High Density Of Tumor-associated Macrophages Research Articles

The prognostic effect of tumor-associated macrophages in stage I-III colorectal cancer depends on T cell infiltration.

Tumor-associated macrophages (TAMs) are associated with unfavorable patient prognosis in many cancer types. However, TAMs are a heterogeneous cell population and subsets have been shown to activate tumor-infiltrating T cells and confer a good patient prognosis. Data on the prognostic value of TAMs in colorectal cancer are conflicting. We investigated the prognostic effect of TAMs in relation to tumor-infiltrating T cells in colorectal cancers. The TAM markers CD68 and CD163 were analyzed by multiplex fluorescence immunohistochemistry and digital image analysis on tissue microarrays of 1720 primary colorectal cancers. TAM density in the tumor stroma was scored in relation to T cell density (stromal CD3+ and epithelial CD8+ cells) and analyzed in Cox proportional hazards models of 5-year relapse-free survival. Multivariable survival models included clinicopathological factors, MSI status and BRAFV600E mutation status. High TAM density was associated with a favorable 5-year relapse-free survival in a multivariable model of patients with stage I-III tumors (p = 0.004, hazard ratio 0.94, 95% confidence interval 0.90-0.98). However, the prognostic effect was dependent on tumoral T-cell density. High TAM density was associated with a good prognosis in patients who also had high T-cell levels in their tumors, while high TAM density was associated with poorer prognosis in patients with low T-cell levels (pinteraction = 0.0006). This prognostic heterogeneity was found for microsatellite stable tumors separately. This study supported a phenotypic heterogeneity of TAMs in colorectal cancer, and showed that combined tumor immunophenotyping of multiple immune cell types improved the prediction of patient prognosis.

Breast Cancer Survival Outcomes and Tumor-Associated Macrophage Markers: A Systematic Review and Meta-Analysis.

Tumor-associated macrophages (TAMs) in breast cancer are associated with a poor prognosis. Early studies of TAMs were largely limited to the pan-macrophage marker CD68, however, more recently, an increasing number of studies have used CD163, a marker expressed by alternatively activated M2 macrophages and TAM subsets. We hypothesized that CD163-positive (CD163+) TAMs would be a better predictor of survival outcomes in breast cancer compared to CD68+ TAMs. We performed a systematic literature search of trials (from 1900 to August 2020) reporting overall survival (OS) or progression-free survival (PFS), breast cancer-specific survival (BCSS), TAM phenotype, and density. Thirty-two studies with 8446 patients were included. Meta-analyses were carried out on hazard ratios (HRs) for survival outcomes of breast cancer patients with a high density of TAMs (CD68+ and/or CD163+) compared to a low density of TAMs. A high density of TAMs (CD68+ and/or CD163+) was associated with decreased OS (HR 1.69, 95% CI 1.37-2.07) and reduced PFS (HR 1.64; 95% CI 1.35-1.99). Subgrouping by CD marker type showed a lower OS for high density of CD163+ TAMs (HR 2.24; 95% CI 1.71-2.92) compared to a high density of CD68+ TAMs (HR 1.5; 95% CI 1.12-2). A high density of TAMs (CD68+ and/or CD163+) in triple-negative breast cancer (TNBC) cases was associated with lower OS (HR 2.81, 95% CI 1.35-5.84). Compared to CD68+ TAMs, a high density of CD163+ TAMs that express a similar phenotype to M2 macrophages are a better predictor of poor survival outcomes in breast cancer.

What is the status of immunotherapy in thyroid neoplasms?

Immunotherapy has changed the treatment of patients with advanced cancer, with different phase III trials showing durable responses across different histologies. This review focuses on the preclinical and clinical evidence of potential predictive biomarkers of response and efficacy of immunotherapy in thyroid neoplasms. Programmed death-ligand 1 (PD-L1) staining by immunohistochemistry has shown higher expression in anaplastic thyroid cancer (ATC) compared to other subtypes. The tumor mutational burden in thyroid neoplasms is low but seems to be higher in ATC. Immune infiltrates in the tumor microenvironment (TME) differ between the different thyroid neoplasm subtypes. In general, differentiated thyroid cancer (DTC) has a higher number of tumor-associated lymphocytes and regulatory T cells (Tregs), while ATC and medullary thyroid cancer (MTC) display a high density of tumor-associated macrophages (TAMs). Nevertheless, results from clinical trials with immunotherapy as monotherapy or combinations have shown limited efficacy. Further investigation into new strategies aside from anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4)/programmed death 1 (PD-1)/PD-L1 antibodies, validation of predictive biomarkers, and better population selection for clinical trials in thyroid neoplasms is more than needed in the near future.

