Small-Cell Lung Cancer: Is Liquid Biopsy a New Tool Able to Predict the Efficacy of Immunotherapy?

Small-cell lung cancer (SCLC) cases represent approximately 15% of all lung cancer cases, remaining a recalcitrant malignancy with poor survival and few treatment options. In the last few years, the addition of immunotherapy to chemotherapy improved clinical outcomes compared to chemotherapy alone, resulting in the current standard of care for SCLC. However, the advantage of immunotherapy only applies to a few SCLC patients, and predictive biomarkers selection are lacking for SCLC. In particular, due to some features of SCLC, such as high heterogeneity, elevated cell plasticity, and low-quality tissue samples, SCLC biopsies cannot be used as biomarkers. Therefore, the characterization of the tumor and, subsequently, the selection of an appropriate therapeutic combination may benefit greatly from liquid biopsy. Soluble factors, circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and extracellular vesicles (EVs) are now useful tools in the characterization of SCLC. This review summarizes the most recent data on biomarkers detectable with liquid biopsy, emphasizing their role in supporting tumor detection and their potential role in SCLC treatment choice.

Microfluidic development and biological evaluation of targeted therapy-loaded biomimetic nano system to improve the metastatic melanoma treatment.

Optimizing current therapies is among next steps in metastatic melanoma (MM) treatment landscape. The innovation of this study is the design of production process by microfluidics of cell membrane (CM)-modified nanoparticles (NPs), as an emerging biomimetic platform that allows for reduced immune clearance, long blood circulation time and improved specific tumor targeting. To achieve melanoma selectivity, direct membrane fusion between synthetic liposomes and CMs extracted from MM cell line was performed by microfluidic sonication approach, then the hybrid liposomes were loaded with cobimetinib (Cob) or lenvatinib (Lenva) targeting agents and challenged against MM cell lines and liver cancer cell line to evaluate homotypic targeting and antitumor efficacy. Characterization studies demonstrated the effective fusion of CM with liposome and the high encapsulation efficiency of both drugs, showing the proficiency of microfluidic-based production. By studying the targeting of melanoma cells by hybrid liposomes versus liposomes, we found that both NPs entered cells through endocytosis, whereas the former showed higher selectivity for MM cells from which CM was extracted, with 8-fold higher cellular uptake than liposomes. Hybrid liposome formulation of Cob and Lenva reduced melanoma cells viability to a greater extent than liposomes and free drug and, notably, showed negligible toxicity as demonstrated by bona fide haemolysis test. The CM-modified NPs presented here have the potential to broaden the choice of therapeutic options in MM treatment.

Circulating extracellular vesicles are monitoring biomarkers of anti-PD1 response and enhancer of tumor progression and immunosuppression in metastatic melanoma.

BACKGROUND: Clinical drawback in checkpoint inhibitors immunotherapy (ICI) of metastatic melanoma (MM) is monitoring clinical benefit. Soluble forms of PD1(sPD1) and PD-L1(sPD-L1) and extracellular vesicles (EVs) expressing PD1 and PD-L1 have recently emerged as predictive biomarkers of response. As factors released in the blood, EVs and soluble forms could be relevant in monitoring treatment efficacy and adaptive resistance to ICI. METHODS: We used pre-therapy plasma samples of 110 MM patients and longitudinal samples of 46 patients. Elisa assay and flow cytometry (FCM) were used to measure sPD-L1 and sPD1 concentrations and the percentage of PD1+ EVs and PD-L1+ EVs, released from tumor and immune cells in patients subsets. Transwell assays were conducted to investigate the impact of EVs of each patient subset on MM cells invasion and interaction between tumor cells and macrophages or dendritic cells. Viability assays were performed to assess EVs effect on MM cells and organoids sensitivity to anti-PD1. FCM was used to investigate immunosuppressive markers in EVs and immune cells. RESULTS: The concentrations of sPD1 and sPD-L1 in pre-treatment and longitudinal samples did not correlate with anti-PD1 response, instead only tumor-derived PD1+ EVs decreased in long responders while increased during disease progression in responders. Notably, we observed reduction of T cell derived EVs expressing LAG3+ and PD1+ in long responders and their increase in responders experiencing progression. By investigating the impact of EVs on disease progression, we found that those isolated from non-responders and from patients with progression disease accelerated tumor cells invasiveness and migration towards macrophages, while EVs of long responders reduced the metastatic potential of MM cells and neo-angiogenesis. Additionally, the EVs of non-responders and of progression disease patients subset reduced the sensitivity of MM cells and organoids of responder to anti-PD1 and the recruitment of dendritic cells, while the EVs of progression disease subset skewed macrophages to express higher level of PDL-1. CONCLUSION: Collectively, we suggest that the detection of tumor-derived PD1 + EVs may represent a useful tool for monitoring the response to anti-PD1 and a role for EVs shed by tumor and immune cells in promoting tumor progression and immune dysfunction.

