Presence Of Tumor-associated Macrophages Research Articles

A pancreatic cancer organoid incorporating macrophages reveals the correlation between the diversity of tumor-associated macrophages and cancer cell survival

Pancreatic ductal adenocarcinoma (PDAC) is a progressive cancer with a poor prognosis. It contains a complex tumor microenvironment (TME) that includes various stromal cell types. Comprehending cellular communications within the TME is difficult due to a lack of research models that can recapitulate human PDAC-TME. Previously, we recapitulated, in part, the PDAC-TME containing a diversity of cancer-associated fibroblasts (CAFs) in vitro. This was done by establishing a PDAC organoid by co-culturing patient-derived cancer cells with human induced pluripotent stem cell (hiPSC)-derived mesenchymal and endothelial cells, which was designated the fused pancreatic cancer organoid (FPCO). We further incorporated macrophages derived from the THP-1 cell line, which are the source of tumor-associated macrophages (TAMs), a major TME component, into FPCO, which was designated M0-FPCO. Bulk RNA sequencing (RNAseq) analysis revealed that macrophages in M0-FPCO (FPCO-Mac) lost their pro-inflammatory features but acquired pro-angiogenic features. Consistently, the formation of an endothelial cell network was enhanced in M0-FPCO. Single-cell RNA-seq (scRNA-seq) analysis revealed that M0-FPCO contained five TAM subpopulations similar to the corresponding TAM in human PDAC tissue in the integrated analysis, including SPP1+-TAM, which has been correlated with tumor angiogenesis and cell proliferation. Focusing on PDAC cells, we found that they could survive longer within the organoid in the presence of TAM. Consistent with the prolonged proliferation and survival of PDAC cells, PDAC subclusters were characterized by proliferative features, such as increased M0-FPCO. Therefore, by establishing a PDAC organoid with macrophages, we recapitulated the diversity of TAMs and identified the role of TAM in endothelial network formation as well as in the modulation of PDAC cell properties. SignificancePDAC organoids, including macrophages using hiPSC, showed that PDAC-TAM has angiogenic features and contributes to PDAC cell survival.

Discovery of a novel small molecule as CD47/SIRPα and PD-1/PD-L1 dual inhibitor for cancer immunotherapy

BackgroundTargeting the tumor microenvironment (TME) has emerged as a promising strategy in cancer treatment, particularly through the utilization of immune checkpoint blockade (ICB) agents such as PD-1/PD-L1 inhibitors. Despite partial success, the presence of tumor-associated macrophages (TAMs) contributes to an immunosuppressive TME that fosters tumor progression, and diminishes the therapeutic efficacy of ICB. Blockade of the CD47/SIRPα pathway has proven to be an effective intervention, that restores macrophage phagocytosis and yields substantial antitumor effects, especially when combined with PD-1/PD-L1 blockade. Therefore, the identification of small molecules capable of simultaneously blocking CD47/SIRPα and PD-1/PD-L1 interactions has remained imperative.MethodsSMC18, a small molecule with the capacity of targeting both SIRPα and PD-L1 was obtained using MST. The efficiency of SMC18 in interrupting CD47/SIRPα and PD-1/PD-L1 interactions was tested by the blocking assay. The function of SMC18 in enhancing the activity of macrophages and T cells was tested using phagocytosis assay and co-culture assay. The antitumor effects and mechanisms of SMC18 were investigated in the MC38-bearing mouse model.ResultsSMC18, a small molecule that dual-targets both SIRPα and PD-L1 protein, was identified. SMC18 effectively blocked CD47/SIRPα interaction, thereby restoring macrophage phagocytosis, and disrupted PD-1/PD-L1 interactions, thus activating Jurkat cells, as evidenced by increased secretion of IL-2. SMC18 demonstrated substantial inhibition of MC38 tumor growths through promoting the infiltration of CD8+ T and M1-type macrophages into tumor sites, while also priming the function of CD8+ T cells and macrophages. Moreover, SMC18 in combination with radiotherapy (RT) further improved the therapeutic efficacy.ConclusionOur findings suggested that the small molecule compound SMC18, which dual-targets the CD47/SIRPα and PD-1/PD-L1 pathways, could be a candidate for promoting macrophage- and T-cell-mediated phagocytosis and immune responses in cancer immunotherapy.

