Immunocompetent Rats Research Articles

Development and Biocompatibility Assessment of Decellularized Porcine Uterine Extracellular Matrix-Derived Grafts.

Biomaterials derived from biological matrices have been widely investigated due to their great therapeutic potential in regenerative medicine, since they are able to induce cell proliferation, tissue remodeling, and angiogenesis in situ. In this context, highly vascularized and proliferative tissues, such as the uterine wall, present an interesting source to produce acellular matrices that can be used as bioactive materials to induce tissue regeneration. Therefore, this study aimed to establish an optimized protocol to generate decellularized uterine scaffolds (dUT), characterizing their structural, compositional, and biomechanical properties. In addition, in vitro performance and invivo biocompatibility were also evaluated to verify their potential applications for tissue repair. Results showed that the protocol was efficient to promote cell removal, and dUT general structure and extracellular matrix composition remained preserved compared with native tissue. In addition, the scaffolds were cytocompatible, allowing cell growth and survival. In terms of biocompatibility, the matrices did not induce any signs of immune rejection invivo in a model of subcutaneous implantation in immunocompetent rats, demonstrating an indication of tissue integration after 30 days of implantation. In summary, these findings suggest that dUT scaffolds could be explored as a biomaterial for regenerative purposes, which is beyond the studies in the reproductive field.

Unraveling devitalization: its impact on immune response and ectopic bone remodeling from autologous and allogeneic callus mimics.

Endochondral bone regeneration is a promising approach in regenerative medicine. Callus mimics (CMs) are engineered and remodeled into bone tissue upon implantation. The long-term objective is to fabricate a sustainable off-the-shelf treatment option for patients. Devitalization was introduced to facilitate storage and using allogeneic (donor) cells would further propel the off-the-shelf approach. However, allogeneic CMs for bone regeneration pose a potential antigenicity concern. Here, we explored the impact of devitalization on antigenicity and osteoinductive bone formation when implanting syngeneic or allogeneic CM in a vital or devitalized state. For this, we implanted chondrogenically differentiated rat-derived mesenchymal stromal cells using an allogeneic immunocompetent ectopic rat model. Vital syngeneic CMs demonstrated the highest bone formation, and vital allogeneic CMs showed the lowest bone formation, while both devitalized CMs showed comparable intermediate levels of bone formation. Preceding bone formation, the level of tartrate-resistant acid phosphatase staining at 7 and 14 days was proportional to the level of eventual bone formation. No differences were observed for local innate immune responses at any time point before or after bone formation. In contrast, allogeneic CMs elicit a mild adaptive immune response, which still permits bone formation in an immunocompetent environment, albeit at a reduced rate compared to the autologous living counterpart. Overall, devitalization delays bone formation when autologous CMs are implanted, whereas it accelerates bone formation in allogeneic CMs, highlighting the potential of this approach for achieving off-the-shelf treatment.

Intracavitary cisplatin-fibrin followed by irradiation improved tumor control compared to the single treatments in a mesothelioma rat model

ObjectiveTo test the safety and efficacy of combination treatment for pleural mesothelioma (PM) with intracavitary cisplatin-fibrin (cis-fib) plus hemithoracic irradiation (IR) applied after lung-sparing surgery in an orthotopic immunocompetent rat model. MethodsWe randomized male F344 rats into 5 groups: cis-fib (n = 9), 10 Gy IR (n = 6), 20 Gy IR (n = 9), cis-fib+10 Gy IR (n = 6), and cis-fib+20 Gy IR (n = 9). Subpleural tumor implantation was performed on day 0 with 1 million syngeneic rat mesothelioma cells (IL45-luciferase). Tumors were resected on day 9, followed by treatment with intracavitary cis-fib or vehicle control (NaCl-fib). On day 12, computed tomography–guided local irradiation in a single high dose of the former tumor region was applied. ResultsWe observed only short-term side effects related to 20 Gy radiotherapy. Compared to 20 Gy, 10 Gy IR did not show an impact on tumor growth. At 3 days after treatment with 20 Gy IR (day 15 of the experiment), we detected significantly smaller tumors in the cis-fib+IR group compared to IR alone (mean tumor growth, 252% vs 539%; P = .04). On day 21, there was a significant difference in tumor growth between cis-fib–treated and cis-fib+IR– treated tumors (mean tumor growth, 2295% vs 660%; P = .01). ConclusionsLocalized treatment after tumor resection in PM aims to improve local tumor control. Irradiation applied in combination with intracavitary cis-fib in rats is safe up to a dosage of 20 Gy and shows an additive effect on tumor growth delay compared to the single treatments.

