Kidney Capsule Research Articles

Modulation Effects of Eugenol on Nephrotoxicity Triggered by Silver Nanoparticles in Adult Rats.

The use of silver nanoparticles (AgNPs) is expanding. This study evaluates the modulator effect of eugenol (Eug) on AgNP-induced nephrotoxicity in rats. Sixty male rats were separated into six groups: control, Eug, AgNPs low-dose, AgNPs high-dose, Eug + AgNPs low-dose, and Eug + AgNPs high-dose. After 30 days, kidney function, antioxidative and proinflammatory status, histopathological, histomorphometrical, and immunohistochemical assessments were performed. AgNPs markedly induced oxidative stress in renal tissues, characterized by increased levels of blood urea nitrogen, creatinine, uric acid, kidney injury molecule-1, the total oxidant capacity, malondialdehyde, tumor necrosis factor-alpha (TNF-α), and interleukin-6, as well as decreased levels of the total antioxidant capacity, superoxide dismutase, catalase, reduced glutathione, and glutathione peroxidase. Moreover, the normal renal architecture was destroyed, and the thickness of the renal capsules, cortex, and medulla, alongside the diameter and quantity of the normal Malpighian corpuscles and the proximal and distal convoluted tubules were decreased. Immunoreactivity for P53, caspase-3, and TNF-α reactive proteins were significantly increased; however, Bcl-2 immunoreactivity was decreased. Eug reversed most biochemical, histological, histomorphometrical, and immunohistochemical changes in AgNP-treated animals. This study demonstrated that nephrotoxicity in AgNP-treated rats was mitigated by an Eug supplementation. Eug's antioxidant, antiapoptotic, and anti-inflammatory capabilities were the key in modulating AgNPs nephrotoxicity.

Human stem cell derived beta-like cells engineered to present PD-L1 improve transplant survival in NOD mice carrying human HLA class I.

There is a critical need for therapeutic approaches that combine renewable sources of replacement beta cells with localized immunomodulation to counter recurrence of autoimmunity in type 1 diabetes (T1D). However, there are few examples of animal models to study such approaches that incorporate spontaneous autoimmunity directed against human beta cells rather than allogenic rejection. Here, we address this critical limitation by demonstrating rejection and survival of transplanted human stem cell-derived beta-like cells clusters (sBCs) in a fully immune competent mouse model with matching human HLA class I and spontaneous diabetes development. We engineered localized immune tolerance toward transplanted sBCs via inducible cell surface overexpression of PD-L1 (iP-sBCs) with and without deletion of all HLA class I surface molecules via beta-2 microglobulin knockout (iP-BKO sBCs). NOD.HLA-A2.1 mice, which lack classical murine MHC I and instead express human HLA-A*02:01, underwent transplantation of 1,000 human HLA-A*02:01 sBCs under the kidney capsule and were separated into HLA-A2 positive iP-sBC and HLA-class I negative iP-BKO sBC groups, each with +/- doxycycline (DOX) induced PD-L1 expression. IVIS imaging showed significantly improved graft survival in mice transplanted with PD-L1 expressing iP-sBC at day 3 post transplantation compared to controls. However, luciferase signal dropped below in vivo detection limits by day 14 for all groups in this aggressive immune competent diabetes model. Nonetheless, histological examination revealed significant numbers of surviving insulin+/PD-L1+ sBCs cells for DOX-treated mice at day 16 post-transplant despite extensive infiltration with high numbers of CD3+ and CD45+ immune cells. These results show that T cells rapidly infiltrate and attack sBC grafts in this model but that significant numbers of PD-L1 expressing sBCs manage to survive in this harsh immunological environment. This investigation represents one of the first in vivo studies recapitulating key aspects of human autoimmune diabetes to test immune tolerance approaches with renewable sources of beta cells.

Functional Human iPSC-Derived Thymic Epithelial Progenitor Cells Reconstitute T Cell Development and Function in an In Vivo Model of Thymic Aplasia

