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C. Mascaux
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MS 26 - Re-Modeling Microenvironment Mimicking Human Cancer (ID 548)
- Event: WCLC 2017
- Type: Mini Symposium
- Track: Biology/Pathology
- Presentations: 5
- Moderators:P. Yang, C. Mascaux
- Coordinates: 10/18/2017, 14:30 - 16:15, Room 502
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MS 26.01 - Cancer Stem Cell (ID 7764)
14:30 - 14:50 | Presenting Author(s): Hideyuki Saya
- Abstract
- Presentation
Abstract:
Cancer stem cells (CSCs) are a subset of tumor cells that are responsible for initiating and maintaining the disease. In the clinical point of view, the most important characteristics of CSCs include their resistance to various therapeutic interventions[1)]. However, the underlying mechanisms of the resistance remain unclear. CD44 has been identified as a cell surface marker associated with cancer stem cells (CSCs) in several types of epithelial tumor. We have recently found that expression of CD44, in particular variant forms of CD44 (CD44v), contributes to the defense against reactive oxygen species (ROS) by promoting the synthesis of reduced gluathione (GSH), a primary intracellular antioxidant. CD44v interacts with and stabilizes xCT, a subunit of a glutamate-cystine transporter, and thereby promotes the uptake of cystine for GSH synthesis[2)]. Therefore, ablation of CD44 reduced GSH levels and increased ROS levels, leading to suppression of tumor growth and metastasis in both transgenic and xenograft tumor models[3,4)]. Our findings reveal a novel function for CD44v in protection of CSCs from high levels of ROS in the tumor microenvironment[5)]. Expression of CD44v and xCT is associated with tumorigenesis and therapeutic resistance[6,7)]. Based on these preclinical findings, we conducted clinical trials using an xCT inhibitor, sulfasalazine, for cancer patients having advanced gastric cancer and lung cancer. The clinical trials for gastric cancers revealed that sulfasalazine treatment reduceed the number of CD44v-positive cells in post-treatment tumor tissue[8,9)]. In terms of the clinical trial for lung cancer, chemotherapy-naive patients with advanced non-squamous nonsmall cell lung cancer were enrolled in a dose-escalation study (standard 3 + 3 design) of SASP in combination with cisplatin and pemetrexed[10)]. Fifteen patients were enrolled in the study and dose-limiting toxicity was observed in one of six patients at a SASP dose of 1.5 g/day, two of five patients at 3 g/day, and two of three patients at 4.5 g/day. The maximum tolerated dose was thus 3 g/day, and the recommended dose was 1.5 g/day. The overall response rate was 26.7% and median progression-free survival (PFS) was 11.7 months, much longer than that for cisplatin–pemetrexed alone in previous studies. It is possible that the prolonged PFS was due to a sulfasalazine-induced reduction in the number of CD44v-positive CSCs that are the origin of disease recurrence. References: 1) Sugihara E and Saya H: Complexity of cancer stem cells (Review article). Int J Cancer 132:1249-1259, 2013 2) Ishimoto T, Nagano O, Yae T, Tamada M, Motohara T, Oshima H, Oshima M, Ikeda T, Asaba R, Yagi H, Masuko T, Shimizu T, Ishikawa T, Kai K, Takahashi E, Imamura Y, Baba Y, Ohmura M, Suematsu M, Baba H and Saya H: CD44 variant regulates redox status in cancer cells by stabilizing the xCT subunit of system xc- and thereby promotes tumor growth. Cancer Cell 19: 387-400, 2011 3) Yae T, Tsuchihashi K, Ishimoto T, Motohara T, Yoshikawa M, Yoshida GJ, Wada T, Masuko T, Mogushi K, Tanaka H, Osawa T, Kanki Y, Minami T, Aburatani H, Ohmura M, Kubo A, Suematsu M, Takahashi K, Saya H and Nagano O: Alternative splicing of CD44 mRNA by ESRP1 enhances lung colonization of metastatic cancer cell. Nat Commun 3: 883, 2012 4) Yoshikawa M, Tsuchihashi K, Ishimoto T, Yae T, Motohara T, Sugihara E, Onishi N, Masuko T, Yoshizawa K, Kawashiri S, Mukai M, Asoda S, Kawana H, Nakagawa T, Saya H and Nagano O: xCT inhibition depletes CD44v-expressing tumor cells that are resistant to EGFR-targeted therapy in head and neck squamous cell carcinoma. Cancer Res 73: 1855-1866, 2013 5) Nagano O, Okazaki S and Saya H: Redox regulation in stem-like cancer cells by CD44 variant isoforms (Review article). Oncogene 32: 5191-5198, 2013 6) Seishima R, Wada