Mauro Truini

Inhibition of angiogenesis and vascular tumor growth by interferon-producing cells: A gene therapy approach

We developed an in vivo gene therapy approach to characterize and optimize the anti-angiogenic activity of class I interferons (IFNs), using packaging cell lines producing an amphotropic LXSN-based retrovirus expressing either IFN-alpha1 (alpha1Am12), IFN-beta (betaAm12) murine cDNAs, or the vector alone (neoAm12). Pretreatment of endothelial-like Eahy926 cells in vitro with conditioned media (CM) from alpha1Am12 or betaAm12 cells for 48 hours significantly inhibited their migration and invasion as compared to neoAm12-CM-treated cells. betaAm12-CM also inhibited the formation of capillary-like structures on Matrigel by EAhy926 cells. In vivo, inclusion of the betaAm12 cells strongly inhibited, and alpha1Am12 partially inhibited, the angiogenic response in the Matrigel sponge model in both immune-competent and athymic nude mice. Electron microscopy showed a reduction of host cell infiltration in alpha1Am12- and betaAm12-containing sponges and reduction of invading tubular clefts of host cells as compared to controls. Finally, inoculation of either alpha1Am12 or betaAm12 cells (10%) along with a highly angiogenic Kaposis sarcoma cell line (90%) resulted in a powerful reduction of tumor growth in nude mice in vivo, as did infection with the interferon-alpha-producing retroviruses. These data suggest that a gene therapy approach using class I interferons can effectively inhibit tumor angiogenesis and growth of vascular tumors.

Tumor heterogeneity and lesion-specific response to targeted therapy in colorectal cancer

UNLABELLED How genomic heterogeneity associated with acquired resistance to targeted agents affects response to subsequent therapy is unknown. We studied EGFR blockade in colorectal cancer to assess whether tissue and liquid biopsies can be integrated with radiologic imaging to monitor the impact of individual oncogenic alterations on lesion-specific responses. Biopsy of a patients progressing liver metastasis following prolonged response to cetuximab revealed a MEK1(K57T) mutation as a novel mechanism of acquired resistance. This lesion regressed upon treatment with panitumumab and the MEK inhibitor trametinib. In circulating tumor DNA (ctDNA), mutant MEK1 levels declined with treatment, but a previously unrecognized KRAS(Q61H) mutation was also identified that increased despite therapy. This same KRAS mutation was later found in a separate nonresponding metastasis. In summary, parallel analyses of tumor biopsies and serial ctDNA monitoring show that lesion-specific radiographic responses to subsequent targeted therapies can be driven by distinct resistance mechanisms arising within separate tumor lesions in the same patient. SIGNIFICANCE Molecular heterogeneity ensuing from acquired resistance drives lesion-specific responses to subsequent targeted therapies. Analysis of a single-lesion biopsy is inadequate to guide selection of subsequent targeted therapies. ctDNA profiles allow the detection of concomitant resistance mechanisms residing in separate metastases and assessment of the effect of therapies designed to overcome resistance.

Development of sarcomas in mice implanted with mesenchymal stem cells seeded onto bioscaffolds

Bone marrow-derived mesenchymal stem cells (MSCs) are precursors of bone, cartilage and fat tissue. MSC can also regulate the immune response. For these properties, they are tested in clinical trials for tissue repair in combination with bioscaffolds or injected as cell suspension for immunosuppressant therapy. Experimental data, however, indicate that MSC can undergo or induce a tumorigenic process in determined circumstances. We used a modified model of ectopic bone formation in mice by subcutaneously implanting porous ceramic seeded with murine MSC. In this new model, host-derived sarcomas developed when we implanted MSC/bioscaffold constructs into syngeneic and immunodeficient recipients, but not in allogeneic hosts or when MSCs were injected as cell suspensions. The bioscaffold provided a tridimensional support for MSC to aggregate, thus producing the stimulus for triggering the process eventually leading to the transformation of surrounding cells and creating a surrogate tumor stroma. The chemical and physical characteristics of the bioscaffold did not affect tumor formation; sarcomas developed either when a stiff porous ceramic was used or when the scaffold was a smooth collagen sponge. The immunoregulatory function of MSC contributed to tumor development. Implanted MSC expanded clones of CD4+CD25+ T regulatory lymphocytes that suppressed hosts antitumor immune response.

