Development and characterization of NILK-2301, a novel CEACAM5xCD3 κλ bispecific antibody for immunotherapy of CEACAM5-expressing cancers.

BACKGROUND: T-cell retargeting to eliminate CEACAM5-expressing cancer cells via CEACAM5xCD3 bispecific antibodies (BsAbs) showed limited clinical activity so far, mostly due to insufficient T-cell activation, dose-limiting toxicities, and formation of anti-drug antibodies (ADA). METHODS: We present here the generation and preclinical development of NILK-2301, a BsAb composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format). RESULTS: NILK-2301 binds CD3ɛ on T-cells with its lambda light chain arm with an affinity of ≈100 nM, and the CEACAM5 A2 domain on tumor cells by its kappa light chain arm with an affinity of ≈5 nM. FcγR-binding is abrogated by the "LALAPA" mutation (Leu234Ala, Leu235Ala, Pro329Ala). NILK-2301 induced T-cell activation, proliferation, cytokine release, and T-cell dependent cellular cytotoxicity of CEACAM5-positive tumor cell lines (5/5 colorectal, 2/2 gastric, 2/2 lung), e.g., SK-CO-1 (Emax = 89%), MKN-45 (Emax = 84%), and H2122 (Emax = 97%), with EC50 ranging from 0.02 to 0.14 nM. NILK-2301 binds neither to CEACAM5-negative or primary colon epithelial cells nor to other CEACAM family members. NILK-2301 alone or in combination with checkpoint inhibition showed activity in organotypic tumor tissue slices and colorectal cancer organoid models. In vivo, NILK-2301 at 10 mg/kg significantly delayed tumor progression in colon- and a pancreatic adenocarcinoma model. Single-dose pharmacokinetics (PK) and tolerability in cynomolgus monkeys at 0.5 or 10 mg/kg intravenously or 20 mg subcutaneously showed dose-proportional PK, bioavailability ≈100%, and a projected half-life in humans of 13.1 days. NILK-2301 was well-tolerated. Data were confirmed in human FcRn TG32 mice. CONCLUSIONS: In summary, NILK-2301 combines promising preclinical activity and safety with lower probability of ADA-generation due to its format compared to other molecules and is scheduled to enter clinical testing at the end of 2023.

A tumor microenvironment-based prognostic index for osteosarcoma.

BACKGROUND: The tumor microenvironment (TME) has a central role in the oncogenesis of osteosarcomas. The composition of the TME is essential for the interaction between tumor and immune cells. The aim of this study was to establish a prognostic index (TMEindex) for osteosarcoma based on the TME, from which estimates about patient survival and individual response to immune checkpoint inhibitor (ICI) therapy can be deduced. METHODS: Based on osteosarcoma samples from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database, the ESTIMATE algorithm was used to estimate ImmuneScore and StromalScore. Combined differentially expressed gene analysis, weighted gene co-expression network analyses, the Least Absolute Shrinkage and Selection Operator regression and stepwise regression to construct the TMEindex. The prognostic role of TMEindex was validated in three independent datasets. The molecular and immune characteristics of TMEindex and the impact on immunotherapy were then comprehensively investigated. The expression of TMEindex genes in different cell types and its effects on osteosarcoma cells were explored by scRNA-Seq analysis and molecular biology experiments. RESULTS: Fundamental is the expression of MYC, P4HA1, RAMP1 and TAC4. Patients with high TMEindex had worse overall survival, recurrence-free survival, and metastasis-free survival. TMEindex is an independent prognostic factor in osteosarcoma. TMEindex genes were mainly expressed in malignant cells. The knockdown of MYC and P4HA1 significantly inhibited the proliferation, invasion and migration of osteosarcoma cells. A high TME index is related to the MYC, mTOR, and DNA replication-related pathways. In contrast, a low TME index is related to immune-related signaling pathways such as the inflammatory response. The TMEindex was negatively correlated with ImmuneScore, StromalScore, immune cell infiltration, and various immune-related signature scores. Patients with a higher TMEindex had an immune-cold TME and higher invasiveness. Patients with a low TME index were more likely to respond to ICI therapy and achieve clinical benefit. In addition, the TME index correlated with response to 29 oncologic drugs. CONCLUSIONS: The TMEindex is a promising biomarker to predict the prognosis of patients with osteosarcoma and their response to ICI therapy, and to distinguish the molecular and immune characteristics.

