Abstract Human cancer tissue is a complex ecosystem with heterogeneous cell types that communicate with each other dynamically to promote tumor progression and immune evasion. Hence, experimental systems that destroy or select for cancer cells in culture have often failed to accurately predict clinical responses of new drugs. Therefore, a technology that can retain the intact tumor microenvironment (TME) and accurately predict cancer patient’s response to anti-cancer treatments, including immunotherapies, will greatly benefit patients and dramatically reduce the cost of new drug development. E-slice is a proprietary 3D human tumor culture platform that maintains individual patient tumor’s unique ecosystem and the TME ex vivo for up to 30 days. It is generated by making thin sections of intact, fresh tumor tissues and culturing them in serum-free, defined media. As such, immune components and the TME in E-slices faithfully recapitulate human tumors ex vivo. We demonstrate that E-slices can be used to: 1) measure viability changes upon anti-cancer agent treatment longitudinally; 2) retain the native TME and tissue architecture since E-slices are never dissociated or artificially reconstituted; 3) culture any solid tumor types from patient needle biopsy cores or surgical samples, PDX, or mouse models; 4) perform high content drug screening on human tissues; 5) discover secreted biomarkers and to 6) discover or validate molecular mechanisms of action or therapy resistance; and 7) measure immunotherapy responses ex vivo from human tissues. Importantly, it has been shown to accurately predict individual patient treatment responses to chemotherapies and targeted therapies in 4-8 days, paving the way for evidence-based personalized treatment selections in a clinically actionable time frame. Furthermore, single cell RNA-sequencing analysis demonstrates that E-slices can retain the viability and molecular phenotypes of tumor infiltrating immune cells for up to 8 days ex vivo, providing a unique platform to study human tumor-immune interactions and evaluate efficacy of immune modulators on human tumor tissues ex vivo. Citation Format: Viridiana Leyva-Aranda, Thomas Gallup, Jose Maldonado, Corina Margain, Sang Yun, David Gallup, Min Kim, Kyuson Yun. A novel 3D culture platform, E-slice, retains intact tumor microenvironment and tumor infiltrating immune cells [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 4200.

Abstract Immunotherapy is a promising treatment modality for GBM; however, clinical trials thus far have failed to provide significant clinical benefit to most patients. GBM is one of the “immune cold” tumors characterized by poor infiltration of T cells and the abundance of immune-suppressive myeloid cells that block T cell infiltration and/or activation. A major bottleneck to developing more effective immunotherapies is the paucity of faithful preclinical models to discover and validate new treatments. Here, we report multi-dimensional analyses of six different transplantable mouse glioma models in the C57BL6/J background that represent the three human GBM molecular subtypes. We performed whole exome sequencing, STR fingerprinting, immune phenotyping, and single-cell RNA-sequencing of each model and confirmed that the new models exhibit highly heterogeneous and immune suppressive TME, similar to human GBM. In addition, we performed cross-species comparisons of glioma and immune cell subtypes between human and mouse GBMs at the single-cell level and demonstrate strong concordances in molecular phenotypes and cellular heterogeneity. These result in qualitative and quantitative differences in the cell: cell communication among different stromal cells and glioma cells in each model, and we propose that these interactions shape the local niche and immune suppressive neighborhoods. Notably, in both human tissues and mouse models, PD1/PDL1 expression is low or sporadic, and, unlike the GL261 model, the new mouse models are resistant to anti-PD1 and CTLA4 treatment in vivo. Finally, we discovered that VISTA is a widely expressed immune checkpoint in both human and mouse GBM and demonstrate the efficacy of anti-VISTA and anti-PD1 combination therapy in the most immune-suppressive mouse model. In summary, we demonstrate the utility of these immune-competent mouse models and nominate anti-VISTA+PD1 treatment as a promising novel combination therapy for GBM.

Single cell RNA-sequencing (scRNA-seq) yields valuable insights into the molecular heterogeneity of multiple cell types in normal and cancer tissues. scRNA-seq approach has the potential to not only answer basic research questions but also enhance clinical studies and clinical trials and accelerate drug development and testing. However, a consistent and reproducible method for isolating viable single cells and generating high quality single cell data from needle biopsy cores has been challenging to develop. Based on our experience isolating single cells from a large number of surgical tumor tissues, we set out to optimize a dissociation and processing protocol to generate viable single cell suspensions in adequate quantity for scRNA-seq from needle biopsy cores. A total of 17 de-identified 18-gauge needle biopsy samples were collected in collaboration with MD Anderson Cancer Center investigators. Samples were from liver, lymph node, pelvis, abdominal, neck, and lung tissues and included matched pre- and post-treatment samples. Patient biopsies were kept in RPMI medium on ice and transferred to EMPIRI (time to capture ranged from 3 to 6.5 hours after biopsy collection). Samples were dissociated using mechanical and enzymatic methods to achieve a single cell suspension. Cells were washed and viability and cell counts were verified before capture via 10x Chromium Connect. Captured cells were processed through the 10x Genomics Chromium Next GEM Single Cell workflow before sequencing on Illumina NovaSeq 6000 sequencer. Raw sequencing data were processed through EMPIRI’s computational pipeline that includes QC steps, doublet removal, and Seurat and other downstream analyses. Recovery of viable cells from individual 18-gauge biopsy cores ranged from 2,000-300,000 cells per core, depending on the tissue type and cellularity. Cell viability was strictly monitored and was maintained above 80% for 16 of 17 samples (most samples >90%). Cell counts after sequencing and pipeline analysis averaged 6,466 cells per sample. The median number of UMI counts per cell was 5,580, and an average of 1,699 genes were identified per cell. Average sequencing saturation was 73%. Here, we demonstrate the feasibility of integrating scRNA-seq analysis into clinical trials or clinical studies to obtain rapid insights into on-target drug effects and anticipated cellular responses to therapies, including immunotherapies, within 3 weeks of biopsy collection. We have established a method for isolating high quality single cells for scRNA-seq from tissue biopsy core, as small as a single 18-gauge needle core. We tested our protocol on 17 human samples and showed successful isolation of highly viable cells from lung, lymph node, pelvis, abdominal, neck, and lung tissues. We were able to generate scRNA-seq data sets from all samples for analysis. Acknowledgements: We thank our MDACC collaborators, particularly Drs. Scott Kopetz and Van Morris. Citation Format: Thomas Gallup, Sang Yun, Dave Gallup, Kyuson Yun. Gain early insights from single cell RNA-sequencing of clinical trial needle biopsy cores. [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 5461.

