducibility have been highlighted as advantages of MIPs. The biological applications utilizing MIPs discussed here include enzyme-linked assays, sensors, in vivo applications, drug delivery, cancer diagnostics and more. Indeed, there are numerous examples of how MIPs can be used as recognition elements similar to natural antibodies

Increased exposition to blue light may induce many changes in cell behavior and significantly affect the critical characteristics of cells. Here we show that multimodal holographic microscopy (MHM) within advanced image analysis is capable of correctly distinguishing between changes in cell motility, cell dry mass, cell density, and cell death induced by blue light. We focused on the effect of blue light with a wavelength of 485 nm on morphological and dynamical parameters of four cell lines, malignant PC-3, A2780, G361 cell lines, and the benign PNT1A cell line. We used MHM with blue light doses 24 mJ/cm(2), 208 mJ/cm(2) and two kinds of expositions (500 and 1000 ms) to acquire real-time quantitative phase information about cellular parameters. It has been shown that specific doses of the blue light significantly influence cell motility, cell dry mass and cell density. These changes were often specific for the malignant status of tested cells. Blue light dose 208 mJ/cm(2) x 1000 ms affected malignant cell motility but did not change the motility of benign cell line PNT1A. This light dose also significantly decreased proliferation activity in all tested cell lines but was not so deleterious for benign cell line PNT1A as for malignant cells. Light dose 208 mJ/cm(2) x 1000 ms oppositely affected cell mass in A2780 and PC-3 cells and induced different types of cell death in A2780 and G361 cell lines. Cells obtained the least damage on lower doses of light with shorter time of exposition.

Cancer cells often have an increased amount of glycans, such as sialic acid (SA), on the cell surface, which normally play an important role in cell growth, proliferation and differentiation. In this study, SA expression is determined by fluorescent nanoprobes, molecularly imprinted polymers, SA-MIPs. The nanoprobes are synthesized with an imprinting approach to produce tailor-made fluorescent core-shell particles with high affinity for cell surface SA. Inflammation and cytokine production are well known tumor promoters, modulating the cellular microenvironment, including an aberrant cell surface glycan pattern. The recombinant cytokines IL-4, IL-6, IL-8 and a cocktail of cytokines collected from stimulated T leukemia Jurkat cells were used to induce in vitro inflammation in two cell lines, and thereafter analyzed with the use of SA-MIPs and flow cytometry. One of cell lines showed a different binding pattern of SA-MIPs after treatment with recombinant cytokines and the cytokine cocktail. This study shows that SA-MIPs can be an important tool in the investigation of overexpressed glycans in the tumor microenvironment.

Digital holographic cytometry (DHC) is a state-of-the-art quantitative phase imaging (QPI) method that permits time-lapse imaging of cells without induced cellular toxicity. DHC platforms equipped with semi-automated image segmentation and analysis software packages for assessing cell behavior are commercially available. In this study we investigate the possible uptake of nanoprobes in macrophages in vitro over time.

Cancer är en term som används för cirka 200 olika sjukdomar. Det de alla har gemensamt är att cellerna i kroppen börjar delas okontrollerat. 2018 fanns det 18 miljoner cancerfall över hela världen. I Sverige kommer ungefär varje tredje person att diagnostiseras med en cancersjukdom någon gång under sin livstid.

Det är en stor utmaning för vetenskapen att hitta sätt att diagnostisera och behandla dessa sjukdomar. På Malmö universitet arbetar en ny generation kemister, fysiker och biologer tillsammans i två internationella nätverk, BioCapture och GlycoImaging. Deras forskning fokuserar på att utforma antikroppar, plastkulor, som ska upptäcka cancerceller i ett tidigt skede. Kulorna är dessutom billiga att producera.

De två projekten samordnas av Börje Sellergren och Anette Gjörloff Wingren, som utbildar och handleder 19 doktorander. Utställningen Fighting Cancer with Plastic Bullets belyser doktorandernas tvärvetenskapliga arbete och deras betydelse för cancerforskningen. Biblioteket har fått bidrag av Sten K Johnsons stiftelse för att producera utställningen.

Review on TNFRSF9 (CD137), with data on DNA, on the protein encoded, and where the gene is implicated.

Review on CDK2, with data on DNA, on the protein encoded, and where the gene is implicated.

