Targeted PDT agent eradicates TrkC expressing tumors via photodynamic therapy (PDT)

Abstract

This contribution features a small molecule that binds TrkC (tropomyosin receptor kinase C) receptor that tends to be overexpressed in metastatic breast cancer cells but not in other breast cancer cells. A sensitizer for (1)O2 production conjugated to this structure gives 1-PDT for photodynamic therapy. Isomeric 2-PDT does not bind TrkC and was used as a control throughout; similarly, TrkC- cancer cells were used to calibrate enhanced killing of TrkC+ cells. Ex vivo, 1- and 2-PDT where only cytotoxic when illuminated, and 1-PDT, gave higher cell death for TrkC+ breast cancer cells. A 1 h administration-to-illumination delay gave optimal TrkC+/TrkC--photocytotoxicity, and distribution studies showed the same delay was appropriate in vivo. In Balb/c mice, a maximum tolerated dose of 20 mg/kg was determined for 1-PDT. 1- and 2-PDT (single, 2 or 10 mg/kg doses and one illumination, throughout) had similar effects on implanted TrkC- tumors, and like those of 2-PDT on TrkC+ tumors. In contrast, 1-PDT caused dramatic TrkC+ tumor volume reduction (96% from initial) relative to the TrkC- tumors or 2-PDT in TrkC+ models. Moreover, 71% of the mice treated with 10 mg/kg 1-PDT (n = 7) showed full tumor remission and survived until 90 days with no metastasis to key organs.

Keywords: histochemistry; imaging; metastatic breast cancer; photodynamic therapy (PDT); theranostic; tropomyosin receptor kinase C (TrkC).

Figure 1

Fundamentals of active targeting. (A) mAb conjugates have limited cell permeabilities, but (B) many small molecule conjugates can. (C) Structures of the targeted compounds featured in this work, 1-F and 1-PDT, and the parent iodinated BODIPY, I₂-BODIPY.

Figure 2

Compound 1-F stains in TrkC⁺ tumor tissue and is internalized TrkC+ cells. (A) Histochemical stains for a library of 96 breast tissue slices were performed using 1-F (top) and anti-TrkC antibody as control (bottom), and the three illustrative ones shown here illustrate staining of the malignant tumor, whereas normal tissue is not stained. No staining was observed in the tissues without the small molecule probe or mAb. (B) Cell imaging on 4T1 cells shows 1-F was internalized into lysosomes just as the natural TrkC ligand NT3 is.

Figure 3

1-PDT is photocytotoxic in TrkC+ cell lines. (A) Photocytotoxicities for 1-PDT are more for the following breast cells, murine metastatic 4T1 and human metastatic, HS578t; compared with the following breast cell lines, murine nonmetastatic, 67NR; human immortalized MCF-10A. (B) Photocytotoxicities on the 4T1 and HS578t cells were enhanced for 1-PDT compared to the scramble control 2-PDT featuring an isomer of the targeting fragment that does not adhere to TrkC⁺ cells and control I₂-BODIPY. (C) Structure of 2-PDT. (D) Photocytotoxicities for 1-PDT on 4T1 and HS578t cells are dose dependent (red bars) and suppressed by fixed concentrations of competing: (i) natural ligand NT3 (blue) and (ii) targeting ligand without a PDT group “IY-IY-TEG”. Data shown are mean ± SEM of three independent experiments. *, p < 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 vs control using One-Way ANOVA ((A) TrkC^– cell line, (B) I₂-BODIPY, (D) red bars).

Figure 4

1-PDT was not toxic to mice at 20 mg/kg. Healthy 7–8 weeks old Balb/c female mice were administered intravenously via tail vein respectively with 1-PDT and 2-PDT at 20, 30, and 100 mg/kg (I₂-BODIPY content equivalent to 6.25, 10, and 30 mg/kg, respectively, i.e., corrected for MW), and the parent I₂-BODIPY (30 mg/kg). The mice were then kept in the dark and observed for 16 days. Data represent the average body weight (grams) of two mice/treatment group.

Figure 5

1-PDT demonstrated significant and prolonged accumulation in tumor tissue for up to 6 h and cleared from the body 72 h postadministration. 4T1-tumor bearing female Balb/c mice were treated at 10 mg/kg via the tail vein. Mice (n = 3) were sacrificed at 0, 0.25, 1, 3, 6, 24, 48, 72 h. (A) Organs and tissues (tumor, draining lymph nodes, spleen, kidney, liver, lung, skin, and eye) were harvested, and (B) fluorescence intensities in each organ were imaged using an in vivo imager (data represent mean ± SEM of three mice at each time point). * p < 0.05; ** p < 0.01; for 1-PDT vs 2-PDT.

Figure 6

1-PDT effectively suppressed the growth of TrkC+ (4T1) tumor but not in TrkC– (67NR). (A) Regrowth of TrkC+ 4T1 tumor (yellow arrow) in female Balb/C mice receiving 2-PDT (10 mg/kg), I₂-BODIPY (3.0 mg/kg), and saline controls but not in mice receiving 1-PDT (10 mg/kg). (B) Significant dose dependent mean tumor volume reduction and delayed tumor regrowth in TrkC+ 4T1 tumor bearing mice receiving 2 and 10 mg/kg 1-PDT as compared to rapid tumor growth in mice receiving the control substances. (C) 1-PDT gave impermanent and nonselective antitumor effect (resembled that with 2-PDT) in mice bearing TrkC– 67NR tumor. Photoactivation was conducted at 100 J/cm² with a fluence rate of 0.16 W/cm² 1 h after intravenous injection of the compounds. All graphs showed mean tumor volume ± SEM (n = 7). * p < 0.05, ** p < 0.005, for I₂-BODIPY vs 1-PDT and 2-PDT group using One-Way ANOVA. (D) There were no tumor metastases in 1-PDT treated survivor mice post 90 d. Mice treated with 10 mg/kg 1-PDT that survived up to 90 d with no palpable primary tumor found were metastases free in all the major organs assessed (liver, lung, draining lymph node, and spleen, representative histological images). Control (tumor free healthy and 4T1 tumor burden mice) results were included for comparison (yellow arrow = 4T1 tumor metastases). Scale bar: 100 μm. The current results had been verified by certified veterinary pathologist.