The endothelial glycocalyx (EG) is degraded early during sepsis, and currently available treatments are not effective in promptly restoring it. Here, we created liposomal nanocarriers of preassembled glycocalyx (LNPG) by synthesizing glycosylated syndecan-1 and inserting it into the lipid membrane of unilamellar liposomes. We hypothesized that LNPG would fuse with the endothelial cells where EG is degraded and restore EG in sepsis. We induced endotoxemia in C57BL/6J mice using lipopolysaccharides (LPS) and treated them with LNPG, saline, syndecan-1, or liposomes. LNPG significantly prolonged the survival time of LPS-treated mice compared with the other treatments. Immunostaining of en face mesenteric arteries of LPS-treated mice showed that syndecan-1 was fully restored after LNPG administration. In addition, EG height in microvasculature of mouse cremaster muscle was monitored using sidestream dark field imaging. LNPG restored the perfused boundary region (PBR), which is inversely related to EG dimensions, to the control level after LPS administration. Furthermore, flow-induced dilation in isolated mouse mesenteric arterioles was fully recovered after LNPG treatment in LPS-treated mice. In summary, our findings provide evidence of the therapeutic efficacy of LNPG in the LPS-induced mouse model of sepsis, achieved by expeditiously restoring EG through fusion of LNPG with the endothelial plasma membrane and recovery of endothelial function.NEW & NOTEWORTHY Vascular endothelial cells represent the first line of exposure to bacterial endotoxins. Here, we propose a novel therapeutic strategy using liposomes to deliver preassembled glycocalyx to vascular endothelial cell surface and consequently restore endothelial glycocalyx (EG). We tested liposomal nanocarriers of preassembled glycocalyx (LNPG) in vivo and ex vivo to establish for the first time their expeditious therapeutic efficacy in improving survival of lipopolysaccharides (LPS)-treated mice, as achieved by the restoration of EG and recovery of endothelial function.

Despite the exciting progresses in the treatment of breast cancer, the effectiveness of the current therapeutic modalities is still restricted by drug toxicity, resistance, and lack of predictive and prognostic biomarkers. Breast cancer continues to be the second leading cause of cancer death among women in the U.S. Therefore, the development of new therapeutic targets and further understanding of the tumor microenvironment is extremely critical for accelerating the progress against breast cancer. Human AC133+/CD34+ stem cells are a highly promising and novel therapeutic option for targeting tumor angiogenesis. We and others have described the incorporation of bone marrow derived AC133+/CD34+/KDR+ cells in the neovasculature around implanted tumors supporting their growth and metastasis. Many mediators have been involved in the cross talk between AC133+/CD34+ cells, endothelial cells, and the tumor cells, but most of these have insufficient clinical benefits as reported by several trials. In this study, we evaluated the secretome of the AC133+/CD34+ stem cells that were isolated by positive selection from human umbilical cord blood and their role in breast cancer progression. Using flow cytometry, we show that the high proliferative AC133+/CD34+ stem cells maintain their capacity to differentiate in to AC133+/CD34+/KDR+ endothelial progenitor cells even after long period of in vitro expansion. In order to evaluate the effect of AC133+/CD34+ stem cells on breast cancer cells, a proliferation (XTT) assay was performed using conditioned medium (CM) from AC133+/CD34+ stem cells and examined on MCF-7 and MDA-MB-231 proliferation. As anticipated, CM significantly induced breast cancer cells proliferation. This effect was in part due to the high expression of a large range of proinflammatory and proangiogenic cytokines in the CM of the AC133+/CD34+ cells. In particular, angiogenin, GRO, IL-8, MCP, and TIMP2. Next, we examined if exosomes, a component of paracrine secretion are involved in the paracrine effect of the AC133+/CD34+ stem cells. Surprisingly, exosomes from AC133+/CD34+ stem cells significantly increased MCF-7 and MDA-MB-231 proliferation at a comparable level as the CM. Further analysis of the exosomes using miRNA array screen reveals that exosomes of AC133+/CD34 cells are highly enriched with oncogenic miRNAs including miR-21-5p, miR-142-3p, and miR-223-3p. These miRNAs are up-regulated in breast cancer. Several studies have confirmed their role in mediating breast cancer cells invasiveness. However, miR-142-3p and miR-223-3p are exclusively expressed in hematopoietic cells. Therefore, we propose that shuttling of the exosomes between AC133+/CD34+cells and breast cancer cells induces breast cancer invasiveness. The analysis of the paracrine interactive mediators between breast cancer cells and AC133+/CD34+ cells is likely to yield viable novel clinically translatable therapeutic targets.Citation Format: Ghada Ben Rahoma, Rachana Maniyar, Sanjukta Chakraborty, Sarnath Singh, Anitha Srinivasan, Abraham Mittelman, Jan Geliebter, Raj K. Tiwari. Exosomes secreted by AC133+/CD34+cells harbor invasion potentiating miRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3562.

