Microtumor Model Research Articles

Tumor-Infiltration Mimicking Model of Contaminated Ovarian Tissue as an Innovative Platform for Advanced Cancer Research.

The development of advanced preclinical models is crucial for the evaluation and validation of novel therapeutic strategies in oncology. Three-dimensional (3D) microtumor models, which incorporate both cancer and stromal cells within biomimetic hydrogels, have emerged as powerful tools that more accurately replicate the complex tumor microenvironment compared to traditional two-dimensional (2D) cell culture systems. In this context, our study aims to develop 3D microtumor models by integrating cancer and stromal cells within an extracellular-matrix-mimetic hydrogel, as a physiologically accurate microtumor model that can serve as an innovative platform for advanced cancer research and drug screening. Microtumors composed of varying ratios of leukemia cells (HL-60) to healthy ovarian stromal cells (SCs) (1:1, 1:10, 1:100, or 1:1000) were encapsulated in PEGylated fibrin hydrogel and cultured for 5days. The proliferation and dynamics of cancerous and healthy cell populations were evaluated using CD43/Ki67 immunofluorescence double staining. Our findings indicate that tumor development and malignancy progression can be influenced by adjusting cell culture ratios and incubation time. Notably, the HL-60:SCs ratio of 1:100 closely replicated leukemia cell invasion in ovarian tissue, demonstrating detectable malignancy on the third and fifth days without significant changes in total cell density dynamics. This 3D leukemia microtumor model offers superior physiological relevance compared to traditional 2D in vitro assays and shows promising potential for applications in cellular analysis and drug screening.

Protrusion force and cell-cell adhesion play a critical role in collective tumor cluster migration.

While collective migration is shown to enhance invasive and metastatic potential in cancer, the mechanisms driving this behavior and regulating tumor migration plasticity remain poorly understood. This study provides a mechanistic framework explaining the emergence of different modes of collective migration under hypoxia-induced secretome. We focus on the interplay between cellular protrusion force and cell-cell adhesion using collectively migrating three-dimensional microtumors as models with well-defined microenvironment. Large microtumors show directional migration due to intrinsic hypoxia, while small microtumors exhibit radial migration in response to hypoxic secretome. Here, we developed the minimal multi-scale microtumor model (MSMM) to elucidate underlying mechanisms. We identified distinct migration modes within specific regions of protrusion force and cell-cell adhesion parameter space. We show that sufficient cellular protrusion force is crucial for both, radial and directional collective microtumor migration. Radial migration emerges when sufficient cellular protrusion force is generated, driving neighboring cells to move collectively in diverse directions. Within migrating tumors, strong cell-cell adhesion enhances the alignment of cell polarity, breaking the symmetric angular distribution of protrusion forces, and leading to directional microtumor migration. The integrated results from the experimental and computational models provide fundamental insights into collective migration in response to different microenvironment stimuli.

A novel bioprinted microtumour model for studying acute-leukemia-cell- contaminated in ovarian tissue

Microtumor models, combining cancer and stromal cells within 3D hydrogels, are vital for testing anticancer therapies. Bioprinting hydrogel scaffolds allows tailored in vitro models. We created a 3D microtumor model using a bioprinter, with varying ratios of ovarian stromal cells and leukemia cells (HL-60). PEGylated fibrinogen and alginate hydrogel were used. Cell dynamics and proliferation were assessed via immunofluorescence staining. Microtumors with different HL-60 ratios (1:1, 1:10, 1:100) were cultured for 5 days. Results showed tumor development modulation by cell ratios and culture time. A significant cell density increase occurred in 1:1 ratio microtumors, indicating rapid cancer cell proliferation. No HL-60 cells were found in 1:100 ratio microtumors by day 5. The 1:10 ratio closely mimicked leukemia invasion in ovarian tissue, showing detectable cancer cells by days 3 and 5 without altering total cell density dynamics significantly. This bioprinted leukemia microtumor model offers better physiological relevance than 2D assays, promising applications in cellular analysis and drug screening.

