Platelets disrupt vasculogenic mimicry by cancer cells

Tumour vasculature supports the growth and progression of solid cancers with both angiogenesis (endothelial cell proliferation) and vasculogenic mimicry (VM, the formation of vascular structures by cancer cells themselves) predictors of poor patient outcomes. Increased circulating platelet counts also predict poor outcome for cancer patients but the influence of platelets on tumour vasculature is incompletely understood. Herein, we show with in vitro assays that platelets did not influence angiogenesis but did actively inhibit VM formation by cancer cell lines. Both platelet sized beads and the releasates from platelets were partially effective at inhibiting VM formation suggesting that direct contact maximises the effect. Platelets also promoted cancer cell invasion in vitro. B16F10 melanomas in Bcl-xPlt20/Plt20 thrombocytopenic mice showed a higher content of VM than their wildtype counterparts while angiogenesis did not differ. In a xenograft mouse model of breast cancer with low-dose aspirin to inactivate the platelets, the burden of MDA-MB-231-LM2 breast cancer cells was reduced and the gene expression profile of the cancer cells was altered; but no effect on tumour vasculature was observed. Taken together, this study provides new insights into the action of platelets on VM formation and their involvement in cancer progression.

Notably, in tumours, platelet activation largely occurs at sites of vascular hyperpermeability where plasma leakage permits contact between collagen and the cancer cells 18 .
Aspirin, a widely used anti-platelet and non-steroidal anti-inflammatory drug (NSAID) 19 , has emerged as a promising drug for cancer prevention 20,21 . This drug has long been characterized as an irreversible cyclooxygenase (COX) inhibitor that reduces the synthesis of prostanoids, such as prostaglandin E2 and thromboxane A2, from arachidonic acid 22 . There is also building evidence that aspirin has COX-independent mechanisms of action in cancer cells causing changes in NFκB, RUNX1 and apoptosis [23][24][25] . These broad acting reactions are attributed to a dosing effect with 75 mg being anti-platelet, 325-600 mg being analgesic and 1.2 g being anti-inflammatory 26 . Etulain and colleagues reported that following thrombin activation of platelets, the ensuing platelet releasate promotes angiogenesis in vitro, and that this process can be inhibited by aspirin independently of VEGF 27 . In contrast, an earlier report from Abdelrahim and coworkers showed that other COX inhibitors (not aspirin) directly suppress VEGF expression by cancer cells to reduce angiogenesis 28 . Clearly, understanding the molecular mechanisms by which platelets and aspirin mediate tumour vascularisation is key to fighting cancer.
There is increasing evidence that tumour vasculature is comprised of not only of EC-lined vessels, but also non-EC lined vascular-like structures formed by cancer cells via a process called vasculogenic mimicry (VM) 29,30 . This process was initially identified in uveal melanoma 30 with a recent meta-analysis of 5 year survival of >3600 patients across 11 different cancer types confirming VM in most solid tumours and correlating with poor prognosis 31 . Laser capture and gene expression profiling of VM-competent melanoma has identified genes associated with angiogenesis, stem cells, the extracellular matrix and hypoxia-related signalling pathways 32 , but a single VM defining biomarker is yet to be identified. VM networks are often contiguous with the EC-lined vasculature and both provide passage for erythrocytes and leukocytes as confirmed via Doppler imaging, intravital microscopy and magnetic resonance imaging [33][34][35][36] . The effect of platelets on VM in cancer remains to be determined.
In this study we use melanoma and breast cancer cell lines in in vitro assays to investigate the role of platelets in VM formation. We examine whether established VM can be influenced by the addition of platelets and whether platelet releasates are equally effective in modulating VM. We investigate VM formation in vivo by melanoma cells in mice with persistent thrombocytopenia. We also use the MDA-MB-231-LM2 cells in a xenograft model of breast cancer to monitor tumour growth, metastasis and the VM gene profile in mice treated without or with the platelet-inactivating aspirin.

Results involvement of platelets in angiogenesis and vasculogenic mimicry by cancer cells in vitro.
