Justin J. Jeffery

COX-2 modulates mammary tumor progression in response to collagen density

BackgroundHigh breast density is linked to an increased risk of breast cancer, and correlates with changes in collagen. In a mouse model of mammary carcinoma in the context of increased collagen deposition, the MMTV-PyMT/Col1a1tm1jae, there is accelerated mammary tumor formation and progression. Previous gene expression analysis suggests that increased collagen density elevates expression of PTGS2 (prostaglandin-endoperoxide synthase 2), the gene for cyclooxygenase-2 (COX-2).MethodsTo understand the role of COX-2 in tumor progression within a collagen-dense microenvironment, we treated MMTV-PyMT or MMTV-PyMT/Col1a1tm1jae tumors prior to and after tumor formation. Animals received treatment with celecoxib, a specific COX-2 inhibitor, or placebo. Mammary tumors were examined for COX-2, inflammatory and stromal cell components, and collagen deposition through immunohistochemical analysis, immunofluorescence, multiplex cytokine ELISA and tissue imaging techniques.ResultsPyMT/Col1a1tm1jae tumors were larger, more proliferative, and expressed higher levels of COX-2 and PGE2 than PyMT tumors in wild type (WT) mice. Treatment with celecoxib significantly decreased the induced tumor size and metastasis of the PyMT/Col1a1 tumors, such that their size was not different from the smaller PyMT tumors. Celecoxib had minimal effect on the PyMT tumors. Celecoxib decreased expression levels of COX-2, PGE2, and Ki-67. Several cytokines were over-expressed in PyMT/Col1a1 compared to PyMT, and celecoxib treatment prevented their over-expression. Furthermore, macrophage and neutrophil recruitment were enhanced in PyMT/Col1a1 tumors, and this effect was inhibited by celecoxib. Notably, COX-2 inhibition reduced overall collagen deposition. Finally, when celecoxib was used prior to tumor formation, PyMT/Col1a1 tumors were fewer and smaller than in untreated animals.ConclusionThese findings suggest that COX-2 has a direct role in modulating tumor progression in tumors arising within collagen-dense microenvironments, and suggest that COX-2 may be an effective therapeutic target for women with dense breast tissue and early-stage breast cancer.

Multi-Modal Imaging with a Toolbox of Influenza A Reporter Viruses

Reporter viruses are useful probes for studying multiple stages of the viral life cycle. Here we describe an expanded toolbox of fluorescent and bioluminescent influenza A reporter viruses. The enhanced utility of these tools enabled kinetic studies of viral attachment, infection, and co-infection. Multi-modal bioluminescence and positron emission tomography–computed tomography (PET/CT) imaging of infected animals revealed that antiviral treatment reduced viral load, dissemination, and inflammation. These new technologies and applications will dramatically accelerate in vitro and in vivo influenza virus studies.

Neutrophils drive accelerated tumor progression in the collagen-dense mammary tumor microenvironment

BackgroundHigh mammographic density has been correlated with a 4-fold to 6-fold increased risk of developing breast cancer, and is associated with increased stromal deposition of extracellular matrix proteins, including collagen I. The molecular and cellular mechanisms responsible for high breast tissue density are not completely understood.MethodsWe previously described accelerated tumor formation and metastases in a transgenic mouse model of collagen-dense mammary tumors (type I collagen-α1 (Col1α1)tm1Jae and mouse mammary tumor virus - polyoma virus middle T antigen (MMTV-PyVT)) compared to wild-type mice. Using ELISA cytokine arrays and multi-color flow cytometry analysis, we studied cytokine signals and the non-malignant, immune cells in the collagen-dense tumor microenvironment that may promote accelerated tumor progression and metastasis.ResultsCollagen-dense tumors did not show any alteration in immune cell populations at late stages. The cytokine signals in the mammary tumor microenvironment were clearly different between wild-type and collagen-dense tumors. Cytokines associated with neutrophil signaling, such as granulocyte monocyte-colony stimulated factor (GM-CSF), were increased in collagen-dense tumors. Depleting neutrophils with anti-Ly6G (1A8) significantly reduced the number of tumors, and blocked metastasis in over 80 % of mice with collagen-dense tumors, but did not impact tumor growth or metastasis in wild-type mice.ConclusionOur study suggests that tumor progression in a collagen-dense microenvironment is mechanistically different, with pro-tumor neutrophils, compared to a non-dense microenvironment.

