Cancer-associated fibroblasts promote an immunosuppressive microenvironment through the induction and accumulation of protumoral macrophages.
ABSTRACT: Stromal cells in the tumor microenvironment (TME) closely interact with tumor cells and affect tumor cell behavior in diverse manners. We herein investigated the mechanisms by which cancer-associated fibroblasts (CAFs) affect the functional polarization of tumor-associated macrophages (TAMs) in oral squamous cell carcinoma (OSCC) in vitro and in human cancer samples. The expression of CD68, CD14, CD163, CD200R, CD206, HLA-G, CD80, and CD86 was higher in CD14-positive cells co-cultured with the culture supernatants of CAFs established from OSCC specimens (CAF-educated cells) than in control cells. The gene expression level of ARG1, IL10, and TGFB1 was increased in CAF-educated cells. CAF-educated cells suppressed T cell proliferation more strongly than control cells, and the neutralization of TGF-? IL-10, or arginase I significantly restored T cell proliferation. We then investigated the relationship between the infiltration of CAFs and TAMs using tissue samples obtained from patients with OSCC. The infiltration of CAFs was associated with the numbers of CD68-positive and CD163-positive macrophages. It also correlated with lymphatic invasion, vascular invasion, lymph node involvement, and the TNM stage. The infiltration of CAFs was identified as an independent prognostic factor in OSCC. Our results indicate that CAFs play important roles in shaping the tumor immunosuppressive microenvironment in OSCC by inducing the protumoral phenotype of TAMs. Therapeutic strategies to reverse CAF-mediated immunosuppression need to be considered.
Project description:Tumor-associated macrophages (TAMs) can be polarized into antitumoral M1 and protumoral and immunosuppressive M2 macrophages. This study investigated the clinical relevance of TAM infiltration in oral squamous cell carcinoma (OSCC), evaluating CD68 (M1 and M2 macrophage marker) and CD163 expression (M2 macrophage marker) in the tumor nests and surrounding stroma. Immunohistochemical analysis of both stromal/tumoral CD68+ and CD163+ TAMs was performed in paraffin-embedded tissue specimens from 125 OSCC patients, and correlated with clinical data. Potential relationships with the expression of cancer stem cell (CSC) markers and PD-L1 in the tumors were also assessed. Stromal CD163+ infiltration was significantly associated with the tumor location in the tongue, and stromal and tumoral CD68+ and CD163+-infiltrating TAMs were more abundant in nonsmokers and non-alcohol-drinkers. Strikingly, this study uncovers an inverse relationship between CD68+ and CD163+ TAMs and CSC marker expression (NANOG and SOX2) in OSCC. High infiltration of CD163+ TAMs in both tumor and stroma was strongly and significantly correlated with the absence of NANOG expression. Moreover, infiltration of both CD68+ and CD163+ TAMs was also significantly associated with high tumor expression of PD-L1. Our results suggest that there is a link between TAM infiltration and immune escape in OSCC.
Project description:The polarization of tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs), especially from the antitumoral phenotype to the protumoral phenotype under certain conditions, has an important influence on the progression of tumors. However, the interactions and combined prognosis of these cells are poorly known. Here, we detected the infiltration of CD68+ TAMs, CD163+ TAMs, and CD66b+ TANs in the specimens from 662 patients with GC by immunohistochemistry. The results showed that the infiltration of each of CD163+ , CD68+ , and CD66b+ cells in GC tissue was significantly increased and independently associated with GC prognosis. Strong collinearity (r = 0.690, P < 0.001) was found between the infiltration of CD163+ and CD68+ cells in GC, and multivariate Cox analysis confirmed the infiltration of CD163+ cells was a better predictor for prognosis than that of CD68+ cells. The combination of the infiltration of CD163+ and CD66b+ cells provided more accurate survival prediction than any individual marker. Patient subgroups with CD66blow CD163low (hazard ratio (HR) = 2.161; 95% confidence interval (CI) = 1.266-3.688; P < 0.001), CD66bhigh CD163high (HR = 3.575; 95% CI = 2.155-5.933; P < 0.001), and CD66blow CD163high (HR = 7.514; 95% CI = 4.583-12.312; P < 0.001) were gradually associated with shorter DFS when compared with the subgroup with CD66bhigh CD163low . The similar result was also for DSS among the subgroups. Moreover, the two-marker model could more effectively discriminate the prognosis among the patients with chemotherapy than that among those without chemotherapy. We concluded that CD163+ TAMs were a more valuable prognostic marker than CD68+ TAMs, and CD163+ TAMs combined with CD66b+ TANs could more precisely predict the prognosis of patients with GC.
