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Most human cells require anchorage for survival. Cell-substrate adhesion activates diverse signaling pathways, without which cells undergo anoikis – a form of programmed cell death1. Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cancer cells often lose firm attachment to surrounding tissue2–5. In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs6–13. Bleb function has been thoroughly investigated in the context of amoeboid migration but is far less examined in other scenarios14–19. Here we show by quantitative subcellular 3D imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signaling hubs that confer anoikis resistance. Specifically, we discovered in melanoma cells that blebbing generates plasma membrane contours, which recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signaling hubs activate ERK and PI3K – canonical promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils an unanticipated morphological requirement for mutant NRAS to operate as an effective oncoprotein. Moreover, we find that though some BRAF mutant melanoma do not rely on this survival pathway in a basal state, BRAF/MEK inhibition strongly sensitizes them to both bleb and septin inhibition. Importantly, we demonstrate that fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harboring oncogenic mutations. These data define a role for blebs as potent signaling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance.

The heterogeneity of cancer necessitates developing a multitude of targeted therapies. We propose the view that cancer drug discovery is a low rank tensor completion problem. We implement this vision by using heterogeneous public data to construct a tensor of drug-target-disease associations. We show the validity of this approach computationally by simulations, and experimentally by testing drug candidates. Specifically, we show that a novel drug candidate, SU11652, controls melanoma tumor growth, including BRAFWT melanoma. Independently, we show that another molecule, TC-E 5008, controls tumor proliferation on ex vivo ER+ human breast cancer. Most importantly, we identify these chemicals with only a few computationally selected experiments as opposed to brute-force screens. The efficiency of our approach enables use of ex vivo human tumor assays as a primary screening tool. We provide a web server, the Cancer Vulnerability Explorer (accessible at https://cavu.biohpc.swmed.edu), to facilitate the use of our methodology.

Researchers often aim to incorporate microenvironmental variables such as the dimensionality and composition of the extracellular matrix into their cell-based assays. A technical challenge created by introduction of these variables is quantification of single-cell measurements and control of environmental reproducibility. Here, we detail a methodology to quantify viability and proliferation of melanoma cells in 3D collagen-based culture platforms by automated microscopy and 3D image analysis to yield robust, high-throughput results of single-cell responses to drug treatment.

We introduce a cost-effective and easily implemented scan unit which enables any camera-based microscope to perform projection imaging from diverse viewing angles. We demonstrate this capability on Lattice Light-Sheet and Oblique Plane Microscopy by rapidly delivering projection images with an uncompromised lateral resolution and high optical contrast. By imaging the sample from one or multiple perspectives, our method enables visualization of rapid biological processes, real time stereoscopic imaging as well as three-dimensional particle localization throughout a cellular volume from just two images. Furthermore, because our projection imaging technique provides intuitive three-dimensional renderings in real-time, it improves microscope usability, allows users to more-readily optimize instrument performance and identify biological phenomena of interest on-the-fly, while also reducing data overhead by a factor of >100. We leverage our rapid projection method to image cancer cell morpho-dynamics and calcium signaling in cultured neurons, to perform three-dimensional localization of genetically encoded nanoparticles, as well as to image orthogonal views of an embryonic Zebrafish heart simultaneously.

Actin assembly supplies the structural framework for cell morphology and migration. Beyond structure, this actin framework can also be engaged to drive biochemical signaling programs. Here, we describe how the hyperactivation of Rac1 via the P29S mutation (Rac1(P29S)) in melanoma hijacks branched actin network assembly to coordinate proliferative cues that facilitate metastasis and drug resistance. Upon growth challenge, Rac1(P29S)-harboring melanoma cells massively upregulate lamellipodia formation by dendritic actin polymerization. These extended lamellipodia form a signaling microdomain that sequesters and phospho-inactivates the tumor suppressor NF2/Merlin, driving Rac1(P29S) cell proliferation in growth suppressive conditions. These biochemically active lamellipodia require cell-substrate attachment but not focal adhesion assembly and drive proliferation independently of the ERK/MAPK pathway. These data suggest a critical link between cell morphology and cell signaling and reconcile the dichotomy of Rac1’s regulation of both proliferation and actin assembly by revealing a mutual signaling axis wherein actin assembly drives proliferation in melanoma.

