In vivo genome-wide CRISPR screening identifies CITED2 as a driver of prostate cancer bone metastasis.

Most cancer deaths are due to metastatic dissemination to distant organs. Bone is the most frequently affected organ in metastatic prostate cancer and a major cause of prostate cancer deaths. Yet, our partial understanding of the molecular factors that drive bone metastasis has been a limiting factor for developing preventative and therapeutic strategies to improve patient survival and well-being. Although recent studies have uncovered molecular alterations that occur in prostate cancer metastasis, their functional relevance for bone metastasis is not well understood. Using genome-wide CRISPR activation and inhibition screens we have identified multiple drivers and suppressors of prostate cancer metastasis. Through functional validation, including an innovative organ-on-a-chip invasion platform for studying bone tropism, our study identifies the transcriptional modulator CITED2 as a novel driver of prostate cancer bone metastasis and uncovers multiple new potential molecular targets for bone metastatic disease.

Graphene-Based Coatings for Surface Modification and Their Applications in Fixed Orthodontics: A Scoping Review.

BACKGROUND: Surface coating technology can assist fixed appliances by reducing friction, improving antibacterial characteristics, and increasing corrosion resistance. The application of functional coatings composed of graphene onto the surfaces of orthodontic brackets and archwires has been shown to enhance their mechanical qualities. The objective of the current study was to carry out a scoping analysis of published recent evidence on the utilization of graphene as a covering material in metallic orthodontic accessories, such as brackets and archwires; Methods: A scoping review was undertaken following the PRISMA-ScR guidelines. PubMed, Embase, the Cochrane Library, Dentistry and Oral Science Source, and Google Scholar were searched between 2003 and 2023; Results: In total, 38 potential references were detected, from which 10 were selected for this review. These articles addressed the benefits of the application of graphene-oxide functional coatings onto the surface of archwires and brackets during fixed orthodontic treatment. Orthodontic graphene-oxide-based coatings provide improved surface characteristics (e.g., reduced friction and anticorrosive effects), antibacterial capabilities, and biocompatibility. These characteristics can increase the effectiveness of orthodontic therapy, improve patient comfort, and lower the likelihood of problems; Conclusion: Orthodontists should be aware of and comprehend the prerequisites for using graphene-oxide-coated archwires and brackets to fulfill needs throughout their clinical practice.

OncoLoop: A Network-Based Precision Cancer Medicine Framework.

Prioritizing treatments for individual patients with cancer remains challenging, and performing coclinical studies using patient-derived models in real time is often unfeasible. To circumvent these challenges, we introduce OncoLoop, a precision medicine framework that predicts drug sensitivity in human tumors and their preexisting high-fidelity (cognate) model(s) by leveraging drug perturbation profiles. As a proof of concept, we applied OncoLoop to prostate cancer using genetically engineered mouse models (GEMM) that recapitulate a broad spectrum of disease states, including castration-resistant, metastatic, and neuroendocrine prostate cancer. Interrogation of human prostate cancer cohorts by Master Regulator (MR) conservation analysis revealed that most patients with advanced prostate cancer were represented by at least one cognate GEMM-derived tumor (GEMM-DT). Drugs predicted to invert MR activity in patients and their cognate GEMM-DTs were successfully validated in allograft, syngeneic, and patient-derived xenograft (PDX) models of tumors and metastasis. Furthermore, OncoLoop-predicted drugs enhanced the efficacy of clinically relevant drugs, namely, the PD-1 inhibitor nivolumab and the AR inhibitor enzalutamide. SIGNIFICANCE: OncoLoop is a transcriptomic-based experimental and computational framework that can support rapid-turnaround coclinical studies to identify and validate drugs for individual patients, which can then be readily adapted to clinical practice. This framework should be applicable in many cancer contexts for which appropriate models and drug perturbation data are available. This article is highlighted in the In This Issue feature, p. 247.

Assessment of Pediatricians' Knowledge, Practices, and Attitudes on Oral Health/Care in Children in the Last Decade: A Systematic Scoping Review and Critical Reflection.

