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Supplementary Movie 1 from An Ets2-Driven Transcriptional Program in Tumor-Associated Macrophages Promotes Tumor Metastasis

Supplementary Figure 4 from Fibroblasts Isolated from Common Sites of Breast Cancer Metastasis Enhance Cancer Cell Growth Rates and Invasiveness in an Interleukin-6–Dependent Manner

Supplementary Figure Legends 1-2 from Direct Evidence for Epithelial-Mesenchymal Transitions in Breast Cancer

Supplementary Figures 1-2 from Direct Evidence for Epithelial-Mesenchymal Transitions in Breast Cancer

Common sites of breast cancer metastasis include the lung, liver, and bone, and of these secondary metastatic sites, estrogen receptor α (ERα)–positive breast cancer often favors bone. Within secondary organs, cancer cells would predictably encounter tissue-specific fibroblasts or their soluble factors, yet our understanding of how tissue-specific fibroblasts directly affect cancer cell growth rates and survival remains largely unknown. Therefore, we tested the hypothesis that mesenchymal fibroblasts isolated from common sites of breast cancer metastasis provide a more favorable microenvironment with respect to tumor growth rates. We found a direct correlation between the ability of breast, lung, and bone fibroblasts to enhance ERα-positive breast cancer cell growth and the level of soluble interleukin-6 (IL-6) produced by each organ-specific fibroblast, and fibroblast-mediated growth enhancement was inhibited by the removal or inhibition of IL-6. Interestingly, mice coinjected with MCF-7 breast tumor cells and senescent skin fibroblasts, which secrete IL-6, developed tumors, whereas mice coinjected with presenescent skin fibroblasts that produce little to no IL-6 failed to form xenograft tumors. We subsequently determined that IL-6 promoted growth and invasion of breast cancer cells through signal transducer and activator of transcription 3–dependent up-regulation of Notch-3, Jagged-1, and carbonic anhydrase IX. These data suggest that tissue-specific fibroblasts and the factors they produce can promote breast cancer disease progression and may represent attractive targets for development of new therapeutics. [Cancer Res 2008;68(21):9087–95]

Supplementary Methods, Figures 1-2 from Zoledronic Acid Reduces Bone Loss and Tumor Growth in an Orthotopic Xenograft Model of Osteolytic Oral Squamous Cell Carcinoma

We developed stromal- and epithelial-specific cre-transgenic mice to directly visualize epithelial-mesenchymal transition (EMT) during cancer progression in vivo. Using three different oncogene-driven mouse mammary tumor models and cell-fate mapping strategies, we show in vivo evidence for the existence of EMT in breast cancer and show that myc can specifically elicit this process. Hierarchical cluster analysis of genome-wide loss of heterozygosity reveals that the incidence of EMT in invasive human breast carcinomas is rare, but when it occurs it is associated with the amplification of MYC. These data provide the first direct evidence for EMT in breast cancer and suggest that its development is favored by myc-initiated events. [Cancer Res 2008;68(3):937–45]

Supplementary Movie 3 from An Ets2-Driven Transcriptional Program in Tumor-Associated Macrophages Promotes Tumor Metastasis

Supplementary Figure and Movie Legends from An Ets2-Driven Transcriptional Program in Tumor-Associated Macrophages Promotes Tumor Metastasis

Supplementary Figures 1-7 from An Ets2-Driven Transcriptional Program in Tumor-Associated Macrophages Promotes Tumor Metastasis

Adult T-cell /lymphomaleukemia (ATLL) is caused by human T-cell lymphotropic virus type 1 (HTLV-1). Approximately 80% of ATLL patients develop humoral hypercalcemia of malignancy (HHM), a life-threatening complication leading to a poor prognosis. Parathyroid hormone–related protein (PTHrP) and macrophage inflammatory protein-1α (MIP-1α) are important factors in the pathogenesis of HHM in ATLL and the expression of PTHrP can be activated by nuclear factor κB (NF-κB). NF-κB is constitutively activated in ATLL cells and is essential for leukemogenesis including transformation of lymphocytes infected by HTLV-1. Our goal was to evaluate the effects of NF-κB disruption by a proteasomal inhibitor (PS-341) and osteoclastic inhibition by zoledronic acid (Zol) on the development of ATLL and HHM using a novel bioluminescent mouse model. We found that PS-341 decreased cell viability, increased apoptosis, and down-regulated PTHrP expression in ATLL cells in vitro. To investigate the in vivo efficacy, nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice were xenografted with ATLL cells and treated with vehicle control, PS-341, Zol, or a combination of PS-341 and Zol. Bioluminescent imaging and tumor cell count showed a significant reduction in tumor burden in mice from all treatment groups. All treatments also significantly reduced the plasma calcium concentrations. Zol treatment increased trabecular bone volume and decreased osteoclast parameters. PS-341 reduced PTHrP and MIP-1α expression in tumor cells in vivo. Our results indicate that both PS-341 and Zol are effective treatments for ATLL and HHM, which are refractory to conventional therapy. [Cancer Res 2007;67(24):11859–66]

