Clonal analysis of human mammary cell transformation and X-ray sensitivity

Intra-tumoural biological, transcriptional and genomic heterogeneity are hallmarks of human breast cancers. However, tumour propagating activity appears confined to subsets of cells within each tumour. This finding is thought to indicate a persistence of mechanisms that maintain a hierarchical growth and differentiation structure in the normal mammary gland. Because little is known about the responses of either primary normal or malignant human mammary cells to existing therapies, this thesis sought to examine the intrinsic sensitivity of different purified human mammary colony-forming cell (CFC) types and tumours derived from them to ionizing radiation. Luminal progenitor (LP) CFCs were found to be ~1.5-fold more radioresistant than basal cell (BC) CFCs and LPs also showed evidence of checkpoint adaptation, slower repair activity and greater predisposition of γH2AX foci accumulation. Two human breast cancer cell lines (MDA-MB231 and SUM149) and a non-tumorigenic human mammary cell line (MCF-10A) were all found to be more radioresistant than the normal LP-CFCs. CFCs isolated from 8-week tumours generated in mice transplanted with normal human BCs or LPs transduced with KRASG¹²D showed even greater radioresistance and this was further increased in serially passaged derivative lines with more aggressive growth properties. To examine the responsiveness of malignant cells with tumor-initiating cell (TIC) activity in vivo, a dose-response analysis was first undertaken of their frequencies in the MDA-MB231 and SUM149 cells. Limiting dilution analysis (LDA) and single-cell transplants showed the frequency of TICs in both to be very high (<10%), but with increasingly marked inhibition of their detection in tumours initiated from innocula containing 2x10⁴ cells or more. Analogous LDA measurements of the TICs in KRASG12D-transduced BCs and LPs, yielded frequencies of ~0.2% and ~0.07%, respectively, in contrast to the frequencies of 0.02-0.5% for BCs and 0.01-0.6% for LPs for tumours initiated from 3x10⁴-10⁶ cells. These results demonstrate the heterogeneity of treatment responses in normal human mammary cells with innate proliferative ability that can be heightened by transformation. They also reveal the complex clonal dynamics operative in the growth of TICs in vivo that may confound interpretation of treatment effects assessed only by measuring immediate changes in tumor size.Medicine, Faculty ofGraduat

Single-cell analysis of autophagy activity in normal and de novo transformed human mammary cells

International audienceAssessment of autophagy activity has historically been limited to investigations of fixed tissue or bulk cell populations. To address questions of heterogeneity and relate measurements to functional properties of viable cells isolated from primary tissue, we created a lentiviral (RFP-GFP-MAP1LC3B) vector that allows the autophagosome and autolysosome content of transduced cells to be monitored at the single-cell level. Use of this strategy to analyze purified subsets of normal human mammary cells showed that both the luminal progenitor-containing (LP) subset and the basal cells (BCs) display highly variable but overall similar autophagic flux activity despite differences suggested by measurements of the proteins responsible (i.e., LC3B, ATG7 and BECLIN1) in bulk lysates. Autophagosome content was also highly variable in the clonogenic cells within both the LPs and BCs, but the proliferative response of the BCs was more sensitive to autophagy inhibition. In addition, use of this vector showed cells with the lowest autophagosome content elicited the fastest tumor growth in 2 different models of human mammary tumorigenesis. These results illustrate the utility of this vector to define differences in the autophagy properties of individual cells in primary tissue and couple these with their responses to proliferative and oncogenic stimuli

Radiation-induced effects on micro-scratch of ultra high molecular weight polyethylene biocomposites

Sterilization of ultra-high molecular weight polyethylene (UHMWPE) based composites used for acetabular cup liner via UV or gamma-irradiation before surgery is inevitable. Thus, understanding the alteration of mechanical properties and wear resistance of cup liner via irradiation process requires investigation. This paper aims to understand the effect of UV (0.03 J/cm(2)) and gamma (25 kGy) irradiation on mechanical properties and scratch resistance of compression molded UHMWPE composites reinforced with alumina (Al2O3), hydroxyapatite (HAp) and carbon nanotubes (CNTs). After irradiation, nearly 100% increased crystallinity of polyethylene, due to recrystallization, helped in enhancing the hardness and elastic modulus by similar to 1.5 times compared to that of as-processed UHMWPE (Hardness: similar to 70 MPa and Elastic modulus: similar to 1.25 GPa). The recrystallization was not favored by the presence of nano-reinforcements in irradiated UHMWPE based nanocomposites, and caused deterioration in the mechanical properties. The micro-scratch results revealed the remarkable wear resistance (i.e., 1.5 to 2 times lower wear rate) of irradiated samples than that of as-processed samples. Mouse fibroblast L929 in vitro cell culture test confirmed the cytocompatibility of irradiated samples. This study indicated that the UV and gamma irradiated UHMWPE nanocomposites are the challenging candidates as acetabular cup liner. (c) 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

The effect of 3T3 cell CM and selected factors on fetal and adult basal and luminal mammary cell production of CFCs.

