Abstract

   Colorectal cancer (CRC) is among the top five leading cancers
   worldwide. Preoperative concurrent chemoradiotherapy is recommended for
   locally advanced CRC. Radiotherapy (RT), a traditional cancer
   treatment, not only controls local tumor growth but also potentially
   induces immunogenic cell death, initiating systemic immune responses.
   Given the poor radiosensitivity of CRC, improving RT sensitization is a
   critical unmet need. Despite advances in intraoperative imaging,
   achieving complete resection of colorectal tumors with clear margins in
   real time remains a significant clinical challenge. This study
   introduces RVLu@ICG, a novel multifunctional fluorescent nanoprobe
   emitting in the second near‐infrared (NIR‐II) range. It's demonstrated
   that RVLu@ICG has tumor‐specific targeting due to modification with
   cyclic arginine‐glycine‐aspartic acid (c(RGDfK)) pentapeptide and
   induces augmented reactive oxygen species (ROS) production under
   ionizing radiation exposure. This synergistic mechanism not only
   potentiates radiosensitization efficacy but also facilitates
   radiation‐induced remodeling of the tumor immune microenvironment.
   Additionally, NIR‐II fluorescence image guidance facilitates precise
   surgical navigation in microtumor models, intramuscular tumor invasion
   models, and peritoneal metastasis models. Notably, the nanoprobe
   demonstrates excellent biocompatibility both in vitro and in vivo.
   Thus, RVLu@ICG establishes a robust precision therapy platform for the
   treatment of CRC.

   Keywords: colorectal cancer, immune microenvironment remodeling, NIR‐II
   fluorescence imaging, precise discrimination of surgical margins,
   radiotherapy sensitization
     __________________________________________________________________

   Enhancing radiosensitization, modulating the tumor immune
   microenvironment, and ensuring complete resection are crucial for
   colorectal cancer (CRC) treatment. This study presents a
   multifunctional NIR‐II fluorescent nanoprobe, RVLu@ICG, which enhances
   radiosensitization, remodels the tumor immune microenvironment, and
   enables precise image‐guided resection in CRC models. Demonstrating
   excellent biocompatibility, RVLu@ICG offers a promising platform for
   integrated radio‐immunotherapy and surgical management of CRC.

   graphic file with name ADVS-12-e10136-g009.jpg

1. Introduction

   According to Global Cancer Statistics 2022, colorectal cancer (CRC)
   ranks among the most prevalent malignancies globally, constituting 9.6%
   of total cancer diagnoses and 9.3% of cancer‐related mortality.^[
   [44]^1 ^] Neoadjuvant radiotherapy or chemoradiotherapy (RT/CRT)
   represents a clinically recommended neoadjuvant therapeutic regimen for
   patients with locally advanced CRC. This intervention achieves the
   reduction of tumor volume to enable surgical resection, particularly
   for locally advanced or anatomically constrained lesions.^[ [45]^2 ^]
   Emerging evidence indicates that, beyond direct tumoricidal effects, RT
   exerts pleiotropic immunomodulatory effects on the tumor
   microenvironment (TME). Notably, ionizing radiation promotes the
   presentation of tumor‐associated antigens (TAAs) and triggers the
   release of damage‐associated molecular patterns (DAMPs) in the TME.^[
   [46]^3 , [47]^4 ^] This process facilitates their recognition by
   dendritic cells (DCs)‐expressed pattern recognition receptors, thereby
   initiating a signaling cascade culminating in immunogenic cell death
   (ICD) of malignant cells.^[ [48]^5 , [49]^6 ^]

   Due to the moderate radiosensitivity of CRC and the anoxic
   microenvironment of the tumor, higher and potentially harmful radiation
   doses are often required to achieve adequate tumoricidal effects.^[
   [50]^7 ^] Paradoxically, the therapeutic intensification may negatively
   impact normal tissues by disrupting vascular permeability, inducing
   fibrosis, and worsening hypoxia.^[ [51]^8 , [52]^9 ^] An effective
   strategy to address these limitations involves augmenting the
   therapeutic efficacy of radiotherapy (RT) while concurrently amplifying
   RT‐induced ICD through the incorporation of radiosensitizing agents
   within the TME. High atomic number (Z) elements—such as gold,^[ [53]^10
   ^] selenium,^[ [54]^11 ^] and gadolinium,^[ [55]^12 ^]—exhibit
   promising radiosensitizing properties by enhancing X‐ray absorption and
   retention. Upon irradiation, these metals emit secondary electrons that
   facilitate intracellular water radiolysis, leading to the generation of
   free radicals capable of inducing DNA double‐strand breaks (DSBs), with
   reduced off‐target toxicity.^[ [56]^13 ^] Rare earth based
   nanoparticles have been widely proven to be effective as radiation
   sensitizers. Compared to the elements reported above, the atomic number
   of lutetium (Lu) is higher (Z = 71), exhibiting greater sensitivity to
   RT. This fundamental property endows Lu‐based nanosensitizers with
   superior translational potential in precision RT, particularly for
   deep‐seated malignancies requiring optimized radiation dose deposition.

   Emerging evidence suggests that the efficacy of radiosensitization
   arises from its capacity to remodel the TME, augment cellular
   susceptibility to ionizing radiation, and potentiate antitumor immune
   activation. However, these strategies remain insufficient for complete
   eradication of tumor tissues. Consequently, radical surgical resection
   remains a core component in the treatment of locally advanced CRC. By
   surgically removing all visible tumor tissue and ensuring negative
   margins (R0 resection), the risks of postoperative local recurrence and
   distant metastasis can be significantly reduced, thereby improving
   patients' long‐term survival rates.^[ [57]^14 ^] And the intraoperative
   determination of tumor margins currently relies primarily on
   pathological diagnosis through frozen section analysis.^[ [58]^15 ^]
   However, this analysis could not provide real‐time intraoperative
   identification of margins, and limited sampling points could neither
   comprehensively reflect the whole tumor margin.^[ [59]^16 ^]
   Approximately 5% of colorectal cancer (CRC) cases present with positive
   surgical margins, a rate that significantly increases with tumor
   progression—reaching up to 28% in cases of locally advanced
   disease—thereby contributing to elevated recurrence risk.^[ [60]^17 ,
   [61]^18 , [62]^19 ^] Additionally, peritoneal metastases (PM) occur in
   an estimated 3–20% of CRC patients, further complicating clinical
   outcomes.^[ [63]^20 ^]

   Encouragingly, fluorescence‐based imaging offers non‐invasive and
   repeatable in vivo visualization, delivering real‐time feedback with
   exceptional temporal and spatial resolution. It's expected to achieve
   the task of differentiating malignant tissue from benign tissue.^[
   [64]^21 , [65]^22 ^] Over the last few years, FDA‐approved
   near‐infrared regtion (NIR, 650–1700 nm) fluorophore indocyanine green
   (ICG) has been extensively investigated for image‐guided surgery within
   the second near‐infrared (NIR‐II, 1000–1700 nm) window due to its
   favorable optical properties. NIR‐II offers less tissue absorption,
   autofluorescence, and photon scattering, deeper tissue penetration,
   better spatial resolution, and an improved tumor‐to‐background ratio
   (TBR).^[ [66]^23 , [67]^24 , [68]^25 , [69]^26 ^] But concerns remain
   regarding ICG's limited anti‐photobleaching and tumor tissue
   recognition capabilities.^[ [70]^27 , [71]^28 ^] Fortunately, our
   previous work demonstrated that loading ICG into the mesopores of
   nanocarriers significantly enhances its photostability performance.^[
   [72]^29 ^] Consequently, the design of mesoporous nanocarriers capable
   of responding to the TME for ICG loading presents a promising strategy
   for enabling NIR‐II fluorescence‐guided surgical interventions.

   Molecular image‐guided cancer therapy has gained considerable attention
   as a promising strategy for enhancing treatment outcomes.^[ [73]^30 ^]
   This approach enables accurate tumor localization and facilitates
   sustained in vivo accumulation of therapeutic agents. In the pursuit of
   more efficient image‐guided modalities, the development of
   multifunctional therapeutic agents has become a focal point. Various
   inorganic nanomaterials—such as iron oxide‐based nanoparticles, gold
   nanostructures,^[ [74]^31 ^] gold nanostructures,^[ [75]^32 ^] and
   Gadolinium‐Bismuth composites^[ [76]^33 ^]—have been investigated.
   Despite their potential, clinical translation of many of these
   nanoplatforms remains limited due to concerns over biostability,
   biocompatibility, and synthetic complexity. Notably, our previous work
   introduced a degradable, virus‐mimetic hollow nanoparticle based on
   neodymium (Nd), which disassembles into ∼5 nm Nd[2]O[3] nanogranules
   under physiological conditions. Remarkably, approximately 40% of these
   nanoprobes are renally cleared within 24 hours, highlighting their
   favorable in vivo safety profile.

