Tumor-associated Immune Cells Research Articles

Abstract 6539: RNAi mediated silencing of STAT3 in tumor-associated immune cells induces robust anti-tumor effects in immunotherapy resistant tumors

Abstract Malignant tumors are often associated with an immunosuppressive tumor microenvironment (TME), rendering most of them resistant to standard-of-care immune checkpoint inhibitors (CPIs). Signal transducer and activator of transcription 3 (STAT3), a ubiquitously expressed transcription factor, has well-defined immunosuppressive functions in several leukocyte populations within the TME. Since the STAT3 protein has been challenging to target using conventional pharmaceutical modalities, we investigated the feasibility of applying systemically delivered RNA interference (RNAi) agents to silence its mRNA directly in tumor-associated immune cells. In preclinical murine tumor models, chemically stabilized acetylated small-interfering RNAs (siRNAs) selectively silenced Stat3 mRNA in multiple relevant cell types in TME and tumor-draining lymph nodes (TDLN), reduced STAT3 protein levels, and increased cytotoxic T-cell infiltration. In murine models of CPI-resistant tumors, RNAi-mediated Stat3 silencing resulted in tumor growth inhibition, which was further enhanced and resulted in tumor regressions in combination with CPIs. Our findings suggest that targeting STAT3 directly and reducing STAT3 protein in the suppressive immune cell populations in the TME and TDLN may overcome some of the current limitations of immunotherapy and be of therapeutic benefit especially for cancer patients who failed to respond to standard immunotherapy treatments alone. A human active STAT3 conjugate is currently in Phase 1 clinical trial for treatment of immunotherapy refractory cancer types (NCT06098651). Citation Format: Shanthi Ganesh, Minju Kim, Jenny Lee, Xudong Feng, Harini Sivagurunatha Krishnan, Rene Rijnbrand, Amber Beaudry, Jennifer A. Lockridge, Vinita Uttamsingh, John Hanrahan. RNAi mediated silencing of STAT3 in tumor-associated immune cells induces robust anti-tumor effects in immunotherapy resistant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6539.

Abstract 2902: CA-4948 (emavusertib) improves treatment response of preclinical metastatic brain melanoma to anti-PD-1 immune checkpoint blockade

Abstract Background: Although recent advances in immunotherapy for metastatic melanoma have transformed the treatment paradigm for a disease that once carried a grim prognosis, nearly 50% of patients fail to reach a complete response to immune checkpoint blockade (ICB) due to multiple mechanisms of tumor-induced immune resistance. These challenges are amplified in the CNS tumor microenvironment (TME) where metastatic brain melanoma (MBM) demonstrates diminished clinical benefit to ICB treatment. A deeper understanding of these mechanisms are required to improve therapeutic response to ICB for these patients. Our group has previously identified that aberrant activation of the myddosomal cascade through downstream interleukin (IL)-1 receptor-associated kinase (IRAK-4) signaling results in chronic stimulation of MAPK and NF-κB in MBM. Further investigation reveals myddosome signaling emanates in large part from the TME, resulting in reflexive immune suppression. CA-4948 (emavusertib), a small molecule inhibitor of IRAK-4, is capable of reaching therapeutic dose levels in the brain where it demonstrates on-target inhibition of this pathway. These data highlight a unique therapeutic opportunity where selective inhibition of immune-suppressive signaling via targeted IRAK-4 blockade may sensitize MBM to anti-PD-1 ICB. Methods Multispectral imaging of patient MBM samples were analyzed for myddosome expression in the TME. Melanoma preclinical models, including both primary site (cutaneous) and metastatic brain disease, were evaluated for tumor burden and survival in response to CA-4948 treatment alone and in combination with anti-PD-1 ICB. Immunological responses of tumor to treatment were evaluated in two transgenic reporter models, CCR2RFPCX3CR1GFP and IFNγIRES-EYFP, to determine both the distribution and activation of infiltrating immune cells. Tumor-associated myeloid cells were assessed for phenotypic markers by multiparameter flow cytometry. Results Myddosome signaling was found to be elevated in tumor-associated immune cells in patient MBM specimens. Treatment of preclinical melanoma with CA-4948 combined with anti-PD-1 ICB resulted in significantly reduced tumor growth and improved survival in three independent models. CA-4948 treatment was associated with reduced tumor recruitment of peripherally derived tumor-associated macrophages, as well as increased lymphocyte infiltration and improved pro-inflammatory signaling measured by increased IFNγ expression in the combinatorial setting. Conclusion Targeted inhibition of IRAK-4 with CA-4948 improves immune surveillance and activation in preclinical models when combined with anti-PD-1 ICB, resulting in reduced tumor growth and increased survival. Thus, CA-4948 may be an effective adjuvant treatment strategy alongside anti-PD-1 ICB for the treatment of metastatic melanoma. Citation Format: Christina A. von Roemeling, Bently P. Doonan, Amanda M. Acevedo, Jeet A. Patel, Savannah L. Carpenter, Alisha Bhatia, Reinhard W. von Roemeling, Jamison Hoffman, Maureen Lane, Duane A. Mitchell. CA-4948 (emavusertib) improves treatment response of preclinical metastatic brain melanoma to anti-PD-1 immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2902.

