Targeting CD3L1-NRP2 disarms myeloid-driven tumor immune evasion.

Tumor-associated macrophages (TAMs) are present in and are important components of the tumor microenvironment (TME). TAMs differentiate into 2 functionally distinct morphologies, classically activated (M1)-type TAMs and alternatively activated (M2)-type TAMs, when stimulated by different cytokines. The 2 types of TAMs exhibit distinct properties and functions. M1 TAMs secrete high levels of pro-inflammatory and chemotactic factors, exerting proinflammatory, antitumor effects. Conversely, M2 TAMs alter the extracellular matrix, facilitate cellular immune escape, and stimulate tumor angiogenesis, thereby promoting anti-inflammatory responses and tumor growth. The ratio of M1 TAMs to M2 TAMs in the TME is closely related to the prognosis of the tumor. Tumor cells and other cells in the TME can regulate the polarization of TAMs and thus promote tumor progression through the secretion of various substances; however, polarized TAMs can also act on various cells in the TME through the secretion of exosomes, thus forming a positive feedback loop. Therefore, modulating the phenotype of TAMs in the TME or blocking the polarization of M2 TAMs might be a new approach for cancer treatment. However, the intracellular signaling pathways involved in the polarization of TAMs are poorly understood. The AKT signaling pathway is an important signaling pathway involved in the polarization, growth, proliferation, recruitment, and apoptosis of TAMs, as well as the action of TAMs on other cells within the TME. This paper reviews the AKT signaling pathway in the polarization of TAMs and the regulation of the TME and provides new ideas for tumor immunotherapy.

BackgroundCrosstalk between pancreatic cancer cells and tumor-associated macrophages (TAMs) is a critical driver of malignant progression, and plays an important role in the low response rate to immunotherapy in patients with for pancreatic cancer. Although it is known that cancer cells induce TAM infiltration and M2 polarization, the underlying mechanisms remain elusive. Herein, we identified matrix metalloproteinase 28 (MMP28), a highly expressed protein, as a key regulator of this process.MethodsImmunohistochemical staining and qRT-PCR were used to validate MMP28 as a potential marker for the prognosis of patients with pancreatic cancer. We evaluated the tumor-promoting effect of MMP28 in vitro with CCK-8, Transwell, and EdU assay and Western blotting and explored the potential mechanism of MMP28-induced M2 polarization of TAMs with a coculture system, immunofluorescence staining and flow cytometry. A subcutaneous graft tumor model was constructed to assess the tumor-promoting effect of MMP28 and its ability to induce M2 TAM infiltration.ResultsThe relevant results of this study revealed a strong correlation between MMP28 expression and TAM infiltration, with a predominance of M2-polarized TAMs in pancreatic cancer tissues. Mechanistic investigations demonstrated that MMP28 promotes the secretion of multiple cytokines, including IL-8 and VEGFA through the activation of the MAPK/JNK signaling pathway. These cytokines act as potent chemoattractants and polarizing factors for TAMs. Additionally, we discovered an interaction between MMP28 and ANXA2, which contributes to the regulation of TAM recruitment and polarization. In vivo studies confirmed the critical role of MMP28 in tumor growth and TAM infiltration. Depletion of macrophages, inhibition of JNK, or neutralization of IL-8 and VEGFA significantly suppressed tumor progression. Transcriptomic analysis suggested that IL-8 and VEGFA induce M2 TAM polarization by modulating TAM amino acid metabolism.ConclusionsCollectively, our findings elucidate a novel mechanism by which pancreatic cancer cells manipulate the tumor microenvironment through MMP28-dependent cytokine secretion, promoting TAM infiltration and M2 polarization. These results highlight MMP28 as a promising therapeutic target for pancreatic cancer.Graphical Schematic overview of the mechanisms by which MMP28 promotes the migration and polarization of TAMs. High levels of MMP28 promote the secretion of IL-8 and VEGFA by cancer cells by mediating the phosphorylation of the MAPK/JNK signalling pathway and then recruiting TAMs. IL-8 and VEGFA subsequently induce amino acid metabolism alterations in TAMs by binding to relevant receptors on TAMs, which ultimately promote the polarization of TAMs to the M2 phenotype. In addition, ANXA2 increases MMP28-mediated M2 TAM infiltration by interacting with MMP28.