Total glucosides of paeony inhibit breast cancer growth by inhibiting TAMs infiltration through NF-κB/CCL2 signaling

PurposeThe high density of tumor-associated macrophages (TAMs) and inflammatory factors are crucial elements leading to tumor immune tolerance. Previously, we found that total glucosides of paeony (TGP) have strong inhibitory effects on the release of various inflammatory factors; however, it is unclear whether the inhibitory effects can improve the inflammatory microenvironment of tumors. Therefore, in the present study, we investigated the mechanism via which TGP depresses tumor growth and metastasis via modulation of TAM infiltration in breast cancer. MethodsWe assessed the effects of TGP on various mouse models of tumor. Lung metastasis was detected using hematoxylin and eosin staining. T cell (CD3+CD4+ and CD3+CD8+) effector and memory subsets, and TAM (CD45+CD11b+F4/80+) populations in the tumor microenvironment (TME) were examined using flow cytometry. Lipopolysaccharide (LPS)-stimulated macrophage experiments were used to investigate the TGP anti-inflammatory effects in vitro. Furthermore, conditional medium (CM) was added to detect 4T1 breast cancer cell growth using a Real-Time Cell Analyzer (RTCA) xCELLigence system. Inflammatory cytokine and chemokine levels were measured using cytometric bead array (CBA) kits and quantitative polymerase chain reaction (qPCR). NF-κB expression in the nucleus was examined by immunofluorescence and Western blot analysis. ResultsTGP suppressed tumor growth and lung metastasis, decreased CD45+CD11b+F4/80+ (TAMs) population obviously, and increased CD44LowCD62LHi (T memory stem cells) and CD44HiCD62LHi (central memory cells) populations in the tumor-infiltrating CD4+ and CD8+ T cells. In addition, TGP reduced inflammatory factor levels in tumors, thus inhibiting the infiltration of TAMs to improve the inflammation immunosuppressive microenvironment. In the in vitro experiment, TGP inhibited IL-10 and C-C Motif Chemokine Ligand 2 (CCL2) secretion and mRNA expression in LPS-stimulated macrophages to inhibit 4T1 cell growth and restrain macrophages M2 polarization. In addition, TGP can directly inhibit 4T1 cell proliferation by restraining autocrine CCL2 and IL-10. Further mechanistic studies reavealed that TGP inhibited CCL2 secretion by inhibiting NF-κB accumulation in the nucleus in macrophages. ConclusionTGP reduced TAM recruitment mainly through the NF-κB/CCL2 signaling pathway, thereby promoting T cell infiltration in the TME. TGP has a unique advantage in balancing the inflammatory response. Furthermore, our results present novel insights on the mechanisms underlying TAM infiltration that were inhibited by TGP, with potential application in development of novel therapies targeting CCL2 pathways.

The Overexpression of Programmed Death-Ligand 2 in Uterine Adenosarcoma: Correlation with High-Grade Morphology, Mutant Type TP53 Expression and Clinical Outcomes.

Immunotherapy involving the programmed death-1 (PD-1)/the programmed death-ligand (PD-1/PD-L) blockade is an understudied tumor therapy approach in cases of adenosarcoma. PD-L1 and PD-L2, and tumor protein p53 (p53) were examined in 20 uterine adenosarcoma cases, and tumor-infiltrating lymphocytes and tumor-associated macrophages were counted in tumor tissue using immunohistochemistry. While CPS PD-L1 positivity with 1% and 10% cut-off values was observed in 40% and 10% of tumors, respectively, CPS PD-L2 positivity with 1%, 10% and 50% cut-off values was observed in 100%, 85% and 50% of the tumors, respectively. The CPS PD-L2 positivity with a 50% cut-off value was positively correlated with tumor grade and the presence of sarcomatous overgrowth and lymphovascular invasion (LVI) (p = 0.025, p = 0.025, and p = 0.025, respectively). Nine of 11 high-grade adenosarcomas and none of the low-grade adenosarcomas showed mutant type p53 expression (p = 0.000). However, PD-L1 expression and tumor-infiltrating immune cells did not correlate with clinicopathological parameters. The CPS PD-L2 positivity with a 50% cut-off value was also positively correlated with mutant type p53 expression (p = 0.024) and tumor-associated macrophages density (p = 0.024). The CPS PD-L2 positivity with a 50% cut-off value and mutant type p53 expression were associated with shorter disease-free survival and shorter overall survival. The high density of tumor-associated macrophages and low density of tumor-infiltrating lymphocytes were also associated with shorter disease-free survival and overall survival (p < 0.05).These results suggested that the CPS PD-L2 positivity with a 50% cut-off value, p53 mutation and tumor microenvironment played an essential role in the progression of uterine adenosarcomas.

