Generation of Immunoprotection Against Squamous Cell Carcinomas by In Vitro Cultivation and a Possible Mechanism of Action

Author Institution: Departments of Medical Technology and Biological Sciences, Bowling Green State UniversityThe immunogenicity of individual diethylnitrosamine (DEN)-induced forestomach carcinomas in female BALB/c mice was investigated following in vitro and in vivo cultivation. Of the five transplantable tumor lines studied, (DEN1? DEN3, DEN6, DEN8, and DEN^ only two (DEN6 and DEN8) showed some degree of immunogenicity. DENX, E»EN3, and DEN9 were highly tumorigenic with very little immunogenic potency as judged by tumor transplantation-excision assay, Winn neutralization, and antibody binding tests. These three tumors grew rapidly and showed a high degree of malignancy. DENX and DEN3 also metastasized readily. Cell lines from DEN6 and DEN9 lost their tumorigenicity at the 5th and 50th passage of culture, respectively. Although DENa and DEN3 did not lose their tumorigenicity, the number of tumor cells required to produce tumors increased substantially and their ability to metastasize was lost. Tumor transplantation studies, with these cultured cell lines in normal and x-irradiated recipients, suggested that the decrease in tumorigenicity may be immunologically mediated. Mice immunized with the in vitro lines demonstrated transplantation resistance against the respective in vitro and in vivo lines. The treatment of in vivo or in vitro propagated cells with periodic acid or neuraminidase enhanced antigen-antibody binding significantly. The effect of these chemicals became less pronounced as in vitro culture continued. It appears that during in vivo cultivation the antigenic determinants are masked or modulated by some glycoprotein or glycolipid molecules which render them non-, or very weakly, immunogenic

Frequency and Distribution of Pseudomonas aeruginosa Serotypes 03, 06, 011 in Three Northwestern Ohio Hospitals as Determined by ELISA Using Specific Monoclonal Antibodies

Author Institution: Medical Technology and Biological Sciences, Bowling Green State UniversityHybridoma producing monoclonal antibodies (mAbs), specific for three clinically significant Pseudomonas aeruginosa (serotypes 03, 06, and Oil), were generated to investigate the prevalence of these serotypes in three Northwestern Ohio hospitals. Fusion products reacting with bacterial cells or membrane extracts were detected by enzyme-linked-immunosorbent assay (ELISA). Three mAbs designated: 72C, 11 H (IgM) and HE (IgG2b), were selected. These mAbs reacted with approximately 40% of the clinical isolates of P. aeruginosa in each hospital. The incidence of serotype Oil varied in these hospitals, ranging from 13.2%-23.8%. Serotype Oil predominated in two of the three hospitals. The prevalence of serotype 06 was similar in all three hospitals (13.6-15%). In one of the hospitals (Hospital 2), the occurrence of serotype 06 was slightly higher (15%) than serotype Oil (13-2%). Serotype 03 occurred less frequently (1.5%) in one of the hospitals than in the other two (10-11%). None of the serotypes showed clear predilection toward any body site. The mAbs did not react with other strains of P. aeruginosa, nor with other gram-negative or gram-positive organisms. The results of Immunofluorescence and Western blot correlated well with ELISA. However, ELISA showed a higher sensitivity, indicating the usefulness of this technique for serotyping P. aeruginosa

Non-immunological enhancement of tumour transplantability in x-irradiated host animals.

MSC-10 tumour cells (derived from a chemically induced pulmonary squamous-cell carcinoma in DBA/2 mice) were inoculated intramuscularly into thymectomized, X-irradiated isogeneic mice, either 48 h or 6 weeks after thymectomy and X-irradiation. Normal mice and immunologically reconstituted mice served as controls. A marked enhancement in frequency of tumour takes was observed in all groups of animals inoculated with tumour cells 48 h after whole:-body X-irradiation, whether thymectomized, immunologically reconstituted or not. The TD50 decreased to less than 1/10 of that observed in unirradiated controls. When mice were inoculated with tumour cells 6 weeks after X-irradiation, the incidence of tumour takes was similar to that of unirradiated controls, including the thymectomized-irradiated group, which was still severely immunodeficient as measured by antibody formation and skin graft rejection. The experiments indicate that whole-body X-irradiation creates a condition that favours tumour cell survival or growth. This "permissive state" exists only shortly after X-irradiation and is not correlated with the host's level of immunocompetence

