Heterogeneous Cell Populations Research Articles

Single-Cell RNA Sequencing in Organ and Cell Transplantation

Single-cell RNA sequencing is a high-throughput novel method that provides transcriptional profiling of individual cells within biological samples. This method typically uses microfluidics systems to uncover the complex intercellular communication networks and biological pathways buried within highly heterogeneous cell populations in tissues. One important application of this technology sits in the fields of organ and stem cell transplantation, where complications such as graft rejection and other post-transplantation life-threatening issues may occur. In this review, we first focus on research in which single-cell RNA sequencing is used to study the transcriptional profile of transplanted tissues. This technology enables the analysis of the donor and recipient cells and identifies cell types and states associated with transplant complications and pathologies. We also review the use of single-cell RNA sequencing in stem cell implantation. This method enables studying the heterogeneity of normal and pathological stem cells and the heterogeneity in cell populations. With their remarkably rapid pace, the single-cell RNA sequencing methodologies will potentially result in breakthroughs in clinical transplantation in the coming years.

Considering the cellular landscape in marrow stimulation techniques for cartilage repair.

Marrow stimulation is a common reparative approach to treat injuries to cartilage and other soft tissues (e.g., rotator cuff). It involves the recruitment of bone marrow elements and mesenchymal stem cells (MSCs) into the defect, theoretically initiating a regenerative process. However, the resulting repair tissue is often weak and susceptible to deterioration with time. The populations of cells at the marrow stimulation site (beyond MSCs), and their contribution to inflammation, vascularity, and fibrosis, may play a role in quality of the repair tissue. In this review, we accomplish three goals: 1) systematically review clinical trials on the augmentation of marrow stimulation and evaluate their assumptions on the biological elements recruited; 2) detail the cellular populations in bone marrow and their impact on healing; and 3) highlight emerging technologies and approaches that could better guide these specific cell populations towards enhanced cartilage or soft tissue formation. We found that most clinical trials do not account for cell heterogeneity, nor do they specify the regenerative element recruited, and those that do typically utilize descriptions such as "clots", "elements", and "blood". Furthermore, our review of bone marrow cell populations demonstrates a dramatically heterogenous cell population, including hematopoietic cells, immune cells, fibroblasts, macrophages, and only a small population of MSCs. Finally, the field has developed numerous innovative techniques to enhance the chondrogenic potential (and reduce the anti-regenerative impacts) of these various cell types. We hope this review will guide approaches that account for cellular heterogeneity and improve marrow stimulation techniques to treat chondral defects.

Transmembrane Protein TMEM230, Regulator of Glial Cell Vascular Mimicry and Endothelial Cell Angiogenesis in High-Grade Heterogeneous Infiltrating Gliomas and Glioblastoma.

High-grade gliomas (HGGs) and glioblastoma multiforme (GBM) are characterized by a heterogeneous and aggressive population of tissue-infiltrating cells that promote both destructive tissue remodeling and aberrant vascularization of the brain. The formation of defective and permeable blood vessels and microchannels and destructive tissue remodeling prevent efficient vascular delivery of pharmacological agents to tumor cells and are the significant reason why therapeutic chemotherapy and immunotherapy intervention are primarily ineffective. Vessel-forming endothelial cells and microchannel-forming glial cells that recapitulate vascular mimicry have both infiltration and destructive remodeling tissue capacities. The transmembrane protein TMEM230 (C20orf30) is a master regulator of infiltration, sprouting of endothelial cells, and microchannel formation of glial and phagocytic cells. A high level of TMEM230 expression was identified in patients with HGG, GBM, and U87-MG cells. In this study, we identified candidate genes and molecular pathways that support that aberrantly elevated levels of TMEM230 play an important role in regulating genes associated with the initial stages of cell infiltration and blood vessel and microchannel (also referred to as tumor microtubule) formation in the progression from low-grade to high-grade gliomas. As TMEM230 regulates infiltration, vascularization, and tissue destruction capacities of diverse cell types in the brain, TMEM230 is a promising cancer target for heterogeneous HGG tumors.

Stem cell dynamics and cellular heterogeneity across lineage subtypes of castrate resistant prostate cancer.

