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.

79.10 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.

79.08 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.

 

79.04 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.
 

79.05 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. 

79.06 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.

 

79.02 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.

 

79.03 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.

 

78.20 Microglia Activation In Spinal Cord Exposed To Amniotic Fluid In RA-induced Spina Bifida Rats

Posted on January 18, 2018

M. Oria1, R. L. Figueira1, F. Scorletti1, L. Sbragia1, F. Y. Lim1, J. L. Peiro1  1Cincinnati Children’s Hospital Medical Center,Center For Fetal, Cellular And Molecular Therapy,Cincinnati, OH, USA

Introduction:
Spina bifida aperta is the most common permanently disabling birth defects. However, the pathophysiological mechanism by astrocyte proliferation with reactive astrogliosis and neuroinflammation related to microglial activation are poorly understood. We hypothesized that microglia are activated in the exposed neural tissue in spina bifida, and this activation may be associated with impairment of CD200-CD200R-mediated microglia silencing in the exposed spinal cord, which could lead to the irreversible neurological alterations after birth. 

Methods:
Spinal cord exposed and spinal cord non-exposed to the amniotic fluid in spina bifida pups were collected at day 20 of gestation in retinoic acid-induced spina bifida rats and spinal cord from sham-treated pups (control) (n=6 moms/group). RNA was isolated and pellets were partially re-extracted, precipitated, DNAseI-digested and cleaned. A 1-µg cDNA sample was used to set up RT-qPCR using TaqManR gene expression assay. CD200 and CD200R expression were assessed by flow cytometry. Protein expression were analyzed by immunofluorescence, western blot for target proteins and multiplex technology was used for cytokine tissue expression (IL1β , IL6, and INFγ).

Results:
Spina bifida samples present typical alterations with open posterior arc, dysraphic spinal cord, lack of dura and, exposed spinal cord to the amniotic fluid. Exposed neural tissue shows reactive astrocytes and activated microglia (p<0.05) located in the exposed external layers. These activated microglia exhibited disruption of the inhibitory immune ligand-receptor system (CD200-CD200R) in the lesion with neural tissue loss down-expressing CD200 and stimulating neuroinflammation up-regulating CD200R (p<0.05). The spinal cord lesion induced neuroinflammation with increased tissue water content compared wiith the non-exposed spinal cord (p<0.05) and cytokine production (IL1β, IL6, and INFγ)(p<0.05).

Conclusion:
Our study analyzed the spinal cord alterations in RA-induced spina bifida aperta in rats. For first time, we discern the relationship of the activate microgliosis by disruption of the endogenous inhibitory system (CD200-CD200R) and neural tissue loss in the spinal cord exposed to the amniotic fluid in spina bifida. 
 

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.

78.18 Experimental Diffuse Traumatic Brain Injury Increases Glucocorticoid-Receptors in the Amygdala

Posted on January 18, 2018

S. Ogle1,2,3, C. Hair1,3, B. Tallent1,3, P. D. Adelson1,3, J. Lifshitz1,3, T. C. Thomas1,3, S. B. Johnson2  1University Of Arizona College Of Medicine,Child Health,Phoenix, AZ, USA 2Banner University Medical Center,Surgery,Phoenix, AZ, USA 3Barrow Neurological Institute At Phoenix Children’s Hospital,Translational Neurotrauma Research Program,Phoenix, AZ, USA

Introduction: Investigations of the etiology of psychiatric morbidity (e.g. post-traumatic stress disorder) after traumatic brain injury (TBI) have suggested a role for circuit reorganization in brain regions associated with emotional processing, such as the amygdala. The fundamental mechanism behind this process is still unknown; however post-traumatic hormonal dysregulation of the hypothalamic-pituitary-adrenal axis (HPA) has been postulated to be involved in dendritic hypertrophy in the amygdala. Previously, we demonstrated increased complexity of glutamatergic projection and inter-neurons in the basolateral amygdala after experimental diffuse TBI up to 28 days post-injury (DPI) and a decrease in plasma corticosterone (rodent equivalent of human cortisol; CORT) at 56 DPI.  Decreased cortisol levels in patients with psychiatric morbidity after TBI have been postulated to be a response, in part, to glucocorticoid-receptor (GR) hypersensitivity, GR polymorphism, and dysregulation of the HPA axis; supporting a role for GRs in the pathogenesis of psychiatric morbidity in this population. We therefore hypothesized that experimental diffuse TBI would result in increased GR protein levels in the amygdala in response to late-onset low levels of circulating CORT.

