Hypercholesterolemic Dysregulation of Calpain in Lymphatic Endothelial Cells Interferes With Regulatory T-Cell Stability and Trafficking.

BACKGROUND: Although hypercholesterolemia reportedly counteracts lymphocyte trafficking across lymphatic vessels, the roles of lymphatic endothelial cells (LECs) in the lymphocyte regulations remain unclear. Previous studies showed that calpain-an intracellular modulatory protease-interferes with leukocyte dynamics in the blood microcirculation and is associated with hypercholesterolemic dysfunction in vascular endothelial cells. METHODS: This study investigated whether the calpain systems in LECs associate with the LEC-lymphocyte interaction under hypercholesterolemia using gene-targeted mice. RESULTS: Lipidomic analysis in hypercholesterolemic mice showed that several lysophospholipids, including lysophosphatidic acid, accumulated in the lymphatic environment. Lysophosphatidic acid enables the potentiation of calpain systems in cultured LECs, which limits their ability to stabilize regulatory T cells (Treg) without altering Th1/Th2 (T helper type1/2) subsets. This occurs via the proteolytic degradation of MEKK1 (mitogen-activated protein kinase kinase kinase 1) and the subsequent inhibition of TGF (transforming growth factor)-ß1 production in LECs. Targeting calpain systems in LECs expanded Tregs in the blood circulation and reduced aortic atherosclerosis in hypercholesterolemic mice, concomitant with the reduction of proinflammatory macrophages in the lesions. Treg expansion in the blood circulation and atheroprotection in calpain-targeted mice was prevented by the administration of TGF-ß type-I receptor inhibitor. Moreover, lysophosphatidic acid-induced calpain overactivation potentiated the IL (interleukin)-18/NF-κB (nuclear factor κB)/VCAM1 (vascular cell adhesion molecule 1) axis in LECs, thereby inhibiting lymphocyte mobility on the cells. Indeed, VCAM1 in LECs was upregulated in hypercholesterolemic mice and human cases of coronary artery disease. Neutralization of VCAM1 or targeting LEC calpain systems recovered afferent Treg transportation via lymphatic vessels in mice. CONCLUSIONS: Calpain systems in LECs have a key role in controlling Treg stability and trafficking under hypercholesterolemia.

Expression of macrophage/dendritic cell-related molecules in lymph node sinus macrophages.

The role of sinus macrophages (SMs) in anticancer immune responses has received considerable interest in recent years, but the types of molecules that are expressed in human SMs have not yet been clarified in detail. We therefore sought to identify dendritic cell (DC)- or macrophage-related molecules in SMs in human lymph nodes (LNs). SMs are strongly positive for Iba-1, CD163, CD169, and CD209. CD169 (clone SP216) reacted with almost all SMs, mainly in the cell surface membrane, while CD169 (clone HSn 7D2) reacted with a subpopulation of SMs, mainly in the cytoplasm, with a significant increase observed after IFN-α stimulation. The immunoreactivity of clone HSn 7D2 was markedly reduced after transfection with small interfering RNA against CD169, while that of clone SP216 was slightly reduced. The induction of CCL8 and CXCL10 messenger RNA (mRNA) expression by IFN-α was confirmed using cultured macrophages and RT-qPCR, but fluorescence in situ hybridization did not detect CCL8 and CXCL10 mRNA expression in SMs. Single-cell RNA sequence data of LNs indicated that the highest level of CXCL10 gene expression occurred in monocytes. In conclusion, we found that CD209, also known as DC-related molecule, was expressed in human SMs. The heterogeneity observed in CD169 reacted with cone HSn 7D2 and SP216 was potentially due to the modification of CD169 protein by IFN stimulation. Further, no expression of CXCL10 mRNA in SMs suggested that SMs might be resident macrophages.

Naringenin potentiates anti-tumor immunity against oral cancer by inducing lymph node CD169-positive macrophage activation and cytotoxic T cell infiltration.

