Cancer Biology Research Research Articles

The Current Status and Future Directions on Nanoparticles for Tumor Molecular Imaging.

Molecular imaging is an advanced technology that utilizes specific probes or markers in conjunction with cutting-edge imaging techniques to observe and analyze the localization, distribution, activity, and interactions of biomolecules within living organisms. Tumor molecular imaging, by enabling the visualization and quantification of molecular characteristics of tumor cells, facilitates a deeper and more comprehensive understanding of tumors, providing valuable insights for early diagnosis, treatment monitoring, and cancer biology research. However, the image quality of molecular imaging still requires improvement, and nanotechnology has significantly propelled the advancement of molecular imaging. Currently, nanoparticle-based tumor molecular imaging technologies encompass radionuclide imaging, fluorescence imaging, magnetic resonance imaging, ultrasound imaging, photoacoustic imaging, and multimodal imaging, among others. As our understanding of the tumor microenvironment deepens, the design of nanoparticle probes for tumor molecular imaging has also evolved, offering new perspectives and expanding the applications of tumor molecular imaging. Beyond diagnostics, there is a marked trend towards integrated diagnosis and therapy, with image-guided treatment playing a pivotal role. This includes image-guided surgery, photodynamic therapy, and chemodynamic therapy. Despite continuous advancements and innovative developments in molecular imaging, many of these remain in the experimental stage and require breakthroughs before they can be fully integrated into clinical practice.

Aptamer-guided Selective Delivery of Therapeutics to Breast Cancer Cells Expressing Specific Biomarkers

Abstract: Cancer biomarkers or tumor-associated antigens (TAA) are the focus area of current research in cancer biology for diagnosis, prognosis, screening, and targeted treatments. Breast cancer is the second most common type of cancer, affecting women more than men. Conventional methods and antibody-targeted therapies are less effective and suffer systemic cytotoxicity, poor tissue sensitivity, low penetration capacity, and reduced accumulation of the drug in tumor cells that limit its application and sometimes result in treatment failure. Opting for aptamer-mediated targeted delivery of various anti-cancer agents (drugs, siRNA, miRNA, shRNA and peptides) could possibly overcome these limitations by utilizing aptamer as a targeting ligand. The purpose of this article is to review the novel indicative biomarkers of breast cancer and also describe current applications of aptamer-guided active targeting systems in breast cancer therapy in vivo and in vitro.

Abstract PO3-10-09: The allocation of US $2.6 billion in global funding for breast cancer research between 2016 – 2020: are we investing wisely for breast cancer patients?

Abstract Background: Breast cancer is a global health challenge and the highest incident cancer globally, with over 2 million new cases and 685,000 deaths in 2020, making it the highest incident cancer globally. Furthermore, it is the most prevalent cancer, resulting in the loss of more disability-adjusted life years than any other malignancy. Research is thus crucial to improving outcomes and reducing suffering for breast cancer patients worldwide. This study aimed to analyse global patterns of funding for breast cancer research. Methods: We have previously created a comprehensive database of awards for human cancer research in this period using publicly available data on research funding awards. From this database we extracted all awards relating to breast cancer research, and categorised these by cancer type, cross-cutting research theme and phase of research. Findings: We identified 7051 awards with a total investment of $2.6 billion in 2016-2020, representing 11.2% of all global public and philanthropic cancer research funding during this period. Breast cancer research funding fell year on year, from $773.4 million in 2016 to $243.8 million in 2020. The majority of the investment was in pre-clinical research ($1.9 billion, 73.9%), with clinical trials ($276 million, 10.7%) and public health ($269.2 million, 10.5%) receiving roughly equivalent amounts of funding. The USA accounted for 66.9% of all global breast cancer research investment, with China ($110.4 million, 4.3%) and the UK ($96.6 million, 3.8%) the next largest funders. By research theme, cancer biology research received most funding ($926.2 million, 37.5% of investment), with drug treatment the next best funded research area ($600.5 million, 23.4%), and diagnosis, screening and monitoring third ($396 million, 15.4%). Surgery ($65.2 million, 2.5%) and radiotherapy ($35.3 million, 1.4%) accounted for only a small proportion of research investment. There was minimal investment into breast cancer as a global health issue ($8.1 million, 0.3%). Research into metastatic disease received a greater proportion of investment in breast cancer than in the overall cancer portfolio (23.2% in breast cancer, $597.4 million, compared with 11.7% across all cancers). Conclusions: Breast cancer public and philanthropic research funding is largely invested in pre-clinical research in high-income countries, with the main areas of investment being cancer biology research and drug treatment. The direct benefit to patients from such research is likely to be low and only apparent after a substantial time lag (if at all). Despite their pivotal role in the treatment of the majority of breast cancer patients, surgery and radiotherapy research received a tiny proportion of the overall research investment. Furthermore, relatively little investment is directed towards cancer prevention and early detection, despite their acknowledged key role in cancer control globally. Finally, more equitable research funding is required to align with the global burden of breast cancer, with increased funding for low- and middle-income countries, which account for an increasing proportion of this burden. These data should be considered by funders and policymakers in the allocation of finite resources, which must be invested wisely. Citation Format: Stuart McIntosh, Ellen Copson, Ramsey Cutress, Michael Head. The allocation of US $2.6 billion in global funding for breast cancer research between 2016 – 2020: are we investing wisely for breast cancer patients? [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 PO3-10-09.

