Multiplex Flow Cytometry Research Articles

Abstract B025: MHC class II and CD1d expressing conventional dendritic cells with immunoglobulin therapy drives anti-tumor abscopal effect by recruiting Natural Killer T cells to the tumor microenvironment

Abstract Background Dendritic cells (DC) are the primary antigen presenting cells in the body responsible for processing and presenting MHC Class I and II antigens to initiate adaptive T cell response against cancer. In addition to MHC, DC also express non-classical receptors such as CD1d to activate invariant TCR α chain natural killer T (iNKT) cells. The conventional type-I dendritic cells (cDC1) presenting MHC class-II tumor associated antigens within tumor microenvironment (TME) have shown robust efficacy in mouse models of breast cancer (BC). Here, we have extended prior knowledge to deliver DC-based therapeutics, co-presenting class II antigens and CD1d directly to TME to show the potential of co-stimulation of multiple effector T cells in combination of tumor specific antibody (IgG1) mediating antibody dependent cellular cytotoxicity (ADCC). We hypothesize that merger of adaptive and innate responses will attain greater pathologic response in pre-clinical and clinical settings to design futuristic therapeutics against BC. Methods Present study uses variety of pre-clinical mouse models of BC and other types of cancer to test the therapeutic efficacy of intratumoral delivery of mouse bone marrow-derived conventional type I (CD103+ CD1d+) polarized DC pulsed with MHC class II HER2-derived peptides (HER2-DC1) in combination with systemic delivery of tumor specific IgG1 (aSEMA4D) to evoke ADCC. Cellular mechanism of response was evaluated using downstream methods including multiplex immunofluorescence, flow cytometry, and transcriptomics assays. Results We exhibit that specifically intratumoral combination therapy of HER2-DC1 expressing CD1d and aSEMA4D primes both innate and adaptive immune responses to completely regress tumor in 75-80% of mice with significant survival advantage in comparison to mice that received no therapy or monotherapies. The early CD4 Th1 response through interferon gamma combined with ADCC played a critical role in the decrease of pro-tumorigenic myeloid cells and increase in anti-tumor effector cells with a major contribution from classical CD4+ NKT cells within the TME. Interestingly, intratumoral combination therapy intensified DC1 migration from primary tumor to secondary tumor sites along with dynamic changes in subpopulations of DC with an associated increase in NKT cells at untreated tumor sites to display abscopal effect. Likewise, early data from phase I/II clinical trial illustrated great potential of cDC1 and IgG1 combination therapy to recruit anti-tumor effector cells such CD8 and NKT cells to mediate cancer eradication in HER2+ BC setting. Conclusions Specifically, intratumoral delivery of HER2-DC1 in combination with anti-tumor antibody mediating ADCC shows great promise for future BC clinical trials and improved patient outcomes. Furthermore, present study highlighted the role of NKT cells in favorable anti-tumor response. Our future studies are focused on understanding the crosstalk between DC and NKT to modulate the function of various cell types in TME to design improved cancer immunotherapies. Citation Format: Saurabh K Garg, Colin Snyder, Ganesan Ramamoorthi, Elizabeth E Evans, Amy L Aldrich, Namrata Gautam, Doris Wiener, Marie C Lee, Hyo S Han, Koski K Gary, Brian J Czerniecki. MHC class II and CD1d expressing conventional dendritic cells with immunoglobulin therapy drives anti-tumor abscopal effect by recruiting Natural Killer T cells to the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B025.

Immune checkpoint CD161/LLT1-associated immunological landscape and diagnostic value in oral squamous cell carcinoma.

An active host adaptive response is characterized by the existence of programmed cell death protein 1 (PD-1)+ /IFN-γ+ cytotoxic T cells and IFN-γ-induced PD-L1+ tumor cells (TCs), which predicts high response rate to anti-PD-1/L1 therapy. Recently, CD161 and its ligand LLT1 (CLEC2D) have been identified as an emerging checkpoint for immunotherapy. Clarifying its heterogeneous clinical expression pattern and its immune landscape is a prerequisite for maximizing the response rate of CD161 blockade therapy in a specific population of oral squamous cell carcinoma (OSCC) patients. Here, we investigated the expression pattern of CD161/LLT1 and its association with major immunocytes (T cells, B cells, NK cells, and macrophages) by multiplex immunofluorescence, immunohistochemistry, and flow cytometry in 109 OSCC tissues and 102 peripheral blood samples. TCs showed higher LLT1 levels than tumor infiltrating lymphocytes (TILs), whereas CD161 was highly expressed in CD8+ T cells at the tumor front, which was decreased in paracancerous tissue. High expression of TC-derived LLT1 (LLT1TC ) conferred poor clinical outcomes, whereas higher CD161+ and LLT1+ TILs were associated with better prognosis. Meanwhile, patients with high LLT1TC showed a decreased ratio of CD8+ /Foxp3+ T cells in situ, but CD161+ TILs correlated with more peripheral CD3+ T cells. Interestingly, treatment of OSCC patients with nivolumab (anti-PD-1) could restore tumoral CD161/LLT1 signal. Furthermore, an OSCC subgroup characterized by high LLT1+ TCs and low CD161+ CD8+ T cells showed fewer peripheral T cells and a higher risk of lymph node metastasis, leading to a shorter 5-year survival time (29%). More LLT1TC at the invasive front was another risk characteristic of exhausted T cells. In conclusion, in view of this heterogeneity, the LLT1/CD161 distribution pattern should be determined before CD161-based immunotherapy.

