Subtype Composition Research Articles

Single-cell transcriptomics link gene expression signatures to clinicopathological features of gonadotroph and lactotroph PitNET

BackgroundPituitary neuroendocrine tumors (PitNET) are among the most common intracranial tumors. Despite a frequent benign course, aggressive behavior can occur. Tumor behavior is known to be under the influence of the tumor microenvironment (TME). However, the relationship between TME cells and aggressive tumor behavior has not been adequately explored in PitNET.MethodsWe performed differential expression analysis as well as gene expression program identification based on single-cell RNA sequencing to comparatively characterize the transcriptome of seven gonadotroph and three lactotroph PitNET and correlate it with clinical features using bulk RNA-seq data from an independent cohort of 134 PitNET. Tumor immune infiltration was quantified via immunostaining on tissue sections of gonadotroph and lactotroph PitNET.ResultsIn lactotroph PitNET, we detect a highly proliferative gene profile with significantly increased expression levels in aggressively growing tumors within bulk RNA-seq data of an independent cohort of 134 PitNET samples. We also report high intratumoral heterogeneity in gonadotroph PitNET (GoPN) and lactotroph PitNET (LaPN) and identify signatures of epithelial, endocrine, and immunological gene networks in both subtypes. A comparison of their TME composition shows enrichment of SPP1+ macrophages and CD4+ T cells in GoPN, as well as enrichment of CD4/CD8 double-negative T cells (DN) and natural killer cells (NK) in LaPN. Also notable is the presence of proliferative lymphocytes, the occurrence of which positively correlates with more aggressive tumor behavior in the bulk RNA-seq cohort. However, increased CD8+ T and NK cell abundances correlate significantly with reduced aggressiveness indicating potential anti-tumoral effects.ConclusionsOur study expands the knowledge of the differences in cellular composition of gonadotroph and lactotroph PitNET subtypes. It lays the foundation for further studies on the influence of lymphoid cells on the variable aggressive behavior of PitNET. Regarding the treatment of drug-resistant lactotroph PitNET, proliferative lymphocytes, CD8+ T, and NK cells could represent potentially valuable targets for developing new cancer immunotherapies.

Quantification of Histone H1 Subtypes Using Targeted Proteomics.

Histone H1 is involved in the regulation of chromatin structure. Human somatic cells express up to seven subtypes. The variability in the proportions of somatic H1s (H1 complement) is one piece of evidence supporting their functional specificity. Alterations in the protein levels of different H1 subtypes have been observed in cancer, suggesting their potential as biomarkers and that they might play a role in disease development. We have developed a mass spectrometry-based (MS) parallel reaction monitoring (PRM) assay suitable for the quantification of H1 subtypes. Our PRM method is based on the quantification of unique peptides for each subtype, providing high specificity. Evaluation of the PRM performance on three human cell lines, HeLa, K562, and T47D, showed high reproducibility and sensitivity. Quantification values agreed with the electrophoretic and Western blot data, indicating the accuracy of the method. We used PRM to quantify the H1 complement in peripheral blood samples of healthy individuals and chronic myeloid leukemia (CML) patients. In CML, the first line of therapy is a tyrosine kinase inhibitor, imatinib. Our preliminary data revealed differences in the H1 complement in CML patients between imatinib responders and non-responders. These results support further research to determine if the H1 content or subtype composition could help predict imatinib response.

53 Associations between body composition, ClearCode34, and clinical outcomes in localized clear cell renal cell carcinoma

Abstract Background Emerging evidence suggests that pre-surgical body composition is a prognostic factor for cancer outcomes. Lower skeletal muscle quantity and radiodensity have been associated with renal cell carcinoma (RCC) outcomes in other studies, while reports of the relationship between visceral adipose tissue and RCC outcomes have been inconsistent. Mechanisms underlying these associations are unclear but may relate to tumor biology. We leveraged a large molecular epidemiology study of localized clear cell renal cell carcinoma (ccRCC) patients to examine how pre-surgical body composition features were associated with survival post-nephrectomy as well as ClearCode34 status, a previously validated prognostic gene expression signature. Methods The RESOLVE study at Memorial Sloan Kettering Cancer Center is a retrospective study of 1,239 patients with stage I-III ccRCC undergoing nephrectomy without systemic therapy. Pre-surgical CT scans at the third lumbar vertebrae were segmented for the cross-sectional areas and radiodensities of skeletal muscle, subcutaneous adipose and visceral adipose tissues using Automatica software. Formalin-fixed, paraffin-embedded tumor tissue samples were available for 929 patients, which were sent to the University of North Carolina at Chapel Hill and assayed for gene expression. RNA was extracted from these samples and analyzed using the Nanostring nCounter system on a custom panel of 356 genes, including the 34 genes used in the ClearCode34 assay. Of the 929 patients with tumor tissue available, gene expression data from 837 patients (90.1%) passed RNA extraction and data QC. Samples were classified into either ccA or ccB ClearCode34 classes using a reference data set and PAM (prediction analysis of microarrays) centroid-based classification. We estimated hazard ratios (HRs) and 95% confidence intervals (CIs) for disease-free survival (DFS) within five years of diagnosis using Cox proportional hazards models for both body composition variables and ClearCode34 subtype. We also estimated odds ratios (ORs) and 95% CIs for associations between ClearCode34 subtype and body composition with logistic regression. All multivariable models were adjusted for age, sex, and all body composition variables. Results Of the 837 patients in our analytic sample, 219 (26%) had ccB tumors. Men, patients with high stage or grade tumors, and patients with chronic kidney disease prior to nephrectomy were more likely to present with ccB tumors. Lower skeletal muscle density (SMD) was significantly associated with lower five-year DFS [HR (95% CI) per 10 HU decrease in SMD: 1.7 (1.2, 2.3)]. While no other body composition features were significantly associated, we also observed non-significantly increased HRs for VATI and VATD [HR (95% CIs): 1.3 (0.8, 2.0) and 1.5 (0.9, 2.7), respectively]. ClearCode34 subtype was significantly associated with five-year DFS, where patients with ccB tumors had poorer DFS [HR (95% CI): 1.7 (1.1, 2.5)] compared to patients with ccA tumors. Notably, we observed no significant associations between any body composition feature and ClearCode34 in univariable analyses. However, in multivariable models that accounted for all body composition variables simultaneously as well as age and sex, lower SMD emerged as significantly associated with ccB subtype [OR (95% CI) per 10 HU decrease in SMD: 1.3 (1.0, 1.7)]. Conclusions Our findings suggest that the associations observed between body composition features and clinical outcomes in ccRCC may be related to tumor biology. Lower pre-surgical SMD, which may represent myosteatosis and metabolic dysregulation in ccRCC patients, was associated with both decreased five-year DFS and an increased likelihood of presenting with a more aggressive ccB tumor. Future studies should investigate body composition features related to ccRCC prognosis and additional markers of tumor biology, such as immune-related gene expression patterns, to extend these findings.

