Alterations in the red blood cell rheology in pancreatic cancer

Abstract

Aim

This study aimed to investigate the effect of pancreatic ductal adenocarcinoma (PDAC) on the RBCs rheological properties at the time of diagnosis and to examine their potential relationship with tumor stage.

Material and Methods

We studied 32 patients with confirmed PDAC.The aggregation and deformability of RBCs were evaluated using a LORCA.The following parameters specific to the aggregation process were estimated: the aggregation index (AI), the aggregation half-time (t_1/2), and the threshold shear rate (γ_thr).RBC deformability expressed as erythrocyte elongation (EI), was measured from 0.3 Pa to 60 Pa shear stresses.

Results

All measured RBC aggregation parameters among PDAC subjects differed from those in controls. The AI (P < 0.03) was significantly higher in the PDAC group, whereas t_1/2 (P < 0.001) and AMP (P < 0.001) were significantly lower compared to the control group. No significant differences in RBC deformability were observed between the PDAC and control groups.There were significant correlations between some RBC aggregation parameters and PDAC tumor staging (T).There was that T correlated positively with t_1/2, and negatively with AI and γ_thr.

Conclusion

PDAC is associated with alterations in RBC rheology behavior. Results indicated a higher tendency for RBC aggregation and aggregate stability associated with the rigidity of RBC at lower shear stress.

Keywords

Red blood cell aggregation aggregability pancreatic cancer

Introduction

Pancreatic carcinoma (PC) is a highly malignant tumor with a low 5-year survival rate and is the most significant cause of cancer mortality in Europe¹ and the United States.² Approximately 95% of PCs arise from the exocrine portion of the ductal epithelium, acinar cells, or connective tissue. The most common type of PC is pancreatic ductal adenocarcinoma (PDAC), which accounts for 85-90% of all PCs. PDAC is highly malignant due to locoregional invasion, distant metastasis, high recurrence rates, and low resection rates.³ Only 15-20% of cases are diagnosed at a stage when curative surgery can be considered due to a lack of symptoms or vague symptoms when the cancer is still localized.⁴

Independent of cancer aetiology, tumor growth and progression are associated with extensive neovascularization, including microvessel growth and vascular network remodelling, in which angiogenesis and vascular remodelling play an important role. Hypoxia induce the production of signalling molecules and cytokines and upregulates certain microRNAs important for cancer progression, altering the expression of tumor suppressor genes, promoting angiogenesis, and regulating tumor immune escape under hypoxic conditions.⁵ VEGF is the key mediator of angiogenesis in cancer and is upregulated by oncogene expression, variety growth factors, and hypoxia.⁶ Recent study indicated that PDAC is associated with blood and tumor VEGF and HIF-1α hypoxia-inducible factor 1α overexpression.^7,8 The literature indicates that HIF-1α is a principal regulator of tumor hypoxia and plays a critical role in promoting PDAC,⁹ and it has been considered for prognostic stratification¹⁰ and survival of patients with PDAC.¹¹ Other studies have shown relationships between RBCs rheology disturbances and angiogenesis status, but the physiological importance of these relationship is unclear.^12,13

Rheological properties of RBC expressed by their deformation and aggregation are a significant determinant of shear-dependent behaviour of blood viscosity and affect blood-flow to minimize the viscous resistance in capillaries. Shear stress causes disaggregation and deformation of RBC leading to a decrease in blood viscosity and improving blood-flow.

Figure 1.

Correlation between tumor stage and: A. with t_1/2^⃰, B. with AI ^⃰ and C. with γ_thr⃰ ⃰

It is well recognized that disturbances of RBC aggregation are associated with medical conditions such as cardiovascular disease, metabolic disease, haematological disease, or infection.14 Studies have indicated disturbances in the blood's rheological behaviour, such as blood hyperviscosity, enhanced RBC aggregation, and evidence of a lower ability to disaggregate, in some cancers.^15,16 To date, limited information is available on the interactions between pancreatic cancer and RBC rheological behaviour. Here, we address the hypothesis that PDAC may be associated with RBC rheological disturbances.

This study aimed to investigate the effect of PDAC on RBCs’ rheological properties at the time of diagnosis and their potential relationship with tumor stage.

