Intra-arterial Delivery Research Articles

PET imaging of distinct brain uptake of a nanobody and similarly-sized PAMAM dendrimers after intra-arterial administration.

We have recently shown that intracerebral delivery of an anti-VEGF monoclonal antibody bevacizumab using an intra-arterial (IA) infusion is more effective than intravenous administration. While antibodies are quickly emerging as therapeutics, their disadvantages such as large size, production logistics and immunogenicity motivate search for alternatives. Thus we have studied brain uptake of nanobodies and polyamidoamine (PAMAM) dendrimers. Nanobodies were conjugated with deferoxamine (DFO) to generate NB(DFO)2. Generation-4 PAMAM dendrimers were conjugated with DFO, and subsequently primary amines were capped with butane-1,2-diol functionalities to generate G4(DFO)3(Bdiol)110. Resulting conjugates were radiolabeled with zirconium-89. Brain uptake of 89ZrNB(DFO)2 and 89ZrG4(DFO)3(Bdiol)110 upon carotid artery vs tail vein infusions with intact BBB or osmotic blood-brain barrier opening (OBBBO) with mannitol in mice was monitored by dynamic positron emission tomography (PET) over 30min to assess brain uptake and clearance, followed by whole-body PET-CT (computed tomography) imaging at 1h and 24h post-infusion (pi). Imaging results were subsequently validated by ex-vivo biodistribution. Intravenous administration of 89ZrNB(DFO)2 and 89ZrG4(DFO)3(Bdiol)110 resulted in their negligible brain accumulation regardless of BBB status and timing of OBBBO. Intra-arterial (IA) administration of 89ZrNB(DFO)2 dramatically increased its brain uptake, which was further potentiated with prior OBBBO. Half of the initial brain uptake was retained after 24h. In contrast, IA infusion of 89ZrG4(DFO)3(Bdiol)110 resulted in poor initial accumulation in the brain, with complete clearance within 1h of administration. Ex-vivo biodistribution results reflected those on PET-CT. IA delivery of nanobodies might be an attractive therapeutic platform for CNS disorders where prolonged intracranial retention is necessary.

Development of radiotherapeutic rhenium-188 liposomes in alginate microspheres (Rhe-LAMs) for treatment of liver tumors and technetium-99m liposomes in alginate microspheres (Tech-LAMs) for image-guided treatment planning.

e15599 Background: Radio-embolic agents such as beta-emitting yttrium-90 spheres have been widely adopted as a modality for liver cancer therapy; however, their production can be timely and costly, shunting to the lungs may occur, and post-procedural visualization is limited. Alginate, a polysaccharide which can easily be formed into microspheres, has already been investigated for drug delivery applications; however, we propose utilizing alginate to manufacture radioembolic microspheres for intra-arterial delivery to liver tumors: Rhenium-188/186-labeled liposomes in alginate microspheres (Rhe-LAMS) as a radioembolic agent for the treatment of liver tumors and technetium-99m labeled liposomes in alginate microspheres (Tech-LAMs) as an agent for nuclear image-guided pre-treatment planning for liver cancer patients. Methods: Liposomes were manufactured and labeled with either Re-188/186 or Tc-99m. The liposomes were then mixed with alginate solution and then cross-linked with CaCl2 to form microspheres. Microsphere diameter was evaluated via light microscopy, and retention of radioactivity was measured over time via dosimeter. Microspheres containing free Re-186/Tc-99m (i.e. no liposomes) were also constructed for control comparison. To test in vivo stability, Tech-LAMs were intra-arterially injected into the liver of rabbits for nuclear imaging. Results: 2 ml batches of Rhe-LAMS/Tech-LAMS of 20-80 microns could be manufactured in 3 hours. Radiolabeling efficiency of the liposomes reached 85% and retention of radioactivity in microspheres reached 75%. After overnight incubation, 90% activity was retained. Control microspheres showed a retention of < 5%. In vivo imaging revealed absent activity in the lungs and high embolic activity in the liver. Conclusions: Our novel method demonstrated success regarding radioactivity retention and embolization capabilities. We envision this method to be a quickly-producible, cost-efficient, and effective means for radioembolization of liver tumors that could be adopted by any radiopharmacy.

