Tumor-associated Macrophages Density Research Articles

Correlation of tumor-associated macrophage density and proportion of M2 subtypes with the pathological stage of colorectal cancer.

Colorectal cancer (CRC) is a prevalent global malignancy with complex prognostic factors. Tumor-associated macrophages (TAMs) have shown paradoxical associations with CRC survival, particularly concerning the M2 subset. We aimed to establish a simplified protocol for quantifying M2-like TAMs and explore their correlation with clinicopathological factors. A cross-sectional study included histopathological assessment of paraffin-embedded tissue blocks obtained from 43 CRC patients. Using CD68 and CD163 immunohistochemistry, we quantified TAMs in tumor stroma and front, focusing on M2 proportion. Demographic, histopathological, and clinical parameters were collected. TAM density was significantly higher at the tumor front, with the M2 proportion three times greater in both zones. The tumor front had a higher M2 proportion, which correlated significantly with advanced tumor stage (P = 0.04), pathological nodal involvement (P = 0.04), and lymphovascular invasion (LVI, P = 0.01). However, no significant association was found between the M2 proportion in the tumor stroma and clinicopathological factors. Our study introduces a simplified protocol for quantifying M2-like TAMs in CRC tissue samples. We demonstrated a significant correlation between an increased M2 proportion at the tumor front and advanced tumor stage, nodal involvement, and LVI. This suggests that M2-like TAMs might serve as potential indicators of disease progression in CRC, warranting further investigation and potential clinical application.

Macrophage-Induced Carboxypeptidase A4 Promotes the Progression of Anaplastic Thyroid Cancer.

Background: The density of tumor-associated macrophages in the tumor microenvironment of anaplastic thyroid cancer (ATC) is associated with poor prognosis. However, the crosstalk between macrophages and ATC cells is poorly understood. This study aimed to examine the impact of macrophages on cancer cell phenotypes. We found a new mediator between M2 macrophages and ATC cells through proteomics analysis. Methods: The role of macrophages in proliferation, migration, and invasion of ATC cells was evaluated using coculture assay and conditioned medium (CM). Secretory factors in the CM from single or coculture were identified using liquid chromatography-tandem mass spectrometry proteomics analysis. We evaluated the role of the secretory factor in proliferation, migration, and invasion of cancer cells. In vivo xenograft model was used to evaluate the effect of the factor. Results: M2 macrophages significantly increased the proliferation, migration, and invasion of ATC cells, whereas M1 macrophages decreased the proliferation, migration, and invasion of ATC cells. Based on proteomic analysis of CM, we identify carboxypeptidase A4 (CPA4) as a mediator of the crosstalk between macrophages and ATC cells. CPA4 was only detected in the coculture media of M2 macrophage/8505C, and its expression in cancer cells increased by M2 macrophage. The expression of CPA4 protein was significantly higher in human thyroid cancers, particularly in ATCs, than normal and benign tissues. A bioinformatics analysis of public data revealed that CPA4 expression was associated with poor prognosis and dedifferentiation of thyroid cancer. Knockdown of CPA4 suppressed proliferation, colony formation, migration, and invasion of ATC cells, consistent with the decrease of STAT3, ERK, and AKT/mTOR phosphorylation and epithelial-mesenchymal transition (EMT) marker expression. In addition, the increased expression of CPA4 in cancer cells by M2 macrophage stimulation induced the polarization of macrophages to the M2 phenotype, which formed a positive feedback loop. Xenograft tumors did not develop after CPA4 knockdown. Conclusions: Our data suggest that CPA4 stimulates the progression of thyroid cancer by mediating between M2 macrophages and ATC cells. CPA4 can be a new therapeutic target for the treatment of patients with ATC.

Spatial distribution of tumor-associated macrophages in an orthotopic prostate cancer mouse model.

