Limits...
ALDH activity selectively defines an enhanced tumor-initiating cell population relative to CD133 expression in human pancreatic adenocarcinoma.

Kim MP, Fleming JB, Wang H, Abbruzzese JL, Choi W, Kopetz S, McConkey DJ, Evans DB, Gallick GE - PLoS ONE (2011)

Bottom Line: These cancer stem cells (CSCs), or tumor-initiating cells (TICs), exhibit properties of normal stem cells and are associated with resistance to current therapies.Although cell populations enriched for CD133 expression may alone possess tumorigenic potential, they are significantly less tumorigenic than ALDH(high) cell populations.We have thus identified a distinct population of TICs that should lead to identification of novel targets for pancreatic cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.

ABSTRACT

Background: Multiple studies in recent years have identified highly tumorigenic populations of cells that drive tumor formation. These cancer stem cells (CSCs), or tumor-initiating cells (TICs), exhibit properties of normal stem cells and are associated with resistance to current therapies. As pancreatic adenocarcinoma is among the most resistant human cancers to chemo-radiation therapy, we sought to evaluate the presence of cell populations with tumor-initiating capacities in human pancreatic tumors. Understanding which pancreatic cancer cell populations possess tumor-initiating capabilities is critical to characterizing and understanding the biology of pancreatic CSCs towards therapeutic ends.

Methodology/principal findings: We have isolated populations of cells with high ALDH activity (ALDH(high)) and/or CD133 cell surface expression from human xenograft tumors established from multiple patient tumors with pancreatic adenocarcinoma (direct xenograft tumors) and from the pancreatic cancer cell line L3.6pl. Through fluorescent activated cell sorting (FACs)-mediated enrichment and depletion of selected pancreatic cancer cell populations, we sought to discriminate the relative tumorigenicity of cell populations that express the pancreatic CSC markers CD133 and aldehyde dehydrogenase (ALDH). ALDH(high) and ALDH(low) cell populations were further examined for co-expression of CD44 and/or CD24. We demonstrate that unlike cell populations demonstrating low ALDH activity, as few as 100 cells enriched for high ALDH activity were capable of tumor formation, irrespective of CD133 expression. In direct xenograft tumors, the proportions of total tumor cells expressing ALDH and/or CD133 in xenograft tumors were unchanged through a minimum of two passages. We further demonstrate that ALDH expression among patients with pancreatic adenocarcinoma is heterogeneous, but the expression is constant in serial generations of individual direct xenograft tumors established from bulk human pancreatic tumors in NOD/SCID mice.

Conclusions/significance: We conclude that, in contrast to some previous studies, cell populations enriched for high ALDH activity alone are sufficient for efficient tumor-initiation with enhanced tumorigenic potential relative to CD133(+) and ALDH(low) cell populations in some direct xenograft tumors. Although cell populations enriched for CD133 expression may alone possess tumorigenic potential, they are significantly less tumorigenic than ALDH(high) cell populations. ALDH(high)/CD44(+)/CD24(+) or ALDH(low)/CD44(+)/CD24(+) phenotypes do not appear to significantly contribute to tumor formation at low numbers of inoculated tumor cells. ALDH expression broadly varies among patients with pancreatic adenocarcinoma and the apparent expression is recapitulated in serial generations of direct xenograft tumors in NOD/SCID. We have thus identified a distinct population of TICs that should lead to identification of novel targets for pancreatic cancer therapy.

Show MeSH

Related in: MedlinePlus

ALDH activity and expression in L3.6pl pancreatic cancer cells.(A) Representative example of L3.6pl cells stained with Aldefluor reagent with and without the DEAB inhibitor as described in Materials and Methods. Analysis by flow cytometry demonstrated high ALDH activity in approximately 16% of cells. (B) L3.6pl cells stained with directly conjugated CD133 antibodies failed to reveal a significant CD133+ population relative to isotype controls. (C) L3.6pl cells form spheres after plating on ultra-low attachment plates (average of 12 spheres/10,000 cells). (D) Cross sections of spheres were made and stained for ALDH1. A minority of cells contained within spheroids demonstrated strong ALDH1expression. (E) Staining for ALDH1 in sections of indirect xenograft tumors formed from L3.6pl cells in nude mice. Note small clusters of ALDH positive cells. Scale bar  = 20 µm (D), 50 µm (E).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3113804&req=5

