Development of a novel and economical agar-based non-adherent three-dimensional culture method for enrichment of cancer stem-like cells.

BACKGROUND: Non-adherent or ultra-low attachment three-dimensional (3D) culture, also called sphere formation assay, has been widely used to assess the malignant phenotype and stemness potential of transformed or cancer cells. This method is also popularly used to isolate the cancer stem-like cells (CSCs) or tumor-initiating cells based on their unique anchorage-independent growth or anoikis-resistant capacity. Different non-adhesive coating agents, such as poly-2-hydroxyethyl methacrylate (poly-HEMA) and synthetic hydrogels, have been used in this non-adherent 3D culture. However, preparation of non-adherent culture-ware is labor-intensive and technically demanding, and also costs of commercial non-adherent culture-ware prepared with various coating agents are relatively expensive and the culture-ware cannot be used repeatedly. METHODS: In this study, we developed a non-adherent 3D culture method based on agar coating for growing tumor spheres derived from various cancer cell lines and primary prostate cancer tissues under a non-adherent and serum-free condition. The tumor spheres generated by this 3D culture method were analyzed on their expression profiles of CSC-associated markers by reverse transcription quantitative polymerase chain reaction, presence and relative proportion of CSCs by fluorescence-activated cell sorting (CD133+/CD44+ cell sorting) and also a CSC-visualizing reporter system responsive to OCT4 and SOX2 (SORE6), and in vivo tumorigenicity. The repeated use of agar-coated plates for serial passages of tumor spheres was also evaluated. RESULTS: Our results validated that the multicellular tumor spheres generated by this culture method were enriched of CSCs, as evidenced by their enhanced expression profiles of CSC markers, presence of CD133+/CD44+ or SORE6+ cells, enhanced self-renewal capacity, and in vivo tumorigenicity, indicating its usefulness in isolation and enrichment of CSCs. The agar-coated plates could be used multiple times in serial passages of tumor spheres. CONCLUSIONS: The described agar-based 3D culture method offers several advantages as compared with other methods in isolation of CSCs, including its simplicity and low-cost and repeated use of agar-coated plates for continuous passages of CSC-enriched spheres.

Critical assessment of the efficiency of CD34 and CD133 antibodies for enrichment of rabbit hematopoietic stem cells.

Rabbits have many hereditary diseases common to humans and are therefore a valuable model for regenerative disease and hematopoietic stem cell (HSC) therapies. Currently, there is no substantial data on the isolation and/or enrichment of rabbit HSCs. This study was initiated to evaluate the efficiency of the commercially available anti-CD34 and anti-CD133 antibodies for the detection and potential enrichment of rabbit HSCs from peripheral blood. PBMCs from rabbit and human blood were labelled with different clones of anti-human CD34 monoclonal antibodies (AC136, 581, and 8G12) and rabbit polyclonal CD34 antibody (pCD34) and anti-human CD133 monoclonal antibodies (AC133 and 293C3). Flow cytometry showed a higher percentage of rabbit CD34+ cells labelled by AC136 in comparison to the clone 581 and pCD34 (P < 0.01). A higher percentage of rabbit CD133+ cells were also detected by 293C3 compared to the AC133 clone (P < 0.01). Therefore, AC136 clone was used for the indirect immunomagnetic enrichment of rabbit CD34+ cells using magnetic-activated cell sorting (MACS). The enrichment of the rabbit CD34+ cells after sorting was low in comparison to human samples (2.4% vs. 39.6%). PCR analyses confirmed the efficient enrichment of human CD34+ cells and the low expression of CD34 mRNA in rabbit positive fraction. In conclusion, the tested antibodies might be suitable for detection, but not for sorting the rabbit CD34+ HSCs and new specific anti-rabbit CD34 antibodies are needed for efficient enrichment of rabbit HSCs. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1278-1289, 2018.

Assessment of neutralizing interleukin-4 effect on CD133 gene expression in colon cancer cell line.

