Multiplex Immunohistochemistry Research Articles

PCDHGA10 as a potential prognostic biomarker and correlated with immune infiltration in gastric cancer

BackgroundGastric cancer (GC) is one of the most common malignant tumors and is associated with poor prognosis. To improve the prognosis of GC patients, an effective immune-related prognostic biomarker is urgent. Here, we aim to explore the correlation between the expression of procalcitonin gamma subfamily A, 10 (PCDHGA10) and clinicopathological characteristics, especially its relation with tumor-infiltrating immune cells (TILs) in GC.MethodsThe differential mRNA expression of PCDHGA10 between GC tissues and normal gastric mucosa and prognostic potential were assessed from The Cancer Genome Atlas (TCGA). Then, based on tissue microarrays (TMAs) with multiplex immunohistochemistry (mIHC) from GC patients, we statistically assess the correlation between PCDHGA10 protein expression and the clinical profiles and prognosis of the patients. Additionally, with IHC and mIHC, we applied the machine-learning algorithms to evaluate the localization and expression levels of TILs and immune checkpoints in the tumor microenvironment. We analyzed the relationship between PCDHGA10 protein expression and TILs and immune checkpoints.ResultsThrough the database and TMA analysis, the expression of PCDHGA10 was significantly higher in GC tissues compared with normal tissues. High PCDHGA10 expression independently predicted poor prognosis in GC. Additionally, elevated PCDHGA10 expression was positively associated with the number of CD8+ T cells, CD68+ macrophages, Foxp3+ T cells, and CD4+ T cells in GC tissues and the stromal region. Besides, the expression of PCDHGA10 was positively correlated with immune checkpoints, including CTLA-4, LAG3, and PD-L1.ConclusionsPCDHGA10 might be a potential prognostic marker and an immunological therapeutic target for GC.

Dynamics in the Prostate Immune Microenvironment Induced by Androgen Deprivation Therapy.

The influence of testosterone on the prostate's immune microenvironment remains unclear. This study aims to elucidate the dynamics of immune cells in the prostate following androgen deprivation therapy (ADT). We retrospectively compared prostate needle biopsy and radical prostatectomy specimens from 33 patients who underwent both procedures, along with neoadjuvant ADT at a single institution. Immune cell infiltration in the cancer and stroma areas was assessed using multiplex fluorescence immunohistochemistry. Post-ADT, all immune cells, including CD4+ T cells, CD8+ T cells, Foxp3+ regulatory T cells, CD204+ macrophages, and CD20+ B cells, significantly increased in the prostatectomy specimen. However, few immune cells were detected in the biopsy of the same patients (p < 0.001). The number of CD20+ B cells in the cancer area was significantly lower post-ADT in high-risk cases according to the NCCN classification (p = 0.020). This difference was significantly associated with the Gleason Grade Group, rather than PSA levels or T classification (p < 0.001). However, no significant difference was observed in the recurrence rate between Grade Groups 1, 2, 3 and 4, 5 (p = 0.991). There was no significant difference in immune cells other than CD20+ B cells when divided into NCCN classifications. The marked increase in immune cells following ADT suggests an intensified immune response against prostate cancer.

An Optimized Protocol for Multiple Immunohistochemical Staining of Fragile Tissue Samples.

