Pharmacological Polarization of Tumor‐Associated Macrophages Toward a CXCL9 Antitumor Phenotype

Abstract Tumor‐associated macrophages (TAM) are a diverse population of myeloid cells that are often abundant and immunosuppressive in human cancers. CXCL9Hi TAM has recently been described to have an antitumor phenotype and is linked to immune checkpoint response. Despite the emerging understanding of the unique antitumor TAM phenotype, there is a lack of TAM‐specific therapeutics to exploit this new biological understanding. Here, the discovery and characterization of multiple small‐molecule enhancers of chemokine ligand 9 (CXCL9) and their targeted delivery in a TAM‐avid systemic nanoformulation is reported. With this strategy, it is efficient encapsulation and release of multiple drug loads that can efficiently induce CXCL9 expression in macrophages, both in vitro and in vivo in a mouse tumor model. These observations provide a window into the molecular features that define TAM‐specific states, an insight a novel therapeutic anticancer approach is used to discover.

The first goal of the current project was to determine whether small-molecules could be used to polarize macrophages toward the newly defined CXCL9 Hi phenotype.A secondary goal was to formulate any such hits into a TAM-targeting preparation to enhance treatment efficacy and reduce systemic toxicity.Therefore, we performed a screen of known small-molecules targeting some Overview of myeloid cell screening approach.A) TAM are bone marrow-derived, abundant in many cancers, and mostly immunosuppressive and thus pro-tumorigenic.B, C) This phenotype is driven primarily by IL10 and hypoxia (SPP1) signaling.We hypothesized that it should be possible to polarize macrophages to an antitumorigenic phenotype by increasing CXCL9 signaling. [4]Yet, no effective pharmaceutical strategies have emerged to do this effectively while retaining TAM specificity.D) In this research, we used BMDM from Rex3 reporter mice (expressing CXCL9-RFP) to screen for smallmolecules and combinations that could induce CXCL9 in myeloid cells.Top hits from the screen were then encapsulated into TAM-avid nanoparticles to elicit a CXCL9 phenotype in vivo.
of the above pathways to determine whether they induce CXCL9 production in bone marrow-derived macrophages (BMDM).We reasoned that a scalable, in vitro, approach for testing CXCL9 induction in macrophages would lead to a therapeutic combination.We hypothesized that dual and triple combinations of different small-molecules could synergize, prevent compensatory resistance mechanisms, and then be formulated into a single TAM-avid drug delivery system.We discovered a triple combination that indeed increased CXCL9 production by an order of magnitude above traditional CXCL9-inducing stimuli and much more efficiently than IFNg alone.
These findings are significant as they offer a promising avenue for developing novel therapeutic strategies targeting TAM.The ability to augment CXCL9 production in TAM can potentially increase lymphocytic infiltration, which drives antitumor immunity.This approach may hold promise for advancing cancer immunotherapy and improving treatment outcomes for cancer.

Optical Screens in Freshly Isolated Target Cells
Pharmacological modulation of TAM in vivo has been challenging due to several reasons: first, the lack of cost-effective methods to combinatorially screen drugs that can polarize TAM; second, the still limited knowledge of key regulators of TAM program-ming; and third, the availability of efficient and selective delivery vehicles with high drug payloads.To address these challenges, we have developed optically resolved screening approaches that more efficiently and rapidly identify therapeutic combinations that induce CXCL9 and other interferon-stimulated genes (ISG) (Figure 1).In this study, we performed the screening in primary isolated bone marrow-derived cells (BMDC) from CXCL9 red fluorescent protein (RFP) and CXCL10 blue fluorescent protein (BFP) reporter mice to identify potential small-molecule compound hits capable of inducing RFP expression.Freshly obtained BMDC from these mice were cultured with macrophage colonystimulating factor (M-CSF) for 7 days, at which time they typically have low levels of baseline CXCL9 expression.Upon the addition of potential modulators fluorescence microscopy and flow cytometry can be used to measure increases in RFP (Figure 1).

