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Pesquisar educação na contemporaneidade exige, acima de tudo, respeito e comprometimento com o ambiente educacional, com a experiência dos sujeitos envolvidos; e, no caso desta pesquisa, com a experiência vinculada à prática docente. Entender o universo educacional em suas diversidades, valorizar as diferenças e compreender que, para o ensinar e o aprender não há regras fechadas, engessadas, fórmulas prontas e acabadas, mas sim a capacidade da escuta atenta, do diálogo, da formação docente, que alia teoria e prática em busca de novas aprendizagens e novos saberes docentes. Para a construção desta pesquisa, estabeleceu-se como questão problematizadora: Em que medida os professores de classes regulares constroem seus saberes docentes frente à educação inclusiva? Partindo do ponto de que esses saberes docentes são, eminentemente, formativos e entendidos tanto como a formação inicial quanto a formação continuada. O objetivo geral desta investigação é: desvelar, a partir das histórias narradas por professores de uma escola regular da rede municipal de Aparecida de Goiânia, como se dá o processo formativo para a construção de saberes frente à educação inclusiva. E, os específicos são: conhecer as vivências dos professores e suas experiências com a inclusão; analisar o processo de formação docente, inicial e continuada, vivida por professores frente à temática da educação inclusiva; identificar, junto aos professores, a partir daquilo que eles já experienciaram em sua trajetória educacional, quais fatores interferem, facilitam e\ou dificultam na realização de práticas inclusivas; promover a ação reflexiva dos professores no âmbito da educação inclusiva baseada na memória de suas próprias histórias de vida. A metodologia utilizada nesse processo foi a abordagem qualitativa com aproximações fenomenológicas. Os autores que sustentaram a proposta foram: Mazzotta (1998); Mantoan (2003; 2017); Reis (2013), Santos e Reis (2016); Pietro (2006); Teixeira (2002), Freire (1996); Nóvoa (2002; 2007; 2009); Tardif (2012); Tardif, Lessard (2007), dentre outros. O texto foi organizado em cinco seções: na primeira apresenta-se a trajetória acadêmico profissional da pesquisadora enfatizando as razões da escolha do tema; a segunda traça o processo metodológico da pesquisa e apresenta a caracterização da escola e dos sujeitos participantes; a terceira seção destaca os principais conceitos que sustentam este estudo, bem como o histórico das políticas públicas inclusivas; a quarta apresenta os dados e análise da pesquisa a partir das narrativas dos participantes e a quinta apresenta as considerações finais. A pesquisa apontou a necessidade de um maior empenho e compromisso por parte do poder público na implementação de políticas públicas que favoreçam a educação inclusiva em suas várias dimensões, principalmente a formação de professores. Comprovou-se, ainda, que a construção dos saberes docentes perpassa a formação inicial e os conhecimentos teóricos, porém, é na prática que essa formação se consolida, é na experiência e na troca delas que os professores vão cada vez mais adquirindo uma bagagem formativa para lidar com as especificidades que vão surgindo. Researching education in contemporary times demands, above all, respect and commitment to the educational environment, to the experience of the individuals involved; and, in the case of this research, to the experience linked to teaching practice. Understanding the educational universe in its diversities, valuing differences, and understanding that for teaching and learning there are no closed, cast rules, or ready and finished formulas, but rather, the capacity for attentive listening, dialogue, and teacher education that allies theory and practice in search of new learning and new teaching knowledge. For the construction of this research, it was established as a problematizing question: To what extent do teachers of regular classrooms build their teaching knowledge facing inclusive education? Starting from the point that these teaching knowledges are eminently formative and understood both as initial training and continuing education. The overall goal of this research is: to unveil, from the stories told by teachers of a regular school in the municipal system of Aparecida de Goiânia, how the formation process take place for the construction of knowledge regarding inclusive education. And, the specific ones are: to know the experiences of teachers and their experiences with inclusion; to analyze the process of teacher training, both initial and continuing, experienced by teachers in relation to inclusive education; to identify, together with the teachers, based on what they have experienced in their educational career, which factors interfere, facilitate, and/or hinder the implementation of inclusive practices; to promote the reflective action of teachers in inclusive education based on the memory of their own life stories. The methodology used in this process was the qualitative approach with phenomenological approaches. The authors who supported the proposal were: Mazzotta (1998); Mantoan (2003; 2017); Reis (2013), Santos and Reis (2016); Pietro (2006); Teixeira (2002), Freire (1996); Nóvoa (2002; 2007; 2009); Tardif (2012); Tardif, Lessard (2007), among others. The text was organized into four chapters: the first presents the academic and professional trajectory of the researcher emphasizing the reasons for choosing the theme; the second outlines the methodological process of the research and presents the characterization of the school and the participating individuals; the third chapter highlights the main concepts that support this study, as well as the history of inclusive public policies; and, the last chapter presents the data and research analysis from the participants' narratives. The research pointed out the need for greater effort and commitment by the government in the implementation of public policies that favor inclusive education in its various dimensions, especially teacher training. It was also proven that the construction of teachers' knowledge goes through initial education and theoretical knowledge, but it is in practice that this education is consolidated, it is in experience and in the exchange of experience that teachers increasingly acquire a formative baggage to deal with the specificities that arise.

