Intratumoral Delivery of Crystallised IDO1 Inhibitors Fails to Overcome Tumor Immunosuppression Due to Metabolic Adaptation

Intratumoral Delivery of Crystallised IDO1 Inhibitors Fails to Overcome Tumor Immunosuppression Due to Metabolic Adaptation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Intratumoral Delivery of Crystallised IDO1 Inhibitors Fails to Overcome Tumor Immunosuppression Due to Metabolic Adaptation Arnau Solé Casaramona, Anish Ghimire, Martin F. Bachmann, Mona O. Mohsen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7138169/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Tryptophan (Trp) is required by both immune and tumor cells for protein synthesis and key metabolic pathways. While immune cells rely on Trp for proliferation and function, tumor cells exploit IDO1-mediated Trp degradation to evade immune surveillance. However, clinical trials have demonstrated limited efficacy of IDO1 inhibitors, likely due to insufficient tumor targeting and immune evasion strategies within the tumor microenvironment (TME). Here, we explored a novel approach using intratumoral (IT) delivery of a crystallised IDO1 inhibitor designed for depot-based slow release. Using aggressive murine solid tumor models, we combined in vivo assessments of tumor growth with metabolic profiling to investigate treatment effects. Despite increased metabolite abundance in tumors, IT administration of the crystallised IDO1 inhibitor failed to control tumor growth compared to soluble formulations (subcutaneously or intravenously). Immune profiling confirmed that IDO1 blockade alone did not reverse immunosuppression in the TME. Interestingly, IT treatment with IDO1 inhibitor crystals significantly elevated nicotinamide riboside (NR) levels, suggesting tumor-intrinsic metabolic adaptation via the NAD⁺ salvage pathway. Similarly, local delivery of crystallised Trp did not suppress tumor progression. On the contrary, an excess presence of Trp enhanced tumor growth, demonstrating that Trp is also essential for tumor growth. These findings underscore the metabolic plasticity of tumors and highlight the limited therapeutic potential of IDO1 inhibition, even with local, sustained-release strategies. Biological sciences/Cancer Health sciences/Oncology IDO1 inhibitor Crystalisation Tryptophan Anti-Tumor Response Solid Murine Tumors Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The essential amino acid tryptophan (Trp) plays important roles in a variety of physiological processes in the body. A small proportion of free Trp is used for protein synthesis as well as for the production of neurotransmitters and neuromodulators. [ 1 ] About 95% of the free Trp is degraded in the Kynurenine Pathway, generating different metabolites involved in distinct biological activities. [ 2 ] Indoleamine-2,3-dioxygenase 1 (IDO1) is a rate-limiting enzyme in this pathway that degrades Trp to N-formylkynurenine. [ 2 ] It has also been shown that IDO1 is constitutively expressed in immune privileged sites such as the placenta and epidermis to block T cell activation by restricting access to Trp. [ 3 ] IDO1 is over expressed in about 60% of tumors such as melanoma, colon cancer and cervical cancer. [ 2 , 4 ] IDO1 expression has been associated with poor prognosis and outcome. [ 5 ] Trp degradation regulates T regulatory cells and other immune cells in tumors. In cervical cancer draining lymph nodes, FOXP3 + Treg cells have been found in direct contact with dendritic cells expressing IDO1. [ 6 ] Previous studies have shown that the concentration of kynurenine (Kyn) metabolites in the blood of patients with melanoma, colorectal cancer or cervical cancer was less frequently elevated than within tumors, pointing out that changes in Kyn and its downstream metabolites may be more restricted and localized in the TME. Accumulating body of evidence suggests that Trp metabolism and degradation can support tumor progression by suppressing T cell responses. [ 7 , 8 ] Several preclinical studies are based on the critical role of IDO1 in inducing immune tolerance and promoting tumor growth, targeting IDO1 has been an attractive approach in cancer immunotherapy. Different strategies have been developed to target IDO1 using IDO1 expression inhibitors, IDO1 enzymatic inhibitors, IDO1 effector modulators and IDO1 peptide vaccines. [ 7 , 9 , 10 ] Allison et al have shown anti-tumor efficacy of IDO1 inhibitor when combined with the cytotoxic T lymphocyte antigen-4 (CTLA4) immune-checkpoint inhibitor in B16F10 murine melanoma model, (IDO1 inhibitor was given subcutaneously (SC) or orally. [ 11 ] In 4T1 mammary carcinoma murine model, IDO1 ablation (IDO1 ⁻/⁻) was sufficient to reduce the pathological neovascularization in lung metastases. [ 12 ] Diverse clinical trials are currently ongoing testing the combination of IDO1 inhibitors with radiation, chemotherapy or immunotherapy. [ 2 ] Opposing the preclinical data, clinical studies have shown limited and disappointing efficacy of IDO1 inhibitor as a systemic monotherapy; however combination of IDO1 inhibitors with immunotherapies such as immune-check point inhibitors or vaccines showed more promising results. [ 13 ] For example, a phase I study combining PD1 with IDO1 inhibitor showed an objective response in 12 out of 22 melanoma patients. [ 14 ] On the contrary, phase III clinical trial of this combination did not improve the progression-free survival compared to anti-PD1 immunotherapy alone. [ 15 ] Overall, IDO1 inhibition has yielded mixed and sometimes contradictory outcomes in both preclinical and clinical studies, which could be attributed in part to the route of administration. In this context, we hypothesized that inhibiting IDO1 locally at the tumor site may offer a more rational and targeted approach. Hence, we developed a novel crystalline formulation of an IDO1 inhibitor designed for in situ IT administration, aiming to achieve localized inhibition of IDO1 activity. To evaluate its therapeutic potential, we compared this approach to systemic delivery of the same IDO1 inhibitor in soluble form administered SC or intravenously (IV). Our results revealed that although the overall abundance of metabolites increased in tumors, the IT administration of a crystallised IDO1 inhibitor did not induce an anti-tumor response, neither did the soluble formulations that were administered either SC or IV. Immune profiling further indicated that IDO1 blockade alone was insufficient to overcome immunosuppression within the TME. Interestingly, IT treatment with IDO1 inhibitor crystals resulted in a significant increase in nicotinamide riboside (NR) levels, suggesting tumor-intrinsic metabolic adaptation via the NAD⁺ salvage pathway. Similarly, direct IT injection of crystallised Trp failed to impede tumor progression. These findings emphasise the adaptability of tumor metabolism and call into question the therapeutic value of IDO1 inhibition, even when delivered locally via a sustained-release depot. Results Successful Crystallization of IDO1 Inhibitor Enables Direct Intratumoral Delivery Clinical trials assessing the effectiveness of IDO1 inhibitors have seen limited success, possibly influenced by the administration route (typically SC or oral). Delivering the drug directly into the TME as crystals for depot-slow release is a novel and promising strategy that has not been explored before. Accordingly, we proceeded to crystallise the IDO1 inhibitor (IDO1 inh), as outlined in the Methods section and in Fig. 1 a. To do so, 1 g of IDO1 inhibitor was dissolved at its maximum solubility (30 mg/mL) in 70% ethanol and vortexed thoroughly. Ultrapure water was then added dropwise while gently mixing, resulting in visible precipitation of crystalline nanoparticles. We successfully crystallised the IDO1 inhibitor and visualised the approximately 5–10 µm IDO1 inhibitor crystals using light microscopy (Fig. 1 b and c). Although IT delivery of IDO1 crystals introduces some variability in crystal size and distribution, our formulation protocol aimed to minimize these differences. Crystals were suspended in PBS, and equal injection volumes were administered across all treated tumors to standardize dosing. To further ensure even distribution within the tumor, injections were performed at multiple sites per tumor. To approximate clinically relevant exposure, we administered 500 µg of the IDO1 inhibitor in mice, equivalent to ~ 25 mg/kg based on average mouse body weight. Using standard body surface area normalization (K_m conversion), this corresponds to a human equivalent dose of ~ 2.0 mg/kg, or ~ 142 mg for a 70 kg adult. This aligns with oral doses used in clinical trials of IDO1 inhibitors like epacadostat (100–300 mg BID) and linrodostat (100–120 mg QD). Overall, we successfully developed a novel crystalline formulation of the IDO1 inhibitor, which can potentially be tailored for alternative administration routes in addition to directly into the tumor, such as intraperitoneal injection, although this was not explored in the present study. IDO1 Inhibitor Fails to Provide Anti-tumor Protection Regardless of Administration Route or Formulation Next, we aimed to evaluate the anti-tumor efficacy of our newly developed crystallized IDO1 inhibitor administered IT in solid tumors, with the goal of limiting Trp degradation and alleviating immune cell suppression. As controls, we used an IDO1 inhibitor solution that was administered either SC or IV. PBS treated groups were used as a baseline control. We employed the aggressive 4T1 triple-negative mammary carcinoma (TNBC) cell line, SC inoculated into the flank of wild-type (WT) Balb/c mice to allow convenient access to the tumor site. The following groups were generated: a control group injected with PBS SC; IDO1 inhibitor-treated groups injected either SC or IV with IDO1 inhibitor solution or IT with IDO1 inhibitor crystals. Given the importance of generating a TME before initiating crystal IDO1 inhibitor IT therapy, all treatments were started 5 days after 4T1 inoculation (Fig. 2 a). Tumor volume was monitored throughout the experiment, during which treatment was administered three times (days 5, 7 and 11) (Fig. 2 a). The tumor volume and weight measurements showed that the IDO1 inhibitor was ineffective in inhibiting 4T1 tumor growth when administered as crystals for depot-slow release upon IT administration, neither as solution formulation administered SC or IV (Fig. 2 b-d). In a separate experiment, we measured the density of immune cells in the groups treated with PBS or crystalized IDO1 inhibitor administered into the tumor. We specifically calculated the density of CD4 + and CD8 + T cells as well as myeloid cells characterized by CD11b+. Our results showed no statistical significance between the two groups (p = 0.4952, 0.6020 and 0.0971 respectively) (Suppl. Figure 1). Additionally, we evaluated the effects of administering IDO1 inhibitor crystals IT in the aggressive, low-mutational burden B16F10 murine melanoma model, as illustrated in Fig. 2 e. In brief, B16F10 melanoma cells were injected into Recombination-Activating Gene knockout “RAG⁻/⁻” mice, and tumors were harvested after 14 days, once they had reached a volume of approximately 1 cm³. The tumors were cut into fragments measuring ~ 8 mm² and implanted into the flanks of anaesthetized WT C57BL/6 mice. Treatments began three days post-implantation and were administered IT three times over 14 days (days 3, 7 and 10) (Fig. 2 e). The results also showed no significant difference between the groups treated IT with PBS or crystallised IDO1 inhibitor (p = 0.7319) (Fig. 2 f-g). These results are consistent with our previous observations in the TNBC model and confirm that IT delivery of IDO1 inhibitor crystals has no significant impact. In our previously published work, we demonstrated that formulating icosahedral T = 3 virus-like nanoparticles (VLPs) packaging Toll-like receptor (TLR) ligands with microcrystalline L-tyrosine significantly produces a potent immune-enhancer that formed a depot in treated tumors and enhanced local and systemic immune responses. [ 16 ] Building on these findings, we hypothesized that formulating VLPs with IDO1 crystals would further potentiate the anti-tumor immune response. Accordingly, we recombinantly expressed our CuMVtt-VLPs and confirmed their successful formation into 30 nm VLPs as shown in Fig. 2 i and j. Next, we formulated CuMVtt VLPs with IDO1 crystals by mixing them at room temperature, which