- Email: [email protected]
40 Dichotomous Signaling Mechanisms for Activin-Induced Growth Suppression and Migration in Colon Cancer Cells
AGA Abstracts
(MLM) after (avg, 28 months) radical surgery; of 49 MSI CRC (31N0, 18N+) pts, only 3 (1N0, 2N+) developed MLM. By a software-scanning system, we quantified the percent of the area of CD3+ cells at the invasion front (expressed as the avg percent ± S.D. of immunoreactive cell area, out of 3 fields). Non-parametric methods were used for statistical analyses. RESULTS. MSI CRC had a higher percentage of CD3+ lymphocytes (10.6 ± 9.33) than MSS CRC (6.2 ± 5.9; p=0.006). CRC from pts with MLM harbored a lower amount of CD3+ cells (3.0 ± 3.0) than those from pts with no MLM (9.2 ± 8.3; p=0.001). In the MSS group, CRC from pts without MLM had more CD3+ lymphocytes (7.16 ± 6.1) than their MLM counterpart (2.4 ± 3.2; p=0.005). In the MSI group, we did not detect differences between cases with/without MLM, even due to low number of the latters. Among MSS CRC, the observed difference was mainly contributed by CRC with CD3+ density ≤1% (6/11, 54%, in MLM cases vs 5/42, 12%, in non-MLM CRC; p=0.005). Upon multivariate logistic regression analysis, CRC with ≤1% CD3+ cells had a high risk of MLM (OR 6.6, 95% CI 1.5-20; p=0.003), independent of molecular, demographic, and pathological features. CONCLUSIONS. In pT3 MSS CRC, a low CD3+ density at the invasion front was associated with a high frequency of MLM in our retrospective study. Quantification of CD3+ is a promising tool for discriminating CRC pts without distant organ metastases at diagnosis who can undergo hepatic progression within 2-3 years. Further studies assessing the predictive value of this marker in stage II CRC pts are warranted.
increased sensitivity to PI3K inhibition either with LY294002 or wild-type PTEN overexpression. iii) Transient Akt2 overexpression had a significant impact on cell metabolism by increasing glucose consumption ~ 2-fold. iv) In Vivo studies showed that only cells with a concurrent mutation of PTEN and Akt2 overexpression metastasized to the liver and surrounding lymph nodes. CONCLUSIONS: Taken together, these results suggest that PTEN mutation and Akt2 amplification mediate different cellular events and act in concert to stimulate a more aggressive phenotype in colon cancer cells. 42 Loss of CDx2 Increases Migration and Induces Changes Consistent with Epithelial to Mesenchymal Transition in Human Colon Cancer Cells Sanjay R. Hegde, Long H. Dang, Duyen Dang, John P. Lynch Introduction: The intestine specific homeobox gene Cdx2 has been implicated in the progression of colon cancer. It has been reported to modulate cell proliferation and apoptosis; however its effects on cell migration are not well explored. Previously we have shown that increased expression of Cdx2 is associated with increased cellular compaction, increased cell-cell adhesive junctions, and decreased migration. These changes may be related to an increase in E-cadherin function. In this study we aim to evaluate the effects of loss of Cdx2 expression on cell migration and E-cadherin function in colon cancer cells. Methods: Immunoblotting studies on whole cell lysates of LOVOCdx2 +/+ and LOVOCdx2 -/- cells were performed on components of the adherens junction complex (E-cadherin, Beta-catenin, and p120). Localization of components of the E-cadherin complex (E-cadherin and Betacatenin) was evaluated by immunofluoresence. Boyden chamber assay was used to quantify cell migration using fluorometric analysis of calcein AM uptake. shRNA mediated knockdown of Cdx2 in T-84 cells was performed and cell morphology and migration were examined. Results: Morphologically, the LOVOCdx2-/- cells do not appear to form strong cell-cell adhesive interactions in culture. This subjective observation is supported objectively by Boyden chamber assays in which the LOVOcdx2-/- cells demonstrated a 2-fold increase in migration compared to LOVOCdx2+/+ cells (p=0.024). Adherens junction formation is impaired in the LOVOcdx2-/- cells, as E-cadherin and beta-catenin localize to the cytoplasm rather than cell-cell borders as is seen in the LOVOCdx2+/+ on immunofluorescence. Total beta-catenin levels are increased in the LOVOCdx2-/- compared to LOVOCdx2 +/+ cells. In contrast, levels of E-cadherin and p120 are unchanged. Preliminarily, in T84 cells in which Cdx2 protein levels are reduced by shRNA targeting, we observe increased cell spreading, a more spindle-form morphology, poorer cell-cell adhesive interactions, and increased migration on Boyden chamber assays compared to control cells. Conclusions: Our data suggest that Cdx2 loss in LOVO and T84 colon cancer cells is associated with a change in morphology, reduced cell-cell adhesion, and an increase in cell migration. In LOVO cells we also observed loss of the E-cadherin complex from cell-cell junctions, and increased levels of beta-catenin. Loss of E-cadherin from the cell surface and increased cell migration are markers of epithelial to mesenchymal transition (EMT), an important process in tumor progression. We propose that Cdx2 loss enhances EMT in colon cancer by reducing Ecadherin dependent cell-cell adhesion.
