Fig 1: Receiver operating characteristic (ROC) curves demonstrating the incremental utility of incorporating CXCL9 into prognostic models for the prediction of osteoporotic hip fracture in men (A) and women (B). Model 1: Logistic regression model incorporating age at blood draw (years, continuous), dialect group (Hokkien, Cantonese). Model 2: All factors in model 1 + BMI (kg/m2, continuous), history of smoking (never, ever), history of diabetes mellitus (yes, no). Model 3: All factors in model 2 + CXCL9 [log(pg/mL), continuous].
Fig 2: Cellular effects in macrophages(A) Cellular uptake of CANDI470-AF647 into iMACs. 100% of all cells internalize the CANDI at concentrations of 1 mg/well (incubation time was 2.5 h).(B) Dose response for eliciting Cxcl9 in bone marrow-derived macrophages (BMDMs). BMDMs were obtained from Cxcl9-RFP donor mice and incubated with varying amounts of CANDI470 for 24 h, after which RFP fluorescence was measured.(C–F) Dose response for downregulating Spp1 in BMDMs. BMDMs were obtained from Spp1-tdTomato donor mice and incubated with varying amounts of CANDI470 for 24 h after which tdTomato fluorescence was measured. Cellular toxicity of empty nanoparticle (D), CANDI470 (E), and free drugs (F) following a 2.5 h incubation with cells. The free drug dose refers to the payload-equivalent dose within CANDI470. Note that the empty CANDI has no appreciable toxicity at the concentrations tested. Cellular toxicity at high concentrations is primarily due to small-molecule payloads and is overall lower upon CANDI470 encapsulation. Results are represented as mean ± standard error of the mean (SEM).
Fig 3: Tumor-associated macrophage polarization in PDAC is characterized by a distinctly low CSR compared to other solid tumors(A) Publicly available single-cell RNA sequencing datasets from 87 PDAC tumors across seven independent cohorts (totaling 96,717 cells) were analyzed. Uniform manifold approximation and projection (UMAP) visualization of all cell types in the data.(B) Macrophages were subset from the integrated dataset, and their relative expression of CXCL9 and SPP1 was used to compute a CXCL9/SPP1 ratio (CSR) for each cell. UMAP visualization of all macrophages pooled across patients, colored by CSR.(C) Dot plot showing the mean macrophage CSR for each patient, ordered from lowest to highest.(D) Violin plots showing the distribution of CSR across 11 tumor types on a log scale. Median values are displayed above each plot. Data were derived from bulk RNA-seq analysis of 5,601 patients across 11 tumor types (The Cancer Genome Atlas [TCGA]). Fib, fibroblasts; Mo, monocytes; Neu, neutrophils; Mac, macrophages; EC, endothelial cells; DC, dendritic cells; pDC, plasmacytoid dendritic cells; PC, plasmocytes; MC, mastocytes; NK, natural killer cells; TuEpi, tumor cells; Esoph, esophagus; HNC, head and neck.(E) To address CSR in TAMs, we developed a TAM-targeted nanoformulation containing a triple small-molecule payload to modulate CSR polarization. The triple payload consisted of a TLR7/8 agonist (R848), a CSF1R inhibitor (PLX3397), and a STING agonist prodrug (MSA-2p).
Fig 4: Effect of CANDI470 on BMDMs(A) BMDMs were obtained from BL6 mice and treated with CANDI470 or CANDIE as control (1 mg/mL; 2 h). Note the upregulation of immunostimulatory genes, including Cxcl9, Cxcl10, Ccl5, Marco, Cd40, and IL12, among others. Downregulation of Trem2, Cd163, and Slc46a3, among others.(B) Similar to experiment in (A), but with an empty CANDI. Note that the CANDI particle by itself has no therapeutic efficacy.(C) List of top 20 upregulated IFNg response genes with CANDI470 treatment ranked by fold change. Control is PBS.(D) List of 20 downregulated genes with CANDI470 treatment. Many genes are related to M2 and Spp1 polarization. Control is PBS.
Fig 5: Pharmacodynamics of CANDI470 effects in vivoSerial intravital imaging (before, immediately after i.v. administration, and at 24 and 48 h) of CANDI470 in KPC-YFP tumor-bearing mice (either expressing Cxcl9-RFP or Spp1-Tdtomato).(A) Combined colors from (B) and (C) show KPC-YFP tumor cells (green), AF647-labeled CANDI470 (white), and Cxcl9-expressing cells (red).(B) Fluorescently labeled CANDI was initially observed in the tumor neovasculature but was completely cell associated at 24 and 48 h (see Figure 5 for cell types).(C) Using Cxcl9-RFP reporter mouse, massive Cxcl9 upregulation was observed in the tumor by 48 h post-injection; most Cxcl9+ cells were also positive for CANDI uptake.(D) Via Spp1-Tdtomato reporter mouse, Spp1 downregulation was also detected at a later time point. Scale bar, 200 μm. Results are represented as mean ± standard error of the mean (SEM). p values from one-way ANOVA are shown. p < 0.05 was considered significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001. n.s. denotes not significant.
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