Multiplex genetic manipulations in Clostridium butyricum and Clostridium sporogenes to secrete recombinant antigen proteins for oral-spore vaccination.

BACKGROUND: Clostridium spp. has demonstrated therapeutic potential in cancer treatment through intravenous or intratumoral administration. This approach has expanded to include non-pathogenic clostridia for the treatment of various diseases, underscoring the innovative concept of oral-spore vaccination using clostridia. Recent advancements in the field of synthetic biology have significantly enhanced the development of Clostridium-based bio-therapeutics. These advancements are particularly notable in the areas of efficient protein overexpression and secretion, which are crucial for the feasibility of oral vaccination strategies. Here, we present two examples of genetically engineered Clostridium candidates: one as an oral cancer vaccine and the other as an antiviral oral vaccine against SARS-CoV-2. RESULTS: Using five validated promoters and a signal peptide derived from Clostridium sporogenes, a series of full-length NY-ESO-1/CTAG1, a promising cancer vaccine candidate, expression vectors were constructed and transformed into C. sporogenes and Clostridium butyricum. Western blotting analysis confirmed efficient expression and secretion of NY-ESO-1 in clostridia, with specific promoters leading to enhanced detection signals. Additionally, the fusion of a reported bacterial adjuvant to NY-ESO-1 for improved immune recognition led to the cloning difficulties in E. coli. The use of an AUU start codon successfully mitigated potential toxicity issues in E. coli, enabling the secretion of recombinant proteins in C. sporogenes and C. butyricum. We further demonstrate the successful replacement of PyrE loci with high-expression cassettes carrying NY-ESO-1 and adjuvant-fused NY-ESO-1, achieving plasmid-free clostridia capable of secreting the antigens. Lastly, the study successfully extends its multiplex genetic manipulations to engineer clostridia for the secretion of SARS-CoV-2-related Spike_S1 antigens. CONCLUSIONS: This study successfully demonstrated that C. butyricum and C. sporogenes can produce the two recombinant antigen proteins (NY-ESO-1 and SARS-CoV-2-related Spike_S1 antigens) through genetic manipulations, utilizing the AUU start codon. This approach overcomes challenges in cloning difficult proteins in E. coli. These findings underscore the feasibility of harnessing commensal clostridia for antigen protein secretion, emphasizing the applicability of non-canonical translation initiation across diverse species with broad implications for medical or industrial biotechnology.

Impaired mitochondrial respiration in upper compared to lower body differentiated human adipocytes and adipose tissue.

CONTEXT: Abdominal obesity is associated with increased cardiometabolic disease risk, while lower body fat seems to confer protection against obesity-related complications. The functional differences between upper and lower body adipose tissue (AT) remain poorly understood. OBJECTIVE: We aimed to examine whether mitochondrial respiration is impaired in abdominal as compared to femoral differentiated human multipotent adipose-derived stem cells (hMADS; primary outcome) and AT in postmenopausal women. DESIGN: In this cross-sectional study, 23 postmenopausal women with normal weight or obesity were recruited at the University of Birmingham/Queen Elizabeth Hospital Birmingham (Birmingham, UK). We collected abdominal and femoral subcutaneous AT biopsies to determine mitochondrial oxygen consumption rates in differentiated abdominal and femoral hMADS. Furthermore, we assessed OXPHOS protein expression and mtDNA content in abdominal and femoral AT as well as hMADS. Finally, we explored in vivo fractional oxygen extraction and carbon dioxide release across abdominal and femoral subcutaneous AT in a subgroup of the same individuals with normal weight or obesity. RESULTS: We found lower basal and maximal uncoupled mitochondrial oxygen consumption rates in abdominal compared to femoral hMADS. In line, in vivo fractional oxygen extraction and carbon dioxide release were lower across abdominal than femoral AT. OXPHOS protein expression and mtDNA content did not significantly differ between abdominal and femoral differentiated hMADS and AT. CONCLUSION: The present findings demonstrate that in vitro mitochondrial respiration and in vivo oxygen fractional extraction are lower in upper compared to lower body differentiated hMADS and AT, respectively, in postmenopausal women.

Promising Diagnostic and Therapeutic Approaches Based on VHHs for Cancer Management.