Supplementary Figure 1 from Fibroblasts Isolated from Common Sites of Breast Cancer Metastasis Enhance Cancer Cell Growth Rates and Invasiveness in an Interleukin-6–Dependent Manner

Supplementary Figure 2 from Fibroblasts Isolated from Common Sites of Breast Cancer Metastasis Enhance Cancer Cell Growth Rates and Invasiveness in an Interleukin-6–Dependent Manner

Supplementary Table 1 from A Novel Bioluminescent Mouse Model and Effective Therapy for Adult T-Cell Leukemia/Lymphoma

Supplementary Figure 3 from Fibroblasts Isolated from Common Sites of Breast Cancer Metastasis Enhance Cancer Cell Growth Rates and Invasiveness in an Interleukin-6–Dependent Manner

The addition of flavorings to food and beverages provides practically unlimited opportunities for innovation, for maintaining and enhancing palatability, and is one essential element of a stable supply of nutritious consumer products. A safety evaluation by the Flavor and Extract Manufacturers Association (FEMA) Expert Panel provides a pathway for flavor producers and users to achieve regulatory authority to use for substances under the conditions of intended use as a flavoring. This chapter describes the factors that contribute to the safety assessment process that is conducted by the Expert Panel, and provides examples of specific flavorings and types of flavorings that are considered. The chapter also describes future issues and opportunities likely to be encountered within the context of the FEMA generally recognized as safe assessment of flavorings.

The Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) applies its procedure for the safety evaluation of natural flavor complexes (NFCs) to re-evaluate the safety of Asafetida Oil (Ferula assa-foetida L.) FEMA 2108, Garlic Oil (Allium sativum L.) FEMA 2503 and Onion Oil (Allium cepa L.) FEMA 2817 for use as flavoring in food. This safety evaluation is part of a series of evaluations of NFCs for use as flavoring ingredients conducted by the Expert Panel that applies a scientific procedure published in 2005 and updated in 2018. Using a group approach that relies on a complete chemical characterization of the NFC intended for commerce, the constituents of each NFC are organized into well-defined congeneric groups and the estimated intake of each constituent congeneric group is evaluated using the conservative threshold of toxicological concern (TTC) concept. Data on the metabolism, genotoxic potential and toxicology for each constituent congeneric group are reviewed as well as studies on each NFC. Based on the safety evaluation, Asafetida Oil (Ferula assa-foetida L.), Garlic Oil (Allium sativum L.) and Onion Oil (Allium cepa L.) were affirmed as generally recognized as safe (GRASa) under their conditions of intended use as flavor ingredients.

Summary Objective ‘Carbon stress’ is a newly found mechanism that links obesity and dysregulated metabolism. It is defined as the cellular accumulation of metabolites during obesity post-translationally modifying metabolic proteins and decreasing their enzymatic activity. The objective of this study was to investigate if ‘carbon stress’ also occurs in cartilage and contributes to obesity associated OA development. Methods We histologically evaluated for OA pathology in wild-type (WT) and hyperphagic mice (Pomc-neuron specific enhancer one deficient, PomcΔ1) that were subjected to standard chow (Chow, n = 6 for both genotypes) or high-fat feeding (HFD, n = 7 for both genotypes). Joints were stained and quantified for ‘carbon stress’ markers, including succinyl-lysine (SCK), malonyl-lysine (MAK), and acetyl-lysine (ACK). Lastly, we used a mouse model with deletion of Sirt5 (n = 7), which is an enzyme that removes SCK and MAK, to test if changing the abundance of ‘carbon stress’ would affect OA pathogenesis. Results Both HFD and Pomc deficiency associated obesity induced cartilage degeneration as well as greater abundance of SCK and MAK in the cartilage. PomcΔ1-HFD mice did not have exacerbated OA pathology as compared to PomcΔ1-Chow mice. ACK was mildly increased in the obese groups comparing to WT-Chow. Sirt5−/− mice developed early-OA like phenotype at 40 weeks of age as characterized by cartilage fibrillation and more hypertrophic chondrocytes. Cartilage from Sirt5−/− mice also had increased SCK and MAK, while ACK remained unchanged comparing to WT mice. Conclusion Our data suggests that carbon stress also occurs in cartilage tissue during obesity and can potentially contribute to obesity-associated OA.