Resistance to targeted therapies remains an obstacle in curing cancers. A reason for this is testing of therapeutic agents in 2D culture conditions that do not recapitulate growth conditions of tumors in vivo. A critical component for defining the cell survival in vivo is the extracellular matrix (ECM). The ECM not only modulates cell-matrix and cell-cell interactions, but also determines drug accessibility to the tumor tissue. Many labs thus evaluate drug perturbations in 3D cell culture platforms using Matrigel and collagen scaffolds. A caveat with these models is the simplistic use of only basement membrane proteins or individual ECM proteins that, again, do not efficiently mimic the tumor microenvironment (TME). Although in vivo mouse models can be a better alternative, the time to conduct experiments, the associated cost, and inability to recapitulate patient TME remain a crucial challenge. Thus, we aim to regenerate an in vitro melanoma TME by engineering a melanoma extracellular matrix. We aim to do this by first identifying the patient melanoma ECM. On conducting proteomics for ECM proteins on patient tissues we identified collagen VI to be the most dominant ECM protein. However, prior work from other labs reports collagen I as the most abundant protein. This information was used to construct a platform through which we can recapitulate the melanoma ECM. We did this by generating cell-derived matrices (CDM) using human foreskin fibroblasts. On comparing the CDM from HFF1 to patient ECM, we could detect ~80 of ~120 core ECM proteins. Upon performing proteomic studies on CDMs, we again identified collagen VI as the dominant ECM protein. We also tested CDM deposition with cancer-associated fibroblasts, yielding similar results. This brought us to the question of whether collagen VI plays a role in drug resistance in melanoma. To address the role of cell-derived matrices and collagen VI, we created collagen VI KO fibroblasts through CRISPR/Cas9, which were then used to generate these matrices and will be used to evaluate its effect on drug treatments. While building this in vitro melanoma TME, we understood the need for a 3D assay to have a robust readout on effect of drugs in 3D compared to 2D. Although many viability and proliferation assays are used by various labs, these assays do not look at the effect of treatments at a single-cell resolution and neither distinguishes if the drug is cytostatic or cytotoxic. Towards that goal, we established a 3D assay by culturing melanoma cells in collagen and tested for Raf-Mek combination therapies. We made comparisons of cells either cultured in 3D as single cells or spheroids. Imaging of these platforms was performed on epifluorescence and light-sheet microscopes. We tested the platform for both viability and proliferation and the images were analyzed through individual Z planes of the 3D image. While we observed differences in viability and proliferation in 2D vs. 3D upon drug treatments, there was no difference in 3D single cells vs. spheroids. Citation Format: Vasanth Siruvallur Murali, Justin Cillay, Erik Welf, Gaudenz Danuser, Murat Can Cobanoglu. Engineering a 3D melanoma microenvironment and identifying novel therapeutic targets [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A27.

Cancer cells use a variety of mechanisms to subvert growth regulation and overcome environmental challenges. Often, these same mechanisms enable cancer cells to also develop resistance to targeted therapies. Here, we describe how a hyperactivating mutation of the Rac1 GTPase (Rac1P29S) harnesses Rac1’s role as a regulator of actin polymer assembly to sustain cell cycle progression in growth limiting conditions. This proliferative advantage supports metastatic colonization of melanoma cells and confers insensitivity to inhibitors of the mitogen-activated protein kinase (MAPK) pathway, a frequent target for melanoma treatment. Rac1P29S bypasses the MAPK axis through a mechanism that necessitates cell-matrix attachment, however, does not depend on integrin-mediated focal adhesion assembly and focal adhesion kinase signaling. Even without involvement of canonical adhesion signaling, cells carrying the Rac1P29S mutation show elevated traction upon drug treatment and require mechanical resistance from their surrounding matrix to gain a proliferative advantage. We describe an alternative arm for cell mechanosensing, whereby actin polymerization against a matrix of minimal rigidity organizes biochemical cues to drive proliferative signals. Hyperactivation of Rac1 by the P29S mutation channels this pathway in melanoma through Arp 2/3-dependent formation of a constrained actin brush network that results in the inactivation of tumor suppressor NF2/Merlin. These data suggest an alternative mechanism for mechanosensitive growth regulation that can be hijacked by cancer cells to circumvent the adverse conditions of foreign microenvironments or drug treatment.

