bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2926: Histologically defined intratumoral sequencing uncovers evolutionary cues into conserved molecular events driving gliomagenesis
Authors: Prabhu Antony , Kesarwani Pravin , Kant Shiva , Graham Stewart , Chinnaiyan Prakash
Published: July 2017

Glioblastoma (GBM) represents an archetypal example of a heterogeneous malignancy, harboring regions of invasion, necrosis, and vascularization. To begin to understand the diverse molecular consequences of this complex tumor ecology, we analyzed RNA-seq data generated from commonly identified intratumoral structures in GBM that were isolated and enriched using laser capture micro-dissection. Structures included the infiltrative, leading edge, central tumor core, and cellular regions in areas of necrosis. From this RNA-seq database consisting of 119 structural regions from 37 individual tumors generated from ~12,000 hematoxylin and eosin histologic images, we validated significant intra-tumoral heterogeneity in GBM, with cells derived from the infiltrating leading edge almost exclusively harboring the proneural molecular subtype, while cells in regions of necrosis displaying the mesenchymal subtype. In addition, we made the striking observation that when evaluating the tumors transcriptional profiles in the context of their derived structural regions, there was a relative small amount of inter-tumoral heterogeneity in GBM, with significant clustering of transcriptional profiles of specific structural regions from different tumors. To begin to understand the specific molecular events driving regional growth and their contributory role in gliomagenesis, we analyzed the transcriptional programs in the context of evolutionary progression. This level of analysis identified numerous pathways associated with neuronal receptor signaling, autophagy, and fatty acid metabolism as early events in tumorigenesis. Traditional cancer related signaling pathways emerged as key mediators driving progression, with glioma and p53 signaling ranking highest on the list. As the tumor progressed to harbor regions of necrosis, in addition to expected changes in VEGF and tumor signaling, we uncovered global metabolic changes associated with progression, with metabolic pathways comprising 10 of the top 20 pathways unique to this region. Collectively, these findings suggest that the observed intra-tumoral heterogeneity in GBM may be a conserved, predictable consequence to its complex microenvironment and therapeutic strategies rationally designed to target these unique, unequivocally present tumor cell biomes may lead to therapeutic gains in this otherwise fatal malignancy.

Citation Format: Antony Prabhu, Pravin Kesarwani, Shiva Kant, Stewart Graham, Prakash Chinnaiyan. Histologically defined intratumoral sequencing uncovers evolutionary cues into conserved molecular events driving gliomagenesis [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 2926. doi:10.1158/1538-7445.AM2017-2926
Abstract 2930: Niche microenvironments essential for tumor heterogeneity of primary cancer cells
Authors: Fujino Shiki , Miyoshi Norikatsu , Ohue Masayuki , Takayashi Yusuke , Yasui Masayoshi , Takahashi Hidekazu , Haraguchi Naoyuki , Nishimura Jhunichi , Hata Taishi , Mizushima Tsunekazu , Doki Yuichiro , Mori Masaki
Published: July 2017

Primary culture of cancer cells derived from each patient’s tumor can provide important information of the “individual tumor.” It is general to use cell lines in basic research filed. However, cell lines are quite different from clinical cancers. Clinical cancer tissues are composed of not only cancer cells but also tumor microenvironments such as stromal cells and tumor vessels. The primary culture method of clinical cancer with tumor niche microenvironments has not been optimized. We have developed a simple 2D-culture method for primary colorectal cancer (CRC). We obtained 30 samples from surgically resected tumor. They were mechanically and enzymatically digested and fibrotic tissue and bacteria were excluded using customized two size filters. And we cultured the obtained cells on a matrigel-coated plate with embryonic stem (ES) cells culture medium. We named these cultured cancer cells, “isolated-tumor derived Cancer Cells (iCCs).” All iCCs grew and about 80 % of iCCs were successfully passaged. Twenty-three iCCs were transplanted into the subcutaneous layer of NOD-SCID mice, and the tumor growth and histology of iCCs were examined. The morphology was similar to each parental clinical tumor. And microarray analysis showed that RNA expression of iCCs was similar to each parental tumor. Furthermore, we examined the culture medium; our modified ES culture medium (ES-cultured iCCs) and 10% FBS medium (serum-cultured iCCs). The expression of surface markers regarding cancer stem cells such as CD44 and CD24 were different between ES-cultured iCCs and serum-cultured iCCs, and drug sensitivity of iCCs were also different. FACS analysis and immunocytochemistry revealed that iCCs contained PDGFR-positive cells. The results of multi-drug sensitivity assay were different between iCCs and cell lines especially in PDGFR inhibitor. Strong correlations were observed between the results of multi-drug sensitivity assay of iCCs and clinical outcomes of chemotherapy. We report an innovative primary culture method and the in vivo and in vitro analyses of iCCs, leading to the future application.

