Abstract
Pancreatic ductal adenocarcinoma (PDAC) tumours carry multiple gene mutations and respond poorly to treatments. There is currently an unmet need for drug carriers that can deliver multiple gene cargoes to target high solid tumour burden like PDAC. Here, we report a dual targeted extracellular vesicle (dtEV) carrying high loads of therapeutic RNA that effectively suppresses large PDAC tumours in mice. The EV surface contains a CD64 protein that has a tissue targeting peptide and a humanized monoclonal antibody. Cells sequentially transfected with plasmid DNAs encoding for the RNA and protein of interest by Transwell®-based asymmetric cell electroporation release abundant targeted EVs with high RNA loading. Together with a low dose chemotherapy drug, Gemcitabine, dtEVs suppress large orthotopic PANC-1 and patient derived xenograft tumours and metastasis in mice and extended animal survival. Our work presents a clinically accessible and scalable way to produce abundant EVs for delivering multiple gene cargoes to large solid tumours.
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Introduction
Pancreatic ductal adenocarcinoma (PDAC) is difficult to treat and has a low 5-year survival rate of <10%1. GTPase KRAS mutations occur in >90% of PDAC2 and the only clinically available drugs targeting the RAS family are inhibitors for KRASG12C mutation in non-small cell lung cancer (LUMAKRAS™, sotorasib)3. Most patients at the time of diagnosis have advanced unresectable tumors that carry multiple gene mutations4. In 70% of these cases, KRASG12D mutations co-occur with mutated TP53 tumor suppressor5. Therefore, there is a need for new treatments that target multiple mutations in PDAC. However, delivering multiple genetic agents precisely to such advanced tumors is a significant challenge. Liposomal nanoparticles (LNPs), while popular, are unsuitable for solid tumor treatments because they do not penetrate tissues or traverse physiological barriers well6, while viral deliver efficiently but are highly immunogenic7.
Extracellular vehicles (EVs) such as exosomes and microvesicles are attractive alternatives for gene delivery because they have low immunogenicity and cytotoxicity and can cross physiological barriers8,9,10. Exosomes containing exogenous silencing RNA for KRASG12D (siKRASG12D) have been shown to suppress small, non-advanced, orthotopic PANC-1 PDAC tumors in mouse models11. We have also previously used EVs carrying endogenous phosphatase and tensin homolog (PTEN) mRNA to slow down the growth of small tumors in glioblastoma mice12. While promising, for these EVs-based therapies to be clinically relevant, they must be able to carry multiple gene cargoes, precisely target, penetrate, and treat large solid PDAC tumors and metastases. Furthermore, EV production must be scalable and readily available. Current methods involve either inserting synthetic RNAs into pre-isolated EVs11,13, which is difficult to load large mRNA cargoes, or genetically modifying donor cells14 which are complicated and face the risk of off-target genomic integration.
In this work, we report easily scalable, dual-targeted therapeutic EVs (dtEVs) containing high copy numbers of TP53 mRNA or siKRASG12D that can suppress large solid PDAC tumors (Fig. 1a). Our dtEV surface contains a CD64 (Fc-gamma receptor 1) protein engineered at the N-terminus with a CKAAKNK (CK) tissue homing peptide that targets pancreatic tumor tissue15. This engineered protein (CD64ck), which acts as a universal anchor, binds with high affinity to any clinically available therapeutic humanized monoclonal antibodies (hmAb)16 to form a second targeting ligand on the EV surface. We use humanized anti-receptor tyrosine kinase-like orphan receptor 1 (αROR1, clone: 2A2) antibodies as the second ligand to target ROR1 receptors commonly found on tumors but not normal tissues17. Plasmid DNAs to produce CD64ck protein and either TP53 mRNA or siKRASG12D are sequentially delivered into mouse embryonic fibroblast (MEF) cells or human bone marrow stem cells (hBMSCs) by Transwell®-based asymmetric cell electroporation (TACE)—a clinically accessible and scalable method using affordable Transwell® inserts. Sequentially delivering the CD64ck plasmid DNA and the TP53 or siKRASG12D plasmid DNA by TACE promote the release of abundant EVs with high TP53 mRNA inside the EVs and CD64ck protein on the EV surface. After affinity binding with humanized αROR1 antibodies, our dtEVs together with a first-line chemotherapy drug, Gemcitabine (GEM), suppress large orthotopic PANC-1 and patient-derived xenograft (PDX) tumors and metastasis in mice and extended animal survival through strong gene regulation, cancer cell cycle arrest, and reduced chemodrug resistance. Our cellular trafficking results suggest that dual targeting promote receptor-mediated tissue penetration, tumor cell uptake, and cytosol RNA release of EVs. Our work demonstrates a simple, low-cost way to produce abundant targeted EVs carrying high loads of genetic cargoes that can effectively treat advanced cancer in animal models.
