# Oral Squamous Cell Carcinoma Is Associated with a Low Thrombosis Risk Due to Storage Pool Deficiency in Platelets

Pierre Haen, Lydie Crescence, Diane Mege, Alexandre Altié, Christophe Dubois, Laurence Panicot-Dubois

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## ► To cite this version:

Pierre Haen, Lydie Crescence, Diane Mege, Alexandre Altié, Christophe Dubois, et al., Oral Squamous Cell Carcinoma Is Associated with a Low Thrombosis Risk Due to Storage Pool Deficiency in Platelets. *Biomedicines*, 2021, 9 (3), pp.228. 10.3390/biomedicines9030228. *hal-03222253*

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Article

# Oral squamous cell carcinoma is associated with a low thrombosis risk due to storage pool deficiency in platelets.

Pierre Haen<sup>1,2</sup>, Lydie Crescence<sup>1,3</sup>, Diane Mege<sup>1,4</sup> Alexandre Altié<sup>1</sup>, Christophe Dubois<sup>1,3</sup> and Laurence Panicot-Dubois<sup>1,3,*</sup>

Received: date; Accepted: date; Published: date

**Abstract:** Venous thrombo-embolism (VTE) disease is the second most common cause of mortality in cancer patients, and evaluation and prevention of thrombosis risk is essential. VTE-associated risk varies according to the type of tumor disease. Oral cancer is the most frequent type of head and neck cancer, and it represents approximately 2.1% of all cancers worldwide. Most tumors are squamous cell carcinomas and are mainly due to tobacco and alcohol abuse. VTE risk associated with oral squamous cell carcinoma (OSCC) is low. However, many studies have shown that OSCC has the following biological features of cancers associated with a high thrombosis risk: modified thrombosis and fibrinolysis mechanisms; strong expression of procoagulant proteins; secretion of procoagulant microparticles; and production of procoagulant cytokines. Using an original mouse model of tongue squamous cell carcinoma, our study aimed to clarify this paradoxical situation. First, we showed that OSCC tumors have a pro-aggregatory phenotype and a high local thrombosis risk. Second, we found that tongue tumor mice do not have an elevated systemic thrombosis risk (the risk of an "at distance" thrombosis event such as lower extremity deep venous thrombosis or pulmonary embolism) and even show a reduction in risk. Third, we demonstrated that tongue tumor mice show a reduction in platelet reactivity, which explains the low systemic thrombosis risk. Finally, we found that tongue tumor mice present granule pool deficiency, thereby explaining the reduction in platelet reactivity and systemic thrombosis risk.

**Keywords:**

## 1. Introduction

The association between venous thrombo-embolism and malignancy was first described by Bouillaud and Trousseau[1,2] in the 19th century. Currently, it has been clearly shown that the association exists for a hypercoagulable state associated with malignancy and that thrombosis risk differs according to the cancer features (histological features, localization, and stages) and cancer treatments used (chemotherapy, surgery, and radiotherapy)[3].

Venous thrombo-embolism disease, especially pulmonary embolism, is the second most common cause of mortality in cancer patients. Thus, evaluation and prevention of thrombosis risk is essential[4]. Anticoagulant therapy is indicated in the prevention of venous thromboembolism (VTE), but its use must be limited in high-risk patients because it may also be responsible for complications, such as hemorrhage or immuno-allergic reactions[3].

Oral cancer is the most frequent type of head and neck cancer, and it represents approximately 2.1% of all cancers worldwide[5,6]. Anatomically, oral localization corresponds to the lip, tongue,

<footnote n="1">¹ Aix Marseille Université, INSERM 1263, INRAE, C2VN, Marseille, France.</footnote>

<footnote n="2">² Department of Oral and Maxillofacial Surgery, Laveran military and academic hospital, Marseille, France.</footnote>

<footnote n="3">³ Aix Marseille Université, PIVMI, Marseille, France.</footnote>

<footnote n="4">⁴ Department of digestive Surgery, Timone University Hospital, Marseille France.</footnote>

<footnote>* Correspondence: Laurence.panicot-dubois@univ-amu.fr</footnote>
hard palate, gingivae and internal side of the cheek. Oral tongue cancer is the most frequent sublocalization[7]. Histologically, squamous cell carcinoma accounts for 90% of all malignancies in the oral cavity. Tobacco use and excessive alcohol consumption are the most common risk factors. Papillomavirus infection has also been shown to be a risk factor, especially for tongue cancer, for which epidemiology is thus slightly different (younger patients without alcohol or tobacco abuse)[8]. Thrombosis risk associated with oral squamous cell carcinoma (OSCC) has been empirically shown to be low or nonexistent[9,10]. For some authors, however, this risk is demonstrable[11,12], and many studies have shown that OSCC has the following biological features of cancers associated with a high thrombosis risk: modified thrombosis and fibrinolysis mechanisms[13,14]; strong expression of procoagulant proteins, such as factor tissue (FT)[15,16]; secretion of procoagulant microparticles[17]; and production of procoagulant cytokines[18]. As we have previously suggested in our review[19], a paradoxical situation exists for OSCC tumors, since these tumors present all the features of high thrombosis risk associated cancer but show a low associated systemic thrombosis risk. Our present study aimed to clarify this situation. First, our study highlighted this paradox by confirming the pro-thrombotic profile of OSCC tumors and by exploring the associated systemic venous thrombo-embolism (VTE) risk, which is the risk of a "at distance" thrombosis event such as lower extremity deep venous thrombosis or pulmonary embolism. Thereafter, we identified the potential mechanisms that may explain these facts using an original mouse model of tongue squamous cell carcinoma.

