SLFN11 and ATR as targets for overcoming cisplatin resistance in ovarian cancer cells.

The levels and activities of the DNA/RNA helicase schlafen11 (SLFN11) and the serine/threonine-protein kinase ataxia telangiectasia and Rad3-related protein (ATR) may determine cancer cell sensitivity to DNA damaging agents, including platinum drugs. Here, we studied the roles of SLFN11 and ATR in cisplatin resistance of ovarian cancer using cell lines displaying acquired or intrinsic cisplatin resistance. W1CR, the cisplatin-resistant subline of W1 ovarian cancer cells, displayed reduced SLFN11 levels. HDAC inhibition using entinostat returned an epigenetic downregulation of SLFN11 in W1CR cells, caused SLFN11 re-expression and re-sensitized these cells to cisplatin. Moreover, entinostat also sensitized intrinsically resistant EFO21 ovarian cancer cells to cisplatin by upregulating SLFN11. However, SLFN11 was not involved in cisplatin resistance in all other cell models. Thus, SLFN11 expression is not a general cisplatin resistance marker in ovarian cancer. In contrast, inhibition of the DNA damage repair master regulator ATR using sub-toxic concentrations of elimusertib sensitized parental cell lines as well as intrinsically resistant EFO21 cells to cisplatin, and fully reversed acquired cisplatin resistance in cisplatin-adapted sublines W1CR, A2780cis, and KuramochirCDDP2000. Mechanisms underlying ATR-mediated cisplatin resistance differed between the cell lines and included CHK1/WEE1 signaling and induction of homologous recombination. In conclusion, SLFN11 and ATR are involved in ovarian cancer cisplatin resistance. Although our data identify ATR as key target for tackling cisplatin resistance in ovarian cancer, future studies are needed to identify biomarkers that indicate, which individual ovarian cancers benefit from SLFN11 re-activation and/or ATR inhibition.

Diversity Oriented Synthesis of Novel Xanthones Reveal Potent Doxorubicin-Inspired Analogs.

Inspired by potent antiproliferative xanthone natural products and so far limited examples of derived bioactive agents, a structure activity study of architecturally novel types of xanthones is reported. Their preparation was enabled in a short and divergent manner by a modular chlorination in combination with optimized protocols for a polar condensation and a hetero-cyclization. Application of these procedures allowed for the synthesis of various polyhalogenated representatives (including mixed bromo/chloro xanthones) that were obtained in up to fourfold improved yields as compared to previous procedures. Subsequent Suzuki coupling of either halide enabled access to phenyl- and chloro-bearing xanthones, which may be functionalized at four out of five non-hydroxylated positions. Antiproliferative assays against breast cancer cell lines revealed potent activities of some of these simplified analogs that are in the range of pharmaceutically used anticancer drug doxorubicin.

The Role of CD36/GPIV in Platelet Biology.

CD36 (also known as platelet glycoprotein IV) is expressed by a variety of different cell entities, where it possesses functions as a signaling receptor, but additionally acts as a transporter for long-chain fatty acids. This dual function of CD36 has been investigated for its relevance in immune and nonimmune cells. Although CD36 was first identified on platelets, the understanding of the role of CD36 in platelet biology remained scarce for decades. In the past few years, several discoveries have shed a new light on the CD36 signaling activity in platelets. Notably, CD36 has been recognized as a sensor for oxidized low-density lipoproteins in the circulation that mitigates the threshold for platelet activation under conditions of dyslipidemia. Thus, platelet CD36 transduces atherogenic lipid stress into an increased risk for thrombosis, myocardial infarction, and stroke. The underlying pathways that are affected by CD36 are the inhibition of cyclic nucleotide signaling pathways and simultaneously the induction of activatory signaling events. Furthermore, thrombospondin-1 secreted by activated platelets binds to CD36 and furthers paracrine platelet activation. CD36 also serves as a binding hub for different coagulation factors and, thus, contributes to the plasmatic coagulation cascade. This review provides a comprehensive overview of the recent findings on platelet CD36 and presents CD36 as a relevant target for the prevention of thrombotic events for dyslipidemic individuals with an elevated risk for thrombosis.

Development of First-in-Class Dual Sirt2/HDAC6 Inhibitors as Molecular Tools for Dual Inhibition of Tubulin Deacetylation.

