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. 2008 Jun 18;3(6):e2428.
doi: 10.1371/journal.pone.0002428.

Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy

Scott J Dylla  1 , Lucia Beviglia, In-Kyung Park, Cecile Chartier, Janak Raval, Lucy Ngan, Kellie Pickell, Jorge Aguilar, Sasha Lazetic, Stephanie Smith-Berdan, Michael F Clarke, Tim Hoey, John Lewicki, Austin L Gurney
Affiliations

Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy

Scott J Dylla et al. PLoS One. .

Erratum in

  • PLoS ONE. 2008;3(8). doi: 10.1371/annotation/2aa6a20a-e63c-49b6-aeea-aae62435617f

Abstract

Background: Patients generally die of cancer after the failure of current therapies to eliminate residual disease. A subpopulation of tumor cells, termed cancer stem cells (CSC), appears uniquely able to fuel the growth of phenotypically and histologically diverse tumors. It has been proposed, therefore, that failure to effectively treat cancer may in part be due to preferential resistance of these CSC to chemotherapeutic agents. The subpopulation of human colorectal tumor cells with an ESA(+)CD44(+) phenotype are uniquely responsible for tumorigenesis and have the capacity to generate heterogeneous tumors in a xenograft setting (i.e. CoCSC). We hypothesized that if non-tumorigenic cells are more susceptible to chemotherapeutic agents, then residual tumors might be expected to contain a higher frequency of CoCSC.

Methods and findings: Xenogeneic tumors initiated with CoCSC were allowed to reach approximately 400 mm(3), at which point mice were randomized and chemotherapeutic regimens involving cyclophosphamide or Irinotecan were initiated. Data from individual tumor phenotypic analysis and serial transplants performed in limiting dilution show that residual tumors are enriched for cells with the CoCSC phenotype and have increased tumorigenic cell frequency. Moreover, the inherent ability of residual CoCSC to generate tumors appears preserved. Aldehyde dehydrogenase 1 gene expression and enzymatic activity are elevated in CoCSC and using an in vitro culture system that maintains CoCSC as demonstrated by serial transplants and lentiviral marking of single cell-derived clones, we further show that ALDH1 enzymatic activity is a major mediator of resistance to cyclophosphamide: a classical chemotherapeutic agent.

Conclusions: CoCSC are enriched in colon tumors following chemotherapy and remain capable of rapidly regenerating tumors from which they originated. By focusing on the biology of CoCSC, major resistance mechanisms to specific chemotherapeutic agents can be attributed to specific genes, thereby suggesting avenues for improving cancer therapy.

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Conflict of interest statement

Competing Interests: MFC is a founder and member of the paid advisory board of OncoMed Pharmaceuticals Inc., and has an equity position in the company. All other authors are employees of OncoMed Pharmaceuticals Inc., a biotechnology company that has applied for patents related to this study.