Mounting evidence suggests that the immune landscape within prostate tumors influences progression, metastasis, treatment response, and patient outcomes. In this study, we investigated the spatial density of innate immune cell populations within NOD.SCID orthotopic prostate cancer xenografts following microinjection of human DU145 prostate cancer cells. Our laboratory has previously developed nanoscale liposomes that attach to leukocytes via conjugated E-selectin (ES) and kill cancer cells via TNF-related apoptosis inducing ligand (TRAIL). Immunohistochemistry (IHC) staining was performed on tumor samples to identify and quantify leukocyte infiltration for different periods of tumor growth and E-selectin/TRAIL (EST) liposome treatments. We examined the spatial-temporal dynamics of three different immune cell types infiltrating tumors using QuPath image analysis software. IHC staining revealed that F4/80+ tumor-associated macrophages (TAMs) were the most abundant immune cells in all groups, irrespective of time or treatment. The density of TAMs decreased over the course of tumor growth and decreased in response to EST liposome treatments. Intratumoral versus marginal analysis showed a greater presence of TAMs in the marginal regions at 3 weeks of tumor growth which became more evenly distributed over time and in tumors treated with EST liposomes. TUNEL staining indicated that EST liposomes significantly increased cell apoptosis in treated tumors. Additionally, confocal microscopy identified liposome-coated TAMs in both the core and periphery of tumors, highlighting the ability of liposomes to infiltrate tumors by "piggybacking" on macrophages. The results of this study indicate that TAMs represent the majority of innate immune cells within NOD.SCID orthotopic prostate tumors, and spatial density varies widely as a function of tumor size, duration of tumor growth, and treatment of EST liposomes.

Prognostic and clinical significance of tumor-associated macrophages in esophageal squamous cell carcinoma after surgery: do biomarkers and distributions matter?

The role of tumor-associated macrophages (TAMs) in patients with esophageal squamous cell carcinoma (ESCC) following surgery remains controversial. Hence, we performed the present study to systematically analyze the prognostic and clinical significance of distinct TAMs biomarkers and distributions in ESCC patients underwent surgery. PubMed, Web of Science, and EMBASE databases were searched up to March 31, 2023. The pooled analysis was conducted to evaluate the effects of TAMs on overall survival (OS), disease-free survival (DFS), and clinicopathological characteristics using fixed-effects or random-effect model. Involving a total of 2,502 ESCC patients underwent surgery from 15 studies, the results suggested that the total count of CD68+ TAMs was inversely associated with OS and DFS in ESCC patients, which was also noticed in the relationship of CD68+ TAMs in tumor islet (TI) with OS (all P<0.05), although no association between CD68+ TAMs in tumor stroma (TS) and OS (P>0.05). Moreover, either islet or stromal CD163+ TAMs density was a prognostic factor ESCC (all P<0.05). Similarly, an elevated CD204+ TAMs density in TI predicted a poor DFS (P<0.05), although CD204+ TAMs in TI had no relationship with OS (P>0.05). Besides, a high CD68+ TAMs density was significantly associated with lymphatic vessel invasion, vascular invasion, and lymph node metastasis (all P<0.05). Our results demonstrated the prognostic and clinical significance of TAMs in ESCC patients underwent surgery. TAMs should be considered a target that could improve prognostic stratification and clinical outcomes in ESCC after surgery.

293 The Impact of Tumor Mutational Status on Protoporphyrin IX Accumulation in Tumor-Associated Macrophages in Glioblastoma Patients

INTRODUCTION: Immunotherapies provide promising new treatment options for lung cancer and melanoma, however success in glioblastoma (GBM) patients has been much rarer and uneven. Identifying candidates with GBM that are most likely to benefit from immunotherapy remains a challenge. The abundance of tumor-associated macrophages (TAMs) correlates response to immunotherapy in GBM patients. Here we leverage a new imaging technique for quantifying TAMs via tandem stimulated Raman histology (SRH) and two-photon excitation fluorescence microscopy (TPEF) to assess the impact of genetic classification on the abundance of TAMs. METHODS: We conducted tandem SRH and TPEF on specimens from 79 patients undergoing high-grade glioma resection with 5-ALA. We trained a MaskR-CNN deep neural network to identify TAMs and compute their density in each specimen. Corresponding clinical, demographic, genomic and methylomic data were collected for each patient. Independent sample t-tests were used to identify significant correlations between genetic markers, treatment, and TAM density. RESULTS: We analyzed 3,121,524 300 x 300 micron fields of view, from 151 fresh specimens, enabling segmentation of 611,789 TAMs. IDH-mutant tumors had significantly lower TAM density than IDH-wildtype tumors (p &lt; 0.01). The absence of MGMT promoter hypermethylation was associated with significantly higher TAM density (p = 0.03). Tumors with EGFR amplification averaged significantly lower TAM density than EGFR wildtype specimens (p &lt; 0.01). Recurrent tumors possessed 31.5% lower TAM density than primary resections. CONCLUSIONS: Tandem SRH and TPEF can estimate variations in TAM density in human GBMs. We found TAM density to be associated with IDH status, MGMT promoter methylation, and EGFR amplification. This imaging technique creates a new avenue for forecasting response to immunotherapy in GBM patients.

