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Background:Pazopanib achieved the end point of clinical activity in pretreated patients with urothelial cancer in a single-group, phase 2 trial. The objective was to identify biological predictors of clinical benefit to pazopanib in these patients.Methods:EDTA blood samples were collected at baseline (T0) and after 4 weeks (T1) of treatment, together with radiological imaging in all 41 patients to analyse plasma circulating angiogenic factor levels by multiplex ELISA plates. Changes from T0 to T1 in marker levels were matched with response with the covariance analysis. Univariable and multivariable analyses evaluated the association with overall survival (OS), adjusted for prespecified clinical variables. Net reclassification improvement (NRI) tested the performance of the recognised Cox model.Results:Increasing IL8T1 level associated with lower response probability at covariance analysis (P=0.010). Both IL8T0 (P=0.019) and IL8T1 (P=0.004) associated with OS and the prognostic model, including clinical variables and IL8T1 best-predicted OS after backward selection. The NRI for this model was 39%.When analysed as a time-varying covariate, IL8T1 level<80 pg ml−1 portended significantly greater response (∼80%) and 6-month OS (∼60%) probability than level80.Conclusion:IL8-level changes during pazopanib allowed for a prognostic improvement and were associated with response probability. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is caused by the absence of the full form of the dystrophin protein, which is essential for maintaining the structural integrity of muscle cells, including those in the heart and respiratory system. Despite progress in understanding the molecular mechanisms associated with DMD, myocardial insufficiency persists as the primary cause of mortality, and existing therapeutic strategies remain limited. This study investigates the hypothesis that a dysregulation of the biological communication between infiltrating macrophages (MPs) and neurocardiac junctions exists in dystrophic cardiac tissue. In a mouse model of DMD (mdx), this phenomenon is influenced by the over-release of chondroitin sulfate proteoglycan-4 (CSPG4), a key inhibitor of nerve sprouting and a modulator of the neural function, by MPs infiltrating the cardiac tissue and associated with dilated cardiomyopathy, a hallmark of DMD. Givinostat, the histone deacetylase inhibitor under current development as a clinical treatment for DMD, is effective at both restoring a physiological microenvironment at the neuro-cardiac junction and cardiac function in mdx mice in addition to a reduction in cardiac fibrosis, MP-mediated inflammation, and tissue CSPG4 content. This study provides novel insight into the pathophysiology of DMD in the heart, identifying potential new biological targets. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

CD28 and 4-1BB costimulatory endodomains included in chimeric antigen receptor (CAR) molecules play a critical role in promoting sustained antitumor activity of CAR-T cells. However, the molecular events associated with the ectopic and constitutive display of either CD28 or 4-1BB in CAR-T cells have been only partially explored. In the current study, we demonstrated that 4-1BB incorporated within the CAR leads to cell cluster formation and cell death in the forms of both apoptosis and necroptosis in the absence of CAR tonic signaling. Mechanistic studies illustrate that 4-1BB sequesters A20 to the cell membrane in a TRAF-dependent manner causing A20 functional deficiency that in turn leads to NF-κB hyperactivity, cell aggregation via ICAM-1 overexpression, and cell death including necroptosis via RIPK1/RIPK3/MLKL pathway. Genetic modulations obtained by either overexpressing A20 or releasing A20 from 4-1BB by deleting the TRAF-binding motifs of 4-1BB rescue cell cluster formation and cell death and enhance the antitumor ability of 4-1BB-costimulated CAR-T cells., (© 2024. The Author(s).)

