Lung endothelial PEAR1 induces tumor cell dormancy

Abstract

In many cancer patients, distant metastases develop after years of dormancy. Understanding how disseminated tumor cells (DTCs), which are often found in proximity to the microvasculature, remain dormant and what regulates their reactivation is one of the major challenges in tumor biology. In a screen for endothelial secreted and plasma membrane proteins able to regulate tumor cell dormancy, we identified the transmembrane protein platelet and endothelial aggregation receptor 1 (PEAR1). Human and murine endothelial cells lacking PEAR1 lost the ability to promote dormancy of different tumor cells, and the extracellular part of PEAR1 was able to rescue this effect. Similarly, in mice lacking PEAR1 in endothelial cells, tumor cell dormancy in the lung was reduced and tumor metastasis was increased. We found that PEAR1 induces tumor cell dormancy by binding lysyl oxidase like 2 (LOXL2) and cathepsin D (CTSD), which both inhibit tumor cell dormancy and promote tumor growth and metastasis. Tumor cells with suppressed CTSD expression showed increased dormancy and decreased metastatic potential in vivo. Our data identify a mechanism underlying tumor cell dormancy and suggest CTSD and LOXL2 as targets for approaches to promote dormancy.

Keywords: CTSD; Dormancy; Endothelial; LOXL2; Metastasis; PEAR1; p27.

Fig. 1

Endothelial cells induce tumor cell dormancy in vitro. A-E, Human MDA-MB-231 breast cancer cells co-expressing mCherry-luciferase and mVenus-p27K⁻ were cultured alone or together with HUVECs for 96 h in a direct co-culture system. Shown in (A) is a representative immunohistochemical staining with Hoechst 33,342 and with antibodies directed against mCherry to detect tumor cells (TC), against mVenus to detect the mVenus-p27K⁻ fusion protein (p27) and against Ki67 to detect proliferating cells with magnified areas indicated by a box with dashed lines. (B-E) show the quantification of tumor cell count (B), percentage of Ki67-positive cells (C), percentage of p27-positive and Ki67-negative cells (D) as well as percentage of live cells as indicated by 7-AAD and annexin V negativity (E) after 1–4 days of culture (n = 3 independent experiments). Scale bars: 100 μm. F-I, E0771 mouse tumor cells co-expressing mCherry-luciferase and mVenus-p27K⁻ were cultured alone or together with mouse lung endothelial cells (MLECs) for 4 days. Shown are the quantifications of the tumor cell number (F), the percentage of Ki67-positive (F), the percentage of p27-positive and Ki67-negative cells (H), and the percentage of alive, 7-AAD-negative and annexin V-negative cells (I) after 1–4 days of culture (n = 3 independent experiments). J, K, MDA-MB-231 tumor cells co-cultured with HUVECs were sorted to obtain cells positive and negative for mVenus-p27K⁻, and both cell fractions were cultured again in the absence of endothelial cells. Shown is the sorting strategy (J) as well as the cell number per well (K) during 7 days of culture (n = 3 independent experiments). L-O, E0771 and B16F10 mouse tumor cells co-expressing mCherry-luciferase and mVenus-p27K⁻ and co-cultured with MLECs for 4 days were sorted to obtain cells negative or positive for mVenus-p27K⁻. Both cell fractions were then cultured alone for 7 days, and cells per well were counted (L, M), or both fractions were injected orthotopically into the mammary fat pad of wild-type mice (N) or subcutaneously into wild-type animals (O), and tumor growth was monitored for 18 and 14 days, respectively (n = 4 independent experiments (L, M); n = 4 mice per group (N, O)). Shown are mean values ± S.E.M.; ^*, P ≤ 0.05; ^**, P ≤ 0.01; ^***, P ≤ 0.001; n.s., non-significant (2-way ANOVA with Bonferroni’s post-hoc test (B-I, K-L, N); 2-way ANOVA repeated measures with Bonferroni's post-hoc test (M, O))

