Nociceptor neurons affect cancer immunosurveillance.

Solid tumours are innervated by nerve fibres that arise from the autonomic and sensory peripheral nervous systems1-5. Whether the neo-innervation of tumours by pain-initiating sensory neurons affects cancer immunosurveillance remains unclear. Here we show that melanoma cells interact with nociceptor neurons, leading to increases in their neurite outgrowth, responsiveness to noxious ligands and neuropeptide release. Calcitonin gene-related peptide (CGRP)-one such nociceptor-produced neuropeptide-directly increases the exhaustion of cytotoxic CD8+ T cells, which limits their capacity to eliminate melanoma. Genetic ablation of the TRPV1 lineage, local pharmacological silencing of nociceptors and antagonism of the CGRP receptor RAMP1 all reduced the exhaustion of tumour-infiltrating leukocytes and decreased the growth of tumours, nearly tripling the survival rate of mice that were inoculated with B16F10 melanoma cells. Conversely, CD8+ T cell exhaustion was rescued in sensory-neuron-depleted mice that were treated with local recombinant CGRP. As compared with wild-type CD8+ T cells, Ramp1-/- CD8+ T cells were protected against exhaustion when co-transplanted into tumour-bearing Rag1-deficient mice. Single-cell RNA sequencing of biopsies from patients with melanoma revealed that intratumoral RAMP1-expressing CD8+ T cells were more exhausted than their RAMP1-negative counterparts, whereas overexpression of RAMP1 correlated with a poorer clinical prognosis. Overall, our results suggest that reducing the release of CGRP from tumour-innervating nociceptors could be a strategy to improve anti-tumour immunity by eliminating the immunomodulatory effects of CGRP on cytotoxic CD8+ T cells.

Metabolic reprogramming from glycolysis to fatty acid uptake and beta-oxidation in platinum-resistant cancer cells.

Increased glycolysis is considered as a hallmark of cancer. Yet, cancer cell metabolic reprograming during therapeutic resistance development is under-studied. Here, through high-throughput stimulated Raman scattering imaging and single cell analysis, we find that cisplatin-resistant cells exhibit increased fatty acids (FA) uptake, accompanied by decreased glucose uptake and lipogenesis, indicating reprogramming from glucose to FA dependent anabolic and energy metabolism. A metabolic index incorporating glucose derived anabolism and FA uptake correlates linearly to the level of cisplatin resistance in ovarian cancer (OC) cell lines and primary cells. The increased FA uptake facilitates cancer cell survival under cisplatin-induced oxidative stress by enhancing beta-oxidation. Consequently, blocking beta-oxidation by a small molecule inhibitor combined with cisplatin or carboplatin synergistically suppresses OC proliferation in vitro and growth of patient-derived xenografts in vivo. Collectively, these findings support a rapid detection method of cisplatin-resistance at single cell level and a strategy for treating cisplatin-resistant tumors.

Transcriptional neural-like signaling contributes to an immune-suppressive tumor microenvironment.

Tumor innervation has recently been documented and characterized in various settings and tumor types. However, the role that nerves innervating tumors play in the pathogenesis of cancer has not been clarified. In this study, we searched for neural signaling from bulk RNA sequencing from The Cancer Genome Atlas (TCGA) dataset and looked for patterns of interactions between different cell types within the tumor environment. Using a presynapse signature (PSS) as a probe, we showed that multiple stromal cell types crosstalk and/or contribute to neural signals. Based on the correlation and linear regression, we hypothesized that neural signals contribute to an immune-suppressive tumor microenvironment (TME). To test this hypothesis, we performed in vitro dorsal root ganglion (DRG)/macrophage coculture experiments. Compared to the M2 macrophage monoculture, the DRG/M2 macrophage coculture prevented anti-inflammatory M2 to pro-inflammatory M1 polarization by LPS stimulation. Finally, a survey of different TCGA tumor types indicated that higher RNA neural signature is predictive of poor patient outcomes in multiple tumor types.

