Tumor-specific cell-cycle decoy by Salmonella typhimurium A1-R combined with tumor-selective cell-cycle trap by methioninase overcome tumor intrinsic chemoresistance as visualized by FUCCI imaging

Abstract

We previously reported real-time monitoring of cell cycle dynamics of cancer cells throughout a live tumor intravitally using a fluorescence ubiquitination cell cycle indicator (FUCCI). Approximately 90% of cancer cells in the center and 80% of total cells of an established tumor are in G0/G1 phase. Longitudinal real-time FUCCI imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface and, in contrast, and had little effect on the quiescent cancer cells. Resistant quiescent cancer cells restarted cycling after the cessation of chemotherapy. Thus cytotoxic chemotherapy which targets cells in S/G2/M, is mostly ineffective on solid tumors, but causes toxic side effects on tissues with high fractions of cycling cells, such as hair follicles, bone marrow and the intestinal lining. We have termed this phenomenon tumor intrinsic chemoresistance (TIC). We previously demonstrated that tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) decoyed quiescent cancer cells in tumors to cycle from G0/G1 to S/G2/M demonstrated by FUCCI imaging. We have also previously shown that when cancer cells were treated with recombinant methioninase (rMETase), the cancer cells were selectively trapped in S/G2, shown by cell sorting as well as by FUCCI. In the present study, we show that sequential treatment of FUCCI-expressing stomach cancer MKN45 in vivo with S. typhimurium A1-R to decoy quiescent cancer cells to cycle, with subsequent rMETase to selectively trap the decoyed cancer cells in S/G2 phase, followed by cisplatinum (CDDP) or paclitaxel (PTX) chemotherapy to kill the decoyed and trapped cancer cells completely prevented or regressed tumor growth. These results demonstrate the effectiveness of the praradigm of "decoy, trap and shoot" chemotherapy.

Keywords: FUCCI; Salmonella typhimurium A1-R; cancer; cell-cycle; cisplatinum; decoy; methioninase; nude mice; paclitaxel; stomach cancer; trap.

Figure 1.

S. typhimurium A1-(R) decoyed quiescent cancer cells to cycle. S. typhimurium A1-R targeted quiescent cancer cells and decoyed cell cycle transit from G₀/G₁ to S/G₂/M phases. (A) Representative images of control HeLa-FUCCI cancer cells and HeLa-FUCCI cancer cells in monolayer culture treated with S. typhimurium A1-R. (B) Histogram shows cell cycle distribution in control and S. typhimurium A1-R-treated cultures. Scale bar: 500 mm. (C) S. typhimurium A1-R stimulated cell-cycle transit from G₀/G₁ to S/G₂ phase in quiescent tumor spheres formed from MKN45-FUCCI cells in vitro. Representative images of control tumor spheres and and tumor spheres treated with S. typhimurium A1-R. (D) Histogram shows cell-cycle distribution in control and S. typhimurium A1-R-treated tumor spheres. (E) S. typhimurium A1-R decoyed the cell-cycle transit of quiescent cancer cells in MKN45-FUCCI tumors in vivo. Representative images of cross sections of FUCCI-expressing MKN45 tumor xenografts treated with S. typhimurium A1-R or untreated control. (F) Histograms show the cell-cycle phase distribution of FUCCI-expressing cells within the tumors treated with S. typhimurium A1-R or untreated control. The cells in G₀/G₁, S, or G₂/M phases appear red, yellow, or green, respectively.

Figure 2.

rMETase traps cancer cells in S/G₂ phase. Time-course imaging of HeLa-FUCCI cells treated with rMETase. After seeding on 35 mm glass dishes and culture overnight, HeLa-FUCCI cells were treated with rMETase at a dose of 1.0 unit/ml. (A) Kinetics of rMETase trapping of cells in S/G₂. (B) Maintenance of rMETase trap in S/G₂ over time. All images were acquired with the FV1000 confocal microscope (Olympus, Tokyo, Japan). The cells in G₀/G₁, S, or G₂/M phases appear red, yellow, or green, respectively.

Figure 3.

Decoy, trap and shoot chemotherapy with CDDP. (A) Treatment schedule. FUCCI-expressing MKN45 cells (5 × 10⁶ cells/mouse) were injected subcutaneously into the left flank of nude mice. When the tumors reached approximately 8 mm in diameter (tumor volume, 300 mm³), mice were administered iv S. typhimurium A1-R alone (5 × 10⁷ CFU/mouse, iv, qW × 4); or cisplatinum (CDDP) alone (5 mg/kg, ip, q3d); or S. typhimurium A1-R followed by CDDP; or S. typhimurium A1-R, rMETase (200 units/mouse, ip, q d for 3 d × 4) and CDDP in that order. (B) Macroscopic photographs of FUCCI-expressing tumors: untreated control; S. typhimurium A1-R-treated; CDDP-treated; S. typhimurium A1-R and CDDP-treated; or treated with the sequential combination of S. typhimurium A1-R, rMETase and CDDP. (C) Waterfall plot indicating fold change in tumor volume: untreated control; CDDP-treated; S. typhimurium A1-R-treated; S. typhimurium A1-R and CDDP-treated; or treated with the sequential combination of S. typhimurium A1-R, rMETase and CDDP. (D) Representative images of cross-sections of FUCCI-expressing MKN45 subcutaneous tumors: untreated control; S. typhimurium A1-R-treated; CDDP-treated; S. typhimurium A1-R and CDDP-treated; or treated with the sequential combination of S. typhimurium A1-R, rMETase and CDDP.

Figure 4.

Prolonged administration of rMETase induced mitotic catastrophe after late S/G2 phase blocking. (A) Time-lapse imaging of HeLa-FUCCI cells treated with rMETase. After seeding on 35 mm glass dishes and culture overnight, HeLa-FUCCI cells were treated with rMETase at a dose of 1.0 unit/ml for 80 hours. All images were acquired with the FV1000 confocal microscope (Olympus, Tokyo, Japan). The cells in G₀/G₁, S, or G₂/M phases appear red, yellow, or green, respectively. (B) High magnificent image of A. Arrowheads refer to a cell dying from mitotic catastrophe.

Figure 5.

Decoy, trap and shoot chemotherapy with PTX. A). Treatment schedule. FUCCI-expressing MKN45 cells (5 × 10⁶ cells/mouse) were injected subcutaneously into the left flank of nude mice. When the tumors reached approximately 8 mm in diameter (tumor volume, 300 mm³), mice were administered S. typhimurium A1-R alone (5 × 10⁷ CFU/mouse, iv, qW × 4), or PTX alone (6 mg/kg, ip, q3d × 4); or S. typhimurium A1-R followed by PTX, or S. typhimurium A1-R, rMETase (200 units/mouse, ip, q d for 3 d × 4) and PTX sequentially. (B) Macroscopic photographs of FUCCI-expressing tumors: untreated control; S. typhimurium A1-R-treated; PTX-treated; S. typhimurium A1-R in combination with PTX-treated; or treated with the sequential combination of S. typhimurium A1-R, rMETase and PTX. Scale bars, 10 mm. (C) Waterfall plot indicating fold change in tumor volume: untreated control; PTX-treated; S. typhimurium A1-R-treated; S. typhimurium A1-R in combination with PTX-treated; or treated with the sequential combination of S. typhimurium A1-R, rMETase and PTX. (D) Representative images of cross-sections of FUCCI-expressing MKN45 subcutaneous tumors: untreated control; S. typhimurium A1-R-treated; PTX-treated; S. typhimurium A1-R in combination with PTX-treated; or treated with the sequential combination of S. typhimurium A1-R, rMETase and PTX.