Stromal endothelial cells directly influence cancer progression

Abstract

Cancer growth and metastasis are regulated in part by stromal cells such as fibroblasts and immune cells within the tumor microenvironment. Endothelial cells (ECs) are also ubiquitous within tumors because tumors are vascular, and yet, the impact of tumor-resident ECs is less well understood. Through paracrine regulation, ECs modulate a diverse spectrum of pathophysiologic processes in normal and hyperplastic tissues. We hypothesized that ECs offer similar paracrine regulatory control of cancer biology. Indeed, secretions from quiescent ECs muted the proliferative and invasive phenotype of lung and breast cancer cells in vitro and reduced cancer cell protumorigenic and proinflammatory signaling. EC perlecan silencing significantly changed this regulatory relationship, eliminating the ability of ECs to inhibit cancer cell invasiveness via increased interleukin-6 secretion. Moreover, implanting ECs embedded within porous matrices slowed adjacent xenograft tumor growth and prevented architectural degeneration, with a concomitant reduction in proliferative and tumorigenic markers. Finally, lung carcinoma cells pretreated with intact EC-conditioned media, but not media conditioned with perlecan-silenced ECs, exhibited reduced micrometastatic burden after tail vein injection. These findings add to an emerging appreciation of EC-regulatory effects that transcend their structural roles and pave the way for improved characterization and control of EC-cancer cross-talk interactions for diagnosis, prognosis, and treatment of cancer.

Fig. 1

Quiescent endothelial cells (ECs) secrete factors that suppress cancer cell proliferation. (A) Growth of MDA-MB-231 breast and A549 lung carcinoma cells for 4 days in unconditioned (control) or EC-conditioned media. (B) Expression of proliferating cell nuclear antigen (PCNA) protein in cancer cells by Western blot. (C) Ki-67 nuclear expression via immunofluorescence staining in the same groups. *P < 0.05 versus control by t test. Error bars show SEM.

Fig. 2

Quiescent ECs secrete factors that suppress cancer cell invasiveness. (A) Invasiveness of MDA-MB-231 breast and A549 lung carcinoma cells after 4 days of culture in unconditioned (control) or EC-conditioned media. (B) Selected matrix-regulating gene expression (qRT-PCR) of both lines under the same treatment conditions. *P < 0.05 versus control by t test. Error bars show SEM.

Fig. 3

Signaling through protumorigenic and proinflammatory pathways is attenuated when cancer cells are cultured with media conditioned by quiescent ECs. (A) Phosphorylation of S6RP and STAT3β and total expression of NF-κB p65 in MDA-MB-231 and A549 cells after 4 days of culture in EC-conditioned media, with β-actin as a loading control. (B) Nuclear localization of NF-κB p65 by immunofluorescence staining of both cell types. *P < 0.05 versus control by t test. Error bars show SEM.

Fig. 4

EC perlecan expression is required for EC-mediated suppression of cancer cell invasiveness. (A and B) Proliferation (black bars) and invasiveness (white bars) of MDA-MB-231 (A) and A549 (B) cells after 4 days of culture in unconditioned (control) media, media conditioned by normal ECs, and media conditioned by perlecan-silenced ECs (EC_shPerl). *P < 0.05 (black versus control, gray versus EC) by t test. Error bars show SEM.

Fig. 5

Perlecan knockdown abrogates EC suppression of cancer cell invasiveness via increased IL-6 release. (A) Quantification of cytokine arrays showing ratios of different cytokines in perlecan-silenced ECs (EC_shPerl) versus control ECs. (B and C) Effects of IL-6 neutralization (neutralizing anti-body, 50 μg/ml) in media conditioned by ECs and EC_shPerl on the regulation of proliferation (black bars) and invasiveness (white bars) of MDA-MB-231 (B) and A549 (C). *P < 0.05, ⁺P < 0.005, ⁺⁺P < 0.001 by t test (black versus control, gray versus EC media). Error bars show SEM.

Fig. 6

Implantation of matrix-embedded ECs (MEECs) adjacent to xenograft tumors reduces tumor growth and aggressiveness. (A) Schematic of xenograft tumor model with adjacent MEEC implantation. (B) Kinetic growth curves for A549 xenograft tumors in nude mice with control (acellular matrix) or MEEC implants. (C to E) Ki-67 percent nuclear staining (C), cystic mass fraction (D), and p-S6RP percent staining (E) of tumor parenchyma in the above groups. *P < 0.05 versus control group by t test. Error bars show SEM.

Fig. 7

A549 cells cultured in media conditioned by intact ECs, but not perlecan-silenced ECs, were less metastatic than control cells. (A) Increase in lung weights, relative to tumor-free animals, of A549 cells cultured for 4 days in unconditioned (control) media, media conditioned by intact ECs, and media conditioned by perlecan-silenced ECs (EC_shPerl). (B) Metastatic index (see Materials and Methods) of lung cryosections in the above groups. *P < 0.05 versus control group by t test. Error bars show SEM.