Results
Design of dual-targeted extracellular vesicles
To achieve dtEVs carrying TP53 mRNA and siKRASG12D, we transfected MEF or hBMSC donor cells with wild-type CD64 (CD64wt) or CD64ck (with CKAAKNK peptide), TP53, and small hairpin KRASG12D (shKRASG12D) containing plasmids by TACE11 (Fig. 1a). A monolayer of cells was grown on the membrane surface of the Transwell® insert and DNA plasmids were loaded below the membrane. This setup topologically divides the cell surface into a small transfection site equivalent to the 1 μm Transwell® membrane pore beneath the cell and a large EV secretion area covering the rest of the cell surface. Electric pulses delivered to the Transwell® insert drive the negatively charged plasmids into the cells electrophoretically and non-endocytically via the Transwell® pores. Non-endocytic delivery of plasmids by TACE is more efficient than conventional electroporation (bulk electroporation, BEP) because BEP mainly creates pores on cell membrane to facilitate plasmid-membrane binding, but plasmids still need to be up-taken via endocytosis18. We measured human CD64 protein expression in MEF cells at different time points (6, 12, 18, and 24 h) following either BEP or TACE transfection. The electroporated cells exhibited CD64 expression as early as 6 h after TACE electroporation, while it took over 18 h for stable CD64 expression by BEP (Supplementary Fig. 1).
Because electric field strength is much lower outside the pores than inside (Supplementary Note 1), cell damage at the secretion site is mild, allowing abundant EVs to be secreted. After TACE, we measured the release of EVs and vesicular RNA or protein of interest every 4 h for 24 h. The EVs collected over 24 h were purified by size exclusion chromatography (qEV columns) and characterized. Compared to untreated MEFs, TACE produced >30-fold EVs, with a maximum of ~1 × 106 EVs/cell around 16 h (Fig. 1b). CD64ck proteins on the EV surface was ~28 molecules/EV on average and >40 molecules/EV at the peak between 12 and 16 h. Meanwhile, there were ~4 copies of TP53 mRNA per EV on average and >8 copies/EV at the peak of around 4 h (Fig. 1c). Antisense siKRASG12D level was ~1000 copies/EV on average and reached a maximum of ~1900 copies/EV at ~16 h. No hairpin-type preliminary shKRASG12D was detected in the EVs. Quantification of each cargo in dtEVs was performed by using calibration curves with recombinant protein of CD64, synthetic TP53 mRNA and siRNA of KRASG12D as standards. The cargoes per EV was estimated by the total amount of each molecule divided by the dtEV number (Supplementary Fig. 2). The anchor protein of CD64wt or CD64ck were observed in both exosome and MV fractions which allows the preloaded human IgG (hIgG) for further targeting purposes (Fig. 1d).
