Unlocking the secrets: exploring the influence of the aryl hydrocarbon receptor and microbiome on cancer development.

Gut microbiota regulates various aspects of human physiology by producing metabolites, metabolizing enzymes, and toxins. Many studies have linked microbiota with human health and altered microbiome configurations with the occurrence of several diseases, including cancer. Accumulating evidence suggests that the microbiome can influence the initiation and progression of several cancers. Moreover, some microbiotas of the gut and oral cavity have been reported to infect tumors, initiate metastasis, and promote the spread of cancer to distant organs, thereby influencing the clinical outcome of cancer patients. The gut microbiome has recently been reported to interact with environmental factors such as diet and exposure to environmental toxicants. Exposure to environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) induces a shift in the gut microbiome metabolic pathways, favoring a proinflammatory microenvironment. In addition, other studies have also correlated cancer incidence with exposure to PAHs. PAHs are known to induce organ carcinogenesis through activating a ligand-activated transcriptional factor termed the aryl hydrocarbon receptor (AhR), which metabolizes PAHs to highly reactive carcinogenic intermediates. However, the crosstalk between AhR and the microbiome in mediating carcinogenesis is poorly reviewed. This review aims to discuss the role of exposure to environmental pollutants and activation of AhR on microbiome-associated cancer progression and explore the underlying molecular mechanisms involved in cancer development.

Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi.

The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.

Exploring the Role of microRNAs in Glioma Progression, Prognosis, and Therapeutic Strategies.

Gliomas, which arise from glial cells in the brain, remain a significant challenge due to their location and resistance to traditional treatments. Despite research efforts and advancements in healthcare, the incidence of gliomas has risen dramatically over the past two decades. The dysregulation of microRNAs (miRNAs) has prompted the creation of therapeutic agents that specially target them. However, it has been reported that they are involved in complex signaling pathways that contribute to the loss of expression of tumor suppressor genes and the upregulation of the expression of oncogenes. In addition, numerous miRNAs promote the development, progression, and recurrence of gliomas by targeting crucial proteins and enzymes involved in metabolic pathways such as glycolysis and oxidative phosphorylation. However, the complex interplay among these pathways along with other obstacles hinders the ability to apply miRNA targeting in clinical practice. This highlights the importance of identifying specific miRNAs to be targeted for therapy and having a complete understanding of the diverse pathways they are involved in. Therefore, the aim of this review is to provide an overview of the role of miRNAs in the progression and prognosis of gliomas, emphasizing the different pathways involved and identifying potential therapeutic targets.

Stressed target cancer cells drive nongenetic reprogramming of CAR T cells and solid tumor microenvironment.

The poor efficacy of chimeric antigen receptor T-cell therapy (CAR T) for solid tumors is due to insufficient CAR T cell tumor infiltration, in vivo expansion, persistence, and effector function, as well as exhaustion, intrinsic target antigen heterogeneity or antigen loss of target cancer cells, and immunosuppressive tumor microenvironment (TME). Here we describe a broadly applicable nongenetic approach that simultaneously addresses the multiple challenges of CAR T as a therapy for solid tumors. The approach reprograms CAR T cells by exposing them to stressed target cancer cells which have been exposed to the cell stress inducer disulfiram (DSF) and copper (Cu)(DSF/Cu) plus ionizing irradiation (IR). The reprogrammed CAR T cells acquire early memory-like characteristics, potent cytotoxicity, enhanced in vivo expansion, persistence, and decreased exhaustion. Tumors stressed by DSF/Cu and IR also reprogram and reverse the immunosuppressive TME in humanized mice. The reprogrammed CAR T cells, derived from peripheral blood mononuclear cells of healthy donors or metastatic female breast cancer patients, induce robust, sustained memory and curative anti-solid tumor responses in multiple xenograft mouse models, establishing proof of concept for empowering CAR T by stressing tumor as a promising therapy for solid tumors.