Human natural killer T cells (NKTs) are innate-like T lymphocytes increasingly used for cancer immunotherapy. Here we show that human NKTs expressing the pro-inflammatory cytokine interleukin-12 (IL-12) undergo extensive and sustained molecular and functional reprogramming. Specifically, IL-12 instructs and maintains a Th1-polarization program in NKTs in vivo without causing their functional exhaustion. Furthermore, using CD62L as a marker of memory cells in human NKTs, we observe that IL-12 maintains long-term CD62L-expressing memory NKTs in vivo. Notably, IL-12 initiates a de novo programming of memory NKTs in CD62L-negative NKTs indicating that human NKTs circulating in the peripheral blood possess an intrinsic differentiation hierarchy, and that IL-12 plays a role in promoting their differentiation to long-lived Th1-polarized memory cells. Human NKTs engineered to co-express a Chimeric Antigen Receptor (CAR) coupled with the expression of IL-12 show enhanced antitumor activity in leukemia and neuroblastoma tumor models, persist long-term in vivo and conserve the molecular signature driven by the IL-12 expression. Thus IL-12 reveals an intrinsic plasticity of peripheral human NKTs that may play a crucial role in the development of cell therapeutics., (© 2024. The Author(s).)

Selection of the best tumor antigen is critical for the therapeutic success of chimeric antigen receptor (CAR) T cells in hematologic malignancies and solid tumors. The anaplastic lymphoma kinase (ALK) receptor is expressed by most neuroblastomas while virtually absent in most normal tissues. ALK is an oncogenic driver in neuroblastoma and ALK inhibitors show promising clinical activity. Here, we describe the development of ALK.CAR-T cells that show potent efficacy in monotherapy against neuroblastoma with high ALK expression without toxicity. For neuroblastoma with low ALK expression, combination with ALK inhibitors specifically potentiates ALK.CAR-T cells but not GD2.CAR-T cells. Mechanistically, ALK inhibitors impair tumor growth and upregulate the expression of ALK, thereby facilitating the activity of ALK.CAR-T cells against neuroblastoma. Thus, while neither ALK inhibitors nor ALK.CAR-T cells will likely be sufficient as monotherapy in neuroblastoma with low ALK density, their combination specifically enhances therapeutic efficacy., Competing Interests: Declaration of interests D.A. is an employee of Celldex Therapeutics, a company that developed anti-ALK antibodies for therapeutic applications. E.B., W.-T.T., and R.C. filed a patent related to this work covering the development of ALK.CAR-T cells., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (© 2023. The Author(s).)

Chimeric antigen receptor (CAR)-modified T-cell therapy has shown enormous clinical promise against blood cancers, yet efficacy against solid tumors remains a challenge. Here, we investigated the potential of a new combination cell therapy, where tumor-homing induced neural stem cells (iNSCs) are used to enhance CAR-T-cell therapy and achieve efficacious suppression of brain tumors. Using in vitro and in vivo migration assays, we found iNSC-secreted RANTES/IL-15 increased CAR-T-cell migration sixfold and expansion threefold, resulting in greater antitumor activity in a glioblastoma (GBM) tumor model. Furthermore, multimodal imaging showed iNSC delivery of RANTES/IL-15 in combination with intravenous administration of CAR-T cells reduced established orthotopic GBM xenografts 2538-fold within the first week, followed by durable tumor remission through 60 days post-treatment. By contrast, CAR-T-cell therapy alone only partially controlled tumor growth, with a median survival of only 19 days. Together, these studies demonstrate the potential of combined cell therapy platforms to improve the efficacy of CAR-T-cell therapy for brain tumors., Competing Interests: Shawn D. Hingtgen has an ownership interest in Falcon Therapeutics, Inc., which has licensed aspects of iNSC technology from UNC‐CH. All remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

