Platelet-Rich Plasma Inhibits Breast Cancer Proliferation

Abstract

Background:

Platelet-rich plasma (PRP) helps promote wound healing, but it is unclear whether it stimulates breast cancer cell proliferation, which restricts its application in breast cancer patients. This article explored the effect of PRP on breast cancer cell proliferation through preclinical experiments.

Method:

We cultivated MDA-MB-231 breast cancer cells with PRP in vitro. Subsequently, we employed Cell Counting Kit-8 (CCK-8) assays to assess their proliferation ability, wound healing assays to evaluate their migration ability, and Transwell assays to detect their invasion ability. Mouse breast cancer 4T1 cells were subcutaneously inoculated into nude mice. After tumor formation, PRP was injected around each tumor. Tumor size was measured. After 15 days, the tumors were surgically removed. Immunohistochemistry was used to detect key proteins involved in proliferation and apoptosis.

Results:

PRP inhibited the proliferation, migration, and invasion of MDA-MB-231 in vitro. After PRP was injected around tumors in nude mice, tumor growth slowed, Ki67 and phospho-histone H3 (pHH3) expression decreased, and Caspase 3 and Poly (adenosine diphosphate-ribose) polymerase 1 (PARP1) expression increased.

Conclusion:

The PRP inhibits the proliferation of breast cancer MDA-MB-231 and 4T1 cells.

Introduction

Breast cancer is currently the most common malignant tumor in women. In 2020, more than 2.26 million new cases of breast cancer were reported in 185 countries/regions worldwide. ¹ Surgery is the primary treatment for breast cancer, but it also comes with numerous complications, such as seroma, non-healing wounds, and pain. These complications can impact the progress of postoperative adjuvant treatment and diminish the quality of life for patients.^2,3

Platelet-rich plasma (PRP) is a platelet concentrate extracted from whole blood. It contains high concentrations of platelets, white blood cells, fibrin, and various growth factors, including platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), and vascular endothelial growth factor (VEGF). PRP is a safe and effective treatment to enhance wound healing and is widely used in orthopedics, dermatology, gynecology, plastic surgery, and other fields.^4-9 Calisir et al ¹⁰ applied PRP to the surgical wounds of rats undergoing mastectomy and axillary lymph node dissection, which suppressed the formation of seroma, enhanced the formation and growth of new blood vessels, and increased fibrocyte density. Eichler et al performed PRP treatment on 35 breast cancer patients after removing the subcutaneous venous access device. After a median follow-up of 45 months, there was no cancer recurrence in the scar area of the subcutaneous venous access device. ¹¹

It can be seen that PRP may be a potential measure to promote wound healing after breast cancer surgery. However, growth factors and cytokines also promote the progression of cancer by stimulating tumor cell proliferation, inducing epithelial-mesenchymal transition, maintaining the stem cell characteristics of tumor cells, facilitating tumor angiogenesis, and aiding tumor cells in evading immune responses. ¹² If cancer tissue remains in the wounds after breast cancer surgery, will using growth factor-rich PRP to treat these wounds promote cancer cell proliferation and metastasis? This is a cause for concern. ¹³ In this study, an in vitro culture model of breast cancer and an in vivo tumor model of nude mice were constructed, and PRP was used to treat the tumor to explore the effect of PRP on breast cancer proliferation.

Materials and Methods

Preparation process for human PRP

This study was conducted at the First Hospital of Jiaxing in China from February 2021 to December 2023. The experiment conformed to the guidelines set by the Declaration of Helsinki, and written informed consent was obtained from the volunteer. Inclusion criteria for volunteers: female; aged over 18 years; absence of any diseases indicated by the results of the full-body examination; willingness to participate in this study. Exclusion criteria: unclear surface veins and difficulty in drawing blood. Thirty-six milliliters of venous blood was extracted from one healthy adult female volunteer using a PRP preparation kit (WEGO, Type I, Shandong WEGO New Life Medical Devices Co., Ltd.). The anticoagulant included in the kit was added to the blood. The blood was then centrifuged twice according to the instructions to obtain PRP. Specifically, the first centrifugation speed was 4000 rpm for 10 minutes, and the red blood cells at the bottom were discarded after centrifugation. The second centrifugation speed was 4000 rpm for 10 minutes, and the supernatant was discarded after centrifugation to obtain PRP. To ensure the biological activity of PRP, subsequent experiments were performed within 30 minutes after preparing PRP.

