Cell Electrokinetic Fingerprint: A Novel Approach Based on Optically Induced Dielectrophoresis (ODEP) for In-Flow Identification of Single Cells.

A novel optically induced dielectrophoresis (ODEP) system that can operate under flow conditions is designed for automatic trapping of cells and subsequent induction of 2D multi-frequency cell trajectories. Like in a "ping-pong" match, two virtual electrode barriers operate in an alternate mode with varying frequencies of the input voltage. The so-derived cell motions are characterized via time-lapse microscopy, cell tracking, and state-of-the-art machine learning algorithms, like the wavelet scattering transform (WST). As a cell-electrokinetic fingerprint, the dynamic of variation of the cell displacements happening, over time, is quantified in response to different frequency values of the induced electric field. When tested on two biological scenarios in the cancer domain, the proposed approach discriminates cellular dielectric phenotypes obtained, respectively, at different early phases of drug-induced apoptosis in prostate cancer (PC3) cells and for differential expression of the lectine-like oxidized low-density lipoprotein receptor-1 (LOX-1) transcript levels in human colorectal adenocarcinoma (DLD-1) cells. The results demonstrate increased discrimination of the proposed system and pose an additional basis for making ODEP-based assays addressing cancer heterogeneity for precision medicine and pharmacological research.

The Impressive Anti-Inflammatory Activity of Cerium Oxide Nanoparticles: More than Redox?

Cerium oxide nanoparticles (CNPs) are biocompatible nanozymes exerting multifunctional biomimetic activities, including superoxide dismutase (SOD), catalase, glutathione peroxidase, photolyase, and phosphatase. SOD- and catalase-mimesis depend on Ce3+/Ce4+ redox switch on nanoparticle surface, which allows scavenging the most noxious reactive oxygen species in a self-regenerating, energy-free manner. As oxidative stress plays pivotal roles in the pathogenesis of inflammatory disorders, CNPs have recently attracted attention as potential anti-inflammatory agents. A careful survey of the literature reveals that CNPs, alone or as constituents of implants and scaffolds, strongly contrast chronic inflammation (including neurodegenerative and autoimmune diseases, liver steatosis, gastrointestinal disorders), infections, and trauma, thereby ameliorating/restoring organ function. By general consensus, CNPs inhibit inflammation cues while boosting the pro-resolving anti-inflammatory signaling pathways. The mechanism of CNPs' anti-inflammatory effects has hardly been investigated, being rather deductively attributed to CNP-induced ROS scavenging. However, CNPs are multi-functional nanozymes that exert additional bioactivities independent from the Ce3+/Ce4+ redox switch, such as phosphatase activity, which could conceivably mediate some of the anti-inflammatory effects reported, suggesting that CNPs fight inflammation via pleiotropic actions. Since CNP anti-inflammatory activity is potentially a pharmacological breakthrough, it is important to precisely attribute the described effects to one or another of their nanozyme functions, thus achieving therapeutic credibility.

Safe-Shields: Basal and Anti-UV Protection of Human Keratinocytes by Redox-Active Cerium Oxide Nanoparticles Prevents UVB-Induced Mutagenesis.

Cerium oxide nanoparticles (nanoceria), biocompatible multifunctional nanozymes exerting unique biomimetic activities, mimic superoxide-dismutase and catalase through a self-regenerating, energy-free redox cycle driven by Ce3+/4+ valence switch. Additional redox-independent UV-filter properties render nanoceria ideal multitask solar screens, shielding from UV exposure, simultaneously protecting tissues from UV-oxidative damage. Here, we report that nanoceria favour basal proliferation of primary normal keratinocytes, and protects them from UVB-induced DNA damage, mutagenesis, and apoptosis, minimizing cell loss and accelerating recovery with flawless cells. Similar cell-protective effects were found on irradiated noncancerous, but immortalized, p53-null HaCaT keratinocytes, with the notable exception that here, nanoceria do not accelerate basal HaCaT proliferation. Notably, nanoceria protect HaCaT from oxidative stress induced by irradiated titanium dioxide nanoparticles, a major active principle of commercial UV-shielding lotions, thus neutralizing their most critical side effects. The intriguing combination of nanoceria multiple beneficial properties opens the way for smart and safer containment measures of UV-induced skin damage and carcinogenesis.

Androgen Deprivation Freezes Hormone-Sensitive Prostate Cancer Cells in a Reversible, Genetically Unstable Quasi-Apoptotic State, Bursting into Full Apoptosis upon Poly(ADP-ribose) Polymerase Inhibition.