Figures

Figure 1
Figure 1. CoCSC phenotype cells preferentially survive CPA chemotherapy.
A) Phenotypic profile of UM-C4 colorectal tumors for ESA, CD44 and CD166, following exclusion of mLin− cells. B) Formalin fixed, paraffin embedded tumor sections from CPA- or vehicle-treated control mice stained with Hematoxylin & Eosin (H&E), for proliferating Ki-67+, or for TUNEL+ dead cells. Black bar = 100 µm. C) Following randomization to normalize treatment groups at 400 mm3 at day 0, twice weekly administration of vehicle (green boxes) or 38 mg/kg CPA (red circles) commenced and tumors were measured periodically. D) Phenotypic analysis of individual tumors, displaying the percentage of human tumor cells with the ESA+CD44+ phenotype as a function of tumor size. E) Representative overlay histogram displaying CD166 surface expression on human ESA+CD44+ tumor cells from vehicle- or CPA-treated animals. The black line represents isotype control staining of ESA+CD44+ tumor cells.
Figure 2
Figure 2. Tumorigenic UM-C4 cells are enriched in residual tumors following CPA administration.
A) Limiting dilution analysis of unfractionated UM-C4 tumor cells was used to calculate TG cell frequency using Poisson distribution statistics (±95% confidence level; *P = 0.0016). B) Percentage of human ESA+CD44+ and ESA+CD44+CD166+ cells in secondary tumors arising from residual tumorigenic cells transplanted serially in limiting dilution following vehicle or CPA treatment regimens.
Figure 3
Figure 3. ALDH1 enzyme activity demarcates a subpopulation of CoCSC.
A) Phenotypic profile of human ESA+ UM-C4 tumor cells for ALDH1 enzymatic activity. Gates demarcate tumor subpopulations isolated by FACS for tumorigenicity studies, wherein B) growth kinetics of CD44+ALDH+ (red circles), CD44+ALDH− (orange boxes) and CD44−ALDH+ (green triangles) populations are plotted. Measurements reflect only mice with palpable tumors. Data representative of n = 3 independent experiments. C) Taqman qRT-PCR data displaying relative expression of ALDH1A1 (1A1), ALDH3A1 (3A1) and ALDH5A1 (5A1) in tumorigenic (TG; black) versus non-tumorigenic (NTG; white) cells (n≥2) from 2 patient-derived xenogeneic colorectal tumor lines (UM-C4 & UM-C6). Data reflects Mean±SEM, is normalized versus GUSB and displayed relative to UM-C4 NTG expression for each gene.
Figure 4
Figure 4. CoCSC with high ALDH1 activity are more prevalent following CPA therapy.
A) Ratio of ALDH+ tumor cells among the human ESA+CD44+ population in tumors from vehicle-treated control or CPA-treated mice. Data reflects the paired Mean of 5 independent experiments using 3 different xenogeneic colorectal tumor lines (UM-C4, UM-C6 & OMP-C8) and n≥5 mice per experiment. B) Taqman qRT-PCR data for the denoted genes using TG and NTG populations isolated from (V) vehicle- or CPA-treated UM-C4 tumors. Data represents Mean±SEM (n≥2). C) Immunofluorescence or immunoperoxidase staining of frozen or formalin-fixed, paraffin embedded tumors for ALDH1 or c-Myc, respectively, from vehicle- and CPA-treated mice. Black bar = 100 µm.
Figure 5
Figure 5. CoCSC phenotype cells are also enriched following Irinotecan treatment.
A) Upon randomization to normalize treatment groups at 400 mm3 at day 43, once weekly administration of vehicle (green boxes) or 15 mg/kg Irinotecan (red triangles) commenced and tumors were measured twice weekly. Growth curves representing the Mean±SEM are shown. B) Percentage of human ESA+CD44+CD166+ CoCSC phenotype cells in residual tumors is shown. Data reflects n≥4 mice per treatment group.
Figure 6
Figure 6. In vitro maintenance and expansion of CoCSC.
Human ESA+CD44+CD166+ cells were plated in limiting dilution and cultured for fourteen days in serum-free maintenance conditions. Colorectal tumor colonies were then either analyzed for A) ESA expression by IHC, or B) ESA, CD44 and CD166 expression by flow cytometry. C) Cellular phenotype of single colony-derived tumors, showing human ESA+ cell subpopulations expressing CD44 and CD166. D) Tumor growth curves are shown for either 2,000 CoCSC phenotype cells or an equal number of cells with all other phenotypes (Other), which were isolated from in vitro colony-derived tumors (tumors/animals injected). Inset shows lentiviral insertion band obtained by inverse PCR of 1) human xenograft tumor cells, 2) ESA+CD44+CD166+ (CoCSC) cells or 3) ESA+CD44− (Other) cells isolated by FACS. Phenotypic and morphological analysis of single-cell derived tumors from serially transplanted CoCSC show that the diverse E) phenotype and F) histological makeup of xenogeneic colorectal tumors are maintained following brief in vitro culture in limiting dilution. Black bar = 100 µm.
Figure 7
Figure 7. ALDH1 enzyme inhibition sensitizes colorectal tumor cells to CPA in vitro.
In vitro cell viability measurements of human colorectal tumor cells following 4 hours of exposure to A) varying concentrations of 4-HC and/or the ALDH1-specific inhibitor DEAB, or B) 20 µg/mL 4-HC in the presence or absence of 75 µM DEAB and/or 1 µM ATRA. C) Cell viability measurements following 7 days of exposure to varying concentrations of Irinotecan in the presence or absence of 75 µM DEAB. All data is expressed as the Mean±SEM of triplicate measurements and is normalized versus vehicle-treated controls. All data is representative of n≥2 independent experiments using either UM-C4 or UM-C6 tumor cells. *P<0.015. **P = 0.003.
Figure 8
Figure 8. Knockdown of ALDH1A1 gene expression sensitizes tumors to CPA in vivo.
UM-C6 tumor cells transduced with Luciferase- (black) or ALDH1A1-targeted shRNA (white) were A) assessed by Taqman™ qRT-PCR for relative expression of ALDH1A1, ALDH3A1 and ALDH5A1, or B) transplanted into mice at 400 cells/mouse to initiate tumors. Following randomization when tumors reached a Mean volume of 175 mm3, mice were treated twice weekly with either vehicle or 38 mg/kg CPA. The Mean difference in tumor volume between CPA- and vehicle-treated mice is plotted for Luciferase shRNA control (open circles) or ALDH1A1 shRNA-containing (triangles) cells. C) Percentage of human ESA+CD44+CD166+ CoCSC phenotype cells in residual tumors is shown. Data reflects n≥5 mice per treatment group.
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