Response In Tumor Microenvironment Research Articles

Immunotherapy drives mesenchymal tumour cell state shift and TME immune response in glioblastoma patients.

Glioblastoma is a highly aggressive type of brain tumour for which there is no curative treatment available. Immunotherapies have shown limited responses in unselected patients, and there is an urgent need to identify mechanisms of treatment resistance to design novel therapy strategies. Here we investigated the phenotypic and transcriptional dynamics at single-cell resolution during nivolumab immune checkpoint treatment of glioblastoma patients. We present the integrative paired single-cell RNA-seq analysis of 76 tumour samples from patients in a clinical trial of the PD-1 inhibitor nivolumab and untreated patients. We identify a distinct aggressive phenotypic signature in both tumour cells and the tumour microenvironment in response to nivolumab. Moreover, nivolumab-treatment was associated with an increased transition to mesenchymal stem-like tumour cells, and an increase in TAMs and exhausted and proliferative T cells. We verify and extend our findings in large external glioblastoma dataset (n = 298), develop a latent immune signature and find 18% of primary glioblastoma samples to be latent immune, associated with mesenchymal tumour cell state and TME immune response. Finally, we show that latent immune glioblastoma patients are associated with shorter overall survival following immune checkpoint treatment (p = 0.0041). We find a resistance mechanism signature in a quarter of glioblastoma patients associated with a tumour-cell transition to a more aggressive mesenchymal-like state, increase in TAMs and proliferative and exhausted T cells in response to immunotherapy. These patients may instead benefit from neuro-oncology therapies targeting mesenchymal tumour cells.

Abstract PO4-28-05: Prolactin drives labile iron transfer from macrophages to breast cancer cells through CD44

Abstract Cancer cells are “ferrophilic” and exhibit great dependence on iron. While iron metabolism is closely related to tumor development, the source of iron and the mechanisms cancer cells adopt to actively acquire iron is not well understood. In the present study, we discovered a novel function of the lactating hormone, prolactin, in the regulation of iron transport. In breast cancer cells using mouse triple negative breast cancer cell lines EO771 and Py230 for demonstration, prolactin stimulation increased intracellular labile iron pool. Analyses of gene expression involved in iron transport revealed dramatic induction of CD44, a surface receptor for iron bound hyaluronan. Other genes involved in iron uptake including transferrin receptor (TFRC), CD163 and divalent metal transporter 1 (DMT1) were not changed. The utilization of neutralizing antibodies against CD44 significantly blocked prolactin mediated labile iron pool accumulation in breast cancer cells. In contrast to what was found in breast cancer cells, prolactin had the opposite impact on immune macrophages. Prolactin treatment increased ferroportin (FPN I, iron exporter) but had no influence on all iron uptake transporters leading to net iron release by macrophages. To further determine if the iron released by macrophages can be a direct source of iron utilized by breast cancer cells, we conducted co-culture work of macrophages with pre-stained labile iron pool and breast cancer cells without any iron staining and found a gradual increase of fluorescent iron pool developed in breast cancer cells over time. In conclusion, our work presents a multidisciplinary regulation of iron transport among prolactin, macrophages, and cancer cells. The novel regulatory role of prolactin to drive iron flow can provide new information on fine-tuning immune responses in tumor microenvironment and potentially benefit the development of novel therapeutics. Citation Format: Reagan Farrell, Nicholas Pascuzzi, Yi-Ling Chen, Miguel Torres, Mary Kim, Lauren Gollahon, Kuan-Hui Chen. Prolactin drives labile iron transfer from macrophages to breast cancer cells through CD44 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-05.

Cancer Nanovaccines: Nanomaterials and Clinical Perspectives.

Cancer nanovaccines represent a promising frontier in cancer immunotherapy, utilizing nanotechnology to augment traditional vaccine efficacy. This review comprehensively examines the current state-of-the-art in cancer nanovaccine development, elucidating innovative strategies and technologies employed in their design. It explores both preclinical and clinical advancements, emphasizing key studies demonstrating their potential to elicit robust anti-tumor immune responses. The study encompasses various facets, including integrating biomaterial-based nanocarriers for antigen delivery, adjuvant selection, and the impact of nanoscale properties on vaccine performance. Detailed insights into the complex interplay between the tumor microenvironment and nanovaccine responses are provided, highlighting challenges and opportunities in optimizing therapeutic outcomes. Additionally, the study presents a thorough analysis of ongoing clinical trials, presenting a snapshot of the current clinical landscape. By curating the latest scientific findings and clinical developments, this study aims to serve as a comprehensive resource for researchers and clinicians engaged in advancing cancer immunotherapy. Integrating nanotechnology into vaccine design holds immense promise for revolutionizing cancer treatment paradigms, and this review provides a timely update on the evolving landscape of cancer nanovaccines.

[The effect of PHD inhibitor on tumor microenvironment and tumor immune response].

