Estimation of Cell Cycle States of Human Melanoma Cells with Quantitative Phase Imaging and Deep Learning

Visualization and classification of cell cycle stages in live cells requires the introduction of transient or stably expressing fluorescent markers. This is not feasible for all cell types, and can be time consuming to implement. Labelling of living cells also has the potential to perturb normal cellular function. Here we describe a computational strategy to estimate core cell cycle stages without markers by taking advantage of features extracted from information-rich ptychographic time-lapse movies. We show that a deep-learning approach can estimate the cell cycle trajectories of individual human melanoma cells from short 3-frame (~23 minute) snapshots, and can identify cell cycle arrest induced by chemotherapeutic agents targeting melanoma driver mutations

Macrophage-derived IL-1β and TNF-α regulate arginine metabolism in neuroblastoma

© 2018 American Association for Cancer Research. Neuroblastoma is the most common childhood solid tumor, yet the prognosis for high-risk disease remains poor. We demonstrate here that arginase 2 (ARG2) drives neuroblastoma cell proliferation via regulation of arginine metabolism. Targeting arginine metabolism, either by blocking cationic amino acid transporter 1 (CAT-1)-dependent arginine uptake in vitro or therapeutic depletion of arginine by pegylated recombinant arginase BCT-100, significantly delayed tumor development and prolonged murine survival. Tumor cells polarized infiltrating monocytes to an M1-macrophage phenotype, which released IL1b and TNFa in a RAC-alpha serine/threonine-protein kinase (AKT)-dependent manner. IL1b and TNFa established a feedback loop to upregulate ARG2 expression via p38 and extracellular regulated kinases 1/2 (ERK1/2) signaling in neuroblastoma and neural crest-derived cells. Proteomic analysis revealed that enrichment of IL1b and TNFa in stage IV human tumor microenvironments was associated with a worse prognosis. These data thus describe an immune-metabolic regulatory loop between tumor cells and infiltrating myeloid cells regulating ARG2, which can be clinically exploited

Proliferation Index: A Continuous Model to Predict Prognosis in Patients with Tumours of the Ewing's Sarcoma Family

The prognostic value of proliferation index (PI) and apoptotic index (AI), caspase-8, -9 and -10 expression have been investigated in primary Ewing's sarcoma family of tumours (ESFT). Proliferating cells, detected by immunohistochemistry for Ki-67, were identified in 91% (91/100) of tumours with a median PI of 14 (range 0–87). Apoptotic cells, identified using the TUNEL assay, were detected in 96% (76/79) of ESFT; the median AI was 3 (range 0–33). Caspase-8 protein expression was negative (0) in 14% (11/79), low (1) in 33% (26/79), medium (2) in 38% (30/79) and high (3) in 15% (12/79) of tumours, caspase-9 expression was low (1) in 66% (39/59) and high (3) in 34% (20/59), and caspase-10 protein was low (1) in 37% (23/62) and negative (0) in 63% (39/62) of primary ESFT. There was no apparent relationship between caspase-8, -9 and -10 expression, PI and AI. PI was predictive of relapse-free survival (RFS; p = 0.011) and overall survival (OS; p = <0.001) in a continuous model, whereas AI did not predict outcome. Patients with tumours expressing low levels of caspase-9 protein had a trend towards a worse RFS than patients with tumours expressing higher levels of caspase-9 protein (p = 0.054, log rank test), although expression of caspases-8, -9 and/or -10 did not significantly predict RFS or OS. In a multivariate analysis model that included tumour site, tumour volume, the presence of metastatic disease at diagnosis, PI and AI, PI independently predicts OS (p = 0.003). Consistent with previous publications, patients with pelvic tumours had a significantly worse OS than patients with tumours at other sites (p = 0.028); patients with a pelvic tumour and a PI≥20 had a 6 fold-increased risk of death. These studies advocate the evaluation of PI in a risk model of outcome for patients with ESFT

Expression analysis of the MCPH1/BRIT1 and BRCA1 tumor suppressor genes and telomerase splice variants in epithelial ovarian cancer.