Leukemic inhibitory factor (LIF) is an IL-6 family member involved in embryonic stem cell maintenance and protection from maternal immune-attack. Emerging evidence indicates that LIF biology is hijacked by some tumors to promote immunosuppressive tumor associated macrophages, cancer initiating cells and epithelial-mesenchymal transition (EMT). In patients treated with anti-PD-1/anti-PD-L1 antibodies (IO), baseline high LIF expression correlated with poor outcome (Loriot et al. 2021). In preclinical models, LIF blockade has separately been reported to sensitize tumors to IO and to chemotherapy (chemo), but the impact of LIF blockade on chemo/IO combination activity is unknown. We therefore evaluated the hypothesis that LIF blockade sensitizes tumors to chemo/IO combinations. AZD0171 is a monoclonal antibody that binds to LIF and prevents signaling through LIF receptor. A murine surrogate of AZD0171 (mAZD0171) was generated and tested in vivo using three syngeneic murine cancer models, and AZD0171 was tested in patient derived xenograft models. Monotherapy mAZD0171 induced tumor gene expression changes consistent with its proposed mechanism of action in the syngeneic CT26 colon tumor model; including elevated CD8 attracting chemokines (CXCL9/10), cDC1 transcription factor (Batf3) and reduced stem cell (CD44), EMT (CY61) and hypoxic/angiogenic signatures, including CXCL12. Flow cytometry analysis highlighted changes consistent with suppressive macrophage re-education (reduced CD206/elevated MHCII). mAZD0171 sensitized mouse tumors to both IO and chemo. mAZD0171 + anti-PD-L1 treatment reversed CD8 T cell exclusion in the D2A1-m2 model; a LIF expressing cancer associated fibroblast rich, anti-PD-L1 resistant mouse mammary tumor syngeneic model (Jungwirth et al. 2018). mAZD0171 + anti-PD-L1 treatment further inhibited D2A1-m2 growth versus (vs) anti-PD-L1 treatment alone. In a non-small cell lung carcinoma patient derived xenograft model, AZD0171 improved the tumor growth inhibitory effect of Carboplatin + Paclitaxel. The triplet combination of mAZD0171 plus chemo and IO had the most marked impact on both tumor microenvironment and tumor growth. mAZD0171 addition to oxaliplatin (OHP) or Docetaxel (DTX) chemotherapy plus anti-PD-L1 significantly elevated CD8 abundance and granzyme B expression in MC38 colon tumors. mAZD0171 + anti-PD-L1 + OHP or DTX improved inhibition of MC38 tumor growth (63% for OHP triplet vs 34% for anti-PD-L1 + OHP; 49% for DTX triplet vs 36% for anti-PD-L1 + DTX) and extended median survival times (P Citation Format: Juliana Candido, Gozde Kar, Bairu Zhang, Grace Opoku-Ansah, Amit Grover, Elizabeth A. Kuczynski, Tim Slidel, Doug Palmer, Robert W. Wilkinsom, Nadia Luheshi, Jim Eyles. AZD0171 (anti-LIF) combines productively with chemotherapy and anti-PD-L1 in mouse models of cancer [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 1293.

