Cell Clonality Research Articles

SLC7A9 suppression increases chemosensitivity by inducing ferroptosis via the inhibition of cystine transport in gastric cancer

SLC7A9 is responsible for the exchange of dibasic amino acids and cystine (influx) for neutral amino acids (efflux). Cystine/cysteine transport is related to ferroptosis. Sanger sequencing detected TP53 status of cancer cells. Transcriptomic sequencing and untargeted metabolome profiling were used to identify differentially expressed genes and metabolites, respectively, upon SLC7A9 overexpression. CCK8, cell clonality, and EdU assays were used to observe cell proliferation. Cystine probes, glutathione (GSH) probes, and lipid ROS probes were used to examine cystine, GSH, and lipid ROS levels. 13C metabolic flow assays were used to monitor cellular cystine and GSH metabolism. Patient-derived organoids (PDO), immunocompetent MFC mice allograft models and patient-derived xenograft (PDX) models were used to evaluate SLC7A9 impact on chemotherapeutic response and to observe therapeutic effect of SLC7A9 knockdown. Elevated SLC7A9 expression levels in gastric cancer cells were attributed to p53 loss. SLC7A9 knockdown suppressed the proliferation and increased the chemotherapy sensitivity of the cells. Chemotherapy was more effective in PDX and immunocompetent mice models upon SLC7A9 knockdown. Differentially expressed genes and metabolites between the SLC7A9 overexpression and control groups were associated with ferroptosis and GSH metabolism. SLC7A9 knockdown reduced cystine transport into cells, hampered intracellular cystine and GSH metabolic flow, decreased GSH synthesis, and increased lipid ROS levels in gastric cancer cells. Erastin was more effective at inducing ferroptosis in PDO and PDX models upon SLC7A9 knockdown. SLC7A9 promotes gastric cancer progression by acting as a suppressor of ferroptosis, independent of SLC7A11, which is negatively regulated by p53. This work was supported by National Natural Science Foundation of China, Innovation Promotion Program of NHC and Shanghai Key Labs SIBPT, and Shanghai Academy of Science & Technology.

MicroRNA-450b-5p modulated RPLP0 promotes hepatocellular carcinoma progression via activating JAK/STAT3 pathway

Hepatocellular carcinoma (HCC) is distinguished by its insidious onset, difficult treatment, and poor prognosis. Ribosomal Protein Lateral Stalk Subunit P0 (RPLP0) is implicated in numerous tumor progression processes. Nevertheless, the regulatory mechanism of RPLP0 in HCC progression remains unclear. Our study suggested that RPLP0 exhibits high expression levels in HCC and possesses promising diagnostic capabilities, as indicated by its area under the curve (AUC) of 0.908. Further analysis showed that RPLP0 was a significant independent prognostic factor, and elevated expression levels of RPLP0 were linked with poorer overall survival (OS) and progression-free interval (PFI) outcomes. Additionally, reducing RPLP0 levels led to a decrease in HCC cell proliferation, clonality, invasion, migration, and xenograft tumor growth, as well as an increase in apoptosis. Furthermore, our findings indicated that microRNA(miR)-450b-5p induced downregulation of RPLP0, leading to the suppression of the JAK/STAT3 pathway and consequently hindering the advancement of HCC. The study indicates that RPLP0 plays a role as a carcinogenic factor in HCC and carries important diagnostic and prognostic implications. Targeting the miR-450b-5p/RPLP0/JAK/STAT3 axis has potential clinical value in treating HCC.

A-286 T cell receptor beta constant 1 as a potential marker for the assessment of T cell clonality in skin disease

A-286 T cell receptor beta constant 1 as a potential marker for the assessment of T cell clonality in skin disease

Tissue determinants of the human T cell receptor repertoire.

98% of T cells reside in tissues, yet nearly all human T cell analyses are performed from peripheral blood. We single-cell sequenced 5.7 million T cells from ten donors' autologous blood and tonsils and sought to answer key questions about T cell receptor biology previously unanswerable by smaller-scale experiments. We identified distinct clonal expansions and distributions in blood compared to tonsils, with surprisingly low (1-7%) clonal sharing. These few shared clones exhibited divergent phenotypes across bodily sites. Analysis of antigen-specific CD8 T cells revealed location as a main determinant of frequency, phenotype, and immunodominance. Finally, diversity estimates from the tissue recalibrates current repertoire diversity estimates, and we provide a refined estimate of whole-body repertoire. Given the tissue-restricted nature of T cell phenotypes, functions, differentiation, and clonality revealed by this dataset, we conclude that tissue analyses are crucial for accurate repertoire analysis and monitoring changes after perturbing therapies.