Review on PIP5K1A, with data on DNA, on the protein encoded, and where the gene is implicated.

To understand complex biological processes, scientists must gain insight into the function of individual living cells. In contrast to the imaging of fixed cells, where a single snapshot of the cell’s life is retrieved, live-cell imaging allows investigation of the dynamic processes underlying the function and morphology of cells. Label-free imaging of living cells is advantageous since it is used without fluorescent probes and maintains an appropriate environment for cellular behavior, otherwise leading to phototoxicity and photo bleaching. Quantitative phase imaging (QPI) is an ideal method for studying live cell dynamics by providing data from noninvasive monitoring over arbitrary time scales. The effect of drugs on migration, proliferation, and apoptosis of cancer cells are emerging fields suitable for QPI analysis. In this review, we provide a current insight into QPI applied to cancer research.

In this study, an epitope-imprinting strategy was employed for the dynamic display of bioactive ligands on a material interface. An imprinted surface was initially designed to exhibit specific affinity towards a short peptide (i.e., the epitope). This surface was subsequently used to anchor an epitope-tagged cell-adhesive peptide ligand (RGD: Arg-Gly-Asp). Owing to reversible epitope-binding affinity, ligand presentation and thereby cell adhesion could be controlled. As compared to current strategies for the fabrication of dynamic biointerfaces, for example, through reversible covalent or host–guest interactions, such a molecularly tunable dynamic system based on a surface-imprinting process may unlock new applications in in situ cell biology, diagnostics, and regenerative medicine.

Digital holographic (DH) microscopy is a digital high-resolution holographic imaging technique with the capacity of quantification of cellular conditions without any staining or labeling of cells. The unique measurable parameters are the cell number, cell area, thickness, and volume, which can be coupled to proliferation, migration, cell cycle analysis, viability, and cell death. The technique is cell friendly, fast and simple to use and has unique imaging capabilities for time-lapse investigations on both the single cell and the cell-population levels. The interest for analyzing specifically cell volume changes with DH microscopy, resulting from cytotoxic treatments, drug response, or apoptosis events has recently increased in popularity. We and others have used DH microscopy showing that the technique has the sensitivity to distinguish between different cells and treatments. Recently, DH microscopy has been used for cellular diagnosis in the clinic, providing support for using the concept of DH, e.g., screening of malaria infection of red blood cells (RBC), cervix cancer screening, and sperm quality. Because of its quick and label-free sample handling, DH microscopy will be an important tool in the future for personalized medicine investigations, determining the optimal therapeutic concentration for both different cancer types and individual treatments.

Single-cell studies using noninvasive imaging is a challenging, yet appealing way to study cellular characteristics over extended periods of time, for instance to follow cell interactions and the behavior of different cell types within the same sample. In some cases, e.g., transplantation culturing, real-time cellular monitoring, stem cell studies, in vivo studies, and embryo growth studies, it is also crucial to keep the sample intact and invasive imaging using fluorophores or dyes is not an option. Computerized methods are needed to improve throughput of image-based analysis and for use with noninvasive microscopy such methods are poorly developed. By combining a set of well-documented image analysis and classification tools with noninvasive microscopy, we demonstrate the ability for long-term image-based analysis of morphological changes in single cells as induced by a toxin, and show how these changes can be used to indicate changes in biological function. In this study, adherent cell cultures of DU-145 treated with low-concentration (LC) etoposide were imaged during 3 days. Single cells were identified by image segmentation and subsequently classified on image features, extracted for each cell. In parallel with image analysis, an MTS assay was performed to allow comparison between metabolic activity and morphological changes after long-term low-level drug response. Results show a decrease in proliferation rate for LC etoposide, accompanied by changes in cell morphology, primarily leading to an increase in cell area and textural changes. It is shown that changes detected by image analysis are already visible on day 1 for [Formula: see text] etoposide, whereas effects on MTS and viability are detected only on day 3 for [Formula: see text] etoposide concentration, leading to the conclusion that the morphological changes observed occur before and at lower concentrations than a reduction in cell metabolic activity or viability. Three classifiers are compared and we report a best case sensitivity of 88% and specificity of 94% for classification of cells as treated/untreated.