In vitro cell culture and grouping

MDA-MB-231 human breast cancer cells (Procell Life Science & Technology Co., Ltd.) were divided into 2 groups and placed in the lower chamber of a Transwell chamber (CORNING, 354480). The culture medium consisted of Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, 11995065) + 10% fetal calf serum (Gibco, 10099141) + 1% penicillin‒streptomycin double antibody (Gibco, 15140122). Experimental group: mix the prepared human PRP (0.2 mL) with culture medium and put it into the upper chamber of the Transwell chamber. Control group: place the culture medium into the upper chamber of the Transwell chamber (Figure 1A). They were cocultured at 37°C with 5% CO₂. Afterward, the cells in the lower chamber were collected for the next experiment. This is a modified cell culturing method designed to facilitate high-quality experiments. We will elaborate on the rationale for employing this method in the “Discussion” section.

Figure 1.

PRP inhibits proliferation, migration, and invasion of MDA-MB-231 in vitro. (A) Coculture mode. (B) PRP inhibited cell viability. (C) PRP inhibited cell migration. (D) PRP inhibited cell invasion.

Cell Counting Kit-8 assay

Cells were collected at 0, 12, 24, 48, and 72 hours when cocultured with PRP. They were then seeded in a 96-well plate, with 5 duplicate wells in each group. Next, 10 μL of Cell Counting Kit-8 (CCK-8) reagent (BBI Life Sciences, E606335-0500) was added to each well. The plate was incubated in a cell culture incubator in the dark for 1 hour, and the absorbance at 450 nm was measured using a microplate reader. The culture medium consisted of DMEM (Gibco, 11995065) + 10% fetal calf serum (Gibco, 10099141) + 1% penicillin‒streptomycin double antibody (Gibco, 15140122).

Wound healing assay

Cells were cocultured with PRP for 48 hours. Afterward, they were collected and seeded into 6-well plates at a density of 3 × 10⁵ cells per well, with 2 duplicate wells per group. The culture medium consisted of DMEM (Gibco, 11995065) + 10% fetal calf serum (Gibco, 10099141) + 1% penicillin‒streptomycin double antibody (Gibco, 15140122). A microinjection tip was used to draw lines along the central axis of the wells. The cells that were detached during wounding were washed away with phosphate-buffered saline (PBS). The culture was maintained, and images of the wound were taken at 0, 24, and 48 hours and visualized under a microscope; the migration distance of the cells was measured at 3 different positions. Migration rate in Nh = (scratch width at 0 hour—scratch width at Nh)/scratch width at 0 hour.

Transwell assay

Cells were cocultured with PRP for 48 hours. Afterward, they were collected and seeded in the upper chamber of the Transwell insert (Corning, 354480, pre-coated with Matrigel). Then serum-free DMEM (Gibco, 11995065) was added to the upper chamber. Next, 600 µL of medium containing 20% FBS was added to the lower chamber, and the cells were cultured in the incubator for 24 hours. Then, the old culture medium was discarded, and the cells were washed twice with PBS. The cells were fixed with formaldehyde (BBI Life Sciences, A500684) for 30 minutes. Then, the chamber was released and allowed to air dry. The cells were stained with 0.1% crystal violet (BBI Life Sciences, E607309) for 30 minutes. The nonmigrated cells were gently removed from the upper chamber using a cotton swab, and the cells were washed 3 times with PBS. Three fields of view were randomly selected, and the cells were observed under a microscope and counted.

Preparation process for mouse PRP

The experiment was conducted in accordance with the ARRIVE guidelines (https://arriveguidelines.org), and steps were taken to minimize the pain and discomfort experienced by the animals during the experiment. ¹⁴ Six BALB/c mice (Beijing HFK Bioscience Co., Ltd.) were used to collect blood from the eyeballs and add anticoagulants. Centrifuge at 300 × g for 10 minutes, and the supernatant should be collected. The supernatant was transferred to a new tube and centrifuged at 3000 × g for 10 minutes. The supernatant was discarded, and the PRP was obtained. To ensure the biological activity of PRP, follow-up experiments were performed within 30 minutes of preparing PRP.

