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Improvement of organotypic tissue slice viability. a Manually sliced tumor slices were incubated for 2 hours in the presence of EU (Ethynyl Uridine) before fixation. During this time penetration of EU is limited to 10–20 cell layers from the edge of the slice. Automatically sliced (300 μm) tumor slices display EU incorporation across the entire depth of the slice within a 2-hour labeling period. b Constant orbital movement (60 rpm) significantly increased the number of EdU positive cells after 48 hours incubation compared to static culture conditions. c EdU incorporation after 96 hours of culturing under constant movement. d Prolonged culture of 300 μm tumor slices from one individual tumor using continuous movement and Medium I. Blue = DAPI, Red = EdU, Green lines indicate the edge of the tumor slice. Scale bars indicate 100 μm
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Background
The high incidence of breast cancer has sparked the development of novel targeted and personalized therapies. Personalization of cancer treatment requires reliable prediction of chemotherapy responses in individual patients. Effective selection can prevent unnecessary treatment that would mainly result in the unwanted side effects of the...
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Colon cancer is a major malignant neoplasm with a low survival rate for late-stage patients. Therefore, the investigation of molecules regulating colon cancer progression and the discovery of novel therapeutic targets is critical. Mitochondria play a vital role in maintaining the homeostasis of cells. Abnormal mitochondrial metabolism alterations a...
Citations
... One of the most highly upregulated mRNAs was IL-11, which was also prominently increased in the study examining PCTS of normal tissues 8,16 . IL-11 is a member of the IL-6 family of cytokines and has been implicated in the pathogenesis of fibrosis and solid malignancy, as well as inflammation 17,18 . ...
With cancer immunotherapy and precision medicine dynamically evolving, there is greater need for pre-clinical models that can better replicate the intact tumor and its complex tumor microenvironment (TME). Precision-cut tumor slices (PCTS) have recently emerged as an ex vivo human tumor model, offering the opportunity to study individual patient responses to targeted therapies, including immunotherapies. However, little is known about the physiologic status of PCTS and how culture conditions alter gene expression. In this study, we generated PCTS from head and neck cancers (HNC) and mesothelioma tumors (Meso) and undertook transcriptomic analyses to understand the changes that occur in the timeframe between PCTS generation and up to 72 h (hrs) in culture. Our findings showed major changes occurring during the first 24 h culture period of PCTS, involving genes related to wound healing, extracellular matrix, hypoxia, and IFNγ-dependent pathways in both tumor types, as well as tumor-specific changes. Collectively, our data provides an insight into PCTS physiology, which should be taken into consideration when designing PCTS studies, especially in the context of immunology and immunotherapy.
... To be able to analyze the progenitor and stem cell population in all samples, p63 (isoform ΔNp63) as a marker of epithelial progenitor cells was combined with EdU or 53BP1 staining. Immunostaining was performed as described previously [20]. ...
... TUNEL assay was performed using In Situ Cell Death Detection Kit (Roche Life Sciences) as described previously [20]. ...
Background: Radiotherapy in the head-and-neck area is one of the main curative treatment options. However, this comes at the cost of varying levels of normal tissue toxicity, affecting up to 80% of patients. Mucositis can cause pain, weight loss and treatment delay leading to worse outcomes and decreased quality of life. Therefore, there is an urgent need for an approach to predict normal mucosa response in patients prior to treatment. Methods: We here describe an assay to detect irradiation response in patient healthy oral mucosa tissue. Mucosa specimens from the oral cavity were obtained after surgical resection, cut into thin slices, irradiated, and cultured for three days. Seven samples were irradiated with X-ray and three additional samples were irradiated with both X-ray and protons. Results: Healthy oral mucosa tissue slices maintained normal morphology and viability for three days. We measured a dose-dependent response to X-ray irradiation and compared X-ray and proton irradiation in the same mucosa sample, using standardized automated image analysis. Furthermore, increased levels of inflammation inducing factors - major drivers in mucositis development - could be detected after irradiation. Conclusion: This model can be utilized for investigating mechanistic aspects of mucositis development and can be developed into an assay to predict radiation-induced toxicity in normal mucosa.
