Alterations In Tumor Microenvironment Research Articles

Comprehensive understanding of glioblastoma molecular phenotypes: classification, characteristics, and transition.

Among central nervous system-associated malignancies, glioblastoma (GBM) is the most common and has the highest mortality rate. The high heterogeneity of GBM cell types and the complex tumor microenvironment frequently lead to tumor recurrence and sudden relapse in patients treated with temozolomide. In precision medicine, research on GBM treatment is increasingly focusing on molecular subtyping to precisely characterize the cellular and molecular heterogeneity, as well as the refractory nature of GBM toward therapy. Deep understanding of the different molecular expression patterns of GBM subtypes is critical. Researchers have recently proposed tetra fractional or tripartite methods for detecting GBM molecular subtypes. The various molecular subtypes of GBM show significant differences in gene expression patterns and biological behaviors. These subtypes also exhibit high plasticity in their regulatory pathways, oncogene expression, tumor microenvironment alterations, and differential responses to standard therapy. Herein, we summarize the current molecular typing scheme of GBM and the major molecular/genetic characteristics of each subtype. Furthermore, we review the mesenchymal transition mechanisms of GBM under various regulators.

Abstract PO3-25-02: Circadian Rhythm Disruption Accelerates Aggressive Mammary Tumorigenesis by Altering Tumor Microenvironment

Abstract \Breast cancer is the most common cancer in women worldwide, leading to over 650,000 deaths worldwide. The circadian rhythm is a 24-hour biological rhythm that controls most cellular, hormonal, and behavioral processes in living organisms, which is why perturbation of these rhythms is linked to several devastating diseases such as cardiovascular diseases, diabetes, and cancer. In the mammary gland, circadian clocks regulate the rhythmic expression of numerous genes. Circadian rhythm disruption (CRD), which has been termed a "carcinogen" by the International Agency for Research in Cancer, is caused by different factors, one of them being shift work. Several epidemiological studies have revealed that women who work night shifts are predisposed to breast cancer. The aim of this study is to investigate the effect of circadian rhythm disruption on aggressive mammary tumorigenesis. To do this, we used genetically engineered mouse models of mammary tumors (HER2-tumor and triple-negative breast tumor, or TNBC). Chronic jetlag, which mimics shift work in mice and is known to cause CRD, was introduced in one group of mice by perturbing the normal light and dark cycles with an 8-hour advance every three days. Wheel-running, which is represented by an actogram, was used to confirm the circadian disruption in the CRD mice. CRD was found to accelerate tumor initiation with a significant increase in weight in the CRD group of mice. CRD enhanced tumor burden significantly compared to LD mice. Our results also showed high metastatic foci in the lungs of CRD mice compared to LD mice. A flow cytometry study showed a higher cancer stem cell population in CRD tumors. Mammosphere assays with tumor-derived cells showed that CRD increased the number and size of the mammospheres compared to the control tumors. The tumor cells were analyzed using single-cell RNA sequencing, which showed that CRD alters the tumor microenvironment by developing more immunosuppressive and "cold" tumors. The tumor-derived organoids (tumoroid) showed resistance to chemotherapeutics. Finally, bioinformatics analysis using single-cell RNA seq data from human breast tumors (TNBC, ER/PR, and HER2) showed that the malignant cells in TNBC tumors had a significantly higher circadian rhythm disrupted score (CRD score) compared to ER/PR tumors. Taken together, our data showed how circadian rhythm disruption affects TNBC tumorigenesis and drug response by altering the tumor microenvironment. Citation Format: Olajumoke Ogunlusi, Mrinmoy Sarkar, Tristan Nguyen, Devon Boland, James Cai, Arhit Chakrabarti, Bani Mallick, Alex Keene, Tapasree Roy Sarkar. Circadian Rhythm Disruption Accelerates Aggressive Mammary Tumorigenesis by Altering Tumor Microenvironment [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 PO3-25-02.

Abstract PO3-26-08: Genetic modification of tumor associated macrophages using CRISPR lipid nanoparticle platform altered tumor microenvironment and induced cancer cell elimination

