Characterization of Side Populations in HNSCC: Highly Invasive, Chemoresistant and Abnormal Wnt Signaling

Side Population (SP) cells, a subset of Hoechst-low cells, are enriched with stem cells. Originally, SP cells were isolated from bone marrow but recently have been found in various solid tumors and cancer cell lines that are clonogenic in vitro and tumorigenic in vivo. In this study, SP cells from lymph node metastatic head and neck squamous cell carcinoma (HNSCC) cell lines were examined using flow cytometry and Hoechst 3342 efflux assay. We found that highly metastatic HNSCC cell lines M3a2 and M4e contained more SP cells compared to the low metastatic parental HNSCC cell line 686LN. SP cells in HNSCC were highly invasive in vitro and tumorigenic in vivo compared to non-SP cells. Furthermore, SP cells highly expressed ABCG2 and were chemoresistant to Bortezomib and etoposide. Importantly, we found that SP cells in HNSCC had abnormal activation of Wnt/β-catenin signaling as compared to non-SP cells. Together, these findings indicate that SP cells might be a major driving force of head and neck tumor formation and metastasis. The Wnt/β-catenin signaling pathway may be an important target for eliminating cancer stem cells in HNSCC

Clinical significance of side population in ovarian cancer cells

Recently, accumulating evidence has suggested that tumors, including ovarian cancer, are composed of a heterogeneous cell population with a small subset of cancer stem cells (CSCs) that sustain tumor formation and growth. The emergence of drug resistance is one of the most difficult problems in the treatment of ovarian cancer, which has been explained recently by the potential of CSCs to have superior resistance against anti-cancer drugs than conventional cancer cells. In this study, we expanded this line of study to examine whether this phenomenon is also observed in clinical specimens of ovarian cancer cells. In total we could analyze 28 samples out of 60 obtained from ovarian cancer patients. The clinical samples were subjected to testing of the expression of side population (SP) as a CSC marker, and according to the presence of SP (SP+) or absence of SP (SP−), clinicopathological significances were analyzed. Although there was no statistical significance, there were more SP+s in recurrent cases as well as in ascitic and peritoneal dissemination than in primary tumor of the ovary. There was no correlation between SP status and FIGO staging. In 19 cases of those who could be followed more than 6 months from initial therapy, there were 8 cases of recurrence or death from disease, and all of these were SP+. On the other hand, in 11 cases of disease-free survivors, 6 were SP+. There was a significant difference in prognosis between SP+ and SP− (p = 0.017). Although this study was limited, it revealed that SP could be contained more in recurrent or metastatic tumors than in primary tumors, and also that the presence of SP could be a risk factor of recurrence in ovarian cancer. Therefore, a novel therapeutic strategy targeting SP could improve the prognosis of ovarian cancer

Ulnar-sided wrist pain. Part I: anatomy and physical examination

Ulnar-sided wrist pain is a common complaint, and it presents a diagnostic challenge for hand surgeons and radiologists. The complex anatomy of this region, combined with the small size of structures and subtle imaging findings, compound this problem. A thorough understanding of ulnar-sided wrist anatomy and a systematic clinical examination of this region are essential in arriving at an accurate diagnosis. In part I of this review, ulnar-sided wrist anatomy and clinical examination are discussed for a more comprehensive understanding of ulnar-sided wrist pain

Small Non-coding RNAs Govern Mammary Gland Tumorigenesis

Small non-coding RNAs include siRNA, miRNA, piRNA and snoRNA. The involvement of miRNAs in the regulation of mammary gland tumorigenesis has been widely studied while the role for other small non-coding RNAs remains unclear. Here we summarize the involvement of miRNA in breast cancer onset and progression through regulating the cell cycle and cellular proliferation. The regulation of breast cancer stem cells and tumor regeneration by miRNA is reviewed. In addition, the emerging evidence demonstrating the involvement of piRNA and snoRNA in breast cancer is briefly described

Targeting and killing of glioblastoma with activated T cells armed with bispecific antibodies

Abstract Background Since most glioblastomas express both wild-type EGFR and EGFRvIII as well as HER2/neu, they are excellent targets for activated T cells (ATC) armed with bispecific antibodies (BiAbs) that target EGFR and HER2. Methods ATC were generated from PBMC activated for 14 days with anti-CD3 monoclonal antibody in the presence of interleukin-2 and armed with chemically heteroconjugated anti-CD3×anti-HER2/neu (HER2Bi) and/or anti-CD3×anti-EGFR (EGFRBi). HER2Bi- and/or EGFRBi-armed ATC were examined for in vitro cytotoxicity using MTT and 51Cr-release assays against malignant glioma lines (U87MG, U118MG, and U251MG) and primary glioblastoma lines. Results EGFRBi-armed ATC killed up to 85% of U87, U118, and U251 targets at effector:target ratios (E:T) ranging from 1:1 to 25:1. Engagement of tumor by EGFRBi-armed ATC induced Th1 and Th2 cytokine secretion by armed ATC. HER2Bi-armed ATC exhibited comparable cytotoxicity against U118 and U251, but did not kill HER2-negative U87 cells. HER2Bi- or EGFRBi-armed ATC exhibited 50—80% cytotoxicity against four primary glioblastoma lines as well as a temozolomide (TMZ)-resistant variant of U251. Both CD133– and CD133+ subpopulations were killed by armed ATC. Targeting both HER2Bi and EGFRBi simultaneously showed enhanced efficacy than arming with a single BiAb. Armed ATC maintained effectiveness after irradiation and in the presence of TMZ at a therapeutic concentration and were capable of killing multiple targets. Conclusion High-grade gliomas are suitable for specific targeting by armed ATC. These data, together with additional animal studies, may provide the preclinical support for the use of armed ATC as a valuable addition to current treatment regimens

Phenotypic and Functional Characterization of Human Mammary Stem/Progenitor Cells in Long Term Culture

Background: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages. Methodology: Single cell suspensions derived from human breast `organoids' were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres) were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres. Principal Findings: We show that primary mammospheres contain a distinct side-population (SP) that displays a CD24(low)/CD44(low) phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high)/CD24(low) cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1) mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated beta-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture. Conclusions: Thus, the self-renewal potential of human breast stem cells is exhausted within five in vitro passages of mammospheres, suggesting the need for further improvisation in culture conditions for their long-term maintenance

Neuroblastoma Cell Lines Contain Pluripotent Tumor Initiating Cells That Are Susceptible to a Targeted Oncolytic Virus

Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers.Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice.These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus