Distribution Of Tumor-associated Macrophages Research Articles

M2-like tumor-associated macrophages promote epithelial-mesenchymal transition through the transforming growth factor β/Smad/zinc finger e-box binding homeobox pathway with increased metastatic potential and tumor cell proliferation in lung squamous cell carcinoma.

Epithelial-mesenchymal transition (EMT) promotes primary tumor progression toward a metastatic state. The role of tumor-associated macrophages (TAMs) in inducing EMT in lung squamous cell carcinoma (LUSC) remains unclear. We aimed to clarify the significance of TAMs in relation to EMT in LUSC. We collected 221 LUSC specimens from patients who had undergone surgery. Immunohistochemistry was performed to evaluate M1-like and M2-like TAM distribution and EMT by E-cadherin and vimentin staining. Human LUSC cell lines (H226 and EBC-1) and a human monocyte cell line (THP-1) were used for in vitro experiments. M2-like polarization of TAMs and EMT marker expression in LUSC cells were evaluated by western blotting. The biological behavior of LUSC cells was evaluated by migration, invasion, and cell proliferation assays. Immunohistochemical analysis showed that 166 (75.1%) tumors were E-cadherin-positive and 44 (19.9%) were vimentin-positive. M2-like TAM density in the tumor stroma was significantly associated with vimentin positivity and worse overall survival. Western blotting demonstrated higher levels of CD163, CD206, vascular endothelial growth factor, and transforming growth factor beta 1 (TGF-β1) in TAMs versus unstimulated macrophages. Furthermore, increased TGF-β1 secretion from TAMs was confirmed by ELISA. TAM-co-cultured H226 and EBC-1 cells exhibited EMT (decreased E-cadherin, increased vimentin). Regarding EMT-activating transcriptional factors, phosphorylated Smad3 and ZEB-family proteins were higher in TAM-co-cultured LUSC cells than in parental cells. TAM-co-cultured H226 and EBC-1 cells demonstrated enhanced migration and invasion capabilities and improved proliferation. Overall, the present study suggests that TAMs can induce EMT with increased metastatic potential and tumor cell proliferation in LUSC.

Abstract 5829: Differential expression of PD-L1 and PD-L2 is associated with M2 tumor-associated macrophages and tumor differentiation in non-small cell lung cancer

Abstract Objectives: To improve the treatment strategy of immune-checkpoint inhibitors for non-small cell lung cancer (NSCLC), a comprehensive analysis of programmed death-ligand (PD-L)1 and PD-L2 expression is clinically important. The expression of PD-L1 and PD-L2 on tumor cells (TCs) and tumor-infiltrating immune cells (ICs) was investigated with respect to M2 tumor-associated macrophages (TAMs), which are key components of the tumor microenvironment. Methods: We performed immunohistochemical studies to evaluate the M2 TAM distribution by CD163 staining, PD-L1 expression on TCs and ICs by the Ventana SP263 assay, and PD-L2 expression on TCs and ICs, using the Ventana BenchMark GX system, in 175 patients with resected NSCLC. Results: M2 TAM density revealed high levels of variation among the 175 tumor tissues (mean ± standard deviation (SD), 382.5 ± 381.9 cells per mm2). The percentage of PD-L1-positive TCs and PD-L1-positive ICs varied greatly among the 175 tumor tissues (mean ± SD; 15.6 ± 27.0% and 9.4 ± 10.9%, respectively). In addition, the percentage of PD-L2-positive TCs and PD-L2-positive ICs also ranged widely among the 175 tumor tissues (mean ± SD, 14.6 ± 22.9% and 12.5 ± 18.4%, respectively). M2 TAM density was significantly associated with the expression of PD-L1 on TCs and ICs (P < 0.0001 and P < 0.0001, respectively). M2 TAM density was also significantly associated with the expression of PD-L2 on TCs and ICs (P = 0.0452 and P = 0.0125, respectively). The percentage of PD-L1-positive TCs was significantly correlated with the percentage of PD-L1-positive ICs (r = 0.396; P < 0.0001). In addition, the percentage of PD-L2-positive TCs also was significantly correlated with the percentage of PD-L2-positive ICs (r = 0.488; P < 0.0001). However, there was no correlation between the percentage of PD-L1-positive TCs and the percentage of PD-L2-positive TCs (r = 0.019; P = 0.8049). Meanwhile, tumor differentiation was significantly associated with PD-L1 expression on TCs and ICs (P = 0.0002 and P < 0.0001, respectively). By contrast, tumor differentiation was inversely associated with PD-L2 expression on TCs and ICs (P = 0.0260 and P = 0.0326, respectively). Conclusions: PD-L1 and PD-L2 expression on TCs and ICs was associated with M2 TAMs in NSCLC, although there was no correlation between PD-L1 and PD-L2 expression on TCs. The combined evaluation of PD-L1 and PD-L2 expression could be clinically important in the treatment strategy of immune-checkpoint inhibitors in patients with NSCLC. In particular, the evaluation of PD-L2 expression may be necessary for patients with PD-L1-negative NSCLC. Citation Format: Ryota Sumitomo, Huang Cheng-long, Hiroshi Date. Differential expression of PD-L1 and PD-L2 is associated with M2 tumor-associated macrophages and tumor differentiation in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5829.

