Colorectal cancer (CRC) has become one of the major threats to public health. Surgical operation combined with chemotherapy intervention is currently the main clinical approach for treating malignant CRC. The progression of this malignancy is frequently associated with the disruptions to the intestinal microbiota and an immunosuppressive landscape within the tumor microenvironment, both of which contribute to an increased propensity for tumor recurrence and metastatic spread. However, traditional treatment strategies, as well as emerging tumor immunotherapies, often result in severe gastrointestinal adverse events (AEs) and a dysregulated immune microenvironment, which fail to make significant progress in the efficacy of CRC treatment. It is essential to explore a holistic approach that incorporates modulation of the microbial and immune microenvironments for the treatment of CRC. In this study, we provide an alternative transabdominal photothermal therapy (PTT) for CRC that utilizes a novelty designed photothermal functional assembled drug (MnBV@DPHA NPs) administered orally. We employ PTT to precisely ablate orthotopic tumors, and achieved a cascade synergistic modulation of the intestinal microbiota and immune microenvironment, thereby enhanced the therapeutic efficacy of CRC. The oral assembled drug effectively targeted the tumor, precisely thermo-ablated cancer lesion while sparing healthy tissue. The aforementioned treatment strategy significantly reduced the presence of pathogenic bacteria and increased the proportion of probiotics within the intestinal microenvironment, thereby restoring the intestinal microbiota’s homeostasis. Furthermore, it reshaped the immunosuppressive microenvironment of CRC, thereby maintaining an immune homeostasis. Further research has confirmed that the cascade synergistic between the intestinal microbiota and the immune microenvironment enhances the efficacy of PTT and inhibits the recurrence and metastasis of CRC.
Research Article
Cascade-Synergistic Modulation of Intestinal Microbiota and Immune Microenvironment in Photothermal Orthotopic Colorectal Cancer Therapy
https://doi.org/10.21203/rs.3.rs-5000938/v1
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Colorectal Cancer
Intestinal Microbiota
Immune Microenvironment
Photothermal Therapy
Assembled Drug
Colorectal cancer (CRC) has become a kind of serious cancer with high global morbidity and mortality in dire need of improved treatment strategies.1,2 The current clinical therapeutic schedule for CRC mainly involves surgical excision, chemotherapy (e.g., 5-fluorouracil, oxaliplatin or irinotecan) or immunotherapy.3 However, the recurrence rate of CRC patients after standardized treatment is alarmingly high at 30–40%, with a median recurrence interval of 1.6 years.4 The reason is that CRC exhibits high invasiveness and a strong tendency for metastasis, making the complete removal of the tumor and metastases by surgical intervention almost impossible. Postoperative chemotherapy and emerging immunotherapy protocols are expected to maximize the clearance of residual lesions and inhibit the distal tumor spread and growth. However, according to current clinical statistics, the comprehensive therapeutic effects and prognosis of CRC have been consistently unsatisfactory.5 Therefore, it is necessary to delve into the underlying mechanisms of CRC pathogenesis and cure, and to develop effective therapeutic interventions.
It has now become abundantly clear that intestinal microbiota are crucial for the onset and progression of CRC.2,6 For instance, a large proportion of CRC patients, particularly those in advanced stages, typically show dramatic alterations in the microbial composition of the tumor and the adjacent mucosa, which are indicative of severe microbiota dysbiosis.7 Clinical data have confirmed that oral antibiotic use increases the likelihood of CRC development towards the disruption of the intestinal microbiota.8 Likewise, modulating the intestinal microbiota through administration of probiotics and/or prebiotics can be advantageous for the prevention and therapy of CRC.2,9 Additionally, as the most direct method for altering intestinal microbiota composition, fecal microbiota transplantation (FMT) has achieved positive advancement in preclinical and clinical researches on gastrointestinal and nongastrointestinal cancers(NCT04729322 and NCT03341143). Conversely, the transplantation of feces from CRC patients to germ-free mxice has been demonstrated to promote tumorigenesis when the mice are exposed to azoxymethane.10 The composition of the intestinal microbiota impacts susceptibility to intestinal diseases, yet its intrinsic relationship to CRC remains confusing.
