Abstract 6312: AI-Driven Identification of Drug-Tolerant Persister Cells in Lung Cancer

Micro-RNAs are approximately 21-25-nucleotide-long noncoding RNAs that regulate gene expression primarily at the post-transcriptional level in animals. Here, we report that micro-RNA-1 (miR-1), abundant in the cardiac and smooth muscles, is expressed in the lung and is down-regulated in human primary lung cancer tissues and cell lines. In situ hybridization demonstrated localization of miR-1 in bronchial epithelial cells. The tumor suppressor C/EBPalpha, frequently suppressed in lung cancer, reactivated miR-1 expression in the lung cancer cells. Repressed miR-1 was also activated in lung cancer cells upon treatment with a histone deacetylase inhibitor. These observations led us to examine the antitumorigenic potential of miR-1 in lung cancer cells. Expression of miR-1 in nonexpressing A549 and H1299 cells reversed their tumorigenic properties, such as growth, replication potential, motility/migration, clonogenic survival, and tumor formation in nude mice. Exogenous miR-1 significantly reduced expression of oncogenic targets, such as MET, a receptor tyrosine kinase, and Pim-1, a Ser/Thr kinase, frequently up-regulated in lung cancer. Similarly, the levels of two additional targets, FoxP1, a transcription factor with oncogeneic property, and HDAC4 that represses differentiation-promoting genes, were reduced in miR-1-expressing cells. Conversely, depletion of miR-1 facilitated N417 cell growth with concomitant elevation of these targets. Further, ectopic miR-1 induced apoptosis in A549 cells in response to the potent anticancer drug doxorubicin. Enhanced activation of caspases 3 and 7, cleavage of their substrate PARP-1, and depletion of anti-apoptotic Mcl-1 contributed to the sensitivity of miR-1-expressing cells to doxorubicin. Thus, miR-1 has potential therapeutic application against lung cancers.

Cell type prediction is one of the most challenging goals in single-cell RNA sequencing (scRNA-seq) data. Existing methods use unsupervised learning to identify signature genes in each cluster, followed by a literature survey to look up those genes for assigning cell types. However, finding potential marker genes in each cluster is cumbersome, which impedes the systematic analysis of single-cell RNA sequencing data. To address this challenge, we proposed a framework based on regularized multi-task learning (RMTL) that enables us to simultaneously learn the subpopulation associated with a particular cell type. Learning the structure of subpopulations is treated as a separate task in the multi-task learner. Regularization is used to modulate the multi-task model (e.g., W1, W2, … Wt) jointly, according to the specific prior. For validating our model, we trained it with reference data constructed from a single-cell RNA sequencing experiment and applied it to a query dataset. We also predicted completely independent data (the query dataset) from the reference data which are used for training. We have checked the efficacy of the proposed method by comparing it with other state-of-the-art techniques well known for cell type detection. Results revealed that the proposed method performed accurately in detecting the cell type in scRNA-seq data and thus can be utilized as a useful tool in the scRNA-seq pipeline.

