The transcriptional landscape of cancer stem-like cell functionality in breast cancer.

BACKGROUND: Cancer stem-like cells (CSCs) have been extensively researched as the primary drivers of therapy resistance and tumor relapse in patients with breast cancer. However, due to lack of specific molecular markers, increased phenotypic plasticity and no clear clinicopathological features, the assessment of CSCs presence and functionality in solid tumors is challenging. While several potential markers, such as CD24/CD44, have been proposed, the extent to which they truly represent the stem cell potential of tumors or merely provide static snapshots is still a subject of controversy. Recent studies have highlighted the crucial role of the tumor microenvironment (TME) in influencing the CSC phenotype in breast cancer. The interplay between the tumor and TME induces significant changes in the cancer cell phenotype, leading to the acquisition of CSC characteristics, therapeutic resistance, and metastatic spread. Simultaneously, CSCs actively shape their microenvironment by evading immune surveillance and attracting stromal cells that support tumor progression. METHODS: In this study, we associated in vitro mammosphere formation assays with bulk tumor microarray profiling and deconvolution algorithms to map CSC functionality and the microenvironmental landscape in a large cohort of 125 breast tumors. RESULTS: We found that the TME score was a significant factor associated with CSC functionality. CSC-rich tumors were characterized by an immune-suppressed TME, while tumors devoid of CSC potential exhibited high immune infiltration and activation of pathways involved in the immune response. Gene expression analysis revealed IFNG, CXCR5, CD40LG, TBX21 and IL2RG to be associated with the CSC phenotype and also displayed prognostic value for patients with breast cancer. CONCLUSION: These results suggest that the characterization of CSCs content and functionality in tumors can be used as an attractive strategy to fine-tune treatments and guide clinical decisions to improve patients therapy response.

Hypoxia signaling: Challenges and opportunities for cancer therapy.

Hypoxia is arguably the first recognized cancer microenvironment hallmark and affects virtually all cellular populations present in tumors. During the past decades the complex adaptive cellular responses to oxygen deprivation have been largely elucidated, raising hope for new anti cancer agents. Despite undeniable preclinical progress, therapeutic targeting of tumor hypoxia is yet to transition from bench to bedside. This review focuses on new pharmacological agents that exploit tumor hypoxia or interfere with hypoxia signaling and discusses strategies to maximize their therapeutic impact.

Breast Cancer-Delivered Exosomal miRNA as Liquid Biopsy Biomarkers for Metastasis Prediction: A Focus on Translational Research with Clinical Applicability.

Metastasis represents the most important cause of breast cancer-associated mortality. Even for early diagnosed stages, the risk of metastasis is significantly high and predicts a grim outcome for the patient. Nowadays, efforts are made for identifying blood-based biomarkers that could reliably distinguish patients with highly metastatic cancers in order to ensure a closer follow-up and a more personalized therapeutic method. Exosomes are nano vesicles secreted by cancer cells that can transport miRNAs, proteins, and other molecules and deliver them to recipient cells all over the body. Through this transfer, cancer cells modulate their microenvironment and facilitate the formation of the pre-metastatic niche, leading to sustained progression. Exosomal miRNAs have been extensively studied due to their promising potential as prognosis biomarkers for metastatic breast cancer. In this review, we tried to depict an overview of the existing literature regarding exosomal miRNAs that are already validated as potential biomarkers, and which could be immediately available for the clinic. Moreover, in the last section, we highlighted several miRNAs that have proven their function in preclinical studies and could be considered for clinical validation. Considering the lack of standard methods for evaluating exosomal miRNA, we also discussed the challenges and the technical aspects underlying this issue.

Circulating Small EVs miRNAs as Predictors of Pathological Response to Neo-Adjuvant Therapy in Breast Cancer Patients.