To appropriately investigate the effect of platelets on EC angiogenesis, we first confirmed that platelets isolated from the blood of healthy volunteers were 'unstimulated' as defined by a low-level surface expression of P-selectin ( Supplementary Fig. 1). To avoid any influence by growth factors present within our angiogenesis assay, we utilised a growth factor-reduced formulation of Geltrex TM (a Matrigel equivalent of secreted extracellular matrix proteins purified from murine Engelbreth-Holm-Swarm tumour cells 37 ). As shown in Fig. 1A, unstimulated platelets co-cultured with human umbilical vein ECs (HUVEC) for 8 hours did not alter the angiogenic process (i.e. EC branches) formed by HUVEC. In fact, even when 40 platelets were co-cultured with 1 HUVEC, no change in angiogenic performance was observed (Fig. 1A). This observation was somewhat surprising given the initial observation by Pipili-Synetos and colleagues in 1998, that platelets promoted the formation of capillary-like structures by HUVEC on Matrigel in vitro 38 . To address this disparity, we performed additional experiments with lower seeding densities of HUVEC (i.e. 2.5 × 10 3 or 5 × 10 3 cells/well) but again observed no difference in angiogenesis when platelets were added (data not shown). Because the Pipili-Synetos study presented data of HUVEC angiogenesis with and without platelets as 'relative tube area' , rather than the number of EC branches presented here, we reanalysed our data via ImageJ using an threshold mask algorithm to determine well area covered by vascular tubes (i.e. relative tube area). As shown in Supplementary Fig. 2, even at a ratio of 1 HUVEC to 40 platelets, the relative tube area did not differ from HUVEC alone.
With an increasing interest in vasculogenic mimicry (VM) by cancer cells, we next examined whether platelets influenced VM formation by cancer cells in vitro. To this end, we undertook similar experiments to those described above using two human melanoma and two human breast cancer VM-competent cell lines (Fig. 1B, left hand images). In contrast to what we observed with the HUVEC, when C32 melanoma cancer cells were co-cultured with platelets a robust and reproducible inhibition of VM formation was observed. Video footage in the Supplementary Data captures this disruption of VM formation with increasing numbers of platelets. When 40 platelets were added to 1 cancer cell a ~90% reduction in VM formation was observed (Fig. 1B). To address whether platelets can also disrupt established VM networks, platelets were added to the VM assay 4 hours post C32 cancer cell seeding. Supplementary Fig. 3 shows that the addition of platelets to largely established VM structures caused a dissociation of the network suggesting that platelets can, at any time, interfere with the VM process. Interestingly, CHL-1 melanoma cells exhibited a greater resistance to the platelets and for the most part maintained VM competence. For both breast cancer cell lines (HS-578T and MDA-MB-231) platelets significantly inhibited VM formation (Fig. 1B, inhibition of ~56% and ~64%, respectively). Taken together, these results suggest that when cancer cells are exposed to increasing numbers of platelets, their ability to form VM structures, at least in vitro, is perturbed. To determine whether these changes in VM may be attributable to platelets directly affecting cancer cell survival, an alamarBlue viability assay was performed. Repeated experiments confirmed that MDA-MB-231 cancer cells co-cultured with platelets for up to 24 hours (ratio of 1:40) retained a viability equivalent to that of cancer cells alone.

Mechanism of platelet action.
To confirm that the effect of platelets on cancer cell VM was not mediated simply through spatial interference, we performed similar experiments in which inert platelet-sized (i.e. 2 µm) polystyrene microspheres were added to the VM assay. Figure 2A shows that when platelet-sized beads were  cells or HS-578T breast cancer cells at an approximate ratio of 1:40 (cancer cell:releasate volume) and the VM formation monitored as above. As shown in Fig. 2B, the releasate co-cultured with C32 melanoma cells and HS-578T breast cancers cells caused a 38% and 46% reduction in VM formation respectively. These results suggest that for platelet inhibition of VM to be most effective the platelets are ideally in close proximity, or in direct contact, with the cancer cells and that they release soluble factors.

Platelets and cancer cell invasion.
Precisely what factors are released by platelets to perturb VM formation is yet to be determined, but we hypothesized that this disruption of VM network might serve as a means to permit the migration of cancer cells, i.e. metastasis. To address this, we performed an inverse invasion assay wherein C32 melanoma cells without or with platelets co-cultured at a ratio of 1:40 as above, penetrated through an extracellular matrix (Matrigel) towards 10% FCS as a chemoattractant. Figure 3 shows that the cancer cells seeded together with the platelets exhibited a greater invasive potential as demonstrated by them being able to migrate further through the matrix and towards the FCS gradient. Quantitation of cell number relative to distance travelled confirmed that the platelets significantly enhanced the number of cancer cells invading into the matrix as well as their distance travelled (Fig. 3, histogram).

VM formation in melanoma of thrombocytopenic mice.
To investigate the contribution of platelets to VM formation in vivo, we used the syngeneic mouse model of B16F10 melanoma in the thrombocytopenic Bcl-x Plt20/Plt20 mice wherein platelet counts are reduced to ~25% 41 . First, we confirmed the ability of B16F10 melanoma cells to form VM using the in vitro angiogenesis assay (Fig. 4A). Next, we injected B16F10 cells into the flank of wildtype and Bcl-x Plt20/Plt20 mice. Figure 4B shows that the Bcl-x Plt20/Plt20 mice had reduced circulating platelet and white blood cell (WBC) counts both prior to, and at the conclusion of, the experiment. Figure 4C shows that neither tumour size (volume and weight) differed between the two groups.
