Patient-derived Tumor Spheroids Research Articles

Abstract B065: TANK-Binding Kinase 1 (TBK1) as a novel cancer immunotherapy target

Abstract Despite the unprecedented success of immune checkpoint blockade (ICB) in melanoma and other cancers, tackling innate (primary) resistance remains a major challenge and robust biomarkers to guide treatment are lacking. Clinical trials are already underway evaluating novel immune modulatory agents in combination with anti-PD-1/PD-L1 therapies in an effort to overcome innate resistance. Despite increasing reports of ‘rational’ combination strategies, these therapies remain “one size fits all”, due the lack of robust biomarkers to guide clinical decision-making. There is an unmet need for novel approaches, tools, techniques, and methods for pre-clinical and clinical use to better understand mechanisms of response and resistance to immune checkpoint inhibitors and next-generation anti-tumor immune modulatory drugs. We have adapted a 3D microfluidic device to the short-term culture of murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS)for functional ex vivo profiling of PD-1 blockade in a model tumor immune microenvironment (TIME) to facilitate identification of mediators of response and resistance to ICB and to guide development of next-generation immunotherapy combinations. The MDOTS/PDOTS platform provides a window into the complex and dynamic events of the TIME during response (and resistance) to ICB and is an ideal model system for pre-clinical evaluation of novel cancer immunotherapy targets. Focused evaluation of novel treatment combinations using MDOTS identified TANK-binding kinase 1 (TBK1) as a novel cancer immunotherapy target, mirroring in vivoefficacy of this treatment approach. TBK1 is a Ser/Thr kinase involved in innate immune signaling and is an emerging target for anti-cancer therapy. Importantly, independent orthogonal data from two different laboratories has also identified TBK1 as a cancer immunotherapy target.We have confirmed that Tbk1 deletion and/or TBK1 pharmacologic inhibition enhances response to in vivo anti-PD-1 therapy, and have characterized the impact of deletion of Tbk1 (CRISPR) or pharmacologic inhibition of TBK1 (Cmpd1) on the tumor-immune microenvironment.Lastly, we confirmed that resistance to PD-1 blockade can be overcome with pharmacologic TBK1 inhibition using murine- and patient-derived organotypic tumor spheroids in 3D microfluidic culture. These findings confirm TBK1 as a target to overcome resistance to PD-1 blockade, further supporting the pre-clinical and clinical development of this novel combination strategy. REFERENCES 1. Jenkins RW, Barbie DA, Flaherty KT. Mechanisms of resistance to immune checkpoint inhibitors. Br J Cancer. 2018;118(1):9-16. doi: 10.1038/bjc.2017.434. PubMed PMID: 29319049; PMCID: PMC5765236. 2. Manguso RT, Pope HW, Zimmer MD, Brown FD, Yates KB, Miller BC, Collins NB, Bi K, LaFleur MW, Juneja VR, Weiss SA, Lo J, Fisher DE, Miao D, Van Allen E, Root DE, Sharpe AH, Doench JG, Haining WN. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature. 2017. doi: 10.1038/nature23270. PubMed PMID: 28723893. 3. Pan D, Kobayashi A, Jiang P, Ferrari de Andrade L, Tay RE, Luoma AM, Tsoucas D, Qiu X, Lim K, Rao P, Long HW, Yuan GC, Doench J, Brown M, Liu XS, Wucherpfennig KW. A major chromatin regulator determines resistance of tumor cells to T cell-mediated killing. Science. 2018;359(6377):770-5. doi: 10.1126/science.aao1710. PubMed PMID: 29301958. Citation Format: Ajaykumar Vishwakarma, Yi Sun, Amina Fu, Emily Robitschek, Arvin Iracheta-Vellve, Susanna Stinson, Aliasger Salem, Robert Manguso, Russell Jenkins. TANK-Binding Kinase 1 (TBK1) as a novel cancer immunotherapy target [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B065. doi:10.1158/1535-7163.TARG-19-B065