We next confirmed that there were ~11% full-length TP53 mRNA in the total EV RNA obtained from TACE-stimulated MEFs by Bioanalyzer electrophoresis with synthetic TP53 mRNA as standard (Fig. 1e). Since the endogenous mRNAs have longer poly-A tails with various lengths, there is a shift in size between synthetic- and EV-mRNA. Moreover, the integrity of vesicular TP53 mRNA was also confirmed by oligo-dT primed quantitative polymerase chain reaction (qPCR) from both the 5′ end (exons 2 and 3) and 3′ end (exons 10 and 11) of the transcript from two edges of TP53 mRNA (Fig. 1f). By using two independent methods, we confirmed that the integrity of encapsulated mRNA of interest. Current methods of cellular transfection12 or RNA insertion to EVs13 by conventional electroporation and cellular modification14 also increase the EV secretion and RNA loading, but the efficacy is poor with low integrity of RNA cargos12. They are not suitable for clinical uses.
Sequential TACE alters donor cells for dtEV production and RNA loading
Even though CD64ck protein and RNA cargos were both detected in TACE-produced EVs, we observed their expressional peaks were quite different (Fig. 1b, c). The exosomal TP53 mRNA reached peak around 4 h, while the expression of CD64ck protein and siKRASG12D required longer time for translation and maturation after 12 h of TACE. Here, we introduced the sequential TACE (sTACE) by delivering CD64ck plasmid followed by TP53 plasmid with an 8, 16, or 24 h gap between the two plasmids in MEF (Fig. 2a) and BMSC (Supplementary Fig. 3). The optimized sTACE with 16 h gap gave the best coordination of surface CD64ck protein, TP53 mRNA loading, and EV number for our the dtEV formulation. CD64ck and shKRASG12D plasmids could be delivered together since their production profiles were similar. Since dtEVs are comprised of surface CD64ck and RNA cargos, we further used immunofluorescence (anti-CD64) and RNA in-situ hybridization (RNA-ISH) to measure each composition within the precursor of dtEVs, late-endosomal MVBs, in donor cells. Through the proportion of colocalized versus total fluorescent signal, we estimated that ~45% MVBs contained CD64ck protein and ~93% MVBs contained TP53 mRNA at 4 h, while ~55% MVBs contained siKRASG12D at 12 h after TACE (Fig. 2b). The sequence and specificity of the probes and antibodies used for TP53 mRNA, siKRASG12D, CD64 protein, and Rab7 is shown in Supplementary Fig 4.
We further characterized single dtEVs by using an immune lipoplex nanoparticle (ILN)-total internal reflection fluorescence microscopy (TIRFM) biochip assay19. Individual dtEV was captured by antibodies of exosomal markers (CD9 and CD63) tethered on the chip surface, and the TP53 mRNA or siKRASG12D in these captured exosomes are detected by florescent probes encapsulated in immune lipoplex nanoparticles (ILNs)20 (Supplementary Fig. 5). Fluorescent signals from TIRFM were used to quantify the RNA content and its colocalization with anti-CD64 signal. The colocalization ratios of encapsulated TP53 mRNA or siKRASG12D and CD64ck surface protein within dtEV were ~60% (Fig. 2c). The specificity of probes used in ILN for TP53 mRNA, and siKRASG12D is demonstrated in Supplementary Fig. 6. To further realize the distribution of RNA cargos (i.e. TP53 mRNA or siKRASG12D) in entire vesicular fraction, harvested dtEVs were sorted by size extrusion chromatography (qEV columns, IZON) into 20 fractions in which the EV number, averaged size, surface protein markers, and loaded RNA/protein cargo in each fraction were characterized. By real-time PCR analysis for RNA cargos from each fraction, TP53 mRNA was located mainly in exosomal fractions (11–14; high CD63/CD9 expression; Fig. 2d). Interestingly, siKRASG12D was detected in both exosomes and microvesicles, with more copies in MVs (5000–6000 copies/MV, size: 275.2–461.3 nm) that have a larger averaged volume than exosomes (150-250 copies/exosome, size: 61.8–118.0 nm).