CRC is recognized as a quintessential “cold tumor”, and its immunosuppressive microenvironment forms a sophisticated ecosystem.11 Within this immunosuppressive microenvironment, specific cell types, such as regulatory T cells (Tregs), adversely affect cancer treatment and prognosis by suppressing the activity of antitumor effector T cells, natural killer cells, and antigen-presenting cells. It is noteworthy that there is a complex interplay between intestinal microbiota dysbiosis and the immunosuppressive microenvironment, those interactions may collectively promote the development of CRC and the emergence of therapeutic resistance.2,8 On the one hand, the intestinal microbiota closely interacts with the host’s intestinal immune-related cells, significantly influencing the immune profiles of the gastrointestinal tract, which critically controls intestinal carcinogensis. Additionally, the microbiota harbors the potential to shape an inflammatory tumor immune microenvironment.12,13 In the vicinity of CRC lesions, the intestinal mucosa is typically in a damaged state.14 Due to the immunosuppressive properties of the CRC microenvironment, pathogenic bacteria are enriched in this region, resulting in severe inflammation.15 This, in turn, contributes to further exacerbating the deterioration of the tumor microenvironment. These can account for the therapeutic failures in CRC treatment, encompassing both conventional strategies and novel tumor immunotherapies, which often lead to severe gastrointestinal adverse events (AEs) and contribute to a dysregulated intestinal microbiota, but without altering the immunosuppressive CRC tumor microenvironment.16 The occurrence, development, metastasis, and recurrence of CRC are collectively driven by intestinal microbiota, and intestinal immunity. It is essential to explore a holistic approach that incorporates modulation of the microbial and immune microenvironments for the treatment of CRC.
Photothermal therapy (PTT) represents a promising approach to cancer treatment, employing photothermal drugs to eliminate cancer lesions through irradiation.17,18 This emerging strategy offers several inherent advantages, including being mild, non-invasive, highly precise, drug resistance-free and offering integrated imaging functionality. Effective PTT has been proven to ablate primary tumors and, at the same time, reshape the immunosuppressive microenvironment of malignant tumors, turning “cold” tumors into “hot” tumors.19,20 Upon laser ablation of tumor cells, the release of damage-associated molecular patterns (DAMPs) mediates immunogenic cell death (ICD), resulting in the generation of immunogenic cellular fragments, which ultimately triggers a change in the tumor immune microenvironment.21 Currently, PTT for CRC has achieved significant research progress. However, there is a lack of research on reshaping the intestinal microbiota and the tumor microenvironment through PTT to improve the CRC treatment efficacy.22 Here, we propose a cascade-synergistic modulation strategy of intestinal microbiota and tumor immune-suppressive microenvironment based on PTT, using a well-designed near-infrared (NIR) absorbing photothermal assembled drug administered orally.
Our research focuses on how to design and construct assembled drugs, as well as reveal how they achieve cascade synergistic regulation of microbiota and immunity, to eliminate the primary tumor and prevent tumor recurrence and metastasis.
We have optimized the maximum absorption wavelength of the assembled drug utilizing the coordination reaction of endogenous biological pigments with metal ions,
and employed it to orthotopic CRC PTT treatment based on “transabdominal irradiation”. We found that after PTT for tumors, the proportion of pathogenic bacteria accumulated at the tumor site decreased, and the abundance of probiotics was restored. Meanwhile, the “cold” immunosuppressive microenvironment of CRC has been reprogrammed. Further research has unveiled the cascade synergistic mechanism mediated by PTT between the intestinal microbiota and the immune microenvironment, which enhances the therapeutic effect on CRC and effectively inhibits tumor recurrence and metastasis.
Design of MnBV with super-large red-shifted absorption peak
Biliverdin (BV) is a naturally-occurring endogenous pigment known for its excellent biocompatibility and a well-defined metabolic pathway. It exhibits a maximum absorption peak at ~ 680 nm and has been utilized for PTT of superficial tumor-bearing mice in our previous studies.23 By coordinating BV with manganese acetate, we obtained a new complex molecule named manganese biliverdin (MnBV), and successfully achieved a super-large red-shifted absorption peak in the NIR region (Fig. 1a). Firstly, we methylated the two propionic acid terminals of BV to obtain the intermediate product BVDME, which exhibits stronger hydrophobicity. Subsequently, by introducing metal ions into BVDME, a coordination reaction occurred between them, yielding the target product MnBV with a maximum absorption peak at 880nm (Supplementary Figs. 1–2). This absorption redshift of the chromophore greatly facilitates the development of NIR-triggered photothermal drug assemblies, thereby promoting the implementation of PTT on non-superficial tumors, such as CRC.
Notably, the molar extinction coefficient (ε) of MnBV at 880 nm is calculated to be 2.3×105 L mol− 1 m− 1, which is higher than that of BV of 4.7×104 L mol− 1 m− 1 (ref.24). This indicates that MnBV has an enhanced photothermal conversion potential compared to BV. Moreover, we compared the fluorescence quantum yields (Φf) of BVDME and MnBV, which provide a direct comparison for the efficiency of the conversion of absorbed light into emitted light.24 The Φf value of BVDME measured at 680 nm is 0.738%, whereas Φf value of MnBV measured at 880 nm was 0.673% (Fig. 1b-c). It indicates that when MnBV is exposed to laser irradiation, the energy is mainly dissipated through vibration relaxation, thereby exhibiting a higher photothermal conversion capability compared to BVDME.
