79.16 Elevated Expression of High Mobility Group Protein B1 is Associated with Breast Cancer Survival

Posted on January 18, 2018

K. McDonald1, E. Katsuta1, L. Yan1, Q. Qi1,2, X. Peng1,2, K. Takabe1  1Roswell Park Cancer Institute,Department Of Surgery,Buffalo, NY, USA 2State University Of New York Upstate Medical University,Syracuse, NY, USA

Introduction: HMGB1 is an evolutionarily conserved protein present in the nucleus of eukaryotic cells.  HMGB1 release has been found to be higher in breast cancer cells compared to normal tissue counterparts.  The function of released HMGB1 is diverse and compartment specific.  Inside the nucleus it facilitates DNA repair, while outside the nucleus it functions as a damage associated molecular protein (DAMP), regulating processes such as cell proliferation, autophagy, inflammation and immunity.  HMGB1 released outside the cells is detected by the TLR4 receptor. There is a strong correlation between TLR4 expression and expression of pro-inflammatory mediators.  Depending on the tumor type, the HMGB1/TLR4 interaction can promote or inhibit tumorigenesis. The exact role of HMGB1 in breast cancer has not been fully elucidated. We hypothesize that high HMGB1expression is associated with improved breast cancer survival.

Methods:  All data was obtained from the Cancer Genome Atlas (TCGA). Expression patterns of HMGB1 were retrieved from the Genomic Data Commons (GDC) data portal for analyses. After HMGB1 specific thresholds were derived and used to group estrogen receptor (ER) positive and negative breast cancer patients into a high- or low-expression group, survival data was calculated by using the Cox proportional hazard model. 

Results: When evaluating TLR4 expression in breast cancers on survival, there was no survival benefit in ER-positive (n-High = 284, n-low = 526; p=.201) or ER-negative (n-High = 207, n-low = 31; p=.221) cancers in our cohort.  However, high HMGB1 expression was associated with a statistically significant improvement in survival for ER-positive cancers (n-High = 312, n-low = 498; p=.006).  In the sub-analysis, both luminal A and B breast cancers with high HMGB1 expression had a statistically significant improvement in survival (n-High =389, n-low =30 ; p=.023; n-High =145 , n-low =46 ; p=.034 respectively).  On the other hand, a survival advantage in ER-negative cancers that overexpressed HMGB1 was not statistically significant (n-High =112, n-low =126; p=.092).  

Conclusion: By utilizing a big dataset (TCGA) with sufficient statistical power, we found that high expression of HMGB1 in breast tumor samples was associated with better overall survival in ER-positive, luminal A and B breast cancers. Our study supports the novel role of HMGB1 as a tumor suppressor in breast cancer. 

 

79.15 Methods to improve establishment of breast cancer patient-derived xenografts

Posted on January 18, 2018

M. Okano1, T. Kawaguchi1, M. Oshi1, I. Okano1, E. Katsuta1, K. Takabe1  1Roswell Park Cancer Institute,Breast Surgery,Buffalo, NY, USA

Introduction:  Despite massive expenditures in drug development to treat breast cancer, the number of compounds that was proved to be effective in animal models but failed in humans remains high. This is partly because currently used murine models do not mimic human patient cancer biology. Patient-Derived Xenograft (PDX) model, where tumor fragments from the patients are implanted into immunocompromised mice, has emerged as pre-clinical model to address these issues, however, even the optimal site of implantation remain controversial. We hypothesized that patient-derived breast tumor survive and grow better when implanted orthotopically in mammary fat pad (MFP) compared to dorsal subcutaneous space (SQ).

Methods:  We xenografted 9 patient breast cancer tumors into NSG mice. 3 tumors (including 1 brain metastatic tumor) were ER(+)HER2(-) and 6 tumors were triple negative (TN). 2 of the TN samples were previously established PDX tumor provided from University of Utah. Using 2-3 mice per each patient sample, 1mm(3) tumor fragments were implanted surgically into both four sites in SQ and four sites in MFP, the bilateral second and forth fat pads. Tumors were resected at the day when the biggest tumor grew up to 1.5cm and they were passaged to another 2-4 mice as the next generation. The size and weight of the tumor were measured. Tumor “take” was defined as tumorigenesis of palpable tumor after implantation regardless of time it took.

Results: PDX tumors were established in 5 out of 9 patient tumors. Except brain metastasis tumor, the average time for tumor take was significantly longer in 1st generation compared to 2nd or 3rd generation (117.3 days vs 60.5 days, p<0.0001). The overall tumor take rate was 51.8% (156/301 implantation site). Take rate from TN tumors was 57.4% (156/272 site), on the other hand, that from ER positive tumors was 0% (0/39 site). Interestingly, tumor take rate was 89.6% (86/96 site) in brain metastatic tumor although it was ER positive tumor. Tumor take rate was significantly better in MFP implantation compared from SQ (61.5%, 99/161 vs 40.1%, 57/140, p<0.001). Tumor weight were significantly heavier in MFP compared to SQ (0.58g vs 0.097g, p<0.0001). With increase of passage, the weight difference between MFP tumor and SQ tumor significantly increased (weight MFP/SQ of 1st generation vs 2nd and 3rd generation were 3.2 vs 5.3, p<0.0001). Even in brain metastasis tumor, take rate was significantly better in MFP implantation than SQ (100%, 48/48 vs 79.2%, 38/48, p=0.0012), and tumor weight were significantly heavier in MFP compared to SQ (0.21g vs 0.50g, p<0.0001). Time to tumor taken was significantly shorter in metastatic tumor compared from the other tumors (51days vs 74days, p=0.018). 

Conclusion: Triple negative breast cancers were more successful than ER positive tumors in establishing PDX. Brain metastasis tumor had the best take rate. MPF is better implantation site than SQ to develop breast PDX model.

 

79.14 Neurotransmitters Attenuate Glucagon Release in Cultured Pancreatic Islets

Posted on January 18, 2018

A. M. Kressel1, S. Chavan1,2, K. J. Tracey1,2  1Northwell Health,Center For Biomedical Science,Manhasset, NY, USA 2Northwell Health,Center For Bioelectronic Medicine,Manhasset, NY, USA

Introduction:  Diabetes and its sequelae are a significant cause of morbidity and mortality, affecting the lives of millions of people worldwide. Many current therapies for diabetes are aimed at lowering blood glucose. Evidence indicates that inappropriate hyperglucagonemia may contribute to the phenotype of diabetes, and is therefore a potential target for treatment. Under normal conditions, glucagon is released into the blood from α-cells in the pancreas in response to hypoglycemia and induces gluconeogenesis in the liver. Glucagon release from the pancreas may be subject to neural control, as the pancreas is innervated by the vagus nerve. However, the specific neurotransmitters involved are not yet completely understood. Here, we developed a bioassay and analyzed the effects of neurotransmitters on the islet response ex vivo. 

