Acidity and hypoxia of tumor microenvironment, a positive interplay in extracellular vesicle release by tumor cells.

The complex and continuously evolving features of the tumor microenvironment, varying between tumor histotypes, are characterized by the presence of host cells and tumor cells embedded in a milieu shaped by hypoxia and low pH, resulting from the frequent imbalance between vascularity and tumor cell proliferation. These microenvironmental metabolic stressors play a crucial role in remodeling host cells and tumor cells, contributing to the stimulation of cancer cell heterogeneity, clonal evolution, and multidrug resistance, ultimately leading to progression and metastasis. The extracellular vesicles (EVs), membrane-enclosed structures released into the extracellular milieu by tumor/host cells, are now recognized as critical drivers in the complex intercellular communication between tumor cells and the local cellular components in a hypoxic/acidic microenvironment. Understanding the intricate molecular mechanisms governing the interactions between tumor and host cells within a hypoxic and acidic microenvironment, triggered by the release of EVs, could pave the way for innovative strategies to disrupt the complex interplay of cancer cells with their microenvironment. This approach may contribute to the development of an efficient and safe therapeutic strategy to combat cancer progression. Therefore, we review the major findings on the release of EVs in a hypoxic/acidic tumor microenvironment to appreciate their role in tumor progression toward metastatic disease.

The CAIX inhibitor SLC-0111 exerts anti-cancer activity on gastric cancer cell lines and resensitizes resistant cells to 5-Fluorouracil, taxane-derived, and platinum-based drugs.

Gastric cancer (GC) is the fifth most frequent malignancy and the fourth leading cause of worldwide cancer-related death. Despite the usage of multimodal perioperative chemotherapy (pCT), GC progressively gains chemoresistance, thereby, the identification of suitable targets to overcome drug resistance is fundamental. Amongst the potential biomarkers, carbonic anhydrase IX (CAIX) - associated with a poor prognosis of several solid cancers - has gained the most attention. In a cohort of GC patients who received perioperative FLOT (i.e., Leucovorin, 5-Fluouracil, Docetaxel, and Oxaliplatin) or FOLFOX (i.e., Leucovorin, 5-Fluouracil, and Oxaliplatin), non-responder patients showed an increased expression of tumor CAIX compared to responder group. Moreover, GC cell lines induced to be resistant to 5-Fluouracil, Paclitaxel, Cisplatin, or the combination of 5-Fluorouracil, Oxaliplatin, and Docetaxel, overexpressed CAIX compared to the control. Accordingly, CAIX-high-expressing GC cells showed increased therapy resistance compared to low-expressing cells. Notably, SLC0111 significantly improved the therapy response of both wild-type and resistant GC cells. Overall, these data suggest a correlation between CAIX and GC drug resistance highlighting the potential of SLC-0111 in re-sensitizing GC cells to pCT.

Schwann cell insulin-like growth factor receptor type-1 mediates metastatic bone cancer pain in mice.

Insulin growth factor-1 (IGF-1), an osteoclast-dependent osteolysis biomarker, contributes to metastatic bone cancer pain (MBCP), but the underlying mechanism is poorly understood. In mice, the femur metastasis caused by intramammary inoculation of breast cancer cells resulted in IGF-1 increase in femur and sciatic nerve, and IGF-1-dependent stimulus/non-stimulus-evoked pain-like behaviors. Adeno-associated virus-based shRNA selective silencing of IGF-1 receptor (IGF-1R) in Schwann cells, but not in dorsal root ganglion (DRG) neurons, attenuated pain-like behaviors. Intraplantar IGF-1 evoked acute nociception and mechanical/cold allodynia, which were reduced by selective IGF-1R silencing in DRG neurons and Schwann cells, respectively. Schwann cell IGF-1R signaling promoted an endothelial nitric oxide synthase-mediated transient receptor potential ankyrin 1 (TRPA1) activation and release of reactive oxygen species that, via macrophage-colony stimulating factor-dependent endoneurial macrophage expansion, sustained pain-like behaviors. Osteoclast derived IGF-1 initiates a Schwann cell-dependent neuroinflammatory response that sustains a proalgesic pathway that provides new options for MBCP treatment.

Nintedanib-αVß6 Integrin Ligand Conjugates Reduce TGFß-Induced EMT in Human Non-Small Cell Lung Cancer.