To assess tumour vasculature, immunohistochemistry was performed on the harvested tumours. Histologically, EC-lined blood vessels (angiogenesis) are identified by their expression of CD31 while VM structures are CD31-negative but can be visualised using periodic acid-Schiff (PAS) reagent, which stains basement membranes rich in collagen and laminin 30 . Figure 4D shows that the tumours in the Bcl-x Plt20/Plt20 mice contained significantly more VM structures than their wildtype counterparts. No difference in CD31+ EC-lined tumour angiogenesis was observed between the two groups (Fig. 4D). No metastasis was detected in the lungs or livers of the mice (data not shown) and is consistent with this relatively short and subcutaneous B16F10 model 42,43 . Bcl-x Plt20/Plt20 mice prior to, and experimental end (open bars, pre-bleed at day -14, grey bars, end-bleed at day 15). In (C), caliper measurements of B16F10 tumour growth over time and final B16F10 tumour weights at experimental end (open symbols, WT mice; grey symbols, Bcl-x Plt20/Plt20 mice). In (D), representative image of CD31 and PAS stained B16F10 harvested tumour. CD31 + /PAS + EC-lined angiogenic structure (Ang, red arrow head) and CD31 − /PAS + VM structure (VM, green arrow head and pink dotted line). Scale bar is 50 µm. Corresponding quantification of the average angiogenic and VM structures per mm 2 (open bars, WT mice; grey bars, Bcl-x Plt20/Plt20 mice). Data show mean ± SEM for n = 5-7 mice. *p < 0.05, unpaired t-test. (2020) 10:5869 | https://doi.org/10.1038/s41598-020-62648-x www.nature.com/scientificreports www.nature.com/scientificreports/ luciferase tag 44 ). Our experimental plan was to inject the cancer cells as a bolus in growth factor-reduced Matrigel into mice with a subgroup of those mice given a daily gavage of low-dose aspirin (25 mg/kg) to inactivate the platelets. Prior to the in vivo experiments, in vitro experiments confirmed that platelets inhibit VM formation as equally in Matrigel as we had observed in Geltrex (Fig. 5A). We also confirmed that VM by MDA-MB-231 cells was inhibitable by the releasate of α-thrombin activated platelets (Fig. 5B) and investigated whether exposure of MDA-MB-231 cells to aspirin alone would influence VM formation, it did not (Fig. 5C). Similarly, exposure of platelets to aspirin did not alter their inhibition of VM (Fig. 5C). The viability of these breast cancer cells was also not affected but exposure to aspirin or releasate over 24 hours (Fig. 5D).
With these results in hand, MDA-MB-231-LM2 cancer cells were injected into the mammary fat-pad (MFP) and tumour growth was monitored on day one after tumour cell injection, and weekly thereafter (days 8, 15, 22, and 29 post implantation) through bioluminescence imaging (Fig. 6A). As illustrated in Fig. 6B, quantification of the bioluminescence signal revealed a statistical difference in tumour burden between the control and aspirin-treated mice at day 29 post implantation. Heat maps of the excised tumours revealed lower bioluminescence readings from the aspirin-treated mice at experimental end point, day 29 (Fig. 6C). Interestingly, caliper measurements taken throughout the experiment, as well as final tumour weights, showed no significant differences in tumour volume (Fig. 6D,E). With Coupland and colleagues having shown that platelets assist breast cancer metastasis to the lung but not the liver 45 , these two organs were harvested from the aforementioned mice to examine their cancer cell content via luciferin-induced bioluminescence. Figure 7 shows that at experimental end, no significant difference could be detected in metastatic load in either the lungs or liver across the two groups of mice.
Apoptotic potential of aspirin in breast cancer in vivo. To determine why the aspirin-treated mice harboured tumours of the same overall size (i.e. tumour volume, Fig. 6D) but reduced bioluminescence (i.e. www.nature.com/scientificreports www.nature.com/scientificreports/ cancer cell content, Fig. 6C) a histological assessment of the tumours was performed via TUNEL assays to detect apoptotic cells. As shown in Fig. 8A, all tumours harvested from the mice contained necrotic regions with a significant reduction in apoptosis detected in the aspirin-treated mice, an observation that correlates with reduced cancer cell burden by these mice.

Ly6G+ cells in tnBc in vivo.
