Tumor Immune Escape Mechanisms Research Articles

IMMU-52. A NOVEL MHC-INDEPENDENT MECHANISM FOR CD8+ T CELL KILLING

Abstract The long-accepted paradigm for both cellular and antitumor immunity relies upon tumor cell kill by CD8+ T cells recognizing cognate antigens presented in the context of target cell major histocompatibility complex class I (MHC I) molecules. Likewise, a classically described mechanism of tumor immune escape is tumor MHC-I downregulation. Here, in contrast to the decades old model of T cell immunity, we instead report that CD8+ T cells maintain the capacity to kill tumor cells that are entirely devoid of MHC-I expression. This capacity proves to be dependent instead on interactions between T cell NKG2D and tumor NKG2D ligands (NKG2DL), the latter of which are highly expressed on MHC-loss variants. Necessarily, tumor cell kill in these instances is antigen-independent, although prior T cell antigen-specific activation is required and can be furnished by myeloid cells or even neighboring MHC-replete tumor cells. In this manner, adaptive priming can beget innate killing. These mechanisms are active in vivo in mice, as well as in vitro in human tumor systems, and are obviated by NKG2D knockout or blockade. These studies challenge the long-advanced notion that downregulation of MHC-I is a viable means of tumor immune escape, and instead identify the NKG2D/NKG2DL axis as a therapeutic target for enhancing T cell-dependent anti-tumor immunity against MHC loss variants.

Itaconate promotes an unexpected tumor immune escape mechanism

Itaconate is a metabolite produced by macrophages upon infection and acts as an antimicrobial molecule. In this issue of Cancer Cell, Lin etal. find that itaconate produced by tumor-associated macrophages is taken up by cancer cells via the transporter solute carrier family 13 member 3 (SLC13A3), promoting resistance to immune checkpoint inhibitors.

In vivo CRISPR screens identify Mga as an immunotherapy target in triple-negative breast cancer

Immune evasion is not only critical for tumor initiation and progression, but also determines the efficacy of immunotherapies. Through iterative in vivo CRISPR screens with seven syngeneic tumor models, we identified core and context-dependent immune evasion pathways across cancer types. This valuable high-confidence dataset is available for the further understanding of tumor intrinsic immunomodulators, which may lead to the discovery of effective anticancer therapeutic targets. With a focus on triple-negative breast cancer (TNBC), we found that Mga knock-out significantly enhances antitumor immunity and inhibits tumor growth. Transcriptomics and single-cell RNA sequencing analyses revealed that Mga influences various immune-related pathways in the tumor microenvironment. Our findings suggest that Mga may play a role in modulating the tumor immune landscape, though the precise mechanisms require further investigation. Interestingly, we observed that low MGA expression in breast cancer patients correlates with a favorable prognosis, particularly in those with active interferon-γ signaling. These observations provide insights into tumor immune escape mechanisms and suggest that further exploration of MGA's function could potentially lead to effective therapeutic strategies in TNBC.

The progress of tumor vaccines clinical trials in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) remains a significant global health challenge, with high mortality rates and limited treatment options. Tumor vaccines have emerged as a potential therapeutic approach, aiming to stimulate the immune system to specifically target tumor cells. This study screened 283 clinical trials registered on ClinicalTrials.gov through July 31, 2023. After excluding data that did not meet the inclusion criteria, a total of 108 trials were assessed. Data on registered number, study title, study status, vaccine types, study results, conditions, interventions, outcome measures, sponsor, collaborators, drug target, phases, enrollment, start date, completion date and locations were extracted and analyzed. The number of vaccines clinical trials for NSCLC has continued to increase in recent years, the majority of which were conducted in the United States. Most of the clinical trials were at stages ranging from Phase I to Phase II. Peptide-based vaccines accounted for the largest proportion. Others include tumor cell vaccines, DNA/RNA vaccines, viral vector vaccines, and DC vaccines. Several promising tumor vaccine candidates have shown encouraging results in early-phase clinical trials. However, challenges such as heterogeneity of tumor antigens and immune escape mechanisms still need to be addressed. Tumor vaccines represent a promising avenue in the treatment of NSCLC. Ongoing clinical trials are crucial for optimizing vaccine strategies and identifying the most effective combinations. Further research is needed to overcome existing limitations and translate these promising findings into clinical practice, offering new hope for NSCLC patients.

