Mechanisms Of Immunotherapy Resistance Research Articles

PD-L1 blockade immunotherapy rewires cancer-induced emergency myelopoiesis.

Immune checkpoint blockade (ICB) immunotherapy has revolutionized cancer treatment, demonstrating exceptional clinical responses in a wide range of cancers. Despite the success, a significant proportion of patients still fail to respond, highlighting the existence of unappreciated mechanisms of immunotherapy resistance. Delineating such mechanisms is paramount to minimize immunotherapy failures and optimize the clinical benefit. In this study, we treated tumour-bearing mice with PD-L1 blockage antibody (aPD-L1) immunotherapy, to investigate its effects on cancer-induced emergency myelopoiesis, focusing on bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs). We examined the impact of aPD-L1 treatment on HSPC quiescence, proliferation, transcriptomic profile, and functionality. Herein, we reveal that aPD-L1 in tumour-bearing mice targets the HSPCs in the BM, mediating their exit from quiescence and promoting their proliferation. Notably, disruption of the PDL1/PD1 axis induces transcriptomic reprogramming in HSPCs, observed in both individuals with Hodgkin lymphoma (HL) and tumour-bearing mice, shifting towards an inflammatory state. Furthermore, HSPCs from aPDL1-treated mice demonstrated resistance to cancer-induced emergency myelopoiesis, evidenced by a lower generation of MDSCs compared to control-treated mice. Our findings shed light on unrecognized mechanisms of action of ICB immunotherapy in cancer, which involves targeting of BM-driven HSPCs and reprogramming of cancer-induced emergency myelopoiesis.

P2.11B.01 Deciphering Immunotherapy Resistance Mechanisms in Metastatic NSCLC, Insights from Plasma Proteomics Analysis

P2.11B.01 Deciphering Immunotherapy Resistance Mechanisms in Metastatic NSCLC, Insights from Plasma Proteomics Analysis

1986P Prospective evaluation of BCG unresponsive bladder cancer carcinoma in situ identifies genetic mechanisms of immunotherapy resistance and targeted therapy using an ultra-sensitive next generation sequencing minimal residual disease (MRD) assay

1986P Prospective evaluation of BCG unresponsive bladder cancer carcinoma in situ identifies genetic mechanisms of immunotherapy resistance and targeted therapy using an ultra-sensitive next generation sequencing minimal residual disease (MRD) assay

Assessing personalized responses to anti-PD-1 treatment using patient-derived lung tumor-on-chip

There is a compelling need for approaches to predict the efficacy of immunotherapy drugs. Tumor-on-chip technology exploits microfluidics to generate 3D cell co-cultures embedded in hydrogels that recapitulate simplified tumor ecosystems. Here, we present the development and validation of lung tumor-on-chip platforms to quickly and precisely measure exvivo the effects of immune checkpoint inhibitors on Tcell-mediated cancer cell death by exploiting the power of live imaging and advanced image analysis algorithms. The integration of autologous immunosuppressive FAP+ cancer-associated fibroblasts impaired the response to anti-PD-1, indicating that tumors-on-chips are capable of recapitulating stroma-dependent mechanisms of immunotherapy resistance. For a small cohort of non-small cell lung cancer patients, we generated personalized tumors-on-chips with their autologous primary cells isolated from fresh tumor samples, and we measured the responses to anti-PD-1 treatment. These results support the power of tumor-on-chip technology in immuno-oncology research and open a path to future clinical validations.

Current Landscape of Cancer Immunotherapy: Harnessing the Immune Arsenal to Overcome Immune Evasion.

