Over the past decade, cold atmospheric plasmas have shown promising application in cancer therapy. The therapeutic use of plasma-activated media is a topic addressed in an emerging field known as ...plasma pharmacy. In oncology, plasma-activated media are used to harness the therapeutic effects of oxidant species when they come in contact with cancer cells. Among several factors that contribute to the anticancer effect of plasma-activated liquid media (PALM), H
O
and NO derivatives likely play a key role in the apoptotic pathway. Despite the significant amount of literature produced in recent years, a full understanding of the mechanisms by which PALM exert their activity against cancer cells is limited. In this paper, a sealed dielectric-barrier discharge was used to disentangle the effect of reactive nitrogen species (RNS) from that of reactive oxygen species (ROS) on cancer cells. Two cancers characterized by poor prognosis have been investigated: metastatic melanoma and pancreatic cancer. Both tumour models exposed to PALM rich in H
O
showed a reduction in proliferation and an increase in calreticulin exposure and ATP release, suggesting the potential use of activated media as an inducer of immunogenic cell death via activation of the innate immune system.
Standard chemotherapy for soft tissue sarcomas has shown limited efficacy. Here, we sought to evaluate whether β-adrenergic receptor (β-AR) signalling contributed to the progression of sarcomas and ...therapy resistance. To assess the translational potential of β-adrenergic receptors, we performed immunohistochemical detection of β1-AR, β2-AR and β3-AR in leiomyosarcoma, liposarcoma and angiosarcoma tissue specimens, reporting the results scored for the intensity. By using established and patient-derived sarcoma cells, we demonstrated the antitumour potential of the pharmacological targeting of β-ARs with the nonselective β-blocker propranolol in such sarcomas. Of note, pharmacological β-AR inhibition synergized with doxorubicin in inhibiting the cell viability of liposarcoma and leiomyosarcoma cells and increased the response to docetaxel in angiosarcoma- and solitary fibrous tumour (SFT)-patient-derived cells. Notably, the SFT patient was treated with the combination of propranolol and docetaxel, reporting prolonged disease control. Mechanistically, we found that propranolol reduced the activity of the multidrug resistance efflux pump P-gp, thereby increasing the intracellular doxorubicin concentration and antitumour activity. In addition, propranolol attenuated the Akt-dependent survival signal induced by doxorubicin and strongly reduced the activation of the NF-kB/COX-2 pathway, increasing cell sensitivity to docetaxel. Overall, our study highlighted the therapeutic potential of propranolol, alone or in rational combination therapies, for sarcoma treatment.
In the attempt to understand the origin of autoantibody (AAb) production in patients with and at risk for type 1 diabetes (T1D), multiple studies have analyzed and reported alterations in T ...follicular helper (Tfh) cells in presymptomatic AAb
subjects and patients with T1D. Yet, whether the regulatory counterpart of Tfh cells, represented by T follicular regulatory (Tfr) cells, is similarly altered is still unclear. To address this question, we performed analyses in peripheral blood, spleen, and pancreatic lymph nodes (PLN) of organ donor subjects with T1D. Blood analyses were also performed in living AAb
and AAb
subjects. While negligible differences in the frequency and phenotype of blood Tfr cells were observed among T1D, AAb
, and AAb
adult subjects, the frequency of Tfr cells was significantly reduced in spleen and PLN of T1D as compared with nondiabetic control subjects. Furthermore, adoptive transfer of Tfr cells delayed disease development in a mouse model of T1D, a finding that could indicate that Tfr cells play an important role in peripheral tolerance and regulation of autoreactive Tfh cells. Together, our findings provide evidence of Tfr cell alterations within disease-relevant tissues in patients with T1D, suggesting a role for Tfr cells in defective humoral tolerance and disease pathogenesis.
The immunotherapy with immune checkpoints inhibitors (ICI) has changed the life expectancy in metastatic melanoma (MM) patients. Nevertheless, several patients do not respond hence, the ...identification and validation of novel biomarkers of response to ICI is of crucial importance. Circulating extracellular vesicles (EVs) such as PD-L1
EV mediate resistance to anti-PD1, instead the role of PD1
EV is not fully understood.
We isolated the circulating EVs from the plasma of an observational cohort study of 71 metastatic melanoma patients and correlated the amount of PD-L1
EVs and PD1
EVs with the response to ICI. The analysis was performed according to the origin of EVs from the tumor and the immune cells. Subsequently, we analysed the data in a validation cohort of 22 MM patients to assess the reliability of identified EV-based biomarkers. Additionally we assessed the involvement of PD1
EVs in the seizure of nivolumab and in the perturbation of immune cells-mediated killing of melanoma spheroids.
