Exploring the potential of the convergence between extracellular vesicles and CAR technology as a novel immunotherapy approach.

Cancer therapy is a dynamically evolving field, witnessing the emergence of innovative approaches that offer a promising outlook for patients grappling with persistent disease. Within the realm of therapeutic exploration, chimeric antigen receptor (CAR) T cells as well as CAR NK cells, have surfaced as novel approaches, each possessing unique attributes and transformative potential. Immune cells engineered to express CARs recognizing tumour-specific antigens, have shown remarkable promise in treating terminal cancers by combining the precision of antibody specificity with the potent cytotoxic function of T cells. However, their application in solid tumours is still in its nascent stages, presenting unique major challenges. On the same note, CAR NK cells offer a distinct immunotherapeutic approach, utilizing CARs on NK cells, providing advantages in safety, manufacturing simplicity, and a broader scope for cancer treatment. Extracellular vesicles (EVs) have emerged as promising therapeutic agents due to their ability to carry crucial biomarkers and biologically active molecules, serving as vital messengers in the intercellular communication network. In the context of cancer, the therapeutic potential of EVs lies in delivering tumour-suppressing proteins, nucleic acid components, or targeting drugs with precision, thereby redefining the paradigm of precision medicine. The fusion of CAR technology with the capabilities of EVs has given rise to a new therapeutic frontier. CAR T EVs and CAR NK EVs, leveraging the power of EVs, have the potential to alleviate challenges associated with live-cell therapies. EVs are suggested to reduce the side effects linked to CAR T cell therapy and hold the potential to revolutionize the penetrance in solid tumours. EVs act as carriers of pro-apoptotic molecules and RNA components, enhancing immune responses and thereby expanding their therapeutic potential. In this review article, we navigate dynamic landscapes, with our objective being to evaluate comparative efficacy, safety profiles, manufacturing complexities, and clinical applicability.

APR-246 as a radiosensitization strategy for mutant p53 cancers treated with alpha-particles-based radiotherapy.

Radiation therapy (RT) remains a common treatment for cancer patients worldwide, despite the development of targeted biological compounds and immunotherapeutic drugs. The challenge in RT lies in delivering a lethal dose to the cancerous site while sparing the surrounding healthy tissues. Low linear energy transfer (low-LET) and high linear energy transfer (high-LET) radiations have distinct effects on cells. High-LET radiation, such as alpha particles, induces clustered DNA double-strand breaks (DSBs), potentially inducing cell death more effectively. However, due to limited range, alpha-particle therapies have been restricted. In human cancer, mutations in TP53 (encoding for the p53 tumor suppressor) are the most common genetic alteration. It was previously reported that cells carrying wild-type (WT) p53 exhibit accelerated senescence and significant rates of apoptosis in response to RT, whereas cells harboring mutant p53 (mutp53) do not. This study investigated the combination of the alpha-emitting atoms RT based on internal Radium-224 (224Ra) sources and systemic APR-246 (a p53 reactivating compound) to treat tumors with mutant p53. Cellular models of colorectal cancer (CRC) or pancreatic ductal adenocarcinoma (PDAC) harboring mutant p53, were exposed to alpha particles, and tumor xenografts with mutant p53 were treated using 224Ra source and APR-246. Effects on cell survival and tumor growth, were assessed. The spread of alpha emitters in tumors was also evaluated as well as the spatial distribution of apoptosis within the treated tumors. We show that mutant p53 cancer cells exhibit radio-sensitivity to alpha particles in vitro and to alpha-particles-based RT in vivo. APR-246 treatment enhanced sensitivity to alpha radiation, leading to reduced tumor growth and increased rates of tumor eradication. Combining alpha-particles-based RT with p53 restoration via APR-246 triggered cell death, resulting in improved therapeutic outcomes. Further preclinical and clinical studies are needed to provide a promising approach for improving treatment outcomes in patients with mutant p53 tumors.

Acidovorax temperans skews neutrophil maturation and polarizes Th17 cells to promote lung adenocarcinoma development.

