Image-based assessment of natural killer cell activity against glioblastoma stem cells.

Glioblastoma (GBM) poses a significant challenge in oncology and stands as the most aggressive form of brain cancer. A primary contributor to its relentless nature is the stem-like cancer cells, called glioblastoma stem cells (GSCs). GSCs have the capacity for self-renewal and tumorigenesis, leading to frequent GBM recurrences and complicating treatment modalities. While natural killer (NK) cells exhibit potential in targeting and eliminating stem-like cancer cells, their efficacy within the GBM microenvironment is limited due to constrained infiltration and function. To address this limitation, novel investigations focusing on boosting NK cell activity against GSCs are imperative. This study presents two streamlined image-based assays assessing NK cell migration and cytotoxicity towards GSCs. It details protocols and explores the strengths and limitations of these methods. These assays could aid in identifying novel targets to enhance NK cell activity towards GSCs, facilitating the development of NK cell-based immunotherapy for improved GBM treatment.

Quantitative assessment of the CD26+ leukemic stem cell compartment in chronic myeloid leukemia: patient-subgroups, prognostic impact, and technical aspects.

Little is known about the function and phenotype of leukemic stem cells (LSCs) in chronic myeloid leukemia (CML) or about specific markers that discriminate LSCs from normal hematopoietic stem cells (HSCs). CD26 has recently been described as a specific marker of CML LSCs. In the current study, we investigated this marker in a cohort of 31 unselected CML patients. BCR/ABL1 positivity was analyzed in highly enriched stem cell fractions using fluorescence in situ hybridization (FISH) and reverse transcription PCR (RT-PCR). The proportion of CD26+ LSCs and CD26- HSCs varied considerably among the patients analyzed, and the percentage of CD26+ cells correlated with leukocyte count. The CD26 expression robustly discriminated LSCs from HSCs. This required a strict gating of the stem cell compartment. Thus, in patients with very low LSC or HSC numbers, only the highly sensitive RT-PCR method discriminated between clonal and non-clonal cells, while a robust FISH analysis required larger numbers of cells in both compartments. Finally, our data show that the numbers of CD26+ CML LSCs correlate with responses to treatment with BCR-ABL1 inhibitors.

Novel immunofluorescence protocol for multimarker assessment of putative disseminating breast cancer stem cells.

PURPOSE: The phenotypical and functional variety of breast cancer cells is well recognized. This variety is evident in primary tumors and in disseminated tumor cells (DTCs) and solid metastases as shown for recognized prognostic factors, such as estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2/neu and also for cancer stem cell markers such as CD44, CD24, or aldehyde dehydrogenase (ALDH). For the development of new therapeutic strategies, the identification and characterization of disseminated breast cancer cells are needed. This requires the use of multiple antibodies (ie, cytokeratin, Her2/neu, ALDH1, CD44, and CD24) labeled with fluorochromes of different colors and spectral image analysis to separate different color spectra. METHODS: We have focused here on putative breast cancer stem cell markers and evaluated the feasibility of triple and quadruple labeling of breast cancer cells. Using breast cancer cell lines we have developed a method optimized for multimarker analysis by employing novel DyLight Technology. Single marker immunofluorescence was performed in 6 replicates, and reproducible results had to be obtained before proceeding to multimarker immunofluorescence. RESULTS: Three of the markers, CD44, ALDH1, and cytokeratin have been directly conjugated with DyLight dyes. CD24 could not be conjugated directly to the fluorescent dye. A labeled secondary antibody was used for visualization. Single and multimarker immunofluorescence gave consistent results throughout the replicates. CONCLUSIONS: This novel protocol will facilitate detection and phenotypical characterization of disseminated tumor cells. In addition, by adding additional markers, distinct subpopulations could be evaluated for the expression of particular therapeutic targets.

Immunofluorescent assessment of tumour infiltrating cells in laryngeal carcinoma. Application of monoclonal antibodies.

In order to gain some insight into host cell accumulations within primary tumour, frozen sections from surgical specimens of laryngeal carcinoma were subjected to indirect immunofluorescence using a panel of monoclonal antibodies against various human lymphocyte subsets as well as macrophages. In addition, polyclonal antibodies against Ig were used in order to trace B cells. Numerous host cell infiltrates seen at the tumour periphery were composed of T4 (helper) lymphocytes and macrophages. Lymphocytes of OKT8 (suppressor/cytotoxic) and Leu-7 (NK cells) series were intermingled with tumour cells in the case of scanty infiltrates. Infiltrating cells were also linked to the presence of metastases in regional lymph nodes. OKT4-positive abundant infiltrates were usually accompanied by uninvolved nodes, while scanty ones with OKT8 specificity were relatively frequently seen in the patients with evidence of nodal metastases. These differences were not statistically significant, however, B cells as well as plasma cells were infrequently observed and were encountered both in tumour samples with intensive cellular infiltrates as well as in those with scanty ones.