Distribution of tumor-associated macrophages and M1/M2 polarization in different types and grades of ovarian tumors

Ovarian cancer represents one of the major causes of gynaecologic cancer mortality worldwide. Tumor-associated macrophages (TAM) are associated with the development and progression of ovarian cancer. TAMs are mainly represented by two types M1 and M2. We investigated the distribution of total, M1, M2 macrophages, tumor cell/macrophage ratio and M1/M2 ratio in mucinous and serous ovarian tumors. The study results showed that total TAM count is significantly higher in serous ovarian tumors, compared to mucinous tumors and the highest infiltration rate is detected in high-grade serous ovarian carcinomas. In addition, the number of M2 macrophages is significantly increased in higher-grade serous ovarian carcinomas. The evaluation of tumor/cell macrophage ratio could be used as an objective measure of macrophage infiltration in ovarian tumors.

Tumor-Derived C-C Motif Ligand 2 Induces the Recruitment and Polarization of Tumor-Associated Macrophages and Increases the Metastatic Potential of Bladder Cancer Cells in the Postirradiated Microenvironment

Radiation therapy (RT) is mainly used for bladder preservation in patients with muscle-invasive bladder cancer. The response of urothelial tumors to RT remains unsatisfactory. We investigated the interaction of RT and tumor-associated macrophages (TAMs) in the context of bladder cancer radioresistance. We evaluated the therapeutic effects of RT and TAM distribution by establishing an ectopic allograft mouse model. A Transwell coculture system was used to simulate the interaction between TAMs and MB49 bladder cancer cells in the tumor microenvironment. Cytokines and chemokines were analyzed in irradiated MB49 cells. Colony formation and Boyden chamber assays were used to assess the cytotoxic effects and the effects of TAMs on MB49 cell invasion, respectively. Local RT delayed primary tumor growth but promoted pulmonary metastases in C57BL/6 mice. Increased secretion of C-C motif chemokine ligand (CCL2) by irradiated MB49 cells, especially in the presence of M1-type TAMs, contributed to the infiltration of bone marrow-derived C-C motif chemokine receptor 2 (CCR2)-positive myeloid cells and the polarization of M1-type TAMs toward the M2 type to promote MB49 cell invasion. Blockade of CCL2-CCR2 activation by a CCR2 antagonist reversed the phenotypic TAM transformation and suppressed pulmonary metastases. Bladder cancer cells responded to RT by producing CCL2, which recruited TAM precursors from bone marrow and polarized M1-type TAMs toward the M2 type. This phenotypic TAM transformation promoted the pulmonary metastasis of bladder cancer cells after RT. Disrupting the CCL2-CCR2 signaling axis in combination with RT holds promise for improving RT efficacy in bladder cancer.

Giant foam-like macrophages in advanced ovarian cancer

Introduction. Ovarian cancer (OC) is the third most common gynecological cancer with the worst prognosis and highest mortality rate. The progression of OC can be accompanied by the detrimental functions of the components of the tumor microenvironment, including tumor-associated macrophages (TAMs).The purpose of the study to analyze distribution and morphological phenotype of TAMs in tumor tissue of patients with high-grade serous ovarian cancer (HGSOC).Material and methods. Formalin fixed paraffin embedded tissue sections were obtained from ovarian cancer patients after tumor resection. The protein expression of general macrophage marker CD68 and M2-like markers CD206, CD163 and stabilin-1, belonging to scavenger receptors, was analysed by immunohistochemical staining in tumor tissue. Histological assessment of TAM distribution was performed by pathologist. Immunofluorescent analysis/confocal microscopy was applied to establish the co-expression of CD68 with the main macrophage scavenger receptors.Results. We were able to find giant CD 68-positive macrophages with foamy cytoplasm in ovarian tumor tissue. The accumulation of these tams was specific only for patients with advanced stage (IIIC and IV stages). The presence of foamlike tams had a statistical tendency to be associated with ovarian cancer progression, including metastasis and recurrence. The distribution of stabilin-1-positive macrophages was matched to CD68 expression in almost all cases, as was shown by IHC. Confocal microscopy confirmed that stabilin-1 was expressed in at least 50 % of giant tams. If analysis of tumor samples also demonstrated co-expression of other scavenger receptors, CD163 and CD36, in foam-like cells. Similar to IHC, in most samples the expression of CD206 in tams of foam-like morphology was limited.Conclusion. For the first time we demonstrated the accumulation of giant macrophages with fluffy foam cytoplasm in the tumor tissue of treated patients with advanced ovarian cancer. Such macrophages express diverse scavenger receptors (stabilin-1, CD163, CD 36), thus indicating a high clearance activity of giant TAMs.