The CD169+ macrophages in lymph nodes are implicated in cytotoxic T lymphocyte (CTL) activation and are associated with improved prognosis in several malignancies. Here, we investigated the significance of CD169+ macrophages in oral squamous cell carcinoma (OSCC). Further, we tested the anti-tumor effects of naringenin, which has been previously shown to activate CD169+ macrophages, in a murine OSCC model. Immunohistochemical analysis for CD169 and CD8 was performed on lymph node and primary tumor specimens from 89 patients with OSCC. We also evaluated the effects of naringenin on two murine OSCC models. Increased CD169+ macrophage counts in the regional lymph nodes correlated with favorable prognosis and CD8+ cell counts within tumor sites. Additionally, naringenin suppressed tumor growth in two murine OSCC models. The mRNA levels of CD169, interleukin (IL)-12, and C-X-C motif chemokine ligand 10 (CXCL10) in lymph nodes and CTL infiltration in tumors significantly increased following naringenin administration in tumor-bearing mice. These results suggest that CD169+ macrophages in lymph nodes are involved in T cell-mediated anti-tumor immunity and could be a prognostic marker for patients with OSCC. Moreover, naringenin is a new potential agent for CD169+ macrophage activation in OSCC treatment.

Clinical impact of TROP2 in non-small lung cancers and its correlation with abnormal p53 nuclear accumulation.

Tumor-associated calcium signal transducer 2 (TROP2) is a cell-surface glycoprotein involved in the high malignant potential of several cancers. Antibody-drug conjugates that target TROP2 represent a promising approach for the treatment of TROP2-expressing cancers including lung cancer and breast cancer. TROP2 expression was tested by immunohistochemistry in lung adenocarcinoma (ADC) and squamous cell carcinoma samples, and its correlation with clinicopathological factors, including survival rate and p53 mutation, was statistically analyzed. We found that increased TROP2 expression was significantly associated with a poor clinical course in patients with ADC, but not in patients with squamous cell carcinoma. A more significant association with poor outcome was seen in ADC cases with a high histological grade as well as those without the epidermal growth factor receptor (EGFR) mutation. A significant correlation between TROP2 expression and abnormal p53 nuclear accumulation/expression was also found in ADC. In the present study, we discovered a significant correlation between TROP2 expression and p53 mutation in ADC, and that TROP2 expression was a prognostic factor in ADC cases with a high histological grade as well as those without the EGFR mutation. Signals mediated by mutated p53 might influence TROP2 expression in ADC.

Accurate expression of PD-L1/L2 in lung adenocarcinoma cells: A retrospective study by double immunohistochemistry.

The percentage of programmed death ligand 1 (PD-L1) positivity in cancer cells, named as the tumor proportion score, is considered to be a predictive biomarker for anti-PD-1/PD-L1 therapy in lung cancer. PD-L1 is expressed on not only cancer cells but also on immune cells, including macrophages. Although previous studies related to PD-L1/2 expression in cancer tissues have been generally based on single immunohistochemistry (IHC), in the present study, we attempted to evaluate accurate PD-L1/2 expression in cancer cells in lung adenocarcinoma cells using double IHC to also evaluate macrophages. Of the 231 patients, PD-L1 expression was negative in 169 patients (73.2%), 1%-49% positive in 47 patients (20.3%), and ≥50% positive in 15 patients (6.5%). Interestingly, PD-L1 positivity was decreased when using double IHC compared with the estimation by single IHC. High PD-L1 expression was associated with high-grade cancer cells and in higher stage cancer. PD-L2 was negative in 109 patients (47.2%), 1%-49% positive in 50 patients (21.6%), and ≥50% positive in 72 patients (31.2%). The number of PD-L2-positive patients was increased in cases that had an epidermal growth factor receptor (EGFR) mutation and in lower stage cancer. Thirty-five patients (15.2%) were positive for both PD-L1 and PD-L2, whereas 81 patients (35.1%) were negative for both PD-L1 and PD-L2. Log-rank analysis showed that progression-free survival and overall survival were significantly the longest in the PD-L1-negative and PD-L2-positive groups (P < .0001 and P = .0120). We observed lower PD-L1 or PD-L2 expression in lung adenocarcinoma than previously reported. Double IHC for macrophages may help clinicians to evaluate PD-L1 or PD-L2 expression specifically in cancer cells.

CD169-positive sinus macrophages in the lymph nodes determine bladder cancer prognosis.