Targeting tumor–stromal interactions in triple-negative breast cancer using a human vascularized micro-tumor model

Triple-negative breast cancer (TNBC) is highly aggressive with limited available treatments. Stromal cells in the tumor microenvironment (TME) are crucial in TNBC progression; however, understanding the molecular basis of stromal cell activation and tumor–stromal crosstalk in TNBC is limited. To investigate therapeutic targets in the TNBC stromal niche, we used an advanced human in vitro microphysiological system called the vascularized micro-tumor (VMT). Using single-cell RNA sequencing, we revealed that normal breast tissue stromal cells activate neoplastic signaling pathways in the TNBC TME. By comparing interactions in VMTs with clinical data, we identified therapeutic targets at the tumor–stromal interface with potential clinical significance. Combining treatments targeting Tie2 signaling with paclitaxel resulted in vessel normalization and increased efficacy of paclitaxel in the TNBC VMT. Dual inhibition of HER3 and Akt also showed efficacy against TNBC. These data demonstrate the potential of inducing a favorable TME as a targeted therapeutic approach in TNBC.

Establishment of an in Vitro Three-Dimensional Vascularized Micro-Tumor Model and Screening of Chemotherapeutic Drugs.

Breast cancer is the most common malignancy in women worldwide, and major challenges in its treatment include drug resistance and metastasis. Three-dimensional cell culture systems have received widespread attention in drug discovery studies but existing models have limitations, that warrant the development of a simple and repeatable three-dimensional culture model for high-throughput screening. In this study, we designed a simple, reproducible, and highly efficient microencapsulated device to co-culture MCF-7 cells and HUVECs in microcapsules to establish an in vitro vascularized micro-tumor model for chemotherapeutic drug screening. First, to construct a three-dimensional micro-tumor model, cell encapsulation devices were created using three different sizes of flat-mouthed needles. Immunohistochemistry and immunofluorescence assays were conducted to determine vascular lumen formation. Cell proliferation was detected using the Cell Counting Kit-8 assay. Finally, to observe the drug reactions between the models, anticancer drugs (doxorubicin or paclitaxel) were added 12 h after the two-dimensional cultured cells were plated or 7 days after cell growth in the core-shell microcapsules. Vascularized micro-tumors were obtained after 14 days of three-dimensional culture. The proliferation rate in the three-dimensional cultured cells was slower than that of two-dimensional cultured cells. Three-dimensional cultured cells were more resistant to anticancer drugs than two-dimensional cultured cells. This novel sample encapsulation device formed core-shell microcapsules and can be used to successfully construct 3D vascularized micro-tumors in vitro. The three-dimensional culture model may provide a platform for drug screening and is valuable for studying tumor development and angiogenesis.

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research.

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

Design, Synthesis, In Vitro and In Vivo Characterization of CDC42 GTPase Interaction Inhibitors for the Treatment of Cancer.

CDC42 GTPases (RHOJ, CDC42, and RHOQ) are overexpressed in multiple tumor types and activate pathways critical for tumor growth, angiogenesis, and metastasis. Recently, we reported the discovery of a novel lead compound, ARN22089, which blocks the interaction of CDC42 GTPases with specific downstream effectors. ARN22089 blocks tumor growth in BRAF mutant mouse melanoma models and patient-derived xenografts (PDXs) in vivo. ARN22089 also inhibits tumor angiogenesis in three-dimensional vascularized microtumor models in vitro. Notably, ARN22089 belongs to a novel class of trisubstituted pyrimidines. Based on these results, we describe an extensive structure-activity relationship of ∼30 compounds centered on ARN22089. We discovered and optimized two novel inhibitors (27, ARN25062, and 28, ARN24928), which are optimal back-up/follow-up leads with favorable drug-like properties and in vivo efficacy in PDX tumors. These findings further demonstrate the potential of this class of CDC42/RHOJ inhibitors for cancer treatment, with lead candidates ready for advanced preclinical studies.