Leveraging Maker Learning in STEM to Promote Children's Interest in Cancer Research: A Pilot Program.

Children's early awareness about cancer, through exposure to cancer biology and prevention strategies and research principles, is a promising focus of education and learning. It may also benefit the pipeline of people entering into science, technology, engineering, and math (STEM) careers. We describe an educational pilot program for elementary school students, using developmentally appropriate activities focused on cancer at a museum dedicated to children's maker-centered learning and STEM. The program was implemented through a public school in Washington, DC serving students underrepresented in STEM. Program conceptualization, museum and school engagement, and maker learning pedagogy are described, as well as curricular outcomes. A total of N = 111 students (44% female, 75% Black/African American, 5% Latine) participated in a day-long field trip. Museum educators, assisted by cancer center researchers, led a multipart workshop on cancer and the environment and hands-on rotation of activities in microbiology, immunology, and ultraviolet radiation safety; students then completed self-report evaluations. Results indicate that nearly all (> 95%) students practiced activities typical of a STEM professional at the program, and > 70% correctly answered factual questions about topics studied. Importantly, 87-94% demonstrated clear STEM interest, a sense of belonging in the field, and practice implementing skills for success in STEM (e.g., perseverance, imagination, teamwork). This pilot demonstrated acceptability and feasibility in delivering a cancer-focused curriculum to underserved elementary students using maker learning while favorably impacting key objectives. Future scale-up of this program is warranted, with the potential to increase students' motivation to engage in STEM and cancer research.

Chimeric Antigen Receptor T-cell based cellular therapies for cancer: An introduction and Indian perspective.

Using one's own immune system for curing cancer has been an active field of research in cancer biology and therapeutics. One such opportunity in cellular immunotherapy is adoptive cell transfers. With the recent approval of CAR-T therapy as a cancer treatment, a whole new paradigm of cancer treatment has opened-up, with a ray of hope for relapsed/refractory cancer patients. Despite promising clinical outcomes, the therapy is in its early phase and remains out of reach for most patients due to its high cost and logistic challenges. In India, these therapies are unavailable and further confounded by the economic challenges and a large population. In this review, we discuss various aspects of T-cell immunotherapies with a special focus on CAR-T in the Indian scenario. We touch upon the basic scientific aspects, mechanism of action, manufacturing, clinical aspects and commercial aspects of the CAR-Tcell therapies and its future worldwide and in India.

Abstract 2760: Novel GPCR recombinant monoclonal antibodies for cancer biology research