Change within the intratumoral microbiome is a common feature in lung and other cancers and may influence inflammation and immunity in the tumor microenvironment, affecting growth and metastases. We previously characterized the lung cancer microbiome in patients and identified Acidovorax temperans as enriched in tumors. Here, we instilled A. temperans in an animal model driven by mutant K-ras and Tp53. This revealed A. temperans accelerates tumor development and burden through infiltration of proinflammatory cells. Neutrophils exposed to A. temperans displayed a mature, pro-tumorigenic phenotype with increased cytokine signaling, with a global shift away from IL-1ß signaling. Neutrophil to monocyte and macrophage signaling upregulated MHC II to activate CD4+ T cells, polarizing them to an IL-17A+ phenotype detectable in CD4+ and γδ populations (T17). These T17 cells shared a common gene expression program predictive of poor survival in human LUAD. These data indicate bacterial exposure promotes tumor growth by modulating inflammation.

First measurements of radon-220 diffusion in mice tumors, towards treatment planning in diffusing alpha-emitters radiation therapy.

BACKGROUND: Diffusing alpha-emitters radiation therapy ("Alpha-DaRT") is a new method for treating solid tumors with alpha particles, relying on the release of the short-lived alpha-emitting daughter atoms of radium-224 from interstitial sources inserted into the tumor. Alpha-DaRT tumor dosimetry is governed by the spread of radium's progeny around the source, as described by an approximate framework called the "diffusion-leakage model". The most important model parameters are the diffusion lengths of radon-220 and lead-212, and their estimation is therefore essential for treatment planning. PURPOSE: Previous works have provided initial estimates for the dominant diffusion length, by measuring the activity spread inside mice-borne tumors several days after the insertion of an Alpha-DaRT source. The measurements, taken when lead-212 was in secular equilibrium with radium-224, were interpreted as representing the lead-212 diffusion length. The aim of this work is to provide first experimental estimates for the diffusion length of radon-220, using a new methodology. METHODS: The diffusion length of radon-220 was estimated from autoradiography measurements of histological sections taken from 24 mice-borne subcutaneous tumors of five different types. Unlike previous studies, the source dwell time inside the tumor was limited to 30 min, to prevent the buildup of lead-212. To investigate the contribution of potential non-diffusive processes, experiments were done in two sets: fourteen in vivo tumors, where during the treatment the tumors were still carried by the mice with active blood supply, and 10 ex-vivo tumors, where the tumors were excised before source insertion and kept in a medium at 37 ∘ C $37^\circ {\text{C}}$ with the source inside. RESULTS: The measured diffusion lengths of radon-220, extracted by fitting the recorded activity pattern up to 1.5 mm from the source, lie in the range 0.25 - 0.6 mm ${0.25-0.6}\nobreakspace {\text{mm}}$ , with no significant difference between the average values measured in in-vivo and ex-vivo tumors: L R n i n - v i v o = 0.40 ± 0.08 mm $L_{Rn}^{in-vivo}=0.40{\pm }0.08\nobreakspace {\text{mm}}$ versus L R n e x - v i v o = 0.39 ± 0.07 mm $L_{Rn}^{ex-vivo}=0.39{\pm }0.07\nobreakspace {\text{mm}}$ . However, in-vivo tumors display an enhanced spread of activity 2-3 mm away from the source. This effect is not explained by the current model and is much less pronounced in ex-vivo tumors. CONCLUSIONS: The average measured radon-220 diffusion lengths in both in-vivo and ex-vivo tumors are consistent with published data on the diffusion length of radon in water and lie close to the upper limit of the previously estimated range of 0.2 - 0.4 mm $0.2-0.4\nobreakspace {\text{mm}}$ . The observation that close to the source there is no apparent difference between in-vivo and ex-vivo tumors, and the good agreement with the theoretical model in this region suggest that the spread of radon-220 is predominantly diffusive in this region. The departure from the model prediction in in-vivo tumors at large radial distances may hint at potential vascular contribution, which will be the subject of future works.

Identifying p53 Target Genes Using Hypomorphic Variants.

