Development of photodynamic therapy regimens that control primary tumor growth and inhibit secondary disease

Abstract

Effective therapy for advanced cancer often requires treatment of both primary tumors and systemic disease that may not be apparent at initial diagnosis. Numerous studies have shown that stimulation of the host immune system can result in the generation of anti-tumor immune responses capable of controlling metastatic tumor growth. Thus, there is interest in the development of combination therapies that both control primary tumor growth and stimulate anti-tumor immunity for control of metastatic disease and subsequent tumor growth. Photodynamic therapy (PDT) is an FDA-approved anticancer modality that has been shown to enhance anti-tumor immunity. Augmentation of anti-tumor immunity by PDT is regimen dependent, and PDT regimens that enhance anti-tumor immunity have been defined. Unfortunately, these regimens have limited ability to control primary tumor growth. Therefore, a two-step combination therapy was devised in which a tumor-controlling PDT regimen was combined with an immune-enhancing PDT regimen. To determine whether the two-step combination therapy enhanced anti-tumor immunity, resistance to subsequent tumor challenge and T cell activation and function was measured. The ability to control distant disease was also determined. The results showed that the novel combination therapy stimulated anti-tumor immunity while retaining the ability to inhibit primary tumor growth of both murine colon (Colon26-HA) and mammary (4T1) carcinomas. The combination therapy resulted in enhanced tumor-specific T cell activation and controlled metastatic tumor growth. These results suggest that PDT may be an effective adjuvant for therapies that fail to stimulate the host anti-tumor immune response.

Fig. 1

Long-term tumor growth controlfollowing combination therapy. Colo26-HA tumor-bearing mice were injected systemically with HPPH (a; 0.4 μmol/kg) or PII (b; 5 mg/kg); 18–24 h later, mice were subjected to PDT at a total dose of 48 J/cm². Nine days later, treated mice were randomly divided into two groups. One group was injected systemically with HPPH (a; 0.4 μmol/kg) or PII (b; 5 mg/kg); tumor growth was monitored in the second group, which represents the immune-enhancing treatment group. Control mice bearing Colo26-HA tumors of equal size to those regrowing in mice that had been previously treated with PDT at a dose of 48 J/cm² were also injected systemically with HPPH or PII; 18–24 h later, control mice were left untreated (HPPH or PII alone) or treated with PDT (132 J/cm²; tumor-controlling regimen). Mice that had been previously treated with PDT at a dose of 48 J/cm² were retreated with PDT at a dose of 132 J/cm² (combination). Each group contained a minimum of eight animals. Tumor growth was followed until tumors reached 400 mm³ or 60 days

Fig. 2

Combination therapy increases long-term tumor growth control. a, b 4T1 tumor-bearing mice were treated with PII-PDT (5 mg/kg PII) doses of 48 or 132 J/cm². Control animals were treated with PII alone. Each group contained a minimum of five animals. Tumors (a) and tumor-draining lymph nodes (TDLN; b) were collected 4 h after the final treatment. Single cell suspensions were generated, and the number of neutrophils (CD45⁺CD11b⁺Ly6C⁺Ly6G^Hi)/gm of tumor (a) or (CD11b⁺ Ly6C⁺Ly6G^Hi)/TDLN (b) was determined. Error bars represent SEM. *P ≤ 0.05; **P ≤ 0.01 (c) 4T1 tumor-bearing mice were injected systemically with PII (5 mg/kg PII); 18–24 h later, mice were subjected to PDT at a total dose of 48 J/cm². Nine days later, treated mice were randomly divided into two groups. One group was injected systemically with PII (5 mg/kg); tumor growth was monitored in the second group, which represents the immune-enhancing treatment group. Control mice bearing 4T1 tumors of equal size to those regrowing in mice that had been previously treated with PDT at a dose of 48 J/cm² were also injected systemically with PII; 18–24 h later, control mice were left untreated (PII alone) or treated with PDT (132 J/cm²; tumor-controlling regimen). Mice that had been previously treated with PDT at a dose of 48 J/cm² were retreated with PDT at a dose of 132 J/cm² (combination). Each group contained a minimum of eight animals. Tumor growth was followed until tumors reached 400 mm³ or 60 days

Fig. 3

Combination therapy increases T cell activation. Colon26-HA tumor-bearing mice were treated with HPPH or HPPH-PDT (0.47 μmoles/kg HPPH) immune-enhancing (48 J/cm²), tumor-controlling (132 J/cm²) or a combination regimen of immune-enhancing treatment followed by tumor-controlling treatment as described in Fig. 1. a TDLNs were collected 24 h after the final treatment, and single cell suspensions were generated. The number of activated CD8⁺ T cells (CD3⁺CD8⁺CD44^HiCD25⁺) was determined by flow cytometry. A minimum of 14 mice were examined in each group. b TDLNs were collected 24 h after the final treatment, and in vivo cytotoxicity assays were performed as described in Materials and Methods. Results are presented as % specific cytotoxicity. Each symbol represents results obtained from an individual mouse; horizontal lines represent the median. A minimum of ten mice were examined per group

Fig. 4

Combination therapy increases resistance to tumor challenge. Colo26-HA or 4T1 tumor-bearing mice were treated with tumor-controlling, immune-enhancing, or the combination HPPH- or PII-PDT regimens, respectively, as described in Figs. 1 and 2. Mice that remained tumor-free 40 days after treatment were challenged with live tumor cells (a: Colon 26 challenge; b: 4T1 challenge). Tumor growth was monitored for 60 days or until tumors reached 400 mm³

Fig. 5

Combination therapy reduces 4T1 lung metastases. 4T1 tumor-bearing BALB/c (a + b) or scid (b) mice were treated with immune-enhancing PDT as described in Fig. 2. After 9 days, treated mice were re-injected with PII (5 mg/kg); 18–24 h later tumors were either surgically removed (Immune Enhancing + SR) or treated with tumor-controlling PDT (combination). Tumors at an equivalent size to those present in mice treated 10 days prior with the immune-enhancing PDT regimen were treated with PII alone, surgically removed (SR) or treated with the tumor-controlling PDT regimen (tumor controlling). Lungs were collected 14 days later, and the number of metastases/lung was determined. a Results represent the number of tumors tumors/mouse. Each symbol represents an individual mouse. The mean is represented by a line, and the error bars represent the SEM. Each group contains a minimum of ten mice. b Results represent the average number of lung tumors/mouse. Error bars represent SEM; each group contains a minimum of ten mice. *P ≤ 0.05, **P ≤ 0.001 when compared to treatment with PII alone; ^# P ≤ 0.05 when compared to BALB/c