Background & Aims Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC. Methods We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient–derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. Results Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency. Conclusions Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy., Graphical abstract

Emerging evidence suggests that cancer cell metabolism can be regulated by cancer-associated fibroblasts (CAFs), but the mechanisms are poorly defined. Here we show that CAFs regulate malignant cell metabolism through pathways under the control of FAK. In breast and pancreatic cancer patients we find that low FAK expression, specifically in the stromal compartment, predicts reduced overall survival. In mice, depletion of FAK in a subpopulation of CAFs regulates paracrine signals that increase malignant cell glycolysis and tumour growth. Proteomic and phosphoproteomic analysis in our mouse model identifies metabolic alterations which are reflected at the transcriptomic level in patients with low stromal FAK. Mechanistically we demonstrate that FAK-depletion in CAFs increases chemokine production, which via CCR1/CCR2 on cancer cells, activate protein kinase A, leading to enhanced malignant cell glycolysis. Our data uncover mechanisms whereby stromal fibroblasts regulate cancer cell metabolism independent of genetic mutations in cancer cells., Cancer associated fibroblasts (CAFs) have been suggested to regulate cancer cell metabolism, but the mechanisms are not completely elucidated. Here, the authors show that low FAK expression in stromal cells correlates with poor prognosis in breast and pancreatic cancer patients and that FAK-silencing in CAFs promotes tumourigenesis by the paracrine regulation of cancer cell metabolism.

In tumor engineering, 3D approaches are used to model components of the tumor microenvironment and to test new treatments. Pancreatic cancers are a cancer of substantial unmet need and survival rates are lower compared to any other cancer. Bioengineering techniques are increasingly applied to understand the unique biology of pancreatic tumors and to design patient-specific models. Here we summarize how extracellular and cellular elements of the pancreatic tumor microenvironment and their interactions have been studied in 3D cell cultures. We review selected clinical trials, assess the benefits of therapies interfering with the tumor microenvironment and address their limitations and future perspectives.

Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting the DNA damage response (DDR) and replication stress. We show that patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum and PARP inhibitor therapy in vitro and in vivo. We generated a novel signature of replication stress with potential clinical utility in predicting response to ATR and WEE1 inhibitor treatment. Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy.Abstract FigureSTATEMENT OF SIGNIFICANCEWe define therapeutic strategies that target subgroups of PC using novel signatures of DNA damage response deficiency and replication stress. This potentially offers patients with DNA repair defects therapeutic options outside standard of care platinum chemotherapy and is being tested in clinical trials on the Precision-Panc platform.

B cells are salient features of pancreatic ductal adenocarcinoma (PDAC) tumors, yet their role in this disease remains controversial. Murine studies have indicated a protumoral role for B cells, whereas clinical data show tumor-infiltrating B cells are a positive prognostic factor, both in PDAC and other cancers. This disparity needs to be clarified in order to develop effective immunotherapies. In this study, we provide new evidence that reconcile human and mouse data and highlight the importance of using relevant preclinical tumor models when assessing B cell function. We compared B cell infiltration and activation in both a genetic model of murine PDAC (KPC mouse) and an injectable orthotopic model. A pronounced B cell infiltrate was only observed in KPC tumors and correlated with T cell infiltration, mirroring human disease. In contrast, orthotopic tumors exhibited a relative paucity of B cells. Accordingly, KPC-derived B cells displayed markers of B cell activation (germinal center entry, B cell memory, and plasma cell differentiation) accompanied by significant intratumoral immunoglobulin deposition, a feature markedly weaker in orthotopic tumors. Tumor immunoglobulins, however, did not appear to form immune complexes. Furthermore, in contrast to the current paradigm that tumor B cells are immunosuppressive, when assessed as a bulk population, intratumoral B cells upregulated several proinflammatory and immunostimulatory genes, a distinctly different phenotype to that of splenic-derived B cells; further highlighting the importance of studying tumor-infiltrating B cells over B cells from secondary lymphoid organs. In agreement with the current literature, genetic deletion of B cells (μMT mice) resulted in reduced orthotopic tumor growth, however, this was not recapitulated by treatment with B-cell-depleting anti-CD20 antibody and, more importantly, was not observed in anti-CD20-treated KPC mice. This suggests the result from B cell deficient mice might be caused by their altered immune system, rather than lack of B cells. Therefore, our data indicate B cells do not favor tumor progression. In conclusion, our analysis of relevant preclinical models shows B cells to be active members of the tumor microenvironment, producing immunostimulatory factors that might support the adaptive antitumor immune response, as suggested by human PDAC studies.