40 Dichotomous Signaling Mechanisms for Activin-Induced Growth Suppression and Migration in Colon Cancer Cells Barbara H. Jung, Jennifer Cabral, Jessica Gomez, Stayce E. Beck, John M. Carethers BACKGROUND: Activin is a growth suppressive ligand of the transforming growth factor β (TGFβ) family and its signaling pathway is disrupted in colon cancers. Its primary receptor, activin receptor 2 (ACVR2) is mutated in > 80% of microsatellite unstable (MSI) and a subset of microsatellite stable (MSS) colon cancers, preventing expression of ACVR2. Activin binds to ACVR2, ultimately triggering phosphorylation of intracellular SMAD proteins that complex with SMAD4 and translocate to the nucleus to affect transcription of genes that slow cellular growth. Expression of the cell cycle inhibitor p21 is known to be increased by activin signaling, but the mechanism of activation has not been explored in colon cancer cells. Here, we assessed activin-SMAD-p21 activation, and how phosphatidylinositol 3 kinase (PI3K) mitogenic signaling might influence it. METHODS: ACVR2 (WT)/SMAD4 (WT) FET, ACVR2 (WT)/SMAD4 (null) SW480, ACVR2 (null) HCT116, and ACVR2-restored HCT116+chr2 colon cancer cells were utilized to examine activin signaling and its cellular effects. Expression of SMADs and p21-specific transactivation, transcription, and protein stability, as well as cell growth, cell death and migration were assessed. p21-specific siRNA was used to knock down p21 expression, and LY294002 was used to inhibit PI3K activity. RESULTS: Activin increased p21 through protein stabilization without changing transactivation or mRNA levels in ACVR2/SMAD4 wild type cells, and p21 protein stability was prolonged with simultaneous inhibition of PI3K. Activin-induced cell growth was dependent on p21, while p21 induction via activin was dependent on SMAD4. Activin-induced signaling, transactivation, growth suppression and cell cycle arrest were enhanced with concomitant inhibition of PI3K in ACVR2-competent, but not in ACVR2-null colon cancer cells. Activininduced migration was ACVR2-specific, but SMAD4-independent, and was enhanced via PI3kinase signaling. CONCLUSIONS: Activin-induced growth suppression is mediated by an increase in p21 protein stability via SMAD4 signaling, but reduced by PI3K that is often activated in colon cancers. Conversely, activin-induced cell migration occurs in a SMAD4independent fashion and is enhanced by PI3K signaling. The simultaneous inhibition of activin-SMAD-p21 suppressive signaling with activation of an activin-SMAD-independent cell migration pathway, both of which are influenced by PI3K to enhance metastatic behavior, suggest that these activin pathways exist to fine tune normal growth but become unbalanced during tumorigenesis. Further exploration of activin dependent migration may provide targets for intervention of advanced colon cancer.
