Prithy C. Martis

Three-Dimensional Culture of Mouse Renal Carcinoma Cells in Agarose Macrobeads Selects for a Subpopulation of Cells with Cancer Stem Cell or Cancer Progenitor Properties

The culture of tumor cell lines in three-dimensional scaffolds is considered to more closely replicate the in vivo tumor microenvironment than the standard method of two-dimensional cell culture. We hypothesized that our method of encapsulating and maintaining viable and functional pancreatic islets in agarose-agarose macrobeads (diameter 6-8 mm) might provide a novel method for the culture of tumor cell lines. In this report we describe and characterize tumor colonies that form within macrobeads seeded with mouse renal adenocarcinoma cells. Approximately 1% of seeded tumor cells survive in the macrobead and over several months form discrete elliptical colonies appearing as tumor cell niches with increasing metabolic activity in parallel to colony size. The tumor colonies demonstrate ongoing cell turnover as shown by BrdU incorporation and activated caspase-3 and TUNEL staining. Genes upregulated in the tumor colonies of the macrobead are likely adaptations to this novel environment, as well as an amplification of G(1)/S cell-cycle checkpoints. The data presented, including SCA-1 and Oct4 positivity and the upregulation of stem cell-like genes such as those associated with the Wnt pathway, support the notion that the macrobead selects for a subpopulation of cells with cancer stem cell or cancer progenitor properties.

Enhancement of In Vitro and In Vivo Function of Agarose Encapsulated Porcine Islets by Changes in the Islet Microenvironment

The transplantation of porcine islets of Langerhans to treat type 1 diabetes may provide a solution to the demand for insulin-producing cells. Porcine islets encapsulated in agarose–agarose macrobeads have been shown to function in nonimmunosuppressed xenogeneic models of both streptozotocin-induced and autoimmune type 1 diabetes. One advantage of agarose encapsulation is the ability to culture macrobeads for extended periods, permitting microbiological and functional assessment. Herein we describe optimization of the agarose matrix that results in improved islet function. Porcine islets (500 IEQs) from retired breeding sows were encapsulated in 1.5% SeaKem Gold (SG), 0.8% SG, or 0.8% Litex (Li) agarose, followed by an outer capsule of 5% SG agarose. Insulin production by the encapsulated islets exhibited an agarose-specific effect with 20% (0.8% SG) to 50% (0.8% Li) higher initial insulin production relative to 1.5% SG macrobeads. Insulin production was further increased by 40–50% from week 2 to week 12 in both agarose types at the 0.8% concentration, whereas islets encapsulated in 1.5% SG agarose increased insulin production by approximately 20%. Correspondingly, fewer macrobeads were required to restore normoglycemia in streptozotocin-induced diabetic female CD(SD) rats that received 0.8% Li (15 macrobeads) or 0.8% SG (17 macrobeads) as compared to 1.5% SG (19 macrobeads). Islet cell proliferation was also observed during the first 2 months postencapsulation, peaking at 4 weeks, where approximately 50% of islets contained proliferative cells, including β-cells, regardless of agarose type. These results illustrate the importance of optimizing the microenvironment of encapsulated islets to improve islet performance and advance the potential of islet xenotransplantation for the treatment of type 1 diabetes.

Hydrophilic Agarose Macrobead Cultures Select for Outgrowth of Carcinoma Cell Populations That Can Restrict Tumor Growth

Cancer cells and their associated tumors have long been considered to exhibit unregulated proliferation or growth. However, a substantial body of evidence indicates that tumor growth is subject to both positive and negative regulatory controls. Here, we describe a novel property of tumor growth regulation that is neither species nor tumor-type specific. This property, functionally a type of feedback control, is triggered by the encapsulation of neoplastic cells in a growth-restricting hydrogel composed of an agarose matrix with a second coating of agarose to form 6- to 8-mm diameter macrobeads. In a mouse cell model of renal adenocarcinoma (RENCA cells), this process resulted in selection for a stem cell-like subpopulation which together with at least one other cell subpopulation drove colony formation in the macrobeads. Cells in these colonies produced diffusible substances that markedly inhibited in vitro and in vivo proliferation of epithelial-derived tumor cells outside the macrobeads. RENCA cells in monolayer culture that were exposed to RENCA macrobead-conditioned media exhibited cell-cycle accumulation in S phase due to activation of a G(2)/M checkpoint. At least 10 proteins with known tumor suppression functions were identified by analysis of RENCA macrobead-conditioned media, the properties of which offer opportunities to further dissect the molecular basis for tumor growth control. More generally, macrobead culture may permit the isolation of cancer stem cells and other cells of the stem cell niche, perhaps providing strategies to define more effective biologically based clinical approaches to treat neoplastic disease.

