Pro-tumorigenic M2 Macrophages Research Articles

Abstract 1468: Low exposures to amphibole or serpentine asbestos in germline Bap1-mutant mice induce mesotheliomacharacterized by an immunosuppressive tumor microenvironment

Abstract The purpose of this study was to determine if minimal exposures to amphibole (crocidolite) and serpentine (chrysotile) asbestos fibers significantly increase the incidence and rate of malignant mesothelioma (MM) onset in mice with a germline heterozygous Bap1 mutation, and to assess the effect of the resulting gene × environment interactions on the tumor immune microenvironment (TIME). The studies were carried out by chronically injecting intraperitoneally Bap1+/- and Bap1+/+ (wild-type, WT) mice with minimal doses of asbestos (in some experiments, as low as 0.0125 mg x 8 injections; total, 0.1 mg/mouse) and then monitoring the mice for tumor formation for up to 90 weeks. Pro-inflammatory and pro-tumorigenic factors in the TIME were evaluated by immunohistochemistry (IHC) and immunofluorescence (IF) for immune cell markers. With either type of asbestos and at each dose tested, MMs occurred at a significantly higher rate and earlier onset time in Bap1-mutant mice than in WT littermates. MMs from mice injected with chrysotile, but less so with crocidolite, had widespread adhesions in the peritoneal cavity owing to an overwhelming immune response, particularly at the higher dose used. IHC staining demonstrated an increased number of immune cells in granulomas and MMs. In particular, pro-inflammatory M1 macrophages were found mainly in granulomas, whereas pro-tumorigenic M2 macrophages were predominant in MMs. IF staining revealed that the relative number of M2 macrophages was strikingly greater in chrysotile-induced than crocidolite-induced MMs, suggesting that chrysotile induces a more profound immune response. MMs from Bap1-mutant mice showed upregulation of both CD39/CD73-adenosine and Ccl2/Ccr2 pathways, which collectively are known to mechanistically create favorable conditions for neoplastic transformation, invasion, and the development of an immunosuppressive TIME, in part by attracting M2 macrophages. This implies that chemoprevention and immunotherapy strategies targeting these pro-tumorigenic immune pathways could be beneficial in BAP1 mutation carriers. In conclusion, we provide evidence that low-level exposures to either chrysotile or crocidolite in germline Bap1 heterozygous mice result in a markedly increased risk of MM characterized by an immunosuppressive tumor microenvironment. Drawing parallels to humans, these experimental findings suggest that exposure of BAP1 mutation carriers to even minimal doses of different forms of asbestos may be associated with significantly enhanced susceptibility to MM. Citation Format: Yuwaraj Kadariya, Eleonora Sementino, Maggie Ruan, Mitchell Cheung, Andres J. Klein-Szanto, Kathy Cai, Joseph R. Testa. Low exposures to amphibole or serpentine asbestos in germline Bap1-mutant mice induce mesotheliomacharacterized by an immunosuppressive tumor microenvironment [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 1468.

Abstract 5286: M2 to M1 repolarization of tumor-associated macrophages via the lysosomal effect of itraconazole

Abstract Objective: Itraconazole (ITZ), an antifungal agent, has been reported to have tumor agnostic anticancer effects. Previous reports have shown prolonged survival in patients with lung and ovarian cancer who received ITZ. The anticancer mechanisms vary depending on the cancer type and stromal cells. Based on our previous studies, we focused on tumor-associated macrophages (TAMs). Methods: Anti-tumorigenic M1 and pro-tumorigenic M2 macrophages were established from THP-1 cells and their phenotypes were determined based on morphology, cell surface antigens by Western blotting, and secreted proteins by ELISA. Bulk proteomic analysis of cell proteins was conducted using liquid chromatography-tandem mass spectrometry. The viability of CaSki cervical cancer cells was evaluated both in culture with the supernatant and co-culture with M2 macrophages after treatment with ITZ (10-5 M). Single-cell RNA sequencing (scRNA-seq) with surface labeling of M2 macrophages with and without the ITZ was performed. Using LoupeBrowser (10x Genomics, Inc.), ITZ-treated M2 macrophages were divided into two groups, one with newly emerged clusters after ITZ treatment (group 1) and the other (group 2). Differentially expressed genes between group 1 and group 2 were identified based on a two-fold and a 0.05 adjusted p value and were subjected to Reactome pathway analysis. Significant pathways were identified based on both p value < 0.05 and FDR <0.3. A triple-labeling immunofluorescence study was conducted to detect cholesterol, IL-1β, and organelles (the endoplasmic reticulum, phagosome, and lysosome). Results: ITZ changed M2 macrophages to M1-like morphology and protein expression. While co-culture with M2 macrophages promoted cancer cell proliferation, both culture with the supernatant and co-culture with ITZ-treated M2 macrophages significantly inhibited CaSki cell growth. scRNA-seq identified newly emerged clusters (group 1) among M2 macrophages following ITZ treatment. Group 1 had elevated mRNA expression of M1 markers, including IL1β, IL6, CD86, TL4, GAPDH, and RKM, compared to group 2. Group 1 expressed the CD86 and TLR4 surface antigen proteins. Pathway analysis identified 176 significant pathways including lysosome vesicle biogenesis. Immunofluorescence analysis showed that M1-like macrophages in ITZ-treated M2 macrophages had enlarged lysosomes containing cholesterol. Conclusion: ITZ repolarized THP-1 derived M2 macrophage to an M1-like phenotype. The mechanism of repolarization involves intracellular cholesterol accumulation via lysosomal dysfunction. Citation Format: Yumi Takimoto, Hiroshi Tsubamoto, Kazuko Sakata, Roze Isono-Taniguchi, Tomoko Ueda, Hiroaki Shibahara. M2 to M1 repolarization of tumor-associated macrophages via the lysosomal effect of itraconazole [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 5286.

