Tumor-associated Macrophages Density Research Articles

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.

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.

Crosstalk between tumor-associated macrophages and tumor cells promotes chemoresistance via CXCL5/PI3K/AKT/mTOR pathway in gastric cancer

Background5-fluorouracil (5-FU)-based chemotherapy regimen has been widely used for the treatment of gastric cancer, but meanwhile the development of chemotherapeutic resistance remains a major clinical challenge. Tumor microenvironment (TME) frequently correlates with the development of chemoresistance in human cancer. As a major component of TME, the role of tumor-associated macrophages (TAMs) in the chemoresistance of gastric cancer has not been fully elucidated.MethodsImmunohistochemistry (IHC) was applied to detect the density of TAMs in clinical samples of 103 patients with gastric cancer who had undergone 5-FU-based neoadjuvant chemotherapy. 5-FU-resistant gastric cell lines MKN45-R and HGC27-R were established, macrophages were then separately co-cultured with MKN45-R, HGC27-R cells and their parental cells. The effect of gastric cancer cells on the polarization of macrophages, the biological function of M2-polaried macrophages and the mechanism for promoting 5-FU-resistance were investigated. Then the correlation between the expression of CXC motif chemokine ligand 5 (CXCL5) and the infiltration of hemoglobin scavenger receptor (CD163) positive and mannose receptor (CD206) positive macrophages was analyzed, the prognostic value of CXCL5 expression in clinical samples was further explored.ResultsThe high infiltration of macrophages marked by CD68 in gastric cancer samples was significantly associated with the resistance of gastric cancer to chemotherapy. Gastric cancer cells could modulate macrophages to M2-like polarization through indirect co-culture, and chemoresistant cells were more efficient in inducing macrophages polarization to M2 phenotype. Co-culturing M2-polarized macrophages in turn enhanced 5-FU-resistance of gastric cancer cells, and it was further verified that CXCL5 derived from M2-polarized macrophages promoted chemoresistance through activing the PI3K/AKT/mTOR pathway. Besides, high level of CXCL5 could recruit monocytes to form more M2-polarized macrophages. Clinically, high expression of CXCL5 in gastric cancer samples was associated with the high infiltration of CD163 positive macrophages and CD206 positive macrophages, and patients with high expression of CXCL5 presented lower overall survival (OS) rates than those with low expression of CXCL5.ConclusionInteraction between TAMs and gastric cancer cells promoted chemoresistance in gastric cancer via CXCL5/PI3K/AKT/mTOR pathway. Thus, targeting TAMs and blocking the cell–cell crosstalk between TAMs and gastric cancer cells may represent prospective therapeutic strategies for patients with gastric cancer.

Wnt2b and Wnt5a expression is highly associated with M2 TAMs in non‑small cell lung cancer.

Tumor-associated macrophages (TAMs), particularly M2 macrophages, promote tumor progression, while Wnt genes encode a family of multi-functional glycoproteins that serve an important role in tumorigenesis. Immunohistochemical studies were performed to evaluate Wnt2b and Wnt5a expression in tumor and stromal cells in M2 and M1 TAMs and Ki-67 proliferation index in 160 consecutive patients with resected non-small cell lung cancer (NSCLC). Overall, 52 tumors (32.5%) were classified as tumoral Wnt2b-high (Wnt2b-positive tumor cells >30%) and 95 (59.4%) as stromal Wnt2b-high (Wnt2b-positive stromal cells >30%), while 75 (46.9%) were classified as tumoral Wnt5a-high (Wnt5a-positive tumor cells >30%) and 63 (39.4%) as stromal Wnt5a-high (Wnt5a-positive stromal cells >28%). The density of M2 TAMs was significantly higher in the tumoral (P=0.0024) and stromal Wnt2b-high groups (P=0.0054). The density of M2 TAMs was also significantly higher in the tumoral (P=0.0005) and stromal Wnt5a-high groups (P=0.0486). By contrast, no difference in stromal or islet M1 TAM density was observed in relation to tumoral or stromal Wnt2b or Wnt5a status. Furthermore, Ki-67 proliferation index was significantly higher in the tumoral (P=0.0121) and stromal Wnt2b-high (P=0.0019) and tumoral Wnt5a-high (P=0.0088) groups. Overall survival rate was significantly lower in the Wnt2b-high (P=0.0437), Wnt5a-high (P=0.0106) and M2 TAM-high (P=0.0060) groups. Wnt2b and Wnt5a expression in tumor and stromal cells may induce M2 TAMs to produce more aggressive behavior during tumor progression in NSCLC.