Growth factors and cytokines released in the lung cancer microenvironment promote an epithelial-to-mesenchymal transition (EMT) that sustains the progression of neoplastic diseases. TGFß is one of the most powerful inducers of this transition, as it induces overexpression of the fibronectin receptor, αvß6 integrin, in cancer cells which, in turn, is strongly associated with EMT. Thus, αvß6 integrin receptors may be exploited as a target for the selective delivery of anti-tumor agents. We introduce three novel synthesized conjugates, in which a selective αvß6 receptor ligand is linked to nintedanib, a potent kinase inhibitor used to treat advanced adenocarcinoma lung cancer in clinics. The αvß6 integrin ligand directs nintedanib activity to the target cells of the tumor microenvironment, avoiding the onset of negative side effects in normal cells. We found that the three conjugates inhibit the adhesion of cancer cells to fibronectin in a concentration-dependent manner and that αvß6-expressing cells internalized the conjugated compounds, thus permitting nintedanib to inhibit 2D and 3D cancer cell growth and suppress the clonogenic ability of the EMT phenotype as well as intervening in other aspects associated with the EMT transition. These results highlight αvß6 receptors as privileged access points for dual-targeting molecular conjugates engaged in an efficient and precise strategy against non-small cell lung cancer.

Extracellular Acidosis Differentially Regulates Estrogen Receptor ß-Dependent EMT Reprogramming in Female and Male Melanoma Cells.

Clinical outcomes of melanoma patients pointed out a gender disparity that supports a correlation between sex hormone activity on estrogen receptors (ER) and melanoma development and progression. Here, we found that the epithelial-to-mesenchymal transition (EMT) of melanoma cells induced by extracellular acidosis, which is a crucial hallmark of solid cancers, correlates with the expression of ERß, the most representative ER on melanoma cells. Extracellular acidosis induces an enhanced expression of ERß in female cells and EMT markers remain unchanged, while extracellular acidosis did not induce the expression of ERß in male cells and EMT was strongly promoted. An inverse relationship between ERß expression and EMT markers in melanoma cells of different sex exposed to extracellular acidosis was revealed by two different technical approaches: florescence-activated cell sorting of high ERß expressing cell subpopulations and ERß receptor silencing. Finally, we found that ERß regulates EMT through NF-κB activation. These results demonstrate that extracellular acidosis drives a differential ERß regulation in male and female melanoma cells and that this gender disparity might open new perspectives for personalized therapeutic approaches.

Physicochemical aspects of the tumour microenvironment as drivers of vasculogenic mimicry.

Tumour vascularisation is vital for cancer sustainment representing not only the main source of nutrients and oxygen supply but also an escape route for single or clustered cancer cells that, once detached from the primary mass, enter the blood circulation and disseminate to distant organs. Among the mechanisms identified to contribute to tumour vascularisation, vasculogenic mimicry (VM) is gaining increasing interest in the scientific community representing an intriguing target for cancer treatment. VM indeed associates with highly aggressive tumour phenotypes and strongly impairs patient outcomes. Differently from vessels of healthy tissues, tumour vasculature is extremely heterogeneous and tortuous, impeding efficient chemotherapy delivery, and at the meantime hyperpermeable and thus extremely accessible to metastasising cancer cells. Moreover, tumour vessel disorganisation creates a self-reinforcing vicious circle fuelling cancer malignancy and progression. Because of the inefficient oxygen delivery and metabolic waste removal from tumour vessels, many cells within the tumour mass indeed experience hypoxia and acidosis, now considered hallmarks of cancer. Being strong inducers of vascularisation, therapy resistance, inflammation and metastasis, hypoxia and acidosis create a permissive microenvironment for cancer progression and dissemination. Along with these considerations, we decided to focus our attention on the relationship between hypoxia/acidosis and VM. Indeed, besides tumour angiogenesis, VM is strongly influenced by both hypoxia and acidosis, which could potentiate each other and fuel this vicious circle. Thus, targeting hypoxia and acidosis may represent a potential target to treat VM to impair tumour perfusion and cancer cell sustainment.

miR-214-Enriched Extracellular Vesicles Released by Acid-Adapted Melanoma Cells Promote Inflammatory Macrophage-Dependent Tumor Trans-Endothelial Migration.