A human vascularized microtumor model of patient-derived colorectal cancer recapitulates clinical disease

Accurately modeling tumor biology and testing novel therapies on patient-derived cells is critically important to developing therapeutic regimens personalized to a patient's specific disease. The vascularized microtumor (VMT), or "tumor-on-a-chip," is a physiologic preclinical cancer model that incorporates key features of the native human tumor microenvironment within a transparent microfluidic platform, allowing rapid drug screening in vitro. Herein we optimize methods for generating patient-derived VMT (pVMT) using fresh colorectal cancer (CRC) biopsies and surgical resections to test drug sensitivities at the individual patient level. In response to standard chemotherapy and TGF-βR1 inhibition, we observe heterogeneous responses between pVMT derived from 6 patient biopsies, with the pVMT recapitulating tumor growth, histological features, metabolic heterogeneity, and drug responses of actual CRC tumors. Our results suggest that a translational infrastructure providing rapid information from patient-derived tumor cells in the pVMT, as established in this study, will support efforts to improve patient outcomes.

Three-Dimensional Microtumor Formation of Infantile Hemangioma-Derived Endothelial Cells for Mechanistic Exploration and Drug Screening

Infantile hemangioma (IH) is the most prevalent type of vascular tumor in infants. The pathophysiology of IH is unknown. The tissue structure and physiology of two-dimensional cell cultures differ greatly from those in vivo, and spontaneous regression often occurs during tumor formation in nude mice and has severely limited research into the pathogenesis and development of IH. By decellularizing porcine aorta, we attempted to obtain vascular-specific extracellular matrix as the bioink for fabricating micropattern arrays of varying diameters via microcontact printing. We then constructed IH-derived CD31+ hemangioma endothelial cell three-dimensional microtumor models. The vascular-specific and decellularized extracellular matrix was suitable for the growth of infantile hemangioma-derived endothelial cells. The KEGG signaling pathway analysis revealed enrichment primarily in stem cell pluripotency, RAS, and PI3KAkt compared to the two-dimensional cell model according to RNA sequencing. Propranolol, the first-line medication for IH, was also used to test the model's applicability. We also found that metformin had some impact on the condition. The three-dimensional microtumor models of CD31+ hemangioma endothelial cells were more robust and efficient experimental models for IH mechanistic exploration and drug screening.

Establishment and large-scale validation of a three-dimensional tumor model on an array chip for anticancer drug evaluation.

Two-dimensional (2D) tumor model has always poorly predicted drug response of animal model due to the lack of recapitulation of tumor microenvironment. Establishing a biomimetic, controllable, and cost-effective three-dimensional (3D) model and large-scale validation of its in vivo predictivity has shown promise in bridging the gap between the 2D tumor model and animal model. Here, we established a matrigel-based 3D micro-tumor model on an array chip for large-scale anticancer drug evaluation. Compared with the 2D tumor model, the 3D tumor model on the chip showed spheroid morphology, slower proliferation kinetics, and comparable reproducibility. Next, the results of the chemotherapeutic evaluation from 18 drugs against 27 cancer cell lines showed 17.6% of drug resistance on the 3D tumor model. Moreover, the evaluation results of targeted drugs showed expected sensitivity and higher specificity on the 3D tumor model compared with the 2D model. Finally, the evaluation results on the 3D tumor model were more consistent with the in vivo cell-derived xenograft model, and excluded 95% false-positive results from the 2D model. Overall, the matrigel-based 3D micro-tumor model on the array chip provides a promising tool to accelerate anticancer drug discovery.

P4HA2: A link between tumor-intrinsic hypoxia, partial EMT and collective migration

Epithelial-to-mesenchymal transition (EMT), a well-established phenomenon studied across pan-cancer types, has long been known to be a major player in driving tumor invasion and metastasis. Recent studies have highlighted the importance of partial EMT phenotypes in metastasis. Initially thought as a transitional state between epithelial and mesenchymal phenotypic states, partial EMT state is now widely recognized as a key driver of intra-tumoral heterogeneity and phenotypic plasticity, further accelerating tumor metastasis and therapeutic resistance. However, how tumor microenvironment regulates partial EMT phenotypes remains unclear. We have developed unique size-controlled three-dimensional microtumor models that recapitulate tumor-intrinsic hypoxia and the emergence of collectively migrating cells. In this study, we further interrogate these microtumor models to understand how tumor-intrinsic hypoxia regulates partial EMT and collective migration in hypoxic large microtumors fabricated from T47D breast cancer cells. We compared global gene expression profiles of hypoxic, migratory microtumors to that of non-hypoxic, non-migratory microtumors at early and late time-points. Using our microtumor models, we identified unique gene signatures for tumor-intrinsic hypoxia (early versus late), partial EMT and migration (pre-migratory versus migratory phenotype). Through differential gene expression analysis between the microtumor models with an overlap of hypoxia, partial EMT and migration signatures, we identified prolyl 4-hydroxylase subunit 2 (P4HA2), a hypoxia responsive gene, as a central regulator common to hypoxia, partial EMT and collective migration. Further, the inhibition of P4HA2 significantly blocked collective migration in hypoxic microtumors. Thus, using the integrated computational-experimental analysis, we identify the key role of P4HA2 in tumor-intrinsic hypoxia-driven partial EMT and collective migration.