Abstract 2370: Revitalizing anti-tumor immunity through PLT012 monoclonal antibody, targeting CD36 for metabolic rewiring in the tumor microenvironment

Abstract The accumulation of lipid metabolic products in the TME has been extensively documented to exacerbate cancer malignancy by suppressing anti-tumor immunity. This phenomenon is characterized by the induction of immunosuppressive features in various immune cells and the development of exhausted phenotypes in T cells, creating formidable obstacles for effective immunotherapies. Notably, CD36, a fatty acid transporter, has been identified as up-regulated in both malignant cells and various tumor-associated immune cells, including regulatory T cells, tumor-associated macrophages, and CD8+ T cells. This upregulation serves to adjust the metabolic preferences of these cells, enabling adaptation to the lipid-enriched TME. CD36-mediated adaptation not only instigates alterations in metabolic regulations but also significantly influences immune cell properties, contributing to the establishment of an immunosuppressive TME. Consequently, it has been proposed that inhibiting CD36-mediated fatty acid uptake could strategically reshape the host's anti-tumor immunity within the TME, resulting in the reduction of tumor-infiltrating regulatory T cells and the restoration of both survival and functionality in CD8 T cells. These findings underscore the therapeutic potential of anti-CD36 antibodies as a targeted approach to mitigate the immunosuppressive milieu within the TME. Hence, PLT012, a humanized anti-CD36 antibody, was developed via phage display, followed by affinity maturation and developability optimization, and possesses cross-reactivity among species, including murine, non-human primates, and human. Through CryoEM-based structural analysis, we found that PLT012 recognizes the lipid binding domain of CD36 without interfering with the TSP-1 binding site. Moreover, administration of PLT012 significantly reduced oxLDL-mediated M2 macrophage polarization and fatty acid uptake in CD8+ TILs. Notably, HCC-bearing mice treated with PLT012 exhibited a significant reduction in tumor growth with an increased CD8/Treg ratio and CD8 effector functions, consistent with the ex vivo observations of PLT012 treatments in human HCC samples. In addition to murine studies, PLT012 also demonstrated excellent tolerability in NHP studies without any adverse effects. Ongoing studies in translational medicine will further bridge our findings and proposed mechanism of action with clinical observations, highlighting the potential therapeutic outcome in HCC patients particularly. Taken together, these results reveal that blocking CD36-mediated metabolic alterations in Treg and CD8+ TILs with PLT012 treatment can elicit robust tumor growth inhibition and shift the immunosuppressive nature within the TME toward an immunosupportive one. This study further provides the pillar for harnessing immunometabolic targeting in cancer immunotherapy. Citation Format: Yi-Ru Yu, Sheue-Fen Tzeng, Huey-Wen Hsiao, Jaeoh Park, Lana Kandalaft, Yun-Han Lin, Chin-Hsien Tsai, Ping-Chih Ho. Revitalizing anti-tumor immunity through PLT012 monoclonal antibody, targeting CD36 for metabolic rewiring in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2370.

Abstract 5287: Ex vivo pharmacologic inhibition of STAT3 effectively targets tumor and tumor associated suppressor cells in high-grade glioma 3D spheroids

Abstract Gliomas, a type of brain tumor originating from glial cells, comprise a heterogenous group of neoplasms. High-grade gliomas, in particular, present significant therapeutic challenges in the clinic. Unlike other solid tumors, the heterogenous glioma microenvironment is heavily enriched with tumor associated suppressor cells including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), which can account for 30-40% of the cellular composition1. These pro-tumorigenic myeloid cells are thought to contribute to the resistance of glioma to immunotherapy. Efforts to target the suppressive nature of these dominating cell types are in development to yield more therapeutic options for patients with these devastating diseases. Constitutive activation of STAT3 in gliomas has been associated with increased tumor progression and reduced immune response. We have observed in other cancer models that a phenotypic switch occurs from STAT3 pathway activation in 3D spheroid culture to STAT3 inactivation when cultured in 2D. Therefore, we sought to determine the role of STAT3 activation and its role in the interplay between glioma and their immune suppressive environment using a 3D spheroid model. We modified our commercially available test platform, 3D-Predict™ Glioma, such that it is conducive to monitoring immune cell function. Newly diagnosed treatment naive primary samples from HGG patients were dissociated and cryogenically preserved and banked. Samples were profiled for the abundance of tumor associated suppressive myeloid cells using flow cytometry. We determined that relative proportions of tumor suppressor cells were detectable and varied across HGG primary patient samples with TAMs being the predominate immune cell type identified. Pro-tumorigenic TAM markers including CD163 and CD206 were correlated with temozolomide response and were associated with poor patient prognosis. When we evaluated the role of p-STAT in HGG primary samples, p-STAT was detected at baseline and abundance was related back to the presence of tumor associated immune suppressor cells. The impact of STAT3 signaling blockade by the inhibitor WP1066 was determined ex vivo, and efficacy was related back to MGMT promotor methylation status and patient clinical outcome. We detected shifts in cytokine signatures following treatment of HGG 3D spheroid models with WP1066 which are known to target tumor associated suppressor cell functions. This work demonstrates the critical role suppressive immune cells play in HGG clinical response. Given p-STAT can be altered in 2D culture compared to 3D culture, ex vivo therapeutic response profiling of HGG samples is necessary using the more physiologically relevant spheroid culture model. 1.Hambardzumyan et al. The role of microglia and macrophages in glioma maintenance and progression. Nat. Neurosci. 19, 20-27 (2016). Citation Format: Katy A. Lassahn, Ashley K. Elrod, Teresa M. DesRochers, Kathryn M. Appleton. Ex vivo pharmacologic inhibition of STAT3 effectively targets tumor and tumor associated suppressor cells in high-grade glioma 3D spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5287.