Dysregulated expression of ribosomal protein S3A (RPS3A) is associated with the tissue infiltration of immune-related cells in a variety of cancers. However, the role of RPS3A in immune cell infiltration in glioma remains unclear. This study aimed to explore the role of RPS3A in the glioma immune microenvironment.RPS3A expression was detected in tumor tissues from patients with glioma. U251 cells were transfected with RPS3A shRNA (sh-RPS3A) and overexpression vector (pcDNA-RPS3A) and then co-cultured with PMA-induced THP-1 cells. Cell viability, invasion, and apoptosis were detected by Edu staining, Transwell, and flow cytometry, respectively. The expression of tumor-associated macrophage (TAM) M1 and M2 markers was detected with RT-qPCR. Next, the interaction between RPS3A and E4 transcription factor 1 (E4F1) was verified by Co-IP analysis, and the binding of E4F1 to colony-stimulating factor 1 (CSF1) promoter was verified by ChIP analysis. Overexpression vectors of CSF1 and E4F1 were used to treat sh-RPS3A-transfected U251 cells for reversal experiments. Finally, U251 cells transfected with sh-RPS3A adenovirus vectors were subcutaneously injected into nude mice to construct a xenograft tumor model, and the growth and metastasis of glioma in vivo were monitored.RPS3A was significantly upregulated in glioma tissues. Overexpression of RPS3A promoted glioma cell proliferation and invasion and inhibited apoptosis. Moreover, overexpression of RPS3A promoted TAM proliferation, invasion, and M2 polarization. Silencing RPS3A had the opposite effect. Silencing RPS3A inhibited autophagy in U251 cells, whereas rapamycin, an activator of autophagy, reversed the inhibitory effect of RPS3A silencing on TAM M2 polarization. Meanwhile, RPS3A promoted its expression by interacting with E4F1, and E4F1 promoted CSF1 transcriptional activation. Overexpression of CSF1 promoted the proliferation and invasion of U251 cells and reversed the inhibitory effect of RPS3A silencing on TAM proliferation and invasion, but had no effect on TAM M2 polarization. The results of in vivo experiments showed that knockdown of RPS3A significantly inhibited glioma tumor growth and metastasis in vivo.This study revealed that RPS3A recruited TAMs by upregulating E4F1-mediated transcription activation of CSF1, and promoted the M2 polarization of TAMs through autophagy, promoting glioma cell malignant growth and tumor progression.

Pancreatic cancer remains a serious unmet medical need. The majority of pancreatic tumors are composed of stromal cells that make tumors resistant to conventional therapies. We are developing an approach in which a TLR9 agonist, IMO-2125, is administered intratumorally, which induces local Th1 responses and modulates the tumor microenvironment. This approach has shown promising antitumor activity in preclinical models of multiple tumor types in combination with checkpoint inhibitors. We are employing a similar approach to the treatment of pancreatic cancer. We evaluated the local therapeutic effect of IMO-2125 in a murine syngeneic pancreatic ductal adenocarcinoma (Panc02) cancer models. In the first study, C57BL/6 mice were intraperitoneally (i.p.) implanted with Panc02 cells to generate peritoneal metastatic cancer. Tumor-bearing mice (n=10 per group) were then treated with 2.5 mg/kg IMO-2125 either by i.p. or subcutaneous (s.c.) injections for six times in two weeks. In the second study, to establish dual solid tumors in C57BL/6 mice, Panc02 cells were subcutaneously implanted in both the right and left flanks of the animals. When tumors reached to 50-150 mm3, we started the intratumoral (i.t.) administration of IMO-2125 in right tumors at doses of either 0.25 mg/kg, 1 mg/kg or 2.5 mg/kg, or PBS (n=10 per group), 3x/week for three weeks. In the peritoneal metastatic pancreatic tumor model, 80% of IMO-2125 intraperitoneally treated mice survived for more than 88 days, while 90% of the control group died within 29 days. Compared to i.p. treatment, the s.c. treated group survived for only 36 days. Tumor-free mice from the IMO-2125 i.p treatment group (n=7) rejected the same tumor