To resist lineage-dependent therapies such as androgen receptor inhibition, prostate luminal epithelial adenocarcinoma cells often adopt a stem-like state resulting in lineage-plasticity and phenotypic heterogeneity. Castrate resistant prostate adenocarcinoma can transition to neuroendocrine and occasionally to amphicrine, co-expressed luminal and neuroendocrine, phenotypes. We developed CRPC patient-derived organoid models that preserve heterogeneity of the originating tumor, including an amphicrine model displaying a range of luminal and neuroendocrine phenotypes. To gain biological insight and to identify potential treatment targets within heterogeneous tumor cell populations, we assessed the lineage hierarchy and molecular characteristics of various CRPC tumor subpopulations. Transcriptionally similar stem/progenitor cells were identified for all lineage populations. Lineage tracing in amphicrine CRPC showed that heterogeneity originated from distinct subclones of infrequent stem/progenitor cells that produced mainly quiescent differentiated amphicrine progeny. By contrast, adenocarcinoma CRPC progeny originated from stem/progenitor cells and self-renewing differentiated luminal cells. NEPC was composed almost exclusively of self-renewing stem/progenitor cells. Amphicrine subpopulations were enriched for secretory luminal, mesenchymal, and enzalutamide treatment persistent signatures that characterize clinical progression. Finally, the amphicrine stem/progenitor subpopulation was specifically depleted with an AURKA inhibitor, which blocked tumor growth. These data illuminate distinct stem cell characteristics for subtype-specific CRPC in addition to demonstrating a context for targeting differentiation-competent prostate stem cells.

Intracellular acidification and glycolysis modulate inflammatory pathway in senescent cells.

Senescent cells accumulate in various organs with aging, and its accumulation induces chronic inflammation and age-related physiological dysfunctions. Several remodeling of intracellular environments has been identified in senescent cells, including enlargement of cell / nuclear size and intracellular acidification. Although these alterations of intracellular environments were reported to be involved in unique characteristics of senescent cells, the contribution of intracellular acidification to senescence-associated cellular phenotypes is poorly understood. Here, we identified that upregulation of TXNIP and its paralog ARRDC4 as a hallmark of intracellular acidification in addition to KGA-type GLS1. These genes were also upregulated in response to senescence-associated intracellular acidification. Neutralization of the intracellular acidic environment ameliorated not only senescence-related upregulation of TXNIP, ARRDC4, and KGA, but also inflammation-related genes, possibly through suppression of PDK-dependent anaerobic glycolysis. Furthermore, we found that expression of the intracellular acidification-induced genes, TXNIP and ARRDC4, correlated with inflammatory gene expression in heterogeneous senescent cell population in vitro and even in vivo, implying that the contribution of intracellular pH to senescence-associated cellular features, such as SASP.

Multi-Omics Profiling Reveals Phenotypic and Functional Heterogeneity of Neutrophils in COVID-19.

Accumulating evidence has revealed unexpected phenotypic heterogeneity and diverse functions of neutrophils in several diseases. Coronavirus disease (COVID-19) can alter the leukocyte phenotype based on disease severity, including neutrophil activation in severe cases. However, the plasticity of neutrophil phenotypes and their relative impact on COVID-19 pathogenesis has not been well addressed. This study aimed to identify and validate the heterogeneity of neutrophils in COVID-19 and evaluate the functions of each subpopulation. We analyzed public single-cell RNA-seq, bulk RNA-seq, and proteome data from healthy donors and patients with COVID-19 to investigate neutrophil subpopulations and their response to disease pathogenesis. We identified eight neutrophil subtypes: pro-neutrophil, pre-neutrophil, immature neutrophil, and five mature neutrophil subpopulations. The subtypes exhibited distinct features, including diverse activation signatures and multiple enriched pathways. The pro-neutrophil subtype was associated with severe and fatal disease, while the pre-neutrophil subtype was particularly abundant in mild/moderate disease. One of the mature neutrophil subtypes showed consistently large fractions in patients with different disease severity. Bulk RNA-seq dataset analyses using a cellular deconvolution approach validated the relative abundances of neutrophil subtypes and the expansion of pro-neutrophils in severe COVID-19 patients. Cell-cell communication analysis revealed representative ligand-receptor interactions among the identified neutrophil subtypes. Further investigation into transcription factors and differential protein abundance revealed the regulatory network differences between healthy donors and patients with severe COVID-19. Overall, we demonstrated the complex interactions among heterogeneous neutrophil subtypes and other blood cell types during COVID-19 disease. Our work has great value in terms of both clinical and public health as it furthers our understanding of the phenotypic and functional heterogeneity of neutrophils and other cell populations in multiple diseases.