Selective ADA2 inhibition for enhancing anti-tumor immune response in glioma: Insights from computational screening of flavonoid compounds

Brain tumors, particularly gliomas, remain difficult to treat due to their complex and dynamic microenvironment and high mortality rate. The presence of tumor-associated macrophages (TAMs) is considered one of the primary factors contributing to a poor prognosis in Glioma. Previous reports have linked elevated levels of Adenosine deaminase 2 (ADA2) with immunosuppression, tumor progression, and angiogenesis via MAPK, PDGFβ signaling pathway in the glioma microenvironment. In contrast, Adenosine deaminase 1 (ADA1), another type of adenosine deaminase, plays a pivotal role in purine metabolism, which is essential for lymphocyte survival. Hence, selectively targeting ADA2 while preserving ADA1 activity could offer a viable approach for regulating macrophage polarization and enhancing the anti-tumor immune response. In pursuit of this objective, our study employed a computational approach, unveiling the remarkable attributes of Daidzin, characterized by its exceptional specificity, and binding affinity towards ADA2 while displaying minimal affinity towards ADA1. Furthermore, Define Secondary Structure of Proteins (DSSP) analysis revealed that Daidzin elicits conspicuous conformational alterations within the dimerization domain of the ADA2 receptor, which could have a crucial impact on its activity. However, the ADA1 structure remained unaltered. Our study offers the potential use of Daidzin as a specific therapeutic agent for modulating the tumor microenvironment and revolutionizing glioma management.

Attenuated Salmonella carrying siRNA-CD24 improved the effect of oxaliplatin on HCC

Oxaliplatin is a chemotherapy drug currently utilized in the treatment of advanced cancer patients. However, its tolerability poses a limitation to its clinical application. Studies have demonstrated that the presence of tumor-associated macrophages is positively correlated with poor prognosis in various solid tumors, including hepatocellular carcinoma (HCC), and is a significant factor contributing to oxaliplatin resistance. Therefore, targeting tumor-associated macrophages may be an effective strategy to improve the efficacy of oxaliplatin in the treatment of HCC patients. CD24 is a novel target for tumor therapy that can interact with the inhibitory receptor Siglec-10 on tumor-associated macrophages, transmitting immune inhibitory signals and inhibiting macrophage phagocytosis function. In this study, we utilized RNAi technology to inhibit the expression of CD24 in tumor cells and combined it with oxaliplatin, resulting in reduced tumor invasion, migration, and proliferation, as well as increased cell apoptosis. Furthermore, immunofluorescence and flow cytometry results indicated that both the single treatment group and combination treatment group enhanced the infiltration of immune cells. This study presents a novel approach to identifying combination therapy and targets for the clinical treatment of HCC with oxaliplatin.

Spatial oriented single-cell proteomic assay of low grade serous ovarian carcinoma identifies tumor oncogenic pathways associated with brain metastases (233)

Objectives: As the systemic treatment of epithelial ovarian cancer (EOC) has improved, and the survival is longer, the incidence of brain metastases (BM) in EOC patients has increased to about 2-6% and invariably results in death. In order to better understand the molecular mechanisms involved in the development of EOC BM and to characterize the BM architecture and heterogeneity, we recently used multiplex cyclic-immunofluorescence (Cyc-IF) to characterize a set of matched samples from a single ovarian cancer patient. Methods: The samples included a primary tumor, a peritoneal tumor collected at recurrence, and a BM. Cyc-IF is a spatial oriented single-cell proteomic assay that allows the quantification of over 60 proteins in a single FFPE slide. Results: Our analyses indicated that tumor architecture and heterogeneity were highly altered in the BM compared to the primary and recurrent tumors in this patient. Indeed, the primary and recurrent intraperitoneal tumors showed extensive heterogeneity with cancer cells in different cell states related to the cell cycle (cyclins), hormone receptors (PR and ER) and signaling pathways. In contrast, the BM was overall more homogeneous, with no PR receptor expression and low expression of cyclins. EOC cells from the BM harbor higher oncogenic MAPK and PI3K-AKT signaling pathway activity, higher estrogen receptor expression and displayed immunosuppressive properties including increased expression of PD1 and the presence of tumor-associated macrophages (TAM) and regulatory T cells (Tregs). Importantly, we were able to identify cell populations in the primary tumor that had phenotypes similar to that of cells found in the BM. Conclusions: These findings are consistent with the concept that EOC cells with a predilection for BM are present early during the disease course. Therefore, this represents an opportunity to develop a molecular signature for BM prevention and treatment. Objectives: As the systemic treatment of epithelial ovarian cancer (EOC) has improved, and the survival is longer, the incidence of brain metastases (BM) in EOC patients has increased to about 2-6% and invariably results in death. In order to better understand the molecular mechanisms involved in the development of EOC BM and to characterize the BM architecture and heterogeneity, we recently used multiplex cyclic-immunofluorescence (Cyc-IF) to characterize a set of matched samples from a single ovarian cancer patient. Methods: The samples included a primary tumor, a peritoneal tumor collected at recurrence, and a BM. Cyc-IF is a spatial oriented single-cell proteomic assay that allows the quantification of over 60 proteins in a single FFPE slide. Results: Our analyses indicated that tumor architecture and heterogeneity were highly altered in the BM compared to the primary and recurrent tumors in this patient. Indeed, the primary and recurrent intraperitoneal tumors showed extensive heterogeneity with cancer cells in different cell states related to the cell cycle (cyclins), hormone receptors (PR and ER) and signaling pathways. In contrast, the BM was overall more homogeneous, with no PR receptor expression and low expression of cyclins. EOC cells from the BM harbor higher oncogenic MAPK and PI3K-AKT signaling pathway activity, higher estrogen receptor expression and displayed immunosuppressive properties including increased expression of PD1 and the presence of tumor-associated macrophages (TAM) and regulatory T cells (Tregs). Importantly, we were able to identify cell populations in the primary tumor that had phenotypes similar to that of cells found in the BM. Conclusions: These findings are consistent with the concept that EOC cells with a predilection for BM are present early during the disease course. Therefore, this represents an opportunity to develop a molecular signature for BM prevention and treatment.