Global RNA analysis revealed ~2700 genes and >130 microRNAs with altered concentrations in EVs from hBMSCs and MEFs electroporated with TP53 plasmids by TACE (Supplementary Table 1–2, Supplementary Data 1–2, and Supplementary Figs. 7–9). The trend in both cell types is similar. Most of the EVs containing transcripts were probably fragments (Fig. 1e), and their levels were much lower than the TP53 mRNA level encoded by the introduced plasmid. Further, because concentrations of the affected microRNAs were also much lower than the siKRASG12D level, it is unlikely any individual mRNA or microRNA in the EVs will perturb the functions of the recipient cells. However, it is worth examining the collective impact of all native microRNAs in the EVs in the future because their overall concentration is higher than the non-native siKRASG12D.
dtEVs provide strong cancer cell uptake and penetration ability
The dual targeting ability of dtEVs consists of preloaded humanized antibody and additional tissue homing peptides (CK) cloned into the N-terminus of CD64. Using enzyme-linked immunoassay (ELISA) with immobilized human IgG (hIgG) to pull down CD64, we first demonstrated that engineered CD64ck bound to hIgG1 with the same high affinity (Kd) as CD64wt (Fig. 3a). Also, flow cytometry showed that our engineered CD64ck is expressed on the surface of CD63-expressed dtEVs and bound well to hIgG (Fig. 3b). In PANC-1 cells expressing high levels of epidermal growth factor receptor (EGFR) and ROR1 that can be targeted with humanized anti-EGFR (αEGFR, Cetuximab, clone: C225) or anti-ROR1 (αROR1, clone: 2A2)21. Internalization assay using fluorescently labeled EVs revealed better cell uptake with αROR1-targeted EVs than αEGFR-targeted EVs or normal IgG control (Fig. 3c). Adding CK peptides onto the EV surface nearly doubled the uptake of αROR1-targeted EVs in PANC-1 cells. Since IgG exists in healthy human serum at 6–16 g/L22, we evaluated the stability of preloaded humanized antibodies on EVs in 50% human serum for 6 h at 37 °C. No significant loss in targeting ability was seen with EVs carrying either αROR1 or αEGFR (Supplementary Fig. 10a).
Transcytosis of extracellular vesicles23 or antibodies24,25 was previously reported, but whether they could pass the cellular barrier as an entire complex remains unclear. The putative transcytosis of dtEVs was determined by two (top and bottom) monolayers of PANC-1 cells separated by a Transwell® membrane with 5 μm pores (Fig. 3d). Fluorescent EVs and LNPs were applied to the top monolayer (1st recipient, >90% confluence) and their presence in the bottom monolayer (2nd recipient) was measured after 24 h. Adding hmAb on the EV surface enhanced transcytosis by 1–4-fold, with dtEV bearing CD64ck and αROR1 (CD64ck_αROR1) performing the best (Fig. 3e). Compared to LNPs, 2–3-fold more dtEVs were found in the bottom cell monolayer (Supplementary Fig. 10b).
Enhanced endocytosis of dtEVs was observed in single-cell level as well. The faster binding and uptake occurred with CD64ck_ αROR1 dtEVs (mean uptake time: 600.2 ± 31.4 s) than with single-targeted EVs (stEVs) bearing CD64ck alone (805.6 ± 47.5 s) or non-targeted LNPs (956.2 ± 51.4 s) (Supplementary Fig. 11). More counts of dtEVs (18.4 ± 9.5 vesicles/cell) than stEVs (13.0 ± 8.7 vesicles/cell) and LNPs (15.1 ± 12.2 particles/cell) were taken by PANC-1 cells after 45 min (Supplementary Fig. 12). The enhanced uptake of dtEVs was mainly mediated by clathrin- and caveolae-relative endocytosis. Inhibiting clathrin- (Pitstop 10 mM)26 and caveolae- (Methyl-β-cyclodextrin, 10 mM)27 mediated endocytosis, exosomal secretion (neticonazole, 10 mM)28, and intracellular trafficking (Brefeldin A 5 μM, and Exo1 20 μM) in the first recipient cells prevented putative transcytosis of dtEVs (Fig. 3f).