<p>CFC assays were performed on 7-d cultures of 30 EpCAM⁺⁺ fetal cells (green), 60 EpCAM⁺CD49f⁺ adult basal cells (blue), 100 EpCAM⁺⁺CD49f^low/−CD61⁺ adult luminal cells (red), and 300 adult EpCAM⁺ cells (purple) using various additives to determine their effects on CFC outputs. (A) Comparison of the effect of 80% 3T3 cell CM, 160 ng/ml Wnt3a±400 ng/ml R-Spondin 1 (R-Spo), or 16 ng/ml bFGF relative to added 3T3 cells (set = 100%). The concentrations shown are the final concentrations in the 250 µl cultures. Results are pooled from 3–6 experiments. The difference in CFC output between CM and no added 3T3 cells was significant for cultures initiated with all types of cells (<i>p</i><0.05, one-way ANOVA with Bonferroni's multiple comparison test). (B) Effect of Wnt pathway inhibitors: XAV939 (0.8 µM for adult, 4 µM for fetal) or mDKK1 (160 ng/ml) in cultures with irradiated 3T3 cells (set = 100%). Results are pooled from 3–9 experiments. Only the effect of added XAV939 was significant, and only for basal cells (<i>p</i> = 0.04, one-way ANOVA with Bonferroni's multiple comparison test). (C) Effect of 40 ng/ml HGF and 16 ng/ml CSF-1 on adult EpCAM⁺ cells. The difference in CFC output between added HGF or CSF-1 and no added 3T3 cells is not significant (<i>p</i>>0.99, one-way ANOVA with Bonferroni's multiple comparison test).</p

Comparison of CFC and MRU outputs in 7-d Matrigel cultures initiated with single fetal or adult mammary cells.

<p>(A) Correlation between the total number of cells retrieved from individually assessed 7-d cultures with the corresponding number of CFCs detected. (B) Distribution of clonal CFC outputs in 7-d cultures initiated with different types of input cells. The dotted line indicates the median values of positive clones: 1,000 for clones derived from basal adult cells (<i>n</i> = 44), 140 for clones from CD61⁺ adult luminal cells (<i>n</i> = 35), and 13,000 for clones from fetal cells (<i>n</i> = 55). (C) Comparison of MRU outputs determined by LDA in 7-d Matrigel cultures initiated with single adult and fetal mammary cells. The value for fetal cells is significantly higher than either of the adult values (<i>p</i><0.01, Chi-square <i>p</i> value, pair-wise comparison of stem cell frequencies, ELDA, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001630#pbio.1001630.s009" target="_blank">Table S7</a>).</p

Morphology and cellular composition of structures generated in 7-d Matrigel cultures of single fetal and adult mammary cells.

<p>(A) Gross appearance of representative structures from each of the three types of input cells tested. The scale shown indicates 30 µm. (B) Representative photomicrographs of sectioned structures stained with antibodies against the markers shown. Scale, 50 µm. (C) Flow cytometric analysis of dissociated PI⁻ cells obtained from pooled harvests of cultures initiated with the same types of cells.</p

Regenerated glands produced <i>in vivo</i> from MRU generated <i>in vitro</i> from structures generated from single fetal, basal, or luminal cells.

<p>Whole mounts (upper panels) and sections (middle panels) of mammary glands produced in fat pads injected with cells from cultures initiated with single fetal cells (left panel), or single adult basal cells (middle panel) or single adult luminal cells (right panels). Scale bars, 100 µm (whole mounts) and 50 µm (sections). Lower panels show flow cytometry profiles of regenerated mammary glands from cultures initiated with single fetal cells (left panel), or single adult basal (middle panel) or six CD61⁺ luminal cells (right panels).</p

Microarray analysis of differentially expressed genes in MRU-enriched fetal and adult basal subsets of mammary cells.

<p>(A) Comparison of transcripts detected in Agilent arrays of extracts of E18.5 fetal (CD31⁻CD45⁻Ter119⁻EpCAM⁺⁺CD49f⁺) and adult basal (CD31⁻CD45⁻Ter119⁻BP-1⁻EpCAM⁺CD49f⁺) cells. Expression values of both datasets are shown as log₂-normalized values. Shown in pink are the genes (probes) identified as significantly differentially expressed (≥2-fold, <i>p</i>≤0.05). (B) Heat map of the top 1% of differentially expressed genes (probes) that were higher in the fetal dataset and the top 1% of differentially expressed genes (probes) that were higher in the adult basal dataset (shown as log₂ normalized gene expression values from panel A).</p

Frequency of single cells that form structures and generate CFCs and MRUs from different subsets of fetal and adult mammary cells.

<p>Values shown in brackets are the number of positive wells/number of wells assayed.</p>a<p>Based on assessment of wells containing a visible structure.</p>b<p>Calculated assuming MRUs would be found exclusively in wells containing visible structures.</p