Mechanisms and effects of the heterogeneous response of CAIX to hypoxia

Breast cancer is the most common cancer in females and several molecular subtypes have been described. However, intra-tumour heterogeneity exists and is associated with evolution of cancer metastasis and therapy resistance. The presence of regions of low oxygen (hypoxia) is associated with poor prognosis and the hypoxic microenvironment is also associated with resistance to current therapeutic strategies. Moreover, hypoxia and metabolite deficiency can alter the tumour microenvironment and drive heterogeneity. The pH regulating enzyme carbonic anhydrase IX (CAIX) is strongly induced under hypoxic conditions and its overexpression is likewise associated with poor therapeutic outcome. In a previous study, taking advantage of the membranous localization of CAIX, it was shown that under hypoxia a CAIX positive and CAIX negative population is present in MCF7 cells. RNA sequencing suggested an enrichment of stem cell markers in the CAIX positive population as well as a potential symbiotic relationship between these two subpopulations. In this study MCF7CAIX positive and negative populations were further investigated. Additionally, CAIX positive and negative populations were also found in HCT116 cells and subsequently established as stable cell populations. Both population show similar levels of induction of the hypoxia induced transcription factor HIF-1. The underlying mechanism of CAIX regulation was confirmed to rely on epigenetic modifications, as it could be shown that HIF-1 binding to the CAIX promoter is decreased in the CAIX negative population. To investigate the interaction of CAIX positive and negative cells in 3D models, stably labelled cells using either mCherry of GFP were generated. Upon mixing the two populations in a 1:1 ratio an initially even mix of the cells was observed in the spheroid. However, at a 72 h time point CAIX positive cells moved more towards the centre of the spheroid. This was inhibited by knockdown or pharmacological inhibition of CAIX. A key factor in migration and invasion are metalloproteinases/peptidases (MMPs). We found that MMP14 was upregulated in CAIX positive cells in 2D and 3D models, indicating an underlying mechanism for the re-localisation seen. Analysis of co-culture experiments using 2D a trans-well system revealed that under hypoxia the number of the CAIX positive population was increased in the presence of CAIX negative cells but not vice versa. A similar effect was seen using conditioned media, pointing to a secreted factor. Using a FRET based intracellular sensor to measure lactate levels it could be shown that lactate was higher in the CAIX negative cells. After spiking of the media during live imaging the CAIX positive cells quickly absorbed and retained lactate. In comparison, the CAIX negative cells initially showed an uptake of the metabolite, which was followed by a much quicker release. During co-culture, lactate levels in the conditioned media of CAIX negative cells were higher. Incubation of CAIX positive cells with this conditioned media decreased lactate levels, indicating lactate uptake by the cells. Moreover, knockdown of LDHA and/or B decreased the survival benefit CAIX positive cells had from CAIX negative cells. Another metabolic pathway showing marked differences between the two populations was lipid droplet formation. Under hypoxia CAIX positive cells showed an increased production of lipid droplet in comparison to CAIX negative cells and this was partly due to fatty acid uptake. In line with this, mRNA levels of the fatty acid transporter CD36 as well as some members of the FAB proteins were higher in the CAIX positive cells. Following a previous report showing that lipid droplets formed under hypoxia provide a survival benefit upon re-oxygenation, it was confirmed that CAIX positive cells grew indeed better in this set-up. Therefore, the results suggest a metabolic symbiosis between the CAIX positive and negative populations based on a lactate shuttle and differential uptake of fatty acids. Of wider significance, these findings show that tumours are heterogeneous in the response to hypoxia and that cell populations interact with each other in a symbiotic manner. This highlights the need for a better utilisation and combination of biomarkers to assess metabolic pathways and a better stratification of tumours. Moreover, this work highlights a potential effect of therapeutics addressing metabolic symbiosis.