Neutrophil Depletion Enhanced the Clostridium novyi-NT Therapy in Mouse and Rabbit Tumor Models

Background Hypoxia is a prominent feature of solid tumors and can function as fertile environment for oncolytic anaerobic bacteria such as Clostridium novyi-NT (C. novyi-NT) where it can induce tumor destruction in mice and patients. However, two major obstacles have limited its use, namely the host inflammatory response and the incomplete clearance of normoxic tumor areas. Methods In this study, we first used a subcutaneous tumor model of a glioblastoma (GBM) cell line in immunocompetent mice to investigate the local distribution of tumor hypoxia, kinetics of C.novyi-NT germination and spread, and the local host immune response. We subsequently applied the acquired knowledge to develop a C.novyi-NT therapy in an orthotopic rabbit brain tumor model. Results We found that local accumulation of granular leukocytes, mainly neutrophils, could impede the spread of bacteria through the tumor and prevented complete oncolysis. Depletion of neutrophils via anti-Ly6G antibody or bone marrow suppression using hydroxyurea significantly improved tumor clearance. We then applied this approach to rabbits implanted with an aggressive intracranial brain tumor and achieved long term survival in majority of the animals without apparent toxicity. Conclusion These results indicated that depleting neutrophils can greatly enhance the safety and efficacy of C.novyi-NT cancer therapy for brain tumors.

Paired 18F-Fluorodeoxyglucose (18F-FDG), and 64Cu-Copper(II)-diacetyl-bis(N(4)-methylthiosemicarbazone) (64Cu-ATSM) PET Scans in Dogs with Spontaneous Tumors and Evaluation for Hypoxia-Directed Therapy

Hypoxia is associated with neoplastic tissue, protecting cancer cells from death by irradiation and chemotherapy. Identification of hypoxic volume of tumors could optimize patient selection for hypoxia-directed medical, immunological, and radiation therapies. Clostridium novyi-NT (CNV-NT) is an oncolytic bacterium derived from attenuated wild-type Clostridium novyi spores, which germinates exclusively in the anaerobic core of tumors with low-oxygen content. The hypothesis was that 64Cu-ATSM would localize to regions of hypoxia, and that greater hypoxic volume would result in greater germination of Clostridium novyi-NT (CNV-NT). Tumor-bearing companion dogs were recruited to a veterinary clinical trial. Dogs received a CT scan, 18F-FDG PET scan (74 MBq) and 64Cu-ATSM PET scan (74 MBq). Scan regions of interest were defined as the highest 20% of counts/voxel for each PET scan, and regions with voxels overlapping between the two scans. Maximum standardized uptake value (MaxSUV) and threshold volume were calculated. Direct oximetry was performed in select tumors. Tumor types evaluated included nerve sheath tumor (10), apocrine carcinoma (1), melanoma (3) and oral sarcoma (6). MaxSUVATSM ranged from 0.3–6.6. Measured oxygen tension ranged from 0.05–89.9 mmHg. Inverse of MaxSUVATSM had a linear relationship with oxygen tension (R2 = 0.53, P = 0.0048). Hypoxia <8 mmHg was associated with an SUVATSM > 1.0. Hypoxic volume ranged from 0 to 100% of gross tumor volume (GTV) and MaxSUVATSM was positively correlated with hypoxic volume (R = 0.674; P = 0.0001), but not GTV (P = 0.182). Tumor hypoxic volume was heterogeneous in location and distribution. 64Cu-ATSM-avid regions were associated with differential CT attenuation. Hypoxic volume did not predict CNV-NT germination. 64Cu-ATSM PET scanning predicts hypoxia patterns within spontaneously occurring tumors of dogs as measured by direct oxymetry. Total tumor volume does not accurately predict degree or proportion of tumor hypoxia.

Exploration of the Diversity of Clustered Regularly Interspaced Short Palindromic Repeats-Cas Systems in Clostridium novyi sensu lato

