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PIP5K1? 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 PIP5K1? in this process. Here, we show that siRNA-mediated knockdown of PIP5K1? and blockade of PIP5K1? 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 PIP5K1? in CRPC cells results in reduced growth and migratory ability of cancer cells. Further, the xenograft tumors lacking the N-terminal domain of PIP5K1? exhibited reduced tumor growth and aggressiveness in xenograft mice as compared to that of controls. The N-terminal domain of PIP5K1? is required for regulation of mRNA expression and protein stability of PIP5K1?. This suggests that the expression and oncogenic activity of PIP5K1? are in part dependent on its N-terminal domain. We further show that PIP5K1? 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 PIP5K1? and in CRPC cells lacking the N-terminal domain of PIP5K1?. These results indicate that the growth of PIP5K1?-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 PIP5K1?, confirming that PIP5K1? is an intriguing target for cancer treatment, especially for treatment of CRPC.

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.

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.

Digital holographic (DH) microscopy is a unique noninvasive method to analyze living cells. With DH microscopy, in vitro cell cultures can be imaged in 2D and pseudo-3D and measurements of size and morphology of the cells are provided. Here, a description of a novel methodology utilizing DH microscopy for the analysis of spheroids is presented. A cell culture protocol is introduced and morphological parameters of cell spheroids as measured by DH microscopy are presented. The study confirms the use of DH microscopy for the analysis of cell spheroids. In the future, organoids can be analyzed with DH microscopy, and it can also be used for drug response and cell death analyses. Method summary This method aims to optimize DH microscopy for the analysis of morphological parameters of spheroids in an easy and convenient way. Cell spheroids were cultured in culture dishes for 5 days. Then, the lid was replaced with HoloLid. After that, the spheroids were imaged and quantitative morphological information was collected and analyzed.

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.

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

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.

Cancer is a leading cause of death worldwide. Normally the balance betweencell growth and cell death is strongly controlled. Chronic lymphocytic leukemiais an indolent disease that has a highly variable clinical course and is the mostcommon hematological malignancy amongst adults in the Western countries.The protein tyrosine phosphatase SHP-1 is a key regulator that controls theintracellular phosphotyrosine level in lymphocytes by inhibiting the B cell receptorsignals. We have compared the expression and activity of SHP-1 inchronic lymphocytic leukemia cells from lymph nodes with matched peripheralblood samples. The expression levels of SHP-1 were higher in peripheral blood,but the phosphatase activity in lymph nodes and peripheral blood did not differsignificantly. All cells in the body normally present glycans on the cell surface,which are involved in cellular communication and in processes like cell differentiation,proliferation and infection, including protecting the cells from invadersand in cell-cell contacts. Sialic acid occurs on the terminal end of glycans,and the frequency of sialic acid expression is increased on metastatic cancer cellsand overexpression controls tumor cell growth and cell differentiation. Theavailability of specific antibodies against sialic acid is limited. We have beenscreening sialic acid on cancer cells by using a molecular imprinting polymertechnique. Our results show that sialic acid is expressed on chronic lymphocyticleukemia cell lines at different levels at the plasma membrane. Higher expressionof sialic acid in the more aggressive chronic lymphocytic leukemia cell lineswas observed. To analyze morphological changes of death cells, digital holographicmicroscopy was used. Digital holographic microscopy is an approachfor label-free non-invasive 3D imaging of cultured cells. We have analyzed celldeath of adherent cancer cells using digital holographic microscopy and developedit to analyze suspension cells by combining this technique with antibodybased microassays. Digital holographic microscopy can be used for cell-deathinduced cell analysis of both adherent cells and suspension cells. This thesistakes us one step further in cancer research as regards developing techniques forscreening circulating cancer cells in blood as well as for individualized treatmentof cancer patients.

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. Background: Digital holography is a novel technique that has been used for about five years to study living cells. The technique is an innovative, non-destructive method with possibilities to study living cells over time.Materials and methods: The literature in this paper was selected on the basis of already known research groups and companies in the field working with both digital holography and cell studies, as well as PubMed search.Results and conclusions: Digital holography is a method that gives us information about the refractive index of cells, which can change under different circumstances. The unique measurable parameters are the cell number, cell area, thickness and volume, which can be transformed to proliferation, viability and cell death. The technique is cheap, fast and simple to use.

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.