Effect of Heat-Inactivated Clostridium sporogenes and Its Conditioned Media on 3-Dimensional Colorectal Cancer Cell Models

Abstract

Traditional cancer treatments, such as chemotherapy and radiation therapy continue to have limited efficacy due to tumor hypoxia. While bacterial cancer therapy has the potential to overcome this problem, it comes with the risk of toxicity and infection. To circumvent these issues, this paper investigates the anti-tumor effects of non-viable bacterial derivatives of Clostridium sporogenes. These non-viable derivatives are heat-inactivated C. sporogenes bacteria (IB) and the secreted bacterial proteins in culture media, known as conditioned media (CM). In this project, the effects of IB and CM on CT26 and HCT116 colorectal cancer cells were examined on a 2-Dimensional (2D) and 3-Dimensional (3D) platform. IB significantly inhibited cell proliferation of CT26 to 6.3% of the control in 72 hours for the 2D monolayer culture. In the 3D spheroid culture, cell proliferation of HCT116 spheroids notably dropped to 26.2%. Similarly the CM also remarkably reduced the cell-proliferation of the CT26 cells to 2.4% and 20% in the 2D and 3D models, respectively. Interestingly the effect of boiled conditioned media (BCM) on the cells in the 3D model was less inhibitory than that of CM. Thus, the inhibitive effect of inactivated C. sporogenes and its conditioned media on colorectal cancer cells is established.

Figure 1. Fluorescence image of cell viability of C. sporogenes after heat inactivation.

DMAO stains bacterial cells green and EthD-III stains the non-viable bacterial cells red. (a) Bacteria before heat-treatment. (b) Bacterial cells after 80 °C heat inactivation for 2 hours. Scale bar represents 100 μm.

Figure 2. Effect of IB on 2D cell culture.

(a) Cell proliferation with WST-1 assay of 2D culture of CT26 cells exposed to varying concentrations of inactivated bacteria. 0.1 OD = 2.4 × 10⁶ bacterial cells/ml. t-test is a comparison with control. (*p < 0.005) (b) Fluorescence microscopy with Calcein AM and Ethidium homodimer I (EthD-I), of cell viability of CT26 cells after they had been exposed to inactivated bacteria at varying concentrations for 24 hours. Image of cells in top left quadrant of each well. Scale bar represents 200 μm.

Figure 3. Effect of CM on 2D cell culture.

(a) Cell proliferation with WST-1 assay of 2D culture of CT26 exposed to 10% of Conditioned Media (CM) of C. sporogenes and Reinforced Clostridium Media (RCM). t-test is a comparison with control. (*p < 0.005) (b) Fluorescence assay of cell viability of CT26 cells after they had been exposed to 10% of RCM and CM for 24 hours. Image of cells in top left quadrant of each well. Scale bar represents 200 μm.

Figure 4. H&E staining of 3D spheroids of CT26, at 72 hours.

Arrows indicate necrotic regions formed within spheroid, stained only with eosin.

Figure 5. Effect of IB on 3D spheroids.

(a) Cell proliferation with WST-1 assay of CT26 and HCT116 spheroids exposed to 0.1 OD concentration of inactivated bacteria over 72 hours. 0.1 OD = 2.4 × 10⁶ bacterial cells/ml. t-test is a comparison with control. (*p < 0.005) (b) Area of 3D spheroids after incubation with 0.1 OD of inactivated bacteria, at the 0, 24, 48 and 72 hour time points. The area (μm²) of the spheroids in the images was measured using ImageJ software. t-test is a comparison with control (*p < 0.005, at all time points). (c) Effect of 0.1 OD of inactivated C. sporogenes on 3D spheroids, compared with Control. Scale bar represents 1000 μm.

Figure 6. Effect of CM on 3D spheroids.

(a) Cell proliferation with WST-1 assay of CT26 and HCT116 spheroids exposed to 10% of Conditioned Media (CM) of C. sporogenes and Reinforced Clostridium Media (RCM). t-test is a comparison with control. (*p < 0.005). (b) Area of spheroids after incubation with 10% RCM, CM and BCM of C. sporogenes at the 0, 24, 48 and 72 hour time points. The area (μm²) of the spheroids in the images was measured using ImageJ. t-test is a comparison with control (*p < 0.005, at all time points). (c) Effect of 10% CM of C. sporogenes on 3D spheroids, compared with Control, 10% RCM and 10% BCM. Scale bar represents 1000 μm.

Figure 7. Comparison between morphology of CT26 Control spheroid and spheroid exposed to IB after 24 hours.

Images taken with scanning electron microscope (SEM). Arrows indicate elongated cells on the surface and the white arrow indicate deformation in the shape of IB-exposed spheroids.