The prospects of microphysiological systems in modeling platelet pathophysiology in cancer

Abstract The contribution of platelets is well recognized in thrombosis and hemostasis. However, platelets also promote tumor progression and metastasis through their crosstalk with various cells of the tumor microenvironment (TME). For example, several cancer models continue to show that platelet functions are readily altered by cancer cells upon activation leading to the formation of platelet-tumor aggregates, triggering release of soluble factors from platelet granules and altering platelet turnover. Further, activated platelets protect tumor cells from shear forces in circulation and assault of cytotoxic natural killer (NK) cells. Platelet-secreted factors promote proliferation of malignant cells, metastasis, and chemoresistance. Much of our knowledge of platelet biology in cancer has been achieved with animal models, particularly murine. However, this preclinical understanding of the complex pathophysiology is yet to be fully realized and translated to clinical trials in terms of new approaches to treat cancer via controlling the platelet function. In this review, we summarize the current state of knowledge of platelet physiology obtained through existing in vivo and in vitro cancer models, the complex interactions of platelets with cancer cells in TME and the pathways by which platelets may confer chemoresistance. Since the FDA Modernization Act recently passed by the US government has made animal models optional in drug approvals, we critically examine the existing and futuristic value of employing bioengineered microphysiological systems and organ-chips to understand the mechanistic role of platelets in cancer metastasis and exploring novel therapeutic targets for cancer prevention and treatment. Plain Language Summary The recent passage of the FDA Modernization Act by the US government has removed the requirement of the use of animal models in disease modeling and drug discovery process. This has resulted in a much-renewed excitement within engineers, scientists, and industry in applying in vitro cell biosystems as a platform technology that assists or replaces animals in reproducing human biology. The contribution of platelets is well recognized in thrombosis and hemostasis. However, platelets also promote tumor progression and metastasis through their crosstalk with various cells of the tumor microenvironment. In this review, we summarize the current state of knowledge of platelet physiology obtained through existing cancer models and also critically examine the existing and futuristic value of employing bioengineered organ-chips to improve the knowledge of the underlying biology.


Introduction
Platelets are anucleate discoid blood cells known to regulate hemostasis and thrombosis. 1 The association between thrombosis and cancer has long been recognized, with thromboembolic complications often preceding cancer diagnosis. 2Thromboembolism commonly occurs during cancer progression or as a complication of oncologic treatment. 3Thrombocytosis, an elevated platelet count, is frequently observed in 10% of breast cancer cases to as high as 55% of lung and colorectal cancer patients can promote cancer growth and metastasis. 3,4][7][8][9][10] Pretreatment of thrombocytosis is a negative prognostic factor associated with aggressive tumor behavior in cervical, colon, and non-small cell lung cancer. 11,12Conversely, thrombocytopenia, a decreased platelet count, is associated with worse prognosis in patients with bone marrow, spleen, or pancreatic tumors. 13Thus, platelets play a complex role in disease progression and metastasis depending on the cancer type and stage 14 (Figure 1).Platelets contribute to tumor growth, migration, and metastasis by promoting angiogenesis 15 through the transport of proangiogenic factors like vascular endothelial growth factor (VEGF).Recent clinical studies also indicate a strong correlation between platelet count and tumor chemoresistance 16 which is in agreement with in vitro studies (Figure 1).Platelets have been extensively studied for their systemic effects on tumor angiogenesis, progression, and metastasis in cancer patients.However, the direct involvement of platelets in TME remains poorly understood.This is partly because animal studies have some limitations as preclinical model systems in being able to dissect specific signaling pathways and offer simplicity in analyzing individual cells or cell-drug interactions.Recent advancements in organ-onchip microdevices offer a controlled platform to model the human TME and study its interactions with platelets.These bioengineered organ-chip technologies use human cells to create miniaturized models of specific stages of cancer, providing real-time information on tumor progression and blood cell interactions.This review article focuses on the strengths and limitations of organ-chips as a complementary platform to enhance our understanding of platelet biology in cancer.It also explores how platelet function can be modeled with these systems in different stages of tumor progression and highlights the potential of combining anti-cancer and anti-platelet therapies as a strategy to treat cancer.

