Abstract
Spontaneous regression of tumours is a fascinating phenomenon rarely observed in oncological patients. We used a Lewis rat sarcoma model in which subcutaneous tumours developed after inoculation of the R5-28/clone C4 cells. Rats with tumour progression showed splenomegaly and anaemia. Tumour growth was associated with leucocytosis, granulocytosis, decrease in lymphocyte and CD161+ population in peripheral blood and increase in serum MCP1 concentration. Animals with spontaneous regression of tumours initially showed an increase in white blood cells number and proportion of granulocytes. Between the 42nd and 49th day, however, values of these parameters dropped in correlation with reduction of tumour size. In spontaneously regressed tumours, vascularization was higher and on the contrary, progressive tumours had more necrotic areas with a high number of infiltrating granulocytes. In conclusion, progression and spontaneous regression of tumours in the Lewis rat sarcoma model is associated with distinct changes in populations of blood cells and immune cells which participate in these completely different processes of tumourigenesis.
Spontaneous regression (SR) is a rare fascinating phenomenon in oncological patients. The incidence of SR has been observed in patients with various types of tumours, e.g. in renal carcinoma (1, 2), neuroblastoma (3) and breast cancer (4). Other cases of SR of different types of sarcoma such as alveolar soft tissue sarcoma (5), angiosarcoma (6), extraskeletal osteosarcoma (7) and endometrial stromal sarcoma (8) have been also described. SR was documented by many authors as a relatively rare event. Up to 20 years ago, approximately 20-30 cases were reported in the world literature each year, but it is possible that this phenomenon was more frequent than could be recognized clinically at the time (9, 10). In recent years, SR has been recorded more frequently, mainly because of the development of more efficient diagnostic techniques (11, 12).
SR of cancer is defined as the complete or partial disappearance of malignant tumour in the absence of any therapy that is capable of inducing anti-neoplastic effects or in the presence of therapy inadequate to significantly influence the disease (13, 10, 14, 15). Participation of the immune system, chemokines, tumour necrosis and changes in blood supply was suggested in SR. Because detailed and long-term studies of SR in patients are impossible for ethical reasons, suitable animal models are necessary to reveal mechanisms responsible for SR.
A rat sarcoma model, in which progression and SR occurs, was established in our laboratory. The R5-28 cell line was isolated from a spontaneously arising tumour in a female Lewis rat. It was characterized both in vitro and in vivo (16, 17). Three morphologically distinct rat sarcoma clones (C4, C7 and D6) were later derived from the original R5-28 cell line. These newly-derived clones also exhibited distinct behaviour (tumour growth/SR) after inoculation in vivo (18). Subcutaneous inoculation of clone D6 cells and clone C7 cells produced progressively growing tumours, whereas tumours arising from clone C4 cells exhibited SR in 62.5% of inoculated animals. Therefore, for the present study focusing on comparison of progression and SR of tumours, we used cells of the clone C4 as providing the best model.
Cytokines and chemokines produced by tumour cells participate in forming the tumour microenvironment. These molecules can influence tumour growth by three important mechanisms: regulation of tumour-associated angiogenesis, modulation of host antitumour responses, and direct stimulation of tumour cell proliferation (19). Monocyte chemotactic protein 1 (MCP1) significantly stimulates chemotaxis of human peripheral blood T-lymphocytes (CD4+, CD8+) in vitro (20). Purified human MCP1 has the ability to induce CD4+ and CD8+ T-cell migration through an endothelial cell barrier but it has no effect on migration of natural killer CD161+ cells (21). ADP-activated platelets modulate MCP1 levels of endothelial cells via a nuclear factor kappa-light-chain-enhancer of activated B-cells-dependent mechanism (22). Upon activation, platelets release a number of biologically highly active compounds from their granules which exert important reactions within endothelial cells (23). The platelet count remains constant in each individual throughout life, unless altered by non-physiological conditions or disease (24). Splenomegaly is a common feature in patients with cancer (25). It can modify the platelet number in peripheral blood. With increasing splenomegaly, a larger proportion of the body platelet mass is pooled in the spleen (26). Under pathophysiological conditions, platelets might adhere to the intact endothelial monolayer and change the microenvironment of the vessel wall (27, 28). They induce endothelial secretion of MCP1 within the vessel wall. Significantly lower endothelial MCP1 secretion is observed in the presence of non-stimulated platelets (22).