Pan-cancer single-cell dissection reveals phenotypically distinct B cell subtypes

Characterizing the compositional and phenotypic characteristics of tumor-infiltrating B cells (TIBs) is important for advancing our understanding of their role in cancer development. Here, we establish a comprehensive resource of human B cells by integrating single-cell RNA sequencing data of B cells from 649 patients across 19 major cancer types. We demonstrate substantial heterogeneity in their total abundance and subtype composition and observe immunoglobulin G (IgG)-skewness of antibody-secreting cell isotypes. Moreover, we identify stress-response memory B cells and tumor-associated atypical B cells (TAABs), two tumor-enriched subpopulations with prognostic potential, shared in a pan-cancer manner. In particular, TAABs, characterized by a high clonal expansion level and proliferative capacity as well as by close interactions with activated CD4 Tcells in tumors, are predictive of immunotherapy response. Our integrative resource depicts distinct clinically relevant TIB subsets, laying a foundation for further exploration of functional commonality and diversity of B cells in cancer.

Cell atlas of the regenerating human liver after portal vein embolization

The liver has the remarkable capacity to regenerate. In the clinic, regeneration is induced by portal vein embolization, which redirects portal blood flow, resulting in liver hypertrophy in locations with increased blood supply, and atrophy of embolized segments. Here, we apply single-cell and single-nucleus transcriptomics on healthy, hypertrophied, and atrophied patient-derived liver samples to explore cell states in the regenerating liver. Our data unveils pervasive upregulation of genes associated with developmental processes, cellular adhesion, and inflammation in post-portal vein embolization liver, disrupted portal-central hepatocyte zonation, and altered cell subtype composition of endothelial and immune cells. Interlineage crosstalk analysis reveals mesenchymal cells as an interaction hub between immune and endothelial cells, and highlights the importance of extracellular matrix proteins in liver regeneration. Moreover, we establish tissue-scale iterative indirect immunofluorescence imaging for high-dimensional spatial analysis of perivascular microenvironments, uncovering changes to tissue architecture in regenerating liver lobules. Altogether, our data is a rich resource revealing cellular and histological changes in human liver regeneration.

Elucidating differences in concussion subtype symptomatology in sports-related versus non-sports-related concussions: a retrospective cohort study.

Postconcussive symptom questionnaires (PCSQs) are often used in concussion patient assessment, yet there is a lack of knowledge as to whether symptom subtype prevalence is dependent on the mechanism of injury (MOI). These subtypes can be defined as cognitive, atlanto-occipital/cervical spine, autonomic, balance, low energy/fatigue/sleep, emotional changes, eyes, and somatic. Using an institutional PCSQ that quantitatively addressed these subtypes, this retrospective study aimed to provide insight into differences in subtype symptomatology between sports-related (SR) and non-sports-related (NSR) injuries. Consecutive concussion patients with Glasgow Coma Scale (GCS) score ≥ 13 and ≥ 16 years of age who were treated at a concussion clinic affiliated with an academic level I trauma center in the United States between December 2009 and January 2020 were eligible for inclusion. The authors extracted data on MOI, comorbidities, habits, prior injuries, and PCSQ results. Multivariate analysis of covariance was then conducted to determine the correlations between subtype scores and MOI while considering covariates. Of the 194 patients remaining after applying inclusion and exclusion criteria, analysis included 91 patients in the SR group consisting of 54 (59%) males with mean ± SD (range) age of 20.9 ± 7.3 (16-58) years and 103 patients in the NSR group consisting of 38 (37%) males with mean age of 39.2 ± 14.8 (17-71) years. Demographic characteristics differed significantly between groups. Estimated marginal mean scores were significantly lower in the SR injury group compared to the NSR injury group (with comparing main effects) for the cognitive (p < 0.001), autonomic (p < 0.000), balance (p < 0.025), energy (p < 0.006), emotional (p < 0.000), and total score (p < 0.001) subtypes. Multivariate tests identified three comorbidities that contributed to differences in subtype scores between groups: migraines (p < 0.012), vertigo (p < 0.004), and anxiety (p < 0.038). No significant results were found for the remaining comorbidities of (but not limited to) depression, neuropsychiatric disorders, seizures, syncope, sleep disorder, or none. The findings indicate that patients who sustain a concussion via an NSR injury present with more severe symptoms but similar concussion subtype frequency as those presenting with SR concussion. This suggests that the MOI may correlate more closely to symptom severity than concussion subtype composition, although larger patient populations with more definitive control of MOI are needed to further elucidate these claims.