The Distinct Impact of TAM Infiltration on the Prognosis of Patients With Cardia and Non-Cardia Gastric Cancer and Its Association With H. pylori Infection.

BackgroundIn stage III gastric cancer (GC), the role of tumor-associated macrophages (TAMs) and Helicobacter pylori (H. pylori) infection impact tumor progression; however, the specific mechanisms remain controversial. We speculated whether this controversy is caused by differences in the location of TAM infiltration (in the core (CT) and invasive margin (MI) of primary tumors) and the topographical subsites of GC (cardia and non-cardia). Therefore, in this study, we investigated TAMs in different locations and H. pylori infection status as prognostic biomarkers for GC.MethodsImmunohistochemical staining for CD68 (pan-macrophage), CD163 (M2-like macrophage), and H. pylori in 200 samples (100 cases of cardia-GC [CGC] and 100 cases of non-cardia GC [NCGC]) was performed. We compared the number of CD68+ and CD163+ macrophages that infiltrated the CT and MI in patients with the prognosis of CGC and NCGC, respectively. In addition, we analyzed the relationship between H. pylori status and the prognosis of patients with GC in different locations, as well as the correlation with TAM infiltration.ResultsThe distribution of TAMs had distinct characteristics in CGC and NCGC, especially differences between CT and MI subtype. A Kaplan–Meier analysis showed that a high number of CD68+ macrophages that infiltrated the CT in CGC was associated with a better prognosis, whereas infiltration at the MI in NCGC indicated a poor prognosis. Furthermore, a high number of CD163+ macrophages infiltrating the MI resulted in a poor prognosis in CGC and NCGC cohorts. Considering the larger differences in the relationship between the infiltration of CD68+ macrophages at different locations and prognosis, we divided the GC cases into marginal and central GC, based on this difference. This resulted in an accurate estimation of the prognosis. Moreover, positive H. pylori status in central GC was significantly associated with a better prognosis and TAM infiltration.ConclusionTAMs in different locations and H. pylori status were identified as independent prognostic markers, with an obvious correlation between them. Therefore, it is important to clarify the impact of TAM location on the prognosis of patients with GC, which contributes to the development of potential therapeutic strategies.

A hybrid bacterium with tumor-associated macrophage polarization for enhanced photothermal-immunotherapy

Remodeling the tumor microenvironment through reprogramming tumor-associated macrophages (TAMs) and increasing the immunogenicity of tumors via immunogenic cell death (ICD) have been emerging as promising anticancer immunotherapy strategies. However, the heterogeneous distribution of TAMs in tumor tissues and the heterogeneity of the tumor cells make the immune activation challenging. To overcome these dilemmas, a hybrid bacterium with tumor targeting and penetration, TAM polarization, and photothermal conversion capabilities is developed for improving antitumor immunotherapy in vivo. The hybrid bacteria (B.b@QDs) are prepared by loading Ag2S quantum dots (QDs) on the Bifidobacterium bifidum (B.b) through electrostatic interactions. The hybrid bacteria with hypoxia targeting ability can effectively accumulate and penetrate the tumor tissues, enabling the B.b to fully contact with the TAMs and mediate their polarization toward M1 phenotype to reverse the immunosuppressive tumor microenvironment. It also enables to overcome the intratumoral heterogeneity and obtain abundant tumor-associated antigens by coupling tumor penetration of the B.b with photothermal effect of the QDs, resulting in an enhanced immune effect. This strategy that combines B.b-triggered TAM polarization and QD-induced ICD achieved a remarkable inhibition of tumor growth in orthotopic breast cancer.

Immunochemical analysis of iron transporters and M2 macrophages in ovarian endometrioma and clear cell adenocarcinoma.

