Lay Summary

Diffuse intrinsic pontine glioma (DIPG) is a rare form of inoperable cancer arising in the brainstem of children, with poor response to chemotherapy and no improvement in outcomes over the past 3 decades. The unique brainstem location and the invasive nature of DIPG is suboptimally represented in modern therapeutic models and patient derived DIPG cell lines grown via standard culture techniques.  As a result of this mismatch between preclinical models and the native tumor, responses to clinical drug trials have been dismal. Understanding how the tumor cell interacts with the tumor microenvironment (TME) - an area populated with diverse brain-specific cell types and the non-cellular tissue scaffold - is the next critical step towards better understanding the disease process and advancing treatment. Leveraging our ability to grow cells in three-dimensional (3D) culture conditions, we have designed an environment that enables DIPG cells to grow and interact with support cells and the surrounding extra-cellular framework, simulating the tumor’s biology in pediatric patients. In the proposed study, this methodology will allow us to more accurately study the mechanism of cancer cell communication with the microenvironment, and will enable the search for treatment strategies that interrupt this dynamic ecosystem.

DIPG is a universally fatal brainstem cancer in children. Several studies have shown that genetic changes in tumor cells explain a certain portion of the tumor’s aggressive behavior, but these do not fully explain tumor development, growth, and chemotherapy resistance. Tumor growth and response to treatment is highly affected by the specialized niche or microenvironment that confers distinct functions to the cancer cells. It is well recognized that cancer cells and the surrounding environment of the non-cancerous tissue co-evolve with time. This crosstalk between cells in the microenvironment results in the transformation of neighboring non-cancerous cells into cancer-promoting tissue that directly affects the growth and invasion of DIPG.  To improve critical understanding of DIPG invasiveness and dismal treatment response, it is especially important to understand the impact of the dynamic microenvironment. To this end, using a cell culture system to grow and monitor the interaction of multiple cell types to replicate aspects of the microenvironment, we have developed DIPG tumor tissue analogs with color-coded cell types along with a unique model of the structural support tissue around tumor cells which is derived from the human brain that can be maintained for several weeks.  This will allow us to monitor the ability of DIPG and neighboring cells to change in response to their environment, and determine how they influence each other over time.  This phenomenon has been a key deficiency of prior experimental models, and will provide insights that can transform our understanding of DIPG biology, and directly enable better therapeutic drug development.

Executive Summary

Background: Diffuse intrinsic pontine glioma (DIPG) is a rapidly progressive and universally fatal malignancy of the brainstem, affecting children mostly between 5-9 years of age. Median overall survival for DIPG is less than one year after diagnosis. Outcomes have not changed over the past decades, owing in part to chemotherapy resistance, as well as a historical lack of biopsy tissue from this delicate area of the brain.  Although modern neuronavigation now permits safe surgical biopsy, available tissue for study from this disease remains limited in comparison to other tumors of the brain. Decades of clinical trials suggest that a multimodal therapeutic approach utilizing cell-intrinsic, microenvironmental, and immunotherapeutic targets may provide improved treatment avenues for DIPG. Emphasis has been placed on the DIPG tumor microenvironment - a dynamic network of neoplastic cells along with non-neoplastic neuronal and stromal cells. Research has revealed that these cells recruited from the neighboring host tissue, along with the local brain extracellular matrix (BECM) scaffold, establish a favorable niche for the growth and maintenance of malignancy. The interactions between the neoplastic and the non-neoplastic cell types in the developing tumor influence the microenvironment, which in turn contributes to properties of DIPG stemness and promotes a locally invasive phenotype.

To understand malignancy and invasiveness of DIPG it is relevant to fully understand the tumor microenvironment. The infiltrative pathology and the unique ecological niche of these pontine tumors are not adequately represented in current experimental design using animal models and patient derived cell lines. An improved representative model of the tumor microenvironment has been devised, that includes tumor tissue analogs (TTA) formed through 3D co-culture, with monitored size, cell number, and cellular composition.  These TTAs are capable of self-assembling into tissue-like microstructures with environmental characteristics more akin to native tumors.  Exploiting the 3D TTA to elucidate the mechanistic basis of microenvironment-induced changes in the tumor cells will provide insights into chemotherapy resistance mechanisms, and will also help explain the molecular basis of stroma-induced synthetic lethality. The proposed study has the potential to provide therapeutic targets for validation in clinical trials, circumventing the logistical challenges of developing biomarkers identified from the small and heterogeneous pediatric patient population of DIPG and expediting the discovery of treatments and cures. This improved mechanistic approach to modern treatment testing falls well in line with advancing The Cure Starts Now Foundation’s mission of “Homerun Cure.”

