Direct CD32 T-cell cytotoxicity: implications for breast cancer prognosis and treatment

The FcγRII (CD32) ligands are IgFc fragments and pentraxins. The existence of additional ligands is unknown. We engineered T cells with human chimeric receptors resulting from the fusion between CD32 extracellular portion and transmembrane CD8α linked toCD28/ζ chain intracellular moiety (CD32-CR). Transduced T cells recognized three breast cancer (BC) and one colon cancer cell line among 15 tested in the absence of targeting antibodies. Sensitive BC cell conjugation with CD32-CR T cells induced CD32 polarization and down-regulation, CD107a release, mutual elimination, and proinflammatory cytokine production unaffected by human IgGs but enhanced by cetuximab. CD32-CR T cells protected immunodeficient mice from subcutaneous growth of MDA-MB-468 BC cells. RNAseq analysis identified a 42 gene fingerprint predicting BC cell sensitivity and favorable outcomes in advanced BC. ICAM1 was a major regulator of CD32-CR T cell–mediated cytotoxicity. CD32-CR T cells may help identify cell surface CD32 ligand(s) and novel prognostically relevant transcriptomic signatures and develop innovative BC treatments

Enhancement of anti-leukemia activity of NK cells in vitro and in vivo by inhibition of leukemia cell-induced NK cell damage

Acute myeloid leukemia (AML) cells induce, in vitro, NK cell abnormalities (NKCAs) including apoptosis and activating receptor down-regulation. The potential negative impact of AML cells on the therapeutic efficacy of NK cell-based strategies prompted us to analyze the mechanisms underlying NKCAs and to develop approaches to protect NK cells from NKCAs. NKCA induction by the AML leukemia cells target a subpopulation of peripheral blood NK cells and is interleukin-2 independent but is abrogated by a long-term culture of NK (LTNK) cells at 37°C. LTNK cells displayed a significantly enhanced ability to damage AML cells in vitro and inhibited the subcutaneous growth of ML-2 cells grafted into CB17 SCID mice. Actinomycin D restored the susceptibility of LTNK cells to NKCAs while TAPI-0, a functional analog of the tissue inhibitor of metalloproteinase (TIMP) 3, inhibits ML-2 cell-induced NKCAs suggesting that the generation of NK cell resistance to NKCAs involves RNA transcription and metalloproteinase (MPP) inactivation. This conclusion is supported by the reduced susceptibility to AML cell-induced NKCAs of LTNK cells in which TIMP3 gene and protein are over-expressed. This information may contribute to the rational design of targeted strategies to enhance the efficacy of NK cell-based-immunotherapy of AML with haploidentical NK cells

Natural humoral immune response to ribosomal P0 protein in colorectal cancer patients

Tumor associated antigens are useful in colorectal cancer (CRC) management. The ribosomal P proteins (P0, P1, P2) play an important role in protein synthesis and tumor formation. The immunogenicity of the ribosomal P0 protein in head and neck, in breast and prostate cancer patients and the overexpression of the carboxyl-terminal P0 epitope (C-22 P0) in some tumors were reported

Potenziamento dell’efficacia del cetuximab mediante combinazione con cellule T ingegnerizzate con recettori Fc chimerici per il trattamento del carcinoma del colon retto K-ras mutato.

INTRODUZIONE: L’impiego di anticorpi monoclonali (AM) diretti contro il recettore per il fattore di crescita epidermico (EGFR), tra cui il cetuximab, ha rappresentato un significativo avanzamento nel trattamento del carcinoma colorettale metastatico (mCRC). L’effetto anti-tumorale del cetuximab è dovuto a: i) blocco della proliferazione neoplastica; ii) attivazione di processi apoptotici; iii) induzione di citotossicità cellulare dipendente da anticorpo (ADCC) mediante il legame con i recettori Fc. Tuttavia, diversi fattori limitano l’efficacia terapeutica del cetuximab; tra questi la presenza di mutazioni nel gene Kras (Kras-mut) e la scarsa presenza di cellule Natural Killer, capaci di mediare ADCC, nel microambiente tumorale. In questo studio ipotizziamo di superare i limiti terapeutici del cetuximab attraverso la sua combinazione con cellule T ingegnerizzate con recettori chimerici Fc (Fc-CRs). MATERIALI E METODI: Due Fc-CRs, denominati CD32-CR e CD16-CR, sono stati generati dalla fusione della regione extracellulare del FcRIIA (CD32) o del FcRIIIA (CD16) con il dominio transmembrana del CD8a e con i domini intracitoplasmatici del CD28 e del CD3. Linfociti T attivati sono stati trasdotti con le chimere e la corretta espressione è stata valutata mediante citofluorimetria e western blot. I recettori sono stati caratterizzati funzionalmente in vitro attraverso test di binding e saggi ELISA. L’attività anti-tumorale delle cellule T trasdotte in associazione al cetuximab è stata valutata in test di deplezione in vitro e in topi SCID xenotrapiantati con la linea cellulare di CRC Kras-mut HCT116. RISULTATI: Entrambi i Fc-CRs sono espressi correttamente sulla superficie delle cellule T. Il CD32-CR, ma non il CD16-CR, lega in maniera specifica il frammento Fc degli AM anti-EGFR cetuximab e panitumumab. La dose massima di cetuximab necessaria per saturare tutti i CD32-CR che è pari a 10g/ml ed il binding è mantenuto anche in presenza di quantità crescenti di plasma umano. Sia il CD32-CR che il CD16-CR inducono rilascio di citochine in risposta a stimolazione con cetuximab o panitumumab. La combinazione del cetuximab con cellule T CD32-CR+: i) induce l’eliminazione di HCT116 in vitro; ii) riduce significativamente la crescita tumorale in vivo. CONCLUSIONI: In questo studio preclinico dimostriamo che cellule T armate con CD32-CR potenziano l’efficacia del cetuximab contro CRC Kras-mutato

