Imaging of Lymph Flow in Breast Cancer Patients after Microdose Administration of a Near-Infrared Fluorophore: Feasibility Study

Movies and Supplemental Figures

Movie 1. Dynamic lymph trafficking of IC-Green in one lymph channel in the left breast of patient 21 after four intradermal injections of a total of 10 μg IC-Green with use of conventional 27-gauge needles. The lymph trafficking shows propulsion of lymph at a velocity of 0.08 cm/sec ± 0.03 (SD), with an average time interval of 15 seconds between "packets" of IC-Green moving from the sites of injection to the axilla. One second of playing time is equivalent to 7.76 seconds of real time.

Movie 2. Dynamic lymph trafficking of IC-Green in the left breast of patient 16 after four intradermal injections of a total of 10 μg IC-Green with use of microneedle devices. Initially, the incident laser power was set at Class I and increased to Class IIIb, resulting in a brightening of the fluorescence at the injection sites. To avoid oversaturation, the injection sites were covered (as shown in the movie) and laser power was increased until lymph propulsion can be seen. The lymph trafficking shows propulsion of lymph at an effective velocity of 0.32 cm/sec ± 0.08, with time intervals of from 32 to 94 seconds between packets of IC-Green moving from the sites of injection to the axilla. One second of playing time is equivalent to 9.75 seconds of real time.

Movie 3. Dynamic lymph trafficking of IC-Green within two lymph channels in the left breast of patient 19 after four intradermal injections of a total of 100 μg IC-Green with use of microneedle devices. The lymph trafficking shows propulsion of lymph at a velocity of 0.22 cm/sec ± 0.06, with a mean time interval of 22.9 seconds ± 7.8 between packets of IC-Green moving from the sites of injection to the axilla. The brightest portion of the image shows either a site of superficial lymph pooling or a node. The lymph vessels correspond to the lymph anatomy previously reported in the breast (20). One second of playing time is equivalent to 7.03 seconds of real time.

Figure E1. To test whether the acidic radiocolloid solution influenced the optical properties of IC-Green at a 1 μmol/L concentration, we measured absorption (excitation) spectra and emission spectra by setting the emission monochromator at 830 nm and the excitation monochromator at 780 nm. The pink curve is the absorption spectrum and the red curve is the emission spectrum for 1 μmol/L IC-Green in distilled water; the dark blue curve is the absorption spectrum and the light blue curve is the emission spectrum for 1 μM IC-Green in saline; the light green curve is the absorption spectrum and the dark green curve is the emission spectrum for 1 μM IC-Green constituted in cold 99Tc-sulfur colloid. Owing to the degradation of IC-Green under acidic conditions, the absorption and fluorescence are attenuated by an order of magnitude. To make certain no fluorescence was due to the 99Tc-sulfur colloid, its absorption and emission spectra were measured and found to be negligible. These results show that combining of IC-Green with the radiocolloid caused optical imaging performance to deteriorate. Likewise, particle size analysis of the radiocolloid in the presence or absence of IC-Green remained constant, suggesting that the nuclear imaging results would not be impeded with the IC-Green combination. a.u. = arbitrary units.

Figure E2. Patient 16. (a) White light view; (b–d) NIR fluorescence views at (b) reference time point (c) after 1.95 seconds, and (d) after 4.58 seconds; and (e) lateral scintigram of the left breast. The patient received a total of 10 μg IC-Green in four intradermal periaerolar injections of 2.5 μg IC-Green in 100 μL using the RCS. The stick diagram in a illustrates the breast view in a–d, and the yellow circles in b–d demarcate the trafficking of an IC-Green packet. Movie 2 illustrates the dynamic lymph propulsion from the injection site to the axilla, where the lymph drains into the region corresponding to the axillary lymph nodes shown on the scintigram (e). Lymph trafficking of five IC-Green packets occurred with apparent velocities of 0.32 cm/sec ± 0.08 and a period of 66 seconds ± 44. We noted that the packets traveled in pairs and in a total of eight packets found two distinct time intervals of 32.8 seconds ± 6 and 94 seconds ± 16, with a combined average period of 54 seconds ± 35. Two SLNs were resected from this patient 23 hours after IC-Green and radiocolloid administration; one lymph node was radioactive due to radiocolloid, blue due to isosulfan blue, and fluorescent due to IC-Green accumulation, while the remaining lymph node was not radioactive, blue, or fluorescent. The resection of the latter lymph node was directed by palpation, and the node was found to be positive for cancer. Axillary lymph nodes were subsequently dissected and were found to be not fluorescent.

Figure E3. Patient 19. (a) White light view; (b–d) NIR fluorescence frontal view at (b) reference time point, (c) after 8.43 seconds, and (d) after 10.73 seconds; and (e) frontal scintigram of the right breast. The patient received a total of 80 μg IC-Green in four periareolar intradermal injections of 20 μg of IC-Green in 100 μL with use of the needle with limiter microdelivery device. The stick diagram in a illustrates the breast view in a–d, and the yellow circles in b–d demarcate the trafficking of an IC-Green packet. Two crossing lymph channels are shown to propel IC-Green packets into the axilla, as depicted in b–d. Movie 3 shows packet propulsion occurring at an average velocity of 0.22 cm/sec ± 0.06 and at time intervals of 22.9 seconds ± 7.8. Three fluorescent, negative lymph nodes were resected 5 hours after agent administration; all were radioactive, but none were blue.

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