Creation of a Mini-Library for Screening
There is an emerging realization that combination therapies are needed to: i) improve treatment efficacy, ii) lower the dose of single immune-stimulatory agonists, and iii) circumvent immune cell resistance mechanisms.Based on the hypothesis that certain small-molecule combinations may indeed affect distinct cellular programs when delivered specifically to TAM, we curated a small collection of drugs (Figure 2).We focused primarily on known modulators of several major pathways (IFNg, NFkB, TLR, STAT1, and interleukin 10 (IL10).As no direct single agonist of IFNg signaling nor CXCL9 has been reported, we adapted our imaging readout approach to measure dozens of therapeutic combinations at varying dose levels for their ability to boost CXCL9 in macrophages.The selection of these compounds was largely driven by the current understanding of TAM signaling (Figure 2) and the potential of a given compound to be useable clinically.Our collections contained 21 individual small-molecules and another ≈20 combinations, resulting in screens with ≈40 different drug combinations.Figures S2 and S3 (Supporting Information) summarize the synthesis and characterization of some of the compounds in our screen that are not commercially available.

Screening Identifies Drug Combinations that Induce CXCL9
Figure 3 summarizes the primary screening results.All screenings were carried out in triplicates, yielding consistent and reproducible results with minimal variation.The first screening, performed as a control, examined whether any single compound or dual combination thereof could induce CXCL9 production in nonstimulated baseline macrophages.As expected, the screening did not identify any significant hits capable of elevating CXCL9 expression in vitro.
The second screening paralleled the first, yet it incorporated baseline IFNg stimulation to mimic native cytokine exposure within the tumor environment, if any.IFNg is primarily secreted by T-cells and NK cells, instigating macrophage and dendritic cell responses via the interferon-gamma receptor (IFNGR) and JAK/STAT pathways.This time, most single agents induced a mild increase in CXCL9 response, and certain dual combinations induced a moderate increase.However, specific pairings, particularly those including the RIG-1-like receptor agonist KIN-1408, resulted in cellular toxicity and did not induce CXCL9.
Based on these results, we performed a third screening using triple drug combinations to determine whether CXCL9 expression could be further enhanced.Certain combinations with KIN-1408 and CRX527 (an LPS mimetic) were associated with toxicity at the doses applied, leading to their exclusion from subsequent testing.The screening identified several triple combinations that achieved exceptionally high CXCL9 expression.The key finding resulting from the third screen was the combination consisting of RBN2397 (a PARP7 inhibitor), MSA-2 (a STING agonist), and R848 (a TLR7/8 agonist) capable of increasing CXCL9 expression ∼8 fold.Interestingly, the expression of IL12 was also enhanced by this triple combination (Figure 3).Subsequent experiments were then performed to incorporate this potent combination into a TAM-avid nanotherapeutic formulation (CANDI400).

CANDI400 Formulation and Efficacy
We had previously developed a cyclodextrin-adjuvant nanoparticle-drug delivery system (CANDI) that accumu- lates avidly in TAM after systemic injection and enables highly effective, multitarget, and pathway modulation inside TAM and dendritic cells. [9,14]Specifically, versions of this delivery platform had primarily been used to induce IL12 in TAM. [14]Unfortunately, the prior drug combination "HAMT" (LCL161, R848, and Ruxolitinib) had no effect on cellular CXCL9 expression (Figure 3). [14]e first validated the propensity of our hit drug combination to form strong complexes with the bisuccinyl cyclodextrin (sbCD) monomers of CANDI nanoparticle.Initially, we looked for changes in chemical shifts and equimolar complexation in nuclear magnetic resonance (NMR) titration experiments with R848, RBN2397, and MSA-2 individually.These payloads were mixed in a D 2 O solution containing a fixed concentration of the host, sbCD (Figure S4, Supporting Information). [14]From these experiments, we concluded that R848 had a very high binding affinity toward sbCD and potentially multiple binding orientations which favored its high solubility.RBN2397 had optimal complexation at a three-host per guest complexation ratio.Potentially accounting for two aromatic and one aliphatic binding moieties (Figure S4, Supporting Information).Throughout all mea-sured conditions, however, MSA-2 showed very poor affinity to sbCD and remained a stable turbid suspension regardless of the presence or concentration of sbCD, hindering our NMR comparative studies in D 2 O.
