Malignant Prostate Cells Research Articles

Characterization of the Peroxisomal Proteome and Redox Balance in Human Prostate Cancer Cell Lines.

Prostate cancer (PCa) is associated with disruptions in cellular redox balance. Given the intricate role of peroxisomes in redox metabolism, we conducted comprehensive proteomics analyses to compare peroxisomal and redox protein profiles between benign (RWPE-1) and malignant (22Rv1, LNCaP, and PC3) prostate cell lines. Our analyses revealed significant enrichment of the "peroxisome" pathway among proteins notably upregulated in androgen receptor (AR)-positive cell lines. In addition, catalase (CAT) activity was consistently higher in these malignant cell lines compared to RWPE-1, which contrasts with previous studies reporting lower CAT levels and increased H2O2 levels in PCa tissues compared to adjacent normal tissues. To mimic this clinical scenario, we used RNA interference to knock down CAT expression. Our results show that reduced CAT levels enhanced 22Rv1 and LNCaP cell proliferation. R1881-induced activation of AR, a key driver of PCa, increased expression of the H2O2-producing peroxisomal β-oxidation enzymes acyl-coenzyme A oxidase 1 and 3, reduced CAT expression and activity, and elevated peroxisomal H2O2 levels. Considering these changes and other antioxidant enzyme profile alterations, we propose that enhanced AR activity in PCa reduces CAT function, leading to increased peroxisomal H2O2 levels that trigger adaptive stress responses to promote cell survival, growth, and proliferation.

Targeting valine catabolism to inhibit metabolic reprogramming in prostate cancer

Metabolic reprogramming and energetic rewiring are hallmarks of cancer that fuel disease progression and facilitate therapy evasion. The remodelling of oxidative phosphorylation and enhanced lipogenesis have previously been characterised as key metabolic features of prostate cancer (PCa). Recently, succinate-dependent mitochondrial reprogramming was identified in high-grade prostate tumours, as well as upregulation of the enzymes associated with branched-chain amino acid (BCAA) catabolism. In this study, we hypothesised that the degradation of the BCAAs, particularly valine, may play a critical role in anapleurotic refuelling of the mitochondrial succinate pool, as well as the maintenance of intracellular lipid metabolism. Through the suppression of BCAA availability, we report significantly reduced lipid content, strongly indicating that BCAAs are important lipogenic fuels in PCa. This work also uncovered a novel compensatory mechanism, whereby fatty acid uptake is increased in response to extracellular valine deprivation. Inhibition of valine degradation via suppression of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) resulted in a selective reduction of malignant prostate cell proliferation, decreased intracellular succinate and impaired cellular respiration. In combination with a comprehensive multi-omic investigation that incorporates next-generation sequencing, metabolomics, and high-content quantitative single-cell imaging, our work highlights a novel therapeutic target for selective inhibition of metabolic reprogramming in PCa.

State-of-the-art in transposable element modulation affected by drugs in malignant prostatic cancer cells.

Over recent years, the investigation of transposable elements (TEs) has granted researchers a deeper comprehension of their characteristics and functions, particularly regarding their significance in the mechanisms contributing to cancer development. This manuscript focuses on prostate carcinoma cell lines and offers a comprehensive review intended to scrutinize the associations and interactions between TEs and genes, as well as their response to treatment using various chemical drugs, emphasizing their involvement in cancer progression. We assembled a compendium of articles retrieved from the PubMed database to construct networks demonstrating correlations with genes and pharmaceuticals. In doing so, we linked the transposition of certain TE types to the expression of specific transcripts directly implicated in carcinogenesis. Additionally, we underline that treatment employing different drugs revealed unique patterns of TE reactivation. Our hypothesis gathers the current understanding and guides research toward evidence-based investigations, emphasizing the association between antiviral drugs, chemotherapy, and the reduced expression of TEs in patients affected by prostate cancer.