CD169+ macrophages are suggested to play a pivotal role in establishing anti-tumor immunity. They capture dead tumor cell-associated antigens and transfer their information to lymphocsytes, including CD8+ T cells, which is important for successful tumor suppression. This study aimed to determine the prognostic significance of CD169+ macrophages residing in the tumor-draining lymph nodes from cases of bladder cancer. In this retrospective study, 44 bladder cancer patients who received radical cystectomy were examined. The abundance of CD169+ macrophages in the regional lymph nodes and the number of CD8+ T cells in the primary tumor were investigated by immunohistochemistry. A CD169 score was calculated based on the intensity of CD169 staining and the proportion of CD169+ macrophages, and the scores were compared to the patients' clinicopathological parameters. A high CD169 score was significantly associated with low T stage and with a high number of CD8+ T cells infiltrating into the tumor. The group with high CD169 expression had significantly longer cancer-specific survival than the group with low CD169 expression (5-year cancer-specific survival rate: 83.3% vs 31.3%). In a multivariate analysis, the CD169 score was identified as a strong and independent favorable prognostic factor for cancer-specific survival. Our findings suggest that CD169+ macrophages in the lymph nodes enhance anti-tumor immunity by expanding CD8+ T cells in bladder cancer. The CD169 score may serve as a novel marker for the evaluation of bladder cancer prognosis.

High CD169 expression in lymph node macrophages predicts a favorable clinical course in patients with esophageal cancer.

Recent findings indicate CD169-positive lymph node sinus macrophages (LySMs) in the regional lymph nodes (RLNs) play an important role in anti-cancer immunity. In the present study, we investigated the correlation between CD169 expression in RLNs and clinicopathologic factors. Higher CD169 expression in LySMs was significantly associated with longer cancer-specific survival (CSS). The density of tumor-infiltrating lymphocytes (TILs) in the cancer nest and CD169 expression on LySMs were positively associated in patients who underwent pretreatment. As CD169 expression is thought to reflect a high interferon signature in RLNs, we tried to identify immunity-related genes that are up-regulated by interferon in macrophages as well as CD169. Indoleamine 2,3-dioxygenase (IDO1) was found to be elevated by interferon, and expression of IDO1 was tested using immunohistochemistry. IDO1 expression on LySMs was positively correlated with CD169 expression; however, there was no significant correlation between IDO1 and clinicopathologic factors. These results suggest that high expression of CD169 in LySMs reflects a high potential for anti-cancer immune responses in esophageal cancer patients and that monitoring CD169 expression would be useful for evaluating the potential of anti-cancer immune reactions.

CD163-positive cancer cells are potentially associated with high malignant potential in clear cell renal cell carcinoma.

CD163 is preferentially expressed by monocyte/macrophages; however, recent studies using immunohistochemistry (IHC) have reported that some cancer cells also express CD163. In the present IHC study, we investigated CD163 staining of cancer cells and macrophages in clear cell renal cell carcinoma (ccRCC) tissues and determined the relationship between cancer cell CD163 expression and clinical prognosis in patients with ccRCC. IHC for CD163 was performed in ccRCC tissues from 103 patients. CD163-positive cancer cells were detected in 35% of the patients (36/103); however, the positive signals on cancer cells were significantly lower than those on macrophages. CD163-positive cancer cells were preferentially detected in patients with high T classification, and females, and were significantly associated with shortened progression-free survival and a lower overall survival ratio. Notably, a high intensity of CD163-positive macrophage infiltration was detected in the CD163-positive cancer cell-high tumor areas. Although CD163 mRNA was detected in cultured macrophages, no CD163 mRNA was detected in two cultured RCC cell lines. The detailed mechanism by which a positive signal is detected on cancer cells has not been clarified. Detection of the CD163 antigen on cancer cells might be a useful marker for evaluating the clinical course of patients with ccRCC.

Possible functions of CD169-positive sinus macrophages in lymph nodes in anti-tumor immune responses.

The lymph node (LN) is an important immune system in which a number of antigen-presenting cells are present that induce rapid immune responses to foreign antigens. While a great number of macrophages exist in lymph nodes, recent studies using animal models have shown that lymph node sinus macrophages are associated with the induction of anti-tumor immunity, playing a significant role in host immune responses against tumor cells. In colorectal tumor, malignant melanoma, and endometrial tumor, it was shown that a high density of CD169-positive macrophages in the LN sinus was a predictive factor for better clinical prognosis. The observations that the density of CD169-positive macrophages in the LN sinus was positively associated with the density of infiltrating T or NK cells in tumor tissues, indicates the significance of CD169-positive macrophages in anti-tumor immune reactions of tumor patients. Moreover, antigen delivery targeting LN macrophages is also considered to be promising approach for vaccination. In this article, we have summarized the significance of CD169-positive LN macrophages in anti-tumor immunity.