Fig. 2

Identification of PEAR1 and CCL2 as mediators of endothelium-induced tumor cell dormancy. A, HUVECs were transfected with control siRNA or siRNAs against RNAs encoding surface receptors and secreted proteins highly expressed in HUVECs. Thereafter, cells were co-cultured with MDA-MB-231 cancer cells for 96 h and numbers of tumor cells were determined. Shown is the ratio of the cell count of MDA-MB-231 cells co-cultured with HUVECs transfected with a control siRNA and with each of the individual siRNA. The plot shows the ranked average ratios of 4 independent experiments. B, C, HUVECs were transfected with control siRNA or siRNAs directed against MCAM, CCL2, THBS1, BMP6, and PEAR1. 24 h later, cells were counted (B) or the percentage of 7-AAD-negative and annexin V-negative, alive cells was determined (C) (n = 3 independent experiments). D-G, MDA-MB-231 human tumor cells co-expressing mCherry-luciferase and mVenus-p27K⁻ were cultured alone or together with HUVECs transfected with control siRNA or siRNAs directed against MCAM, CCL2, THBS1, BMP6, and PEAR1, and cells were analyzed 4 days later. Shown in (D) are representative immunohistochemical images stained with Hoechst 33,342 and with antibodies directed against mCherry (TC), mVenus-p27K⁻ (p27) and Ki67 with magnified areas indicated by a box with dashed lines. Bar diagrams show the quantification of cell numbers (E), percentage of p27-positive and Ki67-negative (F) and of Ki67-positive tumor cells (G) (n = 16 (E) and 12 (F and G) independent experiments). Scale bars: 100 μm. Shown are mean values ± S.D. (A) or S.E.M. (B, C, E-G); ^***, P ≤ 0.001; n.s., non-significant (one-way ANOVA and Tukey’s multiple comparison test)

Fig. 3

Endothelial loss of PEAR1 reduces tumor cell dormancy and promotes metastasis. A, B16F10, RM1 or E0771 mouse tumor cells were i.v. injected into wild-type, EC-Ccl2-KO or EC-Pear1-KO mice. Lung metastasis was analyzed by quantifying the number of visible nodules 12 days after i.v. injection. Shown is the fold change in the two knock-out lines compared to wild-type animals (n = 14 mice per group (Control); n = 7 mice per group (EC-Ccl2-KO and EC-Pear1-KO). B-D, E0771, RM1 and B16F10 mouse tumor cells co-expressing mCherry-luciferase and mVenus-p27K⁻ and loaded with the cell division tracking dye CellTrace Yellow were i.v. injected into control, EC-Pear1-KO or EC-Ccl2-KO animals. 5 days after the injection, animals were euthanized and tumor cells in lungs were analyzed. Shown in (B) are representative flow cytometric analysis plots of mCherry-positive tumor cells from digested lungs with mVenus-p27K⁻ intensity plotted against the CellTrace Yellow intensity. The bar diagrams in (C) and (D) show the statistical analysis of the percentage of CellTrace Yellow-high and p27-positive tumor cells in EC-Pear1-KO (C) and in EC-Ccl2-KO (D) compared to control animals (n = 4 mice per group (B16F10 for EC-Pear1-KO and RM1 control group in (D)); n = 5 mice per group (B16F10 control group in (C), and B16F10 for EC-Ccl2-KO and corresponding control, RM1 EC-Ccl2-KO, and E0771 control group in (D)); n = 6 mice (E0771 (EC-Ccl2-KO)); n = 8 mice per group (RM1 and E0771 (EC-Pear1-KO and corresponding control))). E, F, The indicated mouse tumor cells loaded with the cell division tracker dye CFSE (F) were i.v. injected into control or EC-Pear1-KO mice, and mouse lungs were analyzed by flow cytometry 5 days after i.v. injection (E) or were analyzed histologically 1 day after i.v. injection. Shown is the percentage of alive, 7-AAD-negative and annexin V-negative, tumor cells (E) or the percentage of extravasated tumor cells after staining of lung sections with DAPI and anti-CD31 antibodies (F) (n = 4 mice (E0771 in E), n = 5 mice (B16F10 in E, B16F10 control in F, RM1 control in E, RM1 in F and E0771 EC-Pear1-KO in E), n = 6 mice (RM1 EC-Pear1-KO in E, B16F10 EC-Pear1-KO in F and E0771 in F)). G-J, The indicated tumor cells co-expressing mCherry-luciferase and mVenusp-27K⁻ were i.v. injected into control or EC-Pear1-KO mice. 1, 5 or 9 days after the injection, mice were euthanized, and isolated lungs were analyzed by bioluminescence imaging. Shown are representative bioluminescent images of lungs (G) at the end of each experiment as well as the statistical analysis of photon flux of lungs at the indicated time points after i.v. injection of B16F10 (H), RM1 (I), or E0771 (J) mouse tumor cells (n = 4 mice per group). Shown are mean values ± S.E.M.; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; n.s., non-significant (one-way ANOVA with Tukey’s multiple comparison test (A); Mann-Whitney U test (C: B16F10, E: E0771); two-tailed, unpaired t-test with Welch’s correction (C: RM1); two-tailed, unpaired t-test (C: E0771, D-F); 2-way ANOVA with Bonferroni’s post-hoc test (H-J))−−−