Owing to the high occurrence of tissue detachment during the sample preparation process, the application of multiplex immunohistochemistry (mIHC) technology is limited in the field of fragile tissue samples, such as tendons, ligaments, and bones. To optimize a method for preparing sections for mIHC on fragile tissue samples, taking the human anterior cruciate ligament as an example, paraffin-embedded continuous sections with a thickness of 4 μm were divided into two groups: baking groups underwent routine section processing, and after being mounted on glass slides, they were baked at 65°C for 4 h, 8 h, or 24 h; ultraviolet (UV) photosensitive cross-linking groups used adhesive-coated slides for mounting and were directly subjected to UV light-induced cross-linking, with the cross-linking time set at 0 s, 20 s, 40 s, 1 min, 2 min, 3 min, 4 min, and 5 min, respectively. After deparaffinization and rehydration, we simulated the microwave step, which was most likely to cause tissue detachment during the mIHC experimental procedure, and then, the sections were stained with eosin. Finally, using the optimal cross-linking time selected from the UV cross-linking groups, mIHC staining of tendon and bone tissues was performed. After deparaffinization and rehydration, both groups were able to maintain the integrity of the tissue structure, except for the slides from the UV-sensitive cross-linking 0 s group, which showed complete tissue detachment. Following the seventh microwave treatment, the baking groups presented significant tissue detachment. The UV cross-linking groups were affected by the cross-linking time, and severe tissue detachment occurred with cross-linking times of 20 s, 40 s, and 5 min, whereas the tissues cross-linked for 1 min, 2 min, 3 min, and 4 min all maintained complete tissue morphology and structure. Finally, after 2 min of cross-linking, the results of spectral imaging revealed that the tissue morphology and structure were intact. During the process of mIHC staining, photocrosslinking with UV irradiation for 1-4 min effectively preserves the integrity of the tissue morphological structure.

Interfacial Engineering of Biocompatible Nanocapsules for Near-Infrared-Triggered Drug Release and Photothermal Therapy.

Chemotherapy is an effective option for cancer treatment. However, its clinical application is often limited by the severe side effects of chemical drugs. To overcome these limitations, a novel drug-loaded phase-change nanocapsule system is developed. These nanocapsules are assembled via one-step electrostatic self-assemblythrough guided interfacial engineering. The phase change material core nanocapsules demonstrate great photothermal-controlled drug release performance and exhibit excellent tumor-targeting drug delivery performance both in vitro and in vivo via the binding of hyaluronic acid shell on the nanocapsule surface with corresponding receptors on the tumor cell membrane. The phototherapy function of the nanocapsules enhances immune activation within the tumor microenvironment, as demonstrated by flow cytometry and multiplex immunohistochemistry. The developed nanocapsules are biocompatible, versatile, and scalable and offer a promising smart delivery platform for controllable near-infrared triggered drug release and photothermal therapy.

Systemic chemokine-modulatory regimen combined with neoadjuvant chemotherapy in patients with triple-negative breast cancer

BackgroundHigher cytotoxic T lymphocyte (CTL) numbers in the tumor microenvironment (TME) predict pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) and positive long-term outcomes in triple-negative breast cancer (TNBC). pCR...

BIOM-56. HEAT SHOCK PROTEINS PREDICT CLINICAL RESPONSE AND POTENTIATE LOCAL IMMUNOGENICITY AFTER LITT FOR GLIOBLASTOMA

Abstract Glioblastoma (GBM) remains one of the deadliest cancers despite advances in therapies. Laser interstitial thermal therapy (LITT) is a minimally invasive technique used to ablate intra-axial brain tumors. Non-ablative LITT-induced hyperthermia (33-43˚C) increases intra-tumoral mutational burden and neoantigen production, promoting neoantigen presentation via heat shock proteins (HSPs). HSPs are protein-folding chaperones that present novel intracellular antigens for immune recognition. We investigated HSPs as predictors of local immune response and clinical outcomes of GBM patients undergoing LITT. We conducted immune deconvolution of bulk RNA-sequencing data from The Cancer Genome Atlas to determine the correlation between 97 unique HSPs and tumor infiltration of CD8+, CD4+, B, macrophage, neutrophil, and dendritic cells. We evaluated the association between HSP expression and overall survival (OS). Pre-LITT biopsies from 9 patients (responders and non-responders) were analyzed via immunohistochemistry (IHC) to identify spatial HSP expression. Next-generation sequencing was utilized to validate findings and identify other predictive biomarkers. We identified two HSPs for further investigation: SACS and HSPA5. SACS was associated with improved survival based on TCGA data (14.9 vs. 11 months, HR:1.3, p&amp;lt;0.0001). HSPA5 was associated with worse outcomes (12.1 vs. 15.0 months, HR:0.73, p&amp;lt;0.0001). Immune deconvolution showed HSPA5 positively correlated with dendritic cell infiltration (r=0.50, p=6.1E-27) and SACS with CD8+ infiltration (r=0.31, p=6.8E-11). IHC of 9 patients demonstrated high SACS expression was associated with improved OS (p&amp;lt;0.05) after LITT. Multiplex IHC illustrated that immune cell infiltration was associated with therapeutic response post-LITT. Our ongoing immunohistochemical and genomic analyses aim to clarify the role of HSPs in enhancing immunogenicity in GBM. Further investigation into SACS and immune cell infiltration may reveal how hyperthermia via LITT improves response through neoantigen presentation.

