Sugar-based drug-free systemic immunoregulating and imageable nanoparticles promote the efficacy of immune checkpoint inhibitors in aggressive cancer

doi:10.21203/rs.3.rs-3399241/v1

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Sugar-based drug-free systemic immunoregulating and imageable nanoparticles promote the efficacy of immune checkpoint inhibitors in aggressive cancer

https://doi.org/10.21203/rs.3.rs-3399241/v1

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Poor drug delivery and an unresponsive tumor microenvironment (TME) hinder the treatment of glioblastoma (GBM) and pancreatic cancer (PC). Targeting macrophage polarization through nanoparticle (NP) internalization represents a promising approach to regulate the TME and enhance immunotherapy. Here, we introduce a galactose -based NP designed for monocarboxylate transporter 4 (MCT-4)-mediated transport in cells, promoting M2-to-M1 macrophage polarization and disrupting tumor cell mitochondrial function to trigger apoptosis. The CG-based NP promotes M1 macrophage accumulation, facilitates the M2-to-M1 transition, and exhibits the potential to overcome the obstacles in GBM and PC treatment. This approach enhances immune checkpoint inhibitor efficacy while overcoming the challenging TME. Additionally, incorporating Fe₃O₄ promotes ferroptosis and enables the use of these NPs in magnetic resonance imaging-based monitoring. Diverging from previous studies that focused on synthesizing nanomaterials, our work has pioneered a synthetic methodology that generates immune-stimulating NPs through a precursor-only hydrothermal reaction and galactose surface engineering. This approach can be meticulously tailored for precise immunotherapeutic applications, which is beneficial for targeting the intricacies of challenging cancers.

Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles

Biological sciences/Biotechnology/Biomaterials/Drug delivery

drug-free

macrophage polarization

iron oxide

sugar-based nanoparticle

ferroptosis

magnetic resonance imaging

glioblastoma

pancreatic cancer

The treatment of glioblastoma (GBM) and pancreatic cancer (PC) remains a formidable challenge due to drug delivery issues and suppressed immune responses^1,2. To address these challenges, numerous researchers are currently focused on developing a versatile therapeutic nanoparticle (NP) that can be applied to several treatment modalities at a specific treatment time interval, such as phototherapy or drug delivery^3-7. NP uptake relies on phagocytosis and specific transporters, and the effects of NPs are shaped by their specific formulation, which includes their molecular payloads⁸. Macrophages are among the first immune cells to come into contact with and internalize NPs during the uptake process⁹. Once the NP is internalized, oxidative phosphorylation in macrophages can prompt mitochondrial dysfunction, impacting cellular polarization dynamics^10,11.

Immune surveillance protects against the development of cancer. Ideally, drug-free NPs that are delivered to the lesion area and designed to specifically target macrophages could promote their M1 polarization. This precision treatment has unprecedented therapeutic potential when combined with immune-checkpoint antibodies, offering a promising strategy for treating various types of cancer. Glycopolymers have the ability to directly target and bind to target cells, and their use in nanoassembles may contribute to effective immunotherapy against tumors by influencing macrophage polarization, but few studies on their effects on transduction and metabolism^12,13. Despite their potential, limited research has been conducted on diverse sugar-based solid NP development for GBM and PC cancer therapy due to the challenges posed by intricate organic synthesis methods and demanding surface immobilization technologies¹⁴.

Our group's objective is to harness the potential of NPs to induce systemic cancer inactivation and immune modulation against GBM and PC. Here, we designed a precursor-only hydrothermal reaction to fabricate condensed galactose (CG) nanomolecules, which is a repeatable and more straightforward process than the organic synthesis of glycopolymers and other sugar-based macromolecules¹⁴. The multivalent interaction of these CG nanomolecules has been shown to promote the polarization of M1-like macrophages, making them valuable for GBM and PC immunotherapy. What sets our approach apart from previous studies that focused on the synthesis of biomedical nanostructures^3-6,8,14 is that the new methodology could extend for surface engineering of iron oxide NPs that incorporate CG (named IONPs@CG). These CG-based NPs have the ability to modulate the immune microenvironment when taken up by macrophages, and disrupt glycolysis in tumor cells, and confer ferroptosis and magnetic resonance imaging (MRI) by IONPs@CG. This study can serve as a reference for the development of advanced sugar-based hybrid NPs.

It has recently been identified that iron oxides are involved in peroxidase function, ferroptosis, and autophagy ^15-18. However, our IONPs@CG used MCT-4 to facilitate their delivery to macrophages. This CG-mediated mechanism promoted the polarization from M2 to M1 macrophages, thereby increasing the presence of immunostimulant cells in the TME, both locally and systemically. Additionally, these well-structured CG-based NPs might disrupt the mitochondrial function of tumor cells, triggering apoptosis signaling. Within the TME, tumor cells characterized by a highly glycolytic phenotype engage in metabolic competition with immune cells. This competitive metabolic process underscores the vital role of MCT-4 in exporting lactate and facilitating the polarization of M2 macrophages. Our study demonstrated the versatile capabilities of CG-enhanced systemic immunomodulation with proof of concept in a GBM model and a highly immunosuppressive PC model.

The characteristics of sugar-based NPs and their immunoregulatory potential.

Fig. 1b shows the transmission electron microscopy (TEM) and EDS mapping images to verify the spherical NPs with C, O, and N elements after carbonization/condensation of ONPG (8.3 mM) via a precursor-only hydrothermal reaction (Fig. 1a). Their diameter was 60.5 ± 6.8 nm (Fig. 1c). These condensed CG NPs possessed carbon-related structures¹⁹ at 1587 cm^-1 (G band) and 1377 cm^-1 (D-band) in the Raman spectra (Supplementary Fig. 1a) and the sp² carbon structure was verified by the C 1s XPS peaks (Fig. 1d). The N 1s peaks (Fig. 1e) at 399.5 eV and 401 eV (C-N) are deduced from the pyrolysis residues of the nitro groups in ONPG²⁰. The presence of these oxidized groups was confirmed by the O 1s (Fig. 1f) peaks at 532.2 eV and 533.2 eV²¹. Supplementary Fig. 2b shows that the FTIR spectra of these particles involved C-O, C=O, and C=C vibrations peaks. The aromatization process produces a low yield (21% of the original amount) at 4 mM, but substantial precipitation occurred at 25 mM (Supplementary Fig. 3). We found that the size of CG NPs increased with increasing reaction time (Supplementary Fig. 4a) due to the high-level carbonization/graphitization (Supplementary Fig. 4b) undergoing dehydration and cross-linking reactions^22,23 of the hexose or nitrophenol block of ONPG. This precursor-only hydrothermal reaction can be applied to the fabrication of sugar-based condensed NPs by using galactose²⁴, 2-nitrophenol (2-NP), and 4-nitrophenyl-beta-d-glucopyranoside (ONPGlu) (Supplementary Fig. 1, 2, 5).

