Immunofluorescence Image Analysis Research Articles

Abstract B006: GSTT2 modulates immune activation and impacts response to BCG immunotherapy in bladder cancer

Abstract Whilst M. bovis Bacillus Calmette-Guérin (BCG) therapy is the gold-standard for treating high risk non-muscle invasive bladder cancer (NMIBC), 30-40% of patients fail therapy, resulting in disease recurrence and progression. Loss of glutathione-S-transferase theta 2 (GSTT2) expression resulting from a promoter deletion, has been associated with modulation of intracellular ROS and BCG survival in BC cells. Retrospective analysis of NMIBC patients revealed that patients with the promoter deletion responded better to fewer instillations of BCG. To understand these responses, wildtype (WT) and GSTT2-knockout (KO) mice were implanted with MB49-PSA BC cells, either subcutaneously or orthotopically, and treated with weekly BCG instillations. Subcutaneous tumors were harvested after 3 BCG instillations for flow cytometry analysis, or after 4 BCG instillations for immunofluorescence (IF) imaging and gene expression analysis. Bladders (orthotopic model) were harvested after 4 BCG instillations for single-cell RNA sequencing. Splenocytes from WT and KO male mice were also stimulated in-vitro with TLR agonists, for 48h, to assess immune responses. In the subcutaneous model, in male mice, the cure rate was higher in WT mice (57%) compared to KO mice (0%). Flow cytometry analysis showed significantly higher numbers of tumor-associated CD8+ T-cells in BCG-treated KO males compared to WT males; however, IF imaging revealed higher penetration of CD8+ T-cells in tumors from WT males. In female mice, the cure rate was similar in WT (25%) and GSTT2-KO (38%) mice. IF imaging showed higher penetration of CD8+ T-cells in KO females. Quantitative real-time PCR (q-PCR) analysis of BCG-treated subcutaneous tumors from female mice revealed lower expression of the immune exhaustion markers, PD-L1 and CTLA-4, in KO mice compared to WT mice. Similarly, in the orthotopic model (female mice), q-PCR analysis of bladders also showed lower expression of PD-L1 and CTLA-4 in KO mice compared to WT mice. Single-cell RNA sequencing of bladders from BCG-treated mice revealed differences in the expression of genes such as Peak1, Hmga2, Scl2a1, Kras, Ahank, Gsta4, Mecom and Sh3gl2, of which Gsta4, Mecom, Sh3gl2, Hmga2 and Peak1 were validated using q-PCR. In-vitro analysis of splenocytes from male mice revealed higher levels of IL-6, IL-1β and IFN-α, after stimulation with TLR7/8 and TLR9 agonists, in KO splenocytes relative to WT splenocytes. Despite the higher levels of cytokine production by splenocytes and higher proportion of tumor-associated CD8+ T-cells in KO male mice, these mice fared poorly, which could be explained by immune exhaustion or lack of immune penetration into the tumor. In females, lower expression of immune exhaustion markers in KO mice did not yield better responses; however, single-cell data showed differences in expression of genes associated with pro- and anti-tumorigenic pathways, which could influence response. Thus, loss of GSTT2 may influence the response to BCG therapy, through modulating downstream signaling pathways and immune responses. Citation Format: Mugdha Vijay Patwardhan, Kane Toh, Edmund Chiong, Ratha Mahendran. GSTT2 modulates immune activation and impacts response to BCG immunotherapy in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B006.

Collagen IV assembly is influenced by fluid flow in kidney cell-derived matrices

Kidney podocytes and endothelial cells assemble a complex and dynamic basement membrane that is essential for kidney filtration. Whilst many components of this specialised matrix are known, the influence of fluid flow on its assembly and organisation remains poorly understood. Using the coculture of podocytes and glomerular endothelial cells in a low-shear stress, high-flow bioreactor, we investigated the effect of laminar fluid flow on the composition and assembly of cell-derived matrix. With immunofluorescence and matrix image analysis we found flow-mediated remodelling of collagen IV. Using proteomic analysis of the cell-derived matrix we identified changes in both abundance and composition of matrix proteins under flow, including the collagen-modifying enzyme, prolyl 4-hydroxylase (P4HA1). To track collagen IV assembly, we used CRISPR-Cas9 to knock in the luminescent marker HiBiT to the endogenous COL4A2 gene in podocytes. With this system, we found that collagen IV was secreted and accumulated consistently under both static and flow conditions. However knockdown of P4HA1 in podocytes led to a reduction in the secretion of collagen IV and this was more pronounced under flow. Together, this work demonstrates the effect of fluid flow on the composition, modification, and organisation of kidney cell-derived matrix and provides an in vitro system for investigating flow-induced matrix alteration in the context of kidney development and disease.

Advancing Biological Research: New Automated Analysis of Immunofluorescence Signals

In recent years, optical imaging and efficient computational approaches have improved the ability to analyse and understand biological phenomena. Immunofluorescence (IF) is a widely used immunochemical technique that provides information about protein localisation and expression levels. However, the manual analysis of IF images can present important limitations, such as operator workload and interpretative bias. Thus, the development of automated tools for IF signal computation is crucial. Several software programs have been proposed to address this challenge, but there is still a need for more accurate and reliable systems. In this work, we present Q-IF, a software for automatically measuring cellular IF signals with an intuitive and easy-to-use interface. We describe the software and validate its results in different biological scenarios using SH-SY5Y neuroblastoma cells, human fibroblasts, and rat brain sections. The Q-IF system automatically carries out the entire process, from IF signal quantification to statistical analysis, thus evading operator biases and speeding up the analysis workflow. Our results demonstrate the accuracy and reliability of the Q-IF system, highlighting its potential as a valuable tool for IF analysis in biological research.

