Heterogeneous Expression Research Articles

A novel glycolysis-related gene signature for predicting the prognosis of multiple myeloma.

Background: Metabolic reprogramming is an important hallmark of cancer. Glycolysis provides the conditions on which multiple myeloma (MM) thrives. Due to MM's great heterogeneity and incurability, risk assessment and treatment choices are still difficult. Method: We constructed a glycolysis-related prognostic model by Least absolute shrinkage and selection operator (LASSO) Cox regression analysis. It was validated in two independent external cohorts, cell lines, and our clinical specimens. The model was also explored for its biological properties, immune microenvironment, and therapeutic response including immunotherapy. Finally, multiple metrics were combined to construct a nomogram to assist in personalized prediction of survival outcomes. Results: A wide range of variants and heterogeneous expression profiles of glycolysis-related genes were observed in MM. The prognostic model behaved well in differentiating between populations with various prognoses and proved to be an independent prognostic factor. This prognostic signature closely coordinated with multiple malignant features such as high-risk clinical features, immune dysfunction, stem cell-like features, cancer-related pathways, which was associated with the survival outcomes of MM. In terms of treatment, the high-risk group showed resistance to conventional drugs such as bortezomib, doxorubicin and immunotherapy. The joint scores generated by the nomogram showed higher clinical benefit than other clinical indicators. The in vitro experiments with cell lines and clinical subjects further provided convincing evidence for our study. Conclusion: We developed and validated the utility of the MM glycolysis-related prognostic model, which provides a new direction for prognosis assessment, treatment options for MM patients.

Association of CAR-T cell secreting CD47 blocking protein with an enhanced antitumor activity in small cell lung cancer.

e20512 Background: Notch ligand family DLL3 has been considered a therapeutic target for small cell lung cancer. CD47/SIRPa is an important immunosuppressive factor in the tumor microenvironment and is considered to be a "checkpoint" of innate immune cells. Here, we described an DLL3 targeting CAR-T cells secreting CD47/SIRPa signal blocking protein. We hypothesized that the armored CAR-T cells, by blocking the CD47/SIRPa signal with specific antibodies may promote macrophages to engulf tumor cells and induce anti-tumor immune response, thus addressing a clinical need of cell therapy in SCLC. Methods: Bioinformatics analysis of immune cell composition in the microenvironment of small cell lung cancer, focusing on macrophage content and phenotypic characteristics. The CAR genes were cloned into the lentiviral vector pWPXLd, the expression, phenotype, activation, proliferation, killing and cytokine release of CAR-T cells were detected by flow cytometry, Western blot, RTCA and ELISA; NSG mice were injected subcutaneously with H446-DLL3 cells or H446-DLL3(+/-) combined with CAR-T cells or armored CAR-T cells. Tumor size was measured every 3rd day. The number and phenotype of macrophages in tumors were detected by FCM. Results: Bioinformatics analysis showed that tumors with high expression of DLL3 were often accompanied by downregulation of antigen presentation-related genes; there is a large amount of macrophage infiltration in SCLC, and the later the stage, the more the infiltration. CAR-T cells co-cultured with target cells could effectively activate T cells, promote CAR-T cell proliferation, secrete IL-2, IFN-γ and TNF-α cytokines, and specifically kill DLL3 positive tumor cells. We have established a solid tumor model with heterogeneous antigen expression. CAR-T cells can effectively inhibit the growth of tumors with positive DLL3 expression in vivo, but the inhibition on tumors with heterogeneous antigens is not obvious. Blocking CD47 can promote phagocytosis of tumor cells by macrophages. Western blot analysis revealed the presence of CD47 blocking protein CV1-Fc in the supernatant of secreted CAR-T cells, which can effectively promote the phagocytic effect of macrophages; Armored CAR-T cells have a better anti-tumor effect in antigen heterogeneous tumor model in vivo, delays tumor progression, and promotes the aggregation of M1 type macrophages. Conclusions: CAR-T cells that specifically target DLL3 can effectively kill antigen-positive tumor cells in vivo and in vitro. Blocking CD47 can promote the non-specific anti-tumor effect of macrophages. CAR-T cells secreting CD47 blocking protein have dual anti-tumor effects, not only killing antigen-positive tumor cells, but also recruit and activate macrophages to exert phagocytosis, and have better anti-tumor effects in antigenic heterogeneous solid tumors.