Treatment of agarose–agarose RENCA macrobeads with docetaxel selects for OCT4+ cells with tumor-initiating capability

The cancer stem cell (CSC) theory depicts such cells as having the capacity to produce both identical CSCs (symmetrical division) and tumor-amplifying daughter cells (asymmetric division). CSCs are thought to reside in niches similar to those of normal stem cells as described for neural, intestinal, and epidermal tissue, are resistant to chemotherapy, and are responsible for tumor recurrence. We recently described the niche-like nature of mouse renal adenocarcinoma (RENCA) cells following encapsulation in agarose macrobeads. In this paper we tested the hypothesis that encapsulated RENCA colonies function as an in vitro model of a CSC niche and that the majority of cells would undergo chemotherapy-induced death, followed by tumor recurrence. After exposure to docetaxel (5 µg/ml), 50% of cells were lost one week post-treatment while only one or two cells remained in each colony by 6 weeks. Surviving cells expressed OCT4 and reformed tumors at 16 weeks post-treatment. Docetaxel-resistant cells also grew as monolayers in cell culture (16–17 weeks post-exposure) or as primary tumors following transplantation to Balb/c mice (6 of 10 mice) or NOD.CB17-Prkdcscid/J mice (9 of 9 mice; 10 weeks post-transplantation or 28 weeks post-exposure). These data support the hypothesis that a rare subpopulation of OCT4+ cells are resistant to docetaxel and these cells are sufficient for tumor recurrence. The reported methodology can be used to obtain purified populations of tumor-initiating cells, to screen for anti-tumor-initiating cell agents, and to investigate the in vitro correlate of a CSC niche, especially as it relates to chemo-resistance and tumor recurrence.

Abstract 1675: MEF2 plays a significant role in the tumor inhibitory effects of agarose encapsulated RENCA cells through the EGF receptor

Identification of the molecular targets involved in the tumor growth inhibition induced by agarose encapsulated mouse renal adenocarcinoma (RENCA) cells will aid in an understanding of the mechanism of action and may allow for the discovery of key targets for therapeutic intervention. The epidermal growth factor receptor (EGFR/ERBB1/HER1) and related family members have been shown to transduce extracellular signals through the mitogen-activated protein kinase (MAPK) pathway to regulate post-translational regulation of myocyte enhancer factor 2 (MEF2) proteins. We have previously reported that suppression of MEF2 isoforms in target tumor cells reduced the growth inhibitory effect of RENCA macrobeads. In this study, we evaluated signaling through EGFR in response to RENCA macrobead conditioned media and examined post-translational activation of MEF2D, using the human prostate cancer, DU145 and the human breast adenocarcinoma, MCF7 cell lines. DU145 and MCF7 cells exposed to naive or conditioned media were evaluated using In-Cell Western™ analysis for the expression of phospho-EGFR Y1068 in parallel with total EGFR. Phospho-MEF2D and total MEF2D were assessed in nuclear extracts using western blotting. In the DU145 cells, exposure to RENCA macrobead conditioned media activates EGFR as evidenced by phosphorylation of the receptor at tyrosine 1068 in parallel with the increasing age and inhibitory capacity of macrobeads. Further, the growth inhibition of DU145 cells exposed to RENCA macrobead conditioned media is accompanied by dephosphorylation of MEF2D at Serine 444 supporting a role for increased MEF2D transcriptional activity in the inhibitory effect. MCF7 cells are less sensitive to RENCA macrobead-induced growth inhibition, EGFR is not phosphorylated by 2 hours following exposure to macrobead conditioned media and MEF2D remains phosphorylated at Serine 444. These findings support the hypothesis that RENCA macrobeads signal, at least in part, through the EGF receptor to differentially regulate MEF2D, which is significant for macrobead-induced tumor growth inhibition. Citation Format: Prithy C. Martis, Melissa A. Laramore, Atira Dudley, Barry H. Smith, Lawrence S. Gazda. MEF2 plays a significant role in the tumor inhibitory effects of agarose encapsulated RENCA cells through the EGF receptor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1675. doi:10.1158/1538-7445.AM2015-1675