The association between iron ions and endometriosis-associated ovarian cancer (EAOC) has been previously investigated to elucidate EAOC carcinogenesis; however, the dynamics of iron deposition in the endometrial epithelium and endometrial stroma of ovarian endometrioma (OE) remains unknown. The present study aimed to determine the expression of iron transporters on the cell surface and the distribution of tumor-associated macrophages (TAMs) englobed with iron in the endometrial stroma. The current retrospective study investigated 20 OE and 18 ovarian clear cell adenocarcinoma (CCC) samples, using Perls Prussian blue staining and immunohistochemistry of iron transporters, including divalent metal transporter 1 (DMT1), transferrin receptor (TfR) and ferroportin (FPN). Additionally, samples were stained for CD68, CD11c, CD163 and CD206, and double-immunostained for iron and CD163 to define the distribution of macrophages. Iron transporters were identified on the endometrial epithelium of OE and CCC tumor cells, and TAMs were englobed with iron in the endometrial stroma of OE and CCC. Histological findings revealed DMT1 upregulation in OE and CCC, whereas lower TfR and FPN expression was observed in OE than in CCC. M2 macrophages were englobed with iron ions in the deep layers of the OE and CCC stroma. The endometrial epithelium located in the endometrial stroma of one patient with OE and the endometrial epithelium adjacent to CCC in two patients with CCC stained positive for the tumor proliferation marker Ki67. Epithelium infiltrating the stroma of OE may become the origin of cancer under the influence of M2 macrophages englobed with iron. These findings provide new perspectives on the malignant transformation of OE into EAOC and its possibility as a precancerous index.

ARG1 mRNA Level Is a Promising Prognostic Marker in Head and Neck Squamous Cell Carcinomas.

Head and neck squamous cell carcinomas (HNSCC) can be induced by smoking or alcohol consumption, but a growing part of cases relate to a persistent high-risk papillomavirus (HPV) infection. Viral etiology has a beneficial impact on the prognosis, which may be explained by a specific immune response. Tumor associated macrophages (TAMs) represent the main immune population of the tumor microenvironment with a controversial influence on the prognosis. In this study, the level, phenotype, and spatial distribution of TAMs were evaluated, and the expression of TAM-associated markers was compared in HPV positive (HPV+) and HPV negative (HPV−) tumors. Seventy-three formalin and embedded in paraffin (FFPE) tumor specimens were examined using multispectral immunohistochemistry for the detection of TAM subpopulations in the tumor parenchyma and stroma. Moreover, the mRNA expression of TAM markers was evaluated using RT-qPCR. Results were compared with respect to tumor etiology, and the prognostic significance was evaluated. In HPV− tumors, we observed more pro-tumorigenic M2 in the stroma and a non-macrophage arginase 1 (ARG1)-expressing population in both compartments. Moreover, higher mRNA expression of M2 markers—cluster of differentiation 163 (CD163), ARG1, and prostaglandin-endoperoxide synthase 2 (PTGS2)—was detected in HPV− patients, and of M1 marker nitric oxide synthase 2 (NOS2) in HPV+ group. The expression of ARG1 mRNA was revealed as a negative prognostic factor for overall survival of HNSCC patients.

Tumor-associated macrophages: Harbingers of aggressiveness in oral squamous cell carcinoma.

Background:The significance of the tumor microenvironment (TME) as a modulator of tumor behavior was acknowledged by Hanahan and Weinberg in 2011 as the emerging hallmarks and enabling characteristics of the hallmarks of cancer. Subsequently, the role of inflammation, in conferring aggressiveness to a tumor, was regarded as a fundamental process in the evolution of the TME. Tumor-associated macrophages (TAMs) are distinctly polarized inflammatory cells and key shapers of a protumorigenic microenvironment.Aims:The aim of the study was to evaluate the distribution of TAMs and the expression of CD-163 as a marker to evince tumor aggressiveness, in oral squamous cell carcinoma (OSCC).Settings and Design:A retrospective institutional study was approved by the institutional ethics committee, and random sampling was carried out. Cases fulfilling the inclusion criteria were subjected to S(site), T(tumor), N( node), M( metastasis), P(pathology) STNMP staging along with immunohistochemical evaluation of CD-163.Methods:Samples for this study included 58 archival cases of OSCC. Demographic details were recorded, and the STNMP stage ascertained, following which, each case was reevaluated histopathologically for the invasive front. Cases with sufficient stroma and demonstrating the invasive front were further subjected to immunohistochemical evaluation of CD-163 for TAMs. The density of CD-163-positive cells was assessed by three pathologists, independently, in a double-blinded evaluation using Image-J©.Statistical Analysis:Descriptive data were evaluated and analyzed statistically using Spearman's/Pearson's correlation tests based on the distribution of data.Results:The density of TAMs was noted to be directly proportional to the STNMP stage. In addition, a strong positive, statistically significant correlation was noted between the density of TAMs and tumor size, nodal status and STNMP stage.Conclusion:The crucial role of the tumor microenvironment must be considered when evaluating OSCC. TAMs prove to be a reliable marker for assessing tumor aggressiveness and could aid in improved prognostication of OSCC, while also being potential targets for therapy.

