Presence Of Prostate Cancer Cells Research Articles

Abstract 2032: Osteoblasts alter their protein expression profile in the presence of prostate cancer cells to facilitate their invasion

Abstract Bone metastasis is the leading cause of death in prostate cancer patients, for which there is currently no effective treatment. Since the bone microenvironment plays an important role in this process, attention has been directed to the interactions between cancer cells and the bone microenvironment, including osteoblasts. Here, we investigated the mechanism of interactions between prostate cancer cells (DU-145 and PC-3) and osteoblast cell line hFOB.1.19. We are investigating the broader effects of four different nucleoside reverse transcriptase inhibitor (NRTI) analogs 5-fluoro-1-((1R,4R)-4-hydroxycyclopent-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (KBMD-E), 3-benzoyl-5-fluoro-1-((1S,4S)-4-((tetrahydro-2H-pyran-2-yl)oxy)cyclopent-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (KBMD-G), 1-((1R,4R)-4-hydroxycyclopent-2-en-1-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (KBMD-H) and 1-(5-hydroxymethyl)-2,5-dihydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (KBMD-S) along with aPKC specific inhibitors ICA-1S and ζ-Stat to develop as potential anti-cancer drugs. Our previous publications suggested that aPKC-ι and ζ are involved in prostate cancer progression and metastasis. Current data suggested that aPKC specific inhibitors along with KBMD compounds induced apoptosis and pyroptosis of DU-145 and PC-3 while having lesser effects on hFOB.1.19 cell line. Our data demonstrate that osteoblasts assist prostate cells to establish in the bone microenvironment by producing metastatic stimulating chemokines while upregulating responsible transcription factors such as c-Jun. In addition, our preliminary data suggested that PKC-ι and ζ are crucial for driving critical steps of the metastatic cascades of prostate cancer cells. Preliminary data suggested that all 6 compounds upregulated interleukin (IL)-18 and IL-1β while downregulating IL-8, IL-6 and CXCL-1. Upregulation of Caspase 8 with cleaved gasdermin D in prostate cells indicates an increase in pyroptosis. Our preliminary results suggest that all 6 compounds can be used to disrupt the main steps of prostate cancer bone metastasis which can be targeted to develop customized, tailored therapies for bone metastatic prostate cancer which merit further research. Citation Format: Aaron J. Todman, Wishrawana S. Ratnayake, Luke Lajmi, Sloan Breedy, Abiral H. Shourav, Kirpal Bisht, Mildred Acevedo-Duncan. Osteoblasts alter their protein expression profile in the presence of prostate cancer cells to facilitate their invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2032.

Heat Shock Protein 27 Affects Myeloid Cell Activation and Interaction with Prostate Cancer Cells

Heat shock proteins are cytoprotective molecules induced by environmental stresses. The small heat shock protein 27 (Hsp27) is highly expressed under oxidative stress conditions, mediating anti-oxidative effects and blocking apoptosis. Since medical gas plasma treatment subjects cancer cells to a multitude of reactive oxygen species (ROS), inducing apoptosis and immunomodulation, probable effects of Hsp27 should be investigated. To this end, we quantified the extracellular Hsp27 in two prostate cancer cell lines (LNCaP, PC-3) after gas plasma-induced oxidative stress, showing a significantly enhanced release. To investigate immunomodulatory effects, two myeloid cell lines (THP-1 and HL-60) were also exposed to Hsp27. Only negligible effects on viability, intracellular oxidative milieu, and secretion profiles of the myeloid cells were found when cultured alone. Interestingly, prostate cancer-myeloid cell co-cultures showed altered secretion profiles with a significant decrease in vascular endothelial growth factor (VEGF) release. Furthermore, the myeloid surface marker profiles were changed, indicating an enhanced differentiation in co-culture upon Hsp27 treatment. Finally, we investigated morphological changes, proliferation, and interaction with prostate cancer cells, and found significant alterations in the myeloid cells, supporting the tendency to differentiate. Collectively, our results suggest an ambiguous effect of Hsp27 on myeloid cells in the presence of prostate cancer cells which needs to be further investigated.

