The liver cancer immune microenvironment: Therapeutic implications for hepatocellular carcinoma

Abstract

The liver is the sixth most common site of primary cancer in humans and the fourth leading cause of cancer-related death in the world. Hepatocellular carcinoma (HCC) accounts for 90% of liver cancers. HCC is a prevalent disease with a progression that is modulated by the immune system. Half of the patients with HCC receive systemic therapies, traditionally sorafenib or lenvatinib, as a first-line therapy. In the last few years, immune-checkpoint inhibitors (ICIs) have revolutionized cancer therapy and have gained an increased interest in the treatment of HCC. In 2020, the combination of atezolizumab (anti-programmed death-ligand 1) and bevacizumab (anti-vascular endothelial growth factor) improved overall survival over sorafenib, resulting in Food and Drug Administration (FDA) approval as a first-line treatment for patients with advanced HCC. Despite these major advances, a better molecular and cellular characterization of the tumor microenvironment is still needed because it has a crucial role in the development and progression of HCC. Inflamed (hot) and noninflamed (cold) HCC tumors and genomic signatures have been associated with response to ICIs. However, there are no additional biomarkers to guide clinical decision-making. Other immune-targeting strategies, such as adoptive T-cell transfer, vaccination, and virotherapy, are currently under development. This review provides an overview on the HCC immune microenvironment, different cellular players, current available immunotherapies, and potential immunotherapy modalities.

Figure 1:. Understand the liver microenvironment

Myeloid and lymphoid progenitors arise from hematopoietic stem cells via intermediate progenitors. In the steady-state, these progenitor cells supply cells to tissues for immune surveillance. Monocyte progenitors produce neutrophils, dendritic cells (DC) and monocytes. Monocytes can be differentiated in macrophages in specific organs. In the liver, there are two categories of macrophages, the ones having an embryonic origin (the Kupffer cells) and the others differentiated from circulating monocytes (the monocyte derived macrophages). In mice, specific markers to discriminate Kupffer cells have been discovered but in humans, they are still unknown. The lymphoblast lineage gives rise to NK cells and lymphocytes (iNKT, T cells and B cells). Representative markers for each cell type are shown.

Figure 2:. Neutrophils: Friends or foes?

Neutrophils are recruited at the tumoral site by the release of CXCL5 and CXCL6. Tumor-associated neutrophils (TANs) are turned into a pro-tumorigenic (N2) phenotype by the tumor. These N2 TANs exhibit strong immunosuppressive functions including expression of PD-L1, release of immunosuppressive cytokines (IL-8, CCL2, CCL17), and NETosis. These features inhibit T cells, promote Treg differentiation, and induce immunosuppressive tumor associated-macrophages (TAMs), leading to sorafenib resistance. There is an important dialogue between tumor cells, cancer-associated fibroblasts (CAFs), and TANs (blue and red arrows). Secretion of cardiotrophin-like cytokine factor 1 (CLCF1) by CAFs mediates tumoral expression of CXCL5, IL-6, and TGF-β, responsible for neutrophil recruitment and “N2” polarization, respectively. Activation of STAT3 leads to PD-L1 expression associated with T cell inhibition and triggers a positive loop to amplify cancer stem cell characteristics with the release of TGF-β and BMP2. Strategies to inhibit the immunosuppressive function of TANs during HCC could involve preventing their recruitment by using immunotherapies (anti-Ly6G or anti-Gr1), blocking the pro-tumorigenic NETosis mechanism with DNAse or cathepsin G inhibitor, or to block some cytokine pathways such as CXC5 or TGF-β.

Figure 3:. Monocytes: Dr Jekyll or Mr Hide?

Monocytes are recruited into the tumor site by the release of tumoral and stromal chemokines, such as CCL2 and CCL15. Monocytes can be polarized into different subtypes such as CD14⁺, CCR1⁺CD14⁺, and myeloid-derived suppressor cells (MDSC). All of these subtypes promote a strong immunosuppressive environment with expression of immune-checkpoint inhibitors (PD-L1/2, B7-H3, TIM-3) and cytokines (IL-10, CXCL2, CXCL8), inhibiting NK cytotoxicity and inducing Tregs. They also interact with neutrophils to promote tumor invasiveness though oncostatin M pathway. Ways to control tumorigenesis through monocytes could be to prevent their recruitment to the tumor by inhibiting the CCL15 pathway, to block their polarization by inhibiting the p38 pathway, or to repress the IL-6 pathway in order to prevent Treg formation.

