Presence Of M2 Macrophages Research Articles

Modulation of the tumor microenvironment in non-muscle-invasive bladder cancer by OncoTherad® (MRB-CFI-1) nanoimmunotherapy: effects on tumor-associated macrophages, tumor-infiltrating lymphocytes, and monoamine oxidases.

Non-muscle-invasive bladder cancer (NMIBC) presents management challenges due to its high recurrence rate and a complex tumor microenvironment (TME). This study investigated the effects of OncoTherad® (MRB-CFI1) nanoimmunotherapy on the TME of BCG-unresponsive NMIBC, focusing on alterations in monoamine oxidases (MAO-A and MAO-B) and immune markers: CD163, FOXP3, CD8, and CX3CR1. A comparative analysis of immunoreactivities was made before and after OncoTherad® treatment and an immune score (IS) was established to evaluate the correlation between immunological changes and clinical outcomes. Forty bladder biopsies of twenty patients were divided into 2 groups (n = 20/group): 1 (pre-treatment biopsies); and 2 (post-treatment biopsies). Our results showed stable MAO-A levels but a significant (p < 0.05) decrease in MAO-B immunoreactivity after treatment, suggesting OncoTherad®'s efficacy in targeting the tumor-promoting and immunosuppressive functions of MAO-B. Significant (p < 0.05) reductions in CD163 and FOXP3 immunoreactivities were seen in post-treatment biopsies, indicating a decreased presence of M2 macrophages and Tregs. Corroborating with these results, we observed reductions in tumor histological grading, focality and size, factors that collectively enhanced recurrence-free survival (RFS) and pathological complete response (PCR). Moreover, elevated IFN-γ immunoreactivities in treated biopsies correlated with increased counts of CD8+ T cells and higher CX3CR1 expression, underscoring OncoTherad®'s enhancement of cytotoxic T cell functionality and overall antitumor immunity. The IS revealed improvements in immune responses post-treatment, with higher scores associated with better RFS and PCR outcomes. These findings validate OncoTherad®'s capability to modify the bladder cancer microenvironment favorably, promoting effective immune surveillance and response.

Crosstalk between GBP2 and M2 macrophage promotes the ccRCC progression.

Clear cell renal cell carcinoma (ccRCC) represents a highly heterogeneous kidney malignancy associated with the poorest prognosis. The metastatic potential of advanced ccRCC tumors is notably high, posing significant clinical challenges. There is an urgent imperative to develop novel therapeutic approaches to address ccRCC metastasis. Recent investigations indicated a potential association between GBP2 and tumor immunity. However, the precise functional role of GBP2 in the progression of ccRCC remains poorly understood. The present study revealed a strong correlation between GBP2 and M2 macrophages. Specifically, our findings demonstrated that the inhibition of GBP2 significantly impedes the migratory and invasive capabilities of ccRCC cells. We observed that the presence of M2 macrophages can reverse the effects of GBP2 knockdown on tumor cell migration and invasion. Mechanistically, we demonstrated that M2 macrophages promote the expression of the GBP2/p-STAT3 and p-ERK axis in tumor cells through the secretion of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), thereby substantially enhancing the migratory and invasive capacities of the tumor cells. Simultaneously, we have identified that GBP2 promotes the polarization of macrophages to the M2 phenotype by stimulating the secretion of interleukin-18 (IL-18). In summary, our investigation anticipates that the GBP2/IL-18/M2 macrophages/IL-10 and the TGF-β/GBP2, p-STAT3, p-ERK loop plays a crucial role in ccRCC metastasis. The collective findings from our research underscore the significant role of GBP2 in tumor immunity and emphasize the potential for modulating GBP2 as a promising therapeutic strategy for targeting ccRCC metastasis.

Novel cell therapy with ex vivo cultured peripheral blood mononuclear cells significantly impacts angiogenesis in the murine ischemic limb model

IntroductionAutologous mononuclear cells (MNCs) have been used in vascular regenerative therapy since the identification of endothelial progenitor cells (EPCs). However, the efficacy of autologous EPC therapy for diseases such as diabetes and connective tissue disorders is limited due to deficiencies in the number and function of EPCs. To address this, we developed a novel RE-01 cells that enriches pro-angiogenic cells from peripheral blood MNCs (PBMNCs). MethodsPBMNCs were collected from healthy volunteers following ethical guidelines. RE-01 cells were cultured in the presence of specific growth factors for 5 days without media change. Flow cytometry was used to analyze cell surface markers. Tube formation assays, EPC culture assays, and mRNA analysis were performed to evaluate angiogenic potential. The efficacy of RE-01 cells upon transplantation into ischemic hind limbs of mice was evaluated. ResultsRE-01 cells exhibited a significant increase in pro-angiogenic cells such as M2 macrophages and angiogenic T cells, in contrast to PBMNCs, while the number of inflammatory cells reduced. In vitro assays demonstrated the enhanced angiogenic abilities of RE-01 cells, supported by increased mRNA expression of angiogenesis-related cytokines. In vivo studies using mouse ischemic hind limb models have shown that blood flow and angiogenesis improved following RE-01 cell transplantation. Transplantations for 3 consecutive days significantly improved the number of pericyte-recruited vessels in the severely ischemic hind limbs of mice. ConclusionsRE-01 cells showed promising results in enhancing angiogenesis and arteriogenesis, possibly owing to the presence of M2 macrophages and angiogenic T cells. These cells also demonstrated anti-fibrotic effects. The efficacy of RE-01 cells has been confirmed in mouse models, suggesting their potential for treating ischemic vascular diseases. Clinical trials are planned to validate the safety and efficacy of RE-01 cell therapy in patients with connective tissue disease and unhealed ulcers. We hope that this new RE-01 cell therapy will prevent many patients from undergoing amputation.