Neo-adjuvant therapy (NAT) is increasingly used in the clinic for the treatment of breast cancer (BC). Pathological response to NAT has been associated with improved patients' survival; however, the current techniques employed for assessing the tumor response have significant limitations. Small EVs (sEVs)-encapsulated miRNAs have emerged as promising new biomarkers for diagnosis and prediction. Therefore, our study aims to explore the predictive value of these miRNAs for the pathological response to NAT in BC. By employing bioinformatic tools, we selected a set of miRNAs and evaluated their expression in plasma sEVs and BC biopsies. Twelve miRNAs were identified in sEVs, of which, miR-21-5p, 221-3p, 146a-5p and 26a-5p were significantly associated with the Miller-Payne (MP) pathological response to NAT. Moreover, miR-21-5p, 146a-5p, 26a-5p and miR-24-3p were independent as predictors of MP response to NAT. However, the expression of these miRNAs showed no correlation between sEVs and tissue samples, indicating that the mechanisms of miRNA sorting into sEVs still needs to be elucidated. Functional analysis of miRNA target genes and drug interactions revealed that candidate miRNAs and their targets, can be regulated by different NAT regimens. This evidence supports their role in governing the patients' therapy response and highlights their potential use as prediction biomarkers.

The Role of miR-375-3p, miR-210-3p and Let-7e-5p in the Pathological Response of Breast Cancer Patients to Neoadjuvant Therapy.

Background and Objectives: Prediction of response to therapy remains a continuing challenge in treating breast cancer, especially for identifying molecular tissue markers that best characterize resistant tumours. Microribonucleic acids (miRNA), known as master modulators of tumour phenotype, could be helpful candidates for predicting drug resistance. We aimed to assess the association of miR-375-3p, miR-210-3p and let-7e-5p in breast cancer tissues with pathological response to neoadjuvant therapy (NAT) and clinicopathological data. Material and methods: Sixty female patients diagnosed with invasive breast cancer at The Oncology Institute "Ion Chiricuța", Cluj-Napoca, Romania (IOCN) were included in this study. Before patients received any treatment, fresh breast tissue biopsies were collected through core biopsy under echographic guidance and processed for total RNA extraction and miRNA quantification. The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) database was used as an independent external validation cohort. Results: miR-375-3p expression was associated with more differentiated tumours, hormone receptor presence and lymphatic invasion. According to the Miller-Payne system, a higher miR-375-3p expression was calculated for patients that presented with intermediate versus (vs.) no pathological response. Higher miR-210-3p expression was associated with an improved response to NAT in both Miller-Payne and RCB evaluation systems. Several druggable mRNA targets were correlated with miR-375-3p and miR-210-3p expression, with upstream analysis using the IPA knowledge base revealing a list of possible chemical and biological targeting drugs. Regarding let-7e-5p, no significant association was noticed with any of the analysed clinicopathological data. Conclusions: Our results suggest that tumours with higher levels of miR-375-3p are more sensitive to neoadjuvant therapy compared to resistant tumours and that higher miR-210-3p expression in responsive tumours could indicate an excellent pathological response.

Blood Genome-Wide Transcriptional Profiles of HER2 Negative Breast Cancers Patients.

Tumors act systemically to sustain cancer progression, affecting the physiological processes in the host and triggering responses in the blood circulating cells. In this study, we explored blood transcriptional patterns of patients with two subtypes of HER2 negative breast cancers, with different prognosis and therapeutic outcome. Peripheral blood samples from seven healthy female donors and 29 women with breast cancer including 14 triple-negative breast cancers and 15 hormone-dependent breast cancers were evaluated by microarray. We also evaluated the stroma in primary tumors. Transcriptional analysis revealed distinct molecular signatures in the blood of HER2- breast cancer patients according to ER/PR status. Our data showed the implication of immune signaling in both breast cancer subtypes with an enrichment of these processes in the blood of TNBC patients. We observed a significant alteration of "chemokine signaling," "IL-8 signaling," and "communication between innate and adaptive immune cells" pathways in the blood of TNBC patients correlated with an increased inflammation and necrosis in their primary tumors. Overall, our data indicate that the presence of triple-negative breast cancer is associated with an enrichment of altered systemic immune-related pathways, suggesting that immunotherapy could possibly be synergistic to the chemotherapy, to improve the clinical outcome of these patients.

PDGF beta targeting in cervical cancer cells suggest a fine-tuning of compensatory signalling pathways to sustain tumourigenic stimulation.

