Immunosuppressive Tumor Immune Microenvironment Research Articles

Immune Checkpoints in B Cells: Unlocking New Potentials in Cancer Treatment.

B cells are crucial component of humoral immunity, and their role in the tumor immune microenvironment (TME) has garnered significant attention in recent years. These cells hold great potential and application prospects in the field of tumor immunotherapy. Research has demonstrated that the TME can remodel various B cell functions, including proliferation, differentiation, antigen presentation, and antibody production, thereby invalidating the anti-tumor effects of B cells. Concurrently, numerous immune checkpoints (ICs) on the surface of B cells are upregulated. Aberrant B-cell IC signals not only impair the function of B cells themselves, but also modulate the tumor-killing effects of other immune cells, ultimately fostering an immunosuppressive TME and facilitating tumor immune escape. Blocking ICs on B cells is beneficial for reversing the immunosuppressive TME and restoring anti-tumor immune responses. In this paper, the intricate connection between B-cell ICs and the TME is delved into, emphasizing the critical role of targeting B-cell ICs in anti-tumor immunity, which may provide valuable insights for the future development of tumor immunotherapy based on B cells.

A nanotheranostics with hypoxia-switchable fluorescence and photothermal effect for hypoxia imaging-guided immunosuppressive tumor microenvironment modulation

Modulating the immunosuppressive tumor immune microenvironment (TIME) is considered a promising strategy for cancer treatment. However, effectively modulating the immunosuppressive TIME within hypoxic zones remains a significant challenge. In this work, we developed a hypoxia-responsive amphiphilic drug carrier using boron-dipyrromethene (BODIPY) dye-modified chitosan (CsB), and then fabricated a hypoxia-targeted nanotheranostic system, named CsBPNs, through self-assembly of CsB and pexidartinib (5-((5-Chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)-N-((6-(trifluoromethyl)pyridin-3-yl)methyl), PLX3397), an immunotherapeutic drug targeting tumor-associated macrophages (TAMs), for synergistic photothermal/immunotherapy and hypoxia imaging. CsBPNs demonstrated uniform size, good stability, and hypoxia-switchable fluorescence and photothermal effects, enabling deep penetration and hypoxia imaging capacities in three-dimensional tumor cell spheres and tumor tissues. In vitro and in vivo experiments showed that CsBPNs under laser irradiation promoted TAMs repolarization, reversed the immunosuppressive TIME, and enhanced the therapeutic outcome of PLX3397 in solid tumors by facilitating deep delivery into hypoxic regions and synergistic photothermal therapy. This work provides a new strategy for detecting and modulating the immunosuppressive TIME in hypoxic zones, potentially enabling more precise and effective photo-immunotherapy in the future.

Fusobacterium nucleatum, immune responses, and metastatic organ diversity in colorectal cancer liver metastasis.

The presence of Fusobacterium nucleatum is associated with an immunosuppressive tumor immune microenvironment (TIM) in primary colorectal cancer (CRC), contributing to tumor progression. Its persistence in CRC liver metastasis tissues raises questions about its role in modulating local and systemic immune responses and influencing recurrence patterns. This retrospective cohort study of 218 patients with CRC liver metastasis investigated the association of F. nucleatum in CRC liver metastasis tissues with systemic inflammation, TIM alterations, and the number of metastatic organs involved in recurrence. Two-step polymerase chain reaction (PCR), including digital PCR, detected F. nucleatum in 42% (92/218) of fresh-frozen specimens of CRC liver metastases. Compared with the F. nucleatum-none group, the F. nucleatum-high group showed higher C-reactive protein levels (0.82 vs. 0.22 mg/dL; Ptrend = 0.02), lower numbers of CD8+ cells (33.2 vs. 65.3 cells/mm2; Ptrend = 0.04) and FOXP3+ cells (11.3 vs. 21.7 cells/mm2; Ptrend = 0.01) in the TIM, and a greater number of metastatic organs involved in recurrence (1.6 vs. 1.1; p < 0.001). The presence of F. nucleatum in CRC liver metastasis tissues was associated with increased systemic inflammation, TIM alterations, and a greater number of metastatic organs involved in recurrence. These findings suggest a potential contribution of F. nucleatum to the metastatic propensity of CRC cells and could inform future research to enhance understanding of the interaction between tumor, host, and microbes in the metastatic process.

IRF8-driven reprogramming of the immune microenvironment enhances anti-tumor adaptive immunity and reduces immunosuppression in murine glioblastoma.

