Koji Morimoto

Pharmacokinetics of pro-FTY and its effect on lymphocytes and tumor suppression in mice. A, BALB/c and control (Ct) mice implanted with the 4T1 breast cancer cell line were treated three times with FTY720 and pro-FTY, and tumor volumes were measured. B, pStat3 expression in representative tumors from BALB/c mice administered the 4T1 breast cancer cell line after treatment with FTY720 or pro-FTY. Scale bars, 25 μm. C and D, Pro-FTY, FTY720, and FTY-P levels in tumor and plasma samples from BALB/c mice were quantified by mass spectrometry on d1 (C) and d3 (D) after the last pro-FTY drug administration. E–N, Blood counts were performed the day after the last dose of drug was administered: white blood cells (WBC; E), red blood cells (RBC; F), hemoglobin (HGB; G), hematocrit (HCT; H), platelets (PLT; I), lymphocytes (LYM; J), neutrophils (NEU; K), eosinophils (EOS; L), basophils (BAS; M), and monocytes (MON; N). O, CD4+ and CD8+ lymphocyte abundances were measured by flow cytometry. P, Serum IL-6 levels were determined by performing ELISAs. Q and R, With the PDX mouse model, the effect of pro-FTY was evaluated by measuring tumor volumes (Q) over time and tumor weights (R) when the mice were euthanized. *, P < 0.05 vs. control. S, Body weights of the mice in each group during the treatment period.

Dose–response curves for the indicated drugs in multidrug-resistant breast cancer cell lines. A–H, Dose–response curves and IC50 values of doxorubicin (A and B), paclitaxel (C and D), FTY720 (E and F), and pro-FTY (G and H) were determined in MCF-7 and MCF-7R cells (A and C–E) and MDA-MB-231 and MDA-MB-231R cells (B, D, F, and H) by performing WST-8 assays. ND, not determined.

Pro-FTY, a novel anticancer prodrug that is converted to its active form (FTY720) after reacting with acrolein, which is overproduced in breast cancer cells. A, Synthesis of pro-FTY. FTY720 was reacted with compound 1 to synthesize pro-FTY. B, Schematic representation of the reaction of pro-FTY with acrolein in cancer cells to produce the active drug, FTY720, which can block S1P signaling. C, Acrolein expression in breast cancer tissue and normal breast tissue. Fluorescence microscopy images of acrolein expression in cancer (c) tissue and normal (n) breast tissue labeled with the CTS probe (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 100 nm. D–G, Dose–response bar graphs for each drug in breast cancer and normal mammary cell lines. The IC50 values of FTY720 (D) and pro-FTY (E) were determined using the MDA-MB-468 breast cancer cell line by performing WST-8 assays, and the IC50 values of FTY720 (F) and pro-FTY (G) were determined in the noncancer, mammary epithelial cell line, MCF-10A.

Effects of pro-FTY and other drugs on PDOs. A, PDOs stained with hematoxylin and eosin (H&E), the CTS probe (red), and 4′,6-diamidino-2-phenylindole (DAPI; blue), along with a merged image (merge). Scale bars, 100 µm. B–M, Effects of doxorubicin (B–D), paclitaxel (E and F), FTY720 (H–J), and pro-FTY (K–M) were determined with PDOs 1–3 by performing CellTiter-Glo 3D cell viability assays. PDOs 1 and 2 were generated from patients with treatment-naïve breast cancer, and PDO 3 was generated from a biopsy specimen from a patient acquired multidrug resistance by the end of treatment.

Dose–response curves for each drug in 10 breast cancer cell lines. A–D, Dose–response curves and IC50 values for doxorubicin (A), paclitaxel (B), FTY720 (C), and pro-FTY (D) were determined in each cell line by performing WST-8 assays.