NDRG1 overcomes resistance to immunotherapy of pancreatic ductal adenocarcinoma through inhibiting ATG9A-dependent degradation of MHC-1

AimsPancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is resistant to immune checkpoint blockade (ICB) therapies. Emerging evidence suggests that NDRG1 may be an important target for the development of new therapies for PDAC. Herein, we investigated the novel roles of NDRG1 and Combretastatin A-4 (CA-4) in the treatment of PDAC ICB resistance. MethodsEnrichment of MHC class I was detected by RNA sequence and verified by RT-qPCR and immunoblotting in NDRG1-knockdown human pancreatic cancer cell lines. The protein degradation mode was found by stimulation with various inhibitors, and the autophagy degradation pathway was found by immunoprecipitation and immunolocalization. The roles of NDRG1 and MHC-I in immunotherapy were investigated by orthotopic solid tumors, histology, immunohistochemistry, multiplex immunofluorescence staining and flow cytometry. ResultsHere, we identified a previously undescribed role of NDRG1 in activating major histocompatibility complex class 1 (MHC-1) expression in pancreatic ductal adenocarcinoma (PDAC) cells through lysosomal-autophagy-dependent degradation. In mouse models of PDAC, either tumor cell overexpression or pharmacologic activation of NDRG1 leads to MHC-1 upregulation in tumor cells, which in turn promotes the infiltration and activity of CD8 + T cells, enhances anti-tumor immunity, and overcomes resistance to ICB therapy. Moreover, combination therapy of CA-4 and ICB overcomes the drug resistance of pancreatic cancer to ICB therapy. In PDAC patients, NDRG1 expression correlates with high MHC-1 expression and better survival. ConclusionOur results reveal NDRG1 in PDAC cancer cells as a tumor suppressor and suggest that pharmaceutically targeting NDRG1 is a promising way to overcome pancreatic cancer resistance to immunotherapy and provides a potential therapeutic strategy for the treatment of pancreatic cancer patients.

Targeting PLCG2 Suppresses Tumor Progression, Orchestrates the Tumor Immune Microenvironment and Potentiates Immune Checkpoint Blockade Therapy for Colorectal Cancer.

Background: Tumor progression and limited benefits of immune checkpoint blockade (ICB) therapy have been two major challenges in the clinical management of colorectal cancer (CRC). The objective of our research was to explore the role of PLCG2 in CRC progression, tumor microenvironment, and potentiating ICB therapy. Methods: Based on bioinformatics analysis and a prospective clinical observational study, the expression, prognostic significance, and clinical relevance of PLCG2 in CRC were unveiled. The single-cell and spatial transcriptome revealed the role of PLCG2 in shaping the heterogeneity of the CRC tumor microenvironment. The biological function of PLCG2 was validated by in vivo and in vitro experiments. The underlying mechanisms were elucidated by RNA-seq, western blotting, qRT-PCR, and multicolor immunofluorescence. The multiplex immunohistochemistry and flow cytometry were adopted to clarify the immunomodulatory role of PLCG2 in facilitating CRC immune escape. The translational value of targeting PLCG2 to potentiate the efficacy of ICB therapy and synergistic therapy to improve prognosis was explored in the preclinical animal models. Results: In CRC, PLCG2 exhibited high expression levels and was strongly associated with poor prognosis and advanced clinicopathological characteristics of patients. The single-cell transcriptome shed light on its important role in cell communication and the development and differentiation of immune cells. The spatial transcriptome described the spatial distribution of PLCG2 in CRC tissues. Further mechanistic analysis demonstrated that PLCG2 could promote proliferation, invasion, metastasis, epithelial-mesenchymal transition, and cell cycle regulation and inhibit apoptosis of CRC cells via the Akt-mTOR pathway activation. Furthermore, PLCG2 was found to contribute greatly to the immunosuppressive microenvironment and enhanced immune escape as it significantly suppressed the infiltration and functional activation of CD8+ T cells and promoted the infiltration of Treg cells as well as PD-1 and PD-L1 expression. Meanwhile, knockdown of PLCG2 could potentiate the efficacy of ICB therapy. Conclusion: In summary, we have identified for the first time that PLCG2 could be considered a precise biomarker and promising therapeutic target for predicting CRC prognosis, optimizing individualized treatment, reversing CRC immune escape, and overcoming resistance to ICB therapy.

Abstract PR002: Single-cell spatial analysis reveals microenvironmental features that contribute to immune discrepancies between adult and pediatric nasopharyngeal carcinomas