Methods: Adult, male Sprague-Dawley rats underwent a single midline fluid percussion or sham injury and biopsies of the amygdala were evaluated at multiple time-points post-injury for protein levels of GR via automated capillary western. A one-way ANOVA with Fischer’s LSD post-hoc analysis was used for statistical analysis.

Results: GR protein levels significantly changed over time in comparison to sham (F(6,24)=19.28, p=<0.0001). Decreased levels of GR were measured at 3 DPI, with rebound increases in GR protein levels at 14 and 56 DPI.

Conclusion: These data indicate that experimental TBI results in a chronic evolving sequaelae of events over at least 2 months post-injury and represent a potential mechanism by which the pathogenesis of psychiatric morbidity develops after TBI.

78.19 Novel Rexinoids Decrease Cancer Stemness in Neuroblastoma Patient Derived Xenografts

Posted on January 18, 2018

A. P. Williams1, L. L. Stafman1, J. Aye1, V. R. Atigadda4, J. E. Stewart1, C. Grubbs2, D. Muccio3, K. J. Yoon5, K. Whelan6, E. A. Beierle1  1University Of Alabama at Birmingham,Pediatric Surgery,Birmingham, Alabama, USA 2University Of Alabama at Birmingham,Surgery,Birmingham, Alabama, USA 3University Of Alabama at Birmingham,Chemistry,Birmingham, Alabama, USA 4University Of Alabama at Birmingham,Dermatology,Birmingham, Alabama, USA 5University Of Alabama at Birmingham,Pharmacology And Toxicology,Birmingham, Alabama, USA 6University Of Alabama at Birmingham,Pediatrics,Birmingham, Alabama, USA

Introduction:  Current treatments for high risk neuroblastoma (NB) include 13-cis-retinoic acid (RA), but nearly half of children treated with RA develop disease recurrence. A subgroup of NB cells expressing the cell surface marker CD133 have been identified that have stem cell-like qualities and are associated with poor prognosis and disease relapse. These stem cell-like cancer cells demonstrate self renewal, avoid apoptosis, and are therefore promising targets for therapy. Recently, two novel rexinoids, UAB116 and 7-Me-UAB30, were developed to meet the goals of improved efficacy and decreased toxicity profiles over those of RA. We hypothesized that these novel rexinoids would affect NB cells in a fashion similar to that seen with RA, and may be used to target NB cancer stem-like cells. 

Methods: Using 2 NB patient derived xenografts (PDXs), COA3 and COA6, UAB116 and 7-Me-UAB30 were evaluated along with RA. Following 72-hour treatment, cell viability was measured using alamarBlue, proliferation was assessed with CellTiter96 assays and fluorescence-associated cell sorting (FACS) analysis was used to detect CD133 expression. Extreme limiting dilution assay (ELDA) was performed to determine the compounds’ impact on tumorsphere formation both in bulk and sorted cell populations. Student’s t-test, extreme limiting dilution analysis, and χ 2 statistics were used with mean ± standard error of the mean reported and p<0.05 significant.

Results: AlamarBlue demonstrated decreased viability following treatment with both UAB116 and 7-Me-UAB30 that was comparable to that seen with RA (Figure) and similar results were obtained for proliferation. Using FACS, we found that CD133 expression decreased following treatment with UAB116 (16% to 5%, treated vs. control, p=<0.001) and 7-Me-UAB30 (16% to 6%, treated vs. control, p=<0.001). Tumorsphere formation was diminished significantly in the NB PDX cells treated with UAB116 or 7-Me-UAB30, decreasing tumorsphere formation in the 1000 cell/well group from 75% to 18% and 42% respectively (p<0.001). When sorted based on CD133 expression, the sphere forming capacity of the CD133 enriched population was significantly decreased after treatment with either compound from 100% in the 500 cell/well group to 58% when treated with UAB116 and 75% when treated with 7-Me-UAB30 (p<0.001).

 

Conclusion: The novel rexinoids, UAB116 and 7-Me-UAB30, affected NB PDX tumor cell survival and stemness. Treatment resulted in decreased cell viability and proliferation, decreased CD133 expression, and decreased tumorsphere formation. These findings indicate that these compounds should be investigated further as potential novel therapeutics for NB.   
 