The platelet-derived growth factor (PDGF) signalling pathway has been reported to play an important role in human cancers by modulating autocrine and paracrine processes such as tumour growth, metastasis and angiogenesis. Several clinical trials document the benefits of targeting this pathway; however, in cervical cancer the role of PDGF signalling in still unclear. In this study, we used siRNA against PDGF beta (PDGFBB) to investigate the cellular and molecular mechanisms of PDGFBB signalling in Ca Ski and HeLa cervical cancer cells. Our results show that PDGFBB inhibition in Ca Ski cells led to rapid alterations of the transcriptional pattern of 579 genes, genes that are known to have antagonistic roles in regulating tumour progression. Concomitantly, with the lack of significant effects on cervical cancer cells proliferation, apoptosis, migration or invasion, these findings suggests that cervical cancer cells shift between compensatory signalling pathways to maintain their behaviour. The observed autocrine effects were limited to cervical cancer cells ability to adhere to an endothelial cell (EC) monolayer. However, by inhibiting PDGFBB on cervical cells, we achieved reduced proliferation of ECs in co-culture settings and cellular aggregation in conditioned media. Because of lack of PDGF receptor expression on ECs, we believe that these effects are a result of indirect PDGFBB paracrine signalling mechanisms. Our results shed some light into the understanding of PDGFBB signalling mechanism in cervical cancer cells, which could be further exploited for the development of synergistic anti-tumour and anti-angiogenic therapeutic strategies.

Regulation of stem cells-related signaling pathways in response to doxorubicin treatment in Hs578T triple-negative breast cancer cells.

Different molecular changes have been previously associated with therapeutic response and recurrent disease, however, the detailed mechanism of action in triple-negative breast cancer subtype remains elusive. In this study, we investigated the cellular and molecular signaling of two claudin-low triple-negative breast cancer cells to doxorubicin and docetaxel treatment. Whole human transcriptomic evaluation was used to identify the subsequent changes in gene expression, while biological effects were measured by means of proliferation and anchorage-independent growth assays. Microarray analysis revealed changes in stem cell-related signaling pathways, suggesting that doxorubicin treatment affects the balance between self-renewal and differentiation. While the treatment reduced the proliferation, aggregation and mammosphere forming ability of stem-like cells derived from Hs578T cell line, stem-like cells derived from MDA-MB-231 cells were not significantly affected. Our results suggest that claudin-low triple-negative breast cancer cells might predominantly alter stem cell-related signaling pathways to promote stem-like cells activity as an innate resistance mechanism to doxorubicin treatment.

Gene expression profiling reveals activation of the FA/BRCA pathway in advanced squamous cervical cancer with intrinsic resistance and therapy failure.

BACKGROUND: Advanced squamous cervical cancer, one of the most commonly diagnosed cancers in women, still remains a major problem in oncology due to treatment failure and distant metastasis. Antitumor therapy failure is due to both intrinsic and acquired resistance; intrinsic resistance is often decisive for treatment response. In this study, we investigated the specific pathways and molecules responsible for baseline therapy failure in locally advanced squamous cervical cancer. METHODS: Twenty-one patients with locally advanced squamous cell carcinoma were enrolled in this study. Primary biopsies harvested prior to therapy were analyzed for whole human gene expression (Agilent) based on the patient's 6 months clinical response. Ingenuity Pathway Analysis was used to investigate the altered molecular function and canonical pathways between the responding and non-responding patients. The microarray results were validated by qRT-PCR and immunohistochemistry. An additional set of 24 formalin-fixed paraffin-embedded cervical cancer samples was used for independent validation of the proteins of interest. RESULTS: A 2859-gene signature was identified to distinguish between responder and non-responder patients. 'DNA Replication, Recombination and Repair' represented one of the most important mechanisms activated in non-responsive cervical tumors, and the 'Role of BRCA1 in DNA Damage Response' was predicted to be the most significantly altered canonical pathway involved in intrinsic resistance (p = 1.86E-04, ratio = 0.262). Immunohistological staining confirmed increased expression of BRCA1, BRIP1, FANCD2 and RAD51 in non-responsive compared with responsive advanced squamous cervical cancer, both in the initial set of 21 cervical cancer samples and the second set of 24 samples. CONCLUSIONS: Our findings suggest that FA/BRCA pathway plays an important role in treatment failure in advanced cervical cancer. The assessment of FANCD2, RAD51, BRCA1 and BRIP1 nuclear proteins could provide important information about the patients at risk for treatment failure.

Mammographic assessment of breast density as a tool for predicting the response to neoadjuvant therapy in breast cancer patients.