Dysregulation of both tubulin deacetylases sirtuin 2 (Sirt2) and the histone deacetylase 6 (HDAC6) has been associated with the pathogenesis of cancer and neurodegeneration, thus making these two enzymes promising targets for pharmaceutical intervention. Herein, we report the design, synthesis, and biological characterization of the first-in-class dual Sirt2/HDAC6 inhibitors as molecular tools for dual inhibition of tubulin deacetylation. Using biochemical in vitro assays and cell-based methods for target engagement, we identified Mz325 (33) as a potent and selective inhibitor of both target enzymes. Inhibition of both targets was further confirmed by X-ray crystal structures of Sirt2 and HDAC6 in complex with building blocks of 33. In ovarian cancer cells, 33 evoked enhanced effects on cell viability compared to single or combination treatment with the unconjugated Sirt2 and HDAC6 inhibitors. Thus, our dual Sirt2/HDAC6 inhibitors are important new tools to study the consequences and the therapeutic potential of dual inhibition of tubulin deacetylation.

Heparins are potent inhibitors of ectonucleotide pyrophosphatase/phospho-diesterase-1 (NPP1) - a promising target for the immunotherapy of cancer.

Introduction: Heparins, naturally occurring glycosaminoglycans, are widely used for thrombosis prevention. Upon application as anticoagulants in cancer patients, heparins were found to possess additional antitumor activities. Ectonucleotidases have recently been proposed as novel targets for cancer immunotherapy. Methods and results: In the present study, we discovered that heparin and its derivatives act as potent, selective, allosteric inhibitors of the poorly investigated ectonucleotidase NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1, CD203a). Structure-activity relationships indicated that NPP1 inhibition could be separated from the compounds' antithrombotic effect. Moreover, unfractionated heparin (UFH) and different low molecular weight heparins (LMWHs) inhibited extracellular adenosine production by the NPP1-expressing glioma cell line U87 at therapeutically relevant concentrations. As a consequence, heparins inhibited the ability of U87 cell supernatants to induce CD4+ T cell differentiation into immunosuppressive Treg cells. Discussion: NPP1 inhibition likely contributes to the anti-cancer effects of heparins, and their specific optimization may lead to improved therapeutics for the immunotherapy of cancer.

A biscarbene gold(I)-NHC-complex overcomes cisplatin-resistance in A2780 and W1 ovarian cancer cells highlighting pERK as regulator of apoptosis.

PURPOSE: Cisplatin resistance is the major obstacle in the clinical treatment of ovarian cancer patients. Molecular mechanisms of cisplatin resistance are multifaceted. Gold(I)-compounds, i.e. N-heterocyclic carbene-gold(I)-complexes (NHC-Au(I)) has been regarded as promising cytotoxic drug candidates. However, their potential to overcome cisplatin resistance has hardly been addressed yet. Here we investigated the activity of the gold(I) drug auranofin and the NHC-Au(I)-compound MC3 in W1CR and A2780cis cisplatin-resistant ovarian cancer cells. METHODS: Cytotoxicity of auranofin and MC3 was detected by MTT assay, correlated with intracellular gold(I) content, analyzed by AAS, and with flow cytometric detection of the cell cycle. Insight into cellular redox balance was provided by fluorimetric ROS-formation assay and western blotting thioredoxin (Trx) and Nrf2. The role of ERK was elucidated by using the inhibitor SCH772984 and its impact on cytotoxicity upon co-treatment with cisplatin and Au(I)-compounds, respectively. RESULTS: MC3 overcomes cisplatin resistance in A2780cis and W1CR, and auranofin in W1CR cells completely, which is neither reflected by intracellular gold levels nor cell cycle changes. Upregulated redox balance appears as a basis for resistance. W1CR cells possess higher Trx levels, whereas A2780cis cells display strong Nrf2 expression as anti-oxidative protection. Nevertheless, overcoming redox balance appears not primary mode of activity comparing cisplatin and gold(I)-compounds. pERK emerges as a critical component and thus a promising target for overcoming resistance, regulating apoptosis differently in response to either gold(I) or cisplatin in A2780 cells. CONCLUSION: These data reflect the complexity of cisplatin resistance in cell models and emphasize NHC-Au(I)-complexes as prospective cytotoxic agents for further investigations in that respect.

The Tissue Factor Pathway in Cancer: Overview and Role of Heparan Sulfate Proteoglycans.

