Paying the price for standing tall: Fluid mechanics of prostate pathology.

BACKGROUND: Age-dependent increase in the incidence of benign prostatic hyperplasia (BPH) and prostate cancer (PCa) are both related to cell proliferation and survival controlled by intraprostatic free testosterone (FT) concentration. Paradoxically, BPH and PCa occur as circulating testosterone levels decrease, so any possible relationship between testosterone levels and development of BPH and PCa remains obscure. RESULTS: In BPH the enlarging prostate is exposed to high testosterone levels arriving directly from the testes at concentrations about hundredfold higher than systemic FT. This occurs because venous blood from the testes is diverted into the prostate due to the elevated hydrostatic pressure of blood in the internal spermatic veins (ISVs). Elevated pressure is caused by the destruction of one-way valves (clinically detected as varicocele), a unique phenomenon related to human erect posture. While standing, human males are ISVs vertically oriented, resulting in high intraluminal hydrostatic pressures-a phenomenon not found in quadrupeds. In this communication, we demonstrate the fluid mechanics' phenomena at the basis of varicocele leading to prostate pathology. CONCLUSIONS: So far, varicocele has been studied mostly for its etiologic role in male infertility and, thus, for its effects on the testes. It is becoming clear that varicocele is a major etiologic factor in BPH and likely also in PCa. Restoring normal testicular venous pressure by treatment of the abnormal ISV's in varicocele has been shown to avert the flow from the prostate with the effect of reducing prostate volume, alleviating symptoms of BPH, and increasing concentrations of circulating FT.

Effects of oxygen on the antigenic landscape of prostate cancer cells.

BACKGROUND: Use of allogeneic cancer cells-based immunotherapy for treatment of established prostate cancer (PCa) has only been marginally effective. One reason for failure could stem from the mismatch of antigenic signatures of vaccine cells and cancer in situ. Hence, it is possible that vaccine cells expressed antigens differently than tumor cells in situ. We hypothesized that cells grown in vitro at low oxygen tension (pO2) provide a better antigen match to tumors in situ and could reveal a more relevant antigenic landscape than cells grown in atmospheric pO2. METHODS: We tested this hypothesis by comparing PCa cells propagated at pO2 = 2 kPa and 20 kPa. To identify potential tumor-associated antigens (TAAs), we prepared PCa cell lysates, resolved them by two-dimensional electrophoresis and immunoblotting using spontaneous antibodies from plasma derived from PCa patients and control subjects. Antibody-labeled spots were analyzed by MALDI-TOF mass spectrometry and validated by ELISA. We selected hypoxia-regulated HSP70 and hnRNP L and hypoxia-independent HSP60 and determined the frequency of plasma samples reacting with these molecules. RESULTS: Frequency of HSP60-reactive plasma was low in healthy controls [1.3 % (1/76)], while it was elevated in PCa patients [13.0 % (7/54); p < 0.05]. These data suggest a humoral immune response to HSP60 in PCa. Levels of autoantibodies to HSP70 did not differ from healthy controls [3.7 % (2/54)] in PCa patients [5.3 % (2/38)]. Similarly, hnRNP L autoantibodies did no differ between healthy controls [6.1 % (3/49)] and PCa patients [5.3 % (2/38)]. CONCLUSIONS: Overall our results suggest the value of hypoxia as a modifier of the cellular and antigenic landscape of PCa cells. By modifying the immune reactivity of PCa cells in culture, manipulation of pO2 can be proposed as a new avenue for improving diagnosis, prognosis and immunotherapy for PCa.

Prognostic value of discs large homolog 7 transcript levels in prostate cancer.