The strong penetration of dtEVs was noted in PANC-1 spheroids (~300 μm) as well. dtEVs penetrated deeper into the core of tumor spheroids than single targeting bearing either αROR1 or CK alone after 24 h of incubation, while LNPs did not penetrate to the core of spheroids (Fig. 3g). A similar trend was seen at 6 h where dtEVs bearing CD64ck and αEGFR could target the spheroids well, but the dtEVs bearing CD64ck and αROR1 showed better internalization in individual cells (Supplementary Fig. 13).
Trafficking of dtEVs in recipient cells
The process of drug delivery within tissues and cells is highly complex. In this study, we present experimental results aimed at investigating the distinctions in cellular trafficking between dtEVs and other nanocarriers. Typically, recipient cells take up nanocarriers through endocytosis, leading to intracellular trafficking from the endosome to the lysosome (Fig. 4a). We treated PANC-1 cells with fluorescent dtEVs, non-targeted IgG-EVs, or LNPs (indicated by red) for 2 h. Subsequently, the cells were fixed and co-stained with anti-Rab5 to visualize the extent of colocalization with early endosomes (indicated by green). We observed similar levels of encapsulation in early endosomes of PANC-1 cells for all three nanocarriers (Fig. 4b, co-localization % (Col%): LNP: 89.0 ± 3.5; non-targeted EV: 88.17 ± 5.2; dtEV: 81.50 ± 3.7, n = 3). In contrast, the levels of colocalization (indicated by yellow) among the three nanocarriers in lysosomes exhibited significant variations, with dtEVs showing the lowest level in lysosomes (green) (Fig. 4c, Col%: LNP: 26.17 ± 4.0; IgG-EV: 13.53 ± 4.87; dtEV: 5.73 ± 1.45, n = 3). These findings suggest a potentially superior ability of dtEVs to escape the endosomal compartment. However, further rigorous experimental analyses are required to confirm this observation. While the exact mechanism of endosomal escape of EVs remains unclear, there is growing evidence suggesting that the release of EV lumens differs from that of conventional liposomal and polymeric nanoparticles29,30. To delve deeper into this phenomenon, we utilized a cytosol fluobody recognition system29 to monitor the interaction of dtEV lumens throughout vesicular trafficking (Fig. 4d). Prior to the experiment, recipient cells were transfected with an anti-GFP fluobody tagged with mCherry (indicated by red), which exhibits widespread expression within the cytosol (see plasmid construct in Supplementary Fig. 14). Following the release of the dtEV lumen, the cytosolic anti-GFP fluobody exhibited recognition of CD63GFP releasing from the dtEV lumens, leading to a transient local accumulation. PANC-1 cells expressing anti-GFP mCherry fluobody were incubated with dtEVs containing CD63GFP for 2, 4, and 8 h. We observed gradual increase in the accumulation of the anti-GFP fluobody from 2 to 4 and 8 h, indicating the potential release of dtEVs lumens (Fig. 4e).
Adeno-associated virus (AAV) vectors are the highly effective gene delivery carriers owing to their strong receptor-mediated clathrin-31 and caveolae-mediated endocytosis32, transcytosis33, and endosomal escape34. Based on our experimental results, it is tentatively suggested that the presence of specific antibody and THP targeting on the dtEV surface may enhance receptor-mediated transcytosis, endocytosis, and endosomal escape, thus resembling the behavior of viral particles.