Recombinant expression of IGF-1 and LR3 IGF-1 fused with xylanase in Pichia pastoris.

Insulin-like growth factor-1 (IGF-1) is a pleiotropic protein hormone and has become an attractive therapeutic target because of its multiple roles in various physiological processes, including growth, development, and metabolism. However, its production is hindered by low heterogenous protein expression levels in various expression systems and hard to meet the needs of clinical and scientific research. Here, we report that human IGF-1 and its analog Long R3 IGF-1 (LR3 IGF-1) are recombinant expressed and produced in the Pichia pastoris (P. pastoris) expression system through being fused with highly expressed xylanase XynCDBFV. Furthermore, purified IGF-1 and LR3 IGF-1 display excellent bioactivity of cell proliferation compared to the standard IGF-1. Moreover, higher heterologous expression levels of the fusion proteins XynCDBFV-IGF-1 and XynCDBFV-LR3 IGF-1 are achieved by fermentation in a 15-L bioreactor, reaching up to about 0.5g/L XynCDBFV-IGF-1 and 1g/L XynCDBFV-TEV-LR3 IGF-1. Taken together, high recombinant expression of bioactive IGF-1 and LR3 IGF-1 is acquired with the assistance of xylanase as a fusion partner in P. pastoris, which could be used for both clinical and scientific applications. KEY POINTS: • Human IGF-1 and LR3 IGF-1 are produced in the P. pastoris expression system. • Purified IGF-1 and LR3 IGF-1 show bioactivity comparable to the standard IGF-1. • High heterologous expression of IGF-1 and LR3 IGF-1 is achieved by fermentation in a bioreactor.

RGX-019-MMAE, a novel MERTK-targeting antibody-drug conjugate (ADC) with robust anti-tumor efficacy in both solid and liquid tumors.

e15103 Background: MERTK, a member of the TAM family of receptor tyrosine kinases, is overexpressed in numerous solid and hematologic cancers. MERTK expression has been implicated in promoting cellular proliferation, survival and drug resistance. MERTK is also expressed on innate immune cells, predominantly on immune-suppressive M2 macrophages, where it mediates phagocytosis of apoptotic cells and regulates expression of immunosuppressive cytokines. Development of MERTK inhibitors has been hampered by observance of retinal degeneration due to inhibition of MERTK signaling in retinal pigment epithelium (RPE). We previously reported that our monoclonal antibody RGX-019 triggers transient activation of MERTK, followed by MERTK receptor internalization and degradation. RGX-019 also reduces colony formation of MERTK-expressing cancer cells. Methods: RGX-019-MMAE is a humanized IgG1-MMAE ADC with high specificity and affinity for human and cynomolgus monkey MERTK that induces efficient internalization of MERTK. These characteristics and the limited expression of MERTK in normal tissue make RGX-019 an ideal candidate for an ADC approach. In addition, MMAE selectively targets actively dividing cells such as tumor cells, while sparing macrophages and RPE, both of which represent terminally-differentiated non-dividing cells. Results: We have observed various degrees of MERTK expression on cancer cells within solid tumors and on primary AML samples. In AML, significantly higher expression was noted in M5 subtype, those with PTPN11 mutation or t(9;11) translocation and higher expression correlated with significantly inferior survival. In contrast, in normal tissue, MERTK expression was almost exclusively limited to macrophages, with virtually no expression on healthy epithelial tissue, except for the retina. Twenty-eight-day treatment with RGX-019-MMAE caused no retinal degeneration in a transgenic mouse model expressing a human version of MERTK. RGX-019-MMAE induced cytotoxicity in MERTK-expressing cancer cells and in xenografts, RGX-019-MMAE demonstrated anti-tumor efficacy including complete tumor regressions in various solid cancers such as TNBC as well as AML and MM. Anti-tumor efficacy was associated with increased overall survival. Notably, even tumors with low and heterogenous MERTK expression responded to RGX-019-MMAE treatment. In vitro studies demonstrated that RGX-019-MMAE can kill MERTK non-expressing cancer cells through bystander effects when co-cultured with MERTK-positive cells, consistent with the activity we observed in MERTK heterogenous tumors. Conclusions: Collectively, RGX-019-MMAE’s robust anti-tumor efficacy in multiple aggressive tumors, and MERTK’s expression profile on cancer cells and normal tissue support further advancement of RGX-019-MMAE as a drug candidate for development in solid and liquid cancers.