Cancer immune evasion represents a leading hallmark of cancer, posing a significant obstacle to the development of successful anticancer therapies. However, the landscape of cancer treatment has significantly evolved, transitioning into the era of immunotherapy from conventional methods such as surgical resection, radiotherapy, chemotherapy, and targeted drug therapy. Immunotherapy has emerged as a pivotal component in cancer treatment, harnessing the body's immune system to combat cancer and offering improved prognostic outcomes for numerous patients. The remarkable success of immunotherapy has spurred significant efforts to enhance the clinical efficacy of existing agents and strategies. Several immunotherapeutic approaches have received approval for targeted cancer treatments, while others are currently in preclinical and clinical trials. This review explores recent progress in unraveling the mechanisms of cancer immune evasion and evaluates the clinical effectiveness of diverse immunotherapy strategies, including cancer vaccines, adoptive cell therapy, and antibody-based treatments. It encompasses both established treatments and those currently under investigation, providing a comprehensive overview of efforts to combat cancer through immunological approaches. Additionally, the article emphasizes the current developments, limitations, and challenges in cancer immunotherapy. Furthermore, by integrating analyses of cancer immunotherapy resistance mechanisms and exploring combination strategies and personalized approaches, it offers valuable insights crucial for the development of novel anticancer immunotherapeutic strategies.

Abstract 2634: CRISPR in vivo screen utilizing removable Cas9 system to identify epigenetic immune response modulators in antigen-sensitive mouse syngeneic models

Abstract The application of CRISPR-Cas9 screen in tumor immunology research has promoted the discovery of potential therapeutic targets and provided a deeper understanding of the mechanisms of immunotherapy resistance. Mouse syngeneic tumor model based CRISPR in vivo screen revealed significant features of immunotherapy by better approaching the tumor immune interaction. However, the immunogenicity of CRISPR-Cas9 has been widely observed in a variety of syngeneic models, which greatly limited for the application of CRISPR in vivo screen. Herein, we used two strategies for cancer cell gene editing, Cas9 protein electroporation and loxP-flanked Cas9 with Cre recombinase, to reduce the immunogenicity of CRISPR-Cas9 in CT26 subcutaneous and Renca orthotropic syngeneic models. Encouragingly, we found out that compared with constitutive Cas9-expressing All-In-One based cell pool models, both CT26 subcutaneous model and Renca orthotropic model showed a parental cell tumor comparable growth kinetics and immune checkpoint blockade ICB response by PD-1 antibody treatment. Further immuno-profiling of tumor immune microenvironment indicated limited changes from the parental cell model, which helped the data interpretation of in vivo screen outcome. We then performed a CRISPR in vivo screen by using a focused library against mouse epigenetic regulators in these two models and will discuss the hits from the screen in future. Citation Format: Yuan He, Yafei Liu, Xiao Wang, Xianyi Li, Wenle Dong, Tongrui Hao, Chenyang Zhu, Wenrong Zhou, Zhengang Peng. CRISPR in vivo screen utilizing removable Cas9 system to identify epigenetic immune response modulators in antigen-sensitive mouse syngeneic models [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 2634.

Abstract 6857: Evolution of myeloid-mediated mechanisms of immunotherapy resistance at single-cell resolution with prostate cancer progression

