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.

A simple, quantitative assay for the detection of viable but non-culturable (VBNC) bacteria

“…The Molecular Probes Bac Light stains are compatible across numerous bacterial species such as Agrobacterium tumefaciens , Clostridium perfringens , Klebsiella pneumoniae , Pseudomonas aeruginosa , Salmonella oranienburg , Staphylococcus aureus and Streptococcus pyogenes ( Hu et al, 2017 ; Lindbäck et al, 2010 ; Park and Kim, 2018 ; Robertson et al, 2019 ; Molecular Probes, 2004 ). The Biotium stains have been used with E. coli, Leptothrix , Lactobacillus fermentum, Lactobacillus reuteri , Pleurotus osreatus , and Salmonella enterica , ( Adamski and Pietr, 2019 ; Bhave et al, 2015 ; Duplantis et al, 2015 ; Furutani et al, 2011 ; Hernandez et al, 2012 ; Kim et al, 2016 ; Rodes et al, 2013 ; Biotium, 2020 ).…”

Bacteria and cancer: Advances, perspectives and applications

“…Bhave et al [28] observed the cytotoxic effects of Clostridium sporogenes content of the plasma in colorectal cancer cell lines in twodimensional platforms (in vitro specimens as Petri dishes) and three dimensional (in mice) and observed the death of 26.2% of the cell's colorectal cancer of the three-dimensional model in the first 72 hours of application.…”

Rapid screening of engineered microbial therapies in a 3D multicellular model

“…Due to the rapid proliferation rate of bacteria, coculture with mammalian cells has been a challenge, limiting assays to short periods or necessitating the use of heat-killed bacteria (18,19). Previous attempts to control the imbalance in growth rates have fluidically controlled excess bacteria, directly injected bacteria into multicellular aggregates, or used obligate anaerobes to prevent overgrowth (20)(21)(22).…”