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Abstract Oral malignancies continue to have severe morbidity with less than 50% long-term survival despite the advancement in the available therapies. There is a persisting demand for new approaches to establish more efficient strategies for their treatment. In this regard, the human topoisomerase II (topoII) enzyme is a validated chemotherapeutics target, as topoII regulates vital cellular processes such as DNA replication, transcription, recombination, and chromosome segregation in cells. TopoII inhibitors are currently used to treat some neoplasms such as breast and small cells lung carcinomas. Additionally, topoII inhibitors are under investigation for the treatment of other cancer types, including oral cancer. Here, we report the therapeutic effect of a tetrahydroquinazoline derivative (named ARN21934) that preferentially inhibits the alpha isoform of human topoII. The treatment efficacy of ARN21934 has been evaluated in 2D cell cultures, 3D in vitro systems, and in chick chorioallantoic membrane cancer models. Overall, this work paves the way for further preclinical developments of ARN21934 and possibly other topoII alpha inhibitors of this promising chemical class as a new chemotherapeutic approach for the treatment of oral neoplasms.

Glioblastoma, a deadly brain tumor, shows limited response to standard therapies like temozolomide (TMZ). Recent findings from the REGOMA trial underscore a significant survival improvement offered by Regorafenib (REGO) in recurrent glioblastoma. Our study aimed to propose a 3D ex vivo drug response precision medicine approach to investigate recurrent glioblastoma sensitivity to REGO and elucidate the underlying molecular mechanisms involved in tumor resistance or responsiveness to treatment. Three-dimensional glioblastoma organoids (GB-EXPs) obtained from 18 patients’ resected recurrent glioblastoma tumors were treated with TMZ and REGO. Drug responses were evaluated using NAD(P)H FLIM, stratifying tumors as responders (Resp) or non-responders (NRs). Whole-exome sequencing was performed on 16 tissue samples, and whole-transcriptome analysis on 13 GB-EXPs treated and untreated. We found 35% (n = 9) and 77% (n = 20) of tumors responded to TMZ and REGO, respectively, with no instances of TMZ-Resp being REGO-NRs. Exome analysis revealed a unique mutational profile in REGO-Resp tumors compared to NR tumors. Transcriptome analysis identified distinct expression patterns in Resp and NR tumors, impacting Rho GTPase and NOTCH signaling, known to be involved in drug response. In conclusion, recurrent glioblastoma tumors were more responsive to REGO compared to TMZ treatment. Importantly, our approach enables a comprehensive longitudinal exploration of the molecular changes induced by treatment, unveiling promising biomarkers indicative of drug response.

Abstract The mouse mammary tumour virus (MMTV) is implicated in the aetiology of murine mammary carcinomas and a variant of it, the type B leukemogenic virus, can cause murine thymic lymphomas. Interestingly, a MMTV-like virus is suspected to be involved in human breast cancer and feline mammary carcinomas. However, to date, no cases of MMTV-like sequence amplifications have been described in lymphoid neoplasms in veterinary literature. The aim of this study was to investigate the presence of env nucleotide sequences and protein 14 (p14) of a MMTV-like virus in fifty-three feline lymphoma samples. Our results show that MMTV-like sequences were detected in 5/53 tumours (9.4%): three gastrointestinal lymphomas (one B-type diffuse large, one B-type small non-cleaved, and one T-type diffuse mixed lymphoma); and two nasal lymphomas (one B-type diffuse small cleaved lymphoma and one B-type diffuse mixed lymphoma). P14 expression was detected in the cytoplasm, and rarely in nuclei, exclusively of neoplastic cells from PCR-positive tumours. The correlation between the presence of the MMTV-env like sequences (MMTVels) and p14 antigen was statistically significant in nasal lymphomas. All cats with MMTVels-positive lymphoma had a history of contact with the outdoor environment and/or catteries, and two deceased subjects shared their environment with cats that also died of lymphoma. In conclusion, this study succeeds in demonstrating the presence of MMTVels and p14 in feline lymphomas. The characterization of the immunophenotype of MMTVels-positive lymphomas could contribute to the understanding of a possible role of a MMTV-like virus in feline tumour aetiology. The significant association between the presence of the viral sequences in lymphoid tumours and their nasal localization, together with the data collected through supplementary anamnesis, should be further analysed in order to understand the epidemiology of the virus.

