Fluctuations in tumor blood perfusion assessed by dynamic contrast-enhanced MRI.

Temporal heterogeneity in blood perfusion is a common phenomenon in tumors, but data characterizing the nature of the blood flow fluctuations are sparse. This study investigated the occurrence of blood flow fluctuations in A-07 melanoma xenografts by using gadopentetate dimeglumine (Gd-DTPA)-based dynamic contrast-enhanced MRI (DCE-MRI). Each tumor was subjected to two DCE-MRI acquisitions separated by 1 hour. The data were processed by Kety analysis and resulted in two E.F images (E is the initial extraction fraction of Gd-DTPA and F is the perfusion) and two lambda images (lambda is the partition coefficient of Gd-DTPA) for each tumor. The E . F images were used to determine the changes in blood perfusion arising in the time between the two imaging sequences. The lambda images were used to control the reproducibility of the experimental procedure. The study showed that DCE-MRI with subsequent Kety analysis is a useful method for detection of blood flow fluctuations in A-07 tumors, and strongly suggested that the peripheral regions of A-07 tumors are more exposed to temporal changes in blood perfusion than are the central regions.     

Development of patient derived organoids for cancer drug screening applications

Cancer is a disease characterised by abnormal cell growth that can invade or spread to other regions of the body. Organoids are three-dimensional ex vivo tissue cultures made from embryonic stem cells, induced pluripotent stem cells, progenitor cells or tissue that serve as a physiological model for cancer research. These are designed to recapitulate the in vivo properties of tumours. Importantly, effective recapitulation of the structure of tissues and function is believed to predict patient response, allowing for the creation of personalised therapy in a timely manner that may be used in the clinic. This Review discusses the pre-clinical model and different types of human organoids as models for the development of high throughput drug screening and also aims to highlight how organoids are shaping the future of cancer research.     

In vivo evaluation of cell mediated immunity against human melanoma

Evaluation of cell mediated immunity against human melanoma target cells was performed in an in vivo model using human tumor xenografts growing in Balb/c athymic mice. Intraperitoneal inoculation of 1 × 10⁷ human melanoma cells produced peritoneal carcinomatosis which lead to death of the animals at 23.8 ± 2.6 days (N = 12). Peripheral blood lymphocytes (PBL) from normal donors were administered to tumor bearing mice, and survival times of 22.0±2.3 days were observed (N = 8). Peripheral blood lymphocytes from four of five normal donors which had been presensitized on monolayers of melanoma tissue culture cells in vitro failed to prolong host survival times. In contrast, PBL obtained from 15 of 20 melanoma patients were found to prolong survival of the tumor bearing nude mice. Of these 15 patients, 8 were undergoing specific active immunotherapy, while 7 had not been sensitized except by the disease process. The ability of PBL obtained from patients to prolong survival of tumor bearing animals did not appear to correlate with either the stage of the disease or the patient's clinical course. The possible mechanisms for the prolonged survival and usefulness of this model are discussed.     

A rat model of leptomeningeal human neoplastic xenografts

Leptomeningeal (LM) cancer spread from either a primarybrain tumor or a systemic cancer is rapidlyfatal. Current therapies are ineffective and highly toxicto normal nervous system tissues. A xenograft modelof LM neoplasia in nude rats using adiversity of tumor cell types was established inorder to evaluate new treatment strategies and tostudy the pharmacokinetics and biological effects of treatmentsadministered into the subarachnoid space. Consistent leptomeningeal engraftmentand progressive tumor growth was seen after intrathecalinjection of 9 of 13 tumor cells lines,including 2 melanomas, 2 neuroblastomas, 2 medulloblastomas, 2gliomas, and 1 breast cancer. Clinical signs rangedfrom steady weight loss commencing from the dayafter tumor implantation to absence of any signsfor three weeks until the sudden occurrence ofmajor neurological deficits or death. Pathologic examination showedonly leptomeningeal tumor growth with some cell linesand severe parenchymal invasion with others. CSF cytologyconsistently demonstrated tumor cells in animals with LMdisease. Cranial magnetic resonance (MR) following intravenous (IV)administration of a contrast agent revealed enhancing lesionsone week following melanoma tumor implantation. Reliable ventricularpuncture was demonstrated by radiography following intraventricular (IVent)injection of an iodinated contrast material. IVent instillationof saline, albumin, or antibodies did not provokeclinical toxicity or an inflammatory response.     