Chimeric antigen receptor (CAR) T cells showed great activity in hematologic malignancies. However, heterogeneous antigen expression in tumor cells and suboptimal CAR-T cell persistence remain critical aspects to achieve clinical responses in patients with solid tumors. Here we show that CAR-T cells targeting simultaneously two tumor-associated antigens and providing transacting CD28 and 4-1BB costimulation, while sharing the sane CD3ζ-chain cause rapid antitumor effects in in vivo stress conditions, protection from tumor re-challenge and prevention of tumor escape due to low antigen density. Molecular and signaling studies indicate that T cells engineered with the proposed CAR design demonstrate sustained phosphorylation of T cell receptor-associated (TCR) signaling molecules and a molecular signature supporting CAR-T cell proliferation and long-term survival. Furthermore, metabolic profiling of CAR-T cells displayed induction of glycolysis that sustains rapid effector T cell function, but also preservation of oxidative functions, which are critical for T cell long-term persistence., Competing Interests: Reporting Summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Background: Chimeric antigen receptor (CAR) T cells have shown considerable promise as a personalized cellular immunotherapy against B cell malignancies. However, the complex and lengthy manufacturing processes involved in generating CAR T cell products ex vivo result in substantial production time delays and high costs. Furthermore, ex vivo expansion of T cells promotes cell differentiation that reduces their in vivo replicative capacity and longevity., Methods: Here, to overcome these limitations, CAR-T cells are engineered directly in vivo by administering a lentivirus expressing a mutant Sindbis envelope, coupled with a bispecific antibody binder that redirects the virus to CD3 + human T cells., Results: This redirected lentiviral system offers exceptional specificity and efficiency; a single dose of the virus delivered to immunodeficient mice engrafted with human peripheral blood mononuclear cells generates CD19-specific CAR-T cells that markedly control the growth of an aggressive pre-established xenograft B cell tumor., Conclusions: These findings underscore in vivo engineering of CAR-T cells as a promising approach for personalized cancer immunotherapy., Competing Interests: Competing interests: TMJ has an equity interest in Dualogics LLC, a company aimed at commercializing bispecific antibodies., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation., (©2021 American Association for Cancer Research.)

Purpose: No effective therapy is available for unresectable soft-tissue sarcomas (STS). This unmet clinical need prompted us to test whether chondroitin sulfate proteoglycan 4 (CSPG4)-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CAR.CIK) are effective in eliminating tumor cells derived from multiple STS histotypes in vitro and in immunodeficient mice., Experimental Design: The experimental platform included patient-derived CAR.CIK and cell lines established from multiple STS histotypes. CAR.CIK were transduced with a retroviral vector encoding second-generation CSPG4-specific CAR (CSPG4-CAR) with 4-1BB costimulation. The functional activity of CSPG4-CAR.CIK was explored in vitro , in two- and three-dimensional STS cultures, and in three in vivo STS xenograft models., Results: CSPG4-CAR.CIK were efficiently generated from patients with STS. CSPG4 was highly expressed in multiple STS histotypes by in silico analysis and on all 16 STS cell lines tested by flow cytometry. CSPG4-CAR.CIK displayed superior in vitro cytolytic activity against multiple STS histotypes as compared with paired unmodified control CIK. CSPG4-CAR.CIK also showed strong antitumor activity against STS spheroids; this effect was associated with tumor recruitment, infiltration, and matrix penetration. CSPG4-CAR.CIK significantly delayed or reversed tumor growth in vivo in three STS xenograft models (leiomyosarcoma, undifferentiated pleomorphic sarcoma, and fibrosarcoma). Tumor growth inhibition persisted for up to 2 weeks following the last administration of CSPG4-CAR.CIK., Conclusions: This study has shown that CSPG4-CAR.CIK effectively targets multiple STS histotypes in vitro and in immunodeficient mice. These results provide a strong rationale to translate the novel strategy we have developed into a clinical setting., (©2020 American Association for Cancer Research.)

Retinoblastoma (RB) is a pediatric retinal tumor that overexpresses the ganglioside GD2. Although it is treatable in patients with early diagnosis, patients may lose one or two eyes. We generated GD2-specific chimeric antigen receptor T lymphocytes (GD2.CAR-Ts) and locally delivered them to mice with an in-situ grafting RB. When used in combination with the local release of interleukin (IL)-15 and an injectable hydrogel, we showed that GD2.CAR-Ts successfully eliminate RB tumor cells without impairment of the mouse vision., Competing Interests: Competing Interests statement The authors declare no conflicts of interest.

