Investigation of the enhanced antitumour potency of STING agonist after conjugation to polymer nanoparticles.

Intravenously administered cyclic dinucleotides and other STING agonists are hampered by low cellular uptake and poor circulatory half-life. Here we report the covalent conjugation of cyclic dinucleotides to poly(ß-amino ester) nanoparticles through a cathepsin-sensitive linker. This is shown to increase stability and loading, thereby expanding the therapeutic window in multiple syngeneic tumour models, enabling the study of how the long-term fate of the nanoparticles affects the immune response. In a melanoma mouse model, primary tumour clearance depends on the STING signalling by host cells-rather than cancer cells-and immune memory depends on the spleen. The cancer cells act as a depot for the nanoparticles, releasing them over time to activate nearby immune cells to control tumour growth. Collectively, this work highlights the importance of nanoparticle structure and nano-biointeractions in controlling immunotherapy efficacy.

Pharmacokinetics and Pharmacodynamics of TAK-164 Antibody Drug Conjugate Coadministered with Unconjugated Antibody.

The development of new antibody-drug conjugates (ADCs) has led to the approval of 7 ADCs by the FDA in 4 years. Given the impact of intratumoral distribution on efficacy of these therapeutics, coadministration of unconjugated antibody with ADC has been shown to improve distribution and efficacy of several ADCs in high and moderately expressed tumor target systems by increasing tissue penetration. However, the benefit of coadministration in low expression systems is less clear. TAK-164, an ADC composed of an anti-GCC antibody (5F9) conjugated to a DGN549 payload, has demonstrated heterogeneous distribution and bystander killing. Here, we evaluated the impact of 5F9 coadministration on distribution and efficacy of TAK-164 in a primary human tumor xenograft mouse model. Coadministration was found to improve the distribution of TAK-164 within the tumor, but it had no significant impact (increase or decrease) on efficacy. Experimental and computational evidence indicates that this was not a result of tumor saturation, increased binding to perivascular cells, or compensatory bystander effects. Rather, the cellular potency of DGN549 was matched with the single-cell uptake of TAK-164 making its IC50 close to its equilibrium binding affinity (KD), and as such, coadministration dilutes total DGN549 in cells below the maximum cytotoxic concentration, thereby offsetting an increased number of targeted cells with decreased ability to kill each cell. These results provide new insights on matching payload potency to ADC delivery to help identify when increasing tumor penetration is beneficial for improving ADC efficacy and demonstrate how mechanistic simulations can be leveraged to design clinically effective ADCs.

Activation of Tumor-Cell STING Primes NK-Cell Therapy.

Activation of the stimulator of interferon genes (STING) pathway promotes antitumor immunity but STING agonists have yet to achieve clinical success. Increased understanding of the mechanism of action of STING agonists in human tumors is key to developing therapeutic combinations that activate effective innate antitumor immunity. Here, we report that malignant pleural mesothelioma cells robustly express STING and are responsive to STING agonist treatment ex vivo. Using dynamic single-cell RNA sequencing of explants treated with a STING agonist, we observed CXCR3 chemokine activation primarily in tumor cells and cancer-associated fibroblasts, as well as T-cell cytotoxicity. In contrast, primary natural killer (NK) cells resisted STING agonist-induced cytotoxicity. STING agonists enhanced migration and killing of NK cells and mesothelin-targeted chimeric antigen receptor (CAR)-NK cells, improving therapeutic activity in patient-derived organotypic tumor spheroids. These studies reveal the fundamental importance of using human tumor samples to assess innate and cellular immune therapies. By functionally profiling mesothelioma tumor explants with elevated STING expression in tumor cells, we uncovered distinct consequences of STING agonist treatment in humans that support testing combining STING agonists with NK and CAR-NK cell therapies.

TAK-676: A Novel Stimulator of Interferon Genes (STING) Agonist Promoting Durable IFN-dependent Antitumor Immunity in Preclinical Studies.