Androgen deprivation therapy (ADT) is a powerful treatment for metastatic hormone-sensitive prostate cancer (mHSPC) patients, but eventually and inevitably, cancer relapses, progressing to the fatal castration-resistant (CR)PC stage. Progression implies the emergence of cells proliferating in the absence of androgen through still elusive mechanisms. We show here for the first time that ADT induces LNCaP mHSPC cells to collectively enter a metastable quasi-apoptotic state (QUAPS) consisting of partial mitochondrial permeabilization, limited BAX and caspase activation, and moderate induction of caspase-dependent dsDNA breaks; despite this, cells maintain full viability. QUAPS is destabilized by poly(ADP)-polymerase inhibition (PARPi), breaking off toward overt intrinsic apoptosis and culture extinction. Instead, QUAPS is rapidly and efficiently reverted upon androgen restoration, with mitochondria rapidly recovering integrity and cells collectively resuming normal proliferation. Notably, replication restarts before DNA repair is completed, and implies an increased micronuclei frequency, indicating that ADT promotes genetic instability. The recovered cells re-acquire insensitivity to PARPi (as untreated LNCaP), pointing to specific, context-dependent vulnerability of mHSPC cells to PARPi during ADT. Summarizing, QUAPS is an unstable, pro-mutagenic state developing as a pro-survival pathway stabilized by PARP, and constitutes a novel viewpoint explaining how ADT-treated mHSPC may progress to CRPC, indicating possible preventive countermeasures.

Peroxisome proliferator-activated receptorα/γ agonist pioglitazone for rescuing relapsed or refractory neoplasias by unlocking phenotypic plasticity.

A series of seven clinical trials on relapsed or refractory (r/r) metastatic neoplasias followed the question: Are networks of ligand-receptor cross-talks that support tumor-specific cancer hallmarks, druggable with tumor tissue editing approaches therapeutically exploiting tumor plasticity? Differential recombinations of pioglitazone, a dual peroxisome-proliferator activated receptorα/γ (PPARα/γ) agonist, with transcriptional modulators, i.e., all-trans retinoic acid, interferon-α, or dexamethasone plus metronomic low-dose chemotherapy (MCT) or epigenetic modeling with azacitidine plus/minus cyclooxygenase-2 inhibition initiated tumor-specific reprogramming of cancer hallmarks, as exemplified by inflammation control in r/r melanoma, renal clear cell carcinoma (RCCC), Hodgkin's lymphoma (HL) and multisystem Langerhans cell histiocytosis (mLCH) or differentiation induction in non-promyelocytic acute myeloid leukemia (non-PML AML). Pioglitazone, integrated in differentially designed editing schedules, facilitated induction of tumor cell death as indicated by complete remission (CR) in r/r non-PML AML, continuous CR in r/r RCCC, mLCH, and in HL by addition of everolimus, or long-term disease control in melanoma by efficaciously controlling metastasis, post-therapy cancer repopulation and acquired cell-resistance and genetic/molecular-genetic tumor cell heterogeneity (M-CRAC). PPARα/γ agonists provided tumor-type agnostic biomodulatory efficacy across different histologic neoplasias. Tissue editing techniques disclose that wide-ranging functions of PPARα/γ agonists may be on-topic focused for differentially unlocking tumor phenotypes. Low-dose MCT facilitates targeted reprogramming of cancer hallmarks with transcriptional modulators, induction of tumor cell death, M-CRAC control and editing of non-oncogene addiction. Thus, pioglitazone, integrated in tumor tissue editing protocols, is an important biomodulatory drug for addressing urgent therapeutic problems, such as M-CRAC in relapsed or refractory tumor disease.

Addressing Genetic Tumor Heterogeneity, Post-Therapy Metastatic Spread, Cancer Repopulation, and Development of Acquired Tumor Cell Resistance.

The concept of post-therapy metastatic spread, cancer repopulation and acquired tumor cell resistance (M-CRAC) rationalizes tumor progression because of tumor cell heterogeneity arising from post-therapy genetic damage and subsequent tissue repair mechanisms. Therapeutic strategies designed to specifically address M-CRAC involve tissue editing approaches, such as low-dose metronomic chemotherapy and the use of transcriptional modulators with or without targeted therapies. Notably, tumor tissue editing holds the potential to treat patients, who are refractory to or relapsing (r/r) after conventional chemotherapy, which is usually based on administering a maximum tolerable dose of a cytostatic drugs. Clinical trials enrolling patients with r/r malignancies, e.g., non-small cell lung cancer, Hodgkin's lymphoma, Langerhans cell histiocytosis and acute myelocytic leukemia, indicate that tissue editing approaches could yield tangible clinical benefit. In contrast to conventional chemotherapy or state-of-the-art precision medicine, tissue editing employs a multi-pronged approach targeting important drivers of M-CRAC across various tumor entities, thereby, simultaneously engaging tumor cell differentiation, immunomodulation, and inflammation control. In this review, we highlight the M-CRAC concept as a major factor in resistance to conventional cancer therapies and discusses tissue editing as a potential treatment.