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.

MRI-derived radiomics assessing tumor-infiltrating macrophages enable prediction of immune-phenotype, immunotherapy response and survival in glioma

BackgroundThe tumor immune microenvironment can influence the prognosis and treatment response to immunotherapy. We aimed to develop a non-invasive radiomic signature in high-grade glioma (HGG) to predict the absolute density of tumor-associated macrophages (TAMs), the preponderant immune cells in the microenvironment of HGG. We also aimed to evaluate the association between the signature, and tumor immune phenotype as well as response to immunotherapy.MethodsIn this retrospective setting, total of 379 patients with HGG from three independent cohorts were included to construct a radiomic model named Radiomics Immunological Biomarker (RIB) for predicting the absolute density of M2-like TAM using the mRMR feature ranking method and LASSO classifier. Among them, 145 patients from the TCGA microarray cohort were randomly allocated into a training set (N=101) and an internal validation set (N=44), while the immune-phenotype cohort (N=203) and the immunotherapy-treated cohort (N=31, patients from a prospective clinical trial treated with DC vaccine) recruited from Huashan Hospital were used as two external validation sets. The immunotherapy-treated cohort was also used to evaluate the relationship between RIB and immunotherapy response. Radiogenomic analysis was performed to find functional annotations using RNA sequencing data from TAM cells.ResultsAn 11-feature radiomic model for M2-like TAM was developed and validated in four datasets of HGG patients (area under the curve = 0.849, 0.719, 0.674, and 0.671) using MRI images of post contrast enhanced T1-weighted (T1CE). Patients with high RIB scores had a strong inflammatory response. Four hub-genes (SLC7A7, RNASE6, HLA-DRB1 and CD300A) expressed by TAM were identified to be closely related to the RIB, providing important evidence for biological interpretation. Only individuals with a high RIB score were shown to have survival benefits from DC vaccine [DC vaccine vs. Placebo: median progression-free survival (mPFS), 10.0 mos vs. 4.5 mos, HR=0.17, P=0.0056, 95%CI=0.041-0.68; median overall survival (mOS), 15.0 mos vs. 7.0 mos, HR=0.17, P =0.0076, 95%CI=0.04-0.68]. Multivariate analyses also confirmed that treatment by DC vaccine was an independent factor for improved survival in the high RIB score group. However, in the low RIB score group, DC vaccine was not associated with improved survival. Furthermore, a radiomic nomogram based on the RIB score and clinical factors could efficiently predict the 1-, 2-, and 3-year survival rates, as confirmed by ROC curve analysis (AUC for 1-, 2- and 3-year survival: 0.705, 0.729 and 0.684, respectively).ConclusionsThe radiomic model could allow for non-invasive assessment of the absolute density of TAM from MRI images in HGG patients. Of note, our RIB model is the first immunological radiomic model confirmed to have the ability to predict survival benefits from DC vaccine in gliomas, thereby providing a novel tool to inform treatment decisions and monitor patient treatment course by radiomics.