Diagnostic utility of cytology in a patient with Epstein-Barr virus-positive mucocutaneous ulcer in palatine tonsils: A case report.

Epstein-Barr virus-positive mucocutaneous ulcer (EBVMCU) is a newly established immunodeficiency-related disease. Herein, we report a case of EBVMCU and focus on its cytological usefulness for diagnosis. An 82-year-old man manifested pharyngalgia, dysphagia, and oral pain. His medical history included rheumatoid arthritis that had been treated with methotrexate. Clinically, peritonsillar abscess was suspected, but since neoplastic lesions, including malignant lymphoma (ML), could not be excluded, a series of cytohistological examination was attempted. Despite some alarming findings (e.g., frequent mitoses), fine-needle aspiration and touch imprint cytology consistently revealed a heterogeneous population of lymphoid and plasmacytoid cells with mild nuclear atypia. The final diagnosis of EBVMCU was established based on the permanent histologic specimen; however, retrospectively, cytology was more representative of the benign nature of the lesion than histology, helping a great deal to differentiate it from ML. Cytology can be a useful tool for the correct diagnosis of EBVMCU.

Immunophenotypes in psychosis: is it a premature inflamm-aging disorder?

Immunopsychiatric field has rapidly accumulated evidence demonstrating the involvement of both innate and adaptive immune components in psychotic disorders such as schizophrenia. Nevertheless, researchers are facing dilemmas of discrepant findings of immunophenotypes both outside and inside the brains of psychotic patients, as discovered by recent meta-analyses. These discrepancies make interpretations and interrogations on their roles in psychosis remain vague and even controversial, regarding whether certain immune cells are more activated or less so, and whether they are causal or consequential, or beneficial or harmful for psychosis. Addressing these issues for psychosis is not at all trivial, as immune cells either outside or inside the brain are an enormously heterogeneous and plastic cell population, falling into a vast range of lineages and subgroups, and functioning differently and malleably in context-dependent manners. This review aims to overview the currently known immunophenotypes of patients with psychosis, and provocatively suggest the premature immune "burnout" or inflamm-aging initiated since organ development as a potential primary mechanism behind these immunophenotypes and the pathogenesis of psychotic disorders.

Abstract 423: Cell state-specific genomics to define plasticity along the epithelial-mesenchymal transitioncontinuum

Abstract The epithelial-to-mesenchymal transition (EMT) is a biological process that allows epithelial cells to convert into a mesenchymal phenotype, acquiring increased mobility and the capacity to migrate. EMT has historically been represented as binary states; however, a growing consensus supports an EMT continuum involving multiple intermediate stable states. While we lack a comprehensive understanding of the EMT continuum, several reports indicate that the late stable states lead to optimal stem cell traits for cancer. In addition, it has been suggested that EMT heterogeneity within tumor cell populations is associated with poor patient prognosis. Hence, there is a clear need to define the genetic drivers of EM plasticity in heterogeneous cell populations along the EMT continuum. To fulfill this need, we will collect and analyze single cell RNA sequencing (scRNA-seq) data from EMT-cell-state specific genetic CRISPR screens on EMT-heterogeneous cell populations. Here, we apply a series of cloning techniques including polymerase chain reaction and Gibson ligation to engineer an adeno-associated virus (AAV) plasmid containing the avian TVA receptor gene under control of the N-cadherin promoter. Since N-cadherin is predominantly expressed in mesenchymal cells, this AAV enables us to generate cell lines that will exclusively express the TVA receptor when in a mesenchymal state. Consequently, we will be able to deliver mesenchymal-specific CRISPR gene edits via an avian retrovirus-derived RCAS system. Citation Format: Destinee K. Williams, Nicholas Hou. Cell state-specific genomics to define plasticity along the epithelial-mesenchymal transitioncontinuum [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 423.

