Tumor Cell Nuclei Research Articles

The addition of celecoxib improves the antitumor effect of cetuximab in colorectal cancer: role of EGFR-RAS-FOXM1-β- catenin signaling axis.

Here we showed that the addition of the COX-2 inhibitor celecoxib improved the antitumor efficacy in colorectal cancer (CRC) of the monoclonal anti-EGFR antibody cetuximab. The addition of celecoxib augmented the efficacy of cetuximab to inhibit cell proliferation and to induce apoptosis in CRC cells. Moreover, the combination of celecoxib and cetuximab was more effective than either treatment alone in reducing the tumor volume in a mouse xenograft model. The combined treatment enhanced the inhibition of EGFR signaling and altered the subcellular distribution of β-catenin. Moreover, knockdown of FOXM1 showed that this transcription factor participates in this enhanced antitumoral response. Besides, the combined treatment decreased β-catenin/FOXM1 interaction and reduced the cancer stem cell subpopulation in CRC cells, as indicated their diminished capacity to form colonospheres. Notably, the inmunodetection of FOXM1 in the nuclei of tumor cells in human colorectal adenocarcinomas was significantly associated with response of patients to cetuximab. In summary, our study shows that the addition of celecoxib enhances the antitumor efficacy of cetuximab in CRC due to impairment of EGFR-RAS-FOXM1-β-catenin signaling axis. Results also support that FOXM1 could be a predictive marker of response of mCRC patients to cetuximab therapy.

Impact of circulating tumor cell (CTC) nucleus size on outcomes with abiraterone acetate (AA) therapy in men with metastatic castration-resistant prostate cancer (mCRPC).

253 Background: CTC enumeration but not CTC morphology has been reported to predict outcomes to treatment in men with mCRPC. Recently Chen JF et. al (Cancer, 2015) showed an association with nuclear size and incidence of visceral disease in metastatic prostate cancer. In this study, we investigate the impact of CTC nucleus size on outcomes in men treated with AA for mCRPC. Methods: In a cohort of men with mCRPC treated with first-line AA, who had CTCs identified by CellSearch (CS) analysis prior to initiating treatment, we retrospectively quantified the nuclear size of CTCs by ImageJ/Fiji 1.46 software and correlated with progression free survival (PFS) on AA. We analyzed with univariate in addition to pre-specified multivariable analysis adjusted for Gleason score and baseline log PSA to assess independent predictive value of CTC nuclear size on PFS. Median PFS was calculated by Kaplan-Meier analysis and p-values were determined from Cox proportional hazards model. Results: 22 men treated with AA for mCRPC were included. Median nucleus size was 23.8 µm. Patients were divided in to 2 cohorts: small nuclear cohort (CTC nucleus size < 23.8 µm) vs large nuclear cohort (CTC nucleus size ≥23.8 µm). There was a non-significant trend towards worsened PFS (5.8 versus 6.8 months) in the larger nuclear size arm (Table). Conclusions: In this cohort of men with CRPC treated with AA, there is a non-significant trend towards decreased PFS associated with larger CTC nucleus size. Data are hypothesis generating and require further interrogation in a larger cohort. [Table: see text]

Higher IGFBP-1 to IGF-1 serum ratio predicts unfavourable survival in patients with nasopharyngeal carcinoma