   Additionally, we found the unique virus‐like mesoporous exhibited a
   higher and faster internalization rate than ordinary mesoporous silica
   nanoparticles.^[ [77]^34 , [78]^35 ^] Therefore, we strategically
   incorporated ICG into a biodegradable Lu[2]O[3] nanoprobe with hollow
   virus‐like morphology (VLu@ICG). Specially, since α[v]β[3] integrin was
   overexpressed in CRC, we conjugated tumor‐targeting peptide, cyclic
   arginine‐glycine‐aspartic acid (c(RGDfK)) motifs (RVLu@ICG), on the
   prepared novel Lu‐based virus‐like mesoporous RT sensitizer, to pursue
   specific binding to α[v]β[3] integrin receptors to improve the precise
   targeting capability of CRC.

   Finally, we successfully developed a multifunctional nanoreagent
   RVLu@ICG for RT enhancement, immune microenvironment remodeling, and
   fluorescence imaging (Figure [79]1 ). CRC can be thoroughly suppressed
   by the highly effective RT and immune activation both in vitro and in
   vivo. What's more, residual tumor tissues were successfully excised
   under NIR‐II fluorescence guidance, with negligible evidence of local
   recurrence or distant metastasis observed within 28 days
   postoperatively. Collectively, these findings underscore the promising
   preclinical utility of a NIR‐II biodegradable radiosensitizer in
   improving therapeutic outcomes through efficacious RT, immune
   microenvironment remodeling, and enhancing precise tumor margin
   detection.

Figure 1.

   Figure 1
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   Schematic representation of the synthesis and application of RVLu@ICG
   nanoprobe for NIR‐II fluorescence‐guided targeted radiosensitization,
   modulation of the immune microenvironment, and precise CRC surgical
   resection. Abbreviations: VSi, virion‐like mesoporous silica
   nanoparticle; Pre‐op, preoperative; After‐op, postoperative.

2. Results

2.1. Fabrication and Physicochemical Profiling of RVLu@ICG

   Virion‐mimicking mesoporous silica nanoparticles (VSi) were fabricated
   via a conventional biphasic synthesis utilizing a hard‐template
   methodology. After 72 hours of mild agitation in a slightly alkaline
   environment, transmission electron microscopy (TEM) revealed the
   successful generation of uniform ≈100 nm particles with distinct
   virus‐like surface architecture (Figure [81]2A). The VSi structure
   served as the core scaffold, while Lu(NO[3])[3]·6H[2]O was employed as
   the Lu source and urotropine acted as the reductant. After a 17‐hour
   reaction at 80/90 °C, homogeneous hollow virion‐like mesoporous
   Lu[2]O[3] nanospheres (VLu) with a diameter of ≈110 nm was successfully
   obtained, displaying a rough surface morphology (Figure [82]2A).
   Scanning electron microscopy (SEM) further revealed that both VSi and
   VLu exhibited a uniform biomimetic virus‐like nanostructure
   (Figure [83]2B). High‐angle annular dark field (HAADF) scanning
   electron microscopy, accompanied by elemental mapping, demonstrated a
   uniform spatial distribution of Lu and oxygen (O) within individual VLu
   nanostructures, verifying the effective construction of the
   Lu‐integrated hollow nanoprobes (Figure [84]2C).

Figure 2.

   Figure 2
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   Synthesis and characterization of the nanoprobe. A) TEM micrographs of
   VSi (left) and VLu (right). B) SEM images of VSi (left) and VLu
   (right). C) Elemental distribution mapping of VLu: left—HAADF image;
   middle—elemental maps showing Lu (red) and O (green); right—merged
   overlay. D) High‐resolution TEM of RVLu. E) UV–vis absorption spectra
   of c(RGDfK), VLu, VLu‐NH₂, and RVLu; blue circled regions highlight the
   distinctive absorption peak at 258 nm corresponding to the c(RGDfK)
   peptide. F) Zeta potential measurements of VLu, VLu‐NH₂, and RVLu (n =
   3; data presented as mean ± SD). G) Standard curve for ICG
   quantification. H) Absorption spectra of free ICG, VLu@ICG, and
   RVLu@ICG. I) Fluorescence emission profiles within the NIR‐II window
   for ICG, VLu@ICG, and RVLu@ICG. J) NIR‐II fluorescence images of ICG
   (top) and RVLu@ICG (bottom) under continuous 808 nm laser excitation
   (0.1 W/cm^2) at 37 °C over time. K) Quantitative fluorescence intensity
   corresponding to panel (J). L) NIR‐II fluorescence images of RVLu@ICG
   supernatants incubated at pH 7.4, 6.5, and 5.5 for varied time
   intervals. M) Fluorescence intensity analysis corresponding to panel
   L). Abbreviation: HAADF, high‐angle annular dark‐field imaging.

   VLu nanospheres were subsequently functionalized with amino groups
   using amino silane (3‐aminopropyl) triethoxysilane (APTES). To achieve
   covalent conjugation of c(RGDfK) peptides onto the VLu surface (RVLu),
   a standard N‐hydroxysuccinimide
   (NHS)/1‐(3‐dimethylaminopropyl)‐3‐ethylcarbodiimide hydrochloride (EDC)
   reaction was performed. As illustrated in Figure [86]S3 (Supporting
   Information), RVLu retained the original surface morphology of VLu.
   High‐resolution transmission electron microscopy (HRTEM) analysis
   further confirmed that RVLu consisted of interpenetrated nanogranules
   (Figure [87]2D), while the absence of lattice fringes indicated their
   amorphous nature. ″Moreover, the UV‐vis absorption spectrum revealed a
   distinct absorption peak at 258 nm, characteristic of the c(RGDfK)
   peptide, verifying its successful attachment to the nanoshell surface
   (Figure [88]2E). Based on the standard calibration curves of c(RGDfK)
   (Figures [89]S4 and [90]S5, Supporting Information), the c(RGDfK)
   loading was quantified to be 17.01%, corresponding to a mass ratio of
   1:11.76 (c(RGDfK) to RVLu@ICG, w/w). Moreover, zeta potential results
   showed that the surface charge varied during different preparation
   stages, again implying the successful construction of RVLu
   (Figure [91]2F). Furthermore, dynamic light scattering (DLS)
   measurements revealed that the hydrodynamic diameters of VLu,
   VLu‐NH[2], and RVLu particles were 112.45 nm, 120.54 nm, and 136.87 nm,
   respectively, thereby validating the effectiveness of the peptide
   conjugation strategy (Figure [92]S6, Supporting Information).

   Subsequently, ICG was encapsulated within the hollow RVLu particles
   (RVLu@ICG), with an ICG loading efficiency of 23.56% according to the
   ICG standard curve (Figure [93]2G). Absorption spectra revealed a
   prominent peak near 780 nm for both RVLu@ICG and free ICG, indicating
   successful encapsulation of ICG within the nanoprobe cavity
   (Figure [94]2H). Moreover, upon 808 nm laser excitation and detection
   through a 1000 nm long‐pass filter, both formulations displayed
   characteristic NIR‐II emission profiles exceeding 1000 nm, affirming
   their optical activity in NIR‐II window (Figure [95]2I). Collectively,
   we have successfully engineered RVLu@ICG for acting a NIR‐II contrast
   agent. And the mole ratio of ICG, Lu and c(RGDfK) in RVLu@ICG was
   figured out by UV‐vis spectrophotometer and inductively coupled plasma
   as 1:330.65:18.54.

