Heterogeneity In Tumor Microenvironment Research Articles

Leveraging advances in immunopathology and artificial intelligence to analyze in vitro tumor models in composition and space.

Cancer is the leading cause of death worldwide. Unfortunately, efforts to understand this disease are confounded by the complex, heterogenous tumor microenvironment (TME). Better understanding of the TME could lead to novel diagnostic, prognostic, and therapeutic discoveries. One way to achieve this involves in vitro tumor models that recapitulate the in vivo TME composition and spatial arrangement. Here, we review the potential of harnessing in vitro tumor models and artificial intelligence to delineate the TME. This includes (i) identification of novel features, (ii) investigation of higher-order relationships, and (iii) analysis and interpretation of multiomics data in a (iv) holistic, objective, reproducible, and efficient manner, which surpasses previous methods of TME analysis. We also discuss limitations of this approach, namely inadequate datasets, indeterminate biological correlations, ethical concerns, and logistical constraints; finally, we speculate on future avenues of research that could overcome these limitations, ultimately translating to improved clinical outcomes.

Dissecting the single-cell transcriptome network in patients with esophageal squamous cell carcinoma receiving operative paclitaxel plus platinum chemotherapy

Esophageal squamous cell carcinoma (ESCC) accounts for 90% of all cases of esophageal cancers worldwide. Although neoadjuvant chemotherapy (NACT-ESCC) improves the survival of ESCC patients, the five-year survival rate of these patients is dismal. The tumor microenvironment (TME) and tumor heterogeneity decrease the efficacy of ESCC therapy. In our study, 113,581 cells obtained from five ESCC patients who underwent surgery alone (SA-ESCC) and five patients who underwent preoperative paclitaxel plus platinum chemotherapy (NACT-ESCC), were used for scRNA-seq analysis to explore molecular and cellular reprogramming patterns. The results showed samples from NACT-ESCC patients exhibited the characteristics of malignant cells and TME unlike samples from SA-ESCC patients. Cancer cells from NACT-ESCC samples were mainly at the ‘intermediate transient stage’. Stromal cell dynamics showed molecular and functional shifts that formed the immune-activation microenvironment. APOE, APOC1, and SPP1 were highly expressed in tumor-associated macrophages resulting in anti-inflammatory macrophage phenotypes. Levels of CD8+ T cells between SA-ESCC and NACT-ESCC tissues were significantly different. Immune checkpoints analysis revealed that LAG3 is a potential immunotherapeutic target for both NACT-ESCC and SA-ESCC patients. Cell–cell interactions analysis showed the complex cell-cell communication networks in the TME. In summary, our findings elucidate on the molecular and cellular reprogramming of NACT-ESCC and ESCC patients. These findings provide information on the potential diagnostic and therapeutic targets for ESCC patients.

Development and characterization of a novel human 3D model of bone metastasis from breast carcinoma <em>in vitro</em> cultured

Breast cancer frequently metastasizes to the skeleton causing significant morbidity. Here, we set-up a novel and advanced ex vivo model by using fresh tissue from human vertebral bone metastasis from breast carcinoma patients able to retain the tumor microenvironment and tumor cells heterogeneity.

Scouting for common genes in the heterogenous hypoxic tumor microenvironment and their validation in glioblastoma.

The online version contains supplementary material available at 10.1007/s13205-021-02987-2.

Metabolic Modifications, Inflammation, and Cancer Immunotherapy.

Cancer immunotherapy has accomplished significant progresses on treatment of various cancers in the past decade; however, recent studies revealed more and more heterogeneity in tumor microenvironment which cause unneglectable therapy resistance. A central phenomenon in tumor malignancy is metabolic dysfunctionality; it reprograms metabolic homeostasis in tumor and stromal cells thus affecting metabolic modifications on specific proteins. These posttranslational modifications include glycosylation and palmitoylation, which usually alter the protein localization, stability, and function. Many of these proteins participate in acute or chronic inflammation and play critical roles in tumorigenesis and progression. Therefore, targeting these metabolic modifications in immune checkpoints and inflammation provides an attractive therapeutic strategy for certain cancers. In this review, we summarize the recent progresses on metabolic modifications in this field, focus on the mechanisms on how glycosylation and palmitoylation regulate innate immune and inflammation, and we further discuss designing new immunotherapy targeting metabolic modifications. We aim to improve immunotherapy or targeted-therapy response and achieve more accurate individual therapy.