In an attempt to further explain why the overall tumour size and weight did not differ between the two groups of mice, we examined whether other cells, such as scavenging neutrophils, may be more or less abundant. As shown in Fig. 8B, Ly6G+ neutrophils could be identified in tumours from both control and aspirin-treated mice with no discernible differences.

Tumour vascularity; angiogenesis and VM.
To assess the impact of aspirin treatment on angiogenesis and VM in breast cancer, immunohistochemistry was performed on the harvested tumours. Quantitation of angiogenic and VM structures revealed no significant difference between the tumours from control and aspirin-treated mice (Fig. 9). Notably, in the tumours of control and aspirin-treated mice, ~64% of the vascular structures were EC-lined (CD31+/PAS+) while the remaining ~36% were VM structures (CD31−/PAS+). These results suggest that at this single time point (day 29 of MDA-MB-231-LM2 breast cancer growth in NOD/SCID mice) there is no discernible difference in tumour vascularity as a result of low-dose aspirin treatment.
Gene expression profile of VM by TNBC cells in vivo following aspirin-treatment. Finally, we assessed whether aspirin treatment of the mice affected the gene expression by the human breast cancer cells. Of particular interest was a select group of genes documented to be upregulated with VM occurrence 29,30 ; namely genes associated with vascular structures (CDH5 (vascular endothelial (VE)-cadherin), EPHA2, KDR (vascular endothelial growth factor receptor 2,VEGFR2), genes associated with the extracellular matrix (laminin, LAMC2) and key matrix-metalloproteases (MMPs) MMP1, MMP2, MMP9, and MMP14. To identify only the changes caused by aspirin on the human cancer cells, we constructed primer sequences to detect only human genes (Supplementary Table 1). As shown in Fig. 10, CDH5 (VE-cadherin) was the most abundantly expressed gene of those tested and did not differ between the tumours harvested from control or aspirin-treated mice. Similarly, gene expression of EPHA2, KDR, LAMC2, MMP1, and MMP14 did not vary between tumours of mice treated without and with aspirin. In contrast, expression levels of both MMP2 and MMP9 were significantly higher in tumours excised from the aspirin-treated mice (Fig. 10).

Discussion
This study provides several novel insights into the role of platelets in cancer. First, we reveal with in vitro angiogenesis assays that platelets do not perturb capillary-like tube formation by HUVEC, but they do inhibit VM by human cancer cells. This inhibition of VM was observed across three out of four cancer cell lines (C32 melanoma as well as HS-578T and MDA-MB-231 breast cancer cells) with one melanoma cell line (CHL-1) exhibiting only a trend for VM inhibition by the platelets. We observed a consensus for 20-40 resting platelets per cancer cell to effectively inhibit VM formation by 56-88% and that platelet sized beads and the releasates from activated platelets could also inhibit VM formation. Our observation that whole platelets were more effective at blocking VM formation than either beads or releasate alone, suggests that direct contact between the platelet and the cancer cell maximises the opportunity for one or more anti-VM factors to be released from the platelets. Our in vitro inverse invasion assay suggests that platelets also promote cancer cell migration which supports the recent publication by Lucotti and colleagues of platelets orchestrating an intravascular metastatic niche to promote tumour cell seeding 43 . Interestingly, the pre-treatment of platelets with aspirin did not prevent them from inhibiting VM in vitro. One explanation for this may be that platelets inherit their functions from their megakaryocyte precursors 41,43 and that exposure of pre-formed platelets to aspirin cannot influence their VM-inhibiting functions. This concept is supported by documentation that aspirin can modulate many megakaryocyte genes (e.g. integrins and www.nature.com/scientificreports www.nature.com/scientificreports/ transcription factors) resulting in platelets containing an 'aspirin response gene signature' which influences their function in vitro and in vivo 46-48 . Our in vitro angiogenesis results concur with those of Kuznetsov and colleagues who elegantly showed that resting/unactivated platelets are not pro-angiogenic for HUVEC in vitro 49 . In contrast, Pipili-Synetos and coworkers claimed that resting platelets promoted angiogenesis by HUVEC in vitro 38 . Notably, their study did not provide evidence that their platelets were not activated (e.g. low in P-selectin expression). The importance of platelet activation for angiogenesis is supported by Etulain and colleagues who published that releasates from thrombin-activated platelets are proangiogenic for human microvascular ECs in vitro 27 ; notably, unactivated platelets were not examined. Here we reveal an effect of platelets on a second form of tumour vasculature, vasculogenic mimicry, with platelets actively disassembling VM structures formed by