The level of PD-L1
EVs released from melanoma and CD8
T cells and that of PD1
EVs irrespective of the cellular origin were higher in non-responders. The Kaplan-Meier curves indicated that higher levels of PD1+ EVs were significantly correlated with poorer progression-free survival (PFS) and overall survival (OS). Significant correlations were found for PD-L1
EVs only when released from melanoma and T cells. The multivariate analysis showed that high level of PD1
EVs, from T cells and B cells, and high level of PD-L1
EVs from melanoma cells, are independent biomarkers of response. The reliability of PD-L1
EVs from melanoma and PD1
EVs from T cells in predicting PFS was confirmed in the validation cohort through the univariate Cox-hazard regression analysis. Moreover we discovered that the circulating EVs captured nivolumab and reduced the T cells trafficking and tumor spheroids killing.
Our study identified circulating PD1
EVs as driver of resistance to anti-PD1, and highlighted that the analysis of single EV population by liquid biopsy is a promising tool to stratify MM patients for immunotherapy.
Aims/hypothesis
Protein tyrosine phosphatase non-receptor 22 (PTPN22) plays a central role in T cell, B cell and innate immune cell signalling. A genetic variation in
Ptpn22
is considered a major ...risk factor for the development of type 1 diabetes and has been the subject of extensive study. While several reports have addressed how
Ptpn22
might predispose to autoimmunity, its involvement in other immune-mediated diseases, such as allograft rejection, has not been explored.
Methods
To address a possible function for
Ptpn22
in allograft rejection, we used a mouse model of pancreatic islet transplantation. We performed transplant tolerance experiments and determined how PTPN22 shapes tolerance induction and maintenance.
Results
Ptpn22
−/−
recipient mice generate higher numbers of alloreactive T cells after allogeneic pancreatic islet transplantation compared with wild-type (WT) mice, but reject grafts with similar kinetics. This is not only due to their well-documented increase in forkhead box protein P3 (FOXP3)
+
T regulatory (Treg) cells but also to the expansion of T regulatory type 1 (Tr1) cells caused by the lack of PTPN22. In addition, a tolerogenic treatment known to induce transplant tolerance in WT mice via Tr1 cell generation is more effective in
Ptpn22
−/−
mice as a consequence of boosting both Tr1 and FOXP3
+
Treg cells.
Conclusions/interpretation
A lack of PTPN22 strengthens transplant tolerance to pancreatic islets by expanding both FOXP3
+
Treg and Tr1 cells. These data suggest that targeting PTPN22 could serve to boost transplant tolerance.
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Reported here is the synthesis and biological evaluation of the asialoglycoprotein receptor (ASGP-R) targeted fourth generation poliamidoamine dendrimer (G(4)-PAMAM) loaded with ...sorafenib. The ASGP-R targeted dendrimer was obtained by conjugation of Lactobionic acid (La) to the G(4)-PAMAM dendrimer, followed by acetylation (Ac) of the free amino groups in order to reduce the non-specific interactions with the cell membrane. Moreover, by additionally grafting fluorescein (FITC), it was easy to characterize the internalization pathway and the intracellular fate of the targeted dendrimer Ac-La-G(4)-PAMAM-FITC. In vitro experiments performed on HepG-2 and HLE cell lines, allowed to study the ability of the dendrimers to affect the cell vitality. Confocal microscopy and cytofluorimetric analysis confirmed higher binding and uptake ability of the Ac-La-G(4)-PAMAM-FITC dendrimer in well differentiated and ASGP-R expressing human liver cancer cell line HepG-2 compared non-expressing HLE cells. Ac-La-G(4)-PAMAM-FITC dendrimer loaded with sorafenib was stable and showed sustained sorafenib release. As evidenced by the cytotoxicity studies, sorafenib included in the dendrimer maintained its effectiveness, and was able to produce a longer lasting effect over the time compared to molar equivalent doses of free sorafenib. This new targeted dendrimer appears to be a suitable carrier for the delivery of sorafenib to liver cancer cells expressing ASGP-R.
Tumor⁻stroma interactions are of key importance for pancreatic ductal adenocarcinoma (PDAC) progression. Our aim was to investigate whether cancer associated fibroblasts (CAFs) and mast cells (MC) ...affected the sensitivity of PDAC cells to gemcitabine/nabpaclitaxel (GEM/NAB). For this purpose, the combination cytotoxicity and the effect on tumor invasion and angiogenesis were evaluated with or without a conditioned medium from the mast cell line HMC-1 (human mast cell line-1 cells) and CAFs. Beside the clinical outcome of a homogenous population of PDAC patients, receiving GEM/NAB, was correlated to the circulating levels of mast cell tryptase and to a panel of inflammatory and immunosuppressive cytokines. CAFs neither affected drugs' cytotoxicity nor the inhibition of angiogenesis, but promoted tumor cell invasion. The MC instead, caused resistance to drugs by reducing apoptosis, by activating the TGF-β signalling and by promoting tumor invasion. Indeed, the inhibition of TβRI serine/threonine kinase activity by galunisertib restored drugs cytotoxicity. Moreover, MC induced the release of TGF-β1, and increased expression of PAR-2, ERK1/2 and Akt activation. Accordingly, TGF-β1, tryptase and other pro-inflammatory and immunosuppressive cytokines increased in the unresponsive patients. In conclusion, MC play a pivotal role in the resistance to GEM/NAB. A correlation between high level of circulating pro-inflammatory/ immunosuppressive cytokines and unresponsiveness was found in PDAC patients.