Hypoxia has been associated with malignant progression, metastasis and resistance to therapy. Hence, we studied expression of hypoxia-regulated genes in 100 prostate cancer (CaP) bulk tissues and 71 adjacent benign tissues. We found 24 transcripts significantly overexpressed (p ≤ 0.02). Importantly, higher transcript levels of disc large (drosophila) homolog-associated protein 5 (DLGAP5)/discs large homolog 7 (DLG7)/hepatoma up-regulated protein (HURP), hyaluronan-mediated motility receptor (HMMR) and cyclin B1 (CCNB1) were associated with higher Gleason score and more advanced systemic progression. Since the products of HMMR and CCNB1 have been identified recently as molecular markers of CaP progression, we postulated that DLG7 has prognostic value too. To test this hypothesis, we measured transcript levels for DLG7 in a 150-pair case-control cohort. The cases (progression to systemic disease within six years of surgery) and controls (no progression within eight years) were matched for clinical and pathologic prognostic variables, including grade, stage, and preoperative serum levels of PSA. The overall prognostic ability of DLG7, as tested in receiver operating characteristic analysis was of 0.74 (95% CI, 0.68 to 0.8). Overall, our data indicate that expression of DLG7, a hypoxia-controlled gene, holds prognostic potential in high-risk CaP; this also demonstrates that variation of oxygen tension may constitute a tool for identification of novel biomarkers for CaP.

Personalizing immunotherapy: Balancing predictability and precision.

Despite great expectations and research efforts, anticancer immunotherapy has not yet become a definitive cure. Perhaps, this is because past reductionist approaches were too simplistic for the patient-specific complex system of co-evolving tumor cells and host immunity. Recent efforts based on a systems-based approach promise improved clinical outcomes engendered by a dynamic modification of personalized therapeutic regimens.

Reconsidering the paradigm of cancer immunotherapy by computationally aided real-time personalization.

Although therapeutic vaccination often induces markers of tumor-specific immunity, therapeutic responses remain rare. An improved understanding of patient-specific dynamic interactions of immunity and tumor progression, combined with personalized application of immune therapeutics would increase the efficacy of immunotherapy. Here, we developed a method to predict and enhance the individual response to immunotherapy by using personalized mathematical models, constructed in the early phase of treatment. Our approach includes an iterative real-time in-treatment evaluation of patient-specific parameters from the accruing clinical data, construction of personalized models and their validation, model-based simulation of subsequent response to ongoing therapy, and suggestion of potentially more effective patient-specific modified treatment. Using a mathematical model of prostate cancer immunotherapy, we applied our model to data obtained in a clinical investigation of an allogeneic whole-cell therapeutic prostate cancer vaccine. Personalized models for the patients who responded to treatment were derived and validated by data collected before treatment and during its early phase. Simulations, based on personalized models, suggested that an increase in vaccine dose and administration frequency would stabilize the disease in most patients. Together, our findings suggest that application of our method could facilitate development of a new paradigm for studies of in-treatment personalization of the immune agent administration regimens (P-trials), with treatment modifications restricted to an approved range, resulting in more efficacious immunotherapies.

Age-dependent response of murine female bone marrow cells to hyperbaric oxygen.

Consequences of age on the effects of hyperbaric oxygen (HBO) on bone marrow (BM) derived stem cells and progenitors (SCPs) are largely unknown. We treated 2- and 18-month old C57BL/6 female mice by HBO. Hematopoietic stem cells and progenitors, enumerated as colony-forming units in culture, were doubled only in peripheral leukocytes and BM cells of young mice receiving HBO. In old mice colony-forming unit fibroblast numbers, a measure of mesenchymal stromal cells (MSCs) from BM, were high but unaffected by HBO. To further explore this finding, in BM-MSCs we quantified the transcripts of adipocyte early-differentiation genes peroxisome proliferator-activated receptor-γ, CCAAT/enhancer binding protein-ß and fatty-acid binding protein 4; these transcripts were not affected by age or HBO. However, osteoblast gene transcripts runt-related transcription factor 2, osterix (OSX) and alkaline phosphatase (AP) were twofold to 20-fold more abundant in MSCs from old control mice relative to those of young control mice. HBO affected expression of osteoblast markers only in old MSCs (OSX gene expression was reduced by twofold and AP expression was increased threefold). Our data demonstrate the impact of aging on the response of BM SCPs to HBO and indicate the potentially different age-related benefit of HBO in wound healing and tissue remodeling.