Abstract 67: Predicting colorectal patient prognoses by functional characterisation of heterogeneous cell types and their spatial interaction using a new technique: Whole slide imaging mass cytometry

Abstract Immunotherapies stimulate T cell function in the tumor microenvironment (TME). CD3+ and CD8+ T cell infiltration can predict disease recurrence in colorectal cancer (CRC) patients and has been validated as Immunoscore® (IS). The TME comprises multiple heterogeneous populations of immune cells, including T cells and cancer-associated fibroblasts (CAFs); CRC tumors are also morphologically diverse. Standard BioTools™ (SBI) has developed a novel whole slide Imaging Mass Cytometry™ technique that allows complete analysis of cell population heterogeneity in the TME using >40 markers. We hypothesised there are multiple heterogeneous subtypes of CAFs within CRC tumors and that they modulate T cell infiltration and patient outcome. In collaboration with SBI, we studied a cohort of CRC patients stratified based on T cell infiltrate and disease recurrence. We identified multiple CAF populations that interact with tumor-infiltrating T cell populations. Slides from 10 CRC patients with high or low CD3+CD8+ T cell infiltrate (IS) were stained with a panel encompassing markers for CAF and T cell heterogeneity, function, and metabolism. Slides were imaged using the Hyperion XTi™ Imaging System at SBI. Using a scan mode called Preview Mode, the whole slide was imaged in minutes to identify CAF and T cell phenotypic regions of interest. Next, the same slide was imaged at 1 μm resolution using Cell Mode (CM). Finally, for each sample, an additional serial section was imaged using a slightly lower resolution called Tissue Mode (TM). TM allowed us to image the samples in several hours instead of days while capturing the full heterogeneity of the entire tissue sample. We used clustering to identify phenotype clusters and neighbourhood analysis to study spatial enrichment. We also compared results between CM and TM. We report: Identification of heterogeneous CAF subtypes in CRC patients with high or low IS; determination of CAF populations that spatially interact with T cells in patients with high or low IS; and examination of consistent spatial interactions between CAF and T cells in CRC, and whether these interactions differ in patients with high or low IS. TM revealed several heterogeneous cellular populations that differed between patients with high or low IS; importantly, low IS tumors had more podoplanin+αSMA+ CAF populations and fewer podoplanin+αSMA- cell populations compared with high IS tumors. High IS tumors had more PD-1+CD8+ T cells compared with low IS tumors; these PD-1+CD8+ T cells spatially interacted with podoplanin+αSMA- CAF populations in high IS tumors. CAF-PD-1+CD8+ T cell interactions were absent from low IS tumors. Thus, CAF-PD-1+CD8+ T cell interactions are important in CRC patients with high IS, and these cellular interactions require further investigation. Citation Format: Rory M. Costello, Sonya Fenton, Helen McGuire, Liang Lim, David Howell, Qanber Raza, Jyh Yun Chwee, Roslyn Kemp. Predicting colorectal patient prognoses by functional characterisation of heterogeneous cell types and their spatial interaction using a new technique: Whole slide imaging mass cytometry [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 67.

Abstract 6549: Mechanistic models of tumor and patient heterogeneity explain and predict clinical outcomes of large B-cell lymphoma (LBCL) treatment