cancer cells. This interference was observed (to varying degrees) across two human breast cancer lines and two human melanoma cell lines. Our observation that VM disassembly by platelets is most effective when platelets come into direct contact with the cancer cells supports the discovery by Pang and colleagues of thrombin-activated platelet membranes needing to bind to the MDA-MB-231 cancer cells via P-selectin and GPIIb/IIIa to promote cancer cell migration and metastasis in vivo 50 . Whether platelets become activated as they inhibit VM formation is still to be determined. The divergent nature of platelet function in vitro has been published by Battinelli and colleagues who showed that human platelets activated with adenosine diphosphate stimulated the release of pro-angiogenic VEGF which promoted HUVEC angiogenesis in vitro, whereas platelets activated with thromboxane A2 released the anti-angiogenic endostatin resulting in inhibition of angiogenesis by HUVEC 51 . Whether these pathways, or www.nature.com/scientificreports www.nature.com/scientificreports/ others, are involved in the regulation of VM by cancer cells is yet to be determined. With increasing interest in targeting VM to combat cancer progression 31,52 , the current study also reveals that co-culture of platelets with cancer cells did not influence cell viability but did promote cancer cell migration. This observation is also supported by Pang and coworkers who published that when MDA-MB-231 cells come into direct contact with the membranes of thrombin-activated platelets, the cells adopt a Matrigel-degrading phenotype via P-selectin and GPIIb/IIIa 50 . In addition, Labelle and colleagues published that platelet-derived TGFβ and direct platelet-tumour cell contacts synergistically activate the TGFβ/Smad and NFκB pathways in cancer cells, resulting in their transition to an invasive mesenchymal-like phenotype and enhanced metastasis in vivo 53 . Based on these observations, we speculate that platelets may also actively dissolve VM structures to promote metastasis. Precisely what soluble factor/s are being released by the platelets, or whether platelet-derived extracellular vesicles perturb VM formation and promote cancer cell migration is yet to be determined. Our discovery that platelets have no effect on angiogenesis but actively disassemble VM structures in vitro and in vivo may provide important information on the differences between these two processes of tumour vascularisation for therapeutic purposes 52 .
While mouse models have clearly demonstrated that platelet depletion attenuates cancer metastasis 45,54 the use of aspirin to inactivate platelets has also yielded promising results 49,55 . For example, administration of medium-dose aspirin (75 mg/kg, equivalent to 360 mg human analgesic) in a MDA-MB-231 xenograft model showed that aspirin prevented tumour growth and induced caspase-3-mediated apoptosis of the cancer cells 55 . Similarly, low-dose aspirin (10-25 mg/kg, equivalent to ≤150 mg human anti-platelet/analgesic) in animal models of neuroblastoma and colorectal cancer reduced tumour growth, metastasis and chemoprotection [56][57][58][59] . The current study has investigated the effects of oral low-dose aspirin (25 mg/kg) on the MDA-MB-231-LM2 xenografted mice and revealed reduced cancer cell burden with decreased necrosis, but no effect on overall tumour size, neutrophil content or tumour vascularisation. Coupland and colleagues reported that in the lungs, platelets play a significant role in anchoring the tumour to the endothelium against the relatively high inherent blood flow velocity. In the liver however, where the blood flow is comparatively slow due to hepatic vessel anatomy, www.nature.com/scientificreports www.nature.com/scientificreports/ interactions between endothelial cell and tumour cell adhesion proteins are sufficient to arrest tumour cells enabling their subsequent extravasation, with platelet assistance not being required 45 . While we observed no significant difference in overall metastases between our groups of mice, a slight reduction in lung metastasis was detected and thus supports the aforementioned Coupland et al. study. A more recent study by this same group revealed the importance of cell-cell contact between thrombin-activated platelets and MDA-MB-231 cells to promote lung metastasis in vivo 50 . The effect of aspirin on cancer progression in humans is also unclear with Marshall  and co-workers documenting that long-term daily aspirin use was associated with an increased risk of estrogen receptor/progesterone receptor (ER/PR)-negative breast cancer but decreased risk of ER/PR-positive breast cancer 60 . More recently, Shaio and colleagues showed that aspirin improved the 5-year disease-free survival rate for patients with Stage II-III TNBC 61 . In conflicting observations by others, low-dose aspirin (100 mg every other day) showed no preventative effect on breast cancer 61,62 . Clearly, a better understanding of the effect of aspirin across the breast cancer subgroups is warranted.