Tumor- and osteoclast-derived NRP2 in prostate cancer bone metastases

Understanding the role of neuropilin 2 (NRP2) in prostate cancer cells as well as in the bone microenvironment is pivotal in the development of an effective targeted therapy for the treatment of prostate cancer bone metastasis. We observed a significant upregulation of NRP2 in prostate cancer cells metastasized to bone. Here, we report that targeting NRP2 in cancer cells can enhance taxane-based chemotherapy with a better therapeutic outcome in bone metastasis, implicating NRP2 as a promising therapeutic target. Since, osteoclasts present in the tumor microenvironment express NRP2, we have investigated the potential effect of targeting NRP2 in osteoclasts. Our results revealed NRP2 negatively regulates osteoclast differentiation and function in the presence of prostate cancer cells that promotes mixed bone lesions. Our study further delineated the molecular mechanisms by which NRP2 regulates osteoclast function. Interestingly, depletion of NRP2 in osteoclasts in vivo showed a decrease in the overall prostate tumor burden in the bone. These results therefore indicate that targeting NRP2 in prostate cancer cells as well as in the osteoclastic compartment can be beneficial in the treatment of prostate cancer bone metastasis.

Electrochemical detection of Flutamide as an anticancer drug with gold nanoparticles modified glassy carbon electrode in the presence of prostate cancer cells

In the current study, a novel electrochemical sensor was constructed based on gold nanoparticles modified glassy carbon electrode (AuNPs/GCE) to detect Flutamide. Field emission scanning electron microscope was used to identify the morphology of the modified electrode. Cyclic voltammetry and differential pulse voltammetry (DPV) were used to investigate the electrochemical activity of the modified electrode. Flutamide concentration, pH, and scan rate were optimized as electrochemical parameters. Flutamide peak currents obtained by DPV were linear within the concentration range of 1–600 µM, and the detection limit was calculated as 1.5 nM. In addition, the fabricated electrochemical sensor was successfully employed to detect Flutamide in a cell culture media containing prostate cancer cells as the biological sample. The stability of the modified electrode within a day showed an appropriate precision.

Targeting Prostate Cancer Using Intratumoral Cytotopically Modified Interleukin-15 Immunotherapy in a Syngeneic Murine Model.

BackgroundThe prostate cancer microenvironment is highly immunosuppressive; immune cells stimulated in the periphery by systemic immunotherapies will be rendered inactive once entering this environment. Immunotherapies for prostate cancer need to break this immune tolerance. We have previously identified interleukin-15 (IL-15) as the only cytokine tested that activates and expands immune cells in the presence of prostate cancer cells. In the current study, we aimed to identify a method of boosting the efficacy of IL-15 in prostate cancer.MethodsWe engineered, by conjugation to a myristoylated peptide, a membrane-localising form of IL-15 (cyto-IL-15) and the checkpoint inhibitor antibodies cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death ligand 1 (PD-L1) (cyto-abs) to enable them to bind to cell surfaces by non-specific anchoring to the phospholipid bilayer. The efficacy of these agents was investigated by intratumoral administration either alone (cyto-IL-15 or cyto-abs) or in combination (cyto-combo) in subcutaneous TRAMP-C2 prostate tumors in C57BL/6J mice and compared with their non-modified equivalents in vivo. Following the survival endpoint, histological analyses and RNA sequencing were performed on the tumors.ResultsIntratumoral injection of cyto-IL-15 or cyto-combo delayed tumor growth by 50% and increased median survival to 28 and 25 days, respectively, compared with vehicle (17 days), whereas non-modified IL-15 or antibodies alone had no significant effects on tumor growth or survival. Histological analysis showed that cyto-IL-15 and cyto-combo increased necrosis and infiltration of natural killer (NK) cells and CD8 T cells in the tumors compared with vehicle and non-modified agents. Overall, the efficacy of cyto-combo was not superior to that of cyto-IL-15 alone.ConclusionWe have demonstrated that intratumoral injection of cyto-IL-15 leads to prostate cancer growth delay, induces tumor necrosis and increases survival. Hence, cytotopic modification in combination with intratumoral injection appears to be a promising novel approach for prostate cancer immunotherapy.

