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Despite significant advances, cancer remains a leading global cause of death. Current therapies often fail due to incomplete tumor removal and nonspecific targeting, spurring interest in alternative treatments. Hyperthermia, which uses elevated temperatures to kill cancer cells or boost their sensitivity to radio/chemotherapy, has emerged as a promising alternative. Recent advancements employ nanoparticles (NPs) as heat mediators for selective cancer cell destruction, minimizing damage to healthy tissues. This approach, known as NP hyperthermia, falls into two categories: photothermal therapies (PTT) and magnetothermal therapies (MTT). PTT utilizes NPs that convert light to heat, while MTT uses magnetic NPs activated by alternating magnetic fields (AMF), both achieving localized tumor damage. These methods offer advantages like precise targeting, minimal invasiveness, and reduced systemic toxicity. However, the efficacy of NP hyperthermia depends on many factors, in particular, the NP properties, the tumor microenvironment (TME), and TME-NP interactions. Optimizing this treatment requires accurate heat monitoring strategies, such as nanothermometry and biologically relevant screening models that can better mimic the physiological features of the tumor in the human body. This review explores the state-of-the-art in NP-mediated cancer hyperthermia, discussing available nanomaterials, their strengths and weaknesses, characterization methods, and future directions. Our particular focus lies in preclinical NP screening techniques, providing an updated perspective on their efficacy and relevance in the journey towards clinical trials.

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Renal cell carcinoma, a leading cause of death in urological malignancies, arises from the nephron. Its characteristics include diversity in disease biology, varied clinical behaviors, different prognoses, and diverse responses to systemic therapies. The term 'organoids' is used to describe structures resembling tissues created through the three-dimensional cultivation of stem cells in vitro. These organoids, when derived from tumor tissues, can retain the diversity of the primary tumor, mirror its spatial tissue structure, and replicate similar organ-like functions. In contrast to conventional two-dimensional cell cultures and the transplantation of tumor tissues into other organisms, organoids derived from tumors maintain the complexity and microenvironment of the original tumor tissue. This fidelity makes them a more reliable model for the development of cancer drugs, potentially accelerating the translation of these drugs to clinical use and facilitating personalized treatment options for patients. This review aims to summarize the recent advancements in the use of organoids for studying renal cell carcinoma, focusing on their cultivation, potential applications, and inherent limitations.

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Solid tumors often contain genetically different populations of cancer cells, stromal cells, various structural and soluble proteins, and other soluble signaling molecules. The American Cancer society estimated 1,958,310 new cancer cases and 609,820 cancer deaths in the United States in 2023. A major barrier against successful treatment of cancer patients is drug resistance. Gain of stem cell-like states by cancer cells under drug pressure or due to interactions with the tumor microenvironment is a major mechanism that renders therapies ineffective. Identifying approaches to target cancer stem cells is expected to improve treatment outcomes for patients. Most of our understanding of drug resistance and the role of cancer stemness is from monolayer cell cultures. Recent advances in cell culture technologies have enabled developing sophisticated three-dimensional tumor models that facilitate mechanistic studies of cancer drug resistance. This review summarizes the role of cancer stemness in drug resistance and highlights the various tumor models that are used to discover the underlying mechanisms and test potentially novel therapeutics.

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Cancer remains a serious burden in society and while the pace in the development of novel and more effective therapeutics is increasing, testing platforms that faithfully mimic the tumor microenvironment are lacking. With a clear shift from animal models to more complex in vitro 3D systems, spheroids emerge as strong options in this regard. Years of development have allowed spheroid-based models to better reproduce the biomechanical cues that are observed in the tumor-associated extracellular matrix (ECM) and cellular interactions that occur in both a cell-cell and cell-ECM manner. Here, we summarize some of the key cellular interactions that drive tumor development, progression and invasion, and how successfully are these interactions recapitulated in 3D spheroid models currently in use in the field. We finish by speculating on future advancements in the field and on how these can shape the relevance of spherical 3D models for tumor modelling.