A potential mechanism of tumor immune escape: Regulation and application of soluble natural killer group 2 member D ligands (Review).

The immune system is integral to the surveillance and eradication of tumor cells. Interactions between the natural killer group 2 member D (NKG2D) receptor and its ligands (NKG2DLs) are vital for activating NKG2D receptor‑positive immune cells, such as natural killer cells. This activation enables these cells to identify and destroy tumor cells presenting with NKG2DLs, which is an essential aspect of tumor immunity. However, tumor immune escape is facilitated by soluble NKG2DL (sNKG2DL) shed from the surface of tumor cells. The production of sNKG2DL is predominantly regulated by metalloproteinases [a disintegrin and metalloproteinases (ADAM) and matrix metalloproteinase (MMP) families] and exosomes. sNKG2DL not only diminish immune recognition on the tumor cell surface but also suppress the function of immune cells, such as NK cells, and reduce the expression of the NKG2D receptor. This process promotes immune evasion, progression, and metastasis of tumors. In this review, an in‑depth summary of the mechanisms and factors that influence sNKG2DL production and their contribution to immune suppression within the tumor microenvironment are provided. Furthermore, due to the significant link between sNKG2DLs and tumor progression and metastasis, they have great potential as novel biomarkers. Detectable via liquid biopsies, sNKG2DLs could assess tumor malignancy and prognosis, and act as pivotal targets for immunotherapy. This could lead to the discovery of new drugs or the enhancement of existing treatments. Thus, the application of sNKG2DLs in clinical oncology was explored, offering substantial theoretical support for the development of innovative immunotherapeutic strategies for sNKG2DLs.

The impact of histone lactylation on the tumor microenvironment and metabolic pathways and its potential in cancer therapy.

The complexity of cancer is intricately linked to its multifaceted biological processes, including the roles of the tumor microenvironment (TME) as well as genetic and metabolic regulation. Histone lactylation has recently emerged as a novel epigenetic modification mechanism that plays a pivotal role in regulating cancer initiation, proliferation, invasion, and metastasis. This review aims to elucidate the role of histone lactylation in modulating various aspects of tumor biology, including DNA repair mechanisms, glycolytic metabolic abnormalities, functions of non-tumor cells in the TME, and the promotion of tumor inflammatory responses and immune escape. Additionally, the review explores potential therapeutic strategies targeting histone lactylation. A comprehensive literature review was performed, analyzing recent findings on histone lactylation and its impact on cancer biology. This involved a systematic examination of studies focusing on biochemical pathways, cellular interactions, and clinical implications related to histone lactylation. Histone lactylation was identified as a critical regulator of tumor cell DNA repair mechanisms and glycolytic metabolic abnormalities. It also significantly influences the functions of non-tumor cells within the TME, promoting tumor inflammatory responses and immune escape. Moreover, histone lactylation acts as a multifunctional biological signaling molecule impacting immune responses within the TME. Various cell types within the TME, including T cells and macrophages, were found to regulate tumor growth and immune escape mechanisms through lactylation. Histone lactylation offers a novel perspective on tumor metabolism and its role in cancer development. It presents promising opportunities for the development of innovative cancer therapies. This review underscores the potential of histone lactylation as a therapeutic target, paving the way for new strategies in cancer treatment.

CircARAP2 controls sMICA-induced NK cell desensitization by erasing CTCF/PRC2-induced suppression in early endosome marker RAB5A

Natural killer cells (NK) are the "professional killer" of tumors and play a crucial role in anti-tumor immunotherapy. NK cell desensitization is a key mechanism of tumor immune escape. Dysregulated NKG2D-NKG2DL signaling is a primary driver of this desensitization process. However, the factors that regulate NK cell desensitization remain largely uncharacterized. Here, we present the first report that circular RNA circARAP2 (hsa_circ_0069396) is involved in the soluble MICA (sMICA)-induced NKG2D endocytosis in the NK cell desensitization model. CircARAP2 was upregulated during NK cell desensitization and the loss of circARAP2 alleviated NKG2D endocytosis and NK cell desensitization. Using Chromatin isolation by RNA purification (ChIRP) and RNA pull-down approaches, we identified that RAB5A, a molecular marker of early endosomes, was its downstream target. Notably, transcription factor CTCF was an intermediate functional partner of circARAP2. Mechanistically, we discovered that circARAP2 interacted with CTCF and inhibited the recruitment of CTCF-Polycomb Repressive Complex 2 (PRC2) to the promoter region of RAB5A, thereby erasing histone H3K27 and H3K9 methylation suppression to enhance RAB5A transcription. These data demonstrate that inhibition of circARAP2 effectively alleviates sMICA-induced NKG2D endocytosis and NK cell desensitization, providing a novel target for therapeutic intervention in tumor immune evasion.