CRISPR activation as a platform to identify interferon stimulated genes with anti-viral function.

Interferon Stimulated Gene (ISG) expression plays a key role in the control of viral replication and development of a robust adaptive response. Understanding this dynamic relationship between the pathogen and host is critical to our understanding of viral life-cycles and development of potential novel anti-viral strategies. Traditionally, plasmid based exogenous prompter driven expression of ISGs has been used to investigate anti-viral ISG function, however there are deficiencies in this approach. To overcome this, we investigated the utility of CRISPR activation (CRISPRa), which allows for targeted transcriptional activation of a gene from its endogenous promoter. Using the CRISPRa-SAM system to induce targeted expression of a panel of anti-viral ISGs we showed robust induction of mRNA and protein expression. We then employed our CRISPRa-SAM ISG panel in several antiviral screen formats to test for the ability of ISGs to prevent viral induced cytopathic cell death (CPE) and replication of Dengue Virus (DENV), Zika Virus (ZIKV), West Nile Virus Kunjin (WNVKUN), Hepatitis A Virus (HAV) and Human Coronavirus 229E (HCoV-229E). Our CRISPRa approach confirmed the anti-viral activity of ISGs like IFI6, IFNβ and IFNλ2 that prevented viral induced CPE, which was supported by high-content immunofluorescence imaging analysis. This work highlights CRISPRa as a rapid, agile, and powerful methodology to identify and characterise ISGs and viral restriction factors.

Pathobiology of Candida auris infection analyzed by multiplexed imaging and single cell analysis.

Fungal organisms contribute to significant human morbidity and mortality and Candida auris (C. auris) infections are of utmost concern due to multi-drug resistant strains and persistence in critical care and hospital settings. Pathogenesis and pathology of C. auris is still poorly understood and in this study, we demonstrate how the use of multiplex immunofluorescent imaging (MxIF) and single-cell analysis can contribute to a deeper understanding of fungal infections within organs. We used two different neutrophil depletion murine models (treated with either 1A8-an anti-Ly6G antibody, or RB6-8C5-an anti-Ly6G/Ly6C antibody; both 1A8 and RB6-8C5 antibodies have been shown to deplete neutrophils) and compared to wildtype, non-neutropenic mice. Following pathologist assessment, fixed samples underwent MxIF imaging using a C. albicans antibody (shown to be cross-reactive to C. auris) and immune cell biomarkers-CD3 (T cells), CD68 (macrophages), B220 (B cells), CD45 (monocytes), and Ly6G (neutrophils) to quantify organ specific immune niches. MxIF analysis highlighted the heterogenous distribution of C. auris infection within heart, kidney, and brain 7 days post-infection. Size and number of fungal abscesses was greatest in the heart and lowest in brain. Infected mice had an increased count of CD3+, CD68+, B220+, and CD45+ immune cells, concentrated around C. auris abscesses. CD68+ cells were predominant in wildtype (non-neutropenic mice) and CD3+/CD45+ cells were predominant in neutropenic mice, with B cells being the least abundant. These findings suggest a Th2 driven immune response in neutropenic C. auris infection mice models. This study demonstrates the value of MxIF to broaden understanding of C. auris pathobiology, and mechanistic understanding of fungal infections.

Prolonged exposure to lactate causes TREK1 channel clustering in rat hippocampal astrocytes

Increased concentrations of lactate (15–30 mM) are associated with and found to be neuroprotective in various brain pathophysiology. In our earlier studies we showed that high levels of lactate can increase TREK1 channel activity and expression within 1 h. TREK1 channels are two pore domain leak potassium ion channels that are upregulated during cerebral ischemia, epilepsy and other brain pathologies. They play a prominent neuroprotective role against excitotoxicity. Although it has been previously shown that chronic application of lactate (6 h) causes increased gene transcription and protein expression, we observe clustering of TREK1 channels that is dependent on time of exposure (3–6 h) and concentration of lactate (15–30 mM). Using immunofluorescence techniques and image analysis, we show that the clustering of TREK1 channels is dependent on the actin cytoskeletal network of the astrocytes. Clustering of TREK1 channels can augment astrocytic functions during pathophysiological conditions and have significant implications in lactate mediated neuroprotection.

Measuring CD8-Positive T-Cells in the Tumor Microenvironment in Classical Hodgkin Lymphoma Using Multiplex Immunofluorescence Staining and Image Analysis: A Possible Prognostic Factor