Methods: Pancreatic islets were isolated from adult mice and maintained in culture. After a recovery period of 24 hours, islets were placed in a hypoglycemic environment and exposed to different neurotransmitters in a range of concentrations. They were then stimulated with acetylcholine to augment glucagon and insulin release. Supernatants were collected one hour later, and insulin and glucagon levels were quantified by ELISA.

Results: GABA and somatostatin were found to inhibit glucagon release from cultured pancreatic islets in a concentration-dependent manner.

Conclusion: These findings demonstrate that exogenous neurotransmitters attenuate glucagon release from pancreatic islets ex vivo in the setting of hypoglycemia and acetylcholine challenge.

79.13 High Amplification of MYC are Associated with Poor Survival in non-Triple Negative Breast Cancer

Posted on January 18, 2018

K. Takabe1, E. Katsuta1, L. Yan2, M. Nagahashi3  1Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA 2Roswell Park Cancer Institute,Department Of Biostatistics And Bioinformatics,Buffalo, NY, USA 3Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata, , Japan

Introduction:  Mutation of MYC that causes elevated expression of this oncogenic transcription factor is found in many cancers. This leads to the unregulated expression of many genes that result in cell proliferation and tumorigenesis. It has been reported that MYC amplification is associated with Triple Negative Breast Cancer (TNBC). However, clinical relevance of MYC amplification in breast cancer remains mostly unexplored. We hypothesized that high amplification of MYC is a prognostic biomarker that represent worse biology and poor survival.  

Methods:  Genomic and clinical data were obtained from the Cancer Genome Atlas (TCGA) through cBioportal. MYC amplification was defined based upon Genomic Identification of Significant Targets in Cancer (GISTIC) copy-number; GISTIC 2 was defined as tumor with amplification, remaining GISTIC -1, 0 and 1 were defined as tumor without amplification. 

Results: Among 1080 patients with DNA copy-number data, 229 tumors (21.2%) showed MYC amplification which was the most common copy-number alteration in whole TCGA cohort of breast cancer. 156 patients (15.5%) were classified as TNBC based on ER, PgR, immunohistochemistry status and HER2 immunohistochemistry and FISH method. In agreement with previous report, there was greater proportion of tumors with high amplification of MYC in TNBC compared to non-TNBC (37% vs 18%, p<0.001). MYC mRNA expression was significantly higher in tumor with amplification compare to tumor without amplification in whole cohort as well as TNBC and non-TNBC (p<0.001, p=0.002 and p<0.001, respectively). There was no significant difference in overall survival (OS) between tumor with vs without amplification in whole breast cohort (p=0.112). To our surprise, tumors with high amplification showed significantly worse prognosis in non-TNBC patients (5-year OS rates: 80.2% vs 86.6%, p<0.037), whereas there was no significant difference in TNBC (p=0.68). These findings imply that altered MYC plays a role to promote cancer progression in non-TNBC. In non-TNBC cohort, there was greater proportion of higher AJCC Stage patients in tumors with MYC amplification group (Stage III/IV: 35.9% vs 24.8%, p<0.007).

Conclusion: Breast tumors with MYC amplification has worse prognosis in non-TNBC, but not in TNBC. MYC amplification may play different role between TNBC and non-TNBC.

 

79.12 Sphingosine-1-phosphate Affects Tumor-associated Immune Cells in Human Breast Cancer Patients

Posted on January 18, 2018

J. Tsuchida1, M. Nagahashi1, K. Moro1, A. Otani1, M. Endo1, M. Ikarashi1, M. Nakajima1, Y. Koyama1, J. Sakata1, T. Kobayashi1, H. Kameyama1, Q. Qi3, L. Yan3, K. Takabe2,4, T. Wakai1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata, NIIGATA, Japan 2Roswell Park Cancer Institute,Breast Surgery,Buffalo, NEW YORK, USA 3Roswell Park Cancer Institute,Department Of Biostatistics And Bioinformatics,Buffalo, NEW YORK, USA 4University At Buffalo Jacobs School Of Medicine And Biomedical Sciences, The State University Of New York,Department Of Surgery,Buffalo, NEW YORK, USA

Introduction: It is known that immune cells such as certain T cells and macrophages assist cancer progression. Those tumor-associated immune cells (TAICs) are considered to be one of the key players in the mechanisms how cancer invades and metastasizes. Sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid mediator, has emerged as a new player in cancer progression. S1P is produced by sphingosine kinases (SPHKs) inside cells and exported out of the cells. Extracellular S1P exerts its functions by binding to S1P specific receptors on the cell surface, and regulates various cellular functions such as cell proliferation, migration, and angiogenesis. Although previous in vivo studies indicated that S1P regulates immune function in many pathological conditions, the role of S1P in tumor-associated immune function has not been fully investigated in patients. Here, we test our hypothesis that S1P signaling affects TAICs in human breast cancer.

Methods: The expression level of each enzyme-encoding gene involved in S1P production was evaluated by retrieving RNA sequencing and gene expression quantification data using the Genomics Data Commons (GDC) data portal of the The Cancer Genome Atlas cohort. Gene expression levels were derived using normalization methods provided in the DESeq2 package. We compared the difference in expression levels of S1P related genes between HER2-negative and sphingosine kinase 1 (SPHK1)-high breast tissue (n = 263), and HER2-negative and SPHK1-low breast tissue (n = 277). We also compared the expression level of TAIC-related genes in between the same groups of the patients.

Results: The expression of some of the S1P-related genes; S1PR1, S1PR2, S1PR4, S1PR5, and SPNS2, were significantly increased in patients with high SPHK1 compared to those with low SPHK1. In contrast, expression of some of the S1P-related genes; S1PR3, ABCG2, SGPL1 and ORMDL3 were significantly decreased in patients with high SPHK1 compared to those with low SPHK1. Importantly, we found a significant increase in the expression of the TAIC-related genes, including CD4, CD8A, CD8B, PTPRC as T cell markers in SPHK1-high HER2-negative breast cancer tissues than in SPHK1-low. Macrophage markers including CCL2, CD163, CD68, CXCL8, IL-10, MSR-1, TGFB1, VEGFA, but not PLIN1, are increased in SPHK1-high HER2-negative breast cancer tissues. Moreover, adepocyte-related genes, such as HOXC9, LEP, SERPINE1, TNF, and TNFRSF9, but not UCP1, AGT, and ADIPOQ, were also increased in SPHK1-high HER2-negative breast cancer tissue. Taken together, these data revealed that SPHK1 is associated with increased expression of most of the TAIC-related genes in HER2-negative breast cancer.

Conclusion: Our results suggest that S1P affects TAICs in HER2-negative breast cancer patients, indicating the important roles of S1P in the complicated immune system related to tumor progression. Further investigations are needed to understand the underlying mechanisms.

79.11 Identification of Unique Immune Cell Subsets During the Progression of Capsular Fibrosis

Posted on January 18, 2018

B. Kuehlmann1, M. Rodrigues1, G. C. Gurtner1  1Stanford University,Department Of Surgery,Palo Alto, CA, USA

Introduction: Capsular fibrosis is the most common long-term complication after breast implant-based augmentation. The cellular alterations that underlie capsular fibrosis are still barely understood. The identification of cell types in the implant area can provide clues in determining the pathophysiology of the fibrotic changes to find a medical cure. Using an in-vivo mouse model with silicone implants we analyze the cell types present in the capsule over time. We use single cell analysis to characterize these cells to determine their involvement in fibrotic capsule development.