Breast cancer is the most common cancer among women worldwide, contributing to 25.4% of newly diagnosed cancer cases in 2018. Triple negative breast cancer (TNBC) accounts for 10-20% of the breast cancer cases and represents an unmet clinical need because of its higher risk of recurrence and metastasis. Adjuvant therapies have often been employed to prevent secondary recurrence. Traditional Chinese medicine (TCM) formulations have long been used in prevention and cure of many types of diseases because of its anti-inflammatory property. Mainstream treatment modalities for TNBC involve surgery, radiation and chemotherapy (adjuvant or neoadjuvant). Unfortunately, chemotherapy in these patients often lead to resistance, which underscores the importance of alternative therapeutic approaches for this cancer. To this end, we wanted to evaluate anti-carcinogenic property of a TCM formulation ASHMITM developed primarily for asthma treatment. ASHMI™ is an extract of 3 traditional Chinese medicinal herbs-Ganoderma lucidum (Ling-Zhi), Sophora flavescens Ait (Ku-Shen) and Glycyrrhiza uralensis Fischer (Gan-Gao). For this study, triple-negative/basal B mammary carcinoma cell line MDA-MB157 was treated with two different concentrations of ASHMI and cellular proliferation was examined by [3H] thymidine incorporation assay. ASHMITM treated cells showed approximately 74% less [3H] thymidine incorporation indicating dampened proliferation. This observation was validated using western blot analysis with whole cell lysate, showing reduced expression of proliferation markers PCNA and NFκB. Interestingly, an increased expression of tumor suppressor protein cyclin-dependent kinase inhibitor 1 C (p57, Kip2) was observed after ASHMITM treatment. Next, we tested anti-proliferative property of the individual constituents and observed Ganoderma lucidum (GL) extract had a comparable efficacy as the whole formulation at a concentration of 100μg/ml. This observation was further confirmed by western blot and the same expression pattern was observed for PCNA, NFκB and p57. GL was further fractionated into 11 sub-components and each component was tested for the their anti-proliferative activity using similar assays. Fraction 2 with Ganoderenic acid D showed dose-dependent inhibition of cell proliferation and was further tested for its activity at lower concentrations. Ganoderenic acid D had similar efficiency as 100μg/ml of whole GL extract at a concentration as low as 2.5μg/ml suggesting this active compound might be primarily responsible for the anti-proliferative effect of GL. Our studies using an anti-inflammatory formulation of TCM have identified an active anti-carcinogenic compound, Ganoderenic acid D, that could be potentially used as an adjuvant in TNBC therapies. Citation Format: Sanjukta Chakraborty, Fei Mo, Martin Walsh, Changda Liu, Mingzhuo Cou, Rachana Maniyar, Ghada Ben Rahoma, Sarnath Singh, Tara Jarboe, Michelle Carnazza, Raj Tiwari, Xiu-min Li. Re-purposing traditional Chinese anti-asthma formula ASHMI for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1855.

The last three decades have seen a steady rise in melanoma, the most aggressive form of skin cancer. The discovery of checkpoint inhibitors has revolutionized the treatment landscape for melanoma; anti-CTLA4 and anti-PD1 showing great successes in clinic. While checkpoint molecules and co-stimulatory molecules are traditionally present on cells involved in immune activation, mounting evidence suggests that tumor cells also express these molecules. Our laboratory has characterized five patient-derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM, and GLM-2 and screened them for the expression of a comprehensive list of 29 co-stimulatory and co-inhibitory molecules compared to normal adult melanocytes. We see a differential mRNA expression of many of these immune-regulatory molecules, including BTLA, HVEM, CD160, CD226 and TIM1. Western blots and immunofluorescence confirmed the presence of these molecules at the protein level. A flow cytometry analysis demonstrated that BTLA, HVEM, CD160, TIM1 and CD226 are present on the membrane of these patient derived melanoma cells; implying that they are capable of engaging their respective ligands and exerting a functional role in immunomodulation. These findings were additionally validated in melanoma tissues, obtained from patients, by immunohistochemistry. Interestingly, treatment of MEL-2, MEL-V, KFM and GLM-2, our BRAFV600E containing patient derived cells, with BRAFV600E inhibitor PLX4032 led to the upregulation of these molecules. This upregulation was coupled with an increase in transcription factor MITF, a binding site for which is present in the promoter region of all the upregulated molecules. Co-culture experiments with immune cells, demonstrate a modulatory role played by the HVEM/BTLA/CD160 axis molecules that are present on tumor cells. The presence of these additional immune-regulatory molecules signify the existence of a robust tolerance mechanism induced by these checkpoint inhibitors. Our studies underscore the importance of characterizing tumor profiles to enable the selection of an optimal treatment regimen combining targeted small molecule inhibitors, and checkpoint inhibitors to maximize T cell killing, and tumor clearance. Citation Format: Rachana R. Maniyar, Sanjukta Chakraborty, Ghada Ben Rahoma, John J. Degliuomini, Marc Wallack, Jan Geliebter, Raj K. Tiwari. Tumor checkpoint inhibitor profiling for an optimal clinical response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3946.

