Bioengineered iPSC-derived human macrophages with increased angiotensin-converting enzyme (ACE) expression suppress solid tumor growth

Figures

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  Fig. 1. Generation of iPSC-derived macrophages (iMac). a Plasmid construct used to create a doxycycline (Dox)-inducible ACE expressing iPSC line (ACE-iPSC). b ACE expression measured by Western blot 48 hours after treatment with the specified Dox concentrations. c Overview of the differentiation process from iPSC to mature macrophage, with microscopic images showing an embryoid body (EB) at day 7, myeloid progenitors at day 10, and mature macrophages at day 15. d Characterization of myeloid progenitors and iMac during iPSC differentiation using flow cytometry. FACS dot plots display populations of Common myeloid progenitor (CMP), Granulocyte-monocyte progenitor (GMP), and Monocyte-dendritic cell progenitor (MDP) at day 10, and mature iMac at day 15. e ACE expression measured by Western blotting. f ACE expression in iMac measured using flow cytometry. g ACE catalytic activity in iMac measured using a substrate-based colorimetry assay. For e–g cells were treated with Dox for 48 h before analysis. Flow cytometry data are presented as mean fluorescence intensity (MFI). Comparisons were analyzed using a two-sided unpaired Student t-test, with data presented as means ± SEM. ****p < 0.0001

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  Fig. 2. Determination of M1 vs. M2 polarization in iMac. a Pro-inflammatory M1 cytokines IL-1β, TNFα, IL-6, and IL-8 measured by ELISA. b Anti-inflammatory M2 cytokines IL-4, IL-10, IL-13, CCL5, CCL17, and CCL20 measured by ELISA. For a, b iMac were cultured in 6-well plates (3 × 106 cells/well). To challenge with tumor factors, iMac were co-cultured with 50% fresh supernatant from SK-MEL-28 cells every 24 h for 3 days. Supernatants were collected during the final 24 h for cytokine measurement. c Activation (phosphorylation) of NF-kB p65, STAT1, STAT3, and STAT6 in iMac was determined using Western blotting after conditioning with melanoma (SK-MEL-28) supernatant for 72 h. Protein band intensity was quantified using Odyssey version 3.0 software. After adjusting for loading using the β-actin band, data are presented as fold change to the iMac (Dox-) group, which is set as 1. For a–c, one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons. Data are presented as means ± SD. Each experiment was performed three times, with triplicate samples each time. *p < 0.05, ***p < 0.001, and ****p < 0.0001

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  Fig. 3. Determination of anti-tumor activity of iMac with induced ACE expression, in vitro. a Proliferation of melanoma (SK-MEL-28), Triple Negative Breast Cancer (TNBC: HCC1806), and FP chemotherapy resistant HNSCC (FaDu/FP-R) cell lines cultured alone or indirectly co-cultured with iMac ( ± Dox) using 3.0 µm pore polyester membrane inserts for 5 days. Total and dead cell counts were determined via trypan blue exclusion on days 1, 3, and 5. b, c Quantification of iNOS and interleukin-12 (IL-12) production in Cnt-iMac and ACE-iMac treated with LPS (500 ng/ml, 12 h), ± Dox. d ROS production in ACE-iMac following LPS stimulation (1 µg/ml, 30 min) ±Dox, assessed by DCFDA (2’,7’-dichlorodihydrofluorescein diacetate) staining. Representative histograms (left) and mean fluorescence intensity (MFI) quantification (right) are shown. Nitric Oxide (NO) production in ACE-iMac stimulated with LPS (75 ng/ml) for 24 h (e), and/or conditioned with SK-MEL-28 supernatant for 24 h (f) ± Dox. g Perforin and IFN-γ levels in human peripheral blood NK and Tc cells, respectively, after co-culture with Cnt-iMac or ACE-iMac pretreated with LPS (24 h), followed by melanoma conditioning (24 h). To induce ACE…

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  Fig. 4. The effect of ACE-expressing iMac treatment on tumor growth in immunocompromised nude mice. To induce ACE expression, myeloid progenitors were differentiated in the presence of 1 µg/ml Dox (termed as ACE-iMac), while control iMac with basal ACE expression were differentiated in the absence of Dox. a Experimental scheme of iMac treatment. Mice were injected intratumorally with PBS, Cnt-iMac (±Dox) or ACE-iMac (±Dox) weekly (3–4 doses) as indicated by arrow. b Growth of melanoma xenografts derived from the SK-MEL-28 cell line. c Growth of breast cancer xenografts derived from the HCC1806 cell line. d Growth of HNSCC xenografts derived from the FaDu/FP-R cell line. For b–d Left panels show plots comparing tumor growth rates, the middle panels show representative images of tumors, and right panels show graphs comparing tumor weight. Following cell administration, Dox+ (ACE-iMac) mouse groups were provided with 150 µg/ml doxycycline in their drinking water throughout the duration of the study. Group comparisons were analyzed using one-way ANOVA with Bonferroni’s correction for multiple comparisons. Data are presented as means ± SEM (n = 5–10). *p < 0.05, **p < 0.01, ***p < 0.001, and…

