ABSTRACT
Keratins are an integral part of cell structure and function. Here, it is shown that ectopic expression of a truncated isoform of keratin 81 (tKRT81) in breast cancer is upregulated in metastatic lesions compared to primary tumors and patient-derived circulating tumor cells, and is associated with more aggressive subtypes. tKRT81 physically interacts with keratin 18 (KRT18) and leads to changes in the cytosolic keratin intermediate filament network and desmosomal plaque formation. These structural changes are associated with a softer, more elastically deformable cancer cell with enhanced adhesion and clustering ability leading to greater in vivo lung metastatic burden. This work describes a novel biomechanical mechanism by which tKRT81 promotes metastasis, highlighting the importance of the biophysical characteristics of tumor cells.
Subject(s)
Breast Neoplasms , Keratins, Hair-Specific , Female , Humans , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Ectopic Gene Expression , Keratins, Hair-Specific/genetics , Keratins, Hair-Specific/metabolism , Protein Isoforms/geneticsABSTRACT
Circulating tumor cells (CTC) can be isolated via a minimally invasive blood draw and are considered a "liquid biopsy" of their originating solid tumors. CTCs contain a small subset of metastatic precursors that can form metastases in secondary organs and provide a resource to identify mechanisms underlying metastasis-initiating properties. Despite technological advancements that allow for highly sensitive approaches of detection and isolation, CTCs are very rare and often present as single cells, posing an extreme challenge for ex vivo expansion after isolation. Here, using previously established patient-derived CTC lines, we performed a small-molecule drug screen to identify compounds that can improve ex vivo culture efficiency for single CTCs. We found that N-acetyl-L-cysteine (NAC) and other antioxidants can promote ex vivo expansion of single CTCs, by reducing oxidative and other stress particularly at the initial stage of single-cell expansion. RNA-seq analysis of growing clones and nongrowing clones confirmed the effect by NAC, but also indicates that NAC-induced decrease in oxidative stress is insufficient for promoting proliferation of a subset of cells with predominant senescent features. Despite the challenge in expanding all CTCs, NAC treatment led to establishment of single CTC clones that have similar tumorigenic features. IMPLICATIONS: Through a small molecule screen and validation study, we found that NAC could improve the success of ex vivo expansion of single CTCs by mitigating the initial stress, with the potential to facilitate the investigation of functional heterogeneity in CTCs.
Subject(s)
Acetylcysteine/pharmacology , Heat-Shock Proteins/metabolism , Neoplastic Cells, Circulating/drug effects , Neoplastic Cells, Circulating/pathology , Scavenger Receptors, Class A/metabolism , Animals , Antioxidants/pharmacology , Breast Neoplasms/blood , Breast Neoplasms/pathology , Cell Growth Processes/drug effects , DNA Copy Number Variations , Female , Heterografts , Humans , Mice , Neoplastic Cells, Circulating/metabolism , Oxidative Stress/drug effectsABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Circulating tumor cells (CTCs) shed from solid tumors can serve as a minimally invasive liquid biopsy for monitoring disease progression. Because CTCs are rare and heterogeneous, their biological properties need to be investigated at the single cell level, which requires efficient ways to isolate and analyze live single CTCs. Current methods for CTC isolation and identification are either performed on fixed and stained cells or need multiple procedures to isolate pure live CTCs. Here, we used the AccuCyte-RareCyte system to develop a Protocol for Integrated Capture and Retrieval of Ultra-pure single live CTCs using Negative and positive selection (PIC&RUN). The positive selection module of PIC&RUN identifies CTCs based on detection of cancer surface markers and exclusion of immune markers. Combined with a two-step cell picking protocol to retrieve ultrapure single CTCs, the positive selection module is compatible for downstream single cell transcriptomic analysis. The negative selection module of PIC&RUN identifies CTCs based on a live cell dye and the absence of immune markers, allowing retrieval of viable CTCs that are suitable for ex vivo culture. This new assay combines the CTC capture and retrieval in one integrated platform, providing a valuable tool for downstream live CTC analyses.
Subject(s)
Biomarkers, Tumor/analysis , Breast Neoplasms/pathology , Neoplastic Cells, Circulating/pathology , Single-Cell Analysis/methods , Adult , Aged , Breast Neoplasms/metabolism , Case-Control Studies , Cell Count , Cell Separation , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Middle Aged , Neoplastic Cells, Circulating/metabolism , Tumor Cells, CulturedABSTRACT
Estrogens attenuate bone turnover by inhibiting both osteoclasts and osteoblasts, in part through antagonizing Runx2. Apparently conflicting, stimulatory effects in osteoblast lineage cells, however, sway the balance between bone resorption and bone formation in favor of the latter. Consistent with this dualism, 17ß-estradiol (E2) both stimulates and inhibits Runx2 in a locus-specific manner, and here we provide evidence for such locus-specific regulation of Runx2 by E2 in vivo. We also demonstrate dual, negative and positive, regulation of Runx2-driven alkaline phosphatase (ALP) activity by increasing E2 concentrations in ST2 osteoblast progenitor cells. We further compared the effects of E2 to those of the Selective Estrogen Receptor Modulators (SERMs) raloxifene (ral) and lasofoxifene (las) and the phytoestrogen puerarin. We found that E2 at the physiological concentrations of 0.1-1â¯nM, as well as ral and las, but not puerarin, antagonize Runx2-driven ALP activity. At ≥10â¯nM, E2 and puerarin, but not ral or las, stimulate ALP relative to the activity measured at 0.1-1â¯nM. Contrasting the difference between E2 and SERMs in ST2 cells, they all shared a similar dose-response profile when inhibiting pre-osteoclast proliferation. That ral and las poorly mimic the locus- and concentration-dependent effects of E2 in mesenchymal progenitor cells may help explain their limited clinical efficacy.