Properties of Lewis Lung Carcinoma Cells Surviving Curcumin Toxicity

The anti-inflammatory agent curcumin can selectively eliminate malignant rather than normal cells. The present study examined the effects of curcumin on the Lewis lung carcinoma (LLC) cell line and characterized a subpopulation surviving curcumin treatments. Cell density was measured after curcumin was applied at concentrations between 10 and 60 μM for 30 hours. Because of the high cell loss at 60 μM, this dose was chosen to select for surviving cells that were then used to establish a new cell line. The resulting line had approximately 20% slower growth than the original LLC cell line and based on ELISA contained less of two markers, NF-κB and ALDH1A, used to identify more aggressive cancer cells. We also injected cells from the original and surviving lines subcutaneously into syngeneic C57BL/6 mice and monitored tumor development over three weeks and found that the curcumin surviving-line remained tumorigenic. Because curcumin has been reported to kill cancer cells more effectively when administered with light, we examined this as a possible way of enhancing the efficacy of curcumin against LLC cells. When LLC cells were exposed to curcumin and light from a fluorescent lamp source, cell loss caused by 20 μM curcumin was enhanced by about 50%, supporting a therapeutic use of curcumin in combination with white light. This study is the first to characterize a curcumin-surviving subpopulation among lung cancer cells. It shows that curcumin at a high concentration either selects for an intrinsically less aggressive cell subpopulation or generates these cells. The findings further support a role for curcumin as an adjunct to traditional chemical or radiation therapy of lung and other cancers

A New Method for Identifying Stem-Like Cells in Esophageal Cancer Cell Lines

Cancer stem cells (CSCs) appear to resist chemo-radiotherapy and initiate tumor recurrence in patients. Isolation and further characterization of this subpopulation is important for targeting CSCs. Flow cytometry using Aldefluor, a fluorescent substrate of aldehyde dehydrogenase, has been used to isolate CSCs from various cancer cell lines. However, newtechniques are needed to locate and identify CSCs in culture for live-cell analyses such as fluorescence microscopy without introducing artifacts during cell sorting and to observe CSC and non-CSC interactions. Previously, we characterized a distinct CSC subpopulation within human esophageal cancer cell lines (ESCC). In this study we introduce the attached-cell Aldefluor method (ACAM) to detect CSCs in ESCC cell lines (KY-5, KY-10, TE-1, TE-8, YES-1, YES-2). To validate this technique, we isolated CSCs from the YES-2 parental line using standard Aldefluor flow cytometry to create a cell line enriched in CSCs (YES-2CSC). This line showed significantly greater ACAM staining and higher CD44 levels than YES-2. ACAMalso showed significantly higher ALDH activity in YES-2CSC than in YES-2S, a cell line that has a diminished CSC subpopulation after having survived treatment with curcumin. ACAM stained cells within tumorspheres made from the CSC-enriched line but not differentiating cells from the tumorspheres. This study also demonstrates a new method for generating and growing tumorspheres without the growth factor supplements normally used in medium to form tumorspheres. ACAM should be evaluated using other cancer cell lines to further substantiate its effectiveness and to characterize CSCs in culture through various imaging techniques. ©Ivyspring International Publisher

Effects of Curcumin on Stem-Like Cells in Human Esophageal Squamous Carcinoma Cell Lines