In Supplementary Fig. 2a, C-OH stretching and OH bending signals of residual hydroxyl groups¹⁵ appeared in the 1000–1300 cm^-1 region. Based on element analysis, ~ 24% of the CG NP structure was composed of oxygen. The galactose structure at 50-80 ppm in the CG NPs was identified by 13C NMR experiments (Fig. 1g). The NMR lineshape analysis showed line broadening in the CG NP structure compared with the sp³ carbon atoms of the ONPG molecule, which is similar to previous analysis results^25,26, which suggested the possible increase of local GC concertation and formation of an amorphous state in polymer NP structures^{18, 19}. In addition to C-sp³ in region I (0-100 ppm), a significant peak at 129 ppm of the graphite structure in the CG NPs was observed, as well as the presence of a β-C signal at 110-120 ppm and an α-C signal at 140.7-156 ppm for hydroxymethylfurfural of a hydrothermal sugar product²⁶.

Cocultivation of M2 macrophages with CG NPs displayed remarkable efficiency in inducing M2-to-M1 macrophage transition, reaching 93.2% at 11 ppm, (Fig. 1h). FITC- and RITC-immobilized CG NPs (Supplementary Fig. 6a-c) showed efficient cellular internalization (Fig. 1i). The reprogramming rate of CG NPs was superior compared with the 51.2% reprogramming rate of Gal NPs in the same coculture experiment (Supplementary Fig. 7), suggesting that the exposure of many galactose moieties facilitated multivalent interactions¹⁴. When CG NPs reacted with galactosidase (24 hour) to cleave the Gal groups (see Fig. 1a), the reprogramming rate decreased to 11.6% (Fig. 1h). Notably, the sp² and sp³ carbon structure and particle size of CG NPs were not affected by their long-term storage in the galactosidase solution or other solutions such as PBS (pH=4), esterase, and GSH (Supplementary Fig. 8-9). The polarization of M2-to-M1 macrophages increased depending as a result of a long hydrothermal reaction time (Supplementary Fig. 10) or a large precursor amount (Fig. 1j, Supplementary Fig. 11). However, the condensed 2-NP (galactose-free) and ONPGlu NPs did not effectively promote M2-to-M1 macrophage polarization (31.2% and 72.6%), elaborating the crucial role of high Gal concentrations of CG NPs in facilitating macrophage polarization.

Intriguingly, introducing tannic acid-coated IONPs²⁷ into the ONPG solution, followed by a hydrothermal reaction (Fig 1k), and yielded IONPs@CG. The XRD pattern of the Fe₃O₄face-centered cubic (fcc) structure could be assigned to the (220), (311), (400), (511), and (440) planes (Fig. 1l). Fig. 1m shows 6.2 nm of a thin organic layer (yellow arrow) coated around the surface of IONP (~ 41.3 nm), and distinct lattice fingerprint structures measuring 3.0 Å (220 plane) and 2.1 Å (400 plane)²⁷ appeared in the core structure of the face-centered cubic Fe₃O₄ crystal structure based on high-resolution TEM (HR-TEM) imagery. They consisted of Fe 2p core-level XPS at 710.8/723.8 eV for Fe(II) and at 713.2/726.2 eV for Fe(III) (Supplementary Fig. 12). The CHO, C-O, and COOH functional groups in IONP@CG were detectable in the FT-IR spectra (Fig. 1n). The XPS peak deconvolutions for the N 1s binding energies of IONPs@CG ere similar to those of CG NPs, and the O 1s of IONP@CG contains a higher ratio of C=O and more O-H from the TNA molecules (Supplementary Fig. 12).

CG NPs and IONPs@CG internalized by SLC16A3 may induce M2-to-M1 macrophage polarization.

Firstly, in addition to IONP material, we also evaluated other metallic materials such as Au and graphene oxide (GO) for loading with CG. As illustrated in Figure Supplementary Fig. 13, we demonstrated the galactosylation reaction with ONPG precursor, which allowed us to create other potential nanomaterials (i.e., Au@TNA and GO) for novel reprogramming of M2-to-M1 macrophages. Considering their application potential, we selected IONP as the raw material for further development. In Fig. 2a, we proposed that CG NPs and IONPs@CG may not only be phagocytosed by macrophages but also transported by SCL16A3 (MCT-4), thereby regulating the STAT3/NF-kB pathways and inducing M1/M2 transition. First, we found no toxicity in either CG NP- or IONPs@CG-treated Raw264.7 cells, THP-1 cells, and BMDMs (Fig. 2b). The hemolysis ratio was relatively low for both types of NPs (Supplementary Fig. 14), further supporting their biocompatibility. The secretion of lactate from cancer cells, mediated by MCT-1/4, is crucial in promoting the polarization of macrophages toward the M2-like state ²⁸. We observed that the increased M2-to-M1 transition effect induced by CG NPs could be diminished by the addition of anti-MCT-4 antibodies, confirming the link between NP internalization and MCT-4 involvement (Fig. 2c). Furthermore, in M2 macrophages differentiated by IL-4/IL-13, the expression of CD11b⁺CD206⁺ was significantly downregulated by both CG NPs and IONPs@CG (Fig. 2d). Conversely, the expression of CD11b⁺CD86⁺ in M1-differentiated BMDMs was markedly upregulated by CG NPs and IONPs@CG (Fig. 2e). These effects were confirmed in other cell line models, including Raw264.7 and THP-1 cells (Supplementary Fig. 15). Moreover, the levels of IL-4 and IL-10 secreted by M2 macrophages were decreased in the collected supernatant (Fig. 2f), while the levels of IL-6 and IL-12 secreted by M1 macrophages were increased (Fig. 2g). Additionally, changes in M1 and M2 marker mRNA expression were observed along with a reduction in the levels of anti-inflammatory cytokines such as IL-4, IL-10, and TGF-β (Fig. 2h & Supplementary Fig. 16). In contrast, the levels of proinflammatory cytokines such as IL-6 (Fig. 2h), IL-12, and IL-1β (Supplementary Fig. 17) were upregulated in both Raw264.7- and BMDM-differentiated M2 cells. Finally, we found that CG and IONPs@CG can decrease STAT3 phosphorylation, leading to a reduction in M2 differentiation (Fig. 2i and Supplementary Fig. 18-19). In contrast, Raw264.7-differentiated M2 cells showed increased NF-κB phosphorylation, suggesting a potential shift toward an M1 phenotype. In summary, CG NPs and IONPs@CG can trigger M2-to-M1 macrophage polarization via MCT-4-mediated transport linked to STAT3 inactivation and NF-κB-mediated transcriptional activation.