BackgroundThe insulin-like growth factor (IGF) system plays an important role in the development and progression of cancer. However, little is known about the expression of the IGF system components and their clinicopathological significance and prognostic value in nasopharyngeal carcinoma (NPC).MethodsIGF system components (IGF-1, IGF-2, IGF-1SR, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4 and IGFBP-6) were quantified from the plasma of NPC patients and healthy individuals using the RayBio Human Cytokine Antibody Array. IGFBP-1 and IGF-1 mRNA levels were quantified by real-time qPCR, and protein expression was detected by western blot in nine NPC cell lines and four immortalized nasopharyngeal epithelial (NPE) cell lines. Tissue-specific expression of IGFBP-1 and IGF-1 was detected by immunohistochemistry in paraffin-embedded NPC tissues. ELISA analysis was used to measure the serum levels of IGFBP-1 and IGF-1 in 142 NPC patients and 128 healthy controls and determine potential correlation with clinicopathological parameters.ResultsSignificantly higher levels of circulating IGFBP-1 and lower levels of IGF-1 and IGF-2 were detected in NPC patients compared to healthy controls by Cytokine Antibody Array analyses (P = 0.034, 0.012, 0.046, respectively). IGFBP-1 expression was detected in the majority of NPC cell lines, but not in NPE cell lines, and was shown to localize to the nucleus of tumour cells, in contrast to the cytoplasmic staining observed in normal cells. Importantly, IGFBP-1 expression was stronger in NPC tumour tissues compared to peritumoural tissues. In contrast, IGF-1 expression was weak or absent in NPC and NPE cell lines, with the exception of the EBV-infected C666 cell line, and was found to be expressed at lower levels in tumour tissues compared to tumour-adjacent normal tissue. Levels of serum IGFBP-1 were shown to be significantly higher in patients with NPCs compared to healthy control individuals (55.23 ± 41.25 μg/L vs. 32.08 ± 29.73 μg/L, P < 0.001), whereas serum levels of IGF-1 were significantly lower in NPC patients compared to healthy controls (98.14 ± 71.48 μg/L vs. 164.01 ± 92.08 μg/L, P = 0.001). Consistently, the IGFBP-1/IGF-1 serum ratio was shown to be significantly higher in NPC patients compared to healthy control individuals (P = 0.002). Serum levels of IGFBP-1 and the IGFBP-1/IGF-1 ratio significantly correlated with age (P = 0.020; P = 0.016), WHO histological classification (P = 0.044; P = 0.048), titre of EA (EB Virus Capsid Antigen-IgA) and NPC (P = 0.015; P = 0.016). In contrast, higher IGFBP-1 serum levels and IGFBP-1/IGF-1 ratio significantly correlated with poor RFS (P = 0.046; P = 0.037) and OS (P = 0.038; P = 0.009). Multivariate analysis revealed that the IGFBP-1/IGF-1 ratio, but not serum IGFBP-1 level, represents an independent risk factor for poor RFS (P = 0.044) and OS (P = 0.035).ConclusionsA higher IGFBP-1/IGF-1 serum ratio is significantly associated with poor prognosis in NPC patients.

Expression of Lysyl Oxidase Predictive of Distant Metastasis of Laryngeal Cancer.

Objective To investigate the prognostic significance of lysyl oxidase (LOX) expression in laryngeal cancer. Study Design Retrospective chart review and histologic analysis. Setting Tertiary referral academic center. Subjects and Methods Patients (N = 100) underwent surgical treatment for laryngeal cancer and had tissue specimens available. Immunohistochemical staining for LOX was performed on laryngeal cancer tissue microarrays, and the proportion and intensity of staining were evaluated. Patients with LOX scores ≤6 were classified into the low LOX group, while those with scores >6 were classified into the high LOX group. We analyzed the correlation between LOX expression and clinical factors as well as prognosis. Results LOX was predominantly expressed in the cytoplasm and nuclei of tumor cells. Kaplan-Meier analysis revealed that the high LOX group had worse overall survival and recurrence-free survival rates than the low LOX group ( P < .05). LOX expression exhibited marginally significant correlation with lymph node metastasis. In the Cox regression analysis, LOX expression and lymph node metastasis were significant factors correlated with overall survival rate (odds ratio [OR] = 3.92, 95% confidence interval [95% CI]: 1.35-11.37, P = .012; OR = 1.96, 95% CI: 0.93-1.43, P = .024, respectively). LOX expression was related to distant metastasis free survival rate (OR = 7.72, 95% CI: 1.02-19.18, P = .048). Conclusion A high expression level of LOX is associated with lymph node and distant metastasis as well as poor prognosis among patients with laryngeal cancer.

Prognostic and predictive value of loss of nuclear RAD51 immunoreactivity in resected non-small cell lung cancer patients

ObjectivesIn response to DNA damage, recombination proteins are relocalized into sub-nuclear complexes that are microscopically detected as RAD51-containing nuclear foci. We aimed for assessing the prognostic and predictive value of loss of nuclear RAD51 immunoreactivity (‘RAD51 loss’) in 2 independent stage I to III non-small cell lung cancer (NSCLC) patient cohorts undergoing surgical resection and eventual perioperative chemo-/radiotherapy (CT/RT). Materials and methodsThe discovery set included 69 evaluable patients (19 adenocarcinomas, ADC, 50 squamous cell carcinomas, SCC) from Palacky University Hospital, 45/69 (65.2%) with additional platinum-based CT. The replication set entailed 845 evaluable patients (446 ADC, 399 SCC) from University Hospital Zurich, 308/845 (36.5%) with platinum based CT or RT. RAD51 loss was defined as ≤20% of tumor cell nuclei having any nuclear RAD51 expression. We assessed the prognostic value of RAD51 loss in all patients and its predictive value in patients receiving CT/RT. ResultsRAD51 loss was observed in 40/69 (58.0%) and 439/845 (51.9%) evaluable tumors in the discovery and replication set, respectively (p=0.34). It was more frequent in ADC compared to SCC (57.2% vs 47.4%, p=0.003). RAD51 loss was significantly associated with worse OS in both the discovery (adjusted HR=2.39, p=0.039) and replication set (adjusted HR=1.31, p=0.008). The unfavourable prognostic effect of RAD51 loss seen in the overall population was not observed in patients receiving perioperative CT (adjusted HR=1.07, p=0.73) or perioperative RT (adjusted HR=1.05, p=0.82). ConclusionRAD51 loss has an unfavourable prognostic impact in NSCLC patients undergoing curative surgical resection, but it may have a favourable predictive value in the subgroup of patients receiving perioperative platinum-based CT or RT, most likely as a consequence of deficient DNA repair.