## 2. Materials and Methods

### 2.1. Antibodies and reagent

Alexa 649 rat anti-mouse GPIb antibody (Emfret) was used *in vitro* for immunofluorescence microscopy and flow cytometry experiments, an *in vivo* for intravital microscopy. A homemade Alexa 488-conjugated mouse anti-fibrin antibody (the hybridoma was generously provided by Prof B. Furie) was used *in vitro* for flow cytometry experiments, an *in vivo* for intravital microscopy. Alexa 649 and Alexa 488 irrelevant antibodies respectively from Beckman and homemade were used *in vitro* for immunofluorescence microscopy. PE Conjugated rat anti-mouse CD41/CD61 (activated form, JON/A Emfret) and microbeads (Biocytex) were used *in vitro* for flow cytometry experiments. ADP (Sigma-Aldrich) and TRAP (NeosystemSA) were used for platelet aggregation experiments.

### 2.2. Cell culture

The AT-84 oral cancer cell line is derived from a murine tumor of the oral mucosa that spontaneously arose in a C3H mice, and it was originally isolated by Hier et al.[20] and was kindly provided by Dr Venuti A. and Dr Paolini F.[21] (Regina Elena National Cancer Institute, Rome-Italy). AT-84 cells were grown in RPMI-1640 medium (Life Technologies) supplemented with 10% FCS (PAA), 100 U/mL penicillin (Life Technologies), 100 mg/mL streptomycin (Life Technologies) and 0.1% fungizone (Life Technologies). Cells were grown at 37°C in a humidified atmosphere with 5% CO2.

The Hight TF-Panc02 cell line was derived from a pancreatic ductal adenocarcinoma induced in a C57BL/6 mouse¹. This cell line is a subclone of Panc02 cells transfected with pcDNA 6.2-GW/EmGFP-miR mock described previously by Mezouar et al.[22]. Hight TF-Panc02 were grown in RPMI-1640 medium (Life Technologies) supplemented with 10% FCS (PAA), 100 U/mL penicillin (Life Technologies), 100 mg/mL streptomycin (Life Technologies) and 0.1% fungizone (Life Technologies). The cells were grown at 37°C in a humidified atmosphere with 5% CO2.

### 2.3. Mice

Wild-type C3H/HeNRj mice were obtained from Janvier Laboratories and were housed under standard conditions. All animal care and experimental procedures were performed as recommended by the European Community guidelines and were approved by the local ethical committee number 14 (number APAFIS#9330-201703017514353).
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## 2.4. TF and TFPI activity assay

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A chromogenic assay (Actichrome TF and TFPI activity assay; Sekisui Diagnostics) was used to analyze TF and TFPI activity according the manufacturer's instructions. 115 000 AT84 Cells (70 000 High TF PancO2 cells, control group) were incubated in a 96-well microplate for 24 hours, then washed and reagents added. The absorbance was read at 405 nm with a microplate reader (MR5000; Dynatech). The TF activity and the TFPI concentration were determined by interpolation from a standard curve constructed using different amounts of lipidated TF and TFPI standards. To standardized results, TF activity and TFPI concentration values were recalculated on the basis of a single cell.

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## 2.5. Mice platelet rich plasma preparation (PRP)

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PRP were obtained from C3H/HeNRj mice under anesthesia (ketamine 125 mg/kg, xylazine 12.5 mg/k, atropine 0.25 mg/kg). blood was collected from the inferior vena cava on citrated tube. Citrated whole blood (1/9) was centrifuged at 90 g during 13 min. The upper phase was considered as platelet-rich plasma (PRP). Platelets were quantified by flow cytometry (Gallios) and adjusted to 125 000 platelet/µL with Tyrode when necessary.

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## 2.6. Induction of platelet aggregation by OSCC cell

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AT-84 cells were dissociated using a non-enzymatic buffer and re-suspended in RPMI-medium not supplemented. Then RPMI-medium, platelet activator (ADP or TRAP) or different amounts of cells (final concentration 2.10⁵ cells/ml) were added to mice PRP and the extent of aggregation of platelets was measured by turbidimetric method using an aggregometer (APACT 4004, labitech).

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## 2.7. Platelet aggregometry

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Platelet aggregation was tested in platelet rich plasma using a light transmission aggregometer (APACT 4004) under constant magnetic stirring at 37 °C. PRP samples were adjusted with Tyrode buffer to standardize platelet concentration in each sample (125 000 platelet/µL). Platelet stimulation was done using ADP (final concentration of 6µM) or using thrombin receptor activating peptide (TRAP, final concentration of 150µM). The concentration of each agonist was selected based on pilot studies that identified minimal concentrations that consistently resulted in approximately 70% aggregation of wild type mouse platelets (data not shown). Aggregation test time was 1200 sec and stimulation occurred at the 100th second.