CNSC-55. UNCOVERING THE ROLE OF COLLAGEN EXTRACELLULAR MATRIX IN GLIOBLASTOMA BRAIN MICROENVIRONMENT TRANSFORMATION

Abstract Glioblastoma (GBM) is a highly malignant brain tumor characterized by diffuse infiltration and significant heterogeneity, without effective treatment. Mesenchymal-like (MES) glioma cells drive tumor aggression by promoting brain invasion and tumor microenvironment remodeling. Previous spatial transcriptomic analyses identified Collagen, particularly COL1A, as a key extracellular matrix (ECM) component, enriched in MES areas termed oncostreams. Depletion of COL1A1 disrupted oncostreams, influencing the tumor microenvironment to support cell survival, and migration. Reactive astrocytes and macrophages, essential components of the brain microenvironment, impact glioma progression. However, the interplay between glioma-secreted collagen and the brain microenvironment remains poorly understood. To elucidate this, we generated orthotopic mouse glioma models with high-collagen (NPD) and low-collagen (NPDshCol1A1). Our data revealed that downregulation of COL1A1 in glioma cells alters the abundance and spatial distribution of reactive astrocytes and macrophages/microglia. Immunofluorescence and flow cytometry analysis showed that tumors with low-collagen had decreased GFAP+ reactive astrocyte cells at the invasive edge and perivascular niche compared to high-collagen gliomas. Moreover, our results shows that high collagen levels may activate astrocytes through distant communication, as evidenced by the higher presence of reactive astrocytes in the contralateral cortex of NPD tumors. However, low-collagen tumors exhibited an increase in astrocytes (GFAP+) in the tumor core. Furthermore, high-collagen gliomas demonstrated increased macrophage infiltration, promoting an immunosuppressive environment. Using multiplex immunohistochemistry assays, we observed that reactive astrocytes along the invasive tumor edge were associated with glioma cells expressing elevated levels of COL1A1 and TGF-β ligand. In summary, our results show that glioma cell-expressed COL1A1 promotes the activation of peritumoral and perivascular brain-resident astrocytes and macrophages. The interactions of reactive astrocyte with glioma cells enriched in collagen form a feedback loop that enhances tumor invasion and vascular proliferation, suggesting that targeting the ECM-astrocyte interactions could disrupt these processes and offer a therapeutic avenue for GBM.

Immune infiltration correlates with transcriptomic subtypes in primary estrogen receptor positive invasive lobular breast cancer

Understanding interplay of breast cancer and microenvironment is critical. Here we identified two transcriptomic subtypes and five immune infiltration patterns from RNA-seq and multiplex immunohistochemistry from 21 ER + /HER2- ILCs. We found proliferative subtype associated with increased suppressive immune infiltration, and defined a signature associated with lower proliferative, pro-inflammatory TAM infiltration, and improved survival in ER+ breast cancer. Our work identified genes related to ILC immune microenvironment and prognosis.

Integrated single-cell transcriptomic analyses identify a novel lineage plasticity-related cancer cell type involved in prostate cancer progression