M6A Classification Combined With Tumor Microenvironment Immune Characteristics Analysis of Bladder Cancer.

BackgroundStudies have shown that N6-methyl adenosine (m6A) plays an important role in cancer progression; however, the underlying mechanism of m6A modification in tumor microenvironment (TME) cell infiltration of bladder cancer remains unclear. This study aimed to investigate the role of m6A modification in TME cell infiltration of bladder cancer.MethodsThe RNA expression profile and clinical data of bladder cancer were obtained from The Cancer Genome Atlas and Gene Expression Omnibus. We assessed the m6A modification patterns of 664 bladder cancer samples based on 20 m6A regulators through unsupervised clustering analysis and systematically linked m6A modification patterns to TME cell infiltration characteristics. Gene ontology and gene set variation analyses were conducted to analyze the underlying mechanism based on the assessment of m6A methylation regulators. Principal component analysis was used to construct the m6A score to quantify m6A modification patterns of bladder cancer.ResultsThe genetic and expression alterations in m6A regulators were highly heterogeneous between normal and bladder tissues. Three m6A modification patterns were identified. The cell infiltration characteristics were highly consistent with the three immune phenotypes, including immune rejection, immune inflammation, and immune desert. The biological functions of three m6A modification patterns were different. Cox regression analyses revealed that the m6A score was an independent signature with patient prognosis (HR = 1.198, 95% CI: 1.031–1.390). Patients with a low-m6A score were characterized by increased tumor mutation burden, PD-L1 expression, and poorer survival. Patients in the low-m6A score group also showed significant immune responses and clinical benefits in the CTLA-4 immunotherapy cohort (p =0.0069).ConclusionsThe m6A methylation modification was related to the formation of TME heterogeneity and complexity. Assessing the m6A modification pattern of individual bladder cancer will improve the understanding of TME infiltration characteristics.

MP48-02 SINGLE CELL RNA SEQUENCING OF UTUC IS FEASIBLE AND REVEALS HETEROGENEITY OF THE TUMOR AND IMMUNE MICROENVIRONMENT