Project description:Fibroblasts turn into cancer associated fibroblasts (CAFs) in the tumour microenvironment. CAFs have recently attracted attention for their function as a regulator of immune cell recruitment and function in addition to their tumour-promoting roles. In this study, we aimed to determine the role of CAFs on monocyte recruitment and macrophage polarization in breast cancer. CAFs, which were ?-SMA expressing fibroblasts in contrast to normal fibroblasts (NFs), effectively recruited monocytes. Recruitment of monocytes by CAFs might be mediated by monocyte chemotactic protein-1 (MCP-1) as well as stromal cell-derived factor-1 (SDF-1) cytokines. CAFs differentiated the recruited monocytes into M2-like macrophages which are capable of exerting their immunosuppressive roles via the PD-1 axis. CAF-educated monocytes exhibited strong immune suppression unlike NF-educated monocytes and enhanced the motility/invasion of breast cancer cells in addition to increasing the expressions of epithelial-mesenchymal transition (EMT)-related genes and vimentin protein in cancer cells. CAF-educated M1 macrophages displayed increased expression of M2 markers and production of anti-inflammatory cytokine IL-10 in contrast to decreased production of pro-inflammatory cytokine IL-12 compared with control M1 macrophages; suggesting that CAFs were also able to induce the trans-differentiation of M1 macrophages to M2 macrophages. We then investigated the relationship between the infiltration of CAFs and tumour associated macrophages (TAMs) using tissue samples obtained from breast cancer patients. High grade of CAFs significantly correlated with the number of TAMs in human breast cancer tissue samples. It was also associated with higher Ki-67 proliferation index, and higher tumour volume. This result is in line with our finding of increased breast cancer cell proliferation due to the effects of CAF-educated monocytes in vitro. Our results concluded that CAFs play pivotal roles in sculpturing the tumour microenvironment in breast cancer, and therapeutic strategies to reverse the CAF-mediated immunosuppressive microenvironment should be taken into consideration.
Project description:Purpose:Tumour-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment and provide a barrier against the cytotoxic effector functions of T cells and natural killer (NK) cells. Recently, TAMs have become increasingly recognised as an attractive target in combination therapy with PD-1/PD-L1 immuno-checkpoint blockades (ICBs). However, the relationship between PD-L1 expression and TAMs remains unknown in nasopharyngeal carcinoma (NPC). Patients and Methods:A total of 212 NPC patients from Nanfang hospital were collected in this study. We evaluated the expression of PD-L1 in tumor cells, CD68 (pan-macrophages), and CD163 (M2-like macrophage) in NPC tissues using immunohistochemical (IHC) staining. Results:The positivity of PD-L1 on tumor cells was 61.3% (130/212). The infiltration densities of CD68+ cells and CD163+ cells in PD-L1-positive NPC tissues were significantly higher than those in PD-L1-negative NPC tissues (P=0.0012 for CD68; P<0.0001 for CD163). Logistic regression analysis showed that high densities of CD68+ macrophages and CD163+ TAMs were significantly associated with increased PD-L1 expression. Subgroup analyses demonstrated that a positive PD-L1 expression on tumor cells in combination with lower CD163+ TAMs density was significantly associated with favorable prognosis, whereas negative PD-L1 expression on tumor cells with higher CD163+ TAMs density was associated with worse prognosis. Conclusion:The PD-L1 expression in tumor cells was positively correlated with TAMs density in tumor microenvironment of NPC, suggesting TAMs as a new target for combination therapy to improve the response rate of ICBs in NPC treatment.
