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MOV file - 314K, Live-imaging of a HCC1806-H2B:GFP spheroid.

MOV file - 73K, Live-imaging of a T47D-H2B:GFP spheroid co-cultured with fibroblasts.

Most ductal breast carcinoma cells are weakly invasive in vitro and in vivo, suggesting that components of their microenvironment may facilitate a transition from in situ to invasive stages during progression. Here, we report that coculture of mammary fibroblasts specifically triggers invasive behavior in basal-type breast cancer cells through a ligand independent mechanism. When cultured alone in organotypic culture, both basal- and luminal-type breast cancer cells formed noninvasive spheroids with characteristics of ductal carcinoma in situ (DCIS). In contrast, when cocultured with mammary fibroblasts, basal-type spheroids exhibited invasive character whereas the luminal-type spheroids retained a benign and noninvasive duct-like architecture. Real-time imaging and functional studies revealed that the specificity of invasion was linked to a unique capacity of basal-type breast cancer cells to move within spheroids. Mammary fibroblasts induced invasion by triggering basal-type breast cancer cells to convert from a noninvasive program of mammary epithelial morphogenesis to an invasive program of sprouting endothelial angiogenesis. Contrary to the existing invasion models, soluble ligands produced by the fibroblasts were not sufficient to trigger invasion. Instead, basal-type invasion relied upon a Cdc42-dependent reorganization of collagen fibers in the extracellular matrix by fibroblasts. Inhibiting basal-type cell movement with clinically relevant drugs blocked invasion both in organotypic culture and in animals, suggesting a new treatment strategy for early-stage patients. Together our findings establish that fibroblast recruitment by basal-type breast cancer cells into early-stage tumors is sufficient to trigger their conversion from a benign, noninvasive DCIS-like stage to a malignant invasive stage. Furthermore, our findings suggest that different subtypes of breast cancer may require distinct types of contributions from the microenvironment to undergo malignant progression. Cancer Res; 71(21); 6857–66. ©2011 AACR.

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MOV file - 50K, Live-imaging of a HCC1954-H2B:GFP spheroid used for tracking cell movement.

MOV file - 2MB, Phase contrast and H2B:mCherry images from live-imaging of a MCFDCIS-H2B:mCherry spheroid.

PDF file - 2MB, Expression of Cdc42 in mammary fibroblasts transfected with siRNAs.

PDF file - 3.9MB, Residual tumor lesions after MEK1/2 inhibitor treatment are composed of MCFDCIS cells.

MOV file - 773K, Live-imaging of MCFDCIS-mCherryCAAX cells invading towards mammary fibroblasts expressing GFP.

MOV file - Live-imaging of a small MCFDCIS-H2B:mCherry spheroid co-cultured with mammary fibroblasts.

PDF file - 7MB, Fibroblast induced collective invasion does not require a loss of epithelial character.

PDF file - 1.6MB, EGFR and ERK1/2 are necessary for HCC1806 cell morphogenic movement and fibroblast induced collective invasion.

PDF file - 5MB, Cancer cells in the spheroids follow the path of the initial invading cells.

PDF file - 2MB, Tracking the movement of breast cancer cells in spheroids.

PDF file - 2MB, MDA-MB-231 breast cancer cells, but not MCFDCIS cells collectively invade in organotypic culture.

MOV file - 105K, Live-imaging of a MCFDCIS-mCherryCAAX spheroid and a mammary fibroblast.

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MOV file - 1.1MB, Live-imaging of a MCFDCIS-H2B:mCherry spheroid co-cultured with mammary fibroblasts.

MOV file - 2MB, Phase contrast and H2B:mCherry images from live-imaging of a MCFDCIS-H2B:mCherry spheroid co-cultured with mammary fibroblasts.

PDF file - 4MB, Fibroblasts are localized within areas of reorganized FITC-Collagen I.