Cervical cancer benefits from trabectedin combination with the ß-blocker propranolol: in vitro and ex vivo evaluations in patient-derived organoids.

Background: Cervical cancer (CC) is characterized by genomic alterations in DNA repair genes, which could favor treatment with agents causing DNA double-strand breaks (DSBs), such as trabectedin. Hence, we evaluated the capability of trabectedin to inhibit CC viability and used ovarian cancer (OC) models as a reference. Since chronic stress may promote gynecological cancer and may hinder the efficacy of therapy, we investigated the potential of targeting ß-adrenergic receptors with propranolol to enhance trabectedin efficacy and change tumor immunogenicity. Methods: OC cell lines, Caov-3 and SK-OV-3, CC cell lines, HeLa and OV2008, and patient-derived organoids were used as study models. MTT and 3D cell viability assays were used for drug(s) IC50 determination. The analysis of apoptosis, JC-1 mitochondrial membrane depolarization, cell cycle, and protein expression was performed by flow cytometry. Cell target modulation analyses were carried out by gene expression, Western blotting, immunofluorescence, and immunocytochemistry. Results: Trabectedin reduced the proliferation of both CC and OC cell lines and notably of CC patient-derived organoids. Mechanistically, trabectedin caused DNA DSBs and S-phase cell cycle arrest. Despite DNA DSBs, cells failed the formation of nuclear RAD51 foci and underwent apoptosis. Under norepinephrine stimulation, propranolol enhanced trabectedin efficacy, further inducing apoptosis through the involvement of mitochondria, Erk1/2 activation, and the increase of inducible COX-2. Notably, trabectedin and propranolol affected the expression of PD1 in both CC and OC cell lines. Conclusion: Overall, our results show that CC is responsive to trabectedin and provide translational evidence that could benefit CC treatment options. Our study pointed out that combined treatment offset trabectedin resistance caused by ß-adrenergic receptor activation in both ovarian and cervical cancer models.

Gene Expression Comparison between Alcohol-Exposed versus Not Exposed Pancreatic Ductal Adenocarcinoma Patients Reveals a Peculiar TGFß-Related Phenotype: An Exploratory Analysis.

Background: Over the past few decades, there has been much debate and research into the link between alcohol consumption and the development and progression of pancreatic ductal adenocarcinoma (PDAC). Objectives: To contribute to the ongoing discussion and gain further insights into this topic, our study analysed the gene expression differences in PDAC patients based on their alcohol consumption history. Methods: To this end, we interrogated a large publicly available dataset. We next validated our findings in vitro. Results: Our findings revealed that patients with a history of alcohol consumption showed significant enrichment in the TGFß-pathway: a signaling pathway implicated in cancer development and tumor progression. Specifically, our bioinformatic dissection of gene expression differences in 171 patients with PDAC showed that those who had consumed alcohol had higher levels of TGFß-related genes. Moreover, we validated the role of the TGFß pathway as one of the molecular drivers in producing massive stroma, a hallmark feature of PDAC, in patients with a history of alcohol consumption. This suggests that inhibition of the TGFß pathway could serve as a novel therapeutic target for PDAC patients with a history of alcohol consumption and lead to increased sensitivity to chemotherapy. Our study provides valuable insights into the molecular mechanisms underlying the link between alcohol consumption and PDAC progression. Conclusions: Our findings highlight the potential significance of the TGFß pathway as a therapeutic target. The development of TGFß-inhibitors may pave the way for developing more effective treatment strategies for PDAC patients with a history of alcohol consumption.