T, Tsuchihashi K, Okazaki S, Yoshikawa M, Oshima H, Oshima M, Sato T, Hasegawa H, Kitagawa Y, Goldenring JR, Saya H and Nagano O: Ink4a/Arf-dependent loss of parietal cells induced by oxidative stress promotes CD44-dependent gastric tumorigenesis. Cancer Prev Res 8: 492-501, 2015 7) Tsuchihashi K, Okazaki S, Ohmura M, Ishikawa M, Sampetrean O, Onishi N, Wakimoto H, Yoshikawa M, Seishima R, Iwasaki Y, Morikawa T, Abe S, Takao A, Shimizu M, Masuko T, Nagane M, Furnari FB, Akiyama T, Suematsu M, Baba E, Akashi K, Saya H and Nagano O: The EGF receptor promotes the malignant potential of glioma by regulating amino acid transport system xc(-). Cancer Res 76: 2954-2963, 2016 8) Shitara K, Doi T, Nagano O, Imamura CK, Ozeki T, Ishii Y, Tsuchihashi K, Takahashi S, Nakajima TE, Hironaka S, Fukutani M, Hasegawa H, Nomura S, Sato A, Einaga Y, Kuwata T, Saya H and Ohtsu A: Dose-escalation study for the targeting of CD44v[+] cancer stem cells by sulfasalazine in patients with advanced gastric cancer (EPOC1205). Gastric Cancer 20: 341-349, 2017 9) Shitara K, Doi T, Nagano O, Fukutani M, Hasegawa H, Nomura S, Sato A, Kuwata T, Asai K, Einaga Y, Tsuchihashi K, Suina K, Maeda Y, Saya H and Ohtsu A: Phase 1 study of sulfasalazine and cisplatin for patients with CD44v-positive gastric cancer refractory to cisplatin (EPOC1407). Gastric Cancer 2017 (in press) 10) Otsubo K, Nosaki K, Imamura CK, Ogata H, Fujita A, Sakata S, Hirai F, Toyokawa G, Iwama E, Harada T, Seto T, Takenoyama M, Ozeki T, Mushiroda T, Inada M, Kishimoto J, Tsuchihashi K, Suina K, Nagano O, Saya H, Nakanishi Y and Okamoto I: Phase I study of salazosulfapyridine in combination with cisplatin and pemetrexed for advanced non-small cell lung cancer. Cancer Sci 108: 1843-1849, 2017
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MS 26.02 - Innate Immune Microenvironment (ID 7765)
14:50 - 15:10 | Presenting Author(s): Ruben Pio
- Abstract
- Presentation
Abstract:
A better understanding of the interaction between tumors and the immune microenvironment has led to the successful development of immunotherapies for tumors traditionally considered poorly immunogenic, such as non-small cell lung cancer (NSCLC). Clinically approved immunotherapies for lung cancer are based on antibodies able to reactivate cytotoxic T cells by targeting the PD-1/PD-L1 immune checkpoint. However, the substantial proportion of tumors refractory to these treatments, together with the limited predictive value of PD-L1 expression, evidences the existence of additional immune suppressive regulatory systems. This calls for a more detailed understanding of the immune cell landscape related to lung tumors. Some studies have begun to dissect the details of immune cell distribution in lung cancer lesions using multiscale immune profiling strategies. Along with adaptive immune alterations in T cells, significant innate immune cell changes have been identified (1). We have recently reviewed the role played by the innate immune system in supporting tumor-promoting activities and an immunosuppressive microenvironment (2). Innate immunity includes soluble components and immune cell populations, such as myeloid cells. Specific subpopulations of myeloid cells have been identified as specialized immunosuppressive cells. These myeloid-derived suppressor cells (MDSCs) are immature myeloid cells arrested in different stages of differentiation. The recognition of these cells as a defined cell lineage remains controversial, but their cancer-specific phenotypic and functional characteristics are manifest (3). Chronic inflammation factors released by tumors induce the accumulation of these immature cells in the bone marrow, which are released to the circulation and recruited to tumors. MDCSs are thought to be major contributors to T-cell exhaustion. They deplete the tumor microenvironment of amino acids essential for T cell proliferation (e.g. arginine) and produce immunosuppressive cytokines (e.g. IL10 and TGFβ). Increased levels of MDSCs have been described in patients with NSCLC, are associated with poor prognosis and can mediate resistance to chemotherapy (4). Interestingly, in tumor models, accumulation of MDSCs has been proposed as a contributor to the incapacity of the anti-PD-1/PD-L1 blockade to completely reverse the suppressive activity (5). Therefore, stimulation of effector T