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Colorectal carcinoma (CRC) presents a formidable medical challenge, demanding innovative therapeutic strategies. Chimeric antigen receptor (CAR) natural killer (NK) cell therapy has emerged as a promising alternative to CAR T-cell therapy for cancer. A suitable tumor antigen target on CRC is carcinoembryonic antigen (CEA), given its widespread expression and role in tumorigenesis and metastasis. CEA is known to be prolifically shed from tumor cells in a soluble form, thus hindering CAR recognition of tumors and migration through the TME. We have developed a next-generation CAR construct exclusively targeting cell-associated CEA, incorporating a PD1-checkpoint inhibitor and a CCR4 chemokine receptor to enhance homing and infiltration of the CAR-NK-92 cell line through the TME, and which does not induce fratricidal killing of CAR-NK-92-cells. To evaluate this therapeutic approach, we harnessed intricate 3D multicellular tumor spheroid models (MCTS), which emulate key elements of the TME. Our results demonstrate the effective cytotoxicity of CEA-CAR-NK-92 cells against CRC in colorectal cell lines and MCTS models. Importantly, minimal off-target activity against non-cancerous cell lines underscores the precision of this therapy. Furthermore, the integration of the CCR4 migration receptor augments homing by recognizing target ligands, CCL17 and CCL22. Notably, our CAR design results in no significant trogocytosis-induced fratricide. In summary, the proposed CEA-targeting CAR-NK cell therapy could offer a promising solution for CRC treatment, combining precision and efficacy in a tailored approach.

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The use of three-dimensional (3D) bioprinting has been proposed for the reproducible production of 3D disease models that can be used for high-throughput drug testing and personalized medicine. However, most such models insufficiently reproduce the features and environment of real tumors. We report the development of bioprinted in vitro 3D tumor models for breast cancer, which physically and biochemically mimic important aspects of the native tumor microenvironment, designed to study therapeutic efficacy. By combining a mix of breast decellularized extracellular matrix and methacrylated hyaluronic acid with tumor-derived cells and non-cancerous stromal cells of biological relevance to breast cancer, we show that biological signaling pathways involved in tumor progression can be replicated in a carefully designed tumor-stroma environment. Finally, we demonstrate proof-of-concept application of these models as a reproducible platform for investigating therapeutic responses to commonly used chemotherapeutic agents.

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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.

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Microfluidic technologies allow the generation of large datasets using smaller quantities of cells and reagents than with traditional well plate assays. Such miniaturized methods can also facilitate the generation of complex 3D preclinical models of solid tumors with controlled size and cell composition. This is particularly useful in the context of recreating the tumor microenvironment for preclinical screening of immunotherapies and combination therapies at a scale, to reduce the experimental costs during therapy development while using physiologically relevant 3D tumor models, and to assess the therapy's efficacy. Here, we describe the fabrication of microfluidic devices and the associated protocols to culture tumor-stromal spheroids for assessing the efficacy of anticancer immunotherapies as monotherapies and as part of combination therapy regimes.

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BACKGROUND: The high recurrence of glioblastoma (GB) that occurs adjacent to the resection cavity within two years of diagnosis urges an improvement of therapies oriented to GB local control. Photodynamic therapy (PDT) has been proposed to cleanse infiltrating tumor cells from parenchyma to ameliorate short long-term progression-free survival. We examined 5-aminolevulinic acid (5-ALA)-mediated PDT effects as therapeutical treatment and determined optimal conditions for PDT efficacy without causing phototoxic injury to the normal brain tissue. METHODS: We used a platform of Glioma Initiation Cells (GICs) infiltrating cerebral organoids with two different glioblastoma cells, GIC7 and PG88. We measured GICs-5-ALA uptake and PDT/5-ALA activity in dose-response curves and the efficacy of the treatment by measuring proliferative activity and apoptosis. RESULTS: 5-ALA (50 and 100 µg/mL) was applied, and the release of protoporphyrin IX (PpIX) fluorescence measures demonstrated that the emission of PpIX increases progressively until its stabilization at 24 h. Moreover, decreased proliferation and increased apoptosis corroborated the effect of 5-ALA/PDT on cancer cells without altering normal cells. CONCLUSIONS: We provide evidence about the effectiveness of PDT to treat high proliferative GB cells in a complex in vitro system, which combines normal and cancer cells and is a useful tool to standardize new strategic therapies.

