Prostate Draining Lymph Nodes Research Articles

CD8 T cells are dispensable for experimental autoimmune prostatitis induction and chronic pelvic pain development.

Chronic Pelvic Pain Syndrome or Chronic Prostatitis (CPPS/CP) is the most prevalent urologic affliction among young adult men. It is a challenging condition to treat, which significantly decreases patient quality of life, mostly because of its still uncertain aetiology. In that regard, an autoimmune origin is a prominent supported theory. Indeed, studies in patients and in rodent models of Experimental Autoimmune Prostatitis (EAP) have provided compelling evidence suggesting a key role of CD4 Th1 cells in disease pathogenesis. However, the implication of other prominent effectors of the immune system, such as CD8 T cells, has yet to be studied. We herein analyzed the induction of prostatitis and the development of chronic pelvic pain in EAP using CD8 T cell-deficient animals. We found similarly elevated PA-specific immune responses, with high frequencies of specific IFNg+CD4+ and IL17+CD4+ T cells in prostate draining lymph nodes from PA-immunized either CD8 KO or wild type animals with respect to controls. Moreover, these peripheral immune responses were paralleled by the development of significant chronic pelvic pain, and accompanied by prostate histological lesions, characterized by hemorrhage, epithelial cell desquamation, marked periglandular leukocyte infiltration, and increased collagen deposition in both, PA-immunized CD8 KO and wild type animals. As expected, control animals did not develop prostate histological lesions. Our results indicate that CD8 T cells do not play a major role in EAP pathogenesis and chronic pelvic pain development. Moreover, our results corroborate the previous notion that a CD4 Th1 associated immune response drives the induction of prostate tissue inflammation and the development of chronic pelvic pain.

Galectin-3 in Prostate Cancer Stem-Like Cells Is Immunosuppressive and Drives Early Metastasis.

Galectin-3 (Gal-3) is an extracellular matrix glycan-binding protein with several immunosuppressive and pro-tumor functions. The role of Galectin-3 in cancer stem-like cells (CSCs) is poorly investigated. Here, we show that prostate CSCs also colonizing prostate-draining lymph nodes of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice overexpress Gal-3. Gal-3 contributes to prostate CSC-mediated immune suppression because either Gal-3 silencing in CSCs, or co-culture of CSCs and T cells in the presence of the Gal-3 inhibitor N-Acetyl-D-lactosamine rescued T cell proliferation. N-Acetyl-D-lactosamine also rescued the proliferation of T cells in prostate-draining lymph nodes of TRAMP mice affected by prostate intraepithelial neoplasia. Additionally, Gal-3 impacted prostate CSC tumorigenic and metastatic potential in vivo, as Gal-3 silencing in prostate CSCs reduced both primary tumor growth and secondary invasion. Gal-3 was also found expressed in more differentiated prostate cancer cells, but with different intracellular distribution as compared to CSCs, which suggests different functions of Gal-3 in the two cell populations. In fact, the prevalent nuclear and cytoplasmic distribution of Gal-3 in prostate CSCs made them less susceptible to apoptosis, when compared to more differentiated prostate cancer cells, in which Gal-3 was predominantly intra-cytoplasmic. Finally, we found Gal-3 expressed in human and mouse prostate intraepithelial neoplasia lesions and in metastatic lymph nodes. All together, these findings identify Gal-3 as a key molecule and a potential therapeutic target already in the early phases of prostate cancer progression and metastasis.

