Macrophage polarization in dynamics of Lewis lung carcinoma growth and metastasis
Summary. Aim: To assess the functional state of macrophages based on various manifestations of their activity at the different stages of metastatic tumor growth in C57Bl mice. Materials and Methods: On days 7, 14, 21 and 28 after Lewis lung carcinoma transplantation to C57Bl mice, macrophages from various anatomic sites were isolated and tested on their cytotoxicity, metabolic activity, NO production and arginase activity. Results: In the populations of peritoneal and splenic macrophages, on days 7 and 21 of tumor growth antitumor (M1) cells prevailed while on days 14 and 28 tumor-promoting (M2) macrophages predominated. In the population of lung macrophages, cells with M1 phenotype were in the majority in the early stages of tumor growth. On days 21 and 28, M1 cells were gradually substituted by cells exhibiting M2 phenotype. This shift correlated with metastasis to lungs. Conclusion: Lewis lung carcinoma growth is accompanied by the gradual change in macrophage polarization from antitumor (M1) towards tumor-promoting (M2) type. These changes were more evident in population of lung macrophages and correlated with the parameters of metastasis.
Submitted: February 28, 2021.
*Correspondence: E-mail: email@example.com
Abbreviations used: Arg — arginase; CTA — cytotoxic activity; CTAI — cytotoxic activity index; IL — interleukin; LLC — Lewis lung carcinoma; Mph — macrophages; NBT — nitroblue tetrazolium; NO — nitric oxide; OD — optical density.
Cancer progression is accompanied by the systemic effects in the host, with the immune system being involved [1, 2]. Tumor tissue is not homogeneous — it consists of different cell types: malignant (forming the main part of tumor or metastases) and normal cells of the body (tumor microenvironment), making tumor stroma. Tumor microenvironment also includes immune cells especially those constituting innate immune system — macrophages (Mph), neutrophils, natural killer cells. Mph representing the considerable part of immune cells in tumor microenvironment affect tumor-microenvironment relations during all the stages of tumor development. Nevertheless, their role in cancer is ambiguous .
Mph belong to the mononuclear phagocyte system. Nowadays, the term mononuclear phagocyte system refers to the family of cells of myeloid origin comprising bone marrow progenitors, blood monocytes, tissue Mph and dendritic cells [4–6]. Mph represent a big heterogeneous cells population. Depending on the tissue of residence and the surrounding microenvironment, Mph can exhibit different properties [7, 8]. Current Mph classification considers their anatomical niches of residence/functioning and activation signals, which allows dividing them into subtypes . In general, these cells are divided into two populations: tissue residential and monocyte-derived infiltrating Mph [10, 11]. Tissue Mph are believed to be the progeny of the myeloid bone marrow cells, which give rise to circulating blood monocytes through consecutive stages of differentiation. The latter constantly migrate into peripheral tissues, where they differentiate into Mph . However, by now it is shown that the majority of tissue-resident Mph population arises in embryonic development (originate from erythromyeloid precursors of the yolk sac) and self-maintains due to local proliferation rather than monocyte extravasation . In particular, it was demonstrated that Mph resident in brain, lungs, liver, abdominal cavity and spleen are not differentiated from monocytes, but originate from embryonic precursors [11, 14]. On the other hand, classical monocytes (proinflammatory, Ly6Chigh monocytes) serve as a source for infiltrating Mph, which function in various pathological settings including cancer [15, 16].
Today, the origin of resident Mph subsets in different compartments of the body is still the subject of discussion. It does not seem to be always correct to classify these cells according to M1 and M2 (respectively, classically activated, proinflammatory and alternatively activated, anti-inflammatory) cell phenotypes . While the mechanisms of M1 and M2 Mph activation and polarization are well described, the problem of functional state and polarization of resident Mph subsets in different anatomical niches during cancer progression remains to be elucidated. We have previously described the changes in the functional activity of Mph isolated from peritoneal cavity, spleen and tumor of mice bearing Ehrlich carcinoma . The aim of this research was to assess the functional state of Mph based on various manifestations of their activity (cytotoxic and metabolic activity, nitric oxide (NO) production, arginase (Arg) activity) at different stages of metastatic Lewis lung carcinoma (LLC) growth.
MATERIALS AND METHODS
Animals. The male C57Bl mice 2–2.5 month old weighting 19–20 g from the breeding facility of R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology were used in the study. The experiments were performed in accordance with standard international rules on biologic ethics, the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes  and the regulations approved by Institutional Animal Care and Use Committee.
Experimental tumor. The strain of metastasizing LLC used in the experiment  was kindly granted by the R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology Bank of Cell Lines from Human and Animal Tissues.