Summary CXCR2 has been suggested to have both tumor-promoting and tumor-suppressive properties. Here we show that CXCR2 signaling is upregulated in human pancreatic cancer, predominantly in neutrophil/myeloid-derived suppressor cells, but rarely in tumor cells. Genetic ablation or inhibition of CXCR2 abrogated metastasis, but only inhibition slowed tumorigenesis. Depletion of neutrophils/myeloid-derived suppressor cells also suppressed metastasis suggesting a key role for CXCR2 in establishing and maintaining the metastatic niche. Importantly, loss or inhibition of CXCR2 improved T cell entry, and combined inhibition of CXCR2 and PD1 in mice with established disease significantly extended survival. We show that CXCR2 signaling in the myeloid compartment can promote pancreatic tumorigenesis and is required for pancreatic cancer metastasis, making it an excellent therapeutic target., Graphical Abstract, Highlights • CXCR2 signaling is upregulated in myeloid cells in human pancreatic cancer • Cxcr2 loss reduces metastasis and inhibition prolongs tumor-free survival in mice • Neutrophils/MDSCs play a key role in the establishment of the metastatic niche • CXCR2 inhibition enhances T cell entry and confers sensitivity to anti-PD1 therapy, Steele et al. show that CXCR2 is important in immune modulation of pancreatic cancer and that inhibition of CXCR2 reduces metastasis and improves response to gemcitabine and anti-PD1. Peptide inhibitor, but not germline deletion of Cxcr2, improved survival, revealing differential effects in early and late tumors.

Tissue engineering technologies have produced controllable and reproducible three-dimensional (3D) models that mimic the architecture and complexity of native tissues. In particular cell biology-based research is driven by the development of cell culture platforms and techniques that allow the analysis of cells cultured in 3D. Here we summarize several easy-to-follow methods for the characterization of cells that have been encapsulated and grown in hydrogels to measure their cell viability, metabolic activity, and mechanical properties of cell-containing hydrogels. We also describe an enzymatic approach for the digestion of cell-containing hydrogels and cell recovery thereby maintaining high cell viability for subsequent analysis.

The majority of human pancreatic cancers have activating mutations in the KRAS proto-oncogene. These mutations result in increased activity of the NF-κB pathway and the subsequent constitutive production of proinflammatory cytokines. Here, we show that inhibitor of κB kinase 2 (Ikk2), a component of the canonical NF-κB signaling pathway, synergizes with basal Notch signaling to upregulate transcription of primary Notch target genes, resulting in suppression of antiinflammatory protein expression and promotion of pancreatic carcinogenesis in mice. We found that in the Kras(G12D)Pdx1-cre mouse model of pancreatic cancer, genetic deletion of Ikk2 in initiated pre-malignant epithelial cells substantially delayed pancreatic oncogenesis and resulted in downregulation of the classical Notch target genes Hes1 and Hey1. Tnf-α stimulated canonical NF-κB signaling and, in collaboration with basal Notch signals, induced optimal expression of Notch targets. Mechanistically, Tnf-α stimulation resulted in phosphorylation of histone H3 at the Hes1 promoter, and this signal was lost with Ikk2 deletion. Hes1 suppresses expression of Pparg, which encodes the antiinflammatory nuclear receptor Pparγ. Thus, crosstalk between Tnf-α/Ikk2 and Notch sustains the intrinsic inflammatory profile of transformed cells. These findings reveal what we believe to be a novel interaction between oncogenic inflammation and a major cell fate pathway and show how these pathways can cooperate to promote cancer progression.