Establishment of orthotopic osteosarcoma animal models in immunocompetent rats through muti-rounds of in-vivo selection

Immunodeficient murine models are usually used as the preclinical models of osteosarcoma. Such models do not effectively simulate the process of tumorigenesis and metastasis. Establishing a suitable animal model for understanding the mechanism of osteosarcoma and the clinical translation is indispensable. The UMR-106 cell suspension was injected into the marrow cavity of Balb/C nude mice. Tumor masses were harvested from nude mice and sectioned. The tumor fragments were transplanted into the marrow cavities of SD rats immunosuppressed with cyclosporine A. Through muti-rounds selection in SD rats, we constructed orthotopic osteosarcoma animal models using rats with intact immune systems. The primary tumor cells were cultured in-vitro to obtain the immune-tolerant cell line. VX2 tumor fragments were transplanted into the distal femur and parosteal radius of New Zealand white rabbit to construct orthotopic osteosarcoma animal models in rabbits. The rate of tumor formation in SD rats (P1 generation) was 30%. After four rounds of selection and six rounds of acclimatization in SD rats with intact immune systems, we obtained immune-tolerant cell lines and established the orthotopic osteosarcoma model of the distal femur in SD rats. Micro-CT images confirmed tumor-driven osteolysis and the bone destruction process. Moreover, the orthotopic model was also established in New Zealand white rabbits by implanting VX2 tumor fragments into rabbit radii and femurs. We constructed orthotopic osteosarcoma animal models in rats with intact immune systems through muti-rounds in-vivo selection and the rabbit osteosarcoma model.

Unveiling Drivers of Retinal Degeneration in RCS Rats: Functional, Morphological, and Molecular Insights.

Retinal degenerative diseases, including age-related macular degeneration and retinitis pigmentosa, significantly contribute to adult blindness. The Royal College of Surgeons (RCS) rat is a well-established disease model for studying these dystrophies; however, molecular investigations remain limited. We conducted a comprehensive analysis of retinal degeneration in RCS rats, including an immunodeficient RCS (iRCS) sub-strain, using ocular coherence tomography, electroretinography, histology, and molecular dissection using transcriptomics and immunofluorescence. No significant differences in retinal degeneration progression were observed between the iRCS and immunocompetent RCS rats, suggesting a minimal role of adaptive immune responses in disease. Transcriptomic alterations were primarily in inflammatory signaling pathways, characterized by the strong upregulation of Tnfa, an inflammatory signaling molecule, and Nox1, a contributor to reactive oxygen species (ROS) generation. Additionally, a notable decrease in Alox15 expression was observed, pointing to a possible reduction in anti-inflammatory and pro-resolving lipid mediators. These findings were corroborated by immunostaining, which demonstrated increased photoreceptor lipid peroxidation (4HNE) and photoreceptor citrullination (CitH3) during retinal degeneration. Our work enhances the understanding of molecular changes associated with retinal degeneration in RCS rats and offers potential therapeutic targets within inflammatory and oxidative stress pathways for confirmatory research and development.

Liver metastatic colonization by invasive cancer cells: a review of potential biomarkers with mitochondrial involvement

The liver, characterized by a unique metabolic and immunosuppressive environment, is also the organ to which invasive malignant cells of many different cancer types most frequently metastasize. The reasons for this organ-specific metastatic process have been investigated for decades. This review first provides an overview of recent breakthroughs in this field, introducing intercellular communication between circulating tumor cells and the heterogeneous cell populations of the liver, and modifications to the extracellular matrix (ECM). Subsequently, to improve the understanding of the molecular mechanisms involved in the metastasis of colorectal cancer to the liver, the second leading cause of cancer-related mortality, the recent literature on this question was analyzed. Among the various parameters involved, the mechanisms behind the activation of hepatic stellate cells, proteins inducing ECM remodeling, specific genomic features of liver metastases, metabolic rewiring, and characteristics of stromal-enriched microenvironments were discussed. To provide more insights into the molecular determinants of liver metastatic colonization, important findings reported on a set of mitochondrial proteins were addressed, the relative abundance of which changed in the liver during the progression stage of an aggressive experimental model of peritoneal malignant mesothelioma in immunocompetent rats. Based on previous studies cross-comparing the liver proteomes from curcumin-treated tumor-bearing rats/untreated tumor-bearing rats/normal rats, data from the literature were reviewed for 25 mitochondrial proteins of interest. Their role in lipid metabolism, heme biosynthesis, the electron transport chain, small molecule transport, mitochondrial dynamics, the tricarboxylic acid cycle, and protection against oxidative stress were analyzed in the context of both cancer and non-malignant liver diseases.

