Immunodominant PstS1 antigen of mycobacterium tuberculosis is a potent biological response modifier for the treatment of bladder cancer

Abstract

Background: Bacillus Calmette Guerin (BCG)-immunotherapy has a well-documented and successful clinical history in the treatment of bladder cancer. However, regularly observed side effects, a certain degree of nonresponders and restriction to superficial cancers remain a major obstacle. Therefore, alternative treatment strategies are intensively being explored. We report a novel approach of using a well defined immunostimulatory component of Mycobacterium tuberculosis for the treatment of bladder cancer. The phosphate transport protein PstS1 which represents the phosphate binding component of a mycobacterial phosphate uptake system is known to be a potent immunostimulatory antigen of M. tuberculosis. This preclinical study was designed to test the potential of recombinant PstS1 to serve as a non-viable and defined immunotherapeutic agent for intravesical bladder cancer therapy.

Methods: Mononuclear cells (PBMCs) were isolated from human peripheral blood and stimulated with PstS1 for seven days. The activation of PBMCs was determined by chromium release assay, IFN-gamma ELISA and measurement of lymphocyte proliferation. The potential of PstS1 to activate monocyte-derived human dendritic cells (DC) was determined by flow cytometric analysis of the marker molecules CD83 and CD86 as well as the release of the cytokines TNF-alpha and IL-12. Survival of presensitized and intravesically treated, tumor-bearing mice was analyzed by Kaplan-Meier curve and log rank test. Local and systemic immune response in PstS1-immunotherapy was investigated by anti-PstS1-specific ELISA, splenocyte proliferation assay and immunohistochemistry.

Results: Our in vitro experiments showed that PstS1 is able to stimulate cytotoxicity, IFN-gamma release and proliferation of PBMCs. Further investigations showed the potential of PstS1 to activate monocyte-derived human dendritic cells (DC). In vivo studies in an orthotopic murine bladder cancer model demonstrated the therapeutic potential of intravesically applied PstS1. Immunohistochemical analysis and splenocyte restimulation assay revealed that local and systemic immune responses were triggered by intravesical PstS1-immunotherapy.

Conclusion: Our results demonstrate profound in vitro activation of human immune cells by recombinant PstS1. In addition, intravesical PstS1 immunotherapy induced strong local and systemic immune responses together with substantial anti-tumor activity in a preclinical mouse model. Thus, we have identified recombinant PstS1 antigen as a potent immunotherapeutic drug for cancer therapy.

Figure 1

Activation of PBMC by PstS1 is dose-dependent. PBMCs were stimulated for 7 days with different PstS1 concentrations in a range from 0.1 μg/ml to 100 μg/ml. A 4-hour chromium release assay against T-24 bladder tumor cells. Effector-target cell ratio of 40:1 B IFN-γ release was determined by ELISA from supernatants of the stimulated PBMCs C PBMC proliferation was measured by overnight ³H-Thymidine incorporation assay (1 μCi/2 × 10⁵ cells). One representative experiment out of 3–6 is shown (mean ± SD). n.d. = not detectable

Figure 2

Time-course of PBMC stimulation. PBMC were stimulated with PstS1 (10μg/ml), BCG (4 × 10⁴ CFU/ml), PHA (2,5 μg/ml) or left unstimulated. Cytotoxicity (A), IFN-γ-production (B) and proliferation (C) of PBMC were measured after 2, 5 and 7 days of stimulation. A Cytotoxicity assay. 4-hour chromium release assay against T-24 bladder tumor cells. Effector-target cell ratio of 40:1. B IFN-γ release measured by ELISA from culture media of PBMC. C PBMC proliferation measured by overnight ³H-Thymidine incorporation assay (1 μCi/2 × 10⁵ cells). For A and B one representative experiment out of 3 is shown (mean ± SD), for C a composite of seven independent experiments is shown (mean ± SEM). For PHA-stimulation one out of two experiments is shown (mean ± SD) . n.d. = not detectable

Figure 3

Activation of human monocyte-derived dendritic cells by PstS1. A Monocytes were differentiated for seven days with GM-CSF and IL-4 (500 U / ml each), stimulated with BCG (MOI = 0.01) or PstS1 (10 μg / ml) for three days and afterwards analyzed by flow cytometry for the expression of CD1a, CD14, CD83 and CD86. Isotype control = filled histograms, specific antibody = open histograms. Cells were gated on intact dendritic cells according to forward scatter and sideward scatter. B Supernatants of the stimulated DCs were collected and ELISAs for TNF-α and IL-12p70 were performed. One representative experiment out of 5 is shown (mean ± SD). n.d. = not detectable.

Figure 4

Intravesical PstS1 immunotherapy in nonsensitized and PstS1-sensitized mice. Ten days before inoculation of MB-49 tumor cells mice were s.c. sensitized by injection of empty L-particles (A/B) or L-particle loaded with PstS1 (C). On days 1, 8, 15 and 22 after tumor challenge, mice were treated by intravesical instillation of PstS1 (A/C) or PBS (B). Mice which received s.c. PBS and intravesical PBS served as control. Survival of mice was analyzed by Kaplan-Meier curve and log rank test.

Figure 5

Intravesical PstS1 treatment after sensitization with PBS, PLG-particles or PLG-particles/BSA. Ten days before inoculation of MB-49 tumor cells mice were s.c. sensitized by injection of PBS (A), L-particles (B) or L-particles loaded with BSA (C). On days 1, 8, 15 and 22 after tumor challenge, mice were treated by intravesical instillation of PstS1 (A/B/C). Mice which received s.c. PBS and intravesical PBS served as control. Survival of mice was analyzed by Kaplan-Meier curve and log rank test.

Figure 6

Splenocyte Restimulation. Treatment of mice was performed as indicated (s.c. sensitization / intravesical instillation). One day after the fourth intravesical PstS1 treatment splenocytes were isolated and restimulated with PBS as control (open circles) or 10 μg/ml PstS1 (filled boxes) for five days and proliferation was analyzed by ³H-Thymidine incorporation assay.