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Enhanced bone destruction is a hallmark of various carcinomas such as breast cancer, where osteolytic bone metastasis is associated with increased morbidity and mortality. Immune cells contribute to osteolysis in cancer growth, but the factors contributing to aggressive bone destruction are not well understood. In this study, we show the importance of myeloid-derived suppressor cells (MDSC) in this process at bone metastatic sites. Because MDSC originate from the same myeloid lineage as macrophages, which are osteoclast precursors, we hypothesized that MDSC may undergo osteoclast differentiation and contribute to enhanced bone destruction and tumor growth. Using an immunocompetent mouse model of breast cancer bone metastasis, we confirmed that MDSC isolated from the tumor-bone microenvironment differentiated into functional osteoclasts both in vitro and in vivo. Mechanistic investigations revealed that nitric oxide signaling was critical for differentiation of MDSC into osteoclasts. Remarkably, osteoclast differentiation did not occur in MDSC isolated from control or tumor-bearing mice that lacked bone metastasis, signifying the essential cross-talk between tumor cells and myeloid progenitors in the bone microenvironment as a requirement for osteoclast differentiation of MDSC. Overall, our results identify a wholly new facet to the multifunctionality of MDSC in driving tumor progression, in this case as a novel osteoclast progenitor that specifically drives bone metastasis during cancer progression. Cancer Res; 73(2); 672–82. ©2012 AACR.

CD8+ T cells are cytolytic effectors of adaptive immunity against invading pathogens, and under various pathological conditions including cancer. In syngeneic, immunocompetent murine models of epithelial cancers, including breast and lung cancer, we observed up-regulation in frequencies of a unique CD8+ T cell population, characterized as CD8+CD25-Foxp3+, referred here as CD8+ natural regulatory T cells. This regulatory cell population was present in very high numbers during initial stages of tumor growth. However, a drastic reduction in CD8+ natural regulatory T cells was observed as tumor burden increased, and also with the onset of metastasis to distant sites, including bone in the case of breast cancer. Upon further investigation of these cells, we observed that they are highly cytotoxic towards the cancer cells. Adoptive transfer of these CD8+ natural regulatory cells obtained from mice with minimal tumor burden resulted in significantly reduced tumor growth in the tumor-bearing recipients. There was also an inverse relationship between CD8+ natural regulatory T cells and plasmacytoid dendritic cells (pDC) as tumor burden increased. Additionally, depletion of pDC, either using depletion antibody or by using a transgenic mouse model lacking pDC, showed increased numbers of CD8+ natural regulatory T cells that resulted in decreased tumor burden. Analysis of a possible molecular mechanism on the role of CD8+ natural regulatory T cells as anti-tumor effectors demonstrated that these cells express PD-1. Furthermore, PD-L1, a ligand for PD-1, is expressed by both pDC and cancer cells in vivo. Ongoing experiments are focused on deciphering a possible role of PD-1 and PD-L1 interaction in determining the fate of CD8+ natural regulatory T cells in the presence of pDC and cancer cells through in vitro assays and using transgenic murine models and depletion antibodies for both PD-1 and PD-L1. Collectively, these data suggest a novel anti-tumor immune population that has a potential to be used as immunotherapy for cancer in conjunction with conventional treatments. Citation Format: Anandi Sawant, Carnella Lee, Cara Schafer, Jessy Deshane, Selvarangan Ponnazhagan. CD8+ natural regulatory T cells as antitumor immune effectors in carcinomas of breast and lung. [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 2794. doi:10.1158/1538-7445.AM2014-2794

Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer deaths among males in the United States. Successful treatments for patients with metastatic PCa are limited suggesting a need for alternate targets. Recently, we identified that antimicrobial peptides in human (LL-37) and mouse (CRAMP) are positively correlated with PCa progression in humans and mice, respectively. As a host defense peptide, LL-37/CRAMP functions against microbial pathogens, and in chemotaxis of leukocytes at sites of inflammation. Preliminary studies pertaining to immunomodulation of PCa cell-derived CRAMP in situ indicated that CRAMP chemoattracts myeloid-derived suppressor cells (MDSC), a heterogeneous population of myeloid precursors that induce T cell suppression, to tumor microenvironment (TME) indicating the significance of CRAMP in pro-tumorigenic immune cell functions during PCa progression. The present study further evaluated role of CRAMP as an immunomodulator during PCa growth using transplantable mouse PCa cell line, TRAMP-C1, containing varying CRAMP expression in wild-type and CRAMP loss of function mouse (Cnlp-/-) models. Results of the study indicated targeted down-regulation of CRAMP in mouse PCa cells delays tumorigenesis in syngenic immunocompetent mouse model suggesting significance of CRAMP in PCa development. Whereas implantation of PCa cells abrogated of CRAMP resulted in retention of neutrophils and macrophages in spleen at significantly elevated levels, mice bearing CRAMP-expressing tumors displayed decreased number of neutrophils and macrophages in spleen, but increased infiltration toward TME. These results suggest tumor-derived CRAMP possibly mediates infiltration of neutrophils and macrophages toward TME from spleen. Whether the infiltrated neutrophils and macrophages correlate to pro-tumorigenic N2 and M2 cells, respectively, needs to be confirmed. Since host immune cells, mainly neutrophils, produce CRAMP and recruit additional innate effectors to inflammatory sites, we further analyzed whether host immune cell-derived CRAMP exert synergistic effects in PCa growth together with tumor-derived CRAMP by using Cnlp-/- mice. Results indicated not only comparable tumor growth kinetics, but also comparable levels of MDSC and neutrophils in TME between Cnlp-/- and wild-type mice, suggesting that tumor is a key source of CRAMP that functions as a chemoattractant of MDSC and neutrophils. Taken together, present study proposes that tumor-produced CRAMP functions as a chmoattractant for infiltration of pro-tumorigenic immune cells; MDSC, neutrophils, and macrophages that facilitate tumor growth. Citation Format: Ha-Ram Cha, Anandi Sawant, Jonathan Hensel, Carnella Lee, Selvarangan Ponnazhagan. Immunomodulatory role of CRAMP (cathelicidin-related antimicrobial peptide) in 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 162. doi:10.1158/1538-7445.AM2014-162

Breast cancer (BCa) is the most common malignancy in United States women, accounting for greater than 40,000 deaths each year. Studies have attributed the presence of cancer stem cells (CSC) with resistance to chemotherapy as a key component of refractories of treatments in BCa. Breast cancer stem cells (BCa-SC) are characterized by CD24lo and CD44hi phenotype with mesenchymal properties, which differ from the epithelial CD24hi and CD44hi phenotype expressed in the majority of BCa cells. Thus, further understanding properties of BCa-SC may lead to the development of novel treatment options towards treating refractory cancer cells. Fundamental to this, further characterization of BCa-SC will be needed to identify therapeutic targets based CSC biology. To this end, we sought to characterize the plasticity of BCa-SC based on cell cycle in vitro and growth kinetics in vivo. A murine BCa cell line, 4T1, was cultured in CSC medium and analyzed every 6 hrs for CD24 and CD44 expression by flow cytometry. Results of this analysis indicated that specific BCa-SC markers peaked every 24 hours. This data supports a possible reprogramming of BCa-SC based on cell cycle regulation, suggesting the use of cell cycle inhibitors to block cells in particular stages of the cell cycle, which may improve therapeutic approaches to induce cell death. The stemness of these cells was further confirmed by real-time PCR. BCa-SC showed increased expression of ALDH1, TWIST, and TGF-β3 with decreased E-Cadherin and CD24 levels correlating to the phenotypic changes observed with CD24 and CD44 expression. To further examine the role of BCa-SC in tumor growth in vivo, BALB/c mice were subcutaneously implanted with populations of 4T1 cells that constituted CSC and non- cancer stem cells (NCSC) in different combinations and tumor growth was analyzed. Results showed that the growth kinetics CSC and NCSC when used alone was significantly lower in vivo compared to mixed populations containing both CSC and NCSC, suggesting CSC may attribute for the repopulation of the disease but an aggressive growth requires differentiated non-stem cells in the tumor microenvironment. Citation Format: Carnella M. Lee, Anandi Sawant, Ha-Ram Cha, Selvarangan Ponnazhagan. Growth characteristics of breast cancer stem cells in vitro and in vivo. [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 250. doi:10.1158/1538-7445.AM2013-250