Abstract More than half of patients with stage IV breast cancer will eventually develop liver metastasis. Due to their nature, liver metastases are hard to treat, and the five-year survival rate for stage IV breast cancer is very low (~11%). In the recent years, vital roles macrophages play in cancer progression and immune evasion have become increasingly clear, making them an appealing target for therapeutic intervention. As one of the most abundant cell populations recruited to the tumor microenvironment, tumor-associated macrophages (TAM) are typically immunosuppressive Arg1+ M2-like macrophages, which support cancer growth, metastasis progression, and drug resistance. Numerous studies, including from our laboratory, have shown that reversal of the macrophage phenotype to the classical inflammatory CD80+ M1-like macrophages is very promising for cancer eradication. In this study, we used CRISPR loaded into lipid nanoparticles (CRISPR-LNP) to modulate macrophage polarization in breast cancer metastasis in vivo. We hypothesize that CRISPR-mediated gene editing through lipid nanoparticle (LNP) delivery of CRISPR enzyme and target gRNA (CRISPR-LNP) is a highly effective and safe approach. Using our CRISPR-LNP platform, we screened multiple genes with significant roles in macrophage polarization and found that targeting the RICTOR gene alone enabled reprograming of pro-tumorigenic M2 TAMs to anti-tumorigenic M1 phenotype in vitro and in vivo. We administered the CRISPR-LNP targeting RICTOR (CRISPR-RIC-LNP) on a syngeneic liver metastasis mouse model via intravenous injection. Single dose studies were conducted to determine biodistribution, treatment efficacy, and shift in immune landscape of the tumor after treatment via immunofluorescence staining, Imaging Mass Cytometry, and single cell RNA sequencing. A spatial map was built using digital pathology analysis for investigation of the interactions amongst immune and tumor cells within the tumor immune microenvironment. In 4T1 breast cancer liver metastatic model, we observed that CRISPR-RIC-LNP treatment: (i) accumulated in the liver metastases lesions in vivo 24 hours after injection. ~0.5% of total injected dose, which corresponds to ~75% of fluorescence from the whole body at 24h time point was confined only in the metastatic regions; (ii) led to modest increase of macrophage number, and (iii) significant shift of macrophage phenotype from immunosuppressive, pro-tumorigenic Arg1+, CD206+ macrophages to inflammatory, anti-tumor CD80+ phenotype (from ~5 to ~9% of total cell in the tumor lesions) 24 hours after injection; (iv) reduced the cancer cell number by almost 80%; and (v) dramatically reduced exhausted T-cell and regulatory T-cell population, as determined by single cell RNA sequencing analysis. We plan to validate our preliminary finding with flow cytometry analysis of metastatic lesions, and evalute the efficacy of CRISPR-RIC-LNP as single and in combinatorial therapies in survival studies. These data showed that reprograming macrophages from cancer-supporting to cancer-eliminating phenotype is a promising tool to suppress cancer cell growth and increase the presence of cytotoxic CD8+ T-cells. We propose that this approach can be efficiently used as single therapy or as combination with immune checkpoint inhibitors to obtain synergistic effects and efficiently eradicate cancer cells. We will assess the safety, optimize the accumulation in TME, and devise methods to limit exposure to healthy cells. In the future, we also aim to expand scope of our candidate therapy as therapeutics for other types of breast cancer metastases. Citation Format: Fransisca Leonard, Tyler Galbraith, Nourhan Abdelfattah, Arturas Ziemys, Chihiro Hashimoto, Harlan Cook, Yitian Xu, Shu-Hsia Chen, Roberto Rosato, Kyuson Yun. Genetic modification of tumor associated macrophages using CRISPR lipid nanoparticle platform altered tumor microenvironment and induced cancer cell elimination [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 PO3-26-08.

CD276 regulates the immune escape of esophageal squamous cell carcinoma through CXCL1–CXCR2 induced NETs

BackgroundCD276 (B7-H3), a pivotal immune checkpoint, facilitates tumorigenicity, invasiveness, and metastasis by escaping immune surveillance in a variety of tumors; however, the underlying mechanisms facilitating immune escape in esophageal squamous...

Deciphering the Prognostic Significance of MYD88 and CD79B Mutations in Diffuse Large B-Cell Lymphoma: Insights into Treatment Outcomes.

The clinical and genetic characteristics, as well as treatment outcomes, of diffuse large B-cell lymphoma (DLBCL) patients with different MYD88 and CD79B mutation status merit further investigation. This study aims to investigate the distinctions in clinical manifestations, genetic characteristics, and treatment outcomes among MYD88-CD79Bco-mut, MYD88/CD79Bsingle-mut, and MYD88-CD79Bco-wt DLBCL patients. Clinical and genetic characteristics, along with treatment outcomes among 2696 DLBCL patients bearing MYD88-CD79Bco-mut, MYD88/CD79Bsingle-mut, and MYD88-CD79Bco-wt treated with R-CHOP/R-CHOP-like regimens from the Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College and six external cohorts were analyzed. Potential molecular mechanisms were investigated through Gene Set Enrichment Analysis and xCell methodology. In the MCD subtype, patients with MYD88-CD79Bco-mut showed comparable progression-free survival (PFS) and overall survival (OS) compared to MYD88/CD79Bsingle-mut or MYD88-CD79Bco-wt. However, in the non-MCD subtype, patients with MYD88-CD79Bco-mut exhibited significantly inferior OS than MYD88/CD79Bsingle-mut or MYD88-CD79Bco-wt, while there was no significant OS difference between MYD88/CD79Bsingle-mut and MYD88-CD79Bco-wt (median OS: 68.8 [95% CI 22-NA] vs NA [95% CI 112-NA] vs 177.7 [95% CI 159-NA]months; MYD88-CD79Bco-mut vs MYD88/CD79Bsingle-mut: p=0.02; MYD88-CD79Bco-mut vs MYD88-CD79Bco-wt: p=0.03; MYD88/CD79Bsingle-mut vs MYD88-CD79Bco-wt: p=0.33). Regarding patients with MYD88-CD79Bco-mut, there was no significant difference in PFS and OS between the MCD and non-MCD subtypes. Within the MYD88-CD79Bco-mut group, patients with PIM1mut had better PFS than PIM1wt (median PFS: 8.34 [95% CI 5.56-NA] vs 43.8 [95% CI 26.4-NA] months; p=0.02). Possible mechanisms contributing to the superior PFS of PIM1mut patients may include activated lymphocyte-mediated immunity and interferon response, a higher proportion of natural killer T cells and plasmacytoid dendritic cells, as well as suppressed angiogenesis and epithelial-mesenchymal transition, along with lower fibroblast and stromal score. In the MCD subtype, patients with MYD88-CD79Bco-mut showed comparable PFS and OS compared to MYD88/CD79Bsingle-mut or MYD88-CD79Bco-wt, while in the non-MCD subtype, they exhibited significantly inferior OS. There was no significant disparity in PFS and OS of MYD88-CD79Bco-mut between the MCD and non-MCD subtypes. The presence of PIM1mut within the MYD88-CD79Bco-mut group correlated with better PFS, which may result from an intricate interplay of immune processes and tumor microenvironment alterations.