Material and methods

Patient characteristics

We studied 32 patients with confirmed PDAC who were qualified for pancreaticoduodenectomy for the head of the pancreas tumor. There were 18 (56%) males and 14 (44%) females in the study, with a mean age of 61.7 ± 12.2 years. We included patients who did not exhibit factors that could potentially influence the observed rheological blood parameters, such as diabetes mellitus, chronic kidney disease, anemia, abnormal coagulation parameters, uncontrolled hypertension, history of leg vein thrombosis, and antithrombotic therapy. The control group consisted of 23 healthy individuals without diabetes mellitus, arterial hypertension, or any of the other above-listed features. There were 14 (61%) men and 9 (39%) women, with a mean age of 56.7 ± 3.03 years. This group was recruited from volunteers from the medical staff. The study protocol was approved by the ethical committee of the Medical University of Silesia, and all participants provided written consent.

Hemorheological measurements

Blood samples were collected from the cubital vein with a syringe for a biochemical examination and anticoagulated with K₃EDTA (1 mg/mL) for rheological tests. Blood withdrawal was performed after a 10-min resting period after a 12-h overnight fast between 8 and 9 a.m. The rheological tests were performed at a stable temperature of 37°C within two hours after the blood was collected. Hematocrit (Hct) was measured with a microhaematometer. Hematocrit was adjusted to a standard Hct of 45%. Given the paramount importance of Htc on RBC aggregation, all samples were adjusted to a 45% Htc to ensure that aggregation results were not confounded by the baseline differences in Htc between groups. The RBC aggregation and kinetics of the red blood cell aggregation were performed by the Laser-assisted Optical Rotational Cell Analyser - LORCA (Mechatronics, Zwaag, The Netherlands). In the erythrocyte aggregation measurements, a laser backscatter light was used, in which the intensity of the reflection depended on whether the erythrocytes formed rouleaux at rest or if they were disaggregated and deformed at high shear rates. A blood sample is initially subjected to a high shear rate to cause cell disaggregation. Next the motion is stopped to cause relaxation of the elongated RBC and forming of rouleaux. The LORCA computer program analyzed the aggregation parameters of RBC, which were based on the syllectogram, i.e., the curve of the relation between the intensity of laser backscattering and time, where light intensity is expressed in arbitrary units (au). The relevant part of the syllectogram reflects both static and kinetic parameters of aggregation and the behavior of the RBC elongation. A normal syllectogram consists of an ascending part and a descending part, representing the progression in rouleaux formation. A decrease in laser backscatter intensity in the descending part of the syllectogram represents the progression of the aggregation process. The size of the ascending part of the syllectogram represents the time of relaxation of the elongated RBC. The syllectogram amplitude depends on the size of both parts of the syllectogram, so it contains information about the aggregation process and the behavior of the elongated RBC, which reflects the state where elongated RBC recovered their normal shape. From the syllectogram, the following parameters to the aggregation process were estimated: the aggregation index (AI in %), amplitude (AMP in au), and aggregation half-time (t_1/2 in s), which expresses the kinetics of the aggregation process and threshold shear rate (γ_thr in s⁻¹). The computer program controls the measurement and analysis of the aggregation parameters of the RBC. The RBC deformability was measured at shear stresses of 0.3 Pa, 3 Pa and 60 Pa. The needs of this device are 25 μl of a blood sample diluted in 5 ml a solution of 0.14 mM polyvinylpyrrolidone in phosphate buffered saline (140 mM NaCl, 18.4 mM Na₂HPO₄, 1.3 mM NaH₂PO₄, pH = 7.4 and viscosity at 37°C: 30 ± 2 mPa.s).

Statistical analysis

Continuous variables are presented as the mean ± SD or the median with the interquartile range if the data were not normally distributed. Categorical variables are presented as absolute numbers and percentages. The Shapiro‒Wilk test was used for all continuous variables to test for a normal distribution. Statistical comparisons were achieved using the unpaired Student's t test and the Mann‒Whitney U test for nonnormally distributed data. Chi2 or Fisher's exact test (in the case of expected numbers smaller than 5) was performed to compare the differences in the categorical data. The association between continuous variables was tested using Person's correlation or Spearman's rank correlation. The effect size for correlations was defined as weak (0.1 ≤ r_xy <0.3), moderate (0.3 ≤ r_xy <0.5), large (0.5 ≤ r_xy <0.7) or very large (0.7 ≤ r_xy <0.9). Results with a P value <0.05 were considered to be significant. The statistical analysis was performed using Statistica 12 (StatSoft, Inc., Tulsa, OK, USA).