IRF8 deficiency-induced myeloid-derived suppressor cell promote immune evasion in lung adenocarcinoma

BackgroundPatients with lung adenocarcinoma (LUAD) have a low response rate to immune checkpoint blockade. It is highly important to explore the tumor immune escape mechanism of LUAD patients and expand the population of patients who may benefit from immunotherapy.MethodsBased on 954 bulk RNA-seq data of LUAD patients and 15 single-cell RNA-seq data, the relationships between tumor immune dysfunction and exclusion (TIDE) scores and survival prognosis in each patient were calculated and evaluated, and the immune escape mechanism affecting the independent prognosis of LUAD patients was identified. Functional enrichment analysis explored the antitumour immune response and biological behavior of tumor cells among different LUAD groups. Single-cell annotation and pseudotemporal analysis were used to explore the target molecules and immune escape mechanisms of LUAD.ResultsMyeloid-derived suppressor cells (MDSCs) and IRF8 were identified as risk and protective factors for the independent prognosis of LUAD patients, respectively. In the tumor microenvironment of patients with high infiltration of MDSCs, the antitumor immune response is significantly suppressed, while tumor cell division, proliferation, and distant metastasis are significantly enhanced. Single-cell RNA-seq analysis revealed that IRF8 is an important regulator of MDSC differentiation in LUAD myeloid cells. In addition, IRF8 may regulate the differentiation of MDSCs through the IL6-JAK-STAT3 signalling pathway.ConclusionsIRF8 deficiency impairs the normal development of LUAD myeloid cells and induces their differentiation into MDSCs, thereby accelerating the immune escape of LUAD cells. IRF8-targeted activation to inhibit the formation of MDSCs may be a new target for immunotherapy in LUAD.

Dual analysis of IL-8 in non-small cell lung cancer: A clinical perspective on protein and RNA evaluation via imaging mass cytometry.

e15068 Background: The cytokine Interleukin-8 (IL-8) plays a pivotal role in the tumorigenesis and progression of non-small cell lung cancer (NSCLC), influencing both the tumor microenvironment and immune escape mechanisms. Given its significance, accurately assessing IL-8 levels at the protein and RNA levels could provide valuable insights into its prognostic value and inform therapeutic targeting strategies. This study utilizes imaging mass cytometry (IMC) to conduct a comprehensive evaluation of IL-8 expression in NSCLC, integrating both protein presence and gene expression analysis within the spatial context of the tumor microenvironment. Methods: Our approach leverages the capabilities of IMC to simultaneously detect protein and RNA levels of IL-8 in NSCLC tissue samples. For protein analysis, antibodies tagged with heavy metal isotopes specifically target IL-8, allowing for precise quantification and spatial localization within the tumor. Concurrently, RNA detection is achieved through hybridization probes designed for IL-8 mRNA, enabling the direct observation of gene expression patterns. This method provides a unique opportunity to correlate IL-8's protein abundance with its mRNA expression, offering insights into the regulatory mechanisms at play and their impact on the tumor's behavior and patient outcomes. Results: Preliminary data demonstrate a marked heterogeneity in IL-8 distribution at both the protein and RNA levels, with distinct patterns emerging that relate to tumor aggressiveness and immune infiltration. High IL-8 expression, identified through our integrated IMC approach, correlates with poor prognosis and reduced response to standard therapies, underscoring its potential as a biomarker for NSCLC management. Furthermore, the spatial analysis afforded by IMC highlights the microenvironmental niches where IL-8-mediated interactions could drive tumor progression and immune evasion. Conclusions: By employing IMC for the dual analysis of IL-8 protein and RNA in NSCLC, our study provides a comprehensive view of this cytokine's role in lung cancer pathology. The ability to visualize and quantify IL-8 expression within the spatial context of the tumor microenvironment opens new avenues for understanding its biological impact and for developing targeted therapies. Our findings advocate for the inclusion of IL-8 in the biomarker panel for NSCLC, with potential implications for patient stratification and personalized treatment planning. Future research will focus on expanding these observations to a larger cohort and exploring the therapeutic modulation of IL-8 as a strategy for improving NSCLC outcomes.