Immunohistochemical staining to identify EC-lined tumour vasculature (CD31+/PAS+) and VM (CD31−/ PAS+) vascular structures showed no differences in tumour vasculature in the breast cancers harvested at experiment completion from mice without or with aspirin. However, the administration of aspirin to tumour bearing mice did influence the known VM gene signature 52 by the cancer cells. Upon examination of VE-cadherin, ephrin A2, VEGFR2, laminin 5γ2, MMP1, MMP2, MMP9 and MMP14 by the human cancer cells, we made the surprise finding that of the genes investigated, VE-cadherin (CDH5) was the most highly expressed. Normally restricted in expression by endothelial cells 63,64 , CDH5 is gaining recognition as a prominent factor involved in VM formation by aggressive cancer cells 52 and its downregulation in melanoma results in a loss of VM formation 65 . We observed no differences in expression of CDH5 in the tumours harvested from mice treated without or with aspirin, however post-translation modifications of VE-cadherin also trigger changes in vascular integrity and leukocyte trafficking 66 . Congruent with this, Delgado-Bellido and colleagues published that phosphorylation of VE-cadherin residue Y658 by focal adhesion kinase (FAK) promoted VM formation by melanoma cells via kaiso-dependent gene expression 67 . Two additional genes known primarily for their contribution to angiogenesis, VEGFR2 (KDR) and Ephrin A2 (EPHA2), were lowly expressed by the human cancer cells and did not change in response to aspirin treatment. Of the four MMPs investigated, MMP1 and MMP14 were the lowest in expression and did not change in response to aspirin treatment. In contrast, MMP2 and MMP9 were highly expressed and these levels were further elevated in the mice treated with aspirin. The proteinases MMP2 and MMP9 are associated with cancer progression with known roles in degradation and remodelling of the surrounding extracellular matrix (ECM) to facilitate tumour angiogenesis, invasion and metastasis 68,69 . These MMPs exhibit elevated expression at the leading edge of invasive tumours 70 . Our observation of aspirin-treated mice harbouring cancer cells with increased MMP2 and MMP9 gene expression is intriguing and whether this has resulted from aspirin acting on the platelets or the cancer cells themselves is still to be determined. The latter is possible with aspirin known to bind to IκB kinase (IKK) β and prevent NFκB activation both in vitro and in vivo 71 to alter the transcriptome of a diverse array of proteins that stimulate proliferation, migration and survival in cancer 24 . However, with Shi and colleagues showing that prostate cancer cells treated with aspirin significantly reduce MMP9 activity without any effect on MMP2 activity 72 , and an aspirin analogue ATL-1 capable of inactivating FAK in ECs 73 , the effect of aspirin on cancer cells is complex and yet to be fully elucidated.
In summary, this study suggests that co-culture of HUVEC with unstimulated platelets does not influence angiogenesis in vitro. In contrast, unstimulated platelets robustly and reproducibly inhibit VM formation by multiple cancer cell lines in vitro. This anti-VM effect by platelets was observed not only during the initial formation of VM structures but also as an active disassembly of existing VM structures. We observed that this was achieved by whole platelets and to a lesser extent by thrombin-induced platelet releasates. The inability of aspirin to block platelet inhibition of VM suggests a pathway/process independent of COX and likely via gene regulation of the megakaryocytes during platelet biogenesis. A role for platelets in VM formation was supported in vivo with the B16F10 tumours in the thrombocytopenic Bcl-x Plt20/Plt20 mice exhibiting increased VM content when compared to wildtype controls. Interestingly, our attempt to inactivate platelets and perturb breast cancer growth in vivo via administration of low-dose aspirin resulted in a significant reduction in tumour burden and corresponded with a decrease in necrotic regions within the tumours without discernible difference in tumour vascularization (angiogenesis or VM) or metastasis at experimental end. Taken together, the in vitro results suggest that targeting platelets may prevent the disassembly of VM structures, prevent cancer cell migration and perturb breast cancer burden in vivo. Precisely how this inhibition of platelet function in cancer is brought to fruition is yet to be determined, but unlikely to be made possible from the administration of aspirin alone. Statistical analysis. Data were expressed as mean ± standard error of the mean (SEM). Statistical analyses and significance were calculated by Student's t test, Mann Whitney U test or ANOVA to determine statistical significance using GraphPad PRISM software (San Diego, CA, USA). In all comparisons, p < 0.05 was considered statistically significant.