Oncology therapies targeting the immune system have improved patient outcomes across a wide range of tumor types, but resistance due to an inadequate T-cell response in a suppressive tumor microenvironment (TME) remains a significant problem. New therapies that activate an innate immune response and relieve this suppression may be beneficial to overcome this hurdle. TAK-676 is a synthetic novel stimulator of interferon genes (STING) agonist designed for intravenous administration. Here we demonstrate that TAK-676 dose-dependently triggers activation of the STING signaling pathway and activation of type I interferons. Furthermore, we show that TAK-676 is a highly potent modulator of both the innate and adaptive immune system and that it promotes the activation of dendritic cells, natural killer cells, and T cells in preclinical models. In syngeneic murine tumor models in vivo, TAK-676 induces dose-dependent cytokine responses and increases the activation and proliferation of immune cells within the TME and tumor-associated lymphoid tissue. We also demonstrate that TAK-676 dosing results in significant STING-dependent antitumor activity, including complete regressions and durable memory T-cell immunity. We show that TAK-676 is well tolerated, exhibits dose-proportional pharmacokinetics in plasma, and exhibits higher exposure in tumor. The intravenous administration of TAK-676 provides potential treatment benefit in a broad range of tumor types. Further study of TAK-676 in first-in-human phase I trials is ongoing. Significance: TAK-676 is a novel systemic STING agonist demonstrating robust activation of innate and adaptive immune activity resulting in durable antitumor responses within multiple syngeneic tumor models. Clinical investigation of TAK-676 is ongoing.

Identification of Novel Carbocyclic Pyrimidine Cyclic Dinucleotide STING Agonists for Antitumor Immunotherapy Using Systemic Intravenous Route.

Stimulator of Interferon Genes (STING) plays an important role in innate immunity by inducing type I interferon production upon infection with intracellular pathogens. STING activation can promote increased T-cell activation and inflammation in the tumor microenvironment, resulting in antitumor immunity. Natural and synthetic cyclic dinucleotides (CDNs) are known to activate STING, and several synthetic CDN molecules are being investigated in the clinic using an intratumoral administration route. Here, we describe the identification of STING agonist 15a, a cyclic dinucleotide structurally diversified from natural ligands with optimized properties for systemic intravenous (iv) administration. Our studies have shown that STING activation by 15a leads to an acute innate immune response as measured by cytokine secretion and adaptive immune response via activation of CD8+ cytotoxic T-cells, which ultimately provides robust antitumor efficacy.

Quantifying ADC bystander payload penetration with cellular resolution using pharmacodynamic mapping.

With the recent approval of 3 new antibody drug conjugates (ADCs) for solid tumors, this class of drugs is gaining momentum for the targeted treatment of cancer. Despite significant investment, there are still fundamental issues that are incompletely understood. Three of the recently approved ADCs contain payloads exhibiting bystander effects, where the payload can diffuse out of a targeted cell into adjacent cells. These effects are often studied using a mosaic of antigen positive and negative cells. However, the distance these payloads can diffuse in tumor tissue while maintaining a lethal concentration is unclear. Computational studies suggest bystander effects partially compensate for ADC heterogeneity in tumors in addition to targeting antigen negative cells. However, this type of study is challenging to conduct experimentally due to the low concentrations of extremely potent payloads. In this work, we use a series of 3-dimensional cell culture and primary human tumor xenograft studies to directly track fluorescently labeled ADCs and indirectly follow the payload via an established pharmacodynamic marker (γH2A. X). Using TAK-164, an anti-GCC ADC undergoing clinical evaluation, we show that the lipophilic DNA-alkylating payload, DGN549, penetrates beyond the cell targeted layer in GCC-positive tumor spheroids and primary human tumor xenograft models. The penetration distance is similar to model predictions, where the lipophilicity results in moderate tissue penetration, thereby balancing improved tissue penetration with sufficient cellular uptake to avoid significant washout. These results aid in mechanistic understanding of the interplay between antigen heterogeneity, bystander effects, and heterogeneous delivery of ADCs in the tumor microenvironment to design clinically effective therapeutics.