ES11.03 Immunotherapy and Endogenous Retroviruses in Small-Cell Lung Cancer

Tumor cell heterogeneity is a key determinant of cancer progression and drug resistance, which is often mediated by mesenchymal cell subpopulations. While these subclones can secrete growth factors, chemokines and cytokines, the immune signaling networks that fuel this pro-tumorigenic state remain incompletely defined. Elucidating what underlies this state would provide insights into tumor biology and inform clinical strategies to improve anti-cancer therapies. Because of their well-defined nature, we used the phenotypically distinct H69M and H69AR Small Cell Lung Cancer (SCLC) mesenchymal subclones to uncover a novel mechanism of dysregulated innate immune signaling as compared with parental neuroendocrine H69 cells. Analysis of gene signatures across TCGA and CCLE databases, functional studies in additional cell lines, and ex vivo testing of patient-derived organotypic tumor spheroids (PDOTS) were conducted to determine the broader relevance across human cancers. We discovered a novel epigenetically regulated subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in mesenchymal cancer subpopulations. Stimulated 3 Prime Antisense Retroviral Coding Sequences (SPARCS) are oriented inversely in 3’UTRs of certain interferon-inducible genes and silenced by EZH2. De-repression of these loci resulted in dsRNA generation following IFNγ exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5’ LTR of the antisense ERV. We found that dsRNA sensing preferentially by MAVS fuels activation of TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction across specific human tumors and cell lines is tightly associated with downregulation of chromatin modifying enzymes, including EZH2, a mesenchymal AXL positive cell state, and B2M and MHC class 1 antigen expression. SPARCS high tumors were marked by immune infiltration, but also exhibited multiple features of tumor immune suppression. IFNγ treatment of PDOTS with de-repressed SPARCS markedly enhanced CXCL10 production and sensitized them to PD-1 blockade. Together, these data unveil a novel subclass of ERVs whose de-repression triggers pathologic innate immune signaling in cancer, with potentially important implications for cancer immunotherapy.

MA12.06 Patient-Derived Organotypic Tumor Spheroids (PDOTS) Facilitate Therapeutic Screening for Malignant Pleural Mesothelioma

MA12.06 Patient-Derived Organotypic Tumor Spheroids (PDOTS) Facilitate Therapeutic Screening for Malignant Pleural Mesothelioma

Abstract 2382: Development and testing of the first in class immunotherapy targeting immuno-suppressive δ1 containing γδ T cells for the treatment of pancreatic ductal adenocarcinoma and other solid tumors

Abstract Background: Targeting and engineering γδ T cells has recently emerged as an orthogonal therapeutic approach in oncology with capacity to effectively modulate both innate and adaptive immune properties. We and others have shown that in solid tumors such as pancreatic ductal adenocarcinoma (PDA), melanoma, glioblastoma, breast cancer etc., γδ1 T cells express immunosuppression-related molecules and possess a pro-tumorigenic capacity. Previously, we have shown that intra-tumoral γδ T cells from patients bearing PDA, colorectal cancer (CRC) and hepatocellular carcinoma (HCC) potently inhibit patients’ αβ T cells, rendering them immunosuppressed. We hypothesized that anti-δ1 monoclonal antibody would be a potent and effective, novel therapeutic. In order to harness the therapeutic potential of γδ1 T cell blockade we have developed a set of highly specific, fully human anti-δ1 T cell receptor (TCR) antibodies. Methods: We have sequenced δ chains from 19 cancer patients with primary PDA, primary CRC and gastric cancer (GC) to identify tumor-specific δ1. A proprietary synthetic, human antibody library was screened via phage display to identify high-affinity antibodies. Surface plasmon resonance and bead-based assays were used to measure binding affinity. We used a panel of ex-vivo experiments to assess the immunosuppressive features of γδ T cells. Antibody efficacy was assayed using patient-derived organotypic tumor spheroids (PDOTS) which recapitulate complex tumor architecture. PDOTS of n = 20 patients (PDA, CRC and liver metastases, HCC) were treated with the antibodies and resulting immune profiles analyzed by flow cytometry. Results: Because the δ1 chains in patients showed diverse CDR3 sequences, we used a selection strategy to identify antibodies that bind diverse δ1 TCRs. Our first-in-class anti-δ1 antibodies have low nanomolar affinity to human δ1 TCRs and show no binding to δ2 TCRs. Furthermore, we chose the lead clinical candidate that showed no preference for the γ chains of the TCR, as patients may harbor a diverse set of γδ heterodimers. Importantly, we show that our lead anti-δ1 antibody achieves reproducible and robust efficacy in the PDOTS system as shown by the up-regulation of pro-inflammatory T cell markers (IFNγ, TNF-α, CD44). Ongoing experiments in γδ knockout mice will investigate the efficacy of combination of γδ1 T cell targeting and existing checkpoint inhibitors (anti- PD1, PDL1, and CTLA4). Conclusion: We have defined a novel therapeutic immuno-oncology strategy and translated it to develop and characterize a lead clinical candidate anti-δ1 monoclonal antibody. Overall, our results demonstrate that we have an efficacious, novel immunotherapy that has the potential to be transformative for the treatment of cancers where γδ1 T cells drive a pro-tumorigenic, immunosuppressive environment. Citation Format: Tatyana Panchenko, Wei Wang, Eric Denbaum, Takamitsu Hattori, Akiko Koide, Aleksandra Filipovic, George Miller, Shohei Koide. Development and testing of the first in class immunotherapy targeting immuno-suppressive δ1 containing γδ T cells for the treatment of pancreatic ductal adenocarcinoma and other solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2382.