Abstract Background: Patients with metastatic castration-resistant prostate cancer (mCRPC) are largely refractory to immune checkpoint inhibitors (ICIs). This resistance is, in part, attributed to the presence of immunosuppressive myeloid cells in tumors. However, attempts to target these cells broadly, such as CSF1R antagonism, have thus far failed clinically primarily due to their heterogeneous nature. Thus, we hypothesized that a more comprehensive understanding of immunosuppressive myeloid subsets and their molecular mechanisms at the single-cell level is critical to enhance the effectiveness of immunotherapy. Methods: We performed single-cell profiling of patient biopsies with either localized, metastatic hormone-sensitive prostate cancer, or mCRPC. We then reverse translated these findings in a syngeneic mouse model of CRPC, where we performed multi-omic single-cell assessment and conducted studies to assess tumor efficacy and gain mechanistic insights. Results: We identified a specific subset of tumor-associated macrophages with elevated hypoxia signatures, including higher SPP1 transcript levels (SPP1hi-TAMs). These macrophages become significantly more abundant as the disease advances, expressing elevated immunosuppressive molecular programs. Notably, CSF1R transcripts were significantly reduced in SPP1hi-TAMs, suggesting their potential role in the clinical ineffectiveness of CSF1R blockade. In a syngeneic mouse CRPC model, we identified an analogous macrophage subset through single-cell profiling and flow cytometry. These macrophages possess the ability to suppress T cell activity in co-culture. Consistent with these findings, adoptive transfer of Spp1hi-TAMs into CRPC promoted ICI resistance and worsened survival in vivo. Importantly, these macrophages proved resistant to anti-CSF1R treatment, underscoring the need to target their immunosuppressive signals as a potential therapeutic approach. Pathway analysis revealed enrichment of gene signatures associated with adenosine signaling in SPP1hi-TAMs both in patients and in mice, implicating this pathway as their immunosuppressive mechanism. Indeed, blocking adenosine signaling using antibodies or pharmacologic inhibitors resulted in a significant reduction in T cell suppression mediated by Spp1hi-TAMs in culture. Furthermore, pharmacologic blockade of adenosine receptors in vivo significantly decreased tumor growth and enhanced the responsiveness of tumor cells to ICI therapies. Conclusions: Our studies demonstrate that SPP1hi-TAMs become prevalent as the disease progresses. They are resistant to CSF1R blockade and play a key role in mediating immunotherapeutic resistance in vivo by activating adenosine signaling pathways. These findings highlight the potential of the associated signals as promising targets for therapeutic intervention. Citation Format: Aram Lyu, Zenghua Fan, Diamond Luong, Ali Setayesh, Alec Starzinski, Laura Valderrábano, Eliezer Van Allen, Lawrence Fong. Evolution of myeloid-mediated mechanisms of immunotherapy resistance at single-cell resolution with prostate cancer progression [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 6857.

Abstract 2065: Development of a genetically validated, cell-based reporter assay for ADAR1 editing activity

Abstract Adenosine Deaminase Acting on RNA (ADAR) enzymes perform adenosine to inosine base editing in RNA, particularly targeting adenosines located within a specific double-stranded stem-loop motif. The ADAR1 gene encodes both a universally expressed isoform (p110) and an interferon-inducible isoform (p150), which plays a role in innate immunity by mediating interferon signaling. In the context of healthy, uninfected cells, ADAR1 performs A-to-I editing on endogenous double-stranded RNA to prevent it from activating downstream dsRNA sensors RIG-I and MDA5, which in-turn activate a pro-inflammatory response. Loss of function mutations in ADAR1 result in aberrant activation of the dsRNA sensors and are involved in autoimmune disorders. ADAR1 dysfunction also impacts cancer cell growth, proliferation, and response to immunotherapy. ADAR1 expression is increased in several types of cancer and ADAR1 knock-out has been demonstrated to improve the response to certain immunotherapies such PD-1/PD-L1 blockade and to circumvent tumor immunotherapy resistance mechanisms, making ADAR1 an attractive target for therapeutic development. We have developed and validated a high-throughput, cell-based assay for measuring ADAR1 editing activity. Several ADAR editing reporter constructs were designed and evaluated for their relative response to ADAR1 over-expression. They all feature an ADAR1 hairpin target with a stop codon (UAG) which is susceptible to ADAR1-mediated editing to a tryptophan (UUG), located upstream of a firefly luciferase gene. We show that increasing transient expression of ADAR1 led to proportional increased activity of the downstream firefly luciferase, indicating dose-dependent transcript editing by ADAR1. Selected reporters were used in conjunction with ADAR2 overexpression to identify an ADAR1-biased reporter. Thus, a reporter with low luciferase activity in response to ADAR2 expression was chosen to establish stable HEK293 cell lines (which express low levels of ADAR1) with and without constitutively expressed ADAR1 protein. ADAR1-overexpressing HEK293 cells displayed high luciferase activity, in accordance with high ADAR1-mediated editing of the reporter transcript, which was decreased in response to siRNA-mediated knockdown of ADAR1 expression or pharmacological inhibition of ADAR1 activity. Sequencing of the reporter RNA confirmed that changes in reporter activity were correlated with editing of the RNA transcript. Finally, these validated ADAR1 editing reporter cell lines were further engineered to constitutively express Renilla luciferase under the control of a CMV promoter, serving as an internal control for the toxicity of ADAR1 inhibitors. In conclusion, our robust cell-based assay provides a well-validated resource to identify modulators of ADAR1 editing activity in a cellular context. Citation Format: Valerie Sapp, Jim Bilakovics, Ava Burr, Fernando Martins, Pavel Shashkin, Veronique Baron, Henry Zhu, Junguk Park. Development of a genetically validated, cell-based reporter assay for ADAR1 editing activity [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 2065.

