Abstract
Multicellular tumor spheroids represent a 3D in vitro model that mimics solid tumor essential properties including assembly and development of extracellular matrix and nutrient, oxygen and proliferation gradients. In the present study, we analyze the impact of 3D spatial organization of HER2-overexpressing breast cancer cells on the response to Trastuzumab. We cultured human mammary adenocarcinoma cell lines as spheroids with the hanging drop method and we observed a gradient of proliferating, quiescent, hypoxic, apoptotic and autophagic cells towards the inner core. This 3D organization decreased Trastuzumab sensitivity of HER2 over-expressing cells compared to monolayer cell cultures. We did not observe apoptosis induced by Trastuzumab but found cell arrest in G0/G1 phase. Moreover, the treatment downregulated the basal apoptosis only found in tumor spheroids, by eliciting protective autophagy. We were able to increase sensitivity to Trastuzumab by autophagy inhibition, thus exposing the interaction between apoptosis and autophagy. We confirmed this result by developing a resistant cell line that was more sensitive to autophagy inhibition than the parental BT474 cells. In summary, the development of Trastuzumab resistance relies on the balance between death and survival mechanisms, characteristic of 3D cell organization. We propose the use of spheroids to further improve the understanding of Trastuzumab antitumor activity and overcome resistance.
Highlights
HER2 is a member of the human epidermal growth factor receptor (HER/ErbB) family of tyrosine kinases which includes EGFR, HER3 and HER4
Since BT474 cells generated regular spheres, we considered diameters as a measure of spheroids growth; on the other hand, as MCF7 spheroids grew irregularly, we chose the area as a measure of growth
Since many reports point that autophagy could play a cytoprotective role countering anti-tumor therapies [18], we investigated if autophagy could be a mechanism involved in BT474 cell escape from apoptosis
Summary
HER2 is a member of the human epidermal growth factor receptor (HER/ErbB) family of tyrosine kinases which includes EGFR, HER3 and HER4. Human breast cancers with overexpression of HER2, occur in about 20% of patients and are associated with poor prognosis [1]. Trastuzumab (Tz, Herceptin), a humanized monoclonal antibody, binds the extracellular region of HER2 and inhibits receptor signaling via several mechanisms [2,3,4]. Even though treatment with Tz is the alternative choice in HER2-positive breast cancer treatment [5], only a PLOS ONE | DOI:10.1371/journal.pone.0137920. Autophagy and Trastuzumab Resistance in Tumor Spheroids Even though treatment with Tz is the alternative choice in HER2-positive breast cancer treatment [5], only a PLOS ONE | DOI:10.1371/journal.pone.0137920 September 11, 2015
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