Abstract 3480: Alterations in the tumor microenvironment and key signaling pathways in naturally-occurring canine gliomas mirror those seen in human patients

Abstract Background: Malignant gliomas are the most common type of primary brain tumors, with patient outcomes that are generally dismal. Recently, pet dogs have been assessed as a valuable model in which to study gliomas. Gliomas in pet dogs occur at similar rates to those seen in humans, and a recent study comparing canine and human gliomas identified high similarity in terms of mutational rates, aneuploidy, the timing of mutations, and methylation patterns. Here, we further compare naturally-occurring canine and human gliomas, focusing on the tumor microenvironment (TME) and key pathways dysregulated in human gliomas. Methods: Spontaneously occurring gliomas from adult and pediatric human, and canine patients were utilized for this study. RNA, whole genome/whole exome, and bisulfite sequencing data from adult and canine samples were downloaded from the Genomic Data Commons and the National Cancer Institute’s Integrated Canine Data Commons, respectively. Pediatric tumor RNA-seq and genomic data were obtained from the Gabriella Miller Kids First database. Normal adult human and canine brain tissue served as control tissue. Only tumors with a clear diagnosis of astrocytoma, oligodendroglioma, or oligoastrocytoma were included in this study. Results: Overall, we found that key canonical pathways that are altered in human gliomas are also altered in canine gliomas. A comparison of high-grade gliomas in pet dogs to normal canine brain tissues showed significant abnormal gene expression in 3 canonical oncogenic pathways of human GBM: the TP53, RB1, and RTK/RAS/PI3K pathways. Similarities between species included increased expression of PDGF, and receptors PDGFRA and PDGFRB. Frequent copy number alterations and mutations were also observed in PDGFRA and EGFR. Differences between species include the mutation rate of TP53, which we found to be mutated frequently in adults but not in pediatric or canine gliomas, however, expression of TP53 as assessed by RNA-seq was significantly increased in both adult and canine gliomas. IDH1, which drives methylation, was significantly overexpressed in adults but underexpressed in canine. Correspondingly, each of the promotor regions of TERT, PD1 and MGMT was hypermethylated in adults, but hypomethylated in canine. Although an analysis of the TME from RNA-seq data revealed an influx of immune cells in adult, pediatric and canine gliomas, there was a concurrent influx of immunosuppressive cells. Gene expression profiles of astrocytomas and oligodendrogliomas show differences in alterations of immune-related and TME-specific pathways. Conclusion: We found that similar TME and pathway alterations were observed between human and canine gliomas, and that TME alterations were different between canine astrocytomas and oligodendrogliomas. The canine TME, like that in humans, appears to be immunosuppressive. Citation Format: Nadia A. Lanman, Sagar Utturkar, Harish Kothandaraman, Deepika Dhawan, Danni Liu, Min Zhang, Matthew Beyers, Deborah W. Knapp, R. Timothy Bentley. Alterations in the tumor microenvironment and key signaling pathways in naturally-occurring canine gliomas mirror those seen in human patients [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 3480.

Abstract 5562: Altered tumor microenvironment in animal model of concomitant GBM and Alzheimer's pathology

Abstract Glioblastoma (GBM) and Alzheimer’s disease (AD) are two devastating central nervous system diagnoses with no known cure. Both diseases are associated with advanced age. The majority of GBM tumors form in the frontal and temporal lobes, two of the brain regions most impacted by amyloid and tau pathology in AD. Females have a higher risk of AD, while males have a higher risk of GBM. Several large case series have established epidemiological evidence that the two diseases are inversely correlated. Nevertheless, it remains unknown whether presence of subclinical amyloid or tau pathology antagonizes the establishment or progression of GBM. The present study sought to characterize GBM growth, progression, and immune response in the setting of concomitant AD pathology using the syngeneic murine GL261 tumor model. To further understand the tumor cell changes that occur in the presence or absence of AD pathology, we engineered GL261 with a dual-tagged vector consisting of RiboTag (to allow for full-length mRNA profiling) and TurboID (to allow for proteomic profiling). GL261 cells were injected intracranially into APPNL-G-F/NL-G-F - MAPThuman/human, APPNL-G-F/+ - MAPThuman/+, and wildtype C57BL/6J mice. Tumors in both wildtype and AD conditions contained ~50% intratumoral myeloid cells (Iba+, P2RY12-). Differential activation of intratumoral myeloid cells will be assessed with immunohistochemistry. Microgliosis (IBA1, P2RY12) and astrogliosis (GFAP) around the tumor in the setting of Alzheimer’s pathology versus normal conditions will be compared. Invasiveness, proliferation, DNA damage (pH2AX), and apoptosis (cleaved PARP, cleaved Caspase 3) will be compared. Tumor cell-specific changes unique to tumors in the AD microenvironment will be assessed transcriptionally with RiboTag profiling as well as proteomically with TurboID purifications. Our preliminary data suggests that the presence of concomitant AD pathology affects GBM growth and progression as well as the immune response to the tumor, and warrants further investigation. Citation Format: David Nascari, Ryan Eghlimi, Angad Beniwal, Drake Alton, John Fryer, Nhan L. Tran. Altered tumor microenvironment in animal model of concomitant GBM and Alzheimer's pathology [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 5562.

