Postmenopausal obesity promotes tumor angiogenesis and breast cancer progression in mice

Obese postmenopausal women have a 50% higher risk of breast cancer than non-obese women. There is not an animal model that mimics postmenopausal obesity related to breast cancer progression. Using age-relevant C57BL/6 mice, this study determined whether postmenopausal obesity increases VEGF expression, tumor angiogenesis, and breast tumor growth. Ovariectomy (OVX) was performed in 12 sixty week-old female mice, then followed by a low-fat (5%, LF, n=6) or a high-fat (60%, HF, n=6) diet for 12 weeks. In the eighth week of the dietary program, 10(6) E0771 (mouse breast cancer) cells were injected in the left fourth mammary gland. Tumor size was monitored for 4 weeks. Body weights were monitored weekly. At the end of the experiment, blood samples, visceral fat and tumors were collected for measuring VEGF expression using ELISA and intratumoral microvessel density (IMD) using CD31 immunochemistry. Body weight was significantly increased in OVX/HF mice, compared to OVX/LF group (55.3±1.7 vs. 41.5±1.5 g; p < 0.01). There was a two-fold increase in the ratio of visceral fat/BW in OVX/HF mice, compared to those in OVX/LF group (0.062±0.005 vs. 0.032±0.003; p < 0.01). Postmenopausal obesity significantly increased breast tumor weight over the control (4.62±0.63 vs. 1.98±0.27 g; p < 0.01) and IMD (173±3.7 vs. 139±4.3 IM#/mm^2; p < 0.01). Tumor VEGF levels were higher in OVX/HF mice, compared to OVX/LF group (73.3±3.8 vs. 49.5±4.3 pg/mg protein; p < 0.01). Plasma VEGF levels (69±7.1 vs. 48±3.5 pg/ml) and visceral fat VEGF levels (424.4±39.5 vs. 208.5±22.4 pg/mg protein) were significantly increased in OVX/HF mice, compared to OVX/LF group, respectively (n=6; p < 0.01). Interestingly, adipose tissue primary culture showed that subcutaneous fat released more VEGF, compared to visceral fat (6.77±1.14 vs. 0.94±0.16 pg/mg tissue; n=6; p < 0.01). These findings support the hypothesis that postmenopausal obesity promotes tumor angiogenesis and breast cancer progression, possibly through increased adipose tissue mass and adipokines such as VEGF that could systemically and locally affect breast cancer progression.     

Clinicopathologic Characteristics of Breast Cancer Stem Cells Identified on the Basis of Aldehyde Dehydrogenase 1 Expression

Purpose

Breast cancer displays varying molecular and clinical features. The ability to form breast tumors has been shown by several studies with aldehyde dehydrogenase 1 (ALDH1) positive cells. The aim of this study is to investigate the association between ALDH1 expression and clinicopathologic characteristics of invasive ductal carcinoma.

Methods

We investigated breast cancer tissues for the prevalence of ALDH1⁺ tumor cells and their prognostic value. The present study included paraffin-embedded tissues of 70 patients with or without recurrences. We applied immunohistochemical staining for the detection of ALDH1⁺ cells. Analysis of the association of clinical outcomes and molecular subtype with marker status was conducted.

Results

ALDH1⁺ and ALDH1^- tumors were more frequent in triple-negative breast cancers and in luminal A breast cancers, respectively (p<0.01). ALDH1 expression was found to exert significant impact on disease free survival (DFS) (ALDH1⁺ vs. ALDH1^-, 53.1±6.7 months vs. 79.2±4.7 months; p=0.03) and overall survival (OS) (ALDH1⁺ vs. ALDH1^-, 68.5±4.7 months vs. 95.3±1.1 months; p<0.01). In triple-negative breast cancer (TNBC) patients, DFS and OS showed no statistical differences according to ALDH1 expression (ALDH1⁺ vs. ALDH1^-, 45.3±9.4 months vs. 81.3±7.4 months, p=0.52; 69.0±7.5 months vs. 91.3±6.3 months, p=0.67). However, non-TNBC patients showed significant OS difference between ALDH1⁺ and ALDH1^- tumors (ALDH1⁺ vs. ALDH1^-, 77.6±3.6 months vs. 98.0±1.0 months; p=0.04) with no statistical difference of DFS (ALDH1⁺ vs. ALDH1^-, 60.5±8.0 months vs. 81.8±4.6 months; p=0.27).