pone-0020636-g001: ALDH activity and expression in L3.6pl pancreatic cancer cells.(A) Representative example of L3.6pl cells stained with Aldefluor reagent with and without the DEAB inhibitor as described in Materials and Methods. Analysis by flow cytometry demonstrated high ALDH activity in approximately 16% of cells. (B) L3.6pl cells stained with directly conjugated CD133 antibodies failed to reveal a significant CD133+ population relative to isotype controls. (C) L3.6pl cells form spheres after plating on ultra-low attachment plates (average of 12 spheres/10,000 cells). (D) Cross sections of spheres were made and stained for ALDH1. A minority of cells contained within spheroids demonstrated strong ALDH1expression. (E) Staining for ALDH1 in sections of indirect xenograft tumors formed from L3.6pl cells in nude mice. Note small clusters of ALDH positive cells. Scale bar  = 20 µm (D), 50 µm (E).

Mentions: We first evaluated ALDH activity and CD133 cell surface expression in a pancreatic cancer cell line (L3.6pl) previously used to enrich CSC populations through CD133 cell surface expression[8]. Flow cytometry demonstrated heterogeneous ALDH activity among L3.6pl cells with an average of 16.2% (median = 13.8%) ALDHhigh cells relative to DEAB control samples, whereas CD133 expression was nearly undetectable (0.02%, median = 0.17) relative to isotype controls (Figure 1A, 1B). When plated in ultra-low attachment plates under conditions favoring the undifferentiated state, an average of 12 /10,000 L3.6pl cells (median = 11.5) possessed the ability to form spheroid structures and serial sections of paraffin-embedded spheres demonstrated the infrequent presence of cells expressing high levels of ALDH1 relative to all other cells (Figures 1C, 1D). Subcutaneously implanted L3.6pl cells in nude mice formed tumors with heterogeneous ALDH1 expression. ALDH1 expression was confined to small clusters of cells distributed throughout the tumors (Figure 1E). CD133 expression was not detected in indirect xenograft tumors formed from L3.6pl cells (data not shown).


ALDH activity selectively defines an enhanced tumor-initiating cell population relative to CD133 expression in human pancreatic adenocarcinoma.

Kim MP, Fleming JB, Wang H, Abbruzzese JL, Choi W, Kopetz S, McConkey DJ, Evans DB, Gallick GE - PLoS ONE (2011)

ALDH activity and expression in L3.6pl pancreatic cancer cells.(A) Representative example of L3.6pl cells stained with Aldefluor reagent with and without the DEAB inhibitor as described in Materials and Methods. Analysis by flow cytometry demonstrated high ALDH activity in approximately 16% of cells. (B) L3.6pl cells stained with directly conjugated CD133 antibodies failed to reveal a significant CD133+ population relative to isotype controls. (C) L3.6pl cells form spheres after plating on ultra-low attachment plates (average of 12 spheres/10,000 cells). (D) Cross sections of spheres were made and stained for ALDH1. A minority of cells contained within spheroids demonstrated strong ALDH1expression. (E) Staining for ALDH1 in sections of indirect xenograft tumors formed from L3.6pl cells in nude mice. Note small clusters of ALDH positive cells. Scale bar  = 20 µm (D), 50 µm (E).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113804&req=5

