12th Tuscany Retreat on Cancer Research and Apoptosis: Genetic profiling, resistance mechanisms and novel treatment concepts in cancer and neurodegeneration.

The 12th Tuscany Retreat on Cancer Research and Apoptosis was held on August 19-26, 2023. The biennial retreat aims to bring together scientists who advance research in cancer, cell death, and neurodegenerative diseases. Topics covered ranged from drug resistance in cancer to insights into novel molecular cell signaling mechanisms and targets, all related to the pathways and molecules that regulate programmed cell death and the diseases that result from the dysregulation of programmed cell death. In this meeting review, we summarize the content of the most recent retreat.

Mimicking Tumor Cell Heterogeneity of Colorectal Cancer in a Patient-derived Organoid-Fibroblast Model.

BACKGROUND & AIMS: Patient-derived organoid cancer models are generated from epithelial tumor cells and reflect tumor characteristics. However, they lack the complexity of the tumor microenvironment, which is a key driver of tumorigenesis and therapy response. Here, we developed a colorectal cancer organoid model that incorporates matched epithelial cells and stromal fibroblasts. METHODS: Primary fibroblasts and tumor cells were isolated from colorectal cancer specimens. Fibroblasts were characterized for their proteome, secretome, and gene expression signatures. Fibroblast/organoid co-cultures were analyzed by immunohistochemistry and compared with their tissue of origin, as well as on gene expression levels compared with standard organoid models. Bioinformatics deconvolution was used to calculate cellular proportions of cell subsets in organoids based on single-cell RNA sequencing data. RESULTS: Normal primary fibroblasts, isolated from tumor adjacent tissue, and cancer associated fibroblasts retained their molecular characteristics in vitro, including higher motility of cancer associated compared with normal fibroblasts. Importantly, both cancer-associated fibroblasts and normal fibroblasts supported cancer cell proliferation in 3D co-cultures, without the addition of classical niche factors. Organoids grown together with fibroblasts displayed a larger cellular heterogeneity of tumor cells compared with mono-cultures and closely resembled the in vivo tumor morphology. Additionally, we observed a mutual crosstalk between tumor cells and fibroblasts in the co-cultures. This was manifested by considerably deregulated pathways such as cell-cell communication and extracellular matrix remodeling in the organoids. Thrombospondin-1 was identified as a critical factor for fibroblast invasiveness. CONCLUSION: We developed a physiological tumor/stroma model, which will be vital as a personalized tumor model to study disease mechanisms and therapy response in colorectal cancer.

Neoadjuvant immunotherapy in gastrointestinal cancers - The new standard of care?

The development of immune checkpoint inhibitors (ICI) offers novel treatment possibilities for solid cancers, with the crucial benefit of providing higher cure rates. These agents have become part of standard treatments in the metastatic and adjuvant setting for select cancers, such as melanoma, non-small cell lung cancer (NSCLC) or urological malignancies. Currently, there is ample clinical interest in employing ICI in a neoadjuvant setting with a curative intent. This approach is especially supported by the scientific rationale that ICI primarily stimulate the host immune system to eradicate tumor cells, rather than being inherently cytotoxic. Aside from tumor downstaging, neoadjuvant immunotherapy offers the potential of an in situ cancer vaccination, leading to a systemic adjuvant immunological effect after tumor resection. Moreover, preclinical data clearly demonstrate a synergistic effect of ICI with radiotherapy (RT), chemoradiotherapy (CRT) or chemotherapy (ChT). This review harmonizes preclinical concepts with real world data (RWD) in the field of neoadjuvant ICI in gastrointestinal (GI) cancers and discusses their limitations. We believe this is a crucial approach, since up to now, neoadjuvant strategies have been primarily developed by clinicians, whereas the advances in immunotherapy primarily originate from preclinical research. Currently there is limited published data on neoadjuvant ICI in GI cancers, even though neoadjuvant treatments including RT, CRT or ChT are frequently employed in locally advanced/oligometastatic GI cancers (i.e. rectal, pancreatic, esophagus, stomach, etc.). Utilizing established therapies in combination with ICI provides an abundance of opportunities for innovative treatment regimens to further improve survival rates.

Analysis of Methylation Dynamics Reveals a Tissue-Specific, Age-Dependent Decline in 5-Methylcytosine Within the Genome of the Vertebrate Aging Model Nothobranchius furzeri.

