Synthetic biology approaches for enhancing safety and specificity of CAR-T cell therapies for solid cancers.

CAR-T cell therapies have been successful in treating numerous hematologic malignancies as the T cell can be engineered to target a specific antigen associated with the disease. However, translating CAR-T cell therapies for solid cancers is proving more challenging due to the lack of truly tumor-associated antigens and the high risk of off-target toxicities. To combat this, numerous synthetic biology mechanisms are being incorporated to create safer and more specific CAR-T cells that can be spatiotemporally controlled with increased precision. Here, we seek to summarize and analyze the advancements for CAR-T cell therapies with respect to clinical implementation, from the perspective of synthetic biology and immunology. This review should serve as a resource for further investigation and growth within the field of personalized cellular therapies.

Microbial community profiling by next-generation DNA sequencing of adenocarcinoma of the prostate with evidence of ochratoxin A producing fungi.

BACKGROUND: Prostatic carcinomas are a leading cancer and leading cause of mortality in the developed world. The etiology is diverse with underlying patient genetics, environmental factors, and microbial associations. Sequencing DNA for microbes allows the detection of potential disease relationships. OBJECTIVE: Targeted 16S (prokaryotic) and 18S (eukaryotic) rDNA sequencing was performed to map the tumor microbial flora. DESIGN: Twelve patients undergoing elective laparoscopic prostatectomy for biopsy proven adenocarcinoma of the prostate were enrolled. PCR and amplicon based sequencing was conducted; a portion of the sequencing results were confirmed by special stains. SETTING: Patients were recruited by the urologist were prospectively scheduled for radical prostatectomy by 'Da Vinci' robotically assisted procedure in an outpatient setting. Samples were portioned in the hospital surgical suite at the time of prostatectomy. PARTICIPANTS: Male patients were requested to enter the study on a first come basis. OUTCOME MEASUREMENT AND STATISTICAL ANALYSIS: Average age of the 12 participants was 64.3 years. RESULTS AND LIMITATIONS: DNA reads were detected and by 'best match' were identified belonging to Perkinsus, Hydrurus, Diversispora and Funneliformis genera, few samples displayed bacteria. Out of the 12 total patients, 11 patients had detectable DNA sequences matching arbuscular mycorrhizal fungi in the Glomeromycetes Class; Funneliformis mosseae and Diversasporum versiformis. Specific PCR for arbuscular mycorrhizal fungi failed to confirm Glomeromycetes Class; in-depth taxonomic analysis suggests a newer fungal grouping, not falling within an accepted Phylum of fungi. Calcoflour white staining of histological sections confirmed potential fungal markers in all 12 cases. Ochratoxin A antigen was identified by immunofluorescence in all 12 patient samples. The study was limited by the low sample volume and disease free normal controls. CONCLUSIONS: Fungi may play a significant role in adenocarcinoma of the prostate.

Evidence for polymicrobial communities in explanted vascular filters and atheroma debris.

