Metabolomic Phenotyping of Gliomas: What Can We Get with Simplified Protocol for Intact Tissue Analysis?

Glioblastoma multiforme is one of the most malignant neoplasms among humans in their third and fourth decades of life, which is evidenced by short patient survival times and rapid tumor-cell proliferation after radiation and chemotherapy. At present, the diagnosis of gliomas and decisions related to therapeutic strategies are based on genetic testing and histological analysis of the tumor, with molecular biomarkers still being sought to complement the diagnostic panel. This work aims to enable the metabolomic characterization of cancer tissue and the discovery of potential biomarkers via high-resolution mass spectrometry coupled to liquid chromatography and a solvent-free sampling protocol that uses a microprobe to extract metabolites directly from intact tumors. The metabolomic analyses were performed independently from genetic and histological testing and at a later time. Despite the small cohort analyzed in this study, the results indicated that the proposed method is able to identify metabolites associated with different malignancy grades of glioma, as well as IDH and 1p19q codeletion mutations. A comparison of the constellation of identified metabolites and the results of standard tests indicated the validity of using the characterization of one comprehensive tumor phenotype as a reflection of all diagnostically meaningful information. Due to its simplicity, the proposed analytical approach was verified as being compatible with a surgical environment and applicable for large-scale studies.

Emerging Nanoparticle Strategies for Modulating Tumor-Associated Macrophage Polarization

Immunotherapy has made great progress in recent years, yet the efficacy of solid tumors remains far less than expected. One of the main hurdles is to overcome the immune-suppressive tumor microenvironment (TME). Among all cells in TME, tumor-associated macrophages (TAMs) play pivotal roles because of their abundance, multifaceted interactions to adaptive and host immune systems, as well as their context-dependent plasticity. Underlying the highly plastic characteristic, lots of research interests are focused on repolarizing TAMs from M2-like pro-tumor phenotype towards M1-like antitumoral ones. Nanotechnology offers great opportunities for targeting and modulating TAM polarization to mount the therapeutic efficacy in cancer immunotherapy. Here, this mini-review highlights those emerging nano-approaches for TAM repolarization in the last three years.

Differences in breast cancer-risk factors between screen-detected and non-screen-detected cases (MCC-Spain study)

Purpose The variation in breast cancer (BC)-risk factor associations between screen-detected (SD) and non-screen-detected (NSD) tumors has been poorly studied, despite the interest of this aspect in risk assessment and prevention. This study analyzes the differences in breast cancer-risk factor associations according to detection method and tumor phenotype in Spanish women aged between 50 and 69. Methods We examined 900 BC cases and 896 controls aged between 50 and 69, recruited in the multicase–control MCC-Spain study. With regard to the cases, 460 were detected by screening mammography, whereas 144 were diagnosed by other means. By tumor phenotype, 591 were HR+, 153 were HER2+, and 58 were TN. Lifestyle, reproductive factors, family history of BC, and tumor characteristics were analyzed. Logistic regression models were used to compare cases vs. controls and SD vs. NSD cases. Multinomial regression models (controls used as a reference) were adjusted for case analysis according to phenotype and detection method. Results TN was associated with a lower risk of SD BC (OR 0.30 IC 0.10–0.89), as were intermediate (OR 0.18 IC 0.07–0.44) and advanced stages at diagnosis (OR 0.11 IC 0.03–0.34). Nulliparity in postmenopausal women and age at menopause were related to an increased risk of SD BC (OR 1.60 IC 1.08–2.36; OR 1.48 IC 1.09–2.00, respectively). Nulliparity in postmenopausal women was associated with a higher risk of HR+ (OR 1.66 IC 1.15–2.40). Age at menopause was related to a greater risk of HR+ (OR 1.60 IC 1.22–2.11) and HER2+ (OR 1.59 IC 1.03–2.45) tumors. Conclusion Reproductive risk factors are associated with SD BC, as are HR+ tumors. Differences in BC-risk factor associations according to detection method may be related to prevailing phenotypes among categories.

Personalized Immunotherapy in Colorectal Cancers: Where Do We Stand?