43 The Role of T Cells in Vagal Protection Against Intestinal Inflammation Caitlin O'Mahony, Hanneke P. van der Kleij, Fergus Shanahan, John Bienenstock, Liam O'Mahony Introduction & Aim: The vagus nerve is a conduit for bi-directional signalling between the brain and the gut and has been shown to involve the α7 subunit of the nicotinic acetylcholine receptor on macrophages which down-regulates inflammation. We and others have demonstrated that colitis is more severe in immuno-competent vagotomised animals compared to sham-vagotomised controls. In this study, we investigated vagal signalling in an immuno-deficient SCID mouse model of colitis. Our aim was to establish if vagal signalling is exclusively at the level of macrophages or if T cells are involved in the vagal antiinflammatory reflex. Methods: Sub-diaphragmatic vagotomy or sham-vagotomy was performed in CB-17/IcrHanHsd SCID animals (n=7/grp). Colitis was induced in the SCIDs by i.p. injection of 1.0 x 106 CD4+CD62L+ donor cells. In addition, sub-diaphragmatic vagotomy (n=3) or sham-vagotomy (n=4) was performed in immuno-competent BALB/c animals. Naïve recipient mice (n>6/grp) were injected with 1.0 x 106 CD4 T cells from vagotomised or sham-vagotomised donor animals. 4% DSS was administered to induce colitis in recipient mice. Colonic tissue was homogenised and the supernatant was assayed spectrophotometrically for MPO activity. In Vitro splenocyte release of cytokines and CD4/CD25/Foxp3 regulatory T cells were measured by flow cytometry. Results:In contrast to immuno-competent mice where vagotomy aggravates DSS colitis, colonic MPO levels are unchanged in vagotomised colitic SCIDs versus sham-vagotomy controls. In addition, vagotomised SCIDs splenocytes do not secrete higher levels of pro-inflammatory cytokines compared to controls. The vagal modulatory effect appears absent in lymphocyte deficient SCID's suggesting T cells as possible cellular targets. Adoptive transfer of splenic CD4 T cells from vagotomised animals exacerbates colitis in naïve recipients reflected in the increased level of colonic MPO activity (p<0.001) and increased secretion of TNF (p<0.01) and MCP-1 (p<0.05) compared to recipient animals that received T cells from sham-vagotomised donors. Also, the percentage of CD4 T cells expressing CD4/CD25/Foxp3 was significantly reduced in colitic animals that received lymphocytes from vagotomised donor animals (p<0.001). Conclusion: While vagotomy aggravates DSS colitis in immuno-competent mice we show that this effect is lost in the SCID animal model. The vagal anti-inflammatory effect is not exclusively mediated at the level of macrophage function - it also involves lymphocytes.
41 Concurrent Mutation of PTEN and Akt2 Overexpression in Colorectal Cancer Increases Micrometastasis Establishment and Growth Piotr G. Rychahou, Jung-Hee Kang, Hung Q. Doan, Dai H. Chung, B. M. Evers Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US; approximately 35-55% of patients with CRC will develop liver metastases during the course of their disease. Previously, we have demonstrated the importance of the phosphatidylinositol 3-kinase (PI3K) pathway, acting through its downstream effector protein, Akt2, in CRC metastasis establishment. The purpose of our present study was to delineate the roles of concurrent mutation of PTEN and Akt2 overexpression on the systemic invasion and metastasis of CRC. METHODS. i) The relative expression of Akt2 in different histotypes of human CRC and between primary CRC and matched metastases in the same patients was determined using immunohistochemical tissue microarray analyses. The relative levels of Akt2 mRNA expression were examined in different stages of CRC and normal colon using real time PCR. ii) To determine whether cancer-associated PTEN mutation promotes CRC survival capacity under stress conditions In Vitro, we subjected mutant PTEN or stable knockdown PTEN cells to growth factor deprivation stress (GFDS). iii) To determine the impact of concurrent mutation of PTEN and Akt2 overexpression on CRC metabolism; we measured glucose consumption in PTEN wild-type or knockdown cells after transient Akt2 overexpression. iv) Next, we transiently expressed Akt2 in non-metastatic human CRC cell lines with wildtype PTEN and mutant PTEN; mice were examined by real-time fluorescence imaging for metastasis formation and growth. RESULTS. i) Marked Akt2 immunoreactivity was detected in 70% of well- or moderately-differentiated human CRC. In 88% (n=32/36 cases), Akt2 expression was greater in the liver metastases than in primary CRCs. Akt2 mRNA expression was increased ~7-fold compared to normal mucosa. Increased Akt2 expression at early CRC stages suggests that additional mechanisms are required for metastatic progression. ii) When subjected to GFDS, the mutant or stable knockdown PTEN cells displayed resistance to GFDS-induced apoptosis relative to wild-type cells. The mutant PTEN cells also showed
AGA Abstracts
A-6
