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Molecular targeting requires assessing several factors that come into play such as the location of the target, the choice of radionuclide, the inertness of the bifunctional chelate and stability of the covalently bound halogens, matching the residence time in the tumor with the physical half-life of the radionuclide, the scale and scope of the disease, and the absorbed dose sensitivity of the targeted tumor compared to normal tissue. The principles of molecular targeting are well established, but a paradigm shift from designing a medium-affinity radiotracer used to determine target density to designing a high-affinity, high-target density radioligand to maximize the target-to-nontarget ratio should increase the probability of detecting lesions smaller than the instrument resolution. Developing and validating a therapeutic radiopharmaceutical for a single target is necessary, but often not sufficient to produce a toxic event because of other mechanisms that are only partially understood. These include nontargeted effects due to radiation emitted from neighboring, targeted cells as well as bystander effects produced by the cellular processing of radiation not necessarily impinging on DNA. Both of these indirect consequences of cellular radiation could make a substantial contribution to the efficacy of targeted radionuclide therapy. These mechanisms should be exploited to optimize the efficacy of targeted radiotherapy and overcome the inefficiency of tumor control due to nonuniform distribution of radiation dose. The design approach to take advantage of the indirect consequences of cellular radiation depends heavily on further elucidation of the indirect effect. The successful combination of these two should lead to more effective nuclear radiotherapy.

For better tumor control, high-precision proton beam radiation therapy is currently being intensively discussed relative to conventional photon therapy. Here, we assumed that radiation type-specific molecular response profiles in more physiological 3D, matrix-based head and neck squamous cell carcinoma (HNSCC) cell cultures can be identified and therapeutically exploited. While proton irradiation revealed superimposable clonogenic survival and residual DNA double strand breaks (DSB) relative to photon irradiation, kinome profiles showed quantitative differences between both irradiation types. Pharmacological inhibition of a subset of radiation-induced kinases, predominantly belonging to the mitogen-activated protein kinase (MAPK) family, failed to sensitize HNSCC cells to either proton or photon irradiation. Likewise, inhibitors for ATM, DNA-PK and PARP did not discriminate between proton and photon irradiation but generally elicited a radio-sensitization. Conclusively, our results suggest marginal cell line-specific differences in the radio-sensitivity and DSB repair without a superiority of one radiation type over the other in 3D grown HNSCC cell cultures. Importantly, radiation-induced activity changes of cytoplasmic kinases induced during the first, acute phase of the cellular radiation response could neither be exploited for sensitization of HNSCC cells to photon nor proton irradiation.

Pancreatic ductal adenocarcinoma (PDAC) is a highly therapy resistant tumor entity of unmet need. Over the last decades, radiotherapy has been considered as additional treatment modality to surgery and chemotherapy. Owing to radiosensitive abdominal organs, high precision proton beam radiotherapy has been regarded superior to photon radiotherapy. To further elucidate the potential of combination therapies, we employed a more physiological 3D, matrix-based cell culture model to assess tumoroid formation capacity after photon and proton irradiation. Additionally, we investigated proton and photon irradiation-induced phosphoproteomic changes for identifying clinically exploitable targets. Here, we show that proton irradiation elicits a higher efficacy to reduce 3D PDAC tumoroid formation and a greater extent of phosphoproteome alterations compared with photon irradiation. Targeting of proteins identified in the phosphoproteome that were uniquely altered by protons or photons failed to cause radiation type-specific radiosensitization. Targeting DNA repair proteins associated with non-homologous endjoing, however, revealed a strong radiosensitizing potential independent from the radiation type. In conclusion, our findings suggest proton irradiation to be potentially more effective in PDAC than photons without additional efficacy when combined with DNA repair inhibitors.