Clinical laboratory and imaging evidence for effectiveness of agarose-agarose macrobeads containing stem-like cells derived from a mouse renal adenocarcinoma cell population (RMBs) in treatment-resistant, advanced metastatic colorectal cancer: Evaluation of a biological-systems approach to cancer therapy (U.S. FDA IND-BB 10091; NCT 02046174, NCT 01053013)

Objective The complexity, heterogeneity and capacity of malignant neoplastic cells and tumors for rapid change and evolution suggest that living-cell-based biological-systems approaches to cancer treatment are merited. Testing this hypothesis, the tumor marker, metabolic activity, and overall survival (OS) responses, to the use of one such system, implantable macrobeads [RENCA macrobeads (RMBs)], in phase I and IIa clinical trials in advanced, treatment-resistant metastatic colorectal cancer (mCRC) are described here. Methods Forty-eight mCRC patients (30 females; 18 males), who had failed all available, approved treatments, underwent RMB implantation (8 RMB/kg body weight) up to 4 times in phase I and phase IIa open-label trials. Physicals, labs [tumor and inflammation markers, lactate dehydrogenase (LDH)] and positron emission tomography-computed tomography (PET-CT) imaging to measure number/volume and metabolic activity of the tumors were performed pre- and 3-month-post-implantation to evaluate safety and initial efficacy (as defined by biological responses). PET-CT maximum standard uptake value (SUVmax) (baseline and d 90; SUVmax ≥2.5), LDH, and carcinoembryonic antigen (CEA) and/or cancer antigen 19-9 (CA 19-9) response (baseline, d 30 and/or d 60) were assessed and compared to OS. Results Responses after implantation were characterized by an at least 20% decrease in CEA and/or CA 19-9 in 75% of patients. Fluorodeoxyglucose (FDG)-positive lesions (phase I, 39; 2a, 82) were detected in 37/48 evaluable patients, with 35% stable volume and stable or decreased SUV (10) plus four with necrosis; 10, increased tumor volume, SUV. LDH levels remained stable and low in Responders (R) (d 0-60, 290.4-333.9), but increased steadily in Non-responders (NR) (d 0-60, 382.8-1,278.5) (d 60, P=0.050). Responders to RMBs, indicated by the changes in the above markers, correlated with OS (R mean OS=10.76 months; NR mean OS=4.9 months; P=0.0006). Conclusions The correlations of the tumor marker, tumor volume and SUV changes on PET-CT, and LDH levels themselves, and with OS, support the concept of a biological response to RMB implantation and the validity of the biological-systems approach to mCRC. A phase III clinical trial is planned.

Abstract B36: RENCA macrobead-induced AKT hyperphosphorylation leads to MEF2 activation and inhibition of the proliferation of human DU145 prostate carcinoma cells

