Tumor Heterogeneity Research Articles

Abstract B015: Targeting CX3CL1 transforms a cold tumor microenvironment into a hot one by enhancing T cells and DCs locally and systemically

Abstract Intratumoral heterogeneity (ITH) is associated with immunotherapy failure and a weaker anti- tumor immune response. Using a series of carcinogen-induced tumor (CIT) squamous cell skin carcinoma cell lines, our lab has sought to identify tumor-intrinsic factors—particularly chemokines and cytokines—which can have a “dominant negative” effect on the immune microenvironment when present within a heterogeneous tumor. We identified CX3CL1 as a one such critical mediator of a cold (T cell low) immune microenvironment and found that tumors containing as few as 10% CX3CL1high tumor cells exhibit a diminished T cell response compared to tumors which lack CX3CL1high tumor cells. We predicted that targeting CX3CL1 could therefore transform a cold tumor microenvironment (TME) into an inflammatory hot (T cell high) TME. Supporting this, we found that CRISPR-Cas9-mediated deletion of CX3CL1 in a tumor cell line that typically gives rise to cold tumors led to higher T cell infiltration and fewer immunosuppressive macrophages in the TME of CX3CL1-/- tumors. Building on this, we further explored T cell populations in the TME and found that CX3CL1-/- tumors had significantly increased activation and decreased exhaustion markers on T cells. Flow cytometry analysis showed higher expression of CD44 on effector CD4 T cells and an increase in the number of activated CD44+ PD1low CD4 T cells. By contrast, the frequency of CD4 T cells which were PD1High TIM3+ and PD1High TIM3+ TIGIT+ which represent a likely exhausted effector CD4 population was lower in CX3CL1-/- tumors than in counterpart control tumors. An interrogation of DC subsets in the TME further revealed a higher number of cDC1 and cDC2 populations in CX3CL1−/− tumors. To determine the effect of tumor-produced CX3CL1 on immune activity in lymphoid organs, we investigated T cell and DC markers in the spleen and tumor-draining lymph nodes during early tumor development. Spleens harvested 10 days after tumor injection showed higher infiltration of cDC1 CCR7+ and cDC2 CCR7+ mature DCs in CX3CL1-/- tumor-bearing mice. Thus, in the absence of tumor-produced CX3CL1, both cDC1 and cDC2 dendritic cell subsets attain more mature phenotypes in both the TME and the spleen. At the same time point, tumor-draining lymph nodes also exhibited a shift in immune cell composition characterized by an increased frequency of T cells, particularly CD4+ T cells, and a decreased frequency of B cells. Moreover, the percentage of PD1+ CD8 cells was also higher, indicating better priming and activation of CD8 T cells via DCs in tumor-draining lymph nodes. These findings indicate that tumor-produced CX3CL1 drives a cold immune microenvironment through both tumor-localized and systemic effects. These studies suggest therapeutically targeting CX3CL1 particularly in tumors where tumor cells produce high levels of CX3CL1 holds promise to transform a cold TME into a hot immune inflammatory TME and potentiate better anti-tumor immunity. Citation Format: Piyush Chaudhary, Savannah Hughes, Joshua Tay, Robert Letchworth, Miho Tanaka, Melissa Reeves. Targeting CX3CL1 transforms a cold tumor microenvironment into a hot one by enhancing T cells and DCs locally and systemically [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B015.

Abstract B001: Intra- and inter-tumoral heterogeneity of melanoma across different metastatic sites