High density of CD204-positive macrophages predicts worse clinical prognosis in patients with breast cancer.

Recent studies have indicated the clinical significance of tumor-associated macrophages (TAM) in several malignant tumors including breast cancer. Although recent studies have focused on CD68-positive or CD163-positive TAM in breast cancer, no study has investigated the significance of CD204-positive TAM in breast cancer. We found that CD204 expression on macrophages was evaluated following stimulation with the conditioned medium (CM) of breast cancer cell lines. Paraffin sections of 149 breast cancer samples which were diagnosed as invasive ductal carcinoma were immunohistochemically analyzed for CD68, CD163 and CD204 expression. The results of analyses indicated that a high number of CD204-positive TAM was associated with worse clinical prognoses, including relapse-free survival, distant relapse-free survival and breast cancer-specific survival; however, neither the numbers of CD68-positive or CD163-positive TAM were associated with clinical courses. Of the clinicopathological factors investigated, estrogen receptor, Ki-67 index, hormone subtype, and histological grade were significantly related to the increased number of CD163-positive and CD204-positive TAM. These data indicate the clinical significance of CD204-positive TAM in breast cancer progression and CD204 is a marker for predicting clinical prognosis in breast cancer.

Prognostic significance of CD169-positive lymph node sinus macrophages in patients with endometrial carcinoma.

Lymph node (LN) macrophages play critical roles in anti-tumor immunity, which develops via the activation of cytotoxic T cells (CTL) and NK cells. The present study aims to determine the prognostic significance of CD169(+) LN macrophages in patients with endometrial carcinoma (EC). The number of CD169(+) cells or the CD169(+) -to-CD68(+) macrophage ratio in regional LN (RLN), and the number of CD8(+) CTL or CD57(+) NK cells in tumor tissues were investigated by immunohistochemistry in paraffin-embedded tissue samples from 79 patients with EC. A high density of CD169(+) cells in the RLN of patients with EC was correlated with an early clinical stage or no LN metastasis. A high number of CD169(+) cells and a high CD169(+) -to-CD68(+) macrophage ratio were significantly associated with longer overall survival in EC. We also found that the density of CD169(+) macrophages was positively correlated with the number of CD8(+) CTL and CD57(+) NK cells that infiltrated into tumor tissues. A high density of CD57(+) cells in EC tissues was associated with a better prognosis, while a high density of CD8(+) cells was not linked to an altered prognosis. The present study showed that the density of CD169(+) macrophages in RLN was associated with an improved prognosis in EC patients. CD169(+) macrophages in RLN might represent a useful marker for assessing clinical prognoses and monitoring anti-tumor immunity in patients with EC.

An IL-27/Stat3 axis induces expression of programmed cell death 1 ligands (PD-L1/2) on infiltrating macrophages in lymphoma.

Immune escape and tolerance in the tumor microenvironment are closely involved in tumor progression, and are caused by T-cell exhaustion and mediated by the inhibitory signaling of immune checkpoint molecules including programmed death-1 (PD-1), cytotoxic T-lymphocyte associated protein 4, and T-cell immunoglobulin and mucin domaincontaining molecule-3. In the present study, we investigated the expression of the PD-1 ligand 1 (PD-L1) in a lymphoma microenvironment using paraffin-embedded tissue samples, and subsequently studied the detailed mechanism of upregulation of PD-L1 on macrophages using cultured human macrophages and lymphoma cell lines. We found that macrophages in lymphoma tissues of almost all cases of adult T-cell leukemia/lymphoma (ATLL), follicular lymphoma and diffuse large B-cell lymphoma expressed PD-L1. Cell culture studies showed that the conditioned medium of ATL-T and SLVL cell lines induced increased expression of PD-L1/2 on macrophages, and that this PD-L1/2 overexpression was dependent on activation of signal transducer and activator of transcription 3 (Stat3). In vitro studies including cytokine array analysis showed that IL-27 (heterodimer of p28 and EBI3) induced overexpression of PD-L1/2 on macrophages via Stat3 activation. Because lymphoma cell lines produced IL-27B (EBI3) but not IL-27p28, it was proposed that the IL-27p28 derived from macrophages and the IL-27B (EBI3) derived from lymphoma cells formed an IL-27 (heterodimer) that induced PD-L1/2 overexpression. Although the significance of PD-L1/2 expressions on macrophages in lymphoma progression has never been clarified, an IL-27-Stat3 axis might be a target for immunotherapy for lymphoma patients.