CG NPs and IONPs@CG induced apoptosis, ferroptosis, and mitochondrial impairment.

We proposed that the induced toxicity by CG NPs or IONPs@CG is associated with a mitochondria-mediated apoptosis mechanism (Fig. 3a). As shown in Fig. 3b, the toxicity of CG NPs and IONPs@CG was apparent in GL261, U-87 MG, and T98G GBM cell lines. In contrast, human fetal glial cells (SVG-P12) showed no toxicity (Supplementary Fig. 20), suggesting a targeted impact. Interestingly, the toxic effect on GBM cells might be reversed by anti-MCT-4 antibody intervention. Given the expression of c-type lectins and lactate transporters on cancer cells^29,30, we hypothesized that CG NPs and IONPs@CG, exposing galactose molecules, could specifically target and bind to cancer cells. This hypothesis was further supported by the successful infiltration of RITC-labeled CG NPs and IONPs@CG to the bottom of GL261 cells and into the center of GL261 spheres (Fig. 3c-d and Supplementary Fig. 21), as confirmed by fluorescence microscopy analysis. We then aimed to investigate the mechanism by which the NPs induced cell toxicity in GBM cells. Treatment with these CG-based NPs led to annexin-V activation in GL261 cells, indicating apoptotic cell death (Fig. 3e and Supplementary Fig. 22a). Simultaneously, exposure to CG NPs and IONPs@CG resulted in increased Ca²⁺ release and ROS production (Fig. 3f and Supplementary Fig. 22c-d, c). Furthermore, we observed a notable decrease in mitochondrial membrane potential (ΔΨm), along with increased activation of cleaved caspase-9 and cleaved caspase-3 in CG NP- and IONPs@CG-treated GL261 cells (Fig. 3g-h and Supplementary Fig. 22b), suggesting the induction of apoptotic cell death, with cleaved caspase-9 serving as a crucial factor in mitochondrial-dependent apoptosis³¹. We also observed an increase in the colocalization of the ROS signal with a mitochondrial marker in GL261 cells treated with CG NPs and IONPs@CG (Fig. 3i). To gain insight into the role of our NPs in regulating mitochondria, we conducted a Seahorse assay. Treatment with CG NPs led to a rapid decrease in the spare respiratory capacity and glycolytic ability of GL261 cells, which was sustained even 48 hours postadministration (Fig. 3j-k). The GSEA performed on RNA samples showed higher levels of apoptosis and ROS in mice treated with CG NPs compared to nontreated mice (Supplementary Fig. 23a-b), as well as increased glycolysis and oxidative phosphorylation compared to all treatment groups.

Supplementary Fig. 23c results indicated the potential regulation of CG NPs and IONPs@CG on STAT3 signaling. Following treatment with CG NPs and IONPs@CG, a reduction in STAT3 nuclear translocation and dephosphorylation of both STAT3 and NF-κB were observed (Fig. 3l-m and Supplementary Fig. 24a). In summary, the CG NP-mediated antitumor effect is associated with the suppression of the KRAS, STAT3, and NF-kB signaling pathways, which are known to play essential roles in the promotion of GBM. The migration and invasion of GBM cells may also be diminished by CG NP treatment (Supplementary Fig. 25). In conclusion, the tumor inhibitory effects of CG and IONPs@CG can be attributed to the deactivation of the STAT3 and NF-κB pathways and the induction of mitochondria-mediated apoptosis.

IONPs@CG exhibit both ferroptosis-inducing and MRI-visible properties.

IONPs@CG may possess the capacity to trigger ferroptosis and they can also be visualized by MRI (Fig. 4a). The T2* images demonstrated that the signal intensity was dependent on the concentration of IONPs@CG, as shown in Fig. 4b. Both IONPs@CG alone and IONPs@CG-treated GL261 cells exhibited a significant dose-dependent signal intensity change within the TE range from 0 to 5 msec. The uptake of IONPs@CG in GL261 cells was validated by Prussian blue staining (Supplementary Fig. 26). The MR perfusion-weighted images of rCBV demonstrated an enhancement in the signal intensity of GBM post IONPs@CG administration (Fig. 4c). After perfusion scanning, T2-RARE imaging also showed a noticeable accumulation of IONPs@CG within the tumor region. In Fig. 4d, the red arrows highlight an apparent enhancement in the IONP signal in T2*-MGE imaging post-IONPs@CG injection. The positive value of ∆R2* (0.0172 ± 0.007) demonstrated the effective enhancement of IONPs@CG in the ROI area (tumor). IONPs@CG distribution in the tumor area was also confirmed by H&E and Prussian staining (Fig. 4e). The biodistribution study showed that IONPs@CG was predominantly cleared through the spleen, liver, and urine, indicating their suitable clearance properties (Fig. 4f). Furthermore, the signaling activity of ¹²⁵I-IONPs@CG within the brain tumor region was higher than that of free ¹²⁵I, underscoring its heightened targeting efficacy. The labeling efficacy of ¹²⁵I in IONPs@CG was optimized to 90.83% before intravenous injection (Supplementary Fig. 27). Finally, the ferroptosis effect was confirmed by the reduction in GPX-4 protein levels and GSH levels in the cell/tissue model (Fig. 4g-i). These results suggest that IONPs@CG can be utilized as an MR contrast agent and exhibit a Fe(II)-rich-elicited ferroptosis effect on GBM models.