Combined cryoablation and PD-1 inhibitor synergistically enhance antitumor immune responses in Lewis lung adenocarcinoma mice via the PI3K/AKT/mTOR pathway

Cryoablation is a therapeutic modality for lung adenocarcinoma that destroys target tumors using lethal levels of cold, resulting in the release of large amounts of specific antigens that activate immune responses. However, tumor immune checkpoint escape mechanisms prevent these released self-antigens from inducing effective anti-tumor immune responses. To overcome this challenge, we propose the use of immune checkpoint inhibitors to relieve T cell inhibition by immune checkpoints and enhance the anti-tumor immune response mediated by cryoablation. We used bilateral tumor-bearing mouse models and a specific cryoablation instrument to study the efficacy of cryoablation combined with PD-1 inhibitors in Lewis lung adenocarcinoma model mice. We found that cryoablation combined with PD-1 inhibitors significantly inhibited the growth of mouse lung adenocarcinoma, prolonged mouse survival, and enhanced the anti-tumor immune response. Moreover, this combined regimen could synergistically promote the activation and proliferation of T cells via the PI3K/AKT/mTOR pathway. The present study provides a strong theoretical basis for the clinical combination of cryoablation and PD-1 inhibitors.

The effect of gD-derived peptides on T cell immune response mediated by BTLA-HVEM protein complex in melanoma patients.

The effector function of T cells is regulated via immune checkpoints, activating or inhibiting the immune response. The BTLA-HVEM complex, the inhibitory immune checkpoint, may act as one of the tumor immune escape mechanisms. Therefore, interfering with the binding of these proteins can prove beneficial in cancer treatment. Our study focused on peptides interacting with HVEM at the same place as BTLA, thus disrupting the BTLA-HVEM interaction. These peptides' structure and amino acid sequences are based on the gD protein, the ligand of HVEM. Here, we investigated their immunomodulatory potential in melanoma patients. Flow cytometry analyses of activation, proliferation, and apoptosis of T cells from patients were performed. Additionally, we evaluated changes within the T cell memory compartment. The most promising compound - Pep(2), increased the percentages of activated T cells and promoted their proliferation. Additionally, this peptide affected the proliferation rate and apoptosis of melanoma cell line in co-culture with T cells. We conclude that the examined peptide may act as a booster for the immune system. Moreover, the adjuvant and activating properties of the gD-derived peptide could be used in a combinatory therapy with currently used ICI-based treatment. Our studies also demonstrate that even slight differences in the amino acid sequence of peptides and any changes in the position of the disulfide bond can strongly affect the immunomodulatory properties of compounds.

The role of fibrinogen-like protein 1 in immune escape and tumor growth mechanism of Warthin’s tumor

Background: One of the benign tumors of the salivary glands is Warthin’s Tumor (WT), which consists of cystic bilayer papillary epithelial cells accompanied by the presence of a lymphoid stroma. Several cases have been reported to turn malignant. One of the markers developed to identify tumor proteins is fibrinogen-like protein 1 (FGL1). Along with lymphocyte-activation gene 3 (LAG-3), FGL1 establishes an immune checkpoint pathway that plays a role in the mechanism of tumor immune escape. Not much has been reported regarding FGL1 expression in WT, but some studies have reported that its expression is associated with tumor growth. Purpose: This study aims to analyze the location of FGL1 expression in WT and its relation to the mechanism of tumor immune release through the interaction between FGL1 and LAG-3. Methods: Cases of WT (n = 11) and breast cancer (n = 1) were used as positive controls. All cases were stained with hematoxylin-eosin and Recombinant Anti-FGL1 antibody. The FGL1 expression was observed in the cell membrane, cytoplasm, and lymphoid stroma. The results are presented in the form of figures. Results: All cases of WT expressed FGL1 in the cell membrane, cytoplasm, and lymphoid stroma. Its expression in the cell membrane and cytoplasm is possibly related to the process of tumorigenesis and the increasing size of the lesion. Additionally, its expression is seen in the lymphoid stroma, which is closely related to immune escape by inhibiting lymphocytes against tumor cells. Conclusion: Warthin’s tumor cells express FGL1, and this expression plays a role in tumor immune escape mechanisms and tumor growth.