Conclusion

Our findings suggest that the expression of ALDH1 in breast cancer may be associated with TNBC and poor clinical outcomes. On the basis of our findings, we propose that ALDH1 expression in breast cancer could be correlated with poor prognosis, and may contribute to a more aggressive cancer phenotype.     

Proliferation,migration and invasion of triple negative breast cancer cells are suppressed by berbamine via the PI3K/Akt/MDM2/p53 and PI3K/Akt/mTOR signaling pathways

Breast cancer is the second most common cause of cancer-associated mortality among women worldwide, and triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Berbamine (BBM) is a traditional Chinese medicine used for the treatment of leukopenia without any obvious side effects. Recent reports found that BBM has anti-cancer effects. The present study aimed to investigate the effects of BBM on TNBC cell lines and the underlying molecular mechanism. MDA-MB-231 cells and MCF-7 cells, two TNBC cell lines, were treated with various concentrations of BBM. A series of bioassays including MTT, colony formation, EdU staining, apoptosis, trypan blue dye, wound healing, transwell, ELISA and western blotting assays were performed. The results showed that BBM significantly inhibited cell proliferation of MDA-MB-231 cells (P<0.05; IC₅₀=22.72 µM) and MCF-7 cells (P<0.05; IC₅₀=20.92 µM). BBM (20 µM) decreased the apoptosis ratio (percentage of absorbance compared with the control group) by 28.4±3.3% (P<0.05) in MDA-MB-231 cells, and 62.4±24.6% (P<0.05) in MCF-7 cells. In addition, BBM inhibited cell migration and invasion of TNBC cells. Furthermore, the expression levels of PI3K, phosphorylated-Akt/Akt, COX-2, LOX, MDM2 and mTOR were downregulated by BBM, and the expression of p53 was upregulated by BBM. These results indicated that BBM may suppress the development of TNBC via regulation of the PI3K/Akt/MDM2/p53 and PI3K/Akt/mTOR signal pathways. Therefore, BBM might be used as a drug candidate for the treatment of TNBC in the future.     

Endothelial follicle-stimulating hormone receptor expression in invasive breast cancer and vascular remodeling at tumor periphery

Background

Follicle-stimulating hormone receptor (FSHR) is expressed on the endothelial surface of blood vessels associated with solid tumor periphery, where angiogenesis is known to occur. The correlation between FSHR expression and formation of new peritumoral vessels has not been previously investigated.

Methods

We used immunohistochemical techniques involving specific antibodies to detect FSHR and the endothelial markers (CD34, VEGFR2, and D2-40) in tissue samples from 83 patients with lymph node-negative, invasive breast cancer representing four main clinical treatment groups: HR+/HER2-, HR+/HER2+, HR-/HER2+ and triple-negative.

Results

The FSHR+ vessels were exclusively located at breast cancer periphery, in a layer that extended 2 mm into and 5 mm outside of the tumor. The percentage of blood vessels expressing FSHR reached a maximum of 100% at the demarcation line between the tumor and the normal tissue. Common among FSHR+ vessels, regardless of breast cancer type, were the high densities of arterioles and venules (6.4 ± 1.4 and 13.9 ± 2.1 vessels/mm², respectively). These values were 3-fold higher that those noticed for CD34+ arterioles and venules associated with normal breast tissue located at a distance greater than 10 mm outside the tumors. The average density of FSHR+ and CD34+ blood vessels as well as of D2-40+ lymphatic vessels did not differ significantly among breast cancer subgroups. FSHR+ vessels did not express VEGFR2. The endothelial FSHR expression correlated significantly with the peritumoral CD34+ vessels’ density (p < 0.001) and tumor size (p = 0.01).

Conclusion

Endothelial FSHR expression in breast cancer is associated with vascular remodeling at tumor periphery.     