Expression of Oct4 and Sox2 and their clinical significance in tongue squamous cell carcinoma

Objective: To investigate Oct4 and Sox2 protein expression in tongue squamous cell carcinoma (TSCC) and the relationships between the expressions of Oct4 and Sox2 and clinical pathological characteristics and survival of patients. Methods: The paraffin imbedded tissue specimens of 51 patients with histologically confirmed TSCC were included. Immunohistochemistry was employed to detect the protein expression of Oct4 and Sox2 in 51 TSCC tissue samples. The protein expression levels of Oct4 and Sox2 and their relationships with both clinicopathological features and survival of patients with TSCC were evaluated. Results: In 51 TSCC cases,positive expressions of Oct4 and Sox2 were mainly located in the nucleus of tumor cells. The expression of Oct4 was strongly positive in 27 cases (53%), weakly positive in 16 (31%) and negative in 8 (16%), whereas that of Sox2 was strongly positive in 25 cases (49%), weakly positive in 22 (43%) and negative in 4 (8%). Oct4 and Sox2 expression levels were significantly correlated with the histological grade of TSCC (P=0.004, P=0.006, respectively), not correlated with age, gender, T stage, tobacco smoking and alcohol consumption status (P>0.05), but Oct4 expression level was significantly associated with lymph node metastasis (P=0.001). Sox2 expression level was not associated with lymph node metastasis (P>0.05). The expression of Sox2 was significantly correlated with Oct4 (P<0.001). Oct4 and Sox2 expression was associated with poor overall survival of patients with TSCC (P=0.001, P=0.002, respectively), cases with higher Oct4 and Sox2 expression had the poorest overall survival (P<0.001). Sox2 expression and lymph node metastasis were independent prognostic factors of overall survival in patients with TSCC (P=0.02, P=0.001, respectively). Conclusions: Sox2 had independent prognostic effects on overall survival, suggesting that Sox2 expression may be an usefull indicator for predicting the prognosis of patients with TSCC.

Clinicopathological study of 5 cases of renal cell carcinoma with t(6;11)(p21;q12).

Renal cell carcinoma (RCC) with t(6;11)(p21;q12) has been incorporated into the recent WHO classification. We performed a clinicopathological study of 5 cases with such a tumor. The patients consisted of 4 males and 1 female. The age of patients ranged from 17 to 57 years with a mean age of 38.6 years. Tumor sizes ranged from 2.8 to 11 cm with a mean value of 6.5 cm. Despite immunotherapy and molecular-targeted therapy, one patient died of the disease 28 months after the surgery. Grossly, the cut surface of this tumor showed grayish white color in at least the focal area of all tumors. Furthermore, hemorrhage, daughter nodules and cystic changes were observed in two, three, and two tumors, respectively. Morphologically, all the tumors consisted of two components of large cells and small cells, and the latter surrounded basement membrane-like materials, forming rosette-like structures. Immunohistochemically, nuclei of tumor cells in all cases were positive for TFEB. Fluorescence in situ hybridization study confirmed the TFEB gene break in two tumors. Finally, urologists and pathologists should bear in mind that this tumor may occur in young adults to adults and might behave in an aggressive fashion. Break-apart FISH is useful for the definite diagnosis.