Background: Over 50% of patients with ovarian cancer (OVCA) succumb to their disease within five years from diagnosis, highlighting the need for new approaches to identify therapeutic options. The PARIS® test is a CLIA certified organoid based high throughput drug sensitivity assay that can be used by oncologists to select personalized treatments. In this study our objectives were to: 1) demonstrate the feasibility of organoid based drug testing in the clinical setting; 2) highlight the clinical utility and the predictive value of the PARIS® test; and 3) identify new therapeutic options for OVCA patients. Methods: 93 patients, with a median age at diagnosis of 55 years (range 25-82) were selected as eligible for the PARIS® test at the discretion of the oncologists. The cohort included 74.1% high grade serous tumors, 6.9% low grade serous tumors, 6.0% clear cell carcinomas, 3.4% granulosa cell tumors, 2.5% Mullerian tumors, 1.7% carcinosarcomas and 2.5% fallopian tumors. 16.1% of patients were treatment naïve and 83.9% received one or more previous lines of treatment. 116 patient derived tumor organoids (PDTOs) were derived from surgical excisions, biopsies, or body fluids and subjected to functional testing with a panel of chemo- and targeted therapies that are FDA-approved or in clinical development. Drug responses were evaluated using a proprietary score ranking drugs from exceptional responses to no response. The median turnaround time from biopsy to report delivery was 19 days. Results: The success rate for deriving OVCA organoids was 92%; 81.0% of samples demonstrated exceptional/good response to at least one drug. PDTO drug sensitivities were highly concordant with both retrospective and prospective clinical response. Drugs that frequently showed exceptional/good responses included WEE1 inhibitors (28.4%), HDAC inhibitors (25.0%), and Pan-DNMT inhibitors (11.2%). Further, 12.9% of cases showed responses to the BTK/EGFR inhibitor ibrutinib and 23.3% to HER2 inhibitors lapatinib and neratinib. Two patients with advanced stage, recurrent low grade serous ovarian cancer showed exceptional and durable clinical responses to PARIS® guided treatments, ibrutinib and lapatinib, respectively. Conclusions: This study demonstrates the utility of organoid based drug testing to identify potentially effective drugs and to guide treatment choice for patients with ovarian cancer. Tumor organoid based personalized treatments lead to extraordinary responses in several patients, including a patient in hospice care who responded to ibrutinib monotherapy and is still alive after 27 months. Citation Format: Amanda M. Prechtl, Marwah Al-Aloosi, Payel Chatterjee, Rachele Rosati, Samantha Velazquez, Lauren Appleyard, Grace Durenberger, Cristina Cordero, Robert Diaz, Hallie Swan, Brady Bernard, Siddhartha Mukherjee, Barbara Goff, Elizabeth Swisher, Heidi Gray, Kalyan Banda, Christopher Kemp, Carla Grandori. Tumor organoid based drug sensitivity testing in ovarian cancer identifies exceptional responders to targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3399.

Loss of the Ras GTPase-activating protein neurofibromin promotes the development of aggressive spindle cell neoplasms known as Malignant Peripheral Nerve Sheath Tumors (MPNSTs) in patients with the genetic disorder neurofibromatosis type 1 (NF1). Currently, the available chemotherapeutic regimens and radiotherapy are ineffective against MPNSTs, so the prognosis for patients with these neoplasms is poor. Neurofibromin loss dysregulates multiple Ras proteins in the classic (H-Ras, N-Ras, K-Ras) and R-Ras (R-Ras, R-Ras2/TC21, R-Ras3/M-Ras) subfamilies. Consequently, it is unclear which Ras proteins or pathways regulated by these Ras proteins should be therapeutically targeted in MPNSTs. We have previously shown that classic Ras proteins drive MPNST cell proliferation and survival. However, the role(s) of the R-Ras subfamily of proteins in MPNSTs have not been elucidated. To determine how R-Ras proteins contribute to the pathogenesis of neurofibromin-null MPNSTs, we introduced dominant negative (DN) R-Ras mutants, which are pan-inhibitors of the R-Ras subfamily, into MPNST cells and assessed the impact of R-Ras subfamily inhibition on mitogenesis, migration and the phosphoproteome. A panel of MPNST cell lines (STS-26T, YST-1, ST88-14, 90–8, NMS2, NMS-PC, S462, T265-2c) was used. Methodologies utilized include immunoblotting, PCR, Transwell migration, 3H-thymidine incorporation, and mass spectrometric analysis of phosphoprotein-enriched specimens. We found that R-Ras and R-Ras2 are widely expressed and can be activated in neurofibromin-null MPNST cells. In contrast to classic Ras proteins, we found that R-Ras proteins drive MPNST both mitogenesis and migration. Using mass spectrometry-based phosphoproteomics, we identified thirteen protein networks that were regulated by DN R-Ras, including networks affecting cellular movement via effects on microtubules. We chose to further study changes in ROCK1 phosphorylation and found that R-Ras subfamily proteins function, at least in some part, through the same pathways as ROCK1. We conclude that R-Ras proteins promote tumorigenesis by regulating distinct signaling pathways that regulate MPNST mitogenesis and migration.