cells while blocking the immunosuppressive activity of MDSCs represents a rational immunotherapy combination potentially effective for lung cancer. Several agents used in conventional cancer chemotherapy (e.g. gemcitabine, 5-fluorouracil or cyclophosphamide) have been found to reduce MDSC numbers and can be used to deplete this cell subpopulation. However, the use of these drugs results in a profound and unpredictable remodeling of the myeloid cell compartment in the tumor stroma, and careful evaluation of the appropriate timing and dosing is required (6). An alternative strategy recently proposed by us to successfully reverse the tumor immunosuppressive microenvironment is based on the inhibition of the complement system, another essential element of innate immunity. The complement system has developed as a first defense against pathogens or unwanted host elements. The three major pathways of complement activation (the classical, alternative, and lectin pathways) converge in the cleavage of C3 into C3b. C3b deposition leads to the formation of C3 convertases that amplify both opsonization and the complement response, and eventually promote C5 convertase formation and assembly of the membrane attack complex. In addition, enzymatic cleavage of C3 and C5 releases C3a and C5a, two multifunctional immunomodulators. Traditionally, tumor-associated complement activation has been considered as part of the body’s immunosurveillance against cancer. However significant work in recent years has identified new and surprising activities for complement within the tumor microenvironment. In the context of chronic inflammation, complement elements can promote an immunosuppressive response, induce angiogenesis, and activate cancer-related signaling pathways. In the seminal study that shifted the paradigm, Markiewski et al. reported that complement deficiencies were associated with impaired tumor growth in a syngeneic model of cervical carcinoma (7). This study demonstrated that the immunomodulator C5a, generated after complement activation within the tumors, is able to hamper antitumor CD8 T cell-mediated responses. Importantly, this activity was associated with the accumulation of MDSCs in the tumor stroma. In the case of lung cancer, reduction of tumor growth after blockade of the C5a receptor-1 (C5aR1) is accompanied by a decrease in the expression of immunosuppressive molecules and, again, a diminution in the percentage of MDSCs (8). These studies reveal the important role played by C5a/C5aR1 signaling in tumor immunity, and point to this pathway as a potential therapeutic target in the context of checkpoint inhibition. To support this hypothesis, we have recently evaluated the therapeutic efficacy of the combined administration of anti-PD-1 and anti-C5a drugs in a variety of syngeneic models of lung cancer. We demonstrated that the combination of C5a and PD-1 blockade synergistically impairs lung cancer growth and metastasis. This therapeutic effect is accompanied by a negative association between the frequency of CD8 T cells and MDSCs within the tumors, which may result in a more complete reversal of CD8 T-cell exhaustion (9). These studies suggest that inhibition of complement may overcome tumor resistance in cancer immunotherapy, providing support for the clinical evaluation of anti-PD-1 and anti-C5a drugs as a novel combination therapeutic strategy for lung cancer. In conclusion, immunotherapy strategies tailored to restore innate immune modifications might recondition the tumor immunosuppressed niche, strengthening anti-tumor T cell immunity after immune-checkpoint blockade. A more comprehensive knowledge of the dynamic spatiotemporal interactions between the tumors and the microenvironment would be required to predict response, facilitate further investigations in this field, and extend the benefit of immunotherapies to most patients. References 1. Lavin Y et at. Cell 2017;169:750-765. 2. Berraondo P et al. Immunol Rev 2016;274:290-306. 3. Escors T et al. Oncoimmunology 2013;2:e26148. 4. Huang A et al. Cancer Immunol Immunother 2013;62:1439-1451. 5. Youn JI et al. J Immunol 2008;181:5791-5802. 6. Berraondo P et al. Cancer Res 2007;67:8847-8855. 7. Markiewski MM et al. Nat Immunol 2008;9:1225-1235. 8. Corrales L et al. J Immunol 2012;189:4674-4683. 9. Ajona D et al. Cancer Discov 2017;7:694-703.
