Invivo Tumor Research Articles

Positional influence on cellular transcriptional identity revealed through spatially segmented single-cell transcriptomics.

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for describing cell states. Identifying the spatial arrangement of these states in tissues remains challenging, with the existing methods requiring niche methodologies and expertise. Here, we describe segmentation by exogenous perfusion (SEEP), a rapid and integrated method to link surface proximity and environment accessibility to transcriptional identity within three-dimensional (3D) disease models. The method utilizes the steady-state diffusion kinetics of a fluorescent dye to establish a gradient along the radial axis of disease models. Classification of sample layers based on dye accessibility enables dissociated and sorted cells to be characterized by transcriptomic and regional identities. Using SEEP, we analyze spheroid, organoid, and invivo tumor models of high-grade serous ovarian cancer (HGSOC). The results validate long-standing beliefs about the relationship between cell state and position while revealing new concepts regarding how spatially unique microenvironments influence the identity of individual cells within tumors.

Three-Dimensional Magnetic Chip with Extracorporeal Circulation for Circulating Tumor Cell In Vivo Detection and Tumor Growth Inhibition.

Liquid biopsy technology involves taking samples from body fluids in a minimally invasive way and analyzing tumor markers to achieve early diagnosis and efficacy evaluation of tumors. The development of real-time cancer diagnosis and treatment strategies based on liquid biopsy technology is of great significance to cancer management. This paper described an extracorporeal circulation based on a three-dimensional (3D) magnetic chip (3DMC-system) for in vivo detection and real-time monitoring of circulating tumor cells (CTCs). Utilizing biofunctionalized magnetic nanospheres (MNs) with CTC recognition function, this 3DMC-system could effectively achieve the real-time monitoring of CTCs in vivo with good stability and strong anti-interference. Compared with in vitro CTC detection, in vivo detection could not only detect more CTCs but also detect the presence of CTCs in the blood at an early stage of the tumor, when tumor metastasis is not observed in imaging. In addition, due to the flexibility of the chip design, the system can easily add a treatment module to integrate cancer diagnosis and treatment together. With good biocompatibility and high stability, this 3DMC-system is expected to provide a new personalized medical program for cancer patients.

Multifunctional Ultrasmall Theranostic Nanohybrids Developed by Ultrasonic Atomizer for Drug Delivery and Magnetic Resonance Imaging.

Theranostic nanoparticulate systems (TNPs) have shown potential in addressing problems related to spatial localization and temporally controlled release of drugs with the capabilities of real-time imaging to evaluate the progress of therapy. The current study reports the ultrasonic atomization-led synthesis of in vitro and in vivo evaluations of ultrasmall chitosan-based theranostic nanohybrid formulations with encapsulated doxorubicin (DOX) and iron-oxide magnetic nanoparticles. The nanohybrid particles are characterized using transmission electron microscopy, powder X-ray diffraction, FTIR, DOX encapsulation efficiency, in vitro release, cellular uptake, and toxicity. These formulations were also tested for the capability of invivo tumor reduction and simultaneous magnetic resonance imaging using Swiss albino mice. Ultrasonic atomizer-led synthesis resulted in chitosan-magnetic nanohybrids (CMNPs) having sizes of 15 ± 3 nm which comprise MNP of 10 ± 3 nm. The encapsulation of DOX in CMNP was around 25%, resulting in an 80% sustained release over 10 days at pH 5 and 7. CMNP was also found to be an efficient DOX delivery vehicle tested on cancer cells (HeLa). The CMNPs were able to reduce the tumor volume by 60% in 15 days. The inherent magnetic property and nanoscale size of CMNPs also provided for enhanced contrast efficiency in magnetic resonance imaging of tumors. Thus, such multifunctional theranostic nanoparticles can be an efficient tool for targeted diagnostic and therapeutic success.

Prosopis juliflora (Sw.) DC phytochemicals induce apoptosis and inhibit cell proliferation signaling pathways, EMT, migration, invasion, angiogenesis and stem cell markers in melanoma cell lines