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## 2.8. Mouse model of tongue squamous cell carcinoma

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AT-84 cells were cultured to 80% confluence. Once the cells reached the exponential growth phase, they were washed three times with PBS (Phosphate Buffered-Saline 1X, Thermo Fisher) and briefly exposed to nonenzymatic cell dissociation buffer (Thermo Fisher) to dislodge the cells. Cells were carefully washed three times with PBS, resuspended in RPMI medium and diluted to the desired concentration. Ten-week-old C3H mice were anesthetized with isoflurane, and a tumor cell suspension (8x10⁵ AT84 cells in 30 µL of RPMI medium, or 30 µL of RPMI) were injected into the musculature of the mobile tongue. Buprenorphine was subcutaneously administered to mice (0.1 mg/kg) just after the surgery. Mice were observed daily and treated with buprenorphine if a distinctive pain signs were detected. After eight days, mice lost approximately 15-20% of their weight and did not present any pain. At this time, mice had tongue tumors that were approximately a third of the mobile tongue. All experimentations of this study were performed at this stage.

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## 2.9. Intravital microscopy and laser-induced injury

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Mice were anesthetized, and a tracheal tube was inserted. A jugular vein cannula was inserted to infuse into blood circulation (anesthesia and antibodies). Cremaster preparation and intravital video microscopy were performed as previously described using a Slide-Book (Intelligent Imaging
137 Innovation)[23]. Vessel wall injury was induced with a MicroPoint Laser System (Photonics 138 Instruments) focused through the microscope's objective and the parfocal focused on the focal plane 139 and aimed at the vessel wall as previously described[24]. The analyses were performed using 140 SlideBook software as described previously by Dubois et al[25].

141 **2.10. Optical microscopy**

142 Mouse tongues were collected, formalin fixed and embedded in paraffin. Four micrometer 143 paraffin embedded tumors sections were mounted on slides and stained by hematoxylin-eosin-safran 144 (HES). Optical microscopy was performed using a Leica DMI8.

145 **2.11. Fluorescent microscopy**

146 Mouse tongues were collected and stored in Optimal Cutting Temperature (OCT) at -80°C. Four 147 micrometer OCT glue-embedded tumors sections were mounted on slides, acetone fixed, protein 148 saturated by Bovine Serum albumin and submitted to labeling by different antibodies. Tongues 149 section were analyzed in fluorescent microscopy using a Leica DMI8.

150 The fluorescence analysis has been done using Fiji (ImageJ, Bethesda, USA). Native fluorescent 151 images were imported in the software, and a 1500x750 pixels sized rectangle located in the tumor 152 area was analyzed. Sums of pixels' intensity were calculated and compared.

153 **2.12. Transmission electron microscopy (TEM)**

154 TEM analysis was carried out by resuspending mice PRP to an equal volume of 2% 155 glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. overnight. Then sample were centrifuged at 1000 156 × g for 10 min, washed, and post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer for 60 157 min at 4 °C. After that they were dehydrated through a graded ethanol series and embedded in resin. 158 Ultrathin sections were prepared, stained with uranyl acetate and lead citrate, and examined under 159 a transmission electron microscope (JEM 1400 JEOL, Tokyo, Japan).

160 **2.13. Statistics:**

161 Significance was determined by the Mann-Whitney test for the in vivo experiments and Student 162 t test for the in vitro experiments. Differences were considered significant at P <0.05.

163 **3. Results**

164 **3.1. OSCC cells induce platelet aggregation in vitro**

165 To investigate the ability of OSCC cells to induce platelet aggregation, we evaluated murine 166 platelet aggregation (using PRP) after adding suspended AT-84 cells in serum free to RPMI. No 167 aggregation was noted when platelets were incubated alone or with RPMI. At a threshold AT-84 cell 168 density (4.10⁵ cells/ml, other concentrations not shown), significant platelet aggregation occurred on 169 part with that induced by platelet agonist (high concentration of ADP, final concentration of 50 µM, 170 figure 1a, 1b). We observed with the AT84 cells a latent period between cell adjunction and platelet 171 aggregation which has been reported by Chang et al. in a similar experiment using human tongue 172 cancer cells. This latent period was dependent on the final OSCC cell concentration. As illustrated 173 figure 1c, a doubling of the latency time occurred when the cell concentration was decreased by five 174 (5x10⁵ cells/mL versus 1x10⁵ cells/mL). Chang et al. also reported that normal oral cells (e.g. fibroblast 175 buccal and gingival epithelial cells) do not induce platelet aggregation[16].

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<figure><img src="image_8.png" /><figcaption>**Figure 1.** OSCC cells induce platelet aggregation *in vitro*. (a) Mice showed typical platelet aggregation following injection of AT84 cells suspended in RPMI (black arrow) in PRP using a light transmission aggregometer (AT84 final concentration: 5x10⁵ cells/ml in PRP; platelet concentration: 1.25x10⁸ platelet/ml). Platelet aggregation induced by ADP (final concentration: 50 µM) was used as a positive control, and RPMI alone was used as a negative control. (b) Mean maximum platelet aggregation at 1200 seconds (n=3 mice; 3 independent experimentations). (c) representative graph of lag time (sec) as a function of AT84 cells concentration added to the reaction mixture. (d) Mean tissue factor activity (pM) and (e) tissue factor pathway inhibitor concentration (x10⁻⁷ unit/ml) per cell for AT84 and High-TF PancO2 cells (n=4 independent experimentations). The results are expressed as the mean (bar) +/- standard error of mean (error bar). NS = nonsignificant. \*p<0.05 and \*\*\*p<0.001.</figcaption></figure>