Treatment with decitabine induces the expression of stemness markers, PD-L1 and NY-ESO-1 in colorectal cancer: potential for combined chemoimmunotherapy.

BACKGROUND: The mechanism of tumor immune escape and progression in colorectal cancer (CRC) is widely investigated in-vitro to help understand and identify agents that might play a crucial role in response to treatment and improve the overall survival of CRC patients. Several mechanisms of immune escape and tumor progression, including expression of stemness markers, inactivation of immunoregulatory genes by methylation, and epigenetic silencing, have been reported in CRC, indicating the potential of demethylating agents as anti-cancer drugs. Of these, a chemotherapeutic demethylating agent, Decitabine (DAC), has been reported to induce a dual effect on both DNA demethylation and histone changes leading to an increased expression of target biomarkers, thus making it an attractive anti-tumorigenic drug. METHODS: We compared the effect of DAC in primary 1076 Col and metastatic 1872 Col cell lines isolated and generated from patients' tumor tissues. Both cell lines were treated with DAC, and the expression of the NY-ESO-1 cancer-testis antigen, the PD-L1 immunoinhibitory marker, and the CD44, Nanog, KLF-4, CD133, MSI-1 stemness markers were analyzed using different molecular and immunological assays. RESULTS: DAC treatment significantly upregulated stemness markers in both primary 1076 Col and meta-static 1872 Col cell lines, although a lower effect occurred on the latter: CD44 (7.85 fold; ***p = 0.0001 vs. (4.19 fold; *p = 0.0120), Nanog (4.1 fold; ***p < 0.0001 vs.1.69 fold; ***p = 0.0008), KLF-4 (4.33 fold; ***p < 0.0001 vs.2.48 fold; ***p = 0.0005), CD133 (16.77 fold; ***p = 0.0003 vs.6.36 fold; *p = 0.0166), and MSI-1 (2.33 fold; ***p = 0.0003 vs.2.3 fold; ***p = 0.0004), respectively. Interestingly, in the metastatic 1872 Col cells treated with DAC, the expression of both PD-L1 and NY-ESO-1 was increased tenfold (*p = 0.0128) and fivefold (***p < 0.0001), respectively. CONCLUSIONS: We conclude that the upregulation of both stemness and immune checkpoint markers by DAC treatment on CRC cells might represent a mechanism of immune evasion. In addition, induction of NY-ESO-1 may represent an immuno-therapeutic option in metastatic CRC patients. Finally, the combination of DAC and anti-PD-1/anti-PD-L1 antibodies treatment should represent a potential therapeutic intervention for this group of patients.

CAR-cell therapy in the era of solid tumor treatment: current challenges and emerging therapeutic advances.

In the last decade, Chimeric Antigen Receptor (CAR)-T cell therapy has emerged as a promising immunotherapeutic approach to fight cancers. This approach consists of genetically engineered immune cells expressing a surface receptor, called CAR, that specifically targets antigens expressed on the surface of tumor cells. In hematological malignancies like leukemias, myeloma, and non-Hodgkin B-cell lymphomas, adoptive CAR-T cell therapy has shown efficacy in treating chemotherapy refractory patients. However, the value of this therapy remains inconclusive in the context of solid tumors and is restrained by several obstacles including limited tumor trafficking and infiltration, the presence of an immunosuppressive tumor microenvironment, as well as adverse events associated with such therapy. Recently, CAR-Natural Killer (CAR-NK) and CAR-macrophages (CAR-M) were introduced as a complement/alternative to CAR-T cell therapy for solid tumors. CAR-NK cells could be a favorable substitute for CAR-T cells since they do not require HLA compatibility and have limited toxicity. Additionally, CAR-NK cells might be generated in large scale from several sources which would suggest them as promising off-the-shelf product. CAR-M immunotherapy with its capabilities of phagocytosis, tumor-antigen presentation, and broad tumor infiltration, is currently being investigated. Here, we discuss the emerging role of CAR-T, CAR-NK, and CAR-M cells in solid tumors. We also highlight the advantages and drawbacks of CAR-NK and CAR-M cells compared to CAR-T cells. Finally, we suggest prospective solutions such as potential combination therapies to enhance the efficacy of CAR-cells immunotherapy.