RATIONALE: Microbial communities have been implicated in a variety of disease processes and have been intermittently observed in arterial disease; however, no comprehensive unbiased community analysis has been performed. We hypothesize that complex microbial communities may be involved in chronic vascular diseases as well and may be effectively characterized by molecular assays. OBJECTIVE: The main objective is to survey vascular debris, atheroma, and vascular filters for polymicrobial communities consisting of prokaryotic and eukaryotic microbes, specifically eukaryotic microbes. METHODS AND RESULTS: We examined vascular aspirates of atheromatous debris or embolic protection filters in addition to matched peripheral blood samples, from fifteen patients, as well as three cadaveric coronary arteries from two separate patients, for microbial communities. General fluorescence microscopy by Höechst and ethidium bromide DNA stains, prokaryotic and eukaryotic community analysis by Next Generation DNA Sequencing (NGS), and a eukaryotic microbial 9 probe multiplexed quantitative PCR were used to detect and characterize the presence of putative polymicrobial communities. No prokaryotes were detected in peripheral blood; however, in 4 of 9 sequenced filters and in 2 of 7 sequenced atheroma debris samples, prokaryotic populations were identified. By DNA sequencing, eukaryotic microbes were detected in 4 of 15 blood samples, 5 of the 9 sequenced filters, and 3 of the 7 atheroma debris samples. The quantitative multiplex PCR detected sequences consistent with eukaryotic microbes in all 9 analyzed filter samples as well as 5 of the 7 atheroma debris samples. Microscopy reveals putative polymicrobial communities within filters and atheroma debris. The main contributing prokaryotic species in atheroma debris suggest a diverse and novel composition. Additionally, Funneliformis mosseae, an arbuscular mycorrhizal fungus in the Glomeraceae family, was detected in the coronary hard plaque from two patients. Well studied biofilm forming bacteria were not detectable in circulating peripheral blood and were not universally present in atheroma or filters. Analyses of the sequenced eukaryotes are consistent with a diverse of array poorly studied environmental eukaryotes. In summary, out of 15 patients, 6 exhibited molecular evidence of prokaryotes and 14 had molecular evidence of eukaryotic and/or polymicrobial communities in vivo, while 2 post-mortem coronary plaque samples displayed evidence of fungi. CONCLUSION: Prokaryotes are not consistently observed in atheroma debris or filter samples; however, detection of protozoa and fungi in these samples suggests that they may play a role in arterial vascular disease or atheroma formation.

Rapid infectious disease identification by next-generation DNA sequencing.

Currently, there is a critical need to rapidly identify infectious organisms in clinical samples. Next-Generation Sequencing (NGS) could surmount the deficiencies of culture-based methods; however, there are no standardized, automated programs to process NGS data. To address this deficiency, we developed the Rapid Infectious Disease Identification (RIDI™) system. The system requires minimal guidance, which reduces operator errors. The system is compatible with the three major NGS platforms. It automatically interfaces with the sequencing system, detects their data format, configures the analysis type, applies appropriate quality control, and analyzes the results. Sequence information is characterized using both the NCBI database and RIDI™ specific databases. RIDI™ was designed to identify high probability sequence matches and more divergent matches that could represent different or novel species. We challenged the system using defined American Type Culture Collection (ATCC) reference standards of 27 species, both individually and in varying combinations. The system was able to rapidly detect known organisms in <12h with multi-sample throughput. The system accurately identifies 99.5% of the DNA sequence reads at the genus-level and 75.3% at the species-level in reference standards. It has a limit of detection of 146cells/ml in simulated clinical samples, and is also able to identify the components of polymicrobial samples with 16.9% discrepancy at the genus-level and 31.2% at the species-level. Thus, the system's effectiveness may exceed current methods, especially in situations where culture methods could produce false negatives or where rapid results would influence patient outcomes.

Integrin α3ß1 signaling through MEK/ERK determines alternative polyadenylation of the MMP-9 mRNA transcript in immortalized mouse keratinocytes.