resulted in the formation of nanoparticles decorating the IDO1 crystals. We visualised AF488-labelled VLPs bound to IDO1 inhibitor crystals using fluorescent microscopy (Fig. 2 k). We used the same B16F10 murine melanoma model described earlier, and designed three groups for IT treatment, initiated on day 3 post tumor fragment transplantation: the first group was treated with crystalised IDO1 inhibitor; the second group received CuMVtt VLPs alone; and the third group received a combination of CuMVtt VLPs formulated with the IDO1 inhibitor crystals (Fig. 2 l). The results showed a significant reduction in tumor weight in groups treated with IT CuMVtt VLPs as monotherapy or formulated with IDO1 inhibitor crystals, compared to the first group that received IDO1 inhibitor crystals (p = 0.0008 and p = 0.0001, respectively) (Fig. 2 m). No significant difference was detected between the 2nd and 3rd groups (P = 0.6647). These data suggest that the IDO1 inhibitor did not improve the overall acquired antitumor efficiency of our VLPs, indicating the limited efficacy of IDO1 inhibitor approach. Intratumor Delivery Elevates Metabolite Levels but Faces Limitations in Achieving Durable Tryptophan Retention Since local depot-release of the IDO1 inhibitor failed to elicit an anti-tumor response, we were prompted to conduct comprehensive metabolomic profiling of tumor samples. This analysis included 4T1 tumors treated SC or IV with the soluble IDO1 inhibitor, as well as IT with its crystallised form. The aim was to identify potential metabolic factors underlying the limited therapeutic efficacy observed. We conducted targeted analysis of kynurenine pathway metabolites, along with a semi-quantitative targeted search for additional related metabolites. Our focus was on metabolites associated with the Kyn and serotonin pathways including Trp, Kyn, 3-hydroxy-kynurenine, quinolinic acid, nicotinic acid, picolinic acid, Kynurenic acid, xanthurenic acid, 3-hydroxyanthranilic acid (3-HAA), NR, and nicotinamide adenine dinucleotide (NAD). Our data show that IT therapy with crystalized IDO1 inhibitor has the highest average level of metabolites, followed by IV, SC and then PBS control group (Fig. 3 a). The number of identified metabolites overall was consistent across all treatment groups (Fig. 3 b). We measured the coefficient of variance (CVs) across metabolites within each treatment group to evaluate the consistency of the metabolic response. IT-treated samples displayed a moderate spread of CVs, indicating some variability among metabolites, but overall, a more consistent pattern compared to other treatment groups. In contrast, the IV group exhibited a markedly higher degree of variability, with several metabolites showing elevated CVs, suggesting a heterogeneous and less predictable metabolic effect (Fig. 3 c). The cluster dendrogram, which was created using a hierarchical clustering analysis of the metabolites, revealed that the samples could be divided into distinct groups. IT-treated tumors with IDO1 Inhibitor crystals formed well-supported clusters (AU p-values > 95%), which indicates a consistent metabolic response (Fig. 3 d). In contrast, IV and SC samples were more dispersed across the dendrogram, which is consistent with the higher variability observed in CV analysis. These results reinforce the observation that IT administration of crystalised IDO1 inhibitor induces a more defined and reproducible metabolic shift. Heatmap analysis revealed that IT administration of the crystallized IDO1 inhibitor induced the most distinct and widespread metabolic changes, compared to SC and IV administration of the soluble formulation. Notably, metabolites involved in nucleotide biosynthesis and the pentose phosphate pathway (PPP) were upregulated, while serotonin, melatonin, and urea were downregulated, contrary to initial expectations (Fig. 3 e). An additional heatmap showing log₂-transformed intensities of significantly regulated metabolites is provided in Supplementary Fig. 2 and further supports the overall limited divergence between treatment routes. Principal component analysis (PCA) also indicates that IT-treated mice with IDO1 Inhibitor crystals exhibit a distinct metabolic profile, which clearly differentiates them from the PBS control group and the SC- and IV-IDO1 Inhibitor treated groups that were administered the solution formulation (Fig. 3 f). Such findings highlight the potential of IT treatment to alter the metabolic profile of 4T1 TNBC tumors, despite its inability to induce an antitumor response. Additionally, we performed cluster analysis of metabolite profiles in the four different groups which revealed two distinct clusters, indicating clear differences in metabolic responses among treatment groups. Samples from the IT treatment group primarily clustered together within Cluster 1 , forming a distinct group separated from the PBS control, IDO1 inhibitor SC, and IV treated samples, which were predominantly grouped in Cluster 2 . This segregation suggests that IT administration of IDO1 Inhibitor crystals induced a unique metabolic signature not observed with other administration routes; although some overlap was noted, particularly with IDO1 Inhibitor solution formulation administered SC or IV treatments. IDO1 Inhibitor crystals injected IT showed greater internal consistency and separation along the primary dimensions of variation (Dim1 and Dim2), which together captured over 60% of the total variance (Fig. 3 g). Intratumoral IDO1 Inhibition Induces NAD⁺ Salvage Pathway Activation We next evaluated the concentration and relative abundance of the metabolites associated with the kynurenine pathway following treatment in the tumor samples. The targeted metabolic analysis focused on key intermediates involved in essential cellular processes linked to the kynurenine pathway. Additionally, we specifically examined a range of downstream metabolites, including 3-hydroxyanthranilic acid (3-HA), quinolinic acid, nicotinic acid, picolinic acid, xanthurenic acid, melatonin, hydroxyindoleacetic acid (3-HIAA), NR, and NAD⁺(Fig. 4 a). The following metabolites could be detected in the kynurenine pathway: Trp, Kyn, serotonin, 3-HIAA, melatonin, and 3-HA in 4T1 tumor. NR is a vitamin B3 derivative that serves as a precursor for NAD⁺ through the salvage pathway. While not directly part of the kynurenine pathway, both NR and Kyn metabolites contribute to NAD⁺ biosynthesis via distinct routes. [ 17 ] Overall, no significant differences and minimal variation across the different treatment groups were observed for Trp, Kyn, serotonin, 3-HIAA, melatonin or 3-HA metabolites, which remained relatively stable in concentration (Fig. 4 b-g and Supplementary Figs. 3, 4 and 5). These findings suggest that neither systemic administration of the IDO1 inhibitor in soluble form nor local depot-release via IT injection significantly impacted Trp degradation in the 4T1 TNBC murine model. Interestingly, IT treated tumors with IDO1 crystals exhibited significant increased levels of NR (p = 0.0483) in comparison to the other groups, suggesting a possible tumor-intrinsic adaptation via the NAD⁺ salvage pathway (Fig. 4 h). Guanosine level was significantly elevated in tumor treated with IDO1 inhibitor crystals IT as compared to PBS-control (Supplementary Fig. 5v1). This reflect the NAD + depletion caused due to the inhibition of IDO1 activity. [ 18 ] Under NAD + stress, tumor cells can upregulate Purine salvage pathway to increase the GTP accumulation ensuring critical biosynthetic and signalling functions persist despite metabolic stress. [ 19 ] α-Ketoglutarate (α-KG) levels were lower in the IT treated group compared to the control group (Supplementary Fig. 4l1). This observation reflect a reduced oxidative burden in response to IDO1 inhibition. During Trp catabolism, the downstream metabolite 3-hydroxykynurenine is known to generate reactive oxygen species (ROS). [ 20 ] To reduce this oxidative stress, tumor cells upregulate glutamine metabolism, producing α-KG to fuel the TCA cycle and support redox homeostasis through glutathione synthesis. [ 21 ] The reduced α-KG levels observed in the anti-IDO treated group, along with lower glutamine abundance, suggests a decreased ROS demand due to diminished kynurenine pathway activity. Tumor-Driven L-Tryptophan Consumption Impairs Immune Activation Given the limited anti-tumor effect of IDO1 inhibition, likely due to tumor cells benefiting more from Trp than immune cells, we hypothesized that direct Trp administration in the tumor would likewise fail to suppress tumor growth. To test this, we assessed the impact of Trp delivered either as crystals into the TME or as a solution via SC or IV injection. We used the aggressive 4T1 TNBC cell line, inoculating it SC in the flank region of WT Balb/c mice. The following groups were defined: a control group injected with PBS SC; and Trp-treated groups injected either SC or IV with Trp solution, or IT with Trp crystals. Treatment was administered three times (Fig. 5 a), and tumor volume was monitored throughout. Tumor volume and weight measurements revealed no significant differences between the treated groups and the control, suggesting limited anti-tumor efficacy of Trp alone in this model (Fig. 5 b-d). We carried out an immunological assessment focusing on the tumors in the group treated with IDO1 inhibitor crystals via IT and compared the results to those of the control group treated with PBS (Fig. 5 f). Our results revealed overall a trend in increased density of immune cells, specifically CD4 + and CD8 + T cells, albeit not statistically different (p = 0.3233, 0.1590 respectively) (Fig. 5 g-i). No trends have been noticed in the enhanced levels of myeloid-derived cells characterised by CD11b or B cells (Fig. 5 j and k). Our data suggest that supplementing tumors with Trp may preferentially support tumor growth rather than enhance anti-tumor immune responses within the TME. This finding highlights the need to develop more effective strategies to counteract tumor-favoring metabolic pathways and improve immunotherapeutic outcomes. Discussion IDO1 inhibitors were designed to counteract tumor immune evasion by blocking the degradation of Trp into immunosuppressive Kyn, thereby alleviating metabolic constraints on T cell function within the TME. In this study, we assessed the anti-tumor efficacy of a novel crystallized IDO1 inhibitor formulated for IT administration. We hypothesized that sustained local release would achieve prolonged enzyme inhibition, preserving Trp availability and enhancing anti-tumor immunity. Despite achieving pharmacologically relevant doses, as confirmed by human equivalent dose (HED) calculations, no measurable tumor regression was observed following IT, SC, or IV delivery, highlighting the limited therapeutic effect of IDO1 inhibition in two cold murine tumor models. Crystallizing the IDO1 inhibitor for slow local release represents an innovative and consistent approach for localized drug delivery, as supported by moderate metabolite variability and distinct clustering patterns in metabolic profiles. The IT dose of 500 µg corresponds to an HED of approximately 2.0 mg/kg (~142 mg for a 70 kg adult), aligning with clinical doses of oral IDO1 inhibitors. [22,23] Over 95% of free Trp is a substrate for the kynurenine pathway of Trp degradation. [2] Our metabolic profiling revealed that Trp degradation was limited under all conditions, as evidenced by a low Kyn/Trp ratio (~0.033), suggesting either inherently low IDO1 activity in 4T1 tumor model or successful enzyme inhibition. This is consistent with the notion that IDO1 is not uniformly active across all tumor types and may be upregulated only under specific inflammatory stimuli. Our findings echo broader challenges faced in the clinical translation of IDO1 inhibitors. The high-profile failure of the ECHO-301 Phase 3 trial, which combined epacadostat with pembrolizumab in melanoma, suggests that IDO1 monotherapy may be insufficient in the absence of a primed immune microenvironment. It is also possible that compensatory pathways such as TDO, IL4I1, or microbiota-driven Trp metabolism bypass the IDO1 blockade and sustain immunosuppressive signaling via activation of the aryl hydrocarbon receptor (AhR). While we observed a metabolic shift including elevated NR and guanosine suggestive of enhanced NAD+ salvage and nucleotide regeneration, these responses reflect tumor adaptation rather than