BackgroundGlioblastoma (GB) is the most severe form of brain cancer, with a 12-15 month median survival. Surgical resection, temozolomide (TMZ) treatment, and radiotherapy remain the primary therapeutic options for GB, and no new therapies have been introduced in recent years. This therapeutic standstill is primarily due to preclinical approaches that do not fully respect the complexity of GB cell biology and fail to test efficiently anti-cancer treatments. Therefore, better treatment screening approaches are needed. In this study, we have developed a novel functional precision medicine approach to test the response to anticancer treatments in organoids derived from the resected tumors of glioblastoma patients.MethodsGB organoids were grown for a short period of time to prevent any genetic and morphological evolution and divergence from the tumor of origin. We chose metabolic imaging by NAD(P)H fluorescence lifetime imaging microscopy (FLIM) to predict early and non-invasively ex-vivo anti-cancer treatment responses of GB organoids. TMZ was used as the benchmark drug to validate the approach. Whole-transcriptome and whole-exome analyses were performed to characterize tumor cases stratification.ResultsOur functional precision medicine approach was completed within one week after surgery and two groups of TMZ Responder and Non-Responder tumors were identified. FLIM-based metabolic tumor stratification was well reflected at the molecular level, confirming the validity of our approach, highlighting also new target genes associated with TMZ treatment and identifying a new 17-gene molecular signature associated with survival. The number of MGMT gene promoter methylated tumors was higher in the responsive group, as expected, however, some non-methylated tumor cases turned out to be nevertheless responsive to TMZ, suggesting that our procedure could be synergistic with the classical MGMT methylation biomarker.ConclusionsFor the first time, FLIM-based metabolic imaging was used on live glioblastoma organoids. Unlike other approaches, ex-vivo patient-tailored drug response is performed at an early stage of tumor culturing with no animal involvement and with minimal tampering with the original tumor cytoarchitecture. This functional precision medicine approach can be exploited in a range of clinical and laboratory settings to improve the clinical management of GB patients and implemented on other cancers as well.

Abstract Recent evidence suggests that a loss of expression of caveolin in the stromal compartment (sCav-1) of human invasive breast carcinoma (IBC) may be a predictor of disease recurrence, metastasis and poor outcome. At present, there is little knowledge regarding the expression of sCav-1 at the metastatic sites. We therefore studied sCav-1 expression in IBCs and in their axillary lymph nodes to seek a correlation with cancer metastasis. 189 consecutive invasive IBCs (53 with axillary lymph node metastases and 136 without) were studied by immunohistochemistry, using a rabbit polyclonal anti-Cav-1 antibody. In IBCs sCav-1 was evaluated in fibroblasts scattered in the tumor stroma whereas in lymph nodes sCav-1 was assessed in fibroblast-like stromal cells. For the first time, we observed a statistically significant progressive loss of sCav-1 from normal/reactive axillary lymph nodes of tumors limited to the breast to metastatic axillary lymph nodes, through normal/reactive axillary lymph nodes of tumors with axillary metastatic spread. These data indicate that Cav-1 expressed by the stromal compartment of lymph nodes, somehow, may possibly contribute to metastatic spread in IBC.

MiRNAs represent a mechanism that regulates gene expression in many pathological conditions. Exosomes are known to be secreted from all types of cells, and the exosomes-released molecules are crucial messengers that can regulate cellular processes. We investigated the miRNAs content of exosomes released by cancer cells during the invasion . An invasion stimulus has been generated through scratches created on the confluent cells of cancer cell lines: glioblastoma, breast and prostate cancers. Several miRNAs were found to be significantly differentially abundant during the cell invasion , both in common among different cell lines and exclusive. Understanding the language codes among cells involved in invasion can lead to the development of therapies that can inhibit cellular communication, slowing or eventually stopping their activity.

Background: Glioblastoma (GB) is a devastating primary brain malignancy. The recurrence of GB is inevitable despite the standard treatment of surgery, chemotherapy, and radiation, and the median survival is limited to around 15 months. The barriers to treatment include the complex interactions among the different cellular components inhabiting the tumor microenvironment. The complex heterogeneous nature of GB cells is helped by the local inflammatory tumor microenvironment, which mostly induces tumor aggressiveness and drug resistance. Methods: By using fluorescent multiple labeling and a DEPArray cell separator, we recovered several single cells or groups of single cells from populations of different origins from IDH-WT GB samples. From each GB sample, we collected astrocytes-like (GFAP+), microglia-like (IBA1+), stem-like cells (CD133+), and endothelial-like cells (CD105+) and performed Copy Number Aberration (CNA) analysis with a low sequencing depth. The same tumors were subjected to a bulk CNA analysis. Results: The tumor partition in its single components allowed single-cell molecular subtyping which revealed new aspects of the GB altered genetic background. Conclusions: Nowadays, single-cell approaches are leading to a new understanding of GB physiology and disease. Moreover, single-cell CNAs resource will permit new insights into genome heterogeneity, mutational processes, and clonal evolution in malignant tissues.

