Protocol for profiling in vitro intratumor heterogeneity using spatially annotated single-cell sequencing

Here, we present a protocol for spatially annotated single-cell sequencing, a technique for spatially profiling intratumor heterogeneity with deep single-cell RNA sequencing and single-cell resolution. By combining live-cell imaging and photopatterned illumination, we describe steps to identify regions of interest in an in vitro tumor model, label the selected cells with photoactivatable dyes, and isolate and subject them to scRNAseq. This protocol can be applied to a range of cell lines and could be expanded to tissue sections. For complete details on the use and execution of this protocol, please refer to Smit et al. (2022).1</p

Instant processing of large-scale image data with FACT, a real-time cell segmentation and tracking algorithm

Quantifying cellular characteristics from a large heterogeneous population is essential to identify rare, disease-driving cells. A recent development in the combination of high-throughput screening microscopy with single-cell profiling provides an unprecedented opportunity to decipher disease-driving phenotypes. Accurately and instantly processing large amounts of image data, however, remains a technical challenge when an analysis output is required minutes after data acquisition. Here, we present fast and accurate real-time cell tracking (FACT). FACT can segment ∼20,000 cells in an average of 2.5 s (1.9–93.5 times faster than the state of the art). It can export quantifiable features minutes after data acquisition (independent of the number of acquired image frames) with an average of 90%–96% precision. We apply FACT to identify directionally migrating glioblastoma cells with 96% precision and irregular cell lineages from a 24 h movie with an average F1 score of 0.91.</p

Genomic Exploration of Distinct Molecular Phenotypes Steering Temozolomide Resistance Development in Patient-Derived Glioblastoma Cells

Chemotherapy using temozolomide is the standard treatment for patients with glioblastoma. Despite treatment, prognosis is still poor largely due to the emergence of temozolomide resistance. This resistance is closely linked to the widely recognized inter- and intra-tumoral heterogeneity in glioblastoma, although the underlying mechanisms are not yet fully understood. To induce temozolomide resistance, we subjected 21 patient-derived glioblastoma cell cultures to Temozolomide treatment for a period of up to 90 days. Prior to treatment, the cells’ molecular characteristics were analyzed using bulk RNA sequencing. Additionally, we performed single-cell RNA sequencing on four of the cell cultures to track the evolution of temozolomide resistance. The induced temozolomide resistance was associated with two distinct phenotypic behaviors, classified as “adaptive” (ADA) or “non-adaptive” (N-ADA) to temozolomide. The ADA phenotype displayed neurodevelopmental and metabolic gene signatures, whereas the N-ADA phenotype expressed genes related to cell cycle regulation, DNA repair, and protein synthesis. Single-cell RNA sequencing revealed that in ADA cell cultures, one or more subpopulations emerged as dominant in the resistant samples, whereas N-ADA cell cultures remained relatively stable. The adaptability and heterogeneity of glioblastoma cells play pivotal roles in temozolomide treatment and contribute to the tumor’s ability to survive. Depending on the tumor’s adaptability potential, subpopulations with acquired resistance mechanisms may arise.</p

Origins Space Telescope: baseline mission concept

The Origins Space Telescope will trace the history of our origins from the time dust and heavy elements permanently altered the cosmic landscape to present-day life. How did galaxies evolve from the earliest galactic systems to those found in the Universe today? How do habitable planets form? How common are life-bearing worlds? To answer these alluring questions, Origins will operate at mid- and far-infrared (IR) wavelengths and offer powerful spectroscopic instruments and sensitivity three orders of magnitude better than that of the Herschel Space Observatory, the largest telescope flown in space to date. We describe the baseline concept for Origins recommended to the 2020 US Decadal Survey in Astronomy and Astrophysics. The baseline design includes a 5.9-m diameter telescope cryocooled to 4.5 K and equipped with three scientific instruments. A mid-infrared instrument (Mid-Infrared Spectrometer and Camera Transit spectrometer) will measure the spectra of transiting exoplanets in the 2.8 to 20  μm wavelength range and offer unprecedented spectrophotometric precision, enabling definitive exoplanet biosignature detections. The far-IR imager polarimeter will be able to survey thousands of square degrees with broadband imaging at 50 and 250  μm. The Origins Survey Spectrometer will cover wavelengths from 25 to 588  μm, making wide-area and deep spectroscopic surveys with spectral resolving power R  ∼  300, and pointed observations at R  ∼  40,000 and 300,000 with selectable instrument modes. Origins was designed to minimize complexity. The architecture is similar to that of the Spitzer Space Telescope and requires very few deployments after launch, while the cryothermal system design leverages James Webb Space Telescope technology and experience. A combination of current-state-of-the-art cryocoolers and next-generation detector technology will enable Origins’ natural background-limited sensitivity

Spatially Annotated Single Cell Sequencing for Unraveling Intratumor Heterogeneity