78.17 Tranexamic Acid and Plasma Have a Synergistic Effect on Ameliorating the Endotheliopathy of Trauma

Posted on January 18, 2018

J. V. Martin1, D. M. Liberati1, L. N. Diebel1  1Wayne State University,Michael And Marian Ilitch Department Of General Surgery,Detroit, MI, USA

Introduction:
Endothelial dysfunction (endotheliopathy of trauma [EOT]) is an important component of acute traumatic coagulopathy (ATC).  Drivers of ATC include tissue hypoperfusion, sympathoadrenal activation, inflammation, and accompanying hyperfibrinolysis.  Clinical studies suggest that early tranexamic acid (TXA) and plasma administration may ameliorate EOT.  However, the combined effects are unknown.  Microfluidics is a novel technology to study coagulation and cell biology in vitro. A microfluidic device lined by endothelium was used to study the combined effects of TXA and plasma on the EOT under flow conditions.

Methods:
Microfluidic channels lined with human umbilical vein endothelial cells (HUVEC) were exposed to hypoxia/reoxygenation (H/R) and epinephrine (EPI) for 60 minutes under flow conditions.  The preparations were then treated with TXA or TXA and 5% plasma.  Microfluidic perfusate was collected after H/R and at 60 minute intervals thereafter.  Glycocalyx degradation was indexed by hyaluronic acid (HA) and syndecan-1 in the perfusate.  Endothelial activation/injury was indexed by soluble thrombomodulin (TM), and coagulation phenotype by tPA (tissue-plasminogen activator) and PAI-1 (plasminogen activator inhibitor-1) in the microfluidic perfusate.

Results:
See Table

Conclusion:
H/R + EPI treatment of HUVEC under flow conditions that mimic shock conditions resulted in endothelial injury/activation, glycocalyx degradation and a profibrinolytic phenotype.  Treatment with TXA and plasma had a synergistic effect on ameliorating the “EOT” in our model.  Our study supports the clinical impetus for the early use of these therapies in the severely injured patient.
 

78.16 Changes in the Microparticle Milieu Following Traumatic Brain Injury with Concomitant Splenectomy

Posted on January 18, 2018

R. Moran1, G. E. Martin1, A. M. Pugh1, R. Veile1, L. Friend1, A. T. Makley1, C. C. Caldwell1, M. D. Goodman1  1University Of Cincinnati,Surgery,Cincinnati, OH, USA

Introduction: Splenectomy in the setting of concomitant traumatic brain injury (TBI) is associated with increased mortality without a known mechanism. Both TBI and splenectomy induce posttraumatic hypercoagulability resulting in thromboembolic events. Microparticles (MPs) are generated following both splenectomy and TBI, however it remains unknown how these MPs affect coagulation following injury. We hypothesized that post-TBI splenectomy will exacerbate the posttraumatic hypercoagulable state by altering circulating MPs.

Methods: An established weight-drop model was used to induce concussive TBI in anesthetized mice. Splenectomy was performed either seven days prior to TBI or immediately following TBI. Blood samples were collected 6 and 24 hours after TBI. Platelet counts and the total number of MPs were determined. Platelet-derived MPs were identified by the presence of CD41 and calculated as a proportion of the total number of MPs. Platelet contribution to maximum clot formation was measured using rotational thromboelastometry.

Results: No difference was seen in platelet count between any groups. At 6 hours post-TBI, platelet contribution to clot formation was greater in mice undergoing TBI with immediate splenectomy compared to mice with TBI alone (69.2% vs. 63.0%, p<0.05) or with TBI 7 days post-splenectomy (69.2% vs. 56.1%, p<0.05). Despite the total number of MPs 24 hours post-injury being significantly decreased, the proportion of platelet-derived MPs in mice sustaining either TBI alone (27.1% vs. 12.1%, p<0.01) or splenectomy alone (25.9% vs. 12.1%, p<0.05) was higher compared to sham mice. There was no difference in the composition of MPs between mice that sustained TBI alone, splenectomy alone, or combined injury.  

Conclusion: Platelet contribution to clot formation is significantly increased following TBI with immediate splenectomy even without thrombocytosis. Following either TBI or splenectomy, the total number of MPs decreases with a concomitant increase in the proportion of MPs arising from platelets. The addition of splenectomy to TBI, however, does not affect the posttraumatic microparticle milieu. 