The growth of new blood vessels occurs at different stages of normal embryonic and postnatal development. It provides the required oxygen and nutrient to proliferating tissues (29, 30). In tumourigenesis, neovascularization is necessary to supply nutrients and oxygen to tumour cells during exponential tumour growth (31, 32). Subsets of myeloid cells identified by the expression of CD11b and myeloid differentiation antigen Gr1 are producing angiogenic factors, thus they are capable of facilitating tumour growth. They can also prevent tumour cells from being recognized and attacked by immune cells (33). Myeloid cells with CD11b expression are able to down-regulate the immune responses in subtypes of T-cells, including CD4+ and CD8+ cells (34). Analyses of the neoplasms at different stages of growth show that regressing tumours contain higher numbers of lymphocytes, mostly T-cells (35). In patients with SR of lymphoma, significantly higher numbers of T-helper cells cells were found in tumour infiltrates compared to patients with lymphoma progression. No significant differences in cytotoxic/suppressor T-cells or other lymphocyte sub-populations were observed by immunophenotyping (10).
Neutrophil granulocytes, myeloid cells originally considered only as effector cells of the innate immunity against invading microorganisms, are also important players in cancer regulation. Elevated neutrophil counts or neutrophil to lymphocyte ratios in peripheral blood are prognostic factors of poor outcomes in various malignancies (36-38). In agreement with these findings, the number of neutrophils (CD11b+ myeloid cells with segmented nuclei) highly increased with tumour progression in our Lewis rat sarcoma model using R5-28 cell inoculation (16). Tumour-derived chemokines attract circulating neutrophils into tumours (tumour-associated neutrophils). These can support tumour progression by enhancing angiogenesis, invasion and metastasis (39-41). On the contrary, the administration of granulocytes isolated from healthy animals and applied in the vicinity of solid tumours (W256 carcinoma growing in Sprague Dawley rats, Ehrlich ascites tumour growing in BALBs mice) significantly improved the survival of tumour-bearing animals, and even increased the incidence of W256 tumour regression from 17% to 75% (42). This controversial behaviour of granulocytes may be explained by the existence of two distinct tumour-associated neutrophil populations – protumorigenic N2 and anti-tumorigenic N1 neutrophils (43).
In the present study, we used the Lewis rat sarcoma model with inoculation of clone C4 cells to characterize the process of tumour progression and SR. Tumour size, changes of basic haematological and immunological parameters and concentration of MCP1 in serum of rats with tumour progression and SR were monitored at two-weekly intervals. Histology of tumours and their infiltration with various lymphocyte sub-populations was analyzed at the end of the experiment.
Materials and Methods
Cells. The rat sarcoma clone C4 cells (the 4th passage) were cultivated in high-glucose Dulbecco's modified Eagle's medium (Sigma-Aldrich, Czech Republic) supplemented with 10% heat inactivated foetal bovine serum (Lonza, Switzerland), 10 IU penicillin/5 μg streptomycin per 1ml, 10 μM HEPES, 1 mM L-glutamine and 1 mM non-essential amino acids (all from Sigma-Aldrich) in 5% CO2 at 37°C. Growing clone C4 cells (reaching almost monolayer) were washed with PBS, treated with 0.2% trypsin-EDTA (5 min/37°C) and centrifuged (after addition of the same volume of culture medium) at 1500 × g (5 min). Cell suspension with about 0.5×106 clone C4 cells was washed twice in PBS, dispersed in PBS and subcutaneously injected into experimental animals on the dorsal side.
Animals. We used 70-day-old female inbred Lewis rats. They were housed in the animal facility of our Institute, under controlled light-dark cycling, maintained at four in a cage and one rat was housed separate in a cage. Rats were fed by standard pellet diet and with water ad libitum. For subcutaneous inoculation, we applied the clone C4 cells at dose of 5×105 in 0.2 ml Phosphate-buffered saline (PBS) to 21 experimental rats. Four healthy animals without inoculation of sarcoma cells (with subcutaneous injection of 0.2 ml PBS only) served as a control group. The sarcoma progression or SR was evaluated by tumour measurement by Vernier calliper twice a week. The longitudinal and transverse diameters were determined and the tumour area was calculated.