dtEVs carrying siKRAS G12D or TP53 mRNA inhibit pancreatic cancer cells
PANC-1 cells carrying KRASG12D and TP53 mutations (p.R273H, loss of function) display strong resistance to first-line treatment of Gemcitabine (GEM). Treating PANC-1 cells with EVs carrying siKRASG12D at 1 × 106 EVs/cell (~1 × 109 copies/cell or ~16 pg siKRASG12D mass/cell) for 24 h silenced KRASG12D expression at both the mRNA and protein levels, with the targeting formulation of CD64ck_αROR1 showing the best suppression (Fig. 5a and Supplementary Fig. 15). The siKRASG12D carrying EVs slowed down PANC-1 proliferation mainly via cell cycle arrest in the G1 phase (Fig. 5b and Supplementary Fig. 16). The combinative dtEVs containing 50/50 siKRASG12D and TP53 mRNA resulted in the highest apoptotic population (Fig. 5b). The EV dose was also 1×106 total EVs/cell containing ~5x108 copies siKRASG12D and ~4x106 copies of TP53 mRNA/cell or ~8 pg siKRASG12D mass and ~3 pg TP53 mRNA mass/cell). The wild-type TP53 mRNA delivered by CD64ck_ αROR1 dtEVs enhanced the expression of p53 and p21 in the recipient PANC-1 cells (Fig. 5c). Although PANC-1 cells show strong GEM resistance, the expression of wild-type p53 protein in the presence of GEM decreased anti-apoptotic BCL-xl and increased p21 expressions (Fig. 5d). The combined tEV-gene therapy and Gem-chemotherapy effectively killed PANC-1 cells, with the CD64ck_αROR1 dtEVs containing 50/50 siKRASG12D and TP53 mRNA (Fig. 5e) showing the best performance (>65% cancer cell killing efficacy under 24 h treatment) (Fig. 5f and Supplementary Fig. 17). Treatment of PANC-1 cells with dtEVs, both with and without GEM, resulted in an elevation of oncogene-induced senescence, as evidenced by an increase in SA-β-galactosidase activity (Fig. 5g and Supplementary Fig. 18). Although both mRNA and siRNA cargoes could be loaded into the same EVs by co-delivering multiple plasmid DNAs to donor cells via TACE, we prepared dtEVs with either mRNA or siRNA cargo in this study because their release profiles were different (Fig. 1c).
dtEVs suppress orthotopic PANC-1 mice with high tumor burden
We assessed the clinical potential of dtEVs in PANC-1 tumors orthotopically implanted in the pancreas in NOD/SCID mice expressing green fluorescent protein (GFP) and luciferase. Each targeting formulation of dtEVs was fluorescently labeled (PKH26) and intraperitoneally (i.p.) injected into mice. After 24 h, major organs were collected and analyzed (Fig. 6a). Compared to LNPs and other targeted and non-targeted EVs, significantly more CD64ck_αROR1 dtEVs accumulated in the pancreatic tumor (Fig. 6b). Fluorescence imaging (Fig. 6c) and immunohistochemical staining (Fig. 6d) of tumor sections confirm the preferential accumulation of CD64ck_αROR1 dtEVs in the tumor over the liver and spleen.
Over 70% of pancreatic cancer patients were diagnosed at advanced stages with unresectable tumor burden35. We then used NOD/SCID mice for examining the therapeutic efficacy of dtEVs on high tumor burden in vivo. Animals were injected with 5 × 105 luciferase transduced PANC-1 cells per mouse and commenced treatment on Day 25. EV-based gene therapy mainly regulates cancer cell cycle arrest but not directly killing, we administered a low-dose GEM (20 mg/kg per week) to the animals. Controls include animals treated with dtEVs carrying scramble RNA (SCR), GEM alone, and LNPs containing 10-fold more synthetic TP53 mRNA and siKRASG12D than those delivered by CD64ck_αROR1 dtEVs. GEM was delivered once a week, while the rest formulations were delivered three times per week with i.p. injection of 1 × 1011 dtEVs per dose. The estimated RNA cargo was ~1 × 1014 copies (~1.6 µg mass) of siKRASG12D or scramble RNA. Of all treatments, GEM together with CD64ck_αROR1 dtEVs carrying TP53 mRNA and siKRASG12D strongly suppressed tumor growth and prolonging the overall survival of animals (Fig. 6e–g). For the combinative siKRASG12D and TP53 mRNA formulation, 5 × 1010 dtEVs carrying either siKRASG12D or TP53 mRNA were mixed before use. The estimated RNA cargo was ~2 × 1011 copies (~0.3 µg mass) of TP53 mRNA and ~5x1013 copies (~0.8 µg mass) of siKRASG12D. Immunohistochemical staining of residual tumor tissue revealed that dtEV treatment restored TP53 expression and silenced KRAS expression (Fig. 6h and Supplementary Fig. 19). The dtEVs were biocompatible and displayed good blood circulation profiles with minimal cellular toxicity (Supplementary Fig. 20a, b). Unlike GEM or LNP treatment, the EV-treated mice showed much less weight loss (Supplementary Fig. 20c).
