Quantitative characterization of stem cell dynamics in the hypoxic microenvironment with chemotherapy using computer vision

<p>Background: The cancer stem cell hypothesis has implicated a unique subpopulation of cancer cells as the primary drivers of tumorigenesis with capabilities of self-renewal and ability to reconstitute the full spectrum of heterogeneity within a tumour. It has been noted that the proportion of cancer stem cells is increased by hypoxia, which has been implicated as playing a key causative role in metastasis and therapeutic resistance in multiple solid tumours, including breast cancer. Recently it has been also reported that that cytotoxic chemotherapeutics may also induce a stem-like state in cancer cells. The dynamics of cancer stem cell induction by these conditions, and the possibility of synergistic effects, however, have not been accurately quantified. </p><p>Methods: In this work, we present, for the first time, an analysis pipeline for the quantification of cancer stem cell dynamics using time-lapse microscopy and computer-assisted, unbiased digital image processing. The MDA-MB-231 breast cancer cell line, transduced with a NANOG promoter driven GFP reporter gene, thereby reporting a stem-like state, was cultured under varying conditions of deferoxamine (a hypoxia chemo-mimetic) and paclitaxel (a chemotherapeutic reagent). Cellular dynamics were recorded by time-lapse microscopy capturing phase contrast and fluorescent images over a four-day period. Areas of fluorescence and confluence determined from phase images were compared to obtain the time dynamics of the proportion of stem-like cells. These dynamics were then fit to a two-state ordinary differential equation model, and the underlying distribution of parameter sets was studied.</p> <p>Results: Using the obtained dynamics, the time series of the proportion of NANOG-expressing cells was elucidated, and the parameter sets obtained by fitting experimental data to the model revealed shifts in parameter value distributions for the various cases of chemotherapy and deferoxamine concentrations. These shifts indicate that there are likely non-linear effects of these drugs on key underlying biological rates, such as the rates of differentiation and dedifferentiation. </p> <p>Conclusions: Taken together, these results are indicative of a number of relationships between the parameters governing cancer stem cell dynamics when affected by both hypoxia and chemotherapy, as occurs in the setting of a patient. This underscores the need for further characterisation of these dynamics, including a more thorough exploration of the effects of differing concentrations of chemotherapy, deferoxamine, and true hypoxia. Gaining an understanding of these dynamics may ultimately provide avenues to improve the efficacy of chemotherapy and reduce the risk of tumour recurrence, by optimizing the chemotherapy regimen together with manipulating the microenvironment.</p

Five-Part Pentameric Nanocomplex Shows Improved Efficacy of Doxorubicin in CD44+ Cancer Cells

The CD44 receptor is common among many cancer types where overexpression is synonymous with poor prognosis in prostate, glioma, and breast cancer. More notably CD44 overexpression has been shown in a number of different cancer stem cells (CSC) which are present in many solid tumors and drive growth, recurrence, and resistance to conventional therapies. Triple negative breast cancer CSCs correlate to worse prognosis and early relapse due to higher drug resistance and increased tumor heterogeneity and thus are prime targets for anticancer therapy. To specifically target cells overexpressing CD44 receptors, including CSCs, we synthesized a pentameric nanocomplex (PNC) containing gold nanoparticles, doxorubicin (Dox) conjugated to thiolated hyaluronic acid via an acid-labile hydrazone bond, and thiolated poly­(ethylene glycol) DNA CD44 aptamer. In vitro drug release was highest at 8 h time point at acidic pH (pH 4.7) and in 10 mM glutathione. The PNC is almost an order of magnitude more effective than Dox alone in CD44+ cells versus CD44 low cells. Functionally, the PNC reduced CSC self-renewal. The PNC provides a therapeutic strategy that can improve the efficiency of Dox and decrease nontargeted toxicity thereby prolonging its use to individual patients

A 4-miRNA signature to predict survival in glioblastomas

<div><p>Glioblastomas are among the most lethal cancers; however, recent advances in survival have increased the need for better prognostic markers. microRNAs (miRNAs) hold great prognostic potential being deregulated in glioblastomas and highly stable in stored tissue specimens. Moreover, miRNAs control multiple genes representing an additional level of gene regulation possibly more prognostically powerful than a single gene. The aim of the study was to identify a novel miRNA signature with the ability to separate patients into prognostic subgroups. Samples from 40 glioblastoma patients were included retrospectively; patients were comparable on all clinical aspects except overall survival enabling patients to be categorized as short-term or long-term survivors based on median survival. A miRNome screening was employed, and a prognostic profile was developed using leave-one-out cross-validation. We found that expression patterns of miRNAs; particularly the four miRNAs: hsa-miR-107_st, hsa-miR-548x_st, hsa-miR-3125_st and hsa-miR-331-3p_st could determine short- and long-term survival with a predicted accuracy of 78%. Heatmap dendrograms dichotomized glioblastomas into prognostic subgroups with a significant association to survival in univariate (HR 8.50; 95% CI 3.06–23.62; p<0.001) and multivariate analysis (HR 9.84; 95% CI 2.93–33.06; p<0.001). Similar tendency was seen in The Cancer Genome Atlas (TCGA) using a 2-miRNA signature of miR-107 and miR-331 (miR sum score), which were the only miRNAs available in TCGA. In TCGA, patients with O6-methylguanine-DNA-methyltransferase (MGMT) unmethylated tumors and low miR sum score had the shortest survival. Adjusting for age and MGMT status, low miR sum score was associated with a poorer prognosis (HR 0.66; 95% CI 0.45–0.97; p = 0.033). A Kyoto Encyclopedia of Genes and Genomes analysis predicted the identified miRNAs to regulate genes involved in cell cycle regulation and survival. In conclusion, the biology of miRNAs is complex, but the identified 4-miRNA expression pattern could comprise promising biomarkers in glioblastoma stratifying patients into short- and long-term survivors.</p></div