The Low Tumorigenic Risk and Subtypes of Cardiomyocytes Derived from Human-induced Pluripotent Stem Cells

Background: Clinical application of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is a promising approach for the treatment of heart diseases. However, the tumorigenicity of hiPSC-CMs remains a concern for their clinical applications and the composition of the hiPSC-CM subtypes need to be clearly identified. Methods: In the present study, hiPSC-CMs were induced from hiPSCs via modulation of Wnt signaling followed by glucose deprivation purification. The structure, function, subpopulation composition, and tumorigenic risk of hiPSC-CMs were evaluated by single-cell RNA sequencing (scRNAseq), whole exome sequencing (WES), and integrated molecular biology, cell biology, electrophysiology, and/or animal experiments. Results: The high purity of hiPSC-CMs, determined by flow cytometry analysis, was generated. ScRNAseq analysis of differentiation day (D) 25 hiPSC-CMs did not identify the transcripts representative of undifferentiated hiPSCs. WES analysis showed a few newly acquired confidently identified mutations and no mutations in tumor susceptibility genes. Further, no tumor formation was observed after transplanting hiPSC-CMs into NOD-SCID mice for 3 months. Moreover, D25 hiPSC-CMs were composed of subtypes of ventricular-like cells (23.19%) and atrial-like cells (66.45%) in different cell cycle stages or mature levels, based on the scRNAseq analysis. Furthermore, a subpopulation of more mature ventricular cells (3.21%) was identified, which displayed significantly up-regulated signaling pathways related to myocardial contraction and action potentials. Additionally, a subpopulation of cardiomyocytes in an early differentiation stage (3.44%) experiencing nutrient stress-induced injury and heading toward apoptosis was observed. Conclusions: This study confirmed the biological safety of hiPSC-CMs and described the composition and expression profile of cardiac subtypes in hiPSC-CMs which provide standards for quality control and theoretical supports for the translational applications of hiPSC-CMs.

Abstract PR003: T cell receptor repertoire and diversity are prognostic markers in bladder cancer

Abstract T cells represent essential effector cells in the intrinsic antitumor immune response. Cancer-specific T cells can recognize cancer neoantigens through their T cell receptors (TCRs). The expansion of these clones is believed to be an early response to malignancy. We hypothesized that the T cell landscape offers insights into immune competency. We aimed to characterize the TCR repertoire in patients with bladder cancer and explore correlations with disease outcomes. We analyzed the TCR landscape in a cohort of 119 patients with muscle-invasive bladder cancer (MIBC) treated with chemotherapy followed by radical cystectomy. Peripheral and tumor TCR repertoires were produced using ultradeep amplicon-based sequencing of the TCR beta chain. T cell fractions were inferred from whole exome sequencing data of blood DNA using TcellExTRECT. Latent viral DNA was investigated in plasma whole genome sequencing data using Kraken2. Antigen targets were estimated using GLIPH2. The T cell subtype compositions in tumor and blood were investigated in four patients with bladder cancer using the Chromium Single Cell Immune profiling kit from 10X Genomics. Low pre-treatment peripheral TCR diversity was associated with worse overall survival (HR = 2.3, p = 0.024, n = 119) in patients with MIBC, particularly when combined with a low fraction of circulating T cells (HR= 4.7, p = 0.00049, n = 67). We validated these findings in two independent cohorts of patients with MIBC (p = 0.01, n = 107; p = 0.042, n = 79). The low-diversity TCR repertoires were characterized by large expanded T cell clones that persisted over time. Longitudinal analysis indicated a potential adverse impact of treatment, evidenced by a reduction in TCR diversity and circulating T cells over time in patients with initially high-diversity repertoires. TCR target annotation revealed that expanded T cell clones disproportionately targeted latent viral infections and cytomegalovirus DNA detection was strongly associated with low TCR diversity (p = 5.3 × 10−5). We observed a notable disparity between tumor and peripheral blood TCR repertoires. Single-cell sequencing revealed that regulatory T cells were prevalent in the tumor, whereas a combination of cytotoxic and naïve T cells dominated the blood. More explicitly, expanded clones in the blood were commonly annotated as terminally differentiated effector memory T cells expressing high levels of cytotoxic and exhausted genes. Our results suggest that high peripheral TCR diversity and high T cell fraction are markers of general immune competence, reflecting the ability to eradicate cancer and other diseases. In contrast, the expanded persistent clones predominating the low peripheral TCR diversity repertoires are likely exhausted and less likely to exhibit tumor specificity or effective disease-fighting capabilities. Our findings underline the crucial role of the immune system in determining disease outcomes and highlight the potential for improving immune health as a promising approach for future treatment and prevention. Citation Format: Nanna Kristjánsdóttir, Asbjørn Kjær, Iver Nordentoft, Randi I Juul, Karin Birkenkamp-Demtröder, Johanne Ahrenfeldt, Trine Strandgaard, Deema Radif, Darren Hodgson, Christopher Abbosh, Hugo JWL Aerts, Mads Agerbæk, Jørgen B Jensen, Nicolai J. Birkbak, Lars Dyrskjøt. T cell receptor repertoire and diversity are prognostic markers in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR003.

