Abstract
Background/Aim: Tumor interstitial fluid (TIF), a component of the tumor microenvironment, is a valuable source of molecules and substances that help in diagnosis and prognosis of solid tumors. There is still no consensus on the optimal method for collecting TIF. Therefore, this study aimed to evaluate the effectiveness of a new method of collecting TIF in invasive ductal carcinoma (IDC) samples for cytokine interleukin 1β (IL1β) quantification. Materials and Methods: Forty women allowed the collection of TIF using absorbent paper strips during the performance of the core biopsy. The samples were stored at a temperature of –80°C and then analyzed using an enzyme-linked immunoassay. Results: The mean values for IL1β and total protein were 11.39 mg/ml and 2.15 mg/ml, respectively. Conclusion: it was possible to quantify the cytokine IL1β and the total protein concentration present in the tumor tissue through TIF collection with the use of absorbent paper filters, demonstrating the effectiveness of this new method in oncology.
Tumor interstitial fluid (TIF), the fluid bathing tumor cells, is part of the tumor microenvironment and has attracted the interest of the scientific community, especially with the emergence of proteomics technologies as it is a valuable source of molecules and substances such as proteins, cytokines, enzymes and immune cells, which can function as biomarkers and thus provide new insights into tumor biology and aspects related to antitumor therapy (1-5). In this context, it is important to use appropriate methodologies that allow the collection of TIF in a reliable and representative way. To date, there is no consensus on which method should be used to obtain this fluid. Although lymph is accepted as a measure of interstitial fluid, the literature has shown that lymphatic vessels present in tumor tissue seem to be non-functional. In other words, they do not drain any fluid, at least in the central areas of the tumor. In addition, tumor lymphatics cannot be cannulated, making lymph sampling inapplicable (3, 6, 7).
Therefore, there is an increasing need for alternative methods for collecting TIF. Different obtaining methods are available and widely used, such as glass capillary, implanted chambers, implanted wicks, microdialysis, capillary ultrafiltration, tissue centrifugation, and tissue elution. These techniques are generally well accepted and applicable. Some have greater applicability in animal models than in humans, and during collection and processing, they may lead to sampling a mixture of cytoplasmic fluids with interstitial fluid by breaking cells, which may therefore interfere with the results of biochemical analysis (2-4, 8-10).
Existing methodologies have both advantages and disadvantages. The centrifugation method, despite being widely used in several types of cancer, has some disadvantages such as the low amount obtained, which makes it difficult to use in hard tissues, in addition to tissue damage that can occur with increased centrifugation speed. In relation to the tissue elution technique, two main problems have been found: the isolated fluid is usually quite dilute and, during the cutting of tissue fragments, there may be cell damage and the action of proteases. Fluid isolated by the glass capillary technique can be contaminated by intracellular components and proteins, while the implanted chambers method, despite allowing several analyses over time, has been associated with inflammation that can interfere with the fluid content. In the wick implant procedure, the disadvantages are bleeding, inflammation, and cell damage. The main problems of microdialysis are the low uptake of proteins with high molecular weight and the inflammatory response triggered by the insertion of the device into the tissue. In capillary ultrafiltration, selective filtration of proteins and tissue-membrane interface induced by negative pressure within the capillary cause insufficient pressure for protein recovery, which is considered a disadvantage of the technique (2-4,11).
Dentistry investigations, especially in periodontics, commonly use absorbent paper strips to collect gingival crevicular fluid, a serous transudate that can become an inflammatory exudate in active periodontal disease. In this fluid, it is common to find different cell types and inflammatory mediators such as cytokines, proteins, proteases, interleukins (IL) and enzymes. Thus, gingival crevicular fluid is a rich source of markers used in diagnosis and prognosis of periodontal diseases. The collection of gingival fluid using absorbent paper strips is a quick, simple, practical, non-invasive, and low-cost method. The technique consists of gently inserting strips of absorbent paper into the gingival sulcus, waiting approximately 30 seconds, then removing and storing until analysis using different biochemical methods. Extrapolating this method of obtaining tumor fluid to other health science methodologies can significantly contribute to scientific research, diagnosis, prognostic analysis and therapeutic approaches to other diseases (12-14).
Thus, this study aimed to evaluate the effectiveness of collecting TIF in invasive ductal carcinoma samples using absorbent paper strips to quantify the cytokine IL1β.
