Deep immunophenotyping at the single-cell level identifies a combination of anti-IL-17 and checkpoint blockade as an effective treatment in a preclinical model of data-guided personalized immunotherapy.

BACKGROUND: Although immune checkpoint blockade is effective for several malignancies, a substantial number of patients remain refractory to treatment. The future of immunotherapy will be a personalized approach adapted to each patient's cancer-immune interactions in the tumor microenvironment (TME) to prevent suppression of antitumor immune responses. To demonstrate the feasibility of this kind of approach, we developed combination therapy for a preclinical model guided by deep immunophenotyping of the TME. METHODS: Gastric cancer cell lines YTN2 and YTN16 were subcutaneously inoculated into C57BL/6 mice. YTN2 spontaneously regresses, while YTN16 grows progressively. Bulk RNA-Seq, single-cell RNA-Seq (scRNA-Seq) and flow cytometry were performed to investigate the immunological differences in the TME of these tumors. RESULTS: Bulk RNA-Seq demonstrated that YTN16 tumor cells produced CCL20 and that CD8+ T cell responses were impaired in these tumors relative to YTN2. We have developed a vertical flow array chip (VFAC) for targeted scRNA-Seq to identify unique subtypes of T cells by employing a panel of genes reflecting T cell phenotypes and functions. CD8+ T cell dysfunction (cytotoxicity, proliferation and the recruitment of interleukin-17 (IL-17)-producing cells into YTN16 tumors) was identified by targeted scRNA-Seq. The presence of IL-17-producing T cells in YTN16 tumors was confirmed by flow cytometry, which also revealed neutrophil infiltration. IL-17 blockade suppressed YTN16 tumor growth, while tumors were rejected by the combination of anti-IL-17 and anti-PD-1 (Programmed cell death protein 1) mAb treatment. Reduced neutrophil activation and enhanced expansion of neoantigen-specific CD8+ T cells were observed in tumors of the mice receiving the combination therapy. CONCLUSIONS: Deep phenotyping of YTN16 tumors identified a sequence of events on the axis CCL20->IL-17-producing cells->IL-17-neutrophil-angiogenesis->suppression of neoantigen-specific CD8+ T cells which was responsible for the lack of tumor rejection. IL-17 blockade together with anti-PD-1 mAb therapy eradicated these YTN16 tumors. Thus, the deep immunological phenotyping can guide immunotherapy for the tailored treatment of each individual patient's tumor.

Combinatory use of distinct single-cell RNA-seq analytical platforms reveals the heterogeneous transcriptome response.

Single-cell RNA-seq is a powerful tool for revealing heterogeneity in cancer cells. However, each of the current single-cell RNA-seq platforms has inherent advantages and disadvantages. Here, we show that combining the different single-cell RNA-seq platforms can be an effective approach to obtaining complete information about expression differences and a sufficient cellular population to understand transcriptional heterogeneity in cancers. We demonstrate that it is possible to estimate missing expression information. We further demonstrate that even in the cases where precise information for an individual gene cannot be inferred, the activity of given transcriptional modules can be analyzed. Interestingly, we found that two distinct transcriptional modules, one associated with the Aurora kinase gene and the other with the DUSP gene, are aberrantly regulated in a minor population of cells and may thus contribute to the possible emergence of dormancy or eventual drug resistance within the population.

Vertical flow array chips reliably identify cell types from single-cell mRNA sequencing experiments.

Single-cell mRNA sequencing offers an unbiased approach to dissecting cell types as functional units in multicellular tissues. However, highly reliable cell typing based on single-cell gene expression analysis remains challenging because of the lack of methods for efficient sample preparation for high-throughput sequencing and evaluating the statistical reliability of the acquired cell types. Here, we present a highly efficient nucleic reaction chip (a vertical flow array chip (VFAC)) that uses porous materials to reduce measurement noise and improve throughput without a substantial increase in reagent. We also present a probabilistic evaluation method for cell typing depending on the amount of measurement noise. Applying the VFACs to 2580 monocytes provides 1967 single-cell expressions for 47 genes, including low-expression genes such as transcription factors. The statistical method can distinguish two cell types with probabilistic quality values, with the measurement noise level being considered for the first time. This approach enables the identification of various sub-types of cells in tissues and provides a foundation for subsequent analyses.

Coherent anti-Stokes Raman scattering hyperspectral imaging of cartilage aiming for state discrimination of cell.

Noninvasive cell analyses are increasingly important in the medical field. A coherent anti-Stokes Raman scattering (CARS) microscope is the noninvasive imaging equipment and enables to obtain images indicating molecular distribution. However, due to low-signal intensity, it is still challenging to obtain images of the fingerprint region, in which many spectrum peaks correspond to compositions of a cell. Here, to identify cell differentiation by using multiplex CARS, we investigated hyperspectral imaging of the fingerprint region of living cells. To perform multiplex CARS, we used a prototype of a compact light source generating both pump light and broadband Stokes light. Assuming application to regenerative medicine, we chose a cartilage cell, whose differentiation is difficult to be identified by change of the cell morphology. Because one of the major components of cartilage is collagen, we focused on distribution of proline, which accounts for approximately 20% of collagen. The spectrum quality was improved by optical adjustments of the power branching ratio and divergence of Stokes light. Periphery of a cartilage cell was highlighted in a CARS image of proline, and this result suggests correspondence with collagen generated as an extracellular matrix. The possibility of noninvasive analyses by using CARS hyperspectral imaging was indicated.

