Alterations in the epigenetic landscape are now recognized as a hallmark of cancer. Among these, histone posttranslational modifications (HPTMs) represent a critical control layer, playing a significant role in chromatin remodeling and gene expression regulation. The comprehensive mapping of these modifications on a genome-wide scale is crucial for identifying regulatory regions and understanding downstream pathways, thereby providing a deeper and more integrated understanding of cancer biology. CUT&RUN (Cleavage Under Targets and Release Using Nuclease), combined with next-generation sequencing, is emerging as a highly reliable, low-background, and user-friendly technology for mapping antibody-bound chromatin regions in a genome-wide manner. In this chapter, we provide a comprehensive overview of an optimized protocol for targeting HPTMs in frozen tissue samples, emphasizing the crucial role of nucleus purification in our methodology.

Studying metabolism in the different cellular compartments of the tumor microenvironment (TME) is crucial to identify the specific metabolic signatures that contribute to tumor progression. Additionally, TME-driven metabolic changes can disrupt the essential metabolic processes involved in immune cell function, hindering immunotherapy success. Extracellular flux analysis (Seahorse) is a well-established approach used to characterize cellular metabolism. When combined with flow cytometry-based measurements, it offers a comprehensive view of the metabolic signatures within the different cellular compartments of the TME. Here we provide a detailed description on the procedures of Seahorse analysis and complementary flow cytometry-based metabolic readouts on tumor cells and T cells in the context of cancer, which can also be applied to other physiological and pathological situations.

Natural killer (NK) cells exert a pivotal role in cancer pathology due to their ability to recognize and eliminate virus-infected, stressed, or malignant transformed cells. Although adaptive immunity recognizes cancer cells by detecting tumor-associated antigens, NK cells detect target cells in an antigen-specific independent manner, relying on a complex array of activating and inhibitory receptors. Despite their potential, NK cell-mediated antitumor activity faces challenges such as tumor resistance mechanisms and limited infiltration into tumor tissues. Herein, we describe a colony formation-based assay to evaluate NK cell-mediated tumor cytotoxicity in vitro, providing insights into NK cell responses and tumor cell resistance mechanisms, offering a valuable tool for studying and optimizing NK cell-based immunotherapy for cancer treatment. This method evaluates relative survival of tumor target cells to NK effector cells, and it can be complemented with flow cytometry analyses of NK cell immunophenotype or functional studies of soluble factors in supernatants, such as an Enzyme-Linked ImmunoSorbent Assay (ELISA). This allows the elucidation of molecules and signaling pathways that regulate NK cell anticancer activity.

The tumor microenvironment (TME) is a heterogeneous and continuously evolving system. Besides malignant cells, it is composed of numerous immune cells, astrocytes and other stromal cells, blood vessels, and extracellular matrix. Interactions of tumor with other cells produce an immunosuppressive milieu, thus impeding antitumor immunity. The advancements of single-cell techniques provide powerful means to scrutinize the tumor and TME at high resolution, shedding light on the pathogenic mechanisms and dysfunctions of antitumor immunity. Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) enables simultaneously single-cell RNA sequencing (scRNAseq) and the acquisition of both quantitative and qualitative information about surface proteins using oligonucleotide-tagged antibodies. By combining proteomic and transcriptomic data from the same single cell, CITE-seq has become one of the leading approaches for phenotyping discrete cell populations. The method has been successfully used to detect over a hundred epitopes per cell, adding an additional layer of information that, when integrated with transcriptomic data, enables the identification of cellular states and functions, as well as the prediction of cell trajectories, maturity, and interactions with other cells.

Multicolor fluorescence-activated cell sorting (FACS) has allowed cancer researchers to gain insights into the role of immune cells within the tumor microenvironment. According to this, flow cytometry is widely used to characterize and quantify immune cell infiltration during tumor development in a given tumor implant. In preclinical mouse models of peritoneal carcinomatosis, FACS is usually used to study the peritoneal lavages. However, one of the most relevant tissues to analyze in peritoneal metastases, the omentum, often goes unnoticed. Previous works have demonstrated that flow cytometry analysis of the omentum is critical to understanding the anti-pro-tumoral response of gynecological or gastrointestinal cancer. Here, we set up a detailed protocol to study different cell populations in omental metastasis with specific multicolor FACS panel design, including T cells, natural killer cells, natural killer T cells, and B cells in peritoneal carcinomatosis mice models. In cancer, these cell populations exhibit unique phenotypic profiles and are known to contribute to tumor growth and immune evasion. Nevertheless, immunomodulation of the omentum through intraperitoneal immunotherapy strategies has been demonstrated to be crucial in controlling peritoneal carcinomatosis. The protocol described in this chapter lists the steps to be taken for the localization, extraction, processing, and characterization of different immune cell populations of omental tumor implants in peritoneal carcinomatosis mice models using multicolor flow cytometry. In conclusion, we consider that the immune characterization of omentum cell infiltration is relevant to predicting the antitumor response of different locoregional immunotherapy strategies.