Methods: Capsular fibrosis was induced by inserting a customized silicone implant (same shape&filling as a human breast implant) in C57/BL6 mice. A paravertebral incision was performed on the dorsum of the mice, the implant was placed in a subcutaneous pocket. Explantation was done on day 15, day 30 &day 90 by resecting the implant with the surrounding capsule en-block for SEM. The capsule itself was digested. Cells were immunostained with CD45, EpCam, CD31, CD11b&F4/80. Cells were subjected to further analysis by fluorescence-activated cell scanning (FACS). Single cells were sorted and subjected to single cell transcriptional analysis. H&E+Trichrome staining was performed on the capsules.

Results: FACS analysis revealed that the major cell types in fibrotic capsules across all time-points were immune cells, not fibroblasts or endothelial cells. The immune cells could be classified as: (i)CD45+/CD11b+/F4/80+ (macrophages), (ii)CD45+/CD11b+/F480- (myeloid cells) and (iii)CD45+/CD11b-/F4/80- cells. On day 15, the maximum number of cells was CD45+/CD11b+/F4/80+ (mean:76.61%, SEM:5.13%) and there were no cells that were CD45+/CD11b-/F4/80-. On day 30 the macrophages were reduced and the CD45+/CD11b+/F4/80- cells were the statistically significant population (mean:57.35%, SEM:8.78%). The most interesting finding was that CD45+/CD11b-/F4/80- cells that were not found at earlier time-points were seen on day 90 (mean:27.2%, SEM:3.67%) and there was a further reduction in the macrophage population. SEM&TEM were used to confirm the presence of these immune cells. We are currently using single cell analysis to understand the identity of the CD45+/CD11b-/F4/80- cells that arise in the implant at day 90.

Conclusion: For the first time, our results reveal that the most prominent cells at every time-point in the capsule are immune cells. Macrophages comprise the largest subset of immune cells at day 15 but the macrophage numbers decrease subsequently. At day 30 and day 90 CD45+/CD11b+/F4/80- myeloid cells comprise the largest immune subset. Importantly on day 90, CD45+/CD11b-/F4/80- cells arise in the capsule. We are currently conducting single cell and functional studies on these immune subsets to understand their identity and role in capsule formation over the course of time. Our findings have promising therapeutic implications for the treatment of capsular fibrosis.

79.10 Higher CD73 Expression is Associated with Poor Prognosis in Breast Cancer

Posted on January 18, 2018

E. Katsuta1, L. Yan2, K. Takabe1  1Roswell Park Cancer Institute,Surgical Oncology,Buffalo, NY, USA 2Roswell Park Cancer Institute,Department Of Biostatistics And Bioinformatics,Buffalo, NY, USA

Introduction:  CD73 is a surface enzyme that converts AMP into adenosine. Accumulated extracellular adenosine in tumor microenvironment generates immunosuppression and pro-angiogenic environment that promotes the onset and progression of cancer. Further, tumors that express high levels of CD73 have worse prognosis in some types of malignancies. However the impact of CD73 expression levels on breast cancer patient survival remains controversial. It was also reported that the impact of CD73 expression on patients survival was different among the subtype of breast cancer.

Methods:  Gene expression was compared between cancer and non-cancer tissue using GENT (Gene Expression across Normal and Tumor tissue). Overall survival (OS) and disease-free survival (DFS) were compared between CD73 high and low expression groups based upon RNA-seq data of The Cancer Genome Atlas (TCGA) as the treatment- naïve cohort. Gene set enrichment analysis (GSEA) was conducted between CD73 high and low patients in TCGA. Relapse-free survival (RFS) was compared between CD73 high and low expression patients who received neoadjuvant chemotherapy using GSE25066 cohort. Gene expression was compared between before and after chemotherapy using GSE28844 cohort.

Results: CD73 expression level was significantly lower in cancer than normal breast tissue (p<0.001). Patients were classified as Luminal A (n=419), Luminal B (n=194), Her2 (n=67), Basal (n=140) and normal (n=24) by PAM50 classification. The expression of CD73 was significantly higher in Normal and Basal subtype compare to Her2, Luminal A, and Luminal B. Patients with high expression of CD73 showed worse survival compared with low expression group in both OS (5-year OS rate: 60.5% vs 83.4%, p<0.001) and DFS (5-year DFS rate: 71.5% vs 82.1%, p=0.049) in whole cohort, as well as in Luminal A+B patients OS (5-year OS rate: 54.5% vs 85.9%, p<0.001).  However, there was no significant difference between these two groups neither in Her2 (p=0.180), Basal (p=0.962) or Normal (p=0.172) subtypes. High expression of CD73 group also showed worse relapse-free survival in neoadjuvant chemotherapy patients cohort (3-year RFS rate: 72.2% vs 83.6%, p=0.003). CD73 expression was significantly elevated in tumors after chemotherapy compared from before the treatment (p<0.001). It may imply that CD73 high expressed cells were resistant to chemotherapy. GSEA results revealed that epithelial-mesenchymal transition (EMT) and angiogenesis related gene sets were significantly enriched in CD73 high patient tumors.

Conclusion: Tumors with high expression of CD73 have worse prognosis in treatment-naïve patients as well as patients who underwent neoadjuvant chemotherapy. The worse prognosis of the patients with high expression of CD73 may be able to be explained by metastatic potential with up-regulated EMT as well as promoted angiogenesis.

79.09 Intrinsic heterogeneity of triple-negative breast cancer cells triggers vascular mimicry in 3D culture

Posted on January 18, 2018

A. MAITI1, A. MAITI1  1Roswell Park Cancer Institute,Breast Surgery,Buffalo, NY, USA

Introduction: Within the same tumor microenvironment phenotypic and functional heterogeneity arise due to plasticity of cancer cells as a consequence of environmental differences, genetic changes and reversible epigenetic changes. Individual tumor cells growing in culture also display heterogeneity in their intrinsic ability to progress and metastasize, however, molecular mechanism is still unknown. Tumor growth and metastasis are thought to be angiogenesis related processes. Recent reports suggested that cancer cells feed themselves by an angiogenesis independent pathway, known as Vacsulogenic mimicry (VM). We have examined the ability of matrigel to stimulate complex cell behavior by its heterogeneous composition.

Methods: Cells are mixed with matrigel and plated in low attachment plates. In order to understand differential gene expression pattern we stained MDA MB 231cells in formaldehyde fixed 3D matrigel matrix environment. In order to under the differential gene expression pattern, we isolated two phenotypically different groups of cells (Vessel and Tumorsphere forming cells) from the 3D matrigel culture by using microscopic suction procedure for gene expression analysis by qPCR. Epigenetic mechanisms mediated suppression of tumor suppressors or anti-angiogenesis marker genes are hall mark of VM formation and cancer progression,  we sought to examine whether re-expression of those genes with Entinostat (MS-275), a selective inhibitor of class I histone deacetylase (HDAC) could abolish VM structures in 3D matrigel cell culture.