Abstract 6851: The genomic, transcriptomic, and immunologic landscape of TEM8 (ANTXR1) in neuroendocrine neoplasms (NENs)

Abstract Introduction: TEM8 (coded by ANTXR1) is overexpressed in malignant tissues, with oncolytic viruses such as SVV-01 uniquely binding to this receptor on tumor-associated angiogenic endothelial cells, pericytes, fibroblasts, and immune inflammatory cells. Recent pre-clinical data suggest that TEM8-targeting therapies may convert immunologically “cold” tumor microenvironments (TME) into “hot” milieu more amenable to treatment with immune checkpoint inhibitors (ICIs). Methods: NextGen sequencing of DNA (592 genes or WES) and RNA (WTS) was performed on neuroendocrine neoplasms (NENs; N = 1724), excluding small-cell lung cancer, submitted to Caris Life Sciences (Phoenix, AZ). Mutations were defined as pathogenic SNVs/indels. ANTXR1 expression was divided by ANTXR1-expression quartiles (transcripts per million [TPM];Q4:H;Q1:L). PD-L1 expression (SP142; Positive (+): ≥2+,≥%5) was assessed by IHC. High tumor mutational burden (TMB-H) was defined as ≥10 mutations per MB. Cell infiltration in the TME was estimated by QuantiSEQ. Gene expression profiles were analyzed for transcriptional signatures predictive of response to immunotherapy (T cell-inflamed) and MAPK pathway activation score (MPAS). Overall survival (OS) data was obtained from insurance claims, and Kaplan-Meier estimates were calculated for molecularly defined subpopulations of patients. Mann-Whitney U and X2/Fisher-Exact tests were applied where appropriate, with p-values adjusted for multiple comparisons (p<0.05). Results: NENs from the adrenal gland had the highest median ANTXR1 expression (50 TPM), followed by those from the colon and rectum (43.5), genitourinary organs (43.3), and small bowel (excluding appendix) (39.2). The lowest median ANTXR1 expression was observed in NENs from the pancreas (31.5). No pathogenic mutations were significantly associated with ANTXR1H vs L tumors (p>0.05 for all), along with no differences in the prevalence of TMB-H (8.6% vs 12.6%, p=1) or PD-L1+ (5.8%: vs 3.1%, p=1). A greater proportion of B cells (5.34 % vs 3.78%, p<0.001), M1 (1.46% vs 0.15%, p<0.001) and M2 macrophages (3.92% vs 2.67%, p<0.001), and T-regulatory cells (1.77% vs 1.08%, p<0.001) was observed in ANTXR1H TMEs, which were more frequently classified as T cell-inflamed compared to ANTXR1L (42% vs 8%, p<0.001). ANTXR1H tumors also had higher MPAS compared to ANTXR1L (1.16 vs -1.61 arbitrary units, p<0.001). There was no significant difference in median OS between ANTXR1H vs L tumors (18.8 mo. vs 21.2 months, Hazard Ratio:1.18, p=0.114). Conclusion: The increased immune cell infiltrate and prevalence of T-cell inflamed status among ANTXR1H NENs suggests these tumors might be more responsive to treatment with ICIs. As trials incorporating intratumoral injections of SVV-01 in combination with ICIs are underway, further investigation of clinical and molecular associations with ANTXR1 expression in NENs is warranted. Citation Format: Samuel A. Kareff, Harris Krause, Andrew Elliott, Peter Hosein, Emil Lou, Heloisa Soares, Matthew Oberley, Aman Chauhan. The genomic, transcriptomic, and immunologic landscape of TEM8 (ANTXR1) in neuroendocrine neoplasms (NENs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6851.

Single-cell transcriptomic sequencing identifies subcutaneous patient-derived xenograft recapitulated medulloblastoma.