Abstract 177: Ex vivo 3D culture of adenoid cystic carcinoma PDX models recapitulate disease biomarkers and predict drug response

Abstract Adenoid cystic carcinoma (ACC) is a rare, glandular cancer whose incidence rate and limited 2D cell culture capability make it difficult to study primary patient samples. Although well characterized ACC patient-derived xenografts (PDX) have proven to be a useful model to study disease mechanisms and therapeutic sensitives, animal drug studies are relatively low throughput, costly, and take months to accomplish. Ex vivo 3D cell culture can provide a high-throughput, less costly, and significantly faster platform for these drug studies but are hampered by the rarity of tissue. To address this unmet need for models of rare tumor types we developed 3D spheroid (3D-XPDXs™) and microtumor (3D-XPDXmt™) models of ACC using PDX as the primary tissue source. ACC PDX cells readily formed spheroids in our 3D-XPDXs™ platform, remained viable for up to 14 days, and maintained disease-relevant biomarkers such as MYB and c-kit. 3D-XPDXmt™ represent a more complex model of ACC by incorporating extracellular matrix. ACC 3D-XPDXmt™ displayed tumor-like morphologies concordant with the parental tumors and exhibited MYB and c-kit biomarker expression for up to 56 days in culture. Drug response profiling (DRP) was performed using the 3D-XPDXs™ ACC model with KIYATEC’s validated KIYA-PREDICT™ DRP platform. The screening panel consisted of drugs and drug-like compounds currently in use or under investigation for use in ACC, including broad-spectrum chemotherapies and targeted agents. Individualized drug responses were noted for each model as they exhibited differential sensitivities to DNA-damaging agents, microtubule stabilizers, and c-kit targeting kinase inhibitors mirroring the diversity of clinical outcomes. KIYA-PREDICT™ also identified drugs and drug-like compounds that uniformly inhibited viability. This is significant because there is currently no standard of care drugs for ACC, as few, if any have demonstrated homogenous responses in test populations. Monensin has been shown to inhibit activity of the MYB transcription factor, making it an attractive candidate drug for the treatment of ACCs which have a high prevalence of MYB activating alterations. In our study, monensin was effective in all three models with IC50 concentrations in the low micromolar range. These results correlate well with immunohistochemical staining of MYB in ACC 3D-XPDXs™ and 3D-XPDXmt™. Taken together, this data represents a new ex vivo 3D cell culture platform for the study of ACC biology and potential therapies. Citation Format: Melissa Millard, Teresa M. DesRochers, Michael J. Wick, Jeffrey Kaufman, Nicole Spardy Burr. Ex vivo 3D culture of adenoid cystic carcinoma PDX models recapitulate disease biomarkers and predict drug response [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 177.

Structure-based design of CDC42 effector interaction inhibitors for the treatment of cancer.

SUMMARYCDC42 family GTPases (RHOJ, RHOQ, CDC42) are upregulated but rarely mutated in cancer and control both the ability of tumor cells to invade surrounding tissues and the ability of endothelial cells to vascularize tumors. Here, we use computer-aided drug design to discover a chemical entity (ARN22089) that has broad activity against a panel of cancer cell lines, inhibits S6 phosphorylation and MAPK activation, activates pro-inflammatory and apoptotic signaling, and blocks tumor growth and angiogenesis in 3D vascularized microtumor models (VMT) in vitro. Additionally, ARN22089 has a favorable pharmacokinetic profile and can inhibit the growth of BRAF mutant mouse melanomas and patient-derived xenografts in vivo. ARN22089 selectively blocks CDC42 effector interactions without affecting the binding between closely related GTPases and their downstream effectors. Taken together, we identify a class of therapeutic agents that influence tumor growth by modulating CDC42 signaling in both the tumor cell and its microenvironment.

