Jonathan A. Hensel

Depletion of Plasmacytoid Dendritic Cells Inhibits Tumor Growth and Prevents Bone Metastasis of Breast Cancer Cells

Elevated levels of plasmacytoid dendritic cells (pDC) have been reported in breast cancer patients, but the significance remains undefined. Using three immunocompetent mouse models of breast cancer bone metastasis, we identified a key role for pDC in facilitating tumor growth through immunosuppression and aggressive osteolysis. Following infiltration of macrophages upon breast cancer dissemination, there was a steady increase in pDC within the bone, which resulted in a sustained Th2 response along with elevated levels of regulatory T cells and myeloid-derived suppressor cells. Subsequently, pDC and CD4+ T cells, producing osteolytic cytokines, increased with tumor burden, causing severe bone damage. Microcomputed tomography and histology analyses of bone showed destruction of femur and tibia. The therapeutic significance of this finding was confirmed by depletion of pDC, which resulted in decreased tumor burden and bone loss by activating tumor-specific cytolytic CD8+ T cells and decreasing suppressor cell populations. Thus, pDC depletion may offer a novel adjuvant strategy to therapeutically influence breast cancer bone metastasis.

Therapeutic potential of adult bone marrow-derived mesenchymal stem cells in prostate cancer bone metastasis.

Purpose: Current evidence indicates that an osteoblast lesion in prostate cancer is preceded by osteolysis. Thus, prevention of osteolysis would reduce complications of bone metastasis. Bone marrow–derived mesenchymal stem cells have the ability to differentiate into osteoblast and produce osteoprotegerin, a decoy receptor for the receptor activator for nuclear factor κB ligand, naturally. The present study examined the potential of unmodified mesenchymal stem cells to prevent osteolytic bone lesions in a preclinical mouse model of prostate cancer. Experimental Design: The human prostate cancer cell line PC3 was implanted in tibiae of severe combined immunodeficient mice. After establishment of the tumor, either unmodified or genetically engineered mesenchymal stem cells overexpressing osteoprotegerin was injected at the site of tumor growth. The effects of therapy were monitored by bioluminescence imaging, micro–computed tomography, immunohistochemistry, and histomorphometry. Results: Data indicated significant (P < 0.001) inhibition of tumor growth and restoration of bone in mice treated with unmodified and modified mesenchymal stem cells. Detailed analysis suggested that the donor mesenchymal stem cell inhibited tumor progression by producing woven bone around the growing tumor cells in the tibiae and by preventing osteoclastogenesis. Conclusions: Overcoming the limitation of the number of mesenchymal stem cells available in the bone can provide significant amelioration for osteolytic damage without further modification. (Clin Cancer Res 2009;15(23):7175–85)

LL-37 as a therapeutic target for late stage prostate cancer

The antimicrobial peptide, leucine–leucine‐37 (LL‐37), stimulates proliferation, angiogenesis, and cellular migration, inhibits apoptosis and is associated with inflammation. Since these functional processes are often exaggerated in cancer, the aim of the present study was to investigate the expression and role of LL‐37 in prostate cancer (PCa) and establish its value as a therapeutic target.

Anterior Gradient Protein-2 Is a Regulator of Cellular Adhesion in Prostate Cancer

Anterior Gradient Protein (AGR-2) is reported to be over-expressed in many epithelial cancers and promotes metastasis. A clear-cut mechanism for its observed function(s) has not been previously identified. We found significant upregulation of AGR-2 expression in a bone metastatic prostate cancer cell line, PC3, following culturing in bone marrow-conditioned medium. Substantial AGR-2 expression was also confirmed in prostate cancer tissue specimens in patients with bone lesions. By developing stable clones of PC3 cells with varying levels of AGR-2 expression, we identified that abrogation of AGR-2 significantly reduced cellular attachment to fibronectin, collagen I, collagen IV, laminin I and fibrinogen. Loss of cellular adhesion was associated with sharp decrease in the expression of α4, α5, αV, β3 and β4 integrins. Failure to undergo apoptosis following detachment is a hallmark of epithelial cancer metastasis. The AGR-2-silenced PC3 cells showed higher resistance to Tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) induced apoptosis in vitro. This observation was also supported by significantly reduced Caspase-3 expression in AGR-2-silenced PC3 cells, which is a key effector of both extrinsic and intrinsic death signaling pathways. These data suggest that AGR-2 influence prostate cancer metastasis by regulation of cellular adhesion and apoptosis.