Citation Format: Shiki Fujino, Norikatsu Miyoshi, Masayuki Ohue, Yusuke Takayashi, Masayoshi Yasui, Hidekazu Takahashi, Naoyuki Haraguchi, Jhunichi Nishimura, Taishi Hata, Tsunekazu Mizushima, Yuichiro Doki, Masaki Mori. Niche microenvironments essential for tumor heterogeneity of primary cancer cells [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 2930. doi:10.1158/1538-7445.AM2017-2930
Abstract 2931: Targeting membrane-bound carbonic anhydrases in breast cancer to intervene in the metastatic phenotype
Authors: Mboge Mam Y. , Chen Zhijuan , Mahon Brian P. , Tu Chingkkuang , Wolff Alyssa S. , Mathias John V. , Carta Fabrizio , Supuran Claudiu T. , McKenna Rob , Frost Susan C.
Published: July 2017

Breast cancer is the second leading cause of cancer related deaths among women in the United States. Despite the tremendous progress that has been made towards treating localized tumors, nearly 40,000 women die each year, predominantly from metastatic drug resistance. The tumor microenvironment plays a pivotal role in determining tumor growth, invasion, metastasis, and therapeutic success or failure. Therefore, a therapy targeting the tumor microenvironment is needed to sufficiently preserve the quality of life of cancer patients, by inhibiting metastasis. Elevated levels of Carbonic anhydrase IX (CAIX) expression in primary breast cancers is a marker for highly aggressive and metastatic tumors, especially of the triple negative subtype (TNBC). It is also associated with hypoxia, extracellular acidification, and poor prognosis. Low pH (values of ~6.5-6.8) is toxic to normal cells in the tumor microenvironment while enhancing cancer cell proliferation and tumor growth. Our goal was to compare the structure of a CAIX-mimic bound to ureidosulfonamide inhibitors with the biological activity of these inhibitors in triple negative and estrogen receptor positive (ER+) breast cancer cell lines. CAIX is a reversible enzyme and at low pH (high proton concentration), the enzyme will consume protons, raising pH. Our hypothesis is that CAIX inhibition, in the context of an acidic microenvironment, will dysregulate its ability to maintain the acidic pH preferred by cancer cells which favors their growth and migration. In this study, we have shown the interaction of sulfonamide-based inhibitors with a CAIX-mimic using X-ray crystallography. These structures show that the inhibitors make multiple contacts within the active site cavity. This is consistent with the inhibitor-induced decrease in CAIX activity and to some extent expression. We have also investigated the effect of CA inhibition on breast cancer cell growth, proliferation, activation of cell death pathways and migration. This reveals that, although CA inhibition with the sulfonamide-based compounds inhibits cell growth and migration, it does not activate apoptotic pathways. In total, these observations indicate that CAIX is a viable small molecular drug target for the treatment of metastatic breast cancer.

Citation Format: Mam Y. Mboge, Zhijuan Chen, Brian P. Mahon, Chingkkuang Tu, Alyssa S. Wolff, John V. Mathias, Fabrizio Carta, Claudiu T. Supuran, Rob McKenna, Susan C. Frost. Targeting membrane-bound carbonic anhydrases in breast cancer to intervene in the metastatic phenotype [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 2931. doi:10.1158/1538-7445.AM2017-2931
Abstract 2935: Sex disparity in the response of the immune microenvironment to colon cancer liver metastases
Authors: Milette Simon , Wang Ni , Perrino Stéphanie , Brodt Pnina
Published: July 2017

Background Metastasis remains the most life-threatening aspect of malignant disease and a major clinical challenge. Myeloid-derived suppressor cells (MDSC) and regulatory T lymphocytes (Tregs), which are frequently found in the tumor microenvironment, are known to impair immune surveillance and promote a state of immune tolerance that leads to metastatic expansion. Previously, we have shown that the incidence of colon carcinoma liver metastases was reduced in TNF receptor 2 (TNFR2) deficient female mice, as a consequence of reduced recruitment of MDSCs and Tregs into the liver. Intriguingly however, no reduction in liver metastasis or MDSC/Treg accumulation was observed in TNFR2 deficient male mice, suggesting that their recruitment and survival were distinctly regulated. The objective of this study was, therefore, to elucidate the mechanisms underlying the sex disparity observed in the response of the immune microenvironment to liver metastases, in particular the role of sex hormones in immune response regulation in this context.

Methods Hormone-ablated female mice were generated using surgical ovariectomy (OVX). Estrogen-deprived mice, sham-operated controls and hormone reconstituted OVX mice were injected with syngeneic murine colon carcinoma MC-38 cells via the intrasplenic/portal route and experimental liver metastases enumerated 2 weeks later. MDSCs and Tregs recruited into the livers of tumor-bearing mice were analyzed 7-10 days post-tumor injection using flow cytometry, as well as IHC performed on liver cryostat sections.

Results We found that the number of experimental liver metastases was markedly reduced in ovariectomized female mice, as compared to sham-operated controls but was restored in estradiol supplemented OVX mice. Reduced incidence of hepatic metastases in OVX mice correlated with reduced accumulations of CD11b+Gr-1+ myeloid cells (MDSCs) and CD4+Foxp3+ cells (Tregs) at metastatic sites. IHC also revealed an increased CD8+ : CD4+Foxp3+ (i.e. cytotoxic T cell : Treg) ratio around hepatic metastases in OVX mice as compared to sham-operated controls. Estradiol reconstitution in OVX mice restored MDSC and Treg cell accumulation to the levels observed in control mice.

Conclusion Together, our data identify female sex hormones as major players in the regulation of the tumor immune microenvironment and a potential contributing factor in the patients’ response to immune-based cancer therapy.

Supported by the Canadian Institute for Health Research, MDEIE and FRSQ.