... Sectioning was performed with ice-cold sterile balanced salt solution in the buffer tray to assist in tissue sectioning and collection. Slicing speed was set at 0.6 mm/s and vibration amplitude at 3.0 mm [18]. Three tumours grown from individual mice were employed for this experiment. ...
Background
5-Fluorouracil (5-FU) remains a core component of systemic therapy for colorectal cancer (CRC). However, response rates remain low, and development of therapy resistance is a primary issue. Combinatorial strategies employing a second agent to augment the therapeutic effect of chemotherapy is predicted to reduce the incidence of treatment resistance and increase the durability of response to therapy.
Methods
Here, we employed quantitative proteomics approaches to identify novel druggable proteins and molecular pathways that are deregulated in response to 5-FU, which might serve as targets to improve sensitivity to chemotherapy. Drug combinations were evaluated using 2D and 3D CRC cell line models and an ex vivo culture model of a patient-derived tumour.
Results
Quantitative proteomics identified upregulation of the mitosis-associated protein Aurora B (AURKB), within a network of upregulated proteins, in response to a 24 h 5-FU treatment. In CRC cell lines, AURKB inhibition with the dihydrogen phosphate prodrug AZD1152, markedly improved the potency of 5-FU in 2D and 3D in vitro CRC models. Sequential treatment with 5-FU then AZD1152 also enhanced the response of a patient-derived CRC cells to 5-FU in ex vivo cultures.
Conclusions
AURKB inhibition may be a rational approach to augment the effectiveness of 5-FU chemotherapy in CRC.
... Microscopic preparations were examined for the presence of cells undergoing apoptosis. As indicators of apoptosis, cell nuclei with morphological changes were used -pyknosis (irreversible condensation of chromatin causing the nuclei to decrease in size), karyorrhexis (destructive fragmentation of a pyknotic nucleus) and karyolysis (nuclear fading caused by the dissolution of chromatin) [21]. ...
Ex situ conservation of Bulgarian endemic species Achillea thracica offers a chance for development of a new antitumor drug. This study aimed to evaluate the anticancer effects of water extracts of ex vitro established A. thracica grown in Bulgaria against fibrosarcoma cells. Our preliminary study determined the cytotoxicity of hot and cold water extracts using Triticum root elongation test and Allium cepa assay. In vitro cytotoxicity and pro-apoptotic activity of hot extract (HE) were estimated using HT1080 cell line fibrosarcoma cells. The change in the cell number, the cell viability, and the cell population growth determined cytotoxicity. The pro-apoptotic activity was assessed by the score of cell nuclei with morphological changes. The result of the root elongation test showed that the HEs exerted a stronger inhibitory effect than the cold extracts (CEs). Respectively, the EC50 value of the hot water extract is 9.85 g/l and of the CE – 12.71 g/l. The extracts tested at a concentration equal to ½ ??50 and EC50 values showed a significant mitodepressive effect on A. cepa meristematic cells. The in vitro tests revealed a significant negative impact on cancer cell viability and a promising pro-apoptotic activity of hot water extract.
... It is difficult to ensure the survival of cells at the center of the sample and the process lacks repeatability. 45 The other method is vibratome technology, which can generate slices from tens to hundreds of microns. Tissue slices generated by the vibratome are precisely cut to obtain a relatively regular shape, making them superior for culture and testing. ...
... Moreover, slices thinner than 100 μm may result in a high percentage of injured cells caused by slicing. 45 Slices of 200-300 μm thickness are the most commonly reported in publications to date. In addition, the media used for these tissue slices are easier to use than that for organoid culture, considering that they do not require differentiation or self-organization processes. ...
