Tumor-Secreted GRP78 Promotes the Establishment of a Pre-metastatic Niche in the Liver Microenvironment

Abstract

The liver is an immunologically tolerant organ and a common site of distant metastasis for various cancers. The expression levels of glucose-regulated protein 78 (GRP78) have been associated with tumor malignancy. Secretory GRP78 (sGRP78) released from tumor cells contributes to the establishment of an immunosuppressive tumor microenvironment by regulating cytokine production in macrophages and dendritic cells (DCs). However, the role of sGRP78 on tumor cell colonization and metastasis in the liver remains unclear. Herein, we found that GRP78 was expressed at higher levels in the liver compared to other tissues and organs. We performed intravital imaging using a sGRP78-overexpressing breast cancer cell line (E0771) and found that sGRP78 interacted with dendritic cells (DCs) and F4/80⁺ macrophages in the liver. Importantly, sGRP78 overexpression inhibited DC activation and induced M2-like polarization in F4/80⁺ macrophages. Moreover, sGRP78 overexpression enhanced TGF-β production in the liver. In conclusion, sGRP78 promotes tumor cell colonization in the liver by remodeling the tumor microenvironment and promoting immune tolerance. The ability of sGRP78-targeting strategies to prevent or treat liver metastasis should be further examined.

Keywords: dendritic cells; immunomodulation; liver pro-metastatic niche; macrophages; natural killers; tumor-secreted GRP78.

FIGURE 1

Tumor-secreted GRP78 promotes tumor invasion and liver metastasis. (A) sGRP78-overexpressing E0771-sGRP78-mCherry cells were identified by confocal microscopy. Scale bar, 20 μm. (B) The levels of sGRP78 in the cell culture supernatant were determined by ELISA. (C) Tumor growth curves. (D) The migration ability of tumor cells was assessed by wound healing assay. (E) Frequencies of mCherry-positive cells in the liver 4 and 7 days after cell inoculation. Representative flow cytometry dot plots (left) and percentage of positive cells (right) are shown. (F) Representative images of H&E-stained liver sections from mice sacrificed 20 days after tumor cell intrasplenic injection. Scale bar, 2,000 μm. (G) Quantification of liver weight (left panel) and metastatic burden (right panel) in livers (n = 5 mice per group). (H) Representative images of organ tumor burden 20 days after tumor cell intrasplenic injection. (I) Survival curves (n = 6). Error bars represent SEM. Ns, not significant; ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001. Statistical significance was determined by Student’s t-test (E,G right), by Welch’s ANOVA (B,C,G left) or by log-rank Mantel-Cox test (I).

FIGURE 2

Tumor-secreted GRP78 induces tolerogenic phenotypes in hepatic monocytes/macrophages. (A) Gating strategy followed to distinguish the hepatic myeloid cells. Frequencies of hepatic F4/80⁺ macrophages and DC cells, as well as their expressions of CD86 and MHC-II four (B) and seven (C) days after tumor cell intrasplenic injection. Expression of CD86 and MHC-II on LSEC cells four (D) and seven (E) days after tumor cell intrasplenic injection. Graphs represent the mean ± SEM. Ns, not significant; ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001. Statistical significance was determined by Student’s t-test.

FIGURE 3

Tumor-secreted GRP78 interacts with hepatic DC and F4/80⁺ macrophages. Mice liver sections showing sGRP78 interacting with CD11c⁺ cells (A) and F4/80⁺ cells (B). Scale bar, 100 μm. (C) Numbers of infiltrated DC cells and F4/80⁺ cells (upper), as well as of mCherry⁺ DC and F4/80⁺ cells (lower). sGRP78 was detected to interact with CX3CR1⁺ cells (D). Scale bar, 50 μm. (E) Numbers of mCherry⁺ CX3CR1⁺ cells. Error bars represent SEM. Ns, not significant; ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001. Statistical significance was determined by Student’s t-test (C) or by Welch’s ANOVA (E).

FIGURE 4

Tumor-secreted GRP78 promotes myeloid CX3CR1⁺ cell recruitment in metastatic lesions. In vivo imaging of the livers four (A) and seven (B) days after E0771-mCherry (left) and E0771-sGRP78-mCherry (right) injection, using confocal microscopy. Trajectories (C) and motion parameters of CX3CR1⁺ cells four (D) and seven (E) days after tumor cell intrasplenic injection. Each data point represents a single cell, and the red bars indicate mean values. ^∗∗P < 0.01; ^∗∗∗P < 0.001; ns: not significant. The data from 3–5 mice in two independent experiments were pooled. Statistical significance was determined by Student’s t-test.

FIGURE 5

Tumor-secreted GRP78 inhibits hepatic NK cell activation. Liver tissues were excised and their mass was measured at days 4 and 7 after injection. The liver was digested and then filtered. The cell pellet containing leukocytes was collected by Percoll centrifugation. Then cells were stained with antibodies for FCM analysis. (A) Gating strategy followed to distinguish the hepatic immune cells. (B) Frequencies of immune cell subsets in the liver four (upper panel) and seven (lower panel) days after tumor cell intrasplenic injection. (C–E) Frequencies of CD69⁺ and PD-L1⁺ cells among CD4, CD8 subsets, as well as of CD69⁺ and Ki-67⁺ cells in NK1.1 cells. Representative flow cytometry dot plots (C) and percentage of positive cells (D,E) are shown. (F,G) Detection of cytokines. Graphs represent the mean ± SEM. Ns, not significant; ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001. Statistical significance was determined by Student’s t-test (B,D,E) or by Welch’s ANOVA (F,G).

FIGURE 6

Schematic description of the role of tumor-secreted GRP78 in the pre-metastatic niche. Tumor-secreted GRP78 interacts with hepatic DCs and macrophages in the metastatic niche to induce tolerogenic phenotypes. These impair NK cell recruitment and activation, thereby promoting the establishment of a pre-metastatic niche, which fosters tumor cell colonization, invasion, and metastasis.