   The significant photobleaching of ICG has greatly limited its clinical
   utility. To evaluate its stability in comparison to ICG, we
   investigated the photostability of RVLu@ICG dispersed in PBS. As
   depicted in Figure [96]2J,K, following continuous irradiation with an
   808 nm laser at room temperature, RVLu@ICG owned a superior
   photobleaching resistance with approximately 100% fluorescence
   intensity maintaining, while that of ICG only maintained 28.74%.
   Because ICG is unstable in aqueous solutions, the NIR‐II fluorescence
   intensity of ICG and RVLu@ICG (dispersed in PBS for different time at
   room temperature) were latterly measured. As a result, free ICG lost
   approximately 84.27% of its initial fluorescence intensity over 12
   hours. In contrast, RVLu@ICG retained more than 75% of its initial
   fluorescence intensity after 12 hours (Figures [97]S8 and [98]S9,
   Supporting Information). These findings clearly indicate that the
   aqueous stability of ICG was significantly enhanced when encapsulated
   in RVLu. The fluorescence results collectively confirmed the potential
   of RVLu@ICG as a NIR‐II contrast agent for prolonged surgical
   navigation under laser irradiation. As we reported previously,^[
   [99]^34 ^] these distinctive hollow virus‐like nanoparticles were
   formed through the surface assembly of metal oxide nanogranules,
   indicating that RVLu possessed pH‐sensitive decomposition performance.
   As displayed in Figure [100]S10 (Supporting Information), the nanoprobe
   gradually degraded in a normal physiological environment (pH 7.4),
   maintaining relatively structural stability for up to 36 hours. In
   contrast, RVLu demonstrated progressive degradation over time under
   acidic conditions representative of the intracellular milieu (pH 5.5)
   and the TME (pH 6.5), achieving complete decomposition within 6 and 12
   hours, respectively. As anticipated, a large number of Lu‐containing
   nanogranules with diameters of < 5 nm were observed by high‐resolution
   TEM (HRTEM) following a 6‐hour incubation at pH 5.5 (Figure [101]S11,
   Supporting Information), suggesting that the interparticle interactions
   within RVLu nanoparticles become destabilized under mildly acidic
   conditions, resulting in structural disassembly. Notably, the resulting
   small nanogranules are rapidly excreted via renal metabolism.^[
   [102]^35 ^] To evaluate the pH‐responsive release behavior of ICG from
   RVLu@ICG under varying physiological and acidic conditions (pH 7.4,
   6.5, and 5.5), release kinetics were systematically assessed. As
   depicted in Figure [103]2L,M and Figure [104]S12 (Supporting
   Information), incubation at pH 7.4 for 12 hours resulted in the
   liberation of less than 35% of the encapsulated ICG, indicating minimal
   leakage under neutral conditions. In contrast, a significant release of
   ICG from RVLu@ICG was observed when the pH was reduced to 6.5. Within 2
   hours of incubation at pH 5.0, the release of ICG reached up to 90%.
   These results proved that our TME RVLu@ICG facilitated the rapid
   diffusion of ICG within the tumor and demonstrated very high biological
   safety.

2.2. Assessment of Cancer Cell‐Specific Targeting Efficiency of RVLu@ICG in
Vitro

   Cancer cell precise recognition is essential to precise treatment of
   tumor tissues successfully. RVLu@ICG was utilized for tumor cell
   targeting studies using flow cytometry and confocal laser scanning
   microscopy (CLSM). Prior to assessing cellular internalization, the
   biocompatibility of the nanoprobes was evaluated through hemolysis and
   cell proliferation assays. The results from the hemolysis experiment
   (Figure [105]3A) show no significant hemolysis, with the hemolysis
   ratio of RVLu@ICG at all concentrations (0–80 µM) remaining below 5%,
   indicating good blood compatibility. Additionally, as shown in Figure
   [106]S13 (Supporting Information), the viability of CT26 cells remained
   above 87% after incubation with RVLu@ICG (0‐40 µM), suggesting that
   RVLu@ICG exhibits no obvious toxicity of RVLu@ICG.

Figure 3.

   Figure 3
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   Cell cytotoxicity, cellular uptake and RT sensitivity of RVLu@ICG in
   vitro. A) Representative images and quantitative analysis of hemolysis
   for various treatments (n = 3). B) Flow cytometry quantification of
   CT26 cell internalization of ICG, VLu@ICG, and RVLu@ICG at 0.25, 0.5,
   1, 2, and 4 hours post‐incubation. C) CLSM images of CT26 cells after
   2‐hour incubation with ICG, VLu@ICG, RVLu@ICG, and RVLu@ICG following
   c(RGDfK) pretreatment. D) Viability of CT26 cells treated with PBS or
   RVLu@ICG, with or without 4 Gy X‐ray irradiation; non‐irradiated PBS
   and RVLu@ICG served as controls (n = 3). E) Colony formation assays of
   CT26 cells exposed to various formulations. F) Apoptosis analysis by
   flow cytometry in CT26 cells treated with PBS, RVNd@ICG, RVGd@ICG, and
   RVLu@ICG under 4 Gy irradiation; corresponding non‐irradiated groups
   served as controls (n = 3). G) Intracellular ROS levels measured by
   flow cytometry in CT26 cells after PBS or RVLu@ICG treatment
   with/without 4 Gy X‐ray exposure. H, I) CLSM images and MFI analysis of
   ROS levels in CT26 cells following different treatments. J, K) CLSM
   images and MFI analysis of calreticulin (CRT) surface exposure in CT26
   cells following different treatments. L, M) CLSM visualization and MFI
   quantification of HMGB1 release from treated CT26 cells. Scale bars
   represent 25 µm. Statistical significance was determined by two‐way
   ANOVA and two‐tailed unpaired t‐tests: **P < 0.01; ***P < 0.001; ****P
   < 0.0001. Data are presented as mean ± SD (n = 3).

   Meanwhile, the cellular uptake efficiency of the CT26 cell line was
   conducted after treatment with different formulations of nanoprobes.
   According to previous reports, the unique virus‐like spikes on the
   surface of nanoparticles can enhance cellular uptake,^[ [108]^36 ,
   [109]^37 ^] possibly because these protrusions impose greater
   mechanical stress on the cell membrane compared to smooth‐surfaced
   particles, thereby strengthening their interaction with biological
   systems.^[ [110]^38 ^] To validate this hypothesis, we have
   successfully fabricated ≈110 nm normal mesoporous Lu[2]O[3]&NPs
   (MLu@ICG) (Figure [111]S14, Supporting Information). As anticipated,
   the distinct virus‐like mesoporous structure of VLu@ICG demonstrated a
   significantly higher internalization rate compared to MLu@ICG,
   highlighting its superior cellular uptake efficiency. This enhanced
   recognition capability suggests improved targeting of cancerous tissues
   (Figure [112]S14, Supporting Information). Later, we were encouraged to
   evaluate the CT26 tumor recognition ability of the biodegradable NIR‐II
   nanoprobes. Flow cytometry results demonstrated that RVLu@ICG achieved
   approximately twice the cellular uptake compared to VLu@ICG lacking
   c(RGDfK) functionalization (Figure [113]3B; Figure [114]S15, Supporting
   Information), with a statistically significant difference (P < 0.001).
   Finally, the endocytosis of CT26 cells was further performed by CLSM.
   After a 4‐hour incubation with tumor cells, the RVLu@ICG group
   exhibited significantly higher red fluorescence (ICG) intensity, with a
   mean fluorescence intensity (MFI) three times stronger than that of the
   VLu@ICG group (Figure [115]3C; Figure [116]S16, Supporting Information;
   P < 0.01). These findings validate the improved cellular uptake and
   selective tumor‐targeting properties of the nanoprobes conferred by
   c(RGDfK) functionalization. Additionally, a competitive inhibition
   assay was performed by pre‐incubating cells with an excess of c(RGDfK)
   peptide. In this pre‐treated group, RVLu@ICG uptake was notably
   reduced, as evidenced by both flow cytometry analysis (Figures [117]S17
   and [118]S18, Supporting Information; P < 0.001) and CLSM imaging
   (Figure [119]3C; Figure [120]S16, Supporting Information; P < 0.0001).
   These findings further validate that the Lu‐based nanoprobes
   functionalized with c(RGDfK) can accurately recognize and target tumor
   cells.