Impact of vitamin d on the colon tumor immune microenvironment: Results of a randomized clinical trial of preoperative vitamin D supplementation in patients with stage I-III colon cancer.

3607 Background: Although the anti-tumor properties of vitamin D in colorectal cancer (CRC) have been broadly reported, its potential role as a tumor microenvironment (TME) immunomodulator has not been well established. Vitamin D receptor (VDR) and CYP27B1 (1-α-hydroxylase; required to convert vitamin D to its active form) are critical factors in the vitamin D signaling pathway and harbor prognostic significance for CRC. Pre-clinical studies suggest that higher stromal VDR expression enhances chemotherapy efficacy. To characterize the effects of vitamin D on the TME in humans, we conducted a randomized placebo-controlled trial of preoperative high-dose vitamin D supplementation in stage I-III colon cancer patients. Methods: Forty-two patients were randomized to receive vitamin D3 50,000 IU/day x 7 days followed by 10,000 IU/day versus placebo prior to resection. Paired pre-treatment biopsy specimens and post-treatment surgical resection specimens from 19 patients who completed the assigned treatment and had sufficient tissue for analysis were collected for spatially-resolved immune cell profiling and evaluation of VDR and CYP27B1 expression using custom multiplex immunofluorescence. Blood samples were collected pre- and post-treatment to assess plasma 25-hydroxyvitamin D [25(OH)D] levels. Linear mixed effects models were used to compare tissue parameters; plasma 25(OH)D level changes were assessed using the Wilcoxon rank-sum test. Results: Patients randomized to high-dose vitamin D had increased post-treatment plasma 25(OH)D (median 18.0 to 63.6 (ng/ml)) whereas placebo-randomized patients did not (median 19.1 to 18.6 (ng/ml)) ( p <0.001). Analysis of both biopsy and resection tissue revealed highly heterogeneous VDR and CYP27B1 expression. VDR expression was highest in tumor cells, followed by immune and then stromal cells. Although resection tissue from vitamin D-treated patients showed lower VDR expression than specimens from placebo-treated patients in all cells ( p= 0.03), VDR was preferentially expressed by tumor cells at the tumor invasive front compared to tumor center in the vitamin D arm, while the opposite was observed in the placebo arm. Vitamin D supplementation did not alter CYP27B1 expression. While treatment did not change total immune cell density, resection tissue from vitamin D-treated patients had higher overall CD3+CD8+ T cell density ( p= 0.04), particularly CD3+CD8+ memory T cell density ( p= 0.02). Additionally, vitamin D supplementation led to greater co-localization between CD3+CD8+ T cells and tumor cells ( p< 0.001) compared to placebo. Conclusions: In patients with stage I-III CRC, vitamin D supplementation led to changes in vitamin D pathway signaling and evidence of an anti-tumor effect in the TME. Further investigation into the underlying mechanism of action is warranted. Clinical trial information: NCT02172651 .

Single-Cell Heterogeneity in Snake Venom Expression Is Hardwired by Co-Option of Regulators from Progressively Activated Pathways.

The ubiquitous cellular heterogeneity underlying many organism-level phenotypes raises questions about what factors drive this heterogeneity and how these complex heterogeneous systems evolve. Here, we use single-cell expression data from a Prairie rattlesnake (Crotalus viridis) venom gland to evaluate hypotheses for signaling networks underlying snake venom regulation and the degree to which different venom gene families have evolutionarily recruited distinct regulatory architectures. Our findings suggest that snake venom regulatory systems have evolutionarily co-opted trans-regulatory factors from extracellular signal-regulated kinase and unfolded protein response pathways that specifically coordinate expression of distinct venom toxins in a phased sequence across a single population of secretory cells. This pattern of co-option results in extensive cell-to-cell variation in venom gene expression, even between tandemly duplicated paralogs, suggesting this regulatory architecture has evolved to circumvent cellular constraints. While the exact nature of such constraints remains an open question, we propose that such regulatory heterogeneity may circumvent steric constraints on chromatin, cellular physiological constraints (e.g., endoplasmic reticulum stress or negative protein-protein interactions), or a combination of these. Regardless of the precise nature of these constraints, this example suggests that, in some cases, dynamic cellular constraints may impose previously unappreciated secondary constraints on the evolution of gene regulatory networks that favors heterogeneous expression.

