Necessary, Legendary and Detrimental Components of Human Colorectal Organoid Culture Medium: Raising Awareness to Reduce Experimental Bugs Roberto Benelli Cancers, 2026 The creation of a specific culture medium for colorectal organoids in 2011 heralded a new era in human primary cultures by enabling the indefinite expansion of normal and pathological epithelial organoids. The original formula has been used ever since, with only minor, lab-specific modifications. The goal of culturing organoids from different tissues has relied on saving and propagating the pluripotent stem cell. The “magic bullet” and all its subsequent derivatives have pursued this goal. Consequently, agonist and antagonist signals are chronically activated in the organoid medium, forcing organoid cells (as well as any other co-cultured cellular model) into constrained signaling pathways. This extremely artificial condition is often overlooked in experimental approaches and may bias the results. Furthermore, some molecules in the organoid medium have unpredictable off-target effects that significantly impact the behavior and maturation of certain cell populations. Nicotinamide, gastrin and PGE2 inhibit immune responses. SB202190, A83-01 and vanadate (from advanced DMEM-F12) modify intracellular signaling. N-AcetylCysteine and Primocin modify the redox response and mitochondrial metabolism, respectively. Thus, the unintentional addition of these molecules to the organoid medium introduces biases under specific experimental settings. While the original organoid medium formula is the gold standard for propagating organoids in vitro, more focused, reliable conditions are necessary for specific organoid-based tests.
Developing a 3D Model Culture of an EBV+/CD30+ B-Anaplastic Large Cell Lymphoma Cell Line to Assay Brentuximab Vedotin Treatment Paolo Giannoni, Gabriella Pietra, Orlando Izzo, Giuseppina Fugazza, Roberto Benelli, et al. Antibodies, 2025 Background/Objectives: Three-dimensional (3D) in vitro cell culture models have recently stimulated great interest since they may have more pre-clinical value than conventional in vitro 2D models. In fact, 3D culture models may mimic the in vivo biophysical 3D structure of tumors and cell-to-cell interaction, thereby representing a more useful approach to testing drug responses. In this study we have developed a 3D culture model of an EBV+/CD30+cell line, D430B, previously characterized as an Anaplastic Large Cell Lymphoma of B phenotype (B-ALCL), to determine the cytotoxic activity of the antibody–drug conjugate Brentuximab Vedotin. Methods: By using of ultra-low attachment plates, we developed D430B spheroids that appeared particularly homogenous in terms of growth and size. Results: Brentuximab Vedotin treatment (1 to 20 μg/mL) turned out to be significantly cytotoxic to these cells, while the addition of the anti-CD20 chimeric antibody Rituximab (10 μg/mL) appeared almost ineffective, even though these cells express CD20. Moreover, when we co-cultured D430B cells with stromal cells (HS5), to re-create a microenvironment representative of neoplastic cell/mesenchymal cell interactions within the lymph node, we observed a significant, although faint, protective effect. Conclusions: This simple and reproducible method of generating D430B-ALCL spheroids to evaluate their response to Brentuximab Vedotin treatment, as here described, may provide a valuable preliminary tool for the future pre-clinical screening of patients’ primary lymphoma cells or the development of novel therapies for this type of pathology and related diseases.