In vitro and in vivo models play integral roles in preclinical drug research, evaluation, and precision medicine. In vitro models primarily involve research platforms based on cultured cells, typically in the form of two‐dimensional (2D) cell models. However, notable disparities exist between 2D cultured cells and in vivo cells across various aspects, rendering the former inadequate for replicating the physiologically relevant functions of human or animal organs and tissues. Consequently, these models failed to accurately reflect real‐life scenarios post‐drug administration. Complex in vitro models (CIVMs) refer to in vitro models that integrate a multicellular environment and a three‐dimensional (3D) structure using bio‐polymer or tissue‐derived matrices. These models seek to reconstruct the organ‐ or tissue‐specific characteristics of the extracellular microenvironment. The utilization of CIVMs allows for enhanced physiological correlation of cultured cells, thereby better mimicking in vivo conditions without ethical concerns associated with animal experimentation. Consequently, CIVMs have gained prominence in disease research and drug development. This review aimed to comprehensively examine and analyze the various types, manufacturing techniques, and applications of CIVM in the domains of drug discovery, drug development, and precision medicine. The objective of this study was to provide a comprehensive understanding of the progress made in CIVMs and their potential future use in these fields.
... This primary tissue culture approach has been well-established in preserving the cellular TME of the original tumor [4][5][6][7]. While TSCs have been used to evaluate the effects of chemotherapy agents in primary tumor specimens [8][9][10], only a limited number of recent studies have leveraged them to determine the consequences of immunotherapies such as anti-PD-1, anti-TIM-3, anti-IL-10 and CAR-T cells [11,12]. ...
Background
Understanding the mechanistic effects of novel immunotherapy agents is critical to improving their successful clinical translation. These effects need to be studied in preclinical models that maintain the heterogenous tumor microenvironment (TME) and dysfunctional cell states found in a patient’s tumor. We investigated immunotherapy perturbations targeting co-stimulatory molecule GITR and co-inhibitory immune checkpoint TIGIT in a patient-derived ex vivo system that maintains the TME in its near-native state. Leveraging single-cell genomics, we identified cell type-specific transcriptional reprogramming in response to immunotherapy perturbations.
Methods
We generated ex vivo tumor slice cultures from fresh surgical resections of gastric and colon cancer and treated them with GITR agonist or TIGIT antagonist antibodies. We applied paired single-cell RNA and TCR sequencing to the original surgical resections, control, and treated ex vivo tumor slice cultures. We additionally confirmed target expression using multiplex immunofluorescence and validated our findings with RNA in situ hybridization.
Results
We confirmed that tumor slice cultures maintained the cell types, transcriptional cell states and proportions of the original surgical resection. The GITR agonist was limited to increasing effector gene expression only in cytotoxic CD8 T cells. Dysfunctional exhausted CD8 T cells did not respond to GITR agonist. In contrast, the TIGIT antagonist increased TCR signaling and activated both cytotoxic and dysfunctional CD8 T cells. This included cells corresponding to TCR clonotypes with features indicative of potential tumor antigen reactivity. The TIGIT antagonist also activated T follicular helper-like cells and dendritic cells, and reduced markers of immunosuppression in regulatory T cells.
Conclusions
We identified novel cellular mechanisms of action of GITR and TIGIT immunotherapy in the patients’ TME. Unlike the GITR agonist that generated a limited transcriptional response, TIGIT antagonist orchestrated a multicellular response involving CD8 T cells, T follicular helper-like cells, dendritic cells, and regulatory T cells. Our experimental strategy combining single-cell genomics with preclinical models can successfully identify mechanisms of action of novel immunotherapy agents. Understanding the cellular and transcriptional mechanisms of response or resistance will aid in prioritization of targets and their clinical translation.
... At the end of the experiment, xenograft tissues were removed and weighted. Sections will be subjected to Haematoxylin and Eosin (H&E), and IHC stains of USP22, USP7, CD31: endothelial marker for angiogenesis, Ki67: cell proliferation marker; and apoptosis marker: cleaved caspase-3 etc. Quantitative analysis and scoring of quantity and intensity in relation to positive and negative control was performed microscopically using FIJI image analysis software [42,43]. Microvessel density (MVD) in lung cancer tissues was evaluated after immunostaining endothelial cells with antibody against mouse CD31 (The JC70 Mab from DAKO), and MVD count was carried out on three fields (×100) chosen within the whole vascularized areas. ...