Genes that are normally biased towards expression in the testis are often induced in tumor cells. These gametogenic genes, known as cancer-testis antigens (CTAs), have been extenstively investigated as targets for immunotherapy. However, despite their frequent detection, the degree to which CTAs support neoplastic invasion is poorly understood. Here, we find that the CTA genes SPANX-A/C/D and CTAG2 are coordinately induced in breast cancer cells and regulate distinct features of invasive behavior. Our functional analysis revealed that CTAG2 interacts with Pericentrin at the centrosome and is necessary for directional migration. Conversely, SPANX-A/C/D interacts with Lamin A/C at the inner nuclear membrane and is required for the formation of actin-rich cellular protrusions that reorganize the extracellular matrix. Importantly, SPANX-A/C/D was required for breast cancer cells to spontaneously metastasize to the lung, demonstrating that CTA reactivation can be critical for invasion dependent phenotypes in vivo. Moreover, elevated SPANX-A/C/D expression in breast cancer patient tumors correlated with poor outcome. Together, our results suggest that distinct CTAs promote tumor progression by regulating complementary cellular functions that are integrated together to induce invasive behavior.

Tumor cells can engage in a process called collective invasion, in which cohesive groups of cells invade through interstitial tissue. Here, we identified an epigenetically distinct subpopulation of breast tumor cells that have an enhanced capacity to collectively invade. Analysis of spheroid invasion in an organotypic culture system revealed that these “trailblazer” cells are capable of initiating collective invasion and promote non-trailblazer cell invasion, indicating a commensal relationship among subpopulations within heterogenous tumors. Canonical mesenchymal markers were not sufficient to distinguish trailblazer cells from non-trailblazer cells, suggesting that defining the molecular underpinnings of the trailblazer phenotype could reveal collective invasion-specific mechanisms. Functional analysis determined that DOCK10, ITGA11, DAB2, PDFGRA, VASN, PPAP2B, and LPAR1 are highly expressed in trailblazer cells and required to initiate collective invasion, with DOCK10 essential for metastasis. In patients with triple-negative breast cancer, expression of these 7 genes correlated with poor outcome. Together, our results indicate that spontaneous conversion of the epigenetic state in a subpopulation of cells can promote a transition from in situ to invasive growth through induction of a cooperative form of collective invasion and suggest that therapeutic inhibition of trailblazer cell invasion may help prevent metastasis.

In glioblastoma (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Indeed, patients respond poorly to the current therapies of tumor resection followed by chemotherapy with radiotherapy and have only a 16-month median survival. It is therefore imperative to develop novel therapies. RSK2 is a kinase that regulates proliferation and adhesion and can promote metastasis. We demonstrate that active RSK2 regulates GBM cell adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. RSK2 control of adhesion and migration is mediated in part by its effects on integrin-Filamin A complexes. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. In agreement with the in vitro data, using public datasets, we find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