Abstract A11: Targeting prostate cancer using novel cytotopically modified immunotherapies intratumorally in a syngeneic murine model

Abstract The immunosuppressive prostate cancer microenvironment renders most infiltrating effector immune cells anergic or regulatory. Immunotherapeutic agents localized to the prostate can break this immune tolerance, leading to greater efficacy and less toxicity than systemically administered drugs. We have previously shown that cytotopic modification of interleukin-15 (cyto-IL-15) can activate NK and CD8+ T-cells in the presence of prostate cancer cells. In the present study, we used this cyto-IL-15 and two immuno-checkpoint blocking antibodies modified in a similar approach (these antibodies have previously exhibited antitumor response in clinical trials) to investigate their efficacy when used alone or in combination in a syngeneic murine model of prostate cancer. IL-15 and antibodies were modified by conjugation to a myristoylated peptide that enables them to adhere to cell membranes (patents pending), and thus localize to any potential site of injection. The cytotopically modified IL-15 and antibodies were injected intratumorally either alone (cyto-IL-15 or cyto-abs) or in combination (cyto-combo) in subcutaneous TRAMP C2 prostate tumors in C57BL/6 mice and compared with their nonmodified equivalents and with a vehicle-treated control group. Histopathologic analysis and immunohistochemistry were performed on sections from excised frozen tumors in order to quantify necrosis (haematoxylin and eosin staining) and immune cell infiltration. Intratumoral injection of cyto-IL15, combo or cyto-combo significantly delayed tumor growth at day 14 post-treatment by 50%. However, only cyto-IL15 and cyto-combo significantly increased median survival after treatment to 28 (p < 0.05) and 24 days (p < 0.05) respectively, compared with 17 days in the vehicle group. Nonmodified IL15 or antibodies alone had no effect on tumor growth or survival. Mice showed no adverse effects or weight loss with either of the treatments. Moreover, histologic analysis showed that cyto-IL-15 increased necrosis and infiltration of NK cells and CD8+ T cells in the tumors compared with the vehicle- and nonmodified-IL-15-treated groups. We have demonstrated that cytotopically modified immunotherapeutic agents can lead to tumor growth delay in an in vivo murine model of prostate cancer, induce tumor cell death and increase mouse survival. Hence, cytotopic modification in combination with direct injection of the agents directly into the tumor site appears to be a promising novel approach for prostate cancer immunotherapy. Citation Format: Efthymia Papaevangelou, Dorota Smolarek, Oussama Elhage, Richard A. Smith, Prokar Dasgupta, Christine Galustian. Targeting prostate cancer using novel cytotopically modified immunotherapies intratumorally in a syngeneic murine model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A11.

Prostate cancer cells enhance interleukin-15-mediated expansion of NK cells.

To identify cytokines that can activate and expand NK cells in the presence of prostate cancer cells in order to determine whether these agents may be useful in future intra-tumoural administration in pre-clinical and clinical prostate cancer trials. Lymphocytes isolated from normal donor blood were set up in co-cultures with either cancer or non-cancerous prostate cell lines, together with each of the cytokines interleukin (IL)-2, IL-12, IL-15, interferon (IFN)-γ or IL-21 for a period of 7days. Then, expansion of NK cells, NKT cells and CD8 T cells was measured by flow cytometry and compared with the expansion of the same cells in the absence of prostate cells. The cytotoxic activity of NK cells, as measured by perforin and tumour cell killing, was also assessed. NK cell receptors and their corresponding ligands on prostate tumour cells were analysed to determine whether any of these were modulated by co-culture. The role of the tumour-secreted heat shock proteins HSP90 and HSP70 in the expansion of NK cells in the co-cultures was also investigated because of their effects on NK and CD8 T-cell activation. We showed that, among a panel of cytokines known to cause NK cell activation and expansion, only IL-15 could actively induce expansion of NK, NKT and CD8 T cells in the presence of prostate cancer cell lines. Furthermore, the expansion of NK cells was far greater (up to 50% greater) in the presence of the cancer cells (LNCaP, PC3) than when lymphocytes were incubated alone. In contrast, non-cancerous cell lines (PNT2 and WPMY-1) did not exert any expansion of NK cells. The cytolytic activity of the NK cells, as measured by perforin, CD107a and killing of tumour cells, was also greatest in co-cultures with IL-15. Examination of NK cell receptors shows that NKG2D is upregulated to a greater degree in the presence of prostate cancer cells, compared with the upregulation with IL-15 in lymphocytes alone. However, blocking of NKG2D does not inhibit the enhanced expansion of NK cells in the presence of tumour cells. Among a panel of NK cell-activating cytokines, IL-15 was the only cytokine that could stimulate expansion of NK cells in the presence of prostate cancer cells; therefore IL-15 may be a good candidate for novel future intra-tumoural therapy of the disease.