We developed a novel FTY720 prodrug (pro-FTY) that specifically inhibits sphingosine-1-phosphate signaling in cancer cells using a novel drug delivery system that reacts with acrolein. Our objective was to evaluate the efficacy and safety of pro-FTY in preclinical experiments. Ten breast cancer cell lines, two multidrug-resistant cell lines, and one normal mammary cell line were used to compare the IC50 values of pro-FTY with those of other drugs. Patient-derived organoids (PDO) were established and utilized for IC50 value comparisons. Drug efficacy was tested in mice bearing either syngeneic 4T1 cell tumors or patient-derived xenograft tumors, and blood analysis (including mass spectrometry) was performed. FTY720 and pro-FTY inhibited the survival of all breast cancer cell lines, including multidrug-resistant cells resistant to paclitaxel or doxorubicin. Unlike pro-FTY, FTY720 inhibited the survival of normal breast cell lines, suggesting that pro-FTY does not affect normal breast cells. Pro-FTY showed reproducible activity against multidrug-resistant PDOs, whereas paclitaxel and doxorubicin did not. Mass spectrometric analysis of pro-FTY–treated mice showed that FTY720 accumulated in tumors but was barely detectable in blood. Importantly, lymphocytopenia occurred in FTY720-treated mice but not in pro-FTY–treated mice. Furthermore, intravenous pro-FTY treatment significantly suppressed tumor growth in mice bearing patient-derived xenograft tumors generated from multidrug-resistant PDOs. In conclusion, pro-FTY inhibited breast cancer, including multidrug-resistant breast cancer, while avoiding lymphocytopenia, highlighting its clinical potential.Significance:Pro-FTY selectively inhibits sphingosine-1-phosphate signaling in cancer cells using a novel acrolein-responsive drug delivery system that reacts with acrolein. Pro-FTY does not inhibit normal cell growth, thus avoiding lymphocytopenia. Pro-FTY is effective against multidrug-resistant breast cancer with a unique mechanism of action, highlighting its translational and therapeutic potential.

Hematoxylin and eosin (HE)-stained specimens of heart, kidney, liver, lung, and spleen in control mice, and FTY720 or pro-FTY-treated mice.

We developed a novel FTY720 prodrug (pro-FTY) that specifically inhibits sphingosine-1-phosphate signaling in cancer cells using a novel drug delivery system that reacts with acrolein. Our objective was to evaluate the efficacy and safety of pro-FTY in preclinical experiments. Ten breast cancer cell lines, two multidrug-resistant cell lines, and one normal mammary cell line were used to compare the IC50 values of pro-FTY with those of other drugs. Patient-derived organoids (PDO) were established and utilized for IC50 value comparisons. Drug efficacy was tested in mice bearing either syngeneic 4T1 cell tumors or patient-derived xenograft tumors, and blood analysis (including mass spectrometry) was performed. FTY720 and pro-FTY inhibited the survival of all breast cancer cell lines, including multidrug-resistant cells resistant to paclitaxel or doxorubicin. Unlike pro-FTY, FTY720 inhibited the survival of normal breast cell lines, suggesting that pro-FTY does not affect normal breast cells. Pro-FTY showed reproducible activity against multidrug-resistant PDOs, whereas paclitaxel and doxorubicin did not. Mass spectrometric analysis of pro-FTY–treated mice showed that FTY720 accumulated in tumors but was barely detectable in blood. Importantly, lymphocytopenia occurred in FTY720-treated mice but not in pro-FTY–treated mice. Furthermore, intravenous pro-FTY treatment significantly suppressed tumor growth in mice bearing patient-derived xenograft tumors generated from multidrug-resistant PDOs. In conclusion, pro-FTY inhibited breast cancer, including multidrug-resistant breast cancer, while avoiding lymphocytopenia, highlighting its clinical potential. Significance: Pro-FTY selectively inhibits sphingosine-1-phosphate signaling in cancer cells using a novel acrolein-responsive drug delivery system that reacts with acrolein. Pro-FTY does not inhibit normal cell growth, thus avoiding lymphocytopenia. Pro-FTY is effective against multidrug-resistant breast cancer with a unique mechanism of action, highlighting its translational and therapeutic potential.