Targeting and therapeutic efficacy of dtEVs in subcutaneous PDX mouse models
We then tested the targeting and therapeutic performance of dtEVs in patient-derived xenograft (PDX) PDAC tumors inoculated subcutaneously (subQ) into the back of NOD/SCID mice. We selected a subQ mouse model first, because the animal viability and quality in the orthotopic PDX NOD/SCID mice was very poor due to the major surgery in animal model preparation. One subQ animal was intratumourally injected with 1 × 1011 fluorescent-labeled IgG_EVs and CD64ck_αROR1 dtEVs. Consistent with the transcytosis and spheroid penetration results in Fig. 3g, dtEVs (CD64ck_αROR1) spread more broadly throughout the tumor than IgG_EVs (Fig. 7a). Biodistribution in subQ mice via i.p. injection also showed stronger dtEV accumulation in tumor comparing to non-targeted IgG_EVs and non-targeted LNPs (Fig. 7b, c). Encouraged by these results, we further investigated the dtEVs properties in a genotype matching PDX model (ROR1++, KRASG12D, and TP53A138V mutations). We subcutaneously inoculated a 3 × 3 ×3 mm3 PDX tissue into the back of NOD/SCID mouse. By three-week treatment, dtEVs containing TP53-mRNA and siKRASG12D (dtEVs: 1 × 1011/100 μl, i.p. injection, three-time/week with ~2 × 1011 copies or ~0.3 µg mass of TP53 mRNA and ~5 x 1013 copies or ~0.8 µg mass of siKRASG12D per injection) greatly inhibited the large tumor growth, while combining a low dose of GEM (20 mg/kg, once/week) could further suppress the tumor activity (Fig. 7d,e). KRAS knockdown using Western blot on the collected tumor tissues was observed (Supplementary Fig. 21). Hematoxylin and eosin staining of various tissues obtained from the treated animals revealed cancer metastasis in the lung, pancreas, and fat tissue (Supplementary Fig. 22). However, animals treated with dtEVs containing TP53 mRNA and siKRASG12D had substantially fewer and smaller metastatic lesions, particularly with GEM (Fig. 7f, g and Supplementary Fig. 23). It is well-established that aberrant activation of mutant KRAS can override oncogene-induced senescence (OIS), thereby promoting the survival and proliferation of cancer cells36,37. In the tumor tissues and metastatic lung lesions of mice treated with dtEV or dtEV+GEM, we observed an increase in β-galactosidase activity and a decrease in K-i67 expression (Fig. 7h, i). These findings indicate a reactivation of OIS by dtEVs to suppress tumor growth.