Abstract Recombinant technology is becoming the preferred production mechanism for creation of antibody reagents used in academic and clinical cancer biology research. Recombinant antibodies (rAbs) are superior to standard polyclonal and hybridoma-generated monoclonal reagents as they offer reproducible performance and consistent, scalable supply. A major focus of antibody manufacturers is to leverage rAb production strategies to generate antibodies against challenging targets for which there are few, if any, reliable antibodies. G-protein coupled receptors (GPCRs) are encoded by more than 820 human genes and represent the largest membrane protein superfamily in the genome. These seven-pass transmembrane domain receptors are involved in a multitude of physiological and pathophysiological processes related to various chronic diseases, including an array of malignancies. Perhaps 30-40% of all therapeutics are directed against GPCR activity, and ~350 of the non-olfactory GPCRs are thought to be potential candidates for drug development. However, for a slew of technical reasons, the development and validation of high-quality antibodies against GPCRs has proven difficult. In an effort to facilitate further research into GPCRs, GeneTex is combining its recombinant antibody production platform with enhanced validation techniques to make specific monoclonal antibodies against GPCRs with direct relevance to cancer and other diseases. GeneTex’s rAb production protocol employs a multi-parameter fluorescence-activated single cell sorting (FACS)-based methodology to identify and select antigen-specific IgG+ memory B cells from an immunized rabbit (Starkie et al., 2016). The heavy and light chain variable region genes from single cells are amplified, cloned, and co-expressed in mammalian cells to generate a monoclonal IgG. Application-specific testing is performed in conjunction with knockout/knockdown (KO/KD), differential expression comparison in cells and tissues, cell fraction enrichment (i.e., membrane extracts versus whole cell extracts), and other methodologies. Antibodies directed against extracellular GPCR domains are validated for specificity using GPCR arrays (CDI Labs, Baltimore, MD). This workflow has resulted in the production of specific antibodies for a number of GPCRs studied by cancer biologists. These include novel recombinant monoclonal antibodies to the dopamine D2 receptor (DRD2), retinoic acid-induced protein 3 (RAI3), CXC motif chemokine receptor 2 (CXCR2), CXCR4, and CXCR7. All of these antibodies were validated for at least two applications, while the specificity of three (i.e., RAI3, CXCR4, and CXCR7) is supported by KO/KD data. GPCR array data argues for the specificity of the other two antibodies (i.e., DRD2 and CXCR2). This same production/validation approach will be applied as GeneTex targets the remaining GPCRs. Citation Format: Alexander Ball, Yung Lin Hsieh, Chun Kai Huang, Sega Huang, Shang Ru Wu, Yue Yun Chang, Jiming Liu, Po Shin Peng, Chia Yi Lin. Novel GPCR recombinant monoclonal antibodies for cancer biology research [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 2760.

Abstract 1291: ASPIRE: Advocates and Scientists Partner in Research Education

Abstract UNC Lineberger Comprehensive Cancer Center (LCCC) has a robust and growing cancer patient advocacy community. UNC Lineberger is the flagship publicly funded comprehensive cancer center in North Carolina. We are constantly looking for ways to educate patient and community advocates on recent advances in cancer biology research and clinical trials. Our advocates are also valuable partners for our basic and clinical researchers that may be early in their career. We developed and implemented an inaugural research education program for new advocates. We model this program partly on the highly successful and popular AACR Scientist Survivor Program that recently celebrated it’s 25th anniversary. Some of the UNC LCCC advocates participate in the SSP and were motivated to develop our own local program. We will showcase our pilot 2024 ASPIRE education program that brings together cancer patient and community advocates and cancer researchers to learn from each other. The basic itinerary of the ASPIRE program is: 1.Three separate one-hour online presentations by UNC Lineberger researchers with a patient advocate moderator. The topics covered are: Cancer 101 : cancer biology, diagnosis, staging, treatment. Clinical Trials 101: clinical trials, phases, patient protection, progress with for example accrual of representative populations. Research 101: bench research, bioinformatic, AI, how research works and how it may lead to new treatments. To showcase a tissue donation program being developed by UNC Patient Advocates that will benefit researchers. 2.The lecture series is followed by a breakfast roundtable discussion for all the participants to discuss their experiences. There will be short presentations and discussions in small groups of researchers and cancer patient advocates. We will summarize our ASPIRE pilot experience, how our participation in the SSP program motivated this local program, and how we plan to incorporate feedback from participants to inform future programs. We hope this will lead to more informed patient advocates that can then be even more powerful in research advocacy. Citation Format: Laurie Betts, Jennifer Potter, Veronica Carlisle, Patricia A. Spears. ASPIRE: Advocates and Scientists Partner in Research Education [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 1291.

Mesenchyme instructs growth while epithelium directs branching in the mouse mammary gland.

The mammary gland is a unique organ that undergoes dynamic alterations throughout a female's reproductive life, making it an ideal model for developmental, stem cell and cancer biology research. Mammary gland development begins in utero and proceeds via a quiescent bud stage before the initial outgrowth and subsequent branching morphogenesis. How mammary epithelial cells transit from quiescence to an actively proliferating and branching tissue during embryogenesis and, importantly, how the branch pattern is determined remain largely unknown. Here, we provide evidence indicating that epithelial cell proliferation and onset of branching are independent processes, yet partially coordinated by the Eda signaling pathway. Through heterotypic and heterochronic epithelial-mesenchymal recombination experiments between mouse mammary and salivary gland tissues and ex vivo live imaging, we demonstrate that unlike previously concluded, the mode of branching is an intrinsic property of the mammary epithelium whereas the pace of growth and the density of ductal tree are determined by the mesenchyme. Transcriptomic profiling and ex vivo and in vivo functional studies in mice disclose that mesenchymal Wnt/ß-catenin signaling, and in particular IGF-1 downstream of it critically regulate mammary gland growth. These results underscore the general need to carefully deconstruct the different developmental processes producing branched organs.

