Enhanced bacterial cancer therapy delivering therapeutic RNA interference of c-Myc.

BACKGROUND: Bacterial cancer therapy was first trialled in patients at the end of the nineteenth century. More recently, tumour-targeting bacteria have been harnessed to deliver plasmid-expressed therapeutic interfering RNA to a range of solid tumours. A major limitation to clinical translation of this is the short-term nature of RNA interference in vivo due to plasmid instability. To overcome this, we sought to develop tumour-targeting attenuated bacteria that stably express shRNA by virtue of integration of an expression cassette within the bacterial chromosome and demonstrate therapeutic efficacy in vitro and in vivo. RESULTS: The attenuated tumour targeting Salmonella typhimurium SL7207 strain was modified to carry chromosomally integrated shRNA expression cassettes at the xylA locus. The colorectal cancer cell lines SW480, HCT116 and breast cancer cell line MCF7 were used to demonstrate the ability of these modified strains to perform intracellular infection and deliver effective RNA and protein knockdown of the target gene c-Myc. In vivo therapeutic efficacy was demonstrated using the Lgr5creERT2Apcflx/flx and BlgCreBrca2flx/flp53flx/flx orthotopic immunocompetent mouse models of colorectal and breast cancer, respectively. In vitro co-cultures of breast and colorectal cancer cell lines with modified SL7207 demonstrated a significant 50-95% (P < 0.01) reduction in RNA and protein expression with SL7207/c-Myc targeted strains. In vivo, following establishment of tumour tissue, a single intra-peritoneal administration of 1 × 106 CFU of SL7207/c-Myc was sufficient to permit tumour colonisation and significantly extend survival with no overt toxicity in control animals. CONCLUSIONS: In summary we have demonstrated that tumour tropic bacteria can be modified to safely deliver therapeutic levels of gene knockdown. This technology has the potential to specifically target primary and secondary solid tumours with personalised therapeutic payloads, providing new multi-cancer detection and treatment options with minimal off-target effects. Further understanding of the tropism mechanisms and impact on host immunity and microbiome is required to progress to clinical translation.

Characterization of peritoneal reactive ascites collected from acute appendicitis and small bowel obstruction patients.

BACKGROUND: Pathological abdominal adhesions can cause bowel obstructions. A history of appendectomy (appy) increases patient rehospitalization risk directly related to adhesions. To potentially identify strategies for adhesion treatment, we characterized reactive ascites (rA) collected during appy or adhesiolysis for small bowel obstruction (SBO). METHODS: This is a non-randomized, prospective observational study recruiting patients with non-perforated appendicitis or SBO from three Level 1 trauma centers in the United States. rA were analyzed via liquid chromatography-mass spectrometry (LC-MS) (n = 31), bead-based quantification cytokines and chemokines (n = 32) and soluble receptors (n = 30), and LC-MS metabolomics (n = 18). RESULTS: LC-MS showed that samples contained albumin, apolipoprotein A1, and transthyretin and that metabolites increased in SBO vs appy rA were biomarkers of oxidative stress. Multi-plex analyses showed levels of 17 cytokines/chemokines and 6 soluble receptors were significantly different in appy vs SBO rA. Top increased proteins in appy compared to SBO rA by 20.14-, 11.53-, and 8.18-fold were granulocyte-colony stimulating factor, C-X-C motif chemokine ligand 10, and interleukin-10, respectively. CONCLUSIONS: These data further define pro- and anti-inflammatory mediators and metabolites that may drive formation or perpetuate chronic abdominal adhesions. Future research is to further explore whether attenuation of these factors may decrease pathologic adhesion formation.

C1 esterase inhibitor-mediated immunosuppression in COVID-19: Friend or foe?