rechallenge, suggesting establishment of durable tumor-specific immune response. In the dual solid pancreatic tumor model, intratumoral IMO-2125 treatment led to a dose dependent antitumor activity in both injected and distant tumors. Tumors treated with 0.25, 1 and 2.5 mg/kg doses of IMO-2125 showed reductions in tumor volume by 66% (p = 0.004), 74% (p = 0.002) and 95% (p = 0.003), respectively. Moreover, the distant tumors exhibited a reduction of 0% (p = 0.65), 64% (p = 0.006) and 69% (p = 0.001), respectively. Antitumor activity was associated with increased T-cell infiltration and upregulation of the gene expression of various checkpoints in both treated and distant tumors. In an independent study, treatment with a control IMO showed minimal antitumor activity. All treatments were well tolerated. In summary, local treatment with IMO-2125 exerts antitumor activity in preclinical pancreatic cancer models, associated with an increase in T-cell infiltration in the tumor microenvironment, providing a novel approach to the treatment of pancreatic cancer. The clinical trial of intratumoral IMO-2125 is in progress in patients with melanoma. Citation Format: Daqing Wang, Evren Kocabas Argon, Fugang Zhu, Sudhir Agarwal. Local treatment with novel TLR9 agonist IMO-2125 demonstrates antitumor activity in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5659. doi:10.1158/1538-7445.AM2017-5659

Gastric cancer (GC) is often diagnosed at an advanced stage due to the absence of early symptoms and low screening rates, resulting in poor prognosis. The progression of GC is closely related to the immune response within the tumor microenvironment (TME). Tumor-associated macrophages (TAMs), particularly the M2 subtype, are the most prevalent inflammatory cells in the TME and play a crucial role in GC. Tumor cells also evade immune surveillance by upregulating CD274. OTUB2, a deubiquitinase, regulates tumor progression by deubiquitinating substrate proteins. However, the role of OTUB2 in TAMs polarization and immune evasion in GC remains unclear. Stable OTUB2 overexpression and knockdown cells were cocultured with M0 macrophages to study TAMs polarization. Flow cytometry was used to analyze M2 TAMs and CD274 expression on GC cells. Cytokine secretion was evaluated by ELISA. T cell killing assays were performed by co-culturing GC cells with CD8+ T cells. Co-immunoprecipitation and Western blotting assessed the ubiquitination levels of YAP, TAZ and CD274. In vivo studies were conducted to evaluate OTUB2’s effect on TAMs polarization, immune evasion and GC progression. Immunohistochemistry of GC tissues was performed to investigate the correlation between OTUB2 and TME components. OTUB2 overexpression activated YAP/TAZ to increase TGF-β1 and M2 TAMs polarization by inhibiting SMAD7. It also enhanced CD274 expression, promoting immune evasion. OTUB2 deubiquitinated YAP, TAZ, and CD274, preventing their degradation. In vivo, OTUB2 increased M2 TAMs polarization and CD274 expression, exacerbating GC progression. Immunohistochemistry confirmed a positive correlation between OTUB2, M2 TAMs infiltration and CD274 levels and a negative correlation with CD8+ T cell infiltration. Kaplan–Meier analysis showed reduced overall survival in GC patients with high OTUB2 expression. OTUB2 promotes M2 TAMs polarization and immune evasion in GC. Targeting OTUB2 offers a promising strategy to reshape the GC TME and improve the efficacy of immune checkpoint inhibitors.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with a high degree of malignancy and poor prognosis. Tumor-associated macrophages (TAMs) have been identified as significant contributors to the growth and metastasis of TNBC through the secretion of various growth factors and chemokines. Salvianolic acid A (SAA) has been shown to have anti-cancer activities. However, the potential activity of SAA on re-polarized TAMs remains unclear. As there is a correlation between the TAMs and TNBC, this study investigates the effect of SAA on TAMs in the TNBC microenvironment. For that purpose, M2 TAM polarization was induced by two kinds of TNBC-conditioned