PLOD Family: A Novel Biomarker for Prognosis and Personalized Treatment in Soft Tissue Sarcoma.

Despite various treatment attempts, the heterogenous group of soft tissue sarcomata (STS) with more than 100 subtypes still shows poor outcomes. Therefore, effective biomarkers for prognosis prediction and personalized treatment are of high importance. The Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase (PLOD) gene family, which is related to multiple cancer entities, consists of three members which encode important enzymes for the formation of connective tissue. The relation to STS, however, has not yet been explored. In this study, data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases were used to analyze the role of PLOD1-3 in STS. It was found that an overexpression of PLOD family members correlates with poor prognosis, which might be due to an increased infiltration of immune-related cells in the tumor microenvironment. In STS, the expression of PLOD genes could be a novel biomarker for prognosis and a personalized, more aggressive treatment in these patients.

Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase Family: Novel Prognostic Biomarkers and Tumor Microenvironment Regulators for Lower-Grade Glioma.

Lower-grade glioma (LGG) is a group of tumors arising from the cells of the central nervous system. Although various therapy interventions are used, the prognosis remains different. Novel biomarkers are needed for the prognosis of disease and novel therapeutic strategies in LGG. The procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD) family contains three members and is related to multiple cancers, yet it was not investigated in LGG. Data from the Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) cohorts were used to analyze the role of PLOD in LGG. As the PLOD family is involved in processes, such as tumor formation and cancer metastasis, we focused on its relationship to the tumor microenvironment (TME) in LGG. A high expression of the PLOD family relates to poor prognosis and high infiltration of immune cells within the TME. The expression level of the PLOD family might become a novel biomarker for prognosis and is a potential target for individual treatment decisions in LGG.

Identification of Tumor Antigens and Immune Subtypes for the Development of mRNA Vaccines and Individualized Immunotherapy in Soft Tissue Sarcoma.

Soft tissue sarcomas (STS) are a rare disease with high recurrence rates and poor prognosis. Missing therapy options together with the high heterogeneity of this tumor type gives impetus to the development of individualized treatment approaches. This study identifies potential tumor antigens for the development of mRNA tumor vaccines for STS and explores potential immune subtypes, stratifying patients for immunotherapy. RNA-sequencing data and clinical information were extracted from 189 STS samples from The Cancer Genome Atlas (TCGA) and microarray data were extracted from 103 STS samples from the Gene Expression Omnibus (GEO). Potential tumor antigens were identified using cBioportal, the Oncomine database, and prognostic analyses. Consensus clustering was used to define immune subtypes and immune gene modules, and graph learning-based dimensionality reduction analysis was used to depict the immune landscape. Finally, four potential tumor antigens were identified, each related to prognosis and antigen-presenting cell infiltration in STS: HLTF, ITGA10, PLCG1, and TTC3. Six immune subtypes and six gene modules were defined and validated in an independent cohort. The different immune subtypes have different molecular, cellular, and clinical characteristics. The immune landscape of STS reveals the immunity-related distribution of patients and intra-cluster heterogeneity of immune subtypes. This study provides a theoretical framework for STS mRNA vaccine development and the selection of patients for vaccination, and provides a reference for promoting individualized immunotherapy.

A Human Pan-Cancer System Analysis of Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase 3 (PLOD3).

The overexpression of the enzymes involved in the degradation of procollagen lysine is correlated with various tumor entities. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3) expression was found to be correlated to the progression and migration of cancer cells in gastric, lung and prostate cancer. Here, we analyzed the gene expression, protein expression, and the clinical parameters of survival across 33 cancers based on the Clinical Proteomic Tumor Analysis Consortium (CPTAC), function annotation of the mammalian genome 5 (FANTOM5), Gene Expression Omnibus (GEO), Genotype-Tissue Expression (GTEx), Human Protein Atlas (HPA) and The Cancer Genome Atlas (TCGA) databases. Genetic alteration, immune infiltration and relevant cellular pathways were analyzed in detail. PLOD3 expression negatively correlated with survival periods and the infiltration level of CD8+ T cells, but positively correlated to the infiltration of cancer associated fibroblasts in diverse cancers. Immunohistochemistry in colon carcinomas, glioblastomas, and soft tissue sarcomas further confirm PLOD 3 expression in human cancer tissue. Moreover, amplification and mutation accounted for the largest proportion in esophageal adenocarcinoma and uterine corpus endometrial carcinoma, respectively; the copy number alteration of PLOD3 appeared in all cancers from TCGA; and molecular mechanisms further proved the effect of PLOD3 on tumorigenesis. In particular, PLOD3 expression appears to have a tumor immunological effect, and is related to multiple immune cells. Furthermore, it is also associated with tumor mutation burden and microsatellite instability in various tumors. PLOD3 acts as an inducer of various cancers, and it could be a potential biomarker for prognosis and targeted treatment.