The thymus is an essential primary lymphoid organ in which T cells mature, proliferate, and acquire a broad and self-tolerant T cell receptor repertoire. In the thymus, specialized thymic epithelial cells (TECs) select T cells capable of recognizing foreign antigens, and eliminate self-reactive T cells to prevent autoimmunity. In addition, the thymus also contributes to the differentiation of regulatory T cells which confine immune responses to actively preserve central tolerance. During thymic development, the interactions between hematopoietic lymphoid progenitor cells and thymic epithelial progenitor cells (TEPCs) provides indispensable signals to the development of the thymic epithelium. In humans, unlike in mice, the development of the thymus is completed before birth. There is no evidence of thymic stem cells that could contribute to a sustained regeneration of the thymic epithelium throughout life. Accordingly, the thymus has no endogenous capacity to regenerate. The thymus peaks in size during infancy and involutes with age, leading to the weakened immune function of the elderly known as immune senescence. Allogeneic hematopoietic stem cell transplant (HSCT) is a curative strategy for many malignant and non-malignant hematopoietic diseases, however, often at the cost of causing severe thymic dysfunction due to conditioning medications and graft-versus-host disease (GVHD). This can severely impair T cell immune reconstitution post-HSCT leading to life-threatening complications. In addition, genetic forms of congenital athymia (e.g. 22q11 Deletion Syndrome, TBX1, TBX2, PAX1, and CHD7 deficiency) can result in absence of T cell development. Complete congenital athymia is fatal early in life unless immune reconstitution is achieved by allogeneic thymus transplantation. However, the shortage of transplantable donor thymus tissue and lack of HLA-matching between donor and recipient limits the use of allogeneic thymus transplantation to a small number of children with complete congenital athymia. Thus, there is a critical unmet clinical need to provide HLA-matched thymic tissues for a larger group of patients with irreversible thymic injury and severe thymic dysfunction. Creating a regenerative thymus replacement therapy by generating functional TEPCs from induced-pluripotent stem cells (iPSCs) has been a longstanding aspiration of the transplant community. We have taken a novel approach by developing a robust in vitro TEPC differentiation protocol that sequentially induces all stages of human thymic ontogeny including bipotent third (III) pharyngeal pouch endoderm (PPEIII), ventralization of the PPE (PPEv), and TEPC commitment, usually lymphoid dependent. Here, we show that TEPCs from various iPSC-lines (tissue source, donor age and sex) express key markers of thymic epithelial fate and function, including FOXN1, PAX1, PAX9, HLA-DR. To examine their functional capacity, we transplanted iPSC-derived TEPCs under the kidney capsule of humanized (hCD34+ HSCs engrafted) athymic NSG-nude mice (NOD.scid.Il2Rγcnull, Foxn1null knockout allele). We observed robust hCD3+ development (40-60% out of hCD45 population) with CD4+ and CD8+ T cells (±70% and ±25%, respectively) at different stages of maturation (17% CD45RA+ CD45RO-, naïve; 80% CD45RA- CD45RO+, memory). Similar levels of T cells were found in mice that received human fetal thymus transplants, while T cells were not detected in NSG-nude mice without any thymus graft (primary or iPSC-derived). Mice with iPSC-derived thymus grafts had no clinical signs of autoimmunity or GVHD. In vivo matured grafts express levels of FOXN1, PAX1, PAX9, DLL4, PSMB11 and CD205 protein that are comparable to primary thymus tissue grafts. Altogether, our protocol reliably produces functional TEPCs from a diverse set of iPSCs that support T cell differentiation in vivo. We are currently characterizing the transcriptomic and epigenomic profile of iPSC-derived TEPCs on a single cell level before and after transplant. In addition, we are examining in vivo T cell function by analyzing the TCR repertoire and response to T-cell dependent antigens. We foresee that iPSC-derived TEPCs will offer a safe alternative as an HLA-matched thymic replacement therapy that will benefit a large cohort of patients, i.e. patients undergoing HSCT and patients receiving solid organ transplants in addition to patients with congenital thymic aplasia.

Multiparametric Magnetic Resonance Imaging and Metabolic Characterization of Patient-Derived Xenograft Models of Clear Cell Renal Cell Carcinoma.

Patient-derived xenografts (PDX) are high-fidelity cancer models typically credentialled by genomics, transcriptomics and proteomics. Characterization of metabolic reprogramming, a hallmark of cancer, is less frequent. Dysregulated metabolism is a key feature of clear cell renal cell carcinoma (ccRCC) and authentic preclinical models are needed to evaluate novel imaging and therapeutic approaches targeting metabolism. We characterized 5 PDX from high-grade or metastatic ccRCC by multiparametric magnetic resonance imaging (MRI) and steady state metabolic profiling and flux analysis. Similar to MRI of clinical ccRCC, T2-weighted images of orthotopic tumors of most PDX were homogeneous. The increased hyperintense (cystic) areas observed in one PDX mimicked the cystic phenotype typical of some RCC. The negligible hypointense (necrotic) areas of PDX grown under the highly vascularized renal capsule are beneficial for preclinical studies. Mean apparent diffusion coefficient (ADC) values were equivalent to those of ccRCC in human patients. Hyperpolarized (HP) [1-13C]pyruvate MRI of PDX showed high glycolytic activity typical of high-grade primary and metastatic ccRCC with considerable intra- and inter-tumoral variability, as has been observed in clinical HP MRI of ccRCC. Comparison of steady state metabolite concentrations and metabolic flux in [U-13C]glucose-labeled tumors highlighted the distinctive phenotypes of two PDX with elevated levels of numerous metabolites and increased fractional enrichment of lactate and/or glutamate, capturing the metabolic heterogeneity of glycolysis and the TCA cycle in clinical ccRCC. Culturing PDX cells and reimplanting to generate xenografts (XEN), or passaging PDX in vivo, altered some imaging and metabolic characteristics while transcription remained like that of the original PDX. These findings show that PDX are realistic models of ccRCC for imaging and metabolic studies but that the plasticity of metabolism must be considered when manipulating PDX for preclinical studies.