The characteristic of stem-related genes with pancreatic carcinoma cell after irradiation

PurposeTo investigate stem-related differentially expressed genes (DEGs) and their potential mechanism in pancreatic cancer cells (MIAPaCa-2) exposed to x-ray and proton radiation, as well as how these factors affected the prognosis of patients with pancreatic adenocarcinoma (PADC). MethodsThe stem-related DEGs were screened using the online tool Stemchecker after protons and x-rays were used to irradiate MIAPaCa-2 cells. Analysis was done on the probable processes and prognostic significance of the DEGs in PAC patients. ResultsFour datasets containing 401 DEGs were filtered, and the stem-related DEGs for each irradiation type indicated a variety of radiobiological characteristics. In pancreatic cancer cells, a number of stem-related DEGs may serve as biomarkers of radiation effects. Patients with pancreatic cancer demonstrated predictive significance for GRB7, B2M, and PMAIP1. ConclusionsMIAPaCa-2 cells exposed to x-rays and protons repeatedly displayed heterogeneous expression of stem-related DEGs involved in complex radiosensitivity, radio-resistance, and radio-induced mortality pathways. GRB7 and B2M were considered potential radiation sensitivity indicators for pancreatic cancer.

Tenofovir alters the immune microenvironment of pregnant women with hepatitis B virus infection: Evidence from single-cell RNA sequencing.

Mother-to-child is the main route of the transmission of hepatitis B virus (HBV) infection. Tenofovir fumarate (TDF) antiviral treatment has become the most extensive choice worldwide. However, the effects of TDF treatment on the immune function of pregnant women remains unclear. Here we investigate the effect of TDF treatment on the immune microenvironment of pregnant women with HBV infection using single-cell RNA sequencing (scRNA-seq). Three HBV-infected pregnant women were treated with TDF and six samples were collected before and after the treatment. In total, 68,200 peripheral blood mononuclear cells (PBMCs) were extracted for 10×scRNA-seq. The cells were clustered using t-distributed stochastic neighbor embedding (t-SNE) and unbiased computational informatics analysis. The analysis identified four-cell subtypes, including T cells, monocytes, natural killer (NK) cells, and B cells, and unraveled the developmental trajectory and maturation of CD4+ T and CD8+ T cell subtypes. The cellular state and molecular features of the effector/memory T cells revealed a significant increase in the inflammatory state of CD4+ T cells and the cytotoxic characteristics of CD8+ T cells. Additionally, after TDF treatment, the monocytes showed a tendency for M1 polarization, and the cytotoxicity of NK cells was enhanced. Furthermore, the analysis of intercellular communication revealed the interaction of various subtypes of cells and the heterogeneous expression of key signal pathways. The findings of this study reveal significant differences in cellular subtypes and molecular characteristics of PBMCs of pregnant women with HBV infection before and after TDF treatment and demonstrate the recovery of immune response after treatment. These findings could help develop immune intervention measures to control HBV during pregnancy and the puerperium period.

ScLiverDB: a Database of Human and Mouse Liver Transcriptome Landscapes at Single-Cell Resolution.