Abstract Background: Nasopharyngeal carcinoma (NPC) is an EBV-positive malignancy with high immune infiltrates but also high immunosuppressive activities. Pediatric NPC only constitutes 0.1-1% of the total NPC incidences and exhibits superior treatment responses and prognosis compared to adult NPC. Clinically, we consider that the differences in the tumor landscape between adult and pediatric NPC patients are the vital driver of such immune discrepancies. Therefore, we apply scRNA-seq and Visium spatial sequencing to primary pediatric NPC tissues and paired peripheral blood samples, and integrate our data with multiple independent adult NPC scRNA-seq cohorts. Furthermore, we establish a bioinformatics analysis pipeline that can calculate spatial cellular compositions and biological activities. Here, we demonstrate that low lipid metabolism, disrupted interactions between NPC cells and regulatory T cells (Tregs)/CD8+ T cells, and high infiltration of central stem memory T cells, contribute to more potent and enduring anti-tumor immunity in pediatric NPC patients, and these microenvironmental features might serve as new immunotherapeutic vulnerabilities. Method: 5' scRNA-seq coupled with TCR/BCR profiling was performed on 5 primary and treatment-naïve pediatric NPC tissues with 5 paired peripheral blood samples. Visium spatial-seq was performed on 4 paired pediatric and 7 adult NPC tissues. We incorporated our scRNA-seq cohort with three independent adult NPC cohorts. Our computational framework was integrated with existing analysis packages, including Seurat, Spacexr, and SpaCET, to analyze single-cell-referenced spatial data. The key results in our study were validated by multiplex IHC staining and flow cytometry on patient samples. Results: We established a multi-center NPC scRNA spatial cohort containing 503,021 cells from 69 samples, and 15,222 spatial spots from 11 samples. We developed a computational framework to calculate spatial co-localization and biological activities. We unveiled that lipid metabolism was elevated in the tumor-T cell core in adult NPC. Strong co-localizations between NPC cells and Tregs/CD8+ T cells were found in adult NPC, with spatially enriched CD70-CD27 and LGALS9-TIM3 interactions. This finding was corroborated by multiplex IHC staining of KRT19+ NPC cells, CD4+/FOXP3+ Tregs, and CD8+/PD-1/TIM-3+ exhausted T cells. We also identified and characterized novel IL7R+ central stem memory T cells in pediatric NPC tissues and blood, with strong resilience to immunosuppressive cues, including TGF-β, PEG2, and cyclic AMP, and could lead to enduring immunity and anti-PD-1/PD-L1 responses. Conclusion: We demonstrate that higher T-cell immunosuppression and exhaustion caused by aberrant lipid metabolism, tumor-T interactions, and a lower age-associated central stem memory T-cell pool, are vital drivers of immune discrepancies in adult and pediatric NPC patients. Thus, screening and targeting these molecular microenvironmental characteristics might help stratify immunotherapy responders and generate therapeutic benefits in NPC patients. Citation Format: Lanqi Gong, Yu Zhang, Grace Guan, Beibei Ru, Peng Jiang. Single-cell spatial analysis reveals microenvironmental features that contribute to immune discrepancies between adult and pediatric nasopharyngeal carcinomas [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR002.

Phase 1 Study of Autologous Sirpα-Low Macrophages (SIRPant-M) Administered By Intratumoral Injection Alone or in Combination with External-Beam Radiotherapy in Patients with Relapsed or Refractory Non-Hodgkin Lymphoma (NCT05967416)

Background and Significance. While a sentinel role of macrophages in channeling immune responses has long been recognized, the opportunity to harvest, harness and deploy macrophages for therapeutic potential has been underappreciated clinically. Most currently marketed and development-stage cell therapies target a single tumor-associated antigen, permitting activation of resistance and tumor escape mechanisms that frequently lead to relapse. High rates of CRS and neurotoxicity (ICANS) associated with CAR-T therapies complicate usage, with associated product labeling that includes a Risk Evaluation and Mitigation Strategy (REMS), mandating hospitalization and post-therapy monitoring with attendant administrative burden that precludes widespread use in community-based settings. Moreover, many subtypes of lymphoma (NHL) lack a readily druggable target. The current first-in-human Phase 1 study advances a novel paradigm in cell-therapy, deploying SIRPα-depleted activated macrophages, manufactured from an autologous mononuclear apheresis (SIRPant-M). In the context of macrophages, SIRPα acts as a strong negative regulator of (i) phagocytosis and (ii) productive pro-inflammatory T cell stimulation; therefore abrogation of SIRPα-activity serves as a necessary precondition for robust neo-antigen-specific anti-tumor responses. Requiring no genetic modification, in pre-clinical models these SIRPα-depleted macrophages orchestrated an immune response that targets multiple individual-specific and tumor-unique neo-antigens in parallel. SIRPant-M elicits a multi-pronged polyclonal immune response that transforms the tumor microenvironment (TME) by fostering a pro-inflammatory milieu that provides for unencumbered and orchestrated targeting of cancer cells via both cellular and humoral effector mechanisms. Manufactured and subsequently frozen as 3 aliquots from a single apheresis, activation of macrophages is brought about by conditioning the macrophages during ex vivo culture with a proprietary cytokine cocktail (PhagoAct™) with the added effect of modulating activity of SIRPα. This combination of product attributes permits the same cell product to have potential clinical applications in both solid tumor and hematologic malignancies. While this initial Phase 1 study administers the macrophages by intra-tumoral injection (ITI), subsequent studies aim to explore IV-dosing. Study design, methods and patients. This is a multicenter open-label Phase 1 study of SIRPant-M monotherapy, with sequential cohorts receiving low [90×10 6] and high [300×10 6] ITI cell doses. Cell product is administered alone or combined with focal external-beam radiotherapy (XRT). Each cell dose is administered in 3 ITI-fractions on days 1, 3, and 5 over 1 cycle. Upon confirmation of safety as a single agent, subsequent cohorts receive cell product concurrent with focally directed radiation in three 2.5 Gy fractions (7.5 Gy total). The primary end point, Dose Limiting Toxicity, is assessed over 30 days and dose escalation is gated by a standard 3+3 dose escalation design, overseen by a Safety Monitoring Committee. The enrollment target is 12-24 DLT-evaluable patients. The study enrolls an “all comers” B- and T-cell NHL population, including systemic and cutaneous histologies (CTCL), with relapsed or progressive disease following a minimum of 2- but no maximum number of prior therapies. Minimum age is 18, with no upper age limit. Prior CAR-T, bi-specific antibody and/or autologous/allogeneic transplant is allowed after recovery from treatment-related AEs. Patients must not receive immuno-suppression for GVHD and must have at least 1 target lesion that is 1.5 to 5 cm in diameter and accessible for safe ITI, ECOG ≤ 2, life expectancy > 3 months, renal/hepatic/coagulation parameters and blood counts that are standard for Phase 1 studies at baseline. Secondary and Exploratory Endpoints. Tumor response, including abscopal response, is evaluated by Lugano criteria or other Workgroup criteria as appropriate for the tumor type at the end of the DLT-period (Day 30), Week 12 and per SOC thereafter. Serial blood samples and a tumor biopsy before and 16 days after treatment are collected for multi-parameter analyte detection including pro-inflammatory cytokines, multiplex flow cytometry, scRNA sequencing, and ctDNA analysis to investigate pharmacodynamics and mechanism of action.