The X-ray Photoelectron Spectroscopy (XPS) analysis results indicate that the spin energy separation between the 2p1/2 and 2p3/2 orbital of the manganese ion is 11.75 eV, while the spin energy separation between the 3sA and 3sB orbitals is 5.30 eV, inferring that manganese is trivalent (III) in MnBV (Fig. 1d-e) (ref.25). Further, to represent electron transitions simply and efficiently of BVDME and MnBV, the electron transition difference map (EDDM) was plotted based on density functional theory (DFT) calculations. As depicted in the EDDM (Fig. 1f), the green region signifies an area of electron outflow, whereas the blue region represents an area of electron inflow area. During the electronic transition, electrons from BVDME (the ligand) migrate to the 3d orbital of Mn(III). The highest occupied molecular orbital (HOMO)–lowest unoccupied molecular orbital (LUMO) energy gap (ΔEH–L) of BVDME was calculated to be 2.11 eV, whereas for MnBV is 1.75 eV. This process results in a reduction of the energy difference between the excited state and the ground state, consequently causing an absorption redshift.
Construction of NIR-triggered assembled drug for transabdominal PTT
Self-assembly provides a flexible strategy for constructing photothermal functional drugs with tailored properties and performance through the organization of designed molecular building blocks.26–28 To enhance the targeted tumor delivery and accumulation of MnBV, assembled drug (MnBV@DPHA NPs) entities with CRC-targeting capabilities for oral administration have been constructed (Fig. 2a). The nanoassemblies are formed through the co-assembly of two molecules, the MnBV molecule, which possesses NIR photothermal conversion ability, and the phospholipid polymer conjugate with tumor-targeting functionality (DSPE-PEG-HA, DPHA). Here, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (poly(ethylene glycol)) (DSPE-PEG, an FDA approved PEGylated lipid) possesses excellent amphoteric affinity, creating optimal conditions for drug encapsulation and delivery.29 DSPE-PEG has been modified with hyaluronic acid (HA) to target the overexpressed CD44 on the tumor cell membrane.30
By adjusting assembly parameters, such as the ratio of good/poor solvent and the concentration of assembly building blocks, we have optimized the nanoassemblies with a uniform size distribution (Supplementary Fig. 3). Transmission electron microscopy (TEM) shows that the optimized assemblies exhibit a uniform particulate morphology, while MnBV exhibits an aggregated state (Fig. 2b). Dynamic light scattering (DLS) measurement reveals that the particles are uniformly distributed with an average diameter of 95.5 ± 8.9 nm (Fig. 2c). Zeta potential measurements indicate that MnBV aggregates possess a positive charge of 1.62 ± 0.13 mV, while MnBV@DPHA NPs display a negative charge of -20.47 ± 0.60 mV (Fig. 2d). The negatively charged nanostructures usually lead to favorable blood circulation and ideal tumor accumulation.31 Energy dispersive X-ray spectroscopy (EDS) images show the presence of manganese and phosphorus elements within MnBV@DPHA NPs (Supplementary Fig. 4). The XPS analysis reveals that the mass ratio of MnBV in MnBV@DPHA NPs is 8.72%, whereas the mass ratio of DPHA is 91.28%. This suggests a successful assembly between MnBV and DPHA.
Photothermal performance of the assembled drugs determines the PTT treatment outcomes,27,32 therefore, we evaluated their photothermal conversion efficiency and photostability. Firstly, the UV-Vis spectrum indicates that the assembly process does not alter the NIR absorption of MnBV (Fig. 2e). Subsequently, the MnBV@DPHA NPs (in 1.0 mL water) were irradiated with 880nm laser, and the solution’s temperature increased by 25 ℃ within 10 minutes at a power density of 1.0 W cm− 2. Additionally, the temperature values can be precisely controlled by adjusting the laser power density (Fig. 2f). The photothermal conversion efficiency was calculated to be 58.1% (Fig. 2g). Continuous irradiation-cooling measurements were carried out to further investigate the photothermal stability of MnBV@DPHA NPs. Results exhibited that these NPs maintained a high photothermal effect throughout all five irradiation-cooling cycles (Fig. 2h).