Pediatricians are primary health care professionals who supervise the growth and development and treat infants and children during the first years of life. Thus, they should possess knowledge regarding oral health care, to provide anticipatory guidance, as well as dental education to parents in order to make appropriate clinical decisions. For many years, several surveys have been performed worldwide to assess the pediatricians' knowledge, awareness, and experience regarding oral health care and prevention. This work aimed to scope the existing literature and summarize the most relevant evidence about knowledge, practices, and attitudes on oral health/care among pediatricians worldwide. PubMed, Cochrane Library, Google Scholar, and Dentistry & Oral Sciences Source were explored. Under a structured PCC question and eligibility criteria, for relevant clinical trials and observational studies, published during the last decade. Titles and abstracts were screened. Full-text articles were critically reviewed for bias risk and a data charting table was constructed. A total of 44 references were initially identified, and 37 titles remained for abstract screening after removing duplicates; then, 27 potential full-text articles were carefully reviewed. Finally, 25 relevant and most informative studies were included. The selected studies were conducted in India, Lebanon, Saudi Arabia, and Paraguay, Europe, Australia, Qatar, Iran, Turkey, United Arab Emirates, Nigeria, Brazil, Chile, Germany, Taiwan, Canada, and the USA. Through included surveys, researchers have reported different levels of knowledge, practice involvement, and attitude on children's oral health among pediatricians. In general, unsatisfactory knowledge of oral health was reported. The main impediments for a better professional involvement or practice include inappropriate education, poor auto-confidence, and lack of time. So, it has been suggested that some oral health training or clinical guidelines should be included in the current medical curricula.

Synthesis of a PVA drug delivery system for controlled release of a Tramadol-Dexketoprofen combination.

The local administration of analgesic combinations by means of degradable polymeric drug delivery systems is an alternative for the management of postoperative pain. We formulated a Tramadol-Dexketoprofen combination (TDC) loaded in poly(vinyl alcohol) (PVA) film. Films were prepared by the solvent casting method using three different molecular weights of PVA and crosslinking those films with citric acid, with the objective of controlling the drug release rate, which was evaluated by UV-vis spectrometry. Non-crosslinked PVA films were also evaluated in the experiments. Differential scanning calorimetry (DSC) analysis of samples corroborated the crosslinking of PVA by the citric acid. Blank and loaded PVA films were tested in vitro for its impact on blood coagulation prothrombin time (PT) and partial thromboplastin time (PTT). The swelling capacity was also evaluated. Crosslinked PVA films of higher-molecular weight showed a prolonged release rate compared with that of the lower-molecular-weight films tested. Non-crosslinked PVA films released 11-14% of TDC. Crosslinked PVA films released 80% of the TDC loaded (p < 0.05). This suggests that crosslinking films can modify the drug release rate. The blank and loaded PVA films induced PT and PTT in the normal range. The results showed that the polymeric films evaluated here have the appropriate properties to allow films to be placed directly on surgical wounds and have the capacity for controlled drug release to promote local analgesia for the control of postoperative pain.

Longitudinal Immune Profiling Reveals Unique Myeloid and T-cell Phenotypes Associated with Spontaneous Immunoediting in a Prostate Tumor Model.

The theory of cancer immunoediting, which describes the dynamic interactions between tumors and host immune cells that shape the character of each compartment, is foundational for understanding cancer immunotherapy. Few models exist that facilitate in-depth study of each of the three canonical phases of immunoediting: elimination, equilibrium, and escape. Here, we utilized NPK-C1, a transplantable prostate tumor model that we found recapitulated the three phases of immunoediting spontaneously in immunocompetent animals. Given that a significant portion of NPK-C1 tumors reliably progressed to the escape phase, we were able to delineate cell types and mechanisms differentially prevalent in equilibrium versus escape phases. Using high-dimensional flow cytometry, we found that activated CD4+ effector T cells were enriched in regressing tumors, highlighting a role for CD4+ T cells in antitumor immunity. CD8+ T cells were also important for NPK-C1 control, specifically, central memory-like cytotoxic CD8+ T cells. Regulatory T cells (Treg), as a whole, were counterintuitively enriched in regressing tumors; however, high-dimensional analysis revealed their significant phenotypic diversity, with a number of Treg subpopulations enriched in progressing tumors. In the myeloid compartment, we found that iNOS+ dendritic cell (DC)-like cells are enriched in regressing tumors, whereas CD103+ DCs were associated with late-stage tumor progression. In total, these analyses of the NPK-C1 model provide novel insights into the roles of lymphoid and myeloid populations throughout the cancer immunoediting process and highlight a role for multidimensional, flow-based analyses to more deeply understand immune cell dynamics in the tumor microenvironment.