Abstract 5222: Biomarkers associated with complete metabolic response (CMR) to nivolumab and brentuximab vedotin (nivo + BV) for the treatment of children, adolescents, and young adults (CAYA) with relapsed or refractory (R/R) classical Hodgkin lymphoma (cHL) in the CheckMate 744 study

Abstract Introduction: CAYA with R/R cHL without CMR have suboptimal outcomes. Biomarkers associated with poor outcomes in cHL include PD-L1 overexpression and presence of tumor-associated macrophages (TAMs). In the phase 2 CheckMate 744 study (NCT02927769), CAYA with R/R cHL treated with a risk-adapted approach using nivo + BV induction showed a CMR rate per BICR of 59% (26/44) in the standard risk cohort; 41% of patients (pts) did not achieve CMR (non-CMR). Here we compared biomarker data from pts achieving CMR vs non-CMR. Methods: Pts aged 5-30 y with R/R cHL received 4 cycles of nivo + BV induction. Tumor biopsy and peripheral blood samples were collected at baseline (BL) and 4 prespecified timepoints during induction. CD163+ CD68+ TAMs were quantified using multiplex immunohistochemistry. Cytokine expression in pts with CMR vs non-CMR was assessed by the limma workflow and a linear model based on changes from BL. Immune cells were analyzed by flow cytometry. Statistical testing was adjusted for repeated measures across visits; values are reported from 39/39 cytokines and 13/16 cell subsets tested. Cytokine ratios to BL were compared across all timepoints and between response groups by ANOVA. Results: Pts with CMR (n = 24/26) after nivo + BV induction had more CD163+ CD68+ TAMs than pts with non-CMR (n = 17/18) in the BL tumor microenvironment (P = 0.018). Hierarchical clustering of cytokine fold changes from BL to the indicated timepoint, between pts with CMR vs non-CMR, showed reduced levels of haptoglobin, RANTES, CRP, PARC, EN-RAGE, and TARC/CCL17, and increased levels of MCP-2, IFN-γ, IP-10, and MIG. In all pts, median concentrations of TARC, IL-2Rα, CRP, and ICAM-1 decreased significantly (all P < 0.05); a greater reduction in TARC and CRP was observed in early cycles in pts with CMR vs non-CMR. In contrast, IP-10 levels increased from BL in all pts (P = 0.0012). Four cytokines showed higher concentrations in pts with CMR vs non-CMR: TNFR2, MCP-2, vWF, and TIMP-1 (all P < 0.05). Increased numbers of lymphocytes and monocytes and decreased numbers of granulocytes were observed after induction in all pts (all P < 0.01). Pts with non-CMR had more naive (CCR7+ CD45RA+) CD4, and naive CD8 T cells than pts with CMR at BL, and more overall CD4 (CD3+ CD4+) T cells at the end of cycle 4 (all P < 0.05). Conclusions: This biomarker analysis from CheckMate 744 suggests that different immunological parameters are associated with CMR vs non-CMR to nivo + BV induction in CAYA with R/R cHL. These include increased TAMs at BL in pts with CMR, changes in cytokine levels associated with macrophage biology, and greater numbers of naive lymphocytes and CD4 T cells in pts with non-CMR. TAMs have been reported to express high PD-L1 levels; thus, increased TAM numbers may sensitize pts to nivo + BV. Citation Format: Charlie Garnett-Benson, Ron Ammar, Russell Crowe, Stephen Francis, Sahar Ansari, Alev Akyol. Biomarkers associated with complete metabolic response (CMR) to nivolumab and brentuximab vedotin (nivo + BV) for the treatment of children, adolescents, and young adults (CAYA) with relapsed or refractory (R/R) classical Hodgkin lymphoma (cHL) in the CheckMate 744 study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5222.