Anaplastic thyroid cancer(ATC) is a rare but extremely aggressive form of endocrine malignancy that accounts for only 1-2% of total thyroid cancer cases but responsible for 20-30% of annual mortality from thyroid cancer in the USA. Genetic lesion landscape in ATC is not conducive to multiple targeted therapies. BRAF mutation is one of the dominant genetic lesions observed in 30-40% of ATC cases and 98% of them are BRAFV600E positive. Due to the limited therapeutic efficacy of BRAFV600E inhibitor vemurafenib, identification of novel therapeutic candidates would provide a viable alternative. Immunecheckpoint therapies have showed huge promise in recent years but are relatively underexplored in ATC patients. To this end, 4 ATC cell lines 8505C, T238, SW1736 and HTh74; 3 PTC cell lines TPC-1, BCPAP and K1 and 1 FTC cell line CGTH-W-1 were screened for expression of 29 immune-checkpoint molecules by qRT PCR with or without 10μM vemurafenib treatment for 24 hrs. Initial screening revealed a differential expression level of the transcripts among the cells. Vemurafenib treatment further up regulated expression of CD160, HVEM, BTLA, TIM3 and galectin9 in cell lines positive for BRAFV600E mutation (8505C, SW1736, BCPAP) and the ones harboring an additional PIK3CA mutation (T238, K1). This observation was further confirmed by immunocytochemistry. CD160 was not detected in any of the cell lines except for K1, TPC-1 and NTHY at protein level. Flow cytometry confirmed presence of BTLA, HVEM and TIM3 on the surface of these tumor cells which would enable them to engage their cognate ligands on T cells and suppress antitumor immune response. Western blots confirmed upregulation of BTLA, HVEM, TIM3 and galectin 9 in response to vemurafenib in both BRAFV600E positive cell lines and HTh74. Expression of these molecules were also validated by immunohistochemistry in patient samples. In an effort to evaluate the functional activity of these immunecheckpoint molecules, co-culture studies were done. Both HVEM and BTLA showed immunomodulatory capability and were capable of redirecting T cell differentiation towards a suppressive phenotype. Preclinical studies with combination of BRAFV600E or PI3K inhibitor and antagonistic antibodies against these molecules are currently underway. This study has identified two novel immune checkpoint molecules in ATC and might provide viable therapeutic targets. Citation Format: Sanjukta Chakraborty, Rachana R. Maniyar, Sina Dadafarin, Ghada Ben Rahoma, Sarnath Singh, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari. Combinatorial immune checkpoint inhibitor therapy in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3237.

Metastatic melanoma, leads to the highest number of skin cancer related deaths. Checkpoint inhibitor therapy has witnessed a high success rate in melanoma patients with anti-CTLA-4 and anti-PD-1. However, checkpoint inhibitor molecules and their compensatory stimulatory counterparts are widely expressed on T cells and antigen presenting cells suggesting a robust redundancy in these molecules as clinical targets. Some of these molecules that include CTLA-4, PD-1, HVEM, VISTA, 41-BB, OX-40 and CD226 are also expressed on tumor cells. Their role in regulating an immune response whether it is cell killing or immune evasion remains to be elucidated. We isolated and characterized five primary patient derived melanoma cell lines: MEL-2, MEL-V, 3MM, KFM and GLM2. We screened these cells for the expression of a comprehensive panel of twenty-five co-stimulatory and co-inhibitory molecules by RT-PCR which revealed significant heterogeneity in expression of these molecules compared to normal adult melanocytes under normal conditions; underscoring the importance of understanding tumor tissue pleiotropy prior to designing a therapeutic regimen. Surprisingly, inhibitory molecules including PD-1, VISTA and LAIR1 and stimulatory molecules including 4-1BB, HVEM and ICOS were upregulated differentially in these cell lines by metabolic stress brought on by starvation conditions. Some of these molecules were restored to basal levels of expression on treatment with 10μM vemurafenib (PLX4032), a BRAFV600E inhibitor, for 24 hours. However the treatment led to concurrent upregulation of molecules such as LAG3, BTLA, CD226 and TIM1, suggesting a compensatory mechanism that could aid melanoma adaptation and escape from immune recognition. Exposing melanoma cells to classical activated dendritic cell cytokines, IL-6 and IL-12, led to a differential expression of these molecules. Additionally, experiments using tumor lysate loaded dendritic cells to study activation revealed an ability to modulate immune activation correlating with unique stimulatory and inhibitory molecule expression profile of each primary cell line. Our results underscore the importance of understanding the profile of co-stimulatory and co-inhibitory molecules expressed in tumor cells. With eighty percent of melanoma patients being positive for the BRAFV600E lesion, we make a case for designing a combinatorial therapeutic regimen, with targeted immunotherapies as well as targeting specific genetic lesions with small molecule inhibitors. Citation Format: Rachana R. Maniyar, Sanjukta Chakraborty, Neha Y. Tuli, Ghada Ben Rahoma, Sarnath Singh, Marc Wallack, Jan Geliebter, Raj K. Tiwari. A case for combining immunotherapy and targeted small molecule inhibitors: Immunoregulation by primary melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 714.