Abstract PO5-16-07: Unveiling the Landscape of PD-L1 Expression and Tumor-Infiltrating Lymphocyte Subtypes in Advanced Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC) is known for its aggressive behavior with poor survival outcomes. Influenced by different expression levels of Programmed Death-Ligand 1 (PD-L1) and several subtypes of tumor-infiltrating lymphocytes (TILs), tumor microenvironment (TME) plays a crucial role in TNBC response to treatment and progression. Objective: To assess the prevalence and prognostic role of PD-L1 expression in advanced TNBC, defined as unresectable stage III or stage IV disease, in patients treated with a standard cytotoxic chemotherapy regimen. TILs composition was also evaluated as an exploratory objective. Methods: The internal database of the Brazilian National Cancer Institute was queried out for women diagnosed and treated with advanced TNBC from January 2018 to December 2022. Formalin-fixed paraffin-embedded samples of a maximum of four years old were analyzed. PD-L1 expression using 22C3 pharmDX assay was estimated through combined positive score (CPS) dichotomization (CPS &amp;lt; 10 vs. CPS≥10). Tissue microarrays comprising the samples of biopsies were subjected to immunohistochemistry, targeting specific markers including CD3, CD4, CD8, CD56, CD68, CD117, FOXP3, PD-1, the immune cell profiles were carefully examined and their correlation with the PD-L1 CPS was determined. Results: A total of 150 patients were included. The expression of PD-L1 was undetermined in 2 cases. The median age was 51.5 years (IQR 41.8-60.2) and 20.9% of the cases had CPS≥10. Most patients were &amp;lt; 65 years (73.0%), were postmenopausal (56.8%) and belonged to non-white ethnicity (70.3%). Postmenopausal women predominated in the CPS≥10 subgroup (74.2%) (Table 1). No significant differences in demographic characteristics and clinicopathological variables were observed based on PD-L1 subgroups, 117 patients (79.1%) had CPS &amp;lt; 10, with 91 (77.8%) being de novo stage IV metastatic disease. CD3+ (p=0.037), CD4+ (p=0.005) and CD8+ (p=0.001) TILs had higher expression in tumors with PD-L1 CPS≥10 (Table 2). According to the treatment received, almost half of the patients only received first-line chemotherapy (47%), as the following second line was applied to 28.8% of the total group. The third and fourth lines were administered in 12.9% and 9.1% of patients, respectively. Only 2 patients went through five palliative chemotherapy lines, and only one received up to 6 treatment lines. Between the subgroups CPS≥10 versus CPS &amp;lt; 10 no statistically significant differences were observed in the median progression-free survival (PFS) (5.1 vs. 5.0 months, p=0.89) and overall survival (OS) (8.7 vs. 8.8 months, p=0.6). Conclusion: This study offers insights into the expression patterns of PD-L1 and TILs subtypes in advanced TNBC cases in Brazil. PD-L1 CPS did not influence survival outcomes for this cohort of patients treated with cytotoxic chemotherapy only. The composition of TILs subtypes within the TME appears to vary based on PD-L1 CPS. Table 1. Advanced TNBC population characteristic by CPS (Nf148). Table 2. Tumor Infiltrating Lymphocytes characterization (CD3, CD4, CD8, CD56, CD68, CD 117, FOXP3 and PD-1) and its correlation with CPS status. Citation Format: Alexssandra Lima Siqueira dos Santos, Jesse Lopes da Silva, Lucas Zanetti de Albuquerque, Antônio Lucas Araújo Neto, Cecília Ferreira da Silva, Luana Aguiar Mesquita Cerva, Isabele Ávila Small, Cicera Pimenta Marcelino, Paula de Mendonça Batista, Maria Aparecida do Carmo Rego, Maria Amélia Carlos Souto Maior Borba, Andreia Cristina de Melo. Unveiling the Landscape of PD-L1 Expression and Tumor-Infiltrating Lymphocyte Subtypes in Advanced Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-16-07.

Abstract PO4-20-04: A Randomized Window of Opportunity Study of Preoperative Letrozole and Simvastatin Versus Letrozole Alone in Stage I-III Hormone Receptor Positive, HER2 Negative Breast Cancer

