Pancreatic Cancer constitutes one of the most lethal diseases, with an average 5-year survival rate of less than 10% [1]. About 60–80% of patients are diagnosed with advanced cancer as it invades the surrounding tissues (locally advanced) or has disseminated outside the pancreas (metastatic) [26]. KRAS mutations comprise the most frequent alterations observed in pancreatic ductal adenocarcinoma (PDAC), therefore this type of cancer is frequency, is considered one of the most the most RAS-dependent of all cancers [27][28]. As the disease shows a very high mortality rate, it is imperative to discover novel and, more effective treatments, especially for those carrying the KRAS mutation.
The presence of desmoplasia is a feature of the pathogenesis and evolution of PC that represents 60–90% of the total tumor mass [29] and can be recognized mainly in both primary and metastatic areas of the tumor [30]. Fibroblasts are the predominant cell type within the TME, where they form a heterogeneous group of cells that contribute to ECM formation and support tumor growth [31]. Pancreatic stellate cells represent a subset of cancer-related fibroblasts present in a dormant state in healthy pancreatic tissue. Once activated, i.e., as a result of inflammation, injury, or tumor formation, they adopt a myofibroblast-type phenotype with high proliferative capacity and increased extracellular matrix protein secretion [32]. TME exerts various actions ranging from tumor formation, cancer spread and resistance to treatment [33]. Conventional drugs, such as gemcitabine, are not able to penetrate the rich and thick layer of TME and therefore lead to drug tolerance. TME is further characterized by severe hypoxia and, when combined with vascular compression induced by desmoplasia, triggers the process of angiogenesis to support the tumor's constant need for nutrients [34].
In our study, the PC line PANC-1 was selected, among other reasons, because this type of cells exhibit stromal characteristics and can provide a descent simulation of the real conditions of pancreatic cancer concerning the TME [35],[36]. Taking into account that LMWHs can reduce fibrin formation and contribute to ECM degradation the result is the weakening of the TME. It could be hypothesized that when combined with drugs such as Nab-paclitaxel and gemcitabine that can penetrate the cancer cells more efficiently and eliminate them, there might be a synergistic effect with the action of LMWHs, something that is reflected in our in vivo results.
Tumors release vascular growth factors, such as VEGF and fibroblast basal growth factor (bFGF), which, along with other cytokines, stimulate angiogenesis by interacting with their high-affinity intracellular activity receptors. In humans, therapeutic doses of UFH actually cause increased levels of growth factors, such as plasma bFGF [18]. Heparin affects the activity of other factors involved in angiogenesis and tumor growth, in addition to VEGF and bFGF [37]. Growth factor TGF is a potent immunosuppressive and essential regulator of the growth, differentiation and adhesion of various cells. It is expressed in cancer cells and its overproduction is associated with an unfavorable prognosis.
LMWHs, on the other hand, can inhibit the binding of growth factors to their receptors while they are able to negatively affect the rate of angiogenesis [38]. The reduction of angiogenesis has been shown in an experimental model of human colon cancer, where tinzaparin administration 24 hours after angiogenesis stimulation by VEGF led to a reduction of the angiogenic index to the control level [39]. Tinzaparin exerts its anti-neoangiogenic activity as it appears to stimulate more production of Tissue Factor Pathway Inhibitor (TFPI) by epithelial cells than any other Low Molecular Heparin, inhibiting Tissue Factor (TF) and consequently VEGFR. However, LMWHs are known to inhibit heparanase, an endoglycosidase rarely expressed in normal tissue, while it is overexpressed in pancreatic tumors. The activity of LMWHs is demonstrated through the inhibition of VEGF-A and FGF-2 as well as the increased release of TFPI, suggesting an antagonistic role of LMWHs in the angiogenesis due to heparanase. Vascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development [40].
Τhere are several reports about the role of Tinzaparin in various types of cancer, where it appears to attenuate tumor growth but -most importantly-the metastatic process. Using a mouse pancreatic cancer cell line injected to mouse models, tinzaparin was able to inhibit tumor growth without affecting the thrombotic phenotype [41]. Another study demonstrated Tinzaparin’s ability to upregulate the expression of E-cadherin in pancreatic tumor cells, a marker of decreased disseminating capacity and reduced metastatic potential [42]. Tinzaparin administration in mice injected with melanoma cells was capable of diminishing the metastatic cascade to distant organs by disturbing the P-, L-selectin and VLA-4/VCAM-1 interconnections. In addition, Tinzaparin suppressed the binding of malignant cells expressing the C-X-C chemokine receptor type 4- to their specific ligand on normal tissue, resulting in a substantial reduction in the dissemination of human breast cancer cells to the lung [43]. The antimetastatic properties of tinzaparin have also been observed in a B16 melanoma cell lung metastasis model in mice [15]. Administration of tinzaparin prior to the injection of melanoma cells resulted in lung tumor formation by 89% compared with controls whereas daily administration of tinzaparin for 2 consecutive weeks reached an additional reduction in lung tumor formation, reaching almost 96% [44].
Our results demonstrate, that tinzaparin reduces the expression of VEGFR2 resulting in fewer vessels formation in the tumor and, therefore, lower potential for nutrition. VEGFR2 activation stimulates cell proliferation and survival, activities that were inhibited in our experiments. We showed an important down-regulation of VEGFR2 and suppressed cell proliferation, observed with PCNA marker staining. Also, our data revealed a reduction in the survival through the mechanism of apoptosis, as seen by the in vitro and in vivo experiments using caspase-3.
PaCT (Pancreatic Cancer & Tinzaparin) is a retrospective observational study that collects data regarding progression-free survival (PFS) in advanced or metastatic PC patients who received thromboprophylaxis with tinzaparin during chemotherapy with Nab-paclitaxel and gemcitabine. In this study, the median PFS was 7.9 months. Out of 14 similar studies (involving 2994 patients) identified via systematic search, it was settled that the weighted PFS of patients receiving Nab-P and G but no anticoagulation was 5.6 months. Therefore, patients receiving tinzaparin had 39.54% higher PFS than patients without thromboprophylaxis (p < 0.05). Tinzaparin was administrated in a “hyper-prophylactic” of 10.000 Anti-Xa IU (175 UI/Kg) or even higher full treatment doses. Possible reasons for the increase in PFS were the treatment of venous thromboembolism, a prevalent condition in pancreatic cancer and the possible antitumor role of tinzaparin [45].
It should be noted here that the dose of tinzaparin administered (250UI / Kg) to the mice was the highest possible without bleeding, so it can be considered hyper-prophylactic as used in the PaCT study [41]. A clinical study using tinzaparin in patients with Non-Small Cell Lung Cancer did not have the expected PFS results, probably because the tinzaparin dose was 100UI / Kg, lower than the 175 UI / Kg used in the PaCT study [46].