Can Kidney Organoid Xenografts Accelerate Therapeutic Development for Genetic Kidney Disorders?

A number of genetic kidney diseases can now be replicated experimentally, using kidney organoids generated from human pluripotent stem cells. This methodology holds great potential for drug discovery. Under in vitro conditions, however, kidney organoids remain developmentally immature, develop scarce vasculature, and may contain undesired off-target cell types. Those critical deficiencies limit their potential as disease-modeling tools. Orthotopic transplantation under the kidney capsule improves the anatomic maturity and vascularization of kidney organoids, while reducing off-target cell content. The improvements can translate into more accurate representations of disease phenotypes and mechanisms in vivo . Recent studies using kidney organoid xenografts highlighted the unique potential of this novel methodology for elucidating molecular mechanisms driving monogenic kidney disorders and for the development ofnovel pharmacotherapies.

Association between decreased ipsilateral renal function and aggressive behavior in renal cell carcinoma

BackgroundTo assess prognostic value of pre-operative ipsilateral split renal function (SRF) on disease-free survival (DFS) and its association with aggressive pathological features in renal cell carcinoma (RCC) patients. MethodsWe examined patients registered in SNUG-RCC-Nx who underwent partial or radical nephrectomy at Seoul National University Hospital between January 1, 2010 and December 31, 2020. Patients with the following criteria were excluded from the study. 1) non-kidney origin cancer or benign renal tumor, 2) no pre-operative Tc 99 m-DTPA renal scan, 3) single kidney status or previous partial or radical nephrectomy, and 4) bilateral renal mass. Finally, 1,078 patients were included.ResultsAmong 1,078 patients, 899 (83.4%) showed maintained ipsilateral SRF on DTPA renal scan; 179 patients (16.6%) showed decreased SRF. The decreased SRF group showed significantly large tumor size (maintained vs. decreased SRF; 3.31 ± 2.15 vs. 6.85 ± 3.25, p < 0.001), high Fuhrman grade (grade 3–4) (41.7% vs. 55.6%, p < 0.001), and high T stage (T stage 3–4) (9.0% vs. 20.1%, p < 0.001). Pathological invasive features, including invasion of the renal capsule, perirenal fat, renal sinus fat, vein, and collecting duct system, were associated with low SRF of the ipsilateral kidney. Univariate Cox regression analysis identified higher SSIGN (The stage, size, grade, and necrosis) score and decreased ipsilateral SRF as significant risk factors, while multivariate analysis showed SSIGN (5–7) (hazard ratio [HR] 11.9, p < 0.001) and SSIGN (8–10) (HR 69.2, p < 0.001) were significantly associated with shortened DFS, while decreased ipsilateral SRF (HR 1.75, p = 0.065) showed borderline significance. Kaplan–Meier analysis showed that decreased ipsilateral SRF (< 45%) group had shorter DFS than the other group (median DFS: 90.3 months vs. not reached, p < 0.001).ConclusionsAmong unilateral RCC patients, those with low ipsilateral SRF showed poor prognosis with pathologically invasive features. Our novel approach may facilitate risk stratification in RCC patients, helping formulate a treatment strategy.

Co-transplantation of pancreatic islets and microvascular fragments effectively restores normoglycemia in diabetic mice

Insufficient revascularization of pancreatic islets is one of the major obstacles impairing the success of islet transplantation. To overcome this problem, we introduce in the present study a straightforward strategy to accelerate the engraftment of isolated islets. For this purpose, we co-transplanted 250 islets and 20,000 adipose tissue-derived microvascular fragments (MVF) from donor mice under the kidney capsule as well as 500 or 1000 islets with 40,000 MVF into the subcutaneous space of diabetic mice. We found that the co-transplantation of islets and MVF markedly accelerates the restoration of normoglycemia in diabetic recipients compared with the transplantation of islets alone. In fact, the transplantation of 250 islets with 20,000 MVF under the kidney capsule reversed diabetes in 88% of mice and the subcutaneous transplantation of 500 or 1000 islets with 40,000 MVF restored normoglycemia in 100% of mice. Moreover, diabetic mice receiving islets and MVF exhibited plasma insulin levels similar to nondiabetic control animals. Additional immunohistochemical analyses of the grafts revealed a significantly higher number of islet cells and microvessels in the co-transplantation groups. These findings demonstrate that the co-transplantation of islets and MVF is a promising strategy to improve the success rates of islet transplantation, which could be easily implemented into future clinical practice.