Materials and Methods
Study population. Forty women from the Department of Gynecology and Obstetrics, of the Clinical Hospital of the Medical School of Ribeirão Preto - University of São Paulo participated in the study, as volunteers, between August and December 2016. The study protocol was approved by the Institutional Ethics Committee (protocol 37030514.7.0000.5419). All participants signed an informed consent form in accordance with the 1964 Declaration of Helsinki (revised in 2013), containing detailed information about the research (objectives, benefits, risks, and discomforts).
Inclusion and exclusion criteria. Inclusion criteria were as follows: Female, 35 years of age or older, imaging tests suggestive of carcinoma, and absence of other systemic diseases besides cancer. Exclusion criteria were: Previous history of breast or other cancer, history of chemotherapy or radiotherapy, mastectomy, pregnancy, immunosuppression, and absence of histopathological diagnosis of breast cancer.
Experimental design. Women with a diagnostic suspicion of breast cancer were invited to participate in the study at the time of the biopsy to confirm the disease. During the biopsy, samples of fluid from the tumor microenvironment were collected. Until then, no antitumor therapy had been instituted, such as chemotherapy, radiotherapy or surgery. The samples were stored at –80°C until measurement of IL1β.
TIF collection. During the core biopsy by the Hospital’s medical team, fragments of the breast tumor tissue were removed with a large-caliber needle attached to a special pistol. This procedure was ultrasound-guided and performed under local anesthesia.
After location of the biopsy area, the skin was sterilized and then the biopsy needle path was anesthetized. Subsequently, a small incision was made in the skin with a scalpel to facilitate the introduction of the biopsy needle. Tissue fragments were obtained by moving the needle inside the lesion at least four times; at each incursion, the needle was removed and the fragment collected in a single tube. After collection of adequate material, local compression and a bandage were applied to avoid bleeding from the biopsy area. This procedure did not require hospitalization.
Before storing each tissue fragment in a formalin-containing tube, the biopsied material was placed on sterile gauze and TIF samples were obtained using Periopaper® strips (Oralflow Inc., Amityville, NY, USA) (Figure 1). This was performed for each fragment, totaling four samples per patient. The absorbent paper strips were gently placed on the tissue fragment for 15 seconds and then only the absorbent part of the four strips were placed in a single sterilized Eppendorf tube. These samples were stored at –80°C until laboratory processing.
Periopaper® strips (Oralflow Inc., Amityville, NY, USA) used in this study.
Biochemical analysis. The test of sample viability was conducted by fluid extraction from the absorbent paper strips was followed by total protein measurement. The samples were thawed and 2 μl of protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA) in a 0.01% Tween 20 solution with 200 μl of buffered saline solution (PBS; Gibco-Invitrogen, Grand Island, NY, USA) were added. The Eppendorf tubes were left for 30 minutes on an orbital plate shaker. Two centrifugations were subsequently carried out at 11,200 × g for 15 min at 4°C, with tube placement exchanged between centrifugations, to collect the supernatant. At the end of extraction, it was possible to obtain approximately 200 μl of TIF in total from the four strips. For analysis, 5 μl of each concentration of the standard curve, a blank and each sample were pipetted into 96-well plates (Costar 3595, Corning Inc., Corning, NY, USA), in duplicate. Then a commercial kit for labeling the proteins present in the wells (Bio-Rad, Hercules, CA, USA) was used. Briefly, 25 μl of reagent A (Bio-Rad DCTM Protein Assay) was added to all wells, followed by 200 μl of reagent B (Bio-Rad Reagent B). The plate was left for 15 minutes in the dark at room temperature for later reading in a μQuant plate reader (BioTek, Winooski, VT, USA). Data reading was performed at 750 nm and results are presented in mg/ml.
An enzyme-linked immunosorbent assay (R&D Systems, Inc., Minneapolis, MN, USA) was used to quantify IL1β. The 96-well plate was incubated overnight and washed twice with phosphate-buffered saline (PBS)/Tween solution and once with PBS. Standard cytokine samples, control samples and TIF samples were added to the wells while the plate was incubated for 1 hour to allow antigen binding by the IL1β-specific capture antibody. The plate was then washed twice with PBS/Tween and once with PBS before adding the detection antibody, and incubated for 2 h. The plate was again washed twice with PBS/Tween and once with PBS. Streptavidin-conjugated horseradish peroxidase was added to the wells and places were incubated for a further 40 minutes to bind to the detection antibody. After washing twice with PBS/Tween and once with PBS to remove horseradish peroxidase conjugate excess, a substrate solution was added to the wells and converted to a detectable product (color signal). Absorbance was recorded on a SpectraMax® Paradigm® multimode microplate detection platform (Molecular Devices, San Jose, CA, USA) at 450 nm. The intensity of the colored product was directly proportional to the concentration of antigen in the original sample. Results are presented in mg/ml IL1β. The limit of detection of this assay was 1 pg/ml and of quantification was 3.9 to 250 pg/ml. All assays were performed according to the manufacturer’s instructions.