Chimeric antigen receptor (CAR) T-cell therapy has been acclaimed as a revolution in cancer treatment following the impressive results in hematologic malignancies. Unfortunately, in patients with solid tumors, objectives responses to CAR T cells are still anecdotal, and important issues are driven by on-target but off-tumor activity of CAR T cells and by the extremely complex biology of solid tumors. Here, we will review the recent attempts to challenge the therapeutic impediments to CAR T-cell therapy in solid tumors. We will focus on the most promising strategies of antigen targeting to improve tumor specificity and address the tumor heterogeneity, efforts to circumvent the physical barriers of the tumor architecture such as subverted tumor vasculature, impediments of CAR T-cell trafficking and immune suppressive microenvironment., (©2020 American Association for Cancer Research.)

Cetuximab and panitumumab bind the human epidermal growth factor receptor (EGFR). Although the chimeric cetuximab (IgG1) triggers antibody-dependent-cellular-cytotoxicity (ADCC) of EGFR positive target cells, panitumumab (a human IgG2) does not. The inability of panitumumab to trigger ADCC reflects the poor binding affinity of human IgG2 Fc for the FcγRIII (CD16) on natural killer (NK) cells. However, both human IgG1 and IgG2 bind the FcγRII (CD32A) to a similar extent. Our study compares the ability of T cells, engineered with a novel low-affinity CD32A 131R -chimeric receptor (CR), and those engineered with the low-affinity CD16 158F -CR T cells, in eliminating EGFR positive epithelial cancer cells (ECCs) in combination with cetuximab or panitumumab. After T-cell transduction, the percentage of CD32A 131R -CR T cells was 74 ± 10%, whereas the percentage of CD16 158F -CR T cells was 46 ± 15%. Only CD32A 131R -CR T cells bound panitumumab. CD32A 131R -CR T cells combined with the mAb 8.26 (anti-CD32) and CD16 158F -CR T cells combined with the mAb 3g8 (anti-CD16) eliminated colorectal carcinoma (CRC), HCT116 FcγR+ cells, in a reverse ADCC assay in vitro. Crosslinking of CD32A 131R -CR on T cells by cetuximab or panitumumab and CD16 158F -CR T cells by cetuximab induced elimination of triple negative breast cancer (TNBC) MDA-MB-468 cells, and the secretion of interferon gamma and tumor necrosis factor alpha. Neither cetuximab nor panitumumab induced Fcγ-CR T antitumor activity against Kirsten rat sarcoma (KRAS)-mutated HCT116, nonsmall-cell-lung-cancer, A549 and TNBC, MDA-MB-231 cells. The ADCC of Fcγ-CR T cells was associated with the overexpression of EGFR on ECCs. In conclusion, CD32A 131R -CR T cells are efficiently redirected by cetuximab or panitumumab against breast cancer cells overexpressing EGFR., (© 2019 UICC.)

T-cell receptor (TCR) gene transfer redirects T cells to target intracellular antigens. However, the potential autoreactivity generated by TCR mispairing and occurrence of graft-versus-host disease in the allogenic setting due to the retention of native TCRs remain major concerns. Natural killer T cells (NKT) have shown promise as a platform for adoptive T-cell therapy in cancer patients. Here, we showed their utility for TCR gene transfer. We successfully engineered and expanded NKTs expressing a functional TCR (TCR NKTs), showing HLA-restricted antitumor activity in xenogeneic mouse models in the absence of graft-versus-mouse reactions. We found that TCR NKTs downregulated the invariant TCR (iTCR), leading to iTCR + TCR + and iTCR - TCR + populations. In-depth analyses of these subsets revealed that in iTCR - TCR + NKTs, the iTCR, although expressed at the mRNA and protein levels, was retained in the cytoplasm. This effect resulted from a competition for binding to CD3 molecules for cell-surface expression by the transgenic TCR. Overall, our results highlight the feasibility and advantages of using NKTs for TCR expression for adoptive cell immunotherapies. NKT-low intrinsic alloreactivity that associated with the observed iTCR displacement by the engineered TCR represents ideal characteristics for "off-the-shelf" products without further TCR gene editing., (©2019 American Association for Cancer Research.)