medium (TNBC-TCM) in the absence or presence of SAA. The gene and protein expression of TAM markers were analyzed by qPCR, FCM, IF, ELISA, and Western blot. The protein expression levels of ERK and p-ERK in M2-like TAMs were analyzed by Western blot. The migration and invasion properties of M2-like TAMs were analyzed by Transwell assays. Here, we demonstrated that SAA increased the expression levels of CD86, IL-1β, and iNOS in M2-like TAMs and, conversely, decreased the expression levels of Arg-1 and CD206. Moreover, SAA inhibited the migration and invasion properties of M2-like TAMs effectively and decreased the protein expression of TGF-β1 and p-ERK in a concentration-dependent manner, as well as TGF-β1 gene expression and secretion. Our current findings for the first time demonstrated that SAA inhibits macrophage polarization to M2-like TAMs by inhibiting the ERK pathway and promotes M2-like TAM re-polarization to the M1 TAMs, which may exert its anti-tumor effect by regulating M1/M2 TAM polarization. These findings highlight SAA as a potential regulator of M2 TAMs and the possibility of utilizing SAA to reprogram M2 TAMs offers promising insights for the clinical management of TNBC.

Objectives: The more aggressive behavior of type 2 than type 1 endometrial cancers may be due to differences in their tumor microenvironments. In other solid cancers, tumor associated macrophages (TAMs) play a pivotal role in orchestrating the microenvironment. TAMs differentially polarize into M1 or M2 macrophages with distinct cytokine secretion patterns and actions. The aim of our work is to characterize the density, subtype and distribution of TAMs in endometrial hyperplasia and cancer. Methods: Five patients with complex atypical hyperplasia (hyperplasia), 5 patients with grade 1, non-invasive endometrioid adenocarcinoma (type 1) and 5 patients with uterine papillary serous carcinoma (type 2) were included. Formalin-fixed, paraffin-embedded sections from each specimen were stained with anti-CD68 and anti-CD163 monoclonal antibodies as markers for total TAMs and M2 TAMs, respectively. M1 macrophages were estimated by subtraction of CD163+ from CD68+ densities. Macrophages were counted at 40x magnification in 10 fields per slide by 4 observers. Repeated measure models, accounting for intra- and inter-observer correlation were contstructed and the compound symmetry covariance method was used to determine the relationship between macrophages and cancer. To establish an in vitro model, immortalized histiocytic lymphoma (U937) cells were differentiated to macrophages and treated with LPS to induce a M1 phenotype. Supernatants were applied to Cytokine C1000 microarray (RayBiotech) to screen for differentially expressed cytokines. Results: The correlation between the 4 observers was robust (r=0.71). Most TAMs were located in the stromal (mean=41.0/field) compared to epithelial (mean=11.0) or luminal (mean=11.6) compartments (p<0.0001). The mean total TAM density was highest in patients with type 2 cancers (mean=90.1/field), followed by type 1 cancers (mean=51.4) and hyperplasias (mean=41.1, p<0.0001). The calculated density of M1 macrophages was higher in type 2 cancers (mean=71.7) compared to type 1 cancers (mean =36.4, p<0.0001). This difference was only observed in the stromal compartment (p<0.0001). In the in vitro M1 macrophage model, ENA-78, a neutrophil chemo-attractant, and IL-6 were expressed at the highest concentrations. Conclusions: Type 2 cancers have nearly twice the TAM density of type 1 cancers. This difference is due to M1 macrophage predominance in the stroma of type 2 cancers. We postulate that cytokines produced by M1 TAMs within the tumor stroma contribute to the aggressive biology of these cancers. Future experiments will determine how well the in vitro model mimics M1 TAMs in the tumor microenvironment. Citation Format: Michael G. Kelly, Antonio M.C. Francisco, Adela Cimic, Anne Wofford, Nora Fitzgerald, Jie Yu, Robert N. Taylor. Type 2 endometrial cancer is associated with an M1 subtype, tumor associated macrophage polarization in the stromal compartment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1090. doi:10.1158/1538-7445.AM2014-1090