Design of experiment. On day 0, C57Bl mice (n = 20) were injected with LLC cells in the hind foot (4 × 105 cells/mouse) and followed till day 28 after tumor transplantation. The frequency (%) and latent period of tumor appearance, the tumor volume (mm3), proportion of mice with metastases (%), number and volume of metastases (mm3) were recorded . Immunological testing was performed on days 7, 14, 21 and 28 after tumor challenge. Mph from different anatomical niches were obtained and functionally tested. Peritoneal, spleen and lung Mph were assessed for NO production and Arg activity. Peritoneal and spleen Mph were tested for the metabolic and cytotoxic activities too. Intact mice of the same strain, age and sex (n = 12) were used as the control group (referred to as the intact control).
Isolation of Mph. Peritoneal and spleen Mph were isolated as described in . In order to obtain lung Mph, aseptically removed lungs were homogenized with Potters homogenizer in medium 199 supplemented with 10% of bovine serum (Sigma, USA). Cell suspensions were centrifuged (550 g, 10 min) and resuspended in medium 199. After counting, 1 × 106 cells were placed on flat-bottomed plates and cultured for 2 h (37 °C, 5% CO2, 100% humidity). Thereafter, non-adherent cells were removed, and the adherent cells were washed twice with 0.9% NaCl and taken for further investigation.
Cytotoxic activity (CTA) assay. CTA was determined by МТТ assay . LLC cells were used as a target. In brief, target cells (2 × 104 cell/well) in RPMI 1640 medium supplemented with 10% fetal bovine serum (all reagents from Sigma, USA) and antibiotics were placed in a flat-bottom 96-well plates where Mph (4 × 105 cell/well) were adhered beforehand, and incubated for 18 h at 37 °С in a 100% humidity atmosphere with 5% СО2. Control wells contained target cells or adhered Mph only. Then 0.01 ml of МТТ solution/well (5 mg/ml, Sigma, USA) was added, and incubation continued for 2 h. Then the plates were centrifuged (550 g for 15 min) and washed twice with 0.9% NaCl solution. After that, 0.12 ml of 2 M КОН and 0.14 ml of dimethyl sulfoxide (50% solution) were added into each well. Optical density (OD) was measured at λ = 545 nm vs λ = 630 nm using a microplate ELISA reader (StatFax-2100, USA). Each sample was done in triplicate. CTA index (CTAI, %) was calculated by the formula:
CTAI = [1 – (ODmph+tc – ODmph)/(ODtc – ODblank)] • 100%,
where ODmph — optical density (OD) in wells containing only adhered Mph; ODtc — OD in wells containing only tumor cells; ODmph+tc — OD in wells wherein tumor cells and Mph where incubated; ODblank — OD in wells with the culture medium only.
Metabolic activity examination. Mph metabolic activity was assessed by nitroblue tetrazolium (NBT) test as described previously with some modification . In brief, Mph (1 × 106 cell/ml, 0.2 ml/well) were incubated with 0.2% NBT solution (0.02 ml/well, Sigma, USA). After incubation (1 h, 5% СО2, 37 °С), the plates were washed two times with 0.9% NaCl solution. The 2 M KOH solution (0.06 ml/well) and 50% DMSO solution (0.07 ml/well) were used to dissolve diformazan granules. OD was measured at λ = 630 nm with the use of a micro ELISA reader (StatFax-2100, USA). Each sample was done in triplicate. The results are presented as OD units (OU).
NO production was measured by standard Griess reaction . In brief, cell suspensions (2 × 106 cell/well) were placed in a volume of 200 μl in 96-well flat-bottom tissue culture plates and cultured for 24 h. Each cell sample was investigated in duplicate. At the end of the incubation period, supernatants were collected and NO production was assessed by the accumulation of nitrite (as stable metabolite of NO) by Griess reaction. An aliquot of culture supernatant (100 μl) was mixed with an equal volume of Griess reagent (Acros Organics, Belgium) and incubated for 1 h at room temperature in the dark. The reaction products were colorimetrically quantified at λ = 550 nm. The standard curve plotted by the results of measurements of the solutions containing known concentration of NaNO2 was used for converting the absorbance to micromolar concentrations of NO expressed in μM NO2 per 106 cells.