Ultrasensitive Vibrational Imaging of Retinoids by Visible Preresonance Stimulated Raman Scattering Microscopy.

High-sensitivity chemical imaging offers a window to decipher the molecular orchestra inside a living system. Based on vibrational fingerprint signatures, coherent Raman scattering microscopy provides a label-free approach to map biomolecules and drug molecules inside a cell. Yet, by near-infrared (NIR) pulse excitation, the sensitivity is limited to millimolar concentration for endogenous biomolecules. Here, the imaging sensitivity of stimulated Raman scattering (SRS) is significantly boosted for retinoid molecules to 34 micromolar via electronic preresonance in the visible wavelength regime. Retinoids play critical roles in development, immunity, stem cell differentiation, and lipid metabolism. By visible preresonance SRS (VP-SRS) imaging, retinoid distribution in single embryonic neurons and mouse brain tissues is mapped, retinoid storage in chemoresistant pancreatic and ovarian cancers is revealed, and retinoids stored in protein network and lipid droplets of Caenorahbditis elegans are identified. These results demonstrate VP-SRS microscopy as an ultrasensitive label-free chemical imaging tool and collectively open new opportunities of understanding the function of retinoids in biological systems.

Room-Temperature Phosphorescence and Low-Energy Induced Direct Triplet Excitation of Alq3 Engineered Crystals.

Crystal engineering is a practical approach for tailoring material properties. This approach has been widely studied for modulating optical and electrical properties of semiconductors. However, the properties of organic molecular crystals are difficult to control following a similar engineering route. In this Letter, we demonstrate that engineered crystals of Alq3 and Ir(ppy)3 complexes, which are commonly used in organic light-emitting technologies, possess intriguing functional properties. Specifically, these structures not only process efficient low-energy induced triplet excitation directly from the ground state of Alq3 but also can show strong emission at the Alq3 triplet energy level at room temperatures. We associate these phenomena with local deformations of the host matrix around the guest molecules, which in turn lead to a stronger host-guest triplet-triplet coupling and spin-orbital mixing.

Functionalized NIR-II Semiconducting Polymer Nanoparticles for Single-cell to Whole-Organ Imaging of PSMA-Positive Prostate Cancer.

Development of molecular probes holds great promise for early diagnosis of aggressive prostate cancer. Here, 2-[3-(1,3-dicarboxypropyl) ureido] pentanedioic acid (DUPA)-conjugated ligand and bis-isoindigo-based polymer (BTII) are synthesized to formulate semiconducting polymer nanoparticles (BTII-DUPA SPN) as a prostate-specific membrane antigen (PSMA)-targeted probe for prostate cancer imaging in the NIR-II window. Insights into the interaction of the imaging probes with the biological targets from single cell to whole organ are obtained by transient absorption (TA) microscopy and photoacoustic (PA) tomography. At single-cell level, TA microscopy reveals the targeting efficiency, kinetics, and specificity of BTII-DUPA SPN to PSMA-positive prostate cancer. At organ level, PA tomographic imaging of BTII-DUPA SPN in the NIR-II window demonstrates superior imaging depth and contrast. By intravenous administration, BTII-DUPA SPN demonstrates selective accumulation and retention in the PSMA-positive tumor, allowing noninvasive PA detection of PSMA overexpressing prostate tumors in vivo. The distribution of nanoparticles inside the tumor tissue is further analyzed through TA microscopy. These results collectively demonstrate BTII-DUPA SPN as a promising probe for prostate cancer diagnosis by PA tomography.

Multiplex Stimulated Raman Scattering Imaging Cytometry Reveals Lipid-Rich Protrusions in Cancer Cells under Stress Condition.

