Path Analysis of intra-metastatic hypoxia in breast cancer

Hypoxia (low O2) signals into the nucleus of cancer cells through hypoxia-inducible factor (HIF)-1-dependent transcription triggering proliferative, metabolic and vascular adaptations linked to therapy resistance and mortality due to overt metastasis. In contrast with the wealth of molecular data on primary intra-tumoral hypoxia, there is a dearth of statistical modelling studies addressing the mechanisms of intra-metastatic hypoxia. In this study, we used path analysis to model intra-metastatic hypoxia (Hx), HIF-1 expression and microvascular area (MVA) as functions of metastatic cross-sectional area (MCSA) in an advanced mouse breast cancer model; in this context, we tested the effect of conventional, maximum-tolerated dose (MTD) or low-dose metronomic (LDM) chemotherapy. Iterative analysis of 34 non-isomorphic paths yielded four well-fitting, configuration-invariant models [{chi}2(6,171) [≥] 6.12; P [≥] 0.328; CFI [≥] 0.998; RMSEA [≤] 0.07]. All four models contained HIF-1 as a mediating variable within the MCSA{leftrightarrow}Hx{leftrightarrow}MVA path, as well as significant Hx{leftrightarrow}MVA interactions. LDM disrupted the HIF-1{leftrightarrow}MCSA[->]Hx and HIF-1{leftrightarrow}MVA paths; furthermore, all LDM and MTD combinations impaired Hx{leftrightarrow}HIF-1. These results confirmed well-established hypoxic interactions, whilst uncovering possible differential effects of chemotherapeutic modalities upon metastatic size, hypoxia, HIF-1 and vascularisation. Our data indicate that MVA can act as a downstream readout, rather than an adaptive angiogenic mechanism alleviating intra-metastatic hypoxia. Moreover, well-fitting path models locate HIF-1 activity either upstream or downstream of hypoxia, thereby allowing us to posit the existence of bi-directional feedback loops driving vascularisation and growth in metastatic tumours, of relevance for targeted therapies.