The role of unpaired image-to-image translation for stain color normalization in colorectal cancer histology classification.

BACKGROUND: Histological assessment of colorectal cancer (CRC) tissue is a crucial and demanding task for pathologists. Unfortunately, manual annotation by trained specialists is a burdensome operation, which suffers from problems like intra- and inter-pathologist variability. Computational models are revolutionizing the Digital Pathology field, offering reliable and fast approaches for challenges like tissue segmentation and classification. With this respect, an important obstacle to overcome consists in stain color variations among different laboratories, which can decrease the performance of classifiers. In this work, we investigated the role of Unpaired Image-to-Image Translation (UI2IT) models for stain color normalization in CRC histology and compared to classical normalization techniques for Hematoxylin-Eosin (H&E) images. METHODS: Five Deep Learning normalization models based on Generative Adversarial Networks (GANs) belonging to the UI2IT paradigm have been thoroughly compared to realize a robust stain color normalization pipeline. To avoid the need for training a style transfer GAN between each pair of data domains, in this paper we introduce the concept of training by exploiting a meta-domain, which contains data coming from a wide variety of laboratories. The proposed framework enables a huge saving in terms of training time, by allowing to train a single image normalization model for a target laboratory. To prove the applicability of the proposed workflow in the clinical practice, we conceived a novel perceptive quality measure, which we defined as Pathologist Perceptive Quality (PPQ). The second stage involved the classification of tissue types in CRC histology, where deep features extracted from Convolutional Neural Networks have been exploited to realize a Computer-Aided Diagnosis system based on a Support Vector Machine (SVM). To prove the reliability of the system on new data, an external validation set composed of N = 15,857 tiles has been collected at IRCCS Istituto Tumori "Giovanni Paolo II". RESULTS: The exploitation of a meta-domain consented to train normalization models that allowed achieving better classification results than normalization models explicitly trained on the source domain. PPQ metric has been found correlated to quality of distributions (Fréchet Inception Distance - FID) and to similarity of the transformed image to the original one (Learned Perceptual Image Patch Similarity - LPIPS), thus showing that GAN quality measures introduced in natural image processing tasks can be linked to pathologist evaluation of H&E images. Furthermore, FID has been found correlated to accuracies of the downstream classifiers. The SVM trained on DenseNet201 features allowed to obtain the highest classification results in all configurations. The normalization method based on the fast variant of CUT (Contrastive Unpaired Translation), FastCUT, trained with the meta-domain paradigm, allowed to achieve the best classification result for the downstream task and, correspondingly, showed the highest FID on the classification dataset. CONCLUSIONS: Stain color normalization is a difficult but fundamental problem in the histopathological setting. Several measures should be considered for properly assessing normalization methods, so that they can be introduced in the clinical practice. UI2IT frameworks offer a powerful and effective way to perform the normalization process, providing realistic images with proper colorization, unlike traditional normalization methods that introduce color artifacts. By adopting the proposed meta-domain framework, the training time can be reduced, and the accuracy of downstream classifiers can be increased.

Probing Single-Cell Fermentation Fluxes and Exchange Networks via pH-Sensing Hybrid Nanofibers.

The homeostatic control of their environment is an essential task of living cells. It has been hypothesized that, when microenvironmental pH inhomogeneities are induced by high cellular metabolic activity, diffusing protons act as signaling molecules, driving the establishment of exchange networks sustained by the cell-to-cell shuttling of overflow products such as lactate. Despite their fundamental role, the extent and dynamics of such networks is largely unknown due to the lack of methods in single-cell flux analysis. In this study, we provide direct experimental characterization of such exchange networks. We devise a method to quantify single-cell fermentation fluxes over time by integrating high-resolution pH microenvironment sensing via ratiometric nanofibers with constraint-based inverse modeling. We apply our method to cell cultures with mixed populations of cancer cells and fibroblasts. We find that the proton trafficking underlying bulk acidification is strongly heterogeneous, with maximal single-cell fluxes exceeding typical values by up to 3 orders of magnitude. In addition, a crossover in time from a networked phase sustained by densely connected "hubs" (corresponding to cells with high activity) to a sparse phase dominated by isolated dipolar motifs (i.e., by pairwise cell-to-cell exchanges) is uncovered, which parallels the time course of bulk acidification. Our method addresses issues ranging from the homeostatic function of proton exchange to the metabolic coupling of cells with different energetic demands, allowing for real-time noninvasive single-cell metabolic flux analysis.

circPVT1 and PVT1/AKT3 show a role in cell proliferation, apoptosis, and tumor subtype-definition in small cell lung cancer.

