OBJECTIVE Cancer vaccines represent a formidable strategy that harnesses the immune system to recognize and combat cancerous cells. Spatiotemporal visualization of cancer vaccine trafficking aids dosage optimization, localizes distribution, and visually informs interactions with the immune system. Here, we developed a gold quantum cluster (AuQC) nanoprobe stabilized by mammalian milk-derived alpha-lactalbumin (α-LA), an FDA-designated GRAS (generally recognized as safe) protein as an investigational vaccine against breast cancer currently in clinical phases. Ultrasmall-sized AuQCs (< 3 nm) display intrinsic near-infrared fluorescence (NIRF) and can be radiolabeled without chelators through anti-galvanic catalytic reduction of radioactive 64Cu2+ for positron emission tomography (PET), enabling lymph node (LN) imaging by complementary dual-modalities. We hypothesize that AuQCs can precisely delineate tumor-draining LNs with dual contrasts, including sentinel lymph nodes (SLN), and function as therapeutic and prophylactic vaccines by interacting with the immune system. We aim to evaluate the imaging performances and identify the specific type of immune cells with clearly elucidated vaccination mechanisms. METHODS We first synthesized ultrasmall AuQCs with emission in the NIR region as previously and characterized them by scanning transmission electron microscopy (STEM). The chelator-free labeling of AuQCs with 64Cu2+ or NatCu2+ in chlorides was achieved under ambient conditions. The interaction between AuQCs and Cu2+ was thoroughly studied by a series of techniques, including dynamic light scattering (DLS), biolayer interferometry (BLI), and X-ray photoelectron spectroscopy (XPS). The room-temperature radiolabeling efficiency and stability were evaluated using instant thin-layer chromatography (ITLC). Dual-modality lymphatic imaging was carried out following footpad injection in wild-type mice in a time-lapse manner. After harvesting LNs, we performed ex vivo imaging and verified probe uptake in LNs of ipsilateral and contralateral sides. SLN detection was estimated after peritumoral injection of 64Cu-AuQCs in small-cell lung cancer (SCLC) patient-derived xenograft (PDX) mice. To investigate how the agent is fated in the bloodstream, we quantified the radioactivity in individual excised organs using an automated gamma counter with reference to an intravenous injectate. Next, we designed multicolor panels of fluorescence-activated cell sorting (FACS) to identify the primary cells in LNs that interact with AuQCs. Finally, we assessed the in vivo therapeutic and prophylactic effects in immunocompetent mice bearing allografted tumors of murine melanoma and breast cancer.    RESULTS AuQCs have a core size below 3 nm by STEM and a hydrodynamic size around 3.5 nm, which is minimally affected by low Cu2+ concentrations and only slightly increases above 4 nm at 500 μM Cu2+. BLI sensorgrams reveal distinctly different binding kinetics and mechanisms of α-LA and AuQCs with Cu2+ in the light of two-active-site binding and Michaelis-Menten enzyme-substrate catalysis, respectively. After screening the valence states by XPS and 3,5-DiBr-PAESA colorimetric assays, we found the majority of Cu species were metallic Cu(0) with only minor Cu+/Cu2+, validating the anti-galvanic catalytic reduction by AuQCs. The overall chelator-free 64Cu radiolabeling efficiency was calculated to be >60% within 1 h at room temperature, maintaining high stability in 54 h. Of the versatile immune cell landscape, dendritic cells (DCs) and macrophages were identified as the dominant populations that interact with AuQCs. In addition to macrophage phenotypic polarization from M2 to M1 toward the anti-cancer direction, AuQCs primarily regulated DCs by promoting their maturation and migration, resulting in an enhanced activity to process and present tumor antigens for naïve T cell priming. Both in vivo and ex vivo imaging disclosed restricted retention in the ipsilateral side rather than the contralateral side, involving major LNs of axillary, inguinal, popliteal, and iliac LNs. PET imaging pre-surgically pinpointed SLNs, while companion NIRF image-guided resection precisely delineated node contours contrasted to surrounding dense connective stroma and collagenous fibrous tissues. Biodistribution demonstrated predominant radioactivities in the