Over the past several years there has been much debate with regards to the prognostic and clinical significance of pancreatic ductal adenocarcinoma (PDAC) with lymph nodes metastasis. The PDAC gene ...expression knowledge and the biologic alterations underlying the lymph node involvement convey a clinical implication in dealing with the theranostic window. To this end, we provide an original bioinformatic dissection of the gene expression differences of PDAC according to the nodal involvement from a large public available dataset. Comprehensive transcriptomic analysis from 143 RNA-seq patient's derived samples indicated that WNT increased activation and a peculiar immune microenvironment identify subjects with nodal involvement. In frame of this thinking, we validated the WNT pathway role in increasing the likelihood of lymphatic dissemination in vitro. Moreover, we demonstrated for the first time in a PDAC model the potential therapeutic window that XAV-939-a specific WNT pathway inhibitor-has in re-educating a tumor-permissive immune system. Finally, we outline the potential implication on bystander molecular drivers exerted by WNT molecular inhibition, providing a picture of the proteomic oncogenic landscape changes elicited by XAV-939 on PDAC cells and their clinical implication. Our findings hold the promise to identify novel immune-based therapeutic strategies targeting WNT to enhance PDAC cytotoxicity and restore anti-PDAC immunity in node-positive disease.
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•Notch boosts MAPK signaling in mutated uveal and cutaneous melanoma.•Notch inhibition counteracts GNAQ-induced MAPK activation.•Galunisertib prolongs the efficacy of cobimetinib in ...GNAQQ209L UM cells.•Simultaneous inhibition of Notch and MEK induces senescence of GNAQQ209L UM cells.
The crosstalk between Notch and MAPK pathway plays a role in MEK inhibitor resistance in BRAFV600E metastatic melanoma (MM) and promotes migration in GNAQQ209L uveal melanoma (UM) cells. We determined the cytotoxicity of combinatorial inhibition of MEK and Notch by cobimetinib and γ-secretase inhibitor (GSI) nirogacestat, in BRAFV600E and BRAF wt MM and GNAQQ209L UM cells displaying different Erk1/2 and Notch activation status, with the aim to elucidate the impact of Notch signaling in the response to MEK inhibitor. Overall the combination was synergic in BRAFV600E MM and GNAQQ209L UM cells and antagonistic in BRAF wt one. Focusing on UM cells, we found that cobimetinib resulted in G0/G1 phase arrest and apoptosis induction, whereas the combination with GSI increased treatment efficacy by inducing a senescent-like state of cells and by blocking migration towards liver cancer cells. Mechanistically, this was reflected in a strong reduction of cyclin D1, in the inactivation of retinoblastoma protein and in the increase of p27KIP1 expression levels. Of note, each drug alone prevented Notch signaling activation resulting in inhibition of c-jun(Ser63) and Hes-1 expression. The combination achieved the strongest inhibition on Notch signaling and on both c-jun(Ser63) and Erk1/2 activation level. In conclusion we unveiled a coordinate action of MAPK and Notch signaling in promoting proliferation of BRAFV600E MM and GNAQQ209L UM cells. Remarkably, the simultaneous inhibition of MEK and Notch signaling highlighted a role for the second pathway in protecting cells against senescence in GNAQQ209L UM cells treated with the MEK inhibitor.
Tr1 cell therapy is considered an emerging approach to improve transplant tolerance and enhance allogeneic graft survival. However, it remains unclear how Tr1 cells promote transplant tolerance and ...whether they will be safe and stable in the face of an acute viral infection. By employing a mouse model of pancreatic islet transplantation, we report that Tr1 cell therapy promoted transplant tolerance via de novo induction of Tr1 cells in the recipients. Acute viral infection with lymphocytic choriomeningitis virus (LCMV) had no impact on Tr1 cell number and function, neither on the Tr1 cells infused nor on the ones induced, and that was reflected in the robust maintenance of the graft. Moreover, Tr1 cell immunotherapy had no detrimental effect on CD8 and CD4 anti‐LCMV effector T‐cell responses and viral control. Together, these data suggest that Tr1 cells did not convert to effector cells during acute infection with LCMV, maintained transplant tolerance and did not inhibit antiviral immunity.
Tr1 cell immunotherapy induces transplant tolerance to pancreatic islets via the de novo induction of Tr1 cells in the spleen. Tolerance established via Tr1 cell immunotherapy does not inhibit acute antiviral T‐cell responses. Viral Infection does not break transplant tolerance established by Tr1 cell immunotherapy.
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