Human tissue cultures of lung cancer predict patient susceptibility to immune-checkpoint inhibition.

Despite novel immunotherapies being approved and established for the treatment of non-small cell lung cancer (NSCLC), ex vivo models predicting individual patients' responses to immunotherapies are missing. Especially immune modulating therapies with moderate response rates urge for biomarkers and/or assays to determine individual prediction of treatment response and investigate resistance mechanisms. Here, we describe a standardized ex vivo tissue culture model to investigate individual tumor responses. NSCLC tissue cultures preserve morphological characteristics of the baseline tumor specimen for up to 12 days ex vivo and also maintain T-cell function for up to 10 days ex vivo. A semi-automated analysis of proliferating and apoptotic tumor cells was used to evaluate tissue responses to the PD-1 inhibitor nivolumab (n = 12), from which two cases could be successfully correlated to the clinical outcome. T-cell responses upon nivolumab treatment were investigated by flow cytometry and multispectral imaging. Alterations in the frequency of the Treg population and reorganization of tumor tissues could be correlated to nivolumab responsiveness ex vivo. Thus, our findings not only demonstrate the functionality of T cells in NSCLC slice cultures up to 10 days ex vivo, but also suggests this model for stratifying patients for treatment selection and to investigate in depth the tumor-associated T-cell regulation.

PD-1 inhibition in patient derived tissue cultures of human gastric and gastroesophageal adenocarcinoma.

Emerging immunotherapies quest for better patient stratification in cancer treatment decisions. Moderate response rates of PD-1 inhibition in gastric and esophagogastric junction cancers urge for meaningful human model systems that allow for investigating immune responses ex vivo. Here, the standardized patient-derived tissue culture (PDTC) model was applied to investigate tumor response to the PD-1 inhibitor Nivolumab and the CD3/CD28 t-lymphocyte activator ImmunoCultTM. Resident t-lymphocytes, tumor proliferation and apoptosis, as well as bulk gene expression data were analyzed after 72 h of PD-1 inhibition either as monotherapy or combined with Oxaliplatin or ImmunoCultTM. Individual responses to PD-1 inhibition were found ex vivo and combination with chemotherapy or t-lymphocyte activation led to enhanced antitumoral effects in PDTCs. T-lymphocyte activation as well as the addition of pre-cultured peripheral blood mononuclear cells improved PDTC for studying t-lymphocyte and tumor cell communication. These data support the potential of PDTC to investigate immunotherapy ex vivo in gastric and esophagogastric junction cancer.

Integrative and Comprehensive Pancancer Analysis of Regulator of Chromatin Condensation 1 (RCC1).

Regulator of Chromatin Condensation 1 (RCC1) is the only known guanine nucleotide exchange factor that acts on the Ras-like G protein Ran and plays a key role in cell cycle regulation. Although there is growing evidence to support the relationship between RCC1 and cancer, detailed pancancer analyses have not yet been performed. In this genome database study, based on The Cancer Genome Atlas, Genotype-Tissue Expression and Gene Expression Omnibus databases, the potential role of RCC1 in 33 tumors' entities was explored. The results show that RCC1 is highly expressed in most human malignant neoplasms in contrast to healthy tissues. RCC1 expression is closely related to the prognosis of a broad variety of tumor patients. Enrichment analysis showed that some tumor-related pathways such as "cell cycle" and "RNA transport" were involved in the functional mechanism of RCC1. In particular, the conducted analysis reveals the relation of RCC1 to multiple immune checkpoint genes and suggests that the regulation of RCC1 is closely related to tumor infiltration of cancer-associated fibroblasts and CD8+ T cells. Coherent data demonstrate the association of RCC1 with the tumor mutation burden and microsatellite instability in various tumors. These findings provide new insights into the role of RCC1 in oncogenesis and tumor immunology in various tumors and indicate its potential as marker for therapy prognosis and targeted treatment strategies.

FOXM1 Inhibition in Ovarian Cancer Tissue Cultures Affects Individual Treatment Susceptibility Ex Vivo.