Historically, the only focus on tissue factor (TF) in clinical pathophysiology has been on its function as the initiation of the extrinsic coagulation cascade. This obsolete vessel-wall TF dogma is now being challenged by the findings that TF circulates throughout the body as a soluble form, a cell-associated protein, and a binding microparticle. Furthermore, it has been observed that TF is expressed by various cell types, including T-lymphocytes and platelets, and that certain pathological situations, such as chronic and acute inflammatory states, and cancer, may increase its expression and activity. Transmembrane G protein-coupled protease-activated receptors can be proteolytically cleaved by the TF:FVIIa complex that develops when TF binds to Factor VII (PARs). The TF:FVIIa complex can activate integrins, receptor tyrosine kinases (RTKs), and PARs in addition to PARs. Cancer cells use these signaling pathways to promote cell division, angiogenesis, metastasis, and the maintenance of cancer stem-like cells. Proteoglycans play a crucial role in the biochemical and mechanical properties of the cellular extracellular matrix, where they control cellular behavior via interacting with transmembrane receptors. For TFPI.fXa complexes, heparan sulfate proteoglycans (HSPGs) may serve as the primary receptor for uptake and degradation. The regulation of TF expression, TF signaling mechanisms, their pathogenic effects, and their therapeutic targeting in cancer are all covered in detail here.

Exostosin 1 Knockdown Induces Chemoresistance in MV3 Melanoma Cells by Upregulating JNK and MEK/ERK Signaling.

Heparan sulfate proteoglycans (HSPGs) possess various functions driving malignancy of tumors. However, their impact on tumor cell sensitivity to cytotoxic treatment is far less understood. Aiming to investigate this, we depleted HSPGs by downregulating Exostosin 1 (EXT1), a key enzyme in HS formation, or upregulating heparanase in human MV3 human melanoma cells, and investigated their response to cytotoxic drugs. Cytotoxicity of trametinib, doxorubicin, and mitoxantrone was detected by MTT assay. Insights into intracellular signaling was provided by kinome protein profiler array, and selected kinases were inhibited to investigate their impact on cell sensitization and migratory dynamics. EXT1 knockdown (EXT1kd) in MV3 cells affected the activity of doxorubicin and mitoxantrone, significantly increasing EC50 values two- or fourfold, respectively. Resistance formation was scarcely related to HSPG deficiency, suggested by enzymatic cleavage of HSPG in control cells. Notably, EXT1kd induced an upregulation of EGFR signaling via JNK and MEK/ERK, and hence blocking these kinases returned resistance to a sensitive level. JNK appeared as a key signal component, also inducing higher migratory activity of EXT1kd cells. Furthermore, EXT1kd upregulated thrombotic properties of MV3 cells, indicated by tissue factor and PAR-1 expression, functionally reflected by a stronger activation of platelet aggregation. EXT1 was confirmed to act as a tumor suppressor, shown here for the first time to affect chemosensitivity of melanoma cells.

The Heparan Sulfate Proteoglycan Syndecan-1 Triggers Breast Cancer Cell-Induced Coagulability by Induced Expression of Tissue Factor.

Syndecan-1 (Sdc-1) upregulation is associated with poor prognosis in breast cancer. Sdc-1 knockdown results in reduced angiogenesis and the dysregulation of tissue factor (TF) pathway constituents. Here, we evaluate the regulatory mechanisms and functional consequences of the Sdc-1/TF-axis using Sdc-1 knockdown and overexpression approaches in MCF-7 and MDA-MB-231 breast cancer cells. Gene expression was analyzed by means of qPCR. Thrombin generation and cell migration were detected. Cell-cycle progression and apoptosis were investigated using flow cytometry. In MDA-MB-231 cells, IL6, IL8, VEGF, and IGFR-dependent signaling affected TF pathway expression depending on Sdc-1. Notably, Sdc-1 depletion and TF pathway inhibitor (TFPI) synergistically affected PTEN, MAPK, and STAT3 signaling. At the functional level, the antiproliferative and pro-apoptotic effects of TFPI depended on Sdc-1, whereas Sdc-1's modulation of cell motility was not affected by TFPI. Sdc-1 overexpression in MCF-7 and MDA-MB-231 cells led to increased TF expression, inducing a procoagulative phenotype, as indicated by the activation of human platelets and increased thrombin formation. A novel understanding of the functional interplay between Sdc-1 and the TF pathway may be compatible with the classical co-receptor role of Sdc-1 in cytokine signaling. This opens up the possibility of a new functional understanding, with Sdc-1 fostering coagulation and platelet communication as the key to the hematogenous metastatic spread of breast cancer cells.

