Development and activity of canine B7-H3-CAR.CIK lymphocytes against sarcomas: preclinical evidence and perspectives for human clinical translation Raffaella De Maria, Chiara Donini, Sonia Capellero, Annamaria Massa, Federica Galvagno, Alessia Proment, Letizia Vitali, Elisa Vigna, Elisa Landoni, Gianpietro Dotti, Valeria Leuci, Paolo Accornero, Luiza Cesar Conti, Matteo Olimpo, Enrico Berrino, Giovanni Grignani, Anna Sapino, Dario Sangiolo, Luca Aresu Cancer Immunology Immunotherapy, 2025 Advanced-stage sarcomas pose a major challenge in oncology, as they are often resistant to conventional therapies and associated with poor prognosis. CAR-based cellular immunotherapy is emerging as a very promising therapeutic option; however, clinically relevant animal models are urgently needed to accelerate the clinical development of these approaches. B7-H3 molecule, due to its low expression in normal tissue, high prevalence in multiple human cancers, and association with cancer stemness and aggressiveness, represents one of the most attractive targets for CAR-immunotherapy. In this study, we established a preclinical cellular immunotherapy platform based on canine cytokine-induced killer cells (CIK) redirected by an antihuman B7-H3 CAR against canine sarcoma cells and 3D sarcoma spheroids. B7-H3 was consistently detected across all analyzed canine sarcoma subtypes, including osteosarcoma, soft tissue sarcoma, and hemangiosarcoma, although with variable levels of expression intensity. We successfully generated canine B7-H3-CAR.CIK, achieving a mean CAR expression of 39% ± 2 with an immune phenotype unmodified (NTD) control (CD3 = 92% ± 3, CD8 = 87% ± 7; CD4 = 49% ± 5; CD5 = 77% ± 0.1; NKp46 = 83% ± 5). Canine B7-H3-CAR.CIK efficiently killed canine sarcoma cell lines compared with NTD.CIK, even at low effector/target (E/T) ratios (B7-H3-CAR.CIK: 45% vs 8%; E:T 1:1; 48 h; N = 7, n = 8; p < 0.0001), and demonstrated significant cytotoxicity against 3D sarcoma spheroids (58% vs 13%; E:T 2:1; 48 h; N = 3, n = 4; p < 0.01). Our findings establish a clinically relevant and translationally valuable platform for evaluating B7-H3-CAR.CIK therapy in dogs with incurable sarcomas, providing a bridge toward the development of novel CAR-based immunotherapies for human incurable sarcomas.
The cGAS-STING pathway is a master regulator of OCT4 expression in persistent sarcoma cells and enhances cellular immunotherapy with NK and CIK lymphocytes Giorgia Giordano, Alessandra Merlini, Federica Capozzi, Giulio Ferrero, Cristina Tucciarello, Shahab Majidi, Simona Comparato, Giulia Mesiano, Elisabetta Liuzzi, Federica Galvagno, Annamaria Massa, Silvia Brusco, Valeria Leuci, Chiara Riganti, Santina Cutrupi, Michele De Bortoli, Lorenzo D’Ambrosio, Dario Sangiolo, Giovanni Grignani, Ymera Pignochino Cancer Immunology Immunotherapy, 2025 Advanced sarcomas have a poor prognosis and limited therapeutic options. Disease recurrence is caused by persistent cells that survive drug treatments. The alkylating agent trabectedin, when combined with the poly (ADP-ribose) polymerase 1 (PARP1) inhibitor olaparib, exhibits variable antitumor effects in advanced sarcomas. In this study, we demonstrate that the expression of the transcription factor OCT4 is upregulated in persistent cells that survive treatment with trabectedin and olaparib, through the cGAS-STING-IRF3-IFNβ pathway. This route also leads to the upregulation of natural killer (NK) and cytokine-induced killer (CIK) lymphocyte activating ligands. These molecular events enhance the antitumor efficacy of immunotherapy with NK and CIK cells, targeting both the bulk population and residual drug-tolerant cells. In conclusion, the activation of the cGAS-STING pathway has a double-edged effect, enriching the OCT4+ persistent cell population while increasing the expression of NK/CIK ligands. The addition of olaparib to trabectedin potentiates the cGAS-STING pathway activation and the upregulation of NKG2DLs, while simultaneously counteracting the OCT4 overexpression. Therefore, sequential treatment with trabectedin and olaparib followed by NK/CIK immunotherapy represents a promising strategy against advanced sarcomas and warrants further investigation.