Non-small cell lung cancer (NSCLC) remains a major cause of cancer-related deaths worldwide and is often driven by mutations in the KRAS oncogene. Interestingly, smokers most often bear KRASG12C mutations, whereas KRASG12D mutations are more common in non-smokers. KRASG12C inhibitors were FDA-approved in 2021 and have revolutionized personalized therapeutic approaches for lung cancer. However, resistance is widespread, comprising genetic alterations in RAS, gene amplification, and compensatory pathway activation, along with changes in the tumor microenvironment (TME). For KRASG12D inhibitors now emerging for clinical testing, therapeutic resistance is also predicted. To understand both tumor microenvironmental changes induced by oncogenic KRAS and TME-dependent resistance mechanisms, we developed a new orthotopic model of KRASG12D-driven lung cancer, derived from our published genetically engineered mouse model of KRASG12D NSCLC (Lasse-Opsahl and Barravecchia et al., JCI Insight 2025). Single-cell RNA sequencing data from lung tumor tissue of mice in which KRAS was inhibited, either genetically or pharmacologically with the pan-RAS inhibitor RMC-7977, demonstrated major changes in several cell clusters. Not surprisingly, both genetic and therapeutic inhibition of oncogenic KRAS reduced proliferating tumor cells. In fact, 95% of proliferating cells were lost in lungs where KRASG12D expression was turned ‘OFF’ and 90% were lost in RMC-7977-treated mice. Gene set enrichment analysis (GSEA) of the proliferating tumor cell cluster confirmed strong oncogenic KRAS signaling activity in these cells and thus, dependency on oncogenic KRAS. Despite loss of most proliferating tumor cells, further sub-clustering revealed that a small subset of tumor cells persisted. These ‘persister’ cells displayed a low GSEA ‘hallmark oncogenic KRAS signaling’ signature in ‘ON’ tumors, and thereby likely allowed evasion of KRAS-targeted elimination. However, upon KRAS inhibition, persister cells were activated and exhibited dramatic upregulation of the GSEA hallmark KRAS signaling pathway signature (39% in OFF and 16% in RMC-7977-treated tumors, respectively). Furthermore, persister cells expressed multiple RAS/MAPK signaling genes, including Raf and Cdc42, likely contributing to evasion and resistance to KRAS inhibition. Genetic and pharmacological inhibition of oncogenic KRAS eliminated not only tumor cells, but also IFN-responsive CD8+ cytotoxic T-cells. Loss of these CTLs may have been caused by reduced antigen presentation and inflammatory cues associated with the dramatic loss of tumor cells observed upon KRAS inhibition. In contrast, KRAS inhibition drove a marked expansion of CTLA4+ CD4+ T cells, indicating adaptive resistance in the TME to KRAS inhibition. In conclusion, our findings reveal major changes in both tumor and T-cell composition following KRASG12D inhibition and potential TME-dependent mechanisms of resistance to KRAS inhibitors. Citation Format: Niloofar Khairkhah, Ali Namvar, Emily L. Lasse-Opsahl, Mostafa M H. Ibrahim, Carlos E. Espinoza, Megan Faunce, Lily Rober, Marina Pasca di Magliano, Stefanie Galban. Identification of TME-dependent resistance and emergence of persister cells to KRAS inhibition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RAS Oncogenesis and Therapeutics; 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_1):Abstract nr B023.

Osteoporosis is a common disease, especially among the elderly. This study aimed to comprehensively examine the roles of immune microenvironment in osteoporosis pathogenesis. Expression profiles of GSE35959, GSE7158, and GSE13850 datasets were used to analyze differential expression and identify hub genes related to immune features. Based on the single-cell RNA sequencing (scRNA-seq) data of an osteoporosis patient, different cell types were classified and the relation between immune environment and osteoporosis was explored. Twelve hub genes significantly associated with immune features were selected and 11 subgroups were defined using scRNA-seq data. The expression of two hub genes (CDKN1A and TEFM) was greatly altered during the transformation from mesenchymal stem cells (MSCs) to osteoblasts. Chemokines and chemokine receptors were differentially enriched in different cell types. CXCL12 was high-expressed in MSCs. This study emphasized that immune microenvironment played a critical role in the pathogenesis of osteoporosis. Chemokines and chemokine receptors can modify cell development and affect the interactions among different cell types, leading to unbalanced bone remodeling.

Transcriptomic analysis across species is increasingly used to reveal conserved gene regulations which implicate crucial regulators. Cross-species analysis of single-cell RNA sequencing (scRNA-seq) data provides new opportunities to identify the cellular and molecular conservations, especially for cell types and cell type-specific gene regulations. However, few methods have been developed to analyze cross-species scRNA-seq data to uncover both molecular and cellular conservations. Here, we built a tool called CACIMAR, which can perform cross-species analysis of cell identities, markers, regulations, and interactions using scRNA-seq profiles. Based on the weighted sum models of the conserved features, we developed different conservation scores to measure the conservation of cell types, regulatory networks, and intercellular interactions. Using publicly available scRNA-seq data on retinal regeneration in mice, zebrafish, and chick, we demonstrated four main functions of CACIMAR. First, CACIMAR allows to identify conserved cell types even in evolutionarily distant species. Second, the tool facilitates the identification of evolutionarily conserved or species-specific marker genes. Third, CACIMAR enables the identification of conserved intracellular regulations, including cell type-specific regulatory subnetworks and regulators. Lastly, CACIMAR provides a unique feature for identifying conserved intercellular interactions. Overall, CACIMAR facilitates the identification of evolutionarily conserved cell types, marker genes, intracellular regulations, and intercellular interactions, providing insights into the cellular and molecular mechanisms of species evolution.

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130(Cas). Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130(Cas). WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.