Introduction: Classical Hodgkin lymphoma (cHL) is histologically composed of Hodgkin Reed-Sternberg (HRS) cells and the tumor microenvironment (TME) of the surrounding T-cells and macrophages, in which the expression of PD-1 and PD-L1 play critical roles in the immune response against cHL. Recently, multiplex immunofluorescence (MIF) staining and image analysis have enabled the evaluation of TME in cHL. However, the relationship between the characteristics of TME and clinical features of cHL is not fully understood. Therefore, we analyzed the pathology of patients with cHL and their clinical outcomes using MIF technology to evaluate the clinical features and the variations in TME. Methods: We retrospectively evaluated the clinical records and pathological samples for the diagnosis of patients with primary or relapsed cHL treated in our institution between 2010 and 2021. MIF staining and image analysis of TME were performed sequentially per the following process. Multiplexed immunofluorescence labeling against CD4, CD 8, CD30, CD68, CD163, PD-1, and PD-L1 was performed using the Opal 6-Plex Manual Detection Kit (Akoya Biosciences), and the image was captured using the Quantitative Pathology Workstation (Mantra2, Akoya). After image capture, the visual images were analyzed using supervised machine learning algorithms in Inform 2.6.0 (Akoya), which assigns phenotypes to all cells. To identify the structure of TME, the Euclidean distance from each HRS cell to other phenotype cell was calculated and identified, defined as area A within a radius of 30 µm from the HRS cells, area B from 30 µm to 60 µm, and area C from 60 µm to 120 µm. Thereafter, we analyzed the relationship between these parameters in TME at the initial diagnosis and the 3-year progression-free survival (PFS) calculated with Kaplan-Meyer methods. If the sample at the initial diagnosis was not available, the data from the sample at the first relapse were substituted. Results: We obtained the data from 34 patients with 45 tissues, consisting of the pathological samples at the initial diagnosis (n = 32), the first relapse (n = 12), and the second relapse (n = 1). The median patient age was 46 years (range: 16-82). In the initial diagnosis, 18 and 16 patients had localized and advanced stages, respectively. All patients were initially treated with ABVD or BV-AVD therapy. During the median observation period of 61 months, 12 patients relapsed, and the 5-year PFS rate was 73%. All 45 tissues showed the expected patterns of cellular staining, with anti-CD30 delineating cells with HRS cells, anti-CD68 with macrophages (defined as anti-CD68-only expression as M1-macrophages (M1M) and co-expression of anti-CD68 and anti-CD163 as M2-macrophages (M2M)), and CD4 positive T-cells co-expressing anti-PD-1 and macrophages co-expressing anti-PD-L1. Comparisons of the characteristics of TME among areas A, B, and C revealed that CD8-positive T-cells, PD-1-positive CD4-positive T-cells, and PD-L1-positive macrophages were significantly more common in areas A and B than in area C, whereas PD-1 negative T-cells and PD-L1 negative macrophages exhibited no particular pattern in all areas. Thus, we identified that TME structures within 60μm are crucial for their immune effect on HRS cells. In the 34 patients at the initial diagnosis, we compared the TME within 60 µm and the PFS. As a result, TME containing higher numbers of CD8-positive T-cells (defined >15 cells) and M2M (defined > 13 cells) in TME were significantly associated with better 3-year PFS as compared with lower numbers of these immune components (100 vs 53%, p = 0.006 and 100 vs 65%, p = 0.012, respectively), whereas such findings were negative for CD4-positive T-cells or M1M. In the TME containing >15 cells of CD8-positive T-cell, significantly more M2M (mean 16 vs 5 cells, p=0.004) and fewer (mean 16 vs 28 cells, p=0.036) PD-1-positive T-cells were observed than in the TME containing ≤15 cells of CD8-positive T-cell. In the comparisons of 32 initial and 13 relapse samples, the number of CD8-positive T-cells TME significantly decreased at the time of relapse compared to the initial diagnosis (mean 7 vs 11 cells, p = 0.035). Conclusion: The findings of the MIF technology suggest that CD8-positive, cytotoxic T-cell enrichment in TME is a favorable prognostic factor for cHL. Such TME could be associated with PD-1/PD-L1-independent immune response against HRS cells. These results warrant further investigations.

Localization of DC-SIGN Expressing Cells in Follicular Lymphoma

The high incidence of unusual mannose-rich glycans on the expressed surface immunoglobulins of follicular lymphoma (FL) suggests a pathogenic role for this modification. While lectin-mediated signaling has been proposed to provide trophic support to FL cells, the localization of the lectins has not been extensively characterized. Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) is a mannose-selective lectin named for its expression on classic marrow-derived dendritic cells. In vitro, recombinant DC-SIGN induces signaling in primary FL cells. The nodules of Follicular lymphoma (nFL) are the neoplastic counterparts of normal germinal centers (GC). Both GC and nFL contain variable numbers of centrocytic and/or centroblastic B cells, T-cells, and follicular dendritic cells (FDCs), but macrophages are typically found only in GC. Although termed “follicular dendritic cells”, FDCs are thought to be derived from non-blood stem-cells, potentially pericytes. Therefore, nFL lack classic dendritic cells and macrophages so it is unclear what cells in FL could express DC-SIGN. Therefore, we systematically characterized FDC and DC- SIGN localization in sixteen FL and nine Follicular hyperplasia (FH) lymph nodes. Using flow cytometry of disaggregated FL nodes, we were not able to identify a specific cell population expressing DC-SIGN. However, using immunohistochemistry and Immunofluorescence methods, we identified nine cases of FH and five cases of FL (n=5/16) with DC-SIGN and FDC co-localization. Immunohistochemical stains of the FDCs using CD23 and CD21 showed that the follicular dendritic structures were disrupted in almost all the FL cases assessed showing either a peripheral crescentic staining or broken FDC meshwork within the follicle. We identified five cases of FL with an average of more than four well visualized and complete FDC structures staining per low power field (4X) while the remaining cases had an average of less than two complete FDC network staining. There was near complete absence of CD21 positive FDCs meshwork identified in 50% of the FL cases compared to only one case lacking CD23 positive FDCs structure staining. In contrast, FH cases (n=9) showed complete staining of the FDC meshwork within the follicles with both CD21/CD23. For FH, the average number of complete FDC staining with CD23 was 18 while an average of 10 complete FDC meshworks stained with CD21. Furthermore, immunofluorescence imaging analysis showed different pattern of localization of CD23 positive FDC with DC-SIGN with between the two conditions. In FH, DC-SIGN and CD23 colocalization was identified mainly in the light zone. For FL, we observed that DC-SIGN overlap with CD23 localized in the intra-follicular areas and co-localized with CD163 (M2 macrophages) in the inter-follicular zones. Confocal imaging confirmed colocalization of CD23 and DC-SIGN on FDCs with close association to MUM1-positive B cells in both GC and nFL. Additional analysis also showed that 80% (n=5) of the cases which had more than four complete FDC structures staining with CD23 co-localized with DC-SIGN in the intra-follicular areas and the cases with complete FDC network disruption did not. There was no significant relationship identified between the extent of FDC networks and the fraction of non-neoplastic B cells in the FL cases. In summary, our data show that DC-SIGN is expressed on FDCs in FL and FH even though FDCs are not the marrow-derived DCs for which DC-SIGN is named. Furthermore, localization of DC-SIGN to the follicle suggests a potential role for the microenvironment in the pathogenesis of FL and possible therapeutic target.