Cancer cell plasticity is the ability of neoplastic cells to alter their identity and acquire new biological properties under microenvironmental pressures. In prostate cancer (PCa), lineage plasticity often results in therapy resistance and trans-differentiation to neuroendocrine (NE) lineage. However, identifying the cancer cells harboring lineage plasticity-related status remains challenging. Based on 13 multi-center human PCa bulk transcriptomic cohorts (samples=3314) and 9 bulk transcriptomic datasets derived from PCa experimental models, we established an integrated lineage plasticity-related gene signature, termed LPSig. Leveraging this gene signature, AUCell enrichment analysis was applied to identify the cell population with high lineage plasticity from a comprehensive single-cell RNA-sequencing (scRNA-seq) meta-atlas assembled by us, which consisted of 10 public human PCa scRNA-seq datasets (samples=93, cells=222,529). Moreover, additional scRNA-seq dataset of human PCa, multiplex immunohistochemistry staining for human PCa tissues, invitro and invivo functional experiments, as well as qPCR and Western blot analyses were employed to validate our findings. We found that LPSig could finely capture the dynamics of tumor lineage plasticity throughout the progression of PCa, accurately estimating the status of lineage plasticity. Based on LPSig, we identified a previously undefined minority population of lineage plasticity-related PCa cells (LPCs) from the human PCa scRNA-seq meta-atlas assembled by this study. Furthermore, in-depth dissection revealed pivotal roles of LPCs in trans-differentiation, tumor recurrence, and poor patient survival during PCa progression. Furthermore, we identified HMMR as a representative cell surface marker for LPCs, which was validated using additional scRNA-seq datasets and multiplexed immunohistochemistry. Moreover, HMMR was transcriptionally inhibited by androgen receptor (AR), and was required for the aggressive adenocarcinoma features and NE phenotype. Our study uncovers a novel population of lineage plasticity-related cells with low AR activity, stemness-like traits, and elevated HMMR expression, that may facilitate poor prognosis in PCa. This work was supported by National Key R&D Program of China (2022YFA0807000), National Natural Science Foundation of China (82160584), Advanced Prostate Cancer Diagnosis and Treatment Technology Innovation Team of Kunming Medical University (CXTD202216), and Reserve Talents of Young and Middle-aged Academic Leaders in Yunnan Province (202105AC160013).

Single-cell analyses reveal metastasis mechanism and microenvironment remodeling of lymph node in intrahepatic cholangiocarcinoma

Background and AimsLymph node metastasis (LNM) is a major determinant of recurrence and prognosis in intrahepatic cholangiocarcinoma (iCCA). LNM disrupts T-cell-mediated cytotoxicity, promotes tumor-specific immune tolerance, and facilitates distant metastasis. Despite its importance, extensive research on LMN in iCCA is lacking. This study aimed to systematically explore the heterogeneity of the LNM-associated microenvironment in iCCA by integrating single-cell and multiomics analyses, identifying metastasis-associated cell subgroups, and validating these findings through multiple cohorts. MethodsWe analyzed single-cell transcriptomics data from primary tumors, cancer-adjacent liver tissues, and tumor-draining lymph nodes of four iCCA patients who underwent radical surgery. Additionally, we collected 81 tumor and matched lymph node tissue sections from iCCA patients. We performed single-cell RNA sequencing and multiplex immunohistochemistry (mIHC), followed by differential gene expression analysis, functional enrichment analysis, single-cell copy number variation assessment, and pseudotime analysis. ResultsOur analysis revealed the complex heterogeneity of the iCCA LNM-associated microenvironment. We found a significant increase in stromal and mature immune cells in the metastatic lymph nodes. T cells constitute the predominant component, with diverse functional subtypes. We identified CD36+ macrophages and SAA1+ tumor cells as key players in the metastatic process. The SAA1-CD36 receptor‒ligand pair may be crucial in forming the LNM-associated microenvironment. ConclusionWe identified several metastasis-associated cell subgroups. These findings provide new insights into the mechanisms underlying LNM in iCCA and lay the groundwork for the development of novel therapeutic strategies. Our study highlights the importance of single-cell technologies in understanding tumor microenvironment complexity and offers valuable resources for future research. Impact and implicationsThe lack of single-cell transcriptome analysis of iCCA lymph node metastases has prevented us from understanding the underlying mechanisms of disease progression. To fill this knowledge gap, we elucidated the unique ecosystem of iCCA lymph node metastases, which is an important advance in clarifying the impact of the immune environment on the development of this disease. The results of this study identified several LNM-related therapeutic targets, which will not only be helpful to basic researchers but also provide potential diagnostic and treatment ideas for physicians, thereby helping patients and their caregivers develop more personalized treatment plans. This finding is highly important for improving the prognosis of patients with advanced iCCA in the future.

Temporal dynamics of immune cell patterns in bladder cancer patients receiving Bacillus Calmette-Guérin therapy.