Drug Repurposing by Tumor Tissue Editing.

The combinatory use of drugs for systemic cancer therapy commonly aims at the direct elimination of tumor cells through induction of apoptosis. An alternative approach becomes the focus of attention if biological changes in tumor tissues following combinatory administration of regulatorily active drugs are considered as a therapeutic aim, e.g., differentiation, transdifferentiation induction, reconstitution of immunosurveillance, the use of alternative cell death mechanisms. Editing of the tumor tissue establishes new biological 'hallmarks' as a 'pressure point' to attenuate tumor growth. This may be achieved with repurposed, regulatorily active drug combinations, often simultaneously targeting different cell compartments of the tumor tissue. Moreover, tissue editing is paralleled by decisive functional changes in tumor tissues providing novel patterns of target sites for approved drugs. Thus, agents with poor activity in non-edited tissue may reveal new clinically meaningful outcomes. For tissue editing and targeting edited tissue novel requirements concerning drug selection and administration can be summarized according to available clinical and pre-clinical data. Monoactivity is no pre-requisite, but combinatory bio-regulatory activity. The regulatorily active dose may be far below the maximum tolerable dose, and besides inhibitory active drugs stimulatory drug activities may be integrated. Metronomic scheduling often seems to be of advantage. Novel preclinical approaches like functional assays testing drug combinations in tumor tissue are needed to select potential drugs for repurposing. The two-step drug repurposing procedure, namely establishing novel functional systems states in tumor tissues and consecutively providing novel target sites for approved drugs, facilitates the systematic identification of drug activities outside the scope of any original clinical drug approvals.

Apoptosis as Driver of Therapy-Induced Cancer Repopulation and Acquired Cell-Resistance (CRAC): A Simple In Vitro Model of Phoenix Rising in Prostate Cancer.

Apoptotic cells stimulate compensatory proliferation through the caspase-3-cPLA-2-COX-2-PGE-2-STAT3 Phoenix Rising pathway as a healing process in normal tissues. Phoenix Rising is however usurped in cancer, potentially nullifying pro-apoptotic therapies. Cytotoxic therapies also promote cancer cell plasticity through epigenetic reprogramming, leading to epithelial-to-mesenchymal-transition (EMT), chemo-resistance and tumor progression. We explored the relationship between such scenarios, setting-up an innovative, straightforward one-pot in vitro model of therapy-induced prostate cancer repopulation. Cancer (castration-resistant PC3 and androgen-sensitive LNCaP), or normal (RWPE-1) prostate cells, are treated with etoposide and left recovering for 18 days. After a robust apoptotic phase, PC3 setup a coordinate tissue-like response, repopulating and acquiring EMT and chemo-resistance; repopulation occurs via Phoenix Rising, being dependent on high PGE-2 levels achieved through caspase-3-promoted signaling; epigenetic inhibitors interrupt Phoenix Rising after PGE-2, preventing repopulation. Instead, RWPE-1 repopulate via Phoenix Rising without reprogramming, EMT or chemo-resistance, indicating that only cancer cells require reprogramming to complete Phoenix Rising. Intriguingly, LNCaP stop Phoenix-Rising after PGE-2, failing repopulating, suggesting that the propensity to engage/complete Phoenix Rising may influence the outcome of pro-apoptotic therapies. Concluding, we established a reliable system where to study prostate cancer repopulation, showing that epigenetic reprogramming assists Phoenix Rising to promote post-therapy cancer repopulation and acquired cell-resistance (CRAC).

Metabolic Reprogramming of Castration-Resistant Prostate Cancer Cells as a Response to Chemotherapy.

Prostate cancer at the castration-resistant stage (CRPC) is a leading cause of death among men due to resistance to anticancer treatments, including chemotherapy. We set up an in vitro model of therapy-induced cancer repopulation and acquired cell resistance (CRAC) on etoposide-treated CRPC PC3 cells, witnessing therapy-induced epithelial-to-mesenchymal-transition (EMT) and chemoresistance among repopulating cells. Here, we explore the metabolic changes leading to chemo-induced CRAC, measuring the exchange rates cell/culture medium of 36 metabolites via Nuclear Magnetic Resonance spectroscopy. We studied the evolution of PC3 metabolism throughout recovery from etoposide, encompassing the degenerative, quiescent, and repopulating phases. We found that glycolysis is immediately shut off by etoposide, gradually recovering together with induction of EMT and repopulation. Instead, OXPHOS, already high in untreated PC3, is boosted by etoposide to decline afterward, though stably maintaining values higher than control. Notably, high levels of EMT, crucial in the acquisition of chemoresistance, coincide with a strong acceleration of metabolism, especially in the exchange of principal nutrients and their end products. These results provide novel information on the energy metabolism of cancer cells repopulating from cytotoxic drug treatment, paving the way for uncovering metabolic vulnerabilities to be possibly pharmacologically targeted and providing novel clinical options for CRPC.