Antioxidant, cytotoxicity, and anti-prostate carcinoma effects of CuO nanoparticles containing Thymus vulgaris

In this work, we have described the green supported of CuO NPs over Thymus vulgaris (CuNPs) as a reducing/stabilizing nanocomposite in alkaline medium. In the cellular and molecular part of the recent study, the treated cells with CuNPs were assessed to determine the cytotoxicity and anti-human prostate carcinoma properties on prostate carcinoma cell lines i.e., LNCaP clone FGC-Luc2, 22Rv1, and NCI-H660. The morphological and physicochemical features of the prepared nanocomposite were determined using several advanced techniques as Field Emission Scanning Electron Microscopes (FE-SEM), Ultraviolet–visible spectroscopy (UV-Vis), and fourier transform infrared spectroscopy (FTIR) studies. In the antioxidant test, the IC50 of CuNPs and butylated hydroxytoluene (BHT) against 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals were 94 and 88 μg/mL, respectively. The IC50 of CuNPs were 191, 275, and 250 μg/mL against LNCaP clone FGC-Luc2, 22Rv1, and NCI-H660 cell lines, respectively. In conclusion, our data suggest that the malignant prostate cell lines viability decreased in the CuNPs presence.

Antiproliferative polycyclic polyprenylated acylphloroglucinols from Garcinia paucinervis

Bioassay-guided isolation of the stems of Garcinia paucinervis led to one new adamantane-type polycyclic polyprenylated acylphloroglucinols (PPAPs), (-)-garpauvinin A (1), and four known analogues (2–5). The structure and absolute configuration of 1 was established via spectroscopic techniques and ECD method. All the isolates displayed moderate antiproliferative activity against HL-60, PC-3 and Caco-2 human cancer cell lines with IC50 values ranging from 0.81 to 19.92 μM, and exhibited low toxicity on WPMY-1 normal human cells, showing selectivity between normal and malignant prostate cells. The biosynthetic pathways of the isolated PPAPs were proposed.

The Journal of Urology® Home Study Course 2023 Volume 209/210.

The Journal of Urology® Home Study Course 2023 Volume 209/210.

The Role of Isoflavones in the Prevention of Breast Cancer and Prostate Cancer

This narrative review summarizes epidemiological studies on breast cancer and prostate cancer with an overview of their global incidence distribution to investigate the relationship between these diseases and diet. The biological properties, mechanisms of action, and available data supporting the potential role of isoflavones in the prevention of breast cancer and prostate cancer are discussed. Studies evaluating the effects of isoflavones in tissue cultures of normal and malignant breast and prostate cells, as well as the current body of research regarding the effects of isoflavones attained through multiple modifications of cellular molecular signaling pathways and control of oxidative stress, are summarized. Furthermore, this review compiles literature sources reporting on the following: (1) levels of estrogen in breast and prostate tissue; (2) levels of isoflavones in the normal and malignant tissue of these organs in European and Asian populations; (3) average concentrations of isoflavones in the secretion of these organs (milk and semen). Finally, particular emphasis is placed on studies investigating the effect of isoflavones on tissues via estrogen receptors (ER).

Post-diagnostic Zinc Supplement Use and Prostate Cancer Survival Among Men With Nonmetastatic Prostate Cancer.

