Treatment-mediated Changes Research Articles

Abstract 6782: 3D-EXpress - an ex vivo platform using viably cryopreserved tumoroids for rapid assessment of targeted therapeutic outcomes

Abstract Background: it has been a significant challenge to select, combine, and sequence the multiple FDA-approved IO therapies and pharmaceutical agents, highlighting the need to develop a precision oncology platform to explore novel combination treatments. 3D-EXpress is a unique ex vivo therapeutics testing platform using a biorepository of fresh patient tumoroids that were never dissociated, propagated, or reassembled to retain an intact microenvironment. Therefore, these specimens provide an excellent ex vivo platform to investigate the efficacy of therapeutics targeting the tumor microenvironment. Here, we employed the 3D-EXpress platform to compare the efficacy of different therapeutic modalities targeting tumor antigens and immune checkpoint ex vivo. Methods: Tumoroids measuring 150 μm in size retaining tumor cell heterogeneity, tumor-resident immune cells, stromal components, and extracellular matrix interaction were prepared from human endometrial and colorectal tumors. Tumoroids were cryopreserved and were selected based on tumor antigen status. Samples were treated for 72 hours ex vivo with antibody-drug conjugates, chimeric antigen receptor (CAR)T cells, or bispecific antibodies alone and in combinations. Treatment-mediated changes in tumor cell killing and tumor immune microenvironment were investigated. Results: 3D high throughput confocal imaging and Nilogen Oncosystem’s proprietary algorithm were used to detect and analyze treatment-induced tumor cell killing activity in tumoroids. The impact of ex vivo treatment by different modalities in combination with chemotherapeutic agents upon tumor resident immune cell populations was monitored by deep immune phenotyping and multiplex cytokine release assay. Based on the ex vivo responses tumors were assigned to treatment sensitive and resistant groups. Correlative analysis was performed and compared with detailed clinicopathologic data that is readily available for Biorepository tumors. Conclusions: The 3D-EXpress platform using cryopreserved 3D tumoroids with intact tumor microenvironment is an effective tool for the pre-clinical assessment of rational drug combinations for treatment of solid tumors. Furthermore, the 3D-EXpress platform provides unique insight into the microenvironment of both treatment responsive and non-responsive tumors and can aid in the development of patient-centered therapeutic regimens. Citation Format: Vanessa Garrido, Michelle Ataya, Nisha R. Dhanushkodi, Seth Currlin, Alliyah Humphrey, Jared Ehrhart, Rikhia Chakraborty. 3D-EXpress - an ex vivo platform using viably cryopreserved tumoroids for rapid assessment of targeted therapeutic outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6782.

Abstract 4552: 3D-EXplore platform of fresh patient tumoroids with intact TME allows assessment of the efficacy of drugs targeting the tumor stroma on ex vivo tumor immunotherapy

Abstract Introduction: Cancer associated fibroblasts (CAFs) are a major component of the tumormicroenvironment (TME) and exhibit diverse tumorigenic functions such as immunosuppressionand extracellular matrix (ECM) remodeling. Treatment of solid tumors is often hindered by acomplex TME, which persists despite effective tumor-cell directed therapies. Therefore,treatment of solid tumors in combination with stromal-targeting therapies is essential toovercome CAF-facilitated tumor growth, and immunosuppression. Herein we interrogate theimpact of tumor stroma targeting therapeutics, in combination with nivolumab, on the efficacy oftumor cell killing (TCK) in a 3D human tumoroid ex vivo culture model (3D-EXplore). Resultingdata suggests that impairing the recruitment and activation of CAFs within the TME leads toenhanced TCK when combined with immune checkpoint blockade therapy. Materials and Methods: All patients tumor samples were collected with patient consent andrelevant IRB approval. 3D tumoroids measuring 150 microns in size were generated from freshpatient tumors including endometrial, ovarian, and colorectal cancer tissues. Tumoroids werethen treated with stromal targeting strategies for TGF-beta (galunisertib), the FGF pathway(Dovitinib), FAK inhibitors (defactinib), and cell adhesion modulators (plerixafor) alone or incombination with nivolumab for 72-hours ex vivo. Tumor responses to ex vivo treatments wereassessed using a proprietary tumor cell killing assay and 21-color flow cytometry analysis. Results and Summary: Here we evaluated the impact of stromal targeting therapeutics onCAFs and associated tumor cell killing as well as overcoming immune evasion mechanismspreserved in the tumoroid models. Furthermore, we used a multicolor flow panel to analyzewhether combination of stromal targeting drugs enhances nivolumab’s effect on the activation oftumor resident CD4, CD8 T-cells, NKT, and NK cell populations as well as on macrophagepolarization. Treatment-mediated changes in the tumor immune microenvironment was furthercorroborated by a multiplex cytokine release assay detecting GM-CSF, sCD137, IFNγ, sFas,sFasL, Granzyme A, Granzyme B, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, MIP-1α, MIP-1β, TNF-α,Perforin in tumoroid culture media and correlated with clinicopathologic findings and PD-L1expression for individual tumnors. Further, this 3D-tumoroid platform provides unique insightinto the microenvironment of both treatment responsive and non-responsive tumors and can aidin the development of patient-centered therapeutic regimens. Citation Format: Seth Currlin, Brittney Ruedlinger, Sharon Camacho, Angie Rivera, Jasmin D'Andrea, Jared Ehrhart, Soner Altiok. 3D-EXplore platform of fresh patient tumoroids with intact TME allows assessment of the efficacy of drugs targeting the tumor stroma on ex vivo tumor immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4552.