Results:When MDA-MB-231 cells are mixed with matrigel and cultured in low attachment plates,  around 80% cells formed vessel like phenotype known as vascular mimicry (VM) and  20% cells form spheroids. Since CD44, a stem cell marker of enhances tumor cell plasticity, we examine CD44 expression in MDA-MB-231 cells grown in 3D matrigel matrix environment. We have observed that VM forming cells are stained CD44 positive while spheroid forming cells are negative in 3D matrigel culture. Both group of cells stained positive for VEGFc and HIF1α . Gene expression data suggested that VM forming cells have more expression of CD44 and HIF1α compared to spheroid forming cells. When we treated cells with MS-275, VM structure is totally abolished. QPCR data suggested that MS-275 treatment epigenetically re-express anti-angiogenic genes; SERPINF1, THBS1 and THBS2 and tumor suppressor genes; APC, PTEN and p21. While MS-275 treatment also downregulated Vimentin, VEGF-A and CD44 .

Conclusion:Our results suggest that the VM phenotype arises in a subpopulation of cells from a conserved transcriptional response in 3D matrigel environment. Epigenetically re-expression of anti-angiogenic genes  could be a mechanism to control VM formation in triple-negative breast cancer cells.

79.08 Ceramides Are Elevated with Activation of Ceramide Biosynthesis Pathways in Human Breast Cancer

Posted on January 18, 2018

K. Moro1,4, T. Kawaguchi2, J. Tsuchida1, E. Gabriel2, Q. Yan3, L. Yan3, N. Sato4, T. Wakai1, K. Takabe2,5, M. Nagahashi1  1Niigata University Graduate School Of Medical And Dental Sciences,Digestive And General Surgery,Niigata, NIIGATA, Japan 2Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NEW YORK, USA 3Roswell Park Cancer Institute,Department Of Biostatistics And Bioinformatics,Buffalo, NEW YORK, USA 4Niigata Cancer Center Hospital,Surgery,Niigata, NIIGATA, Japan 5University At Buffalo Jacobs School Of Medicine And Biomedical Sciences,Surgery,Buffalo, NEW YORK, USA

Introduction: Sphingolipids have emerged as key regulatory molecules that control various aspects of cell biology. Among them, sphingosine-1-phosphate (S1P) and ceramide are known to form a “rheostat” where former promote cell growth and survival, and the latter apoptosis in cancer. There are three biosynthesis pathways that generate ceramide; De novo pathway; Sphingomyelin pathway; and Salvage pathway, and it is metabolized to S1P through Catabolic pathway. Despite their critical roles, the levels of sphingolipids have never been measured in patients due to lack of methods to precisely quantify them until recently. We have recently published high levels of S1P not only in breast tumor, but also in tumor microenvironment, such as tumor interstitial fluid, and reported that S1P plays pivotal roles in breast cancer progression. On the other hand, the levels of ceramide, a bioactive metabolite of S1P, in breast cancer patients have not yet well investigated to date. The aim of this study is to clarify the ceramide levels and its biosynthesis pathways in breast cancer patients.

Methods: Breast cancer, peri-tumor normal breast defined as tissue within 1 cm from the gross edge of cancer and normal breast tissue samples were collected from surgical specimens from a series of 44 patients with breast cancer. Sphingolipids, including ceramides (C14:0, C16:0, C18:1, C18:0, C20:0, C22:0, C24:1, C24:0, C26:0) and their metabolites of monohexosylceramides, dihydroceramide and sphingomyelin in the tissue samples were determined by mass spectrometry. Results were analyzed for statistical significance with the Kruskal-Wallis test. The Cancer Genome Atlas (TCGA) was used to analyze gene expressions related to the sphingolipid metabolism.

Results: Ceramide levels were higher in breast cancer compared from both normal and peri-tumor breast tissue. Substrates and enzymes that generate ceramide were significantly increased in all three ceramide biosynthesis pathways in cancer; Monohexocylceramide and glucosylceramides beta in Salvage pathway; Sphingomyelin and sphingomyelin phosphodiesterase 2 and 4 in Sphingomyelin pathway; Dihydroceramide and dihydroceramide desaturase 1 in De novo pathway. Sphingosine and ceramide synthases 2, 4, 5 and 6 in Catabolic pathway were also significantly elevated in cancer. On the contrary, gene expression of enzymes that catalyze ceramide, sphingomyelin synthase 2 and ceramide kinase were significantly suppressed, all contribute to ceramide increase in cancer.

Conclusion: This is the first study to reveal the clinical relevance of ceramide metabolism in breast cancer patients. We demonstrated that ceramide levels in breast cancer tissue are significantly higher than those in normal tissue, with activation of the three ceramide biosynthesis pathways. Our finding is in agreement with the classic notion that apoptotic cell destruction signal is activated in cancer, however, it is not enough to overcome its proliferative drive.

 

79.07 Triple-negative Breast Cancer that expresses high level of Annexin A1 have worse prognosis

Posted on January 18, 2018

M. Okano1, E. Katsuta1, M. Oshi1, K. Takabe1  1Roswell Park Cancer Institute,Breast Surgery,Buffalo, NY, USA

Introduction:  Annexin A1 (ANXA1) is a calcium-dependent phospholipid-linked protein, involved in anti-inflammatory effects, regulation of cellular differentiation, proliferation and apoptosis. Recently, we reported that ANXA1 is associated with triple-negative breast cancer (TNBC) and its poor prognosis. It was also reported that ANXA1 relates to epithelial mesenchymal transition (EMT). We hypothesized that ANXA1 expression associate with EMT that leads to poor prognosis of TNBC. 

Methods:  Clinical and RNA-seq data were all obtained from the Cancer Genome Atlas (TCGA). Patients were classified as either high or low expression of ANXA1 determined by automated scanning and selecting the threshold yielding the lowest p-value. Overall survival (OS) and Gene set enrichment analysis (GSEA) were conducted comparing high and low expression group. To validate the relationship between ANXA1 expression and survival, ANXA1 protein expression was assessed by Immunohistochemistry (IHC) in 48 TNBC patients. Patients were classified into either positive or negative based upon IHC score. Clinicopathological factors and survival were compared between them. 

Results: TNBC patients had significantly higher levels of ANXA1 expression compare to non-TNBC patients in TCGA cohort (p<0.001). ANXA1 high and low expression group were 140 and 20 patients in TNBC, and 540 and 245 in non-TNBC in TCGA cohort, respectively. High expression of ANXA1 group showed significantly worse OS (5-year OS rate: 68.6% vs 100%, p=0.035) in TNBC patients. On the contrary, high expression of ANXA1 demonstrated better OS in non-TNBC patients (5-year OS rate: 88.4% vs 78.7%, p=0.004). This finding was validated in protein expression level by IHC. Among 48 cases of TNBC patients 17 cases (35.4%) were classified as ANXA1 positively group. OS was significantly shorter in patients with ANXA1 positive tumors compared with ANXA1 negative tumor (p=0.008). To explore the mechanism of worse survival of TNBC patients with ANXA1 high expression, GSEA was conducted between ANXA1 high and low expression group. GSEA demonstrated that high expression of ANXA1 group enriched not only EMT related genes (NES=1.916, p=0.004), but also IL2/STAT5 (NES=2.04, p<0.001) and TNF alpha signaling (NES=2.02, p<0.001) related genes as well.