The understanding of the molecular mechanisms leading to melanoma dissemination is urgently needed in view of the identification of new targets and the development of innovative strategies to improve patients' outcomes. Within the complexity of tumor intercellular communications leading to metastatic dissemination, extracellular vesicles (EV) released by tumor cells are central players. Indeed, the ability to travel through the circulatory system conveying oncogenic bioactive molecules even at distant sites makes EV capable of modulating recipient cells to facilitate metastatic dissemination. The dynamic remodeling of the tumor microenvironment might influence, along with a number of other events, tumoral EV release. We observed that, in melanoma, extracellular acidosis increases the release of EV enriched in miR-214, an onco-miRNA involved in melanoma metastasis. Then, miR-214-enriched EV were found to induce a state of macrophage activation, leading to an overproduction of proinflammatory cytokines and nitric oxide. Such an inflammatory microenvironment was able to alter the endothelial cell permeability, thereby facilitating the trans-endothelial migration of melanoma cells, a crucial step in the metastatic cascade. The use of synthetic miR-214 inhibitors and miR-214 overexpression allowed us to demonstrate the key role of miR-214 in the EV-dependent induction of macrophage activation. Overall, our in vitro study reveals that the release of tumor miR-214-enriched EV, potentiated by adapting tumor cells to extracellular acidosis, drives a macrophage-dependent trans-endothelial migration of melanoma cells. This finding points to miR-214 as a potential new therapeutic target to prevent melanoma intravasation.

An Oleocanthal-Enriched EVO Oil Extract Induces the ROS Production in Gastric Cancer Cells and Potentiates the Effect of Chemotherapy.

Oleocanthal, a minor polar compound in extra-virgin olive (EVO) oil, contains anticancer properties, which should be encouraged in its use in oncology. Gastric Cancer (GC), a very aggressive human cancer, is often diagnosed at advanced stages, when surgery is substituted or supported by chemotherapy (CT). However, CT frequently fails due to the patient's resistance to the treatment. Thus, the aim of this study is to verify whether an OC-enriched EVO oil extract fraction (OCF) may be useful in order to overcome a resistance to GC. We evaluated the OCF effects on an AGS gastric adenocarcinoma cell line wild type (AGS wt) and on its subpopulations resistant to 5-fluorouracil (5FUr), Paclitaxel (TAXr) or cisplatin (CISr). We found that a 60 µM dose of the OCF acts on the AGS wt, 5FUr and TAXr, leading to the cell cycle inhibition and to a ROS production, but not on CISr cells. Resistance of CISr to the OCF seems to be due to higher levels of antioxidant-enzymes that can counteract the OCF-induced ROS production. Moreover, using the OCF plus 5-fluorouracil, Paclitaxel or cisplatin, we found a potentiating effect compared with a mono-treatment in all resistant GC cells, including CISr. In conclusion, the use of the OCF in the management of GC has shown very interesting advantages, opening-up the possibility to evaluate the efficacy of the OCF in vivo, as a valid adjuvant in the treatment of resistant GC.

Nintedanib-Containing Dual Conjugates Targeting αVß6 Integrin and Tyrosine Kinase Receptors as Potential Antifibrotic Agents.

αVß6 Integrin plays a fundamental role in the activation of transforming growth factor-ß (TGF-ß), the major profibrotic mediator; for this reason, αVß6 ligands have recently been forwarded to clinical phases for the therapy of fibrotic diseases. Herein, we report the synthesis and in vitro biological evaluation as antifibrotic agents of three new covalent conjugates, constituted by c(AmpLRGDL), an αVß6 integrin-recognizing small cyclopeptide, and nintedanib, a tyrosine kinase inhibitor approved for idiopathic pulmonary fibrosis (IPF) treatment. One of these conjugates recapitulates optimal in vitro antifibrotic properties of the two active units. The integrin ligand portion within the conjugate plays a role in inhibiting profibrotic stimuli, potentiating the nintedanib effect and favoring the selective uptake of the conjugate in cells overexpressing αVß6 integrin. These results may open a new perspective on the development of dual conjugates in the targeted therapy of IPF.

Design, synthesis and evaluation of RGD peptidomimetic - Gold nanostar conjugates as M21 cell adhesion inhibitors.