Sialic acid (SA) is normally expressed on the cell membranes and is located at the terminal position of the sugar chains. SA plays an important role for regulation of the innate immunity, function as markers of the cells and can be recognized by a variety of receptors. Interestingly, the level of SA expression is increased on metastatic cancer cells. The availability of specific antibodies against SA is limited and, therefore, biomarker tools for detection of SA are lacking. We have recently presented a novel method for specific fluorescence labeling of SA molecular imprinted polymers (MIP). Here, we have performed an extended screening of SA expression by using SA-MIP and included four different chronic lymphocytic leukemia (CLL) cell lines, conveniently analyzed by flow cytometry and fluorescence microscopy. SA expression was detected in four cell lines at different levels, and the SA expression were verified with lectin-FITC. These results show that SA-MIP can be used as a plastic antibody for detection of SA using both flow cytometry and fluorescence microscopy. We suggest that SA-MIP can be used for screening of different tumor cells of various stages, including CLL cells.

Adult diffuse large B cell lymphoma (DLBCL) is a heterogeneous form of hematopoietic cancer and difficult to treat. In order to find a better diagnostic indication for the disease, we analyzed low molecular weight protein tyrosine phosphatase (LMWPTP) that in humans is encoded by the ACP1 gene. LMWPTP is an enzyme shown to counteract protein tyrosine kinases (PTK) and was suggested to be a negative growth factor regulator. However, the 18 kDa PTP can also have a positive effect on cell growth and proliferation, indicating a controversial role in the tumorigenic process. LMWPTP exists in different isoforms which are electrophoretically, kinetically and immunologically distinct. We have studied two subgroups of DLBCL consisting of a germinal center B cell like (GCB) and a non-germinal center B cell like (non-GCB) group. The two subgroups have been defined by gene-expressing profiling and are associated with differential outcome. The expression levels of LMWPTP protein was compared and showed significant differences between the GCB and non-GCB subgroups (p=0.012). Interestingly, when the samples were divided into survivors and non-survivors, and thereafter analyzed for LMWPTP expression, the samples from patients with a higher survival rate showed increased staining intensity, whereas the samples from patients with lower intensity of LMWPTP did not survive the disease (p=0.001). In conclusion, we have shown that DLBCL patients with worse outcome express LMWPTP with a lower intensity, suggesting a tumor suppressor role for this form of the enzyme.

One mechanism of resistance of prostate cancer (PCa) to enzalutamide (MDV3100) treatment is the increased expression of AR variants lacking the ligand binding-domain, the best characterized of which is AR-V7. We have previously reported that Phosphatidylinositol-4-phosphate 5-kinase alpha (PIP5Kα), is a lipid kinase that links to CDK1 and AR pathways. The discovery of PIP5Kα inhibitor highlight the potential of PIP5K1α as a drug target in PCa. In this study, we show that AR-V7 expression positively correlates with PIP5K1α in tumor specimens from PCa patients. Overexpression of AR-V7 increases PIP5K1α, promotes rapid growth of PCa in xenograft mice, whereas inhibition of PIP5K1α by its inhibitor ISA-2011B suppresses the growth and invasiveness of xenograft tumors overexpressing AR-V7. PIP5K1α is a key co-factor for both AR-V7 and AR, which are present as protein-protein complexes predominantly in the nucleus of PCa cells. In addition, PIP5K1α and CDK1 influence AR-V7 expression also through AKT-associated mechanism dependent on PTEN-status. ISA-2011B disrupts protein stabilization of AR-V7 which is dependent on PIP5K1α, leading to suppression of invasive growth of AR-V7-high tumors in xenograft mice. Our study suggests that combination of enzalutamide and PIP5K1α may have a significant impact on refining therapeutic strategies to circumvent resistance to antiandrogen therapies

It remains poorly understood how the haematopoietic stem/progenitor cells (HSPC) are attracted to their niches and the functional consequences of such interaction. In the present study, we show that the cell cycle regulator cyclin A1 in association with vascular endothelial growth factor receptor 1 (VEGFR1), is required for HSPC and their niches to maintain their function and proper interaction. In the absence of cyclin A1, the HSPC in the BM are increased in their frequency and display an increased migratory and homing ability. Concomitantly, the ability of the endosteal and central BM niche zones to attract and home the wild-type HSPC is significantly reduced in cyclin A1-null mice as compared to the wild-type controls. The impaired proliferation and homing of HSPC in the BM of cyclin A1-null mice are attributed to the increased density of microvessels in the endosteal and central BM niche zones, which is associated with the increased VEGFR1 expression. Thus, modulation of cyclin A1 and VEGFR1 in HSPC and their niches may provide new insights into therapeutic approaches.