The pregnancy-associated protein glycodelin as a potential sex-specific target for resistance to immunotherapy in non-small cell lung cancer

Lung cancer has been shown to be targetable by novel immunotherapies which reactivate the immune system and enable tumor cell killing. However, treatment failure and resistance to these therapies is common. Consideration of sex as a factor influencing therapy resistance is still rare. We hypothesize that the success of the treatment is impaired by the presence of the immunosuppressive pregnancy-associated glycoprotein glycodelin that is expressed in patients with non-small-cell lung cancer (NSCLC). We demonstrate that the glycan pattern of NSCLC-derived glycodelin detected by a lectin-based enrichment assay highly resembles amniotic fluid-derived glycodelin A, which is known to have immunosuppressive properties. NSCLC-derived glycodelin interacts with immune cells in vitro and regulates the expression of genes associated with inflammatory and tumor microenvironment pathways. In tumor microarray samples of patients, high glycodelin staining in tumor areas results in an impaired overall survival of female patients. Moreover, glycodelin colocalizes to tumor infiltrating CD8+ T cells and pro-tumorigenic M2 macrophages. High serum concentrations of glycodelin prior to immunotherapy are associated with a poor progression-free survival (p < 0.001) of female patients receiving PD-(L)1 inhibitors. In summary, our findings suggest that glycodelin not only is a promising immunological biomarker for early identification of female patients that do not benefit from the costly immunotherapy, but also represents a promising immunotherapeutic target in NSCLC to improve therapeutic options in lung cancer.

Alterations in tumor immune microenvironment and progressive immune exhaustion with advancing penile squamous cell carcinoma using multiplex immunofluorescence and image analysis.

9 Background: Penile squamous cell carcinoma (PSCC) is a rare and aggressive malignancy with limited treatment options in the advanced or recurrent settings. Immunotherapy can yield clinical responses in PSCC, but less than 20% of patients benefit. Herein, using multiplex immunofluorescence (MIF), we analyzed the composition of the tumor immune microenvironment as it varies with human papillomavirus (HPV) status and across advancing disease stages with an aim to further our understanding of how immune composition may impact clinical outcome, potential role in disease progression and response to immune therapies. Methods: Formalin-fixed and paraffin-embedded tissue samples from men with PSCC treated at Moffitt cancer center (Tampa, FL) were immunostained for CD3, CD4, CD8, CD68, PD-1, PD-L1, CD163 and CD 206 using OPAL TM 7 kit (AKOYA Biosciences) for MIF. Densities of various cell phenotypes (expressed as cells/mm2) were quantified using an automated quantitative image analysis system (InForm). Immune cell phenotype densities were stratified by HPV status and by tumor stage. We will apply the nearest neighbor cell analysis to identify the spatial orientation of immune cells in the tumor microenvironment. Results: 57 PSCC patients (median age, 60 years [IQR (interquartile range) 31-92]; 60% HPV negative) had MIF analysis. More than half (31/57) had lymph nodes involved (6 N1, 25 N2-3). The immune composition did not differ significantly with HPV status. When investigated by individual clinical stage, we observed an increase in median density of total (CD3+), helper (CD3+CD4+), cytotoxic (CD3+CD8+) and CD3+PD-L1+ T cells from N0 to N1 stage (pN0: CD3+ 44.23, CD3+CD4+ 14.77, CD3+CD8+ 16.15 cells/mm2; pN1: CD3+ 188.36, CD3+CD4+ 71.69, CD3+CD8+ 40.22 cells/mm2) followed by a decrease in N2-N3 stage (CD3+CD4+ 18.45 and CD3+CD8+ 28.22 cells/mm2).The median density of total macrophages increased with stage (pN0: 306.38; pN1: 502.47; pN2-3: 648.27 cells/mm2). While the activated M1 macrophages increased from N0 to N1 and decreased in the more advanced N2-3 stage, M2 macrophages steadily increased across stages and became the dominant type in N2-3. Conclusions: This study describes the interplay of T cells and macrophages across disease stages using MIF. We observed an initial T cell and myeloid immune response in the early locoregional stage, followed by the emergence of immune exhaustion, marked by a decline in the density of cytotoxic T cells, rising PD-L1 expression, and the progressive replacement of anti-tumor M1 macrophages with pro-tumorigenic M2 macrophages across stages. Our findings can inform treatments utilizing immune manipulation. Geospatial analysis exploring the proximity relationships of different immune cell types to each other and to the tumor is ongoing and will be presented at the meeting.

Defining the spatial distribution of extracellular adenosine revealed a myeloid-dependent immunosuppressive microenvironment in pancreatic ductal adenocarcinoma

BackgroundThe prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) remains extremely poor. It has been suggested that the adenosine pathway contributes to the ability of PDAC to evade the immune...

TCGA RNA-Seq and Tumor-Infiltrating Lymphocyte Imaging Data Reveal Cold Tumor Signatures of Invasive Ductal Carcinomas and Estrogen Receptor-Positive Human Breast Tumors.