Abstract 2007: Neurolysin: A novel architect of the prostate cancer microenvironment and therapeutic resistance

Abstract Introduction: Prostate cancer is the second leading cause of cancer-related death in American men, primarily driven by its ability to evade therapeutic interventions. This study uncovers a novel paradigm where neurolysin, a peptidase previously ascribed to neuronal function, orchestrates the tumor microenvironment (TME) by interfacing with cellular motor proteins. This interaction precipitates a series of events that not only fortify the cancer cells' survival but also underpin the resistance to antiandrogen therapies. Experimental Procedures: We deployed advanced prostate cancer models to delve into the extra-neuronal influence of neurolysin, probing its role in the orchestration of the TME. An exhaustive suite of transcriptomic and proteomic analyses, complemented by comprehensive patient cohort studies, laid the foundation for unraveling the complex interplay between neurolysin, motor proteins, and the TME dynamics. Results: Our data elucidates that neurolysin's depletion correlates with an altered TME, conducive to cancer proliferation and resilience against targeted therapies. This shift is mediated through neurolysin's interaction with cellular motor proteins, a liaison that appears to be a cornerstone in TME modulation. A significant subset of prostate cancer cases exhibits a neurolysin deficiency, paralleling more aggressive disease and therapeutic defiance, highlighting its role as a critical biomarker and a novel target for intervention. Conclusions: Neurolysin steps into the limelight as a pivotal modulator of the TME, operating through mechanisms distinct from its known neuronal functions. Its interplay with motor proteins opens a novel avenue in prostate cancer biology, providing a unique perspective on the regulation of the TME and subsequent therapeutic resistance. Targeting this newly discovered regulatory axis presents a promising therapeutic strategy that could potentially redefine the clinical approach to managing prostate cancer and may have implications for other malignancies driven by similar TME alterations. Citation Format: Choushi Wang, Ping Mu. Neurolysin: A novel architect of the prostate cancer microenvironment and therapeutic resistance [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 2007.

Candida albicans-myeloid cells-T lymphocytes axis in the tumor microenvironment of oral tumor-bearing mice

Candida albicans (C. albicans) is associated with the development of oral cancer. Here, we report the altered tumor microenvironment in oral tumor-bearing mice caused by C. albicans infection. Single-cell RNA sequencing showed that C. albicans infection influenced the tumor microenvironment significantly. Specifically, C. albicans infection reduced the CD8+ T cells but increased the IL-17A+ CD4+ T cells and IL-17A+ γδ T cells in oral tumor. The neutralization of IL-17A or TCR γ/δ alleviated the tumor progression caused by C. albicans infection. Additionally, C. albicans infection promoted the infiltration of myeloid-derived suppressor cells (MDSCs) into tumor, especially polymorphonuclear (PMN)-MDSCs, which infiltration was reduced after the neutralization of CCL2. Thus, our findings reveal the myeloid cells-T lymphocytes axis in oral tumor microenvironment with C. albicans infection, which helps to understand the mechanisms for C. albicans promoting oral cancer from the perspective of immune microenvironment.

The role of companion animal models in radiopharmaceutical development and translation.

Advancements in molecular imaging and drug targeting have created a renaissance in the development of radiopharmaceuticals for therapy and theranostics. While some radiopharmaceuticals, such as Na[131I]I, have been used clinically for decades, new agents are being approved using small-molecules, peptides, and antibodies for targeting. As these agents are being developed, the need to understand dosimetry and biologic effects of the systemically delivered radiotherapy becomes more important, particularly as highly potent radiopharmaceuticals using targeted alpha therapy become clinically utilized. As the processes being targeted become more complex, and the radiobiology of different particulate radiation becomes more diverse, models that better recapitulate human cancer and geometry are necessary. Companion animals develop many of the same types of cancer, carrying many of the same genetic drivers as those seen in people, and the scale and geometry of tumours in dogs more closely mimics those in humans than murine tumour models. Key translational challenges in oncology, such as alterations in tumour microenvironment, hypoxia, heterogeneity, and geometry are addressed by companion animal models. This review paper will provide background on radiopharmaceutical targeting techniques, review the use of radiopharmaceuticals in companion animal oncology, and explore the translational value of treating these patients in terms of dosimetry, treatment outcomes, and normal tissue complication rates.