You have accessJournal of UrologyBladder Cancer: Upper Tract Transitional Cell Carcinoma II (MP48)1 Sep 2021MP48-02 SINGLE CELL RNA SEQUENCING OF UTUC IS FEASIBLE AND REVEALS HETEROGENEITY OF THE TUMOR AND IMMUNE MICROENVIRONMENT Peter Reisz, Andrew Tracey, Fengshen Kuo, Jasmine Thomas, Quinlan Demac, Tim Clinton, Hong Truong, Andrew Lenis, Lucas Nogueira, Karan Nagar, Taha Merghoub, Bernard Bochner, Eugene Pietzak, David Solit, Kwanghee Kim, and Jonathan Coleman Peter ReiszPeter Reisz More articles by this author , Andrew TraceyAndrew Tracey More articles by this author , Fengshen KuoFengshen Kuo More articles by this author , Jasmine ThomasJasmine Thomas More articles by this author , Quinlan DemacQuinlan Demac More articles by this author , Tim ClintonTim Clinton More articles by this author , Hong TruongHong Truong More articles by this author , Andrew LenisAndrew Lenis More articles by this author , Lucas NogueiraLucas Nogueira More articles by this author , Karan NagarKaran Nagar More articles by this author , Taha MerghoubTaha Merghoub More articles by this author , Bernard BochnerBernard Bochner More articles by this author , Eugene PietzakEugene Pietzak More articles by this author , David SolitDavid Solit More articles by this author , Kwanghee KimKwanghee Kim More articles by this author , and Jonathan ColemanJonathan Coleman More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002074.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Upper tract urothelial carcinoma (UTUC) has unique clinical and molecular characteristics compared to bladder UC. Prior investigations have used bulk profiling of tumor tissue to identify molecular subtypes, classifying the majority of UTUC as luminal and T-cell depleted. Single-cell RNA sequencing (scRNA-seq) allows examination of intra-tumoral heterogeneity and the immune tumor microenvironment (TME). We applied scRNA-seq to better characterize UTUC. METHODS: scRNA-seq was performed on 16 UTUC tissue specimens using an established institutional process and the 10X Genomics platform. Sequencing reads were aligned/captured by 10X Cellranger, R/Seurat package. We assessed the composition of each specimen with known marker genes for molecular subtypes. We then assessed the composition of immune cells in each specimen using known marker genes. We compared high- (HG) and low-grade (LG) specimens by subtype composition and immune cell infiltrates. RESULTS: Table 1 describes cohort characteristics and sequencing metrics. Lineage density analyses demonstrate intra-tumoral heterogeneity with luminal subtype predominance across all samples. CD45+ cell analysis revealed significant heterogeneity of immune cell infiltrates (Figure 1). There were no significant differences in immune cell populations in HG vs. LG. Increased interferon gamma (IFNg) expression in tumor cells is associated with significantly increased immune cell infiltration (Spearman’s rank correlation r = 0.71, p=0.009). CONCLUSIONS: Our scRNA-seq data confirmed luminal subtype predominance and described the heterogeneous TME in UTUC. The statistically significant relationship of tumor cell IFNg with increased overall immune cell infiltration is notable and may have implications for systemic therapy response. Further high-resolution studies are needed to expand our knowledge of the molecular biology and TME of UTUC with implications for treatment selection and disease surveillance. Source of Funding: Thompson Family Foundation © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e863-e863 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Peter Reisz More articles by this author Andrew Tracey More articles by this author Fengshen Kuo More articles by this author Jasmine Thomas More articles by this author Quinlan Demac More articles by this author Tim Clinton More articles by this author Hong Truong More articles by this author Andrew Lenis More articles by this author Lucas Nogueira More articles by this author Karan Nagar More articles by this author Taha Merghoub More articles by this author Bernard Bochner More articles by this author Eugene Pietzak More articles by this author David Solit More articles by this author Kwanghee Kim More articles by this author Jonathan Coleman More articles by this author Expand All Advertisement Loading ...

Salubrinal Enhances Cancer Cell Death during Glucose Deprivation through the Upregulation of xCT and Mitochondrial Oxidative Stress.

Cancer cells have the metabolic flexibility to adapt to heterogeneous tumor microenvironments. The integrated stress response (ISR) regulates the cellular adaptation response during nutrient stress. However, the issue of how the ISR regulates metabolic flexibility is still poorly understood. In this study, we activated the ISR using salubrinal in cancer cells and found that salubrinal repressed cell growth, colony formation, and migration but did not induce cell death in a glucose-containing condition. Under a glucose-deprivation condition, salubrinal induced cell death and increased the levels of mitochondrial reactive oxygen species (ROS). We found that these effects of salubrinal and glucose deprivation were associated with the upregulation of xCT (SLC7A11), which functions as an antiporter of cystine and glutamate and maintains the level of glutathione to maintain redox homeostasis. The upregulation of xCT did not protect cells from oxidative stress-mediated cell death but promoted it during glucose deprivation. In addition, the supplementation of ROS scavenger N-acetylcysteine and the maintenance of intracellular levels of amino acids via sulfasalazine (xCT inhibitor) or dimethyl-α-ketoglutarate decreased the levels of mitochondrial ROS and protected cells from death. Our results suggested that salubrinal enhances cancer cell death during glucose deprivation through the upregulation of xCT and mitochondrial oxidative stress.

Single-Cell Analysis Reveals Spatial Heterogeneity of Immune Cells in Lung Adenocarcinoma.