AZD9150, a gen2.5 antisense oligonucleotide (ASO) targeting human STAT3, has improved drug-like properties compared to previous generation ASO therapeutics, including increased stability and resistance to nucleases, reduced proinflammatory effects, and enhanced potency. We have previously reported that in tumors, STAT3 ASOs are taken up preferentially in stromal and immune cells of the tumor microenvironment (TME). Since AZD9150 is selective for human STAT3, we used a surrogate ASO (muSTAT3 ASO) to explore the pharmacodynamics of ASO-mediated STAT3 inhibition in syngeneic and genetically engineered mouse (GEM) tumor models, focusing on effects in the TME. In mice bearing subcutaneous CT-26 tumors, treatment with muSTAT3 ASO at 50 mg/kg, s.c., on a qdx5/wk schedule decreased STAT3 levels in immune cell subsets in the tumor and in circulating leukocytes by 40 - 60%, similar to the decrease in STAT3 achievable in circulating leukocytes in human patients after AZD9150 treatment. In a Nanostring analysis (nCounter mouse immunology panel) of CT-26 tumors from muSTAT3 ASO treated mice, CD163 (M2 immune suppressive macrophage marker) was the gene most consistently and significantly downregulated, by an average of 84% in three independent experiments, and was confirmed by immunohistochemistry (IHC). Flow cytometry analysis of myeloid subpopulations - tumor associated macrophages (F4/80+ TAMs), monocytic myeloid derived suppressor cells, and granulocytic cells - showed a decrease in TAMs averaging 69% across three independent experiments. The analysis was extended to include IHC for arginase (Arg, a marker of functional immune suppression activity). Subpopulations of cells identified included Arg+, CD163+, and Arg+CD163+. Treatment with muSTAT3 ASO decreased these populations by 79%, 88% and 97% respectively, compared to control treatment. These populations were also analyzed in two GEM tumor models - the KPC pancreatic cancer model, and a PTEN -/- prostate cancer model - which have a TME more representative of that found in tumors in the clinic. While the specific changes varied across the models, likely reflecting differences in TME makeup, a reduction in immune suppressive cell populations was present in both GEM models, including a decrease in CD163+ cells of 79% (along with modest antitumor activity) in the PTEN -/- prostate model after muSTAT3 ASO treatment. These results indicate that selective STAT3 inhibition can reduce immune suppressive cell populations in the TME, and suggest that STAT3 inhibition has the potential to enhance the antitumor activity of T-cell targeted therapies, such as those targeting the PD1-PDL1 axis. In support of this hypothesis, we observed that addition of muSTAT3 ASO to anti-PD-L1 Ab treatment significantly enhanced the antitumor activity of PD-L1 Ab treatment in two subcutaneous syngeneic tumor models, CT-26 and A20. Citation Format: Rich Woessner, Vasu Sah, Patricia McCoon, Shaun Grosskurth, Nanhua Deng, Rachel DuPont, Deborah Lawson, Lourdes Pablo, Corinne Reimer, Marco A. De Velasco, Hirotsugu Uemura, Juliana Candido, Paul Lyne. Inhibition of STAT3 by antisense oligonucleotide treatment decreases the immune suppressive tumor microenvironment in syngeneic and GEM tumor models [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 3684. doi:10.1158/1538-7445.AM2017-3684

e14612 Background: Amphiregulin is an EGFR ligand which contributes to many of the hallmarks of cancer including angiogenesis and metastasis. Recent studies demonstrate that Amphiregulin promotes an immunosuppressive tumour microenvironment by modulating the expression of T regulatory cells. Although its expression has been described on a variety of immune cells and tumour cells, there is no published data on Amphiregulin expression by monocytes to date. Previous work by this group has demonstrated that monocytes from non-small cell lung cancer patients are phenotypically and genomically different compared to healthy volunteers. This study aims to further investigate the differing genomic profile of monocytes in non-small cell lung cancer. Methods: Gene expression profiling was performed on CD14++CD16- (classical) monocytes of newly diagnosed patients with Stage IV non-small cell lung cancer (n = 6) and age-matched healthy volunteers (n = 6) using Affymetrix Human U133 Plus 2.0 array. Validation of differentially expressed genes of interest (including Amphiregulin) at mRNA level was performed using real-time PCR with TaqMan gene expression assays on independent patient (n = 6) and healthy volunteer cohorts (n = 6). Differential protein expression of Amphiregulin from the monocytes of healthy volunteers (n = 3) and non-small cell lung cancer patients (n = 3) was validated using ELISA technology. Results: Amphiregulin is one of the most upregulated genes on classical monocytes in non-small cell lung cancer patients compared to healthy volunteers (p = 0.001). The increased expression of Amphiregulin was confirmed on qPCR validation in an independent cohort of non-small cell lung cancer patients (p < 0.05) as well as in the original group (p < 0.001). Amphiregulin was also found to be increased at protein level on CD14++CD16- monocytes of non-small cell lung cancer compared to healthy volunteers. (p = 0.37). Conclusions: Amphiregulin is overexpressed by classical monocytes in non-small cell lung cancer and this over-expression is confirmed at mRNA and protein level. The production of Amphiregulin has not been ascribed to human monocytes in non-small cell lung cancer making its overexpression in this study a unique finding.