Medulloblastoma (MB) is one of the most common malignant brain tumors that mainly affect children. Various approaches have been used to model MB to facilitate investigating tumorigenesis. This study aims to compare the recapitulation of MB between subcutaneous patient-derived xenograft (sPDX), intracranial patient-derived xenograft (iPDX), and genetically engineered mouse models (GEMM) at the single-cell level. We obtained primary human sonic hedgehog (SHH) and group 3 (G3) MB samples from six patients. For each patient specimen, we developed two sPDX and iPDX models, respectively. Three Patch+/- GEMM models were also included for sequencing. Single-cell RNA sequencing was performed to compare gene expression profiles, cellular composition, and functional pathway enrichment. Bulk RNA-seq deconvolution was performed to compare cellular composition across models and human samples. Our results showed that the sPDX tumor model demonstrated the highest correlation to the overall transcriptomic profiles of primary human tumors at the single-cell level within the SHH and G3 subgroups, followed by the GEMM model and iPDX. The GEMM tumor model was able to recapitulate all subpopulations of tumor microenvironment (TME) cells that can be clustered in human SHH tumors, including a higher proportion of tumor-associated astrocytes and immune cells, and an additional cluster of vascular endothelia when compared to human SHH tumors. This study was the first to compare experimental models for MB at the single-cell level, providing value insights into model selection for different research purposes. sPDX and iPDX are suitable for drug testing and personalized therapy screenings, whereas GEMM models are valuable for investigating the interaction between tumor and TME cells.

Isolation and high-dimensional flow cytometric analysis of tumor-infiltrating leukocytes in a mouse model of colorectal cancer.

Colorectal cancer (CRC) is a complex and heterogeneous disease characterized by dysregulated interactions between tumor cells and the immune system. The tumor microenvironment plays a pivotal role in cancer initiation as well as progression, with myeloid immune cells such as dendritic cell and macrophage subsets playing diverse roles in cancer immunity. On one hand, they exert anti-tumor effects, but they can also contribute to tumor growth. The AOM/DSS colitis-associated cancer mouse model has emerged as a valuable tool to investigate inflammation-driven CRC. To understand the role of different leukocyte populations in tumor development, the preparation of single cell suspensions from tumors has become standard procedure for many types of cancer in recent years. However, in the case of AOM/DSS-induced colorectal tumors, this is still challenging and rarely described. For one, to be able to properly distinguish tumor-associated immune cells, separate processing of cancerous and surrounding colon tissue is essential. In addition, cell yield, due to the low tumor mass, viability, as well as preservation of cell surface epitopes are important for successful flow cytometric profiling of tumor-infiltrating leukocytes. Here we present a fast, simple, and economical step-by-step protocol for isolating colorectal tumor-associated leukocytes from AOM/DSS-treated mice. Furthermore, we demonstrate the feasibility of this protocol for high-dimensional flow cytometric identification of the different tumor-infiltrating leukocyte populations, with a specific focus on myeloid cell subsets.

RNAi mediated silencing of STAT3/PD-L1 in tumor-associated immune cells induces robust anti-tumor effects in immunotherapy resistant tumors

Malignant tumors are often associated with an immunosuppressive tumor microenvironment (TME), rendering most of them resistant to standard-of-care immune checkpoint inhibitors (CPIs). Signal transducer and activator of transcription 3 (STAT3), a ubiquitously expressed transcription factor, has well-defined immunosuppressive functions in several leukocyte populations within the TME. Since the STAT3 protein has been challenging to target using conventional pharmaceutical modalities, we investigated the feasibility of applying systemically delivered RNA interference (RNAi) agents to silence its mRNA directly in tumor-associated immune cells. In preclinical rodent tumor models, chemically stabilized acetylated small-interfering RNAs (siRNAs) selectively silenced Stat3 mRNA in multiple relevant cell types, reduced STAT3 protein levels, and increased cytotoxic T-cell infiltration. In a murine model of CPI-resistant pancreatic cancer, RNAi-mediated Stat3 silencing resulted in tumor growth inhibition, which was further enhanced in combination with CPIs. To further exemplify the utility of RNAi for cancer immunotherapy, this technology was used to silence Cd274, the gene encoding the immune checkpoint protein programmed death-ligand 1 (PD-L1). Interestingly, silencing of Cd274 was effective in tumor models that are resistant to PD-L1 antibody therapy. These data represent the first demonstration of systemic delivery of RNAi agents to the TME and suggest applying this technology for immuno-oncology applications.

Sialic Acid Conjugate-Modified Cationic Liposomal Paclitaxel for Targeted Therapy of Lung Metastasis in Breast Cancer: What a Difference the Cation Content Makes.

