Abstract 4121: Autophagy influences the development of the pre-metastatic niche

Abstract

Abstract Background: Autophagy is a lysosomal degradation process that is characterized by cellular self-consumption. Autophagy allows for the recycling and removal of damaged cytoplasmic material. Many types of cancer have elevated autophagic activity in order to sustain a high rate of metabolism and circumvent stress-induced cell death. However, little is known of autophagy's role in metastasis. Therefore, we tested the effect of autophagy modulation in different mouse models and cell based assays that reflect different points along the metastatic cascade. Methods: Pharmacologic autophagy inhibition in the murine mammary carcinoma cell line 4T1 was achieved with 10uM chloroquine (CQ) or 1nM bafilomycin A1 (Baf A1). Knockdown of autophagy critical genes Becn1 or Atg7 by lentiviral delivery of shRNA was also used as a means of inhibition. Proliferation, invasion, migration, and anchorage independent growth of cells were assessed in the presence or absence of active autophagy. For an experimental model of metastasis, Balb/c mice were challenged with 4T1 cells expressing luciferase (4T1-luc) injected via the tail vein. Mice were pre-treated with CQ (60 mg/kg, qd, i.p.) and monitored until the development of luciferase positive metastases. Mice were also challenged with 4T1 Becn1 knockdowns or cells pre-treated with CQ for 2h to assess effect of cellular autophagy impairment. An orthotopic model was also utilized by implanting 4T1-luc cells into the mammary fat pad, with tumors resected at 100-200 mm3 and mice followed until metastasis development. CQ was given either adjuvantly, 24h after surgery, or neoadjuvantly, 24h after implantation. Autophagy stimulation, 2% Trehalose in drinking water, was also given as a neoadjuvant therapy. Bone marrow derived cells (BMDCs), mediators of pre-metastatic niche formation, were measured by flow cytometry in the lungs and blood of treated mice prior to the arrival of tumor cells. Results: Autophagy inhibition was able to reduce cell proliferation, but did not significantly delay metastasis in the experimentally induced model. Similarly, CQ was not effective as an adjuvant therapy. Yet, as a neoadjuvant therapy both CQ and Becn1 knockdown were able to significantly delay metastases. Autophagy stimulation actually sped up metastasis development. Autophagy modulation had no effect on invasion, migration, or anchorage independent growth. Conversely, altering autophagy did impact the number of BMDCs present in the lung and blood. Trehalose treated mice had more than either vehicle or CQ. In the blood, CQ treated mice had even less BMDCs present than vehicle treated mice. Conclusions: Autophagy does not seem to be required for metastatic colonization and survival. Nor does it seem to affect the metastatic capability of cells. Rather, autophagy may be influencing the pre-metastatic microenvironment by impacting BMDCs. Thus autophagy appears to be most essential before the arrival of tumor cells to the site of metastasis. Citation Format: Rebecca A. Barnard, Daniel Regan, Ryan J. Hansen, Paola Maycotte, Andrew Thorburn, Daniel L. Gustafson. Autophagy influences the development of the pre-metastatic niche. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4121. doi:10.1158/1538-7445.AM2015-4121