Ethnopharmacological relevanceProsopis juliflora (Sw.), DC is a xerophytic plant species that extensively grow in Asia, Africa, Australia, and Brazil. From ancient time P. juliflora is being utilized in various folk remedies for example in wound healing, fever, inflammation, measles, excrescences, diarrhea and dysentery. Traditionally, gum, paste, and smoke obtained from the leaves and pods are applied for anticancer, antidiabetic, anti-inflammatory, and antimicrobial purposes. Aim of the studyOur previous studies have demonstrated the promising potential of Prosopis Juliflora leaves methanol extract (PJLME) against breast cancer, and suggested its possible integration as a complementary medicine for the effective management of breast cancer. However, evidence against how PJLME mechanistically target the cancer proliferative pathways and other targets is poorly understood. The basic aim of the present study was to understand the anti-melanoma potential of PJLME against B16f10 cells with possible mechanisms of action. Materials and methodsMTT assay was used to determine cell viability. Wound and transwell migration assay was performed to check migration potential of cells after PJLME treatment, while clonogenic assay was carried out to understand its colony inhibition actvity. Flow cytometry was used to perform annexin V/PI assay (apoptosis assay), ROS assay, cell cycle analysis. In-vitro angiogenesis assay was performed to check formation of capillary like vascular structure after PJLME treatment. Apoptotic genes, signaling pathways markers, EMT markers and stem cell markers were determined by western blotting. In-vivo BALB/C mice xenograft model study was performed to check the effect of PJLME on in-vivo melanoma tumor growth. ResultsThe experimental outcome of the present study has clearly demonstrated the inhibition of growth, migration, invasion, colony formation and apoptosis inducing potential of PJLME against mouse melanoma cancer cells. Treatment of B16F10 melanoma cells with PJLME resulted in arrest of cell cycle at G0/G1 phase. Annexin V-FITC/PI assay confirmed the apoptosis inducing potential of PJLME in B16F10 and A375 melanoma cells. Furthermore, Western blot experiments confirmed that the treatment of PJLME downregulates the expression of anti-apoptotic gene like Bcl2 and increase the expression profile of pro-apoptotic genes like Bax, Bad, and Bak in B16F10 melanoma cells. HUVEC (Human umbilical vein endothelial cells) tube formation assay clearly demonstrated the anti-angiogenic potential of PJLME. The study also revealed that PJLME has potential to inhibit the Akt and Erk signaling pathways which are participating in cancer cell proliferation, migration, invasion etc. The outcome of qRT-PCR and immunoblotting analysis clearly unveiled that PJLME treatment leads to downregulation of epithelial-mesenchymal transition (EMT) as well as stem cell markers. Finally, the in-vivo animal xenograft model study also revealed the anti-melanoma potential of PJLME by significantly inhibiting the B16F10 melanoma tumor growth in BALB/c mice model. The LC-ESI-MS/MS analysis of PJLME showed the presence of variety of bioactive molecules associated with anticancer effects. ConclusionThe outcome of the present investigation clearly demonstrated the anti-melanoma potential of PJLME against B16f10 melanoma cells. PJLME can be explored as an adjuvant or complementary therapy against melanoma cancer, however further studies are required to understand the clinical efficacy of PJLME. Nevertheless, it can be further explored as a promising resource for identification of novel anticancer candidate drug.

Abstract 4672: In-vivo tumor implantation site exhibits differential immune response in solid tumors

Abstract Successful discovery and development of cancer targeted therapeutics depends on testing these agents in the clinically most relevant translational models. Emerging evidence highlights the importance of the local tumor microenvironment in evaluating the efficacy of new therapeutics, especially for immunotherapies. Historically, patient-derived xenograft (PDX) modeling involves subcutaneous implantation which minimally represents the actual human tumor site. Here, we use PBMC humanized PDX models to demonstrate differences in the tumor microenvironment, overall immune response, and the pharmacological outcome, between subcutaneous and orthotopic PDX models. These findings highlight the importance of testing cancer therapeutics in the most physiologically relevant setting for greater translation into clinical success. Citation Format: Rajeev Shrimali, Jasmine Borroel, Carmen Sunico, Bridget Corcoran, Bianca Carapia, Rowan Prendergast, Jantzen Sperry, Jonathan Nakashima. In-vivo tumor implantation site exhibits differential immune response in solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4672.

Abstract 818: Preclinical studies of the cabazitaxel nanoformulation MPB-1734 support therapeutic application in chemo-resistant solid tumors and synergistic benefit in combination with immune checkpoint inhibitor