High grade breast ductal carcinomas have high density of tumor-associated macrophages.

The role of tumor-associated macrophages (TAMs) is double-natured and still controversial. Depending on different settings, macrophages may suppress or promote tumor growth. TAM density may be one of the predictive factors of treatment outcome in cancer patients. To evaluate the density of tumor-associated macrophages in breast cancer and its relationship with various histopathologic findings. 55patients with invasive ductal carcinoma of breast who underwent mastectomy were enrolled. Sections of tumor samples were stained and the density of CD68+ cells was evaluated. There was anassociation between estrogen receptor (ER) expression and CD68density (p = 0.010) as the higher densities of CD68were seen inER negative tumors. Moreover, there was a significant relationship between histological grade and CD68density (p =0.006). The higher TAM density is associated with higher tumor grade and negative ER expression in breast cancer tissues. These findings revealed that inflammation could have an important role in malignancies.

Tumor-Infiltrating Cytotoxic T Cells and Tumor-Associated Macrophages Correlate With the Outcomes of Neoadjuvant Chemoradiotherapy for Locally Advanced Rectal Cancer.

BackgroundTumor-infiltrating immune cells (TIICs) play a key role in immunoregulatory networks and are related to tumor development. Emerging evidence shows that these cells are associated with sensitivity to chemotherapy and radiotherapy. However, the predictive role of TIICs in the outcomes of neoadjuvant chemoradiotherapy (nCRT) for locally advanced rectal cancer (LARC) is unclear.MethodsImaging mass cytometry (IMC) was performed to comprehensively assess the immune status before nCRT in 6 patients with LARC (3 achieved pathological complete response (pCR), 3 did not) with matched clinicopathological parameters. Immunohistochemistry (IHC) for CD8, CD163 and Foxp3 on biopsy samples from 70 patients prior to nCRT and logistic regression analysis were combined to further evaluate its predictive value for treatment responses in an independent validation group.ResultsA trend of increased CD8+ cytotoxic T lymphocytes (CTLs) and decreased CD163+ tumor-associated macrophages (TAMs) and Foxp3+ regulatory T cells (Tregs) in the pCR group was revealed by IMC. In the validation group, CTLs and TAMs were strong predictors of the clinical response to nCRT. High levels of CTLs were positively associated with the pCR ratio (OR=1.042; 95% CI: 1.015~1.070, p=0.002), whereas TAMs were correlated with a poor response (OR=0.969; 95% CI: 0.941~0.998, p=0.036). A high density of TAMs was also associated with an advanced cN stage.ConclusionCTLs in the tumor microenvironment (TME) may improve the response to nCRT, whereas TAMs have the opposite effect. These results suggest that these cells might be potential markers for the clinical outcomes of nCRT and aid in the clinical decision-making of LARC for improved clinical outcomes.

DMF Activates NRF2 to Inhibit the Pro-Invasion Ability of TAMs in Breast Cancer.

Tumor-associated macrophages (TAMs) account for more than 50% of the cells in the tumor immune microenvironment of patients with breast cancer. A high TAM density is associated with a poor clinical prognosis. Targeting TAMs is a promising therapeutic strategy because they promote tumor growth, development, and metastasis. In this study, we found that dimethyl formamide (DMF) significantly inhibited the tumor invasion-promoting ability of TAMs in the co-culture system and further showed that DMF functioned by reducing reactive oxygen species (ROS) production in TAMs. The orthotopic 4T1 cell inoculation model and the spontaneous mouse mammary tumor virus-polyoma middle tumor-antigen tumor model were used to evaluate the antitumor effect of DMF. The results showed that DMF significantly inhibited tumor metastasis and increased T-cell infiltration into the tumor microenvironment. Mechanistically, NRF2 activation was necessary for DMF to exert its function, and DMF can play a role in breast cancer as an anticancer drug targeting TAMs.