Unveiling the Role of Endoplasmic Reticulum Stress Pathways in Canine Demodicosis.

Canine demodicosis is a prevalent skin disease caused by overpopulation of a commensal species of Demodex mite, yet its precise cause remains unknown. Research suggests that T-cell exhaustion, increased immunosuppressive cytokines, induction of regulatory T cells and increased expression of immune checkpoint inhibitors may contribute to its pathogenesis. This study aimed to gain a deeper understanding of the molecular changes occurring in canine demodicosis using mass spectrometry and pathway enrichment analysis. The results indicate that endoplasmic reticulum stress promotes canine demodicosis through regulation of three linked signalling pathways: eIF2, mTOR, and eIF4 and p70S6K. These pathways are involved in the modulation of Toll-like receptors, most notably TLR2, and have been shown to play a role in the pathogenesis of skin diseases in both dogs and humans. Moreover, these pathways are also implicated in the promotion of immunosuppressive M2 phenotype macrophages. Immunohistochemical analysis, utilising common markers of dendritic cells and macrophages, verified the presence of M2 macrophages in canine demodicosis. The proteomic analysis also identified immunological disease, organismal injury and abnormalities and inflammatory response as the most significant underlying diseases and disorders associated with canine demodicosis. This study demonstrates that Demodex mites, through ER stress, unfolded protein response and M2 macrophages contribute to an immunosuppressive microenvironment, thereby assisting in their proliferation.

Abstract 2180: Modulation of immune infiltration patterns in inflammation-associated colorectal cancer through rice bran-based dietary strategies

Abstract The correlation between gut microbiota dysbiosis and inflammation-associated colorectal cancer (CRC) has generated considerable interest in dietary interventions targeting immune modulation. Our prior study demonstrated the colon cancer protective effects of rice bran (RB) and ex vivo Bifidobacterium longum fermented rice bran (FRB) diet intervention against azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC in gut microbiota-intact specific pathogen-free mice (SPF). RB showed higher levels of protection compared to FRB against CRC, as evidenced by diminished neoplastic lesion size and restored colonic epithelial integrity. The major objective of the present study was to leverage cutting-edge multispectral imaging technique for simultaneous detection of multiple immune cells markers and for a comprehensive analysis of distinct immune landscapes. We also performed spatial correlations within the tumor microenvironment of AOM/DSS-SPF mice undergoing RB and FRB dietary interventions. The primary focus was on T cell subtypes including cytotoxic T cells, helper T cells, regulatory T (Treg) cells, natural killer (NK) cells, NKT cells, B cells and macrophages (MΦ) subtypes (M1 MΦ, and M2 MΦ) to uncover the intricacies of the immune landscape and the interplay for CRC prevention through dietary intervention. The results indicated that RB intervention for 15 weeks led to reductions in overall T cell subtypes in the colonic tissue, particularly Treg cells, which are associated with protecting tumors against immune surveillance in CRC. On the other hand, FRB intervention also resulted in reductions in overall T cell subtypes but exhibited higher infiltration of NK cells and proinflammatory M1 macrophages in the colonic tissue after 15 weeks. While an increased presence of NK cells might be beneficial in countering CRC, the infiltration of M1 macrophages could potentially lessen the impact of FRB compared to RB intervention in CRC protection. The outcomes suggest that the better protective efficacy of non-fermented RB diets is linked to the modulation of Treg cell infiltration, whereas the efficacy of the FRB diet is to some extent limited by the presence of M1 macrophages. Overall, this study provides valuable insights into the complex immune landscape influenced by RB and FRB interventions in a murine model of inflammation-induced CRC, and a novel mechanism for the immunomodulatory effects of dietary RB interactions with gut microbiota. In the future, observing immune modulation throughout diet intervention would enhance our understanding of the role played by these immune cells. Citation Format: Robin Kumar, Lakshmi Sai Pratyusha Bugata, Md. Imtiazul Kabir, Rajesh Agarwal, Elizabeth P. Ryan, Komal Raina. Modulation of immune infiltration patterns in inflammation-associated colorectal cancer through rice bran-based dietary strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2180.

Transient receptor potential channels as predictive marker and potential indicator of chemoresistance in colon cancer.