Abstract Introduction: Heterogeneity between patients and within tumors poses challenges to effective cancer treatment. Drug combinations can overcome heterogeneity by increasing the probability that a patient will have response to one or more drugs. This approach is successful in numerous disease settings, but technological advances have led to new approach to addressing heterogeneity: precision approaches to stratify patients into subpopulations with more uniform, stronger responses to specific drugs. These strategies can be implemented together to improve patient outcomes. We have developed computational mechanistic models of cancer treatment in the context of both sources of heterogeneity which explain and predict clinical outcomes of treatment of LBCL with combination therapy and CAR T-cell therapy. Combination therapy: Our novel population tumor kinetics (pop-TK) model simulates clinical combination therapy outcomes by implementing multidrug dose response functions in heterogeneous populations of tumor cells, within heterogeneous cohorts of patients. Informed by the outcomes of novel drugs in phase 2 trials in relapsed/refractory (r/r) patients, the pop-TK model accurately predicted of outcomes of seven first-line phase 3 trials of novel combinations. Most notably, informed by treatment of r/r LBCL with polatuzumab-vedotin (pola), the pop-TK predicted the success of modifying the standard treatment regimen (RCHOP) with pola before the clinical trial results were published. The pola combination improves overall survival (OS) and PFS in Activated B-Cell (ABC) LBCL (hazard ratio (HR): OS 0.3, PFS 0.4), but reduces OS (HR: 1.6) and does not improve PFS (HR: 1.0) in Germinal Center B-Cell (GCB) LBCL. The subtype specific difference in PFS was also predictable based on subtype results of r/r trials (predicted HR: ABC 0.3, GCB 0.9). CAR T-cell therapy: We used a population pharmacokinetic/pharmacodynamic (pop-PK/PD) model of CAR T-cell therapy to reproduce the PFS distribution and the distribution CAR T-cell abundance for CAR T therapy in r/r LBCL patients. The impact of tumor size on patient outcome in our model and in clinical studies led us to computationally explore the impact treating smaller tumors with CAR T-cell therapy on treatment outcomes. We found that using detectable circulating tumor DNA (ctDNA) as a biomarker for administering CAR T-cell therapy immediately after RCHOP increased the proportion of simulated patients without a second disease progression event by 30% (HR: 0.43 [0.34, 0.55]). Conclusions: The pop-TK model supports LBCL subtype biomarker for pola performance, and the pop-PK/PD model demonstrates that detectable ctDNA is a potential biomarker for early CAR T-cell administration. These examples of model-driven precision trial design have the potential to improve outcomes using readily-available biomarkers and existing therapies. Citation Format: Amy E. Pomeroy, Adam C. Palmer. Mechanistic models of tumor and patient heterogeneity explain and predict clinical outcomes of large B-cell lymphoma (LBCL) treatment [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 6549.

Abstract 3646: A spatially resolved single cell proteomic atlas of small bowel adenocarcinoma

Abstract Small bowel adenocarcinoma (SBA) is a rare malignancy associated with poor prognosis. The cellular and proteomic heterogeneity within the tumor immune microenvironment (TIME) of SBA is a likely driver of prognosis, disease progression and response to therapy. There is, however, a major gap in our knowledge of tumor and immune interactions in SBA. Cyclic Immunofluorescence (CycIF), an antibody-based, highly multiplexed imaging technology for spatially resolved single cell level proteomic profiling, is well suited to map the proteomic heterogeneity and organization of TIME in SBA. Here, using CycIF, we have generated a spatially resolved single-cell proteomic atlas of TIME for 136 tumor and normal samples from clinically and genomically annotated SBA patients (N=37). The SBA TIME Atlas covers information on expression levels, spatial position, and cell morphology for > 40 proteomic markers of tumor-intrinsic processes and states, diverse immune cell types, immune checkpoints, and tumor vascularization. In total, we collected proteomics data from > 600,000 cells. Using a hierarchical decision tree of cell type markers, we have generated the cell identity annotations for > 350,000 cells, with positional information. A comprehensive computational analysis of the SBA atlas revealed various spatial and heterogeneity features that may impact the TIME organization in fine detail. Using those features, we have compared the proteomic heterogeneity in tumor vs. normal cell populations, identifying spatial and single-cell correlates of key clinical variables (e.g., patient survival, age, sex, disease stage, metastatic status). We have also performed differential analyses of spatial features involving protein markers and cell types with respect to genomic alteration states and have mapped the spatial enrichment of likely actionable immune check proteins to immune and tumor cells. Such mapping may guide future therapeutic decisions. Overall, we expect that the SBA TIME Atlas will provide a useful resource and inform future translational studies, as well as basic research for better mechanistic understanding and treatment of this disease. Citation Format: Zeynep Dereli, Behnaz Bozorgui, Huamin Wang, John Weinstein, Michael Overman, Anil Korkut. A spatially resolved single cell proteomic atlas of small bowel 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 3646.