Janus Microcarriers for Magnetic Field-controlled Combination Chemotherapy of Hepatocellular Carcinoma†.

Combination chemotherapy administering multiple chemo-agents is widely exploited for the treatment of various cancers in the clinic. Specially for hepatocellular carcinoma (HCC), one of the most common malignancies, a co-administration of combinational cytostatic multi-kinase inhibitors and cytotoxic chemo-agents has been suggested as a potential curative approach. Here, Janus microcarriers were developed for the controlled local combination chemotherapy of HCC. The Janus microcarriers are composed of polycaprolactone (PCL) compartment and magnetic nanoparticles-loaded poly(lactide-co-glycolic acid) (PLGA) compartment which contain hydrophobic regorafenib and hydrophilic doxorubicin, respectively. Exploiting the magnetic anisotropy, rotational motion of the Janus microcarriers is controlled with magnetic field, which enables the active co-release of dual chemo-agents. Furthermore, Janus microcarriers exhibit magnetic resonance (MR) contrast effect, supporting the successful transcatheter intra-arterial delivery of the combination chemo-agents loaded the microcarriers to the targeted tumor. This Janus microcarriers potentially serve as a general combinational chemo-therapeutic platform for the co-delivery of various combinations of multi-chemo-agents.

Allergen-induced histaminergic and non-histaminergic activation of itch C-fiber nerve terminals in mouse skin

Acute cutaneous exposure to allergen often leads to itch, but seldom pain. The effect of mast cell activation on cutaneous C-fibers was studied using innervated isolated mouse skin preparation that allows for intra-arterial delivery of chemicals to the nerve terminals in the skin. Allergen (ovalbumin) injection into the isolated skin of actively sensitized mice strongly stimulated chloroquine (CQ)-sensitive C-fibers (also referred to as “itch” nerves); on the other hand, CQ-insensitive C-fibers were activated only modestly, if at all. The histamine H1 receptor antagonist pyrilamine abolished itch C-fibers response to histamine, but failed to significantly reduce the response to ovalbumin. Ovalbumin also strongly activated itch C-fibers in skin isolated from Mrgpr-cluster Δ−/− mice. When pyrilamine was studied in the Mrgpr-cluster Δ−/− mice thereby eliminating the influence of both histamine H1 and Mrgpr receptors (MrgprA3 and C11 are selectively expressed by itch nerves), the ovalbumin response was very nearly eliminated. The data indicate that the acute activation of itch C-fibers in mouse skin is largely secondary to the combined effect of activation of histamine H1 and Mrpgr receptors.

Toward a Better Understanding of The Mechanism Of Action for Intra-Arterial Delivery of Irinotecan From Dc Bead (TM) (DEBIRI)

DC Bead is designed for the embolization of liver malignancies combined with local sustained chemotherapy delivery. It was first demonstrated around a decade ago that irinotecan could be loaded into DC Bead and used in a transarterially directed procedure to treat colorectal liver metastases, commonly referred to as drug-eluting bead with irinotecan (DEBIRI). Despite numerous reports of its safe and effective use in treating colorectal liver metastases patients, there remains a perceived fundamental paradox as to how this treatment works. This review of the mechanism of action of DEBIRI provides a rationale for why intra-arterial delivery of this prodrug from an embolic bead provides for enhanced tumor selectivity, sparing the normal liver while reducing adverse side effects associated with the irinotecan therapy.

Combining vascular targeting and the local first pass provides 100-fold higher uptake of ICAM-1-targeted vs untargeted nanocarriers in the inflamed brain