Abstract Melanoma, a highly metastatic skin cancer, exhibits variations in prognosis and response to therapy based on the site of metastasis. Despite the success of immunotherapy and targeted therapies in melanoma, over half of metastatic melanoma patients will experience disease progression due to therapy resistance. The heterogeneity and plasticity of melanoma cells contribute to metastatic dissemination and therapy resistance. Our aim is to determine whether distinct clones and/or their transcriptional cell states can predict the formation of tumors in various organs and assess how these clones change over time. To identify melanoma clones across different metastatic sites, NOD scid gamma (NSG) mice and C57BL/6 mice were injected subcutaneously, intravenously or intracranially with the same pool of cells of barcoded luciferase expressing YUMMER1.7 murine melanoma cells. Transduction conditions ensured that one DNA barcode integrated into cell genomes at one barcode per cell, serving as a lineage tag. Bioluminescence imaging was performed once a week to monitor tumor growth of mice injected intravenously and intracranially. Subcutaneous tumors were measured by calliper. Mice were euthanized at different time points; day 8, 15, 22, 29 post-implantation and at ethical endpoints. Tumors were harvested and DNA sequencing was performed to identify barcodes expressed by the tumor cells. All mice developed tumors, with 100% penetrance in NSG mice. However, in C57BL/6 mice, 10% of mice intravenously injected and 27% of subcutaneously implanted mice showed complete lesion regression, suggesting that the immune system may be responsible for tumor regression. Analysis of barcodes allowed us to assess the heterogeneity of melanoma tumors at different metastatic sites and their evolution over time. Lineage tracing and clonal heterogeneity will be evaluated using the state of art technology, SPLINTR (Single-cell Profiling and LINeage Tracing), enabling us to match a cells evolution with changes in transcriptional states. Barcode analysis performed before implanting the cells and, at different timepoints in subcutaneous and lung tumors showed that dominant subclones at the baseline were also dominant in subcutaneous and lung tumors in immunocompromised mice. In contrast, dominant subclones in immunocompetent mice was those present in lower proportion at baseline. Additionally, greater variability of subclones was observed especially in lung and brain tumors, across immunocompetent mice, likely as a mechanism of resistance, enabling these tumors to overcome immunoediting. Understanding the variability of clonality between different metastatic sites and over time will improve our comprehension of the role of different subclones in organ- specific metastasis and their transcriptional cell states. Citation Format: Veronica L. Aedo Lopez, Reem Saleh, Benjamin Blyth, Xin Du, Dane Vassiliadis, Katie Fennell, Davide Moi, Roberta Mazzieri, Riccardo Dolcetti, Karen E. Sheppard, Grant A. McArthur. Intra- and inter-tumoral heterogeneity of melanoma across different metastatic sites [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B001.

Abstract B039: Exploring the impact of neoantigen expression on lung tumor development

Abstract Lung adenocarcinoma (LUAD) stands as a formidable challenge in oncology, despite the emergence of immunotherapy as a promising treatment avenue. The dynamic interplay between the immune system and cancer plays a pivotal role in shaping cancer development and progression. Central to this process are neoantigens, tumor-specific antigens recognized by cytotoxic T cells, which trigger the elimination of tumor cells. It has been implicated that the presence of T cells also decreases tumor grade. However, the precise impact of neoantigen expression on LUAD development and progression remains poorly understood. Our lab previously developed a genetically engineered mouse model called the iNversion-INduced Joined neoAntigen (NINJA) system with the ability to induce expression of a fluorescent neoantigen in oncogenic KRAS, P53-null, tdTomato-expressing lung adenocarcinoma cells (KPT-NINJA). Using this system, we track tumor initiation, progression, and advanced LUAD development across 20 weeks in both immune-deficient and -sufficient settings. Further, tumors initiated in this system give rise to heterogeneous (neoantigen+ and -) tumors, enabling the study of T cell-driven tumor selection. Through multiplexed immunofluorescence, we identified that neoantigen+ tumors experience increased immune infiltration (CD3+, CD4+, CD8+, B220+) and reduced volume, ultimately leading to prolonged animal survival. Surprisingly, advanced lung tumors maintain neoantigen+ cells, indicating that endogenous lung T cells are ineffective at clearing neoantigen-expressing tumors. Yet, we detected that infiltrated tumors exhibit reduced p-ERK expression, suggesting that T cells may slow tumor progression through a non-cytotoxic mechanism. Transferring stimulated neoantigen-specific T cells to tumor-bearing mice largely eliminated neoantigen+ tumor cells. All these data together suggest complex mechanisms for the role of endogenous T cells in controlling lung tumors. Through molecular characterization of tumor progression and T cell-mediated tumor suppression, this study reveals specific pathways that LUAD cells undergo during the process of development and progression, providing valuable insights into the immune response against neoantigen-expressing tumors. The characterization of early neoantigen-expressing tumor cells is pivotal in validating its utility for studying cancer immunotherapy and identifying key molecular changes during tumor development. Ultimately, by unraveling the intricate interplay between neoantigen expression, immune surveillance, and tumor progression, this project aims to contribute significantly to our understanding of how neoantigens shape the dynamics of tumor biology. Citation Format: Jennifer L. Loza, Ishan Bansal, Brian Hunt, Kelli Connolly, Advait Jeevanandam, Srividhya Venkatesan, Nikhil Joshi. Exploring the impact of neoantigen expression on lung tumor development [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B039.