Therapeutic efficacy of dtEVs in orthoptic PDX mouse models
Because the orthotopic and subcutaneous PDAC pathology is quite different, especially the physiological barriers, we further prepared an orthotopic tumor by stitching a 3 x 3 x 3 mm3 PDX tissue from a different PDAC patient to the pancreatic tail of BALB/c-Nude mice which are much more robust than NOD/SCID mice in the animal model preparation. We also increased the suggested GEM dose from 20 to 50 mg/kg per week because of the different mouse types. The genoty** of PDX tissue carried both KRASG12D and TP53R196* gene mutations. TP53R196* mutation has worse prognosis due to the loss of the entire cancer suppression function38. Furthermore, this PDX tissue also expressed a high level of the Glioma-associated homologue-1 (Gli1) protein known to regulate the Hedgehog pathway. Overexpression of Gli1 accelerates KRAS-initiated pancreatic tumourigenesis39,Histology and immunohistochemistry analysis All tissues were fixed in formalin and processed for paraffin embedding. Histological analyses on the tumor tissue and other organs, including the liver, lung, heart, spleen, and kidney were performed using H&E staining. For immunostaining, tissue sections were subjected to antigen retrieval. Then the slides were incubated with 0.3% H2O2 and blocked in 5% bovine serum albumin. The following primary antibodies were used for staining: anti-rabbit p-ERK p44/p42 MAPK (ERK1/2) (Thr202/Tyr204), anti-rabbit Ki-67, Anti-rabbit p21, Anti Human CD64, and RAS (G12D Mutant) antibody. Data are presented as mean ± SD of triplicates unless otherwise indicated. Statistical analysis was performed using a two-tailed Student’s t test or one-way ANOVA with post-hoc tests, as appropriate. For the mouse survival study, the log-rank test was used to test the difference between the two survival curves. The Holm procedure was applied to adjust for multiplicity. The overall family-wise type I error rate was controlled at α = 0.05. SAS version 9.3 was used for all statistical analyses (SAS Institute, Inc). For immunoblots and treatment assays, the experiments have been repeated at least three with similar results and representative data was shown. Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.Statistics and reproducibility
Reporting summary
Data availability
The non-coding RNA profiling data in this study are available in the GEO database under the accession code GSE223409. All other data supporting the findings of this study are available within the article and its supplementary files. Any additional requests for information can be directed to the corresponding author (L.J.L.). Source data are provided with this paper.
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Acknowledgements
This work was partially supported by The Ministry of Education, Higher Education SPROUT Project for Cancer and Immunology Research Center (112W31101) (L.J.L./C-Y.F.H.), the Yushan Fellow Program (L.J.L.) and NSTC 112-2321-B-A49-019 (C-Y.F.H./Y-S.S./L.J.L.) in Taiwan, National Science and Technology Council (NSTC) and Taiwan Ministry of Health and Welfare (MOHW), grant number NSTC 111-2327-B-006-008, NSTC 112-2321-B-006-010, MOHW112-TDU-B-221-124015 and MOHW112-TDU-B-211-144003 for Y-S.S., Veterans General Hospitals and University System of Taiwan Joint Research Program (VGHUST110-G1-3-2) and Yen T**g Ling Medical Foundation (CI-110-3) for Y-S.C., and National Natural Science Foundation of China (NSFC 32271401 and NSFC 31971264) and Program of Shanghai Academic/Technology Research Leader in 2022 for Y.Y. We are grateful to Ruey-Hwa Chen. from Academia Sinica in Taiwan for valuable suggestions to this work. We thank A.L. Chun of Science Storylab for critically reading and editing the manuscript.
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L.J.L., C-L.C., and Y.M. designed the study and all experiments with feedback from A.S.L., C-Y.F.H., Y-S.S., M.H., and Y.Y.; C-L.C., Y.M., and J.P. designed and produced ACE/TEP-based dtEVs with help from S-Y.C. and T-S.C.; A.L., Y-S.S., M.H., A.S.L., and R.L. led the animal studies; Y-C.H., Y.M., C-L.C., M-H.C., Z-X.Z., W-H.H., S-Y.C., H-C.L., L.S., and C.Z. conducted animal studies and analysis; C-L.C., Y.M., and J.P. conducted EV characterization with help from X.W., J.Z., H.L., and E.R.; C-L.C., J.P., and Y.M. conducted in vitro experiments; I.L., S.F., and K.W. conducted EV RNA sequencing and analysis; X-Y.C. and Y-S.C. conducted confocal microscopy for single-cell EV endocytosis; L.J.L., C-L.C., Y.M., J.P., and Y-C.H. wrote the paper with feedback from A.S.L., Y-S.S., C-Y.F.H., M.H., Y.Y., K.W., W.J., and B.Y.S.K.
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L.J.L. and A.S.L. are shareholders at Spot Biosystems Ltd. All other co-authors declare no competing interests.
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Chiang, CL., Ma, Y., Hou, YC. et al. Dual targeted extracellular vesicles regulate oncogenic genes in advanced pancreatic cancer. Nat Commun 14, 6692 (2023). https://doi.org/10.1038/s41467-023-42402-3
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DOI: https://doi.org/10.1038/s41467-023-42402-3
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