New advances in intra-arterial (IA) catheters offer clinically proven local interventions in the brain. Here we tested the effect of combining local IA delivery and vascular immunotargeting. Microinjection of tumor necrosis factor alpha (TNFα) in the brain parenchyma causes cerebral overexpression of Inter-Cellular Adhesion Molecule-1 (ICAM-1) in mice. Systemic intravenous injection of ICAM-1 antibody (anti-ICAM-1) and anti-ICAM-1/liposomes provided nearly an order of magnitude higher uptake in the inflamed vs normal brain (from ~0.1 to 0.8%ID/g for liposomes). Local injection of anti-ICAM-1 and anti-ICAM-1/liposomes via carotid artery catheter provided an additional respective 2-fold and 5-fold elevation of uptake in the inflamed brain vs levels attained by IV injection. The uptake in the inflamed brain of respective untargeted IgG counterparts was markedly lower (e.g., uptake of anti-ICAM-1/liposomes was 100-fold higher vs IgG/liposomes). These data affirm the specificity of the combined effect of the first pass and immunotargeting. Intravital real-time microscopy via cranial window revealed that anti-ICAM-1/liposomes, but not IgG/liposomes bind to the lumen of blood vessels in the inflamed brain within minutes after injection. This straightforward framework provides the basis for translational efforts towards local vascular drug targeting to the brain.

Image-Guided Transarterial Directed Delivery of Human Mesenchymal Stem Cells for Targeted Gastrointestinal Therapies in a Swine Model

PurposeTo evaluate the feasibility of catheter-directed intra-arterial stem cell delivery of human mesenchymal stem cells (MSCs) to the small bowel in a porcine model. Materials and MethodsThe cranial mesenteric artery of 6 Yucatan minipigs was selectively catheterized under fluoroscopic guidance following cut-down and carotid artery access. A proximal jejunal branch artery was selectively catheterized for directed delivery of embolic microspheres (100–300 μm) or MSCs (0.1–10 million cells). The pigs were euthanized after 4 hours and specimens collected from the proximal duodenum and the targeted segment of the jejunum. The Chiu/Park system for scoring intestinal ischemia was used to compare hematoxylin and eosin–stained sections of jejunum and duodenum. ResultsSuccessful delivery of microspheres or MSCs in a proximal jejunal branch artery of the cranial mesenteric artery was achieved in all subjects. Radiopaque microspheres and post-delivery angiographic evidence of stasis in the targeted vessels were observed on fluoroscopy after delivery of embolics. Preserved blood flow was observed after MSC delivery in the targeted vessel. The Chiu/Park score for intestinal ischemia in the targeted proximal jejunal segments were similar for microspheres (4, 4; n = 2) and MSCs (4, 4, 4, 3; n = 4), indicating moderate ischemic effects that were greater than for control duodenal tissue (3, 1; 0, 0, 3, 3). ConclusionsSelective arteriographic deployment of MSCs in swine is feasible for study of directed intestinal stem cell delivery. In this study, directed therapy resulted in intestinal ischemia.

Efficacy of Sonothrombolysis Using Microbubbles Produced by a Catheter-Based Microfluidic Device in a Rat Model of Ischemic Stroke.

Limitations of existing thrombolytic therapies for acute ischemic stroke have motivated the development of catheter-based approaches that utilize no or low doses of thrombolytic drugs combined with a mechanical action to either dissolve or extract the thrombus. Sonothrombolysis accelerates thrombus dissolution via the application of ultrasound combined with microbubble contrast agents and low doses of thrombolytics to mechanically disrupt the fibrin mesh. In this work, we studied the efficacy of catheter-directed sonothrombolysis in a rat model of ischemic stroke. Microbubbles of 10-20µm diameter with a nitrogen gas core and a non-crosslinked albumin shell were produced by a flow-focusing microfluidic device in real time. The microbubbles were dispensed from a catheter located in the internal carotid artery for direct delivery to the thrombus-occluded middle cerebral artery, while ultrasound was administered through the skull and recombinant tissue plasminogen activator (rtPA) was infused via a tail vein catheter. The results of this study demonstrate that flow focusing microfluidic devices can be miniaturized to dimensions compatible with human catheterization and that large-diameter microbubbles comprised of high solubility gases can be safely administered intraarterially to deliver a sonothrombolytic therapy. Further, sonothrombolysis using intraarterial delivery of large microbubbles reduced cerebral infarct volumes by approximately 50% vs. no therapy, significantly improved functional neurological outcomes at 24h, and permitted rtPA dose reduction of 3.3 (95% CI 1.8-3.8) fold when compared to therapy with intravenous rtPA alone.