Developing Methods to Improve Vascularization of Nephron Progenitor Cell Grafts Beneath the Kidney Capsule Through the Use of Pro-Angiogenic Uniformly Porous Templated Hydrogel Scaffolds

Developing Methods to Improve Vascularization of Nephron Progenitor Cell Grafts Beneath the Kidney Capsule Through the Use of Pro-Angiogenic Uniformly Porous Templated Hydrogel Scaffolds

Effect of Thioketal Antioxidants on Islet Cell Transplantation

AbstractPancreatic islet transplantation is an effective strategy for restoring glucose regulation for highly selected patients with type 1 diabetes. However, an undesirable level of islet cell death is caused by oxidative stress that occurs during islet isolation, culture, and transplantation. The loss of islets throughout the transplantation procedure often necessitates multiple human donors per recipient to achieve insulin independence. Administration of exogenous antioxidants has shown promise in both preserving islet cell viability and functionality post‐transplantation. Herein, thioketal (TK) antioxidant is evaluated using neonatal porcine islets and Beta‐TC‐6 cells exposed to H2O2 for changes in membrane integrity, oxygen consumption rates, and lipid peroxidation. Diabetic BALB/c mice are transplanted with a marginal dose of syngeneic islets, ±48‐h TK pre‐treatment, under the kidney capsule. Graft function is measured by nonfasting blood glucose and glucose tolerance testing. It is found that 200 µM of TK was not cytotoxic, reduced reactive oxygen species‐mediated oxidative islet damage, preserved in vitro islet cell functionality, and facilitated superior murine syngeneic marginal islet mass engraftment. These results demonstrate that the antioxidant attributes of TK reduce the deleterious effects of reactive oxygen species experienced in vitro and enhance marginal islet mass transplant outcomes.

The Value of Myocardium and Kidney Histopathological and Immunohistochemical Findings in Accidental Hypothermia-Related Fatalities.

Background and Objectives: The post-mortem diagnosis of hypothermia is challenging in forensics. The aim of our study was to detect the kidney and heart histopathological changes that occurred in a group of hypothermia-related fatalities. Materials and Methods: The cohort included 107 cases identified in the database of our department between 2007 and 2021, which have been associated with extreme cold stress. Demographic and clinicopathological data were collected from the medico-legal reports. Archived tissue samples were evaluated to identify the histopathological features, in routine haematoxylin-eosin (H&E), Periodic acid-Schiff (PAS), and Masson's trichrome stainings, while cardiac sirtuin1 (SIRT1) and renal ubiquitin (Ub) immunostaining have been performed. Results: The majority of cases exposed to low temperatures were males (76%) from rural regions (68.2%) during the cold season. Paradoxical undressing was documented in 9.3% of cases. The common comorbidities included alcoholism (50.5%), neuropsychiatric diseases (10.3%), diabetes mellitus (3.7%), and lung tuberculosis (4.7%). The microscopic heart exam revealed areas of myocardial degeneration (100%), contraction bands (95.3%), fatty change (13.1%) and focal wavy contractile myocardial cells. Basal vacuolisation of renal tubular epithelial cells (Armanni-Ebstein lesions) (21.5%), focal tubular necrosis (7.5%), tubular renal cysts (7.5%), interstitial haemorrhages (5.6%), diabetic kidney disease (3.7%), background benign nephroangiosclerosis (42.1%), variable thickening of tubules and corpuscles basement membranes, capsular space amorphous material, and intratubular casts were identified in kidney tissue samples. Myocardial cells displayed SIRT1 weak expression, with a loss of immunopositivity correlated with areas with contraction bands, while a variable Ub expression was observed in renal corpuscles capsules, proximal, distal, and collecting renal tubules, Henle's loops, urothelium, and intratubular casts. Conclusions: In the context of the current concept that death associated with hypothermia is still a diagnosis of exclusion, our findings suggest that the microscopic exam provides relevant data that support the diagnosis of hypothermia-related fatalities in appropriate circumstances of death. A deeper insight into the histopathologic findings in hypothermic patients may lead to new therapeutic approaches in these cases.

The Role of Macrophages in Sertoli Cell Transplant Survival Without the Use of Immunosuppressive Drugs

INTRODUCTION: Islet transplantation is an innovative method to treat diabetes; however, the immunosuppressive drugs necessary to prevent transplant rejection make patients susceptible to infections, cancer, and are even detrimental to the transplanted islet function. Sertoli cells (SC) are immune-privileged testicular cells that have potential as an alternative to immunosuppressive therapy, because they have been shown to protect co-transplanted islets without the use of chronic immunosuppressive drugs. However, the mechanisms by which SC extend graft survival remain unknown. Because the successful SC/islet co-grafts contain macrophages, we examined the role of macrophages in SC transplant survival. METHODS: C57BL6 SC or MSC-1 cells (rejecting control mouse SC line) (donors) were transplanted under the left kidney capsule of control or macrophage-depleted (MOKO) BALB/c mice (recipients). Macrophages were depleted using chlorate liposome injections. Grafts were collected at day 20 post-transplantation and analyzed for cell survival by immunohistochemistry (IHC) and macrophage depletion by IHC and flow cytometry. RESULTS: Macrophages were present in control recipients and confirmed depleted in MOKO recipients. In control recipients, 100% of the SC grafts survived, but 100% of the MSC-1 grafts were rejected. In MOKO recipients, SC grafts exhibited 100% survival. Surprisingly, 56% of MSC-1 grafts survived, which is especially interesting because normally they are rejected. CONCLUSION: These results indicate that macrophages are not required for SC survival; however, macrophages may be involved in graft rejection. Understanding the role of macrophages in transplantation could lead to improved graft survival and increased viability of islet transplants.

