Targeting immunosuppressive myeloid cells using nanocarriers to improve cancer immunotherapy

In the last decade, different cancer immunotherapy approaches have been proved to produce objective clinical responses and survival benefits in cancer patients who failed conventional treatment options. However, the efficacy of cancer immunotherapy is known to be limited by tumor-induced expansion of immunosuppressive and tumor-promoting immune populations, including cells of myeloid origin (such as tumor associated macrophages –TAMs-, and myeloid-derived suppressor cells –MDSCs-). The development of increasingly powerful and widely applicable immunotherapies is therefore dependent on the availability of supporting treatments able to reduce tumor-associated immunosuppression, with good efficacy and low toxicity. Drug delivery nanosystems have been shown to improve pharmacological proprieties of anti-cancer drugs by increasing drug half-life, enhancing accumulation into tumor tissues and reducing off-target toxicity. In addition, the use of RNA delivery nanosystems is currently being explored for the development of RNA intereference (RNAi)-based anti-cancer therapeutics.The use of drug and RNA delivery nanosystems to target tumor-associated immune cells, besides tumor cells, is a far less explored approach, which is currently showing promiseas tool to improve cancer immunotherapy. In the present work we investigated two different nanotechnology-based tools to modulate the presence and function of tumor-associated myeloid cells:a modified form of gemcitabine encapsulated into lipid nanocapsules (LNC-GemC12), and ployarginine-coated nanocapsules (PolyArgNCs) loaded with short hairpin (sh) RNAs, for in vivo RNAi-based gene silencing. LNC-GemC12 and free gemcitabine hydrochloride (GemHCl, the current standard gemcitabine formulation)were found to selectively deplete both splenic and tumor-infiltrating monocytic (M-) MDSCs, following administration at a very low drug dose to EG7-OVA tumor bearing mice. Remarkably, LNC-GemC12 administration was associated with a stronger and more durable reduction of tumor-infiltrating M-MDSCs as compared withGemHCl, which resulted in the attenuation of MDSC suppressive activity towards T cells. More importantly,treatment of EG7-OVA tumor bearing mice with LNC-GemC12,prior to adoptive cell transfer therapy (ACT) with OVA-specific T lymphocytes,significantly extended mouse survival as compared to mice receiving ACT alone. Conversely, preconditioning with GemHCl at the same dose did not result in a similar survival benefit. PolyArg NCs were loaded with a fluorinated shRNA specific for mouse C/EBPβ transcription factor, which is known to be required for tumor-induced MDSC expansion and acquisition of immunosuppressive functions.PolyArgNCs loaded with the C/EBPβ-specific shRNA (NC-shC/EBPβ) efficiently down-regulated target gene expression in an immortalized MDSC cell line.In vivo, we reported a significant reduction of C/EBPβ mRNA levels in splenic and tumor-infiltrating myeloid cells, following repeated administration of NC-shC/EBPβ to mice bearing MCA203 subcutaneous sarcomas. The present datasupport the use of LNC-GemC12 as MDSC-targeted agent in cancer immunotherapy, notably in combination with ACT. As compared with standard MDSC-depleting chemotherapeutic drug formulations, LNC-GemC12 bears the potential of achieving significant effects at very low, likely not toxic, drug doses. Moreover, the use of a drug already employed in clinical oncology, combined with a biocompatible delivery nanosystem, might facilitate clinical translation.We also provided an initial evidence that shRNA-loaded PolyArg NCs allow to downregulate target gene expression in myeloid cellsin vivo, and could be exploited to therapeutically modulate tumor-associated myeloid cells.

T Cell Cancer Therapy Requires CD40-CD40L Activation of Tumor Necrosis Factor and Inducible Nitric-Oxide-Synthase-Producing Dendritic Cells

Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We\ua0found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8(+) cytotoxic T\ua0cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1(+) myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies

Canagliflozin and Cardiovascular and Renal Outcomes in Type 2 Diabetes Mellitus and Chronic Kidney Disease in Primary and Secondary Cardiovascular Prevention Groups

