CD68-positive Cell Infiltration Research Articles

Prognostic significance of immunohistochemical classification utilizing biopsy specimens in patients with extensive-disease small-cell lung cancer treated with first-line chemotherapy and immune checkpoint inhibitors

PurposeAlthough immune checkpoint inhibitors (ICIs), together with cytotoxic chemotherapy (chemoimmunotherapy), have been adapted for the initial treatment of extensive-disease small-cell lung cancer (ED-SCLC), they have achieved limited success. In ED-SCLC, a subtype of SCLC, the expression of immune-related molecules and clinical data are not well understood in relation to ICI treatment efficiency.MethodsWe examined lung biopsy specimens from patients diagnosed with ED-SCLC treated with chemoimmunotherapy or chemotherapy. SCLC subtype, expression of HLA class I, and infiltration of CD8-positive cells were examined using immunohistochemistry (IHC). Subsequently, the association between clinical factors, IHC results, and progression-free survival or overall survival was assessed.ResultsMost of the cases showed the achaete-scute homolog 1 (ASCL1) subtype. Among the 75 SCLC cases, 29 expressed high levels of HLA class I, while 46 showed low levels or a negative result; 33 patients were characterized as CD8-high, whereas 42 were CD8-low. In the chemoimmunotherapy cohort, multivariate analysis revealed a correlation between CD8-high and improved survival. Specifically, patients in the CD8-high group of the chemoimmunotherapy cohort experienced enhanced survival compared to those in the chemotherapy cohort, which was attributed to ICI addition. IHC subtype analysis demonstrated a survival advantage in the SCLC-I and SCLC-A groups when ICI was combined with chemotherapy compared to chemotherapy alone.ConclusionOur study highlights the predictive value of IHC-classified subtypes and CD8-positive cell infiltration in estimating outcomes for patients with ED-SCLC treated with chemoimmunotherapy as a first-line therapy. These findings have practical implications for daily clinical assessments and treatment decisions.

Xihuang pill ameliorates colitis in mice by improving mucosal barrier injury and inhibiting inflammatory cell filtration through network regulation

Ethnopharmacological relevanceThe prevalence of colitis is on the rise, and effective treatment options are currently lacking. Xihuang pill (XHP) is a traditional Chinese medicine formula mentioned in the “Volume 4 of Surgical Evidence and Treatment of the Whole Life” authored by the renowned doctor Hong-Xu Wang during the Qing Dynasty. It is now part of the “Volume 9 of Chinese medicine formula preparation in Drug Standard.” XHP and its primary ingredients have been demonstrated anti-inflammatory properties against colitis. However, the specific effects and underlying mechanisms of XHP in treating colitis remain unknown. Aim of the studyThis study aimed to investigate the potential impact of XHP on colitis and uncover the underlying mechanisms involved. Materials and methodsAn acute colitis model was developed in C57BL/6N mice, and the effects on weight loss, colon length, the permeability of the colonic mucosa barrier, Claudin-5 and Occludin expression, number of both infiltrating MPO-positive cells and CD68-positive cells, and the content of pro-inflammatory cytokines (IL-6, IL-22, IL-1β, and TNF-α) in the colon tissue were investigated. Low-, medium-, and high-dose XHP (0.45, 0.9, and 1.8 g/kg/day) (batch number: z21021222) were administered to the mice by gavage over the course of two weeks. Additionally, the protein expression levels in colon tissue from the control group, colitis group, and XHP low-dose administration group mice were analyzed by quantitative proteomics techniques. The comprehensive profiling and characterization of absorbed components in mice blood following oral administration of XHP were identified by HPLC/Q-TOF-MS techniques, and the absorbed components in blood were combined with proteomics to reveal the mechanism of enteritis inhibition by XHP. ResultsOur findings indicated that XHP enhanced weight loss and colonic shortening of colitis mice. Additionally, XHP reduced the increase in permeability of the colonic mucosa barrier and decreased expression of Claudin-5 and Occludin, while significantly reducing the number of infiltrating MPO-positive cells and CD68-positive cells in the colon tissue. We found that XHP reduced the production of pro-inflammatory cytokines, including IL-6, IL-22, IL-1β, and TNF-α in colon tissue. Pharmacokinetic analysis suggested that XHP contained 24 blood-entering prototype ingredients, which improved colitis through the regulation of various proteins (e.g., Ctsb, Sting1, and Abat) linked to mucosal barrier injury and inflammation. ConclusionXHP improved intestinal mucosal barrier injury and reduced MPO-positive cells and CD68-positive cell infiltration through multiple targets and pathways, providing support for XHP as a promising therapy for colitis.