The expression and clinical value of tumor infiltrating dendritic cells in tumor tissues of patients with esophageal cancer

BackgroundAs dendritic cells (DCs) are the major antigen-presenting cells of the immune system, understanding their role in esophageal cancer is essential for the development of preventative and treatment strategies. This study investigated the expression level and clinical value of tumor infiltrating dendritic cells (TIDCs) in tumor tissues of patients with esophageal cancer.MethodsFrom January 2019 to January 2021, 184 patients with esophageal cancer treated were prospectively enrolled as the observation group and 184 patients with benign esophageal tumors were selected as the control group. Tumor tissue samples were obtained and the expression level and phenotypes of the TIDCs were analyzed. The correlation between TIDC expression and clinical characteristics of patients with esophageal cancer was investigated.ResultsThe density of the TIDCs in the observation group was lower than that in the control group (8.76±2.25 vs. 9.97±2.19; P=0.000). Furthermore, the percentage of major histocompatibility complex-II (MHC-II) positive DCs and the percentage of CD54 positive DCs were relatively lower in the observation group compared to the control group (6.60%±2.12% vs. 9.34%±2.41%; P=0.000 and 7.41%±2.36% vs. 9.98%±2.47%; P=0.000, respectively). Esophageal cancer patients with lymph node metastasis had lower TIDC density, lower percentage of MHC-II positive DCs, and lower percentage of CD54 positive DCs compared to patients without node metastasis (P<0.05). Patients with stage III esophageal cancer also showed significantly lower TIDC density, lower percentage of MHC-II positive DCs, and lower percentage of CD54 positive DCs compared to patients with stage I/II esophageal cancer (P<0.05). Esophageal cancer patients with tumor diameter ≥4 cm presented with decreased TIDC density, decreased percentage of MHC-II positive DCs, and decreased percentage of CD54 positive DCs compared to patients with tumor diameter <4 cm (P<0.05). In addition, the density of TIDCs, the percentage of MHC-II positive DCs, and the percentage of CD54 positive DCs were significantly negatively correlated with the percentage of CD4⁺ T-lymphocytes and positively correlated with the percentage of CD8⁺ T-lymphocytes (P<0.05).ConclusionsPatients with esophageal cancer had low expression and function of TIDCs, and this was related to the imbalance of T-lymphocyte subsets, lymph node metastasis, TNM stage, and lesion size.     

Mammographic density and risk of breast cancer by age and tumor characteristics

Introduction

Understanding whether mammographic density (MD) is associated with all breast tumor subtypes and whether the strength of association varies by age is important for utilizing MD in risk models.

Methods

Data were pooled from six studies including 3414 women with breast cancer and 7199 without who underwent screening mammography. Percent MD was assessed from digitized film-screen mammograms using a computer-assisted threshold technique. We used polytomous logistic regression to calculate breast cancer odds according to tumor type, histopathological characteristics, and receptor (estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor (HER2)) status by age (<55, 55–64, and ≥65 years).

Results

MD was positively associated with risk of invasive tumors across all ages, with a two-fold increased risk for high (>51%) versus average density (11-25%). Women ages <55 years with high MD had stronger increased risk of ductal carcinoma in situ (DCIS) compared to women ages 55–64 and ≥65 years (P_{age-interaction} = 0.02). Among all ages, MD had a stronger association with large (>2.1 cm) versus small tumors and positive versus negative lymph node status (P’s < 0.01). For women ages <55 years, there was a stronger association of MD with ER-negative breast cancer than ER-positive tumors compared to women ages 55–64 and ≥65 years (P_{age-interaction} = 0.04). MD was positively associated with both HER2-negative and HER2-positive tumors within each age group.

Conclusion

MD is strongly associated with all breast cancer subtypes, but particularly tumors of large size and positive lymph nodes across all ages, and ER-negative status among women ages <55 years, suggesting high MD may play an important role in tumor aggressiveness, especially in younger women.     

Lack of cyclin E immunoreactivity in non-malignant breast and association with proliferation in breast cancer.