Abstract 1453: Development and evaluation of subcutaneous and orthotopic PC3M metastatic prostate cancer xenograft model in SRG rats for therapeutic assessment

Abstract Introduction: Prostate cancer is the most prevalent cause of cancer deaths in American males after lung cancer. The establishment of clinically relevant prostate cancer models is crucial for advancing our understanding of the disease and evaluating potential therapeutics. Oncology studies in rats provide a secondary species to mice with the advantage of extensive longitudinal blood sampling and tumor biopsies in pharmacodynamics analysis. This in vivo study evaluates the behavior of subcutaneous and orthotopic luciferase (luc) tagged metastatic PC3M-luc cells in immunodeficient SRG rats (Hera Biolabs) for tumor growth kinetics, metastasis, and response to paclitaxel and carboplatin alone and in combination. Methods: PC3M-luc cells were implanted subcutaneously into the right flank or orthotopically into the prostate of SRG rats. Subcutaneous tumor progression and treatment responses were monitored using calipers and bioluminescent imaging (BLI), with the capacity to detect developing metastases prior to necropsy. Orthotopic implants were monitored exclusively using BLI. Tolerability was evaluated based on biweekly changes in body weight, daily observations for signs of tumor- or treatment-related clinical signs, necropsy results, and complete blood count (CBC) and clinical chemistry marker comparisons with immunocompetent CD rats. Results: PC3M-luc prostate carcinoma was successfully established orthotopically and subcutaneously in SRG rats, with similar growth rates and the development of spontaneous metastases to distant organs, modeling metastases observed in prostate cancer patients. Paclitaxel treatment was less tolerated in SRG rats in comparison to nude rats, enhanced toxicity was observed when combined with carboplatin. Comparison of CBC and clinical chemistry blood markers in naïve immunocompetent CD and immunodeficient SRG rats differed mainly in White Blood Cell (WBC) counts that were significantly lower in SRG rats, as expected due to the lack of mature B, T, and NK cells in these animals. Conclusion: The development of orthotopic and subcutaneous PC3M-luc prostate cancer models in rats provides a valuable alternative platform for assessing the effectiveness of established and novel therapies. This rat-based models offers a secondary species for comparison of prostate cancer behavior in mice, successfully mimics the clinical development of metastases, and provides additional insights into disease progression and therapeutic strategies. Future studies will focus on the assessment of tolerability and combination therapy strategies for improved treatment outcomes. Citation Format: Kyle Draheim, Elizabeth Rainbolt, Bincy John, Cara Clouse, Karyn Shinn, Justin Avery, Koh Meng Aw Yong, Chassidy Hall, Patrick Fadden, Anya Avrutskaya. Development and evaluation of subcutaneous and orthotopic PC3M metastatic prostate cancer xenograft model in SRG rats for therapeutic assessment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1453.

Development of an immunosuppressed orthotopic hepatocellular carcinoma rat model for the evaluation of chemo- and radioembolization therapies