Drug resistance mechanism and reversal strategy in lung cancer immunotherapy.

Among all malignant tumors, lung cancer has the highest mortality and morbidity rates. The non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) are the most common histological subtypes. Although there are a number of internationally recognized lung cancer therapy regimens, their therapeutic effects remain inadequate. The outlook for individuals with lung carcinoma has ameliorated partly thanks to the intensive study of the tumor microenvironment and immune checkpoint inhibitors. Numerous cancers have been effectively treated with immunotherapy, which has had positive therapeutic results. Global clinical trials have validated that PD-1/PD-L1 inhibitors are effective and safe for treating lung cancer either independently or in combination, and they are gradually being recommended as systemic treatment medications by numerous guidelines. However, the immunotherapy resistance restricts the immunotherapy efficacy due to the formation of tumor immunosuppressive microenvironment and tumor mutations, and immunotherapy is only effective for a small percentage of lung cancer patients. To summarize, while tumor immunotherapy is benefiting an increasing number of lung cancer patients, most of them still develop natural or acquired resistance during immunotherapy. Consequently, a crucial and urgent topic is understanding and tackling drug resistance triggered by immunotherapy in lung cancer treatment. This review will outline the presently recognized mechanisms of immunotherapy resistance and reversal strategies in lung cancer.

Machine learning-based integration develops a metabolism-derived consensus model for improving immunotherapy in pancreatic cancer

BackgroundPancreatic cancer (PAC) is one of the most malignant cancer types and immunotherapy has emerged as a promising treatment option. PAC cells undergo metabolic reprogramming, which is thought to modulate...

Multi-omics Analysis Identifies Hypoxia Subtypes and S100A2 as an Immunosuppressive Factor in Cervical Cancer.

Cervical cancer is a common gynecological oncology. Growing evidence indicates hypoxia plays an important role in tumor progression and immunity. However, no study has examined the hypoxia landscape in cervical cancer. In this study, using hierarchical clustering, we identified three hypoxia subtypes in cervical cancer samples from The Cancer Genome Atlas dataset according to formerly described hypoxia-related genes. The overall survival time, hypoxic features, genomics, and immunological characteristics of these subtypes existed distinct differences. We also created a hypoxia score by principle component analysis for dimension reduction. The hypoxiaScore was an effective prognostic biomarker validated by GSE44001 and was associated with immunotherapy response. Furthermore, combined with single-cell RNA-sequence (scRNA-seq) and experiments, S100A2 was identified as an immunosuppressive factor induced by hypoxia and regulated expression of PD-L1. S100A2 also served as an oncogene promoting the proliferation and migration of cervical cancer cells. These findings depicted a new hypoxia-based classification and identified S100A2 as a potential therapeutic target for cervical cancer, thereby advancing the understanding of immunotherapy resistance mechanisms and cervical cancer genetic markers.

Molecular mechanisms of immunotherapy resistance in triple-negative breast cancer.

The emergence of immunotherapy has profoundly changed the treatment model for triple-negative breast cancer (TNBC). But the heterogeneity of this disease resulted in significant differences in immunotherapy efficacy, and only some patients are able to benefit from this therapeutic modality. With the recent explosion in studies on the mechanism of cancer immunotherapy drug resistance, this article will focus on the processes of the immune response; summarize the immune evasion mechanisms in TNBC into three categories: loss of tumor-specific antigen, antigen presentation deficiency, and failure to initiate an immune response; together with the aberrant activation of a series of immune-critical signaling pathways, we will discuss how these activities jointly shape the immunosuppressive landscape within the tumor microenvironment. This review will attempt to elucidate the molecular mechanism of drug resistance in TNBC, identify potential targets that may assist in reversing drug resistance, and lay a foundation for research on identifying biomarkers for predicting immune efficacy and selection of breast cancer populations that may benefit from immunotherapy.