(MLM) after (avg, 28 months) radical surgery; of 49 MSI CRC (31N0, 18N+) pts, only 3 (1N0, 2N+) developed MLM. By a software-scanning system, we quantified the percent of the area of CD3+ cells at the invasion front (expressed as the avg percent ± S.D. of immunoreactive cell area, out of 3 fields). Non-parametric methods were used for statistical analyses. RESULTS. MSI CRC had a higher percentage of CD3+ lymphocytes (10.6 ± 9.33) than MSS CRC (6.2 ± 5.9; p=0.006). CRC from pts with MLM harbored a lower amount of CD3+ cells (3.0 ± 3.0) than those from pts with no MLM (9.2 ± 8.3; p=0.001). In the MSS group, CRC from pts without MLM had more CD3+ lymphocytes (7.16 ± 6.1) than their MLM counterpart (2.4 ± 3.2; p=0.005). In the MSI group, we did not detect differences between cases with/without MLM, even due to low number of the latters. Among MSS CRC, the observed difference was mainly contributed by CRC with CD3+ density ≤1% (6/11, 54%, in MLM cases vs 5/42, 12%, in non-MLM CRC; p=0.005). Upon multivariate logistic regression analysis, CRC with ≤1% CD3+ cells had a high risk of MLM (OR 6.6, 95% CI 1.5-20; p=0.003), independent of molecular, demographic, and pathological features. CONCLUSIONS. In pT3 MSS CRC, a low CD3+ density at the invasion front was associated with a high frequency of MLM in our retrospective study. Quantification of CD3+ is a promising tool for discriminating CRC pts without distant organ metastases at diagnosis who can undergo hepatic progression within 2-3 years. Further studies assessing the predictive value of this marker in stage II CRC pts are warranted.
increased sensitivity to PI3K inhibition either with LY294002 or wild-type PTEN overexpression. iii) Transient Akt2 overexpression had a significant impact on cell metabolism by increasing glucose consumption ~ 2-fold. iv) In Vivo studies showed that only cells with a concurrent mutation of PTEN and Akt2 overexpression metastasized to the liver and surrounding lymph nodes. CONCLUSIONS: Taken together, these results suggest that PTEN mutation and Akt2 amplification mediate different cellular events and act in concert to stimulate a more aggressive phenotype in colon cancer cells. 42 Loss of CDx2 Increases Migration and Induces Changes Consistent with Epithelial to Mesenchymal Transition in Human Colon Cancer Cells Sanjay R. Hegde, Long H. Dang, Duyen Dang, John P. Lynch Introduction: The intestine specific homeobox gene Cdx2 has been implicated in the progression of colon cancer. It has been reported to modulate cell proliferation and apoptosis; however its effects on cell migration are not well explored. Previously we have shown that increased expression of Cdx2 is associated with increased cellular compaction, increased cell-cell adhesive junctions, and decreased migration. These changes may be related to an increase in E-cadherin function. In this study we aim to evaluate the effects of loss of Cdx2 expression on cell migration and E-cadherin function in colon cancer cells. Methods: Immunoblotting studies on whole cell lysates of LOVOCdx2 +/+ and LOVOCdx2 -/- cells were performed on components of the adherens junction complex (E-cadherin, Beta-catenin, and p120). Localization of components of the E-cadherin complex (E-cadherin and Betacatenin) was evaluated by immunofluoresence. Boyden chamber assay was used to quantify cell migration using fluorometric analysis of calcein AM uptake. shRNA mediated knockdown of Cdx2 in T-84 cells was performed and cell morphology and migration were examined. Results: Morphologically, the LOVOCdx2-/- cells do not appear to form strong cell-cell adhesive interactions in culture. This subjective observation is supported objectively by Boyden chamber assays in which the LOVOcdx2-/- cells demonstrated a 2-fold increase in migration compared to LOVOCdx2+/+ cells (p=0.024). Adherens junction formation is impaired in the LOVOcdx2-/- cells, as E-cadherin and beta-catenin localize to the cytoplasm rather than cell-cell borders as is seen in the LOVOCdx2+/+ on immunofluorescence. Total beta-catenin levels are increased in the LOVOCdx2-/- compared to LOVOCdx2 +/+ cells. In contrast, levels of E-cadherin and p120 are unchanged. Preliminarily, in T84 cells in which Cdx2 protein levels are reduced by shRNA targeting, we observe increased cell spreading, a more spindle-form morphology, poorer cell-cell adhesive interactions, and increased migration on Boyden chamber assays compared to control cells. Conclusions: Our data suggest that Cdx2 loss in LOVO and T84 colon cancer cells is associated with a change in morphology, reduced cell-cell adhesion, and an increase in cell migration. In LOVO cells we also observed loss of the E-cadherin complex from cell-cell junctions, and increased levels of beta-catenin. Loss of E-cadherin from the cell surface and increased cell migration are markers of epithelial to mesenchymal transition (EMT), an important process in tumor progression. We propose that Cdx2 loss enhances EMT in colon cancer by reducing Ecadherin dependent cell-cell adhesion.