Abstract Hormone receptor positive (HR+), HER2 negative (HER2-) breast cancers are poor responders to immunotherapy. There is a need for innovative approaches to improve immune responses in these tumors. The combination of statins and aromatase inhibitors have demonstrated antiproliferative and immunomodulatory properties. However, this combination has never been studied in the preoperative setting; thus, our ongoing study represents an opportunity to investigate this approach. We designed a randomized two-arm presurgical “window of opportunity” trial to evaluate the effects of letrozole and simvastatin versus letrozole alone 14 days prior to surgery. Tissue and blood will be obtained at baseline and 14 days after completion of therapy at the time of surgery. Inflammatory markers in the blood and multiplex immunohistochemistry (IHC) in tissue will be assessed. To be eligible for the trial, participants must be postmenopausal women with histologically confirmed stage I-III HR+, HER2- invasive breast cancer with baseline ki-67 ≥10% that have not received prior chemotherapy, endocrine therapy, and/or immunotherapy within 3 months prior to trial enrollment. They should also not have received any cholesterol lowering medication within 3 months prior to trial enrollment. The primary objective is to determine if the addition of simvastatin to letrozole compared to letrozole alone will result in a decrease in geometric mean % change in ki-67 from pre-surgical baseline to 14 days following preoperative therapy. Ki-67 is a validated surrogate marker for disease-free survival in HR+, HER2- breast cancer. The main secondary objective is to determine if the addition of simvastatin to letrozole compared to letrozole alone will result in increased immune activation from pre- to post-treatment based on the evaluation of the immune subtype composition in tissue via multiplex immunofluorescence. We aim to enroll 16 patients in each arm to achieve 90% power and detect a minimum difference of 7.5% in ki-67 using a two-sided Mann-Whitney U or Wilcoxon Rank-Sum test. After accounting for a possible drop off rate of 20%, the study’s target accrual will be 40 participants. Statistical analyses will be performed using SAS 9.4. the significance level will be set at alpha = 0.1. Descriptive statistics will be applied to tissue and blood biomarkers of interest at 2 designated time points, prior to preoperative therapy and following completion of preoperative therapy. The absolute change or percentage change of those biomarkers will also be calculated and compared between the 2 arms using the nonparametric Mann-Whitney U test. The correlation among biomarkers will be described by Pearson correlation coefficient with 95% confidence interval. The p-value will be adjusted by Benjamini-Hochberg procedure to control the false discovery rate. All adverse events experienced data will be described by summary statistics and will be assessed according to CTCAE version 5.0. Currently, the study has accrued 2 participants. After completing accrual, we will assess whether the tumor-immune milieu has undergone modulation, resulting in a more immunogenic environment. If an immunogenic effect is demonstrated, this will provide rationale for a future multi-center trial assessing the combination of letrozole, simvastatin, and immunotherapy. Citation Format: Ruth Sacks, Elizabeth Haas, Jade Jones, Manali Bhave, Keerthi Gogineni, Jane Meisel, Demetria Smith-Graziani, Suchi Pakkala, Sarah Friend, Cletus Arciero, Lauren Postlewait, Clara Farley, Cathy Graham, Toncred Styblo, Monica Rizzo, Rhonda Pickett, Nicholas Crasta, Lori Brown, Xiaoxian Li, Sunil Badve, Madhav Dhodapkar, Kevin Kalinsky. A Randomized Window of Opportunity Study of Preoperative Letrozole and Simvastatin Versus Letrozole Alone in Stage I-III Hormone Receptor Positive, HER2 Negative Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-20-04.

Impact of different tissue dissociation protocols on endothelial cell recovery from developing mouse lungs.

Flow cytometry and fluorescence-activated cell sorting are widely used to study endothelial cells, for which the generation of viable single-cell suspensions is an essential first step. Two enzymatic approaches, collagenase A and dispase, are widely employed for endothelial cell isolation. In this study, the utility of both enzymatic approaches, alone and in combination, for endothelial cell isolation from juvenile and adult mouse lungs was assessed, considering the number, viability, and subtype composition of recovered endothelial cell pools. Collagenase A yielded an 8-12-fold superior recovery of viable endothelial cells from lung tissue from developing mouse pups, compared to dispase, although dispase proved superior in efficiency for epithelial cell recovery. Single-cell RNA-Seq revealed that the collagenase A approach yielded a diverse endothelial cell subtype composition of recovered endothelial cell pools, with broad representation of arterial, capillary, venous, and lymphatic lung endothelial cells; while the dispase approach yielded a recovered endothelial cell pool highly enriched for one subset of general capillary endothelial cells, but poor representation of other endothelial cells subtypes. These data indicate that tissue dissociation markedly influences the recovery of endothelial cells, and the endothelial subtype composition of recovered endothelial cell pools, as assessed by single-cell RNA-Seq.

Single-cell analysis identifies critical regulators of spermatogonial development and differentiation in cattle-yak bulls

Spermatogenesis is a continuous process in which functional sperm are produced through a series of mitotic and meiotic divisions and morphological changes in germ cells. The aberrant development and fate transitions of spermatogenic cells cause hybrid sterility in mammals. Cattle-yak, a hybrid animal between taurine cattle (Bos taurus) and yak (Bos grunniens), exhibits male-specific sterility due to spermatogenic failure. In the present study, we performed single-cell RNA sequencing analysis to identify differences in testicular cell composition and the developmental trajectory of spermatogenic cells between yak and cattle-yak. The composition and molecular signatures of spermatogonial subtypes were dramatically different between these 2 animals, and the expression of genes associated with stem cell maintenance, cell differentiation and meiotic entry was altered in cattle-yak, indicating the impairment of undifferentiated spermatogonial fate decisions. Cell communication analysis revealed that signaling within different spermatogenic cell subpopulations was weakened, and progenitor spermatogonia were unable to or delayed receiving and sending signals for transformation to the next stage in cattle-yak. Simultaneously, the communication between niche cells and germ cells was also abnormal. Collectively, we obtained the expression profiles of transcriptome signatures of different germ cells and testicular somatic cell populations at the single-cell level and identified critical regulators of spermatogonial differentiation and meiosis in yak and sterile cattle-yak. The findings of this study shed light on the genetic mechanisms that lead to hybrid sterility and speciation in bovid species.