The Role of LAIR1 as a Regulatory Receptor of Antitumor Immune Cell Responses and Tumor Cell Growth and Expansion Alessandro Poggi, Serena Matis, Chiara Rosa Maria Uras, Lizzia Raffaghello, Roberto Benelli, et al. Biomolecules, 2025 It is becoming evident that the therapeutic effect of reawakening the immune response is to limit tumor cell growth and expansion. The use of immune checkpoint inhibitors, like blocking antibodies against programmed cell death receptor (PD) 1 and/or cytotoxic T lymphocyte antigen (CTLA) 4 alone or in combination with other drugs, has led to unexpected positive results in some tumors but not all. Several other molecules inhibiting lymphocyte antitumor effector subsets have been discovered in the last 30 years. Herein, we focus on the leukocyte-associated immunoglobulin (Ig)-like receptor 1 (LAIR1/CD305). LAIR1 represents a typical immunoregulatory molecule expressed on almost all leukocytes, unlike other regulatory receptors expressed on discrete leukocyte subsets. It bears two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the intracytoplasmic protein domain involved in the downregulation of signals mediated by activating receptors. LAIR1 binds to several ligands, such as collagen I and III, complement component 1Q, surfactant protein D, adiponectin, and repetitive interspersed families of polypeptides expressed by erythrocytes infected with Plasmodium malariae. This would suggest LAIR1 involvement in several cell-to-cell interactions and possibly in metabolic regulation. The presence of both cellular and soluble forms of LAIR would indicate a fine regulation of the immunoregulatory activity, as happens for the soluble/exosome-associated forms of PD1 and CTLA4 molecules. As a consequence, LAIR1 appears to play a role in some autoimmune diseases and the immune response against tumor cells. The finding of LAIR1 expression on hematological malignancies, but also on some solid tumors, could open a rationale for the targeting of this molecule to treat neoplasia, either alone or in combination with other therapeutic options.
Colorectal Organoids: Models, Imaging, Omics, Therapy, Immunology, and Ethics Martina Taglieri, Linda Di Gregorio, Serena Matis, Chiara Rosa Maria Uras, Massimo Ardy, et al. Cells, 2025 Colorectal epithelium was the first long-term 3D organoid culture established in vitro. Identification of the key components essential for the long-term survival of the stem cell niche allowed an indefinite propagation of these cultures and the modulation of their differentiation into various lineages of mature intestinal epithelial cells. While these methods were eventually adapted to establish organoids from different organs, colorectal organoids remain a pioneering model for the development of new applications in health and disease. Several basic and applicative aspects of organoid culture, modeling, monitoring and testing are analyzed in this review. We also tackle the ethical problems of biobanking and distribution of these precious research tools, frequently confined in the laboratory of origin or condemned to destruction at the end of the project.
Epidermal Growth Factor Receptor Targeting in Colorectal Carcinoma: Antibodies and Patient-Derived Organoids as a Smart Model to Study Therapy Resistance Samuele Tardito, Serena Matis, Maria Raffaella Zocchi, Roberto Benelli, Alessandro Poggi International Journal of Molecular Sciences, 2024 Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. Therefore, the need for new therapeutic strategies is still a challenge. Surgery and chemotherapy represent the first-line interventions; nevertheless, the prognosis for metastatic CRC (mCRC) patients remains unacceptable. An important step towards targeted therapy came from the inhibition of the epidermal growth factor receptor (EGFR) pathway, by the anti-EGFR antibody, Cetuximab, or by specific tyrosine kinase inhibitors (TKI). Cetuximab, a mouse–human chimeric monoclonal antibody (mAb), binds to the extracellular domain of EGFR thus impairing EGFR-mediated signaling and reducing cell proliferation. TKI can affect the EGFR biochemical pathway at different steps along the signaling cascade. Apart from Cetuximab, other anti-EGFR mAbs have been developed, such as Panitumumab. Both antibodies have been approved for the treatment of KRAS-NRAS wild type mCRC, alone or in combination with chemotherapy. These antibodies display strong differences in activating the host immune system against CRC, due to their different immunoglobulin isotypes. Although anti-EGFR antibodies are efficient, drug resistance occurs with high frequency. Resistant tumor cell populations can either already be present before therapy or develop later by biochemical adaptations or new genomic mutations in the EGFR pathway. Numerous efforts have been made to improve the efficacy of the anti-EGFR mAbs or to find new agents that are able to block downstream EGFR signaling cascade molecules. Indeed, we examined the importance of analyzing the anti-EGFR antibody–drug conjugates (ADC) developed to overcome resistance and/or stimulate the tumor host’s immunity against CRC growth. Also, patient-derived CRC organoid cultures represent a useful and feasible in vitro model to study tumor behavior and therapy response. Organoids can reflect tumor genetic heterogeneity found in the tissue of origin, representing a unique tool for personalized medicine. Thus, CRC-derived organoid cultures are a smart model for studying the tumor microenvironment and for the preclinical assay of anti-EGFR drugs.