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Cervical cancer is one of the most common malignant diseases in women worldwide. Despite the global introduction of a preventive vaccine against the leading cause of cervical cancer, human papillomavirus (HPV) infection, the incidence of this malignant disease is still very high, especially in economically challenged areas. New advances in cancer therapy, especially the rapid development and application of different immunotherapy strategies, have shown promising pre-clinical and clinical results. However, mortality from advanced stages of cervical cancer remains a significant concern. Precise and thorough evaluation of potential novel anti-cancer therapies in pre-clinical phases is indispensable for efficient development of new, more successful treatment options for cancer patients. Recently, 3D tumor models have become the gold standard in pre-clinical cancer research due to their capacity to better mimic the architecture and microenvironment of tumor tissue as compared to standard two-dimensional (2D) cell cultures. This review will focus on the application of spheroids and patient-derived organoids (PDOs) as tumor models to develop novel therapies against cervical cancer, with an emphasis on the immunotherapies that specifically target cancer cells and modulate the tumor microenvironment (TME).

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Glioblastoma is a rapidly progressing tumor quite resistant to conventional treatment. These features are currently assigned to a self-sustaining population of glioblastoma stem cells. Anti-tumor stem cell therapy calls for a new means of treatment. In particular, microRNA-based treatment is a solution, which in turn requires specific carriers for intracellular delivery of functional oligonucleotides. Herein, we report a preclinical in vitro validation of antitumor activity of nanoformulations containing antitumor microRNA miR-34a and microRNA-21 synthetic inhibitor and polycationic phosphorus and carbosilane dendrimers. The testing was carried out in a panel of glioblastoma and glioma cell lines, glioblastoma stem-like cells and induced pluripotent stem cells. We have shown dendrimer-microRNA nanoformulations to induce cell death in a controllable manner, with cytotoxic effects being more pronounced in tumor cells than in non-tumor stem cells. Furthermore, nanoformulations affected the expression of proteins responsible for interactions between the tumor and its immune microenvironment: surface markers (PD-L1, TIM3, CD47) and IL-10. Our findings evidence the potential of dendrimer-based therapeutic constructions for the anti-tumor stem cell therapy worth further investigation.

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Breast cancer is still the leading cause of women's death due to relapse and metastasis. In vitro tumor models are considered reliable tools for drug screening and understanding cancer-driving mechanisms due to the possibility of mimicking tumor heterogeneity. Herein, a 3D breast cancer model (3D-BCM) is developed based on enzymatically-crosslinked silk fibroin (eSF) hydrogels. Human MCF7 breast cancer cells are encapsulated into eSF hydrogels, with and without human mammary fibroblasts. The spontaneously occurring conformational change from random coil to ß-sheet is correlated with increased eSF hydrogels' stiffness over time. Moreover, mechanical properties analysis confirms that the cells can modify the stiffness of the hydrogels, mimicking the microenvironment stiffening occurring in vivo. Fibroblasts support cancer cells growth and assembly in the eSF hydrogels up to 14 days of culture. Co-cultured 3D-BCM exhibits an upregulated expression of genes related to extracellular matrix remodeling and fibroblast activation. The 3D-BCM is subjected to doxorubicin and paclitaxel treatments, showing differential drug response. Overall, these results suggest that the co-culture of breast cancer cells and fibroblasts in eSF hydrogels allow the development of a mimetic in vitro platform to study cancer progression. This opens up new research avenues to investigate novel molecular targets for anti-cancer therapy.