Dendritic Cells Coordinate the Development and Homeostasis of Organ-Specific Regulatory T Cells

Although antigen recognition mediated by the T cell receptor (TCR) influences many facets of Foxp3(+) regulatory T (Treg) cell biology, including development and function, the cell types that present antigen to Treg cells in vivo remain largely undefined. By tracking a clonal population of Aire-dependent, prostate-specific Treg cells in mice, we demonstrated an essential role for dendritic cells (DCs) in regulating organ-specific Treg cell biology. We have shown that the thymic development of prostate-specific Treg cells required antigen presentation by DCs. Moreover, Batf3-dependent CD8α(+) DCs were dispensable for the development of this clonotype and had negligible impact on the polyclonal Treg cell repertoire. In the periphery, CCR7-dependent migratory DCs coordinated the activation of organ-specific Treg cells in the prostate-draining lymph nodes. Our results demonstrate that the development and peripheral regulation of organ-specific Treg cells are dependent on antigen presentation by DCs, implicating DCs as key mediators of organ-specific immune tolerance.

Immunosuppression via Tenascin-C.

Immunosuppression via Tenascin-C.

Pim Kinases Promote Migration and Metastatic Growth of Prostate Cancer Xenografts

Background and methodsPim family proteins are oncogenic kinases implicated in several types of cancer and involved in regulation of cell proliferation, survival as well as motility. Here we have investigated the ability of Pim kinases to promote metastatic growth of prostate cancer cells in two xenograft models for human prostate cancer. We have also evaluated the efficacy of Pim-selective inhibitors to antagonize these effects.ResultsWe show here that tumorigenic growth of both subcutaneously and orthotopically inoculated prostate cancer xenografts is enhanced by stable overexpression of either Pim-1 or Pim-3. Moreover, Pim-overexpressing orthotopic prostate tumors are highly invasive and able to migrate not only to the nearby prostate-draining lymph nodes, but also into the lungs to form metastases. When the xenografted mice are daily treated with the Pim-selective inhibitor DHPCC-9, both the volumes as well as the metastatic capacity of the tumors are drastically decreased. Interestingly, the Pim-promoted metastatic growth of the orthotopic xenografts is associated with enhanced angiogenesis and lymphangiogenesis. Furthermore, forced Pim expression also increases phosphorylation of the CXCR4 chemokine receptor, which may enable the tumor cells to migrate towards tissues such as the lungs that express the CXCL12 chemokine ligand.ConclusionsOur results indicate that Pim overexpression enhances the invasive properties of prostate cancer cells in vivo. These effects can be reduced by the Pim-selective inhibitor DHPCC-9, which can reach tumor tissues without serious side effects. Thus, Pim-targeting therapies with DHPCC-9-like compounds may help to prevent progression of local prostate carcinomas to fatally metastatic malignancies.

Tenascin-C Protects Cancer Stem-like Cells from Immune Surveillance by Arresting T-cell Activation.

Precociously disseminated cancer cells may seed quiescent sites of future metastasis if they can protect themselves from immune surveillance. However, there is little knowledge about how such sites might be achieved. Here, we present evidence that prostate cancer stem-like cells (CSC) can be found in histopathologically negative prostate draining lymph nodes (PDLN) in mice harboring oncogene-driven prostate intraepithelial neoplasia (mPIN). PDLN-derived CSCs were phenotypically and functionally identical to CSC obtained from mPIN lesions, but distinct from CSCs obtained from frank prostate tumors. CSC derived from either PDLN or mPIN used the extracellular matrix protein Tenascin-C (TNC) to inhibit T-cell receptor-dependent T-cell activation, proliferation, and cytokine production. Mechanistically, TNC interacted with α5β1 integrin on the cell surface of T cells, inhibiting reorganization of the actin-based cytoskeleton therein required for proper T-cell activation. CSC from both PDLN and mPIN lesions also expressed CXCR4 and migrated in response to its ligand CXCL12, which was overexpressed in PDLN upon mPIN development. CXCR4 was critical for the development of PDLN-derived CSC, as in vivo administration of CXCR4 inhibitors prevented establishment in PDLN of an immunosuppressive microenvironment. Taken together, our work establishes a pivotal role for TNC in tuning the local immune response to establish equilibrium between disseminated nodal CSC and the immune system.