The Roles of Tumor-Associated Macrophages in Prostate Cancer.

The morbidity of prostate cancer (PCa) is rising year by year, and it has become the primary cause of tumor-related mortality in males. It is widely accepted that macrophages account for 50% of the tumor mass in solid tumors and have emerged as a crucial participator in multiple stages of PCa, with the huge potential for further treatment. Oftentimes, tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) behave like M2-like phenotypes that modulate malignant hallmarks of tumor lesions, ranging from tumorigenesis to metastasis. Several clinical studies indicated that mean TAM density was higher in human PCa cores versus benign prostatic hyperplasia (BPH), and increased biopsy TAM density potentially predicts worse clinicopathological characteristics as well. Therefore, TAM represents a promising target for therapeutic intervention either alone or in combination with other strategies to halt the “vicious cycle,” thus improving oncological outcomes. Herein, we mainly focus on the fundamental aspects of TAMs in prostate adenocarcinoma, while reviewing the mechanisms responsible for macrophage recruitment and polarization, which has clinical translational implications for the exploitation of potentially effective therapies against TAMs.

Machine learning models demonstrate that clinicopathologic variables are comparable to gene expression prognostic signature in predicting survival in uveal melanoma

PurposeSince molecular assays are not accessible to all uveal melanoma patients, we aim to identify cost-effective prognostic tool in risk stratification using machine learning models based on routine histologic and clinical variables. Experimental designWe identified important prognostic parameters in a discovery cohort of 164 enucleated primary uveal melanomas from 164 patients without prior therapies. We then validated the prognostic prediction of top important parameters identified in the discovery cohort using 80 uveal melanomas from the Tumor Cancer Genome Atlas database with available gene expression prognostic signature (GEPS). The performance of three different survival analysis models (Cox proportional hazards (CPH), random survival forest (RSF), and survival gradient boosting (SGB)) was compared against GEPS using receiver operating curves (ROC). ResultsIn all three selection methods, BAP1 status, nucleoli size, age, mitotic rate per 1 mm2, and ciliary body infiltration were identified as significant overall survival (OS) predictors; and BAP1 status, nucleoli size, largest basal tumor diameter, tumor-infiltrating lymphocyte density, and tumor-associated macrophage density were identified as significant progression-free survival (PFS) predictors. ROC plots for the median survival time point showed that significant parameters in SGB studied model can predict OS better than GEPS. For PFS, SGB model performed similarly to GEPS. The time-dependent area under the curve (AUC) showed SGB model performing better than GEPS in predicting OS and metastatic risk. ConclusionsOur study shows that routine histologic and clinical variables are adequate for patient risk stratification in comparison with not readily accessible GEPS.

M-CSFR expression in the embryonal component of hepatoblastoma and cell-to-cell interaction between macrophages and hepatoblastoma.

Tumor-associated macrophages (TAMs) have protumor functions in various cancers. However, their significance in hepatoblastoma, the most common liver tumor in children, remains unclear. The aim of this study was to explore the potential roles of TAMs in hepatoblastoma. Immunohistochemical analysis revealed that the density of CD204-positive TAMs was significantly higher in the embryonal component than in other histological subtypes of hepatoblastoma. An in vitro co-culture study with Huh6 cells and human monocyte-derived macrophages (HMDMs) showed that macrophage-colony-stimulating factor receptor (M-CSFR) was strongly up-regulated in the Huh6 cells that were directly co-cultured with HMDMs. The expressions of M-CSFR ligands (interleukin-34 and M-CSF) were also increased by co-culture with HMDMs. The proliferation of HepG2 cells (another hepatoblastoma cell line expressing M-CSFR) was inhibited by an M-CSFR inhibitor. M-CSFR was found to be highly expressed in the embryonal component and in recurrent lesions. The number of CD204-positive macrophages was also higher in the M-CSFR-positive areas than in the M-CSFR-negative areas. Thus, M-CSFR expression appeared to be induced by cell-cell contact with macrophages in hepatoblastoma cells, and M-CSFR inhibitor is potentially effective against M-CSFR-positive hepatoblastoma, especially recurrent cases.

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.

Tumor-associated macrophages and risk of recurrence in stage III colorectal cancer.

