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To date, no specific marker exists for the detection of circulating tumor cells (CTC) from different types of sarcomas, though tools are available for detection of CTCs in peripheral blood of patients with cancer for epithelial cancers. Here, we report cell-surface vimentin (CSV) as an exclusive marker on sarcoma CTC regardless of the tissue origin of the sarcoma as detected by a novel monoclonal antibody. Utilizing CSV as a probe, we isolated and enumerated sarcoma CTCs with high sensitivity and specificity from the blood of patients bearing different types of sarcoma, validating their phenotype by single cell genomic amplification, mutation detection, and FISH. Our results establish the first universal and specific CTC marker described for enumerating CTCs from different types of sarcoma, thereby providing a key prognosis tool to monitor cancer metastasis and relapse. Cancer Res; 74(6); 1645–50. ©2014 AACR.

It is well known that the interleukin (IL)-27 receptor WSX1 is expressed in immune cells and induces an IL-27–dependent immune response. Opposing this conventional dogma, this study reveals a much higher level of WSX1 expression in multiple types of epithelial tumor cells when compared with normal epithelial cells. Expression of exogenous WSX1 in epithelial tumor cells suppresses tumorigenicity in vitro and inhibits tumor growth in vivo. Different from the role of WSX1 in immune cells, the antitumor activity of WSX1 in epithelial tumor cells is independent of IL-27 signaling but is mainly dependent on natural killer (NK) cell surveillance. Deficiency of either the IL-27 subunit EBV-induced gene 3 or the IL-27 receptor WSX1 in the host animals had no effect on tumor growth inhibition induced by WSX1 expression in tumor cells. Expression of WSX1 in epithelial tumor cells enhances NK cell cytolytic activity against tumor cells, whereas the absence of functional NK cells impairs the WSX1-mediated inhibition of epithelial tumor growth. The underlying mechanism by which WSX1 expression in tumor cells enhances NK cytolytic activity is dependent on up-regulation of NKG2D ligand expression. Our results reveal an IL-27–independent function of WSX1: sensitizing NK cell-mediated antitumor surveillance via a NKG2D-dependent mechanism. [Cancer Res 2009;69(13):5505–13]

Ras activation plays an important role in Squamous Cell Carcinomas (SCC) development. Additionally, there is large evidence confirming the role of inflammation during cancer initiation. However, the interaction between inflammation and oncogene activation is poorly understood in tumorigenesis. We found that IL27 promotes papilloma formation in a 2 step skin carcinogenesis (DMBA followed by benzoyl peroxide) model where DMBA causes Ras mutation. Because K15-positive follicular stem cells contribute to papillomas in the two-step skin carcinogenesis model, we used an inducible oncogenic K-RasG12D under the K15 promoter, which marks follicular stem cells. Indeed, treating mice with IL27 increased papilloma incidence earlier in these K15-K-RASG12D mice. Mechanistically, we see that ETAR+ macrophages are significantly increased in IL27-treated K15-K-RasG12D mice. Additionally, the ETAR ligand ET1, and the downstream factor of ETAR, COX2, are upregulated in these K15-KRasG12D mice by IL27, suggesting that ET1/ETAR pathway is involved in IL27 mediated papilloma formation. Indeed, IL27-mediated papilloma formation was reversed by using the ETAR inhibitor ZD4054. Therefore, the data shown here suggest that IL27 drives accumulation of ETAR+ macrophages, ET1, and Cox-2 in the skin and ultimately causes faster papilloma formation in K-Ras mutated follicular stem cells. These findings propose an unrecognized role of IL27 during initiation of skin carcinogenesis. Citation Format: Denada Dibra, Xueqing Xia, Melissa Newman, Camille Keenan, Shulin Li. IL27 promotes papilloma formation via inducing a pro-inflammatory milieu in the skin. [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 1082. doi:10.1158/1538-7445.AM2014-1082

Circulating tumor cells (CTC) released from primary tumor tissues into bloodstream or lymphatic vessels are emerging as novel tools in the detection and prognosis of several types of metastatic cancers. At present, CTC markers are limited to epithelial cancers and there are no specific markers available to detect mesenchymal and epithelial-mesenchymal transformed (EMT) CTCs. EMT cells are characterized by increased motility and invasiveness and are known to escape detection technologies that use EpCAM and cytokeratins as CTC markers. Here we report a novel antibody, 84-1 for detection of mesenchymal and EMT CTC. Antibodies were raised in mice after injecting metastatic cancer cells and clones were selected that were positive only for CTC and negative for PBMCs and normal cells. 84-1 was the only antibody that was unique in its ability to detect CTCs from mesenchymal tumors and EMT CTC from epithelial tumors. Studies for characterization of the epitope for 84-1 antibody are ongoing. We then analyzed the sensitivity and specificity of the antibody by utilizing spiking assay and were able to specifically detect and isolate as low as one cell from 1 ml of blood. In a pilot analysis, we were able to detect CTCs from patients with colon cancer, hepatocellular carcinoma, osteosarcoma, and leukemia with very high specificity, while CTCs were not observed in healthy subjects. CTCs were characterized based on the size of the nucleus and for 84-1 positivity and CD45 negativity using fluorescence microscopy. The CTCs from cancer patients were isolated and cultured for mutational analysis. CTCs were also detectable in the blood of mice with tumors of both epithelial (colon) and non-epithelial (osteosarcoma) origin and were isolated and cultured in vitro, which formed colonies of cells that were used for further analysis to confirm the identity of CTCs. Furthermore, we also evaluated CTCs in mutant p53 mice that developed spontaneous tumors of both mesenchymal and epithelial origin. Collectively, our observations provide a novel technology to selectively detect and isolate both mesenchymal and EMT CTC. Detection and molecular characterization of CTCs is one of the fastest growing areas of translational cancer research. Utilizing our antibody, we can enumerate and isolate rare CTCs that will aid in the early detection of tumors, metastasis, and relapse and will contribute to the development of specific targeted therapies, an ultimate goal of personalized medicine. Citation Format: Arun Satelli, Abhisek Mitra, Jeffery J. Cutrera, Xia Xeuqing, Dibra Denada, Dina Lev Chelouche, Michael James Overman, Ellis Lee, Shulin Li. Specific detection tool for mesenchymal and epithelial-mesenchymal transformed circulating tumor cells. [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 1469. doi:10.1158/1538-7445.AM2013-1469