Diagnosis in an advanced state is a major hallmark of ovarian cancer and recurrence after first line treatment is common. With upcoming novel therapies, tumor markers that support patient stratification are urgently needed to prevent ineffective therapy. Therefore, the transcription factor FOXM1 is a promising target in ovarian cancer as it is frequently overexpressed and associated with poor prognosis. In this study, fresh tissue specimens of 10 ovarian cancers were collected to investigate tissue cultures in their ability to predict individual treatment susceptibility and to identify the benefit of FOXM1 inhibition. FOXM1 inhibition was induced by thiostrepton (3 µM). Carboplatin (0.2, 2 and 20 µM) and olaparib (10 µM) were applied and tumor susceptibility was analyzed by tumor cell proliferation and apoptosis in immunofluorescence microscopy. Resistance mechanisms were investigated by determining the gene expression of FOXM1 and its targets BRCA1/2 and RAD51. Ovarian cancer tissue was successfully maintained for up to 14 days ex vivo, preserving morphological characteristics of the native specimen. Thiostrepton downregulated FOXM1 expression in tissue culture. Individual responses were observed after combined treatment with carboplatin or olaparib. Thus, we successfully implemented a complex tissue culture model to ovarian cancer and showed potential benefit of combined FOXM1 inhibition.

Examination of ex-vivo viability of human adipose tissue slice culture.

Obesity is associated with significantly higher mortality rates, and excess adipose tissue is involved in respective pathologies. Here we established a human adipose tissue slice cultures (HATSC) model ex vivo. HATSC match the in vivo cell composition of human adipose tissue with, among others, mature adipocytes, mesenchymal stem cells as well as stroma tissue and immune cells. This is a new method, optimized for live imaging, to study adipose tissue and cell-based mechanisms of obesity in particular. HATSC survival was tested by means of conventional and immunofluorescence histological techniques, functional analyses and live imaging. Surgery-derived tissue was cut with a tissue chopper in 500 µm sections and transferred onto membranes building an air-liquid interface. HATSC were cultured in six-well plates filled with Dulbecco's Modified Eagle's Medium (DMEM), insulin, transferrin, and selenium, both with and without serum. After 0, 1, 7 and 14 days in vitro, slices were fixated and analyzed by morphology and Perilipin A for tissue viability. Immunofluorescent staining against IBA1, CD68 and Ki67 was performed to determine macrophage survival and proliferation. These experiments showed preservation of adipose tissue as well as survival and proliferation of monocytes and stroma tissue for at least 14 days in vitro even in the absence of serum. The physiological capabilities of adipocytes were functionally tested by insulin stimulation and measurement of Phospho-Akt on day 7 and 14 in vitro. Viability was further confirmed by live imaging using Calcein-AM (viable cells) and propidium iodide (apoptosis/necrosis). In conclusion, HATSC have been successfully established by preserving the monovacuolar form of adipocytes and surrounding macrophages and connective tissue. This model allows further analysis of mature human adipose tissue biology ex vivo.

The Chk1 inhibitor SAR-020106 sensitizes human glioblastoma cells to irradiation, to temozolomide, and to decitabine treatment.