The most common target organ for toxicity in the endocrine system is the adrenal gland, and its function is dependent upon the hypothalamus and pituitary gland. Histopathologic examination of the adrenal glands and pituitary gland is routinely performed in toxicity studies. However, the function of the adrenal gland is not routinely assessed in toxicity studies. Assessment of adrenal cortical function may be necessary to determine whether a histopathologic finding in the adrenal cortex results in a functional effect in the test species. As juvenile toxicity studies are more commonly performed in support of pediatric indications for pharmaceuticals, it is important to establish historical control data for adrenal gland function. In this study, adrenal cortical function was assessed in control neonatal and weanling beagle dogs as part of an ongoing juvenile toxicology program. Measurements of serum adrenocorticotropic hormone (ACTH), cortisol prior to and following administration of exogenous ACTH, and aldosterone were conducted beginning at 2 weeks of age continuing through 26 weeks of age. Serum electrolyte concentrations were determined at 4, 13, and 26 weeks of age. Dogs as young as 2 weeks of age synthesize and secrete adrenal cortical hormones and exhibit a functional hypothalamic pituitary adrenal axis.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions) Project ( www.toxpath.org/inhand.asp ) is a joint initiative of the societies of toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying lesions observed in most tissues and organs from the dog used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet ( http://www.goreni.org/ ). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions, lesions induced by exposure to test materials, and relevant infectious and parasitic lesions. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.

Limitations in workforce size and access to resources remain perennial challenges to greater progress in academic veterinary medicine and engagement between human and veterinary medicine (One Health). Ongoing resource constraints occur in part due to limited public understanding of the role veterinarians play in improving human health. One Health interactions, particularly through interdisciplinary collaborations in biomedical research, present constructive opportunities to inform resource policies and advance health care. To this end, inter-institutional partnerships between individual veterinary medical education programs (VMEPs) and several National Institutes of Health (NIH) intramural research programs have created synergies beyond those provided by individual programs. In the NIH Comparative Biomedical Scientist Training Program (CBSTP), interdisciplinary cross-training of veterinarians consisting of specialty veterinary medicine coupled with training in human disease research leading to a PhD, occurs collaboratively on both VMEP and NIH campuses. Pre-doctoral veterinary student research opportunities have also been made available. Through the CBSTP, NIH investigators and national biomedical science policy makers gain access to veterinary perspective and expertise, while veterinarians obtain additional opportunities for NIH-funded research training. CBSTP Fellows serve as de facto ambassadors enhancing visibility for the profession while in residence at NIH, and subsequently through a variety of university, industry, and government research appointments, as graduates. Thus, the CBSTP represents an inter-institutional opportunity that not only addresses critical needs for veterinarian-scientists in the biomedical workforce, but also simultaneously exposes national policy makers to veterinarian-scientists’ specialized training, leading to more effective realization of One Health goals to benefit human and animal health.