Abstract Exciting advances in the treatment of breast cancer have occurred over the past decade. However, the efficacy of the current therapeutic modalities is still limited by drug toxicity, resistance, and lack of predictive and prognostic biomarkers. Breast cancer remains the second leading cause of cancer death among women in the U.S. Thus, the development of new therapeutic targets and further understanding of the tumor microenvironment is extremely crucial for accelerating the progress against breast cancer. Human AC133+/CD34+ stem cells serve as a highly promising and novel therapeutic option for targeting tumor angiogenesis. We and others have demonstrated the incorporation of bone marrow-derived AC133+/CD34+/KDR+ endothelial progenitor cells in the neovasculature around implanted tumors supporting their growth and spread. Many mediators have been implicated in the crosstalk between AC133+/CD34+/KDR+ endothelial progenitor cells, endothelial cells, and the tumor cells, but most of these have limited clinical benefits as reported by several trials. In this study, we analyzed the secretome of the AC133+/CD34+ stem cells that were isolated by positive selection from human umbilical cord blood and evaluated their role in breast cancer progression. Our results show that AC133+/CD34+ stem cells exhibited significant growth potential that was manifested as seventy-five fold increase in cell number after 10 days in culture. Flow cytometry demonstrated that AC133+/CD34+ stem cells preserve their capacity to differentiate into AC133+/CD34+/KDR+ endothelial progenitor cells even after long term in vitro expansion. In order to evaluate the effect of AC133+/CD34+ stem cells on breast cancer cells, we performed a simple proliferation (XTT) assay using conditioned medium (CM) from AC133+/CD34+ stem cells and tested on MCF-7 and MDA-MB-231 proliferation. As expected, CM significantly induced proliferation of breast cancer cells. This effect was in part due to the high expression of a repertoire of proinflammatory and proangiogenic cytokines in the CM of the AC133+/CD34+ stem cells. In particular, angiogenin, GRO, IL-8, MCP, PDGF.BB, TIMP2. Further, we examined if exosomes, a component of paracrine secretion are involved in the paracrine effect of the AC133+/CD34+ stem cells. Interestingly, exosomes from AC133+/CD34+ stem cells significantly enhanced MCF-7 and MDA-MB-231 proliferation at a comparable level as the CM. Further analysis of the exosomes reveals that the pro-proliferative miR-141-3p, miR-182-5p, miR-200b-3p, and miR-203a are highly expressed in AC133+/CD34+ exosomes. The analysis of the paracrine interactive mediators between breast cancer cells, AC133+/CD34+ stem cells, and AC133+/CD34+/KDR+ endothelial progenitor cells is likely to yield viable novel clinically translatable therapeutic targets. Citation Format: Ghada Ben Rahoma, Neha Tuli, Rachana Maniyar, Sanjukta Chakraborty, Sarnath Singh, Abraham Mittelman, Jan Gelibter, Raj K. Tiwari. Exosomes from AC133+/CD34+ stem cells mediate a paracrine effect in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2053.

The majority of breast cancers (90–95%) arise due to mediators distinct from inherited genetic mutations. One major mediator of breast cancer involves chronic inflammation. M1 macrophages are an integral component of chronic inflammation and the breast cancer tumor microenvironment (TME). Previous studies have demonstrated that up to 50% of the breast tumor comprise of tumor-associated macrophages (TAMs) and increased TAM infiltration has been associated with poor patient prognosis. Furthermore, breast cancer associated deaths are predominantly attributed to invasive cancers and metastasis with epithelial-mesenchymal transition (EMT) being implicated. In this study, we investigated the effects of cellular crosstalk between TAMs and breast cancer using an in vitro model system. M1 polarized THP-1 macrophage conditioned media (CM) was generated and used to evaluate cellular and functional changes of breast cancer lines T47D and MCF-7. We observed that T47D and MCF-7 exhibited a partial EMT phenotype in the presence of activated THP-1 CM. Additionally, MCF-7 displayed a significant increase in migratory and invasive properties. We conclude that M1 secretory factors can promote a partial EMT of epithelial-like breast cancer cells. The targeting of M1 macrophages or their secretory components may inhibit EMT and limit the invasive potential of breast cancer.

Abstract Thyroid cancer is the most rapidly increasing cancer in the US with anaplastic thyroid cancer (ATC) being the rarest and most aggressive form. ATC patients display a higher mutational burden and are refractory to current mainstream treatments including second generation kinase inhibitors like vemurafenib (PLX4032). BRAFV600E is one of the most common mutations associated with this phenotype. Presence of BRAFV600E has been associated with an immunosuppressive microenvironment in thyroid cancer that presumably facilitates immune evasion. With recent advancements in immunotherapy, well defined targeted treatment plans can address the unmet medical need of ATC. In an effort to define potential immunotherapeutic targets that can be combined with small molecule inhibitors in this subtype, we evaluated the expression of immune checkpoint molecules in four thyroid cancer cell lines TPC-1 (papillary), BCPAP (BRAFV600E positive papillary), 8505C (BRAFV600E positive anaplastic) and CGTH-W-1 (follicular) by qRT PCR at the basal level and after treatment with vemurafenib and mTORC1 inhibitor rapamycin. Prominent co-stimulatory molecules like CD27, CD30, 4-1BB and DR3 were significantly downregulated at the transcript level in ATC (8505C) as compared to the other thyroid cancer subtypes. We observed a higher baseline expression of co-inhibitory molecules like full length CTLA4 (mCTLA4), soluble CTLA4 (sCTLA4), LAG3, 2B4, PD-1 and PD-L1 in ATC (8505C) compared to the other subtypes of thyroid cancer. Although vemurafenib treatment decreased the expression of PD-L1 as expected, interestingly, it enhanced the expression of co-inhibitory molecules LAIR1, 2B4, mCTLA4 and PD-1 by approximately 1.5 folds. These results suggest an inherent redundancy in inhibitory immune checkpoint molecules that presumably compensate for each other, functionally, making them novel targets in ATC. With our study we identified a distinct group of co-inhibitory molecules in ATC that can be targeted by antagonistic antibodies in a combinatorial treatment regimen with small molecule inhibitors like vemurafenib. Citation Format: Sanjukta Chakraborty, Rachana R. Maniyar, Neha Y. Tuli, Ghada Ben Rahoma, Sarnath Singh, Ameet Kamat, Craig Berzofsky, Cameron Budenz, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari. Identification of novel immunotherapeutic targets in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2762.

