Antisense targeting of FOXP3+ Tregs to boost anti-tumor immunity

Research tools used in this peer-reviewed study.

AUMsilence™ sdASO™ (self-delivering antisense oligonucleotide)

Experimental methodology

The methodology across the two publications removes the usual barriers to primary cell gene silencing. In the FOXP3 study, healthy donor PBMCs and clinical cancer samples were cultured in DMEM with 3% FBS, with ammonium chloride and arsenic added to enhance gymnosis, and AUMsilence FOXP3 sdASO was added directly at 1.5-2.5 μM, with no lipofection reagents, no electroporation, no viral transduction. Cells were stimulated with CD3/CD28 microbeads at 0.3 beads per cell and incubated for 5 days. The microscopy study followed the uptake: fluorescent-labeled AUMsilence sdASO reached approximately 80% of primary murine splenocytes within 15 minutes and 100% of both cancer cell lines at the same time point, with a diffuse signal in nucleus and cytoplasm. Cell preparation used a simplified approach: rather than cytospin centrifugation, whose centrifugal force the paper says may disrupt cytoskeletal structures or mask subtle morphological changes in activated lymphocytes, researchers used heat-dried smears on Superfrost Plus slides. Both preparations preserved cellular morphology and yielded comparable cell numbers; what the smears showed and the cytospins did not was a polarized accumulation of β-actin at one pole of activated cells. For adherent cells, a parafilm chamber punched and heat-sealed onto a microscope slide replaced a commercial chamber slide. Validation included flow cytometry (measuring Treg numbers, FOXP3 protein levels, exhaustion markers), qPCR (FOXP3 mRNA, down 54.9% in PBMC and 64.7% in cancer samples), functional assays (Treg suppression measured by CFSE dilution, falling to 66.4% of Scramble), and microscopy (visualizing ASO uptake dynamics and intracellular distribution). The workflow: add ASO to medium, wait, harvest, analyze, with no transfection optimization and no expensive equipment. The screening criterion the authors applied was no more than a 10% decrease in cell viability against the Scramble control, and no more than 20% inhibition of CD4+ and CD8+ T cell division.

Results achieved

The approach produced results in every group the paper reports, though not evenly: the three rejected transplant lungs, used as a control, downregulated their Treg numbers but were less sensitive than the distant tumor-free lung and the other cancer samples. In healthy donor PBMC, FOXP3 mRNA decreased by 54.9% and Treg numbers fell by 41.0%, with no effects on cell viability or cell division. Cancer samples were more sensitive: 64.7% FOXP3 mRNA reduction and 60.0% Treg depletion, all p < 0.0001, and the paper concludes from this that intratumoral Tregs were more sensitive to ASO FOXP3 than peripheral blood Tregs. Isolated Tregs incubated with AUMsilence FOXP3 sdASO for 3.5 hours suppressed at 66.4% of the Scramble control when tested against autologous or allogeneic responders, a loss of about a third. The functional consequences extended beyond Treg numbers: the leftover Tregs that were still FOXP3+ after 5 days carried less FOXP3 protein per cell, more so in cancer samples than in PBMC samples, and downregulated the Treg-associated marker CD39. In the cancer samples, mRNA expression of five of the nine tested exhaustion markers fell significantly (CTLA-4, Tim-3, PD-1, LAG-3 and TIGIT), and mRNA expression of 7 of 11 inflammatory cytokines rose, those seven being IL-2, IL-6, IL-7, IL-10, IL-18, TNF-α and Perforin-1. In vivo, in TC1 and MC38 tumor-bearing mice, tumor growth was significantly inhibited in both models, against Scramble at p = 0.0007 for TC1 and p = 0.0040 for MC38, and 22% of TC1 tumors and 13.6% of MC38 tumors were completely resorbed. Intratumoral FOXP3 mRNA decreased ~50%. In draining lymph nodes, FOXP3 mRNA and Treg numbers were similar between treated and control mice. In spleens, FOXP3 mRNA did not change but Treg numbers rose, significantly so on Figure 8C. So the targeting is selective, and it does not deplete Tregs systemically. The paper is inconsistent on this point, its abstract saying no change in splenic Treg numbers where its results section and Figure 8 both report an increase; the results section is the reading this page follows. The microscopy study quantified cellular uptake kinetics, reporting that AUMsilence sdASO reached approximately 80% of primary cells and 100% of both cancer cell lines within 15 minutes, with intracellular presence sustained for at least 180 minutes. All of it without a transfection reagent, an electroporator or a viral vector.

Quote from paper

Page reference

Akimova et al., Frontiers in Immunology 2024, Page 1 (Abstract); Bartosh et al., Cells 2025, Figures 5-9

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What each figure measured, and which of the two papers it is from.