SUMMARY: The reduced tumor suppression activity of hypomorphic variants of the TP53 gene was used by Indeglia and colleagues to corroborate PADI4 as a p53 target. The study makes a noteworthy advancement in comprehending the downstream implications of TP53-PDI4, including potential predictions of survival and the efficacy of immunotherapy. See related article by Indeglia et al., p. 1696 (4).

Diffusing alpha-emitters radiation therapy in combination with temozolomide or bevacizumab in human glioblastoma multiforme xenografts.

Glioblastoma multiforme (GBM) is at present an incurable disease with a 5-year survival rate of 5.5%, despite improvements in treatment modalities such as surgery, radiation therapy, chemotherapy [e.g., temozolomide (TMZ)], and targeted therapy [e.g., the antiangiogenic agent bevacizumab (BEV)]. Diffusing alpha-emitters radiation therapy (DaRT) is a new modality that employs radium-224-loaded seeds that disperse alpha-emitting atoms inside the tumor. This treatment was shown to be effective in mice bearing human-derived GBM tumors. Here, the effect of DaRT in combination with standard-of-care therapies such as TMZ or BEV was investigated. In a viability assay, the combination of alpha radiation with TMZ doubled the cytotoxic effect of each of the treatments alone in U87 cultured cells. A colony formation assay demonstrated that the surviving fraction of U87 cells treated by TMZ in combination with alpha irradiation was lower than was achieved by alpha- or x-ray irradiation as monotherapies, or by x-ray combined with TMZ. The treatment of U87-bearing mice with DaRT and TMZ delayed tumor development more than the monotherapies. Unlike other radiation types, alpha radiation did not increase VEGF secretion from U87 cells in culture. BEV treatment introduced several days after DaRT implantation improved tumor control, compared to BEV or DaRT as monotherapies. The combination was also shown to be superior when starting BEV administration prior to DaRT implantation in large tumors relative to the seed size. BEV induced a decrease in CD31 staining under DaRT treatment, increased the diffusive spread of 224Ra progeny atoms in the tumor tissue, and decreased their clearance from the tumor through the blood. Taken together, the combinations of DaRT with standard-of-care chemotherapy or antiangiogenic therapy are promising approaches, which may improve the treatment of GBM patients.

MPAPASS software enables stitched multiplex, multidimensional EV repertoire analysis and a standard framework for reporting bead-based assays.

Extracellular vesicles (EVs) of various types are released or shed from all cells. EVs carry proteins and contain additional protein and nucleic acid cargo that relates to their biogenesis and cell of origin. EV cargo in liquid biopsies is of widespread interest owing to its ability to provide a retrospective snapshot of cell state at the time of EV release. For the purposes of EV cargo analysis and repertoire profiling, multiplex assays are an essential tool in multiparametric analyte studies but are still being developed for high-parameter EV protein detection. Although bead-based EV multiplex analyses offer EV profiling capabilities with conventional flow cytometers, the utilization of EV multiplex assays has been limited by the lack of software analysis tools for such assays. To facilitate robust EV repertoire studies, we developed multiplex analysis post-acquisition analysis (MPAPASS) open-source software for stitched multiplex analysis, EV database-compatible reporting, and visualization of EV repertoires.

MEK1/2 inhibition transiently alters the tumor immune microenvironment to enhance immunotherapy efficacy against head and neck cancer.