Effective targeting of αvß3 integrin is of high relevance in cancer research as this protein is overexpressed on several types of tumor cells, making such receptor ideal for the development of therapeutics and of diagnostic imaging agents. In this paper, the synthesis of a novel functionalized triazole-based RGD peptidomimetic and its covalent conjugation on pegylated gold nanostars is reported. These highly stable nanoconstructs showed a multivalent effect in binding αvß3 integrin receptors and proved to inhibit M21 cell adhesion at 25 pM concentration. Thanks to their peculiar surface plasmon resonance in the "NIR transparent window", targeted gold nanostars may represent a promising agent for anticancer multi-modality treatments. 2009 Elsevier Ltd. All rights reserved.

Modular synthesis of 2,4-diaminoanilines as CNS drug-like non-covalent inhibitors of asparagine endopeptidase.

Asparagine endopeptidase (AEP), also called legumain, is a pH-dependent endolysosomal cysteine protease that cleaves its substrates after asparagine residues. Recent studies showed that it possesses δ-secretase activity and that it is implicated in numerous neurological diseases such as Alzheimer's disease (AD). Following evidence of aryl-morpholines as useful asparagine endopeptidase inhibitors, a series of morpholinoanilines with diverse substituents at ortho position were synthesized in view of improving the potency and scope of this molecular scaffold, allowing to identify ethyl 2-isonipecotate-4-morpholinoaniline possessing inhibition potency in the nanomolar range. CNS MPO (CNS MultiParameter Optimization) calculations revealed that most of the compounds developed in this work show physicochemical parameters in the desirable range for CNS drug candidates.

Lactate Rewires Lipid Metabolism and Sustains a Metabolic-Epigenetic Axis in Prostate Cancer.

Lactate is an abundant oncometabolite in the tumor environment. In prostate cancer, cancer-associated fibroblasts (CAF) are major contributors of secreted lactate, which can be taken up by cancer cells to sustain mitochondrial metabolism. However, how lactate impacts transcriptional regulation in tumors has yet to be fully elucidated. Here, we describe a mechanism by which CAF-secreted lactate is able to increase the expression of genes involved in lipid metabolism in prostate cancer cells. This regulation enhanced intracellular lipid accumulation in lipid droplets (LD) and provided acetyl moieties for histone acetylation, establishing a regulatory loop between metabolites and epigenetic modification. Inhibition of this loop by targeting the bromodomain and extraterminal protein family of histone acetylation readers suppressed the expression of perilipin 2 (PLIN2), a crucial component of LDs, disrupting lactate-dependent lipid metabolic rewiring. Inhibition of this CAF-induced metabolic-epigenetic regulatory loop in vivo reduced growth and metastasis of prostate cancer cells, demonstrating its translational relevance as a therapeutic target in prostate cancer. Clinically, PLIN2 expression was elevated in tumors with a higher Gleason grade and in castration-resistant prostate cancer compared with primary prostate cancer. Overall, these findings show that lactate has both a metabolic and an epigenetic role in promoting prostate cancer progression. SIGNIFICANCE: This work shows that stromal-derived lactate induces accumulation of lipid droplets, stimulates epigenetic rewiring, and fosters metastatic potential in prostate cancer.

Identification of a Common Pharmacophore for Binding to MMP2 and RGD Integrin: Towards a Multitarget Approach to Inhibit Cancer Angiogenesis and Metastasis.

During tumor angiogenesis different growth factors, cytokines and other molecules interact closely with each other to facilitate tumor cell invasion and metastatic diffusion. The most intensively studied as molecular targets in anti-angiogenic therapies are vascular endothelial growth factor (VEGF) and related receptors, integrin receptors and matrix metalloproteinases (MMPs). Considering the poor efficacy of cancer angiogenesis monotherapies, we reasoned combining the inhibition of αvß3 and MMP2 as a multitarget approach to deliver a synergistic blockade of tumor cell migration, invasion and metastasis. Accordingly, we identified a common pharmacophore in the binding cavity of MMP2 and αvß3, demonstrating such approach with the design, synthesis and bioassays of tyrosine-derived peptidomimetics carrying the necessary functional groups to bind to key pharmacophoric elements of MMP2 and αvß3 RGD integrin.