Conclusion: High expression of ANXA1 in TNBC patients associated with worse OS. It may be able to be explained by its metastatic potential with up-regulated EMT signaling and aggressive characteristics with up-regulated TNF alpha and IL2/STAT5 signaling.

 

79.06 Metabolic Inhibition of Anaplastic Thyroid Cancer with 3-BP Depends on Hexokinase II Expression

Posted on January 18, 2018

M. A. Nehs1, S. Aggarwal1, B. Pollard1, A. Aggarwal1  1Brigham And Women’s Hospital,Department Of Surgery,Boston, MA, USA

Introduction: Anaplastic thyroid cancer (ATC) is a fatal malignancy, and current therapies are ineffective.  The glycolytic enzyme Hexokinase II (HK2) is over expressed in many cancers and represents a potential novel target.  We therefore hypothesized that inhibition of hexokinase II with 3-bromopyruvate (3BP) would inhibit cell proliferation in ATC cell lines.

Methods: We performed cell proliferation assays using 3 ATC cell-lines (8505c, JL16, and JL30) and one thyroid cancer cell line (TPC-1). We cultured the cell-lines in high (25mM) or low (3mM) glucose concentrations with the administration of 200uM of 3BP with or without supplemental Betahydroxybutarate (BHB).  We analyzed HK2 expression by Fluorescent in-situ hybridization (FISH).  Continuous variables were analyzed by One-way ANOVA and Student’s T tests.

Results:We found that 3BP significantly inhibited proliferation in cell lines that over expressed HK2 (JL30 and TPC-1) (P<0.002) but did not significantly inhibit proliferation in the cell lines with low HK2 expression (8505c and JL16).  A low glucose environment significantly decreased proliferation in JL30 and TPC-1 (p<0.02), but not JL16 or 8505c cells.  The combination of 3BP, BHB, and a low glucose environment significantly decreased proliferation in all four cell lines (p<0.01).

Conclusion:ATC proliferation was inhibited by 3BP in cells lines that overexpress HK2.  This effect was augmented with the addition of BHB and a low glucose environment.  Further studies are warranted to see if metabolic inhibition of glycolysis with 3BP, BHB, and a low glucose environment may be an effective treatment for anaplastic thyroid carcinoma.

 

79.05 Breast Tumors that Express CCL5 and CXCL10 Attract CTLs and are Associated with Improved Survival

Posted on January 18, 2018

E. Katsuta1, L. Yan2, P. Kalinski3, K. Takabe1  1Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA 2Roswell Park Cancer Institute,Department Of Biostatistics And Bioinformatics,Buffalo, NY, USA 3Roswell Park Cancer Institute,Department Of Medicine,Buffalo, NY, USA

Introduction:  Cytotoxic T-lymphocytes (CTLs) infiltration into tumor of has been shown to predict better prognosis in breast cancer. Triple negative breast cancer (TNBC) has been shown to have higher immunogenicity and to show enhanced CTL attraction. However, the mechanism of CTL attraction to breast cancer remains elusive. CD8A is a surface marker of CTLs and Granzyme B (GZMB) is a serine protease, secreted by CTLs to mediate apoptosis of the target cells. Chemokines such as CCL5 and CXCL10 are known to attract CTLs into other types of cancer. However, the expression of CCL5 and CXLC10 and their role in the CTL attraction to breast cancer remain unknown. Therefore, we hypothesized that breast cancers that express CCL5 and CXLC10 attract CTLs and have better survival.

Methods:  RNA-seq and clinical data were obtained from the Cancer Genome Atlas (TCGA). CD8A and GZMB expression was analyzed as CTL markers. Overall survival (OS) was analyzed based upon gene expression of RNA-seq data. The cutoff value was determined by automated scanning and selecting the threshold yielding the lowest p-value.

Results: We observed that high expression of CTL markers showed significantly better OS; 5-year OS rates of CD8A high and low expression: 83.5% vs 74.7%, p<0.001, 5-year OS rates of GZMB high and low expression: 82.2% vs 66.0%, p<0.001. High expression of chemokines was also associated with better OS; 5-year OS rates in CCL5 high vs low cohorts was 82.2% vs 72.1%, p<0.001, 5-year OS rates in CXCL10 high vs low cohorts: 95.3% vs 80.8%, p=0.020. We found that CCL5 expression highly correlated with CD8A (R=0.793, p<0.001) as well as GZMB expression (R=0.756, p<0.001), while CXCL10 expression showed a weaker, although highly-significant correlation with CD8A (R=0.340, p<0.001) and GZMB (R=0.495, p<0.001) expression, indicating the roles of these chemokines in CTL attraction. These results support our notion, thus we further hypothesized that tumor that express high CCL5 and CXCL10 as well as CD8A and GZMB should have best survival. Tumors with high expression of CD8A, GZMB, CCL5 and CXCL10 were classified as high CTL infiltrating tumor (high-CTL), low expression of all of them as low-CTL, and others as middle-CTL. High-CTL group showed significantly better prognosis than other two groups, and middle-CTL group showed better prognosis than low-CTL group, with 89.3%, 81.2% and 61.8% of 5-year OS rates, respectively (high vs middle: p=0.016, middle vs low: p<0.001). There was greater proportion of high-CTL tumor in TNBC compare to non-TNBC patients (24.3% vs 4.4%, p<0.001). Mutation count was significantly higher in high-CTL tumors compare to other two groups, as well as mid-CTL vs low-CTL (Mutation count: 74, 63, 30, respectively, p<0.001).

Conclusion: Breast tumors with high mutation count and TNBC tumors highly express CCL5 and CXCL10, and attract CTLs, that result in better prognosis and may predict their responsiveness to immunotherapies. 