Glioblastoma (GBM) has a highly immunosuppressive tumor immune microenvironment (TIME), largely mediated by myeloid-derived suppressor cells (MDSCs). Here, we utilized a retroviral replicating vector (RRV) to deliver Interferon Regulatory Factor 8 (IRF8), a master regulator of type 1 conventional dendritic cell (cDC1) development, in a syngeneic murine GBM model. We hypothesized that RRV-mediated delivery of IRF8 could "reprogram" intratumoral MDSCs into antigen-presenting cells (APCs) and thereby restore T-cell responses. Effects of RRV-IRF8 on survival and tumor growth kinetics were examined in the SB28 murine GBM model. Immunophenotype was analyzed by flow cytometry and gene expression assays. We assayed functional immunosuppression and antigen presentation by ex vivo T-cell-myeloid co-culture. Intratumoral injection of RRV-IRF8 in mice bearing intracerebral SB28 glioma significantly suppressed the tumor growth and prolonged survival. RRV-IRF8 treated tumors exhibited significant enrichment of cDC1s and CD8+ T-cells. Additionally, myeloid cells derived from RRV-IRF8 tumors showed decreased expression of the immunosuppressive markers Arg1 and IDO1 and demonstrated reduced suppression of naïve T-cell proliferation in ex vivo co-culture, compared to controls. Furthermore, DCs from RRV-IRF8 tumors showed increased antigen presentation compared to those from control tumors. In vivo treatment with azidothymidine (AZT), a viral replication inhibitor, showed that IRF8 transduction in both tumor and non-tumor cells is necessary for survival benefit, associated with a reprogrammed, cDC1- and CD8 T-cell-enriched TIME. Our results indicate that reprogramming of glioma-infiltrating myeloid cells by in vivo expression of IRF8 may reduce immunosuppression and enhance antigen presentation, achieving improved tumor control.

Interaction between VEGF-A and immune checkpoint targets in triple-negative breast cancer and mechanisms of immune evasion and tumor progression.

1096 Background: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment triple negative breast cancer (TNBC). However, not all patients with TNBC benefit from the addition of ICIs due to factors such as intrinsic and acquired resistance to ICIs, and complex interplay between tumor and immune cells in the tumor microenvironment (TME). Vascular endothelial growth factor (VEGF) promotes angiogenesis and potentially modulates tumor immune evasion. A phase I clinical trial has shown that a bispecific antibody to VEGF-A and PD-L1 produces encouraging tumor responses in advanced TNBC. In this study, we investigated the interaction between VEGF and various immune gene expression signatures including PD-L1 in a real-world breast cancer patient population. Methods: Comprehensive immune profiling, including the RNA-seq based gene expression assessment of 395 immune-associated genes, was performed on 120 formalin-fixed paraffin-embedded breast cancer samples. PD-L1 expression (Combined Positive Score, CPS ≥ 10%) was measured by immunohistochemistry (IHC; 22C3). Clinicopathologic variables were collected. Statistical associations between quantitative biomarkers, including 28 immune checkpoint genes, were calculated using Pearson correlations (r), and comparisons of quantitative biomarkers between groups were performed using the proportions test. For statistical significance, p&lt;0.05 was required. Results: The study cohort included 29 patients (24.2%) with TNBC and 91 (75.2%) with non-TNBC (including ER+/HER2- and HER2+). Most patients had metastatic tumors (n=92, 76.7%) and invasive ductal carcinoma (n=83, 69.2%). Among the TNBC group, 21 patients had PD-L1 positive tumors (72.4%), defined by CPS ≥ 10.In non-TNBC, VEGF has significant, though weak (r&lt;0.35) correlations with the expression of three checkpoint genes: AKT1 (r=0.35, p=0.00063), ICOS (r=0.34, p=0.00099), and TIM3 (r=0.29, p=0.0061). In TNBC, VEGF has significant, strong (r&gt;0.47) correlations with the expression of three other checkpoint genes: PD-1 (r=0.47, p=0.0094), PD-L1 (r=0.57, p=0.0012), and PIK3CA (r=0.6, p=0.00051) However, in TNBC, VEGF gene expression does not have a statistical association with PD-L1 IHC (CPS ≥/&lt; 10 (p=0.9)). Conclusions: Our findings suggest that there is an interplay between angiogenesis and an immunosuppressive tumor immune microenvironment in TNBC to a greater degree than in non-TNBCs. This supports the notion that angiogenesis mediators such as VEGF-A may enhance the expression of immunosuppressive checkpoint molecules, leading to immune evasion and tumor progression. Combination treatments of ICIs, especially anti-PD-1 therapy, with VEGF antibodies may be promising approaches to enhance the immune responses in TNBC, especially in tumors with CPS&lt;10, where the current frontline treatment is cytotoxic chemotherapy.

