Cellular memory of hypoxia elicits neuroblastoma metastasis and enables invasion by non-aggressive neighbouring cells

Abstract

Therapies targeting cancer metastasis are challenging owing to the complexity of the metastatic process and the high number of effectors involved. Although tumour hypoxia has previously been associated with increased aggressiveness as well as resistance to radio- and chemotherapy, the understanding of a direct link between the level and duration of hypoxia and the individual steps involved in metastasis is still missing. Using live imaging in a chick embryo model, we have demonstrated that the exposure of neuroblastoma cells to 1% oxygen for 3 days was capable of (1) enabling cell migration towards blood vessels, (2) slowing down their velocity within blood vessels to facilitate extravasation and (3) promoting cell proliferation in primary and secondary sites. We have shown that cells do not have to be hypoxic anymore to exhibit these acquired capabilities as a long-term memory of prior hypoxic exposure is kept. Furthermore, non-hypoxic cells can be influenced by neighbouring hypoxic preconditioned cells and be entrained in the metastatic progression. The acquired aggressive phenotype relies on hypoxia-inducible factor (HIF)-dependent transcription of a number of genes involved in metastasis and can be impaired by HIF inhibition. Altogether, our results demonstrate the need to consider both temporal and spatial tumour heterogeneity because cells can 'remember' an earlier environment and share their acquired phenotype with their close neighbours. As a consequence, it is necessary to monitor the correct hypoxic markers to be able to predict the consequences of the cells' history on their behaviour and their potential response to therapies.

Artificial neural network classifier predicts neuroblastoma patients’ outcome

“…The importance of hypoxia in conditioning tumor aggressiveness is documented by an extensive literature [30, 32–34, 36, 37, 57]. Studies on the relationship between hypoxia inducible factors and neuroblastoma aggressiveness showed that high HIF-2 alpha expression correlated with disseminated disease (for review see [58]).…”

“…The characterization of the tumor at diagnosis is indispensable for deciding the treatment and the NB-hypo classifier poor outcome prediction may identify the tumors that, as a result of the hypoxic status, express high genetic instability [69], contain undifferentiated or cancer stem cells [32, 40] or a higher metastatic potential [33, 34]. Therapeutic agents are being developed to target hypoxia (for review see [59]) and are being tested in the clinic.…”

“…Hypoxia activates specific genes encoding angiogenic, metabolic and metastatic factors [31, 34, 35] and contributes to the acquisition of the tumor aggressive phenotype [31, 36–38]. We derived a 62-probe set neuroblastoma hypoxia signature (NB-hypo) [29, 39] and we demonstrated that NB-hypo is an independent risk factor for neuroblastoma patients [12].…”

“…We derived a 62-probe set neuroblastoma hypoxia signature (NB-hypo) [29, 39] and we demonstrated that NB-hypo is an independent risk factor for neuroblastoma patients [12]. The importance of hypoxia and hypoxia inducible genes in the progression, differentiation and spreading of neuroblastoma has been the subject of several reports [12, 34, 40–42]. …”

Assessing Estrogen-Induced Proliferative Response in an Endometrial Cancer Cell Line Using a Universally Applicable Methodological Guide

“…RL95-2 cells were labeled with a lentivirus-expressing tGFP (pGFP-C-shLenti plasmid) (Origene, Rockville, MD) and selected using puromycin antibiotic selection. Ishikawa and HEC1Aviral transfection and egg preparation were performed according to Carter et al 20 Cells were added to the CAM at embryonic day 7 (E7) 21 ; the resulting tumors were imaged at E14, using a standard Leica M165-FC fluorescent microscope in situ, and after excision (Fig. 1B), the resulting tumors were fixed in 10% (vol/vol) NBF and embedded in paraffin wax.…”