Since the approval of HIF-PH inhibitors, HIF-PH inhibitors have been used clinically, and many studies and clinical case reports have been reported in Japan. A lot of information has been accumulated on clinical usage. However, HIF-PH inhibitors require careful administration for cancer patients due to their action mechanism through upregulating hypoxia-inducible factors (HIFs) level. In cancer cells, HIFs affect tumor progression and contribute to chemo- and radio-resistance. On the other hand, upregulation of HIFs in immune cells is associated with inflammation and suppress tumor progression. However, these controversial effects are not clear in in vivo model. It is needed to reveal whether upregulating HIFs level is beneficial for cancer therapy or not. We have previously reported that HIF-PH inhibitor treatment in tumor bearing mice model led to reconstitute tumor blood vessel and inhibit tumor growth. In addition, these phenomena were caused by tumor infiltrated macrophages and they altered these phenotypes. In this review, we will describe our findings on the mechanism of tumor growth suppression by HIF-PH inhibitors. We also want to mention the risks and benefits of future HIF-PH inhibitors.

ScRNA-seq characterizing the heterogeneity of fibroblasts in breast cancer reveals a novel subtype SFRP4+ CAF that inhibits migration and predicts prognosis.

Cancer-associated fibroblasts (CAFs) are a diverse group of cells that significantly impact the tumor microenvironment and therapeutic responses in breast cancer (BC). Despite their importance, the comprehensive profile of CAFs in BC remains to be fully elucidated. To address this gap, we utilized single-cell RNA sequencing (scRNA-seq) to delineate the CAF landscape within 14 BC normal-tumor paired samples. We further corroborated our findings by analyzing several public datasets, thereby validating the newly identified CAF subtype. Additionally, we conducted coculture experiments with BC cells to assess the functional implications of this CAF subtype. Our scRNA-seq analysis unveiled eight distinct CAF subtypes across five tumor and six adjacent normal tissue samples. Notably, we discovered a novel subtype, designated as SFRP4+ CAFs, which was predominantly observed in normal tissues. The presence of SFRP4+ CAFs was substantiated by two independent scRNA-seq datasets and a spatial transcriptomics dataset. Functionally, SFRP4+ CAFs were found to impede BC cell migration and the epithelial-mesenchymal transition (EMT) process by secreting SFRP4, thereby modulating the WNT signaling pathway. Furthermore, we established that elevated expression levels of SFRP4+ CAF markers correlate with improved survival outcomes in BC patients, yet paradoxically, they predict a diminished response to neoadjuvant chemotherapy in cases of triple-negative breast cancer. This investigation sheds light on the heterogeneity of CAFs in BC and introduces a novel SFRP4+ CAF subtype that hinders BC cell migration. This discovery holds promise as a potential biomarker for refined prognostic assessment and therapeutic intervention in BC.

Abstract LB349: Cysteine restriction enhances antitumor immunity in colorectal cancer

Abstract Background: Colon cancer is the second leading cause of cancer mortality worldwide, however, there are still issues with multidrug resistance and severe adverse reactions. Previous studies have shown that sulfur-containing amino acids are involved in cancer development, drug resistance, and immune evasion. Here, Our experimental findings suggested that restricting the intake of sulfur-containing amino acids may present a novel strategy for treating colorectal cancer. Methods: We assessed the impact of sulfur-containing amino acid restriction (SAR) on distinct cellular subpopulations within the tumor microenvironment through single-cell sequencing on colon carcinogenesis induced by azoxymethane/dextran sodium sulfate (AOM/DSS). Employing our custom-produced neutralizing antibodies against SLC7A11 to restrict cysteine intake, we examined the therapeutic implications and underlying molecular mechanisms by RNA-seq, Multiplexed immunofluorescence staining and Western blotting. Results: In the AOM/DSS model, SAR led to a slight reduction in mouse body weight compared to the normal diet. However, this dietary restriction significantly enhanced the survival rate of mice and effectively suppressed tumor growth. The single-cell sequencing results indicated a significant increase in the proportion of T cells within the tumor microenvironment in response to SAR. This suggested that restricting the intake of sulfur-containing amino acids may be a promising anti-tumor strategy. Previous studies have indicated high expression of the SLC7A11, a cysteine transporter protein, in various cancers, including colorectal cancer. To further investigate the role of cysteine in colorectal cancer, we developed the neutralizing antibody 1A4 against SLC7A11. Our results indicated that 1A4 markedly inhibited colorectal cancer development in mice. As known, 85% of colorectal cancers are microsatellite stable, which hardly benefit from treatment with immune checkpoint inhibitors (ICIs). Compared to anti-PD-1 and anti-CTLA4, 1A4 significantly inhibited the growth of CT26 in vivo. Multiplexed immunofluorescence staining results demonstrated that 1A4 significantly increased the proportion of CD8+ T cells in colorectal cancer tissues. Conclusion: Limiting cysteine intake significantly inhibits the growth of colorectal cancer, increases the proportion of CD8+ T cells, and enhances anti-tumor immunity. Citation Format: Jichang Li, Kang Xia, Zeruo Yang, Xiaoxue Pan, Xiaojing Yang, Pengyuan Wang, Shanwen Chen. Cysteine restriction enhances antitumor immunity in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB349.

Microenvironment, systemic inflammatory response and tumor markers considering consensus molecular subtypes of colorectal cancer.

