We thank Ivan Donati for providing of HA, Leonardo Amadio, Gabriella Zito (Department of Gynaecology of IRCCS &#34;Burlo Garofolo&#34;, Trieste, Italy) and Andrea Romano (Operative Clinical Unit of Anatomy and Pathological Histology, Cattinara Hospital, Trieste, Italy) for the tissue sample collection. We thank also Nicol&#242; Morosini for the help in the video preparation and Alex Coppola, the voice. This work was supported by grants from the Institute for Maternal and Child Health, IRCCS &#34;Burlo Garofolo&#34;, Trieste, Italy (RC20/16) and Fondazione Cassa di Risparmio Trieste to R.Bulla.

Tissue samples from patients were collected after informed consent following approval of the ethical considerations by the Institutional Board of the University Hospital of Trieste, Italy.
1. Tumor cell isolation and culture (Day 1)
<ol>
	<li>Isolate human mesothelioma cells from MPM solid specimens. Finely mince the tissue with a cutter to obtain fragments of about 2-3 mm2 in size and incubate in 5 mL of digestion solution composed of Hanks&#39; Balanced Salt Solution (HBSS) supplemented with 0.5 mM Ca2+/Mg2+, 0.5% trypsin and 50 &#181;g/mL DNase I, overnight at 4 &#176;C.</li>
	<li>Tumor cell isolation and culture (Day 2)
	<ol>
		<li>After overnight incubation, place the digested tissue for 30 min in a 37 &#176;C incubator with gentle shaking.</li>
		<li>Replace the digestion solution upon centrifugation (250 x g) with 3 mg/mL collagenase type 1 dissolved in 5 mL of Medium 199 with HBSS and further incubate for 30 min at 37 &#176;C with gentle shaking.</li>
		<li>Block the enzymatic reaction by adding 10% heat-inactivated fetal bovine serum (FBS). Resuspend the cells very carefully with a 5 mL pipet to ensure that most of the cells are released from the tissue. Then, filter the cell suspension through a 100 &#181;m pore filter.</li>
		<li>Seed the cells in a 12.5 cm2 flask and culture them at 37 &#176;C with human endothelial cells serum-free medium (HESF), with 10% FBS and supplemented with EGF (10 ng/mL), basic FGF (20 ng/mL), and 1% penicillin- streptomycin. 
		NOTE: Replace with fresh medium every 2-3 days.</li>
	</ol>
	</li>
</ol>
2. HA coating (Day 1)
<ol>
	<li>Resuspend high molecular weight HA (1.5 kDa) in double distilled water at the concentration of 1 mg/mL6.</li>
	<li>Dilute HA stock solution to 50 &#181;g/mL in carbonate/bicarbonate buffer (0.1 M, pH 9.6) with gentle&#160;pipetting.</li>
	<li>Coat the 96-well plate with 100 &#181;L of dilute HA stock solution per well overnight at 4 &#176;C. 
	NOTE: Hyaluronic acid was a kind gift from Professor Ivan Donati, Department of Life Sciences, University of Trieste7.</li>
</ol>
3. C1q coating (Day 2)
<ol>
	<li>After overnight incubation, vacuum aspirate the treated wells and wash the 96-well plate with 100 &#181;L of Dulbecco&#39;s PBS (dPBS) per well.</li>
	<li>Allow C1q (25 &#181;g/mL or different concentrations for dose response experiments) or BSA (as a negative control) to bind to plastic immobilized-HA by incubating these proteins at a concentration of 25 &#181;g/mL in 100 &#181;L of dPBS + 0.5% BSA and 0.7 mM Ca2+/Mg2+. Then incubate overnight at 4 &#176;C.</li>
	<li>Vacuum aspirate the wells and wash the 96-well plate with 100 &#181;L/well of dPBS.</li>
</ol>
4. Cell labeling with FAST DiI
<ol>
	<li>Resuspend 1 x 105 tumor cells in 500 &#181;L of dPBS containing 10 &#181;g/mL of the fluorescent dye FAST DiI. Incubate for 15 min at 37 &#176;C in a 5% v/v CO2 incubator for the labelling, mixing manually after 5 min intervals.