Changes in the immune landscape of TNBC after neoadjuvant chemotherapy: correlation with relapse.

Patients with high-risk, triple negative breast cancer (TNBC) often receive neoadjuvant chemotherapy (NAC) alone or with immunotherapy. Various single-cell and spatially resolved techniques have demonstrated heterogeneity in the phenotype and distribution of macrophages and T cells in this form of breast cancer. Furthermore, recent studies in mice have implicated immune cells in perivascular (PV) areas of tumors in the regulation of metastasis and anti-tumor immunity. However, little is known of how the latter change during NAC in human TNBC or their impact on subsequent relapse, or the likely efficacy of immunotherapy given with or after NAC. We have used multiplex immunofluorescence and AI-based image analysis to compare the immune landscape in untreated and NAC-treated human TNBCs. We quantified changes in the phenotype, distribution and intercellular contacts of subsets of tumor-associated macrophages (TAMs), CD4+ and CD8+ T cells, and regulatory T cells (Tregs) in PV and non-PV various areas of the stroma and tumor cell islands. These were compared in tumors from patients who had either developed metastases or were disease-free (DF) after a three-year follow up period. In tumors from patients who remained DF after NAC, there was a marked increase in stromal CD163+ TAMs, especially those expressing the negative checkpoint regulator, T-cell immunoglobulin and mucin domain 3 (TIM-3). Whereas CD4+ T cells preferentially located to PV areas in the stroma of both untreated and NAC-treated tumors, specific subsets of TAMs and Tregs only did so only after NAC. Distinct subsets of CD4+ and CD8+ T cells formed PV clusters with CD163+ TAMs and Tregs. These were retained after NAC. Quantification of stromal TIM-3+CD163+ TAMs in tumor residues after NAC may represent a new way of identifying patients at high risk of relapse. PV clustering of immune cells is highly likely to regulate the activation and function of T cells, and thus the efficacy of T cell-based immunotherapies administered with or after NAC.

Abstract 18550: Impaired Microtubule Mediated Protein Trafficking as a Mechanism of PLN R14del Dilated Cardiomyopathy

Introduction: Phospholamban (PLN) regulates calcium handling and contractility in the heart by altering its phosphorylation status, thereby activating or inhibiting the calcium ATPase SERCA2a. A pathogenic mutation in PLN at the Arginine 14 codon (R14del) results in dilated cardiomyopathy characterized by ventricular arrythmias and sudden cardiac death. Cytoplasmic aggregation of PLN is a hallmark of the disease. However, exactly how the R14del mutation causes DCM remains largely unknown. As PLN is trafficked to the cell membrane before it localizes to the t-tubules, we hypothesize that PLN protein trafficking may be impaired, contributing to the PLN aggregation and DCM. Methods: Induced pluripotent stem cell derived cardiomyocytes (iPSC- iPSC-CMs) from patients carrying the PLN R14del mutation and isogenic controls generated by CRISPR-Cas9 genome editing were tagged with biotin to quantify membrane-bound protein expression. Western blotting and mass spectrometry was used to identify differences membrane protein expression, and immunocytochemistry was used to image microtubule layout, PLN and junctional protein expression at the cell borderintercalated disc (do you mean the intercalated disk?) in micropatterned iPSC-CMs. Results: Quantification of membrane-bound proteins showed a significant decrease of PLN at the cell membrane and connexin 43 expression in the intercalated disk of R14del iPSC-CMs compared to isogenic controls. Additionally, we observed significantly reduced levels of EB1, the microtubule tip protein, at the cell border of PLN R14del iPSC-CMs by immunofluorescence imaging and western blot analysis. Finally, disruption of microtube polymerization by Nocodazole treatment, induced PLN aggregate formation in iPSC-CMs. Conclusion: Our data suggest that the transportation of PLN and Cx43 proteins by microtubules is impaired in PLN R14del iPSC-CMs, which results in PLN accumulation in the cytoplasm and a decrease of Cx43 expression in the intercalated disc. These findings provide a novel mechanism for the pathogenesis of PLN R14del cardiomyopathy and a possible therapeutic target.