Citation Format: Simon Milette, Ni Wang, Stéphanie Perrino, Pnina Brodt. Sex disparity in the response of the immune microenvironment to colon cancer liver metastases [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 2935. doi:10.1158/1538-7445.AM2017-2935
Abstract 2938: Tumour-associated macrophage polarisation and re-education with oncolytic viruses
Authors: Muthana Munitta , Murphy Emer , Kwan Amy , Winder Natalie , Conner Joe
Published: July 2017

Tumor-associated macrophages (TAMs) promote key processes in tumour progression through their plasticity and capacity to become polarised by tumours to have an immunosuppressive phenotype. However, the plasticity of these cells provides an opportunity for therapeutic ‘reprogramming’ towards a classically activated inflammatory phenotype thus preventing tumour-associated immunosuppression and inducing anti-tumour immunity. Oncolytic viruses (OV) represent a new class of anti-cancer therapeutics that promote anti-tumour responses through selective tumour cell killing and the induction of systemic anti-tumour immunity. We propose that TAMs can be targeted using an OV, leading to altered macrophage polarization and a reduction in tumour growth and metastasis. Experimental Design: In-vitro, cytotoxicity, viral replication and cytokine expression was assessed in a panel of primary human and murine macrophages following exposure to Sephrevir, an ICP34.5-deleted oncolytic herpes-simplex virus currently in Phase 1/2a trials. In-vivo, murine TS1-PyMT cells were implanted into the mammary fat pads of female FVB mice to establish a syngeneic immunocompetent mouse model of breast cancer. Tumour growth and overall survival were assessed following administration of Sephrevir given either by intratumoural (I.T) or intravenous injection (I.V). Immune cell enumeration and protective immunity were assessed post-mortem by immunofluorescence and flow cytometry. Results: Both primary human and mouse macrophages served as a host for oncolytic viral replication. Sephrevir was also found to attenuate TAM phenotypes as evidenced by increased “M1” receptor signatures (e.g. CD40, CD80, CD86, MHC II), increased pro-inflammatory gene expression (e.g. IL-1, IL-6, IL-8, CXCL10, TNF-a) and increased nitric oxide production. I.T and I.V administration of Sephrevir led to marked tumour shrinkage as well as the recruitment of cytotoxic T cells. Moreover, TAM polarisation was evident based on a change in signature with TAMs becoming skewed to IL-12hi/IL-10lo and a change in the iNOS/arginase1 ratio. Conclusions: Our results demonstrate that Sephrevir is associated with in vivo anti-tumour effects and importantly can be used for manipulating the phenotype of the abundant macrophage population located within the tumour microenvironment.

Citation Format: Munitta Muthana, Emer Murphy, Amy Kwan, Natalie Winder, Joe Conner. Tumour-associated macrophage polarisation and re-education with oncolytic viruses [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 2938. doi:10.1158/1538-7445.AM2017-2938
Abstract 2942: Immunophenotypes in young-onset colorectal cancer
Authors: Herk M E. van , Lee M , Rytterdahl M , Kop M E. , Ramalheiro A F. , Breggen R van der , Wezel T van , Morreau H , Jordanova E S. , Miranda N F. de
Published: July 2017

Colorectal cancer accounts for ten percent of new cancer cases and is the fourth most frequent cause of cancer-related deaths worldwide. In recent years, mainly due to screening programs, more patients are diagnosed in early stages of tumor progression, leading to a higher survival rate. However, the incidence of young (<50) patients diagnosed with colorectal cancer has been on the rise. These patients often have a poor prognosis due to the fact that tumors are diagnosed at advanced stages of tumor progression.

To our knowledge, no study has yet characterized immunophenotypes and immune evasive mechanisms specifically in young-onset colorectal cancers. To that end we have investigated the expression of HLA class I and PD-L1 in over 200 colorectal cancers derived from young-onset (< 50 years-old) patients. Furthermore, we applied a novel multispectral immunofluorescence technology to perform multiparameter immunophenotyping with CD3, CD8, PD-1, PD-L1, CD163, and Ki-67 in the same cohort.

We describe that HLA class I expression is maintained in the large majority of tumors allowing thus the development of neo-antigen targeted therapies. Interestingly, reduced expression of HLA class I but not total loss was associated with liver metastases, which suggests a specific selective pressure at this organ that might warrant tailored immune therapeutic interventions. As previous studies on colorectal cancer demonstrated, PD-L1 expression is limited and often restricted to immune cell compartments. The presence of immune cell infiltrates was related to the mutation background of tumors but also to their HLA class I phenotype: tumors with altered HLA class I expression were more likely to present traces of lymphocyte-mediated anti-tumor immunity.

Retained HLA class I expression in the majority of colorectal cancers associated with low infiltration by effector immune cells suggests the therapeutic induction of anti-tumor immune responses in young-onset colorectal cancers, for instance, by means of neo-antigen-targeted therapies. We are currently assessing the frequency of natural recognition of neo-antigens in an autologous setting in young-onset, late-stage colorectal cancers.