Hepatocellular carcinoma (HCC) is widely known to be chemo-resistant and presents with significant liver disease resulting in low tolerability to systemic chemotherapy. As a counter measure, more targeted therapies such as trans-arterial chemoembolization (TACE) and trans-arterial radioembolization (TARE) have been developed. To further optimize these therapies, animal models are critical in elucidating the molecular events in disease progression and test new treatment options. The present study focuses on the development of a hepatoma bearing rat model. N1S1 rat hepatoma cells were transfected by a lentiviral method and injected into the liver of Sprague Dawley (SD) and Rowett Nude (RNU) rats. Longitudinal tumor growth was observed by bioluminescence imaging (BLI) and liver/tumor histology. In both models, tumors were visible within 4 days post cell inoculation. Tumor take rates were 52 % and 73 % for male and female SD rats, respectively, and 100 % for male RNU rats. By day 12 and 15 post inoculation, we recorded complete tumor regression in male and female SD rats. Liver histology showed advanced fibrosis in the tumor regressed SD rats, whilst RNU rats exhibited the characteristic sheet pattern of Novikoff tumor with mild liver fibrosis. Increased CD3 and TUNEL staining observed in SD rat livers may be key factors for tumor regression. Our data reveal that the immunocompetent SD rats are not recommended as a model for therapeutic investigations. The immunosuppressed RNU rats, however, are characterized by consistent and reliable tumor growth and thus a desirable model for future therapeutic investigations.

Engineering 3D-Printed Bioresorbable Scaffold to Improve Non-Vascularized Fat Grafting: A Proof-of-Concept Study.

Autologous fat grafting is the gold standard for treatment in patients with soft-tissue defects. However, the technique has a major limitation of unpredictable fat resorption due to insufficient blood supply in the initial phase after transplantation. To overcome this problem, we investigated the capability of a medical-grade poly L-lactide-co-poly ε-caprolactone (PLCL) scaffold to support adipose tissue and vascular regeneration. Deploying FDM 3D-printing, we produced a bioresorbable porous scaffold with interconnected pore networks to facilitate nutrient and oxygen diffusion. The compressive modulus of printed scaffold mimicked the mechanical properties of native adipose tissue. In vitro assays demonstrated that PLCL scaffolds or their degradation products supported differentiation of preadipocytes into viable mature adipocytes under appropriate induction. Interestingly, the chorioallantoic membrane assay revealed vascular invasion inside the porous scaffold, which represented a guiding structure for ingrowing blood vessels. Then, lipoaspirate-seeded scaffolds were transplanted subcutaneously into the dorsal region of immunocompetent rats (n = 16) for 1 or 2 months. The volume of adipose tissue was maintained inside the scaffold over time. Histomorphometric evaluation discovered small- and normal-sized perilipin+ adipocytes (no hypertrophy) classically organized into lobular structures inside the scaffold. Adipose tissue was surrounded by discrete layers of fibrous connective tissue associated with CD68+ macrophage patches around the scaffold filaments. Adipocyte viability, assessed via TUNEL staining, was sustained by the presence of a high number of CD31-positive vessels inside the scaffold, confirming the CAM results. Overall, our study provides proof that 3D-printed PLCL scaffolds can be used to improve fat graft volume preservation and vascularization, paving the way for new therapeutic options for soft-tissue defects.

In vitro and in vivo activity of cefiderocol against Achromobacter spp. and Burkholderia cepacia complex, including carbapenem-non-susceptible isolates.

Achromobacter spp. and Burkholderia cepacia complex (Bcc) are rare but diverse opportunistic pathogens associated with serious infections, which are often multidrug resistant. This study compared the in vitro antibacterial activity of the siderophore antibiotic cefiderocol against Achromobacter spp. and Bcc isolates with that of other approved antibacterial drugs, including ceftazidime-avibactam, ciprofloxacin, colistin, imipenem-relebactam, and meropenem-vaborbactam. Isolates were collected in the SIDERO multinational surveillance program. Among 334 Achromobacter spp. isolates [76.6% from respiratory tract infections (RTIs)], cefiderocol had minimum inhibitory concentration (MIC)50/90 of 0.06/0.5 µg/mL overall and 0.5/4 µg/mL against 52 (15.6%) carbapenem-non-susceptible (Carb-NS) isolates. Eleven (3.3%) Achromobacter spp. isolates overall and 6 (11.5%) Carb-NS isolates were not susceptible to cefiderocol. Among 425 Bcc isolates (73.4% from RTIs), cefiderocol had MIC50/90 of ≤0.03/0.5 µg/mL overall and ≤0.03/1 µg/mL against 184 (43.3%) Carb-NS isolates. Twenty-two (5.2%) Bcc isolates overall and 13 (7.1%) Carb-NS isolates were not susceptible to cefiderocol. Cumulative MIC distributions showed cefiderocol to be the most active of the agents tested in vitro against both Achromobacter spp. and Bcc. In a neutropenic murine lung infection model and a humanized pharmacokinetic immunocompetent rat lung infection model, cefiderocol showed significant bactericidal activity against two meropenem-resistant Achromobacter xylosoxidans strains compared with untreated controls (P < 0.05) and vehicle-treated controls (P < 0.05), respectively. Meropenem, piperacillin-tazobactam, ceftazidime, and ciprofloxacin comparators showed no significant activity in these models. The results suggest that cefiderocol could be a possible treatment option for RTIs caused by Achromobacter spp. and Bcc.