“Micro-introducer access” intra-arterial chemotherapy for retinoblastoma in an 8-week-old infant

Abstract Intra-arterial chemotherapy (IAC) for retinoblastoma has emerged as a standard therapy to facilitate globe salvage in patients with retinoblastoma. However, transfemoral endovascular catheterization of the ophthalmic artery is typically reserved for infants aged 3 months or older due to size of the patient and vascular anatomy. Here, the authors report a successful case of transfemoral intra-arterial chemotherapy delivery performed for group E retinoblastoma in an infant at 8 weeks of age who weighed only 4.1 kg using “micro-introducer sheath” technique to minimize the size of the arteriotomy. The patient received Melphalan, Topotecan, and Carboplatin via infusion through a microcatheter placed in the right ophthalmic artery. The procedure was tolerated well, and the patient went on to have two additional IAC procedures. Follow-up examinations under anesthesia showed tumor regression in response to chemotherapy. While typically reserved for older and larger patients, with proper technical expertise, transfemoral intra-arterial chemotherapy can be considered in such small infants with highly advanced disease.

Localized Intra-Arterial Gene Delivery Using AAV.

In vivo gene therapy is a tremendous tool for a wide variety of genetic modifications. However, often a specific and precise local administration of the viral vector is necessary to deliver the genetic payload in vivo. For many animal studies using viral vectors, such as those investigating neurological disorders, the vector is targeted directly into the tissue/organ of interest. On the other hand, in vascular disease research, viral vectors are administered systemically, either via a tail vein injection or through catheter-mediated infusion, which results in off-target transduction of cells and tissues. Targeting cells in the vascular wall without off-target activity, however, requires localized delivery in order to efficiently target cells of the internal vasculature. Here we describe a novel murine in vivo targeted intra-arterial viral vector delivery method, which has been developed in order to be able to perform more intricate studies in cardiovascular disease.

Intra-arterial Drug Delivery to the Ischemic Brain in Rat Middle Cerebral Artery Occlusion Model.

Rat transient middle cerebral artery occlusion (tMCAO) model is one of the most commonly used animal models in ischemic stroke studies. In the model, increasing safety and efficacy of therapeutic agent administration, such as stem cells and drugs directly to the ischemic brain using the internal carotid artery (ICA) is essential, because using the common carotid artery (CCA) for injection can close CCA completely and cause many complications after tMCAO surgery. Also, the pterygopalatine artery (PPA) is an arterial branch of the ICA that supplies blood circulation of the external part of the brain and removing the blood circulation of the PPA is required for more complete induction of ischemia to the brain. Herein, we present the insertion of intra-arterial catheter in the ICA via the external carotid artery (ECA) after the PPA in rats subjected to tMCAO surgery.

Single-cell, high-throughput analysis of cell docking to vessel wall

Therapeutic potential of mesenchymal stem cells (MSCs) has been reported consistently in animal models of stroke, with mechanism mainly through immunomodulation and paracrine activity. Intravenous injection has been a prevailing route for MSCs administration, but cell quantities needed when scaling-up from mouse to human are extremely high putting into question feasibility of that approach. Intra-arterial delivery directly routes the cells to the brain thus lowering the required dose. Cell engineering may additionally improve cell homing, further potentiating the value of intra-arterial route. Therefore, our goal was to create microfluidic platform for screening and fast selection of molecules that enhance the docking of stem cells to vessel wall. We hypothesized that our software will be capable of detecting distinct docking properties of naïve and ITGA4-engineered MSCs. Indeed, the cell flow tracker analysis revealed positive effect of cell engineering on docking frequency of MSCs (42% vs. 9%, engineered vs. control cells, p < 0.001). These observations were then confirmed in an animal model of focal brain injury where cell engineering resulted in improved homing to the brain. To conclude, we developed a platform to study the docking of cells to the vessel wall which is highly relevant for intraarterial cell targeting or studies on neuroinflammation.

Real-Time MRI Guidance for Reproducible Hyperosmolar Opening of the Blood-Brain Barrier in Mice.