Biomathematical modeling and cancer immunotherapy: report of the workshop held at Bol, Island of Brac, Croatia, 20-21 June 2011.

At the end of June 2011, mathematicians, physicians and biomedical scientists met for a 2-day workshop at Bol, Island of Brac, Croatia. The purpose of the event was to evaluate the role of mathematical models as practical tools for optimizing cancer immunotherapy. Participants reviewed recent advances in the field and addressed potential causes of the lack of predictable clinical outcomes. They agreed that there is a need for treatments tailored to the individual patient based on the patient's ability to respond. Dynamic mathematical models based on the available understanding of interactions between the tumor, immunity and therapeutic intervention and 'trained' on the actual clinical data could help stratify patients before treatment and predict individual responses to ongoing treatment. In addition, mathematical models could assist in selecting the most beneficial individualized treatment modifications. Participants established a collaborative network to test some of the ideas discussed at the workshop and decided to review progress at the next meeting in Croatia in 2012.

Predicting outcomes of prostate cancer immunotherapy by personalized mathematical models.

BACKGROUND: Therapeutic vaccination against disseminated prostate cancer (PCa) is partially effective in some PCa patients. We hypothesized that the efficacy of treatment will be enhanced by individualized vaccination regimens tailored by simple mathematical models. METHODOLOGY/PRINCIPAL FINDINGS: We developed a general mathematical model encompassing the basic interactions of a vaccine, immune system and PCa cells, and validated it by the results of a clinical trial testing an allogeneic PCa whole-cell vaccine. For model validation in the absence of any other pertinent marker, we used the clinically measured changes in prostate-specific antigen (PSA) levels as a correlate of tumor burden. Up to 26 PSA levels measured per patient were divided into each patient's training set and his validation set. The training set, used for model personalization, contained the patient's initial sequence of PSA levels; the validation set contained his subsequent PSA data points. Personalized models were simulated to predict changes in tumor burden and PSA levels and predictions were compared to the validation set. The model accurately predicted PSA levels over the entire measured period in 12 of the 15 vaccination-responsive patients (the coefficient of determination between the predicted and observed PSA values was R(2) = 0.972). The model could not account for the inconsistent changes in PSA levels in 3 of the 15 responsive patients at the end of treatment. Each validated personalized model was simulated under many hypothetical immunotherapy protocols to suggest alternative vaccination regimens. Personalized regimens predicted to enhance the effects of therapy differed among the patients. CONCLUSIONS/SIGNIFICANCE: Using a few initial measurements, we constructed robust patient-specific models of PCa immunotherapy, which were retrospectively validated by clinical trial results. Our results emphasize the potential value and feasibility of individualized model-suggested immunotherapy protocols.

Immunosuppressive CD14+HLA-DRlow/- monocytes in prostate cancer.

OBJECTIVE: To determine if the levels of circulating myeloid-derived suppressor cells increase with progression of prostate cancer (PCa); to determine if such cells could contribute to the relative inefficiency of PCa immunotherapy. MATERIALS AND METHODS: We analyzed peripheral blood mononuclear cells isolated from untreated PCa patients (uPCa; N = 18; mean age +/- SD: 72.1 +/- 6.9 years), tPCa (N = 22; 72.8 +/- 9.8 years) and age matched controls (AMC; N = 12; 68.8 +/- 7.5 years). We quantified surface marker phenotype, differentiation potential, effects on T cell proliferation and intracellular cytokines. RESULTS: We observed an unexpectedly high percentage of a type of myeloid-derived suppressor cells, CD14(+)HLA-DR(low/-) monocytes, in tPCa (30.7 +/- 15.0% of CD14(+) cells) relative to AMC (4.1 +/- 6.5%, P < 0.0001) and uPCa (10.6 +/- 14.3%, P = 0.0001). The levels of CD14(+) HLA-DR(low/-) cells were significantly correlated with circulating PSA levels and treatment with LHRH-agonist leuprolide in combination with either an antiandrogen or dexamethasone. Monocytes from tPCa inhibited autologous T cell proliferation statistically significantly more effectively than AMC monocytes and were defective in their ability to differentiate into phenotypically mature dendritic cells. Isolated CD14(+)HLA-DR(low/-) cells expressed higher levels of intracellular interleukin-10 and suppressed T cell proliferation more effectively than isolated CD14(+)HLA-DR(+) cells. CONCLUSIONS: This is the first report of CD14(+) cells exhibiting reduced expression of HLA-DR molecules in PCa patients. These cells suppress immune cell function in vitro and, plausibly, in vivo, a finding that must be factored into the design of immunotherapy protocols for PCa patients.