BackgroundEvery biological experiment requires a choice of throughput balanced against physiological relevance. Most primary drugs screens neglect critical parameters such as microenvironmental conditions, cell-cell heterogeneity, and specific readouts of cell fate for the sake of throughput.MethodsHere we describe a methodology to quantify proliferation and viability of single cells in 3D culture conditions by leveraging automated microscopy and image analysis to facilitate reliable and high-throughput measurements. We detail experimental conditions that can be adjusted to increase either throughput or robustness of the assay, and we provide a stand alone image analysis program for users who wish to implement this 3D drug screening assay in high throughput.ResultsWe demonstrate this approach by evaluating a combination of RAF and MEK inhibitors on melanoma cells, showing that cells cultured in 3D collagen-based matrices are more sensitive than cells grown in 2D culture, and that cell proliferation is much more sensitive than cell viability. We also find that cells grown in 3D cultured spheroids exhibit equivalent sensitivity to single cells grown in 3D collagen, suggesting that for the case of melanoma, a 3D single cell model may be equally effective for drug identification as 3D spheroids models. The single cell resolution of this approach enables stratification of heterogeneous populations of cells into differentially responsive subtypes upon drug treatment, which we demonstrate by determining the effect of RAK/MEK inhibition on melanoma cells co-cultured with fibroblasts. Furthermore, we show that spheroids grown from single cells exhibit dramatic heterogeneity to drug response, suggesting that heritable drug resistance can arise stochastically in single cells but be retained by subsequent generations.ConclusionIn summary, image-based analysis renders cell fate detection robust, sensitive, and high-throughput, enabling cell fate evaluation of single cells in more complex microenvironmental conditions.

Polyethylene glycol (PEG) and related polymers are often used in the functionalization of carbon nanomaterials in procedures that involve sonication. However, PEG is very sensitive to sonolytic degradation and PEG degradation products can be toxic to mammalian cells. Thus, it is imperative to assess potential PEG degradation to ensure that the final material does not contain undocumented contaminants that can introduce artifacts into experimental results. Described here is a simple and inexpensive polyacrylamide gel electrophoresis method to detect the sonolytic degradation of PEG. The method was used to monitor the integrity of PEG phospholipid constructs and branched chain PEGs after different sonication times. This approach not only helps detect degraded PEG, but should also facilitate rapid screening of sonication parameters to find optimal conditions that minimize PEG damage.

Polyethylene glycol (PEG) and related polymers are often used in the solubilization and noncovalent functionalization of carbon nanomaterials by sonication. For example, carbon nanotubes are frequently sonicated with PEG-containing surfactants of the Pluronic® series or phospholipid-PEG polymers to noncovalently functionalize the nanotubes. However, PEG is very sensitive to degradation upon sonication and the degradation products can be toxic to mammalian cells and to organisms such as zebrafish embryos. It is therefore useful to have a simple and inexpensive method to determine the extent of potential PEG sonolysis, as described in this chapter. Intact PEG polymers and degraded fragments are resolved on sodium dodecyl sulfate polyacrylamide gels by electrophoresis and visualized by staining with barium iodine (BaI2). Digitized images of gels are acquired using a flatbed photo scanner and the intensities of BaI2-stained PEG bands are quantified using ImageJ software. Degradation of PEG polymers after sonication is readily detected by the reduction of band intensities in gels compared to those of non-sonicated, intact PEG polymers. In addition, the approach can be used to rapidly screen various sonication conditions to identify those that might minimize PEG degradation to acceptable levels.

