Engineering of Vaginal Lactobacilli to Express Fluorescent Proteins Enables the Analysis of Their Mixture in Nanofibers

Lactobacilli are a promising natural tool against vaginal dysbiosis and infections. However, new local delivery systems and additional knowledge about their distribution and mechanism of action would contribute to the development of effective medicine. This will be facilitated by the introduction of the techniques for effective, inexpensive, and real-time tracking of these probiotics following their release. Here, we engineered three model vaginal lactobacilli (Lactobacillus crispatus ATCC 33820, Lactobacillus gasseri ATCC 33323, and Lactobacillus jensenii ATCC 25258) and a control Lactobacillus plantarum ATCC 8014 to express fluorescent proteins with different spectral properties, including infrared fluorescent protein (IRFP), green fluorescent protein (GFP), red fluorescent protein (mCherry), and blue fluorescent protein (mTagBFP2). The expression of these fluorescent proteins differed between the Lactobacillus species and enabled quantification and discrimination between lactobacilli, with the longer wavelength fluorescent proteins showing superior resolving power. Each Lactobacillus strain was labeled with an individual fluorescent protein and incorporated into poly (ethylene oxide) nanofibers using electrospinning, as confirmed by fluorescence and scanning electron microscopy. The lactobacilli retained their fluorescence in nanofibers, as well as after nanofiber dissolution. To summarize, vaginal lactobacilli were incorporated into electrospun nanofibers to provide a potential solid vaginal delivery system, and the fluorescent proteins were introduced to distinguish between them and allow their tracking in the future probiotic-delivery studies.

Quantitative assessment of near-infrared fluorescent proteins

Recent progress in fluorescent protein development has generated a large diversity of near-infrared fluorescent proteins, which are rapidly becoming popular probes for a variety of imaging applications. To assist end-users with a selection of the right near-infrared fluorescent protein for a given application, we will conduct a quantitative assessment of intracellular brightness, photostability, and oligomeric state of 19 near-infrared fluorescent proteins in cultured mammalian cells. The top-performing proteins will be further validated for in vivo imaging of neurons in C. elegans, zebrafish, and mice. We will also assess the applicability of the selected NIR FPs for expansion microscopy and two-photon imaging.

iRFP (near-infrared fluorescent protein) imaging of subcutaneous and deep tissue tumours in mice highlights differences between imaging platforms

Background In vivo imaging using fluorescence is used in cancer biology for the detection, measurement and monitoring of tumours. This can be achieved with the expression of fluorescent proteins such as iRFP, which emits light at a wavelength less attenuated in biological tissues compared to light emitted by other fluorescent proteins such as GFP or RFP. Imaging platforms capable of detecting fluorescent tumours in small animals have been developed but studies comparing the performance of these platforms are scarce. Results Through access to three platforms from Xenogen, Bruker and Li-Cor, we compared their ability to detect iRFP-expressing subcutaneous tumours as well as tumours localised deeper within the body of female NSG mice. Each platform was paired with proprietary software for image analyse, but the output depends on subjective decisions from the user. To more objectively compare platforms, we developed an ‘in house’ software-based approach which results in lower measured variability between mice. Conclusions Our comparisons showed that all three platforms allowed for reliable detection and monitoring of subcutaneous iRFP tumour growth. The biggest differences between platforms became apparent when imaging deeper tumours with the Li-Cor platform detecting most tumours and showing the highest dynamic range.

Quantitative in vivo bioluminescence imaging of orthotopic patient-derived glioblastoma xenografts

Despite extensive research, little progress has been made in glioblastoma therapy, owing in part to a lack of adequate preclinical in vivo models to study this disease. To mitigate this, primary patient-derived cell lines, which maintain their specific stem-like phenotypes, have replaced established glioblastoma cell lines. However, due to heterogenous tumour growth inherent in glioblastoma, the use of primary cells for orthotopic in vivo studies often requires large experimental group sizes. Therefore, when using intracranial patient-derived xenograft (PDX) approaches, it is advantageous to deploy imaging techniques to monitor tumour growth and allow stratification of mice. Here we show that stable expression of near-infrared fluorescent protein (iRFP) in patient-derived glioblastoma cells enables rapid direct non-invasive monitoring of tumour development without compromising tumour stemness or tumorigenicity. Moreover, as this approach does not depend on the use of agents like luciferin, which can cause variability due to changing bioavailability, it can be used for quantitative longitudinal monitoring of tumour growth. Notably, we show that this technique also allows quantitative assessment of tumour burden in highly invasive models spreading throughout the brain. Thus, iRFP transduction of primary patient-derived glioblastoma cells is a reliable, cost- and time-effective way to monitor heterogenous orthotopic PDX growth.