Angiogenic inhibitor pre-administration improves the therapeutic effects of immunotherapy.

In lung cancer, immune checkpoint inhibitors (ICIs) are often inadequate for tumor growth inhibition. Angiogenic inhibitors (AIs) are required to normalize tumor vasculature for improved immune cell infiltration. However, in clinical practice, ICIs and cytotoxic antineoplastic agents are simultaneously administered with an AI when tumor vessels are abnormal. Therefore, we examined the effects of pre-administering an AI for lung cancer immunotherapy in a mouse lung cancer model. Using DC101, an anti-vascular endothelial growth factor receptor 2 (VEGFR2) monoclonal antibody, a murine subcutaneous Lewis lung cancer (LLC) model was used to determine the timing of vascular normalization. Microvessel density (MVD), pericyte coverage, tissue hypoxia, and CD8-positive cell infiltration were analyzed. The effects of an ICI and paclitaxel after DC101 pre-administration were investigated. On Day 3, increased pericyte coverage and alleviated tumor hypoxia represented the highest vascular normalization. CD8+ T-cell infiltration was also highest on Day 3. When combined with an ICI, DC101 pre-administration significantly reduced PD-L1 expression. When combined with an ICI and paclitaxel, only DC101 pre-administration significantly inhibited tumor growth, but simultaneous administration did not. AI pre-administration, and not simultaneous administration, may increase the therapeutic effects of ICIs due to improved immune cell infiltration.

Immune Profile of BRAF-Mutated Metastatic Colorectal Tumors with Good Prognosis after Palliative Chemotherapy.

Simple SummaryThe present study demonstrated that the distinct subgroup of BRAF-MT CRC showed a good response after palliative chemotherapy. Based on the immune profile analysis, higher PD-L1 expression and CD8-positive cell infiltration were shown in this study population. Furthermore, the assessment of the immune profile of BRAF-MT tumors can be helpful to better understand tumor biology and the different clinical outcomes of BRAF-MT CRC. Background: BRAF-mutated colorectal cancers (BRAF-MT CRCs) are known to have poor prognoses. BRAF-MT CRC was reported to be possibly related to the immune-activated phenotype. Objectives: This study aimed to investigate the association between the immune microenvironment and prognosis of BRAF-MT CRC. Methods: We evaluated clinical outcomes and investigated the immune profile of the BRAF-MT CRC tumors using the multiplex immunohistochemistry of immune-related markers: cytokeratin, programmed death ligand-1 (PD-L1), programmed cell death protein-1 (PD-1), and a cluster of differentiation 8 (CD8). Results: Out of 2313 tumors, 123 were BRAF-MT tumors. Among them, 86 tumors with available tissue were included. Out of 86 patients, 75 patients were non-good responders (GR), whereas 11 patients were GR. Median progression-free survival after first-line chemotherapy (4.6 vs. 12.4 months, p = 0.008) and overall survival (11.8 vs. 45.0 months) were longer in the GR group (p < 0.001). Median CD8+ T cell (254.29 vs. 656.0, p = 0.092), PD-L1+ tumor cell (0.95 vs.15.56, p = 0.050), PD-L1+ stromal cell (3.17 vs. 72.38, p = 0.025), PD-L1+ tumor and stromal cell (5.08 vs. 74.92, p = 0.032), and PD-1+ stromal cell (45.08 vs. 325.40, p = 0.046) counts were greater in the GR group. Conclusion: The clinical outcomes of unselected patients with BRAF-MT CRC were generally similar to those in previous studies. Based on the immune profile analysis, higher PD-L1 expression and CD8-positive cell infiltration were observed in BRAF-MT tumors with a good prognosis.