Comparative studies of immune-active hot and immune-deserted cold tumors are critical for identifying therapeutic targets and strategies to improve immunotherapy outcomes in cancer patients. Tumors with high tumor-infiltrating lymphocytes (TILs) are likely to respond to immunotherapy. We used the human breast cancer RNA-seq data from the cancer genome atlas (TCGA) and classified them into hot and cold tumors based on their lymphocyte infiltration scores. We compared the immune profiles of hot and cold tumors, their corresponding normal tissue adjacent to the tumor (NAT), and normal breast tissues from healthy individuals from the Genotype-Tissue Expression (GTEx) database. Cold tumors showed a significantly lower effector T cells, lower levels of antigen presentation, higher pro-tumorigenic M2 macrophages, and higher expression of extracellular matrix (ECM) stiffness-associated genes. Hot/cold dichotomy was further tested using TIL maps and H&E whole-slide pathology images from the cancer imaging archive (TCIA). Analysis of both datasets revealed that infiltrating ductal carcinoma and estrogen receptor ER-positive tumors were significantly associated with cold features. However, only TIL map analysis indicated lobular carcinomas as cold tumors and triple-negative breast cancers (TNBC) as hot tumors. Thus, RNA-seq data may be clinically relevant to tumor immune signatures when the results are supported by pathological evidence.

Abstract 2884: Cancer-associated fibroblasts potentiate subcutaneous tumor growth in a novel bladder cancer mouse model with a genotype dependency

Abstract Introduction Interrogation of the relationship of genotype (mutation in relevant gene) and phenotype (sensitivity to therapy) in targeted therapy would be facilitated by the use of isogenic in vitro and in vivo models. The PI3K/AKT/mTOR pathway is widely activated but heretofore untargeted in bladder cancer. We sought to develop an immunocompetent CRISPR-edited bladder cancer mouse model that recapitulates common PI3K/AKT/mTOR mutations to study a genotype-phenotype relationship. Our in vitro CRISPR-edited organoids are immunogenic in vivo, as such, we explored utilizing Cancer-Associated Fibroblasts (CAFs) to mediate immunosuppression and promote a pro-tumorigenic environment. Methods and Results We isolated bladder urothelial cells from Rosa26 LSL-Cas9/GFP transgenic mice and grew them as organoids. Trp53 and Rb1 were co-edited to establish ‘base organoids’. As proof of concept, Pten was edited in base organoids in anticipation of developing a series of organoids with different mTOR pathway genotypes. We found that Pten-KO base organoids were 9x more sensitive to ipatasertib than Pten WT organoids. The edited urothelial cells were engrafted into mouse bladder to further test kinase inhibitor susceptibility as preclinical evidence to support human clinical trials. However, the cells successfully engrafted but disappeared after 3-4 weeks before tumors formed. Subcutaneous allografts were also rejected after 3-4 weeks and showed CD45+ immune infiltration during rejection. When cells were injected subcutaneously with CAFs, tumors persisted for 12 weeks, but only in organoids with both Trp53 loss and loss of murine chromosome 4qC4 (which is homologous to 9p21 loss in humans). 9p21 contains 3 genes which are co-deleted in ~40% of bladder cancers: MTAP, CDKN2A, and CDKN2B. Histopathological analysis of these masses confirmed their malignant nature and revealed an immune-excluded architecture. IHC staining of 9p21/Trp53 KO tumors for CD206 and CD11c revealed greater recruitment of M2 macrophages than M1 macrophages. Conclusions CAFs remodel the tumor-microenvironment in bladder cancer via the recruitment of pro-tumorigenic M2 macrophages. This may occur in a genotype-specific manner and 9p21 loss may be a requirement for some genotypes to be tumorigenic. Citation Format: Henkel Lee Valentine, Uttam Satyal, Alexander Metz, Laura Bukavina, Philip H. Abbosh. Cancer-associated fibroblasts potentiate subcutaneous tumor growth in a novel bladder cancer mouse model with a genotype dependency [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 2884.

Abstract PD1-06: Black patients with triple negative breast cancer (TNBC) have enriched stromal tumor infiltrating lymphocytes (sTILs) and receive preferential benefit from neoadjuvant immunotherapy