2.3. Radiation Sensitization in Vitro

   Subsequently, we were inspired to evaluate the sensitizing effect of
   RVLu@ICG in RT. Under X‐ray irradiation (4 Gy), the cytotoxicity of
   nanoparticles (Lu^3⁺ concentration = 20 µM) was notably higher than
   that of radiation alone, indicating that nanoparticle‐mediated RT
   significantly increased radiation‐induced cell death in tumor cells (P
   < 0.001) (Figure [121]3D). Additionally, colony formation assays were
   performed to evaluate the prolonged radiation sensitization effect of
   RVLu@ICG. CT26 tumor cells treated with RT and nanoparticles formed
   fewer viable cell colonies compared to the RT group, with a significant
   difference (P < 0.001) (Figure [122]3E; Figure [123]S19, Supporting
   Information). Additionally, the impact of RVLu@ICG + RT on apoptosis
   was assessed. Flow cytometry analysis demonstrated a significantly
   higher apoptosis rate in the RVLu@ICG + RT group compared to RT alone
   (P < 0.001) (Figure [124]3F; Figure [125]S21, Supporting Information).
   Moreover, the radiosensitization effect of Lu (RVLu@ICG) was markedly
   superior to that of Gd (RVGd@ICG) and Nd (RVNd@ICG) (Figure [126]3F;
   Figure [127]S21, Supporting Information; P < 0.001). TEM analysis
   revealed that the morphologies of RVNd@ICG and RVGd@ICG were highly
   similar to that of RVLu@ICG (Figure [128]S22, Supporting Information).
   This finding indicates that the observed differences in
   radiosensitizing efficacy are unlikely to result from morphological
   disparities, and are more likely associated with the higher atomic
   number of Lu. Further, we have also investigated the cancerous cell
   killing by RVLu@ICG + RT using a live/dead cell‐stained assay.
   Analogous to apoptosis findings, most of the tumor cells were stained
   with red fluorescent dyes when they were treated with RVLu@ICG + RT
   (Figure [129]S23, Supporting Information). Ionizing radiation induces
   tumor cell apoptosis primarily via intracellular reactive oxygen
   species (ROS)‐mediated DNA damage.^[ [130]^11 , [131]^39 ^] To assess
   ROS levels, 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA)
   staining was performed. Post‐irradiation, CT26 cells exposed to
   RVLu@ICG exhibited markedly enhanced green fluorescence relative to the
   PBS control, accompanied by a significant elevation in MFI (P < 0.0001)
   (Figure [132]3H,I; Figure [133]S24, Supporting Information).
   Fluorescence signals from both the non‐irradiated PBS and RVLu@ICG
   groups were minimal (Figure [134]3H,I; Figure [135]S24, Supporting
   Information). Flow cytometry results revealed that ROS levels in CT26
   cells treated with RVLu@ICG combined with RT were approximately
   eightfold greater than those observed in the RT group (Figure [136]3G;
   Figure [137]S25, Supporting Information), suggesting that RVLu@ICG
   significantly enhanced intracellular ROS production under X‐ray
   irradiation that substantially induced CRC cell apotosis/necrosis.
   These results further verified that RVLu@ICG possessed a powerful
   radiosensitization effect that exhibited significant impediments toward
   cancerous cell proliferation, and can significantly induce the death of
   cancer cells, making it a prospective radiotherapeutic nanoagent.

2.4. RT‐induced Immune Activation in Vitro

   Extensive research has demonstrated that RT facilitates hydroxyl
   radical (•OH) generation upon ionizing radiation, thereby initiating
   ICD and stimulating antitumor immune responses. One hallmark of ICD is
   the translocation of calreticulin (CRT) from the endoplasmic reticulum
   to the plasma membrane. To assess this, immunofluorescence staining
   using Alexa Fluor 594‐conjugated anti‐CRT antibody was conducted. As
   depicted in Figure [138]3J,K, a significant increase in red
   fluorescence intensity was observed in CT26 cells following X‐ray
   exposure, indicating enhanced CRT surface expression. Notably, the
   RVLu@ICG + RT group exhibited even stronger CRT membrane localization
   compared to RT alone, attributable to the radiosensitizing effect of
   Lu. In parallel, the release of high mobility group box 1 (HMGB1),
   another key ICD marker, was investigated via immunofluorescence
   analysis. As ROS generated by RT compromise nuclear membrane integrity,
   HMGB1 is translocated to intercellular substance. Interestingly, an
   inverse trend was detected for HMGB1 relative to CRT expression
   (Figure [139]3L,M); the RVLu@ICG + RT group displayed the lowest
   intracellular green fluorescence signal among all treatments.
   Quantitative CLSM confirmed these findings, revealing that this
   combination therapy significantly reduced nuclear HMGB1 retention while
   promoting CRT externalization. Collectively, these results provide
   compelling evidence that RVLu@ICG, under X‐ray irradiation, induces
   robust ICD in vitro, thereby enhancing tumor immunogenicity and
   potentially contributing to a systemic (abscopal) antitumor response.

2.5. Biosafety, Cancer Targeting, and Biodistribution of RVLu@ICG in Vivo

   Before in vivo fluorescent imaging studies, in vivo safety was first
   investigated. Healthy female BALB/c mice were randomly assigned to four
   experimental groups. Similar to the PBS‐treated group, during the
   observation period of four weeks, no body weight alternation was
   recorded in the RVLu@ICG group (Figure [140]4A). Venous blood samples
   were serially sampled at day = 0/1/7/28 for liver and kidney function
   assessments, along with routine hematological analysis. The data
   indicated stable levels of creatinine (CREA), alanine aminotransferase
   (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN)
   (Figure [141]4B). Furthermore, no notable changes were detected in
   platelet (PLT), red blood cell (RBC), lymphocyte (Lymph), or white
   blood cell (WBC) counts (Figure [142]4C). Histological examination of
   hematoxylin‐eosin (HE)‐stained organ sections revealed no apparent
   pathological changes in either group (Figure [143]4D). These findings
   suggest that RVLu@ICG exhibited minimal toxicity in mice, supporting
   its potential safety for future clinical applications.

Figure 4.

   Figure 4
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   The safety and tumor targeting efficiency estimation of RVLu@ICG in
   vivo. A) Body weight monitoring of mice following intravenous
   administration of PBS or RVLu@ICG over 28 days. B) Biochemical blood
   panel analysis at multiple time points to assess hepatic and renal
   function after tail vein injection of RVLu@ICG (n = 3). C)
   Hematological profiles measured at different intervals post‐injection
   to evaluate effects on blood cell populations (n = 3). D)
   Representative HE stained sections of major organs from BALB/c mice
   after intravenous injection of PBS and RVLu@ICG for 1, 7, and 28 days,
   respectively (n = 3). E) Serial NIR‐II fluorescence imaging of mice
   following intravenous injection of ICG (top), VLu@ICG (middle), and
   RVLu@ICG (bottom) at various time points (n = 3). The blue dotted
   ellipses indicate the tumors. F) TBR quantification in tumor‐bearing
   mice injected with ICG, VLu@ICG, or RVLu@ICG over time (n = 3). G)
   Comparative analysis of tumor‐targeting efficiency among ICG, VLu@ICG,
   and RVLu@ICG at peak TBR. Statistical significance determined by
   two‐tailed unpaired t‐test: *P < 0.05; data expressed as mean ± SD (n =
   3). H) Representative NIR‐II fluorescence images of the RVLu@ICG
   targeting group and c(RGDfK) blocking group (40 mg kg^−1) at 48 hours
   post‐injection. The blue dotted ellipses indicate the tumors. I) Ex
   vivo bright‐field (top) and NIR‐II fluorescence images (bottom) of
   harvested organs (1‐8: brain, heart, liver, spleen, lung, kidney,
   tumor, muscle) collected 48 hours after RVLu@ICG administration. J)
   Quantification of MFI based on panel (I) (n = 3).

   To evaluate the in vivo targeting capability of RVLu@ICG, CT26‐Luc
   tumor cells were subcutaneously inoculated into BALB/c mice following
   the demonstration of efficient tumor cell identification in vitro and
   low systemic toxicity in vivo. Tumor‐bearing mice were administered
   ICG, VLu@ICG, or RVLu@ICG via the caudal vein. NIR‐II fluorescence
   imaging was conducted at various time intervals employing an 808 nm
   excitation wavelength (100 mW/cm^2) alongside a 1000 nm long‐pass
   filter. As illustrated in Figure [145]4E, fluorescence signals in the
   tumor area were scarcely observable in the ICG‐treated group. In
   contrast, the RVLu@ICG group exhibited a strong tumor‐specific
   fluorescence signal, reaching its peak intensity 48 hours
   post‐injection (Figure [146]4F). Furthermore, the maximum TBR of the
   RVLu@ICG group (6.50 ± 1.10) was remarkably higher than the VLu@ICG
   group (4.49 ± 0.63) and ICG group (2.49 ± 1.08) (Figure [147]4G, P <
   0.05). The fluorescence signals gradually diminished, becoming
   undetectable by 96 hours post‐injection. Significantly, the RVLu@ICG
   group exhibited sustained and elevated tumor fluorescence intensity
   compared to both VLu@ICG and ICG groups across all measured time
   points, underscoring the improved tumor‐targeting efficiency attributed
   to c(RGDfK) conjugation (Figure [148]4F). Based on the Rose criterion,
   a TBR exceeding 5 is considered adequate for precise tumor
   visualization.^[ [149]^40 ^] Thus, RVLu@ICG demonstrated superior tumor
   tissue discrimination compared to VLu@ICG and ICG. To further validate
   its targeting specificity, an in vivo blocking study was conducted by
   pre‐injecting c(RGDfK). Fluorescence imaging conducted 48 hours after
   administration demonstrated markedly greater signal intensity in the
   RVLu@ICG‐treated group relative to the receptor‐blocked control group
   (Figure [150]4H; Figure [151]S26, Supporting Information; 7.65 ± 1.61
   vs. 2.40 ± 0.44, P < 0.01). Collectively, these findings highlight the
   precise CRC‐targeting capability of RVLu@ICG. To further assess the
   biodistribution of the nanoprobe in vivo, mouse organs were harvested
   and subjected to fluorescence imaging 48 hours post‐injection, as
   illustrated in Figure [152]4I,J, these images and MFI revealed a high
   uptake in tumor as well as the reticuloendothelial system organs
   (including liver, kidney and lung).