79.04 Breast Cancer Cell Metabolism is Regulated by Sphingosine Kinases

Posted on January 18, 2018

M. Nagahashi1, M. Nakajima1, M. Abe2, T. Saito3, M. Komatsu3, T. Soga4, J. Tsuchida1, K. Yuza1, K. Takabe5,6, K. Sakimura2, T. Wakai1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata City, NIIGATA, Japan 2Brain Research Institute, Niigata University,Department Of Cellular Neurobiology,Niigata City, NIIGATA, Japan 3Niigata University Graduate School Of Medical And Dental Sciences,Department Of Biochemistry,Niigata City, NIIGATA, Japan 4Keio University,Institute For Advanced Biosciences,Tsuruoka City, YAMAGATA, Japan 5Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA 6University At Buffalo Jacobs School Of Medicine And Biomedical Sciences, The State University Of New York,Department Of Surgery,Buffalo, NY, USA

Introduction:
Cancer cells reprogram their metabolism to promote proliferation, survival, and long-term maintenance. The common feature of this altered metabolism is the increased glucose uptake and fermentation of glucose to lactate, which has been known as “Warburg effect”. However, the mechanism how cancer cells regulate their metabolism has not yet revealed. Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that regulates many physiological and pathological processes. S1P exerts its function either intracellularly or extracellularly after produced by sphingosine kinases (SphK1 and SphK2) inside the cells. Previously our group and others have demonstrated that S1P and SphKs play important roles in cancer cell survival. We have recently published that expression of SphK1 associates with worse prognosis of breast cancer patients. Thus, we hypothesized that SphKs regulates cancer cell-specific metabolism, including “Warburg effect”.

Methods:
SphK1 or SphK2 knock-out (KO) E0771 murine breast cancer cell lines were generated with a CRISPR/Cas9 mediated targeted deletion of the SphK1 or SphK2 gene. Proliferation was assessed by WST-8. Metabolic changes in SphK1KO and SphK2KO E0771 cells were analyzed using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) between SphK1/SphK2KO and their corresponding control E0771 cells.

Results:
Proliferation assays revealed significantly less cell proliferation of SphK1KO E0771 cells compared to the control cells. On the other hand, SphK2KO E0771 cells showed significantly more proliferation than the control cells. CE-TOFMS analysis revealed the metabolomics profiles of both SphK1KO and SphK2KO E0771 cells, which were dramatically changed in the glycolysis pathway and tricarboxylic acid (TCA) cycle compared to the control cells. Interestingly, SphK1KO E0771 cells contained lower amount of glutathione (GSH) than control cells, while SphK2KO E0771 cells contained significantly higher amount of GSH than control cells. Considering that GSH plays roles in oxidative stress and drug resistance, our findings indicate an important role of SphK1 and SphK2 in not only cell survival, but also oxidative stress and drug resistance.

Conclusion:
Our results indicate that SphKs play pivotal roles in cancer specific metabolism, which strengthen resistance to oxidative stress and cancer cell survival. SphKs will be a promising target for patients with breast cancer.
 

79.03 Antiproliferative effects of marine natural compounds on neuroendocrine tumor cells

Posted on January 18, 2018

Z. Aburjania1, S. Jang1, A. W. Chang1, J. Ou3, A. Subedi2, S. Velu2, H. Chen1, R. Jaskula-Sztul1  1University Of Alabama at Birmingham,Surgery,Birmingham, Alabama, USA 2University Of Alabama at Birmingham,Chemistry,Birmingham, Alabama, USA 3University Of Alabama at Birmingham,Biomedical Engineering,Birmingham, Alabama, USA

Introduction:  

Neuroendocrine tumor (NET) is a heterogeneous group of cancers derived from the neural crest lineage. The incidence of NET has increased more than four-fold since 1973 that is attributed mainly to diagnosing earlier stage of cancer. Surgery is curative for localized tumors. However, there are limited therapeutic options for metastatic NETs. We screened fifty-two natural and synthetic chemical compounds on NET cell lines to find a viable treatment option for patients with NETs. We then characterized the antitumor activities of two most potent drug candidates.

Methods:  

Medullary thyroid cancer (TT, MZ) and pulmonary carcinoid (H727) cell lines were treated with increasing doses of natural and synthetic compounds. The cell cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay, and the most potent drugs were selected based on IC50. Flow cytometry was used to detect Annexin V binding on cell surface to quantify cells undergoing apoptosis. Western blotting was used to detect changes of NET, cell cycle, and apoptotic markers. We utilized real time polymerase chain reaction (RTPCR) in PrimePCR panels to evaluate the effect of the drug candidates on genes frequently expressed in NETs.

Results: 

The screening process led to the selection of two natural product analogs (DHN-II-84 and DHN-III-14), originating from marine sponges, with the lowest IC50 values (500 nM and 4 uM). Flow cytometry showed dose-dependent increase of apoptotic population in all cell lines. Induction of apoptosis with these compounds was also supported by the increase of Cleaved PARP and the decrease of MCL-1 and XIAP detected by Western blot. Treatment decreased neuroendocrine markers Chromogranin A and Achaete-scute homolog 1 in dose-dependent manner. RTPCR showed decrease of oncogenic genes expression such as c-KIT, PLK1, PRC1, KIAA0101, C-MET. 

Conclusion: 

Two natural product analogs DHN-II-84 and DHN-III-14 effectively killed thyroid and pulmonary neuroendocrine cancer cell lines. The presumed mechanism of cell cytotoxicity was mediated by apoptosis. Decrease of five oncogenes showed possible mechanisms of drug cytotoxicity, and these mechanisms will direct future experiments in further characterizing these natural product analogues.

 

79.02 Novel marine compound demonstrates anticancer properties in thyroid cancer cells

Posted on January 18, 2018

B. A. Hijaz4, S. Jang4, D. Carmona-Matos4, A. Chang4, Z. Aburjania4, R. Jaskula-Sztul4, H. Chen4  4University Of Alabama at Birmingham,Department Of Surgery,Birmingham, Alabama, USA

Introduction:  Effective treatments are desperately needed against aggressive thyroid cancers including anaplastic and poorly differentiated thyroid cancers. Natural products remain one of the best sources for drug leads, and they have made a significant impact on FDA approved anticancer agents. Recently, we screened novel natural compounds for their anticancer properties and selected the most potent compounds, DHN-II-84 and DHN-III-14 derived from marine sponges. The purpose of this study was to test their therapeutic efficacy on metastatic human follicular and anaplastic thyroid cancer cells lines.

Methods:  Follicular (FTC236) and anaplastic (8505C and HTh7) thyroid cancer cell lines were treated with DHN-II-84 and DHN-III-14. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the IC50 values were determined. In order to study the mechanism of cell viability reduction, Western blot was used to detect markers of apoptosis and cell cycle arrest. Cell cycle progression and apoptotic mechanisms were analyzed with flow cytometry 48 hours after treatment. Finally, thyrocyte differentiation markers were analyzed with quantitative real-time PCR to evaluate the compound’s ability to induce re-differentiation in the cancer cell lines.

Results: DHN-II-84 demonstrated an IC50 of 3.71, and 1.41 uM while DHN-III-14 showed 0.92, and 0.70 uM against Hth7 and 8505C, respectively. Therefore, DHN-III-14 was selected for further analysis. DHN-III-14 treatment dose-dependently increased cell cycle regulatory proteins p21/WAF1 and p27/Kip while cyclin D1 decreased. Markers of apoptosis were induced in a dose dependent manner. Finally, DHN-III-14 was able to effectively induce thryocyte specific genes in all three cell lines, resulting in an increase in thyroid transcription factors 1 (TTF1), TTF2, paired box gene 8 (PAX8), and sodium iodide symporter (NIS).