Fig. 4

Loss of endothelial PEAR1 promoted lung metastasis from primary tumors. A, E0771 of B16F10 tumor cells co-expressing mCherry-luciferase and mVenus-p27K− and isolated from stand-alone cultures were injected orthotopically or subcutaneously, respectively, into EC-Pear1-KO or control animals to allow for primary tumor formation for 18 and 14 days, respectively. After resection of the primary tumor, animals were kept for another 6 weeks. Thereafter, mice were euthanized and analyzed. B, C, Shown are quantifications of the growth of E0771 (B) and B16F10 (C) primary tumors using a microcaliper (n = 9 mice per group in (B); n = 10 mice per group in (C)). D-G, Shown are representative images of lungs 6 weeks after E0771 primary tumor resection, the quantification of macrometastatic nodules (n = 12 mice per group) (D), representative bioluminescent images of lungs 6 weeks after resection of E0771 (E) and B16F10 primary tumors (F) and the statistical analysis of photon flux of lungs (G) (n = 8 mice per group (E0771 for all groups, B16F10 (control group); n = 7 mice (B16F10 (EC-Pear1-KO)). H, Flow cytometric quantification of the percentage of p27-positive tumor cells in lung digests from EC-Pear1-KO and control animals 6 weeks after primary tumor resection (n = 8 mice per group (E0771 for all groups, B16F10 (control group); n = 7 mice (B16F10 (EC-Pear1-KO)). I, J, Cryosections of whole lungs taken 6 weeks after tumor resection were stained with DAPI and antibodies against mVenus-p27K⁻, against mCherry to identify tumor cells, and against Ki67, and a comprehensive analysis of whole lung sections was conducted. Tumor cells (TC) were grouped into three categories: solitary (1 cell) and those present in micrometastasis (2–8 cells) and macrometastasis (more than 8 cells). The bar graphs illustrate the total counts of proliferating (Ki67-positive) and dormant (Ki67-negative and p27-positive) E0771 (I) and B16F10 (J) tumor cells per area that have metastasized to the lung 6 weeks after resection in both EC-Pear1-KO and control mice (n = 6 mice per group; 3 sections per mice). Shown are mean values ± S.E.M.; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; n.s., non-significant (2-way ANOVA repeated measures with Bonferroni’s post-hoc test (B-C); Mann-Whitney U test, unpaired, one-tailed (D); Mann-Witney U test, unpaired, two-tailed (H: E0771) unpaired t-test; two-tailed with Welch’s correction (G); unpaired t-test, two-tailed (H: B16F10); 2-way ANOVA with Bonferroni’s post-hoc test (I: solitary TCs, J: solitary TCs and micrometastasis) and Kruskal-Wallis with Dunn’s multiple comparison test (I: microtetastasis and macrometatsis, J: macrometastasis))