Preclinical in vivo antitumor activity of vinflunine, a novel fluorinated Vinca alkaloid

Vinflunine, or 20′,20′-difluoro-3′,4′-dihydrovino‐relbine, is a novel Vinca alkaloid obtained by hemisynthesis using superacidic chemistry. The most impressive structural modification of this vinorelbine derivative was the selective introduction of two fluorine atoms at the 20′ position, a part of the molecule previously inaccessible by classic chemistry. The antitumor activity of vinflunine was evaluated against a range of transplantable murine and human tumors. Vinflunine exhibited marked activity against murine P388 leukemia grafted i.v. when given i.p. in single or multiple doses according to various schedules or in single i.v. or p.o. doses. Increases in life span achieved with vinflunine, as assessed by T/C ratios, ranged from 200% to 457% and proved markedly superior to those of 129–186% obtained with the other Vinca alkaloids tested. Against s.c.-implanted B16 melanoma, multiple i.p. administration of vinflunine proved active in terms of both survival prolongation and tumor growth inhibition, with optimal T/C values and relative areas under the tumor growth curves (rAUC) being 24% and 36%, respectively. The extent of this activity was superior to that noted for vinorelbine under the same experimental conditions. Growth inhibition of human tumor xenografts LX-1 (lung) and MX-1 (breast) was also observed following four weekly i.p. injections of vinflunine as reflected by optimal T/C values of 23% and 26%, respectively, and significant differences in the rAUCs noted for treated versus control animals. It was also noticeable that vinflunine induced considerably more prolonged inhibitory effects on tumor growth than did vinorelbine. These results demonstrate that vinflunine is well tolerated and is definitively active against a range of experimental animal tumor models. Vinflunine activity has been documented in terms of both survival prolongation and tumor growth inhibition, with definite superiority over vinorelbine being shown in each tumor model evaluated. Received: 13 July 1997 / Accepted: 21 October 1997     

Melanoma patient derived xenografts acquire distinct Vemurafenib resistance mechanisms

Variable clinical responses, tumor heterogeneity, and drug resistance reduce long-term survival outcomes for metastatic melanoma patients. To guide and accelerate drug development, we characterized tumor responses for five melanoma patient derived xenograft models treated with Vemurafenib. Three BRAF^V600E models showed acquired drug resistance, one BRAF^V600E model had a complete and durable response, and a BRAF^V600V model was expectedly unresponsive. In progressing tumors, a variety of resistance mechanisms to BRAF inhibition were uncovered, including mutant BRAF alternative splicing, NRAS mutation, COT (MAP3K8) overexpression, and increased mutant BRAF gene amplification and copy number. The resistance mechanisms among the patient derived xenograft models were similar to the resistance pathways identified in clinical specimens from patients progressing on BRAF inhibitor therapy. In addition, there was both inter- and intra-patient heterogeneity in resistance mechanisms, accompanied by heterogeneous pERK expression immunostaining profiles. MEK monotherapy of Vemurafenib-resistant tumors caused toxicity and acquired drug resistance. However, tumors were eradicated when Vemurafenib was combined the MEK inhibitor. The diversity of drug responses among the xenograft models; the distinct mechanisms of resistance; and the ability to overcome resistance by the addition of a MEK inhibitor provide a scheduling rationale for clinical trials of next-generation drug combinations.     