Calcium hydroxide/iodoform nanoparticles as an intracanal filling medication: synthesis, characterization, and in vitro study using a bovine primary tooth model.

The aims of this in vitro study were to synthesize, characterize, and evaluate the efficacy of a Calcium Hydroxide/Iodoform nanoparticles (CHIN) paste compared with Ultrapex as intracanal filling medication using an experimental model of bovine primary teeth. CH nanoparticle synthesis was performed via the simple hydrolysis technique of reacting calcium nitrate with sodium hydroxide. SEM-EDS and FT-IR analyses were used to characterize the obtained product. 30% of CH nanoparticles were combined with 40% of iodoform and 30% silicone oil to prepare an intracanal filling paste (CHIN). All endodontic procedures were performed on 34 uniradicular primary bovine teeth. Every root canal was instrumented with K files (up to #35) and obturated with the nanoparticle paste (experimental) or Ultrapex® (control). Three outcome variables were studied: penetration depth through the root dentinal tubules, Ca2+ ion release, and filling paste dissolution rate. The obtained data were analyzed by Student's t test. The X-ray diffraction pattern of CH nanoparticles showed characteristic peaks at CH, as confirmed by FT-IR analyses in which an intense signal was observed at 3643 cm-1, characteristic of CH. In the morphological characterization, CH particles could be detected at the nanosize scale. When applied as intracanal filling, the CHIN paste exhibited a higher level of penetration through the root dentin tubules. The global mean penetration measures were 500 µm for the experimental paste and 380 µm for the control paste (p < 0.05). The release of Ca2+ ions (up to the seventh day) and the dissolution rate were significantly higher in the experimental paste group than in the control group. No significant differences were observed between the groups regarding pH levels. The findings of this study suggest the potential suitability of CHI nanoparticles as an alternative intracanal filling medication for infected or devitalized primary teeth.

Antifungal biomaterial for reducing infections caused by Candida albicans in edentulous patients.

INTRODUCTION: Polymethylmethacrylate (PMMA) acrylic resins are used to make dentures for edentulous patients. OBJECTIVE: To find out the prevalence of Candida species in patients with and without removable prostheses from a dental clinic in León, Guanajuato, as well as to assess the antifungal effect and biological behavior of an experimental PMMA with silver nanoparticles for its possible application in prostheses. METHOD: To identify Candida species, smear samples were obtained from the palatal mucosa of 140 patients aged ≥ 60 years. The experimental PMMA with silver nnoparticles was placed in Candida albicans cultures, which were stained with the Live/Dead® kit for analysis under confocal microscopy; subsequently, it was implanted in Wistar rats in order to know its behavior in the surrounding tissues. RESULTS: Candida albicans was the most prevalent species in the evaluated patients, followed by Candida tropicalis and Candida krusei. The acrylic resin with silver nanoparticles significantly decreased the presence of Candida albicans. In the animal model, a discrete and controlled inflammatory reaction was found, which indicated biocompatibility of the acrylic resin that was used. CONCLUSIONS: It is possible for the nanostructured material with antifungal effect to be used in order to promote the reduction of oral Candida infections in edentulous patients.

INTRODUCCIÓN: Las resinas acrílicas de polimetilmetacrilato (PMMA) son utilizadas para elaborar dentaduras para pacientes edéntulos. OBJETIVO: Conocer la prevalencia de las especies de Candida en pacientes con y sin prótesis removibles de una clínica de odontología en León, Guanajuato; así como valorar el efecto antifúngico y el comportamiento biológico de un PMMA experimental con nanopartículas de plata para su posible aplicación en prótesis. MÉTODO: Para identificar las especies de Candida se obtuvieron muestras para frotis de la mucosa palatina de 140 pacientes con edad ≥ 60 años. El PMMA experimental con nanopartículas de plata fue colocado en cultivos de Candida albicans, los cuales fueron teñidos con el kit Live/Dead® para su análisis bajo microscopia confocal; posteriormente, se implantó en ratas Wistar para conocer su comportamiento en los tejidos circundantes. RESULTADOS: Candida albicans fue la especie más prevalente en los pacientes valorados, seguida de Candida tropicalis y Candida krusei. La resina acrílica con nanopartículas de plata disminuyó significativamente la presencia de Candida albicans. En el modelo animal se encontró reacción inflamatoria discreta y controlada, lo cual indicó la biocompatibilidad de la resina acrílica utilizada. CONCLUSIONES: Es posible utilizar el material nanoestructurado con efecto antifúngico para promover la reducción de infecciones orales por Candida en pacientes edéntulos.