Assessment of Immune Status in Patients with Mismatch Repair Deficiency Endometrial Cancer.

This study introduced a novel subtype classification method for endometrial cancer (EC) with mismatch repair deficiency (MMRd) by employing immune status and prognosis as the foundational criteria. The goal was to enhance treatment guidance through precise subtype delineation. Study Cohort: This study encompassed a cohort of 119 patients diagnosed with MMRd-EC between 2015 and 2022. Analyses using t-tests and Mann-Whitney U-tests were performed to assess prognostic markers and peripheral blood immune cell profiles in patients with MutS deficiency (MutS-d) versus those with MutL deficiency (MutL-d). Logistic regression analysis was used to identify independent risk factors. Bioinformatics Analysis: An online database was used to assess the prognostic implications, immune cell infiltration, and immune checkpoint involvement associated with the deficiency of MutS versus MutL in EC. Patients with MutL-d exhibited heightened risk factors, including elevated cancer grade and increased myometrial invasion, leading to poorer prognosis and shorter overall survival and progression-free survival. Regarding systemic immune status, patients with MutL-d demonstrated decreased peripheral blood lymphocyte percentage, lymphocyte count, and CD8+ T cell percentage. For local immunity, the infiltration of natural killer cells, CD8+ T cells, and cytotoxic T lymphocytes in the tumor tissue was reduced in patients with MutL-d. Additionally, patients with MutL-d exhibited lower expression of immune checkpoint markers. The composition of immune subtypes and survival outcomes also indicate that patients with MutL-d have a poorer immune status and prognosis than the patients with MutS-d. Patients with MMRd-EC can be subclassified according to MutS or MutL deficiency. Patients with MutS-d exhibited better immune status, prognosis, and immunotherapy benefits than those with MutL-d. These results can help guide patients to a more precise treatment.

Abstract 7567: In-depth mass cytometry analysis reveals different immune cell composition of unique molecular subtypes in extensive stage small-cell lung cancer

Abstract Background: A novel molecular subtype classification was fashionable for small cell lung cancer (SCLC). However, the distribution of SCLC molecular subtype classification in Chinese patients were unknown, especially extensive stage (ES)-SCLC. It should validate and define their associated microenvironments across subtypes in ES-SCLC. Method: We used immunohistochemical (IHC) to evaluate SCLC markers of the subtypes including ASCL1, NEUROD1, POU2F3, SCLC-A/N/P in 135 ES-SCLC tumors. Peripheral blood of 24 ES-SCLC patients was test by mass cytometry using time-of-flight (CyTOF) to evaluate the differences in immune subsets regard to the subtype tumor microenvironment (TME). Validation was performed using flow cytometry and multiplex IF. Results: ASCL1, NEUROD1, POU2F3, and Inflamed were account for 60.7%, 28.9%, 7.4%, and 18.5% of SCLC tumors, respectively. Significant intratumoral heterogeneity was observed with 15.6% of the tumors being both positive for SCLC-A and -N subtype markers, respectively. The SCLC-I type possessed more CD8+ T-cells infiltration and had higher frequency an ‘inflamed’ immunophenotyped. CyTOF data showed greater CD161+CD127+CD8+T cells proportion in SCLC-I than SCLC-N subtypes before and after anti-immunotherapy. Furthermore, we validated that lung tissue of SCLC-I were infiltrated more CD161+CD127+CD8+T cells compared to SCLC-N subpopulation(P=0.0281). Flow cytometry further showed the stronger immune activation of CD161+CD127+CD8+T cells in SCLC-I types induced higher expression of GZMB and GNLY and lower-expression of T-cell exhaustion markers PD-1,TIGIT and LAG3 in comparison with SCLC-N types. We further confirmed that the high level of CD161+CD127+CD8+T associated with a favorable treatment response and prolonged overall survival. Conclusion: The present study validated the molecular subtypes in ES-SCLC patients. Furthermore, SCLC-I subtype has longer PFS and OS than SCLC-A, -N and -P patients. Our research not only provides an insight into the immune landscape of ES-SCLC but also sheds light on a special subset of CD161+CD127+CD8+T cells with prognostic and therapeutic significance. Citation Format: Yuekang Li, Jingjing Qu, Binggen Wu, Qian Shen, Wenjia Sun, Lijun Chen, Bo Wang, Lixiong Ying, Jianya Zhou, Jianying Zhou. In-depth mass cytometry analysis reveals different immune cell composition of unique molecular subtypes in extensive stage small-cell lung 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 7567.