The impacts of the tumor microenvironment (TME) on tumor evolvability remain unclear. A challenge for nearly all cancer types is spatial heterogeneity, providing substrates for the emergence and evolvability of drug resistance and leading to unfavorable prognosis. Understanding TME heterogeneity among different tumor sites would provide deeper insights into personalized therapy. We found 9,992 cell profiles of the TME in human lung adenocarcinoma (LUAD) samples at a single-cell resolution. By comparing different tumor sites, we discovered high TME heterogeneity. Single-sample gene set enrichment analysis (ssGSEA) was utilized to explore functional differences between cell subpopulations and between the core, middle and edge of tumors. We identified 8 main cell types and 27 cell subtypes of T cells, B cells, fibroblasts and myeloid cells. We revealed CD4+ naive T cells in the tumor core that express high levels of immune checkpoint molecules and have a higher activity of immune-exhaustion signaling. CD8+ T cell subpopulations in the tumor core correlate with the upregulated activity of transforming growth factor-β (TGF-β) and fibroblast growth factor receptor (FGFR) signaling and downregulated T cell activity. B cell subtypes in the tumor core downregulate cytokine production. In this study, we revealed that there was immunological heterogeneity in the TME of patients with LUAD that have different ratios of immune cells and stromal cells, different functions, and various degrees of activation of immune-related pathways in different tumor parts. Therefore, clarifying the spatial heterogeneity of the tumor in the immune microenvironment can help clinicians design personalized treatments.

Current Insights and Advancements in Head and Neck Cancer: Emerging Biomarkers and Therapeutics with Cues from Single Cell and 3D Model Omics Profiling.

Head and neck cancer (HNC) is among the ten leading malignancies worldwide, with India solely contributing one-third of global oral cancer cases. The current focus of all cutting-edge strategies against this global malignancy are directed towards the heterogeneous tumor microenvironment that obstructs most treatment blueprints. Subsequent to the portrayal of established information, the review details the application of single cell technology, organoids and spheroid technology in relevance to head and neck cancer and the tumor microenvironment acknowledging the resistance pattern of the heterogeneous cell population in HNC. Bioinformatic tools are used for study of differentially expressed genes and further omics data analysis. However, these tools have several challenges and limitations when analyzing single-cell gene expression data that are discussed briefly. The review further examines the omics of HNC, through comprehensive analyses of genomics, transcriptomics, proteomics, metabolomics, and epigenomics profiles. Patterns of alterations vary between patients, thus heterogeneity and molecular alterations between patients have driven the clinical significance of molecular targeted therapies. The analyses of potential molecular targets in HNC are discussed with connotation to the alteration of key pathways in HNC followed by a comprehensive study of protein kinases as novel drug targets including its ATPase and additional binding pockets, non-catalytic domains and single residues. We herein review, the therapeutic agents targeting the potential biomarkers in light of new molecular targeted therapies. In the final analysis, this review suggests that the development of improved target-specific personalized therapies can combat HNC’s global plight.

Remodeling of Tumor Microenvironment by Tumor-Targeting Nanozymes Enhances Immune Activation of CAR T Cells for Combination Therapy.

Targeting B7-H3 chimeric antigen receptor (CAR) T cells has antitumor potential for therapy of non-small cell lung cancer (NSCLC) in preclinical studies. However, CAR T cell therapy remains a formidable challenge for the treatment of solid tumors due to the heterogeneous and immunosuppressive tumor microenvironment (TME). Nanozymes exhibit merits modulating the immunosuppression of the tumor milieu. Here, a synergetic strategy by combination of nanozymes and CAR T cells in solid tumors is described. This nanozyme with dual photothermal-nanocatalytic properties is endowed to remodel TME by destroying its compact structure. It is found that the B7-H3 CAR T cells infused in mice engrafted with the NSCLC cells have superior antitumor activity after nanozyme ablation of the tumor. Importantly, it is found that the changes altered immune-hostile cancer environment, resulting in enhanced activation and infiltration of B7-H3 CAR T cells. The first evidence that the process of combination nanozyme therapyeffectively improves the therapeutic index of CAR T cells is presented. Thus, this study clearly supports that the TME-immunomodulated nanozyme is a promising tool to improve the therapeutic obstacles of CAR T cells against solid tumors.

The Renin-Angiotensin System in the Tumor Microenvironment of Glioblastoma.