Introduction: Colorectal carcinomas (CRC) are one of the most frequent malignancies worldwide. Based on gene expression profile analysis, CRCs can be classified into four distinct subtypes also known as the consensus molecular subtypes (CMS), which predict biological behaviour. Besides CMS, several other aspects of tumor microenvironment (TME) and systemic inflammatory response (SIR) influence the outcome of CRC patients. TME and inflammation have important role in the immune (CMS1) and mesenchymal (CMS4) subtypes, however, the relationship between these and systemic inflammation has not been assessed yet. Our objective was to evaluate the connection between CMS, TME and SIR, and to analyze the correlation between these markers and routinely used tumor markers, such as CEA (Carcinoembryonic Antigen) and CA19-9 (Carbohydrate Antigen 19-9). Methods: FFPE (Formalin Fixed Paraffin Embedded) samples of 185 CRC patients were collected. TME was described using tumor-stroma ratio (TSR), Klintrup-Makinen (KM) grade, and Glasgow Microenvironment Score (GMS). CMS classification was performed on tissue microarray using MLH1, PMS2, MSH2 and MSH6, and pan-cytokeratin, CDX2, FRMD6, HTR2B and ZEB1 immunohistochemical stains. Pre-operative tumor marker levels and inflammatory markers [C-reactive protein - CRP, albumin, absolute neutrophil count (ANC), absolute lymphocyte count (ALC), absolute platelet count (APC)] and patient history were retrieved using MedSolution database. Results: Amongst TME-markers, TSR correlated most consistently with adverse clinicopathological features (p < 0.001) and overall survival (p < 0.001). Elevated CRP and modified Glasgow Prognostic Score (mGPS) were associated with worse outcome and aggressive phenotype, similarly to tumor markers CEA and CA19-9. Stroma-Tumor Marker score (STM score), a new combined score of CA19-9 and TSR delivered the second best prognostication after mGPS. Furthermore, CMS4 showed association with TSR and several laboratory markers (albumin and platelet derived factors), but not with other SIR descriptors. CMS did not show any association with CEA and CA19-9 tumor markers. Conclusion: More routinely available TME, SIR and tumor markers alone and in combination deliver reliable prognostic data for choosing the patients with higher risk for propagation. CMS4 is linked with high TSR and poor prognosis, but in overall, CMS-classification showed only limited effect on SIR- and tumor-markers.

Pan-cancer analyses of immunogenic cell death-derived gene signatures: Potential biomarkers for prognosis and immunotherapy.

Immunogenic cell death (ICD) is a type of regulated cell death that is capable of initiating an adaptive immune response. Induction of ICD may be a potential treatment strategy, as it has been demonstrated to activate the tumor-specific immune response. The biomarkers of ICD and their relationships with the tumor microenvironment, clinical features, and immunotherapy response are not fully understood in a clinical context. Therefore, we conducted pan-cancer analyses of ICD gene signatures across 33 cancer types from The Cancer Genome Atlas database. We identified key genes that had strong relationships with survival and the tumor microenvironment, contributing to a better understanding of the role of ICD genes in cancer therapy. In addition, we predicted therapeutic agents that target ICD genes and explored the potential mechanisms by which gemcitabine induce ICD. Moreover, we developed an ICD score based on the ICD genes and found it to be associated with patient prognosis, clinical features, tumor microenvironment, radiotherapy access, and immunotherapy response. A high ICD score was linked to the immune-hot phenotype, while a low ICD score was linked to the immune-cold phenotype. We uncovered the potential of ICD gene signatures as comprehensive biomarkers for ICD in pan-cancer. Our research provides novel insights into immuno-phenotypic assessment and cancer therapeutic strategies, which could help to broaden the application of immunotherapy to benefit more patients.

Tumor microenvironment and immune response: A gateway to novel therapies in gastrointestinal cancers

Communication between cancer cells and the surrounding environment affects cancer development by numerous mechanisms leading to the inhibition or weakening of the immune system activity. Despite substantial evidence addressing the role of immune cells and the immune response, immunotherapy dedicated for patients with cancers is manifested by limited effectiveness. This review discusses selected immune-related aspects such as Th2 cell action or specific modification of immune cells and how they can be employed to design new and more effective approaches in the immunotherapy of gastrointestinal cancers.

Pathway Analysis of Tumor Immunity by TIGIT and Recent Scientific Research Results

The T cellular immune receptor with immunoglobulin and TIGIT, an immunoreceptor tyrosine-based inhibitory motif, is a recently identified immune checkpoint molecule primarily found on the outer membrane of T cells and natural killer (NK) cells. It is mediated by binding to ligands such as CD155 inhibits T and NK cells' Immune response and influences of tumor microenvironment. At present, many studies have identified the main pathway and mechanism of TIGIT control, and some drug companies have developed anti-Tigit drugs and put them into clinical trials. The application of TIGIT antibody to the treatment of malignant tumors has shown broad prospects in tumor immunotherapy. However, many studies have limitations, The research on TIGIT is less extensive and detailed compared to PD-1/PD-L1, and there is still uncertainty regarding the impact of the TIGIT pathway on various cancers, which requires further investigation by researchers. This study examined previous research on the structure and pathway of TIGIT, and successfully identified the structure of TIGIT and the ligands associated with its primary pathway. Based on the research on its signaling pathway and the results of its clinical trials in some cancers, the paper summarized its four main pathways, three mechanisms of action and recent research results. On this basis, I put forward my personal speculation. This paper focuses on the structural composition, ligand types and pathway analysis of TIGIT, providing a basic reference for further exploration of the action pathway of TIGIT in the future. However, the summary of the TIGIT pathway is not complete and needs further exploration by researchers.