Abstract The incidence of thyroid cancer (TCa) has doubled in the last decade. Papillary thyroid cancer (PTC), that comprises of 80% of all TCas, is treatable with great outcomes. However, undifferentiated anaplastic thyroid cancer (ATC), with very poor prognosis, is considered a clinical challenge and currently a losing battle. Moreover, the molecular mechanisms responsible for various forms of TCa are largely unknown. We believe that the cells of histopathologically distinct regions of a heterogeneous thyroid tumor are governed by different sets of master gene regulators. The smart manipulation of such master genes will be able to selectively destroy cancer cells but not normal tissue, opening a novel and much more effective avenue in the thyroid cancer targeted gene therapy. Our analysis established the gene hierarchical governance in each region based on their Gene Commanding Height (GCH). GCH is a measure that combines the gene expression coordination with other genes and the expression stability among biological replicas provided by the internal homeostatic mechanisms. Here, we provide experimental evidence that standard papillary (BCPAP) and anaplastic (8505C) human thyroid cancer cell lines have different master regulators. We identified the master regulators of BCPAP and 8505C, and determined their GCH. Transfection of master gene regulators of a particular cell line has significantly larger effects on the cell line they command than on other cells. We found that the stable transfection with TMEM194A, a nuclear envelope protein, regulated twice more genes in BCPAP than in 8505C cells. The analysis using human thyroid cancer cells reaffirmed our hypothesis of the existence of hierarchical master gene regulators and that the phenotypic changes can be manipulated with the introduction of these genes. We further validate these concepts using human thyroid biopsy samples. We found substantial differences in the GCH scores of cancer versus normal tissue of a surgically removed 32.0mm papillary carcinoma from the left lobe of a 33y old male. Because of such differences between the cancer region and the normal tissue, manipulation of cancer regulators is expected to affect the cancer cells in a greater degree than the normal cells. These results suggest that we have defined a master gene regulator hierarchy in thyroid cancer and extrapolation of this analysis to compare anaplastic and papillary thyroid cancer will lead to novel gene therapeutic modalities. Our long-term goal is to identify master regulators of cancer nodules for each patient and develop personalized cancer therapy targeting these master regulators. Citation Format: Neha Yashpal Tuli, Craig Berzofsky, Rachana Maniyar, Sanjukta Chakraborty, Ghada Ben Rahoma, Sarnath Singh, Jan Geliebter, Raj K. Tiwari, Sanda Iacobas, Dumitru A. Iacobas. Hierarchical gene master regulators of papillary and anaplastic thyroid cancer phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3134. doi:10.1158/1538-7445.AM2017-3134

Melanoma is one of the deadliest forms of skin cancer, with a dim prognosis when metastasized, leading to the highest number of skin cancer related deaths. In recent years there has been a focus on the use of checkpoint inhibitor therapies like anti-CTLA-4, anti-PD-1 and anti-PD-L1 to treat melanoma. PD-L1, HVEM and VISTA expression on cancer cells has been shown to promote immune evasion and tumor survival. CTLA-4, when engaged on tumor cells leads to their apoptosis and LIGHT signaling leads to recruitment of T cells and effective tumor clearance. Thus, since these molecules do not function in isolation, we need to consider tissue pleiotropy and the expression of co-stimulatory and co-inhibitory molecules on tumor tissue. Our laboratory has characterized and established five primary patient derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM and GLM-2. In an effort to understand tumor tissue pleiotropy, we conducted a comprehensive expression pattern screening of eight co-inhibitory and ten co-stimulatory molecules by RT-PCR. Among others, VISTA, a CD4+ T cell suppressor, HVEM, an immune evasion regulator, LAG3 and TIM3, potential immune checkpoint targets, were seen to be differentially expressed in these primary cell lines. Current therapies target CTLA-4, PD-1 and PD-L1, which also exhibit varying expression in our primary cell lines, pointing to the importance of considering tissue expression of these molecules when administering these novel immunotherapies. Additionally, 80% of melanoma patients are positive for the BRAFV600E genetic lesion and are administered vemurafenib (PLX4032), an inhibitor of the overactive mutated BRAF. Our in vitro studies show that treatment with PLX4032, changed the expression of CTLA-4 and PD-L1 in these primary cells. We observed a decrease in PD-L1 protein expression in MEL-V and GLM-2 and an increase in PD-L1 protein expression in MEL-2 and KFM, the four BRAFV600E positive cell lines. CTLA-4 protein expression demonstrated an upward trend on treatment with PLX4032. This synergy observed between treatment with drugs targeting genetic lesions and the expression of immunomodulatory molecules warrants characterization of tumor biopsies prior to designing an effective combinatorial therapy regime. Our long term goal is to optimize combinatorial immune and drug therapies directed against both co-stimulators and checkpoint inhibitors. Citation Format: Rachana R. Maniyar, Sanjukta Chakraborty, Neha Tuli, Ghada Ben Rahoma, Sarnath Singh, Jan Geliebter, Marc Wallack, Raj K. Tiwari. Implication for checkpoint therapeutics: Expression of co-stimulatory and co-inhibitory molecules in melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3656. doi:10.1158/1538-7445.AM2017-3656