Bacillus Calmette-Guérin (BCG) is capable of enhancing the infiltration of immune cells into the tumour. However the temporal dynamics of immune cell patterns in patients receiving BCG instillation remains unclear. Ninety-six patients who underwent intravesical BCG therapy, comprising 46 responders and 50 non-responders, were retrospectively enroled to explore the evolving immune landscape. This study involved a detailed examination of sequential samples collected before, during, and after BCG treatment to assess BCG's influence on the immune microenvironment, employing techniques such as immunohistochemistry, fluorescent multiplex immunohistochemistry, and mass spectrometry techniques. Our study found that initial BCG instillation leads to enhanced immune cell infiltration, correlating with treatment efficacy, with responders exhibiting more pronounced increases. Non-responders experience a rise in immune cell infiltration and PD-L1 expression during the first instillation, which returns to baseline after treatment. In non-responders, BCG re-challenge fail to further increase immune cell infiltration into the tumour or improve patient outcomes. Strikingly, proteomics data revealed that GBP1 expression was induced by BCG treatment in non-responders. Our findings demonstrated the induction of tumour PD-L1 expression by BCG in non-responders, and therefore provide insights for the combination of BCG and anti-PD1/anti-PD-L1 therapy.

Crucial immunological roles of the invasion front in innate and adaptive immunity in cervical cancer.

The immunostimulatory actions of innate and adaptive immune responses play a crucial role in the cancer-immunity cycle. Although cervical cancer (CC) exhibits a high recurrence rate, the relation with lymphocytes in the tumor tissue have not been analyzed. We analyzed NKT, NK, and T cells, not only in peripheral blood (PB), but also tumor tissue through histological analysis from 23 patients with CC collected before treatment. A correlation of them between PB and the tumor tissue were assessed. We detected functional NKT and NKG2Dhi NK cells and effector CD4+ Tregs in PB. In the tumor, we detected the infiltration of LAG-3+ TIM-3+ CD4+ and CD8+ T cells rather than NK cells particularly in the invasion front (IF) by fluorescent multiplex immunohistochemistry. The heatmap and correlation analysis revealed that LAG-3+ TIM-3+ CD8+ T cells are highly associated with CD69+ CD103- exhausted CD8+ T cells. We identified the statistical relationship between CD4+Tregs in PB and the number of LAG-3+ TIM-3+ CD4+ T cells in the IF, which may be related to recurrence in patients with CC. LAG-3+ TIM-3+ T cells located in the IF may play a key role in regulation of the tumor immune microenvironment.

Spatial immunogenomic patterns associated with lymph node metastasis in lung adenocarcinoma

BackgroundLung adenocarcinoma (LUAD) with lymph node (LN) metastasis is linked to poor prognosis, yet the underlying mechanisms remain largely undefined. This study aimed to elucidate the immunogenomic landscape associated with LN metastasis in LUAD.MethodsWe employed broad-panel next-generation sequencing (NGS) on a cohort of 257 surgically treated LUAD patients to delineate the molecular landscape of primary tumors and identify actionable driver-gene alterations. Additionally, we used multiplex immunohistochemistry (mIHC) on a propensity score-matched cohort, which enabled us to profile the immune microenvironment of primary tumors in detail while preserving cellular metaclusters, interactions, and neighborhood functional units. By integrating data from NGS and mIHC, we successfully identified spatial immunogenomic patterns and developed a predictive model for LN metastasis, which was subsequently validated independently.ResultsOur analysis revealed distinct immunogenomic alteration patterns associated with LN metastasis stages. Specifically, we observed increased mutation frequencies in genes such as PIK3CG and ATM in LN metastatic primary tumors. Moreover, LN positive primary tumors exhibited a higher presence of macrophage and regulatory T cell metaclusters, along with their enriched neighborhood units (p < 0.05), compared to LN negative tumors. Furthermore, we developed a novel predictive model for LN metastasis likelihood, designed to inform non-surgical treatment strategies, optimize personalized therapy plans, and potentially improve outcomes for patients who are ineligible for surgery.ConclusionsThis study offers a comprehensive analysis of the genetic and immune profiles in LUAD primary tumors with LN metastasis, identifying key immunogenomic patterns linked to metastatic progression. The predictive model derived from these insights marks a substantial advancement in personalized treatment, underscoring its potential to improve patient management.