For this reason, we opted to design a more lipophilic prodrug of MSA-2 with enhanced inclusion complexation ability with sbCD, termed MSA-2p.The synthesis of MSA-2p was achieved in moderate yields activating MSA-2 with 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC).Using an anhydrous mixture of acetonitrile and methanol resulted in the highest yields (56%).MSA-2p was the main product from this reaction (>85% conversion) and interestingly, the structure differed significantly from the other MSA-2 prodrug analogs.MSA-2p is a peculiar cyclic lactone locked in a chiral conformation as shown on the aliphatic region of the 1 H-NMR spectra (Figure S3, Supporting Information).
Solution experiments with MSA-2p showed a fast decrease in turbidity when quasi-equimolar concentrations of sbCD were present, indicating a strong affinity between MSA-2p and sbCD.In the absence of the host, MSA-2p had low water solubility (<3.6 mg mL −1 , Figure 4).We then examined the hydrolysis rate The left bottom graph indicates the percent of free/bound drug to sbCD, determined as turbidity value (measured at 550 nm, average of 500 s time-lapse, N = 3).Bulk studies using MSA-2p in buffer (pH 7.4) showed that complexation to sbCD leads to a significant reduction in the hydrolysis rates of MSA-2p (right).D. Turbidity assay to measure loading and stability of the CANDI400 formulation assessed by an increase in absorbance at 550 nm.We determined the loading capacity for CANDI400 to be ≈0.15mg of payloads per mg of CANDI nanoparticle.E. Release kinetics of the triple small-molecule combo from CANDI400 using a porous membrane (3 kDa) in PBS (1×) at 37 °C.The release rates for R848, MSA-2, and RBN2397 are depicted as the dissociation rates (k off ) and complex half-life (t 1/2 ), respectively determined in a 7 h time lapse (see Figure S6, Supporting Information, N = 3).
of MSA-2p to MSA-2.We used the spectrally unique 325 nm absorbance peak to quantify the formation of MSA-2 in buffered solutions.We studied the hydrolysis rates of MSA-2p from absorbance scans acquired at equilibrium and assessed that the complexation of MSA-2p to sbCD had a significant effect on the hydrolysis rates.When dissolved in phosphate-buffered saline (PBS), regardless of pH, MSA-2p exhibited very fast hydrolysis (0.22% s −1 ).Adding an increasing amount of sbCD to six equivalents dropped the hydrolysis rate to (0.018% s −1 ≈11.8-fold).These results indicate that the strong binding with sbCD hinders the nucleophilic attack of the water molecule, suggesting that the inclusion complexation inserts around the cyclic chiral lactone (details on the proposed hydrolysis mechanism can be found in Figure S5, Supporting Information).
In the next set of experiments, we performed typical nanoparticle characterization experiments of the triple-loaded CANDI400 formulation (Figure S6, Supporting Information).Upon loading, the nanoparticle size was 19.8 ± 1.1 nm, and the zeta potential was −6.15 mV.Transmission electron microscopy revealed spherical polymeric structures with matching size distribution to our dynamic light scattering (DLS) measurements.We determined the drug loading capacity to be around 0.15 mg of drug combo (R848, RBN2397, and MSA-2p) per mg of particle before it reaches saturation.In a closed-dialysis set-up mimicking physiological conditions, we determined the release rates for each drug in the CANDI400 formulation.The release of each released drug was quantified simultaneously using an optimized liquid chromatography method coupled to mass spectrometry (LCMS), which resulted in similar dissociation rates k off (0.46, 0.39, and 0.19 h −1 for R848, MSA-2, and RBN2397) and a complexation half-life of t 1/2 = 1.5, 1.8 and 3.6 h respectively (Figure 4 and Figure S6, Supporting Information).