Tumor‐associated macrophages (TAMs) have a unique favorable effect on the prognosis of colorectal cancer (CRC), although their association with stage‐specific outcomes remains unclear. We assessed the densities of CD68+ and CD163+ TAMs at the invasive front of resected CRC stage III CRC from 236 patients, 165 of whom received post‐surgical FOLFOX treatment, and their relationship with disease‐free survival (DFS). Associations between macrophage mRNAs and clinical outcome were investigated in silico in 59 stage III CRC and FOLFOX‐treated patients from The Cancer Genome Atlas (TCGA). Biological interactions of SW480 and HT29 cells and macrophages with FOLFOX were tested in co‐culture models. Low TAM densities were associated with shorter DFS among patients receiving FOLFOX (CD68+, p = 0.0001; CD163+, p = 0.0008) but not among those who were untreated. By multivariate Cox analysis, only low TAM (CD68+, p = 0.001; CD163+, p = 0.002) and nodal status (CD68+, p = 0.009; CD163+, p = 0.007) maintained an independent predictive value. In the TCGA cohort, high CD68 mRNA levels were associated with better outcome (p = 0.02). Macrophages enhanced FOLFOX cytotoxicity on CRC cells (p < 0.01), and drugs oriented macrophage polarization from M2‐ to M1‐phenotype. Low TAM densities identify stage III CRC patients at higher risk of recurrence after adjuvant therapy, and macrophages can augment the chemo‐sensitivity of micro‐metastases.

Proliferative Potential, and Inflammatory Tumor Microenvironment in Meningioma Correlate with Neurological Function at Presentation and Anatomical Location-From Convexity to Skull Base and Spine.

Simple SummaryThe World Health Organization (WHO) classification grades meningiomas exclusively due to their histopathological features. Meningiomas are predominantly benign intracranial entities, and surgical resection represents the therapy of choice. However, risk of progression and tailored scheduling of follow-up appointments are significantly influenced by various items, such as immunohistochemistry (e.g., MIB-1 index). Emerging evidence focuses attention on the anatomic location of meningiomas, especially regarding the differentiation between skull base and non-skull base meningiomas. In the present study, we therefore investigated demographic, histopathological, and laboratory variables regarding their association with the anatomic location. We found that spinal meningiomas have a significantly lower proliferative activity, less density of macrophage infiltrates, and a longer time to tumor progression. Moreover, increased MIB-1 indices are significantly associated with location-specific baseline symptoms (e.g., convexity: seizure burden, medial skull base: decreased vision, spinal: ambulatory ability). Therefore, anatomic location might be considered as a future subclassification in the grading of the prognosis of meningiomas.Emerging evidence emphasizes the prognostic importance of meningioma location. The present investigation evaluates whether progression-free survival (PFS), proliferative potential, World Health Organization (WHO) grades, and inflammatory burden differ between anatomical locations (skull base, non-skull base, and spinal) meningiomas. Five-hundred-forty-one patients underwent Simpson grade I or II resection for WHO grade 1 or 2 meningiomas. Univariable analysis revealed that spinal meningioma patients are significantly older, had a worse baseline Karnofsky Performance Status (KPS), higher acute-phase protein levels, lower incidence of WHO grade 2, lower mitotic counts, lower MIB-1 index, and less CD68+ macrophage infiltrates. Multivariable analysis identified WHO grade 2 (OR: 2.1, 95% CI: 1.1–3.7, p = 0.02) and cranial location (OR: 3.0, 95% CI: 1.8–4.9, p = 0.001) as independent predictors of diffuse CD68+ macrophage infiltrates. The mean PFS in cranial meningiomas was 115.9 months (95% CI: 107.5–124.3), compared to 162.2 months (95% CI: 150.5–174.0; log-rank test: p = 0.02) in spinal meningiomas. Multivariable Cox regression analysis revealed cranial location as an independent predictor (HR: 4.7, 95% CI: 1.0–21.3, p = 0.04) of shortened PFS. Increased MIB-1 indices ≥5% were significantly associated with location-specific deficits at presentation, such as decreased vision and seizure burden. Spinal meningiomas have a significantly longer PFS time and differ from the cranial meningiomas regarding MIB-1 index and density of tumor-associated macrophages.

Differential expression of PD‑L1 and PD‑L2 is associated with the tumor microenvironment of TILs and M2 TAMs and tumor differentiation in non‑small cell lung cancer.

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 both tumor cells (TCs) and tumor-infiltrating immune cells (ICs) was investigated, with respect to tumor-infiltrating lymphocytes (TILs) and M2 tumor-associated macrophages (TAMs), which are key components of the tumor microenvironment, in 175 patients with resected NSCLC. The TIL and M2 TAM densities were associated with the expression of PD-L1 on the two TCs (both P<0.0001) and ICs (both P<0.0001). The TIL and M2 TAM densities were also associated with the expression of PD-L2 on both TCs (P=0.0494 and P=0.0452, respectively) and ICs (P=0.0048 and P=0.0125, respectively). 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 the PD-L1 expression on TCs and ICs (P=0.0002 and P<0.0001, respectively). By contrast, tumor differentiation was inversely associated with the PD-L2 expression on both TCs and ICs (P=0.0260 and P=0.0326, respectively). In conclusion, 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.