Tissue factor (TF) is a key regulator of platelet aggregation and hemostasis *in vivo*. Even if many biological mechanisms of tumor cell-induced platelet aggregation exist[26], TF activity enhancement by tumor cells is considered as the predominant mechanism implicated in cancer-induced hypercoagulability[27]. Indeed, TF is the first molecule of the extrinsic coagulation cascade. The presence or expression of TF alone is not sufficient to exert an effect on platelet aggregation. TF must be in its active or decrypted form to initiate the coagulation cascade. Thus, the level of TF activity reflects the level of hypercoagulability of a cancer cell line[28]. Tissue factor pathway inhibitor (TFPI) is the specific inhibitor of TF and consequently the coagulation cascade and platelet aggregation. The balance between TF and TFPI activity supported by a cancer cell reflects the real risk of thrombosis associated with cancer cells [22,29]. Using TF and TFPI activity assays, we measured and compared the TF and TFPI activities of AT-84 cells to those of a mouse pancreatic cancer cell line (Hight TF PancO2 cells) which as elevated TF activity and low to moderate TFPI activity. [22]. AT-84 cells presented significantly higher TF activity (figure 1d, p<0.05) than did the Hight TF PancO2 cell line. Moreover, AT-84 cells presented significantly lower TFPI activity (figure 1e, p<0.001) than did the Hight TF PancO2 cells [22]. Taken together, these results highlighted the strong ability of AT-84 cells to induce platelet aggregation and confirmed their proaggregatory phenotype.

3.2. Platelet aggregates and fibrin deposits are locally detected at the site of the primary squamous cell carcinoma tumors
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After confirmation of the pro-aggregatory phenotype of OSCC cells *in vitro*, we investigated this phenomenon *in vivo* by measuring platelet aggregation and fibrin generation (final product of aggregation) in tongue tumors. We used our mouse model of tongue OSCC generated by intralingual AT-84 cell injection. After eight days of tumor growth (figure 2a right), mice were sacrificed, and tongues were collected, fixed, and sliced. Slides were stained using standard dyes (hematoxylin, eosin and safranin) and observed under an optical microscope. Optical examination (figure 2a middle panel) revealed spindle-shaped cells with a pronounced storiform or sarcomatoid pattern. Nuclei were elongated into an oval shape, and they were plump with multiple chromocenters and often exhibited one or more prominent eosinophilic nucleoli. The cytoplasm was moderately abundant, slightly granular and lightly eosinophilic (figure 2a right panel). Some necrotic areas were also observed. Histological features confirmed the presence of tongue squamous cell carcinoma (SCC) and more specifically sarcomatoid carcinoma.

Another set of slides was incubated with an Alexa 649-conjugated rat anti-mouse CD42b antibody to highlight platelet aggregates and an Alexa 488-conjugated mouse anti-fibrin antibody to highlight fibrin deposits. Similar procedures were also performed on control mouse tongues as well as performed with nonspecific antibodies to confirm the specificity of our results.

Fluorescence microscopy (figure 2b and 2c) showed cluster of high fluorescence intensity clusters in tumor areas indicative of the presence of platelets and fibrin deposits, which were not observed in the same regions of tongues without tumors. Fiji analysis showed a significant elevation in the fluorescence intensity in tongues with tumors compared to tongues without tumors (figure 2d and 2e). The fluorescence intensity of slides stained with nonspecific antibodies was low and was from the platelets and fibrin fluorescence intensity thus validating the specificity of the findings. Taken together, these data confirmed the existence of platelet and fibrin deposits at the site of primary SCC tumors, and they confirmed the ability of AT84 cells to induce platelet aggregation *in vivo*.

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Tumor free mice

Tongue tumor mice

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Tongue tumor mice

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**Figure 2.** Localization of platelet aggregates and fibrin deposits in tongue SCC *in vivo*. (a, left panel) Macroscopic (optical magnification x10) view of tongue squamous cell carcinoma eight days following intra lingual AT84 cell injection in C3H mice. Black arrows show tumor. (a, middle and right panel) Microscopic view (optical magnification x50: middle panel) of a mouse tongue section with SCC (white arrows) with hematoxylin eosin safran staining. The right panel represent an enlargement (optical magnification X200) of a mouse tongue section with SCC (b) Fluorescence microscopic imaging of mouse tongue section without (left side) and with (right side) SCC stained with platelet fluorescently labeled antibody (red) (optical magnification x20 (upper pictures) and X200 (lower pictures)). (c) Same views with fibrin staining (green). (d) Histogram showing results of platelet fluorescence analysis using Fiji (ImageJ, Bethesda, USA) of a 1500x750 pixel sized rectangle located in the tumor area (magnification x200). The results represent the sum of pixel intensity using platelet antibodies for tumor-free mice and mice with tongue tumors (n=3 mice in each group with one pixel intensity measurement per mouse). (e) Histogram showing results of fibrin fluorescence analysis (n=3 mice in each group with one pixel intensity measurement per mouse). The results are expressed as the mean (bar) +/- standard error of mean (error bar). ***p<0.001.