Synthetic transcription elongation factors license transcription across repressive chromatin

Chemical control of transcription Friedreichs ataxia, a devastating neurodegenerative disease with no effective therapy, is caused by an expansion of intronic repeats and hence a reduced expression of the FXN gene. Erwin et al. synthesized a molecule that specifically targets the expanded repressive repeats. This molecule thereby licenses productive transcription elongation and restores FXN expression to normal levels. In the future, similar interventions may be effective in a diverse array of diseases caused by unstable expansions in microsatellite repeats. Science, this issue p. 1617 A synthetic molecule can surmount barriers to transcription elongation at repressive microsatellite repeats that cause Friedreich’s ataxia. The release of paused RNA polymerase II into productive elongation is highly regulated, especially at genes that affect human development and disease. To exert control over this rate-limiting step, we designed sequence-specific synthetic transcription elongation factors (Syn-TEFs). These molecules are composed of programmable DNA-binding ligands flexibly tethered to a small molecule that engages the transcription elongation machinery. By limiting activity to targeted loci, Syn-TEFs convert constituent modules from broad-spectrum inhibitors of transcription into gene-specific stimulators. Here we present Syn-TEF1, a molecule that actively enables transcription across repressive GAA repeats that silence frataxin expression in Friedreich’s ataxia, a terminal neurodegenerative disease with no effective therapy. The modular design of Syn-TEF1 defines a general framework for developing a class of molecules that license transcription elongation at targeted genomic loci.

Level-specific amputations and resulting regenerative outcomes in the mouse distal phalanx

Mouse digit tip regeneration involves an intricate coordinated regrowth of the terminal phalanx, nail, dermis and epidermis. During this time, regenerating digits undergo wound healing, blastema formation, and differentiation. However, the regenerative response of the digit is dependent on the level of the amputation. Amputation of <30% of the distal phalanx (P3), with part of the base nail remaining, results in extensive digit regeneration. In contrast, >60% P3 removal results in no regeneration. This level‐dependent regenerative ability of the mouse digit provides a comparative model between regeneration and non‐regeneration that may enable identification of specific factors critical to regeneration. Although the ability to create regenerating and non‐regenerating conditions has been well established, the regenerative response between these regions (“intermediate” zone) has received less scrutiny, and may add insight to the regenerative processes, including the degree of histolysis, and the level of blastema formation. The objective of this study is then to compare the regeneration capacity between amputation levels within the regenerating (<30%), intermediate (40–59%), and non‐regenerating (>60%) regions. Results indicated that regenerative and intermediate amputations led to significant histolysis and blastema formation of the distal phalanx 14 days post‐amputation. Unlike the regenerating digits, intermediate amputations led to incomplete regeneration whereby regrowth of the digits were not to the levels of the intact or regenerating digits. Non‐regenerating amputations did not exhibit significant histolysis or blastema formation. Remarkably, the histolytic process resulted in day 14 P3 lengths that were similar regardless of the initial amputation over 19%. The differences in histolysis, blastema formation and injury outcomes were also marked by changes in the number of proliferating cells and osteoclasts. Altogether, these results indicate that although intermediate amputations result in histolysis and blastema formation similar to regenerating digits, the resulting cellular composition of the blastema differs, contributing to incomplete regeneration.

Absence of the Vitamin D Receptor Inhibits Atherosclerotic Plaque Calcification in Female Hypercholesterolemic Mice.