Background Osteoblastic bone metastasis represents the most common complication in men with prostate cancer (PCa). During progression and bone metastasis, PCa cells acquire properties similar to bone cells in a phenomenon called osteomimicry, which promotes their ability to metastasize, proliferate, and survive in the bone microenvironment. The mechanism of osteomimicry resulting in osteoblastic bone metastasis is unclear. Methods We developed and characterized a novel canine prostatic cancer cell line (LuMa) that will be useful to investigate the relationship between osteoblastic bone metastasis and osteomimicry in PCa. The LuMa cell line was established from a primary prostate carcinoma of a 13-year old mixed breed castrated male dog. Cell proliferation and gene expression of LuMa were measured and compared to three other canine prostatic cancer cell lines (Probasco, Ace-1, and Leo) in vitro. The effect of LuMa cells on calvaria and murine preosteoblastic (MC3T3-E1) cells was measured by quantitative reverse-transcription polymerase chain reaction and alkaline phosphatase assay. LuMa cells were transduced with luciferase for monitoring in vivo tumor growth and metastasis using different inoculation routes (subcutaneous, intratibial [IT], and intracardiac [IC]). Xenograft tumors and metastases were evaluated using radiography and histopathology. Results After left ventricular injection, LuMa cells metastasized to bone, brain, and adrenal glands. IT injections induced tumors with intramedullary new bone formation. LuMa cells had the highest messenger RNA levels of osteomimicry genes (RUNX2, RANKL, and Osteopontin [OPN]), CD44, E-cadherin, and MYOF compared to Ace-1, Probasco, and Leo cells. LuMa cells induced growth in calvaria defects and modulated gene expression in MC3T3-E1 cells. Conclusions LuMa is a novel canine PCa cell line with osteomimicry and stemness properties. LuMa cells induced osteoblastic bone formation in vitro and in vivo. LuMa PCa cells will serve as an excellent model for studying the mechanisms of osteomimicry and osteoblastic bone and brain metastasis in prostate cancer.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavoring ingredients in food. In this publication, tenth in the series, NFCs containing a high percentage of at least one naturally occurring allylalkoxybenzene constituent with a suspected concern for genotoxicity and/or carcinogenicity are evaluated. In a related paper, ninth in the series, NFCs containing anethole and/or eugenol and relatively low percentages of these allylalkoxybenzenes are evaluated. The Panel applies the threshold of toxicological concern (TTC) concept and evaluates relevant toxicology data on the NFCs and their respective constituent congeneric groups. For NFCs containing allylalkoxybenzene constituent(s), the estimated intake of the constituent is compared to the TTC for compounds with structural alerts for genotoxicity and when exceeded, a margin of exposure (MOE) is calculated. BMDL10 values are derived from benchmark dose analyses using Bayesian model averaging for safrole, estragole and methyl eugenol using EPA's BMDS software version 3.2. BMDL10 values for myristicin, elemicin and parsley apiole were estimated by read-across using relative potency factors. Margins of safety for each constituent congeneric group and MOEs for each allylalkoxybenzene constituent for each NFC were determined that indicate no safety concern. The scope of the safety evaluation contained herein does not include added use in dietary supplements or any products other than food. Ten NFCs, derived from basil, estragon (tarragon), mace, nutmeg, parsley and Canadian snakeroot were determined or affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavor ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

The FEMA Expert Panel program to re-evaluate the safety of natural flavor complexes (NFCs) used as flavoring ingredients in food has resulted in the publication of an updated constituent-based procedure as well as publications on the safety evaluation of many botanical-derived NFCs. This publication, ninth in the series and related to the ninth publication, describes the affirmation of the generally recognized as safe (GRAS) status for NFCs with propenylhydroxybenzene and allylalkoxybenzene constituents under their conditions of intended use as flavoring ingredients added to food. The Panel's procedure applies the threshold of toxicological concern (TTC) concept and evaluates relevant data on absorption, metabolism, genotoxic potential and toxicology for the NFCs themselves and their respective constituent congeneric groups. For NFCs containing allylalkoxybenzene constituent(s) with suspected genotoxic potential, the estimated intake of the individual constituent is compared to the TTC for compounds with structural alerts for genotoxicity and if exceeded, a margin of exposure is calculated using BMDL10 values derived from benchmark dose analyses using Bayesian model averaging, as presented in the tenth article of the series. Safety evaluations for NFCs derived from allspice, anise seed, star anise, sweet fennel seed and pimento leaves were conducted and their GRAS status was affirmed for use as flavoring ingredients. The scope of the safety evaluation contained herein does not include added use in dietary supplements or any products other than food.

Simple Summary Adult T-cell leukemia (ATL) Leukemia is an aggressive, peripheral blood (T-cell) neoplasm associated with human T-cell leukemia virus type 1 (HTLV-1) infection. Recent studies have implicated dysregulated histone deacetylases in ATL pathogenesis. ATL modulates the bone microenvironment of patients and activates osteoclasts (bone resorbing cells) that cause severe bone loss. The objective of this study was to assess the individual and dual effects of AR-42 (HDACi) and zoledronic acid (Zol) on the growth of ATL cells in vitro and in vivo. AR-42 and Zol reduced the viability of ATL cells in vitro. Additionally, Zol and Zol/AR-42 decreased ATL tumor growth and halted osteolysis in bone tumor xenografts in immunodeficient mice in vivo. Our study suggests that dual targeting of ATL cells (using HDACi) and bone osteoclasts (using bisphosphonates) may be exploited as a valuable approach to reduce bone tumor burden and improve the life quality of ATL patients. Abstract Adult T-cell leukemia/lymphoma (ATL) is an intractable disease affecting nearly 4% of Human T-cell Leukemia Virus Type 1 (HTLV-1) carriers. Acute ATL has a unique interaction with bone characterized by aggressive bone invasion, osteolytic metastasis, and hypercalcemia. We hypothesized that dual tumor and bone-targeted therapies would decrease tumor burden in bone, the incidence of metastasis, and ATL-associated osteolysis. Our goal was to evaluate dual targeting of both ATL bone tumors and the bone microenvironment using an anti-tumor HDACi (AR-42) and an osteoclast inhibitor (zoledronic acid, Zol), alone and in combination. Our results showed that AR-42, Zol, and AR-42/Zol significantly decreased the viability of multiple ATL cancer cell lines in vitro. Zol and AR-42/Zol decreased tumor growth in vivo. Zol ± AR-42 significantly decreased ATL-associated bone resorption and promoted new bone formation. AR-42-treated ATL cells had increased mRNA levels of PTHrP, ENPP2 (autotaxin) and MIP-1α, and TAX viral gene expression. AR-42 alone had no significant effect on tumor growth or osteolysis in mice. These findings indicate that Zol adjuvant therapy has the potential to reduce growth of ATL in bone and its associated osteolysis.