An unusual metastasis of atypical pulmonary carcinoid to the renal adipose capsule mimicking primary renal cell carcinoma.

An unusual metastasis of atypical pulmonary carcinoid to the renal adipose capsule mimicking primary renal cell carcinoma.

Effects of hypoxia-preconditioned HucMSCs on neovascularization and follicle survival in frozen/thawed human ovarian cortex transplanted to immunodeficient mice

BackgroundThe massive loss of follicles in the early stage of ovarian tissue transplantation is considered a significant restriction to the efficacy of ovarian tissue cryopreservation (OTC) and transplantation (OT). The use of mesenchymal stem cells (MSCs) before transplantation of ovarian fragments shortened the hypoxic period and boosted neovascularization. Hypoxia-preconditioned MSCs can enhance the potential of angiogenesis. Can hypoxia-preconditioned human umbilical cord mesenchymal stem cell (HucMSCs) and ovarian tissue co-xenotransplantation improve more neovascularization and subsequently more follicle survival in human ovarian tissue?MethodsFrozen-thawed cortical pieces from 4 patients were transplanted into the bilateral renal capsule of immune-deficient nude mice without HucMSCs or normoxia/hypoxia-preconditioned HucMSCs. Sixty-four mice were randomly distributed into 4 groups. In each group, the mice were euthanized for blood and/or graft retrieval on post-transplantation days 3 (n = 8) and 7 (n = 8), respectively. Non-grafted frozen-thawed ovarian fragment was taken for non-grafted control. Grafts were histologically processed and analysed for follicle density and atretic follicles by HE, neovascularization by CD34 and CD31 immunohistochemical staining, primordial follicle growth by Ki67 staining, and apoptosis of stromal cell and follicles by immunofluorescence using TUNEL. The ROS and TAC levels of grafted and non-grafted tissue were assessed. We evaluated the protein expression of HIF1α, VEGFA, pAkt, Akt, and GDF9 in grafted and non-grafted ovarian tissue. E2, Prog, AMH, and FSH levels in the plasma of mice were measured after 3 and 7 days of OT.ResultsHypoxia-preconditioned HucMSCs positively protect the grafted ovarian tissue by significantly decreasing the apoptosis and increasing higher expression of CD31, CD34, and VEGFA for earlier angiogenesis. They are crucial to preserving the resting primordial follicle pool by modulation of follicle death.ConclusionThis is the first study to demonstrate that co-transplantation of hypoxia-preconditioned HucMSC with ovarian tissue improved earlier vascularization of ovarian grafts in the early post-grafting period, which correlates with increased follicle survival and reduced apoptosis. The HIF1α/VEGFA signal pathways may play an important role in elucidating the mechanisms of action of hypoxia-preconditioned HucMSCs with regard to OT and clinical implementation.

Effects of human induced pluripotent stem cell-derived intestinal organoids on colitis-model mice

IntroductionUlcerative colitis (UC) is an inflammatory bowel disease characterized by repeated remissions and relapses. Immunosuppressive drugs have facilitated the induction and maintenance of remission in many patients with UC. However, immunosuppressive drugs cannot directly repair impaired intestinal mucosa and are insufficient for preventing relapse. Therefore, new treatment approaches to repair the damaged epithelium in UC have been attempted through the transplantation of intestinal organoids, which can be differentiated into mucosa by embedding in Matrigel, generated from patient-derived intestinal stem cells. The method, however, poses the challenge of yielding sufficient cells for UC therapy, and patient-derived cells might already have acquired pathological changes. In contrast, human induced pluripotent stem (iPS) cells generated from healthy individuals are infinitely proliferated and can be differentiated into target cells. Recently developed human iPS cell-derived intestinal organoids (HIOs) aim to generate organoids that closely resemble the adult intestine. However, no study till date has reported HIOs injected into in vivo inflammatory models, and it remains unclear whether HIOs with cells that closely resemble the adult intestine or with intestinal stem cells retain the better ability to repair tissue in colitis.MethodsWe generated two types of HIOs via suspension culture with and without small-molecule compounds: HIOs that include predominantly more intestinal stem cells [HIO (A)] and those that include predominantly more intestinal epithelial and secretory cells [HIO (B)]. We examined whether the generated HIOs engrafted in vivo and compared their ability to accelerate recovery of the damaged tissue.ResultsFindings showed that the HIOs expressed intestinal-specific markers such as caudal-type homeobox 2 (CDX2) and villin, and HIOs engrafted under the kidney capsules of mice. We then injected HIOs into colitis-model mice and found that the weight and clinical score of the mice injected with HIO (A) recovered earlier than that of the mice in the sham group. Further, the production of mucus and the expression of cell proliferation markers and tight junction proteins in the colon tissues of the HIO (A) group were restored to levels similar to those observed in healthy mice. However, neither HIO (A) nor HIO (B) could be engrafted into the colon.ConclusionsEffective cell therapy should directly repair tissue by engraftment at the site of injury. However, the difference in organoid property impacting the rate of tissue repair in transplantation without engraftment observed in the current study should be considered a critical consideration in the development of regenerative medicine using iPS-derived organoids.