Abstract 7041: Tumor-immune interactions and cisplatin resistance in localized muscle-invasive bladder cancer

Abstract Background: Cisplatin-based chemotherapy followed by radical cystectomy remains the standard-of-care treatment for patient with localized muscle-invasive bladder cancer (MIBC). Neoadjuvant clinical trials studying the combination of cisplatin-based chemotherapy and immune checkpoint blockade (ICB) in MIBC reported encouraging pathologic complete response rates. However, the molecular mechanisms and cellular programs involved in response and resistance to cisplatin and ICB in MIBC remain incompletely characterized. Our goal is to define cellular transcriptional programs and chromatin accessible regions in cancer cells and infiltrating immune cells associated with response and resistance to neoadjuvant systemic therapy. Methods: We performed single-cell Multiome ATAC and Gene expression sequencing (simultaneous identification of gene expression and open chromatin regions within the same cell) of tumors that had a complete response – “responders” – and tumors that had no response or progressed – “non-responders” – through neoadjuvant cisplatin-based chemotherapy (n=15). Single cell suspensions were prepared following the 10x Genomics protocol. Cell Ranger ARC was used for sequence alignment, peak calling, and generation of count matrices. Downstream single-cell gene expression analysis was performed with Scanpy and ATAC-seq with Seurat. Results: We identified two distinct urothelial cell populations in "responders" vs "non-responders" based on single cell gene expression and genome wide chromatin accessibility regions. Cancer cells in “non-responders” preferentially express basal cell gene expression signatures. “Non-responders” were significantly enriched in suppressive tumor-associated macrophages (TAMs). Interestingly, SPP1+ TAMs known to have a pro-tumoral role and promote resistance to ICB were exclusively identified in “non-responders”. Conclusion: Single cell and genome wide chromatin accessibility regions identify heterogenous urothelial cell populations expressing basal cell markers in tumors resistant to neoadjuvant cisplatin-based chemotherapy. “Non-responders” are significantly enriched with TAMs implicated in resistance to ICB. Future studies will better define the implications of these findings in cancer therapeutics for patients with MIBC. Citation Format: Filipe La Fuente De Carvalho, Jihyun Lee, Kevin Bi, Breanna Titchen, Konrad Stawiski, Jihye Park, Amanda Garza, Daniel Michaud, Jennifer Guerriero, Elizabeth Mittendorf, Kent Mouw, Eliezer Van Allen. Tumor-immune interactions and cisplatin resistance in localized muscle-invasive bladder 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 7041.

Abstract 548: Exploiting pronounced selection pressure from therapeutic bottlenecks to identify orthogonal immunotherapy targets

Abstract Evolutionary processes play a crucial role in the development of cancer, where tumor cells continuously adapt, leading to the dominance of cell populations over their non-tumor counterparts, evading treatment and ultimately resulting in death. Effective cancer therapies induce significant and targeted selection pressures. During and in response to the induced bottleneck by therapy, the heterogeneous cancer cell population either diminishes or evolves, causing a subsequent selective sweep and the emergence of cells with resistance traits. However, this bottleneck phase presents a unique opportunity to predictably reduce the diversity of tumor cells. In this context, we propose a framework to quantify the selection on variants before, during, and after the therapeutic bottleneck, enhancing our understanding of the mechanisms underlying the acquisition of therapeutic resistance. Furthermore, we combine measures of selection intensity with the prediction of neoantigens in a patient cohort to identify potential alternative targets for immunotherapy. The outcome is a strategy that exploits the predictability introduced by the primary therapeutic bottleneck to eliminate the remaining tumor cell population. Finally, we illustrate the efficacy of this strategy in the context of several cancer types and empirically identify potential cancer type specific and pan-cancer immunotherapy targets orthogonal to primary, first-line therapies. Citation Format: J. Nic Fisk, Jeffrey P. Townsend. Exploiting pronounced selection pressure from therapeutic bottlenecks to identify orthogonal immunotherapy targets [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 548.

