Docetaxel facilitates lymphatic-tumor crosstalk to promote lymphangiogenesis and cancer progression

Abstract

Background: Infiltration into lymphatic vessels is a critical step in breast cancer metastasis. Lymphatics undergo changes that facilitate metastasis as a result of activation of the cells lining lymphatic vessels, lymphatic endothelial cells (LECs). Inhibition of activation by targeting VEGFR3 can reduce invasion toward lymphatics. To best benefit patients, this approach should be coupled with standard of care that slows tumor growth, such as chemotherapy. Little is known about how chemotherapies, like docetaxel, may influence lymphatics and conversely, how lymphatics can alter responses to therapy.

Methods: A novel 3D in vitro co-culture model of the human breast tumor microenvironment was employed to examine the contribution of LECs to tumor invasion and viability with docetaxel and anti-VEGFR3, using three cell lines, MDA-MB-231, HCC38, and HCC1806. In vivo, the 4T1 mouse model of breast carcinoma was used to examine the efficacy of combinatorial therapy with docetaxel and anti-VEGFR3 on lymph node metastasis and tumor growth. Lymphangiogenesis in these mice was analyzed by immunohistochemistry and flow cytometry. Luminex analysis was used to measure expression of lymphangiogenic cytokines.

Results: In vitro, tumor cell invasion significantly increased with docetaxel when LECs were present; this effect was attenuated by inhibition of VEGFR3. LECs reduced docetaxel-induced cell death independent of VEGFR3. In vivo, docetaxel significantly increased breast cancer metastasis to the lymph node. Docetaxel and anti-VEGFR3 combination therapy reduced lymph node and lung metastasis in 4T1 and synergized to reduce tumor growth. Docetaxel induced VEGFR3-dependent vessel enlargement, lymphangiogenesis, and expansion of the LEC population in the peritumoral microenvironment, but not tumor-free stroma. Docetaxel caused an upregulation in pro-lymphangiogenic factors including VEGFC and TNF-α in the tumor microenvironment in vivo.

Conclusions: Here we present a counter-therapeutic effect of docetaxel chemotherapy that triggers cancer cells to elicit lymphangiogenesis. In turn, lymphatics reduce cancer response to docetaxel by altering the cytokine milieu in breast cancer. These changes lead to an increase in tumor cell invasion and survival under docetaxel treatment, ultimately reducing docetaxel efficacy. These docetaxel-induced effects can be mitigated by anti-VEGFR3 therapy, resulting in a synergism between these treatments that reduces tumor growth and metastasis.

Keywords: Cancer cell invasion; Docetaxel; Lymphangiogenesis; Taxane chemotherapy; Tissue engineered cell culture models; Triple-negative breast cancer; Tumor microenvironment; Tumor-associated lymphatics; VEGFC/VEGFR3.

Fig. 1

Docetaxel induces invasion of multiple human breast cancer cell lines toward lymphatics in vitro in a VEGFR3-dependent manner. a Schematic of the in vitro tissue engineered model of the tumor-lymphatic interface in the human breast cancer microenvironment. Our model contains mammary stromal fibroblasts and TNBC cells in a collagen I matrix. LECs are seeded along the underside of the insert system through which tumor cells transmigrate from a basal to luminal fashion. Physiologically relevant flow (1 μm/s) is applied via a pressure head of media to yield delivery of docetaxel. Schematic depicts experimental groups. b Fold change in invasion of MDA-MB-231 tumor cells across the porous membrane in our 3D microenvironment system +/− docetaxel treatment (0.1 μM), +/− MAZ51 (1 μM), and/or in the presence or absence of LECs. c Fold change in invasion of HCC38 tumor cells across the porous membrane in our 3D microenvironment system +/− docetaxel treatment (1 μM), +/− MAZ51 (1 μM), and/or in the presence or absence of LECs. d Fold change in invasion of HCC1806 tumor cells across the porous membrane in our 3D microenvironment system +/− docetaxel treatment (0.1 μM), +/− MAZ51 (1 μM), and/or in the presence or absence of LECs. Fold change calculated as compared to no docetaxel/with LEC control. n ≥ 3 biological replicates. *p < 0.05; **p < 0.01