Figure 4:. Macrophages: the tough ones

Macrophage-derived monocytes can be recruited by CCL2 to the tumor site. The pro-inflammatory (M1) / anti-inflammatory (M2) nomenclature is controversial but it is still used to describe macrophage activity. HCCs educate tumor-associated macrophages (TAMs) to become immunosuppressive, notably through the secretion of CCL2 and osteopontin (SPP1), and the release of extracellular vesicles. CSF1 expression by macrophages leads to tumoral PD-L1 expression and increases the immunosuppressive environment. Moreover, M2 cells release cytokines such as IL-10, IL-6, and VEGF, leading to T and NK cell inhibition, macrophage immunosuppression, and Treg differentiation responsible for resistance to therapies. Also, TAMs secrete HGF and IGF-1, leading to tumor cell proliferation and monocyte recruitment. Strategies to block their recruitment (anti-CCL2) or prevent their pro-tumorigenic functions (inhibition of VEGF, HGF, c-MET or IL-6 pathway, IFN-α vaccine) could lead to reduced tumor burden.

Figure 5:. Dendritic cells get the T cells rolling

A. The dendritic cell (DC) – T cell synapse is defined by three regulated steps: first, DCs present the antigen on MHC class II molecules to CD4⁺ T helper cells and on MHC class I molecules to CD8⁺ T cells. Then, interaction of costimulatory molecules of the immunoglobulin superfamily (CD80 and CD86, which bind to CD28 on T cells) and the TNF superfamily (CD40L/CD40, 4–1BBL/4–1BB, CD27/CD70, CD30L/CD30, and HVEM/LIGHT) occur to trigger cytokine release and T cell activation and differentiation. B. Plasmacytoid DCs (pDCs) can induce type 1 Tregs (Tr1) from naive CD4⁺ T cells through ICOS-L-ICOS interaction, allowing production of IL-10 from Tregs. LAMP3⁺ DCs (mDCs) can also express inhibitory ligands such as PD-L1, Gal9 (ligand of TIM3), MHC-II (for LAG3), and CD86 and CD80 (for CTLA4). These mechanisms lead to an immunosuppressive environment. Artificial activation of DCs (immunotherapy, vaccination…) could lead to an immune competent environment favoring an anti-tumor response.

Figure 6:. NK cells shoot to kill

A. NK cells express inhibitory receptors such as the inhibitory killer immunoglobulin-like receptors (iKIRs) and the C-type lectin-like receptor NKG2A, binding MHC-I and the non-classical MHC-I complex, HLA-E, respectively. Healthy cells inhibit NK cells by binding to NKG2A and iKIRs. However, absence of these ligands on a cell engages activating NK receptors (NKG2C/D, NKp30 and NKp44) that bind their ligands (MICA-B, B7H6, NKP46L, respectively) expressed on infected or tumor-transformed cells. After binding, perforin and granzyme B are release and kill the cell. However, an immunosuppressive tumor microenvironment – through IL-10 signaling - leads to the expression of NKG2A in NK cells. NKG2A associated to the expression of PD-1 induces NK inactivation and tumor growth. B. Tumors inhibit NK cells, but 6 different ways to reactivate them during tumorigenesis have been uncovered: the use of modified NK cells (CAR-NK), stimulation by IL15, targeting TGF-β, using antibodies binding the tumor cells and NK cells (ADCC and BiKE/TriKE), and inhibition of NKG2A or CD96 to suppress the immunosuppressive NK population.

Figure 7:. T cells lead the anti-tumor charge

A. The immunosuppressive environment, notably through the secretion of cytokines such as TGF-β, leads to Treg differentiation, associated with the expression of immune-checkpoint ligands by the tumor cell (PD-L1/2), which cause inhibition of CD8⁺ T cells. B. Different generations of CAR-T cells have been designed to recognize the tumor cell, kill it, and remain “non-exhausted” (PD-1 deletion).

Figure 8:. B cells join forces to promote or inhibit cancer

A. Naïve B cell interaction with tumor cells triggers B cell differentiation into an activated plasma cell able to kill the tumor cell via the secretion of specific antibodies. Thus, it promotes NK cell activation though the ADCC mechanism, as well as T cell activation. B. CD20⁺ B cells can limit growth of established tumors by secreting TNF-α. However, IgA⁺ B cells and regulatory B cells (Bregs) inhibit T cells by secreting IL-10 and TGF-β. This immunosuppressive environment also triggers polarization of monocytes and macrophages to reinforce it.