Location of tumor affects local and distant immune cell type and number

Tumors comprise heterogeneous populations of cells, including immune infiltrates that polarize during growth and metastasis. Our preclinical studies on breast cancer (BCa) identified functional differences in myeloid‐derived suppressor cells based on tumor microenvironment (TME), prompting variations in host immune response to tumor growth, and dissemination based on tissue type.

Prostate cancer-derived cathelicidin-related antimicrobial peptide facilitates macrophage differentiation and polarization of immature myeloid progenitors to protumorigenic macrophages.

A growing body of evidence indicates a positive correlation between expression of human antimicrobial peptide leucin leucin 37 (LL‐37) and progression of epithelial cancers, including prostate cancer (PCa). Although the molecular mechanisms for this correlation has not yet been elucidated, the primary function of LL‐37 as a chemotactic molecule for innate immune effector cells suggests its possible association in coordinating protumorigenic mechanisms, mediated by tumor‐infiltrating immune cells.

Immature myeloid cells are critical for enhancing bone fracture healing through angiogenic cascade

Bone fractures heal with overlapping phases of inflammation, cell proliferation, and bone remodeling. Osteogenesis and angiogenesis work in concert to control many stages of this process, and when one is impaired it leads to failure of bone healing, termed a nonunion. During fracture repair, there is an infiltration of immune cells at the fracture site that not only mediate the inflammatory responses, but we hypothesize they also exert influence on neovasculature. Thus, further understanding the effects of immune cell participation throughout fracture healing will reveal additional knowledge as to why some fractures heal while others form nonunions, and lead to development of novel therapeutics modulating immune cells, to increase fracture healing and prevent nonunions. Using novel femoral segmental and critical-size defect models in mice, we identified a systemic and significant increase in immature myeloid cell (IMC) infiltration during the initial phase of fracture healing until boney union is complete. Using gemcitabine to specifically ablate the IMC population, we confirmed delayed bone healing. Further, adoptive transfer of IMC increased bone growth in a nonunion model, signifying the role of this unique cell population in fracture healing. We also identified IMC post-fracture have the ability to increase endothelial cell migration, and tube formation, signaling the essential communication between the immune system and angiogenesis as a requirement for proper bone healing. Based on this data we propose that IMC may play a significant role in fracture healing and therapeutic targeting of IMC after fracture would minimize the chances of eventual nonunion pathology.

Effects of Cellular Methylation on Transgene Expression and Site-Specific Integration of Adeno-Associated Virus

DNA methylation is a major epigenetic event that affects not only cellular gene expression but that also has the potential to influence bacterial and viral DNA in their host-dependent functions. Adeno-associated virus (AAV) genome contains a high degree of CpG sequences capable of methylation in its terminal repeat sequences, which are the sole elements retained in AAV-based vectors used in gene therapy. The present study determined the influence of methylation status of the host cell on wild type (wt) AAV integration and recombinant (r) AAV transgene expression in HeLa cells. Results of the study indicated that hypo-methylation significantly enhanced both wtAAV chromosomal integration and transgene expression of rAAV. A direct influence of methylation on AAV integration was further confirmed by methylating the AAVS1 integration sites prior to viral infection with DNA trans-complementation assay. These results signify the importance of epigenetic status of target cells as one of the key factors in long-term transgene expression in AAV gene therapy.

Characterization of immune cell subtypes in three commonly used mouse strains reveals gender and strain-specific variations

