(T1030-06-37) Engineering Tumor and HEK Cells to Produce Multi-Targeted Exosomes: A New Frontier in Targeted Checkpoint Therapy

Purpose: Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by high recurrence rates and the development of multidrug resistance, particularly to doxorubicin (DOX). Effective treatment strategies remain limited. In this study, we explored a novel two-pronged exosome platform: (1) Trop2-targeted cancer-derived exosomes functioning as vaccine-like agents, and (2) HEK293T-derived PD-L1-targeted exosomes loaded with paclitaxel and STAT3 siRNA for immune checkpoint blockade.

Methods: MDA-MB-231 DOX RT cells were transfected with a pDisplay vector encoding a Trop2 nanobody sequence. Exosomes were isolated via ultracentrifugation and characterized by nanoparticle tracking analysis (NTA), western blotting. Their efficacy was evaluated against LTLT-Ca (Letrozole resistant) cells. Parallelly a sequence-optimized anti-PD-L1 scFv (241 amino acids) was engineered based on the heavy and light chain sequences of Atezolizumab, cloned into a recombinant pDisplay plasmid (ThermoFisher Scientific) and used to stably transfect HEK293T cells. Stable clones expressing anti-PD-L1 were selected using geneticin and confirmed via western blotting with a Myc-tag. These HEK293T-derived exosomes (PDL-E) were isolated by ultracentrifugation, characterized by NTA, fluorescent NTA, flow cytometry, and flow nanoanalyzer (NanoFCM) to confirm size, purity, and surface anti-PD-L1 expression. STAT3 siRNA was loaded into PDL-E by electroporation to generate PDL-ESi, and loading efficiency was quantified using a QuickDrop spectrophotometer. Cytotoxicity assays were conducted on TNBC cells. Molecular effects were assessed via western blotting and RT-PCR. In vivo efficacy studies were conducted in BALB/c nude mice bearing subcutaneous MDA-MB-231 DOX RT xenografts, with intraperitoneal treatments administered every two days for two weeks. Tumor volume and molecular marker changes were evaluated.

Results: Western blotting confirmed the successful transfection of Trop2 in MDA-MB-231 DOX RT cells (Figure 1A). NB60 exosomes (Figure 1B) derived from Trop2-transfected MDA-MB-231 DOX RT cells exhibited a mean particle size of 103.5 nm, a concentration of 6.2 × 10¹¹ particles/mL, and a zeta potential of –36.47 ± 0.98 mV (Figure 1D). Early functional studies showed NB60 exosomes led to a promising 9.8–13.9% reduction in LTLT-Ca cell viability at 48 hours, suggesting the potential of Trop2-expressing exosomes to initiate anti-tumor responses (Figure 1E). Further investigations into their direct immunogenic and cytotoxic capabilities are ongoing, offering exciting prospects for vaccine-like therapies against resistant TNBC. PDL-E exhibited a mean particle size of 112.5 ± 3.5 nm, a particle concentration of 5.1E+10 particles/mL, and a zeta potential of –32.22 ± 2.30 mV, with 81.25% purity confirmed by fluorescent NTA (Figure 2(I)). PDL-E induced a 40% (p < 0.001) PD-L1 knockdown in vitro and in vivo in breast cancer tumors, 50% (p < 0.001) knockdown in Pancreatic (Mia PaCa-2) cells, and a 70% (p < 0.0001) knockdown in lung (H1975) tumors (Figure 2(II)). Loading with STAT3 siRNA and co-treatment with paclitaxel reduced the IC50 of paclitaxel by 4-fold (2D cultures: 31.44 ± 0.56 nM to 8.14 ± 0.87 nM) and 8-fold (3D spheroids: 102.76 ± 6.4 nM to 13.52 ± 0.45 nM) against MDA-MB-231 DOX RT and MCJB 2 (PDX) cells (Figure 2(III)). In vivo, the combination group (PDL-ESi + paclitaxel) demonstrated a 3-fold reduction in tumor volume (Figure 3(III)) compared to controls (p < 0.001). Western blot analyses of tumor lysates revealed substantial downregulation of PD-L1 (p < 0.001), PD-1 (p < 0.001), mTOR (p < 0.0001), PARP (p < 0.001), STAT3 (p < 0.0001), and NF-κB (p < 0.0001) in tumors treated with PDL-ESi and paclitaxel. Additionally, caspase-9 expression was significantly upregulated (p < 0.001), indicating enhanced induction of apoptosis (Figure 3(I, II)). These findings suggest that the PDL-ESi exosomes not only effectively knockdown PD-L1 expression but also restore apoptotic pathways while suppressing critical pro-survival and immune-suppressive signaling pathways. Imaging studies in 3D spheroids and tumor xenografts demonstrated efficient tumor targeting by PDL-E, confirming their time-dependent accumulation within tumor tissues (Figure 3(IV)). Future work will focus on enhancing the immunogenic properties of Trop2 exosomes, optimizing siRNA loading strategies, and evaluating long-term anti-tumor memory responses. Additionally, detailed evaluations of immune cell infiltration, including CD8⁺ cytotoxic T cells, CD4⁺ helper T cells, regulatory T cells, and myeloid-derived suppressor cells, will be conducted using flow cytometry and immunohistochemistry. Cytokine profiling will assess systemic immune activation, while further investigations will explore the effects of PDL E on T cell exhaustion markers (e.g., TIM-3, LAG-3) and memory T cell responses.