Colorectal cancer may be maintained by cancer stem-like cells (CSCs) that express the cell surface marker CD133. CSCs (CD133+cells) exhibits greater resistance to the chemotherapy and this resistance may be mediated in part by an autocrine response to IL4. The aim of the study was to assess the effect of anti-IL4 antibody alone or in combination with chemotherapy on the CD133 expression andthe tumor growth. We used Caco cell line in our experiments and the samples were as the following; untreated colorectal cell line, cells treated by chemotherapy, cells treated by anti-IL4 antibody in 3doses (2.5, 5, 10µg/ml), cells treatedby combination of chemotherapy and anti-IL4 antibody in 3 doses. Results of our in vitro studies demonstrated that anti-IL4 inhibited growth of Caco cell line in a dose-dependent manner revealing a 32.11% inhibition at the highest concentration (10µg/ml). There was further significant inhibition by combination of anti IL4 and chemotherapy in a dose response manner when compared to group treated by chemotherapy only. These effects were associated with decreased expression of CD133 in tumor cells also. Lastly, anti-IL4 antibody stimulated apoptosis. Our study suggested that neutralizing of IL4 by anti IL4 antibody affect the CD133+ cells may be by increasing their apoptosis. The effects of anti IL4 antibody either, alone or in combination with chemotherapy, inhibited the tumor growth and decreased the viable tumor cells. Furthermore, neutralizing of IL4 increased the efficacy of chemotherapy treatment.

Assessment of immune reconstitution status in recipients of a successful hematopoietic stem cell transplant from peripheral blood after reduced intensity conditioning.

OBJECTIVE: To document immune reconstitution status after hematopoietic stem cell transplantation (HSCT) for malignant hematologic diseases. METHODS: Hematology patients who received a reduced intensity conditioning (RIC) were followed after successful allogeneic or autologous HSCT. Patients had at least 100days post-transplant. T, B and NK cells in peripheral blood (PB), and CD34+, CD133+ progenitor cells in bone marrow (BM) and peripheral blood (PB) were determined by flow cytometry. RESULTS: Twenty-seven HSCT recipients, 19 allogeneic and 8 autologous, were studied at a median 155 (100-721) days post-transplant. In the whole group the median value of CD34+ cells was 1.03% in the bone marrow and 0.04% in PB, whereas values for CD133+ cells were 0.39% and 0.13%, respectively, without statistical differences between autologous and allogeneic recipients. Significantly more B cells (CD3-/CD56-/CD19+) were found in the autologous compared to the allogeneic group, 12.6 vs. 5.01, p=0.04. An increased number of CD8+ lymphocytes with a 0.63 CD4:CD8 relationship was documented in PB. CONCLUSION: In clinically recovered autologous and allogeneic HSCT recipients BM and PB CD34+/CD133+ hematoprogenitor homeostasis is maintained within normal ranges, with better B-cell reconstitution in the autologous group.

CD133/CD166/Ki-67 triple immunofluorescence assessment for putative cancer stem cells in colon carcinoma.

BACKGROUND & AIMS: Colorectal cancer represents the third most common malignancy and the fourth most common cause of cancer death worldwide. The existence of drug-resistant colon cancer stem cells is thought to be one of the most important reasons behind treatment failure in colon cancer, their existence putatively leading to metastasis and recurrences. The aim of our study was to investigate the immunoexpression patterns of CD133 and CD166 in colon carcinoma, both individually and in combination, assessing their significance as prognostic markers. METHODS: A total of 45 retrospective colon adenocarcinoma cases were investigated by enzymatic and multiple fluorescence immunohistochemistry for their CD133 and CD166 expression and colocalization. RESULTS: Both CD133 and CD166 were expressed to different extents in all cancer specimens, with a predominant cytoplasmic pattern for CD133 and a more obvious membranous-like pattern for CD166. Overall, when comparing their reactivity for the tumoral tissue, CD166 expression areas seemed to be smaller than those of CD133. However, there was a direct correlation between CD133 and CD166 expression levels throughout the entire spectrum of lesions, with higher values for dysplastic lesions. Colocalization of CD133/CD166 was obvious at the level of cells membranes, with higher coefficients in high grade dysplasia, followed by well and moderate differentiated tumours. : CD133/CD166 colocalization is an early event occurring in colon tumorigenesis, with the highest coefficients recorded for patients with high grade dysplasia, followed by well differentiated tumours. Thus, we consider that the coexpression of these two markers could be useful for further prognostic and therapeutically stratification of patients with colon cancer.