This work was supported by the Fight Colorectal Cancer-Michael’s Mission-AACR Fellowship in Young-Onset, Late-Stage Colorectal Cancer Research awarded to N.F.C.C. de Miranda (15-40-1645-DEMI)

Citation Format: M E. van Herk, M Lee, M Rytterdahl, M E. Kop, A F. Ramalheiro, R van der Breggen, T van Wezel, H Morreau, E S. Jordanova, N F. de Miranda. Immunophenotypes in young-onset colorectal 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 2942. doi:10.1158/1538-7445.AM2017-2942
Abstract 2945: Impact of intratumoral mature dendritic cells on prognosis of the patients with esophageal squamous cell carcinoma
Authors: Nishimura Junya , Tanaka Hiroaki , Miki Yuichiro , Tamura Tatsuro , Fukuoka Tatsunari , Ohira Go , Shibutani Masatsune , Yamazoe Sadaaki , Kimura Kenjiro , Toyokawa Takahiro , Nagahara Hisashi , Amano Ryosuke , Muguruma Kazuya , Maeda Kiyoshi , Hirakawa Kosei , Ohira Masaichi
Published: July 2017

[Background] Promising efficacy of the immunotherapy with anti-PD-1 antibody for esophageal squamous cell carcinoma (ESCC) is expected. Tumor-infiltrating lymphocytes (TILs)have been found to be associated with favorable outcome of patients in many cancers including ESCC. However, the contributing factor of TILs in the primary tumor remains unclear. Dendritic cells (DCs) are the most potent antigen-presenting cells to induce cytotoxic T lymphocytes. [Purpose] The aim of this study was to examine the correlation of mature dendritic cells with CD8+ cells in the tumor in order to clarify the impact of DCs on locally infiltrated lymphocytes in ESCC. [Object and Methods] Formalin-fixed, paraffin-embedded blocks of primary lesions were collected from 80 patients with ESCC who underwent surgical treatment at Osaka City University Hospital. Immunohistochemical analysis using anti-LAMP-3 antibody and CD8 antibody was used to evaluate intratumoral DCs and TILs. The number of LAMP-3+DCs and TILs were counted at ×400 magnification. We divided the 80 cases into two groups according to the median number of intratumoral DCs and TILs to examine the clinicopathological features. [Results] LAMP-3+DCs were predominantly observed in peritumoral area and sparsely found in intratumoral area, whereas CD8+T cells distributed between both peritumoral and intratumoral areas. The number of DCs was significantly associated with increasing TNM stage. For example, the median number of DCs was 59.9 in pathological T1, 34.2 in T2/3, 62.4 in pN1, 34.1 in pN1/2/3. In addition, many patients with high infiltaraion of DCs had positive lymphatic invasion (Ly). The number of intratumoral DCs was significantly correlated with the number of TILs which were observed in intratumoral regions. Patients in the high infiltration of DCs group showed a significant better prognosis than patients in the low infiltration group. Double staining showed that LAMP3+DC-CD8+T cell clusters were seen in peritumoral area.

Citation Format: Junya Nishimura, Hiroaki Tanaka, Yuichiro Miki, Tatsuro Tamura, Tatsunari Fukuoka, Go Ohira, Masatsune Shibutani, Sadaaki Yamazoe, Kenjiro Kimura, Takahiro Toyokawa, Hisashi Nagahara, Ryosuke Amano, Kazuya Muguruma, Kiyoshi Maeda, Kosei Hirakawa, Masaichi Ohira. Impact of intratumoral mature dendritic cells on prognosis of the patients with esophageal squamous cell carcinoma [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 2945. doi:10.1158/1538-7445.AM2017-2945
Abstract 295: CASC-578, a novel Chk1 inhibitor, is active as a single agent in solid tumors and displays synergistic anti-tumor activity in combination with Wee1 inhibition
Authors: Vo Alex , Taylor Janelle , Rosler Robert , Piasecki Julia , Leviten Dina , Sierra Teresa , Dozier Ashley , Klucher Kevin , Boyle Bob , Boyce Rich , Peterson Scott
Published: July 2017

Background: Checkpoint kinase 1 (Chk1) is a serine/threonine protein kinase that regulates cell division in response to genotoxic stress by arresting cell cycle progression in the S & G2 phases. Pharmacological inhibition of Chk1 is proposed to target tumor cells with increased DNA replication stress, resulting in the uncoupling of DNA replication checkpoint function and the induction of DNA damage and cell death. These properties make Chk1 inhibition a novel therapeutic approach as a single agent in cancers with high replication stress that is driven by oncogenic signaling and loss of parallel DNA damage response pathway function.

Methods and Results: This report highlights the activity of the orally bioavailable, selective small molecule Chk1 inhibitor, CASC-578, in solid tumor derived cell lines. CASC-578 is a sub-nanomolar enzymatic inhibitor of Chk1 with limited off-target activity against a panel of protein kinases. When evaluated in a large cell line panel in vitro, CASC-578 demonstrated a broad potency range as a single agent in solid tumor derived cells lines, with IC50s ranging from 30 nM to greater than 50 μM. Several solid tumor types demonstrated enriched sensitivity to CASC-578 in vitro, including gastric, non-small cell lung and ovarian cancers. Treatment of sensitive cell lines with CASC-578 resulted in the induction of DNA damage, as measured by phosphorylated histone H2AX, and the induction of cell death. CASC-578 was active as a single agent in SK-MES-1 and NCI-H727 NSCLC tumor xenograft models in vivo with minimal effects on body weight in treated mice. In addition to the potent single agent activity of CASC-578, combination with the Wee1 inhibitor AZD-1775 was highly synergistic in vitro in multiple solid tumor cell lines and the combination was more efficacious than either agent alone in NSCLC tumor xenograft models. These data support the advancement of CASC-578 into clinical development as a potential therapeutic agent for the treatment of solid tumor diseases. Experiments are ongoing to identify biomarkers associated with sensitivity to CASC-578 as a single agent in solid tumor cell lines to prospectively identify tumor genotypes that are more responsive to the drug.