Background: Canagliflozin reduces the risk of kidney failure in patients with type 2 diabetes mellitus and chronic kidney disease, but effects on specific cardiovascular outcomes are uncertain, as are effects in people without previous cardiovascular disease (primary prevention). Methods: In CREDENCE (Canagliflozin and Renal Events in Diabetes With Established Nephropathy Clinical Evaluation), 4401 participants with type 2 diabetes mellitus and chronic kidney disease were randomly assigned to canagliflozin or placebo on a background of optimized standard of care. Results: Primary prevention participants (n=2181, 49.6%) were younger (61 versus 65 years), were more often female (37% versus 31%), and had shorter duration of diabetes mellitus (15 years versus 16 years) compared with secondary prevention participants (n=2220, 50.4%). Canagliflozin reduced the risk of major cardiovascular events overall (hazard ratio [HR], 0.80 [95% CI, 0.67-0.95]; P=0.01), with consistent reductions in both the primary (HR, 0.68 [95% CI, 0.49-0.94]) and secondary (HR, 0.85 [95% CI, 0.69-1.06]) prevention groups (P for interaction=0.25). Effects were also similar for the components of the composite including cardiovascular death (HR, 0.78 [95% CI, 0.61-1.00]), nonfatal myocardial infarction (HR, 0.81 [95% CI, 0.59-1.10]), and nonfatal stroke (HR, 0.80 [95% CI, 0.56-1.15]). The risk of the primary composite renal outcome and the composite of cardiovascular death or hospitalization for heart failure were also consistently reduced in both the primary and secondary prevention groups (P for interaction >0.5 for each outcome). Conclusions: Canagliflozin significantly reduced major cardiovascular events and kidney failure in patients with type 2 diabetes mellitus and chronic kidney disease, including in participants who did not have previous cardiovascular disease

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Targeting immunosuppressive myeloid cells using nanocarriers to improve cancer immunotherapy