Vitamin C alleviates alcoholic liver injury by suppressing neutrophil infiltration in senescence marker protein 30-knockout mice irrespective of its antioxidant effects

AimsAlcoholic liver disease (ALD) comprises an important component in chronic liver diseases, and its clinical significance has increased due to the high consumption of alcohol worldwide. Vitamin C is a potent antioxidant, and several previous studies have suggested that its therapeutic role in ALD is derived from its antioxidant role. However, its anti-inflammatory role in ALD remains to be elucidated. Especially, the relationship between vitamin C and infiltration of neutrophils in ALD has not been discussed to date. For the reason, the present study investigated the precise role of vitamin C in neutrophil infiltration in ALD. Main methodsIn the present study, wild-type C57BL/6 and vitamin C-deficient senescence marker protein 30-knockout mice were pair-fed with a Lieber–DeCarli control or ethanol diet. Ethanol-fed groups were fed with increasing concentrations of EtOH (Lieber–DeCarli control diet for 5 days, 3% EtOH diet for a week, and 5% diet for 2 weeks) with or without vitamin C supplementation. Key findingsVitamin C dramatically attenuated the ethanol-mediated liver disease in the vitamin C-deficient ethanol-fed mice group by suppressing the infiltration of neutrophils accompanied by less CD68-positive cell infiltration. This attenuating role of vitamin C in neutrophil infiltration in the liver is associated with its protective effect for the ethanol-mediated intestinal damage in vitamin C-deficient ethanol-fed mice. SignificanceThis study provides a novel possibility of vitamin C to be used as an anti-inflammatory therapeutic agent associated with neutrophil infiltration in ALD, thereby helping to establish strategies for attenuating ALD.

The NLRP3 inhibitor dapansutrile attenuates folic acid induced nephrotoxicity via inhibiting inflammasome/caspase-1/IL axis and regulating autophagy/proliferation

AimsChemical renal toxicity is common and has limited therapeutic interventions. The NLRP3 inhibitor dapansutrile (DAPA) undergoes clinical phase II trials and it shows promising beneficial effects in various inflammatory diseases. The current study aims at evaluating the effect of DAPA on folic acid (FA) induced acute kidney injury (AKI) and its possible transition to chronic injury. Materials and methodsTwo treatment protocols were studied depending on DAPA injection timing. A prophylactic protocol involving the injection of DAPA (0.2 mg/kg) daily for seven days before FA challenge and a therapeutic protocol where DAPA was injected after FA. Each protocol included four groups of rats: control group, DAPA group, FA group and DAPA+FA group. Serum creatinine, urea and uric acid were measured. Also, kidney injury, necrosis and fibrosis percentage in addition to infiltration of CD68 positive cells were evaluated. Activation markers of inflammasome and the expression of Ki-67 and LC-3 were measured. Key findingsResults showed an improvement in renal tissue integrity and a significant decrease in kidney function biomarkers, caspase-1, IL-1β and IL-18 by DAPA injection (p < 0.05). In addition, DAPA decreased the proliferation marker Ki-67 and the autophagic marker LC-3 (p < 0.01). SignificanceDAPA potentially alleviates FA induced nephrotoxicity through targeting inflammasome/caspase-1/IL axis. Moreover, it shows a regulatory effect on renal regeneration and autophagy.

Multitarget molecule, PTUPB, to treat diabetic nephropathy in rats.

Diabetic nephropathy is a common complications related to high morbidity and mortality in type 2 diabetes. We investigated the action of the dual modulator, PTUPB, a soluble epoxide hydrolase and cyclooxygenase-2 inhibitor against diabetic nephropathy. Sixteen-week-old type 2 diabetic and proteinuric obese ZSF1 rats were treated with vehicle, PTUPB or enalapril for 8weeks. Measurements were made of epoxyeicosatrienoic acids, thromboxane B2 (TBX2 ) and prostaglandin E2 (PGE2 ) in the kidney of these and lean ZSF1 rats along with their blood pressure. Obese ZSF1 rats were diabetic with fivefold higher fasting blood glucose levels and markedly higher HbA1c levels compared with lean ZSF1 rats. PTUPB nor enalapril reduced fasting blood glucose or HbA1c but alleviated the development of diabetic nephropathy. In PTUPB-treated obese ZSF1 rats, glomerular nephrin expression was preserved. Enalapril also alleviated diabetic nephropathy. Diabetic renal injury in obese ZSF1 rats was accompanied by renal inflammation with six to sevenfold higher urinary MCP-1 (CCR2) level and renal infiltration of CD-68 positive cells. PTUPB and enalapril significantly reduced urinary MCP-1 levels and renal mRNA expression of cytokines. Both PTUPB and enalapril lowered blood pressure. PTUPB but not enalapril decreased hyperlipidaemia and liver injury in obese ZSF1 rats. Overall, the dual modulator PTUPB does not treat hyperglycaemia but can effectively alleviate hypertension, diabetic nephropathy, hyperlipidaemia and liver injury in type 2 diabetic rats. Our data further demonstrate that the renal actions of PTUPB are comparable with a current standard diabetic nephropathy treatment.