Tumor-Associated Macrophages as Multifaceted Regulators of Breast Tumor Growth.

Breast cancer is the most commonly occurring cancer in women of Western countries and is the leading cause of cancer-related mortality. The breast tumor microenvironment contains immune cells, fibroblasts, adipocytes, mesenchymal stem cells, and extracellular matrix. Among these cells, macrophages or tumor-associated macrophages (TAMs) are the major components of the breast cancer microenvironment. TAMs facilitate metastasis of the breast tumor and are responsible for poor clinical outcomes. High TAM density was also found liable for the poor prognosis of breast cancer. These observations make altering TAM function a potential therapeutic target to treat breast cancer. The present review summarizes the origin of TAMs, mechanisms of macrophage recruitment and polarization in the tumor, and the contributions of TAMs in tumor progression. We have also discussed our current knowledge about TAM-targeted therapies and the roles of miRNAs and exosomes in re-educating TAM function.

Infiltration by CXCL10 Secreting Macrophages Is Associated With Antitumor Immunity and Response to Therapy in Ovarian Cancer Subtypes.

Ovarian carcinomas (OCs) are poorly immunogenic and immune checkpoint inhibitors (ICIs) have offered a modest benefit. In this study, high CD3+ T-cells and CD163+ tumor-associated macrophages (TAMs) densities identify a subgroup of immune infiltrated high-grade serous carcinomas (HGSCs) with better outcomes and superior response to platinum-based therapies. On the contrary, in most clear cell carcinomas (CCCs) showing poor prognosis and refractory to platinum, a high TAM density is associated with low T cell frequency. Immune infiltrated HGSC are characterized by the 30-genes signature (OC-IS30) covering immune activation and IFNγ polarization and predicting good prognosis (n = 312, TCGA). Immune infiltrated HGSC contain CXCL10 producing M1-type TAM (IRF1+pSTAT1Y701+) in close proximity to T-cells. A fraction of these M1-type TAM also co-expresses TREM2. M1-polarized TAM were barely detectable in T-cell poor CCC, but identifiable across various immunogenic human cancers. Single cell RNA sequencing data confirm the existence of a tumor-infiltrating CXCL10+IRF1+STAT1+ M1-type TAM overexpressing antigen processing and presentation gene programs. Overall, this study highlights the clinical relevance of the CXCL10+IRF1+STAT1+ macrophage subset as biomarker for intratumoral T-cell activation and therefore offers a new tool to select patients more likely to respond to T-cell or macrophage-targeted immunotherapies.

Prognosis of Macrophage Density in the Absence of Neutrophils in Differentiated Thyroid Cancer

BackgroundDespite the advances in treatment of differentiated thyroid cancer (DTC), predicting prognosis remains a challenge. Immune cells in the tumor microenvironment may provide an insight to predicting recurrence. Therefore, the objective of this study was to investigate the association of tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) with recurrence in DTC and to identify serum cytokines that correlate with the presence of these immune cells in the tumor. Materials and methodsForty-two DTC tissues from our institutional neoplasia repository were stained for immunohistochemistry markers for TAMs and TANs. In addition, cytokine levels were analyzed from these patients from preoperative blood samples. TAM and TAN staining were compared with clinical data and serum cytokine levels. ResultsNeither TAM nor TAN scores alone correlated with tumor size, the presence of lymph node metastases, multifocal tumors, lymphovascular or capsular invasion, or the presence of BRAFV600E mutation (all P > 0.05). There was no association with recurrence-free survival (RFS) in TAN density (mean RFS, 169.1 versus 148.1 mo, P = 0.23) or TAM density alone (mean RFS, 121.3 versus 205.2 mo, P = 0.54). However, when scoring from both markers were combined, patients with high TAM density and TAN negative scores had significantly lower RFS (mean RFS, 50.7 versus 187.3 mo, P = 0.04) compared with the remaining cohort. Patients with high TAM/negative TAN tumors had significantly lower serum levels of interleukin 12p70, interleukin 8, tumor necrosis factor alpha, and tumor necrosis factor beta. ConclusionsIn DTCs, high density of TAMs in the absence of TANs is associated with worse outcome. Assessment of multiple immune cell types and serum cytokines may predict outcomes in DTC.