A dose-response curve was obtained with BMDM from CXCL9-RFP mice, yielding an effective EC 50 of 3.3 ng ml −1 .As shown in Figure 5, there was uniform CANDI400 uptake and concomitant CXCL9 production in BMDM.Additional cytokine screening experiments were conducted (Figure S1, Supporting Information) to ascertain that cellular cytokine production was due to the CANDI payload and not the nanoparticle drug carrier itself.At this dose, there was no discernible cellular toxicity with CANDI400.These results confirmed the efficacy of the triple combination nanoparticle to increase CXCL9 production.Subsequent experiments were therefore conducted to show in vivo efficacy.

Intravital Imaging Reveals Drug Action in the Tumor Microenvironment and Antitumor Efficacy
We first determined the vascular half-life of CANDI400 by serial imaging of the microvasculature in the mouse ear (Figure S9, Supporting Information).This showed a vascular half-life of approximately 2.3 h.Cellular uptake could be identified as early as 1 h but was more pronounced by 4 h after IV administration.To determine whether the CANDI400 formulation indeed accumulated within TAM in vivo following systemic administration and elicited CXCL9 expression, we performed intravital microscopic imaging in live mice.CXCL9-RFP transgenic mice were implanted with dorsal window chambers into which MC38-GFP tumors were grown.After 8 to 10 days, we performed serial microscopic examinations of the TME both before and after intravenous systemic administration of CANDI400 labeled with AF647 (Figure 6 and Figure S8, Supporting Information).Baseline expression of CXCL9 pre-treatment was very low, with sparse cellular expression within the TME.Remarkably, within 24 to 48 h postinfusion of CANDI400, we observed a considerable induction of CXCL9 throughout the tumor.Using a fluorescent analog of our particles labeled with AF647, we confirmed that these CXCL9-expressing cells had incorporated the nanoformulation (Figure 6 and Figure S8, Supporting Information).
To determine whether these changes translate to antitumor efficacy, we performed tumor growth experiments in mouse models (Figure S10, Supporting Information).We observed remarkable efficacy in the MC38 tumor model, with all tumors disap-pearing after two systemic administrations of CANDI400.60 days after tumor inoculation, all mice that received CANDI400 were still alive, in contrast to a median survival time of 28 days in the control group.Figure S11 (Supporting Information) summarizes our understanding of how this therapeutic combination works in vivo.These results demonstrate that a tumor CXCL9 Hi phenotype can be controlled through combinatorial nanotherapeutics to inflame the TME.

Discussion
Developing strategies to enhance immune cell trafficking to tumor sites is critical to improving immunotherapy responses.[26][27] This renewed interest is driven in part by the realization and subsequent dissection of different myeloid cell subsets [28] each with its own pro-and antitumor phenotypes. [4,29] major interest has been the identification of abundant TAM subsets that correlate clinically with improved outcomes.One such subset is CXCL9 Hi expressing TAM [8,30,31] and/or CXCL9 Hi /SPP1 Lo ratio in TAM.[4,7] Based on this new understanding of biology, we set out to identify and develop translatable therapies that could pharmacologically mimic and enhance endogenous pathways of antitumor myeloid cells.
The expression of CXCL9, related chemokines (CXCL10), and other ISG is primarily driven by IFNGR signaling through IFNg produced by lymphocytes (e.g., through IL12, IL18, or antigen Eight days after tumor implantation, CANDI400 was administered by tail vein, and serial repeat imaging was performed.CANDI400 was labeled with AF647 and contained the triple small-molecule cocktail (RBN2397, MSA-2p, and R848) to induce CXCL9 in myeloid cells in the TME.The top row (scale bars 50 μm) shows MC38-H2B-GFP tumor cells (green, 488 nm channel), the middle row CANDI400 AF647 (white, 647 nm channel), and the lower row CXCL9-RFP (red, 550 nm channel).Immediately after intravenous injection of CANDI400, the nanotherapeutic drug is largely confined to vessels.Within several hours, the CANDI is later taken up by TAM.Note the high CXCL9 induction within 24 h after systemic CANDI400 administration.See Figure S8 (Supporting Information) for high-resolution images.stimulation). [32,33]Within macrophages, IFNg response is mediated by JAK1/2, STAT1 signaling, which leads to transcription factor binding to gamma interferon activation site (GAS) elements and activation of IFNg programs in macrophages (Figure S11, Supporting Information).Additional pathways are being revealed by ongoing research.For example, in a recent study, CRISPR-Cas9 screening identified numerous positive regulators of CXCL9, including Dnttip1, Prdm14, Zfp431, Klf6, Arid1a, Socs1 and Smarcd1. [20]Interestingly, epigenetic regulation through the SWI/SNF-PRC2 axis (e.g., by inhibiting EED or Irf1) led to up-regulation of CXCL9.Thus, additional CXCL9 drug targets may become available in the future.