Neoadjuvant Chemoimmunotherapy Increases Tumor Immune Lymphocytes Infiltration in Resectable Non-small Cell Lung Cancer.

Neoadjuvant chemoimmunotherapy treatment (NCIT) has achieved great success for non-small cell lung cancer (NSCLC); however, the intrinsic mechanism underlying this treatment remains unclear. Thirty-two patients with stage IIA-IIIC NSCLC who underwent surgery after NCIT were included in this retrospective study. Multiplex immunofluorescence (mIF) staining and image analysis assays were performed on the samples collected before and after NCIT for each patient. RNA analyses was applied to confirm the mIF results. Among the enrolled patients, 14 achieved major pathological response or pathological complete response (pCR) and were defined as the 'response' group, whereas 18 patients did not respond well to NCIT and were defined as the 'nonresponse' group. The results of the mIF assays revealed an overall increase in tumor immune lymphocytes (TILs) after NCIT in the stroma area (p=0.03) rather than the tumor area (p=0.86). The percentage of CD8+ T cells and tertiary lymphoid structure counts in both the response and nonresponse groups increased significantly after NCIT compared with before NCIT. CD3+ T cells and FOXP3+ cells decreased significantly in the response group but remained unchanged or increased in the nonresponse group. A comparison of the response and nonresponse groups showed that CD3, FOXP3+ and CD8+/PD-1+ cells before NCIT may serve as predictors of the response to neoadjuvant immunotherapy. The RNA analyses confirmed the mIF results that TILs were elevated after NCIT. The infiltration of immune cells before NCIT was correlated with pathologic complete response, which enhanced the TILs as a promising predictor for selecting patients who were more likely to benefit from NCIT.

Abstract B030: Form and function in intratumoral immune organization: Understanding the cellular composition of TCF1+ CD8+ T cell niches in human cancer

Abstract Tumor infiltrating T cells are a positive prognosticator in many tumor types, and our prior work demonstrated that in renal cell carcinoma, patients with higher CD8 T cell infiltration have improved survival (Jansen Nature 2019). We described that this T cell response is supported by TCF1+ stem-like CD8 T cells, which reside within dense regions of closely clustered antigen presenting cells within the tumor. Interestingly, aggregations of immune cells have also been described in other settings and termed ‘tertiary lymphoid structures’ (TLS), raising an important question of whether the described immune niches are similar to these TLS or if they represent a distinct type of peripheral immune organization. In the work presented here, we use quantitative immunofluorescence image analysis to examine the cellular composition and organization of intratumoral immune niches, TLS, and secondary lymphoid tissue. The novel data presented here describes how immune niches are distinct from TLS and are more similar to the organization of T cell zones in secondary lymphoid tissue. Immune niches are T cell dominant structures, with T cell and antigen presenting cell compartments similar to T cell zones, whereas these niches profoundly lack the B cell presence seen in TLS and B cell zones of secondary lymphoid tissue. Immune niches are comparatively small in size (50-200um), while TLS are much larger (often >1mm), and, importantly, immune niches are found inside the tumor border, while TLS are located peripherally in tumor tissue. Notably, we find that immune niches have a marked presence of aSMA+ stromal cells, which are similar to those found in T cell zones but absent in B cell zones and TLS. When probed by flow cytometry and RNAseq, we find that aSMA+ stroma in immune niches are myofibroblast-like, similar those known to maintain the structure of secondary lymphoid tissues, and are distinct from those found in healthy kidney tissue. These data suggest these aSMA+ cells may play an important role in the organization of immune niches in tumor tissue. As we have shown that a robust T cell response is beneficial for patient outcomes, it is crucial to understand the mechanisms which support that response. Recently, our group defined that CD8 T cell activation in cancer is comprised of two phases, with an initial priming phase in the lymph node and a second phase of effector program acquisition in the tumor tissue (Prokhnevska Immunity 2023). We suggest that these immune niches represent an important hub for this development of T cell function, which is required for the robust, effective anti-tumor T cell response that supports improved patient outcomes and is the basis for the response to immunotherapy. Advancing our understanding of intratumoral immune organization will allow for enhanced biomarker discovery, improvement in existing therapies, and innovation in developing novel therapeutic strategies that will convert immunologically “cold” tumors to be immunologically “hot” and enhance anti-tumor immunity. Citation Format: Caroline S. Jansen, BaoHan Vo, Ewelina Sobierajska, Rachel Greenwald, Patrick Mullane, Nataliya Prokhnevska, Maria Cardenas, Mehmet Asim Bilen, Adeboye O. Osunkoya, Viraj A. Master, Haydn T. Kissick. Form and function in intratumoral immune organization: Understanding the cellular composition of TCF1+ CD8+ T cell niches in human cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B030.

