Fundamental Cancer Research Research Articles

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle.

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between cancer and immune cells, the so-called cancer-immunity cycle. In vitro co-culture systems combined with multiparametric flow cytometry (mFC) and tumor-on-a-chip microfluidic devices (ToCs) enable simple, fast, and reliable monitoring and characterization of each step of the cancer-immunity cycle and lead to the identification of the mechanisms responsible for tipping the balance between cancer immunosurveillance and immunoevasion. A thorough understanding of the dynamic interplays between cancer and immune cells provides critical insights to outsmart tumors and will accelerate the pace of therapeutic personalization and optimization in patients. Specifically, here we detail a straightforward mFC- and ToC-assisted protocol for unraveling the dynamic complexities of each step of the cancer-immunity cycle in murine cancer cell lines and mouse-derived immune cells and focus on immunosurveillance. Considering the time- and cost-related features of this protocol, it is certainly feasible on a large scale. Moreover, with minor variations, this protocol can be both adapted to human cancer cell lines and human peripheral-blood-derived immune cells and combined with genetic and/or pharmacologic inhibition of specific pathways in order to identify biomarkers of immune response.

3D modeling of normal skin and cutaneous squamous cell carcinoma. A comparative study in 2D cultures, spheroids, and 3D bioprinted systems.

The current cancer research and drug testing are primarily based on 2D cell cultures and animal models. However, these methods have limitations and yield distinct drug response patterns. This study addressed this gap by developing an innovativein vitrohuman three-dimensional (3D) normal skin model and a multicellular model of human cutaneous squamous cell carcinoma (cSCC) using 3D bioprinting technology. Comparative analyzes were performed between bioprinted 3D-cSCC model, consisting of HaCaT keratinocytes, primary normal human dermal fibroblasts and A431 cancer cells (tricellular), bioprinted 3D-A431 model composed of A431 cancer cells only (monocellular), A431 cancer cell spheroids, and conventional 2D models. The models were structurally characterized by light microscopy, immunofluorescence (LIVE/DEAD assay, confocal microscopy) and immunohistochemistry (hematoxylin/eosin, p63, vimentin, Ki67, epidermal growth factor receptor stainings). The spatial arrangement of the 3D models was analyzed using the ARIVIS scientific image analysis platform. All models were also functionally assessed by cetuximab (CTX) response testing with the MTS assay. 3D-cSCC models were maintained for up to 16 weeks. Morphological and histological examinations confirmed the presence of skin-like layers in the bioprinted 3D models of normal skin, and the intricate and diverse features of the bioprinted skin cancer model, replicating the criticalin vivocharacteristics. In both mono- and tricellular bioprinted tumor constructs, there was a gradual formation and continuous growth of spheroid-like clusters of cancer cells, significantly influencing the morphology of the models. Cancer cells in the 3D bioprinted constructs showed reduced sensitivity to CTX compared to spheroids and 2D cultures. This study underscores the potential of 3D multicellular models in elucidating drug responses and gaining a better understanding the intricate interplay of cellular components within the tumor microenvironment. Developing the multicellular 3D tumor model paves the way for new research critical to advancing fundamental cancer research and future clinical applications, particularly drug response testing.

Volume imaging to interrogate cancer cell-tumor microenvironment interactions in space and time.

Volume imaging visualizes the three-dimensional (3D) complexity of tumors to unravel the dynamic crosstalk between cancer cells and the heterogeneous landscape of the tumor microenvironment (TME). Tissue clearing and intravital microscopy (IVM) constitute rapidly progressing technologies to study the architectural context of such interactions. Tissue clearing enables high-resolution imaging of large samples, allowing for the characterization of entire tumors and even organs and organisms with tumors. With IVM, the dynamic engagement between cancer cells and the TME can be visualized in 3D over time, allowing for acquisition of 4D data. Together, tissue clearing and IVM have been critical in the examination of cancer-TME interactions and have drastically advanced our knowledge in fundamental cancer research and clinical oncology. This review provides an overview of the current technical repertoire of fluorescence volume imaging technologies to study cancer and the TME, and discusses how their recent applications have been utilized to advance our fundamental understanding of tumor architecture, stromal and immune infiltration, vascularization and innervation, and to explore avenues for immunotherapy and optimized chemotherapy delivery.

