Correction: Combined IFN-γ and TNF-α treatment enhances the susceptibility of breast cancer cells and spheroids to Natural Killer cell-mediated killing (Cell Death & Disease, (2025), 16, 1, (729), 10.1038/s41419-025-08021-0) Francesca Barberini, Riccardo Pietroni, Simone Ielpo, Valeria Lucarini, Daniela Nardozi, Ombretta Melaiu, Monica Benvenuto, Chiara Focaccetti, Camilla Palumbo, Federica Rossin, Doriana Fruci, Daniel Olive, Laura Masuelli, Roberto Bei, Loredana Cifaldi Cell Death and Disease, 2026 In this article, section "Materials and Methods" typos need to be corrected:"BC cell lines cultured in 2D were treated with a cytokine cocktail containing 50 g/mL IFN- (285-IF, R&D) and 20 g/mL TNF- (210-TA, R&D) for 48 h." with this:"BC cell lines cultured in 2D were treated with a cytokine cocktail containing 50 ng/mL IFN- (285-IF, R&D) and 20 ng/mL TNF- (210-TA, R&D) for 48 h.""BC spheroids were either untreated or treated with a low-dose, non-toxic cytokine cocktail, with cytokine concentration previously established by apoptotic dose-response assays (75 g/mL IFN- and 30 g/mL TNF-)" with this: "BC spheroids were either untreated or treated with a low-dose, non-toxic cytokine cocktail, with cytokine concentration previously established by apoptotic dose-response assays (75 ng/mL IFN- and 30 ng/mL TNF-)"
Lactate-mediated NK cell dysfunction as a prognostic marker and therapeutic target in breast cancer Simone Ielpo, Francesca Barberini, Alice Gaiba, Camilla Baronti, Marco Greppi, Valentina Obino, Silvia Ravera, Nicole Bussola, Adele De Ninno, Luca Businaro, Valentina Mussi, Silvia Pomella, Emanuele Agolini, Monica Benvenuto, Chiara Focaccetti, Emanuela Marcenaro, Roberto Bei, Giovanni Barillari, Silvia Pesce, Loredana Cifaldi, Ombretta Melaiu Cell Death Discovery, 2026 Lactate is recognized as a crucial signalling molecule within the tumor microenvironment, where it shapes immune responses by modulating various cell populations, including T cells and macrophages. However, its effect on natural killer (NK) cells, key effectors of early antitumor immunity, remains poorly understood. This study investigates how intratumoral lactate accumulation affects NK cell function in breast cancer, a neoplasm characterized by elevated glycolytic flux. An in-silico analysis of 882 breast cancer patients revealed that high lactate metabolism is inversely correlated with NK cell activation genes and is associated with poor prognosis. To corroborate these findings, NK cells from healthy donors were cultured under lactate-rich or control conditions. Lactate exposure impaired NK cell proliferation, downregulated activation markers and cytotoxic molecules, disrupted mitochondrial bioenergetics, and induced lipid accumulation, as demonstrated by flow cytometry, metabolic profiling, and Raman spectroscopy. Functional assays using microfluidic devices and degranulation tests revealed that lactate-exposed NK cells exhibited reduced chemotaxis and diminished cytotoxicity against MCF-7 and MDA-MB-231 breast cancer spheroids, accompanied by decreased CXCL9 and CXCL10 production. Pharmacologic inhibition of lactate transport, via Syrosingopine or MSC-4381 and AZD3965 combination, restored NK cell cytotoxicity in tumor co-cultures, as shown by increased NK cell degranulation, caspase-3/7–mediated tumor apoptosis, and spheroid shrinkage. Finally, GPR81 deletion mirrored these effects, enhancing NK cell activity. These findings identify lactate as a driver of NK cell suppression and highlight lactate transport and receptor targeting as a strategy to enhance NK cell–based immunotherapies in breast cancer and other lactate-rich tumors.