Most ductal breast carcinoma cells are weakly invasive in vitro and in vivo, suggesting that components of their microenvironment may facilitate a transition from in situ to invasive stages during progression. Here, we report that coculture of mammary fibroblasts specifically triggers invasive behavior in basal-type breast cancer cells through a ligand independent mechanism. When cultured alone in organotypic culture, both basal- and luminal-type breast cancer cells formed noninvasive spheroids with characteristics of ductal carcinoma in situ (DCIS). In contrast, when cocultured with mammary fibroblasts, basal-type spheroids exhibited invasive character whereas the luminal-type spheroids retained a benign and noninvasive duct-like architecture. Real-time imaging and functional studies revealed that the specificity of invasion was linked to a unique capacity of basal-type breast cancer cells to move within spheroids. Mammary fibroblasts induced invasion by triggering basal-type breast cancer cells to convert from a noninvasive program of mammary epithelial morphogenesis to an invasive program of sprouting endothelial angiogenesis. Contrary to the existing invasion models, soluble ligands produced by the fibroblasts were not sufficient to trigger invasion. Instead, basal-type invasion relied upon a Cdc42-dependent reorganization of collagen fibers in the extracellular matrix by fibroblasts. Inhibiting basal-type cell movement with clinically relevant drugs blocked invasion both in organotypic culture and in animals, suggesting a new treatment strategy for early-stage patients. Together our findings establish that fibroblast recruitment by basal-type breast cancer cells into early-stage tumors is sufficient to trigger their conversion from a benign, noninvasive DCIS-like stage to a malignant invasive stage. Furthermore, our findings suggest that different subtypes of breast cancer may require distinct types of contributions from the microenvironment to undergo malignant progression. Cancer Res; 71(21); 6857–66. ©2011 AACR.

Neurofibromin, the tumor suppressor encoded by the neurofibromatosis type 1 (NF1) gene, potentially suppresses the activation of H-Ras, N-Ras, and K-Ras. However, it is not known whether these classic Ras proteins are hyperactivated in NF1-null nerve sheath tumors, how they contribute to tumorigenesis, and what signaling pathways mediate their effects. Here we show that H-Ras, N-Ras, and K-Ras are coexpressed with their activators (guanine nucleotide exchange factors) in neurofibromin-null malignant peripheral nerve sheath tumor (MPNST) cells, and that all 3 Ras proteins are activated. Dominant negative (DN) H-Ras, a pan-inhibitor of the classic Ras family, inhibited MPNST proliferation and survival, but not migration. However, NF1-null MPNST cells were variably dependent on individual Ras proteins. In some lines, ablation of H-Ras, N-Ras, and/or K-Ras inhibited mitogenesis. In others, ablation of a single Ras protein had no effect on proliferation; in these lines, ablation of a single Ras protein resulted in compensatory increases in the activation and/or expression of other Ras proteins. Using mass spectrometry-based phosphoproteomics, we identified 7 signaling networks affecting morphology, proliferation, and survival that are regulated by DN H-Ras. Thus, neurofibromin loss activates multiple classic Ras proteins that promote proliferation and survival by regulating several distinct signaling cascades.

Diverse genetic and epigenetic traits can promote variant behaviors in subpopulations of cells within primary tumors. In some cases, these heterogeneous subpopulations may compete against each other for clonal dominance to improve tumor fitness. By comparison, the extent to which tumor cell subpopulations cooperate during malignant progression is not well understood. Here, we find that a commensal relationship can exist between epigentically distinct subpopulations of tumor cells during collective invasion. Using quantitive phenotypic analysis and live-cell imaging, we discovered that a subpopulation of cells, termed trailblazer cells, has an enhanced ability to collectively invade. Trailblazer phenotype was heritable and trailblazer subpopulations were derived from 5 triple-negative breast cancer cell lines. The percentage of trailblazers cells within a population ranged from between 0.5% and 90% of the parental population. Gene expression profiling identified a 29-gene expression signature that distinguished trailblazer cells from non-trailblazer cells and identified patients with poor outcome, A cohort of genes trailblazer signature genes were necessary for the formation of actin-rich projections that facilitate path generation during collective invasion. In addition, the trailblazer gene DOCK10 was necessary for spontaneous metastasis to the lung. The path generating function of a small subpopulation of trailblazer cells was sufficient to induce the collective invasion of large population of naturally occurring non-trailblazer cells in organotypic culture. Trailblazer cells were also sufficient to induce the invasion of a mouse model of ductal carcinoma in situ. Taken together, our results suggest that a rare subpopulation of tumor cells with an enhanced ability to collectively invade can promote metastatic progression through cell autonomous and non-cell autonomous mechanisms. Thus, our results support a paradigm shift towards identifying both the cell autonomous and the non cell-autonomous regulatory mechanisms of cell biological processes within heterogeneous tumors to understand neoplastic progression and develop new intervention modalities. Citation Format: Amanda Prechtl, Jill Westcott, Tuyen Dang, Erin Maine, Gray Pearson. Non-cell autonomous regulation of collective invasion be an epigenetically distinct subpopulation of tumor 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 3161. doi:10.1158/1538-7445.AM2014-3161