Abstract Introduction TNBC is overrepresented in Black women, and Black patients with TNBC have worse clinical outcomes compared to non-Black patients. This disparity likely results from racial differences in clinical, biological, and demographic features of TNBC and social determinants of health. Neoadjuvant chemoimmunotherapy is current standard of care for high-risk TNBC. However, Black patients have been poorly represented in immunotherapy TNBC trials, making it difficult to assess comparative efficacy of immunotherapy in Black patients. Methods We utilized two TNBC neoadjuvant trials to assess racial differences in the tumor immune microenvironment composition and evaluate impact of race on response to chemotherapy vs chemoimmunotherapy. NeoSTOP trial (NCT02413320) randomized 100 stage I-III TNBC patients to receive neoadjuvant carboplatin/paclitaxel + doxorubicin/cyclophosphamide (CbP+AC) or carboplatin/docetaxel (CbD). NeoPACT trial (NCT03639948) enrolled 120 patients with stage I-III TNBC who received neoadjuvant CbD + pembrolizumab (CbD+P). sTILs were centrally quantified, and RNA extracted from pretreatment tissue was subjected to next-generation sequencing. Relative leukocyte fractions were computed by CIBERSORTx. Factors were tested as predictors of pathologic complete response (pCR) using logistic regression analysis. Event-free survival (EFS) was estimated by the Kaplan-Meier method and compared between groups by log-rank test, followed by Cox regression analysis. Results The study population includes 197 patients with known race, sTILs, and gene expression data (84 patients from NeoSTOP, 113 from NeoPACT). 15/84 (18%) patients in NeoSTOP and 20/113 (18%) patients in NeoPACT self-reported Black race. There was no significant difference in age, T or N stage, or germline BRCA1/2 mutation status by race in either study. Black patients had significantly higher sTILs than non-Black patients (median 40% vs 15%, P=0.048) and were more likely to have ≥20% sTILs than non-Black patients (66% vs 44%, P=0.026). There was no significant difference in pCR by race in NeoSTOP (OR=0.60, 95% CI 0.19-1.84, P=0.37; pCR 47% for Black vs 59% for non-Black). In contrast, in NeoPACT, Black patients had a significantly higher pCR compared to non-Black patients (OR=3.27, 95% CI 1.01-10.64, P=0.049; pCR 79% for Black vs 53% for non-Black). In NeoSTOP, EFS was similar for Black and non-Black patients (3-year EFS 92% and 94%, respectively, HR=0.88, 95% CI 0.11-7.28, P=0.90). In NeoPACT, EFS was numerically higher in Black vs non-Black patients (3-year EFS 93% and 81%, respectively, HR=0.43, 95% CI 0.05-3.36, P=0.40); NeoPACT survival follow-up is ongoing at the time of this report. On CIBERSORTx analysis, Black patients had relative depletion of immunosuppressive pro-tumorigenic M2 macrophages (P=0.005) and CD4+ memory resting T cells (P=0.021) compared to non-Black patients. Conclusions Compared to non-Black patients, Black patients with TNBC are more likely to have immune-enriched tumors with lower relative abundance of immunosuppressive leukocytes. These findings suggest potential for higher relative magnitude of benefit from checkpoint inhibitor therapy in Black compared to non-Black patients. Supporting this biological hypothesis, we noted that Black and non-Black patients had equivalent rates of pCR with neoadjuvant chemotherapy; however, pCR rate among Black patients was significantly higher than in non-Black patients when treated with neoadjuvant chemoimmunotherapy. These findings should be confirmed in other studies and can optimize utilization of neoadjuvant chemoimmunotherapy. Our findings also underscore the importance of efforts to address disparity in access and use of immunotherapy in Black patients. Citation Format: Shane R. Stecklein, Rachel Yoder, Roberto Salgado, Joshua M. Staley, Anne O’Dea, Lauren Nye, Manana Elia, Deepti Satelli, Gregory Crane, Richard McKittrick, Andrew K. Godwin, Qamar Khan, Priyanka Sharma. Black patients with triple negative breast cancer (TNBC) have enriched stromal tumor infiltrating lymphocytes (sTILs) and receive preferential benefit from neoadjuvant immunotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD1-06.

Abstract 6103: Reshaping the myeloid-dependent pro-tumorigenic microenvironment in PDAC by targeting the extracellular adenosine pathway: A therapeutic opportunity

Abstract The prognosis for patients with pancreatic adenocarcinoma (PDAC) remains extremely poor. PDAC is resistant to both conventional therapies and emerging immunotherapies (IOT), apart from tumors with mismatch repair deficiency. This may be explained in part by its low tumor mutational burden (TMB) but also by its immunosuppressive tumor microenvironment (TME). It has been suggested that CD73, a member of the adenosine pathway, expressed on cancer cells contributes to immune escape and resistance to cytotoxic/radiotherapy treatment. The adenosine pathway converts the immune activator ATP, released by dying cells during cell turnover or after treatment, to extracellular Adenosine (eAdo), which is immunosuppressive. Using syngeneic, in vivo models by s.c. implantation of KPC-derived cell lines (courtesy of Ben Stanger, UPenn) with differential immune infiltration and response to IOT [resistant (IOTResi) or responsive (IOTResp)], we showed by flow cytometry that the adenosine pathway is enriched in the tumor-infiltrating immune cells (in particular myeloid populations) which co-express CD39 and CD73, enabling the formation of eAdo. Mass Spec Imaging (MSI) revealed that adenosine distribution is heterogeneous in the tumors with high concentrations in the hypoxic margins that surround necrotic areas. Subpopulations of myeloid cells infiltrating the lesions are a target for eAdo, expressing high levels of adenosine receptor Adora2a. We discovered that pro-tumorigenic M2 macrophages have the highest expression of the receptor and significantly higher in the IOTResi model. Blocking the in vivo formation and function of eAdo in IOTResi tumors, using a combination of anti-CD73 antibody (2C5, murine IgG1-Fc) and an inhibitor of Adora2a (AZD4635) reduced the presence of eAdo, slowed tumor growth and reduced the lung metastatic burden. The combination remodeled the TME, reducing the infiltration by M2 macrophages, particularly those that are PD-L1 positive and diminished the frequency of infiltrating Tregs. Bulk RNAseq analysis demonstrated a profound dependency of the TME on the presence of eAdo. Genes related to cytokine/chemokine signaling, immunosuppression/inflammation, hypoxia, metastasis and collagen production are strongly downregulated following administration of anti-CD73Ab/Adora2ai. In addition, blocking the adenosine pathway improved the efficacy of combinations of cytotoxics (gemcitabine/ATR inhibitor) and immunotherapy (aCD40/anti-PDL1Ab). The formation of eAdo appears to be a factor in the development of the immunosuppressive TME in PDAC, contributing to its resistance to conventional and novel therapies. Therefore, inhibition of the adenosine pathway using a CD73Ab and an Adora2ai may represent a strategy to modulate the PDAC stroma and improve therapy response in patients with PDAC. Citation Format: Vincenzo Graziano, Andreas Dannhorn, Kate Williamson, Heather Hulme, Hannah Buckley, Sheng Y. Lee, Sabita Islam, James E. Thaventhiran, Richard Goodwin, Rebecca Brais, Simon J. Dovedi, Alwin Schuller, Jim Eyles, Duncan I. Jodrell. Reshaping the myeloid-dependent pro-tumorigenic microenvironment in PDAC by targeting the extracellular adenosine pathway: A therapeutic opportunity [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 6103.