Emperipolesis: An Unreported Novel Phenomenon in Oral Squamous Cell Carcinoma

Emperipolesis is a phenomenon characterized by engulfment of hematopoietic cells by megakaryocytes, monocytes, fibroblasts, and malignant cells within their cytoplasm. This phenomenon has been reported in various physiological and pathological conditions including malignancies. However, emperipolesis has never been reported in oral squamous cell carcinoma (OSCC) till date. We have analyzed histopathological slides of 56 cases of OSCC to see lymphocyte engulfment by tumor cells. Five cases showing features of this phenomenon were observed. Lymphocytes were typically identified as small round cells with oval nuclei and scanty cytoplasm. Both partial and complete engulfment of lymphocytes by tumor cells was appreciated. Nuclei of the host tumor cells were crescent shaped and illustrated small concave indentation, so as to accommodate the internalized lymphocyte. The intercellular bridges were not seen between the internalized cell and the host cell. There were no signs of degeneration appreciable in either cell, thus ruling out the possibility of cannibalism phenomenon. Although emperipolesis is a well-known phenomenon in pathology, this is the first report showing its evidence in OSCC.

Increased expression of YAP1 in prostate cancer correlates with extraprostatic extension.

Objective:Yes associated protein 1 (YAP1) is a member of the Hippo pathway, acting as a transcriptional coactivator. To elucidate the role of YAP1 and phosphorylated (p)YAP1 in prostate cancer (PCa) tumorigenesis, we investigated their expression in clinical samples of PCa and cell lines.Methods:Fifty-four tumor, adjacent nontumor, and prostate intraepithelial neoplasia (PIN) tissues from patients with PCa after radical prostatectomy were selected from a retrospective cohort and studied using immunohistochemistry (IHC). Protein and mRNA expression levels of YAP1 were evaluated by Western blot analysis and quantitative real-time reverse transcription PCR, respectively, in cancer cell lines. Publicly available gene expression datasets were downloaded to analyze YAP1 mRNA and protein levels in PCa tissue samples.Results:IHC analysis of PCa tissues revealed that YAP1 staining intensities were moderate to weak in the nucleus and cytoplasm of tumor cells, whereas adjacent normal epithelia showed strong staining. We observed that benign prostates were characterized by higher expression levels of both nuclear (P=0.004) and cytosolic (P=0.005) YAP1. pYAP1 staining was weak in the cytoplasm and absent in the nucleus of all the tissues investigated. YAP1 expression was an indicator of extraprostatic extension (EPE). The level of YAP1 was negatively correlated with the level of the androgen receptor (AR) in The Cancer Genome Atlas dataset and Western blot analysis of cell lines. Conclusions:Our study suggested that YAP1 expression is heterogeneous in PCa tissue samples; therefore, YAP1 might play different roles in different aspects of PCa progression. This might involve AR–YAP1 interplay in PCa.

Evaluation of Nuclear Morphometry in Oral Squamous Cell Carcinoma: A Retrospective Study

ABSTRACT Aim To evaluate and compare various nuclear morphometric parameters in different histopathological grades of oral squamous cell carcinoma (OSCC) by using computerized image analysis and also to correlate it with regional lymph node metastasis. Materials and methods This retrospective study was conducted on paraffin blocks of 40 tissue specimens of OSCC cases treated with neck dissection, which were retrieved from the archives of the Department of Oral Pathology and Microbiology, Mahatma Gandhi Mission’s Dental College and Hospital, Navi Mumbai, India. All cases were histopathologically graded as well, moderately, and poorly differentiated OSCC. Further, they were also categorized based on pathological lymph node status as with or without lymph node metastasis. Sections from tumor proper were subjected to Feulgen nuclear staining technique. Images of 10 microscopic fields at the deepest invading part of tumor were captured randomly and 100 nuclei of tumor cells with clear, complete, nonoverlapping outlines were selected in each case. Nuclear morphometric parameters, such as large diameter, small diameter, nuclear area, and nuclear perimeter were measured for each of the 100 cells. Results An increase in mean nuclear area coefficient of variation (NACV) was observed in OSCC cases with lymph node metastasis pN(+) than in those without lymph node metastasis pN(–), (p = 0.67). A significant increase in nuclear area and perimeter was observed in pN(+) cases (p &lt; 0.01). A significant decrease in circular rate and increase in largest to smallest nuclear diameter (L/S) ratio (p &lt; 0.01) was observed in pN(+) cases. On comparing the nuclear morphometric parameters with different histopathological grades of OSCC, we found an increase in mean NACV values from well-differentiated OSCC to moderately differentiated and poorly differentiated OSCC (p = 0.612). An increase in mean nuclear area and perimeter was noted as grades of OSCC advanced (p &gt; 0.01). The mean circular rate was found to be lowest in poorly differentiated OSCC (p = 0.362). A significant increase in mean L/S ratio was observed within different histopathological grades of OSCC (p = 0.044), which when further confirmed using least significant difference (LSD) post hoc test, indicated a difference only between well-differentiated and poorly differentiated cases of OSCC (p = 0.132). Conclusion Our observations reveal that tumor cells with greater nuclear dimension and more elliptical shape tend to show increased incidence of nodal metastasis. Also, a positive inclination was observed in nuclear size and shape with increasing histopathological grades of OSCC. However, our data warrant a large-scale study to establish nuclear morphometry as a quantitative objective parameter and also for the rational application of the same. How to cite this article Narayanan N, Pathak J, Patel S, Swain N. Evaluation of Nuclear Morphometry in Oral Squamous Cell Carcinoma: A Retrospective Study. J Contemp Dent 2017;7(2):107-113.