Development of the first non-hydroxamate selective HDAC6 degraders.

The targeted degradation of histone deacetylase 6 (HDAC6) by heterobifunctional degraders constitutes a promising approach to treat HDAC6-driven diseases. Previous HDAC6 selective degraders utilised a hydroxamic acid as a zinc-binding group (ZBG) which features mutagenic and genotoxic potential. Here we report the development of a new class of selective HDAC6 degraders based on a difluoromethyl-1,3,4-oxadiazole warhead as ZBG.

Heparan sulfate dependent binding of plasmatic von Willebrand factor to blood circulating melanoma cells attenuates metastasis.

Heparan sulfate (HS), a highly negatively charged glycosaminoglycan, is ubiquitously present in all tissues and also exposed on the surface of mammalian cells. A plethora of molecules such as growth factors, cytokines or coagulation factors bear HS binding sites. Accordingly, HS controls the communication of cells with their environment and therefore numerous physiological and pathophysiological processes such as cell adhesion, migration, and cancer cell metastasis. In the present work, we found that HS exposed by blood circulating melanoma cells recruited considerable amounts of plasmatic von Willebrand factor (vWF) to the cellular surface. Analyses assisted by super-resolution microscopy indicated that HS and vWF formed a tight molecular complex. Enzymatic removal of HS or genetic engineering of the HS biosynthesis showed that a reduced length of the HS chains or complete lack of HS was associated with significantly reduced vWF encapsulation. In microfluidic experiments, mimicking a tumor-activated vascular system, we found that vWF-HS complexes prevented vascular adhesion. In line with this, single molecular force spectroscopy suggested that the vWF-HS complex promoted the repulsion of circulating cancer cells from the blood vessel wall to counteract metastasis. Experiments in wild type and vWF knockout mice confirmed that the HS-vWF complex at the melanoma cell surface attenuated hematogenous metastasis, whereas melanoma cells lacking HS evade the anti-metastatic recognition by vWF. Analysis of tissue samples obtained from melanoma patients validated that metastatic melanoma cells produce less HS. Transcriptome data further suggest that attenuated expression of HS-related genes correlate with metastases and reduced patients' survival. In conclusion, we showed that HS-mediated binding of plasmatic vWF to the cellular surface can reduce the hematogenous spread of melanoma. Cancer cells with low HS levels evade vWF recognition and are thus prone to form metastases. Therefore, therapeutic expansion of the cancer cell exposed HS may prevent tumor progression.

The GPIb-IX complex on platelets: insight into its novel physiological functions affecting immune surveillance, hepatic thrombopoietin generation, platelet clearance and its relevance for cancer development and metastasis.

The glycoprotein (GP) Ib-IX complex is a platelet receptor that mediates the initial interaction with subendothelial von Willebrand factor (VWF) causing platelet arrest at sites of vascular injury even under conditions of high shear. GPIb-IX dysfunction or deficiency is the reason for the rare but severe Bernard-Soulier syndrome (BSS), a congenital bleeding disorder. Although knowledge on GPIb-IX structure, its basic functions, ligands, and intracellular signaling cascades have been well established, several advances in GPIb-IX biology have been made in the recent years. Thus, two mechanosensitive domains and a trigger sequence in GPIb were characterized and its role as a thrombin receptor was deciphered. Furthermore, it became clear that GPIb-IX is involved in the regulation of platelet production, clearance and thrombopoietin secretion. GPIb is deemed to contribute to liver cancer development and metastasis. This review recapitulates these novel findings highlighting GPIb-IX in its multiple functions as a key for immune regulation, host defense, and liver cancer development.

Tumour cell-activated platelets modulate the immunological activity of CD4+, CD8+, and NK cells, which is efficiently antagonized by heparin.

