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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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Higher clonal efficiency and colony forming potential of CD44hi cells and their CSC molecular markers expression in comparison to CD44lo cells.The sorted cells CD44hi and CD44lo from MPE samples were analyzed in triplicates for their (A) clonal efficiency (Sample M-1: colonies CD44hi = 35.8 (SD = 5.04) vs CD44lo = 21.7 (SD = 6.2) (P = 0.03); Sample M-2 colonies CD44hi = 59.8 (SD = 3.2) vs CD44lo = 40.6 (SD = 4.1) (P = 0.003); Sample M-3 colonies CD44hi = 53.4 (SD = 5.3) vs CD44lo = 33.9 (SD = 3.6) (P = 0.006)); The mean effect of CD44hi versus CD44lo is 17.6 (95% CI: 8.31, 26.89: p = 0.015). (B) colony forming ability in soft agar. (Sample M-1: colonies CD44hi = 16.6 (SD = 1.1) vs CD44lo = 8 (SD = 1.1) (P = 0.0006); Sample M-2: colonies CD44hi = 27 (SD = 7) vs CD44lo = 12 (SD = 3) (P = 0.02); Sample M-3: colonies CD44hi = 24.3 (SD = 6.1) vs CD44lo = 12.6 (SD = 2.5) (P = 0.03)). The mean effect of CD44hi versus CD44lo is 11.8 (95% CI: 3.41, 20.14; P = 0.026). Columns, mean from three independent experiment; SD, *, P<0.001, compared with the CD44lo groups, (student’s t test). (C) Soft agar colonies derived from CD44hi cells (100×) and (D) CD44lo cells (100×). (E) Expression profile of BMI-1, hTERT, SUZ12, EZH2 and OCT-4 in sorted CD44hi and CD44lo cells were analyzed by reverse transcriptase-qPCR. In sample M-3 only BMI-1 is expressed at high level in CD44hi population than the CD44lo cell population. In sample M-2 slight higher expression of hTERT in CD44hi cells than CD44lo cells. The CD44hi population of sample M-1 expressed high level of BMI-1, hTERT, SUZ12, EZH2 and OCT-4, than CD44lo population.
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pone-0073195-g002: Higher clonal efficiency and colony forming potential of CD44hi cells and their CSC molecular markers expression in comparison to CD44lo cells.The sorted cells CD44hi and CD44lo from MPE samples were analyzed in triplicates for their (A) clonal efficiency (Sample M-1: colonies CD44hi = 35.8 (SD = 5.04) vs CD44lo = 21.7 (SD = 6.2) (P = 0.03); Sample M-2 colonies CD44hi = 59.8 (SD = 3.2) vs CD44lo = 40.6 (SD = 4.1) (P = 0.003); Sample M-3 colonies CD44hi = 53.4 (SD = 5.3) vs CD44lo = 33.9 (SD = 3.6) (P = 0.006)); The mean effect of CD44hi versus CD44lo is 17.6 (95% CI: 8.31, 26.89: p = 0.015). (B) colony forming ability in soft agar. (Sample M-1: colonies CD44hi = 16.6 (SD = 1.1) vs CD44lo = 8 (SD = 1.1) (P = 0.0006); Sample M-2: colonies CD44hi = 27 (SD = 7) vs CD44lo = 12 (SD = 3) (P = 0.02); Sample M-3: colonies CD44hi = 24.3 (SD = 6.1) vs CD44lo = 12.6 (SD = 2.5) (P = 0.03)). The mean effect of CD44hi versus CD44lo is 11.8 (95% CI: 3.41, 20.14; P = 0.026). Columns, mean from three independent experiment; SD, *, P<0.001, compared with the CD44lo groups, (student’s t test). (C) Soft agar colonies derived from CD44hi cells (100×) and (D) CD44lo cells (100×). (E) Expression profile of BMI-1, hTERT, SUZ12, EZH2 and OCT-4 in sorted CD44hi and CD44lo cells were analyzed by reverse transcriptase-qPCR. In sample M-3 only BMI-1 is expressed at high level in CD44hi population than the CD44lo cell population. In sample M-2 slight higher expression of hTERT in CD44hi cells than CD44lo cells. The CD44hi population of sample M-1 expressed high level of BMI-1, hTERT, SUZ12, EZH2 and OCT-4, than CD44lo population.

Mentions: To investigate whether the CD44hi cells are functionally different from the CD44lo cells in colony forming efficiency, we sorted and cultured these subsets from the three samples (M-1, M-2 and M-3). 100–500 cells of CD44hi or CD44lo cells were plated in individual wells of 12-well plates. Although we are unable to detect significant differences in initial plating efficiency, but we do observe that CD44hi cells are more competent at forming holoclones than the CD44lo cells (t test and ANOVA: P<0.05) (Figure 2A). Thus, an intrinsic biological difference between CD44hi and CD44lo cells seems to an inherent differential competency in forming holoclones.