Biopolymer-based tumor microenvironment-responsive nanomedicine for targeted cancer therapy.

Nanomedicine has opened up new avenues for cancer treatment by enhancing drug solubility, permeability and targeted delivery to cancer cells. Despite its numerous advantages over conventional therapies, nanomedicine may exhibit off-target drug distribution, harming nontarget regions. The increased permeation and retention effect of nanomedicine in tumor sites also has its limitations, as abnormal tumor vasculature, dense stroma structure and altered tumor microenvironment (TME) may result in limited intratumor distribution and therapeutic failure. However, TME-responsive nanomedicine has exhibited immense potential for efficient, safe and precise delivery of therapeutics utilizing stimuli specific to the TME. This review discusses the mechanistic aspects of various TME-responsive biopolymers and their application in developing various types of TME-responsive nanomedicine.

Molecular Engineering of Electrosprayed Hydrogel Microspheres to Achieve Synergistic Anti-Tumor Chemo-Immunotherapy with ACEA Cargo.

Molecular engineering of drug delivering platforms to provide collaborative biological effects with loaded drugs is of great medical significance. Herein, cannabinoid receptor 1 (CB1)- and reactive oxygen species (ROS)-targeting electrosprayed microspheres (MSs) are fabricated by loading with the CB1 agonist arachidonoyl 2'-chloroethylamide (ACEA) and producing ROS in a photoresponsive manner. The synergistic anti-tumor effects of ACEA and ROS released from the MSs are assessed. ACEA inhibits epidermal growth factor receptor signaling and altered tumor microenvironment (TME) by activating CB1 to induce tumor cell death. The MSs are composed of glycidyl methacrylate-conjugated xanthan gum (XGMA) and Fe3+, which form dual molecular networks based on a Fe3+-(COO-)3 network and a C═C addition reaction network. Interestingly, the Fe3+-(COO-)3 network can be disassembled instantly under the conditions of lactate sodium and ultraviolet exposure, and the disassembly is accompanied by massive ROS production, which directly injures tumor cells. Meanwhile, the transition of dual networks to a single network boosts the ACEA release. Together, the activities of the ACEA and MSs promote immunogenic tumor cell death and create a tumor-suppressive TME by increasing M1-like tumor-associated macrophages and CD8+ T cells. In summation, this study demonstrates strong prospects of improving anti-tumor effects of drug delivering platforms through molecular design.

Application of plasma circulating KRAS mutations as a predictive biomarker for targeted treatment of pancreatic cancer.

Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in circulating tumor deoxyribonucleic acid (ctDNA) have been reported as representative noninvasive prognostic markers for pancreatic ductal adenocarcinoma (PDAC). Here, we aimed to evaluate single KRAS mutations as prognostic and predictive biomarkers, with an emphasis on potential therapeutic approaches to PDAC. A total of 128 patients were analyzed for multiple or single KRAS mutations (G12A, G12C, G12D, G12R, G12S, G12V, and G13D) in their tumors and plasma using droplet digital polymerase chain reaction (ddPCR). Overall, KRAS mutations were detected by multiplex ddPCR in 119 (93%) of tumor DNA and 68 (53.1%) of ctDNA, with a concordance rate of 80% between plasma ctDNA and tumor DNA in the metastatic stage, which was higher than the 44% in the resectable stage. Moreover, the prognostic prediction of both overall survival (OS) and progression-free survival (PFS) was more relevant using plasma ctDNA than tumor DNA. Further, we evaluated the selective tumor-suppressive efficacy of the KRAS G12C inhibitor sotorasib in a patient-derived organoid (PDO) from a KRAS G12C-mutated patient using a patient-derived xenograft (PDX) model. Sotorasib showed selective inhibition invitro and invivo with altered tumor microenvironment, including fibroblasts and macrophages. Collectively, screening for KRAS single mutations in plasma ctDNA and the use of preclinical models of PDO and PDX with genetic mutations would impact precision medicine in the context of PDAC.

Adverse clinical outcomes and immunosuppressive microenvironment of RHO-GTPase activation pattern in hepatocellular carcinoma

BackgroundEmerging evidence suggests that Rho GTPases play a crucial role in tumorigenesis and metastasis, but their involvement in the tumor microenvironment (TME) and prognosis of hepatocellular carcinoma (HCC) is not well understood.MethodsWe aim to develop a tumor prognosis prediction system called the Rho GTPases-related gene score (RGPRG score) using Rho GTPase signaling genes and further bioinformatic analyses.ResultsOur work found that HCC patients with a high RGPRG score had significantly worse survival and increased immunosuppressive cell fractions compared to those with a low RGPRG score. Single-cell cohort analysis revealed an immune-active TME in patients with a low RGPRG score, with strengthened communication from T/NK cells to other cells through MIF signaling networks. Targeting these alterations in TME, the patients with high RGPRG score have worse immunotherapeutic outcomes and decreased survival time in the immunotherapy cohort. Moreover, the RGPRG score was found to be correlated with survival in 27 other cancers. In vitro experiments confirmed that knockdown of the key Rho GTPase-signaling biomarker SFN significantly inhibited HCC cell proliferation, invasion, and migration.ConclusionsThis study provides new insight into the TME features and clinical use of Rho GTPase gene pattern at the bulk-seq and single-cell level, which may contribute to guiding personalized treatment and improving clinical outcome in HCC.