Abstract 246: Application of the 3d fresh patient tumoroid platform 3d-explore to assess the immunomodulatory and phagocytic activity of the receptor tyrosine kinase inhibitor, Sunitinib

Abstract Background: The receptor tyrosine kinase inhibitor (TKI) Sunitinib has proven useful in the treatment of a variety of tumors. Sunitinib has shown therapeutic activity in patients with metastatic renal cell carcinoma and currently represents a frontline therapy for this disease. The immune modulatory effect of Sunitinib on immune cells within the tumor microenvironment has been well documented. Here, we developed a novel 3D ex-vivo platform (3D-EXplore) using fresh patient tumor samples with intact stromal components and tumor immune microenvironment to assess the therapeutic efficacy of Sunitinib. Methods: All tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids with intact TME measuring 150 µm in size were prepared from fresh tumor samples of renal cell carcinoma using a proprietary technology developed at Nilogen Oncosystems. Tumoroids prepared from each patient’s tumor sample were pooled to represent the tumor heterogeneity and treated ex vivo with Sunitinib for 48h to detect treatment-mediated changes in tumor immune cell composition including CD4 and CD8 T-cells, NK cells, and macrophages. Additionally, we analyzed treatment-mediated changes in T-cell activation and phagocytic activity of myeloid cells. Results: Multiparameter flow analysis revealed that Sunitinib treatment led to an increase in CD8+CD25+ central memory-like phenotype and increased granzyme B expression in CD4 and CD8+ T cells indicating T cell activation. Furthermore, multiparameter flow cytometry analysis showed increased EdU uptake by CD8 and NK cells with sunitinib treatment, while cytokine release assays demonstrated increased IFN-γ and IL-2 production accompanied by downregulation of IL-10 and IL-6 cytokines. We observed Sunitinib mediated phagocytotic activity by tumor resident myeloid macrophages, monocytes, and dendritic cells using a quantitative phagocytosis assay. Conclusions: These results demonstrate that 3D-EXplore using fresh tumor samples provides a clinically relevant platform to assess drug response and serves as a critical tool for reflecting the true intact tumor microenvironment for novel immune-oncology drug development. Citation Format: Brittany Bunch, Krithika N. Kodumudi, Jared Ehrhart, Matt Weitzman, Olivia MacIntosh, Kelly Sussman, Soner Altiok. Application of the 3d fresh patient tumoroid platform 3d-explore to assess the immunomodulatory and phagocytic activity of the receptor tyrosine kinase inhibitor, Sunitinib [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 246.

Abstract 239: Assessment of immunotherapeutic efficacy of entinostat by using the 3d-explore ex vivo platform utilizing tumoroids of fresh patient tumor samples with intact tumor microenvironment

Abstract Background: Entinostat, a selective histone deacetylase 1/3 inhibitor has been shown to promote a robust anti-tumor response and increased neoantigen expression and antigen-specific T cell activation in preclinical tumor models. Entinostat plays a role in neutralizing myeloid derived suppressor cells (MDSC) to improve anti-tumor immune response. Entinostat is currently being tested in later-stage clinical trials in various types of cancer. Using a novel 3D ex-vivo platform with fresh patient tumor samples we assessed the therapeutic efficacy of Entinostat with intact stromal components and tumor immune microenvironment. Methods: All tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids with intact TME measuring 150 µm in size were prepared from fresh tumor samples from renal cell carcinoma (RCC) patients using a proprietary technology developed at Nilogen Oncosystems. Tumoroids prepared from each patient’s tumor sample were pooled to represent the tumor heterogeneity and treated ex vivo with Entinostat for 48h to detect treatment-mediated changes in tumor immune cell composition including lymphoid and myeloid markers and their activation status. Results: Tumoroids treated with Entinostat increased NK, NKT, CD4 and CD8+T cells compared to untreated control. Ex vivo treatment with Entinostat led to an increase in CD69 and Granzyme B expression in tumor resident CD4+ T cells. Immune checkpoint receptors, 41BB and PD-1 expression was increased in CD8+T cells followed by Entinostat treatment. Decreased monocytic MDSC was seen in Entinostat treated tumoroids compared to untreated control and this was associated with decreased GM-CSF, IL-4, IL-6 cytokine levels and increased IFN-γ and IL-2, quantified by multiplex cytokine assay. Interestingly, the tumor cell killing study revealed a higher percentage of death in early stage MDSC (eMDSC) indicating a direct effect of Entinostat on MDSC survival. Conclusions: These results demonstrate that Entinostat treatment leads to a broad range of immune cell activation and MDSC inhibition in RCC. These data provide a mechanistic rationale utilizing our 3D tumoroid platform for discovery of potential biomarkers of drug sensitivity and to identify novel immunotherapeutic approaches. Clinical applications of 3D-EXplore may help to identify RCC patients who may likely benefit from Entinostat treatment. Citation Format: Krithika Nandakumar Kodumudi, Brittany Bunch, Jared Ehrhart, Matt Weitzman, Olivia MacIntosh, Kelly Sussman, Soner Altiok. Assessment of immunotherapeutic efficacy of entinostat by using the 3d-explore ex vivo platform utilizing tumoroids of fresh patient tumor samples with intact tumor microenvironment [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 239.