We have previously reported that suppression of the isoforms of myocyte enhancer factor (MEF2) in target tumor cells in vitro reduces the demonstrated proliferation-inhibitory effect of agarose-agarose encapsulated mouse renal adenocarcinoma cells (RENCA macrobeads), suggesting that it is a critical component of a pathway by which the inhibitory action, now being evaluated also in Phase IIb human clinical trials, is mediated. Importantly, MEF2 originally described as active in myogenesis, has been found to be a key regulator of many developmental pathways and to have both pro- and anti-growth regulatory functions in murine and human tumor cells. Recent studies have supported a molecular intersection between components of the PI3K/Akt and the canonical Wnt signaling (carcinogenic) pathways, converging at the glycogen synthase kinase 3β (GSK3β) node. In skeletal and cardiac muscle cells, GSK3β regulates MEF2 activity indirectly through reciprocal regulation of the p38 mitogen-activated protein kinase (MAPK) pathway. To determine, therefore, whether PI3K/Akt is also involved in the anti-proliferative effect of the RENCA macrobeads, we evaluated signaling through PI3K/Akt in response to macrobead-conditioned media and examined post-translational modification of MEF2D in two human cell lines DU145 (prostate cancer) and MCF7 (breast adenocarcinoma). Using In-Cell Western analysis, we determined the expression of phospho-Akt T308 and S473 in parallel with total Akt in cells exposed to naive or conditioned media. Nuclear extracts for phospho-MEF2D and total MEF2D were assessed using western blotting. In the DU145 cells, exposure to RENCA macrobead-conditioned medium resulted in Akt hyperphosphorylation (≥ 48 hours) in parallel with the increasing age and inhibitory capacity of macrobeads. Further, the growth inhibition of DU145 cells exposed to RENCA conditioned medium (36-40% at 12-16 weeks of age; 45-48% at 20-24 weeks of age) was accompanied by de-phosphorylation of MEF2D at Serine 444, supporting a role for increased MEF2D transcriptional activity in the inhibitory effect. In the MCF7 cells, which are less sensitive to RENCA macrobead inhibition (8-12% at 12-16 weeks of age; 17-21% at 20-24 weeks of age), Akt phosphorylation diminished one hour following exposure to conditioned media, and MEF2D remained phosphorylated at Serine 444. These findings support the hypothesis that RENCA macrobeads, as biological cell systems with the in vitro and in vivo (model and clinical) capability of inhibiting cancer cell proliferation and tumor growth, achieve this effect, at least in part, by signaling through Akt to differentially regulate MEF2D. Citation Format: Prithy C. Martis, Melissa A. Laramore, Atira T. Dudley, Barry H. Smith, Lawrence S. Gazda. RENCA macrobead-induced AKT hyperphosphorylation leads to MEF2 activation and inhibition of the proliferation of human DU145 prostate carcinoma cells. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B36.

Possible role of the systemic inflammatory reaction in defining tumor responder vs. nonresponder in cancer macrobead therapy.

572 Background: Peritoneal implantation of mouse renal adenocarcinoma cell-containing (RENCA) Macrobead (MB) represents a cell-system-based approach to the treatment of advanced, mCRC that has been evaluated to date in Phase IIa trials. The data indicate that there are “responders” (R) and “non-responders”(NR) as reflected in overall survival (OS), where “response” is defined as a >20% decrease in either/both CEA or CA19-9 during the first 30 days after MB implantation. We analyzed whether the “response” is due to a post-implant systemic inflammatory response (SIR) or rather a direct inhibitory effect of the MB. Methods: Thirty-four treatment-resistant mCRC patients (pts) were implanted laparoscopically at least once with RENCA MB. Pts were considered R (n=25), or NR (n=9), based on tumor marker responses within the first 30 days. CRP, IL-6, TNF-alpha, and ESR, as measures of SIR, were measured at Day 14 and 30. Results: All 34 pts showed SIR to MB implantation, as indicated by transient rises in CRP, IL-...

Abstract 2486: Docetaxel resistant cells display cancer stem cell properties and regulate growth via PI3/Akt signaling