Natural Killer and CAR-NK immunotherapy in breast cancer: Bridging promise and the solid-tumor barrier Loredana Cifaldi, Monica Benvenuto, Chiara Focaccetti, Doriana Fruci, Laura Masuelli, Roberto Bei Oncologie, 2026 Breast cancer (BC) represents an immunotherapy paradox: despite the development of precise immune engineering protocols in hematologic malignancies, metastatic BC rarely becomes curable, and long-term immune control is uncommon. Natural killer (NK) cells, particularly chimeric antigen receptor (CAR)-modified NK cells, offer a pragmatic alternative to T cell–centric adoptive platforms because NK cells combine innate “missing-self/induced-self” recognition with antibody-dependent cell-mediated cytotoxicity (ADCC) and can be employed in allogeneic, “off-the-shelf” formats. However, the obstacles that blunt NK cell efficacy in solid tumors are not primarily about whether NK cells can kill BC cells in vitro . There is a concern about whether NK cells can access tumor nests, persist long enough, and remain functional within the suppressive and metabolically aggressive tumor microenvironment (TME). Here, we synthesize key biological and translational limitation points as phenotype alteration during manufacturing (including loss of CD16/ADCC competence), licensing and donor KIR/HLA variables, TGF-β and cytokine-driven paralysis, metabolic mismatch, and limited persistence of CAR–NK cells, and propose a BC-focused experimental approach. Priorities include, i) NK cell-engineering and CAR design optimization to enhance efficacy of antibody-based therapy of BC, ii) manufacturing standardization and quality metrics, iii) BC TME modulation, combination strategies and treating trafficking as a first-class endpoint, v) pairing NK cells with rational partners (antibodies, engagers, NK cell checkpoints), and vi) redesigning early trials around immune-functional endpoints that reflect BC metastatic biology.
Chronic low-dose cadmium exposure accelerates the onset and metastasis of invasive mammary carcinoma and promotes immunosuppression in the tumor microenvironment in BALB–neuT mice Chiara Focaccetti, Daniela Nardozi, Monica Benvenuto, Valeria Lucarini, Nicla Cristina, Eleonora Leti Maggio, Valentina Angiolini, Martino Tony Miele, Manuel Scimeca, Francesca Servadei, Alessandro Mauriello, Zein Mersini Besharat, Agnese Po, Alessandra Fabi, Michele Milella, Silvia Migliaccio, Elisabetta Ferretti, Camilla Palumbo, Loredana Cifaldi, Laura Masuelli, Roberto Bei Ecotoxicology and Environmental Safety, 2026 Cadmium (Cd), a heavy metal known to act as an endocrine disruptor, has been implicated in breast cancer (BC) via mechanisms that involve both estrogen receptor (ER)-dependent and -independent pathways. We used BALB-neuT transgenic mice, a model of aggressive ErbB2-driven mammary carcinogenesis, to evaluate the effects of chronic exposure to an environmentally relevant, low-dose of Cd on tumor onset, multiplicity, progression, immune microenvironment, and metastasis. Cd exposure significantly accelerated tumor onset and increased tumor multiplicity and weight, reducing both tumor-free and overall survival. Cd-exposed mice displayed elevated serum estrogen levels, and increased expression of progesterone receptor and tumor progression markers, including CD31, Ki67, and phosphorylated Akt, in tumor tissues. Despite the increased recruitment of CD4⁺ and CD8⁺ T cells to the peritumoral stroma, Cd exposure fostered an immunosuppressive microenvironment with elevated Tregs and PD-1⁺ exhausted T cells, both locally and systemically. Notably, lung metastases were threefold more frequent in Cd-exposed mice. Our results demonstrate that low-dose Cd exposure promotes the development and progression of ErbB2-driven breast tumors through hormonal, proliferative, and immune-modulating mechanisms. These findings reveal that exposure to environmentally relevant doses of Cd not only accelerates ErbB2-driven breast cancer but also reshapes the immune landscape toward dysfunction and immunosuppression, likely compromising both anti-tumor immunity and response to immunotherapies. These data advocate for tighter regulation of Cd exposure in populations at risk for breast cancer.