Preclinical Antitumor Activity and Biodistribution of a Novel Anti-GCC Antibody-Drug Conjugate in Patient-derived Xenografts.

Guanylyl cyclase C (GCC) is a unique therapeutic target with expression restricted to the apical side of epithelial cell tight junctions thought to be only accessible by intravenously administered agents on malignant tissues where GCC expression is aberrant. In this study, we sought to evaluate the therapeutic potential of a second-generation investigational antibody-dug conjugate (ADC), TAK-164, comprised of a human anti-GCC mAb conjugated via a peptide linker to the highly cytotoxic DNA alkylator, DGN549. The in vitro binding, payload release, and in vitro activity of TAK-164 was characterized motivating in vivo evaluation. The efficacy of TAK-164 and the relationship to exposure, pharmacodynamic marker activation, and biodistribution was evaluated in xenograft models and primary human tumor xenograft (PHTX) models. We demonstrate TAK-164 selectively binds to, is internalized by, and has potent cytotoxic effects against GCC-expressing cells in vitro A single intravenous administration of TAK-164 (0.76 mg/kg) resulted in significant growth rate inhibition in PHTX models of metastatic colorectal cancer. Furthermore, imaging studies characterized TAK-164 uptake and activity and showed positive relationships between GCC expression and tumor uptake which correlated with antitumor activity. Collectively, our data suggest that TAK-164 is highly active in multiple GCC-positive tumors including those refractory to TAK-264, a GCC-targeted auristatin ADC. A strong relationship between uptake of 89Zr-labeled TAK-164, levels of GCC expression and, most notably, response to TAK-164 therapy in GCC-expressing xenografts and PHTX models. These data supported the clinical development of TAK-164 as part of a first-in-human clinical trial (NCT03449030).

Increased Tumor Penetration of Single-Domain Antibody-Drug Conjugates Improves In Vivo Efficacy in Prostate Cancer Models.

Targeted delivery of chemotherapeutics aims to increase efficacy and lower toxicity by concentrating drugs at the site-of-action, a method embodied by the seven current FDA-approved antibody-drug conjugates (ADC). However, a variety of pharmacokinetic challenges result in relatively narrow therapeutic windows for these agents, hampering the development of new drugs. Here, we use a series of prostate-specific membrane antigen-binding single-domain (Humabody) ADC constructs to demonstrate that tissue penetration of protein-drug conjugates plays a major role in therapeutic efficacy. Counterintuitively, a construct with lower in vitro potency resulted in higher in vivo efficacy than other protein-drug conjugates. Biodistribution data, tumor histology images, spheroid experiments, in vivo single-cell measurements, and computational results demonstrate that a smaller size and slower internalization rate enabled higher tissue penetration and more cell killing. The results also illustrate the benefits of linking an albumin-binding domain to the single-domain ADCs. A construct lacking an albumin-binding domain was rapidly cleared, leading to lower tumor uptake (%ID/g) and decreased in vivo efficacy. In conclusion, these results provide evidence that reaching the maximum number of cells with a lethal payload dose correlates more strongly with in vivo efficacy than total tumor uptake or in vitro potency alone for these protein-drug conjugates. Computational modeling and protein engineering can be used to custom design an optimal framework for controlling internalization, clearance, and tissue penetration to maximize cell killing. SIGNIFICANCE: A mechanistic study of protein-drug conjugates demonstrates that a lower potency compound is more effective in vivo than other agents with equal tumor uptake due to improved tissue penetration and cellular distribution.

MM-131, a bispecific anti-Met/EpCAM mAb, inhibits HGF-dependent and HGF-independent Met signaling through concurrent binding to EpCAM.