Dendritic cell microvilli: a novel membrane structure associated with the multifocal synapse and T-cell clustering.

Polarizing effects of productive dendritic cell (DC)-T-cell interactions on DC cytoskeleton have been known in some detail, but the effects on DC membrane have been studied to a lesser extent. We found that T-cell incubation led to DC elongation and segregation of characteristic DC veils to the broader pole of the cell. On the opposite DC pole, we observed a novel membrane feature in the form of bundled microvilli. Each villus was approximately 100 nm in diameter and 600 to 1200 nm long. Microvilli exhibited high density of antigen-presenting molecules and costimulatory molecules and provided the physical basis for the multifocal immune synapse we observed during human DC and T-cell interactions. T cells preferentially bound to this site in clusters often contained both CD4(+) and CD8(+) T cells.

Rebuilding immunity in cancer patients.

Rebuilding and maintaining immunity are paramount to the success of cancer immunotherapy and hematopoietic stem cell transplantation. If immune surveillance indeed can protect from cancer, the very manifestation of malignancy means that the disease has prevailed over immunity. Yet, often, tumor-specific T cells can be found in cancer patients irrespective of vaccination. Interestingly, patients suffering from malignancy often harbor unexpectedly high levels of immature CD14(+)HLA-DR(-) monocytes, although the abundance of CD4(+) cells, CD8(+) cells and CD4(+)CD25(high) cells may be normal. It is plausible that in cancer such cells suppress T cell function, analogous to CD14(+)HLA-DR(-) cells in sepsis and major trauma, in addition to their likely failure to re-present tumor-associated antigens once dendritic cells have initiated the T cell response. Recent evidence indicates that tumor-borne adenosine, lactate and hypoxia in the tumor environment may modulate tumor-specific immunity to a significant extent, but their effects on myeloid cell function are unclear. Thus, understanding and controlling these factors may appreciably impact the success of rebuilding and maintaining immunity in cancer patients.

Plasmacytoid dendritic cells in inflamed muscle of patients with juvenile dermatomyositis.

OBJECTIVE: To examine whether dendritic cells (DCs) are constituents of muscle inflammation in juvenile dermatomyositis (DM). METHODS: The types, numbers, and activation state of DC subsets in inflamed muscle tissue from patients with juvenile DM and in noninflamed muscle tissue from control subjects were examined by multicolor immunofluorescence. Chemokine expression of the muscle-infiltrating cells was examined by laser capture microdissection and quantitative polymerase chain reaction. RESULTS: Plasmacytoid DCs were the predominant component of the inflamed muscle tissue from patients with juvenile DM. These cells were identified by coexpression of CD4 and CD123, but not CD11c, and also expressed CD83, indicating maturity of the cells. In contrast, in noninflamed muscle, plasmacytoid DCs were scarce and did not express CD83. Mononuclear cells surrounding the blood vessels of inflamed muscle contained abundant transcripts of CCL19 and CCL21, but very little CCL18 transcripts. In contrast, cells from noninflamed muscle contained negligible amounts of CCL19 and CCL21, but had high amounts of CCL18. Both the inflamed and noninflamed muscle tissue had equivalent levels of CXCL12 transcripts, but inflamed muscle contained more transcripts of the CXCL12 receptor CXCR4. CONCLUSION: These findings are consistent with the idea that plasmacytoid DCs are mediators of muscle inflammation in juvenile DM. The abundance of CD83+ plasmacytoid DCs in perivascular areas and the overexpression of CCL19 and CCL21 in perivascular cellular foci suggest that in situ activation and maturation of resident plasmacytoid DCs are central to the initiation and perpetuation of muscle inflammation in juvenile DM.