Abstract A031: ECM architecture drives distinct evolutionary patterns of tumor immune escape and elimination in breast cancer

Abstract Cancer progression poses a clinical hurdle due to tumor heterogeneity that contributes to treatment resistance and immune evasion. While the adaptive immune system can effectively eliminate cancer in certain instances, tumor escape remains challenging. The immune microenvironment plays an important role in this process. One unanswered question is the role of ECM geometry, also known as Tumor Associated Collagen Signatures (TACS), and its effects on tumor immune recognition. In solid malignancies, ECM geometry has been associated with disease stage and T cell infiltration. Specifically, empirically observed ECM topologies are frequently categorized based on fiber arrangement: random fibers (TACS1), circumferentially aligned fibers (TACS2), and radially arranged fibers (TACS3). Even though a clear negative correlation between TACS and patient survival has been established, the specific roles and extent of TACS in T cell-driven cancer evolution remain uncharacterized. The precise mechanisms underlying how TACS influences cell movement are still not fully elucidated, with divergent opinions on whether and how the ECM mediates immune cell infiltration. At present, we currently lack a physical model relating the impact of TACS on the spatial co-evolution between an adaptive immune repertoire and a heterogeneous population of evading cancer cells. Here, we developed the EVO-ACT (EVOlutionary Agent-based Cancer T cell interaction) model, which is based on the Gillespie algorithm, to study the effects of TACS on tumor evolution and dynamical tumor-T cell interactions. Our analysis of tumor-T cell interactions across three TACS types reveals distinct migration patterns, with TACS3 showing the highest efficiency, TACS1 intermediate, and TACS2 the lowest. TACS impacts T cells more than tumors, with stronger chemokine gradients aiding T cell infiltration. Our model suggests that despite TACS2's limited efficiency, it cannot fully impede T cell infiltration. Varied migration efficiencies lead to diverse T cell infiltration patterns and immunoediting levels. TACS3 shows a benefit to immune recognition and higher survival, contrary to lower survival in TACS3 patients observed clinically in breast cancer. Our results indicate that only after introducing phenotypic changes, such as Epithelial-Mesenchymal Transition (EMT), and noting that EMT occurs exclusively after the TACS2-TACS3 progression, can our findings successfully explain the clinically observed trends. Considering mesenchymal tumor cells' ability to upregulate PD-1/PD-L1, we explore TACS-specific tumor evolution's impact on PD-1/PD-L1 inhibitor responses and patient survival. We observe higher tumor escape rates and lower survival in TACS3 compared to TACS2. Thus, TACS3 alone does not account for lower survival in breast cancer; TACS3-associated late-stage cancer byproducts like EMT and elevated checkpoint expression significantly decrease survival. Citation Format: Yijia Fan, Jason Tomas George. ECM architecture drives distinct evolutionary patterns of tumor immune escape and elimination in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A031.

Abstract C048: Intrapatient heterogeneity and targeting microenvironment protection mechanisms in melanoma metastasis

Abstract Intrapatient tumor heterogeneity contributes to immunotherapy and targeted therapy resistance in cutaneous metastatic melanoma patients. More than 50% of melanoma patients develop brain metastases (MBM) during disease progressionand these patients have a very poor prognosis with a median overall survival of 4 months. To assess how tumors persist through treatments and adapt to and rely on organ-specific microenvironments, we utilized samples from a patient who had received anti-CTLA4, BRAFi/MEKi, and BRAFi/MEKi+CDK4/6i treatments during the LOGIC2 clinical trial. Eight short-term melanoma cultures (STMCs) were established from different metastatic site biopsies at chronological time points throughout the patient’s treatment and subsequent post-mortem resection of the bone, ovary, and brain metastatic sites. While the initial treatment naïve metastatic tumor in the breast was BRAF V600E mutant, NRAS Q61 mutations were acquired after treatment with CDK4/6i in the bone and ovary tumors. We performed single-cell RNA sequencing and identified that each STMC consists of 2-10 different subpopulations. By cell viability assay, we determined all STMCs were resistant to CDK4/6i, however had varying degrees of response to BRAFi and MEKi in vitro. Two STMCs (MM150604, MM150859) were sensitive to both BRAFi and MEKi. We identified HER3 was upregulated in the sensitive STMCs and more specifically, HER3 expression was elevated in the MBM STMC and in other MBM patient tumors in publicly available datasets. In publicly available data of the MBM tumor microenvironment (TME), neurons produce NRG1 and NRG2 while astrocytes and pericytes produce NRG2, exogenous sources of NRGs in the MBM TME that act as ligands for the HER3 receptor. Treatment with recombinant NRG1 partially rescued the growth of MBM cells and restored MAPK and AKT-mTOR signaling in the presence of BRAFi/MEKi suggesting that the brain microenvironment may play a role in drug tolerance. This novel finding demonstrates a unique role of HER3 in MBM and targeting the NRG-HER3 signaling axis could have therapeutic potential in melanoma patients with active brain metastases. Citation Format: Haley P. Wilson, Glenn L. Mersky, Jelan I. Haj, Jessica L.F. Teh, Phil F. Cheng, Signe Caksa, Manal Mustafa, Isabella Trachtenberg, Francis J. Waltrich Jr, Casey D. Stefanski, Vivian Chua, Dan A. Erkes, Mitchell P. Levesque, Reinhard Dummer, Timothy J. Purwin, Claudia Capparelli, Andrew E. Aplin. Intrapatient heterogeneity and targeting microenvironment protection mechanisms in melanoma metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C048.