Antibody–Drug Conjugate Made of Zoledronic Acid and the Anti-CD30 Brentuximab–Vedotin Exert Anti-Lymphoma and Immunostimulating Effects Feliciana Morelli, Serena Matis, Roberto Benelli, Laura Salvini, Maria Raffaella Zocchi, et al. Cells, 2024 Relevant advances have been made in the management of relapsed/refractory (r/r) Hodgkin Lymphomas (HL) with the use of the anti-CD30 antibody–drug conjugate (ADC) brentuximab–vedotin (Bre–Ved). Unfortunately, most patients eventually progress despite the excellent response rates and tolerability. In this report, we describe an ADC composed of the aminobisphosphonate zoledronic acid (ZA) conjugated to Bre–Ved by binding the free amino groups of this antibody with the phosphoric group of ZA. Liquid chromatography–mass spectrometry, inductively coupled plasma–mass spectrometry, and matrix-assisted laser desorption ionization–mass spectrometry analyses confirmed the covalent linkage between the antibody and ZA. The novel ADC has been tested for its reactivity with the HL/CD30+ lymphoblastoid cell lines (KMH2, L428, L540, HS445, and RPMI6666), showing a better titration than native Bre–Ved. Once the HL-cells are entered, the ADC co-localizes with the lysosomal LAMP1 in the intracellular vesicles. Also, this ADC exerted a stronger anti-proliferative and pro-apoptotic (about one log fold) effect on HL-cell proliferation compared to the native antibody Bre–Ved. Eventually, Bre–Ved–ZA ADC, in contrast with the native antibody, can trigger the proliferation and activation of cytolytic activity of effector-memory Vδ2 T-lymphocytes against HL-cell lines. These findings may support the potential use of this ADC in the management of r/r HL.
EGFR-Targeted Antibody–Drug Conjugate to Different Aminobisphosphonates: Direct and Indirect Antitumor Effects on Colorectal Carcinoma Cells Leila Pisheh, Serena Matis, Martina Taglieri, Linda Di Gregorio, Roberto Benelli, et al. Cancers, 2024 Antibody––drug conjugates (ADCs) are a promising delivery system that involves linking a monoclonal antibody (mAb) to a specific drug, such as a cytotoxic agent, to target tumor cells. This new class of antitumor therapy acts as a “biological missile” that can destroy tumor cells while increasing the therapeutic index and decreasing toxicity. One of the most critical factors in ADC design is selecting a target antigen that is highly expressed on the surface of cancer cells. In this study, we conjugated Cetuximab (Cet), a monoclonal antibody that targets the epidermal growth factor receptor (EGFR), to aminobisphosphonates (N-BPs) such as ibandronate (IBA) or risedronate (RIS) or zoledronate (ZA). Cetuximab is administered to patients with metastatic colorectal carcinoma (mCRC) with a wild-type (WT) EGFR transduction pathway. Also, it is well established that N-BPs can trigger the antitumor activity of Vδ2 T cells in both in vitro and in vivo experimental models. The resulting ADCs were added in co-culture to assess the effect on CRC cell line proliferation and sensitivity to Vδ2 T antitumor lymphocytes in comparison with the native antibody. These assays have been performed both in conventional and 3D spheroid cultures. We found that all three ADCs can increase the inhibitory effect on cell proliferation of the WT-EGFR cell line Caco-2 while only Cet-RIS and Cet-ZA can increase the cytotoxicity mediated by Vδ2 T cells against both WT and EGFR-mutated CRC cell lines (Caco-2, DLD-1, and HCT-116). Also, the ADCs can trigger the cell proliferation of Vδ2 T cells present in peripheral blood and tumor specimens. Our findings indicate that anti-EGFR antibodies bound to N-BPs can improve the antitumor effects of the native antibody possibly increasing the therapeutic effect.