Implantable SDF-1α-loaded silk fibroin hyaluronic acid aerogel sponges as an instructive component of the glioblastoma ecosystem: Between chemoattraction and tumor shaping into resection cavities

In view of inevitable recurrences despite resection, glioblastoma (GB) is still an unmet clinical need. Dealing with the stromal-cell derived factor 1-alpha (SDF-1α)/CXCR4 axis as a hallmark of infiltrative GB tumors and with the resection cavity situation, the present study described the effects and relevance of a new engineered micro-nanostructured SF-HA-Hep aerogel sponges, made of silk fibroin (SF), hyaluronic acid (HA) and heparin (Hep) and loaded with SDF-1α, to interfere with the GB ecosystem and residual GB cells, attracting and confining them in a controlled area before elimination. 70 µm-pore sponges were designed as an implantable scaffold to trap GB cells. They presented shape memory and fit brain cavities. Histological results after implantation in brain immunocompetent Fischer rats revealed that SF-HA-Hep sponges are well tolerated for more than 3 months while moderately and reversibly colonized by immuno-inflammatory cells. The use of human U87MG GB cells overexpressing the CXCR4 receptor (U87MG-CXCR4+) and responding to SDF-1α allowed demonstrating directional GB cell attraction and colonization of the device in vitro and in vivo in orthotopic resection cavities in Nude rats. Not modifying global survival, aerogel sponge implantation strongly shaped U87MG-CXCR4+ tumors in cavities in contrast to random infiltrative growth in controls. Overall, those results support the interest of SF-HA-Hep sponges as modifiers of the GB ecosystem dynamics acting as “cell meeting rooms” and biocompatible niches whose properties deserve to be considered toward the development of new clinical procedures. Statement of SignificanceBrain tumor glioblastoma (GB) is one of the worst unmet clinical needs. To prevent the relapse in the resection cavity situation, new implantable biopolymer aerogel sponges loaded with a chemoattractant molecule were designed and preclinically tested as a prototype targeting the interaction between the initial tumor location and its attraction by the peritumoral environment. While not modifying global survival, biocompatible SDF1-loaded hyaluronic acid and silk fibroin sponges induce directional GB cell attraction and colonization in vitro and in rats in vivo. Interestingly, they strongly shaped GB tumors in contrast to random infiltrative growth in controls. These results provide original findings on application of exogenous engineered niches that shape tumors and serve as cell meeting rooms for further clinical developments.

Neuroprotective potential of intranasally delivered L-myc immortalized human neural stem cells in female rats after a controlled cortical impact injury

Efficacious stem cell-based therapies for traumatic brain injury (TBI) depend on successful delivery, migration, and engraftment of stem cells to induce neuroprotection. L-myc expressing human neural stem cells (LMNSC008) demonstrate an inherent tropism to injury sites after intranasal (IN) administration. We hypothesize that IN delivered LMNSC008 cells migrate to primary and secondary injury sites and modulate biomarkers associated with neuroprotection and tissue regeneration. To test this hypothesis, immunocompetent adult female rats received either controlled cortical impact injury or sham surgery. LMNSC008 cells or a vehicle were administered IN on postoperative days 7, 9, 11, 13, 15, and 17. The distribution and migration of eGFP-expressing LMNSC008 cells were quantified over 1 mm-thick optically cleared (CLARITY) coronal brain sections from TBI and SHAM controls. NSC migration was observed along white matter tracts projecting toward the hippocampus and regions of TBI. ELISA and Nanostring assays revealed a shift in tissue gene expression in LMNSC008 treated rats relative to controls. LMNSC008 treatment reduced expression of genes and pathways involved in inflammatory response, microglial function, and various cytokines and receptors. Our proof-of-concept studies, although preliminary, support the rationale of using intranasal delivery of LMNSC008 cells for functional studies in preclinical models of TBI and provide support for potential translatability in TBI patients.