Molecular mechanism of FGFR activation and resulting differences in the tumor-immune environment of bladder cancer.

432 Background: Activation of FGFR signals in bladder cancer is frequently observed. Previous study reported that infiltration of tumor immune cells is suppressed in FGFR3 mutated bladder cancer. In this study, we elucidate the relationship between molecular mechanism of FGFR activation and tumor immune environment in bladder cancer. Methods: We analyzed 103 tissues of bladder cancer patients. Detection of somatic mutations were performed by target sequencing and gene expressions were analyzed by RNA-seq. Immunohistochemical staining of CD8 was performed for evaluating tumor-infiltrating lymphocytes. We compared the relationship between molecular mechanism of FGFR activation and tumor immune environment. Results: FGFR3 activated mutation or fusion was found in 37 cases (36%). FGFR1 overexpression was found in 10 cases (10%). FGFR3 activated mutation or fusion and FGFR1 overexpression were mutually exclusive. Tumors harboring FGFR3 mutation showed less infiltration of CD8 positive cells ( p = 0.01) and low PD-L1 expression ( p = 0.08) compared to those without. On the other hand, tumors with FGFR1 overexpression showed more infiltration of CD8 positive cells ( p = 0.06) and high PD-L1 expression ( p = 0.02). Conclusions: Clinicopathological features and intratumor immune environment of bladder cancer is different depending on the molecular mechanism of FGFR activation. These results suggest a possible association between molecular mechanism of FGFR activation and sensitivity of immunotherapy.

Oncolytic activity of HF10 in head and neck squamous cell carcinomas

Recent developments in therapeutic strategies have improved the prognosis of head and neck squamous cell carcinoma (HNSCC). Nevertheless, 5-year survival rate remains only 40%, necessitating new therapeutic agents. Oncolytic virotherapy entails use of replication-competent viruses to selectively kill cancer cells. We aimed to explore the potential of HF10 as an oncolytic virus against human or mouse HNSCC cell lines, and primary-cultured HNSCC cells. HF10 replicated well in all the HNSCC cells, in which it induced cytopathic effects and cell killing. Next, we investigated the oncolytic effects of HF10 in ear tumor models with human or mouse tumor cells. We detected HF10-infected cells within the ear tumors based on their expression of green fluorescent protein. HF10 injection suppressed ear tumor growth and prolonged overall survival. In the syngeneic model, HF10 infection induced tumor necrosis with infiltration of CD8-positive cells. Moreover, the splenocytes of HF10-treated mice released antitumor cytokines, IL-2, IL-12, IFN-alpha, IFN-beta, IFN-gamma, and TNF-alpha, after stimulation with tumor cells in vitro. The HF10-treated mice that survived their original tumor burdens rejected tumor cells upon re-challenge. These results suggested that HF10 killed HNSCC cells and induced antitumoral immunity, thereby establishing it as a promising agent for the treatment of HNSCC patients.

MON-316 DPYSL4 Regulates Oxygen Consumption Associating with Mitochondrial Supercomplexes

Metabolic regulatory function of p53 can play a role in maintaining homeostasis and protecting cells from damage. Here we have identified Dihydropyrimidinase-like 4 (DPYSL4) is a p53-inducible regulator of energy metabolism using RNA sequencingin both cancer cells and normal cells, such as adipocytes. DPYSL4 was localized in the mitochondria in response to DNA damage or hypoxia. Mitochondria was subjected to 2-D BN/SDS-PAGE followed by immunoblotting. DPYSL4 was detected in the same vertical position as complexes I,IIIand IV. To obtain direct evidence of a functional role in energy regulation, we examined the oxygen consumption rate (OCR) using flux analyzer. The knockdown of p53 or DPYSL4 by the CRISPR/Cas9 system showed reduction in OCR in preadipocytes. OCR of HCT116 p53+/+cellswas higher than p53-/-cells and OCR of p53 or DPYSL4 silencing were lower than p53+/+cells with treated by control siRNA. The overexpression of DPYSL4 in H1299 led to elevation of OCR and the overexpression of DPYSL4 deletion mutantdid not lead to elevation of OCR. Overexpression of DPYSL4 in H1299 cells led to a significant reduction matrigel invasion and canceled by DPYSL4 deletion mutant. In line with its loss of function in energy regulation and tumor suppressor function, DPYSL4 deletion mutant abolished its interaction with supercomplexes.Immunohistology of adipose tissues demonstrated that p53 was significantly upregulated in obese patients compared with non-obese patients. This is concomitant with marked infiltration of CD68-positive cells in adipocytes of obese patients. Taken together, these data indicate that DPYSL4 has a tumor suppressor function associated with OXPHOS regulation and may be concerned with pathogenesis of cancer and obesity regulated under p53.