Peripheral blood was obtained from the vena caudalis of each experimental and control animal at two-week intervals until day 42 and because of the visible tumour spontaneous regression between days 42 and 49 the next blood sampling was after one week on day 49 in the experiment. The first blood sampling was before the inoculation on day −5 (D=0 is the day of inoculation) until the end of the study D=49. Animals with progressing tumours were sacrificed on day 42 and animals with SR tumours were sacrificed on day 49 in order to collect tumour samples for further analyses. All experiments were performed in accordance with the Project of Experiment number 099/2011, approved by the Animal Science Committee of the IAPG, following the rules of the European Convention for the Care and Use of Laboratory Animals.
Haematology and flow cytometry of peripheral blood. About 200 μl of peripheral blood from the tail vena caudalis was collected into K3-EDTA tubes (Vacuette; Greiner Bio-One GmbH,Kremsmünster, Austria) for detection of basic haematological parameters by Vet ABC haematology analyser (ABX Hematologie, Montpellier, France) and blood smear staining (with May-Grünwald and Giemsa-Romanovski solutions). For flow cytometry, 1 ml of Easy Lyse working solution (Dako Cytomation, Glostrup, Denmark) was added to 100 μl of whole blood sample. After 10 min incubation, the cell suspensions were washed (twice in PBS with 0.2% cold water fish skin gelatine and 0.1% sodium azide) and incubated (refrigerator, 30 min) with mouse anti-rat antibodies [anti-rat granulocyte/fluorescein isothiocyanate (FITC), anti-CD4/phycoerythrin (PE), anti-CD8α/PE and anti-CD161/PE] for detection of granulocytes and main lymphocyte subpopulations. The anti-granulocyte/FITC antibody (eBioscience, San Diego, CA, USA) was diluted 1:300, and all other antibodies (AbD Serotec, Kiglington, UK) were diluted 1:30 with washing solution. Samples were washed again and treated with propidium iodide solution to detect dead cells. The stained cell suspensions were immediately analysed by FACS Calibur (Becton-Dickinson, Franklin Lakes, NJ, USA). Data from the cytometer were evaluated using FlowJo software (Tree Star, Ashland, OR, USA). The proportion of granulocytes was evaluated from leukogate and the populations of CD4-, CD8- and CD161-stained cells were evaluated from lymfogate.
Immunohistochemistry. Samples from progressive and SR sarcomas were excised after euthanasia, immediately frozen in liquid nitrogen and stored at −80°C for further analysis. Tumour cryosections (7 μm) were prepared using a Leica CM 1850 cryostat (Leica Instruments Gmb, Wetzlar, Germany) for detection of various types of immune cells by indirect immunofluorescence using the following primary antibodies: mouse anti-rat granulocyte (eBioscience) and anti-endothelial cell antibody (RECA-1; Abcam, Cambridge, UK) was applied to monitor blood vessel distribution. Secondary antibodies Alexa Fluor 555 goat anti-mouse IgG2a and AlexaFluor 488 goat anti-mouse IgG1 (both Invitrogen, Grand Island, NY, USA) were used to detect the bound primary antibodies. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, Prague, Czech Republic).
Cryosections were fixed in acetone (−20°C, 15 min), washed with PBS (3×5 min) and blocked with 10% rat serum [room temperature (RT), 60 min]. Then the sections were treated with the rat-specific antibodies (refrigerator, overnight), washed with PBS (3×5 min), covered with the appropriate secondary antibody (RT, 60 min) and again washed with PBS (3×5 min). DAPI was applied (RT, 5 min) for counterstaining of nuclei and it was followed with washing (2×5 min with PBS, then distilled water shortly). The stained sections were embedded in Mowiol (prepared according to the technical datasheet No. 777; Polysciences, Inc., Warrington, PA, USA) with n-propyl gallate (at final concentration of 5 mg/ml; Sigma-Aldrich). Mouse IgG1 isotype control/FITC and mouse IgG2a Isotype control/FITC (both Invitrogen) were used in control sections. The primary antibodies were diluted 1:100, secondary antibodies 1:1500 and isotype controls 1:100 with 10% goat serum. A BX51 fluorescence microscope (Olympus Czech Group, s.r.o., Prague, Czech Republic) with an Infinity 2 CCD Monochrome Microscopy Camera (Lumenera Corp., Ottawa, ON, Canada) and QuickPhoto Micro 2.3 software (Promicra s.r.o., Prague, Czech Republic) in the pseudo-colour mode were used for evaluation of the immunohistochemically stained sections.