BACKGROUND: Although the mitogen-activated protein kinases (MAPK) pathway is hyperactive in head and neck cancer (HNC), inhibition of MEK1/2 in HNC patients has not shown clinically meaningful activity. Therefore, we aimed to characterize the effect of MEK1/2 inhibition on the tumor microenvironment (TME) of MAPK-driven HNC, elucidate tumor-host interaction mechanisms facilitating immune escape on treatment, and apply rationale-based therapy combination immunotherapy and MEK1/2 inhibitor to induce tumor clearance. METHODS: Mouse syngeneic tumors and xenografts experiments were used to analyze tumor growth in vivo. Single-cell cytometry by time of flight, flow cytometry, and tissue stainings were used to profile the TME in response to trametinib (MEK1/2 inhibitor). Co-culture of myeloid-derived suppressor cells (MDSC) with CD8+ T cells was used to measure immune suppression. Overexpression of colony-stimulating factor-1 (CSF-1) in tumor cells was used to show the effect of tumor-derived CSF-1 on sensitivity to trametinib and anti-programmed death- 1 (αPD-1) in mice. In HNC patients, the ratio between CSF-1 and CD8A was measured to test the association with clinical benefit to αPD-1 and αPD-L1 treatment. RESULTS: Using preclinical HNC models, we demonstrated that treatment with trametinib delays HNC initiation and progression by reducing tumor cell proliferation and enhancing the antitumor immunity of CD8+ T cells. Activation of CD8+ T cells by supplementation with αPD-1 antibody eliminated tumors and induced an immune memory in the cured mice. Mechanistically, an early response to trametinib treatment sensitized tumors to αPD-1-supplementation by attenuating the expression of tumor-derived CSF-1, which reduced the abundance of two CSF-1R+CD11c+ MDSC populations in the TME. In contrast, prolonged treatment with trametinib abolished the antitumor activity of αPD-1, because tumor cells undergoing the epithelial to mesenchymal transition in response to trametinib restored CSF-1 expression and recreated an immune-suppressive TME. CONCLUSION: Our findings provide the rationale for testing the trametinib/αPD-1 combination in HNC and highlight the importance of sensitizing tumors to αPD-1 by using MEK1/2 to interfere with the tumor-host interaction. Moreover, we describe the concept that treatment of cancer with a targeted therapy transiently induces an immune-active microenvironment, and supplementation of immunotherapy during this time further activates the antitumor machinery to cause tumor elimination.

Proteomic analysis of necroptotic extracellular vesicles.

Necroptosis is a regulated and inflammatory form of cell death. We, and others, have previously reported that necroptotic cells release extracellular vesicles (EVs). We have found that necroptotic EVs are loaded with proteins, including the phosphorylated form of the key necroptosis-executing factor, mixed lineage kinase domain-like kinase (MLKL). However, neither the exact protein composition, nor the impact, of necroptotic EVs have been delineated. To characterize their content, EVs from necroptotic and untreated U937 cells were isolated and analyzed by mass spectrometry-based proteomics. A total of 3337 proteins were identified, sharing a high degree of similarity with exosome proteome databases, and clearly distinguishing necroptotic and control EVs. A total of 352 proteins were significantly upregulated in the necroptotic EVs. Among these were MLKL and caspase-8, as validated by immunoblot. Components of the ESCRTIII machinery and inflammatory signaling were also upregulated in the necroptotic EVs, as well as currently unreported components of vesicle formation and transport, and necroptotic signaling pathways. Moreover, we found that necroptotic EVs can be phagocytosed by macrophages to modulate cytokine and chemokine secretion. Finally, we uncovered that necroptotic EVs contain tumor neoantigens, and are enriched with components of antigen processing and presentation. In summary, our study reveals a new layer of regulation during the early stage of necroptosis, mediated by the secretion of specific EVs that influences the microenvironment and may instigate innate and adaptive immune responses. This study sheds light on new potential players in necroptotic signaling and its related EVs, and uncovers the functional tasks accomplished by the cargo of these necroptotic EVs.

Cancer Cells Shuttle Extracellular Vesicles Containing Oncogenic Mutant p53 Proteins to the Tumor Microenvironment.

Extracellular vesicles (EVs) shed by cancer cells play a major role in mediating the transfer of molecular information by reprogramming the tumor microenvironment (TME). TP53 (encoding the p53 protein) is the most mutated gene across many cancer types. Mutations in TP53 not only result in the loss of its tumor-suppressive properties but also results in the acquisition of novel gain-of-functions (GOF) that promote the growth of cancer cells. Here, we demonstrate that GOF mutant p53 proteins can be transferred via EVs to neighboring cancer cells and to macrophages, thus modulating them to release tumor supportive cytokines. Our data from pancreatic, lung, and colon carcinoma cell lines demonstrate that the mutant p53 protein can be selectively sorted into EVs. More specifically, mutant p53 proteins in EVs can be taken up by neighboring cells and mutant p53 expression is found in non-tumor cells in both human cancers and in non-human tissues in human xenografts. Our findings shed light on the intricate methods in which specific GOF p53 mutants can promote oncogenic mechanisms by reprogramming and then recruiting non-cancerous elements for tumor progression.