Here we identified a triple-drug combination that works synergistically in upregulating CXCL9.The combination involves RBN2397, a PARP7 inhibitor in clinical trials, a STING prodrug (MSA-2), and R848, a TLR 7/8 agonist.The ADPribosyltransferase PARP7 modulates protein function by conjugating ADP-ribose to the side chains of acceptor amino acids.PARP7 is expressed in various cells and can affect tumor growth through multiple mechanisms.For example, RBN2397mediated inhibition has been shown to induce both cancer cellautonomous effects and antitumor immunity via enhanced type I IFN signaling.It negatively regulates tank binding kinase 1 activity, which restrains phosphorylation and activation of the transcription factor IRF3, [19] inhibiting androgen-induced ADPribosylation of the androgen receptor in prostate cancer [34] and trapping PARP7 within the nucleus.MSA-2 is an orally available non-nucleotide STING agonist. [35]Activation of STING by cyclic dinucleotide (CDN) ligands in human monocytes induces a type I IFN response and production of pro-inflammatory cytokines associated with the induction of massive cell death. [36]We show that a CANDI encapsulated prodrug formulation using prodrug MSA-2p had similar effects in BMDM.Finally, R848 is a TLR7/8 agonist that activates the canonical NFkB pathway leading to IL12 secretion, [37] which then stimulates T-cells to produce IFNg. [38]s we based our screening on the compounds' ability to increase CXCL9 in IFNg-exposed macrophages, this latter mechanism is important for triggering IFNg in vivo and maximizing the full efficacy of the compounds identified.Despite the different mechanisms of action of the small-molecule modulators, the unifying theme was that they acted synergistically, presumably in part because they were delivered to TAM in an efficient manner.In prior work, it had been shown that up to 10% of injected dose/g tissue of CANDI partitions to tumors and where it is almost exclusively localized to myeloid cells. [14]These works collectively emphasize the therapeutic potential of multiple pathway targeting for TAM re-education in vivo.
Despite the design of the first efficient small-molecule myeloid CXCL9 inducer system in this study, there were some limitations.First, to identify actionable hits rapidly, we quickly focused on the triple combination.Additional formulations, admixtures, and compounds may have similar or even stronger effects, and therapeutics beyond small-molecules could activate CXCL9 phenotypes.Second, we started with a small library of compounds known to affect specific pathways.It is possible to perform unbiased screens of much larger libraries to identify new compounds with similar or even new mechanisms of action.Third, we show cellular uptake of CANDI400 in TAM, and prior work has quantitated tumor uptake. [14]We show that ≈70% of all CD11b positive cells in tumors contain CANDI400, whereas tumor cells and lymphoid cells do not.It is theoretically possible to further increase TAM uptake by targeting ligands on CANDI400, an area that awaits exploration.[41] Fourth, while we showed initial proof of concept, additional work will be needed to ascertain and compare the effectiveness of CANDI400 in different types of cancers and combination therapies.It remains to be investigated how TAMtargeted therapies influence tumor-specific T-cell responses and tumor control, and whether signals such as C/S ratio are key indicators of a tumor's immune "hot" or "cold" status.Despite these limitations, our initial results are extremely encouraging in developing a new class of myeloid cell-directed therapeutics that capitalize upon the polarization of TAM toward CXCL9 to drive antitumor immunity.