Evaluation of the Bond Strength and Marginal Seal of Indirect Restorations of Composites Bonded with Preheating Resin.

OBJECTIVE: Preheated resins (PR) are considered a cementing agent option for indirect adhesive restorations of composite inlays and onlays. The objective of this in vitro study was to evaluate the marginal sealing, adhesive interface, and microtensile bond strength of indirect adhesive restorations of composites in terms of dentin cemented with PR. MATERIALS AND METHODS: Standardized Class II preparations were performed on 30 extracted human premolars, impressions were taken, and indirect composite restorations were manufactured. In total, 15 restorations were cemented with PR (ENA HRi, SYNCA), and 15 restorations were cemented with self-adhesive resinous cement (RC) (Relyx U200, 3M ESPE), followed by a thermocycling regime. After that, these were segmented sagittally and longitudinally to evaluate the marginal sealing and the adhesive interface with scanning electron microscopy and confocal microscopy. Microtensile bond strength was assessed with a mechanical device (TA. XT Plus C, Stable Micro System). STATISTICAL ANALYSIS: Statistical analysis was conducted using the two-sample Student's t-test. RESULTS: The results showed that there is no statistically significant difference in the degree of microfiltration using PR or RC; however, microtensile bond strength is greater when the restoration is cemented with RC (278.75 N/cm3) than with PR (144.49 N/cm3), and better adjustment and sealing were observed for composite restorations with PR. CONCLUSION: PR comprise an alternative cementing agent for indirect composite restorations in Class II cavities in premolars.

A MYC and RAS co-activation signature in localized prostate cancer drives bone metastasis and castration resistance.

Understanding the intricacies of lethal prostate cancer poses specific challenges due to difficulties in accurate modeling of metastasis in vivo. Here we show that NPK EYFP mice (for Nkx3.1 CreERT2/+ ; Pten flox/flox ; Kras LSL-G12D/+ ; R26R-CAG-LSL-EYFP/+) develop prostate cancer with a high penetrance of metastasis to bone, thereby enabling detection and tracking of bone metastasis in vivo and ex vivo. Transcriptomic and whole-exome analyses of bone metastasis from these mice revealed distinct molecular profiles conserved between human and mouse and specific patterns of subclonal branching from the primary tumor. Integrating bulk and single-cell transcriptomic data from mouse and human datasets with functional studies in vivo unravels a unique MYC/RAS co-activation signature associated with prostate cancer metastasis. Finally, we identify a gene signature with prognostic value for time to metastasis and predictive of treatment response in human patients undergoing androgen receptor therapy across clinical cohorts, thus uncovering conserved mechanisms of metastasis with potential translational significance.

Genetically Engineered Mouse Models of Prostate Cancer in the Postgenomic Era.

Recent genomic sequencing analyses have unveiled the spectrum of genomic alterations that occur in primary and advanced prostate cancer, raising the question of whether the corresponding genes are functionally relevant for prostate tumorigenesis, and whether such functions are associated with particular disease stages. In this review, we describe genetically engineered mouse models (GEMMs) of prostate cancer, focusing on those that model genomic alterations known to occur in human prostate cancer. We consider whether the phenotypes of GEMMs based on gain or loss of function of the relevant genes provide reliable counterparts to study the predicted consequences of the corresponding genomic alterations as occur in human prostate cancer, and we discuss exceptions in which the GEMMs do not fully emulate the expected phenotypes. Last, we highlight future directions for the generation of new GEMMs of prostate cancer and consider how we can use GEMMs most effectively to decipher the biological and molecular mechanisms of disease progression, as well as to tackle clinically relevant questions.

Calcium sustained release, pH changes and cell viability induced by chitosan-based pastes for apexification.