A phase 1/2 study of pepinemab in children, adolescents, or young adults with recurrent or refractory solid tumors: A children's oncology group consortium report (ADVL1614).

Pepinemab, a humanized IgG4 monoclonal antibody, targets the SEMA4D (CD100) antigen to inhibit binding to its high-affinity receptors (plexin B1/PLXNB1, plexin B2/PLXNB2) and low-affinity receptor (CD72). SEMA4D blockade leads to increased cytotoxic T-cell infiltration, delayed tumor growth, and durable tumor rejection in murine tumor models. Pepinemab was well tolerated and improved T cell infiltration in clinical studies in adults with refractory tumors. SEMA4D was identified as a strong candidate proto-oncogene in a model of osteosarcoma. Based on these preclinical and clinical data, we conducted a phase 1/2 study to determine the recommended phase 2 dose (RP2D), pharmacokinetics, pharmacodynamics, and immunogenicity, of pepinemab in pediatric patients with recurrent/refractory solid tumors, and activity in osteosarcoma. Pepinemab was administered intravenously on Days 1 and 15 of a 28-day cycle at 20mg/kg, the adult RP2D. Part A (phase 1) used a Rolling 6 design; Part B (phase 2) used a Simon 2-stage design in patients with osteosarcoma. Pharmacokinetics and target saturation were evaluated in peripheral blood. Pepinemab (20mg/kg) was well tolerated and no dose-limiting toxicities were observed during Part A. There were no objective responses. Two patients with osteosarcoma achieved disease control and prolonged stable disease. Pepinemab pharmacokinetics were similar to adults. Pepinemab (20mg/kg) is safe, well tolerated and resulted in adequate and sustained target saturation in pediatric patients. Encouraging disease control in two patients with osteosarcoma warrants further investigation with novel combination strategies to modulate the tumor microenvironment and antitumor immune response. This trial is registered as NCT03320330 at Clinicaltrials.gov. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Abstract 3894: The temporal influence of the tumor microenvironment in response to checkpoint blockade

Abstract Immune checkpoint inhibition (ICI) has produced a paradigm shift in treatment across a range of cancer subtypes. However, our understanding of why only certain patients respond, as well as our ability to predict this response, is still quite limited. This is exacerbated by the complexity of the tumor microenvironment (TME), which contains cancer cells, immune cells, and stromal cells. These differing cell types can have context-dependent roles in either promoting or inhibiting anti-tumor immune responses, making it challenging to identify features which reliably correlate with patient outcome. In addition, the roles of these diverse cell types are often influenced by their location within the TME, meaning that assays without spatial information cannot fully resolve this complexity. To address these gaps, we mapped the spatial distribution and phenotype of 21 cell populations across 117 patients with metastatic triple negative breast cancer that received nivolumab (anti-PD1) through the TONIC clinical trial (NCT02499367). We collected metastatic samples prior to and during treatment to understand how anti-PD1 reshaped the TME. We also collected archival material from the original primary tumor for each patient. We generated multiplexed imaging data from these samples, identified the location and phenotype of each cell, and then quantified the spatial distribution, diversity, and functional marker status of all cell populations in the TME. We detected numerous features indicative of a productive immune response that correlated positively with clinical benefit, whereas features indicative of a suppressive microenvironment correlated negatively with clinical benefit. We observed significant temporal effects, where many pre-treatment predictive features were not predictive on-treatment; similarly, many on-treatment predictive features had no predictive power prior to treatment. Intriguingly, we found features in the original primary tumors which predicted subsequent (metastatic) response to immunotherapy. We observed substantial differences in predictive accuracy of multivariate models based on timepoint, with on-treatment samples exhibiting the best performance and primary tumor samples exhibiting the worst. Of note, we observed substantially worse predictive performance when using bulk DNA sequencing data to predict outcome from the same patients, highlighting the importance of spatial information. Taken together, we show that the features associated with a productive anti-tumor immune response are temporally structured, and differ based on what phase of response is profiled. Furthermore, using the primary tumor to assess whether metastatic TNBC patients are good candidates for ICI may miss important predictive signals. These findings shed new light on the determinants of ICI outcome, and may shape the design of subsequent trials to better understand the temporal aspects of ICI. Citation Format: Noah F. Greenwald, Iris Nederlof, Hugo Horlings, Marleen Kok, Christina Curtis, Michael Angelo. The temporal influence of the tumor microenvironment in response to checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3894.

Abstract 639: Evaluation of individual drug response in tumor microenvironment to cisplatin treatment in precision-cut tumor slices of ovarian cancer