The liver is critical for the digestive and immune systems. Although the physiology and pathology of liver have been well studied and many scRNA-seq data are generated, a database and landscape for characterizing cell types and gene expression in different liver diseases or developmental stages at single-cell resolution are lacking. Hence, scLiverDB is developed, a specialized database for human and mouse liver transcriptomes to unravel the landscape of liver cell types, cell heterogeneity and gene expression at single-cell resolution across various liver diseases/cell types/developmental stages. To date, 62 datasets including 9,050 samples and 1,741,734 cells is curated. A uniform workflow is used, which included quality control, dimensional reduction, clustering, and cell-type annotation to analyze datasets on the same platform; integrated manual and automatic methods for accurate cell-type identification and provided a user-friendly web interface with multiscale functions. There are two case studies to show the usefulness of scLiverDB, which identified the LTB (lymphotoxin Beta) gene as a potential biomarker of lymphoid cells differentiation and showed the expression changes of Foxa3 (forkhead box A3) in liver chronic progressive diseases. This work provides a crucial resource to resolve molecular and cellular information in normal, diseased, and developing human and mouse livers.

89Zr]-Atezolizumab-PET Imaging Reveals Longitudinal Alterations in PDL1 during Therapy in TNBC Preclinical Models.

Triple-negative breast cancers (TNBCs) currently have limited treatment options; however, PD-L1 is an indicator of susceptibility to immunotherapy. Currently, assessment of PD-L1 is limited to biopsy samples. These limitations may be overcome with molecular imaging. In this work, we describe chemistry development and optimization, in vitro, in vivo, and dosimetry of [89Zr]-Atezolizumab for PD-L1 imaging. Atezolizumab was conjugated to DFO and radiolabeled with 89Zr. Tumor uptake and heterogeneity in TNBC xenograft and patient-derived xenograft (PDX) mouse models were quantified following [89Zr]-Atezolizumab-PET imaging. PD-L1 expression in TNBC PDX models undergoing therapy and immunohistochemistry (IHC) was used to validate imaging. SUV from PET imaging was quantified and used to identify heterogeneity. PET/CT imaging using [89Zr]-Atezolizumab identified a significant increase in tumor:muscle SUVmean 1 and 4 days after niraparib therapy and revealed an increased trend in PD-L1 expression following other cytotoxic therapies. A preliminary dosimetry study indicated the organs that will receive a higher dose are the spleen, adrenals, kidneys, and liver. [89Zr]-Atezolizumab PET/CT imaging reveals potential for the noninvasive detection of PD-L1-positive TNBC tumors and allows for quantitative and longitudinal assessment. This has potential significance for understanding tumor heterogeneity and monitoring early expression changes in PD-L1 induced by therapy.

HER2 Intratumoral Heterogeneity in Breast Cancer, an Evolving Concept.

Amplification and/or overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is associated with an adverse prognosis. The introduction of anti-HER2 targeted therapy has dramatically improved the clinical outcomes of patients with HER2-positive breast cancer. Unfortunately, a significant number of patients eventually relapse and develop distant metastasis. HER2 intratumoral heterogeneity (ITH) has been reported to be associated with poor prognosis in patients with anti-HER2 targeted therapies and was proposed to be a potential mechanism for anti-HER2 resistance. In this review, we described the current definition, common types of HER2 ITH in breast cancer, the challenge in interpretation of HER2 status in cases showing ITH and the clinical applications of anti-HER2 agents in breast cancer showing heterogeneous HER2 expression. Digital image analysis has emerged as an objective and reproducible scoring method and its role in the assessment of HER2 status with ITH remains to be demonstrated.

MetaTiME integrates single-cell gene expression to characterize the meta-components of the tumor immune microenvironment

Recent advances in single-cell RNA sequencing have shown heterogeneous cell types and gene expression states in the non-cancerous cells in tumors. The integration of multiple scRNA-seq datasets across tumors can indicate common cell types and states in the tumor microenvironment (TME). We develop a data driven framework, MetaTiME, to overcome the limitations in resolution and consistency that result from manual labelling using known gene markers. Using millions of TME single cells, MetaTiME learns meta-components that encode independent components of gene expression observed across cancer types. The meta-components are biologically interpretable as cell types, cell states, and signaling activities. By projecting onto the MetaTiME space, we provide a tool to annotate cell states and signature continuums for TME scRNA-seq data. Leveraging epigenetics data, MetaTiME reveals critical transcriptional regulators for the cell states. Overall, MetaTiME learns data-driven meta-components that depict cellular states and gene regulators for tumor immunity and cancer immunotherapy.