Spatial Metrics of Interaction between CD163-Positive Macrophages and Cancer Cells and Progression-Free Survival in Chemo-Treated Breast Cancer.

Simple SummaryA majority of breast cancer deaths are caused by aggressive molecular subtypes that are at high risk of progression. Patients with high-risk breast cancer commonly receive first-line systemic chemotherapy. Chemotherapy exerts direct cytotoxic effects on proliferating cancer cells. In addition, significant effects of chemotherapy are mediated through immune-boosting anti-cancer mechanisms that counteract immunosuppressive tumor-associated macrophages (TAMs). The aim of our study was to evaluate the potential prognostic value of the prevalence and the spatial localization of CD TAMs in tumor tissue from breast cancer patients treated with chemotherapy after surgery. We developed a novel algorithm that identifies CD TAMs in an objective manner and quantifies spatial interactions between CD TAMs and cancer cells using distance-based metrics. Our results demonstrate that close spatial proximity of CD TAMs to cancer cells and the average number of CD cells either directly adjacent to or within communicating distance of each cancer cell are independent predictors of unfavorable prognosis in breast cancer.Tumor-associated macrophages (TAMs) promote progression of breast cancer and other solid malignancies via immunosuppressive, pro-angiogenic and pro-metastatic effects. Tumor-promoting TAMs tend to express M2-like macrophage markers, including CD163. Histopathological assessments suggest that the density of CD163-positive TAMs within the tumor microenvironment is associated with reduced efficacy of chemotherapy and unfavorable prognosis. However, previous analyses have required research-oriented pathologists to visually enumerate CD TAMs, which is both laborious and subjective and hampers clinical implementation. Objective, operator-independent image analysis methods to quantify TAM-associated information are needed. In addition, since M2-like TAMs exert local effects on cancer cells through direct juxtacrine cell-to-cell interactions, paracrine signaling, and metabolic factors, we hypothesized that spatial metrics of adjacency of M2-like TAMs to breast cancer cells will have further information value. Immunofluorescence histo-cytometry of CD TAMs was performed retrospectively on tumor microarrays of 443 cases of invasive breast cancer from patients who subsequently received adjuvant chemotherapy. An objective and automated algorithm was developed to phenotype CD TAMs and calculate their density within the tumor stroma and derive several spatial metrics of interaction with cancer cells. Shorter progression-free survival was associated with a high density of CD TAMs, shorter median cancer-to-CD nearest neighbor distance, and a high number of either directly adjacent CD TAMs (within juxtacrine proximity <12 μm to cancer cells) or communicating CD TAMs (within paracrine communication distance <250 μm to cancer cells) after multivariable adjustment for clinical and pathological risk factors and correction for optimistic bias due to dichotomization.

Evaluation of Number Density of Tumor-Associated Macrophages by Immunohistochemistry and Semiquantitative Scoring in Invasive Breast Cancer: An Indian Study.

Tumor microenvironment is emerging as a critical factor for progression of breast cancer. Tumor-associated macrophages (TAMs) play an important role in promoting tumor growth. This study was aimed at correlation of number density (ND) of TAMs with invasive ductal carcinoma (IDC) grading utilizing an image morphometric technique. We also sought to compare the TAMs and ND in the tumoral area and stromal region. We also explored the relationship between the clinical and pathological prognostic parameters. The study included 75 cases of IDC that had undergone modified radical mastectomy. The Institutional Ethics Committee approved the study. Samples were classified as Grade 1, 2, and 3. Cases were graded as per the modified Bloom and Richardson criterion. Mean with standard deviation was calculated for each group. We utilized CD68 and CD163 immunostained sections for determining the ND of TAMs. TAMs were evaluated using computerized digital photomicrograph system with image analyzing software. ND was defined as the number of TAMs in total number of TAMs in five high-power fields/total area of five fields. ND was calculated separately in tumor and tumor stroma (TS). Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2/neu (HER2/neu) were scored in accordance with recommendations. Ki-67 was scored as per the recommended guidelines. Data were tabulated in Microsoft Excel. SPSS version 20.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. To determine the relationship between macrophage density and clinicopathologic parameters, we used the independent t-test. To determine the differences in the parameters, analysis of variance (ANOVA) was utilized. Age of the patients ranged from 34 to 58 years (mean: 55.5). One-way ANOVA between various grades of tumor indicating significant differences in terms of CD68 and CD163 densities in tumor and stroma (P < 0.0001). i.e., significant increased density of CD68 and CD163 was observed in Grade 3 tumor as compared to other two groups. A greater histological grade, ER, PR negative status, and a high Ki-67 index were all associated with TAM ND. There was no relation to HER2/neu status. Result of unpaired t-test indicates increased density in stroma as compared to tumor among various grades of IDC. We analyzed images with a software using photographs of the stained slides. This helped in quantitative analysis of TAMs on the CD68 and CD163 stained sections. This approach standardizes and reproducibly counts TAMs per unit area. We found significant difference between the number densities of TAMs in grades of invasive breast carcinoma. There were statistically significant differences in numerical densities of TAMs with ER, PR negativity, and Ki-67. There was no correlation with HER2/neu. Densities of CD68 and CD163 densities are more prevalent in TS as compared to intratumoral region.