Background: Many cancers contain cell subpopulations that display characteristics of stem cells. Because these cancer stem cells (CSCs) appear to provide resistance to chemo-radiation therapy, development of therapeutic agents that target CSCs is essential. Curcumin is a phytochemical agent that is currently used in clinical trials to test its effectiveness against cancer. However, the effect of curcumin on CSCs is not well established. The current study evaluated curcumin-induced cell death in six cancer cell lines derived from human esophageal squamous cell carcinomas. Moreover, these cell lines and the ones established from cells that survived curcumin treatments were characterized.Methods: Cell loss was assayed after TE-1, TE-8, KY-5, KY-10, YES-1, and YES-2 cells were exposed to 20-80 μM curcumin for 30 hrs. Cell lines surviving 40 or 60 μM curcumin were established from these six original lines. The stem cell markers aldehyde dehydrogenase-1A1 (ALDH1A1) and CD44 as well as NF-κB were used to compare CSC-like subpopulations within and among the original lines as well as the curcumin-surviving lines. YES-2 was tested for tumorsphere-forming capabilities. Finally, the surviving lines were treated with 40 and 60 μM curcumin to determine whether their sensitivity was different from the original lines.Results: The cell loss after curcumin treatment increased in a dose-dependent manner in all cell lines. The percentage of cells remaining after 60 μM curcumin treatment varied from 10.9% to 36.3% across the six lines. The cell lines were heterogeneous with respect to ALDH1A1, NF-κB and CD44 expression. KY-5 and YES-1 were the least sensitive and had the highest number of stem-like cells whereas TE-1 had the lowest. The curcumin-surviving lines showed a significant loss in the high staining ALDH1A1 and CD44 cell populations. Tumorspheres formed from YES-2 but were small and rare in the YES-2 surviving line. The curcumin-surviving lines showed a small but significant decrease in sensitivity to curcumin when compared with the original lines.Conclusion: Our results suggest that curcumin not only eliminates cancer cells but also targets CSCs. Therefore, curcumin may be an effective compound for treating esophageal and possibly other cancers in which CSCs can cause tumor recurrence. © 2012 Almanaa et al.; licensee BioMed Central Ltd

Development of Circadian Oscillators in Neurosphere Cultures during Adult Neurogenesis

Circadian rhythms are common in many cell types but are reported to be lacking in embryonic stem cells. Recent studies have described possible interactions between the molecular mechanism of circadian clocks and the signaling pathways that regulate stem cell differentiation. Circadian rhythms have not been examined well in neural stem cells and progenitor cells that produce new neurons and glial cells during adult neurogenesis. To evaluate circadian timing abilities of cells undergoing neural differentiation, neurospheres were prepared from the mouse subventricular zone (SVZ), a rich source of adult neural stem cells. Circadian rhythms in mPer1 gene expression were recorded in individual spheres, and cell types were characterized by confocal immunofluorescence microscopy at early and late developmental stages in vitro. Circadian rhythms were observed in neurospheres induced to differentiate into neurons or glia, and rhythms emerged within 3–4 days as differentiation proceeded, suggesting that the neural stem cell state suppresses the functioning of the circadian clock. Evidence was also provided that neural stem progenitor cells derived from the SVZ of adult mice are self-sufficient clock cells capable of producing a circadian rhythm without input from known circadian pacemakers of the organism. Expression of mPer1 occurred in high frequency oscillations before circadian rhythms were detected, which may represent a role for this circadian clock gene in the fast cycling of gene expression responsible for early cell differentiation

Development of Circadian Oscillators in Neurosphere Cultures during Adult Neurogenesis

Circadian rhythms are common in many cell types but are reported to be lacking in embryonic stem cells. Recent studies have described possible interactions between the molecular mechanism of circadian clocks and the signaling pathways that regulate stem cell differentiation. Circadian rhythms have not been examined well in neural stem cells and progenitor cells that produce new neurons and glial cells during adult neurogenesis. To evaluate circadian timing abilities of cells undergoing neural differentiation, neurospheres were prepared from the mouse subventricular zone (SVZ), a rich source of adult neural stem cells. Circadian rhythms in mPer1 gene expression were recorded in individual spheres, and cell types were characterized by confocal immunofluorescence microscopy at early and late developmental stages in vitro. Circadian rhythms were observed in neurospheres induced to differentiate into neurons or glia, and rhythms emerged within 3–4 days as differentiation proceeded, suggesting that the neural stem cell state suppresses the functioning of the circadian clock. Evidence was also provided that neural stem progenitor cells derived from the SVZ of adult mice are self-sufficient clock cells capable of producing a circadian rhythm without input from known circadian pacemakers of the organism. Expression of mPer1 occurred in high frequency oscillations before circadian rhythms were detected, which may represent a role for this circadian clock gene in the fast cycling of gene expression responsible for early cell differentiation