Impact of APC mutations on prognosis and tumor microenvironment in colorectal signet ring cell carcinoma.

e15500 Background: Colorectal signet ring cell carcinoma (CRC-SRC) is a malignant tumour characterized by its aggressive nature and unfavourable prognosis. In spite of the fact that numerous studies have examined the molecular profile and therapeutic targets of CRC-SRC, the prognostic biomarkers for this malignancy continue to be unknown. Methods: We conducted a comprehensive analysis of multi-omics in 35 cases of CRC-SRC and 10 cases of colorectal adenocarcinomas (CRC-AC). This analysis involved various techniques, including immunohistochemistry, DNA sequencing, and RNA sequencing. Results: In CRC-SRC, despite a lower frequency of APC mutations at the genomic level (P = 0.01), they rank as the second most prevalent mutations (40%), surpassed only by TP53 mutations (65.71%). The findings revealed that 13 genes associated with immunotherapy response were found to be upregulated in CRC-AC. Additionally, the levels of CD3+, CD8+, and CD3+ &amp; CD8+ T cells were found to be similar in tumor regions in CRC-SRC. These results suggest that CRC-SRC is more likely to have an immunosuppressive tumor immune microenvironment (TiME) compared to CRC-AC. Multivariate Cox proportional hazard regression analysis (hazard ratio: 3.54, 95% confidence interval: 3.93-117.3; p-value = 0.0004), in particular, shows that the presence of APC mutations is associated with a worse prognosis. In contrast, APC-mutant cases of CRC-AC have the same overall survival (OS) rate as APC-wildtype patients in the TCGA cohort. On RNA level, APC-mutant tumors exhibited higher expression levels of VEGFA ( P = 0.028) and Mki67 (P = 0.014), which are implicated in tumour invasion and proliferation. In contrast, APC-mutant CRC-AC did not exhibit differential expression on these genes within the TCGA cohort. GSEA showed that only the E2F hallmark was significantly enriched in APC-mutant CRC-SRC patients ( P = 0.0072). On protein expression level, APC-mutant patients had a significantly lower CD3 or CD68 positive ratio in the tumour region (CD3: P = 0.035; CD68: P = 0.039). This indicated that lymphocyte infiltration is decreased in APC-mutant CRC-SRC. Conclusions: To summarize, APC mutations may have distinct functions in CRC-AC and CRC-SRC. Colorectal cancer (CRC) with APC mutations showed a more unfavourable prognosis and a distinct tumour immune microenvironment compared to CRC with normal APC. Clinical trial information: ChiCTR2300072881.

Understanding the dynamics of TKI-induced changes in the tumor immune microenvironment for improved therapeutic effect

BackgroundThe dynamic interplay between tyrosine kinase inhibitors (TKIs) and the tumor immune microenvironment (TME) plays a crucial role in the therapeutic trajectory of non-small cell lung cancer (NSCLC). Understanding the...

Impact of environmental temperature on the survival outcomes of breast cancer: A SEER-based study

BackgroundExperimental evidence in tumor-bearing mouse models shows that exposure to cool, that is, sub-thermoneutral environmental temperature is associated with a higher tumor growth rate and an immunosuppressive tumor immune microenvironment than seen at thermoneutral temperatures. However, the translational significance of these findings in humans is unclear. We hypothesized that breast cancer patients living in warmer climates will have better survival outcomes than patients living in colder climates.MethodsA retrospective population-based analysis was conducted on 270,496 stage I-III breast cancer patients, who were retrieved from the Surveillance, Epidemiology and End Results (SEER) over the period from 1996 to 2017. The average annual temperature (AAT) was calculated based on city level data from the National Centers for Environmental Information.ResultsA total of 270, 496 patients were analyzed. Temperature as assessed in quartiles. After adjusting for potential confounders, patients who lived in the 3rd and 4th quartile temperature regions with AAT 56.7–62.5°F (3rd quartile) and > 62.5°F (4th quartile) had a 7% increase in the OS compared to patients living at AAT < 48.5°F (1st quartile) (HR 0.93, 95% CI 0.90–0.95 and HR 0.93, 95% CI 0.91–0.96, respectively). For DSS, When comparing AAT quartiles, patients living with AAT in the range of 56.7–62.5°F and > 62.5°F demonstrated a 7% increase each in DSS after adjustment (HR 0.93, 95% CI 0.90–0.96 and HR 0.93, 95% CI 0.90–0.96).ConclusionsHigher environmental temperatures are associated with significantly better OS and DSS in breast cancer patients. Future research is warranted to confirm this observation using large datasets to elucidate the underlying mechanisms and investigate novel therapeutic strategies to minimize this geographic disparity in clinical outcomes.