A novel source of viable peripheral blood mononuclear cells from leukoreduction system chambers.

BACKGROUND: Buffy coats are becoming less available as a source of research-grade peripheral blood mononuclear cells (PBMNCs). Therefore, alternative sources of these cells were investigated. STUDY DESIGN AND METHODS: PBMNCs isolated from the cells retained in leukoreduction system chambers (LRSCs) and those eluted from white blood cell filters were compared. From LRSCs (1.88 +/- 0.40) x 10(9) PBMNCs (n = 13) versus (0.43 +/- 0.15) x 10(9) PBMNCs were isolated from leukofilter eluates (LFEs, n = 8; p < 0.0001). RESULTS: Cells from LRSCs and LFEs produced similar numbers of burst-forming unit-erythroid, colony-forming unit (CFU)-granulocyte-macrophage, and CFU-granulocyte-erythrocyte-monocyte-macrophage-megakaryocyte colonies. The percentages of cells positive for CD3, CD4, CD8, CD14, CD19, and CD56 in the PBMNCs isolated from LRSCs and LFEs were indistinguishable. Cells isolated from LRSCs expressed higher levels of CD69 and CD25 in reaction to staphylococcal enterotoxin B than the cells isolated from LFEs. The source of cells affected neither the yield and purity of immunomagnetically isolated CD3+ cells, CD14+ cells, and CD56+ cells nor the function of T cells, natural killer cells, and in vitro matured dendritic cells (DCs). DC yield from LRSC-derived CD14+ cells, however, was higher. CONCLUSION: LRSCs are a novel source of fully functional PBMNCs that can replace the more traditional sources of research-grade cellular products.

Preparing clinical-grade myeloid dendritic cells by electroporation-mediated transfection of in vitro amplified tumor-derived mRNA and safety testing in stage IV malignant melanoma.

BACKGROUND: Dendritic cells (DCs) have been used as vaccines in clinical trials of immunotherapy of cancer and other diseases. Nonetheless, progress towards the use of DCs in the clinic has been slow due in part to the absence of standard methods for DC preparation and exposure to disease-associated antigens. Because different ex vivo exposure methods can affect DC phenotype and function differently, we studied whether electroporation-mediated transfection (electrotransfection) of myeloid DCs with in vitro expanded RNA isolated from tumor tissue might be feasible as a standard physical method in the preparation of clinical-grade DC vaccines. METHODS: We prepared immature DCs (IDCs) from CD14+ cells isolated from leukapheresis products and extracted total RNA from freshly resected melanoma tissue. We reversely transcribed the RNA while attaching a T7 promoter to the products that we subsequently amplified by PCR. We transcribed the amplified cDNA in vitro and introduced the expanded RNA into IDCs by electroporation followed by DC maturation and cryopreservation. Isolated and expanded mRNA was analyzed for the presence of melanoma-associated tumor antigens gp100, tyrosinase or MART1. To test product safety, we injected five million DCs subcutaneously at three-week intervals for up to four injections into six patients suffering from stage IV malignant melanoma. RESULTS: Three preparations contained all three transcripts, one isolate contained tyrosinase and gp100 and one contained none. Electrotransfection of DCs did not affect viability and phenotype of fresh mature DCs. However, post-thaw viability was lower (69 +/- 12 percent) in comparison to non-electroporated cells (82 +/- 12 percent; p = 0.001). No patient exhibited grade 3 or 4 toxicity upon DC injections. CONCLUSION: Standardized preparation of viable clinical-grade DCs transfected with tumor-derived and in vitro amplified mRNA is feasible and their administration is safe.