Scoping Pleiotropy of CK2 in Musculoskeletal Disorders for a Novel Targeting Approach

Protein kinase CK2 (CK2) influences one-fifth of the cellular phosphoproteome. It regulates almost all cellular pathways and is thus a critical switch between biological processes within a cell. Inhibition of CK2 reverses oncogene addiction of tumor and alters tumor microenvironment. The success of this strategy and its clinical translation opens new opportunities. Targeting CK2 in musculoskeletal disorders is promising. Clinical manifestations of these disorders include dysfunctional inflammation, dysregulated cell differentiation, and senescence. Processes regulated by CK2 include all of these. Its emerging role in senescence also indicates its function’s centrality in cellular metabolism. This review summarizes considerations for targeting CK2 in musculoskeletal disorders. We have discussed the implications of CK2-regulated processes in musculoskeletal disorders.

Multi-Dimensional Molecular and Tumor-Microenvironment Analysis of Classic Hodgkin Lymphoma

Introduction Classic Hodgkin Lymphoma (CHL) is currently classified into histological subtypes based on morphology and antigen marker expression. However, additional biological features might help guide treatment strategies and inform prognosis. Here, we aimed to uncover disease heterogeneity and biologic subtypes of CHL based on multi-dimensional molecular profiling ( Fig A) capturing somatic gene mutations, malignant cell expression phenotypes and altered tumor microenvironment (TME) architecture. METHODS We performed exome/targeted sequencing on flow-sorted or micro-dissected HRS cells from fresh-frozen CHL biopsies (n=116) of patients treated at BC Cancer. In addition, we constructed tissue microarrays (TMA) from this cohort on which we performed GeoMx® Whole Transcriptome Assay (WTA) to obtain gene expression profiles from CD30+ HRS cells and multiplexed imaging analyses using multi-colour immunofluorescence (mIF) to delineate the spatial TME ecosystem. Cox regression analysis was performed to identify outcome differences according to molecular features in CHL patients treated with ABVD-like chemotherapy (n=104). Results Mutational and copy number analyses identified known recurrent driver events including mutations and copy number changes in SOCS1, STAT6, TNFAIP3, B2M, REL and the PDL1/PDL2 locus. These mutational findings provided a statistically powered framework to investigate the association between genomic alterations with pathological and clinical disease parameters including outcomes. Mutations affecting BCL7A, TNFAIP3, and 2p15 (REL) amplifications were strongly enriched in EBV- CHL compared to EBV+ CHL. In line with “thymic mutations” reported in grey zone lymphoma, STAT6, GNA13, ITPKB, and TBL1XR1 mutations were significantly enriched in CHL with involvement of the anterior mediastinum. ZNF217, SPEN and CD58 mutations were significantly associated with progression-free survival (PFS) (all P < .01). When investigating outcome correlation according to age group, genomic alteration in STAT6 including both single nucleotide variants and STAT6 amplification, was the most significant feature associated with unfavorable PFS in patients <45 years of age ( P = .013). Alterations in the antigen recognition system, including loss of major histocompatibility complex (MHC) is one of the most studied mechanisms of immune escape in CHL. Mutations in CSF2RB, GNA13, TNFAIP3, and STAT6 were significantly associated with MHC-I loss. Strikingly, all cases harboring CSF2RB mutations (n=23) also showed loss of MHC-I. GeoMx analysis revealed that IFN-a and IFN-g pathways were significantly upregulated in MHC-I+ HRS cells. Mutations in HIST1H1E and BCL7A were significantly enriched in cases with loss of both MHC-I and MHC-II expression. To define mutational subtypes of CHL, we next applied non-negative matrix factorization (NMF) consensus clustering and discovered four robust subsets of tumors (clusters) using recurrent genomic events. Cluster1 (C1) is characterized by mutations in TNFAIP3, CSF2RB and GNA13, younger age, loss of MHC-I, and up-regulation of GCB and fatty acid metabolism signatures. C2 was associated with old age at initial diagnosis, EBV positivity and significant upregulation of the IFN-g pathway. C3 is characterized by REL and STAT6 amplification andexpression of a DNA repair signature. C4 includes samples from mostly younger patients with STAT6 and B2M mutations and high expression of CCL17 by HRS cells. MIF analyses further identified correlations between each mutational NMF cluster and TME composition ( Fig B), and in particular specific CD4+ T cell subsets. The TME of tumors was significantly enriched by C1: FOXP3+Tregs, C2: LAG3+CD4+ cells, and C3: PD1+CD4+ cells. C4 tumors showed no positive correlation with any of these immune cell compartments. C2 tumors were also significantly associated with the abundance of CD68+ macrophages. Conclusion Our multi-dimensional profiling approach enabled us to delineate molecular profiles of HRS cells that are linked to distinct TME patterns. These linkages have implications for current pathogenesis models, molecular subtyping of CHL, and identification of cellular vulnerabilities that might be therapeutically exploitable via targeting of HRS cell phenotypes and/or immune escape mechanisms.