In situ measurement of cellular metabolites is still a challenge in biology. Conventional methods, such as mass spectrometry or fluorescence microscopy, would either destroy the sample or introduce strong perturbations to target molecules. Here, we present multiplex stimulated Raman scattering (SRS) imaging cytometry as a label-free single-cell analysis platform with chemical specificity and high-throughput capabilities. Using SRS imaging cytometry, we studied the metabolic responses of human pancreatic cancer cells under stress by starvation and chemotherapeutic drug treatments. We unveiled protrusions containing lipid droplets as a metabolic marker for stress-resistant cancer cells. Furthermore, by spectroscopic SRS mapping, we unveiled that triglyceride in lipid droplets are used for local energy production through lipolysis, autophagy, and ß-oxidation. Our findings demonstrate the potential of targeting lipid metabolism for selective treatment of stress-resistant cancers. Collectively, these results highlight SRS imaging cytometry as a powerful label-free tool for biological discoveries with a high-throughput, high-content capacity.

Quantitative imaging of intraerythrocytic hemozoin by transient absorption microscopy.

Hemozoin, the heme detoxification end product in malaria parasites during their growth in the red blood cells (RBCs), serves as an important marker for diagnosis and treatment target of malaria disease. However, the current method for hemozoin-targeted drug screening mainly relies on in vitro ß-hematin inhibition assays, which may lead to false-positive events due to under-representation of the real hemozoin crystal. Quantitative in situ imaging of hemozoin is highly desired for high-throughput screening of antimalarial drugs and for elucidating the mechanisms of antimalarial drugs. We present transient absorption (TA) imaging as a high-speed single-cell analysis platform with chemical selectivity to hemozoin. We first demonstrated that TA microscopy is able to identify ß-hematin, the artificial form of hemozoin, from the RBCs. We further utilized time-resolved TA imaging to in situ discern hemozoin from malaria-infected RBCs with optimized imaging conditions. Finally, we quantitatively analyzed the hemozoin amount in RBCs at different infection stages by single-shot TA imaging. These results highlight the potential of TA imaging for efficient antimalarial drug screening and drug mechanism investigation.

Electronic Preresonance Stimulated Raman Scattering Imaging of Red-Shifted Proteorhodopsins: Toward Quantitation of the Membrane Potential.

Voltage imaging allows mapping of the membrane potential in living cells. Yet, current intensity-based imaging approaches are limited to relative membrane potential changes, missing important information conveyed by the absolute value of the membrane voltage. This challenge arises from various factors affecting the signal intensity, such as concentration, illumination intensity, and photobleaching. Here, we demonstrate electronic preresonance hyperspectral stimulated Raman scattering (EPR-hSRS) for spectroscopic detection of the membrane voltage using a near-infrared-absorbing microbial rhodopsin expressed in E. coli. This newly developed near-infrared active microbial rhodopsin enables electronic preresonance SRS imaging at high sensitivity. By spectral profiling, we identified voltage-sensitive SRS peaks in the fingerprint region in single E. coli cells. These spectral signatures offer a new approach for quantitation of the absolute membrane voltage in living cells.

Label-free quantitation of glycated hemoglobin in single red blood cells by transient absorption microscopy and phasor analysis.

As a stable and accurate biomarker, glycated hemoglobin (HbA1c) is clinically used to diagnose diabetes with a threshold of 6.5% among total hemoglobin (Hb). Current methods such as boronate affinity chromatography involve complex processing of large-volume blood samples. Moreover, these methods cannot measure HbA1c fraction at single-red blood cell (RBC) level, thus unable to separate the contribution from other factors such as RBC lifetime. Here, we demonstrate a spectroscopic transient absorption imaging approach that is able to differentiate HbA1c from Hb on the basis of their distinct excited-state dynamics. HbA1c fraction inside a single RBC is derived quantitatively through phasor analysis. HbA1c fraction distribution of diabetic blood is apparently different from that of healthy blood. A mathematical model is developed to derive the long-term blood glucose concentration. Our technology provides a unique way to study heme modification and to derive clinically important information void of bloodstream glucose fluctuation.

High-Speed Spectroscopic Transient Absorption Imaging of Defects in Graphene.

