Boosting Interleukin-12 Antitumor Activity and Synergism with Immunotherapy by Targeted Delivery with isoDGR-Tagged Nanogold.

The clinical use of interleukin-12 (IL12), a cytokine endowed with potent immunotherapeutic anticancer activity, is limited by systemic toxicity. The hypothesis is addressed that gold nanoparticles tagged with a tumor-homing peptide containing isoDGR, an αvß3-integrin binding motif, can be exploited for delivering IL12 to tumors and improving its therapeutic index. To this aim, gold nanospheres are functionalized with the head-to-tail cyclized-peptide CGisoDGRG (Iso1) and murine IL12. The resulting nanodrug (Iso1/Au/IL12) is monodispersed, stable, and bifunctional in terms of αvß3 and IL12-receptor recognition. Low-dose Iso1/Au/IL12, equivalent to 18-75 pg of IL12, induces antitumor effects in murine models of fibrosarcomas and mammary adenocarcinomas, with no evidence of toxicity. Equivalent doses of Au/IL12 (a nanodrug lacking Iso1) fail to delay tumor growth, whereas 15 000 pg of free IL12 is necessary to achieve similar effects. Iso1/Au/IL12 significantly increases tumor infiltration by innate immune cells, such as NK and iNKT cells, monocytes, and neutrophils. NK cell depletion completely inhibits its antitumor effects. Low-dose Iso1/Au/IL12 can also increase the therapeutic efficacy of adoptive T-cell therapy in mice with autochthonous prostate cancer. These findings indicate that coupling IL12 to isoDGR-tagged nanogold is a valid strategy for enhancing its therapeutic index and sustaining adoptive T-cell therapy.

Invariant NKT cells contribute to chronic lymphocytic leukemia surveillance and prognosis.

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant CD5+ B lymphocytes in blood, bone marrow, and lymphoid organs. CD1d-restricted invariant natural killer T (iNKT) cells are innate-like T lymphocytes strongly implicated in tumor surveillance. We investigated the impact of iNKT cells in the natural history of the disease in the Eµ-Tcl1 (Tcl1) CLL mouse model and 68 CLL patients. We found that Tcl1-CLL cells express CD1d and that iNKT cells critically delay disease onset but become functionally impaired upon disease progression. In patients, disease progression correlates with high CD1d expression on CLL cells and impaired iNKT cells. Conversely, disease stability correlates with negative or low CD1d expression on CLL cells and normal iNKT cells, suggesting indirect leukemia control. iNKT cells indeed hinder CLL survival in vitro by restraining CD1d-expressing nurse-like cells, a relevant proleukemia macrophage population. Multivariable analysis identified iNKT cell frequency as an independent predictor of disease progression. Together, these results support the contribution of iNKT cells to CLL immune surveillance and highlight iNKT cell frequency as a prognostic marker for disease progression.

Long non-coding RNAs as novel therapeutic targets in cancer.

Thanks to impressive technology advancements, pervasive expression of non-coding RNAs (ncRNAs) has been recently identified in the genome of numerous cancers. Long ncRNAs (lncRNAs) belong to a new class of ncRNAs including tens of thousands different species. A fraction of these molecules shows a striking cancer-enriched expression pattern, suggesting an essential role in tumor cells and, possibly, a utility in therapeutic terms. This review aims at summarizing current knowledge for the identification and validation of lncRNAs as therapeutics targets in tumors. Both in-silico and wet-biology resources are presented in relation to the many challenges that the scientific community still needs to address in terms of lncRNA identification, stratification, patient personalization, drug delivery and toxicity.

Induction of T-cell memory by a dendritic cell vaccine: a computational model.

MOTIVATION: Although results from phase III clinical trials substantially support the use of prophylactic and therapeutic vaccines against cancer, what has yet to be defined is how many and how frequent boosts are needed to sustain a long-lasting and protecting memory T-cell response against tumor antigens. Common experience is that such preclinical tests require the sacrifice of a relatively large number of animals, and are particularly time- and money-consuming. RESULTS: As a first step to overcome these hurdles, we have developed an ordinary differential equation model that includes all relevant entities (such as activated cytotoxic T lymphocytes and memory T cells), and investigated the induction of immunological memory in the context of wild-type mice injected with a dendritic cell-based vaccine. We have simulated the biological behavior both in the presence and in the absence of memory T cells. Comparing results of ex vivo and in silico experiments, we show that the model is able to envisage the expansion and persistence of antigen-specific memory T cells. The model might be applicable to more complex vaccination schedules and substantially in any biological condition of prime-boosting. AVAILABILITY AND IMPLEMENTATION: The model is fully described in the article.