CG NPs and IONPs@CG may sensitize GBM to anti-PD-L1 therapy

The treatment efficacy of CG NPs and IONPs@CG combined with an anti-PD-L1 antibody, 10F.9G2 (Fig. 5a and Supplementary Fig. 28a), was validated in GL261-bearing mice. The results in Fig. 5b-c and Supplementary Fig. 27b clearly show that combining CG NPs or IONPs@CG with 10F.9G2 led to significant tumor inhibition and prolonged survival (Supplementary Table 1-2). The combination therapy group showed a reduction in the progression rate of GBM, as validated by MRI (Fig. 5d and Supplementary Fig. 28c). To confirm the role of MCT-4, we combined CG NPs, 10F.9G2 and an anti-MCT-4 antibody (Fig. 5e, Supplementary Fig. 29) and the results showed that the blockage of MCT-4 diminished the anti-GBM effect of CG NPs plus anti-PD-L1. While the anti-MCT-4 approach has been proposed to enhance the function of anti-PD-L1 therapy²⁴, our work suggested that disrupting the uptake of CG NPs by MCT-4 may interfere with the immune regulation mediated by the NPs, potentially reducing their antitumor efficacy. The proposed immunoregulation of CG NPs is presented in Fig. 5f. GBM cells pretreated with CG NPs and IONPs@CG were cocultured with mouse peripheral blood mononuclear cells (PBMCs), and the proliferation of T cells, the induction of CD62^-CD44⁺ memory T cells³² and the activation of CD8⁺IFN-g⁺ cells were observed (Fig. 5g-i and Supplementary Fig. 30). Fig. 5j and Supplementary Figs. 31-32 demonstrate the increased infiltration of CD8⁺IL-2⁺ or CD8⁺IFN-γ⁺ cytotoxic T lymphocytes (CTLs) in the tumor. The increased proportions of CD8⁺IFN-g⁺ T and CD8⁺IL-2⁺ T cells in tumor-draining lymph nodes (TDLNs) in response to CG NPs or IONPs@CG combined with 10F.9G2 was accompanied by increased secretion of IL-12 in mouse serum (Supplementary Fig. 30d). The critical role of CTLs in CG NP treatment was also validated by an immunodeficiency model (Supplementary Fig. 33). Only the slightly tumor inhibition was found in combination of CG NPs with 10F.9G2, which may come from the regulation of macrophage. The reduction in M2 macrophages and induction of M1 macrophages were significantly observed upon combining CG or IONPs@CG with 10F.9G2 in TILs, bone marrow, and the spleen (Fig. 5k and Supplementary 34-36). However, the blockage of MCT-4 may diminish the infiltration of CD8⁺ T cells and the M2-to-M1 transition induced by CG and IONP@CG (Fig. 5l-m and Supplementary Fig. 37a, b). Interestingly, we also observed that the accumulation of DCs, which are involved in the host immune response³³, triggered by CG NPs plus 10F.9G2 treatment may be reduced by the addition of anti-MCT-4 (Fig. 5n and Supplementary Fig. 37c).

Conversely, CG NPs or IONPs@CG combined with 10F.9G2 significantly reduced the population of regulatory T cells (CD4⁺CD25⁺FOXP3⁺) in the SP and TDLN (Supplementary Fig. 38-39). Furthermore, it was discovered that CG NPs or IONPs@CG with 10F.9G2 could inhibit immunosuppressive MDSCs in both mouse BM and SP (Supplementary Fig. 40-41). This effect may also be reversed by anti-MCT-4 (Supplementary Fig. 42-43). The infiltration of CD86⁺ M1, CD8⁺ T cells, and CD11c+ DCs by CG NPs or IONPs@CG plus 10F.9G2 was also validated by IF staining, as indicated in Fig. 5o-p and Supplementary Fig. 43a-f. Moreover, these effects could be reversed by anti-MCT-4. The reduction in CD206⁺ M2 macrophage and Treg (CD4⁺FOXP3⁺) numbers by CG NPs and IONPs@CG was also confirmed in tumor tissue (Supplementary Fig. 44g-h).

We observed that the combination treatment led to the activation of apoptosis factors (cleaved caspase-3, -9) and M1 markers (CD86) in tumor tissues (Supplementary Fig. 45), while the M2 marker CD206 and the mitochondrial function marker COX-IV³⁴ decreased. Additionally, we also observed CD8+ cell accumulation in the tumor region when CG NPs were combined with 10F.9G2, resulting in decreased levels of immunosuppressive factors such as VEGF, FOXP3, and IDO^35,36. These results suggest that combination therapy has the potential to shift the TME toward an immunostimulatory and antitumor state. Pathology images, measurements of body weight, and biochemistry analysis of the mice revealed the toxicity of TMZ, which was not observed in the groups treated with CG NPs or IONPs@CG combined with 10F.9G2 (Supplementary Fig. 46-47 and Supplementary Table 3). We also found that the liver and spleen primarily mediated the metabolism of IONPs@CG. These results suggest that compared to TMZ, the combination therapy involving CG NPs or IONPs@CG and anti-PD-L1 has a favorable safety profile and may be a promising strategy for treating GBM.

A similar trend of CG NP therapy was observed in pancreatic tumor models.

To evaluate the success of CG NPs in the treatment of various cancer types, we evaluated their effect on another malignant tumor type, pancreatic ductal adenoma carcinoma (PDAC), using Pan02-Luc orthotopic mouse models³⁷. The PDAC TME is known to be immunosuppressive, with substantial M2-type macrophage numbers, and unresponsive to immune checkpoint blockade^38,39. Orthotopic Pan02-Luc tumor model mice were treated with PBS (control) or 1500 ppm CG NPs once a week by intravenous injection and/or 12.5 mg/kg anti-PD1 (RMP1-14) through intraperitoneal injection twice per week. As expected, the anti-PD1 group showed no meaningful response 14 days posttreatment (Fig. 6a). Although CG NP monotherapy slightly inhibited tumor growth on day 14, the difference was not significant. Notably, combining CG NPs with anti-PD1 treatment effectively suppressed tumor growth with the smallest IVIS bioluminescence value in pancreatic sites and tumor weight after mouse sacrifice (Fig. 6b). We also observed a notably visible area of cell death within Pan02-Luc pancreatic tumors in the combination of CG NPs and anti-PD1 group (Fig. 6c). Once again, none of these therapies affected mouse body weight or the organ morphology of the heart, lung, liver, spleen, and kidney, which suggested their biosafety (Supplementary Fig. 46).