Investigation into Intraclonal Heterogeneity of CXCR4 DimCD5 Bright Chronic Lymphocytic Leukemia Cells Identifies Distinct Activation Signatures

Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease associated with diverse clinical and molecular profiles, suggesting long-term clonal evolution driven by genetic diversification from a small, often undetected, subset of leukemic cells endowed with superior growth capacities. More importantly, this diversification process enhances the opportunity of CLL subclones to survive selective pressure, such as BTK or PLCG2 mutations that can abolish the effect of BTK inhibitors. While this evolutionary process is inevitable in leukemic cells, therapeutic intervention targeting the cells that have just replicated their DNA, and hence could have accumulated new genomic mutation, would be an effective strategy to significantly delay or abort the emergence of deleterious mutations. In CLL, a series of studies involving consumption of 2H 2O determined that CXCR4 DimCD5 Bright cells (proliferative fraction, PF) of CLL clones contain most of the recently divided cells, identified by the presence of 2H-DNA. This conclusion was supported by finding elevated numbers of Ki67 + and MCM6 + cells in the same fraction, when compared to other CLL cells. Furthermore, PF is superior in: lymphoid tissue homing in xenograft models; activating T cells; the capacity to alter tumor microenvironment (TME) by Th2 skewing; and expression of AICDA which can increase the likelihood of off-target genomic mutations. To permit a focused analysis of PF that would allow us to intercept this evolutionary loop, we characterized the single cell transcriptomes of PF from 4 U-CLL and 4 M-CLL patients immediately prior to ibrutinib treatment. Consistent with previous reports, the majority of PF (herein APC-PF) cells express certain antigen presentation molecules with a gene signature suggestive of T cell regulation and cell-cell interactions. Additionally, we discovered a small subpopulation with an upregulation of MIR155HG, HSP90AB, CCL3, CCL4, MYC and a concomitant MYC activation signature (herein MYC-PF). Since miR-155 expression can be induced by CD40L or B-cell-activating factors, this PF subset may reflect prior BCR ligation and/or T cell co-stimulation. However, the strongest BCR activation signature was found in another PF subpopulation (herein BCR-PF) with an enrichment of germinal center-related genes (e.g., BACH2, PAX5, FOXP1) and CLL-associated molecules ( BCL2, ROR1, MSI2, PLCG2). Furthermore, the BCR-PF cells are least likely to be resting (in G1/G0) compared to others. Collectively, these findings suggest effective BCR engagement, either from the TME or cell-autonomously. Corroborating these findings with our previous studies documenting the CXCR4/CD5 fractions and the expression of protein proliferation markers, our transcriptomic analyses suggest that the BCR-PF may be the most immediate emigrant from the proliferation centers (PC) within CLL lymph nodes (LN). To probe this possibility, spatial transcriptomic analysis was performed on FFPE LN tissues from surgical biopsies from 3 U-CLL and 1 M-CLL patients. Single cell deconvolution was used to determine the relative abundance of cells within the anatomically defined PCs that resemble the three PF subpopulations transcriptomically. Our results suggest that PCs are more enriched in cells resembling BCR-PF, followed by cells bearing the APC-PF signature. To extend on these observations, we have documented that the unique surface markers expressed in these PF subfractions positively correlate with our transcriptomic results. Taken together, these results not only highlighted the molecular heterogeneity of CLL, but also lead to the identification of two PF subpopulations that may provide novel actionable therapeutic targets to significantly halt CLL clonal evolution.

Clinical and Molecular Features of Patients with Double/Triple Hit Large B-Cell Lymphoma