Cyclin E is a G1 cyclin that is essential for the transition from G1 to S phase in the cell cycle. Alterations to cyclin E expression or regulation could be important in tumorigenesis. Previous immunohistochemical and immunoblotting studies have investigated the expression of cyclin E in breast carcinomas. In this study, cyclin E has been investigated in a range of non-malignant and malignant breast using immunohistochemistry. Normal and benign tissue from pre- and post-menopausal women (39 cases), non-involved tissue from cancer-containing breasts (47 cases), ductal carcinoma in situ (22 cases) and invasive breast carcinomas (109 cases) have been examined. There was no reactivity in any of the non-malignant breast. Only one ductal carcinoma in situ contained more than 5% reactive cells. A total of 28% of invasive carcinomas had > 5% of reactive cells (range 0-88% positive cells, mean 12.59%, median 1.0%). A significant association was found with poorer differentiation (P < 0.001), high MIB1 index (P < 0.001), lack of oestrogen receptor (0.05 > P > 0.025) and the presence of p53 protein (0.05 > P > 0.025). Virtually all cases with cyclin E and p53 were poorly differentiated. The presence of cyclin E is therefore only found in breast malignancies and is associated with more aggressive features, including high proliferation.     

The usefulness of F-18 FDG PET/CT-mammography for preoperative staging of breast cancer: comparison with conventional PET/CT and MR-mammography

Background

The objective of the study was to compare the diagnostic efficacy of an integrated Fluorine-18 fluorodeoxyglucose (F-18 FDG) PET/CT-mammography (mammo-PET/CT) with conventional torso PET/CT (supine-PET/CT) and MR-mammography for initial assessment of breast cancer patients.

Patients and methods

Forty women (52.0 ± 12.0 years) with breast cancer who underwent supine-PET/CT, mammo-PET/CT, and MR-mammography from April 2009 to August 2009 were enrolled in the study. We compared the size of the tumour, tumour to chest wall distance, tumour to skin distance, volume of axillary fossa, and number of meta-static axillary lymph nodes between supine-PET/CT and mammo-PET/CT. Next, we assessed the difference of focality of primary breast tumour and tumour size in mammo-PET/CT and MR-mammography. Histopathologic findings served as the standard of reference.

Results

In the comparison between supine-PET/CT and mammo-PET/CT, significant differences were found in the tumour size (supine-PET/CT: 1.3 ± 0.6 cm, mammo-PET/CT: 1.5 ± 0.6 cm, p < 0.001), tumour to thoracic wall distance (1.8 ± 0.9 cm, 2.2 ± 2.1 cm, p < 0.001), and tumour to skin distance (1.5 ± 0.8 cm, 2.1 ± 1.4 cm, p < 0.001). The volume of axillary fossa was significantly wider in mammo-PET/CT than supine-PET/CT (21.7 ± 8.7 cm³ vs. 23.4 ± 10.4 cm³, p = 0.03). Mammo-PET/CT provided more correct definition of the T-stage of the primary tumour than did supine-PET/CT (72.5% vs. 67.5%). No significant difference was found in the number of metastatic axillary lymph nodes. Compared with MR-mammography, mammo-PET/CT provided more correct classification of the focality of lesion than did MR-mammography (95% vs. 90%). In the T-stage, 72.5% of cases with mammo-PET/CT and 70% of cases with MR-mammography showed correspondence with pathologic results.

Conclusions

Mammo-PET/CT provided more correct definition of the T-stage and evaluation of axillary fossa may also be delineated more clearly than with supine-PET/CT. The initial assessment of mammo-PET/CT would be more useful than MR-mammography because the mammo-PET/CT indicates similar accuracy with MR-mammography for decision of T-stage of primary breast tumour and more correct than MR-mammography for defining focality of lesion.     

Regression of glioma tumor growth in F98 and U87 rat glioma models by the Nitrone OKN-007

Background

Glioblastoma multiforme, a World Health Organization grade IV glioma, has a poor prognosis in humans despite current treatment options. Here, we present magnetic resonance imaging (MRI) data regarding the regression of aggressive rat F98 gliomas and human U87 glioma xenografts after treatment with the nitrone compound OKN-007, a disulfonyl derivative of α-phenyl-tert-butyl nitrone.