CD163 as a Biomarker in Colorectal Cancer: The Expression on Circulating Monocytes and Tumor-Associated Macrophages, and the Soluble Form in the Blood.

The macrophage-associated molecule CD163 has been reported as a prognostic biomarker in different cancer types, but its role in colorectal cancer (CRC) is unclear. We studied CD163 in the tumor microenvironment and circulation of patients with CRC in relation to clinicopathological parameters. An enzyme-linked immunosorbent assay (ELISA) was used to measure the serum sCD163 levels and multiparameter flow cytometry was used to study the peripheral blood monocytes and their CD163 expression in CRC patients (N = 78) and healthy donors (N = 50). The distribution of tumor-associated macrophages (TAMs) was studied in primary colorectal tumors with multiplex immunofluorescence. We showed that CRC patients with above-median sCD163 level had a shorter overall survival (OS, p = 0.035) as well as disease-free survival (DFS, p = 0.005). The above-median sCD163 remained significantly associated with a shorter DFS in the multivariate analysis (p = 0.049). Moreover, a shorter OS was observed in CRC patients with an above-median total monocyte percentage (p = 0.007). The number and phenotype of the stromal and intraepithelial TAMs in colorectal tumors were not associated with clinical outcome. In conclusion, sCD163 and monocytes in the circulation may be potential prognostic biomarkers in CRC patients, whereas TAMs in the tumor showed no association with clinical outcome. Thus, our results emphasize the importance of the innate systemic immune response in CRC disease progression.

The prognostic and clinicopathological value of tumor-associated macrophages in patients with colorectal cancer: a systematic review and meta-analysis.

There is a growing literature on the significance of tumor-associated macrophages (TAMs) in colorectal cancer (CRC). However, the role of TAMs in predicting the prognosis of CRC remains controversial. The current study aims to determine the prognostic and clinicopathological value of different types and distribution of TAMs in CRC. A comprehensive literature search of PubMed, Embase, and Cochrane Library databases was conducted from the inception to 1 September 2019. The correlations of TAMs with overall survival (OS), disease-free survival (DFS), cancer-specific survival (CSS), and clinicopathological characteristics were analyzed. A total of 5,575 patients from 29 studies were included in this meta-analysis. The pooled hazard ratios (HRs) indicated that high density of pan-macrophages in tumor invasive margin (IM) was associated with better OS (HR = 0.57, 95%CI = 0.38-0.85), DFS (HR = 0.32, 95%CI = 0.19-0.52), and CSS (HR = 0.56, 95%CI = 0.41-0.77). Moreover, the high density of pan-macrophages in tumor center (TC) was correlated with better DFS (HR = 0.66, 95%CI = 0.45-0.96). However, high expression of M2 macrophages in TC was associated with poor DFS (HR = 2.42, 95%CI = 1.45-4.07) and CSS (HR = 1.74, 95%CI = 1.24-2.44). High M2 macrophages density in IM was also associated with short DFS (HR = 2.81, 95%CI = 1.65-4.77). In addition, the results showed that high density of pan-macrophages in IM was associated with no tumor metastasis, while high M2 macrophages density in TC was correlated with poor tumor differentiation. High Pan-TAMs density in IM has a positive effect on the prognosis of CRC patients, while high density M2 macrophage infiltration in TC is a strong indicator of poor prognosis.

Increased Vascular Adhesion Protein 1 (VAP-1) Levels are Associated with Alternative M2 Macrophage Activation and Poor Prognosis for Human Gliomas.