Somatic nuclear mitochondrial DNA insertions are prevalent in the human brain and accumulate over time in fibroblasts.

The transfer of mitochondrial DNA into the nuclear genomes of eukaryotes (Numts) has been linked to lifespan in nonhuman species and recently demonstrated to occur in rare instances from one human generation to the next. Here, we investigated numtogenesis dynamics in humans in 2 ways. First, we quantified Numts in 1,187 postmortem brain and blood samples from different individuals. Compared to circulating immune cells (n = 389), postmitotic brain tissue (n = 798) contained more Numts, consistent with their potential somatic accumulation. Within brain samples, we observed a 5.5-fold enrichment of somatic Numt insertions in the dorsolateral prefrontal cortex (DLPFC) compared to cerebellum samples, suggesting that brain Numts arose spontaneously during development or across the lifespan. Moreover, an increase in the number of brain Numts was linked to earlier mortality. The brains of individuals with no cognitive impairment (NCI) who died at younger ages carried approximately 2 more Numts per decade of life lost than those who lived longer. Second, we tested the dynamic transfer of Numts using a repeated-measures whole-genome sequencing design in a human fibroblast model that recapitulates several molecular hallmarks of aging. These longitudinal experiments revealed a gradual accumulation of 1 Numt every ~13 days. Numtogenesis was independent of large-scale genomic instability and unlikely driven by cell clonality. Targeted pharmacological perturbations including chronic glucocorticoid signaling or impairing mitochondrial oxidative phosphorylation (OxPhos) only modestly increased the rate of numtogenesis, whereas patient-derived SURF1-mutant cells exhibiting mtDNA instability accumulated Numts 4.7-fold faster than healthy donors. Combined, our data document spontaneous numtogenesis in human cells and demonstrate an association between brain cortical somatic Numts and human lifespan. These findings open the possibility that mito-nuclear horizontal gene transfer among human postmitotic tissues produces functionally relevant human Numts over timescales shorter than previously assumed.

Identification of prostate cancer bone metastasis related genes and potential therapy targets by bioinformatics and invitro experiments.

The aetiology of bone metastasis in prostate cancer (PCa) remains unclear. This study aims to identify hub genes involved in this process. We utilized machine learning, GO, KEGG, GSEA, Single-cell analysis, ROC methods to identify hub genes for bone metastasis in PCa using the TCGA and GEO databases. Potential drugs targeting these genes were identified. We validated these results using 16 specimens from patients with PCa and analysed the relationship between the hub genes and clinical features. The impact of APOC1 on PCa was assessed through invitro experiments. Seven hub genes with AUC values of 0.727-0.926 were identified. APOC1, CFH, NUSAP1 and LGALS1 were highly expressed in bone metastasis tissues, while NR4A2, ADRB2 and ZNF331 exhibited an opposite trend. Immunohistochemistry further confirmed these results. The oxidative phosphorylation pathway was significantly enriched by the identified genes. Aflatoxin B1, benzo(a)pyrene, cyclosporine were identified as potential drugs. APOC1 expression was correlated with clinical features of PCa metastasis. Silencing APOC1 significantly inhibited PCa cell proliferation, clonality, and migration invitro. This study identified 7 hub genes that potentially facilitate bone metastasis in PCa through mitochondrial metabolic reprogramming. APOC1 emerged as a promising therapeutic target and prognostic marker for PCa with bone metastasis.

CD4+CD8αlow T cells in rheumatoid arthritis are clonally expanded and dependent on co-stimulation.