Gene Expression Profiling of FFPE Samples: A Titration Test.

The gene expression analysis of formalin-fixed paraffin-embedded (FFPE) tissues is often hampered by poor RNA quality, which results from the oxidation, cross-linking and other chemical modifications induced by the inclusion in paraffin. Yet, FFPE samples are a valuable source for molecular studies and can provide great insights into disease progression and prognosis. With the advancement of genomic technologies, new methods have been established that offer reliable and accurate gene expression workflows on samples of poor quality. NanoString is a probe-based technology that allows the direct counting of the mRNA transcripts and can be applied to degraded samples. Here, we have tested 2 RNA extraction methods for FFPE samples, and we have performed a titration experiment to evaluate the impact of RNA degradation and RNA input on the gene expression profiles assessed using the NanoString IO360 panel. We have selected FFPE samples of different DV200 values and assessed them on the nCounter platform with 2 different amounts of input RNA. This study concludes that the nCounter is a robust and reliable platform to assess the gene expression of RNA samples with DV200 > 30%; its robustness and ease of use could be of particular benefit to clinical settings.

Understanding the Mechanism of Diabetes Mellitus in a LRBA-Deficient Patient.

The scope of this study is to show that DM in a LRBA-deficient patient with a stop codon mutation (c.3999 G > A) was not mediated through autoimmunity. We have evaluated the ability of the proband's T cells to be activated by assessing their CTLA-4 expression. A nonsignificant difference was seen in the CTLA-4 expression on CD3+ T cells compared to the healthy control at basal level and after stimulation with PMA/ionomycin. Blood transcriptomic analysis have shown a remarkable increase in abundance of transcripts related to CD71+ erythroid cells. There were no differences in the expression of modules related to autoimmunity diseases between the proband and pooled healthy controls. In addition, our novel findings show that siRNA knockdown of LRBA in mouse pancreatic ß-cells leads reduced cellular proinsulin, insulin and consequently insulin secretion, without change in cell viability in cultured MIN6 cells.

MicroRNA Expression Profile Distinguishes Glioblastoma Stem Cells from Differentiated Tumor Cells.

Glioblastoma (GBM) represents the most common and aggressive tumor of the brain. Despite the fact that several studies have recently addressed the molecular mechanisms underlying the disease, its etiology and pathogenesis are still poorly understood. GBM displays poor prognosis and its resistance to common therapeutic approaches makes it a highly recurrent tumor. Several studies have identified a subpopulation of tumor cells, known as GBM cancer stem cells (CSCs) characterized by the ability of self-renewal, tumor initiation and propagation. GBM CSCs have been shown to survive GBM chemotherapy and radiotherapy. Thus, targeting CSCs represents a promising approach to treat GBM. Recent evidence has shown that GBM is characterized by a dysregulated expression of microRNA (miRNAs). In this study we have investigated the difference between human GBM CSCs and their paired autologous differentiated tumor cells. Array-based profiling and quantitative Real-Time PCR (qRT-PCR) were performed to identify miRNAs differentially expressed in CSCs. The Cancer Genome Atlas (TCGA) data were also interrogated, and functional interpretation analysis was performed. We have identified 14 miRNAs significantly differentially expressed in GBM CSCs (p < 0.005). MiR-21 and miR-95 were among the most significantly deregulated miRNAs, and their expression was also associated to patient survival. We believe that the data provided here carry important implications for future studies aiming at elucidating the molecular mechanisms underlying GBM.

Cancer Stem Cells Are Possible Key Players in Regulating Anti-Tumor Immune Responses: The Role of Immunomodulating Molecules and MicroRNAs.