Background: Glioblastoma (GBM) is the most frequent and malignant primary brain tumor in adults and despite the progress in surgical procedures and therapy options, the overall survival remains very poor. Glutamate and α-KG are fundamental elements necessary to support the growth and proliferation of GBM cells. Glutamate oxidative deamination, catalyzed by GLUD2, is the predominant pathway for the production of α-KG. Methods: GLUD2 emerged from the RNA-seq analysis of 13 GBM patients, performed in our laboratory and a microarray analysis of 77 high-grade gliomas available on the Geo database. Thereafter, we investigated GLUD2 relevance in cancer cell behavior by GLUD2 overexpression and silencing in two different human GBM cell lines. Finally, we overexpressed GLUD2 in-vivo by using zebrafish embryos and monitored the developing central nervous system. Findings: GLUD2 expression was found associated to the histopathological classification, prognosis and survival of GBM patients. Moreover, through in-vitro functional studies, we showed that differences in GLUD2 expression level affected cell proliferation, migration, invasion, colony formation abilities, cell cycle phases, mitochondrial function and ROS production. In support of these findings, we also demonstrated, with in-vivo studies, that GLUD2 overexpression affects glial cell proliferation without affecting neuronal development in zebrafish embryos. Interpretation: We concluded that GLUD2 overexpression inhibited GBM cell growth suggesting a novel potential drug target for control of GBM progression. The possibility to enhance GLUD2 activity in GBM could result in a blocked/reduced proliferation of GBM cells without affecting the survival of the surrounding neurons. Keywords: Glioblastoma, GLUD2, Glutamate, Mitochondrial metabolism, Tumor progression, Zebrafish

In the last few years MMTV-like nucleotide sequences were detected in some feline and canine mammary tumours. Due to the confirmed role of cats in the epidemiology of the MMTV-like virus, the aim of this study was to investigate the main pathological features of positive feline mammary carcinomas (FMCs). Twenty-four FMCs were collected at the University of Bologna, submitted to laser microdissection and analysed by nested fluorescence-PCR using primer sets specific for MMTV env sequence. For immunohistochemistry, an antibody against MMTV protein 14 (p14) was used. MMTV-like sequences were detected in three out of 24 FMCs (12.5%), one tubular carcinoma, one tubulopapillary carcinoma and one ductal carcinoma. All PCR-positive tumours were also positive for p14. Multiple nucleotide alignment has shown similarity to MMTV ranging from 98% to 100%. All the 102 examined FMCs were submitted to immunohistochemistry for molecular phenotyping. Of the nine MMTV-like positive FMCs, six were basal-like and three luminal-like. Our results demonstrate MMTV-like sequences and protein in FMCs of different geographic areas. Molecular phenotyping could contribute to understand the possible role of MMTV-like virus in FMC tumor biology.

Abstract The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter‐cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12α, also abbreviated as stromal‐derived factor‐1 (SDF‐1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by α‐latrotoxin. CXCL12α acts via binding to the neuronal CXCR4 receptor. A CXCL12α‐neutralizing antibody or a specific CXCR4 inhibitor strongly delays recovery from motor neuron degeneration in vivo. Recombinant CXCL12α in vivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons in vitro. These findings indicate that the CXCL12α‐CXCR4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage.