The polycomb group protein enhancer of zeste 2 is a novel therapeutic target for cervical cancer

Enhancer of zeste 2 (EZH2), a polycomb histone methyltransferase, is overexpressed in various cancers, including cervical cancer. Gene expression analysis revealed that increased expression of EZH2 is associated with cervical cancer progression, particularly the progression to invasive squamous cell carcinoma. Enhancer of zeste 2 is known to trimethylate lysine 27 on histone H3, leading to gene silencing that contributes to the progression of tumours into a more aggressive form of cancer. However, the specific molecular mechanisms by which EZH2 contributes to the development of cervical cancer remain largely unknown. Recently, an EZH2 inhibitor was reported to selectively inhibit trimethylated lysine 27 on histone H3 and to reactivate silenced genes in cancer cells. In this study, we found that GSK343 (a specific inhibitor of EZH2 methyltransferase) induces phenotypic reprogramming of cancer cells from mesenchymal to epithelial cells, reducing proliferation and cell motility and blocking the invasion of cervical cancer cell lines both in vitro and in vivo. Treatment with the EZH2 inhibitor led to increased levels of the epithelial marker E‐cadherin and decreased levels of mesenchymal markers such as N‐cadherin and vimentin. The observed reprogramming is associated with restrained cervical cancer progression and provides direct evidence in support of EZH2 as a therapeutic target.     

In vivo anticancer activity of rhomboidal Pt(II) metallacycles

The development of novel antitumor agents that have high efficacy in suppressing tumor growth, have low toxicity to nontumor tissues, and exhibit rapid localization in the targeted tumor sites is an ongoing avenue of research at the interface of chemistry, cancer biology, and pharmacology. Supramolecular metal-based coordination complexes (SCCs) have well-defined shapes and geometries, and upon their internalization, SCCs could affect multiple oncogenic signaling pathways in cells and tissues. We investigated the uptake, intracellular localization, and antitumor activity of two rhomboidal Pt(II)-based SCCs. Laser-scanning confocal microscopy in A549 and HeLa cells was used to determine the uptake and localization of the assemblies within cells and their effect on tumor growth was investigated in mouse s.c. tumor xenograft models. The SCCs are soluble in cell culture media within the entire range of studied concentrations (1 nM–5 µM), are nontoxic, and showed efficacy in reducing the rate of tumor growth in s.c. mouse tumor xenografts. These properties reveal the potential of Pt(II)-based SCCs for future biomedical applications as therapeutic agents.Molecular assemblies of nanoscale-size and well-defined geometries have recently emerged as an interesting new paradigm in drug design and drug delivery. To date, liposomes, the self-assembled lipid nanoparticles held together by weak interactions, are among the most widely studied and clinically successful nanoparticle-based drug carriers. Their use allows the drug to achieve sustained plasma levels while encapsulated, with the size preventing the fast clearance by the kidneys that often occurs with the free drug. However, liposomes themselves do not produce a therapeutic effect and their application as drug carriers for medical purposes has often been hindered by poor loading capacity (<5 wt %) and the inability to pass through biological barriers (1, 2). Inorganic and hybrid porous materials, such as molecular organic frameworks (MOFs), have also shown promise due to their higher loading capacities (>25 wt %) (3–5), but MOFs have poor hydrolytic stability (6, 7). Recent studies on materials from Institut Lavoisier (MIL)-100(Cr) and MIL-100(Fe), however, suggest that MOFs can persist in biologically relevant environments and can act as vehicles for some anticancer and antiviral agents (8–10). These early findings have prompted further investigations into the biomedical applications of supramolecular coordination complexes (SCCs) (11–24). SCCs preserve the attractive properties of MOFs, such as building block modularity (22, 23, 25), yet afford an increased solubility in the biological milieu and lend themselves to small-molecule characterization techniques due to their well-defined structure.Although development of SCCs for biomedical applications is in its infancy, some SCCs, such as trigonal prisms self-assembled from p-cymene and ruthenium-based metal fragments with pyridyl donors, have shown the ability to act as effective carriers of some chemotherapeutic agents (26–28). Moreover, a library of cytotoxic to cancer cells p-cymene ruthenium-based polygons and cages has also been developed (11). For biomedical applications, the information about the cellular uptake, delivery of a guest, and metabolism of the drug delivery vehicle is critical, although currently the fate of SCCs in biological environments is not well understood. In a rare report, a systematic investigation of the structural stability of a water-soluble, hexacationic ruthenium-based trigonal prism was performed; however, it was determined that the ruthenium-based trigonal prisms decompose in the presence of amino acids histidine, lysine, and arginine (29).An intriguing approach is the design of tumor-targeted modalities that combine detection and treatment through the self-assembly of emissive, metal-based coordination complexes. Such modalities can be especially valuable as they often do not require photoexcitation to elicit cytotoxicity. Recently Gray, Gross, and Medina-Kauwe and coworkers reported HerGa, a self-assembled tumor-targeted particle that bears the Ga(III)-metalated derivative of the sulfonated corrole (30, 31). The particle, which contained Ga(III)-corrole noncovalently bound to the tumor-targeting cell penetration protein HerPBK10, provided both tumor detection and elimination. Systemic injection of this protein–corrole complex resulted in tumor accumulation, which can be visualized in vivo due to the red corrole fluorescence. Cytotoxic and cytostatic properties of these targeted Ga(III) corroles were found to be cell-line dependent, with the ability to induce late M-phase arrest in several cancer cell lines (32).Despite the well-known cytotoxic properties of mono- and multinuclear platinum complexes (33–35), studies of the antitumor properties of platinum-based SCCs are rare (17, 36). Moreover, recent reports have demonstrated that platinum-based SCCs can act as effective hosts for guests and have interesting photophysical properties (37–42). In particular, highly emissive rhomboids based on aniline-containing donors and Pt-based metal acceptors have been developed that display different photophysical properties from those of their constituent subunits (40). These assemblies are interesting targets to investigate the cytotoxicity of organoplatinum SCCs, whereas their emission spectra could be used for interrogating the structural integrity in vitro. Here, for the first time to our knowledge, we report the uptake of SCCs in vitro in cell-based assays, determined by using laser-scanning confocal microscopy, and an in vivo assessment of the anticancer activity of SCCs in mouse s.c. tumor xenograft models.     