Stability and Targeting Efficiency of MnBV@DPHA NPs
The colloidal stability of the assembled drugs is a prerequisite for achieving tumor targeting and accumulation, which in turn directly influences the efficiency of photothermal tumor therapy.33 Our results demonstrate that MnBV@DPHA NPs exhibit robust resistance to a 20-fold dilution and retain their stability when incubated in physiological culture medium at 37°C for a period of 7 days (Extended Data Fig. 1a-c). Given its intended oral administration, the photothermal drug must exhibit a stable retention profile in the gastrointestinal environment. Consequently, we assessed its stability in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) over a 7-day period. No significant alterations were observed in the DLS profiles, and their UV-Vis spectral changes were minimal, with the exception of a slight attenuation attributed to particle sedimentation (Extended Data Fig. 1d-e).
To investigate the cellular internalization of nanoassemblies, MnBV@DPHA NPs were labeled with the fluorescent dye Rhodamine B (RhB), and then co-cultured with CT26 cells in culture medium (Extended Data Fig. 2a). After a 24-hour incubation, the cell membrane and nucleus were stained with Alexa Fluor 488 (presented as green) and Hoechst 33342 (presented as blue), respectively, for visualization with a confocal microscope. Red fluorescence within cytoplasm suggests a time-dependent endocytosis of these nanoparticles by CT26 cells. Quantitative analysis of fluorescence intensity demonstrated that CT26 cells exhibited significantly enhanced internalization of MnBV@DPHA NPs compared to non-targeted MnBV@DP NPs (Extended Data Fig. 2b). This in vitro result highlights the improved targeting capability due to HA modification, indicative of the specific affinity of MnBV@DPHA NPs for tumor cells.
Subsequently, we established orthotopic mouse models of CRC by injecting CT26-luc cells into the colonic mucosa of mice, and the in vivo targeting ability of MnBV@DPHA NPs was assessed. To visualize the NPs in vivo, we utilized Cy5 fluorescence-labeled DSPE-PEG for the co-assembly of nanodrug formulations. We administered Cy5 fluorescence-labeled NPs to parallel orthotopic CRC mice by gavage at equimolar doses. At predetermined time intervals, we performed autopsies on the mice and conducted ex vivo fluorescence imaging of the intestinal tissues and vital organs (Extended Data Fig. 2c). The results indicated that the MnBV@DPHA NPs can effectively target orthotopic CRC, exhibiting an optimal retention time of 12 hours. Notably, these nanodrugs did not exhibit significant enrichment in any of the dissected organs at 48 h, including liver, heart, spleen, lung and kidney (Extended Data Fig. 2d).
We co-cultured MnBV@DPHA NPs with CT26 cells for 24 hours and employed the standard MTT assay to assess the cytotoxicity of the nanodrug. The results showed that the nanodrug did not exhibit significant toxicity to CT26 cells without laser irradiation, confirming its excellent biosafety. However, when irradiated with an 880 nm laser, the survival rate of CT26 cells decreased significantly, and the half maximal inhibitory concentration (IC50) was calculated to be approximately 35.00 µg ml− 1 (Supplementary Fig. 5). The assembled nanodrugs have collectively exhibited superior stability, precise targeting capabilities, and high safety, complemented by their intrinsic NIR absorption properties. These characteristics establish a robust foundation for the application of transabdominal PTT in the treatment of CRC.
In vivo transabdominal PTT on orthotopic mouse of CRC
We proceeded to assess the in vivo transabdominal PTT efficacy of MnBV@DPHA NPs in an orthotopic CRC mouse model (Fig. 3a). Our study juxtaposed the transabdominal PTT efficacy of these nanodrugs against that of a PBS control and MnBV@DP NPs, both in the presence and absence of a NIR laser irradiation source. CT26-luc cells were cultured and harvested, subsequently mixed with matrix glue for injection into the cecal serosa layer of female BALB/c mice. Upon reaching the 7th day post-injection, the tumor had developed to meet the stipulated experimental criteria. Subsequently, the drugs were administered via oral gavage. Following a 12-hour interval post-administration, the cecal region of the mice was subjected to targeted laser irradiation at a power density of 1.0 W cm− 2, with a wavelength of 880 nm, for a duration of 10 minutes. The abdominal temperatures were monitored by an infrared thermography camera (Fig. 3b). The results indicate that the maximum temperature of colorectal tissues in the MnBV@DPHA NPs treatment cohort attained a remarkable 55 ℃. Comparatively, colorectal tissues in the PBS and MnBV@DP NPs treatment cohorts registered maximum temperatures of 36 ℃ and 44 ℃, respectively (Fig. 3c).