Pushing tumor cells towards a malignant phenotype: stimuli from the microenvironment, intercellular communications and alternative roads.

The tumor microenvironment produces different types of stimuli capable of endowing tumor cells with an aggressive behavior that is characterized by increased motility, invasiveness and propensity to metastasize, gain of a tumor-initiating phenotype, and drug resistance. The following classes of stimuli have been reported to promote such a malignant phenotype: (i) solid- or fluid-induced stress; (ii) altered composition of the extracellular matrix; (iii) hypoxia and low pH; (iv) innate and adaptive immune responses; (v) antitumor drugs. The simultaneous presence of more than one of these stimuli, as likely occurs in vivo, may lead to synergistic interactions in the induction of malignant traits. In many cases, the gain of a malignant phenotype is not the result of a direct effect of the stimuli on tumor cells but, rather, a stimulus-promoted cross-talk between tumor cells and other cell types within the tumor microenvironment. This cross-talk is mainly mediated by two classes of molecules: paracrine factors and adhesion receptors. Stimuli that promote a malignant phenotype can promote additional outcomes in tumor cells, including autophagy and cell death. We summarize here the available evidence about the variables that induce tumor cells to take one or the other of these roads in response to the same stimuli. At the end of this review, we address some unanswered questions in this domain and indicate future directions of research.

Targeting TNF-α to neoangiogenic vessels enhances lymphocyte infiltration in tumors and increases the therapeutic potential of immunotherapy.

Abnormal tumor vasculature impairs T lymphocyte adhesion to endothelial cells and lymphocyte extravasation into neoplastic tissues, limiting the therapeutic potential of both active and adoptive immunotherapies. We have found that treatment of tumor-bearing mice with NGR-TNF, a Cys-Asn-Gly-Arg-Cys peptide-TNF fusion product capable of altering the endothelial barrier function and improving drug penetration in tumors, associated with the intratumor upregulation of leukocyte-endothelial cell adhesion molecules, the release of proinflammatory cytokines and chemokines, and the infiltration of tumor-specific effector CD8(+) T cells. As a result, NGR-TNF enhanced the therapeutic activity of adoptive and active immunotherapy, delaying tumor growth and prolonging survival. Furthermore, we have found that therapeutic effects of these combinations can be further increased by the addition of chemotherapy. Thus, these findings might be relevant for the design of novel immunotherapeutic approaches for cancer patients.

Automated radiosynthesis and preclinical evaluation of two new PSMA-617 derivatives radiolabelled via [18F]AlF2+ method.

BACKGROUND: In the last decade the development of new PSMA-ligand based radiopharmaceuticals for the imaging and therapy of prostate cancer has been a highly active and important area of research. The most promising derivative in terms of interaction with the antigen and clinical properties has been found to be "PSMA-617", and its lutetium-177 radiolabelled version has recently been approved by EU and USA regulatory agencies for therapeutic purposes. For the above reasons, the development of new derivatives of PSMA-617 radiolabelled with fluorine-18 may still be of great interest. This paper proposes the comparison of two different PSMA-617 derivatives functionalized with NODA and RESCA chelators, respectively, radiolabelled via [18F]AlF2+ complexation. RESULTS: The organic synthesis of two PSMA-617 derivatives and their radiolabelling via [18F]AlF2+ complexation resulted to proceed efficiently and successfully. Moreover, stability in solution and in plasma has been evaluated. The whole radiosynthesis procedure has been fully automated, and the final products have been obtained with radiochemical yield and purity potentially suitable for clinical studies. The biodistribution of the two derivatives was performed both in prostate cancer and glioma tumour models. Compared with the reference [18F]F-PSMA-1007 and [18F]F-PSMA-617-RESCA, [18F]F-PSMA-617-NODA derivative showed a higher uptake in both tumors, faster clearance in non-target organs, and lower uptake in salivary glands. CONCLUSION: PSMA-617 NODA and RESCA derivatives were radiolabelled successfully via [18F]AlF2+ chelation, the former being more stable in solution and human plasma. Moreover, preclinical biodistribution studies showed that [18F]F-PSMA-617-NODA might be of potential interest for clinical applications.