15P - Using a multiplexed immunofluorescence assay to detect immunosuppressive cells and their mechanisms in the pancreatic tumor microenvironment

Circular RNAs (circRNAs) are reported to be involved in the tumorigenesis of lung adenocarcinoma (LUAD). Here, this study focused on studying the function and mechanism of circHSPB6 in LUAD progression. Levels of genes and proteins were tested using qRT-PCR and western blotting analyses. The 5-ethynyl-2'-deoxyuridine (EdU), colony formation, flow cytometry, and transwell assays were adopted for in vitro assays. In vivo assay was conducted using mouse xenograft models. The binding between let-7a-2-3p and circHSPB6 or CCL2 was validated using RIP and dual-luciferase reporter assays. The M2 polarization of tumor-associated macrophages (TAMs) was analyzed by flow cytometry. LUAD tissues and cells showed high circHSPB6 expression, knockdown of circHSPB6-suppressed LUAD cell proliferation, migration, invasion, and induced cell apoptosis in vitro, as well as hindered tumor growth in vivo. Mechanistically, circHSPB6/let-7a-2-3p/CCL2 forms a feedback loop. CircHSPB6 could regulate CCL2 expression via sponging let-7a-2-3p. Further rescue assays showed that the effects of circHSPB6 silencing on LUAD cells were reversed by let-7a-2-3p inhibition or CCL2 overexpression. Moreover, circHSPB6 promoted the M2 polarization and infiltration of TAMs by CCL2. Functionally, circHSPB6 knockdown in A549 and H1299 cells inhibited TAM M2 polarization and then suppressed cell proliferation, migration, invasion, and emergency medical technicians (EMT) progression, while these effects were reversed by CCL2 up-regulation CircHSPB6 induced TAM M2 polarization to promote LUAD cell proliferation, migration, invasion, and EMT progression through let-7a-2-3p/CCL2 axis.

Tumor-associated macrophages (TAMs) differentiate into two main types based on signals from their microenvironment: the classically activated M1 type and the alternatively activated M2 type. An increased presence of M2-type TAMs has been identified in colorectal cancer patients, and the laminin alpha-1 chain (LAMA1), a key structural component of the extracellular matrix, plays a pivotal role in tumor metastasis. However, the exact mechanism by which LAMA1 promotes the polarization of TAMs towards the M2 phenotype and induces colorectal cancer remains elusive. In this work, we investigated the levels of LAMA1 and M2 TAMs in peripheral serum and tissue specimens from patients with colorectal cancer by utilizing Western Blotting, ELISA, and multicolor immunofluorescence. Peripheral blood samples from 20 patients were collected for ELISA analysis, with samples from 5 healthy individuals serving as controls. Additionally, tissue samples from 5 tumor patients and 5 normal controls were used to assess LAMA1 protein levels. Furthermore, statistical methods were employed to analyze the relationship between LAMA1 levels, M2 TAMs infiltration, and the pathological characteristics of colorectal cancer. Then we utilized Western Blotting, qPCR, and immunofluorescence to detect M2 TAMs markers and polarization mechanism after treatment with exogenous LAMA1 protein. Moreover, we established a mouse subcutaneous tumor model and utilized Western Blotting, immunohistochemistry and multicolor immunofluorescence to assess LAMA1 levels, tumor proliferation and proportion of M2 TAMs. Our analysis revealed that LAMA1 is overexpressed in the tumor microenvironment (TME) of colorectal cancer patients, which correlates with increased infiltration of M2-type TAMs. Furthermore, we found that the EGFR/AKT/CREB signaling pathway contributes to the polarization of TAMs toward the M2 subtype. Both in vitro and in vivo experiments demonstrated that LAMA1 promotes M2 macrophage polarization and facilitates tumor growth. Overall, these findings highlight a central role for LAMA1 in regulating macrophage polarization through the EGFR/AKT/CREB signaling pathway. Consequently, this process contributes to immune suppression and promotes tumor progression.