Activation of the Met receptor tyrosine kinase, either by its ligand, hepatocyte growth factor (HGF), or via ligand-independent mechanisms, such as MET amplification or receptor overexpression, has been implicated in driving tumor proliferation, metastasis, and resistance to therapy. Clinical development of Met-targeted antibodies has been challenging, however, as bivalent antibodies exhibit agonistic properties, whereas monovalent antibodies lack potency and the capacity to down-regulate Met. Through computational modeling, we found that the potency of a monovalent antibody targeting Met could be dramatically improved by introducing a second binding site that recognizes an unrelated, highly expressed antigen on the tumor cell surface. Guided by this prediction, we engineered MM-131, a bispecific antibody that is monovalent for both Met and epithelial cell adhesion molecule (EpCAM). MM-131 is a purely antagonistic antibody that blocks ligand-dependent and ligand-independent Met signaling by inhibiting HGF binding to Met and inducing receptor down-regulation. Together, these mechanisms lead to inhibition of proliferation in Met-driven cancer cells, inhibition of HGF-mediated cancer cell migration, and inhibition of tumor growth in HGF-dependent and -independent mouse xenograft models. Consistent with its design, MM-131 is more potent in EpCAM-high cells than in EpCAM-low cells, and its potency decreases when EpCAM levels are reduced by RNAi. Evaluation of Met, EpCAM, and HGF levels in human tumor samples reveals that EpCAM is expressed at high levels in a wide range of Met-positive tumor types, suggesting a broad opportunity for clinical development of MM-131.

Immunocapture-LC/MS-Based Target Engagement Measurement in Tumor Plasma Membrane.

For targeted therapies, immunocapture-liquid chromatography mass spectrometry (IC-LC/MS) technology has become an important tool for determination of both drug exposures, target antigen densities, and engagement in the systemic circulation and/or in total target tissue homogenates. Although the information collected from the circulation and tissue homogenates is useful in establishing the correlations of the exposure and target engagement with the pharmacodynamic response and efficacy of a therapy, the measurement at the cell plasma membrane within the target tissue is preferred, since it is the primary action site for antigen and the target drug. However, to the best of our knowledge, IC-LC/MS-based methodologies to conduct the assays at the plasma membrane from tissue sample has been quite limited. In this paper, we reported an IC-LC/MS-based assay platform for assessing the target engagement in tumor plasma membrane prepared from the tumor tissue samples. In addition, tumor samples with guanylyl cyclase C (GCC) expression after fully human IgG1 monoclonal antibody-based antibody-drug conjugate (ADC) treatment were used as a case study. The methodology can differentiate between the total and target-drug bound fraction of GCC with minimal potential equilibrium shift between in-cell surface protein and organelle protein in tumor samples to calculate in vivo target engagement. This approach to determine in vivo target engagement in tumor plasma membrane will provide better understanding of pharmacokinetic/pharmacodynamic relationship to achieve the desired antitumor efficacy.

Dual-Isotope Cryoimaging Quantitative Autoradiography: Investigating Antibody-Drug Conjugate Distribution and Payload Delivery Through Imaging.

In vitro properties of antibody-drug conjugates (ADCs) such as binding, internalization, and cytotoxicity are often well characterized before in vivo studies. Interpretation of in vivo studies might be significantly enhanced by molecular imaging tools. We present here a dual-isotope cryoimaging quantitative autoradiography (CIQA) methodology combined with advanced 3-dimensional imaging and analysis allowing for the simultaneous study of both antibody and payload distribution in tissues of interest in a preclinical setting. Methods: TAK-264, an investigational ADC targeting anti-guanylyl cyclase C (GCC), was synthesized using tritiated monomethyl auristatin E. The tritiated ADC was then conjugated to diethylenetriaminepentaacetic acid, labeled with 111In, and evaluated in vivo in animals bearing GCC-positive and GCC-negative tumors. Results: CIQA revealed the time course of drug release from ADC and its distribution into various tumor regions that are less accessible to the antibody. For GCC-positive tumors, a representative section obtained 96 h after tracer injection showed only 0.8% of the voxels to have colocalized signal, versus over 15% of the voxels for a GCC-negative tumor section, suggesting successful and specific cleaving of the toxin in the GCC-positive lesions. Conclusion: The combination of a veteran established autoradiography technology with advanced image analysis methodologies affords an experimental tool that can support detailed characterization of ADC tumor penetration and pharmacokinetics.