Site-Specific Quadruple-Functionalised Antibodies.

Antibody-drug conjugates (ADCs) are a growing class of chemotherapeutic agents that have yielded striking clinical successes. However, the efficacy of ADCs often suffers from issues associated with tumor heterogeneity and resistance. To overcome these problems, a new generation of ADCs comprising a single monoclonal antibody with multiple different payloads attached, termed multi-payload ADCs, have been developed. Here we deploy multiple orthogonal site-specific protein modification strategies to generate highly homogeneous multi-functionalised antibody conjugates comprising up to four different functionalities installed at four unique sites on the antibody. This work, which includes the use of a site-specific cyclopropenone (CPO)-based reagent, represents the first example of a homogeneous multi-payload ADC with a payload count greater than two, and thereby facilitates the development of the next generation of ADCs.

Site‐Specific Quadruple‐Functionalised Antibodies

AbstractAntibody–drug conjugates (ADCs) are a growing class of chemotherapeutic agents that have yielded striking clinical successes. However, the efficacy of ADCs often suffers from issues associated with tumor heterogeneity and resistance. To overcome these problems, a new generation of ADCs comprising a single monoclonal antibody with multiple different payloads attached, termed multi‐payload ADCs, have been developed. Here we deploy multiple orthogonal site‐specific protein modification strategies to generate highly homogeneous multi‐functionalised antibody conjugates comprising up to four different functionalities installed at four unique sites on the antibody. This work, which includes the use of a site‐specific cyclopropenone (CPO)‐based reagent, represents the first example of a homogeneous multi‐payload ADC with a payload count greater than two, and thereby facilitates the development of the next generation of ADCs.

Tumor heterogeneity and cooperating cancer hallmarks driven by divergent EMT programs.

Epithelial-to-mesenchymal transition (EMT) is known to play roles in orchestrating cellular plasticity across many physiological and pathological contexts. Partial EMT, wherein cells maintain both epithelial and mesenchymal features, is gaining recognition for its functional importance in cancer in recent years. There are many factors regulating both partial and full EMT, and the precise mechanisms underlying these processes vary depending on the biological context. Furthermore, how different EMT states cooperate to create a heterogeneous tumor population and promote different pro-malignant features remains largely undefined. In a recent study published in Nature Cancer, Youssef and colleagues described how two disparate EMT programs, active in either organ fibrosis or embryonic development, are utilized within different cells within the same murine mammary tumor model. This work provides mechanistic insight into the development of intratumoral heterogeneity, providing evidence for the cooperation between the two EMT trajectories.

Loss of XIST lncRNA unlocks stemness and cellular plasticity in ovarian cancer

Plasticity, a key hallmark of cancer, enables cells to transition into different states, driving tumor heterogeneity. This cellular plasticity is associated with cancer progression, treatment resistance, and relapse. Cancer stem cells (CSCs) play a central role in this process, yet the molecular factors underlying cancer cell stemness remain poorly understood. In this study, we explored the role of XIST (X-inactive specific transcript) long noncoding RNA in ovarian cancer stemness and plasticity through in silico and in vitro analyses. We found that XIST is significantly down-regulated in ovarian tumors, with low XIST expression linked to a higher stemness index and lower overall survival. Knocking down XIST in ovarian cancer cells enhanced stemness, particularly increasing mesenchymal-like CSCs, and under hypoxic conditions, it promoted epithelial-like CSC markers. Our findings suggest that XIST loss leads to CSC enrichment and cellular plasticity in ovarian cancer, pointing to potential therapeutic targets for patients with low XIST expression.

Ro 90-7501 suppresses colon cancer progression by inducing immunogenic cell death and promoting RIG-1-mediated autophagy.