Tumor-associated macrophages (TAMs) usually display an antiinflammatory M2-like phenotype to facilitate tumor growth. However, what drives M2 polarization of TAMs and how TAMs suppress antitumor immunity within the tumor microenvironment (TME) remain largely undefined. Using several murine tumor models, we showed that hedgehog (Hh) signaling in myeloid cells is critical for TAM M2 polarization and tumor growth. We also found that tumor cells secrete sonic hedgehog (SHH), an Hh ligand, and that tumor-derived SHH drives TAM M2 polarization. Furthermore, Hh-induced functional polarization in TAMs suppresses CD8+ T cell recruitment to the TME through the inhibition of CXCL9 and CXCL10 production by TAMs. Last, we demonstrated that Krüppel-like factor 4 (Klf4) mediates Hh-dependent TAM M2 polarization and the immunosuppressive function. Collectively, these findings highlight a critical role for tumor-derived SHH in promoting TAM M2 polarization, a mechanism for TAM-mediated immunosuppression, and may provide insights into the design of new cancer immunotherapeutic strategies.

Histone lysine acetyltransferases KAT6A and KAT6B are frequently dysregulated in cancer and are mechanistically linked to key oncogenic processes. Small-molecule inhibitors of KAT6 have shown promising clinical efficacy, particularly in ER/HER2- metastatic breast cancer. However, KAT6 inhibitors remain largely unexplored in other solid tumors despite strong biological and mechanistic rationale for KAT6 as a potential target. Here, we report for the first time that the inhibition of KAT6 using OP-3136, a highly selective KAT6 inhibitor currently in Phase 1 clinical trials, demonstrates robust anti-tumor activity in preclinical models of prostate cancer, ovarian cancer, and non-small cell lung cancer (NSCLC), both in vitro and in vivo. In vitro proliferation assays were conducted on KAT6A-expressing or overexpressing cell lines: VCAP (prostate cancer), OAW28 (ovarian cancer, KAT6A-amplified), OVCAR3 (ovarian cancer), and LCLC-97TM1 (NSCLC). In vivo efficacy was evaluated in xenograft models using OVCAR3 and LCLC-97TM1 cells in immunocompromised mice.OP-3136 inhibited proliferation across all tested prostate cancer, ovarian cancer and NSCLC cell lines. In the OVCAR3 xenograft ovarian cancer model, oral administration of OP-3136 as monotherapy at 0.5 and 1 mg/kg resulted in tumor regression with tumor growth inhibition (TGI) of 104.5% and 105.4%, respectively. In the LCLC-97TM1 NSCLC xenograft model, monotherapy with OP-3136 at 1 mg/kg achieved a TGI of 62%. These findings highlight the potential of OP-3136 monotherapy to treat prostate, ovarian, and NSCLC tumors and strongly support expanding the clinical investigation of KAT6 inhibitors beyond ER+/HER2- breast cancer. Citation Format: Gopinath S. Palanisamy, Susanna A. Barratt, Chelsea Hope, Srinivasan Jayaraman, Brandon Robello, Guadalupe Peña, Raymond A. Ng, David C. Myles. OP-3136, a selective KAT6 inhibitor, demonstrates anti-tumor activity in prostate, ovarian, and non-small cell lung cancer preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB166.