Methods
Platelet purification and validation. Blood from healthy donors (aged 18-65 years, non-smokers and aspirin/NSAIDs free for at least 1 month) was collected into acid-citrate dextrose (ACD, pH 4.5) anticoagulant and platelets purified as described elsewhere 74 . Briefly, blood was collected from single donors, rested at RT for 15 min prior to 180 g centrifugation, the platelet-rich-plasma was then centrifuged at 1,100 g for 15  Cell viability assay. MDA-MB-231 cells were seeded at 1.5 × 10 4 cells/well in 96-well plate 24 hours prior to treatment without or with 100 μM Aspirin (Sigma, diluted in 100% ethanol (Sigma)) for up to 24 hours. Similarly, MDA-MB-231 cells were treated with platelets ± Aspirin (100 μM, 10 min prior to adding platelets to wells) at a 1:40 ratio of cancer cells to platelets. Cells were incubated at 37 °C, 5% CO 2 for 24 hours. All treatments were performed in triplicate. AlamarBlue Cell Viability Reagent (Thermo Fisher) was added at 100 μl/well and incubated at 37 °C for 60 min prior to being read at 590 nm (FLUOstar Omega microplate reader (BMG Labtech, Mornington, Vic., Australia)).
Angiogenesis and VM assays. HUVECs were isolated from human umbilical cords by collagenase digestion as previously described elsewhere 75  In vitro angiogenesis assays of HUVEC or cancer cells was performed using Geltrex (Thermo Fisher Scientific, Waltham, MA, USA) or Matrigel (BD Biosciences) in the Angiogenesis µ-slides (Ibidi, Munich, Germany). Briefly, cells were seeded on a layer of Geltrex at a density of 1 × 10 4 HUVEC; 1.3-2 × 10 4 MDA-MB-231, HS-578T, C32, CHL-1 or B16F10 cells without or with platelet concentrations adjusted with Tyrode's wash buffer to result in a platelet:cell ratio of 1:2, 1:5, 1:10, 1:20, and 1:40 (cells: platelets). Images covering the entire well were captured after 4-6 hours using an inverted imaging microscope (EVOS XL, Life Technologies), merged using Adobe Photoshop and tube-like structures manually counted using Image J software Cell Counter plugin (1.48pv, NIH, Bethesda, MD, USA). Percent relative tube area was also calculated for the HUVEC angiogenesis assays using a threshold mask on the capillary-like tubes across an entire well and measuring the percent area covered. In similar experiments, platelet-sized beads (Polysciences Inc., Warrington, PA, USA) or releasates collected from α-thrombin activated platelets were added to equal either platelet number or representative volume. In other experiments, platelets were pretreated ± 100 μM of Aspirin (or equivalent ethanol as vehicle control) 10 min prior to being combined with MDA-MB-231 at 1:40 ratio then seeded into the aforementioned VM assay. Representative videos of C32 melanoma cells treated without and with platelets at increasing ratios to cancer cells (i.e. cancer cell:platelet of 1:2, 1:5 and 1:20) over 8 hours via disk confocal live microscopy (CV100, Olympus, Tokyo, Japan). For established VM experiments, 2 × 10 4 C32 cells were seeded, VM formation imaged at 4 hours prior to control buffer or platelets added at 1:40 ratio of cells:platelets and imaged again after 2 hours. Vascular-like structures were defined as multi-cellular elongated arrangements tightly aligned and extending between collections of cells as previously described 76 . Inverse invasion assay. Adapted from Hennigan et al. 77 , 100 µl of growth factor-reduced Matrigel diluted 1:1 in cold PBS was added into an 8.0 µm pore sized Transwell (Corning Inc., NY, USA) and allowed to set. Transwells were then inverted and 4 × 10 4 MDA-MB-231 cells were seeded onto the underside of the membrane. Four hours later, the unbound cells were rinsed and Transwells immersed right-way up in serum-free HUVEC media without or with 16 × 10 6 /mL platelets and 10% fetal calf serum (FCS) was added to the upper chamber as chemoattractant, and cells allowed to migrate upward into the Matrigel for 48 hours. Transwells were paraformaldehyde fixed, RNAse-treated (100 µg/ml, Thermo Fisher) and stained with 0.05 mg/ml of propidium iodide (Thermo Fisher); all steps were carried out for 30 minutes with two PBS washes between each. Transwells were imaged at fixed intervals (10 μm) starting at the membrane and in a direction towards the chemoattractant using z-stack setting of Zeiss LSM 700 confocal microscope with a 20x objective (Carl Zeiss AG, Oberkochen, Germany). Cells from 3 fields of view per slice were quantified using ImageJ software, through threshold adjustment and counting particles (cells), this was then averaged.

B16F10 mouse model of melanoma.