Combination therapy using microwave ablation and d-mannose-chelated iron oxide nanoparticles inhibits hepatocellular carcinoma progression

Despite being a common therapy for hepatocellular carcinoma (HCC), insufficient thermal ablation can leave behind tumor residues that can cause recurrence. This is believed to augment M2 inflammatory macrophages that usually play a pro-tumorigenic role. To address this problem, we designed d-mannose-chelated iron oxide nanoparticles (man-IONPs) to polarize M2-like macrophages into the antitumor M1 phenotype. In vitro and in vivo experiments demonstrated that man-IONPs specifically targeted M2-like macrophages and accumulated in peri-ablation zones after macrophage infiltration was augmented under insufficient microwave ablation (MWA). The nanoparticles simultaneously induced polarization of pro-tumorigenic M2 macrophages into antitumor M1 phenotypes, enabling the transformation of the immunosuppressive microenvironment into an immunoactivating one. Post-MWA macrophage polarization exerted robust inhibitory effects on HCC progression in a well-established orthotopic liver cancer mouse model. Thus, combining thermal ablation with man-IONPs can salvage residual tumors after insufficient MWA. These results have strong potential for clinical translation.

MiR-18a Mediates Immune Evasion in ER-Positive Breast Cancer through Wnt Signaling.

ER-positive (ER+) breast cancer is considered immunologically ‘silent’ with fewer tumor-infiltrating immune cells. We have previously demonstrated the role of miR-18a in mediating invasion and poor prognosis in ER+ breast cancer by activation of the Wnt signaling pathway. Here, we explored the immune-modulatory functions of high levels of miR-18a in these tumors. A microarray-based gene expression analysis performed in miR-18a over-expressed ER+ breast cancer cell lines demonstrated dysregulation and suppression of immune-related pathways. Stratification of the ER+ tumor samples by miR-18a levels in the TCGA and METABRIC cohort and immune cell identification performed using CIBERSORT and Immune CellAI algorithms revealed a higher proportion of T-regulatory cells (p < 0.001) and a higher CD4/CD8 ratio (p < 0.01). miR-18a over-expressed MCF7 co-cultured with THP-1 showed decreased antigen presentation abilities and increased invasiveness and survival. They also promoted the differentiation of pro-tumorigenic M2 macrophages. Inhibition of the Wnt pathway in miR-18a over-expressed cells brought about the restoration of TAP-1, a protein critical for antigen presentation. Examination of tumor specimens from our case series showed that miR-18a high ER+ tumors had a dense lymphocyte infiltrate when compared to miR-18a low tumors but expressed a higher CD4/CD8 ratio and the M2 macrophage marker CD206, along with the invasive marker MMP9. We report for the first time an association between miR-18a-mediated Wnt signaling and stromal immune modulation in ER+ tumors. Our results highlight the possibility of formulating specific Wnt pathway inhibitors that may be used in combination with immune checkpoint blockers (ICB) for sensitizing ‘immune-cold’ ER+ tumors to immunotherapy.

The miRNA-21-5p Payload in Exosomes from M2 Macrophages Drives Tumor Cell Aggression via PTEN/Akt Signaling in Renal Cell Carcinoma.

M2 macrophages in the tumor microenvironment are important drivers of cancer metastasis. Exosomes play a critical role in the crosstalk between different cells by delivering microRNAs or other cargos. Whether exosomes derived from pro-tumorigenic M2 macrophages (M2-Exos) could modulate the metastatic behavior of renal cell carcinoma (RCC) is unclear. This study found that M2-Exos promotes migration and invasion in RCC cells. Inhibiting miR-21-5p in M2-Exos significantly reversed their pro-metastatic effects on RCC cells in vitro and in the avian embryo chorioallantoic membrane in vivo tumor model. We further found that the pro-metastatic mechanism of miR-21-5p in M2-Exos is by targeting PTEN-3′UTR to regulate PTEN/Akt signaling. Taken together, our results demonstrate that M2-Exos carries miR-21-5p promote metastatic features of RCC cells through PTEN/Akt signaling. Reversing this could serve as a novel approach to control RCC metastasis.