Doxycycline Restores Gemcitabine Sensitivity in Preclinical Models of Multidrug-Resistant Intrahepatic Cholangiocarcinoma Annamaria Massa, Francesca Vita, Caterina Peraldo-Neia, Chiara Varamo, Marco Basiricò, Chiara Raggi, Paola Bernabei, Jessica Erriquez, Francesco Leone, Massimo Aglietta, Giuliana Cavalloni, Serena Marchiò Cancers, 2025 Background/Objectives: Intrahepatic cholangiocarcinoma (iCCA) is a malignant liver tumor with a rising global incidence and poor prognosis, largely due to late-stage diagnosis and limited effective treatment options. Standard chemotherapy regimens, including cisplatin and gemcitabine, often fail because of the development of multidrug resistance (MDR), leaving patients with few alternative therapies. Doxycycline, a tetracycline antibiotic, has demonstrated antitumor effects across various cancers, influencing cancer cell viability, apoptosis, and stemness. Based on these properties, we investigated the potential of doxycycline to overcome gemcitabine resistance in iCCA. Methods: We evaluated the efficacy of doxycycline in two MDR iCCA cell lines, MT-CHC01R1.5 and 82.3, assessing cell cycle perturbation, apoptosis induction, and stem cell compartment impairment. We assessed the in vivo efficacy of combining doxycycline and gemcitabine in mouse xenograft models. Results: Treatment with doxycycline in both cell lines resulted in a significant reduction in cell viability (IC50 ~15 µg/mL) and induction of apoptosis. Doxycycline also diminished the cancer stem cell population, as indicated by reduced cholangiosphere formation. In vivo studies showed that while neither doxycycline nor gemcitabine alone significantly reduced tumor growth, their combination led to marked decreases in tumor volume and weight at the study endpoint. Additionally, metabolic analysis revealed that doxycycline reduced glucose uptake in tumors, both as a monotherapy and more effectively in combination with gemcitabine. Conclusions: These findings suggest that doxycycline, especially in combination with gemcitabine, can restore chemotherapy sensitivity in MDR iCCA, providing a promising new strategy for improving outcomes in this challenging disease.
Three-dimensional dynamics of mesothelin-targeted CAR.CIK lymphocytes against ovarian cancer peritoneal carcinomatosis Federica Galvagno, Valeria Leuci, Annamaria Massa, Chiara Donini, Ramona Rotolo, Sonia Capellero, Alessia Proment, Letizia Vitali, Andrea Maria Lombardi, Valentina Tuninetti, Lorenzo D’Ambrosio, Alessandra Merlini, Elisa Vigna, Giorgio Valabrega, Luca Primo, Alberto Puliafito, Dario Sangiolo Cancer Immunology Immunotherapy, 2025 Intraperitoneal cellular immunotherapy with CAR-redirected lymphocytes is an intriguing approach to target peritoneal carcinomatosis (PC) from ovarian cancer (OC), which is currently evaluated in clinical trials. PC displays a composite structure with floating tumor cells within ascites and solid-like masses invading the peritoneum. Therefore, a comprehensive experimental model is crucial to optimize CAR-cell therapies in such a peculiar environment. Here, we explored the activity of cytokine-induced killer lymphocytes (CIK), redirected by CAR against mesothelin (MSLN-CAR.CIK), within reductionistic 3D models resembling the structural complexity of both liquid and solid components of PC. MSLN-CAR.CIK effectively killed and were functionally efficient against OC targets. In a "floating-like" 3D context with floating OC spheroids, both tumor localization and killing by MSLN-CAR.CIK were significantly boosted by fluid flow. In a "solid-like" context, MSLN-CAR.CIK were recruited through the extracellular matrix on embedded tumor aggregates, with variable kinetics depending on the effector-target distance. Furthermore, MSLN-CAR.CIK penetrated the inner levels of OC spheroids exerting effective tumor killing. Our findings provide currently unknown therapeutically relevant information on intraperitoneal approaches with CAR.CIK, supporting further developments and improvements for clinical studies in the context of locoregional cell therapy approaches for patients with PC from OC.