Epidemiological and clinical data suggest adverse cardiovascular outcomes with respect to vitamin D deficiency. Here, we explored the effects of vitamin D in atherosclerotic plaque calcification in vivo by utilizing vitamin D receptor (Vdr)‐deficient mice in an Apoe−/− background. Animals were fed a high‐fat diet (HFD) for either 12 or 18 weeks and then examined for atherosclerotic plaque development. In order to prevent calcium deficiency, Vdr−/− and Apoe−/−;Vdr−/− animals were fed a high‐calcium rescue diet prior to initiation of the HFD feeding and supplemented with high‐calcium water during HFD feeding. Although calcium supplementation improved bone mass in Vdr−/− and Apoe−/−;Vdr−/− mice, neither strain was fully rescued. Systemic inflammatory responses observed in the absence of VDR were exaggerated in Apoe−/− mice. Whereas, hyperlipidemic profiles seen in Apoe−/− mice were ameliorated in the absence of VDR. Micro‐computed tomography (µCT) analysis revealed that six out of eight Apoe−/− animals developed atherosclerotic plaque calcification following 12 weeks of HFD feeding and 100% of the mice developed plaque calcification after 18 weeks. In contrast, although atherosclerotic lesions were evident in Apoe−/−;Vdr−/− mice at 12 and 18 weeks of HFD challenge, none of these animals developed plaque calcification at either time point. The active vitamin D hormone, 1,25(OH)2D3 likely increased calcification in aortic smooth muscle cells perhaps by directly modulating expression of Alpl, Rankl, and Opg. Our data suggest that the absence of VDR inhibits atherosclerotic plaque calcification in hypercholesterolemic Apoe−/− mice, providing additional insight into the role of vitamin D in atherosclerotic plaque calcification. J. Cell. Biochem. 118: 1050–1064, 2017.

Radiomanganese PET Detects Changes in Functional β-Cell Mass in Mouse Models of Diabetes

The noninvasive measurement of functional β-cell mass would be clinically valuable for monitoring the progression of type 1 and type 2 diabetes as well as the viability of transplanted insulin-producing cells. Although previous work using MRI has shown promise for functional β-cell mass determination through voltage-dependent Ca2+ channel (VDCC)–mediated internalization of Mn2+, the clinical utility of this technique is limited by the cytotoxic levels of the Mn2+ contrast agent. Here, we show that positron emission tomography (PET) is advantageous for determining functional β-cell mass using 52Mn2+ (t1/2: 5.6 days). We investigated the whole-body distribution of 52Mn2+ in healthy adult mice by dynamic and static PET imaging. Pancreatic VDCC uptake of 52Mn2+ was successfully manipulated pharmacologically in vitro and in vivo using glucose, nifedipine (VDCC blocker), the sulfonylureas tolbutamide and glibenclamide (KATP channel blockers), and diazoxide (KATP channel opener). In a mouse model of streptozotocin-induced type 1 diabetes, 52Mn2+ uptake in the pancreas was distinguished from healthy controls in parallel with classic histological quantification of β-cell mass from pancreatic sections. 52Mn2+-PET also reported the expected increase in functional β-cell mass in the ob/ob model of pretype 2 diabetes, a result corroborated by histological β-cell mass measurements and live-cell imaging of β-cell Ca2+ oscillations. These results indicate that 52Mn2+-PET is a sensitive new tool for the noninvasive assessment of functional β-cell mass.

Sensitivity and Isoform Specificity of18F-Fluorofuranylnorprogesterone for Measuring Progesterone Receptor Protein Response to Estradiol Challenge in Breast Cancer

The purpose of this study was to evaluate the ability of 21-18F-fluoro-16α,17α-[(R)-(1′-α-furylmethylidene)dioxy]-19-norpregn-4-ene-3,20-dione (18F-FFNP) to measure alterations in progesterone receptor (PR) protein level and isoform expression in response to 17β-estradiol (E2) challenge. Methods: T47D human breast cancer cells and female mice bearing T47D tumor xenografts were treated with E2 to increase PR expression. 18F-FFNP uptake was measured using cell uptake and tissue biodistribution assays. MDA-MB-231 breast cancer clonal cell lines were generated that express the A or B isoform of human PR. PR protein levels, transcriptional function, and subcellular localization were determined. In vitro 18F-FFNP binding was measured via saturation and competitive binding curves. In vivo 18F-FFNP uptake was measured using tumor xenografts and PET. Statistical significance was determined using ANOVA and t tests. Results: After 48 and 72 h of E2, 18F-FFNP uptake in T47D cells was maximally increased compared with both vehicle and 24 h of E2 treatment (P < 0.0001 vs. ethanol; P = 0.02 and P = 0.0002 vs. 24 h for 48 and 72 h, respectively). T47D tumor xenografts in mice treated with 72 h of E2 had maximal 18F-FFNP uptake compared with ethanol-treated mice (percentage injected dose per gram: 11.3 ± 1.4 vs. 5.2 ± 0.81, P = 0.002). Corresponding tumor-to-muscle uptake ratios were 4.1 ± 0.6, 3.9 ± 0.5, and 2.3 ± 0.4 for 48 h of E2, 72 h of E2, and ethanol-treated mice, respectively. There was no significant preferential 18F-FFNP binding or uptake by PR-A versus PR-B in the PR isoform–specific cell lines and tumor xenografts. Conclusion: 18F-FFNP is capable of measuring estrogen-induced shifts in total PR expression in human breast cancer cells and tumor xenografts, with equivalent isoform binding.