Heterogeneous expression of stearoyl-acyl carrier protein desaturase genes SAD1 and SAD2 from Linum usitatissimum enhances seed oleic acid accumulation and seedling cold and drought tolerance in Brassica napus

Flax (Linum usitatissimum L.) is a versatile crop and its seeds are a major source of unsaturated fatty acids. Stearoyl-acyl carrier protein desaturase (SAD) is a dehydrogenase enzyme that plays a key role in oleic acid biosynthesis as well as responses to biotic and abiotic stresses. However, the function of SAD orthologs from L. usitatissimum has not been assessed. Here, we found that two LuSAD genes, LuSAD1 and LuSAD2, are present in the genome of L. usitatissimum cultivar ‘Longya 10’. Heterogeneous expression of either LuSAD1 or LuSAD2 in Arabidopsis thaliana resulted in higher contents of total fatty acids and oleic acid in the seeds. Interestingly, ectopic expression of LuSAD2 in A. thaliana caused altered plant architecture. Similarly, the overexpression of either LuSAD1 or LuSAD2 in Brassica napus also resulted in increased contents of total fatty acids and oleic acid in the seeds. Furthermore, we demonstrated that either LuSAD1 or LuSAD2 enhances seedling resistance to cold and drought stresses by improving antioxidant enzyme activity and nonenzymatic antioxidant levels, as well as reducing membrane damage. These findings not only broaden our knowledge of the LuSAD functions in plants, but also offer promising targets for improving the quantity and quality of oil, and the abiotic stress tolerance of oil-producing crops, through molecular manipulation.

The immunopeptidome landscape associated with T cell infiltration, inflammation and immune editing in lung cancer

One key barrier to improving efficacy of personalized cancer immunotherapies that are dependent on the tumor antigenic landscape remains patient stratification. Although patients with CD3+CD8+ T cell-inflamed tumors typically show better response to immune checkpoint inhibitors, it is still unknown whether the immunopeptidome repertoire presented in highly inflamed and noninflamed tumors is substantially different. We surveyed 61 tumor regions and adjacent nonmalignant lung tissues from 8 patients with lung cancer and performed deep antigen discovery combining immunopeptidomics, genomics, bulk and spatial transcriptomics, and explored the heterogeneous expression and presentation of tumor (neo)antigens. In the present study, we associated diverse immune cell populations with the immunopeptidome and found a relatively higher frequency of predicted neoantigens located within HLA-I presentation hotspots in CD3+CD8+ T cell-excluded tumors. We associated such neoantigens with immune recognition, supporting their involvement in immune editing. This could have implications for the choice of combination therapies tailored to the patient’s mutanome and immune microenvironment.

NanoBeacon.AI: AI-Enhanced Nanodiamond Biosensor for Automated Sensitivity Prediction to Oxidative Phosphorylation Inhibitors.

Spalt-like transcription factor 4 (SALL4) is an oncofetal protein that has been identified to drive cancer progression in hepatocellular carcinoma (HCC) and hematological malignancies. Furthermore, a high SALL4 expression level is correlated to poor prognosis in these cancers. However, SALL4 lacks well-structured small-molecule binding pockets, making it difficult to design targeted inhibitors. SALL4-induced expression of oxidative phosphorylation (OXPHOS) genes may serve as a therapeutically targetable vulnerability in HCC through OXPHOS inhibition. Because OXPHOS functions through a set of genes with intertumoral heterogeneous expression, identifying therapeutic sensitivity to OXPHOS inhibitors may not rely on a single clear biomarker. Here, we developed a workflow that utilized molecular beacons, nucleic-acid-based, activatable sensors with high specificity to the target mRNA, delivered by nanodiamonds, to establish an artificial intelligence (AI)-assisted platform for rapid evaluation of patient-specific drug sensitivity. Specifically, when the HCC cells were treated with the nanodiamond-medicated OXPHOS biosensor, high sensitivity and specificity of the sensor allowed for improved identification of OXPHOS expression in cells. Assisted by a trained convolutional neural network, drug sensitivity of cells toward an OXPHOS inhibitor, IACS-010759, could be accurately predicted. AI-assisted OXPHOS drug sensitivity assessment could be accomplished within 1 day, enabling rapid and efficient clinical decision support for HCC treatment. The work proposed here serves as a foundation for the patient-based subtype-specific therapeutic research platform and is well suited for precision medicine.