Glioblastoma (GBM) is associated with high mortality in patients due in large part to the highly migratory and invasive nature of glioma cells. The 90kDa ribosomal S6 Kinases (RSKs) are a family of serine/theronine kinases that are an integral component of the extracellular signal regulated kinase (ERK)/ mitogen activated protein kinase (MAPK) pathway, which is highly active in many types of cancer. The RSK family contains four human isoforms (RSK1-4) that regulate a variety of cellular processes including cell proliferation, survival, and motility, however a role for individual RSK isoforms in GBM cell invasion has not clearly been defined. Here, we analyze the roles of RSK1-4 in glioblastoma cell invasion. Using transwell assays and a novel three-dimensional tumor spheroid assay, we found that the highly specific RSK inhibitor, BI-D1870, decreases glioblastoma cell motility and invasion. To identify which RSK isoform was associated with this decrease in invasion, we knocked down each RSK isoform using lentiviral mediated shRNA transduction and found that different RSK isoforms could decrease invasion depending on the cell line, without significantly affecting proliferation. Additionally, RSK2 knockdown in primary patient-derived glioblastoma cells decreased cell migration, and Kaplan-Meier analysis revealed that patients overexpressing RSK2 had a poorer prognosis compared to those expressing normal levels of RSK2. Our studies support a differential role for individual RSKs in glioblastoma invasion, suggesting that targeting RSKs may provide an effective means for the treatment of glioblastoma. Citation Format: Amanda Prechtl, Florian Sulzmaier, Pengfei Jiang, Santosh Kesari, Joe Ramos. RSK isoform regulation of migration and invasion in glioblastoma. [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 5007. doi:10.1158/1538-7445.AM2014-5007

The invasion of tumor cells out of ductal epithelium is a poorly understood process that dramatically increases the odds of patient death. We have defined a new non cell-autonomous control mechanism for neoplastic invasion that trigger tumors to switch from a benign to a malignant stage of growth. Using high resolution live-imaging and functional studies we show that an epigenetically distinct and autonomously invasive subpopulation of tumor cells induces the invasion of a large bulk population of autonomously noninvasive tumor cells by adopting migratory principles observed during sprouting angiogenesis. The molecular dissection of this cooperative interaction revealed a new “invasion signature” of 29 genes that is distinct from traditional epithelial-to-mesenchymal transition markers. Patients expressing this invasion signature had a worse clinical outcome, which suggests that this commensal relationship between tumor cell subpopulations contributes to tumor progression. Functional analysis showed that multiple components of the invasion signature were specifically required for the cooperative induction of invasion, but not cell motility, thus revealing new roles for these genes as non-cell autonomous regulators of invasion. These results are of broad interest because they are the first demonstration of a commensal relationship between tumor cell subpopulations during the induction of invasion. Further, these findings establish a mechanistic underpinning that obviates the notion that the exclusive role of heterogeneity in tumors is to act as a source for unique traits that can be selected for and expanded to improve the cell autonomous fitness of neoplastic cell populations. Thus, our results support a paradigm shift towards identifying both the cell autonomous and the non cell-autonomous regulatory mechanisms of cell biological processes within heterogeneous tumors to understand neoplastic progression and develop new intervention modalities. Citation Format: Gray Pearson, Amanda Prechtl, Jill Westcott, Tuyen Dang, Erin Maine. A commensal relationship between tumor cell subpopulations promotes collective invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4930. doi:10.1158/1538-7445.AM2013-4930 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.