IMMU-48. SOLUBLE CIRCULATING IMMUNE CHECKPOINT MOLECULES IN MALIGNANT BRAIN TUMORS

Abstract Immune checkpoint therapy has been shown to be effective in several types of cancer, and tumor-directed immune activation is also a promising strategy in glioblastoma (GBM) patients. Our goal was to discover alterations in the circulating immunome of GBM patients and other cerebral tumor entities to identify soluble immune biomarkers and novel therapeutic targets. We analyzed 30 markers in plasma of 208 patients and healthy donors, using bead-based multiplex assays and flow cytometry. In a smaller cohort we further applied a high-sensitivity discovery biomarker platform with 368 markers. We included patients with 1) primary GBM (n=68), 2) recurrent GBM (n=25), 3) cerebral metastases (NSCLC, n=61; breast cancer, n=19), 4) meningioma (n=15) as well as 5) healthy donors (HD, n=20). We detected a distinct immune signature in the peripheral blood of patients with GBM, which was defined by an increased prevalence of soluble immune checkpoint markers (sCD27, sPD-L2, sGal-9), whereas soluble chemokines (CX3CL, CXCL12) were underrepresented. This signature was similar in primary and recurrent GBM, but differed markedly from other cerebral tumor entities as well as from HD. Primary GBM, but not their recurrent counterparts, additionally exhibited increased levels of neurotrophic factors (BDNF, b-NGF) and signs of immune activation with significantly increased levels of IL-18. The biomarker discovery platform further identified 76 proteins that were specifically regulated in GBM compared to HD, offering the potential to serve as biomarkers for disease activity. GBM patients with higher levels of BDNF displayed a significantly shorter survival than patients with lower levels (9.33 vs. 21.90 months; p=0.0001). Likewise patients with high concentrations of VEGF survived significantly shorter than those with low concentrations (12.97 vs. 21.90 months, p=0,0166). Taken together, we were able to delineate a specific immune profile of soluble markers in GBM patients compared to other cerebral tumor entities and to healthy individuals.

Single-cell transcriptomics reveals tumor-infiltrating B cell function after neoadjuvant pembrolizumab and chemotherapy in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most pervasive lung cancer subtype. Recent studies have shown that immune checkpoint inhibitors achieved favorable clinical benefits in resectable NSCLC; however, the associated mechanism remains unclear. The role of T cells in antitumor immunity has received considerable attention, while the antitumor effects of tumor-infiltrating B cells (TIBs) in NSCLC remain poorly understood. Here, we conducted a single-cell RNA sequencing analysis of immune cells isolated from 12 patients with stage IIIA NSCLC to investigate B cell subtypes and their functions following neoadjuvant chemoimmunotherapy. We confirmed the simultaneous existence of the 4 B cell subtypes. Among them, memory B cells were found to be associated with a positive therapeutic effect to neoadjuvant chemoimmunotherapy. Furthermore, we found that G protein-coupled receptor 183 was most prevalent in memory B cells and associated with a positive therapeutic response. Multiplex immunofluorescence and flow cytometry experiments in an additional cohort of 22 treatment-naïve and 30 stage IIIA/IIIB NSCLC patients treated with neoadjuvant chemoimmunotherapy verified these findings. Overall, our analysis revealed the functions of TIBs and their potential effect on clinical treatment in NSCLC.

Overcoming Radio-Immunotherapy Treatment Resistance through ILT4 Blockade and Reversal of HFRT Induced CXCL1-CXCR2 Axis Activation and Tumor-Associated Macrophage Immunosuppression