Prostate cancer (PCa) is the most common type of cancer among males in the United States. However, even with advanced diagnostic capacity and conventional therapies such as surgery and chemotherapy, the 5-year survival rate for disseminated disease is approximately 36 months suggesting the need for finding alterate targets and therapies to increase patient survival. We have recently demonstrated that LL-37, an antimicrobial peptide that plays an important role in innate immune response against microbial pathogens, and its mouse orthologue CRAMP (Cathelicidin-related antimicrobial peptide) plays an important role in the progression of PCa both in humans and in a spontaneously developing mouse PCa model, respectively. Over-expression of CRAMP has also been recently reported in a few human epithelial cancers including carcinomas of the breast and lung. Results of our recent studies also demonstrated that targeted knock-down of CRAMP expression in a mouse PCa cell line, derived from the transgenic adenocarcinoma mouse prostate (TRAMP) model, TRAMP C1, resulted in decreased level of myeloid-derived suppressor cells (MDSC) in the spleen upon syngeneic transplantation. Based on these findings, the present study determined the role of LL-37/CRAMP in immune modulation during PCa progression. C57BL/6 mice were subcutaneously implanted with 3 different PCa cell lines; TRAMP C1 (CRAMPhigh), TRAMP C1scrambled (CRAMPhigh) and TRAMP C1CRAMPshRNA (CRAMPlow). Mice in TRAMP C1 and TRAMP C1scrambled groups developed measurable tumors starting from 45 days post-implantation, whereas mice implanted with TRAMP C1CRAMPshRNA showed measurable tumors from 120 days post-implantation suggesting that high levels of CRAMP expression in TRAMP C1 and TRAMP C1scrambled groups promote prostate tumor growth in vivo. Immune cell phenotyping was performed on cells collected from spleen and tumor at day 50 and 55 post-implantation to identify modulation in infiltrating immune cells as a surrogate for decrease in tumor growth due to abrogation of CRAMP expression. The result indicated a higher number of neutrophils infiltrating to the tumor site from the spleen during the initial stages of tumor growth in TRAMP C1 and TRAMP C1scrambled groups, while high numbers of neutrophils remained in the spleen of TRAMP C1CRAMPshRNA group during that time point. Elevated numbers of MDSC were observed both in spleen and in tumor microenvironment in TRAMP C1 and TRAMP C1scrambled groups suggesting that MDSC may further promote PCa growth whereas mice in the TRAMP C1CRAMPshRNA group showed significantly low level of MDSC in spleen, even when compared to control mice, at earlier time point. The number of plasmacytoid dendritic cells (pDC) both in spleen and tumor in TRAMP C1 and TRAMP C1scrambled groups was low while significantly high numbers of pDC were observed in spleen in TRAMP C1CRAMPshRNA group suggesting that high numbers of pDC may suppress the tumor growth by exerting pro-inflammatory response through cytokines such as IL-12. Altogether, the data suggest that CRAMP promotes PCa growth and plays an important role in immunomodulation during PCa progression. The CRAMP induces infiltration of neutrophils to tumor site and increased number of MDSC in both spleen and tumor, which may induce immunosuppression. Since CRAMP triggers protumorigenic stimuli including angiogenesis, invasion and key immune modulation to promote the tumor growth, LL-37/CRAMP may be used as a possible therapeutic target for PCa treatment. Ongoing tumor challenge studies in CRAMP knockout mouse model will shed more light to confirm these observations and to possibly extend the findings in human patients as a potential therapeutic target. Citation Format: Ha-Ram Cha, Anandi Sawant, Carnella Lee, Jonathan Hensel, George Tsuladze, Selvarangan Ponnazhagan. Immunomodulatory effects of LL-37/CRAMP in prostate cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B86.