Cationic lipids play a pivotal role in developing novel drug delivery systems for diverse biomedical applications, owing to the success of mRNA vaccines against COVID-19 and the Phase III antitumor agent EndoTAG-1. However, the therapeutic potential of these positively charged liposomes is limited by dose-dependent toxicity. While an increased content of cationic lipids in the formulation can enhance the uptake and cytotoxicity toward tumor-associated cells, it is crucial to balance these advantages with the associated toxic side effects. In this work, we synthesized the cationic lipid HC-Y-2 and incorporated it into sialic acid (SA)-modified cationic liposomes loaded with paclitaxel to target tumor-associated immune cells efficiently. The SA-modified cationic liposomes exhibited enhanced binding affinity toward both RAW264.7 cells and 4T1 tumor cells in vitro due to the increased ratios of cationic HC-Y-2 content while effectively inhibiting 4T1 cell lung metastasis in vivo. By leveraging electrostatic forces and ligand-receptor interactions, the SA-modified cationic liposomes specifically target malignant tumor-associated immune cells such as tumor-associated macrophages (TAMs), reduce the proportion of cationic lipids in the formulation, and achieve dual objectives: high cellular uptake and potent antitumor efficacy. These findings highlight the potential advantages of this innovative approach utilizing cationic liposomes.

Abstract B047: Epigenetic reprogramming by CBP/P300 bromodomain inhibition of triple-negative breast cancer and the immune microenvironment

Abstract Triple-negative breast cancer (TNBC) is a biologically heterogeneous and clinically important breast cancer subtype because if considered a distinct disease, TNBC would rank as the 5th leading cause of cancer deaths in women. TNBC patients resistant to standard-of-care therapies have poor survival. Tumor-associated immune cells such as neutrophils and macrophages can display a pro-tumoral phenotype and contribute to therapy resistance. Epigenetic enzymes have been shown to reprogram both the tumor cells and tumor-associated immune cells. Thus, targeting epigenetic enzymes is a potential strategy to improve the response of TNBC to standard-of-care therapies. Previously, our laboratory performed an epigenetic drug screen and identified several epigenetic inhibitors. We selected IACS-70654, a novel selective p300/CBP BRD inhibitor, to be the primary focus of this study. We determined the effects of IACS-70654 in our unique syngeneic TNBC mouse models. In a neutrophil-enriched luminal-like TNBC model 2208L, IACS-70654 as a single agent initially caused tumor regression and then durably prevented tumor growth, which is not usually observed for single agents in these aggressive TNBC models. IACS-70654 also substantially decreased infiltrated neutrophils. In the peripheral blood and bone marrow of 2208L tumor-bearing mice, IACS-70654 reduced neutrophils to the same level as non-tumor-bearing mice. Based on this finding, we treated 2208L tumors with IACS-70654 in combination with immune checkpoint blockade anti-PD1 and docetaxel. After 27 days, all treatments besides anti-PD1 were stopped, and the combination-treated tumors grew slower than the ones only treated with IACS-70654. More importantly, in tumors treated with IACS-70654 in combination with anti-PD1, there was a substantial increase in CD8+ T cell infiltration. In addition, B cell infiltration was induced in the tumor stroma, indicating that IACS-70654 stimulated immune response in the tumor microenvironment. In contrast, IACS-70654 failed to significantly reduce the growth of T12, a Claudin-low macrophage-enriched model. Therefore, we hypothesized that the infiltrated immune profiles, specifically myeloid cell infiltration, might partially account for this differential response. Accordingly, we studied the effects of IACS-70654 in two additional models, basal-like T6 and luminal-like 2250L tumors, which also have relatively high neutrophil infiltration. However, one of the principal differences in immune profiles between the two models and 2208L is that they also are infiltrated with twice as many macrophages. The response of infiltrated immune cells to IACS-70654 in those two models differed from those of 2208L tumors. IACS-70654 reduced monocytic myeloid-derived suppressor cells and pro-tumor macrophages. These results illustrate the immune heterogeneity among our tumor models. We are currently performing ChIP-seq and single-cell ATAC-seq to elucidate potential mechanisms and pathways employed by IACS-70654 in both tumor and tumor-associated immune cells. Citation Format: Xueying Yuan, Michael Soth, Na Zhao, Philip Jones, Jeffrey Rosen. Epigenetic reprogramming by CBP/P300 bromodomain inhibition of triple-negative breast cancer and the immune 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 B047.

Immune cells in the B-cell lymphoma microenvironment: From basic research to clinical applications.

B-cell lymphoma is a group of hematological malignancies characterized by variable genetic and biological features and clinical behaviors. The tumor microenvironment (TME) is a complex network in tumors, which consists of surrounding blood vessels, extracellular matrix, immune and non-immune cells, and signaling molecules. Increasing evidence has shown that the TME, especially immune cells within, is a double-edged sword, acting either as a tumor killer or as a promoter of tumor progression. These pro-tumor activities are driven by subpopulations of immune cells that express typical markers but have unique transcriptional characteristics, making tumor-associated immune cells good targets for human anti-cancer therapy by ablating immunosuppressive cells or enhancing immune-activated cells. Thus, exploring the role of immune cells in the TME provides distinct insights for immunotherapy in B-cell lymphoma. In this review, we elucidated the interaction between immune cells and tumor cells and their function in the initiation, progression, and prognosis of B-cell lymphoma, from preclinical experiments to clinical trials. Furthermore, we outlined potential therapeutic approaches and discussed the potential clinical value and future perspectives of targeting immune cells in patients with B-cell lymphoma.