Deciphering the Multifaceted Immune Landscape of Unresectable Primary Liver Cancer to Predict Immunotherapy Response.

Immunotherapies employing PD-1/PD-L1 immune checkpoint inhibitors (ICIs) are vital for primary liver cancer (PLC), but response rates remain unsatisfying. Accurate differentiation of responders from non-responders to immunotherapy is imperative. Here, single-cell-scaled mass cytometry analysis on sequential peripheral blood mononuclear cells (PBMCs) from ICI-treated PLC patients is conducted, and tissue residence of immune subpopulations is assessed via multiplex immunohistochemistry. In the discovery cohort (n = 24), responders have lower baseline B cell and HLA-DR+CD8+T cell, and higher CD14+CD16- classical monocyte (CM) proportions. CMs decrease more in responders PBMCs, while HLA-DR+CD8+T cells conformably amplify after ICI-exposure. Responsive individuals display upregulated exhaustion and activation markers in peripheral immune lineages. In the expanded cohort of 77 patients, the augment of the B cells in non-responders is re-confirmed. Responders demonstrate much higher enrichment of B cells or tertiary lymphoid structures in tumor compared to non-responders. A prospective model that excelled in early discrimination of responders is developed using generalized linear model and achieves a satisfactory AUC over 0.9 in all three independent cohorts. Integratedly, the study unveils dynamic immune landscapes in PLC patients undergoing ICI-based therapy, aiding in PLC patient stratification for ICI-based treatment and fostering new response monitoring strategies.

Measurement of Four Uterine NK Cell Subtypes Using Multiplexed Fluorescent Immunohistochemical Staining in Women with Repeated Implantation Failure.

Immunohistochemistry (IHC) plays a crucial role in biological research and clinical diagnosis, serving as the most commonly used method for identifying and visualizing tissue antigens. However, traditional IHC staining methods have limitations in distinguishing various subtypes of immune cells. This challenge has driven scientists to explore new technologies and methodologies for precise identification and differentiation of immune cell subtypes. In recent years, multiplex IHC has emerged as a solution, enabling the simultaneous detection of multiple antigens and their visualization within the same tissue sample. Uterine natural killer (uNK) cells play a pivotal role in early pregnancy processes, including decidualization, remodeling of uterine spiral arteries, and embryo implantation. Different subtypes of uNK cells exhibit different functions, allowing them to coordinate various biological events for successful embryo development and pregnancy. Therefore, in-depth research on uNK cell subtypes is essential for elucidating immune regulation mechanisms during pregnancy. Such studies provide valuable insights and novel approaches for addressing related conditions such as infertility and recurrent reproductive failure. This paper introduces a detailed multiplex IHC staining protocol for studying the density of four subtypes of uNK cells in endometrial specimens during the window of implantation (WOI). The protocol includes sample preparation, optimization of subtype markers, microscopic imaging, and data analyses. This multiplex IHC staining protocol offers high specificity and sensitivity, enabling simultaneous detection of different uNK cell subtypes, thus providing researchers with a powerful tool to explore the intricacies and mechanisms of immune regulation during pregnancy.

Topical Losartan Dosage Response and Corneal Toxicity at Higher Concentrations.