Abstract 473: GeneMasking: A new approach for inferring the functional role of cancer driver genes and its application in prostate cancer

Abstract Introduction: Genomic events in cancer driver genes such as mutations and copy number alterations play critical roles in cancer onset and progression. Here we apply a novel biologically inspired deep learning classification method called GeneMasking to perform prostate cancer (PC) classification high accuracy. The important key novelty of GeneMasking is that it marks the genomic events that determine the classification of a given sample in a sample-specific manner. This provides us with an exciting opportunity to investigate two fundamental cancer research questions: 1.For a specific tumor sample, which cancer driver genes are functionally important, playing a detrimental role in its classification? 2.For a specific tumor sample, does an established cancer driver gene act as oncogene or tumor suppressor gene (TSG) in that sample? Methods: We applied GeneMasking to study these questions by analyzing the somatic mutation and copy number alteration (CNA) data of 1011 PC patient samples recently published by Elmarakeby et al. [Nature 598, 348-352 (2021)] and classify as primary or metastatic tumors. GeneMasking achieves an overall AUC (area under the receiver operating characteristic curve) of 0.958 in this classification task. Additionally and importantly, for each sample, GeneMasking provides a list of genes with mutation and/or CNA events that are important in classifying it. We focus on 61 known COSMIC cancer driver genes marked as important in ≥ 20 samples. A driver gene that is marked as important is inferred as oncogenic-acting in that given sample if it is amplified or mutated by an activating mutation, and vice versa for inferring it as a TSG-acting in that sample. For each gene, we compute the oncogene/TSG ratio of its inferred function across all samples where it was marked ‘important’. We then investigate whether this ratio is higher than 1 as expected for drivers traditionally labeled oncogenes, or lower, as expected for those known as TSGs. Results: Our key findings are: 1) For 44 drivers (including 5 PC-specific genes), the overall inferred oncogene/TSG ratios reassuringly match their conventional annotations as oncogenes or TSGs in COSMIC. 2) For 8 drivers, however, their inferred oncogene/TSG ratios are opposite from those listed in COSMIC. Those include MUC4, XPO1, FANCD2 etc. 3) 5 drivers are annotated as both oncogenes and TSGs in COSMIC and here we infer their specific roles in PC. Conclusions: We provide a first-of-its-kind computational approach to annotate cancer driver genes as oncogenes or TSGs in a patient cohort. This lays the basis for two future exciting applications: (a) Identifying the contexts in which a driver acts as oncogene or TSG, and (b) Help advance precision oncology, targeting actionable mutations of drivers only when they are truly oncogenic. Citation Format: Saugato Rahman Dhruba, Niv Amitay, Lior Wolf, Eytan Ruppin. GeneMasking: A new approach for inferring the functional role of cancer driver genes and its application in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 473.

Instructive Hydrogels for Primary Tumor Cell Culture: Current Status and Outlook.

Primary tumor organoids (PTOs) growth in hydrogels have emerged as an important in vitro model that recapitulates many characteristics of the native tumor tissue, and have important applications in fundamental cancer research and for the development of useful therapeutic treatment. This paper begins with reviewing the methods of isolation of primary tumor cells. Then, recent advances on the instructive hydrogels as biomimetic extracellular matrix for primary tumor cell culture and construction of PTO models are summarized. Emerging microtechnology for growth of PTOs in microscale hydrogels and the applications of PTOs are highlighted. This paper concludes with an outlook on the future directions in the investigation of instructive hydrogels for PTO growth.

Super-Multiplex Nonlinear Optical Imaging Unscrambles the Statistical Complexity of Cancer Subtypes and Tumor Microenvironment.