CD4+CD8+ T cells are increased in patients with rheumatoid arthritis (RA). They are not only associated with joint erosions in established disease, but are also present in the pre-clinical stages of RA. This study aims to further investigate their expansion in the context of T cell clonality in patients with RA, as well as their responsiveness to T cell targeted treatment. Single-cell-(sc)RNA- and scTCR-sequencing data were used to determine co-receptor expression and T cell receptor sequences to assess clonality of CD4+CD8+ T cells in RA (n=3) patients and healthy controls (n=2). Peripheral CD4+CD8+ T cells and their subpopulations were measured in patients with RA (n=53), PsA (n=52) and healthy donors (n=50) using flow cytometry. In addition, changes in CD4+CD8+ T cell frequency were prospectively followed in RA patients receiving therapy with abatacept for 12 weeks. We observed an increase of CD4+ T cells expressing CD8α in RA patients, both in comparison to PsA patients and to healthy controls. Clonality analysis revealed, that these CD4+CD8αlow T cells are part of large T cell clones, which cluster separately from CD4+CD8- T cell clones in the scRNA-seq gene expression analysis. Treatment with abatacept significantly reduced the frequency of peripheral CD4+CD8αlow T cells, and this was linked to reduction in disease activity. In RA, clonal expansion of CD4+ T cell clones culminates in the emergence of peripheral CD4+CD8αlow T cells, which are associated with disease activity and diminished upon abatacept treatment, and which could contribute to disease pathogenesis.

Single-cell RNA-sequencing identifies unique cell-specific gene expression profiles in high-grade cardiac allograft vasculopathy.

Cardiac allograft vasculopathy (CAV), a diffuse thickening of the intima of the coronary arteries and microvasculature, is the leading cause of late graft failure and mortality after heart transplantation (HT). Diagnosis involves invasive coronary angiography, which carries substantial risk, and minimally-invasive approaches to CAV diagnosis are urgently needed. Using single-cell RNA-sequencing in peripheral blood mononuclear cells (PBMCs), we sought to identify cell-specific gene expression profiles in CAV. Whole blood was collected from 22 HT recipients with angiographically-confirmed CAV and 18 HT recipients without CAV. PBMCs were isolated and subjected to single-cell RNA-sequencing using a 10X Genomics microfluidic platform. Downstream analyses focused on differential expression of genes, cell compositional changes, and T cell receptor repertoire analyses. Across 40 PBMC samples, we isolated 134,984 cells spanning 8 major clusters and 31 subclusters of cell types. Compositional analyses showed subtle, but significant increases in CD4+ T central memory cells, and CD14+ and CD16+ monocytes in high-grade CAV (CAV-2 and CAV-3) as compared to low-grade or absent CAV. After adjusting for age, gender, and prednisone use, 745 genes were differentially expressed in a cell-specific manner in high-grade CAV. Weighted gene co-expression network analyses showed enrichment for putative pathways involved in inflammation and angiogenesis. There were no significant differences in T cell clonality or diversity with increasing CAV severity. Unbiased whole transcriptomic analyses at single-cell resolution identify unique, cell-specific gene expression patterns in CAV, suggesting the potential utility of peripheral gene expression biomarkers in diagnosing CAV.

A hybrid case of eosinophilic folliculitis and eosinophilic cellulitis associated with hypereosinophilic syndrome

AbstractEosinophilic cellulitis or Wells syndrome encompasses distinct histopathological features but can also be associated with eosinophilic related conditions like hyper eosinophilic syndrome (HES) or eosinophilic granulomatosis with polyangiitis (EGPA) (Churg–Strauss syndrome). We report a case of a Turkish 41‐year‐old female who presented in clinic with pruritus and tenderness on her chest and breasts, having received several courses of antibiotics for recurrent abscess formation. A year before she had been diagnosed with HES with multiorgan involvement that included biopsy proven eosinophilic folliculitis, and prompted further investigation including bone marrow aspiration that revealed T cell clonality. Biopsy of her rash revealed eosinophilic infiltration of the dermis with flame figures. Ongoing respiratory symptoms and a history of childhood asthma were suggestive of EGPA. This case highlights important associations that should be considered in the investigation of Wells syndrome.

A phase 2 trial of IO102-IO103 and nivolumab-relatlimab fixed-dose combination in previously untreated, unresectable melanoma.