### 3.3. Tongue SCC is associated with a reduction in systemic thrombosis risk in living mice.

After demonstrating the proaggregatory phenotype of AT84 cells *in vitro* and *in vivo*, we next explored the systemic thrombotic risk associated with AT84 tongue tumors *in vivo*. Systemic thrombotic risk was considered the general risk of occurrence of a thrombotic event as a lower extremity deep venous thrombosis (phlebitis) or pulmonary embolism, which are always known to have a high incidence in malignancies. First, we examined whole blood samples to best characterize our mouse model of tongue tumors, especially to ensure that there were no issues with the number of blood cells potentially implicated in hemostasis. Whole blood examination was performed by flow cytometry, which did not identify any difference between mice with and mice without tongue tumors (Table 1). Moreover, we measured the tail bleeding time to test primary hemostasis. Although there was a trend of slightly elevated bleeding times in mice with tongue tumors, no significant difference was identified between the two groups (Table 1). These results were in accordance with the clinical observation of mice, which did not present any spontaneous sign of hemostasis perturbation (ecchymosis, hematoma, or abnormal bleeding). To further evaluate the systemic thrombotic risk associated with OSCC, we investigated thrombosis using real-time intravital microscopy protocol. We examined the kinetics of arteriolar thrombus development (platelet accumulation and fibrin generation) in real-time in the cremaster microcirculation of mice with and mice without tongue tumors. Thrombus formation was initiated by laser-induced injury of the arteriolar vessel wall. In tumor-free mice, platelets adhered to and accumulated at the site of injury (figure 3a upper panel, 3b right panel). The thrombus rapidly increased in size between 15 and 30 seconds after injury. The

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thrombus size then slightly decreased before increasing to its maximal value between 100 and 150 seconds after injury, and the thrombus then stabilized and decreased until the end of the measurement period. In mice with tongue tumors, platelets first accumulated at the site of injury (at approximately the same time between 15 and 30 seconds after injury), but the thrombus size quickly decreased and stayed low to the end of the experiment. (figure 3a upper panel, 3b right panel). Comparative analysis of the median area under the curve of the integrated fluorescent intensity and maximum integrated fluorescent intensity highlighted a significant difference (p<0.001) with a decrease in platelet accumulation (figure 3b, middle and right panel) between mice bearing tongue tumors and tumor-free mice.

<table><caption>**Table 1.** Whole blood cells and tail bleeding time examination. Platelet, neutrophil, monocyte and lymphocyte concentrations in whole blood for tongue tumor and tumor-free mice. Examinations were performed using flow cytometry (n=12 in each group). Tail bleeding time measurement for tongue tumor and tumor-free mice (n=10 in each group). SD: standard deviation, NS: nonsignificant.</caption><thead><tr><th></th><th>Tumor-free mice<br>(WT)<br>Mean (SD)</th><th>Tongue tumor mice<br>(AT84)<br>Mean (SD)</th><th>p<br>(Wilcoxon)</th></tr></thead><tbody><tr><td>Platelets (nbx10<sup>3</sup>/µL)</td><td>883.2 (399.6)</td><td>846.5 (254)</td><td>0.893 (NS)</td></tr><tr><td>Neutrophils (nb/µL)</td><td>1238 (486)</td><td>1381 (479)</td><td>0.588 (NS)</td></tr><tr><td>Monocytes (nb/µL)</td><td>159.8 (104.8)</td><td>242.7 (185.5)</td><td>0.168 (NS)</td></tr><tr><td>Lymphocytes (nb/µL)</td><td>3095 (1851)</td><td>2682 (1571)</td><td>0.685 (NS)</td></tr><tr><td>Tail bleeding time<br>(seconds)</td><td>128.9 (28.48)</td><td>146.2 (48.62)</td><td>0.361 (NS)</td></tr></tbody></table>

In the tumor-free mice, fibrin generation started almost immediately following laser injury and increased with a nearly consistent slope to the end of the recording period (figure 3 a, and 3 c), which was similar to previous reports [22,30]. With regard to fibrin generation, in the tongue tumor group, a slope obtained was shallower than that obtained from tumor-free mice (figure 3 a, and 3 c). Comparative analysis of the median area under the curve of integrated fluorescent intensity and maximum integrated fluorescent intensity highlighted a significant difference (p<0.001) with a decrease in platelet accumulation and fibrin generation in mice with tongue tumors (figure 3 c, middle and right panel). These data indicated a reduction in the systemic thrombosis-associated risk in mice with tongue SCC mice using this vessel injury model. Taken together, our results indicated that AT84 cells have a proaggregatory phenotype *in vitro* and locally *in vivo* but that the systemic thrombotic risk associated with tongue tumors is not present in our mouse model. Moreover, this risk seems to be lower in mice with tongue tumors mice than in tumor-free mice according to the real-time thrombosis study protocol.