Erratum to “Prostate Osteoblast-Like Cells: A Reliable Prognostic Marker of Bone Metastasis in Prostate Cancer Patients”

[This corrects the article DOI: 10.1155/2018/9840962.].

Prostate Osteoblast-Like Cells: A Reliable Prognostic Marker of Bone Metastasis in Prostate Cancer Patients.

The main aim of this study was to investigate the putative association among the presence of prostate cancer cells, defined as prostate osteoblast-like cells (POLCs), and showing the expression of typical morphological and molecular characteristics of osteoblasts, the development of bone metastasis within 5 years of diagnosis, and the uptake of 18F-choline evaluated by PET/CT analysis. To this end, prostate biopsies (n = 110) were collected comprising 44 benign lesions and 66 malignant lesions. Malignant lesions were further subdivided into two groups: biopsies from patients that had clinical evidence of bone metastasis (BM+, n = 23) and biopsies from patients that did not have clinical evidence of bone metastasis within 5 years (BM−, n = 43). Paraffin serial sections were obtained from each specimen to perform histological classifications and immunohistochemical (IHC) analysis. Small fragments of tissue were used to perform ultrastructural and microanalytical investigations. IHC demonstrated the expression of markers of epithelial-to-mesenchymal transition (VIM), bone mineralization, and osteoblastic differentiation (BMP-2, PTX-3, RUNX2, RANKL, and VDR) in prostate lesions characterized by the presence of calcium-phosphate microcalcifications and high metastatic potential. Ultrastructural studies revealed the presence of prostate cancer cells with osteoblast phenotype close to microcalcifications. Noteworthy, PET/CT analysis showed higher uptake of 18F-choline in BM+ lesions with high positivity (≥300/500 cells) for RUNX2 and/or RANKL immunostaining. Although these data require further investigations about the molecular mechanisms of POLCs generation and role in bone metastasis, our study can open new and interesting prospective in the management of prostate cancer patients. The presence of POLCs along with prostate microcalcifications may become negative prognostic markers of the occurrence of bone metastases.

The BMP antagonist Noggin is produced by osteoblasts in response to the presence of prostate cancer cells.

Bone metastasis is a key event responsible for morbidity in prostate cancer patients. Interactions between prostate cancer cells and the bone microenvironment facilitate survival of tumor cells and alter bone turnover, a process that is thought to enhance the growth of metastases in this site. This study aimed to test the hypothesis that the presence of tumors cells increases transforming growth factor beta (TGF-β) signaling in bone and that this regulates the proliferation and differentiation of osteoblastic lineage cells in metastatic sites. Initial studies showed that factors produced by prostate cancer cells increased the proliferation of osteoblastic cells and suppressed the early phase of their differentiation. We subsequently showed that interactions between prostate cancer and osteoblastic cells affected the expression of TGF-β superfamily genes in the latter. Noggin was expressed and secreted by prostate cancer cells but expressed at very low levels by osteoblastic cells when these cells were grown alone. This pattern changed when osteoblasic cells were treated with conditioned medium derived from prostate cancer cells or were cocultured with the latter, with strong induction of Noggin being demonstrated. Immunohistochemical examination of prostate cancer xenografts showed strong Noggin protein staining on endosteal bone surfaces and in bone lining cells in close proximity to tumor foci. These studies support previous work that suggest Noggin is an important suppressor of the differentiation of osteoblast lineage cells in bone metastases. Importantly, we have now also shown that this protein can be induced in bone cells themselves by factors derived from prostate cancer cells.

Protein Mimetic and Anticancer Properties of Monocyte-Targeting Peptide Amphiphile Micelles.