Results

The baseline characteristics of the study population are presented in Table 1. There were no significant differences between the groups in the sex distribution (p = 0.55 and p = 0.47), median body mass (p = 0.194), or age; however, patents with PDAC tended to be older compared to control group (p = 0.06). Significant differences were observed for some blood count parameters between both groups. Hematocrit and hemoglobin levels were significantly lower in the PDAC, whereas PTL levels were significantly higher compared with the control group (Table 1). There were no differences in white blood cell count or CRP. As expected levels of the CA 19-9 antigen were significantly higher in the PDAC group than in the control group. There were no differences in CEA or AFP levels between the groups.

Table 1.

Baseline characteristic of the study group.

	PDAC (n = 32)	Control (n = 23)	P
Age (years)	61.7 ± 12.2	56.7 ± 3.03	0.06
Female Male	14 (43.75%) 18 (56.25%)	9 (39.1%) 14 (60.9%)	0.55 0.47
Weight (kg)	65 (61–75)	73 (61–85)	0.19
RBC (10⁶/μl)	4.44 ± 0.62	4.67 ± 0.31	0.07
Hb (g/dl)	12.7 ± 1.5	13.7 ± 1,6	0.023
Hct (%)	37.2 ± 4.1	41.8 ± 4.52	< 0.0001
WBC (10³/μl)	7.36 ± 1.84	6.3 ± 1.9	0.12
PLT (10³/μl)	317.15 ± 75.1	2464 ± 50.3	0.0003
CRP (mg/l)	3.2 (1–8)	2.9 (1.3–7.1)	0.86
Bilirubin, total (umol/l)	27 (9.29–77.36)	9.9 (6.6–13.2)	0.002
CA 19–9 (U/ml)	173.4 (5.61–606.1)	7.36 (2.05–30.27)	< 0.0001
CEA (ng/ml)	2.98 (2.37–11.76)	2.1 (1.8–2.36)	0.29
PDAC pathological stage
T
T1	3 (9.4%)
T2	14 (43.7%)
T3	12 (37.5%)
T4	3 (9.4%)
N
N0	5 (15.6%)
N1	15 (46.9%)
N2	12 (37.5%)

RBC, red blood cell; Hb, hemoglobin; Hct, hematocrit; WBC, white blood cells; PTL, platelets; CEA, carcinoembryonic antigen; CA 19–9 carbohydrate antigen 19–9; T, primary tumor staging; N, regional lymph nodes.

All measured RBC aggregation parameters among PDAC subjects differed from those in controls (Table 2). The AI (P < 0.03) was significantly higher in the PDAC, whereas the t_1/2 (P < 0.001) and the AMP (P < 0.001) were lower compared with the control group. RBC deformability, expressed by the elongation index, did not differ significantly between PDAC patients and controls. No significant differences were found across any of the shear stress levels between the two groups. There were significant correlations between some RBC aggregation parameters and PDAC tumor staging (T). There were that T correlated positively with t_1/2 (r = 0.41; p = 0.018), and negatively with AI (r = -0.51; p = 0.0028) and γ_thr (r = -0.49; p = 0.0037). The effect size of the correlation was found to be moderate to large (Figure 1).

Table 2.

Changes in RBC properties of blood between the pancreatic cancer and control groups.

	PDAC (n = 32)	Control (n = 23)	P
AI (%)	64.56 ± 11.3	57.86 ± 8.5	0.028
AMP (au)	20.47 (18.16–22.9)	23.6 (20.6–25.6)	0.001
t_1/2 (s)	1.79 (1.48- 2.78)	3.12 (2.06–3.74)	0.0009
γthr (s⁻¹)	200 (100–275)	100 (100–100)	0.003
EI (0.3 Pa)	0.060 ± 0.022	0.056 ± 0.01	0.54
EI (3 Pa)	0.363 ± 0.034	0.360 ± 0.01	0.6
EI (60 Pa)	0.622 ± 0.02	0.633 ± 0.01	0.17

AI, aggregation index; AMP, amplitude; t_1/2, aggregation half-time; γ_thr, threshold shear rate; au, arbitrary units; EI, elongation index.