Conclusion: This study establishes a strong foundation for two complementary exosome platforms targeting resistant TNBC. Trop2-expressing tumor-derived exosomes demonstrated early potential as immunogenic, vaccine-like agents, while PD-L1-targeted HEK293T exosomes co-loaded with STAT3 siRNA and paclitaxel showed potent synergistic effects, enhancing tumor suppression and reversing immune resistance. Together, these bioengineered exosomes offer a novel, multi-pronged strategy for precise tumor targeting and immune system activation. Future work will refine siRNA delivery and evaluate immune modulation and long-term therapeutic responses.


Figure 1 (A) Western blot analysis confirming the expression of Trop2 protein in MDA-MB-231 DOX RT cells. (B) Western blot analysis confirms the expression of Trop2 protein on Exosomes. C) NTA of exosomes derived from untransfected cells. D) NTA of exosomes derived from MDA-MB-231 DOX RT TROP2 transfected cells. E) Cytotoxicity of Trop2 exosomes against LTLT-Ca cells.


Figure 2: I) Generation of stable anti-PD-L1 cell line, isolation, and characterization of transfected exosomes. A) Western blot represents expression of anti-PD-L1-Myc in HEK293T cells post transfection. B) Size and Zeta Potential of HEK-293 T exosomes C) Size and Zeta potential of Anti-PD-L1 exosomes D) Fluorescent NTA of transfected exosomes E) Expression of anti-PD-L1 on the surface of exosomes isolated from transfected HEK 293T cells. F) NanoFCM analysis for quantifying the number of copies of anti-PD-L1 expressed relative to the total number of exosomes. G) FACS analysis showing anti-PD-L1 presence on the surface of transfected exosomes. II) PD-L1 knockdown in different tumors post-treatment with PDL E. A) MDA-MB-231 DOX RT cells. B) MDA-MB-231 DOX RT xenograft tumors. C) H1975 OSM RT xenograft tumors. D) MIA PACA 2 cells. III) In-vitro cytotoxicity assessment of transfected exosomes. In-vitro 2D and 3D cytotoxicity assay using MDA-MB-231 DOX RT cell line and MCJB70C PDX TNBC cell line.


Figure 3: I) Assessment of apoptosis and relevant apoptotic proteins in MDA-MB-231 spheroids and xenografts after respective treatment. A) Fluorescence microscopy images of MDA-MB-231 spheroids stained with ethidium bromide (top row), acridine orange (middle row), and merged images (bottom row). Ethidium bromide (red) stains dead cells, while acridine orange (green) stains live cells. The merged images indicate the relative proportions of live and dead cells in each spheroid, demonstrating increased cell death in the combination treatment group (PDL ESi + Pac). B) Western blot analysis of protein expression levels in MDA-MB-231 xenografts treated with the indicated treatment. Blots were probed for markers associated with apoptosis (Cleaved PARP, Cleaved caspase 3), survival (BCL 2, pAKT, SOD2), and key signaling pathways (pMTOR, and pSTAT 3). β-actin was used as a loading control. Quantification of protein expression was performed by normalizing the intensity of each target protein band to its corresponding β-actin. II) Representative western blots showing activation of immune mediators. Treatment of MDA-MB-231 DOX RT cells with PD L1 targeting exosomes (PDL E) leads to activation of T cells and downregulation of IL-1β and TGF-β in the tumor microenvironment, which further acts on tumor cells and activates apoptotic pathways. β-actin was used as a loading control. Quantification of protein expression was performed by normalizing the intensity of each target protein band to its corresponding β-actin. B) qPCR depicting the mRNA expression levels. III) Evaluation of tumor-targeted exosome uptake and therapeutic efficacy in vivo. A) Fluorescent microscopy images of Cy5 PDL E (red) incubated with MDA-MB-231 DOX RT cells. (Top) Cy5-DSPE shows lower uptake by TNBC cells, while (Bottom) PDL E shows significantly enhanced cellular internalization. Scale bars represent 20 μm. Quantitative analysis of the mean fluorescence intensity in MDA-MB-231 DOX RT cells treated with Cy5-DSPE vs Cy5 PDL E demonstrates enhanced uptake of targeted exosomes (p < 0.05). B) In vivo fluorescent imaging of mice with MDA-MB-231 DOX RT tumors after administration of Cy5 PDL E. (Top panel) Mice treated with Cy5-DSPE show minimal tumor accumulation, whereas (Bottom panel) mice treated with Cy5 PDL E exhibit pronounced exosome accumulation at the tumor site.