Cancer cells endowed with stemness properties and representing a rare population of cells within malignant lesions have been isolated from tumors with different histological origins. These cells, denominated as cancer stem cells (CSCs) or cancer initiating cells (CICs), are responsible for tumor initiation, progression and resistance to therapies, including immunotherapy. The dynamic crosstalk of CSCs/CICs with the tumor microenvironment orchestrates their fate and plasticity as well as their immunogenicity. CSCs/CICs, as observed in multiple studies, display either the aberrant expression of immunomodulatory molecules or suboptimal levels of molecules involved in antigen processing and presentation, leading to immune evasion. MicroRNAs (miRNAs) that can regulate either stemness properties or their immunological profile, with in some cases dual functions, can provide insights into these mechanisms and possible interventions to develop novel therapeutic strategies targeting CSCs/CICs and reverting their immunogenicity. In this review, we provide an overview of the immunoregulatory features of CSCs/CICs including miRNA profiles involved in the regulation of the interplay between stemness and immunological properties.

Exploiting B-cell Receptor Stereotypy to Design Tailored Immunotherapy in Chronic Lymphocytic Leukemia.

PURPOSE: Approximately 30% of patients with chronic lymphocytic leukemia (CLL) can be grouped into subsets with stereotyped B-cell receptor immunoglobulin (BcR IG) displaying remarkable similarity in the heavy complementarity-determining region 3 (VH CDR3). Here, we investigated whether the consensus VH CDR3 sequences from CLL stereotyped subsets can be exploited for immunotherapy approaches. EXPERIMENTAL DESIGN: Immunogenic epitopes from the consensus VH CDR3 sequence of the clinically aggressive subsets #1 and #2 and from Eµ-TCL1 mice, which spontaneously develop CLL with BcR IG stereotypy, were identified and used to generate specific HLA class I- and II-restricted T cells in vitro. T-cell reactivity was assayed in vitro as IFNγ production. Bone marrow-derived dendritic cells loaded with the peptides were used as vaccination strategy to restrain leukemia development in the Eµ-TCL1 mouse model. RESULTS: These stereotyped epitopes were naturally processed and presented by CLL cells to the VH CDR3-specific T cells. Furthermore, we validated the efficacy of VH CDR3 peptide-based immunotherapy in the Eµ-TCL1 transplantable mouse model. Immunization of mice against defined VH CDR3 peptide epitopes, prior to the challenge with the corresponding leukemia cells, resulted in the control of CLL development in a significant fraction of mice, and increased overall survival. CONCLUSIONS: Our data highlight the immunogenicity of stereotyped VH CDR3 sequences and support the feasibility and efficacy of their use for novel cancer vaccine in CLL. Such approach has the advantage to generate "off-the-shelf" therapeutic vaccines for relevant groups of patients belonging to stereotyped subsets.See related commentary by Seiffert, p. 659.

SITC cancer immunotherapy resource document: a compass in the land of biomarker discovery.

Since the publication of the Society for Immunotherapy of Cancer's (SITC) original cancer immunotherapy biomarkers resource document, there have been remarkable breakthroughs in cancer immunotherapy, in particular the development and approval of immune checkpoint inhibitors, engineered cellular therapies, and tumor vaccines to unleash antitumor immune activity. The most notable feature of these breakthroughs is the achievement of durable clinical responses in some patients, enabling long-term survival. These durable responses have been noted in tumor types that were not previously considered immunotherapy-sensitive, suggesting that all patients with cancer may have the potential to benefit from immunotherapy. However, a persistent challenge in the field is the fact that only a minority of patients respond to immunotherapy, especially those therapies that rely on endogenous immune activation such as checkpoint inhibitors and vaccination due to the complex and heterogeneous immune escape mechanisms which can develop in each patient. Therefore, the development of robust biomarkers for each immunotherapy strategy, enabling rational patient selection and the design of precise combination therapies, is key for the continued success and improvement of immunotherapy. In this document, we summarize and update established biomarkers, guidelines, and regulatory considerations for clinical immune biomarker development, discuss well-known and novel technologies for biomarker discovery and validation, and provide tools and resources that can be used by the biomarker research community to facilitate the continued development of immuno-oncology and aid in the goal of durable responses in all patients.