From asymptomatic to severe, SARS-CoV-2, causative agent of COVID-19, elicits varying disease severities. Moreover, understanding innate and adaptive immune responses to SARS-CoV-2 is imperative since variants such as Omicron negatively impact adaptive antibody neutralization. Severe COVID-19 is, in part, associated with aberrant activation of complement and Factor XII (FXIIa), initiator of contact system activation. Paradoxically, a protein that inhibits the three known pathways of complement activation and FXIIa, C1 esterase inhibitor (C1-INH), is increased in COVID-19 patient plasma and is associated with disease severity. Here we review the role of C1-INH in the regulation of innate and adaptive immune responses. Additionally, we contextualize regulation of C1-INH and SERPING1, the gene encoding C1-INH, by other pathogens and SARS viruses and propose that viral proteins bind to C1-INH to inhibit its function in severe COVID-19. Finally, we review the current clinical trials and published results of exogenous C1-INH treatment in COVID-19 patients.

Induction of ADAM10 by Radiation Therapy Drives Fibrosis, Resistance, and Epithelial-to-Mesenchyal Transition in Pancreatic Cancer.

Stromal fibrosis activates prosurvival and proepithelial-to-mesenchymal transition (EMT) pathways in pancreatic ductal adenocarcinoma (PDAC). In patient tumors treated with neoadjuvant stereotactic body radiation therapy (SBRT), we found upregulation of fibrosis, extracellular matrix (ECM), and EMT gene signatures, which can drive therapeutic resistance and tumor invasion. Molecular, functional, and translational analysis identified two cell-surface proteins, a disintegrin and metalloprotease 10 (ADAM10) and ephrinB2, as drivers of fibrosis and tumor progression after radiation therapy (RT). RT resulted in increased ADAM10 expression in tumor cells, leading to cleavage of ephrinB2, which was also detected in plasma. Pharmacologic or genetic targeting of ADAM10 decreased RT-induced fibrosis and tissue tension, tumor cell migration, and invasion, sensitizing orthotopic tumors to radiation killing and prolonging mouse survival. Inhibition of ADAM10 and genetic ablation of ephrinB2 in fibroblasts reduced the metastatic potential of tumor cells after RT. Stimulation of tumor cells with ephrinB2 FC protein reversed the reduction in tumor cell invasion with ADAM10 ablation. These findings represent a model of PDAC adaptation that explains resistance and metastasis after RT and identifies a targetable pathway to enhance RT efficacy. SIGNIFICANCE: Targeting a previously unidentified adaptive resistance mechanism to radiation therapy in PDAC tumors in combination with radiation therapy could increase survival of the 40% of PDAC patients with locally advanced disease.See related commentary by Garcia Garcia et al., p. 3158 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3255/F1.large.jpg.

Requirement of zebrafish pcdh10a and pcdh10b in melanocyte precursor migration.

Melanocytes derive from neural crest cells, which are a highly migratory population of cells that play an important role in pigmentation of the skin and epidermal appendages. In most vertebrates, melanocyte precursor cells migrate solely along the dorsolateral pathway to populate the skin. However, zebrafish melanocyte precursors also migrate along the ventromedial pathway, in route to the yolk, where they interact with other neural crest derivative populations. Here, we demonstrate the requirement for zebrafish paralogs pcdh10a and pcdh10b in zebrafish melanocyte precursor migration. pcdh10a and pcdh10b are expressed in a subset of melanocyte precursor and somatic cells respectively, and knockdown and TALEN mediated gene disruption of pcdh10a results in aberrant migration of melanocyte precursors resulting in fully melanized melanocytes that differentiate precociously in the ventromedial pathway. Live cell imaging analysis demonstrates that loss of pchd10a results in a reduction of directed cell migration of melanocyte precursors, caused by both increased adhesion and a loss of cell-cell contact with other migratory neural crest cells. Also, we determined that the paralog pcdh10b is upregulated and can compensate for the genetic loss of pcdh10a. Disruption of pcdh10b alone by CRISPR mutagenesis results in somite defects, while the loss of both paralogs results in enhanced migratory melanocyte precursor phenotype and embryonic lethality. These results reveal a novel role for pcdh10a and pcdh10b in zebrafish melanocyte precursor migration and suggest that pcdh10 paralogs potentially interact for proper transient migration along the ventromedial pathway.