The in vivo imaging systems (IVIS) was leveraged to perform a continuous assessment of the spatial distribution and dimensions of murine tumors throughout the therapeutic intervention (Fig. 3d-f). The results indicate that in the group where mice were orally administered MnBV@DPHA NPs and subsequently subjected to transabdominal laser irradiation (G6), the tumors were effectively ablated, while the intestinal integrity remains intact. In comparison, the tumors in the cohorts subjected to orally administered PBS (G1), orally administered PBS with concurrent laser irradiation (G2), orally administered MnBV@DP NPs (G3), and orally administered MnBV@DPHA NPs without laser (G4) did not display a notable inhibition of tumor growth and instead disseminated throughout the intestines, viscera, and other regions. Mice that were given MnBV@DP NPs orally and then treated with transabdominal laser irradiation (G5) displayed an inhibition of tumor growth. The tumors in groups (G1 to G5) did not ultimately receive effective treatment, and by the end of the observation period, it was observed that tumors had disseminated throughout the intestines, viscera, and other regions (Supplementary Fig. 6–7).
Intestinal tissues were collected and subjected to hematoxylin-eosin (H&E) staining for histological examination (Fig. 3g). The stained sections revealed that the tumor within group G6 undergone severe photothermal damage, while the integrity of the intestinal villi was preserved. Conversely, in the other cohorts, the intestinal tissues suffered significant invasion attributable to tumor progression, culminating in considerable intestinal injury and the virtual obliteration of intestinal villi. By employing NIR-triggered assembled drug oral administration coupled with transabdominal laser irradiation, the orthotopic CRC was effectively managed, thereby extending the survival period of the mice to 120 days (Fig. 3h). Mice that did not undergo effective treatment succumbed within a brief interval. Throughout the treatment and observation period, we monitored the body weight of the mice subjected to various therapeutic regimens. The data consistently demonstrated that there was no significant weight reduction attributable to the medication or transabdominal PTT. Notably, the body weight of the mice in Group G6, which received MnBV@DP HA NPs and laser irradiation, remained stable. In contrast, the body weight of mice in other groups exhibited a marked increase, primarily due to the proliferation of tumor mass (Supplementary Fig. 8).
Manipulating the homeostasis of the intestinal microbiota
The progression of CRC frequently coincides with dysbiosis of the intestinal microbiota. A growing corpus of evidence suggests that the modulation of the intestinal microbiota may improve the efficacy of oncological therapies. Furthermore, by meticulously monitoring the microbial composition, the therapeutic effectiveness and prognostic outcomes of CRC can be accurately assessed.34 To examine the intestinal microbiota characteristics in healthy mice as well as in mice with CRC, and to evaluate the effects of various therapeutic interventions, 16S ribosomal DNA (rDNA) gene sequencing followed by clustering of short sequences into operational taxonomic units (OTUs) was performed.34 The principal component analysis (PCA) plot of beta-diversity revealed statistically significant dissimilarities in the microbial composition between the healthy mice and the CRC mice, indicating the intestinal microbiota dysbiosis triggered by the progression of malignant CRC tumors (Fig. 4a). The further experimental outcomes have delineated a noteworthy amelioration in the intestinal microbiota dysbiosis within the cohort of mice harboring tumors (G6), subsequent to the oral administration of MnBV@DPHA NPs, followed by the application of transabdominal PTT. However, the intestinal microbial assemblage within other groups (G1 to G5) persisted in its original configuration, exhibiting no significant differences. We conducted an advanced analysis of the intestinal microbiota’s alpha-diversity, across all groups, to assess the microbial species richness. The results revealed that the CRC tumors diminished the species richness of the intestinal microbiota in mice. In the group that received efficacious treatment (G6), the species richness was restored. Conversely, in the group that did not receive effective intervention (G1 to G5), the progression of the tumor was accompanied by a further reduction in microbial species diversity (Fig. 4b).
Subsequently, we illustrated the alterations in the relative abundance of the top 15 dynamically fluctuating intestinal microbiota (in family level) before and after transabdominal PTT, by the community heat map analysis (Fig. 4c).
For mice (in G1 to G5) did not receive efficacious therapy, there was a substantial increase in the abundance of pathogenic bacteria in the gut, such as the Bacteroidaceae (Fig. 4d), the Enterobacteriaceae (Fig. 4e), and Prevotellaceae (Fig. 4f), which are associated with the progression and poor prognosis of CRC. Conversely, in the cohort of mice (G6) that underwent optimal therapeutic intervention, there was a significant reduction in the abundance of pathogenic bacteria within the intestinal microbiota. Simultaneously, there was a marked increase in the abundance of probiotic bacteria, such as Ruminococcaceae (Fig. 4g), Lactobacillaceae (Fig. 4h) and Rikenellaceae (Fig. 4i). This shift in microbial composition towards a more beneficial balance is indicative of the positive impact of effective treatment strategies on the intestinal ecosystem.