Both normal organogenesis and tumor development follow a Gompertzian growth curve. An understanding of the mechanisms operating in these apparently discordant situations, in which growth rate slows as mass increases, may be useful for the treatment of cancer. We have previously shown that encapsulating murine renal adenocarcinoma (RENCA) cells in a double layer of agarose to form spherical macrobeads, undergo Gompertzian growth regulation as tumor colonies within the confines of the inner agarose matrix. As the colonies reach maximal size, the RENCA macrobeads (MBs) secrete factors that also inhibit the proliferation of freely growing target cells outside the MB. In the current study, we report the ability of the tumor colonies within the MB to re-form following the debulking of tumor mass with docetaxel (0.5 μg/ml or 5.0 μg/ml). Docetaxel-resistant cells were assessed for the stem cell marker OCT4 using immunohistochemistry (Abcam ab19857), RT-PCR (Qiagen Cancer Stem Cell PCR Array) and cell migration/invasion (Corning Cell Migration Chemotaxis and Invasion Assay). Docetaxel-resistant cells are shown to be a discrete population of cells that exhibit stem cell-like properties, including prolonged quiescence (>16 wk), OCT4 staining, stem cell gene expression, increased migration and invasion, and in vivo tumor induction. These cancer stem-like properties are dose and time post-treatment dependent with the greatest expression observed in the few surviving cells at 6 wk post-treatment with 5 μg/ml. Tumor growth is observed in syngeneic BALB/c mice following transplantation of a single docetaxel-resistant cell at higher frequencies (≥ 6 of 9) as compared to the grafting of single monolayer cells (0 of 8) at 5 wk when using cells recovered at 6 wk from the 5 μg/ml dose treated MBs. To understand the mechanism of colony growth regulation within the MB, we hypothesized that tumor cells external to the MB would use similar growth control signaling when exposed to RENCA MBs or MB conditioned media. We have previously shown an increase in the activity of the transcription factor MEF2 in cells exposed to RENCA MB conditioned media. To investigate PI3/Akt signaling, a known pathway for MEF2 regulation, in human DU145 prostate tumor cells exposed to MB conditioned media, we used In-Cell and Western blotting. Exposure to RENCA MBs resulted in Akt hyperphosphorylation (≥ 48 hours) and de-phosphorylation of MEF2D in DU145 target cells. These findings support the hypothesis that RENCA MBs, as biological cell systems with the in vitro capability of inhibiting cancer cell proliferation, achieve this effect, at least in part, by signaling through Akt to regulate MEF2D. This effect is being investigated in colorectal patients with progressive disease, following informed consent, who underwent laparoscopic intraperitoneal implantations of RENCA MBs as part of ongoing clinical trials. Citation Format: Prithy C. Martis, Melissa A. Laramore, Hunter L. Gazda, Atira Dudley, Pradeep R. Dumpala, Allyson J. Ocean, Nathaniel Berman, Tapan Parikh, Zoe P. Andrada, Angelica Nazarian, Joanne Thomas, Eugene Akahoho, George Stoms, Alex Yaroshinsky, Thomas J. Fahey, David J. Wolf, Lawrence S. Gazda, Barry H. Smith. Docetaxel resistant cells display cancer stem cell properties and regulate growth via PI3/Akt signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2486.

Abstract 4609: MEF2 plays a critical role in RENCA macrobead-induced tumor cell growth inhibition

Regulation of cell proliferation and cell death is often dependent on the coupling of extracellular signals to the activation or repression of specific intracellular signaling pathways. We have previously demonstrated the capacity of mouse renal adenocarcinoma (RENCA) cells encapsulated in agarose macrobeads to form numerous small tumor colonies within the macrobeads over a period of several months. As the encapsulated tumor colonies mature and reach a maximum size, the macrobeads themselves, or conditioned media from cultured macrobeads, markedly inhibit the in vitro and in vivo growth of epithelial-derived tumor cells outside the macrobead microenvironment. Myocyte enhancer factor 2 (MEF2) is a family of four transcription factors that have been shown to play a central role in linking signaling pathways to the genes responsible for cell division, differentiation and death. In this study, we evaluated the expression and role of MEF2 in regulating cell proliferation in mouse (RENCA) and human cell lines (J82, MCF7) in response to RENCA macrobeads. Freely growing RENCA target cells transiently transfected with a MEF2 transcriptional response element and exposed to conditioned media from variously aged macrobeads demonstrated increased response element activation in parallel with the increasing age of the macrobeads. Similarly, growth inhibition of the freely growing RENCA cells increased with older macrobeads. Suppression of the expressed MEF2 isoforms in target RENCA cells, either individually (MEF2a, MEF2b, and MEF2d) or in combination (MEF2pool) using synthetic small interfering RNA (siRNA) markedly reduced the growth inhibitory effects of RENCA macrobeads. In J82 and MCF7 cell lines, MEF2D expression was responsive to replete media from RENCA macrobeads, increasing 2.0-fold and 2.7-fold respectively. Degradation of MEF2D mRNA transcripts in the human cell lines studied resulted in abatement of the growth inhibitory effect of the RENCA macrobeads. These findings reveal an essential role for MEF2 in RENCA macrobead-induced cancer cell growth inhibition and raise interesting questions about the molecular basis of this response. Citation Format: Prithy C. Martis, Atira Dudley, Melissa A. Laramore, Barry H. Smith, Lawrence S. Gazda. MEF2 plays a critical role in RENCA macrobead-induced tumor cell growth inhibition. [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 4609. doi:10.1158/1538-7445.AM2014-4609