Cancer-associated fibroblasts modulate macrophage phenotype in PDAC through hypoxia-inducible factor 2 (HIF2)-dependent metabolite secretion
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic stroma which induces profound intratumoral hypoxia within its tumor microenvironment (TME). PDAC has low levels of effector T cell infiltration and increased numbers of immunosuppressive macrophages and regulatory T cells, which is thought to be a consequence of the hypoxic TME. We recently demonstrated that cancer-associated fibroblasts (CAFs) within spontaneous pancreatic tumors regulate macrophage migration and polarization through a paracrine mechanism dependent upon hypoxia inducible factor-2 (HIF2) expression. Here we use genetic and biochemical techniques to identify the putative secreted factors mediating this HIF2-dependent CAF-macrophage crosstalk.
Methods
We used a dual-recombinase mouse model to generate spontaneous pancreatic tumors with a conditional allele of HIF2 expressed in alpha-SMA+ fibroblasts that was abrogated by ex vivo infection with an adenovirus to overexpress Cre (AdCre) or GFP (AdGFP). HIF2 was also inhibited with the small molecule PT2399. Hypoxia experiments were performed in a chamber which maintained 0.5% O2.
Results
Following Cre overexpression via adenovirus, HIF2 was confirmed to be deleted in CAFs through PCR, qPCR, and western blot. HIF2-KO (AdCre) CAFs and HIF2-WT (AdGFP) CAFs were then cultured under normoxia (21% O2) or hypoxia (0.5% O2) for 48 hours and conditioned media (CM) was collected. We analyzed cytokines and chemokines within the CM using a cytokine array to identify proteins secreted in a hypoxia and HIF2-dependent fashion. Among several proteins, insulin-like growth factor-binding protein 3 (IGFBP3) was significantly increased in CM from HIF2-WT CAFs cultured in hypoxia compared to HIF2-KO CAFs. We confirmed this HIF2-dependent secretion in patient-derived pancreatic stellate cells (hPSCs) in hypoxia with either vehicle (DMSO) or 2µM PT2399. IGFBP3 was greatly increased in CM from hPSCs treated with vehicle compared to PT2399 treatment (1166 vs 413.7 pg/mL, p=0.0052). qPCR analysis revealed that IGFBP3 was the most hypoxia-responsive and HIF2-dependent member of the IGFBP family (IGFBP1-IGFBP7). Furthermore, macrophages cultured with CM from HIF2-WT CAFs increased Mrc1 gene expression, a marker of M2 macrophage polarization, while CM from HIF2-KO CAFs did not increase Mrc1.
Conclusion
Our results suggest that HIF2-dependent secretion of IGFBP3 from CAFs in the hypoxic pancreatic cancer TME contributes to its immunosuppressive nature. Future work will focus on verifying that IGFBP3 mediates HIF2-dependent modulation of macrophages and will explore other paracrine factors which may be involved in CAF-macrophage crosstalk.