A novel CSF-1R neutralizing antibody, DCB-AB21, as a potential therapeutics for tumors.

e15006 Background: Tumor-associated macrophages (TAMs) represent the major cell components of immune infiltrate in solid tumors, and it has been reported that high TAMs density is associated with poor clinical prognosis. Within the tumor microenvironment, TAMs rely on signaling through colony stimulating factor 1 receptor (CSF-1R)/CSF-1 axis for various tumor promoting processes such as tumor growth, drug resistance, angiogenesis, and metastasis. Therefore, decrease of TAMs density in tumors through CSF-1R inhibition is considered an attractive therapeutic target for drug development. Methods: DCB-AB21 was screened from immunized mouse hybridoma clones. ELISA-based and flow cytometry-based affinity screening is performed and then the growth inhibition test by cell-based CSF-1-dependent cell line were further used as antibody screening assay. Results: A novel anti-CSF-1R monoclonal antibody DCB-AB21 which specifically binds to CSF-1R and inhibits CSF-1R downstream signaling. Our results showed DCB-AB21 can block the CSF-1 and IL-34-induced cell proliferation. Potent inhibition of CSF-1R-dependent signaling was observed in the Ba/F3 CSF-1R driven model cells, and in human THP-1 leukemia monocytic cell lines. Humanization engineering further improves this antibody’s affinity and anti-proliferative efficacy. The discovery of novel binding epitope of DCB-AB21 differentiates itself from the current CSF-1R targeting antibodies testing in clinical trials. In vivo efficacy of DCB-AB21 showed AB21 alone has moderate anti-cancer activity and it further potentiate anti-PD-1’s efficacy. Conclusions: These data demonstrate that DCB-AB21 can potently inhibit CSF-1R in cellular contexts and in vivo model, and has the potential to induce a phenotypic effect on macrophages. These data suggest that DCB-AB21 is a promising new agent with possibility to combine with immune checkpoint inhibitors to relief macrophage-dependent immune suppression and would yield clinical benefit.

Tumor-associated macrophage infiltration and prognosis in colorectal cancer: systematic review and meta-analysis.

Tumor-associated macrophages (TAMs) are key components of colorectal cancer (CRC) microenvironment, but their role in CRC prognosis is not fully defined. This study aimed to evaluate prognostic value of different types and distribution of TAMs in CRC. Total 27 studies with 6115 patients were searched from PubMed and Embase and analyzed to determine the association between TAMs, including distinct TAM subsets and infiltration location, and CRC survival. The prognostic impact of TAMs on CRC was further stratified by tumor type and mismatch repair system (MMR) status. A pooled analysis indicated that high density of TAMs in CRC tissue was significantly associated with favorable 5-year overall survival (OS) but not with disease-free survival (DFS). CD 68+ TAM subset correlated with better 5-year OS, while neither CD68+NOS2+ M1 subset nor CD163+ M2 subset was correlated with 5-year OS. Increased CD68+ TAM infiltration in tumor stroma but not in tumor islet predicted improved 5-year OS. Stratification by tumor type and MMR status showed that in colon cancer or MMR-proficient CRC, elevated TAM density was associated with better 5-year OS. High infiltration of CD68+ TAMs could be a favorable prognostic marker in CRC. Future therapies stimulating CD68+ TAM infiltration may be promising in CRC treatment.

LIPOXINS SELECTIVELY CHANGE M2‐LIKE PHENOTYPE OF TUMORASSOCIATED MACROPHAGES BY CHANGING THEIR UNIQUE METABOLIC PROFILE AND IMPAIRING TUMOR PROGRESSION

