A Magnetic Resonance Imaging-Chemical Exchange Saturation Transfer (MRI-CEST) Method for the Detection of Water Cycling across Cellular Membranes.

Water cycling across the membrane transporters is considered a hallmark of cellular metabolism and it could be of high diagnostic relevance in the characterization of tumors and other diseases. The method relies on the response of intracellular proton exchanging molecules to the presence of extracellular Gd-based contrast agents (GBCAs). Paramagnetic GBCAs enhances the relaxation rate of water molecules in the extracellular compartment and, through membrane exchange, the relaxation enhancement is transferred to intracellular molecules. The effect is detected at the MRI-CEST (Magnetic Resonance Imaging - Chemical Exchange Saturation Transfer) signal of intracellular proton exchanging molecules. The magnitude of the change in the CEST response reports on water cycling across the membrane. The method has been tested on Red Blood Cells and on orthotopic murine models of breast cancer with different degree of malignancy (4T1, TS/A and 168FARN). The distribution of voxels reporting on membrane permeability fits well with the cells' aggressiveness and acts as an early reporter to monitor therapeutic treatments.

Assessment of BoHV-4-based vector vaccine intranasally administered in a hamster challenge model of lung disease.

Introduction: Bovine herpesvirus 4 (BoHV-4) is a bovine Rhadinovirus not associated with a specific pathological lesion or disease and experimentally employed as a viral vector vaccine. BoHV-4-based vector (BoHV-4-BV) has been shown to be effective in immunizing and protecting several animal species when systemically administrated through intramuscular, subcutaneous, intravenous, or intraperitoneal routes. However, whether BoHV-4-BV affords respiratory disease protection when administered intranasally has never been tested. Methods: In the present study, recombinant BoHV-4, BoHV-4-A-S-ΔRS-HA-ΔTK, was constructed to deliver an expression cassette for the SARS-CoV-2 spike glycoprotein, and its immunogenicity, as well as its capability to transduce cells of the respiratory tract, were tested in mice. The well-established COVID-19/Syrian hamster model was adopted to test the efficacy of intranasally administered BoHV-4-A-S-ΔRS-HA-ΔTK in protecting against a SARS-CoV-2 challenge. Results: The intranasal administration of BoHV-4-A-S-ΔRS-HA-ΔTK elicited protection against SARS-CoV-2, with improved clinical signs, including significant reductions in body weight loss, significant reductions in viral load in the trachea and lungs, and significant reductions in histopathologic lung lesions compared to BoHV-4-A-S-ΔRS-HA-ΔTK administered intramuscularly. Discussion: These results suggested that intranasal immunization with BoHV-4-BV induced protective immunity and that BoHV-4-BV could be a potential vaccine platform for the protection of other animal species against respiratory diseases.

The Role of the Toll-like Receptor 2 and the cGAS-STING Pathways in Breast Cancer: Friends or Foes?

Breast cancer stands as a primary malignancy among women, ranking second in global cancer-related deaths. Despite treatment advancements, many patients progress to metastatic stages, posing a significant therapeutic challenge. Current therapies primarily target cancer cells, overlooking their intricate interactions with the tumor microenvironment (TME) that fuel progression and treatment resistance. Dysregulated innate immunity in breast cancer triggers chronic inflammation, fostering cancer development and therapy resistance. Innate immune pattern recognition receptors (PRRs) have emerged as crucial regulators of the immune response as well as of several immune-mediated or cancer cell-intrinsic mechanisms that either inhibit or promote tumor progression. In particular, several studies showed that the Toll-like receptor 2 (TLR2) and the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathways play a central role in breast cancer progression. In this review, we present a comprehensive overview of the role of TLR2 and STING in breast cancer, and we explore the potential to target these PRRs for drug development. This information will significantly impact the scientific discussion on the use of PRR agonists or inhibitors in cancer therapy, opening up new and promising avenues for breast cancer treatment.

Immunotherapy against the Cystine/Glutamate Antiporter xCT Improves the Efficacy of APR-246 in Preclinical Breast Cancer Models.