We explored chitosan-based sustained release pastes for apexification. The study aimed to formulate chitosan-based pastes loaded with calcium hydroxide (CH) or with calcium chloride (CC), and to evaluate the sustained release of Ca2+ and pH changes in deionized water as well as the effect of the pastes on cell viability. The pastes were formulated by dissolution of the chitosan in 1% or 2% acetic acid (AAC) plus the addition of CH or CC, then were suspended in deionized water for 50 days; the released Ca(II) and pH were measured with an electrode probe. The effect of the pastes on viability of human dental pulp cells was evaluated with a MTS assay. The results showed that the pastes prepared with 1% and 2% AAC and loaded with CH released a 74.9% and a 76.1% of the Ca2+ content, respectively, while the pastes prepared with 1% and 2% AAC loaded with CC released a content of Ca2+ of 90.8% and 76.6%, respectively. A control paste (CH and polyethylene glycol) released a 95.4%; significant statistical differences were found between the percentage of the experimental pastes and the control. The CH-loaded pastes caused an alkaline pH at the starting of the study, but the pH became neutral at the ending. The pH of the CC-loaded pastes was neutral at the starting and was acid at the ending. The pastes no affected on the cell viability. The chitosan-based pastes showed a suitable sustained release profile and cytocompatibility.

Case Report in Clinical Pediatric Dentistry: How to Review, Organize and Prepare a Manuscript.

A case report is a useful type of publication to describe information on unusual clinical diseases or syndromes, new management techniques, potential risk factors/oral disease associations, and uncommon side effects or responses to traditional dental treatments. In the practice of Dentistry and Medicine, the publication of case reports has the purpose of sharing new clinical experiences and knowledge with interested colleagues. Case reports in the field of Evidence-Based Pediatric Dentistry convey unique contributions to the clinical practice and help improve the process of clinical decision making in the form of a brief written communication. Additionally, case reports are potential resources of new hypotheses for more complex methodological designs in clinical research studies and are one of the best ways to get started in scholarly writing. The purposes of the present report were to comment on the role, relevance, and main limitations of case reports in Clinical Pediatric Dentistry, to describe the reasons for writing a case report and some recommendations for critically reviewing a published case report, and finally, to provide the fundamentals of preparing a case report, and finally, to provide the fundamentals of preparing a case report manuscript in a structured manner.

NSD2 is a conserved driver of metastatic prostate cancer progression.

Deciphering cell-intrinsic mechanisms of metastasis progression in vivo is essential to identify novel therapeutic approaches. Here we elucidate cell-intrinsic drivers of metastatic prostate cancer progression through analyses of genetically engineered mouse models (GEMM) and correlative studies of human prostate cancer. Expression profiling of lineage-marked cells from mouse primary tumors and metastases defines a signature of de novo metastatic progression. Cross-species master regulator analyses comparing this mouse signature with a comparable human signature identifies conserved drivers of metastatic progression with demonstrable clinical and functional relevance. In particular, nuclear receptor binding SET Domain Protein 2 (NSD2) is robustly expressed in lethal prostate cancer in humans, while its silencing inhibits metastasis of mouse allografts in vivo. We propose that cross-species analysis can elucidate mechanisms of metastasis progression, thus providing potential additional therapeutic opportunities for treatment of lethal prostate cancer.

Metallothionein 1G promotes the differentiation of HT-29 human colorectal cancer cells.

Metallothioneins (MTs) are a family of low-molecular-weight, cysteine-rich proteins involved in zinc and redox metabolism, that are epigenetically downregulated during colorectal cancer (CRC) progression, but may be re-induced with a variety of agents. Since loss of MT expression is associated with a worse prognosis, in the present study we investigated the effects of overexpression of the most significantly downregulated isoform in CRC, namely MT1G, on the HT-29 cell line. Overexpression of MT1G resulted in xenograft tumors with an aberrant morphology, characterized by an evident increase in mucin-containing cells that were identified as goblet cells under electron microscopy. Immunohistochemical detection of CDX2 and cytokeratin 20 was also increased, as were goblet­cell and enterocyte-specific genes by qRT-PCR. Microarray analysis of gene expression confirmed the alteration of several differentiation signaling pathways, including the Notch pathway. Using sodium butyrate and post-confluent growth as inducers of differentiation, we demonstrated that MT1G does indeed play a functional role in promoting goblet over enterocyte differentiation in vitro. Labile zinc is also induced upon differentiation of CRC cells, functionally contributing to enterocyte over goblet differentiation, as revealed using zinc­specific chelating agents. Overall, our results uncover a new tumor-suppressor activity of MT1G in promoting the differentiation of at least some CRC tumors, and implicate MTs and zinc signaling as new players in colorectal differentiation. This further contributes to the hypothesis that re-induction of MTs may have therapeutic value by diminishing the aggressiveness of CRC tumors.

Cell viability and hemocompatibility evaluation of a starch-based hydrogel loaded with hydroxyapatite or calcium carbonate for maxillofacial bone regeneration.