Measurement of MCP1. For serum isolation, about 500 μl of peripheral blood without any additive was taken and left to coagulate at room temperature for 1 h. Blood samples were then centrifuged (1,500 × g for 20 min at 4°C) and the obtained sera stored at −80°C. Serum concentration of MCP1 was determined by enzyme-linked immunosorbent assay (ELISA) technique with RayBio Rat MCP1 ELISA Kit (RayBiotech, Norcross, GA, USA) according to the manufacturer's instructions. Absorbance was measured using a Synergy HT microplate reader (BioTek Instruments, Winooski, VT, USA) and data were analysed with Gen5 software (BioTek Instruments).
Statistics. Data values were calculated by MS Excel (Microsoft, Washington, DC, USA) and expressed as the mean±standard deviation (SD). The statistical comparisons between groups with differences in tumour growth, and tumour cell infiltration were performed by Students t-test. The differences in haematological and flow cytometric analyses were performed by using the analysis of variance (ANOVA). We performed the test of normality to decide whether to use parametric tests. Analyses were performed using the statistical software STATISTICA 12 Cz (StatSoft Inc., Tulsa, OK, USA). Differences were considered statistically significant at p<0.05.
Results
Characterization of tumour growth. Experimental rats were divided according to the tumour growth into three groups with approximately the same number of animals. Progressive subcutaneous tumours developed after C4 cell inoculation in six rats (28.6%), SR occurred in eight rats (38.1%) and seven rats (33.3%) did not develop any tumour. No significant changes appeared in the healthy controls during the three weeks after cell inoculation. The first tumours were observed macroscopically on day 21. Once the progressive tumours appeared, they grew extensively, reaching large sizes (1111±293 mm2) on day 42. The animals bearing progressive tumours also became obviously cachectic. The SR tumours appeared at the same time as progressive tumours (i.e. day 21). Their size increased continuously until day 42 (488±217 mm2). They then started to regress spontaneously so that they had markedly diminished to about half (277±66 mm2) their size on day 49. Generally, the SR tumours exhibited slower growth than the progressive ones. All rats with progressive and rats with SR tumours were sacrificed on day 42 and day 49, respectively, to collect tumour samples for further analyses (Figure 1).
Tumour growth in rats with progression and spontaneous regression (SR). Data are the mean±SD. aFirst visible difference in the group of rats with progressing and spontaneously regressing tumours (day 37). bSignificantly different at p<0.05 in comparison to animals with progressive tumours and animals with SR tumours (day 42).
All subcutaneous progressive and SR tumours were freely movable. No tumour invasion into other surrounding or abdominal tissues was observed. Tumours were clearly encapsulated by connective tissue. About 75-90% of the tumour tissue was necrotic in the progressive tumours and cystic formation was observed occasionally. The SR tumours regressed at several locations at once; they consisted mostly of necrotic tissue and no cystic formations were observed. The contrast in necrotic tissue was difficult to distinguish between progressive and SR tumour.
The size of spleen correlated well with tumour growth. Splenomegaly (2.3±0.9 g) was found only in animals with progressive tumours (Figure 2). In animals with no tumour development (n=7), with SR (n=8) and in controls (n=4) the mean weight of spleen was very similar and significantly lower (about 1 g).
Haematology. Tumour growth correlated well with an increase in the number of white blood cells (WBC). The first increase in WBC counts was observed one week after the macroscopic appearance of tumours (day 28) in rats with SR tumours and with progressive tumours. The WBC number increased continuously, being significantly higher than that of control rats and rats with no tumour development on day 42 (p<0.05). The tumour growth and increase of WBC count in rats with progressive course and rats with SR tumours were similar until day 42, when the SR of tumours was clear. A slight (non-significant) decrease of WBC number was observed on day 49 in rats with SR tumours.