therapeutic benefit. Moreover, local Trp metabolism is increasingly recognized as a critical modulator of tumor-immune interactions. For instance, Trp-derived microbial metabolites like indole-3-aldehyde (I3A) can activate AhR and suppress tumor growth via modulation of innate lymphoid cells. [24] Whether such pathways are engaged following IDO1 inhibition remains an open question. Interestingly, the minimal Trp-to-Kyn conversion observed here suggests that additional blockade of TDO may not be necessary in this model, although this may vary depending on tumor type and immune context. [25] Importantly, our data highlight both the potential and the limitations of our novel localized IDO1 inhibition. While the metabolic response to IT delivery showed some potentials in altering the metabolic profile in the tumor, it did not translate into measurable tumor control. Importantly, direct feeding of Trp crystals to the tumors resulted in enhanced growth. This further highlights the need for innovative therapeutic modalities to better control tumor growth. Methods Production of IDO-1 inhibitor crystals IDO5L nanoparticles/crystals were prepared by dissolving IDO5L (CAS registry no.: 914471-09-3) at its maximum solubility of 30 mg/mL in 70% ethanol (ETOH). Specifically, 1 g of IDO5L was dissolved in approximately 33 µL of ETOH, and the mixture was thoroughly vortexed until complete dissolution. Subsequently, ultrapure water (ddH₂O) was carefully added dropwise to the solution while gently mixing, until precipitation occurred and the solution became visibly milky, indicating crystalline nanoparticle formation. Typically, for 1 g of IDO5L, this precipitation was achieved with approximately 4–5 drops (40–50 µL) of ddH₂O. Murine cell lines The 4T1 mammary carcinoma cell line (ATCC CRL-2539) was obtained from the American Type Culture Collection. The B16F10 melanoma cell line was generously provided by the Ochsenbein laboratory. Both cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Once cultures reached approximately 80% confluence, cells were washed three times with PBS to remove residual medium, then incubated with 1× trypsin for 10 minutes at 37°C to facilitate detachment. The detached cells were subsequently collected, resuspended in complete medium, and kept on ice until further use. Mice Female wild-type (WT) Balb/cOlaHsd and female C57BL/6 mice (7-12 weeks old, were purchased from Harlan). RAG1−/− mice, backcrossed onto the C57BL/6 background, were kindly provided by the Ochsenbein laboratory and maintained in our pathogen-free animal facility at the University of Bern's animal facility. All in vivo experiments were conducted using female mice aged (7-12 weeks). All animal procedures complied with the Swiss Animal Act (455.109.1, September 2008, Article 5), approved by the University of Bern and performed under license no. BE30/2024, and the study was conducted in accordance with the relevant guidelines and regulations. This study is reported in accordance with the ARRIVE guidelines (https://arriveguidelines.org). In vivo tumor experiment and dosing Female Balb/c mice (7–12 weeks old, Harlan) were SC inoculated with 1×10⁶ 4T1 cells per mouse on day 0. [26] Tumor growth was monitored regularly. On day 5, when the tumors became palpable, the mice were divided into experimental groups. Treatments were conducted three times on days 5, 7 and 11. The study endpoint was set for day 17. One million B16F10 melanoma cells were SC injected into the flanks of 7–12-week-old C57BL/6 RAG1⁻/⁻ mice. After 12 days, the resulting tumors were harvested and transplanted into the flanks of WT C57BL/6 mice (7–12 weeks old; Harlan). Tumor growth was monitored regularly. When the tumors became palpable on day 5, the mice were divided into experimental groups. Treatments were conducted three times on days 3, 7 and 10. The endpoint of the study was set on day 14. Mice from all groups were euthanized once their tumors reached the humane endpoint in accordance with the Swiss Animal Act. Euthanasia of animals was carried out using CO 2 and decapitation. Tumor volume in mm 3 was monitored using callipers and calculated using the formula V = (W × W × L)/2, where V is volume, L is tumor length and W is tumor width. Tumor volumes at endpoint ranged between 150 mm³ and 1000 mm³. The IDO1 inhibitor was administered at a dose of 500 µg per injection, while the VLPs were given at a dose of 100 µg per injection. Tumor analysis and staining Single-cell suspensions of tumors (TILs) were generated as follows. Tumors were digested using Collagenase D (1mg/mL in DMEM-Medium, Roche) at 37°C for 45 minutes. The digested tumors were smashed through a 70 μm cell strainer (Sigma Aldrich). During the mechanical processing, Rosewell Park Memorial Institute (RPMI) medium was continuously added to a final volume of 5mL. The collected cells were centrifuged at 300 g for 5 minutes at 4°C. The cells were resuspended in a fresh medium. The cells were washed once with FACS-Buffer (PBS + 2% FBS). In Table 1, the used antibodies are listed. Cells were then stained with FC-block (1:100) for 15 minutes in the dark. The cells were centrifuged at 300 g 4°C for 5 minutes. The surface staining antibody mix was added and incubated for 30 minutes in the dark. After the surface staining, the cells were washed 3 times with FACS-Buffer. The 96-Well U bottom plate was acquired using the Aurora 5L of the FACS facility of the DBMR of the University of Bern. The FCS files were analyzed using FlowJo (V10.10), and the graphs were created using GraphPad Prism (V10.4.1). Target Fluorochrome Clone Dilution CD45 BUV737 30-F11 1:300 CD8 BUV563 53-6,7 1:300 Viability Zombie Aqua - 1:1000 CD11b PerCP-Cy5.5 M1/70 1:300 CD4 APC-Cy7 RPA-T4 1:300 VLPs expression, production and purification CuMVtt VLPs were expressed and produced as described previously in prvious papers [ 16, 27 ]. Endotoxin levels were measured and confirmed to be below 1000 EU/mL. CuMVtt VLPs naturally package single-stranded RNA (ssRNA) during expression in E. coli , which serves as a TLR7/8 ligand to effectively activate innate immune cells. [28] Dynamic light scattering (DLS) Analysis was performed on a CuMVtt VLP solution at a concentration of 1 mg/mL using a Zetasizer Nano ZS instrument (Malvern Instruments, UK). Data from three independent measurements were analyzed using DTS software (Malvern, version 6.32). Electron Microscopy The physical stability and structural integrity of CuMVtt VLPs were assessed by transmission electron microscopy (TEM) using a Philips CM12 instrument. For sample preparation, glow-discharged grids were incubated with 5 µL of VLP solution for 30 seconds. The grids were then washed three times with double-distilled water and negatively stained with 5 µL of 5% uranyl acetate for 30 seconds. Excess stain was removed by pipetting, and the grids were air-dried for 10 minutes. Images were acquired at magnifications of ×84,000 and ×110,000. CuMVtt VLPs and IDO1 inhibitor crystals formulation Nanoparticles (CuMVtt VLPs) were formulated with IDO1 inhibitor crystals by mixing the two components on a shaker at 500 rpm at room temperature for 1 hour. This process facilitates the adsorption of CuMVtt onto the crystals. For visualization, CuMVtt VLPs (3 mg/mL) were labeled with Alexa Fluor 488 (AF488) following the manufacturer’s instructions and then mixed with IDO1 inhibitor crystals. A 10 µL aliquot of the mixture was placed on a glass slide, covered with a cover slip, and imaged using a Zeiss AxioImager A2 fluorescence microscope equipped with Plan-NEOFLUAR ×20 and ×40 objectives (Zeiss). Metabolomics Frozen tumor samples (-80°C) were transferred into PowerBead Tubes Ceramic 1.4 mm (Qiagen), and metabolites were extracted by adding nine volumes of methanol (MeOH). Homogenization was performed using the PowerLyzer 24 Homogenizer (Qiagen) in three cycles of 10 seconds at 2500 rpm. The tubes were then centrifuged at 10,000 × g for 10 minutes. A 10 µL aliquot of the resulting supernatant was transferred to a new tube, followed by the addition of 10 µL of an isotopically labeled internal standard mixture and 80 µL of methanol. After vortexing, the samples were centrifuged again at 1000 × g for 10 minutes, and 90 µL of the clarified extract was transferred into HPLC glass vials for LC-MS analysis. Chromatographic separation of metabolites was carried out on an ACQUITY Premier BEH Z-HILIC column (1.7 µm, 2.1 × 100 mm; Waters) using a gradient elution. Mobile phase A consisted of 0.15% formic acid and 10 mM ammonium formate in water, while mobile phase B consisted of 0.15% formic acid and 10 mM ammonium formate in 85% acetonitrile. The total analysis time was 18 minutes, with a flow rate of 0.4 mL/min, an injection volume of 2 µL, and a column temperature of 40 °C. Mass spectrometric analysis was performed on an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific), operated in both positive and negative electrospray ionization (ESI) modes. Full scan acquisition was conducted over an m/z range of 50–600 with a resolving power of 60,000. The ESI spray voltage was set to 3.5 kV for positive mode and 2.5 kV for negative mode. Additional MS settings included a capillary temperature of 350 °C, a gas heater temperature of 400 °C, an auxiliary gas flow rate of 12 arbitrary units, and a sheath gas flow rate of 50 arbitrary units. Quantification was based on seven-point calibration curves using internal standardization. LC-MS data acquisition, processing, and quantification were performed using TraceFinder 5.1 General Quant software (Thermo Fisher Scientific). Metabolite identification was assigned confidence level A based on accurate mass, retention time, and comparison with authentic reference standards previously validated on the same analytical platform. For relative quantification, acquired data files were evaluated for the following metabolites: 3-hydroxyanthranilic acid, quinolinic acid, nicotinic acid, picolinic acid, xanthurenic acid, NR, NAD⁺, hydroxyindoleacetic acid, and melatonin. Accurate m/z values were calculated for each compound, and metabolite detection was performed using a mass accuracy threshold of 10 ppm. Production of Tryptophan crystals L-Tryptophan (Sigma-Aldrich, T0254) was suspended in sterile PBS obtaining a concentration of 5 mg/ml for the solution fomulation or prepared as crystals as described in IDO1 inhibitor method. Statistics Data are presented as mean±SEM. Comparisons between more than two groups were performed by one-way analysis of variance; comparisons between two groups were performed by Welch’s t-test. P values were ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05. Declarations Acknowledgment: Biogenity has contributed by performing the data analysis, including figures, tables, and reports. Metabonet has contributed by performing the metabolomics sample analysis. Author contribution: Design of experiments, acquisition of data, interpretation, and analysis of data: ASC, AG, MFB and MOM. Writing, revision and editing of manuscript: ASC, AG, MFB and MOM. Technical, material and tool support: MFB and MOM. Study supervision: MOM and MFB. All authors read and approved the final manuscript. Data availability statment: All data supporting the findings of this study are contained within the article and its supplementary content. The raw data can be obtained from the corresponding author upon reasonable request. Funding : This research was funded by Swiss National Foundation (SNF): (Grant Number: 320030-228094), Swiss Cancer Research (Grant Number KFS-5246-02-2021-R) and Jubiläumsstiftung von Swiss Life. Illustrations were generated with BioRender.com. Competing interests: MOM and MFB have financial interests as shareholders in DeepVax GmbH, a company that specializes in the development of vaccines. DeepVax GmbH is a spinoff from the University of Bern. References Schwarcz R, Stone TW. The kynurenine pathway and the brain: Challenges, controversies and promises. Neuropharm 2017; 112 (Pt B):237-247. Platten M, Nollen EAA, Röhrig UF, Fallarino F, Opitz CA. Tryptophan metabolism as a common therapeutic target in cancer, neurodegeneration and beyond. Nat Rev Drug Discov 2019; 18 (5):379-401. Munn DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B, Brown C, Mellor AL. Prevention of allogeneic fetal rejection by tryptophan catabolism. Science 1998; 281 (5380):1191-3. Théate I, van Baren N, Pilotte L, Moulin P, Larrieu P, Renauld JC, Hervé C, Gutierrez-Roelens I, Marbaix E, Sempoux C, Van den Eynde BJ. Extensive profiling of the expression of the indoleamine 2,3-dioxygenase 1 protein in normal and tumoral human tissues. Cancer Immunol Res 2015; 3 (2):161-72 Platten M, von Knebel Doeberitz N, Oezen I, Wick W, Ochs K. Cancer Immunotherapy by Targeting IDO1/TDO and Their Downstream Effectors. Front Immunol 2015; 12 ;5:673. Nakamura T, Shima T, Saeki A, Hidaka T, Nakashima A, Takikawa O, Saito S. 