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  Fig. 5. Analysis of anti-tumor macrophage activation in the TME post-iMac treatment. Tumors were harvested and single-cell suspensions were prepared for flow cytometry analysis as detailed in the methods section (refer to Fig. 4). a Gating strategy for distinguishing M1 and M2 macrophages using flow cytometry based on surface marker immunostaining. b Assessment of CD86+ (M1) and CD206+ (M2) expression in tumor associated macrophages (TAMs) in SK-MEL-28-derived xenografts (±Dox). The bar plot presents FACS quantification of M1 and M2 populations (n = 5/group). c Assessment of arginase expression in TAMs. d ACE expression in TAMs. e–g Measurement of proinflammatory and anti-tumor markers IFN-γ, IL-12, and iNOS (left: representative histogram; right: mean fluorescence intensity) in TAMs in melanoma xenografts (n = 5/group). Statistical analysis was performed using a two-sided unpaired Student’s t-test for comparisons in (b, d, g), and one-way ANOVA with Bonferroni’s correction for multiple comparisons in (c, e, f). Data are presented as means ± SEM. *p < 0.05 and ****p < 0.0001

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  Fig. 6. Analysis of NK cell and B cell activation in the TME post-iMac treatment. Tumors were harvested, and single-cell suspensions were prepared for flow cytometry analysis. a Representative gating strategy for identifying activated NK cells, B cells, and neutrophils in tumor xenografts using flow cytometry. b Percentage of activated NK cells (CD122+CD107a+) in melanoma, breast cancer, and HNSCC xenografts (n = 5/group). c Measurement of IFN-γ-positive NK cells in Cnt-iMac and ACE-iMac melanoma tumors (±Dox) (n = 5/group) in SK-MEL-28-derived xenografts. d Percentage of activated B cells (CD19+CD21/CD35+) in melanoma, breast cancer, and HNSCC xenografts (n = 5/group). Statistical analysis was performed using a one-way ANOVA with Bonferroni’s correction for multiple comparisons in (b–d). Data are presented as means ± SEM. *p < 0.05 and ****p < 0.0001

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  Fig. 7. The effect of ACE-iMac treatment on human immune cells in a humanized mouse tumor model. a Growth of SK-MEL-28 tumors in humanized BLT-NSG mice; comparing tumor volumes between PBS, iMac and ACE-iMac treated groups. b Representative images of tumors are shown (see Supplementary Fig. 6a for additional images). c Final tumor volumes on Day 28 post-implantation. d Tumor weight on Day 28 post-implantation. Flow cytometry analysis of anti-tumor activation of human CD8 T cells in the TME, measured by intracellular IFN-γ (e) and perforin (f) in tumor-infiltrating CD3+CD8+ T cells at sacrifice on Day 28 post-implantation. g Flow cytometry analysis of anti-tumor activation of NK cells, measured by intracellular IFN-γ in tumor-infiltrating CD45+CD56+ NK cells in the TME on Day 28. The flow cytometry gating strategy for identifying human immune cells is provided in Supplementary Fig. 6b. One-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons. Data are presented as means ± SEM (n = 9–10). *p < 0.05, **p < 0.01, and ****p < 0.0001

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  Fig. 8. Transcriptomic and pathway analysis of ACE-iMac under basal conditions. ACE-iPSCs were differentiated into macrophages (iMac) with or without 1 µg/ml doxycycline (Dox) treatment, as described in the “Methods”. a Volcano plots showing significantly altered mRNA expression in iMac. Red dots indicate upregulated genes. Differential expression was defined by p < 0.01 and fold change > 5. b Ingenuity Pathway Analysis (IPA) of differentially expressed genes. Upregulated and downregulated clusters were identified, classified, and ranked according to z-scores of canonical pathway enrichment. c Bubble charts depicting significant canonical pathways based on −log (p-values). Broad categories are shown on the y-axis, with more specific subcategories indicated within bubbles. Bubble color denotes pathway activation status: red (z-score > 0, activated) or blue (z-score < 0, inhibited). d Heatmap of gene expression highlighting the macrophage classical activation signaling pathway. e Expression levels of key RNAs within the macrophage classical activation signaling gene cluster. f GO analysis of differentially expressed genes. Upregulated (red bars) and downregulated (blue bars) clusters were…

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  Fig. 9. Transcriptomic profiling and pathway analysis of ACE-iMac following tumor-stimulation. ACE-iPSCs were differentiated into iMac ± 1 µg/ml Dox. iMac were then stimulated with SK-MEL-28 conditioned medium for 12 h prior to RNA extraction and sequencing. a Volcano plot showing significantly altered mRNA expression in ACE-iMac versus iMac. b IPA of differentially expressed genes. c Bubble charts illustrating significant canonical pathways. d Heatmap of gene expression highlighting the macrophage classical activation signaling pathway. e Expression levels of key RNAs within the macrophage classical activation signaling gene cluster. f GO analysis of differentially expressed genes, with upregulated (red bars) and downregulated (blue bars) clusters ranked by pathway enrichment. g Heatmap showing differential expression of genes involved in primary metabolic processes. h Expression levels of representative RNAs from metabolic gene clusters. Statistical analyses were performed using a two-sided unpaired Student’s t-test in (a, e, h). Data are presented as means ± SEM. ****p < 0.0001. For details, refer to Fig. 8