Erosion of the epigenetic DNA methylation landscape is a widely recognized hallmark of aging. Emerging advances in high throughput sequencing techniques, in particular DNA methylation data analysis, have resulted in the establishment of precise human and murine age prediction tools. In vertebrates, methylation of cytosine at the C5 position of CpG dinucleotides is executed by DNA methyltransferases (DNMTs) whereas the process of enzymatic demethylation is highly dependent on the activity of the ten-eleven translocation methylcytosine dioxygenase (TET) family of enzymes. Here, we report the identification of the key players constituting the DNA methylation machinery in the short-lived teleost aging model Nothobranchius furzeri. We present a comprehensive spatio-temporal expression profile of the methylation-associated enzymes from embryogenesis into late adulthood, thereby covering the complete killifish life cycle. Data mining of the N. furzeri genome produced five dnmt gene family orthologues corresponding to the mammalian DNMTs (DNMT1, 2, 3A, and 3B). Comparable to other teleost species, N. furzeri harbors multiple genomic copies of the de novo DNA methylation subfamily. A related search for the DNMT1 recruitment factor UHRF1 and TET family members resulted in the identification of N. furzeri uhrf1, tet1, tet2, and tet3. Phylogenetic analysis revealed high cross-species similarity on the amino acid level of all individual dnmts, tets, and uhrf1, emphasizing a high degree of functional conservation. During early killifish development all analyzed dnmts and tets showed a similar expression profile characterized by a strong increase in transcript levels after fertilization, peaking either at embryonic day 6 or at the black eye stage of embryonic development. In adult N. furzeri, DNA methylation regulating enzymes showed a ubiquitous tissue distribution. Specifically, we observed an age-dependent downregulation of dnmts, and to some extent uhrf1, which correlated with a significant decrease in global DNA methylation levels in the aging killifish liver and muscle. The age-dependent DNA methylation profile and spatio-temporal expression characteristics of its enzymatic machinery reported here may serve as an essential platform for the identification of an epigenetic aging clock in the new vertebrate model system N. furzeri.

From threat to cure: understanding of virus-induced cell death leads to highly immunogenic oncolytic influenza viruses.

Oncolytic viruses constitute an emerging strategy in immunomodulatory cancer treatment. The first oncolytic virus, Talimogene laherparepvec (T-VEC), based on herpes simplex virus 1 (HSV-1), was approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) in 2015. The field of oncolytic virotherapy is still in its beginnings, since many promising viruses remain only superficially explored. Influenza A virus causes a highly immunogenic acute infection but never leads to a chronic disease. While oncolytic influenza A viruses are in preclinical development, they have not made the transition into clinical practice yet. Recent insights into different types of cell death caused by influenza A virus infection illuminate novel possibilities of enhancing its therapeutic effect. Genetic engineering and experience in influenza A virus vaccine development allow safe application of the virus in patients. In this review we give a summary of efforts undertaken to develop oncolytic influenza A viruses. We discuss strategies for targeting viral replication to cancerous lesions and arming them with immunogenic transgenes. We furthermore describe which modes of cell death are induced by influenza A virus infection and how these insights may be utilized to optimize influenza A virus-based oncolytic virus design.

Histone deacetylase inhibitors valproic acid and vorinostat enhance trastuzumab-mediated antibody-dependent cell-mediated phagocytosis.

BACKGROUND: The monoclonal antibody (mAb) trastuzumab is part of the standard of care for patients with human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer. Antibody-dependent cell-mediated phagocytosis (ADCP) and cytotoxicity (ADCC) are major mechanisms of action of the mAb trastuzumab. Histone deacetylase inhibitors (HDACi), such as valproic acid (VPA) or vorinostat (SAHA), exert several immunostimulatory properties, which contribute at least in part to their anticancer effect. However, the impact of HDACi-induced immunostimulatory effects on trastuzumab-mediated anti-tumor immune response is not well characterized. METHODS: We analyzed the ADCP and ADCC activity of peripheral blood mononuclear cells (PBMCs) from age and gender-matched healthy volunteers (n=5) against HDACi-treated HER2-overexpressing breast cancer cells (SKBR3), using a well-established in vitro three-color imaging flow cytometry and flow cytometry approach. RESULTS: VPA and SAHA enhanced trastuzumab-mediated ADCP and trastuzumab-independent cytotoxicity. Mechanistically, VPA upregulated the activating antibody-binding receptor Fc-gamma receptor (FcγR) IIA (CD32A) on monocytes (CD14+). Moreover, VPA and SAHA downregulated the anti-apoptotic protein myeloid leukemia cell differentiation 1 (MCL1) in breast cancer cells. Additionally, VPA and SAHA induced an immunogenic cell death, characterized by the exposure of calreticulin (CALR), as well as decreased the "do not eat me" signal CD47 on tumor cells. CONCLUSIONS: HDACi VPA and SAHA increase trastuzumab-mediated phagocytosis and trastuzumab-independent cytotoxicity. The immunomodulatory activities of those HDACi support a rationale combined treatment approach with mAb for cancer treatment.