Integrin-Mediated Targeted Cancer Therapy Using c(RGDyK)-Based Conjugates of Gemcitabine.

c(RGDyK)-based conjugates of gemcitabine (GEM) with the carbonate and carbamate linkages in the 6-OH group of GEM were synthesized for the targeted delivery of GEM to integrin αvß3, overexpressing cancer cells to increase the stability as well as the tumor delivery of GEM and minimize common side effects associated with GEM treatment. Competitive cell uptake experiments demonstrated that conjugate TC113 could be internalized by A549 cells through integrin αvß3. Among the synthesized conjugates, TC113 bearing the carbamate linker was stable in human plasma and was further assessed in an in vivo pharmacokinetic study. TC113 appeared to be relatively stable, releasing GEM slowly into blood, while it showed potent antiproliferative properties against WM266.4 and A549 cells. The encouraging data presented in this study with respect to TC113 provide a promising keystone for further investigation of this GEM conjugate with potential future clinical applications.

uPAR Controls Vasculogenic Mimicry Ability Expressed by Drug-Resistant Melanoma Cells.

Malignant melanoma is a highly aggressive skin cancer characterized by an elevated grade of tumor cell plasticity. Such plasticity allows adaptation of melanoma cells to different hostile conditions and guarantees tumor survival and disease progression, including aggressive features such as drug resistance. Indeed, almost 50% of melanoma rapidly develop resistance to the BRAFV600E inhibitor vemurafenib, with fast tumor dissemination, a devastating consequence for patients outcomes. Vasculogenic mimicry (VM), the ability of cancer cells to organize themselves in perfused vascular-like channels, might sustain tumor spread by providing vemurafenib-resistant cancer cells with supplementary ways to enter into circulation and disseminate. Thus, this research aims to determine if vemurafenib resistance goes with the acquisition of VM ability by aggressive melanoma cells, and identify a driving molecule for both vemurafenib resistance and VM. We used two independent experimental models of drug-resistant melanoma cells, the first one represented by a chronic adaptation of melanoma cells to extracellular acidosis, known to drive a particularly aggressive and vemurafenib-resistant phenotype, the second one generated with chronic vemurafenib exposure. By performing in vitro tube formation assay and evaluating the expression levels of the VM markers EphA2 and VE-cadherin by Western blotting and flow cytometer analyses, we demonstrated that vemurafenib-resistant cells obtained by both models are characterized by an increased ability to perform VM. Moreover, by exploiting the CRISPR-Cas9 technique and using the urokinase plasminogen activator receptor (uPAR) inhibitor M25, we identified uPAR as a driver of VM expressed by vemurafenib-resistant melanoma cells. Thus, uPAR targeting may be successfully leveraged as a new complementary therapy to inhibit VM in drug-resistant melanoma patients, to counteract the rapid progression and dissemination of the disease.

Oleuropein-Rich Leaf Extract as a Broad Inhibitor of Tumour and Macrophage iNOS in an Apc Mutant Rat Model.

Oleuropein, the major compound found in olive leaves, has been reported to exert numerous pharmacological properties, including anti-inflammatory, anti-diabetic and anti-cancer effects. The purpose of this study was to evaluate, for the first time, the effect of oleuropein-rich leaf extracts (ORLE) in already-developed colon tumours arising in Apc (adenomatous polyposis coli) mutated PIRC rats (F344/NTac-Apcam1137). Here, we were able to investigate in parallel the anti-cancer effect of ORLE, both in vivo and in vitro, and its anti-inflammatory effect on macrophages, representing a critical and abundant population in most solid tumour microenvironment. We found that in vivo ORLE treatment promoted apoptosis and attenuated iNOS activity both in colon tumours as in peritoneal macrophages of PIRC rats. We this confirmed in vitro using primary RAW264.7 cells: ORLE reduced iNOS activity in parallel with COX-2 and pro-inflammatory cytokines, such as IL-1ß, IL-6 and TGF-ß. These findings suggest that ORLE possess a strong anti-inflammatory activity, which could be crucial for dampening the pro-tumourigenic activity elicited by a chronic inflammatory state generated by either tumour cells or tumour-associated macrophages.

New 4-Aminoproline-Based Small Molecule Cyclopeptidomimetics as Potential Modulators of α4ß1 Integrin.