Abstract A36: Building of a large B-cell lymphoma (LBCL) patient-derived xenograft (PDX) repository

Abstract Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous B-cell malignancy that consists of no fewer than 5 genetic subtypes. Cell lines have been a central model system for DLBCL but fail to fully recapitulate the diversity of genetic alterations that have been observed in large patient cohorts. Approximately 40% of DLBCLs will be refractory to or relapse following first-line R-chemo regimens, with many of these patients destined to receive CD19 CAR T-cells in the third line or later. Due to the relatively recent introduction of CD19 CAR T-cells into standard of care for relapsed/refractory DLBCL, there is a paucity of models in the CAR T relapsed setting. Together, these factors led us to embark upon an effort to build a substantive DLBCL patient-derived xenograft (PDX) repository that encompasses the major DLBCL genetic subtypes and includes CAR T relapsed models. From September 2019 to March 2022, we implanted 231 biopsies with diagnoses of DLBCL (n=120), HGBL (n=17), PMBL (n=2), other high grade B-cell lymphomas (n=87) and other low grade B-cell lymphoma (n=5). Our approaches were modeled from those utilized by the Public Repository of Xenografts (PRoXe: Townsend et al., Cancer Cell 2016), employing implantation of intact core needle biopsies into the renal capsule of NSG mice. Of 206 models that have been monitored for up to 6 months, we have generated PDX tumors from 52 tumors originating from 50 patients (25.2% take rate). The average time from implantation to signs of illness was 8 weeks (2 min, 22 max). The average number of preceding lines of therapy for these models was 3 (0 min, 8 max), with 22 models being generated from biopsies taken subsequent to progression from CD19 CAR T-cells. This included paired biopsies from two patients generated from biopsies taken immediately prior to receiving and following progression from CD19 CAR T-cells. All PDX models were confirmed to be EBV negative by EBER-ISH and subjected to histopathologic and immuno-phenotypical examination to compare their characteristics to an alternative core taken alongside the biopsy used to generate the PDX model. We additionally performed extensive molecular characterization, including RNA, whole-exome (WES), low-pass whole-genome (lpWGS), ATAC and ChIP sequencing for 7 histone post-translational modifications using the Mint-ChIP approach. Epigenetic characterization will be reported at a later date. RNA-seq confirmed both ABC (n=16) and GCB (n=24) subtypes. WES identified cardinal genetic features of all major genetic subtypes of DLBCL, including coexistent MYD88 L265P and CD79B mutations (MCD subtype); EZH2, CREBBP and KMT2D mutations (EZB subtype); NOTCH2 truncating mutations (N2 subtype); SGK1 and TET2 mutations (ST2 subtype); and NOTCH1 mutations (N1 subtype). In summary, we have established and characterized a large LBCL PDX model repository to serve as a resource for mechanistic and pre-clinical studies that will improve our understanding of lymphoma etiology and mechanisms of response/resistance to therapy. Citation Format: Haopeng Yang, Vida Ravanmehr, Jordan C Showell, Estela M Rojas-Neira, Jared M Henderson, Jason R Westin, Michael R Green. Building of a large B-cell lymphoma (LBCL) patient-derived xenograft (PDX) repository [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A36.

Nestin-GFP transgene labels immunoprivileged bone marrow mesenchymal stem cells in the model of ectopic foci formation.