Colon cancer is one of the leading causes of cancer-related mortality worldwide. Current treatments, including surgery, chemotherapy, and radiation therapy, often have limited efficacy due to tumor heterogeneity and resistance mechanisms. Therefore, there is a critical need for novel therapeutic strategies that can enhance the immune response against colon cancer cells while promoting their efficient clearance. In this study, we investigated the anti-tumor effects of Ro 90-7501, a specific small molecule, in colon cancer cell lines (HCT8 and SW480) and in vivo models. MTS, EdU, Scratch Wound and Transwell assays were performed to detect the cell proliferation and metastasis. Additionally, flow cytometry and immunofluorescence staining were used to analyze changes in apoptosis and necrosis. Furthermore, we examined its ability to induce immunogenic cell death (ICD) and promote retinoic acid-inducible gene I (RIG-I)-mediated autophagy. The expression levels of key molecules involved in ICD and autophagy were assessed using Western blot analysis, immunofluorescence staining, and enzyme-linked immunosorbent assay (ELISA). Our findings demonstrated that Ro 90-7501 significantly suppressed colon cancer cell proliferation and metastasis, inducing apoptosis and necrosis. Mechanistically, Ro 90-7501 triggered ICD by upregulating the exposure of calreticulin (CRT) on the cell surface and increasing the release of high mobility group box 1 (HMGB1) and extracellular ATP levels. Concurrently, it promoted RIG-I-mediated autophagy via the MAVS-TRAF6 signaling axis, evidenced by increased expression of autophagy-related genes, such as microtubule-associated protein 1 light chain 3 (LC3) and Beclin 1 proteins, coupled with a reduction in P62 protein. Additionally, Ro 90-7501 treatment activated the RIG-I-MAVS-TRAF6 signaling axis in colon cancer cells. Furthermore, Ro 90-7501 enhanced the secretion of pro-inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFα), thereby activating the immune response. In conclusion, Ro 90-7501 exhibits potent anti-tumor activity against colon cancer by inducing ICD and promoting RIG-I-mediated autophagy. These results suggest that Ro 90-7501 may serve as a promising therapeutic candidate for colon cancer treatment by enhancing both intrinsic cellular processes and adaptive immune responses. Further studies are warranted to elucidate the detailed mechanisms underlying these effects and to evaluate its therapeutic potential in clinical settings.

Current biological implications and clinical relevance of metastatic circulating tumor cells.

Metastatic disease and cancer recurrence are the primary causes of cancer-related deaths. Circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) are the driving forces behind the spread of cancer cells. The emergence and development of liquid biopsy using rare CTCs as a minimally invasive strategy for early-stage tumor detection and improved tumor management is a promising advancement in recent years. However, before blood sample analysis and clinical translation, precise isolation of CTCs from patients' blood based on their biophysical properties, followed by molecular identification of CTCs using single-cell multi-omics technologies is necessary to understand tumor heterogeneity and provide effective diagnosis and monitoring of cancer progression. Additionally, understanding the origin, morphological variation, and interaction between CTCs and the primary and metastatic tumor niche, as well as and regulatory immune cells, will offer new insights into the development of CTC-based advanced tumor targeting in the future clinical trials.

Molecular imaging of renal cell carcinomas: ready for prime time.

The clinical diagnosis of renal cell carcinoma (RCC) is constantly evolving. Diagnostic imaging of RCC relying on enhanced computed tomography (CT) and magnetic resonance imaging (MRI) is commonly used for renal mass characterization and assessment of tumour thrombosis, whereas pathology is the gold standard for establishing diagnosis. However, molecular imaging is rapidly improving the clinical management of RCC, particularly clear-cell RCC. Molecular imaging aids in the non-invasive visualization and characterization of specific biomarkers such as carbonic anhydrase IX and CD70 within the tumours, which help to assess tumour heterogeneity and status. Target-specific molecular imaging of RCCs will substantially improve the diagnostic landscape of RCC and will further facilitate clinical decision-making regarding initial staging and re-staging, monitoring of recurrence and metastasis, patient stratification and selection, and the prediction and evaluation of treatment responses.

A systematic literature review on clonal evolution events preceding relapse in multiple myeloma.

Despite considerable treatment advances, multiple myeloma (MM) remains an incurable hematological cancer due to treatment resistance. A systematic literature search was conducted to identify determinants for clonal evolution driving relapse and drug resistance in MM. A total of 631 non-duplicate publications were screened of which 28 articles were included for data extraction. Genetic alterations, mutational signatures, evolutionary trajectories, and non-genetic determinants were identified as key topics to characterize clonal evolution in relapsed MM. A variety of factors led to clonal diversification and increased tumor mutation burden, such as MAPK-Ras mutations and incremental changes related to chromosomal bands 1 and 17, while mutational signature analyses revealed that APOBEC activity and melphalan treatment leave a distinct impact on the clonal composition in MM genomes. To capture and dissect tumor heterogeneity, our review suggests combining methods or using technical approaches with high resolution to assess the impact of clonal evolution.