Solid tumors consist of neoplastic cells, non-malignant stromal cells, and migratory hematopoietic cells. Complex interactions between the cell types in this microenvironment regulate tumor growth, progression, metastasis, and angiogenesis. The cells and mediators of inflammation form a major part of the epithelial tumor microenvironment. In some cancers, inflammatory conditions precede development of malignancy; in others, oncogenic change drives a tumor-promoting inflammatory milieu. Whatever its origin, this “smoldering” inflammation aids proliferation and survival of malignant cells, stimulates angiogenesis and metastasis, subverts adaptive immunity, and alters response to hormones and chemotherapy. Cytokines are major mediators of communication between cells in the inflammatory tumor microenvironment. It is known that neoplastic cells often over-express proinflammatory mediators including proteases, eicosanoids, cytokines, and chemokines. Several cytokines such as macrophage migratory inhibitory factor (MIF), TNF-α, IL-6, IL-17, IL-12, IL-23, IL-10, and TGF-β have been linked with both experimental and human cancers and can either promote or inhibit tumor development. MIF is a major cytokine in many cancers and there is evidence that the cytokine is produced by both malignant cells and infiltrating leukocytes. In this article we will discuss the role of cancer-associated inflammation and the particular role of MIF in malignant disease.

11071 Background: Monocytes are intrinsic members of the innate immune system and play an important role in immunity and inflammation. Human monocytes are subdivided into three populations depending on cell surface CD14 and CD16 expression: Classical (CD14++ CD16-), intermediate (CD14+CD16- ) and non-classical (CD14- CD16++). These populations have diverse functions and have been postulated to play both anti and pro-inflammatory roles in a variety of diseases including atherosclerosis, sarcoidosis and other rheumatological conditions. However, the prevalent monocyte populations in cancer have not as yet been identified. We aim to define the prevalent monocyte populations in non-small cell lung cancer as well as further characterising them using flow cytometry and Affymetrix technology. Methods: Blood was obtained from 24 newly diagnosed patients with advanced non-small cell lung cancer and 12 age matched healthy donors. Monocyte subpopulations were sorted using flow cytometry. Gene expression profiling was performed using Affymetrix Human U133 Plus 2.0 array. Results: The classical (CD14++CD16-) monocyte population is significantly increased in non-small cell lung cancer patients when compared to healthy donors (p+CD16+) and non-classical (CD14- CD16++) populations are unchanged. Analysis of the gene expression profile of the classical monocyte subset identified 265 up-regulated and 261 down-regulated genes in cancer patients compared to healthy donors (p2). These genes were assigned to biological processes with gene ontology annotation. Functional annotation reveals a strong association of regulated genes with the G.O term “inflammation” (p=0.009). Among these inflammatory genes are a cluster of chemokines including CXCL2 and CXCR4. Conclusions: By identifying the prevalent monocyte subsets as well as characterising their function in cancer, there is the potential to target detrimental effects and promote their beneficial functions. As such, monocytes subsets may be used as a possible therapeutic target.