Methods

MRI was used to assess tumor volumes in F98 and U87 gliomas, and bioluminescence imaging was used to measure tumor volumes in F98 gliomas encoded with the luciferase gene (F98_luc). Immunohistochemistry was used to assess angiogenesis (vascular endothelial growth factor [VEGF] and microvessel density [MVD]), cell differentiation (carbonic anhydrase IX [CA-IX]), hypoxia (hypoxia-inducible factor-1α [HIF-1α]), cell proliferation (glucose transporter 1 [Glut-1] and MIB-1), proliferation index, and apoptosis (cleaved caspase 3) markers in F98 gliomas. VEGF, CA-IX, Glut-1, HIF-1α, and cleaved caspase 3 were assessed in U87 gliomas.

Results

Animal survival was found to be significantly increased (P < .001 for F98, P < .01 for U87) in the group that received OKN-007 treatment compared with the untreated groups. After MRI detection of F98 gliomas, OKN-007, administered orally, was found to decrease tumor growth (P < .05). U87 glioma volumes were found to significantly decrease (P < .05) after OKN-007 treatment, compared with untreated animals. OKN-007 administration resulted in significant decreases in tumor hypoxia (HIF-1α [P < .05] in both F98 and U87), angiogenesis (MVD [P < .05], but not VEGF, in F98 or U87), and cell proliferation (Glut-1 [P < .05 in F98, P < .01 in U87] and MIB-1 [P < .01] in F98) and caused a significant increase in apoptosis (cleaved caspase 3 [P < .001 in F98, P < .05 in U87]), compared with untreated animals.

Conclusions

OKN-007 may be considered as a promising therapeutic addition or alternative for the treatment of aggressive human gliomas.     

Prognostic role of matrix metalloproteinase 9 in early breast cancer

MMP9 is involved in extracellular matrix degradation during various physiological and pathological conditions, including tumorigenesis. The present study aimed to assess the prognostic role of intratumoral MMP9 and to determine its association with circulating tumor cells (CTCs) in patients with early breast cancer. A total of 318 patients with primary breast cancer (PBC) were enrolled into the present study. Specimens were subjected to immunohistochemistry analysis, using the MMP9 monoclonal antibody. MMP9 expression was scored using a weighted histoscore (WH). The results demonstrated that the mean WH ± SEM for MMP9 expression was significantly higher in breast tumor cells compared with tumor associated stromas (132.0±5.2 vs. 50.8±3.7; P<0.00001). Furthermore, a positive association was observed between MMP9 expression, the hormone positive status and proliferation index of analysed breast cancer tumour cells. Notably, the prognostic role of MMP9 was not observed in tumor cells [hazard ratio (HR) =0.96; 95% confidence interval (CI), 0.58–1.59; P=0.864] or tumor associated stroma (HR=1.29; 95% CI, 0.60–2.78; P=0.547). Subgroup analysis demonstrated that patients that were HR negative or triple negative, with low MMP9 expression in tumor cells and stroma had a significantly improved disease-free survival than patients with high MMP9 expression. Taken together, the results of the present study demonstrated that high MMP9 expression in PBC was associated with favorable tumor characteristics. However, the prognostic value of MMP9 was limited to only the HR negative and CTC epithelial-to-mesenchymal transition positive subgroups. Thus, analyzing MMP9 tumor expression may help identify patients with increased risk of disease recurrence in these subgroups.     

International study on inter-reader variability for circulating tumor cells in breast cancer

Introduction

Circulating tumor cells (CTCs) have been studied in breast cancer with the CellSearch® system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement.

Methods

CellSearch® images (N = 272) of either CTCs or white blood cells or artifacts from 109 non-metastatic (M0) and 22 metastatic (M1) breast cancer patients from reported studies were sent to 22 readers from 15 academic laboratories and 8 readers from two Veridex laboratories. Each image was scored as No CTC vs CTC HER2- vs CTC HER2+. The 8 Veridex readers were summarized to a Veridex Consensus (VC) to compare each academic reader using % agreement and kappa (κ) statistics. Agreement was compared according to disease stage and CTC counts using the Wilcoxon signed rank test.