40 Dichotomous Signaling Mechanisms for Activin-Induced Growth Suppression and Migration in Colon Cancer Cells Barbara H. Jung, Jennifer Cabral, Jessica Gomez, Stayce E. Beck, John M. Carethers BACKGROUND: Activin is a growth suppressive ligand of the transforming growth factor β (TGFβ) family and its signaling pathway is disrupted in colon cancers. Its primary receptor, activin receptor 2 (ACVR2) is mutated in > 80% of microsatellite unstable (MSI) and a subset of microsatellite stable (MSS) colon cancers, preventing expression of ACVR2. Activin binds to ACVR2, ultimately triggering phosphorylation of intracellular SMAD proteins that complex with SMAD4 and translocate to the nucleus to affect transcription of genes that slow cellular growth. Expression of the cell cycle inhibitor p21 is known to be increased by activin signaling, but the mechanism of activation has not been explored in colon cancer cells. Here, we assessed activin-SMAD-p21 activation, and how phosphatidylinositol 3 kinase (PI3K) mitogenic signaling might influence it. METHODS: ACVR2 (WT)/SMAD4 (WT) FET, ACVR2 (WT)/SMAD4 (null) SW480, ACVR2 (null) HCT116, and ACVR2-restored HCT116+chr2 colon cancer cells were utilized to examine activin signaling and its cellular effects. Expression of SMADs and p21-specific transactivation, transcription, and protein stability, as well as cell growth, cell death and migration were assessed. p21-specific siRNA was used to knock down p21 expression, and LY294002 was used to inhibit PI3K activity. RESULTS: Activin increased p21 through protein stabilization without changing transactivation or mRNA levels in ACVR2/SMAD4 wild type cells, and p21 protein stability was prolonged with simultaneous inhibition of PI3K. Activin-induced cell growth was dependent on p21, while p21 induction via activin was dependent on SMAD4. Activin-induced signaling, transactivation, growth suppression and cell cycle arrest were enhanced with concomitant inhibition of PI3K in ACVR2-competent, but not in ACVR2-null colon cancer cells. Activininduced migration was ACVR2-specific, but SMAD4-independent, and was enhanced via PI3kinase signaling. CONCLUSIONS: Activin-induced growth suppression is mediated by an increase in p21 protein stability via SMAD4 signaling, but reduced by PI3K that is often activated in colon cancers. Conversely, activin-induced cell migration occurs in a SMAD4independent fashion and is enhanced by PI3K signaling. The simultaneous inhibition of activin-SMAD-p21 suppressive signaling with activation of an activin-SMAD-independent cell migration pathway, both of which are influenced by PI3K to enhance metastatic behavior, suggest that these activin pathways exist to fine tune normal growth but become unbalanced during tumorigenesis. Further exploration of activin dependent migration may provide targets for intervention of advanced colon cancer.