Feasibility and expected effectiveness of approach/strategy: The anticipated success of this project is facilitated by the multidisciplinary team of cancer and neuro-oncology researchers, along with broad institutional support that will provide ready access to resources and core support far beyond those conventionally available. The proposed study will employ a range of complementary strategies including cutting edge molecular and imaging platforms along with diverse computational analysis to interrogate the permissive TME in 3D TTA. The cost-effective access to these cutting-edge resources has been guaranteed/supported by leadership (Dr. Rex Moats) at The Saban Research Institute (TSRI) of Children’s Hospital Los Angeles (CHLA). Based on our preliminary understanding in the development of assay systems for semi-quantitative analyses in 3D models (Dr. Upreti), including tumor permissive microenvironment representative of Breast and Lung cancer [1-6], together with the proficiency in DIPG biology [7-9], DIPG clinical care, and access to human brain tissue samples by Dr. Chiarelli (Co-PI), we expect a higher level of refinement in the characterization of the DIPG niche. The expertise in quantitative multi-feature phenotypic image analysis for 3D tissue culture models available at CHLA/TSRI, combined with the depth of our preliminary work, make the proposed experiments in the study highly feasible.

Hypothesis: Investigation of tumor cell biology and treatment response to new therapeutic targets is more informative when performed using a representative model of the native tumor niche, including stromal components and scaffold microenvironment that exist in human pediatric tumors.

We have identified the following three specific aims to test our hypothesis:

Aim 1: Address tumor-stromal cell interactions by implementation of a 3D tumor tissue analog model.

Design: Mechanistic insights into the complex and dynamic tumor-stromal interactome in DIPG is crucial for the identifying of novel markers capable of serving as therapeutic targets and used as prognostic factors.

Methods: We have devised a 3D co-culture system to enable self-assembly of multiple cell types into tumor tissue analogs of DIPG in vitro. The spatio-temporal interactions of the fluorescently labeled tumor and stromal cell types in the DIPG TTA will be monitored using chronic, live and episodic imaging techniques. An integrated multi-omic (Protein and RNA) approach will be used to reveal global rewiring of gene expression both at the transcript (RNA-seq) and protein (Mass Spectrometry) level during the co-culture of different cell types in the 3D TTA.

Aim 2: Investigate the stromal modulation of DIPG resistance to chemotherapy and targeted therapies. 

Design: Exploiting the benefits of the third dimension in regaining the tissue-related functions of DIPG utilizing color-coded florescent tumor and stromal cell types and cutting-edge imaging techniques is essential for increasing the predictive power of therapeutic assays in vitro.

Methods: Utilizing mechanistic endpoints of morphometric and imaging modality, we will investigate aspects of microenvironmental regulation of chemoresistance (Temozolomide) and response to H3K27M targeted therapies (Panabinostat) in the 3D TTA along with the interrogation of molecular markers

Aim 3: Examine the role of native brain extracellular matrix in regulating DIPG cell migration, malignant phenotype, and molecular interactions using decellularized human brain-derived tissue.

Design: Repopulating the decellularized human brain matrix with 3D TTA comprised of tumor and stromal cell types will provide us with insights into the behavior of DIPG cells in relation to the mechanical and biochemical signals in the native stem cell niche.

Methods: IRB approval for procuring discarded brain tissue during live human neurosurgical procedures requiring neocortical resection (e.g. epilepsy surgery) has been obtained (CHLA-21-00088). The tissue will be decellularized and repopulated with the 3D TTA to monitor and track the fluorescently labeled tumor and stromal cell types. Phenotypic endpoints will be used for molecular and biochemical analysis.

Significance: DIPG is among the most devastating of childhood cancers, with no effective treatment for cure or long-term improvement in quality of life. The underrepresentation of the TME reminiscent of the disease in patients in current experimental designs is a deficiency that has impeded optimal understanding of the unique stem cell properties and maintenance of the undifferentiated state of cancer cells within the dysregulated DIPG tissue. The proposed study addresses a significant need in DIPG research by utilizing a novel in vitro 3D co-culture pre-clinical platform that mimics many aspects of DIPG in-vivo. The 3D TTA engaged in this study overcomes critical limitations of existing research in the development of effective therapeutic interventions for DIPG for the following reasons: Inclusion of stromal support cell types of human origin empowers it to capture the dynamic interaction of complex cellular and acellular components in the DIPG niche that contribute to “stemness” of DIPG cells and disease progression. Such an understanding, in turn, facilitates better drug screening and development. 