Citation Format: Alex Vo, Janelle Taylor, Robert Rosler, Julia Piasecki, Dina Leviten, Teresa Sierra, Ashley Dozier, Kevin Klucher, Bob Boyle, Rich Boyce, Scott Peterson. CASC-578, a novel Chk1 inhibitor, is active as a single agent in solid tumors and displays synergistic anti-tumor activity in combination with Wee1 inhibition [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 295. doi:10.1158/1538-7445.AM2017-295
Abstract 2951: The tumor microenvironment in cholangiocarcinoma is dominated by an immunosuppressive infiltrate
Authors: Figueroa Nathania M. , Belt Brian , Patel Ankit , Han Booyeon , Hill Margaret , Alexander William , O'Dell Michael , Murthy Aditi , Connolly Kelli , Gerber Scott A. , Hezel Aram , Linehan David
Published: July 2017

Purpose: To characterize the dynamics of the immune response to cholangiocarcinoma (CCA) in a genetically engineered mouse model of CCA in order to identify immune pathways susceptible to targeted immunotherapies.

Experimental Design: Genetically modified mice with targeted Kras activation and loss of p53 (Alb-Cre/LSL-KRASG12D /p53Lox/+) in the liver spontaneously develop CCA recapitulating the histopathologic features of human disease. To evaluate the in vivo immune response to CCA, tumors from Kras-p53 mice and normal livers were excised and processed for histologic examination, flow cytometry, and gene expression analysis. Immunohistochemistry (IHC) and flow cytometry were performed on myeloid and T cell markers to distinguish immune cell subsets. Cell lines established from isolated CCA tumors were used to assess the functional impact between the immune system and tumor cells in co-culture in-vitro studies.

Results: Kras-p53 murine hepatic tumors were highly desmoplastic with a heavy fibrotic stromal compartment. IHC analysis demonstrated a prominent inflammatory leukocyte infiltrate compared to normal liver. Flow cytometry of single cell tumor suspensions showed the immune reaction was dominated by CD11b+ monocytic (Ly6C+) and granulocytic (Ly6G+) myeloid cells, and Foxp3+ regulatory T cells (T reg). In vitro studies revealed tumor educated myeloid cells expressed higher levels of genes associated with an immunosuppressive phenotype, including Arginase 1 (p=<0.007). Invasion assays analyzing the migration potential of CCA cell lines co-cultured with CD11b+ cells showed increased invasive properties in these tumor cells when compared to naive controls (p=<0.0001). Additionally, increased tumor-initiating properties were seen by qRT-PCR in tumor cell lines having undergone similar co-culture experiments. These data suggest the immune response to CCA is predominantly immunosuppressive and tumor supportive.

Conclusion: CCA tumors from Alb-Cre/LSL-KRASG12D /p53Lox/+ mice have a prominent immunosuppressive infiltrate recapitulating features of the immune reaction in human disease. Thus, Kras-p53 mice provide an ideal model to test targeted immunotherapy for the treatment of CCA.

Citation Format: Nathania M. Figueroa, Brian Belt, Ankit Patel, Booyeon Han, Margaret Hill, William Alexander, Michael O'Dell, Aditi Murthy, Kelli Connolly, Scott A. Gerber, Aram Hezel, David Linehan. The tumor microenvironment in cholangiocarcinoma is dominated by an immunosuppressive infiltrate [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 2951. doi:10.1158/1538-7445.AM2017-2951
Abstract 2952: The novel cathepsin L/K inhibitors KGP94 and KGP207 prevent M0 to M2 macrophage differentiation and macrophage-mediated pro-tumor functions
Authors: Dykes Samantha S. , Siemann Dietmar W.
Published: July 2017

Tumor-associated macrophages play many important roles in tumor progression, including facilitating tumor cell invasion and angiogenesis. Due to their numerous pro-tumor functions, tumor-associated macrophages represent an important cell population in the design of targeted anti-cancer therapies. Interleukin-4-mediated activation of macrophages resulted in an M2-like, or pro-tumor macrophage phenotype, often associated with increased expression of proteases including the lysosomal cathepsin L. Cathepsin L is important in both macrophage and tumor cell invasion, whereby secreted cathepsin L degrades the extracellular matrix, allowing for cell infiltration. The present study examined the role of cathepsin L in M0 to M2 differentiation and macrophage-mediated tumor cell invasion using the novel cathepsin L/K inhibitors KGP94 and KGP207 [Dr. Kevin Pinney, Baylor University]. KGP94 and KGP207 prevented M2 macrophage motility and invasion in in vitro migration and invasion assays. Moreover, KGP94 and KGP207 reduced macrophage-stimulated invasion of 4T1 murine breast cancer cells. Together, these data suggested that cathepsin L was necessary for macrophage invasion and motility and for macrophage-mediated invasion of breast cancer cells. KGP94 and KGP207 treatment partially prevented IL-4-stimulated M0 to M2 differentiation of macrophages as determined by a decrease in the IL-4-induced expression of the M2 marker Arginase-1 upon drug treatment. Additionally, exogenous recombinant cathepsin L partially stimulated the expression of Arginase-1 in M0 macrophages. Together, these data suggest that cathepsin L plays a role in macrophage M0 to M2 differentiation. In conclusion, the novel cathepsin L/K inhibitors KGP94 and KGP207 prevented M0 to M2 differentiation, macrophage invasion, and macrophage-stimulated invasion of breast cancer cells. These data highlight the importance of cathepsin L in macrophage functions and suggest that cathepsin L inhibition is a viable approach for the treatment of tumors associated with high macrophage infiltration.