Prognostic significance of annexin A2 and tumor associated macrophages (TAMs) in metastatic renal cell carcinoma and their relation to Sunitinib resistance

ABSTRACT Sunitinib, an antiangiogenic tyrosine kinase inhibitor, is the main treatment for metastatic renal cell carcinoma (mRCC). Development of resistance is a major obstacle against therapy success. The aim of this study was to assess annexin A2 and CD163+ tumor associated macrophages (TAMs) immunohistochemical expression in 50 mRCC cases as regard to patients’ prognosis and Sunitinib response. Also, to assess the correlation between annexin A2 and TAMs expression. High annexin A2 expression and TAMs density were associated with serum calcium level (P = 0.024 and 0.037, respectively), larger tumor size (P < 0.001), high tumor grade (P = 0.014 and <0.001, respectively), and the presence of tumor necrosis (P < 0.001). High annexin A2 and TAMs expressions were related to shorter patients’ overall survival (P = 0.009 and 0.001, respectively) and progression-free survival (P = 0.003 and 0.001, respectively). Annexin A2 was correlated with TAMs density (r = 0.890). Annexin A2 and TAMs are associated with poor prognostic parameters in mRCC patients, including high nuclear grade, increased tumor size, and the presence of tumor necrosis, together with shorter patients’ survivals and poor response to Sunitinib. Annexin A2 expression is correlated with TAMs density suggesting immunomodulatory role of annexin A2.

TMIC-84. A DEEP LEARNING-BASED METHOD FOR RAPID, PATIENT-SPECIFIC ASSAY OF MACROPHAGE INFILTRATION IN HIGH-GRADE GLIOMA USING LABEL-FREE STIMULATED RAMAN HISTOLOGY

Abstract BACKGROUND 5-Aminolevulinic acid (5-ALA), the most widely used fluorophore in image-guided glioma surgery, identifies tumor-associated macrophages (TAM) in high-grade glioma (HGG) tissue. Infiltrating macrophages, the predominant immune cells in glioma, are highly implicated in tumor progression, recurrence, and treatment response – particularly in immunotherapy. Using a unique, paired dataset of label-free stimulated Raman histology (SRH) and two-photon microscopy (TPEF) images sharing one-to-one spatial resolution, we developed a deep-learning approach to automatically quantify TAM infiltration from intraoperatively acquired SRH images. METHODS We compiled a dataset of 906 paired whole-slide SRH/TPEF images from 79 patients with HGG. We first trained a pix2pix generative adversarial network to convert raw, label-free SRH into synthetic TPEF with identifiable macrophages. Next, we trained a MaskR-CNN model to locate and segment TAMs. The pix2pix network was trained using 5,531 hand-picked 300-by-300-pixel fields-of-view (FOV) best exemplifying TAMs. We used a human-in-the-loop approach to train the segmentation network on 1,000 hand-labeled FOVs. RESULTS Macrophage segmentation from purely histologic data was near-identical to the fluorescence-based ground truth, with a mean dice score of 90.3%. Predicted TAM density was highly correlated with r=0.735. Subgroup analyses of TAM density in 346,836 non-overlapping FOVs revealed significantly lower TAM density in IDH-mutant (p&amp;lt; 0.01) and MGMT-hypermethylated (p=0.03) tumors. Our end-to-end algorithm uses fresh, unlabeled tissue specimens in the operating room and takes just three minutes to analyze whole-slide SRH images. CONCLUSION We demonstrate how deep neural networks can be used to rapidly and quantitatively evaluate macrophage infiltration in HGG in the operating room. Our software enables analysis of glioma patients’ tumor immune environment without immunohistochemistry or fluorescent labels, and may be leveraged to study the effects of immune-modulating therapies in the tumor microenvironment. Future work will focus on evaluating macrophage density and distribution as biomarkers for response to immunotherapies showing efficacy in glioma patients.

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.

Tumour-associated macrophages as a potential target to improve natural killer cell-based immunotherapies.

Adoptive transfer of natural killer (NK) cells has been proposed as a novel immunotherapy for malignant tumours resistant to current therapeutic modalities. Several clinical studies have demonstrated that the NK cell-infusion is well tolerated without severe side effects and shows promising results in haematological malignancies. However, patients with malignant solid tumours do not show significant responses to this therapy. Such disappointing results largely arise from the inefficient delivery of infused NK cells and the impairment of their functions in the tumour microenvironment (TME). Tumour-associated macrophages (TAMs) are the most abundant stromal cells in the TME of most solid tumours, and a high TAM density correlates with poor prognosis of cancer patients. Although our knowledge of the interactions between TAMs and NK cells is limited, many studies have indicated that TAMs suppress NK cell cytotoxicity against cancer cells. Therefore, blockade of TAM functions can be an attractive strategy to improve NK cell-based immunotherapies. On the other hand, macrophages are reported to activate NK cells under certain circumstances. This essay presents our current knowledge about mechanisms by which macrophages regulate NK cell functions and discusses possible therapeutic approaches to block macrophage-mediated NK cell suppression.