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**Figure 3.** Kinetics of arterial thrombosis associated risk with tongue SCC in living mice. (a) Representative images of thrombus formation obtained by intravital microscopy in tumor-free mice and tongue tumor mice. Following laser-induced injury, the kinetics of thrombosis were evaluated based on the platelet (depicted in red) and fibrin (depicted in green) accumulation at different time points during thrombus formation by infusion of an anti-mouse platelet antibody and anti-fibrin antibody. (b) Graph on the left depicts the median integrated fluorescence intensity of platelets as a function of time in thrombi after laser-induced injury in tumor-free mice (37 thrombi in 3 mice) and tongue tumor mice (32 thrombi in 3 mice). Graph in the middle shows the area under the curve of integrated fluorescence intensity of platelets (each point corresponds to a thrombus) in both groups of mice. Graph on the right shows the median of the maximum integrated fluorescence intensity of platelets for both groups. (c) Each graph depicts the same results for fibrin accumulation. The results are expressed as median (bar in blue). \*\*\*p<0.001.

### 3.4. Tongue tumors are associated with a decrease in platelet reactivity

In our model of intravital microscopy with vessel laser injury, the thrombosis mechanism is thrombin dependent[31]. Briefly, laser injury induces endothelial cell activation and neutrophil recruitment (interaction of ICAM 1/LFA1). Neutrophils are the first cells to be recruited at the site of injury and are the main source of tissue factor (TF) in the laser injury model [30]. TF activates the coagulation cascade and induces thrombin generation, platelet activation (depending on thrombin and ADP release from platelets) and so thrombus formation[32]. Fibrin formation is due to coagulation activation and is independent of platelet accumulation [33]. Therefore, we investigated the influence of neutrophil accumulation in thrombus formation. We were not able to detect any difference in neutrophil accumulation at the site of injury in mice with tongue tumors compared to tumor-free mice (Median of area under curve of integrated fluorescence intensity of neutrophils, tumor-free mice: 2.9x10⁻⁹AU, tongue tumor mice: 3.6x10⁻⁹AU; P=0,2278). Moreover, we verified that

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334 the blood neutrophil and platelet concentrations in mice with tongue tumors were not significantly 335 different from those in tumor-free animals (Table 1). Then, we focused on platelet functions, 336 including activation and aggregation abilities. With PRP, we studied platelet aggregation in mice 337 with tongue tumors using two different major activators implicated in our laser injury model as 338 already published and validated by our team, namely thrombin and ADP[24,25,32]. Thrombin and 339 ADP are also the main agonists involved in thrombosis associated with tumor [2]. We observed 340 hypoaggregability of platelets from mice with tongue tumors compared to tumor free mice. A 341 significant reduction in platelet aggregation was found in mice with tongue tumors following 342 activation by ADP (6 µM) and following activation by a low concentration of TRAP (75 µM) (figure 343 4a and 4b). In contrast, no difference in platelet aggregation was observed with activation by a high 344 concentration of TRAP (300 µM) (figure 4c). To the same purpose, we detected the activated form 345 of αIIbβ3 in platelets issued from mice with tongue tumors and tumor-free mice before and after 346 activation by a high concentration of ADP (50 mM) or low concentration of TRAP (75 µM). In the 347 presence of each agonist, the platelets from mice with tongue tumors presented a decrease in αIIbβ3 348 activation on their surface compared to that in platelets from tumor-free mice in presence of each 349 agonist (figure 4d). This decrease in platelet reactivity in mice with tongue tumors may explain the 350 reduction in the general thrombosis risk in the laser injury model.
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<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

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d.

<figure><img src="image_36.png" /></figure>

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<caption>**Figure 4.** Platelet reactivity in mouse model of tongue SCC. (a) Tumor-free mice (WT) and tongue tumor mice (AT84) platelet aggregation following treatment (at 100 seconds) with ADP (final concentration of 6 µM) in a light transmission aggregometer. Graphs in the middle and on the right side depict difference of platelet aggregation between tumor-free mice and tongue SCC mice at 600 and 1000 seconds following ADP activation (n=8 mice in each group). (b) Same experimentation with</caption>
357 low concentration (75 µM) TRAP activation (n=5 mice in each group) and (c) high concentration (300 µM) TRAP activation (n=5 mice in each group). (d) Proportion of platelets expressing activated αIIbβ3 following ADP (50 µM, on the left) or TRAP (75 µM, on the right) activation. Data represent the percentage compared to the expression before activation. The results are expressed as the mean (bar) +/- standard error of mean (error bar). \*p<0.05, \*\*p<0.01, \*\*\*p<0.001, and NS: nonsignificant.

362 3.5. *Tongue tumors are associated with granule pool deficiency*

363 Tongue SCC is associated with a low risk of systemic thrombosis due to the reduction in platelet reactivity after an ADP and TRAP stimulation, with a small increase in activated αIIbβ3. The main source of ADP in platelets is dense granules [34]. This ADP pool is necessary to achieve a full platelet aggregation. We hypothesized that platelets from lingual tumor-bearing mice could present morphologic differences compared to platelets issued from tumor-free mice. Then, we studied the morphology of platelets of mice bearing tongue tumors using transmission electron microscopy (TEM). Platelets from the mice with tongue tumors were rounder in shape than those from tumor-free mice. These observations suggested that the granules in the platelets from mice with tongue tumors, were modified (figure 5a). Platelet delta-granules or dense granules contain mostly small molecules, including calcium, adenine nucleotides (ATP and ADP), αIIbβ3, serotonin, and pyrophosphate. We counted the number of dense granules in (sliced) platelets from mice with tongue tumors and tumor-free mice. A significant (p<0.0001) decrease in number of dense and alpha granules was found in platelets from mice with tongue tumors (mean of 0.48+/- 0.03 dense granules and 4.4 +/- 0.13 alpha granules per platelet) compared to platelets from tumor-free mice (mean of 1.6 +/- 0.05 dense granules and 5.3 +/- 0.15 alpha granules per platelet) (figure 5b).