Platelets, key players in haemostasis, are progressively investigated with respect to their role in immunity and inflammation. Although the platelet support to haematogenous cancer cell metastasis has been the subject of multiple studies, their impact on anti-cancer immunity remains unaddressed. Here, we investigated the immunomodulatory potential of platelets upon their activation by MDA-MB-231 breast cancer cells in various in vitro approaches. We provide evidence that platelets as well as their tumour cell-induced releasates increased the ratio of regulatory T cells, shaping an immunosuppressive phenotype in isolated CD4+ cultures. The influence on CD8+ T cells was assessed by detecting the expression of activation markers CD25/CD69 and release of cytolytic and pro-inflammatory proteins. Notably, the platelet preparations differentially influenced CD8+ T cell activation, while platelets were found to inhibit the activation of CD8+ T cells, platelet releasates, in contrast, supported their activation. Furthermore, the NK cell cytolytic activity was attenuated by platelet releasates. Low molecular weight heparin (LMWH), the guideline-based anticoagulant for cancer-associated thrombotic events, is known to interfere with tumour cell-induced platelet activation. Thus, we aimed to investigate whether, unfractionated heparin, LMWH or novel synthetic heparin mimetics can also reverse the immunosuppressive platelet effects. The releasate-mediated alteration in immune cell activity was efficiently abrogated by heparin, while the synthetic heparin mimetics partly outperformed the commercial heparin derivatives. This is the first report on the effects of heparin on rebalancing immunosuppression in an oncological context emerging as a novel aspect in heparin anti-tumour activities.

Design, synthesis, and characterization of PROTACs targeting the androgen receptor in prostate and lung cancer models.

Although the androgen receptor (AR) is a validated target for the treatment of prostate cancer, resistance to antiandrogens necessitates the development of new therapeutic modalities. Exploiting the ubiquitin-proteasome system with proteolysis-targeting chimeras (PROTACs) has become a practical approach to degrade specific proteins and thus to extend the portfolio of small molecules used for the treatment of a broader spectrum of diseases. Herein, we present three subgroups of enzalutamide-based PROTACs in which only the exit vector was modified. By recruiting cereblon, we were able to demonstrate the potent degradation of AR in lung cancer cells. Furthermore, the initial evaluation enabled the design of an optimized PROTAC with a rigid linker that degraded AR with a DC50 value in the nanomolar range. These results provide novel AR-directed PROTACs and a clear rationale for further investigating AR involvement in lung cancer models.

Balancing Histone Deacetylase (HDAC) Inhibition and Drug-likeness: Biological and Physicochemical Evaluation of Class I Selective HDAC Inhibitors.

Herein we report the structure-activity and structure-physicochemical property relationships of a series of class I selective ortho-aminoanilides targeting the "foot-pocket" in HDAC1&2. To balance the structural benefits and the physicochemical disadvantages of these substances, we started with a set of HDACi related to tacedinaline (CI-994) and evaluated their solubility, lipophilicity (log D7.4 ) and inhibition of selected HDAC isoforms. Subsequently, we selected the most promising "capless" HDACi and transferred its ZBG to our previously published scaffold featuring a peptoid-based cap group. The resulting hit compound 10 c (LSH-A54) showed favorable physicochemical properties and is a potent, selective HDAC1/2 inhibitor. The following evaluation of its slow binding properties revealed that LSH-A54 binds tightly to HDAC1 in an induced-fit mechanism. The potent HDAC1/2 inhibitory properties were reflected by attenuated cell migration in a modified wound healing assay and reduced cell viability in a clonogenic survival assay in selected breast cancer cell lines.

Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells.

Tumor cell crosstalk with platelets and, subsequently, their activation are key steps in hematogenous tumor metastasis. MACC1 is an oncogene involved in molecular pathogenesis of colorectal cancer (CRC) and other solid tumor entities, mediating motility and metastasis, making MACC1 an accepted prognostic biomarker. However, the impact of MACC1 on platelet activation has not yet been addressed. Here, we investigated the activation of platelets by human CRC cells upon MACC1 modulation, indicated by platelet aggregation and granule release. These approaches led to the identification of insulin-like growth factor binding protein-2 (IGFBP2) as a functional downstream molecule of MACC1, affecting communication with platelets. This was confirmed by an shRNA-mediated IGFBP2 knockdown, while maintaining MACC1 activity. Although IGFBP2 displayed an attenuated platelet activation potential, obviously by scavenging IGF-I as a platelet costimulatory mediator, the MACC1/IGFBP2 axis did not affect the thrombin formation potential of the cells. Furthermore, the IGFBP2/MACC1-driven cell migration and invasiveness was further accelerated by platelets. The key role of IGFBP2 for the metastatic spread in vivo was confirmed in a xenograft mouse model. Data provide evidence for IGFBP2 as a downstream functional component of MACC1-driven metastasis, linking these two accepted oncogenic biomarkers for the first time in a platelet context.