Abstract Ovarian cancer is a complex and heterogeneous disease and the major cause of death among women with gynecological cancers. While patients usually respond well to the platinum-based first-line chemotherapy, the disease often becomes increasingly resistant to the treatment. The tumor microenvironment (TME) plays an important role in tumor development and drug resistance. Characterizing the TME of ovarian cancer after drug treatment is essential to identify molecular mechanisms that allow residual tumor cells to survive chemotherapy in ovarian cancer. We have established a preclinical model using precision-cut tumor slices (PCTS) with 250 µm thickness which preserves the TME of solid tumors with their heterogeneous cell composition during cultivation. The tumor morphology, viability and heterogeneity in terms of tumor and stromal cells as well as the immune compartment are preserved within the system. Based on the PCTS model, we also established a method to perform single-cell RNA sequencing (scRNA-seq) of the PCTS after cultivation and drug treatment to characterize the cellular features and dynamic relationships of different cell populations in the TME after drug treatment. Over 20 cell subtypes including different clusters of epithelial cells, immune cells and fibroblasts can be identified in PCTS after cisplatin treatment through the scRNA-seq analysis. This enables further deeper analysis of each cell subgroup in the TME after drug treatment. In response to cisplatin treatment, patients PCTS showed an individual induction of PD-L1 in different cell types. Additionally, multiplex immunofluorescence (mIF) stainings of the PCTS were performed to spatially trace the response of the TME to drug treatment. The mIF staining allows the simultaneous detection of up to 6 different markers on one FFPE tissue section. Images were analyzed using a machine-learning based workflow, which allows the comparison of biomarker expression and immune cell spatial distribution in the PCTS stromal and tumor areas before and after treatment. Combining the PCTS as a preclinical model with scRNA-seq and mIF staining analysis allows us to bridge the cellular characteristics and cellular spatial distribution with treatment response in the TME of solid tumors. It enables the systematic analysis of residual cancer populations after cisplatin treatment within the complex TME and further identification of predictive markers and targets for an individualized combination of chemotherapy with compounds targeting these mechanisms. The individual drug response of patients can be deeply evaluated and efficacious therapies can be further developed. Citation Format: Julia Thiel, Adrian Kneer, Weimeng Yu, Bernd Winkler, Georg Sauer, German Ott, Walter E. Aulitzky, Thomas E. Mürdter, Matthias Schwab, Chunguang Liang, Meng Dong. Evaluation of individual drug response in tumor microenvironment to cisplatin treatment in precision-cut tumor slices of ovarian cancer [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 639.

Abstract 1582: Cabozantinib changes the tumor microenvironment to increase the efficacy of immunotherapy in refractory metastatic pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. Tumors evade immune surveillance by expressing PD-L1 that interacts with the T cell protein PD-1, subsequently inhibiting CD8+ cytotoxic T lymphocyte proliferation and effector function. A plausible reason for this observation may be that other, redundant, immune suppressive mechanisms are at play. Our research team has shown that elevated infiltration of polymorphonuclear (PMN)-MDSCs within the PDAC tumor microenvironment (TME) inhibit T cell effector function, regardless of PD-1/PD-L1 inhibition (Holokai et al., 2020). Objective: A Phase II clinical trial was developed to identify the potential therapeutic benefit of combinatorial anit-PD-L1/PD-1 and cabozantinib (cabo) treatment in therapy-resistant (refractory and metastatic) PDAC patients. Methods: NanoString CosMx™ spatial molecular transcriptomic imaging (CosMx™ SMI) was performed using FFPE sections collected from a liver or lung metastatic target lesions at visit 1 (pretreatment) and visit 2 (9 weeks on combinatorial treatment) from individual patients. FFPE sections were prepared from the same target lesion, and fields of view were chosen on the H&amp;E in consultation with GI pathologist. Seurat V 4.0 was used for the analysis and visualization by spatial mapping of each FOV showing identified cell population indicated by color coding in visit 1 and visit 2 within the same patient. Multiplex immunofluorescence (MxIF) of patient FFPE tissues and single cell RNA sequencing (scRNAseq) of PDAC organoids (PDOs) generated from the core biopsies collected at visit 1 and matched visit 2 from each patient were used to validate the CosMx™ SMI data. Results: A uniform manifold approximation and projection showed loss of tumor cell populations within the PDAC TME in response to combinatorial treatment compared to pretreatment visit 1. Comparison between spatial maps of visit 1 to visit 2 demonstrated a shift in cell populations within defined NICHES (local cellular TME) whereby there was 1) a loss of tumor cell populations, 2) increase in CTL infiltration, 3) increase in endothelial cells, 4) decrease immunosuppressive MDSCs, 5) decrease in antigen presenting CAFs, and 6) change in myCAFs to an inflammatory CAF phenotype. Within the iCAF population IL-6, CXCL1, LIFR, ACTA2 and FAK were expressed; correlating with an increase in CD8 perforin and granzyme B positive cells in response to cabo and checkpoint inhibition. MxIF, spectral flow cytometry and scRNAseq analyses of the matched in vitro PDO model showed tumor cell death that correlated with decreased MDSC viability, increased CTL proliferation and a shift in CAF phenotypes. Conclusion: These findings suggest that cabo induces a TME conducive to increased response to immunotherapy in PDAC patients with refractory metastatic disease. Citation Format: Jayati Chakrabarti, Pritha Adhikary, Jiang Wang, Davendra Sohal, Syed A. Ahmad, Junaid Arshad, Aaron J. Scott, Rachna T. Shroff, Yana Zavros. Cabozantinib changes the tumor microenvironment to increase the efficacy of immunotherapy in refractory metastatic pancreatic ductal adenocarcinoma [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 1582.