The number of red blood cells (RBC), haemoglobin and haematocrit decreased in rats with progression as well as those with SR tumours, but these changes were not statistically significant (p>0.05) (Figure 3).
The number of platelets increased significantly in the peripheral blood of rats with SR of tumours compared to rats with tumour progression, controls and rats with no tumour development on day 42 (p<0.05).
Analysis of blood smears showed an increased number of polymorphonuclear (PMN) cells in rats with progressive tumours compared to control rats (Figure 4). In rats with SR of tumours, we also observed an increase of PMN cells during the initial period of tumour progression, but the PMN levels returned to normal pre-inoculation levels after tumour absorption. The PMN cells exhibited segmented nuclei and pink cytoplasm, thus corresponding to neutrophil granulocytes.
Dynamics of leukocyte sub-populations in peripheral blood. Flow cytometry was used to assess the proportion of CD4+ (T-helper), CD8+ (T-cytotoxic), CD161+ (natural killer) cells and granulocytes in peripheral blood. Significant changes were observed in lymphocyte sub-populations during the course of the experiment (Figure 5). The percentages of CD+, CD8+ and CD161+ cells gradually decreased in rats with progressive tumours and SR tumours. They were significantly lower in tumour-bearing rats than in control rats (p<0.05) on day 42.
In contrast to lymphocyte sub-populations, the proportion of granulocytes increased with tumour growth. In rats with SR tumours, granulocytes increased until the beginning of SR (day 42). With regression of tumours, granulocytes decreased to normal levels. A significant increase in the proportion of granulocytes was found in rats with progressive tumours compared to rats with SR (after the onset of the process of SR) and to controls and rats without tumour development (p<0.05).
Vascularization of subcutaneous tumours. Staining for endothelial cells indicated that vascularization was more pronounced in SR tumours than in progressive ones (Figure 6). Granulocytes were unevenly distributed in necrotic tissue. In tissue without necrosis, granulocytes were only observed in veins.
MCP1 expression. The concentration of MCP1 in serum (determined by ELISA) showed a clear increase associated with progressive tumour growth. On day 42, the MCP1 concentration was higher in the serum of rats with progressive tumours than in animals with SR tumours or in controls (Figure 7, p<0.05).
Discussion
In human oncological patients, SR of tumours is very rare, may take place covertly and if revealed cannot be monitored long-term from an ethical viewpoint because the patient should start an appropriate anticancer therapy. To study the entire process of SR in detail over time it is essential to utilize animal models. Mice and rats of various inbred strains with inoculated tumour cells are often applied as suitable models of various cancer types (16, 44). In the present study, we used Lewis rats with subcutaneous inoculation of the R5-28/clone C4 rat sarcoma cells. Tumours developing from the clone C4 cells either spontaneously regress or progress. It allows these two pathways of tumorigenesis to be studied in detail well. The tumours arising from these cells occurred around day 21 and SR was clearly apparent in around one-third of experimental animals at day 42 after inoculation. Tumour progression was observed in approximately the same number of rats as SR. Progressive tumours reached their maximum size at day 42 when the animal had to be euthanized to avoid animal death due tumour progression. The remaining one-third of animals showed no tumour development, suggesting effective immune response against the clone C4 tumour cells. In another study by Holubová et al. testing various clones of the R5-28 rat tumour cells, the subcutaneous tumours from clone C4 also exhibited progression or SR (18). However, SR was observed in more than half of the experimental animals and tumours appeared one week later than in our experiment here. In comparison with Holubová et al., we used more animals in our experimental group (21 versus 8) so that our results can confirm the previous finding.
Spleen enlargement in a rat with progressive tumour (left) in comparison with the spleen of a control animal (right).