Abstract 4312: KRAS oncogenic activation in Gramd2 + AT1 cells form lung adenocarcinoma with distinct transcriptomic and tumor microenvironment signatures

Abstract Lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer and presents with a high degree of biological heterogeneity and distinct clinical outcomes. The current paradigm of LUAD etiology recognizes alveolar epithelial type II (AT2) cells as the primary cell of origin, while the role of AT1 cells in LUAD oncogenesis remains relatively unknown. Here, we examined transformation via activation of the oncogenic KRASG12D mutation in a Gram-domain containing 2 (Gramd2)+ AT1 cell lineage tracing model system, which resulted in multifocal LUAD, primarily of papillary histology. Spatial transcriptomic sequencing revealed significant differences of transcriptomic signatures and signaling activities between Gramd2+ AT1 cell-derived and Sftpc+ AT2 cell-derived LUAD. Single-cell RNA sequencing of macrodissected LUAD lesions demonstrated heterogeneous cell composition of the tumor microenvironment (TME), with Gramd2+ AT1 cell-derived LUAD fostering a proinflammatory, immunoreactive TME and Sftpc+ AT2-derived LUAD presenting with an immunosuppressive TME. Collectively, our study suggests that cell of origin for LUAD is associated with histologic subtype, transcriptomic aberrations, and immune cell composition within LUAD that may influence response to therapy. Citation Format: Minxiao Yang, Jonathan Castillo, Edgar Gonzalez, Chunli Yan, Evanthia Roussos Torres, Zea Borok, Crystal Marconett. KRAS oncogenic activation in Gramd2 + AT1 cells form lung adenocarcinoma with distinct transcriptomic and tumor microenvironment signatures [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 4312.

Abstract 3873: Understanding spatial organization of cellular plasticity in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is notorious for poor survival rates, underscoring the urgency of understanding factors that drive tumor progression. A key player in this process is epithelial-mesenchymal transition (EMT), known for its role in enhancing cancer cell adaptability, resistance to treatment, and metastasis. Our study focuses on EMT within PDAC, using single-cell spatial transcriptomics to explore how the tumor microenvironment influences changes in cellular states. We introduce a novel method involving Gaussian mixture models (GMM) to categorize EMT stages in single-cell data and utilize deep learning for label transfer of these tumor cell states, including intermediate cell states, onto spatial transcriptomic data with limited gene panels and sparse count profiles. We find strong spatial autocorrelation of classical cells and a dispersion of basal cells alongside other cells at intermediate stages of the EMT spectrum. By leveraging the spatial proximity of clonally related cells, we confirm that cells transition along the EMT spectrum in a gradient and infer transition rates. We next leverage density gradients of tumor cells in different states to identify distinct neighborhoods with varying EMT subtype composition. We observe significant variations in cell density among different EMT states. A focus on cancer-associated fibroblasts (CAFs) and macrophages reveals differences in these cells across density contours. We also examine differential ligand-receptor interactions across cell state gradients to better understand the cellular interactions that drive EMT transitions and alter the tumor environment. By leveraging spatial transcriptomics, this research aims to provide new biological insights into PDAC intratumoral heterogeneity, potentially leading to the identification of novel therapeutic targets to disrupt treatment resistance. Citation Format: Izabella Zamora, Milan Parikh, Bidish K. Patel, Lynn Bi, Doron Haviv, Jingyi Cao, Carina Shiau, Martin Hemberg, William L. Hwang, Nir Hacohen, David T. Ting, Arnav Mehta. Understanding spatial organization of cellular plasticity in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3873.

BAP1 is required prenatally for differentiation and maintenance of postnatal murine enteric nervous system.

Epigenetic regulatory mechanisms are underappreciated, yet are critical for enteric nervous system (ENS) development and maintenance. We discovered that fetal loss of the epigenetic regulator Bap1 in the ENS lineage caused severe postnatal bowel dysfunction and early death in Tyrosinase-Cre Bap1fl/fl mice. Bap1-depleted ENS appeared normal in neonates; however, by P15, Bap1-deficient enteric neurons were largely absent from the small and large intestine of Tyrosinase-Cre Bap1fl/fl mice. Bowel motility became markedly abnormal with disproportionate loss of cholinergic neurons. Single-cell RNA sequencing at P5 showed that fetal Bap1 loss in Tyrosinase-Cre Bap1fl/fl mice markedly altered the composition and relative proportions of enteric neuron subtypes. In contrast, postnatal deletion of Bap1 did not cause enteric neuron loss or impaired bowel motility. These findings suggest that BAP1 is critical for postnatal enteric neuron differentiation and for early enteric neuron survival, a finding that may be relevant to the recently described human BAP1-associated neurodevelopmental disorder.

Implication of m6A Methylation Regulators in the Immune Microenvironment of Bronchopulmonary Dysplasia.

N6-methyladenosine (m6A) regulates gene expression and governs many important biological processes. However, the function of m6A in the development of bronchopulmonary dysplasia (BPD) remains poorly characterized. Thus, the purpose of this investigation was to evaluate the effects of m6A RNA methylation regulators on the development of BPD. BPD-related transcriptome data were downloaded from the GEO database. Differentially expressed m6A methylation regulators between BPD and control group were identified. Consensus clustering was conducted for the classification of BPD and association between clusters and BPD phenotypes were explored. Analysis of differentially expressed genes (DEGs) and immune-related DEGs was performed. The GSEA, GO and KEGG analyses were used to interpret the functional enrichments. The composition of immune cell subtypes in BPD subsets was predicted by CIBERSORT analysis. Compared with the control group, expression of most m6A regulators showed significant alteration, especially for IGF2BP1/2/3. BPD was classified into 2 subsets, and cluster 1 was correlated with severe BPD. Furthermore, the results of functional enrichment analyses showed a disturbed immune-related signaling pathway. Based on CIBERSORT analysis, we found that the proportion of immune cell subsets changed between cluster 1 and cluster 2. Our study revealed the implication of m6A methylation regulators in the development of BPD, which might provide a novel insight for the diagnosis and treatment of BPD.