In situ administration of STING-activating hyaluronic acid conjugate primes anti-glioblastoma immune response

Glioblastoma (GBM) is an aggressive brain tumor, with a highly immunosuppressive tumor immune microenvironment (TIME). In this work, we investigated the use of the STimulator of INterferon Genes (STING) pathway as an effective means to remodel the GBM TIME through the recruitment of both innate and adaptive immune cell populations. Using hyaluronic acid (HA), we developed a novel polymer-drug conjugate of a non-nucleotide STING agonist (MSA2), called HA-MSA2 for the in situ treatment of GBM. In JAWSII cells, HA-MSA2 exerted a greater increase of STING signaling and upregulation of STING-related downstream cyto-/chemokines in immune cells than the free drug. HA-MSA2 also elicited cancer cell-intrinsic immunostimulatory gene expression and promoted immunogenic cell death of GBM cells. In the SB28 GBM model, local delivery of HA-MSA2 induced a delay in tumor growth and a significant extension of survival. The analysis of the TIME showed a profound shift in the GBM immune landscape after HA-MSA2 treatment, with higher infiltration by innate and adaptive immune cells including dendritic, natural killer (NK) and CD8 T cell populations. The therapeutic potential of this novel polymer conjugate warrants further investigation, particularly with other chemo-immunotherapeutics or cancer vaccines as a promising combinatorial therapeutic approach.

Abstract 6872: A spatio-temporal approach to mapping the dynamics of cutaneous squamous cell carcinoma progression and immunotherapy response: A journey through TiME

Abstract Background: Cutaneous squamous cell carcinoma (cSCC), arising from proliferation of malignant epidermal keratinocytes, is the second most common non-melanoma skin cancer. cSCC accounts for 75% of all skin cancer related deaths excluding melanoma, and metastatic and immunotherapy-resistant cases pose an emerging global threat. Tumor development is a gradual process characterized by high mutational burden and an immunosuppressive tumor immune microenvironment (TiME); therefore, identifying the underlying changes across the spatial and temporal landscape of these tumors will be crucial to understanding the key cellular and molecular drivers of disease progression and response. Methods: In this longitudinal study, we used a single-cell, whole-slide approach to mapping key proteins of immune lineage, tissue architecture, cellular metabolism, and stress to study the spatial landscape of cSCC at baseline (pre-treatment) and multiple time points over the course of immunotherapy with the aim to develop a temporal atlas of the tumor. An ultra-high plex antibody panel encompassing over 50 major determinants in the tumor microenvironment was employed on the PhenoCycler®-Fusion spatial biology platform. Deep bioinformatic analyses was performed to identify cellular phenotypes, spatial neighborhoods, heterogenous functional states and cellular interactions by spatial proximity determinations. Results: Previous studies in our laboratory have identified unique phenotypes correlating with response and resistance to immunotherapies in pre-treatment cSCC biopsies. Our results expand on these findings with temporal data tracking the dynamic changes in the TiME over the course of immune checkpoint inhibitor therapy. Apart from differences in immune cell composition and spatial localization patterns, we also saw differences in macrophage polarization and expression of metabolic markers in the responder and non-responder cohorts. Conclusions: Overall, ultrahigh-plex spatio-temporal monitoring of cSCC revealed distinct signatures of response and resistance and identified key features in the TiME that reveal deeper insights into the pathobiology of the tumor. Our discovery-based approach identifies novel biomarkers for patient stratification and therapeutic modulation. Citation Format: Niyati Jhaveri, Bassem Ben Cheikh, Dmytro Klymyshyn, Ning Ma, Aditya Pratapa, James Monkman, Nadine Nelson, Arutha Kulasinghe. A spatio-temporal approach to mapping the dynamics of cutaneous squamous cell carcinoma progression and immunotherapy response: A journey through TiME [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 6872.

Abstract 2663: Determining the role of tumor mutational burden in a pre-clinical model of glioblastoma