KEGG pathway enriched for mRNAs predicted to be targeted by miRNAs in the 4-miRNA signature.

<p>KEGG pathway enriched for mRNAs predicted to be targeted by miRNAs in the 4-miRNA signature.</p

Short-(STS) and long-term (LTS) glioblastoma survivors have different microRNA (miRNA) profiles.

<p><b>(A)</b> Heatmap of the ten most deregulated miRNAs in STS and LTS. STS and LTS are grouped into two overall patterns as shown by the dendrograms. Pattern one (red bar) was characterized by STS whereas pattern two (green bar) mostly characterized LTS (18 LTS and 7 STS). In the heatmap, red represents upregulated miRNAs and green represents downregulated miRNAs. <b>(B)</b> Kaplan Meier plot showing a significant separation in overall survival between the two patterns. <b>(C)</b> Volcano plot illustrating that no miRNAs were significantly deregulated above the two-fold threshold. Blue represent normal fold changes and p-values while red represent permutated values. The four miRNAs included in the signature are indicated with arrows.</p

The two miRNA patterns and multivariate analysis.

<p>The two miRNA patterns and multivariate analysis.</p

TCGA and multivariate analysis.

<p>TCGA and multivariate analysis.</p

Glioblastoma patient characteristics.

<p>Glioblastoma patient characteristics.</p

Transferrin receptor-1 and ferritin heavy and light chains in astrocytic brain tumors: Expression and prognostic value

<div><p>Astrocytic brain tumors are the most frequent primary brain tumors. Treatment with radio- and chemotherapy has increased survival making prognostic biomarkers increasingly important. The aim of the present study was to investigate the expression and prognostic value of transferrin receptor-1 (TfR1) as well as ferritin heavy (FTH) and light (FTL) chain in astrocytic brain tumors. A cohort of 111 astrocytic brain tumors (grade II-IV) was stained immunohistochemically with antibodies against TfR1, FTH, and FTL and scored semi-quantitatively. Double-immunofluorescence stainings were established to determine the phenotype of cells expressing these markers. We found that TfR1, FTH, and FTL were expressed by tumor cells in all grades. TfR1 increased with grade (p<0.001), but was not associated with prognosis in the individual grades. FTH and FTL were expressed by tumor cells and cells with microglial/macrophage morphology. Neither FTH nor FTL increased with malignancy grade, but low FTH expression by both tumor cells (p = 0.03) and microglia/macrophages (p = 0.01) correlated with shorter survival in patients anaplastic astrocytoma. FTL-positive microglia/macrophages were frequent in glioblastomas, and high FTL levels correlated with shorter survival in the whole cohort (p = 0.01) and in patients with anaplastic astrocytoma (p = 0.02). Double-immunofluorescence showed that TfR1, FTH, and FTL were co-expressed to a limited extent with the stem cell-related marker CD133. FTH and FTL were also co-expressed by IBA-1-positive microglia/macrophages. In conclusion, TfR1 was highly expressed in glioblastomas and associated with shorter survival in the whole cohort, but not in the individual malignancy grades. Low levels of FTH-positive tumor cells and microglia/macrophages were associated with poor survival in anaplastic astrocytomas, while high amounts of FTL-positive microglia/macrophages had a negative prognostic value. The results suggest that regulation of the iron metabolism in astrocytic brain tumors is complex involving both autocrine and paracrine signaling.</p></div

Normal brain tissue from subventricular zone and neocortex as well as DAs immunohistochemically stained with TfR1, FTH, and FTL.

<p>Cells in the ependymal layer expressed TfR1 <b>(A)</b> and FTH (<b>B</b>). No expression of FTL <b>(C)</b> in the ependymal layer was seen. A few cells beneath the ependymal layer were positive for FTH <b>(B)</b> and FTL <b>(C)</b> (arrows). In the neocortex, there was a weak TfR1 neuronal staining <b>(D)</b>, but a stronger neuronal staining for FTH <b>(E)</b> and FTL <b>(F)</b>, which also stained the neuropil. In DAs, TfR1was mainly expressed by the endothelium (arrow) <b>(G)</b>. The FTH <b>(H)</b> and FTL <b>(I)</b> stainings were pronounced in many DAs with staining of both tumor cells (arrows) and microglia (arrowheads). <i>Abbreviations</i>: DA diffuse astrocytoma, FTH ferritin heavy chain, FTL ferritin light chain, TfR1 transferrin receptor-1. Scale bar: 100 μm.</p