Functional Interrogation of Ca2+ Signals in Human Cancer Cells In Vitro and Ex Vivo by Fluorescent Microscopy and Molecular Tools.

Genetically encoded calcium indicators (GECIs) and high-resolution confocal microscopy enable dynamic visualization of calcium signals in cells and tissues. Two-dimensional and 3D biocompatible materials mimic the mechanical microenvironments of tumor and healthy tissues in a programmable manner. Cancer xenograft models and ex vivo functional imaging of tumor slices reveal physiologically relevant functions of calcium dynamics in tumors at different progression stages. Integration of these powerful techniques allows us to quantify, diagnose, model, and understand cancer pathobiology. Here, we describe detailed materials and methods used to establish this integrated interrogation platform, from generating transduced cancer cell lines that stably express CaViar (GCaMP5G + QuasAr2) to in vitro and ex vivo calcium imaging of the cells in 2D/3D hydrogels and tumor tissues. These tools open the possibility for detailed explorations of mechano-electro-chemical network dynamics in living systems.

Human cancer cells generate spontaneous calcium transients and intercellular waves that modulate tumor growth.

Electrically excitable cells such as neurons transmit long-distance calcium or electrical signals to regulate their physiological functions. While the molecular underpinnings and down-stream effects of these intercellular communications in excitable cells have been well appreciated, little is known about whether and how non-excitable cancer cells spontaneously initiate and transmit long-distance intercellular signals. Here we report that non-excitable human colon and prostate cancer cells spontaneously initiate and spread intercellular calcium waves, in vitro and ex vivo. Xenograft model studies suggest that these calcium signals promote the growth rate of tumors in mice. Pharmacological studies elucidated that the inositol-trisphosphate-receptor (IP3R)-regulated calcium release from endoplasmic reticulum (ER), which is activated by the Gq-PLC-IP3R pathway, is a major cause for the initiation of spontaneous calcium transients. Further, the spatial-temporal characteristics of calcium dynamics can be tuned by the culture substrates of different mechanical stiffnesses. Our results provide evidence that calcium dynamics enables long-distance functional communication in non-excitable cancer cells and offer the potential to modulate calcium signaling for new cancer therapies.

Automatic Multi-functional Integration Program (AMFIP) towards all-optical mechano-electrophysiology interrogation.

Automatic operations of multi-functional and time-lapse live-cell imaging are necessary for the biomedical science community to study active, multi-faceted, and long-term biological phenomena. To achieve automatic control, most existing solutions often require the purchase of extra software programs and hardware that rely on the manufacturers' own specifications. However, these software programs are usually non-user-programmable and unaffordable for many laboratories. To address this unmet need, we have developed a novel open-source software program, titled Automatic Multi-functional Integration Program (AMFIP), as a new Java-based and hardware-independent system that provides proven advantages over existing alternatives to the scientific community. Without extra hardware, AMFIP enables the functional synchronization of the µManager software platform, the Nikon NIS-Elements platform, and other 3rd party software to achieve automatic operations of most commercially available microscopy systems, including but not limited to those from Nikon. AMFIP provides a user-friendly and programmable graphical user interface (GUI), opening the door to expanding the customizability for myriad hardware and software systems according to user-specific experimental requirements and environments. To validate the intended purposes of developing AMFIP, we applied it to elucidate the question whether single cells, prior to their full spreading, can sense and respond to a soft solid substrate, and if so, how does the interaction depend on the cell spreading time and the stiffness of the substrate. Using a CRISPR/Cas9-engineered human epithelial Beas2B (B2B) cell line that expresses mNeonGreen2-tagged mechanosensitive Yes-associated protein (YAP), we show that single B2B cells develop distinct substrate-stiffness-dependent YAP expressions within 10 hours at most on the substrate, suggesting that cells are able to sense, distinguish, and respond to mechanical cues prior to the establishment of full cell spreading. In summary, AMFIP provides a reliable, open-source, and cost-free solution that has the validated long-term utility to satisfy the need of automatic imaging operations in the scientific community.

Normal tissue and tumor microenvironment adaptations to aerobic exercise enhance doxorubicin anti-tumor efficacy and ameliorate its cardiotoxicity in retired breeder mice.