Impaired dynamic X-chromosome inactivation maintenance in T cells is a feature of spontaneous murine SLE that is exacerbated in female-biased models

ObjectiveSystemic lupus erythematosus (SLE) is a highly female-biased systemic autoimmune disease, but the molecular basis for this female bias remains incompletely elucidated. B and T lymphocytes from patients with SLE and female-biased mouse models of SLE exhibit features of epigenetic dysregulation on the X chromosome which may contribute to this strong female bias. We therefore examined the fidelity of dynamic X-chromosome inactivation maintenance (dXCIm) in the pathogenesis of two murine models of spontaneous lupus—NZM2328 and MRL/lpr—with disparate levels of female-bias to determine whether impaired dXCIm contributes to the female bias of disease. MethodsCD23+ B cells and CD3+ T cells were purified from age-matched C57BL/6 (B6), MRL/lpr, and NZM2328 male and female mice, activated in vitro, and processed for Xist RNA fluorescence in situ hybridization, H3K27me3 immunofluorescence imaging, qPCR, and RNA sequencing analyses. ResultsThe dynamic relocalization of Xist RNA and the canonical heterochromatin mark, H3K27me3, to the inactive X chromosome was preserved in CD23+ B cells, but impaired in activated CD3+ T cells from the MRL/lpr model (p < 0.01 vs. B6), and even more impaired in the heavily female-biased NZM2328 model (p < 0.001 vs. B6; p < 0.05 vs. MRL/lpr). RNAseq of activated T cells from NZM2328 mice revealed the female-biased upregulation of 32 X-linked genes distributed broadly across the X chromosome, many of which have roles in immune function. Many genes encoding Xist RNA-interacting proteins were also differentially expressed and predominantly downregulated, which may account for the observed mislocalization of Xist RNA to the inactive X chromosome. ConclusionsAlthough evident in T cells from both the MRL/lpr and NZM2328 models of spontaneous SLE, impaired dXCIm is more severe in the heavily female-biased NZM2328 model. The aberrant X-linked gene dosage in female NZM2328 mice may contribute towards the development of female-biased immune responses in SLE-prone hosts. These findings provide important insights into the epigenetic mechanisms contributing to female-biased autoimmunity.

Personal immune profiles: Diversity and prognostic value for oral tongue squamous cell carcinoma evaluated by comprehensive immune parameter analyses with multiplex immunofluorescence

ObjectivesUnderstanding the tumor immune microenvironment is becoming increasingly necessary for risk prediction and treatment selection. In particular, oral cancer has various immunosuppressive characteristics in the tumor microenvironment. Therefore, we comprehensively assessed the immune profiles of oral tongue squamous cell carcinoma (OTSCC). Materials and methodsMultiplex immunofluorescence and tissue imaging analyses were performed to evaluate immune profiles at the invasive tumor front of 60 OTSCC surgical specimens. We analyzed 58 immune parameters including the density and proportion (%) of total leukocytes (Leu) and T cells, six subsets of T and myeloid cells, and the expression of programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1). ResultsThe density, proportion, and location of CD45+ Leu, three T cell subsets (CD8+, Foxp3–CD4+ conventional, and Foxp3+CD4+ regulatory T cells), CD163-CD68+ M1 and CD163+CD68+ M2 macrophages, and neutrophils were highly variable at the individual level. The density and proportion of M2 macrophages were significantly lower in the T1 stage group. Risk prediction analyses for recurrence and/or metastasis (R/M) showed that R/M (+) T1 cases had significantly higher M2 density and percentages. ConclusionsThe immune profiles of OTSCC patients are diverse and cannot be predicted from clinicopathological information alone. The M2 macrophage abundance is a potential candidate biomarker for R/M in the early stage of OTSCC. Personal immune profiling may provide beneficial information for risk prediction and treatment selection.

373 Identification of potential targets for immunotherapy in a cynomolgus macaque model of Ebola virus disease

OBJECTIVES/GOALS: Ebola virus infection causes severe disease and liver injury in humans. Macrophages contribute to inflammatory signaling and are prevalent in the liver. We assessed the activation status, including therapeutic target expression, of hepatic macrophages. METHODS/STUDY POPULATION: We compared formalin-fixed, paraffin-embedded liver tissue from terminal Ebola virus-infected and uninfected control cynomolgus macaques, a gold-standard model for human disease. We characterized region-specific protein and whole transcriptome expression in these tissues using GeoMx Digital Spatial Profiling. Macrophage (CD68+) and leukocyte (CD45+) accumulation in liver tissue was quantified by immunofluorescence image analysis using digital pathology software. RESULTS/ANTICIPATED RESULTS: Macrophage-specific (CD68+) regions in the liver of Ebola virus-infected macaques demonstrated a shift towards an inflammatory gene expression profile, as compared to those from healthy control tissue. These regions showed differential expression of monocyte/macrophage differentiation, antigen presentation, and T cell activation gene sets, which were associated with decreased MHC-II allele expression. Moreover, macrophage-specific regions in the infected macaques showed enriched expression of genes or proteins associated with known immunomodulatory therapeutics, including S100A9, IDO1, and CTLA-4. DISCUSSION/SIGNIFICANCE: These data demonstrate that hepatic macrophages express an inflammatory phenotype, that their ability to present antigens to the adaptive immune system may be impaired, and that they express therapeutically targetable markers for immunomodulation of these cells during Ebola virus infection.

Development of the oncolytic virus, CF33, and its derivatives for peritoneal-directed treatment of gastric cancer peritoneal metastases

BackgroundGastric cancer (GC) that metastasizes to the peritoneum is fatal. CF33 and its genetically modified derivatives show cancer selectivity and oncolytic potency against various solid tumors. CF33-hNIS and CF33-hNIS-antiPDL1 have...