Vascular stress and angiogenesis
Inflammation is one of the determinants of morbidity and mortality of cancer patients. 17Inflammatory molecules and platelets have reciprocal signaling transduction and mechanisms, ultimately triggering thrombi formation, leukocyte recruitment, endothelial activation, and angiogenesis in the tumor microenvironment. 18Various cell adhesion molecules (CAMs) and integrins like ICAM-1, VCAM-1, LFA-1, and Mac-1 facilitate adhesion of leukocytes to endothelial cells (ECs) through P-selectin and CD40L receptors disrupting vascular integrity and promoting platelet activation and adhesion. 19Activated platelets interact with leukocytes and eventually induce thrombus formation. 20P-selection present on both ECs and platelets contributes to tumor progression 21 and metastasis. 22ancer cells stimulate platelets to promote angiogenesis for preserving integrity of leaky blood vessels. 23Stored proangiogenic factors like VEGF, FGF, EGF, and anti-angiogenic factors namely ANGPT1, S1P, TSP1, and endostatin 24 are released by platelets based on signals from the tumor niche. 25A xenograft breast cancer model showed that platelets sequester cancer cell-secreted cytokines and deliver them to tumor sites, promoting angiogenesis. 26Tumorderived VEGF activates ECs, induces platelet aggregation, and promotes thrombin generation thus facilitating angiogenesis. 27latelet accumulation on activated endothelial surfaces enhances EC survival and migration. 28Cancer patient-derived platelet microparticles (PMPs) contain growth factors, cytokines, and inflammatory molecules aiding angiogenesis. 29,30These PMPs carry microRNAs contributing to neovascularization and enhance plateletdependent endothelial tube formation thus inhibiting Thrombospondin-1 (TSP1) and increasing tumorigenicity.Platelets play dual role in tumor progression.They promote pathological angiogenesis while being involved in tumor inhibition by blood vessel normalization.Platelets recruit and mature endothelial progenitor cells, support pericyte coverage and release factors like angiopoietin-1 and serotonin to promote vessel maturation. 31educed vessel density and impaired vessel maturation were observed in tumors from thrombocytopenic mice. 32,33Initially, platelets are recruited to restore vessel integrity in TME but upon activation by tumor-released factors, initiate pathological angiogenesis.Further research is needed to understand the specific signaling cascades, conditions, and events favoring platelet-induced angiogenesis.

Metastasis
Cancer metastasis involves complex interactions between cancer cells and the host. 34Only small fraction of circulating tumor cells (CTCs) survive the high shear in circulation evading immune system to form metastatic foci. 35Platelets are the first to interact and protect CTCs from natural killer (NK) cells via thrombi formation. 36,37Platelet count is an influential factor in cancer metastasis.The condition of elevated platelet count, thrombocytosis, is associated with enhanced pro-metastatic functions resulting in increased tumor metastasis and poor prognosis in lung, ovarian, and colorectal cancers. 38,39Conversely, induced thrombocytopenia, a low platelet count, or impaired platelet activation in preclinical models have shown reduced metastatic ability of primary tumors. 33,40Activated platelets in association with fibrinogen mask cancer cells from immune surveillance by transferring major histocompatibility class I complex (MHC 1) to tumor cells and preventing cytolysis by cytotoxic NK cells. 41,42Plateletmediated shedding of Natural Killer Group 2D (NKG2D) ligands compromises tumor cell recognition by NK cells, facilitating immune evasion. 43Tumor cells release factors like ADP, thromboxane A2 (TXA2), and high-mobility group box 1 (HMGB1) 44 activate platelets resulting in granule secretion, ATP release, and endothelial junction disruption.Tissue factor (TF) expressed on cancer cell membranes, 45 tumor cell-derived matrix metalloproteinases (MMPs) and TF detected on tumor-derived vesicles 46 can also induce platelet activation.Platelets also play a crucial role in tumor growth and metastasis by releasing transforming growth factor-β (TGF-β), which suppresses immune responses creating an immunosuppressive TME. 47Platelets secreted TGF-β downregulates NKG2D receptor on NK cells, inhibiting anti-tumor activity.Elevated TGF-β levels are associated with reduced NKG2D expression in lung cancer patients. 48Labelle et al. showed that the inhibition of TGFβ1 expression in platelets and megakaryocytes prevents metastasis and lung colonization by tumor cells. 49Forming thrombi, platelets confer anoikis (homelessness, form of programmed cell death) resistance to CTCs by releasing growth factors, cytokines, and allowing integrin αIIbβ3 receptors to interact with cancer cell integrins, enabling survival, proliferation, and colonization of tumor in distant organs.0][51] Labelle and Hynes demonstrated that platelets and granulocytes are sequentially recruited to disseminated tumor cells forming "early metastatic niches."Inhibiting platelet-granulocyte interactions could be a potential therapy for preventing metastasis alongside targeting tumor cells. 52However, limited knowledge of platelet supporting tumor cells at secondary sites and CTC survival mechanisms exists and needs further investigation.