Engineering Endogenous Tumor-Associated Macrophage-Targeted Biomimetic Nano-RBC to Reprogram Tumor Immunosuppressive Microenvironment for Enhanced Chemo-Immunotherapy.

Immunotherapy has shown encouraging results in various cancers, but the response rates are relatively low due to the complex tumor immunosuppressive microenvironment (TIME). The presence of tumor-associated macrophages (TAMs) and tumor hypoxia correlates significantly with potent immunosuppressive activity. Here, a hemoglobin-poly(ε-caprolactone) (Hb-PCL) conjugate self-assembled biomimetic nano red blood cell (nano-RBC) system (V(Hb)) is engineered to deliver chemotherapeutic doxorubicin (DOX) and oxygen for reprogramming TIME. The Hb moiety of V(Hb)@DOX can bind to endogenous plasma haptoglobin (Hp) and specifically target the M2-type TAMs via the CD163 surface receptor, and effectively kill the cells. In addition, the O2 released by the Hb alleviates tumor hypoxia, which further augments the antitumor immune response by recruiting fewer M2-type macrophages. TAM-targeting depletion and hypoxia alleviation synergistically reprogram the TIME, which concurrently downregulate PD-L1 expression of tumor cells, decrease the levels of immunosuppressive cytokines such as IL-10 and TGF-β, elevate the immunostimulatory IFN-γ, enhance cytotoxic T lymphocyte (CTL) response, and boost a strong memory response. The ensuing TAM-targeted chemo-immunotherapeutic effects markedly inhibit tumor metastasis and recurrence. Taken together, the engineered endogenous TAM-targeted biomimetic nano-RBC system is a highly promising tool to reprogram TIME for cancer chemo-immunotherapy.

Tumor-Associated Macrophages: Combination of Therapies, the Approach to Improve Cancer Treatment.

Macrophages are one of the most important cells of the innate immune system and are known for their ability to engulf and digest foreign substances, including cellular debris and tumor cells. They can convert into tumor-associated macrophages (TAMs) when mature macrophages are recruited into the tumor microenvironment. Their role in cancer progression, metastasis, and therapy failure is of special note. The aim of this review is to understand how the presence of TAMs are both advantageous and disadvantageous in the immune system.

Immunohistochemical Expression of CD68 in Triple Negative Invasive Ductal Carcinoma of the Breast and its Correlation with Clinicopathological Parameters

Introduction: Triple negative breast cancer (TNBC) is an aggressive form of breast cancer associated with a poor prognosis. No targeted treatment is available for this subtype. Tumor microenvironment (TME) has been increasingly considered a diagnostic and a prognostic biomarker and a therapeutic target for breast cancer. Tumor associated macrophages (TAM) are a pivotal member of TME and have been proposed as potential targets of therapy. Material and Methods: The immunohistochemical expression of CD68+ve TAM was studied in both tumor stroma (TS) and tumor nest (TN) in 50 cases of triple negative invasive ductal carcinoma as well as in 10 control cases of benign breast lesions. Results: The cases were divided into high or low density groups according to the median. The median in CD68+ve TAM in TS was (61.88), while in CD68+ve TAM in TN was (49.88). The expression of CD68+ve TAM in TS was low in 22 cases and high in 28 cases, while its expression in TN was low in 35 cases and high in 15 cases. There was no statistical association between high CD68+ve TAM in TN and different clinicopathological parameters, meanwhile a statistically significant association was found between high CD68 +ve TAM in TS and tumor grade, lymph/vascular invasion and lymph node metastasis. Conclusions: High expression of TAM in TS, but not in TN, is of clinical significance in patients with TNBC and highlights the importance of analyzing the localization rather than merely the presence of TAM as a marker for prognosis and a potential target for future treatment of triple negative breast cancer.