Particle Synthesis: The synthesis of CANDI was further developed from a previously reported method. [9]sbCD to produce smaller nanoparticles (17 nm vs 37 nm) are employed.sbCD de novo with a well-defined degree of substitution (DS) of 2.5 [14,42] addressing variability and high cost observed in commercially available products is synthesized.The resulting compound was then used to prepare CANDI nanoparticles, activated with EDC and NHS in MES buffer. [14]Briefly, L-lysine was added drop-wise, and the reaction was allowed to stir for 18 h.The particles were precipitated with ice-cold ethanol, purified, and characterized by DLS and Zeta potential before storage at −20 °C.
Characterization-Small-Molecule Loading of Nanoparticles: A solution of empty CANDI (CANDI E ; 5 mg) in PBS (0.5×, 90 μL) was used for payload loading to a final DMSO concentration of 10%.The following nanoparticle compounds were prepared: CANDI400 containing MSA-2p (0.26 mg), RBN2397 (0.1 mg), and R848 (0.22 mg).The solutions were vortexed rapidly until the complete dissolution of the drugs.All solutions were filtered through a 0.22 μm sterile filter (VWR) and used immediately for characterization, in vitro assays, or stored at −20 °C until further use (Table S1, Supporting Information).tive drug release was determined as the ratio of the integrated area under the curve for each eluted peak to the total area under the curve of chro-matographs obtained from the non-membrane controls.All experiments were performed in triplicates (N = 3).
Characterization-Transmission Electron Microscopy: CANDI400 particles were freshly prepared (50 mg mL −1 , PBS 1×) and diluted with water to a final concentration of 0.1 mg mL −1 .The particle solution was charged on a TEM grid for 1 min and treated with a 2% aqueous uranyl acetate solution for 15 min, followed by three washing steps with ultra-pure water (×3).Imaging was performed in a transmission electron microscope (JEOL 2100).
Characterization-Nanoparticle Tracking Analysis (NTA): A stock of pharmaceutically loaded CANDI400 was prepared in freshly filtered PBS (50 mg mL −1 ).Next, the total particle count, size distribution, and homogeneity of four dilutions (×62.5, ×125, ×250, ×500, N = 3) are estimated.The particle counts obtained from each concentration were multiplied by the dilution factor and averaged to obtain the total particle count depicted in Figure S6 (Supporting Information).All experiments and analyses were performed using a Panalytical NanoSight LN10 (Malvern) nanoparticle characterization system.All nanoparticle tracking analyses (NTA) were done with identical experiment settings.
In Vitro Experiments-Immortalized Cell Lines: The immortalized murine bone marrow-derived macrophages (iMACs) [45] were acquired from Charles L. Evavold (Ragon Institute, Harvard University) and used to assess toxicity (Table S4, Supporting Information).Briefly, iMAC cells were plated and grown in Dulbecco's Modified Eagle Medium (DMEM, Corning) supplemented with 10% Fetal Bovine Serum (FBS, Corning) and 1% Penicillin Streptomycin (Corning) at 37 °C and 5% CO 2 and MC38 cells were cultured in Iscove's Modification of DMEM (Corning).Upon reaching confluency, cells were split using 0.05% Trypsin / 0.53 mM EDTA (Corning), and all in vitro assays were performed after the cells reached 90% confluency.Prior to cell culture application, all CANDI preparations were filtered through a 0.22 μm sterile filter (VWR).
In Vitro Experiments-Bone Marrow-Derived Cells: Murine BMDC were isolated from CXCL9-RFP/CXCL10-BFP reporter mice, IL12-eYFP reporter mice, or wild-type C57BL/6J mice.BMDC of reporter mice were employed for flow cytometry and live-cell microscopy analyses, while BMDC of wildtype cells were utilized to evaluate cytokine induction.To obtain the whole bone marrow, femurs were prepared and flushed with sterile PBS using syringes and a 28-gauge needle.RBC Lysis Buffer (BioLegend) was then used according to the manufacturer's instructions to lyse red blood cells.The remaining cells were counted using a Neubauer chamber and seeded into either transparent (NEST, flow cytometry analysis) or black (ibidi, glass bottom for imaging) 96 well plates at a density of 1.25 × 10 5 cells per well.For cytokine assays, cells were seeded into transparent 6-well plates (Corning) at a density of 1 × 10 6 cells per well.BMDM were differentiated by adding 50 ng mL −1 recombinant murine M-CSF (BioLegend) to cell culture media for 7 days.New media was added every 3-4 days.