Current tumor neoantigen calling algorithms primarily rely on epitope/major histocompatibility complex (MHC) binding affinity predictions to rank and select for potential epitope targets. These algorithms do not predict for epitope immunogenicity using approaches modeled from tumor-specific antigen data. Here, we describe peptide-intrinsic biochemical features associated with neoantigen and minor histocompatibility mismatch antigen immunogenicity and present a gradient boosting algorithm for predicting tumor antigen immunogenicity. This algorithm was validated in two murine tumor models and demonstrated the capacity to select for therapeutically active antigens. Immune correlates of neoantigen immunogenicity were studied in a pan-cancer data set from The Cancer Genome Atlas and demonstrated an association between expression of immunogenic neoantigens and immunity in colon and lung adenocarcinomas. Lastly, we present evidence for expression of an out-of-frame neoantigen that was capable of driving antitumor cytotoxic T-cell responses. With the growing clinical importance of tumor vaccine therapies, our approach may allow for better selection of therapeutically relevant tumor-specific antigens, including nonclassic out-of-frame antigens capable of driving antitumor immunity., (©2019 American Association for Cancer Research.)

Purpose: A delay in encountering the cognate antigen while in the circulation, and the suboptimal costimulation received at the tumor site are key reasons for the limited activity of chimeric antigen receptor-redirected T cells (CAR-T) in solid tumors. We have explored the benefits of incorporating the IL15 cytokine within the CAR cassette to provide both a survival signal before antigen encounter, and an additional cytokine signaling at the tumor site using a neuroblastoma tumor model., Experimental Design: We optimized the construct for the CAR specific for the NB-antigen GD2 without (GD2.CAR) or with IL15 (GD2.CAR.15). We then compared the expansion, phenotype, and antitumor activity of T cells transduced with these constructs against an array of neuroblastoma cell lines in vitro and in vivo using a xenogeneic metastatic model of neuroblastoma., Results: We observed that optimized GD2.CAR.15-Ts have reduced expression of the PD-1 receptor, are enriched in stem cell-like cells, and have superior antitumor activity upon repetitive tumor exposures in vitro and in vivo as compared with GD2.CAR-Ts. Tumor rechallenge experiments in vivo further highlighted the role of IL15 in promoting enhanced CAR-T antitumor activity and survival, both in the peripheral blood and tissues. Finally, the inclusion of the inducible caspase-9 gene (iC9) safety switch warranted effective on demand elimination of the engineered GD2.CAR.15-Ts., Conclusions: Our results guide new therapeutic options for GD2.CAR-Ts in patients with neuroblastoma, and CAR-T development for a broad range of solid tumors., (©2019 American Association for Cancer Research.)

Background: Interactions between cancer cells and the surrounding microenvironment are crucial determinants of cancer progression. During this process, bi-directional communication among tumor cells and cancer associated fibroblasts (CAF) regulate extracellular matrix (ECM) deposition and remodeling. As a result of this dynamic process, soluble ECM proteins can be released into the bloodstream and may represent novel circulating biomarkers useful for cancer diagnosis. The aim of the present study was to measure the levels of three circulating ECM related proteins (COL11A1, COL10A1 and SPARC) in plasma samples of lung cancer patients and in healthy heavy-smokers controls and test whether such measurements have diagnostic or prognostic value., Methods: Gene expression profiling of lung fibroblasts isolated from paired normal and cancer tissue of NSCLC patients was performed by gene expression microarrays. The prioritization of the candidates for the study of circulating proteins in plasma was based on the most differentially expressed genes in cancer associated fibroblasts. Soluble ECM proteins were assessed by western blot in the conditioned medium of lung fibroblasts and by ELISA assays in plasma samples., Results: Plasma samples from lung cancer patients and healthy heavy-smokers controls were tested for levels of COL11A1 and COL10A1 (n = 57 each) and SPARC (n = 90 each). Higher plasma levels of COL10A1 were detected in patients (p ≤ 0.001), a difference that was driven specifically by females (p < 0.001). No difference in COL11A1 levels between patients and controls was found. SPARC levels were also higher in plasma patients than controls (p < 0.001) with good performance in discriminating the two groups (AUC = 0.744). No significant association was observed between plasma proteins levels and clinicopathological features or survival., Conclusion: Soluble factors related to proficient tumor-stroma cross-talk are detectable in plasma of primary lung cancer patients and may represent a valuable complementary diagnostic tool to discriminate lung cancer patients from healthy heavy-smokers individuals as shown for the SPARC protein.