Vertical Growth Phase as a Prognostic Factor for Sentinel Lymph Node Positivity in Thin Melanomas: A Systematic Review and Meta-Analysis.

BACKGROUND: The 2010 American Joint Committee on Cancer guidelines recommended consideration of sentinel lymph node biopsy for thin melanoma (Breslow thickness <1.0 mm) with aggressive pathologic features such as ulceration and/or high mitotic rate. The therapeutic benefit of biopsy-based treatment remains controversial. The authors conducted a meta-analysis to estimate the risk and outcomes of sentinel lymph node positivity in thin melanoma, and examined established and potential novel predictors of positivity. METHODS: Three databases were searched by two independent reviewers for sentinel lymph node positivity in patients with thin melanoma. Study heterogeneity, publication bias, and quality were assessed. Data collected included age, sex, Breslow thickness, mitotic rate, ulceration, regression, Clark level, tumor-infiltrating lymphocytes, and vertical growth phase. Positivity was estimated using a random effects model. Association of positivity and clinicopathologic features was investigated using meta-regression. RESULTS: Ninety-three studies were identified representing 35,276 patients with thin melanoma who underwent sentinel lymph node biopsy. Of these patients, 952 had a positive sentinel lymph node biopsy, for an event rate of 5.1 percent (95 percent CI, 4.1 to 6.3 percent). Significant associations were identified between positivity and Breslow thickness greater than 0.75 mm but less than 1.0 mm, mitotic rate, ulceration, and Clark level greater than IV. Seven studies reported on vertical growth phase, which was strongly associated with positivity (OR, 4.3; 95 percent CI, 2.5 to 7.7). CONCLUSIONS: To date, this is the largest meta-analysis to examine predictors of sentinel lymph node biopsy positivity in patients with thin melanoma. Vertical growth phase had a strong association with biopsy positivity, providing support for its inclusion in standardized pathologic reporting.

Brachyury identifies a class of enteroendocrine cells in normal human intestinal crypts and colorectal cancer.

Normal homeostasis of adult intestinal epithelium and repair following tissue damage is maintained by a balance of stem and differentiated cells, many of which are still only poorly characterised. Enteroendocrine cells of the gut are a small population of differentiated, secretory cells that are critical for integrating nutrient sensing with metabolic responses, dispersed amongst other epithelial cells. Recent evidence suggests that sub-sets of secretory enteroendocrine cells can act as reserve stem cells. Given the link between cells with stem-like properties and cancer, it is important that we identify factors that might provide a bridge between the two. Here, we identify a sub-set of chromogranin A-positive enteroendocrine cells that are positive for the developmental and cancer-associated transcription factor Brachyury in normal human small intestinal and colonic crypts. Whilst chromogranin A-positive enteroendocrine cells are also Brachyury-positive in colorectal tumours, expression of Brachyury becomes more diffuse in these samples, suggesting a more widespread function in cancer. The finding of the developmental transcription factor Brachyury in normal adult human intestinal crypts may extend the functional complexity of enteroendocrine cells and serves as a platform for assessment of the molecular processes of intestinal homeostasis that underpins our understanding of human health, cancer and aging.

Cdon promotes neural crest migration by regulating N-cadherin localization.