Abstract 66: A new mechanism of tumor immune escape: TCF19 promotes the development of novel terminally exhausted T cells through E2F and MYC transcription family

Abstract Introduction: ICB (immune checkpoint blocking) has been a promising therapy in cancer treatment, but there are still some patients do not response to this therapy. And it is well known that the majority cells reactivated to ICB are TCF1+ precursor exhausted T cells. Thus, studying the formation and heterogeneity of exhausted T cells are very necessary for improving the efficacy of ICB therapy. In this research, we identified a new population as TCF19+PD1+TIM3+ exhausted CD8+ T cells, which blocked the responds to ICB therapy. Methods: We collected single cell sequencing data, including colorectal cancer, gastric cancer, melanoma, and lung cancer, and drew tumor immune cells map through cluster analysis. Then using bioinformatics tools CytoTRACE. T and CCAT. A to calculate the differentiation of exhausted T cell populations, which discovered a new subset of highly proliferative and poorly differentiated terminal exhausted T cells. Subsequently, the existence of this group of cells was verified through in vitro experiments, and their formation mechanism and correlation with immunotherapy were further studied. Results: We utilized CytoTRACE.T and CCAT.A to predict differentiation states of terminally exhausted T cells. Then GEO and KEGG pathway gene set enrichment analysis were used. After analyzing, we found out that there was a subset of cells in terminally exhausted T cells defined as TCF19+PD1+TIM3+ Tex cells, which presented low differentiated and high proliferative potential.Then we performed multiple immunofluorescence staining and flow cytometry to verified the existence of TCF19+PD1+TIM3+ Tex cells in vitro. Clarifing the generation of these cell subset through regulating the expression of E2F and MYC transcription factor families.By collecting pathological sections of patients with immunotherapy information, we found that the amount of TCF19+PD1+TIM3+ Tex cells was inversely proportional to the efficacy of ICB, through immunofluorescence staining and immunohistochemical analysis as well as organoid models cocultured with T cells. Conclusion: In this research, we found a new subpopulation of terminal Tex cells, which showed high proliferation and low differentiation. For cells, TCF19 mediated the generation of stemness terminal Tex cells may through regulating the expression of E2F and MYC. Finally, we discovered that the existence of TCF19+PD1+TIM3+ Tex cells can predict the efficacy of ICB, that the more TCF19+PD1+TIM3+ Tex cells contained, the less efficacy of ICB in patients.This finding may provide a new research direction for immunotherapy. Thus, more attention should be paid to this new cell subset. Citation Format: Fuhui Wang, Nan Dong, Changhua Zhang. A new mechanism of tumor immune escape: TCF19 promotes the development of novel terminally exhausted T cells through E2F and MYC transcription family [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 66.

Abstract 4586: ITF3756 increases NKG2D expression and cytotoxic activity of activated human CD8 T cells

Abstract Effector cytolytic CD8 T cells play a major role in the recognition and killing of cancer cells. These cells are short living due to the loss of their self-renewal capacity and rapid exhaustion. Enduring antitumor immune response relies on the capacity of regenerating effector cells upon antigen re-encounter by memory cells. Focusing on molecules that could modulate the differentiation of T cells towards a memory subset with a prolonged anti-tumor activity is strategic for an efficient anti-tumor immunotherapy. Thus, we developed a selective HDAC6 inhibitor currently in Phase I clinical trial, ITF3756, which in vitro drives the differentiation of human CD8 T cells towards a central memory phenotype with reduced expression of exhaustion markers and higher expression of IFNγ and Granzyme b upon tumor cells coculture. This suggests that ITF3756-treated CD8 T cells could have a greater tumor killing activity. Moreover, if this augmented cytotoxic activity is directed not only towards tumor cells expressing MHC I but also towards MHC I negative cells, the anti-tumor efficacy could be improved. We thus selected the MHC I negative K562 leukemic cell line as target in an in vitro model of CD8 T cell killing assay. CD8 T cells were stimulated with anti CD3CD28 beads with or without ITF3756, and then co-cultivated with K562 cells for 4h. ITF3756 treatment improved significantly CD8 mediated K562 killing over this short period of time. However, since the effectiveness of memory cells is based on their capacity of maintaining a long-lasting immune response, we monitored the cytolytic activity over a period of 2 weeks. After 14 days of co-culture, ITF3756 treated CD8 T cells showed a higher cytotoxic capacity compared to control cells. These results indicate that ITF3756-treated CD8 T cells can kill a target tumor cell in a more efficient way independently of MHC I restricted tumor recognition, both after short and long term in vitro co-culture. Since it has been described that CD8 T cells can also kill MHC I-negative tumor cells through the NKG2D-NKG2DL axis, we assessed if ITF3756 could modulate the expression of NKG2D on CD8 T cells. Indeed, ITF3756-treated CD8 T cells had a significantly higher expression of NKG2D compared to untreated cells. In conclusion, we show that ITF3756 promotes the differentiation of cells that possess better effector activity when co-cultured with tumor cells for both short and long periods of time. ITF3756 increased the expression of NKG2D and this could be a driver of the increased cytolytic activity. One mechanism of tumor immune escape is the downregulation of MHC I. Our results suggest that ITF3756 could be used to improve the efficacy of T cell therapy by enhancing the cytolytic activity even in the absence of MHC I. Citation Format: Chiara Ripamonti, Valeria Spadotto, Christian Steinkuhler, Gianluca Fossati. ITF3756 increases NKG2D expression and cytotoxic activity of activated human CD8 T cells [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 4586.