TPS9605 Background: One emerging strategy to augment immune checkpoint inhibitor efficacy is to eliminate immunosuppressive cell populations in the tumor microenvironment (TME) through vaccination. IO102-IO103 is an investigational cancer vaccine that targets both tumor and immune-suppressive cells in the TME. IO102-IO103 treatment promotes inflammation and potentiates anti-tumor activity via activation and expansion of T cells against IDO1 and/or PD-L1 positive cells. IO102-IO103 plus nivolumab demonstrated an impressive objective response rate (ORR) of 80% in the phase 1/2 MM1636 trial of 30 patients with previously untreated, unresectable melanoma, and a randomized phase 3 trial of pembrolizumab with or without IO102-IO103 (NCT05155254) is ongoing. No prior studies have evaluated IO102-IO103 in combination with dual PD-1 and LAG-3 blockade with nivolumab-relatlimab (nivo-rela), and to our knowledge there are no existing clinical data for other investigational agents in combination with nivo-rela. Methods: NCT05912244 is a single-arm, investigator-initiated trial testing IO102-IO103 in combination with nivo-rela in patients with previously untreated, unresectable melanoma. Twelve of forty-three planned patients have been enrolled. All patients will be treated with nivo-rela every four weeks for up to two years. IO102-IO103 will be administered subcutaneously every two weeks for the initial eight weeks, and then every four weeks for up to two years. An interim efficacy analysis for futility will be performed after response data are available for 21 patients. Eligible patients must have histologically confirmed, unresectable stage III or stage IV non-uveal melanoma, measurable disease by RECIST v1.1, an ECOG performance status of 0 or 1, and definitive treatment of any brain metastases. Prior systemic treatment in the neoadjuvant or adjuvant setting is allowed if completed at least six months prior to trial enrollment. Available pre-treatment melanoma tissue is required, and all patients will undergo an on-treatment study biopsy between the second and third dose of nivo-rela. Peripheral blood mononuclear cells will be collected prior to the first three doses of nivo-rela. The primary endpoint is ORR by RECIST v1.1, weighted by PD-L1 expression. Secondary endpoints include safety assessed by Common Terminology Criteria for Adverse Events v 5.0, progression-free survival (PFS) by RECIST v1.1, duration of disease response, and disease control rate. Exploratory correlative endpoints include assessment of changes in PD-L1 and IDO-expressing cells in the TME on paired tumor biopsies, identification of peripheral PD-L1 and IDO-reactive cells in the peripheral blood by ELISpot, and assessment of peripheral pre-treatment and on-treatment T cell clonality by TCR sequencing. Clinical trial information: NCT05912244 .

Cell proliferation and carcinogenesis: an approach to screening for potential human carcinogens.

Cancer arises from multiple genetic errors occurring in a single stem cell (clonality). Every time DNA replicates, mistakes occur. Thus, agents can increase the risk of cancer either by directly damaging DNA (DNA-reactive carcinogens) or increasing the number of DNA replications (increased cell proliferation). Increased cell proliferation can be achieved either by direct mitogenesis or cytotoxicity with regenerative proliferation. Human carcinogens have a mode of action of DNA reactivity, immunomodulation (mostly immunosuppression), increased estrogenic activity (mitogenesis), or cytotoxicity and regeneration. By focusing on screening for these four effects utilizing in silico, in vitro, and short-term in vivo assays, a biologically based screening for human chemical carcinogens can be accomplished with greater predictivity than the traditional 2-year bioassay with considerably less cost, less time, and the use of fewer animals.

Cytomegalovirus drives Vδ1+ γδ T cell expansion and clonality in common variable immunodeficiency

The function and phenotype of γδ T cells in the context of common variable immunodeficiency (CVID) has not been explored. CVID is a primary immunodeficiency disorder characterized by impaired antibody responses resulting in increased susceptibility to infections. γδ T cells are a subset of unconventional T cells that play crucial roles in host defence against infections. In this study, we aim to determine the roles and functions of γδ T cells in CVID. We observe a higher frequency of Vδ1+ γδ T cells compared to healthy controls, particularly in older patients. We also find a higher proportion of effector-memory Vδ1+ γδ T cells and a more clonal T cell receptor (TCR) repertoire in CVID. The most significant driver of the Vδ1+ γδ T cell expansion and phenotype in CVID patients is persistent cytomegalovirus (CMV) viremia. These findings provide valuable insights into γδ T cell biology and their contribution to immune defence in CVID.