Small cell lung cancer (SCLC) is treated as a homogeneous disease, although the expression of NEUROD1, ASCL1, POU2F3, and YAP1 identifies distinct molecular subtypes. The MYC oncogene, amplified in SCLC, was recently shown to act as a lineage-specific factor to associate subtypes with histological classes. Indeed, MYC-driven SCLCs show a distinct metabolic profile and drug sensitivity. To disentangle their molecular features, we focused on the co-amplified PVT1, frequently overexpressed and originating circular (circRNA) and chimeric RNAs. We analyzed hsa_circ_0001821 (circPVT1) and PVT1/AKT3 (chimPVT1) as examples of such transcripts, respectively, to unveil their tumorigenic contribution to SCLC. In detail, circPVT1 activated a pro-proliferative and anti-apoptotic program when over-expressed in lung cells, and knockdown of chimPVT1 induced a decrease in cell growth and an increase of apoptosis in SCLC in vitro. Moreover, the investigated PVT1 transcripts underlined a functional connection between MYC and YAP1/POU2F3, suggesting that they contribute to the transcriptional landscape associated with MYC amplification. In conclusion, we have uncovered a functional role of circular and chimeric PVT1 transcripts in SCLC; these entities may prove useful as novel biomarkers in MYC-amplified tumors.

BRAFV600E;K601Q metastatic melanoma patient-derived organoids and docking analysis to predict the response to targeted therapy.

The V600E mutation in BRAF is associated with increased phosphorylation of Erk1/2 and high sensitivity to BRAFi/MEKi combination in metastatic melanoma. In very few patients, a tandem mutation in BRAF, V600 and K601, causes a different response to BRAFi/MEKi combination. BRAFV600E;K601Q patient-derived organoids (PDOs) were generated to investigate targeted therapy efficacy and docking analysis was used to assess BRAFV600E;K601Q interactions with Vemurafenib. PDOs were not sensitive to Vemurafenib and Cobimetinib given alone and sensitive to their combination, although not as responsive as BRAFV600E PDOs. The docking analysis justified such a result showing that the tandem mutation in BRAF reduced the affinity for Vemurafenib. Tumor analysis showed that BRAFV600E;K601Q displayed both increased phosphorylation of Erk1/2 at cytoplasmic level and activation of Notch resistance signaling. This prompted us to inhibit Notch signaling with Nirogacestat, achieving a greater antitumor response and providing PDOs-based evaluation of treatment efficacy in such rare metastatic melanoma.

Circulating extracellular vesicles expressing PD1 and PD-L1 predict response and mediate resistance to checkpoint inhibitors immunotherapy in metastatic melanoma.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Magnetic implants in vivo guiding sorafenib liver delivery by superparamagnetic solid lipid nanoparticles.

HYPOTHESIS: Solid lipid nanoparticles (SLNs), co-encapsulating superparamagnetic iron oxide nanoparticles and sorafenib, have been exploited for magnetic-guided drug delivery to the liver. Two different magnetic configurations, both comprising two small magnets, were under-skin implanted to investigate the effect of the magnetic field topology on the magnetic SLNP accumulation in liver tissues. A preliminary simulation analysis was performed to predict the magnetic field topography for each tested configuration. EXPERIMENTS: SLNs were prepared using a hot homogenization approach and characterized using complementary techniques. Their in vitro biological behavior was assessed in HepG-2 liver cancer cells; wild-type mice were used for the in vivo study. The magnet configuration that resulted in a higher magnetic targeting efficiency was investigated by evaluating the iron content in homogenated murine liver tissues. FINDINGS: SLNs, characterized by an average size smaller than 200 nm, retained their superparamagnetic behavior and relevant molecular resonance imaging properties as negative contrast agents. The evaluation of iron accumulation in the liver tissues was consistent with the magnetic induction profile of each magnet configuration, concurring with the results predicted by simulation analysis and obtained by measurements in living mice.