Cascade-synergistic modulation of intestinal microbiota and immune microenvironment
The CRC tumor microenvironment is characteristically replete with substantial inflammatory infiltrates, comprising neutrophils, macrophages, natural killer cells (NKs), and dendritic cells (DCs).35 Upon the induction of necrosis in tumor cells through PTT, the extrusion of intracellular contents prompts an interaction with the immune cell milieu, potentially transforming the tumor immunosuppressive microenvironment (Fig. 5a) (ref36).
We conducted a detailed assessment of the immunological dynamics within CRC, employing flow cytometry to analyze the variations in immune cell populations before and after therapeutic intervention (Supplementary Fig. 9). In the instances of tumors subjected to photothermal ablation, post-treatment analysis was performed on the residual neoplastic elements and the surrounding intestinal tissue.
The highest populations of CD3+CD8+ T cells were observed in MnBV@DPHA NPs-orally administered and transabdominal laser irradiated group (G6), which was 1.61-fold higher relative to those for the control group (G1). No discernible disparities were observed among the various treatment groups (G2 to G5) in contrast to the control cohort (Fig. 5b, Supplementary Fig. 10). The PTT-induced tumor ablation, facilitated by the triggered assembled drugs, enhances the activity of CD8+ T cells, signifying a pronounced augmentation in the immunological anti-tumor response.
The immunosuppressive activity mediated by regulatory T (Treg) cells constitutes an essential regulatory mechanism that exerts a dampening effect on immune-mediated inflammation, playing a pivotal role in the pathophysiology of CRC.37 To delve into the immunosuppressive landscape of the tumor microenvironment in CRC, we conducted a quantitative assessment of Treg cells (identified by the phenotype CD3+ CD4+ CD25+ FoxP3+) and IFN-γ (characterized as CD3+ CD8+ IFN-γ+). Our analysis revealed a notable reduction in the prevalence of Treg cells within the G6 group, exhibiting a 46.0% decrease relative to the control group (Fig. 5c, Supplementary Fig. 11). Concurrently, there was a marked escalation in the frequency of IFN-γ expression, with an 87.5% increase noted in comparison to the control group (Fig. 5d, Supplementary Fig. 12). No significant variations were detected among the other study groups. These findings intimate that the PTT ablation of the tumor may ameliorate immunosuppressive conditions and invigorate the antitumor immune cascade in CRC.
To elucidate the role of intestinal microbiota in the immunoregulation by transabdominal PTT of tumors, we conducted a controlled experiment in CRC-bearing germ-free mice. Initially, the mice were administered an antibiotic cocktail (ABx), a mixture comprising ampicillin, neomycin, metronidazole, and vancomycin, via oral gavage for a period of one month.38 Subsequently, CT26-luc cells were cultured, harvested, and suspended in a matrix gel, which was then inoculated into the cecal serosa of mice. Following a seven-day period for tumor establishment, the mice were subjected to pharmacological intervention and laser therapy, after which the tumor microenvironment was examined using flow cytometry. In comparison to the Control group (G1), the frequency of CD3+CD8+ T cells in G6 group exhibited no significant alteration (a non-significant decrease of 15.2% relative to G1 group, Fig. 5e and Supplementary Fig. 13). The Treg cell frequency was similarly invariant (a minimal reduction of 11.5% compared to G1 group, Fig. 5f and Supplementary Fig. 14), as was the case for IFN-γ levels, which registered a negligible decrease of 1.8% relative to G1 group (Fig. 5g, Supplementary Fig. 15). These findings intimate that alterations in the intestinal microbiota’s composition may substantially influence the immune cell status. The absence of the intestinal microbiota post-antibiotic treatment results in a diminished capacity for MnBV@DPHA NPs-mediated transabdominal PTT to reverse the immunosuppressive microenvironment. This foretells an intricate interplay between healthy gut microbiota and the homeostasis of the tumor immune microenvironment, underscoring the significance of their symbiotic relationship in CRC oncological dynamics.13,39