Abstract 6661: Profiling response to oncolytic adenovirus and immune checkpoint inhibitors by mass cytometry in a triple negative breast cancer mouse model

Abstract Immune checkpoint inhibitors (ICIs) based immunotherapy has found success in treating breast cancers but for more immunosuppressive cancer types, especially most of triple negative breast cancers (TNBCs), response rates generally are low. Also, combination immunotherapy with oncolytic adenoviruses (OAVs) has been shown to induce a more immune inflamed phenotype in many pre-clinical studies. Here, by using a 40-antibody panel for mass cytometry, we profiled comprehensive changes of cell phenotypes in a TNBC mouse model both systemically and in the tumor microenvironment in response to the treatment of ICIs and/or a new systemically delivered, TGFβ signaling inhibiting OAV, mHAdLyp.sT. The samples in this study were collected on day 16 of tumor cell inoculation, while mHAdLyp.sT were applied by tail vein on day 7 and 9, and anti-PD-1 and anti-CTLA-4 antibodies were injected Intraperitoneally on day 8, 10, 12, 14. After sample processing and data acquisition on Helios system, a standard workflow was applied for data preprocessing including normalization, compensation, transformation, diagnostic analysis, marker ranking, cell population identification, visual representation, differential analysis, and statistical analysis. In blood samples, for T cell clusters, percentages of effector CD8+ T cells, naïve CD8+ T cells, and CD44lowCD62Llow CD8+ T cells among total cells were increased by mHAdLyp.sT, anti-PD-1, and anti-CTLA-4 combination when compared to the buffer group but none of these significant changes were observed in groups treated with mHAdLyp.sT or ICI antibodies alone. Combination treatment also increased percentages of clusters of CD272highCD44high DCs, CD44highCD62Llow B cells, and CD44high macrophages significantly. In spleen, combination treatment significantly increased several T cell clusters, including TCRβhighCD38highRORγthigh T cells, TCRβhigh CD4+ T cells, effector CD8+ T cells, TCRβhighCD44lowCD62Llow CD8+ T cells, and CD11bhigh CD8+ T cells when compared to the buffer group. Interestingly, while ICIs treatment increased several macrophage clusters, such as CD44highLy-6G/Chigh macrophages, CD11blow macrophages, and CD44highCD11bhigh macrophages, they are not significantly increased in the combination treatment group. In the tumor, we did not detect any significant changes by the combination treatment in the clusters we identified now although the average percentages of several clusters were either increased or decreased. We will refine these algorithm-generated clusters from tumors once we obtain our data from day 24 sample collection point for advanced analysis. In short, our current results suggested that the combination therapy is more potent in generating cytotoxic T cells and other anti-tumor immune cell populations systemically in the early stage of the treatment and tumor progression. Citation Format: Weidong Xu, Qiaoshan Lin, Yan Li, Beniamin Filimon, Soon Cheon Shin, Michael Olson, Mengjie Chen. Profiling response to oncolytic adenovirus and immune checkpoint inhibitors by mass cytometry in a triple negative breast cancer mouse model [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 6661.

Abstract 1529: Development of lymphoid aggregates in metastatic uveal melanoma tumors treated with tebentafusp

Abstract Tebentafusp (tebe), targeting gp100, is the first TCR-CD3 bispecific to demonstrate overall survival (OS) benefit and is approved for the treatment of metastatic uveal melanoma (mUM). We have previously shown B cell recruitment into the tumor associates with OS on tebe [1]. We explored further the formation of lymphoid aggregates (LA) within the tumor microenvironment in response to tebe. HLA-A*02:01+ 2L+ adult patients (pts) with mUM were treated with tebe (NCT02570308). Paired tumor biopsies were collected at baseline (n=70), after 3 doses of tebe (n=48), and at radiographical progression (n=17). Presence of B cells, T cells and macrophages was assessed by immunohistochemistry, using antibodies against CD20, CD3 and CD163 respectively, and digitally quantified. Using digital density analysis, aggregates of ≥10 CD20+ B cells within CD3+ T cell area in a 100µm radius were defined as LA. At progression pts were categorized by OS ≥12 or &amp;lt;12 months. Mean fold change (FC) was used. At baseline, B cells were detected in 90% of pts (median 5 B cells/mm2) with those pts having no more than 2 LA/mm2 (17% pts with 1 LA/mm2 and 3% pts with 2 LA/mm2). The median number of B cells in the biggest LA per sample was 24.B cells were recruited into the tumor after 3 doses of tebe (median 32 B cells/mm2, FC 3, p=6.5e-05). The LA density increased up to 6 LA/mm2 (23% pts with 1 LA/mm2, 8% pts with 2 LA/mm2, 6% pts with 3 LA/mm2 and 2% pts with 6 LA/mm2) with a trend for an increase in LA size (median B cell number in biggest LA = 30, p = 0.67). Tumors with a low baseline CD163+ macrophage to CD3+ T cell ratio, which was previously associated with longer OS on tebe, tended to have more LA/mm2 at baseline and on-treatment than those with a high ratio. At progression, pts with long OS had more B cells in the tumor compared to short OS (median 20 vs 2 B cells/mm2; FC 5, p=0.05). Additionally, more pts with long OS had LA than pts with short OS (6/12 pts vs 1/5 pts respectively) with a density not exceeding 1 LA/mm2. LA size was larger in pts with long OS compared to short OS (median B cell number in biggest LA 30 vs 10 respectively). LA in the short OS patient was found in the normal adjacent tissue compared to the LA in long OS patients which were located in the tumor and invasive margin. Tebe enhanced the formation of LA in the tumor microenvironment, notably in the low immunosuppressed tumors, which is consistent with the hypothesis that it results in epitope spread. These observations provide deeper insight into the mechanism of action of tebe and helps to explain prolonged survival of mUM pts beyond radiographical progression. [1] Greenshields-Watson A. et al., 619, SITC 2022 Citation Format: Takami Sato, Camille Britton-Rivet, Sophie Khakoo, Alistair Easton, Anastasiya Kazachenka, Laura Collins, Emma Leach, Adel Benlarech, Sarah Stanhope, Marcus O. Butler. Development of lymphoid aggregates in metastatic uveal melanoma tumors treated with tebentafusp [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 1529.