Photodynamic activation as a molecular switch to promote osteoblast cell differentiation via AP-1 activation.

In photodynamic therapy (PDT), cells are impregnated with a photosensitizing agent that is activated by light irradiation, thereby photochemically generating reactive oxygen species (ROS). The amounts of ROS produced depends on the PDT dose and the nature of the photosensitizer. Although high levels of ROS are cytotoxic, at physiological levels they play a key role as second messengers in cellular signaling pathways, pluripotency, and differentiation of stem cells. To investigate further the use of photochemically triggered manipulation of such pathways, we exposed mouse osteoblast precursor cells and rat primary mesenchymal stromal cells to low-dose PDT. Our results demonstrate that low-dose PDT can promote osteoblast differentiation via the activation of activator protein-1 (AP-1). Although PDT has been used primarily as an anti-cancer therapy, the use of light as a photochemical "molecular switch" to promote differentiation should expand the utility of this method in basic research and clinical applications.

Selective treatment and monitoring of disseminated cancer micrometastases in vivo using dual-function, activatable immunoconjugates.

Drug-resistant micrometastases that escape standard therapies often go undetected until the emergence of lethal recurrent disease. Here, we show that it is possible to treat microscopic tumors selectively using an activatable immunoconjugate. The immunoconjugate is composed of self-quenching, near-infrared chromophores loaded onto a cancer cell-targeting antibody. Chromophore phototoxicity and fluorescence are activated by lysosomal proteolysis, and light, after cancer cell internalization, enabling tumor-confined photocytotoxicity and resolution of individual micrometastases. This unique approach not only introduces a therapeutic strategy to help destroy residual drug-resistant cells but also provides a sensitive imaging method to monitor micrometastatic disease in common sites of recurrence. Using fluorescence microendoscopy to monitor immunoconjugate activation and micrometastatic disease, we demonstrate these concepts of "tumor-targeted, activatable photoimmunotherapy" in a mouse model of peritoneal carcinomatosis. By introducing targeted activation to enhance tumor selectively in complex anatomical sites, this study offers prospects for catching early recurrent micrometastases and for treating occult disease.

Epidermal growth factor receptor-targeted photosensitizer selectively inhibits EGFR signaling and induces targeted phototoxicity in ovarian cancer cells.

Targeted photosensitizer delivery to EGFR-expressing cells was achieved in the present study using a high purity, targeted photoimmunoconjugate (PIC). When the PDT agent, benzoporphyrin derivative monoacid ring A (BPD) was coupled to an EGFR-targeting antibody (cetuximab), we observed altered cellular localization and selective phototoxicity of EGFR-positive cells, but no phototoxicity of EGFR-negative cells. Cetuximab in the PIC formulation blocked EGF-induced activation of the EGFR and downstream signaling pathways. Our results suggest that photoimmunotargeting is a useful dual strategy for the selective destruction of cancer cells and also exerts the receptor-blocking biological function of the antibody.

UVB radiation-induced ß-catenin signaling is enhanced by COX-2 expression in keratinocytes.