Next, to verify the effects of CG NP treatment on M1 macrophage polarization and TME immunomodulation in the pancreatic cancer model, we analyzed the quantity of individual immune cell populations and the secretion of several cytokines in Pan02-Luc pancreatic tumor samples after CG treatment. Similarly, CG NP monotherapy significantly increased the proportion of M1 macrophages and decreased the proportion of M2 macrophages within the pancreatic TME (Fig. 6d). After combining with anti-PD1 treatment, the above effect was augmented, followed by an increase in the proportion of mature DCs (CD11c⁺CD24⁺MHCII⁺), CTLs (CD8⁺IL-2⁺IFN-γ⁺), and NK cells (CD3^-NK1.1⁺CD49b⁺) and a decrease in the proportion of Tregs (CD4⁺CD25⁺Foxp3⁺). The combination group also exhibited the highest expression levels of immunogenic cytokines (TNF-α, IFN-γ, and IL-12) and the lowest secretion levels of immunosuppressive cytokines (IL-4, IL-10, and IL-13) (Fig. 6e). Importantly, the anti-PD1 group showed no effect on the above phenomenon, which suggested that CG NPs might reduce ICB resistance in the immunosuppressive pancreatic TME and convert immunologically “cold” tumors into “hot” tumors via immunogenic remodeling. Moreover, we directly observed significantly enhanced M1 macrophage (CD86⁺), DC (CD11c⁺), and CTL (CD8⁺) populations in the CG NPs and anti-PD1 combination group (Fig. 6f). Altogether, these results indicated that CG NPs remodeled the pancreatic TME from “cold” to “hot” and strengthened the antitumor efficacy of anti-PD-1 therapy via M1 macrophage polarization.

In conclusion, the CG NPs synthesized solely from precursor materials were successfully delivered to macrophages through MCT-4-mediated transport. Within macrophages, they promoted M1 macrophage accumulation and facilitated the transition from M2 to M1, enhancing macrophage infiltration into the tumor areas of GBM and PC. This immunomodulating nanoadjuvant, featuring a CG structure, enhances the efficacy of immune checkpoint inhibitors while addressing the challenges posed by the tumor microenvironment (TME). Furthermore, it induces apoptosis and ferroptosis in cancer cells while disrupting mitochondrial function. Additionally, CG-integrated IONPs exhibit immunoregulatory and antitumor effects and can serve for MRI-based monitoring. This multifunctional strategy holds great promise for advancing the treatment of GBM and PC, meriting further investigation in preclinical and clinical settings.

Synthesis of hydrothermal condensed galactose nanoparticles (CG NPs)

25 mg of ortho-nitrophenyl-β-galactoside (ONPG) was dissolved in 10 mL of deionized (DI) water to yield a stock solution with a concentration of 8.3 mM. The solution was transferred to a Teflon-lined stainless-steel autoclave. The autoclave was cooled to room temperature after a hydrothermal reaction in an oven at 200℃ for 24 hours. Afterward, the resulting CG NP product was collected by centrifugation at 10000 rpm and redispersed with DI water three times. The purified CG NP sample was dissolved in 1 mL DI water for storage.

Synthesis of rhodamine B isothiocyanate (RITC)- and fluorescein isothiocyanate (FITC)-CG NPs

CG NPs (100 ppm) were mixed with 2 mL of RITC (0.02 mM). After a gentle spin at 35 rpm in a 4 ℃ environment, the RITC-CG NP product was obtained by centrifugation at 10000 rpm. The redispersion of CG NPs with DI water was repeated more than three times. The purified RITC-CG NPs were redispersed in 2 mL DI water for storage before use. The FITC-CG NPs were synthesized by the same experimental method used for RITC-CG NP synthesis. Fluorescence was measured by a microplate reader (Synergy H1, BioTek).

Synthesis of IONPs@CG

The synthesis of IONPs was carried out according to our previously published article²¹. The IONPs (25000 ppm_[Fe]) and 25 mg of ONPG were dissolved in 10 mL of DI water. The solution was transferred to a Teflon-lined stainless-steel autoclave. After heating in an oven at 200 ℃ for 24 hours, the autoclave was cooled to room temperature. The IONPs@CG were collected by centrifugation at 10000 rpm and redispersed with DI water three times. The purified IONPs@CG was dispersed in 1 mL DI water for storage. The formula used for comparing the iron ion concentration (mg/mL) and solid content of IONP@CG was X ppm [Fe] = 0.002X mg/mL (IONP@CG)

CG NP stability

The CG NPs (100 ppm) were separated into different buffer solutions (H₂O, PBS, medium, and H₂O₂). The solution was subjected to UV‒Vis spectral measurements over time (0, 4, 7, and 14 days). The size and morphology were observed by TEM and DLS on day 14.

Intracellular localization using fluorescence imaging

Macrophages were treated with RITC-CG and FITC-CG NPs for 24 hours. The macrophages were washed with PBS three times and fixed with 4% paraformaldehyde at 4°C for 10 min and 1% Triton X-100 for 15 min. Afterward, the cells were washed three times with PBS and incubated with DAPI (DNA nucleic acid stain) for 5 min. The stained macrophages were observed under a fluorescence microscope (Olympus IX73).

Cell culture

Raw264.7 (mice), THP-1 (human), bEnd.3 (human brain endothelial), GL261 (murine GBM), U-87 MG, T98G (mammalian GBM), and Pan02-Luc (murine PC) cells were purchased from American Type Culture Collection or Prof. Yan-Shen Shan (Dean, College of Medicine, National Cheng Kung University). These cell lines were subsequently cultured in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS). SVG-p12 (human microglia) cells were cultured in Eagle's minimum essential medium supplemented with 10% FBS, 1% nonessential amino acids, and 1% PS. All cell lines were maintained in a 37°C incubator with a humidified 5% CO₂ atmosphere.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

Raw264.7, THP-1, mouse bone marrow-derived macrophage (BMDM), GL261, and SVG-p12 cells were cultured in 24-well plates at a density of 3×10⁴ cells/well with 100 μl DMEM overnight. Cells were treated with different concentrations of CG or IONPs@CG (0, 5, 10, 20, 40, and 80 ppm) for 48 hours. After treatment, the cells were washed with fresh medium, and 200 μl of medium containing 0.5 mg/mL MTT was added to each well. After 2 hours of incubation at 37°C, 200 μl of DMSO was added to the wells to dissolve the MTT formazan for 5 min, and the supernatant was extracted and centrifuged at 1500 rpm for 5 min. Finally, the absorbance of each well was determined using an ELISA reader (Power Wave X340; Bio-Tek Instrument Inc., Winooski, VT, USA) at an excitation wavelength of 570 nm.