The blood-brain barrier (BBB) prevents effective delivery of most therapeutic agents to the brain. Intra-arterial (IA) infusion of hyperosmotic mannitol has been widely used to open the BBB and improve parenchymal targeting, but the extent of BBB disruption has varied widely with therapeutic outcomes often being unpredictable. In this work, we show that real-time MRI can enable fine-tuning of the infusion rate to adjust and predict effective and local brain perfusion in mice, and thereby can be allowed for achieving the targeted and localized BBB opening (BBBO). Both the reproducibility and safety are validated by MRI and histology. The reliable and reproducible BBBO we developed in mice will allow cost-effective studies on the biology of the BBB and drug delivery to the brain. In addition, the IA route for BBBO also permits subsequent IA delivery of a specific drug during the same procedure and obtains high targeting efficiency of the therapeutic agent in the targeted tissue, which has great potential for future clinical translation in neuro-oncology, regenerative medicine and other neurological applications.

Gene Silencing in the Right Place at the Right Time.

Gene Silencing in the Right Place at the Right Time.

A Distinct Advantage to Intraarterial Delivery of 89Zr-Bevacizumab in PET Imaging of Mice With and Without Osmotic Opening of the Blood-Brain Barrier.

Glioblastoma multiforme (GBM) is the most aggressive and common type of brain cancer. Five-year survival rates are below 12%, even with the most aggressive trimodal therapies. Poor blood-brain barrier (BBB) permeability of therapeutics is a major obstacle to efficacy. Intravenous administration of bevacizumab is the standard treatment for GBM. It has been recently demonstrated that a single intraarterial infusion of bevacizumab provides superior therapeutic outcomes in patients with recurrent GBM. Further GBM treatment benefits can be achieved through opening of the BBB before intraarterial infusion of bevacizumab. However, a rationale for intraarterial delivery and BBB opening when delivering antibodies is lacking. A method facilitating quantification of intraarterial delivery of bevacizumab is needed for more effective and personalized GBM treatment. Here, we demonstrate such a method using PET imaging of radiolabeled bevacizumab. Methods: Bevacizumab was conjugated with deferoxamine and subsequently radiolabeled with 89Zr. 89Zr-bevacizumab deferoxamine (89Zr-BVDFO) was prepared with a specific radioactivity of 81.4 ± 7.4 MBq/mg (2.2 ± 0.2 μCi/mg). Brain uptake of 89Zr-BVDFO on carotid artery and tail vein infusion with an intact BBB or with BBB opening with mannitol was initially monitored by dynamic PET, followed by whole-body PET/CT at 1 and 24 h after infusion. Th ex vivo biodistribution of 89Zr-BVDFO was also determined. Results: Intraarterial administration of 89Zr-BVDFO resulted in gradual accumulation of radioactivity in the ipsilateral hemisphere, with 9.16 ± 2.13 percentage injected dose/cm3 at the end of infusion. There was negligible signal observed in the contralateral hemisphere. BBB opening with mannitol before intraarterial infusion of 89Zr-BVDFO resulted in faster and higher uptake in the ipsilateral hemisphere (23.58 ± 4.46 percentage injected dose/cm3) and negligible uptake in the contralateral hemisphere. In contrast, intravenous infusion of 89Zr-BVDFO and subsequent BBB opening did not lead to uptake of radiotracer in the brain. The ex vivo biodistribution results validated the PET/CT studies. Conclusion: Our findings demonstrate that intraarterial delivery of bevacizumab into the brain across an osmotically opened BBB is effective, in contrast to the intravenous route.

Delivery of immunoglobulin G antibodies to the rat nervous system following intranasal administration: Distribution, dose-response, and mechanisms of delivery