Retention of Human iPSC-Derived or Primary Cells Following Xenotransplantation into Rat Immune-Privileged Sites.

In regenerative medicine, experimental animal models are commonly used to study potential effects of human cells as therapeutic candidates. Although some studies describe certain cells, such as mesenchymal stromal cells (MSC) or human primary cells, as hypoimmunogenic and therefore unable to trigger strong inflammatory host responses, other studies report antibody formation and immune rejection following xenotransplantation. Accordingly, the goal of our study was to test the cellular retention and survival of human-induced pluripotent stem cell (iPSCs)-derived MSCs (iMSCs) and primary nucleus pulposus cells (NPCs) following their xenotransplantation into immune-privileged knee joints (14 days) and intervertebral discs (IVD; 7 days) of immunocompromised Nude and immunocompetent Sprague Dawley (SD) rats. At the end of both experiments, we could demonstrate that both rat types revealed comparably low levels of systemic IL-6 and IgM inflammation markers, as assessed via ELISA. Furthermore, the number of recovered cells was with no significant difference between both rat types. Conclusively, our results show that xenogeneic injection of human iMSC and NPC into immunoprivileged knee and IVD sites did not lead to an elevated inflammatory response in immunocompetent rats when compared to immunocompromised rats. Hence, immunocompetent rats represent suitable animals for xenotransplantation studies targeting immunoprivileged sites.

Umbilical Cord Stem Cell Lysate: A New Biologic Injectate for the Putative Treatment of Acute Temporomandibular Joint Inflammation.

To compare in vivo, the acute anti-inflammatory effects of a lysate derived from human umbilical perivascular mesenchymal cells with the cells themselves in both an established hind-paw model of carrageenan-induced inflammation and also in the inflamed temporomandibular joint. Human umbilical cord perivascular cells were harvested and cultured in xeno- and serum-free conditions to P3. In addition, P3 cells were used to prepare a proprietary 0.22 micron filtered lysate. First, CD1 immunocompetent mice underwent unilateral hind-paw injections of carrageenan for induction of inflammation, followed immediately by treatment with saline (negative control), 1% cell lysate, or viable cells. The contralateral paw remained un-injected with carrageenan. Paw circumference was measured prior to injections and 48 hr later and myeloperoxidase and TNF-alpha concentrations were measured post-sacrifice in excised tissue. Second, immunocompetent Male Wistar rats underwent unilateral intra-articular temporomandibular (TMJ) injections from the same treatment groups and were sacrificed at 4 and 48 hr post-injection. The contralateral TMJ remained un-injected with carrageenan. Articular tissue and synovial aspirates, from the treated TMJ were obtained for histologic and leukocyte infiltration analyses. The lysate and cell-treated hind-paw demonstrated reduced tissue edema, and significantly lower concentrations of myeloperoxidase and TNF-alpha at 48 hr compared to untreated controls. Treated TMJs demonstrated lower concentrations of leukocytes in the synovium compared to controls and histologic evidence, in the peri-articular tissue, of reduced inflammation. In this preliminary study, both the human umbilical perivascular cells and a highly diluted lysate produced therefrom were anti-inflammatory.

Radical Tumor Denervation Activates Potent Local and Global Cancer Treatment.

This preliminary study seeks to determine the effect of R&P denervation on tumor growth and survival in immunocompetent rats bearing an aggressive and metastatic breast solid tumor. A novel microsurgical approach was applied "in situ", aiming to induce R&P denervation through the division of every single nerve fiber connecting the host with the primary tumor via its complete detachment and re-attachment, by resecting and reconnecting its supplying artery and vein (anastomosis). This preparation, known as microsurgical graft or flap, is radically denervated by definition, but also effectively delays or even impedes the return of innervation for a significant period of time, thus creating a critical and therapeutic time window. Mammary adenocarcinoma cells (HH-16.cl4) were injected into immunocompetent Sprague Dawley adult rats. When the tumors reached a certain volume, the subjects entered the study. The primary tumor, including a substantial amount of peritumoral tissue, was surgically isolated on a dominant artery and vein, which was resected and reconnected using a surgical microscope (orthotopic tumor auto-transplantation). Intending to simulate metastasis, two or three tumors were simultaneously implanted and only one was treated, using the surgical technique described herein. Primary tumor regression was observed in all of the microsurgically treated subjects, associated with a potent systemic anticancer effect and prolonged survival. In stark contrast, the subjects received a close to identical surgical operation; however, with the intact neurovascular connection, they did not achieve the therapeutic result. Animals bearing multiple tumors and receiving the same treatment in only one tumor exhibited regression in both the "primary" and remote- untreated tumors at a clinically significant percentage, with regression occurring in more than half of the treated subjects. A novel therapeutic approach is presented, which induces the permanent regression of primary and, notably, remote tumors, as well as, evidently, the naturally occurring metastatic lesions, at a high rate. This strategy is aligned with the impetus that comes from the current translational research data, focusing on the abrogation of the neuro-tumoral interaction as an alternative treatment strategy. More data regarding the clinical significance of this are expected to come up from a pilot clinical trial that is ongoing.