Concordance among three programmed death-ligand 1 (PD-L1) scoring methods and their association with clinical outcomes of tislelizumab (TIS) monotherapy in esophageal squamous cell carcinoma (ESCC).

390 Background: Multiple scoring methods and cutoffs have been developed to evaluate tumor PD-L1 expression status in patients with ESCC, and PD-L1 expression level has been associated with the degree of response to anti-programmed cell death protein 1 (PD-1)/PD-L1 therapy. Here, we retrospectively investigated concordance between three PD-L1 scoring methods and their association with clinical outcomes in RATIONALE-302, a phase 3 study of the anti-PD-1 antibody TIS vs investigator-chosen chemotherapy (ICC) as second-line treatment for advanced unresectable/metastatic ESCC (NCT03430843). Methods: Patients enrolled in RATIONALE-302 with evaluable PD-L1 expression by the tumor area positivity (TAP) score (based on visual estimation of positive tumor cells [TCs] and tumor-associated immune cells [ICs]) using the VENTANA PD-L1 (SP263) assay were categorized at a 10% cutoff. Stained slides from those patients were rescored post hoc using both combined positive score (CPS; based on counting positive TCs and ICs) at cutoff 10 and TC (based on counting positive TCs only) score at a 1% cutoff, thresholds currently used in ESCC for anti-PD-(L)1 therapy. Concordance at these thresholds was investigated. Clinical efficacy (overall survival [OS]) for PD-L1 subgroups was assessed. Results: Of 512 pts enrolled, 364 had evaluable TAP scores (TIS, n=180; ICC, n=184), of whom 355 had evaluable post-hoc CPS and TC scores (TIS, n=175; ICC, n=180). TAP score and CPS showed high concordance in terms of overall percentage agreement (OPA; 90% [95% confidence interval (CI): 86, 93]) and Cohen’s Kappa (0.79 [95% CI: 0.72, 0.85]), while TAP and TC scores had lower concordance (OPA: 78% [95% CI: 73, 82]; Cohen’s Kappa: 0.56 [95% CI: 0.47, 0.64]). OS benefit with TIS vs ICC in PD-L1 subgroups defined by TAP, CPS, and TC score cutoffs were generally similar (Table). Conclusions: OS subgroup analysis showed comparable treatment effect by TAP score at 10% cutoff, CPS at cutoff 10, and TC score at 1% cutoff based on SP263 staining. TAP score and CPS at these cutoffs exhibited substantial concordance. The results indicate that the less time-consuming, visually estimated TAP score and CPS may be interchangeable for clinical measurement of PD-L1 expression in patients with ESCC. Clinical trial information: NCT03430843 . [Table: see text]

Prediction of lymph node status in stage T1 esophageal squamous cell carcinoma.

263 Background: With the development of endoscopic therapy and the introduction of immunotherapies, it is more important for preserving the esophagus and uninvaded lymph nodes to avoid the damages involving the long-term quality of life and normal immune function caused by esophagectomy with radical lymphadenectomy. Methods: We collected patients with T1 ESCC who underwent endoscopic resection or esophagectomy from 2014 to 2021 in three cancer centers. The primary end point was presence of lymph node metastasis (LNM) in surgical resection specimens or detection of metastases during follow-up. Univariate and multivariate logistic regression were used to screen the clinicopathological features related to LNM. Using the function 'lrm' constructed the prediction model. The area under the curve (AUC) of receiver operating characteristic and calibration graphs were used to assess the performance of the model. An external validation was conducted to evaluate the generalization of the model. We then prospectively collected 28 surgically resected tissue samples of T1 ESCC to further supplement the model from the cellular and molecular level, and used 6 markers (CK, CD34, D2-40, CD4, CD8, FOXP3) and DAPI dye to process multiple immunofluorescence (mIF) staining. Afterwards, we analyzed the density, spatial distribution of microvessels, lymphatic capillaries and tumor-associated immune cells and the interaction between them and malignant cells in the tumor microenvironment of T1 ESCC to explore the relationship between tumor microenvironment and risk of LNM. Results: 1109 patients with T1 ESCC were included. 653 patients from two centers constituted the training cohort, and 456 patients from the third center served as the validation cohort. Multivariate logistic regression based on the training cohort showed that tumor invasion into the submucosa, poor histological grade, and lymphovascular invasion were risk factors for LNM in T1 ESCC. Subsequently, we constructed a model based on three variables to predict the risk of LNM in T1 ESCC. The model demonstrated good discriminative ability (AUC 0.858, 95%CI 0.826-0.890). After mIF staining of 28 prospectively collected T1 ESCC tissue samples, we observed that microvessels and lymphatic capillaries were abundant in the mucosa in lymph node-positive samples but not in lymph node-negative samples. In addition, the average density of FOXP3+ T cells in samples with and without metastasis was 253/mm2 and 26/mm2 (P < 0.001), and the average density of CD8+ T cells was 112/mm2 and 432/mm2 (P < 0.001) in quantitative analysis, respectively. Conclusions: This study (RENMIN-237) developed a well-performing LNM risk stratification model of T1 ESCC to reduce both overtreatment and undertreatment, and we found that FOXP3+ T cells in the tumor microenvironment are a risk factor for LNM, while CD8+ T cells can serve as a protective factor against LNM.