Project description:<b>Background:</b> The aggressive biology and treatment refractory nature of pancreatic ductal adenocarcinoma (PDAC) significantly limits long-term survival. Examining the tumor microenvironment (TME) of long-term survivors (LTS) of PDAC offers the potential of unveiling novel biological insights and therapeutic targets. <b>Methods:</b> We performed an integrated approach involving immunophenotyping, stromal scoring and histomorphological profiling of a cohort of 112 PDAC-cases, including 25 long-term survivors (LTSs, OS ? 60 months). Mutational frequencies were assessed using targeted next generation sequencing. Finally, we validated our findings <i>in silico</i> using an external cohort of microarray data from PDAC patients. <b>Results:</b> LTS cases exhibit a largely quiescent population of cancer-associated fibroblasts (CAFs). Immune profiling revealed key differences between LTS and NON-LTS cases in the intratumoral and stromal compartments. In both compartments, LTS cases exhibit a T cell inflamed profile with higher density of CD3<sup>+</sup> T cells, CD4<sup>+</sup> T cells, iNOS<sup>+</sup> leukocytes and strikingly diminished numbers of CD68<sup>+</sup> total macrophages, CD163<sup>+</sup> (M2) macrophages and FOXP3<sup>+</sup> Tregs. A large proportion of LTS cases exhibited tertiary lymphoid tissue (TLT) formation, which has been observed to be a positive prognostic marker in a number of tumor types. Using a Random-Forest variable selection approach, we identified the density of stromal iNOS<sup>+</sup> cells and CD68<sup>+</sup> cells as strong positive and negative prognostic variables, respectively. In an external cohort, computational cell-type deconvolution revealed a higher abundance of T cells, B lymphocytes and dendritic cells (DCs) in patients with long-term OS compared to short-term survivors. Thus, <i>in silico</i> profiling of long-term survivors in an external cohort, strongly corroborated the T cell-inflamed TME observed in our LTS group. <b>Conclusions:</b> Collectively, our findings highlight the prognostic importance of TME profiles in PDAC, underlining the crucial role of tumor associated macrophages (TAMs) and the potential interdependence between immunosuppressive TAMs and activated CAFs in pancreatic cancer. Additionally, our data has potential for precision medicine and patient stratification. Patients with a T cell inflamed TME might derive benefit from agonistic T cell antibodies (e.g., OX40 or CD137 agonists). Alternately, patients with activated CAFs and high infiltration of immunosuppressive TAMs are highly likely to exhibit therapeutic responses to macrophage targeted drugs (e.g., anti-CSF1R) and anti-CAF agents.
Project description:<b>Background:</b> The interaction and crosstalk between tumor-associated macrophages (TAMs) and epithelial-mesenchymal transition (EMT) has been demonstrated to play a critical role in the progression and metastasis of multiple cancers. However, the roles of the M2-polarized TAMs in different tumor location in EMT and prognosis of colorectal cancer (CRC) have not been elucidated. Therefore, the present study was designed to set up a reliable ratio of CD163<sup>+</sup>/CD68<sup>+</sup> to assess M2-polarized TAMs infiltration in the tumor center (TC) and tumor invasive front (TF) and to further evaluate their prognostic value and biological effects on tumor cells in CRC. <b>Methods:</b> TAMs markers (CD68 and CD163) and EMT markers (E-cadherin and Vimentin) expression were evaluated by immunohistochemistry in 81 patients with CRC. Circulating tumor cells (CTCs) of peripheral blood from above patients was also isolated. The correlation of CD163<sup>+</sup>/CD68<sup>+</sup> ratio in different locations, EMT and CTCs counts were further analyses. Kaplan-Meier and the model analyses of univariate Cox proportional hazards were utilized to compare the survival of patients with high CD163<sup>+</sup>/CD68<sup>+</sup> ratio with those with low CD163<sup>+</sup>/CD68<sup>+</sup> ratio. Furthermore, the effects of the M2-polarized TAMs on growth, migration and invasion of CRC cells were explored <i>in vivo</i> and <i>in vitro</i> co-culture system. <b>Results</b>: The results showed that the level of CD163<sup>+</sup>/CD68<sup>+</sup> ratio in TF was significant higher than that in TC, and higher CD163<sup>+</sup>/CD68<sup>+</sup> <sub>TF</sub> ratio were closely correlated with enhanced lymphovascular invasion, tumor invasion and TNM stage. Interestingly, higher CD163<sup>+</sup>/CD68<sup>+</sup> <sub>TF</sub> ratio were also significantly associated with EMT program and CTCs counts. Meanwhile, Kaplan-Meier analysis showed that CD163<sup>+</sup>/CD68<sup>+</sup> <sub>TF</sub> was associated with both recurrence-free survival (RFS) and overall survival (OS) of patients with CRC. Multivariate Cox regression analyses demonstrated that CD163<sup>+</sup>/CD68<sup>+</sup> <sub>TF</sub> remained an independent prognostic factor for RFS and OS. Further receiver operating characteristic (ROC) curve analysis found that CD163<sup>+</sup>/CD68<sup>+</sup> <sub>TF</sub> was a better prognosticator compared with CD68<sup>+</sup> <sub>TF</sub> and CD163<sup>+</sup> <sub>TF</sub> for CRC patients. What's more, M2-polarized TAMs secreted TGF-? to facilitate the EMT, growth, proliferation and invasion of CRC cells by <i>in vivo</i> and <i>in vitro</i> experiments. <b>Conclusions:</b> Our studies preliminarily elucidated the prognostic value of CD163<sup>+</sup>/CD68<sup>+</sup> ratio in different tumor locations and the biological functions of M2-polarized TAMs in CRC progression via TGF-?.