Abstract 288: An ex-vivo 3D tumoroid model of fresh patient tissue (3D-EXplore) to assess transcriptional and compositional changes of the immune landscape in intact tumor microenvironment using single-cell proteogenomics

Abstract Background: Molecular changes underlying immune cell signaling in response to new therapy development are crucial to validate the clinical efficacy of immunotherapeutics. There are several preclinical tools including patient derived cell lines, organoid and xenograft (PDX) models to assess the efficacy of drugs. However, it is important to recapitulate the complexity of human malignancy and immune contexture within the tumor microenvironment. Here we report, an ex vivo platform (3D-EXplore) using fresh patient tumor samples with intact stromal and immune cell components to assess treatment-mediated changes in molecular and transcriptional profiles of tumor resident immune cells using a Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) platform. Methods: All tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids measuring 150 µm in size with intact tumor immune microenvironment were prepared from fresh tumor samples of colon and lung tissues using proprietary technology developed at Nilogen Oncosystems. No enzymatic digestion, propagation or reassembly was used during the preparation of the tumoroids. Hundreds of tumoroids originated from different parts of each patient’s tumor sample were pooled to represent the tumor heterogeneity and treated ex vivo with or without immunostimulatory agents for 48h. Here, we applied multi-modal CITE-seq profiling using the 10X Genomics platform to interrogate cellular responses to ex vivo treatment. Culture supernatants were collected for multiplex analysis of cytokine release in media. Additionally, flow cytometry was used to assess the activation profile of resident immune cells. Results: Following sequencing of FACS sorted viable CD45 populations, the FAST-Q output files were uploaded into Cell Ranger for analysis with Loupe Browser. Multimodal analysis of transcriptomes or proteomics at the single-cell level revealed significant changes in the top 100 gene expression levels specific to CD4 and CD8+ T cells, NK cells and B cells signaling and activation upon stimulation with immunostimulatory agents. In addition, several pro and anti-inflammatory chemokines and cytokines were upregulated in response to ex vivo treatment in both colon and lung tumoroids, which coincided with marked changes in the activation status of tumor resident immune cells, as detected by multiparameter flow cytometry analysis. Conclusions: These results demonstrate Nilogen’s 3D fresh tumoroid model reflects the significant impact on activation and proliferation of tumor resident immune cells within the intact tumor microenvironment at the transcriptional level. We believe this model system can serve as a clinically relevant tool for accelerating immuno-oncology drug development. Citation Format: Jonathan P. Waterman-Smith, Jared Ehrhart, Brittany Bunch, Krithika Kodumudi, Matt Weitzman, Olivia MacIntosh, Kelly Sussman, Soner Altiok. An ex-vivo 3D tumoroid model of fresh patient tissue (3D-EXplore) to assess transcriptional and compositional changes of the immune landscape in intact tumor microenvironment using single-cell proteogenomics [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 288.

Functional connectivity and neurotransmitter impairments of the salience brain network in chronic low back pain patients: a combined resting-state functional magnetic resonance imaging and 1 H-MRS study.

Functional reorganisation of the salience network (SN) has been proposed as one of the key pathomechanisms associated with central nociceptive processing in the chronic pain state. Being associated with an altered functional connectivity within the SN, these processes have been hypothesized to result from a loss of inhibitory function leading to node hyperexcitability and spontaneous pain. Combined resting-state BOLD functional magnetic resonance imaging (MRI) and 1 H-MR spectroscopy was applied to chronic back pain patients and healthy subjects to assess deviations from functional integrity (weighted closeness centrality [wCC], derived from resting-state functional MRI), oscillatory BOLD characteristics (spectral power), and neurotransmitter levels (GABA + , glutamate+glutamine) in 2 key SN nodes, anterior insular (aIns R ) and anterior mid-cingulate cortices. In addition, examinations were repeated in chronic back pain patients after a 4-week interdisciplinary multimodal pain treatment and in healthy subjects after 4 weeks to explore longitudinal, treatment-mediated changes in target variables. The aIns R and, to a lesser extent, the anterior mid-cingulate of patients exhibited significantly reduced wCC accompanied by a spectral power shift from a lower to a higher frequency band, indicating a desynchronization of their neuronal activity within the SN, possibly because of increased spontaneous activations. Without revealing neurotransmitter differences, patients alone showed significant positive associations between local GABA + levels and wCC in aIns R , suggesting a stronger dependence of node synchronization on the inhibitory tone in the chronic pain state. However, this needs to be explored in the future using magnetic resonance spectroscopy techniques that are more sensitive to detecting subtle neurotransmitter changes and also allow multifocal characterization of neurotransmitter tone.

Abstract 2649: An ex vivo 3D tumoroid model of fresh patient tissue (3D-EX) to assess the efficacy of anti-angiogenic compounds in renal cell carcinoma