In tumor microenvironments, most tumor‐associated macrophages (TAM) exhibit a characteristic M2 phenotype that favors tumor development, with deficient production of NO and ROS, key features of pro‐inflammatory M1 cytotoxic macrophages. Evidences have shown a positive correlation between high TAM density and poor prognosis. The balance between the different tumor derived mediators may modulate the outcome of the Mϕ response and consequent tumor development. Lipoxins (LX) are specialized pro‐resolving lipid mediators, able to shift inflammatory monocytes and macrophages towards M2‐like profile, but their effects on TAMs remained non‐elucidated. We have shown that, in vitro, LX selectively induce a switch of TAM M2‐like phenotype towards an M1‐like profile, increasing ROS and NO production, and enabling these cells to promote tumor cell apoptosis. LX did not affected the M2 profile of IL‐4‐stimulated macrophages. In vivo, the treatment of tumor‐bearing mice with LX, diminished the population of LY6Chi monocytes (TAM precursors) in spleen, blood and bone marrow, decreasing macrophage infiltration into the tumor, reducing the M2 markers expression on TAMs, and impairing tumor progression, increasing mice survival. The differential activation of macrophages is supported by profound intracellular metabolic changes, being well accepted that the pro‐inflammatory M1/M(LPS+IFN‐γ) phenotype rely on aerobic glycolysis, while anti‐inflammatory M2/M(IL‐4) macrophages depend on oxidative metabolism. However, little is known about TAM metabolism. We investigated the metabolic profile and requirements of TAMs, analyzing the lipoxin effects on their metabolic pathways. We show for the first time that despite TAMs present high expression of M2 markers, similarly to the M1, but distinct from M2 cells, have high glycolytic activity, with high lactate secretion, mTOR/Akt activity and increased GLUT‐1 expression. Inhibition of glycolysis, but not the oxidative phosphorylation or PPP, decreased M2 markers expression in TAMs. The inhibition of PPP impairs lipoxin to shift the M2‐profile and restore cytotoxic properties in TAMs. In conclusion, TAMs although phenotyped as M2, are metabolically distinct from these cells, being rather similar to M1, depending on the glycolytic metabolism to support their profile and functions, that lipoxin effects rely on PPP activation.Support or Funding Informationby: FAPERJ # E‐26/202.782/2017; CAPES: Finantial code # 001; CAPES/PROEX # 0574/2018; CNPq # 302413/2017‐0; 118566/2017‐2, Brazil.

Immune overdrive signature in colorectal tumor subset predicts poor clinical outcome.

The prognostic value of immune cell infiltration within the tumor microenvironment (TME) has been extensively investigated via histological and genomic approaches. Based on the positive prognostic value of T cell infiltration, Immunoscore has been developed and validated for predicting risk of recurrence for colorectal cancer (CRC). Also, association between a consensus T helper 1 (Th-1) immune response and favorable clinical outcomes has been observed across multiple cancer types. Here, we reanalyzed public genomic data sets from The Cancer Genome Atlas (TCGA) and NCBI Gene Expression Omnibus (NCBI-GEO) and performed multispectral immunohistochemistry (IHC) on a cohort of colorectal tumors. We identified and characterized a risk group, representing approximately 10% of CRC patients, with high intratumoral CD8+ T cell infiltration, but poor prognosis. These tumors included both microsatellite instable (MSI) and stable (MSS) phenotypes and had a high density of tumor-associated macrophages (TAMs) that expressed CD274 (programmed death-ligand 1 [PD-L1]), TGF-β activation, and an immune overdrive signature characterized by the overexpression of immune response and checkpoint genes. Our findings illustrate that CRC patients may have poor prognosis despite high CD8+ T cell infiltration and provide CD274 as a simple biomarker for identifying these patients.

P04.19 Analysis of tumor immune microenvironment and immune checkpoint modulators across infantile and pediatric pilocytic astrocytomas to elucidate the role of immunotherapy in these tumors