Abstract Background: Cabazitaxel (CBZ) is a second-generation taxane that has the potential to overcome taxane-resistance due to the low affinity for the P-glycoprotein efflux pump. However, with the high systemic toxicity such as neutropenia and hypersensitivity, the clinical use is restricted to patients with metastatic castration-resistant prostate cancer who show progression after docetaxel-based chemotherapy. MPB-1734 is a CBZ nanoformulation which is currently being evaluated as monotherapy in patients with advanced solid tumors in a Phase 1/2a trial (NCT04643418). It is developed to overcome the limitations associated with CBZ application and as potential therapeutic agent for chemo-resistant cancers. This work outlines the results of preclinical studies that support the tumor targeting ability, improved safety profiles, significant therapeutic effect, and synergistic benefit in combination with immune checkpoint inhibitor. Methodology: A series of nonclinical studies of MPB-1734 were conducted including toxicology evaluations in two species and the antitumor activity evaluations in a range of xenograft mice models as cervical cancer (KB), ovarian cancer (OVCAR3) and platinum-resistant human ovarian cancer patient-derived-xenograft (PDX) model (OV0276). The antitumor activity of MPB1734 combined with anti-PD-1 antibody were also evaluated in the syngenetic 4T1 breast cancer mice model. Results: Toxicology evaluations of MPB-1734 demonstrated the safety profiles of CBZ were significantly improved, and the tolerated doses of MPB-1734 are at least 2 folds higher than commercial formulation. An in-vivo tumor targeting study showed that MPB1734 mainly accumulated in the tumor sites, rather than in the vital organs. MPB1734 exhibited a better efficacy compared to commercial formulation in KB and OVCAR3 models. For platinum-resistant PDX model, MPB-1734 demonstrated significant anti-tumor efficacy (TGI:100%, p<0.001). For 4T1 mice model, MPB-1734 combined with anti-PD-1 antibody demonstrated synergistic benefit (TGI: 78.6%) compared to anti-PD-1 antibody monotherapy (TGI: 2.8%) and MPB-1734 monotherapy (TGI: 52.9%). This synergistic benefit may result from increased CD8+ T cells in tumor region after MPB-1734 treatment. Conclusion: MPB-1734 can reduce the major dose-limiting side effect (neutropenia) of CBZ and increase the tolerated dose. With tumor targeting ability, superior antitumor effects were demonstrated as compared with commercial formulation in a range of cancer models. MPB-1734 combined with anti-PD-1 antibody showed synergistic benefit in tumor inhibition. These preclinical results support the potential use of MPB-1734 as therapeutic agent for chemo-resistant cancer with monotherapy or combination therapy with immuno-oncology agents. Citation Format: Yuan Hung Hsu, Chih-Lung Chen, Jhih-Yun Jian, Ming-Cheng Wei, Wen-Yuan Hsieh, Chia-Wen Huang, Yung-Chu Chen, Tzu-Hsin Liao, Shian-Jy Wang. Preclinical studies of the cabazitaxel nanoformulation MPB-1734 support therapeutic application in chemo-resistant solid tumors and synergistic benefit in combination with immune checkpoint inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 818.

MP20-20 CHAPERONE-MEDIATED AUTOPHAGY PROMOTES PCA SURVIVAL DURING ARPI THROUGH SELECTIVE PROTEOME AND PATHWAY REMODELING

You have accessJournal of UrologyCME1 Apr 2023MP20-20 CHAPERONE-MEDIATED AUTOPHAGY PROMOTES PCA SURVIVAL DURING ARPI THROUGH SELECTIVE PROTEOME AND PATHWAY REMODELING Nicholas Nikesitch, Eliana Beraldi, Fan Zhang, Hans Adomat, Robert Bell, Kotaro Suzuki, Ladan Fazli, Sonia Kung, Christopher Wells, Nicholas Pinette, Neetu Saxena, Yuzhuo Wang, and Martin Gleave Nicholas NikesitchNicholas Nikesitch More articles by this author , Eliana BeraldiEliana Beraldi More articles by this author , Fan ZhangFan Zhang More articles by this author , Hans AdomatHans Adomat More articles by this author , Robert BellRobert Bell More articles by this author , Kotaro SuzukiKotaro Suzuki More articles by this author , Ladan FazliLadan Fazli More articles by this author , Sonia KungSonia Kung More articles by this author , Christopher WellsChristopher Wells More articles by this author , Nicholas PinetteNicholas Pinette More articles by this author , Neetu SaxenaNeetu Saxena More articles by this author , Yuzhuo WangYuzhuo Wang More articles by this author , and Martin GleaveMartin Gleave More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.20AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The androgen receptor (AR) plays an important role in PCa metabolism, with androgen receptor pathway inhibition (ARPI) subjecting PCa cells to acute metabolic stress caused by reduced biosynthesis and energy production. Defining acute stress response mechanisms that alleviate ARPI stress and therefore mediate prostate cancer (PCa) treatment resistance will help improve therapeutic outcomes of patients treated with ARPI. METHODS: CMA induction and activity was studied within several in-vitro, in-vivo and ex-vivo PCa models using QPCR, Western Blot and CMA-reporter assays. CMA upregulation/downregulation on PCa growth was measured in-vitro and in-vivo via cell confluence or tumour volume/PSA levels. Proteome profiling of PCa cells was perfromed by TMT mass spec, with metabolic assays performed using western blot, colormetric and flow cytometry. RESULTS: We identified the up-regulation of chaperone-mediated autophagy (CMA) in response to acute ARPI stress, which persisted in castration-resistant PCa (CRPC); previously undefined in PCa. CMA is a selective protein degradation pathway and a key stress response mechanism up-regulated under several stress stimuli, including metabolic stress. Through selective protein degradation, CMA orchestrates the cellular stress response by regulating cellular pathways through selective proteome remodeling. Through broad-spectrum proteomic analysis, CMA coordinates metabolic reprogramming of PCa cells to sustain PCa growth and survival during ARPI; through the upregulation of mTORC1 signaling and pathways associated with PCa biosynthesis and energetics. This not only promoted PCa growth during ARPI, but also promoted the emergence of CRPC in-vivo. During CMA inhibition, PCa metabolism is compromised, leading to ATP depletion, resulting in a profound anti-proliferative effect on PCa cells, and is enhanced when combined with ARPI. Furthermore, CMA inhibition prevented in-vivo tumour formation, and also re-sensitized enzalutamide-resistant cell lines in-vitro. The profound anti-proliferative effect of CMA inhibition was attributed to cell cycle arrest mediated through p53 transcriptional repression of E2F target genes. CONCLUSIONS: In summary, CMA is an acute ARPI stress response mechanism, essential in alleviating ARPI induced metabolic stress, essential for ensuring PCa growth and survival. CMA plays a critical role in the development of ARPI resistance in PCa. Source of Funding: Terry Fox New Frontiers Program Project (1062) and PCFBC Grant-In-Aid Program © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e283 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nicholas Nikesitch More articles by this author Eliana Beraldi More articles by this author Fan Zhang More articles by this author Hans Adomat More articles by this author Robert Bell More articles by this author Kotaro Suzuki More articles by this author Ladan Fazli More articles by this author Sonia Kung More articles by this author Christopher Wells More articles by this author Nicholas Pinette More articles by this author Neetu Saxena More articles by this author Yuzhuo Wang More articles by this author Martin Gleave More articles by this author Expand All Advertisement PDF downloadLoading ...