The purpose of this study was to evaluate the efficacy and safety of higher dosages of topical losartan in an alkali-burn fibrosis model in rabbits. In total, 18 rabbits had standardized alkali burns that trigger stromal fibrosis. Six eyes per group were treated with topical losartan (0.8 mg/mL, 8 mg/mL, or 40 mg/mL) 6 times per day. Slit-lamp photographs were obtained, and multiplex immunohistochemistry was performed for myofibroblast marker alpha-smooth muscle actin (α-SMA), mesenchymal cell marker vimentin, and basement membrane marker laminin alpha-5. Topical losartan at 40 mg/mL 6 times per day produced severe discomfort and ocular surface toxicity in all rabbits, and treatment was discontinued at nine days in this group. Topical losartan at 8 mg/mL 6 times per day caused less rabbit discomfort on application, but there were persistent epithelial defects and marked stromal opacity in 5 of 6 eyes after 1 month of treatment. Topical losartan 0.8 mg/mL was well tolerated by rabbits, and corneal opacity was markedly reduced at 1 month in 5 of 6 corneas compared with corneas in the 8 mg/mL and 40 mg/mL losartan groups. A persistent epithelial defect with opacity was noted in 1 cornea in the 0.8 mg/mL losartan group. Both total SMA-positive stromal cells per section (14.5 ± 2.8 vs. 3.5 ± 0.7, P = 0.04) and total stromal vimentin intensity units (310 ± 64 vs. 132 ± 35, P = 0.02) were significantly greater after 1 month of treatment in corneas treated with 8 mg/mL than corneas treated with 0.8 mg/mL of topical losartan. Topical losartan dosages over 0.8 mg/mL should be used cautiously in patient eyes. In eyes with a current epithelial defect, it is recommended that 0.2 mg/mL losartan 6 times per day be used until the epithelium closes.

Systemic longitudinal immune profiling identifies proliferating Treg cells as predictors of immunotherapy benefit: biomarker analysis from the phase 3 CONTINUUM and DIPPER trials

The identification of predictors for immunotherapy is often hampered by the absence of control groups in many studies, making it difficult to distinguish between prognostic and predictive biomarkers. This study presents biomarker analyses from the phase 3 CONTINUUM trial (NCT03700476), the first to show that adding anti-PD-1 (aPD1) to chemoradiotherapy (CRT) improves event-free survival (EFS) in patients with locoregionally advanced nasopharyngeal carcinoma. A dynamic single-cell atlas was profiled using mass cytometry on peripheral blood mononuclear cell samples from 12 pairs of matched relapsing and non-relapsing patients in the aPD1-CRT arm. Using a supervised representation learning algorithm, we identified a Ki67+ proliferating regulatory T cells (Tregs) population expressing high levels of activated and immunosuppressive molecules including FOXP3, CD38, HLA-DR, CD39, and PD-1, whose abundance correlated with treatment outcome. The frequency of these Ki67+ Tregs was significantly higher at baseline and increased during treatment in patients who relapsed compared to non-relapsers. Further validation through flow cytometry (n = 120) confirmed the predictive value of this Treg subset. Multiplex immunohistochemistry (n = 249) demonstrated that Ki67+ Tregs in tumors could predict immunotherapy benefit, with aPD1 improving EFS only in patients with low baseline levels of Ki67+ Tregs. These findings were further validated in the multicenter phase 3 DIPPER trial (n = 262, NCT03427827) and the phase 3 OAK trial of anti-PD-L1 immunotherapy in NSCLC, underscoring the predictive value of Ki67+ Treg frequency in identifying the beneficiaries of immunotherapy and potentially guiding personalized treatment strategies.

Semaphorin 3D promotes pancreatic ductal adenocarcinoma progression and metastasis through macrophage reprogramming.

Axon guidance molecules are frequently altered in pancreatic ductal adenocarcinoma (PDA) and influence PDA progression. However, the molecular mechanism remained unclear. Using genetically engineered mouse models to examine semaphorin 3D (SEMA3D), we identified a dual role for tumor- and nerve-derived SEMA3D in the malignant transformation of pancreatic epithelial cells and invasive PDA development. Pancreatic-specific knockout of the SEMA3D gene from the KRASG12D and TP53R172H mutation knock-in, PDX1-Cre(KPC) mouse model demonstrated delayed tumor initiation, prolonged survival, absence of metastasis, and reduced M2 macrophage expression. Mechanistically, tumor- and nerve-derived SEMA3D indirectly reprograms macrophages through KRASMUT-dependent ARF6 signaling in PDA cells, resulting in increased lactate production, which is sensed by GPCR132 on macrophages to stimulate protumorigenic M2 polarization. Multiplex immunohistochemistry demonstrated increased M2-polarized macrophages proximal to nerves in SEMA3D-expressing human PDA tissue. This study suggests that altered SEMA3D expression leads to an acquisition of cancer-promoting functions, and nerve-derived SEMA3D is "hijacked" by PDA cells to support growth and metastasis in a KRASMUT-dependent manner.