MA17.02 Genome-Wide Copy Number and Mutational Analysis in Longitudinal Biopsies of Matched Primary and Metastatic Pulmonary Adenocarcinomas

There are still limited data on the extent of intratumoral heterogeneity of cancer gene mutations and genome-wide copy number aberrations between primary tumors and metastases in non-small cell lung cancers (NSCLC). Deconvolution of the intermixture of tumor and stromal components remains a major challenge for such analysis. To overcome these limitations, we applied a refined nuclei flow sorting approach on matched longitudinal biopsies (primary/metastasis) from pulmonary adenocarcinomas. Multiparameter Ploidy Profiling (MPP) comprises the isolation of nuclei from frozen or formalin-fixed and paraffin embedded (FFPE) tissues, followed by multiparameter flow sorting by DAPI for DNA content (ploidy) and TTF1 as a lineage marker to enrich for tumor cell nuclei. Homogenous TTF1 expression was ascertained by immunohistochemistry. Sorted populations were subjected to genomic profiling by high resolution aCGH and NGS with the Ion Torrent™ Comprehensive Cancer Panel. This approach allows for the detection of genome-wide copy number aberrations and provides all exon-coverage of 409 well-known cancer genes. Sequencing was performed with a mean depth of 965x. MPP was successfully applied on 44 frozen or FFPE tissue specimens from 19 patients. Clonally unrelated secondary primaries were found in three patients, defined by the absence of both shared copy number (CN) transition and somatic mutations. The concordance rate between primary tumor and corresponding metastases was 65.2% and reached 85.5% for mutations and copy number amplifications/deletions in the top 12 affected genes (including CDKN2A, KRAS, ATM, KEAP1, EGFR and STK11). The correlation of the allele frequencies between primary tumors and metastases was linear (r=0.87, p<0.001), irrespective of the time interval between the tissue resections. Overall, ploidy was not different between primary tumors and metastases. Additionally, the metastases did not bear a higher burden of private events (CN transitions and somatic mutations) than the primary tumors. MPP is a powerful method to increase the precision of downstream analysis due to unprecedented purity of tumor DNA. Our data argue for a high concordance rate of mutations and CN transitions between primary tumors and their corresponding metastases. Intriguingly, the ploidy remains remarkably stable during progression even after long time-periods, which suggests chromosomal stability with a limited degree of macroevolutionary shifts over time and space. Taken together, our data suggest the presence of at least two evolutionary patterns: 1) early/branched and 2) late/linear progression, with a continuum from high to low genetic divergence of the primary tumor and metastases to their most recent common ancestor.

High expression of HMGA2 predicts poor survival in patients with clear cell renal cell carcinoma.

High-mobility group AT-hook 2 (HMGA2) is involved in a wide spectrum of biological processes and is upregulated in several tumors, but its role in renal carcinoma remains unclear. The aim of this study was to examine the expression of HMGA2 and its relationship to the overall survival (OS) of patients with non-metastatic clear cell renal cell carcinoma (ccRCC) following surgery. The expression of HMGA2 was evaluated retrospectively by immunohistochemistry (IHC) in 162 patients with ccRCC who underwent nephrectomy in 2003 and 2004. An IHC analysis revealed that HMGA2 was expressed in the nuclei of tumor cells in 146 (90.1%) patients with ccRCC. The level of HMGA2 was positively correlated with tumor size, lymph node metastasis, and Fuhrman Grade. A Kaplan–Meier analysis with log-rank test found that patients with high HMGA2 expression had a poor outcome and that patients with low HMGA2 expression had better survival. Cox regression analysis showed that HMGA2 expression could serve as an independent prognostic factor for ccRCC patients. The efficacy of the following prognostic models was improved when HMGA2 expression was added: tumor node metastasis stage, UCLA Integrated Scoring System, Mayo Clinic stage, size, grade, and necrosis score. In summary, this study showed that HMGA2 expression is an independent prognostic factor for OS in patients with ccRCC. HMGA2 was found to be a valuable biomarker for ccRCC progression.