Simple SummaryGlioblastoma (GB) is the most aggressive brain cancer in humans. Patient survival outcomes have remained dismal despite intensive research over the past 50 years, with a median overall survival of only 14.6 months. We highlight the critical role of the renin–angiotensin system (RAS) on GB cancer stem cells and the tumor microenvironment which, in turn, influences cancer stem cells in driving tumorigenesis and treatment resistance. We present recent developments and underscore the need for further research into the GB tumor microenvironment. We discuss the novel therapeutic targeting of the RAS using existing commonly available medications and utilizing model systems to further this critical investigation.Glioblastoma (GB) is an aggressive primary brain tumor. Despite intensive research over the past 50 years, little advance has been made to improve the poor outcome, with an overall median survival of 14.6 months following standard treatment. Local recurrence is inevitable due to the quiescent cancer stem cells (CSCs) in GB that co-express stemness-associated markers and components of the renin–angiotensin system (RAS). The dynamic and heterogeneous tumor microenvironment (TME) plays a fundamental role in tumor development, progression, invasiveness, and therapy resistance. There is increasing evidence showing the critical role of the RAS in the TME influencing CSCs via its upstream and downstream pathways. Drugs that alter the hallmarks of cancer by modulating the RAS present a potential new therapeutic alternative or adjunct to conventional treatment of GB. Cerebral and GB organoids may offer a cost-effective method for evaluating the efficacy of RAS-modulating drugs on GB. We review the nexus between the GB TME, CSC niche, and the RAS, and propose re-purposed RAS-modulating drugs as a potential therapeutic alternative or adjunct to current standard therapy for GB.

Establishment and characterization of patient-derived head and neck cancer models from surgical specimens and endoscopic biopsies

BackgroundHead and neck squamous cell carcinoma (HNSCC) is heterogeneous in etiology, phenotype and biology. Patient-derived xenografts (PDX) maintain morphology and molecular profiling of the original tumors and have become a standard “Avatar” model for human cancer research. However, restricted availability of tumor samples hindered the widespread use of PDX. Most PDX-projects include only surgical specimens because reliable engraftment from biopsies is missing. Therefore, sample collection is limited and excludes recurrent and metastatic, non-resectable cancer from preclinical models as well as future personalized medicine.MethodsThis study compares the PDX-take rate, -growth, histopathology, and molecular characteristics of endoscopic specimens with surgical specimens. HNSCC samples (n = 55) were collected ad hoc, fresh frozen and implanted into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice.ResultsEngraftment was successful in both sample types. However, engraftment rate was lower (21 vs. 52%) and growth delayed (11.2 vs. 6.7 weeks) for endoscopic biopsies. Following engraftment, growth kinetic was similar. Comparisons of primary tumors and corresponding PDX models confirmed preservation of histomorphology (HE histology) and molecular profile (Illumina Cancer Hotspot Panel) of the patients’ tumors. Accompanying flow cytometry on primary tumor specimens revealed a heterogeneous tumor microenvironment among individual cases and identified M2-like macrophages as positive predictors for engraftment. Vice versa, a high PD-L1 expression (combined positive score on tumor/immune cells) predicted PDX rejection.ConclusionIncluding biopsy samples from locally advanced or metastatic lesions from patients with non-surgical treatment strategies, increases the availability of PDX for basic and translational research. This facilitates (pre-) clinical studies for individual response prediction based on immunological biomarkers.

Comparing syngeneic and autochthonous models of breast cancer to identify tumor immune components that correlate with response to immunotherapy in breast cancer

BackgroundThe heterogeneity of the breast tumor microenvironment (TME) may contribute to the lack of durable responses to immune checkpoint blockade (ICB); however, mouse models to test this are currently lacking. Proper selection and use of preclinical models are necessary for rigorous, preclinical studies to rapidly move laboratory findings into the clinic.MethodsThree versions of a common syngeneic model derived from the MMTV-PyMT autochthonous model were generated by inoculating 1E6, 1E5, or 1E4 cells derived from the MMTV-PyMT mouse into wildtype recipient mice. To elucidate how tumor latency and TME heterogeneity contribute to ICB resistance, comprehensive characterization of the TME using quantitative flow-cytometry and RNA expression analysis (NanoString) was performed. Subsequently, response to ICB was tested. These procedures were repeated using the EMT6 breast cancer model.ResultsThe 3 syngeneic versions of the MMTV-PyMT model had vastly different TMEs that correlated to ICB response. The number of cells used to generate syngeneic tumors significantly influenced tumor latency, infiltrating leukocyte populations, and response to ICB. These results were confirmed using the EMT6 breast cancer model. Compared to the MMTV-PyMT autochthonous model, all 3 MMTV-PyMT syngeneic models had significantly more tumor-infiltrating lymphocytes (TILs; CD3+, CD4+, and CD8+) and higher proportions of PD-L1-positive myeloid cells, whereas the MMTV-PyMT autochthonous model had the highest frequency of myeloid cells out of total leukocytes. Increased TILs correlated with response to anti-PD-L1 and anti-CTLA-4 therapy, but PD-L1expression on tumor cells or PD-1 expression of T cells did not.ConclusionsThese studies reveal that tumor cell number correlates with tumor latency, TME, and response to ICB. ICB-sensitive and resistant syngeneic breast cancer models were identified, in which the 1E4 syngeneic model was most resistant to ICB. Given the lack of benefit from ICB in breast cancer, identifying robust murine models presented here provides the opportunity to further interrogate the TME for breast cancer treatment and provide novel insights into therapeutic combinations to overcome ICB resistance.