Immunotherapy combined with radiotherapy (iRT) has unlimited potential, but up to 60% of cancer patients do not benefit from it. Enhancing the anti-tumor immune stimulatory effect triggered by radiotherapy is the key to overcome iRT resistance. Immunoglobulin-like transcript (ILT) 4 is a potential immune checkpoint molecule, highly expressed in various tumor cells, but its role in radiotherapy is still unknown. This study confirmed the role and molecular mechanism of ILT4 in suppressing radiotherapy immunosuppressive microenvironment formation and promoting tumor radiotherapy resistance. We propose a new therapeutic strategy that block ILT4 to enhance the efficacy of radiotherapy, and cooperate with radiotherapy to reverse immunotherapy resistance. Using multiplex immunohistochemistry, we analyzed ILT4 expression, tumor-associated macrophage (TAM) /T cell phenotype and quantity in tumor patient treated with SBRT. Using mice subcutaneous tumor models, Single-cell RNA sequencing and multiplex flowcytometry, we assessed the role of ILT4 inhibition and hyper-fractionated radiotherapy (HFRT) on preventing tumor growth and immune escape. The molecular signaling and cytokines regulated by ILT4 under HFRT were analyzed by transcriptome sequencing and further verified by molecular experiments. By establishing cancer cell/TAM co-culture system in vitro, using CXCL1 protein or CXCR2 inhibitor and macrophage/CD8+ T cell deletion antibody in vivo, we identified the downstream pathway and cytokine of ILT4 to enhancing HFRT -induced TAM immune response. In the tumor specimens of NSCLC patients treated with SBRT, we found that high ILT4 expression predicted poor progression-free survival and more M2-TAM recruitment. Among the C57BL/6 mice model, ILT4 inhibition in cancer cells reduced HFRT mediated M2-TAMs accumulation, and to sustain activation and proliferation of CD8+ T cells, and eventually suppressed tumor progression. Mechanistically, RT promoted ILT4 expression, which subsequently induced NF-κB pathway activation and CXCL1 secretion to enhance M2-TAMs migration in vitro. Using CXCL1 protein or CXCR2 inhibitor administration, inferring that ILT4 promotes TAMs migration via NF-κB-CXCL1-CXCR2 axis. Consistently, depletion of TAMs blocked the T cell function impairment and radiotherapy resistance induced by ILT4 in vivo. Importantly, targeting ILT4 potentiated the effect of radiotherapy, overcomes radio-immunotherapy treatment resistance. ILT4 mediates HFRT-induced M2-like TAMs recruitment and subsequently T cell response impairment by regulating NF-κB-CXCL1-CXCR2 axis. ILT4 is an attractive drug target for enhancing radiotherapy and overcomes radio-immunotherapy treatment resistance.

Fungal Depletion Bolsters Anti-Tumor Immune Response Elicited by Anti-PD1 Alone and in Combination with Radiation Therapy

Pembrolizumab in combination with chemotherapy has become the standard of care treatment for both metastatic and early-stage triple negative breast cancer (TNBC). Clinical trials are currently underway investigating the use of pembrolizumab with radiation in the neoadjuvant setting in early TNBC. Several groups have described a link between the microbiome and the efficacy of chemotherapy and anti-PD1 immune checkpoint inhibitors (ICIs) in preclinical models. Recent work from our lab has shown that targeting commensal fungi in the microbiome enhances the radiation induced antitumor immune response. Therefore, we hypothesized that fungal depletion might positively impact anti-PD1 therapy and combination treatment with anti-PD1 and radiation therapy (RT). This study utilized an orthotopic syngeneic breast tumor model in which the syngeneic cell line E0771 was injected into the mammary fat pad of female C57BL/6 mice. Tumor-bearing mice were then treated with and without the antifungal fluconazole, anti-PD1, and radiation (16 Gy single fraction) using the X-RAD SmART platform with CT guidance. Tumor volumes were compared using 2-way ANOVA and survival curves analyzed using log rank. In a separate set of experiments, tumor-infiltrating immune cells were isolated and analyzed by high-dimensional multiplex flow cytometry. We found that fungal depletion with fluconazole prior to treatment with anti-PD1 reduced the tumor volume and significantly improved survival in comparison to those treated with anti-PD1 alone (P = 0.0016). To identify what changes in the tumor immune microenvironment is driving this increased anti-tumor response, we performed flow cytometry on immune cells isolated from the tumors. We found that the use of fluconazole prior to anti-PD1 treatment reduced the proportion of CD11b+F4/80+ tumor-associated macrophages (TAMs) (P = 0.01) and increased tumor infiltrating cytotoxic T cell population (P = 0.04) when compared with the use of anti-PD1 alone. We also evaluated the effect of fungal depletion on combination therapy with RT and anti-PD1. Strikingly, we found that mice depleted of fungi with fluconazole prior to radiation and anti-PD1 therapy, have decreased tumor burden and significantly increased survival when compared to their fungally-intact counterparts (P = 0.043). Our data indicates that the depletion of the gut fungal populations induces an increased antitumor response following anti-PD1 alone and in combination with radiation. This increased antitumor immune response is associated with an increase in the cytotoxic CD8+ T cell compartment with concomitant decrease in immunosuppressive tumor associated macrophages.

Identification of optimal conditions for human placental explant culture and extracellular vesicle release

Extracellular vesicles (EVs) can mediate intercellular communication, including signaling between the placenta and maternal tissues. Human placental explant culture is a versatile invitro model system to investigate placental function. We performed systematic studies in different tissue culture media types and oxygen tensions to identify a defined serum-free culture condition that supports high trophoblast viability and metabolism, as well as the release of similar populations of EVs, compared to traditional undefined conditions that contain media additives potentially contaminated with exogenous EVs. We also determined the time frame in which trophoblast viability and functionality remain optimal. Multiplex vesicle flow cytometry with classical EV and placenta-specific markers revealed three separate populations of explant-derived EVs: small CD63+ EVs; large PLAP+ EVs; and CD63-/PLAP- EVs. These culture and analytical approaches will enable invitro modeling of short-term effects of environmental perturbations associated with pregnancy complications on placental function and EV release.