Abstract 6765: A patient-derived hepatocellular carcinoma multicellular spheroid platform harnessing autologous patient serum to model the tumor microenvironment and treatment response

Abstract Hepatocellular carcinoma (HCC) is the third leading and fastest rising cause of cancer-related death worldwide. The discovery and preclinical development of compounds targeting HCC is hampered by the absence of authentic tractable systems recapitulating the heterogeneity of patient HCC tumors and the tumor microenvironment (TME). Here, we established a novel, simple and robust patient-derived multicellular tumorspheroid model based on clinical HCC tumor tissues combined with the autologous patient serum. The model was robust and reproducible independently from cancer etiology and grade recapitulating key functional features of original tissue. We show that the use of autologous patient serum was essential for the TME function. The tumorspheroids comprised the main cell compartments including epithelial cancer cells as well as all major cell populations of the TME (cancer-associated fibroblasts, macrophages and T cells). Cell type proportions were variable between tumorspheroids reflecting HCC heterogeneity. Moreover, we observed differential responses to FDA HCC approved drugs (including tyrosine kinase and checkpoint inhibitors as well as VEGF-targeting agents) between donors as in patients. This patient HCC serum-tumorspheroid model provides novel opportunities for drug discovery and development as well mechanism of action studies including compounds targeting the TME. In conclusion, this HCC serum-tumorspheroid platform will contribute to improve the dismal outcome of patients with HCC. Citation Format: Emilie Crouchet, Nuno Almeida, Sarah C. Durand, Marie Parnot, Marine A. Oudot, Fabio Giannone, Cloé Gadenne, Sara Cherradi, Natascha Roehlen, Antonio Saviano, Emanuele Emanuele Felli, Patrick Pessaux, Kazuaki Chayama, Hong Tuan F. Duong, Thomas F. Baumert, Catherine Schuster. A patient-derived hepatocellular carcinoma multicellular spheroid platform harnessing autologous patient serum to model the tumor microenvironment and treatment response [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 6765.

Abstract 2488: The penile cancer tumor microenvironment and immunotherapy response: Results from the PERICLES trial

Abstract Introduction: The phase II PERICLES trial investigated the efficacy of atezolizumab (anti-PD-L1) for advanced penile cancer, with or without radiotherapy. As previously published, durable responses (progression-free at 12 months) were observed in 4 out of 32 enrolled patients. Here, we present the final overall survival (OS) results and an analysis of potential biomarkers for immunotherapy response. Methods: Preplanned analysis projected that at 27/32 events, the study would have 80% power (2-sided α=0.05) to assess whether the OS for the entire cohort significantly differed from the hypothesized historical median for stage IV patients. Baseline tumor tissue samples were subjected to bulk-RNA sequencing (n=31), fluorescent (n=28) and chromogenic (n=30) multiplex immunohistochemistry. We quantified the densities of CD3+CD8+ cytotoxic T-cells, CD3+CD8−FoxP3− T-helper cells, CD3+CD8−FoxP3+ regulatory T-cells, CD68+ macrophages and CD20+ B-cells in both the tumor and stromal compartments and assessed spatial relationships. We also quantified CD8+PD1+ (potentially enriched for tumor reactivity) and CD8+PD1− T-cells. Differential expression and gene set enrichment analysis (GSEA) were performed using the hallmarks gene sets. Results: The median OS for the entire study cohort was 11.3 months (95% CI, 5.5-16.7), which is an improvement compared to the hypothesized historical median of 7.8 months for stage IV patients (one sample log-rank p=0.033). Biomarker analyses showed an increased density of stromal macrophages (p&amp;lt;0.001) and intratumoral cytotoxic T-cells (p=0.035) in durable responders compared to non-responders. Additionally, we observed an increased density of intratumoral CD8+PD1+ in durable responders (p&amp;lt;0.001). In contrast, intratumoral CD8+PD1− densities were comparable between durable and non-responders (p=0.057). Patients with a high density of intratumoral CD8+PD1+ also had an improved progression-free survival (PFS) (log-rank, p&amp;lt;0.001) and OS (log-rank, p=0.002). Analysis of the spatial relationships in the tumor microenvironment (TME) showed that durable responders had larger distances between cytotoxic T-cells and T-helper cells (p=0.034). GSEA revealed significant (FDR≤5%) up-regulation of IFN-α/γ responses in durable responders. In contrast, non-responders showed up-regulation of epithelial-mesenchymal transition, KRAS signaling and angiogenesis, suggesting potential barriers to an anti-tumor response. Conclusion: In the final clinical analysis, OS was improved with atezolizumab compared to the historical population of stage IV patients. CD8+PD1+ T-cells, a common proxy for tumor-reactive T-cells, and spatial relationships in the TME might serve as biomarkers of durable response to immunotherapy in advanced penile cancer. Citation Format: Tynisha S. Rafael, Alberto Gil-Jimenez, Hielke M. de Vries, Iris M. Seignette, Elise Bekers, Marta Lopez-Yurda, Dennis Peters, Erik Hooijberg, Annegien Broeks, Oscar R. Brouwer, Eva Schaake, Daniel J. Vis, Tanja D. de Gruijl, Lodewyk F.A. Wessels, Michiel S. van der Heijden. The penile cancer tumor microenvironment and immunotherapy response: Results from the PERICLES trial [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 2488.