Resistance to therapy
Chemotherapy resistance is a major challenge in cancer treatment resulting in relapse and metastasis.Tumors that initially respond to chemotherapy can develop resistance and continue growth. 53hemoresistance is associated with an increased risk of thromboembolic disorders in patients. 54Studies have shown a strong correlation between platelet count and tumor chemoresistance, 53 where higher platelet counts contribute to resistance against drugs like paclitaxel and 5-fluorouracil 55 and decreased platelet counts enhanced effectiveness of drugs like doxorubicin and paclitaxel. 56,57Glycoprotein VI (GPVI) on platelet surface is responsible for tumor inhibition, and using antibody fragment (JAQ1 F(ab)2) to inhibit GPVI has shown promising result in reducing tumor growth and enhancing accumulation of Doxil and paclitaxel. 56Focal adhesion kinase (FAK) regulates platelet migration into TME, while FAK-deficient platelets prevent tumor relapse upon discontinuation of anti-angiogenic therapy. 58oluble factors released by activated platelets alter the balance between anti-apoptotic and pro-apoptotic genes, counteract cell cycle arrest induced by anti-cancer agents, promote tumor growth, enhance phosphorylation of DNA repair proteins like Chk1, BRCA1, and Mre11, and inhibit the cytotoxic effects of drugs like sorafenib and regorafenib. 59Platelets being the primary source of TGF-β1 in blood induce epithelial-mesenchymal transition, a process associated with tumor progression and metastasis. 49Recent studies have described the unique roles of platelets after withdrawal of anti-angiogenic therapy, followed by antiangiogenic effects of aspirin, and predicted that antiangiogenesis escape by tumors may be arrested by investigating mechanisms and combining other drugs. 58,60,61Therefore, preclinical models will be beneficial in exploring the potential of combining antiplatelet drugs with chemotherapeutic and antiangiogenic agents to arrest cancer metastasis and overcome chemoresistance.Such combinatorial approaches could offer new therapeutic strategies to improve clinical outcomes for cancer patients.