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The stiffness of tumors and their host tissues is much higher than most hydrogels, which are conventionally used to study in vitro cancer progression. The tumoroid assay is an engineered 3D in vitro tumor model that allows investigation of cancer cell invasion in an environment that is biomimetic in terms of extracellular matrix (ECM) composition and stiffness. Using this model, the change in matrix stiffness by epithelial colorectal cancer cells is systematically characterized by atomic force microscopy indentation tests. Less invasive epithelial cancer cells stiffen the tumor microenvironment while highly aggressive epithelial cancer cells show significant softening of the tumor microenvironment. Changes in stiffness are attributed to both cell-generated active forces as well as ECM degradation and remodeling. The degradation is in part attributed to the enzymatic activity of matrix metalloproteinases (MMPs) as demonstrated by the significant expression of MMP-2 and MMP-9 at both gene and protein levels. Targeting MMP activity through broad-spectrum drug inhibition (BB-94) reverses the changes in stiffness and also decreases cancer cell invasion. These results promote the idea of using mechano-based cancer therapies such as MMP inhibition.

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Patient-derived organoids are promising tumor models for functional validation of next-generation sequencing-based therapy recommendations. In times of rapidly advancing precision oncology approaches in everyday clinical processes, reliable and valid tumor models are required. Tumor organoids consist of tumor "stem" cells, differentiated (epithelial) tumor, and stroma cells. The cellular architecture and interactions closely mimic the original patient tumor. These organoids can be implanted into immunodeficient mice, generating patient-derived organoid-derived xenografts, thus enabling in vitro to in vivo transfer. Most importantly, the high clinical relevance of PDO models is maintained in this conversion. This protocol describes in detail the methods and techniques as well as the materials necessary to generate in vitro PDO and in vivo PDO-derived xenograft models. The elaborate process description starts with the processing of freshly obtained tumor tissue. The proceedings include tissue processing, organoid culturing, PDO implantation into immunodeficient mice, tumor explantation, and finally tumor preservation. All these proceedings are described in this timely chronological order. This protocol will enable researchers to generate PDO models from freshly received tumor tissue and generate PDO-derived xenografts. Models generated according to these methods are suitable for a very broad research spectrum.

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Immunotherapy has revolutionized cancer treatment with the advent of advanced cell engineering techniques aimed at targeted therapy with reduced systemic toxicity. However, understanding the underlying immune-cancer interactions require development of advanced three-dimensional (3D) models of human tissues. In this study, we fabricated 3D tumor models with increasing complexity to study the cytotoxic responses of CD8+T cells, genetically engineered to express mucosal-associated invariant T (MAIT) cell receptors, towards MDA-MB-231 breast cancer cells. Homotypic MDA-MB-231 and heterotypic MDA-MB-231/human dermal fibroblast tumor spheroids were primed with precursor MAIT cell ligand 5-amino-6-D-ribitylaminouracil (5-ARU). Engineered T cells effectively eliminated tumors after a 3 d culture period, demonstrating that the engineered T cell receptor recognized major histocompatibility complex class I-related (MR1) protein expressing tumor cells in the presence of 5-ARU. Tumor cell killing efficiency of engineered T cells were also assessed by encapsulating these cells in fibrin, mimicking a tumor extracellular matrix microenvironment. Expression of proinflammatory cytokines such as interferon gamma, interleukin-13, CCL-3 indicated immune cell activation in all tumor models, post immunotherapy. Further, in corroborating the cytotoxic activity, we found that granzymes A and B were also upregulated, in homotypic as well as heterotypic tumors. Finally, a 3D bioprinted tumor model was employed to study the effect of localization of T cells with respect to tumors. T cells bioprinted proximal to the tumor had reduced invasion index and increased cytokine secretion, which indicated a paracrine mode of immune-cancer interaction. Development of 3D tumor-T cell platforms may enable studying the complex immune-cancer interactions and engineering MAIT cells for cell-based cancer immunotherapies.