Monocyte chemoattractant protein-1 (MCP-1) stimulates the migration of monocytes to inflammatory sites, leading to the progression of many diseases. Recently, we described a monocyte-targeting peptide amphiphile micelle (MCP-1 PAM) incorporated with the chemokine receptor CCR2 binding motif of MCP-1, which has a high affinity for monocytes in atherosclerotic plaques. We further report here the biomimetic components of MCP-1 PAMs and the influence of the nanoparticle upon binding to monocytes. We report that MCP-1 PAMs have enhanced secondary structure compared to the MCP-1 peptide. As a result, MCP-1 PAMs displayed improved binding and chemoattractant properties to monocytes, which upregulated the inflammatory signaling pathways responsible for monocyte migration. Interestingly, when MCP-1 PAMs were incubated in the presence of prostate cancer cells in vitro, the particle displayed anticancer efficacy by reducing CCR2 expression. Given that monocytes play an important role in tumor cell migration and invasion, our results demonstrate that PAMs can improve the native biofunctional properties of the peptide and may be used as an effective inhibitor to prevent chemokine-receptor interactions that promote disease progression.

Abstract 555: Prostate cancer cell-induced differentiation of human monocytes into MDSCs ex vivo is inhibited by targeting STAT3

Abstract Background: The transcription factor Signal Transducer and Activator of Transcription Factor 3 (STAT3) is implicated in acquired drug resistance, metastases and immune suppression in prostate cancer and is a potential target in drug-resistant prostate cancer. Today, the treatment options for metastatic drug-resistant prostate cancer are very limited and existing immunotherapies have so far shown low efficacy. Cancer cells may avoid immune responses by expressing immunosuppressive markers or activating immunosuppressive cells. Myeloid-derived suppressor cells (MDSC) play a major role in the suppression of antitumor immunity and active STAT3 signaling has been shown to be involved in the immunosuppressive functions of these cells. Elevated levels of MDSC have been found in the peripheral blood and at the tumor site of prostate cancer patients and to correlate with disease progression. Aims: In this study we aimed to investigate if human monocytes could be induced to differentiate into immunosuppressive cells in the presence of prostate cancer cells and if this effect could be blocked by the STAT3 inhibitor GPA500 (galiellalactone). Methods: Monocytes were isolated from peripheral blood mononuclear cells from healthy human donors using a magnetic monocyte enrichment kit. Monocytes were co-cultured with the prostate cancer cell lines DU145, LNCaP or longterm IL6 treated LNCaP cells (LNCaP-IL6+) in transwells allowing cancer cells and monocytes to share culture medium. The co-cultures were grown for three days with or without the STAT3 inhibitor GPA500 (5 uM or 10 uM) with subsequent analysis by flow cytometry for cell surface markers of MDSC differentiation (CD14+ HLA-DRlo). The presence of active STAT3 (pSTAT3) in the prostate cancer cell lines was determined by Western blot. Results: Monocytes in the presence of DU145 and LNCaP-IL6+ cells showed a significantly increased population of CD14+ HLA-DRlo cells. In the presence of GPA500 the induction of this population was inhibited in a dose dependent manner compared to untreated cells. Co-culture with LNCaP cells that lack pSTAT3 did not induce this population in monocytes after three days in culture. Furthermore, GPA500 induced a dendritic cell-like population in monocytes in the absence of prostate cancer cell co-culture. This population was also observed in monocytes when co-cultured with LNCaP-IL6+ cells, but not in DU145 co-cultures, with GPA500 present. Conclusion: The prostate cancer cell lines DU145 and LNCaP-IL6+ were able to induce a monocyte population displaying MDSC markers and this induction could be inhibited by the STAT3 inhibitor GPA500. Impact: This study demonstrates that inhibiting the transcription factor STAT3 in prostate cancer could potentially reverse the immunosuppressive mechanisms caused by MDSC activation and that STAT3 inhibitors, alone or in combination with other therapies, could be a new treatment option for prostate cancer. Citation Format: Rebecka Hellsten, Karin Leandersson, Anders Bjartell, Martin E. Johansson. Prostate cancer cell-induced differentiation of human monocytes into MDSCs ex vivo is inhibited by targeting STAT3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 555.