Molecular and Immune Biomarkers for Cutaneous Melanoma: Current Status and Future Prospects.

Advances in the genomic, molecular and immunological make-up of melanoma allowed the development of novel targeted therapy and of immunotherapy, leading to changes in the paradigm of therapeutic interventions and improvement of patients' overall survival. Nevertheless, the mechanisms regulating either the responsiveness or the resistance of melanoma patients to therapies are still mostly unknown. The development of either the combinations or of the sequential treatment of different agents has been investigated but without a strongly molecularly motivated rationale. The need for robust biomarkers to predict patients' responsiveness to defined therapies and for their stratification is still unmet. Progress in immunological assays and genomic techniques as long as improvement in designing and performing studies monitoring the expression of these markers along with the evolution of the disease allowed to identify candidate biomarkers. However, none of them achieved a definitive role in predicting patients' clinical outcomes. Along this line, the cross-talk of melanoma cells with tumor microenvironment plays an important role in the evolution of the disease and needs to be considered in light of the role of predictive biomarkers. The overview of the relationship between the molecular basis of melanoma and targeted therapies is provided in this review, highlighting the benefit for clinical responses and the limitations. Moreover, the role of different candidate biomarkers is described together with the technical approaches for their identification. The provided evidence shows that progress has been achieved in understanding the molecular basis of melanoma and in designing advanced therapeutic strategies. Nevertheless, the molecular determinants of melanoma and their role as biomarkers predicting patients' responsiveness to therapies warrant further investigation with the vision of developing more effective precision medicine.

Cancer Immunotherapy Using Chimeric Antigen Receptor Expressing T-Cells: Present and Future Needs of Clinical Cancer Centers.

Chimeric Antigen Receptor-T cells (CAR-T) are considered novel biological agents, designed to selectively attack cancer cells expressing specific antigens, with demonstrated clinical activity in patients affected with relapsed/refractory B-cell malignancies. In consideration of their complexity, the use of CAR-T requires dedicated clinical setting and health care practitioners with expertise in the selection, treatment, and management of toxicities and side effects. Such issue appears particularly important when contextualized in the rapid progress of CAR-T cell treatment, translating into a constant need of updating and evolution. Moreover, the clinical grade manufacturing of CAR-T cells is complex and implies articulated regulatory and organizational aspects. The main goal of this review is to summarize and provide an accurate analysis of the clinical, logistic, and regulatory requirements of CAR-T cell centers. Finally, we describe a new occupational figure called "CAR-T specialist" devoted to the establishment and coordination of the required facilities and regulatory landscape in the context of cancer centers.

Proceedings From the First International Workshop at Sidra Medicine: "Engineered Immune Cells in Cancer Immunotherapy (EICCI): From Discovery to Off-the-Shelf Development", 15th-16th February 2019, Doha, Qatar.

The progress in the isolation and characterization of tumor antigen (TA)-specific T lymphocytes and in the genetic modification of immune cells allowed the clinical development of adoptive cell therapy (ACT). Several clinical studies highlighted the striking clinical activity of T cells engineered to express either Chimeric Antigen (CAR) or T Cell (TCR) Receptors to target molecularly defined antigens expressed on tumor cells. The breakthrough of immunotherapy is represented by the approval of CAR-T cells specific for advanced or refractory CD19+ B cell malignancies by both the Food and Drug Administration (FDA) and the European Medicinal Agency (EMA). Moreover, advances in the manufacturing and gene editing of engineered immune cells contributed to the selection of drug products with desired phenotype, refined specificity and decreased toxicity. An important step toward the optimization of CAR-T cell therapy is the development of "off-the shelf" T cell products that allow to reduce the complexity and the costs of the manufacturing and to render these drugs available for a broad number of cancer patients. The Engineered Immune Cells in Cancer Immunotherapy (EICCI) workshop hosted in Doha, Qatar, renowned experts, from both academia and industry, to present and discuss the progress on both pre-clinical and clinical development of genetically modified immune cells, including advances in the "off-the-shelf" manufacturing. These experts have addressed also organizational needs and hurdles for the clinical grade production and application of these biological drugs.