Sex differences in renal cell carcinoma: a single-cell analysis reveals exhausted CD8+ T-cells highly infiltrated in males

BackgroundAlthough sex bias has been reported in the development and progression of renal cell carcinoma (RCC), the underlying mechanisms remain enigmatic. Here, we investigated the sex differences in the tumor microenvironment (TME) of RCC and explored a promising combination drug regimen to enhance the efficacy of immunotherapy.MethodsSingle-cell RNA sequencing (scRNA-seq) data from four published datasets were analyzed to investigate the sex differences in RCC patients, and tumor tissues were collected to validate the sex differences using multiplex immunofluorescence (MxIF) and flow cytometry (FCM). The function of the androgen–androgen receptor axis in sex differences was explored in vivo and in vitro experiments.ResultsOur analysis of scRNA-seq data from 220,156 cells, as well as MxIF and FCM assays, revealed that CD8+ T-cells infiltrated highly in the TME of male RCC, but were mostly in an exhausted and dysfunctional state. In vitro and in vivo experiments indicated that the dysfunction and exhaustion of CD8+ T-cells in male TME were induced by androgen. Clinically, higher serum androgen was significantly associated with a worse prognosis in male RCC patients receiving immunotherapy. Androgen receptor inhibitors could activate tumor-infiltrating CD8+ T-cells and enhance the efficacy of immunotherapy of RCC in vivo.ConclusionsOur study delineated the difference in TME between male and female patients with RCC, and demonstrated that the androgen–androgen receptor axis plays an important role in immunosuppression in male RCC. Our findings suggest that androgen receptor inhibitors in combination with immunotherapy may be a promising treatment option for male RCC patients.

Impact of sleep deprivation on neurocognition and inflammation in rhesus macaques

Sleep deprivation in humans is associated with both cognitive impairment and immune dysregulation. An animal model of neuropathogenesis may provide insight to understand the effects of sleep deprivation on the brain. Human neurocognition is more closely mirrored by nonhuman primates (NHP) than other animals. As such, we developed an NHP model to assess the impact of sleep deprivation on neurocognition and markers of systemic immune activation. Six male rhesus macaques underwent three rounds of sleep deprivation (48 h without sleep) at days 0, 14, and 28. We performed domain specific cognitive assessments using the Cambridge Neuropsychological Test Automated Battery (CANTAB) via a touch screen before and after 24 and 48 h of sleep deprivation. Immune activation markers were measured in the blood by multiplex assay and flow cytometry. Although we observed variability in cognitive performance between the three rounds of sleep deprivation, cognitive impairments were identified in all six animals. We noted more cognitive impairments after 48 h than after 24 h of sleep deprivation. Following 48 h of sleep deprivation, elevations in markers of immune activation in the blood were observed in most animals. The observed impairments largely normalized after sleep. The co-occurrence of systemic immune alterations and cognitive impairment establishes this model as useful for studying the impact of sleep deprivation on neurobehavior and immune perturbations in rhesus macaques.

Analyzing Flow Cytometry or Targeted Gene Expression Data Influences Clinical Discoveries-Profiling Blood Samples of Pancreatic Ductal Adenocarcinoma Patients.

Monitoring the therapeutic response of pancreatic ductal adenocarcinoma (PDAC) patients is crucial to determine treatment strategies. Several studies have examined the effectiveness of FOLFIRINOX as a first-line treatment in patients with locally advanced pancreatic cancer, but little attention has been paid to the immunologic alterations in peripheral blood caused by this chemotherapy regimen. Furthermore, the influence of the measurement type (e.g., flow cytometry and targeted gene expression) on the clinical discoveries is unknown. Therefore, we aimed to scrutinize the influence of using flow cytometry or targeted immune gene expression to study the immunological changes in blood samples of PDAC patients who were treated with a single-cycle FOLFIRINOX combined with lipegfilgrastim (FFX-Lipeg). Whole-blood samples from 44 PDAC patients were collected at two time points: before the first FOLFIRINOX cycle and 14 days after the first cycle. EDTA blood tubes were used for multiplex flow cytometry analyses to quantify 18 immune cell populations and for complete blood count tests as the standard clinical routine. The flow cytometry data were analyzed with FlowJo software. In addition, Tempus blood tubes were used to isolate RNA and measure 1230 immune-related genes using NanoString Technology®. Data quality control, normalization, and analysis were performed using nSolver™ software and the Advanced Analysis module. FFX-Lipeg treatment increased the number of neutrophils and monocytes, as shown by flow cytometry and complete blood count in concordance with elevated gene expression measured via targeted gene expression profiling analysis. Interestingly, flow cytometry analysis showed an increase in the number of B and T cells after treatment, while targeted gene expression analysis showed a decrease in B and T cell-specific gene expression. Targeted gene expression complements flow cytometry analysis to provide a comprehensive understanding of the effects of FFX-Lipeg. Flow cytometry and targeted gene expression showed increases in neutrophils and monocytes after FFX-Lipeg. The number of lymphocytes is increased after treatment; nevertheless, their cell-specific gene expression levels are downregulated. This highlights that different techniques influence clinical discoveries. Therefore, it is important to carefully select the measurement technique used to study the effect of a treatment.

CD44+ and CD31+ extracellular vesicles (EVs) are significantly reduced in polytraumatized patients with hemorrhagic shock - evaluation of their diagnostic and prognostic potential.