Pan-cancer analysis of STAT3 indicates its potential prognostic value and correlation with immune cell infiltration in prostate cancer.

Targeting the STAT3 signaling pathway is a promising therapeutic approach for cancer patients. However, the association between STAT3 expression, the tumor immune microenvironment, and genetic variation remains unclear across human cancers, especially prostate cancer. We used R software and other tools to analyze pan-cancer and mutation data from publicly available databases statistically. A comprehensive investigation was performed to assess the genetic heterogeneity and clinical relevance of STAT3 in various malignancies, with a specific focus on its role in the immune landscape and prognostic significance in prostate cancer. The findings were validated through immunohistochemistry (IHC) and multiplex immunofluorescence (mIF). STAT3 expression is abnormal in the majority of cancer tissues, which is strongly correlated with these patients' prognosis. Eight measures of tumor heterogeneity and six measures of tumor stemness of multiple tumor types showed a strong correlation with STAT3 expression. Furthermore, in individuals with prostate cancer, STAT3 expression indicated the degree of immune cell infiltration and the advancement of the disease. IHC analysis revealed that STAT3 was down-regulated in prostate tumor tissues, while mIF analysis demonstrated that STAT3 signaling (p-STAT3) was extensively active in tumor tissues and positive lymph node tissues. STAT3 may serve as a valuable prognostic biomarker and therapeutic target across various cancers, with particular relevance to prostate cancer.

From Images to Genes: Radiogenomics Based on Artificial Intelligence to Achieve Non-Invasive Precision Medicine in Cancer Patients.

With the increasing demand for precision medicine in cancer patients, radiogenomics emerges as a promising frontier. Radiogenomics is originally defined as a methodology for associating gene expression information from high-throughput technologies with imaging phenotypes. However, with advancements in medical imaging, high-throughput omics technologies, and artificial intelligence, both the concept and application of radiogenomics have significantly broadened. In this review, the history of radiogenomics is enumerated, related omics technologies, the five basic workflows and their applications across tumors, the role of AI in radiogenomics, the opportunities and challenges from tumor heterogeneity, and the applications of radiogenomics in tumor immune microenvironment. The application of radiogenomics in positron emission tomography and the role of radiogenomics in multi-omics studies is also discussed. Finally, the challenges faced by clinical transformation, along with future trends in this field is discussed.

Nanomaterials: breaking the bottleneck of breast cancer drug resistance

Drug resistance poses a significant challenge in the treatment of breast cancer. In recent years, a variety of nanomaterials have been discovered and synthesized that can selectively target tumor cells and play a crucial role in the advancement of breast cancer therapies. As our understanding of tumor heterogeneity deepens, the emerging potential of nanomaterials in addressing drug resistance has garnered considerable attention. These materials not only selectively target tumor cells but also possess unique properties that make them promising options for cancer treatment, including low toxicity, excellent biocompatibility, ease of preparation, the ability to carry antitumor drugs, and customizable surface functions. In this review, we will comprehensively summarize two key developments in breast cancer treatment: the application of antitumor drugs and nanomaterials. We will explore the mechanisms by which nanomaterials improve drug resistance in breast cancer, targeted nanotherapy strategies to mitigate this resistance, and recent research advancements in anticancer nanomaterials. This overview aims to highlight the significant role of nanomaterials in breast cancer treatment and provide a theoretical framework for identifying optimal treatment strategies in the future.

AR (CAG)n Microsatellite and APEX1 c.444T>G (p.Asp148Glu) Polymorphisms as Independent Prognostic Biomarkers in Prostate Cancer: Insights from an Argentinian Cohort

Background/Objectives: Prostate cancer (PCa) is the leading malignancy and the third most common cause of cancer-related death in Argentinian men. Predicting outcomes in localized PCa remains difficult due to tumor heterogeneity. In this study, we assessed the impact of AR (CAG)n and APEX1 c.444T>G polymorphisms on biochemical relapse in Argentine patients with localized PCa. Methods: We genotyped blood samples from 123 PCa patients for AR (CAG)n and APEX1 p.Asp148Glu (c.444T>G) polymorphisms. Associations with clinicopathological parameters and biochemical relapse-free survival (BRFS) were assessed. Results: AR (CAG)20–23 was associated with a family history of breast/ovarian cancer (p = 0.0469). The combination of AR (CAG)20–23 and APEX1 c.444TT/GG correlated with a 2.89 times higher risk of biochemical relapse (log-rank p = 0.006). Multivariable analysis confirmed AR and APEX1 polymorphisms as independent predictors of biochemical relapse (HR = 3.95, p = 0.002). In patients with PSA levels <10 ng/mL, combined AR (CAG)20–23 and APEX1 c.444TT/GG genotypes were significantly associated with an increased risk of biochemical relapse (HR = 2.61, p = 0.044). Multivariable analysis confirmed the prognostic significance of these genotypes (HR = 3.44, p = 0.02). Conclusions: This study has identified AR (CAG)n and APEX1 c.444T>G polymorphisms as independent predictors of PCa relapse in Argentinian patients, suggesting their potential use in improving prognostic models.