The discovery of the distinctive structure of heavy chain-only antibodies in species belonging to the Camelidae family has elicited significant interest in their variable antigen binding domain (VHH) and gained attention for various applications, such as cancer diagnosis and treatment. This article presents an overview of the characteristics, advantages, and disadvantages of VHHs as compared to conventional antibodies, and their usage in diverse applications. The singular properties of VHHs are explained, and several strategies that can augment their utility are outlined. The preclinical studies illustrating the diagnostic and therapeutic efficacy of distinct VHHs in diverse formats against solid cancers are summarized, and an overview of the clinical trials assessing VHH-based agents in oncology is provided. These investigations demonstrate the enormous potential of VHHs for medical research and healthcare.

Targeting serine/glycine metabolism improves radiotherapy response in non-small cell lung cancer.

BACKGROUND: Lung cancer is the most lethal cancer, and 85% of cases are classified as non-small cell lung cancer (NSCLC). Metabolic rewiring is a cancer hallmark that causes treatment resistance, and lacks insights into serine/glycine pathway adaptations upon radiotherapy. METHODS: We analyzed radiotherapy responses using mass-spectrometry-based metabolomics in NSCLC patient's plasma and cell lines. Efficacy of serine/glycine conversion inhibitor sertraline with radiotherapy was investigated by proliferation, clonogenic and spheroid assays, and in vivo using a serine/glycine dependent NSCLC mouse model by assessment of tumor growth, metabolite and cytokine levels, and immune signatures. RESULTS: Serine/glycine pathway metabolites were significantly consumed in response to radiotherapy in NSCLC patients and cell models. Combining sertraline with radiotherapy impaired NSCLC proliferation, clonogenicity and stem cell self-renewal capacity. In vivo, NSCLC tumor growth was reduced solely in the sertraline plus radiotherapy combination treatment group. Tumor weights linked to systemic serine/glycine pathway metabolite levels, and were inhibited in the combination therapy group. Interestingly, combination therapy reshaped the tumor microenvironment via cytokines associated with natural killer cells, supported by eradication of immune checkpoint galectin-1 and elevated granzyme B levels. CONCLUSION: Our findings highlight that targeting serine/glycine metabolism using sertraline restricts cancer cell recovery from radiotherapy and provides tumor control through immunomodulation in NSCLC.

A deep-learning assisted bioluminescence tomography method to enable radiation targeting in rat glioblastoma.

Objective. A novel solution is required for accurate 3D bioluminescence tomography (BLT) based glioblastoma (GBM) targeting. The provided solution should be computationally efficient to support real-time treatment planning, thus reducing the x-ray imaging dose imposed by high-resolution micro cone-beam CT.Approach. A novel deep-learning approach is developed to enable BLT-based tumor targeting and treatment planning for orthotopic rat GBM models. The proposed framework is trained and validated on a set of realistic Monte Carlo simulations. Finally, the trained deep learning model is tested on a limited set of BLI measurements of real rat GBM models.Significance. Bioluminescence imaging (BLI) is a 2D non-invasive optical imaging modality geared toward preclinical cancer research. It can be used to monitor tumor growth in small animal tumor models effectively and without radiation burden. However, the current state-of-the-art does not allow accurate radiation treatment planning using BLI, hence limiting BLI's value in preclinical radiobiology research.Results. The proposed solution can achieve sub-millimeter targeting accuracy on the simulated dataset, with a median dice similarity coefficient (DSC) of 61%. The provided BLT-based planning volume achieves a median encapsulation of more than 97% of the tumor while keeping the median geometrical brain coverage below 4.2%. For the real BLI measurements, the proposed solution provided median geometrical tumor coverage of 95% and a median DSC of 42%. Dose planning using a dedicated small animal treatment planning system indicated good BLT-based treatment planning accuracy compared to ground-truth CT-based planning, where dose-volume metrics for the tumor fall within the limit of agreement for more than 95% of cases.Conclusion. The combination of flexibility, accuracy, and speed of the deep learning solutions make them a viable option for the BLT reconstruction problem and can provide BLT-based tumor targeting for the rat GBM models.

Predicting the Tumour Response to Radiation by Modelling the Five Rs of Radiotherapy Using PET Images.