RNA sequencing methods enable immune cell gene expression profiling and clonality determination

Abstract The adaptive immune system defends the human body from pathogens through B/T cell functions in various organs. Abnormality of these cells may reflect immunological disorder, presence of neoplastic tissue, or response to infectious agents. High-throughput sequencing technology has facilitated understanding gene expression and related cell functions. Still, an in-depth understanding of B and T cell phenotype and clonotype profiling is an emerging application that has yet to take full advantage of sequencing technology for disease diagnosis and prognosis. Here, we share results of RNA sequencing of immune system tissues, including healthy donor peripheral blood mononuclear cells (PBMC), lymph nodes, bone marrow, spleen, thyroid, thymus, and colon, as well as diseased PBMC and colon samples. Sequencing libraries were constructed and sequenced for B and T cell-specific gene expression and full-length immune repertoire profiling. We have identified tissue-specific B/T cell phenotype and clonality patterns related to tissue functions and disease progression. RNA-seq enables highly multiplex immunophenotyping and clonality determination for high-abundant clones that correlate with Immune-seq, with the latter targeted approach providing more in-depth clonality signatures. Integrating both RNA sequencing and immune repertoire sequencing approaches allows accurate phenotype and clonotype determination for the immune landscape in various tissues.

Streamlined 96-well plate method for simultaneous single-cell TCR immune receptor repertoire analysis and immunophenotyping

Abstract This study describes an efficient immune profiling method by FACS-sorting single cells in a 96-well plate to assess clonotype repertoire diversity, paired-chain information, and cell phenotypes in a single, multiplex RT-PCR-based assay without the use of any specialized equipment. It is a cost-effective alternative to conventional single-cell technologies such as microwell arrays or droplet microfluidics. The 96-well plate contains primers for either T-cell receptor (TCR) / or TCR / chains, together with primers for 30 key T-cell markers. T cells from PBMCs are sorted in a single-cell-per-well format. We conduct RT-PCR library prep using the DriverMapTM Adaptive Immune Receptor (AIR) Repertoire Profiling Assay and sequence the CDR3 region. The results show clonotype frequencies, chain pairing details for / and / chains, and T-cell subtype classifications based on gene expression profiling. This enables in-depth analysis of TCR gene rearrangements at the single-cell level, enhancing understanding of T cell development, proliferation, and clonality, which are important in studying cancer, immunodeficiencies, and autoimmune disorders. Additionally, we can effectively identify and characterize γδ T cells by integrating single-cell TCR sequencing with RNA sequencing data, potentially useful in the development of γδ cancer immunotherapies. Overall, this method provides an efficient way to simultaneously analyze clonotypes and immunophenotypes in a single assay.

Novel regulation of kidney function by gastrointestinal hormones

The control of whole-body energy metabolism involves intricate crosstalk between multiple organ systems including the gut and the brain that play well-recognized roles in this process. However, the important contributions and mechanisms by other organs are poorly understood. Errors in metabolic regulation can lead to human metabolic diseases including diabetes and obesity that affect 75% of the population and can lead to significant comorbidities. Improving our mechanistic understanding of multi-organ metabolic crosstalk in health and disease is essential for developing highly effcient and new therapeutic approaches. Macula densa (MD) cells in the kidney are chief regulators of renal blood flow and tissue remodeling. We hypothesized that the kidney is a major player in body metabolism via a new mechanism of gut-kidney crosstalk that involves the gut hormones gastrin and cholecystokinine (CCK) activating intrarenal, MD-specific mechanisms to increase blood flow and filtration, progenitor cells and tissue growth factors. High-resolution MD bulk and single-cell transcriptome analysis was performed from freshly isolated, enriched MD cells using MD-GFP mouse kidneys. Single cell MD Ca2+ signaling was analyzed in vivo using intravital multiphoton microscopy (MPM) of Sox2-GT mice that express the ratiometric Ca2+ reporter GCaMP6f/tdTomato in all renal cells. The altered expression of MD specific markers in response to gastrin/CCK8 stimuli was studied using western blot and the novel MDgeo immortalized cell line cultured in vitro. RNA sequencing and transcriptome analysis identified the gastrin/CCK receptor Cckbr as one of the highest expressed genes in MD versus control kidney cells. Intravital multiphoton imaging of Sox2-GCaMP6 reporter mouse kidneys revealed robust and entirely MD cell-specific calcium signaling in response to systemic gastrin/CCK injection. Using the novel MDgeo immortalized cell line cultured in vitro, CCK8 treatment led to 3-fold increases in MD cell synthesis of Prostaglandin E2 (PGE2)-generating COX2 and CCN1 that are established mediators of MD-induced glomerular hyperfiltration and tissue remodeling, respectively. Chronic treatment of Ren1d-Confetti mice with Darunavir, an HIV protease inhibitor significantly increased plasma CCK levels, Ren+ mesenchymal progenitor cell number and clonality, renal cell proliferation and tissue hypertrophy. This study uncovered new regulatory mechanisms of systemic metabolism especially the role of MD cells in the kidney. Repurposing the commonly used HIV drug Darunavir for kidney and metabolic diseases may provide therapeutic benefit for millions of patients worldwide. DK064234, DK123564, DK135290. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2663: Determining the role of tumor mutational burden in a pre-clinical model of glioblastoma