Biological and experimental evidence support restoration of normal zinc levels in malignant prostate cells as a promising prostate cancer treatment, yet the influence of zinc supplementation after diagnosis on prostate cancer survival in a human population is unknown. We prospectively assessed post-diagnostic zinc supplementation in relation to prostate cancer survival among 5,788 men with nonmetastatic prostate cancer in the Health Professionals Follow-up Study (1986-2019). We used Cox regression models to estimate the multivariable hazard ratios and 95% confidence intervals of lethal prostate cancer (distant metastases or prostate cancer-specific death) and all-cause mortality according to post-diagnostic zinc supplement use and dosage. During a median follow-up of 11 years, we documented 527 lethal prostate cancer events and 3,198 all-cause deaths. Fifteen percent of men reported zinc supplement use post-diagnosis. Compared to nonusers, post-diagnostic zinc supplement use was associated suggestively with a lower risk of lethal prostate cancer (HR [95% CI], 0.82 [0.60-1.13]) and significantly with all-cause mortality (0.84 [0.74-0.96]). The inverse association was mostly observed among men who used post-diagnostic zinc supplements of 1-24 mg/d (lethal prostate cancer: 0.55 [0.32-0.96]; all-cause mortality: 0.77 [0.64-0.93]), while higher dosage did not show a lower risk. Post-diagnostic low-dose zinc supplement use among nonmetastatic prostate cancer patients was associated with lower risk of lethal prostate cancer and all-cause mortality. A potential benefit of low-dose post-diagnostic zinc supplement for prostate cancer survival merits further study.

Vitamin D-Dimer: A Possible Biomolecule Modulator in Cytotoxic and Phagocytosis Processes?

Background: Vitamin D3 complexed to deglycosylated vitamin D binding protein (VitD-dgVDBP) is a water-soluble vitamin D dimeric compound (VitD-dgVDBP). It is not clear how VitD-dgVDBP affects circulating monocytes, macrophages, other immune cell systems, including phagocytosis and apoptosis, and the generation of reactive oxygen species (ROS) compared to dgVDBP. Methods: Flow cytometry was used to measure superoxide anion radical (O2*−) levels and macrophage activity in the presence of VitD-dgVDBP or dgVDBP. VitD-dgVDBP was incubated with normal human lymphocytes (nPBMCs), and several clusters of determination (CDs) were estimated. dgVDBP and VitD-dgVDBP apoptosis was estimated on malignant prostatic cells. Results: The macrophage activity was 2.8-fold higher using VitD-dgVDBP (19.8·106 counts) compared to dgVDBP (7.0·106 counts), but O2*− production was 1.8-fold lower in favor of VitD-dgVDBP (355·103 counts) compared to dgVDBP (630·106 counts). The calculated ratio of the radical/macrophage activity was 5-fold lower compared to that of dgVDBP. Only VitD-dgVDBP activated caspase-3 (8%), caspase-9 (13%), and cytochrome-C (11%) on prostatic cancer cells. PE-Cy7-labeled VitD-dgVDBP was found to bind to cytotoxic suppressor cells, monocytes/macrophages, dendritic and natural killer cells (CD8+), and helper cells (CD4+). After 12 h of co-incubation of nPBMCs with VitD-dgVDBP, significant activation and expression were measured for CD16++/CD16 (0.6 ± 0.1% vs. 0.4 ± 0.1%, p < 0.05), CD45k+ (96.0 ± 6.0% vs. 84.7 ± 9.5%, p < 0.05), CD85k+ (24.3 ± 13.2% vs. 3.8 ± 3.2%, p < 0.05), and CD85k+/CD123+ (46.8 ± 8.1% vs. 3.5 ± 3.7%, p < 0.001) compared to the control experiment. No significant difference was found using CD3+, CD4+, CD8+, CD4/CD8, CD4/CD8, CD16+, CD16++, CD14+, or CD123+. A significant decline in CD14+/CD16+ was obtained in the presence of VitD-dgVDBP (0.7 ± 0.2% vs. 3.1 ± 1.7%; p < 0.01). Conclusion: The newly developed water-soluble VitD3 form VitD-dgVDBP affected cytotoxic suppressor cells by activating the low radical-dependent CD16 pathway and seemed to induce apoptosis in malignant prostatic cells.

Biochemical recurrence related metabolic novel signature associates with immunity and ADT treatment responses in prostate cancer.