In the last decade, different cancer immunotherapy approaches have been proved to produce objective clinical responses and survival benefits in cancer patients who failed conventional treatment options. However, the efficacy of cancer immunotherapy is known to be limited by tumor-induced expansion of immunosuppressive and tumor-promoting immune populations, including cells of myeloid origin (such as tumor associated macrophages –TAMs-, and myeloid-derived suppressor cells –MDSCs-). The development of increasingly powerful and widely applicable immunotherapies is therefore dependent on the availability of supporting treatments able to reduce tumor-associated immunosuppression, with good efficacy and low toxicity. Drug delivery nanosystems have been shown to improve pharmacological proprieties of anti-cancer drugs by increasing drug half-life, enhancing accumulation into tumor tissues and reducing off-target toxicity. In addition, the use of RNA delivery nanosystems is currently being explored for the development of RNA intereference (RNAi)-based anti-cancer therapeutics.The use of drug and RNA delivery nanosystems to target tumor-associated immune cells, besides tumor cells, is a far less explored approach, which is currently showing promiseas tool to improve cancer immunotherapy. In the present work we investigated two different nanotechnology-based tools to modulate the presence and function of tumor-associated myeloid cells:a modified form of gemcitabine encapsulated into lipid nanocapsules (LNC-GemC12), and ployarginine-coated nanocapsules (PolyArgNCs) loaded with short hairpin (sh) RNAs, for in vivo RNAi-based gene silencing. LNC-GemC12 and free gemcitabine hydrochloride (GemHCl, the current standard gemcitabine formulation)were found to selectively deplete both splenic and tumor-infiltrating monocytic (M-) MDSCs, following administration at a very low drug dose to EG7-OVA tumor bearing mice. Remarkably, LNC-GemC12 administration was associated with a stronger and more durable reduction of tumor-infiltrating M-MDSCs as compared withGemHCl, which resulted in the attenuation of MDSC suppressive activity towards T cells. More importantly,treatment of EG7-OVA tumor bearing mice with LNC-GemC12,prior to adoptive cell transfer therapy (ACT) with OVA-specific T lymphocytes,significantly extended mouse survival as compared to mice receiving ACT alone. Conversely, preconditioning with GemHCl at the same dose did not result in a similar survival benefit. PolyArg NCs were loaded with a fluorinated shRNA specific for mouse C/EBPβ transcription factor, which is known to be required for tumor-induced MDSC expansion and acquisition of immunosuppressive functions.PolyArgNCs loaded with the C/EBPβ-specific shRNA (NC-shC/EBPβ) efficiently down-regulated target gene expression in an immortalized MDSC cell line.In vivo, we reported a significant reduction of C/EBPβ mRNA levels in splenic and tumor-infiltrating myeloid cells, following repeated administration of NC-shC/EBPβ to mice bearing MCA203 subcutaneous sarcomas. The present datasupport the use of LNC-GemC12 as MDSC-targeted agent in cancer immunotherapy, notably in combination with ACT. As compared with standard MDSC-depleting chemotherapeutic drug formulations, LNC-GemC12 bears the potential of achieving significant effects at very low, likely not toxic, drug doses. Moreover, the use of a drug already employed in clinical oncology, combined with a biocompatible delivery nanosystem, might facilitate clinical translation.We also provided an initial evidence that shRNA-loaded PolyArg NCs allow to downregulate target gene expression in myeloid cellsin vivo, and could be exploited to therapeutically modulate tumor-associated myeloid cells.  Nell’ultimo decennio l’impiego di diversi tipi di immunoterapia dei tumori ha consentito di ottenere risposte cliniche e un miglioramento della sopravvivenza in pazienti non rispondenti alle terapie convenzionali. Tuttavia, l’efficacia dell’immunoterapia del cancro è limitata dalla presenza di popolazioni di cellule immunitarie con proprietà immunosoppressive e pro-tumorali che si espandono durante la patogenesi della malattia. Tali cellule comprendono popolazioni di origine mieloide, quali i macrofagi associati al tumore (tumor associated macrophages, TAMs), e cellule soppressive di derivazione mieloide (myeloid derived suppressor cells, MDSCs). Pertanto, per lo sviluppo di protocolli d’immunoterapia ottimali sono necessari trattamenti di supporto, con buona efficacia e bassa tossicità, in grado di contrastare l’immunosoppressione indotta dal tumore. Diversi studi preclinici e clinici hanno dimostrato che i nanosistemi per il trasporto di farmaci antitumorali sono in grado di migliorarne le proprietà farmacologiche aumentandone l’emivita, favorendone l’accumulo nel sito tumorale e limitando la tossicità a carico di tessuti sani. Inoltre, i nanosistemi per il trasporto di RNA sono attualmente impiegati nello sviluppo di terapie antineoplastiche che utilizzano approcci di silenziamento genico in vivo basati sul meccanismo dell’Interferenza a RNA (RNAi). L’uso di nanosistemi per il trasporto di farmaci ed RNA che hanno come target le cellule sistema immunitario, oltre alle cellule tumorali, sembra essere un nuovo e promettente approccio per potenziare l’efficienza dell’immunoterapia dei tumori. In questo lavoro abbiamo valutato l’efficaciadi due diverse formulazioni di nanotrasportatori, caricati con farmaci o RNA, come agenti immunomodulanti per alterare la presenza e/o funzione delle cellule mieloidi associate al tumore. La prima formulazione consiste in una forma modificata di gemcitabina incapsulata in nanocapsule lipidiche (LNC-GemC12); la seconda è costituita da nanocapsule rivestite di poliarginina (PolyArgNCs) caricate con short hairpin (sh) RNA, per il silenziamento genico in vivo mediante RNAi. Nel modello tumorale murino EG7-OVA, la somministrazione di LNC-GemC12 e di gemcitabina idrocloruro (GemHCl, l’attuale formulazione standard per la gemcitabina), ad un dosaggio molto basso, causa una deplezione selettiva delle MDSC monocitarie (M-MDSCs) nella milza e nel sito tumorale. In particolare, alla somministrazione di LNC-GemC12, rispetto alla GemHCl, si associa una riduzione più intensa e prolungata delle M-MDSCs tumorali ed una conseguente attenuazione dell’attività soppressiva delle MDSC infiltranti il tumore nei confronti dei linfociti T. Inoltre, nello stesso modello, il trattamento con LNC-GemC12 prima della somministrazione di una terapia di trasferimento cellulare adottivo (ACT) con linfociti T OVA-specifici, risulta in un miglioramento della sopravvivenza rispetto al solo trattamento mediante ACT. In confronto, il pre-trattamento con GemHCl alla stessa dose non si associa ad un’estensione della sopravvivenza dopo ACT. Nella seconda parte di questo lavoro, le PolyArg NC sono state caricate con un shRNA fluorinato specifico per il fattore di trascrizione murino C/EBPβ, la cui espressione è necessaria per l’espansione e l’induzione di funzioni immunosoppressive nelle MDSC. Le PolyArg NC caricate con l’shRNA C/EBPβ-specifico (NC-shC/EBPβ) sono state impiegate con successo per ridurre l’espressione del gene bersaglio in una linea cellulare di MDSC immortalizzate. Successivamente, abbiamo valutato il funzionamento di questo sistema in vivo in un modello murino di sarcoma sottocutaneo. In questo modello abbiamo riportato una riduzione significativa nei livelli di mRNA di C/EBPβ nelle cellule mieloidi spleniche e intratumorali a seguito della somministrazione ripetuta di NC-shC/EBPβ. I dati ottenuti in questo lavoro supportano la validità dell’uso delle LNC-GemC12 come agente per la modulazione delle MDSC nell’immunoterapia del cancro, in particolare in combinazione con l’ACT. Rispetto alle formulazioni standard di agenti chemioterapici in grado di ridurre la frequenza delle MDSC, le LNC-GemC12 hanno il potenziale di fornire un’efficacia terapeutica a dosaggi di farmaco molto bassi e verosimilmente non tossici. In aggiunta, l’utilizzo di un farmaco già in uso nella pratica clinica, combinato con una formulazione di nano capsule lipidiche biocompatibili, potrebbe facilitare la traslazione in ambito clinico di questo approccio. Nel presente studio, abbiamo inoltre fornito una prima validazione dell’efficacia in vivo delle PolyArg NC caricate con shRNA come strumento per ridurre l’espressione di geni bersaglio nelle cellule mieloidi associate al tumore, potenzialmente impiegabile come agente immunomodulante nella terapia del cancro