Conclusion: The natural marine compound, DHN-III-14, markedly suppresses proliferation in follicular and anaplastic thyroid cancer cells by inducing apoptosis. In addition, DHN-III-14 induces re-differentiation, which could sensitize these poorly differentiated tumors radioactive iodine therapy. Therefore, this novel compound may be a promising agent against aggressive thyroid cancers.

 

79.01 Whole-Exome Sequencing Identifies Distinct Mutation Profiles in Adrenocortical Cancer Cell Lines

Posted on January 18, 2018

N. G. Nicolson1, R. Korah1, T. Carling1  1Yale University School Of Medicine,Yale Endocrine Neoplasia Laboratory, Department Of Surgery,New Haven, CT, USA

Introduction:
Adrenocortical cancer (ACC) is a rare disease with a poor prognosis. Recent genetic analyses confirmed dysregulated Wnt and p53 pathways in these aggressive tumors, which may represent an opportunity for development of targeted therapies. Investigators rely on two widely used ACC cell lines, NCI-H295R and SW-13, for in vitro analysis of novel therapeutic agents. Both cell lines harbor loss-of-function TP53 mutations, and unlike non-hormone-producing SW-13 cells, NCI-H295R cells produce steroid hormones and are known to harbor a CTNNB1 mutation. However, the lack of comprehensive knowledge about the genomic landscape of these cell lines limits their utility in developing molecular targets for ACC treatment. The aim of the present study was to characterize the mutational profile of these cell lines via whole exome sequencing analysis, to confirm their validity as model systems for the development of novel targeted therapeutics.

Methods:
Genomic DNA harvested from NCI-H295R and SW-13 cells was subjected to whole exome sequencing via an established exome library preparation protocol and Illumina sequencing platform. Sequences were filtered and enriched for damaging, non-synonymous single nucleotide variants and insertion/deletion events. Predicted mutations were matched with known patient derived ACC driver genes and validated cancer driver genes in other cancer types. Cancer driver genes identified in our cohort were subjected to gene ontology and protein pathway analysis.

Results:
Whole exome analysis revealed 672 and 1123 non-synonymous gene variants in NCI-H295R and SW-13, respectively. Of these, 36 and 59 were known cancer driver genes, of which 14 were shared between the cell lines, and the remainder private mutations. Of the 23 potential ACC driver gene mutations identified in earlier patient exome studies, 4 were found to be mutated in NCI-H295R, and 3 in SW-13. Gene interaction analysis revealed distinct mutation networks – Wnt pathway in NCI-H295R and chromatin remodeling in SW-13 – potentially complementing dysregulated TP53-promoted malignancy signaling in these ACC cell lines. 

Conclusion:
ACC cell line whole exome analysis correlates with similar analyses of ACC tumors in vivo, and this genetic and functional characterization of the mutational landscapes of the 2 commonly used cell lines will help to elucidate potential novel molecular therapeutic targets in ACC. This study confirms that the NCI-H295R and SW-13 cell lines represent distinct molecular pathways in adrenocortical carcinogenesis, and that these distinct mutational profiles reflect the genomic landscapes of previously established subsets of ACCs in patient-based studies.

59.10 Psychosocial Stress and Dysbiosis Alter Intestinal Immunity in C57BL/6

Posted on January 18, 2018

S. Deas1, J. Neilson1, K. Brawner1, C. Morrow2, C. Martin1  1University Of Alabama at Birmingham,Department Of Surgery,Birmingham, Alabama, USA 2University Of Alabama at Birmingham,Department Of Cell, Developmental, And Integrative Biology,Birmingham, Alabama, USA

Introduction:  It is widely accepted that stress can negatively impact intestinal immune function. IgA is an established regulator of intestinal homeostasis and has been demonstrated to regulate bacterial translocation. The precise mechanism of how chronic psychological stress and bacterial dysrugulation affect the immune system and intestinal homeostasis is not known, but may be due to cortisol release by the hypothalamic-pituitary-adrenal (HPA) axis. We hypothesize that chronic stress and a state of dysbiosis impair intestinal immune function, perhaps mediated by dysregulation of the gut-brain immune axis.

Methods:  8-week-old C57BL/6 littermates were assigned to one of three experimental groups over a 7-day period. Group 1 was subjected to daily psychological stress via a well-established restraint model for one hour daily. Group 2 was given 1mg/mL vancomycin and 0.1mg/mL gentamicin in their drinking water to induce dysbiosis. Group 3 was subjected to both daily psychological stress and the antibiotic cocktail. The control group experienced no stress or antibiotic treatment. After the 7-day period, mesenteric lymph nodes (MLN) were harvested and cultured on Schaedler agar and incubated at 37°C for 3.5 days. Select bacterial colonies from our Group 3 MLNs were analyzed via 16S PCR for species identification. Enzyme-linked immunosorbent assay (ELISA) was utilized to measure fecal IgA and serum corticosterone levels. 

Results: Microbial culture of MLN from the control group revealed minimal bacterial growth, with an average of 1.5 pinpoint colony forming units (CFUs) per plate. CFUs were difficult to quantify from other groups due to morphology. All experimental groups were characterized by irregular, raised, undulate growth. The 16S PCR analysis of isolates from Group 3 revealed that the predominant phylum involved in MLN translocation was Proteobacteria. Finally, the Group 2 had significantly less fecal IgA compared to the control group; P-value = 0.045*. The Group 3 had significantly less fecal IgA compared to Group 1; P-value = 0.0045# (Figure 1). There was no significant difference in serum corticosterone between the control and experimental groups.

Conclusion: 
The combination of chronic stress and dysbiosis severely impairs the intestinal immune response, demonstrated by decreased fecal IgA. Serum corticosterone levels were not significantly different between control and experimental groups, suggesting that dysregulation of the HPA axis may not be the mechanism explaining the observed dampening of intestinal immunity. Bacterial translocation, as evidenced by MLN culture, indicates that this may be due to direct barrier dysfunction. Future experiments will seek to further elucidate this mechanism.

59.09 Prrx1 Identifies the Fibroblast Sub-Population Responsible for Scarring in the Ventral Dermis

Posted on January 18, 2018

M. S. Hu1, T. Leavitt1, R. Ransom1, J. Garcia1, U. Litzenburger1, G. Walmsley1, C. Marshall1, L. Barnes1, A. Moore1, E. Zielins1, C. Chan1, D. Wan1, P. Lorenz1, H. Chang1, M. Longaker1  1Stanford University,Division Of Plastic Surgery, Department Of Surgery,Palo Alto, CA, USA

Introduction:
Scarring and fibrosis are an enormous public health concern, resulting in excessive morbidity and mortality, in addition to countless lost health care dollars. Currently, there are many treatments for cutaneous scarring available, but few have proven successful, and there is an estimated $12 billion annual market in the United States for such treatments. We previously identified a sub-population of fibroblasts responsible for the bulk of connective tissue deposition in the dorsal dermis during embryonic development, cutaneous wound healing, and melanoma stroma formation. Whether these findings translate to the ventral dermis has yet to be elucidated.