Infiltration of tumor-associated macrophages is involved in CD44 expression in clear cell renal cell carcinoma.

Cancer stem-like cells (CSC) or cancer-initiating cells are now considered to be an important cell population related to cancer recurrence and the resistance to anti-cancer therapy. Tumor-associated macrophages (TAM) are a main component of stromal cells and are related to cancer progression in clear cell renal cell carcinoma (ccRCC). Because the detailed mechanisms allowing the maintenance of CSC in cancer tissues remain unclear, we investigated the relationship between TAM and CD44-expressing cancer cells in ccRCC. CD44 was used as a marker for CSC, and CD163 and CD204 were used as markers for TAM. CD44-positive cancer cells were detected in 37 of the 103 cases. Although statistical analysis showed no relationship between CD44-positive cancer cells and the clinical course, the distribution of CD44-positive cancer cells was significantly associated with a high density of TAM. Our in vitro study using RCC cell lines and human macrophages demonstrated that CD44 expression was upregulated by direct co-culture with macrophages. Silencing of TNF-alpha on macrophages abrogated the upregulation of CD44 expression in cancer cells. Macrophage-induced CD44 overexpression was also suppressed by NF-κB inhibitors. These results suggest that TNF-alpha derived from TAM is linked to CD44 overexpression via NF-κB signaling in ccRCC.

Role of tumor-associated macrophages in hematological malignancies.

The tumor microenvironment consists of many non-tumor cells such as leukocytes, endothelial cells, and fibroblasts, and phenotypic changes in a tumor microenvironment are believed to be involved in tumor progression and resistance to anticancer treatments. In hematological malignancies, tumor-associated macrophages (TAMs) that have infiltrated lymphoma or leukemia tissues may be involved in tumor progression, and many researchers have studied phenotypic changes in TAMs. This review article summarizes the publications related to TAMs in hematological malignancies, with an emphasis on CD163(+) protumoral TAMs, which seem to be associated with disease progression. Cell-cell interactions between protumoral TAMs and lymphoma or leukemia cells may play an important role in lymphoma or leukemia microenvironments. Although detailed molecular mechanisms of these cell-cell interactions have not yet been clarified, phenotypic characterization of TAMs is thought to be a useful approach for evaluating clinical prognosis. In addition, targeting TAMs may be a new strategy for treating malignant hematological diseases.

CXCL10 Expression in Human Colorectal Cancer Tissue and its Correlation With Serum Levels of CXCL10.

BACKGROUND/AIM: CXCL10, a member of the CXC chemokine family, plays a crucial role in immune response by facilitating the chemotaxis of CXCR3-positive immune cells. We examined the expression of CXCL10 to unravel its functional significance in colorectal cancer. MATERIALS AND METHODS: Bioinformatics analysis was performed to investigate CXCL10 expression and its clinicopathological relevance. Subsequently, we examined the correlation between the serum levels of CXCL10 and its expression within cancer tissues. RESULTS: Analysis of the TCGA database revealed that elevated CXCL10 expression in CRC tissues correlates with improved long-term survival and is inversely associated with lymph node infiltration and metastasis. Insights from Gene Ontology and Kyoto Encyclopedia of Genes and Genomes further established a connection between increased CXCL10 and co-regulated gene expression with enhanced immune activation and regulation, mediated by the inhibition of the NOD-like receptor signaling pathway. Single-cell analysis pinpointed myeloid cells and macrophages as the primary sources of CXCL10. Immunohistochemical assessments revealed that a subset of cancer cells and macrophages are positive for CXCL10 expression. CXCL10-positive cells are predominantly located at the invasive front of the tumor. Intriguingly, our findings reveal an inverse correlation between serum CXCL10 levels and its expression in cancer tissues. CONCLUSION: The expression of CXCL10 may play a role in mediating the inflammatory responses at the invasive front in colorectal cancer and is observed to be inversely correlated with serum CXCL10 levels. It is pivotal to elucidate the distinct roles of CXCL10 in colorectal cancer, particularly different functions of cancer-tissue CXCL10 from serum CXCL10.