Deciphering Cancer Cell Behavior From Motility and Shape Features: Peer Prediction and Dynamic Selection to Support Cancer Diagnosis and Therapy.

Cell motility varies according to intrinsic features and microenvironmental stimuli, being a signature of underlying biological phenomena. The heterogeneity in cell response, due to multilevel cell diversity especially relevant in cancer, poses a challenge in identifying the biological scenario from cell trajectories. We propose here a novel peer prediction strategy among cell trajectories, deciphering cell state (tumor vs. nontumor), tumor stage, and response to the anticancer drug etoposide, based on morphology and motility features, solving the strong heterogeneity of individual cell properties. The proposed approach first barcodes cell trajectories, then automatically selects the good ones for optimal model construction (good teacher and test sample selection), and finally extracts a collective response from the heterogeneous populations via cooperative learning approaches, discriminating with high accuracy prostate noncancer vs. cancer cells of high vs. low malignancy. Comparison with standard classification methods validates our approach, which therefore represents a promising tool for addressing clinically relevant issues in cancer diagnosis and therapy, e.g., detection of potentially metastatic cells and anticancer drug screening.

Polylactic is a Sustainable, Low Absorption, Low Autofluorescence Alternative to Other Plastics for Microfluidic and Organ-on-Chip Applications.

Organ-on-chip (OOC) devices are miniaturized devices replacing animal models in drug discovery and toxicology studies. The majority of OOC devices are made from polydimethylsiloxane (PDMS), an elastomer widely used in microfluidic prototyping, but posing a number of challenges to experimentalists, including leaching of uncured oligomers and uncontrolled absorption of small compounds. Here we assess the suitability of polylactic acid (PLA) as a replacement material to PDMS for microfluidic cell culture and OOC applications. We changed the wettability of PLA substrates and demonstrated the functionalization method to be stable over a time period of at least 9 months. We successfully cultured human cells on PLA substrates and devices, without coating. We demonstrated that PLA does not absorb small molecules, is transparent (92% transparency), and has low autofluorescence. As a proof of concept of its manufacturability, biocompatibility, and transparency, we performed a cell tracking experiment of prostate cancer cells in a PLA device for advanced cell culture.

A Camera Sensors-Based System to Study Drug Effects On In Vitro Motility: The Case of PC-3 Prostate Cancer Cells.

Cell motility is the brilliant result of cell status and its interaction with close environments. Its detection is now possible, thanks to the synergy of high-resolution camera sensors, time-lapse microscopy devices, and dedicated software tools for video and data analysis. In this scenario, we formulated a novel paradigm in which we considered the individual cells as a sort of sensitive element of a sensor, which exploits the camera as a transducer returning the movement of the cell as an output signal. In this way, cell movement allows us to retrieve information about the chemical composition of the close environment. To optimally exploit this information, in this work, we introduce a new setting, in which a cell trajectory is divided into sub-tracks, each one characterized by a specific motion kind. Hence, we considered all the sub-tracks of the single-cell trajectory as the signals of a virtual array of cell motility-based sensors. The kinematics of each sub-track is quantified and used for a classification task. To investigate the potential of the proposed approach, we have compared the achieved performances with those obtained by using a single-trajectory paradigm with the scope to evaluate the chemotherapy treatment effects on prostate cancer cells. Novel pattern recognition algorithms have been applied to the descriptors extracted at a sub-track level by implementing features, as well as samples selection (a good teacher learning approach) for model construction. The experimental results have put in evidence that the performances are higher when a further cluster majority role has been considered, by emulating a sort of sensor fusion procedure. All of these results highlighted the high strength of the proposed approach, and straightforwardly prefigure its use in lab-on-chip or organ-on-chip applications, where the cell motility analysis can be massively applied using time-lapse microscopy images.

A Computational Model of Tumor Growth and Anakoinosis.