Facilitating long-term cell examinations and time-lapse recordings in cell biology research with CO2 mini-incubators

In recent years, microscopy has revolutionized the study of dynamic living cells. However, performing long-term live cell imaging requires stable environmental conditions such as temperature, pH, and humidity. While standard incubators have traditionally provided these conditions, other solutions, like stagetop incubators are available. To further enhance the accessibility of stable cell culture environments for live cell imaging, we developed a portable CO2 cell culture mini-incubator that can be easily adapted to any x–y inverted microscope stage, enabling long-term live cell imaging. This mini-incubator provides and maintains stable environmental conditions and supports cell viability comparable to standard incubators. Moreover, it allows for parallel experiments in the same environment, saving both time and resources. To demonstrate its functionality, different cell lines (VERO and MDA-MB-231) were cultured and evaluated using various assays, including crystal violet staining, MTT, and flow cytometry tests to assess cell adhesion, viability, and apoptosis, respectively. Time-lapse imaging was performed over an 85-h period with MDA-MB-231 cells cultured in the mini-incubator. The results indicate that this device is a viable solution for long-term imaging and can be applied in developmental biology, cell biology, and cancer biology research where long-term time-lapse recording is required.

Machine learning-based model for prediction of deep vein thrombosis after gynecological laparoscopy: A retrospective cohort study.

Successful monitoring of deep vein thrombosis (DVT) remains a challenging problem after gynecological laparoscopy. Thus, this study aimed to create and validate predictive models for DVT with the help of machine learning (ML) algorithms. A total of 489 patients from the Cancer Biology Research Center, Tongji Hospital were included in the study between January 2017 and February 2023, and 35 clinical indicators from electronic health records (EHRs) were collected within 24h of patient admission. Risk factors were identified using the least absolute shrinkage and selection operator (LASSO) regression. Then, the three commonly used DVT prediction models are random forest model (RFM), generalized linear regression model (GLRM), and artificial neural network model (ANNM). In addition, the predictive performance of various prediction models (i.e. the robustness and accuracy of predictions) is evaluated through the receiver operating characteristic curve (ROC) and decision curve analysis (DCA), respectively. We found postoperative DVT in 41 (8.38%) patients. Based on the ML algorithm, a total of 13 types of clinical data were preliminarily screened as candidate variables for DVT prediction models. Among these, age, body mass index (BMI), operation time, intraoperative pneumoperitoneum pressure (IPP), diabetes, complication and D-Dimer independent risk factors for postoperative DVT and can be used as variables in ML prediction models. The RFM algorithm can achieve the optimal DVT prediction performance, with AUC values of 0.851 (95% CI: 0.793-0.909) and 0.862 (95% CI: 0.804-0.920) in the training and validation sets, respectively. The AUC values of the other two prediction models (ANNM and GLRM) range from 0.697 (95% CI: 0.639-0.755) and 0.813 (95% CI: 0.651-0.767). In summary, we explored the potential risk of DVT after gynecological laparoscopy, which helps clinicians identify high-risk patients before gynecological laparoscopy and make nursing interventions. However, external validation will be needed in the future.

Establishment of the microscope incubation system and its application in evaluating tumor treatment effects through real-time live cellular imaging.

Introduction: Long-term imaging of live cells is commonly used for the study of dynamic cell behaviors. It is crucial to keep the cell viability during the investigation of physiological and biological processes by live cell imaging. Conventional incubators that providing stable temperature, carbon dioxide (CO2) concentration, and humidity are often incompatible with most imaging tools. Available commercial or custom-made stage-top incubators are bulky or unable to provide constant environmental conditions during long time culture. Methods: In this study, we reported the development of the microscope incubation system (MIS) that can be easily adapted to any inverted microscope stage. Incremental PID control algorithm was introduced to keep stable temperature and gas concentration of the system. Moreover, efficient translucent materials were applied for the top and bottom of the incubator which make it possible for images taken during culture. Results: The MIS could support cell viability comparable to standard incubators. When used in real time imaging, the MIS was able to trace single cell migration in scratch assay, T cell mediated tumor cells killing in co-culture assay, inflation-collapse and fusion of organoids in 3D culture. And the viability and drug responses of cells cultured in the MIS were able to be calculated by a label-free methods based on long term imaging. Discussion: We offer new insights into monitoring cell behaviors during long term culture by using the stage adapted MIS. This study illustrates that the newly developed MIS is a viable solution for long-term imaging during in vitro cell culture and demonstrates its potential in cell biology, cancer biology and drug discovery research where long-term real-time recording is required.

Life-Threatening Endocrinological Immune-Related Adverse Events of Immune Checkpoint Inhibitor Therapy.