Glioma is characterized by a high heterogeneity in the brain tumor. Abundant tumor-associated macrophages (TAMs) exist as neoplastic tissues, implicating tumor plasticity and thus leading to therapeutic challenges. Vascular adhesion protein (VAP-1) potentially serves as a mediator for TAM immunity in tumor milieu. We previously demonstrated that VAP-1 could contribute to tumor malignancy, but its characteristics in TAM immunity of glioma progression are still unclear. This study explored the association of VAP-1 expression with TAM distribution as well as the resulting clinical significance and prognostic value in human gliomas. An in-depth analysis of AOC3 (VAP-1) gene expression was performed using 695 glioma samples derived from the cancer genome atlas (TCGA)-lower grade glioma and glioblastoma (GBMLGG) cohort. Bioinformatic analysis confirmed that VAP-1 expression is associated with poor prognosis of glioma patients (p = 0.0283). VAP-1 and TAM biomarkers (CD68, iNOS, and CD163) were evaluated by immunohistochemistry in 108 gliomas from Kaohsiung Medical University Hospital. VAP-1+ was expressed in 56 (51.85%) cases and this phenotype revealed a significant association with overall survival in Kaplan–Meier analysis (p < 0.0001). Immunohistochemical double staining showed that VAP-1 immunoreactivity was present around CD163+ M2 infiltration location, including aggressive lesions and neighboring neovasculature. We demonstrated that high VAP-1 expression levels positively correlated with CD163+ M2 activation and coexpression of these two proteins was associated with worse survival in gliomas (p < 0.0001). Multivariate analysis indicated that VAP-1 alone and co-expressed with CD163 were the significantly independent indicators (both p < 0.0001). Furthermore, VAP-1/CD163 coexpression exhibited excellent diagnostic accuracy in gliomas (AUC = 0.8008). In conclusion, VAP-1 and TAM CD163 M2 coexpression was found in glioma tissues belonging to a highly malignant subgroup that was associated with poor prognosis. These results implied VAP-1 abundance is closely linked to alternative M2 activation during glioma progression. From the aforementioned data, a reasonable inference is that VAP-1 combined with targeting M2 immunity might be an effective therapeutic target for human gliomas.

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.

Imaging and histopathologic correlates of plasma cell-free DNA concentration and circulating tumor DNA in adult patients with newly diagnosed glioblastoma.

BackgroundPlasma cell-free DNA (cfDNA) concentration is lower in glioblastoma (GBM) compared to other solid tumors, which can lead to low circulating tumor DNA (ctDNA) detection. In this study, we investigated the relationship between multimodality magnetic resonance imaging (MRI) and histopathologic features with plasma cfDNA concentration and ctDNA detection in patients with treatment-naive GBM.MethodsWe analyzed plasma cfDNA concentration, MRI scans, and tumor histopathology from 42 adult patients with newly diagnosed GBM. Linear regression analysis was used to examine the relationship of plasma cfDNA concentration before surgery to imaging and histopathologic characteristics. In a subset of patients, imaging and histopathologic metrics were also compared between patients with and without a detected tumor somatic mutation.ResultsTumor volume with elevated (>1.5 times contralateral white matter) rate transfer constant (Kep, a surrogate of blood–brain barrier [BBB] permeability) was independently associated with plasma cfDNA concentration (P = .001). Histopathologic characteristics independently associated with plasma cfDNA concentration included CD68+ macrophage density (P = .01) and size of tumor vessels (P = .01). Patients with higher (grade ≥3) perivascular CD68+ macrophage density had lower volume transfer constant (Ktrans, P = .01) compared to those with lower perivascular CD68+ macrophage density. Detection of at least 1 somatic mutation in plasma cfDNA was associated with significantly lower perivascular CD68+ macrophages (P = .01).ConclusionsMetrics of BBB disruption and quantity and distribution of tumor-associated macrophages are associated with plasma cfDNA concentration and ctDNA detection in GBM patients. These findings represent an important step in understanding the factors that determine plasma cfDNA concentration and ctDNA detection.

Macrophage spatial heterogeneity in gastric cancer defined by multiplex immunohistochemistry

Tumor-associated macrophages (TAMs), one of the most abundant immune components in gastric cancer (GC), are difficult to characterize due to their heterogeneity. Multiple approaches have been used to elucidate the issue, however, due to the tissue-destructive nature of most of these methods, the spatial distribution of TAMs in situ remains unclear. Here we probe the relationship between tumor context and TAM heterogeneity by multiplex immunohistochemistry of 56 human GC cases. Using distinct expression marker profiles on TAMs, we report seven predominant populations distributed between tumor and non-tumor tissue. TAM population-associated gene signatures reflect their heterogeneity and polarization in situ. Increased density of CD163+ (CD206−) TAMs with concurrent high CD68 expression is associated with upregulated immune-signaling and improved patient survival by univariate, but not multivariate analysis. CD68-only and CD206+ TAMs are correlated with high PDL1 expression.