Spatial Dynamics of T and B Cell Response Predicts Clinical Outcome of Resectable and Unresectable Hepatocellular Carcinoma.

Immunotherapies have led to a paradigm shift in the treatment of hepatocellular carcinoma (HCC). Studies have revealed the single-cell catalogues of tumor-infiltrating immune cells and the trajectories of their differentiation. Nevertheless, the spatial distribution of these immune cells with distinct phenotypes in tumor microenvironment and their clinicopathological significance in resectable and unresectable HCCs are still largely unclear. We analyzed the spatial dynamics of intratumoral CD4 and CD8 T cells and their association with B and plasma cells using 283 surgically resected HCCs, 58 unresectable HCC samples before combined immunotherapy (atezolizumab plus bevacizumab [Atezo+Bev]), and autopsy specimens from 50 cases of advanced-stage HCCs through multiplex immunohistochemistry combined with transcriptomic and driver gene mutation analysis. Classification based on the spatial dynamics of T and B cell responses (refined immunosubtype) was developed and its clinicopathological significance was analyzed. We found that stem-like CD4 and CD8 T cells were mainly observed in T cell aggregates. Differentiation of T follicular helper cells were associated with the development of tertiary lymphoid structures, whereas differentiation of CD4 TCXCL13 cells with phenotype resembling T peripheral helper cells were associated with the development of lymphoplasmacytic microenvironment. The refined immunosubtype could predict clinical outcomes of resectable HCC after surgery and unresectable HCC after Atezo+Bev therapy. The immune microenvironment of metastatic lesions tended to reflect those of primary lesions. We revealed the spatial dynamics of T and B cell response in HCC, which is closely associated with the clinical outcome after surgical resection or Atezo+Bev therapy.

The spatial impact of a Western diet in enriching Galectin-1-regulated Rho, ECM, and SASP signaling in a novel MASH-HCC mouse model

BackgroundHepatocellular carcinoma (HCC) arising from metabolic dysfunction-associated steatohepatitis (MASH) presents a significant clinical challenge, particularly given the prevalence of the Western diet (WD). The influence of diet on the tumor microenvironment remains poorly understood. Galectin-1 (Gal-1) is a biomarker for HCC and has a crucial role in liver carcinogenesis. Our previous studies demonstrated that silencing Gal-1 effectively treats mouse HCC. However, the impacts of a WD on Gal-1 signaling on MASH to HCC progression are unknown, and this study addresses these knowledge gaps.MethodsWe developed a novel MASH-HCC mouse model. Using spatial transcriptomics and multiplex immunohistochemistry (IHC), we studied the effects of a WD on the liver and tumor microenvironment. By modulating Gal-1 expression through silencing and overexpression, we explored the location-specific impacts of WD on Gal-1 signaling.ResultsPathways such as Rho signaling, extracellular matrix (ECM) remodeling, and senescence-associated secretory phenotypes (SASP) were prominently activated in WD-induced metabolic dysfunction-associated fatty liver disease (MAFLD) and MASH-HCC, compared to healthy livers controls. Furthermore, Rho GTPase effectors, ECM remodeling, neutrophil degranulation, cellular stress, and cell cycle pathways were consistently enriched in human and mouse MASH-HCC. Spatially, these pathways were enriched in the tumor and tumor margins of mouse MASH-HCC. Additionally, there was a notable increase in CD11c and PD-L1-positive cells from non-tumor tissues to the tumor margin and inside the tumor of MASH-HCC, suggesting compromised immune surveillance due to WD intake. Moreover, MASH-HCC exhibited significant Gal-1 induction in N-Cadherin-positive cells, indicating enhanced epithelial-to-mesenchymal transition (EMT). Modulating Gal-1 expression in MASH-HCC further established its specific roles in regulating Rho signaling and SASP in the tumor margin and non-tumor tissues in MASH-HCC.ConclusionWD intake significantly influences vital cellular processes involved in Gal-1-mediated signaling, including Rho signaling and ECM remodeling, in the tumor microenvironment, thereby contributing to the development of MASH-HCC.