BACKGROUND: Glioblastoma is the most common and aggressive brain tumour in adults with a median overall survival of only 14 months after standard therapy with radiation therapy (IR) and temozolomide (TMZ). In a novel multimodal treatment approach we combined the checkpoint kinase 1 (Chk1) inhibitor SAR-020106 (SAR), disrupting homologue recombination, with standard DNA damage inducers (IR, TMZ) and the epigenetic/cytotoxic drug decitabine (5-aza-2'-deoxycitidine, 5-aza-dC). Different in vitro glioblastoma models are monitored to evaluate if the impaired DNA damage repair may chemo/radiosensitize the tumour cells. METHODS: Human p53-mutated (p53-mut) and -wildtype (p53-wt) glioblastoma cell lines (p53-mut: LN405, T98G; p53-wt: A172, DBTRG) and primary glioblastoma cells (p53-mut: P0297; p53-wt: P0306) were treated with SAR combined with TMZ, 5-aza-dC, and/or IR and analysed for induction of apoptosis (AnnexinV and sub-G1 assay), cell cycle distribution (nuclear PI staining), DNA damage (alkaline comet or gH2A.X assay), proliferation inhibition (BrdU assay), reproductive survival (clonogenic assay), and potential tumour stem cells (nestinpos/GFAPneg fluorescence staining). Potential treatment-induced neurotoxicity was evaluated on nestin-positive neural progenitor cells in a murine entorhinal-hippocampal slice culture model. RESULTS: SAR showed radiosensitizing effects on the induction of apoptosis and on the reduction of long-term survival in p53-mut and p53-wt glioblastoma cell lines and primary cells. In p53-mut cells, this effect was accompanied by an abrogation of the IR-induced G2/M arrest and an enhancement of IR-induced DNA damage by SAR treatment. Also TMZ and 5-aza-dC acted radioadditively albeit to a lesser extent. The multimodal treatment achieved the most effective reduction of clonogenicity in all tested cell lines and did not affect the ratio of nestinpos/GFAPneg cells. No neurotoxic effects were detected when the number of nestin-positive neural progenitor cells remained unchanged after multimodal treatment. CONCLUSION: The Chk1 inhibitor SAR-020106 is a potent sensitizer for DNA damage-induced cell death in glioblastoma therapy strongly reducing clonogenicity of tumour cells. Selectively enhanced p53-mut cell death may provide stronger responses in tumours defective of non-homologous end joining (NHEJ). Our results suggest that a multimodal therapy involving DNA damage inducers and DNA repair inhibitors might be an effective anti-tumour strategy with a low risk of neurotoxicity.

Tumor-associated macrophages and individual chemo-susceptibility are influenced by iron chelation in human slice cultures of gastric cancer.

Purpose: Presence of tumor-associated macrophages (TAM) and high levels of ferritin and lipocalin 2 (Lcn2) in the tumor microenvironment are associated with poor prognosis in many types of cancer. Here we investigate whether iron deprivation influences TAM phenotype and chemotherapy resistance in tumor slice cultures (TSC) of gastric cancer. Results: TAM remained morphologically and functionally stable for four DIV. DFO treatment for 72 h decreased ferritin expression in TAM and in the tumor stroma but did not alter Lcn2 expression. TAM phenotype was altered after 72 h of cisplatin or DFO treatment compared with control conditions. Single DFO treatment and combined treatment with cytotoxic drugs significantly increased tumor cell apoptosis in TSC of gastric cancer. Methods: TSC were manufactured by cutting tissue of gastric cancer resection specimens in 350 µm thick slices and cultivating them under standard conditions on a filter membrane, at an air-liquid interface. After 24 h ex vivo, TSC were treated with irinotecan (100 nM) or cisplatin (10 µM) alone and in combination with deferoxamine (DFO; 10 µM, 100 µM), respectively, for 72 h. After four days in vitro (DIV) the TSC were fixated with paraformaldehyde, paraffin embedded and analyzed by immunohistochemistry for apoptosis (cPARP), proliferation (Ki67), TAM (CD68, CD163), ferritin, and Lcn2 expression. Conclusions: TAM are well preserved and can be studied in TSC of gastric cancer. Iron deprivation significantly increased tumor cell apoptosis.

Individual Susceptibility Analysis Using Patient-derived Slice Cultures of Colorectal Carcinoma.

BACKGROUND: Nonresponse to chemotherapy in colorectal carcinoma (CRC) is still a clinical problem. For most established treatment regimens, no predictive biomarkers are available. Patient-derived tumor slice culture may be a promising ex vivo technology to assess the drug susceptibility in individual tumors. METHODS: Patient-derived slice cultures of CRC specimens were prepared according to a standardized protocol and treated with different concentrations of 5-fluorouracil (5-FU) and an adapted FOLFOX regimen (5-FU and oxaliplatin) to investigate histologic response. Additionally, a semi-automatized readout using fluorescent stain-specific segmentation algorithms for Image J was established to quantify changes in tumor proliferation. Nonresponse to chemotherapy was defined as persisting tumor cell proliferation. RESULTS: Slices treated with 5-FU showed lower tumor cell fractions and dose-dependent alterations of proliferating tumor cells compared with controls (1 µM, Δ +3%; 10 µM, Δ -9%; 100 µM, Δ -15%). Individual tumor samples were examined and differences in chemotherapy susceptibility could be observed. Untreated slice cultures contained an average tumor cell fraction of 31% ± 7%. For all samples, the histopathologic characteristics exhibited some degree of intratumoral heterogeneity with regard to tumor cell morphology and distribution. The original tumor matched the features found in slices at baseline and after 3 days of cultivation. CONCLUSIONS: Patient-derived slice cultures may help to predict response to clinical treatment in individual patients with CRC. Future studies need to address the problem of tumor heterogeneity and evolution. Prospective correlation of ex vivo results with the clinical course of treated patients is warranted.