Abstract P2-21-11: A high content imaging and analysis approach to screen compounds for novel drug targets in 3D breast cancer co-culture model

Abstract The desired effect of cancer therapeutics can only be conceived when applied to complex multicellular structures with different cell types and extracellular matrix (ECM) in three-dimensional (3D) space. Therefore, 3D cell culture systems have become very popular in drug screening and discovery. There is an immense demand for highly efficient and easy methods to produce 3D spheroids in any cell format. However, heterogeneity in size, extended cultivation times, and reproducibility for high throughput assays are limiting factors in the generation of spheroids. Recently we have established a naturally transformed breast cancer cell line, KAIMRC1 (1), from ductal breast carcinoma. We have developed a novel and easy method to produce spheroids (2) from KAIMRC1 cells in vitro, which can be used as a 3D model to study proliferation, differentiation, metabolism, cell death, and drug response of cells in the tumor microenvironment. We developed an in-vitro high content imaging (HCI) based cellular assay using commercially available compound panels to perform initial drug screening in the KAIMRC1 spheroids model. Our approach allows rapid screening of a panel of drugs to assess inhibitory effects on the growth of tumor cells in 3D cultures. To mimic the actual in-vivo tumor microenvironment, we are now developing an HCI-based assay utilizing 3D KAIMRC1 spheroids co-cultured with patient-derived fibroblast cells. 1. Ali, Rizwan, et al. “Isolation and Characterization of a New Naturally Immortalized Human Breast Carcinoma Cell Line, KAIMRC1.” BMC Cancer 17 (2017) 2. Rizwan Ali et al., (2021) New Born Calf Serum (NBCS) can induce spheroid formation in breast cancer KAIMRC1 cell line. Front. Mol. Biosci. DOI: 10.3389/fmolb.2021.769030 Citation Format: Rizwan Ali, Sarah Huwaizi, Arwa Alsubait, Mohamed Boudjelal. A high content imaging and analysis approach to screen compounds for novel drug targets in 3D breast cancer co-culture model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-11.

Abstract P3-04-04: Correlation of Mammographic Microcalcifications with Final Surgical Pathology after Neoadjuvant Chemotherapy for Breast Cancer

Abstract Introduction: Benefits of neoadjuvant chemotherapy (NAC) in breast cancer include de-escalating surgical management, treating occult systemic metastases and the assessment of in-vivo tumor response. Guidelines for post-NAC imaging to assess response lack specificity on appropriateness and utility of individual imaging modalities for surgical planning. Mammographic microcalcifications are a confounder that are not well studied. We examined the correlation between the mammographic extent of microcalcifications present post-NAC, corresponding MRI lesions, and definitive surgical pathology. Methods: In this retrospective cohort study, patients with calcifications on pre-NAC mammograms were collected from a database of consecutive breast cancer patients receiving NAC at an academic center between January 1st 2014 – December 31st 2019. The primary objective was to determine how the maximum diameter of post-NAC calcifications correlates with final surgical pathology, stratified by tumor receptor subtype. The secondary objectives were to assess correlation of the maximum diameter on final pathology with the maximum diameter of (1) post-NAC mammographic mass (if present) and (2) post-NAC MRI, mass (ME) and non-mass enhancement (NME). Parameters on final surgical pathology included the diameters of invasive disease, ductal carcinoma in-situ (DCIS) and the tumor bed (TB). Pearson’s correlation coefficient was used to evaluate statistical significance and considered strong if R2 ≥70%, moderate if R2 = 25-70% and weak if R2 ≤25%. Results: 343 patients received NAC, of which 157 were excluded for lack of calcifications (n=147), lack of imaging reports (n=8) or inflammatory breast cancer (n=2). 186 patients met the inclusion criteria with a mean age of 49.9 years. 34 (18.3%) patients had triple negative breast cancers (TNBCs). Mammographic calcifications correlated poorly with residual invasive disease (R2= 10.8%), overestimating by 57%. TNBCs demonstrated the strongest correlation between microcalcifications and invasive disease (R2=83%), but calcifications overestimated pathology by 41%. Mammographic calcifications correlated moderately with the TB (R2= 50.3%) and poorly with DCIS (R2 = 3.4%). By subtype, both correlations were strong in TNBCs (R2 = 76.3% and 77.9% respectively). Similarly, focal mass on mammography correlated poorly with pathology except in TNBCs, which correlated moderately with all three pathology parameters (Table 1). In patients with calcifications on mammography, MRI ME and NME correlated weakly with invasive disease, except in TNBCs, where correlation was moderate in ME (R2 = 37.7%) and NME (R2 = 28.4%). Conclusion: In breast cancer patients with microcalcifications, current post-NAC imaging modalities appear to overestimate the extent of residual disease. Surgical excision of all residual microcalcifications may maximize oncologic safety but may also represent over-treatment. Ongoing feasibility trials on surgical omission rely on post-NAC imaging to determine trial candidacy. Our data suggests that potentially eligible patients are excluded on the basis of post-NAC imaging overestimating the extent of residual disease. Further studies are needed to determine the most accurate imaging correlate of residual disease after NAC to plan better surgeries, identify candidates for surgical omission and establish optimal post-NAC imaging guidelines. Current imaging modalities appear most accurate for TNBCs and may be more reliable at identifying exceptional responders for surgical omission in this subtype. Table 1. Summary of Correlation Analyses in Overall and Triple Negative Breast Cancer (TNBC) cohorts. * Average % difference in maximal diameter calculated as (pathology – radiology)/radiology Citation Format: Riordan M. Azam, Nicole Look Hong, David Lim. Correlation of Mammographic Microcalcifications with Final Surgical Pathology after Neoadjuvant Chemotherapy for Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-04-04.

