Summary Background Onychomycosis affects almost 6% of the world population. Topical azoles and systemic antifungal agents are of low efficacy and can have undesirable side effects. An effective, non‐invasive therapy for onychomycosis is an unmet clinical need. Objective Determine the efficacy threshold of non‐thermal atmospheric plasma (NTAP) to treat onychomycosis in an in vitro model. Methods A novel toe/nail‐plate model using cadaver nails and agarose media inoculated with Candida albicans was exposed to a range of NTAP doses. Results Direct exposure of C albicans and Trichophyton mentagrophytes to 12 minutes of NTAP results in complete killing at doses of 39 and 15 kPulses, respectively. Onset of reduced viability of C albicans to NTAP treatment through the nail plate occurs at 64 kPulses with 10× and 100× reduction at 212 and 550 kPulses, respectively. Conclusions NTAP is an effective, non‐invasive therapeutic approach to onychomycosis that should be evaluated in a clinical setting.

Only 2% of thyroid cancer (TC) cases are anaplastic thyroid cancer (ATC), but this rare, undifferentiated cancer with a highly inflamed tumor microenvironment (TME) presents universally as stage IV with a 5-year survival rate of 3%. Due to its loss of differentiation, ATC is resistant to radioiodine therapy, refractory to external beam radiation, and insufficiently treated by chemotherapy or immunotherapy. Berberine (BBR), a natural plant alkaloid, has vast pharmacological activities, including anti-inflammatory and anti-cancer roles. BBR blocks proliferation, induces cell cycle arrest, promotes apoptosis, and hinders invasion and metastasis. BBR may also inhibit cell proliferation through miRNA interactions and regulate the TME through its anti-inflammatory and antioxidant properties. Research in ATC using BBR is limited, specifically investigation on how BBR reprograms the inflammatory TME via altering miRNA cargo of secretory exosomes and polarization of tumor associated macrophages (TAMs). ATC tissues have extensive infiltration of a mixed TAM population. In ATC, infiltration with classically anti-tumorigenic, inflammatory M1 macrophages has a pro-tumorigenic role. We assessed the secretory profile of ATC cells and found a high expression of pro-inflammatory cytokines, such as TNFα, IL-6, IL-1, MCP-1/2, and MIP-1α/β in ATC conditioned media (CM), supporting the existence of an inflammatory TME. As this chronic inflammation is partially mediated by the presence of M1 TAMs in the TME, we activated monocyte cell line U937 with TPA and polarized with IFNγ and LPS into an M1 phenotype. In the presence of BBR at the activation and polarization stages, inflammatory mediators IL-1β, IL-6, IL-6R, CXCL9, MIP-1α/β, and TNFR1/2 were downregulated in the CM compared to M1 macrophages activated and polarized without BBR. BBR also appeared to prevent some level of activation and polarization of these cells into the M1 phenotype altogether. BBR may have an essential role in lessening the burden of inflammation in ATC through remodeling its tumor immune infiltrates, potentially priming it for greater success in combination therapy. Intervention prior to metastasis warrants secretome analysis to define ATC disease progression. Exosomal cargo, particularly miRNA, secreted by ATC cells was evaluated as exosomes have potential as biomarkers to detect ATC earlier improving prognosis, guide treatment, and avoid unneeded surgeries. Distinct miRNA expression from ATC-secreted exosomes compared to papillary thyroid cancer (PTC)-secreted exosomes revealed six miRNAs specifically downregulated in ATC-secreted exosomes that are known tumor suppressors. We are currently evaluating how BBR alters the miRNA profile of ATC- and PTC-secreted exosomes. The ability of BBR to alleviate inflammation in ATC and remodel its TME releases its chronic hold on ATC progression and may prime the tumor for increased responsiveness to therapy. Citation Format: Tara Jarboe, Kaci Kopec, Nicole R. DeSouza, Sarnath Singh, Augustine Moscatello, Jan Geliebter, Xiu-Min Li, Raj K. Tiwari. Berberine eases inflammation in the anaplastic thyroid cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 243.

Anaplastic thyroid cancer's (ATC) undifferentiated, inflammatory nature makes it one of the most aggressive cancers, with a five-year survival rate of only 4% when metastatic. ATC is a rare cancer that is refractory to conventional therapeutic modalities. Without high expression of targetable genetic lesions, small molecule inhibitors alone have been insufficient in trials. Treatment of ATC could benefit from a holistic approach that reprograms the inflammatory tumor microenvironment (TME). Berberine (BBR), a natural plant-derived alkaloid used extensively in Traditional Chinese Medicine, is a compound shown to exhibit anti-microbial, anti-inflammatory, and anti-cancer properties. Our work aims to exploit the anti-inflammatory activity of BBR in ATC. As the inflammatory status of ATC defines its intractable nature, remodeling its secretome, including its cytokine and chemokine profile and its exosomal cargo, in the TME fundamentally targets ATC's progressive determinant - inflammation. Exosomes, membrane-bound extracellular vesicles, are secreted by cells in the TME, including activated tumor-associated macrophages and ATC cells. Exosomal cargo primarily consists of miRNAs. We observed distinct miRNA expression from ATC-secreted exosomes when compared to papillary thyroid cancer (PTC)-secreted exosomes. Comparative analysis revealed ten miRNAs specifically downregulated in exosomes secreted from anaplastic-like 8505C compared to papillary BCPAP, including: hsa-miR-26b-5p, hsa-miR-125b-5p, hsamiR-138-5p, hsa-miR-148a-5p, hsamiR-152-5p, hsa-miR-191-5p, hsa-miR-9-5p, hsa-miR-21-5p, hsa-miR-134-5p, and hsa-miR-379-5p. The first six miRNAs listed are tumor suppressors, and as such, their downregulation may be contributory to the metastatic propensity and aggressiveness of ATC and its refractory nature towards conventional treatments. In activated macrophage-derived exosomes, miR-21-5p and miR-138-5p were also upregulated. ATC-secreted exosomes activate macrophages, subsequently priming the TME to be pro-tumorigenic and pro-inflammatory. This reciprocal interaction between inflammatory macrophages and ATC cells mediated by exosomal miRNAs defines its metastatic and inflammatory phenotype. We also observed that BBR significantly downregulates phosphorylation of MEK, ERK, and ribosomal protein S6 in proliferating ATC cells with as low as 10 μM BBR treatment. These are important downstream regulators of the pro-proliferative, pro-survival, and metabolic MAPK and PI3K-PTEN-AKT signaling pathways. Overall, the ability for BBR to alleviate the pro-inflammatory phenotype of ATC and remodel its immune environment, while simultaneously depressing overactive signaling in these cell survival pathways, may mark it as an important agent to make ATC amenable to combination therapy with small molecule inhibitors (MEKi) or other immunotherapeutics. Citation Format: Tara Jarboe, Nicole DeSouza, Sarnath Singh, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari, Xiu-Min Li. Berberine-mediated reprogramming of the inflammatory environment in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 317.

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

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