Prostate cancer (PCa) is a unique cancer from a metabolic perspective. Androgen receptor assumes a vital part in normal and malignant prostate cells regarding almost all aspects of cell metabolism, such as glucose, fat, amino acids, nucleotides, and so on. We used The Cancer Genome Atlas database as training set, Memorial Sloan-Kettering Cancer Center cohort as validation set, and Gene Expression Omnibus database (GSE70769) as test set to identify the optimal prognostic signature. We evaluated the signature in terms of biochemical progression-free survival (bPFS), ROC curve, clinicopathological features, independent prognostic indicators, tumor microenvironment, and infiltrating immune cells. Nomogram was built dependent on the results of cox regression analyses. GSEA algorithm was used to evaluate differences in metabolism. The signature's prediction of androgen deprivation therapy (ADT) response was validated based on two groups of basic cytological experiments treat with ADT (GSE143408 and GSE120343) and the transcriptional information of pre-ADT/post-ADT of six local PCa patients. We finally input four screened genes into the stepwise regression model to construct metabolism-related signature. The signature shows good prediction performance in training set, verification set, and test set. A nomogram based on the PSA, Gleason score, T staging, and the signature risk score could predict 1-, 3-, and 5-year bPFS with the high area under curve values. Based on gene-set enrichment analysis, the characteristics of four genes signature could influence some important metabolic biological processes of PCa and were serendipitously found to be significantly related to androgen response. Subsequently, two cytological experimental data sets and our local patient sequencing data set verified that the signature may be helpful to evaluate the therapeutic response of PCa to ADT. Our systematic study definite a metabolism-related gene signature to foresee prognosis of PCa patients which might add to individual prevention and treatment.

Rhenium(I) conjugates as tools for tracking cholesterol in cells.

Cholesterol is vital to control membrane integrity and fluidity, but is also a precursor to produce steroid hormones, bile acids, and vitamin D. Consequently, altered cholesterol biology has been linked to many diseases, including metabolic syndromes and cancer. Defining the intracellular pools of cholesterol and its trafficking within cells is essential to understand both normal cell physiology and mechanisms of pathogenesis. We have synthesized a new cholesterol mimic (ReTEGCholestanol), comprising a luminescent rhenium metal complex and a cholestanol targeting unit, linked using a tetraethylene glycol (TEG) spacer. ReTEGCholestanol demonstrated favourable imaging properties and improved water solubility when compared to a cholesterol derivative, and structurally related probes lacking the TEG linker. A non-malignant and three malignant prostate cell lines were used to characterize the uptake and intracellular distribution of ReTEGCholestanol. The ReTEGCholestanol complex was effectively internalized and mainly localized to late endosomes/lysosomes in non-malignant PNT1a cells, while in prostate cancer cells it also accumulated in early endosomes and multivesicular bodies, suggesting disturbed cholesterol biology in the malignant cells. The ReTEGCholestanol is a novel imaging agent for visualizing endosomal uptake and trafficking, which may be used to define cholesterol related biology including membrane integration and altered lipid trafficking/processing.

177Lu-DOTA-TLX591 safety, biodistribution and dosimetry study (ProstACT-SELECT).

