Androgen receptor signaling in circulating tumor cells as a marker of hormonally responsive prostate cancer

Abstract

Androgen deprivation therapy (ADT) is initially effective in treating metastatic prostate cancer, and secondary hormonal therapies are being tested to suppress androgen receptor (AR) reactivation in castration-resistant prostate cancer (CRPC). Despite variable responses to AR pathway inhibitors in CRPC, there are no reliable biomarkers to guide their application. Here, we used microfluidic capture of circulating tumor cells (CTC) to measure AR signaling readouts before and after therapeutic interventions. Single-cell immunofluorescence analysis revealed predominantly "AR-on" CTC signatures in untreated patients, compared with heterogeneous ("AR-on, AR-off, and AR-mixed") CTC populations in patients with CRPC. Initiation of first-line ADT induced a profound switch from "AR-on" to "AR-off" CTCs, whereas secondary hormonal therapy in CRPC resulted in variable responses. Presence of "AR-mixed" CTCs and increasing "AR-on" cells despite treatment with abiraterone acetate were associated with an adverse treatment outcome. Measuring treatment-induced signaling responses within CTCs may help guide therapy in prostate cancer.

Significance: Acquired resistance to first-line hormonal therapy in prostate cancer is heterogeneous in the extent of AR pathway reactivation. Measurement of pre- and posttreatment AR signaling within CTCs may help target such treatments to patients most likely to respond to second-line therapies.

Figure 1

Multiparameter single cell immunofluorescence assay to measure changes in AR activity in cultured prostate cancer cells. (a) Western blot for PSA, PSMA, and alpha-tubulin in LNCaP cells treated with 1 nM R1881 after being cultured under androgen-deprived conditions (left panel), or treated with 10μM bicalutamide after being cultured under standard conditions (right panel). (b) Merged immunofluorescence images of LNCaP cells dual stained with antibodies against PSA (green) and PSMA (red) after treatment with 1 nM R1881 or 10 μM bicalutamide. (c) Pseudocolor density plots of multiparameter immunofluorescence profiles of LNCaP cells treated with 1 nM R1881, imaged using an automated fluorescence microscopy scanning system. X- and y- axes represent “area-pixel” single cell signal intensity measurements for PSMA and PSA, respectively. The total fraction of PSA+/PSMA- (AR-on), PSA-/PSMA+ (AR-off), and PSA+/PSMA+ (AR-mixed) cells is shown in the bar graph. (d) Comparable analysis for LNCaP cells treated with 10 μM bicalutamide after being cultured under standard conditions.

Figure 2

Single cell measurements of AR signaling identify predominantly AR-on CTCs in castration-sensitive prostate cancer versus heterogeneous signatures in castration-resistant prostate cancer. (a) Pseudocolor density plots of multiparameter immunofluorescence profiles of CTCs from patient with castration-sensitive prostate cancer (left panel) and castration-resistant prostate cancer (right panel). X- and y- axes represent “area-pixel” single cell signal intensity measurements for PSMA and PSA, respectively. Representative images are depicted of an “AR-on” (PSA+/PSMA-) CTC (top row on right), an “AR-mixed” (PSA+/PSMA+) CTC (middle row), and an “AR-off” (PSA-/PSMA+) CTC (bottom row), with CD45 (FITC), PSMA (Cy3), and PSA (Cy5). Contaminating CD45+ leukocytes are depicted for comparison in the middle and bottom rows. Scale bars, 10 μm. (b) Bar graphs showing proportional distribution of AR signaling phenotypes in CTCs from patients with CSPC or CRPC prior to initiation of therapy. Patient samples are ordered according to relative percentage of “AR-on” %PSA+ only CTCs. (c) Box plots demonstrating the relative proportions of AR signaling phenotypes in CTCs from patients with CSPC compared to CRPC prior to initiation of therapy (P = 0.012 for %PSA+/PSMA-; P = 0.071 for %PSA+/PSMA+; P = 0.076 for %PSA-/PSMA+).

Figure 3

ADT-induced AR signaling changes in CTCs from patients with castration-sensitive metastatic prostate cancer. (a) Pseudocolor density plots of multiparameter immunofluorescence AR signaling profiles of CTCs in a patient with castration-sensitive prostate cancer before and after ADT with leuprolide showing transformation of CTCs from the “AR-on” (PSA+/PSMA-) phenotype to the “AR-off” (PSA-/PSMA+) phenotype. (b) Bar graphs showing proportional distribution of AR signaling phenotypes in CTCs from this patient before and after ADT. Corresponding CTC numbers and serum PSA levels are shown for pretreatment (pre) and after therapy. (c) Box plots showing composite data for relative proportions of AR signaling phenotypes in CTCs from patients with castration-sensitive prostate cancer (n = 4) pretreatment and after 4 weeks of ADT (P = 0.028 for %PSA+/PSMA-; P = 0.41 for %PSA+/PSMA+; P = 0.64 for %PSA-/PSMA+).

Figure 4

AR signaling in CTCs from CRPC patients treated with abiraterone acetate. (a) Pseudocolor density plots of multiparameter immunofluorescence AR signaling profiles of CTCs in a patient with CRPC, showing a decrease in the proportion of PSA+/PSMA- “AR-on” CTCs after initiation of abiraterone acetate. (b) Bar graphs depicting the results for this patient at serial time points following treatment. Corresponding CTC numbers and serum PSA levels are shown for pretreatment (pre) and at weeks following therapy. (c) Box plots showing composite data for relative proportions of AR signaling phenotypes in CTCs from patients that exhibit stable or declining proportion of “AR-on” CTCs after initiation of therapy (P = 0.56 for %PSA+ only; P = 0.12 for %PSA+/PSMA+; P = 0.14 for %PSMA+ only). (d) Increase in the proportion of PSA+/PSMA- “AR-on” CTCs observed in a patient with CRPC after treatment with abiraterone acetate. (e) Bar graphs depicting the results for this patient. (f) Box plots showing composite data for relative proportions of AR signaling phenotypes in CTCs from patients that exhibit an increasing proportion of “AR-on” CTCs after initiation of therapy (P = 0.67 for %PSA+ only; P = 0.67 for %PSA+/PSMA+; P = 0.67 for %PSMA+ only). (g) Kaplan-Meier curves for overall survival in patients with CRPC treated with abiraterone acetate, according to baseline percentage of >10% “AR-mixed” CTCs (red) versus <10% “AR-mixed” CTCs (blue) (logrank P = 0.048).