Abstract Introduction: Renal Cell Carcinoma (RCC) is highly resistant to systemic chemotherapy; however, targeting pro-angiogenic pathways has shown significant clinical benefit. The development of targeted anti-angiogenetic agents has been hampered by the lack of clinically relevant models for screening. We have previously reported that the generation of 3D-tumoroids containing the unaltered tumor stroma could effectively be used to detect tumor response to small molecule inhibitors as well as antibody-based therapeutic agents. In this study we described a novel ex vivo platform to develop anti-angiogenic therapeutic strategies using fresh patient tumoroids of hepatocellular carcinoma and renal cell carcinoma. Materials and Methods: All fresh tumor samples were obtained with patient consent and relevant IRB approval. For the ex vivo assays, 3D tumoroids measuring 150 µm in size were treated with tyrosine kinase inhibitors (TKIs) sunitinib, sorafenib or axitinib. Treatment-mediated changes in endothelial cell viability was assessed by the confocal-based high-content real time imaging platform using fluorescent labeled anti-CD31 and anti-VEGFR2 antibodies, combined with custom image analysis algorithms. Additionally, treatment-mediated changes in tumor immune cell composition including CD4 and CD8 T-cells, Tregs, NK cells, macrophages, cell surface expression of checkpoint proteins as well as T-cell activation were evaluated by multiplex flow cytometry. Results: Our data shows that endothelial cells and capillary structures could clearly be visualized within3D tumoroids by confocal microscopy. Furthermore, we were able to quantify apoptotic cell death in endothelial cells induced by TKI inhibitors. Flow cytometry analysis demonstrated significant changes in T-cell activation upon treatment with TKIs that closely correlated with increased proinflammatory cytokine release. Observations found that the combination of anti-angiogenic treatments and immuno-modulatory agents significantly improved treatment responses. Conclusion: The ex vivo platform described in this study allowed assessment of the effect of anti-angiogenetic TKIs on tumor capillary endothelial cells as well as on tumor resident immune cell populations in intact RCC tumoroids of fresh patient tumor samples. We believe this clinically relevant approach can be used to identify the most effective anti-angiogenic and immunotherapeutic drugs and drug combinations in RCC and may improve personalized therapy for individual patients in clinical studies. Citation Format: Jared Ehrhart, Mibel M. Pabón, Stephen Iwanowycz, Zhisong Tong, Tina Pastoor, Soner Altiok. An ex vivo 3D tumoroid model of fresh patient tissue (3D-EX) to assess the efficacy of anti-angiogenic compounds in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2649.

Abstract 1566: Monitoring the impact of IL15 in combination with Sting agonists and nivolumab on activation and proliferation of tumor resident immune cells in 3D-EX platform of intact patient tumoroids

Abstract Background: The major goal of many immune-oncology (IO) therapeutics is improving the functionality of cytotoxic T cells through inhibiting checkpoints or through targeting stimulatory pathways. Tumor infiltrating myeloid cells are often educated within the tumor microenvironment (TME) to adopt regulatory phenotypes and become a major barrier to effective immune responses. Generation of an optimal immune response likely requires a combination of agents designed to promote anti-tumor T cell activation and to recondition the TME to support T cell functions. We generated patient derived 3D-tumoroids which retain the tumors stroma and suppressive immune landscape. This platform allowed for the screening of immune agonists targeting the IL15, Sting, or 41BB pathways alone or in combination with checkpoint inhibitors to identify the most effective combination in fresh patient tumoroid samples ex vivo. Methods: 3D-tumoroids approximately 150 µm in size were generated from fresh patient tumor samples which were obtained with informed consent and relevant IRB approval. Tumoroids were treated ex vivo with an IL15 agonist or Sting pathway agonists alone or in combination with nivolumab. Multi-parameter flow cytometry was used to analyze treatment-mediated changes inCD4/CD8 and NK cell activation. Additionally, we directly measured proliferation of T-cells, Tregs and myeloid cell populations in response to ex vivo treatments through EDU incorporation assay by flowcytometry. Immune cell function was further assessed by multiplex cytokine analysis and treatment-induced tumor cell killing was determined by high content confocal imaging. Results: Our data showed that IL15 treatment alone and select Sting agonists in combination and nivolumab had significant impact on activation and proliferation of tumor resident T-cells in their intact TME. Additionally, by using the 3D-EX platform, somewhat unexpectedly, we also observed meaningful changes in Tregs and multiple tumor infiltrating myeloid subsets including macrophages and dendritic cells upon ex vivo treatment with the IO drugs used in this study. Enhancement in pro-inflammatory cytokines production and immune cell functional phenotype were further correlated with tumor cell killing ex vivo. Conclusion: Our data reveals the power of the 3D-EX platform to identify combinations of immunotherapy agents capable of overcoming the unique suppressive environment developed by an individual tumor. Furthermore, the 3D-EX platform provides unique insight into the microenvironment to better identify compensatory mechanisms limiting the efficacy of IO therapeutics that may help to develop rational combination strategies. We believe the 3D-EX platform is clinically relevant and capable of significantly accelerating immuno-oncology drug discovery. Citation Format: Mibel M. Pabón, Jared Ehrhart, Stephen Iwanowycz, Zhisong Tong, Tina Pastoor, Soner Altiok. Monitoring the impact of IL15 in combination with Sting agonists and nivolumab on activation and proliferation of tumor resident immune cells in 3D-EX platform of intact patient tumoroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1566.

Abstract 935: Optimization of checkpoint inhibitor-TKI combinations in renal cell carcinoma using an ex vivo 3D tumor organoid model of fresh patient tissue with intact TME