Neural crest cells (NCCs) are essential embryonic progenitor cells that are unique to vertebrates and form a remarkably complex and coordinated system of highly motile cells. Migration of NCCs occurs along specific pathways within the embryo in response to both environmental cues and cell-cell interactions within the neural crest population. Here, we demonstrate a novel role for the putative Sonic hedgehog (Shh) receptor and cell adhesion regulator, cdon, in zebrafish neural crest migration. cdon is expressed in developing premigratory NCCs but is downregulated once the cells become migratory. Knockdown of cdon results in aberrant migration of trunk NCCs: crestin positive cells can emigrate out of the neural tube but stall shortly after the initiation of migration. Live cell imaging analysis demonstrates reduced directedness of migration, increased velocity and mispositioned cell protrusions. In addition, transplantation analysis suggests that cdon is required cell-autonomously for directed NCC migration in the trunk. Interestingly, N-cadherin is mislocalized following cdon knockdown suggesting that the role of cdon in NCCs is to regulate N-cadherin localization. Our results reveal a novel role for cdon in zebrafish neural crest migration, and suggest a mechanism by which Cdon is required to localize N-cadherin to the cell membrane in migratory NCCs for directed migration.

Brachyury regulates proliferation of cancer cells via a p27Kip1-dependent pathway.

The T-box transcription factor Brachyury is expressed in a number of tumour types and has been demonstrated to have cancer inducing properties. To date, it has been linked to cancer associated induction of epithelial to mesenchymal transition, tumour metastasis and expression of markers for cancer stem-like cells. Taken together, these findings indicate that Brachyury plays an important role in the progression of cancer, although the mechanism through which it functions is poorly understood. Here we show that Brachyury regulates the potential of Brachyury-positive colorectal cancer cells to proliferate and reduced levels of Brachyury result in inhibition of proliferation, with features consistent with the cells entering a quiescent-like state. This inhibition of proliferation is dependent upon p27Kip1 demonstrating that Brachyury acts to modulate cellular proliferative fate in colorectal cancer cells in a p27Kip1-dependent manner. Analysis of patient derived colorectal tumours reveals a heterogeneous localisation of Brachyury (in the nucleolus, nucleus and cytoplasm) indicating the potential complexity of the regulatory role of Brachyury in solid colorectal tumours.

The evolution of an osmotically inducible dps in the genus Streptomyces.

Dps proteins are found almost ubiquitously in bacterial genomes and there is now an appreciation of their multifaceted roles in various stress responses. Previous studies have shown that this family of proteins assemble into dodecamers and their quaternary structure is entirely critical to their function. Moreover, the numbers of dps genes per bacterial genome is variable; even amongst closely related species - however, for many genera this enigma is yet to be satisfactorily explained. We reconstruct the most probable evolutionary history of Dps in Streptomyces genomes. Typically, these bacteria encode for more than one Dps protein. We offer the explanation that variation in the number of dps per genome among closely related Streptomyces can be explained by gene duplication or lateral acquisition, and the former preceded a subsequent shift in expression patterns for one of the resultant paralogs. We show that the genome of S. coelicolor encodes for three Dps proteins including a tailless Dps. Our in vivo observations show that the tailless protein, unlike the other two Dps in S. coelicolor, does not readily oligomerise. Phylogenetic and bioinformatic analyses combined with expression studies indicate that in several Streptomyces species at least one Dps is significantly over-expressed during osmotic shock, but the identity of the ortholog varies. In silico analysis of dps promoter regions coupled with gene expression studies of duplicated dps genes shows that paralogous gene pairs are expressed differentially and this correlates with the presence of a sigB promoter. Lastly, we identify a rare novel clade of Dps and show that a representative of these proteins in S. coelicolor possesses a dodecameric quaternary structure of high stability.

Optimized solvent-exchange synthesis method for C60 colloidal dispersions.

An existing solvent exchange method used to produce aqueous suspensions of fullerene C(60) aggregates (nC(60)) using the solvents toluene, tetrahydrofuran, acetone, and water, has been optimized for producing 75 nm diameter particles. Numerous synthesis parameters were evaluated for their effects on colloid yield and particle size distribution. Varying the relative volumes used of the intermediate solvents relative to the initial toluene volume allowed the controlled tuning of the resulting particle size up to a diameter of 210 nm. The resulting suspensions produced 10-20 ppm concentrations and reduced the residual organic solvents to less than the detection limit of 1 ppm.