Concurrent allorecognition has a limited impact on posttransplant vaccination.

Transplantation of allogeneic hematopoietic stem cells with or without immunocompetent lymphocytes has proved a successful strategy in the treatment of hematological malignancies. We have recently shown that this approach can also cure mouse prostate cancer, provided that it is combined with tumor-specific vaccination. Whether the response to alloantigens acts by providing helper function to enhance vaccine-specific responses or in other ways impinges on vaccine immunogenicity remains to be clarified, and this question is of clinical relevance. In this study, we have addressed this issue by comparing the immunogenicity of dendritic cells pulsed with a peptide derived from a tumor/viral model Ag in recipients of donor cells either syngeneic to the host or differing for either Y-encoded or multiple minor H antigens. We report that vaccination elicits comparable proliferation and differentiation of peptide-specific CD8(+) T cells despite concurrent expansion and differentiation of minor H antigen-specific IFN-γ effector T cells. Depletion of alloreactive CD4(+) T cells reduced alloreactivity but not vaccine-induced CD8(+) T cell priming, suggesting that alloresponses do not provide helper functions in peripheral lymphoid tissues. Vaccine-mediated T cell priming was also preserved in the case of multiple minor H antigen disparities, prone to graft-versus-host disease. Thus, in the context of nonmyeloablative allotransplantation aimed at restoring an effective tumor-specific T cell repertoire, minor H antigen-specific T cells do not interfere with vaccine-induced T cell priming, supporting the notion that posttransplant vaccination is a valuable strategy to boost tumor and pathogen-specific protective immunity.

Dietary and microbiome evidence in multiple myeloma and other plasma cell disorders.

Multiple Myeloma (MM) remains an incurable plasma cell neoplasm. Although little is known about the etiology of MM, several metabolic risk factors such as obesity, diabetes mellitus, diet, and the human intestinal microbiome have been linked to the pathogenesis of MM. In this article, we provide a detailed review of dietary and microbiome factors involved in the pathogenesis of MM and their impact on outcomes. Concurrent with treatment advancements that have improved survival in MM, focused efforts are needed to reduce the burden of MM as well as improve MM specific and overall outcomes once MM is diagnosed. The findings presented in this review will provide a comprehensive guide on the evidence available to date of the impact of dietary and other lifestyle interventions on the gut microbiome and on MM incidence, outcomes, and quality of life. Data generated from such studies can help formulate evidence-based guidelines for healthcare providers to counsel individuals at risk such as those with Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM) as well as MM survivors with respect to their dietary habits.

Prostate cancer, tumor immunity and a renewed sense of optimism in immunotherapy.

The recent FDA approval of the first therapeutic vaccine against prostate cancer has revitalized the public interest in the fields of cancer immunology and immunotherapy. Yet, clinical results are modest. A reason for this limited success may reside in the capacity of the tumor to convert inflammation in a tumor-promoting condition and eventually escape immune surveillance. Here we present the main known interactions between the prostate tumor and the immune system, showing how the malignancy can dodge the immune system by also exerting several immunosuppressive mechanisms. We also discuss experimental and clinical strategies proposed to counteract cancer immune evasion and emphasize the importance of implementing appropriate murine models like the transgenic adenocarcinoma of the mouse prostate model for investigating the biology of prostate cancer and novel immunotherapy approaches against it.

The Insider: Impact of the Gut Microbiota on Cancer Immunity and Response to Therapies in Multiple Myeloma.