378 Thus, these findings suggest a deficiency of granule storage in mice with tongue tumors resulting in a lower activibility of platelets in bearing lingual tumor mice, independent of the agonist used. This could lead to a lower level of activated αIIbβ3 at the platelet surface.

381 Higher magnification TEM images highlighted the following ultrastructural modifications in platelets from mice tongue tumors. Platelets had a rounded shape with many large empty vacuoles. These TEM findings indicated that the deficiency in the granule storage pool may explain the decreased platelet reactivity and the decreased risk of systemic thrombosis highlighted in the laser injury model.

a.

<figure><img src="image_38.png" /><figcaption>Tumor-free mice</figcaption></figure>

<figure><img src="image_39.png" /><figcaption>Tongue tumor mice</figcaption></figure>
b.

<figure><img src="image_40.png" /></figure>

c.

<figure><img src="image_41.png" /><figcaption>**Figure 5.** Morphological analysis of tongue tumor mouse platelets. (a) Transmission electron microscopy (TEM) examination of platelet (PRP) at 8000-fold magnification from tumor-free mice on left side and tongue tumor mice on right side. Blue arrows indicate granules. (b) Graphs depict the average number of dense granules per platelet (left panel) and the average number of alpha granules per platelet (right panel) for tumor-free mice and tongue tumor mice (n= 4 mice in each group; mean was calculated per 100 counted platelets; mean (bar) +/- standard error of mean (error bar); ****p<0.0001). (c) TEM examination of platelet (PRP) at 20,000-fold magnification from tumor-free mice on left side and mice with tongue tumors on right side.</figcaption></figure>

4. Discussion

Thrombosis risk associated with malignancy has been extensively studied for specific cancers (colon, lung, and pancreas), but thrombosis risk for OSCC has been poorly studied[19]. To the best of our knowledge, our study is the first to specifically explore the risk of thrombosis in tongue SCC using a live animal model. Malignancy is usually associated with elevated thrombosis risk, but the thrombosis risk associated with OSCC is low or even nonexistent[35]. In the literature, few nonsolid tumors, such as leukemia, have been reported to be associated with a poor thrombosis risk [36], or to deficiency in the dense granule pool (chronic lymphocytic leukemia) [37]. However, solid tumors are usually associated with an elevated risk of the thrombosis [3].

Even so, few studies have reported that a solid cancer is associated with a low specific risk of thrombosis, due to detailed associated mechanisms, such as acquired hemophilia [38] or acquired von Willebrand disease [39]. Our study indicates that the lack of thrombosis risk in a solid cancer could be due to a deficiency in the storage pool.

In the clinic, patients with cancer usually benefit from anticoagulation medication, especially with surgery or chemotherapy, which would enhance the thrombosis risk[3]. For OSCC patients and some head and neck cancer patients, recommendations for anticoagulation medication are unclear. Some authors propose a systematic anticoagulation therapy [40], whereas others limit this approach to patients with high risk of thrombosis due to other comorbidities[41]. These treatments are not

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415 without risk (hemorrhage and immune/allergic response), and their use must be considered
416 thoroughly. Our study provides additional arguments to limit the administration of anticoagulant
417 therapy in OSCC cancer patients.

418 OSCC is even more unique than originally thought because we showed that OSCC tumors had
419 a proaggregatory phenotype with a high risk of local thrombosis without enhancement of the risk of
420 systemic thrombosis. AT84 cells had characteristics of cancer cells with high risk of thrombosis, and
421 they induced platelet aggregation. In addition, AT84 cells presented high tissue factor activity and
422 low tissue factor pathway inhibitor activity. Moreover, signs of coagulation activation were observed
423 in OSCC tumors with platelet aggregation and fibrin deposits. All of these features are generally
424 found in cancer associated with high risk of thrombosis[42]. However, this was not the case for our
425 model for which systemic thrombosis risk was not enhanced due to decreased platelet reactivity with
426 delta-storage pool deficiency, which counteracted the pro-aggregatory characteristics of OSCC.
427 Despite this reduction in platelet reactivity, the risk of local thrombosis remained high in OSCC
428 tumors because there was high local expression of procoagulant proteins, resulting in platelet
429 aggregation. Moreover, the platelets from mice with tongue tumors maintained normal aggregation
430 abilities in response to a strong activation resulting by a high concentration of TRAP.