PD-L1 expression on tumor-infiltrating immune cells is highly associated with M2 TAM and aggressive malignant potential in patients with resected non-small cell lung cancer

ObjectivesPD-L1 expression on tumor cells (TCs) and tumor-infiltrating immune cells (ICs) plays important roles in regulating the antitumor T cell response. However, the mechanistic and clinical significance of the effect of PD-L1 on TCs versus ICs remains unclear. On the other hand, tumor-associated macrophages (TAMs), M2 macrophages in particular, can promote tumor progression. MethodsWe evaluated PD-L1 expression on TCs and ICs using Ventana SP263 assay and the stromal M2 TAM distribution using CD163 staining in 160 consecutive patients with resected non-small cell lung cancer (NSCLC). ResultsPD-L1 expression on TCs and ICs was significantly higher in stromal M2 TAM-high group than in stromal M2 TAM-low group (p < 0.001 and p < 0.001, respectively). Regarding the clinical significance of PD-L1, PD-L1 expression on TCs was significantly associated with histology (p = 0.001), tumor differentiation (p < 0.001) and nodal status (p = 0.029). Furthermore, PD-L1 expression on ICs was significantly associated with histology (p < 0.001), tumor differentiation (p < 0.001), tumor status (p = 0.024), nodal status (p = 0.016), and pathologic stage (p = 0.004). The disease-free survival rate was significantly lower in patients with PD-L1-positive TC than in those with PD-L1-negative TC (p = 0.023), as well as in patients with PD-L1-positive IC than in those with PD-L1-negative IC (p < 0.001). Furthermore, the overall survival rate was significantly lower in patients with PD-L1-positive IC than in those with PD-L1-negative IC (p = 0.023). ConclusionsDuring tumor progression in NSCLC, the presence of M2 TAMs might affect PD-L1 expression both on TCs and ICs. In patients with NSCLC, PD-L1 expression both on TCs and ICs was associated with malignant behaviors, which was more in case of ICs.

Abstract 1138: Non-invasive detection and quantification of tumor-associated macrophage density with magnetic particle imaging

Abstract Background: There is increasing interest in the role of tumor-associated macrophages (TAMs) in promoting cancer growth and metastatic potential.(Obeid 2013) Increased TAM density has been correlated with poor prognosis and they can contribute up to 50% of the mass in breast tumors.(Bingle 2002) Previous studies have used cellular MRI to indirectly image the spatial distribution of TAMs, through the in situ uptake of superparamagnetic iron oxide (SPIO) nanoparticles (Makela 2017). However, iron-based MRI is limited by low specificity and challenging quantification. Magnetic Particle Imaging (MPI) is an emerging cellular imaging technique that can directly detect and quantify SPIO in vivo (Zheng 2016). MPI produces a positive contrast signal that is not attenuated by biological tissue. In this study, we investigated the first application of MRI and MPI to detect and differentiate between murine breast cancer models with varying metastatic potentials. Methods: Female BALB/c mice were implanted with 300,000 4T1 (n=3) or 168FARN (n=3) into the inguinal mammary fat pad. Tumors were grown for 3 weeks. 24 hours prior to imaging, 100μL of ferucarbotran (Magnetic Insight) was administered IV. One mouse from each group was first imaged on a 3T clinical MRI (GE Healthcare) with a custom-gradient insert located at Robarts Research Institute. A balanced steady-state free precession pulse sequence was used with imaging time of 30mins. The mice were then sacrificed and fixed in formalin. MPI was performed at Stanford University with the MPI system (Magnetic Insight) alongside three reference fiducials of known concentration. Iron quantification was performed with VivoQuant (inviCRO). Tumor tissue was extracted for histology and ex vivo imaging. Results: MRI signal voids were observed throughout the 4T1 tumor, but predominately in the tumor periphery. Fewer voids were observed in the 168FARN model, consistent with previous studies.3 MPI signal was observed in two out of three 4T1 tumors. Signal is seen across the entire tumor since the MPI is a sagittal projection compared to a single MRI slice. MPI quantification of SPIO in the 4T1 tumors indicated a consistent uptake, of 4.7 and 5.9 μg of iron. Signal was also detectable in the liver and tail at the site of injection. Fewer voids were observed in the 168FARN model, consistent with previous studies (Makela 2017). MPI signal was not detected in the tumors of the 168FARN group, consistent with the low iron uptake observed in the MRI. Histological analysis will be performed to verify the SPIO and macrophage distribution in tumor samples. Conclusions: By combining the high spatial resolution of MRI with the accurate quantification and specificity of MPI, additional information can be obtained on TAM presence and distribution. In addition to aiding therapeutic development, this information could be utilized to more accurately grade cancer. Citation Format: Jeffrey M. Gaudet, Ashley V. Makela, Paula J. Foster. Non-invasive detection and quantification of tumor-associated macrophage density with magnetic particle imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1138.