Arg activity was determined based on urea measurement [24, 25]. Mph were lysed by double freezing and melting. Then 50 μl of 50 mM Tris-HCl (рН 7.4) and 10 μl of 50 mM MnCl2 were added to each sample. Samples were incubated at 56 °C for 10 min, and upon addition of 100 μl of 0.5 M L-arginine (pH 9.7) further heated for 30 min (37 °С). The reaction was stopped with 800 μl of acidic mixture (1:3:7, 96% H2SO4 : 85% H3PO4 : H2O). Then 40 μl of α-isonitrosopropriophenone (Sigma-Aldrich, USA) was added to the solution, which was heated for 30 min (95 °С) and incubated for 30 min at 4 °С. Urea concentration was measured spectrophotometrically at λ = 550 nm. Values of optical density were converted to mass of urea based on calibration curve of standard urea solution. Arg activity was calculated as described in . One unit of Arg activity means the amount of the enzyme hydrolyzing 1 μM of arginine per 1 min. Results are expressed as units/106 cells.
Statistical analysis. Statistical significance between groups was evaluated by nonparametric Mann — Whitney U test using Prism software Version 4.0. and assessed at p < 0.05. Correlation analysis between metastases volume, metastases number and NO production by lung Mph was determined by Pearson`s correlation coefficient using Prism software Version 4.0. Results are presented in box plots where whiskers mean maximum and minimum values, upper and lower borders of rectangles match the third and first quartiles respectively.
RESULTS AND DISCUSSION
Throughout the experiment, the standard parameters of tumor growth (frequency, latent period of tumor appearance, tumor volume) and metastasis (proportion of mice with metastases, number and volume of metastases) were analyzed. The tumor yield reached 100% with the latency of 9.3 ± 1.2 days. The dynamics of tumor growth and metastasis in the lung within 28 days of observation were characteristic of this cancer model (Table).
Table. Tumor volume and metastasis parameters in LLC-bearing C57Bl mice
In the intact mice, CTA of peritoneal or splenic Mph was almost the same with cytotoxicity index of 14.4% and 13.0%, respectively (Fig. 1). In the tumor-bearing mice, Mph isolated from different anatomical niches exhibited different functional activity depending on the day of tumor growth and the isolation source. CTA of the peritoneal Mph was elevated on day 7 of tumor growth (p < 0.05) and sharply decreased a week later (1.4 times lower as compared to the intact control). CTA of the splenic Mph on day 7 and 14 of tumor growth was 1.2 and 2.9 times lower (p < 0.05) as compared to the intact control level. On day 21, the CTA of peritoneal Mph was elevated as compared to the intact control, splenic Mph — increased to the level of intact control. On day 28, independently of the isolation source, CTA of Mph was below the intact control level (see Fig. 1, a and b).
Fig 1. Changes in the cytotoxic activity of splenic (a) and peritoneal (b) Mph in LLC-bearing C57Bl mice. P-value were calculated as compared to intact control
The metabolic activity of Mph, examined by the NBT-reduction test, is depicted in Fig. 2. The metabolic activity of Mph changed in the same way whichever was the source of their isolation. As compared to the intact control, metabolic activity of peritoneal and splenic Mph was elevated on day 7 of tumor growth and gradually declined at the end of the experiment.
Fig 2. Changes in the metabolic activity in NBT-test of splenic (a) and peritoneal (b) Mph in LLC-bearing C57Bl mice. P-value were calculated as compared to intact control
As it is known, Mph play ambiguous role in cancer progression. During all the stages of cancer processes, they regulate the interplay between cancer cells and tumor microenvironment and are able to acquire different properties depending on the signals of the functioning environment. Depending on tissue microenvironment, Mph can polarize towards M1 or M2 phenotype, possessing pro-inflammatory or anti-inflammatory activities, respectively. We assessed the functional state of Mph based on their Arg- or NO-producing activities that allows us to distinguish between Mph polarization type.
In the intact C57Bl mice, Mph produced high levels of NO but low amounts of Arg independently of the isolation niche. NO/Arg ratio was 25.6, 26.8 and 27.8 for, respectively, peritoneal, spleen and lung Mph (Fig. 3). In the tumor-bearing mice, peritoneal and spleen Mph exhibited similar changes in their NO- and Arg-producing activities. On day 14 and 28 of tumor growth, production of NO by peritoneal Mph dropped by 31.7% and 28.6% comparing with the intact control. Splenic Mph production of NO was reduced by 27.1% on day 14 comparing with the intact control. It worth mentioning that on day 21 of tumor growth the production of NO by splenic Mph increased (р < 0.05) 1.1 times as compared to the intact control group. The highest Arg activity, on the contrary to NO production, was registered on days 14 and 28 of tumor growth. Lung Mph exhibited different dynamics of changes in NO and Arg production. On the early stages of tumor growth (days 7 and 14), NO/Arg ratio was 28.3 and 33.6 respectively. At further time points (day 21 and 28 of tumor growth) NO/Arg ratio was significantly declining (р < 0.05) due to the increase in Arg activity and statistically significant decrease in NO production.