Abstract Despite advances in care in many cancer types, patients with glioblastoma (GBM) have a grim prognosis of 15 to 21 months and unchanged standard therapy since 2005. These tumors have a profoundly immunosuppressive tumor immune microenvironment. Immunotherapy, including the checkpoint inhibitors (ICIs) targeting PD-1 and CTLA-4, has transformed the treatment of many cancers but has failed in trials of both newly diagnosed and recurrent GBM. Tumor mutational burden (TMB) has been proposed as a predictor of response to ICI treatment, presumably through an increase in neoantigens that can be recognized by cytotoxic CD8+ T cells leading to tumor rejection. GBM typically has a low TMB, a feature speculated as a contributor to poor response to ICI treatment. However, cases with an extremely high TMB (i.e., patients with the biallelic mismatch repair deficiency syndrome) do show response to immunotherapy. To begin to investigate the impact of mutational burden on immune response in GBM, we created a murine syngeneic tumor model using CRISPR-Cas9 to knockout (KO) the mismatch repair protein Msh2, the lynchpin to mismatch recognition, in the SB28 GBM model that has a low TMB (108 mutations) at baseline. Msh2 KO increased the TMB from 2 to 12-fold depending on single cell sorted clone, resulting in between 150 and 200 predicted strong MHC-I binding neoantigens estimated by the NetMHC algorithm. Preliminary studies have demonstrated that in vitro proliferation is similar amongst low TMB and high TMB SB28 clones. Flank implantation of high TMB SB28 clones revealed slower growth compared to the low TMB SB28 tumors. Furthermore, treatment with ICIs did provide a survival advantage in this flank model. However, no survival advantage was noted when the high TMB clones were implanted intracranially either with or without ICI treatment. These results suggested that either there is poor immune cell trafficking to the intracranial tumors, or a robust immune response was causing increased intracranial pressure that is prematurely causing death. Use of dexamethasone starting at day 11 to control cerebral edema has shown some survival improvement with ICI treatment, supporting that an inflammatory response is at least partially responsible for the lack of improved survival in early studies. We are currently evaluating CD4+ and CD8+ T cell clonality, earlier timepoints to evaluate the immune infiltrate and anti-tumor immune response, and alterations in chemokines/cytokines in the tumor microenvironment. By creating high mutation burden clones from a GBM with a low mutational burden, we will be able to interrogate potential mechanisms of heightened immunogenicity that has been reported in other cancers. Citation Format: Kevin Breen, Tuesday Haynes, Masashi Watanabe, Mark Gilbert. Determining the role of tumor mutational burden in a pre-clinical model of glioblastoma [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 2663.

Abstract 6846: Tumor cell metaplastic programs and failure of T cell remodeling drive resistance and progression to frontline chemoimmunotherapy in advanced gastric cancer