The ERRα-VDR axis promotes calcitriol degradation and estrogen signaling in breast cancer cells, while VDR-CYP24A1-ERRα overexpression correlates with poor prognosis in patients with basal-like breast cancer.

Vitamin D is used to reduce cancer risk and improve the outcome of cancer patients, but the vitamin D receptor (VDR; also known as the calcitriol receptor) pathway needs to be functionally intact to ensure the biological effects of circulating calcitriol, the active form of vitamin D. Besides estrogen receptor alpha (ERα), estrogen-related receptor alpha (ERRα) has also been shown to interfere with the VDR pathway, but its role in the antitumor and transactivation activity of calcitriol is completely unknown in breast cancer (BC). We observed that ERRα functionally supported the proliferation of BC cell lines and acted as a calcitriol-induced regulator of VDR. As such, ERRα deregulated the calcitriol-VDR transcription by enhancing the expression of CYP24A1 as well as of both ERα and aromatase (CYP19A1) in calcitriol-treated cells. ERRα knockdown limited the effect of calcitriol by reducing calcitriol-induced G0/G1 phase cell cycle arrest and by affecting the expression of cyclin D1 and p21/Waf. The interactome analysis suggested that Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-α (PGC-1α) and Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) are key players in the genomic actions of the calcitriol-VDR-ERRα axis. Evaluation of patient outcomes in The Cancer Genome Atlas (TCGA) dataset showed the translational significance of the biological effects of the VDR-ERRα axis, highlighting that VDR, CYP24A1, and ERRα overexpression correlates with poor prognosis in basal-like BC.

Natural Bovine Coronavirus Infection in a Calf Persistently Infected with Bovine Viral Diarrhea Virus: Viral Shedding, Immunological Features and S Gene Variations.

The evolution of a bovine coronavirus (BCoV) natural infection in a calf persistently infected with bovine viral diarrhea virus (BVDV) was described. The infected calf developed intermittent nasal discharge, diarrhea and hyperthermia. The total number of leukocytes/mL and the absolute differential number of neutrophils and lymphocytes resulted within the normal range, but monocytes increased at T28 (time 28 post-infection). Flow-cytometry analysis evidenced that the CD8+ subpopulation increased at T7 and between T28 and T35. BCoV shedding in nasal discharges and feces was detected up to three weeks post infection and high antibody titers persisted up to T56. The RNA BCoV load increased until T14, contrary to what was observed in a previous study where the fecal excretion of BCoV was significantly lower in the co-infected (BCoV/BVDV) calves than in the calves infected with BCoV only. We can suppose that BVDV may have modulated the BCoV infection exacerbating the long viral excretion, as well as favoring the onset of mutations in the genome of BCoV detected in fecal samples at T21. An extensive study was performed to verify if the selective pressure in the S gene could be a natural mode of variation of BCoV, providing data for the identification of new epidemic strains, genotypes or recombinant betacoronaviruses.

Salting-Out Approach Is Worthy of Comparison with Ultracentrifugation for Extracellular Vesicle Isolation from Tumor and Healthy Models.

The role of extracellular vesicles (EVs) has been completely re-evaluated in the recent decades, and EVs are currently considered to be among the main players in intercellular communication. Beyond their functional aspects, there is strong interest in the development of faster and less expensive isolation protocols that are as reliable for post-isolation characterisations as already-established methods. Therefore, the identification of easy and accessible EV isolation techniques with a low price/performance ratio is of paramount importance. We isolated EVs from a wide spectrum of samples of biological and clinical interest by choosing two isolation techniques, based on their wide use and affordability: ultracentrifugation and salting-out. We collected EVs from human cancer and healthy cell culture media, yeast, bacteria and Drosophila culture media and human fluids (plasma, urine and saliva). The size distribution and concentration of EVs were measured by nanoparticle tracking analysis and dynamic light scattering, and protein depletion was measured by a colorimetric nanoplasmonic assay. Finally, the EVs were characterised by flow cytometry. Our results showed that the salting-out method had a good efficiency in EV separation and was more efficient in protein depletion than ultracentrifugation. Thus, salting-out may represent a good alternative to ultracentrifugation.