Human complement factor H (CFH), a central complement control protein, is a member of the regulators of complement activation family. Recent studies suggested that CFH may play a key role in the resistance of complement-mediated lysis in various cancer cells. In this study, we investigated the role of CFH in human lung cancer. Expression of CFH was analyzed in lung cancer cell lines by RT-PCR, Western blotting and immunofluorescence. In primary lung tumors, the protein expression of CFH was evaluated by immunohistochemistry (IHC) on tissue microarray (TMA). Binding of CFH to lung cancer cells was detected by flow cytometry. mRNA expression of CFH was detected in 6 out of 10 non-small cell lung cancer (NSCLC) cell lines, but in none of the small cell lung cancer (SCLC) cell lines. In line with Western blotting, immunofluorescence analysis demonstrated CFH protein expression in 3 NSCLC cell lines, and the immunoreaction was mainly associated with cell cytoplasm and membrane. In primary lung tumors, 54 out of 101 samples exhibited high expression of CFH and high expression was significantly correlated with lung adenocarcinoma (p=0.009). Also, in adenocarcinoma of the lung, Kaplan-Meier survival analysis showed a tendency that CFH-positive tumors had worse prognosis in comparison to CFH-negative tumors (p=0.082). Additionally, shorter survival time of patients with adenocarcinoma (<20 months) was associated with higher staining of CFH (p=0.033). Our data showed that non-small cell lung cancer cells expressed and secreted CFH. CFH might be a novel diagnostic marker for human lung adenocarcinoma.

MIG6 loss increased RET inhibitor tolerant persister cells in RET-rearranged non-small cell lung cancer

The fatty acid binding protein (FABP) family of proteins plays a role in multiple myeloma cell survival and disease progression in vitro and in silico, and thus blocking the FABPs could have anti-cancer effects and prove beneficial for myeloma patients.

This study aims to investigate the impacts of SLC12A8 on the invasion, migration, and epithelial-mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC) cells. GEPIA database was employed to examine SLC12A8 expression pattern in lung cancer cells. Subsequently, qRT-PCR and Western blot analyses were conducted to assess SLC12A8 expression in NSCLC tissues and cell lines. The overall prognosis of NSCLC patients was evaluated using Kaplan-Meier plot and univariate and multivariate COX regression curves. The knockdown of SLC12A8 was established using lentivirus-mediated shRNA in A549 and H1299 cells. Cell proliferation, invasion, migration, and apoptosis were evaluated using CCK-8 assay, transwell, and flow cytometry techniques, respectively. Western blot analysis was performed to measure the expression levels of EMT-related proteins (E-cadherin and vimentin). The expression level of SLC12A8 was found to be significantly higher in both NSCLC cell lines and tissues. High SLC12A8 expression was correlated with a poor prognosis in NSCLC patients. Knocking down SLC12A8 led to a significant decrease in proliferation, migration, and invasion abilities, while promoting apoptosis in NSCLC cells. Additionally, SLC12A8 knockdown resulted in decreased levels of N-cadherin and vimentin, along with increased E-cadherin expression. The results indicate that reducing SLC12A8 expression may suppress the malignant phenotype of NSCLC cells, as well as the EMT. SLC12A8 may serve as a target for the clinical management of NSCLC progression.

Cancer-associated fibroblasts (CAFs) have long been known as one of the most important players in tumor initiation and progression. Even so, there is an incomplete understanding of the identification of CAFs among tumor microenvironment cells as the list of CAF marker genes varies greatly in the literature, therefore it is imperative to find a better way to identify reliable markers of CAFs. To this end, we summarized a large number of single-cell RNA-sequencing data of multiple tumor types and corresponding normal tissues. As a result, for 9 different types of cancer, we identified CAF-specific gene expression signatures and found 10 protein markers that showed strongly positive staining of tumor stroma according to the analysis of IHC images from the Human Protein Atlas database. Our results give an insight into selecting the most appropriate combination of cancer-associated fibroblast markers. Furthermore, comparison of different approaches for studying differences between cancer-associated and normal fibroblasts (NFs) illustrates the superiority of transcriptome analysis of fibroblasts obtained from fresh tissue samples. Using single-cell RNA sequencing data, we identified common differences in gene expression patterns between normal and cancer-associated fibroblasts, which do not depend on the type of tumor.