Activating mutations of K-ras are one of the most common molecular alterations associated with lung cancer development. Several attempts to develop therapies directly targeting K-ras have failed thus far, clearly state the need for new strategies to bring clinical benefits to patients displaying such molecular profile. Using a conditional K-ras mutated lung cancer mouse model (CC-LR) we previously showed that K-ras mutated lung tumors have intrinsic inflammatory characteristics and we also demonstrated that chronic obstructive pulmonary disease (COPD)-like airway inflammation, an independent risk factor for lung cancer, promotes lung tumor in this model. CC-LR mice shows high protein level of interleukin 6 (IL-6) and increased STAT3 (downstream signal to IL6) gene expression, and we have further shown that genetic ablation of IL-6 in CC-LR mice results in significant tumor reduction. High levels of IL-6 and up-regulation of STAT3 and its target genes have been observed in the lungs of smokers with COPD, and in 50% of human lung adenocarcinomas. These indicate an essential role for IL-6 in lung cancer promotion, and introduce IL-6 as a potential druggable target for lung cancer patients who harbor K-ras mutations. This prompted us to block IL-6 pathway using a monoclonal anti-IL-6 immunotherapy in our K-ras mutated lung cancer mouse model. Six week old CC-LR mice were injected intraperitoneally (I.P.) with 20 mg/kg dose of an anti-IL-6 monoclonal IgG1 antibody, twice a week, for a period of eight weeks. Flowcytometry analysis of total lung inflammatory cell population in non-treated CC-LR mice showed 2-fold increase in the population of tumor associated macrophage (TAM) (CD45+/CD11b+/GR1+/F4/80+/MCSFR+) during tumor progression from age 6 weeks to 14 weeks. qPCR analysis of total lung RNA from non-treated CC-LR mice showed that there is an increase in the level of Arginase-1 (3 fold) with no change in the level of inducible Nitric Oxide Synthetize (NOS2). This suggests that epithelial signal initiated by K-ras mutation may dictate the fate of lung microenvironment by creating an inflammatory pro-tumor type 2 response. Then we found that anti-IL-6 treatment suppresses tumor progression by ~78% (4.6-fold) in CC-LR mice which is associated with a significant reduction in the quantity of inflammatory cells in bronchoalveolar lavage fluid (BALF). It also decreased the expression of proliferation marker: Ki-67, and pro angiogenic factors: VEGF, MMP9 and CD31 and phospho-STAT3 in lung tissue. ELISA analysis of BALF from CC-LR mice also showed that anti-IL-6 treatment reduces TGFβ and KC protein levels with no change in level of IL-17. qPCR and flowcytometry analysis of total lung homogenate of CC-LR mice further showed that IL-6 blockade induces a significant decrease in Arginase1 expression (3-fold) and TAM population (5-fold). Taking together here we showed that K-ras mutated lung tumor drives an immunosuppressive pro-tumor microenvironment, with an M2 TAM polarization and blocking the suppressive actions of IL-6 shifts this to an anti-tumor lung microenvironment. Therefore we propose pharmacological targeting of IL-6 as a potential therapeutic strategy alone or in combination with conventional cytotoxic therapy for lung cancer patients with K-ras mutation. Funded by: American Cancer Society, RSG-11-115-01-CNE, and American Lung Association/LUNGevity Foundation, LCD-114696-N Citation Format: Mauricio S. Caetano, Amber M. Cumpian, Lei Gong, Cesar E. Ochoa, Seon H. Chang, Huiyuan Zhang, Stephanie Watowich, Cinthya Sternberg, Carlos G. Ferreira, Seyed J. Moghaddam. Pharmacologic targeting of IL-6 suppresses tumor progression in a non-small cell lung cancer mouse model with K-ras mutation through re-educating the tumor microenvironment. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A13.