2.6. Molecular Mechanism Analysis in Vivo

   Having confirmed the excellent tumor targeting ability, we subsequently
   explored the potential biological effects and antitumor mechanisms of
   RVLu@ICG + RT from the RNA level. RNA sequencing (RNA‐seq) analysis of
   tumor tissues receiving PBS or RVLu@ICG + RT irradiation treatments was
   conducted to identify the differentially expressed genes and
   corresponding signaling pathways. Results indicated that RVLu@ICG + RT
   irradiation induced many genes expression changes, especially the
   upregulated expression of immune activation‐related genes (such as
   Slc8a1, Tnk1, Ncam1 and Nr4a1) and down‐regulated expression of
   immunosuppressive genes (such as Hsd11b2, Itih4, and Zfp286) (Figure
   [153]5A,B), which posed impacts on the immune activation‐associated
   biological processes, cellular components, and molecular functions
   (Figure [154]5C). Correspondingly, the Reactome pathway enrichment
   analysis also uncovered significant up‐regulation of immune
   activation‐related pathways, such as signaling by MST1, AKT
   phosphorylates targets in the nucleus, arachidonic acid metabolism, and
   platelet calcium homeostasis (Figure [155]5D). Especially, the
   transmembrane receptor protein tyrosine kinase activity pathways were
   effectively activated, strongly implying the provoking of immune
   response during treatment of RVLu@ICG + RT irradiation (Figure
   [156]S28, Supporting Information). Moreover, the Estimation of STromal
   and Immune cells in MAlignant Tumor tissues using Expression data
   (ESTIMATE) score heatmap indicates that the Immune Score in the
   RVLu@ICG + RT group is higher than that in the PBS group, suggesting
   that the RVLu@ICG + RT group has greater immune cell infiltration and
   stronger immune activity (Figure [157]5E). Given the above results, we
   could conclude that RVLu@ICG + RT irradiation possessed effective
   immunomodulation ability on the TME during therapy.

Figure 5.

   Figure 5
   [158]Open in a new tab

   Molecular mechanism analysis of RVLu@ICG + RT in vivo. A) Volcano plot
   of differentially expressed genes. B) Clustering heatmap of individual
   genes in different treatment groups (Orange: up‐regulation; green:
   down‐regulation). C) Gene Ontology (GO) enrichment analysis of tumor
   tissues after receiving different treatments. D) Top 20 significantly
   enriched reactome pathways which were up‐regulated. E) Heatmap of TME
   ESTIMATE Scores in different treatment groups (Orange: up‐regulation;
   green: down‐regulation).

2.7. Radio‐Immunotherapy Antitumor Effect of RVLu@ICG in Vivo

   Leveraging the X‐ray activated Compton scattering properties of Lu^3+,
   tumor growth inhibition was evaluated in RVLu@ICG‐treated mice through
   intravenous administration. CT26 tumor‐bearing mice were randomly
   allocated into four groups: PBS, RVLu@ICG, RT, and RVLu@ICG + RT.
   Representative fluorescence images (Figure [159]S29, Supporting
   Information) confirmed the probe's selective targeting of CRC,
   prompting further investigation into its role in RT. As depicted in
   Figure [160]6A,D,E, tumor growth remained largely unrestrained in both
   nonirradiated PBS‐treated and RVLu@ICG‐treated mice. However, upon
   X‐ray exposure, RVLu@ICG‐treated mice exhibited significantly enhanced
   RT sensitization, leading to substantial tumor volume reduction
   compared to those receiving RT alone (Figure [161]6A,F,G). Furthermore,
   weight of resected tumors in RVLu@ICG + RT group is the lightest on day
   18, confirmed substantial tumor elimination and growth suppression in
   RT‐sensitized animals compared to the other groups (Figure [162]6B,C).
   Six days post‐X‐ray radiation, the body weight of the mice slightly
   decreased but returned to normal levels by day 10 (Figure [163]6H).

Figure 6.

   Figure 6
   [164]Open in a new tab

   Radiosensitization of RVLu@ICG in CT26‐tumor‐bearing mice. A) Tumor
   growth curves of mice treated with PBS or RVLu@ICG, with or without 8
   Gy X‐ray irradiation on day 2 (n = 5). B) Representative images of
   excised tumors from groups receiving PBS (I), RVLu@ICG (II), RT (III),
   and RVLu@ICG + RT (IV) treatments (n = 5). C) Tumor weights measured on
   day 18 post‐treatment (n = 5). Individual tumor volume progression for
   PBS D), RVLu@ICG E), RT F), and RVLu@ICG + RT G) groups (n = 5). H)
   Body weight monitoring of mice under different treatment regimens (n =
   5). I) Representative histological staining of tumor sections for HE,
   Ki67 proliferation marker, and cleaved caspase‐3 apoptosis marker,
   collected 18 days after treatments (n = 3). Scale bars, 100 µm. J, K)
   Quantitative analysis of Ki67 and caspase‐3 positive cells from panel
   (I). Data are presented as mean ± SD. Statistical significance was
   determined by one‐way ANOVA with Tukey's post hoc test: *P < 0.05, **P
   < 0.01, ***P < 0.001.

   To further evaluate the antitumor efficacy of radiotherapy,
   histological examination was conducted on tumor samples collected from
   various treatment groups after 18 days. Ki67 staining, a standard
   indicator of cellular proliferation, was utilized to assess in vivo
   therapeutic effects. Findings revealed a marked decrease in
   Ki67‐positive cells in the RVLu@ICG combined with RT group relative to
   all other cohorts (Figure [165]6I,J). Concurrently, caspase‐3 activity
   was markedly elevated in the RVLu@ICG + RT group relative to the
   remaining groups (Figure [166]6I,K). These findings indicate that
   RVLu@ICG + RT treatment effectively suppressed tumor cell proliferation
   while promoting apoptosis through the activation of effector caspase‐3
   and its downstream pathways. Additionally, a TUNEL assay was conducted
   to assess apoptosis rates in tumor sections. Tumors exposed to
   radiation monotherapy exhibited only marginally increased apoptosis
   compared to those treated with PBS or RVLu@ICG alone. Conversely, the
   combination of RVLu@ICG with radiotherapy induced substantially
   elevated apoptosis rates relative to both PBS and RVLu@ICG groups
   (Figures [167]S30 and [168]S31, Supporting Information). These
   immunohistochemical data strongly indicate that RVLu@ICG combined with
   RT effectively enhances tumor cell apoptosis and suppresses tumor
   progression, corroborating the observations from HE staining analyses.