Nude mouse tumor model

The experiment was conducted in accordance with the ARRIVE guidelines (https://arriveguidelines.org), ¹⁴ and steps were taken to minimize the pain and discomfort experienced by the animals during the experiment. The experiments were conducted in an animal experiment center that met Specific Pathogen-Free (SPF) standards. The mice were fed for 1 week after arriving at the center, and the experiment was started only after confirming that the physiological activity of the mice was normal. Twelve BALB/c nude mice (Beijing HFK Bioscience Co., Ltd.), 6 weeks old, healthy, and wild type, were inoculated subcutaneously with 4T1 cells (Chinese Academy of Sciences) under the armpits of the forelimbs, and the number was 1×106/mouse. The sample size was determined based on statistical significance, resource limitations, reproducibility, animal welfare, and results from a preliminary experiment (6 mice). The RAND() function in Excel software was used to generate random numbers between 0 and 1. Subsequently, the mice were randomly allocated to either the experimental group or the control group in a 1:1 ratio. The 2 groups were housed under the same conditions, and the experimenters and analysts were blinded to the grouping to reduce observation and analysis bias. The inoculation site and the condition of the mice were observed every other day after inoculation. After successful tumor-bearing was confirmed on the seventh day of inoculation, 6 mice (experimental group) were injected with mouse PRP at 4 points around the tumor (the platelet count at each injection point was 1 × 10⁷ with 10 μL), and 6 mice (control group) were injected with the same volume of normal saline in the same way. The mice’s activity level, eating habits, breathing, hydration status, and so on were observed every other day. Their body weights were measured, and the length and diameter of the tumor were assessed using a vernier caliper. The primary outcome measure was the length of the tumors. Humane endpoints for mice include body weight loss of more than 20%, decreased activity, decreased appetite, abnormal body posture, difficulty breathing, tumor weight exceeding 10% of the mouse’s body weight, tumor diameter exceeding 20 mm, ulceration, infection, and necrosis of the skin on the tumor surface. On the 15th day, the mice were euthanized, and the tumors were surgically removed. The euthanasia process was as follows: place the mice in a sealed container, inject low-concentration CO₂ (starting from 0%) into the container, and increase the CO₂ concentration by about 20% per minute. Monitor the mice’s vital signs. When the mice lose their ability to move, their pupils dilate, and their breathing ceases, continue observation for another 5 minutes to confirm the mice’s death. The length, diameter, and weight of the tumors were measured. The tumor volume = length × diameter × diameter × 0.52. The tumors were then fixed in tissue fixative (ServileBio, G1101-15).

Hematoxylin and eosin (HE) staining

The tumor tissue was placed in an automatic tissue dehydrator (DIAPATH, Donatello) for dehydration. The reagents used for dehydration include 4% paraformaldehyde (ServiceBio, G1101-15), 75% to 100% ethanol (Sinopharm Chemical Reagent, 100092183), xylene (Sinopharm Chemical Reagent, 1330-20-7), and paraffin (Sinopharm Chemical Reagent, 8012-95-1). Then embedded in paraffin. The paraffin-embedded tissue was then cut into 3 to 5 μm slices and dewaxed with toluene (Sinopharm Chemical Reagent, 108-88-3), xylene, 100% to 70% ethanol, and distilled water. Stain the sections with hematoxylin for 1 minute and then stain with eosin for 1 minute (ServiceBio, G1076-500). ¹⁵

Immunohistochemistry

Tumor sections were deparaffinized and hydrated. H₂O₂ (3%) blocks and inactivates endogenous peroxidase. The sections were boiled in sodium citrate buffer for 3 minutes, and 10% goat serum was added dropwise to block nonspecific sites. Primary antibody (Table 1) was added and incubated overnight at 4°C. The secondary antibody enhancer (BSA, Servicebio, G5001) and secondary antibody (HRP-conjugated Affinipure Goat Anti-Mouse IgG [H + L], Proteintech, SA00001-1) were added. The chromogenic reagent was added for 1 to 2 minutes, and the slides were soaked in hematoxylin for counterstaining, dehydrated, and sealed.

Table 1.

Primary antibody.

Primary antibody	Manufacturer	Number
Anti-Caspase 3 Rabbit pAb	Servicebio	GB11009
Anti-Ki67 Rabbit pAb	Servicebio	GB111499
Anti-PARP1 Rabbit pAb	Servicebio	GB111500
Histone H3 (phospho Ser10) Polyclonal Antibody	Immunoway	YP0129

For immunohistochemistry scoring, 5 nonoverlapping fields of view were randomly selected under higher magnification, and the staining intensity and proportion of positive cells were scored as follows. Staining intensity: 0 indicates no cell staining, 1 indicates mild staining, 2 indicates moderate staining, and 3 indicates strong staining. Proportion of positive cells: 0 indicates no cell staining, 1 indicates that the proportion of positive cells is ⩽ 25%, 2 indicates that the proportion of positive cells is 26% to 49%, and 3 indicates that the proportion of positive cells is ⩾ 50%. ¹⁶ Scores were independently assessed by 2 experienced pathologists. For accurate and consistent results, they collaborated to review the sections and engage in discussions before providing their findings. The final result was determined by multiplying the staining intensity score by the positive cell proportion score.^17-19