Transient receptor potential (TRP) channels are strongly associated with colon cancer development and progression. This study leveraged a multivariate Cox regression model on publicly available datasets to construct a TRP channels-associated gene signature, with further validation of signature in real world samples from our hospital treated patient samples. Kaplan-Meier (K-M) survival analysis and receiver operating characteristic (ROC) curves were employed to evaluate this gene signature's predictive accuracy and robustness in both training and testing cohorts, respectively. Additionally, the study utilized the CIBERSORT algorithm and single-sample gene set enrichment analysis to explore the signature's immune infiltration landscape and underlying functional implications. The support vector machine algorithm was applied to evaluate the signature's potential in predicting chemotherapy outcomes. The findings unveiled a novel three TRP channels-related gene signature (MCOLN1, TRPM5, and TRPV4) in colon adenocarcinoma (COAD). The ROC and K-M survival curves in the training dataset (AUC = 0.761; p = 1.58e-05) and testing dataset (AUC = 0.699; p = 0.004) showed the signature's robust predictive capability for the overall survival of COAD patients. Analysis of the immune infiltration landscape associated with the signature revealed higher immune infiltration, especially an increased presence of M2 macrophages, in high-risk group patients compared to their low-risk counterparts. High-risk score patients also exhibited potential responsiveness to immune checkpoint inhibitor therapy, evident through increased CD86 and PD-1 expression profiles. Moreover, the TRPM5 gene within the signature was highly expressed in the chemoresistance group (p = 0.00095) and associated with poor prognosis (p = 0.036) in COAD patients, highlighting its role as a hub gene of chemoresistance. Ultimately, this signature emerged as an independent prognosis factor for COAD patients (p = 6.48e-06) and expression of model gene are validated by public data and real-world patients. Overall, this bioinformatics study provides valuable insights into the prognostic implications and potential chemotherapy resistance mechanisms associated with TRPs-related genes in colon cancer.

THU499 The Mesenchymal Stem Cells Induce Immunosuppressive Microenvironment In Pituitary Tumors

Abstract Disclosure: D. Marrero-Rodríguez: None. V. Cortes-Morales: None. K. Taniguchi-Ponciano: None. A. Valenzuela-Perez: None. S. Vela-Patiño: None. A. Cano-Zaragoza: None. F. Martinez-Mendoza: None. J. Kerbel: None. S. Andonegui-Elguera: None. J. Montesinos: None. M. Mercado: None. The tumor microenvironment (TME) includes diverse cellular components such as mesenchymal stem cells (MSC) and immune cells. MSC are a subset of heterogeneous cell populations characterized by the expression of CD105, CD73, and CD90 that can differentiate into chondrocytes, osteoblasts, and adipocytes in vitro. They are known to have immunomodulatory capabilities. Our aim was to isolate and characterize the immunomodulatory capabilities of MSC from pituitary tumors (PT) and non-tumoral gland as well as to describe the immune TME. We isolated and cultivated MSC from LH/FSH-PT and ACTH-PT, as well as non-tumoral pituitary glands. The MSC obtained from PT and non-tumoral gland were co-cultured in the presence of monocytes from healthy donors and their ability to transform into M0, M1 or M2 macrophages was assessed by flow cytometry. The results showed that pituitary tumors derived MSC induce an immunosuppressive M2 macrophage state, by increasing the expression of CD14 and CD206 markers, and decreasing HLA-DR. Upon co-culturing them with naïve T cells, PT MSC were capable inducing a Treg phenotype. To identify PT TME, we performed tumor whole transcriptome characterization and cellular deconvolution using CIBERSORT. Our analysis was indicative of a strong presence of M2 macrophages, TCD4+ and TCD8+ cells, which was confirmed by immunofluorescence, suggesting an immunosuppressive TME. Thus, the MSC immunosuppressive induction correlates directly with immune TME. ACTH-PT derived MSC showed a stronger anti-inflammatory activity than LH/FSH-PT. We also characterized the transcriptomic differences between non-tumoral and PT derived MSC by means of RNAseq. We found differences between the non-tumoral MSC and pituitary tumor derived MSC, but mainly between ACTH- and LH/FSH-PT derived MSC. In conclusion our data suggest that the presence of MSC influence an immunosuppressive tumor microenvironment alongside with tumor cells. Presentation: Thursday, June 15, 2023

M2 macrophage inhibits the antitumor effects of Lenvatinib on intrahepatic cholangiocarcinoma.

