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The CD44(high) tumorigenic subsets in lung cancer biospecimens are enriched for low miR-34a expression.

Basak SK, Veena MS, Oh S, Lai C, Vangala S, Elashoff D, Fishbein MC, Sharma S, Rao NP, Rao D, Phan R, Srivatsan ES, Batra RK - PLoS ONE (2013)

Bottom Line: Progression can be associated with emergence of cells that exhibit high phenotypic plasticity (including "de-differentiation" to primitive developmental states), and aggressive behavioral properties (including high tumorigenic potentials).The colony forming efficiency of CD44(hi) cells, characteristic property of CSC, can be inhibited by mir-34a replacement in these samples.In addition the highly tumorigenic CD44(hi) cells are enriched for cells in the G2 phase of cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Stem Cell Institute, Veterans Affairs Greater Los Angeles Healthcare System (VAGLAHS), Los Angeles, California, United States of America ; Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America.

ABSTRACT
Cellular heterogeneity is an integral part of cancer development and progression. Progression can be associated with emergence of cells that exhibit high phenotypic plasticity (including "de-differentiation" to primitive developmental states), and aggressive behavioral properties (including high tumorigenic potentials). We observed that many biomarkers that are used to identify Cancer Stem Cells (CSC) can label cell subsets in an advanced clinical stage of lung cancer (malignant pleural effusions, or MPE). Thus, CSC-biomarkers may be useful for live sorting functionally distinct cell subsets from individual tumors, which may enable investigators to hone in on the molecular basis for functional heterogeneity. We demonstrate that the CD44(hi) (CD44-high) cancer cell subsets display higher clonal, colony forming potential than CD44(lo) cells (n=3) and are also tumorigenic (n=2/2) when transplanted in mouse xenograft model. The CD44(hi) subsets express different levels of embryonal (de-differentiation) markers or chromatin regulators. In archived lung cancer tissues, ALDH markers co-localize more with CD44 in squamous cell carcinoma (n=5/7) than Adeno Carcinoma (n=1/12). MPE cancer cells and a lung cancer cell line (NCI-H-2122) exhibit chromosomal abnormalities and 1p36 deletion (n=3/3). Since miR-34a maps to the 1p36 deletion site, low miR-34a expression levels were detected in these cells. The colony forming efficiency of CD44(hi) cells, characteristic property of CSC, can be inhibited by mir-34a replacement in these samples. In addition the highly tumorigenic CD44(hi) cells are enriched for cells in the G2 phase of cell cycle.

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Morphologically variant cells in MPE samples and absence of morphological changes between CD44hi and CD44lo cells.The tumor cells from M-1, M-2 and M-3. (A) 100× (2–3 weeks). (B) 400× (2–3 weeks). (C) Later stages of culture 100× (6–10 weeks). (D) CD44- FACS expression pattern and MFI. (E) Sorting of CD44hi and CD44lo cells (5–10%). The sorted cells CD44hi and CD44lo were washed and plated out in PCM for 2–3 days to evaluate their morphological differences. (F) Morphology of sorted CD44hi cells and sorted (G) CD44lo cells were similar (100×). The purity of the CD44hi and CD44lo cells were ≥98%, as revealed by post sort analysis (data not shown).
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pone-0073195-g001: Morphologically variant cells in MPE samples and absence of morphological changes between CD44hi and CD44lo cells.The tumor cells from M-1, M-2 and M-3. (A) 100× (2–3 weeks). (B) 400× (2–3 weeks). (C) Later stages of culture 100× (6–10 weeks). (D) CD44- FACS expression pattern and MFI. (E) Sorting of CD44hi and CD44lo cells (5–10%). The sorted cells CD44hi and CD44lo were washed and plated out in PCM for 2–3 days to evaluate their morphological differences. (F) Morphology of sorted CD44hi cells and sorted (G) CD44lo cells were similar (100×). The purity of the CD44hi and CD44lo cells were ≥98%, as revealed by post sort analysis (data not shown).

Mentions: The primary cultures from three different MPE-samples (M-1, M-2 and M-3), contain morphological variants (flat, oval and rounded shapes) by light microscopy (Figure 1A, B). By the 4th week of culture, the adherent tumor cells display a more homogeneous morphology pattern in culture (Figure 1C). Cultured cells uniformly express CD44 in all three tumor samples (Figure 1D), but the labeling intensity is highly variable both between and within the same sample. Thus, compared to cells labeled with secondary antibody alone, the samples are 96%, 99% and 98% positive for CD44; however, the Mean Fluorescence Intensities (MFIs) of CD44 labeling are 10861, 5295 and 2120 respectively. Thus, the surface labeling intensity of CD44 expression may vary from 2 to 5 fold among tumor samples, and there is typically a large variance in average surface CD44 labeling within individual samples.


