Rapid differentiation of estrogen receptor status in patient biopsy breast cancer aspirates with an optical nanosensor

Abstract

Breast cancer is a substantial source of morbidity and mortality worldwide. It is particularly more difficult to treat at later stages, and treatment regimens depend heavily on both staging and the molecular subtype of the tumor. However, both detection and molecular analyses rely on standard imaging and histological method, which are costly, time-consuming, and lack necessary sensitivity/specificity. The estrogen receptor (ER) is, along with the progesterone receptor (PR) and human epidermal growth factor (HER-2), among the primary molecular markers which inform treatment. Patients who are negative for all three markers (triple negative breast cancer, TNBC), have fewer treatment options and a poorer prognosis. Therapeutics for ER+ patients are effective at preventing disease progression, though it is necessary to improve the speed of subtyping and distribution of rapid detection methods. In this work, we designed a near-infrared optical nanosensor using single-walled carbon nanotubes (SWCNT) as the transducer and an anti-ERα antibody as the recognition element. The nanosensor was evaluated for its response to recombinant ERα in buffer and serum prior to evaluation with ER- and ER+ immortal cell lines. We then used a minimal volume of just 10 μL from 26 breast cancer biopsy samples which were aspirated to mimic fine needle aspirates. 20 samples were ER+, while 6 were ER-, representing 13 unique patients. We evaluated the potential of the nanosensor by investigating several SWCNT chiralities through direct incubation or fractionation deployment methods. We found that the nanosensor can differentiate ER- from ER+ patient biopsies through a shift in its center wavelength upon sample addition. This was true regardless of which of the three SWCNT chiralities we observed. Receiver operating characteristic area under the curve analyses determined that the strongest classifier with an AUC of 0.94 was the (7,5) chirality after direct incubation and measurement, and without further processing. We anticipate that further testing and development of this nanosensor may push its utility toward field-deployable, rapid ER subtyping with potential for additional molecular marker profiling.

Figure 1:. Nanosensor scheme and synthesis

(A) Schematic of the ERα antibody-based NIR fluorescent nanosensor detection concept. (B) NIR fluorescent spectra of the construct nanosensor in PBS. Success of the antibody conjugation to base construct was assessed by (C) comparison of decay in correlation coefficient as a function of time for SWCNT-(TAT)₆-NH₂ and ERα antibody (Ab) conjugated to SWCNT-(TAT)₆, (D) Change in zeta potential for ssDNA-SWCNT compared to the ERα nanosensor. Difference in means = 9.1 mV, p = 0.006.

Figure 2:. ERα nanosensor response in vitro.

(A) Response of the nanosensor (7,6) chirality to 250 nM recombinant ERα in PBS. Difference in means = 3.5 nm, p = 0.004. (B) Response of the nanosensor (7,6) chirality to 250 nM recombinant ERα in 10% FBS. Difference in means = 1 nm, p = 0.002. (C) Change in center wavelength of the (7,5) chirality for the nanosensor incubated with ER− or ER+ cells. Difference in means = 5.5 nm, p = 0.04).

Figure 3:. Nanosensor response to aspirated patient biopsy samples.

(A) Schematic of direct nanosensor response measurement to ER+ breast cancer patient cells. (B) Change in center wavelength of the nanosensor (7,5) chirality after incubation with ER− or ER+ breast cancer biopsy aspirates. Difference in means = 2.3 nm, p = 0.0011. (C) Change in center wavelength of the nanosensor (7,6) chirality after incubation with ER− or ER+ breast cancer biopsy aspirates. Difference in means = 1.6 nm, p = 0.0002. (D) Change in center wavelength of the nanosensor (9,4) chirality after incubation with ER− or ER+ breast cancer biopsy aspirates. Difference in means = 1.2 nm, p = 0.0001. (E) Receiver operating characteristic evaluation of the ability of each chirality to differentiate ER− from ER+ biopsy samples. AUC is area under the curve. p(7,5) = 0.0020, (7,6) = 0.017, (9,4) = 0.0029.

Figure 4:. Comparison of fractionation method versus direct measurement.

(A) Schematic representing the fractionation method to measure the change in center wavelength for bound vs. unbound nanosensor. (B) Change in center wavelength of the nanosensor each chirality of the “Washed Bound” fraction. Only the (7,6) chirality (center) exhibited a significant change. Difference in the means for this sample = 3.1 nm, p = 0.025. (C) Change in center wavelength of the nanosensor each chirality of the “Washed Unbound” fraction. No differences were statistically significant. (D) ROC comparison of the three measurements (from Figure 3 & 4) and the ability of each chirality to differentiate ER− from ER+ cells. Only the Unwashed method was statistically significant (please refer to Figure 3 for AUC and p values for each).