Dendritic cells coordinate the development and homeostasis of organ-specific regulatory T cells (LYM2P.730)

Abstract Foxp3+ regulatory T cells (Tregs) are critical for the maintenance of tolerance to self. However, the antigen presenting cells (APCs) controlling the thymic development and peripheral homeostasis of these cells have yet to be clearly defined. Previously, we identified a monoclonal population of prostate-specific Tregs, termed MJ23 Tregs, that require Autoimmune Regulator (Aire) for their thymic development. Using a variety of mixed bone marrow chimera experiments, we sought to determine the contributions of various thymic APC subsets towards MJ23 Treg development. We demonstrate that the thymic development of MJ23 Tregs requires both MHC-II and B7.1/.2 on bone-marrow derived cells. Moreover, development is abolished by the depletion of CD11c-expressing cells. Together, these data indicate that the antigen recognized by MJ23 Tregs in the thymus must be transferred from Aire-expressing medullary thymic epithelial cells to CD11c-expressing dendritic cells to coordinate MJ23 Treg development. In the periphery, we demonstrate that the depletion of CD11c-expressing cells eliminates the enrichment of MJ23 Tregs in the prostate-draining lymph nodes, demonstrating that dendritic cells also coordinate the anatomical distribution of organ-specific Tregs in the secondary lymphoid organs. In all, our results reveal a critical role for dendritic cells in orchestrating the development and homeostasis of Aire-dependent, organ-specific Treg cells.

Interleukin-30 Expression in Prostate Cancer and Its Draining Lymph Nodes Correlates with Advanced Grade and Stage

The interleukin (IL)-27 cytokine subunit p28, also called IL-30, has been recognized as a novel immunoregulatory mediator endowed with its own functions. These are currently the subject of discussion in immunology, but completely unexplored in cancer biology. We set out to investigate the role of IL-30 in prostate carcinogenesis and its effects on human prostate cancer (hPCa) cells. IL-30 expression, as visualized by immunohistochemistry and real-time reverse transcriptase PCR on prostate and draining lymph nodes from 125 patients with prostate cancer, was correlated with clinicopathologic data. IL-30 regulation of hPCa cell viability and expression of selected gene clusters was tested by flow cytometry and PCR array. IL-30, absent in normal prostatic epithelia, was expressed by cancerous epithelia with Gleason ≥ 7% of 21.3% of prostate cancer stage I to III and 40.9% of prostate cancer stage IV. IL-30 expression by tumor infiltrating leukocytes (T-ILK) was higher in stage IV that in stage I to III prostate cancer (P = 0.0006) or in control tissue (P = 0.0011). IL-30 expression in prostate draining lymph nodes (LN)-ILK was higher in stage IV than in stage I to III prostate cancer (P = 0.0031) or in control nodes (P = 0.0023). The main IL-30 sources were identified as CD68(+) macrophages, CD33(+)/CD11b(+) myeloid cells, and CD14(+) monocytes. In vitro, IL-30 stimulated proliferation of hPCa cells and also downregulated CCL16/LEC, TNFSF14/LIGHT, chemokine-like factor (CKLF), and particularly CKLF-like MARVEL transmembrane domain containing 3 (CMTM3) and greatly upregulated ChemR23/CMKLR. We provide the first evidence that IL-30 is implicated in prostate cancer progression because (i) its expression by prostate cancer or T- and LN-ILK correlates with advanced disease grade and stage; and (ii) IL-30 exerts protumor activity in hPCa cells.

Abstract 452: Murine prostate tumors recruit endogenous, thymic-derived regulatory T cells which are redistributed following castration.