Background: High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) is a recently defined category in the latest revision of the World Health Organization (WHO) classification, which comprises approximately 5-15% of newly diagnosed DLBCL. This subtype of lymphoma poses numerous challenges from diagnosis to treatment of patients. On the one hand, diagnosis of HGBL-DH/TH requires the accomplishment of fluorescent in-situ hybridization (FISH) to identify the translocations of MYC(8q24) and BCL2(18q21) or/and BCL6(3q27), with no consensus being reached regarding the most applicable population for FISH tests due to high cost and low prevalence. On the other hand, HGBL-DH/TH displays aggressive features, including a high incidence of advanced disease at diagnosis, and inferior efficacy to standard-induced chemical immunotherapy with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Despite several treatment strategies including intensified protocols and hematopoietic cell transplantation (HCT) have been explored, there is no standard regimen to improve outcomes for HGBL patients, and participation in clinical trials is encouraged. Aims: This study aims to investigate the clinical characteristics and prognostic outcomes of HGBL-DH/TH patients, and performed genomic and transcriptomic analyses to link the oncogenic mutations and tumor microenvironment alterations to different DLBCL subgroups. Method:In this study, 107 patients with newly diagnosed HGBL-DH/TH were analyzed. Histological diagnoses were established according to the WHO classification. These HGBL-DH/THs were termed DHL-BCL2 (MYC and BCL2 translocation), DHL-BCL6 (MYC and BCL6 translocation), and THL in our study. DNA sequencing was carried out on 95 patients, for detection of genetic aberrations. Results: Patients with DHL-BCL2 (n=44) and THL (n=15) have similar clinical features, including elevated serum lactate dehydrogenase (LDH), increased proportion of double expression (DE) as well as increased prevalence of non-GCB subtype compared to those with DHL-BCL6 (n=48). With the median follow-up time of 19.3 months (2.5-114.2 months), patients in the DHL-BCL2 and THL group had dismal survival (2-year PFS: 49.3% and 34.2%; 2-year OS: 64.0% and 55.6%) when compared with those in the DHL-BCL6 group (2-year PFS: 63.5%; 2-year OS: 89.3%) (Figure A). Moreover, intensive chemoimmunotherapy or combination with novel targeted agents seemed to improve outcomes in patients with DHL/THL compared with those who received a standard R-CHOP regime. The use of autologous stem cell transplant (ASCT) did not yield a significant improvement in survival for patients who achieved remission after receiving first-line R-DA-EDOCH therapy. However, it is worth noting that there was a trend towards prolonging progression-free survival (PFS) and overall survival (OS) in patients who achieve complete remission (CR) through RCHOP-based treatment and undergo ASCT (Figure B). Compared to DHL-BCL6, the primary genetic lesions in DHL-BCL2 and THL were alterations associating epigenetic regulators like EZH2 and CREBBP, while the primary genetic lesions in DHL-BCL6 were alterations associating cellular differentiation and transcription factors like BTG2, PTPN6, CD70, and BCL6. Correlatively, the EZB genotype was more frequently observed in patients with DHL-BCL2 and THL, while the BN2 genotype was more frequently observed in patients with DHL-BCL6. Conclusion: DHL-BCL2/THL exhibits similar clinical characteristics, prognostic outcomes, and molecular features that set it apart from DHL-BCL6. This implies the need for testing novel agents or therapeutic strategies to expedite treatment development for patients with DHL-BCL2/THL.

Hypoxia-inducible factor 1α modulates interstitial pneumonia-mediated lung cancer progression

BackgroundThe prognosis of patients with lung cancer accompanied by interstitial pneumonia is poorer than that of patients with lung cancer but without interstitial pneumonia. Moreover, the available therapeutic interventions for lung cancer patients with interstitial pneumonia are limited. Therefore, a new treatment strategy for these patients is required. The aim of the present study was to investigate the pathophysiological relationship between interstitial pneumonia and lung cancer and explore potential therapeutic agents.MethodsA novel hybrid murine model of lung cancer with interstitial pneumonia was established via bleomycin-induced pulmonary fibrosis followed by orthotopic lung cancer cell transplantation into the lungs. Changes in tumor progression, lung fibrosis, RNA expression, cytokine levels, and tumor microenvironment in the lung cancer with interstitial pneumonia model were investigated, and therapeutic agents were examined. Additionally, clinical data and samples from patients with lung cancer accompanied by interstitial pneumonia were analyzed to explore the potential clinical significance of the findings.ResultsIn the lung cancer with interstitial pneumonia model, accelerated tumor growth was observed based on an altered tumor microenvironment. RNA sequencing analysis revealed upregulation of the hypoxia-inducible factor 1 signaling pathway. These findings were consistent with those obtained for human samples. Moreover, we explored whether ascorbic acid could be an alternative treatment for lung cancer with interstitial pneumonia to avoid the disadvantages of hypoxia-inducible factor 1 inhibitors. Ascorbic acid successfully downregulated the hypoxia-inducible factor 1 signaling pathway and inhibited tumor progression and lung fibrosis.ConclusionsThe hypoxia-inducible factor 1 pathway is critical in lung cancer with interstitial pneumonia and could be a therapeutic target for mitigating interstitial pneumonia-mediated lung cancer progression.

The Special Role of JAK/STAT, and Notch Signaling Pathways in Cancer Pathogenesis

Cancer is essentially a genetic disease, characterized by unclear etiology in many cases, complex pathogenic mechanisms, since in the majority of cancer cases only risk or predisposing factors have been recognized, and unclarified observations concerning organs that are not often affected by malignant tumors such as the small intestine, spleen, heart, and the neurons of the Central Nervous System. A tumor’s appearance and progression presupposes a series of genetic and non-genetic alterations that characterize tumor cells, and are related to self-sufficiency in growth stimuli, insensitivity to anti-growth signals, unlimited replicative potential, angiogenesis promotion, ability to infiltrate surrounding tissues and induce distant metastases, and resistance to apoptosis. The mentioned characteristics are associated with signaling pathways that are involved in cell survival, cell death, cell growth and division, cell motility, and can be considered in the context of abnormalities of wider signaling networks that support cancer progression, such as tumor microenvironment alterations, angiogenesis, and inflammation. Among others, two signaling pathways, JAK/STAT and Notch, seem to play essential roles in tumorigenesis, despite the fact that are implicated in diverse cellular functions such as tissue formation and cell differentiation, proliferation, apoptosis, inflammation, self-renewal, antitumor immune response suppression, motility, stress response and other responses depending on the target tissue. Hyperactivation of those signaling pathways caused by mutations are able to transform cellular proto-oncogenes to oncogenes, whereas inactivation of tumor suppressor genes eradicates crucial negative regulators of signaling. Various anticancer agents target those signaling pathways in an attempt to inhibit those pathways including monoclonal antibodies and other agents in the context of targeted cancer therapy.