Mutant KRAS triggers functional reprogramming of tumor-associated macrophages in colorectal cancer

Oncogenic KRAS has been previously identified to act in a cell-intrinsic manner to modulate multiple biological functions of colorectal cancer (CRC). Here, we demonstrate a cell-extrinsic role of KRAS, where KRAS engages with the tumor microenvironment by functional reprogramming of tumor-associated macrophages (TAMs). In human CRC specimens, mutant KRAS positively correlates with the presence of TAMs. Mutationally activated KRAS in tumor cells reprograms macrophages to a TAM-like phenotype via a combination effect of tumor-derived CSF2 and lactate. In turn, KRAS-reprogrammed macrophages were shown to not only promote tumor progression but also induce the resistance of tumor cells to cetuximab therapy. Mechanistically, KRAS drives the production of CSF2 and lactate in tumor cells by stabilizing hypoxia-inducible factor-1α (HIF-1α), a transcription factor that controls the expression of CSF2 and glycolytic genes. Mutant KRAS increased the production of reactive oxygen species, an inhibitor of prolyl hydroxylase activity which decreases HIF-1α hydroxylation, leading to enhanced HIF-1α stabilization. This cell-extrinsic mechanism awards KRAS a critical role in engineering a permissive microenvironment to promote tumor malignancy, and may present new insights on potential therapeutic defense strategies against mutant KRAS tumors.

A narrative review of tumor-associated macrophages in lung cancer: regulation of macrophage polarization and therapeutic implications.

Lung cancer is the deadliest malignancy worldwide. An inflammatory microenvironment is a key factor contributing to lung tumor progression. Tumor-Associated Macrophages (TAMs) are prominent components of the cancer immune microenvironment with diverse supportive and inhibitory effects on growth, progression, and metastasis of lung tumors. Two main macrophage phenotypes with different functions have been identified. They include inflammatory or classically activated (M1) and anti-inflammatory or alternatively activated (M2) macrophages. The contrasting functions of TAMs in relation to lung neoplasm progression stem from the presence of TAMs with varying tumor-promoting or anti-tumor activities. This wide spectrum of functions is governed by a network of cytokines and chemokines, cell-cell interactions, and signaling pathways. TAMs are promising therapeutic targets for non-small cell lung cancer (NSCLC) treatment. There are several strategies for TAM targeting and utilizing them for therapeutic purposes including limiting monocyte recruitment and localization through various pathways such as CCL2-CCR2, CSF1-CSF1R, and CXCL12-CXCR4, targeting the activation of TAMs, genetic and epigenetic reprogramming of TAMs to antitumor phenotype, and utilizing TAMs as the carrier for anti-cancer drugs. In this review, we will outline the role of macrophages in the lung cancer initiation and progression, pathways regulating their function in lung cancer microenvironment as well as the role of these immune cells in the development of future therapeutic strategies.

Co-culture hydrogel micro-chamber array-based plate for anti-tumor drug development at single-element resolution

In response to the need for reliable cellular models that reflect complex tumor microenvironmental properties, and enable more precise testing of anti-cancer therapeutics effects on humans, a co-culture platform for in-vitro model that enhances the physiology of breast cancer (BC) microenvironment is presented. A six well imaging plate wherein each macro-well contains several separate compartments was designed. Three-dimensional (3D) cancer spheroids are generated and cultured in the inner compartment which is embossed with an array of nano-liter micro-chambers made of hydrogel. Stromal cells are cultured in the outer chambers. The two cell types are cultured side-by-side, sharing a common space, thus enabling extra-cellular communication via secreted molecules. As proof of concept, a model of BC tumor microenvironment was recapitulated by co-cultivating 3D MCF7 spheroids in the presence of tumor-associated macrophages (TAMs). The presence of TAMs induced an aggressive phenotype by promoting spheroid growth, enhancing survivin expression levels and enabling invasive behavior. Moreover, TAMs influenced the response of BC spheroids to cytotoxic treatment as well as hormonal drug therapy, and enhanced the effects of nitric oxide donor. The platform enables time-lapse imaging and treatment without losing spatial location of the measured spheroids, thereby allowing measurements and analysis at individual-object resolution in an easy and efficient manner.

The Role of Tumor-Associated Myeloid Cells in Modulating Cancer Therapy.

Myeloid cells include various cellular subtypes that are distinguished into mononuclear and polymorphonuclear cells, derived from either common myeloid progenitor cells (CMPs) or myeloid stem cells. They play pivotal roles in innate immunity since, following invasion by pathogens, myeloid cells are recruited and initiate phagocytosis and secretion of inflammatory cytokines into local tissues. Moreover, mounting evidence suggests that myeloid cells may also regulate cancer development by infiltrating the tumor to directly interact with cancer cells or by affecting the tumor microenvironment. Importantly, mononuclear phagocytes, including macrophages and dendritic cells (DCs), can have either a positive or negative impact on the efficacy of chemotherapy, radiotherapy as well as targeted anti-cancer therapies. Tumor-associated macrophages (TAMs), profusely found in the tumor stroma, can promote resistance to chemotherapeutic drugs, such as Taxol and Paclitaxel, whereas the suppression of TAMs can lead to an improved radiotherapy outcome. On the contrary, the presence of TAMs may be beneficial for targeted therapies as they can facilitate the accumulation of large quantities of nanoparticles carrying therapeutic compounds. Tumor infiltrating DCs, however, are generally thought to enhance cytotoxic therapies, including those using anthracyclines. This review focuses on the role of tumor-infiltrating and stroma myeloid cells in modulating tumor responses to various treatments. We herein report the impact of myeloid cells in a number of therapeutic approaches across a wide range of malignancies, as well as the efforts toward the elimination of myeloid cells or the exploitation of their presence for the enhancement of therapeutic efficacy against cancer.