Abstract 2582: Cytotopic IL-15 as a novel therapeutic for prostate cancer

Abstract Introduction & Objectives: Prostate cancer progression can arise by tolerance to, and evasion of tumor cells from immune effector cells such as CD8 T cells and NK cells due to the immunosuppressive tumor microenvironment. Immunotherapeutic drugs can eliminate tumors by breaking this tolerance, and such drugs that can be localized to the prostate will have greater efficacy and less systemic toxicity than drugs administered systemically. It has also been shown that a localized stimulation of the immune system can boost immunity all over the body to clear distant metastases. However, in the highly immunosuppressive prostate cancer microenvironment, most activated effector immune cells are rendered anergic or even regulatory. We have previously shown that IL-15, unlike other therapeutic cytokines such as IL-2 and IL-12, can stimulate expansion and activity of CD8 and NK cells when they are exposed to prostate cancer cells. Therefore, we decided to create a localizable form of this cytokine to study its activity on NK and CD8 T cells in the presence and absence of prostate cancer cells. Material & Methods: The cytokine IL-15 was produced in a modified form to enable its conjugation to a cytotopic “tail” molecule. The “tail” consists of three regions - a thiol reactive region which interacts with free cysteines on the protein of interest, a cationic region, which binds to the negatively charged cell membrane surface, and a hydrophobic region that enters the cell membrane. The cytotopic IL-15 can therefore adhere to cell membranes and can thus localize to any lesion where it is potentially injected. The tailed IL-15 was then compared to non-tailed IL-15 and other cytokines in assays to measure T cell proliferation, and NK and CD8 T cell expansion in the presence and absence of prostate cancer cells. Results: Compared to non-tailed forms of IL-15, the tailed IL-15 induced greater proliferation of a murine T cell line, CTLL-2 and PBMCs . Proliferation levels were up to 20 times greater with the tailed IL-15. In initial experiments, activity of the tailed IL-15 was also greater or equivalent to non tailed IL-15 in its ability to expand NK cells in the presence of a prostate cancer cell line (TRAMP-C1). Conclusion: We have identified a cytokine, IL-15, that can break tolerance to immunity in the prostate cancer microenvironment. A tailed form of this molecule, has equivalent or greater activity in expanding immune effector cells in the presence or absence of prostate cancer cells and its potential for regioselective action makes it therefore a promising new agent for immunotherapy of prostate cancer. Citation Format: Dorota A. Smolarek, Christina A. Sakellariou, Oussama Elhage, Osamu Ukimura, Inderbir Gill, Angus G. Dalgleish, Prokar Dasgupta, Richard A. G. Smith, Christine Galustian. Cytotopic IL-15 as a novel therapeutic for prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2582. doi:10.1158/1538-7445.AM2014-2582

Prostate Cancer Detected by Methylated Gene Markers in Histopathologically Cancer-Negative Tissues from Men with Subsequent Positive Biopsies

The goal of this retrospective, multicenter study was to evaluate the ability of a newly developed refinement of a quantitative methylation-specific PCR assay to detect prostate cancer in histopathologically negative biopsy samples collected from men who were later positively diagnosed during a follow-up biopsy procedure. Biomarkers tested in the assay included the much-studied glutathione-S-transferase P1 gene and others reported to be frequently methylated in prostate cancer. Core biopsy tissue from subjects with serial negative biopsies served as a negative control to assess assay specificity. As a positive control, biopsy core tissue from patients histopathologically diagnosed with prostate cancer was used to gauge true marker sensitivity in known cancer-containing specimens. Testing was completed in 971 archived paraffin-embedded tissue blocks from 264 men screened for prostate cancer. More samples were initially tested, but due to the advanced age of the paraffinized tissue, DNA quality for quantitative methylation-specific PCR analysis was insufficient in 34% of the available blocks. The glutathione-S-transferase P1 gene has been confirmed as a powerful indicator of the presence of prostate cancer cells. A sensitivity of 52% was observed in the "potentially false-negative first biopsies," with a corresponding specificity of 85% and the sensitivity in biopsy tissue cores containing histopathologically confirmed prostate cancer was 95%. An even higher sensitivity can be reached with RAR-2beta (84%) and APC (72%), with respective specificities of 48% and 50%. Gene methylation was detected in initial, negative biopsy tissue in men who were later diagnosed with prostate cancer. Testing for methylation in histopathologically negative biopsies could improve the early detection of prostate cancer.