Tumor Microenvironment Properties are Associated With Low CD68-positive Cell Infiltration and Favorable Disease-free Survival in EGFR-mutant Lung Adenocarcinoma

BackgroundThe benefits of immune checkpoint inhibitors for first-line treatment in patients with lung adenocarcinoma harboring EGFR mutations are unclear. The effects of ICIs depend on the tumor microenvironment (TME). Differences in TME properties between mutant and wild-type EGFR have not been fully characterized. Patients and MethodsWe collected 105 surgically resected (50 EGFR mutated and 55 EGFR wild-type), treatment-naïve lung adenocarcinoma tissues with clinical data to investigate the landscape and compartmentalization of tumor-infiltrating immune cells with respect to EGFR status by immunohistochemistry. The normalized FPKM values of data for 531 patients were obtained from The Cancer Genome Atlas (TCGA) Data Portal (https://portal.gdc.cancer.gov/). ResultsCD68-positive cells within the tumor niche exhibited more intensive infiltration in wild-type EGFR than in mutations, and was related to lymph node invasion. In the RNA-Seq analysis, MMP9 and VEGFA showed higher levels in wild-type EGFR than in mutant cases. The EGFR mutation independently predicted a favorable disease-free survival. ConclusionThe CD68-positive cells play a crucial role in discriminating the TME between different EGFR statuses.

Tumor-associated macrophage infiltration is highly associated with PD-L1 expression in gastric adenocarcinoma

Programmed death ligand 1 (PD-L1) is a key protein upregulated by tumor cells to suppress immune responses. Tumor-associated macrophages (TAMs) play a major role in this immunosuppression, but the relationship between PD-L1 expression and TAMs remains unclear in gastric adenocarcinoma (GAC). We simultaneously examined expression of PD-L1 and TAMs in GAC. We performed immunohistochemical staining for PD-L1, CD68 (pan-macrophage), and CD163 (M2-like macrophage) in 217 GAC samples using a tissue microarray. Expression of PD-L1 and CD68- and CD163-positive cells was evaluated using the Cytoplasmic V2.0 algorithm in Aperio ImageScope software, and logistic regression analysis was used to compare expression patterns between groups. Thirty-one samples (14%) were positive for PD-L1 expression. The mean (± standard error) rates of infiltration were 6.83±0.38% for CD68-positive cells and 6.16±0.29% for CD163-positive cells. The mean rate of CD163-positive cell infiltration was significantly higher in diffuse GAC than in intestinal GAC (diffuse n=111, 6.91%; intestinal n=91, 5.26%; p=0.006), but the mean rate of CD68-positive cell infiltration was similar between these types (p=0.38). The mean infiltration rates of CD68- and CD163-positive cells in PD-L1-positive GAC were significantly higher than in PD-L1-negative GAC (CD68 p=0.0002; CD163 p<0.0001). In multivariate logistic regression analyses, CD163-positive cell infiltration was associated with PD-L1 expression (odds ratio 1.13; 95% confidence interval 1.02-1.25; p=0.021). M2-like macrophage infiltration is highly associated with PD-L1 expression in GAC cells, suggesting that macrophage infiltration can serve as a potential therapeutic target.

Involvement of infiltrating macrophage-derived activin A in the progression of renal damage in MRL-lpr mice.

Lupus nephritis is a life-threatening complication of systemic lupus erythematosus (SLE). Various growth factors, cytokines, and chemokines are implicated in the development of SLE. However, the pathophysiological processes involved in the development of lupus nephritis still remain unclear. In this study, we examined the involvement of activin A, a member of the transforming growth factor β (TGF-β) superfamily, in the progression of renal damage in lupus-prone MRL-lpr mice. Activin A was not expressed in the kidneys of control MRL-MpJ mice but was detectable in perivascular infiltrating cluster of differentiation 68 (CD68)-positive cells in the kidneys of MRL-lpr mice. Urinary activin A, which was also absent in MRL-MpJ mice, was detectable in MRL-lpr mice from 16 wk onward. Urinary activin A levels were significantly correlated with the number of perivascular inflammatory cell layers, the number of crescentic glomeruli, and the percentage of Elastica van Gieson (EVG)-positive fibrotic areas, but not with urinary protein levels or serum activin A. When activin action was blocked in vivo by the intraperitoneal administration of an activin antagonist, follistatin, the number of crescentic glomeruli, percentage of EVG-positive fibrotic areas, CD68-positive cell infiltration, and proteinuria were significantly reduced in a dose-dependent manner. These data suggest that infiltrating macrophage-derived activin A is involved in the progression of renal damage in MRL-lpr mice.