Isolation of bone marrow-derived macrophages

Mice were sacrificed by anesthesia overdose. Both the left and right femurs were isolated and placed on ice. Bone marrow in the femur bone was then added to a centrifuge tube with 5 mL of PBS using a 26 G needle. Red blood cells were lysed by ammonium-chloride-potassium lysing buffer, and a 70 μm strainer was used to filter the remaining cells. Following 1200 rpm centrifugation for 10 min, cells (1×10⁶ cell/well) were seeded in 6-well plates filled with 2 mL DMEM and MCS-F (2 ng/mL) and stored in a 37°C incubator with 5% CO₂.

CD11b/CD86/CD206 (M1/M2 macrophage) staining

Raw264.7, THP-1, and naive BMDMs were seeded in 6-well plates at a density of 2×10⁵cells/well with 2 ml DMEM overnight. LPS (20 ng/ml) and IFN-γ (20 ng/ml) were used to differentiate Raw264.7 into the M1 macrophages. After being induced into the M2 type by IL-13 (20 ng/ml) and IL-4 (20 ng/ml), Raw264.7-differentiated M2 macrophages were treated with CG (10 and 40 ppm), IONPs@CG (20, and 40 ppm) combined with anti-MCT-4 (1 mg) or not, respectively, for 72 hours. Cells were then collected for CD86 and CD206 (BD Pharmingen, San Diego, CA, USA) staining and incubated for 1 hour at 37°C. After staining, the intensity of CD11b, CD86, and CD206 was evaluated by flow cytometry.

Detection of reactive oxygen species (ROS) by immunofluorescence (IF) staining

GL261 cells (2×10⁴/well) were seeded in chamber slides and treated with different concentrations of CG NPs or IONPs@CG (40 ppm) for 48 hours. Chamber slides were mounted with a mitochondria tracer (CellLight™ ER-RFP, BacMam 2.0) overnight. After treatment, cells were fixed with 4% formaldehyde. Then, the slides were permeabilized with 0.1% Triton-X100 for 10 min using the ROS peroxide-sensitive fluorescent probe 2′,7′-dichlorofluorescein diacetate (1:300, DCFH-DA, Molecular Probes) at room temperature in the dark for 1 hour and then covered with DAPI Fluoromount-G dye. Finally, the slides were photographed using a fluorescence microscope (ECHO Revolve).

Erythrocyte hemolysis

Blood was collected from the hearts of mice (C57BL/6) after anesthesia overdose. In brief, 200 ml of the erythrocyte stock dispersion was added to 200 ml of saline containing different concentrations of CG NPs (10 and 40 ppm) and IONPs@CG (20 and 40 ppm) and incubated in an Eppendorf tube at a mixing frequency of 414 rpm at 37°C for 1 hour. Intact erythrocytes were lysed by centrifugation at 10000 rpm for 5 min. Saline solution (0.9% NaCl) was employed as a negative control (0% lysis), and distilled water was employed as a positive control (100% lysis).

The equation of hemolysis rate:

where Dt, Dnc, and Dpc are the absorbance of the tested sample, negative control, and positive control, respectively⁴⁰.

Enzyme-linked immunosorbent assay (ELISA)

Raw264.7 cells were seeded in 24-well plates at a density of 1×10⁵ cells/well with 1 ml DMEM overnight, and the induction procedure was previously described. After being differentiated into M2 macrophages, cells were then treated with CG NPs or IONPs@CG for 72 hours; the supernatant was collected and used in the ELISA following the manufacturer’s procedures. Optical density was assessed at 450 nm and 570 nm. In the in vivo experiment, the supernatants from tumor homogenates were centrifuged in 3K centrifuge tubes (Pall Corporation) and analyzed with mouse TNF-alpha (TNF-α), IFN-gamma (IFN-γ), IL-12, IL-4, IL-10, and IL-13 DuoSet ELISA kits (R&D Systems) according to the manufacturer’s guidelines.

In vitro blood–brain barrier model

bEnd.3 brain-derived endothelial cells were seeded in the upper Transwell chamber (Franklin Lakes, NJ, USA) at a density of 1×10⁵/well with 500 μl DMEM overnight to form monolayer cells. GL261 cells were seeded in the lower chamber at a density of 1×10⁵/well with 500 μl MEM for one day. On the next day, FITC-CG or FITC-IONPs@CG NPs were added to the upper chamber and incubated for 48 hours⁴¹. After treatment, the GL261 cells were imaged under a fluorescence microscope (ECHO Revolve).

Multicellular tumor spheroid (MCTS)

GL261 cells were seeded in a MCTS chip at a density of 1×10⁶ cells/well with 1 ml DMEM for one day and treated with different concentrations of CG NPs or IONPs@CG (40 ppm) for 24 hours. After treatment, the cells were fixed with 4% formaldehyde at room temperature for 15 min. Then, the cells were permeabilized with 0.1% Triton X-100 for 10 min and stained with 1 μg of DAPI in 300 μl of PBS for 20 min in the dark at room temperature⁴². Then, to hyalinize the cells, 300 μl of SCALEVIVEW (FUJIFILM) was added to the cells for 4 hours and incubated in a 37°C incubator. Finally, chips were photographed using a confocal system (ANDOR, Dragonfly 200) and quantified by ImarisViewer and ImageJ version 1.5.

Seahorse assay

GL261 cells were seeded at 2×10⁴ cell/well in 24-well plates with 1 ml DMEM for 24 hours and treated with different concentrations of ONPG NPs (0 and 40 ppm) for 0 and 48 hours, and in 24-well plates, A1, B4, C3, and D6 cells were not seeded, as the temperature background correction on the machine. Then, following the procedure of the XF Mito Stress Test Kit, oligomycin (1 mM), FCCP (1 mM and 2 mM), and rotenone/antimycin A (0.5 mM) were added. Finally, the Seahorse XFe24 Extracellular Flux Analyzer was used to obtain the results⁴³.