Trastuzumab is an effective therapeutic treatment for Her2-like breast cancer; despite this most of these tumors develop resistance to therapy due to specific gene mutations or alterations in gene expression. Understanding the mechanisms of resistance to Trastuzumab could be a useful tool in order to identify combinations of drugs that elude resistance and allow a better response for the treated patients. Twelve primary biopsies of Her2+/hormone receptor negative (ER-/PgR-) breast cancer patients were selected based on the specific response to neoadjuvant therapy with Trastuzumab and their whole exome was sequenced leading to the identification of 18 informative gene mutations that discriminate patients selectively based on response to treatment. Among these genes, we focused on the study of the ANKRD44 gene to understand its role in the mechanism of resistance to Trastuzumab. The ANKRD44 gene was silenced in Her2-like breast cancer cell line (BT474), obtaining a partially Trastuzumab-resistant breast cancer cell line that constitutively activates the NF-kb protein via the TAK1/AKT pathway. Following this activation an increase in the level of glycolysis in resistant cells is promoted, also confirmed by the up-regulation of the LDHB protein and by an increased TROP2 protein expression, found generally associated with aggressive tumors. These results allow us to consider the ANKRD44 gene as a potential gene involved in Trastuzumab resistance.

Laser capture microdissection (LCM) coupled with RNA-seq is a powerful tool to identify genes that are differentially expressed in specific histological tumor subtypes. To better understand the role of single tumor cell populations in the complex heterogeneity of glioblastoma, we paired microdissection and NGS technology to study intra-tumoral differences into specific histological regions and cells of human GBM FFPE tumors. We here isolated astrocytes, neurons and endothelial cells in 6 different histological contexts: tumor core astrocytes, pseudopalisading astrocytes, perineuronal astrocytes in satellitosis, neurons with satellitosis, tumor blood vessels, and normal blood vessels. A customized protocol was developed for RNA amplification, library construction, and whole transcriptome analysis of each single portion. We first validated our protocol comparing the obtained RNA expression pattern with the gene expression levels of RNA-seq raw data experiments from the BioProject NCBI database, using Spearman's correlation coefficients calculation. We found a good concordance for pseudopalisading and tumor core astrocytes compartments (0.5 Spearman correlation) and a high concordance for perineuronal astrocytes, neurons, normal, and tumor endothelial cells compartments (0.7 Spearman correlation). Then, Principal Component Analysis and differential expression analysis were employed to find differences between tumor compartments and control tissue and between same cell types into distinct tumor contexts. Data consistent with the literature emerged, in which multiple therapeutic targets significant for glioblastoma (such as Integrins, Extracellular Matrix, transmembrane transport, and metabolic processes) play a fundamental role in the disease progression. Moreover, specific cellular processes have been associated with certain cellular subtypes within the tumor. Our results are promising and suggest a compelling method for studying glioblastoma heterogeneity in FFPE samples and its application in both prospective and retrospective studies.

Background and objectives: Cancer stem cells (CSCs) have been implicated in tumor initiation, recurrence, metastatic spread and poor survival in multiple tumor types, breast cancers included. CSCs selectively overexpress key mitochondrial-related proteins and inhibition of mitochondrial function may represent a new potential approach for the eradication of CSCs. Because mitochondria evolved from bacteria, many classes of FDA-approved antibiotics, including doxycycline, actually target mitochondria. Our clinical pilot study aimed to determine whether short-term pre-operative treatment with oral doxycycline results in reduction of CSCs in early breast cancer patients.Methods: Doxycycline was administered orally for 14 days before surgery for a daily dose of 200 mg. Immuno-histochemical analysis of formalin-fixed paraffin-embedded (FFPE) samples from 15 patients, of which 9 were treated with doxycycline and 6 were controls (no treatment), was performed with known biomarkers of “stemness” (CD44, ALDH1), mitochondria (TOMM20), cell proliferation (Ki67, p27), apoptosis (cleaved caspase-3), and neo-angiogenesis (CD31). For each patient, the analysis was performed both on pre-operative specimens (core-biopsies) and surgical specimens. Changes from baseline to post-treatment were assessed with MedCalc 12 (unpaired t-test) and ANOVA.Results: Post-doxycycline tumor samples demonstrated a statistically significant decrease in the stemness marker CD44 (p-value < 0.005), when compared to pre-doxycycline tumor samples. More specifically, CD44 levels were reduced between 17.65 and 66.67%, in 8 out of 9 patients treated with doxycycline. In contrast, only one patient showed a rise in CD44, by 15%. Overall, this represents a positive response rate of nearly 90%. Similar results were also obtained with ALDH1, another marker of stemness. In contrast, markers of mitochondria, proliferation, apoptosis, and neo-angiogenesis, were all similar between the two groups.Conclusions: Quantitative decreases in CD44 and ALDH1 expression are consistent with pre-clinical experiments and suggest that doxycycline can selectively eradicate CSCs in breast cancer patients in vivo. Future studies (with larger numbers of patients) will be conducted to validate these promising pilot studies.