Colorectal cancer (CRC) is the second leading cause of cancer death in the world. Immunotherapy using monoclonal antibodies, immune-checkpoint inhibitors, adoptive cell therapy, and cancer vaccines has raised great hopes for treating poor prognosis metastatic CRCs that are resistant to the conventional therapies. However, high inter-tumor and intra-tumor heterogeneity hinder the success of immunotherapy in CRC. Patients with a similar tumor phenotype respond differently to the same immunotherapy regimen. Mutation-based classification, molecular subtyping, and immunoscoring of CRCs facilitated the multi-aspect grouping of CRC patients and improved immunotherapy. Personalized immunotherapy using tumor-specific neoantigens provides the opportunity to consider each patient as an independent group deserving of individualized immunotherapy. In the recent decade, the development of sequencing and multi-omics techniques has helped us classify patients more precisely. The expansion of such advanced techniques along with the neoantigen-based immunotherapy could herald a new era in treating heterogeneous tumors such as CRC. In this review article, we provided the latest findings in immunotherapy of CRC. We elaborated on the heterogeneity of CRC patients as a bottleneck of CRC immunotherapy and reviewed the latest advances in personalized immunotherapy to overcome CRC heterogeneity.

CNTM-02. Regulation of glioma-network integration by tumor mediated secretion of TSP-1

Glioma exists in the complex neural circuitry of the brain, making the interface between neoplastic and healthy neurons and glia potentially damaging to long range neural networks and stimulatory to tumor growth. Thrombospondin-1 (TSP-1), an astrocyte derived neurogenic factor expressed by glia of the normal brain, has been found to be upregulated in intratumoral regions with high network functional connectivity (HFC). This modified cell signaling represents cancer cell hijacking of normal physiology with direct impact on tumor biology. There is emerging evidence that neuronal activity influences glioma proliferation and gliomas promote neuronal hyperexcitability. In humans, we have recently shown that bidirectional cellular interactions between gliomas and neurons alter cognitive circuit dynamics and ultimately patient survival. Previously, a subpopulation of human high-grade glioma cells which are enriched for tumor cells with synaptogenic potential were identified (HFC-IHDwtGBM). We plan to study the mechanisms of TSP1 signaling in three different established glioma models (1) HFC-IDHwtGBM hippocampal neuron co-culture, (2) HFC-IDHwtGBM + induced neuron organoids, (3) patient derived xenografts (PDX) for in vivo GCaMP calcium imaging. This project aims to test the hypothesis that increased TSP-1 secretion from HFC-IHDwtGBM cells plays a central role in the maintenance of an invasive and proliferative tumor phenotype when compared with LFC-IHDwtGBM PDX. We hope our study guides future work focused on preventing the infiltration of tumor cells into healthy brain tissues.

885 Targeting FPR2 as a novel approach for immunotherapy in pancreatic cancer female patients - studies of sexual immune dimorphism in the tumor microenvironment

BackgroundImmunotherapy for pancreatic cancer (PC) is inefficient due to a highly immune-suppressive tumor microenvironment (TME) orchestrated by myeloid suppressor cells, which limit the infiltration and function of cytotoxic immune cells. We have evidence that accumulation of a subpopulation of myeloid cells in human pancreatic lesions is associated with immune-exclusive tumor phenotype and effector T cell exhaustion by mechanisms involving the G-coupled protein receptor formyl peptide receptor 2 (FPR2), exclusively in women. We hypothesize that female FPR2+ myeloid cells in tumors induce immune exhaustion and contribute to immune-cold tumor phenotype.MethodsTo test our hypothesis, we first investigated the FPR2 RNA and protein expression in PC transcriptomic data and in murine and human PC tissues. Further, in vitro cytokine differentiated, alternatively tumor conditioned myeloid cells (TCM) were co-cultured with T cells to mimic their interaction in the TME. In vivo, PC cells were injected subcutaneously in FPR2 WT and KO mice to study tumor progression and the immune landscape in male vs. female mice. Later, human myeloid cells were treated with FPR2 agonists and antagonists to study the interaction mechanisms in detail.ResultsWe found high FPR2 expression in tumor compared to healthy tissues and higher in women compared to men. In mice and human, FPR2+ myeloid cells were associated with immune cold-exclusive and cold-ignored tumor phenotype in women and men, respectively. Notably, analysis in PC and other gastrointestinal (GI)-tract cancers revealed a significant association of FPR2 expression and poor survival only in women, emerging the potential impact of sex factors in the TME. Such sexual dimorphism in the TME was associated with T cell exhaustion apparent by high expression of TIM3 and PD1. In vitro, FPR2-agonist treated myeloid-suppressive cells induced TIM3 and PD1 expression in T cells specifically in female T cells. However, a significant repression of TIM3 and a trend of PD1 expression was observed in T cells when interacting with FPR2-inhibited or -deficient myeloid cells. Finally, tumor progression was significantly slower in FPR2 KO female mice compared to WT and male FPR2 WT and KO mice.ConclusionsIn this study, we have shown that sex differences are involved in shaping the TME in PC, where sexual dimorphism is still a largely unknown area allowing novel personalized/sex-specific immunotherapies. We found that FPR2 is highly involved in T cell exhaustion and can potentially be a therapeutic target for immunotherapy in women developing PC and other GI-tract cancers.Ethics ApprovalThe study was approved by the regional ethics review board in Stockholm (Dnr2020-06587 and Dnr2013.977-31.1) and the Swedish Board of Agriculture and regional ethical committee (10681-2020).