Aerobic exercise is receiving increased recognition in oncology for its multiple purported benefits. Exercise is known to induce physiologic adaptations that improve patient quality-of-life parameters as well as all-cause mortality. There also is a growing body of evidence that exercise may directly alter the tumor microenvironment to influence tumor growth, metastasis, and response to anticancer therapies. Furthermore, the physiologic adaptations to exercise in normal tissues may protect against treatment-associated toxicity and allow for greater treatment tolerance. However, the exercise prescription required to induce these beneficial tumor-related outcomes remains unclear. This study characterized the aerobic adaptations to voluntary wheel running in normal tissues and the tumor microenvironment. Female, retired breeder BALB/c mice and syngeneic breast adenocarcinoma cells were utilized in primary tumor and metastasis models. Aerobic exercise was found to induce numerous adaptations across various tissues in these mice, although primary tumor growth and metastasis were largely unaffected. However, intratumoral hypoxia and global metabolism were altered in the tumors of exercising hosts relative to non-wheel running controls. Doxorubicin chemotherapy also was found to be more efficacious at delaying tumor growth with adjuvant aerobic exercise. Additionally, doxorubicin-induced cardiac toxicity was ameliorated in exercising hosts relative to non-wheel running controls. Taken together, these data suggest that the normal tissue and tumor microenvironment adaptations to aerobic exercise can improve doxorubicin efficacy while simultaneously limiting its toxicity.

All-optical Mechanobiology Interrogation of Yes-associated Protein in Human Cancer and Normal Cells using a Multi-functional System.

Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. However, manual control of various equipment produced by different manufacturers is labor-intensive and time-consuming, potentially decreasing the accuracy, reproducibility, and quality of acquired data. Therefore, an all-in-one and user-programmable system that enables automatic, multi-functional, and long-term image acquisition and is compatible with most fluorescent microscopy platforms can benefit the scientific community. This paper introduces the complete operating protocols of utilizing a novel integrated software system that consists of (1) a home-built software program, titled "Automatic Multi-functional Integration Program (AMFIP)," which enables automatic multi-channel imaging acquisition, and (2) a suite of quantitative imaging analysis and cell traction computation packages. This integrated system is applied to reveal the previously unknown relationship between the spatial-temporal distribution of mechano-sensitive Yes-associated protein (YAP) and the cell mechanics, including cell spreading and traction, in CRISPR/Cas9-engineered human normal cells (B2B) and lung cancer cells (PC9). Leveraging this system's capability of multi-channel control and readout, the result shows: (1) B2B normal cells and PC9 cancer cells show a distinct relationship between YAP expression, traction, and cell dynamics during cell spreading and migration processes; and (2) PC9 cancer cells apply noticeable peri-nuclear forces on substrates. In summary, this paper presents a detailed stepwise protocol on how to utilize an integrated user-programmable system that enables automatic multi-functional imaging and analysis to elucidate YAP mechano-sensitivity. These tools open the possibility for detailed explorations of multifaceted signaling dynamics in the context of cell physiology and pathology.

Impact of the SRC inhibitor dasatinib on the metastatic phenotype of human prostate cancer cells.

SRC, a non-receptor tyrosine kinase, is frequently over-expressed and highly activated in blood as well as solid tumors in various organs, including prostate, and has been associated with aggressive disease and a poor patient prognosis. Prostate cancer patients with a high risk of developing metastases have few treatment options, none of which can result in a durable cure. Therefore, the aim of the present study was to examine the impact of a SRC inhibitor, dasatinib, on the ability of human prostate cancer cell to complete key steps in the metastatic process, including invasion and angiogenesis. Dasatinib treatment impaired the metastatic phenotypes of the human prostate cancer cell lines, PC-3, DU-145, and LNCaP, by significantly reducing migration and invasion in modified Boyden chambers. Inhibition of phosphorylation, and therefore enhanced activation, of SRC and key downstream signaling pathway elements, including FAK, STAT3, Paxillin, and Akt, as determined by Western blotting, also was observed. This suggests that dasatinib interferes with critical cell functions associated with the metastatic cascade. Dasatinib also had direct effects on the ability of microvascular endothelial cells to form tubes in vitro and impaired the ability of PC-3 cells to induce angiogenesis in vivo. In conclusion, the present findings suggest that SRC inhibition by dasatinib may have utility in reducing the metastatic spread of prostate cancer cells.

Support of a free radical mechanism for enhanced antitumor efficacy of the microtubule disruptor OXi4503.