Abstract 6797: CCR5/CCL4 axis in lung cancer clinicopathological significance and its therapeutic application

Abstract Lung cancer (LC) is the most prevalent type of cancer globally and the leading cause of cancer-related deaths. Based on the histology and type of treatments, LC is divided into small-cells and non-small cell lung cancer (NSCLC) carcinomas, which are more aggressive and less responsive to chemotherapy. Since LC is known to be a highly heterogeneous cell population, treatment is challenging. It has been observed that chemokine receptor-ligand interaction is essential for cancer cells homing to distant sites. Most malignancies, including LC, have established disease status indicators concerning elevated levels of CCL4, a structurally unique chemokine, and its receptor (CCR5). Therefore, treatment strategies that inhibit the LC cell's tumor environment may improve patients' quality of life. However, the CCR5/CCL4 interface in LC invasion and metastasis is poorly understood. In this study, we examined the expression and function of the CCR5/CCL4 axis in the development and invasiveness of LC patient's clinical tissues and cell lines. We conducted immunohistochemistry (IHC) dual staining of CCR5/CCL4 antibodies in LC tissues. Our findings demonstrated significantly (p < 0.001) higher expression of CCR5 and CCL4 in advanced-stage malignant tissues than in normal tissues. The in-vitro analysis of the human LC cell lines NCI-H1437, NCI-H1573, and NCI-H1975 revealed CCL4 activation expressing higher CCR5, determined by immunofluorescence, western blots, flow cytometry, 3D-invasion assay, and RT-qPCR methods. Further, a calcium mobilization assay confirmed the direct interaction between CCR5 and CCL4. However, treating with Maraviroc, a small molecule inhibitor of CCR5, to LC cells blocks their ability to proliferate, demonstrating the crucial role that CCR5-CCL4 chemotaxis plays in LC metastasis. Our findings show both the biological and clinical significance of CCR5 and CCL4 expression, which may serve as potential indicators for metastatic LC. Therefore, inhibiting the CCR5/CCL4 interaction may offer a more effective therapeutic approach for managing the progression of LC. Citation Format: Santosh K. Singh, Rajesh Singh. CCR5/CCL4 axis in lung cancer clinicopathological significance and its therapeutic application [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 6797.

Liver metastatic colonization by invasive cancer cells: a review of potential biomarkers with mitochondrial involvement

The liver, characterized by a unique metabolic and immunosuppressive environment, is also the organ to which invasive malignant cells of many different cancer types most frequently metastasize. The reasons for this organ-specific metastatic process have been investigated for decades. This review first provides an overview of recent breakthroughs in this field, introducing intercellular communication between circulating tumor cells and the heterogeneous cell populations of the liver, and modifications to the extracellular matrix (ECM). Subsequently, to improve the understanding of the molecular mechanisms involved in the metastasis of colorectal cancer to the liver, the second leading cause of cancer-related mortality, the recent literature on this question was analyzed. Among the various parameters involved, the mechanisms behind the activation of hepatic stellate cells, proteins inducing ECM remodeling, specific genomic features of liver metastases, metabolic rewiring, and characteristics of stromal-enriched microenvironments were discussed. To provide more insights into the molecular determinants of liver metastatic colonization, important findings reported on a set of mitochondrial proteins were addressed, the relative abundance of which changed in the liver during the progression stage of an aggressive experimental model of peritoneal malignant mesothelioma in immunocompetent rats. Based on previous studies cross-comparing the liver proteomes from curcumin-treated tumor-bearing rats/untreated tumor-bearing rats/normal rats, data from the literature were reviewed for 25 mitochondrial proteins of interest. Their role in lipid metabolism, heme biosynthesis, the electron transport chain, small molecule transport, mitochondrial dynamics, the tricarboxylic acid cycle, and protection against oxidative stress were analyzed in the context of both cancer and non-malignant liver diseases.

Abstract 6896: Biomimetic hydrogels at physiological stiffness reprogram patient derived melanoma cells