Therapeutic potential of extracellular vesicles derived from cardiac progenitor cells in rodent models of chemotherapy-induced cardiomyopathy.

Current treatments of chemotherapy-induced cardiomyopathy (CCM) are of limited efficacy. We assessed whether repeated intravenous injections of human extracellular vesicles from cardiac progenitor cells (EV-CPC) could represent a new therapeutic option and whether EV manufacturing according to a Good Manufacturing Practices (GMP)-compatible process did not impair their bioactivity. Immuno-competent mice received intra-peritoneal injections (IP) of doxorubicin (DOX) (4 mg/kg each; cumulative dose: 12 mg/kg) and were then intravenously (IV) injected three times with EV-CPC (total dose: 30 billion). Cardiac function was assessed 9-11 weeks later by cardiac magnetic resonance imaging (CMR) using strain as the primary end point. Then, immuno-competent rats received 5 IP injections of DOX (3 mg/kg each; cumulative dose 15 mg/kg) followed by 3 equal IV injections of GMP-EV (total dose: 100 billion). Cardiac function was assessed by two dimensional-echocardiography. In the chronic mouse model of CCM, DOX + placebo-injected hearts incurred a significant decline in basal (global, epi- and endocardial) circumferential strain compared with sham DOX-untreated mice (p = 0.043, p = 0.042, p = 0.048 respectively) while EV-CPC preserved these indices. Global longitudinal strain followed a similar pattern. In the rat model, IV injections of GMP-EV also preserved left ventricular end-systolic and end-diastolic volumes compared with untreated controls. Intravenously-injected extracellular vesicles derived from CPC have cardio-protective effects which may make them an attractive user-friendly option for the treatment of CCM.

91-OR: AdoShell Islet, a New Biomaterial for Allogenic Islet Encapsulation Shows Six Months Insulin Secretion in Diabetic Immunocompetent Rats

Islet transplantation is an effective cure for T1D but the requirement for immunosuppression makes its application limited. AdoShell® is an implantable and fully retrievable scaffold for islet transplantation without immunosuppression. It is based on a permselective hydrogel film that allows the diffusion of insulin and prevents immune cell invasion. AdoShell® is a soft biomaterial with 95% water, synthetized from non-degradable polymers cross-linked by bio-orthogonal click chemistry. Encapsulated human islets displayed a gluco-responsive insulin secretion comparable to naked islets in perifusion experiments after 7 days and 1 month (N=3 donors 7 days / N=2 donors 1 month). Wistar rat allogenic islets encapsulated in AdoShell® were implanted in immunocompetent STZ-induced diabetic rats. Insulin levels in vivo were on average 121pM/day over 41 days (n=22 rats), a significant increase versus negative control (45pM/day, n=9 rats) (M.W. t-test, p&amp;lt;0.0001). AdoShell® Islets led to physiological weight gain, reduction of fasting hyperglycemia, and sustained C-peptide and insulin secretion for over 6 months in diabetic rats. It was well tolerated and did not trigger inflammatory reaction nor fibrosis after the 6-month implantation period in rat peritoneal cavity. Neither biodegradation, nor immune cell penetration were observed. Disclosure A.Geissler: Employee; Adocia, Stock/Shareholder; Adocia. O.Jouannot: Employee; ADOCIA, Stock/Shareholder; ADOCIA. A.Gaffuri: Employee; ADOCIA, Stock/Shareholder; ADOCIA. C.Gautier: Employee; Centre européen d'étude du diabète, Biomeostasis, Charles Rivers Laboratories, Institut de recherche contre les cancers de l'appareil digestif, GenOway, NAMSA. Y.P.Courbebaisse: None. K.Bouzakri: Consultant; Adocia, Stock/Shareholder; ILONOV. R.Eloy: Stock/Shareholder; Adocia. O.Soula: Employee; Adocia, Stock/Shareholder; Adocia.