Abstract 1551: First in class immunotherapy targeting Galectin-9 promotes T-cell activation and anti-tumor response against pancreatic cancer and other solid tumors

Abstract Background: Pancreatic adenocarcinoma (PDA) is one of the deadliest forms of cancer, with a five-year survival rate at or below seven percent. Current immunotherapies show promise in treating other cancers, but success remains elusive for pancreatic cancer. Poor prognosis in pancreatic cancer is invariably linked to immune dysfunction. We have shown that in PDA, Galectin-9 (Gal-9) plays a significant role in facilitating a tumor-permissive immune microenvironment. Specifically, Gal-9 modulates immunosuppression via binding to multiple partner molecules (TIM-3, Dectin-1, CD44, CD206 etc). In light of these findings, we have developed and characterized first-in-class, therapeutic monoclonal antibodies to target Gal-9 and impede tumor growth. Methods: A proprietary synthetic, human antibody library was screened via phage display to identify high-affinity antibodies cross-reactive with human, cynomolgus and rodent Gal-9, to enable efficacy and toxicity studies. We used surface plasmon resonance and bead-based assays to measure antibody binding affinity. ELISA and cell-based approaches were used to demonstrate antibody blockade of Gal-9 binding to known ligands, inferring potential therapeutic efficacy. Efficacy in multiple IgG formats were assayed using KPC (Pdx1Cre; KrasG12D; Tp53R172H)-derived cancer cells implanted orthotopically into the pancreas of C57BL/6 mice. Patient-derived organotypic tumor spheroids (PDOTs) derived from PDA and other GI tumors, which recapitulate complex tumor architecture, were treated with anti-Gal-9 antibodies and resulting immune profile was analyzed via flow cytometry. Results: Our lead clinical candidate Gal-9 antibodies exhibit specific binding towards mouse, cynomolgus and human Gal-9 with KDvalues in the sub nanomolar range. These antibodies potently block Gal-9 interactions with known ligands such as CD206 and Dectin-1. These antibodies also inhibit Gal-9 mediated apoptosis of Jurkat cells. Mice bearing orthotopic pancreatic tumors treated with Gal-9 antibodies induced significant reduction in tumor size (p<0.005) compared with relevant controls. Immune profiling showed increased T cell activation and modulation of the monocytic compartment towards an immune permissive state. PDOTs showed similar immune profiles including an increase in type 1 inflammatory cytokines and effector T cell activation. Conclusions: Our Gal-9 lead therapeutic antibody has been identified, characterized and is being further assessed in expanding pre-clinical efficacy, molecular and safety studies in preparation for an IND filing. Our data demonstrate novel cancer immune biology with therapeutic implications of a first-in-class monoclonal antibody against Gal-9 to reverse immunosuppressive mechanisms contributing to tumor growth immune escape in pancreatic cancer and other solid tumors. Citation Format: Linxiao Chen, Wei Wang, Akiko Koide, Joseph Bolen, George Miller, Shohei Koide. First in class immunotherapy targeting Galectin-9 promotes T-cell activation and anti-tumor response against pancreatic cancer and other solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1551.

Oncolytic Ad co-expressing decorin and Wnt decoy receptor overcomes chemoresistance of desmoplastic tumor through degradation of ECM and inhibition of EMT.