P‑STAT3 influences doxorubicin and etoposide resistance of A549 cells grown in an invitro 3D culture model.

Tumor microenvironment undoubtedly has a significant impact on therapeutic responses. Abundant evidence suggests that the 3D invitro culture holds great promise for drug discovery and development by bridging the gap between conventional 2D culture and animal models. The present study described 3D basement membrane culture of A549 cells, which mimics the complex 3D arrangement of tumors invivo and elucidates the underlying mechanisms of microenvironmental influences on cellular functions and therapeutic efficacy. A549 cells cultured in 3D undergo G0/G1 phase arrest and decreased migratory and invasive capacity, indicating dormant cell characteristics. Hypoxia, apoptosis and stemness were demonstrated in the A549 cells in 3D architecture compared with the 2D‑cultured counterparts. More importantly, cells in the 3D environment exhibited increased resistance to different classes of anticancer agents. Western blotting revealed changes in the levels of key cancer‑associated pathways, phosphorylated (p)‑STAT3, p‑ERK, and p‑Akt, in response to 3D culture compared with 2D monolayer culture. Notably, mechanistic analysis using specific inhibitors showed that the STAT3 inhibitor overcomes the 3D culture‑induced doxorubicin and etoposide resistance. These results implicated an important role of p‑STAT3 in conferring chemoresistance in 3D‑cultured A549 cells, as well as the use of STAT3 inhibitor as a potential chemosensitizer to improve drug sensitivity. Thus, 3D culture systems, that more closely resemble invivo tumor biology, may be more effective models in searching for novel chemotherapeutic agents and therapeutic targets for cancer treatment.

Imaging Guided Endogenic H2 -Augmented Electrochemo-Sonodynamic Domino Cotherapy of Tumor in Vivo.

Precise and on-demand release of sufficient hydrogen (H2 ) to the tumor sites remains a key challenge for emerging H2 -oncotherapy, and little is known about the physiological effects of "abundant" H2 on complex tumor microenvironments (TME). Here, we report a highly efficient and cost-effective imaging-guided/-assessed H2 -therapy of tumors based on a joint electrochemo-sonodynamic treatment (H2 -EC/SD cotherapy) with strong "domino effect" triggered by endogenous H2 generation at tumor sites to speedily eliminate tumor tissue (≤80 mm3 ) within 1 day. Adequate H2 was controllably generated in tumor sites through mild electrochemistry in vivo due to acidic TME, by using clinic acupuncture Fe needle as electrodes. Besides starvation damage due to gas blockage destruction for vessels, nano-/micro-bubbles of H2 formed in-situ can elevate its internal temperature and burst vessels to further destroy tumor under ultrasound irradiation. Remarkably, vulnerable homeostasis of TME was disturbed as H2 also participates in physiological activity of tumor cells, leading to the tumor dysfunction. Last but not least, the inflammatory response of body from cancer was reduced after the treatment, beneficial for the body's immune system for post-treatment recovery. Based on all these merits, the H2 -EC/SD cotherapy provides a potentially safe and viable therapeutic strategy for future clinical applications. This article is protected by copyright. All rights reserved.

A 3D Ex Vivo Tumor-Immune Coculture System Mimicking In Vivo Tumor Environmental Stress on CD8+ T Cells Exhaustion.