Clinical significance of CD163⁺ tumor-associated macrophages in patients with adult T-cell leukemia/lymphoma.

In several malignant tumors including lymphoma, macrophages that infiltrate tumor tissues are called tumor-associated macrophages (TAMs). We discovered that TAMs, especially the CD163⁺ alternatively activated phenotype (M2), were closely involved with progression of adult T-cell leukemia/lymphoma (ATLL). We used CD68 (a pan-macrophage marker) and CD163 (an M2 marker) to immunostain 58 ATLL samples. Statistical analyses showed that a high number of CD68⁺ TAMs and an increased percentage of CD163⁺ cells among the TAMs were associated with a worse clinical prognosis; multivariate analysis indicated that the percentage of CD163⁺ cells was an independent prognostic factor. We also carried out in vitro coculture experiments with ATLL cell lines (ATN-1 and TL-Mor) and monocyte-derived macrophages and found that direct coculture with M2 macrophages significantly increased BrdU incorporation into ATLL cell lines. A cytokine array analysis showed that macrophage-derived soluble factors including C5a, tumor necrosis factor-α, growth-related oncogene-α, CCL1/I-309, and interleukin-6 stimulated ATLL cell lines. CD163 expression in macrophages was strongly induced by direct contact with ATN-1 cells, and downregulation of CD163 in macrophages significantly suppressed growth of cocultured ATN-1 cells. These results suggest that interaction between M2 macrophages and lymphoma cells may be an appropriate target in treatment of patients with ATLL.

CD169-positive macrophages in regional lymph nodes are associated with a favorable prognosis in patients with colorectal carcinoma.

CD169 (sialoadhesin) is a sialic acid receptor that is expressed on specific macrophages such as lymph node sinus macrophages. Animal studies have suggested that CD169(+) macrophages have a pro-inflammatory property, however, the role of these cells in human diseases has not been clarified. In our in vitro experiments with human macrophages, pro-inflammatory cytokines, such as type 1 interferon, induced strong expression of CD169, suggesting that CD169 might be a specific marker of inflammatory macrophages. To examine the role of CD169 in antitumor immunity, we examined the expression of CD169 in regional lymph nodes (RLNs) and its association with overall survival in colorectal carcinoma (CRC). In a clinicopathological analysis on 83 CRC patients, paraffin-embedded specimens were evaluated for CD169 expression of RLN macrophages by immunohistochemistry. We found, for the first time, a high density of CD169(+) macrophages was significantly associated with longer overall survival; multivariate analysis showed that the ratio of CD169(+) cells to CD68(+) cells was an independent prognostic factor. The majority of CD169(+) macrophages were in direct contact with CD8(+) T cells expressing CD43, a major ligand of CD169. We also found that the density of CD169(+) macrophages had a positive correlation with the number of CD8(+) cytotoxic T cells infiltrating tumor tissues. These data suggest that CD169(+) macrophages in RLNs promote CD8(+) T-cell-mediated antitumor immunity and are associated with a better prognosis for CRC patients. CD169(+) macrophages in RLNs could be a useful marker for assessing clinical prognosis and monitoring antitumor immunity in patients with CRC.

CD169+ sinus macrophages in regional lymph nodes do not predict mismatch-repair status of patients with colorectal cancer.