Recent advances in cancer immunotherapy have accomplished clinical successes in certain cancer models over the past decade. However, cancer treatments with adoptive cell transfer or immune checkpoint blockade (ICB) have shown critical limitations against solid tumors, which comprise the majority of human cancers. Thus, novel cancer immunotherapy which harnesses innate immunity process may be required in these tumor types. Cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, one of the innate immune sensors, has gained interest in the field of immuno-oncology as activation of this pathway can drive both innate and adaptive immune responses among immunosuppressive tumor microenvironments. Recently, various cGAS-STING-activating strategies have been intensively investigated to achieve durable and widespread therapeutic responses in in vivo models. These meaningful preclinical outcomes have enabled several clinical trials. This review discusses agents targeting various aspects of the cGAS-STING pathway in cancer immunotherapy from benchtop to bedside. Moreover, various approaches to improve the clinical feasibility of cGAS-STING-activating strategies are delineated.

Combining radiotherapy (RT), a standard of care treatment for many cancer types, with immunotherapeutic agents has been gaining interest due to improved efficacy. One such approach is RT in combination with high-dose recombinant interleukin 2 (IL-2), which elicits anti-tumor immune responses by stimulating T-cell and NK cell populations. Although effective for some patients, the overall clinical benefit of recombinant IL-2 is limited by toxicity at high doses and the expansion of regulatory T cells (Tregs) at low doses, effects thought to be mediated by preferential binding to IL2Rα (i.e. CD25). In this study we examined RT in combination with NL-201, a highly potent and stable CD25-independent IL-2/IL-15 agonist with enhanced affinity for the IL-2Rβγ heterodimeric receptor. Single-agent NL-201 was well tolerated in mice, yielded tumoricidal activity, expanded peripheral T cells, and enhanced the infiltration of effector T cells and dendritic cells (DCs) into murine glioblastoma (GL261 and SB28). In combination with RT, NL-201 enhanced activation of the cytosolic DNA sensor cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, resulting in increased type I interferon (IFN) production in DCs and, consequently, greater tumor infiltration by T cells and more efficient priming of antigen-specific T cells by professional antigen presenting cells (APCs). The immune stimulatory mechanisms triggered by the NL-201 and RT combination resulted in superior tumor growth inhibition. We demonstrated that the CD25-independent IL-2 and IL-15 receptor agonist, NL-201, in combination with RT is well-tolerated and elicits robust anti-tumor activity through both innate and adaptive responses, including in checkpoint resistant tumors. Furthermore, we identify a unique mechanism in which NL-201 synergizes with RT. Taken together, the results provided herein support further preclinical and clinical investigation of this novel combination regimen.

Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults and is virtually incurable. Immunotherapy is a promising new approach to treat GBM as it harnesses one’s own immune system to recognize and kill aberrant cancer cells. Unfortunately ongoing trials with immunotherapies show disappointing results in most glioma patients. GBM has highly immune-suppressive microenvironment. Consistently, mesenchymal subtype, the subtype with worst prognosis, has a strong immune signature. We recently reported that S100A4 is necessary for human and mouse glioma initiating cell (GIC) self-renewal and tumor growth, and that S100A4 is a master regulator of mesenchymal transition in GBM. Importantly, we report that S100A4 regulates expression of cytokines that affect TAM infiltration and polarization towards tumor-promoting phenotype. Consistently, TCGA and IVY-GAP data analyses indicate that S100A4 expression is strongly correlated with GBM patient survival, the mesenchymal subtype, and tumor-promoting TAM (tumor-associated macrophage) and MDSC (myeloid-derived suppressor cell) marker expression (such as CD163, CD204/MSR1, IL10, CD11b, S100A8, and S100A9). S100A4 expression and TAM marker expression strikingly overlap in perivascular and perinecrotic regions, previously reported niches for GICs. Interestingly there is no correlation between S100A4 expression and markers of microglia. Through single cell RNA-sequencing analyses of human GBM samples, we now have evidence that S100A4 is expressed in both glioma cells and infiltrating myeloid cells. When S100a4 is knocked down in mouse glioma cells and transplanted into syngeneic mice, tumor promoting myeloid cell numbers are significantly reduced. We are currently testing the role of S100A4 in bone marrow derived myeloid cells and elucidating the molecular mechanism of S100A4 function. Our unpublished observations strongly suggest that S100A4 is a critical regulator of GBM immune landscape and may be key node that links GICs, mesenchymal transition, and the myeloid cell infiltration.