Dissection of exhaustion trajectories of immune cells under tumor selection pressure in the tumor microenvironment (TME) elucidates the underlying machinery in anti-tumor immunity, which still lacks easy-to-use models to decipher. Herein,gelatin methacryloyl (GelMA)-poly (ethylene oxide) (PEO) based 3D hydrogel microspheroids are constructed with non-immunogenicity and controllable macroporous structure to establish a tumor-immune cell coculture (3D-HyGTIC) system. In 3D-HyGTIC system, when immune cells embarked, stepwise up-regulation of main immune checkpoints (ICs) molecules is observed with compromised cytokine production in CD8+ T cells, the trajectory of which is in lineage correlation with in vivo grafted tumors. Reinvigoration of CD8+ T cells is more obvious with the addition of an anti-PD-1 regimen at the early time point, which is recapitulated during the coculture of patient-derived tumor fragments (PDTF) and autologous T cells. Moreover, the upregulation of LAG-3 on CD8+ T cells after anti-PD-1 treatment is uncovered. Sequential addition of anti-LAG-3 successfully rescues the otherwise failed reactivation of CD8+ T cells. Therefore, the 3D-HyGTIC system is not only inclined to mimic the early differentiation trajectories of tumor-infiltrating CD8+ T cells but also may facilitate an evaluation of the efficacy of IC blockades and guide the designing of combination immunotherapy.

Chaperone-mediated autophagy promotes PCa survival during ARPI through selective proteome remodeling.

The androgen receptor (AR) plays an important role in PCa metabolism, with androgen receptor pathway inhibition (ARPI) subjecting PCa cells to acute metabolic stress caused by reduced biosynthesis and energy production. Defining acute stress response mechanisms that alleviate ARPI stress and therefore mediate prostate cancer (PCa) treatment resistance will help improve therapeutic outcomes of patients treated with ARPI. We identified the up-regulation of chaperone-mediated autophagy (CMA) in response to acute ARPI stress, which persisted in castration-resistant PCa (CRPC); previously undefined in PCa. CMA is a selective protein degradation pathway and a key stress response mechanism up-regulated under several stress stimuli, including metabolic stress. Through selective protein degradation, CMA orchestrates the cellular stress response by regulating cellular pathways through selective proteome remodeling. Through broad-spectrum proteomic analysis, CMA coordinates metabolic reprogramming of PCa cells to sustain PCa growth and survival during ARPI; through the upregulation of mTORC1 signaling and pathways associated with PCa biosynthesis and energetics. This not only promoted PCa growth during ARPI, but also promoted the emergence of CRPC in-vivo. During CMA inhibition, PCa metabolism is compromised, leading to ATP depletion, resulting in a profound anti-proliferative effect on PCa cells, and is enhanced when combined with ARPI. Furthermore, CMA inhibition prevented in-vivo tumour formation, and also re-sensitized enzalutamide-resistant cell lines in-vitro. The profound anti-proliferative effect of CMA inhibition was attributed to cell cycle arrest mediated through p53 transcriptional repression of E2F target genes. In summary, CMA is an acute ARPI stress response mechanism, essential in alleviating ARPI induced metabolic stress, essential for ensuring PCa growth and survival. CMA plays a critical role in the development of ARPI resistance in PCa.

Novel acridone derivatives probed using DFT, including design, synthesis, characterization with anti-oxidant and anti-mitotic screening

Applying the Ullmann synthesis, substituted aniline and o-halobenzoic acids are combined with potassium carbonate and copper powder to produce N- (substituted phenyl) anthranilic acids. Under the action of strong acids, the N-(substituted phenyl) anthranilic acids cyclize into acridone or substituted Acridones. In-vitro human cancer cell lines, in-vivo tumor xenograft models and genetically modified animal models are a few of the preclinical screening techniques that were used to evaluate the anticancer efficacy of the drug. The substance with a high binding score was produced, identified by the binding site for molecular docking and ligand binding was provided through the model's CASTp web server-revealed active site. Characterization was done using TLC, IR, NMR and mass spectroscopy.Their antioxidant and antimitotic properties were also analysed. DFT was used at the B3LYP/6-31G(d) level under solvent ethanol and a good correlation with experimental data was obtained. The synthesized compound showed significant activity.

Targeting IRS-1/2 in Uveal Melanoma Inhibits In Vitro Cell Growth, Survival and Migration, and In Vivo Tumor Growth.

Uveal melanoma originating in the eye and metastasizing to the liver is associated with poor prognosis and has only one approved therapeutic option. We hypothesized that liver-borne growth factors may contribute to UM growth. Therefore, we investigated the role of IGF-1/IGF-1R signaling in UM. Here, we found that IRS-1, the insulin receptor substrate, is overexpressed in both UM cells and tumors. Since we previously observed that IGF-1R antibody therapy was not clinically effective in UM, we investigated the potential of NT157, a small molecule inhibitor of IRS-1/2, in blocking this pathway in UM. NT157 treatment of multiple UM cell lines resulted in reduced cell growth and migration and increased apoptosis. This treatment also significantly inhibited UM tumor growth in vivo, in the chicken egg chorioallantoic membrane (CAM) and subcutaneous mouse models, validating the in vitro effect. Mechanistically, through reverse phase protein array (RPPA), we identified significant proteomic changes in the PI3K/AKT pathway, a downstream mediator of IGF-1 signaling, with NT157 treatment. Together, these results suggest that NT157 inhibits cell growth, survival, and migration in vitro, and tumor growth in vivo via inhibiting IGF-1 signaling in UM.