TPS5109 Background: The cell surface glycoprotein prostate-specific membrane antigen (PSMA) has proven to be an ideal therapeutic target in prostate cancer (PC) as it is highly expressed by malignant prostate cells.1,2 177Lu-DOTA-HuJ591-CHO (TLX591) is a radioimmunoconjugate comprised of the humanized IgG1 mAb rosopatamab, linked to the low energy beta-emitting radioisotope lutetium-177 (177Lu) via the bifunctional chelating agent DOTA-NHS ester. Chinese Hamster Ovary (CHO) cell line has been selected for the manufacture of the recombinant mAb. There is a strong rationale for investigation of TLX591 as a potential radioligand therapy for the treatment of PC, supported by previous clinical evidence of the safety of the antibody, as unconjugated and as a DOTA conjugate, and of the specificity of the antibody for PC tumors. 3,4,5,6. This multicenter Phase 1 radiomics study (ClinicalTrials.gov Identifier: NCT04786847) is designed to evaluate the safety, tolerability, biodistribution and dosimetry of TLX591 administered with best SoC to patients with PSMA-expressing, metastatic castration-resistant prostate cancer (mCRPC) progressing despite prior treatment with a novel androgen axis drug (NAAD). At the time of this abstract the study has commenced recruitment in Australia. This trial is sponsored by Telix Pharmaceuticals. Methods: The study will consist of 2 cohorts: Cohort 1: Five patients will be recruited for evaluation of biodistribution of TLX591 administered in combination with SoC. These patients will receive a single tracer (27 mCi) intravenous (IV) infusion of TLX591, and SPECT images and pharmacokinetic blood samples acquired at several time points until Day 13. A qualitative comparison of biodistribution of tracer level TLX591, as demonstrated by SPECT, with 68Ga-PSMA-11 on PET imaging will be performed to ensure equivalent (or improved) radiopharmaceutical tumour targeting. Dosimetry analysis will also be performed. If safety is confirmed by independent DSMB review, each individual patient in Cohort 1 will proceed 14 days following the initial tracer dose to a second administration of TLX591. SoC therapy will continue according to standard practice. Cohort 2: All further enrolled patients will receive two administrations of 76 mCi TLX591 for further evaluation of safety, tolerability and biodistribution, and efficacy in combination with SoC. 1. Pan M-H et al. 2009 2. Dorff TB et al., 2019 3. Tagawa, Milowsky et al., 2013 4. Tagawa et al., 2019 5. Tagawa, Whang et al., 2014 6. Niaz et al., 2020. Clinical trial information: NCT04786847.

Experimental and Numerical Investigation of Parameters Affecting High-Frequency Irreversible Electroporation for Prostate Cancer Ablation.

While the primary goal of focal therapy for prostate cancer (PCa) is conserving patient quality of life by reducing oncological burden, available modalities use thermal energy or whole-gland radiation which can damage critical neurovascular structures within the prostate and increase risk of genitourinary dysfunction. High-frequency irreversible electroporation (H-FIRE) is a promising alternative ablation modality that utilizes bursts of pulsed electric fields (PEFs) to destroy aberrant cells via targeted membrane damage. Due to its nonthermal mechanism, H-FIRE offers several advantages over state-of-the-art treatments, but waveforms have not been optimized for treatment of PCa. In this study, we characterize lethal electric field thresholds (EFTs) for H-FIRE waveforms with three different pulse widths as well as three interpulse delays in vitro and compare them to conventional irreversible electroporation (IRE). Experiments were performed in non-neoplastic and malignant prostate cells to determine the effect of waveforms on both targeted (malignant) and adjacent (non-neoplastic) tissue. A numerical modeling approach was developed to estimate the clinical effects of each waveform including extent of nonthermal ablation, undesired thermal damage, and nerve excitation. Our findings indicate that H-FIRE waveforms with pulse durations of 5 and 10 μs provide large ablations comparable to IRE with tolerable levels of thermal damage and minimized muscle contractions. Lower duration (2 μs) H-FIRE waveforms exhibit the least amount of muscle contractions but require increased voltages which may be accompanied by unwanted thermal damage.

Stage-specific differential expression of zinc transporter SLC30A and SLC39A family proteins during prostate tumorigenesis.