In Vitro Experiments-Cytokine Screen: To determine the effect of Nanoparticle drug loading on broader cytokine induction, wild-type C57BL/6J BMDM were seeded and stimulated with 50 ng mL −1 IFNg.Subsequently, CANDI400 formulation (5 μg mL −1 ) or empty control nanoparticles were added for 24 h.The conditioned media were then collected for cytokine array analysis.Cytokine array analysis was performed using the Proteome Profiler Mouse Cytokine Array Kit, Panel A (R&D, ARY006) according to the manufacturer's instructions.Images of the membranes were obtained (Azure Sapphire Biomolecular Imager) and quantified using ImageJ.
In Vitro Experiments-Toxicity: iMACs were seeded in 96 well plates at a density of 15 × 10 3 cells per well and incubated for 24 h at 37 °C and 5% CO 2 before use.Stock solutions of different CANDI nanoparticles (Table S1, Supporting Information) were prepared and then diluted in cell culture medium to desired concentrations (0.09 μg mL −1 to 10 mg mL −1 , DMSO 0.5%).Cells were incubated for 2.5 h with nanoparticles before the medium was exchanged.Cells were further incubated for 48 h at 37 °C and 5% CO 2 before adding MTT (3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl-2H-tetrazolium bromide) solution (5 g L −1 in FluoroBrite DMEM, 10% final) to each well.After 3 h, the supernatant was carefully removed, and metabolized formazan was dissolved with isopropyl alcohol.Plates were shaken at 500 rpm on a microplate shaker (VWR) for 30 min, and the ab-

Figure 1 .
Figure1.Overview of myeloid cell screening approach.A) TAM are bone marrow-derived, abundant in many cancers, and mostly immunosuppressive and thus pro-tumorigenic.B, C) This phenotype is driven primarily by IL10 and hypoxia (SPP1) signaling.We hypothesized that it should be possible to polarize macrophages to an antitumorigenic phenotype by increasing CXCL9 signaling.[4]Yet, no effective pharmaceutical strategies have emerged to do this effectively while retaining TAM specificity.D) In this research, we used BMDM from Rex3 reporter mice (expressing CXCL9-RFP) to screen for smallmolecules and combinations that could induce CXCL9 in myeloid cells.Top hits from the screen were then encapsulated into TAM-avid nanoparticles to elicit a CXCL9 phenotype in vivo.

Figure 2 .
Figure 2. Small-molecule compounds tested.A) Summary of the chemical structure of the 21 small-molecule compounds tested.The colored dots represent individual compounds for identification across figures.B) Different classes of compounds are considered according to the current understanding of CXCL9 regulation in macrophages.C) Summary of single compound screens, dual compound screens, and triple compound screens.The colors of the dots represent the molecular structures shown in panel A.

Figure 3 .
Figure 3. Screening results.Three separate screens were performed, each one informing the design of the next screen.A) Screen 1 was performed without IFNg stimulation of BMDM and explored CXCL9 TAM expression after single or dual agent exposure, as shown.Note the lack of efficacy of any of the compounds, indicating that baseline levels of IFNg are likely required for pharmacological CXCL9 induction.Screen 2 repeated the same screen but with baseline stimulation of IFNg.Note that some of the dual combinations yielded elevated levels of CXCL9.Screen 3 largely explored triple drug combinations with IFNg stimulation.Note the highest CXCL9 expression of combinations involving RBN2397, CRX527, R848, and/or MSA-2.In parallel experiments, drug toxicity was determined.Drugs or combinations with a limited therapeutic window are shown in light brown.Given these results, the top hit emerging from the screen was the triple combination of RBN2397+MSA-2+R848 which was then formulated into the CANDI400 formulation shown in subsequent figures.B. Representative images (550 nm channel, RFP) obtained from screens 2 (dual compound R848+RBN2397) and screen 3 (Bottom triple compound RBN2397+MSA-2+R848).The black inserts represent the negative controls without drugs and IFNg stimulation alone.Scale bar 200 μm.See Figure S7 (Supporting Information) for other compounds and cytokines.