The Cross Talk between Cancer Stem Cells/Cancer Initiating Cells and Tumor Microenvironment: The Missing Piece of the Puzzle for the Efficient Targeting of these Cells with Immunotherapy.

Cancer Stem Cells/Cancer Initiating Cells (CSCs/CICs) is a rare sub-population within a tumor that is responsible for tumor formation, progression and resistance to therapies. The interaction between CSCs/CICs and tumor microenvironment (TME) can sustain "stemness" properties and promote their survival and plasticity. This cross-talk is also pivotal in regulating and modulating CSC/CIC properties. This review will provide an overview of the mechanisms underlying the mutual interaction between CSCs/CICs and TME. Particular focus will be dedicated to the immunological profile of CSCs/CICs and its role in orchestrating cancer immunosurveillance. Moreover, the available immunotherapy strategies that can target CSCs/CICs and of their possible implementation will be discussed. Overall, the dissection of the mechanisms regulating the CSC/CIC-TME interaction is warranted to understand the plasticity and immunoregulatory properties of stem-like tumor cells and to achieve complete eradications of tumors through the optimization of immunotherapy.

Reverting Immune Suppression to Enhance Cancer Immunotherapy.

Tumors employ strategies to escape immune control. The principle aim of most cancer immunotherapies is to restore effective immune surveillance. Among the different processes regulating immune escape, tumor microenvironment-associated soluble factors, and/or cell surface-bound molecules are mostly responsible for dysfunctional activity of tumor-specific CD8+T cells. These dynamic immunosuppressive networks prevent tumor rejection at several levels, limiting also the success of immunotherapies. Nevertheless, the recent clinical development of immune checkpoint inhibitors or of molecules modulating cellular targets and immunosuppressive enzymes highlights the great potential of approaches based on the selective disruption of immunosuppressive networks. Currently, the administration of different categories of immunotherapy in combination regimens is the ultimate modality for impacting the survival of cancer patients. With the advent of immune checkpoint inhibitors, designed to mount an effective antitumor immune response, profound changes occurred in cancer immunotherapy: from a global stimulation of the immune system to a specific targeting of an immune component. This review will specifically highlight the players, the mechanisms limiting an efficient antitumor response and the current immunotherapy modalities tailored to target immune suppressive pathways. We also discuss the ongoing challenges encountered by these strategies and provide suggestions for circumventing hurdles to new immunotherapeutic approaches, including the use of relevant biomarkers in the optimization of immunotherapy regimens and the identification of patients who can benefit from defined immune-based approaches.

Tailoring cells for clinical needs: Meeting report from the Advanced Therapy in Healthcare symposium (October 28-29 2017, Doha, Qatar).

New technologies and therapies designed to facilitate development of personalized treatments are rapidly emerging in the field of biomedicine. Strikingly, the goal of personalized medicine refined the concept of therapy by developing cell-based therapies, the so-called "living drugs". Breakthrough advancements were achieved in this regard in the fields of gene therapy, cell therapy, tissue-engineered products and advanced therapeutic techniques. The Advanced Therapies in Healthcare symposium, organized by the Clinical Research Center Department of Sidra Medicine, in Doha, Qatar (October 2017), brought together world-renowned experts from the fields of oncology, hematology, immunology, inflammation, autoimmune disorders, and stem cells to offer a comprehensive picture of the status of worldwide advanced therapies in both pre-clinical and clinical development, providing insights to the research phase, clinical data and regulatory aspects of these therapies. Highlights of the meeting are provided in this meeting report.