Recruitment of a Host's Osteoprogenitor Cells Using Exogenous Mesenchymal Stem Cells Seeded on Porous Ceramic

The contribution of the hosts circulating progenitor cells after implantation of mesenchymal stem cells (MSC)/bioscaffold combinations for repairing bone defects has not been elucidated, although this issue affects the clinical application of the tissue engineering approach. We implanted blocks of hydroxyapatite loaded with murine MSCs into syngenic, allogenic, and immunocompromised recipients. After 8 weeks, we found that bone tissue was formed in syngenic and immunocompromised animals. The implanted cells appeared pivotal in the early stages of tissue development, but cells of the recipients origin finally made bone. In this system, osteoprogenitors migrated from the recipient to the implant, whereas the implanted cells left the scaffold and entered the circulatory flow. We observed rapid destruction of implanted cells when allogenic MSC/bioscaffold combinations were grafted onto immunocompetent recipients without immunosuppressant therapy. This destruction blocked the recruitment process and did not allow the formation of new bone tissue. The possibility that the implanted exogenous MSCs could engage the hosts osteoprogenitor cells to form new bone tissue could open new perspectives for the tissue engineering approach to bone repair, including the opportunity of using allogenic cells combined with a temporary immunosuppressant therapy, stimulating the replacement of the exogenous cells with autologous cells.

IL-8 induces exocytosis of arginase 1 by neutrophil polymorphonuclears in nonsmall cell lung cancer.

Arginase 1 (ARG1) inhibits T‐cell proliferation by degrading extracellular arginine, which results in decreased responsiveness of T cells to CD3/TCR stimulation. In humans, ARG1 is stored in inactive form within granules of polymorphonuclear neutrophils (PMNs) and gets activated on release. We studied the role of PMNs‐related ARG1 activity in nonsmall cell lung cancer (NSLC), in which tumor‐infiltrating lymphocytes showed reduced proliferation in response to CD3/TCR triggering. Patients with NSCLC had increased ARG1 plasma levels as compared to healthy controls. Furthermore, immunohistochemistry showed that tumor‐infiltrating PMNs display reduced intracellular ARG1, in comparison to intravascular or peritumoral PMNs, suggesting a role of tumor microenvironment in ARG1 release. Indeed, supernatants of NSCLC cell lines induced exocytosis of ARG1 from PMNs. All (4/4) NSCLC cell lines and all (7/7) CD14− cell samples from NSCLC expressed interleukin (IL)‐8 mRNA, whereas TNFα mRNA was expressed by 1 cell line and by 2 tumor specimens. Furthermore, all NSCLC cell lines secreted immunoreactive IL‐8, albeit at different levels. IL‐8 was as effective as TNFα in triggering ARG1 release and the 2 cytokines acted synergistically. Secreted ARG1 was biologically active and catabolized extracellular arginine. The supernatant of IL‐8 gene‐silenced NSCLC cells did not mediate ARG1 release by PMNs. Altogether these findings demonstrate a role of IL‐8 in ARG1 exocytosis by PMNs and indicate that, due at least in part to IL‐8 secreted by NSCLC cells, PMNs infiltrating NSCLC release ARG1. This phenomenon could contribute to local immune suppression.

NCR + ILC3 concentrate in human lung cancer and associate with intratumoral lymphoid structures

Tertiary lymphoid structures (TLSs) are a common finding in non-small cell lung cancer (NSCLC) and are predictors of favourable clinical outcome. Here we show that NCR(+) innate lymphoid cell (ILC)-3 are present in the lymphoid infiltrate of human NSCLC and are mainly localized at the edge of tumour-associated TLSs. This intra-tumoral lymphocyte subset is endowed with lymphoid tissue-inducing properties and, on activation, produces IL-22, TNF-α, IL-8 and IL-2, and activates endothelial cells. Tumour NCR(+)ILC3 may interact with both lung tumour cells and tumour-associated fibroblasts, resulting in the release of cytokines primarily on engagement of the NKp44-activating receptor. In patients, NCR(+)ILC3 are present in significantly higher amounts in stage I/II NSCLC than in more advanced tumour stages and their presence correlate with the density of intratumoral TLSs. Our results indicate that NCR(+)ILC3 accumulate in human NSCLC tissue and might contribute to the formation of protective tumour-associated TLSs.