Changes in amount and composition of extracellular matrix (ECM) are considered a hallmark of tumor development. We tested the hypothesis that abnormal production of ECM components leads to blood-released ECM molecules representing tumor circulating biomarkers. Candidate genes were selected through class comparison in two publicly available datasets and confirmed in paired normal and tumor associated fibroblasts from breast carcinoma (BC) specimens. Production and release of ECM molecules were evaluated in normal human dermal fibroblasts (NHDFs) treated with conditioned media from three BC cell lines. Plasma samples from healthy donors and from patients with malignant or benign breast disease were tested by ELISA for the presence of collagen 11a1 (COL11A1), collagen oligomeric matrix protein (COMP), and collagen 10a1 (COL10A1). Selected ECM molecules were investigated by IHC in malignant and benign specimens. In silico analysis of gene expression profiles identified 11 ECM genes significantly up-regulated in tumor versus normal tissue. Western blot analyses revealed increased levels of molecules encoded by three of these genes, COL11A1, COMP, and COL10A1, in cell lysates and supernatants of conditioned NHDFs. Class comparison and class prediction analyses of two independent series of human plasma samples identified the combination of COL11A1, COMP, and COL10A1 as potentially informative in discriminating BC patients from those with benign disease. The three molecules resulted expressed in the stroma of BC tissue samples. Our results indicate that circulating COL11A1, COMP, and COL10A1 may be useful in diagnostic assessment of suspicious breast nodules and ECM molecules could represent an avenue to biomarker identification., (© 2018 Wiley Periodicals, Inc.)

Background: Fibroblasts are crucial mediators of tumor-stroma cross-talk through synthesis and remodeling of the extracellular matrix and production of multiple soluble factors. Nonetheless, little is still known about specific determinants of fibroblast pro-tumorigenic activity in lung cancer. Here, we aimed at understanding the role of miRNAs, which are often altered in stromal cells, in reprogramming fibroblasts towards a tumor-supporting phenotype., Methods: We employed a co-culture-based high-throughput screening to identify specific miRNAs modulating the pro-tumorigenic potential of lung fibroblasts. Multiplex assays and ELISA were instrumental to study the effect of miRNAs on the secretome of both primary and immortalized lung fibroblasts from lung cancer patients and to evaluate plasmatic levels of HGF in heavy smokers. Direct mRNA targeting by miRNAs was investigated through dual-luciferase reporter assay and western blot. Finally, the pro-tumorigenic activity of fibroblasts and their conditioned media was tested by employing in vitro migration experiments and mouse xenografts., Results: We identified miR-16 as a master regulator of fibroblast secretome and showed that its upregulation reduces HGF secretion by fibroblasts, impairing their capacity to promote cancer cell migration. This effect is due to a pleiotropic activity of miR-16 which prevents HGF expression through direct inhibition of FGFR-1 signaling and targeting of HGF mRNA. Mechanistically, miR-16 targets FGFR-1 downstream mediator MEK1, thus reducing ERK1/2 activation. Consistently, chemical or genetic inhibition of FGFR-1 mimics miR-16 activity and prevents HGF production. Of note, we report that primary fibroblast cell lines derived from lungs of heavy smokers express reduced miR-16 levels compared to those from lungs not exposed to smoke and that HGF concentration in heavy smokers' plasma correlates with levels of tobacco exposure. Finally, in vivo experiments confirmed that restoration of miR-16 expression in fibroblasts reduced their ability to promote tumor growth and that HGF plays a central role in the pro-tumorigenic activity of fibroblasts., Conclusions: Overall, these results uncover a central role for miR-16 in regulating HGF production by lung fibroblasts, thus affecting their pro-tumorigenic potential. Correlation between smoking exposure and miR-16 levels could provide novel clues regarding the formation of a tumor-proficient milieu during the early phases of lung cancer development.