PurposeThe purpose of this study is to determine whether mouse mammary tumor virus (MMTV)-associated human breast cancer has the same or similar histology to MMTV-associated mouse mammary tumors. Such associations may indicate a role for MMTV in human breast cancer.MethodsImmunohistochemical techniques (using antibodies directed against the signal peptide p14 of the envelope precursor protein of MMTV) and polymerase chain reaction (PCR) analyses were used to identify MMTV proteins and MMTV-like envelope gene sequences in a series of breast cancers from Australian women. The histological characteristics of these human breast cancer specimens were compared with MMTV positive mouse mammary tumors. The same methods were used to study benign breast tissues which 1–11 years later developed into breast cancer.ResultsMMTV p14 proteins were identified in 27 (54%) of 50 human breast cancers. MMTV env gene sequences were identified by PCR in 12 (27%) of 45 human breast cancers. There was a significant correlation between the presence of MMTV (identified by p14 immunohistochemistry) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.001). There was a non-significant correlation between the presence of MMTV env gene sequences (identified by PCR) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.290). MMTV p14 proteins were identified in 7 (54%) of 13 benign breast specimens that later developed into human breast cancers. MMTV by PCR was identified in two benign specimens one of whom later developed MMTV positive breast cancer.DiscussionThese observations offer evidence that MMTV may be associated with characteristic human breast cancer histology. p14-based immunohistochemistry appears to be a more reliable technique than PCR for the identification of MMTV in human breast cancer. Identification of MMTV-associated p14 proteins in benign breast tissues confirms prior PCR-based studies that MMTV infection occurs before the development of MMTV positive breast cancer.ConclusionMany MMTV positive human breast cancers have similar histology to MMTV positive mouse mammary tumors. MMTV infection identified in benign breast tissues precedes development of MMTV positive human breast cancer. When considered in the context of prior studies, these observations indicate a likely role for MMTV in human breast cancer.

Mouse mammary tumour virus-like (MMTV-like) is suspected to be involved in human breast cancer and it has been hypothesized that companion animals might have a role in viral transmission. The aim of our study was to investigate the presence of MMTV-like nucleotide sequences and viral protein in a larger number of feline (FMCs) and canine mammary carcinomas (CMCs) by nested PCR and immunohistochemistry. Results showed that the presence of MMTV-like env sequence in FMCs was 7% (6/86), while all the CMCs and canine dysplastic lesions scored negative. All PCR-positive FMCs scored positive for the MMTV p14 signal peptide of the envelope precursor protein of the virus. In contrast, all PCR-negative FMCs and canine mammary lesions were also negative for immunohistochemistry analysis. Canine and feline normal mammary gland tissues scored negative for both PCR and MMTV-p14 protein. Multiple nucleotide alignment of MMTV-like env gene sequences isolated from cat showed 97% and 99% similarity with HMTV and MMTV, respectively, while the others two presented some polimorphisms. Particularly the sequences of one of these two tumors showed a polymorphism (c.7575 A> G), that causes a previously unreported amino acid substitution (Thr > Ala). In conclusion, the results of our study showed the presence of MMTV-like sequences and viral protein in some FMCs. Further studies are needed to understand whether this virus does play a role in the development of FMCs, if MMTV-like is an exogenous virus as these data suggest and, in such a case, how and from whom this virus was acquired.

Papillary thyroid carcinoma is the most frequent histologic type of thyroid tumor. Few studies investigated the role of c-KIT expression in thyroid tumors, suggesting a role for this receptor and its ligand in differentiation and growth control of thyroid epithelium and a receptor loss following malignant transformation. We investigated and correlated c-KIT expression levels and two known markers of thyrocytes differentiation, PAX8 and TTF-1, in malignant and benign cytological thyroid samples. Moreover, we performed functional studies on human papillary thyroid carcinoma cell line to associated c-KIT expression to thyrocytes differentiation and tumor proliferation. c-KIT and PAX8 expression resulted higher in benign samples compared to the malignant ones, and the expression levels of these two genes were significantly correlated to each other. We also observed that c-KIT overexpression led to an increase of PAX8 expression level together with a decrease of proliferation. Furthermore, c-KIT overexpressing cells showed a regression of typical morphological features of malignancy. Taken together these results suggest that c-KIT could be involved in the differentiation of thyroid cells and in tumor progression.