Abstract BACKGROUND Pilocytic astrocytomas are the most common central nervous system tumors in pediatric age-group. Although grade I, some of the cases show recurrence and progression, and few might not be amenable to surgery due to location or size, and hence have a less favorable prognosis. Drugs blocking immune check-point interactions such as those including programmed cell death ligand-1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) are now in clinical use for certain tumors. We performed this study to understand the potential candidature of pilocytic astrocytomas in infants and children for immunotherapy by analyzing the expression of immune checkpoint proteins and immune infiltrate, and correlating with clinical details, wherever possible. MATERIALS AND METHODS Cases with adequate tissue (2010–2017) diagnosed in pediatric age-group (&lt;18 years) were retreived from the archives of Department of Pathology, AIIMS, New Delhi. Immunohistochemistry for PD-L1 (SP263, Ventana), CTLA-4, CD3, CD8, CD4 and CD68 was performed. Quantification of cytotoxic lymphocytes was done using digital imaging in the core of the tumor. RESULTS A total of 50 pilocytic astrocytomas were included, 14 of them were &lt;3 years (infants), while 36 were of pediatric age-group (3–18 years). Overall, male preponderance was noted. Cerebellum was the most common location, followed by 4th venrticle, optic pathway, hypothalamus, cerebrum and thalamus. Almost all CD3 lymphocytes were cytotoxic T-lymphocyes (CD8 positive, CTLs). Helper T-lymphocyte infiltration was not seen. Median CTL density/mm3 was 13/mm3(Range:1–85/mm3). CTLA-4 was positive in 4 cases, positivity ranged from 1–4 cells/lpf. PD-L1 was found to be positive in 7 cases, and the positivity ranged from 1+ to 2+ in 1 to 5% of tumor cells. A median TAM (tumor associated macrophages) density of 44/hpf (range: 1–98/hpf) was noted. There was no correlation of CTL density with PD-L1 or CTLA-4 expression, and neither with TAM density. On correlation with clinical parameters, a higher density of CTLs and TAMs was noted in infants, and a higher proportion of cases revealed PD-L1 positivity, though not statistically significant. There was no correlation of TILs or TAMs with the tumor location. CONCLUSION Immune check point blockade using PD-(L)1 or CTLA4 inhibitors may not be a potential therapeutic option for unresectable or recurrent pilocytic astrocytomas, as low positivity rate as well as extremely low percentage of tumor/ immune cells found to be positive. However, alternate forms of immunotherapy might be helpful as most of the cases showed immune infiltrates and a high density of tumor-associated macrophages (TAMs). Large scale studies with larger numbers and longer follow-up periods including in-vitro and clinical studies are warranted for decoding the tumor immunogram.

Breast Phyllodes Tumors Recruit and Repolarize Tumor-Associated Macrophages via Secreting CCL5 to Promote Malignant Progression, Which Can Be Inhibited by CCR5 Inhibition Therapy.

Malignant phyllodes tumor (PT) is a fast-progression neoplasm derived from periductal stromal cells of the breast, which currently still lack effective treatment strategies. Our previous studies showed that the high density of tumor-associated macrophages (TAM) plays an important role in the malignant progression of PTs. TAMs secreted large amount of CCL18 to promote myofibroblast differentiation and invasion via binding to its receptor PIPTNM3 on myofibroblasts. Herein, we investigate the mechanism of how TAMs are recruited and repolarized by PTs to drive the malignant progression. The cytokines secreted by PTs were identified by the cytokine array. The clinical and pathologic correlations of the cytokine with PTs were estimated with IHC. The mechanisms of the cytokine that recruited and polarized the macrophage were explored with a coculture model of primary PT cells and macrophages in vitro and in vivo. The patient-derived xenografts (PDX) of malignant PTs were used to evaluate the therapeutic effect of CCR5 inhibitor. A high level of malignant PT-secreted CCL5 correlated with poor outcome of PTs and could be an independent prognostic factor of PTs. CCL5 bound to its receptor, CCR5, on macrophages thus activated AKT signaling to recruit and repolarize TAMs. Subsequently, the TAMs released CCL18 to further promote the aggressive phenotype of malignant PTs by enhancing and maintaining the myofibroblast differentiation and invasion in vitro and in vivo. In a murine PDX model of human malignant PTs, the CCL5-CCR5 axis blocked by maraviroc, an FDA-proved CCR5 inhibitor, prevented recruitment of monocytes to the tumor and dramatically suppressed tumor growth. Our findings indicate that malignant PTs recruit and repolarize TAMs through a CCL5-CCR5-driven signaling cascade. Thus, a positive feedback loop of CCL5-CCR5 and CCL18-PIPTNM3 between myofibroblast and TAMs is constituted to drive the malignant progression of PTs. Furthermore, targeting CCR5 with maraviroc represents a potential clinically available strategy to treat malignant PTs.