Aims The aim of this study was to explore the correlation of hTERT splice variant expression with MCPH1/BRIT1 and BRCA1 expression in epithelial ovarian cancer (EOC) samples. Background Telomerase activation can contribute to the progression of tumors and the development of cancer. However, the regulation of telomerase activity remains unclear. MCPH1 (also known as BRIT1, BRCT-repeat inhibitor of hTERT expression) and BRCA1 are tumor suppressor genes that have been linked to telomerase expression. Methods qPCR was used to investigate telomerase splice variants, MCPH1/BRIT1 and BRCA1 expression in EOC tissue and primary cultures. Results The wild type α+/β+ hTERT variant was the most common splice variant in the EOC samples, followed by α+/β− hTERT, a dominant negative regulator of telomerase activity. EOC samples expressing high total hTERT demonstrated significantly lower MCPH1/BRIT1 expression in both tissue (p = 0.05) and primary cultures (p = 0.03). We identified a negative correlation between MCPH1/BRIT1 and α+/β+ hTERT (p = 0.04), and a strong positive association between MCPH1/BRIT1 and both α−/β+ hTERT and α−/β− hTERT (both p = 0.02). A positive association was observed between BRCA1 and α−/β+ hTERT and α−/β− hTERT expression (p = 0.003 and p = 0.04, respectively). Conclusions These findings support a regulatory effect of MCPH1/BRIT1 and BRCA1 on telomerase activity, particularly the negative association between MCPH1/BRIT1 and the functional form of hTERT (α+/β+)

A. Kaplan Meier survival plots comparing the overall and relapse-free survival of patients with a tumour volume ≥200 ml with that of patients with a tumour volume <200 ml, p = 0.019 and 0.0133 respectively; log rank test. Crosses indicate censored events.

<p>B. Kaplan Meier survival plots to compare the overall and relapse-free survival of patients with pelvic tumours to that of patients with tumours at other sites, p = 0.0185 and 0.0088 respectively; log rank test. Crosses indicate censored events.</p

A. Immunohistochemistry for Ki-67 performed on primary ESFT (5 µm).

<p>Ki-67 protein was visualised with the DAB substrate, proliferating cells are identified by Ki-67 positive nuclei (see arrows). Immunohistochemistry performed in the absence of primary antibody was included to control for non-specific binding of the secondary antibody (No primary AB). Magnification ×400. B. TUNEL on primary ESFT (5 µm). Apoptotic cells were identified by TUNEL positive nuclei (see arrows). The TUNEL assay was performed in the absence TdT enzyme to control for non-specific staining (No enzyme). Magnification ×400.</p

Immunohistochemistry on primary ESFT (5 µm) for caspases-8, -9 and -10.

<p>Immunohistochemistry in the absence of primary antibody was to control for non-specific binding of the secondary antibody (No primary AB). Magnification of images = ×400. Caspase expression was scored as negative (0), low (1), medium (2) or high (3). A. Caspase-8 protein expression. B. Caspase-10 protein expression. C. Caspase-9 protein expression.</p

Macrophage IL-1B and TNF-a create an immune-metabolic loop regulating Arginase2 in neuroblastoma

International audienceNeuroblastoma is the most common solid tumour of childhood, yet the prognosis for high risk disease remains poor. We demonstrate that arginine metabolism via Arginase 2 (ARG2) drives neuroblastoma cell proliferation. Targeting arginine metabolism by blocking Cationic Amino Acid Transporter 1 (CAT-1) dependent arginine uptake in vitro or therapeutic depletion of arginine by pegylated-recombinant arginase BCT-100 significantly delays tumour development and prolongs murine survival. Tumour cells polarise infiltrating monocytes to a M1- macrophage phenotype, which release IL-1 and TNF-a in an RAC-alpha serine/threonine-protein kinase (AKT)-dependent manner. Il-1 and TNF- signal-back to upregulate ARG2 expression via p38 and Extracellular regulated kinases 1/2 (ERK1/2) signalling in neuroblastoma and neural crest-derived cells. Proteomic analysis reveal Stage IV human tumour microenvironments are enriched in IL-1 and TNF-a, which is associated with a worse prognosis. Thus we describe an immune-metabolic regulatory loop between tumour cells and infiltrating myeloid cells regulating ARG2, which could clinically exploited. Statement of significance: Neuroblastoma polarised macrophages released IL-1and TNF which signal back to regulate Arginase2 in tumour cells and drive their proliferatio