Fig. 1. Cy5-Lys is the major fluorescent LUM015 metabolite. (A) Expected LUM015 cleavage products: Fragment 1 contains the quencher QSY21, and optically active fragments 2 and 3 contain the Cy5 fluorophore. (B) In vitro protease (0.5 μM) activation of LUM015 (5 μM). Fluorescence was measured at multiple time points with peak excitation at 650 nm and fluorescence emission collection at 675 nm (n = 6 replicates per enzyme). (C and D) HPLC analysis of pure LUM015 (25 ng/ml), fragment 2 (25 ng/ml), and fragment 3 (2.5 ng/ml) (C) and a representative patient plasma sample collected from patient 14 at 8 hours after intravenous administration of LUM015 (1.5 mg/kg) (D). (E and F) Plasma profiles of fragment 3 in three patients (E) and in tumor-bearing and non–tumor-bearing mice (n = 6 mice per group) (F) each injected with LUM015 (1.5 mg/kg). P value determined by unpaired t test.
Fig. 2. LUM015 fluorescently labels tumor cells in mouse models of STS and breast cancer. (A and B) Mean measured fluorescence in primary sarcoma (n = 16) (A) or orthotopic breast cancer (n = 23) (B) and muscle. P values determined by unpaired t test. Representative fluorescence images of resected normal muscle and tumors are shown along with corresponding hematoxylin and eosin (H&E) histology. The same contrast scale was applied to both fluorescence images in each pair. Scale bars, 5 mm for fluorescence images; 500 μm for H&E images in (A); 100 μm for H&E images in (B). (C) Representative flow cytometric analysis of resected mouse tumors expressing tumor cell–specific YFP after intravenous administration of phosphate-buffered saline (PBS) or LUM015 (3.5 mg/kg). Of the tumors from mice treated with LUM015, Cy5+ cells (red box, cells with fluorescence >2 × 102) and YFP+ tumor cells (yellow box) were sorted and quantified (nPBS = 2 mice, nLUM015 = 3 mice). In the Cy5+ cells, the proportions of YFP+ tumor cells and CD11b+ tumor–associated macrophages were further quantified (n = 3 mice). P values were determined by unpaired t test. (D) The correlation between residual fluorescence within the sarcoma tumor bed measured before wound closure and local recurrence–free survival in mice. P value determined by log-rank test.
Fig. 3. Comparative LUM015 pharmacokinetics in mouse and human subjects. (A) LUM015 plasma clearance profile in mouse and human subjects administered a 1.5 mg/kg dose of LUM015. [LUM015]plasma is given as a fraction of the maximum concentration and shown on a log scale. (B) Tumor fluorescence in patients and mice at the 6- and 30-hour imaging time points (n = 5 mice and 3 humans per dose cohort). P = 0.02 for imaging time, as determined by two-way analysis of variance (ANOVA). (C) Tumor/normal fluorescence ratio in mice and humans at the 6- and 30-hour imaging time points. P = 0.6 for imaging time, as determined by two-way ANOVA. (D) Tumor/normal fluorescence ratio measured in humans and mice by dose (nmouse-1.5, 3.5 = 5; nhuman-0.5, 1.0 = 3; nhuman-1.5 = 2). P values for dose cohort comparisons determined by Tukey’s multiple comparisons test. P = 0.02 for dose effect on human subset determined by one-way ANOVA.
Fig. 4. Tumor-selective fluorescence in patients receiving intravenous LUM015 before resection. (A to C) Representative human subjects with UPS of the thigh (A, patient 2), myxofibrosarcoma of the thigh (B, patient 12), and IDC of the breast (C, patient 9). From left to right: gadolinium-enhanced magnetic resonance imaging, gross tissue resection containing tumor (T) and normal muscle (M), H&E histology of imaged tumor tissue, fluorescence image of tumor tissue obtained with the LUM device, H&E staining of imaged muscle, fluorescence image of muscle obtained with the LUM device. (D) Tumor tissue fluorescence and fluorescence measured from adjacent normal tissue in the same patient (n = 14). P value determined by paired t test. (E) Distribution of tumor, muscle, and adipose tissue fluorescence across all patients (ntumor = 14, nmuscle = 10, nadipose = 11). P = 0.04 for tissue type effect determined by one-way ANOVA. P values for tumor to muscle and adipose comparison determined by unpaired t test.