Prostate cancer (PCa) initiation and progression uniquely modify the prostate milieu to aid unrestrained cell proliferation. One salient modification is the loss of the ability of prostate epithelial cells to accumulate high concentrations of zinc; however, molecular alterations associated with loss of zinc accumulating capability in malignant prostate cells remain poorly understood. Herein, we assessed the stage-specific expression of zinc transporters (ZNTs) belonging to the ZNT(SLC30A) and Zrt- and Irt-like protein (ZIP) (SLC39A) solute-carrier family in the prostate tissues of different genetically engineered mouse models (GEMM) of PCa (TMPRSS2-ERG.Ptenflox/flox , Hi-Myc+/- , and transgenic adenocarcinoma ofmouseprostate), their age-matched wild-type controls, and 104 prostate core biopsies from human patients with different pathological lesions. Employing immunohistochemistry, differences in the levels of protein expression and spatial distribution of ZNT were evaluated as a function of the tumor stage. Results indicated that the expression of zinc importers (ZIP1, ZIP2, and ZIP3), which function to sequester zinc from circulation and prostatic fluid, was low to negligible in the membranes of the malignant prostate cells in both GEMM and human prostate tissues. Regarding zinc exporters (ZNT1, ZNT2, ZNT9, and ZNT10) that export excess zinc into the extracellular spaces or intracellular organelles, their expression was low in normal prostate glands of mice and humans; however, it was significantly upregulated in prostate adenocarcinoma lesions in GEMM and PCa patients. Together, our findings provide new insights into altered expression of ZNTs during the progression of PCa and indicate that changes in zinc homeostasis could possibly be an early-initiation event during prostate tumorigenesis and a likely prevention/intervention target.

Genomic Features and Clinical Implications of Intraductal Carcinoma of the Prostate.

Intraductal carcinoma of the prostate (IDC-P) is a rare and unique form of aggressive prostate carcinoma, which is characterized by an expansile proliferation of malignant prostatic epithelial cells within prostatic ducts or acini and the preservation of basal cell layers around the involved glands. The vast majority of IDC-P tumors result from adjacent high-grade invasive cancer via the retrograde spreading of tumor cells into normal prostatic ducts or acini. A subset of IDC-P tumors is rarely derived from the de novo intraductal proliferation of premalignant cells. The presence of IDC-P in biopsy or surgical specimens is significantly associated with aggressive pathologic features, such as high Gleason grade, large tumor volume, and advanced tumor stage, and with poor clinical courses, including earlier biochemical recurrence, distant metastasis, and worse survival outcomes. These architectural and behavioral features of IDC-P may be driven by specific molecular properties. Notably, IDC-P possesses distinct genomic profiles, including higher rates of TMPRSS2–ERG gene fusions and PTEN loss, increased percentage of genomic instability, and higher prevalence of germline BRCA2 mutations. Considering that IDC-P tumors are usually resistant to conventional therapies for prostate cancer, further studies should be performed to develop optimal therapeutic strategies based on distinct genomic features, such as treatment with immune checkpoint blockades or poly (adenosine diphosphate–ribose) polymerase inhibitors for patients harboring increased genomic instability or BRCA2 mutations, as well as genetic counseling with genetic testing. Patient-derived xenografts and tumor organoid models can be the promising in vitro platforms for investigating the molecular features of IDC-P tumor.

Intermittent Hormone Therapy Models Analysis and Bayesian Model Comparison for Prostate Cancer

The prostate is an exocrine gland of the male reproductive system dependent on androgens (testosterone and dihydrotestosterone) for development and maintenance. First-line therapy for prostate cancer includes androgen deprivation therapy (ADT), depriving both the normal and malignant prostate cells of androgens required for proliferation and survival. A significant problem with continuous ADT at the maximum tolerable dose is the insurgence of cancer cell resistance. In recent years, intermittent ADT has been proposed as an alternative to continuous ADT, limiting toxicities and delaying time-to-progression. Several mathematical models with different biological resistance mechanisms have been considered to simulate intermittent ADT response dynamics. We present a comparison between 13 of these intermittent dynamical models and assess their ability to describe prostate-specific antigen (PSA) dynamics. The models are calibrated to longitudinal PSA data from the Canadian Prospective Phase II Trial of intermittent ADT for locally advanced prostate cancer. We perform Bayesian inference and model analysis over the models’ space of parameters on- and off-treatment to determine each model’s strength and weakness in describing the patient-specific PSA dynamics. Additionally, we carry out a classical Bayesian model comparison on the models’ evidence to determine the models with the highest likelihood to simulate the clinically observed dynamics. Our analysis identifies several models with critical abilities to disentangle between relapsing and not relapsing patients, together with parameter intervals where the critical points’ basin of attraction might be exploited for clinical purposes. Finally, within the Bayesian model comparison framework, we identify the most compelling models in the description of the clinical data.