Presence of prostate cells in bone marrow biopsies as a sign of micrometastasis in cancer patients

The presence of prostate cancer cells in bone marrow of patients with clinically localized disease is associated with increased chance of disease recurrence. The presence of prostate specific antigen (PSA) in bone marrow aspirates has been used to indicate the presence of micrometastasis. The aim of this study was to present a prospective study of prostate cancer patients to determine the presence of cells that express PSA in aspirates taken from bone marrow and to compare with bone marrow biopsy samples. Results indicated a significant difference between the frequency of cells detected in bone marrow aspirate and biopsy samples (P<0.0002) when all patients were considered. There was no difference between the frequencies of cells detected in bone marrow aspirate and biopsy of patients analyzed before treatment. However, there was a significant (P<0.003) difference between them after treatment. There was also a significant difference in the frequency of PSA positive cells detected (P<0.005) in Stages I to IV as well as in the frequency of cells detected (P<0.0002) when analyzed according to Gleason score. The present results explain the lack of correlation between positive aspirates and prognosis in numerous clinical cases.

6.01] Prostate cancer diagnostics using spatially resolved fluorescence-based optical imaging

Objective Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men. An ideal imaging modality should identify the location of the aggressive cancer cells with 100% accuracy. So far this has not been achieved. The method we propose suggests the use of “smart probes” that specifically activate multiple fluorophores when found in the presence of PCa cells. Fluorescence detection can be performed with high sensitivity using fairly cheap equipment compared to other imaging modalities. Material and methods Fluorophores emitting between 600 and 850 nm are ideal labels. The tissue absorbs little and scatters most of this light, exhibiting a high signal-to-noise ratio. Different wavelengths offer a deeper, spatially resolved resolution. Optical imaging is impaired by the penetration depth of the excitation light and the geometrical restrictions of the detectors. Results Determination of the optical properties of the involved tissue is necessary for precise calculations of the light propagation. Goniometric measurements retrieve the scattering function and collimated transmission experiments provide the attenuation coefficient. Forward model: the light propagation through tissue is determined analytically using the diffusion model approximation and the a priori known optical properties. Results are available for simple parallelepiped geometry. The calculations will be generalized using a Finite element model (FEM) for more complex geometries found in the real environment. Inverse problem: derives the locations of the tumor, i.e. the fluorophores, within the prostate. The numerical 3D-reconstruction algorithm is based on the Adjoint theorem that extracts the exact locations of the fluorescing light sources by a single step linear calculation. The method relies on the equivalent readings when the detector and the light source exchange positions in space. Using the simplified parallelepiped model, the exact locations of the fluorescent light sources have been identified analytically for a determined system and partially determined for an underdetermined system. Conclusion The absolute determination of the tumor locations is dependent on the information gathered by the recorded data. For a determined system, i.e. the number of detectors equals the number of fluorescent light sources in the phantom; the locations have been extracted accurately. In general, the number of unknowns (i.e. fluorescent locations) is greater than the number of detectors; so the system is underdetermined. In this case the data have to be linearly independent so maximum information output is achieved. In the real world we are faced with underdetermined or ill-posed systems. Mathematical methods like Tikhonov regularization are employed for the calculation of the inverse solution. Depending on the sought spatial resolution, the percentage of the retrieved locations can be 100% or less. The spatial arrangement for detectors and the choice of emission wavelength of the fluorophores are the key factors in obtaining higher information content for the measured data.

Interferon regulatory factor 8 mediates tumor-induced inhibition of antigen processing and presentation by dendritic cells