Abstract B075: Spatial genomics identifies cancer cell cytokines regulating ovarian cancer immunity

Abstract Ovarian cancer (OvCa) is among the most aggressive and lethal cancers with a poor response to immune checkpoint blockade (ICB) despite its modest tumor mutational burden and expression of known cancer antigens. Consequently, it is critical to understand the molecular mechanisms of tumor immune escape and resistance to immunotherapies. Intercellular signaling between cancer cells and the tumor microenvironment (TME) is a crucial determinant of tumor immunity and given the immunosuppressive nature of OvCa TME, it is of utmost importance to dissect how this immunosuppressed TME is established. To begin to determine which cancer-cell genes may function cell externally in controlling of OvCa immunity, we employed a first-of-its-kind spatial functional genomics approach we developed, called Perturb-map, which enables dozens of CRISPR gene knock-out (KO) cells to be resolved in a tissue/tumor by imaging, along with cells in the local TME. We used Perturb-map to determine how 35 different genes, identified through ligand-receptor analysis of ovarian tumors, influenced tumor response to anti-PD-1 treatment in an OvCa mouse model. Strikingly, Il4 KO tumors were unique amongst the 35 gene KOs making the tumors significantly more responsive to anti-PD-1 treatment despite not impacting tumor growth in the absence of immunotherapy. Further experiments with single CRISPR KO tumors validated the Perturb-map finding, where Il4 KO combined with anti-PD-1 treatment resulted in a major reduction in tumor growth and a significant increase in survival. Cyclic immunofluorescence (CyCIF) multiplex imaging revealed that loss of cancer cell-derived IL-4 led to a significant reduction in pro-tumor macrophages and an increase in dendritic cell (DC) subsets as well as both progenitor and terminally exhausted CD8 T cells. Upon anti-PD-1 treatment, Il4 KO tumors had significant enrichment for activated CD8 T cells and B cells. Importantly, spatial analysis of the tumor samples identified enrichment of T follicular helper cells, B cells, and DCs in tertiary lymphoid structures in tumors with combined IL-4 loss and PD-1 inhibition. Further, we analyzed a human OvCa single-cell transcriptomics dataset and demonstrated that an IL-4 response signature is expressed in tumor-associated macrophages, indicating active IL-4 signaling in human ovarian tumors. Importantly, we employed a CRISPR-knock-in expression reporter in mouse tumors as well as immunohistochemistry of both human and mouse tumor samples to confirm that IL-4 is expressed by cancer cells. Thus, we identified cancer cell-secreted IL-4 as a potent regulator of OvCa TME and response to ICB. Given that an anti-IL-4-receptor antibody is already in clinical use to treat immune diseases, our findings have the potential to be translated to the clinical management of this dreadful disease. Citation Format: Gurkan Mollaoglu, Alexander Tepper, Hunter Potak, Luisanna Pia, Angelo Amabile, Chiara Falcomata, Jaime Mateus-Tique, Noam Rabinovich, Rachel Brody, Lindsay Browning, Jia-Ren Lin, Peter Sorger, Sandro Santagata, Miriam Merad, Alessia Baccarini, Brian Brown. Spatial genomics identifies cancer cell cytokines regulating ovarian cancer immunity [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B075.