Abstract Despite advances in care in many cancer types, patients with glioblastoma (GBM) have a grim prognosis of 15 to 21 months and unchanged standard therapy since 2005. These tumors have a profoundly immunosuppressive tumor immune microenvironment. Immunotherapy, including the checkpoint inhibitors (ICIs) targeting PD-1 and CTLA-4, has transformed the treatment of many cancers but has failed in trials of both newly diagnosed and recurrent GBM. Tumor mutational burden (TMB) has been proposed as a predictor of response to ICI treatment, presumably through an increase in neoantigens that can be recognized by cytotoxic CD8+ T cells leading to tumor rejection. GBM typically has a low TMB, a feature speculated as a contributor to poor response to ICI treatment. However, cases with an extremely high TMB (i.e., patients with the biallelic mismatch repair deficiency syndrome) do show response to immunotherapy. To begin to investigate the impact of mutational burden on immune response in GBM, we created a murine syngeneic tumor model using CRISPR-Cas9 to knockout (KO) the mismatch repair protein Msh2, the lynchpin to mismatch recognition, in the SB28 GBM model that has a low TMB (108 mutations) at baseline. Msh2 KO increased the TMB from 2 to 12-fold depending on single cell sorted clone, resulting in between 150 and 200 predicted strong MHC-I binding neoantigens estimated by the NetMHC algorithm. Preliminary studies have demonstrated that in vitro proliferation is similar amongst low TMB and high TMB SB28 clones. Flank implantation of high TMB SB28 clones revealed slower growth compared to the low TMB SB28 tumors. Furthermore, treatment with ICIs did provide a survival advantage in this flank model. However, no survival advantage was noted when the high TMB clones were implanted intracranially either with or without ICI treatment. These results suggested that either there is poor immune cell trafficking to the intracranial tumors, or a robust immune response was causing increased intracranial pressure that is prematurely causing death. Use of dexamethasone starting at day 11 to control cerebral edema has shown some survival improvement with ICI treatment, supporting that an inflammatory response is at least partially responsible for the lack of improved survival in early studies. We are currently evaluating CD4+ and CD8+ T cell clonality, earlier timepoints to evaluate the immune infiltrate and anti-tumor immune response, and alterations in chemokines/cytokines in the tumor microenvironment. By creating high mutation burden clones from a GBM with a low mutational burden, we will be able to interrogate potential mechanisms of heightened immunogenicity that has been reported in other cancers. Citation Format: Kevin Breen, Tuesday Haynes, Masashi Watanabe, Mark Gilbert. Determining the role of tumor mutational burden in a pre-clinical model of glioblastoma [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 2663.

Translational and oncologic significance of tertiary lymphoid structures in pancreatic adenocarcinoma.

Pancreatic adenocarcinoma (PDAC) is an aggressive tumor with poor survival and limited treatment options. PDAC resistance to immunotherapeutic strategies is multifactorial, but partially owed to an immunosuppressive tumor immune microenvironment (TiME). However, the PDAC TiME is heterogeneous and harbors favorable tumor-infiltrating lymphocyte (TIL) populations. Tertiary lymphoid structures (TLS) are organized aggregates of immune cells that develop within non-lymphoid tissue under chronic inflammation in multiple contexts, including cancers. Our current understanding of their role within the PDAC TiME remains limited; TLS are complex structures with multiple anatomic features such as location, density, and maturity that may impact clinical outcomes such as survival and therapy response in PDAC. Similarly, our understanding of methods to manipulate TLS is an actively developing field of research. TLS may function as anti-tumoral immune niches that can be leveraged as a therapeutic strategy to potentiate both existing chemotherapeutic regimens and potentiate future immune-based therapeutic strategies to improve patient outcomes. This review seeks to cover anatomy, relevant features, immune effects, translational significance, and future directions of understanding TLS within the context of PDAC.

Spatial and temporal heterogeneity of tumor immune microenvironment between primary tumor and brain metastases in NSCLC

BackgroundBrain metastasis is a common outcome in non-small cell lung cancer, and despite aggressive treatment, its clinical outcome is still frustrating. In recent years, immunotherapy has been developing rapidly, however, its therapeutic outcomes for primary lung cancer and brain metastases are not the same, suggesting that there may be differences in the immune microenvironment of primary lung cancer and brain metastases, however, we currently know little about these differences.MethodsSeventeen paired samples of NSCLC and their brain metastases and 45 other unpaired brain metastases samples were collected for the current study. Immunohistochemical staining was performed on all samples for the following markers: immune checkpoints CTLA-4, PD-1, PD-L1, B7-H3, B7-H4, IDO1, and EphA2; tumor-infiltrating lymphocytes (TILs) CD3, CD4, CD8, and CD20; tumor-associated microglia/macrophages (TAMs) CD68 and CD163; and tumor proliferation index Ki-67. The differences in expression of these markers were compared in 17 paired samples, and the effect of the expression level of these markers on the prognosis of patients was analyzed in lung adenocarcinoma brain metastases samples. Subsequently, multiplex immunofluorescence staining was performed in a typical lung-brain paired sample based on the aforementioned results. The multiplex immunofluorescence staining results revealed the difference in tumor immune microenvironment between primary NSCLC and brain metastases.ResultsIn 17 paired lesions, the infiltration of CTLA-4+ (P = 0.461), PD-1+ (P = 0.106), CD3+ (P = 0.045), CD4+ (P = 0.037), CD8+ (P = 0.008), and CD20+ (P = 0.029) TILs in brain metastases were significantly decreased compared with primary tumors. No statistically significant difference was observed in the CD68 (P = 0.954) and CD163 (P = 0.654) TAM infiltration between primary NSCLC and paired brain metastases. In all the brain metastases lesions, the expression of PD-L1 is related to the time interval of brain metastases in NSCLC. In addition, the Cox proportional hazards regression models showed high expression of B7-H4 (hazard ratio [HR] = 3.276, 95% confidence interval [CI] 1.335–8.041, P = 0.010) and CD68 TAM infiltration (HR = 3.775, 95% CI 1.419–10.044, P = 0.008) were independent prognosis factors for lung adenocarcinoma brain metastases patients.ConclusionsBoth temporal and spatial heterogeneity is present between the primary tumor and brain metastases of NCSLC. Brain metastases lesions exhibit a more immunosuppressive tumor immune microenvironment. B7-H4 and CD68+ TAMs may have potential therapeutic value for lung adenocarcinoma brain metastases patients.