Anti-bevacizumab idiotype antibody vaccination is effective in inducing vascular endothelial growth factor-binding response, impairing tumor outgrowth.

Tumors require blood supply and, to overcome this restriction, induce angiogenesis. Vascular endothelial growth factor (VEGF) plays an important role in this process, which explains the great number of antiangiogenic therapies targeting VEGF. The research and development of targeted therapy has led to the approval of bevacizumab, a humanized anti‐VEGF monoclonal antibody (mAb), in clinical settings. However, side effects have been reported, usually as a consequence of bolus‐dose administration of the antibody. This limitation could be circumvented through the use of anti‐idiotype (Id) antibodies. In the present study, we evaluated the efficacy of an active VEGF‐binding immune response generated by an anti‐bevacizumab idiotype mAb, 10.D7. The 10.D7 anti‐Id mAb vaccination led to detectable levels of VEGF‐binding anti‐anti‐Id antibodies. In order to examine whether this humoral immune response could have implications for tumor development, 10.D7‐immunized mice were challenged with B16‐F10 tumor cells. Mice immunized with 10.D7 anti‐Id mAb revealed reduced tumor growth when compared to control groups. Histological analyses of tumor sections from 10.D7‐immunized mice showed increased necrotic areas, decreased CD31‐positive vascular density and reduced CD68‐positive cell infiltration. Our results encourage further therapeutic studies, particularly if one considers that the anti‐Id therapeutic vaccination maintains stable levels of VEGF‐binding antibodies, which might be useful in the control of tumor relapse.

Influence of blood pressure variability on the life of arteriovenous fistulae in maintenance hemodialysis patients.

Although numerous risk factors for arteriovenous fistulae (AVF) dysfunction have been identified, these risk factors do not explain all cases of AVF dysfunction. Because of the importance of blood pressure variability (BPV) in vascular injury, the predictive value of BPV for AVF dysfunction, was evaluated in this prospective cohort study. Twenty-four-hour BP monitoring at the intervals of dialysis was recorded every 3 months in 137 patients. The expression of smooth muscle actin (SMA) and the infiltration of mononuclear cells and T lymphocytes were determined by immunohistochemistry on the specimens of fistula vessels. Eighteen patients developed AVF dysfunction. Cox proportional hazards multivariate analysis revealed a significant relationship between fistula dysfunction and daytime systolic-BPV (d-SBPV), nighttime systolic-BPV (n-SBPV), diabetes mellitus, and initial venous diameter. Patients with AVF dysfunction were observed to have increased SMA expression and more infiltration of inflammatory cells in venous walls compared with the controls. A significant correlation between SBPV and the infiltration of CD68-positive cells was observed. Our study showed that the degrees of SBPV were significantly associated with the risk of AVF dysfunction. Potentially, the increase of SBPV will aggravate venous wall inflammation and may play a role in AVF dysfunction.

Glucagon-like peptide-1 prevented abdominal aortic aneurysm development in rats.

To demonstrate the protective effect of glucagon-like peptide 1 (GLP-1) signaling on the cardiovascular system, we conducted this study to show that the GLP-1 receptor analog (lixisenatide) could inhibit abdominal aortic aneurysm (AAA) development in rats. Lixisenatide was injected subcutaneously 7days after aneurysm preparation. We evaluated reactive oxygen species (ROS) expression by dihydroethidium staining and 8-hydroxydeoxyguanosine (8-OHdG; the oxidation product of DNA) by immunohistochemical staining. We also analyzed the effect of GLP-1 signaling on the inflammatory response. Histopathological examination was done on day 28, and the AAA dilatation ratio was calculated. On day 14, ROS expression and 8-OHdG-positive cells in the aneurysm walls were seen to have been significantly decreased by lixisenatide treatment. Western blot analysis showed decreased ERK expression. There was significantly reduced tumor necrosis factor-α mRNA expression in the aneurysm walls and CD68-positive cell infiltration in the aneurysm walls. On day 28, it was evident that the lixisenatide had dramatically reduced aneurysm development in the rats. GLP-1 elevation inhibits AAA development in rats through its anti-oxidant and anti-inflammatory effects. Thus, GLP-1 could be a potent pharmacological target for AAA treatment.