Fig. 5

Endothelium-specific KO of Pear1 promoted lung metastasis in the spontaneous MMTV-PyMT mouse model. A, Schematic representation of the experimental strategy. MMTV-PyMT mice without (control) or with endothelial Pear1 deficiency (EC-Pear1-KO), which in addition express the Flp recombinase under the control of the MMTV promoter as well as the mCherry-expressing Flp reporter RC::FrePe, which allows to mark disseminated MMTV-PyMT breast cancer cells with mCherry fluorescence, were generated. Once MMTV-PyMT mice reached the humane endpoint, circulating tumor cells (CTC) as well as lung metastases were analyzed. B, C, Shown are the tumor-free survival rate (B) and the overall time after tumor occurrence until the humane endpoint was reached (C) (n = 15 mice (control group); n = 12 mice (EC-Pear1-KO)). D-F, Upon reaching the humane endpoint, mice were euthanized, blood samples were taken intracardially, and lungs were collected for analysis using epifluorescence imaging for mCherry. Shown in (D) is the number of mCherry-positive tumor cells per milliliter blood obtained from EC-Pear1-KO and control PyMT mice (n = 6 mice per group). Representative epifluorescent images of the lungs and the statistical analysis of mCherry epifluorescence at the endpoint are shown in (E) and (F) respectively. G, H, Lung tissues were digested and analyzed by flow cytometry. Shown are the results from flow cytometric analysis, which quantified the total number of mCherry-positive breast cancer cells present in the lung (G) and the percentage of Ki67-positive, mCherry-positive breast cancer cells (H) (n = 6 mice per group). Shown are mean values ± S.E.M.; ^**, P ≤ 0.01; n.s., non-significant (two-sided log-rank test for Kaplan-Meier curves (B, C); two-tailed, unpaired t-test (D); Mann-Whitney U test (F, H); two-tailed, unpaired t-test with Welch’s correction (G))

Fig. 6

PEAR1 sequesters CTSD and LOXL2 to promote tumor cell dormancy. A, MDA-MB-231 cells were treated with increasing concentrations of the extracellular domain of recombinant human PEAR1 containing a His-tag and immobilized on Ni-NTA beads. Cells incubated with Ni-NTA beads only served as a control group. Cell count was determined by flow cytometry analysis (n = 3 independent experiments). B, MDA-MB-231 cells were treated daily for 4 days with PEAR1 immobilized on Ni-NTA beads, and cells were analyzed by immunoblotting using anti-GAPDH and anti-p27 antibodies. Shown is a representative immunoblot and the statistical analysis (n = 3 independently performed experiments). C, D, MDA-MB-231 cells expressing mVenus-p27K⁻ were transfected with control siRNA or siRNA directed against SVEP1 or LOXL2 and were incubated in the absence or presence of PEAR1 (100 nM). After 36 h, the cell count (C) and the percentage of p27-positive cells (D) was determined (n = 3 independent experiments). E, Procedure to search for interaction partners of PEAR1 within the conditioned medium of tumor cells using a pull-down assay with PEAR1 immobilized on Ni-NTA beads. Control Ni-NTA beads and the recombinant extracellular part of human epidermal growth factor receptor immobilized on Ni-NTA beads served as controls. F, The plot shows the proteins that were specifically co-precipitated with PEAR1 compared to control samples. Proteins only found with PEAR1 are indicated by green dots, and proteins found with both PEAR1 and EGFR are represented by grey dots. G, Recombinant CTSD, SEMA6D or LOXL2 were incubated alone or together with purified His-tagged PEAR1. PEAR1 was precipitated with Ni-NTA beads, and precipitates were analyzed by immunoblotting. Shown are representative of 3 independently performed experiments. H, I, MDA-MB-231 cells co-expressing mCherry-luciferase and mVenus-p27K⁻ were co-cultured with HUVECs for the indicated time periods, and cells were treated daily without or with CTSD (100 nM), LOXL2 (100 nM) or SEMA6D (1000 nM). The cell count (H) was assessed using flow cytometry. At the end of the experiment, the percentage of p27-positive cells (l) was determined by flow cytometry (n = 3 independent experiments). J, K, Co-cultures of HUVECs and MDA-MD-231 cells were incubated for the indicated time periods with 100 nM PEAR1 alone or in combination with 100 nM CTSD or LOXL2. Assessment of cell count (J) was conducted utilizing flow cytometry, and, after 4 days, the percentage of p27-positive cells (K) was analyzed by flow cytometry (n = 3 independent experiments). L, M, MDA-MB-231 cells co-expressing mCherry and mVenus-p27K⁻ were transfected with control siRNA or siRNA directed against CTSD, LOXL2 or both, and were cultured in the absence or presence of the recombinant extracellular part of human PEAR1 (rhPEAR1; 100 nM). After 96 h, cells were counted (L), and p27 expression was determined (M) (n = 3 independently performed experiments). Shown are mean values ± S.E.M.; ^*, p ≤ 0.05; ^**, p ≤ 0.01; ^***, p ≤ 0.001; n.s., non-significant (2-way ANOVA and Bonferroni’s post-hoc test (A-D, H, J, L, M); multiple two-tailed t-test, Bayesian moderated, Benjamin, Krieger, and Yekutieli-corrected, false discovery rate = 1% (F); one-way ANOVA with Tukey’s multiple comparison test (I, K))