Relationship between tumor microenvironment (TME)-based histomic TGFβ signature (TGFBs), stromal fibroblast recruitment, and exclusion of immune cells as immunotherapy resistance mechanisms.

2585 Background: TGF-beta activates fibroblasts within the TME and may be associated with poor response to immunotherapy.However, objective and reliable assessment of the TGFBs by gene expression profiling (GEP) is challenging. Using H&E whole-slide images (WSI) from The Cancer Genome Atlas (TCGA), we developed an artificial intelligence (AI) model to predict a normalized TGFBs, applying it to real-world datasets (RWDs) of advanced solid tumor patients treated with immunotherapy. Methods: The TGFBs was defined using the mean GEP values of the 'Hallmark TGF-beta signaling' gene set. H&E WSIs of 23 carcinoma types from TCGA (n=6,945) were used to discover the association of the TGFBs with various cell types, including fibroblasts and tumor-infiltrating lymphocytes (TILs) detected by an AI model, Lunit SCOPE IO (AI-1). Another custom AI model (AI-2) to predict TGFBs was developed by integrating self-supervised and supervised features and other semantic content extracted by AI-1 with multilayered perceptron classifiers. TGFBs prediction scores from the AI-2 were applied to 5 different RWDs containing 1,792 immunotherapy-treated patients (> 16 primary tumor types). The immune-excluded phenotype (IEP) was defined by high stromal TIL and low intratumoral TIL density. Results: In the TCGA dataset, TGFBs and fibroblast density detected by AI-1 were closely correlated (R=0.19, p<0.001) compared to other cell types, with both highest in pancreatic adenocarcinoma (PAC)(median [interquartile range, IQR] 0.73 [0.66-0.80], 1306 [985-1588]/mm2, respectively) and CMS4 colorectal cancer (0.61 [0.54-0.67], 868 [582-1128]/mm2) compared to other cancers (0.57 [0.47-0.66], 577 [295-890]/mm2). The AI-2 predicted TGFBs with area-under-the-curve of 0.781 on cross-validation within the TCGA dataset. The TGFBs of immunotherapy-treated RWDs were inferred from their corresponding H&E WSI, with scores comparable to those from the TCGA (0.42 [0.16-0.60] vs 0.58 [0.48-0.67]), with PAC having the highest TGFBs (0.65 [0.45-0.77]) in the RWD as well. Interestingly, TGFBs inferred scores and fibroblast density were significantly correlated (R=0.7, p<0.001) in the RWD and also significantly correlated with the IEP (0.52 [0.30-0.66]) compared to other immune phenotypes (0.42 [0.16-0.60], p<0.001). In the RWD, patients with inferred TGFBs in the upper 75% quartile had a poor clinical response to immunotherapy (objective response rate: 15.1% vs 20.7%, median progression-free survival 3.0 m vs 4.0 m, hazard ratio 1.21, 95% confidence interval 1.07-1.36, p=0.002). Conclusions: TGFBs is closely correlated with fibroblast density within the TME and may confer resistance to immunotherapy. Unique morphologic features of the TGFBs can be captured by AI, enabling scalable application to various datasets for clinical and translational research.

Methods for assessment of the tumour microenvironment and immune interactions in non-small cell lung cancer. A narrative review.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer death worldwide. Immunotherapy with immune checkpoint inhibitors (ICI) has significantly improved outcomes in some patients, however 80-85% of patients receiving immunotherapy develop primary resistance, manifesting as a lack of response to therapy. Of those that do have an initial response, disease progression may occur due to acquired resistance. The make-up of the tumour microenvironment (TME) and the interaction between tumour infiltrating immune cells and cancer cells can have a large impact on the response to immunotherapy. Robust assessment of the TME with accurate and reproducible methods is vital to understanding mechanisms of immunotherapy resistance. In this paper we will review the evidence of several methodologies to assess the TME, including multiplex immunohistochemistry, imaging mass cytometry, flow cytometry, mass cytometry and RNA sequencing.