Parathyroid hormone-related protein (PTHrP) and macrophage inflammatory protein-1α (MIP-1α) are important factors that increase bone resorption and hypercalcemia in Adult T-cell Leukemia (ATL). We investigated the role of PTHrP and MIP-1α in the development of local osteolytic lesions in T-cell leukemia through overexpression in Jurkat T-cells. Injections of Jurkat-PTHrP and Jurkat-MIP-1α into the tibia and the left ventricle of NSG mice were performed to evaluate tumor growth and metastasis in vivo. Jurkat-pcDNA tibial neoplasms grew at a significantly greater rate and total tibial tumor burden was significantly greater than Jurkat-PTHrP neoplasms. Despite the lower tibial tumor burden, Jurkat-PTHrP bone neoplasms had significantly greater osteolysis than Jurkat-pcDNA and Jurkat-MIP-1α neoplasms. Jurkat-PTHrP and Jurkat-pcDNA cells preferentially metastasized to bone following intracardiac injection, though the overall metastatic burden was lower in Jurkat-PTHrP mice. These findings demonstrate that PTHrP induced pathologic osteolysis in T-cell leukemia but did not increase the incidence of skeletal metastasis.

Human Dickkopf-1 (Dkk-1) upregulates a noncanonical Wnt/JNK pathway, resulting in osteoclast stimulation, cell proliferation, and epithelial-to-mesenchymal transition (EMT) of cancer cells. Ace-1-Dkk-1, a canine prostate cancer (PCa) cell line overexpressing Dkk-1, was used to investigate Wnt signaling pathways in PCa tumor growth. SP600125, a JNK inhibitor, was used to examine whether it would decrease tumor growth and bone tumor phenotype in canine PCa cells in vitro and in vivo. Ace-1-VectorYFP-Luc and Ace-1-Dkk-1YFP-Luc cells were transplanted subcutaneously, while Ace-1-Dkk-1YFP-Luc was transplanted intratibially into nude mice. The effects of Dkk-1 and SP600125 on cell proliferation, in vivo tumor growth, and bone tumor phenotype were investigated. The mRNA expression levels of Wnt/JNK-related genes were measured using RT-qPCR. Dkk-1 significantly increased the mRNA expression of Wnt/JNK-signaling-related genes. SP600125 significantly upregulated the mRNA expression of osteoblast differentiation genes and downregulated osteoclastic-bone-lysis-related genes in vitro. SP600125 significantly decreased tumor volume and induced spindle-shaped tumor cells in vivo. Mice bearing intratibial tumors had increased radiographic density of the intramedullary new bone, large foci of osteolysis, and increased cortical lysis with abundant periosteal new bone formation. Finally, SP600125 has the potential to serve as an alternative adjuvant therapy in some early-stage PCa patients, especially those with high Dkk-1 expression.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients, mostly consisting of a variety of essential oils and botanical extracts. This publication, seventh in the series, re-evaluates NFCs with constituent profiles dominated by phenolic derivatives including carvacrol, thymol and related compounds using a constituent-based procedure first published in 2005 and updated in 2018. The procedure is based on the chemical characterization of each NFC as intended for commerce and the estimated intake of the constituent congeneric groups. The procedure applies the threshold of toxicological concern (TTC) concept and evaluates relevant data on absorption, metabolism, genotoxic potential and toxicology of the constituent congeneric groups and the NFC under evaluation. Herein, the FEMA Expert Panel affirmed the generally recognized as safe (GRAS) status of seven phenolic derivative-based NFCs, Origanum Oil (Extractive) (FEMA 2828), Savory Summer Oil (FEMA 3013), Savory Summer Oleoresin (FEMA 3014), Savory Winter Oil (FEMA 3016), Savory Winter Oleoresin (FEMA 3017), Thyme Oil (FEMA 3064) and Thyme White Oil (FEMA 3065) under their conditions of intended use as flavor ingredients.