The cancer/testis antigen MAGEC2 promotes amoeboid invasion of tumor cells by enhancing STAT3 signaling.

The biological function of MAGEC2, a cancer/testis antigen highly expressed in various cancers, remains largely unknown. Here we demonstrate that expression of MAGEC2 induces rounded morphology and amoeboid-like movement of tumor cells in vitro and promotes tumor metastasis in vivo. The pro-metastasis effect of MAGEC2 was mediated by signal transducer and activator of transcription 3 (STAT3) activation. Mechanistically, MAGEC2 interacts with STAT3 and inhibits the polyubiquitination and proteasomal degradation of STAT3 in the nucleus of tumor cells, resulting in accumulation of phosphorylated STAT3 and enhanced transcriptional activity. Notably, expression levels of MAGEC2 and phosphorylated STAT3 are positively correlated and both are associated with incidence of metastasis in human hepatocellular carcinoma. This study not only reveals a previously unappreciated role of MAGEC2 in promoting tumor metastasis, but also identifies a new molecular mechanism by which MAGEC2 sustains hyperactivation of STAT3 in the nucleus of tumor cells. Thus, MAGEC2 may represent a new antitumor metastasis target for treatment of cancer.

Comparison of therapeutic efficacy of lipo-doxorubicin and doxorubicin in treating bladder cancer

ObjectivesDoxorubicin is commonly used in the treatment of superficial bladder cancer, but more side effects and shorter intracellular retention time hamper its clinical application. Since lipo-doxorubicin (Lipodox) has the advantages of longer half-life and lower clearance rate than doxorubicin, it should improve the efficacy of tumor therapy and reduce the normal tissue toxicity of doxorubicin. Materials and MethodsIn this study, we compared the cytotoxicity of Lipodox and doxorubicin in different treatment durations on bladder cancer cells by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Drug distribution was tracked under fluorescence microscopy. The metabolic rate after treatment was measured by serial flow cytometry. Finally, an in vivo orthotopic MBT-2 bladder tumor model was established for comparing the differences of therapeutic efficacy, including tumor weight and survival rate. ResultsThe 50% inhibitory concentration (IC50) of doxorubicin and Lipodox for MBT-2 cells was 0.62 μg/mL and 130 μg/mL, respectively, after 48 hours treatment. Lipo-dox presented higher cytotoxicity than doxorubicin at 6 hours (93% vs 73%) and 12 hours (93% vs 80%) treatment. After drug treatment, Lipodox fluorescence distribution was observed mostly in the cell membrane, lysosomes, and nuclei of tumor cells, while doxorubicin was concentrated in the nuclei. Initial fluorescence intensity of doxorubicin was 27.3 times that of Lipodox (p < 0.001) at time of treatment. The fluorescence intensity of doxorubicin decreased to 12% after 24 hours culture but that of Lipodox remained at 81%. In an orthotopic model, the average tumor weight and survival were: control group: 1.0 ± 0.3 g, 25%; doxorubicin treatment group: 0.7 ± 0.05 g, 43%; and Lipodox treatment group: 0.2 ± 0.1 g, 57%. ConclusionOur study demonstrated that Lipodox can enhance doxorubicin cytotoxicity in bladder cancer cells and inhibit tumor growth in orthotopic bladder cancer with improved survival rate. Therefore, we suggest Lipodox may act as an alternative to doxorubicin in the treatment of local bladder cancer.

Combinatorial nanocarrier based drug delivery approach for amalgamation of anti-tumor agents in breast cancer cells: an improved nanomedicine strategy.

Combination therapy of multiple drugs through a single system is exhibiting high therapeutic effects. We investigate nanocarrier mediated inhibitory effects of topotecan (TPT) and quercetin (QT) on triple negative breast cancer (TNBC) (MDA-MB-231) and multi drug resistant (MDR) type breast cancer cells (MCF-7) with respect to cellular uptake efficiency and therapeutic mechanisms as in vitro and in vivo. The synthesized mesoporous silica nanoparticle (MSN) pores used for loading TPT; the outer of the nanoparticles was decorated with poly (acrylic acid) (PAA)-Chitosan (CS) as anionic inner-cationic outer layer respectively and conjugated with QT. Subsequently, grafting of arginine-glycine-aspartic acid (cRGD) peptide on the surface of nanocarrier (CPMSN) thwarted the uptake by normal cells, but facilitated their uptake in cancer cells through integrin receptor mediated endocytosis and the dissociation of nanocarriers due to the ability to degrade of CS and PAA in acidic pH, which enhance the intracellular release of drugs. Subsequently, the released drugs induce remarkable molecular activation as well as structural changes in tumor cell endoplasmic reticulum, nucleus and mitochondria that can trigger cell death. The valuable CPMSNs may open up new avenues in developing targeted therapeutic strategies to treat cancer through serving as an effective drug delivery podium.