Abstract Introduction: Renal Cell Carcinoma (RCC) is highly resistant to systemic chemotherapy. Different tyrosine kinase inhibitors (TKIs), such as sunitinib, sorafenib and axitinib have been introduced in the treatment of RCC with improved outcomes in the subsets of RCC patients. Tumor microenvironment (TME) plays a critical role in response to TKIs as well as to immuno-oncology drugs including the PD1 inhibitor nivolumab. Given the heterogeneity of the TME in each patient's tumor, optimization of combination treatments is highly desirable. Here, we employed an ex vivo 3D-tumor organoid model with unaltered TME to identify the most effective TKI-nivolumab combinations in individual RCC tumors and further explored the impact on both tumor cell viability and immune cell activation. Materials and Methods: All RCC tumor samples were obtained with patient consent and relevant IRB approval. For the ex vivo assays 3D organoids measuring 150 micron in size were treated with sunitinib, sorafenib, axitinib alone or in combination with nivolumab ex vivo. Treatment-mediated changes in tumor immune cell composition including CD4 and CD8 T-cells, Tregs, NK cells, macrophages, cell surface expression of checkpoint proteins as well as T-cell activation were evaluated by multiplex flow cytometry. Multiplex cytokine assays were performed to assess drug-induced changes in cytokine release. High content confocal analysis was used to quantify tumor cell killing. Results: We showed that TKI and nivolumab treatments alone had limited immune cell activation and tumor cell killing efficacy in RCC while their combination showed significant variations in T-cell activation by flow cytometry analysis and tumor cell killing by high content confocal imaging among different patient samples. We correlated tumor response to ex vivo treatments with tumor characteristics and further characterized the impact of treatment on TME in each tumor sample in an attempt to identify markers associated with drug responsiveness. Conclusion: In this comprehensive study we used a physiologically relevant 3D tumor organoid model to demonstrate the impact of the heterogeneity of TME on TKI/nivolumab treatments in renal cell carcinoma. This approach can be used to identify the most effective drug and drug combinations in RCC and may improve personalized immunotherapy for individual patients in clinical studies. Citation Format: Jared C. Ehrhart, Mibel M. Pabón, Vijayendra Agrawal, Tina Pastoor, Jenny M. Kreahling, Soner Altiok. Optimization of checkpoint inhibitor-TKI combinations in renal cell carcinoma using an ex vivo 3D tumor organoid model of fresh patient tissue with intact TME [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 935.

Abstract 34: A high-content imaging platform for quantitative assessment of immunogenic tumor cell killing and T-cell proliferation in 3D tumoroids of fresh patient tumor samples

Abstract Introduction: Challenges in the development of immunotherapeutics still remain due to the lack of clinically relevant drug screening platforms that recapitulate the complexity of the tumor immune microenvironment. T-cell activation and immunogenic tumor cell death have been the most desired assay readouts to fully explore the efficacy of immuo-oncology drugs and to develop most effective rational drug combinations. Conventional cytotoxicity assays such as Cr51 and LDH release assays are limited in providing clinically relevant data about treatment mediated tumor cell killing by immune cells. Here, we describe a novel 3D live tumor explant model of fresh patient tumor tissue (3D-EXplore) to simultaneously and cost effectively evaluate T-cell activation and tumor cell killing by immuno-oncology drugs. Materials and Methods: All human tumor samples were obtained with patients’ consent and relevant IRB approval. Fresh patient tumor samples of various cancer types (lung, bladder, kidney) were processed to generate unpropagated 3D tumoroids of standard size (100-150 microns). 3D tumoroids were treated with pembrolizumab (Keytruda) and atezolizumab (Tecentriq) alone or in combination with conventional chemotherapeutic drugs. Various combinations of viability, vitality, cell proliferation and cell death fluorogenic dyes were utilized to label tumor cells prior to high content confocal imaging. Treatment-mediated changes in tumor cell death and T-cell activation were quantified by Nilogen’s QTDI-algorithm. The results were corroborated by multiplex flow cytometry and cytokine relase assays. Results: 3D-tumoroids were successfully prepared from different tumor types. Tumor cell viability and immune cell composition including CD4 and CD8 T-cells, Tregs, NK cells and M1/M2 macrophages were monitored over 5 days to ensure obtaining reliable results. Using high-content image analysis and flow cytometric analysis, in selected tumors we observed a significant increase in T-cell activation and tumor cell killing upon ex vivo treatment with pembrolizumab or atezolizumab alone, which was enhanced in the presence of carboplatin. Conclusion: Here we have described a cost-effective and rapid drug testing platform using clinically relevant unpropogated fresh patient tumor tissue for rapid testing of multiple drugs and drug combinations based on tumor cell killing and T-cell activation to accelerate drug discovery, which can be applied in clinical studies to improve personalized immunotherapy for individual patients. Citation Format: Melanie Mediavilla-Varela, Vijayendra Agrawal, Melba Marie Page, Jenny Kreahling, Soner Altiok. A high-content imaging platform for quantitative assessment of immunogenic tumor cell killing and T-cell proliferation in 3D tumoroids of fresh patient tumor samples [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 34.

Abstract 2546: Impact of CDK4/6 inhibition on tumor immune microenvironment and immunomodulatory effects PD-L1 blockade in a physiologically relevant 3D-tumoroid model of fresh patient tumor tissue ex vivo

Abstract Introduction: In mammalian cells, exposure to growth factors triggers de novo synthesis of D-type cyclins, which then associate with their catalytic partners, cyclin dependent kinase CDK4 or CDK6 to regulate the cell cycle. Recent studies implied the role of CK4/6 inhibitors such as Abemaciclib in modulating tumor immune microenvironment and response to PD1/PD-L1 inhibitors. However, the mechanisms by which CD4/6 inhibitors affect the tumor microenvironment is not well understood. Here we investigated the effect of CDK4/6 inhibition by Abemaciclib on the tumor immune microenvironment and response to PD1/PD-L1 inhibitors using Nilogen’s 3D-EX ex vivo drug screening platform utilizing un-propagated 3D tumoroids of fresh patient tumor tissue with an intact tumor microenvironment. Materials and Methods: All non-small cell lung carcinoma tumor samples were obtained with patient consent and relevant IRB approval. For the ex vivo assays 3D tumoroids measuring 100-150 micron in size were treated with Abemaciclib, pembrolizumab (Keytruda) alone or in combination. Treatment-mediated changes in tumor immune cell composition including CD4 and CD8 T-cells, Tregs, NK cells, macrophages and cell surface expression of checkpoint proteins as well as T-cell activation were evaluated by multiplex flow cytometry. Multiplex cytokine assays were performed to assess drug-induced changes in cytokine release. High-content confocal analysis was used to quantify tumor cell killing. Results: We showed that drug treatments had significant impact on immune cell compositions, specifically on M1/M2 macrophage polarization as well as on CD4 and CD8 lymphocyte populations in selected tumors, which was accompanied by significant changes in pro-inflammatory cytokine release. We further characterized pharmacological interaction between Abemaciclib and pembrolizumab and correlated with tumor cell killing in quantitative imaging studies. Conclusion: In this comprehensive study we documented the pharmacological effect of CDK4/6 inhibition on tumor immune microenvironment in a physiologically relevant 3D tumoroid model of ex vivo drug testing. We further characterized the pharmacological interaction between Abemaciclib and pembrolizumab to better understand the mechanisms by which CDK4/6 inhibition may enhance therapeutic response to PD1 blockade in patient tumors. Citation Format: Melba Marie Page, Melanie Mediavilla Varela, Vijayendra Agrawal, Jenny Kreahling, Soner Altiok. Impact of CDK4/6 inhibition on tumor immune microenvironment and immunomodulatory effects PD-L1 blockade in a physiologically relevant 3D-tumoroid model of fresh patient tumor tissue ex vivo [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 2546.