Abstract Studies in a variety of solid tumors have shown a correlation between high frequencies of Foxp3+ regulatory T cells (Tregs) and poor patient prognosis. Thus, due to their central role in regulating immune responses, the modulation of Tregs has been the focus of many emerging cancer immunotherapies. While promising, the catastrophic autoimmunity associated with widespread Treg ablation has significantly limited such approaches. Thus, strategies must be developed which specifically target unique aspects of the life-cycle and function of tumor-associated Tregs (taTregs). In order to identify such novel targets, it will be essential to elucidate the fundamental biology behind the development, specificity and function of taTregs in the tumor environment. To better understand endogenous taTreg responses, we analyzed the T Cell Receptor (TCR) repertoire within autochthonous prostate tumors of TRAMP mice. Using this approach, we identified a TCR specificity that was recurrently expressed by a significant proportion of Tregs within the tumor, and was exclusively found in the Treg subset. We then generated transgenic mice expressing this TCR, coined “MJ23," in order to facilitate the study of the antigenic specificity, developmental origins and effects of castration on the anatomical distribution of an endogenous taTreg population. We first determined that MJ23 T cells show poor “induced” Treg development in the periphery, develop into “natural” Tregs in the thymus, and become enriched in the prostate-draining lymph nodes of both tumor-free and tumor-bearing mice, suggesting reactivity to a prostate self antigen. These results support a model in which tumors enrich for thymic-derived Tregs specific for antigens found within the tissue of cancer origin, rather than promoting the differentiation or induction of Foxp3neg CD4+ T cells reactive to tumor neoantigens. Next, to determine the impact of castration (a first line treatment given for locally advanced prostate cancer) on the prostate taTreg population MJ23, one of three treatments was administered: castration, sham castration or castration followed by testosterone restoration. Using this approach, we discovered that castration led to a dramatic increase in thymic cellularity, a selective expansion of thymic MJ23 Treg frequency, and the anatomical redistribution of MJ23 Tregs. Thus, MJ23 Tregs are impacted by the loss of testosterone following castration, leading to systemic redistribution away from the prostate draining lymph nodes. In future studies it will be critical to determine if the effects observed are due to the loss of self antigens or if Tregs themselves are androgen sensitive. Additionally, utilizing this model we will focus on elucidating the in situ function, antigen specificity, and cognate antigen presenting cells within prostate tumors. Citation Format: Daniel Leventhal, Sven Malchow, Yi Cai, Donald Vander Griend, Peter Savage. Murine prostate tumors recruit endogenous, thymic-derived regulatory T cells which are redistributed following castration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 452. doi:10.1158/1538-7445.AM2013-452

Breaking immune tolerance to prostate-associated tumor antigens through Tim-1 receptor (162.20)

Abstract It has been suggested that one factor limiting cancer immunotherapy in clinical trials are immunological checkpoints that impede vaccine-induced anti-tumor responses. We postulate that manipulating lymphocytes and dendritic cells through the Tim-1 receptor, a membrane protein that regulates the vigor and differentiation of these cells, using an agonist monoclonal antibody could enhance cytotoxic lymphocyte (CTL) responses to prostate tumor-associated antigens. We used an approach that combines concomitant immunization against a model prostate tumor-associated antigen (TAA) SV40 Tag and treatment with an agonist anti-Tim1 antibody and assessed Tag-specific CTL responses two weeks later. Ab treatment of TRAMP mice resulted in elevated Tag-specific CTL in both spleen and prostate-draining lymph nodes, indicating that manipulating Tim1 results in circumventing immune tolerance to Tag antigen. We then tested this strategy for human epitopes derived from the prostate cancer-specific antigen ERG and SIM2 by combining anti-Tim1 antibody with an ERG- and SIM2-derived HLA-A2.1-restricted peptide vaccine in HLA-A2.1 transgenic mice and showed that CTL responses to ERG and SIM2 epitopes are elevated in the presence of anti-Tim1 Ab. Further characterization of lymphocyte differentiation post-immunization/anti-Tim1 treatment revealed that major shifts in T cell differentiation occur. Our findings suggest that targeting Tim1 could be successfully combined with prostate cancer vaccines.