 

78.13 Evaluating Endothelial Dysfunction in Burn Patients with Syndecan-1 as Marker of Glycocalyx Shedding

Posted on January 18, 2018

R. L. Ball1,3, M. C. Bravo2, K. Brummel-Ziedins2, T. Orfeo2, L. Moffatt3, J. W. Shupp1,3  1Washington Hospital Center,Washington, DC, USA 2University Of Vermont College Of Medicine / Fletcher Allen Health Care,Burlington, VT, USA 3Firefighters’ Burn And Surgical Research Laboratory,Washington, DC, USA

Introduction:  Endotheliopathy is a complex process that can be initiated by insults such as tissue damage and leads to poor outcomes for critically ill patients. The role of endothelial dysfunction in burn pathophysiology is largely unknown. Syndecan-1 is a component of the endothelial glycocalyx released into the bloodstream upon endothelial disruption and serves as a potential biomarker for endotheliopathy. This study aims to investigate the relationship between total body surface area (TBSA) affected and syndecan-1 levels at multiple time points during a burn patient’s hospitalization.

Methods:  Blood samples were serially collected from 30 burn patients with varying degrees of TBSA.  Baseline samples were collected within 4 hours of the injury using IRB approved protocols.  Additional blood samples were collected approximately 4, 8, and 12 hours after the baseline.  Syndecan-1 levels were quantified by ELISA.  For analysis, patients were grouped by extent of TBSA: <10% (n=12), 10-30% (n=10), and >30% (n=8).   ANOVA and t-tests were performed.

Results: At baseline, mean syndecan-1 levels were higher in groups with more severe burn injury: <10% TBSA = 19.4 ng/mL, 10-30% TBSA = 36.0, >30% TBSA = 51.5.  However, the differences were not statistically significant (p = 0.198).  Intragroup variability of baseline syndecan-1 levels was captured by large standard deviations (11.7, 53.0, and 42.8 respectively).  Similarly at hours 4 and 8, mean syndecan-1 levels increased with increasing TBSA, but the differences were not significant with a large intragroup variability.  At hour 12, significant differences in mean syndecan-1 levels were observed with increasing TBSA: <10% TBSA = 26.6 ±14.9 ng/mL, 10-30% TBSA = 56.2±43.7, >30% TBSA = 76.3±42.9, (p = 0.012).  

Conclusion: Thermal injury itself is impressively heterogeneous, which is evidenced by the large intragroup variation in syndecan-1 levels observed in this study. Although a consistent pattern from the earliest time points of increasing syndecan-1 levels with increasing TBSA was observed, it was not until hour 12 when syndecan-1 levels were related to injury size in burn patients with statistical significance. Additional markers of endothelial dysfunction need to be evaluated along with a thorough review of outcomes of these patients to establish the clinical relevance of these findings.

 

78.14 Failure of Emergency Myelopoiesis in the Elderly in a Clinically Relevant Murine Sepsis Model

Posted on January 18, 2018

J. Stortz1, M. Hollen1, H. Horiguchi1, E. Miller1, R. Hawkins1, S. Raymond1, D. Nacionales1, R. Ungaro1, M. Dirain1, B. Weiss1, C. Leeuwenburgh2, F. Moore1, S. Brakenridge1, L. Moldawer1, A. Mohr1, P. A. Efron1  1University Of Florida,Surgery,Gainesville, FL, USA 2University Of Florida,Institute On Aging,Gainesville, FL, USA

Introduction:  In order to make improvements in human sepsis outcomes, it is necessary to use improved translational animal models that better imitate the human condition. In adults, sepsis primarily occurs in the elderly, who have distinct immune responses vs younger cohorts. Since debate still exists as to whether the worsened outcomes of the aged is an “over response” vs an “under response” to severe infection, we sought to examine the emergency myeloid response, a component vital to the host’s survival to severe infection, in young vs elderly sepsis patients in a clinically relevant animal model.

Methods:  A prospective observational cohort study following 248 patients with sepsis (CMS definition) in the SICU (Loftus TJ, BMJ Open, 2017), blood (BL) was used to determine the neutrophil & monocytes levels of post-septic patients. Next, B6 old(18-24mo) & young(8-12wk) mice were subjected to an improved and clinically relevant cecal ligation & puncture(CLP) model of abdominal sepsis. We have previously demonstrated that old mice have increased mortality with CLP (Nacionales DC, J Immunol, 2014). After CLP with a 25g needle, these mice received 72hrs of saline resuscitation(1ml) & antibiotics(imipenem, 25mg/kg) every 12hrs, with the first dose being 12 hours after CLP. Animals were sacrificed at post-operative days(POD) 1, 3 & 5 (n=2-5/group per day) post-CLP, & their bone marrow (BM), BL & splenic (SP) leukocytes were stained & analyzed by flow cytometry for the myeloid cell markers CD11b & Gr1.