High-Throughput Examination of Therapy-Induced Alterations in Redox Metabolism in Spheroid and Microtumor Models.

The capacity of cancer cells to adjust their metabolism to thrive in new environments and in response to treatments has been implicated in the acquisition of treatment resistance. To optimize therapeutic strategies such as photodynamic therapy (PDT)-based combination treatments, methods to characterize the plasticity of cancer metabolism in response to treatments are required. This protocol provides a method for high-throughput and label-free tracking of metabolic redox states in cancer tissues, leveraging the autofluorescent properties of nicotinamide dinucleotide (NAD(P)H) and oxidized flavoprotein adenine dinucleotide (FAD). The methodology is optimized to be applied to 3D spheroid/microtumor/organoid cultures, regardless of the culture type (e.g., adherent or suspension cultures) and morphology. The exploitation of these methods may elucidate mechanisms of metabolic adaptation and perturbations in redox homeostasis, and chart the overall tumor health in both 3D culture models and ex vivo tissues following cancer therapies, such as PDT.

Microtumor Models as a Preclinical Investigational Platform for Photodynamic Therapy.

Classic preclinical investigations on the mechanisms and effects of photodynamic therapy (PDT) are typically performed in two-dimensional cell cultures that have some, albeit limited, relevance to cancer biology. Bioengineered three-dimensional (3D) culture models of cancer are gaining traction in translational oncology as microtumors recapitulate the tumor architectures and cellular heterogeneity more faithfully than conventional 2D cultures. These 3D models bridge a gap between highly relevant but low-throughput in vivo animal models and high-throughput two-dimensional cultures with low clinical relevance, and thus hold promise as preclinical testing platforms in PDT research. Here, we discuss the potential applications of organotypic cancer models for PDT research and provide two well-established methodologies for generating 3D cultures of cancer: a liquid-suspended spheroid model and an adherent microtumor culture model grown on extracellular matrix scaffolds. Particular emphasis is given to harvesting the cultures for the purpose of immunoblotting and flow cytometry.

Analysis of Treatment Effects on Structurally Complex Microtumor Cultures Using a Comprehensive Image Analysis Procedure.

As three-dimensional (3D) culture models are attractive platforms to assess treatment response and expedite the development of new therapeutic regimens, appropriate methodologies to extract quantitative data from these models are required. Here, we present a live/dead staining protocol together with a recently developed analysis methodology for the multiparametric assessment of treatment effects on 3D culture models (CALYPSO: Comprehensive image AnaLYsis Procedure for Structurally complex Organoids). This methodology can process up to thousands of individual organoids within a single experiment and provides multiple informative readouts for each individual microtumor. Moreover, this protocol utilizes conventional fluorescence microscopy and commercially available dyes, allowing it to be easily implemented in most laboratories. Taken together, the methodology presented here encourages the use of microtumor models by enabling the high-throughput assessment of treatment effects, regardless of 3D culture type or microtumor architectures.

Generating Large Numbers of Pancreatic Microtumors on Alginate-Gelatin Hydrogels for Quantitative Imaging of Tumor Growth and Photodynamic Therapy Optimization.

The emerging use of 3D culture models of cancer has provided novel insights into the therapeutic mechanisms of photodynamic therapy on a mesoscopic scale. Especially microscale tumors grown on scaffolds of extracellular matrix can provide statistically robust data on the effects of photosensitizers and photodynamic therapy by leveraging high-throughput imaging-based assays. Although highly informative, the use of such 3D cultures can be impractical due to the high costs and inter-batch variability of the extracellular matrix scaffolds that are necessary to establish such cultures. In this study, we therefore provide a protocol to generate inexpensive and defined hydrogels composed of sodium alginate and gelatin that can be used for culturing 3D microtumors in a manner that is compatible with state-of-the-art imaging assays. Our results reveal that the alginate-gelatin hydrogels can perform similarly to a commercially available ECM scaffold in terms of facilitating microtumor growth. We then applied these microtumor models to quantify the uptake and dark toxicity of benzoporphyrin derivative encapsulated in liposomes with either an anionic or a cationic surface charge. The results indicate that cationic liposomes achieve the highest level of uptake in the microtumors, yet also exert minor toxicity. Moreover, we reveal that there is typically a significant positive correlation between microtumor size and liposome uptake. In conclusion, alginate-based hydrogels are inexpensive and effective scaffolds for 3D culture models of cancer, with versatile applications in research toward photodynamic therapy.