Abstract Thyroid cancer is the most common type of endocrine malignancy that has an escalating global frequency. Although most well differentiated thyroid cancers (WDTC) are manageable and respond to current therapeutic modalities, undifferentiated anaplastic thyroid cancers (ATC) exhibit a dramatically different clinical behavior and poor prognosis. With the recent development of immunotherapies, targeted, well-defined treatment plans can demonstrate promising treatment outcomes in ATC patients. Precise immunological targets in ATCs with potential clinical relevance are unknown. Major progress has been made in last 5 years toward development of immune checkpoint inhibitors using anti-CTLA-4 and anti-PD-1/PD-L1 antibodies for cancer treatment which has made immunotherapies one of the mainstream treatment choices. Few additional members of the immunoglobulin superfamily of receptors, like LAG3, TIM3 and VISTA have recently been identified as potential checkpoint targets. Interestingly some of these molecules including TIM3 and PD-L1 promote tumor progression and immune escape. Identification of specific immunotherapeutic targets requirs a better understanding of the immune microenvironment in ATC. To this end we evaluated the expression of prominent co-stimulatory and co-inhibitory cell surface molecules by RT-PCR in three thyroid cancer cell lines - TPC-1 (papillary), CGTH-W-1 (follicular) and 8505C (anaplastic). We observed that many co-inhibitory molecules were upregulated in all three tumor cell lines. CTLA4, interestingly, had the highest expression in 8505C. Additionally we observed differential expression of BTLA, LAIR1, TIM3 and VISTA between TPC-1 and 8505C. LAG3, PD-1 and PD-L1 were also upregulated in 8505C compared to TPC-1. Similar pattern was observed with the expression of co-stimulatory molecules, CD40L and GITR . GITR has been shown to have a tumor suppressor function in multiple myeloma. Another co-stimulatory molecule OX40, which has shown promise in tumor recession when targeted, was upregulated in all three cell lines and 8505C showed the highest expression. Our findings suggest that the aggressive and less immunogenic phenotype of ATC might be attributed to the differential expression of these molecules. Targeting these immunomodulatory molecules in ATC warrants a better understanding of the crosstalk between them and it might provide an efficient means for the disease management. Citation Format: Sanjukta Chakraborty, Rachana R. Maniyar, Neha Y. Tuli, Ghada Ben Rahoma, Cameron Budenz, Sarnath Singh, Jan Geliebter, Raj Tiwari. Functional pairing of immunomodulatory targets in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5676. doi:10.1158/1538-7445.AM2017-5676

Abstract Breast cancer affects one in eight women in the USA. Early diagnosis and newer treatment modalities have rendered breast cancer manageable. However, triple negative breast cancer is still difficult to treat and warrantes a search for newer targets. One strategy that has emerged in cancer research involves targeting of tumor associated blood vessels which provide growing tumors with oxygenated blood and growth factors necessary for maintenance and metastasis. Antiangiogenic drug therapy is transient and has not been able to gain mainstream therapeutic modality. We discovered that endothelial progenitor cells (EPCs) are mobilized from the bone marrow to the tumor site and contribute to the development of breast tumor vessel formation in an estrogen dependent manner. Therefore, characterization of tumor associated endothelial progenitor cells in breast cancer may provide a more specific antivascular therapy. Using the highly proliferative human umbilical cord blood derived EPCs, having the phenotype (CD133+, CD34+, VEGFR-2+), the effect of growth factor and chemokine rich EPCs conditioned medium (CM) was assessed in luminal (MCF-7), and post-EMT (MDA-MB-231) breast carcinoma cell lines. We observed an initial halt in cellular proliferation in MCF-7 followed by a significant increase in proliferation after forty eight hours of treatment. On the other hand, MDA-MB-231 showed decreased proliferation even after forty eight hours of treatment. Treating the EPCs with breast cancer conditioned medium resulted in morphological and cellular growth changes in the EPCs. MDA-MB-231 CM resulted in an increase of the EPCs proliferation and differentiation by increasing the number of spindle shaped attaching cells, and MCF-7 CM resulted only in an increase in the differentiation rate by increasing the number of cell clusters. This increase in EPCs proliferation and differentiation associated with MDA-MB-231 CM treatment might explain the invasiveness of this breast cancer cells through the increase in the tumor associated neovascularization. The analysis of the paracrine interaction between breast cancer cells and EPCs along with the associated cellular changes will facilitate identification of the interactive mediators and subsequent development of effective antivascular therapy. Citation Format: Ghada Ben Rahoma, Neha Tuli, Rachana Maniyar, Sanjukta Chakraborty, Sarnath Singh, Abraham Mittelman, Raj K. Tiwari. Human endothelial progenitor cells: A new target for anti-vascular therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 793. doi:10.1158/1538-7445.AM2017-793

Abstract The incidence of thyroid cancer in the United States is on a rise with an appreciably high disease recurrence rate of 20-30%. Tumor associated macrophages (TAMs) release cytokines, chemokines and other secretory components like exosomes which aid in thyroid tumor progression and metastasis. In our previous study on thyroid tumor microenvironment, we established that M1 polarized pro-inflammatory macrophages modulate thyroid cancer phenotype. TAMs secretome, consisting of pro-inflammatory cytokines such as TGF-β, IL6, TNF-α, IL-1β, amongst others, induce epithelial to mesechymal transition (EMT) in thyroid cancer cells promoting tumor metastasis and propagation. In our present study, we used an in vitro model system to assess the effects of secretory molecules called exosomes, in the thyroid tumor microenvironment. We used thyroid cancer cell lines: BCPAP (papillary), 8505C (anaplastic) and CGTHW-1 (follicular) to represent the spectrum of clinically observed thyroid cancers. THP-1 monocyte/macrophage cell line was used to denote the inflammatory component. Thyroid cancer cells treated with activated M1 polarized THP-1 macrophage exosomes showed halt in proliferation, transformation to mesenchymal phenotype as well as modulation in expression of EMT markers, such as vimentin and NFk-B, indicative of induction of EMT in thyroid cancer cells. Interestingly, secretory exosomes from anaplastic thyroid cancer cells led to activation of THP-1 monocytes. Since the exosomal cargo is the reflection and fingerprint of its originating parental cells, we profiled exosomal miRNA derived from thyroid cancer cells and THP1 cells. Modulation of various miRNA in follicular and anaplastic thyroid cancer exosomes was observed, when compared to papillary thyroid cancer exosomes. We detected a down regulation of miR-138-5p, miR-146a-5p, miR-26a-5p, miR-26b-5p, miR-34a-5p and miR-31-5p in anaplastic and follicular cancer exosomes, which may play a role in promoting cancer invasion and metastasis. In comparison to papillary thyroid cancer exosomes, an upregulation of miR-214-3p, miR-200a-3p, miR-298, miR-299-3p, and miR-302a-3p was ascertained in follicular thyroid cancer exosomes. Conversely, we noted a down regulation of these miRNAs in anaplastic thyroid cancer exosomes. These distinct miRNA expressions in cancer secretome will provide new insights into the tumor development and dissemination. Our findings suggest an important crosstalk between the secretome of thyroid cancer cells and inflammatory cells in tumor microenvironment, defining thyroid cancer phenotype. These exosomal miRNA serve as early diagnostic markers of thyroid cancer differentiation as well as targets for novel therapies specifically for anaplastic thyroid cancer. Citation Format: Neha Yashpal Tuli, Ameet Kamat, Rachana Maniyar, Ghada Ben Rahoma, Sanjukta Chakraborty, Sarnath Singh, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari. Role of thyroid tumor microenvironment secretome in cancer initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3973. doi:10.1158/1538-7445.AM2017-3973