A novel urokinase plasminogen activator receptor-targeted peptide-based probe for in-vivo molecular imaging of glioblastoma.

The urokinase plasminogen activator receptor (uPAR) is a promising biomarker for cancer diagnosis and therapy. We herein fabricated a new type of uPAR-targeted imaging probe Al18F-NOTA-VC and preliminarily evaluated its potential application in PET imaging of the glioma model in vivo. Peptide VC was synthesized and identified by MALDI-TOF-MS. The IC50 between VC/precursor NOTA-VC and uPAR was then determined before the synthesis and purification of Al18F-NOTA-VC, followed by further studies of in-vitro properties of Al18F-NOTA-VC. Meanwhile, the AE105-based probe followed a similar procedure in-vitro test. Finally, the PET imaging properties, including uPAR-targeting ability and the metabolism of Al18F-NOTA-VC, were investigated. The VC and NOTA-VC were obtained successfully and demonstrated a good affinity with uPAR. Followed by Al18F labeling successfully, excellent properties, including the serum stability, water solubility, and specificity of Al18F-NOTA-VC, were obtained in-vitro test compared with AE105 based probe. An excellent tumor uptake and renal excretion data of Al18F-NOTA-VC were acquired from in-vivo U87MG tumor model PET imaging, consistent with the subsequent biodistribution study. In addition to the excellent specificity and high tumor/normal tissue contrast for uPAR-targeted PET imaging of U87MG tumor, Al18F-NOTA-VC possessed promising clearance ability by renal system route. These excellent properties facilitated Al18F-NOTA-VC to be a promising imaging agent for uPAR high-expressing tumors and, thus, provided a paradigm for developing peptide-based probes for uPAR-associated disease diagnosis.

Mitochondrial uncoupler DNP induces coexistence of dual-state hyper-energy metabolism leading to tumor growth advantage in human glioma xenografts.

Cancer bioenergetics is an essential hallmark of neoplastic transformation. Warburg postulated that mitochondrial OXPHOS is impaired in cancer cells, leading to aerobic glycolysis as the primary metabolic pathway. However, mitochondrial function is altered but not entirely compromised in most malignancies, and that mitochondrial uncoupling is known to increase the carcinogenic potential and modifies treatment response by altering metabolic reprogramming. Our earlier study showed that transient DNP exposure increases glycolysis in human glioma cells (BMG-1). The current study investigated the persistent effect of DNP on the energy metabolism of BMG-1 cells and its influence on tumor progression in glioma xenografts. BMG-1 cells were treated with 2,4-dinitrophenol (DNP) in-vitro, to establish the OXPHOS-modified (OPM-BMG) cells. Further cellular metabolic characterization was carried out in both in-vitro cellular model and in-vivo tumor xenografts to dissect the role of metabolic adaptation in these cells and compared them with their parental phenotype. Chronic exposure to DNP in BMG-1 cells resulted in dual-state hyper-energy metabolism with elevated glycolysis++ and OXPHOS++ compared to parental BMG-1 cells with low glycolysis+ and OXPHOS+. Tumor xenograft of OPM-BMG cells showed relatively increased tumor-forming potential and accelerated tumor growth in nude mice. Moreover, compared to BMG-1, OPM-BMG tumor-derived cells also showed enhanced migration and invasion potential. Although mitochondrial uncouplers are proposed as a valuable anti-cancer strategy; however, our findings reveal that prolonged exposure to uncouplers provides tumor growth advantage over the existing glioma phenotype that may lead to poor clinical outcomes.

HPB O06 Developing a patient-derived model of cholangiocarcinoma using Precision Cut Tissue Slices (PCTS)