Abstract LB050: Modulation of innate and adaptive immunity in blood and tumor of patients receiving the SHP2 inhibitor RMC-4630

Abstract RMC-4630 is a potent, selective, orally bioavailable allosteric inhibitor of SHP2, a central node in the RAS signaling pathway. In preclinical models, SHP2 inhibition not only directly inhibited tumor growth through suppression of tumor-intrinsic RAS signaling, but also resulted in transformation of the tumor immune microenvironment, characterized by an increase in CD8+T cell infiltrates and selective depletion of pro-tumorigenic M2 macrophages. In this study, we evaluated pharmacodynamic biomarkers in blood and tumors from patients in the RMC-4630 phase I monotherapy clinical trial (NCT 03634982) by using flow cytometry and immunohistochemistry (IHC). Safety, PK and efficacy data are reported in a separate abstract. Longitudinal analysis of immune cell phenotyping in blood was conducted in 35 patients. There was a trend for lower pre-study monocytic myeloid-derived suppressor cell (mMDSC) to be associated with a better clinical outcome on RMC-4630 therapy. While the proportion of circulating T cell and B cell populations did not change, both blood mMDSC and total monocytes were significantly reduced during RMC-4630 administration. Furthermore, tumor volumes changes, and the proportion of patients with SD versus PD, positively correlated with the ratio of mMDSCs to total monocytes on RMC-4630 treatment. Inhibition of pERK was observed in a subset of patients. Three paired tumor biopsies from efficacy-evaluable patients, including 1 PR, 1 SD and 1 PD, were available for tumor microenvironment analysis by multiplexed-IHC assays. Increase in tumor infiltrating T cells in the tumors of one patient with a PR and another with SD was observed on RMC-4630 therapy. Inhibition of tumor PD-L1 expression and a decrease in M2 macrophages was also observed on treatment in the tumor biopsy of the PR patient. Collectively, the preliminary clinical biomarker data supports the preclinical observations that SHP2 inhibition with RMC-4630 modulates both innate and adaptive anti-tumor immunity. Citation Format: Ariel Yung-Chia Chen, Eric Haura, Jose Pacheco, Marianna Koczywas, Michael Gordon, Susanna Ulahannan, Howard A. Burris, Sai-Hong Ignatius Ou, Judy S. Wang, Jonathan W. Riess, Caroline McCoach, Anna Capasso, Elsa Quintana, Josie Hayes, Richa Dua, Bojena Bitman, Martha Guerra, Hongfang Wang, Xiaolin Wang, Pasi A. Janne. Modulation of innate and adaptive immunity in blood and tumor of patients receiving the SHP2 inhibitor RMC-4630 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB050.

A Novel Blockade CD47 Antibody With Therapeutic Potential for Cancer.

Macrophages as components of the innate immune system play a critical role in antitumor responses. Strategies for targeting CD47 are becoming a hot spot for cancer therapy. The expression of CD47 is exercised by macrophages to make a distinction between “self” or “nonself.” Anti-CD47 antibodies block the interaction between macrophage signal regulatory protein-α (SIRPα) and tumor surface CD47. In this study, we report and assess a novel anti-CD47 blocking antibody named 2C8, which exhibits high affinity and tremendous anticancer effects. More concretely, 2C8 significantly induces macrophages, including protumorigenic subtype M2 macrophages killing tumor cells in vitro, and is revealed to be more effective than commercially available anti-CD47 mAb B6H12.2. In vivo, 2C8 controls tumor growth and extends survival of xenograft mice. The antitumor ability of 2C8 might be applicable to many other cancers. The generation of a novel CD47 antibody contributes to consolidating clinical interest in targeting macrophages for the treatment of malignancy and, moreover, as a supplement therapy when patients are resistant or refractory to other checkpoint therapies or relapse after such treatments.

Macrophage polarization contributes to the efficacy of an oncolytic HSV-1 targeting human uveal melanoma in a murine xenograft model

Uveal melanoma (UM), the most common primary malignant tumor of the eye in adults, is difficult-to-treat. UM has a relatively high mortality secondary to distant metastasis and poor overall survival with existing therapies. The oncolytic virus herpes simplex virus type-1 (HSV-1) has been approved for clinical use in melanoma. This double-stranded DNA virus was suspected to directly activate lysis specifically in neoplastic cells. We tested the antitumor efficacy of recombinant oncolytic HSV-1-EGFP (oHSV-EGFP) in UM and characterized the local and systemic antitumor innate immune response in a murine xenograft model. We first determined the efficacy of the oncolytic virus in 92.1, MUM2B and MP41 cell lines. In murine xenograft models, oHSV-EGFP reduced intraocular tumors as well as systemic subcutaneous tumors. A significant change in cytokines was observed in viral infected cells, especially a rise in IFNγ. In vivo analyses showed increased anti-tumorigenic M1 macrophages and decreased pro-tumorigenic M2 macrophages in peripheral blood, and intraocular and distant tumors after intravitreal viral treatment. Increased infiltration of natural killer cells and mature dendritic cells was also detected after viral treatment. In addition, no virus was detected in major organs after the treatment. Our data support that oHSV-EGFP is effective, neoplasm specific, immune active and safe, providing possible clinical translatable options to treat ocular and metastatic UM.