The intranasal route has been hypothesized to circumvent the blood-brain and blood-cerebrospinal fluid barriers, allowing entry into the brain via extracellular pathways along olfactory and trigeminal nerves and the perivascular spaces (PVS) of cerebral blood vessels. We investigated the potential of the intranasal route to non-invasively deliver antibodies to the brain 30 min following administration by characterizing distribution, dose-response, and mechanisms of antibody transport to and within the brain after administering non-targeted radiolabeled or fluorescently-labeled full length immunoglobulin G (IgG) to normal adult female rats. Intranasal [125I]-IgG consistently yielded highest concentrations in the olfactory bulbs, trigeminal nerves, and leptomeningeal blood vessels with their associated PVS. Intranasal delivery also resulted in significantly higher [125I]-IgG concentrations in the CNS than systemic (intra-arterial) delivery for doses producing similar endpoint blood concentrations. Importantly, CNS targeting significantly increased with increasing dose only with intranasal administration, yielding brain concentrations that ranged from the low-to-mid picomolar range with tracer dosing (50 μg) up to the low nanomolar range at higher doses (1 mg and 2.5 mg). Finally, intranasal pre-treatment with a previously identified nasal permeation enhancer, matrix metalloproteinase-9, significantly improved intranasal [125I]-IgG delivery to multiple brain regions and further allowed us to elucidate IgG transport pathways extending from the nasal epithelia into the brain using fluorescence microscopy. The results show that it may be feasible to achieve therapeutic levels of IgG in the CNS, particularly at higher intranasal doses, and clarify the likely cranial nerve and perivascular distribution pathways taken by antibodies to reach the brain from the nasal mucosae.

Protein synthesis observed in vivo using injected deuterated amino acids and Raman scattering

Combining intra-arterial delivery of deuterated amino acids with stimulated Raman scattering microscopy to observe in vivo protein metabolism in mouse models.

Fate of mesoangioblasts in a vaginal birth injury model: influence of the route of administration

Currently cell therapy is considered as an experimental strategy to assist the healing process following simulated vaginal birth injury in rats, boosting the functional and morphologic recovery of pelvic floor muscles and nerves. However, the optimal administration route and dose still need to be determined. Mesangioblasts theoretically have the advantage that they can differentiate in skeletal and smooth muscle. We investigated the fate of mesoangioblasts transduced with luciferase and green fluorescent protein reporter genes (rMABseGFP/fLUC) using bioluminescence, immunofluorescence and RT-PCR in rats undergoing simulated birth injury. rMABseGFP/fLUC were injected locally, intravenously and intra-arterially (common iliacs and aorta). Intra-arterial delivery resulted in the highest amount of rMABseGFP/fLUC in the pelvic organs region and in a more homogeneous distribution over all relevant pelvic organs. Sham controls showed that the presence of the injury is important for recruitment of intra-arterially injected rMABseGFP/fLUC. Injection through the aorta or bilaterally in the common iliac arteries resulted in comparable numbers of rMABseGFP/fLUC in the pelvic organs, yet aortic injection was faster and gave less complications.

Abstract 4162: Lentivirally delivered shRNA knockdown of CXCL12 is effective at preventing radiation fibrosis in normal tissues