Label‐free nonlinear optical imaging (NLOI) has made tremendous inroads toward unscrambling the microcosmic complexity of cancers. However, harmonic and Raman microscopy offers throughput without redox information to reveal metabolic differentiation, and fluorescence lifetime microscopy lacks the vibrational response of molecules to visualize specific molecular constituents such as lipid. Here, a flexible, robust simultaneous multi‐nonlinear imaging and cross‐modality system that combines complementary imaging contrast mechanisms is demonstrated. This system, utilizing multiplexed ultrashort pulses, ingeniously integrates typical nonlinear processes, and high‐dimension lifetime extension in a single setup to enhance the imaging dimensions and quality. Using this system, the authors perform label‐free comprehensive evaluation of clinicopathological tissues of ovarian carcinoma due to its statistical complexity. The results show that the technology provides statistically rich, insightful information with high accuracy, sensitivity, and specificity, in contrast to standard histopathology, and can potentially be a powerful tool for fundamental cancer research and clinical applications.

Facile One Step Formation and Screening of Tumor Spheroids Using Droplet-Microarray Platform.

Tumor spheroids or microtumors are important 3D in vitro tumor models that closely resemble a tumor's in vivo "microenvironment" compared to 2D cell culture. Microtumors are widely applied in the fields of fundamental cancer research, drug discovery, and precision medicine. In precision medicine tumor spheroids derived from patient tumor cells represent a promising system for drug sensitivity and resistance testing. Established and commonly used platforms for routine screenings of cell spheroids, based on microtiter plates of 96- and 384-well formats, require relatively large numbers of cells and compounds, and often lead to the formation of multiple spheroids per well. In this study, an application of the Droplet Microarray platform, based on hydrophilic-superhydrophobic patterning, in combination with the method of hanging droplet, is demonstrated for the formation of highly miniaturized single-spheroid-microarrays. Formation of spheroids from several commonly used cancer cell lines in 100 nL droplets starting with as few as 150 cells per spheroid within 24-48 h is demonstrated. Established methodology carries a potential to be adopted for routine workflows of high-throughput compound screening in 3D cancer spheroids or microtumors, which is crucial for the fields of fundamental cancer research, drug discovery, and precision medicine.

Label-free molecular profiling for identification of biomarkers in carcinogenesis using multimodal multiphoton imaging.

Label-free molecular profiling, imaging, and analysis are of particular interest in cancer biology for detecting subtle biochemical changes during cancer progression and potentially during cancer treatment. Multimodal, multiphoton imaging that combines diverse molecular contrasts derived from different physical mechanisms can improve our understanding of the tumor microenvironment. A label-free optical molecular profiling technique has been developed based on penta-modal multiphoton imaging to investigate mammary tumor progression in a pre-clinical rat model. Pulses from a coherent supercontinuum were tailored for two-photon (2PF) and three-photon fluorescence (3PF), second (SHG) and third harmonic generation (THG), and hyperspectral coherent anti-Stokes Raman scattering (CARS)-based imaging. A graphic multiphoton molecular profiling model was constructed to intuitively combine the co-registered quantitative, chemical, functional, and structural tissue information, enabling longitudinal in situ biomolecular analysis. Over a 9-week period of tumor progression, and even before the formation of solid tumor, we observed lipid-protein transitions, microenvironmental reorganization, and a shift from FAD to NAD(P)H fluorescence, which reflects the reprogramming of cellular metabolism in carcinogenesis. Multimodal multiphoton imaging reveals and interrelates diverse carcinogenic signatures, identifying biomarkers that could serve as early molecular indicators for breast cancer diagnosis. This quantitative multimodal imaging methodology for molecular profiling of associated cancer biomarkers may have a broader impact in fundamental cancer research and future clinical applications.