Despite the intensive use of radiotherapy in clinical practice, its effectiveness depends on several factors. Several studies showed that the tumour response to radiation differs from one patient to another. The non-uniform response of the tumour is mainly caused by multiple interactions between the tumour microenvironment and healthy cells. To understand these interactions, five major biologic concepts called the "5 Rs" have emerged. These concepts include reoxygenation, DNA damage repair, cell cycle redistribution, cellular radiosensitivity and cellular repopulation. In this study, we used a multi-scale model, which included the five Rs of radiotherapy, to predict the effects of radiation on tumour growth. In this model, the oxygen level was varied in both time and space. When radiotherapy was given, the sensitivity of cells depending on their location in the cell cycle was taken in account. This model also considered the repair of cells by giving a different probability of survival after radiation for tumour and normal cells. Here, we developed four fractionation protocol schemes. We used simulated and positron emission tomography (PET) imaging with the hypoxia tracer 18F-flortanidazole (18F-HX4) images as input data of our model. In addition, tumour control probability curves were simulated. The result showed the evolution of tumours and normal cells. The increase in the cell number after radiation was seen in both normal and malignant cells, which proves that repopulation was included in this model. The proposed model predicts the tumour response to radiation and forms the basis for a more patient-specific clinical tool where related biological data will be included.

Polyphenols as Potential Protectors against Radiation-Induced Adverse Effects in Patients with Thoracic Cancer.

Radiotherapy is one of the standard treatment approaches used against thoracic cancers, occasionally combined with chemotherapy, immunotherapy and molecular targeted therapy. However, these cancers are often not highly sensitive to standard of care treatments, making the use of high dose radiotherapy necessary, which is linked with high rates of radiation-induced adverse effects in healthy tissues of the thorax. These tissues remain therefore dose-limiting factors in radiation oncology despite recent technological advances in treatment planning and delivery of irradiation. Polyphenols are metabolites found in plants that have been suggested to improve the therapeutic window by sensitizing the tumor to radiotherapy, while simultaneously protecting normal cells from therapy-induced damage by preventing DNA damage, as well as having anti-oxidant, anti-inflammatory or immunomodulatory properties. This review focuses on the radioprotective effect of polyphenols and the molecular mechanisms underlying these effects in the normal tissue, especially in the lung, heart and esophagus.

Treating colorectal peritoneal metastases with an injectable cytostatic loaded supramolecular hydrogel in a rodent animal model.

Patients with peritoneal metastases (PM) of colorectal cancer have a very poor outcome. Intraperitoneal delivery of chemotherapy is the preferred route for PM treatment. The main limitation of the treatment options is the short residence time of the cytostatic, with subsequent short exposure of the cancer cells. To address this, a supramolecular hydrogel has been developed that allows both local and slow release of its encapsulated drug, mitomycin C (MMC) or cholesterol-conjugated MMC (cMMC), respectively. This experimental study investigates if drug delivery using this hydrogel improves the therapeutic efficacy against PM. PM was induced in WAG/Rij rats (n = 72) by intraperitoneally injecting syngeneic colon carcinoma cells (CC531) expressing luciferase. After seven days, animals received a single intraperitoneal injection with saline (n = 8), unloaded hydrogel (n = 12), free MMC (n = 13), free cMMC (n = 13), MMC-loaded hydrogel (n = 13), or cMMC-loaded hydrogel (n = 13). Primary outcome was overall survival with a maximum follow-up of 120 days. Intraperitoneal tumor development was non-invasive monitored via bioluminescence imaging. Sixty-one rats successfully underwent all study procedures and were included to assess therapeutic efficacy. After 120 days, the overall survival in the MMC-loaded hydrogel and free MMC group was 78% and 38%, respectively. A trend toward significance was found when comparing the survival curves of the MMC-loaded hydrogel and free MMC (p = 0.087). No survival benefit was found for the cMMC-loaded hydrogel compared to free cMMC. Treating PM with our MMC-loaded hydrogel, exhibiting prolonged MMC exposure, seems effective in improving survival compared to treatment with free MMC.

The Modulatory Effects of Fatty Acids on Cancer Progression.

Cancer is the second leading cause of death worldwide and the global cancer burden rises rapidly. The risk factors for cancer development can often be attributed to lifestyle factors, of which an unhealthy diet is a major contributor. Dietary fat is an important macronutrient and therefore a crucial part of a well-balanced and healthy diet, but it is still unclear which specific fatty acids contribute to a healthy and well-balanced diet in the context of cancer risk and prognosis. In this review, we describe epidemiological evidence on the associations between the intake of different classes of fatty acids and the risk of developing cancer, and we provide preclinical evidence on how specific fatty acids can act on tumor cells, thereby modulating tumor progression and metastasis. Moreover, the pro- and anti-inflammatory effects of each of the different groups of fatty acids will be discussed specifically in the context of inflammation-induced cancer progression and we will highlight challenges as well as opportunities for successful application of fatty acid tailored nutritional interventions in the clinic.

Qualitative Study on Diversity, Equity, and Inclusion Within Radiation Oncology in Europe.