Neutrophils are the predominant WBC type in peripheral blood, with a dual function in tumourigenesis. In some types of cancer, they may induce SR (48). They also play a role in antitumour immune response because of elimination of tumour tissue by phagocytic activity (49). On the contrary, neutrophils are also capable of preventing the immune system from attacking tumour cells. They exhibit a direct immunosuppressive effect against T-cells, inducing apoptosis and suppressing an effective antitumour immune response. They are also attracted into hypoxic and necrotic areas of tumours where they can stimulate angiogenesis by their own production of cytokines (50). In the present study, neutrophils in peripheral blood clearly correlated with tumour growth. Their proportion in peripheral blood and tumour tissue (determined by flow cytometry) and number in blood smears showed a significant increase in rats with progressive tumours compared to controls. In rats with SR of tumours, neutrophils initially also increased but numbers fell after day 42 with reduction in tumour size. Progressive and SR tumour tissues were infiltrated with granulocytes in the same manner, which were accumulated mainly in the necrotic areas. On the basis of our results, we can only speculate about the role of neutrophils in tumour progression or SR in our Lewis rat model with inoculation of clone C4 cells. Neutrophils can exhibit phagocytosis in necrotic tumour tissue. Moreover, tumour-associated neutrophils can be polarized to N1 (antitumoral) or N2 (protumoral) phenotype depending on the tumour microenvironment (51). We cannot distinguish these two subtypes of neutrophils with the analyses used herein. It is possible that expanded populations of neutrophils in progressive and SR tumours are heterogeneous, with various proportions of N1 and N2 neutrophils depending on the cytokine milieu. They could then support (N1<<N2) or depress (N1>>N2) tumour development.
Dynamics of basic haematological parameters at different days after inoculation of tumour cells: number of white blood cells (WBC; A), number of red blood cells (RBC; B), haematocrit (HCT; C), haemoglobin (HGB; D) and number of platelets (PLT; E) in rats with tumour progression, spontaneous regression (SR), controls (Ctrl) and rats with no tumour development. Average±SD haematological values are given. Significantly different at p<0.05 in comparison to acontrol animals and animals with no tumour development, banimals with progressive tumours, control animals and animals with no tumour development.
Blood smear on day 42 from rat with progressive tumour (B) and control rat (A) stained by May-Grünwald and Giemsa-Romanovski solutions. Highly increased number of neutrophil granulocytes can be seen in the animal with progressive tumour. Magnification ×100.
The number of WBC increased from day 28 in animals with SR and returned to a normal level during tumour absorption. In rats with progressive tumours, WBC were similarly increased at day 42 after inoculation. This leucocytosis can be caused in both cases by the increase of granulocytes in peripheral blood (shown by flow cytometry and blood smears) as has been already reported in some experimental studies (16, 48). Our results also showed that SR of tumour arising from clone C4 cells is associated with changes in immune cell populations around day 42 after inoculation. Poor vascularization of the tumour (52, 53) together with the rapid tumour growth may be responsible for the formation of necrotic tumour tissue. Indeed, the vascularization was better developed in SR tumours than in progressive ones.
Anaemia is a frequent phenomenon in clinical practice. It can be caused by the malignant disease (45) or by the age of patients (46). An anaemic period also exists in healthy animals during early postnatal development. In male rats, the number of RBC, haematocrit and concentration of haemoglobin was reduced at about 15 days of age. In the period of 70-340 days of age, however, these parameters remain relatively constant (47). During our experiment, we observed that number of RBC, haematocrit and concentration of haemoglobin slightly gradually decreased in rats with progressive and SR tumours. Because these parameters were almost stable in the group with no tumour development and in controls, their small (although statistically non-significant) changes in tumour-bearing animals were probably related to tumour growth, indicating an initial phase of anaemia. We had to euthanize the rats from an ethical viewpoint before anaemia could fully develop.