Receptor for hyaluronan-mediated motility (RHAMM) defines an invasive niche associated with tumor progression and predicts poor outcomes in breast cancer patients.

Breast cancer invasion and metastasis result from a complex interplay between tumor cells and the tumor microenvironment (TME). Key oncogenic changes in the TME include aberrant synthesis, processing, and signaling of hyaluronan (HA). Hyaluronan-mediated motility receptor (RHAMM, CD168; HMMR) is an HA receptor enabling tumor cells to sense and respond to this aberrant TME during breast cancer progression. Previous studies have associated RHAMM expression with breast tumor progression; however, cause and effect mechanisms are incompletely established. Focused gene expression analysis of an internal breast cancer patient cohort confirmed that increased RHAMM expression correlates with aggressive clinicopathological features. To probe mechanisms, we developed a novel 27-gene RHAMM-related signature (RRS) by intersecting differentially expressed genes in lymph node (LN)-positive patient cases with the transcriptome of a RHAMM-dependent model of cell transformation, which we validated in an independent cohort. We demonstrate that the RRS predicts for poor survival and is enriched for cell cycle and TME-interaction pathways. Further analyses using CRISPR/Cas9-generated RHAMM-/- breast cancer cells provided direct evidence that RHAMM promotes invasion in vitro and in vivo. Immunohistochemistry studies highlighted heterogeneous RHAMM protein expression, and spatial transcriptomics associated the RRS with RHAMM-high microanatomic foci. We conclude that RHAMM upregulation leads to the formation of 'invasive niches', which are enriched in RRS-related pathways that drive invasion and could be targeted to limit invasive progression and improve patient outcomes. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Transforming RNA-Seq gene expression to track cancer progression in the multi-stage early to advanced-stage cancer development.

Cancer progression can be tracked by gene expression changes that occur throughout early-stage to advanced-stage cancer development. The accumulated genetic changes can be detected when gene expression levels in advanced-stage are less variable but show high variability in early-stage. Normalizing advanced-stage expression samples with early-stage and clustering of the normalized expression samples can reveal cancers with similar or different progression and provide insight into clinical and phenotypic patterns of patient samples within the same cancer. This study aims to investigate cancer progression through RNA-Seq expression profiles across the multi-stage process of cancer development. RNA-sequenced gene expression of Diffuse Large B-cell Lymphoma, Lung cancer, Liver cancer, Cervical cancer, and Testicular cancer were downloaded from the UCSC Xena database. Advanced-stage samples were normalized with early-stage samples to consider heterogeneity differences in the multi-stage cancer progression. WGCNA was used to build a gene network and categorized normalized genes into different modules. A gene set enrichment analysis selected key gene modules related to cancer. The diagnostic capacity of the modules was evaluated after hierarchical clustering. Unnormalized RNA-Seq gene expression failed to segregate advanced-stage samples based on selected cancer cohorts. Normalization with early-stage revealed the true heterogeneous gene expression that accumulates across the multi-stage cancer progression, this resulted in well segregated cancer samples. Cancer-specific pathways were enriched in the normalized WGCNA modules. The normalization method was further able to stratify patient samples based on phenotypic and clinical information. Additionally, the method allowed for patient survival analysis, with the Cox regression model selecting gene MAP4K1 in cervical cancer and Kaplan-Meier confirming that upregulation is favourable. The application of the normalization method further enhanced the accuracy of clustering of cancer samples based on how they progressed. Additionally, genes responsible for cancer progression were discovered.

Pro- and anti-tumour activities of CD146/MCAM in breast cancer result from its heterogeneous expression and association with epithelial to mesenchymal transition.