Immunophenotypes and Tumor Immune Microenvironment in Hepatocellular Carcinoma With Macrotrabecular Massive and Vessels Encapsulating Tumor Clusters.

Recently, vessels encapsulating tumor clusters (VETC) pattern and macrotrabecular massive (MTM) pattern of hepatocellular carcinoma (HCC) have been reported as aggressive histological types. These histological patterns showed an immunosuppressive tumor immune microenvironment (TIME). Since there have been no reports on the differences of these two subtypes simultaneously, this study examined the immunophenotypes and TIME of MTM-HCC and VETC-HCC immunohistochemically. Seventy-four cases of previously diagnosed HCC, including 32 MTM-HCCs, 21 VETC-HCCs, and 21 conventional HCCs, were enrolled in immunohistochemical analysis. We conducted immunohistochemical analysis. We found that MTM-HCC showed less frequent expression of HepPar-1, which is one of the most common hepatocytic markers. In MTM-HCC, the frequency of high expression levels of Keratin19, carbonic anhydrase (CA) IX, and PD-L1 was higher compared to VETC-HCC and conventional HCC. PD-L1 expression was found in 34.4% of MTM-HCC, 0% of VETC-HCC, and 19.0% of conventional HCC. The rate of PD-L1 expression in MTM-HCC was significantly higher than the others (p=0.0015). PD-L1 expression was significantly associated with epithelial cell adhesion molecules and CA IX expression, which are representative markers of tumor stemness and hypoxic conditions, respectively. The CD8 infiltration in VETC-HCC was significantly lower than that in conventional HCC. MTM-HCC had different immunophenotypes and TIMEs compared to HCC with the VETC pattern. Although both had immunosuppressive TIME, the elements forming TIME were quite different. To enhance the immune checkpoint inhibitor efficacy, changing TIME from a suppressive to an active form is essential.

Abstract LB_C19: Suppression of the antitumoral activity of natural killer cells by cancer-associated fibroblasts in a pancreatic TIME-on-Chip model

Abstract Background: Recently, natural killer (NK) cells emerged as a treatment option for various solid tumors. However, the immunosuppressive tumor immune microenvironment (TIME) can reduce the cytotoxic ability of NK cells in pancreatic ductal adenocarcinoma. Cancer-associated fibroblasts within the tumor stroma can suppress immune surveillance by dysregulating factors involved in the cellular activity of NK cells. Herein, the effect of activated pancreatic stellate cells (aPSCs) on NK cell-mediated anticancer efficacy under three-dimensional (3D) coculture conditions was investigated. Methods: 3D cocultures of PANC-1 tumor spheroids (TSs) with aPSCs and NK-92 cells in a collagen matrix were optimized to identify the occurring cellular interactions and differential cytokine profiles in conditioned media using microchannel chips. PANC-1 TSs and aPSCs were indirectly cocultured, whereas NK-92 cells were allowed to infiltrate the TS channel using convective medium flow. Results: Coculture with aPSCs promoted PANC-1 TSs growth and suppressed the antitumor cytotoxic effects of NK-92 cells. Mutual inhibition of cellular activity without compromising migration ability was observed between aPSCs and NK-92 cells. Moreover, the reduced killing activity of NK-92 cells was found to be related with reduced granzyme B expression in NK cells. Conclusions: Herein, a novel TIME-on-chip model based on the coculture of PANC-1 TSs, aPSCs, and NK-92 cells was described. This model may be useful for studying the detailed mechanisms underlying NK cells dysregulation and for exploring future therapeutic interventions to restore NK cell activity in the tumor microenvironment. Citation Format: Hyun-Ah Kim, Hyunsoo Kim, Min-Kyung Nam, Jong Kook Park, Moo-Yeal Lee, Seok Chung, Kyung-Mi Lee, Hyo-Jeong Kuh. Suppression of the antitumoral activity of natural killer cells by cancer-associated fibroblasts in a pancreatic TIME-on-Chip model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C19.