The lack of consensus on bone marrow (BM) and splenic immune cell profiles in preclinical mouse strains complicates comparative analysis across different studies. Although studies have documented relative distribution of immune cells from peripheral blood in mice, similar studies for BM and spleen from naïve mice are lacking. In an effort to establish strain- and gender-specific benchmarks for distribution of various immune cell subtypes in these organs, we performed immunophenotypic analysis of BM cells and splenocytes from both genders of three commonly used murine strains (C57BL/6NCr, 129/SvHsd, and BALB/cAnNCr). Total neutrophils and splenic macrophages were significantly higher in C57BL/6NCr, whereas total B cells were lower. Within C57BL/6NCr female mice, BM B cells were elevated with respect to the males whereas splenic mDCs and splenic neutrophils were reduced. Within BALB/cAnNCr male mice, BM CD4+ Tregs were elevated with respect to the other strains. Furthermore, in male BALB/cAnNCr mice, NK cells were elevated with respect to the other strains in both BM and spleen. Splenic CD4+ Tregs and splenic CD8+ T cells were reduced in male BALB/c mice in comparison to female mice. Bone marrow CD4+ T cells and mDCs were significantly increased in 129/SvHsd whereas splenic CD8+ T cells were reduced. In general, males exhibited higher immature myeloid cells, macrophages, and NK cells. To our knowledge, this study provides a first attempt to systematically establish organ-specific benchmarks on immune cells in studies involving these mouse strains.This study evaluated both innate and adaptive cell types of myeloid and lymphoid lineage from both genders of three major immunocompetent mouse strains used in pre-clinical research. Results of the study underscore that both innate and cell-mediated immune profile varies dramatically based on mouse strain and gender, which will significantly impact on interpretation of experimental outcome in a wide spectrum of studies, including cancer.

Abstract 434: Prostate cancer cell-derived cathelicidin-related antimicrobial peptide regulates MCP-1 and CXCL1/2 through autocrine signaling

Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer deaths among males in the United States. Even with conventional therapies PCa metastasizes to advanced stage in many patients, suggesting a need for alternate therapeutic targets to increase patient survival. We have recently shown that elevated levels of the human antimicrobial peptides, leucin leucin-37 (LL-37) and its murine orthologue, cathelicidin-related antimicrobial peptide (CRAMP), were positively associated with PCa progression. In addition, we have observed that PCa-derived CRAMP chemoattracts protumorigenic immune cells, including macrophages, neutrophils, and myeloid-derived suppressor cells (MDSCs) to the tumor microenvironment. To further understand possible protumorigenic immune mechanisms of CRAMP during PCa growth, we studied a relationship between CRAMP and formyl peptide receptor 2 (FPR2) in mouse PCa cell lines. Interestingly, we observed that TRAMP-C1 cells, which express high-level of CRAMP, also express high levels of its receptor, FPR2, whereas its cloned derivative, TRAMP-C1shRNA cells with targeted down-regulation of CRAMP, display decreased level of FPR2 both in mRNA and protein levels. We further characterized downstream target genes of CRAMP-FPR2 signaling pathway. Quantitative real-time PCR results indicated that TRAMP-C1 cells have increased expression of both monocyte chemotactic protein-1 (MCP-1), known to chemoattract monotyes/macrophages, and CXCL1/2, known to chemoattract neutrophils and MDSCs, whereas TRAMP-C1shRNA cells exhibited low levels of MCP-1 and CXCL1/2 mRNA. Moreover, results indicated elevated gene expression of FPR2, MCP-1, and CXCL1/2 in TRAMP-C1shRNA cells following stimulation with exogenous CRAMP peptide in culture. In a separate experiment, we also observed down-regulation of MCP-1 and CXCL1/2 in TRAMP-C1 cells after the blockade of FPR2 by FPR2 inhibitor, suggesting a possible association between CRAMP and FPR2 in TRAMP-C1 cells, influencing the levels of MCP-1 and CXCL1/2 during PCa progression. Chemokines including MCP-1 and CXCL1/2 play a significant role in PCa development by recruiting protumorigenic immune effectors, such as MDSCs, tumor-associated macrophages and neutrophils to the tumor microenvironment. Thus, understanding the role of CRAMP in stimulating PCa cells to produce protumorigenic chemokines will define the immunomodulatory role of CRAMP during PCa progression. Altogether, the data suggest that PCa-derived CRAMP triggers pro-tumorigenic stimuli by influencing levels of protumorigenic chemokines through autocrine and paracrine FPR2 signaling. Ongoing studies to identify transcriptional regulators influenced by CRAMP-FPR2 signaling will further define the precise mechanisms of action of CRAMP, to possibly extend the findings in PCa patients as a potential therapeutic target. Citation Format: Ha-Ram Cha, Jonathan Hensel, Selvarangan Ponnazhagan. Prostate cancer cell-derived cathelicidin-related antimicrobial peptide regulates MCP-1 and CXCL1/2 through autocrine signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 434. doi:10.1158/1538-7445.AM2015-434