Abstract 1657 Introduction: In vitro stimulation of macrophages can produce classically activated (M1) or alternatively activated (M2) phenotypes. In various solid tumors, tumor associated macrophages (TAM) have been described as approximating an M2 phenotype and are potentially pro-tumoral. We are testing the hypothesis that TAM play an important role in the progression of NHL, and might therefore constitute a rational and effective therapeutic target. Our aim is to investigate tumor-macrophage interactions by manipulating macrophage numbers and phenotype in a transplantable mouse lymphoma. Experimental approach: We used as a model system B-cell lymphoma arising in Em-myc/bcl-2 transgenic mice, which, when intravenously injected into C57BL/6 mice produces disseminated lymphoma. Lymphoma growth was assessed by measuring lymph node weight, and cross-sectional tumor area in tissue sections, as well as by flow cytometry. Gene expression changes were determined by real-time PCR in whole lymph nodes or sorted macrophages in control mice or mice with lymphoma, and following interventions to manipulate macrophage populations. Changes to the cellular composition of the immune microenvironment were assessed by flow cytometry of lymph nodes and blood, and by immunohistochemistry. Methods and results: The consequences of widespread monocyte and macrophage depletion were first investigated using intravenous injection of the liposome-encapsulated bisphosphonate, sodium clodronate (Liposomal Clodronate, LC). Intravenous LC successfully depleted macrophages in the bone marrow, lymph nodes, liver and spleen, and correlated with significantly reduced lymphoma mass by weight and flow cytometry, compared to controls (p=0.0058). Next we examined MaFIA mice (Macrophage Fas Induced Apoptosis, Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6) that combine the expression of an enhanced green fluorescent protein on cells with the macrophage-specific csf-1 receptor, with a drug (dimerizer, AP20187) inducible suicide gene leading to Fas-mediated apoptosis in resting and cycling cells of the macrophage lineage. We observed the effects of this non-pharmacological method of macrophage depletion on lymphoma progression. Lymph node macrophage numbers are not reduced in normal healthy mice treated with the dimerizer, but were significantly reduced in the infiltrated lymph nodes of mice with lymphoma given the dimerizer. This was associated with a reduced lymphoma mass (p=0.044). Subsequent studies employed adoptive transfer of syngeneic bone marrow derived macrophages (BMDM), in vitro polarized to M1, M2 and model-specific TAM phenotypes; the purpose being to test whether supplementing the TAM population might invigorate lymphoma growth, and whether administering M1 phenotype macrophages might lead to anti-tumor effects. Adoptive transfer of M1-polarized BMDM attenuated lymphoma growth, and transfer of TAM augmented lymphoma growth (p=0.0003). Having gathered evidence that reducing TAM numbers might slow lymphoma growth, we explored a novel approach to targeting this macrophage sub-population in lymphoma, with translational potential for human studies. More specific targeting of TAM, and relative sparing of physiological monocytes and resident tissue macrophages, was attempted by targeting the CSF1 receptor with an orally available highly selective small molecule inhibitor. Current literature suggests this might reduce both monocyte trafficking to areas of malignancy, and maturation of monocytes to TAM. This strategy was used against growing lymphomas, resulting in an attenuated TAM population (p=0.0009) and reduced lymphoma mass (p=0.0036), whilst not depleting the circulating monocyte pool. In vitro cytotoxicity studies confirmed that neither LC, nor the CSF1R inhibitor nor AP20187 dimerizer caused significant direct lymphoma cell death, confirming that the observed in vivo attenuation of lymphoma growth likely resulted from indirect phenomena, such as TAM depletion. Conclusion: By evaluating a variety of pharmacological, genetic and cellular therapy strategies we have generated a strong body of evidence supporting a causal relationship between TAM abundance and lymphoma progression in this model. These data support the hypothesis that targeting TAM may well provide an attractive therapeutic opportunity to improve outcome in human B-cell lymphomas. Disclosures: Hagemann: Astra Zeneca and the Medical Research Council: In receipt of a joint studentship with Astra Zeneca and the UK Medical Research Council. Gribben:Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

Simple Summary Kallikrein-related peptidases have tumour-biological roles and are dysregulated in many cancers. Only a few studies have reported their upregulation in pancreatic cancer and linked them to poor prognosis. By interrogating publicly available and our own datasets, we studied their expression in patient-derived tissues and pancreatic cancer cells. We found several kallikrein-related peptidases that were upregulated, in particular kallikrein-related peptidase 6 at the forefront of the tumour area. We then tested the effect of a kallikrein-related peptidase 6 inhibitor on cancer cell functions. Because the majority of patients present with inoperable disease, a targeted therapeutic intervention may have a positive impact on the survival of this patient population. Abstract As cancer-associated factors, kallikrein-related peptidases (KLKs) are components of the tumour microenvironment, which represents a rich substrate repertoire, and considered attractive targets for the development of novel treatments. Standard-of-care therapy of pancreatic cancer shows unsatisfactory results, indicating the need for alternative therapeutic approaches. We aimed to investigate the expression of KLKs in pancreatic cancer and to inhibit the function of KLK6 in pancreatic cancer cells. KLK6, KLK7, KLK8, KLK10 and KLK11 were coexpressed and upregulated in tissues from pancreatic cancer patients compared to normal pancreas. Their high expression levels correlated with each other and were linked to shorter survival compared to low KLK levels. We then validated KLK6 mRNA and protein expression in patient-derived tissues and pancreatic cancer cells. Coexpression of KLK6 with KRT19, αSMA or CD68 was independent of tumour stage, while KLK6 was coexpressed with KRT19 and CD68 in the invasive tumour area. High KLK6 levels in tumour and CD68+ cells were linked to shorter survival. KLK6 inhibition reduced KLK6 mRNA expression, cell metabolic activity and KLK6 secretion and increased the secretion of other serine and aspartic lysosomal proteases. The association of high KLK levels and poor prognosis suggests that inhibiting KLKs may be a therapeutic strategy for precision medicine.