Classified as the genospecies Clostridium novyi sensu lato and distributed into four lineages (I–IV), Clostridium botulinum (group III), Clostridium novyi, and Clostridium haemolyticum are clostridial pathogens that cause animal diseases. Clostridium novyi sensu lato contains a large mobilome consisting of plasmids and circular bacteriophages. Here, we explored clustered regularly interspaced short palindromic repeats (CRISPR) arrays and their associated proteins (Cas) to shed light on the link between evolution of CRISPR-Cas systems and the plasmid and phage composition in a study of 58 Clostridium novyi sensu lato genomes. In 55 of these genomes, types I-B (complete or partial), I-D, II-C, III-B, III-D, or V-U CRISPR-Cas systems were detected in chromosomes as well as in mobile genetic elements (MGEs). Type I-B predominated (67.2%) and was the only CRISPR type detected in the Ia, III, and IV genomic lineages. Putative type V-U CRISPR Cas14a genes were detected in two different cases: next to partial type-IB CRISPR loci on the phage encoding the botulinum neurotoxin (BoNT) in lineage Ia and in 12 lineage II genomes, as part of a putative integrative element related to a phage-inducible chromosomal island (PICI). In the putative PICI, Cas14a was associated with CRISPR arrays and restriction modification (RM) systems as part of an accessory locus. This is the first time a PICI containing such locus has been detected in C. botulinum. Mobilome composition and dynamics were also investigated based on the contents of the CRISPR arrays and the study of spacers. A large proportion of identified protospacers (20.2%) originated from Clostridium novyi sensu lato (p1_Cst, p4_BKT015925, p6_Cst, CWou-2020a, p1_BKT015925, and p2_BKT015925), confirming active exchanges within this genospecies and the key importance of specific MGEs in Clostridium novyi sensu lato.

The oncolytic activity of Clostridium novyi nontoxic spores in breast cancer

Introduction: Hypoxia context is highly specific for tumors and represents a unique niche which is not found elsewhere in the body. Clostridium novyi is an obligate anaerobic bacterium. It has a potential to treat tumors. The aim of this study was to produce the C. novyi nontoxic spores and to investigate its oncolytic effect on breast cancer in mice model. Methods: Primarily, the lethal toxin gene in C. novyi type B was removed. Colonies were isolated using PCR testing. To assure the removal of alpha-toxin, plasmid extraction and in vivo assay were conducted. Next, to treat breast cancer model in different sizes of tumors, a single dose of spores of C. novyi nontoxic was tested. Results: The results denoted that C. novyi nontoxic lost lethal toxin and a­­ppeared to be safe. For smaller than 1000 mm3 tumors, a single dose of C. novyi nontoxic was able to cure 100% of mice bearing breast tumors. Hence the mice remained free of tumor relapse. Tumors larger than 1000 mm3 were not cured by a single dose­ of C. novyi nontoxic treatment. Conclusion: The experiment concluded that the C. novyi nontoxic might be a suitable and safe candidate, a novel therapeutic approach to encounter such hypoxic regions in the center of tumors. Research also showed that bacteriolytic therapy by C. novyi nontoxic could lead to regression in small tumor.

Large Clostridial Toxins: Mechanisms and Roles in Disease

Large clostridial toxins (LCTs) are a family of bacterial exotoxins that infiltrate and destroy target cells. Members of the LCT family include Clostridioides difficile toxins TcdA and TcdB, Paeniclostridium sordellii toxins TcsL and TcsH, Clostridium novyi toxin TcnA, and Clostridium perfringens toxin TpeL.

Methods and Techniques to Facilitate the Development of Clostridium novyi NT as an Effective, Therapeutic Oncolytic Bacteria

The tumor microenvironment is characterized by anomalous vascularization, hypoxia, and acidity at the core of solid tumors that culminates in concentrated necrosis and immune system dysregulation among other effects. While this environment presents several challenges for the development of oncotherapeutics that deliver their activity via the enhanced permeability and retention (EPR) effect of the leaky blood vessels around a tumor, oncolytic bacteria, or a class of bacteria with a noted capacity to lyse solid tumors, are attracted to the very environment found at the center of solid tumors that confounds other therapeutics. It is this capacity that allows for a potent, active penetration from the tumor margins into the core, and subsequent colonization to facilitate lysis and immune reactivation. Clostridium novyi in particular has recently shown great promise in preclinical and clinical trials when administered directly to the tumor. These studies indicate that C. novyi is uniquely poised to effectively accomplish the long sought after “holy grail” of oncotherapeutics: selective tumor localization via intravenous delivery. This study reports the development of efficient methods that facilitate experimental work and therapeutic translation of C. novyi including the ability to work with this obligate micro-anaerobe on the benchtop. Additionally, this study seeks to utilize this newfound experimental flexibility to address several gaps in the current knowledge regarding the efficacy of CRIPSR/Cas9-mediated gene insertion in this species to further develop this oncolytic bacteria and the genetic customization of bacteria in general.