Citation Format: Samantha S. Dykes, Dietmar W. Siemann. The novel cathepsin L/K inhibitors KGP94 and KGP207 prevent M0 to M2 macrophage differentiation and macrophage-mediated pro-tumor functions [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 2952. doi:10.1158/1538-7445.AM2017-2952
Abstract 2953: Tumor-associated macrophages infiltration highly associated with PD-L1 expression in gastric cancer
Authors: Harada Kazuto , Estrella Jeannelyn S , Correa Arlene M , Ma Lang , Dong Xiaoquan , Xu Yan , Hofstette Wayne L , Sudo Kazuki , Onodera Hisashi , Suzuki Koyu , Suzuki Akihiro , Johnson Randy L , Zhenning Zhenning , Song Shumei , Ajani Jaffer A , Onodera Hisashi
Published: July 2017

Objectives: programmed death ligand 1 (PD-L1) is key protein for tumor to acquire the evacuation form T cell immune response and PD-L1 inhibitors are useful for an immune checkpoint blockade therapy. Tumor-associated macrophages (TAM) facilitate not only tumor cell progression, but also immunosuppression, which prevent tumor cell attack by T cell. Therefore, TAM is considered to be associated with PD-L1 expression, however it remain unclear in gastric cancer (GC).

Methods: We performed immunohistochemical staining of PD-L1, CD68 (pan-macrophage) and CD163 (M2 macrophage) in 217 GC sample tissue microarray. The area of CD68 and CD163 positive cells were evaluated by using by the Cytoplasmic V2.0 algorithm on the Aperio ImageScope software.

Results: 31 samples (14.3%) were positive for PD-L1 expression. The mean of rate for CD68 and CD163 positive area were 6.8±0.38% and 6.2±0.30%, respectively. CD163 positive macrophage infiltration is significantly more in diffuse type tumor than in intestinal type tumor (diffuse (n=111): 6.9%, intestinal (n=91): 5.3%, p=0.0056), but CD68 positive macrophage infiltration is similar in both type (p=0.38). Interestingly, PD-L1 expression tumor have significantly more CD68 and CD163 positive macrophage infiltration (CD68: PD-L1 negative 6.3%, positive 10.4%, p=0.0002, CD163: PD-L1 negative 5.7%, positive 9.2%, p<0.0001). PD-L1 expression and CD68 and CD163 positive area had no relationship between any clinicopathological features.

Conclusions: Macrophages infiltration in GC is highly associated with PD-L1 expression of tumor cells. M2 macrophage infiltration is significantly associated with diffused type of GAC. This result suggests that TAM

infiltration is associated with the immunosuppression through upregulating PD-L1 expression in GC cells. Thus, macrophages infiltration can serve as a potential therapeutic target.

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Citation Format: Kazuto Harada, Jeannelyn S Estrella, Arlene M Correa, Lang Ma, Xiaoquan Dong, Yan Xu, Wayne L Hofstette, Kazuki Sudo, Hisashi Onodera, Koyu Suzuki, Akihiro Suzuki, Randy L Johnson, Zhenning Zhenning, Shumei Song, Jaffer A Ajani, Hisashi Onodera. Tumor-associated macrophages infiltration highly associated with PD-L1 expression in gastric 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 2953. doi:10.1158/1538-7445.AM2017-2953
Abstract 2954: The role of hedgehog signaling in breast cancer progression through macrophage polarization
Authors: Hanna Ann , Shevde Lalita A.
Published: July 2017

The tumor microenvironment is comprised of stromal cells that constantly crosstalk with the cancer cells. Stromal cells, specifically tumor infiltrating immune cells, secrete cytokines and chemokines that regulate host responses to cancer through tumor-suppressing or tumor-promoting mechanisms. Macrophages within the tumor microenvironment are very plastic and can modulate different functions that promote tumor growth or tumor suppression, depending on their phenotype. Classically activated macrophages (M1) suppress tumor growth by mounting a pro-inflammatory immune response; while alternatively activated macrophages (M2) promote tumor progression by inducing an anti-inflammatory response which suppresses immune system function.

Hedgehog (Hh) signaling is essential for normal mammalian embryonic development by modulating vital functions such as cell proliferation, differentiation, and angiogenesis. Although the Hh signaling pathway is tightly controlled, it often is deregulated, thus promoting tumorigenesis and tumor progression. Aberrant Hh signaling has particularly been implicated in breast cancer progression and metastasis.

In this project, we investigate the role of Hh signaling in polarizing cancer associated macrophages toward the tumor-promoting M2 phenotype in a breast cancer model. We have discovered that using small molecule inhibitors to inhibit Hh signaling attenuates the cytokine profile associated with M2 macrophages, while adding recombinant Hh ligand potentiates it. Our data thus far supports a role for Hh signaling in alternatively polarizing macrophages. We hypothesize that Hh inhibitors will reduce the abundance of M2 macrophages and prevent metastasis.