Background and aims: Breast cancer (BC) is the most frequently diagnosed cancer and the leading cause of cancer-related death among women worldwide. For locally advanced diseases and high-risk tumors, neoadjuvant therapy (NAT) is the treatment of choice. Some studies show that mammographic density (MD) tumor margins and the presence of microcalcifications play a prognostic role in BC patients. Hence, the objective of this retrospective study was to assess if MD could predict the response to NAT among different molecular subtypes of BC patients undergoing NAT at The "Prof. Dr I. Chiricuta" Oncology Institute of Cluj-Napoca, Romania (IOCN). Furthermore, the association between MD, tumor margins and the presence of microcalcifications with clinico-pathological data was analyzed. Methods: Eighty-four breast cancer patients diagnosed and treated at IOCN were included in this study. The morphological characteristics of the tumors were framed according to the BIRADS lexicon. The presence or absence of microcalcifications was also assessed. First, the significance of associations between breast density, margins and microcalcifications and clinico-pathological parameters of the patients were tested with Fisher or Fisher-Freeman-Halton Exact Test. Next, using multinomial logistic regression, we modelled the associations between the pathological response measured by Miller Payne and Residual cancer burden (RCB) systems and the BI-RADS. Variables having significant univariate tests were selected as candidates for the multivariable analysis (adjusted model). Results: Breast densities were significantly associated with the age of the patients (p=0.01), number of positive lymph nodes (p=0.037), margins (p=0.002) and combined categories of Miller-Payne (p=0.034) and RCB pathological response (p=0.021). Margins was significantly associated with ki67 proliferation index (p=0.029), estrogen receptor (ER) (p=0.007), progesterone receptor (PR) (p=0.019), molecular subtype (p<0.001) and the number of clinically observed positive lymph nodes at diagnosis (p=0.019). Conclusions: In our cohort, BC patients with lower MD had higher odds of achieving pCR following NAT, suggesting the role of MD as a clinical prognostic marker. Larger multicenter studies are warranted to validate the prognostic value of MD, which could aid in patients stratification based on their likelihood to respond to NAT.

Scavenger receptor-mediated uptake of cell-penetrating peptide nanocomplexes with oligonucleotides.

Cell-penetrating peptides (CPPs) are short cationic peptides that penetrate cells by interacting with the negatively charged plasma membrane; however, the detailed uptake mechanism is not clear. In contrary to the conventional mode of action of CPPs, we show here that a CPP, PepFect14 (PF14), forms negatively charged nanocomplexes with oligonucleotides and their uptake is mediated by class-A scavenger receptors (SCARAs). Specific inhibitory ligands of SCARAs, such as fucoidin, polyinosinic acid, and dextran sulfate, totally inhibit the activity of PF14-oligonucleotide nanocomplexes in the HeLa pLuc705 splice-correction cell model, while nonspecific, chemically related molecules do not. Furthermore, RNA interference (RNAi) knockdown of SCARA subtypes (SCARA3 and SCARA5) that are expressed in this cell line led to a significant reduction of the activity to <50%. In line with this, immunostaining shows prevalent colocalization of the nanocomplexes with the receptors, and electron microscopy images show no binding or internalization of the nanocomplexes in the presence of the inhibitory ligands. Interestingly, naked oligonucleotides also colocalize with SCARAs when used at high concentrations. These results demonstrate the involvement of SCARA3 and SCARA5 in the uptake of PF14-oligonucleotide nanocomplexes and suggest for the first time that some CPP-based systems function through scavenger receptors, which could yield novel possibilities to understand and improve the transfection by CPPs.

Assessing the Layer-by-Layer Assembly of Cellulose Nanofibrils and Polyelectrolytes in Pancreatic Tumor Spheroid Formation.

Three-dimensional (3D) tumor spheroids are regarded as promising models for utilization as preclinical assessments of chemo-sensitivity. However, the creation of these tumor spheroids presents challenges, given that not all tumor cell lines are able to form consistent and regular spheroids. In this context, we have developed a novel layer-by-layer coating of cellulose nanofibril-polyelectrolyte bilayers for the generation of spheroids. This technique builds bilayers of cellulose nanofibrils and polyelectrolytes and is used here to coat two distinct 96-well plate types: nontreated/non-sterilized and Nunclon Delta. In this work, we optimized the protocol aimed at generating and characterizing spheroids on difficult-to-grow pancreatic tumor cell lines. Here, diverse parameters were explored, encompassing the bilayer count (five and ten) and multiple cell-seeding concentrations (10, 100, 200, 500, and 1000 cells per well), using four pancreatic tumor cell lines-KPCT, PANC-1, MiaPaCa-2, and CFPAC-I. The evaluation includes the quantification (number of spheroids, size, and morphology) and proliferation of the produced spheroids, as well as an assessment of their viability. Notably, our findings reveal a significant influence from both the number of bilayers and the plate type used on the successful formation of spheroids. The novel and simple layer-by-layer-based coating method has the potential to offer the large-scale production of spheroids across a spectrum of tumor cell lines.