Physiologically-based pharmacokinetic and pharmacodynamic models for gemcitabine and birinapant in pancreatic cancer xenografts.

The anticancer effects of combined gemcitabine and birinapant were demonstrated as synergistic in PANC-1 cells in vitro. In this study, pharmacokinetic information derived from experiments and the literature was utilized to develop full physiologically-based pharmacokinetic (PBPK) models that characterize individual drugs. The predicted intra-tumor drug concentrations were used as the driving force within a linked PBPK/PD model for treatment-mediated changes in tumor volume in a xenograft mouse model. The efficacy of the drug combination in vivo was evaluated mathematically as exhibiting additivity. The network model developed for drug effects in the in vitro cell cultures was applied successfully to link the in vivo tumor drug concentrations with tumor growth inhibition, incorporating more mechanistic features and accounting for disparate drug interaction outcomes in vitro and in vivo.

Abstract LB-347: Integrated Comprehensive Analysis of Immune Cell Subsets and Assessment of Checkpoint Inhibitor Response in Head and Neck Squamous Cell Carcinoma, Urothelial Carcinoma and Renal Cell Carcinoma 3D Ex Vivo

Abstract Background: Since FDA approval in 2014, PD-1 blocking antibodies including Keytruda (pembrolizumab) have been used in the treatment of different types of malignancies including head and neck squamous cell, urothelial and renal cell carcinoma. Each solid tumor type not only varies patient-to-patient, but also varies in its heterogeneity, including but not limited to, its immune cell infiltrate. The impact of checkpoint inhibitor treatment on tumor immune landscape is not fully understood in solid tumors. In this study, we used an integrated comprehensive strategy to interrogate tumor immune cell compositions and T-cell activation in intact tumors before and after ex vivo treatment with checkpoint inhibitors utilizing Nilogen Oncosystems' 3D-EX drug screening platform. Methods: For the 3D-EX platform, 3D tumor microspheres were produced from fresh head and neck squamous cell carcinoma, urothelial carcinoma and renal cell carcinoma tumor tissue obtained from consented patients at the time of surgical resection. Flow cytometry, Nanostring's PanCancer Immune Profiling Panel and bioplex multiplex human cytokine assay were used to analyze tumor immune microenvironment and treatment-mediated changes by checkpoint inhibitors. Results: The flow cytometry analysis revealed a significant heterogeneity in immune cell composition (TILs, macrophages and MDSCs), T-cell activation status and checkpoint expressions between different tumor types. In our studies tumor responses to treatment ex vivo varied significantly for each tumor types, as assessed by activation of CD8 and CD4 T-cells by flow. Furthermore, Nanostring analysis revealed increased expression of genes involved in T-cell activation as well as genes with compensatory regulatory functions such as IDO1, LAG3, TIM3 and PD-L1 in tumor samples responsive to treatment ex vivo. Bioplex cytokine assays performed on the media demonstrated increased release of cytokines such as IFNg, TNFa, IL2, IL1b, IL6, GM-CSF and MIP1 that closely correlated with T-cell activation. Conclusion: 3D-EX platform can successfully detect checkpoint inhibitor-mediated activation in T-cells within microspheroids prepared from surgical samples obtained from fresh patient tumors. 3D-EX platform is versatile and provide valuable information on mechanisms involved in drug sensitivity and resistance. This approach might aid in development of rational mechanism-based combination strategies to block compensatory signaling mechanisms to impart clinical benefit. Citation Format: Melba Marie Page, Melanie Mediavilla Varela, Jenny Kreahling, Soner Altiok. Integrated Comprehensive Analysis of Immune Cell Subsets and Assessment of Checkpoint Inhibitor Response in Head and Neck Squamous Cell Carcinoma, Urothelial Carcinoma and Renal Cell Carcinoma 3D Ex Vivo [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 LB-347.