Transcriptomics based multi-dimensional characterization and drug screen in esophageal squamous cell carcinoma.

BackgroundEsophageal squamous cell carcinoma (ESCC) remains one of the deadly cancer types. Comprehensively dissecting the molecular characterization and the heterogeneity of ESCC paves the way for developing more promising therapeutics.MethodsExpression profiles of multiple ESCC datasets were integrated. ATAC-seq and RNA-seq were combined to reveal the chromatin accessibility features. A prognosis-related subtype classifier (PrSC) was constructed, and its association with the tumor microenvironment (TME) and immunotherapy was assessed. The key gene signature was validated in clinical samples. Based on the TME heterogeneity of ESCC patients, potential subtype-specific therapeutic agents were screened.FindingsThe common differentially expressed genes (cDEGs) in ESCC were identified. Up-regulated genes (HEATR1, TIMELESS, DTL, GINS1, RUVBL1, and ECT2) were found highly important in ESCC cell survival. The expression alterations of PRIM2, HPGD, NELL2, and TFAP2B were associated with chromatin accessibility changes. PrSC was a robust scoring tool that was not only associated with the prognosis of ESCC patients, but also could reflect the TME heterogeneity. TNS1high fibroblasts were associated with immune exclusion. TG-101348 and Vinorelbine were identified as potential subtype-specific therapeutic agents. Besides, the application of PrSC into two immunotherapy cohorts indicated its potential value in assessing treatment response to immunotherapy.InterpretationOur study depicted the multi-dimensional characterization of ESCC, established a robust scoring tool for the prognosis assessment, highlighted the role of TNS1high fibroblasts in TME, and identified potential drugs for clinical use.FundingA full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Targeting AXL in NSCLC.

State-of-the-art cancer precision medicine approaches involve targeted inactivation of chemically and immunologically addressable vulnerabilities that often yield impressive initial anti-tumor responses in patients. Nonetheless, these responses are overshadowed by therapy resistance that follows. AXL, a receptor tyrosine kinase with bona fide oncogenic capacity, has been associated with the emergence of resistance in an array of cancers with varying pathophysiology and cellular origins, including in non-small-cell lung cancers (NSCLCs). Here in this review, we summarize AXL biology during normal homeostasis, oncogenic development and therapy resistance with a focus on NSCLC. In the context of NSCLC therapy resistance, we delineate AXL’s role in mediating resistance to tyrosine kinase inhibitors (TKIs) deployed against epidermal growth factor receptor (EGFR) as well as other notable oncogenes and to chemotherapeutics. We also discuss the current understanding of AXL’s role in mediating cell-biological variables that function as important modifiers of therapy resistance such as epithelial to mesenchymal transition (EMT), the tumor microenvironment and tumor heterogeneity. We also catalog and discuss a set of effective pharmacologic tools that are emerging to strategically perturb AXL mediated resistance programs in NSCLC. Finally, we enumerate ongoing and future exciting precision medicine approaches targeting AXL as well as challenges in this regard. We highlight that a holistic understanding of AXL biology in NSCLC may allow us to predict and improve targeted therapeutic strategies, such as through polytherapy approaches, potentially against a broad spectrum of NSCLC sub-types to forestall tumor evolution and drug resistance.

Interleukin-2 inducible T-cell kinase: a potential prognostic biomarker and tumor microenvironment remodeling indicator for hepatocellular carcinoma.