Fig. 3. Changes in NO/Arg ratio for various residential peritoneal (a), splenic (b) and pulmonary (c) Mph isolated from LLC-bearing C57Bl mice. P-value were calculated as compared to intact control
Thus, analysis of the changes in the indices characterizing functional activity of Mph isolated from various anatomical niches (peritoneal cavity, spleen and lung) on different stages of LCC growth pointed to the gradual change in Mph polarization. In peritoneal and splenic Mph, antitumor (M1) Mph prevailed on days 7 and 21 of tumor growth while on days 14 and 28 tumor-promoting (M2) Mph came out on top. In the population of lung Mph, obtained data indicated the presence of M1 cells on the early stages of tumor growth (up to day 14); later (till day 28) their gradual polarization to the M2 phenotype was registered. Changes in functional polarization of lung Mph (from anti-tumor towards tumor-promoting) correlated with the metastasis initiation that is proved by Pearson correlation coefficient. Metastases volume and number inversely correlated with NO production by lung Mph (r = –0.92 and –0.89 respectively). Correlation between metastasis parameters and the activity of peritoneal and splenic Mph was insignificant.
It is known that the tumor process is accompanied by the development of local and systemic inflammation with the expression of proinflammatory transcription factors in tumor cells (such as NF-κB, STAT3, HIF-1α), which determine the production of spectrum of cytokines, chemokines and inflammatory enzymes in the tumor microenvironment, as well as the influence of these factors on the immune response.
As the effectors of the innate immune response, Mph are able to response on wide spectrum of stimuli: viral, bacterial, parasitic antigens, immune complexes, antigens of apoptotic or necrotic cells, soluble mediators produced by other cells. The direction of resident Mph polarization depends on the signals of the local microenvironment, such as components of damaged cells, cytokines and chemokines of activated lymphocytes, microbial products. In particular, affected by lipopolysaccharide, interferon-gamma and granulocyte-macrophage colony-stimulating factor Mph are polarized to M1 phenotype and acquire the ability to secrete significant amounts of proinflammatory cytokines (interleukin (IL)-1-beta, tumor necrosis factor, IL-12, IL-18 and IL-23), which promotes the involvement in the immune response of lymphocytes taking part in antigen-specific inflammatory reactions (Th1 and Th17) [2, 27, 28]. Such gradual, associated with changes in production of humoral factors, mutual activation of lymphocytes and Mph possibly explains the increase in number of M1-polarized cells on day 21 of tumor growth registered in this experiment. Later, due to the increase in production of tumor-promoting cytokines (IL-6, IL-17, IL-23, epidermal growth factor receptor, transforming growth factor-β), Mph polarization towards M2 phenotype emerged on day 28 of tumor growth.
Gradual polarization from M1 toward M2 phenotype was more evident for lung Mph and correlated strongly with the metastasis parameters. This finding can underline new approaches to reducing the metastasis by influencing the direction of Mph polarization.
Інститут експериментальної патології, онкології та радіобіології ім. Р.Є. Кавецького НАН України, Київ 03022, Україна
Мета: Оцінити стан макрофагів мишей лінії С57Вl за різними проявами їх функціональної активності на різних стадіях росту метастазуючої пухлини. Матеріали та методи: У дослідженні використовували мишей лінії C57Bl з епідермоїдною метастазуючою карциномою легені Льюїс. На 7-, 14-, 21- та 28-й день після перещеплення пухлини макрофаги з різних ніш були виділені та піддані функціональному аналізу. Результати: Макрофаги, отримані з різних біологічних ніш у інтактних тварин, за дослідженими показниками функціональної активності мали фенотип М1. У популяціях макрофагів, отриманих з селезінки та перитонеальної порожнини мишей лінії С57Вl на 7-і 21-шу доби росту карциноми легені Льюїс переважали клітини з протипухлинними властивостями (М1), на 14- та 28-му добу — клітини з пропухлинними властивостями (М2). У популяції легеневих макрофагів на ранніх стадіях пухлинного процесу переважали клітини з фенотипом М1, до 28-ї доби спостерігали поступову їх поляризацію до фенотипу М2. Такі зміни корелювали з показниками метастазування в легені в ці терміни. Висновок: Aналіз показників функціональної активності макрофагів свідчить про їх поступову поляризацію від клітин з проти- (М1) до клітин з пропухлинними (М2) властивостями в динаміці росту карциноми легені Льюїс. Виявлені зміни були найбільш вираженими в популяції легеневих макрофагів та мали кореляційний зв’язок з показниками метастазування.
Ключові слова: карцинома легені Льюїс, миші лінії C57Bl, макрофаги, функціональна активність, метастазування.
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