Abstract Over 200,000 cases of breast cancer are diagnosed within the United States, resulting in over 40,000 deaths annually. Of the many lifestyle habits that are associated with cancer development, various epidemiological observations have provided a strong link between increased alcohol consumption and breast cancer progression. Specifically, this observation was made in women on estrogen replacement therapy thus suggesting a possible synergistic link between alcohol and estrogen. Our laboratory has demonstrated that estrogen increases breast cancer neo-vascularization both in vivo and in vitro. Provided that alcohol and estrogen can potentially synergize to promote breast cancer progression, we hypothesize that this progression is attributed to an increase in tumor neo-vascularization. In the current study, Tg-1 murine mammary carcinoma cells were treated with varying concentrations of alcohol +/- estrogen and analyzed for angiogenesis markers, such as VEGF and eNOS, and the pro-proliferation marker, MEK. At 24 hours post treatment, VEGF and eNOS were upregulated in response to both estrogen and alcohol compared to untreated cells and cells treated with either alcohol or estrogen alone. MEK levels remained constant through the conditions with a marginal upregulation in presence of alcohol and estrogen when compared to untreated cells. To further characterize neo-vascularization, we performed a scratch wound assay using the murine endothelial cell line, SVEC4-10, which was cultured in Tg1-1 conditioned media. The scratch wound assay results indicated enhanced migration of endothelial cells in response to conditioned media from Tg1-1 cells cultured in the presence of both alcohol and estrogen. SVEC4-10, treated with Tg-1-1 conditioned media were processed by Western blot analyses for MEK expression. MEK was upregulated in SVEC4-10 cells cultured in the media obtained from cells cultured with both alcohol and estrogen, indicating enhanced proliferation. Our results indicate a pro-neovasculogenic effect elicited by alcohol and estrogen, as determined by enhanced proliferation and migration of endothelial as well as increased expression of the pro-vasculogenic markers, VEGF and eNOS. Future experiments will be aimed to characterize the expression of other essential pro-vasculogenic markers, such as basic fibroblast growth factor (bFGF) and angiopoietin (Ang). The identification of these cellular and metabolic markers will establish a biochemical link between estrogen activity and signal transduction induced cellular effects. Citation Format: Rachana R. Maniyar, Robert B. Bednarczyk, Ghada M. Ben Rahoma, Neha Tuli, Abraham Mittelman, Raj K. Tiwari, Robert Suriano. Provasculogenic effects of alcohol and estrogen: implications for breast cancer development. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3365.

Tumor angiogenesis refers to the sprouting and cooption of proliferating endothelial cells (EC’s) from adjacent pre-existing host vasculature, and is a key target of cancer therapy. Tumor cells exploit their microenvironment by releasing cytokines and growth factors to promote and support angiogenesis. Within this complex tumor microenvironment, we and others have shown that tumors can recruit bone marrow derived endothelial progenitor cells that differentiate into mature bone marrow-derived endothelial cells and incorporate into sprouting tumor neovessels. Under pathological circumstances, such as breast cancer, a clear association between estrogen receptor expression by EC’s, angiogenic activity, and/or tumor invasiveness has been made. Approximately, 80% of breast cancers are hormone-receptor-positive cancers, thus enabling tamoxifen as the mainstay of breast cancer therapy. The roles of the anti-estrogens fulvestrant (ICI) and the dietary supplement 3, 3’-diindolylmethane (DIM) on cell-cell interaction and angiogenesis have not been fully elucidated. This study is designed to evaluate and compare the effect of these antiestrogens on angiogenesis at the cellular and molecular levels using tube formation of human umbilical vein endothelial cells (HUVEC) as an in vitro angiogenesis model. HUVEC cells were treated with serial dilutions of either DIM or ICI in presence and absence of (3nM) estrogen, and subjected to in vitro tube formation, proliferation, migration, and angiogenesis antibody array assays. We report that HUVEC cells are more sensitive to DIM than ICI. At 25 μM concentration, DIM significantly inhibited the crucial steps of angiogenesis including HUVEC cells proliferation, migration, cytokine release, and tube formation in an estrogen independent manner. On the other hand, at 1 μM concentration, ICI significantly exerted an antiangiogenic effects inhibiting HUVEC cells proliferation, migration, and tube formation, but this effect was totally dependent on the presence of estrogen. These results are validated by our observation that HUVEC cells express estrogen receptor beta (ER-β) and not estrogen receptor alpha (ER-α). A correlative effect between the antiangiogenic activity of DIM and ER-β upregulation was noted. We believe that the anti-estrogenic activity of DIM is mediated through the genomic and non-genomic activity of ER-β in endothelial cells predicting a new target for DIM to manifest its antiangiogenic effect. Citation Format: Ghada M. Ben Rahoma, Neha Y. Tuli, Robert B. Bednarczyk, Rachana R. Maniyar, Abraham Mittelman, Jan Geliebter, Raj Tiwari. Dietary supplement 3, 3’-diindolylmethane (DIM) as an antiangiogenic agent in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3263.