Galectin-9 in cancer therapy: from immune checkpoint ligand to promising therapeutic target.

Galectin-9 (Gal-9) is a vital member of the galectin family, functioning as a multi-subtype galactose lectin with diverse biological roles. Recent research has revealed that Gal-9's interaction with tumors is an independent factor that influences tumor progression. Furthermore, Gal-9 in the immune microenvironment cross-talks with tumor-associated immune cells, informing the clarification of Gal-9's identity as an immune checkpoint. A thorough investigation into Gal-9's role in various cancer types and its interaction with the immune microenvironment could yield novel strategies for subsequent targeted immunotherapy. This review focuses on the latest advances in understanding the direct and indirect cross-talk between Gal-9 and hematologic malignancies, in addition to solid tumors. In addition, we discuss the prospects of Gal-9 in tumor immunotherapy, including its cross-talk with the ligand TIM-3 and its potential in immune-combination therapy.

Characteristics and immune checkpoint status of radioiodine-refractory recurrent papillary thyroid carcinomas from Ukrainian Chornobyl Tissue Bank donors.

The radioiodine-refractory (RAI-R) recurrent papillary thyroid carcinomas (PTCs) are more frequent in elderly patients and have an unfavorable prognosis. Data on the prevalence and characteristics of RAI-R recurrent PTCs in patients of young and middle age with or without a history of radiation exposure in childhood are poorly described. The aim of the current study was: i) to determine the frequency of RAI-R recurrent PTCs among donors of the Chornobyl Tissue Bank (CTB) and analyze the clinicopathological features of primary tumors (PTs), primary metastases (PMTSs), recurrent metastases (RMTSs) and risk factors for RMTS, and ii) to determine the immune checkpoint status (ICS) of the RAI-R recurrent PTCs and to assess the factors associated with ICS positivity. Sixty RAI-R recurrent PTCs (46 exposed to radiation and 14 non-exposed, 2.5% of all cases registered with the CTB) from the Ukrainian patients aged up to 48 years were identified. The clinicopathological characteristics of the PTs moderately to weakly resembled those of the PMTS and RMTS from the same patients while the metastatic tissues were highly similar. The multivariate model of RMTS included the dominant solid-trabecular growth pattern of the PT, cystic changes, N1b metastases, and the probability of a causation (POC) of PTC by radiation as risk factors. Among these factors, the lateral PMTS (N1b) had the strongest effect. The longer period of latency (a POC component) was the second statistically significant characteristic. ICS percent agreement between the PT and RAI-R RMTS was 91.5%; 23.7% of PTs and 28.8% of RMTSs had positive ICS (positive PD-L1 tumor epithelial cells (TECs) and positive PD-L1/PD1 tumor-associated immune cells). ICS positivity of PTs was associated with pronounced oncocytic changes and high density of the p16INK4A-positive TECs in the invasive areas of PTs. In RMTSs, ICS positivity was associated with pronounced oncocytic changes and Ki-67 labeling index ≥ 4.5% of PTs, and the dominant solid-trabecular growth pattern, Ki-67 labeling index ≥ 7.6% and p16INK4A-positivity of RMTS. The findings are of clinical relevance and may be useful for developing individual treatment approaches for patients with RAI-R recurrent PTCs possibly involving immunotherapy.

Neutrophil-to-Lymphocyte Ratio (NLR) in NSCLC, Gastrointestinal, and Other Solid Tumors: Immunotherapy and Beyond.

In the era of immunotherapy, identifying biomarkers of immune system activation has become a high-priority challenge. The blood neutrophil-to-lymphocyte ratio (NLR) has been largely investigated as a biomarker in several cancer types. NLR values have been shown to mirror the tumor-induced inflammatory status and have been demonstrated to be a reliable prognostic tool across stages of disease and therapeutic approaches. When integrated with other biomarkers of response to immunotherapy, such as PD-L1, tumor mutational burden, and tumor-associated immune cells, the NLR may allow to further stratify patients with different likelihoods of deriving a significant clinical benefit. However, despite its accessibility, low cost, and easy interpretation, the NLR is still poorly used as a prognostic tool in daily clinical practice. In this review, we analyze the role of the NLR in defining the relationship between cancer and the immune system, its usefulness in daily clinical practice, and its relationship with other established or emerging biomarkers of immunotherapy outcomes.

AIRE is expressed in breast cancer TANs and TAMs to regulate the extrinsic apoptotic pathway and inflammation.