Potential involvement of oncostatinM in the immunosuppressive tumor immune microenvironment in hepatocellular carcinoma with vessels encapsulating tumor clusters.

Vessels encapsulating tumor clusters (VETC) represents an adverse prognostic morphological feature of hepatocellular carcinoma (HCC), which is associated with an immunosuppressive tumor immune microenvironment (TIM). However, the underlying factors characterizing the TIM in HCC with a VETC pattern (VETC-positive HCC) remain uncertain. OncostatinM (OSM), a pleiotropic cytokine of the interleukin-6 family, regulates various biological processes, including inflammation, proliferation, and invasiveness of tumor cells. We aimed to test a hypothesis that OSM is associated with the immunosuppressive TIM of VETC-positive HCC. A total of 397 consecutive HCC patients with curative-intent hepatectomy were included. OSM-positive cells and inflammatory cells including CD4-, CD8-, CD163-, and FOXP3-positive cells were immunohistochemically evaluated. We compared VETC-positive and VETC-negative HCCs in terms of the number of these cells. We found the VETC pattern in 62 patients (15.6%). Our analysis revealed a significant decrease in the expression of arginase-1, a marker associated with mature hepatocyte differentiation, in VETC-positive HCC (p=0.046). The number of tumor-infiltrating OSM-positive cells was significantly low in VETC-positive HCC (p=0.0057). Notably, in VETC-positive HCC, the number of OSM-positive cells was not associated with vascular invasion, whereas in VETC-negative HCC, an increase in the number of OSM-positive cells was associated with vascular invasion (p=0.042). We identified an association between a decrease in OSM-positive cells and the VETC pattern. Additionally, our findings indicate that VETC-positive HCC is characterized by low hepatocyte differentiation and OSM-independent vascular invasion. These findings highlight the potential interaction between VETC-positive HCC cells and their TIM through the reduction of OSM-expressing cells.

CSIG-16. PTEN LOSS IN GLIOMA CELL LINES FACILITATES IMMUNE EVASION BY UPREGULATING EXTRACELLULAR VESICLE BIOGENESIS AND PD-L1 CARGO

Abstract Phosphatase and tensin homologue (PTEN) is deleted or mutated in over 50% of all glioblastoma (GBM) cases. Recently, PTEN loss has been implicated as a prognostic biomarker for patients likely to be non-responders treated with immune checkpoint inhibitor (ICI) therapy. Indeed, a growing body of evidence has highlighted the loss of PTEN with immuno-modulatory functions including the upregulation of the programmed death ligand-1 (PD-L1) and the development of an immunosuppressive tumor immune microenvironment (TIME), likely the result of an altered secretome. This raised the question of whether the loss of PTEN impacts the biogenesis and function of extracellular vesicles (EVs), given their functional contributions to immunosuppression and tumor growth. Here we show that glioma cell lines with no or reduced PTEN expression produce elevated numbers of EVs, as measured through nanoparticle tracking analysis. Pharmacological inhibition of PI3K reversed this observation, indicating that this phenomenon occurs in a PI3K-dependent manner. Moreover, we show through flow cytometry and immunoblot analysis that the ability for the inflammatory cytokine interferon-γ (IFN-γ) to upregulate PD-L1 at the cell surface and as EV-associated cargo is markedly increased in cells lacking PTEN. Lastly, we show that in response to IFN-γ, EVs derived from glioma cells lacking PTEN exhibit an enhanced ability to inhibit TCR signaling in a Jurkat reporter system. Taken together, these findings shed new light on how the loss of a major tumor suppressor, PTEN, can alter EV biogenesis and function and contribute to an immunosuppressive extracellular milieu. Future studies will be directed toward exploring whether loss of PTEN function alters additional EV-associated immuno-modulatory proteins relevant to other immune cell populations, toward gaining a further understanding of how EVs shape the “cold” TIME of GBM and the potential therapeutic benefit of combining PI3K inhibitors with ICIs.

Receptor-interacting Protein Kinase 2 Is an Immunotherapy Target in Pancreatic Cancer.

PDAC is resistant to almost all available therapies, including immune checkpoint blockade. Through in vivo CRISPR screen, we identified that RIPK2 plays a crucial role in facilitating immune evasion by impeding antigen presentation and cytotoxic T-cell killing. Targeting tumor-intrinsic RIPK2 either genetically or pharmacologically improves PDAC to anti-PD-1 immunotherapy. See related commentary by Liu et al., p. 208 . This article is featured in Selected Articles from This Issue, p. 201.