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Figure 1: Characterization and room-temperature, chelator-free ⁶⁴Cu labeling of AuQCs. a, Representative STEM images of AuQCs with a magnified individual probe shown in the inset. b, Hydrodynamic sizes, c, polydispersity indexes and zeta potentials, d, fluorescence emission spectra, e, derived NIRF quenching dynamics of AuQCs after interaction with different concentrations of ^NatCu²⁺. NIRF phantom imaging of AuQCs in the presence of gradient concentrations of f, ^NatCu²⁺ and g, radioactive ⁶⁴Cu²⁺. Corresponding NIRF emission curves for g with ⁶⁴Cu²⁺. BLI sensorgrams of i, α-LA and j, AuQCs in the presence of gradient concentrations of ^NatCu²⁺, resulting in (k) distinctly different fitted binding kinetics. l, XPS spectra of ^NatCu²⁺-AuQCs in the Cu 2p region. m, iTLC chromatograms of AuQCs after interaction with ⁶⁴Cu²⁺ for different durations at room temperature. n, Quantified relative radioactivity of bound and unbound ⁶⁴Cu species after different interaction time periods. o, Ambient stability of ⁶⁴Cu-AuQCs by iTLC at different time points. p, Time-bound radioactivity correlation as a function of time from the chromatograms in o.

Figure 2: Dual-modality LN imaging by PET and NIRF in living mouse models. a, NIRF imaging for detecting draining LNs 1 h after footpad injection of AuQCs in wild-type mice. b, PET/CT imaging at different time points following footpad injection of ⁶⁴Cu-labeled AuQCs in wild-type mice. c, The time-activity curve (TAC) of the footpad from PET/CT images for quantitative analysis. d, TAC curves of major LNs of axillary, inguinal, popliteal, and iliac LNs with PET signals. e, TAC curves of the liver and kidneys. f, Ex vivo NIRF imaging and g, signal quantification of dissected LNs 1 h post footpad injection. h, Quantified associated and dissociated ⁶⁴Cu species in the liver, kidneys, and blood. i, iTLC chromatograms of tissue homogenates from indicated organs. Ex vivo NIRF-imaging and H&E staining of j, surgically resected SLN under image guidance and k, the magnified region of interest showing LN margins. Coronal and MIP PET/CT images showing LN detection in xenograft mice bearing SCLC PDX tumors l, after footpad injection, and m and n, after peritumoral injection with ⁶⁴Cu-AuQCs. o, Biodistribution of systemically circulating nanoprobes after intravenous injection, calculated by a gamma counter.

Figure 3: AuQCs as a cancer vaccine. a, Uptake of AuQCs in different immune cells in LNs. b, A schematic diagram showing the maturation of bone marrow-derived dendritic cells (BMDCs) regulated by AuQCs. c, Phase-contrast microscopic images of BMDCs with or without treatment of AuQCs. d, A schematic diagram showing the antigen-presenting process to naïve T cells by DCs. e, Flow cytometry analysis of maturation and antigen presentation of DCs influenced by AuQCs. f, Quantitative analysis of matured DC and APC populations. g, Flow cytometry analysis of MHC class I presented antigen levels. h, The experimental scheme evaluating AuQCs as a cancer vaccine for therapy in B16F10-OVA allograft mouse models. i, Tumor growth curves, j, tumor weights, k, the photograph of tumors, and l, survival curves of allograft mice receiving therapeutic cancer vaccines of AuQCs and OVA_257-264. m, The experimental scheme using AuQCs vaccines in combination with anti-PD-L1 ICI therapy. i, Tumor growth curves and j, tumor weights harvested from allograft mice. p, The experimental scheme using AuQCs as a prophylactic cancer vaccine. q, Individual tumor growth curves in different groups of B16F10-OVA allograft mice. r, The experimental scheme using AuQCs as vaccines in combination with anti-PD-1 antibody ICI therapy in 4T1 allograft mouse models. s, The photograph, t, tumor weights, and u, tumor growth curves of primary orthotopic tumors. v, Quantification of metastasized lung nodules.

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