ADGRL4/ELTD1 Expression in Breast Cancer Cells Induces Vascular Normalization and Immune Suppression

Abstract

ELTD1/ADGRL4 expression is increased in the vasculature of a number of tumor types and this correlates with a good prognosis. Expression has also been reported in some tumor cells with high expression correlating with a good prognosis in hepatocellular carcinoma (HCC) and a poor prognosis in glioblastoma. Here we show that 35% of primary human breast tumors stain positively for ELTD1, with 9% having high expression that correlates with improved relapse-free survival. Using immunocompetent, syngeneic mouse breast cancer models we found that tumors expressing recombinant murine Eltd1 grew faster than controls, with an enhanced ability to metastasize and promote systemic immune effects. The Eltd1-expressing tumors had larger and better perfused vessels and tumor-endothelial cell interaction led to the release of proangiogenic and immune-modulating factors. M2-like macrophages increased in the stroma along with expression of programmed death-ligand 1 (PD-L1) on tumor and immune cells, to create an immunosuppressive microenvironment that allowed Eltd1-regulated tumor growth in the presence of an NY-ESO-1-specific immune response. Eltd1-positive tumors also responded better to chemotherapy which could explain the relationship to a good prognosis observed in primary human cases. Thus, ELTD1 expression may enhance delivery of therapeutic antibodies to reverse the immunosuppression and increase response to chemotherapy and radiotherapy in this subset of tumors. ELTD1 may be useful as a selection marker for such therapies. IMPLICATIONS: ELTD1 expression in mouse breast tumors creates an immunosuppressive microenvironment and increases vessel size and perfusion. Its expression may enhance the delivery of therapies targeting the immune system.

Figure 1. Expression of mcoEltd1 in breast tumours increases tumour growth and metastasis.

(A) Syngeneic tumour growth curves of E0771 control and E0771 mcoEltd1 tumours. Tumour sizes were compared at endpoint by un-paired t-test, n=8. Individual and merged graphs shown. (B) IVIS fluorescent imaging of perfused tumours and (C) quantification. (D) H&E staining of lungs collected at experiment end point. Representative images taken on a Nikon Eclipse E800 microscope at 4× magnification. (E) Quantification of tumour area and tumour number in lungs. Data analysed by un-paired t-test. (F) Weight of lungs at experiment end point. *p <0.05; **p <0.005; ***p <0.0005. n=5 biological replicates.

Figure 2. Eltd1 expression in tumours increases vessel size and decreases hypoxia.

Representative immunohistochemistry and quantification of (A) Endomucin vessel staining, (B) a-SMA staining for pericyte coverage, (C) Ki67 proliferation, (D) H&E Staining for necrosis, (E) Hypoxyprobe staining for hypoxia. Data analysed by un-paired t-test *p <0.05; **p <0.005; ***p <0.0005. n=5 biological replicates.

Figure 3. Eltd1 expressing tumour cells proliferate in the presence of endothelial cells and modifies their secretome.

(A) Appearance of 4T1 tumour cells (GFP positive) when co-cultured with mouse endothelial cells for 48hrs at 4x magnification and quantification of GFP positive cells (B) Quantification of a mouse cytokine array performed using the cell supernatants collected after 48hrs of co-culture. Dot intensity was quantified using Fiji software. (C) QPCR of Ccl20 and (D) Ccl17 from RNA extracted from mouse endothelial cells (sEnd.1) and 4T1 tumour cells cultured alone or after co-culture and subsequent FACs sorting. Data were analysed by the unpaired t-test *p <0.05; **p <0.005; ***p <0.0005. # p <0.05, ### p <0.0005

Figure 4. Eltd1 expression alters immune infiltration and PD-L1 expression within 4T1 tumours.

FACS analysis of the tumour cell populations at day 22 (A). Data are presented as mean percentage of CD45+ cells. (B). Pie charts represent the proportion of CD45⁺ (all leukocytes) and CD45^- (tumour cells and others) from the tumours. Representative scatter plots showing the differences in leukocyte populations within the tumour, (C). Ly6G⁺Ly6C^- Myeloid cells, Neutrophils, Monocytes (D), M2 Macrophages (E) CD4+ and CD8+ lymphocytes. Percentage PD-L1 expression in (F) tumour cells, (G) leukocytes, (H) myeloid cells. Data analysed by the unpaired t-test (n=10 independent tumour samples analysed) *p <0.05.

Figure 5. Expression of Eltd1 supresses the immune mediated tumour cell rejection of NY-ESO-1.

(A) Individual growth curves for 4T1-NY-ESO-1 control and mcoEltd1 expressing tumours (B) Merged data. Tumour sizes were compared at endpoint by the unpaired t-test (C) Total number of immune cells in the spleen of the controls and mcoEltd1 tumour allografts at day 22. A group of naïve/healthy mice were used as control. (D) Mean percentage of total CD8+ CD44+ active T CD8 cells ± SEM. Data were analysed by the unpaired t-test (n = 5 independent mice analysed). Data were analysed by the unpaired t-test (n = 5 independent mice analysed) *p < 0.05; **p < 0.005; ***p < 0.0005.

Figure 6. Breast cancer cells can express ELTD1 and high expression correlates with improved relapse free survival (RFS).

(A) Representative images of low (score 0) to high (score 3) tumour ELTD1 expression. (B) Histogram of tumour cell IHC expression in larger patient cohort (C) Survival plot showing improved RFS with higher tumour ELTD1 expression. (D) Histogram of tumour cell ELTD1 IHC expression in primary breast tumours and regional node metastases. (E) Comparison of tumour cell ELTD1 IHC expression between primaries and regional node metastases. Data analysed by un-paired t-test. (F) Tumour cell ELTD1 expression changes. Black arrows indicate upward expression from primary tumour to metastatic node in the same patient; blue arrows indicate downwards expression in the same patient.

Figure 7. Eltd1 expressing tumours respond better to chemotherapy.

(A) Syngeneic tumour growth curves of E0771 control and E0771 mcoEltd1 tumours treated with or without doxorubicin (n=7). (B) Untreated tumour growth (C) Control +/− doxorubicin (D) mcoEltd1 +/− doxorubicin. Tumour sizes were compared between groups at day 8, 10, 12 and 15 and the data were analysed by the unpaired t-test *p <0.05; **p <0.005; ***p <0.0005.