Deubiquitinases (DUBs) play important roles in various human cancers and targeting DUBs is considered as a novel anticancer therapeutic strategy. Overexpression of ubiquitin specific protease 7 and 22 (USP7 and USP22) are associated with malignancy, therapy resistance, and poor prognosis in many cancers. Although both DUBs are involved in the regulation of similar genes and signaling pathways, such as histone H2B monoubiquitination (H2Bub1), c-Myc, FOXP3, and p53, the interdependence of USP22 and USP7 expression has never been described. In the study, we found that targeting USP7 via either siRNA-mediated knockdown or pharmaceutical inhibitors dramatically upregulates USP22 in cancer cells. Mechanistically, the elevated USP22 occurs through a transcriptional pathway, possibly due to desuppression of the transcriptional activity of SP1 via promoting its degradation upon USP7 inhibition. Importantly, increased USP22 expression leads to significant activation of downstream signal pathways including H2Bub1
and c-Myc, which may potentially enhance cancer malignancy and counteract the anticancer efficacy of USP7 inhibition. Importantly, targeting USP7 further suppresses the in vitro proliferation of USP22-knockout (USP22-Ko)
A549 and H1299 lung cancer cells and induces a stronger activation of p53 tumor suppressor signaling pathway. In addition, USP22-Ko cancer cells are more sensitive to a combination of cisplatin and USP7 inhibitor. USP7 inhibitor treatment further suppresses in vivo angiogenesis and tumor growth and induced more apoptosis in USP22-Ko cancer xenografts. Taken together, our findings demonstrate that USP7 inhibition can dramatically upregulate USP22 in cancer cells; and targeting USP7 and USP22 may represent a more effective approach for targeted cancer therapy,
which warrants further study.
Keywords Deubiquitinase, USP7, USP22, SP1, c-Myc, p53, Targeted anticancer therapy
... At the end of the experiment, xenograft tissues were removed and weighted. Sections will be subjected to Haematoxylin and Eosin (H&E), and IHC stains of USP22, USP7, CD31: endothelial marker for angiogenesis, Ki67: cell proliferation marker; and apoptosis marker: cleaved caspase-3 etc. Quantitative analysis and scoring of quantity and intensity in relation to positive and negative control was performed microscopically using FIJI image analysis software [42,43]. Microvessel density (MVD) in lung cancer tissues was evaluated after immunostaining endothelial cells with antibody against mouse CD31 (The JC70 Mab from DAKO), and MVD count was carried out on three fields (×100) chosen within the whole vascularized areas. ...
Deubiquitinases (DUBs) play important roles in various human cancers and targeting DUBs is considered as a novel anticancer therapeutic strategy. Overexpression of ubiquitin specific protease 7 and 22 (USP7 and USP22) are associated with malignancy, therapy resistance, and poor prognosis in many cancers. Although both DUBs are involved in the regulation of similar genes and signaling pathways, such as histone H2B monoubiquitination (H2Bub1), c-Myc, FOXP3, and p53, the interdependence of USP22 and USP7 expression has never been described. In the study, we found that targeting USP7 via either siRNA-mediated knockdown or pharmaceutical inhibitors dramatically upregulates USP22 in cancer cells. Mechanistically, the elevated USP22 occurs through a transcriptional pathway, possibly due to desuppression of the transcriptional activity of SP1 via promoting its degradation upon USP7 inhibition. Importantly, increased USP22 expression leads to significant activation of downstream signal pathways including H2Bub1 and c-Myc, which may potentially enhance cancer malignancy and counteract the anticancer efficacy of USP7 inhibition. Importantly, targeting USP7 further suppresses the in vitro proliferation of USP22-knockout (USP22-Ko) A549 and H1299 lung cancer cells and induces a stronger activation of p53 tumor suppressor signaling pathway. In addition, USP22-Ko cancer cells are more sensitive to a combination of cisplatin and USP7 inhibitor. USP7 inhibitor treatment further suppresses in vivo angiogenesis and tumor growth and induced more apoptosis in USP22-Ko cancer xenografts. Taken together, our findings demonstrate that USP7 inhibition can dramatically upregulate USP22 in cancer cells; and targeting USP7 and USP22 may represent a more effective approach for targeted cancer therapy, which warrants further study.