Figure 2G-I (FOXP3 paper): Cancer samples show enhanced sensitivity to AUMsilence™ FOXP3 sdASO™

Flow cytometry analysis from 5 experiments encompassing 5 healthy donor PBMCs, 4 tumor samples (3 lung cancer + 1 mesothelioma), 4 tumor-free lung tissue, 3 pleural effusions, 3 draining lymph nodes, and 4 cancer patient PBMCs, all treated with 1.5 μM ASO 16 for 5 days. Cells were stimulated with CD3/CD28 microbeads at 0.3 beads per cell. FOXP3 expression measured in viable CD45+ cells and CD4+ T cells, with data normalized as relative percent change: 100×(ASO result - Scramble)/Scramble. Statistical analysis used one-sample t-tests (mean=0) for PBMC samples and Wilcoxon rank test (median=0) for cancer samples, plus one-way ANOVA with Tukey's multiple comparisons across sample types. Panel H shows representative flow cytometry plots of FOXP3+ expression in CD4+ cells, four of them.

Figure 3E-F (FOXP3 paper): ASO FOXP3 treatment lowers exhaustion markers in human cancer samples

Two panels, measuring different things in different sample sets. Figure 3E is mRNA: the cancer samples of Figure 2E, treated with 1.5 μM of Scramble or ASO FOXP3 for 5 days, evaluated by TaqMan qPCR for the expression of exhaustion markers. Five of the nine markers tested were significantly downregulated, with the p values printed on the panel: CTLA-4 p = 0.0034, Tim-3 p = 0.0001, PD-1 p < 0.0001, LAG-3 p = 0.0048 and TIGIT p = 0.0284. Figure 3F is protein: the samples of Figures 2G-I, supplemented by 3 transplant lungs and 6 Treg-depleted samples, quantified by multi-parameter flow cytometry across CD4+ T cells, CD8+ T cells and CD4-CD8- non-T cells, with data calculated as percentage points, that is expression in the ASO 16 treated sample minus expression in Scramble. The six Treg-depleted samples showed almost no changes in their expression of exhaustion markers, and slightly increased expression for some of them, which the authors take to suggest that the downregulation follows from the effect of ASO FOXP3 on Tregs rather than from a direct effect on the other cells. Analysis of co-expression showed that the combination of BTLA and CD160, with or without LAG-3, gave the maximum downregulation in cancer samples.

Figure 4E-F (FOXP3 paper): ASO FOXP3 alone inhibits tumor growth, with complete responses in some mice

In vivo efficacy in two syngeneic mouse tumor models in wild-type C57BL/6 mice: TC1 adenocarcinoma (N=165 mice across 7 independent experiments) and MC38 colon adenocarcinoma (N=65 mice across 3 experiments). Mice received subcutaneous tumor injection (1.2×10⁶ cells), then were randomized on day 7 to PBS, Scramble control, or murine ASO 6B (50 mg/kg i.p. daily for 14-16 days). Tumor volumes measured every 2-3 days with the formula (3.14 × long axis × short axis × short axis)/6. Data from different experiments combined using 0-100 normalization within each experiment to account for inter-experimental variability. Area under the curve analysis compared cumulative tumor burden. Kruskal-Wallis test with Dunn's multiple comparisons showed significant separation of ASO-treated groups from controls. Non-normalized data from one TC1 and one MC38 experiment are shown in Supplementary Figures 4A, B.

Figures 6-9 (microscopy paper): Time-series visualization of AUMsilence™ sdASO™ cellular uptake across cell types

Time-series microscopy analysis of far-red fluorescent-labeled AUMsilence Scramble sdASO uptake in primary murine splenocytes and two adherent cancer cell lines (A549 lung adenocarcinoma, and HEp-2, which the paper notes was long thought to come from a laryngeal carcinoma but is a HeLa derivative). Cells pre-stained with FITC-anti-CD45 (splenocytes) or CFSE (cell lines) to visualize cell boundaries, then incubated with 1.5 μM ASO in DMEM + 3% FBS + 3 mM NH₄Cl + 1 μM arsenic (gymnosis enhancers). The two preparations are different protocols and are reported separately in the paper. For the splenocytes in suspension, aliquots were withdrawn after 15, 30, 60, 90 and 120 minutes, smeared on Superfrost Plus slides, heat-dried (55-60°C, 20 min), fixed with IC Fixation Buffer, and mounted with DAPI. The two adherent lines were grown in the parafilm wells and run as a series from 15 to 180 minutes, with the longest incubation started first so that every well finished at the same moment, then rinsed, dried, fixed and stained on the slide. Quantitative analysis assessed at least 100 cells per timepoint across 3-4 microscopic fields, scoring no ASO uptake, a few discrete ASO spots, diffuse uptake at low or high intensity, diffuse uptake with small high-intensity spots, and diffuse uptake with relatively large granules. Cell lines showed differential patterns: A549 cells accumulated small ASO granules at the cytoplasmic periphery as early as 15 min, and at 30 and 60 min the majority showed a nearly uniform bright nuclear signal, which remained the predominant pattern during the first 120 min. HEp-2 cells instead showed a diffuse low-intensity signal in nucleus and cytoplasm at all times up to 180 min, with bright nuclear staining rising from 14% at 90 min to 39% at 150 min, and accumulated ASO in the cytoplasm as large granules, especially at 15-120 min. Partial nuclear exclusion of ASO was uncommon in both lines and the paper quantifies it: in A549 it peaked at 5.6% of cells at 60 min, fell to 3% at 150 min and was no longer detectable by 180 min; in HEp-2 it was 1.5% at 60 min, rose to 4% between 90 and 150 min and fell to 1.8% at 180 min. Granule counts in the two lines were compared by two-sided Fisher's exact test at each of the seven time points, with Bonferroni correction (Figure 9C).