Microfluidic preparation and in vitro evaluation of iRGD-functionalized solid lipid nanoparticles for targeted delivery of paclitaxel to tumor cells.

Solid lipid nanoparticles (SLNs) can combine the advantages of different colloidal carriers and prevent some of their disadvantages. The production of nanoparticles by means of microfluidics represents a successful platform for industrial scale-up of nanoparticle manufacture in a reproducible way. The realisation of a microfluidic technique to obtain SLNs in a continuous and reproducible manner encouraged us to create surface functionalised SLNs for targeted drug release using the same procedure. A tumor homing peptide, iRGD, owning a cryptic C-end Rule (CendR) motif is responsible for neuropilin-1 (NRP-1) binding and for triggering extravasation and tumor penetration of the peptide. In this study, the Paclitaxel loaded-SLNs produced by microfluidics were functionalized with the iRGD peptide. The SLNs proved to be stable in aqueous medium andwere characterized by a Z-average under 150 nm, a polydispersity index below 0.2, a zeta-potential between -20 and -35 mV and a drug encapsulation efficiency around 40%. Moreover, in vitro cytotoxic effects and cellular uptake have been assessed using 2D and 3D tumour models of U87 glioblastoma cell lines. Overall, these results demonstrate that the surface functionalization of SLNs with iRGD allow better cellular uptake and cytotoxicity ability.

Behind the Scene: Exploiting MC1R in Skin Cancer Risk and Prevention.

Melanoma and non-melanoma skin cancers (NMSCs) are the most frequent cancers of the skin in white populations. An increased risk in the development of skin cancers has been associated with the combination of several environmental factors (i.e., ultraviolet exposure) and genetic background, including melanocortin-1 receptor (MC1R) status. In the last few years, advances in the diagnosis of skin cancers provided a great impact on clinical practice. Despite these advances, NMSCs are still the most common malignancy in humans and melanoma still shows a rising incidence and a poor prognosis when diagnosed at an advanced stage. Efforts are required to underlie the genetic and clinical heterogeneity of melanoma and NMSCs, leading to an optimization of the management of affected patients. The clinical implications of the impact of germline MC1R variants in melanoma and NMSCs' risk, together with the additional risk conferred by somatic mutations in other peculiar genes, as well as the role of MC1R screening in skin cancers' prevention will be addressed in the current review.

The Pharmaceutical Technology Approach on Imaging Innovations from Italian Research.

Many modern therapeutic approaches are based on precise diagnostic evidence, where imaging procedures play an essential role. To date, in the diagnostic field, a plethora of agents have been investigated to increase the selectivity and sensitivity of diagnosis. However, the most common drawbacks of conventional imaging agents reside in their non-specificity, short imaging time, instability, and toxicity. Moreover, routinely used diagnostic agents have low molecular weights and consequently a rapid clearance and renal excretion, and this represents a limitation if long-lasting imaging analyses are to be conducted. Thus, the development of new agents for in vivo diagnostics requires not only a deep knowledge of the physical principles of the imaging techniques and of the physiopathological aspects of the disease but also of the relative pharmaceutical and biopharmaceutical requirements. In this scenario, skills in pharmaceutical technology have become highly indispensable in order to respond to these needs. This review specifically aims to collect examples of newly developed diagnostic agents connoting the importance of an appropriate formulation study for the realization of effective products. Within the context of pharmaceutical technology research in Italy, several groups have developed and patented promising agents for fluorescence and radioactive imaging, the most relevant of which are described hereafter.

New Oxaliplatin-Pyrophosphato Analogs with Improved In Vitro Cytotoxicity.