Radiation and chemotherapy are the main pillars of the current multimodal treatment concept for cancer patients. However, tumor recurrences and resistances still hamper treatment success regardless of advances in radiation beam application, particle radiotherapy, and optimized chemotherapeutics. To specifically intervene at key recurrence- and resistance-promoting molecular processes, the development of potent and specific molecular-targeted agents is demanded for an efficient, safe, and simultaneous integration into current standard of care regimens. Potential targets for such an approach are integrins conferring structural and biochemical communication between cells and their microenvironment. Integrin binding to extracellular matrix activates intracellular signaling for regulating essential cellular functions such as survival, proliferation, differentiation, adhesion, and cell motility. Tumor-associated characteristics such as invasion, metastasis, and radiochemoresistance also highly depend on integrin function. Owing to their dual functionality and their overexpression in the majority of human malignancies, integrins present ideal and accessible targets for cancer therapy. In the following chapter, the current knowledge on aspects of the tumor microenvironment, the molecular regulation of integrin-dependent radiochemoresistance and current approaches to integrin targeting are summarized.

Human epidermal growth factor receptor type 3 (HER3) is a transmembrane receptor tyrosine kinase belonging to the HER (ErbB) receptor family. Membranous expression of HER3 is associated with trastuzumab resistance in breast cancer and the transition to androgen independence in prostate cancer. Imaging of HER3 expression in malignant tumors may provide important diagnostic information that can influence patient management. Affibody molecules with low picomolar affinity to HER3 were recently selected. The aim of this study was to investigate the feasibility of HER3 imaging using radiolabeled Affibody molecules. A HER3-binding Affibody molecule, Z(08699), with a HEHEHE-tag on N-terminus was labeled with Tc-99m(CO)(3) using an IsoLink kit. In vitro and in vivo binding specificity and the cellular processing of the labeled binder were evaluated. Biodistribution of Tc-99m(CO)(3)-HEHEHE-Z(08699) was studied over time in mice bearing HER3-expressing xenografts. HEHEHE-Z(08699) was labeled with Tc-99m(CO)(3) with an isolated yield of > 80 % and a purity of > 99 %. Binding of Tc-99m(CO)(3)-HEHEHE-Z(08699) was specific to BT474 and MCF7 (breast cancer), and LS174T (colon cancer) cells. Cellular processing showed rapid binding and relatively quick internalization of the receptor/Affibody molecule complex (70 % of cell-associated radioactivity was internalized after 24 h). The tumor targeting was receptor mediated and the excretion was predominantly renal. Receptor-mediated uptake was also found in the liver, lung, stomach, intestine, and salivary glands. At 4 h pi, tumor-to-blood ratios were 7 +/- 3 for BT474, and 6 +/- 2 for LS174T xenografts. LS174T tumors were visualized by microSPECT 4 h pi. The results of this study suggest the feasibility of HER3-imaging in malignant tumors using Affibody molecules., QC 20140714

Understanding the behavior of multifunctional colloidal nanoparticles capable of biomolecular targeting remains a fascinating challenge in materials science with dramatic implications in view of a possible clinical translation. In several circumstances, assumptions on structure–activity relationships have failed in determining the expected responses of these complex systems in a biological environment. The present Review depicts the most recent advances about colloidal nanoparticles designed for use as tools for cellular nanobiotechnology, in particular, for the preferential transport through different target compartments, including cell membrane, cytoplasm, mitochondria, and nucleus. Besides the conventional entry mechanisms based on crossing the cellular membrane, an insight into modern physical approaches to quantitatively deliver nanomaterials inside cells, such as microinjection and electroporation, is provided. Recent hypotheses on how the nanoparticle structure and functionalization may affect the interactions at the nano–bio interface, which in turn mediate the nanoparticle internalization routes, are highlighted. In addition, some hurdles when this small interface faces the physiological environment and how this phenomenon can turn into different unexpected responses, are discussed. Finally, possible future developments oriented tosynergistically tailor biological and chemical properties of nanoconjugates to improve the control over nanoparticle transport, which could open new scenarios in the fi eld of nanomedicine, are addressed.