Depletion of Jmjd1c impairs adipogenesis in murine 3T3-L1 cells.

Differentiation of adipocytes is a highly regulated process modulated by multiple transcriptional co-activators and co-repressors. JMJD1C belongs to the family of jumonji C (jmjC) domain-containing histone demethylases and was originally described as a ligand-dependent co-activator of thyroid hormone and androgen receptors. Here, we explored the potential role of Jmjd1c in white adipocyte differentiation. To investigate the relevance of Jmjd1c in adipogenesis, murine 3T3-L1 preadipocyte cells with transient knock-down of Jmjd1c (3T3_Jmjd1c) were generated. Depletion of Jmjd1c led to the formation of smaller lipid droplets, reduced accumulation of triglycerides and maintenance of a more fibroblast-like morphology after adipocyte differentiation. Concomitantly, insulin stimulated uptake of glucose and fatty acids was significantly reduced in 3T3_Jmjd1c adipocytes. In line with these observations we detected lower expression of key genes associated with lipid droplet formation (Plin1, Plin4, Cidea) and uptake of glucose and fatty acids (Glut4, Fatp1, Fatp4, Aqp7) respectively. Finally, we demonstrate that depletion of Jmjd1c interferes with mitotic clonal expansion (MCE), increases levels of H3K9me2 (dimethylation of lysine 9 of histone H3) at promotor regions of adipogenic transcription factors (C/EBPs and PPARγ) and leads to reduced induction of these key regulators. In conclusion, we have identified Jmjd1c as a modulator of adipogenesis. Our data suggest that Jmjd1c may participate in MCE and the activation of the adipogenic transcription program during the induction phase of adipocyte differentiation in 3T3-L1 cells.

Long-Term Tissue Culture of Adult Brain and Spleen Slices on Nanostructured Scaffolds.

Long-term tissue culture of adult mammalian organs is a highly promising approach to bridge the gap between single cell cultures and animal experiments, and bears the potential to reduce in vivo studies. Novel biomimetic materials open up new possibilities to maintain the complex tissue structure in vitro; however, survival times of adult tissues ex vivo are still limited to a few days with established state-of-the-art techniques. Here, it is demonstrated that TiO2 nanotube scaffolds with specific tissue-tailored characteristics can serve as superior substrates for long-term adult brain and spleen tissue culture. High viability of the explants for at least two weeks is achieved and compared to tissues cultured on standard polytetrafluoroethylene (PTFE) membranes. Histological and immunohistochemical staining and live imaging are used to investigate tissue condition after 5 and 14 d in vitro, while environmental scanning electron microscopy qualifies the interaction with the underlying scaffold. In contrast to tissues cultured on PTFE membranes, enhanced tissue morphology is detected in spleen slices, as well as minor cell death in neuronal tissue, both cultured on nanotube scaffolds. This novel biomimetic tissue model will prove to be useful to address fundamental biological and medical questions from tissue regeneration up to tumor progression and therapeutic approaches.

Optimized polyethylenimine (PEI)-based nanoparticles for siRNA delivery, analyzed in vitro and in an ex vivo tumor tissue slice culture model.

The non-viral delivery of small RNA molecules like siRNAs still poses a major bottleneck for their successful application in vivo. This is particularly true with regard to crossing physiological barriers upon systemic administration. We have previously established polyethylenimine (PEI)-based complexes for therapeutic RNA formulation. These nanoplexes mediate full RNA protection against nucleolytic degradation, delivery to target tissues as well as cellular uptake, intracellular release and therapeutic efficacy in preclinical in vivo models. We herein present data on different polyplex modifications for the defined improvement of physicochemical and biological nanoparticle properties and for targeted delivery. (i) By non-covalent modifications of PEI polyplexes with phospholipid liposomes, ternary complexes ("lipopolyplexes") are obtained that combine the favorable features of PEI and lipid systems. Decreased cytotoxicity and highly efficient delivery of siRNA is achieved. Some lipopolyplexes also allow prolonged storage, thus providing formulations with higher stability. (ii) Novel tyrosine modifications of low molecular weight PEI offer further improvement of stability, biocompatibility, and knockdown efficacy of resulting nanoparticles. (iii) For ligand-mediated uptake, the shielding of surface charges is a critical requirement. This is achieved by PEI grafting with polyethylene glycol (PEG), prior to covalent coupling of anti-HER1 antibodies (Erbitux®) as ligand for targeted delivery and uptake. Beyond tumor cell culture, analyses are extended towards tumor slice cultures from tumor xenograft tissues which reflect more realistically the in vivo situation. The determination of siRNA-mediated knockdown of endogenous target genes, i.e., the oncogenic survival factor survivin and the oncogenic receptor tyrosine kinase HER2, reveals nanoparticle penetration and biological efficacy also under intact tissue and stroma conditions.