Malignant neoplasms are currently one of the leading causes of morbidity and mortality worldwide, posing a major public health challenge. However, recent advances in research in cancer biology and immunity have led to the development of immunotherapy, which is now used on an everyday basis in cancer treatment in addition to surgical treatment, classical cytostatics, and radiotherapy. The efficacy of immunotherapy has promoted the great popularity of this treatment among patients, as well as significant research interest. The increasing number of patients being treated with immunotherapy not only reassures physicians of the efficacy of this technique but also shows the wide spectrum of side effects of this therapy, which has not been considered before. Immune-related adverse events may affect many systems and organs, such as digestive, cardiovascular, respiratory, skin, or endocrine organs. Most complications have a mild or moderate course, but there are life-threatening manifestations that are essential to be aware of because if they are not properly diagnosed and treated on time, they can have fatal consequences. The purpose of this paper was to present the results of a literature review on the current state of knowledge on life-threatening endocrine side effects (such as adrenal crisis, thyroid storm, myxoedema crisis, diabetic ketoacidosis, and severe hypocalcaemia) of immune checkpoint inhibitors to provide information on symptoms, diagnostics, and management strategies.

Pan-Cancer Profiling of Intron Retention and Its Clinical Significance in Diagnosis and Prognosis

Alternative splicing can produce transcripts that affect cancer development and thus shows potential for cancer diagnosis and treatment. However, intron retention (IR), a type of alternative splicing, has been studied less in cancer biology research. Here, we generated a pan-cancer IR landscape for more than 10,000 samples across 33 cancer types from The Cancer Genome Atlas (TCGA). We characterized differentially retained introns between tumor and normal samples and identified retained introns associated with survival. We discovered 988 differentially retained introns in 14 cancers, some of which demonstrated diagnostic potential in multiple cancer types. We also inferred a large number of prognosis-related introns in 33 cancer types, and the associated genes included well-known cancer hallmarks such as angiogenesis, metastasis, and DNA mutations. Notably, we discovered a novel intron retention inside the 5′UTR of STN1 that is associated with the survival of lung cancer patients. The retained intron reduces translation efficiency by producing upstream open reading frames (uORFs) and thereby inhibits colony formation and cell migration of lung cancer cells. Besides, the IR-based prognostic model achieved good stratification in certain cancers, as illustrated in acute myeloid leukemia. Taken together, we performed a comprehensive IR survey at a pan-cancer level, and the results implied that IR has the potential to be diagnostic and prognostic cancer biomarkers, as well as new drug targets.

Hybrid Cellular Automata Modeling Reveals the Effects of Glucose Gradients on Tumour Spheroid Growth

Purpose: In recent years, mathematical models have become instrumental in cancer research, offering insights into tumor growth dynamics, and guiding the development of pharmacological strategies. These models, encompassing diverse biological and physical processes, are increasingly used in clinical settings, showing remarkable predictive precision for individual patient outcomes and therapeutic responses. Methods: Motivated by these advancements, our study introduces an innovative in silico model for simulating tumor growth and invasiveness. The automated hybrid cell emulates critical tumor cell characteristics, including rapid proliferation, heightened motility, reduced cell adhesion, and increased responsiveness to chemotactic signals. This model explores the potential evolution of 3D tumor spheroids by manipulating biological parameters and microenvironment factors, focusing on nutrient availability. Results: Our comprehensive global and local sensitivity analysis reveals that tumor growth primarily depends on cell duplication speed and cell-to-cell adhesion, rather than external chemical gradients. Conversely, tumor invasiveness is predominantly driven by chemotaxis. These insights illuminate tumor development mechanisms, providing vital guidance for effective strategies against tumor progression. Our proposed model is a valuable tool for advancing cancer biology research and exploring potential therapeutic interventions.

SCAR: Single-cell and Spatially-resolved Cancer Resources.

Advances in sequencing and imaging technologies offer a unique opportunity to unravel cell heterogeneity and develop new immunotherapy strategies for cancer research. There is an urgent need for a resource that effectively integrates a vast amount of transcriptomic profiling data to comprehensively explore cancer tissue heterogeneity and the tumor microenvironment. In this context, we developed the Single-cell and Spatially-resolved Cancer Resources (SCAR) database, a combined tumor spatial and single-cell transcriptomic platform, which is freely accessible at http://8.142.154.29/SCAR2023 or http://scaratlas.com. SCAR contains spatial transcriptomicdata from 21 tumor tissues and single-cell transcriptomic data from 11 301 352 cells encompassing 395 cancer subtypes and covering a wide variety oftissues, organoids, and cell lines. This resource offers diverse functional modules to address key cancer research questions at multiple levels, including the screening of tumor cell types, metabolic features, cell communication and gene expression patterns within the tumor microenvironment. Moreover, SCAR enables the analysis of biomarker expression patterns and cell developmental trajectories. SCAR also provides a comprehensive analysis of multi-dimensional datasets based on 34 state-of-the-art omics techniques, serving as an essential tool for in-depth mining and understanding of cell heterogeneity and spatial location. The implications of this resource extend to both cancer biology research and cancer immunotherapy development.