Figure 4 .
Figure 4. Formulation of small-molecule hits into TAM-avid nanoparticle (CANDI400). A. Synthetic strategy to form ≈17 nm nanoparticles consisting of sbCD cross-linked by L-lysine linkers via EDC/NHS chemistry.B. Transmission electron microscopy images revealed that the nanoparticles retained a spherical form and were able to carry the proposed three small-molecule payloads.C. Synthesis of MSA-2p from MSA-2.The left bottom graph indicates the percent of free/bound drug to sbCD, determined as turbidity value (measured at 550 nm, average of 500 s time-lapse, N = 3).Bulk studies using MSA-2p in buffer (pH 7.4) showed that complexation to sbCD leads to a significant reduction in the hydrolysis rates of MSA-2p (right).D. Turbidity assay to measure loading and stability of the CANDI400 formulation assessed by an increase in absorbance at 550 nm.We determined the loading capacity for CANDI400 to be ≈0.15mg of payloads per mg of CANDI nanoparticle.E. Release kinetics of the triple small-molecule combo from CANDI400 using a porous membrane (3 kDa) in PBS (1×) at 37 °C.The release rates for R848, MSA-2, and RBN2397 are depicted as the dissociation rates (k off ) and complex half-life (t 1/2 ), respectively determined in a 7 h time lapse (see FigureS6, Supporting Information, N = 3).

Figure 5 .
Figure 5. Cellular properties of CANDI400.A. BMDM obtained from Rex3 reporter mice were incubated with CANDI400 for 24 h and then observed by microscopy.Cellular nuclei were stained with SYTO™ 11 Green (green, 473 nm channel), CANDI400 was revealed by labeling the NP with AF647 (white, 633 nm channel).CXCL9 (red) and CXCL10 (cyan) were imaged by endogenous fluorescent protein expression (RFP, 559 nm channel and BFP, 405 nm channel respectively).Note the high CXCL9 levels in all cells containing CANDI400.Scale bar: 20 μm.B. Comparative measurements of key cytokines and TAM activation makers using flow cytometry.Note the high CXCL9 expression with CANDI400, even higher than with IFNg and LPS stimulation of cells.C. Dose-response curve with increasing concentration of CANDI400, revealing EC 50 values of 3.4 ng mL −1 for CXCL9 and 3.2 ng mL −1 for CXCL10, respectively.D. Cellular toxicity using the MTT assay.Note the lack of toxicity at drug concentrations <0.1 mg mL −1 .C and D were used to estimate drug concentrations for in vivo experiments (green-shaded area).

Figure 6 .
Figure 6.Intravital microscopy of CXCL9 induction in a tumor mouse model.A. Serial imaging of the TME in the colorectal MC38-H2B-GFP tumor model.Eight days after tumor implantation, CANDI400 was administered by tail vein, and serial repeat imaging was performed.CANDI400 was labeled with AF647 and contained the triple small-molecule cocktail (RBN2397, MSA-2p, and R848) to induce CXCL9 in myeloid cells in the TME.The top row (scale bars 50 μm)shows MC38-H2B-GFP tumor cells (green, 488 nm channel), the middle row CANDI400 AF647 (white, 647 nm channel), and the lower row CXCL9-RFP (red, 550 nm channel).Immediately after intravenous injection of CANDI400, the nanotherapeutic drug is largely confined to vessels.Within several hours, the CANDI is later taken up by TAM.Note the high CXCL9 induction within 24 h after systemic CANDI400 administration.See FigureS8(Supporting Information) for high-resolution images.