Methods:
Prrx1-derived fibroblasts were traced by crossing Prrx1Cre and ROSA26mTmG mice. Prrx1-derived fibroblasts were characterized using flow cytometry, histology, and ATAC-seq analysis at various stages of embryonic development.

 

Results:
Lineage tracing of fibroblasts within the ventral dermis revealed a sub-population, labeled by the embryonic expression of Prrx1, acting as the key contributor to connective tissue deposition during scar formation. This lineage increased as a proportion of total fibroblasts within the ventral dermis over the course of gestation, associated with the transition from scarless to scarring repair. Differential patterns of chromosomal accessibility based on ATAC-seq data further demonstrated the heterogeneic nature of fibroblasts within the ventral dermis.

Conclusion:
Analogous to findings in the dorsal dermis, fibroblasts of the ventral dermis show functional heterogeneity. Prrx1 identifies the fibroblast sub-population with fibrogenic potential in the ventral dermis. As in the dorsal dermis, selectively ablating this fibroblast sub-population may lead to decreased cutaneous scarring and a novel therapeutic to decrease scarring and fibrosis.

 

59.08 Skin Fibrosis is Decreased by Local Application of Doxycycline Without Compromising Tensile Strength

Posted on January 18, 2018

R. E. Jones1,2, A. L. Moore2,3, M. P. Murphy2, D. S. Foster2, S. Mascharak2, B. Duoto2,4, D. Irizarry2,5, E. A. Brett2, G. Wernig6, M. T. Longaker2  1University Of Texas Southwestern Medical Center,General Surgery,Dallas, TX, USA 2Stanford University,Department Of Surgery,Palo Alto, CA, USA 3Brigham And Women’s Hospital,Department Of Surgery,Boston, MA, USA 4San Jose State University,San Jose, CA, USA 5Beth Israel Deaconess Medical Center,Department Of Surgery,Boston, MA, USA 6Stanford University,Department Of Pathology,Palo Alto, CA, USA

Introduction: Cutaneous scarring represents a challenging clinical problem which results in disability, deformity, and psychosocial dysfunction. Matrix metalloproteinase inhibitors are important in regulating scar tissue deposition and extracellular matrix remodeling. Doxycycline is an antibiotic approved by the Federal Drug Administration known to function as a matrix metalloproteinase inhibitor. Given this chemical quality, we theorized that doxycycline may positively affect wound healing. We tested the effect of locally-applied doxycycline on skin fibrosis and bacterial contamination.

Methods: Experiments were conducted with C57BL/J6 mice to test scar thickness, tensile strength, and bacterial colony load in wounds treated with doxycycline or phosphate buffered sodium (PBS). All mice underwent dorsal excisional skin wounding in accordance with previously published work. Doxycycline or PBS was then injected into the base of acute wounds at varying concentrations. Wounds were harvested 15 days postoperatively. Histologic analyses were performed to measure scar thickness and collagen content, including staining with hematoxylin and eosin, trichrome, and picrosirius red. The tensile strength of healed wounds was tested. Wounds were swabbed and cultured, and bacterial colonies were tabulated. Results were compared utilizing the Mann-Whitney U test.

Results: Scar thickness was significantly decreased by 37% in wounds treated with 2 mg/mL doxycycline versus PBS (*p = 0.0107). Doxycycline treated wounds exhibited significantly decreased proportions of mature collagen as compared to control wounds (*p = 0.0470). Bacterial loads were similar at postoperative days 1, 3, 5, 7 and 9 in doxycycline and control mice (p > 0.05). Finally, tensile strength was not significantly different in treatment versus control mice (p = 0.6334).

Conclusions: We show that local application of 2 mg/mL doxycycline decreases scarring in comparison to PBS treated wounds without compromising tensile strength. Additionally, bacterial contamination in treated versus control wounds was not significantly different, suggesting that doxycycline functions as a vulnerary agent to reduce fibrosis in non-antimicrobial manner. Given the longstanding safety profile of doxycycline, this application could be rapidly translated for use in acute injury, postoperatively, hypertrophic scars and keloids, and burns. 

59.07 Ischemia-Reperfusion Injury Induces Neuronal Cell Division in the Murine Enteric Nervous System

Posted on January 18, 2018

C. J. Greig1,2, C. J. Park1, S. J. Armenia1, L. Zhang1, R. A. Cowles1  1Yale University School Of Medicine,New Haven, CT, USA 2University Of Massachusetts Medical School – Baystate Medical Center,Springfield, MA, USA

Introduction:

The enteric nervous system (ENS) has long been considered a static environment, lacking significant neurogenesis under steady-state conditions. Recent evidence suggests, however, that certain stimuli can induce neurogenesis originating from enteric neuronal precursors. While much about this process remains unknown, emerging data suggest the generation of new neurons may occur via two different mechanisms: differentiation from existing neuronal precursors or generation of new mature neurons via cell division. Previous experiments suggested that bowel resection could induce the generation of new mature neurons, but whether this occurred due to cell division was unclear. In the current study, we hypothesized that ischemia-reperfusion injury would induce neuronal precursor cells to generate new neurons via cell division.

Methods:

Wild type (WT) C57Bl/6 controls and Nestin_CreER/eGFP bitransgenic mice expressing eGFP in a tamoxifen-inducible manner and under the control of the actin promoter were used for experiments. Following tamoxifen induction, bitransgenic mice underwent ischemia-reperfusion (IR) injury via superior mesenteric artery occlusion (T=30 and 45 min). WT and bitransgenic controls received tamoxifen injections alone without IR, and bitransgenic sham laparotomy animals received the same surgical procedure as IR animals without SMA clamping. IR animals received injections of EdU at the start of the reperfusion period and again at day 5. After the reperfusion period (3 or 10 days), distal ileal segments were obtained from all animals. Whole mounts of the myenteric and submucosal plexuses were created by mechanically peeling these layers after fixation. Peeled specimens were then stained with fluorescent antibodies targeting a marker of mature neurons, HuC/D, and GFP. A click chemistry assay was used to detect Edu in the whole mount specimens.

Results:

WT and bitransgenic controls demonstrated no evidence of GFP-expressing mature neurons, suggesting no demonstrable neurogenesis occurred at baseline. Bitransgenic sham laparotomy mice demonstrated a low incidence of neurogenesis, as evidenced by mature neurons expressing GFP in one of four high-powered fields. In IR animals, we found evidence of extensive neurogenesis with GFP-expressing mature neurons in nearly all high-powered fields. Moreover, EdU was detected in most mature, GFP-expressing neurons, suggesting that these neurons had been generated from precursors via cell division.

Conclusion:

Ischemia-reperfusion injury appears to induce a substantial mitotic response in the ENS of the murine ileum, and this process appears to occur via cell division of neuronal precursors. Interestingly, anesthesia and laparotomy alone appear to represent a sufficient stimulus to induce neurogenesis, but to a lesser extent than ischemia-reperfusion. While preliminary in nature, these data provide insight for future study into the mechanisms of enteric neurogenesis. 

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