The tunable bioengineered platform integrating microenvironmental cues of the human-derived extracellular brain scaffold facilitates “continuous” or long-term cell culture for simulating tumor-stromal progression over time in the native DIPG tissue microenvironment.

• The potential of the proposed 3D platform utilizing DIPG TTA lends itself to controlled manipulation in a reproducible manner, and is a powerful tool with an enhanced predictive power and potential to accelerate drug development.
• Labeling tumor and stromal cell types with fluorescent markers and performing molecular analysis of histological parameters in the 3D tumor tissue analogs will assist in delineating the spatio-temporal organization of DIPG niche and facilitate quantitative (image-based) tracking of different cell types in culture. This is essential for improved preclinical understanding of novel and combination therapies for clinical translation.
• Engaging a range of complementary strategies including molecular (omics) and high-resolution imaging/tracking in real time for extended duration followed by a comprehensive analysis using advanced computational programs will assist in delineating the extracellular architectural aspects of DIPG, along with the associated dynamic molecular interactions.

• High-throughput and high-content (image-based) screening allow for multiparametric quantitative analysis of the tumor-stromal interactions. The ability of high throughput multiparametric assay systems proposed to provide quantitative, reproducible read-outs with focused and mechanistically simple interpretations should simplify the evaluation and validation processes for DIPG research.

Overall goal: The proposed study utilizes a 3D in-vitro platform incorporating critical microenvironment characteristics of DIPG to interpret the complex multifactorial cooperation between tumor and stromal cell types in the DIPG niche during tumor progression and disease management.

Future Directions. The results of this work will form the basis of additional studies and extramural funding. We plan to translate aspects of this work to screen and validate potential multimodal/combinatorial therapeutic regimens, in collaboration with national resources including the Pediatric Brain Tumor Consortium (PBTC) and the Pacific Pediatric Neuro-Oncology Consortium (PNOC). Our ultimate goal is to apply this knowledge to the clinic and develop targeted therapies that disrupt essential tumor-microenvironment interactions for the benefit of patients.

References:

1.         Chan R, Sethi P, Jyoti A, McGarry R, Upreti M: Investigating the Radioresistant Properties of Lung Cancer Stem Cells in the Context of the Tumor Microenvironment. Radiation research 2016,              185(2):169-181.

2.         Jyoti A, Fugit KD, Sethi P, McGarry RC, Anderson BD, Upreti M: An in vitro assessment of liposomal topotecan simulating metronomic chemotherapy in combination with radiation in tumor-                endothelial spheroids. Scientific reports 2015, 5:15236.

3.         Sethi P, Jyoti A, Swindell EP, Chan R, Langner UW, Feddock JM, Nagarajan R, O'Halloran TV, Upreti M: 3D tumor tissue analogs and their orthotopic implants for understanding tumor-targeting              of microenvironment-responsive nanosized chemotherapy and radiation. Nanomedicine : nanotechnology, biology, and medicine 2015, 11(8):2013-2023.

4.         Stocke NA, Sethi P, Jyoti A, Chan R, Arnold SM, Hilt JZ, Upreti M: Toxicity evaluation of magnetic hyperthermia induced by remote actuation of magnetic nanoparticles in 3D micrometastasic              tumor tissue analogs for triple negative breast cancer. Biomaterials 2017, 120:115-125.

5.         Upreti M: Tumor Tissue Analogs for the Assessment of Radioresistance in Cancer Stem Cells. Methods in molecular biology (Clifton, NJ) 2018, 1692:117-128.

6.         Upreti M, Jamshidi-Parsian A, Koonce NA, Webber JS, Sharma SK, Asea AA, Mader MJ, Griffin RJ: Tumor-Endothelial Cell Three-dimensional Spheroids: New Aspects to Enhance Radiation                    and  Drug Therapeutics. Translational oncology 2011, 4(6):365-376.

7.         Chiarelli PA, Hauptman JS, Browd SR: Machine Learning and the Prediction of Hydrocephalus: Can Quantitative Image Analysis Assist the Clinician? JAMA pediatrics 2018, 172(2):116-118.

8.         Chiarelli PA, Kievit FM, Zhang M, Ellenbogen RG: Bionanotechnology and the future of glioma. Surgical neurology international 2015, 6(Suppl 1):S45-58.

9.         Stephen ZR, Chiarelli PA, Revia RA, Wang K, Kievit F, Dayringer C, Jeon M, Ellenbogen R, Zhang M: Time-Resolved MRI Assessment of Convection-Enhanced Delivery by Targeted and                            Nontargeted Nanoparticles in a Human Glioblastoma Mouse Model. Cancer research 2019, 79(18):4776-4786.

Description of Research Proposal

Budget

Collaborations and Conflicts of Interest