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Liu M, Wang X, Wang L, Ma X, Gong Z, Zhang S, Li Y. Targeting the IDO1 pathway in cancer: from bench to bedside. J Hematol Oncol 2018; 11 (1):100. Holmgaard RB, Zamarin D, Munn DH, Wolchok JD, Allison JP. Indoleamine 2,3-dioxygenase is a critical resistance mechanism in antitumor T cell immunotherapy targeting CTLA-4. J Exp Med 2013; 210 (7):1389-402. Mondal A, Smith C, DuHadaway JB, Sutanto-Ward E, Prendergast GC, Bravo-Nuevo A, Muller AJ. IDO1 is an Integral Mediator of Inflammatory Neovascularization . J EBioM 2016; 14 :74-82. Blair AB, Kleponis J, Thomas DL 2nd, Muth ST, Murphy AG, Kim V, Zheng L. IDO1 inhibition potentiates vaccine-induced immunity against pancreatic adenocarcinoma. J Clin Invest 2019; 129 (4):1742-1755. Mitchell TC, Hamid O, Smith DC, Bauer TM, Wasser JS, Olszanski AJ, Luke JJ, Balmanoukian AS, Schmidt EV, Zhao Y, Gong X, Maleski J, Leopold L, Gajewski TF. Epacadostat Plus Pembrolizumab in Patients With Advanced Solid Tumors: Phase I Results From a Multicenter, Open-Label Phase I/II Trial (ECHO-202/KEYNOTE-037). J Clin Oncol 2018; 36 (32):3223-3230. Long GV, Dummer R, Hamid O, Gajewski TF, Caglevic C, Dalle S, Arance A, Carlino MS, Grob JJ, Kim TM, Demidov L, Robert C, Larkin J, Anderson JR, Maleski J, Jones M, Diede SJ, Mitchell TC. Epacadostat plus pembrolizumab versus placebo plus pembrolizumab in patients with unresectable or metastatic melanoma (ECHO-301/KEYNOTE-252): a phase 3, randomised, double-blind study. Lancet Oncol 2019; 20 (8):1083-1097. Mohsen MO, Heath M, Kramer MF, Velazquez TC, Bullimore A, Skinner MA, Speiser DE, Bachmann MF. In situ delivery of nanoparticles formulated with micron-sized crystals protects from murine melanoma. J Immunother Cancer 2022 Sep; 10 (9):e004643. Covarrubias AJ, Perrone R, Grozio A, Verdin E. NAD + metabolism and its roles in cellular processes during ageing. Nat Rev Mol Cell Biol 2021 Feb; 22 (2):119-141. Zhang J, Tao J, Ling Y, Li F, Zhu X, Xu L, Wang M, Zhang S, McCall CE, Liu TF. Switch of NAD Salvage to de novo Biosynthesis Sustains SIRT1-RelB-Dependent Inflammatory Tolerance. Front Immunol 2019; 10 :2358. Wang H, He X, Li Z, Jin H, Wang X, Li L. Guanosine primes acute myeloid leukemia for differentiation via guanine nucleotide salvage synthesis. Am J Cancer Res 2022; 12 (1):427-444. Wang Q, Zhang M, Ding Y, Wang Q, Zhang W, Song P, Zou MH. Activation of NAD(P)H oxidase by tryptophan-derived 3-hydroxykynurenine accelerates endothelial apoptosis and dysfunction in vivo. Circ Res 2014; 114 (3):480-92. Serrano JJ, Medina MÁ. Metabolic Reprogramming at the Edge of Redox: Connections Between Metabolic Reprogramming and Cancer Redox State . Int J Mol Sci 2025; 26 (2):498. Beatty GL, O'Dwyer PJ, Clark J, Shi JG, Bowman KJ, Scherle PA, Newton RC, Schaub R, Maleski J, Leopold L, Gajewski TF. First-in-Human Phase I Study of the Oral Inhibitor of Indoleamine 2,3-Dioxygenase-1 Epacadostat (INCB024360) in Patients with Advanced Solid Malignancies. Clin Cancer Res 2017; 23 (13):3269-3276. Siu LL, Gelmon K, Chu Q, Pachynski R, Alese O, Basciano P, Desai J. BMS-986205, an optimized indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor, is well tolerated with potent pharmacodynamic (PD) activity, alone and in combination with nivolumab (nivo) in advanced cancers in a phase 1/2a trial. Cancer Res 2017; 77 (13 Suppl):CT116. Cui L, Wang Z, Guo Z, Zhang H, Liu Y, Zhang H, Jin H, Xu F, Wang X, Xie C, Guo H, Wang T, Lin Y, Zhao Q, Zhou P, Tan J, Bei JX, Huang P, Liu J, Xia X. Tryptophan Metabolite Indole-3-Aldehyde Induces AhR and c-MYC Degradation to Promote Tumor Immunogenicity. Adv Sci (Weinh) 2025; e09533. Heimberger AB, Lukas RV. The kynurenine pathway implicated in patient delirium: possible indications for indoleamine 2,3 dioxygenase inhibitors . J Clin Invest 2023; 133 (2):e164577 Mohsen MO, Balke I, Zinkhan S, Zeltina V, Liu X, Chang X, Krenger PS, Plattner K, Gharailoo Z, Vogt AS, Augusto G, Zwicker M, Roongta S, Rothen DA, Josi R, Costa JJD, Sobczak JM, Nonic A, Brand LA, Nuss K, Martina B, Speiser DE, Kündig T, Jennings GT, Walton SM, Vogel M, Zeltins A, Bachmann MF. A scalable and highly immunogenic virus-like particle-based vaccine against SARS-CoV-2. Allergy 2022; 77 (1):243-257. Josi R, Ogrina A, Rothen D, Balke I, Casaramona AS, de Brot S, Mohsen MO. Intranodal Injection of Immune Activator Demonstrates Antitumor Efficacy in an Adjuvant Approach. Vaccines (Basel) 2024; 12 (4):355. Chang X, Krenger P, Krueger CC, Zha L, Han J, Yermanos A, Roongta S, Mohsen MO, Oxenius A, Vogel M, Bachmann MF. TLR7 Signaling Shapes and Maintains Antibody Diversity Upon Virus-Like Particle Immunization. Front Immunol 2022 Jan 19; 12 :827256. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFig.1.jpg SupplementaryFig.2.jpg SupplementaryFig.3.jpg SupplementaryFig.4.jpg SupplementaryFig.5.jpg SupplementaryFigures.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7138169","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":499588423,"identity":"f5238347-9dab-4fd3-9b92-8eeaaec86c20","order_by":0,"name":"Arnau Solé Casaramona","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYDADPgbmAwwMBkDMwEOkFjYGtgSStfAYACkitPDPPnvwMw9DnRwbe8836YKCO3IM7GcP4NUicS4vWZqH4bAxG8/ZbdIzDJ4ZM/DkJeC35gyPAVDLgcQ2idxt0jwGhxMbJMAuxA3kz/AY/wY6LLFN/s0zkJZ6gloMzvCYAW1hBtrCwwbSksBASIshUIvlHAOQX9KMrXkMnhm28eTg1yIHdNiNNxV1cvzshx/e5vlzR56f/Qx+LVDnIbHZiFA/CkbBKBgFo4AAAADQcjeLhYc2/QAAAABJRU5ErkJggg==","orcid":"","institution":"University Hospital of Bern","correspondingAuthor":true,"prefix":"","firstName":"Arnau","middleName":"Solé","lastName":"Casaramona","suffix":""},{"id":499588424,"identity":"e1d0caa6-316d-4b61-af99-e68f346a6e0b","order_by":1,"name":"Anish Ghimire","email":"","orcid":"","institution":"University Hospital of Bern","correspondingAuthor":false,"prefix":"","firstName":"Anish","middleName":"","lastName":"Ghimire","suffix":""},{"id":499588425,"identity":"76831d5a-8d6f-4cb8-896b-d2ef8f1a5ab5","order_by":2,"name":"Martin F. Bachmann","email":"","orcid":"","institution":"University Hospital of Bern","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"F.","lastName":"Bachmann","suffix":""},{"id":499588426,"identity":"1f0b5c32-0017-4a64-b381-11ed2a4a79de","order_by":3,"name":"Mona O. Mohsen","email":"","orcid":"","institution":"University Hospital of Bern","correspondingAuthor":false,"prefix":"","firstName":"Mona","middleName":"O.","lastName":"Mohsen","suffix":""}],"badges":[],"createdAt":"2025-07-16 09:23:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7138169/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7138169/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89044841,"identity":"a9d5eee1-d48e-4af7-82f9-7da029a04382","added_by":"auto","created_at":"2025-08-14 06:29:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":263056,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea) \u003c/strong\u003eSchematic illustration of the preparation of IDO1 inhibitor crystalline nanoparticles. \u003cstrong\u003eb) and c)\u003c/strong\u003e Light microscopy images (20× and 40× magnifications) illustrating the successfully crystallized IDO1 inhibitor. The crystals measure approximately 5–10 µm.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138169/v1/e99ecf7a39af3a815e651726.jpg"},{"id":89044838,"identity":"37d85e79-fa2c-4757-9f71-191de4a0622d","added_by":"auto","created_at":"2025-08-14 06:29:55","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":363942,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea) \u003c/strong\u003eA schematic illustration of the experimental strategy and treatment timeline. Mice were inoculated SC with 1×10⁶ 4T1 cells on day 0. Treatments were administered on days 5, 7, and 11 once tumors became palpable. Treatment groups included: PBS SC (CTRL) (Group 1), soluble IDO1 inhibitor administered SC (Group 2), soluble IDO1 inhibitor administered IV (Group 3), and crystallized IDO1 inhibitor administered IT (Group 4). Tumors were collected for analysis on day 17.\u003cstrong\u003e b)\u003c/strong\u003e\u0026nbsp;Tumor growth kinetics following administration of IDO1 inhibitor in the different groups or control PBS. Tumor volumes were measured expressed as mean ± SEM (mm³).\u003cstrong\u003e c)\u003c/strong\u003e Tumor volume mm\u003csup\u003e3\u003c/sup\u003e\u0026nbsp;of individual mice per group. \u003cstrong\u003ed)\u003c/strong\u003e Tumor weight (mg) on day 17. \u003cstrong\u003ee)\u003c/strong\u003e Schematic illustration of the experimental strategy and timeline. 1×10\u003csup\u003e6\u003c/sup\u003e\u0026nbsp;B16F10 melanoma cell line into the flank of C57BL/6\u0026nbsp;\u003cem\u003eRAG1\u003c/em\u003e\u003csup\u003e−/−\u003c/sup\u003e\u0026nbsp;mice. Twelve to thirteen days later the growing tumors are collected and processed for transplantation of ~8 mm\u003csup\u003e3\u003c/sup\u003e\u0026nbsp;into the flank of C57BL/6 WT mice. Treatments were administered on days 3, 7 and 10 once tumors became palpable. Treatment groups included: PBS IT CTRL (Group 1) and crystallized IDO1 inhibitor administered IT Group 2). Tumors were collected for analysis on day 14. \u003cstrong\u003ef)\u003c/strong\u003e Tumor growth kinetics following administration of IDO1 inhibitor IT or control PBS IT. Tumor volumes were measured expressed as mean ± SEM (mm³). \u003cstrong\u003eg)\u003c/strong\u003e Tumor volume mm\u003csup\u003e3\u003c/sup\u003e\u0026nbsp;of individual mice. \u003cstrong\u003eh)\u003c/strong\u003e Tumor weight (mg). \u003cstrong\u003ei)\u003c/strong\u003e The integrity of CuMVtt-VLPs was confirmed with electron microscopy, images showed purified CuMVtt-VLPs, ~30 mm in size. \u003cstrong\u003ej)\u003c/strong\u003e DLS analysis of purified CuMVtt-VLPs. \u003cstrong\u003ek) \u003c/strong\u003eCuMVtt-VLPs labelled with AF488 and formulated with MCT micron-sized adjuvant, x20 objective scale bar at 50 mm. \u003cstrong\u003el)\u003c/strong\u003e A schematic illustration of the experimental strategy and timeline as explained in e. Treatments were administered on days 3,7 and 10 once tumors became palpable. Treatment groups included: crystallized IDO1 inhibitor administered IT (Group 1), CuMVtt-VLPs administered IT (Group 2) and a combination group where CuMVtt-VLPs was formulated with crystalised IDO1 inhibitor (Group 3). Tumors were collected for analysis on day 14. \u003cstrong\u003em)\u003c/strong\u003e Tumor weight (mg) of the different groups. Statistical analysis with one-way ANOVA or Welch’s t test. The sample size for each group was\u0026nbsp;\u003cem\u003en\u003c/em\u003e = 5 or 6.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138169/v1/ddd3514efad797c610f41efc.jpg"},{"id":89045022,"identity":"2d55beab-f772-4001-8487-74e4b615bc16","added_by":"auto","created_at":"2025-08-14 06:37:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":467415,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea)\u003c/strong\u003e Total abundance of identified metabolites (log-transformed sum of intensity) across treatment groups in 4T1 tumor model. \u003cstrong\u003eb)\u003c/strong\u003e Number of identified metabolites per group across all treatment conditions. \u003cstrong\u003ec)\u003c/strong\u003e Violin plot of coefficient of variation (CV) for each group \u003cstrong\u003e(d)\u003c/strong\u003e Hierarchical clustering dendrogram of samples based on global metabolite profiles, showing grouping of samples according to treatment condition. \u003cstrong\u003e(e)\u003c/strong\u003e Heatmap of significantly altered metabolites across pairwise group comparisons, with hierarchical clustering of both metabolites and comparisons. \u003cstrong\u003e(f)\u003c/strong\u003e Principal component analysis (PCA) showing partial separation of groups, with PC1 explaining 53% of total variance. \u003cstrong\u003e(g)\u003c/strong\u003e K-means clustering (k=2) based on metabolite intensity data, showing partial grouping by treatment and visualization of cluster overlap using a 2D PCA plot.