Hemorrhagic shock (HS) is responsible for approximately 2 million deaths per year worldwide and is caused in 80% by polytrauma. These patients need a precise and quick diagnostic, which should be based on a combination of laboratory markers and radiological data. Extracellular vesicles (EVs) were described as potential new markers and mediators in trauma. The aim of the present study was to analyze, whether the surface epitopes of plasma-EVs reflect HS in polytraumatized patients and whether cell-specific EV subpopulations are useful diagnostic tools. Plasma samples from polytraumatized patients (ISS ≥16) with HS (n=10) and without (n=15), were collected at emergency room (ER) and 24h after trauma. Plasma-EVs were isolated via size exclusion chromatography and EV-concentrations were detected by Coomassie Plus (Bradford) Assay. The EVs subpopulations were investigated by a bead-based multiplex flow cytometry measurement of surface epitopes and were compared with healthy controls (n=10). To investigate the diagnostic and prognostic potential of EVs subpopulations, results were correlated with clinical outcome parameters documented in the electronical patients' record. We observed a significant reduction of the total amount of plasma EVs in polytrauma patients with HS, as compared to polytrauma patients without HS and healthy controls. We found significant reduction of CD42a+ and CD41b+ (platelet-derived) EVs in all polytrauma patients, as well as a reduction of CD29+ EVs compared to healthy volunteers (*p<0.05). CD44+ and CD31+ EVs were specifically altered in patients with HS (*p<0.05). Both EV populations showed a moderate correlation (r² = 0.42) with the transfusion of erythrocyte concentrate, were associated with non-survival and the need for catecholamines (*p<0.05). Our data reveal that polytrauma patients with a hemorrhagic shock are characterized by a reduction of CD44+ and CD31+ plasma-EVs. Both EV populations showed a moderate correlation with the need of erythrocyte transfusion, were associated with non-survival and the need for catecholamines.

Antitumor effect of anti-vascular therapy with STING agonist depends on the tumor microenvironment context.

Targeting tumor vasculature is an efficient weapon to fight against cancer; however, activation of alternative pathways to rebuild the disrupted vasculature leads to rapid tumor regrowth. Immunotherapy that exploits host immune cells to elicit and sustain potent antitumor response has emerged as one of the most promising tools for cancer treatment, yet many treatments fail due to developed resistance mechanisms. Therefore, our aim was to examine whether combination of immunotherapy and anti-vascular treatment will succeed in poorly immunogenic, difficult-to-treat melanoma and triple-negative breast tumor models. Our study was performed on B16-F10 melanoma and 4T1 breast tumor murine models. Mice were treated with the stimulator of interferon genes (STING) pathway agonist (cGAMP) and vascular disrupting agent combretastatin A4 phosphate (CA4P). Tumor growth was monitored. The tumor microenvironment (TME) was comprehensively investigated using multiplex immunofluorescence and flow cytometry. We also examined if such designed therapy sensitizes investigated tumor models to an immune checkpoint inhibitor (anti-PD-1). The use of STING agonist cGAMP as monotherapy was insufficient to effectively inhibit tumor growth due to low levels of STING protein in 4T1 tumors. However, when additionally combined with an anti-vascular agent, a significant therapeutic effect was obtained. In this model, the obtained effect was related to the TME polarization and the stimulation of the innate immune response, especially activation of NK cells. Combination therapy was unable to activate CD8+ T cells. Due to the lack of PD-1 upregulation, no improved therapeutic effect was observed when additionally combined with the anti-PD-1 inhibitor. In B16-F10 tumors, highly abundant in STING protein, cGAMP as monotherapy was sufficient to induce potent antitumor response. In this model, the therapeutic effect was due to the infiltration of the TME with activated NK cells. cGAMP also caused the infiltration of CD8+PD-1+ T cells into the TME; hence, additional benefits of using the PD-1 inhibitor were observed. The study provides preclinical evidence for a great influence of the TME on the outcome of applied therapy, including immune cell contribution and ICI responsiveness. We pointed the need of careful TME screening prior to antitumor treatments to achieve satisfactory results.

Cutaneous squamous cell carcinoma-derived extracellular vesicles exert an oncogenic role by activating cancer-associated fibroblasts

Cutaneous squamous cell carcinoma (cSCC) is a fast-increasing cancer with metastatic potential. Extracellular vesicles (EVs) are small membrane-bound vesicles that play important roles in intercellular communication, particularly in the tumor microenvironment (TME). Here we report that cSCC cells secrete an increased number of EVs relative to normal human epidermal keratinocytes (NHEKs) and that interfering with the capacity of cSCC to secrete EVs inhibits tumor growth in vivo in a xenograft model of human cSCC. Transcriptome analysis of tumor xenografts by RNA-sequencing enabling the simultaneous quantification of both the human and the mouse transcripts revealed that impaired EV-production of cSCC cells prominently altered the phenotype of stromal cells, in particular genes related to extracellular matrix (ECM)-formation and epithelial-mesenchymal transition (EMT). In line with these results, co-culturing of human dermal fibroblasts (HDFs) with cSCC cells, but not with normal keratinocytes in vitro resulted in acquisition of cancer-associated fibroblast (CAF) phenotype. Interestingly, EVs derived from metastatic cSCC cells, but not primary cSCCs or NHEKs, were efficient in converting HDFs to CAFs. Multiplex bead-based flow cytometry assay and mass-spectrometry (MS)-based proteomic analyses revealed the heterogenous cargo of cSCC-derived EVs and that especially EVs derived from metastatic cSCCs carry proteins associated with EV-biogenesis, EMT, and cell migration. Mechanistically, EVs from metastatic cSCC cells result in the activation of TGFβ signaling in HDFs. Altogether, our study suggests that cSCC-derived EVs mediate cancer-stroma communication, in particular the conversion of fibroblasts to CAFs, which eventually contribute to cSCC progression.

Effects of sevoflurane on metalloproteinase and natural killer group 2, member D (NKG2D) ligand expression and natural killer cell-mediated cytotoxicity in breast cancer: an in vitro study.