Fig. 2

Blockade of VEGFR3 synergizes with docetaxel to reduce tumor growth and docetaxel-enhanced metastasis in 4T1 breast cancer. a Top panel, Representative images of 4T1 breast tumor cells (red) in the inguinal lymph nodes of mice treated with systemic docetaxel, 8 mg/kg IV (or vehicle control) and/or anti-VEGFR3 antibody, 400 μg total over 2 doses, IP (or control IgG) as detected by histological analysis of RFP-expressing tumor cells. Scale bar = 500 μm. Bottom panel, magnified images from boxed regions in top panel. Dotted white lines outline lymph node border. Scale bar = 100 μm. b Quantification of lymph node metastasis from whole lymph node scans as percent coverage of RFP+ area in whole lymph node sections. (n ≥ 4/group) (c) Tumor volume of treated mice (mm³) of 4T1 mice treated as described above via caliper measurements. Blue dashed arrow indicates dosing of anti-VEGFR3 antibody or control IgG and red dashed arrow indicates dosing of docetaxel or vehicle control. Curve was analyzed by MANOVA (n = 5/group) with *p < 0.05, **p < 0.01

Fig. 3

Lymphatic endothelial cells diminish cancer cell response to docetaxel. a Cancer cell death (% dead cells of total) of MDA-MB-231 cells in 3D microenvironment system +/− docetaxel treatment (10 μM) +/− LECs as measured by live/dead stain in flow cytometry. b Cancer cell death (% dead cells of total) of HCC38 cells in 3D microenvironment system +/- docetaxel treatment (10 μM) +/− LECs. c Cancer cell death (% dead cells of total) of HCC1806 cells in 3D microenvironment system +/- docetaxel treatment (10 μM) +/− LECs. d LEC-conditioned media (LEC CM) was administered to MDA-MB-231, HCC38, or HCC1806 cells followed by 1 μM docetaxel and viability assessed by CCK8 analysis. Results displayed as measured absorbance. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Docetaxel induces VEGFR3-dependent morphological changes of tumor-draining lymphatics in vivo. Representative images of 4T1 tumor-bearing fat pad tissue sections from mice treated as outlined in Fig. 1. Sections were immunostained for lymphatic marker podoplanin (black) and nuclei (green). Left panel (scale bar = 4 mm) shows whole tissue sections and middle panel (scale bar = 500 μm) shows peritumoral stroma. Red dashed lines show border of inguinal lymph node, blue dashed line shows border of tumor, and black arrows indicate lymphatic vessels. Right panel (scale bar = 125 μm) shows representative images of morphology of individual lymphatic vessels

Fig. 5

Docetaxel induces enlargement and expansion of tumor-draining lymphatics in vivo that can be attenuated by VEGFR3 blockade. a Quantified lymphatic vessel perimeter in both tumor-bearing mammary fat pad of treated 4T1 mice and contralateral naïve (non-tumor bearing) fat pad. b Quantified lymphatic vessel area in both tumor-bearing mammary fat pad of treated 4T1 mice and contralateral naïve (non-tumor bearing) fat pad. c Quantified lymphatic vessel density of single podoplanin+ lymphatic vessels per stromal area throughout sections. d Total lymphatic endothelial cells per weight of fat pad (live CD45⁻CD31⁺gp38⁺) as determined by flow cytometry. e Fluid drainage from tumor to axillary lymph node as determined after Evans blue injection into tumor-bearing mammary fat pad 24 h after treatment with docetaxel. (n = 5–9 mice per group). f Quantified lymphatic vessel density displayed as number of peritumoral lymphatic vessels per mm² of stromal tissue after 0, 1, 2, and 3 doses of docetaxel (8 mg/kg, IV, 3 days apart). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

Docetaxel treatment increases expression of pro-lymphangiogenic cytokines in 4T1 tumors. a Heat map representation of expression of chemokines associated with lymphangiogenesis (left) and corresponding known roles in cancer and lymphangiogenesis (right). Results obtained by flow cytometry analysis of 4T1 tumors treated as outlined in Fig. 2 (n = 4/group). Log-transformed data displayed as fold change and heat map generated using MatLab software. (*) indicates analysis by ELISA. Docetaxel abbreviated as DTX and anti-VEGFR3 therapy abbreviated as α-V3 in figure. b Proposed mechanism by which docetaxel results in lymphatic activation and the resulting effect on cancer cell response to therapy. (1) Docetaxel induces production of pro-lymphangiogenic factors in the breast tumor microenvironment. (2) Docetaxel-induced lymphangiogenic factors like VEGFC and TNFa result in VEGFR3-dependent enlargement of lymphatics (brown) and lymphangiogenesis. (3) Docetaxel-activated lymphatics promote VEGFR3-mediated tumor cell (green) invasion and (4) reduce docetaxel efficacy