Immune privileges are demonstrated for different types of quiescent stem cells of adult mammalian organisms. Mesenchymal stem cells (MSCs) are believed to have immune privileges; however, an accurate experimental confirmation hasn’t been presented. Here, we provide direct experimental evidence that MSCs of C57Black/6J murine bone marrow (BM) are immune privileged in vivo and retain their functionality after prolonged exposure to the uncompromised immune system. The BM of Nes-Gfp transgenic mice was implanted as a tissue fragment under the kidney capsule in isogenic C57Black/6J immunocompetent recipients. Nestin-Gfp strain provides a fluorescent immunogenic marker for a small fraction of BM cells, including GFP+CD45– MSCs. Despite the exposure of xenogenically marked MSCs to the fully-functional immune system, primary ectopic foci of hematopoiesis formed. Six weeks after implantation, multicolor fluorescence cytometry revealed both GFP+CD45– and GFP+CD45+ cells within the foci. GFP+CD45– cells proportion was 2.0 × 10–5 ×÷9 and it didn’t differ significantly from syngenic Nes-GFP transplantation control. According to current knowledge, the immune system of the recipients should eliminate GFP+ cells, including GFP+ MSCs. These results show that MSCs evade immunity. Primary foci were retransplanted into secondary Nes-GFP recipients. The secondary foci formed, in which CD45–GFP+ cells proportion was 6.7 × 10–5 ×÷2.2, and it didn’t differ from intact Nes-GFP BM. The results demonstrate that MSCs preserve self-renewal and retain their functionality after prolonged immune exposure. The success of this study relied on the implantation of BM fragments without prior dissociation of cells and the fact that the vast majority of implanted cells were immunologically equivalent to the recipients.

P12.11: Immunomodulatory Microporous Annealed Particle (MAP) Gel Protects Delivery of Islets to Promote Islet Transplantation Outcomes

Introduction: To investigate if coating dissociated or whole islets with microporous annealed particle (MAP) gel could support cell function and viability and improve islet transplantation outcomes. Methods: a) C57BL/6 mouse islets were mixed with MAP gel and cultured in 24 well plates at 37°C. The viability of islets was scored after Propidium Iodide-Fluorescein Diacetate (PI/FDA) staining and Glucose stimulated insulin secretion (GSIS) assay performed on Day 1, 3, 5, and 7 post-culture. The experiment was done thrice. b) Single cells from C57BL/6 mouse dissociated islets were mixed with MAP gel, and cultured in 24 well plates at 37°C. Viability of cells was scored after PI/FDA staining and GSIS assay performed 1, 24, 48, and 72 hours post-culture. c) 100 C57BL/6 mouse islets were dissociated into single cells by Trypsin, mixed with MAP gel, and transplanted into syngeneic diabetic recipients under the kidney capsule. Blood glucose (BG) was measured daily. Results: Mouse islets protected with MAP gel displayed similar GSIS and viability scores when compared to uncoated islets. Both groups retained above 95% viability, with the stimulation index (SI) of MAP gel-protected islets 3.2 and that of uncoated islets is 2.4. Viability of dissociated islets protected with MAP gel was 84% with a SI=8.5 at 72 hours post culture. In contrast, uncoated dissociated islets were nearly dead with only 35% viability and an SI=0.38 at 72 hours post culturing. In MAP scaffold, dissociated islet cell viability was density dependent − cell viability in the 50 and 12.5 dissociated islet conditions was on average >78%, while dissociated cells from 2.5 islets demonstrated 56% viability after 72 hours. In in vivo studies, no significant difference was observed in the ability to return Streptozotocin-induced diabetic mice to normoglycemia when either MAP gel coated or uncoated whole islets (75-100 islets) were transplanted under the mouse kidney capsule. C57BL/6 mouse islets (100 islets) that were dissociated to single cells and co-transplanted with MAP gel under the kidney capsule, permanently returned transplanted mice to normoglycemia, with glucose levels below 200 mg/dL within 18 days post-transplantation (n=6). In contrast, uncoated dissociated islets failed to restore normoglycemia in tranplanted mice. Treatment efficacy continued 40 days post-transplantation (time mice were monitored). The average blood glucose measured at Day 30 was 163 ± 38.2 mg/dL. Recipient mice transplanted with cells from dissociated islets without MAP gel remained hyperglycemic (n=6/group). Conclusion: The MAP scaffold protects delivery of dissociated islet cells, and helps promote islet transplantation outcomes. Moreover, MAP scaffold provides a possible platform for delivering stem cell-derived insulin-secreting cells. LaunchPad Ignite. NIH grant: NIBIB R21 Trailblazer [1R21EB028971-01A1].

P12.05: Bionic Pancreas - The First Results of Functionality of 3D-Bioprinted Bionic Tissue Model Transplantation With Pancreatic Islets