Results: Human patients >55yo had a trend towards a neutropenia (12 (8,19) vs 15(11,19) 103cells/mm3, p=0.08, 12hrs) and a significant monocytopenia (0.8 (0.5,1.0) vs 0.9 (0.6,1.4), p=0.02, 12hrs; 0.6(0.4,1.0) vs 0.7(0.6,1.1), p=0.03, 24hrs) acutely after sepsis which subsequently resolved. Murine BL myeloid cells followed a similar pattern as their human counterparts. Interestingly, the % of splenic CD11b+Gr1+ cells were lower in old mice after sepsis: (mean±SEM), POD1 -4.5±1.9 vs 5.9±1.7; POD3 – 9.6±1.9 vs 12.9±1.9; POD5 – 5.1±2.1 vs 12.3±2.1; (p=0.02, two-way ANOVA). Although the total number of myeloid cells present in old BM was greater vs young, the % of CD11b+Gr1+ cells present in the old BM by POD5 was deficient: 69.5±4.9 vs 88.0±4.9 (p<0.02; two-way ANOVA).

Conclusion: Our mouse CLP model recapitulates human sepsis & displays a resulting similar leukocyte phenotype as human elderly septic patients. The results of this clinically relevant work illustrates that sepsis induces an acute & subacute systemic failure of emergency myelopoiesis in elderly mice not present in the young. Immunomodulation of elderly emergency myelopoiesis, rather than blunting the innate immune response, will be a key component to improving sepsis outcomes as precision medicine becomes more prevalent.

 

78.15 Acute Lung Injury In Sepsis Patients Is Associated With Increased Available Circulating Heme

Posted on January 18, 2018

T. Cyr1, P. Waltz1, S. Shiva1, S. Ofori-Acquah1, B. Zuckerbraun1  1University Of Pittsburgh,Surgery,Pittsburgh, PA, USA

Introduction.  Erythroid danger associated molecular pattern molecules (eDAMPs) may amplify vascular and endothelial injury in sepsis.  Free heme is a known DAMP and may be increased in sepsis to promote microvascular and subsequent organ injury.  The purpose of these experiments was to test the hypothesis that sepsis results in increased available heme, which promotes vascular and organ injury.

 

Methods. Serum was collected from patients with intra-abdominal  sources of sepsis that required source control.  Free heme, HO-1, bilirubin, and hemopexin was measured.  Serum from 10 patients that did and 10 patients that did not develop acute lung injury/MODS was evaluated for ability to activate cultured wild type or TLR4 knockout endothelial cells or macrophages.  Additionally, the ‘available’ levels of heme were measured by a heme reporter assay system.

 

Results. Serum from patients taken on presentation and within 24 hours that went on to develop ALI/MODS demonstrated an increased endothelial cell or macrophage activation.  TLR4 knockout cells were activated to a significantly lower extent than wild types.  Lipopolysaccharide deactivation did not significantly decrease cell activation.  HO-1 over-expression or hemopexin treatment ameliorated  cell activation.  Measurable heme levels did not show any significant differences between groups, however ‘available’ heme levels were higher in the ALI/MODS group by reporter assay.

 

Conclusion.  Serum from sepsis patients can increase cellular injury through a TLR4 pathway independent of LPS levels and dependent upon heme signaling pathways.  Further investigation is warranted to investigate this complex signaling pathway and possible therapeutic development. 

78.11 Microparticles Generated Following Traumatic Brain Injury Induce Observed Platelet Dysfunction

Posted on January 18, 2018

G. E. Martin1, A. M. Pugh1, R. Moran1, R. Veile1, L. Friend1, A. T. Makley1, C. C. Caldwell1, M. D. Goodman1  1University Of Cincinnati,Surgery,Cincinnati, OH, USA

Introduction: Traumatic brain injury (TBI) can result in an acute coagulopathy including platelet dysfunction that contributes to ongoing intracranial hemorrhage. Microparticles (MPs) are generated by many cell types and have been shown to play a role in posttraumatic coagulopathy following TBI. We hypothesized that TBI-induced MPs would affect platelet aggregation in a murine head injury model. 