Extracellular Vesicle-Derived microRNAs: Novel Non-Invasive Biomarkers for Gastrointestinal Malignancies Daniela Nardozi, Valeria Lucarini, Valentina Angiolini, Nicole Feverati, Monica Benvenuto, Chiara Focaccetti, Letizia Del Conte, Olga Buccitti, Camilla Palumbo, Loredana Cifaldi, Elisabetta Ferretti, Roberto Bei, Laura Masuelli International Journal of Molecular Sciences, 2026 Gastrointestinal (GI) cancers represent a heterogeneous group of malignant neoplasms arising from the digestive tract, including gastric, colorectal, hepatic, pancreatic, and biliary cancers. These tumors represent a major public health challenge due to their aggressive nature and poor prognosis. Although significant progress has been made in diagnostic imaging, endoscopy, and multimodal therapies, early detection remains difficult. Conventional serum biomarkers often lack sufficient sensitivity and specificity for reliable diagnosis, prompting a growing interest in identifying novel, minimally invasive biomarkers. In this context, liquid biopsy is emerging as a revolutionary tool in oncology. Among its components, extracellular vesicles (EVs) have gained increasing attention because they carry a wide range of molecular cargoes that reflect the biological state of their tumor of origin. In particular, EV-associated microRNAs (miRNAs) hold great promise as biomarkers for early cancer detection, real-time monitoring of disease progression, and assessment of therapeutic response. This review discusses the diagnostic and prognostic potential of EVs as novel biomarkers in GI cancers, emphasizing EV-contained miRNAs as a key resource for the development of personalized and precision medicine strategies.
Combined IFN-γ and TNF-α treatment enhances the susceptibility of breast cancer cells and spheroids to Natural Killer cell-mediated killing Francesca Barberini, Riccardo Pietroni, Simone Ielpo, Valeria Lucarini, Daniela Nardozi, Ombretta Melaiu, Monica Benvenuto, Chiara Focaccetti, Camilla Palumbo, Federica Rossin, Doriana Fruci, Daniel Olive, Laura Masuelli, Roberto Bei, Loredana Cifaldi Cell Death and Disease, 2025 NK cell-based immunotherapy of solid tumors has been shown to be increasingly successful, but much effort is still needed to optimize its efficacy. This study explores the effects of treatment with low, non-toxic doses of IFN-γ and TNF-α on the susceptibility of breast cancer (BC) cell lines (MCF-7, MDA-MB-231, and MDA-MB-468) cultured in 2D and 3D as spheroids, to NK cell-mediated antitumor function. We evaluated the expression of (i) ligands for NK cell-activating receptors on BC cells, (ii) death and adhesion molecules on BC cells, and (iii) the expression of NK cell-receptors on NK cells infiltrating BC spheroids. Cytokine treatment significantly increased the expression of FAS, TRAIL-R2, and ICAM-1 in all BC cell lines, enhancing NK cell-mediated apoptosis and promoting NK cell-tumor cell conjugate formation. Differently, the expression of ligands for activating receptors remained essentially unchanged. In BC spheroids, the treatment with IFN-γ and TNF-α enhanced NK-cell infiltration, with increased expression of activating receptors (NKG2D, DNAM-1, NKp30, and NKp46) on infiltrating NK cells. However, regardless of treatment, markers of NK cell exhaustion, such as PD-1 and CTLA-4, were also upregulated, the latter especially in triple-negative BC (TNBC) MDA-MB-231 spheroids, thus suggesting an exhausted phenotype of NK cells infiltrating spheroids despite activation. Cytokine treatment resulted in a significant NK cell-mediated reduction in spheroid size, accompanied by an increased apoptotic state, with effects more pronounced in MCF-7 than in MDA-MB-231 spheroids. These results indicate that, although the treatment with IFN-γ and TNF-α improves NK cell-mediated tumor interaction and apoptosis, its therapeutic efficacy may be dependent on the BC subtype, with TNBC spheroids showing greater resistance. These findings highlight the importance of the tumor microenvironment (TME) in shaping NK cell responses and suggest that combining IFN-γ and TNF-α treatments with NK-cell-based immunotherapeutic strategies may improve treatment outcomes, particularly for more aggressive BC subtypes.