Data analyses. All data were tabulated in Excel (Microsoft Corporation, Redmond, WA, USA). Descriptive analyses (mean and standard deviation) were performed with SPSS 22.0 software (Statistical Package for Social Sciences; IBM, Armonk, NY, USA) and the dot-plot graphs were obtained with GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA). The individual patient was considered as the evaluation unit (n=40).
Results
All women had a primary diagnosis of invasive ductal carcinoma and absence of metastasis at the time of collection. Table I shows the descriptive analysis of the variables evaluated. The determination of IL1β and total protein in TIF was possible through the collection of TIF with absorbent paper filters. Figure 2 shows the dot-plot graph of the concentrations of total protein and IL1β in TIF samples of invasive ductal carcinoma.
Descriptive analysis of evaluated variables.
Total protein and interleukin 1β (IL1β) concentrations in samples of tumor interstitial fluid of invasive ductal carcinoma (mg/ml). Data are shown as the mean±standard deviation.
Discussion
The results of the present study show that the collection of breast tumor fluid using absorbent paper strips provided sufficient amounts of TIF for analysis of IL1β in samples of invasive ductal carcinoma.
Generally, during core biopsy, the amount of tumor sampled is restricted due to the patient’s anatomical and pathophysiological conditions, which requires complementary measures to assist histopathological examination, final diagnosis, evaluation of the prognosis, and the decision-making of antitumor therapy to be adopted. In this regard, the method proposed in the present study can be of great value in assisting methodologies already used by the scientific community, as the technique is simple, non-invasive, low cost and allows different biochemical analyses. Moreover, this type of biopsy grants access to the central parts of tumors, thus providing valuable information from the tumor microenvironment.
In 2016, Espinoza et al. profiled cytokines in breast TIF obtained by centrifugation and compared them to serum and normal interstitial fluid, using Multiplex Luminex xMAP technology (15). The authors assessed several cytokines significantly expressed in TIF, including IL1β, IL7, IL10, IL13, IL1RA and IL12 when compared to normal interstitial fluid. In addition, positive correlation was found for IL1β, IL7 and IL10 between TIF and serum samples. These results have important implications for cancer immunotherapy research. Through enzyme-linked immunosorbent assay analysis, Haslene-Hox et al. evaluated biomarker concentrations in the ovarian tumor microenvironment using TIF obtained by centrifugation and then compared them with plasma levels. The results showed significantly increased biomarkers in the TIF (16). In addition, microRNAs have also been detected in the interstitial fluid of breast tumors to identify potential diagnostic and prognostic markers (17).
Knowledge of the level of inflammatory markers, including IL1β, in the tumor microenvironment and other diseases can contribute to selection of therapeutic strategies (17, 18). This cytokine has been commonly related to the immunopathogenesis of breast cancer (19-21). In this context, members of the IL1 family (IL1α and IL1β) are often expressed in breast cancer cell lines as well in the tumor microenvironment. These cytokines act by autocrine and paracrine mechanisms that contribute to pro-tumorigenic activities such as angiogenesis, proliferation and local tumor invasion. Moreover, the promotion of tumor growth and metastasis by inducing several pro-metastatic genes has been observed (19-21). Interleukins IL1β and IL6 also stimulate proliferation of malignant breast cells by activating catalytic enzymes in the tissue, leading to estrogen synthesis. This type of cancer is hormone-dependent, which favors its development (19-22).
Recently, an in vitro and in vivo study by Eyre et al. observed that IL1β plays an essential role in breast cancer metastasis to bone (23). Hence, the authors provided strong arguments to consider the use of signaling pathway inhibitors involving IL1β as an adjuvant therapeutic strategy and thus prevent bone metastasis. The findings of Tulotta et al. are aligned with the data mentioned above as the authors agreed that high levels of IL1β are associated with the worst prognosis of breast cancer due to their involvement in pro-metastatic mechanisms (24).