Suppression of dendritic cells (DCs) is a crucial mechanism by which tumor cells escape immune recognition and elimination. We have recently reported that MHC class I antigen processing machinery (APM) component expression in human DCs is down-regulated by tumor-derived gangliosides. However, the molecular mechanisms underlying this abnormality were not identified. Thus, the aim of this work was to analyze the role of interferon regulatory factor 8 (IRF-8) in APM protein expression and the antigen presenting capacity of DCs developed in the tumor microenvironment. We demonstrate that the expression of several MHC class I APM components, including delta, MB-1, LMP-10, ERp57, and tapasin, is significantly decreased in murine DCs generated in the presence of prostate cancer cells. APM component down-regulation was associated with decreased ability of DCs to present model antigen to antigen-specific T cells. Notable, impaired antigen-presenting activity of DCs co-cultured with tumor cells was accompanied by decreased levels of IRF-8. Transduction of DCs with the silencing RNA for the IRF-8 gene also led to reduced expression of APM components in DCs and decreased antigen presenting function. Together, our data suggest that tumor-induced inhibition of antigen processing and presenting function of DCs is mediated by IRF-8, a member of the interferon regulatory factor family. These results provide a new molecular target for optimizing the generation of efficient DC vaccines for cancer therapy.

Mechanisms Underlying the Development of Androgen-Independent Prostate Cancer

Prostate cancer continues to be the most common lethal malignancy diagnosed in American men and the second leading cause of male cancer mortality. The American Cancer Society estimates that during 2005, ∼232,090 new cases of prostate cancer will be diagnosed in the United States and 30,350 men

Simultaneous Quantification of Prostate-specific Antigen and Human Glandular Kallikrein 2 mRNA in Blood Samples from Patients with Prostate Cancer and Benign Disease

Detection or quantification of circulating cancer cells has been proposed as an aid in detection and monitoring of several solid tumors. We investigated the classification accuracy of prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) mRNA copy numbers in blood for the differentiation of patients with prostate cancer (PC) and benign disease. PSA and hK2 mRNA expression was studied in blood samples from 51 men with PC and 19 men with benign disease. Among the PC patients, 10 had organ-confined disease (pT1-pT2). We used a multiplexed reverse transcription-PCR assay with two highly target-like mRNA internal standards for the simultaneous quantification of PSA and hK2 mRNA. An external calibration curve covered the range of 10(2)-10(6) mRNA copies. PSA and hK2 mRNA were detected in 41 of 51 (median, 1200 copies/0.5 mL of blood) and 43 of 51 (median, 3800 copies/0.5 mL of blood) patients with PC, respectively, whereas only 1 of 19 men with benign disease was positive for both mRNAs (1500 PSA and 3100 hK2 mRNA copies/0.5 mL of blood; P <0.0001, Mann-Whitney U-test). Of the 10 patients with organ-confined PC, only 3 with low Gleason scores (< or =5) were negative for both PSA and hK2 (P = 0.02, Mann-Whitney U-test). The presence of PC cells in the blood circulation is an early event in PC progression, and quantitative assays for PSA and hK2 mRNA discriminate benign from PC cases. Further studies are needed to determine the diagnostic accuracy and prognostic value of the assays.

Proliferation of prostate cancer cells in the bone marrow predicts recurrence in patients with localized prostate cancer.

Reverse-transcription polymerase chain reaction (RT-PCR) amplification of prostate specific antigen (PSA) mRNA has been used to detect the presence of prostate cancer cells in the peripheral blood and bone marrow of patients with clinically localized disease. Some studies have demonstrated a correlation between detection of PSA-mRNA and disease recurrence. However, many RT-PCR-positive patients remain disease-free. We propose that phenotypic characterization of individual micrometastatic cells may provide more prognostic information than mere detection of such cells. We studied 58 patients undergoing radical prostatectomy for clinically localized disease whose bone marrow had been found to contain PSA-mRNA by RT-PCR. Immunohistochemical detection and phenotypic characterization of micrometastatic cells was performed using a two-color technique: cytokeratin antibody for detection and the MIB-1 antibody for proliferation. The clinical endpoint was disease recurrence. One or more micrometastatic cells were proliferating in 36.2% of the patients; the disease-free survival rate was 76.2% in this group. In contrast, in the patients with non-proliferating cells, 97.3% remained disease-free (P = 0.025). Multivariate analysis demonstrated that the presence of proliferating cells was the only preoperative variable that correlated with disease-free survival (P = 0.05). Determination of the phenotype of individual micrometastatic cells can contribute prognostic information above and beyond the mere determination of their presence or absence. Phenotypic characterization of individual micrometastatic cells may ultimately be used to select patients for systemic therapy given either alone or in combination with local therapy.