Background: The heterogeneous tumor microenvironment (TME) contributes to poor prognosis of hepatocellular carcinoma (HCC). However, determining the modulation of TME during HCC progression remains a challenge.Methods: Herein, the stromal score and immune score of HCC samples from The Cancer Genome Atlas database were calculated using the ESTIMATE algorithm and differentially expressed genes (DEGs) were obtained. Key DEGs were identified based on a protein-protein interaction network and survival analysis. Immunohistochemistry was carried out using primary samples to evaluate key DEGs expression. The CIBERSORT algorithm was applied to evaluate immune components. Gene Set Enrichment Analysis (GSEA) and correlation analysis were carried out to determine the relationship between key DEGs and tumor-infiltrating immune cells (TICs).Results: The stromal score, immune score and estimate score correlated significantly with 1-year recurrence-free survival of patients with HCC. Interleukin-2 inducible T-cell kinase (ITK) was identified as the most prognostic DEG for patients with HCC. GSEA revealed that genes in the high ITK subgroup were enriched in inflammatory-immunological terms. CIBERSORT analysis identified nine TIC subsets that correlated with ITK expression.Conclusion: We identified ITK as a novel indicator for early post-surgery tumor recurrence and microenvironment remodeling in HCC, providing a potential therapeutic target to treat HCC.

Single-cell RNAseq and longitudinal proteomic analysis of a novel semi-spontaneous urothelial cancer model reveals tumor cell heterogeneity and pretumoral urine protein alterations.

Bladder cancer, one of the most prevalent malignancies worldwide, remains hard to classify due to a staggering molecular complexity. Despite a plethora of diagnostic tools and therapies, it is hard to outline the key steps leading up to the transition from high-risk non–muscle-invasive bladder cancer (NMIBC) to muscle-invasive bladder cancer (MIBC). Carcinogen-induced murine models can recapitulate urothelial carcinogenesis and natural anti-tumor immunity. Herein, we have developed and profiled a novel model of progressive NMIBC based on 10 weeks of OH-BBN exposure in hepatocyte growth factor/cyclin dependent kinase 4 (R24C) (Hgf-Cdk4R24C) mice. The profiling of the model was performed by histology grading, single cell transcriptomic and proteomic analysis, while the derivation of a tumorigenic cell line was validated and used to assess in vivo anti-tumor effects in response to immunotherapy. Established NMIBC was present in females at 10 weeks post OH-BBN exposure while neoplasia was not as advanced in male mice, however all mice progressed to MIBC. Single cell RNA sequencing analysis revealed an intratumoral heterogeneity also described in the human disease trajectory. Moreover, although immune activation biomarkers were elevated in urine during carcinogen exposure, anti-programmed cell death protein 1 (anti-PD1) monotherapy did not prevent tumor progression. Furthermore, anti-PD1 immunotherapy did not control the growth of subcutaneous tumors formed by the newly derived urothelial cancer cell line. However, treatment with CpG-oligodeoxynucleotides (ODN) significantly decreased tumor volume, but only in females. In conclusion, the molecular map of this novel preclinical model of bladder cancer provides an opportunity to further investigate pharmacological therapies ahead with regards to both targeted drugs and immunotherapies to improve the strategies of how we should tackle the heterogeneous tumor microenvironment in urothelial bladder cancer to improve responses rates in the clinic.

OTEH-2. High-dimensional analysis of spatial immune cell heterogeneity in glioblastoma reveals differences between contrast-enhancing and non-contrast-enhancing tumor rims

