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Expression data of CD24-CD44+ and ALDH+ cells

ABSTRACT: It has been suggested that breast cancers are driven and maintained by a cellular subpopulation with stem cell properties. These breast cancer stem cells (BCSCs) mediate metastasis and by virtue of their resistance to radiation and chemotherapy, contribute to relapse. Although several BCSC markers have been described, it is unclear whether these markers identify the same or independent BCSC populations. Based on established breast cancer cell lines, as well as primary tumor xenografts, we show that BCSCs exist in distinct mesenchymal-like (epithelial-mesenchymal transition, EMT) and epithelial-like (mesenchymal-epithelial transition, MET) states characterized by expression of distinct markers, proliferative capacity and invasive characteristics. The gene expression profiles of mesenchymal-like and epithelial-like BCSCs are remarkably similar across the different molecular subtypes of breast cancer and resemble those of distinct basal and luminal stem cells found in the normal breast. We propose that the plasticity of BCSCs allowing them to transition between EMT- and MET-like states endows these cells with the capacity for tissue invasion, dissemination and growth at metastatic sites. Breast cancer cell lines, primary xenografts and normal breast cells from patient were sorted using flow cytometry to select for cells that were CD24-,CD44+ and ALDH+. Gene expression profiles of CD24-CD44+ cells were compared with non-CD24-CD44+ cells. Gene expression profiles of ALDH+ cells were compared with ALDH- cells.

ORGANISM(S): Homo sapiens

SUBMITTER: CONG yang

PROVIDER: S-ECPF-GEOD-52262 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:BackgroundSTAT3 activation is frequently detected in breast cancer and this pathway has emerged as an attractive molecular target for cancer treatment. Recent experimental evidence suggests ALDH-positive (ALDH(+)), or cell surface molecule CD44-positive (CD44(+)) but CD24-negative (CD24(-)) breast cancer cells have cancer stem cell properties. However, the role of STAT3 signaling in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells is unknown.Methods and resultsWe examined STAT3 activation in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells by sorting with flow cytometer. We observed ALDH-positive (ALDH(+)) cells expressed higher levels of phosphorylated STAT3 compared to ALDH-negative (ALDH(-)) cells. There was a significant correlation between the nuclear staining of phosphorylated STAT3 and the expression of ALDH1 in breast cancer tissues. These results suggest that STAT3 is activated in ALDH(+) subpopulations of breast cancer cells. STAT3 inhibitors Stattic and LLL12 inhibited STAT3 phosphorylation, reduced the ALDH(+) subpopulation, inhibited breast cancer stem-like cell viability, and retarded tumorisphere-forming capacity in vitro. Similar inhibition of STAT3 phosphorylation, and breast cancer stem cell viability were observed using STAT3 ShRNA. In addition, LLL12 inhibited STAT3 downstream target gene expression and induced apoptosis in ALDH(+) subpopulations of breast cancer cells. Furthermore, LLL12 inhibited STAT3 phosphorylation and tumor cell proliferation, induced apoptosis, and suppressed tumor growth in xenograft and mammary fat pad mouse models from ALDH(+) breast cancer cells. Similar in vitro and tumor growth in vivo results were obtained when ALDH(+) cells were further selected for the stem cell markers CD44(+) and CD24(-).ConclusionThese studies demonstrate an important role for STAT3 signaling in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells which may have cancer stem cell properties and suggest that pharmacologic inhibition of STAT3 represents an effective strategy to selectively target the cancer stem cell-like subpopulation.

Project description:Tumorigenic breast cancer cells characterized by CD44 expression and low or undetectable CD24 levels (CD44+/CD24-/low) may be resistant to chemotherapy and therefore responsible for cancer relapse. Paired breast cancer core biopsies before and after neoadjuvant chemotherapy or lapatinib were obtained and as single cell suspensions stained using antibodies against CD24, CD44, and lineage markers, and then analyzed by flow cytometry. Mammosphere (MS) formation in culture was compared before and after treatment. Global gene expression differences between cancer cells bearing CD44+/CD24-/low cells and all other sorted cells, and between cancer MS and the primary bulk invasive cancers were analyzed. We report that CD44+/CD24-/low tumorigenic breast cancer cells were intrinsically chemoresistant ─ chemotherapy led to increased CD44+/CD24-/low cells, increased self-renewal capacity on MS assays, and enhanced tumorigeneicity in immunocompromised SCID/Beige mice. Conversely, in patients with HER2 overexpressing tumors, the EGFR/HER2 tyrosine kinase inhibitor, lapatinib decreased CD44+/CD24-/low cells, with the majority of these patients after conventional therapy achieving pathologic complete response, a validated surrogate marker for long-term survival. Gene transcription pathways that underlie chemoresistant, MS-forming CD44+/CD24-/low cells involve genes belonging to stem cell self-renewal, Wnt signaling, and early development pathways. Experiment Overall Design: Human breast tumor samples were sorted using flow cytometry to select for cells that were CD44+ and CD24-. Gene expression profiles of these cells were compared with profiles of the other sorted cells (CD24+ and CD44-/CD24-). Experiment Overall Design: Core biopsies of primary breast tumors were taken and placed immediately in cold RPMI-1640 supplemented with 10% heat-inactivated newborn calf serum (HINCS, Invitrogen, Carlsbad, CA). Within an hour, the samples were minced and then digested in 10-15 mL of MEGM with 250-300 units/mL collagenase at 370C. The samples were filtered, washed, and then subjected to hypotonic shock to lyse red blood cells. About 106 single cells were re-suspended, incubated for 15 min at 40C with anti-CD44 (APC), anti-CD24 (FITC), and anti-lineage cocktail antibodies (PE-conjugated anti-CD2, CD3, CD10, CD16, CD18, CD31 and CD 140B) (Pharmingen, San Diego, CA) using the manufacturer’s suggested concentrations. The cells were then washed twice, re-suspended with the viability dye propidium iodide, and analyzed using Dako MoFlo flow cytometry. Side- and forward- scatter were used to eliminate debris and cell doublets, and the Lin- cells were further analyzed by CD44 and CD24 markers.

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Expression data of CD24-CD44+ and ALDH+ cells

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