Anakoinosis is a new cancer treatment paradigm that posits a key role for communicative reprogramming within tumor systems. To date no mathematical or computational models of anakoinosis have been developed. Here we outline the NEATG_A system, a first computational model of communicative reprogramming. The model recapitulates key features of real tumor systems and responses to both traditional cytotoxic treatments and biomodulatory/anakoinotic treatments. Results are presented and discussed, particularly with respect to the implications for future cancer treatment protocols.

Learning Cancer-Related Drug Efficacy Exploiting Consensus in Coordinated Motility Within Cell Clusters.

OBJECTIVE: The ability of cells to collectively move is essential in various biological contexts including cancer metastasis. In this paper, we propose an automatic video analysis tool to correlate the cell movement inhibition with replication block induced by dose-dependent chemotherapy administration. METHODS: The novel approach combines individual and collective cell kinematic analysis performed over time-lapse microscopy video frames. Cells are first localized and tracked, and then kinematic descriptors are extracted for each track. Selective track identification is performed assuming diversified cell roles within the same cluster (spontaneously forming groups of cells), and finally individual results are grouped exploiting consensus of coordinated motility within cell clusters. RESULTS: Recognition performance of three different experimental conditions (no drug, 0.5-5 µM merged in the same condition, and 50 µM) reached an average accuracy value of 88% over 958 different tracks collected in 36 clusters of diverse dimensions in eight independent experiments. CONCLUSION: An extensive application of this methodology could give a different point of view of the cancer mechanisms.

Anakoinosis: Correcting Aberrant Homeostasis of Cancer Tissue-Going Beyond Apoptosis Induction.

The current approach to systemic therapy for metastatic cancer is aimed predominantly at inducing apoptosis of cancer cells by blocking tumor-promoting signaling pathways or by eradicating cell compartments within the tumor. In contrast, a systems view of therapy primarily considers the communication protocols that exist at multiple levels within the tumor complex, and the role of key regulators of such systems. Such regulators may have far-reaching influence on tumor response to therapy and therefore patient survival. This implies that neoplasia may be considered as a cell non-autonomous disease. The multi-scale activity ranges from intra-tumor cell compartments, to the tumor, to the tumor-harboring organ to the organism. In contrast to molecularly targeted therapies, a systems approach that identifies the complex communications networks driving tumor growth offers the prospect of disrupting or "normalizing" such aberrant communicative behaviors and therefore attenuating tumor growth. Communicative reprogramming, a treatment strategy referred to as anakoinosis, requires novel therapeutic instruments, so-called master modifiers to deliver concerted tumor growth-attenuating action. The diversity of biological outcomes following pro-anakoinotic tumor therapy, such as differentiation, trans-differentiation, control of tumor-associated inflammation, etc. demonstrates that long-term tumor control may occur in multiple forms, inducing even continuous complete remission. Accordingly, pro-anakoinotic therapies dramatically extend the repertoire for achieving tumor control and may activate apoptosis pathways for controlling resistant metastatic tumor disease and hematologic neoplasia.

Clinical Efficacy of a Novel Therapeutic Principle, Anakoinosis.

Classic tumor therapy, consisting of cytotoxic agents and/or targeted therapy, has not overcome therapeutic limitations like poor risk genetic parameters, genetic heterogeneity at different metastatic sites or the problem of undruggable targets. Here we summarize data and trials principally following a completely different treatment concept tackling systems biologic processes: the principle of communicative reprogramming of tumor tissues, i.e., anakoinosis (ancient greek for communication), aims at establishing novel communicative behavior of tumor tissue, the hosting organ and organism via re-modeling gene expression, thus recovering differentiation, and apoptosis competence leading to cancer control - in contrast to an immediate, "poisoning" with maximal tolerable doses of targeted or cytotoxic therapies. Therefore, we introduce the term "Master modulators" for drugs or drug combinations promoting evolutionary processes or regulating homeostatic pathways. These "master modulators" comprise a broad diversity of drugs, characterized by the capacity for reprogramming tumor tissues, i.e., transcriptional modulators, metronomic low-dose chemotherapy, epigenetically modifying agents, protein binding pro-anakoinotic drugs, such as COX-2 inhibitors, IMiDs etc., or for example differentiation inducing therapies. Data on 97 anakoinosis inducing schedules indicate a favorable toxicity profile: The combined administration of master modulators, frequently (with poor or no monoactivity) may even induce continuous complete remission in refractory metastatic neoplasia, irrespectively of the tumor type. That means recessive components of the tumor, successively developing during tumor ontogenesis, are accessible by regulatory active drug combinations in a therapeutically meaningful way. Drug selection is now dependent on situative systems characteristics, to less extent histology dependent. To sum up, anakoinosis represents a new substantive therapy principle besides novel targeted therapies.