Fig. 5. Tumor-selective distribution and activation of LUM015. (A) Tumor fluorescence as a function of [fragment 3]tumor and [fragment 2]tumor (nhuman = 14, nmouse = 16). P values determined by F test on the linear regression model. (B) The FAP was determined using the following equation: ([fragment 2 (nM)]tissue + [fragment 3 (nM)]tissue)/([LUM015 (nM)]tissue + [fragment 2 (nM)]tissue + [fragment 3 (nM)]tissue). FAP data are shown for muscle and tumor tissue samples from human (n = 5, patients 8 and 11 to 14) and mouse (n = 18) STS subjects and from all human subjects imaged at 6 and 30 hours (n = 14). P values determined by unpaired t test. (C) Correlation between the tumor: muscle fluorescence ratio and the FAP ratio in the mouse and human STS cohorts (nhuman = 5, nmouse = 16). P value determined by F test on the linear regression model. (D) The [TP] in the tissue (in nM) was determined using the following equation: [LUM015]tissue + [fragment 2]tissue + [fragment 3]tissue. [TP] data are shown for muscle and tumor tissue samples from human (n = 5, patients 8 and 11 to 14) and mouse (n = 18) STS subjects. P values determined by unpaired t test. (E and F) Tumor fluorescence versus [TP] in the mouse and human STS cohorts (nhuman = 5, nmouse = 16) (E) and in tumor and normal tissue samples from all human subjects (n = 14 per tissue type) (F). P values determined by F test on the linear regression models. (G) Tumor/muscle fluorescence ratios measured in mice 6 hours after injection with equimolar amounts of LUM015 or LUM033 (nLUM015 = 11, nLUM033 = 9). P value determined by unpaired t test.
Fig. 6. Visualizing tumor-selective LUM015 biodistribution. (A) PEG immunofluorescence of human tumor samples from LUM015-injected patients (+LUM015 representative image from patient 2) and from uninjected patients (−LUM015 representative image from n = 3 patients) with UPS. (B) PEG immunofluorescence of margin tissue from LUM015-injected breast cancer patient 15. T, tumor tissue; N, normal breast tissue. The corresponding H&E histology is shown. (C) Immunohistochemistry for PEG in normal muscle tissue, normal breast tissue, and tumor tissue. Representative images of normal muscle and UPS (patient 2) as well as normal breast and IDC (patient 9). (D) Quantification of PEG staining intensity in tumors and normal tissues (n = 14 per tissue type; 1 tumor and 1 normal tissue sample from each patient except patient 10). P value determined by Mann-Whitney test.
- Table 1. Enrolled patients in first-in-human phase 1 clinical trial of LUM015.
Imaging time was measured as the number of hours elapsed between LUM015 injection and imaging of the resected tissues with the LUM device. Histological descriptors are based on clinical surgical pathology reports. ER, estrogen receptor; PR, progesterone receptor; RT, radiation therapy.