Supramolecular Nanofibrillar Thermoreversible Hydrogel for Growth and Release of Cancer Spheroids

Growth of three-dimensional cancer spheroids (CSs) in man-made hydrogels mimicking natural extracellular matrix is an important and challenging task. Herein, we report on a supramolecular temperature-responsive hydrogel designed for the growth and subsequent release of CSs. A filamentous hydrogel was formed at 37 °C from an aqueous suspension of cellulose nanocrystals surface-functionalized with temperature-responsive polymer molecules. The encapsulation of cells in the hydrogel enabled effective growth of CSs with dimensions determined by the concentration of cellulose nanocrystals in the hydrogel. On demand release of CSs without loss of cell viability and spheroid integrity was achieved upon hydrogel cooling. The tumorigenic properties of the released CSs were examined by encapsulating and re-growing them in fibrin hydrogel. The results in this work can be used in fundamental cancer research and in cancer drug screening.

Supramolecular Nanofibrillar Thermoreversible Hydrogel for Growth and Release of Cancer Spheroids

AbstractGrowth of three‐dimensional cancer spheroids (CSs) in man‐made hydrogels mimicking natural extracellular matrix is an important and challenging task. Herein, we report on a supramolecular temperature‐responsive hydrogel designed for the growth and subsequent release of CSs. A filamentous hydrogel was formed at 37 °C from an aqueous suspension of cellulose nanocrystals surface‐functionalized with temperature‐responsive polymer molecules. The encapsulation of cells in the hydrogel enabled effective growth of CSs with dimensions determined by the concentration of cellulose nanocrystals in the hydrogel. On demand release of CSs without loss of cell viability and spheroid integrity was achieved upon hydrogel cooling. The tumorigenic properties of the released CSs were examined by encapsulating and re‐growing them in fibrin hydrogel. The results in this work can be used in fundamental cancer research and in cancer drug screening.

Mouse models in oncoimmunology.

Fundamental cancer research and the development of efficacious antineoplastic treatments both rely on experimental systems in which the relationship between malignant cells and immune cells can be studied. Mouse models of transplantable, carcinogen-induced or genetically engineered malignancies - each with their specific advantages and difficulties - have laid the foundations of oncoimmunology. These models have guided the immunosurveillance theory that postulates that evasion from immune control is an essential feature of cancer, the concept that the long-term effects of conventional cancer treatments mostly rely on the reinstatement of anticancer immune responses and the preclinical development of immunotherapies, including currently approved immune checkpoint blockers. Specific aspects of pharmacological development, as well as attempts to personalize cancer treatments using patient-derived xenografts, require the development of mouse models in which murine genes and cells are replaced with their human equivalents. Such 'humanized' mouse models are being progressively refined to characterize the leukocyte subpopulations that belong to the innate and acquired arms of the immune system as they infiltrate human cancers that are subjected to experimental therapies. We surmise that the ever-advancing refinement of murine preclinical models will accelerate the pace of therapeutic optimization in patients.

Effects of mechanical properties on tumor invasion: insights from a cellular model.

Understanding the regulating mechanism of tumor invasion is of crucial importance for both fundamental cancer research and clinical applications. Previous in vivo experiments have shown that invasive cancer cells dissociate from the primary tumor and invade into the stroma, forming an irregular invasive morphology. Although cell movements involved in tumor invasion are ultimately driven by mechanical forces of cell-cell interactions and tumor-host interactions, how these mechanical properties affect tumor invasion is still poorly understood. In this study, we use a recently developed two-dimensional cellular model to study the effects of mechanical properties on tumor invasion. We study the effects of cell-cell adhesions as well as the degree of degradation and stiffness of extracellular matrix (ECM). Our simulation results show that cell-cell adhesion relationship must be satisfied for tumor invasion. Increased adhesion to ECM and decreased adhesion among tumor cells result in invasive tumor behaviors. When this invasive behavior occurs, ECM plays an important role for both tumor morphology and the shape of invasive cancer cells. Increased stiffness and stronger degree of degradation of ECM promote tumor invasion, generating more aggressive tumor invasive morphologies. It can also generate irregular shape of invasive cancer cells, protruding towards ECM. The capability of our model suggests it a useful tool to study tumor invasion and might be used to propose optimal treatment in clinical applications.