Breast Cancer Cell Type and Biomechanical Properties of Decellularized Mouse Organs Drives Tumor Cell Colonization.

Tissue engineering has emerged as an indispensable tool for the reconstruction of organ-specific environments. Organ-derived extracellular matrices (ECM) and, especially, decellularized tissues (DCL) are recognized as the most successful biomaterials in regenerative medicine, as DCL preserves the most essential organ-specific ECM properties such as composition alongside biomechanics characterized by stiffness and porosity. Expansion of the DCL technology to cancer biology research, drug development, and nanomedicine is pending refinement of the existing DCL protocols whose reproducibility remains sub-optimal varying from organ to organ. We introduce a facile decellularization protocol universally applicable to murine organs, including liver, lungs, spleen, kidneys, and ovaries, with demonstrated robustness, reproducibility, high purification from cell debris, and architecture preservation, as confirmed by the histological and SEM analysis. The biomechanical properties of as-produced DCL organs expressed in terms of the local and total stiffness were measured using our facile methodology and were found well preserved in comparison with the intact organs. To demonstrate the utility of the developed DCL model to cancer research, we engineered three-dimensional tissue constructs by recellularization representative decellularized organs and collagenous hydrogel with human breast cancer cells of pronounced mesenchymal (MDA-MB-231) or epithelial (SKBR-3) phenotypes. The biomechanical properties of the DCL organs were found pivotal to determining the cancer cell fate and progression. Our histological and scanning electron microscopy (SEM) study revealed that the larger the ECM mean pore size and the smaller the total stiffness (as in lung and ovary), the more proliferative and invasive the mesenchymal cells became. At the same time, the low local stiffness ECMs (ranged 2.8-3.6 kPa) did support the epithelial-like SKBR-3 cells' viability (as in lung and spleen), while stiff ECMs did not. The total and local stiffness of the collagenous hydrogel was measured too low to sustain the proliferative potential of both cell lines. The observed cell proliferation patterns were easily interpretable in terms of the ECM biomechanical properties, such as binding sites, embedment facilities, and migration space. As such, our three-dimensional tissue engineering model is scalable and adaptable for pharmacological testing and cancer biology research of metastatic and primary tumors, including early metastatic colonization in native organ-specific ECM.

Extracellular matrix remodelling and stiffening contributes to tumorigenesis of salivary carcinoma ex pleomorphic adenoma——A study based on patient-derived organoids

BackgroundSalivary carcinoma ex pleomorphic adenoma (CXPA) is defined as a carcinoma that develops from benign pleomorphic adenoma (PA). Abnormally activated Androgen signaling pathway and amplification of HER-2/neu(ERBB-2) gene are known to be involved in CXPA tumorigenesis. Recent progress in tumour microenvironment research has led to identification that extracellular matrix (ECM) remodelling and increased stiffness act as critical contributing role in tumour carcinogenesis. This study examined ECM modifications to elucidate the mechanism underlying CXPA tumorigenesis.ResultsPA and CXPA organoids were successfully established. Histological observation, immunohistochemistry (IHC), and whole-exome sequencing demonstrated that organoids recapitulated phenotypic and molecular characteristics of their parental tumours. RNA-sequencing and bioinformatic analysis of organoids showed that differentially expressed genes are highly enriched in ECM-associated terms, implying that ECM alternations may be involved in carcinogenesis. Microscopical examination for surgical samples revealed that excessive hyalinized tissues were deposited in tumour during CXPA tumorigenesis. Transmission electron microscopy confirmed that these hyalinized tissues were tumour ECM in nature. Subsequently, examination by picrosirius red staining, liquid chromatography with tandem mass spectrometry, and cross-linking analysis indicated that tumour ECM was predominantly composed of type I collagen fibers, with dense collagen alignment and an increased level of collagen cross-linking. IHC revealed the overexpression of COL1A1 protein and collagen-synthesis-related genes, DCN and IGFBP5 (p < 0.05). Higher stiffness of CXPA than PA was demonstrated by atomic force microscopy and elastic imaging analysis. We utilized hydrogels to mimic ECM with varying stiffness degrees in vitro. Compared with softer matrices (5Kpa), CXPA cell line and PA primary cells exhibited more proliferative and invasive phenotypes in stiffer matrices (50Kpa, p < 0.01). Protein-protein interaction (PPI) analysis of RNA-sequencing data revealed that AR and ERBB-2 expression was associated with TWIST1. Moreover, surgical specimens demonstrated a higher TWIST1 expression in CXPA over PA. After knocking down TWIST1 in CXPA cells, cell proliferation, migration, and invasiveness were significantly inhibited (p < 0.01).ConclusionDeveloping CXPA organoids provides a useful model for cancer biology research and drug screening. ECM remodelling, attributed to overproduction of collagen, alternation of collagen alignment, and increased cross-linking, leads to increased ECM stiffness. ECM modification is an important contributor in CXPA tumorigenesis.