Highlights of This Issue

Tumor-associated macrophages (TAM) initiate and maintain a chronic inflammation in malignant tumors, which promotes tumor growth and metastasis. It becomes increasingly important to monitor the quantity and distribution of TAM in human tumors as new TAM-targeted cancer immunotherapies are developed. To solve this unmet clinical need, Aghighi and colleagues developed an MR imaging test for detection of TAM in sarcomas and lymphomas using the iron supplement ferumoxytol “off label” as an MRI-detectable TAM biomarker. In a first-in-human proof-of-concept clinical trial, the authors show that tumor ferumoxytol enhancement on MR images correlates with TAM quantities on histopathology.Bezrookove and colleagues examine the role of copy number elevations in the PHIP gene in melanoma progression using fluorescence in situ hybridization in three independent cohorts. The authors demonstrate an independent prognostic role for PHIP in primary melanoma, identify molecular subtypes of melanoma in which PHIP is enriched, and show that PHIP copy number is monotonically increased in the transition from primary to nodal to distant metastatic melanoma. Taken together with prior work showing that PHIP promotes melanoma metastasis in murine models, these results demonstrate a critical role for PHIP in melanoma progression, identifying PHIP as a promising target for therapy.To date, the epigenomic underpinnings of pituitary adenomas remains largely unexplored. Here, Salomon and colleagues leverage a multiomics study design to investigate the role of epigenetic alterations among PA subtypes. Genome-wide methylation analysis reveals that each pituitary adenoma subtype displays a disease-specific DNA methylation pattern. Furthermore, integrating DNA methylation and gene expression data revealed that hypomethylation of promoter regions drive increased expression of GH1 and SSTR5 genes in GH-secreting adenomas, and POMC gene in ACTH-secreting adenomas. The study suggests that epigenetic differences among pituitary adenoma subtypes play a crucial role and could be important in diagnosis and treatment decisions.Oncolytic viruses are demonstrating increasing promise as anticancer agents, with adenovirus (Ad) -based vectors proving popular. Efficacy could be enhanced through rational refinement of the capsid to maximize sequestration in cancerous cells. To achieve this, Uusi-Kerttula and colleagues redesigned the capsid to prevent uptake via all known native infectious routes and inserted a targeting peptide highly specific to the tumor-selective αvβ6 integrin. The resultant vector, Ad5NULL-A20, demonstrated exquisitely tumor-selective infection, minimal uptake in off-target organs, and significant efficacy in a model of ovarian cancer, thus providing a promising platform for future translational immuno-oncolytic applications.

Polarization and Distribution of Tumor-Associated Macrophages and COX-2 Expression in Basal Cell Carcinoma of the Ocular Adnexae

ABSTRACTPurpose: Basal cell carcinoma (BCC) is a locally invasive skin tumor which can be subdivided into a circumscribed nodular and an invasive fibrosing subtype. There is increasing evidence that macrophages play an important role in interacting between tumor cells and their microenvironment, thereby affecting not only the invasive potential but also the patients’ prognosis. Thus, we wanted to compare these two BCC variants with regard to tumor-related inflammation, COX-2 expression, distribution, and polarization of tumor-associated macrophages.Material and Methods: 30 BCCs (nodular: n = 15; fibrosing: n = 15) of the ocular adnexae were investigated by histopathology and immunohistochemistry. The grade of inflammation was evaluated on hematoxylin and eosin stains (score: 0–3). Immunohistochemical stains for CD68 (macrophages), Ki67 (proliferative activity), and COX-2 as well as immunofluorescence stains for CD68 and CD163 (to distinguish M1 and M2 macrophage subtypes) were performed. SPSS was used for statistical analysis.Results: Fibrosing BCCs were predominantly located on the lower lid, while nodular BCCs showed a broader distribution (p = 0.013). The fibrosing BCC subtype was associated with a higher degree of inflammation (p < 0.001) and revealed a higher COX-2 immunoreactivity than nodular BCC (p = 0.012). COX-2-positive cells were predominantly located on the infiltrating edge of the tumor. Macrophage polarization was balanced regarding M1 and M2 macrophage subtypes. There was no difference in macrophage number (p = 0.389) or polarization (p = 0.161) between nodular and fibrosing BCC.Conclusions: The findings indicate that COX-2 represents a factor for invasion of BCC. Macrophage polarization did not play a major role for aggressive behavior. However, other inflammatory components than tumor-associated macrophages seem to be involved in tissue destruction and thereby an invading growth pattern since fibrosing BCC revealed a significantly more intense inflammatory reaction in the surrounding tissue.