We are using the label-free technique of holographic microscopy to analyze cellular parameters including cell number, confluence, cellular volume and area directly in the cell culture environment. We show that death-induced cells can be distinguished from untreated counterparts by the use of holographic microscopy, and we demonstrate its capability for cell death assessment. Morphological analysis of two representative cell lines (L929 and DU145) was performed in the culture flasks without any prior cell detachment. The two cell lines were treated with the anti-tumour agent etoposide for one to three days. Measurements by holographic microscopy showed significant differences in average cell number, confluence, volume and area when comparing etoposide-treated with untreated cells. The cell volume of the treated cell lines was initially increased at early time-points. By time, cells decreased in volume, especially when treated with high doses of etoposide. In conclusion, we have shown that holographic microscopy allows label-free and completely non-invasive morphological measurements of cell growth, viability and death. Future applications could include real-time monitoring of these holographic microscopy parameters in cells in response to clinically relevant compounds.

Background: We introduce the combination of digital holographic microscopy (DHM) and antibody microarrays as a powerful tool to measure morphological changes in specifically antibody-captured cells. The aim of the study was to develop DHM for analysis of cell death of etoposide-treated suspension cells. Result/Methodology: We demonstrate that the cell number, mean area, thickness, and volume were non-invasively measured by using DHM. The cell number was stable over time, but the two cell lines showed changes of cell area and cell irregularity after treatment. The cell volume in etoposide-treated cells was decreased, whereas untreated cells showed stable volume. Conclusions: Our results provide proof of concept for using DHM combined with antibody-based microarray technology for detecting morphological changes in captured cells.

The expression of cell surface glycans terminating with sialic acid (SA) residues has been found to correlate with various disease states there among cancer. We here report a novel strategy for specific fluorescence labeling of such motifs. This is based on sialic acid imprinted core-shell nanoparticles equipped with nitrobenzoxadiazole (NBD) fluorescent reporter groups allowing environmentally sensitive fluorescence detection at convenient excitation and emission wavelengths. Imprinting was achieved exploiting a hybrid approach combining reversible boronate ester formation between p-vinylphenylboronic acid and SA, the introduction of cationic amine functionalities and the use of an NBD-appended urea-monomer as a binary hydrogen bond donor targeting the SA carboxylic acid and OH functionalities. The monomers were grafted from 200 nm RAFT modified silica core particles using ethyleneglycol dimethacrylate (EGDMA) as crosslinker resulting in a shell thickness of ca 10 nm. The particles displayed strong affinity for SA in methanol/water mixtures (K = 6.6 x 105 M-1 in 2% water, 5.9 x 103 M-1 in 98% water, Bmax ≈ 10 μmol g–1) whereas binding of the competitor glucuronic acid (GA) and other monosaccharides was considerably weaker (K (GA) = 1.8 x 103 M-1 in 98% water). In cell imaging experiments the particles selectively stained different cell lines in correlation with the SA expression level. This was further verified by enzymatic cleavage of SA and by staining using a FITC labeled SA selective lectin.

Review on ZAP70, with data on DNA, on the protein encoded, and where the gene is implicated.

Digital holographic microscopy (DHM) has emerged as a powerful non-invasive tool for cell analysis. It has the capacity to analyse multiple parameters simultaneously, such as cell- number, confluence and phase volume. This is done while cells are still adhered and growing in their culture flask. The aim of this study was to investigate whether DHM was able to monitor drug-induced cell cycle arrest in cultured cells and thus provide a non-disruptive alternative to flow cytometry. DHM parameters from G1 and G2/M cell cycle arrested L929 mouse fibroblast cells were collected. Cell cycle arrest was verified with flow cytometry. This study shows that DHM is able to monitor phase volume changes corresponding to either a G1 or G2/M cell cycle arrest. G1-phase arrest with staurosporine correlated with a decrease in the average cell phase volume and G2/M-phase arrest with colcemid and etoposide correlated with an increase in the average cell phase volume. Importantly, DHM analysis of average cell phase volume was of comparable accuracy to flow cytometric measurement of cell cycle phase distribution as recorded following dose-dependent treatment with etoposide. Average cell phase volume changes in response to treatment with cell cycle arresting compounds could therefore be used as a DHM marker for monitoring cell cycle arrest in cultured mammalian cells.