Most cancer-related deaths arise from metastasis produced by circulating tumor cells (CTCs). A new concept of this research is that circulating cancer stem cell (CSC)-platelet aggregates (CSCPA) represent the most aggressive subset of CTCs responsible for breast cancer metastasis. The goal of this proposal is to identify and count CSCPAs in vivo in preclinical models of metastatic breast cancer, and to define the correlation of CSCPA amount with metastasis development. We developed a novel, in vivo multicolor photoacoustic (PA) flow cytometry (PAFC) platform for the detection of CSCPAs using the principle of flow cytometry, negative and positive PA contrasts, multispectral high-pulse-repetition-rate lasers, bioconjugated nanoparticles and a mouse model of human breast cancer. Using this approach, we provided a proof-of-concept for highly sensitive detection of CSCPAs and individual CSCs in real biological environments in a whole body in vivo. For the first time, we demonstrated the ability of CSCs to form aggregates with platelets (CSCPAs) in blood circulation of tumorbearing mice. Furthermore we showed that the number of CSCPAs increased during development of metastatic disease. Obtained preclinical results can advance general understanding of cancer stem cell biology and metastasis progression as well as will be used as the basis for initiating highly innovative clinical research in cancer patients., The original document contains color images.

Bone disease is a common complication of metastatic solid tumors but also of primary hematological malignancies such as multiple myeloma. Our understanding of the molecular mechanisms underlying the development of bone disease by solid tumors and multiple myeloma has been significantly improved. A complex inter-dependence exists between bone disease and malignant cell growth, creating a vicious cycle of extensive bone destruction and tumor progression. Although myeloma and solid tumors share a number of common molecular pathogenetic mechanisms, they involve distinct pathophysiological pathways, resulting in osteoclastic bone resorption and inhibition of bone formation. In this review, we analyze the molecular mechanisms, involved in tumor-induced bone disease and discuss the current therapeutic approaches and the most recent clinical developments of emerging targeted therapies.

The expression of proteoglycans (PGs), essential macromolecules of the tumor microenvironment, is markedly altered during malignant transformation and tumor progression. Synthesis of stromal PGs is affected by factors secreted by cancer cells and the unique tumor-modified extracellular matrix may either facilitate or counteract the growth of solid tumors. The emerging theme is that this dual activity has intrinsic tissue specificity. Matrix-accumulated PGs, such as versican, perlecan and small leucine-rich PGs, affect cancer cell signaling, growth and survival, cell adhesion, migration and angiogenesis. Furthermore, expression of cell-surface-associated PGs, such as syndecans and glypicans, is also modulated in both tumor and stromal cells. Cell-surface-associated PGs bind various factors that are involved in cell signaling, thereby affecting cell proliferation, adhesion and motility. An important mechanism of action is offered by a proteolytic processing of cell-surface PGs known as ectodomain shedding of syndecans; this facilitates cancer and endothelial cell motility, protects matrix proteases and provides a chemotactic gradient of mitogens. However, syndecans on stromal cells may be important for stromal cell/cancer cell interplay and may promote stromal cell proliferation, migration and angiogenesis. Finally, abnormal PG expression in cancer and stromal cells may serve as a biomarker for tumor progression and patient survival. Enhanced understanding of the regulation of PG metabolism and the involvement of PGs in cancer may offer a novel approach to cancer therapy by targeting the tumor microenvironment. In this minireview, the implication of PGs in cancer development and progression, as well as their pharmacological targeting in malignancy, are presented and discussed.

Recently, the PI3K/Akt pathway has been found to be a significant factor in the development and progression of prostate cancer. It is the authors' belief that the PI3K/Akt pathway is the critical pathway that is maintaining survival by blocking apoptosis in the absence of hormonal stimulation. They will use molecular targeting to inhibit the phosphorylation of Akt. Celecoxib is a FDA approved COX-2 inhibitor. What is unique to celecoxib is its ability to inhibit the phosphorylation of Akt. This effectively turns off the PI3k/Akt pathway leading to apoptosis. Celecoxib has been shown to induce apoptosis in a number of different malignancies. Unfortunately, the half maximal inhibitory concentration (IC(sub 50)) of celecoxib is less than is usually obtainable clinically. Therefore, in an attempt to improve upon Akt activity and decrease the IC(sub 50) concentration to clinically obtainable levels, Chin et al. synthesized multiple 2nd and 3rd generation compounds. These newer compounds have significantly lower IC(sub 50) levels, thus, therapeutic levels can be obtained clinically. The authors will use celecoxib and these newer compounds to evaluate the effects of combined PI3K/Akt inhibition and androgen ablation.

