Reporters to mark and eliminate basal or luminal epithelial cells in culture and in vivo

The contribution of basal and luminal cells to cancer progression and metastasis is poorly understood. We report generation of reporter systems driven by either keratin-14 (K14) or keratin-8 (K8) promoter that not only express a fluorescent protein but also an inducible suicide gene. Transgenic mice express the reporter genes in the right cell compartments of mammary gland epithelia and respond to treatment with toxins. In addition, we engineered the reporters into 4T1 metastatic mouse tumor cell line and demonstrate that K14+ cells, but not K14- or K8+, are both highly invasive in three-dimensional (3D) culture and metastatic in vivo. Treatment of cells in culture, or tumors in mice, with reporter-targeting toxin inhibited both invasive behavior and metastasis in vivo. RNA sequencing (RNA-seq), secretome, and epigenome analysis of K14+ and K14- cells led to the identification of amphoterin-induced protein 2 (Amigo2) as a new cell invasion driver whose expression correlated with decreased relapse-free survival in patients with TP53 wild-type (WT) breast cancer

Generation and characterization of transgenic mice expressing K14 and K8 reporters.

<p>(A) Flow cytometry analysis for stromal, basal, and luminal compartments of cells from mammary glands of control or K14.tRPT/K8.tGPD double-positive transgenic mouse. For each type of mouse, the first dot plot shows the total population in each cell compartment, whereas the second plot shows only the cells that are positive for the reporters. Gates were set based on the negative control and dots pseudocolored to represent the reporter-positive cells. (B) Fluorescent IHC showing colocalization of tGFP with endogenous K8 in the lung of a K8.tGPD-positive mouse (upper panels). Lower panel shows the control staining on a WT mouse in which no tGFP was detected; scale bar 10 μm. (C) Positive and negative mice were injected i.p. with either high (“H”; GCV = 100 μg/g; DT = 50 ng/g) or low (“L”; GCV = 20 μg/g; DT = 10 ng/g) doses at indicated time points (days). DAPI, 4’,6-diamidino-2-phenylindole; DT, diphtheria toxin; GCV, ganciclovir; IHC, immunohistochemistry; i.p., intraperitoneally; K8.tGPD, keratin-8 promoter followed by turbo green fluorescent protein and diphtheria toxin receptor; K14.tRPT, keratin-14 promoter followed by a turbo red fluorescent protein and herpes simplex virus thymidine kinase; tGFP, turbo green fluorescent protein; WT, wild-type</p

K14+ cells secrete more Col6a1 and express higher levels of Amigo2 than K14− cells.

<p>(A) Anti-Col6a1 immunoblots of secreted proteins from K14.GFP+ or K14.GFP− reporter cell lines. CM were concentrated and analyzed for levels of secreted Col6a1. ITGB1, also present in the secretome, was used for loading control. (B) RT-PCR for <i>Amigo2</i> mRNA level on K14+ and K14− cells. Results show the mean ± SD of 3 independent experiments, <i>p</i> = 0.0126 by paired <i>t</i> test. (C) Amigo2 protein level was detected in K14+ and K14− cell lysates by western blot. Quantification of 3 independent experiments, <i>p</i> < 0.0001 by paired <i>t</i> test; mean ± SD is shown. (D) Chip for H3K27Ac shows the magnitude of the peaks for K14+ and K14− replicates at the <i>Amigo2</i> locus. (E) Cartoon of self-inactivating lentiviral K14.tRPT (upper cartoon) and K8.tGPD reporters (lower cartoon). (F) RT-PCR analysis of <i>Amigo2</i> mRNA expression in K14+ or K14− human breast cancer cell line HCC1143. Quantification of independent experiments in triplicates <i>p</i> = 0.0148 by paired <i>t</i> test; mean ± SD is shown. (G) Kaplan-Meier plot in TP53 mutant and TP53 WT breast cancer show relationship between <i>Amigo2</i> expression and relapse-free survival. Amigo2, amphoterin-induced protein 2; Chip, chromatin immunoprecipitation; CM, conditioned medium; Col6a1, Collagen VI subunit A; DTR, diphtheria toxin receptor; <i>EF-1α</i>, <i>elongation factor 1α</i>; GFP, green fluorescent protein; H3K27Ac, histone 3 lysine 27; ITGB1, integrin β-1; K, cytokeratin; K8.tGPD, keratin-8 promoter followed by turbo green fluorescent protein and diphtheria toxin receptor; K14.tRPT, keratin-14 promoter followed by a turbo red fluorescent protein and herpes simplex virus thymidine kinase; LTR, long terminal repeat; RT-PCR, real-time PCR; tBFP, turbo blue fluorescent protein; tGFP, turbo green fluorescent protein; TK, thymidine kinase; tRFP, turbo red fluorescent protein; WT, wild-type.</p

Characterization of K14.GFP reporter and relationship between K14+ and K14− status and invasive behavior of cells in culture.