Immunotherapy resistance in non-small-cell lung cancer: From mechanism to clinical strategies.

The high primary resistance incidence and unavoidable secondary resistance are the major clinical obstacle to lasting long-term benefits in Non-small-cell lung cancer (NSCLC) patients treated with immunotherapy. The mechanisms of immunotherapy resistance in NSCLC are complex, mainly involving tumor cells and tumor microenvironment (TME) infiltrating immune cells, including TAMs, B cells, NK cells, and T cells. The selection of clinical strategies for NSCLC progression after immunotherapy resistance should depend on the progressive mode. The progression pattern of NSCLC patients after immunotherapy resistance can be divided into oligo-progression and systemic/multiple progression, which should be considered for further treatment selection. In the future, it needs to explore how to optimize the combined therapy and explore strategies to reprogram infiltrating immune cells under various genetic backgrounds of tumor cells and timely reshape TME during antitumor treatments.

A metabolomic and proteomic study to elucidate the molecular mechanisms of immunotherapy resistance in patients with oesophageal squamous cell carcinoma.

Systemic chemotherapy, the standard first-line treatment option for patients with advanced oesophageal squamous cell carcinoma (OSCC), results in a median survival of ~1 year. Immune checkpoint inhibitors are a breakthrough oncology treatment option; however, most patients with advanced OSCC develop primary and acquired resistance to programmed death receptor-1 (PD-1) monoclonal antibody, severely affecting their prognosis. Therefore, there is an urgent need to investigate the molecular mechanism underlying resistance to treatment. The present study aimed to explore the mechanism of resistance to PD-1 monoclonal antibody. Plasma samples were collected from patients with OSCC treated with immunotherapy, who achieved pathological response/partial response (CR/PR) or stable disease/progressive disease (SD/PD) after the fourth treatment cycle. TM-widely targeted metabolomics, widely targeted lipidomics, and DIA proteomics assays were performed. Differential metabolites were screened based on fold change (FC) ≥1.5 or ≤0.67 and a VIP ≥1; differential proteins were screened based on FC >1.5 or <0.67 and P<0.05. The identified metabolites were annotated and mapped using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases. The differential proteins were annotated to the Gene Ontology and KEGG pathway databases. A correlation network diagram was drawn using differential expressed proteins and metabolites with (Pearson correlation coefficient) r>0.80 and P<0.05. Finally, 197 and 113 differential metabolites and proteins were screened, respectively, in patients with CR/PR and SD/PD groups. The KEGG enrichment analysis revealed that all of these metabolites and proteins were enriched in cholesterol metabolism and in the NF-κB and phospholipase D signalling pathways. The present study is the first to demonstrate that PD-1 inhibitor resistance may be attributed to cholesterol metabolism or NF-κB and phospholipase D signalling pathway activation. This finding suggests that targeting these signalling pathways may be a promising novel therapeutic approach in OSCC which may improve prognosis in patients undergoing immunotherapy.

Tumor microenvironment-associated lactate metabolism regulates the prognosis and precise checkpoint immunotherapy outcomes of patients with lung adenocarcinoma