Expression of CD68+ Tumor associated macrophages in relation to β-catenin in carcinoma stomach.

With no unified system for tumor associated macrophages (TAMs) density assessment, limited information is available on their relationship with β-catenin expression. To evaluate the density of CD68+ TAMs in gastric adenocarcinoma samples by immunohistochemistry and correlate it with grade, stage, invasion, and beta-catenin. Formalin fixed paraffin embedded (FFPE) blocks from gastrectomy specimens of proven gastric adenocarcinoma were prospectively and retrospectively were studied over a period of two years. Immunohistochemistry with CD68 and β-catenin was performed. TAM density was qualitatively compared in "tumor" versus "stroma" and "tumor" versus "non-tumor" regions. Quantitative CD68+ TAM density was assessed using different methods and compared. Cases were classified as high and low TAM based on the median value and correlated with histologic type, location, grade, stage and β-catenin expression pattern. Spearman's rank correlation test was used to compare the different methods of TAM density evaluation. The categorical variables were studied using Pearson's Chi-square or Fisher's exact test. CD68+ TAM density and β-catenin expression were correlated by analysis of variance. A P value ≤ 0.05 was taken as statistically significant. The CD68+ TAMs in the "tumor" versus "non-tumor" area (p = 0.34) and "tumor" versus "stroma distribution" (p = 0.81) did not show any statistical significance. All methods of TAM density were found to be comparable. High TAM group is significantly associated with lymphovascular invasion, tumor depth, lymph node metastasis, and abnormal β-catenin expression. TAMs density plays an important role in the tumor stage. Macrophages may possibly induce gastric cancer invasiveness by activating β-catenin pathway.

Tumor-infiltrating lymphocytes and tumor-associated macrophages as potential predictors of lymph node metastases in major salivary gland cancers.

The purpose of this study is to define if tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) could represent potential predictors of lymph node metastases (LNM) in salivary gland cancers (SGC). A selected number of immunohistochemical markers related to TILs (CD3, CD4, CD68, and FOXP3) and TAMs (CD68 and CD163) were investigated on major salivary gland cancers. TIL and TAM densities were measured on digital images using the open-source QuPath both in the tumor interior (TI) and invasive margin (IM). Correlation with pathologic N classification and follow-up clinical data was investigated. A total of 25 consecutive patients (men: 11; median age: 62.0) were included. Densities of CD3+ IM (OR = 7.7, 95% CI 1.2-51.2), CD8+ TI (OR = 7.7, 95% CI 1.2-51.2), CD8+ IM (OR = 7.7, 95% CI 1.2-51.2), FOXP3+ TI (OR = 24.0, 95% CI 2.2-255.9), CD68+ TI (OR = 7.7, 95% CI 1.2-51.2), and CD163+ IM (OR = 7.7, 95% CI 1.2 - 51.2), and the Immunoscore CD8/CD3 (OR = 1.9, 95% CI 1.1-3.4) were significantly associated with LNM (p < 0.05). CD3+ TI density was significantly associated with tumor recurrence and death (HR = 5.8, 95% CI 1.5-22.6; p < 0.05). A high density of specific TIL and TAM subpopulations might be correlated with a higher probability of LNM in SGC.

Prognostic and predictive value of CD163 expression and the CD163/CD68 expression ratio for response to adjuvant chemotherapy in patients with surgically resected lung squamous cell carcinoma.