Two new Pt(II)-pyrophosphato complexes containing the carrier ligands cis-1,3-diaminocyclohexane (cis-1,3-DACH) and trans-1,2-diamine-4-cyclohexene (1,2-DACHEX), variants of the 1R,2R-diaminocyclohexane ligand present in the clinically used Pt-drug oxaliplatin, have been synthesized with the aim of developing new potential antitumor drugs with high bone tropism. The complexes are more stable at physiological pH than in acid conditions, with Na2[Pt(pyrophosphato)(cis-1,3-DACH)] (1) slightly more stable than [Pt(dihydrogenpyrophosphato)(1,2-DACHEX)] (2). The greater reactivity at acidic pH ensures a greater efficacy at the tumor site. Preliminary NMR studies indicate that 1 and 2 react slowly with 5'-GMP (used as a model of nucleic acids), releasing the pyrophosphate ligand and affording the bis 5'-GMP adduct. In vitro cytotoxicity assays performed against a panel of four human cancer cell lines have shown that both compounds are more active than oxaliplatin. Flow cytometry studies on HCT116 cells showed that the pyrophosphato compounds with the non-classical 1,3- and 1,4-diaminocyclohexane ligands (1 and 4) are the most capable to induce cells' death by apoptosis and necrosis.

The Interaction between Reactive Peritoneal Mesothelial Cells and Tumor Cells via Extracellular Vesicles Facilitates Colorectal Cancer Dissemination.

Advanced colorectal cancer (CRC) is highly metastatic and often results in peritoneal dissemination. The extracellular vesicles (EVs) released by cancer cells in the microenvironment are important mediators of tumor metastasis. We investigated the contribution of EV-mediated interaction between peritoneal mesothelial cells (MCs) and CRC cells in generating a pro-metastatic environment in the peritoneal cavity. Peritoneal MCs isolated from peritoneal lavage fluids displayed high CD44 expression, substantial mesothelial-to-mesenchymal transition (MMT) and released EVs that both directed tumor invasion and caused reprogramming of secretory profiles by increasing TGF-ß1 and uPA/uPAR expression and MMP-2/9 activation in tumor cells. Notably, the EVs released by tumor cells induced apoptosis by activating caspase-3, peritoneal MC senescence, and MMT, thereby augmenting the tumor-promoting potential of these cells in the peritoneal cavity. By using pantoprazole, we reduced the biogenesis of EVs and their pro-tumor functions. In conclusion, our findings provided evidence of underlying mechanisms of CRC dissemination driven by the interaction of peritoneal MCs and tumor cells via the EVs released in the peritoneal cavity, which may have important implications for the clinical management of patients.

uPAR+ extracellular vesicles: a robust biomarker of resistance to checkpoint inhibitor immunotherapy in metastatic melanoma patients.

BACKGROUND: Emerging evidence has highlighted the importance of extracellular vesicle (EV)-based biomarkers of resistance to immunotherapy with checkpoint inhibitors in metastatic melanoma. Considering the tumor-promoting implications of urokinase-type plasminogen activator receptor (uPAR) signaling, this study aimed to assess uPAR expression in the plasma-derived EVs of patients with metastatic melanoma to determine its potential correlation with clinical outcomes. METHODS: Blood samples from 71 patients with metastatic melanoma were collected before initiating immunotherapy. Tumor-derived and immune cell-derived EVs were isolated and analyzed to assess the relative percentage of uPAR+ EVs. The associations between uPAR and clinical outcomes, sex, BRAF status, baseline lactate dehydrogenase levels and number of metastatic sites were assessed. RESULTS: Responders had a significantly lower percentage of tumor-derived, dendritic cell (DC)-derived and CD8+ T cell-derived uPAR +EVs at baseline than non-responders. The Kaplan-Meier survival curves for the uPAR+EV quartiles indicated that higher levels of melanoma-derived uPAR+ EVs were strongly correlated with poorer progression-free survival (p<0.0001) and overall survival (p<0.0001). We also found a statistically significant correlation between lower levels of uPAR+ EVs from both CD8+ T cells and DCs and better survival. CONCLUSIONS: Our results indicate that higher levels of tumor-derived, DC-derived and CD8+ T cell-derived uPAR+ EVs in non-responders may represent a new biomarker of innate resistance to immunotherapy with checkpoint inhibitors. Moreover, uPAR+ EVs represent a new potential target for future therapeutic approaches.