CD146, also known as melanoma cell adhesion molecule (MCAM), is expressed in numerous cancers and has been implicated in the regulation of metastasis. We show that CD146 negatively regulates transendothelial migration (TEM) in breast cancer. This inhibitory activity is reflected by a reduction in MCAM gene expression and increased promoter methylation in tumour tissue compared to normal breast tissue. However, increased CD146/MCAM expression is associated with poor prognosis in breast cancer, a characteristic that is difficult to reconcile with inhibition of TEM by CD146 and its epigenetic silencing. Single cell transcriptome data revealed MCAM expression in multiple cell types, including the malignant cells, tumour vasculature and normal epithelium. MCAM expressing malignant cells were in the minority and expression was associated with epithelial to mesenchymal transition (EMT). Furthermore, gene expression signatures defining invasiveness and a stem cell-like phenotype were most strongly associated with mesenchymal-like tumour cells with low levels of MCAM mRNA, likely to represent a hybrid epithelial/mesenchymal (E/M) state. Our results show that high levels of MCAM gene expression are associated with poor prognosis in breast cancer because they reflect tumour vascularisation and high levels of EMT. We suggest that high levels of mesenchymal-like malignant cells reflect large populations of hybrid E/M cells and that low CD146 expression on these hybrid cells is permissive for TEM, aiding metastasis.

Nanomedicine sheds new light on cancer immunotherapy.

Cancer immunotherapy comprising of immune checkpoint blockade (ICB) therapy, immune cell therapies, cancer vaccines and many others represents a profound arsenal in the fight against different types of cancers. However, their overall clinical objective response rates, particularly against most solid tumors, are still not sufficient owing to a variety of reasons including the heterogenous expression of tumor antigens, limited tumor infiltration of effector immune cells, acquired tumor immunosuppression and some other factors. In recent years, various nanomedicine strategies have been proposed to assist cancer immunotherapy via distinct mechanisms, presenting new promises in many published studies. This perspective willthus provide a brief overview regarding the development of nanomedicine platforms for improving cancer immunotherapy.

Abstract LB273: An emerging paradigm of heterogeneous midkine expression in thyroid cancer

Abstract Midkine (MDK) is a pleiotropic heparin-binding growth factor, contributing to both normal tissue homeostasis and disease development. MDK expression is gradually increased during carcinogenesis, acting as a mediator for the acquisition of cancer hallmarks, such as invasion/metastasis, and immunosuppression. Tissue-wide gene expression analysis using publicly available databases reveal that MDK is significantly upregulated in most human carcinomas. In the thyroid, MDK has been associated with increased metastatic potential in the context of papillary thyroid cancer, which is an otherwise relatively non-aggressive thyroid tumor. We thus theorized that more aggressive types of thyroid cancers may be linked to increased MDK expression/function from an earlier onset. To gain such insights, we developed a digital pathology infrastructure to investigate MDK expression in a characterized mouse model of Anaplastic Thyroid Carcinoma (ATC), the most aggressive and lethal type of thyroid cancer with low but increased prevalence in the human population, and an intrinsic resistance to available therapeutic options. Mice with conditional ablation of tumor suppressors, Pten and p53, in thyrocytes [Pten, p53]thyr-/− develop thyroid carcinomas with mixed follicular and anaplastic components, and histological hallmarks of high aggressiveness, including giant cells, bone metaplasia, and muscular/tracheal invasion, which are all recapitulated in human disease. MDK was more highly expressed in anaplastic, when compared to follicular components of the same animal tumors. When the above hallmarks were topographically and microanatomically demarcated within the anaplastic component, it was found that MDK expression was higher near and around giant cell islets, as well as within the anaplastic lesions invading beyond the cartilaginous rings of tracheal mucosal epithelium. Based on recent evidence that MDK does not solely function in the extracellular space, but can also be endocytosed within tumor cells, whereby it exerts tumor-promoting functions, we further quantified the intra/extranuclear MDK fraction using intensity thresholding immunofluorescence. Of interest, MDK+ follicular lesions, were primarily characterized by nuclear MDK expression, whereas MDK was mostly found secreted in the extracellular space of anaplastic lesions, thus implying different modes of MDK trafficking, secretion, and possibly function in them. The take home message is to work towards establishing and developing gain- and loss-of-function experiments, to elucidate mechanistic underpinnings on the role of MDK in the development of aggressive ATC lesions. Citation Format: Dimitra P. Anastasiadou, Antonio Di Cristofano, George S. Karagiannis. An emerging paradigm of heterogeneous midkine expression in thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB273.