The relationship between the tumor microenvironment and the network of key signaling pathways in cancer plays a key role in the occurrence and development of tumors. Tumor-associated macrophages (TAMs) are important inflammatory cells in the tumor microenvironment and play an important role in tumorigenesis and progression. Macrophages in malignant tumors, mainly the M2 subtype, promote tumor progression by producing cytokines and down-regulating anti-inflammatory immune responses. Several articles have investigated the effect of macrophages on the sensitivity of cancer chemotherapeutic agents, but few such articles have been reported in cholangiocarcinoma, so we investigated the effect of M2 macrophage on the sensitivity of cholangiocarcinoma cells to Lenvatinib compared to M1. THP-1 monocytes were polarized to M0 macrophage by phorbol 12-myristate 13-acetate (PMA) and then induced to differentiate into M1 and M2 macrophages by LPS, IFN-γ and IL-4 and IL-13, respectively. Macrophages and cholangiocarcinoma cells were co-cultured prior to 24 hours of Lenvatinib administration, cancer cell apoptosis was detected by western-blot, FACS analysis of Annexin V and PI staining. Furthermore, we use xCELLigence RTCA SP Instrument (ACEA Bio-sciences) to monitor cell viability of Lenvatinib administration in co-culture of cholangiocarcinoma cells and macrophages. After tumorigenesis in immunodeficient mice, Lenvatinib was administered, and the effects of M2 on biological characteristics of cholangiocarcinoma cells were investigated by immuno-histochemistry. mRNA and protein expression of M1 and M2 markers confirmed the polarization of THP-1 derived macrophages, which provided a successful and efficient model of monocyte polarization to TAMs. Lenvatinib-induced apoptosis of cholangiocarcinoma cells was significantly reduced when co-cultured with M2 macrophage, whereas apoptosis of cholangiocarcinoma cells co-cultured with M1 macrophage was increased. In the CDX model, Lenvatinib-induced cancer cell apoptosis was markedly reduced, and proliferative cells increased in the presence of M2 macrophages. Angiogenesis related factors was significantly increased in cholangiocarcinoma cells co-cultured with M2. Compared with M1, M2 macrophages can inhibit the anti-tumor effect of Lenvatinib on cholangiocarcinoma through immune regulation, which may be related to the tumor angiogenesis factor effect of M2 macrophage.

Soluble CD163 predicts outcome in both chemoimmunotherapy and targeted therapy-treated mantle cell lymphoma.

The outcome for patients with mantle cell lymphoma (MCL) has drastically improved with new treatments directed toward the tumor immune microenvironment, where macrophages play an important role. In MCL, the presence of M2 macrophages defined by CD163 expression in diagnostic biopsies has been associated with a worse prognosis. An alternative way to assess the abundance of M2 macrophages is by measuring the level of soluble CD163 in serum (sCD163). We aimed to investigate the prognostic value of sCD163 in 131 patients with MCL. We found that high sCD163 at diagnosis was associated with shorter progression-free survival (PFS) and shorter overall survival (OS) in 81 patients who were newly diagnosed and subsequently treated with chemoimmunotherapy. The same was seen in a cohort of 50 patients with relapsed MCL that were mainly treated within the phase 2 Philemon-trial with rituximab, ibrutinib, and lenalidomide. In patients who were newly diagnosed and had low levels of sCD163, 5-year survival was 97%. There was a moderate correlation between sCD163 and tissue CD163. The association with a poor prognosis was independent of MCL international prognostic index, Ki67, p53 status, and blastoid morphology, as assessed in a multivariable Cox proportional hazards model. In this study, high sCD163 was associated with both shorter PFS and shorter OS, showing that high levels of the M2 macrophage marker sCD163 is an independent negative prognostic factor in MCL, both in the chemoimmunotherapy and ibrutinib/lenalidomide era. In addition, low sCD163 levels identify patients with MCL with a very good prognosis.

P-316 Involvement of collective cell migration in endometrial invasiveness in uterine adenomyosis and the role of M2 macrophages

Abstract Study question How do endometrial cells manage to invade surrounding myometrium to establish adenomyotic lesions and what is the role of macrophages in this process? Summary answer M2 macrophage-mediated invasiveness and resistance of endometrial cells to physiological cell death appear to contribute to the pathogenesis of adenomyosis. What is known already Despite the high prevalence and debilitating symptoms of uterine adenomyosis, its pathogenesis has not yet been elucidated. To date, the most acceptable theory to explain disease development suggests invasion of the myometrium by eutopic endometrial cells. Recent data suggest aberrant infiltration of immune cells but not platelets in adenomyosis, eventually leading to enhanced cell motility via collective cell migration (CCM) and resistance to physiological cell death during disease pathogenesis. Study design, size, duration A retrospective immunohistochemistry-based study was conducted on formalin-fixed paraffin-embedded uterine tissue from 17 women (8 adenomyosis patients and 9 healthy subjects). Fresh endometrial biopsies were retrieved from 16 women (6 adenomyosis patients and 10 healthy subjects) for in vitro culture of epithelial and stromal cells, and co-culture with THP-1 monocyte-derived M2 macrophages. Participants/materials, setting, methods Double immunofluorescence was performed to detect macrophages of M1 and M2 phenotypes. Invasiveness of endometrial cells upon interaction with macrophages was assessed by in vitro invasion assays and quantitative PCR for genes involved in cell motility and epithelial-mesenchymal transition (EMT). Mechanisms of disease progression were investigated by immunohistochemistry against E-cadherin, N-cadherin and matrix metalloproteinase 9 (MMP9). Caspase 3 and microtubule-associated protein light chain 3 beta (LC3B) were studied as markers of apoptosis and autophagy respectively. Main results and the role of chance Only M2 macrophages were found to accumulate in adenomyosis, in higher numbers in both endometrium and ectopic lesions from adenomyosis patients than healthy controls. Co-culture with M2 macrophages significantly increased invasion capacity in endometrial epithelial and stromal cells from both adenomyosis patients and healthy controls. Epithelial cells from adenomyosis patients were nonetheless more invasive than their healthy counterparts in the presence of M2 macrophages. No gene expression differences pointing to EMT were noted, either between co-cultured and control cells, or between adenomyotic and healthy cells, refuting the implication of this mechanism in adenomyosis invasiveness. E- and N-cadherin protein expression did not differ significantly between eutopic endometrium from adenomyosis subjects and healthy tissue, but N-cadherin expression was stronger in ectopic lesions. In adenomyosis, both E- and N-cadherin were more extensively expressed in basal (also known as invasive) glands than functional glands, suggesting maintenance of intercellular junctions and subsequent CCM. Extracellular matrix-degrading enzyme MMP9 was more abundantly expressed in eutopic stroma from adenomyosis patients compared to healthy tissue. Lower levels of caspase 3 were detected in both endometrium and lesions from adenomyosis patients, while LC3B was found significantly decreased in adenomyotic stroma compared to that of healthy subjects. Limitations, reasons for caution A limitation of the study is the 2D nature of in vitro cultures, which cannot fully reconstitute in vivo cell arrangement and cell-cell interactions, nor allow to observe CCM in real time. Wider implications of the findings M2 macrophages accumulating in endometrium and ectopic lesions from adenomyosis patients boost invasion capacity of endometrial epithelial and stromal cells, as shown by in vitro co-culture experiments. These findings indicate that M2 macrophages play a key role in disease pathogenesis and could be targeted to develop novel therapeutic options. Trial registration number not applicable