Tracer-cocktail injections for combined pre- and intraoperative multimodal imaging of lymph nodes in a spontaneous mouse prostate tumor model

To improve surgical guidance toward prostate draining lymph nodes, we investigate the potential of intraoperative fluorescence imaging and combined pre- and intraoperative multimodality imaging approaches. Transgenic adenocarcinoma mouse prostate mice with spontaneous prostate tumors are injected intratumorally with: 1. a cocktail of patent blue (Pb) and indocyanine green (ICG); 2. a cocktail of albumin radiocolloids (99mTc-NanoColl), Pb, and ICG; or 3. a cocktail of radiolabeled albumin (99mTc-Vasculosis), Pb, and ICG. The distribution of these imaging agents over the lymph nodes (LNs) are studied at different time points after injection. We find that at 60-min postinjection, ICG significantly improves the detection of the LNs compared to Pb, 53 versus 7%, respectively. Moreover, a cocktail of ICG and 99mTc-NanoColl improves the fluorescent detection rate to 86%, equalling that of the clinically applied 99mTc-NanoColl. A similar overlap is observed in our initial clinical pilot data. Fluorescent detection of the LNs using a ICG with 99mTc-Vasculosis gives similar results as "free" ICG (58%; 60 min). A 99mTc-NanoColl, Pb, and cocktail ICG enriches the standard 99mTc-NanoColl approach by adding optical detection of the sentinel lymph nodes. Furthermore, this approach improves fluorescent-based guidance and enables both accurate surgical planning and intraoperative detection, based on a single injection.

Activation of tolerogenic dendritic cells in the tumor draining lymph nodes by CD8+ T cells engineered to express CD40 ligand.

Tolerogenic dendritic cells in the tumor microenvironment can inhibit the generation and maintenance of robust antitumor T cell responses. In this study, we investigated the effects of local delivery of CD40L by tumor-reactive CD8(+) T cells on dendritic cell activation and antitumor T cell responses in the TRAMP model. To increase the immunostimulatory signal, CD40L was engineered, by deleting the majority of the cytoplasmic domain, to increase its levels of expression and duration on the surface of CD8(+) T cells. Tumor-reactive CD8(+) T cells expressing the truncated form of CD40L stimulated maturation of dendritic cells in vitro and in the prostate draining lymph nodes in vivo. Following dendritic cell maturation, a significantly higher fraction of adoptively transferred, tumor-reactive (reporter) CD8(+) T cells was stimulated to express IFN-gamma and infiltrate the prostate tissue. The antitumor CD8(+) T cell response was further enhanced if TRAMP mice were also immunized with a tumor-specific Ag. These findings demonstrate that augmented T cell responses can be achieved by engineering tumor-reactive T cells to deliver stimulatory signals to dendritic cells in the tumor microenvironment.

Overexpression of vascular endothelial growth factor C increases growth and alters the metastatic pattern of orthotopic PC-3 prostate tumors

BackgroundProstate cancer metastasizes to regional lymph nodes and distant sites but the roles of lymphatic and hematogenous pathways in metastasis are not fully understood.MethodsWe studied the roles of VEGF-C and VEGFR3 in prostate cancer metastasis by blocking VEGFR3 using intravenous adenovirus-delivered VEGFR3-Ig fusion protein (VEGFR3-Ig) and by ectopic expression of VEGF-C in PC-3 prostate tumors in nude mice.ResultsVEGFR3-Ig decreased the density of lymphatic capillaries in orthotopic PC-3 tumors (p < 0.05) and inhibited metastasis to iliac and sacral lymph nodes. In addition, tumor volumes were smaller in the VEGFR3-Ig-treated group compared with the control group (p < 0.05). Transfection of PC-3 cells with the VEGF-C gene led to a high level of 29/31 kD VEGF-C expression in PC-3 cells. The size of orthotopic and subcutaneous PC-3/VEGF-C tumors was significantly greater than that of PC-3/mock tumors (both p < 0.001). Interestingly, while most orthotopic PC-3 and PC-3/mock tumors grown for 4 weeks metastasized to prostate-draining lymph nodes, orthotopic PC-3/VEGF-C tumors primarily metastasized to the lungs. PC-3/VEGF-C tumors showed highly angiogenic morphology with an increased density of blood capillaries compared with PC-3/mock tumors (p < 0.001).ConclusionThe data suggest that even though VEGF-C/VEGFR3 pathway is primarily required for lymphangiogenesis and lymphatic metastasis, an increased level of VEGF-C can also stimulate angiogenesis, which is associated with growth of orthotopic prostate tumors and a switch from a primary pattern of lymph node metastasis to an increased proportion of metastases at distant sites.