Polyhydric Corrole and Its Gallium Complex: Synthesis, DNA‐binding Properties and Photodynamic Activities

AbstractA new hydrophilic polyhydric corrole (1) and its Ga(III) complex (1‐Ga) were synthesized and characterized. Corroles 1 and 1‐Ga bind to calf thymus DNA externally and exhibit remarkable photonuclease‐like activity. Singlet oxygen (1O2) was identified as the reactive oxygen species involved. These corroles also exhibit significant photocytotoxicity against HeLa, A549, BEL‐7402 and HepG2 tumor cell lines. They mainly accumulated in the nuclei of tumor cells. ROS‐mediated mitochondrial damage and intracellular DNA disintegration contributed to the observed photodynamic activity.

1011P - High expression of FOXM1 is a potential prognostic marker for oral squamous cell carcinoma patients treated with docetaxel-containing regimens

Forkhead box protein M1 (FOXM1) is an oncoprotein that regulates cell growth and differentiation, angiogenesis, apoptosis and aging; and it is reported to play an important role in progression and drug sensitivity of various cancers. The purpose of this study was to determine whether FOXM1 expression could be a useful prognostic factor for oral squamous cell carcinoma (OSCC). FOXM1 expression was investigated by immunohistochemistry in tissue samples of 56 OSCC patients treated with docetaxel (DOC)-containing regimens. In this study, we investigated the relationship between FOXM1 expression and clinicopathological features of OSCC, as well as the prognosis of above patients. Moreover, we examined the expression of FOXM1 in DOC-resistant human tongue carcinoma cell lines (HSC2/DOC, HSC3/DOC and HSC4/DOC) in vitro. We established these DOC-resistant cell lines by exposing HSC2, HSC3 and HSC4 parental cells to increasing concentrations of DOC over approximately two years. FOXM1 was detected both in nucleus and cytoplasm of OSCC tumor cells. FOXM1 expression in tumor tissues was significantly correlated with N classification (P = 0.0395), stage (P = 0.004), therapeutic efficacy (P = 0.0113) and outcome of patient (P = 0.0134); although there was no correlation between FOXM1 expression and patient's gender, age or T classification. Moreover, high expression of FOXM1 in tumor cells was associated with shorter overall survival (OS, P = 0.0257). Multivariate analysis also revealed that high expression of FOXM1 was a predictor of reduced survival (P = 0.0327). Additionally, DOC-resistant OSCC cell lines showed significantly higher expression of FOXM1 compared to the parental cell lines in vitro. These findings suggest that high expression of FOXM1 in OSCC tumors is correlated with DOC resistance, as well as poor therapeutic effects and worse clinical outcomes in OSCC patients treated with DOC -containing regimen. Therefore, FOXM1 might have prognostic significance in oral squamous cell carcinoma patients.

OS1.6 Tumor Microtubes contribute to resistance against surgical lesions, and chemotherapy in malignant glioma