Early transcriptional pattern of angiogenesis induced by EGCG treatment in cervical tumour cells.

The major green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) has been shown to exhibit antitumour activities in several tumour models. One of the possible mechanisms by which EGCG can inhibit cancer progression is through the modulation of angiogenesis signalling cascade. The tumour cells' ability to tightly adhere to endothelium is a very important process in the metastatic process, because once disseminated into the bloodstream the tumour cells must re-establish adhesive connections to endothelium in order to extravasate into the target tissues. In this study, we investigated the anti-angiogenic effects of EGCG treatment (10 µM) on human cervical tumour cells (HeLa) by evaluating the changes in the expression pattern of 84 genes known to be involved in the angiogenesis process. Transcriptional analysis revealed 11 genes to be differentially expressed and was further validated by measuring the induced biological effects. Our results show that EGCG treatment not only leads to the down-regulation of genes involved in the stimulation of proliferation, adhesion and motility as well as invasion processes, but also to the up-regulation of several genes known to have antagonist effects. We observed reduced proliferation rates, adhesion and spreading ability as well as invasiveness of HeLa tumour cells upon treatment, which suggest that EGCG might be an important anti-angiogenic therapeutic approach in cervical cancers.

Early apoptosis signals induced by a low dose of epigallocatechin 3-gallate interfere with apoptotic and cell death pathways.

Research for natural compounds with novel advantageous biological activities or antineoplastic potential is an important issue in drug discovery. An example of this type of compound is epigallocatechin 3-gallate (EGCG), which is the major polyphenol in green tea. EGCG has therapeutic effects and modulates multiple metabolic pathways, including apoptosis. The present study evaluates the effect of 10 microM EGCG on the expression of genes that are involved in apoptosis and cell death using PCR-array technology and HeLa cells as an in vitro model. The specific modulatory effects of EGCG were demonstrated by significant alterations in the expression levels of the following genes that control the apoptosis pathway: the TNF receptor and its ligand family, the death effector domain family, the Bcl-2 family or TP73. The structure of EGCG suggests that it has the potential to be an anticancer agent and that EGCG serves as a promising starting point for the derivation of novel anticancer drugs. EGCC metabolite products, such as pyrogallol, gallic acid or quinones, may also play important roles in inducing apoptosis. Therefore, quantification of mRNA using PCR-array technology is a valuable tool for screening these anticarcinogenic compounds. By evaluating the action of EGCG on genes that regulate apoptosis, our results suggest that EGCG is as an activator of gene expression with direct implications in cancer therapy. In addition, the limited success in activation of the antiapoptotic gene Bcl-2 may be associated with resistance to cancer treatment.

The Role of Circulating Tumor Cells in the Prognosis of Metastatic Triple-Negative Breast Cancers: A Systematic Review of the Literature.

Breast cancer is one of the leading causes of death in women worldwide. One subtype of breast cancer is the triple-negative, which accounts for 15% of total breast cancer cases and is known for its poor prognosis. The main cause of death is due to metastasis. Circulating tumor cells (CTCs) play a key role in the metastatic process. CTCs arise either by detaching from the primary tumor or from cancer stem cells undergoing an epithelial-to-mesenchymal transition (EMT). This review aims to present up-to-date data concerning the role of CTC numbers in relation to the prognostic and treatment response in metastatic triple-negative breast cancer (mTNBC) patients, and also to discuss the methods used for CTCs' identification. A search in the MEDLINE database was performed. A total of 234 articles were identified. The results of the 24 eligible studies showed that positive CTC status is associated with shorter overall survival (OS) and progression-free survival (PFS) in mTNBC patients. Furthermore, a decrease in number of CTCs during therapy seems to be a favorable prognostic factor, making CTCs' detection an important prognostic tool before and during therapy in mTNBC patients. The methods used for CTC detection are still developing and need further improvement.

Regulation of cellular sterol homeostasis by the oxygen responsive noncoding RNA lincNORS.