Tumor tissue slice cultures as a platform for analyzing tissue-penetration and biological activities of nanoparticles.

The success of therapeutic nanoparticles depends, among others, on their ability to penetrate a tissue for actually reaching the target cells, and their efficient cellular uptake in the context of intact tissue and stroma. Various nanoparticle modifications have been implemented for altering physicochemical and biological properties. Their analysis, however, so far mainly relies on cell culture experiments which only poorly reflect the in vivo situation, or is based on in vivo experiments that are often complicated by whole-body pharmacokinetics and are rather tedious especially when analyzing larger nanoparticle sets. For the more precise analysis of nanoparticle properties at their desired site of action, efficient ex vivo systems closely mimicking in vivo tissue properties are needed. In this paper, we describe the setup of organotypic tumor tissue slice cultures for the analysis of tissue-penetrating properties and biological activities of nanoparticles. As a model system, we employ 350µm thick slice cultures from different tumor xenograft tissues, and analyze modified or non-modified polyethylenimine (PEI) complexes as well as their lipopolyplex derivatives for siRNA delivery. The described conditions for tissue slice preparation and culture ensure excellent tissue preservation for at least 14days, thus allowing for prolonged experimentation and analysis. When using fluorescently labeled siRNA for complex visualization, fluorescence microscopy of cryo-sectioned tissue slices reveals different degrees of nanoparticle tissue penetration, dependent on their surface charge. More importantly, the determination of siRNA-mediated knockdown efficacies of an endogenous target gene, the oncogenic survival factor Survivin, reveals the possibility to accurately assess biological nanoparticle activities in situ, i.e. in living cells in their original environment. Taken together, we establish tumor (xenograft) tissue slices for the accurate and facile ex vivo assessment of important biological nanoparticle properties. Beyond the quantitative analysis of nanoparticle tissue-penetration, the excellent tissue preservation and cell viability also allows for the evaluation of biological activities.

Adeno-Associated Viral Vectors Transduce Mature Human Adipocytes in Three-Dimensional Slice Cultures.

Adipose tissue plays a pivotal role, both in the regulation of energy homeostasis and as an endocrine organ. Consequently, adipose tissue dysfunction is closely related to insulin resistance, morbid obesity, and metabolic syndrome. To study molecular mechanisms and to develop novel therapeutic strategies, techniques are required to genetically modify mature adipocytes. Here, we report on adeno-associated viral (AAV) vectors as a versatile tool to transduce human mature adipocytes in organotypic three-dimensional tissue cultures.

Organotypic slice cultures of human gastric and esophagogastric junction cancer.

Gastric and esophagogastric junction cancers are heterogeneous and aggressive tumors with an unpredictable response to cytotoxic treatment. New methods allowing for the analysis of drug resistance are needed. Here, we describe a novel technique by which human tumor specimens can be cultured ex vivo, preserving parts of the natural cancer microenvironment. Using a tissue chopper, fresh surgical tissue samples were cut in 400 µm slices and cultivated in 6-well plates for up to 6 days. The slices were processed for routine histopathology and immunohistochemistry. Cytokeratin stains (CK8, AE1/3) were applied for determining tumor cellularity, Ki-67 for proliferation, and cleaved caspase-3 staining for apoptosis. The slices were analyzed under naive conditions and following 2-4 days in vitro exposure to 5-FU and cisplatin. The slice culture technology allowed for a good preservation of tissue morphology and tumor cell integrity during the culture period. After chemotherapy exposure, a loss of tumor cellularity and an increase in apoptosis were observed. Drug sensitivity of the tumors could be assessed. Organotypic slice cultures of gastric and esophagogastric junction cancers were successfully established. Cytotoxic drug effects could be monitored. They may be used to examine mechanisms of drug resistance in human tissue and may provide a unique and powerful ex vivo platform for the prediction of treatment response.