Pancreatic cancer is a highly lethal disease. Excessive accumulation of tumor extracellular matrix (ECM) and epithelial-to-mesenchymal transition (EMT) phenotype are two main contributors to drug resistance in desmoplastic pancreatic tumors. To overcome desmoplasia and chemoresistance of pancreatic cancer, we utilized an oncolytic adenovirus (Ad) co-expressing decorin and soluble Wnt decoy receptor (HEmT-DCN/sLRP6). An orthotopic pancreatic xenograft tumor model was established in athymic nude mice using Mia PaCa-2 cells, and the antimetastatic and antitumor efficacy of systemically administered HEmT-DCN/sLRP6 was evaluated. Immunohistochemical analysis of tumor tissues was performed to assess ECM degradation, induction of apoptosis, viral dispersion, and inhibition of the Wnt/β-catenin signaling pathway. HEmT-DCN/sLRP6 effectively degraded tumor ECM and inhibited EMT, leading to enhanced viral distribution, induction of apoptosis, and attenuation of tumor cell proliferation in tumor tissue. HEmT-DCN/sLRP6 prevented metastasis of pancreatic cancer. Importantly, HEmT-DCN/sLRP6 sensitized pancreatic tumor to gemcitabine treatment. Furthermore, HEmT-DCN/sLRP6 augmented drug penetration and dispersion within pancreatic tumor xenografts and patient-derived tumor spheroids. Collectively, these results illustrate that HEmT-DCN/sLRP6 can enhance the dispersion of both oncolytic Ad and a chemotherapeutic agent in chemoresistant and desmoplastic pancreatic tumor, effectively overcoming the preexisting limitations of standard treatments.

Identification and Characterization of AES-135, a Hydroxamic Acid-Based HDAC Inhibitor That Prolongs Survival in an Orthotopic Mouse Model of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, incurable cancer with a 20% 1 year survival rate. While standard-of-care therapy can prolong life in a small fraction of cases, PDAC is inherently resistant to current treatments, and novel therapies are urgently required. Histone deacetylase (HDAC) inhibitors are effective in killing pancreatic cancer cells in in vitro PDAC studies, and although there are a few clinical studies investigating combination therapy including HDAC inhibitors, no HDAC drug or combination therapy with an HDAC drug has been approved for the treatment of PDAC. We developed an inhibitor of HDACs, AES-135, that exhibits nanomolar inhibitory activity against HDAC3, HDAC6, and HDAC11 in biochemical assays. In a three-dimensional coculture model, AES-135 kills low-passage patient-derived tumor spheroids selectively over surrounding cancer-associated fibroblasts and has excellent pharmacokinetic properties in vivo. In an orthotopic murine model of pancreatic cancer, AES-135 prolongs survival significantly, therefore representing a candidate for further preclinical testing.

Abstract 4968: Tumor innate immunity primed by specific interferon stimulated endogenous retroviruses

Abstract Introduction: Tumor cell heterogeneity is a key determinant of cancer progression and drug resistance, which is often mediated by mesenchymal cell subpopulations. While these subclones can secrete growth factors, chemokines and cytokines, the immune signaling networks that fuel this pro-tumorigenic state remain incompletely defined. Elucidating what underlies this state would provide insights into tumor biology and inform clinical strategies to improve anti-cancer therapies. Methods: Because of their well-defined nature, we used the phenotypically distinct H69M and H69AR Small Cell Lung Cancer (SCLC) mesenchymal subclones to uncover a novel mechanism of dysregulated innate immune signaling as compared with parental neuroendocrine H69 cells. Analysis of gene signatures across TCGA and CCLE databases, functional studies in additional cell lines, and ex vivo testing of patient-derived organotypic tumor spheroids (PDOTS) were conducted to determine the broader relevance across human cancers. Results: We discovered a novel epigenetically regulated subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in mesenchymal cancer subpopulations. Stimulated 3 Prime Antisense Retroviral Coding Sequences (SPARCS) are oriented inversely in 3'UTRs of certain interferon-inducible genes and silenced by EZH2. De-repression of these loci resulted in dsRNA generation following IFNγ exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5' LTR of the antisense ERV. We found that dsRNA sensing preferentially by MAVS fuels activation of TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction across specific human tumors and cell lines is tightly associated with downregulation of chromatin modifying enzymes, including EZH2, a mesenchymal AXL positive cell state, and B2M and MHC class 1 antigen expression. SPARCS high tumors were marked by immune infiltration, but also exhibited multiple features of tumor immune suppression. IFNγ treatment of PDOTS with de-repressed SPARCS markedly enhanced CXCL10 production and sensitized them to PD-1 blockade. Conclusions: Together, these data unveil a novel subclass of ERVs whose de-repression triggers pathologic innate immune signaling in cancer, with potentially important implications for cancer immunotherapy. Citation Format: David A. Barbie. Tumor innate immunity primed by specific interferon stimulated endogenous retroviruses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4968.