Our experiments have demonstrated a positive correlation between the reconstruction of the immunosuppressive microenvironment and the reduction of pathogenic bacteria coupled with the increase of probiotics within the gut in CRC tumor-bearing mice, following oral administration of MnBV@DPHA NPs and transabdominal PTT (Fig. 5). Pathogenic bacteria primarily influence the progression and prognosis of CRC by altering intestinal permeability and secreting specific toxins.40 In our study, we observed a significant increase in the populations of three pathogenic bacterial families post-tumoral inoculation: Bacteroidaceae, Enterobacteriaceae, and Prevotellaceae (Fig. 4c). Bacteroidaceae and Enterobacteriaceae are predominantly known for inducing intestinal inflammation through the release of inflammatory cytokines, exacerbating the disequilibrium of immune homeostasis.41 Prevotellaceae, on the other hand, is associated with tumor-induced immunosuppression, potentially through the activation of the TLR2 receptor inducing Th17 cell polarization and increasing the frequency of Treg cells via TGF-β, thereby fostering an immunosuppressive microenvironment.42 Following effective intervention, the abundance of pathogenic bacteria was downregulated, accompanied by a reshaping of the immunosuppressive microenvironment (Fig. 4d-f). Conversely, probiotics can maintain intestinal immune balance by secreting prebiotic substances that alter the intestinal environment and inhibit the growth of pathogenic bacteria.43 We noted a significant decrease in the populations of nearly all probiotics post-CRC tumor inoculation, coinciding with the formation of an immunosuppressive microenvironment (Fig. 4c, Fig. 5b-d). Upon receiving effective treatment, the abundance of Ruminococcaceae, Lactobacillaceae, and Rikenellaceae was restored (Fig. 4g-i). Ruminococcaceae, a typical butyrate-producing bacteria, can suppress the expression of various interferon signaling pathways (such as IL-2, IL-4, IL-6, IL-10, and IL-12 signaling pathways), reducing inflammatory levels.44 Lactobacillaceae maintains intestinal immune balance through the activation of the Wnt/β-catenin signaling pathway, 45 while Rikenellaceae can stimulate immune cells to secrete IL-10, stabilizing intestinal inflammatory levels.46 Parallel experiments conducted using germ-free mouse models also confirmed the aforementioned cascade regulatory mechanism between the intestinal microbiota and the immunosuppressive microenvironment (Fig. 5e-g).
Our work presents an innovative CRC treatment strategy, predicated on the cascade-synergistic modulation of intestinal microbiota and immune microenvironment, based on novelty designed oral assembled drug (MnBV@DPHA NPs) and transabdominal PTT method. We initially designed manganese biliverdin dimethyl ester (MnBV) molecule with NIR absorption, achieving a super-large red-shifted in the absorption spectrum of the endogenous pigment BV. Utilizing self-assembly strategies, we further developed a targeted assembled system (MnBV@DPHA NPs), which, when administered orally and combined with transabdominal PTT, has demonstrated a profound suppression of tumor growth and a substantial extension in the survival of CRC mice in vivo. Furthermore, the dysbiosis of the intestinal microbiota in CRC mice post-treatment was reversed, with a decrease in the abundance of pathogenic bacteria and an increase in that of probiotics. Concurrently, there was a notable elevation in the frequency of CD8+ T cells, enhanced expression of IFN-γ level, and a pronounced reduction in the frequency of Treg cells in CRC mice, leading to the reshaping of the immunosuppressive tumor microenvironment and the re-establishment of immune homeostasis. Our use of germ-free mice in parallel experiments further substantiated the cascade synergistic mechanism between intestinal microbiota modulation and immunosuppressive tumor microenvironment reconstruction. This work presents a novel perspective on enhancing the therapeutic efficacy and improving the prognosis of CRC through the synergistic regulation of microbiota and immune homeostasis using PTT, which underscores the significant potential of our functional assembled drugs in CRC treatment.
Acknowledgments
This work was financially supported by the National Science Fund for Distinguished Young Scholars of China (No. 22025207), the Innovation Research Community Science Fund of China (No. 21821005), National Natural Science Foundation of China (Nos. 22232006, 22072154 and 22377127), the Youth Innovation Promotion Association of Chinese Academy of Sciences (No. 2021048), and the Beijing Nova Program (No. 20230484352).
Author contributions
K.C., R.X.and X.Y. conceived the project and designed the experiments. K.C., Y. L., and M. S. performed the experiments, and collected and analysed the data. L.Z. performed the DFT calculation. K.C., Y.L., R.X. and X.Y. co-wrote the manuscript. All authors discussed the results and reviewed the manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Extended data is available for this paper at https://doi.org/xxxxxx.
Supplementary information The online version contains supplementary material available at https://doi.org/xxxxxx.
Correspondence and requests for materials should be addressed to Xuehai Yan.