2.8. Immune Activation Analysis in Vivo

   The pronounced antitumor efficacy observed with RVLu@ICG + RT is
   largely attributable to the synergistic interaction between ionizing
   radiation and immune modulation. To further elucidate the mechanism of
   immune activation, we evaluated immune cell populations and cytokine
   expression levels using flow cytometry, immunofluorescence staining,
   and ELISA assays. Maturation of DCs within tumor‐draining lymph nodes
   was assessed, revealing elevated proportions of CD80⁺CD86⁺ mature DCs
   in both RT‐treated groups, with the RVLu@ICG + RT cohort exhibiting the
   most pronounced increase, indicative of enhanced antigen‐presenting
   activity (Figure [169]7A,D; Figure [170]S34, Supporting Information).
   Subsequent analysis of tumor‐infiltrating lymphocytes showed
   significantly increased frequencies of both CD8⁺CD3⁺ and CD4⁺CD3⁺ T
   cells in the RVLu@ICG + RT group, suggesting potent activation of
   cellular and adaptive immune responses (Figure [171]7B,C,E,F; Figure
   [172]S35, Supporting Information). These findings were further
   corroborated by immunofluorescence imaging, which demonstrated a marked
   accumulation of CD8⁺ T cells and mature DCs within the tumor
   microenvironment (Figure [173]7G–I). Cytokine profiling further
   confirmed the immunostimulatory effect of the combined therapy. The
   RVLu@ICG + RT group exhibited the highest concentrations of
   proinflammatory cytokines—including IL‐6, IFN‐γ, and TNF‐α—alongside a
   notable suppression of the immunosuppressive cytokine IL‐10
   (Figure [174]7J–M). Specifically, RT alone elevated IFN‐γ secretion
   compared to PBS and RVLu@ICG groups, confirming its role in inducing
   IFN‐γ production. The addition of RVLu@ICG to RT further augmented
   IFN‐γ levels, underscoring that RVLu@ICG can further amplify
   radiotherapy‐associated immune activation through its radiosensitizing
   effects. IFN‐γ as well as other immune response together confirmed that
   the RVLu@ICG could efficiently sensitizing RT and provoke potent
   antitumor immune response.

Figure 7.

   Figure 7
   [175]Open in a new tab

   Immune activation analysis of RVLu@ICG + RT in vivo. A) Flow cytometry
   representative plots showing mature DCs (CD80^+CD86^+ gated on CD11c^+)
   in tumor‐draining lymph nodes. B, C) Representative flow cytometric
   profiles of CD8^+ (CD8^+CD3^+ gated on CD45^+) and CD4^+ (CD4^+CD3^+
   gated on CD45^+) T lymphocytes within tumor tissues. D–F) Quantitative
   analyses of mature DCs in lymph nodes, and CD8^+ and CD4^+ T cells in
   tumors across treatment groups (n = 5). G) Immunofluorescence images of
   tumor sections stained for nuclei (blue) and CD8^+ or CD86^+ cells
   (red) following various treatments. H, I) Quantification of MFI for
   CD8^+ and CD86^+ markers. J–M) ELISA measurements of serum cytokine
   concentrations for IL‐6, IL‐10, IFN‐γ, and TNF‐α post‐treatment (n =
   3). Data are shown as mean ± SD. Statistical significance: **P < 0.01,
   ***P < 0.001, ****P < 0.0001.

2.9. NIR‐II Fluorescence Image‐Guided Tumor Localization and Surgical
Resection in a Microtumor Model

   The highest tumor‐to‐background ratio (TBR) was observed at 48 hours
   post‐injection, identifying this as the optimal time for NIR‐II
   fluorescence‐guided tumor resection. To assess the nanoprobe's capacity
   for intraoperative detection of small tumors, a multiple microtumor
   model was established. The intraoperative fluorescence signals closely
   correlated with bioluminescence imaging results (Figure [176]8A),
   demonstrating the accuracy of NIR‐II fluorescence imaging in
   delineating tumor margins. Corresponding HE staining confirmed that the
   diameters of seven microtumors excised under NIR‐II guidance ranged
   from approximately 0.5 to 3 mm, with clear differentiation of tumor
   boundaries from adjacent normal tissue (Figure [177]8B; Figure
   [178]S36, Supporting Information). Importantly, microtumors T3, T4, and
   T5, each under 1 mm in diameter, were accurately identified,
   highlighting the effectiveness of RVLu@ICG fluorescence imaging for
   detecting small residual lesions during surgery.

Figure 8.

   Figure 8
   [179]Open in a new tab

   Surgery navigation of RVLu@ICG in the microtumor model, intramuscular
   tumor‐invasion model and the abdominal transfered carcinoma model. A)
   Representative preoperative bioluminescence, as well as pre‐ and
   post‐operative NIR‐II fluorescence images, of a mouse bearing multiple
   microtumors following RVLu@ICG injection. B) HE staining of tumor and
   adjacent muscle tissues corresponding to panel (A). C) Sequential
   imaging (white light and NIR‐II fluorescence) before, during, and after
   tumor excision in an intramuscular tumor‐infiltration model treated
   with RVLu@ICG (pre‐op: I, IV; intra‐op: II, V; post‐op: III, VI). D)
   Similar imaging of tumor resection guided by ICG in the same model,
   including bioluminescence, white light, and fluorescence views. E)
   Post‐surgical NIR‐II fluorescence images of two resected tumors (P1,
   P2) and adjacent muscle from panel (C). F) HE histology of excised
   tissues shown in panel (E). G) Kaplan–Meier survival analysis comparing
   outcomes among groups receiving RVLu@ICG‐, ICG‐, or white‐light‐guided
   surgery (n = 5); statistical significance determined using the Log‐rank
   test (P < 0.05). H) NIR‐II fluorescence‐guided pre‐ and post‐operative
   imaging of mice with peritoneally disseminated tumors. I) Corresponding
   HE‐stained sections of tumor and surrounding muscle. All scale bars
   represent 1000 µm.

2.10. NIR‐II Fluorescence‐Guided Real‐Time Tumor Resection in Intramuscular
Tumor‐Invasion Model

   An intramuscular tumor‐invasion model was constructed to evaluate the
   feasibility of our RVLu@ICG‐based NIR‐II nanoprobes for precisely
   identifying the tumors with a fluorescence imaging system. First,
   CT26‐Luc cells were implanted intramuscularly into BALB/c mice until
   tumor volume reached approximately 300–400 mm^3, which was confirmed by
   the bioluminescence imaging (Figures [180]S37–S39, Supporting
   Information). Then, the tumor‐bearing mice were anesthetized by
   isoflurane under aseptic conditions. As shown in Figure [181]8C, 48
   hours after RVLu@ICG administration, tumor margins were distinctly
   visualized via the NIR‐II fluorescence imaging system (Figure [182]8C
   I,IV). Initial excision of the primary tumor (P1) was performed under
   white light, followed by assessment of the surgical margins using
   NIR‐II imaging (Figure [183]8C II,V). Residual NIR‐II fluorescence
   detected in the surgical bed prompted additional resection (P2) in
   certain cases. The resection area was subsequently re‐examined under
   808 nm laser excitation to verify the complete absence of fluorescence
   signals, confirming thorough tumor clearance (Figure [184]8C III,VI).
   Peritumoral normal tissue was collected as a standard control. Ex vivo
   NIR‐II fluorescence imaging revealed that both P1 and P2 exhibited
   strong fluorescence signals, whereas the muscle tissue remained dark
   (Figure [185]8E). These findings were consistent with intraoperative
   fluorescence imaging results (Figure [186]8C IV,V). HE staining
   confirmed that the resected tissues exhibiting fluorescence signals
   were tumor tissues, while non‐fluorescent tissues were identified as
   normal tissue (Figure [187]8F). This unequivocally demonstrated that
   RVLu@ICG nanoprobes effectively differentiate tumors from normal
   tissues, assisting surgeons in achieving negative surgical margins. In
   contrast, in the ICG group, most residual tumors lacked fluorescence
   signals, underscoring the tumor‐targeting and retention limitations of
   ICG, which is commonly used in surgical oncology (Figure [188]8D).

   To evaluate tumor recurrence and metastatic spread, bioluminescence
   imaging was performed 14 days post‐surgery. Remarkably, no local tumor
   relapse or distant metastasis was observed in the RVLu@ICG group
   following NIR‐II fluorescence‐guided resection (Figure [189]S39,
   Supporting Information). Conversely, 80% of mice in the ICG‐treated
   group and all mice in the control cohort exhibited tumor regrowth at
   the original site (Figures [190]S38 and [191]S37, Supporting
   Information). These results underscore the superior efficacy of
   RVLu@ICG nanoprobes in achieving thorough tumor removal. Animals were
   monitored bi‐daily for survival assessment until tumor volumes reached
   2000 mm^3 or death occurred. Kaplan–Meier analysis (Figure [192]8G)
   revealed a significant survival advantage in the RVLu@ICG‐guided
   surgery group relative to ICG‐ and white light‐guided groups.

