HER2 expression identifies dynamic functional states within circulating breast cancer cells

Circulating tumor cells (CTCs) in women with advanced estrogen receptor-positive/HER2-negative breast cancer acquire a HER2-positive subpopulation following multiple courses of therapy1,2. In contrast to HER2-amplified primary breast cancer, which is highly sensitive to HER2-targeted therapy, the clinical significance of acquired HER2 heterogeneity during the evolution of metastatic breast cancer is unknown. Here, we analyzed CTCs from 19 ER+/HER2− patients, 84% of whom had acquired CTCs expressing HER2. Cultured CTCs maintain discrete HER2+ and HER2− subpopulations: HER2+ CTCs are more proliferative but not addicted to HER2, consistent with activation of multiple signaling pathways. HER2− CTCs show activation of Notch and DNA damage pathways, exhibiting resistance to cytotoxic chemotherapy, but sensitivity to Notch inhibition. HER2+ and HER2− CTCs interconvert spontaneously, with cells of one phenotype producing daughters of the opposite within four cell doublings. While HER2+ and HER2− CTCs have comparable tumor initiating potential, differential proliferation favors the HER2+ state, while oxidative stress or cytotoxic chemotherapy enhances transition to the HER2− phenotype. Simultaneous treatment with paclitaxel and Notch inhibitors achieves sustained suppression of tumorigenesis in orthotopic CTC-derived tumor models. Together, these results point to distinct yet interconverting phenotypes within patient-derived CTCs, contributing to progression of breast cancer and acquisition of drug resistance

Efficient Differentiation of Steroidogenic and Germ-Like Cells from Epigenetically-Related iPSCs Derived from Ovarian Granulosa Cells

To explore restoration of ovarian function using epigenetically-related, induced pluripotent stem cells (iPSCs), we functionally evaluated the epigenetic memory of novel iPSC lines, derived from mouse and human ovarian granulosa cells (GCs) using c-Myc, Klf4, Sox2 and Oct4 retroviral vectors. The stem cell identity of the mouse and human GC-derived iPSCs (mGriPSCs, hGriPSCs) was verified by demonstrating embryonic stem cell (ESC) antigen expression using immunocytochemistry and RT-PCR analysis, as well as formation of embryoid bodies (EBs) and teratomas that are capable of differentiating into cells from all three germ layers. GriPSCs’ gene expression profiles associate more closely with those of ESCs than of the originating GCs as demonstrated by genome-wide analysis of mRNA and microRNA. A comparative analysis of EBs generated from three different mouse cell lines (mGriPSCs; fibroblast-derived iPSC, mFiPSCs; G4 embryonic stem cells, G4 mESCs) revealed that differentiated mGriPSC-EBs synthesize 10-fold more estradiol (E2) than either differentiated FiPSC- or mESC-EBs under identical culture conditions. By contrast, mESC-EBs primarily synthesize progesterone (P4) and FiPSC-EBs produce neither E2 nor P4. Differentiated mGriPSC-EBs also express ovarian markers (AMHR, FSHR, Cyp19a1, ER and Inha) as well as markers of early gametogenesis (Mvh, Dazl, Gdf9, Boule and Zp1) more frequently than EBs of the other cell lines. These results provide evidence of preferential homotypic differentiation of mGriPSCs into ovarian cell types. Collectively, our data support the hypothesis that generating iPSCs from the desired tissue type may prove advantageous due to the iPSCs’ epigenetic memory

Genomic Instability Is Induced by Persistent Proliferation of Cells Undergoing Epithelial-to-Mesenchymal Transition