Macrophages infiltrating the tumor microenvironment are defined as tumor-associated macrophages (TAMs). TAMs can be polarized into different phenotypes, that is, proinflammatory M1 macrophages or anti-inflammatory M2 macrophages. Particularly, M2 macrophages promote angiogenesis, wound healing, and tumor growth. This study aimed to evaluate whether M2 TAMs can serve as a useful marker to predict prognosis and benefit from adjuvant chemotherapy in patients with surgically resected lung squamous cell carcinomas (SCCs). We examined 104 patients with SCC. Tissue microarrays were constructed, and the density of TAMs was analyzed by immunohistochemistry for expression of CD68 and CD163. The relationship between CD68 and CD163 expression and the CD163/CD68 expression rate and clinicopathological characteristics including patient outcomes were investigated. In addition, propensity score matching (PSM) analysis was conducted to test the hypothesis that these cells significantly influenced chemotherapy responses. Univariate analysis revealed that pathological stage, CD163 expression, and the CD163/CD68 expression ratio were significant prognostic factors. Multivariate analysis showed that these factors were all independent prognostic factors. Thirty-four pairs were determined by using PSM analysis. Patients with a low CD163/CD68 expression ratio benefited more from adjuvant chemotherapy than those with a high ratio. We suggest that M2 TAMs may be a useful marker to predict prognosis and differential benefit from adjuvant chemotherapy in patients with surgically resected lung SCCs.

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 &amp;lt; 0.0001 and P &amp;lt; 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 &amp;lt; 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 &amp;lt; 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 &amp;lt; 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.

Abstract 2480: Chemotherapy increases invasive cancer stem cells in breast cancer

Abstract Background: Metastasis is the leading cause of breast cancer (BC) death. To successfully metastasize, tumor cells must have both invasive and stem-like phenotypes. BC cells that express MenaINV, an invasive isoform of the actin regulatory protein Mena, have an enhanced ability to migrate and intravasate within the primary tumor, and extravasate at secondary sites. BC cells with a stem-like phenotype can withstand anoikis during hematogenous dissemination and initiate metastatic growth. Therefore, MenaINVHigh cancer stem cells (CSCs) have high metastatic potential and can be considered the most dangerous cells in the tumor. We recently showed that MenaINV and the CSC program can be induced independently in BC cells via direct contact with tumor associated macrophages (TAMs). Though chemotherapy improves survival, recent evidence shows that for some BC patients, chemotherapy may increase metastatic dissemination, though the reasons for this are unclear. Chemotherapy causes extensive cell death and a proinflammatory cytokine surge that recruits macrophages to the primary tumor and enhances the assembly of the tumor cell dissemination portals TMEM doorways. Given that TAM-tumor cell contact induces MenaINV and the CSC program in BC cells, we hypothesized that chemotherapy promotes dissemination by increasing TAM-tumor cell contact, leading to the induction of MenaINVHigh cells, CSCs, and “double positive” MenaINVHigh CSCs. Methods: We used two models of BC (MMTV-PyMT mice bearing spontaneous mammary tumors, and HT17 patient-derived xenografts) which recapitulate metastatic triple-negative BC in mice. Mice were treated with paclitaxel (10 mg/kg, IV) every 5 days for a total of 3 treatments. Mouse subgroups received an additional treatment of liposomes with the macrophage-depleting agent clodronate (200 µL, 18.4 µM concentration, IP), or PBS control liposomes, every 2 days. Mammary tumors were evaluated for: (1) TAM density, (2) number of “single positive” cells - defined as those expressing high levels of Sox9 (CSC marker) or MenaINV (invasion marker) - and (3) the number of “double positive” MenaINVHigh CSCs (Sox9HighMenaINVHigh). Results: We found that chemotherapy increased TAM density, and this corresponded with an increase in “double positive” MenaINVHigh CSCs. Upon macrophage depletion, MenaINVHigh CSCs fell to below baseline levels regardless of chemotherapy treatment. Chemotherapy did not increase “single positive” cells. Conclusion: The induction of prometastatic MenaINVHigh CSCs during chemotherapy is macrophage dependent. The finding that chemotherapy only affected double positive cells suggests that MenaINV and the CSC program are induced together, rather than independently, by TAMs. Further work is required to determine the mechanism of this co-induction. These results lay the groundwork for novel microenvironment-based therapies to alleviate the prometastatic effects of chemotherapy in BC. Citation Format: Madeline Friedman, Camille L. Duran, Ved P. Sharma, John S. Condeelis, Maja H. Oktay, George S. Karagiannis, David Entenberg. Chemotherapy increases invasive cancer stem cells in breast 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 2480.

