Investigating the Heterogeneity of Tumor Associated Macrophages in Human Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a liver cancer and is one of the leading causes of cancer death worldwide. Despite significant efforts in treating HCC, such as with the use of novel immune checkpoint inhibitors, the overall response rate remains low. This may be partly attributed to the lack of a clear understanding of the HCC immune microenvironment. However, a high density of regulatory macrophages is correlated with poor prognosis in HCC patients. Therefore, we hypothesized that macrophages in HCC liver are distinct from their healthy counterparts and exhibit a clear tumour-driven regulatory pro-tumourgenic phenotype. With our access to fresh HCC and healthy liver, we demonstrated with single-cell RNA sequencing and flow cytometry that HCC liver macrophages have a more regulatory, tumour-promoting phenotype. HCC Macrophages are transcriptionally distinct from canonical Kupffer cells and exhibit a higher composition of C1QA+Marcolo population. Macrophages in the HCC liver also had an increased expression of regulatory surface markers (such as CD206 and PDL1) and functional phenotype (decreased TNF-α secretion after LPS stimulation) with increasing proximity toward the tumour. Further, these described phenotypes are distinct from healthy liver macrophages and are also independent of HCC etiology. Next, we used an in vitro model to gain mechanistic insight into the interaction between the tumour and macrophages. We used three HCC cell lines (Huh7, HepaRG, and HepG2) that broadly reflected the heterogeneous nature of HCC and established a co-culture system with primary monocyte-derived macrophages (MoDM). Co-culture with all HCC lines resulted in macrophages that exhibited an HCC-like regulatory CD206hi and low TNF-α secretory phenotype. Secreted factors in the tumour-conditioned media from HCC cell lines were sufficient to modulate MoDM, ranging from well-differentiated macrophages to monocytes towards an HCC-like phenotype. Using cytometric bead array, we identified that growth factors of VEGF, MCSF, PDGF, and EPO were secreted by the three cell lines. Purified recombinant growth factors were sufficient to induce a regulatory phenotype in MoDM. Overall, this work demonstrated the unique surface and functional phenotypes exhibited by macrophages in the HCC liver and identified a potential key secreted tumour-derived growth factor as an important modulator of tumour macrophages.

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