CSPG4 CAR-redirected Cytokine Induced Killer lymphocytes (CIK) as effective cellular immunotherapy for HLA class I defective melanoma Lidia Giraudo, Giulia Cattaneo, Loretta Gammaitoni, Ilenia Iaia, Chiara Donini, Annamaria Massa, Maria Laura Centomo, Marco Basiricò, Elisa Vigna, Alberto Pisacane, Franco Picciotto, Enrico Berrino, Caterina Marchiò, Alessandra Merlini, Luca Paruzzo, Stefano Poletto, Daniela Caravelli, Andrea Michela Biolato, Valentina Bortolot, Elisa Landoni, Marco Ventin, Cristina R. Ferrone, Massimo Aglietta, Gianpietro Dotti, Valeria Leuci, Fabrizio Carnevale-Schianca, Dario Sangiolo Journal of Experimental and Clinical Cancer Research, 2023 Background Even acknowledging the game-changing results achieved in the treatment of metastatic melanoma with the use of immune checkpoint inhibitors (ICI), a large proportion of patients (40–60%) still fail to respond or relapse due to the development of resistance. Alterations in the expression of Human Leukocyte Antigen class I (HLA-I) molecules are considered to play a major role in clinical resistance to ICI. Cellular immunotherapy with HLA-independent CAR-redirected lymphocytes is a promising alternative in this challenging setting and dedicated translational models are needed. Methods In this study, we propose an HLA-independent therapeutic strategy with Cytokine Induced Killer lymphocytes (CIK) genetically engineered with a Chimeric Antigen Receptor (CAR) targeting the tumor antigen CSPG4 as effector mechanism. We investigated the preclinical antitumor activity of CSPG4-CAR.CIK in vitro and in a xenograft murine model focusing on patient-derived melanoma cell lines (Mel) with defective expression of HLA-I molecules. Results We successfully generated CSPG4-CAR.CIK from patients with metastatic melanoma and reported their intense activity in vitro against a panel of CSPG4-expressing patient-derived Mel. The melanoma killing activity was intense, even at very low effector to target ratios, and not influenced by the expression level (high, low, defective) of HLA-I molecules on target cells. Furthermore, CAR.CIK conditioned medium was capable of upregulating the expression of HLA-I molecules on melanoma cells. A comparable immunomodulatory effect was replicated by treatment of Mel cells with exogenous IFN-γ and IFN-α. The antimelanoma activity of CSPG4-CAR.CIK was successfully confirmed in vivo, obtaining a significant tumor growth inhibition of an HLA-defective Mel xenograft in immunodeficient mice. Conclusions In this study we reported the intense preclinical activity of CSPG4-CAR.CIK against melanoma, including those with low or defective HLA-I expression. Our findings support CSPG4 as a valuable CAR target in melanoma and provide translational rationale for clinical studies exploring CAR-CIK cellular immunotherapies within the challenging setting of patients not responsive or relapsing to immune checkpoint inhibitors.
Novel Lymphocyte-Independent Antitumor Activity by PD-1 Blocking Antibody against PD-1+ Chemoresistant Lung Cancer Cells Ramona Rotolo, Valeria Leuci, Chiara Donini, Federica Galvagno, Annamaria Massa, Maria Chiara De Santis, Serena Peirone, Giovanni Medico, Martina Sanlorenzo, Igor Vujic, Loretta Gammaitoni, Marco Basiricò, Luisella Righi, Chiara Riganti, Iris Chiara Salaroglio, Francesca Napoli, Fabrizio Tabbò, Annapaola Mariniello, Elisa Vigna, Chiara Modica, Lorenzo D’Ambrosio, Giovanni Grignani, Riccardo Taulli, Emilio Hirsch, Matteo Cereda, Massimo Aglietta, Giorgio Vittorio Scagliotti, Silvia Novello, Paolo Bironzo, Dario Sangiolo Clinical Cancer Research, 2023 Purpose: Antibodies against the lymphocyte PD-1 (aPD-1) receptor are cornerstone agents for advanced non–small cell lung cancer (NSCLC), based on their ability to restore the exhausted antitumor immune response. Our study reports a novel, lymphocyte-independent, therapeutic activity of aPD-1 against NSCLC, blocking the tumor-intrinsic PD-1 receptors on chemoresistant cells. Experimental Design: PD-1 in NSCLC cells was explored in vitro at baseline, including stem-like pneumospheres, and following treatment with cisplatin both at transcriptional and protein levels. PD-1 signaling and RNA sequencing were assessed. The lymphocyte-independent antitumor activity of aPD-1 was explored in vitro, by PD-1 blockade and stimulation with soluble ligand (PD-L1s), and in vivo within NSCLC xenograft models. Results: We showed the existence of PD-1+ NSCLC cell subsets in cell lines and large in silico datasets (Cancer Cell Line Encyclopedia and The Cancer Genome Atlas). Cisplatin significantly increased PD-1 expression on chemo-surviving NSCLC cells (2.5-fold P = 0.0014), while