We hereby provide the initial portrait of lincNORS, a spliced lincRNA generated by the MIR193BHG locus, entirely distinct from the previously described miR-193b-365a tandem. While inducible by low O2 in a variety of cells and associated with hypoxia in vivo, our studies show that lincNORS is subject to multiple regulatory inputs, including estrogen signals. Biochemically, this lincRNA fine-tunes cellular sterol/steroid biosynthesis by repressing the expression of multiple pathway components. Mechanistically, the function of lincNORS requires the presence of RALY, an RNA-binding protein recently found to be implicated in cholesterol homeostasis. We also noticed the proximity between this locus and naturally occurring genetic variations highly significant for sterol/steroid-related phenotypes, in particular the age of sexual maturation. An integrative analysis of these variants provided a more formal link between these phenotypes and lincNORS, further strengthening the case for its biological relevance.

Publisher Correction: Regulation of cellular sterol homeostasis by the oxygen responsive noncoding RNA lincNORS.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

How reliable and useful are angiogenesis blood mediators for prostate cancer diagnosis?

PURPOSE: To evaluate if a blood panel of genes involved in the modulation of the immune system, angiogenesis and tumor development could be used for prostate cancer detection. METHODS: Gene expression profiling of blood samples was assessed with the human angiogenesis RT² Profiler™ pcr array. The study group was divided into training and a testing/validation set. In total, 36 blood samples from 6 heatlhy men, 19 patients with prostate cancer (PCa) and 11 patients with benign prostate pathology (BP) were included in this study. RESULTS: Transcriptional analysis revealed a supervised signature of 28 genes which discriminated the PCa samples from control on the training set (fold regulation [FR] cut off 1.5, p<0.05). This signature was further validated on the testing set. All 28 genes used for this classification were differentially expressed in the new set of 12 PCa samples compared to control but also compared to benign samples (FR cut off 1.5, p<0.05). CONCLUSIONS: Our data could provide new insight into PCa, as a non-invasive predictive tool which along with other factors could improve PCa diagnosis. However, our findings have to be confirmed in a larger cohort of patients before having a clear picture of how this molecular profile will help to increase the accuracy of diagnosis.

The Many Faces of Long Noncoding RNAs in Cancer.

SIGNIFICANCE: The emerging connections between an increasing number of long noncoding RNAs (lncRNAs) and oncogenic hallmarks provide a new twist to tumor complexity. Recent Advances: In the present review, we highlight specific lncRNAs that have been studied in relation to tumorigenesis, either as participants in the neoplastic process or as markers of pathway activity or drug response. These transcripts are typically deregulated by oncogenic or tumor-suppressing signals or respond to microenvironmental conditions such as hypoxia. CRITICAL ISSUES: Among these transcripts are lncRNAs sufficiently divergent between mouse and human genomes that may contribute to biological differences between species. FUTURE DIRECTIONS: From a translational standpoint, knowledge about primate-specific lncRNAs may help explain the reason behind the failure to reproduce the results from mouse cancer models in human cell-based systems. Antioxid. Redox Signal. 29, 922-935.

New alginate-pullulan-bioactive glass composites with copper oxide for bone tissue regeneration trials.

Composites based on sodium alginate, pullulan, and bioactive SiO2 -CaO-P2 O5 glass-ceramics with copper oxide were prepared as capsules. The obtained samples were structurally characterized by Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), and scanning electron microscopy (SEM), and their bioactivity and biocompatibility properties were also tested both in vitro and in vivo by XRD, FT-IR, SEM, and high-resolution transmission electron microscopy. The fibroblast and osteoblast cell viability assays have shown good proliferation rates for all investigated samples, whereas all composites exhibited a good in vivo tolerance. The recovered composites after 5 weeks' in vivo and in vitro trials evidenced clear macroscopic alterations; particularly, after soaking in simulated body fluid, they have a corn flake aspect, and after their in vivo inoculation, a globular shape is retained. Different crystalline shapes of hydroxyapatite were formed after in vitro and in vivo trials for the glass-ceramic-polymer composites, the in vitro precipitated apatite was found to be nodular, and the in vivo experiment led to needlelike crystallites formation. Histopathological results showed a good biocompatibility with no significant signs of rejection by the host tissue. These assessments performed on the composites indicate that the studied materials can be considered without any doubt suitable candidates for future bone regeneration applications.