The human microbiota is a unique set of microorganisms colonizing the human body and evolving within it from the very beginning. Acting as an insider, the microbiota provides nutrients, and mutualistically interacts with the host's immune system, thus contributing to the generation of barriers against pathogens. While a strong link has been documented between intestinal dysbiosis (i.e., disruption to the microbiota homeostasis) and diseases, the mechanisms by which commensal bacteria impact a wide spectrum of mucosal and extramucosal human disorders have only partially been deciphered. This is particularly puzzling for multiple myeloma (MM), a treatable but incurable neoplasia of plasma cells that accumulate in the bone marrow and lead to end-organ damage. Here we revise the most recent literature on data from both the bench and the bedside that show how the gut microbiota modulates cancer immunity, potentially impacting the progression of asymptomatic monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM) to full blown MM. We also explore the effect of the gut microbiome on hematopoietic stem cell transplantation, chemotherapy, immunomodulating therapy and cancer immunotherapy in MM patients. Additionally, we identify the most cogent area of investigation that have the highest chance to delineate microbiota-related and pathobiology-based parameters for patient risk stratification. Lastly, we highlight microbiota-modulating strategies (i.e., diet, prebiotics, probiotics, fecal microbiota transplantation and postbiotics) that may reduce treatment-related toxicity in patients affected by MM as well as the rates of undertreatment of SMM patients.

A novel expressed prostatic secretion (EPS)-urine metabolomic signature for the diagnosis of clinically significant prostate cancer.

OBJECTIVE: Significant efforts are currently being made to identify novel biomarkers for the diagnosis and risk stratification of prostate cancer (PCa). Metabolomics can be a very useful approach in biomarker discovery because metabolites are an important read-out of the disease when characterized in biological samples. We aimed to determine a metabolomic signature which can accurately distinguish men with clinically significant PCa from those affected by benign prostatic hyperplasia (BPH). METHODS: We first performed untargeted metabolomics using ultrahigh-performance liquid chromatography tandem mass spectrometry on expressed prostatic secretion urine (EPS-urine) from 25 patients affected by BPH and 25 men with clinically significant PCa (defined as Gleason score ≥ 3 + 4). Diagnosis was histologically confirmed after surgical treatment. The EPS-urine metabolomic approach was then applied to a larger, prospective cohort of 92 consecutive patients undergoing multiparametric magnetic resonance imaging for clinical suspicion of PCa prior to biopsy. RESULTS: We established a novel metabolomic signature capable of accurately distinguishing PCa from benign tissue. A metabolomic signature was associated with clinically significant PCa in all subgroups of the Prostate Imaging Reporting and Data System (PI-RADS) classification (100% and 89.13% of accuracy when the PI-RADS was in range of 1-2 and 4-5, respectively, and 87.50% in the more critical cases when the PI-RADS was 3). CONCLUSIONS: A combination of metabolites and clinical variables can effectively help in identifying PCa patients that might be overlooked by current imaging technologies. Metabolites from EPS-urine should help in defining the diagnostic pathway of PCa, thus improving PCa detection and decreasing the number of unnecessary prostate biopsies.

CD4+ T cells sustain aggressive chronic lymphocytic leukemia in Eµ-TCL1 mice through a CD40L-independent mechanism.

Chronic lymphocytic leukemia (CLL) is caused by the progressive accumulation of mature CD5+ B cells in secondary lymphoid organs. In vitro data suggest that CD4+ T lymphocytes also sustain survival and proliferation of CLL clones through CD40L/CD40 interactions. In vivo data in animal models are conflicting. To clarify this clinically relevant biological issue, we generated genetically modified Eµ-TCL1 mice lacking CD4+ T cells (TCL1+/+AB0), CD40 (TCL1+/+CD40-/-), or CD8+ T cells (TCL1+/+TAP-/-), and we monitored the appearance and progression of a disease that mimics aggressive human CLL by flow cytometry and immunohistochemical analyses. Findings were confirmed by adoptive transfer of leukemic cells into mice lacking CD4+ T cells or CD40L or mice treated with antibodies depleting CD4 T cells or blocking CD40L/CD40 interactions. CLL clones did not proliferate in mice lacking or depleted of CD4+ T cells, thus confirming that CD4+ T cells are essential for CLL development. By contrast, CD8+ T cells exerted an antitumor activity, as indicated by the accelerated disease progression in TCL1+/+TAP-/- mice. Antigen specificity of CD4+ T cells was marginal for CLL development, because CLL clones efficiently proliferated in transgenic mice whose CD4 T cells had a T-cell receptor with CLL-unrelated specificities. Leukemic clones also proliferated when transferred into wild-type mice treated with monoclonal antibodies blocking CD40 or into CD40L-/- mice, and TCL1+/+CD40-/- mice developed frank CLL. Our data demonstrate that CD8+ T cells restrain CLL progression, whereas CD4+ T cells support the growth of leukemic clones in TCL1 mice through CD40-independent and apparently noncognate mechanisms.