Breast cancer is the most frequent cancer in women. Despite recent clinical advances, new therapeutic approaches are still required. The cystine-glutamate antiporter xCT, encoded by the SLC7A11 gene, which imports cystine in exchange with glutamate, is a potentially new target for breast cancer therapy, being involved in tumor cell redox balance and resistance to therapies. xCT expression is regulated by the oncosuppressor p53, which is mutated in many breast cancers. Indeed, mutant p53 (mut-p53) can induce xCT post-transcriptional down modulation, rendering mut-p53 tumors susceptible to oxidative damage. Interestingly, the drug APR-246, developed to restore the wild-type function of p53 in tumors harboring its mutation, alters the cell redox balance in a p53-independent way, possibly rendering the cells more sensitive to xCT inhibition. Here, we propose a combinatorial treatment based on xCT immunetargeting and APR-246 treatment as a strategy for tackling breast cancer. We demonstrate that combining the inhibition of xCT with the APR-246 drug significantly decreased breast cancer cell viability in vitro and induced apoptosis and affected cancer stem cells' self-renewal compared to the single treatments. Moreover, the immunetargeting of xCT through DNA vaccination in combination with APR-246 treatment synergistically hinders tumor progression and prevents lung metastasis formation in vivo. These effects can be mediated by the production of anti-xCT antibodies that are able to induce the antibody dependent cellular cytotoxicity of tumor cells. Overall, we demonstrate that DNA vaccination against xCT can synergize with APR-246 treatment and enhance its therapeutic effect. Thus, APR-246 treatment in combination with xCT immunetargeting may open new perspectives in the management of breast cancer.

Toll-like receptor 2 promotes breast cancer progression and resistance to chemotherapy.

Cancer stem cells (CSCs) are the main drivers of disease progression and chemotherapy resistance in breast cancer. Tumor progression and chemoresistance might then be prevented by CSC-targeted therapies. We previously demonstrated that Toll-like Receptor (TLR)2 is overexpressed in CSCs and fuels their self-renewal. Here, we show that high TLR2 expression is linked to poor prognosis in breast cancer patients, therefore representing a candidate target for breast cancer treatment. By using a novel mammary cancer-prone TLR2KO mouse model, we demonstrate that TLR2 is required for CSC pool maintenance and for regulatory T cell induction. Accordingly, cancer-prone TLR2KO mice display delayed tumor onset and increased survival. Transplantation of TLR2WT and TLR2KO cancer cells in either TLR2WT or TLR2KO hosts shows that tumor initiation is mostly sustained by TLR2 expression in cancer cells. TLR2 host deficiency partially impairs cancer cell growth, implying a pro-tumorigenic effect of TLR2 expression in immune cells. Finally, we demonstrate that doxorubicin-induced release of HMGB1 activates TLR2 signaling in cancer cells, leading to a chemotherapy-resistant phenotype. Unprecedented use of TLR2 inhibitors invivo reduces tumor growth and potentiates doxorubicin efficacy with no negative impact on the host immune system, opening new perspectives for the treatment of breast cancer patients.

Are Cancer Stem Cells a Suitable Target for Breast Cancer Immunotherapy?

There is substantial evidence to suggest that complete tumor eradication relies on the effective elimination of cancer stem cells (CSCs). CSCs have been widely described as mediators of resistance to conventional therapies, including chemo- and radiotherapy, as well as of tumor metastasization and relapse in different tumor types, including breast cancer. However, the resistant phenotype of CSCs makes their targeting a tough task, and immunotherapy may therefore be an interesting option. Nevertheless, although immunotherapeutic approaches to cancer treatment have generated great enthusiasm due to recent success in clinics, breast cancer treatment mostly relies on standard approaches. In this context, we review the existing literature on the immunological properties of breast CSC and immunotherapeutic approaches to them. We will thus attempt to clarify whether there is room for the immunotargeting of breast CSCs in the current landscape of breast cancer therapies. Finally, we will provide our opinion on the CSC-targeting immunotherapeutic strategies that could prospectively be attempted.

RGD_PLGA Nanoparticles with Docetaxel: A Route for Improving Drug Efficiency and Reducing Toxicity in Breast Cancer Treatment.