Methods:  TBI was performed using an established weight-drop method in anesthetized mice. Sham mice underwent anesthesia without TBI. Multiplate impedance platelet aggregometry, using both arachidonic acid (AA) and adenosine diphosphate (ADP) as agonists, was used to analyze post-TBI platelet function. Blood was collected 10 minutes, 6 hours, and 24 hours following TBI and a standardized 3.0 x 108 MPs were isolated from platelet-poor plasma. The MPs were mixed with whole blood from uninjured mice and the degree of platelet inhibition was measured. Normal saline was used as a dilution control. Results from platelet aggregometry are reported as Area Under the Curve. The ADP receptor inhibitor, ticagrelor (300nM), was subsequently added to TBI-induced MPs and platelet inhibition was measured using ADP as an agonist. Dimethyl sulfoxide diluted with normal saline was used as a vehicle control. 

Results: Whole blood taken from mice 10 minutes post-TBI demonstrated diminished platelet aggregation induced by ADP compared to sham mice (13.6 vs. 29.5, p < 0.01) but no difference was seen with AA. Similarly, the addition of TBI-induced MPs to uninjured donor whole blood reduced platelet aggregation induced by ADP, but not AA. Post-TBI MPs from 10 minutes and 24 hours (Figure 1) following injury significantly reduced ADP-induced platelet aggregation compared to sham-induced MPs and normal saline. The reduction in ADP-induced platelet aggregation was prevented by the pretreatment of TBI-induced MPs with 300nM Ticagrelor (9.0 vs. 6.0 vs. 31.0, MPs vs. MPs with DSMO control vs. MPs with ticagrelor, p<0.01).  

Conclusion: ADP-induced platelet aggregation is inhibited acutely following TBI in a murine model. This platelet inhibition is reproduced by the introduction of TBI-induced MPs. Furthermore, platelet inhibition is abrogated by MP pretreatment with an ADP receptor inhibitor. Clinically observed post-TBI platelet dysfunction may therefore be explained by the presence of the ADP receptor within TBI-induced MPs and may represent a future therapeutic target for TBI patients.

 

78.12 Crystalloid and Blood Resuscitation Improves Murine Survival in Combined Hemorrhage and Burn Injury

Posted on January 18, 2018

A. D. Jung1, L. Friend1, S. Stevens-Topie1, R. Schuster1, C. C. Caldwell1, T. A. Pritts1  1University Of Cincinnati,Department Of Surgery,Cincinnati, OH, USA

Introduction:  Clinical practice guidelines for resuscitation strategies are well established for either isolated hemorrhagic shock or large burn injuries. A one-to-one resuscitation with packed red blood cells and fresh frozen plasma (1:1) is standard for hemorrhagic shock. The use of crystalloids, such as Ringer’s Lactate (LR), is standard for large burn injury resuscitation. In a military setting, patients frequently sustain combined hemorrhage and burn injury, but the optimal fluid resuscitation strategy in this setting is unknown. We hypothesized that a resuscitation strategy that utilizes both crystalloid and blood products improves survival following hemorrhagic shock and burn injury.

Methods:  Male mice aged 8-10 weeks (n=7) were anesthetized with pentobarbital and subjected to a 30% full thickness scald injury to their dorsal surface. Immediately following the burn, mice received either no crystalloid resuscitation or 1.5 mL of 0.9% normal saline administered intraperitoneally. The mice then underwent femoral artery cannulation and hemorrhage to a systolic blood pressure of 25 mmHg for 30 minutes. Mice were then resuscitated to a target blood pressure with either lactated Ringer’s solution or a 1 to 1 ratio of packed red blood cells to plasma.  Survival was determined and serum was collected at 1 and 4 hours post-resuscitation for electrolyte, and cytokine analysis.

Results: Mice that underwent resuscitation with LR or 1:1 alone died soon after injury (LR: 1.6±0.4 h, 1:1: 2.3±1.2 h).  The addition of intraperitoneal saline provided a significant survival advantage within resuscitation groups (NS+LR: p<0.0001 ; NS+1:1: p=0.0001).  There was no significant difference in survival times between mice that received NS then LR or 1:1 (8.8±9.4 h vs. 12.0±10.3 h ; p=0.3). At 4 hours, mice that received NS then 1:1 demonstrated less severe metabolic acidosis compared to mice that received NS then LR (p<0.01).

Conclusion: In the setting of a simultaneous hemorrhagic shock and large burn injury, a combined resuscitation strategy with crystalloid and blood products resulted in improved survival when compared to either strategy alone. The administration of blood products improved metabolic acidosis during the resuscitation period.

 

« Previous Page
Next Page »