Abstract 1076: Hexavalent HERA-CD40L induces a productive T cell-mediated anti-tumor immune response and shows superior activity in comparison to benchmark CD40 agonistic antibodies

Abstract TNF Receptor Superfamily members (TNF-R-SF), including CD40, are key regulators of the immune system and have been immunotherapeutic targets for over 20 years. CD40 signaling serves as an important co-stimulatory signal for antigen-presenting cells (APC). For the induction of a CD40 driven anti-tumor immune response multiple approaches, - most of them antibody based - are currently under investigation. However, the structural necessity of trimerization within the TNF-R-SF, defines bivalent antibodies generally as unfeasible inducers of signaling events within this protein family. To overcome the known inadequacies of antibodies, we developed HERA-CD40L, composed of two trivalent CD40L-receptor-binding domains, fused to a silenced human IgG1 Fc-domain. This hexavalent CD40 agonist mimics the natural ligand and enables efficient receptor clustering and superior signaling. HERA-CD40L treatment increased the pro-inflammatory state of all CD40-expressing cells examined. It promoted the licensing of dendritic cells (DC), macrophages, B cells and other APC. Comparison to benchmark antibodies revealed that HERA-CD40L elicited a stronger and more rapid activation of NFκB signaling in primary B cells. HERA-CD40L treatment, but not clinical benchmark antibodies triggered immediate NFκB, MAPK, PI3K and STAT-1 signaling in primary monocyte-derived immature DC. As a result, HERA-CD40L induced upregulation of activation markers and co-stimulatory molecules in B cells and DC. Using SEC fractionation followed by a CD40 reporter assay, we could furthermore demonstrate that the activity of a clinical benchmark antibody derived exclusively from antibody aggregates. In vitro HERA-CD40L treatment converted immature phagocytic macrophages into mature/professional APC and induced repolarization of M2- to M1-like macrophages. These findings were confirmed in vivo using a mouse surrogate (mmHERA-CD40L). Upon treatment of MC38-CEA and CT26wt syngeneic mouse models, we observed single agent anti-tumor efficacy. In the CT26wt model, mmHERA-CD40L treatment converted cold into hot tumors by increasing T cell infiltration. Furthermore, mmHERA-CD40L induced a strong dose dependent decrease of tumor associated pro-tumorigenic M2-macrophages indicating a profound reorganization of the tumor microenvironment. Both in vitro (human) and in vivo (mouse), HERA-CD40L increased antigen-specific immune system activation without affecting the non-specific immune cells. These data, together with pilot PD/safety results, demonstrate that the activity of HERA-CD40L is both potent and safe. In conclusion, HERA-CD40L is a potent agonist able to show single agent anti-tumor activity. The biological activity is distinct from and superior to clinical benchmark “agonistic” antibodies. HERA-CD40L has a well-defined mechanism of action, does not depend on Fc gamma receptor-mediated crosslinking and hence functions as a true agonist. Citation Format: Christian Gieffers, David M. Richards, Jaromir Sykora, Christian Merz, Julian P. Sefrin, Katharina Billian-Frey, Karl Heinonen, Mauricio Redondo Müller, Matthias Schröder, Meinolf Thiemann, Oliver Hill. Hexavalent HERA-CD40L induces a productive T cell-mediated anti-tumor immune response and shows superior activity in comparison to benchmark CD40 agonistic antibodies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1076.

Single-Cell RNA-seq Reveals Obesity-Induced Alterations in the Brca1-Mutated Mammary Gland Microenvironment.

Clinical and experimental studies have shown that obesity increases the development and progression of breast cancer. The impact of obesity on the tumor microenvironment plays an important role in tumorigenesis, yet the precise mechanisms underlying obesity-mediated effects on cell-to-cell communication within the tumor microenvironment have been difficult to define. In this study, we conducted single-cell RNA sequencing (scRNA-seq) studies to investigate the impact of high-fat diet (HFD)-induced obesity on transcriptomic landscapes of stromal and immune cells in mammary glands of Brca1−/−; p53+/− mice, an animal breast cancer model. Hierarchical clustering and gene pathway enrichment analyses of scRNA-seq data showed that five different subtypes of stromal fibroblasts existed in mouse Brca1-mutated mammary glands. HFD-induced obesity led to upregulated expression of extracellular matrix (ECM) genes (Col3a1, Col6a3, Eln, and Sparc) and downregulated expression of immunoregulatory genes (Iigp1 and Cxcl10) in these stromal subtype cells. These findings, taken together, suggest that obesity alters the ECM composition and immune ecosystem through modulating the functionality of mammary stromal fibroblasts. Moreover, scRNA-seq analysis of mammary immune cells indicated that HFD-induced obesity promoted the generation and/or recruiting of pro-tumorigenic M2 macrophages in mammary glands. Our studies provide new insight into a mechanistic paradigm wherein obesity modulates the functions of stromal and immune cells to create the tumorigenic microenvironment for promoting breast tumorigenesis.

Allosteric Inhibition of SHP2 Stimulates Antitumor Immunity by Transforming the Immunosuppressive Environment.

The protein tyrosine phosphatase SHP2 binds to phosphorylated signaling motifs on regulatory immunoreceptors including PD-1, but its functional role in tumor immunity is unclear. Using preclinical models, we show that RMC-4550, an allosteric inhibitor of SHP2, induces antitumor immunity, with effects equivalent to or greater than those resulting from checkpoint blockade. In the tumor microenvironment, inhibition of SHP2 modulated T-cell infiltrates similar to checkpoint blockade. In addition, RMC-4550 drove direct, selective depletion of protumorigenic M2 macrophages via attenuation of CSF1 receptor signaling and increased M1 macrophages via a mechanism independent of CD8+ T cells or IFNγ. These dramatic shifts in polarized macrophage populations in favor of antitumor immunity were not seen with checkpoint blockade. Consistent with a pleiotropic mechanism of action, RMC-4550 in combination with either checkpoint or CSF1R blockade caused additive antitumor activity with complete tumor regressions in some mice; tumors intrinsically sensitive to SHP2 inhibition or checkpoint blockade were particularly susceptible. Our preclinical findings demonstrate that SHP2 thus plays a multifaceted role in inducing immune suppression in the tumor microenvironment, through both targeted inhibition of RAS pathway-dependent tumor growth and liberation of antitumor immune responses. Furthermore, these data suggest that inhibition of SHP2 is a promising investigational therapeutic approach. SIGNIFICANCE: Inhibition of SHP2 causes direct and selective depletion of protumorigenic M2 macrophages and promotes antitumor immunity, highlighting an investigational therapeutic approach for some RAS pathway-driven cancers.