Abstract A036: Next gen liquid biopsy: Comprehensive analysis from a single tube of blood

Abstract Introduction There remains a largely untapped potential for utilizing liquid biopsy as a non- invasive, real-time window into tumor biology. In this study, we report on the use of the high- multiplex protein quantitation capability of the OrionTM spatial biology platform (RareCyte®, Inc) and cell picking capability of the CyteFinder® rare cell platform, to perform a comprehensive liquid biopsy analysis of whole blood that provides a deeper understanding of samples. This novel technology was used to characterize circulating tumor cells (CTCs), perform immune profiling of white blood cells (WBCs), and perform targeted mutation profiling of CTCs and cell-free DNA (cfDNA). All of these analytes can be measured from one 7.5 mL draw of blood. This array of tests was applied to cancer samples to demonstrate clinical feasibility. MethodsBlood samples were collected into AccuCyte® blood collection tubes. Buffy coats were processed to microscope slides using AccuCyte® blood processing kits. Slides were fixed and stained with an 18-plex immuno-oncology panel and imaged using the Orion Spatial Biology platform to evaluate protein biomarker expression on CTCs and to profile WBCs. Individual CTCs and WBCs from clinical samples were isolated using the CytePicker® for downstream molecular analysis. cfDNA was isolated from plasma collected during the AccuCyte process. Picked CTCs, WBCs, and cfDNA were sequenced using the CleanPlex® OncoZoom® Cancer Hotspot Kit. ResultsCancer patient blood was tested with a high-plex immunofluorescence assay using the Orion spatial biology system. CTCs were identified and phenotypically characterized in multiple cancer types. On the same slides, immunophenotyping of WBCs was performed. T- cell, B-cell, and macrophage populations were quantified. Additional sub-populations of T-cells (regulatory and memory) and proliferating cells (Ki67+) were enumerated. Expression of checkpoint-specific markers PD-L1 and PD-1 was measured on CTCs and WBCs. Sequencing results from individual picked CTCs showed evidence of minor clones representative of tumor heterogeneity that were not detectable in the cfDNA analysis. Conclusions This novel application of a high-plex spatial biology imaging system to liquid biopsies, in conjunction with the proven capabilities of rare cell detection and genomic analysis of single cells and cfDNA, has the potential to revolutionize liquid biopsy testing. From a single blood sample, this approach allows one to ask important questions about the complex interactions between the immune system and the tumor and to follow these interactions longitudinally in real time over the course of the disease. The goal is to leverage this unprecedented depth of analysis to improve patient outcomes and better understand the biological complexity of disease. Future directions of this work are to study rare types of WBCs that indicate immune activation and response to the tumor, and the detection and longitudinal monitoring of cell therapies such as CAR T cells after infusion. Citation Format: Arturo B Ramirez, Jon Ladd, Erin Bayer, Brock Bartels, Rachel Ponting, Arista Tischner. Next gen liquid biopsy: Comprehensive analysis from a single tube of blood [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A036.

Abstract PR014: Robust disease monitoring using CSF liquid biopsies collected from children undergoing cellular therapy for malignant central nervous system tumors

