Abstract 4971: Quantitative image analysis of androgen receptor (AR) and tubulin biomarker profiles in circulating tumor cells (CTCs) from metastatic castration resistant prostate cancer (mCRPC) patients
Authors: Worroll Daniel , Galletti Giuseppe , Nanus David M. , Tagawa Scott T. , Giannakakou Paraskevi 
Published: July 2016

Background: CTCs represent a real-time, liquid biopsy for analyses of a variety of molecular biomarkers that offer clinically relevant information. In prostate cancer, the AR pathway controls tumor cell growth and survival and is an important therapeutic target. AR becomes biologically active upon translocation from the cytoplasm to the nucleus in a microtubule dependent fashion, resulting in the activation of numerous genes. Real-time analysis of AR and tubulin status in CTCs may provide insight to therapy response in mCRPC patients.

Methods: Androgen sensitive human prostate cancer cells, LNCaP, were treated with and without a synthetic androgen (R1881; 10 nM, 1 hr) and docetaxel, spiked into healthy donor blood, and captured using a prostate specific microfluidic device (GEDI chip), following the same protocol that we use with patient samples. Cells were fixed and stained (N-terminal AR, CD45, CK, DAPI, and tyr-tubulin) directly on the device and imaged using high resolution multiplex confocal microscopy. AR and tubulin status were analyzed using a quantitative image analysis algorithm. Nuclear AR percentage was calculated by integrating fluorescence intensity within regions defining the entire cell and nucleus. H-Score was calculated by multiplying the nuclear AR percentage by the normalized total cell AR intensity. Tubulin status and microtubule bundling were assessed using a variety of quantitative parameters including measurements of tubulin fluorescence intensity and distribution within the cell.

Results: LNCaP cells in the presence of synthetic androgen exhibited higher nuclear AR percentage (p = 0.0004) and H-score (p = 0.003). Docetaxel treatment led to lower nuclear AR percentage (p = 0.04) and lower H-Score (p = 0.03). Cells treated with docetaxel exhibited higher tubulin intensity range (p = 0.008) and standard deviation (p = 0.03), consistent with docetaxel's mechanism of action. We applied these methods of quantitative image analyses to CTCs isolated from a small cohort of mCRPC patients. Single CTC image analysis revealed heterogeneity in AR status. Quantitative evidence for microtubule bundling was observed in patients receiving docetaxel chemotherapy.

Conclusions: We have developed a high throughput quantitative image analysis algorithm to interrogate mCRPC specific biomarkers, such as AR and tubulin status, in single CTCs. These methods of quantitative image analyses will be prospectively validated with CTCs from mCRPC patients followed longitudinally over the course of treatment with AR inhibitors and taxanes, yielding patient specific, clinically relevant information that can guide physicians’ strategies for the clinical management of cancer.

Citation Format: Daniel Worroll, Giuseppe Galletti, David M. Nanus, Scott T. Tagawa, Paraskevi Giannakakou. Quantitative image analysis of androgen receptor (AR) and tubulin biomarker profiles in circulating tumor cells (CTCs) from metastatic castration resistant prostate cancer (mCRPC) patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4971.