Exploiting lymphatic vessels for immunomodulation: Rationale, opportunities, and challenges

Lymphatic vessels are the primary route of communication from peripheral tissues to the immune system; as such, they represent an important component of local immunity. In addition to their transport functions, new immunomodulatory roles for lymphatic vessels and lymphatic endothelial cells have come to light in recent years, demonstrating that lymphatic vessels help shape immune responses in a variety of ways: promoting tolerance to self-antigens, archiving antigen for later presentation, dampening effector immune responses, and resolving inflammation, among others. In addition to these new biological insights, the growing field of immunoengineering has begun to explore therapeutic approaches to utilize or exploit the lymphatic system for immunotherapy. (C) 2017 Published by Elsevier B.V

Lymphangiogenesis-inducing vaccines elicit potent and long-lasting T cell immunity against melanomas

In melanoma, the induction of lymphatic growth (lymphangiogenesis) has long been correlated with metastasis and poor prognosis, but we recently showed it can synergistically enhance cancer immunotherapy and boost T cell immunity. Here, we develop a translational approach for exploiting this “lymphangiogenic potentiation” of immunotherapy in a cancer vaccine using lethally irradiated tumor cells overexpressing vascular endothelial growth factor C (VEGF-C) and topical adjuvants. Our “VEGFC vax” induced extensive local lymphangiogenesis and promoted stronger T cell activation in both the intradermal vaccine site and draining lymph nodes, resulting in higher frequencies of antigen-specific T cells present systemically than control vaccines. In mouse melanoma models, VEGFC vax elicited potent tumor-specific T cell immunity and provided effective tumor control and long-term immunological memory. Together, these data introduce the potential of lymphangiogenesis induction as a novel immunotherapeutic strategy to consider in cancer vaccine design

Low dose gemcitabine-loaded lipid nanocapsules target monocytic myeloid-derived suppressor cells and potentiate cancer immunotherapy

umor-induced expansion of myeloid-derived suppressor cells (MDSCs) is known to impair the efficacy of cancer immunotherapy. Among pharmacological approaches for MDSC modulation, chemotherapy with selected drugs has a considerable interest due to the possibility of a rapid translation to the clinic. However, such approach is poorly selective and may be associated with dose-dependent toxicities. In the present study, we showed that lipid nanocapsules (LNCs) loaded with a lauroyl-modified form of gemcitabine (GemC12) efficiently target the monocytic (M-) MDSC subset. Subcutaneous administration of GemC12-loaded LNCs reduced the percentage of spleen and tumor-infiltrating M-MDSCs in lymphoma and melanoma-bearing mice, with enhanced efficacy when compared to free gemcitabine. Consistently, fluorochrome-labeled LNCs were preferentially uptaken by monocytic cells rather than by other immune cells, in both tumor-bearing mice and human blood samples from healthy donors and melanoma patients. Very low dose administration of GemC12-loaded LNCs attenuated tumor-associated immunosuppression and increased the efficacy of adoptive T cell therapy. Overall, our results show that GemC12-LNCs have monocyte-targeting properties that can be useful for immunomodulatory purposes, and unveil new possibilities for the exploitation of nanoparticulate drug formulations in cancer immunotherapy. (C) 2016 Elsevier Ltd. All rights reserved

Appropriateness of antiplatelet therapy for primary and secondary cardio- and cerebrovascular prevention in acutely hospitalized older people

Aims: Antiplatelet therapy is recommended for the secondary prevention of cardio- and cerebrovascular disease, but for primary prevention it is advised only in patients at very high risk. With this background, this study aims to assess the appropriateness of antiplatelet therapy in acutely hospitalized older people according to their risk profile. Methods: Data were obtained from the REPOSI register held in Italian and Spanish internal medicine and geriatric wards in 2012 and 2014. Hospitalized patients aged ≥65 assessable at discharge were selected. Appropriateness of the antiplatelet therapy was evaluated according to their primary or secondary cardiovascular prevention profiles. Results: Of 2535 enrolled patients, 2199 were assessable at discharge. Overall 959 (43.6%, 95% CI 41.5–45.7) were prescribed an antiplatelet drug, aspirin being the most frequently chosen. Among patients prescribed for primary prevention, just over half were inappropriately prescribed (52.1%), being mainly overprescribed (155/209 patients, 74.2%). On the other hand, there was also a high rate of inappropriate underprescription in the context of secondary prevention (222/726 patients, 30.6%, 95% CI 27.3–34.0%). Conclusions: This study carried out in acutely hospitalized older people shows a high degree of inappropriate prescription among patients prescribed with antiplatelets for primary prevention, mainly due to overprescription. Further, a large proportion of patients who had had overt cardio- or cerebrovascular disease were underprescribed, in spite of the established benefits of antiplatelet drugs in the context of secondary prevention