pone-0020636-g001: ALDH activity and expression in L3.6pl pancreatic cancer cells.(A) Representative example of L3.6pl cells stained with Aldefluor reagent with and without the DEAB inhibitor as described in Materials and Methods. Analysis by flow cytometry demonstrated high ALDH activity in approximately 16% of cells. (B) L3.6pl cells stained with directly conjugated CD133 antibodies failed to reveal a significant CD133+ population relative to isotype controls. (C) L3.6pl cells form spheres after plating on ultra-low attachment plates (average of 12 spheres/10,000 cells). (D) Cross sections of spheres were made and stained for ALDH1. A minority of cells contained within spheroids demonstrated strong ALDH1expression. (E) Staining for ALDH1 in sections of indirect xenograft tumors formed from L3.6pl cells in nude mice. Note small clusters of ALDH positive cells. Scale bar  = 20 µm (D), 50 µm (E).
Mentions: We first evaluated ALDH activity and CD133 cell surface expression in a pancreatic cancer cell line (L3.6pl) previously used to enrich CSC populations through CD133 cell surface expression[8]. Flow cytometry demonstrated heterogeneous ALDH activity among L3.6pl cells with an average of 16.2% (median = 13.8%) ALDHhigh cells relative to DEAB control samples, whereas CD133 expression was nearly undetectable (0.02%, median = 0.17) relative to isotype controls (Figure 1A, 1B). When plated in ultra-low attachment plates under conditions favoring the undifferentiated state, an average of 12 /10,000 L3.6pl cells (median = 11.5) possessed the ability to form spheroid structures and serial sections of paraffin-embedded spheres demonstrated the infrequent presence of cells expressing high levels of ALDH1 relative to all other cells (Figures 1C, 1D). Subcutaneously implanted L3.6pl cells in nude mice formed tumors with heterogeneous ALDH1 expression. ALDH1 expression was confined to small clusters of cells distributed throughout the tumors (Figure 1E). CD133 expression was not detected in indirect xenograft tumors formed from L3.6pl cells (data not shown).

Bottom Line: These cancer stem cells (CSCs), or tumor-initiating cells (TICs), exhibit properties of normal stem cells and are associated with resistance to current therapies.Although cell populations enriched for CD133 expression may alone possess tumorigenic potential, they are significantly less tumorigenic than ALDH(high) cell populations.We have thus identified a distinct population of TICs that should lead to identification of novel targets for pancreatic cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.

ABSTRACT

Background: Multiple studies in recent years have identified highly tumorigenic populations of cells that drive tumor formation. These cancer stem cells (CSCs), or tumor-initiating cells (TICs), exhibit properties of normal stem cells and are associated with resistance to current therapies. As pancreatic adenocarcinoma is among the most resistant human cancers to chemo-radiation therapy, we sought to evaluate the presence of cell populations with tumor-initiating capacities in human pancreatic tumors. Understanding which pancreatic cancer cell populations possess tumor-initiating capabilities is critical to characterizing and understanding the biology of pancreatic CSCs towards therapeutic ends.

Methodology/principal findings: We have isolated populations of cells with high ALDH activity (ALDH(high)) and/or CD133 cell surface expression from human xenograft tumors established from multiple patient tumors with pancreatic adenocarcinoma (direct xenograft tumors) and from the pancreatic cancer cell line L3.6pl. Through fluorescent activated cell sorting (FACs)-mediated enrichment and depletion of selected pancreatic cancer cell populations, we sought to discriminate the relative tumorigenicity of cell populations that express the pancreatic CSC markers CD133 and aldehyde dehydrogenase (ALDH). ALDH(high) and ALDH(low) cell populations were further examined for co-expression of CD44 and/or CD24. We demonstrate that unlike cell populations demonstrating low ALDH activity, as few as 100 cells enriched for high ALDH activity were capable of tumor formation, irrespective of CD133 expression. In direct xenograft tumors, the proportions of total tumor cells expressing ALDH and/or CD133 in xenograft tumors were unchanged through a minimum of two passages. We further demonstrate that ALDH expression among patients with pancreatic adenocarcinoma is heterogeneous, but the expression is constant in serial generations of individual direct xenograft tumors established from bulk human pancreatic tumors in NOD/SCID mice.

Conclusions/significance: We conclude that, in contrast to some previous studies, cell populations enriched for high ALDH activity alone are sufficient for efficient tumor-initiation with enhanced tumorigenic potential relative to CD133(+) and ALDH(low) cell populations in some direct xenograft tumors. Although cell populations enriched for CD133 expression may alone possess tumorigenic potential, they are significantly less tumorigenic than ALDH(high) cell populations. ALDH(high)/CD44(+)/CD24(+) or ALDH(low)/CD44(+)/CD24(+) phenotypes do not appear to significantly contribute to tumor formation at low numbers of inoculated tumor cells. ALDH expression broadly varies among patients with pancreatic adenocarcinoma and the apparent expression is recapitulated in serial generations of direct xenograft tumors in NOD/SCID. We have thus identified a distinct population of TICs that should lead to identification of novel targets for pancreatic cancer therapy.

Show MeSH
Related in: MedlinePlus