Abstract 988: Short-term culture of organotypic tumor spheroids derived from patient xenografts in a novel 3D microfluidic chip predicts in vivo response of targeted therapies

Abstract Introduction: Patient-derived xenografts (PDX) are increasingly adopted to evaluate the efficacy of new treatment approaches, including the development of combination therapies and testing of predictive biomarkers hypotheses. However, PDXs can take months to generate and are impractical for screening large sets of drug combinations. Here we report on a novel 3D microfluidic platform that allows for evaluating ex vivo responses to targeted therapies from genotyped defined patient-derived tumor spheroids (XDOTS) (1). Methods: Non-small cell lung cancer (NSCLC) PDXs with known genotype, including EGFR ex19 del, HER-2 ins, and BRAF V600E, were generated from either core needle biopsies or pleural effusions under an IRB-approved protocol. XDOTS (<100 μm and >40μm) were grown from PDX tumors using DAX-1 3D cell culture chips (AIM, Singapore) and treated with genotype matched inhibitors that are either approved (e.g., erlotinib for EGFR mut) or are being tested in the clinic (neratinib for HER-2 ins) at known peak plasma concentrations for 3 days. Live/death quantification was performed by dual labeling fluorescence microscopy using acridine orange for live and propidium iodide for dead cells. Cell type characterization was performed by immunofluorescence in the devices. Results: We first characterized our XDOT system on models for which we had orthogonal validation. We show by dual labeling fluorescence microscopy that PC9GR4 (EGFR Del19/T790M) cells are sensitive to osimertinib (IC50 = 12nM), but not gefitinib (IC50 >10uM). We further show that HCC827GR6 (Del 19/MET Amp) and DFCI32 (EML4-ALK/EGFR/HER2) cells are sensitive to crizotinib/gefitinib combo, but not to each of the single agents. Results were validated using the IncuCyte Live Cell analysis. We then went on to show that genotype-specific responses can be recapitulated in tumor spheroids generated from PDXs. In DFCI 359, a PDX derived from a HER2 mutant (755_757 LRdelinsPR), we show the antitumor efficacy of afatinib and confirm that effect is specific to tumor cells and not stromal. We observe significant drug efficacy only on cells growing in our microfluidic devices and not on spheroids growing 3D in 384 well plates. Similar results were obtained for additional PDXs with known NSCLC driver mutations. Finally, we evaluate our system on a small cohort of direct patient specimens. Conclusions: Taken together, our data show that short culture experiments using XDOTS are feasible and recapitulate key futures of in vivo response and resistance ex vivo. Reference: 1. Jenkins RW, et al. Cancer Discov DOI:10.1158/2159-8290. Citation Format: Amir Aref, Elena Ivanova, Emily Chambers, Andrew Portell, Russell Jenkins, Mika Lin, Man Xu, Jacob Haworth, Magda Bahcall, Cloud Paweletz, Pasi Janne. Short-term culture of organotypic tumor spheroids derived from patient xenografts in a novel 3D microfluidic chip predicts in vivo response of targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 988.

Patient-derived multicellular tumor spheroids towards optimized treatment for patients with hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide and has poor prognosis. Specially, patients with HCC usually have poor tolerance of systemic chemotherapy, because HCCs develop from chronically damaged tissue that contains considerable inflammation, fibrosis, and cirrhosis. Since HCC exhibits highly heterogeneous molecular characteristics, a proper in vitro system is required for the study of HCC pathogenesis. To this end, we have established two new hepatitis B virus (HBV) DNA-secreting HCC cell lines from infected patients.MethodsBased on these two new HCC cell lines, we have developed chemosensitivity assays for patient-derived multicellular tumor spheroids (MCTSs) in order to select optimized anti-cancer drugs to provide more informative data for clinical drug application. To monitor the effect of the interaction of cancer cells and stromal cells in MCTS, we used a 3D co-culture model with patient-derived HCC cells and stromal cells from human hepatic stellate cells, human fibroblasts, and human umbilical vein endothelial cells to facilitate screening for optimized cancer therapy.ResultsTo validate our system, we performed a comparison of chemosensitivity of the three culture systems, which are monolayer culture system, tumor spheroids, and MCTSs of patient-derived cells, to sorafenib, 5-fluorouracil, and cisplatin, as these compounds are typically standard therapy for advanced HCC in South Korea.ConclusionIn summary, these findings suggest that the MCTS culture system is the best methodology for screening for optimized treatment for each patients with HCC, because tumor spheroids not only mirror the 3D cellular context of the tumors but also exhibit therapeutically relevant pathophysiological gradients and heterogeneity of in vivo tumors.