Abstract Background: Central nervous system (CNS) tumors remain the leading cause of cancer-related mortality in children, necessitating more effective treatment options. Chimeric Antigen Receptor T-cells (CAR-T) have emerged as a promising strategy to treat CNS tumors, with B7H3 (CD276) representing an attractive target due to its high expression across a broad range of entities. B7H3-CAR-T therapy is currently being evaluated in a St. Jude Children’s Research Hospital Phase I clinical trial (NCT05835687) for the treatment of relapse/refractory pediatric CNS tumors. Monitoring disease progression after CAR-T infusion by magnetic resonance imaging (MRI) remains challenging, emphasizing the need for more sensitive biomarkers of treatment response. Detection of circulating tumor DNA (ctDNA) in cerebrospinal fluid (CSF) liquid biopsies holds tremendous potential for minimal residual disease (MRD) monitoring in the clinical setting. Here, we leverage CSF liquid biopsies for longitudinal disease profiling in pediatric CNS tumor patients receiving B7H3-CAR-T immunotherapy. Methods: CSF samples were collected from Ommaya reservoirs of 9 pediatric patients with relapse/refractory CNS tumors undergoing B7H3-CAR-T therapy. CSF was sampled at study enrollment, and weekly intervals pre- and post-infusion. Cell-free DNA (cfDNA) was extracted from 115 longitudinally collected CSF samples and subjected to enzymatic methylation sequencing (EM-seq). Implementation of a custom computational workflow enabled detection of tumor-derived copy number variations (CNVs), estimation of malignant vs. non-malignant fractions, and molecular classification of tumor entity. Results: Tumor-derived CNVs were detected in 7/9 (78%) CSF samples collected at enrollment, prior to immunotherapy. Liquid biopsy-inferred CNV profiles and entity classifications were highly concordant with matched tumor tissue profiles. Chromosomal alterations that were restricted to CSF profiles indicated clonal evolution. Increased ctDNA burden was repeatedly observed after B7H3-CAR-T infusions, suggesting subclinical antitumor activity. Transient subclonal alterations emerging in response to treatment were also identified in a subset of patients. Conclusions: EM-seq is a feasible method for serial tracking of ctDNA burden in patients receiving cellular immunotherapy. CSF liquid biopsies capture the global landscape of tumor heterogeneity and genetic alterations that arise during therapy, providing a sensitive platform for detecting clinical response and disclosing potential mechanisms of treatment resistance. Citation Format: Anna Kostecka, Kyle S Smith, Katie Han, Hong Lin, Deanna Langfitt, Tara Walhart, Stephen Gottschalk, Giedre Krenciute, Christopher DeRenzo, Kelsey Bertrand, Paul A Northcott. Robust disease monitoring using CSF liquid biopsies collected from children undergoing cellular therapy for malignant central nervous system tumors [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR014.

Abstract A055: Tumor muscle invasion promotes tumor heterogeneity and normal muscle reprogramming

Abstract Aggressive human prostate tumors traverse a contractile smooth muscle pseudo-capsule, in a process termed extracapsular extension (ECE), defining the pT3 pathologic stage associating with increased biochemical recurrence, bone metastases, and cancer-specific mortality. While T3 lesions can be detected by non-invasive mpMRI imaging, how the tumor invades into and through the contractile muscle is unknown. Using a mouse xenograft model system of ECE, we investigated the transcriptomic consequences of human tumor invasion into and through the contractile smooth muscle of the mouse diaphragm. Both human and mouse gene transcripts were documented and analyzed. Tumor cells were intraperitoneally injected into male NSG mice and 6 weeks later, three tumor compartments (pre-invasive, muscle-invasive, and super-invasive cells that had reached the superior diaphragm surface) were analyzed for differential bulk human and mouse gene expression. Whole genome transcriptomic sequencing and GO pathway analysis revealed that approximately 414 human genes were differentially expressed between the non- invasive, muscle-resident, and super-invasive tumor populations and at least 5 enriched pathways were involved. The unique expression gene patterns of muscle-resident tumor cells were reversible when compared to the pre-invasive and super-invasive expression patterns. Significant increases in g-H2AX and nuclear deformation were observed in the muscle-resident tumor clusters as compared to the non-invasive tumor mass. No differences in tumor cell proliferation, as detected by Ki67 staining, were found between the tumor clusters in the three compartments. Immunohistochemistry staining for a damage response cytokeratin (KRT6A) and integrin (CD49f) revealed heterogeneity within the muscle resident tumors as compared to the non- invasive cells. Taken together, these data suggest a hostile contractile muscle environment elicits specific responses by the invading tumor. Single cell analysis within each of the tumor compartments is currently underway to define the spatial heterogeneity of gene expression in invasive tumor cell clusters within the contractile muscle. A dynamic reciprocity of the tumor/muscle microenvironment is suggested by the analysis of the bulk transcriptomic sequencing demonstrating a significant increase in mouse muscle bio-synthetic gene transcription as a consequence of the human tumor penetrating the tissue. Taken together, these data indicate that human tumors, during the act of traversing the contractile muscle layer, respond to this unique microenvironment by transiently altering transcription, while sustaining nuclear damage which results in the reprogramming of the muscle. This new information suggests that novel tumor or muscle biomarkers might indicate early muscle invasion events and assist new high-resolution image analysis for precision diagnostic and/or therapeutic decisions. (Supported in part by P30 CA23074; F30 CA143924, UACC Team Science Award). Citation Format: Kendra D Marr, Beatrice S Knudsen, Rafael Sainz, Kelvin W Pond, Noel E Warfel, Belinda E Sun, Anne E Cress. Tumor muscle invasion promotes tumor heterogeneity and normal muscle reprogramming [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A055.