Biological monitoring of a promissory xenogenic pin for biomedical applications: a preliminary intraosseous study in rats

Objectives: Over the last years, it is known that in some cases metal devices for biomedical applications present some disadvantages suggesting absorbable materials (natural or synthetic) as an alternative of choice. Here, our goal was to evaluate the biological response of a xenogenic pin, derived from bovine cortical bone, intraosseously implanted in the femur of rats. Material and methods: After 10, 14, 30 and 60 days from implantation, the animals (n=5/period) were killed and the femurs carefully collected and dissected out under histological demands. For identifying the osteoclastogenesis level at 60 days, we performed the immunohistochemisty approach using antibody against RANKL. Results: Interestingly, our results showed that the incidence of neutrophils and leukocytes was observed only at the beginning (10 days). Clear evidences of pin degradation by host cells started at 14 days and it was more intensive at 60 days, when we detected the majority of the presence of giant multinucleated cells, which were very similar to osteoclast cells contacting the implanted pin. To check osteoclastogenesis at 60 days, we evaluated RANKL expression and it was positive for those resident multinucleated cells while a new bone deposition was verified surrounding the pins in all evaluated periods. Conclusions: Altogether, our results showed that pins from fully processed bovine bone are biocompatible and absorbable, allowing bone neoformation and it is a promissory device for biomedical applications. To cite this article :
Zambuzzi WF, Oliveira RC, Subitoni BL, Menezes R, Taga R, Granjeiro, JM. Biological monitoring of a promissory xenogenic pin for biomedical applications: a preliminary intraosseous study in rats.
Clin. Oral Impl. Res. 23 , 2012; 367–372.
doi: 10.1111/j.1600‐0501.2010.02143.x