In vitro synergistic effect of AXL, FAK and ErbB receptors inhibitors for head and neck cancer Valeria Lucarini, Valentina Angiolini, Daniela Nardozi, Monica Benvenuto, Chiara Focaccetti, et al. Biology Direct, 2025 The prognosis for patients with head and neck cancer (HNC) is usually poor, highlighting the need for new therapeutic strategies. To this end, this study aims to evaluate the antitumor efficacy of a combined treatment with low doses of different molecular targeted drugs, i.e. Y15, a FAK inhibitor, Afatinib (AFA) an ErbB inhibitor and TP-0903, an Axl inhibitor, on HNC. Human cell lines from salivary gland, tongue and pharynx HNC, cultured in 2D and 3D (spheroids) conditions, were used to evaluate the antitumor effects of Y15, AFA and TP-0903, alone or in combination. Cell survival, death and migration were evaluated. Western blotting and immunofluorescence analysis were performed to investigate the expression and activation of proteins involved in signal transduction and epithelial to mesenchymal transition. The combined treatment with low doses of Y15, AFA and TP-0903, was more effective than the individual and dual drug treatments in reducing survival, increasing cell death and reducing migration of HNC cells. The three inhibitors in combination had a synergistic effect in reducing survival of HNC cell lines in both 2D and 3D conditions. Moreover, as compared to the individual inhibitors and their pairwise combinations, the triple drug combination was the only able to simultaneously downregulate Axl, FAK, and N-cadherin while upregulating E-cadherin expression levels. The results reported herein provide compelling preliminary evidence supporting the combined use of Y15, AFA and TP-0903 as a novel therapeutic strategy for HNCs.
The CD64/CD28/CD3ζ chimeric receptor reprograms T-cell metabolism and promotes T-cell persistence and immune functions while triggering antibody-independent and antibody-dependent cytotoxicity Sara Caratelli, Francesca De Paolis, Domenico Alessandro Silvestris, Silvia Baldari, Illari Salvatori, Apollonia Tullo, Giulia Lanzilli, Aymone Gurtner, Alberto Ferri, Cristiana Valle, Simona Padovani, Valeriana Cesarini, Tommaso Sconocchia, Loredana Cifaldi, Roberto Arriga, Giulio Cesare Spagnoli, Soldano Ferrone, Adriano Venditti, Piero Rossi, Graziano Pesole, Gabriele Toietta, Giuseppe Sconocchia Experimental Hematology and Oncology, 2025 Background Recent studies have shown that CD32/CD8a/CD28/CD3ζ chimeric receptor cells directly kill breast cancer cells, suggesting the existence of cell surface myeloid FcγR alternative ligands (ALs). Here, we investigated the metabolism, ALs, cytotoxicity, and immunoregulatory functions of CD64/CD28/CD3ζ in colorectal cancer (CRC) and squamous cell carcinoma of the head and neck. Methods The CD64/CD28/CD3ζ -SFG retroviral vector was used to produce viruses for T-cell transduction. T-cell expansion and differentiation were monitored via flow cytometry. Gene expression was assessed by RNA-seq. Bioenergetics were documented on a Seahorse extracellular flux analyzer. CD64/CD28/CD3ζ polarization was identified via confocal microscopy. Cytotoxicity was determined by MTT assay and bioluminescent imaging, and flow cytometry. Tridimensional antitumor activity of CD64/CD28/CD3ζ T cells was achieved by utilizing HCT116-GFP 3D spheroids via the IncuCyte S3 Live-Cell Analysis system. The intraperitoneal distribution and antitumor activity of NIR-CD64/CD28/CD3ζ and NIR-nontransduced T cells were investigated in CB17-SCID mice bearing subcutaneous FaDu Luc + cells by bioluminescent and fluorescent imaging. IFNγ was assessed by ELISA. Results Compared to CD16/CD8a/CD28/CD3ζ T cells, CD32/CD8a/CD28/CD3ζ T cells, and non-transduced T cells, CD64/CD28/CD3ζ T cells