Abstract P5-06-06: TROP2 and HER2 expression by liquid biopsy in women with mTNBC

Abstract Introduction A blood-based biopsy can inform the prognosis for MBC patients when conventional tissue biopsies are not feasible within often biologically heterogeneous sites of metastatic disease. Several anti-Trophoblast cell-surface antigen 2 (TROP2) and HER2 targeted therapies are now available, either as approved therapy options or in clinical trials for MBC patients. Tumor expression of TROP2 is prominent in metastatic cancers, such as HER2-negative MBC and TNBC, with limited treatment options. Technical feasibility data of blood-based TROP2 and HER2 immunofluorescence assays demonstrate the utility of the Epic Sciences platform for this cohort of metastatic TNBC (mTNBC) patients. Methods Cultured cancer cells (expressing TROP2, HER2, or neither) were added to Healthy Donor (HD) blood, creating the model system used in assay development studies. Blood from 11 confirmed mTNBC patients was collected to analyze this clinically relevant population. Immunofluorescence staining and image analysis were performed on replicate blood-based biopsy slides to assess expression for TROP2 and HER2. CTCs were identified and characterized using Epic Sciences digital imaging and machine learning algorithms. Results Three cancer cell lines of various TROP2 expression levels (HEK293, low; MDA-MB-231, intermediate; and A431, high) exhibited immunofluorescence signal ranges of 168 MFI, 2147 MFI, and 26982 MFI, respectively. A fluorescence cutoff of 218 MFI was established following assay optimization to distinguish TROP2 positive CTCs based on a 95% confidence level. Within the cohort of 11 mTNBC patients, 100% of patients with mTNBC had detectable CTCs. 64% of mTNBC patients had TROP2 positivity (MFI, mean: 1328, range: 37-38281)). On the other hand, 0% of mTNBC patients had HER2 positivity (MFI, mean: 128, range: 40-361)). To date, studies with biomarker expression for these two drugs have been limited to tissue biopsy, which may not always yield contemporaneous sampling in the metastatic setting. These results offer a potential liquid biopsy test identifying pts more responsive to trop2 and her2 directed therapies. Discussion Here we report on a liquid biopsy profile combining protein expression of TROP2 and HER2. Blood-based assessment of TROP2 and HER2 expression is a potential marker for selecting MBC patients likely to respond to anti-TROP2 targeted therapies such as Sacituzumab, or Trastuzumab Deruxtecan has shown to improve survival in mTNBC in the ASCENT trial and DESTINY-04 studies. Recent data on the Destiny-04 trial, which allocated HER2 expressing MBC patients into T-DXd treatment, transformed the definition of TNBC. The development of quantitative, reproducible, and more sensitive immunofluorescence assays is becoming crucial for assigning patients whose disease continually evolves to targeted therapies by increasing clinical trial options. Analysis of the clinical utility of the blood-based cell analysis in guiding patient selection strategies for novel anti-TROP2, HER2, and other targeted therapies treatment in MBC is ongoing. Citation Format: David Bourdon, Alessandra Cunsolo, Brandon Guillory, Megan M. Slade, Giuseppe Di Caro, Alisa Tubbs, Toshiaki Iwase, Naoto T. Ueno. TROP2 and HER2 expression by liquid biopsy in women with mTNBC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-06-06.

A multi-targeting bionanomatrix coating to reduce capsular contracture development on silicone implants

BackgroundCapsular contracture is a critical complication of silicone implantation caused by fibrotic tissue formation from excessive foreign body responses. Various approaches have been applied, but targeting the mechanisms of capsule formation has not been completely solved. Myofibroblast differentiation through the transforming growth factor beta (TGF-β)/p-SMADs signaling is one of the key factors for capsular contracture development. In addition, biofilm formation on implants may result chronic inflammation promoting capsular fibrosis formation with subsequent contraction. To date, there have been no approaches targeting multi-facted mechanisms of capsular contracture development.MethodsIn this study, we developed a multi-targeting nitric oxide (NO) releasing bionanomatrix coating to reduce capsular contracture formation by targeting myofibroblast differentiation, inflammatory responses, and infections. First, we characterized the bionanomatrix coating on silicon implants by conducting rheology test, scanning electron microcsopy analysis, nanoindentation analysis, and NO release kinetics evaluation. In addition, differentiated monocyte adhesion and S. epidermidis biofilm formation on bionanomatrix coated silicone implants were evaluated in vitro. Bionanomatrix coated silicone and uncoated silicone groups were subcutaneously implanted into a mouse model for evaluation of capsular contracture development for a month. Fibrosis formation, capsule thickness, TGF-β/SMAD 2/3 signaling cascade, NO production, and inflammatory cytokine production were evaluated using histology, immunofluorescent imaging analysis, and gene and protein expression assays.ResultsThe bionanomatrix coating maintained a uniform and smooth surface on the silicone even after mechanical stress conditions. In addition, the bionanomatrix coating showed sustained NO release for at least one month and reduction of differentiated monocyte adhesion and S. epidermidis biofilm formation on the silicone implants in vitro. In in vivo implantation studies, the bionanomatrix coated groups demonstrated significant reduction of capsule thickness surrounding the implants. This result was due to a decrease of myofibroblast differentiation and fibrous extracellular matrix production through inhibition of the TGF-β/p-SMADs signaling. Also, the bionanomatrix coated groups reduced gene expression of M1 macrophage markers and promoted M2 macrophage markers which indicated the bionanomatrix could reduce inflammation but promote healing process.ConclusionsIn conclusion, the bionanomatrix coating significantly reduced capsular contracture formation and promoted healing process on silicone implants by reducing myfibroblast differentiation, fibrotic tissue formation, and inflammation.Graphical A multi-targeting nitric oxide releasing bionanomatrix coating for silicone implant can reduce capsular contracture and improve healing process. The bionanomatrix coating reduces capsule thickness, α-smooth muscle actin and collagen synthesis, and myofibroblast differentiation through inhibition of TGF-β/SMADs signaling cascades in the subcutaneous mouse models for a month.