UVB radiation is the major carcinogen responsible for skin carcinogenesis, thus elucidation of the molecular pathways altered in skin in response to UVB would reveal novel targets for therapeutic intervention. It is well established that UVB leads to upregulation of cyclooxygenase 2 (COX-2) in the skin which contributes to skin carcinogenesis. Overexpression of COX-2 has been shown to promote colon cancer cell growth through ß-catenin signaling, however, little is known about the connection between UVB, COX-2, and ß-catenin in the skin. In the present study, we have identified a novel pathway in which UVB induces ß-catenin signaling in keratinocytes, which is modulated by COX-2 expression. Exposure of the mouse 308 keratinocyte cell line (308 cells) and primary normal human epidermal keratinocytes (NHEKs) to UVB resulted in increased protein levels of both N-terminally unphosphorylated and total ß-catenin. In addition, we found that UVB-enhanced ß-catenin-dependent TOPflash reporter activity and expression of a downstream ß-catenin target gene. We demonstrated that UVB-induced ß-catenin signaling is modulated by COX-2, as treatment of keratinocytes with the specific COX-2 inhibitor NS398 blocked UVB induction of ß-catenin. Additionally, ß-catenin target gene expression was reduced in UVB-treated COX-2 knockout (KO) MEFs compared to wild-type (WT) MEFs. Furthermore, epidermis from UVB-exposed SKH-1 mice exhibited increased N-terminally unphosphorylated and total ß-catenin protein levels and increased staining for total ß-catenin, and both responses were reduced in COX-2 heterozygous mice. Taken together, these results suggest a novel pathway in which UVB induces ß-catenin signaling in keratinocytes which is enhanced by COX-2 expression.

Killing hypoxic cell populations in a 3D tumor model with EtNBS-PDT.

An outstanding problem in cancer therapy is the battle against treatment-resistant disease. This is especially true for ovarian cancer, where the majority of patients eventually succumb to treatment-resistant metastatic carcinomatosis. Limited perfusion and diffusion, acidosis, and hypoxia play major roles in the development of resistance to the majority of front-line therapeutic regimens. To overcome these limitations and eliminate otherwise spared cancer cells, we utilized the cationic photosensitizer EtNBS to treat hypoxic regions deep inside in vitro 3D models of metastatic ovarian cancer. Unlike standard regimens that fail to penetrate beyond ∼150 µm, EtNBS was found to not only penetrate throughout the entirety of large (>200 µm) avascular nodules, but also concentrate into the nodules' acidic and hypoxic cores. Photodynamic therapy with EtNBS was observed to be highly effective against these hypoxic regions even at low therapeutic doses, and was capable of destroying both normoxic and hypoxic regions at higher treatment levels. Imaging studies utilizing multiphoton and confocal microscopies, as well as time-lapse optical coherence tomography (TL-OCT), revealed an inside-out pattern of cell death, with apoptosis being the primary mechanism of cell killing. Critically, EtNBS-based photodynamic therapy was found to be effective against the model tumor nodules even under severe hypoxia. The inherent ability of EtNBS photodynamic therapy to impart cytotoxicity across a wide range of tumoral oxygenation levels indicates its potential to eliminate treatment-resistant cell populations.

Quantitative imaging reveals heterogeneous growth dynamics and treatment-dependent residual tumor distributions in a three-dimensional ovarian cancer model.

Three-dimensional tumor models have emerged as valuable in vitro research tools, though the power of such systems as quantitative reporters of tumor growth and treatment response has not been adequately explored. We introduce an approach combining a 3-D model of disseminated ovarian cancer with high-throughput processing of image data for quantification of growth characteristics and cytotoxic response. We developed custom MATLAB routines to analyze longitudinally acquired dark-field microscopy images containing thousands of 3-D nodules. These data reveal a reproducible bimodal log-normal size distribution. Growth behavior is driven by migration and assembly, causing an exponential decay in spatial density concomitant with increasing mean size. At day 10, cultures are treated with either carboplatin or photodynamic therapy (PDT). We quantify size-dependent cytotoxic response for each treatment on a nodule by nodule basis using automated segmentation combined with ratiometric batch-processing of calcein and ethidium bromide fluorescence intensity data (indicating live and dead cells, respectively). Both treatments reduce viability, though carboplatin leaves micronodules largely structurally intact with a size distribution similar to untreated cultures. In contrast, PDT treatment disrupts micronodular structure, causing punctate regions of toxicity, shifting the distribution toward smaller sizes, and potentially increasing vulnerability to subsequent chemotherapeutic treatment.