For syngeneic studies, 7-8 week-old female or male, wildtype or Bcl-x Plt20/Plt20 mice 41 on C57BL/6 background were used 78 . 1 × 10 6 B16F10 cells in 50% growth factor-reduced Matrigel were injected subcutaneously into the flank. Caliper measurements of the tumours were taken every 2-3 days and after the animals were euthanized, their primary tumour, lungs and liver were harvested for histology. Blood was collected pre-and post-experiment from the retro-orbital sinus into Microtainer tubes containing EDTA, and circulating platelet and white blood cells (WBC) counts were performed using an ADVIA 2120 (2020) 10:5869 | https://doi.org/10.1038/s41598-020-62648-x www.nature.com/scientificreports www.nature.com/scientificreports/ haematological analyser (Seimens, Munich Germany). Mice were rested for 2 weeks following bleeding prior to tumour cell injection.
Preparation and administration of aspirin to mice. The mouse dose equivalent to 100-150 mg/60kg human low dose aspirin was calculated as Human equivalency dose (HED) = animal dose (mg/kg) × (Animal Km)/ (Human Km), where mouse Km factor is 3, and human Km factor is 37 79 . Aspirin (Sigma Aldrich) was prepared at 2.5 mg/mL to administer 10 µl/g body weight to deliver a dose of 25 mg/kg daily via oral gavage, with control mice receiving the same volume of corn oil (Sigma Aldrich).
Orthotopic mouse model of breast cancer. For orthotopic studies, 5-6 week-old female mice were used and anesthetised before injections of 1 × 10 6 MDA-MB-231-LM2 cells in 50 μl of 50% Matrigel (BD Biosciences) into the fourth mammary fat pad. Four weeks following inoculation, bioluminescence imaging was performed using the Xenogen IVIS-100 imaging system (Perkin Elmer, Waltham, MA, USA). Mice were injected intraperitoneally with 30 mg/ml of D-Luciferin (in PBS, Cayman Chemical, Ann Arbor, MI, USA) 10 min before imaging. Dorsal images of the primary tumour were collected before the animals were humanely killed and their primary tumour, lung and liver harvested for ex vivo imaging. Photon emission was quantified using the Living Image Software (Perkin Elmer).
Histology and immunohistochemistry staining on tumours. Primary tumours were fixed in 10% buffered formalin for 24 hours before processing and embedding in paraffin. Sections (4 µm) were cut and subjected to heat-induce epitope retrieval (microwaved at 900W for 4 min, then 350W for 15 min) in 10mM citrate buffer pH 6.0 (for CD31 and Ly6G staining). Sections were allowed to cool to RT and quenched with 1-3% H 2 O 2 prior to incubation with anti-CD31 antibody (1:250, Bethyl lab, Montgomery, TX, USA), an anti-Ly6G antibody (1:1000, BioLegend, San Diego, CA, USA) or an isotype IgG control overnight at 4 °C. Sections were then incubated for 30 min with avidin-biotinylated-horseradish peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA, USA) and visualized using peroxidase substrate solution (ImmPACT ™ DAB, Vector Laboratories). Ly6G-stained sections were immediately counterstained with Ehlrich's hematoxylin and mounted in DPX, while CD31-stained sections were further stained using a PAS staining kit from Merck Millipore (Burlington, MA, USA) according to manufacturer's instructions before counterstaining and mounting. Stained sections were imaged using an Olympus BX40 microscope fitted with a DP70 digital camera and operated through the Olympus Image Analysis Cell B software program (Olympus). Ly6G-stained slides were scanned by the whole slide image (WSI) scanner (Hamamatsu NanoZoomer Slide scanner) and quantitated via ImageJ software. EC-lined blood vessels (CD31+/PAS+) and VM structures (CD31−/PAS+) were defined by the presence of RBCs or WBCs in the lumen. Structures were manually quantified in 4-6 fields of view (FOV) per tumour using ImageJ counter plugin software, and represented as average structures/FOV. RNA extraction, reverse transcription and quantitative PCR (qPCR). RNA was extracted from cell pellets using the RNeasy Micro Plus kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Reverse transcription was performed on 1μg of purified RNA using a Superscript III enzyme (Life Technologies) following manufacturer's instructions. Primers for PCR and qPCR (Supplementary Table 1) were either sourced from the literature or designed using Primer Blast (NCBI), synthesised (GeneWorks, Hindmarsh, SA, Australia) and validated for species specificity with Primer Blast (NCBI). qPCR was performed using QuantiTectTM SYBR Green master mix (Qiagen) on a Rotor-Gene thermocycler (Corbett Research, NSW, Australia) with reaction parameters: 15 minutes at 95 °C, then cycling of 10 seconds at 95 °C, 20 seconds at 57-60 °C, 30 seconds at 72 °C; for 45 cycles followed by a melt phase. Relative gene expression levels were calculated using the comparative quantitation method available in the Rotor-Gene Software (Corbett Research) and normalised against housekeeper genes GAPDH, ACTB, and CYCA validated using the geNorm algorithm (M value < 1.5).