Although the genetic components that drive carcinogenesis and the stem cell involvement in cancer initiation have been established, the mechanism through which the immune response/cytokine milieu within the tumor microenvironment promotes cancer formation and stem cell behavior is an exciting yet understudied area of research. This statement is specifically true with an important regulator of the immune response, IL27, which is known to have both pro- and anti-inflammatory properties. During an inflammatory response, IL27 has been shown to regulate two canonical cytokines IL10 and IL17, and to affect multiple inflammatory and infectious diseases. Yet, the function of IL27 at physiological levels during the instigation of carcinogenesis remains unknown. To determine the role of IL27 during the early stages of cancer formation, the well-established two step skin carcinogenesis model (DMBA followed by the promoter benzoyl peroxide (BP)) was used. Interestingly, mice that lack IL27 signaling (IL27RA-/-) are protected during the initiation of carcinogenesis as they have lower incidence of papilloma formation over time when compared to wildtype mice. Additionally, wildtype mice treated with IL27 via gene therapy have a higher incidence and develop more papillomas when compared to control counterparts. Bone marrow transfer studies showed that IL27 signaling in both hematopoietic and non-hematopoietic cells is needed to drive skin carcinogenesis. Additionally, IL27 signaling induces loss of epithelial stem cell homeostasis/maintenance. In mice treated with DMBA/BP, 23% of the wildtype mice treated had ulcerations that failed to heal over time, while none of IL27RA-/- showed any defects in the skin. In mice that went under bone marrow transfer followed by DMBA/BP, 40% of wildtype ones developed severe skin ulcerations, while none of the IL27RA-/- developed any skin abnormalities. And lastly, mice undergoing the tumorigenesis protocol that were treated with IL27 developed hair loss, which was mirrored by loss of hair follicles. These phenotypes are like due to the loss of epithelial stem cell maintenance and/or homeostasis by IL27. These finding propose an unrecognized role of IL27 in promoting papilloma formation and disrupting epithelial cell maintenance and/or homeostasis. Citation Format: Denada Dibra, Melissa Newman, Jeffry Cutrera, Shulin Li. The role of IL27 as a driver of skin carcinogenesis. [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 1430. doi:10.1158/1538-7445.AM2013-1430

Our analysis of the interleukin (IL) 27 receptor WSX1 in a variety of human epithelial cells showed that WSX1 is expressed in human tumor epithelial cells. A few cell lines like HT29 and UM-SCC17A showed a 6.9-8.4-fold lower expression of WSX1 when compared to a normal epithelial cell line, NCM460, while most of the cell lines such as Hela, HCT116, and UM-SCC11A showed much higher levels of expression (ranging from 13-78 times higher than NCM460). The role of wsx1 expression in tumors has not been reported by others. In the oral squamous cell carcinoma cell line AT84 and the cervical cancer cell line TC1, we illustrated that overexpression of WSX1 resulted in inhibition of tumor growth via an NKG2D-depent mechanism (Can Res, 2009); however, our most recent results show that WSX1 plays a dual role. Overexpression of WSX1 in Lewis lung carcinoma (LLC) cells enhanced tumor growth in vivo, but it reduced tumor cell proliferation and clonogenic ability of these cells in vitro, which is also observed in the TC1 and AT84 cell lines. The observed phenomenon in LLC tumors is not dependent on the NKG2D pathway, as WSX1 overexpression does not induce NKG2D ligand expression. Considering that WSX1 is a receptor for IL27, we asked whether IL27 is indeed the factor that promotes tumor growth. Since IL27 could signal in both tumor and immune cells to promote tumor growth, we used either truncated WSX1 lacking an intracellular domain (IL27 unable to signal into tumors) or TCCR−/− mice (IL27 unable to signal into immune cells) to understand how IL27 affects WSX1-mediated tumor growth. Our data indicates that overexpression of truncated WSX1 promotes tumor growth even further than full length WSX1, demonstrating that IL27 signaling in tumors does not promote tumor growth. Moreover, IL27 signaling in immune cells does not explain how WSX1 promotes tumor growth, as LLC-WSX1 grows faster than GFP control in wildtype and TCCR−/− mice. Finally, treatment of LLC-WSX1-tumor bearing mice with IL27 inhibits rather than promotes tumor growth which fully excludes the role of IL27. Although WSX1-mediated promotion of LLC tumor growth is independent of NKG2D or IL27 pathways, it inhibits adaptive immune responses: overexpression of WSX1 inhibits accumulation of CD4+, CD8+, and dendritic cells in the tumor microenvironment. WSX1 does not only affect immune cells via paracrine signaling in the local tumor microenvironment, but also in an endocrine manner in distal organs such as the spleen, suggesting the release of secretable factors. Moreover, in immune-deficient mice (SCID), WSX1-positive tumor cells grow at the same rate as GFP control, once more confirming the role of WSX1 in attenuating adaptive immune responses to promote LLC tumor growth as the underlying mechanism. In conclusion, the presence of WSX1 in tumors attenuates the adaptive immune response via a mechanism independent of IL27 and the NKG2D pathway. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3821.