Tamoxifen-Based Chemical Probes
G
Cell Culture and Bioconjugate Incubation
Acknowledgements
R.O.F. thanks Dr Anthony Reeder (CMCA-UWA) for help with mass
spectra acquisition and Dr Gavin Flematti for assistance with HPLC, and is
grateful for financial support from the Australian Research Council
(DE180100112), Cancer Council Western Australia through a Susan
Cavanagh ECI grant, and the University of Western Australia ReEntry
Fellowship. R.A.M. is supported by the Australian Research Council
(CE140100003 and DP150104660), Breast Cancer Research Centre WA,
and a Premier’s Research and Industry Fund grant provided by the South
Australian Government Department for Industry and Skills.
Breast cancer cells (ER positive MCF7 and ER negative
MDA231) were cultured in RPMI media containing 10 % fetal
bovine serum (Life Technologies). Cells were grown at 378C
in a humidifying incubator and then were sub-cultured onto
glass coverslips in sterile six-well plates at a concentration of
4 ꢀ 105 cells mLꢁ1. Sub-confluent cultures were incubated for
one hour in either 4 or 5 diluted in culture media to 10 mg mLꢁ1
.
Prior to imaging, cultures were rinsed and replaced into normal
growth media. Live stained cells were mounted under buffered
saline and visualised using a Nikon A1Si fluorescent confocal
microscope. Sequential fields were captured in the z-plane to
confirm internal cellular localisation.
References
[1] E. A. Specht, E. Braselmann, A. E. Palmer, Annu. Rev. Physiol. 2017,
[2] Y. Fu, N. S. Finney, RSC Adv. 2018, 8, 29051. doi:10.1039/
Imaging Experiments
[3] H.-W. Liu, L. Chen, C. Xu, Z. Li, H. Zhang, X.-B. Zhang, W. Tan,
[4] V. C. Jordan, S. Koerner, Eur. J. Cancer 1975, 11, 205. doi:10.1016/
[5] R. M. O’Regan, V. C. Jordan, Lancet Oncol. 2002, 3, 207. doi:10.1016/
[6] V. C. Jordan, Endocr. Relat. Cancer 2014, 21, R235. doi:10.1530/
[7] Ahmad I. Shagufta, Eur. J. Med. Chem. 2018, 143, 515. doi:10.1016/
The imaging needle was fabricated as described by Scolaro
et al.[24] Briefly, the device consisted of a length of double-clad
fibre (SM-9/105/125-20A, Nufern, USA). The distal end was
terminated with focusing optics fabricated by fusion-splicing a
short section of large-core step-index fibre which served as a
beam-expanding spacer, a section of GRIN fibre (100/125
GIMM, Draka Communications, USA) which functioned as a
lens, and an angle-polished section of no-core fibre to redirect the
light beam perpendicular to the fibre. This was enclosed within a
small glass capillary to maintain a fibre–air interface at the angle
polished surface and ensure total-internal reflection of the light
beam. The fibre was inserted into a 23-gauge stainless steel
needle (outer diameter 640 mm) and aligned such that the light
beam was directed out through a small hole that had been elec-
trochemically etched into the side of the needle, and then glued in
place using optical adhesive. The imaging needle was fusion
spliced to a double-clad fibre coupler (Castor Optics, Canada),
which allowed the OCT light and fluorescence excitation to be
inserted into the imaging needle, and the backscattered OCT
light and fluorescence emission tobe separated upon return. OCT
imaging was performed using a custom-built swept-source
OCTsystem, basedon a 50 kHzrepetitionrate wavelength-swept
laser source (Axsun Technologies Inc., USA). Fluorescence
excitation used 488 nm light from a frequency-doubled semi-
conductor laser (Sapphire SF 488, Coherent Inc., USA). The
fluorescence emission was detected using a photo-multiplier
tube (PMT) (9136B, Electron Tubes, United Kingdom).