Introduction: Triple-negative breast cancers (TNBCs) are defined as tumors that are negative for both estrogen, progesterone and HER-2 receptors. It is a heterogeneous subtype of breast cancer with a high propensity for systemic metastases and poor survival with only chemotherapy available for treatment. Compared to other hormone-positive breast cancer subtypes, TNBC features a unique tumor microenvironment (TME) characterized by a large number of tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) (1), and the increase of immune infiltrate with high levels of TILs predicts response to neoadjuvant chemotherapy as well as improved survival. TAMs can be described as classically activated M1 types with pro-inflammatory antitumor functions, versus alternatively activated M2 types with immunosuppressive pro-tumor functions. It has been reported that the presence of M2 TAMs positively correlates with TNBC, hormone receptor negativity, as well as higher tumor proliferation (2-3). However, the mechanisms by which TAMs interact with TNBC cells and their phenotypes in TNBC versus estrogen receptor positive (ER+) patients is not well understood. Experimental Procedures: Using MultiOmyx, a proprietary, immunofluorescence (IF) multiplexing assay that enables visualization and characterization of up to 60 biomarkers on a single FFPE section (4), we have characterized TIL phenotypes, tumor proliferation and TAM activation in 15 FFPE tumors from TNBC and 5 tumors from ER+ patients. Results: Using a multiplex panel of 9 markers we found a 10-fold higher number of TILs and a 5-fold higher number of proliferating tumor cells in TNBC versus ER+ tumors. Furthermore, the proportion of T helper cells (CD3+CD4+) that were Tregs (CD3+CD4+FoxP3+) was increased from 26% to 43% in TNBC tumors. Interestingly, when analyzing TAMs we found that while ER+ tumors had a significantly higher proportion of M1 TAMs (CD68+HLA-DR+) than M2 TAMs (CD68+CD163+), this expression pattern was reversed in TNBC. When analyzing a possible correlation between the activation state of TAMs and the proliferation of tumor cells, we found a positive significant correlation between the presence of M2 TAMs and proliferating tumor cells (Pearson's correlation p<0.05), only in TNBC and not in ER+ tumors. Conversely, the presence of M1 tumors showed a weak negative correlation to tumor cell proliferation, in both ER+ and TNBC tumors. Conclusion: These data are suggestive of a possible pathway in which an increase in alternatively activated immunosuppressive M2 TAMs in hormone receptor negative breast cancer tumors, are responsible for providing a suitable environment for tumor proliferation.

Introduction: Triple-negative breast cancers (TNBCs) are defined as tumors that are negative for both estrogen, progesterone and HER-2 receptors. It is a heterogeneous subtype of breast cancer with a high propensity for systemic metastases and poor survival with only chemotherapy available for treatment. Compared to other hormone-positive breast cancer subtypes, TNBC features a unique tumor microenvironment (TME) characterized by a large number of tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) (1), and the increase of immune infiltrate with high levels of TILs predicts response to neoadjuvant chemotherapy as well as improved survival. TAMs can be described as classically activated M1 types with pro-inflammatory antitumor functions, versus alternatively activated M2 types with immunosuppressive pro-tumor functions. It has been reported that the presence of M2 TAMs positively correlates with TNBC, hormone receptor negativity, as well as higher tumor proliferation (2-3). However, the mechanisms by which TAMs interact with TNBC cells and their phenotypes in TNBC versus estrogen receptor positive (ER+) patients is not well understood. Experimental Procedures: Using MultiOmyx, a proprietary, immunofluorescence (IF) multiplexing assay that enables visualization and characterization of up to 60 biomarkers on a single FFPE section (4), we have characterized TIL phenotypes, tumor proliferation and TAM activation in 15 FFPE tumors from TNBC and 5 tumors from ER+ patients. Results: Using a multiplex panel of 9 markers we found a 10-fold higher number of TILs and a 5-fold higher number of proliferating tumor cells in TNBC versus ER+ tumors. Furthermore, the proportion of T helper cells (CD3+CD4+) that were Tregs (CD3+CD4+FoxP3+) was increased from 26% to 43% in TNBC tumors. Interestingly, when analyzing TAMs we found that while ER+ tumors had a significantly higher proportion of M1 TAMs (CD68+HLA-DR+) than M2 TAMs (CD68+CD163+), this expression pattern was reversed in TNBC. When analyzing a possible correlation between the activation state of TAMs and the proliferation of tumor cells, we found a positive significant correlation between the presence of M2 TAMs and proliferating tumor cells (Pearson9s correlation p Conclusion: These data are suggestive of a possible pathway in which an increase in alternatively activated immunosuppressive M2 TAMs in hormone receptor negative breast cancer tumors, are responsible for providing a suitable environment for tumor proliferation.