2.11. NIR‐II Fluorescence‐Guided Real‐Time Tumor Resection in Peritoneal
Metastasis Model

   We further explored the potential of RVLu@ICG‐based NIR‐II nanoprobes
   for intraoperative imaging in a CRC peritoneal metastases model.
   Initially, bioluminescence imaging confirmed tumor presence and
   provided an approximate localization (Figure [193]S42, Supporting
   Information). Under sterile conditions, mice were anesthetized with
   isoflurane, and tumor excision was performed sequentially guided by
   NIR‐II fluorescence imaging (Figure [194]8H; Figure [195]S40,
   Supporting Information). The tumor boundaries (1–8) were clearly
   visualized using NIR‐II imaging (Figure [196]8H; Figures [197]S40 and
   [198]S41, Supporting Information). HE staining of resected tissues
   verified the accurate delineation of tumor margins (Figure [199]8I). To
   assess potential residual recurrence, bioluminescence imaging was
   performed on day 14 post‐surgery, revealing no signs of local
   recurrence or metastasis (Figure [200]S42, Supporting Information).

3. Discussion

   Neoadjuvant radiotherapy has established itself as a cornerstone in the
   multidisciplinary management of patients with locally advanced
   colorectal cancer. Although conventional RT has demonstrated certain
   benefits in improving local tumor control and patient survival, its
   therapeutic efficacy remains limited by several factors, including
   treatment‐related toxicities and intrinsic radioresistance of tumor
   cells. A growing body of evidence suggests that RT induces ICD,
   promoting the release of TAAs and DAMPs, which play a critical role in
   initiating antitumor immune responses. Nevertheless, CRC frequently
   exhibit an immunologically “cold” TME, characterized by scarce
   infiltration of immune effector cells and subdued adaptive immune
   activation,^[ [201]^41 ^] thereby limiting the immunostimulatory
   effects of RT. Additionally, tumor cell radioresistance further
   compromises RT's capacity to induce potent immune responses.
   Consequently, overcoming these challenges through radiosensitization
   strategies that can remodel the tumor immune microenvironment, enhance
   tumor immunogenicity, and potentiate antitumor immunity has become a
   key scientific objective in improving the therapeutic outcomes of RT in
   CRC.

   Over the last decade, a variety of nanomaterials have been engineered
   and evaluated for their potential to enhance radiosensitivity, with
   high atomic number (Z) lanthanide nanoparticles gaining significant
   research interest. These materials have shown promise due to their
   ability to enhance radiation therapy by increasing the production of
   ROS, improving tumor targeting, and amplifying the overall therapeutic
   effect. Radiosensitization effects were observed in high Z lanthanide
   nanoparticles constructed of Neodymium (Nd) and Gadolinium (Gd).^[
   [202]^42 , [203]^43 ^] Unfortunately, the RT sensitization effect of Nd
   and Gd is not ideal and still requires a relatively high radiation
   dose due to their lower atomic numbers. In particular, Lu has a higher
   atomic number than Gd, exhibiting a better RT sensitization effect.
   Compared with reported Gd‐based radiation sensitizers like
   R&HV‐Gd@ICG,^[ [204]^34 ^] NPs‐Bev^[ [205]^44 ^] or VGd@ICG‐FA,^[
   [206]^29 ^] Our RVLu@ICG probe demonstrates superior radiosensitization
   efficacy, significantly enhanced local tumor control, and potential
   prognostic improvement capabilities compared to conventional
   modalities.

   Furthermore, integrin α[v]β[3] is abundantly expressed on neovascular
   endothelial cells as well as CRC cells^[ [207]^45 , [208]^46 ^] making
   it a widely recognized tumor biomarker.^[ [209]^47 , [210]^48 ^] And
   c(RGDfK) can specifically bind to integrin α[v]β[3]. In this study,
   NIR‐II nanoparticles RVLu@ICG was synthesized by integrating ICG into a
   c(RGDfK) modified biodegradable hollow virus‐like Lu[2]O[3] nanoprobe.
   In addition to passive accumulation mediated by the enhanced
   permeability and retention (EPR) effect,^[ [211]^49 ^] conjugating with
   specific tumor‐targeting elements c(RGDfK) could specificity and
   excellently enable RVLu@ICG to target cancer cells in vitro and
   identify tumor in vivo actively. ICG are useful for NIR‐II imaging,
   facilitating the tracking of NPs accumulation within tumors and more
   precision tumor RT. Blood analysis and histological staining of major
   organs jointly supported the reliable biocompatibility of RVLu@ICG,
   making them candidates for anti‐tumor therapy. Following the evaluation
   of the exceptional performance of RVLu@ICG, our in vitro experiments
   revealed their remarkable capacity to enhance RT efficacy and further
   activated RT‐related ICD response by increasing ROS production,
   intensifying oxidative stress, and destroying the integrity of the
   nucleus which collectively improve the therapeutic response to
   radiation.^[ [212]^50 ^] In vivo studies also suggested that the
   RVLu@ICG + RT effectively enhanced tumor cells apoptosis and further
   activated ICD response induced by RT, which could promote more
   secretion of cytokine and recruitment and infiltration of CTLs.
   Following this, DCs and macrophages migrated to phagocytose the
   necrotic tumor cells, facilitating the presentation of tumor‐derived
   antigenic peptides to T cells. This process activated tumor‐specific T
   cell responses, resulting in the elimination of subcutaneous tumors and
   the promotion of immunogenic cell death, thereby eliciting a potent
   antitumor immune reaction.

   In addition to preoperative neoadjuvant therapy, precise intraoperative
   resection of tumor lesions plays a critical role in determining the
   prognosis of CRC patients. Although ICG has been utilized for NIR‐II
   imaging‐guided surgery,^[ [213]^51 ^] its clinical application remains
   challenging due to severe photobleaching. The encapsulation of ICG
   within inorganic nanoparticles significantly enhances its
   photostability. In both in vitro and in vivo experiments presented
   herein, thanks to the accurate tumor targeting ability, RVLu@ICG
   exhibited superior NIR‐II imaging performance, characterized by strong
   fluorescence emission beyond 1000 nm. Remarkably, free ICG fluorescence
   rapidly diminished within 40 minutes of continuous laser irradiation,
   whereas RVLu@ICG maintained nearly 100% of its fluorescence intensity,
   demonstrating outstanding photostability and highlighting its promising
   potential for applications in surgical navigation. By comparing to the
   VLu@ICG, RVLu@ICG achieved a high TBR of ≈6.5 for in vivo tumor imaging
   sufficiently to discriminate tumor tissue intraoperatively. More
   importantly, the tumor tissue retained a significant fluorescent signal
   (TBR = 4.5) even 72 hours post‐injection, demonstrating the prolonged
   imaging window of RVLu@ICG. The 24–72 hour time frame offers
   considerable flexibility for scheduling probe injections and planning
   surgical procedures, making it advantageous for clinical applications
   where precise timing is crucial. The nanoprobe further demonstrated the
   capability to detect microtumors as small as 1 mm^3 in volume within
   microtumor models and effectively guided accurate and complete tumor
   resection in both intramuscular invasion and peritoneal metastasis
   models under NIR‐II fluorescence imaging, facilitated by the c(RGDfK)
   modification. Collectively, these results establish RVLu@ICG as a
   promising tool for precise surgical intervention with strong potential
   for clinical translation.

   Despite the favorable therapeutic efficacy and fluorescence imaging
   capabilities of the developed Lu‐based nanoprobes, their fabrication
   process remains intricate, involving sequential steps such as VLu
   synthesis, ICG incorporation, and c(RGDfK) functionalization.
   Undoubtedly, the purification required at each stage is
   labor‐intensive, making comprehensive optimization of all components
   challenging. Moreover, comprehensive studies are still required to
   elucidate the long‐term biosafety, metabolic implications, and in vivo
   fate of the nanoprobes. Future investigations involving large primate
   models are essential to assess systemic distribution and targeted
   delivery to colorectal tumors.

   In conclusion, we engineered an innovative multifunctional NIR‐II
   emitting degradable fluorescent nanoprobe RVLu@ICG with excellent
   biocompatibility and high photostability, for the RT sensitization,
   immune microenvironment remodeling and accurate navigation of surgery.
   Owing to the pronounced X‐ray absorption efficiency of the high atomic
   number element Lu to generate ROS, RVLu@ICG enhanced the
   radio‐sensitization of CRC tumors while further remodeling the tumor
   immune microenvironment through the radiosensitizing effect, thereby
   stimulating immunity to amplify immune killing effects. In addition,
   the probe could be used for accurate tumor removal under the guidance
   of NIR‐II fluorescence imaging leading to radical resection and improve
   overall survival in various tumor‐bearing models. More encouragingly,
   this nanoprobe is non‐toxic and does not cause organ damage or acute
   harm, highlighting its promising potential for future clinical
   translation. This multifunctional probe enables a comprehensive
   precision treatment strategy for CRC. In the preoperative phase, it
   facilitates maximal tumor cell eradication and tumor volume reduction
   through neoadjuvant RT combined with immune microenvironment
   remodeling. Subsequently, it allows for the precise intraoperative
   resection of residual microscopic lesions under NIR‐II fluorescence
   guidance. This integrated approach holds significant promise for
   improving patient survival and prognosis, and may serve as a viable
   candidate for clinical application in cancer therapy.