Stepwise optimization of the procedure for assessment of circulating progenitor cells in patients with myocardial infarction.

BACKGROUND: The number and functional activity of circulating progenitor cells (CPCs) is altered in diabetic patients. Furthermore, reduced CPC count has been shown to independently predict cardiovascular events. Validation of CPCs as a biomarker for cardiovascular risk stratification requires rigorous methodology. Before a standard operation protocol (SOP) can be designed for such a trial, a variety of technical issues have to be addressed fundamentally, which include the appropriate type of red blood cell lysis buffer, FMO or isotype controls to identify rare cell populations from background noise, optimal antibody dilutions and conditions of sample storage. We herein propose improvements in critical steps of CPC isolation, antigenic characterization and determination of functional competence for final application in a prospective investigation of CPCs as a biomarker of outcome following acute myocardial infarction. METHODS AND FINDINGS: In this validation study, we refined the standard operating procedure (SOP) for flow cytometry characterisation and functional analysis of CPCs from the first 18 patients of the Progenitor Cell Response after Myocardial Infarction Study (ProMIS). ProMIS aims to verify the prognostic value of CPCs in patients with either ST elevation or non-ST elevation myocardial infarction with or without diabetes mellitus, using cardiac magnetic resonance imaging (MRI) for assessment of ventricular function as a primary endpoint. Results indicate crucial steps for SOP implementation, namely timely cell isolation after sampling, use of appropriate lysis buffer to separate blood cell types and minimize the acquisition events during flow cytometry, adoption of proper fluorophore combination and antibody titration for multiple antigenic detection and introduction of counting beads for precise quantification of functional CPC activity in migration assay. CONCLUSION AND SIGNIFICANCE: With systematic specification of factors influencing the enumeration of CPC by flow cytometry, the abundance and migration capacity of CPCs can be correctly assessed. Adoption of validated SOP is essential for refined comparison of patients with different comorbidities in the analysis of risk stratification.

Collection and analysis of hematopoietic progenitor cells from cynomolgus macaques (Macaca fascicularis): assessment of cross-reacting monoclonal antibodies.

Previous studies have shown that hematopoietic progenitor cells can be isolated from human or nonhuman primate bone marrow (BM) cells. In the present study, we studied the cross-reactivity of 13 anti-human CD34, two anti-human c-Kit, and one anti-human CD133 monoclonal antibodies (mAbs) with cynomolgus macaque (Macaca fascicularis) BM cells, using flow cytometric analysis, cell enrichment, and clonogenic assay. Among the 13 anti-human CD34 mAbs assessed, six cross-reacted as previously reported by other groups. However, only three of these six mAbs (clones 561, 563, and 12.8) recognized cynomolgus CD34+ cells that formed progenitor colonies when grown in methylcellulose culture. Similarly, of the two anti-human c-Kit mAbs (clones NU-c-kit and 95C3) that were previously reported to cross-react with cynomolgus BM cells, only one (clone NU-c-kit) resulted in a similar outcome. The anti-human CD133 mAb (clone AC133) also cross-reacted with cynomolgus BM cells, although these cells did not give rise to colonies when grown in culture. These results suggest that antibodies that cross-react with nonhuman primate cells may not identify the hematopoietic cells of interest. In addition, while the CD34 mAb (clone 561) results in the selection of hematopoietic progenitor cells of all lineages when assessed in methylcellulose culture, the c-Kit(high) fraction (NU-c-kit) exclusively identifies erythroid-specific progenitor cells after growth in culture. It is important to consider these findings when selecting cross-reacting mAbs to identify cells of hematopoietic lineages in macaque species.