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138169/v1/690d8c93b8db4704bc4185c8.jpg"},{"id":89045874,"identity":"3e0db76b-2c11-4174-814c-a5b890c4c537","added_by":"auto","created_at":"2025-08-14 06:45:55","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":487984,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea)\u003c/strong\u003e A schematic of the Trp metabolic pathway highlighting serotonin, melatonin and Kyn–NAD⁺ branches. NR contributes to NAD⁺ synthesis via the salvage pathway. \u003cstrong\u003eb)\u003c/strong\u003e LC-MS-based quantification of Trp pathway metabolites in breast tumor tissues. Metabolites were extracted from frozen samples and analyzed using HILIC chromatography coupled to Orbitrap MS. Data were analyzed using one-way ANOVA.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138169/v1/6a9ee8d0095441255c8950a7.jpg"},{"id":89044850,"identity":"b9028160-8ef6-4026-8b40-c909469253bd","added_by":"auto","created_at":"2025-08-14 06:29:55","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":817901,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea) \u003c/strong\u003eA schematic illustration of the experimental strategy and treatment timeline. Mice were inoculated SC with 1×10⁶ 4T1 cells on day 0. Treatments were administered on days 5, 7, and 11 once tumors became palpable. Treatment groups included: PBS SC CTRL (Group 1), soluble Trp administered SC (Group 2), soluble Trp administered IV (Group 3), and crystallized Trp administered IT (Group 4). Tumors were collected for analysis on day 17.\u003cstrong\u003e b) \u003c/strong\u003eTumor growth kinetics following administration of Trp in the different groups or control PBS. Tumor volumes were measured expressed as mean ± SEM (mm³). \u003cstrong\u003ec)\u003c/strong\u003e Tumor volume mm\u003csup\u003e3\u003c/sup\u003e\u0026nbsp;of individual mice per group. \u003cstrong\u003ed)\u003c/strong\u003e Tumor weight (mg). \u003cstrong\u003ee)\u003c/strong\u003e The gating strategy for tumor samples involved a sequential selection of lymphocytes, singlets, live/dead cells, CD45+ cells, CD8+ and CD4+ T cells or CD11b+ and B220+ cells. \u003cstrong\u003ef) \u003c/strong\u003eTumor volume mm\u003csup\u003e3\u003c/sup\u003e\u0026nbsp;of individual mice in CTRL and IT treated groups. \u003cstrong\u003eg-k) \u003c/strong\u003eDensities of CD45+, CD4+ T cells, CD8+ T cells, CD11b+ cells and B220+ B cells in tumors on day 17. Cell density was measured by dividing the total number of cells in each tumor by its weight. Statistical analysis with one-way ANOVA or Welch’s t test. The sample size for each group was\u0026nbsp;\u003cem\u003en\u003c/em\u003e = 5 or 6.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138169/v1/895aecac99991a7387b85df0.jpg"},{"id":97673998,"identity":"461b7bf6-337c-45aa-a518-b91c47efcc53","added_by":"auto","created_at":"2025-12-08 09:42:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3269572,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7138169/v1/9aa2415d-a88a-48eb-b255-faf31ad3600d.pdf"},{"id":89044837,"identity":"2e99bf37-8723-43d0-8ffc-11f5ecbb9a59","added_by":"auto","created_at":"2025-08-14 06:29:55","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":178839,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138169/v1/f9aa57ccbc27e90c2472416e.jpg"},{"id":89045024,"identity":"bb0c2025-8a9c-4450-8ca5-99e20691de2b","added_by":"auto","created_at":"2025-08-14 06:37:55","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":309697,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138169/v1/e7ae175fda9a7cca9a5b9a71.jpg"},{"id":89044847,"identity":"0fb0a2c8-850c-47e4-8b24-e2a70bcb78f9","added_by":"auto","created_at":"2025-08-14 06:29:55","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":334378,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138169/v1/d2d1672a16511a949f08d4ee.jpg"},{"id":89047074,"identity":"a9faeaf4-af6e-41be-81fc-b255932d6d80","added_by":"auto","created_at":"2025-08-14 06:54:03","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":327837,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138169/v1/4b584b46b2d423f07de44084.jpg"},{"id":89044852,"identity":"ebdd83c1-28af-4e01-a9d4-76056b7becee","added_by":"auto","created_at":"2025-08-14 06:29:55","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":337498,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138169/v1/f3f6ea09e1a94e4dc36da899.jpg"},{"id":89047073,"identity":"73aede13-659d-48c5-aa05-579c60f0b7d6","added_by":"auto","created_at":"2025-08-14 06:54:03","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":14457,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-7138169/v1/07ebe7ea410389049d1a2869.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Intratumoral Delivery of Crystallised IDO1 Inhibitors Fails to Overcome Tumor Immunosuppression Due to Metabolic Adaptation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe essential amino acid tryptophan (Trp) plays important roles in a variety of physiological processes in the body. A small proportion of free Trp is used for protein synthesis as well as for the production of neurotransmitters and neuromodulators.\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e About 95% of the free Trp is degraded in the Kynurenine Pathway, generating different metabolites involved in distinct biological activities.\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e Indoleamine-2,3-dioxygenase 1 (IDO1) is a rate-limiting enzyme in this pathway that degrades Trp to N-formylkynurenine.\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e It has also been shown that IDO1 is constitutively expressed in immune privileged sites such as the placenta and epidermis to block T cell activation by restricting access to Trp.\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e IDO1 is over expressed in about 60% of tumors such as melanoma, colon cancer and cervical cancer.\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e IDO1 expression has been associated with poor prognosis and outcome.\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e Trp degradation regulates T regulatory cells and other immune cells in tumors. In cervical cancer draining lymph nodes, FOXP3\u003csup\u003e+\u003c/sup\u003e Treg cells have been found in direct contact with dendritic cells expressing IDO1.\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e Previous studies have shown that the concentration of kynurenine (Kyn) metabolites in the blood of patients with melanoma, colorectal cancer or cervical cancer was less frequently elevated than within tumors, pointing out that changes in Kyn and its downstream metabolites may be more restricted and localized in the TME. Accumulating body of evidence suggests that Trp metabolism and degradation can support tumor progression by suppressing T cell responses.\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eSeveral preclinical studies are based on the critical role of IDO1 in inducing immune tolerance and promoting tumor growth, targeting IDO1 has been an attractive approach in cancer immunotherapy. Different strategies have been developed to target IDO1 using IDO1 expression inhibitors, IDO1 enzymatic inhibitors, IDO1 effector modulators and IDO1 peptide vaccines.\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e Allison et al have shown anti-tumor efficacy of IDO1 inhibitor when combined with the cytotoxic T lymphocyte antigen-4 (CTLA4) immune-checkpoint inhibitor in B16F10 murine melanoma model, (IDO1 inhibitor was given subcutaneously (SC) or orally.\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e In 4T1 mammary carcinoma murine model, IDO1 ablation (IDO1 ⁻/⁻) was sufficient to reduce the pathological neovascularization in lung metastases.\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eDiverse clinical trials are currently ongoing testing the combination of IDO1 inhibitors with radiation, chemotherapy or immunotherapy.\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e Opposing the preclinical data, clinical studies have shown limited and disappointing efficacy of IDO1 inhibitor as a systemic monotherapy; however combination of IDO1 inhibitors with immunotherapies such as immune-check point inhibitors or vaccines showed more promising results.\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e For example, a phase I study combining PD1 with IDO1 inhibitor showed an objective response in 12 out of 22 melanoma patients.\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e On the contrary, phase III clinical trial of this combination did not improve the progression-free survival compared to anti-PD1 immunotherapy alone.\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eOverall, IDO1 inhibition has yielded mixed and sometimes contradictory outcomes in both preclinical and clinical studies, which could be attributed in part to the route of administration. In this context, we hypothesized that inhibiting IDO1 locally at the tumor site may offer a more rational and targeted approach. Hence, we developed a novel crystalline formulation of an IDO1 inhibitor designed for \u003cem\u003ein situ\u003c/em\u003e IT administration, aiming to achieve localized inhibition of IDO1 activity. To evaluate its therapeutic potential, we compared this approach to systemic delivery of the same IDO1 inhibitor in soluble form administered SC or intravenously (IV). Our results revealed that although the overall abundance of metabolites increased in tumors, the IT administration of a crystallised IDO1 inhibitor did not induce an anti-tumor response, neither did the soluble formulations that were administered either SC or IV. Immune profiling further indicated that IDO1 blockade alone was insufficient to overcome immunosuppression within the TME. Interestingly, IT treatment with IDO1 inhibitor crystals resulted in a significant increase in nicotinamide riboside (NR) levels, suggesting tumor-intrinsic metabolic adaptation via the NAD⁺ salvage pathway. Similarly, direct IT injection of crystallised Trp failed to impede tumor progression. These findings emphasise the adaptability of tumor metabolism and call into question the therapeutic value of IDO1 inhibition, even when delivered locally via a sustained-release depot.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eSuccessful Crystallization of IDO1 Inhibitor Enables Direct Intratumoral Delivery\u003c/b\u003e\u003c/p\u003e\u003cp\u003eClinical trials assessing the effectiveness of IDO1 inhibitors have seen limited success, possibly influenced by the administration route (typically SC or oral). Delivering the drug directly into the TME as crystals for depot-slow release is a novel and promising strategy that has not been explored before. Accordingly, we proceeded to crystallise the IDO1 inhibitor (IDO1 inh), as outlined in the Methods section and in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. To do so, 1 g of IDO1 inhibitor was dissolved at its maximum solubility (30 mg/mL) in 70% ethanol and vortexed thoroughly. Ultrapure water was then added dropwise while gently mixing, resulting in visible precipitation of crystalline nanoparticles. We successfully crystallised the IDO1 inhibitor and visualised the approximately 5\u0026ndash;10 \u0026micro;m IDO1 inhibitor crystals using light microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and c). Although IT delivery of IDO1 crystals introduces some variability in crystal size and distribution, our formulation protocol aimed to minimize these differences. Crystals were suspended in PBS, and equal injection volumes were administered across all treated tumors to standardize dosing. To further ensure even distribution within the tumor, injections were performed at multiple sites per tumor. To approximate clinically relevant exposure, we administered 500 \u0026micro;g of the IDO1 inhibitor in mice, equivalent to ~\u0026thinsp;25 mg/kg based on average mouse body weight. Using standard body surface area normalization (K_m conversion), this corresponds to a human equivalent dose of ~\u0026thinsp;2.0 mg/kg, or ~\u0026thinsp;142 mg for a 70 kg adult. This aligns with oral doses used in clinical trials of IDO1 inhibitors like epacadostat (100\u0026ndash;300 mg BID) and linrodostat (100\u0026ndash;120 mg QD). Overall, we successfully developed a novel crystalline formulation of the IDO1 inhibitor, which can potentially be tailored for alternative administration routes in addition to directly into the tumor, such as intraperitoneal injection, although this was not explored in the present study.