Fig. 7

Suppression of CTSD in tumor cells increased tumor cell dormancy both in vitro and in vivo. A, B, The indicated tumor cells co-expressing mCherry-luciferase and mVenusp27K⁻ were subjected to stable transduction with either a scrambled control shRNA (shScr) or an shRNA targeting Ctsd (shCtsd). Subsequently, tumor cells were co-cultured with MLECs and treated daily with either a vehicle control or recombinant mouse CTSD (100 nM) over a period of 96 h, and the percentages of p27-positive E0771 (A) and B16F10 (B) cells were quantified (n = 3 independent experiments). C-J, The indicated tumor cells without or with stable knock-down of CTSD (shScr or shCtsd, respectively) co-expressing mCherry-luciferase and mVenus-p27K⁻ were i.v. injected into wild-type animals. Lung metastasis was assessed by determining the number of visible metastatic nodules 12 days after injection of E0771 (C) or B16F10 (D) as well as by analyzing isolated lungs by bioluminescence imaging (E and F) (n = 4 mice per group). The proportion of p27-positive tumor cells present in lung digests collected 1, 5, and 12 days after i.v. injection of E0771 (G) or B16F10 (H) was determined by flow cytrometry (n = 4 mice per group). Cryosections of whole lungs were stained with Hoechst 33342, and antibodies against mVenus, mCherry and Ki67. Thereafter, entire lung sections were imaged, enabling the categorization of tumor cells into three distinct groups: solitary tumor cells (TC) (1 cell) and those present in micrometastasis (2–8 cells) and macrometastasis (> 8 cells). The bar graphs show the total numbers of proliferating (Ki67-positive) and dormant (Ki67-negative and p27-positive) tumor cells per area for E0771 (I) and B16F10 cells (J) without (shScr) or with CTSD knock-down (shCtsd) (n = 4 mice per group; 3 sections per mice). K, Schematic representation of the proposed role of endothelial PEAR1 in promoting tumor cell dormancy. EC, endothelial cell; TC, tumor cell. Shown are mean values ± S.E.M.; ^*, P ≤ 0.05; ^**, P ≤ 0.01; ^***, P ≤ 0.001; n.s., non-significant (2-way ANOVA and Bonferroni’s post-hoc test (A, B, G, H, I (solitary TCs and micrometastasis and J); two-tailed, unpaired t-test with Welch’s correction (C); two-tailed, unpaired t-test (D, E); Mann-Whitney U test, unpaired, two-tailed (F); Kruskal-Wallis test with Dunn’s multiple comparison test (I (macrometastasis))