180 T cell phenotype drives restructuring of tumor microenvironment to balance T cell longevity and tumor control: insights from multiplexed imaging and multi-scale agent based modeling

BackgroundImmune cell therapies continue to have success in treatment of cancers yet face challenges of complexity, cost, toxicity, and low solid-tumor efficacy. Much work has focused on the phenotype characterization and control of ex vivo expanded cells; however, little is known about its relationship to changes in the tumor microenvironment in vivo. Thus, we imaged tumors treated with different phenotype tumor-specific CD8+ T cells with CODEX multiplexed imaging1–4 that is able to visualize 42 antibodies at the same tissue in the tissue (figure 1A). To further probe this data in a systems immunology approach we created a multiscale agent-based model including critical circuits from the T cell-tumor microenvironment interactions (figure 1B).MethodsWe initialized our agent-based models various percentages of either PD1+, PD1-, PDL1+, or PDL1- phenotypes and ran simulations for 72 hours. We also treated PMEL CD8+ T cells with or without 2 hydroxycitrate as a metabolic inhibitor during activation to achieve different input phenotypes of CD8+ T cells for therapeutic adoptive transfer on day 10 following B16-F10 tumors had been established. We performed neighborhood analysis on CODEX multiplexed imaging data by clustering neighboring cell types using a sliding window for neighborhood analysis.ResultsInterestingly, the agent-based modeling indicated that the tumor phenotype switch to decrease proliferation was more effective than direct T cell killing. We observed spatially restricted inflammatory immune fronts when simulating with different initial percentages of PD1+ T cells and also from our CODEX multiplexed imaging. Quantitatively we observe that there is a drastic increase in the PDL1+, MHCI+, Ki67- tumor phenotype that increases with metabolically inhibited T cells. Neighborhood analysis indicated that metabolically treated T cells were able to create distinct immune cell environments that supported productive T cell-tumor interactions and also helped maintain T cell phenotype.ConclusionsThis indicates there is a balance for therapeutic T cell to mitigate chronic tumor exposure while controlling tumor growth through killing and by changing tumor phenotype. We observe T cells create distinct tumor microenvironments that differs significantly based on the starting T cell phenotype. Controlling T cell phenotype to promote productive immune-tumor structures will be critical to maintain T cell functionality and efficacy. In the future we will investigate T cell recruitment of immune structures by similar systems biology technologies.AcknowledgementsJ.W.H. is funded by an ACS Postdoctoral Fellowship (PF-20-032-01-CSM).ReferencesGoltsev Y, Samusik N, Kennedy-Darling J, Bhate S, Hale M, Vazquez G, Black S and Nolan GP, Deep profiling of mouse splenic architecture with CODEX multiplexed imaging. Cell, 174(4):968–981.Schürch CM, Bhate SS, Barlow GL, Phillips DJ, Noti L, Zlobec I, Chu P, Black S, Demeter J, McIlwain DR and Samusik N. Coordinated cellular neighborhoods orchestrate antitumoral immunity at the colorectal cancer invasive front. Cell 182(5):1341–1359.Black S, Phillips D, Hickey JW, Kennedy-Darling J, Venkataraaman VG, Samusik N, Goltsev Y, Schürch CM. and Nolan GP. CODEX multiplexed tissue imaging with DNA-conjugated antibodies. Nature Protocols 1–36.Kennedy-Darling J, Bhate SS, Hickey JW, Black S, Barlow GL, Vazquez G, Venkataraaman VG, Samusik N, Goltsev Y, Schürch CM and Nolan GP. Highly multiplexed tissue imaging using repeated oligonucleotide exchange reaction. European Journal of Immunology 51(5):1262–1277.Ethics ApprovalAll studies involving mice were approved under Stanford’s APLAC protocol 33502.