Combined MEK inhibition and tumor-associated macrophages depletion suppresses tumor growth in a triple-negative breast cancer mouse model

Tumor-associated macrophages (TAMs) are closely related to poor prognosis in triple-negative breast cancer (TNBC). Thus, gaining insight into how TAMs support cancer progression could contribute to effective therapies. We utilized the 4 T1 murine TNBC cell line and murine bone marrow-derived macrophages to assess TAM-mediated pro-proliferative effects in vivo and in vitro. Further, Transcriptional analysis was performed to identify pathways activated in TAM-stimulated 4 T1 cells. We also explored the therapeutic efficacy of combining a mitogen-activated protein kinase kinase (MEK) inhibitor with TAM-targeted therapy using a TNBC mouse model. We found that the presence of TAMs was significantly associated with proliferating cancer cells in a TNBC mouse model. Moreover, RNA sequencing analysis showed that TAMs could enhance mitogen-activated protein kinase (MAPK) pathway activation in 4 T1 cells compared to that in control cells. Further, the depletion of TAMs by clodronate liposomes significantly reduced MAPK pathway activation in vivo. In addition, the blockade of MAPK signaling by a MEK inhibitor repressed TAM-mediated cancer cell proliferation. Most importantly, MEK inhibition combined with macrophage depletion significantly suppressed tumor growth and increased T lymphocyte infiltration in a TNBC model. Our study suggests the possibility that TAM-induced MAPK pathway activation promotes cancer cell proliferation. Thus, MEK inhibition combined with macrophage depletion might represent an effective treatment for TNBC.

Improved anti-tumor efficacy of paclitaxel in combination with MicroRNA-125b-based tumor-associated macrophage repolarization in epithelial ovarian cancer.

In epithelial ovarian cancers, the presence of tumor-associated macrophages (TAMs) is well correlated with the poor disease outcomes. TAMs are know to suppress the immune system, induce pro-tumoral functions and inhibit anti-tumor responses associated with chemotherapy. In this study, we have evaluated the synergistic efficacy of TAM repolarization and intraperitoneal paclitaxel in epithelial ovarian cancers. We demonstrate that hyaluronic acid-based nanoparticles encapsulating miR-125b (HA-PEI-miR-125b) can specifically target TAMs in the peritoneal cavity of a syngeneic ID8-VEGF ovarian cancer mouse model and can repolarize macrophages to an immune-activating phenotype. These HA-PEI-miR-125b nanoparticles in combination with intraperitoneal paclitaxel can enhance the anti-tumor efficacy of paclitaxel during the later stages of disease progression as seen by the significant reduction in the ascitic fluid and peritoneal VEGF levels. Furthermore, these HA-PEI-miR-125b nanoparticles do not induce systemic toxicity and thus warrant a further evaluation in the clinical setting.

LIF regulates CXCL9 in tumor-associated macrophages and prevents CD8+ T cell tumor-infiltration impairing anti-PD1 therapy

Cancer response to immunotherapy depends on the infiltration of CD8+ T cells and the presence of tumor-associated macrophages within tumors. Still, little is known about the determinants of these factors. We show that LIF assumes a crucial role in the regulation of CD8+ T cell tumor infiltration, while promoting the presence of protumoral tumor-associated macrophages. We observe that the blockade of LIF in tumors expressing high levels of LIF decreases CD206, CD163 and CCL2 and induces CXCL9 expression in tumor-associated macrophages. The blockade of LIF releases the epigenetic silencing of CXCL9 triggering CD8+ T cell tumor infiltration. The combination of LIF neutralizing antibodies with the inhibition of the PD1 immune checkpoint promotes tumor regression, immunological memory and an increase in overall survival.