Recent perspective on CAR and Fcγ-CR T cell immunotherapy for cancers: Preclinical evidence versus clinical outcomes.

The chimeric antigen receptor Tcell (CAR-T cell) immunotherapy currently represents a hot research trend and it is expected to revolutionize the field of cancer therapy. Promising outcomes have been achieved using CAR-T cell therapy for haematological malignancies. Despite encouraging results, several challenges still pose eminent hurdles before being fully recognized. Directing CAR-T cells to target a single tumour associated antigen (TAA) as the case in haematological malignancies might be much simpler than targeting the extensive inhibitory microenvironments associated with solid tumours. This review focuses on the basic principles involved in development of CAR-T cells, emphasizing the differences between humoral IgG, T-cell receptors, CAR and Fcγ-CR constructs. It also highlights the complex inhibitory network that is usually associated with solid tumours, and tackles recent advances in the clinical studies that have provided great hope for the future use of CAR-T cell immunotherapy. While current Fcγ-CR T cell immunotherapy is in pre-clinical stage, is expected to provide a sound therapeutic approach to add to existing classical chemo- and radio-therapeutic modalities

Aspirin inhibits cancer stem cells properties and growth of glioblastoma multiforme through Rb1 pathway modulation.

Several clinical studies indicated that the daily use of aspirin or acetylsalicylic acid reduces the cancer risk via cyclooxygenases (Cox-1 and Cox-2) inhibition. In addition, aspirin-induced Cox-dependent and -independent antitumor effects have also been described. Here we report, for the first time, that aspirin treatment of human glioblastoma cancer (GBM) stem cells, a small population responsible for tumor progression and recurrence, is associated with reduced cell proliferation and motility. Aspirin did not interfere with cell viability but induced cell-cycle arrest. Exogenous prostaglandin E significantly increased cell proliferation but did not abrogate the aspirin-mediated growth inhibition, suggesting a Cox-independent mechanism. These effects appear to be mediated by the increase of p21 and p27 , associated with a reduction of Cyclin D1 and Rb1 protein phosphorylation, and involve the downregulation of key molecules responsible for tumor development, that is, Notch1, Sox2, Stat3, and Survivin. Our results support a possible role of aspirin as adjunctive therapy in the clinical management of GBM patients

A possible interplay between HR-HPV and stemness in tumor development: an in vivo investigation of CD133 as a putative marker of cancer stem cell in HPV18-infected KB cell line

High-risk HPVs (HR-HPVs) are DNA viruses considered as primary etiologic factors in malignancies of the low female genital tract. Their presence has also been documented in oropharyngeal and laryngeal cancers. However, HPV infection is considered a necessary but not sufficient cause of tumoral development; meantime, increasing evidences on the tumorigenic role of cancer stem cells (CSCs) have been documented in the literature. CSCs represent a small subpopulation of neoplastic cells with self-renewal potential, capable of maintaining tumor growth and cell differentiation, also involved in metastatic process, recurrence, and resistance to chemotherapeutic agents. In the present study, performed on KB cell lines, we evaluated the tumor forming potential of CSCs, and their relationship with the HPV infection status. We started our study by identifying the most aggressive cell line on the minimal number of cells being able of growth in vivo in a model of athymic nude mice (BALB/c nu/nu). We used an oral-derived KB cell line separated in the KB-CD133+ and KB-CD133- populations, by using immunomagnetic beads and fluorescence-activated cell sorting (FACS). The separated populations were injected in athymic nude mice (BALB/c nu/nu). Xenograft tumors have been analyzed for tumor size, CD133 expression by immunohistochemistry (IHC) and for DNA HR-HPV integration by in situ hybridization (ISH), comparing CD133-enriched xenograft tumors versus the CD133 non-enriched ones. On standard conditions, the KB cell line has a poor population of glycosylated CD133 marker (<5.0%) when investigated with antibodies versus CD133, and more specifically its glycosylated epitope (AC133). Enriched CD133 KB cells possess a higher capacity of tumor growth in xenograft models of nude mice when compared to KB CD133-negative cells. We observed that the AC133 epitope, extensively used to purifying hematopoietic stem cells, is able to select an epithelial subpopulation of cancer stem cells with aggressive behavior. We retain that CD133 may be a useful target in anticancer strategies including pharmacological and immunological therapies