Chapter Nine - Life is Three Dimensional—As In Vitro Cancer Cultures Should Be

For many decades, fundamental cancer research has relied on two-dimensional in vitro cell culture models. However, these provide a poor representation of the complex three-dimensional (3D) architecture of living tissues. The more recent 3D culture systems, which range from ridged scaffolds to semiliquid gels, resemble their natural counterparts more closely. The arrangement of the cells in 3D systems allows better cell-cell interaction and facilitates extracellular matrix secretion, with concomitant effects on gene and protein expression and cellular behavior. Many studies have reported differences between 3D and 2D systems as regards responses to therapeutic agents and pivotal cellular processes such as cell differentiation, morphology, and signaling pathways, demonstrating the importance of 3D culturing for various cancer cell lines.

Emergent Behaviors from a Cellular Automaton Model for Invasive Tumor Growth in Heterogeneous Microenvironments

Understanding tumor invasion and metastasis is of crucial importance for both fundamental cancer research and clinical practice. In vitro experiments have established that the invasive growth of malignant tumors is characterized by the dendritic invasive branches composed of chains of tumor cells emanating from the primary tumor mass. The preponderance of previous tumor simulations focused on non-invasive (or proliferative) growth. The formation of the invasive cell chains and their interactions with the primary tumor mass and host microenvironment are not well understood. Here, we present a novel cellular automaton (CA) model that enables one to efficiently simulate invasive tumor growth in a heterogeneous host microenvironment. By taking into account a variety of microscopic-scale tumor-host interactions, including the short-range mechanical interactions between tumor cells and tumor stroma, degradation of the extracellular matrix by the invasive cells and oxygen/nutrient gradient driven cell motions, our CA model predicts a rich spectrum of growth dynamics and emergent behaviors of invasive tumors. Besides robustly reproducing the salient features of dendritic invasive growth, such as least-resistance paths of cells and intrabranch homotype attraction, we also predict nontrivial coupling between the growth dynamics of the primary tumor mass and the invasive cells. In addition, we show that the properties of the host microenvironment can significantly affect tumor morphology and growth dynamics, emphasizing the importance of understanding the tumor-host interaction. The capability of our CA model suggests that sophisticated in silico tools could eventually be utilized in clinical situations to predict neoplastic progression and propose individualized optimal treatment strategies.

French Angiogenesis Society partners with Targeted Oncology

The French Angiogenesis Society (Societe Francaise d’Angiogenese) and the journal Targeted Oncology recently decided to become partners in their shared goals of promoting and disseminating clinical and fundamental cancer research. Angiogenesis corresponds to the formation of new blood vessels. It is critically important during embryonic development and throughout life, this process is also associated with several diseases. The theory that tumors depend on oxygen and blood supply was clearly and formally conceptualized in the 1970s. In the following years, the search for a “tumor angiogenic factor” and for endothelial-specific growth factors led to the discovery of vascular endothelial growth factor (VEGF) and many other angiogenic factors. We quickly learned that VEGF played a central role in angiogenesis and, more recently, VEGF was the first angiogenic factor against which a targeted therapy proved to be beneficial to cancer patients. This opened the novel path of so-called “anti-angiogenic” therapies, and several such therapies targeting VEGF and its receptors are now commonly used in the clinic; more will come, aimed at various other molecular and cellular agents of tumor angiogenesis. The French Angiogenesis Society gathers clinicians and research scientists who show interest in the general field of angiogenesis. Although tumor angiogenesis is one of its major topics of interest, the Society is not strictly focused on cancer. Several members are working on other aspects of angiogenesis, such as embryonic development, retinal vessels, endothelial barriers, genetics, and gene regulation. Our next meeting will be held in southern France in May 2010, and we will be honored by presentations in English from several internationally recognized speakers. The readers of Targeted Oncology are most welcome to visit our website (www.angiogenese.fr) and, if interested, find their way to our next meetings. The partnership with Targeted Oncology is clearly pertinent. In upcoming years, the Society will advertise its meetings and publish its reports in the journal. We sincerely hope that the readers of Targeted Oncology will find as much interest in our work as our members do in reading this journal. Should our partnership promote collaborations and the emergence of new, efficient therapies against cancer, both the journal and the Society would reach an important common goal.