Citation Format: Ann Hanna, Lalita A. Shevde. The role of hedgehog signaling in breast cancer progression through macrophage polarization [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 2954. doi:10.1158/1538-7445.AM2017-2954
Abstract 2955: Gr1-MDSCs and Tregs modulate the prostate cancer progression
Authors: Kumar Sanjay , Stokes James , Singh Udai , Gunn Karyn Scissum , Manne Upender , Ponnazhagan Selvarangan , Mishra Manoj K.
Published: July 2017

Myeloid-derived suppressor cells (MDSCs) and Regulatory T cells (Tregs) are the major components of immunosuppressive network that play a critical role in tumor immune evasion. The mechanisms by which tumors induce expansion of suppressive cells and the crosstalk between Gr1-MDSCs (Gr1+, CD11b+, F4/80+) and Tregs relative to TGF-β secretion remain incompletely defined. Prior studies have suggested that MDSCs may contribute to Treg recruitment in cancer. Herein, the goal of this investigation is to examine the role of TGF-β mediated generation of Tregs and Gr1-MDSCs during prostate cancer progression and clearance. To achieve this goal, matrigel system was used along with transgenic adenocarcinoma of mouse prostate (TRAMP-C1, C2 and C3 cell) in C57BL/6 mice. Interestingly, TRAMP-C3 cells are characterized as non-tumorigenic; however, TRAMP-C1 and TRAMP-C2 cells do form tumor. Mice were administered with serial log concentration of TRAMP (C1, C2 and C3) cells along with the matrigel. After three weeks, matrigels along with or without tumor were excised from tumor bearing mice, single cell suspensions was prepared and cells were flow analyzed for GR1-MDSCs and Tregs. Additionally, the TGF-β level was also estimated (colorimetric) in culture supernatant in context to MDSCs and Treg expansion. Our initial findings suggest that the expression of Gr1-MDSCs (Gr1+, CD11b+, F4/80+) and Foxp3+ Tregs increased in TRAMP-C1 and C2 as compared to TRAMP-C3 bearing C57BL/6 mice. These findings indicate that the modulation of Gr1-MDSCs and Tregs could help in tumor suppression.

Citation Format: Sanjay Kumar, James Stokes, Udai Singh, Karyn Scissum Gunn, Upender Manne, Selvarangan Ponnazhagan, Manoj K. Mishra. Gr1-MDSCs and Tregs modulate the prostate cancer progression [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 2955. doi:10.1158/1538-7445.AM2017-2955
Abstract 2963: Inhibition of stromal p38MAPK abrogates breast cancer metastasis
Authors: Stewart Sheila A. , Murali Bhavna , Ren Qiaho , Su Xinming , Flannagan Kevin , Luo Xianmin , Sponagel Jasmine , Fu Yujie , Alspach Elise , Leahy Kathlees , Faccio Roberta , Weilbaecher Kathleen , Monahan Joseph
Published: July 2017

Bone metastasis is a devastating and fatal complication of breast cancer for which we lack effective therapies. Thus, identifying therapies that effectively limit metastases will significantly reduce comorbidities and improve long-term survival. Recently we demonstrated that the p38MAPK pathway sustains the pro-tumorigenic senescence-associated secretory phenotype (SASP) and targeting this pathway limits the tumor-promoting capabilities of senescent cells and cancer-associated fibroblasts (CAFs). Because we found that a significant percentage of p38MAPK-dependent SASP factors are expressed in the stroma associated with breast cancer lesions, we asked whether targeting p38MAPK could limit primary and metastatic breast cancer growth. While p38MAPK inhibition modestly limited primary tumor growth, we found that its inhibition significantly reduced breast cancer bone metastases by specifically targeting the stromal compartment. Further, p38MAPK inhibition was as effective as paclitaxel at limiting tumor growth in the bone but in contrast to paclitaxel, which failed to protect from cancer-induced bone loss, p38MAPK inhibition also protected against devastating bone loss. This contrasts our p38MAPK approach from zoledronic acid, which limits bone loss but fails to slow tumor growth in already engrafted tumors. Analysis of the mechanism(s) responsible for this reduced metastasis suggests that p38MAPK inhibition targets reactive and/or senescent osteoblasts within the bones of animals harboring metastatic lesions. Because we find that senescent osteoblasts are present in human bone, we postulate that they promote metastatic outgrowth and thus p38MAPK inhibition limits the pro-metastatic activities of these cells. Finally, we will present recent data from our preclinical model that demonstrates that inhibition of the p38MAPK pathway can drastically reduce metastasis from the primary site. We propose that p38MAPK is an important stromal-specific therapy for breast cancer metastasis to the bone.