43 The Role of T Cells in Vagal Protection Against Intestinal Inflammation Caitlin O'Mahony, Hanneke P. van der Kleij, Fergus Shanahan, John Bienenstock, Liam O'Mahony Introduction & Aim: The vagus nerve is a conduit for bi-directional signalling between the brain and the gut and has been shown to involve the α7 subunit of the nicotinic acetylcholine receptor on macrophages which down-regulates inflammation. We and others have demonstrated that colitis is more severe in immuno-competent vagotomised animals compared to sham-vagotomised controls. In this study, we investigated vagal signalling in an immuno-deficient SCID mouse model of colitis. Our aim was to establish if vagal signalling is exclusively at the level of macrophages or if T cells are involved in the vagal antiinflammatory reflex. Methods: Sub-diaphragmatic vagotomy or sham-vagotomy was performed in CB-17/IcrHanHsd SCID animals (n=7/grp). Colitis was induced in the SCIDs by i.p. injection of 1.0 x 106 CD4+CD62L+ donor cells. In addition, sub-diaphragmatic vagotomy (n=3) or sham-vagotomy (n=4) was performed in immuno-competent BALB/c animals. Naïve recipient mice (n>6/grp) were injected with 1.0 x 106 CD4 T cells from vagotomised or sham-vagotomised donor animals. 4% DSS was administered to induce colitis in recipient mice. Colonic tissue was homogenised and the supernatant was assayed spectrophotometrically for MPO activity. In Vitro splenocyte release of cytokines and CD4/CD25/Foxp3 regulatory T cells were measured by flow cytometry. Results:In contrast to immuno-competent mice where vagotomy aggravates DSS colitis, colonic MPO levels are unchanged in vagotomised colitic SCIDs versus sham-vagotomy controls. In addition, vagotomised SCIDs splenocytes do not secrete higher levels of pro-inflammatory cytokines compared to controls. The vagal modulatory effect appears absent in lymphocyte deficient SCID's suggesting T cells as possible cellular targets. Adoptive transfer of splenic CD4 T cells from vagotomised animals exacerbates colitis in naïve recipients reflected in the increased level of colonic MPO activity (p<0.001) and increased secretion of TNF (p<0.01) and MCP-1 (p<0.05) compared to recipient animals that received T cells from sham-vagotomised donors. Also, the percentage of CD4 T cells expressing CD4/CD25/Foxp3 was significantly reduced in colitic animals that received lymphocytes from vagotomised donor animals (p<0.001). Conclusion: While vagotomy aggravates DSS colitis in immuno-competent mice we show that this effect is lost in the SCID animal model. The vagal anti-inflammatory effect is not exclusively mediated at the level of macrophage function - it also involves lymphocytes.
41 Concurrent Mutation of PTEN and Akt2 Overexpression in Colorectal Cancer Increases Micrometastasis Establishment and Growth Piotr G. Rychahou, Jung-Hee Kang, Hung Q. Doan, Dai H. Chung, B. M. Evers Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US; approximately 35-55% of patients with CRC will develop liver metastases during the course of their disease. Previously, we have demonstrated the importance of the phosphatidylinositol 3-kinase (PI3K) pathway, acting through its downstream effector protein, Akt2, in CRC metastasis establishment. The purpose of our present study was to delineate the roles of concurrent mutation of PTEN and Akt2 overexpression on the systemic invasion and metastasis of CRC. METHODS. i) The relative expression of Akt2 in different histotypes of human CRC and between primary CRC and matched metastases in the same patients was determined using immunohistochemical tissue microarray analyses. The relative levels of Akt2 mRNA expression were examined in different stages of CRC and normal colon using real time PCR. ii) To determine whether cancer-associated PTEN mutation promotes CRC survival capacity under stress conditions In Vitro, we subjected mutant PTEN or stable knockdown PTEN cells to growth factor deprivation stress (GFDS). iii) To determine the impact of concurrent mutation of PTEN and Akt2 overexpression on CRC metabolism; we measured glucose consumption in PTEN wild-type or knockdown cells after transient Akt2 overexpression. iv) Next, we transiently expressed Akt2 in non-metastatic human CRC cell lines with wildtype PTEN and mutant PTEN; mice were examined by real-time fluorescence imaging for metastasis formation and growth. RESULTS. i) Marked Akt2 immunoreactivity was detected in 70% of well- or moderately-differentiated human CRC. In 88% (n=32/36 cases), Akt2 expression was greater in the liver metastases than in primary CRCs. Akt2 mRNA expression was increased ~7-fold compared to normal mucosa. Increased Akt2 expression at early CRC stages suggests that additional mechanisms are required for metastatic progression. ii) When subjected to GFDS, the mutant or stable knockdown PTEN cells displayed resistance to GFDS-induced apoptosis relative to wild-type cells. The mutant PTEN cells also showed
AGA Abstracts
A-6