Synergistic enhancement of carboplatin efficacy with photodynamic therapy in a three-dimensional model for micrometastatic ovarian cancer.

Metastatic ovarian cancer (OvCa) frequently recurs due to chemoresistance, highlighting the need for nonoverlapping combination therapies that mechanistically synergize to eradicate residual disease. Photodynamic therapy (PDT), a photochemistry-based cytotoxic modality, sensitizes ovarian tumors to platinum agents and biologics and has shown clinical promise against ovarian carcinomatosis. We introduce a three-dimensional (3D) model representing adherent ovarian micrometastases and high-throughput quantitative imaging methods to rapidly screen the order-dependent effects of combining benzoporphyrin-derivative (BPD) monoacid A-based PDT with low-dose carboplatin. 3D ovarian micronodules grown on Matrigel were subjected to BPD-PDT either before or after carboplatin treatment. We developed custom fluorescence image analysis routines to quantify residual tumor volume and viability. Carboplatin alone did not eradicate ovarian micrometastases at a dose of 400 mg/m2, leaving surviving cores that were nonsensitive or impermeable to chemotherapy. BPD-PDT (1.25 µmol/L·J/cm2) created punctate cytotoxic regions within tumors and disrupted micronodular structure. Treatment with BPD-PDT prior to low-dose carboplatin (40 mg/m2) produced a significant synergistic reduction [P<0.0001, analysis of covariance (ANCOVA)] in residual tumor volume [0.26; 95% confidence interval (95% CI), 0.19-0.36] compared with PDT alone (0.76; 95% CI, 0.63-0.92) or carboplatin alone (0.95; 95% CI, 0.83-1.09), relative to controls. This synergism was not observed with the reverse treatment order. Here, we demonstrate for the first time the use of a 3D model for micrometastatic OvCa as a rapid and quantitative reporter to optimize sequence and dosing regimens of clinically relevant combination strategies. This approach combining biological modeling with high-content imaging provides a platform to rapidly screen therapeutic strategies for a broad array of metastatic tumors.

PuraMatrix encapsulation of cancer cells.

Increasing evidence suggests that culturing cancer cells in three dimensions more accurately recapitulates the complexity of tumor biology. Many of these models utilize reconstituted basement membrane derived from animals which contain a variable amount of growth factors and cytokines that can influence the growth of these cell culture models. Here, we describe in detail the preparation and use of PuraMatrix, a commercially available self assembling peptide gel that is devoid of animal-derived material and pathogens to encapsulate and propagate the ovarian cancer cell line, OVCAR-5. We begin by describing how to prepare the PuraMatrix prior to use. Next, we demonstrate how to properly mix the PuraMatrix and cell suspension to encapsulate the cells in the hydrogel. Upon the addition of cell culture media or injection into a physiological environment, the peptide component of PuraMatrix rapidly self assembles into a 3D hydrogel that exhibits a nanometer scale fibrous structure with an average pore size of 5-200 nm(1). In addition, we demonstrate how to propagate cultures grown in encapsulated PuraMatrix. When encapsulated in PuraMatrix, OVCAR-5 cells assemble into three dimensional acinar structures that more closely resemble the morphology of micrometastatic nodules observed in the clinic than monolayer in vitro models. Using confocal microscopy we illustrate the appearance of representative OVCAR-5 cells encapsulated in PuraMatrix on day 1, 3, 5, and 7 post plating. The use of PuraMatrix to culture cancer cells should improve our understanding of the disease and allow us to assess treatment response in more clinically predictive model systems.