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Cancer is a complex disease arising from a homeostatic imbalance of cell-intrinsic and microenvironment-related mechanisms. A multimodal approach to treat cancer that includes surgery, chemotherapy, and radiation therapy often fails in achieving tumor remission and produces unbearable side effects including secondary malignancies. Novel strategies have been implemented in the past decades in order to replace conventional chemotherapeutics with targeted, less toxic drugs. Up to now, scientists have relied on results achieved in animal research before proceeding to clinical trials. However, the high failure rate of targeted drugs in early phase trials leaves no doubt about the inadequacy of those models. In compliance with the need of reducing, and possibly replacing, animal research, studies have been conducted in vitro with advanced cellular models that more and more mimic the tumor in vivo. We will here review those methods that allow for the 3D reconstitution of the tumor and its microenvironment and the implementation of the organ-on-a-chip technology to study minimal organ units in disease progression. We will make specific reference to the usability of these systems as predictive cancer models and report on recent applications in high-throughput screenings of innovative and targeted drug compounds.

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A tumor microenvironment is characterized by its altered mechanical properties. However, most models remain unable to faithfully recreate the mechanical properties of a tumor. Engineered models based on the self-assembly method have the potential to better recapitulate the stroma architecture and composition. Here, we used the self-assembly method based on a bladder tissue model to engineer a tumor-like environment. The tissue-engineered tumor models were reconstituted from stroma-derived healthy primary fibroblasts (HFs) induced into cancer-associated fibroblast cells (iCAFs) along with an urothelium overlay. The iCAFs-derived extracellular matrix (ECM) composition was found to be stiffer, with increased ECM deposition and remodeling. The urothelial cells overlaid on the iCAFs-derived ECM were more contractile, as measured by quantitative polarization microscopy, and displayed increased YAP nuclear translocation. We further showed that the proliferation and expression of epithelial-to-mesenchymal transition (EMT) marker in the urothelial cells correlate with the increased stiffness of the iCAFs-derived ECM. Our data showed an increased expression of EMT markers within the urothelium on the iCAFs-derived ECM. Together, our results demonstrate that our tissue-engineered tumor model can achieve stiffness levels comparable to that of a bladder tumor, while triggering a tumor-like response from the urothelium.

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The tumor microenvironment is highly complex owing to its heterogeneous composition and dynamic nature. This makes tumors difficult to replicate using traditional 2D cell culture models that are frequently used for studying tumor biology and drug screening. This often leads to poor translation of results between in vitro and in vivo and is reflected in the extremely low success rates of new candidate drugs delivered to the clinic. Therefore, there has been intense interest in developing 3D tumor models in the laboratory that are representative of the in vivo tumor microenvironment and patient samples. 3D bioprinting is an emerging technology that enables the biofabrication of structures with the virtue of providing accurate control over distribution of cells, biological molecules, and matrix scaffolding. This technology has the potential to bridge the gap between in vitro and in vivo by closely recapitulating the tumor microenvironment. Here, a brief overview of the tumor microenvironment is provided and key considerations in biofabrication of tumor models are discussed. Bioprinting techniques and choice of bioinks for both natural and synthetic polymers are also outlined. Lastly, current bioprinted tumor models are reviewed and the perspectives of how clinical applications can greatly benefit from 3D bioprinting technologies are offered.

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Bladder cancer is a common and highly heterogeneous malignancy with a relatively poor outcome. Patient-derived tumor organoid cultures have emerged as a preclinical model with improved biomimicity. However, the impact of the different methods being used in the composition and dynamics of the models remains unknown. This study aims to systematically review the literature regarding patient-derived organoid models for normal and cancer tissue of the bladder, and their current and potential future applications for tumor biology studies and drug testing. A PRISMA-compliant systematic review of the PubMED, Embase, Web of Sciences, and Scopus databases was performed. The results were analyzed based on the methodologies, comparison with primary tumors, functional analysis, and chemotherapy and immunotherapy testing. The literature search identified 536 articles, 24 of which met the inclusion criteria. Bladder cancer organoid models have been increasingly used for tumor biology studies and drug screening. Despite the heterogeneity between methods, organoids and primary tissues showed high genetic and phenotypic concordance. Organoid sensitivity to chemotherapy matched the response in patient-derived xenograft (PDX) models and predicted response based on clinical and mutation data. Advances in bioengineering technology, such as microfluidic devices, bioprinters, and imaging, are likely to further standardize and expand the use of organoids.