Discussion

PDAC belongs to the most aggressive solid tumors in human, with a high mortality and poor survival rate. PDAC is characterized by an immunosuppressive tumor microenvironment that dynamically evolves during tumor progression, accompanied by the significant expression of myeloid-derived suppressor cells and tumor-associated macrophages.^17,18,19,20 Independent of cancer aetiology, adequate angiogenesis by upregulating VEGF is necessary for tumor growth because the tumor requires efficient blood perfusion for supplementation of nutrition and oxygen in newly enlarged areas.²¹ On the other hand, accumulating studies have indicated that that HIF-1α is a principal regulator of microenvironment tumor hypoxia and plays a critical role in promoting PDAC,^22,23 and it has been considered for prognostic stratification²⁴ and survival of patients with PDAC.¹¹

Tissue perfusion, including solid tumor, depends on interrelated rheological factors that determine the blood flow in microcirculation. The studies have clearly shown that RBC aggregation plays an important role in determining low shear blood viscosity²⁵ and contribute to blood flow.^26,27 Blood flow through microcirculation is possible due to the unique ability of erythrocytes to deform under the influence of shear forces and the phenomenon of erythrocyte axial accumulation. Axial accumulation is caused by the axial migration of RBCs in the center of the flow stream, which results in the concentration of RBCs at the core and the development of a peripheral cell-free layer near the vessel wall that minimalizes the resistance of blood flow.²⁸ The cell-free layer thickness increases with RBC aggregation and deformability, resulting in higher viscosity in a denser central core; however, the global apparent viscosity tends to decrease because of the lubricating effects generated in the larger cell-free layer. Enhanced RBC rigidity induces a less dense RBC core and a thinner cell-free layer; thus, viscosity increases with decreased RBC deformability²⁹

Slight RBC rigidity may cause substantial changes in the flow-through of the microcirculation.

These results confirmed that PDAC is associated with alterations in the RBC rheology, expressed by an increased total aggregation extent and spontaneous ability to aggregate.

We observed that the aggregation index and the syllectogram's amplitude were significantly higher in the PDAC patients. Alterations in the kinetics of RBC aggregation expressed by shortening aggregation half time indicate that RBC aggregates form aggregates and rouleaux at a shorter time, after their disaggregation than in control group. These results indicated that PDAC is associated with hyperaggregation of RBCs and increased spontaneous ability to aggregate compare to healthy individuals. The γ_thr represents the minimal shear rate needed to prevent RBC aggregation and determines the ability of erythrocyte rouleaux. An elevated γ_thr indicates a greater aggregate stability in PDAC patients. Our study indicates that PDAC is not associated with changes in RBC deformability. The elongation index in the PDAC group was found to be comparable to that of the control group. The deformability of RBCs is a critical rheological factor in microcirculatory blood flow. RBC deformation results in the shear-dependent behavior of blood viscosity and optimizes flow by minimizing viscous resistance within capillaries. The phenomenon of axial accumulation depends on the ability of erythrocytes to deform under shear forces. Shear stress induces the deformation of RBCs, leading to a decrease in blood viscosity, which subsequently improves blood flow. The axial migration of RBCs toward the center of the flow stream leads to the development of a peripheral cell-free layer near the vessel wall, which minimizes flow resistance.^28,29 While the thickness of the cell-free layer increases with RBC deformability—resulting in higher viscosity within the denser central core—the global apparent viscosity actually decreases. McHedlishvili suggested that this flow structure arises from a specifically ordered arrangement of RBCs surrounded by plasma within the lumen of capillaries and adjacent arterioles and venules up to approximately 15–20 μm in diameter.²⁵ Our study indicates that PDAC affects RBC aggregation but not deformability. No significant differences in RBC deformability, as expressed by the elongation index, were observed between the PDAC and control groups. It is possible that PDAC impacts RBC aggregation through an as-yet unknown mechanism rather than through deformability, or that it represents one of the regulatory mechanisms affecting the stability of blood viscosity. While these findings do not allow for broad generalizations, they provide a valuable comparison between the two groups and remain consistent with the phenomenon of shear-dependent blood viscosity.

PADC patients exhibit a high ability to aggregate and aggregability of RBC, which may contribute to problems with blood-flow conditions, especially in the microcirculation, and result in regions of local hypoxia. This may results decrease tissue blood perfusion and predispose to tumor hypoxia and are known to be altered in PDAC.³⁰ The results, which were intriguing, demonstrated significantly strong correlations between RBC aggregation parameters and the extend of PDAC tumor stage (T). We found that T correlated positively with t_1/2, and negatively with AI and γ_thr. On one side, tumor extension is associated with hyperaggregability of RBC expressed by positive correlations between T and shortening t_1/2. On the other side, negative correlations with γ_thr suggest that the minimal shear rate needed to prevent RBC aggregation indicates a lower aggregate stability.