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Synthesis
Biliverdin IX Dimethyl Ester (BVDME): Initially, 5 mL of 98% H2SO4 was slowly added to 100 mL cooled methanol while stirred. After cooling, biliverdin IX hydrochloride (116 mg, 0.2 mmol) was added to the above solution and stirred under argon at 60 ℃ for 2 h. After the reaction, 200mL of water was added into the mixture. Subsequently Na2CO3 was added to make the solution weakly alkaline. The precipitates were extracted with 80mL ethyl acetate for 3 times. Then the organic phase was collected and dried with anhydrous sodium sulfate. The solvent was then evaporated on a rotary evaporator at room temperature. The residue was dried before dissolved in ethyl acetate. The solution was then loaded on silica gel and purified via column chromatography eluting with 25% n-hexane in ethyl acetate yielding over 95%. MALDI-TOF m/z 611.458, calcd for 611.453.
Manganese(III) Biliverdin IX Dimethyl Ester (MnBV): The synthesis method was modified from the literature.47 Accordingly, 50 mg of biliverdin IX dimethyl ester was dissolved in 10mL of chloroform and then 150 mL of extra dry methanol was added. The solution was heated to 60 ℃ under argon and 15 times of manganese acetate (0.3 g, dissolved in 10 mL of extra dry methanol) was added. After reaction for 5 min, the solvent was removed under reduced pressure, and the crude material dissolved in 50 mL of chloroform. The above mixture was washed with water three times. The solution was removed under reduced pressure followed by the addition of 10mL chloroform. The solution was then loaded on neutral alumina and purified via column chromatography eluting with 1% to 5% methanol (gradually increasing proportion) in chloroform. The yield was over 75%. MALDI-TOF m/z 662.19365, calcd for 662.19317.
Preparation of the NPs
MnBV@DP NPs: Initially, 1.5 mg DSPE-PEG was dissolved with 900 μL water. Then the solution was quickly mixed with 100 µL DMSO solution of manganese(III) biliverdin IX dimethyl ester (5 mg mL-1). The formed MnBV@DP NPs were aged for 24 h before centrifugal separation under 300 g. The residue was suspended with 1mL water.
MnBV@DPHA NPs: MnBV@DPHA NPs were prepared by the same method for MnBV@DP NPs except that 1.2 mg DSPE-PEG and 0.3 mg DSPE-PEG-HA were dissolved with 900 μL water.
Cy5.5-labeled MnBV@DP NPs: Cy5.5-labeled MnBV@DP NPs were prepared by the same method for MnBV@DP NPs except that 1.5 mg DSPE-PEG and 0.03 mg DSPE-PEG-Cy5.5 were dissolved with 900 μL water.
Cy5.5-labeled MnBV@DPHA NPs: Cy5.5-labeled MnBV@DPHA NPs were prepared by the same method for MnBV@DP NPs except that 1.2 mg DSPE-PEG, 0.3 mg DSPE-PEG-HA, and 0.03 mg DSPE-PEG-Cy5.5 were dissolved with 900 μL water.
Photothermal Efficiency
The photothermal conversion was measured by irradiating the HANPs in a quartz cuvette (1.0 mL) with a laser (880 nm, 1.0 mW/cm2) for 10 min. The temperature was recorded by a thermocouple probe with a digital thermometer. Pure water was used as a control. The photothermal conversion efficiency (η) of the NPs was calculated according to a reported method.48
Orthotopic Colorectal Tumor Model
All the animal experiments were conducted in accordance with the Institute’s Guide for the Care and Use of Laboratory Animals and were approved by the ethical committee of Institute of Process Engineering, Chinese Academy of Sciences (approval no. IPEAEC2021602, approved on June 2021). The maximal tumor size/burden permitted by our institutional review board is 15% of the body weight (combined burden if more than one mass present) and mean tumor diameter of >20 mm in adult mice (~25 g). The maximum tumor size/burden permitted by our institutional review board was not exceeded.
Female BALB/c mice (14-16g, 5 weeks, Beijing HFK Bioscience Co. Ltd.) were housed in an environmentally controlled animal facility. CT26-luc cells were collected and suspended in PBS (6 × 107 cells mL-1). Then we established an in situ colon cancer model. In short, 10 μL CT26-luc tumor cells were dispersed in 40 μL Matrigel matrix. The mixture were surgically injected into the submucosa of the cecum wall using a 29G BD insulin syringe. The cecum was gently restored to its original position, and cecotomy was sutured with 7-0 silk sutures. The mice was disinfected with iodine.
Statistical Analysis
T-test was utilized for single comparisons and one-way analysis of variance (ANOVA) was utilized for multiple comparisons. All statistical analyses were performed using GraphPad Prism 8 software. Data were presented as the average values (mean) ± standard deviation (SD) for all reported statistical groups. Significant differences were defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
References
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The authors declare no competing interests.
- Supportinginformation.pdf
Supporting Information
- ExtendedData.docx