Metastatic brain cancer is the most common intracranial cancer among adults, often leading to poor survival outcomes. The incidence of brain metastases is on the rise, likely associated with advanced systemic therapeutics which improve patient survival, providing sufficient time for micrometastases to develop into fully established metastases. Therefore, brain metastasis remains an unmet clinical need. The brain metastatic tumor microenvironment is a complex ecosystem composed of numerous brain, stromal, and immune cells with unique adaptations that represent a formidable challenge for treatment. Importantly, brain metastases are enriched in immune cells, especially myeloid resident and recruited cells considered highly plastic. Thus, it is critical to assess the immune landscape and functional phenotypes longitudinally and across treatment conditions in brain metastasis in a non-biased, comprehensive manner. Here, we outline a protocol to assess immune cell populations in murine brain metastasis samples, using a 23-color panel for spectral flow cytometry.

The relationship between regional pathological lymph node status and the benefit of postoperative radiotherapy for gastric cancer remains controversial.

The perioperative period is characterized by psychological stress and inflammatory reactions that can contribute to disease recurrence or metastatic spread. These reactions are mediated particularly by catecholamines and prostaglandins. The PROSPER trial aimed to evaluate whether a perioperative drug repurposing with a non-selective betablocker (propranolol) and a COX-2 inhibitor (etodolac) is feasible and safe in the setting of pancreatic cancer surgery.

In the present study, the synergistic effects and mechanism of recombinant viral vector-mediated co-expression plasmids stat1 and stat3-siRNA on glioma were investigated in vivo and in vitro.

In the present study, we aim to identify novel molecular targets for sensitizing Breast cancer stem cells (BCSCs) to common antitumor treatments. MicroRNAs (miRNAs) play key roles in pivotal cellular processes. Therefore, modulating the expression of these miRNAs may lead to increased sensitivity of BCSCs to current treatments or overcome their therapeutic resistance. Due to their pivotal roles in the regulation of apoptosis (via BCL2) and chemoresistance (via ABCG2) and their differential expression in BCSCs (compared to non-BCSCs), miR-34a and miR-328 were selected for analysis.

Osteosarcoma (OSC) and Ewing sarcoma (EWS) of the skull are rare malignancies in children and adolescents. This study aimed to analyze the demographic, clinical, treatment, and survival characteristics of these tumors using a population-based approach.

Anti-PD-L1 immunotherapy is recommended as standard of care for patients with extensive stage small cell lung cancer (ES-SCLC); however, there are no reliable biomarkers guiding patient selection and the survival benefit of PD-L1 inhibitors in the overall population is limited. In this study, we retrospectively analyzed a total number of 61 cases of ES-SCLC who underwent anti-PD-L1 immunotherapy. Patient demographic characteristics and laboratory findings were processed for univariate and multivariate analysis. Subgrouping of SCLC was performed on IHC platform using antibodies against ASCL1, NEUROD1 and POU2F3. The tumor microenvironment (TME) of ES-SCLC was evaluated by CD8 + T cell infiltration, granzyme B production and PD-L1 expression. We found limited efficacy of defined variable factors conferring therapeutic outcomes of anti-PD-L1 immunotherapy in patients with ES-SCLC. Intriguingly, there was a profound difference in TME and response to anti-PD-L1 immunotherapy when classifying SCLC into A/N/P/I subgroups. Although accounted for a small proportion of SCLC, the SCLC-P and SCLC-I subtypes manifested as T cell-enriched "hot" tumor and elicited more favorable response to immunotherapy, whereas the SCLC-A and SCLC-N subgroups were T cell-absent "cold" tumor. There was also a significant difference in progression free survival and overall survival across these subsets. Moreover, we found the SCLC-P and SCLC-I tumors revealed features of low neuroendocrine (NE) differentiation and showed clinicopathologic features overlapping with the SCLC non-NE lineage. These findings may aid clinicians to select ES-SCLC patients who were more likely to gain higher response rate and longer survival to anti-PD-L1 immunotherapy. Revisiting SCLC according to A/N/P/I subtyping and NE/non-NE differentiation is a reliable approach to guide therapeutic strategy in patients with ES-SCLC.