[8] P. Y. Maximov, R. E. McDaniel, V. C. Jordan, P. Y. Maximov,
R. E. McDaniel, V. C. Jordan, Tamoxifen: Pioneering Medicine in
Breast Cancer 2013 (Springer: Basel).
[9] E. L. Rickert, S. Oriana, C. Hartman-Frey, X. Long, T. T. Webb,
K. P. Nephew, R. V. Weatherman, Bioconjug. Chem. 2010, 21, 903.
[10] F. Abendroth, M. Solleder, D. Mangoldt, P. Welker, K. Licha,
M. Weber, O. Seitz, Eur. J. Org. Chem. 2015, 2157. doi:10.1002/
[11] L. A. Ho, E. Thomas, R. A. McLaughlin, G. R. Flematti, R. O. Fuller,
[12] T. Yogo, K. Umezawa, M. Kamiya, R. Hino, Y. Urano, Bioconjug.
[13] H. Zhu, J. Fan, J. Du, X. Peng, Acc. Chem. Res. 2016, 49, 2115. doi:10.
[15] P. A. Valde´s, A. Kim, M. Brantsch, C. Niu, Z. B. Moses, T. D.
Tosteson, B. C. Wilson, K. D. Paulsen, D. W. Roberts, B. T. Harris,
[16] M. L. Gabriele, G. Wollstein, H. Ishikawa, L. Kagemann, J. Xu,
L. S. Folio, J. S. Schuman, Invest. Ophthalmol. Vis. Sci. 2011, 52,
During imaging experiments, the imaging needle was
mounted on a two-axis motorised translation stage and posi-
tioned a few hundred micrometres above the coverslips that held
the cell cultures. The needle was scanned parallel to the cover-
slip over a 5 mm ꢀ 5 mm field of view, with 4 mm ꢀ 4 mm
spacing between measurements. OCT and fluorescence mea-
surements were intrinsically co-registered as they were acquired
simultaneously. Data was subsequently processed and visua-
lised using in-house software developed in C11 and Matlab
(MathWorks, USA).
[17] G. J. Tearney, E. Regar, T. Akasaka, T. Adriaenssens, P. Barlis,
H. G. Bezerra, B. Bouma, N. Bruining, J.-m. Cho, S. Chowdhary,
M. A. Costa, R. de Silva, J. Dijkstra, C. Di Mario, D. Dudeck, E. Falk,
M. D. Feldman, P. Fitzgerald, H. Garcia, N. Gonzalo, J. F. Granada,
G. Guagliumi, N. R. Holm, Y. Honda, F. Ikeno, M. Kawasaki,
J. Kochman, L. Koltowski, T. Kubo, T. Kume, H. Kyono, C. C. S. Lam,
G. Lamouche, D. P. Lee, M. B. Leon, A. Maehara, O. Manfrini,
G. S. Mintz, K. Mizuno, M.-a. Morel, S. Nadkarni, H. Okura, H. Otake,
A. Pietrasik, F. Prati, L. Ra¨ber, M. D. Radu, J. Rieber, M. Riga,
A. Rollins, M. Rosenberg, V. Sirbu, P. W. J. C. Serruys, K. Shimada,
T. Shinke, J. Shite, E. Siegel, S. Sonada, M. Suter, S. Takarada,
A. Tanaka, M. Terashima, T. Troels, S. Uemura, G. J. Ughi,
H. M. M. van Beusekom, A. F. W. van der Steen, G.-A. van Es,
G. van Soest, R. Virmani, S. Waxman, N. J. Weissman, G. Weisz,
[18] M. J. Suter, S. K. Nadkarni, G. Weisz, A. Tanaka, F. A. Jaffer, B. E.
Bouma, G. J. Tearney, JACC Cardiovasc. Imaging 2011, 4, 1022.
Supplementary Material
Additional confocal imaging for both the MCF-7 and MDA-231
cell lines as well as characterisation data, including HRMS,
1
HPLC chromatogram, H NMR for compounds 4 and 5 are
available on the Journal’s website.
Conflicts of Interest
The authors declare no conflicts of interest.