BackgroundDespite the wide clinical application of checkpoint inhibitor immunotherapy in lung adenocarcinoma, its limited benefit to patients remains puzzling to researchers. One of the mechanisms of immunotherapy resistance may be the dysregulation of lactate metabolism in the immunosuppressive tumor microenvironment (TME), which can inhibit dendritic cell maturation and prevent T-cell invasion into tumors. However, the key genes related to lactate metabolism and their influence on the immunotherapeutic effects in lung adenocarcinoma have not yet been investigated in depth.MethodsIn this study, we first surveyed the dysregulated expression of genes related to lactate metabolism in lung adenocarcinoma and then characterized their biological functions. Using machine learning methods, we constructed a lactate-associated gene signature in The Cancer Genome Atlas cohort and validated its effectiveness in predicting the prognosis and immunotherapy outcomes of patients in the Gene Expression Omnibus cohorts.ResultsA 7-gene signature based on the metabolomics related to lactate metabolism was found to be associated with multiple important clinical features of cancer and was an independent prognostic factor.ConclusionsThese results suggest that rather than being simply a metabolic byproduct of glycolysis, lactate in the TME can affect immunotherapy outcomes. Therefore, the mechanism underlying this effect of lactate is worthy of further study.

Abstract C012: Dissecting the reorganization of pancreatic tumor microenvironments after radiation and immunotherapy reveals insights into immunotherapy resistance

Abstract Immune checkpoint blockade (ICB) has revolutionized the treatment of many cancers but has been ineffective for the treatment of microsatellite stable (MSS) PDAC. The lack of efficacy of immunotherapies in PDAC is due to: 1) a desmoplastic tumor microenvironment (TME); 2) the presence of suppressive cells, including myeloid derived suppressor cells and regulatory T cells; and 3) the lack of antigen-presenting dendritic cells (DCs) that are important in priming an effective immune response to generate functionally effective tumor antigen-specific T cells. We recently completed a pilot study of dual ICB (Ipilumamab and Nivolumab) with radiation therapy (SBRT 8Gy for 3 fractions) in a cohort of 25 metastatic PDAC patients that had progressed on conventional chemotherapy; this combination conferred an impressive 18% ORR and 29% disease control rate measured on non-irradiated lesions (historical 0% ORR with ICB in PDAC). This led to a phase 2 study in 30 metastatic PDAC patients using this dual modality treatment paradigm. To understand the role of radiation and ICB in altering the PDAC tumor microenvironment we performed single-cell RNA-sequencing and TCR-sequencing (&amp;gt;180k cells), and single-nucleus RNA-sequencing (&amp;gt;300k cells) on 36 tumor biopsies (23 pre-treatment, 13 paired on-treatment between day 10 and 21) from patients undergoing treatment in our phase 2 study. Tumor tissue was taken from distinct tissue sites, including primary tumors in the pancreas, and liver and abdominal wall metastases. We identified distinct tumor cell state distributions within different tissues, and a redistribution of cells from basal/mesenchymal states to classical states after radiation. We identified several state-specific interferon stimulated gene programs thus cataloging distinct responses of epithelial cells with different transcriptional states. Importantly, we found a redistribution of T cells states towards proliferating and exhausted T cells with unique clonality after radiation. Additionally, the myeloid compartment after radiation was enriched for C1QC+ and MHCII+ macrophage subsets, as well as infiltrating CD16/CD16 monocytes and CD14 monocytes, each showing induction of unique sets of interferon stimulated genes (ISGs). We next sought to better understand immunotherapy resistance mechanisms within these PDAC patients despite finding strong ISG induction in several subsets. We analyzed covarying gene programs and identified multicellular communities of cells before and after radiation that underlie interaction networks associated with radiation. Together our data provides the most comprehensive single-cell atlas of paired biopsies to study tumor and immune cell states in the context of radiation and ICB response. Citation Format: Arnav Mehta, Aparna Parikh, Milan Parikh, Ryan Park, Moshe Sade-Feldman, Lynn Bi, Nicole Carzo, Tarin M. Grillo, Islam Baiev, Olanike Asupoto, Irena Gushterova, Tom LaSalle, Anna Gonye, Emily Blaum, Sebastien Vigneau, Ronan Chaligne, Ana Lako, Thomas Lila, David Nelson, Caroline Porter, Orr Ashenberg, Karthik Jagadesh, William L. Hwang, Christopher Smillie, David P. Ryan, David T. Ting, Theodore Hong, Dana Pe'er, Nir Hacohen. Dissecting the reorganization of pancreatic tumor microenvironments after radiation and immunotherapy reveals insights into immunotherapy resistance [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C012.