Results

For CTC definition (No CTC vs CTC), the median agreement between academic readers and VC was 92% (range 69 to 97%) with a median κ of 0.83 (range 0.37 to 0.93). Lower agreement was observed in images from M0 (median 91%, range 70 to 96%) compared to M1 (median 98%, range 64 to 100%) patients (P < 0.001) and from M0 and <3CTCs (median 87%, range 66 to 95%) compared to M0 and ≥3CTCs samples (median 95%, range 77 to 99%), (P < 0.001). For CTC HER2 expression (HER2- vs HER2+), the median agreement was 87% (range 51 to 95%) with a median κ of 0.74 (range 0.25 to 0.90).

Conclusions

The inter-reader agreement for CTC definition was high. Reduced agreement was observed in M0 patients with low CTC counts. Continuous training and independent image review are required.     

Angiogenesis is associated with the onset of hyperplasia in human ductal breast disease

Background:

The precise timing of the angiogenic switch and the role of angiogenesis in the development of breast malignancy is currently unknown.

Methods:

Therefore, the expression of CD31 (pan endothelial cells (ECs)), endoglin (actively proliferating ECs), hypoxia-inducible factor-1 (HIF-1α), vascular endothelial growth factor-A (VEGF) and tissue factor (TF) were quantified in 140 surgical specimens comprising normal human breast, benign and pre-malignant hyperplastic tissue, in situ and invasive breast cancer specimens.

Results:

Significant increases in angiogenesis (microvessel density) were observed between normal and benign hyperplastic breast tissue (P<0.005), and between in situ and invasive carcinomas (P<0.0005). In addition, significant increases in proliferating ECs were observed in benign hyperplastic breast compared with normal breast (P<0.05) cancers and in invasive compared with in situ cancers (P<0.005). Hypoxia-inducible factor-1α, VEGF and TF expression were significantly associated with increases in both angiogenesis and proliferating ECs (P<0.05). Moreover, HIF-1α was expressed by 60–75% of the hyperplastic lesions, and a significant association was observed between VEGF and TF in ECs (P<0.005) and invasive tumour cells (P<0.01).

Conclusions:

These findings are the first to suggest that the angiogenic switch, associated with increases in HIF-1α, VEGF and TF expression, occurs at the onset of hyperplasia in the mammary duct, although the greatest increase in angiogenesis occurs with the development of invasion.     

Multiple primary cancers of breast and cervix uteri: An epidemiological approach to analysis

Index sites of breast and cervix uteri were selected from populationbased data held at the West Midlands and Birmingham Regional Cancer Registry, and the expected numbers of second primary cancers in cervix and breast were computed (sequence analyses). In the breast series (17,756 patients) a small deficit of cervical tumours was observed (O = 16, E = 2·119, O/E = 0·76, P > 0·05), while in the cervix series (4817 patients) a small excess of breast tumours was found (O = 29, E = 23·38, O/E = 1·24, P > 0·05) over a period of 15 years.     

Local anesthetics inhibit kinesin motility and microtentacle protrusions in human epithelial and breast tumor cells

In breast cancer, there is a correlation between tissue factor (TF) expression, angiogenesis and disease progression. TF stimulates tumour angiogenesis, in part, through up-regulation of vascular endothelial growth factor (VEGF). Therefore, this study aimed to establish whether TF stimulates angiogenesis and tumour progression directly and independent of VEGF up-regulation. Initially, the effects of TF and VEGF were assessed on endothelial cell migration (Boyden chamber) and differentiation (tubule formation on Matrigel). Subsequently, MDA-MB-436 breast cancer cells, which produce high levels of both TF and VEGF (western blot analysis), were established in vivo, following which tumours were treated three times per week for 3 weeks with intra-tumoural injections of either anti-VEGF siRNA, anti-TF shRNA, the two treatments combined, or relevant controls. Both VEGF and TF significantly stimulated endothelial cell migration and tubule formation (P < 0.02). Breast cancer xenografts (MDA-MB-436) treated with TF or VEGF-specific agents demonstrated significant inhibition in tumour growth (VEGFsiRNA 61%; final volume: 236.2 ± 23.2 mm³ vs TFshRNA 89%; 161.9 ± 17.4 mm³ vs combination 93%; 136.3 ± 9.2 mm³ vs control 400.4 ± 32.7 mm³; P < 0.005). Microvessel density (MVD), a measure of angiogenesis, was also significantly inhibited in all groups (MVD in control = 29 ± 2.9; TFshRNA = 18 ± 1.1; VEGFsiRNA = 16.7 ± 1.5; both = 12 ± 2.1; P < 0.004), whereas the proliferative index of the tumours was only reduced in the TFshRNA-treated groups (control = 0.51 ± 0.011; TFshRNA = 0.41 ± 0.014; VEGFsiRNA = 0.49 ± 0.013; both = 0.41 ± 0.004; P < 0.008). These data suggest that TF has a direct effect on primary breast cancer growth and angiogenesis, and that specific inhibition of the TF-signalling pathway has potential for the treatment of primary breast cancer.     