Abstract 1020: Characterization of the immune landscape and analysis of tumor response after anti-PD-1 blockade in a 3D ex vivo system of non-small lung cancer

Abstract Background: Immuno-oncology drugs face the challenge that cell lines and animal models cannot recapitulate the full intimacies of the tumor microenvironment. Although these models have aided in the discovery of new medical modalities, only 8% of these models have had a successful translation. Furthermore, the phenotype of the microenvironment of various tumor types has not been assessed in depth. Nilogen Oncosystems' proprietary 3D-EX vivo drug screening platform analyzes fresh patient tumor tissue that remains embedded in its natural environment. With our model, the characteristics of the tumor microenvironment can be accurately revealed in response to checkpoint inhibitors in non-small cell lung cancer (NSCLC) and correlates can be found among different platforms. Methods: For the 3D-EX vivo platform, microspheres were produced from procured fresh tumor tissue from NSCLC cancer patients. They were then treated with Keytruda ex vivo and treatment-mediated changes in TIL subpopulations were analyzed using flow cytometric analysis, cytokine release by Bio-Rad's 17-plex cytokine assay as well as gene expression by NanoString's 770 gene Immune Panel. Results: Ex vivo treatment of the 3D microspheres with Keytruda, showed significant changes in T-cell activation and immune cell populations in 26% of NSCLC tumors. This was observed by the simultaneous increase in IFN-γ and TNF-α upon cytokine analysis. Furthermore, we found a differential expression of signature genes such as CD8, CXCL10, CXCL9, EOMES, Granzyme A/B, IFN-γ, related to T-cell subpopulations via Nanostring analysis, which was accompanied by an increase in Granzyme B via flow cytometry. Conclusion: The positive and negative associations between expression of immune function genes, TIL activation, and cytokine production by ex vivo treatment shows that Nilogen Oncosystems' 3D-Ex vivo platform can accurately recapitulate the tumor microenvironment and exhibits the importance of a comprehensive analysis of the tumor immune microenvironment for a better understanding of the mechanism of action of immuno-oncology drugs that may aid in developing biomarkers that can be used for patient selection. Citation Format: Melanie Mediavilla-Varela, Melba Marie Page, Jenny Kreahling, Soner Altiok. Characterization of the immune landscape and analysis of tumor response after anti-PD-1 blockade in a 3D ex vivo system of non-small lung cancer [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 1020.

The neurobiology and neural circuitry of cognitive changes in Parkinson's disease revealed by functional neuroimaging

Patients with Parkinson's disease (PD) often develop a spectrum of cognitive symptoms that can evolve into dementia. Dopamine (DA) replacement medications, though improving motor symptoms, can exert both positive and negative effects on cognitive ability, depending on the severity of the disease and the specific skill being tested. By considering the behavioral and clinical aspects of disease- and treatment-mediated changes in cognition alongside the pathophysiology of PD, an understanding of the factors that govern the heterogeneous expression of cognitive impairment in PD is beginning to emerge. Here, we review the neuroimaging studies revealing the neural correlates of cognitive changes after DA loss and DA replacement as well as those that may accompany the conversion from milder stages of cognitive impairment to frank dementia.

Network correlates of the cognitive response to levodopa in Parkinson disease

Cognitive dysfunction is common in Parkinson disease (PD), even early in its clinical course. This disease manifestation has been associated with impaired verbal learning performance as well as abnormal expression of a specific PD-related cognitive spatial covariance pattern (PDCP). It is not known, however, how this metabolic network relates to the cognitive response to dopaminergic therapy on the individual patient level. We assessed treatment-mediated changes in verbal learning and PDCP expression in 17 patients with PD without dementia who underwent cognitive testing and metabolic imaging in the unmedicated and levodopa-treated conditions. We also determined whether analogous changes were present in 12 other patients with PD without dementia who were evaluated before and during the treatment of cognitive symptoms with placebo. Levodopa-mediated changes in verbal learning correlated with concurrent changes in PDCP expression (r = -0.60, p < 0.01). The subset of patients with meaningful cognitive improvement on levodopa (n = 8) exhibited concurrent reductions in PDCP expression (p < 0.01) with treatment; network modulation was not evident in the remaining subjects. Notably, the levodopa cognitive response correlated with baseline PDCP levels (r = 0.70, p = 0.002). By contrast, placebo did not affect PDCP expression, even in the subjects (n = 7) with improved verbal learning during treatment. These findings suggest that cognitive dysfunction in PD may respond to treatment depending upon the degree of baseline PDCP expression. Quantification of treatment-mediated network changes can provide objective information concerning the efficacy of new agents directed at the cognitive manifestations of this disease.

Differentially Expressed Genes in Window Trials are Influenced by the Wound-Healing Process: Lessons Learned from a Pilot Study with Anastrozole

Perioperative window trials provide an opportunity to obtain intact tumor samples at two different time-points for evaluation of potential surrogate biomarkers. We report results of a pilot trial designed to determine if treatment-mediated changes in gene expression can be detected in formalin-fixed paraffin-embedded (FFPE) samples after 10-d exposure to anastrozole in estrogen receptor (ER)-positive breast cancer compared with untreated controls. Paired tumor samples (biopsy, surgical) were obtained from 26 postmenopausal women with ER-positive breast cancer. Patients were assigned anastrozole (1 mg/d) for 10 d immediately prior to surgery (13 cases) or no treatment (13 controls). Five hundred two cancer-related genes were examined by the Illumina cDNA-mediated annealing, selection, extension, and ligation, FFPE cDNA array (moderated t-test, P ≤ 0.005). Surrogate biomarkers reflecting changes in gene expression were examined by immunohistochemistry (Wilcoxon rank-based test, P < 0.05). Sufficient RNA was available from 19 paired samples (8 controls, 11 cases). Frozen tissue and FFPE showed good correlation (r = 0.82). Within each group, 18 genes, reflecting roles in proliferation, angiogenesis, and apoptosis, showed differential expression from biopsy to surgery (P < 0.005). Estrogen-related genes were dysregulated in the treated group only. A reduction in Ki-67 was observed in 7 (54%) treated cases and in 1 (7.7%) control patient. 10-d exposure to anastrozole resulted in dysregulation of 18/502 cancer-related genes, and Ki-67 was reduced in 54% of cases. FFPE samples demonstrated good correlation with frozen samples. However, changes in gene expression and increased Ki-67 in the control group suggest local effects of wound healing may represent a confounding factor in the interpretation of perioperative window trials.