Citation Format: Sheila A. Stewart, Bhavna Murali, Qiaho Ren, Xinming Su, Kevin Flannagan, Xianmin Luo, Jasmine Sponagel, Yujie Fu, Elise Alspach, Kathlees Leahy, Roberta Faccio, Kathleen Weilbaecher, Joseph Monahan. Inhibition of stromal p38MAPK abrogates breast cancer metastasis [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 2963. doi:10.1158/1538-7445.AM2017-2963
Abstract 2981: Identification of circulating stroma-related biomarkers for pancreatic ductal adenocarcinoma
Authors: Resovi Andrea , Porcu Luca , Anastasia Alessia , Allavena Paola , Cappello Paola , Falanga Anna , Taraboletti Giulia , Bani Mariarosa , Belotti Dorina , Giavazzi Raffaella
Published: July 2017

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive epithelial malignancies characterized by a relevant amount of tumor stroma. It is usually diagnosed late and has limited treatment options. Plasmatic markers capable of detecting the disease in its early stages and monitoring treatment effectiveness might change the fate of PDAC patients. This study investigates the potential value of circulating stroma-related molecules as PDAC biomarkers capable to detect the disease and to monitor treatment response. Thirty-eight stroma-related candidate biomarkers were selected from published proteomic studies on PDAC. Levels of these molecules were tested by ELISA and Multiplex Luminex assays in a first cohort of plasma samples from PDAC patients and healthy subjects. Thirteen molecules -extracellular matrix proteins and proteolytic fragments, matrix-degrading enzymes and their inhibitors, growth factors and adhesion molecules-, were found to be differentially expressed in PDAC plasma compared to healthy subjects. A second analysis in an independent cohort of PDAC patients, chronic pancreatitis patients and healthy controls confirmed the previous findings and allowed restricting the number of biomarkers up-regulated in the plasma of PDAC patients. The plasma analysis of two genetically engineered mouse models of pancreatic cancer [Pdx-1-Cre;LSL-KrasG12D and Pdx-1-Cre;LSL-KrasG12D/+;LSL-Trp53R172H/+] revealed an up-regulation of four markers in association with PanIN development, indicating their potential role in the early diagnosis of pancreatic cancer. These markers were also elevated in the plasma of mice bearing patient derived orthotopic PDAC xenografts (PDAC-PDX), but not in a mouse model of caerulein-induced chronic pancreatitis. Their level was associated with PDAC-PDX tumor growth and response to gemcitabine combined with nab-paclitaxel. In conclusion this analysis has identified a panel of stroma-related potential biomarkers associated with tumor progression and drug response of PDAC.

Supported by Associazione Italiana per la Ricerca sul Cancro (AIRC 5 per mille grant n. 12182) and Fondazione “Eugenio Morandi” ONLUS per lo studio e la cura dei tumori del pancreas.

Citation Format: Andrea Resovi, Luca Porcu, Alessia Anastasia, Paola Allavena, Paola Cappello, Anna Falanga, Giulia Taraboletti, Mariarosa Bani, Dorina Belotti, Raffaella Giavazzi. Identification of circulating stroma-related biomarkers for pancreatic ductal adenocarcinoma [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 2981. doi:10.1158/1538-7445.AM2017-2981
Abstract 2997: The tetravalent bispecific antibody AFM13 engages and primes innate immune cells for anti-cancer immunity
Authors: Pahl Jens , Koch Joachim , Reusch Uwe , Gantke Thorsten , Cerwenka Adelheid , Treder Martin
Published: July 2017

AFM13 is a tetravalent bispecific antibody with bivalent binding to both CD30 and CD16A. It has been shown to engage NK-cells through CD16A with high affinity and specificity, resulting in strong NK-cell cytotoxicity, and is currently being tested in Phase 2 monotherapy and in combination with pembrolizumab in Phase 1b clinical trials. We have previously shown that AFM13-dependent activation of NK-cell cytotoxicity towards CD30+ tumor cells is more pronounced than that of anti-CD30 mAbs. In addition, AFM13 enhances NK-cell sensitivity to low doses of IL-2 and IL-15, leading to an increased NK-cell proliferative potential. Here, we have extended the panel of phenotypic markers on NK-cells that are modulated after exposure to CD30+ tumor cells in the presence of AFM13. Targeting some of these markers may enable the development of novel combination therapies. Moreover, we have analyzed the kinetics of NK-cell responses to AFM13 exposure. Even though short-term exposure to AFM13 significantly enhanced NK-cell cytotoxicity, long-term exposure led to a partial, transient functionally exhausted phenotype in vitro, which could be fully restored by cytokine stimulation for several days in the absence of AFM13. Importantly, these recovered cells displayed high cytotoxicity towards CD30+ target cells in the presence of AFM13. Interestingly, the transient NK-cell exhaustion was not related to the expression of typical exhaustion markers or insufficient levels of perforin and granzyme. These data may warrant the development of novel metronomic application regimens of AFM13. Further studies imply that immune cells other than NK-cells are able to inhibit growth of CD30+ tumor cells in an AFM13-dependent manner. This appears to be strictly dependent on CD16A and a specific cytokine milieu. Taken together, AFM13 specifically enhances the cytotoxic, proliferative and cytokine-producing potential of NK-cells, parameters that can be utilized to monitor NK-cell responses during AFM13 therapy. Moreover, based on our data, engagement of CD16A+ cells to the tumor site might enable several innate immune effector functions within the tumor microenvironment for synergistic anti-tumor activity.

Citation Format: Jens Pahl, Joachim Koch, Uwe Reusch, Thorsten Gantke, Adelheid Cerwenka, Martin Treder. The tetravalent bispecific antibody AFM13 engages and primes innate immune cells for anti-cancer immunity [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 2997. doi:10.1158/1538-7445.AM2017-2997