Sulforaphane Impacts Invasion Capacity and Proliferation of Triple Negative Breast Cancer Cells Grown in the Presence of Tumor-associated Macrophages (P05-003-19)

ObjectivesTriple negative breast cancer (TNBC) accounts for 12% to 24% of all breast cancer cases and is characterized by higher proliferation rates and an increased likelihood of tissue invasion. Tumor cells and cells in the tumor microenvironment (TME), specifically tumor associated macrophages (TAMs), interact through signaling (e.g., cytokines) to promote cancer progression by increasing proliferation and invasion capacity. Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables (i.e., broccoli) that has shown promising results in hindering TNBC progression. SFN reduced proliferation in breast cancer cells and altered cytokine signaling between breast cancer cells and cells in the TME (adipocytes). SFN also decreased invasion markers in TNBC cells grown in isolation. However, TAMs promote tumor cell aggression, and SFN’s effect in multicellular environments is unclear. To determine SFN’s potential in cancer treatment, it is critical to investigate SFN’s effect on proliferation and invasion capacity of TNBC cells grown under TAMs influence, not just grown in isolation. The objective of this study was to determine if SFN can reduce proliferation and invasion capacity of TNBC cells grown in TAM secretions. MethodsFor cell proliferation, TNBC cells (MDA-MB-231) were exposed to TAM secretions using conditioned media and were then treated with SFN (10 μM) or DMSO vehicle control for 24 and 48 hours. Proliferation was measured using a MTT-based assay and cell counts. For invasion, TAMS and TNBC cells were co-cultured for 48 hours in transwell plates. Prior to co-culture period, cells were treated with SFN (15 μM) or vehicle control. Invasion capacity was measured through a transwell invasion assay with collagen representing the tumor basement membrane. ANOVA and t-tests were used to determine statistical differences with significance at P = 0.05. ResultsPreliminary analysis revealed significant reductions in proliferation for TAM exposed TNBC cells after 24 hours (P = 0. 0132) and 48 hours (P = 0. 0190) of SFN treatment. ConclusionsSFN reduced proliferation and can potentially reduce invasion capacity of TNBC cells influenced by TAM secretions thus enhancing future utilization of SFN in TNBC treatment regimens. Funding SourcesCalifornia State University, Chico.

Abstract 4742: Role of tumor-associated macrophages in esophageal cancer: PD-L1 expression and prognosis

Abstract Introduction: It has become clear that macrophages are important cells forming cancer microenvironment and tumor-associated macrophages (TAM) promote their proliferation in various cancer types. In particularly, M2-type macrophages have been reported to promote the production of anti-inflammatory factors and the infiltration of regulatory T cells and suppress anti-tumor immunity, and new antitumor therapies that control tumor growth by controlling TAM have been proposed. In addition, "anti-PD-1 antibody" has attracted attention also in esophageal cancer, but the relationship between TAM and PD-L1 expression has not been clarified. Methods: A retrospective study was performed on 301 patients who underwent esophagectomy for esophageal cancer between March 2005 and June 2013. We performed immunohistochemistry and evaluation of macrophage markers (CD163 and CD204) and PD-L1. In addition, we performed in vitro experiments to clarify the cellular malignancy and relationship with PD-L1 expression. Results: Out of 301 cases of esophageal cancer, 156 cases showed CD163 high expression (51.8%) while 146 cases showed CD204 high expression (48.5%). We divided into three groups of TAM status based on CD204 and CD163 expression (CD163 and CD204 both positive: TAM status high (n=100), either one positive: moderate (n=102), both negative: low (n=99)). In TAM high group, there were more advanced cancers and adenocarcinoma cases (P<0.0001, P=0.042, respectively). Interestingly, TAM status correlated with PD-L1 expression (P=0.0005). We found that there was a significant difference in overall survival, cancer specific survival and disease-free survival among these three groups (log rank P = 0.0062, P= 0.0012, P= 0.0006, respectively), and TAM status correlated with poor prognosis. We performed double immunostaining of PD-L1 and CD163, there is a part where PD-L1 is expressed around TAM, suggesting that the expression of PD-L1 and the presence of TAM are correlated. We performed co-culture of esophageal cancer cell lines (TE-8 and KYSE30) and activated macrophage. In real time PCR PD-L1 expression were elevated at co-culture with activated macrophage. Fluorescence activated cell sorting (FACS) revealed PD-L1 expression positive cells were increased at co-culture with activated macrophage. In invasion assay, when co-cultured with activated macrophages, the number of infiltrating cells were significantly increasing (P=0.00021). In timelapse imaging showed increased cell activity by co-culture with activated macrophage. Conclusion: This study suggested that TAM correlated with poor prognosis in patients with esophageal cancer. In addition, TAM correlated with invasive capacity and migratory capacity. Importantly, TAM correlated PD-L1 expression in esophageal cancer cell lines. Citation Format: Taisuke Yagi, Yoshifumi Baba, Yuki Koga, Tomoyuki Uchihara, Yuki Kiyozumi, Hiroshi Sawayama, Yukiharu Hiyoshi, Masaaki Iwatsuki, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. Role of tumor-associated macrophages in esophageal cancer: PD-L1 expression and prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4742.

