Although three studies two by marker of CD68 [ 15 , 33 ] and one by markers of CD68 and CD [ 20 ] in human breast cancer have included the locations of TAMs, two of which used the TMA samples to put it into practice [ 15 , 20 ]. We used full block-face tissue sections in the study to overcome the limitation of core materials that only contain small volume of tumor tissue may suffer from bias in the assessment of TAMs due to tumor heterogeneity.
Full block-face tissue sections allow the selection of tumor areas with abundant TAMs for accurate scoring in tumor nest and in tumor stroma separately. Medrek et al. Mahmoud et al. Although the methods and molecular subtypes of breast cancer patients were different in these studies, the results suggest that the TAMs infiltrates into tumor nest is not an important prognostic factor for patient outcome.
TAM promotes tumor progression through several mechanisms [ 37 ]. It suggested that TAMs may affect the prognosis of BLBC mainly through modulating the immune response by contacting with tumor-infiltrating lymphocytes in TS, rather than through the direct interaction between macrophages and cancer cells in TN, such as phagocytosis of cancer cells. However, the specific underlying mechanisms need further study. We also got similar conclusions in multivariate Cox regression analyses using continuous variables, one-quarter and median as the cutoffs, i.
It indicates that in contrast to the pan-macrophage marker CD68, macrophages expressing the anti-inflammatory CD marker M2 macrophages are probably the effect TAMs that facilitate the poor prognosis of BLBC and serve as an independent prognostic predictor. A number of potential macrophage-centred therapeutic strategies are being explored [ 37 ]. Either manipulating TAMs localization at tumor sites, or targeting the signal pathways affecting TAMs polarization and functions is proved to improve the outcomes of breast cancer [ 38 ].
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The pro-tumor M2 macrophage, recognized by CD, is reversible [ 39 , 40 ]. Duluc et al. Similar to the result, Guiducci et al. And classically activated M1 macrophage can kill cancer cells and induce tumor-destructive reaction [ 43 ]. Therefore, blocking key molecular determinants of macrophage polarization may result in reorientation of macrophage polarization and activate the anti-tumor activity [ 39 , 44 , 45 ]. Although TAM-centered strategies have not been yet introduced in clinical practice in terms of effectiveness and tolerability in breast cancer patients, reducing number or reeducating of TAM seems to be a promising strategy and should be further investigated due to its clinical significance in BLBC.
There are limitations in the study. Although CD was regarded as a highly special M2 macrophage marker, it can also be expressed by monocyte-derived dendritic cells MDCs [ 47 ]. They attributed this to a CDexpressing subset of immature myeloid derived suppressor cells with prognostic impact although no direct data was available [ 20 ]. Further investigation to identify their roles in sub-classifying TAMs into different prognosis-related biological groups will be meaningful and will be one of the focuses of our future studies.
However, further work is still warranting to validate the findings. Cancer-related inflammation. Neutralizing tumor-promoting chronic inflammation: a magic bullet? Pollard JW. Macrophages define the invasive microenvironment in breast cancer. Journal of Leukocyte Biology. Tang X. Tumor-associated macrophages as potential diagnostic and prognostic biomarkers in breast cancer.
Cancer letters. The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies.
J Pathol. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol. Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer. Perez EA. The Oncologist. Targeted therapy for breast cancer prevention. Front Oncol. Gucalp A, Traina TA.
'Priming' immune cells against cancer
Triple-negative breast cancer: adjuvant therapeutic options. Chemother Res Pract. Tumour-associated macrophages in breast cancer and their prognostic correlations. The Breast. Proliferating macrophages associated with high grade, hormone receptor negative breast cancer and poor clinical outcome. Breast cancer research and treatment. Cancer cell. Expression of vascular notch ligand delta-like 4 and inflammatory markers in breast cancer.
The American journal of pathology. Tumour-infiltrating macrophages and clinical outcome in breast cancer. Journal of clinical pathology. Clinical evaluation and mastectomy. Following a thorough clinical examination, female dogs were categorized by a complete staging determined from information on the size tumor T , involvement of regional lymph node N and the presence or absence of distant metastases M TNM system Owen Macroscopic evaluation of the inguinal and axillary lymph nodes was performed by palpation.
Neoplastic involvement was confirmed by histopathological examination of the lymph nodes following mastectomy. Radiological examinations of the chest in three planes, laterolateral right and left LL and ventrodorsal VD , were performed to investigate lung metastasis. All dogs were subjected to unilateral radical mastectomy, including the removal of the inguinal lymph nodes. Dogs were fitted with a compression bandage and monitored for 48 hours post-surgery. Histological classification and grading. Duplicate slides were prepared and analyzed by two veterinary pathologists.
All tumor samples were identified according to the histological classification established by the World Health Organization WHO Misdorp et al. Morphological and morphometric analysis of inflammatory infiltrate. The inflammatory response corresponding to each tumor sample was characterized according to intensity discrete, moderate or intense and distribution focal, multifocal and diffuse. Lymphocytes, macrophages, plasma cells, neutrophils and eosinophils were identified according to morphological characteristics and then quantified Estrela-Lima et al. Cell counts were obtained by totaling the eight fields laterolateral right and left analyzed and mean values were used to determine intensity.