RNA sequencing

RNA was isolated from each tumor sample after treatment to identify the involved genes, molecules and mechanistic pathways. cDNA was prepared for sequencing based on the Geo-seq protocol. Sequencing libraries were generated by the TruePrep DNA Library Prep Kit V2 from Illumina (Vazyme) and evaluated by a Bioanalyzer (DNA HS kit, Agilent). RNA-seq data were then mapped to the GRCm38 murine genome by HISAT2 (version 2.1.0) with default parameters for murine samples. Differential expression and pathway analyses were conducted, with a focus on immune regulation. Gene set enrichment analysis (GSEA) was performed by GSEA software developed by UC San Diego and the Broad Institute⁴⁴.

T-cell proliferation and activation analysis

Mouse PBMCs were cocultured with GL261 cells pretreated with CG NPs (40 ppm) or IONPs@CG (40 ppm) for 5 days. PBMCs were stained with CellTrace™ Violet reagent (Thermo Fisher) before coculture. The culture medium contained IL-2 (3.3 ng/ml) to promote T-cell proliferation. PBMCs supplemented with a CD3/CD28 stimulator were used as an activation positive control. Suspended immune cells were finally stained with CD8, CD4, CD62L, and CD44 to determine the proliferation of cytotoxic T cells or memory T cells. The percentages of these cell types were obtained using FACSCalibur flow cytometry, and data were analyzed by FlowJo software.

In vitro and in vivo phantom MRI

Based on our previously published work, IONPs@CG and cell phantoms were imaged using a Bruker PharmaScan 7T MRI scanner and a volume coil (inner diameter =72 mm, Bruker Biospin, Billerica, MA, USA). Localizers were used to verify the phantom and animal positioning in the center of the images. Multiecho T2*-weighted images with the same geometry were acquired using the fast, low-angle shot (FLASH) sequence with 30-35 echo sequences. The sequence parameters were as follows: repetition time (TR), 300 ms; first echo time (TE), 3.02 ms; echo spacing, 3.44 ms; in-plane matrix size, 256 × 256; and flip angle, 15°. All MRI data were processed using customized MATLAB scripts (The Mathworks Inc., Natick, MA, USA). We also calculated ΔR2* values using MR images. A region of interest (ROI) was drawn within the GBM margin in the visible tumor area of all tissue samples. The mean intensity levels of the T2* signal, weighted by area, were averaged across the measured samples for each pre- and post-IONPs@CG (3000 ppm) treatment image. The reciprocals of pre- and posttreatment T2* values were subtracted to obtain the ΔR2* value.

Perfusion-weighted MRI (PW MRI) analysis

Cerebral blood volume (CBV) was measured using T2* dynamic susceptibility-weighted contrast-enhanced perfusion-weighted magnetic resonance imaging (PW MRI). A gradient-echo echo-planar imaging sequence was used with the following parameters: FOV= 20 × 20 mm², matrix size 128 × 128, TR/TE = 800/12 ms. A total of 75 time points were collected in one minute. The signal intensity change in the time series was converted into the relaxation rate by the equation ∆S(t)=TE^-1{-ln[S(t)/S₀]}, where S(t) is the signal intensity at time t and S₀ is the precontrast signal intensity⁴⁵. The effects of recirculation and contrast material leakage were reduced by fitting a gamma-variate function to the measured ∆S(t) curve^46,47. Whole-brain CBV was calculated by fitting individual voxel time courses to a gamma variate function. To minimize the possible intersubject differences in those PW MRI-derived parameters caused by factors, such as brain topography and temperature, that might affect the absolute PW MRI-derived parameters, the change in CBV was expressed as a ratio of the lesion to the contralateral area (i.e., relative CBV, rCBV). A consistent ROI area was drawn in the healthy contralateral white matter, and the corresponding mean value of CBV in the ROI was calculated as the reference standard. On a pixel-by-pixel basis, the CBV maps were calculated first and then normalized by dividing every pixel CBV value by the reference value of the CBV calculated from the unaffected area to minimize variances in an individual animal.

Systemic and local immune alterations after CG NP or IONPs@CG treatment

After 21 days of treatment, immune cells from the bone marrow, spleen, tumor-draining lymph nodes (TDLNs) and infiltrating lymphocytes (TILs) were isolated from GBM model mice and stained with different markers to determine the proportions of myeloid-derived suppressor cells (MDSCs, CD11b⁺/Gr-1⁺), regulatory T cells (Tregs, CD4⁺/CD25⁺/FOXP3), dendritic cells (DCs, CD11c⁺/CD24⁺/MHCII⁺), natural killer (NK) cells (CD3^-/NK1.1⁺/CD49b⁺), M1/M2 macrophages (CD11b⁺/CD86 or CD206) and cytotoxic T cells (CD8⁺/IL-2⁺ or CD8⁺/IFN-g⁺) and the systemic immune status. Local immune status was also validated by IF analysis of mouse tumor cryosections. For IF staining, brain tissue from mice was fixed with 4% formaldehyde, dehydrated with 30-50% sucrose, frozen at -80°C, embedded in OCT compound, and sectioned with a cryostat microtome. Brain tissue sections were then be stained for CD8, CD86, CD206, IFN-g, CD4, and FOXP3 and imaged by a fluorescence microscope (TissueFAXS-S PLUS).