High Infiltration of CD68+/CD163− Macrophages Is an Adverse Prognostic Factor after Neoadjuvant Chemotherapy in Esophageal and Gastric Adenocarcinoma

Tumor-associated macrophages (TAMs) have emerged as key players in tumor immunology but demonstrate a continuum of functional states being either tumor suppressive or promoting. Moreover, chemotherapeutic agents have been shown to alter the tumor microenvironment. Perioperative chemotherapy is a standard treatment option for resectable esophageal and gastric (EG) adenocarcinoma. The aim of this study was to investigate the influence of neoadjuvant chemotherapy (NAC) on TAMs to improve the prognostication and treatment course for these patients. The study cohort comprised 148 patients, all of whom were diagnosed with resectable EG adenocarcinoma and treated with NAC. Immunohistochemistry was applied to assess the total infiltration and infiltration into tumor nests (TN) of CD68+/CD163−, CD68+/CD163+, and MARCO+ TAMs, on paired biopsies from primary tumors (PT) pre-NAC, and resected PT and lymph node metastases post-NAC. In pre-NAC specimens, high CD68+/CD163+ infiltration into TN was an unfavorable prognostic factor. No association was found between TAM density in PT pre-NAC and histopathological regression. The density of CD68+/CD163+ TAMs was increased in PT post-NAC, while the density of MARCO+ TAMs was decreased. CD68+/CD163− TAM density was not altered. In post-NAC specimens, higher total as well as TN infiltration of CD68+/CD163− TAMs were adverse prognostic factors. In conclusion, these results suggest that NAC may alter certain TAM subsets in EG adenocarcinoma, along with their functional properties and thus their prognostic value.

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.

Prognostic Value of TRPM7 Expression and Factor XIIIa-Expressing Tumor-Associated Macrophages in Gastric Cancer.

Purpose TRPM7 is known to play a key role in tumor progression by regulating cellular proliferation, migration, and invasion in various cancer cell lines. However, there are no comprehensive clinical studies about the effect of TRPM7 expression on gastric cancer (GC) prognosis. In this study, it was aimed at investigating the effect of TRPM7 expression on prognosis in GC patients. Additionally, for the first time, it was investigated whether the density of Factor XIIIa-expressing tumor-associated macrophages (TAMs) in GC has an effect on the biological behaviour of the tumor. Methods TRPM7 expression and Factor XIIIa-expressing TAM density were immunohistochemically evaluated in paraffin-embedded tumor tissues of 204 GC patients undergoing surgery at a single institution. Results Tumor size was clearly higher in cases with high TRPM7 expression than those with low expression (p < 0.001, Mann-Whitney U). TRPM7 overexpression was closely related to high depth of tumor invasion (p < 0.001, ANOVA), increased lymph node metastasis (p < 0.001, ANOVA), and high distant metastasis rate (p < 0.001, Mann-Whitney U). These findings exposed that high TRPM7 expression is effective in the progression and aggressiveness of GC. In addition, while high CD8+ TIL density affects the prognosis positively, it was determined that high Factor XIIIa+ TAM density negatively affects the prognosis of patients with GC. Furthermore, multivariate analyses revealed TRPM7 overexpression was independently related with short overall (HR 9.64, 95% CI 5.74–16.19, p < 0.001) and disease-free survival (HR 5.67, 95% CI 3.61-8.92, p < 0.001) in GC patients. Conclusions Our data suggest that high TRPM7 expression is closely related to progressive tumor behaviour in GC and independently negatively affects survival in patients. In addition, it was determined that a high density of Factor XIIIa+ TAMs negatively affects the prognosis of patients with GC.