Abstract 4639: Single-cell immunoprofiling and spatial analysis of hormone receptor subtypes in HER2+ and HER2low breast tumors using multiplexed immunofluorescence

Abstract Background: Breast cancer is characterized by distinct molecular subtypes based on expression of estrogen and progesterone hormone receptors (ER and PR), and epidermal growth factor receptor 2 (HER2). To investigate presence and location of distinct immune cell populations on single cell level we utilized the multiplexed immunofluorescence (mIF) platform MultiOmyx to investigate the tumor immune microenvironment (TME) in HER2+ breast cancer. We have built on growing data implicating distinct immunophenotypes in the breast cancer TME with breast cancer outcomes by profiling 1) prevalence; 2 location (e.g. intratumoral, stromal); 3) phenotype (e.g. activated, exhausted) of infiltrating immune cells. Methods: We optimized a custom 26-marker MultiOmyx panel interrogating HER2 IF expression, HER2 signaling, and immune markers. This mIF platform leverages serial IF image capture to allow concurrent profiling of all 26 markers on a pathologic section at single cell resolution. We applied the 26-marker panel to a tissue microarray of 208 unique patients with matched tumor/normal tissue cores (1-4 cores/patient; total 333 tumor and 307 normal cores). HER2-positive was defined via ASCO/CAP guidelines; HER2-low was defined as HER2 immunohistochemistry (IHC) 1+/2+ but HER2 in-situ hybridization (ISH) negative. Results: The 208 unique tumors profiled included 88.9% (185/208) HER2-positive and 11.1% (23/208) HER2-low; 62.5% (130/208) hormone receptor (HR) positive and 37.5% (78/208) HR negative. Median follow-up from diagnosis was 143 months. Among HER2-positive patients, 98.9% (n=183/185) received HER2-directed therapy in the (neo)adjuvant or metastatic setting or were diagnosed prior to FDA approval of trastuzumab for early stage disease. In sum, from 1166 regions of interest in 640 total cores, a total of 1,076,700 single cells were profiled via the 26-marker panel. Tumors categorized based on HR and HER2 reflected distinct immunophenotype. Relative to HR+HER2low, HR-HER2low tumors had a significant increase in the density of T cells (CD3) and tumor-associated macrophages (TAMs) (CD68). The increase in T cells was observed for T helper (CD3+CD4+), T regulatory (CD3+CD4+FoxP3+), as well as T cytotoxic cells (CD3+CD8+). Observed increases in pro-tumorigenic M2 TAM density (CD68+CD163+) were observed for both HR negative subtypes when compared to HR+HER2+ and HR+HER2low groups, indicating a negative correlation between M2 TAM infiltration and ER/PR status. HER2low tumors had significantly lower tumor PDL1+ than HER2+ tumors. Additionally, 18 of the 208 patients with equal distribution across the hormone-receptor subtypes underwent whole slide multiplexed analysis to perform a deeper spatial analysis. Conclusions: In a large, clinically annotated cohort of breast cancers distinct immunophenotypes are evident among HR and HER2+ subsets. Citation Format: Anna Juncker-Jensen, David Tallman, Harry Nunns, Heather Lefebvre, Karen Yamamoto, Katharine A. Collier, Mark Vater, Ava Strahan, Ava Willoughby, Olivia Bouchard, Madison Kingsbury, Mathew Cherian, Ashley C. Pariser, Preeti K. Sudheendra, Bhuvaneswari Ramaswamy, Margaret Gatti-Mays, Ainura Kyshtoobayeva, Zaibo Li, Daniel G. Stover. Single-cell immunoprofiling and spatial analysis of hormone receptor subtypes in HER2+ and HER2low breast tumors using multiplexed immunofluorescence. [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 4639.

Prognostic significance of tumor-associated macrophages polarization markers in lung cancer: a pooled analysis of 5105 patients.