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIDO1 Inhibitor Fails to Provide Anti-tumor Protection Regardless of Administration Route or Formulation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNext, we aimed to evaluate the anti-tumor efficacy of our newly developed crystallized IDO1 inhibitor administered IT in solid tumors, with the goal of limiting Trp degradation and alleviating immune cell suppression. As controls, we used an IDO1 inhibitor solution that was administered either SC or IV. PBS treated groups were used as a baseline control. We employed the aggressive 4T1 triple-negative mammary carcinoma (TNBC) cell line, SC inoculated into the flank of wild-type (WT) Balb/c mice to allow convenient access to the tumor site. The following groups were generated: a control group injected with PBS SC; IDO1 inhibitor-treated groups injected either SC or IV with IDO1 inhibitor solution or IT with IDO1 inhibitor crystals. Given the importance of generating a TME before initiating crystal IDO1 inhibitor IT therapy, all treatments were started 5 days after 4T1 inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Tumor volume was monitored throughout the experiment, during which treatment was administered three times (days 5, 7 and 11) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The tumor volume and weight measurements showed that the IDO1 inhibitor was ineffective in inhibiting 4T1 tumor growth when administered as crystals for depot-slow release upon IT administration, neither as solution formulation administered SC or IV (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d). In a separate experiment, we measured the density of immune cells in the groups treated with PBS or crystalized IDO1 inhibitor administered into the tumor. We specifically calculated the density of CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells as well as myeloid cells characterized by CD11b+. Our results showed no statistical significance between the two groups (p\u0026thinsp;=\u0026thinsp;0.4952, 0.6020 and 0.0971 respectively) (Suppl. Figure\u0026nbsp;1). Additionally, we evaluated the effects of administering IDO1 inhibitor crystals IT in the aggressive, low-mutational burden B16F10 murine melanoma model, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ee. In brief, B16F10 melanoma cells were injected into Recombination-Activating Gene knockout \u0026ldquo;RAG⁻/⁻\u0026rdquo; mice, and tumors were harvested after 14 days, once they had reached a volume of approximately 1 cm\u0026sup3;. The tumors were cut into fragments measuring\u0026thinsp;~\u0026thinsp;8 mm\u0026sup2; and implanted into the flanks of anaesthetized WT C57BL/6 mice. Treatments began three days post-implantation and were administered IT three times over 14 days (days 3, 7 and 10) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). The results also showed no significant difference between the groups treated IT with PBS or crystallised IDO1 inhibitor (p\u0026thinsp;=\u0026thinsp;0.7319) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-g). These results are consistent with our previous observations in the TNBC model and confirm that IT delivery of IDO1 inhibitor crystals has no significant impact. In our previously published work, we demonstrated that formulating icosahedral \u003cem\u003eT\u0026thinsp;=\u0026thinsp;3\u003c/em\u003e virus-like nanoparticles (VLPs) packaging Toll-like receptor (TLR) ligands with microcrystalline L-tyrosine significantly produces a potent immune-enhancer that formed a depot in treated tumors and enhanced local and systemic immune responses.\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e Building on these findings, we hypothesized that formulating VLPs with IDO1 crystals would further potentiate the anti-tumor immune response. Accordingly, we recombinantly expressed our CuMVtt-VLPs and confirmed their successful formation into 30 nm VLPs as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ei and j. Next, we formulated CuMVtt VLPs with IDO1 crystals by mixing them at room temperature, which resulted in the formation of nanoparticles decorating the IDO1 crystals. We visualised AF488-labelled VLPs bound to IDO1 inhibitor crystals using fluorescent microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ek). We used the same B16F10 murine melanoma model described earlier, and designed three groups for IT treatment, initiated on day 3 post tumor fragment transplantation: the first group was treated with crystalised IDO1 inhibitor; the second group received CuMVtt VLPs alone; and the third group received a combination of CuMVtt VLPs formulated with the IDO1 inhibitor crystals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003el). The results showed a significant reduction in tumor weight in groups treated with IT CuMVtt VLPs as monotherapy or formulated with IDO1 inhibitor crystals, compared to the first group that received IDO1 inhibitor crystals (p\u0026thinsp;=\u0026thinsp;0.0008 and p\u0026thinsp;=\u0026thinsp;0.0001, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003em). No significant difference was detected between the 2nd and 3rd groups (P\u0026thinsp;=\u0026thinsp;0.6647). These data suggest that the IDO1 inhibitor did not improve the overall acquired antitumor efficiency of our VLPs, indicating the limited efficacy of IDO1 inhibitor approach.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIntratumor Delivery Elevates Metabolite Levels but Faces Limitations in Achieving Durable Tryptophan Retention\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSince local depot-release of the IDO1 inhibitor failed to elicit an anti-tumor response, we were prompted to conduct comprehensive metabolomic profiling of tumor samples. This analysis included 4T1 tumors treated SC or IV with the soluble IDO1 inhibitor, as well as IT with its crystallised form. The aim was to identify potential metabolic factors underlying the limited therapeutic efficacy observed. We conducted targeted analysis of kynurenine pathway metabolites, along with a semi-quantitative targeted search for additional related metabolites.\u003c/p\u003e\u003cp\u003eOur focus was on metabolites associated with the Kyn and serotonin pathways including Trp, Kyn, 3-hydroxy-kynurenine, quinolinic acid, nicotinic acid, picolinic acid, Kynurenic acid, xanthurenic acid, 3-hydroxyanthranilic acid (3-HAA), NR, and nicotinamide adenine dinucleotide (NAD). Our data show that IT therapy with crystalized IDO1 inhibitor has the highest average level of metabolites, followed by IV, SC and then PBS control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The number of identified metabolites overall was consistent across all treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). We measured the coefficient of variance (CVs) across metabolites within each treatment group to evaluate the consistency of the metabolic response. IT-treated samples displayed a moderate spread of CVs, indicating some variability among metabolites, but overall, a more consistent pattern compared to other treatment groups. In contrast, the IV group exhibited a markedly higher degree of variability, with several metabolites showing elevated CVs, suggesting a heterogeneous and less predictable metabolic effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The cluster dendrogram, which was created using a hierarchical clustering analysis of the metabolites, revealed that the samples could be divided into distinct groups. IT-treated tumors with IDO1 Inhibitor crystals formed well-supported clusters (AU p-values\u0026thinsp;\u0026gt;\u0026thinsp;95%), which indicates a consistent metabolic response (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). In contrast, IV and SC samples were more dispersed across the dendrogram, which is consistent with the higher variability observed in CV analysis. These results reinforce the observation that IT administration of crystalised IDO1 inhibitor induces a more defined and reproducible metabolic shift. Heatmap analysis revealed that IT administration of the crystallized IDO1 inhibitor induced the most distinct and widespread metabolic changes, compared to SC and IV administration of the soluble formulation. Notably, metabolites involved in nucleotide biosynthesis and the pentose phosphate pathway (PPP) were upregulated, while serotonin, melatonin, and urea were downregulated, contrary to initial expectations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). An additional heatmap showing log₂-transformed intensities of significantly regulated metabolites is provided in Supplementary Fig.\u0026nbsp;2 and further supports the overall limited divergence between treatment routes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePrincipal component analysis (PCA) also indicates that IT-treated mice with IDO1 Inhibitor crystals exhibit a distinct metabolic profile, which clearly differentiates them from the PBS control group and the SC- and IV-IDO1 Inhibitor treated groups that were administered the solution formulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). Such findings highlight the potential of IT treatment to alter the metabolic profile of 4T1 TNBC tumors, despite its inability to induce an antitumor response. Additionally, we performed cluster analysis of metabolite profiles in the four different groups which revealed two distinct clusters, indicating clear differences in metabolic responses among treatment groups. Samples from the IT treatment group primarily clustered together within \u003cem\u003eCluster 1\u003c/em\u003e, forming a distinct group separated from the PBS control, IDO1 inhibitor SC, and IV treated samples, which were predominantly grouped in \u003cem\u003eCluster 2\u003c/em\u003e. This segregation suggests that IT administration of IDO1 Inhibitor crystals induced a unique metabolic signature not observed with other administration routes; although some overlap was noted, particularly with IDO1 Inhibitor solution formulation administered SC or IV treatments. IDO1 Inhibitor crystals injected IT showed greater internal consistency and separation along the primary dimensions of variation (Dim1 and Dim2), which together captured over 60% of the total variance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eg).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIntratumoral IDO1 Inhibition Induces NAD⁺ Salvage Pathway Activation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe next evaluated the concentration and relative abundance of the metabolites associated with the kynurenine pathway following treatment in the tumor samples. The targeted metabolic analysis focused on key intermediates involved in essential cellular processes linked to the kynurenine pathway. Additionally, we specifically examined a range of downstream metabolites, including 3-hydroxyanthranilic acid (3-HA), quinolinic acid, nicotinic acid, picolinic acid, xanthurenic acid, melatonin, hydroxyindoleacetic acid (3-HIAA), NR, and NAD⁺(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The following metabolites could be detected in the kynurenine pathway: Trp, Kyn, serotonin, 3-HIAA, melatonin, and 3-HA in 4T1 tumor. NR is a vitamin B3 derivative that serves as a precursor for NAD⁺ through the salvage pathway. While not directly part of the kynurenine pathway, both NR and Kyn metabolites contribute to NAD⁺ biosynthesis via distinct routes.\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOverall, no significant differences and minimal variation across the different treatment groups were observed for Trp, Kyn, serotonin, 3-HIAA, melatonin or 3-HA metabolites, which remained relatively stable in concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-g and Supplementary Figs.\u0026nbsp;3, 4 and 5). These findings suggest that neither systemic administration of the IDO1 inhibitor in soluble form nor local depot-release via IT injection significantly impacted Trp degradation in the 4T1 TNBC murine model. Interestingly, IT treated tumors with IDO1 crystals exhibited significant increased levels of NR (p\u0026thinsp;=\u0026thinsp;0.0483) in comparison to the other groups, suggesting a possible tumor-intrinsic adaptation via the NAD⁺ salvage pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). Guanosine level was significantly elevated in tumor treated with IDO1 inhibitor crystals IT as compared to PBS-control (Supplementary Fig.