Rapid tolerization of virus-activated tumor-specific CD8 + T cells in prostate tumors of TRAMP mice

To study T cell responses to tumors in an autochthonous model, we expressed a CD8 T cell epitope SIYRYYGL (SIY) in the prostate of transgenic adenocarcinoma (TRAMP) mice (referred to as TRP-SIY), which spontaneously develop prostate cancer. Naïve SIY-specific CD8 T cells adoptively transferred into TRP-SIY mice became tolerized in the prostate draining lymph nodes. Vaccination of TRP-SIY mice intranasally with influenza virus that expresses the SIY epitope resulted in generation of SIY-specific effector T cells in the lung-draining lymph nodes. These effector T cells expressed TNFalpha and IFNgamma, eliminated SIY peptide-loaded target cells in vivo, and infiltrated prostate tumors, where they rapidly lost the ability to produce effector cytokines. A population of these T cells persisted in prostate tumors but not in lymphoid organs and could be induced to re-express effector functions following cytokine treatment in vitro. These findings suggest that T cells of a given clone can be activated and tolerized simultaneously in different microenvironments of the same host and that effector T cells are rapidly tolerized in the tumors. Our model provides a system to study T cell-tumor interactions in detail and to test the efficacy of cancer immunotherapeutic strategies.

Alendronate decreases orthotopic PC-3 prostate tumor growth and metastasis to prostate-draining lymph nodes in nude mice

BackgroundMetastatic prostate cancer is associated with a high morbidity and mortality but the spreading mechanisms are still poorly understood. The aminobisphosphonate alendronate, used to reduce bone loss, has also been shown to inhibit the invasion and migration of prostate cancer cells in vitro. We used a modified orthotopic PC-3 nude mouse tumor model of human prostate cancer to study whether alendronate affects prostate tumor growth and metastasis.MethodsPC-3 cells (5 × 105) were implanted in the prostates of nude mice and the mice were treated with alendronate (0.5 mg/kg/day in PBS, s.c.) or vehicle for 4 weeks. After sacrifice, the sizes of tumor-bearing prostates were measured and the tumors and prostate-draining regional iliac and sacral lymph nodes were excised for studies on markers of proliferation, apoptosis, angiogenesis and lymphangiogenesis, using histomorphometry and immunohistochemistry.ResultsTumor occurrence in the prostate was 73% in the alendronate-treated group and 81% in the control group. Mean tumor size (218 mm3, range: 96–485 mm3, n = 11) in the alendronate-treated mice was 41% of that in the control mice (513 mm3, range: 209–1350 mm3, n = 13) (p < 0.05). In the iliac and sacral lymph nodes of alendronate-treated mice, the proportion of metastatic area was only about 10% of that in control mice (p < 0.001). Immunohistochemical staining of tumor sections showed that alendronate treatment caused a marked decrease in the number of CD34-positive endothelial cells in tumors (p < 0.001) and an increase in that of ISEL positive apoptotic cells in tumors as well as in lymph node metastases (p < 0.05) compared with those in the vehicle-treated mice. The density of m-LYVE-1-stained lymphatic capillaries was not changed.ConclusionOur results demonstrate that alendronate treatment opposes growth of orthotopic PC-3 tumors and decreases tumor metastasis to prostate-draining lymph nodes. This effect could be at least partly explained by decreased angiogenesis and increased apoptosis. The results suggest that bisphosphonates have anti-tumoral and anti-invasive effects on primary prostate cancer.