Genetic Concepts and Neoplasia. A collection of papers presented at the 23rd Annual Symposium on Fundamental Cancer Research, M. D. Anderson Hospital and Tumor Institute at Houston, Texas. 9 × 6 in. Pp. 620+xiii. IIIustrated. 1970. Edinburgh: E. & S. Livingstone. £8

Genetic Concepts and Neoplasia. A collection of papers presented at the 23rd Annual Symposium on Fundamental Cancer Research, M. D. Anderson Hospital and Tumor Institute at Houston, Texas. 9 × 6 in. Pp. 620+xiii. IIIustrated. 1970. Edinburgh: E. & S. Livingstone. £8

Carcinogenesis: A broad critique. A Collection of Papers presented at the Twentieth Annual Symposium on Fundamental Cancer Research, 1966, published for the University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, Texas. 9 × 6 in. Pp. 774 + xvi. Illustrated. 1967. Baltimore: The Williams & Wilkins Company. (Edinburgh: E. & S. Livingstone Ltd.) £7 7s. 6d

Carcinogenesis: A broad critique. A Collection of Papers presented at the Twentieth Annual Symposium on Fundamental Cancer Research, 1966, published for the University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, Texas. 9 × 6 in. Pp. 774 + xvi. Illustrated. 1967. Baltimore: The Williams & Wilkins Company. (Edinburgh: E. & S. Livingstone Ltd.) £7 7s. 6d

56th Annual Symposium on Fundamental Cancer Research Cancer Immunity: Challenges for the Next Decade

56th Annual Symposium on Fundamental Cancer Research Cancer Immunity: Challenges for the Next Decade

Defects in G1-S cell cycle control in head and neck cancer: a review.

Tumors gradually develop as a result of a multistep acquisition of genetic alterations and ultimately emerge as selfish, intruding and metastatic cells. The genetic defects associated with the process of tumor progression affect control of proliferation, programmed cell death, cell aging, angiogenesis, escape from immune control and metastasis. Fundamental cancer research over the last thirty years has revealed a multitude of genetic alterations which specify more or less separate steps in tumor development and which are collectively responsible for the process of tumor progression. The genes affected play in normal cells a crucial role in control over cell duplication and the interaction between cells, and between cells and their direct surrounding. This is illustrated on control during the G1/S phase of the cell cycle by its ultimate regulators: cyclins and cyclin dependent kinases. These proteins not only control the transition through the G1/S phase of the cell cycle, but also serve as mediators of the interaction between cells, and between cells and their surrounding. Defaults in the regulation of these proteins are associated with tumor progression, and, therefore, serve as targets for therapy. Defaults in those genes are found in various tumor types, although some of those prevail in particular tumor types. In this review emphasis is given to the defaults that occur in head and neck cancer.

Nobel's day on recent advances in cancer research

The June 7-10th Symposium on 'Recent Advances in Cancer Research', organized by Lorenzo Moretta in San Remo, Italy, on the occasion of Nobel's day, designated cancer genetics and tumor immunology as forefront areas in fundamental cancer research. While the central importance of oncogenes is obvious, this is less the case for the immunology of tumors, which has long been an area of descriptive phenomenology, wild speculation and unfulfilled expectation. The recent molecular identification of genes encoding a human melanoma antigen recognized by T cells, and of cytokines involved in the tumor-host interaction justifies renewed oncological interest in this area.