Platelet–endothelium interaction plays an important role in the pathophysiology of inflammation. The role of platelets in MCP1 secretion by endothelial cells has been assessed (22). In our experiment, the number of platelets significantly increased from day 28 in animals with SR compared to control animals, animals with no tumour development and animals with tumour progression. Platelets promote primary tumour growth through angiogenesis; tumour cells deliver physical and mechanical support of the immune system to attack and penetrate into secondary organs and this is the basis for development of metastases (54). Contrary to these findings we observed an increase in platelets in rats with SR of tumour, but an increase of MCP1 in animals with progressive tumours and a higher vascularization in SR tumour tissue. MCP1 plays an important role as chemoattractant in neutrophil migration into tumours. MCP1-mediated neutrophil chemotaxis was recently observed in mice with Escherichia coli infection (55). A higher level of MCP1 in serum of the Lewis rats with progressive tumours in comparison to animals with SR of tumours was also observed after inoculation of R5-28 rat sarcoma cells, showing the same trend for both the parental cell line and its C4 clone (17). Moreover, cytokine analysis of culture medium revealed that MCP1 is secreted by the clone C4 cells (18). Taken together, in vivo the clone C4 cancer cells directly affect the tumour cytokine milieu and neutrophil migration into tumours and are responsible for an increased level of MCP1 in serum. In a study of canine-transmissible venereal sarcoma, tumour regression in adults was observed after two to four months of progressive growth. The tumours growing in adult dogs were infiltrated with great numbers of lymphocytes (56). Analyses of the neoplasms in dogs at different stages of growth, human malignant melanoma and lymphoma showed that regressive tumours contained higher numbers of lymphocytes most of which were T-cells (57, 56). T-Cells are also attracted to tissue by MCP1 (21). We found a different trend - a decrease in the percentage of CD+, CD8+ and CD161+ cells in peripheral blood in rats with SR and progressive tumour compared to controls and rats with no tumour development. In another study, a very similar trend is described, in which the phenotyping of peripheral blood cells in healthy and R5-28 tumour-bearing rats in advanced-stage disease showed a highly increased number of granulocytes compared to a healthy animals and reduced proportion of lymphocytes (16), as observed in our study.
Dynamics of CD4+ (A), CD8+ (B), CD161+ (C) cells and granulocytes (D) as measured by flow cytometry of peripheral blood of rats with tumour progression, spontaneous regression (SR), controls (Ctrl) and rats with no tumour development. Average±SD values are given. Significantly different at p<0.05 in comparison to acontrol animals and animals with no tumour development, banimals with progressive tumours, control animals and animals with no tumour development.
Immunohistochemical staininig of cryosection from progressive (A) and spontaneously regressing tumour (B) for detection of endothelial cells (red) and granulocytes (green). Cell nuclei are counter-stained with 4’,6-diamidin-2-fenylindol (blue). Magnification ×200.
Concentration of serum monocyte chemotactic protein 1 (MCP1), as determined by enzyme-linked immunosorbent assay in rats with tumour progression, spontaneous regression (SR) and controls (Ctrl). Average±SD values are given. aSignificantly different at p<0.05 in comparison to animals with SR tumours and controls.
Tumour progression and SR are associated with distinct changes in haematological parameters and sub-populations of immune cells in peripheral blood and tumour tissue. The process of SR is a very complicated phenomenon, dependent on many factors. The level of vascularization can help the transport of immune cells to the tumourous areas, but on the other hand can also supply nutrition to the tumour tissue. Neutrophil granulocytes are present in the process of SR and can terminate tumour cells by phagocytosis or help tumour progression as a support for angiogenesis; this depends on the cytokine milieu. According to literature, crucial for the process of SR are higher levels of lymphocytes, but did not observe such changes in our model. This could be explained by the time of observation and sample evaluation because sub-populations of lymphocytes can be hidden in tumours and when the SR of tumour begins, they can be transported to blood vessels.
In the Lewis rat model of cancer, tumour growth is associated with clear changes in haematological parameters and sub-populations of immune cells in peripheral blood and tumour tissue. Leucocytosis observed in animals with progressive as well as those with SR tumours was associated with an increased number of granulocytes which were concentrated in necrotic tumourous areas.
In conclusion we can state that this phenomenon is yet to be explained and our experimental model using inoculated rat sarcoma clone C4 cells, presented in this study, could allow further, more detailed analyses of the mechanism of spontaneous tumour regression.
Acknowledgements
This work was supported by the project ExAM from European Regional Development Fund CZ.1.05/2.1.00/03.0124, student grant FAFNR (CUA 21370/1312/3199; CUA 21370/1111/0001) and by RVO (67985904).
The Authors thank Jaroslava Šestáková and Jitka Klučinová for excellent technical assistance.
Footnotes
Conflicts of Interests
The Authors declare that they have no conflicts of interests in relation to this article.
- Received September 26, 2015.
- Revision received October 24, 2015.
- Accepted October 27, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