Project description:Tumor-associated macrophages (TAMs) play an important role in the progression and prognostication of numerous cancers. However, the role and clinical significance of TAM markers in oral squamous cell carcinoma (OSCC) has not been elucidated. The present study was designed to investigate the correlation between the expression of TAM markers and pathological features in OSCC by tissue microarray. Tissue microarrays containing 16 normal oral mucosa, 6 oral epithelial dysplasia, and 43 OSCC specimens were studied by immunohistochemistry. We observed that the protein expression of the TAM markers CD68 and CD163 as well as the cancer stem cell (CSC) markers ALDH1, CD44, and SOX2 increased successively from the normal oral mucosa to OSCC. The expressions of CD68 and CD163 were significantly associated with lymph node status, and SOX2 was significantly correlated with pathological grade and lymph node status, whereas ALDH1 was correlated with tumor stage. Furthermore, CD68 was significantly correlated with CD163, SOX2, and ALDH1 (P < 0.05). Kaplan-Meier analysis revealed that OSCC patients overexpressing CD163 had significantly worse overall survival (P < 0.05). TAM markers are associated with cancer stem cell marker and OSCC overall survival, suggesting their potential prognostic value in OSCC.
Project description:Aims: Tumor associated macrophages (TAMs) play a critical role in the initiation and progression of breast cancer. However, their prognostic significance in the molecular subtype of basal-like breast cancer (BLBC) is poorly understood. The aim of this study was to investigate the extent and patterns of TAMs in BLBC and their associations with clinicopathological features and patient survival. Methods and Results: We evaluated TAMs in 200 cases of BLBC by immunohistochemistry using the M2 macrophage marker CD163 and the pan-macrophage marker CD68 in tumor nest and stroma, and assessed their prognostic significance. The study demonstrated that infiltration of CD163+ and CD68+ macrophages in tumor stroma was of clinical relevance in BLBC, but not those in tumor nest. Increased stromal infiltration of CD68+ or CD163+ macrophages correlated with larger tumor size, higher histological grade, higher 5-year recurrence and 5-year breast cancer mortality. Although both of CD68+ and CD163+ macrophages in tumor stroma were associated with poor recurrence-free survival (RFS) and overall survival (OS), multivariate analysis demonstrated that only CD163+ macrophage was an independent predictor of RFS and OS. Conclusions: Our results highlight the prognostic importance of TAMs' location in BLBC. CD163, a highly specific biomarker for M2 macrophages, is an independent prognostic marker for BLBC patients, and may serve as an indicator or potential target of macrophage-centred therapeutic strategies.
Project description:BACKGROUND: Tumor associated macrophages (TAMs) are alternatively activated macrophages that enhance tumor progression by promoting tumor cell invasion, migration and angiogenesis. TAMs have an anti-inflammatory function resembling M2 macrophages. CD163 is regarded as a highly specific monocyte/macrophage marker for M2 macrophages. In this study we evaluated the specificity of using the M2 macrophage marker CD163 as a TAM marker and compared its prognostic value with the more frequently used pan-macrophage marker CD68. We also analyzed the prognostic value of the localization of CD163(+) and CD68(+) myeloid cells in human breast cancer. METHODS: The extent of infiltrating CD163(+) or CD68(+) myeloid cells in tumor nest versus tumor stroma was evaluated by immunohistochemistry in tissue microarrays with tumors from 144 breast cancer cases. Spearman's Rho and ?(2) tests were used to examine the correlations between CD163(+) or CD68(+) myeloid cells and clinicopathological parameters. Kaplan Meier analysis and Cox proportional hazards modeling were used to assess the impact of CD163(+) and CD68(+) myeloid cells in tumor stroma and tumor nest, respectively, on recurrence free survival, breast cancer specific and overall survival. RESULTS: We found that infiltration of CD163(+) and CD68(+) macrophages into tumor stroma, but not into tumor nest, were of clinical relevance. CD163(+) macrophages in tumor stroma positively correlated with higher grade, larger tumor size, Ki67 positivity, estrogen receptor negativity, progesterone receptor negativity, triple-negative/basal-like breast cancer and inversely correlated with luminal A breast cancer. Some CD163(+) areas lacked CD68 expression, suggesting that CD163 could be used as a general anti-inflammatory myeloid marker with prognostic impact. CD68(+) macrophages in tumor stroma positively correlated to tumor size and inversely correlated to luminal A breast cancer. More importantly, CD68 in tumor stroma was an independent prognostic factor for reduced breast cancer specific survival. CONCLUSION: These findings highlight the importance of analyzing the localization rather than merely the presence of TAMs as a prognostic marker for breast cancer patients.