Organ-Specific Uptake of Extracellular Vesicles Secreted by Urological Cancer Cells.

Simple SummaryExtracellular vesicles (EVs) play an important role in the communication of cancer cells with their local microenvironment and distant organ systems, in order to promote a supportive tumor microenvironment, as well as to prepare premetastatic niches. In this study, we aimed to analyze if the EVs secreted by urological cancer cells are taken up by specific organ systems, depending on their origin. After the intravenous injection of fluorescence-labeled EVs from benign and malignant prostate, kidney, and bladder cells in immunodeficient mice, their organs were harvested and analyzed for the presence of fluorescent EVs. We could show that (i) EVs are taken up not entirely organ-specifically but in different amounts, depending on their origin; (ii) EVs from malignant cells are taken up more efficiently than EVs from benign cells; and (iii) EVs are taken up very fast. These observations hint to an organotropism in EV uptake, which needs to be further investigated.Extracellular vesicles (EVs) secreted by cancer cells have been shown to take a pivotal part in the process of local and systemic tumor progression by promoting the formation of a supportive local tumor microenvironment and preparing premetastatic niches in distant organ systems. In this study, we analyzed the organ-specific uptake of EVs secreted by urological cancer cells using an innovative in-vivo approach. EVs from benign and malignant prostate, kidney, and bladder cells were isolated using ultracentrifugation, fluorescence-labeled and injected intravenously in immunodeficient mice. After 12 or 24 h, the animals were sacrificed, their organs were harvested and analyzed for the presence of EVs by high-resolution fluorescence microscopy. Across all entities, EVs were taken up fast (12 h > 24 h), and EVs from malignant cells were taken up more efficiently than EVs from benign cells. Though not entirely organ-specific, EVs were incorporated in different amounts, depending on the entity (prostate: lung > liver > brain; kidney: brain > lung > liver; bladder: lung > liver > brain). EV uptake in other organs than lung, liver, brain, and spleen was not observed. Our results suggest a role of EVs in the formation of premetastatic niches and an organotropism in EV uptake, which have to be examined in more detail in further studies.

Enhancing cisplatin anticancer effectivity and migrastatic potential by modulation of molecular weight of oxidized dextran carrier

The molecular weight (Mw) of dextran derivatives, such as regioselectively oxidized dicarboxydextran (DXA), is greatly influencing their faith in an organism, which could be possibly used to improve anticancer drug delivery. Here we present a modified method of sulfonation-induced chain scission allowing direct and accurate control over the Mw of DXA without increasing its polydispersity. Prepared DXA derivatives (Mw = 10–185 kDa) have been conjugated to cisplatin and the Mw of the carrier found to have a significant impact on cisplatin release rates, in vitro cytotoxicity, and migrastatic potential. Conjugates with the high-Mw DXA showed particularly increased anticancer efficacy. The best conjugate was four times more effective against malignant prostatic cell lines than free cisplatin and significantly inhibited the ovarian cancer cell migration. This was traced to the characteristics of spontaneously formed cisplatin-crosslinked DXA nanogels influenced by Mw of DXA and amount of loaded cisplatin.