We present a method to study the morphology of living, dividing and dying cells using DHM. DHM is a non-invasive, non-destructive and non-phototoxic method which allows the user to perform both qualitative and quantitative measurements of living cells over time. We show here our results on cell division and cell death in single cells. The morphological analyses performed here show changes caused by cell death and cell division, and indicate the possibilities to discriminate between different types of cell death. Cells dying in an apoptosis-like manner display different cell area and cell thickness profiles over time compared to cells dying in a necrosis-like manner, although their volume profiles are very similar. Dividing cells show a characteristic dip in the volume profile, which makes them easily distinguishable. Also, several previous studies show the versatile abilities of DHM. Different cell types have been studied and the morphology has been used to determine cell functionality as well as changes in morphology related to the environment. Cell morphology parameters can be very useful when following the effects of different treatments, the process of differentiation as well as cell growth and cell death. Cell morphology studied by DHM can be useful in toxicology, stem cell and cancer research.

Src homology 2 domain-containing tyrosine phosphatase-2 (SHP-2) is an important regulator of cell signaling because of its ability to dephosphorylate receptors of growth factors as well as the cytokines and tyrosine-phosphorylated proteins associated with these receptors. In the current study, we used four different prostate cancer cell lines: PC3, DU145, LNCaP and LNCaP-IL6+. Tumor specimens from 122 patients with prostate cancer were analyzed using a tissue microarray. Our data demonstrate that all four prostate cancer cell lines express the SHP-2 protein. Additionally, low staining intensity and SHP-2 expression in the cytoplasm of cancer cells in prostate tumor specimens was inversely correlated with prostate volume (p = 0.041 and p = 0.042, respectively) whereas nuclear staining was positively correlated with extracapsular extension (p = 0.039). In our post-prostatectomy specimens, we found that patients with low SHP-2 expression had less favorable outcomes with respect to biochemical recurrence and clinical progression (p = 0.005 and p = 0.018, respectively). The loss of cytoplasmic SHP-2 expression is associated with increased growth and prostatic cancer progression.

Digital holography microscopy (DHM) has developed into a broad field, and one of all the interesting applications is to study cells without staining, labeling or in any other way affecting them. Both fixed and living, dying or dead cells can be studied. The first DHM images showing living cells were published in 2004 and 2005 (Carl et al. 2004, Marquet et al. 2005), making this field of research rather new. Digital holography makes it possible to easily measure cell properties that previously have been very difficult to study, such as cell thickness and volume (Marquet et al. 2005, Mölder et al. 2008). Two of the major advantages of DHM is the 3-D imaging possibility and measurements over time. Digital holography has ben used to study several types of cells, such as nerve cells, red blood cells, stem cells and cancer cells (Emery et al. 2007, Kemper et al. 2006, Langehanenberg et al. 2009) . It has also been applied for studies of cell proliferation, cell movement, sub-cellular structures and cell morphology (Kemper et al. 2009, Yu et al. 2009). Both 2-D and 3-D cell movement can be determined ( Langehanenberg et al. 2009). Even cell viability status can be determined using DHM. Interestingly, it is possible to study both single cells and entire populations simultaneously, allowing for very nuanced studies. Older, well known techniques often require some degree of cell disturbance such as the fluorescent antibody labeling required for fluorescense or confocal microscopy studies. In this paper we will present some of the studies made possible by DHM. We will compare DHM with previously used techniques and discuss the benefits and drawbacks of digital holography cell measurements.

Bakgrund: Digital holografi är en ny teknik som de senaste fem åren använts för att studera levande celler. Tekniken utgör en innovativ, icke-förstörande metod som möjliggör studier av levande celler över tid. Material och metoder: Litteraturen har valts ut genom att söka på redan kända forskargrupper och företag som arbetar både med digital holografi och cellstudier samt PubMed-sökningar. Resultat och sammanfattning: Digital holografi ger kunskap om cellernas brytningsindex, som kan ändras under olika förhållanden. De parametrar som kan mätas ger unik information om cellantal, cellernas area, tjocklek och volym, vilket kan omvandlas till proliferation, viabilitet och celldöd. Tekniken är relativt billig, snabb och enkel att använda.