Abstract Late Adverse Effects (LAEs) following adjuvant radiotherapy (RT) are common sequelae in free flaps used for breast reconstruction and result in ongoing healthcare burdens during the phase of cancer survivorship. Intra-arterial delivery of gene therapy to an isolated vascular region permits localized, targeted genetic modulation of reconstructed tissue, avoiding potential off-target effects in microscopic residual disease. We studied the immunological component of LAE development in a validated accelerated rodent model recapitulating autologous flap reconstruction, across 90 days after RT (50 Gy/3). CXCL12 was significantly up-regulated from 2-21 days after RT, peaking at 7 days (p&amp;lt;0.001), and corresponded to an increase in CXCR4 expression (p=0.047). Multiplexed confocal imaging co-localised CXCL12 and γH2AX in fibroblast stromal cells after RT, confirming that CXCL12 was a flap-originating signal suitable for our therapy modality. This was associated with increased macrophage ingress and fibroblast activation as characterized by immunohistochemical (p=0.002) and flow cytometric end-points (p=0.035). In vitro work corroborated the relevance of the CXCL12 axis in the development of LAEs, demonstrating significant increase in CXCL12's transcriptome expression (p=0.013) and protein secretion (p=0.03) in primary rat fibroblasts stimulated with RT, high expression of CXCL12's receptors, CXCR4 and CXCR7 on M1 and M2 rat macrophages, and increased fibroblast activation, assayed with αSMA expression, with co-stimulation of CXCL12 and TGFβ. We hypothesized that LAEs in animals with flap-targeted knockdown of CXCL12 would be reduced. Flaps were infected with vascularly-delivered, lentiviral particles encoding shRNA against CXCL12 (LVshCXCL12), scrambled controls (LVSCR) alongside sham (phosphate-buffered saline (PBS)) and un-irradiated controls. Flaps infected with LVshCXCL12 exhibited reversal of LAE-mediated flap contracture and were not significantly different from un-irradiated flaps at 90 days post-RT, but were significantly greater in area compared to sham flaps or those infected with LVSCR (p&amp;lt;0.0001). Radiation Therapy Oncology Group (RTOG) LAE scores were also significantly improved compared with LvSCR (p&amp;lt;0.0001) and PBS groups (p=0.0011). Endpoint assays demonstrated significant vascular preservation (p=0.049) and reduced tissue collagen deposition (p=0.019). In conclusion, targeted knockdown of CXCL12 is effective at preventing the development of LAEs in flap tissue exposed to RT and is a promising strategy for clinical translation. Citation Format: James T. Paget, Martin McLaughlin, Joan N. Kyula, David Mansfield, Henry G. Smith, Victoria Roulstone, Paul A. Harris, Alan A. Melcher, Kevin J. Harrington, Aadil A. Khan. Lentivirally delivered shRNA knockdown of CXCL12 is effective at preventing radiation fibrosis in normal tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4162.

PO-121 Lentivirally-delivered ShRNA knockdown of CXCL12 prevents fibrosis in a rodent model of radiation late adverse effects

IntroductionLate Adverse Effects (LAEs) following adjuvant radiotherapy (RT) are common sequelae in free flaps used for breast reconstruction. Intra-arterial delivery of gene therapy to an isolated vascular region permits localised, targeted genetic modulation of reconstructed tissue, avoiding potential off-target effects in microscopic residual disease. We hypothesised that targeted knockdown of CXCL12 would reduce LAEs.Material and methodsLAE development was studied across 8 time points to 90 days after 50 Gy/3 in 24 male Fischer 344 rats in a validated LAE flap model. RT and control human skin and subcuticular tissue was obtained for validation from the PRADA study.Efficacy of ShCXCL12 against LAEs was assessed in 72 animals. 1 × 108 lentiviral particles encoding ShRNA against CXCL12 (ShCXCL12), RNA overexpressing CXCL12 (OeCXCL12), or scrambled control (SCR), or sham infection with PBS were delivered intra-arterially to the flap tissue 30 days before RT. Acute and Late toxicities were assessed using Radiation Therapy Oncology Group (RTOG) scores with tissue endpoints at 7, 21 and 90 days after RT.Results and discussionsCXCL12 and CXCR4 were up-regulated after RT, peaking at 7 days (p=0.001 and p=0.047 respectively). Confocal imaging co-localised CXCL12 and γH2AX in fibroblast stromal cells after RT, confirming that CXCL12 was flap-originating and suitable for our therapy. This was associated with increased macrophage ingress and fibroblast activation characterised by immunohistochemical and flow cytometric end-points (p=0.035). We corroborated these findings in human RT tissue, demonstrating increased CXCL12 (p=0.035) and CD68 (p=0.027) expression compared to paired controls.ShCXCL12 animals had reduced acute toxicities compared to LVSCR or PBS groups. OeCXCL12 animals had significantly worse toxicity. ShCXCL12 animals exhibited reversal of flap contracture and retained significantly greater flap area overall. RTOG late toxicity scores were significantly improved (p<0.0001).At day 7 and day 21 ShCXCL12 tissue contained fewer macrophages. This was not seen by day 90. Fibroblasts did not differ at day 7, but were reduced by day 21 and at day 90. Endpoint assays confirmed amelioration of the LAE phenotype with reduced tissue collagen deposition and reduced Acta2 expression.ConclusionCXCL12 over-expression post-RT signals an innate immune response mediated primarily by macrophages. Targeted ShCXCL12 can prevent the development of LAEs. Further work aims to understand the mechanism of immune-fibroblast cross-talk.