Abstract Melanoma is a uniquely difficult cancer to treat due to complex tumor heterogeneity and resistance, where upon dissemination, survival drops to less than 17%. Drug resistance has arisen for the current standard of care and combinatorial therapies, expediting the need for new efficacious agents. The lack of physiologically relevant preclinical models has impeded the development of these therapeutics. Potential melanoma drugs are typically assayed using cell lines on tissue culture plastic, then tested for in vivo efficacy and side effects in animals. This paradigm is inherently unsuitable for the generation of effective therapies as typical cell culture platforms fail to replicate the complex microenvironments known to play critical roles in tumor pathology and rodent models are prohibitively expensive for optimizing novel and combination therapies. Thus, our goal is to synthesize tissue-engineered melanoma models using biomimetic polymers that recapitulate patient tumor characteristics to assess combination and novel therapies that attenuate resistance and disrupt metastatic processes. In this study, patient melanoma cells, which were unable to recapitulate drug responses on traditional cell culture platforms, were cultured in a controlled spatial and temporal manner on polyacrylamide hydrogels. The hydrogel scaffolds feature stiffnesses spanning that of physiological tissue, with microcontact printed human fibronectin for biomimetic protein presentation to create a microtumor array. Cells cultured on the hydrogel showed a 2.5-fold increase in expression of the cancer stem cell biomarkers, CD271 and ABCB5, which play a key role in melanoma survival and recurrence, compared to cells cultured on plastic or glass substrates. Further study identified BRAF mutant, drug resistant patient cells, regained resistance to standard of care targeted therapies, trametinib and dabrafenib. Through hydrogel microarrays featuring physiological similarities, the technology has enabled the exploration of heterogeneous cell populations with novel and combinatorial therapeutics. Citation Format: Yi L. Liu, Justyna Gleba, Chantal Kopecky, Matthew Pawlush, John A. Copland, Kristopher A. Kilian. Biomimetic hydrogels at physiological stiffness reprogram patient derived melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6896.

The role of lipids in genome integrity and pluripotency.

Pluripotent stem cells (PSCs), comprising embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), offer immense potential for regenerative medicine due to their ability to differentiate into all cell types of the adult body. A critical aspect of harnessing this potential is understanding their metabolic requirements during derivation, maintenance, and differentiation in vitro. Traditional culture methods using fetal bovine serum often lead to issues such as heterogeneous cell populations and diminished pluripotency. Although the chemically-defined 2i/LIF medium has provided solutions to some of these challenges, prolonged culturing of these cells, especially female ESCs, raises concerns related to genome integrity. This review discusses the pivotal role of lipids in genome stability and pluripotency of stem cells. Notably, the introduction of lipid-rich albumin, AlbuMAX, into the 2i/LIF culture medium offers a promising avenue for enhancing the genomic stability and pluripotency of cultured ESCs. We further explore the unique characteristics of lipid-induced pluripotent stem cells (LIP-ESCs), emphasizing their potential in regenerative medicine and pluripotency research.

Allele-specific transcriptional effects of subclonal copy number alterations enable genotype-phenotype mapping in cancer cells

Subclonal copy number alterations are a prevalent feature in tumors with high chromosomal instability and result in heterogeneous cancer cell populations with distinct phenotypes. However, the extent to which subclonal copy number alterations contribute to clone-specific phenotypes remains poorly understood. We develop TreeAlign, which computationally integrates independently sampled single-cell DNA and RNA sequencing data from the same cell population. TreeAlign accurately encodes dosage effects from subclonal copy number alterations, the impact of allelic imbalance on allele-specific transcription, and obviates the need to define genotypic clones from a phylogeny a priori, leading to highly granular definitions of clones with distinct expression programs. These improvements enable clone-clone gene expression comparisons with higher resolution and identification of expression programs that are genomically independent. Our approach sets the stage for dissecting the relative contribution of fixed genomic alterations and dynamic epigenetic processes on gene expression programs in cancer.

Immunosuppressive effects of the mycotoxin patulin in macrophages.

Patulin (PAT) is a fungi-derived secondary metabolite produced by numerous fungal species, especially within Aspergillus, Byssochlamys, and Penicillium genera, amongst which P. expansum is the foremost producer. Similar to other fungi-derived metabolites, PAT has been shown to have diverse biological features. Initially, PAT was used as an effective antimicrobial agent against Gram-negative and Gram-positive bacteria. Then, PAT has been shown to possess immunosuppressive properties encompassing humoral and cellular immune response, immune cell function and activation, phagocytosis, nitric oxide and reactive oxygen species production, cytokine release, and nuclear factor-κB and mitogen-activated protein kinases activation. Macrophages are a heterogeneous population of immune cells widely distributed throughout organs and connective tissue. The chief function of macrophages is to engulf and destroy foreign bodies through phagocytosis; this ability was fundamental to his discovery. However, macrophages play other well-established roles in immunity. Thus, considering the central role of macrophages in the immune response, we review the immunosuppressive effects of PAT in macrophages and provide the possible mechanisms of action.