AbstractBackground:The current standard for the treatment of astrocytomas, including glioblastomas, consists of tumor resection, followed by radio- and chemotherapy. As we could recently demonstrate, a dense multicellular tumor network formed by intercellular tumor microtubes (TMs) greatly contributes to radioresistance. The neuronal growth-associated protein 43 (GAP-43) is involved in this mechanism. Here we asked whether similar resistance mechanisms can be found with respect to surgical lesions, and chemotherapy.Methods:Cranial windows were implanted into 8–10 weeks old male NMRI nude mice. Human glioblastoma stem-like cells (GBMSCs; stably transduced with GFP, RFP, YFP, shRNA for GAP-43, control vector, and/or H2B-GFP to label tumor cell nuclei) were stereotactically injected into the mouse’s brain. In established tumors, 100mg/kg temozolomide (TMZ) was applied to mice p.o. for three consecutive days (day 0,1,2). For surgical lesion experiments, a regional injury was set to the brain by a 26 gauge Hamilton syringe.Results:Similar to radiotherapy, GBMSCs that were not part of the TM-connected tumor cell network died in relevant numbers 7 days after chemotherapy, while TM-connected tumor cells survived. The number of TM-connected tumor cells even increased twofold after chemotherapy, which speaks for an adapative resistance mechanism. It is currently under investigation whether knock down of GAP-43 increases overall efficacy of TMZ therapy. After surgical resection, the lesioned area was rapidly re-colonized with TM-extending tumor cells, with a striking increase in tumor cells at the site of lesion that by far extended the tumor cell densitiy in unlesioned tumor areas. Qantification revealed that the tumor cell nuclear density increased to numbers five-fold above values before surfical intervention in this area, evident as early as five days (488.3%; P=0.033; 95% CI, 50.3–726.4), and extending to 14 days (523.9%; P=0.03; 95% CI, 66.7–781.1) after surgical procedure. This apparent,,repair response” gradually diminished with increasing distance to the surgical lesion site. Finally, while dexamethasone did not relevantly change these reponses, GAP-43 knock down reduced them significantly.Conclusion:TMs convey primary and adaptive resistance to TMZ chemotherapy, in accordance to what we have shown before for radiotherapy. They are also involved in a strong repair response at the very site of a surgical lesion. Reducing the ability of gliomas to repair themselves, and to resist genotoxic stress by interfering with TM formation and function appears as a promising avenue to improve treatment efficacies in these callenging diseases.

Leakage-free DOX/PEGylated chitosan micelles fabricated via facile one-step assembly for tumor intracellular pH-triggered release

A general strategy was developed to fabricate the chitosan-based micelles as smart drug delivery system (DDS) for anti-cancer drug doxorubicin (DOX), via modulating the feeding ratio of PEG (PEG2000-CHO) and molecular weight of chitosan. Typically, benefiting from the solubility of chitosan in media with different pH values as an “off-on” switch, the DOX/CS100K-PEG2 micelles possess spherical core with a narrow dispersed diameter of 64nm and regular shell with a thickness of 14nm in pH 7.4 PBS. Interestingly, the DOX/CS-PEG micelles exhibited drug leakage-free behavior in a physiological environment, with a cumulative release ratio of only 2.32% under pH 7.4 PBS, while achieving rapid drug release and a cumulative release ratio of 72.76% in the intracellular microenvironment. Moreover, the micelles transformed into the water-soluble CS100K-PEG2 after the pH-triggered intracellular release of DOX, and it makes them easy to be biodegraded and eliminated by metabolic system. As revealed by MTT assays and CLSM images, DOX could be efficiently and rapidly transported into cell nuclei of tumor cells and showed significant cell inhibition. The simple fabrication and excellent properties make them intelligent and favorable on-demand DDS in cancer treatment.

Neoplastic cellularity is associated with clinical and molecular features of high-grade serous ovarian carcinoma

ObjectiveMost molecular analyses of high-grade serous ovarian, peritoneal and fallopian tube carcinomas (HGSC) require ≥70% tumor (neoplastic) cell nuclei. We characterized the distribution of the percentage of neoplastic nuclei (PNN) in a large cohort of HGSC and correlated PNN with clinical outcomes to determine the fraction of cases outside this range and whether this cut-off introduces selection bias. MethodsSubjects were prospectively enrolled and normal and neoplastic tissues were snap-frozen. All subjects had grade 2 to 3 HGSC. Subjects that received neoadjuvant chemotherapy were excluded. PNN was determined by estimating the fraction of neoplastic nuclei relative to non-neoplastic nuclei on a representative hematoxylin and eosin stained frozen section from the primary neoplasm. Germline BRCA mutation status was determined with Sanger or BROCA sequencing. ResultsPNN was <70% in 101 (33%) of 306 cases. PNN was significantly higher among subjects without optimal cytoreduction (P=0.018). 55 subjects had germline BRCA1/BRCA2 mutations. HGSC associated with BRCA2 but not BRCA1 mutations had significantly lower PNN compared to HGSC in non-carriers (54% vs. 70%, P=0.018). Overall survival was not significantly different between subjects with <70% or ≥70% PNN (median survival 51.8 vs. 46.6months, P=0.858). ConclusionsOne-third of HGSC has PNN <70%. Higher PNN is associated with suboptimal cytoreduction, while lower PNN is associated with inherited BRCA2 mutations. Our findings suggest a nonrandom distribution of PNN that may reflect cancer biology. Further studies exploring the stromal microenvironment are needed. Molecular analyses of HGSC selected for high PNN exclude a significant fraction of patients.