The CD44(high) tumorigenic subsets in lung cancer biospecimens are enriched for low miR-34a expression.

Basak SK, Veena MS, Oh S, Lai C, Vangala S, Elashoff D, Fishbein MC, Sharma S, Rao NP, Rao D, Phan R, Srivatsan ES, Batra RK - PLoS ONE (2013)

Morphologically variant cells in MPE samples and absence of morphological changes between CD44hi and CD44lo cells.The tumor cells from M-1, M-2 and M-3. (A) 100× (2–3 weeks). (B) 400× (2–3 weeks). (C) Later stages of culture 100× (6–10 weeks). (D) CD44- FACS expression pattern and MFI. (E) Sorting of CD44hi and CD44lo cells (5–10%). The sorted cells CD44hi and CD44lo were washed and plated out in PCM for 2–3 days to evaluate their morphological differences. (F) Morphology of sorted CD44hi cells and sorted (G) CD44lo cells were similar (100×). The purity of the CD44hi and CD44lo cells were ≥98%, as revealed by post sort analysis (data not shown).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760902&req=5

pone-0073195-g001: Morphologically variant cells in MPE samples and absence of morphological changes between CD44hi and CD44lo cells.The tumor cells from M-1, M-2 and M-3. (A) 100× (2–3 weeks). (B) 400× (2–3 weeks). (C) Later stages of culture 100× (6–10 weeks). (D) CD44- FACS expression pattern and MFI. (E) Sorting of CD44hi and CD44lo cells (5–10%). The sorted cells CD44hi and CD44lo were washed and plated out in PCM for 2–3 days to evaluate their morphological differences. (F) Morphology of sorted CD44hi cells and sorted (G) CD44lo cells were similar (100×). The purity of the CD44hi and CD44lo cells were ≥98%, as revealed by post sort analysis (data not shown).
Mentions: The primary cultures from three different MPE-samples (M-1, M-2 and M-3), contain morphological variants (flat, oval and rounded shapes) by light microscopy (Figure 1A, B). By the 4th week of culture, the adherent tumor cells display a more homogeneous morphology pattern in culture (Figure 1C). Cultured cells uniformly express CD44 in all three tumor samples (Figure 1D), but the labeling intensity is highly variable both between and within the same sample. Thus, compared to cells labeled with secondary antibody alone, the samples are 96%, 99% and 98% positive for CD44; however, the Mean Fluorescence Intensities (MFIs) of CD44 labeling are 10861, 5295 and 2120 respectively. Thus, the surface labeling intensity of CD44 expression may vary from 2 to 5 fold among tumor samples, and there is typically a large variance in average surface CD44 labeling within individual samples.

Bottom Line: Progression can be associated with emergence of cells that exhibit high phenotypic plasticity (including "de-differentiation" to primitive developmental states), and aggressive behavioral properties (including high tumorigenic potentials).The colony forming efficiency of CD44(hi) cells, characteristic property of CSC, can be inhibited by mir-34a replacement in these samples.In addition the highly tumorigenic CD44(hi) cells are enriched for cells in the G2 phase of cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Stem Cell Institute, Veterans Affairs Greater Los Angeles Healthcare System (VAGLAHS), Los Angeles, California, United States of America ; Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America.

ABSTRACT
Cellular heterogeneity is an integral part of cancer development and progression. Progression can be associated with emergence of cells that exhibit high phenotypic plasticity (including "de-differentiation" to primitive developmental states), and aggressive behavioral properties (including high tumorigenic potentials). We observed that many biomarkers that are used to identify Cancer Stem Cells (CSC) can label cell subsets in an advanced clinical stage of lung cancer (malignant pleural effusions, or MPE). Thus, CSC-biomarkers may be useful for live sorting functionally distinct cell subsets from individual tumors, which may enable investigators to hone in on the molecular basis for functional heterogeneity. We demonstrate that the CD44(hi) (CD44-high) cancer cell subsets display higher clonal, colony forming potential than CD44(lo) cells (n=3) and are also tumorigenic (n=2/2) when transplanted in mouse xenograft model. The CD44(hi) subsets express different levels of embryonal (de-differentiation) markers or chromatin regulators. In archived lung cancer tissues, ALDH markers co-localize more with CD44 in squamous cell carcinoma (n=5/7) than Adeno Carcinoma (n=1/12). MPE cancer cells and a lung cancer cell line (NCI-H-2122) exhibit chromosomal abnormalities and 1p36 deletion (n=3/3). Since miR-34a maps to the 1p36 deletion site, low miR-34a expression levels were detected in these cells. The colony forming efficiency of CD44(hi) cells, characteristic property of CSC, can be inhibited by mir-34a replacement in these samples. In addition the highly tumorigenic CD44(hi) cells are enriched for cells in the G2 phase of cell cycle.

Show MeSH
Related in: MedlinePlus