Introduction: Tissue engineering is currently on advanced stage of development which gives a possibilities for novel strategy of personal treatment of type 1 diabetes. Aim: In the following study, a bioink based on ECM derived from decellularization of porcine pancreas was applied for 3D bioprinting. Materials and Methods: The SCID (n=60) and BALB (n=20) mice were used as a model for in vivo study. Porcine islets mixed with bioink were printed on extrusion printer and transplanted on studied animals. Effectiveness of transplanted petals with regard of their insulin secretion was evaluated based on glucose and c-peptide concentration in blood samples of studied animals. Thus, animals were divided into three groups: mice with transplanted islet-laden petals, mice with transplanted islets into kidney capsule and untreated mice. Examination of studied parameters took place at four time points during the experiment, at the beginning and on day 7th, 14th and 28th day of experiment. Results: Group with transplanted petals from day 7th expressed lower mean fasting glucose concentration while compared with untreated group (129 mg/dl, 119 mg/dl, 118 mg/dl vs. 140 mg/dl, 139 mg/dl, 140 mg/dl respectively in 7th, 14th and 28th day post-transplantation; p<0.001). Post-surgery transverse section of petals revealed that connective tissue of studied animals surrounded and stabilized transplanted petals. Fibroblasts infiltration over time resulted in the process of new blood vessels formation within the petals. Hence, presented in the study bioink provides a favorable conditions for islets functionality. The bioprinted construct was stable over time. Furthermore, no pathological conditions of studied animals were observed which indicates that bioprinted petals were biocompatible. Conclusion: Bionic flake transplantation lowered glucose levels significantly.

Predictors of local recurrence of renal cell cancer. Our experience

Risk factors for renal cell cancer (RCC) recurrence, including its local form, include stage and high Fuhrman grading system score, regional lymph node involvement, microvascular invasion, tumor necrosis, positive surgical margin, and sarcomatoid or rhabdoid tumor differentiation. Objective. The study analyzes data from Moscow Research Oncological Institute named by PA Herzen to determine the predictors of local recurrence of kidney cancer based on the data from surgically treated patients with local recurrent RCC. Material and methods. We analyzed retrospectively data from 87 patients who were divided into 2 groups: 1-st, patients with detected local recurrence of kidney cancer (n=43), and 2-nd, control group (n=44). The following predictors were evaluated: tumor size, tumor histotype, tumor stage, Fuhrman grading system, surgical margin status, tumor necrosis, sarcomatoid and rhabdoid changes, microvascular invasion, hemorrhage and invasion of collecting system components (CSS), renal capsule, and perirenal cellular tissue and primary treatment. Results. The risk of local recurrence was higher in the primary tumor, over 40 mm in diameter (OR=5.8, p&lt;0.001), as well as microvascular invasion and focal hemorrhage (OR=15.1, p=0.001 and OR=3.3, p=0.008, respectively). Both univariate and multivariate analyses showed a negative effect on the risk of local RCC recurrence only for tumor necrosis (OR=15.4, p&lt;0.001 and OR=53.6, p=0.002, respectively) and high Fuhrman grade (OR=10.9, p=0.042 and OR=5.7, p=0.032, respectively). The most significant predictors of local renal cancer recurrence are tumor necrosis (p&lt;0.001), microvascular invasion (p=0.019), positive surgical margin (p=0.009), and high Fuhrman grade (p=0.04). High Fuhrman grade (3–4) of malignancy (HR=1.9, p=0.042), tumor diameter (HR=1.0, p=0.054), positive surgical margin (HR=3.5, p=0.001), and tumor necrosis (HR=2.3, p=0.029) were found to be the most significant factors influencing 5-year local recurrence-free survival rate. Conclusion. The course of renal cell cancer is determined by multiple interrelated and independent prognostic factors.

Papillary renal neoplasm with reverse polarity may be a novel renal cell tumor entity with low malignant potential

AimsThis study retrospectively investigated the morphological, immunohistochemical and molecular genetic features of papillary renal neoplasm with reverse polarity (PRNRP), a recently described renal tumor.Methods and resultsEleven cases of PRNRP were collected, and 16 cases of type I and 9 cases of type II papillary renal cell carcinoma were included as a control series. Pathological features were evaluated based on HE staining and immunohistochemistry. KRAS exon 2 and BRAF V600E mutations were detected by Real-time PCR and Sanger sequencing. Fluorescence in situ hybridization was conducted for identification of chromosomal abnormalities.Hemosiderin deposition was found in a small amount of tumor cells in 6 cases. Multifocal or patchy necrosis (5/11), small focal invasion of the pseudocapsules or renal parenchyma (6/11), and breakthrough of renal capsule with nerve invasion (1/11) were revealed, inconsistent with the previous view that the tumor lacks necrosis and intercellular hemosiderin. Immunohistochemical staining (diffusely positive for CK7 and GATA3, negative for CD117 and vimentin, and negative to weakly positive for P504S) and high frequency of KRAS mutations in exon 2 (9/10) supported the identification and inclusion of our cases. Chromosome 7 trisomy (1/7), chromosome 17 trisomy (0/7) and chromosome Y deletion (0/5 male patients) were seldom detected in this tumor.All patients were alive without metastasis or recurrence at the end of the follow-up.ConclusionOur findings may highlight the possibility of a low malignant potential of this emerging entity. We suggest that the tumor be classified as a novel renal cell tumor subtype independent of papillary renal cell carcinoma.