BackgroundGlioblastoma (GBM) is the most common primary malignant brain tumor in adults. GBM remains an incurable disease, with a median survival ~20 months. Complex intercellular interactions within the tumor microenvironment and spatial heterogeneity have challenged and impeded therapeutic efficacy. The non-contrast-enhancing (by T1-weighted MRI) rim of GBM is not always safely resectable and represents a major source of recurrence. We hypothesized that differential immune infiltration is an underlying factor of spatial heterogeneity in GBM, particularly in the non-contrast-enhancing tumor rim.MethodsFive patients with newly diagnosed GBM (ages 53–84) were recruited to a device feasibility study (NCT04545177) utilizing an intraoperative high-resolution MRI-based navigation system coupled with the NICO Myriad (a non-ablative semi-automated resection tool) and a coupled automated biological Tissue Preservation System (NICO APS) to sample spatially mapped regions of tumors in a reproducible and minimally destructive manner. We obtained brain tumor tissue from: (a) tumor core, (b) contrast-enhancing tumor rim and (c) non-contrast-enhancing tumor rim. Downstream processing consisted of digestion of tumor tissue (Miltenyi human tumor digestion kit) for subsequent single-cell isolation, viability assessment and immediate staining for multiparametric flow cytometry for immune profiling.ResultsViability varied across sampled regions (median 85%, range 52–100%). With the exception of 1 sample, viability was >70% in all specimens. High-dimensional analysis with 26 marker flow cytometry revealed spatial heterogeneity in the frequency of myeloid-derived suppressor cell subsets, regulatory T cells, CD8+ T cells, as well as expression of T cell activation and exhaustion markers.ConclusionsSemi-automated, spatially mapped intraoperative sampling of GBM with high viability of specimens is feasible and reproducible with the NICO Myriad and APS devices. High-dimensional analysis of immune cells in the GBM microenvironment captured the spatial heterogeneity of GBM. Future studies will expand on these observations by analyzing more patient specimens in combination with multiple omics assays.

Abstract 2771: Comprehensive analysis of immuno oncology markers in the tumor microenvironment of solid tumor samples using GeoMxTM digital spatial profiler (DSP) and MultiOmyxTM hyperplexed immunofluorescence (IF)

Abstract In recent years, novel immunotherapy strategies have shown remarkable results in treating various advanced cancers. The efficacy of immunotherapeutics has been dictated by the dynamic and spatially heterogeneous tumor microenvironment (TME) which includes immune tumor infiltrates and immune checkpoint protein expression, in addition to tumor molecular profiles, leading to widely variable clinical outcomes and responses in patients. The local milieu as well as complex tumor-TME interactions vary widely between tumors of the same type and also within the same tumor and therefore highlights the need for enhanced analysis of tissue-immune content and their spatial context for predicting response to immunotherapies and biomarker discovery. In this study, we will apply a multi-faceted, highly multiplexed tissue analysis approach to quantitate and better characterize the spatial arrangement of key immuno oncology protein markers in a pan-cancer cohort of up to 35 FFPE samples from breast, head and neck, prostrate, non-small cell lung cancer (NSCLC), endometrial and colorectal indications using NanoString GeoMxTM Digital Spatial Profiler (DSP) and MultiOmyxTM Hyperplexed Immunofluorescence (IF) assay. For DSP analysis, each sample will be profiled for up to 52 immuno oncology (IO) markers included on NanoString Human IO protein panels. Selection of regions of interest will be guided by H&E staining, fluorescent markers (CD45, pan-CK and Syto 13). Protein profiling of tumor and TME regions will be achieved through segmenting by pan-CK+/CD 45+ followed by collection of indexed oligonucleotides and digital counting using the NanoString nCounter system. Data normalization and unsupervised clustering of IO protein expression across the ROIs will be performed to understand the spatial distribution of these IO biomarkers within the TME. An adjacent section of each tumor sample will be also stained and analyzed using MultiOmyx technology. MultiOmyx is a proprietary IF platform for the visualization and characterization of up to 60 protein biomarkers in a single FFPE section. Using the MultiOmyx IF assay in combination with proprietary algorithms, we will study the expression and spatial distribution of 26 IO markers to characterize different subtypes of immune cells and immune checkpoint inhibitors within the TME in the 35 FFPE tumor samples. Combination of MultiOmyx IF with Nanostring DSP provides a complementary and powerful solution to study the IO markers within the TME. Using the DSP nCounter output along with MultiOmyx cell classification and spatial analysis results, the researchers can simultaneously profile the high-plex protein expression, characterize the immunophenotypes and visualize the spatial distribution of tumor infiltrating immune cells at single-cell resolution within the TME. Citation Format: Lakshmi Chandramohan, Qingyan Au, Mate Nagy, Erinn Parnell, Tricia Peters. Comprehensive analysis of immuno oncology markers in the tumor microenvironment of solid tumor samples using GeoMxTM digital spatial profiler (DSP) and MultiOmyxTM hyperplexed immunofluorescence (IF) [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 2771.