<p>(A) Cartoon of the K14 promoter–driven EGFP-P2A-DTR (K14.GFP) reporter construct. (B) IF shows colocalization of endogenous K14 and GFP in K14.GFP+ monolayer, scale bar 20 μm. (C) Stably transfected K14.GFP reporter cells were sorted by FACS and monitored for changes in percentage of cells expressing GFP by flow cytometry for 54 days. (D) Phase contrast images of K14.GFP+, and K14.GFP− and WT cells grown in 2D, scale bar 100 μm. (E) Changes in cell number over time. Graph shows the mean ± SEM of 3 independent experiments. (F) EdU labeling and flow cytometry to identify cells in different stages of cell cycle. Shown is the mean ± SD of triplicates. (G) K14.GFP+, and K14.GFP− and WT cells grown in 3D (scale bar 200 μm) on top of M/Col-I for 4 days. Two days after seeding, cells were treated with DT (5 ng/ml) for 48 hours (lower panels, DT+). (H) Quantification of invasive structures in G, the data shown are means ± SD from independent experiments carried out in triplicates, at least 300 structures/condition were counted; *<i>p</i> < 0.05 by unpaired <i>t</i> test. (I) Phase contrast and GFP overlay images of K14.GFP+ cells grown in 3D in M/Col-I at day 2 and day 4. Black arrow indicates GFP+ cells at the invasive protrusions, white arrows indicate GFP+ in noninvasive 3D structures. DT+ indicates 48-hour exposure to DT, scale bars 100 μm. 3D, three-dimensional; DT, diphtheria toxin; DTR, diphtheria toxin receptor; EdU, 5-Ethynyl-2´-deoxyuridine; EGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; IF, immunofluorescence; K, cytokeratin; M/Col-I, 1:1 mixture of Matrigel/Collagen-I; pA, polyadenylation signal sequence; WT, wild type</p

Genes identified by RNA-seq analysis as most highly expressed in K14+ compared to K14− cells.

<p>Genes identified by RNA-seq analysis as most highly expressed in K14+ compared to K14− cells.</p

K14.GFP+ cells have greater metastatic potential that K14.GFP− cells <i>in vivo</i>.

<p>(A) Primary tumor diameters from mice injected with 4T1 control, or K14.GFP reporter cell lines (K14.GFP+ or K14.GFP−), measured over the course of the experiment. <i>n</i> = 8 (control), 7 (K14.GFP+), and 8 (K14.GFP−); *<i>p</i> < 0.05, **<i>p</i> < 0.001 by one-way ANOVA followed by Newman-Keuls multiple comparisons posttest. (B) Final tumor masses measured after mice were euthanized. (C) Lung metastases were quantified by measuring the average percent area of lung tissue occupied by the tumor in 5 sections (5-μm thick) cut at 200-μm intervals. (D) Representative images of H&E-stained lung sections containing metastases from mice injected with K14.GFP+ or K14.GFP− cell lines. Arrows indicate metastasis; scale bar 1 mm. (E) Correlation analysis of primary tumor mass and lung metastasis. Scatter plot of the percent tumor area in lung tissue compared to the primary tumor mass for each mouse analyzed (<i>n</i> = 23). The slope does not significantly differ from zero by linear regression analysis (<i>p</i> = 0.1097). Quantification of percent of positive cells for Ki67 (F) and CC3 (G) in tumors from K14.GFP+ and K14.GFP− cells. Shown are means ± SD of quantifications of whole tumor sections. (H) Final tumor masses measured after mice were euthanized. For DT treatment, the mice were injected i.p. with DT (25 mg/kg) on days 7, 9, 11, and 13. <i>n</i> = 7 (K14.GFP+; no DT), 4 (K14.GFP+; with DT), 8 (K14.GFP−; no DT), and 4 (K14.GFP−; with DT). <i>p</i> = 0.0425 by unpaired <i>t</i> test. (I) Lung metastases were quantified for the mice described in H. Statistical analysis for (B), (C), and (I) was calculated by one-way ANOVA followed by Tukey’s multiple comparisons posttest; *<i>p</i> < 0.05 or n.s. (J) Fluorescent IHC was performed for K14 and GFP on primary tumors generated from K14.GFP+ cell lines either DT− or DT treated (“DT+”) as described in (H); scale bar 40 μm. (K) Same staining as described in J was carried out on metastatic lung of K14.GFP+-injected mice; scale bar 20 μm. (L) Immunoblots of lysates from primary tumors were analyzed for GFP expression. Every lane represents a different tumor. Blots were also probed with antibodies for ERK1/2 as loading control. If not otherwise indicated, all graphs show mean ± SEM. DT, diphtheria toxin; ERK1/2, extracellular signal-regulated kinase 1/2; GFP, green fluorescent protein; H&E, hematoxylin and eosin; IHC, immunohistochemistry; i.p., intraperitoneally; K, cytokeratin; n.s., not significant</p