Abstract Thyroid cancer is the most prevalent endocrine malignancy in the United States with an unacceptably high recurrence rate of 20-30%. Tumor associated macrophages (TAMs), one of the most critical component of solid tumor microenvironments, promote cancer initiation, growth, progression, metastasis and angiogenesis. These TAMs release cytokines as well as other secretory components like exosomes in the tumor microenvironment. Previously, we found that M1 polarized TAMs modulate thyroid cancer phenotype by inducing epithelial-mesenchymal transition (EMT), facilitating tissue metastasis and dissemination. In our present study, we used an in vitro model system to assess the crosstalk between the secretory components of macrophages and the epithelial cells in the thyroid tumor microenvironment. We used THP-1 monocyte/macrophage cell line along with thyroid cancer cell lines: consisting of two papillary cancer cell lines (BCPAP and TPC-1), one anaplastic cancer cell line (8505C) and one follicular cancer cell line (CGTHW-1). We observed that activated THP-1 macrophages are polarized towards M1 phenotype, secreting pro-inflammatory cytokines such as TGF-β, IL6, TNF-α, IL-1β, amongst others. These cytokines are responsible for halt in proliferation and change in morphology to mesenchymal phenotype promoting EMT in thyroid cancer cells. Similar pattern in phenotypical changes were noted in thyroid cancer cells treated with activated THP-1 macrophage exosomes. We also observed that EMT markers, such as vimentin and NFκ-B, are modulated in response to activated macrophage secreted exosomes. Moreover, secretory components from anaplastic thyroid cancer cells led to enhanced activation of THP-1 cells. These findings support a mutual cooperation between thyroid cancer cells and inflammatory cells in tumor microenvironment in defining thyroid cancer phenotype. Such correlation can identify early markers and prevent thyroid cancer differentiation, and are putative targets for therapy. Citation Format: Neha Yashpal Tuli, Robert B. Bednarczyk, Ghada M. Ben Rahoma, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari. Thyroid tumor microenvironment: mutual interaction between cancer and inflammatory cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4089.

Abstract Tumor angiogenesis refers to the sprouting and cooption of proliferating endothelial cells (EC’s) from adjacent pre-existing host vasculature, and is a key target of cancer therapy. Tumor cells exploit their microenvironment by releasing cytokines and growth factors to promote and support angiogenesis. Within this complex tumor microenvironment, we and others have shown that tumors can recruit bone marrow derived endothelial progenitor cells that differentiate into mature bone marrow-derived endothelial cells and incorporate into sprouting tumor neovessels. Under pathological circumstances, such as breast cancer, a clear association between estrogen receptor expression by EC’s, angiogenic activity, and/or tumor invasiveness has been made. Approximately, 80% of breast cancers are hormone-receptor-positive cancers, thus enabling tamoxifen as the mainstay of breast cancer therapy. The roles of the anti-estrogens fulvestrant (ICI) and the dietary supplement 3, 3’-diindolylmethane (DIM) on cell-cell interaction and angiogenesis have not been fully elucidated. This study is designed to evaluate and compare the effect of these antiestrogens on angiogenesis at the cellular and molecular levels using tube formation of human umbilical vein endothelial cells (HUVEC) as an in vitro angiogenesis model. HUVEC cells were treated with serial dilutions of either DIM or ICI in presence and absence of (3nM) estrogen, and subjected to in vitro tube formation, proliferation, migration, and angiogenesis antibody array assays. We report that HUVEC cells are more sensitive to DIM than ICI. At 25 μM concentration, DIM significantly inhibited the crucial steps of angiogenesis including HUVEC cells proliferation, migration, cytokine release, and tube formation in an estrogen independent manner. On the other hand, at 1 μM concentration, ICI significantly exerted an antiangiogenic effects inhibiting HUVEC cells proliferation, migration, and tube formation, but this effect was totally dependent on the presence of estrogen. These results are validated by our observation that HUVEC cells express estrogen receptor beta (ER-β) and not estrogen receptor alpha (ER-α). A correlative effect between the antiangiogenic activity of DIM and ER-β upregulation was noted. We believe that the anti-estrogenic activity of DIM is mediated through the genomic and non-genomic activity of ER-β in endothelial cells predicting a new target for DIM to manifest its antiangiogenic effect. Citation Format: Ghada M. Ben Rahoma, Neha Y. Tuli, Robert B. Bednarczyk, Rachana R. Maniyar, Abraham Mittelman, Jan Geliebter, Raj Tiwari. Dietary supplement 3, 3’-diindolylmethane (DIM) as an antiangiogenic agent in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3263.