Parkinson's disease tremor-related metabolic network: Characterization, progression, and treatment effects

The circuit changes that mediate parkinsonian tremor, while likely differing from those underlying akinesia and rigidity, are not precisely known. In this study, to identify a specific metabolic brain network associated with this disease manifestation, we used FDG PET to scan nine tremor dominant Parkinson's disease (PD) patients at baseline and during ventral intermediate (Vim) thalamic nucleus deep brain stimulation (DBS). Ordinal trends canonical variates analysis (OrT/CVA) was performed on the within-subject scan data to detect a significant spatial covariance pattern with consistent changes in subject expression during stimulation-mediated tremor suppression. The metabolic pattern was characterized by covarying increases in the activity of the cerebellum/dentate nucleus and primary motor cortex, and, to a less degree, the caudate/putamen. Vim stimulation resulted in consistent reductions in pattern expression (p<0.005, permutation test). In the absence of stimulation, pattern expression values (subject scores) correlated significantly (r=0.85, p<0.02) with concurrent accelerometric measurements of tremor amplitude.To validate this spatial covariance pattern as an objective network biomarker of PD tremor, we prospectively quantified its expression on an individual subject basis in independent PD populations. The resulting subject scores for this PD tremor-related pattern (PDTP) were found to exhibit: (1) excellent test–retest reproducibility (p<0.0001); (2) significant correlation with independent clinical ratings of tremor (r=0.54, p<0.001) but not akinesia-rigidity; and (3) significant elevations (p<0.02) in tremor dominant relative to atremulous PD patients.Following validation, we assessed the natural history of PDTP expression in early stage patients scanned longitudinally with FDG PET over a 4-year interval. Significant increases in PDTP expression (p<0.01) were evident in this cohort over time; rate of progression, however, was slower than for the PD-related akinesia/rigidity pattern (PDRP). We also determined whether PDTP expression is modulated by interventions specifically directed at parkinsonian tremor. While Vim DBS was associated with changes in PDTP (p<0.001) but not PDRP expression, subthalamic nucleus (STN) DBS reduced the activity of both networks (p<0.05). PDTP expression was suppressed more by Vim than by STN stimulation (p<0.05).These findings suggest that parkinsonian tremor is mediated by a distinct metabolic network involving primarily cerebello-thalamo-cortical pathways. Indeed, effective treatment of this symptom is associated with significant reduction in PDTP expression. Quantification of treatment-mediated changes in both PDTP and PDRP scores can provide an objective means of evaluating the differential effects of novel antiparkinsonian interventions on the different motor features of the disorder.

Dopaminergic Suppression of Brain Deactivation Responses during Sequence Learning

Cognitive processing is associated with deactivation of the default mode network. The presence of dopaminoceptive neurons in proximity to the medial prefrontal node of this network suggests that this neurotransmitter may modulate deactivation in this region. We therefore used positron emission tomography to measure cerebral blood flow in 15 Parkinson's disease (PD) patients while they performed a motor sequence learning task and a simple movement task. Scanning was conducted before and during intravenous levodopa infusion; the pace and extent of movement was controlled across tasks and treatment conditions. In normal and unmedicated PD patients, learning-related deactivation was present in the ventromedial prefrontal cortex (p < 0.001). This response was absent in the treated condition. Treatment-mediated changes in deactivation correlated with baseline performance (p < 0.002) and with the val(158)met catechol-O-methyltransferase genotype. Our findings suggest that dopamine can influence prefrontal deactivation during learning, and that these changes are linked to baseline performance and genotype.

Dissociation of Metabolic and Neurovascular Responses to Levodopa in the Treatment of Parkinson's Disease

We compared the metabolic and neurovascular effects of levodopa (LD) therapy for Parkinson's disease (PD). Eleven PD patients were scanned with both [15O]-H2O and [18F]-fluorodeoxyglucose positron emission tomography in the unmedicated state and during intravenous LD infusion. Images were used to quantify LD-mediated changes in the expression of motor- and cognition-related PD covariance patterns in scans of cerebral blood flow (CBF) and cerebral metabolic rate for glucose (CMR). These changes in network activity were compared with those occurring during subthalamic nucleus (STN) deep brain stimulation (DBS), and those observed in a test-retest PD control group. Separate voxel-based searches were conducted to identify individual regions with dissociated treatment-mediated changes in local cerebral blood flow and metabolism. We found a significant dissociation between CBF and CMR in the modulation of the PD motor-related network by LD treatment (p < 0.001). This dissociation was characterized by reductions in network activity in the CMR scans (p < 0.003) occurring concurrently with increases in the CBF scans (p < 0.01). Flow-metabolism dissociation was also evident at the regional level, with LD-mediated reductions in CMR and increases in CBF in the putamen/globus pallidus, dorsal midbrain/pons, STN, and ventral thalamus. CBF responses to LD in the putamen and pons were relatively greater in patients exhibiting drug-induced dyskinesia. In contrast, flow-metabolism dissociation was not present in the STN DBS treatment group or in the PD control group. These findings suggest that flow-metabolism dissociation is a distinctive feature of LD treatment. This phenomenon may be especially pronounced in patients with LD-induced dyskinesia.