Abstract W P73: Inside Intramural Thrombus Formation with Inflammatory Reactions Is Relevant to the Rupture of Cerebral Aneurysms

Background and Purpose: Although recent researches on cerebral aneurysms (CAs) have been performed with the hydrodynamic or the molecular biological technique, the mechanisms of rupture are not fully understood. The aim of this study is to assess the mechanism by a comparison between ruptured and un-ruptured CAs with histopathological and electron-microscopic analysis. Methods: We analyzed 33 CAs (24 ruptured, 9 un-ruptured) collected surgically after neck clipping. As for the ruptured CAs, we operated them within 24 hours from the onset. HE staining, Elastica Masson staining, PTAH staining, and CD68 immunohistochemical staining were performed with paraffin sections. Morphological analysis with Scanning Electron Microscopy (SEM) was performed with 6 CAs (3 ruptured, 3 un-ruptured). Results: The common histopathological finding in both ruptured and un-ruptured CAs was that the aneurysmal wall consisted mostly of thick collagen layer without normal internal elastic lamina and media. The characteristic histopathological finding in ruptured CAs was inside intramural thrombus formation with infiltration of CD68 positive cells at the rupture point. The common SEM finding in both ruptured and un-ruptured CAs was the denudation of endothelial cells and the exposure of a subendothelial amorphous or a fibrous surface. The characteristic SEM finding in ruptured CAs was the cluster formation of thick fibrin fibers with incorporation of macrophages and platelets. Conclusions: While the endothelial denudation, the disappearance of internal elastic lamina and media, and the predominance of collagen layer in the aneurysmal wall were common in both ruptured and un-ruptured CAs, inside intramural thrombus formation with inflammatory reactions was characteristic only in ruptured CAs. This result suggests that thrombo-inflammatory reactions in CAs may act as a trigger for ruptures.

Abstract 4715: OX40 expression improves the prognostic value of CD8 positive lymphocyte density in colorectal carcinoma

Abstract Background: The prognostic role of the immunological microenvironment in colorectal cancer (CRC) is well known. Among immune cell types, CD8 positive T lymphocyte infiltration is an established prognostic marker clearly associated with a favorable clinical course of the disease. In contrast, there is limited information about the prognostic role of OX40 positive immune cell infiltration in CRC. However, increasing evidence suggests that OX40 positive immune cell infiltration, as a single biomarker, has a positive prognostic effect in CRC patients. To the best of our knowledge, the impact of the combination of OX40 positive immune cells and CD8 positive T lymphocytes is unknown. Methods: A tissue microarray composed of 441 biopsies was evaluated for OX40 positive immune cells and CD8 positive T lymphocyte infiltration by immunohistochemistry. The intratumoral number of tumor infiltrating cells positive for OX40 and CD8 antigens was evaluated by 2 independent investigators achieving an optimal concordance rate of 90%. According to the frequency and staining intensity, we identified four subgroups of cell population: OX40low/CD8low, OX40high/CD8low, OX40low/CD8high and OX40high/CD8high. They were correlated with clinicopathological data, and a combined survival analysis was performed. Results: The mean age of the patients cohort was 69.9 ±10.7 years. A total of 230 (52%) biopsies were from female and 211 (48%) from male CRC patients. Both, OX40 and CD8 positive cell infiltration had a significant, prognostic impact on the clinical outcome of the disease as individual markers (p&amp;lt;0.0001 and p=0.007, respectively). Remarkably, the combination of OX40 and CD8 positive cell infiltration (OX40high/CD8high), significantly enhanced the overall survival of these patients (p&amp;lt;0.0001). These results were maintained after a multivariate Cox regression analysis including T and N stages, grade, vascular invasion, tumor border configuration, microsatellite stability and CD8 and OX40 positive cell infiltration suggesting that this combination is an independent prognostic factor in CRC patients. Conclusion: The combination OX40 and CD8 positive cell infiltration significantly enhances the overall survival compared to OX40 and CD8 positive cell infiltration alone. OX40high/CD8high positive cell infiltration is an independent, favorable, prognostic marker in CRC. Citation Format: Benjamin Weixler, Raoul A. Droeser, Roberto Sorge, Tarik Delko, Christian A. Nebiker, Giandomenica Iezzi, Giulio C. Spagnoli, Christoph Kettelhack, Urs W. von Holzen, Daniel Oertli, Luigi Terracciano, Luigi Tornillo, Giuseppe Sconocchia. OX40 expression improves the prognostic value of CD8 positive lymphocyte density in colorectal carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4715. doi:10.1158/1538-7445.AM2014-4715