High incidence and mortality of breast cancer require efforts to identify and understand its biological behavior. In addition, fast, practical, and low-cost new antitumor techniques may be an adjuvant to current tumor treatment strategies to positively contribute to tumor diagnosis, prognosis and therapy.
When comparing commonly used techniques for obtaining TIF, there is no consensus on the best collection technique. However, interstitial fluid analysis is extremely important for oncology. Wiig et al. isolated interstitial fluid from breast tumors in mice by low-speed centrifugation. The authors concluded that this is a reliable and straightforward method to provide information not found in plasma (25). In 2004, Celis et al. extracted TIF from small fragments of invasive ductal carcinoma in 16 women after mastectomy, without any other prior antitumor therapy (26). The results showed that there were more than 1,000 proteins in the fluid that might be potential biomarkers for diagnosis or a target for therapeutic interventions. Stone et al. used an ultrafiltration catheter for TIF collection from patients with head and neck squamous cell carcinoma and identified 525 relevant proteins (27). The authors highlighted significant differences in the cancer fluid proteome when compared with other body fluids. In 2011, Haslene-Hox et al. described a new TIF collection technique by centrifugation in ovarian tumors (28). In a proteomic approach, 124 proteins were detected in the plasma against 769 tumor specifics proteins in the isolated interstitial fluid (six times higher concentration), which can serve as an improved substrate for proteomics. The authors underlined the relevance of the results for diagnostic, therapeutic, and prognostic monitoring purposes.
Recently, a comparative study evaluated the two most commonly used methods, elution and centrifugation, in cutaneous squamous cell carcinoma biopsy samples. The authors found a higher protein concentration in the centrifuged samples and considered this technique as the best choice to study the TIF proteome in skin biopsies. However, both methods provided important information on TIF composition in the evaluated tissue (29).
Some considerations must be made in relation to the proposed method. Further studies are needed to assess whether TIF collected by absorbent paper strips is representative of fluid free from interference from plasma and intracellular proteins. For future perspectives, it is important to compare the tumor fluid with fluid from normal tissues and serum, in addition to expanding the analysis to other biomarkers and testing different detection methods.
In this regard, the present study provides preliminary information on the use of absorbent paper strips as a the new TIF collection technique. This has unveiled a promising resource for complementary analyses of the tumor microenvironment, especially for determination of cytokine levels. Future studies are needed to validate this method.
Conclusion
This study showed that it is possible to quantify a biomarker (IL1β) and the total protein concentration in fluid from tumor using an absorbent paper filter for collection. Further studies are needed to investigate the usefulness of the fluid collection method in detecting several other cytokines such as IL1β, as well as proteins and other molecules, as these biomarkers may be present in higher concentrations at the tumor site when compared to plasma. Assessment of TIF may possibly allow the identification of substances that can be used in the early detection and follow-up of disease, thus expanding the available methodologies.
Acknowledgements
The Authors thank Milla Sprone Tavares Ricoldi for help in biochemical analysis.
Footnotes
Authors’ Contributions
Conceptualization: Felipe Dantas, Francisco Jose dos Reis and Daniela Palioto. Data curation: Felipe Dantas and Pedro Henrique Silva. Formal analysis: Pedro Henrique Silva, Mario Junior and Sérgio Luís de Souza. Investigation: Hélio Humberto Carrara, Francisco Jose dos Reis and Daniela Palioto. Methodology: Felipe Dantas, Francisco Jose dos Reis and Daniela Palioto. Project administration, Francisco Jose dos Reis and Daniela Palioto. Resources: Hélio Humberto Carrara, Francisco Jose dos Reis, Mario Junior, Sérgio Luís de Souza and Daniela Palioto. Supervision: Sérgio Luís de Souza and Daniela Palioto. Validation: Hélio Humberto Carrara and Mario Junior. Visualization: Felipe Dantas, Pedro Henrique Silva, Hélio Humberto Carrara and Daniela Palioto. Writing – original draft: Felipe Dantas and Pedro Henrique Silva. Writing – review and editing: Hélio Humberto Carrara, Francisco Jose dos Reis, Mario Junior, Sérgio Luís de Souza and Daniela Palioto.
Conflicts of Interest
The Authors declare no conflicts of interest exist.
- Received November 21, 2021.
- Revision received January 14, 2022.
- Accepted January 17, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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