Reshaping the tumor microenvironment: extracellular vesicles as messengers of cancer cells.

The tumor microenvironment (TME) comprises an assortment of immune and non-immune cells. The interactions between the cancer cells and their surrounding TME are known to be a cardinal factor in all stages of cancer progression, from initiation to metastasis. Tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) are considered two of the most abundant TME members associated with poor prognosis in various cancer types. Intercellular communication between the cancer cells and TME cells might occur via direct cell-cell contact or achieved through secreted factors such as cytokines, growth factors and extracellular vesicles (EVs). EVs are released by almost every cell type and by cancer cells in particular. EVs are loaded with unique molecular cargos that might include DNA, proteins, RNA and lipids, commonly reflecting the physiological traits of their donor cells. Once released, EVs are capable of initiating short- and long-distance communication in an autocrine, paracrine and endocrine fashion. The molecular cargos within the EVs are able to impart phenotypic changes at the receiving end thus allowing EV-releasing cancer cells to deliver messages to TME cells and tighten their grasp over the cancerous tissue. In this concise review, we aim to document the bidirectional EV-based communication between cancer cell, TAMs and CAFs, tilting the balance in favor of cancer progression and metastasis.

Author Correction: Interaction between the microbiome and TP53 in human lung cancer.

Following publication of the original paper [1], the authors submitted a new Additional file 5 to replace the one containing formatting issues. The updated Additional file 5 is published in this correction.

Potential oncogenic roles of mutant-p53-derived exosomes in the tumor-host interaction of head and neck cancers.

The wide-ranging collection of malignancies arising at the upper aerodigestive tract is categorized as head and neck cancer (HNC), the sixth most prevalent cancer worldwide. Infection with human papillomavirus (HPV) or exposure to carcinogens is the leading causes of HPV+ and HPV- HNCs development, respectively. HPV+ and HPV- HNCs are different in clinical and molecular aspects. Specifically, HPV- HNCs tightly associate with missense mutants of the TP53 gene (encoding for the p53 protein), suggesting a central role for mutant p53 gain-of-function (GOF) in driving tumorigenesis. In contrast, in HPV + HNC, the sequence of TP53 typically remains intact, while the protein is degraded. In tumor cells, the status of the TP53 gene affects the cargo of secreted exosomes. In this review, we describe the accumulated knowledge regarding the involvement of exosomes and p53 on cellular interactions between HPV+ and HPV- HNC cells, and the surrounding tumor microenvironment (TME). Moreover, we envision how TP53 status may determine exosomes cargo in HNC, and, consequently, modify the TME. The potential roles of exosomes described herein are based on both our studies and the studies of others on mutant p53-derived exosomes. Specifically, we showed how exosomes are shed by cancer cells harboring mutant p53 communicate with tumor-associated macrophages in the colon as well as with cancer-associated fibroblasts in the lung, creating immunosuppressive conditions and promoting invasiveness. Altogether, exosomes in HNC in the context of TP53 status are understudied and extensive research is required to shed light on the biology of HPV+ and HPV- HNC.

Determination of Polarization of Resident Macrophages and Their Effect on the Tumor Microenvironment.

Interactions between tumor cells and their microenvironment have been long established as a cardinal hallmark of tumorigenesis and metastasis. To that end, tumor-associated macrophages (TAMs) have been studied extensively and were found to be typically correlated with poor prognosis in various cancers. TAMs are key elements of cancer-associated inflammation promoting cancer progression by increasing angiogenesis, inducing immunosuppression of the tumor tissue, and remodeling the extracellular matrix favoring invasion and metastasis. Since resident macrophages are characterized by substantial diversity and plasticity, understanding their polarization patterns in response to microenvironmental cues is a prime focus in the field. This chapter demonstrates an efficient manner to characterize polarization patterns of macrophages inside tumor tissues.