The key role of sphingolipid metabolism in cancer: New therapeutic targets, diagnostic and prognostic values, and anti-tumor immunotherapy resistance

Biologically active sphingolipids are closely related to the growth, differentiation, aging, and apoptosis of cancer cells. Some sphingolipids, such as ceramides, are favorable metabolites in the sphingolipid metabolic pathway, usually mediating antiproliferative responses, through inhibiting cancer cell growth and migration, as well as inducing autophagy and apoptosis. However, other sphingolipids, such as S1P, play the opposite role, which induces cancer cell transformation, migration and growth and promotes drug resistance. There are also other sphingolipids, as well as enzymes, played potentially critical roles in cancer physiology and therapeutics. This review aimed to explore the important roles of sphingolipid metabolism in cancer. In this article, we summarized the role and value of sphingolipid metabolism in cancer, including the distribution of sphingolipids, the functions, and their relevance to cancer diagnosis and prognosis. We also summarized the known and potential antitumor targets present in sphingolipid metabolism, analyzed the correlation between sphingolipid metabolism and tumor immunity, and summarize the antitumor effects of natural compounds based on sphingolipids. Through the analysis and summary of sphingolipid antitumor therapeutic targets and immune correlation, we aim to provide ideas for the development of new antitumor drugs, exploration of new therapeutic means for tumors, and study of immunotherapy resistance mechanisms.

Abstract 6121: Correlation of tumor immune microenvironment status and driver genes alterations in Chinese non-small-cell lung cancer patients

Abstract Background: Patients with different driver genes show a variety of immunotherapy efficacy for non-small-cell lung cancer (NSCLC). In addition to PD-L1, the evaluation of the immune microenvironment contributes to a deeper understanding of the mechanisms of immunotherapy resistance. Methods: Tumor tissues from 2674 NSCLC patients were collected. Genetic alterations were profiled using targeted next-generation sequencing with 733 cancer-related genes panel. Immunohistochemistry was used to evaluate the PD-L1 tumor proportion score, (TPS) expression on the surface of tumor cells. Tumor-infiltrating immune cells of 120 tumor samples were labeled with multiple immunofluorescence staining. Results: Overall, CD8+ T cell infiltration level was negatively associated with both EGFR copy number gain (CNG, p &amp;lt; 0.001) and ERBB2 CNG (p = 0.021). Also, the presence of EGFR or ERBB2 CNG was related with fewer natural killer cell infiltration (EGFR: p &amp;lt; 0.001; ERBB2: p = 0.036) and a lower M1/M2 tumor-associated macrophages ratio compared to wild-type tumors (EGFR: p = 0.051; ERBB2: p = 0.036), suggesting a relatively weaker anti-tumor ability. In addition, we noted that KRAS CNV was negatively related to M2 macrophages (p = 0.027). Patients carrying ALK translocation (p &amp;lt; 0.001), ROS1 translocation (p = 0.006), KRAS mutations (p &amp;lt; 0.001) and MET (p &amp;lt; 0.001) mutations had the elevated PD-L1 TPS expressions, while those harboring EGFR (p &amp;lt; 0.001) and ERBB2 mutations (p = 0.001) had lower PD-L1 expression. Compared to the other EGFR mutations, PD-L1 expression in EGFR CNG tumors was higher, and no significant differences were found between CNG and other types of mutation in patients harboring ERBB2, KRAS, MET alterations. Conclusion: NSCLC patients with EGFR CNG and ERBB2 CNG had fewer invasion of cytotoxic T cells and NK cells and a lower M1/M2 macrophages ratio. EGFR CNG may intensively modify the tumor immune microenvironment, which is a possible mechanism contributing to EGFR-TKI resistance. Citation Format: Jindong Guo, Liwen Xiong, Haibin Yuan, Mengna Hu, Bei Zhang, Ding Zhang. Correlation of tumor immune microenvironment status and driver genes alterations in Chinese non-small-cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6121.