Digital holography is a novel technique that has been developed recently to study living cells. The technique is an innovative, non-destructive method with possibilities to study living cells over time. We are investigating cell number, growth, viability and death of adherent cells using digital holography, which is a novel, label-free, imaging technique for biological applications. We have recently demonstrated that digital holography is highly comparable to the conventional manual cell counting method using a hemocytometer (Mölder et al., 2008). Digital holography is a method that gives us information about the refractive index of cells, which can change under different circumstances. The technique is cheap, fast and simple to use. The unique measurable parameters are the cell number, cell area, thickness and volume, which can be transformed to proliferation, migration, viability and cell death. The digital holographic images produced can provide both quantitative and qualitative phase information from a single hologram. Future applications can include real-time cell monitoring of various parameters of cells of different diseases in response to clinically relevant compounds.

The protein tyrosine kinase (PTK) receptors and cytosolic signaling proteins as well as the protein tyrosine phosphatases (PTPs) have important roles in regulation of growth of the benign and malignant prostate gland. Here we studied expression of the protein tyrosine phosphatase SHP-1 in prostate cancer cell lines and in human prostatic tissues. SHP-1 is expressed at a high level in LNCaP prostate cancer cells compared with PC3 cells. Silencing of SHP-1 expression with siRNA in LNCaP cells led to an increased rate of proliferation, whereas overexpression of SHP-1 by means of transient transfection in PC3 cells led to a decrease in proliferation. Corresponding changes were observed in cyclin D1 expression. We further demonstrate that LNCaP and PC3 cells respond differently to IL-6 stimulation. SHP-1 overexpression in PC3 cells reversed IL-6 stimulation of proliferation whereas in SHP-1-silenced LNCaP cells IL-6 inhibition of proliferation was not affected. In addition, IL-6 treatment led to higher levels of phosphorylated STAT3 in SHP-1-silenced LNCaP cells than in control cells. Next, SHP-1 expression in human prostate cancer was analyzed by immunohistochemical staining of tissue microarrays comprising tumor specimens from 100 prostate cancer patients. We found an inverse correlation between the tumor level of SHP-1 expression and time to biochemical recurrence and clinical progression among prostate cancer patients. In conclusion, our results suggest that a decreased level of SHP-1 expression in prostate cancer cells is associated with a high proliferation rate and an increased risk of recurrence or clinical progression after radical prostatectomy for localized prostate cancer.

Background Interleukin-6 (IL-6) is a multifunctional cytokine that has been implicated in the modulation of growth and progression of prostate cancer. Decreased expression of the tyrosine phosphatase SHP-1, involved in regulation of cytokine and tyrosine kinase receptor signaling, has been shown to be associated with less favourable outcome for prostate cancer patients. Methods Parental LNCaP and LNCaP-IL6+ subline, derived from parental LNCaP cells by continuous culture of the cells in the presence of recombinant IL-6 were used in the study. Expression of SHP-I, PTEN, STAT3, pSTAT3, ERK, pERK, AKT, pAKT, PTEN and SHP-1 was analysed by immunohistochemistry, Western blot, cDNA microarray quantitative PCR and reverse transcriptase PCR. Proliferation and apoptosis of transfected cells was analysed with caspase3/7 and flow cytometry. Results Phosphorylation of ERK and STAT3 was increased in the LNCaP-IL6+ subline compared to LNCaP cells, whereas pAkt was decresaed. This could be due tore-expression of PTEN in LNCaP-IL6+ cells. Overexpression and inhibition experiments with SHP-1 siRNA showed that SHP-1 reduced proliferation and increased apoptosis in both cell lines. A cDNA analysis revealed 80 up-regulated and 87 down-regulated SHP-1 related genes in the LNCaP-IL6+ cell line compared to LNCaP cells. Conclusions SHP-1 suppresses growth and increases apoptosis in both LNCaP and LNCaP-IL6+ cells which suggests that SHP-1 could provide a therapeutic target for the prostate cancer cells even in the case that have gained an IL-6 related growth advantage.