The in situ local immune response,tumour senescence and proliferation in colorectal cancer

Background:

Immune cell infiltrates are important determinants of colorectal cancer (CRC) outcome. Their presence may be driven by tumour or host-specific factors. From previous studies in mice, senescence, a state of cell cycle arrest, may moderate tumour progression through upregulation of antitumour immune responses. The relationships between senescence and immune infiltrates have not previously been studied in humans. We explore whether a marker of senescence (p16^ink4a) in combination with low level expression of a proliferation marker (ki-67) relate to T cell infiltrates in CRC, and whether p16^ink4a, Ki-67 and immune infiltrates have similar prognostic value.

Methods:

Immunostaining of p16^inka and Ki-67 was performed within a CRC tissue microarray. Nuclear p16^inka and Ki-67 were categorised as high/low. T-cell markers, CD3, CD45RO, CD8 and FOXP3 were scored separately as high/low grade in three areas of the tumour: the invasive margin (IM), tumour stroma and cancer cell nests (CCNs).

Results:

Two hundred and thirty stage I–III cancers were studied. High nuclear p16^ink4a was expressed in 63% and high proliferation (Ki-67 >15%) in 61%. p16^ink4a expression was associated with reduced CD45RO+ cells at the IM (P<0.05) and within the stroma (P<0.05) and reduced CD8+ cells at the IM (P<0.01). A low Ki-67 proliferative index was associated with reduced density of CD3+ cells in CCNs (P<0.01), reduced CD45RO+ cells at the IM (P<0.05) and within the CCNs (P<0.001), reduced FOXP3+ cells at the IM (P<0.001), within the stroma (P=0.001) and within CCNs (P<0.001) and reduced CD8+ cells at the IM (P<0.05) and within the CCNs (P<0.05). Tumours with both a low proliferative index and expression of p16^ink4a demonstrated similar consistent relationships with reduced densities of T-cell infiltrates. On multivariate analysis, TNM stage (P<0.001), low CD3 cells at the IM (P=0.014), low CD8 cells at the IM (P=0.037), low proliferation (Ki-67; P=0.013) and low senescence (p16^ink4a; P=0.002) were independently associated with poorer cancer survival.

Conclusion:

Senescence, proliferation and immune cell infiltrates are independent prognostic factors in CRC. Although related to survival, p16^ink4a-associated senescence is not associated with an upregulation of antitumour T-cell responses.     

SU11248 (sunitinib) sensitizes pancreatic cancer to the cytotoxic effects of ionizing radiation

PURPOSE: SU11248 (sunitinib) is a small-molecule tyrosine kinase inhibitor which targets VEGFR and PDGFR isoforms. In the present study, the effects of SU11248 and ionizing radiation on pancreatic cancer were studied. METHODS AND MATERIALS: For in vitro studies human pancreatic adenocarcinoma cells lines were treated with 1 microM SU11248 1 h before irradiation. Western blot analysis was used to determine the effect of SU11248 on radiation-induced signal transduction. To determine if SU11248 sensitized pancreatic cancer to the cytotoxic effects of ionizing radiation, a clonogenic survival assay was performed using 0-6 Gy. For in vivo assays, CAPAN-1 cells were injected into the hind limb of nude mice for tumor volume and proliferation studies. RESULTS: SU11248 attenuated radiation-induced phosphorylation of Akt and ERK at 0, 5, 15, and 30 min. Furthermore, SU11248 significantly reduced clonogenic survival after treatment with radiation (p < 0.05). In vivo studies revealed that SU11248 and radiation delayed tumor growth by 6 and 10 days, respectively, whereas combined treatment delayed tumor growth by 30 days. Combined treatment with SU11248 and radiation further attenuated Brdu incorporation by 75% (p = 0.001) compared to control. CONCLUSIONS: SU11248 (sunitinib) sensitized pancreatic cancer to the cytotoxic effects of radiation. This compound is promising for future clinical trials with chemoradiation in pancreatic cancer.     