Introduction: Adoptive cell therapy (ACT) is becoming an increasingly successful and widespread form of treatment for different types of cancer. Compared to chemotherapy or monoclonal antibodies, ACT is an active biological strategy, with infused immune cells featuring dynamic migration, expansion and long-term persistence properties. ACT in hematological malignancies offered the initial proof of principle of the feasibility for this innovative 'live-drug'., Areas Covered: In this review, the authors summarize the clinical results achieved with two specific strategies in hematological malignancies: chimeric antigen receptor (CAR) and T cell receptor (transgenic TCR) redirected T cells. Moreover, they discuss the recent pre-clinical studies aimed at increasing the feasibility, safety and efficacy of ACT., Expert Opinion: ACT can promote cancer regression in patients with leukemia, lymphoma and multiple myeloma. Nevertheless, more precise targeting of tumor cells and containment of side effects are needed. Overcoming tumor-associated immunosuppressive mechanisms and preventing tumor escape are also emerging as critical barriers. Finally, simplification in the manufacturing procedures should promote wider application of these technologies outside academic centers. Although the enthusiasm for ACT-based therapies is high, comprehensive and systematic clinical studies are required to advance the field.

Cats are usually spreaders of allergens that are critical for sensitive people; the Siberian cat is a breed supposed to be low level allergenic, according to some breeders' statements. The sequence of the two genes, namely Ch1 and Ch2 , that code for the allergen Fel d 1, the major allergen responsible for outbreaks of allergy symptoms, is not yet known in the Siberian cat, and finding this was the aim of our investigation. Notably, our work is the first survey of the genetic structure of these genes in Siberian cats. The comparison of the sequences of Siberian cats, non-Siberian cats, and sequences present in the National Center for Biotechnology Information database revealed a considerable number of mutations; some of those detected in the Siberian cat, due to their position in exon regions, could affect the Fel d 1 allergenic properties. Therefore, further investigations are recommended to assess if the identified mutations can be responsible for a reduced-allergen synthesis and can be used as markers for selection of low level allergenic cats., Competing Interests: The authors declare no conflict of interest.

Transfer of T-cell receptors (TCRs) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. We developed the TCR gene editing technology that is based on the knockout of the endogenous TCR α and β genes, followed by the introduction of tumor-specific TCR genes, and that proved safer and more effective than conventional TCR gene transfer. Although successful, complete editing requires extensive cell manipulation and 4 transduction procedures. Here we propose a novel and clinically feasible TCR "single editing" (SE) approach, based on the disruption of the endogenous TCR α chain only, followed by the transfer of genes encoding for a tumor-specific TCR. We validated SE with the clinical grade HLA-A2 restricted NY-ESO-1 157-165 -specific TCR. SE allowed the rapid production of high numbers of tumor-specific T cells, with optimal TCR expression and preferential stem memory and central memory phenotype. Similarly to unedited T cells redirected by TCR gene transfer (TCR transferred [TR]), SE T cells efficiently killed NY-ESO-1 pos targets; however, although TR cells proved highly alloreactive, SE cells showed a favorable safety profile. Accordingly, when infused in NSG mice previously engrafted with myeloma, SE cells mediated tumor rejection without inducing xenogeneic graft-versus-host disease, thus resulting in significantly higher survival than that observed in mice treated with TR cells. Overall, single TCR gene editing represents a clinically feasible approach that is able to increase the safety and efficacy of cancer adoptive immunotherapy., (© 2017 by The American Society of Hematology.)