431 This paradoxical situation may seem surprising, but it reflects observations in the clinic. Similar to
432 the murine OSCC cell line in the present study, human OSCC cells have clearly established
433 proaggregatory phenotype *in vivo* and *in vitro*. OSCC tumors are implicated in local thrombosis
434 events, especially internal jugular vein thrombosis[43] and even carotid artery thrombosis [44].
435 However, no augmentation of the systemic thrombosis risk occurs in humans with OSCC[19],
436 suggesting that a mechanism to counteract the thrombotic potential of OSCC exist in humans. This
437 mechanism is actually unknown in human OSCC, and we hypothesize that it could be the same
438 mechanism as the decreased platelet reactivity observed in our animal model. Several studies have
439 reported decreased platelet reactivity in cancer patients, notably hematological cancer[45] and
440 ovarian cancer [46]patients. One could postulate that the presence of cancer cells in the bloodstream
441 induced exhausted platelets which can be encountered in cancerous patients even in the absence of
442 consumption coagulopathy. However, we reported that OSCC tumors express high levels of active
443 TF, inducing a TCIPA when present in blood. Alternatively, tumors could secrete ADP[2]. ADP may
444 in turn activates platelets leading to the desensibilization of the P2Y1 receptor and the secretion of
445 the granule contents, leading to exhausted platelets insensitive to ADP. In this case, activated and not
446 resting platelets will be observed by electron microscopy. Also, this hypothesis does not explain the
447 decrease in the reduced level of activated αIIbβ3 observed. Another possibility is that OSCC tumors
448 will directly or indirectly act on megakaryocytes leading to the generation of deficient platelets.

449 Delta-storage pool deficiency is a thrombopathy defined by a reduction in granule secretion
450 from platelets, which may be due to a quantitative defect (with a decreased number of granules per
451 platelet) or a qualitative defect (abnormal content or abnormal mobilization of dense platelet
452 granules). These defects lead to diminished platelet aggregation owing to the lack of a second wave
453 of aggregation dependent on the release of dense granule content, especially ADP[47]. This
454 phenomenon was observed in our mouse model of tongue SCC with a lack of platelet aggregation in
455 the intravital real-time thrombosis study and with a decrease in platelet aggregation via activation of
456 ADP or a low concentration of TRAP. Clinically, delta-storage pool deficiency manifests itself as
457 hemorrhagic symptoms that are most of time moderate and often only observed following
458 traumatism or a surgery, potentially without increasing the bleeding time[37]. In our animal study,
459 the mice did not present a hemorrhagic profile, and we did not observe a significant elongation in
460 the bleeding time. Thus, our results were not discordant with a delta-storage pool deficiency. The
461 etiology of delta-storage pool deficiency is complex. This deficiency can be congenital or inherited,
462 and it can be either isolated to a specific location or as part of the whole organism response. Delta-
463 storage pool deficiency can also be acquired as has been observed in leukemia and myelodysplastic
464 syndromes[36] as well as in inflammatory diseases, such as rheumatoid arthritis, lupus
465 erythematosus[48] or postural orthostatic tachycardia syndrome[49]. To the best of our knowledge,
466 this is the first report on the association between OSCC and delta-storage pool deficiency as well as
467 the first report on this platelet disorder in a solid cancer. We also reported that this disorder may
468 counteract with the prothrombotic profile of OSCC tumors and protect against a systemic thrombotic
469 event. Even if a genetic disorder relating cellular transporters has been identified in an inherited form,
470 the mechanism involved in acquired delta-storage pool deficiency remains unclear. It has been
471 reported that failure of megakaryocyte maturation[50] occurs in the acquired form, which may be
472 notable due to defective zinc homeostasis[51]. This approach would be interesting because a lack of
473 zinc homeostasis has been reported in some cancers, especially head and neck cancer [52,53].

474 In conclusion, our mouse model of tongue tumors confirmed that OSCC tumors have a
475 proaggregatory phenotype and may be involved in local thrombosis (in the tumor or peritumor area).
476 However, we demonstrated that there was no elevated systemic thrombosis risk in a laser model.
477 Therefore, we concluded that OSCC tumors are the only solid cancer that does not show an elevated
478 risk of systemic thrombosis in the laser model. Moreover, we demonstrated that a delta-storage pool
479 deficiency in mice with tongue tumors could be responsible almost partially for the decreased platelet
480 reactivity, which counteracted the proaggregatory phenotype of OSCC tumors and explains the low
481 risk of systemic thrombosis. This study provides a supplemental argument for limiting the systemic
482 use of anticoagulant therapy in OSCC patients. The next step will be to identify the molecular
483 mechanisms involved in delta-storage pool deficiency and to explore the distance relationship
484 between OSCC tumors and platelets or megakaryocytes to deeply understand its role in the
485 pathogenicity of the disease.

486 Author Contributions: Conceptualization, Pierre Haen, Christophe Dubois and Laurence Panicot-Dubois;
487 Formal analysis, Pierre Haen, Lydie Crescence and Alexandre Altié; Investigation, Pierre Haen; Methodology,
488 Pierre Haen, Lydie Crescence, Diane Mege and Alexandre Altié; Supervision, Christophe Dubois and Laurence
489 Panicot-Dubois; Validation, Diane Mege, Christophe Dubois and Laurence Panicot-Dubois; Writing – original
490 draft, Pierre Haen; Writing – review & editing, Christophe Dubois and Laurence Panicot-Dubois.

491 **Funding:** This research was partly funded by National French Ministry Grants (INSERM and Aix-Marseille
492 University) and by the association "les Gueules Cassées".

493 Acknowledgments: The authors are grateful to Stéphanie Simoncini for her assistance during the fluorescence
494 shooting and Stéphane Robert for his assistance in flow cytometry experiments.

495 **Conflicts of Interest:** The authors declare no conflict of interest.

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<figure><img src="image_42.png" /></figure>

© 2020 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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