Identifying CTH and MAP1LC3B as ferroptosis biomarkers for prognostic indication in gastric cancer decoding

Gastric cancer (GC), known for its high incidence and poor prognosis, urgently necessitates the identification of reliable prognostic biomarkers to enhance patient outcomes. We scrutinized data from 375 GC patients alongside 32 non-cancer controls, sourced from the TCGA database. A univariate Cox Proportional Hazards Model (COX) regression was employed to evaluate expressions of ferroptosis-related genes. This was followed by the application of Least Absolute Shrinkage and Selection Operator (LASSO) and multivariate COX regression for the development of prognostic models. The composition of immune cell subtypes was quantified utilizing CIBERSORT, with their distribution in GC versus control samples being comparatively analyzed. Furthermore, the correlation between the expressions of Cystathionine Gamma-Lyase (CTH) and Microtubule Associated Protein 1 Light Chain 3 Beta (MAP1LC3B) and the abundance of immune cell subtypes was explored. Our bioinformatics findings underwent validation through immunohistochemical analysis. Our prognostic models integrated CTH and MAP1LC3B. Survival analysis indicated that patients categorized as high-risk, as defined by the model, exhibited significantly lower survival rates compared to their low-risk counterparts. Notably, CTH expression inversely correlated with monocyte levels, while MAP1LC3B expression showed an inverse relationship with the abundance of M2 macrophages. Immunohistochemical validation corroborated lower expressions of CTH and MAP1LC3B in GC tissues relative to control samples, in concordance with our bioinformatics predictions. Our study suggests that the dysregulation of CTH, MAP1LC3B, and the accompanying monocyte-macrophage dynamics could be pivotal in the prognosis of GC. These elements present potential targets for prognostic assessment and therapeutic intervention.

Abstract A035: Stromal composition, fibroblast heterogeneity and spatial organization in pancreatic adenocarcinoma

Abstract Background: The pancreatic ductal adenocarcinoma (PDAC) microenvironment is characterized by highly abundant stromal fibroblasts that are hypothesized to influence tumor behavior. While recent studies have identified multiple populations of stromal fibroblasts with divergent roles in PDAC biology, stromal composition remains incompletely characterized in human tumors, including the landscape of fibroblast subtypes and their spatial organization. Design: We designed multiplex immunofluorescence assays to quantify tumor cells (cytokeratin), immune cells (CD45), fibroblasts (αSMA, CD74, FAP, LaminA), other cells (CD31, CD56, CALD1, NG2), and collagen I in fixed tissue sections. Combinatorial fibroblast marker expression identified myofibroblasts (myCAF), inflammatory fibroblasts (iCAF), antigen-presenting fibroblasts (apCAF), and other hybrid fibroblast subtypes. We characterized fibroblast populations in up-front resected and neoadjuvant-treated cohorts of patients with localized PDAC and in patients with biopsies of metastatic lesions. Using digital imaging, supervised machine learning, and spatial proximity metrics, we quantified cell phenotypes, fibroblast subtype composition and spatial organization at the single-cell level and in the context of tumor clinicopathologic features and patient outcomes. Results: Across 301 up-front resected tumors, we cumulatively analyzed 1.7 million cells, including 611,000 fibroblasts. Stromal fibroblast density ranged from 632 to 4150 cells/mm2. Five main fibroblast subtypes constituted more than 85% of the fibroblast population in these tumors: αSMA+ (myCAF), FAP+αSMA+ (FAP+ myCAF), LaminA+ (iCAF), αSMA+ LaminA+ (myCAF/iCAF), and FAP+ αSMA+ LaminA+ (FAP+ myCAF/iCAF). Unexpectedly, 39% of fibroblasts showed co-expression of markers previously considered distinct for major fibroblast subtypes. By unsupervised clustering of fibroblast subtype proportions, we identified 3 fibroblast-defined tumor subgroups, including tumors rich in FAP+ fibroblasts, hybrid fibroblasts, and single positive fibroblasts (myCAFs, iCAFs, and apCAFs), which were associated with patient survival. Furthermore, fibroblast subtypes were spatially organized, with preferential localization of FAP+ myCAFs near tumor cells and iCAFs more distant from tumor cells yet closer to immune cells. FAP+ myCAFs were more abundant in basal-like tumors than in classical tumors, and FAP+ myCAFs were located closer to basal-like tumor cells than classical tumor cells within individual tumors. Compared to up-front resected primary tumors, neoadjuvant FOLFIRINOX treated tumors and metastatic lesions harbored distinct fibroblast profiles. Conclusion: Fibroblasts in the PDAC microenvironment are heterogeneous both in abundance and subtype composition, exhibit distinct spatial organization and are associated with patient outcomes. Treatment with FOLFIRINOX alters the composition of fibroblast subtypes in primary PDAC, and metastatic PDAC lesions have a different fibroblast composition compared to primary tumors. Citation Format: Dalia Elganainy, Andressa Dias Costa, Alexander Jordan, Suryun Kim, Sara A. Väyrynen, Hannah L. Williams, Chen Yuan, Douglas A. Rubinson, Kimberly Perez, Harshabad Singh, Richard F. Dunne, Thomas E. Clancy, David C. Linehan, Daniel T. Chang, Aram F. Hezel, Albert C. Koong, Andrew Aguirre, Brian M. Wolpin, Jonathan A. Nowak. Stromal composition, fibroblast heterogeneity and spatial organization in pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A035.