Abstract Adding aPD1 to 5-FU/platinum in advanced gastroesophageal adenocarcinomas (GEA) has yielded modest and heterogeneous results. Understanding cooperativity between these two treatment modalities will inform novel combination treatments for GEA. Towards this end, we conducted a trial (n = 47) sequentially adding pembrolizumab to 5-FU/platinum in previously untreated advanced GEA. Using serial biopsy of the primary tumor at baseline, after one cycle of 5-FU/platinum, and after the addition of pembrolizumab we transcriptionally profiled 358,067 single cells and obtained TCR-sequences from single tumor-resident T cells. Stratifying patients into slow- (> 6 months) and fast-progressing (< 6 months) groups based on progression-free survival allowed us to quantify differences in immune, stromal and epithelial subpopulations across treatment time points. Using topic modeling with consensus non-negative matrix factorization (cNMF), we identified gene expression programs in each population at each time point and developed a methodology to verify the stability of those programs across the cohort using cross-validation. Faster clinical progression featured decreased M1-like macrophage repolarization and increased MUC5A and MSLN containing metaplasia programs in tumor cells. We next selected features from cell state gene programs and identified trajectories of tumor cells across a spectrum of treatment sensitive and resistant states. We identified several factors that correlated with these trajectories and regulons that may be influencing them. After pembrolizumab we observed slower progression was associated with further increased CD8 T cell infiltration with increased CXCL13+ T cells. We next calculated covarying programs between patient samples at each time point and performed permutation testing to identify which programs (and respective cell types they were expressed in) were significantly correlated to infer the presence of multicellular tumor communities. We found that the tumor cell interferon program co-varied with the CXCL13 T cell program, and the emergence of a covarying module with tumor-reactive CXCL13 program expression happens as early as after the first dose of chemotherapy. To better understand the behavior of tumor reactive T cells within the tumor microenvironment, we leveraged TCR-sequencing data to study T cell clonality and diversity across treatment time points and between fast and slow progressors. Our data provide an invaluable resource to dissect chemoimmunotherapy response in tumor cells, understand T cell behavior and propose strategies to drive anti-tumor immune hub formation to expand the portion of patients benefiting from immunotherapy approaches. Citation Format: Samuel J. Wright, Minae An, Byung Hoon Min, You Jeong Heo, Milan Parikh, Lynn Bi, Hyuk Lee, Taejun Kim, Song-Yi Lee, Jeonghyeon Moon, Ryan J. Park, Matthew R. Strickland, Woong Yang Park, Won Ki Kang, Kyoung-Mee Kim, Seung Tae Kim, Arnav Mehta, Samuel J. Klempner, Jeeyun Lee. Tumor cell metaplastic programs and failure of T cell remodeling drive resistance and progression to frontline chemoimmunotherapy in advanced gastric 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 6846.

Abstract 5667: Intratumoral heterogeneity in gastric cancer related with survival and tumor microenvironment

Abstract Gastric cancer (GC), often diagnosed at an advanced stage, poses a significant therapeutic challenge due to its resistance to treatment. The understanding of intratumoral heterogeneity, crucial for tumor cell survival, remains limited in GC. We conducted a comprehensive analysis of three patient groups (intraperitoneal metastasis [IM], hematogenous metastasis [HM], and both metastasis [HIM]) undergoing immunotherapy (IO) and chemotherapy. Utilizing single-cell RNA sequencing and TCR sequencing, we examined 11 adjacent normal, 10 HM, 8 IM, and 5 HIM primary GC samples. In epithelial cells, three distinct tumor clusters (Tumor_1, Tumor_2, and Tumor_3) were identified. Trajectory analysis revealed the differentiation of chief cells into Tumor_1, subsequently dividing into more malignant cell types, Tumor_2 and Tumor_3. Tumor_2 was enriched in IM, while Tumor_1 was enriched in HM and HIM. Patients grouped by Tumor_1, 2, 3 levels showed varying survival outcomes. Surprisingly, high Tumor_2 levels correlated with increased survival, attributed to elevated HLA and PDL1 expression, promoting immune cell recruitment and robust response to IO therapy. Conversely, Tumor_3 high patients exhibited worse survival, with enrichment in metabolic pathways associated with glycolysis and hypoxia, linking to metastasis. We observed the proportion of T cells that increased presence of CD8 effector cells among patients exhibiting high levels of Tumor_1 and Tumor_2. Through TCR analysis, we found heightened clonality in both CD8 effector cells and exhausted T cells in patients with elevated Tumor_1 and Tumor_2 levels. This suggests that the activated CD8 effector T cells in these patients may enhance their ability to effectively target and attack cancer cells. In summary, this study provides a comprehensive picture of intratumoral heterogeneity in GC can impact the survival and tumor microenvironment. The unexpected high survival in Tumor_2 patients emphasizes the potential of targeting specific pathways for improved therapeutic outcomes. Additionally, the observed T cell dynamics and clonality in Tumor_1 and 2 underscore the importance of understanding the immune microenvironment for effective cancer attack. Citation Format: Do-eon Gu, Woo Sun Kwon, Sun Young Rha, Woong-Yang Park. Intratumoral heterogeneity in gastric cancer related with survival and tumor microenvironment [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 5667.