Compact and fully collinear light source for broadband multiplex CARS microscopy covering the fingerprint region.

Compact and fully collinear light source for multiplex coherent anti-Stokes Raman scattering (CARS) microscopy was proposed and demonstrated. It consists of only a microchip laser, a short photonic crystal fiber, and a longpass filter. It offers performance of sensitivity, bandwidth, and spectral resolution suitable for biomedical applications, especially covering the entire fingerprint region (500-1800 cm(-1)). It can be readily implemented by a commercially available microchip laser and a photonic crystal fiber. It has great potential to expand the utility of CARS microscopy to a wide variety of fields such as endoscopy.

Compact light source for ultrabroadband coherent anti-Stoke Raman scattering (CARS) microscopy.

A compact light source module for ultrabroadband coherent anti-Stoke Raman scattering (CARS) microscopy was developed. It mainly consists of a nanosecond microchip laser, a photonic crystal fiber for Stokes light generation, and a single mode polarization maintaining fiber for pump light propagation. It is alignment-free and relatively low-cost compared with previous light sources of CARS microscopy. By using an assembled module, we successfully observed an ultrabroadband CARS spectrum and a CARS image of a murine adipocyte. The module is expected to greatly spread the CARS microscopy to various fields by its extreme easiness to handle.

Quantitative index of arbitrary molar concentration for coherent anti-Stoke Raman scattering (CARS) spectroscopy and microscopy.

We propose a simple quantitative index for coherent anti-Stoke Raman scattering (CARS) spectroscopy and microscopy. Unlike previous similar indices, it can be applied to samples with arbitrary molar concentration, and it is robust against environmental change. Concentrations of aqueous hydrogen peroxide solution and lipid concentration distribution in a live murine adipocyte were successfully quantified by the new index. The index can be obtained in a broad range of CARS setups and it is readily applicable to quantitative CARS microscopy for deep inspection of samples such as biological specimens.

A highly sensitive and accurate gene expression analysis by sequencing ("bead-seq") for a single cell.

Analyses of gene expressions in single cells are important for understanding detailed biological phenomena. Here, a highly sensitive and accurate method by sequencing (called "bead-seq") to obtain a whole gene expression profile for a single cell is proposed. A key feature of the method is to use a complementary DNA (cDNA) library on magnetic beads, which enables adding washing steps to remove residual reagents in a sample preparation process. By adding the washing steps, the next steps can be carried out under the optimal conditions without losing cDNAs. Error sources were carefully evaluated to conclude that the first several steps were the key steps. It is demonstrated that bead-seq is superior to the conventional methods for single-cell gene expression analyses in terms of reproducibility, quantitative accuracy, and biases caused during sample preparation and sequencing processes.

Hedgehog-Gli activators direct osteo-chondrogenic function of bone morphogenetic protein toward osteogenesis in the perichondrium.

Specification of progenitors into the osteoblast lineage is an essential event for skeletogenesis. During endochondral ossification, cells in the perichondrium give rise to osteoblast precursors. Hedgehog (Hh) and bone morphogenetic protein (BMP) are suggested to regulate the commitment of these cells. However, properties of perichondrial cells and regulatory mechanisms of the specification process are still poorly understood. Here, we investigated the machineries by combining a novel organ culture system and single-cell expression analysis with mouse genetics and biochemical analyses. In a metatarsal organ culture reproducing bone collar formation, activation of BMP signaling enhanced the bone collar formation cooperatively with Hh input, whereas the signaling induced ectopic chondrocyte formation in the perichondrium without Hh input. Similar phenotypes were also observed in compound mutant mice, where signaling activities of Hh and BMP were genetically manipulated. Single-cell quantitative RT-PCR analyses showed heterogeneity of perichondrial cells in terms of natural characteristics and responsiveness to Hh input. In vitro analyses revealed that Hh signaling suppressed BMP-induced chondrogenic differentiation; Gli1 inhibited the expression of Sox5, Sox6, and Sox9 (SRY box-containing gene 9) as well as transactivation by Sox9. Indeed, ectopic expression of chondrocyte maker genes were observed in the perichondrium of metatarsals in Gli1(-/-) fetuses, and the phenotype was more severe in Gli1(-/-);Gli2(-/-) newborns. These data suggest that Hh-Gli activators alter the function of BMP to specify perichondrial cells into osteoblasts; the timing of Hh input and its target populations are critical for BMP function.

Highly sensitive pyrosequencing system with polymer-supported enzymes for high-throughput DNA analysis.

A highly sensitive massively parallel pyrosequencing system employing a gel matrix to immobilize enzymes at high density in microreaction chambers is demonstrated. Reducing the size of microreaction chambers in a DNA analyzer is important to achieve a high throughput utilizing a commercially available detection device or camera. A high-performance system can be attained by detecting signals from one reaction chamber with one photopixel of around several micrometers by utilizing a 1:1 image magnification. However, the use of small beads immobilizing DNA has a disadvantage in detecting luminescence because only small amounts of DNA can be immobilized on the bead surfaces for sequencing. As luminescence intensity could be enhanced by increasing the luciferase density in the chambers, we overcame this difficulty by using a gel matrix to immobilize luciferase at a high concentration in the microreaction chambers. Luminescence 1 order of magnitude higher could be observed with the new method compared to the conventional method. Consequently, the chamber size and bead size immobilizing DNA could be reduced to as small as 6.5 and 4 µm, respectively. This can be successfully applied to achieving small, inexpensive, pyrosequencing systems with high throughput.