Advances in Immunocompetent Mouse and Rat Models.

Rodent models of breast cancer have played critical roles in our understanding of breast cancer development and progression as well as preclinical testing of cancer prevention and therapeutics. In this article, we first review the values and challenges of conventional genetically engineered mouse (GEM) models and newer iterations of these models, especially those with inducible or conditional regulation of oncogenes and tumor suppressors. Then, we discuss nongermline (somatic) GEM models of breast cancer with temporospatial control, made possible by intraductal injection of viral vectors to deliver oncogenes or to manipulate the genome of mammary epithelial cells. Next, we introduce the latest development in precision editing of endogenous genes using in vivo CRISPR-Cas9 technology. We conclude with the recent development in generating somatic rat models for modeling estrogen receptor-positive breast cancer, something that has been difficult to accomplish in mice.

Zika Virus affects neurobehavioral development, and causes oxidative stress associated to blood–brain barrier disruption in a rat model of congenital infection

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with several neurodevelopmental outcomes after in utero infection. Here, we studied a congenital ZIKV infection model with immunocompetent Wistar rats, able to predict disabilities and that could pave the way for proposing new effective therapies. We identified neurodevelopmental milestones disabilities in congenital ZIKV animals. Also, on 22nd postnatal day (PND), blood–brain barrier (BBB) proteins disturbances were detected in the hippocampus with immunocontent reduction of β_Catenin, Occludin and Conexin-43. Besides, oxidative stress imbalance on hippocampus and cortex were identified, without neuronal reduction in these structures. In conclusion, even without pups’ microcephaly-like phenotype, congenital ZIKV infection resulted in neurobehavioral dysfunction associated with BBB and oxidative stress disturbances in young rats. Therefore, our findings highlighted the multiple impact of the congenital ZIKV infection on the neurodevelopment, which reinforces the continuity of studies to understand the spectrum of this impairment and to provide support to future treatment development for patients affected by congenital ZIKV.

Comparison of Decellularized Human Dermal Scaffolds versus Bovine Collagen/Elastin Matrices for Engineering of Soft-Tissue Flaps.

Free flap-based soft-tissue reconstruction comes at the price of donor-site morbidity. The arteriovenous loop (AVL) technique can overcome this issue by allowing for the de novo generation of axially vascularized soft-tissue flaps from vein grafts embedded into different matrices. Application of the AVL technique has been limited by insufficient long-term volume retention and poor tissue stability. The authors investigated the suitability of a novel human dermal scaffold to improve volume retention and tissue stability. AVLs were created in 28 immunocompetent rats and embedded in either decellularized human dermal scaffolds (experimental group, n = 14) (Epiflex) or bovine collagen/elastin matrices (control group, n = 14) (MatriDerm) in subcutaneous polytetrafluoroethylene chambers. The weight and volume of engineered tissues, the extent of angiogenesis, and the proportion of proliferating cells were compared between groups on postoperative days (PODs) 21 and 28 by means of immunohistochemistry and micro-computed tomography. On POD 28, both groups displayed homogeneous microvascular networks on histopathology and micro-computed tomography. Mean microvessel counts and surface areas and the percentage of proliferating cells did not differ between the groups. However, the experimental human scaffold group displayed significantly smaller volume loss and significantly less tissue degradation compared with bovine matrix controls (volume retention, 102% ± 5% versus 27% ± 7% on POD 21, and 79% ± 12% versus 12% ± 7% on POD 28, respectively; P < 0.0001). Compared with bovine matrices, decellularized human scaffolds allow for superior volume retention and tissue stability of de novo engineered soft-tissue AVL flaps in rats. AVLs allow for the de novo generation of vascularized soft-tissue flaps. However, insufficient long-term volume retention is still an issue. The authors' study shows that decellularized human matrices guarantee superior volume stability of de novo grown soft-tissue flaps in rats.