An in vitro vascularized micro-tumor model of ovarian cancer

<h3>Objectives:</h3> Establish and validate a Vascularized Micro-Tumor model of ovarian cancer (ovarian-VMTm) using established cell lines, SKOV3, COV362.4, and A2780. <h3>Methods:</h3> VMTm is an <i>in vitro</i> vascularized microtissue platform in which a tissue is maintained in a 3D-extracellular matrix and depends on survival with nutrient delivery through living, perfused micro-vessels composed of human endothelial and stromal cells. Nutrients and drugs are delivered through micro-vessels in a manner that simulates their delivery in the human body. This addresses the shortcomings of current 2D models that do not accurately represent actual tumor progression by supporting the growth of tumors and vessels in the 3D-extracellular matrix. We have tested the cell lines with the standard doublet chemotherapy used for the treatment of ovarian cancer, carboplatin and paclitaxel. <h3>Results:</h3> Drug response for all three cell lines has been evaluated in 2D to establish a baseline resulted as a half-maximal-inhibitory-concentration (IC50). The three cell lines consistently showed lower IC50 to paclitaxel, with A2780 at lowest IC50 at 1.5 indicating its responsiveness to paclitaxel. Optimal culture conditions and seeding conditions have been identified to establish two of the ovarian cancer cell lines, SKOV3 and COV362.4, in the ovarian-VMTm to examine tumor growth in the presence of a vascular network (Figure 1). <h3>Conclusions:</h3> Ovarian cancer cell lines have been established within the ovarian-VMTm to begin to assess tumor and angiogenic response to chemotherapy. Future plans are to compare drug IC50 results from 2D to those in the ovarian-VMTm and ultimately optimize the ovarian-VMTm for patient-derived ovarian cancer cell lines. Thus, allowing for the evaluation of patient-specific, tumor response to chemotherapy, as well as targeted agents. The ovarian-VMTm more accurately recapitulates tumor growth <i>in vivo</i> and can be an instrumental tool in the drug development process.

Tumor spheroid-based microtumor models for preclinical evaluation of anticancer nanomedicines

Tumor spheroid-based microtumor models for preclinical evaluation of anticancer nanomedicines

Elaiophylin Is a Potent Hsp90/ Cdc37 Protein Interface Inhibitor with K-Ras Nanocluster Selectivity

The natural product elaiophylin is a macrodiolide with a broad range of biological activities. However, no direct target of elaiophylin in eukaryotes has been described so far, which hinders a systematic explanation of its astonishing activity range. We recently showed that the related conglobatin A, a protein–protein interface inhibitor of the interaction between the N-terminus of Hsp90 and its cochaperone Cdc37, blocks cancer stem cell properties by selectively inhibiting K-Ras4B but not H-Ras. Here, we elaborated that elaiophylin likewise disrupts the Hsp90/ Cdc37 interaction, without affecting the ATP-pocket of Hsp90. Similarly to conglobatin A, elaiophylin decreased expression levels of the Hsp90 client HIF1α, a transcription factor with various downstream targets, including galectin-3. Galectin-3 is a nanocluster scaffold of K-Ras, which explains the K-Ras selectivity of Hsp90 inhibitors. In agreement with this K-Ras targeting and the potent effect on other Hsp90 clients, we observed with elaiophylin treatment a submicromolar IC50 for MDA-MB-231 and MIA-PaCa-2 3D spheroid formation. Finally, a strong inhibition of MDA-MB-231 cells grown in the chorioallantoic membrane (CAM) microtumor model was determined. These results suggest that several other macrodiolides may have the Hsp90/ Cdc37 interface as a target site.