Single-walled carbon nanotubes (SWNTs) are used in the near infrared (NIR)-mediated thermal ablation of tumor cells because they efficiently convert absorbed NIR light into heat. Despite the therapeutic potential of SWNTs, there have been no published studies that directly quantify how many SWNTs need be associated with a cell to achieve a desired efficiency of killing, or what is the most efficient subcellular location of SWNTs for killing cells. Herein we measured dose response curves for the efficiency of killing correlated to the measured amounts of folate-targeted SWNTs that were either on the surface or within the vacuolar compartment of normal rat kidney (NRK) cells. Folate-targeted SWNTs on the cell surface were measured after different concentrations of SWNTs in medium were incubated with cells for 30 min at 4 °C. Folate-targeted SWNTs within the vacuolar compartments were measured after cells were incubated with different concentrations of SWNTs in medium for 6 h at 37 °C. It was observed that a SWNT load of ~13 pg/cell when internalized was sufficient to kill 90% of the cells under standard conditions of NIR light irradiation. When ~3.5 pg/cell of SWNTs were internalized within the endosomal/lysosomal compartments, ~50% of the cells were killed, but when ~3.5 pg/cell of SWNTs were confined to the cell surface only ~5% of the cells were killed under the same NIR irradiation condition. The SWNT subcellular locations were verified using Raman imaging of SWNTs merged with fluorescence images of known subcellular markers. To our knowledge, this is the first time that SWNT amounts at known subcellular locations have been correlated with a dose-normalized efficacy of thermal ablation and the results support the idea that SWNTs confined to the plasma membrane are not as effective in NIR-mediated cell killing as an equivalent amount of SWNTs when internalized within the endosomal/lysosomal vesicles.

Background and Objective: Single-walled carbon nanotubes (SWNTs) are readily taken up by cells, strongly absorb in the NIR (700-1100 nm) region, and efficiently convert absorbed NIR into heat. These properties have made SWNTs an interesting facilitator for the NIR mediated thermal ablation of cancer cells. In the application of SWNTs to thermal ablation, it is not known what amount of cell-associated SWNTs is required to achieve a defined level of cell killing upon NIR irradiation. In addition, it has not been quantitatively demonstrated whether SWNTs bound to the plasma membrane or internalized into vesicles are more efficient in killing cells. The overall objective of this project is to assess NIR mediated killing as a function of cell-associated SWNT amount and SWNT subcellular location. Methods: The study was performed in three phases. The first phase involved non-covalent functionalization of SWNTs by bovine serum albumin. Non-targeted uptake and NIR irradiation of cells was performed to find the optimal laser power density, NIR exposure time and SWNT concentration to kill cells. SWNTs associated with cells were then extracted and quantified by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique (Wang et al., 2009, Anal. Chem. 81:2944-2952) and the SWNT amount was correlated with cell killing. The second phase included the non-covalent functionalization of SWNTs with phospholipid-PEG conjugated folic acid (PL-PEG-FA) as a targeting ligand. The SWNTs were internalized by receptor-mediated endocytosis at 37°C, the optimum laser conditions were determined as in the first phase, and the amount of internalized SWNTs were quantified and correlated with NIR-mediated cell killing. In the third phase, cells were incubated with the PL-PEG-FA SWNTs at 4°C to allow binding to the cell surface without internalization, followed by measuring the amount of surface-bound SWNTs. The amount of surface-bound SWNTs was then correlated with NIR-mediated cell death. Results: An increase in killing of cells incubated with SWNTs was observed in both the non-targeted and targeted systems as a function of NIR laser power density, NIR exposure time and incubated SWNT concentration. With the folate targeted system, there was more cell death when SWNTs were internalized into vesicles compared to when an equivalent amount of SWNTs were confined to the cell surface. Conclusion: SWNTs internalized by cells, either by fluid phase endocytosis, or by receptor-mediated endocytosis via the folate receptor, were effective in killing cells upon NIR irradiation. However, SWNTs confined to the cell surface after binding to folate receptors at low temperature were not as efficient in killing cells upon NIR irradiation. This suggests that NIR-mediated thermal ablation therapy will be more effective when SWNTs are targeted to a receptor and are internalized by receptor-mediated endocytosis. Citation Format: Vasanth Siruvallur Murali, Carole Mikoryak, Ruhung Wang, Rockford K. Draper. Effect of carbon nanotube amount and subcellular location on the near infrared (NIR) photothermal ablation of cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5374. doi:10.1158/1538-7445.AM2014-5374