AIMS: Mismatch-repair deficiency and microsatellite instability-high (dMMR/MSI-H) colorectal cancer (CRC) is treated with programmed death (PD)-1 antibody regardless of PD-ligand (L)1 expression in tumor cells. We previously found that abundant CD169+ macrophages in regional lymph node (RLN) sinuses and CD8+ tumor-infiltrating lymphocytes (TILs) positively correlated in CRC and were associated with a favorable prognosis. However, associations between dMMR/MSI-H CRC and CD8+ TILs or prognoses vary among studies. In this study, we attempted to compare the association between MMR status, CD169+ macrophages in RLNs, CD8+ TILs, PD-L1 scores, and prognoses in CRC. METHODS AND RESULTS: We immunostained 83 surgically resected CRC tumors that we previously analyzed for MMR proteins, and identified 9 that were dMMR. The number of CD169+ macrophages in RLNs and CD8+ TILs significantly correlated with overall survival, whereas MMR status did not. The number of cells positive for the TIL markers CD3, CD4, CD8, and TIA-1, and macrophage markers CD68 and CD169 in RLNs did not significantly differ between groups according to MMR status. Furthermore, combined positive scores (CPS) for PD-L1 expression in five of nine dMMR CRCs were all <1. We found that dMMR in CRC did not correlate with numbers of CD169+ macrophages in RLNs or CD8+ TILs. CONCLUSIONS: CRC with CD169+ macrophages in RLNs and abundant CD8+ TILs indicates a better prognosis and it should be immunologically classified as a different antitumor group from dMMR CRC.

Stromal Reprogramming through Dual PDGFRα/ß Blockade Boosts the Efficacy of Anti-PD-1 Immunotherapy in Fibrotic Tumors.

Excess stroma and cancer-associated fibroblasts (CAF) enhance cancer progression and facilitate immune evasion. Insights into the mechanisms by which the stroma manipulates the immune microenvironment could help improve cancer treatment. Here, we aimed to elucidate potential approaches for stromal reprogramming and improved cancer immunotherapy. Platelet-derived growth factor C (PDGFC) and D expression were significantly associated with a poor prognosis in patients with gastric cancer, and PDGF receptor beta (PDGFRß) was predominantly expressed in diffuse-type gastric cancer stroma. CAFs stimulated with PDGFs exhibited markedly increased expression of CXCL1, CXCL3, CXCL5, and CXCL8, which are involved in polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) recruitment. Fibrotic gastric cancer xenograft tumors exhibited increased PMN-MDSC accumulation and decreased lymphocyte infiltration, as well as resistance to anti-PD-1. Single-cell RNA sequencing and spatial transcriptomics revealed that PDGFRα/ß blockade reversed the immunosuppressive microenvironment through stromal modification. Finally, combining PDGFRα/ß blockade and anti-PD-1 treatment synergistically suppressed the growth of fibrotic tumors. These findings highlight the impact of stromal reprogramming on immune reactivation and the potential for combined immunotherapy for patients with fibrotic cancer. SIGNIFICANCE: Stromal targeting with PDGFRα/ß dual blockade reverses the immunosuppressive microenvironment and enhances the efficacy of immune checkpoint inhibitors in fibrotic cancer. See related commentary by Tauriello, p. 655.

Importance of direct macrophage-tumor cell interaction on progression of human glioma.

We previously showed tumor-associated macrophages/microglia (TAMs) polarized to the M2 phenotype were significantly involved in tumor cell proliferation and poor clinical prognosis in patients with high grade gliomas. However, the detailed molecular mechanisms involved in the interaction between TAMs and tumor cells have been unclear. Current results reveal that, in coculture with human macrophages, BrdU incorporation was significantly elevated in glioma cells, and signal transducer and activator of transcription-3 (Stat3) activation was found in both cell types. Direct mixed coculture led to stronger Stat3 activation in tumor cells than did indirect separate coculture in Transwell chamber dishes. Screening with an array kit for phospho-receptor tyrosine kinases revealed that phosphorylation of macrophage-colony stimulating factor receptor (M-CSFR, CD115, or c-fms) is possibly involved in this cell-cell interaction; M-CSFR activation was detected in both cell types. Coculture-induced tumor cell activation was suppressed by siRNA-mediated downregulation of the M-CSFR in macrophages and by an inhibitor of M-CSFR (GW2580). Immunohistochemical analysis of phosphorylated (p)M-CSFR, pStat3, M-CSF, M2 ratio, and MIB-1(%) in high grade gliomas revealed that higher staining of pM-CSFR in tumor cells was significantly associated with higher M-CSF expression and higher MIB-1(%). Higher staining of pStat3 was associated with higher MIB-1(%). High M2 ratios were closely correlated with high MIB-1(%) and poor clinical prognosis. Targeting these molecules or deactivating M2 macrophages might be useful therapeutic strategies for high grade glioma patients.