Direct inhibition of hexokinase activity by metformin at least partially impairs glucose metabolism and tumor growth in experimental breast cancer

Emerging evidence suggests that metformin, a widely used anti-diabetic drug, may be useful in the prevention and treatment of different cancers. In the present study, we demonstrate that metformin directly inhibits the enzymatic function of hexokinase (HK) I and II in a cell line of triple-negative breast cancer (MDA-MB-231). The inhibition is selective for these isoforms, as documented by experiments with purified HK I and II as well as with cell lysates. Measurements of 18F-fluoro-deoxyglycose uptake document that it is dose- and time-dependent and powerful enough to virtually abolish glucose consumption despite unchanged availability of membrane glucose transporters. The profound energetic imbalance activates phosphorylation and is subsequently followed by cell death. More importantly, the “in vivo” relevance of this effect is confirmed by studies of orthotopic xenografts of MDA-MB-231 cells in athymic (nu/nu) mice. Administration of high drug doses after tumor development caused an evident tumor necrosis in a time as short as 48 h. On the other hand, 1 mo metformin treatment markedly reduced cancer glucose consumption and growth. Taken together, our results strongly suggest that HK inhibition contributes to metformin therapeutic and preventive potential in breast cancer.

Transcriptome Meta-Analysis of Lung Cancer Reveals Recurrent Aberrations in NRG1 and Hippo Pathway Genes

Lung cancer is emerging as a paradigm for disease molecular subtyping, facilitating targeted therapy based on driving somatic alterations. Here, we perform transcriptome analysis of 153 samples representing lung adenocarcinomas, squamous cell carcinomas, large cell lung cancer, adenoid cystic carcinomas and cell lines. By integrating our data with The Cancer Genome Atlas and published sources, we analyze 753 lung cancer samples for gene fusions and other transcriptomic alterations. We show that higher numbers of gene fusions is an independent prognostic factor for poor survival in lung cancer. Our analysis confirms the recently reported CD74-NRG1 fusion and suggests that NRG1, NF1 and Hippo pathway fusions may play important roles in tumors without known driver mutations. In addition, we observe exon skipping events in c-MET, which are attributable to splice site mutations. These classes of genetic aberrations may play a significant role in the genesis of lung cancers lacking known driver mutations.

Identification of low intratumoral gene expression heterogeneity in neuroblastic tumors by genome-wide expression analysis and game theory

Neuroblastic tumors (NTs) are largely comprised of neuroblastic (Nb) cells with various quantities of Schwannian stromal (SS) cells. NTs show a variable genetic heterogeneity. NT gene expression profiles reported so far have not taken into account the cellular components. The authors reported the genome‐wide expression analysis of whole tumors and microdissected Nb and SS cells.

Assessment of a HER2 scoring system for colorectal cancer: Results from a validation study

We sought to develop criteria for ERBB2-positivity (HER2) in colorectal cancer to ensure accurate identification of ERBB2-amplified metastatic colorectal cancer patients suitable for enrolment in a phase II trial of ERBB2-targeted therapy (HERACLES trial). A two-step approach was used. In step 1, a consensus panel of pathologists adapted existing protocols for use in colorectal cancer to test ERBB2 expression and amplification. Collegial revision of an archival test cohort of colorectal cancer samples led to specific recommendations for adapting current breast and gastric cancer criteria for scoring ERBB2 in colorectal cancer. In step 2, from September 2012 to January 2015, colorectal-specific ERBB2 testing protocols and ERBB2 scoring criteria were used to centrally screen for ERBB2-positive KRAS wild-type colorectal cancer patients to be enrolled in the HERACLES trial (clinical validation cohort). In both archival test (N=256) and clinical validation (N=830) cohorts, a clinically sizeable 5% fraction of KRAS wild-type colorectal cancer patients was found to be ERBB2-positive according to the colorectal cancer-specific ERBB2 scoring criteria. ERBB2-positive tumors showed ERBB2 immunostaining consisting of intense membranous ERBB2 protein expression, corresponding to homogenous ERBB2 amplification, in >50% of cells. None of the immunohistochemistry 0 or 1+ cases was amplified. Concordance between SISH and FISH was 100%. In conclusion, we propose specific criteria for defining ERBB2-positivity in colorectal cancer (HERACLES Diagnostic Criteria). In a phase II trial of trastuzumab and lapatinib in a cetuximab-resistant population, HERACLES Diagnostic Criteria shaped the selection of patients and defined ERBB2 as a predictive marker for response to ERBB2-targeted therapy in metastatic colorectal cancer.