Matrix stiffness and tumor-associated macrophages modulate epithelial to mesenchymal transition of human adenocarcinoma cells

The tumor microenvironment (TME) is gaining increasing attention in oncology, as it is recognized to be functionally important during tumor development and progression. Tumors are heterogeneous tissues that, in addition to tumor cells, contain tumor-associated cell types such as immune cells, fibroblasts, and endothelial cells. These other cells, together with the specific extracellular matrix (ECM), create a permissive environment for tumor growth. While the influence of tumor-infiltrating cells and mechanical properties of the ECM in tumor invasion and progression have been studied separately, their interaction within the complex TME and the epithelial -to-mesenchymal transition (EMT) is still unclear. In this work, we develop a 3D co-culture model of lung adenocarcinoma cells and macrophages in an interpenetrating network hydrogel, to investigate the influence of the macrophage phenotype and ECM stiffness in the induction of EMT. Rising ECM stiffness increases both tumor cell proliferation and invasiveness. The presence of tumor-associated macrophages and the ECM stiffness jointly contribute to an invasive phenotype, and modulate the expression of key EMT-related markers. Overall, these findings support the utility of in vitro 3D cancer models that allow one to study interactions among key components of the TME.

Abstract P2-04-20: PD-L1 expression in triple negative breast cancer (TNBC) is associated with improved outcomes

Abstract Background: Breast cancer (BC) evolution is influenced by tumor microenvironment. Presence of CD8+ cytotoxic T lymphocytes (TILs) has been proposed as surrogate marker of adaptive immune response, and programmed death ligand-1 (PD-L1) is a negative regulator of the tumor immune microenvironment. However, whether PD-L1 expression adversely affects breast cancer outcome is unknown (Oncotarget 2014; 6:5449). Tumor-associated macrophages (TAMs) in the tumor microenvironment may contribute to BC progression and metastagenicity. We assessed the potential correlations between PD-L1 expression, the presence of TAMs and TILs, and BC outcomes. Methods: 59 primary BCs (16 HR+, 16 HER2+, and 27 TNBC) with known clinical and pathological features and patient (pt) follow-up for a median of 3.9 years were evaluated by immunohistochemistry for expression of CD8, CD68, and PD-L1 within tumor and stroma. The average number of CD8+ cells within 10 high power fields was determined separately for invasive tumor cell nests and for stroma within each sample, and the median number of CD8+ cells within tumor vs stroma was calculated (Breast Cancer Res Treat 2011 128:703–711). Non-lymphocyte mononuclear cells in tumor and stroma were used in counting CD68+ TAMs. BCs were positive for CD8+ TILs (Cell Marque clone #C8144B) or CD68+ TAMs (Cell Marque clone #KP1) if the number of cells positive in the sample was greater than the median. PD-L1 (Dako 28-8 pharmDx) was positive if at least 1% of tumor cells expressed PD-L1. The log-rank was used to compare the survival and progression free survival between groups and Spearman's rank-order correlation tests were conducted to determine associations between CD8, CD68, and PD-L1. Results: 57% of TN, 26% of HER2+ and 13% of HR+ BCs expressed PD-L1 in tumor. TNBC pts received anthracycline/ taxane chemotherapy (62%) or taxane therapy alone (22%). HR+ and HER2+ pts received standard endocrine therapy and trastuzumab-based therapy. Stromal CD8+ TILs were associated with improved OS in the overall population (log rank p=0.026). In TNBC, PD-L1 expression was positively correlated with the presence of TILs (p=0.0002) and TAMs (p=0.0005) as well as with improved 3 year PFS and OS (log rank p=0.04 and p=0.03, respectively). Furthermore, stromal CD8+ TILs and stromal CD68+ TAMs correlated positively with each other in the TNBC group (p=0.0094). Conclusions: PD-L1 expression correlated with TILs and TAMs in TNBC and was associated with a favorable outcome. PD-L1+ TNBCs with high levels of TILs and TAMs may be primed for exceptional response to immunogenic chemotherapy alone. Whether some pts with PD-L1+ TNBCs with high TILs/TAMs will benefit additionally from an anti-PD-L1/PD-1 agent is being investigated currently. Citation Format: Mardones MA, Grosser D, Levin MK, Daoud Y, Palucka K, O'Shaughnessy J, Osborne C. PD-L1 expression in triple negative breast cancer (TNBC) is associated with improved outcomes [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-20.