Establishment of patient-derived tumor spheroids for non-small cell lung cancer.

The prognosis of advanced non-small cell lung cancer (NSCLC) patients is poor. One of the reasons for this hampered progress has been a lack of in vitro models that would faithfully recapitulate the heterogeneity of tumors and response to treatment. In this study, surgically resected tumors were obtained from patients with stage I/II NSCLC during curative-intent surgery. Using a 3D patient-derived tumor spheroids culture system, our results demonstrate successful long-term expansion of primary NSCLC cells in vitro (> 120 days). Patient-derived tumor spheroid (PDS) cultures could be established with a success rate of 100% (3 out of 3 samples). Consistent with their growth in culture and their cancer type, many cells within the tumor spheroids were stained positive for Ki67 and thyroid transcription factor-1. The result of this study supports the establishment of an expandable 3D in vitro NSCLC model for drug screening, and enables the potential long term studies such as the establishment of drug resistant models.

Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids.

Ex vivo systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate ex vivo response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKε inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response in vivo Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens.Significance: Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of ex vivo profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts. Cancer Discov; 8(2); 196-215. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Deng et al., p. 216This article is highlighted in the In This Issue feature, p. 127.

Potent antitumor effect of tumor microenvironment-targeted oncolytic adenovirus against desmoplastic pancreatic cancer.

Pancreatic cancer is a leading cause of cancer-related death. Desmoplastic pancreatic tumors exhibit excessive extracellular matrix (ECM) and are thus highly resistant to anticancer therapeutics, since the ECM restricts drug penetration and dispersion. Here, we designed and generated two hypoxia-responsive and cancer-specific hybrid promoters, H(mT)E and H(E)mT. Transgene expression driven by each hybrid promoter was markedly higher under hypoxic conditions than normoxic conditions. Moreover, H(E)mT-driven transgene expression was highly cancer-specific and was superior to that of H(mT)E-driven expression. A decorin-expressing oncolytic adenovirus (Ad; oH(E)mT-DCN) replicating under the control of the H(E)mT promoter induced more potent and highly cancer-specific cell death compared with its cognate control oncolytic Ad, which harbored the endogenous Ad E1A promoter. Moreover, oH(E)mT-DCN exhibited enhanced antitumor efficacy compared with both the clinically approved oncolytic Ad ONYX-015 and its cognate control oncolytic Ad lacking DCN. oH(E)mT-DCN treatment also attenuated the expression of major ECM components, such as collagen I/III, elastin and fibronectin and induced tumor cell apoptosis, leading to extensive viral dispersion within orthotopic pancreatic tumors and pancreatic cancer patient-derived tumor spheroids. Collectively, these findings demonstrate that oH(E)mT-DCN exhibits potent antitumor efficacy by degrading the ECM and inducing apoptosis in a multifunctional process. This process facilitates the dispersion and replication of oncolytic Ad, making it an attractive candidate for the treatment of aggressive and desmoplastic pancreatic cancer.

Abstract 3972: Ex vivo profiling of PD-1 blockade using organotypic tumor spheroids