exhibited the highest levels of cell expansion and persistence capacity. A total of 235 genes linked to cell division and 52 genes related to glycolysis were overexpressed. The glycolytic phenotype was confirmed by functional in vitro studies accompanied by preferential T-cell effector memory differentiation. Interestingly, oxamic acid was found to inhibit CD64-CR T cell proliferation, indicating the involvement of lactate. Upon CD64/CD28/CD3ζ T-cell conjugation with CRC cells, CD64/CD28/CD3ζ cells polarize at immunological synapses, leading to CRC cell death. CD64/CD28/CD3ζ T cells kill SCCHN cells, and in combination with the anti-B7-H3 mAb (376.96) or anti-EGFR mAb, these cells trigger antibody-dependent cellular cytotoxicity (ADCC) in vitro under 2D and 3D conditions. The 376.96 mAb combined with CD64/CD28/CD3ζ T cells had anti-SCCHN activity in vivo. In addition, they induce the upregulation of PD-L1 and HLA-DR expression in cancer cells via IFNγ. PD-L1 positive SCCHN cells in combination with anti-PD-L1 mAb and CD64-CR T cells were killed by ADCC, which enhanced direct cytotoxicity. These findings indicate that the glycolytic phenotype is involved in CD64-CR T cell proliferation/expansion. These cells mediate long-lasting HLA-independent cytotoxicity and ADCC in CRC and SCCHN cells. Conclusions CD64/CD28/CD3ζ T cells could significantly impact the rational design of personalized studies to treat CRC and SCCHN and the identification of novel FcγR ALs in cancer and healthy cells.
Immunogenic Cell Death Inducers in Cancer Immunotherapy to Turn Cold Tumors into Hot Tumors Valeria Lucarini, Ombretta Melaiu, Paula Gragera, Kamila Król, Valentina Scaldaferri, Verena Damiani, Adele De Ninno, Daniela Nardozi, Luca Businaro, Laura Masuelli, Roberto Bei, Loredana Cifaldi, Doriana Fruci International Journal of Molecular Sciences, 2025 The combination of chemotherapeutic agents with immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment. However, its success is often limited by insufficient immune priming in certain tumors, including pediatric malignancies. In this report, we explore clinical trials currently investigating the use of immunogenic cell death (ICD)-inducing chemotherapies in combination with ICIs for both adult and pediatric cancers. Given the limited clinical data available for pediatric tumors, we focused on recent preclinical studies evaluating the efficacy of these combinations in neuroblastoma (NB). Finally, to address this gap, we propose an innovative strategy to assess the impact of ICD-inducing chemotherapies on antitumor immune responses in NB. Using tumor spheroids derived from a transgenic NB mouse model, we validated our previous in vivo findings concerning how anthracyclines, specifically mitoxantrone and doxorubicin, significantly enhance MHC class I surface expression, stimulate IFNγ and granzyme B production by CD8+ T cells and NK cells, and promote immune cell recruitment. Importantly, these anthracyclines also upregulated PD-L1 expression on NB spheroids. This screening platform yielded results similar to in vivo findings, demonstrating that mitoxantrone and doxorubicin are the most potent immunomodulatory agents for NB. These data suggest that the creation of libraries of ICD inducers to be tested on tumor spheroids could reduce the number of combinations to be tested in vivo, in line with the principles of the 3Rs. Furthermore, these results highlight the potential of chemo-immunotherapy regimens to counteract the immunosuppressive tumor microenvironment in NB, paving the way for improved therapeutic strategies in pediatric cancers. They provide compelling evidence to support further clinical investigations of these combinations to enhance outcomes for children with malignancies.