Synthesis and Anti-Inflammatory Evaluation of a Library of Chiral Derivatives of Xanthones Conjugated with Proteinogenic Amino Acids.

In recent decades, the relationship between drug chirality and biological activity has been assuming enormous importance in medicinal chemistry. Particularly, chiral derivatives of xanthones (CDXs) have interesting biological activities, including enantioselective anti-inflammatory activity. Herein, the synthesis of a library of CDXs is described, by coupling a carboxyxanthone (1) with both enantiomers of proteinogenic amino esters as chiral building blocks (2-31), following the chiral pool strategy. The coupling reactions were performed at room temperature with good yields (from 44 to 99.9%) and very high enantiomeric purity, with most of them presenting an enantiomeric ratio close to 100%. To afford the respective amino acid derivatives (32-61), the ester group of the CDXs was hydrolyzed in mild alkaline conditions. Consequently, in this work, sixty new derivatives of CDXs were synthetized. The cytocompatibility and anti-inflammatory activity in the presence of M1 macrophages were studied for forty-four of the new synthesized CDXs. A significant decrease in the levels of a proinflammatory cytokine targeted in the treatment of several inflammatory diseases, namely interleukin 6 (IL-6), was achieved in the presence of many CDXs. The amino ester of L-tyrosine (X1AELT) was the most effective in reducing IL-6 production (52.2 ± 13.2%) by LPS-stimulated macrophages. Moreover, it was ≈1.2 times better than the D-enantiomer. Indeed, enantioselectivity was observed for the majority of the tested compounds. Thus, their evaluation as promising anti-inflammatory drugs should be considered.

Abstract 5828: Distinct immunosuppressive tumor microenvironment modulates small cell lung carcinoma neuroendocrine phenotypic plasticity and heterogeneity