Patient LUM015 dose
(mg/kg)Imaging time
(hours)Tumor type Tumor
gradeTumor volume
(cm3)Neoadjuvant
therapyTumor site 1 0.5 30.1 Well-differentiated liposarcoma Low 10.5 × 8.5 × 6.2 None Retroperitoneal 2 0.5 28.5 UPS High 13.5 × 8.3 × 6.5 25 × 2 Gy RT Thigh 3 0.5 28.5 UPS High 18.0 × 9.0 × 6.0 25 × 2 Gy RT Upper arm 4 1.0 30.9 Malignant peripheral nerve
sheath tumorIntermediate 2.5 × 1.6 × 1.5 25 × 2 Gy RT Upper arm 5 1.0 26.9 Myxoinflammatory
fibroblastic
sarcoma/hemosiderotic
fibrolipomatous tumorLow 31.0 × 20.0 × 1.3 None Leg 6 1.0 26.5 UPS High 3.6 × 2.9 × 1.2 None Upper arm 7 1.0 4.8 Metastatic clear cell
sarcomaHigh 4.0 × 2.9 × 2.8 None Inguinal lymph node 8 1.0 7.2 UPS High 13.0 × 6.5 × 7.1 25 × 2 Gy RT Upper arm 9 1.0 8.3 IDC: ER+/PR−/Her2+ High 3.7 None Breast 10 1.5 9.6 IDC: ER+/PR+/Her2− Intermediate 7.5 × 2.5 ×1.5 None Breast 11 1.5 5.1 Metastatic spindle cell
sarcomaLow 0.9 None Thigh 12 1.5 7.3 Myxofibrosarcoma High 11.0 × 6.5 × 6.5 None Thigh 13 0.5 9.6 Synovial sarcoma High 20.0 × 12.0 × 6.0 25 × 2 Gy RT Thigh 14 0.5 8.5 Spindle cell sarcoma High 7.5 25 × 2 Gy RT Thigh 15 0.5 7.3 IDC: ER+, PR+, Her2− High 2.4 × 1.6 × 1.4 None Breast
Supplementary Materials
www.sciencetranslationalmedicine.org/cgi/content/full/8/320/320ra4/DC1
Materials and Methods
Fig. S1. In vitro incubation of LUM015 with mouse tissues.
Fig. S2. The percentage of tumor cells that are Cy5+ after administration of LUM015.
Fig. S3. In vivo detection of residual fluorescence within the mouse tumor bed.
Fig. S4. Summary pharmacokinetic data from mice and humans.
Fig. S5. Contribution of protease-activation and LUM015 distribution for tumor-selective fluorescence.
Table S1. No adverse pharmacological activity of LUM015 in humans.
Table S2. Liver function tests.
Table S3. Ex vivo imaging of human tissues.
Table S4. Correlating tissue fluorescence with metabolite concentration.
References (34–40)
Additional Files
- Supplementary Material for:
A mouse-human phase 1 co-clinical trial of a protease-activated fluorescent probe for imaging cancer
Melodi Javid Whitley, Diana M. Cardona, Alexander L. Lazarides, Ivan Spasojevic, Jorge M. Ferrer, Joan Cahill, Chang-Lung Lee, Matija Snuderl, Dan G. Blazer III, E. Shelley Hwang, Rachel A. Greenup, Paul J. Mosca, Jeffrey K. Mito, Kyle C. Cuneo, Nicole A. Larrier, Erin K. O'Reilly, Richard F. Riedel, William C. Eward, David B. Strasfeld, Dai Fukumura, Rakesh K. Jain, W. David Lee, Linda G. Griffith, Moungi G. Bawendi, David G. Kirsch,* Brian E. Brigman
*Corresponding author. E-mail: david.kirsch{at}duke.edu
Published 6 January 2016, Sci. Transl. Med. 8, 320ra4 (2016)
DOI: 10.1126/scitranslmed.aad0293This PDF file includes:
- Materials and Methods
- Fig. S1. In vitro incubation of LUM015 with mouse tissues.
- Fig. S2. The percentage of tumor cells that are Cy5+ after administration of LUM015.
- Fig. S3. In vivo detection of residual fluorescence within the mouse tumor bed.
- Fig. S4. Summary pharmacokinetic data from mice and humans.
- Fig. S5. Contribution of protease-activation and LUM015 distribution for tumor-selective fluorescence.
- Table S1. No adverse pharmacological activity of LUM015 in humans.
- Table S2. Liver function tests.
- Table S3. Ex vivo imaging of human tissues.
- Table S4. Correlating tissue fluorescence with metabolite concentration.
- References (34–40)