Cancer is a major burden of disease around the globe and one of the leading causes of premature death. The key to improve patient outcomes in modern clinical cancer research is to gain insights into dynamics underlying cancer evolution in order to facilitate the search for effective therapies. However, most cancer data analysis tools are designed for controlled trials and cannot leverage routine clinical data, which are available in far greater quantities. In addition, many cancer models focus on single disease processes in isolation, disregarding interaction. This work proposes a unified stochastic modelling framework for cancer progression that combines (stochastic) processes for tumour growth, metastatic seeding, and patient survival to provide a comprehensive understanding of cancer progression. In addition, our models aim to use non-equidistantly sampled data collected in clinical routine to analyse the whole patient trajectory over the course of the disease. The model formulation features closed-form expressions of the likelihood functions for parameter inference from clinical data. The efficacy of our model approach is demonstrated through a simulation study involving four exemplary models, which utilise both analytic and numerical likelihoods. The results of the simulation studies demonstrate the accuracy and computational efficiency of the analytic likelihood formulations. We found that estimation can retrieve the correct model parameters and reveal the underlying data dynamics, and that this modelling framework is flexible in choosing the precise parameterisation. This work can serve as a foundation for the development of combined stochastic models for guiding personalized therapies in oncology.

Primary central nervous system lymphoma (PCNSL) has a poor prognosis, mainly because of the significant challenges with the efficacy and tolerability of induction chemotherapy. This retrospective study aimed to evaluate the efficacy and safety of the R2-MTX regimen in PCNSL patients.

This meta-analysis aims to assess the safety, efficacy, and outcomes of palliative transurethral resection of the prostate (TURP) for relieving urinary obstruction in patients with metastatic prostate cancer.

Liver metastasis remains the predominant cause of mortality in patients with colorectal cancer (CRC). Nevertheless, the mechanisms underlying the initiation of colorectal cancer liver metastasis remain poorly elucidated. During the metastatic process of CRC cells from the primary site to the liver, we performed time-resolved analyses and identified a subset of tumor cells spatially located in the primary tumor and temporally distributed in the early stages of liver metastasis. These cells were termed liver metastasis-initiating cells (LMICs). LMICs exhibit high stemness, low proliferation, active interaction with surrounding stromal components, and a close association with liver metastasis. Notably, we found significant interactions between cancer-associated fibroblasts (CAFs) and LMICs via the SEMA3C-NRP2 receptor-ligand pair. Further in vivo and in vitro experiments confirmed that CAF-secreted SEMA3C could bind to the NRP2 receptor, which activates the MAPK pathway and promotes colorectal cancer liver metastasis. Our findings suggest potential therapeutic strategies for the early prevention of colorectal cancer liver metastasis.

The introduction of adjuvant treatment with immune checkpoint inhibitors (ICI) and BRAF/MEK inhibitors has significantly improved recurrence-free (RFS) and distant metastasis-free survival (DMFS) for melanoma patients. However, a significant improvement in overall survival (OS) has currently not been demonstrated in phase III clinical trials. Furthermore, not all patients benefit from adjuvant systemic therapy. For these patients, perioperative and neoadjuvant systemic therapy represent a promising new therapeutic approach.

Despite adequate treatment the probability of recurrence of malignant melanoma is high in patients with corresponding risk factors. Adjuvant treatment significantly reduces this risk; however, the choice of the appropriate adjuvant treatment can be difficult.

The purpose of this study is to investigate the impact of a high-salt diet (HSD), which is commonly found in Western countries, on the progression of glioma. Our research shows that the alterations in gut microbiota caused by an HSD facilitated the development of glioma. Mice fed an HSD have elevated levels of intestinal propionate, which accelerated the growth of glioma cells. We also find that propionate supplementation enhanced the response of glioma cells to low oxygen levels. Moreover, we identify a link between TGF-β signaling, response to low oxygen levels, and invasion-related pathways. Propionate treatment increases the expression of HIF-1α, leading to an increase in TGF-β1 production. Additionally, propionate treatment promotes glioma cell invasion through TGF-β signaling. Our findings suggest that an HSD-induced increase in propionate plays a crucial role in glioma progression by facilitating invasion through the hypoxic response and TGF-β signaling pathways, thereby establishing a significant connection between gut microbiota and the progression of glioma.

Vascular endothelial growth factor receptor-tyrosine kinase inhibitors (VEGFR-TKIs), alone or in combination with immune checkpoint inhibitors, are part of the standard of care in treating metastatic renal cell carcinoma (mRCC). VEGFR-TKIs are associated with acute, chronic, and potentially dose-limiting toxicities requiring treatment modifications and discontinuations.