The biochemical properties of lung cancer cell lines were investigated. Bombesin-like peptides were present in three small cell lung cancer (SCLC) cell lines examined and three of four lung carcinoids but not in five non-small cell lung cancer (NSCLC) cell lines. Therefore SCLC and some lung carcinoids, but not NSCLC, are enriched in neuroendocrine properties. In contrast, 125I-EGF bound with high affinity to all five NSCLC cell lines and three of four lung carcinoids but not to the three SCLC cell lines examined. For lung carcinoid cell line NCI-H727, 125I-EGF bound with high affinity (Kd = 6 nM) to a single class of sites (Bmax = 110,000/cell). The 125I-EGF bound was rapidly internalized at 37 degrees C but not 4 degrees C. Using Western blot techniques and antiphosphotyrosine antibodies, EGF induced phosphorylation of a major 170 Kd protein. Using immunoprecipitation techniques and anti-EGF receptor antibodies a major 170 Kd protein was labeled. These data indicate that biologically active EGF receptors are present on NSCLC and lung carcinoid cell lines.

Ovarian cancer is one of the five cancer types with highest incidence of death in women. Due to a lack of diagnostic options, timely detection of ovarian cancer is difficult and 75% of patients are diagnosed at a late stage. The standard therapy for ovarian cancer is resection of the affected tissue, followed by chemotherapy. However, patients frequently suffer from relapse of the disease, as some tumor cells persist chemotherapy and grow into a new, more aggressive tumor. To offer therapeutic approaches that prevent relapse by eradicating persister cells we aimed to identify the molecular mechanisms and determinants that are characteristic for persister cells. As initial cellular model system OVCAR-3 cells were used to select ovarian cancer persister cells. Selection was performed analogously to serum levels of chemotherapy patients, i.e. OVCAR-3 cells were incubated twice in the presence of 13 µM Cisplatin for 4 hours. Cisplatin-selection identified clones were collected and in further analyses compared to randomly collected untreated control clones. In order to account the native tumor microenvironment and heterogeneity within the tumor and among patients we cultured precision-cut tissue slices from primary ovarian tumor tissue with 280 µm thickness. Tumor tissue slices were cultured either under control conditions or exposed to 13 µM Cisplatin. Slices were further processed and cultured in Matrigel producing tumor-derived organoids, i.e. tumoroids. In contrast to OVCAR-3 control clones, the Cisplatin-selected OVCAR-3 persister cells showed filopodia-like structures. A scratch assay demonstrated enhanced motility of OVCAR-3-persister cells. Interestingly, cell death analysis (Annexin V) of Cisplatin exposed control and persister clonal cell lines showed the presence of surviving cells in both, persister and control groups. To identify the molecular determinants specific for persister cells we performed RNA sequencing. Data showed that, in line with initial results, the motility associated genes Snail and Vimentin are up regulated in persister clones. We successfully generated tumoroids from patient-derived ovarian cancer tissue slices. Tumoroids were cultured for more than two months and displayed a high viability. After exposure to Cisplatin less tumoroids generated from tumor slices were found. OVCAR-3-derived persister clones show morphological alterations (filopodia-like structures), enhanced motility and expression of motility associated genes. We expect the identification of genetic determinants for persister cell characteristics from comparison of RNAseq results of OVCAR-3 persister clones and tumor-derived organoids. The identification of genetic determinants will direct the development of therapeutics approaches targeting persister cells in ovarian cancer to prevent recurrence of tumors. Citation Format: Kathrin Boepple, Meng Dong, Andrea Gaissler, Bernd Winkler, Markus Kleih, Frank Essmann, Walter E. Aulitzky. Ovarian cancer persister cells: 2D and 3D in-depth characterization and analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 653.

Anchored-fusion enables targeted fusion search in bulk and single-cell RNA sequencing data

Back pain caused by intervertebral disc (IVD) degeneration has a major socio-economic impact in humans, yet historically has received minimal attention in species other than humans, mice and dogs. However, a general growing interest in this unique organ prompted the expansion of IVD research in rats, rabbits, cats, horses, monkeys, and cows, further illuminating the complex nature of the organ in both healthy and degenerative states. Application of recent biotechnological advancements, including single cell RNA sequencing and complex data analysis methods has begun to explain the shifting inflammatory signaling, variation in cellular subpopulations, differential gene expression, mechanical loading, and metabolic stresses which contribute to age and stress related degeneration of the IVD. This increase in IVD research across species introduces a need for chronicling IVD advancements and tissue biomarkers both within and between species. Here we provide a comprehensive review of recent single cell RNA sequencing data alongside existing case reports and histo/morphological data to highlight the cellular complexity and metabolic challenges of this unique organ that is of structural importance for all vertebrates.