A genome-wide shRNA screen uncovers a novel potential ligand for NK cell activating receptors Paolo Romania, Loredana Cifaldi, Paula Gragera, Valerio D’Alicandro, Matteo Caforio, Valentina Folgiero, Valeria Lucarini, Ombretta Melaiu, Roberto Bei, Franco Locatelli, Doriana Fruci Frontiers in Immunology, 2025 IntroductionNatural Killer (NK) cells play a key role in both innate and adaptive immune responses against viruses and tumor cells. Their function relies on the dynamic balance between activating and inhibitory signals, which are mediated by receptors that bind ligands expressed on target cells. While much is known about the function and expression patterns of NK cell activating receptors (NKARs), many of their ligands remain unidentified.MethodsK562 cells were transduced with a shRNA library targeting 15,000 genes and co-cultured with NK cells from healthy donors. Surviving clones were tested in cytotoxicity and degranulation assays. PLAC1 was cloned from JEG3 cells in a lentiviral vector and transfected in K562 cells. PLAC1-related gene expression and survival data were obtained from the TCGA database and analyzed using R. PLAC1 and DSG2 expression in healthy tissues and NK cells was obtained from the HPA database and a GEO dataset.ResultsWe identified ten candidate genes whose downregulation in K562 cells decreased NK cell-mediated cytotoxicity to levels comparable to silencing the MICA gene. The most promising candidates were functionally validated through single-target gene silencing and overexpression. Among them, the placenta-specific 1 (PLAC1) gene stood out, as its inhibition conferred the greatest protection to target cells from NK cell lysis, while overexpression of PLAC1 significantly increased NK cell degranulation. Importantly, PLAC1 was found to interact with NKAR fusion proteins, including NKG2D, DNAM1 NKp44 and NKp30, suggesting its potential involvement in NK cell function. PLAC1 is typically silent in normal tissues, with the exception of placental trophoblasts and testicular germ cells, but is markedly overexpressed in a wide range of tumors. Notably, its prognostic significance appears to be tumor-type specific, associating with either favorable or poor outcomes depending on the cancer context.DiscussionOur study identifies PLAC1 as a novel potential ligand for NKARs, suggesting it could be a valuable target for pharmacological strategies aimed at enhancing NK cell recognition. This finding holds promise for improving the efficacy of NK cell-based immunotherapies and advancing their clinical application.
Virological and immunological features of SARS-COV-2 infected children with distinct symptomatology Nicola Cotugno, Alessandra Ruggiero, Giuseppe Rubens Pascucci, Francesco Bonfante, Maria Raffaella Petrara, Chiara Pighi, Loredana Cifaldi, Paola Zangari, Stefania Bernardi, Laura Cursi, Veronica Santilli, Emma Concetta Manno, Donato Amodio, Giulia Linardos, Livia Piccioni, Maria Antonietta Barbieri, Daniela Perrotta, Andrea Campana, Daniele Donà, Carlo Giaquinto, Carlo Concato, Petter Brodin, Paolo Rossi, Anita De Rossi, Paolo Palma, Lorenza Romani, Paola Pansa, Sara Chiurchiu, Andrea Finocchi, Caterina Cancrini, Laura Lancella, Maia De Luca, Renato Cutrera, Alberto Villani, Elena Morrocchi, Sonia Zicari, Lorenza Putignani, Francesca Calò Carducci, Maria A. De Ioris, Patrizia D'Argenio, Marta Ciofi degli Atti, Carmen D'Amore, and Pediatric Allergy and Immunology, 2021
Polyphenol-mediated autophagy in cancer: Evidence of in vitro and in vivo studies Monica Benvenuto, Loredana Albonici, Chiara Focaccetti, Sara Ciuffa, Sara Fazi, Loredana Cifaldi, Martino Tony Miele, Fernando De Maio, Ilaria Tresoldi, Vittorio Manzari, Andrea Modesti, Laura Masuelli, Roberto Bei International Journal of Molecular Sciences, 2020
Identification of a Genetic Variation in ERAP1 Aminopeptidase that Prevents Human Cytomegalovirus miR-UL112-5p-Mediated Immunoevasion Paolo Romania, Loredana Cifaldi, Benedetta Pignoloni, Nadia Starc, Valerio D’Alicandro, Ombretta Melaiu, Giuseppina Li Pira, Ezio Giorda, Rosalba Carrozzo, Monika Bergvall, Tomas Bergström, Lars Alfredsson, Tomas Olsson, Ingrid Kockum, Ilkka Seppälä, Terho Lehtimäki, Mikko A. Hurme, Hartmut Hengel, Angela Santoni, Cristina Cerboni, Franco Locatelli, Mauro D’Amato, Doriana Fruci Cell Reports, 2017
T and NK cells: Two sides of tumor immunoevasion Doriana Fruci, Elisa Monaco, Loredana Cifaldi, Franco Locatelli, Elisa Tremante, Maria Benevolo, Patrizio Giacomini Journal of Translational Medicine, 2013
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