The autoimmune regulator (AIRE) is a transcriptional regulator expressed in the thymus and is necessary for maintaining immunological self-tolerance. Extrathymic AIRE expression is rare, and a role for AIRE in tumor-associated innate immune cells has not yet been established. In this study, we show that AIRE is expressed in human pro-tumor neutrophils. In breast cancer, AIRE was primarily located to tumor-associated neutrophils (TANs), and to a lesser extent to tumor-associated macrophages (TAMs) and tumor cells. Expression of AIRE in TAN/TAMs, but not in cancer cells, was associated with an adverse prognosis. We show that the functional role for AIRE in neutrophils and macrophages is to regulate expression of immune mediators and the extrinsic apoptotic pathway involving the Fas/TNFR death receptors and cathepsin G. Here, we propose that the role for AIRE in TAN/TAMs in breast tumors is to regulate cell death and inflammation, thus promoting tumor progression.

Human Umbilical Cord Perivascular Cells Prevent Tumor Growth in a Melanoma Tumor-Bearing Mouse Model and Modulate Breast Cancer and Melanoma Cells in a Cell Line-Dependent Manner In Vitro

First trimester (FTM) and term human umbilical cord perivascular cells are promising mesenchymal stromal cell candidates to mitigate side effects of oncotherapy, but their safety for cancer patients remains to be determined. This study was designed to determine if human umbilical cord perivascular cells modulate tumor growth when injected systemically in a tumor-bearing mouse model. Immunodeficient mice-bearing palpable subcutaneous SK-MEL-28 human melanoma tumors were randomized to receive a tail vein injection of three human umbilical cord perivascular cell lines resuspended in hank’s buffer saline solution (vehicle) or vehicle only, as a control. Fibroblast cells were included as a cell control in some experiments. Tumor size was monitored weekly and weighed at 3-weeks postinjection. Cell fate and tumor cell proliferation, apoptosis, vascularization as well as tumor-associated immune cells were assessed using immunostaining and flow cytometry. Serum tumor necrosis factor alpha and C-reactive protein levels were measured using enzyme-linked immunosorbent assays. Transwell coculture models were used to study the paracrine effects of multiple lines of human umbilical cord cells on human melanoma cell lines as well as breast cancer cell lines. Systemic administration of FTM and term human umbilical cord perivascular cells, but not fibroblast cells, prevented melanoma tumor growth in a tumor-bearing animal model by modulating tumor cell proliferation and systemic inflammatory mechanisms. Cancer cell- and donor-dependent paracrine effects on cancer cell growth were observed in vitro. Our preclinical studies thus suggest that, with regards to its effects on tumor growth, systemic administration of FTM and term human umbilical cord perivascular cells may be a safe cell therapy to address the side effects of cancer.

[Multistain deep learning as a prognostic and predictive biomarker in colorectal cancer].

The tumor immune microenvironment (TIME) plays acrucial prognostic and predictive role in solid malignancies such as colorectal cancer (CRC). Nevertheless, scoring systems based on TIME such as the Immunoscore (IS) are rarely used in clinical practice. Among other reasons, this might be due to the additional time required for manual quantification of tumor-associated immune cells or costs associated with proprietary/commercial solutions. To address these issues, we developed amultistain deep learning model (MSDLM) and trained, validated, and tested it on immunohistochemical image data of different immune cell subtypes from over 1000 patients with CRC. Our model showed high prognostic accuracy and outperformed other clinical, molecular, and immune cell-based parameters. It might also be used for therapy response prediction in rectal cancer patients undergoing neoadjuvant therapy. Leveraging artificial intelligence interpretability/explainability methods, we ascertained that the MSDLM's predictions align with recognized antitumor immune response patterns. Consequently, the AImmunoscore (AIS) could emerge as apotential TIME-based decision-making tool for clinicians.

Tumour immune microenvironment as the origin of heterotypic circulating tumour cell clusters and their impact on metastasis: An hypothesis

Cancer metastasis remains a significant challenge in cancer treatment, contributing to most cancer-related deaths. Circulating Tumour Cells (CTCs) are crucial for the formation of metastases, and the presence of heterotypic CTC clusters has been associated with higher metastatic potential. We hypothesize that the tumour immune microenvironment (TIME) serves as a possible site for the origin of heterotypic CTC clusters. The interactions between CTCs and non-malignant cells within the TIME can promote cluster formation, facilitate intravasation, survival in the bloodstream, and successful extravasation at secondary sites. The survival of CTCs in circulation is limited due to immune attacks and shear stress; while the formation of heterotypic clusters provides a survival advantage to CTCs by evading immune recognition. Understanding the origin and survival mechanisms of these CTC clusters in the blood is crucial for developing strategies to prevent and treat metastatic cancer. The hypothesis highlights the role of the TIME in modulating CTC behaviour and suggests that targeting tumour-associated immune cells may help disrupt CTC clusters and potentially inhibit metastasis at its early stage.