Abstract Small cell lung carcinoma (SCLC) is prototypical of cancers with exceptional chemoresistance, and metastatic capabilities fueled by remarkable heterogeneity. The cell-extrinsic factors that govern SCLC heterogeneity are poorly understood especially since non-genetic mechanisms are thought to be major contributors to its intratumoral heterogeneity and plasticity. In order to answer the above question, we sought to inquire about the role of tumor microenvironment (TME) in SCLC heterogeneity which is challenged universally by a lack of patient-derived adequate tumor biopsies. Leveraging large tumor sampling performed at rapid autopsies done on 10 different patients who died from SCLC metastatic disease, we developed a multi-omics approach involving spatial transcriptomics (ST) (Whole transcriptomic atlas), bulk whole genome sequencing, and multiplex immunofluorescence (IF) and standard immunohistochemistry (IHC) to better characterize spatially proximate tumor (36 segments), TME (32 segments) and 4 normal segments. Genomically, SCLC characteristic alterations like TP53 (70%) and RB1 (80%) inactivation and MYC (50%)/MYCL (80%) were evident. ST profiling reveals three distinct tumor subtypes corresponding to the known high-Neuroendocrine (NE) and low-NE SCLC subtypes as well as a novel hybrid-NE state with multiple elevated cancer hallmark capabilities and morphological evidence of nesting and budding. High-NE and hybrid-NE subtypes appear to be driven by ASCL1 and the low-NE subtype resembles the SCLC- inflamed subtype. ST profiling of proximate TMEs reveals similar heterogeneity that is closely linked with SCLC-NE heterogeneity at inter-patient and intra-tumoral levels with distinct features and cellular compositions. Specifically, low-NE TME is marked by increased pro and anti-tumor features including B cells, NK cells, M1-macrophages, and evidence of more checkpoint molecules. High-NE TMEs are predominated by a distinct absence of adaptive immune cells and a strong presence of M2 macrophages. Most strikingly the hybrid-NE TME shows a significant enrichment of immunosuppressive cancer-associated fibroblasts with the absence of T and other adaptive immune cells confirmed using IHC and IF. Next, we attempt to narrow down the fibroblast growth factor receptor (FGF) axis as one of the unique TME-driven factor(s) that could groom non-NE features in SCLC fueling their plasticity and heterogeneity. Subsequently using various SCLC cell lines, we demonstrate that in-vitro modulation of FGFR in SCLC may constrain their plasticity by downregulation of NOTCH signaling, MYC, and its downstream effector REST. We describe the remarkable phenotypic heterogeneity of SCLC tumor and TME and demonstrate their interlinking as well as potential translational avenues of leveraging the cellular crosstalk in order to constrain SCLC heterogeneity. Citation Format: Parth Anil Desai, Nobuyuki Takahashi, Yingying Cao, Christopher Schultz, Darryl Nousome, Desiree Tillo, Lakshya Chauhan, Mayank tandon, Rajesh kumar Bhardwaj, Delphine Lissa, Vinodh Rajapakse, Suresh kumar, Yang Zhang, Elmeskini Rajaa, Donna Butcher, Linda Scuito, Jingjing Gong, Brett Schroder, Andrew Warner, Samantha Nichols, Jordan Kimberly, Alejandro Schaffer, Mohit kumar Jolly, Stephen Hewitt, Anish Thomas. Distinct immunosuppressive tumor microenvironment modulates small cell lung carcinoma neuroendocrine phenotypic plasticity and heterogeneity [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 5828.

Abstract 79: Study of tumor microenvironment of ovarian clear cell carcinoma

Abstract Background: Clear cell ovarian carcinoma (CCOC) is characterized by a distinct histologic and molecular profile, and associated with very poor responses to standard treatment consisting of surgery and carboplatin:taxol chemotherapy. CCOC is chemo-resistant at the time of diagnosis and response to chemotherapy in the recurrent setting is less than 10%. Literature suggests a potential role for immune checkpoint inhibitors (ICI). However, only a subgroup of patients (20%) responds to ICI and little is known about the mechanisms of response and resistance to therapy. We postulate that a better understanding of CCOC tumor microenvironment (TME) could help predict patients’ response to ICI. Objective: The aim of this study is to investigate the relationship between TME immune infiltrate and clinical/outcome in 22 CCOC cases and identify subsets of CCOC who may benefit from immunotherapy. Material &amp; Methods: We characterized the immune landscape of 11 early and 11 advanced CCOC through a multiplex IHC Discovery Platform. Spatial single-cell proteomics analyses (cyclic-IF) and spatially-resolved RNAseq in 10 CCOC cases using an OC tissue microarray (TMA) were performed. Results were corelated with clinical and treatment outcome. Results: The percent of CD8, CD4, CD20 B cells Tregs, PD1, PDL1, monocytes and M2 monocytes and myeloid cells was significantly higher in advanced than early-stage cancers. Recurrent cancers were more immunosuppressive than cases with no recurrence. Tumor infiltrate was dense in 4 cases. Four patients with early-stage disease had a high number of CD8 naïve cells and experienced no recurrence. TME analysis of single case of advanced CCOC (before and after chemotherapy), revealed that hot tumor changed to cold tumor, suggesting the resistance to treatment. Cyclic IF analyses identified 3 tumor phenotype groups in a subset of 10 CCOC present on the OC TMA. The majority of CCOC had high expression of CCNE and high PI3K-AKT-mTor activity, low expression of hormone receptors (AR, ERa, PRg) and low cell cycle activity. Some tumors were also high for HER2. The stromal compartment was enriched in collagen VI, aSMA and PDGFR. The immune monitoring of several of those samples also revealed an immunosuppressive microenvironment, including the presence of M2 macrophages and expression of immune checkpoint proteins PD-L1 and B7-H4. Conclusion: Our study shows that various phenotypes of CCOC are defined by the cancer cell profiles and TME content. Importantly, a strong immunosuppressive microenvironment was detected in many samples, suggesting a potential response to ICI. Furthermore, we detected the expression of several therapeutic targets (MAPK, CCNE, PI3K-AKT-mTOR, HSP90, HER2), including oncogenic signaling pathways (RTK, MAPK). Different tumor phenotypes identified across our CCOC samples suggest that clear cell carcinoma could be subclassified into subtypes that should be treated differently. Citation Format: Tanja Pejovic, Sonali Joshi, Shawn Campbell, Dhanir Tailor, Joanna Pucilowska, Benjamin Tate, Pierre-Valérien Abate, Korina Mouzakitis, Marilyne Labrie, Elizabeth Munro, Jenna Emerson, Sanjay V. Malhotra. Study of tumor microenvironment of ovarian clear cell carcinoma [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 79.