Statistical analysis

The data were processed using IBM SPSS Statistics for Windows, Version 22.0 (IBM Corp., Armonk, NY, USA), and GraphPad Prism for Windows, Version 8.0.2 (GraphPad Software, LLC, Boston, MA, USA). A normality test was conducted to assess whether the data followed a normal distribution. For data that was normally distributed, the mean ± standard deviation was used, and an independent sample t-test was performed. In cases where the data did not adhere to a normal distribution, quartiles were employed, and a non-parametric test was conducted. All statistical tests were 2-sided, and the differences were considered statistically significant at P < .05.

Results

PRP inhibits proliferation, migration, and invasion of MDA-MB-231 in vitro

The MDA-MB-231 cells cultured in vitro were divided into 2 groups: the control group without PRP and the test group cocultured with PRP. The CCK-8 experiment indicated a statistically significant difference in cell viability between the 2 groups at 12 hours after coculture (except at 48 hours), with PRP leading to a reduction in cell viability (Figure 1B). The wound healing assay indicated that PRP reduces cell migration (Figure 1C). The Transwell assay demonstrated that PRP reduces cell invasion (Figure 1D; Supplemental material Table 1).

PRP inhibits the growth of breast cancer in nude mice

We injected PRP around the 4T1 cells in nude mice and used an equal volume of normal saline as a control. All mice completed the experiment and entered the data analysis phase. No adverse events occurred during the experiment. As a result, the tumor in the PRP group exhibited slower growth (Figure 2A), and the tumor volume (Figure 2B) and weight on the 15th day were significantly smaller (Figure 2C; Supplemental material Table 1).

Figure 2.

PRP inhibits the growth of breast cancer in nude mice. (A) Tumors in 2 groups. (B) The tumor volume in the PRP group was smaller. (C) The tumor weight in the PRP group was lighter.

PRP inhibits breast cancer proliferation and promotes apoptosis

The tumors were sectioned and stained with HE. Observation under a microscope showed that tumors had formed in both groups (Figure 3A). Ki67 and phospho-histone H3 (pHH3) levels in the tumors of the PRP group were lower than those in the control group (Figure 3B and C), whereas Caspase 3 and poly ADP-ribose polymerase 1 (PARP1) levels were higher (Figure 3D and E; Supplemental material Table 1).

Figure 3.

PRP inhibits proliferation and promotes apoptosis. (A) The HE staining showed that tumors had formed in both groups. (B) Ki67 in the PRP group was lower. (C) pHH3 levels of the PRP group were lower. (D) Caspase 3 in the PRP group was higher. (E) PARP1 levels in the PRP group were higher. 200×, *P < .05.

Discussion

PRP was obtained through double centrifugation, which is conducive to obtaining high-quality PRP. ²⁰ In the preliminary experiments conducted prior to this study, PRP was mix-cultured with MDA-MB-231 cells in a well plate. A large number of platelets could be seen attached to the surface of the cancer cells under high magnification. This may hinder the exchange of substances between cells and the external environment, thereby decreasing cell viability. Therefore, we innovated the culture method by specifically placing cells in the lower chamber of the Transwell chamber and PRP in the upper chamber. This coculture method can maximize the contact between active substances in PRP and cells while reducing platelet adhesion. Chiu et al believed that PRP has a gelation effect, which can impact the outcomes of cell culture. They developed a 3-chamber coculture device to mitigate the gelation effect. The device consists of 3 subchambers that are used to separate PRP and cells. These chambers are connected by culture medium. This is similar to the mechanism of our coculture method. ²¹

Our research suggests that PRP inhibits the proliferation, migration, and invasion of breast cancer. Andrade et al ²² added PRP at concentrations ranging from 2.5% to 10% to primary tumor cells of patients with luminal A, luminal B, and HER2 + breast cancer, which were cultured in vitro. The results of their study showed that the addition of PRP promoted cell proliferation, which is different from our findings. The reason may because that the PRP concentrations in the 2 experiments were different. And they did not report the effect of higher concentrations of PRP.