... FBS has been used in human cell cultures for decades and contains a variety of growth factors that are important in overall cell proliferation and differentiation, while antibiotics reduce bacterial contamination of cultures by intestinal flora from tissue specimens. In some studies that omitted FBS, other growthpromoting supplements such as B-27 [54,67,68] and epidermal growth factor (EGF) [54,56,67,68] were added to improve viability, although evidence is scarce and inconclusive in studies on non-CRC explants [78,79]. With the recent development of more physiological cell culture media which mimics the composition of human plasma (e.g. ...
Purpose
Whilst the treatment paradigm for colorectal cancer has evolved significantly over time, there is still a lack of reliable biomarkers of treatment response. Treatment decisions are based on high-risk features such as advanced TNM stage and histology. The role of the tumour microenvironment, which can influence tumour progression and treatment response, has generated considerable interest. Patient-derived explant cultures allow preservation of native tissue architecture and tumour microenvironment. The aim of the scoping review is to evaluate the utility of patient-derived explant cultures as a preclinical model in colorectal cancer.
Methods
A search was conducted using Ovid MEDLINE, EMBASE, Web of Science, and Cochrane databases from start of database records to September 1, 2022. We included all peer-reviewed human studies in English language which used patient-derived explants as a preclinical model in primary colorectal cancer. Eligible studies were grouped into the following categories: assessing model feasibility; exploring tumour microenvironment; assessing ex vivo drug responses; discovering and validating biomarkers.
Results
A total of 60 studies were eligible. Fourteen studies demonstrated feasibility of using patient-derived explants as a preclinical model. Ten studies explored the tumour microenvironment. Thirty-eight studies assessed ex vivo drug responses of chemotherapy agents and targeted therapies. Twenty-four studies identified potential biomarkers of treatment response.
Conclusions
Given the preservation of tumour microenvironment and tumour heterogeneity, patient-derived explants has the potential to identify reliable biomarkers, treatment resistance mechanisms, and novel therapeutic agents. Further validation studies are required to characterise, refine and standardise this preclinical model before it can become a part of precision medicine in colorectal cancer.
... An alternative approach to predict sensitivity is direct analysis of tumor response to chemotherapy. Recently, we developed a technology to maintain viability, tissue integrity and proliferation of BC tissue slices ex vivo for up to 1 week [7][8][9] , keeping cells in their natural (micro)environment. This allows testing of tissue responses to various types of treatment 9 . ...
... This allows testing of tissue responses to various types of treatment 9 . With this technique we previously showed that outcome parameters could be developed for the response of these human BC slices to anthracycline-based chemotherapy, platinum salts and taxanes 8,10 . A correlation of these ex vivo sensitivity assays with the in vivo response, however, was still lacking. ...
... Part I: Development of the ex vivo anthracycline-based sensitivity assay A set of previously published tumors was combined with new samples 8 . After surgical removal of the primary BC, viable tumors were sent to the pathology department. ...
We developed a functional ex vivo anthracycline-based sensitivity test. Surgical resection material of primary breast cancer (BC) was used to determine criteria for the ex vivo sensitivity assay based on morphology, proliferation and apoptosis. Subsequently, a proof-of-concept study was performed correlating results of this assay on primary BC biopsies with in vivo response after treatment with anthracycline-containing neoadjuvant chemotherapy (NAC). Cut off values for the ex vivo anthracycline-based sensitivity test were established based on analysis of 21 primary breast tumor samples obtained after surgery. In the proof-of-concept study based on a new set of tumor biopsies, 41 patients were included. Eight biopsies did not contain tumor cells and three patients could not be biopsied for various reasons. In the remaining 30 biopsies, the success rate of the ex vivo test was 77% (23/30); six out of seven failed tests were due to excessive apoptosis, our pre-specified test criteria. Of the 23 patients with a successful ex vivo test result, three patients did not undergo NAC after the biopsy. Here we report the ex vivo anthracycline-based sensitivity assay is feasible on biopsy material and shows 75% concordance between ex vivo outcomes and in vivo MRI response. Unfortunately, the percentage of unsuccessful tests is rather high. This study provides the foundation for further development of ex vivo sensitivity assays.