Mesenchymal Stem/Stromal Cells Derived from Cervical Cancer Promote M2 Macrophage Polarization

Macrophages with the M2 phenotype promote tumor development through the immunosuppression of antitumor immunity. We previously demonstrated the presence of mesenchymal stem/stromal cells (MSCs) in cervical cancer (CeCa-MSCs), suggesting an immune protective capacity in tumors, but to date, their effect in modulating macrophage polarization remains unknown. In this study, we compared the capacities of MSCs from normal cervix (NCx) and CeCa to promote M2 macrophage polarization in a coculture system. Our results demonstrated that CeCa-MSCs, in contrast to NCx-MSCs, significantly decreased M1 macrophage cell surface marker expression (HLA-DR, CD80, CD86) and increased M2 macrophage expression (CD14, CD163, CD206, Arg1) in cytokine-induced CD14+ monocytes toward M1- or M2-polarized macrophages. Interestingly, compared with NCx-MSCs, in M2 macrophages generated from CeCa-MSC cocultures, we observed an increase in the percentage of phagocytic cells, in the intracellular production of IL-10 and IDO, the capacity to decrease T cell proliferation and for the generation of CD4+CD25+FoxP3+ Tregs. Importantly, this capacity to promote M2 macrophage polarization was correlated with the intracellular expression of macrophage colony-stimulating factor (M-CSF) and upregulation of IL-10 in CeCa-MSCs. Furthermore, the presence of M2 macrophages was correlated with the increased production of IL-10 and IL-1RA anti-inflammatory molecules. Our in vitro results indicate that CeCa-MSCs, in contrast to NCx-MSCs, display an increased M2-macrophage polarization potential and suggest a role of CeCa-MSCs in antitumor immunity.

In vivo efficacy of a polymer layered decellularized matrix composite as a cell honing cardiovascular tissue substitute.

Cardiovascular surgery involves reconstruction of tissues that are under cyclical mechanical loading, and in constant contact with pulsatile blood flow. Durable biomaterials for such tissue reconstruction are scarce, as they need to be mechanically strong, hemocompatible, and resist structural deterioration from calcification. While homografts are ideal, they are scarce; xenografts are immunogenic and rendered inactive from glutaraldehyde fixation, causing them to calficy and structurally deteriorate over time; decellularized xenografts are devoid of cells, mechanically weak; and synthetic polymeric scaffolds are thrombogenic or too dense to enable host cell infiltration. In this work, we report the in vivo feasibility of a new polymer-decellularized matrix composite material (decellularized bovine pericardium-polycaprolactone: chitosan) fabricated by electrospinning, which is designed to be mechanically strong and achieve programmed host cell honing to integrate into the host. In a rodent and sheep model, this new material was found to be hemocompatible, and enabled host cell infiltration into the polymer and the decellularized matrix core underlying the polymer. Presence of M2 macrophages and several vascular cell types, with matrix remodeling in the vicinity of the cells was observed in the explanted tissues. In summary, the proposed composite material is a novel approach to create in-situ host integrating tissue substitutes, with better non-thrombogenicity, reduced infections and endocarditis, and potentially the ability to grow with the patient and remodeling into a native tissue structure.

Immunological and prognostic analysis of PSENEN in low-grade gliomas: An immune infiltration-related prognostic biomarker.

Metformin is widely used in the treatment of type 2 diabetes (T2D) and plays a role in antitumor and antiobesity processes. A recent study identified its direct molecular target, PEN2 (PSENEN). PSENEN is the minimal subunit of the multiprotein complex γ-secretase, which promotes the differentiation of oligodendrocyte progenitors into astrocytes in the central nervous system. This study was mainly based on gene expression data and clinical data from the TCGA and CGGA databases. Analysis of differential expression of PSENEN between tissues from 31 cancers and paracancerous tissues revealed that it had high expression levels in most cancers except 2 cancers. Using univariate Cox regression analysis and Kaplan-Meier survival analysis, a high expression level of PSENEN was shown to be a risk factor in low-grade gliomas (LGG). Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analyses indicated that PSENEN is widely involved in immune-related signaling pathways in LGG. PSENEN expression level was significantly associated with TMB, MSI, tumor stemness index, and the expression levels of immunomodulatory genes in LGG. Finally, immune infiltration analysis revealed that PSENEN level was associated with the presence of various immune infiltrating cells, among which PSENEN was strongly associated with the presence of M2 macrophages and played a synergistic pro-cancer role. In conclusion, PSENEN may partially influence prognosis by modulating immune infiltration in patients with LGG, and PSENEN may be a candidate prognostic biomarker for determining prognosis associated with immune infiltration in LGG.