Generation of Novel Diagnostic and Therapeutic Exosomes to Detect and Deplete Protumorigenic M2 Macrophages.

Given their protumorigenic function and prevalence in most malignant tumors with lower survival; early detection, and intervention of CD206-positive M2 macrophages may boost the clinical outcome. To determine in vivo distribution of M2 macrophages, 111In-oxine-based radiolabeling of the targeted exosomes is adopted. When these radiolabeled targeted exosomes are injected into breast tumor-bearing mice, exosomes accumulate at the periphery of the primary tumor, metastatic foci in the lungs, spleen, and liver. Ex vivo quantification of radioactivity also shows similar distribution. Injecting DiI dye-labeled exosomes into the same mice shows adherence of exosomes to the CD206-positive M2 macrophages on ex vivo fluorescent microscopy imaging. In addition, these engineered exosomes are utilized to carry the Fc portion of lgG2b with the intention of augmenting antibody-dependent cell-mediated cytotoxicity. It is demonstrated that M2 macrophage targeting therapeutic exosomes deplete M2 macrophages both in vitro and in vivo, and reduce tumor burden, increasing survival in a metastatic breast cancer model.

Abstract P6-06-12: Upregulation of beige adipocytes in African-American triple negative breast tumors induce tumorigenesis by promoting anti-inflammatory microenvironment

Abstract Background: The complexity of breast cancer (BC) development and progression is heavily influenced by the cross-talk of critical cell types within the tumor microenvironment. While there have been many advancements in BC treatment, triple negative breast cancer (TNBC) still exhibits the lowest rate of response and the nature of its aggressive properties remain poorly understood. African American (AA) women are disproportionally affected and have worse overall survival. Our lab previously reported important role of beige adipocytes in breast tumor growth. Interestingly, we found beige adipocytes were highly up regulated in AA TN breast tumors when compared to TNBC from Caucasian (CA) women. In this study, we have further investigated the mechanisms that increase beige adipocytes in AA TN breast tumors and their influence on breast tumor growth. Methods: Human tumor samples provided by Cooperative Human Tissue Network and National Disease Research Institute were subjected to immunohistochemistry (IHC) and western blot analysis to examine key beige adipocyte markers. To further investigate the mechanisms by which beige adipocytes may contribute tumor growth, xenografts were established from AA TNBC cell lines HCC70, HCC1806 and MDA-MB 468. Tumors excised at various time points were examined for beige adipocyte markers and their key regulators using validated species specific primers to distinguish between host and tumor cell contributions. Boyden chamber assays and ex-vivo experiments were performed to examine whether beige adipocytes were recruited from the host or induced in xenografts, and test whether paracrine secretion of cytokines or contact between various cell types were necessary for adipose browning in these tumors. Possible involvement of lactic acid-mediated adipose browning in these tumors was tested by inhibiting lactate dehydrogenase using sodium oxamate. We further examined whether increased browning in these tumors was associated with increase in anti-inflammatory cytokines that promote M2 macrophage phenotype and angiogenesis, both of which are highly implicated in tumor progression. Results: Both quantitative IHC and western blot analysis showed higher expression of beige adipocyte markers CD137 and TMEM26 in AA TNBC compared to CA TNBC. Expression of beige marker CD137 was detected in all xenografts from AA TNBC by IHC. Quantitative gene expression analysis (qPCR) revealed that host contributed to the origin of beige adipocytes in these tumors. We demonstrate using Boyden Chamber assay that contact between tumor cells and host adipocytes was important for adipose browning. Ex-vivo experiments also showed that breast cancer cells in contact with either host adipocytes or 3T3-L1 mouse adipocyte cell line increased the expression of beige markers that could be attenuated by lactate dehydrogenase inhibitor. We further show that increase in beige adipocytes in AA TNBC was associated with high anti-inflammatory cytokines IL13/IL4 expression, as well as anti-inflammatory pro-tumorigenic M2 macrophages and high angiogenesis. Conclusion: Key beige markers CD137 and TMEM26 are significantly upregulated in AA TNBC compared to the CA TNBC. Host microenvironment contributed to beige adipocyte characteristics in response to lactic acid-mediated adipose browning of tumor cells. Furthermore, beige adipocytes increased anti-inflammatory cytokines that facilitate accumulation of M2 macrophages and promote angiogenesis. Our results implicate that beige adipocytes are key mediators of aggressive tumor development in AA TNBC and could potentially be targeted for therapeutic purposes. Citation Format: Brandis A. Moore, Briana Osorio, Rajan Singh, Shehla Pervin. Upregulation of beige adipocytes in African-American triple negative breast tumors induce tumorigenesis by promoting anti-inflammatory microenvironment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-06-12.