TGF-β1 precursor and CD8 are potential prognostic and predictive markers in operated breast cancer

The transforming growth factor β1 (TGF-β1) and CD8-positive T cells are two important immune factors that function at opposite directions. The purpose of this study was to verify the relationship between the two factors and their associations with long-term effects of adjuvant chemotherapy or endocrine therapy in breast cancer. Expression of TGF-β1 precursor and CD8 was immunohistochemically detected on surgically-obtained tumor samples of 130 (stage I-III) invasive breast carcinomas from Chinese subjects, who were followed up for a mean time of 112 months. Interstitial CD8-positive cells and TGF-β1 precursor-positive cells adjacent to tumor nests were counted. Infiltration of CD8-positive lymphocytes into tumor nests and TGF-β1 precursor expression in tumor cells were observed and survival analysis was performed. Our results showed that density of interstitial CD8-positive lymphocytes was an independent adverse prognostic factor for distant disease-free survival (DDFS) (HR=8.416, 95% CI=1.636-43.292, P=0.011) in hormone receptor-positive patients who were on adjuvant endocrine therapy. For breast cancer patients who did not receive adjuvant chemotherapy, those without infiltration of CD8-positive cells into tumor nests had a shorter overall survival (OS) than their counterparts with CD8-positive cell infiltration into tumor nests (Log-Rank, P=0.003). But OS of patients without infiltration of CD8-positive cells into tumor nests was significantly prolonged by adjuvant chemotherapy (Log-Rank, P=0.013) and paralleled that of patients with CD8-positive cell infiltration. Although OS was shorter in the tumor cell TGF-β1 precursor (t-TGF-β1-pre)-positive patients than in the negative patients in patients without receiving chemotherapy (P=0.053), OS of t-TGF-β1-pre-positive patients was significantly prolonged by adjuvant chemotherapy (P=0.035) and was longer than that of t-TGF-β1-pre-negative patients. Analysis showed that t-TGF-β1-pre was an independent positive prognostic factor for DDFS (HR=0.392 95% CI=0.157-0.978, P=0.045) in patients who received adjuvant chemotherapy. This study suggested that density of interstitial CD8-positive lymphocytes was of prognostic value in hormone receptor-positive patients who received adjuvant endocrine therapy. Our study verified that adverse immunologic signatures consisting of absence of CD8-positive cells in tumor nests or expression of TGF-β1 precursor in tumor cells in breast cancer were associated with worse prognosis and significantly improved long-term survival with adjuvant chemotherapy, respectively.

The density of macrophages in colorectal cancer is inversely correlated to TGF-β1 expression and patients’ survival

The role of macrophages in colorectal cancer tumorogenesis is complex because they can both prevent and promote tumor development. We investigated CD68-positive cell infiltration in tumor tissue and its correlations with proteins of TGF-β1 signaling pathway and survival of the patients after surgical therapy. A non-selected panel of 210 primary tumors of colorectal origin was investigated immunohistochemically with antibodies against CD68, TGF-β1, TGFβRII and Smad4. Lower CD68 infiltration in tumor stroma was associated with expression of TGF-β1 (p = 0.002) and SMAD4 (p = 0.090) in tumor cell cytoplasm and with TGFβRII expression (p = 0.017) in tumor cells membranes. The absence of SMAD4 immune deposits in tumor cell nuclei was more often seen in biopsies with low number of CD68 in the invasive front (p = 0.044). The low number of CD68-positive cells was significantly associated with several adverse clinical and histological tumor characteristics as the presence of metastases in local lymph nodes (p = 0.047), distant metastases (p = 0.0003), advanced tumor stage (p = 0.006), tumor cell invasion of blood, lymph vessels or perineural invasion (p = 0.004), higher histological types (p = 0.0002) and lower grade of inflammatory infiltration in the invasive front (p = 0.002). Moreover, the low grade of CD68 appeared to be significant unfavorable factors of prognosis of the patients with colorectal cancer. The results of our study confirm the prognostic significance of low level of tumor-associated macrophage infiltration in colorectal cancer as unfavorable marker for survival of the patients.