Hsa_circ_0136666 stimulates gastric cancer progression and tumor immune escape by regulating the miR-375/PRKDC Axis and PD-L1 phosphorylation

BackgroundTargeted drugs are not quite effective for prolonging the survival of patients with gastric cancer due to off-target effects as well as tumor immune escape mechanisms. Circular RNAs widely exist in tumor regions as biomarkers and can be developed as effective drug targets.MethodsWestern blot, QRT-PCR, fluorescence in situ hybridization, and flow cytometry were used to investigate the function of hsa_circ_0136666 in promoting the proliferation of gastric cancer cells. Tissue immunofluorescence, enzyme-linked immunosorbent assay (ELISA), as well as flow cytometric analysis, was conducted to explore the process of tumor immune evasion in tumor-bearing mice. The differences of circRNA expression in clinical samples were analyzed through tissue microarray FISH. The effect of siRNA on improving the efficacy of anti-PDL1 drugs and suppressing the immune microenvironment was evaluated by the coadministration model.ResultsWe demonstrated that hsa_circ_0136666 was widely and highly expressed in gastric cancer tissues and cells. Functionally, hsa_circ_0136666 promoted gastric cancer tumor proliferation and tumor microenvironment formation, leading to tumorigenesis immune escape, and this effect was dependent on CD8 + T cells. Mechanistically, we confirmed that hsa_circ_0136666 competitively upregulated PRKDC expression by sponging miR-375-3p, regulating immune checkpoint proteins, prompting phosphorylation of PD-L1 to preventing its degradation, driving PD-L1 aggregation and suppressing immune function, thereby impairing cancer immune responses. In terms of application, we found that LNP-siRNA effectively improved anti-PDL1 drug efficacy and inhibited immune escape.ConclusionOur results reveal an oncogenic role played by hsa_circ_0136666 in gastric cancer, driving PD-L1 phosphorylation via the miR-375/PRKDC signaling axis, prompting immune escape. This work proposes a completely new pathogenic mechanism of gastric cancer, uncovers a novel role for hsa_circ_0136666 as an immune target, and provides a rationale for enhancing the efficacy of anti-PD-L1 therapy for gastric cancer.

Advanced Generation Therapeutics: Biomimetic Nanodelivery System for Tumor Immunotherapy.

Tumor immunotherapy is a safe and effective strategy for precision medicine. However, immunotherapy for most cancer cases still ends in failure, with the root causes of the immunosuppressive and extraordinary heterogeneity of the solid tumors microenvironment. The emerging biomimetic nanodelivery system provides a promising tactic to improve the immunotherapy effect while reducing the adverse reactions on nontarget cells. Herein, we summarize the relationship between tumor occurrence and tumor immune microenvironment, mechanism of tumor immune escape, immunotherapy classification (including adoptive cellular therapy, cytokines, cancer vaccines, and immune checkpoint inhibitors) and recommend target cells for immunotherapy first, and then emphatically introduce the recent advances and applications of the latest biomimetic nanodelivery systems (e.g., immune cells, erythrocytes, tumor cells, platelets, bacteria) in tumor immunotherapy. Meanwhile, we separately summarize the application of tumor vaccines. Finally, the predictable challenges and perspectives in a forward exploration of biomimetic nanodelivery systems for tumor immunotherapy are also discussed.

Abstract B014: Spatial genomics identifies cancer cell cytokines regulating ovarian cancer immunity