Immunohisto(cyto)chemistry: an old time classic tool driving modern oncological therapies.

In the era of precision medicine immunohistochemistry (IHC) and immunocytochemistry (ICC) share some of the highlights in personalized treatment. Survival data obtained from clinical trials shape the cut-offs and IHC scoring that serve as recommendations for patient selection both for targeted and conventional therapies. Assessment of Estrogen and Progesterone Receptors along with HER2 status has been among the first approved immunostaining assays revolutionizing breast cancer treatment. Similarly, ALK positivity predicts the efficacy of ALK inhibitors in patients with non-small cell lung cancer (NSCLC). In recent years, Programmed Death Ligand 1 (PD-L1) IHC assays have been approved as companion or complimentary diagnostic tools predicting the response to checkpoint inhibitors. Anti-PD-L1 and anti-PD-1 monoclonal antibodies have inaugurated a new period in the treatment of advanced cancers, but the path to approval of these biomarkers is filled with immunohistochemical challenges. The latter brings to the fore the significance of molecular pathology as a hub between basic and clinical research. Besides, novel markers are translated into routine practice, suggesting that we are at the beginning of a new exciting period. Unraveling the molecular mechanisms involved in cellular homeostasis unfolds biomarkers with greater specificity and sensitivity. The introduction of GL13 (SenTraGor®) for the detection of senescent cells in archival material, the implementation of key players of stress response pathways and the development of compounds detecting common mutant P53 isoforms in dictating oncological treatments are paradigms for precision oncology.

Senescence and senotherapeutics: a new field in cancer therapy.

Cellular senescence is a stress response mechanism ensuring homeostasis. Its temporal activation during embryonic development or normal adult life is linked with beneficial properties. In contrast, persistent (chronic) senescence seems to exert detrimental effects fostering aging and age-related disorders, such as cancer. Due to the lack of a reliable marker able to detect senescence in vivo, its precise impact in age-related diseases is to a large extent still undetermined. A novel reagent termed GL13 (SenTraGorTM) that we developed, allowing senescence recognition in any type of biological material, emerges as a powerful tool to study the phenomenon of senescence in vivo. Exploiting the advantages of this novel methodological approach, scientists will be able to detect and connect senescence with aggressive behavior in human malignancies, such as tolerance to chemotherapy in classical Hodgkin Lymphoma and Langerhans Cell Histiocytosis. The latter depicts the importance of developing the new and rapidly expanding field of senotherapeutic agents targeting and driving to cell death senescent cells. We discuss in detail the current progress of this exciting area of senotherapeutics and suggest its future perspectives and applications.

Interaction between the microbiome and TP53 in human lung cancer.

BACKGROUND: Lung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier integrity and increases susceptibility to infections. Herein, we hypothesize that somatic mutations together with cigarette smoke generate a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from 33 controls and 143 cancer cases, we conduct 16S ribosomal RNA (rRNA) bacterial gene sequencing, with RNA-sequencing data from lung cancer cases in The Cancer Genome Atlas serving as the validation cohort. RESULTS: Overall, we demonstrate a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue. In squamous cell carcinoma specifically, a separate group of taxa are identified, in which Acidovorax is enriched in smokers. Acidovorax temporans is identified within tumor sections by fluorescent in situ hybridization and confirmed by two separate 16S rRNA strategies. Further, these taxa, including Acidovorax, exhibit higher abundance among the subset of squamous cell carcinoma cases with TP53 mutations, an association not seen in adenocarcinomas. CONCLUSIONS: The results of this comprehensive study show both microbiome-gene and microbiome-exposure interactions in squamous cell carcinoma lung cancer tissue. Specifically, tumors harboring TP53 mutations, which can impair epithelial function, have a unique bacterial consortium that is higher in relative abundance in smoking-associated tumors of this type. Given the significant need for clinical diagnostic tools in lung cancer, this study may provide novel biomarkers for early detection.