\u0026nbsp;5v1). This reflect the NAD\u0026thinsp;+\u0026thinsp;depletion caused due to the inhibition of IDO1 activity.\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e Under NAD\u0026thinsp;+\u0026thinsp;stress, tumor cells can upregulate Purine salvage pathway to increase the GTP accumulation ensuring critical biosynthetic and signalling functions persist despite metabolic stress.\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eα-Ketoglutarate (α-KG) levels were lower in the IT treated group compared to the control group (Supplementary Fig.\u0026nbsp;4l1). This observation reflect a reduced oxidative burden in response to IDO1 inhibition. During Trp catabolism, the downstream metabolite 3-hydroxykynurenine is known to generate reactive oxygen species (ROS).\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e To reduce this oxidative stress, tumor cells upregulate glutamine metabolism, producing α-KG to fuel the TCA cycle and support redox homeostasis through glutathione synthesis.\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e The reduced α-KG levels observed in the anti-IDO treated group, along with lower glutamine abundance, suggests a decreased ROS demand due to diminished kynurenine pathway activity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTumor-Driven L-Tryptophan Consumption Impairs Immune Activation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven the limited anti-tumor effect of IDO1 inhibition, likely due to tumor cells benefiting more from Trp than immune cells, we hypothesized that direct Trp administration in the tumor would likewise fail to suppress tumor growth. To test this, we assessed the impact of Trp delivered either as crystals into the TME or as a solution via SC or IV injection. We used the aggressive 4T1 TNBC cell line, inoculating it SC in the flank region of WT Balb/c mice. The following groups were defined: a control group injected with PBS SC; and Trp-treated groups injected either SC or IV with Trp solution, or IT with Trp crystals. Treatment was administered three times (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), and tumor volume was monitored throughout. Tumor volume and weight measurements revealed no significant differences between the treated groups and the control, suggesting limited anti-tumor efficacy of Trp alone in this model (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-d). We carried out an immunological assessment focusing on the tumors in the group treated with IDO1 inhibitor crystals via IT and compared the results to those of the control group treated with PBS (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). Our results revealed overall a trend in increased density of immune cells, specifically CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells, albeit not statistically different (p\u0026thinsp;=\u0026thinsp;0.3233, 0.1590 respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eg-i). No trends have been noticed in the enhanced levels of myeloid-derived cells characterised by CD11b or B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003ej and k). Our data suggest that supplementing tumors with Trp may preferentially support tumor growth rather than enhance anti-tumor immune responses within the TME. This finding highlights the need to develop more effective strategies to counteract tumor-favoring metabolic pathways and improve immunotherapeutic outcomes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIDO1 inhibitors were designed to counteract tumor immune evasion by blocking the degradation of Trp into immunosuppressive Kyn, thereby alleviating metabolic constraints on T cell function within the TME. In this study, we assessed the anti-tumor efficacy of a novel crystallized IDO1 inhibitor formulated for IT administration. We hypothesized that sustained local release would achieve prolonged enzyme inhibition, preserving Trp availability and enhancing anti-tumor immunity. Despite achieving pharmacologically relevant doses, as confirmed by human equivalent dose (HED) calculations, no measurable tumor regression was observed following IT, SC, or IV delivery, highlighting the limited therapeutic effect of IDO1 inhibition in two cold murine tumor models.\u003c/p\u003e\n\u003cp\u003eCrystallizing the IDO1 inhibitor for slow local release represents an innovative and consistent approach for localized drug delivery, as supported by moderate metabolite variability and distinct clustering patterns in metabolic profiles. The IT dose of 500 \u0026micro;g corresponds to an HED of approximately 2.0 mg/kg (~142 mg for a 70 kg adult), aligning with clinical doses of oral IDO1 inhibitors.\u003csup\u003e[22,23]\u0026nbsp;\u003c/sup\u003eOver 95% of free Trp is a substrate for the kynurenine pathway of Trp degradation.\u003csup\u003e[2]\u0026nbsp;\u003c/sup\u003eOur\u0026nbsp;metabolic profiling revealed that Trp degradation was limited under all conditions, as evidenced by a low Kyn/Trp ratio (~0.033), suggesting either inherently low IDO1 activity in 4T1 tumor model or successful enzyme inhibition. This is consistent with the notion that IDO1 is not uniformly active across all tumor types and may be upregulated only under specific inflammatory stimuli.\u003c/p\u003e\n\u003cp\u003eOur findings echo broader challenges faced in the clinical translation of IDO1 inhibitors. The high-profile failure of the ECHO-301 Phase 3 trial, which combined epacadostat with pembrolizumab in melanoma, suggests that IDO1 monotherapy may be insufficient in the absence of a primed immune microenvironment. It is also possible that compensatory pathways such as TDO, IL4I1, or microbiota-driven Trp metabolism bypass the IDO1 blockade and sustain immunosuppressive signaling via activation of the aryl hydrocarbon receptor (AhR). While we observed a metabolic shift including elevated NR and guanosine suggestive of enhanced NAD+ salvage and nucleotide regeneration, these responses reflect tumor adaptation rather than therapeutic benefit.\u003c/p\u003e\n\u003cp\u003eMoreover, local Trp metabolism is increasingly recognized as a critical modulator of tumor-immune interactions. For instance, Trp-derived microbial metabolites like indole-3-aldehyde (I3A) can activate AhR and suppress tumor growth via modulation of innate lymphoid cells.\u003csup\u003e[24]\u0026nbsp;\u003c/sup\u003eWhether such pathways are engaged following IDO1 inhibition remains an open question. Interestingly, the minimal Trp-to-Kyn conversion observed here suggests that additional blockade of TDO may not be necessary in this model, although this may vary depending on tumor type and immune context.\u003csup\u003e[25]\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImportantly, our data highlight both the potential and the limitations of our novel localized IDO1 inhibition. While the metabolic response to IT delivery showed some potentials in altering the metabolic profile in the tumor, it did not translate into measurable tumor control. Importantly, direct feeding of Trp crystals to the tumors resulted in enhanced growth. This further highlights the need for innovative therapeutic modalities to better control tumor growth.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eProduction of IDO-1 inhibitor crystals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIDO5L nanoparticles/crystals were prepared by dissolving IDO5L (CAS registry no.: 914471-09-3) at its maximum solubility of 30 mg/mL in 70% ethanol (ETOH). Specifically, 1 g of IDO5L was dissolved in approximately 33 \u0026micro;L of ETOH, and the mixture was thoroughly vortexed until complete dissolution. Subsequently, ultrapure water (ddH₂O) was carefully added dropwise to the solution while gently mixing, until precipitation occurred and the solution became visibly milky, indicating crystalline nanoparticle formation. Typically, for 1 g of IDO5L, this precipitation was achieved with approximately 4\u0026ndash;5 drops (40\u0026ndash;50 \u0026micro;L) of ddH₂O.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMurine cell lines\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 4T1 mammary carcinoma cell line (ATCC CRL-2539) was obtained from the American Type Culture Collection. The B16F10 melanoma cell line was generously provided by the Ochsenbein laboratory. Both cell lines were maintained in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Once cultures reached approximately 80% confluence, cells were washed three times with PBS to remove residual medium, then incubated with 1\u0026times; trypsin for 10 minutes at 37\u0026deg;C to facilitate detachment. The detached cells were subsequently collected, resuspended in complete medium, and kept on ice until further use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale wild-type (WT) Balb/cOlaHsd and female C57BL/6 mice (7-12 weeks old, were purchased from Harlan). RAG1\u0026minus;/\u0026minus; mice, backcrossed onto the C57BL/6 background, were kindly provided by the Ochsenbein laboratory and maintained in our pathogen-free animal facility at the University of Bern\u0026apos;s animal facility. All \u003cem\u003ein vivo\u003c/em\u003e experiments were conducted using female mice aged (7-12 weeks). All animal procedures complied with the Swiss Animal Act (455.109.1, September 2008, Article 5), approved by the University of Bern and performed under license no. BE30/2024, and the study was conducted in accordance with the relevant guidelines and regulations.\u0026nbsp;This study is reported in accordance with the ARRIVE guidelines (https://arriveguidelines.org).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;tumor experiment and dosing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale Balb/c mice (7\u0026ndash;12 weeks old, Harlan) were SC inoculated with 1\u0026times;10⁶ 4T1 cells per mouse on day 0.\u003csup\u003e[26]\u0026nbsp;\u003c/sup\u003eTumor growth was monitored regularly. On day 5, when the tumors became palpable, the mice were divided into experimental groups. Treatments were conducted three times on days 5, 7 and 11. The study endpoint was set for day 17. One million B16F10 melanoma cells were SC injected into the flanks of 7\u0026ndash;12-week-old C57BL/6 RAG1⁻/⁻\u0026nbsp;mice. After 12 days, the resulting tumors were harvested and transplanted into the flanks of WT C57BL/6 mice (7\u0026ndash;12 weeks old; Harlan). Tumor growth was monitored regularly. When the tumors became palpable on day 5, the mice were divided into experimental groups. Treatments were conducted three times on days 3, 7 and 10. The endpoint of the study was set on day 14. Mice from all groups were euthanized once their tumors reached the humane endpoint in accordance with the Swiss Animal Act. Euthanasia of animals was carried out using CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eand decapitation. Tumor volume in mm\u003csup\u003e3\u003c/sup\u003e was monitored using callipers and calculated using the formula V = (W \u0026times; W \u0026times; L)/2, where V is volume, L is tumor length and W is tumor width. Tumor volumes at endpoint ranged between 150 mm\u0026sup3; and 1000 mm\u0026sup3;. The IDO1 inhibitor was administered at a dose of 500 \u0026micro;g per injection, while the VLPs were given at a dose of 100 \u0026micro;g per injection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTumor analysis and staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle-cell suspensions of tumors (TILs) were generated as follows. Tumors were digested using Collagenase D (1mg/mL in DMEM-Medium, Roche) at 37\u0026deg;C for 45 minutes. The digested tumors were smashed through a 70 \u0026mu;m cell strainer (Sigma Aldrich). During the mechanical processing, Rosewell Park Memorial Institute (RPMI) medium was continuously added to a final volume of 5mL. The collected cells were centrifuged at 300 g for 5 minutes at 4\u0026deg;C. The cells were resuspended in a fresh medium. The cells were washed once with FACS-Buffer (PBS + 2% FBS). In Table 1, the used antibodies are listed. Cells were then stained with FC-block (1:100) for 15 minutes in the dark. The cells were centrifuged at 300 g 4\u0026deg;C for 5 minutes. The surface staining antibody mix was added and incubated for 30 minutes in the dark. After the surface staining, the cells were washed 3 times with FACS-Buffer. The 96-Well U bottom plate was acquired using the Aurora 5L of the FACS facility of the DBMR of the University of Bern. The FCS files were analyzed using FlowJo (V10.10), and the graphs were created using GraphPad Prism (V10.4.1).