Role of HSP90 in Cancer

HSP90 is a vital chaperone protein conserved across all organisms. As a chaperone protein, it correctly folds client proteins. Structurally, this protein is a dimer with monomer subunits that consist of three main conserved domains known as the N-terminal domain, middle domain, and the C-terminal domain. Multiple isoforms of HSP90 exist, and these isoforms share high homology. These isoforms are present both within the cell and outside the cell. Isoforms HSP90α and HSP90β are present in the cytoplasm; TRAP1 is present in the mitochondria; and GRP94 is present in the endoplasmic reticulum and is likely secreted due to post-translational modifications (PTM). HSP90 is also secreted into an extracellular environment via an exosome pathway that differs from the classic secretion pathway. Various co-chaperones are necessary for HSP90 to function. Elevated levels of HSP90 have been observed in patients with cancer. Despite this observation, the possible role of HSP90 in cancer was overlooked because the chaperone was also present in extreme amounts in normal cells and was vital to normal cell function, as observed when the drastic adverse effects resulting from gene knockout inhibited the production of this protein. Differences between normal HSP90 and HSP90 of the tumor phenotype have been better understood and have aided in making the chaperone protein a target for cancer drugs. One difference is in the conformation: HSP90 of the tumor phenotype is more susceptible to inhibitors. Since overexpression of HSP90 is a factor in tumorigenesis, HSP90 inhibitors have been studied to combat the adverse effects of HSP90 overexpression. Monotherapies using HSP90 inhibitors have shown some success; however, combination therapies have shown better results and are thus being studied for a more effective cancer treatment.

Prognostic Effect of Microenvironment Phenotype in Triple-Negative Breast Cancer: Biomarker Analysis of a Prospective Trial

Background: The microenvironment of triple-negative breast cancer (TNBC) can be divided into three clusters based on bioinformatics-based immunogenomic analysis: the “immune-desert” cluster, the “innate immune-inactivated” cluster, and the “immune-inflamed” cluster. The immune-inflamed cluster is considered as “hot tumor” while the other two are considered as “cold tumor”.Methods: To investigate the prognostic effect of microenvironment phenotypes on TNBC, we compared relapse-free survival (RFS) of different phenotypes in 100 patients with RNA sequencing-based expression data from the PATTERN trial (NCT01216111, published in JAMA Oncol 2020), which indicated a superior efficacy of adjuvant paclitaxel-plus-carboplatin regimen compared to the regimen of cyclophosphamide/epirubicin/fluorouracil followed by docetaxel for TNBC. We also analyzed the efficacy of the two regimens for different immune phenotypes to explore potential treatment strategies.Results: No significant difference in RFS was observed between the “hot tumor” and the “cold tumor” (hazard ratio [HR] = 0.68, 95% confidence interval [CI] 0.28–1.66, P = 0.40). However, the “hot tumor” subtype was associated with significantly longer RFS in node-positive patients (HR = 0.27, 95%CI 0.07–0.97, P = 0.03). Consistently, a similar trend to improved RFS of the “hot tumor” phenotype was detected in patients with stage pT2-3 tumors (HR = 0.29, 95%CI 0.06–1.30, P = 0.08). Furthermore, no significant difference in RFS between the two treatment arms was observed in patients with “hot tumor” (HR = 0.39, 95% CI 0.08–2.01, P = 0.24) or “cold tumor” (HR = 1.05, 95% CI 0.39–2.82, P = 0.92).Conclusion: The microenvironment phenotype in TNBC might have prognostic significance to patients with a high risk of recurrence. The association of the microenvironment phenotypes with the efficacy of adjuvant chemotherapy for TNBC remains to be further studied.

Detachment Stress Mediated Bioenergetic Switch of Malignant Melanoma Cells Into Anti-Warburg Phenotype

One of the biological features of cancer cells was their aerobic glycolysis by extensive glucose fermentation to harvest energy, so called Warburg effect. Melanoma is one of the most aggressive human cancers with poor prognosis and high mortality for its high metastatic ability. During the metastatic process, the metastatic tumor cells should survive under detachment stress. However, whether the detachment stress could affect the tumor phenotype was worthy to investigate. We had established human melanoma A375 cells under detachment stress, which mimicked circulating melanoma. It was shown the detachment stress altered melanoma cell activities, malignancy, and drug sensitivity. In this study, we found that adherent melanoma cells were more sensitive to glucose depletion. However, detachment stress reduced lactate secretion owing to the reduced MCT4 and GLUT1 expressions, the altered glycolytic and respiratory capacities, and the increased superoxide production. Detachment stress also increase the sensitivity of melanoma cells toward blockade of electron transport chains. Investigation of the change in glucose metabolism of melanoma cells under detachment stress would be critical to provide novel molecular mechanism to develop potential therapeutics