The reason why PRP inhibits breast cancer cell proliferation is still unclear. Due to the limited availability of research reports on high concentrations of PRP in cancer cells, we conducted a review on the role of PRP in normal cells. For example, Graziani et al’s ²³ research on osteoblasts and fibroblasts in vitro showed that low-concentration PRP significantly increased proliferation, while high-concentration PRP was not as effective as low-concentration PRP. Berndt et al’s research on fibroblasts found that the proliferative effect of PRP is a dose-dependent bell-shaped curve. The optimal concentration of PRP to promote fibroblast proliferation is 20%. When the concentration is too high, the cell proliferation ability decreases. ²⁴ Mooren et al ²⁵ also discovered a similar phenomenon through research on osteoblast-like cells in vitro. These studies also suggest that PRP has a concentration-specific effect on cell proliferation. There is an optimal concentration of PRP, and concentrations that are too high or too low are not conducive to cell proliferation. As PRP is a mixture of platelets, white blood cells, and various growth factors, it may have different comprehensive effects on cell proliferation depending on the concentration conditions. High-concentration PRP, for example, contains a higher number of leukocytes, which can lead to a decrease in the rate of cell proliferation. This is because leukocytes release proinflammatory substances during the degranulation process. ²⁶

We performed peritumoral injection of PRP to study its effect on breast cancer in nude mice. Then, we found that tumors in the PRP group grew more slowly and weighed less. The expression of Ki67 and pHH3 was lower in tumor tissues, while the expression of Caspase 3 and PARP1 was higher. Ki67 and pHH3 are markers of cell proliferation, and their decrease indicates a low level of cell proliferation.^27,28 Elevated levels of Caspase 3 and PARP1 indicate increased apoptosis.^29,30 Therefore, we speculate that PRP may inhibit the proliferation of breast cancer by promoting cell apoptosis. Similar studies, such as the one conducted by Barbieri et al, involved the application of PRP to wounds after removing tumors from mice that were carrying human fibrosarcoma. The study found that PRP was able to slow the growth of tumors. ³¹ The mechanisms by which PRP affects tumor growth in the body are complex. In addition to the roles of growth factors and leukocyte degranulation in the effects of PRP on tumors, the influence of the immune system must also be considered. For example, Dias et al conducted a study on the role of PRP and Bacillus Calmette-Guérin (BCG) in a rat model of non-muscle-invasive bladder cancer. They concluded that the growth factors found in PRP stimulated the immune system, leading to the inhibition of cancer progression. ³²

Our study suggests that PRP inhibits breast cancer proliferation both in vivo and in vitro. Therefore, we speculate that the application of PRP to wounds after radical resection of breast cancer patients will not increase the risk of tumor recurrence. In fact, there have been some relevant clinical studies. For example, Berná-Serna et al used PRP to treat the wounds of 8 breast cancer patients, and the wounds healed smoothly. During more than 4 years of follow-up, the patients did not experience any tumor recurrence. ³³ Eichler et al reported that PRP was injected into the site of sentinel lymph node biopsy in 163 breast cancer patients between 2015 and 2018. They were followed up for 30 months, and it was found that PRP did not lead to an increase in the local recurrence rate. In fact, PRP showed a tendency to reduce surgical complications. ³⁴

Limitations

This study did not analyze the specific components in PRP, nor did it elucidate the mechanism by which PRP inhibits breast cancer proliferation. We used MDA-MB-231 cells in in vitro experiments and 4T1 cells in animal experiments. These are breast cancer cell lines known for their robust proliferation, migration, and invasion capabilities, representing triple-negative breast cancer in clinical settings (a highly aggressive subtype of breast cancer, constituting approximately 15% of all breast cancer cases). Due to budget constraints, we were unable to incorporate more cell lines in our in vitro and in vivo experiments.

Conclusions

We have demonstrated that PRP inhibits the proliferation of breast cancer MDA-MB-231 and 4T1 cells. Next, we will conduct studies on additional breast cancer cell lines and explore the molecular mechanism through which PRP inhibits breast cancer proliferation.

Supplemental Material

sj-docx-1-onc-10.1177_11795549241298978 – Supplemental material for Platelet-Rich Plasma Inhibits Breast Cancer Proliferation

Supplemental material, sj-docx-1-onc-10.1177_11795549241298978 for Platelet-Rich Plasma Inhibits Breast Cancer Proliferation by Chao Han, Caiping Chen, Ning Lu, Li Xue, Dan Xing, Wanxin Wu, Wang Li and Xiang Lu in Clinical Medicine Insights: Oncology