Background: Plasma miRNAs have the potential as cancer biomarkers but no consolidated guidelines for data mining in this field are available. The purpose of the study was to apply a supervised data analysis strategy in a context where prior knowledge is available, i.e., that of hemolysis-related miRNAs deregulation, so as to compare our results with existing evidence., Results: We developed a structured strategy with innovative applications of existing bioinformatics methods for supervised analyses including: 1) the combination of two statistical (t- and Anderson-Darling) test results to detect miRNAs with significant fold change or general distributional differences in class comparison, which could reveal hidden differential biological processes worth to be considered for building predictive tools; 2) a bootstrap selection procedure together with machine learning techniques in class prediction to guarantee the transferability of results and explore the interconnections among the selected miRNAs, which is important for highlighting their inherent biological dependences. The strategy was applied to develop a classifier for discriminating between hemolyzed and not hemolyzed plasma samples, defined according to a recently published hemolysis score. We identified five miRNAs with increased expression in hemolyzed plasma samples (miR-486-5p, miR-92a, miR-451, miR-16, miR-22)., Conclusions: We identified four miRNAs previously reported in the literature as hemolysis related together with a new one (miR-22).which needs further investigations. Our findings confirm the validity of the proposed strategy and, in parallel, the hemolysis score capability to be used as pre-analytic hemolysis detector. R codes for implementing the approaches are provided.

Breath analysis represents a new frontier in medical diagnosis and a powerful tool for cancer biomarker discovery due to the recent development of analytical platforms for the detection and identification of human exhaled volatile compounds. Statistical and bioinformatic tools may represent an effective complement to the technical and instrumental enhancements needed to fully exploit clinical applications of breath analysis. Our exploratory study in a cohort of 14 breast cancer patients and 11 healthy volunteers used secondary electrospray ionization-mass spectrometry (SESI-MS) to detect a cancer-related volatile profile. SESI-MS full-scan spectra were acquired in a range of 40-350 mass-to-charge ratio (m/z), converted to matrix data and analyzed using a procedure integrating data pre-processing for quality control, and a two-step class prediction based on machine-learning techniques, including a robust feature selection, and a classifier development with internal validation. MS spectra from exhaled breath showed an individual-specific breath profile and high reciprocal homogeneity among samples, with strong agreement among technical replicates, suggesting a robust responsiveness of SESI-MS. Supervised analysis of breath data identified a support vector machine (SVM) model including 8 features corresponding to m/z 106, 126, 147, 78, 148, 52, 128, 315 and able to discriminate exhaled breath from breast cancer patients from that of healthy individuals, with sensitivity and specificity above 0.9.Our data highlight the significance of SESI-MS as an analytical technique for clinical studies of breath analysis and provide evidence that our noninvasive strategy detects volatile signatures that may support existing technologies to diagnose breast cancer.

Metastasis is the main reason for lung cancer-related mortality, but little is known about specific determinants of successful dissemination from primary tumors and metastasis initiation. Here, we show that CD133(+)/CXCR4(+) cancer-initiating cells (CIC) directly isolated from patient-derived xenografts (PDX) of non-small cell lung cancer are endowed with superior ability to seed and initiate metastasis at distant organs. We additionally report that CXCR4 inhibition successfully prevents the increase of cisplatin-resistant CD133(+)/CXCR4(+) cells in residual tumors and their metastatization. Immunophenotypic analysis of lung tumor cells intravenously injected or spontaneously disseminated to murine lungs demonstrated the survival advantage and increased colonization ability of a specific subset of CD133(+)/CXCR4(+) with reduced expression of epithelial cell adhesion molecule (EpCAM(-)), which also shows the greatest in vitro invasive potential. We next prove that recovered disseminated cells from lungs of PDX-bearing mice enriched for CD133(+)/CXCR4(+)/EpCAM(-) CICs are highly tumorigenic and metastatic. Importantly, microenvironment stimuli eliciting epithelial-to-mesenchymal transition, including signals from cancer-associated fibroblasts, are able to increase the dissemination potential of lung cancer cells through the generation of the CD133(+)/CXCR4(+)/EpCAM(-) subset. These findings also have correlates in patient samples where disseminating CICs are enriched in metastatic lymph nodes (20-fold, P = 0.006) and their detection in primary tumors is correlated with poor clinical outcome (disease-free survival: P = 0.03; overall survival: P = 0.05). Overall, these results highlight the importance of specific cellular subsets in the metastatic process, the need for in-depth characterization of disseminating tumor cells, and the potential of therapeutic strategies targeting both primary tumor and tumor-microenvironment interactions., (©2015 American Association for Cancer Research.)