Abstract Ovarian cancer (OvCa) is among the most aggressive and lethal cancers with a poor response to immune checkpoint blockade (ICB) despite its modest tumor mutational burden and expression of known cancer antigens. Consequently, it is critical to understand the molecular mechanisms of tumor immune escape and resistance to immunotherapies. Intercellular signaling between cancer cells and the tumor microenvironment (TME) is a crucial determinant of tumor immunity and given the immunosuppressive nature of OvCa TME, it is of utmost importance to dissect how this immunosuppressed TME is established. To begin to determine which cancer-cell genes may function cell externally in controlling of OvCa immunity, we employed a first-of-its-kind spatial functional genomics approach we developed, called Perturb-map, which enables dozens of CRISPR gene knock-out (KO) cells to be resolved in a tissue/tumor by imaging, along with cells in the local TME. We used Perturb-map to determine how 35 different genes, identified through ligand-receptor analysis of ovarian tumors, influenced tumor response to anti-PD-1 treatment in an OvCa mouse model. Strikingly, Il4 KO tumors were unique amongst the 35 gene KOs making the tumors significantly more responsive to anti-PD-1 treatment despite not impacting tumor growth in the absence of immunotherapy. Further experiments with single CRISPR KO tumors validated the Perturb-map finding, where Il4 KO combined with anti-PD-1 treatment resulted in a major reduction in tumor growth and a significant increase in survival. Cyclic immunofluorescence (CyCIF) multiplex imaging revealed that loss of cancer cell-derived IL-4 led to a significant reduction in pro-tumor macrophages and an increase in dendritic cell (DC) subsets as well as both progenitor and terminally exhausted CD8 T cells. Upon anti-PD-1 treatment, Il4 KO tumors had significant enrichment for activated CD8 T cells and B cells. Importantly, spatial analysis of the tumor samples identified enrichment of T follicular helper cells, B cells, and DCs in tertiary lymphoid structures in tumors with combined IL-4 loss and PD-1 inhibition. Further, we analyzed a human OvCa single-cell transcriptomics dataset and demonstrated that an IL-4 response signature is expressed in tumor-associated macrophages, indicating active IL-4 signaling in human ovarian tumors. Importantly, we employed a CRISPR-knock-in expression reporter in mouse tumors as well as immunohistochemistry of both human and mouse tumor samples to confirm that IL-4 is expressed by cancer cells. Thus, we identified cancer cell-secreted IL-4 as a potent regulator of OvCa TME and response to ICB. Given that an anti-IL-4-receptor antibody is already in clinical use to treat immune diseases, our findings have the potential to be translated to the clinical management of this dreadful disease. Citation Format: Gurkan Mollaoglu, Alexander Tepper, Hunter Potak, Luisanna Pia, Angelo Amabile, Chiara Falcomata, Jaime Mateus-Tique, Noam Rabinovich, Rachel Brody, Lindsay Browning, Jia-Ren Lin, Peter Sorger, Sandro Santagata, Miriam Merad, Alessia Baccarini, Brian Brown. Spatial genomics identifies cancer cell cytokines regulating ovarian cancer immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B014.

Regulated secretion of mutant p53 negatively affects T lymphocytes in the tumor microenvironment

Several studies have demonstrated the role of the oncogenic mutant p53 in promoting tumor progression; however, there is limited information on the effects of secreted oncogenic mutant p53 on the tumor microenvironment and tumor immune escape. In this study, we found that secretion of mutant p53, determined by exosome content, is dependent on its N-terminal dileucine motif via its binding to β-adaptin, and inhibited by the CHK2-mediated-Ser 20 phosphorylation. Moreover, we observed that the mutant p53 caused downregulation and dysfunction of CD4+ T lymphocytes in vivo and downregulated the levels and activities of rate-limiting glycolytic enzymes in vitro. Furthermore, inhibition of mutant p53 secretion by knocking down AP1B1 or mutation of dileucine motif could reverse the quantity and function of CD4+ T lymphocytes and restrain the tumor growth. Our study demonstrates that the tumor-derived exosome-mediated secretion of oncogenic mutant p53 inhibits glycolysis to alter the immune microenvironment via functional suppression of CD4+ T cells, which may be the underlying mechanism for tumor immune escape. Therefore, targeting TDE-mediated p53 secretion may serve as a potential therapeutic target for cancer treatment.

Identification of macrophage polarization phenotypes in agarose matrix using metasurface-based terahertz biosensor of quasi-bound state continuum

Rapid and noninvasive detection of macrophage polarization can facilitate the acquisition of crucial information for pre- and post-evaluation in cancer immunotherapy, unraveling tumor immune escape mechanisms and discovering drugs. Herein, we demonstrate an approach to identify macrophage polarization using the metasurface-based terahertz biosensor of quasi-bound state in the continuum. Non-polarized macrophages (M0) are driven into a pro-inflammatory phenotype (M1) or an anti-inflammatory phenotype (M2) in response to lipopolysaccharide or interleukin-4 stimulation. It is found that the M1 macrophages result in a slight resonance frequency blueshift, while the M2 results in an obvious resonance frequency blueshift as well as a broader resonance linewidth. The permittivity change reveals the aforementioned frequency change. Flow cytometry and gene expression measurements reveal distinct differences of phenotypes between M1 and M2 polarizations, consistent with the results of terahertz biosensing. Our results manifest a cost-effective approach for detecting macrophage polarization status and their functional activations, which holds significant implications for clinical and cellular immunology research.