Figure 3 (Microscopy paper): Heat-dried cell smears show a polarized β-actin pattern that cytospin does not

Direct methodological comparison of cytospin versus cell smear preparation for microscopy analysis of murine splenocytes cultured ±PMA/ionomycin stimulation for 3 hours. Same cell populations split equally and processed via: (1) standard cytospin (1000 RPM, 5 min, 0.25×10⁶ cells in DPBS+3% FBS), or (2) heat-dried smears (10 μL suspension, smeared with pipette tip, dried at 55-60°C for 20 min). Both processed identically thereafter: fixation, permeabilization, blocking, overnight incubation with anti-β-actin or anti-histone H3 antibodies, secondary antibody staining, nuclear counterstaining with propidium iodide, mounting. Both methods preserved cellular morphology and yielded comparable cell numbers. What differed was that activated lymphocytes in the smear preparations showed a pronounced accumulation of β-actin staining at one pole of the cell (arrowed in Figure 3), which the paper reads as potentially reflecting cytoskeletal reorganization in response to stimulation. That polarized staining pattern was not observed in cytospin-prepared cells from identical samples, which the paper takes to suggest that the centrifugal force used during cytospin may disrupt cytoskeletal structures or mask subtle morphological changes. Additionally, cytospin preparation of stimulated cells showed histone H3 staining artifacts with apparent “leakage” of the signal beyond the cellular boundaries (Figure 4, arrows), consistent with the authors' earlier observation that the membranes of some stimulated lymphocytes become fragile and more susceptible to mechanical stress. Those artifacts were not observed in the smears.

Figure 1 (microscopy paper) and Figure 2C-D (FOXP3 paper): a parafilm chamber for multi-well microscopy on one slide

Novel chamber slide system created using single-hole punch (6 mm diameter) applied to parafilm strips cut to microscope slide dimensions, then heat-sealed to Superfrost Plus slides (30-60 seconds on hot plate until parafilm melts). Creates ~30 μL working volume wells for adherent cell culture directly on slides. Validated with A549 and HEp-2 cells seeded at 2000-3000 cells/well, cultured 24h to 70-80% confluency, then treated with 1.5 μM far-red AUMsilence sdASO in time-series (15-180 min). For extended cultures (>24h), slides placed in Petri dishes with buffer reservoir to prevent evaporation (Figure 1D-E). After treatment, the ASO was removed by a short rinse in DPBS, the slides were dried on the hot plate, the parafilm was removed with forceps leaving a hydrophobic boundary (Figure 1F), and the cells were fixed, stained with DAPI and mounted. The paper states that the system is much less expensive than commercially available small-well chambers, and that a single-hole punch of a different diameter changes the well volume as needed. The FOXP3 study's Treg suppression assay (Figure 2C-D) is a separate method and used no slides: isolated Tregs were incubated with 2.5 μM ASO for 3.5h, washed twice, then co-cultured with CFSE-labeled responder PBMCs at serial dilutions (1:1 to 1:16 Treg:PBMC ratio) for 5-6 days with CD3 microbeads, with suppressive function determined as the area under the curve.

1. 01Tatiana Akimova, Liqing Wang, Zhanna Bartosh, Lanette M. Christensen, Evgeniy Eruslanov, Sunil Singhal, Veenu Aishwarya, Wayne W. Hancock. (2024). Antisense targeting of FOXP3+ Tregs to boost anti-tumor immunity. Frontiers in Immunology. DOI: 10.3389/fimmu.2024.1426657
2. 02Bartosh et al. New Approaches to Old Techniques in Cell Handling for Microscopy. Cells 2025;14:1271.