Breast cancer is the leading cause of cancer-related death in women. Although many therapeutic approaches are available, systemic chemotherapy remains the primary choice, especially for triple-negative and advanced breast cancers. Unfortunately, systemic chemotherapy causes serious side effects and requires high doses to achieve an effective concentration in the tumor. Thus, the use of nanosystems for drug delivery may overcome these limitations. Herein, we formulated Poly (lactic-co-glycolic acid) nanoparticles (PLGA-NPs) containing Docetaxel, a fluorescent probe, and a magnetic resonance imaging (MRI) probe. The cyclic RGD tripeptide was linked to the PLGA surface to actively target αvß3 integrins, which are overexpressed in breast cancer. PLGA-NPs were characterized using dynamic light scattering, fast field-cycling 1H-relaxometry, and 1H-nuclear magnetic resonance. Their therapeutic effects were assessed both in vitro in triple-negative and HER2+ breast cancer cells, and in vivo in murine models. In vivo MRI and inductively coupled plasma mass spectrometry of excised tumors revealed a stronger accumulation of PLGA-NPs in the RGD_PLGA group. Targeted PLGAs have improved therapeutic efficacy and strongly reduced cardiac side effects compared to free Docetaxel. In conclusion, RGD-PLGA is a promising system for breast cancer treatment, with positive outcome in terms of therapeutic efficiency and reduction in side effects.

Toll-Like Receptor 2 at the Crossroad between Cancer Cells, the Immune System, and the Microbiota.

Toll-like receptor 2 (TLR2) expressed on myeloid cells mediates the recognition of harmful molecules belonging to invading pathogens or host damaged tissues, leading to inflammation. For this ability to activate immune responses, TLR2 has been considered a player in anti-cancer immunity. Therefore, TLR2 agonists have been used as adjuvants for anti-cancer immunotherapies. However, TLR2 is also expressed on neoplastic cells from different malignancies and promotes their proliferation through activation of the myeloid differentiation primary response protein 88 (MyD88)/nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) pathway. Furthermore, its activation on regulatory immune cells may contribute to the generation of an immunosuppressive microenvironment and of the pre-metastatic niche, promoting cancer progression. Thus, TLR2 represents a double-edge sword, whose role in cancer needs to be carefully understood for the setup of effective therapies. In this review, we discuss the divergent effects induced by TLR2 activation in different immune cell populations, cancer cells, and cancer stem cells. Moreover, we analyze the stimuli that lead to its activation in the tumor microenvironment, addressing the role of danger, pathogen, and microbiota-associated molecular patterns and their modulation during cancer treatments. This information will contribute to the scientific debate on the use of TLR2 agonists or antagonists in cancer treatment and pave the way for new therapeutic avenues.

Immunotargeting of the xCT Cystine/Glutamate Antiporter Potentiates the Efficacy of HER2-Targeted Immunotherapies in Breast Cancer.

Despite HER2-targeted therapies improving the outcome of HER2+ breast cancer, many patients experience resistance and metastatic progression. Cancer stem cells (CSC) play a role in this resistance and progression, thus combining HER2 targeting with CSC inhibition could improve the management of HER2+ breast cancer. The cystine-glutamate antiporter, xCT, is overexpressed in mammary CSCs and is crucial for their redox balance, self-renewal, and resistance to therapies, representing a potential target for breast cancer immunotherapy. We developed a combined immunotherapy targeting HER2 and xCT using the Bovine Herpes virus-4 vector, a safe vaccine that can confer immunogenicity to tumor antigens. Mammary cancer-prone BALB-neuT mice, transgenic for rat Her2, were immunized with the single or combined vaccines. Anti-HER2 vaccination slowed primary tumor growth, whereas anti-xCT vaccination primarily prevented metastasis formation. The combination of the two vaccines exerted a complementary effect by mediating the induction of cytotoxic T cells and of HER2 and xCT antibodies that induce antibody-dependent cell-mediated cytotoxicity and hinder cancer cell proliferation. Antibodies targeting xCT, but not those targeting HER2, directly affected CSC viability, self-renewal, and migration, inducing the antimetastatic effect of xCT vaccination. Our findings present a new therapy for HER2+ breast cancer, demonstrating that CSC immunotargeting via anti-xCT vaccination synergizes with HER2-directed immunotherapy.