Integrin α4ß1 belongs to the leukocyte integrin family and represents a therapeutic target of relevant interest given its primary role in mediating inflammation, autoimmune pathologies and cancer-related diseases. The focus of the present work is the design, synthesis and characterization of new peptidomimetic compounds that are potentially able to recognize α4ß1 integrin and interfere with its function. To this aim, a collection of seven new cyclic peptidomimetics possessing both a 4-aminoproline (Amp) core scaffold grafted onto key α4ß1-recognizing sequences and the (2-methylphenyl)ureido-phenylacetyl (MPUPA) appendage, was designed, with the support of molecular modeling studies. The new compounds were synthesized through SPPS procedures followed by in-solution cyclization maneuvers. The biological evaluation of the new cyclic ligands in cell adhesion assays on Jurkat cells revealed promising submicromolar agonist activity in one compound, namely, the c[Amp(MPUPA)Val-Asp-Leu] cyclopeptide. Further investigations will be necessary to complete the characterization of this class of compounds.

CRISPR/Cas9 uPAR Gene Knockout Results in Tumor Growth Inhibition, EGFR Downregulation and Induction of Stemness Markers in Melanoma and Colon Carcinoma Cell Lines.

uPAR is a globular protein, tethered to the cell membrane by a GPI-anchor involved in several cancer-related properties and its overexpression commonly correlates with poor prognosis and metastasis. We investigated the consequences of uPAR irreversible loss in human melanoma and colon cancer cell lines, knocking out its expression by CRISPR/Cas9. We analyzed through flow cytometry, western blotting and qPCR, the modulation of the most known cancer stem cells-associated genes and the EGFR while we observed the proliferation rate exploiting 2D and 3D cellular models. We also generated uPAR "rescue" expression cell lines as well as we promoted the expression of only its 3'UTR to demonstrate the involvement of uPAR mRNA in tumor progression. Knocking out PLAUR, uPAR-encoding gene, we observed an inhibited growth ratio unexpectedly coupled with a significant percentage of cells acquiring a stem-like phenotype. In vivo experiments demonstrated that uPAR loss completely abrogates tumorigenesis despite the gained stem-like profile. Nonetheless, we proved that the reintroduction of the 3'UTR of PLAUR gene was sufficient to restore the wild-type status validating the hypothesis that such a region may act as a "molecular sponge". In particular miR146a, by binding PLAUR 3' UTR region might be responsible for uPAR-dependent inhibition of EGFR expression.

Synthesis and biological studies of c(RGDyK) conjugates of cucurbitacins.

Cucurbitacins (CUCUs) are triterpenoids known to display potent cytotoxic effects; however, their clinical application is limited due to poor pharmacokinetics and systemic toxicity. This work focuses on the development of c(RGDyK)-CUCU conjugates for the selective delivery of CUCUs to integrin-overexpressing cancer cells. The activity of the conjugates against various cancer cells was studied. They exhibited a mild cytostatic effect to six cancer cell lines and a cytotoxic effect against integrin-overexpressing MCF-7 and A549 cells. Their chemical and metabolic stability was extensively studied using LC-MS analysis. The conjugates maintained high affinity for αvß3 integrin receptors. c(RGDyK) conjugation via a PEG linker was beneficial for CUCU-D and the resulting conjugate was approximately three-times more active than the free CUCU-D in MCF7 cells.

Enhanced Antitumoral Activity and Photoacoustic Imaging Properties of AuNP-Enriched Endothelial Colony Forming Cells on Melanoma.

Near infrared (NIR)-resonant gold nanoparticles (AuNPs) hold great promise in cancer diagnostics and treatment. However, translating the theranostic potential of AuNPs into clinical applications still remains a challenge due to the difficulty to improve the efficiency and specificity of tumor delivery in vivo as well as the clearance from liver and spleen to avoid off target toxicity. In this study, endothelial colony forming cells (ECFCs) are exploited as vehicles to deliver AuNPs to tumors. It is first demonstrated that ECFCs display a great capability to intake AuNPs without losing viability, and exert antitumor activity per se. Using a human melanoma xenograft mouse model, it is next demonstrated that AuNP-loaded ECFCs retain their capacity to migrate to tumor sites in vivo 1 day after injection and stay in the tumor mass for more than 1 week. In addition, it is demonstrated that ECFC-loaded AuNPs are efficiently cleared by the liver over time and do not elicit any sign of damage to healthy tissue.