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MS 26.03 - Animal Model (ID 7766)
15:10 - 15:30 | Presenting Author(s): Kwok-Kin Wong
- Abstract
Abstract not provided
Information from this presentation has been removed upon request of the author.
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MS 26.04 - PDx Model (ID 7767)
15:30 - 15:50 | Presenting Author(s): John Poirier
- Abstract
- Presentation
Abstract:
The tumor microenvironment differs significantly from the controlled environment of in vitro cell culture. Whereas cell lines are typically grown in plastic vessels containing a pH buffered, nutrient rich liquid medium with 10% fetal bovine serum and incubated at 37 ºC and 5% CO~2~, cancer cells in tumors experience different oxygen tension, growth substrate, nutrient, waste, and growth factor gradients, pH, and the presence of stromal support cells and infiltrating immune cells. The process of establishing a new cell line requires adaptation to this environment, which results in loss of genetic heterogeneity as well as irreversible epigenetic reprogramming that is maintained even when cultured cancer cells are re-introduced to an in vivo setting. Patient-derived xenografts (PDX) are direct human tumor xenografts established and maintained exclusively in mouse hosts. While these models can never perfectly recapitulate an autochthonous human tumor, they are increasingly used as tools in cancer research due to their utility in modeling therapeutic response with higher fidelity than either cell lines or cell line xenografts. A growing interest in defining the role of the tumor microenvironment and in testing the efficacy of immunotherapy in vivo has driven advances in PDX model development. The tumor microenvironment consists of stromal cell components including blood vessels, fibroblasts, and infiltrating immune cells as well as extracellular matrix, growth factors, and nutrients. In PDX, components of the tumor microenvironment are either provided by the mouse host or are excreted by the tumor cells themselves. However, paracrine effects between tumor and stroma may not be entirely replicated in a mouse host due to a lack of cross-species cytokine reactivity. The absence of compatible stroma may bias PDX engraftment toward tumors that are less dependent on paracrine factors or which are more adept at recruiting mouse stromal support cells through enhanced expression of mouse reactive factors. It remains a significant challenge to accurately assess the mechanistic activity of therapeutic approaches designed to inhibit these interactions in the absence of human tumor stroma. Anatomical context can also have significant impact on PDX tumor biology. PDX can be established as subcutaneous flank tumors or at any of a variety of orthotopic sites. Lung PDX are amenable to orthotopic growth in the lung, and can thus be used to model lung cancer growth and metastasis within its normal anatomical context. Orthotopic tumors can have vastly altered metastatic potential and organ preference as well as differential response to anti-cancer therapeutics in comparison to subcutaneous flank tumors. One of the primary limitations of PDX as a model for cancer is the need to use immunocompromised mouse hosts. The degree to which the immune system can be modeled with PDX is dependent on the mouse host chosen and the type of human immune cells used to reconstitute the human immune cell component. PDX can be established in a great variety of different strains of immunocompromised mice including athymic nude mice, severe combined immune deficiency (SCID) and non-obese-diabetic (NOD)-SCID strains, Rag null strains, and profoundly immunocompromised strains in which IL2-Rγc has been disrupted (NSG, NOG, BRG). Each of these strains differs with respect to the type and function of hematopoietic cells. For example, athymic nude mice have intact natural killer (NK) cells, whereas NSG mice do not; NSG mice therefore develop primary tumors and metastases at a much faster rate. Humanized mice are immunocompromised mice that have partially reconstituted human immune components for the purposes of modeling the behavior of the human immune system in a cancer context. NSG, NOG, and