Global funding for cancer research between 2016 and 2020: a content analysis of public and philanthropic investments.

Cancer is a leading cause of disease burden globally, with more than 19·3 million cases and 10 million deaths recorded in 2020. Research is crucial to understanding the determinants of cancer and the effects of interventions, and to improving outcomes. We aimed to analyse global patterns of public and philanthropic investment in cancer research. In this content analysis, we searched the UberResearch Dimensions database and Cancer Research UK data for human cancer research funding awards from public and philanthropic funders between Jan 1, 2016, and Dec 31, 2020. Included award types were project and programme grants, fellowships, pump priming, and pilot projects. Awards focused on operational delivery of cancer care were excluded. Awards were categorised by cancer type, cross-cutting research theme, and research phase. Funding amount was compared with global burden of specific cancers, measured by disability-adjusted life-years, years lived with disability, and mortality using data from the Global Burden of Disease study. We identified 66 388 awards with total investment of about US$24·5 billion in 2016-20. Investment decreased year-on-year, with the largest drop observed between 2019 and 2020. Pre-clinical research received 73·5% of the funding across the 5 years ($18 billion), phase 1-4 clinical trials received 7·4% ($1·8 billion), public health research received 9·4% ($2·3 billion), and cross-disciplinary research received 5·0% ($1·2 billion). General cancer research received the largest investment ($7·1 billion, 29·2% of the total funding). The most highly funded cancer types were breast cancer ($2·7 billion [11·2%]), haematological cancer ($2·3 billion [9·4%]), and brain cancer ($1·3 billion [5·5%]). Analysis by cross-cutting theme revealed that 41·2% of investment ($9·6 billion) went to cancer biology research, 19·6% ($4·6 billion) to drug treatment research, and 12·1% ($2·8 billion) to immuno-oncology. 1·4% of the total funding ($0·3 billion) was spent on surgery research, 2·8% ($0·7 billion) was spent on radiotherapy research, and 0·5% ($0·1 billion) was spent on global health studies. Cancer research funding must be aligned with the global burden of cancer with more equitable funding for cancer research in low-income and middle-income countries (which account for 80% of cancer burden), both to support research relevant to these settings, and build research capacity within these countries. There is an urgent need to prioritise investment in surgery and radiotherapy research given their primacy in the treatment of many solid tumours. None.

Abstract 4185: Inclusive basic and advanced translational laboratory research competencies for research in cancer biology and therapeutics