the sequential treatment with anti–PD-1 Ab impaired their recovery after chemotherapy. PD-1 was found to be associated with tumor stemness features. PD-1 expression was enhanced in NSCLC stem-like pneumospheres (P &lt; 0.0001), significantly promoted by stimulation with soluble PD-L1 (+27% ± 4, P &lt; 0.0001) and inhibited by PD-1 blockade (−30% ± 3, P &lt; 0.0001). The intravenous monotherapy with anti–PD-1 significantly inhibited tumor growth of NSCLC xenografts in immunodeficient mice, without the contribution of the immune system, and delayed the occurrence of chemoresistance when combined with cisplatin. Conclusions: We report first evidence of a novel lymphocyte-independent activity of anti–PD-1 antibodies in NSCLC, capable of inhibiting chemo-surviving NSCLC cells and exploitable to contrast disease relapses following chemotherapy. See related commentary by Augustin et al., p. 505
Integrated Antitumor Activities of Cellular Immunotherapy with CIK Lymphocytes and Interferons against KIT/PDGFRA Wild Type GIST Erika Fiorino, Alessandra Merlini, Lorenzo D’Ambrosio, Ilaria Cerviere, Enrico Berrino, Caterina Marchiò, Lidia Giraudo, Marco Basiricò, Annamaria Massa, Chiara Donini, Valeria Leuci, Ramona Rotolo, Federica Galvagno, Letizia Vitali, Alessia Proment, Soldano Ferrone, Alberto Pisacane, Ymera Pignochino, Massimo Aglietta, Giovanni Grignani, Giulia Mesiano, Dario Sangiolo International Journal of Molecular Sciences, 2022 Gastrointestinal stromal tumors (GISTs) are rare, mesenchymal tumors of the gastrointestinal tract, characterized by either KIT or PDGFRA mutation in about 85% of cases. KIT/PDGFRA wild type gastrointestinal stromal tumors (wtGIST) account for the remaining 15% of GIST and represent an unmet medical need: their prevalence and potential medical vulnerabilities are not completely defined, and effective therapeutic strategies are still lacking. In this study we set a patient-derived preclinical model of wtGIST to investigate their phenotypic features, along with their susceptibility to cellular immunotherapy with cytokine-induced killer lymphocytes (CIK) and interferons (IFN). We generated 11 wtGIST primary cell lines (wtGISTc). The main CIK ligands (MIC A/B; ULBPs), along with PD-L1/2, were expressed by wtGISTc and the expression of HLA-I molecules was preserved. Patient-derived CIK were capable of intense killing in vitro against wtGISTc resistant to both imatinib and sunitinib. We found that CIK produce a high level of granzyme B, IFNα and IFNγ. CIK-conditioned supernatant was responsible for part of the observed tumoricidal effect, along with positive bystander modulatory activities enhancing the expression of PD-L1/2 and HLA-I molecules. IFNα, but not In, had direct antitumor effects on 50% (4/8) of TKI-resistant wtGISTc, positively correlated with the tumor expression of IFN receptors. wtGIST cells that survived IFNα were still sensitive to CIK immunotherapy. Our data support the exploration of CIK immunotherapy in clinical studies for TKI-resistant wtGIST, proposing reevaluation for IFNα within this challenging setting.
Paclitaxel Restores Sensitivity to Chemotherapy in Preclinical Models of Multidrug-Resistant Intrahepatic Cholangiocarcinoma Annamaria Massa, Caterina Peraldo-Neia, Francesca Vita, Chiara Varamo, Marco Basiricò, Chiara Raggi, Paola Bernabei, Jessica Erriquez, Ivana Sarotto, Francesco Leone, Serena Marchiò, Giuliana Cavalloni, Massimo Aglietta Frontiers in Oncology, 2022 The treatment of unresectable cholangiocarcinoma (CCA) is limited by the development of resistance to conventional first-line chemotherapy based on gemcitabine (GEM). In addition, a prior treatment with GEM frequently induces cross-resistance to other drugs employed in the second-line. Paclitaxel (PTX) is now emerging as an alternative option for the management of advanced/metastatic CCA. In the present work, we evaluate the antitumor activity of PTX in preclinical models of multidrug-resistant intrahepatic cholangiocarcinoma (iCCA). In vitro, PTX decreases tumor cell viability by affecting the cell cycle and inducing apoptosis and impairs the stem cell compartment. In vivo, a therapeutic regimen containing albumin-bound nanoparticle (Nab)-PTX overcomes drug resistance resulting in delayed tumor growth, impaired organization of the tumor vasculature, and reduced glucose uptake. Together, our results provide a rationale to consider PTX-based regimens in patients with iCCA who became refractory to conventional therapies.
Association between markers of bone loss and urinary lithogenic risk factors in osteopenic postmenopausal women Journal of Biological Regulators and Homeostatic Agents, 2016