Prevalence of hybrid TLR4+M2 monocytes/macrophages in peripheral blood and lung of systemic sclerosis patients with interstitial lung disease Emanuele Gotelli, Stefano Soldano, Carol Feghali-Bostwick, Paola Montagna, Rosanna Campitiello, et al. Frontiers in Immunology, 2024 IntroductionSystemic sclerosis (SSc) is a complex autoimmune connective tissue disease characterized by microvascular damage, immune system reactivity and progressive fibrosis of skin and internal organs. Interstitial lung disease is the leading cause of death for SSc patients (SSc-ILD), and the process of lung fibrosis involves also circulating monocytes and alveolar macrophages.MethodsCurrent study aimed to identify monocyte/macrophage phenotypes in lung and peripheral blood of SSc-ILD patients by immunostaining and flow cytometry, respectively. Single immunostaining was performed using primary antibodies against CD68 (pan-macrophage marker), CD80, CD86, TLR4 (M1 markers), CD163, CD204, and CD206 (M2 markers). Flow cytometry analysis included the evaluation of CD45, CD14, CD16 (monocyte lineage), CD1c (dendritic lineage), together with M1 and M2 activation markers on circulating monocytes. Protein synthesis of TLR4 and M2 markers was also investigated in cultured monocytes-derived macrophages (MDMs) from SSc-ILD patients by Western Blotting.ResultsLung samples were obtained from 9 SSc-ILD patients (50 ± 9 years old) and 5 control non-SSc patients without lung fibrosis (58 ± 23 years old). Alveolar macrophages (CD68+ cells) showed a significantly higher positivity of M1 and M2 markers in SSc-ILD lung samples than in controls (p<0.05 for CD80, p<0.01 for CD86, p<0.001 for CD68, p<0.0001 for TLR4, CD163, CD204 and CD206). In CD68 positive areas of SSc-ILD samples, a significantly higher percentage of TLR4, CD163, CD204, and CD206 positive cells was observed compared to CD80 and CD86 positive cells (p<0.001 in both cases), suggesting the possible presence of hybrid TLR4+M2 macrophages (CD68+CD80-CD86-TLR4+CD163+CD204+CD206+cells) in SSc-ILD samples. A second cohort of 26 SSc-ILD patients (63 ± 14 years old) and 14 SSc patients without ILD (63 ± 19 years old) was recruited for flow cytometry analysis of circulating monocytes. Again, a significantly higher percentage of hybrid TLR4+M2 monocytes (CD1c-CD80-TLR4+CD163+CD204+CD206+cells) was found in SSc-ILD positive than SSc-ILD negative patients (p<0.05). Moreover, the protein synthesis of TLR4 and M2 markers was also found higher in cultured MDMs obtained from SSc-ILD patients than in MDMs from SSc patients without ILD and this increase was significantly higher for CD163 (p<0.05) and CD206 (p<0.01).ConclusionsThe presence of hybrid TLR4+M2 markers on both circulating monocytes and resident lung macrophages in SSc-ILD patients, is reported for the first time. Therefore, the detection of circulating hybrid TLR4+M2 monocytes in SSc-ILD might represent a further potential biomarker of progressive organ fibrosis, to be searched in blood samples of SSc patients.
Angiogenesis and cancer prevention: a vision. Douglas M. Noonan, Roberto Benelli, Adriana Albini Recent Results in Cancer Research Fortschritte Der Krebsforschung Progres Dans Les Recherches Sur Le Cancer, 2007
Erratum: In vitro models of angiogenesis: The use of Matrigel (The International Journal of Biological Markers (1999) 14, 4 (243-246)) International Journal of Biological Markers, 2000
Erratum: Inhibition of angiogenesis by type I interferons in models of Kaposi's sarcoma (The International Journal of Biological Markers (1999) 14, 4 (257-262)) International Journal of Biological Markers, 2000
HIV-1 Tat protein mimicry of chemokines Adriana Albini, Silvano Ferrini, Roberto Benelli, Sabrina Sforzini, Daniela Giunciuglio, et al. Proceedings of the National Academy of Sciences of the United States of America, 1998
Production of angiogenesis inhibitors and stimulators is modulated by cultured growth plate chondrocytes during in vitro differentiation: Dependence on extracellular matrix assembly European Journal of Cell Biology, 1995
Inhibition of AIDS-Kaposi's sarcoma cell induced endothelial cell invasion by TIMP-2 and a synthetic peptide from the metalloproteinase propeptide: Implications for an anti-angiogenic therapy Oncology Research, 1994