Abstract 3976: Single-cell TCRɑ/β and TCRγ/δ immune receptor profiling and immunophenotyping using a 96-well plate sorted-cell approach

Abstract Single-cell immune receptor profiling is a revolutionary approach that allows investigators to combine clonotype repertoire identification with paired-chain information and the phenotype of cells (e.g., cell subtype). Single-cell immune receptor profiling can be performed using a medium-throughput approach (1,000-5,000 cells) using microwell arrays or droplet microfluidics technologies. However, these assays are more complicated to run and require expensive reagents and limited sequencing throughput when compared to bulk immune receptor profiling methods. Here, we describe a low-throughput, single-cell immune profiling strategy using sorted cells in 96-well plates. The plate is pre-aliquoted with either T-cell receptor (TCR) ɑ/β or TCR γ/δ primers along with 30 crucial T-cell markers. We perform multiplex RT-PCR amplification and sequencing of the CDR3 regions. The resulting data provides the abundant clonotype counts along with the chain pairing information for these ɑ/β and γ/δ chains, together with the T-cell subtype information using gene-expression profiles. By analyzing the TCR gene rearrangement at the single-cell level, researchers can better understand T cell development, proliferation, and clonality, which are crucial for studying diseases such as cancer, immunodeficiency, and autoimmunity. Furthermore, single-cell TCR sequencing when combined with RNA sequencing datasets, facilitates the identification of γδ T cells. This method provides a standardized tool for identifying potential γδ T cell-based cancer immunotherapies. The technology's cost-effectiveness and ability to analyze clonotypes and immunophenotypes of cells in a single assay make it a valuable tool for unraveling the immune dynamics in various diseases. Citation Format: Alex Chenchik, Tianbing Liu, Mikhail Makhanov, Dongfang Hu, Lester Kobzik, Khadija Ghias, Paul Diehl. Single-cell TCRɑ/β and TCRγ/δ immune receptor profiling and immunophenotyping using a 96-well plate sorted-cell approach [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 3976.

Abstract 7045: RNA sequencing approaches enable tissue specific B and T cell gene expression and immune repertoire profiling

Abstract The adaptive immune system defends the human body from pathogens through the recognition ability of antigen receptors from B and T cells. Different organs play various immune function roles, and therefore present varied B and T cell gene expression and immune repertoire profiles. Abnormality of these profiles may reflect immunological disorder, presence of neoplastic tissue, or response to infectious agent. High-throughput sequencing technology has facilitated the understanding of gene expression and related cell functions, but in-depth understanding of B and T cell phenotype and clonotype profiling is an emerging application that has yet to take full advantage of sequencing technology for diagnosis and prognosis of disease. Here, we share results of RNA sequencing of immune system tissues; including B cell and T cell specific gene expression and full-length immune repertoire profiling. Total RNA was extracted from various tissue samples including healthy donor peripheral blood mononuclear cells (PBMC), lymph nodes, bone marrow, spleen, thyroid, thymus and colon, as well as diseased PBMC and colon samples. RNA-seq and Immune-seq libraries were made from these samples and sequenced on Illumina NextSeq2000. Gene expression analysis and immune repertoire profiling from RNA-seq data were performed using edgeR and TRUST4. Immune repertoire sequencing data was processed using pRESTO and IgBlast. We correlated RNA-seq with Immune-seq results for B-cell receptor and T-cell receptor clonotypes. Differential expression and clonotype profiling comparisons were also performed between normal and diseased RNA samples. The effect of reference on immune repertoire detection sensitivity and accuracy was further investigated by comparing IMGT reference and AIRR-C Human IG Reference Sets. This study has identified tissue specific B/T cell phenotype and clonality patterns that relate to tissue functions and disease progression. RNA-seq enables highly multiplex immunophenotyping which is traditionally performed by flow cytometry. RNA-seq can detect high-abundant clones that correlate with Immune-seq, with the latter targeted approach providing more in depth clonality signatures. References comparison results show the importance of continued community effort to develop more representative and accurate immunorepertoire germ line references for clonotype annotation in diverse populations. Integrating both RNA sequencing and immune repertoire sequencing approaches allows accurate phenotype and clonotype determination for immune landscape in various tissues. Citation Format: Chen Song, Evan Janzen, Tessa Devoe, Ariel Erijman, Gautam Naishadham, Li Song, Bradley W. Langhorst. RNA sequencing approaches enable tissue specific B and T cell gene expression and immune repertoire profiling [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 7045.