The prognostic significance of tumor-associated macrophages (TAMs) in patients with lung cancer (LCa) remains controversial. We therefore conducted the present study to systematically evaluate the role of different TAMs markers and histologic locations on the prognosis of LCa. Searches of Web of Science, PubMed, and EMBASE databases were performed up to 28 February 2022. The pooled analysis was conducted in random-effect or fixed-effects model with hazard risk (HR) and 95% confidence interval (CI) for survival data including overall survival (OS), and disease-free survival (DFS) from raw or adjusted measures, according to different TAMs markers and histologic locations. Including a total of 5105 patients from 30 eligible studies, the results indicated that the total count of CD68+ TAMs was negatively associated with OS and DFS, which was also observed in the relationship of CD68+ or CD204+ TAMs in tumor stroma (TS) with OS and DFS (all P<0.05). Conversely, higher CD68+ TAMs density in tumor nest (TN) or TN/TS ratio of CD68+ TAMs predicted better OS (all P<0.05). Similarly, higher HLA-DR+ TAMs density was correlated with better OS in TN and TS (all P<0.05). Besides, neither nest CD163+ TAM density nor stromal CD163+ TAM density was a prognostic factor in LCa patients (all P>0.05). Our study indicated that different TAMs markers and histologic locations could bring about different prognostic effects in LCa patients. Great understanding of the infiltration modes of TAMs may contribute to improve outcomes of LCa patients.

PD-L1 immunohistochemistry assay optimization to provide more comprehensive pathological information in classic Hodgkin lymphoma

Overexpression of PD-L1 can be a predictive marker for anti-PD-1 therapeutic efficacy in classic Hodgkin lymphoma (CHL); however, harmonization of different IHC assays remains to be accomplished, and interpretations of PD-L1 immunostaining results remain controversial in CHL. In this study, we sought to optimize the PD-L1 immunohistochemistry (IHC) assay in CHL. All tests were performed on a tumour tissue microarray established from 54 CHL cases. Three IHC antibodies (405.9A11, SP142, 22C3) for detecting PD-L1 expression were compared semi quantitatively with the RNAscope assay (No. 310035, ACD), and the difference in the expression in background immune cells (ICs) between assays and the associations of expression levels with densities of TILs/TAMs were also analysed. 405.9A11 demonstrated best specificity in HRS cells and best sensitivity in ICs. Positive expression of PD-L1 was more frequent in ICs (85.2%) than in HRS cells (48.1%). Different subgroups of background ICs, including tumour-associated macrophages (TAMs), were assessed and scored for CD4, CD8, FOXP3, and CD163 expression. PD-L1 expression on ICs was the factor most associated with the density of TAMs. 405.9A11 provided the most convincing PD-L1 expression results. Pathologists should report PD-L1 expression in a combined manner, including both the status of HRS cells and the percentage of PD-L1-positive ICs.

NLRP3 in tumor-associated macrophages predicts a poor prognosis and promotes tumor growth in head and neck squamous cell carcinoma.

The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome plays cell- and tissue-specific roles in cancer, meaning that its activation in different tumors or cells may play different roles in tumor progression. We have previously described the tumor-promoting function of tumor-intrinsic NLRP3/IL-1β signaling in head and neck squamous cell carcinoma (HNSCC), but its role in immune cells remains unclear. In this study, we found that NLRP3 was highly expressed in tumor-associated macrophages (TAMs) in both mouse and human HNSCC, and the expression of NLRP3 was positively correlated with the density of TAMs according to immunohistochemistry, immunofluorescence, and flow cytometry analyses. Importantly, the number of NLRP3high TAMs was related to worse overall survival in HNSCC patients. Knocking out NLRP3 inhibited M2-like macrophage differentiation in vitro. Moreover, the carcinogenic effect induced by 4-nitroquinoline-1-oxide was decreased in Nlrp3-deficient mice, which had smaller tumor sizes. Genetic depletion of NLRP3 reduced the expression of protumoral cytokines, such as IL-1β, IL-6, IL-10, and CCL2, and suppressed the accumulation of TAMs and myeloid-derived suppressor cells (MDSCs) in mouse HNSCC. Thus, activation of NLRP3 in TAMs may contribute to tumor progression and have prognostic significance in HNSCC.