Abstract 910: Amanitin-based ADCs targeting PSMA as novel therapeutic modality for prostate cancer therapy

Abstract Background: Antibody-targeted amanitin conjugates (ATACs) comprise a new class of antibody-drug conjugates (ADCs) using amanitin as toxic payload. Amanitin binds to the RNA pol II and thereby efficiently inhibits the cellular transcription process. In the current study, in vitro and in vivo data of an ATAC targeting PSMA (prostate specific membrane antigen) are presented. PSMA is a type II integral membrane glycoprotein and used as tumor marker due to its predominant expression on malignant prostate cells in prostate carcinoma. As PSMA expression increases with tumor aggressiveness, metastatic and disease recurrence, it is considered an ideal target for amanitin-based ADCs. Material and methods: Cell lines: PSMA+ cell lines: LNCap, 22RV1; PSMA- cell line: PC3 Antibody: cysteine engineered monoclonal anti-PSMA antibody (Albert Ludwig University Freiburg, medical center; derivatization and production at Heidelberg Pharma) Toxic warhead: Cysteine reactive amanitin-linker constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to engineered cysteine residues of the anti-PSMA antibody yielding ATACs with a DAR of 2. Cell proliferation assay: Quantitative determination of cell viability was performed by BrdU-based chemiluminescent cell proliferation ELISA (Roche). Animal models: Subcutaneous Mouse xenograft tumor models with PSMA-positive cell lines 22RV1 and LNCap were performed in single-dose experiments. Tolerability was assessed in mice and non-human primates (NHP). Results: All anti-PSMA ATACs with optimized amanitin-linker variants for use in solid tumors showed favorable in vitro cytotoxicity with nano- to picomolar activity on PSMA+ cell lines and no cytotoxic activity on PSMA- cells. In mouse xenograft models, the optimized anti-PSMA ATACs caused dose-dependent tumor regression in LNCap s.c. xenografts. Complete remission was achieved after a single i.v. dose of ¼ MTD with 100% overall survival for the full duration of the studies (&amp;gt;160 days). The tolerability of the tested ATACs in mice differed between 15 and &amp;gt; 80 mg/kg Safety profiling in Cynomolgus monkeys revealed a good tolerability and therapeutic index for all selected amanitin-linker variants. Hematology and clinical chemistry parameters were unaffected except liver enzymes and LDH: A moderate and transient increase was observed. Low off target toxicity was confirmed by a non-binding ATAC. Conclusions: Targeted cytotoxic drug delivery to PSMA positive cell lines was achieved by using anti-PSMA ATACs optimized for the use in solid tumors. The mode of action of the payload amanitin led to an efficient anti-tumor potential in vitro and in vivo with good tolerability in non-human primates. Using ATACs in the therapy of PSMA positive prostate cancer is a promising approach, especially by using a cytotoxic agent whose mode of action differs from other commonly used toxins. Citation Format: Alexandra Braun, Aniko Palfi, Christoph Mueller, Torsten Hechler, Andreas Pahl, Michael Kulke. Amanitin-based ADCs targeting PSMA as novel therapeutic modality for prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 910.

Plasma Deposited Polyoxazoline Films Integration Into Spiral Microfluidics for the Targeted Capture of Size Selected Cells.

Biomolecules readily and irreversibly bind to plasma deposited Polyoxazoline thin films in physiological conditions. The unique reactivity of these thin films toward antibodies is driving the development of immunosensing platforms for applications in cancer diagnostics. However, in order for these coatings to be used as advanced immunosensors, they need to be incorporated into microfluidic devices that are sealed via plasma bonding. In this work, the thickness, chemistry and reactivity of the polyoxazoline films were assessed following plasma activation. Films deposited from methyl and isopropenyl oxazoline precursors were integrated into spiral microfluidic devices and biofunctionalized with prostate cancer specific antibodies. Using microbeads as model particles, the design of the spiral microfluidic was optimised to enable the size-based isolation of cancer cells. The device was tested with a mixed cell suspension of healthy and malignant prostate cells. The results showed that, following size-specific separation in the spiral, selective capture was achieved on the immunofunctionalised PPOx surface. This proof of concept study demonstrates that plasma deposited polyoxazoline can be used for immunosensing in plasma bonded microfluidic devices.