Intratumor heterogeneity is a major obstacle to effective cancer treatment. Current methods to study intratumor heterogeneity using single-cell RNA sequencing (scRNA-seq) lack information on the spatial organization of cells. While state-of-the art spatial transcriptomics methods capture the spatial distribution, they either lack single cell resolution or have relatively low transcript counts. Here, we introduce spatially annotated single cell sequencing, based on the previously developed functional single cell sequencing (FUNseq) technique, to spatially profile tumor cells with deep scRNA-seq and single cell resolution. Using our approach, we profiled cells located at different distances from the center of a 2D epithelial cell mass. By profiling the cell patch in concentric bands of varying width, we showed that cells at the outermost edge of the patch responded strongest to their local microenvironment, behaved most invasively, and activated the process of epithelial-to-mesenchymal transition (EMT) to migrate to low-confluence areas. We inferred cell-cell communication networks and demonstrated that cells in the outermost ∼10 cell wide band, which we termed the invasive edge, induced similar phenotypic plasticity in neighboring regions. Applying FUNseq to spatially annotate and profile tumor cells enables deep characterization of tumor subpopulations, thereby unraveling the mechanistic basis for intratumor heterogeneity

Comparison of the source and quality of information on the internet between anterolateral ligament reconstruction and anterior cruciate ligament reconstruction: an Australian experience

Background: The internet is a valuable tool, but concerns exist regarding the quality and accuracy of medical information available online. Purpose: To evaluate the source and quality of information on the internet relating to anterolateral ligament reconstruction (ALLR) compared with anterior cruciate ligament reconstruction (ACLR). Study Design: Cross-sectional study. Methods: A questionnaire was administered to 50 ACLR patients in Australia to determine their use of the internet to research their operation and their familiarity with the anterolateral ligament (ALL) of the knee. The most common search terms were determined, and the first 70 websites returned by the 5 most popular search engines were used to assess the quality of information about ACLR and ALLR. Each site was categorized by type and was assessed for quality and validity using the DISCERN score, the Journal of the American Medical Association (JAMA) benchmark criteria, and a novel specific content score for each procedure. The presence of the Health on the Net Code (HONcode) seal was also recorded. Results: The majority (84%) of ACLR patients used the internet to research their operation. The quality of information available for ALLR was significantly inferior to that for ACLR according to the DISCERN score (37.3 ± 3.4 vs 54.4 ± 4.6; P <.0001) and specific content score (5.3 ± 1.3 vs 11.0 ± 1.5; P <.0001). ACLR websites were predominantly physician produced, while the majority of ALLR websites were academic. In contrast to ACLR websites, the majority of ALLR websites did not provide information on the indication for treatment or potential complications. ALLR websites scored better on the JAMA benchmark criteria due to the predominance of academic websites. A greater proportion of ACLR websites (14.6%) versus ALLR websites (2.5%) provided an HONcode seal. Correlation was demonstrated between the DISCERN score and specific content scores for both ACLR and ALLR but not with JAMA benchmark criteria. The specific content score had high reliability for both ACLR and ALLR. Conclusion: The majority of patients undergoing ACLR in Australia used the internet to research the procedure. The quality of information on the internet relating to ALLR was significantly inferior to information about ACLR. Most ALLR websites failed to include crucial information about the indication or options for treatment, prognosis, and potential complications. Surgeons should be aware of the information to which their patients are exposed through the internet and should be proactive in directing patients to appropriate websites

Genomic exploration of distinct molecular phenotypes steering temozolomide resistance development in patient-derived glioblastoma cells

Chemotherapy using temozolomide is the standard treatment for patients with glioblastoma. Despite treatment, prognosis is still poor largely due to the emergence of temozolomide resistance. This resistance is closely linked to the widely recognized inter- and intra-tumoral heterogeneity in glioblastoma, although the underlying mechanisms are not yet fully understood. To induce temozolomide resistance, we subjected 21 patient-derived glioblastoma cell cultures to Temozolomide treatment for a period of up to 90 days. Prior to treatment, the cells' molecular characteristics were analyzed using bulk RNA sequencing. Additionally, we performed single-cell RNA sequencing on four of the cell cultures to track the evolution of temozolomide resistance. The induced temozolomide resistance was associated with two distinct phenotypic behaviors, classified as "adaptive" (ADA) or "non-adaptive" (N-ADA) to temozolomide. The ADA phenotype displayed neurodevelopmental and metabolic gene signatures, whereas the N-ADA phenotype expressed genes related to cell cycle regulation, DNA repair, and protein synthesis. Single-cell RNA sequencing revealed that in ADA cell cultures, one or more subpopulations emerged as dominant in the resistant samples, whereas N-ADA cell cultures remained relatively stable. The adaptability and heterogeneity of glioblastoma cells play pivotal roles in temozolomide treatment and contribute to the tumor's ability to survive. Depending on the tumor's adaptability potential, subpopulations with acquired resistance mechanisms may arise. </p