4. Experimental Section

Preparation of RVLu@ICG

   RVLu&NPs (10 mg) were immersed in 5 mL ICG solution (2 mg mL^−1) for
   dark‐stirring over 4 h, then subjected to centrifugation (10,000 rpm)
   and triple aqueous rinsing.

ICG and RVLu@ICG Photostabilities

   RVLu@ICG and ICG were suspended in PBS, and their NIR‐II fluorescence
   images were captured under continuous 808 nm laser irradiation at
   various time point. RVLu@ICG and ICG were dissolved in PBS and stored
   at room temperature under dark conditions for different periods. Then
   their NIR‐II fluorescence images were measured at 808 nm. RVLu@ICG was
   dispersed to aqueous solutions with pH 7.4, 6.5, 5.5 for different
   times. Then centrifuge to collect the supernatant, and measure the
   fluorescence intensity at 808 nm.

Cell Culture

   CT26 murine colon carcinoma cells were procured from the American Type
   Culture Collection (ATCC, USA), with CT26‐Luc cells acquired from
   Shanghai Zhong Qiao Xin Zhou Biotechnology. Both lineages were
   propagated in RPMI‐1640 containing 10% FBS and 1%
   penicillin‐streptomycin, incubated at 37 °C under 5% CO[2] humidified
   atmosphere. Based on the morphological characteristics and growth
   behavior of the cells, it was confirmed that they were not
   contaminated.

Radiosensitization of RVLu@ICG in Vitro

   Intracellular reactive oxygen species production was assessed using a
   DCFH‐DA fluorescence assay. Cell slides were prepared in accordance
   with the protocol described above. Samples treated with RVLu@ICG were
   incubated with a final concentration of 20 µM to evaluate ROS levels.
   The RT group received 4 Gy X‐ray irradiation. ROS fluorescence signals
   were observed using a Nikon AI‐MP microscope under the FITC channel.

Immunogenic Cell Death‐Related Assays in Vitro

   CT26 cells were cultured at a density of 5 × 10^5 cells mL^−1 for 24
   hours and then treated with either PBS or RVLu@ICG for an additional 24
   hours. Following triple PBS rinses, the cells underwent X‐ray exposure
   as previously described. The samples were then fixed in 4%
   paraformaldehyde and permeabilized with Triton X‐100. Following
   blocking with bovine serum albumin (BSA), they were incubated overnight
   at 4 °C with primary antibodies against HMGB1 and CRT. Post‐incubation,
   cells were washed 3 times, followed by a 1‐hour staining step and a
   15‐minute incubation with DAPI and goat anti‐rabbit IgG.
   Immunofluorescence imaging was finally performed using CLSM.

RNA Sequencing Analysis

   When CT26 tumors attained a volume near 100 mm^3, mice were randomly
   assigned to two groups (n = 3) and administered either PBS or a
   combination of RVLu@ICG + RT. Forty‐eight hours following treatment,
   tumors were excised, rapidly frozen in liquid nitrogen, and submitted
   to Shanghai OE Biotech Co., Ltd. for transcriptomic profiling via RNA
   sequencing.

RT of RVLu@ICG Nanoprobe in Vivo

   To establish the xenograft model, tumor‐bearing mice with 50 ± 5 mm^3
   lesions were randomized into four cohorts (n = 5): (I) PBS, (II)
   RVLu@ICG, (III) RT, (IV) RVLu@ICG+RT. Cohorts II and IV received tail
   vein administration of 100 µL RVLu@ICG suspension (2 mg/mL), while
   cohorts III and IV underwent fractional X‐ray irradiation (8 Gy, 6 MeV,
   Varian Medical Systems). Physiological parameters (body mass) and
   neoplastic progression (tumor dimensions) were monitored every 48
   hours. And the tumor growth inhibition (TGI) was calculated using the
   formula: TGI = (1 – RTV[C]/RTV[E]) × 100%, where RTV[C] was the
   relative tumor volume of the control group. RTV[E] was the relative
   rumor volume of the experimental group. Terminal biospecimens were
   excised for macroscopic documentation and gravimetric analysis to
   quantitatively evaluate therapeutic outcomes.

Immune Response Evaluation in Vivo

   To assess in vivo immune activation, CT26 tumor‐bearing mice were
   randomly allocated into four groups (n = 5 each) receiving PBS,
   RVLu@ICG, RT, or the combination of RVLu@ICG + RT. Forty‐eight hours
   post‐treatment, tumors and corresponding draining lymph nodes were
   harvested to prepare single‐cell suspensions. For analysis of DCs
   maturation, lymph node‐derived cells were stained with APC‐conjugated
   anti‐CD11c, PE‐labeled anti‐CD86, and FITC‐tagged anti‐CD80 antibodies,
   followed by flow cytometric evaluation. To assess cytotoxic T
   lymphocytes (CTLs) in tumor tissues and spleens, samples were stained
   with FITC anti‐CD45, Brilliant Violet 510™ anti‐CD3, Brilliant Violet
   421™ anti‐CD4, and PE/Cyanine7 anti‐CD8a, then subjected to
   quantitative flow cytometry. Additionally, portions of tumor tissues
   were processed for immunofluorescence staining targeting CD8 and CD86.
   Serum samples were also collected to quantify cytokine levels via
   ELISA.

Microtumor Identification and Resection under NIR‐II Fluorescence‐Guided

   Mice bearing multiple microtumors (n = 4) were imaged using the IVIS‐II
   bioluminescence system (PerkinElmer, Waltham, MA, USA) to precisely
   identify tumor locations. Following localization, RVLu@ICG was
   administered intravenously, and after 48 hours, the animals were
   euthanized for subsequent analysis. NIR‐II fluorescence imaging was
   utilized to facilitate surgical excision of tumors. Resected specimens,
   including adjacent non‐tumorous tissues, were then subjected to ex vivo
   NIR‐II imaging and further examined through histopathological
   evaluation.

NIR‐II Fluorescence‐Guided Real‐Time Tumor Resection in Intramuscular
Tumor‐Invasion Model

   CT26 Luc tumor cells were implanted near the right leg of mice,
   enabling gradual tumor infiltration into the muscle. Tumor presence and
   localization were verified through bioluminescence imaging. Surgical
   resection, conducted under white light to emulate clinical practice,
   involved both visual inspection and palpation of the tumor. In the
   RVLu@ICG and ICG treatment cohorts, NIR‐II fluorescence imaging was
   utilized intraoperatively to detect residual signal within the surgical
   field. Additional tissue removal was carried out until fluorescence was
   no longer observed. Surrounding muscle tissue was also excised for
   comparative purposes. All collected samples were then subjected to
   NIR‐II imaging, followed by histopathological evaluation. In contrast,
   animals in the control group underwent complete tumor resection guided
   solely by standard white‐light visualization. 14 days post‐surgery,
   bioluminescent imaging was conducted on all mice to monitor tumor
   recurrence.

NIR‐II Fluorescence‐Guided Real‐Time Tumor Resection in Peritoneal Metastasis
Model

   Approximately 5×10^5 CT26 Luc cells per dish were initially cultured in
   flasks containing 8 mL of DMEM supplemented with 1% antibiotics and 10%
   FBS, and maintained in a CO₂ atmosphere at 37 °C for 1 day. After
   incubation, the cells were collected by centrifugation, resuspended in
   sterile PBS, and then administered intraperitoneally into female mice
   at a dosage of 5×10^7 cells one mouse. Bioluminescence imaging was used
   to confirm tumor presence and estimate its location. Surgical resection
   was performed 7 days post‐injection, following the same procedure as
   for intramuscular tumor models.

Conflict of Interest

   The authors declare no conflict of interest.

Supporting information

   Supporting Information
   [214]ADVS-12-e10136-s001.docx^ (25.7MB, docx)

Acknowledgements