Spatio-temporal expression profile of sirtuins during aging of the annual fish Nothobranchius furzeri.

The founding member of the sirtuin family, yeast Sir2, was the first evolutionarily conserved gene to be identified as a regulator of longevity. Sirtuins constitute a protein family of metabolic sensors, translating changes in NAD + levels into adaptive responses, thereby acting as crucial regulators of the network that controls energy homeostasis and as such determines healthspan. In mammals the sirtuin family comprises seven proteins, SIRT1-SIRT7, which vary in tissue specificity, subcellular localization, enzymatic activity and targets. Here, we report the identification and a detailed spatio-temporal expression profile of sirtuin genes in the short-lived fish Nothobranchius furzeri, from embryogenesis to late adulthood, mapping its entire life cycle. Database exploration of the recently published N. furzeri genome revealed eight orthologues corresponding to the seven known mammalian sirtuins, including two copies of the sirt5 gene. Phylogenetic analysis showed high cross species similarity of individual sirtuins in both their overall amino acid sequence and catalytic domain, suggesting a high degree of functional conservation. Moreover, we show that N. furzeri sirtuins exhibit ubiquitous and wide tissue distribution with a unique spatial expression pattern for each individual member of this enzyme family. Specifically, we observed a transcriptional down-regulation of several sirtuin genes with age, most significantly sirt1, sirt5a, sirt6 and sirt7 in a wide range of functionally distinct tissues. Overall, this spatio-temporal expression analysis provides the foundation for future research, both into genetic and pharmacological manipulation of this important group of enzymes in Nothobranchius furzeri, an emerging model organism for aging research.

Radiotherapy as a Backbone for Novel Concepts in Cancer Immunotherapy.

Radiation-induced immunogenic cell death has been described to contribute to the efficacy of external beam radiotherapy in local treatment of solid tumors. It is well established that radiation therapy can induce immunogenic cell death in cancer cells under certain conditions. Initial clinical studies combining radiotherapy with immunotherapies suggest a synergistic potential of this approach. Improving our understanding of how radiation reconditions the tumor immune microenvironment should pave the way for designing rational and robust combinations with immunotherapeutic drugs that enhance both local and systemic anti-cancer immune effects. In this review, we summarize irradiation-induced types of immunogenic cell death and their effects on the tumor microenvironment. We discuss preclinical insights on mechanisms and benefits of combining radiotherapy with immunotherapy, focusing on immune checkpoint inhibitors. In addition, we elaborate how these observations were translated into clinical studies and which parameters may be optimized to achieve best results in future clinical trials.

Phylogenetic analysis and expression profiling of the Klotho gene family in the short-lived African killifish Nothobranchius furzeri.

Members of the Klotho gene family have been identified as modulators of the aging process. Deletion of αklotho in the mouse results in a syndrome resembling rapid human aging. Conversely, overexpression of αklotho extends mammalian lifespan. Here, we identify klotho orthologs in the vertebrate aging model Nothobranchius furzeri and provide a detailed spatio-temporal expression profile of both paralogs, α and ßklotho, from embryogenesis until old age spanning the entire life cycle of the organism. Specifically, we observe low levels of expression of both paralogs during embryogenesis followed by a significant transcriptional induction as development proceeds. In adult killifish, αklotho is predominantly expressed in the liver, the kidney, and the developing pharyngeal teeth. Particularly high levels of αKlotho protein were identified in the kidney tubules, closely resembling mammalian expression patterns. Prominent ßklotho expression was detected in the killifish intestine and liver. Overall, qRT-PCR analysis of Klotho members as a function of age revealed steady transcript levels, except for ßklotho expression in the liver which was significantly downregulated with age. This spatio-temporal expression profiling may serve as a useful starting point to further investigate the distinct physiological roles of Klotho members during the aging process.