Microphysiological systems as contemporary models of platelet biology in cancer
Murine models are considered as trustworthy preclinical model systems to predict human biology, as they have majorly contributed to our current understanding of platelet biology in cancer and have shaped numerous successful clinical trials.But along with their strengths, there exists some limitations.For example, in many cases, murine physiology does not translate to humans directly.Secondly, they are complex and while providing a system-level response to stressors and drugs, cannot be dissected for studying individual signaling pathways or describe cell-cell interactions with high specificity.To complement these models, microphysiological systems, also termed as organon-chips (OoC), have provided an in vitro cell culture system by closely mimicking human physiology, enabling researchers to gain meaningful insights into disease states and underlying mechanisms.As an example, these devices replicate blood flow and allow integration of multiple cell types, mirroring the cellular diversity found in organs.In cancer research, OoC technology has spurred investigations in cancer metastasis, 62 intravasation, 63 extravasation, 64,65 and even angiogenesis. 668][69] But recently, studies conducted by Saha et.al. using microfluidic OoC technology revealed that compromised vessel integrity in ovarian TME facilitate extravasation of platelets toward cancer. 70This study revealed similar behaviors of platelets as observed during cancer progression.Soluble factors released from tumor chamber activated the platelets stimulating movement toward cancer-influenced blood endothelial cells, and finally extravasating to the tumor chamber.The data also suggest statins to be a potential drug in preventing cancer growth, partially by blocking direct contact with platelets 70 (Figure 2a-e).Another study by the same group reported successful development of ovarian tumor microenvironment chip (OTME-chip) (Figure 2f-h) demonstrating mechanisms through which platelets promote metastasis and highlighting a novel drug target to augment chemotherapeutic response. 71The study revealed that interaction of platelet GPVI with tumor cell expressing galectin-3 under shear in time-dependent manner results in upregulation of numerous growth factors inducing metastasis.The metastatic potential of tumor was diminished resulting in improved chemosensitivity on administration of DOI: https://doi.org/10.1080/09537104.2023.2247489 Modeling platelets in cancer with organ-chips 3 combinatorial composition of chemotherapeutic agent with antiplatelet drug. 71Gene-edited tumors and next generation RNAseq-on-chip performed in this study revealed temporal dynamics of vascular disintegration (Figure 2i,j), making the vascular tissue leaky and promoting platelet extravasation (Figure 2g,h) in ovarian cancer.Study reported by Crippa et.al. designed human early metastatic niche (EMN)-on-a-chip to mimick transient microenvironment forms in circulation during cancer dissemination and understand the crosstalk among cancer, platelets, leukocytes, and ECs.EMN-on-a-chip exhibited increased transendothelial migration of cancer cells in presence of neutrophils and platelets, along with enhanced expression of epithelial to mesenchymal transition (EMT) markers in cancer cells under the influence of platelets.Use of antiplatelet drugs have demonstrated effectiveness in reducing platelet aggregation and lowering EMT markers. 72These emerging examples of organ-chips integrating blood and vascular components within TME demonstrate the positive value of these approaches in cancer research and drug discovery.By studying the interactions between different cell types in an organ-chip, researchers can gain a deeper understanding of diseases and develop more effective treatment strategies.While not strictly organ-chips, researchers have also developed in vitro microfluidic assays that enable real-time observation and quantification of critical steps in metastasis, such as tumor cell extravasation.These assays feature perfusable microvascular networks, allowing the study of tumor cell dynamics, morphological changes, and screening of therapeutic agents. 64Additionally, the isolation of platelets or leukocytes from human blood and their perfusion through the device with tumor cells enables the investigation of tumor-immune cell interactions independently of other cell types. 64This reductionist approach in the in vitro system offers advantages in studying specific interactions that are challenging to perform in vivo.For example, Papa et al. demonstrated the effects of platelet decoys on platelet adhesion on exposing to physiological flow using microfluidic devices with collagen-coated channels.Platelet decoys mixed with normal platelets partially inhibited platelet adhesion.The antiplatelet effect was reversible upon adding fresh platelets, while the clinical antiplatelet agent abciximab showed a sustained effect. 73In cancer, platelets are activated by inflammatory cytokines released from tumor cells, contributing to the thrombi formation in the presence of procoagulant-like tissue factor also released by tumor cells. 34,74This phenomenon, known as tumor-associated thrombosis, is a common complication in cancer patients.Since platelets are the drivers of thrombosis, cancer-associated thrombosis is also a complex physiology where organ-chips can make significant contributions.Organ-on -a-chip systems utilize microfluidic flow chambers to include endothelial lumens and enable the perfusion of whole blood, allowing visualization of platelet-endothelial interactions. 69hese microphysiological models of thrombosis can mimic the composition and behavior of thrombi in different blood vessels by perfusing whole blood at high arterial and low venous wall shear rates. 75They accurately model anatomical structures observed in vivo like plaques, bifurcations and incorporate vascular cells, enhancing the physiological relevance of thrombosis studies in various diseases. 76hile there are no visible organ chip studies modeling thrombosis in cancer, it has been shown that tumor cells can induce platelet activation and aggregation, leading to microthrombus formation and impaired blood flow within micro-vessels. 77Such findings highlight the potential of organ-on-a-chip platforms to simulate tumor-induced platelet activation and thrombosis, providing insights into underlying mechanisms and facilitating the development of targeted therapeutic interventions.The use of organ-chips and other microfluidic technologies together provide an opportunity to bridge the gap between traditional cell culture and in vivo models, offering a useful representation of human physiology and advancing our knowledge of disease mechanisms.They may provide significant scope to easily visualize and spatiotemporally control specific biological processes in detail to gain a better understanding of the disease progression and employing the platform for drug testing application. 71,78Thus, with more advancement, OoC can evolve as a new in vitro dissectible platform to model the mechanisms of cancer-vascular-hematology nexus and analyze as well as predict personalized therapeutics toward disease management (Figure 3).