PURPOSE: Organizational culture plays a major role in prioritizing diversity, equity, and inclusion (DEI) objectives by aligning individual values of employees with organizational values. However, effective strategies to create an inclusive organizational culture, in which these values are aligned, remain unclear. The European Society for Radiotherapy and Oncology (ESTRO) launched a qualitative study, as a follow-up of the previous project on DEI that highlighted low levels of inclusion and work engagement among radiation oncology (RO) professionals in Europe. The aim of the present study was to gain an understanding of how DEI could be improved within RO departments by creating a more inclusive organizational culture. METHODS AND MATERIALS: A qualitative research study was conducted by enrolling RO professionals from 4 selected European countries through an open call on the ESTRO platform. Respondents who completed an online survey and met the inclusion criteria, such as experiencing low DEI levels at work, were invited for an online semistructured interview. Interview transcripts were analyzed thematically with an abductive approach via concepts in relation to "DEI," "work engagement," "organizational culture," and "professional values." RESULTS: Twenty-six eligible respondents from Great Britain, Italy, Poland, and Switzerland were interviewed. The thematic analysis identified cases in which limited engagement at work emerged when the personal values of RO professionals conflicted with dominant organizational values, hampering DEI. Three conflicts were found between the following personal versus organizational values: (1) self-development versus efficiency, (2) togetherness versus competition, and (3) people-oriented versus task-oriented cultures. CONCLUSIONS: Awareness of how organizational values can conflict with professionals' values should be raised to improve inclusion and engagement in the workplace. Additionally, efforts should be focused on tackling existing power imbalances that hamper effective deliberation on organizational- versus personal-value conflicts.

Sodium perturbs mitochondrial respiration and induces dysfunctional Tregs.

FOXP3+ regulatory T cells (Tregs) are central for peripheral tolerance, and their deregulation is associated with autoimmunity. Dysfunctional autoimmune Tregs display pro-inflammatory features and altered mitochondrial metabolism, but contributing factors remain elusive. High salt (HS) has been identified to alter immune function and to promote autoimmunity. By investigating longitudinal transcriptional changes of human Tregs, we identified that HS induces metabolic reprogramming, recapitulating features of autoimmune Tregs. Mechanistically, extracellular HS raises intracellular Na+, perturbing mitochondrial respiration by interfering with the electron transport chain (ETC). Metabolic disturbance by a temporary HS encounter or complex III blockade rapidly induces a pro-inflammatory signature and FOXP3 downregulation, leading to long-term dysfunction in vitro and in vivo. The HS-induced effect could be reversed by inhibition of mitochondrial Na+/Ca2+ exchanger (NCLX). Our results indicate that salt could contribute to metabolic reprogramming and that short-term HS encounter perturb metabolic fitness and long-term function of human Tregs with important implications for autoimmunity.

Use of 3D Spheroid Models for the Assessment of RT Response in Head and Neck Cancer.

Radiotherapy (RT) is a key player in the treatment of head and neck cancer (HNC). The RT response, however, is variable and influenced by multiple tumoral and tumor microenvironmental factors, such as human papillomavirus (HPV) infections and hypoxia. To investigate the biological mechanisms behind these variable responses, preclinical models are crucial. Up till now, 2D clonogenic and in vivo assays have remained the gold standard, although the popularity of 3D models is rising. In this study, we investigate the use of 3D spheroid models as a preclinical tool for radiobiological research by comparing the RT response of two HPV-positive and two HPV-negative HNC spheroid models to the RT response of their corresponding 2D and in vivo models. We demonstrate that HPV-positive spheroids keep their higher intrinsic radiosensitivity when compared to HPV-negative spheroids. A good correlation is found in the RT response between HPV-positive SCC154 and HPV-negative CAL27 spheroids and their respective xenografts. In addition, 3D spheroids are able to capture the heterogeneity of RT responses within HPV-positive and HPV-negative models. Moreover, we demonstrate the potential use of 3D spheroids in the study of the mechanisms underlying these RT responses in a spatial manner by whole-mount Ki-67 and pimonidazole staining. Overall, our results show that 3D spheroids are a promising model to assess the RT response in HNC.

Roadmap for precision preclinical x-ray radiation studies.

This Roadmap paper covers the field of precision preclinical x-ray radiation studies in animal models. It is mostly focused on models for cancer and normal tissue response to radiation, but also discusses other disease models. The recent technological evolution in imaging, irradiation, dosimetry and monitoring that have empowered these kinds of studies is discussed, and many developments in the near future are outlined. Finally, clinical translation and reverse translation are discussed.