Unlike normal blood vessels, the unique characteristics of an expanding, disorganized and leaky tumor vascular network can be targeted for therapeutic gain by vascular disrupting agents (VDAs), which promote rapid and selective collapse of tumor vessels, causing extensive secondary cancer cell death. A hallmark observation following VDA treatment is the survival of neoplastic cells at the tumor periphery. However, comparative studies with the second generation tubulin-binding VDA OXi4503 indicate that the viable rim of tumor tissue remaining following treatment with this agent is significantly smaller than that seen for the lead VDA, combretastatin. OXi4503 is the cis-isomer of CA1P and it has been speculated that this agent's increased antitumor efficacy may be due to its reported metabolism to orthoquinone intermediates leading to the formation of cytotoxic free radicals. To examine this possibility in situ, KHT sarcoma-bearing mice were treated with either the cis- or trans-isomer of CA1P. Since both isomers can form quinone intermediates but only the cis-isomer binds tubulin, such a comparison allows the effects of vascular collapse to be evaluated independently from those caused by the reactive hydroxyl groups. The results showed that the cis-isomer (OXi4503) significantly impaired tumor blood flow leading to secondary tumor cell death and >95% tumor necrosis 24h post drug exposure. Treatment with the trans-isomer had no effect on these parameters. However, the combination of the trans-isomer with combretastatin increased the antitumor efficacy of the latter agent to near that of OXi4503. These findings indicate that while the predominant in vivo effect of OXi4503 is clearly due to microtubule collapse and vascular shut-down, the formation of toxic free radicals likely contributes to its enhanced potency.

Impact of the Src inhibitor saracatinib on the metastatic phenotype of a fibrosarcoma (KHT) tumor model.

BACKGROUND: Src, a non-receptor tyrosine kinase frequently overexpressed and highly activated in malignancies, has been associated with a poor patient prognosis. The aim of the present studies was to examine the impact of an Src inhibitor (saracatinib) on a highly metastatic murine sarcoma cell line (KHT). MATERIALS AND METHODS: Phosphorylation of Src and downstream effectors was determined using Western immunoblotting. Cell cycle was analyzed by flow cytometry using propidium iodide DNA staining, migration and invasion in modified Boyden chambers, activated MMP-9 by gel zymography, and visualization of pSrc and pFAK by confocal immunofluorescence. The number of KHT lung nodules in saracatinib-treated mice was compared to controls. RESULTS: Saracatinib inhibited major pathways in the metastatic cascade in vitro, including Src and FAK activation. Functions required for metastasis, such as migration and invasion, were reduced when cells were exposed to 0.5 µM and 1.0 µM saracatinib, respectively (p<0.0001). Pretreatment of KHT cells with either 1 µM or 5 µM saracatinib prior to tail vein injection decreased lung colonies in mice from 13.0 to 5.0 (p<0.05) and less than 1.0 (p<0.01), respectively. CONCLUSION: These findings suggest that Src inhibition by saracatinib may reduce the metastatic activity of tumor cells.

Vascular targeting agents enhance chemotherapeutic agent activities in solid tumor therapy.

The utility of combining the vascular targeting agents 5,6-dimethyl-xanthenone-4 acetic acid (DMXAA) and combretastatin A-4 disodium phosphate (CA4DP) with the anticancer drugs cisplatin and cyclophosphamide (CP) was evaluated in experimental rodent (KHT sarcoma), human breast (SKBR3) and ovarian (OW-1) tumor models. Doses of the vascular targeting agents that led to rapid vascular shutdown and subsequent extensive central tumor necrosis were identified. Histologic evaluation showed morphologic damage of tumor cells within a few hours after treatment, followed by extensive hemorrhagic necrosis and dose-dependent neoplastic cell death as a result of prolonged ischemia. Whereas these effects were induced by a range of CA4DP doses (10-150 mg/kg), the dose response to DMXAA was extremely steep; doses < or = 15 mg/kg were ineffective and doses > or = 20 mg/kg were toxic. DMXAA also enhanced the tumor cell killing of cisplatin, but doses > 15 mg/kg were required. In contrast, CA4DP increased cisplatin-induced tumor cell killing at all doses studied. This enhancement of cisplatin efficacy was dependent on the sequence and interval between the agents. The greatest effects were achieved when the vascular targeting agents were administered 1-3 hr after cisplatin. When CA4DP (100 mg/kg) or DMXAA (17.5 mg/kg) were administered 1 hr after a range of doses of cisplatin or CP, the tumor cell kill was 10-500-fold greater than that seen with chemotherapy alone. In addition, the inclusion of the antivascular agents did not increase bone marrow stem cell toxicity associated with these anticancer drugs, thus giving rise to a therapeutic gain.