Limitations of microphysiological systems and future perspective
Platelets play a crucial role in tumor progression and metastasis by interacting with other cells in circulation and stimulating signaling pathways affecting tumor cells and TME, contributing to angiogenesis, immunoevasion, metastasis, and thrombosis.
Targeting platelet-cancer cell interaction is a potential strategy to reduce metastasis and cancer-associated thrombosis, although clinical trials with antiplatelet agents like prasugrel, ticagrelor, or vorapaxar along with aspirin have yielded inconsistent results. 79Further research exploring platelet contribution at different stages of cancer, the balance between pro-and antitumor activities, involved signaling pathways and molecular variants are necessary.Complex microphysiological models continue to evolve aiming to mimic in vivo conditions closely to explore the unanswered role of platelets in cancer (Figure 3).However, these models still have limitations in studying the systemic effects of cancer and platelet evolution as the disease progresses.Despite promising advantages, OoC models are low throughput.Some progress has been made in developing highthroughput systems by parallelizing multiple culture chambers in a single device, allowing studies with numerous replicates. 80igh drug absorption by polydimethylsiloxane (PDMS) in OoC devices is a concern, thus not recapitulating in vivo pharmacokinetics.Use of biologics, non-absorptive coatings, 81 alternative materials like polyurethanes and polycarbonate-thermoplastic elastomer hybrids, 82 and non-absorptive rigid thermoplastics like polystyrene or polycarbonate 83 can mitigate this challenge.Organ-on-chips are yet to meet the regulatory benchmarks set by the IQ Consortium and the FDA to replace animal studies. 84herefore, at the moment, organ-on-chips and animal models can cooperatively leverage their strengths and cross-validate preclinical findings.Despite recent advancements, challenges in microfluidic device designs for modeling thrombosis include channel shape, setup costs, limited 3D capability, and turbulence creation.Advances in 3D printing hold promise for personalized models using patient-derived tissues and stem cells 69,85 aiming to mimic pathophysiology of thrombosis, predict therapeutic responses, improve patient health outcomes, and reduce costs.Finally, organ-on-a-chip platforms focus on local effects within specific tissue or organ.For example, they may lack the inclusion of the bone marrow, the primary site of Modeling platelets in cancer with organ-chips 5 platelet production and maturation.Therefore, the systemic effects of cancer involving interactions with other organs and dynamic changes in platelet biology may not be fully understood with these systems.Nevertheless, organ-on-chips provide a necessary toolbox to study platelet behavior in context of cancer by co-culturing cancer cells and ECs, investigating platelet activation, aggregation, and prothrombotic properties within TME.Integrating multiple models including bone marrow-on-a-chip can furnish a comprehensive understanding of platelet evolution in cancer, bridging the gap between local and systemic effects.Overall, the future prospects of learning platelet physiology in cancer appear to be bright, and microphysiological systems may serve a critical role alongside animal models in deriving new therapeutic and preventive strategies against cancer.

Figure 1 .
Figure 1.Contribution of platelets in cancer.Platelets get activated by soluble factors and chemokines present in the tumor microenvironment and play a vital role in cancer progression.Platelets play crucial role in supporting cancer growth by inducing stable angiogenesis and neovascularization.They also promote epithelial to mesenchymal transition of tumor cells thereby facilitating metastasis.High platelet counts contribute to chemoresistance by countering the inhibitory effect of chemotherapeutics thereby facilitating tumor cell proliferation and disease spread.Created with BioRender.com.

Figure 2 .
Figure 2. Organ-on-chip model of ovarian cancer vessel-platelet interaction, vascular degradation, metastasis and RNA-sequencing data analysis.(a) Schematic showing OvCa-chip components and assembly in detail.(b) Microscopic images showing human ovarian cancer cells, A2780 cultured in the upper microchannel (top) of OvCa-chip (center) and endothelial vessel formed in lower channel (bottom).Bars represent 100 µm.(c) Fluorescence micrographs displaying cross sectional view of the OvCa-chip with A2780 (red, anti-ZO1) cocultured with primary endothelial cells (green, VE-cadherin) to form a blood-perfusing vessel.The tumor-vascular tissue interface distance of cross-sectional 3D view is 10 µm.Bars represent 100 µm.(d) Immunohistochemical images of tumor biopsies obtained from healthy individual and patient with ovarian cancer showing platelets extravasation (CD42b + , brown and marked by arrows) into the tumor stroma in patients with and without statin treatment.Scale bar = 50 µm.Insets: mag = 40×.(e) Quantification of platelets observed with histology.Error bars are means ± SEM; n = 5. (f) Computer aided design of OTME-chip showing two PDMS compartments separated by thin porous membrane reproducing the microarchitecture of tumor-vascular interface.(g) OTME-Chip schematic depicting the influence of extravasated platelets in tumor invasion dynamics.(h) Cross-sectional 3D confocal view of OTME-Chip representing cancer cells (yellow), endothelial cells (red), and platelets (cyan) at 0 hours (left) and 72 hours (right) after platelet extravasation.Scale bars, 100 μm.(i) Volcano plots exhibiting differentially expressed genes in OTME-Chip, Cx-OTME-Chip, CxRx-OTME-Chip, and KO-OTME-Chip respectively relative to control (black dots).Red dots mark the genes regulating EMT and metastasis.(j) KEGG pathway clustering shows differential presence of EMT regulatory pathways for all study groups relative to control-chip (P < .05).Adapted with permission 70 Copyright 2020, ASH publications and copyright 2021, Science publishing.

Figure 3 .
Figure 3. Importance of organ-on-chip (OoC) technologies in platelet cancer research.OoCs are the next generation modeling system facilitating in vitro disease modeling by closely mimicking the in vivo conditions in humans.This tool provides a dissectible platform by introducing flow mechanism and interaction of multiple cell types in real time by forming microchambers.OoC technology can be widely employed to better understand the role of platelets in vascular stress, angiogenesis and metastasis in cancer as well as finding new therapeutic targets and personalized therapeutic strategy for better disease management.Created with BioRender.com.