Integrin α3ß1 is highly expressed in both normal and tumorigenic epidermal keratinocytes where it regulates genes that control cellular function and extracellular matrix remodeling during normal and pathological tissue remodeling processes, including wound healing and development of squamous cell carcinoma (SCC). Previous studies identified a role for α3ß1 in immortalized and transformed keratinocytes in the regulation of genes that promote tumorigenesis, invasion, and pro-angiogenic crosstalk to endothelial cells. One such gene, matrix metalloproteinase-9 (MMP-9), is induced by α3ß1 through a post-transcriptional mechanism of enhanced mRNA stability. In the current study, we sought to investigate the mechanism through which α3ß1 controls MMP-9 mRNA stability. First, we utilized a luciferase reporter assay to show that AU-rich elements (AREs) residing within the 3'-untranslated region (3'-UTR) of the MMP-9 mRNA renders the transcript unstable in a manner that is independent of α3ß1. Next, we cloned a truncated variant of the MMP-9 mRNA which is generated through usage of an alternative, upstream polyadenylation signal and lacks the 3'-UTR region containing the destabilizing AREs. Using an RNase protection assay to distinguish "long" (full-length 3'-UTR) and "short" (truncated 3'-UTR) MMP-9 mRNA variants, we demonstrated that the shorter, more stable mRNA that lacks 3'-UTR AREs was preferentially generated in α3ß1-expressing keratinocytes compared with α3ß1-deficient (i.e., α3-null) keratinocytes. Moreover, we determined that α3ß1-dependent alternative polyadenylation was acquired by immortalized keratinocytes, as primary neonatal keratinocytes did not display α3ß1-dependent differences in the long and short transcripts. Finally, pharmacological inhibition of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway in α3ß1-expressing keratinocytes caused a shift towards long variant expression, while Raf-1-mediated activation of ERK in α3-null keratinocytes dramatically enhanced short variant expression, indicating a role for ERK/MAPK signaling in α3ß1-mediated selection of the proximal polyadenylation site. These findings identify a novel mode of integrin α3ß1-mediated gene regulation through alternative polyadenylation.

Regulation of fibulin-2 gene expression by integrin α3ß1 contributes to the invasive phenotype of transformed keratinocytes.

The laminin-binding integrin α3ß1 is highly expressed in epidermal keratinocytes, where it regulates both cell-autonomous and paracrine functions that promote wound healing and skin tumorigenesis. However, the roles for α3ß1 in regulating gene expression programs that control the behaviors of immortalized or transformed keratinocytes remain underexplored. In the current study, we used a microarray approach to identify genes that are regulated by α3ß1 in immortalized keratinocytes. α3ß1-Responsive genes included several genes that are involved in extracellular matrix proteolysis or remodeling, including fibulin-2 and secreted protein acidic and rich in cysteine. However, α3ß1-dependent induction of specific target genes was influenced by the genetic lesion that triggered immortalization, as α3ß1-dependent fibulin-2 expression occurred in cells immortalized by either SV40 large T antigen or p53-null mutation, whereas α3ß1-dependent expression of secreted protein acidic and rich in cysteine occurred only in the former cells. Interestingly, quantitative PCR arrays did not reveal strong patterns of α3ß1-dependent gene expression in freshly isolated primary keratinocytes, suggesting that this regulation is acquired during immortalization. p53-null keratinocytes transformed with oncogenic RasV12 retained α3ß1-dependent fibulin-2 expression, and RNAi-mediated knockdown of fibulin-2 in these cells reduced invasion, although not their tumorigenic potential. These findings demonstrate a prominent role for α3ß1 in immortalized/transformed keratinocytes in regulating fibulin-2 and other genes that promote matrix remodeling and invasion.

Integrin control of tumor invasion.

Metastasis is the leading cause of death in cancer patients, and strategies to inhibit tumor cell invasion are a major focus of current efforts to develop cancer treatments. The extracellular matrix (ECM) provides both structural support and extracellular cues that regulate invasive tumor growth, and tumor-associated changes in ECM contribute to cancer progression. Integrins, the major receptors for cell adhesion to ECM, are important at every stage of cancer and occupy a critical position as transducers of chemical and mechanical signals that control tumor cell responses to ECM (i.e., outside-in signaling), as well as tumor-mediated changes to ECM that facilitate invasive growth and metastasis (i.e., inside-out signaling). Integrins are therefore attractive therapeutic targets for antagonistic agents. Here, we provide an overview of cancer-promoting functions of integrins on tumor cells, with a focus on roles in regulating cell invasion, ECM remodeling, tumor angiogenesis, and gene expression. We will also discuss how integrin functions are modulated by ECM ligands outside the cell, cytoskeletal/signaling proteins inside the cell, and other cell surface proteins. Finally, we will discuss current progress towards developing integrin antagonists for clinical use, including barriers that must still be overcome before integrins can be fully exploited as therapeutic targets.