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTarget\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFluorochrome\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClone\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDilution\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003eCD45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 198px;\"\u003e\n \u003cp\u003eBUV737\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e30-F11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1:300\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003eCD8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 198px;\"\u003e\n \u003cp\u003eBUV563\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e53-6,7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1:300\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003eViability\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 198px;\"\u003e\n \u003cp\u003eZombie Aqua\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003eCD11b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 198px;\"\u003e\n \u003cp\u003ePerCP-Cy5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003eM1/70\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1:300\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003eCD4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 198px;\"\u003e\n \u003cp\u003eAPC-Cy7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;RPA-T4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1:300\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVLPs expression, production and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCuMVtt VLPs were expressed and produced as described previously in prvious papers [\u003csup\u003e16, 27\u003c/sup\u003e]. Endotoxin levels were measured and confirmed to be below 1000 EU/mL. CuMVtt VLPs naturally package single-stranded RNA (ssRNA) during expression in \u003cem\u003eE. coli\u003c/em\u003e, which serves as a TLR7/8 ligand to effectively activate innate immune cells.\u003csup\u003e[28]\u003c/sup\u003e\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDynamic light scattering (DLS)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis was performed on a CuMVtt VLP solution at a concentration of 1 mg/mL using a Zetasizer Nano ZS instrument (Malvern Instruments, UK). Data from three independent measurements were analyzed using DTS software (Malvern, version 6.32).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectron Microscopy\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The physical stability and structural integrity of CuMVtt VLPs were assessed by transmission electron microscopy (TEM) using a Philips CM12 instrument. For sample preparation, glow-discharged grids were incubated with 5 \u0026micro;L of VLP solution for 30 seconds. The grids were then washed three times with double-distilled water and negatively stained with 5 \u0026micro;L of 5% uranyl acetate for 30 seconds. Excess stain was removed by pipetting, and the grids were air-dried for 10 minutes. Images were acquired at magnifications of \u0026times;84,000 and \u0026times;110,000.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCuMVtt VLPs and IDO1 inhibitor crystals formulation\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Nanoparticles (CuMVtt VLPs) were formulated with IDO1 inhibitor crystals by mixing the two components on a shaker at 500 rpm at room temperature for 1 hour. This process facilitates the adsorption of CuMVtt onto the crystals. For visualization, CuMVtt VLPs (3 mg/mL) were labeled with Alexa Fluor 488 (AF488) following the manufacturer\u0026rsquo;s instructions and then mixed with IDO1 inhibitor crystals. A 10 \u0026micro;L aliquot of the mixture was placed on a glass slide, covered with a cover slip, and imaged using a Zeiss AxioImager A2 fluorescence microscope equipped with Plan-NEOFLUAR \u0026times;20 and \u0026times;40 objectives (Zeiss).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolomics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrozen tumor samples (-80\u0026deg;C) were transferred into PowerBead Tubes Ceramic 1.4 mm (Qiagen), and metabolites were extracted by adding nine volumes of methanol (MeOH). Homogenization was performed using the PowerLyzer 24 Homogenizer (Qiagen) in three cycles of 10 seconds at 2500 rpm. The tubes were then centrifuged at 10,000 \u0026times; g for 10 minutes. A 10 \u0026micro;L aliquot of the resulting supernatant was transferred to a new tube, followed by the addition of 10 \u0026micro;L of an isotopically labeled internal standard mixture and 80 \u0026micro;L of methanol. After vortexing, the samples were centrifuged again at 1000 \u0026times; g for 10 minutes, and 90 \u0026micro;L of the clarified extract was transferred into HPLC glass vials for LC-MS analysis. Chromatographic separation of metabolites was carried out on an ACQUITY Premier BEH Z-HILIC column (1.7 \u0026micro;m, 2.1 \u0026times; 100 mm; Waters) using a gradient elution. Mobile phase A consisted of 0.15% formic acid and 10 mM ammonium formate in water, while mobile phase B consisted of 0.15% formic acid and 10 mM ammonium formate in 85% acetonitrile. The total analysis time was 18 minutes, with a flow rate of 0.4 mL/min, an injection volume of 2 \u0026micro;L, and a column temperature of 40 \u0026deg;C. Mass spectrometric analysis was performed on an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific), operated in both positive and negative electrospray ionization (ESI) modes. Full scan acquisition was conducted over an m/z range of 50\u0026ndash;600 with a resolving power of 60,000. The ESI spray voltage was set to 3.5 kV for positive mode and 2.5 kV for negative mode. Additional MS settings included a capillary temperature of 350 \u0026deg;C, a gas heater temperature of 400 \u0026deg;C, an auxiliary gas flow rate of 12 arbitrary units, and a sheath gas flow rate of 50 arbitrary units. Quantification was based on seven-point calibration curves using internal standardization. LC-MS data acquisition, processing, and quantification were performed using TraceFinder 5.1 General Quant software (Thermo Fisher Scientific). Metabolite identification was assigned confidence level A based on accurate mass, retention time, and comparison with authentic reference standards previously validated on the same analytical platform. For relative quantification, acquired data files were evaluated for the following metabolites: 3-hydroxyanthranilic acid, quinolinic acid, nicotinic acid, picolinic acid, xanthurenic acid, NR, NAD⁺, hydroxyindoleacetic acid, and melatonin. Accurate m/z values were calculated for each compound, and metabolite detection was performed using a mass accuracy threshold of 10 ppm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProduction of Tryptophan crystals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL-Tryptophan (Sigma-Aldrich, T0254) was suspended in sterile PBS obtaining a concentration of 5 mg/ml for the solution fomulation or prepared as crystals as described in IDO1 inhibitor method.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as mean\u0026plusmn;SEM. Comparisons between more than two groups were performed by one-way analysis of variance; comparisons between two groups were performed by Welch\u0026rsquo;s t-test. P values were ****p\u0026lt;0.0001, ***p\u0026lt;0.001, **p\u0026lt;0.01, and *p\u0026lt;0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u0026nbsp;\u003c/strong\u003eBiogenity has contributed by performing the data analysis, including figures, tables, and reports. Metabonet has contributed by performing the metabolomics sample analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution:\u0026nbsp;\u003c/strong\u003eDesign of experiments, acquisition of data, interpretation, and analysis of data: ASC, AG, MFB and MOM. Writing, revision and editing of manuscript: ASC, AG, MFB and MOM. Technical, material and tool support: MFB and MOM. Study supervision: MOM and MFB. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statment:\u0026nbsp;\u003c/strong\u003eAll data supporting the findings of this study are contained within the article and its supplementary content. The raw data can be obtained from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e:\u0026nbsp;This research was funded by Swiss National Foundation (SNF): (Grant Number: 320030-228094), Swiss Cancer Research (Grant Number KFS-5246-02-2021-R) and Jubil\u0026auml;umsstiftung von Swiss Life. Illustrations were generated with BioRender.com.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e MOM and MFB have financial interests as shareholders in DeepVax GmbH, a company that specializes in the development of vaccines. DeepVax GmbH is a spinoff from the University of Bern.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSchwarcz R, Stone TW. The kynurenine pathway and the brain: Challenges, controversies and promises. \u003cem\u003eNeuropharm\u0026nbsp;\u003c/em\u003e2017; \u003cstrong\u003e112\u003c/strong\u003e(Pt B):237-247.\u003c/li\u003e\n \u003cli\u003ePlatten M, Nollen EAA, R\u0026ouml;hrig UF, Fallarino F, Opitz CA. Tryptophan metabolism as a common therapeutic target in cancer, neurodegeneration and beyond. \u003cem\u003eNat Rev Drug Discov\u0026nbsp;\u003c/em\u003e2019; \u003cstrong\u003e18\u003c/strong\u003e(5):379-401.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMunn DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B, Brown C, Mellor AL. 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TLR7 Signaling Shapes and Maintains Antibody Diversity Upon Virus-Like Particle Immunization. \u003cem\u003eFront Immunol\u003c/em\u003e 2022 Jan 19; \u003cstrong\u003e12\u003c/strong\u003e:827256.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"IDO1 inhibitor, Crystalisation, Tryptophan, Anti-Tumor Response, Solid Murine Tumors","lastPublishedDoi":"10.21203/rs.3.rs-7138169/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7138169/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTryptophan (Trp) is required by both immune and tumor cells for protein synthesis and key metabolic pathways. While immune cells rely on Trp for proliferation and function, tumor cells exploit IDO1-mediated Trp degradation to evade immune surveillance. However, clinical trials have demonstrated limited efficacy of IDO1 inhibitors, likely due to insufficient tumor targeting and immune evasion strategies within the tumor microenvironment (TME). Here, we explored a novel approach using intratumoral (IT) delivery of a crystallised IDO1 inhibitor designed for depot-based slow release. Using aggressive murine solid tumor models, we combined \u003cem\u003ein vivo\u003c/em\u003e assessments of tumor growth with metabolic profiling to investigate treatment effects. Despite increased metabolite abundance in tumors, IT administration of the crystallised IDO1 inhibitor failed to control tumor growth compared to soluble formulations (subcutaneously or intravenously). Immune profiling confirmed that IDO1 blockade alone did not reverse immunosuppression in the TME. Interestingly, IT treatment with IDO1 inhibitor crystals significantly elevated nicotinamide riboside (NR) levels, suggesting tumor-intrinsic metabolic adaptation via the NAD⁺ salvage pathway. Similarly, local delivery of crystallised Trp did not suppress tumor progression. On the contrary, an excess presence of Trp enhanced tumor growth, demonstrating that Trp is also essential for tumor growth. These findings underscore the metabolic plasticity of tumors and highlight the limited therapeutic potential of IDO1 inhibition, even with local, sustained-release strategies.\u003c/p\u003e","manuscriptTitle":"Intratumoral Delivery of Crystallised IDO1 Inhibitors Fails to Overcome Tumor Immunosuppression Due to Metabolic Adaptation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-14 06:29:50","doi":"10.21203/rs.3.rs-7138169/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"87e67c3b-afc5-41e5-930d-931500e69edf","owner":[],"postedDate":"August 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53069112,"name":"Biological sciences/Cancer"},{"id":53069113,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2025-12-08T05:23:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-14 06:29:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7138169","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7138169","identity":"rs-7138169","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}