Another structural correction for 1-oxo-1H-phenalene-2,3-dicarbonitriles: Synthesis of a potent BCL-2 inhibiting 7-phenoxy derivative.

Aromatic scaffolds are an important part of biologically active compounds and molecular probes used to study biochemical pathways and the involved targeted proteins of interest. 1-Oxo-1H-phenalene-2,3-dicarbonitrile-based compounds have been described as inhibitors of the BCL-2 family of proteins, and this core structure represents numerous possibilities for modifications that could lead to improved inhibitory potencies. Many studies demonstrated intriguing characteristics of these compounds in terms of reactivity and, interestingly, some contradictory literature reports appeared about reaction outcomes to synthesize them. Here, we initially provide a condensed overview of transformations performed on the phenalene scaffold, followed by the resynthesis of a 6-phenoxy-substituted derivative. We show that the initial determination of this particular structure was wrong and provide two-dimensional nuclear magnetic resonance (NMR) evidence to assign the structure properly. When preparing new derivatives using the same synthetic route, we observed 6- and 7-substituted regioisomers. After confirming their structures by NMR experiments, the ability of these compounds to inhibit BCL-2 was evaluated. The most potent 1-oxo-1H-phenalene-2,3-dicarbonitrile derivatives inhibited BCL-2 in the nanomolar range and showed double-digit micromolar cytotoxicity against four different cancer cell lines.

A Combined Activity of Thrombin and P-Selectin Is Essential for Platelet Activation by Pancreatic Cancer Cells.

Pancreatic cancer patients have an elevated risk of suffering from venous thrombosis. Among several risk factors that contribute to hypercoagulability of this malignancy, platelets possess a key role in the initiation of clot formation. Although single mechanisms of platelet activation are well-known in principle, combinations thereof and their potential synergy to mediate platelet activation is, in the case of pancreatic cancer, far from being clear. Applying an inhibitor screening approach using light transmission aggregometry, dense granule release, and thrombin formation assays, we provide evidence that a combination of tissue factor-induced thrombin formation by cancer cells and their platelet P-selectin binding is responsible for AsPC-1 and Capan-2 pancreatic cancer cell-mediated platelet activation. While the blockade of one of these pathways leads to a pronounced inhibition of platelet aggregation and dense granule release, the simultaneous blockade of both pathways is inevitable to prevent platelet aggregation completely and minimize ATP release. In contrast, MIA PaCa-2 pancreatic cancer cells express reduced levels of tissue factor and P-selectin ligands and thus turn out to be poor platelet activators. Consequently, a simultaneous blockade of thrombin and P-selectin binding seems to be a powerful approach, as mediated by heparin to crucially reduce the hypercoagulable state of pancreatic cancer patients.

Detection of Tumor Cell-Induced Platelet Aggregation and Granule Secretion.

Hematogenous metastatic spread of cancer is strongly dependent on and triggered by an intensive interplay of tumor cells with platelets. Immediately after entering the blood vascular system, tumor cells are surrounded by a platelet cloak, which protects them physically from shear stress and from attacks by the immune surveillance. Furthermore, tumor cell binding activates platelets, which in turn release growth factors and chemokines to recruit myeloid cells into the platelet/tumor cell microemboli, eventually create a permissive microenvironment in the early metastatic niche. Although the molecular mechanisms of tumor cells to activate platelets appear versatile being a matter of further research, interference with platelet activation turns out to be an attractive target to efficiently inhibit tumor metastasis. Some experimental assays are generally recognized to follow tumor cell-induced platelet activation (TCIPA), which provide an insight into the molecular mechanisms of TCIPA and allow searching for potential inhibitors. In this chapter, we describe the two most prominent experimental assays to follow TCIPA, namely platelet aggregation and platelet granule secretion, experimentally realized by dense granules´ ATP quantification. Although light transmission aggregometry and ATP detection from dense granule secretion are two age-old techniques, they are still highly relevant to provide reliable information concerning platelet activation status since all tumor cell-derived molecular triggers are covered and monitored in the experimental outcome.