Study of long-term biocompatibility and bio-safety of implantable nanogenerators

Implantable nanogenerator (i-NG) has shown great promises for enabling self-powered implantable medical devices (IMDs). One essential requirement for practical i-NG applications is its long-term bio-compatibility and bio-safety. This paper presents a systematic study of polydimethylsiloxane (PDMS) and PDMS/Parylene-C packaged Polyvinylidene fluoride (PVDF) NGs implanted inside female ICR (Institute of Cancer Research) mice for up to six months. The PVDF NG had a stable in vitro output of 0.3 V when bended for 7200 cycles and an in vivo output of 0.1V under stretching. Multiple advanced imaging techniques, including computed tomography (CT), ultrasound, and photoacoustic were used to characterize the embedded i-NGs in vivo. The i-NGs kept excellent adhesion to the adjacent muscle surface, and exhibited stable electrical output during the entire examine period. No signs of toxicity or incompatibility were observed from the surrounding tissues, as well as from the whole body functions by pathological analyses and blood and serum test. The PDMS package was also able to effectively insulate the i-NG in biological environment with negligible stray currents at a pA scale. This series of in-vivo and in-vitro study confirmed the biological feasibility of using i-NG in vivo for biomechanical energy harvesting.

Sex as a Biological Variable in Preclinical Imaging Research: Initial Observations with 18F-Fluorothymidine

The study objective was to investigate whether sex influences 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) uptake and tissue distribution in mouse models of cancer. Methods: 18F-FLT biodistribution was measured in 3 strains of male and female mice (129S6/SvEv, athymic nude, and BALB/c). 18F-FDG biodistribution was measured for comparison. 18F-FLT uptake was also measured in female 129S6/SvEv mice bearing estrogen-dependent SSM3 mouse mammary tumors, male athymic nude mice bearing androgen-dependent CWR22 prostate cancer xenografts, and male and female athymic nude mice bearing estrogen-independent MDA-MB-231 human breast cancer xenografts. Ki-67 expression was assayed by immunohistochemistry. PET/CT imaging was performed to visualize 18F-FLT biodistribution and to determine pharmacokinetics. Results: Greater 18F-FLT activity was observed in blood, liver, muscle, heart, kidney, and bone in female than male mice. Pharmacokinetic analysis demonstrated higher early renal 18F-FLT activity and greater accumulation of 18F-FLT in the urinary bladder in male than female mice. The differential pattern of 18F-FLT biodistribution between the sexes seen with 18F-FLT was not observed with 18F-FDG. Increased tumoral 18F-FLT uptake compared with muscle was observed in both the SSM3 mammary tumors (2.4 ± 0.17 vs. 1.6 ± 0.14 percentage injected dose [%ID]/g at 2 h after injection, P = 0.006) and the CWR22 prostate cancer xenografts (0.34 ± 0.08 vs. 0.098 ± 0.033 %ID/g at 2 h after injection, P = 0.03). However, because of higher nonspecific muscle uptake in female mice, tumor-to-muscle uptake ratios were greater for CWR22 tumors than for SSM3 tumors (4.2 ± 0.78 vs. 1.5 ± 0.049 at 2 h after injection, P = 0.008). Sex-dependent differences in 18F-FLT uptake were also observed for MDA-MB-231 xenografts (tumor-to-muscle ratio, 7.2 ± 0.9 for female vs. 16.9 ± 8.6 for male, P = 0.039). Conversely, greater tumoral Ki-67 staining was observed in female mice (71% ± 3% for female vs. 54% ± 2% for male, P = 0.009), and this finding more closely matched the relative differences in absolute 18F-FLT tumor uptake values (4.5 ± 0.99 %ID/g for female vs. 1.9 ± 0.30 %ID/g for male, P = 0.03). Conclusion: Depending on whether female or male mice are used, differences in biodistribution and nonspecific tissue uptake can adversely affect quantitative measures of 18F-FLT uptake. Thus, sex is a potential variable to consider in defining quantitative imaging metrics using 18F-FLT to assess tumor proliferation.