This may suggest a compensatory mechanism in the rheological behaviour of blood to stabilize flow in the microcirculation associated with tumor growth. Enhanced aggregation does not always indicate pathological processes, and changes in RBC rheology related to PDAC are physiological consequences of disease progression and may be an adaptive mechanism.

The studies showed that RBC aggregation promotes the axial accumulation of RBC in ex vivo investigations in tube flow³¹ and the microcirculation of animals models^32,33 results two-phase flow consist with core of aggregates and cell-free layer and that the magnitude of the effect depends on the intensity of RBC aggregation on microcirculation network. The ability of RBCs to aggregate and the slow-moving plasma layer are most important determining factors affecting hydrodynamic resistance at low flow rates in tubes³⁴ and capillaries³⁵ and the dimension of the effect increases with red cell hyperaggregation. Other authors indicated that RBC aggregation may alter the cell-free layer variability, especially at low shear rates.³⁶ Effect of RBC aggregation on tissue blood flow is not one-way side because hemodynamic consequences of RBC aggregation can affect on decreasing or increasing tissue perfusion as a result of interrelated mechanisms.¹⁴ These mechanisms include axial accumulation of RBC, viscosity and resistance in the cell-free layer and hematocrit of the microvessels or wall shear stress, which depends on RBC aggregation behaviour.

Recent have shown that pancreatic ductal adenocarcinoma (PDAC) is associated with various interrelated biochemical, metabolic, and cytokine disturbances.³⁷ At present, it is difficult to determine which of these factors play a primary role due to the limited data available on the relationship between PDAC and blood rheology disturbances. On the other hand, factors such as hypoxia and HIF-1,³⁸ the promotion of angiogenesis via VEGF upregulation,³⁹ and the dysregulation of the glucose transporter (GLUT1)—which facilitates glucose transport across mammalian cell membranes⁴⁰—are known to be associated with numerous disorders, including PDAC.

It remains an open question whether changes in the PDAC microcirculation and the accompanying hypoxia result solely from microenvironmental shifts that secondarily influence red blood cell (RBC) aggregation. Recent literature indicates that hypoxia or HIF-1 promotes vasculogenic mimicry (VM) in multiple tumor types,⁴¹ including pancreatic cancer.⁴² VM is a process in which cancer cells adopt endothelial characteristics, forming tube-like structures and perfusion channels. This process is considered a novel model for blood vessel formation in aggressive tumors, providing a blood supply for tumor growth through microvascular channels composed of tumor cells.

Conversely, changes in RBC aggregation may result from other comorbidities associated with PDAC and secondarily influence microcirculatory flow. This hypothesis appears to be supported by our findings, which show that tumor extension is associated with RBC hyperaggregability. Specifically, we identified a correlation between RBC aggregation parameters and the extent of the PDAC tumor stage (T).

Conclusion

PDAC is associated with alterations in RBC rheology behaviour. The results indicate a higher tendency for RBC aggregation and aggregate stability in PDAC patients; however, this was not associated with increased RBC rigidity. These hemorheology changes may be the cause of blood flow condition in microcirculation. However, results also indicated that tumor expansion may affect RBC aggregation, but did not identify the reasons for this impact. A compensatory mechanism of rheological behaviour of blood is likely associated with PDAC according to disease progression. Further studies are needed to explore the pathophysiological mechanisms that link PDAC with these aggregation indices.

Study limitations

The present manuscript was designed as an initial study to assess the effects of PDAC on the rheological properties of RBCs. Several limitations must be acknowledged. First, the results cannot be extrapolated to the general population due to the relatively small sample size. Furthermore, we examined the impact of PDAC on RBC rheology only at the time of diagnosis to explore potential relationships with tumor stage, but did not assess patients post-surgery. The impact of pancreatic surgery on rheological changes remains an area for future research. Finally, we did not examine other independent variables (aside from tumor stage) associated with PDAC that may influence RBC behavior. Such an exploration would require a more homogenous research model that incorporates surgical intervention.

Footnotes

Ethical considerations

This study was approved by the Ethics Committee of the Medical University of Silesia (application numbers: KNW/0022/KB1/38/III/14/16/17).

Consent to participate

All participants provided written consent.

Consent for publication

Not applicable

Author contributions

Study design, M.W., K.K,; methodology, M.W., K.K., literature search, D.A. Ch.H., D.E.; data curation, M.W., K.K,; manuscript preparation, M.W., D.A. Ch.H.; supervision, D.E.; project administration, M.W, K.K.

All authors have read and approved the final version of the manuscript.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability statement

All data presented within this manuscript are available upon request from the corresponding author.

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