Abstract 1045: A freely expandable 20-plex immuno-oncology antibody core panel for spatial biology experiments on human FFPE cancer sections: REAplex IO Core for MICS technology

Abstract Spatial biology is integral to understanding the dynamics of immune cells, tumor microenvironment (TME), and therapeutic responses in cancer research. Ultra-high-plex immunofluorescence has transformed deep phenotyping and cell neighborhood analysis, providing unprecedented insight into complex and heterogeneous samples such as the TME. Many spatial biology platforms reduce operator hands-on time by automating image acquisition. However, determining the most appropriate markers to distinguish key cell types, finding application-compatible antibody clones and optimizing their dilution remains a tedious manual process. To streamline the panel-building process, we have developed a series of pre-defined, commercially available antibody panels for the MACSima™ Imaging Cyclic Staining (MICS) technology. So far, these have covered the spectrum from ultra-high-plex screening panels with up to 205 antibodies (REAscreen™ MAX) to the more focused REAscreen Immuno Oncology antibody panel with 61 cancer and immune-related antibodies for the comprehensive analysis of various different human FFPE samples. Here, we introduce a new, 20-plex, pre-defined antibody panel. Organized along essential marker groups, it can function as a stand-alone panel that allows the identification of the major tumor-infiltrating leukocyte populations with a focus on T-cells, and providing insight into TME architecture, tumor neovascularization, and cellular activity. Because it can be combined and expanded with any number of antibodies or even entire panel modules, it can be used as a standardized basis for highly multiplexed custom panels optimized for the research question at hand. We demonstrate its performance and broad applicability across several cancer types analyzed with the MICS technology. Finally, preset template examples for panel-specific data processing with MACS iQ View analysis software demonstrate the simplicity yet power of an integrated end-to-end workflow. Citation Format: Julia Femel, Eric C. DiBasio, Katrin Schönborn, Maximilian Büttner, Tim Düking, Markus Habich, Tanya Wantenaar, Stefan Eulitz, Andreas Bosio. A freely expandable 20-plex immuno-oncology antibody core panel for spatial biology experiments on human FFPE cancer sections: REAplex IO Core for MICS technology [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 1045.

Abstract 863: Scratch: A highly modular pipeline for single-cell cancer research

Abstract Background: Single-cell RNA sequencing (scRNA-seq) is a robust approach to facilitate cancer research, revealing insights into tumor heterogeneity, microenvironment, and treatment response. However, scRNA-seq results frequently encounter reproducibility challenges due to i) high data complexity, intensified by human intervention, and ii) insufficient methods standardization, leading to inconsistent findings. Methods: Here, we introduce SCRATCH (Single-Cell RnA-seq Toolkit and Pipeline for Cancer researcH), a Nextflow-based pipeline designed to improve reproducibility through a layered and modular architecture. SCRATCH follows FAIR principles and guidelines provided by the nf-core community. Result/Discussion: The pipeline provides three execution modes: end-to-end, iterative, and custom. In the end-to-end mode, the pipeline processes data from raw input to downstream analyses automatically. This mode employs ranking- and aggregation-based approaches. For instance, the ranking approach leverages benchmark metrics to select the most suitable method in distinct steps, e.g., batch correction. Therefore, ensuring a consistent and data-driven selection. On another hand, the aggregation approach uses multiple predictions to increase confidence levels, such as on CNV inference and malignant cell identification. These strategies minimize human intervention, ideal for beginner users, enabling rapid access to preliminary results and biological insights. Alternatively, the iterative mode allows intermediate users to define workflow breakpoints in a layered-based fashion. Users can pause, review results, and adjust decisions at stages (e.g., TME annotation), facilitating a "semi-supervised" approach for a more tailored analysis while retaining the SCRATCH framework. Thirdly, the custom mode enables precise executions based on modules for similar tasks (e.g., trajectory analysis and cell-cell communication), allowing experienced users to bypass the SCRATCH workflow and use it as an on-demand toolkit. This mode leverages pipeline parallelism for efficient processing, perfect for ongoing single-cell projects. SCRATCH produces HTML reports to ensure traceability and reproducibility. Conclusion: SCRATCH, an evolving project, comprises 05 subworkflows, 18 modules, and 25 tools. We envisage SCRATCH as an open-source tool and invite developers to leverage its modules for their pipelines. For more information, please visit https://break-through-cancer.github.io/btc-scrna-training. Citation Format: Andre F. Fonseca, Guangchun Han, Marcel Ribeiro-Dantas, Enyu Dai, Diljot Grewal, Matthew Zatzman, Eliyahu Havasov, Andrew McPherson, Break Through Cancer, Data Science TeamLab, Michael N. Noble, Rameen Beroukhim, Rachel Karchin, Sohrab P. Shah, Linghua Wang. Scratch: A highly modular pipeline for single-cell cancer research [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 863.