ROCK1 inhibition promotes the self-renewal of a novel mouse mammary cancer stem cell

Abstract

The differentiation of stem-like tumor cells may contribute to the cellular heterogeneity of breast cancers. We report the propagation of highly enriched mouse mammary cancer stem cells that retain the potential to differentiate both in vivo and in culture and their use to identify chemical compounds that influence both self-renewal and differentiation. We identify epithelial tumor-initiating cells (ETICs) that express lineage markers of both basal and luminal mammary cell lineages and retain the potential, from even single cells, to generate heterogeneous tumors similar to the tumor of origin. ETICs can progress through a Rho-associated coiled-coil containing protein kinase 1 dependent, epithelial to mesenchymal transition to generate mesenchymal tumor-initiating cells capable of initiating tumors of limited heterogeneity. The propagation of ETICs may allow for the identification of new therapeutic compounds that may inhibit or prevent progression of some types of breast cancer.

Figure 1. Keratin expression in primary and secondary MMTV-Wnt1 tumors

(A–H) Immunoflourescence images of sections of two independent MMTV-Wnt1 primary tumors labeled WMG316 (A, B) and WMG300 (E, F) and their secondary tumors derived from cultured epithelial tumor initiating cell lines WMG316 (C, D) and WMG300 (G, H) are shown. Ethanol-acetic acid fixed tumor sections were stained for K8, K14 co-localization (A, C, E, G) and vimentin (B, D, F, H) as indicated. The nuclei in all images are visualized by DAPI staining. (I) Cells from dissociated tumors were prepared by cytospin and stained with both K8 and K14 antibodies. Reactivity was assessed on captured images taken at the same exposure time and without digital manipulation. The bars represent the percent of total cells stained with the indicated antibodies from one tumor. Error bars indicate the standard deviation of scoring multiple microscopic fields. (J) Cytospin preparations of the two cultured cell lines were stained and scored as in M. Note the small fraction of K8^+,K14⁺ cells in tumors and the increased population in cell cultures. Scale bars represent 60 μm.

Figure 2. Cultivation and characterization of ETICs

(A) Spheroid forming activity of WMG300 in Matrigel in two different O2 atmospheres. (B, C) Original tumors (B) and cultured ETICs (C) were dissociated to single cells and analyzed by FACS for the expression of surface markers CD29 and CD24. Values for percent double positive cells are indicated in the upper right corner (D, E) WMG316 cultured cells (E) and secondary tumors (D) were dissociated and analyzed by FACS for surface markers EpCam and CD49f. (F, G, H) The four fractions indicated in panel D were recovered and analyzed for expression of K8 and K14 on cytospin preparations (F) and quantified for individual cell types (G). Individual fractions were also examined for colony forming ability in Matrigel (H). (I) Confocal section through a typical spheroid from the WMG300 cell line under hypoxic conditions. Spheroid was stained for K8, K14 and DAPI. Magnification is 100x.

Figure 3. ETIC differentiation in culture

(A–C) ETIC single cells (A) day 1 were grown for up to 3 days in 3% O₂ (B) or air (C) atmospheres supplemented with 5% CO₂. Cells were fixed and stained with antibodies to K8 (red) and K14 (green). Size bar in A–C indicates 50 μm. The number of colonies (one or more cells) and reactivity of individual cells of colonies were determined from eight digital images of each condition taken with same exposure time. (D) Total colony number decreases with time but is similar in the two conditions. (E) The number of colonies with differentiated cells (K8 or K14 single positive cells) increased in normoxia. (F) The percent of colonies with differentiated cells increased in normoxic atmosphere.

Figure 4. Chemical kinase inhibitor screen reveals compounds that stimulate proliferation

(A) Influence of 242 kinase inhibitors on cell number after three days of exposure to 1 μM in a 3% O₂ atmospheric condition. The y-axis represents the average Z-scores for the change in cell growth upon treatment with an individual kinase inhibitor. The Z score = |average value – mean of controls|/std. dev. of all tests. Controls are represented by black squares. Six candidate inhibitors had a Z-score >2, four of which targeted ROCK1 (R1, R2, R3, R4), one inhibitor of GSK3β (G1) and one inhibitor of checkpoint kinase 1 (C1). (B, C) Dose response of fold change in cell number as function of the R1 and G1 inhibitors (mean± sd). (D–F) Stimulation of WMG300 colony forming activity by R1 and G1 in suspension (D), monolayer (E) and Matrigel (F) (mean± sd). Note differences in vertical scale of the three panels.

Figure 5. ROCK1 dependent stimulation of colony forming efficiency and stabilization of ETIC

(A) Stimulation of WMG300 monolayer colony formation by ROCK1 shRNA (902, 903) but not ROCK2 shRNA (919, 922) upon viral transduction. Each column represents the average of three replicates. Cells were plated in the absence of the R1 inhibitor. (B) Immunoblots of whole cell lysates from WMG316 (316), WMG300 (300), WMG300sh902 (sh902), and WMG300sh919 (sh919; separate blot) with ROCK1 and ROCK2 antibodies. Actin serves as the loading control. (C) Monolayer colony formation dependence on the ROCK inhibitor. ROCK1 knock down cells are insensitive to additional suppression with the ROCK1 inhibitor. (D, E) WMG300 ETIC, (spheroid passage 4) were serially cultivated in monolayer in the presence or absence of R1 or G1. Portions of the cells were evaluated by keratin (D) or E-cadherin staining (E) at the indicated monolayer passage. (F) Representative images of WMG300 monolayer colonies showing the immunofluorescence profile of most ETIC colonies in the presence of the ROCK inhibitor (F, 300R1 panel): K8, K14, ECAD, and VIM. MTICs isolated from G1 treatment in (D, E) have little K8, K14 and ECAD antibody reaction but stain strongly for vimentin (F, 300G1 panel). Scale bars represent 60 μm. (G) Colony morphologies of ETIC and MTIC grown in Matrigel.

Figure 6. EMT-Associated gene signature, tumor formation and the evolution of tumor initiating cells

(A) Comparison of ETIC and MTIC RNA levels of selected genes implicated in an epithelial to mesenchymal transition. (B) Histological analysis of tumors from stably transduced ETIC lines, 300shCntrl (cultivated in the presence of the R1 inhibitor) and 300sh902. “p” indicates the number of passages since isolation of the line, K8, K14 and vimentin staining are shown. (C) Schematic representation of a proposed cellular hierarchy responsible for the generation of HER2, luminal A,B and basal-like tumors from a K8⁺,K14⁺ progenitor. Scale bars represent 60 μm.