Secretome of senescent hepatoma cells modulate immune cell fate by macrophage polarization and neutrophil extracellular traps formation

Presence of dysfunctional senescent hepatocytes is a hallmark feature of liver cirrhosis which finally culminates in liver cancer. We now report the presence of senescent hepatocytes (p21 and p53 positive) in the vicinity of infiltrated immune cells in hepatocellular carcinoma tissue specimens by immunohistochemistry. Hence, we evaluated in vitro, the relevance of senescent hepatoma cells in altering the fate of monocytes and neutrophils by assaying for macrophage polarization and extracellular trap (NETs) formation, respectively. Premature senescence was induced in hepatoma cells (HepG2 and Huh7 cells) by treating cells with doxorubicin. Senescent hepatoma cells showed strong inflammatory phenotype with induced expression of cytokines (IL1β, IL6, IL8 and IL13) as evaluated by flow cytometry. The senescent secretome from hepatoma cells when incubated with healthy monocytes caused it to differentiate predominantly towards M2 fate (CD80low CD86low CD163high CD206high) when analysed by flow cytometry. This was corroborated by the finding in clinical samples where human hepatocellular carcinoma harbouring senescent hepatocytes showed presence of M2 macrophages, while M1 macrophages were predominant in non-tumorous region. Additionally, the senescent secretome from Huh7 cells enhanced the NETs formation, while HepG2 secretome had an inhibitory effect. In conclusion, the "pro-inflammatory" senescent secretome drives non-inflammatory type M2 macrophage polarization and modulated neutrophil traps which in turn can influence the tumor microenvironment.

PD-L1 blockade restores CAR T cell activity through IFN-γ-regulation of CD163+ M2 macrophages

BackgroundThe immune suppressive tumor microenvironment (TME) that inhibits T cell infiltration, survival, and antitumor activity has posed a major challenge for developing effective immunotherapies for solid tumors. Chimeric antigen receptor...

M2 macrophages secrete CXCL13 to promote renal cell carcinoma migration, invasion, and EMT

ObjectiveM2 macrophages are associated with a poor prognosis in a variety of malignancies. There are, however, few relevant investigations in clear cell renal cell carcinoma (ccRCC).MethodsThe expression of M2 macrophages in ccRCC tissues was first discovered using immunohistochemistry in this study. Then, M2 macrophages were created in vitro to see how they affected the proliferation, migration, invasion, and EMT of ccRCC cells. Using qPCR and prognostic analysis identifies important chemokine. Antibody neutralization tests confirmed the chemokine’s involvement and function. Pathway inhibitors confirmed the main pathway of M2 macrophages in ccRCC. Finally, qPCR and IHC were used to confirm the expression of chemokine receptors in ccRCC tissues.ResultsThe presence of M2 macrophages was linked to a poor outcome in ccRCC. M2 macrophages enhanced the proliferation, migration, invasion, and EMT of ccRCC lines in vitro. CXCL13 was identified as the main chemokine by prognostic analysis and qPCR tests. CXCL13 neutralizing antibodies can inhibit the stimulation of M2 macrophages in ccRCC lines’ proliferation, migration, invasion, and EMT. M2 macrophages and CXCL13 may activate the Akt pathway in ccRCC lines, and Akt inhibitors decrease ccRCC lines proliferation, migration, invasion, and EMT. CXCR5 expression is a poor prognostic factor for renal cell carcinoma, according to qPCR and immunohistochemistry. In vivo experiments further proved that CXCL13 secreted by M2 macrophages can promote tumor proliferation.ConclusionsM2 macrophages in the immunological milieu secrete CXCL13, which promotes ccRCC proliferation, migration, invasion, and EMT. Our findings contribute to a better understanding of the function of the tumor microenvironment in the incidence and progression of ccRCC, and they may point to novel therapeutic targets for ccRCC.

Vitreous M2 Macrophage-Derived Microparticles Promote RPE Cell Proliferation and Migration in Traumatic Proliferative Vitreoretinopathy.

PurposeTo characterize vitreous microparticles (MPs) in patients with traumatic proliferative vitreoretinopathy (PVR) and investigate their role in PVR pathogenesis.MethodsVitreous MPs were characterized in patients with traumatic PVR, patients with rhegmatogenous retinal detachment (RRD) complicated with PVR, and control subjects by flow cytometry. The presence of M2 macrophages in epiretinal membranes was measured by immunostaining. Vitreous cytokines were quantified by ELISA assay. For in vitro studies, MPs isolated from THP-1 cell differentiated M1 and M2 macrophages, termed M1-MPs and M2-MPs, were used. The effects and mechanisms of M1-MPs and M2-MPs on RPE cell proliferation, migration, and epithelial to mesenchymal transition were analyzed.ResultsVitreous MPs derived from photoreceptors, microglia, and macrophages were significantly increased in patients with traumatic PVR in comparison with control and patients with RRD (PVR), whereas no significance was identified between the two control groups. M2 macrophages were present in epiretinal membranes, and their signature cytokines were markedly elevated in the vitreous of patients with traumatic PVR. Moreover, MPs from M2 macrophages were increased in the vitreous of patients with traumatic PVR. In vitro analyses showed that M2-MPs promoted the proliferation and migration of RPE cells via activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway. However, M2-MPs did not induce the expression of fibrotic proteins, including fibronectin, α-smooth muscle actin, and N-cadherin in RPE cells.ConclusionsThis study demonstrated increased MP shedding in the vitreous of patients with traumatic PVR; specifically, MPs derived from M2 polarized macrophages may contribute to PVR progression by stimulating RPE cell proliferation and migration.