Development of a novel chimeric oncolytic viral platform, CF33 and its derivatives, for peritoneal-directed CF33-OV treatment of gastric cancer peritoneal carcinomatosis.

428 Background: Peritoneal carcinomatosis (PC) is a deadly end-stage manifestation of gastric cancer (GC). CF33, a chimeric oncolytic virus, and its genetically modified derivatives have shown exclusive cancer selectivity and oncolytic potency against various solid tumors. Intravenous and intratumoral CF33-hNIS and CF33-hNIS-antiPDL1 have entered phase I trials to treat triple-negative breast cancer and unresectable solid tumors. Here, we investigated the antitumor activities of CF33-OVs against GCPC. Methods: We infected six GC cell lines, including both intestinal-type (AGS and MKN-74) and diffuse-type (KATO III, MKN-45, SNU-1, and SNU-16) with CF33, CF33-GFP, or CF33-hNIS-antiPDL1 at various multiplicities (MOI 0.01, 0.1, 1.0 and 10.0) to examine viral proliferation and cytotoxicity by using standard plaque assay and cell proliferation assay. We performed immunofluorescence imaging and flow cytometric analysis to verify virus-encoded gene expression and blockade of PD-L1 binding by virus-encoded anti-PD-L1 scFv. A mouse model of peritoneal dissemination xenograft of SNU-16-ffluc cells was set up to evaluate the antitumor activity of intraperitoneal (IP) CF33-hNIS-antiPDL1 treatment (3x105 pfu x 3 doses). Results: CF33-OVs showed dose-dependent infection, replication, and killing of human GC cell lines. Immunofluorescence imaging showed virus-encoded GFP, hNIS, and anti-PD-L1 scFv expression in CF33-OV-infected GC cells. GC cell surface PD-L1 binding was blocked by virus-encoded anti-PD-L1 scFv. In the xenograft model, IP CF33-hNIS-antiPDL1 treatment significantly reduced peritoneal tumors (p&lt;0.0001), decreased amount of ascites (62.5% PBS vs. 25% CF33-hNIS-antiPLD1), and prolonged animal survival (p&lt;0.01). Conclusions: CF33-OVs demonstrate effective antitumor activity and can deliver functional proteins in vitro and in vivo. CF33-hNIS-antiPDL1 treatment induced sustained regression of GCPC and prolonged survival. These promising preclinical results support peritoneal-directed strategies with CF33-hNIS-antiPDL1 in GCPC patients.

Moxibustion improves experimental colitis in rats with Crohn’s disease by regulating bile acid enterohepatic circulation and intestinal farnesoid X receptor

ObjectiveThis study was conducted to explore the mechanism of intestinal inflammation and barrier repair in Crohn’s disease (CD) regulated by moxibustion through bile acid (BA) enterohepatic circulation and intestinal farnesoid X receptor (FXR). MethodsSprague-Dawley rats were randomly divided into control group, CD model group, mild moxibustion group and herb-partitioned moxibustion group. CD model rats induced by 2,4,6-trinitrobenzene sulfonic acid were treated with mild moxibustion or herb-partitioned moxibustion at Tianshu (ST25) and Qihai (CV6). The changes in CD symptoms were rated according to the disease activity index score, the serum and colon tissues of rats were collected, and the pathological changes in colon tissues were observed via histopathology. Western blot, immunohistochemistry (IHC) and immunofluorescence were used to evaluate the improvement of moxibustion on intestinal inflammation and mucosal barrier in CD by the BA-FXR pathway. ResultsMild moxibustion and herb-partitioned moxibustion improved the symptoms of CD, inhibited inflammation and repaired mucosal damage to the colon in CD rats. Meanwhile, moxibustion could improve the abnormal expression of BA in the colon, liver and serum, downregulate the expression of interferon-γ and upregulate the expression of FXR mRNA, and inhibit Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) mRNA. The IHC results showed that moxibustion could upregulate the expression of FXR and mucin2 and inhibit TLR4 expression. Western blot showed that moxibustion inhibited the protein expression of TLR4 and MyD88 and upregulated the expression of FXR. Immunofluorescence image analysis showed that moxibustion increased the colocalization sites and intensity of FXR with TLR4 or nuclear factor-κB p65. In particular, herb-partitioned moxibustion has more advantages in improving BA and upregulating FXR and TLR4 in the colon. ConclusionMild moxibustion and herb-partitioned moxibustion can improve CD by regulating the enterohepatic circulation stability of BA, activating colonic FXR, regulating the TLR4/MyD88 pathway, inhibiting intestinal inflammation and repairing the intestinal mucosal barrier. Herb-partitioned moxibustion seems to have more advantages in regulating BA enterohepatic circulation and FXR activation.Please cite this article as: Shen JC, Qi Q, Han D, Lu Y, Huang R, Zhu Y, Zhang LS, Qin XD, Zhang F, Wu HG, Liu HR. Moxibustion improves experimental colitis in rats with Crohn’s disease by regulating bile acid enterohepatic circulation and intestinal farnesoid X receptor. J Integr Med. 2023; 21(2): 194–204.