Abstract Our Laboratory was established in 1994 at Univ. of Pennsylvania. Lab members demonstrated initial competencies by performing cell culture, western blots, immunofluorescence, and flow cytometry showing induction of p53/p21(WAF1) in cells treated with chemotherapy. Years later, our Laboratory of Translational Oncology &amp; Experimental Cancer Therapeutics moved to Penn State Univ., Fox Chase Cancer Center/Temple Univ. and then Brown Univ. By 2020, with desire for inclusiveness (everyone succeeds), scientific rigor/reproducibility mandated by NIH, and as a training and mentoring activity (lab scientists/trainees/students mentoring others at High School level and beyond), we established a process for onboarding and training new cancer researchers. By Fall of 2022, there were 17 current Brown University undergraduate students (10 receiving research credit and 7 not receiving credit), HS students, 7 graduate students (PhD, masters, MD/PhD), and 6 medical students working with collaborating faculty at our laboratory at Brown’s Legorreta Cancer Center. After completion of biosafety training, and required trainings such as by IACUC, new lab members complete basic competencies in cell culture, cell viability, and western blot analysis that include technical, presentation quality output, and quantitative/statistical rigor to satisfy current standards for journal publication. For cell culture this includes pathogen free conditions, authentication, attention to details of routine procedures, documentation of morphology, freezing, thawing, passaging, seeding density, and managing cell populations to not run out of cells. Cell viability assessment includes attention to culture conditions, synergy analysis, data robustness, and presentation, and for western blots attention to quality of blots, protein quantification, loading, labeling, antibody specificity and sensitivity controls, presentation at 2022 standards, conventions for splicing, and issues with reproducibility including biological replicates, and generalizability. Additional and advanced competencies include RT-PCR, long-term colony assays, 3-D cultures (spheroids, organoids), transfection (overexpression, knockdown, CRISPR), co-culture and triculture with immune cells and fibroblasts, cytokine profiling, in vivo studies, in vivo imaging, immunohistochemistry, flow cytometric analysis, single cell techniques, viral infection, circulating tumor cell isolation, blood immune and cytokine analysis, and work with transgenic organoids and inducible cancer predisposing alleles. Modeling the tumor microenvironment, relevance to human cancer and translational directions are emphasized. Shared online lab resources, protocols, practices, videos, and manuscripts are available for lab members. The framework herein may be of interest to others involved in similar training programs. Citation Format: Wafik S. El-Deiry, Andrew George, Francesca Di Cristofano, Praveen Srinivasan, Lindsey Carlsen, Kelsey E. Huntington, Arielle De La Cruz, Leiqing Zhang, Marina Hahn, Shuai Zhao, Attila Seyhan, Bradley D. DeNardo, Aaron W. Maxwell, Dae Hee Kim, Alex Raufi, Hina Khan, Stephanie L. Graff, Don S. Dizon, Christopher Azzoli, Abbas E. Abbas, Roxanne Wood, Rishi R. Lulla, Howard P. Safran, Benedito A. Carneiro, Arunasalam Navaraj, Xiaobing Tian, Shengliang Zhang, Lanlan Zhou. Inclusive basic and advanced translational laboratory research competencies for research in cancer biology and therapeutics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4185.

Abstract 1325: Novel recombinant rabbit monoclonal antibodies for cancer biology research

Abstract Recombinant monoclonal antibodies (rAbs) are superior tools for cancer biology research as they offer distinct advantages over standard polyclonal and hybridoma-generated monoclonal antibody reagents. Recombinant technology produces antibodies that can be defined at the primary sequence level and are characterized by consistent performance and supply. These rAb features are in stark contrast to traditional polyclonal and hybridoma-generated monoclonal antibodies, whose lack of dependability has been widely documented as a primary reason for data irreproducibility plaguing biomedical research. In an effort to respond to this challenge, GeneTex has established a recombinant antibody production platform combined with more rigorous validation protocols. The goal is to develop reliable recombinant rabbit monoclonal antibodies for reproducible performance in various experimental applications utilized routinely by cancer biologists. GeneTex’s rAb production protocol is based on the approach described by Starkie et al. (2016). This involves a multi-parameter fluorescence-activated single cell sorting (FACS)-based methodology to identify and isolate antigen-specific IgG+ memory B cells obtained from an immunized rabbit. The heavy and light chain variable region genes from selected cells are PCR-amplified and cloned into plasmids to produce full-length heavy and light chains for a single IgG. These constructs are subsequently introduced together into mammalian cells for expression, meaning that natural pairing of the chains is maintained. This manufacturing strategy allows application-specific testing (e.g., for western blot (WB), immunohistochemistry (IHC), and immunocytochemistry (ICC/IF), etc.) of clones during the antibody production process. Validation of antibodies is performed inhouse and, when possible, in relationships with academic or industry/pharma institutions where ideal samples and reagents are frequently more available. This recombinant monoclonal antibody production platform has created a series of well-validated antibodies against many important targets for cancer biology research. This includes reagents to study tumor immunobiology (e.g., PD-L1), hormone receptors (e.g., ER alpha and androgen receptor variants like ARV7), oncogenic RAS protein mutants (e.g., RAS G12D), transcription factors (e.g., NRF2), and epithelial-mesenchymal transition (EMT) (e.g., E-cadherin), among many others. Extensive validation for multiple applications was performed, with knockdown or CRISPR-based knockout cell lysates employed when possible to establish specificity. In summary, GeneTex is utilizing a FACS-based recombinant rabbit monoclonal antibody production approach to generate and thoroughly validate antibody reagents that will hopefully facilitate reproducible cancer biology research. Citation Format: Alexander Ball, Yung Lin Hsieh, Chun Kai Huang, Sega Huang, Ping Kuan Hsieh, Shang Ru Wu, Yue Yun Chang, Jiming Liu, Po Shin Peng, Chia Yi Lin. Novel recombinant rabbit monoclonal antibodies for cancer biology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1325.