Graphene grain boundaries (GBs) and other nanodefects can deteriorate electronic properties. Here, using transient absorption (TA) microscopy we directly visualized GBs by TA intensity increase due to change in density of state. We also observed a faster decay due to defect-accelerated carrier relaxation in the GB area. By line-illumination and parallel detection, we increased the TA intensity imaging speed to 1000 frames per second, which is 6 orders of magnitude faster than Raman microscopy. Combined with a resonant optical delay tuner which scans a 5.3 ps temporal delay within 92 µs, our system enabled spectroscopic TA imaging, at a speed of 50 stacks per second, to probe and characterize graphene nanodefects based on the TA decay rate. Finally, we demonstrate real-time nondestructive characterization of graphene at a rolling speed of 0.3 m/min, which matches the fastest roll-to-roll manufacturing process reported.

Volumetric chemical imaging by stimulated Raman projection microscopy and tomography.

Volumetric imaging allows global understanding of three-dimensional (3D) complex systems. Light-sheet fluorescence microscopy and optical projection tomography have been reported to image 3D volumes with high resolutions and at high speeds. Such methods, however, usually rely on fluorescent labels for chemical targeting, which could perturb the biological functionality in living systems. We demonstrate Bessel-beam-based stimulated Raman projection (SRP) microscopy and tomography for label-free volumetric chemical imaging. Our SRP microscope enables fast quantitation of chemicals in a 3D volume through a two-dimensional lateral scan. Furthermore, combining SRP and sample rotation, we demonstrate the SRP tomography that can reconstruct the 3D distribution of chemical compositions with optical spatial resolution at a higher speed than the Gaussian-beam-based stimulated Raman scattering sectioning imaging can. We explore the potential of our SRP technology by mapping polymer particles in 3D volumes and lipid droplets in adipose cells.

Coherent anti-Stokes Raman scattering imaging under ambient light.

We demonstrate an ambient light coherent anti-Stokes Raman scattering microscope that allows CARS imaging to be operated under environmental light for field use. The CARS signal is modulated at megahertz frequency and detected by a photodiode equipped with a lab-built resonant amplifier, then extracted through a lock-in amplifier. The filters in both the spectral domain and the frequency domain effectively blocked the room light contamination of the CARS image. In situ hyperspectral CARS imaging of tumor tissue under ambient light is demonstrated.

In vivo sub-femtoliter resolution photoacoustic microscopy with higher frame rates.

Microscopy based on non-fluorescent absorption dye staining is widely used in various fields of biomedicine for 400 years. Unlike its fluorescent counterpart, non-fluorescent absorption microscopy lacks proper methodologies to realize its in vivo applications with a sub-femtoliter 3D resolution. Regardless of the most advanced high-resolution photoacoustic microscopy, sub-femtoliter spatial resolution is still unattainable, and the imaging speed is relatively slow. In this paper, based on the two-photon photoacoustic mechanism, we demonstrated a in vivo label free laser-scanning photoacoustic imaging modality featuring high frame rates and sub-femtoliter 3D resolution simultaneously, which stands as a perfect solution to 3D high resolution non-fluorescent absorption microscopy. Furthermore, we first demonstrated in vivo label-free two-photon acoustic microscopy on the observation of non-fluorescent melanin distribution within mouse skin.

3D SERS (surface enhanced Raman scattering) imaging of intracellular pathways.

We demonstrate dynamic surface-enhanced Raman scattering (SERS) imaging with 3 dimensional mapping in a living cell by combining a confocal Raman spectrometer and dual-focus dark-field microscopy. Gold nanoparticles acting as a probe traveling through the living cell, are tracked by dual-focus dark-field imaging, with position feedback to the confocal Raman spectrometer in order to record evolving SERS signals from the same gold nanoparticle moving through the cell. Simultaneous detection of motion and the SERS spectrum enables the visualization of the SERS pathways. Observation of how the SERS spectra change in space and time during biological functions or by changes of the molecular environment provides exciting opportunities for understanding the molecular basis of the cell dynamics.