The objective of this study is to evaluate the cell viability and hemocompatibility of starch-based hydrogels for maxillofacial bone regeneration. Seven starch-based hydrogels were prepared: three loaded with 0.5, 1 and 2% calcium carbonate (Sigma Aldrich, St. Louis, MO, USA); three loaded with 2, 3 and 4% hydroxyapatite (Sigma Aldrich); and one not loaded as a control. A 10 M NaOH was then added to induce hydrogel formation. Human osteoblasts were cultured on each hydrogel for 72 h. An MTS assay (Cell Titer96; PROMEGA, Madison, WI, USA) was used to assess cell viability. Hemocompatibility testing was conducted with normal human blood in the following conditions: 100 mg of each hydrogel in contact with 900 µL of whole blood for 15 min at 37 °C under lateral stirring. Higher percentages of cell viability were observed in starch-based hydrogels loaded with hydroxyapatite as compared with the control. The hemolysis test showed a hemolysis level lower than 2%. Activated partial thromboplastin time and prothrombin time were unchanged, while platelet counting showed a slight decrease when compared with controls.

Metallothionein 1G and zinc sensitize human colorectal cancer cells to chemotherapy.

Metallothioneins (MT) are a family of low molecular weight proteins that are silenced during colorectal cancer progression, mainly through epigenetic mechanisms, and this loss is associated with poor survival. In this article, we show that overexpression of the MT1G isoform sensitizes colorectal cell lines to the chemotherapeutic agents oxaliplatin (OXA) and 5-fluorouracil (5-FU), in part through enhancing p53 and repressing NF-κB activity. Despite being silenced, MTs can be reinduced by histone deacetylase inhibitors such as trichostatin A and sodium butyrate. In fact, this induction contributes to the cytotoxicity of these agents, given that silencing of MTs by siRNAs reduces their growth-inhibitory activities. Zinc ions also potently enhance MT expression and are cytotoxic to cancer cells. We show for the first time that OXA and 5-FU induce higher levels of intracellular labile zinc, as measured using the fluorescent probe FLUOZIN-3, and that such zinc contributes to the activation of p53 and repression of NF-κB. Addition of zinc enhanced growth inhibition by OXA and 5-FU, and was also capable of resensitizing 5-FU-resistant cell lines to levels comparable with sensitive cell lines. This effect was MT independent because silencing MTs did not affect zinc cytotoxicity. In conclusion, we show that MT induction and zinc administration are novel strategies to sensitize colorectal cancer cells to presently utilized chemotherapeutic agents.

Development of a novel methodology for cryopreservation of melanoma cells applied to CSF470 therapeutic vaccine.

CSF470 vaccine is a mixture of four lethally irradiated melanoma cell lines, administered with BCG and GM-CSF, which is currently being tested in a Phase II/III Clinical trial in stage II/III melanoma patients. To prepare vaccine doses, irradiated melanoma cell lines are frozen using dimethyl sulfoxide (Me(2)SO) and stored in liquid nitrogen (liqN(2)). Prior to inoculation, doses must be thawed, washed to remove Me(2)SO and suspended for clinical administration. Avoiding the use of Me(2)SO and storage in liqN(2) would allow future freeze-drying of CSF470 vaccine to facilitate pharmaceutical production and distribution. We worked on the development of an alternative cryopreservation methodology while keeping the vaccine's biological and immunogenic properties. We tested different freezing media containing trehalose suitable to remain as excipients in a freeze-dried product, to cryopreserve melanoma cells either before or after gamma irradiation. Melanoma cells incorporated trehalose after 5 h incubation at 37°C by fluid-phase endocytosis, reaching an intracellular concentration that varied between 70-140 mM depending on the cell line. Optimal freezing conditions were 0.2 M trehalose and 30 mg/ml human serum albumin, at -84°C. Vaccine doses could be frozen in trehalose at -84°C for at least four months keeping their cellular integrity, antigen expression and apoptosis/necrosis profile after gamma-irradiation as compared to Me(2)SO control. Non-irradiated melanoma cell lines also showed comparable proliferative capacity after both cryopreservation procedures. Trehalose-freezing medium allowed us to cryopreserve melanoma cells, either alive or after gamma irradiation, at -84°C avoiding the use of Me(2)SO and liqN(2) storage. These cryopreservation conditions could be suitable for future freeze-drying of CSF470 vaccine.