Abstract Background Cholangiocarcinoma (CCA) is an aggressive malignancy with increasing incidence and persistently poor prognosis. None surgical treatment options for these patients are limited and a preponderance to chemoresistance means the benefits from chemotherapy are modest. The development of new targeted therapeutic strategies that prolong survival and reduce the risk of recurrence post-surgery are required. Accurate models that recapitulate tumour biology are essential to test and identify these novel therapies. Precision-cut tissue slices (PCTS) are patient-derived whole tissue explants that can be cultured ex-vivo. By retaining all aspects of the tumour micro-environment they recapitulate critical aspects of cancer biology, a significant advantage over 3D organoid culture. Our aim is to establish the use of the PCTS technique in CCA as the first step towards creating a patient-derived model of cholangiocarcinoma for use as a drug discovery platform. Methods CCA tissue samples and matched healthy liver tissue were collected from the operating theatre during surgical resection from fully consented patients. Multiple tissue slices (250μm, 5mm diameter) were prepared using a Krumdiek tissue slicer. Tissue slices were randomly incubated in triplicate and cultured for 0–15 days in 24 well plates. Tissue perfusion and air-liquid interface was maintained using Millipore well inserts. Tissue viability was determined at timepoints utilising MTS and ATP viability assays along with histological markers of proliferation (Ki-67) and apoptosis (cleaved-caspase 3). Maintenance of tissue morphology and phenotype during culture was confirmed on histological H&E staining and by staining of tumour specific markers (e.g CK19) on IHC and Western Blot. Results Thus far n=4 patients have had PCTS generated (3=pCCA 1=iCCA) from their tumour along with matched healthy liver. Following optimisation of the experimental protocol viability of the matched normal and tumour tissue slices can be maintained for upto 10 days, as assessed via in-situ MTS assay. Histological assessment of tissue slices harvested at day 0, 1,3,7 of culture shows the morphological and phenotypic structures of the source tumour to be maintained during ex-vivo culture. Viability of the tumour cells was additionally confirmed through the absence of cleave-caspase 3 markers of apoptosis on IHC staining. Conclusions This initial work has shown it is feasible to generate PCTS of CCA from resected surgical specimens with good viability. Further work is still required to ensure that our model retains a robust recapitulation of the in-vivo tumour including examination of the gene-expression profile. However, these initial results provide encouragement for the use of PCTS as a patient-derived model for drug discovery in CCA.

LncRNA NORAD mediates KMT2D expression by targeting miR-204-5p and affects the growth of gastric cancer.

Long non-coding ribonucleic acids (lncRNAs) are a class of non-coding RNAs implicated in the development of many malignancies, including gastric cancer (GC). In this study, we investigated the functions and molecular mechanisms of non-coding RNA activated by deoxyribonucleic acid damage (NORAD) in GC. NORAD expression at the messenger RNA levels was determined by quantitative reverse transcriptase (RT)-polymerase chain reaction assays. Cell proliferation, migration, and invasion were detected by Cell Counting Kit-8 assays, in-vivo tumor formation assays, and Transwell assays. Cell-cycle distribution was detected by a flow cytometry analysis. NORAD location was detected by nucleocytoplasmic fractionation assays. The interaction between NORAD and the microRNA-204-5p (miR-204-5p)/Lysine Methyltransferase 2D (KMT2D) axis was verified by dual-luciferase reporter gene assays and RNA binding protein immunoprecipitation (RIP) assays. Western blot was used to study the phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinases (PI3K)/protein kinase B (AKT) signaling pathway. NORAD was upregulated in the GC tissues and cell lines. The silencing of NORAD repressed cell proliferation and the Growth 2 (G2)/Mitosis (M) cell-cycle transition in GC. NORAD also regulated KMT2D expression by targeting miR-204-5p and mediated PTEN/PI3K/AKT signaling in GC. We found that NORAD acts as an oncogene in GC. Our findings might provide a novel therapeutic target for GC.

Synthesis and Antitumor Evaluation of Glutathione Responsive Self-Immolative Disulphide Linked Camptothecin-Biotin Conjugate.

Camptothecin is a naturally occurring alkaloid obtained from the stem wood of the Chinese tree, Camptotheca acuminata. It exerts pharmacological effects due to its ability to selectively inhibit the type-I topoisomerase DNA nuclear enzyme. Several semisynthetic analogs of camptothecin have been synthesized to date possessing antitumor activity. Camptothecin (CPT) is one of the most promising anticancer drugs but it produces various side effects because of its non-selectivity towards cancer cells. To overcome these adverse effects, we synthesized biotin conjugate of camptothecin, which was linked via a self-immolative disulfide linker (CPT-SS-Biotin). Biotin conjugated camptothecin linked through a disulfide bond was synthesized following schemes, and the structural characterization was carried out. The stability and drug release studies were performed in the presence of glutathione (GSH) while in vitro studies were performed on 4T1 tumor cell lines. In vivo pharmacological investigation was done using an antitumor Wistar rat model. The stability and drug release studies were performed in the presence of glutathione (GSH), and CPT-SSBiotin was found to be physiologically stable moiety and can only be cleaved in the presence of GSH to release free CPT. The CPT-SS-Biotin showed higher toxicity in the biotin-overexpressing 4T1 tumor cell line with a lower IC50 value (8.44 μM) compared to camptothecin alone (IC50 > 30 μM). CPT-SS-Biotin also showed 10.6% higher cellular uptake by cells in comparison to free camptothecin. The CPT-SS-Biotin was delivered to cells by binding to the biotin receptors on the cell surface, followed by energy-dependent endocytosis and internalization to cause cellular toxicity. In-vivo tumor suppression studies and in vitro cell line studies along with serological parameters and histopathological studies showed that conjugate produced a high therapeutic effect and remarkably reduced toxic effects in comparison to free CPT. The results suggested that biotinylation of camptothecin via disulfide linker can be a safe and efficacious method in cancer therapeutics.