Establishment of orthotopic Pan02-Luc pancreatic tumor model

All animal treatments and surgical procedures were performed following the guidelines of the National Cheng Kung University (NCKU) Laboratory Animal Center. C57BL/6 mice (8 weeks, male) were anesthetized using an intraperitoneal Zoletil 100 (Virbac) injection and placed in a face-up position. Then, 2 × 10⁶ Pan02-Luc cells in a solution containing 10 μL of DMEM and 10 μL of Basement Membrane Matrix (BD Biosciences) were surgically implanted into the pancreas using BD 30 G 3/10 cc insulin syringes (BD Biosciences). The wound was sutured using CT204 Chromic Gut (20 mm, 75 cm, Unify Surgical Sutures Mfg. Co.) and NC193 monofilament nylon (19 mm, 45 cm, Unify Surgical Sutures Mfg. Co.) sutures, and the mice were allowed to rest until they fully recovered. The mice with orthotopic Pan02-Luc pancreatic tumors were treated with 100 μL of sterilized PBS (control) or 100 μL 1500 ppm CG NPs in sterilized PBS through intravenous injection once a week. Simultaneously, 12.5 mg/kg InVivoPlus anti-mouse PD-1 mAb (RMP1-14, BioXCell) was administered through intraperitoneal injection twice a week. Afterward, the body weight of the mice was measured, and the tumor growth of Pan02-Luc pancreatic cancer cells was monitored by the Xenogen IVIS Spectrum Noninvasive Quantitative Molecular Imaging System (Caliper Life Sciences) every 3 to 4 days. The maximum tumor burden permitted by the Institution Animal Care and Use Committee (IACUC) of NCKU was that the IVIS bioluminescence should not exceed 1×10⁷ photon/sec/cm2/sr without ascites formation. All experimental mice with orthotopic pancreatic tumors were sacrificed before reaching the maximum tumor burden.

Establishment of the GL261-bearing GBM animal model and treatment procedure

Six-week-old male C57BL/6JNarl mice (20-25 g) were purchased from the National Animal Center and housed at the Animal Center of China Medical University. To establish an immunocompetent model, 1×10⁵ GL261 cells intracerebrally implanted into the mice. Six-week-old male BALB/c nude mice were purchased from the National Animal Center to establish an immunodeficient model. Then, 5×10⁶ GL261 cells were mixed with Matrigel phosphate-buffered saline (7:3) in a total volume of 100 μl and inoculated into the subcutaneous region of the right flank. After the tumor size reached 5 mm³(C57BL/6JNarl mice) or100 mm³(nude mice), the mice were divided into 5 groups: control (0.1% DMSO), CG NP treatment (1500 ppm/treat/intravenous injection), 10F.9G2 (anti-PD-L1, 100 mg/treat/intraperitoneal injection), temozolomide (TMZ) treatment (50 mg/treat/gavage), IONPs@CG treatment (1500 ppm/treat/intravenous injection), CG NPs combined with 10F.9G2 and IONPs@CG combined with 10F.9G2 for 21 days. Additional anti-MCT-4 (0.5 mg/treat/twice per week) was used in combination with CG plus 10F.9G2.

Biodistribution test of IONPs@CG by radioactive isotope

¹²⁵I (1 mL) was added to the IONPs@CG, and the reaction was performed on a 300 rpm shaker at room temperature for 30 min to produce ¹²⁵I-IONPs@CG. Next, purification was performed by centrifugation in a 10k centrifuge tube (Sartorius Vivaspin®, Göttingen, Germany) at 12,000 rpm for 20 min. Afterward, the supernatant was removed, and the particles were suspended in normal saline. The process was repeated once. The radiolabeling yield and radiochemical purity were determined by an instant thin-layer chromatography system (AR-2000 radio-TLC Imaging Scanner, Bioscan Inc., Poway, CA, USA). Mice were anesthetized with isoflurane and intravenously injected with ¹²⁵I-IONPs@CG (3.7 MBq). After 24 and 48 hours of injection, mice were sacrificed by CO₂. The organs were excised, weighed, and assayed for radioactivity by a gamma counter (1470 Wizard, PerkinElmer, Waltham, MA, USA). The tissue activity was displayed as a percentage injected dose (ID) per gram of tissue (%ID/g).

Statistical analysis

Statistical analysis was performed using GraphPad Prism 7.0 software. One-way analysis of variance (ANOVA) with a Tukey's post hoc test was used to determine significant differences between more than two groups. The Student’s t test was used to determine significant differences between two groups. To ensure the reliability and consistency of our results, each group of cells or mice was independently tested with 3~6 separate samples.

Conflict of interest statement

The authors have reported no potential competing interests.

Funding

This study was supported by the Ministry of Science and Technology (MOST), Taipei, Taiwan, R.O.C. (ID: MOST 109-2314-B-039-021-MY3, MOST 111-2113-M-006-015, MOST 111-2314-B-006-004, MOST 111-2321-B-006-011, NSTC 112-2314-B-039-063-MY3, NSTC 112-2926-I-039-501-G, and NSTC 112-2113-M-006-006-MY3, NSTC 112-2321-B-006-010, NSTC 112-2314-B-006-053, and NSTC 112-2314-B-006-108) and by a grant from the Taipei City Hospital (TCH) and the Department of Health, Taipei City Government (TCH No. 1120162006). This work was also financially supported by the Drug Development Center, China Medical University and the Center of Applied Nanomedicine, National Cheng Kung University, The Featured Areas Research Center Program and the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan.

Acknowledgments

We want to express our sincere gratitude to the molecular imaging group of the Laboratory Animal Center at Chang Gung Memorial Hospital for their valuable support in imaging and technical assistance. Experiments and data analysis were performed in part through the use of the Medical Research Core Facilities Center, Office of Research & Development at China Medical University, Taichung, Taiwan, ROC. We are also grateful for the support from the Laboratory Animal Center, College of Medicine, National Cheng Kung University, and the Core Facility of Taiwan Mouse Clinic and Animal Consortium.

Author contribution statement

YTC, YCC, LCC, SCC, and LXY conducted experiments, analyzed data, and contributed to manuscript drafting. LBH, WCS, ITC, and YCL participated in data analysis and performed validation. HSL and SHH provided methodological and software support. WPS, CCH, and FTH conceptualized ideas, provided supervision, acquired funding, oversaw the research, and finalized the manuscript.

Ethics statements

Studies involving animal subjects

The animal study conducted in this research was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of China Medical University, Taichung, Taiwan (ID: CMUIACUC-2020-388) and National Cheng Kung University (NCKU), Tainan, Taiwan (ID: IACUC No. 111136).

Studies involving human subjects

Not applicable.

Inclusion of identifiable human data

No potentially identifiable human images or data were included in this study.

Data availability

The authors state that the data substantiating the conclusions of this study can be found within the article and its accompanying Supplementary Information files. The corresponding author can obtain pertinent data upon reasonable request.

Code availability

The code used in this study for T2* measurement can be provided by corresponding author with reasonable request.

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Sugar-based drug-free systemic immunoregulating and imageable nanoparticles promote the efficacy of immune checkpoint inhibitors in aggressive cancer

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