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)

Higher clonal efficiency and colony forming potential of CD44hi cells and their CSC molecular markers expression in comparison to CD44lo cells.The sorted cells CD44hi and CD44lo from MPE samples were analyzed in triplicates for their (A) clonal efficiency (Sample M-1: colonies CD44hi = 35.8 (SD = 5.04) vs CD44lo = 21.7 (SD = 6.2) (P = 0.03); Sample M-2 colonies CD44hi = 59.8 (SD = 3.2) vs CD44lo = 40.6 (SD = 4.1) (P = 0.003); Sample M-3 colonies CD44hi = 53.4 (SD = 5.3) vs CD44lo = 33.9 (SD = 3.6) (P = 0.006)); The mean effect of CD44hi versus CD44lo is 17.6 (95% CI: 8.31, 26.89: p = 0.015). (B) colony forming ability in soft agar. (Sample M-1: colonies CD44hi = 16.6 (SD = 1.1) vs CD44lo = 8 (SD = 1.1) (P = 0.0006); Sample M-2: colonies CD44hi = 27 (SD = 7) vs CD44lo = 12 (SD = 3) (P = 0.02); Sample M-3: colonies CD44hi = 24.3 (SD = 6.1) vs CD44lo = 12.6 (SD = 2.5) (P = 0.03)). The mean effect of CD44hi versus CD44lo is 11.8 (95% CI: 3.41, 20.14; P = 0.026). Columns, mean from three independent experiment; SD, *, P<0.001, compared with the CD44lo groups, (student’s t test). (C) Soft agar colonies derived from CD44hi cells (100×) and (D) CD44lo cells (100×). (E) Expression profile of BMI-1, hTERT, SUZ12, EZH2 and OCT-4 in sorted CD44hi and CD44lo cells were analyzed by reverse transcriptase-qPCR. In sample M-3 only BMI-1 is expressed at high level in CD44hi population than the CD44lo cell population. In sample M-2 slight higher expression of hTERT in CD44hi cells than CD44lo cells. The CD44hi population of sample M-1 expressed high level of BMI-1, hTERT, SUZ12, EZH2 and OCT-4, than CD44lo population.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760902&req=5

pone-0073195-g002: Higher clonal efficiency and colony forming potential of CD44hi cells and their CSC molecular markers expression in comparison to CD44lo cells.The sorted cells CD44hi and CD44lo from MPE samples were analyzed in triplicates for their (A) clonal efficiency (Sample M-1: colonies CD44hi = 35.8 (SD = 5.04) vs CD44lo = 21.7 (SD = 6.2) (P = 0.03); Sample M-2 colonies CD44hi = 59.8 (SD = 3.2) vs CD44lo = 40.6 (SD = 4.1) (P = 0.003); Sample M-3 colonies CD44hi = 53.4 (SD = 5.3) vs CD44lo = 33.9 (SD = 3.6) (P = 0.006)); The mean effect of CD44hi versus CD44lo is 17.6 (95% CI: 8.31, 26.89: p = 0.015). (B) colony forming ability in soft agar. (Sample M-1: colonies CD44hi = 16.6 (SD = 1.1) vs CD44lo = 8 (SD = 1.1) (P = 0.0006); Sample M-2: colonies CD44hi = 27 (SD = 7) vs CD44lo = 12 (SD = 3) (P = 0.02); Sample M-3: colonies CD44hi = 24.3 (SD = 6.1) vs CD44lo = 12.6 (SD = 2.5) (P = 0.03)). The mean effect of CD44hi versus CD44lo is 11.8 (95% CI: 3.41, 20.14; P = 0.026). Columns, mean from three independent experiment; SD, *, P<0.001, compared with the CD44lo groups, (student’s t test). (C) Soft agar colonies derived from CD44hi cells (100×) and (D) CD44lo cells (100×). (E) Expression profile of BMI-1, hTERT, SUZ12, EZH2 and OCT-4 in sorted CD44hi and CD44lo cells were analyzed by reverse transcriptase-qPCR. In sample M-3 only BMI-1 is expressed at high level in CD44hi population than the CD44lo cell population. In sample M-2 slight higher expression of hTERT in CD44hi cells than CD44lo cells. The CD44hi population of sample M-1 expressed high level of BMI-1, hTERT, SUZ12, EZH2 and OCT-4, than CD44lo population.
Mentions: To investigate whether the CD44hi cells are functionally different from the CD44lo cells in colony forming efficiency, we sorted and cultured these subsets from the three samples (M-1, M-2 and M-3). 100–500 cells of CD44hi or CD44lo cells were plated in individual wells of 12-well plates. Although we are unable to detect significant differences in initial plating efficiency, but we do observe that CD44hi cells are more competent at forming holoclones than the CD44lo cells (t test and ANOVA: P<0.05) (Figure 2A). Thus, an intrinsic biological difference between CD44hi and CD44lo cells seems to an inherent differential competency in forming holoclones.

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