THU479 FOXA1 Loss Remodels The Tumor Immune Microenvironment In Late-stage Prostate Cancer

Abstract Disclosure: L.T. Brea: None. H. Shi: None. V. Keo: None. W. Xie: None. X. Lu: None. G. Gritsina: None. X. Wang: None. S.H. Park: None. J. Zhao: None. J. Yu: None. Background: Castration resistant prostate cancer (CRPC) has shown a poor response to immune checkpoint inhibitors due to its immunosuppressive nature. CRPC is infiltrated by tumor-associated macrophages (TAMs) and T regulatory cells (Tregs), which induce an immunosuppressive tumor immune microenvironment (TIME) and promote tumor progression. A deeper understanding of tumor intrinsic mechanisms shaping the TIME in prostate cancer (PCa) is needed to harness immunotherapies for PCa patients. Previous studies have found that epithelial transcription factor FOXA1 is downregulated in CRPC. In accordance, we have reported that FOXA1 loss induces tumor cell invasion and macrophage recruitment in vitro through HIF1A-CCL2 signaling. However, the extent to which FOXA1 loss regulates the TIME in PCa remains unclear, largely due to the lack of immune-proficient mouse models for the study of FOXA1 function in the context of PCa. Methods: To examine how FOXA1 loss regulates the PCa TIME, we generated a novel genetically engineered mouse model (GEMM) with prostate-specific deletion of Pten and Foxa1 (PbCre4:Ptenf/fFoxa1f/f) to compare with the established Pten-null PCa mouse model (PbCre4:Ptenf/f). We performed immunohistochemistry analyses on the GEMM prostate tumor tissues to characterize histopathology changes and assess tumor immune infiltration. We also performed spatial transcriptomics and scRNA-seq analyses of the GEMMs to comprehensively evaluate the effect of Foxa1 loss on downstream pathways and the surrounding TIME. Finally, we performed bioinformatics analysis of PCa patient datasets to validate the clinical relevance. Results: We found PbCre4:Ptenf/fFoxa1f/f mouse prostate tumors exhibited a more aggressive histopathologic tumor phenotype compared to age-matched PbCre4:Ptenf/f. Notably, immunohistochemistry staining and spatial transcriptomic analysis of the GEMMs revealed striking remodeling of the TIME upon Foxa1 loss, with marked increases in tumor infiltration by Tregs and TAMs. Moreover, CIBERSORTx analysis of patient datasets revealed FOXA1 expression negatively correlates with Treg and M2-macrophage infiltration. We also found pro-tumorigenic inflammatory cytokine signaling genes, linked to the recruitment of immunosuppressive cells, were upregulated in PbCre4:Ptenf/fFoxa1f/f prostate tumors. Furthermore, we confirmed inflammatory pathways were upregulated among FOXA1-low patient samples. Additional studies are ongoing to further elucidate the mechanisms by which FOXA1 regulates inflammatory cytokine signaling and the TIME in PCa.Conclusion: This study presents a novel GEMM for the study of FOXA1 function in an immunocompetent setting. Our data supports FOXA1 as a critical tumor intrinsic regulator of the TIME. This will have important implications on the design of immunotherapeutic approaches for late-stage PCa often with FOXA1 loss. Presentation: Thursday, June 15, 2023

Suppression of the antitumoral activity of natural killer cells under indirect coculture with cancer-associated fibroblasts in a pancreatic TIME-on-chip model

BackgroundRecently, natural killer (NK) cells emerged as a treatment option for various solid tumors. However, the immunosuppressive tumor immune microenvironment (TIME) can reduce the cytotoxic ability of NK cells in pancreatic ductal adenocarcinoma. Cancer-associated fibroblasts within the tumor stroma can suppress immune surveillance by dysregulating factors involved in the cellular activity of NK cells. Herein, the effect of activated pancreatic stellate cells (aPSCs) on NK cell-mediated anticancer efficacy under three-dimensional (3D) coculture conditions was investigated.Methods3D cocultures of PANC-1 tumor spheroids (TSs) with aPSCs and NK-92 cells in a collagen matrix were optimized to identify the occurring cellular interactions and differential cytokine profiles in conditioned media using microchannel chips. PANC-1 TSs and aPSCs were indirectly cocultured, whereas NK-92 cells were allowed to infiltrate the TS channel using convective medium flow.ResultsCoculture with aPSCs promoted PANC-1 TSs growth and suppressed the antitumor cytotoxic effects of NK-92 cells. Mutual inhibition of cellular activity without compromising migration ability was observed between aPSCs and NK-92 cells. Moreover, the reduced killing activity of NK-92 cells was found to be related with reduced granzyme B expression in NK cells.ConclusionsHerein, a novel TIME-on-chip model based on the coculture of PANC-1 TSs, aPSCs, and NK-92 cells was described. This model may be useful for studying the detailed mechanisms underlying NK cells dysregulation and for exploring future therapeutic interventions to restore NK cell activity in the tumor microenvironment.