The intensity of inflammatory infiltrate was classified as follows: i discrete, less than inflammatory cells; ii moderate, when inflammatory cells totaled ,; and iii severe, when more than 1, inflammatory cells were identified. Immunohistochemical analysis. All slides were deparaffinized, then rehydrated in a series of progressively diluted alcohol solutions and subjected to antigen retrieval in pH 6. For enzyme treatment, proteinase K was incubated in a moist chamber for five minutes at room temperature. Negative controls were obtained by replacing the primary antibody with PBS and normal serum.
Immunophenotyping of circulating monocytes by flow cytometry. To evaluate cellular immune response, 4mL of blood from all dogs were collected 30 minutes before performing mastectomy. Peripheral blood was collected into sterile 5mL disposable syringes via jugular venipuncture, then transferred into sterile EDTA-containing tubes kept at room temperature.
Each sample was thoroughly homogenized and incubated at room temperature for 30 minutes, protected from light. After centrifugation, the supernatant was discarded by decanting the tube, and the cell pellet was then homogenized by vortexing at low speed. Next, 3 mL of PBS-W was added, and the cellular suspension was again centrifuged at xg for seven minutes at room temperature; the supernatant was again discarded, and the pellet was resuspended and homogenized thoroughly; this final wash was then repeated once again.
Same-species, same-isotype antibodies were similarly obtained Serotec - Oxford, England and used as negative controls. After selecting the region of interest R1 , the analysis of phenotypic aspects was performed using two different approaches: i by considering the percentage of positive cells expressed graphically in terms of timely dispersion of fluorescence, corresponding to the positive population for the relevant marker; and ii by evaluating mean fluorescence intensity MFI , determined as the density of expression of a particular phenotypic marker displayed by logarithmic histogram.
The first approach was used to quantify cell phenotypes presenting bimodal distribution, i. The second approach used a semi-quantitative analysis of phenotypic marker expression with univariate distribution - i. In these situations, changes in the expression density can occur, promoting the shift of the cell population along the fluorescence intensity axis.
Macrophage Phagocytosis in Cancer Immunotherapy
For each sample analyzed, a control reaction was performed to assess the quality of the cellular profile and to detect possible nonspecific fluorescence. Survival rates. The follow-up for determining overall survival and clinical outcomes was performed by clinical examination and laboratory testing CBC and serum biochemistry - urea, creatinine, ALT and FA every 30 days for at least nine months after surgery. Radiological examinations were carried out every 90 days. Overall survival time was defined as the number of days between the date of surgical excision of the primary tumor and death.
All dogs that died were necropsied to determine cause of death and to detect metastasis. Statistical analysis. Data were submitted to the Kolmogorov-Smirnov test to evaluate distribution normality. Survival curves were estimated using the Kaplan-Meier method and compared by log-rank testing Mantel-Cox or Cox univariate and multivariate analysis by correlating the intensity of macrophage infiltration with clinical and pathological responses.
Analyses were performed using Prism 5. Multicentric tumors were observed in Morphologic, morphometric and immunohistochemical analysis of tumor-associated inflammatory infiltrate.
Morphological evaluation of the samples obtained by excisional biopsy revealed that tumor-associated inflammatory infiltrate exhibited moderately intense infiltration with predominantly mononuclear cells in multifocal distribution in all dog groups Fig. Lymphocytes were the predominant cell population in all groups.
Macrophages were more frequently found in the group of dogs without metastasis in comparison to the group that presented metastasis Fig. Quantitative analysis performed on HE-stained slides confirmed cell morphology and immunostaining with the CD68 antibody. A Carcinoma arising in a mixed tumor CMT associated with focal inflammatory infiltrate.
HE, obj. Composition of inflammatory infiltrate in carcinoma arising in a mixed tumor CMT. Inflammatory cells were characterized by image analysis. Table 1. Monocyte populations were identified by flow.
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Data are expressed as the percentage of positive cells within the captured monocyte population. C Density of macrophage infiltration among the experimental groups.
'Reprogramming' immune cells to attack cancer tumors
No significant differences were seen in clinical examinations and blood counts performed preoperatively, regardless of the presence or absence of lymph node metastasis. During follow-up, 15 of the 22 dogs died. Twelve of these 15 dogs were necropsied, and metastases were identified in the skin, liver, kidneys and lungs, in addition to paraneoplastic syndromes.
Hypovolemic shock was the most frequent cause of death, particularly in the dogs with indicators for chemotherapy treatment high histological grade and metastasis. The minimum survival period after surgery was 60 days, while the longest period was days-within the limits of the study follow-up timeframe Fig. B Survival rates of dogs with carcinoma arising in a mixed tumor CMT. Survival curves were generated using the Kaplan-Meier method, followed by the log-rank test. Table 2. For multivariate analysis, was used logistic regression. The roles played by TAMs in canine mammary carcinomas are not yet fully understood.
As such, correlations between the predominant immunophenotypical features of the macrophage infiltrates and classic prognostic factors may offer some insight into associations between the host immune response and disease progression. It is important to note that these higher frequencies might very likely be a reflection of breed preferences by owners, as observed since the beginning of the last decade i.
Morphological analysis of the tumor-associated inflammatory infiltrates in the present study indicated that lymphocytes were the most frequent cell type observed among immune cells, followed by macrophages. These findings are congruent with other reports in the human Heys et al. Similarly, Heys et al. However, differently than the findings of study with women, the dogs presenting higher density of TAM also had the higher survival rates, even though they also had higher histologic grades.
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Our long-term goal is apply these new findings toward the development of more effective mAb therapies for CLL and other types of cancer. Click for additional information pertaining to this project. Maxed out macs: physiologic cell clearance as a function of macrophage phagocytic capacity.