BRG mice have been engrafted with isolated peripheral blood mononuclear cells (PBMC) or tumor infiltrating lymphocytes (TIL) primarily to study mechanisms of lymphocyte recruitment; however, a major limitation of this approach is the rapid onset of graft versus host disease in the mouse host. Alternatively, different strains of mice can be engrafted with human CD34+ hematopoietic stem cells (HSC), which results in mouse hosts with fully human lymphocytes, monocytes, dendritic cells and in some cases NK cells. A variety of mouse strains engineered to express human cytokines such as IL-3 and GM-CSF have been developed to promote improved functional human immune system components. PDX models of lung cancer are growing in complexity, variety, and sophistication. These in vivo cancer models will be an integral component in a suite of tools for studying many aspects of lung cancer biology in a research environment. Recent advances in the humanization of mouse hosts promises texpand the possibilities of studying cancer immunology and immunotherapy of human tumors in an experimental setting in vivo.
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MS 26.05 - In Vitro Model of Early Progression in SCLC (ID 7768)
15:50 - 16:10 | Presenting Author(s): Kwon-Sik Park
- Abstract
- Presentation
Abstract:
The abundance of somatic alterations and their heterogeneity between SCLC patient tumors present the immense scientific and clinical challenges. Functional characterization of recurrent mutations in SCLC is an essential step towards understanding the pathogenesis. However, it remains extremely difficult due to the lack of systematic and informed ways of defining oncogenic drivers, especially those involved in early stage tumor development. While the inaccessibility of precancerous lesion in the patients has precluded characterization of lung cancer mutations, we have developed a precancerous cell-based model of SCLC development as a streamlined approach for characterizing novel mutations and determining mutation-driven oncogenic pathways [Kim et al. 2016, Genes and Development]. Using compound transgenic mice expressing neuroendocrine specific GFP in the GEMM of SCLC (Chga-GFP/Rb/p53/Rbl2), we isolated pulmonary neuroendocrine cells one month after adenoviral Cre-mediated tumor induction, at which time the lungs did not show macroscopic lesions (Figure 1). Notably, these cells from an early stage of tumor development grew as adherent monolayers in culture and did not form subcutaneous tumors in nude mice, whereas tumor cells formed floating aggregates and formed the subcutaneous tumors. These findings indicated that these neuroendocrine cells, lacking Rb and p53, were immortalized but not transformed. Therefore, we postulated that these cells were precancerous cells of SCLC (preSC). We then tested whether preSC could be transformed by an oncogene, using a retroviral vector carrying cDNA of L-Myc, one of the most frequently amplified genes in SCLC. The preSC transduced with retroviral L-Myc (L-Myc-preSC) formed spheres characteristic of SCLC cells in culture and palpable tumors resemble primary SCLC in the flanks of nude mice, whereas GFP-preSCs (control) were morphologically identical to uninfected preSC and did not form aggregates and subcutaneous tumor (Figure 2A). Comparative expression profiling of preSCs and L-Myc-preSC or tumor cells permitted identification of the genes and pathways related to oncogene-driven tumor progression. Defining the MYC-driven oncogenic pathways led to a preclinical test of an existing drug that successfully targeted human and mouse SCLC tumors. Furthermore, using this preSC-based model in combination with CRISPR/Cas9 methods of gene targeting and in vivo models, we found that several loss-of-function mutations found in the SCLC tumors were sufficient to cause the tumorigenic progression of preSCs (Figure 2B). In conclusion, this engineered preSC-based model facilitates functional validation of the recurrent mutations in SCLC and discovery of biomarkers and molecular pathways that will be further explored for mechanistic elucidation and targeted therapies.Figure 1
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