Abstract Despite the impressive activity of PD-1/PD-L1 immune checkpoint blockade in melanoma, lung, and other cancers, the majority of patients are intrinsically resistant to this therapy. Furthermore, despite years of effort few robust biomarkers of response exist, and tumor PD-L1 expression and mutational load are only partially predictive of activity. Nearly all attempts at biomarker development have relied on fixed tumor tissue or studies in plasma. Meanwhile, emerging functional approaches to study patient derived samples, such as organoid development and circulating tumor cell culture, lack features of the native tumor immune microenvironment and thus are limited in the ability to assess ex vivo anti-PD-1 response. We have developed a novel approach to study short-term ex vivo response to immune checkpoint blockade based upon short term culture of murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS) in a 3-dimensional microfluidic system. Spheroids derived from fresh mouse and human tumor samples through partial collagenase digestion and filtration retain autologous tumor infiltrating immune cells, including PD-1 positive CD8 T lymphocytes. Using murine syngeneic mouse cancer models with known responsiveness or resistance to PD-1 blockade, we demonstrate the ability to recapitulate tumor killing ex vivo or lack thereof. Multiplexed cytokine profiling of conditioned media in this system further enables characterization of the cytokine and chemokine response to PD-1 and/or CTLA-4 blockade. We thus analyzed the secreted cytokine/chemokine response to PD-1 inhibition across a large panel of PDOTS, and observed pronounced induction of CCL19/CXCL13, which associated with % PD-1 positive CD8 T cells. This strategy further uncovered production of a class of myeloid attracting chemokines and cytokines in major proportion of samples, which clustered with the presence of plasmacytoid dendritic cells and myeloid derived suppressor cells. Importantly, the presence of this maladaptive cytokine/chemokine signature correlated with lack of response in the subset of patients that had been treated with PD-1 blockade. Together, these data demonstrate the ability to profile the functional response to tumor PD-1 blockade ex vivo and unveil a novel strategy to advance precision immuno-oncology. Citation Format: David A. Barbie, Russell Jenkins, Kwok-Kin Wong. Ex vivo profiling of PD-1 blockade using organotypic tumor spheroids [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 3972. doi:10.1158/1538-7445.AM2017-3972

TGF-β-independent CTGF induction regulates cell adhesion mediated drug resistance by increasing collagen I in HCC.

Hepatocellular carcinoma (HCC) is resistant to conventional chemotherapeutic agents and remains an unmet medical need. Here, we demonstrate a mechanism of cell adhesion-mediated drug resistance using a variety of HCC spheroid models to overcome environment-mediated drug resistance in HCC. We classified spheroids into two groups, tightly compacted and loosely compacted aggregates, based on investigation of dynamics of spheroid formation. Our results show that compactness of HCC spheroids correlated with fibroblast-like characteristics, collagen 1A1 (COL1A1) content, and capacity for chemoresistance. We also showed that ablation of COL1A1 attenuated not only the capacity for compact-spheroid formation, but also chemoresistance. Generally, connective tissue growth factor (CTGF) acts downstream of transforming growth factor (TGF)-β and promotes collagen I fiber deposition in the tumor microenvironment. Importantly, we found that TGF-β–independent CTGF is upregulated and regulates cell adhesion-mediated drug resistance by inducing COL1A1 in tightly compacted HCC spheroids. Furthermore, losartan, which inhibits collagen I synthesis, impaired the compactness of spheroids via disruption of cell-cell contacts and increased the efficacy of anticancer therapeutics in HCC cell line- and HCC patient-derived tumor spheroids. These results strongly suggest functional roles for CTGF-induced collagen I expression in formation of compact spheroids and in evading anticancer therapies in HCC, and suggest that losartan, administered in combination with conventional chemotherapy, might be an effective treatment for liver cancer.

Dynamic regulation of the cancer stem cell compartment by Cripto-1 in colorectal cancer.

Stemness was recently depicted as a dynamic condition in normal and tumor cells. We found that the embryonic protein Cripto-1 (CR1) was expressed by normal stem cells at the bottom of colonic crypts and by cancer stem cells (CSCs) in colorectal tumor tissues. CR1-positive populations isolated from patient-derived tumor spheroids exhibited increased clonogenic capacity and expression of stem-cell-related genes. CR1 expression in tumor spheroids was variable over time, being subject to a complex regulation of the intracellular, surface and secreted protein, which was related to changes of the clonogenic capacity at the population level. CR1 silencing induced CSC growth arrest in vitro with a concomitant decrease of Src/Akt signaling, while in vivo it inhibited the growth of CSC-derived tumor xenografts and reduced CSC numbers. Importantly, CR1 silencing in established xenografts through an inducible expression system decreased CSC growth in both primary and metastatic tumors, indicating an essential role of CR1 in the regulation the CSC compartment. These results point to CR1 as a novel and dynamically regulated effector of stem cell functions in colorectal cancer.