We investigated the effects of sevoflurane exposure on the expression of matrix metalloproteinase (MMP), expression and ablation of natural killer group 2, member D (NKG2D) ligands (UL16-binding proteins 1-3 and major histocompatibility complex class I chain-related molecules A/B), and natural killer (NK) cell-mediated cytotoxicity in breast cancer cells. Three human breast cancer cell lines (MCF-7, MDA-MB-453, and HCC-70) were incubated with 0 (control), 600 (S6), or 1200 μM (S12) sevoflurane for 4 h. The gene expression of NKG2D ligands and their protein expression on cancer cell surfaces were measured using multiplex polymerase chain reaction (PCR) and flow cytometry, respectively. Protein expression of MMP-1 and -2 and the concentration of soluble NKG2D ligands were analyzed using western blotting and enzyme-linked immunosorbent assays, respectively. Sevoflurane downregulated the mRNA and protein expression of the NKG2D ligand in a dose-dependent manner in MCF-7, MDA-MB-453, and HCC-70 cells but did not affect the expression of MMP-1 or -2 or the concentration of soluble NKG2D ligands in the MCF-7, MDA-MB-453, and HCC-70 cells. Sevoflurane attenuated NK cell-mediated cancer cell lysis in a dose-dependent manner in MCF-7, MDA-MB-453, and HCC-70 cells (P = 0.040, P = 0.040, and P = 0.040, respectively). Our results demonstrate that sevoflurane exposure attenuates NK cell-mediated cytotoxicity in breast cancer cells in a dose-dependent manner. This could be attributed to a sevoflurane-induced decrease in the transcription of NKG2D ligands rather than sevoflurane-induced changes in MMP expression and their proteolytic activity.

Protective effect of the novel calcineurin inhibitor voclosporin in experimental colitis.

Acute severe steroid-refractory ulcerative colitis remains a medically challenging condition with frequent need of surgery. It can be treated with the calcineurin inhibitor cyclosporine A with the need for therapeutic drug monitoring and significant toxicity. Recently, a novel calcineurin inhibitor, voclosporin, has been approved for the treatment of lupus nephritis with no need for therapeutic drug monitoring and an improved long-term safety profile. However, the therapeutic effect of voclosporin in acute severe steroid-refractory ulcerative colitis is still uncertain. We aimed to assess the therapeutic potential of voclosporin to ameliorate inflammation in an experimental model of colitis. We used the dextran sodium sulfate-induced model of colitis in C57BL/6 J wildtype mice treated with either cyclosporine A, voclosporin or solvent control. We employed endoscopy, histochemistry, immunofluorescence, bead-based multiplex immunoassays and flow cytometry to study the therapeutic effect of calcineurin inhibitors in a preventive setting. Acute colitis was induced by dextran sodium sulfate characterized by weight loss, diarrhea, mucosal erosions and rectal bleeding. Both cyclosporine A and voclosporin strongly ameliorated the course of disease and reduced colitis severity in a similar manner. Voclosporin was identified as biologically effective in a preclinical model of colitis and may be a potential therapeutic option in treating acute severe steroid-refractory ulcerative colitis.

Interrogating drug action and immune response in organotypic glioma microenvironment.

2068 Background: The glioma tumor microenvironment is characteristic of a high level of heterogeneity and a profoundly immune suppressive phenotype as driven by the presence of glioma stem cells and abundant innate immune cells of the myeloid lineages, rendering glioblastomas (GBM) recalcitrant to conventional treatments and immunotherapies. To address the full complexity of the brain tumor with its diverse components, we have developed an organotypic slice culture system to overcome several limitations of the other preclinical models and allow interrogation of drug action and immunomodulatory agents in human GBM ex vivo. Methods: We performed extensive characterization of the organotypic glioma slice system and optimization of the ex vivo tissue culture conditions, and determined the longitudinal dynamics of slice cell viability, relative tumor versus immune cellularity, and responses to a selection of therapeutic agents and cytokines by multiplexed flow cytometry and single cell transcriptome (scRNA seq) profiling. Results: Single cell transcriptomic analysis showed that both tumor and immune cell heterogeneity were preserved ex vivo for the two-week observation time, which exhibited tumor-type specific gene expression profiles suggesting cooperative interactions among cell populations. Consistent with the findings in fresh tumor specimens and an immune suppressive microenvironment, we found a scarcity of T lymphocytes in IDH mutant gliomas, whereas abundant T cells were found in recurrent GBM slices that persisted ex vivo. Additional analyses revealed that T cell infiltration in GBM was associated with the mesenchymal-like molecular phenotype, which exhibited remarkable intratumor heterogeneity highlighting the presence of stem-like cells and a unique cell population expressing aberrant cancer genes and antigen presenting molecules. We further found that T cells in organotypic GBM slices acquired a potent effector phenotype in response to cytokine-induced reprogramming of microglia and tumor-associated macrophages. In addition, scRNA seq also revealed a population of cancer-associated fibroblast-like cells that are present in all tumors sequenced, which may function to mediate the interactions between immune and tumor cells as our data suggested. Conclusions: We demonstrated that the organotypic slice culture system we developed is compatible with genomic, pharmacological, and immunotherapeutic manipulations and provided data showing that organotypic slice culture preserves glioma microenvironment ex vivo allowing acquisition of crucial information of response of heterogeneous patient tumor tissue to the agent tested. This method represents a unique mechanism by which therapy development can be facilitated in an ex vivo setting.