TGF-β secreted by tumor stroma induces epithelial-to-mesenchymal transition (EMT) in cancer cells, a reversible phenotype linked to cancer progression and drug resistance. However, exposure to stromal signals may also lead to heritable changes in cancer cells, which are poorly understood. We show that epithelial cells failing to undergo proliferation arrest during TGF-β-induced EMT sustain mitotic abnormalities due to failed cytokinesis, resulting in aneuploidy. This genomic instability is associated with the suppression of multiple nuclear envelope proteins implicated in mitotic regulation and is phenocopied by modulating the expression of LaminB1. While TGF-β-induced mitotic defects in proliferating cells are reversible upon its withdrawal, the acquired genomic abnormalities persist, leading to increased tumorigenic phenotypes. In metastatic breast cancer patients, increased mesenchymal marker expression within single circulating tumor cells is correlated with genomic instability. These observations identify a mechanism whereby microenvironment-derived signals trigger heritable genetic changes within cancer cells, contributing to tumor evolution

Molecular signatures of circulating melanoma cells for monitoring early response to immune checkpoint therapy

A subset of patients with metastatic melanoma have sustained remissions following treatment with immune checkpoint inhibitors. However, analyses of pretreatment tumor biopsies for markers predictive of response, including PD-1 ligand (PD-L1) expression and mutational burden, are insufficiently precise to guide treatment selection, and clinical radiographic evidence of response on therapy may be delayed, leading to some patients receiving potentially ineffective but toxic therapy. Here, we developed a molecular signature of melanoma circulating tumor cells (CTCs) to quantify early tumor response using blood-based monitoring. A quantitative 19-gene digital RNA signature (CTC score) applied to microfluidically enriched CTCs robustly distinguishes melanoma cells, within a background of blood cells in reconstituted and in patient-derived (n = 42) blood specimens. In a prospective cohort of 49 patients treated with immune checkpoint inhibitors, a decrease in CTC score within 7 weeks of therapy correlates with marked improvement in progression-free survival [hazard ratio (HR), 0.17; P = 0.008] and overall survival (HR, 0.12; P = 0.04). Thus, digital quantitation of melanoma CTC-derived transcripts enables serial noninvasive monitoring of tumor burden, supporting the rational application of immune checkpoint inhibition therapies

mGriPSCs are pluripotent.

<p>Using G4-mESCs as a standard <b>(A-D, I)</b>, mGriPSCs are alkaline phosphatase reactive <b>(E)</b> and express stem cell antigens Oct4 <b>(F)</b>, SSEA1 <b>(G)</b> and Nanog <b>(H, I)</b>. mGriPSCS express additional stem cell markers by RT-PCR <b>(I)</b> and are karyotypically normal <b>(J)</b>. mGriPSC express endogenous copies of the introduced reprogramming genes and retroviral [trans] copies are present to varying degrees <b>(K).</b> mGriPSCs form EBs <b>(L)</b> that differentiate into three germ layers—ectodermal neurofilament <b>(M)</b>, and mesodermal SMA <b>(N)</b>, and endoderm alpha-fetoprotein (<b>O,</b> AFP). Teratomas <b>(P)</b> also show differentiation into three germ layers <b>(Q-S)</b>. Scale bars: <b>A-H</b> 20 μm; <b>L</b> 200 μm; <b>M-O, Q-S</b> 10 μm.</p

Genome-wide analysis of mRNA and microRNA confirms efficient pluripotent reprogramming of mGriPSCs and hGriPSCs.

<p>mGriPSC-EBs and mESC-EBs have comparable microarray expression profiles <b>(A)</b>. All ESC or EB samples show minimal steroidogenic mRNA compared to adult ovary samples <b>(B).</b> Principal component analysis (PCA) indicates attached and unattached mGriPSC-EBs are more disparate relative to the analogous mESC-EBs <b>(C)</b>. microRNA heatmaps support efficient stem cell reprogramming of mGriPSCs <b>(D)</b>.</p

Methylation analysis of GC-derived iPS cells shows stem cell reprogramming with retention of target tissue methylation.

<p>The stem cell gene Oct4 in mGriPSC is less methylated than it is in GCs (mGriPSC vs. GC, P<0.001) and is therefore more similar to Oct4 in G4 ESCs <b>(A)</b>. Methylation patterns of ovarian and germ cell genes Foxl2 and Figla are comparable in both mGriPSC and GCs (<b>B</b>, Foxl2, P = 0.84; Figla, P = 0.606).</p