In T-cell Acute Lymphocytic Leukemia (T-ALL), the inhibitors of cyclin-dependent kinases (CDK) 4 and 6, p16 and p15, are inactivated almost universally at the DNA, RNA and protein levels. This suggests that CDK-targeting may be an effective therapeutic approach for T-ALL and other cancers. In this study, we tested 3 inhibitors of CDK4, 3-aminothioacridone (3-ATA), thioacridone (TA), and oxindole, for their effects on DNA synthesis and viability in primary T-ALL. Each compound was an effective inhibitor, with overall IC50s in similar ranges. In colony formation assay, leukemic cells were approximately 10-fold more sensitive to 3-ATA than normal bone marrow cells. When sorted by G1 protein status of T-ALL, p16(+), p15(+) or pRb(-) samples were significantly less sensitive to 3-ATA and TA, but not to oxindole, than p16(-), p15(-) or pRb(+) samples. There was no relationship of sensitivity with ARF expression. Despite their in vitro function as inhibitors of CDK4, 3-ATA did not inhibit pRb phosphorylation or cause G1 arrest, but did cause DNA damage and result in the induction and phosphorylation of p53. We conclude that 3-ATA efficacy can be predicted by p16 status in T-ALL, but the mechanism of action may be distinct from their in vitro ability to regulate CDK4 kinase activity.

In T-cell Acute Lymphocytic Leukemia (T-ALL), the inhibitors of cyclin-dependent kinases (CDK) 4 and 6, p16 and p15, are inactivated almost universally at the DNA, RNA and protein levels. This suggests that CDK-targeting may be an effective therapeutic approach for T-ALL and other cancers. In this study, we tested 3 inhibitors of CDK4, 3-aminothioacridone (3-ATA), thioacridone (TA), and oxindole, for their effects on DNA synthesis and viability in primary T-ALL. Each compound was an effective inhibitor, with overall IC50s in similar ranges. In colony formation assay, leukemic cells were approximately 10-fold more sensitive to 3-ATA than normal bone marrow cells. When sorted by G1 protein status of T-ALL, p16(+), p15(+) or pRb(-) samples were significantly less sensitive to 3-ATA and TA, but not to oxindole, than p16(-), p15(-) or pRb(+) samples. There was no relationship of sensitivity with ARF expression. Despite their in vitro function as inhibitors of CDK4, 3-ATA did not inhibit pRb phosphorylation or cause G1 arrest, but did cause DNA damage and result in the induction and phosphorylation of p53. We conclude that 3-ATA efficacy can be predicted by p16 status in T-ALL, but the mechanism of action may be distinct from their in vitro ability to regulate CDK4 kinase activity.

A crucial issue in the development of molecularly-targeted anticancer therapies is the identification of appropriate molecules whose targeting would result in tumour regression with a minimal level of systemic toxicity. Anaplastic lymphoma kinase (ALK) is a transmembrane receptor tyrosine kinase, normally expressed at low levels in the nervous system. As a consequence of chromosomal translocations involving the alk gene (2p23), ALK is also aberrantly expressed and constitutively activated in similar to 60% of CD30+ anaplastic large cell lymphomas (ALCLs). Due to the selective overexpression of ALK in tumour cells, its direct involvement in the process of malignant transformation and its frequent expression in ALCL patients, the authors recognise ALK as a suitable candidate for the development of molecularly targeted strategies for the therapeutic treatment of ALK-positive lymphomas. Strategies targeting ALK directly or indirectly via the inhibition of the protein networks responsible for ALK oncogenic signalling are discussed.