[18F](2S,4R)-4-Fluoroglutamine as a New Positron Emission Tomography Tracer in Myeloma.

The high glycolytic activity of multiple myeloma (MM) cells is the rationale for use of Positron Emission Tomography (PET) with 18F-fluorodeoxyglucose ([18F]FDG) to detect both bone marrow (BM) and extramedullary disease. However, new tracers are actively searched because [18F]FDG-PET has some limitations and there is a portion of MM patients who are negative. Glutamine (Gln) addiction has been recently described as a typical metabolic feature of MM cells. Yet, the possible exploitation of Gln as a PET tracer in MM has never been assessed so far and is investigated in this study in preclinical models. Firstly, we have synthesized enantiopure (2S,4R)-4-fluoroglutamine (4-FGln) and validated it as a Gln transport analogue in human MM cell lines, comparing its uptake with that of 3H-labelled Gln. We then radiosynthesized [18F]4-FGln, tested its uptake in two different in vivo murine MM models, and checked the effect of Bortezomib, a proteasome inhibitor currently used in the treatment of MM. Both [18F]4-FGln and [18F]FDG clearly identified the spleen as site of MM cell colonization in C57BL/6 mice, challenged with syngeneic Vk12598 cells and assessed by PET. NOD.SCID mice, subcutaneously injected with human MM JJN3 cells, showed high values of both [18F]4-FGln and [18F]FDG uptake. Bortezomib significantly reduced the uptake of both radiopharmaceuticals in comparison with vehicle at post treatment PET. However, a reduction of glutaminolytic, but not of glycolytic, tumor volume was evident in mice showing the highest response to Bortezomib. Our data indicate that [18F](2S,4R)-4-FGln is a new PET tracer in preclinical MM models, yielding a rationale to design studies in MM patients.

Anticancer innovative therapy congress: Highlights from the 10th anniversary edition.

During the Tenth Edition of the Annual Congress on "Anticancer Innovative Therapy" [Milan, 23/24 January 2020], experts in the fields of immuno-oncology, epigenetics, tumor cell signaling, and cancer metabolism shared their latest knowledge on the roles of i] epigenetics, and in particular, chromatin modifiers, ii] cancer metabolism, iii] cancer stem cells [CSCs], iv] tumor cell signaling, and iv] the immune system. The novel therapeutic approaches presented included epigenetic drugs, cell cycle inhibitors combined with ICB, antibiotics and other off-label drugs, small-molecules active against CSCs, liposome-delivered miRNAs, tumor-specific CAR-T cells, and T-cell-based immunotherapy. Moreover, important evidence on possible mechanisms of resistance to these innovative therapies were also discussed, in particular with respect to resistance to ICB. Overall, this conference provided scientists and clinicians with a broad overview of future challenges and hopes to improve cancer treatment reasonably in the medium-short term.

Commensal bacteria promote endocrine resistance in prostate cancer through androgen biosynthesis.

The microbiota comprises the microorganisms that live in close contact with the host, with mutual benefit for both counterparts. The contribution of the gut microbiota to the emergence of castration-resistant prostate cancer (CRPC) has not yet been addressed. We found that androgen deprivation in mice and humans promotes the expansion of defined commensal microbiota that contributes to the onset of castration resistance in mice. Specifically, the intestinal microbial community in mice and patients with CRPC was enriched for species capable of converting androgen precursors into active androgens. Ablation of the gut microbiota by antibiotic therapy delayed the emergence of castration resistance even in immunodeficient mice. Fecal microbiota transplantation (FMT) from CRPC mice and patients rendered mice harboring prostate cancer resistant to castration. In contrast, tumor growth was controlled by FMT from hormone-sensitive prostate cancer patients and Prevotella stercorea administration. These results reveal that the commensal gut microbiota contributes to endocrine resistance in CRPC by providing an alternative source of androgens.