Effect of standard chemotherapy and antiangiogenic therapy on plasma markers and endothelial cells in colorectal cancer

Introduction:

The importance of the endothelium in angiogenesis and cancer is undisputed, and its integrity may be assessed by laboratory markers such as circulating endothelial cells (CECs), endothelial progenitor cells (EPCs), plasma von Willebrand factor (vWf), soluble E selectin, vascular endothelial growth factor (VEGF) and angiogenin. Antiantigenic therapy may be added to standard cytotoxic chemotherapy as a new treatment modality. We hypothesised that additional antiangiogenic therapy acts in a contrasting manner to that of standard chemotherapy on the laboratory markers.

Methods:

We recruited 68 patients with CRC, of whom 16 were treated with surgery alone, 32 were treated with surgery followed by standard chemotherapy (5-flurouracil), and 20 were treated with surgery followed by standard chemotherapy plus anti-VEGF therapy (Avastin). Peripheral blood was taken before surgery, and again 3 months and 6 months later. CD34⁺/CD45⁻/CD146⁺ CECs and CD34⁺/CD45⁻/CD309[KDR]⁺ EPCs were measured by flow cytometry, plasma markers by ELISA.

Results:

In each of the three groups, CECs and EPCs fell at 3 months but were back at pre-surgery levels at 6 months (P<0.05). VEGF was lower in both 3-and 6-month samples in the surgery-only and surgery plus standard chemotherapy groups (P<0.05), but in those on surgery followed by standard chemotherapy plus anti-VEGF therapy, low levels at 3 months (P<0.01) increased to pre-surgery levels at 6 months. In those having surgery and standard chemotherapy, soluble E selectin was lower, whereas angiogenin was higher at 6 months than at baseline (both P<0.05).

Conclusions:

We found disturbances in endotheliod cells regardless of treatment, whereas VEGF returned to levels before surgery in those on antiangiogenic therapy. These observations may have clinical and pathophysiological implications.     

The roles of BTG3 expression in gastric cancer: a potential marker for carcinogenesis and a target molecule for gene therapy

BTG (B-cell translocation gene) can inhibit cell proliferation, metastasis and angiogenesis, cell cycle progression, and induce differentiation in various cells. Here, we found that BTG3 overexpression inhibited proliferation, induced S/G2 arrest, differentiation, autophagy, apoptosis, suppressed migration and invasion in MKN28 and MGC803 cells (p < 0.05). BTG3 transfectants showed a higher mRNA expression of p27, Bax, 14-3-3, Caspase-3, Caspase-9, Beclin 1, NF-κB, IL-1, -2, -4, -10 and -17, but a lower mRNA expression of p21, MMP-9 and VEGF than the control and mock (p < 0.05). At protein level, BTG3 overexpression increased the expression of CDK4, AIF, LC-3B, Beclin 1 and p38 (p < 0.05), but decreased the expression of p21 and β-catenin in both transfectants (p < 0.05). After treated with cisplatin, MG132, paclitaxel and SAHA, both BTG3 transfectants showed lower viability and higher apoptosis than the control in both time- and dose-dependent manners (p < 0.05). BTG3 expression was restored after 5-aza-2′-deoxycytidine or MG132 treatment in gastric cancer cells. BTG3 expression was decreased in gastric cancer in comparison to the adjacent mucosa (p < 0.05), and positively correlated with venous invasion and dedifferentiation of cancer (p < 0.05). It was suggested that BTG3 expression might contribute to gastric carcinogenesis. BTG3 overexpression might reverse the aggressive phenotypes and be employed as a potential target for gene therapy of gastric cancer.