Cholangiocarcinoma-associated macrophages and tumor necrosis impact survival after surgery

Background: Tumor necrosis as well as tumor-associated macrophages (TAMs) in tumor invasive front (TIF) have been suggested to have a prognostic value in solid tumors, inclusive hilar cholangiocarcinoma. However, little is known regarding their influence on tumor progression and prognosis in intrahepatic cholangiocarcinoma (iCC). Methods: We analyzed surgically resected tumor specimens of human iCC (n = 88) for distribution and localization of TAMs, as defined by expression of CD68, formation of necrosis and extent of peritumoral fibrosis. TAMs, tumor necrosis and grade of fibrosis were assessed immunohistochemically and histologically and correlated with clinicopathological characteristics, tumor recurrence, and patients’ survival. Results: Patients with tumors characterized by necrosis or low CD68 density showed a significantly decreased recurrence-free and overall survival. Patients with high density of TAMs in TIF or absence of necrosis showed significantly lower incidence of tumor recurrence (both ρ < 0.05). Absence of tumor necrosis and TAMs in TIF were confirmed as independent prognostic variables in a multivariate analysis (all ρ < 0.05). Conclusion: High levels of TAMs in TIF or absence of tumor necrosis is associated with a significantly improved recurrence free and overall survival. These Results suggest TAMs and necrosis as valuable prognostic markers in routine histopathologic evaluation, and might indicate more individualized therapeutic strategies.

Tumor-driven like macrophages induced by conditioned media from pancreatic ductal adenocarcinoma promote tumor metastasis via secreting IL-8.

Tumor‐associated macrophages (TAMs) are abundant population of inflammatory cells which play an essential role in remodeling tumor microenvironment and tumor progression. Previously, we found the high density of TAMs was correlated with lymph node metastasis and poor prognosis in pancreatic ductal adenocarcinoma (PDAC). Therefore, this study was designed to investigate the mechanisms of interaction between TAMs and PDAC. THP‐1 monocytes were the exposure to conditioned media (CM) produced by PDAC cells; then, monocyte recruitment and macrophage differentiation were assessed. CM from PDAC attracted and polarized THP‐1 monocytes to tumor‐driven like macrophages. mRNA expression cytokine profiling and ELISA identified the IL‐8 secretion was increasing in tumor‐driven like macrophages, and STAT3 pathway was involved. Addition of exogenous recombinant human IL‐8 promoted PDAC cells motility in vitro and metastasis in vivo via upregulating Twist expression, which mediated epithelial‐mesenchymal transition in cancer cells. What is more, IL‐8 expression level in tumor stroma by immunohistochemical analysis was related to lymph node metastasis, the number of tumor CD68 but not CD163 positive macrophages and patient outcome. Taken together, these findings shed light on the important interplay between cancer cells and TAMs in tumor microenvironment and suggested that IL‐8 signaling might be a potential therapeutic target for PDAC.

Tumor necrosis and infiltrating macrophages predict survival after curative resection for cholangiocarcinoma

ABSTRACTBackground. Tumor necrosis as well as tumor-associated macrophages (TAMs) in the tumor invasive front (TIF) have been suggested to have a prognostic value in selected solid tumors, inclusive hilar cholangiocarcinoma. However, little is known regarding their influence on tumor progression and prognosis in intrahepatic cholangiocarcinoma (iCC).Methods. We analyzed surgically resected tumor specimens of human iCC (n = 88) for distribution and localization of TAMs, as defined by expression of CD68, formation of necrosis and extent of peritumoral fibrosis. Abundance of TAMs, tumor necrosis and grade of fibrosis were assessed immunohistochemically and histologically and correlated with clinicopathological characteristics, tumor recurrence and patients' survival. Statistical analysis was performed using SPSS software.Results. Patients with tumors characterized by low levels of TAMs in TIF or necrosis showed a significantly decreased 1-, 3- and 5-y recurrence-free survival and a significantly decreased overall survival, when compared with patients with tumors showing high levels of TAMs in TIF or no necrosis. Patients with high density of TAMs in TIF showed significantly lower incidence of tumor recurrence, as well (p < 0.05). Absence of tumor necrosis and TAMs in TIF were confirmed as independent prognostic variables in the multivariate survival analysis (all p < 0.05).Conclusions. High levels of TAMs in TIF or absence of histologic tumor necrosis are associated with a significantly improved recurrence-free and overall survival of patients with iCC. These results suggest TAMs and necrosis as valuable prognostic markers in routine histopathologic evaluation, and might indicate more individualized therapeutic strategies.