Picomolar fluorescent probes for compound affinity determination to carbonic anhydrase IX expressed in live cancer cells.

Numerous human cancers, especially hypoxic solid tumors, express carbonic anhydrase IX (CAIX), a transmembrane protein with its catalytic domain located in the extracellular space. CAIX acidifies the tumor microenvironment, promotes metastases and invasiveness, and is therefore considered a promising anticancer target. We have designed a series of high affinity and high selectivity fluorescein-labeled compounds targeting CAIX to visualize and quantify CAIX expression in cancer cells. The competitive binding model enabled the determination of common CA inhibitors' dissociation constants for CAIX expressed in exponentially growing cancer cells. All tested sulfonamide compounds bound the proliferating cells with similar affinity as to recombinantly purified CAIX. The probes are applicable for the design of selective drug-like compounds for CAIX and the competition strategy could be applied to other drug targets.

Homologous Recombination Deficiency Scar: Mutations and Beyond-Implications for Precision Oncology.

Homologous recombination deficiency (HRD) is a prevalent in approximately 17% of tumors and is associated with enhanced sensitivity to anticancer therapies inducing double-strand DNA breaks. Accurate detection of HRD would therefore allow improved patient selection and outcome of conventional and targeted anticancer therapies. However, current clinical assessment of HRD mainly relies on determining germline BRCA1/2 mutational status and is insufficient for adequate patient stratification as mechanisms of HRD occurrence extend beyond functional BRCA1/2 loss. HRD, regardless of BRCA1/2 status, is associated with specific forms of genomic and mutational signatures termed HRD scar. Detection of this HRD scar might therefore be a more reliable biomarker for HRD. This review discusses and compares different methods of assessing HRD and HRD scar, their advances into the clinic, and their potential implications for precision oncology.

Fatty Acids as a Tool to Boost Cancer Immunotherapy Efficacy.

Although immunotherapy represents one of the most potent therapeutic anti-cancer approaches, only a limited number of patients shows clinical benefit. Recent evidence suggests that patients' nutritional status plays a major role in immunotherapy outcome. Fatty acids are essential in a balanced diet and well-known to influence the immune response. Moreover, short-chain fatty acids (SCFAs) show beneficial effects in metabolic disorders as well as in cancer and polyunsaturated fatty acids (PUFAs) contribute to body weight and fat free mass preservation in cancer patients. In line with these data, several studies imply a role for SCFAs and PUFAs in boosting the outcome of immunotherapy. In this review, we specifically focus on mechanistic data showing that SCFAs modulate the immunogenicity of tumor cells and we discuss the direct effects of SCFAs and PUFAs on the immune system in the context of cancer. We provide preclinical and clinical evidence indicating that SCFAs and PUFAs may have the potential to boost immunotherapy efficacy. Finally, we describe the challenges and address opportunities for successful application of nutritional interventions focusing on SCFAs and PUFAs to increase the therapeutic potential of immunotherapeutic approaches for cancer.

A deep learning and Monte Carlo based framework for bioluminescence imaging center of mass-guided glioblastoma targeting.

Objective.Bioluminescence imaging (BLI) is a valuable tool for non-invasive monitoring of glioblastoma multiforme (GBM) tumor-bearing small animals without incurring x-ray radiation burden. However, the use of this imaging modality is limited due to photon scattering and lack of spatial information. Attempts at reconstructing bioluminescence tomography (BLT) using mathematical models of light propagation show limited progress.Approach.This paper employed a different approach by using a deep convolutional neural network (CNN) to predict the tumor's center of mass (CoM). Transfer-learning with a sizeable artificial database is employed to facilitate the training process for, the much smaller, target database including Monte Carlo (MC) simulations of real orthotopic glioblastoma models. Predicted CoM was then used to estimate a BLI-based planning target volume (bPTV), by using the CoM as the center of a sphere, encompassing the tumor. The volume of the encompassing target sphere was estimated based on the total number of photons reaching the skin surface.Main results.Results show sub-millimeter accuracy for CoM prediction with a median error of 0.59 mm. The proposed method also provides promising performance for BLI-based tumor targeting with on average 94% of the tumor inside the bPTV while keeping the average healthy tissue coverage below 10%.Significance.This work introduced a framework for developing and using a CNN for targeted radiation studies for GBM based on BLI. The framework will enable biologists to use BLI as their main image-guidance tool to target GBM tumors in rat models, avoiding delivery of high x-ray imaging dose to the animals.