	<ol>
		<li>To remove excess FAST DiI, add 10 mL of dPBS, pipette gently up and down, and centrifuge at 250 x g for 7 min. Resuspend the cell pellet in 1 mL of human endothelial serum free medium containing 0.1% BSA (HESF + 0.1% BSA).</li>
	</ol>
	</li>
</ol>
5. Cell Adhesion on HA/C1q matrices (Day 1)
<ol>
	<li>Vacuum aspirate the wells coated with the different matrixes (wells were coated in step 3.2).</li>
	<li>Distribute 100 &#181;L of the labelled cell suspension to the coated wells and incubate the plate for 35 min at 37 &#176;C in 5% v/v CO2 incubator.</li>
	<li>Remove the non-adherent cells by aspirating the supernatant. Wash once with dPBS containing 0.5% BSA, 0.7 mM Ca2+ and 0.7 mM Mg2+.</li>
	<li>Lyse the adherent cells by adding 200 &#181;L/well of 10 mM Tris-HCl, pH 7.4 + 0.1% v/v SDS and immediately read the 96-well plate with a fluorescence reader (544 nm, emission 590 nm) using a calibration curve generated through the lysis of an increasing number of labelled cells.</li>
</ol>

<ol>
	<li>Zamboni, F., Vieira, S., Reis, R. L., Oliveira, J. M., Collins, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+hyaluronic+acid+in+immunoprotection+and+immunomodulation:+Chemistry,+processing+and+function.">The potential of hyaluronic acid in immunoprotection and immunomodulation: Chemistry, processing and function.</a> Progress in Materials Science. 97, 97-122 (2018).</li><li>Lorusso, G., Ruegg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumor+microenvironment+and+its+contribution+to+tumor+evolution+toward+metastasis.">The tumor microenvironment and its contribution to tumor evolution toward metastasis.</a> Histochemistry and Cell Biology. 130 (6), 1091-1103 (2008).</li><li>Ricklin, D., Hajishengallis, G., Yang, K., Lambris, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement:+a+key+system+for+immune+surveillance+and+homeostasis.">Complement: a key system for immune surveillance and homeostasis.</a> Nature Immunology. 11 (9), 785-797 (2010).</li><li>Bulla, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C1q+acts+in+the+tumour+microenvironment+as+a+cancer-promoting+factor+independently+of+complement+activation.">C1q acts in the tumour microenvironment as a cancer-promoting factor independently of complement activation.</a> Nature Communications. 7, 10346 (2016).</li><li>Winslow, S., Leandersson, K., Edsjo, A., Larsson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+stromal+gene+signatures+in+breast+cancer.">Prognostic stromal gene signatures in breast cancer.</a> Breast Cancer Research. 17, 23 (2015).</li><li>Collins, M. N., Birkinshaw, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronic+acid+solutions:+A+processing+method+for+efficient+chemical+modification.">Hyaluronic acid solutions: A processing method for efficient chemical modification.</a> Journal of Applied Polymer Science. 130 (1), 145-152 (2013).</li><li>Itano, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+primary+structure,+complex+turnover+regulation+and+multiple+roles+of+hyaluronan.">Simple primary structure, complex turnover regulation and multiple roles of hyaluronan.</a> Journal of Biochemistry. 144 (2), 131-137 (2008).</li><li>Agostinis, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+role+of+C1q+in+cell+migration+and+tissue+remodeling:+contribution+to+trophoblast+invasion+and+placental+development.">An alternative role of C1q in cell migration and tissue remodeling: contribution to trophoblast invasion and placental development.</a> Journal of Immunology. 185 (7), 4420-4429 (2010).</li><li>Agostinis, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement+Protein+C1q+Binds+to+Hyaluronic+Acid+in+the+Malignant+Pleural+Mesothelioma+Microenvironment+and+Promotes+Tumor+Growth.">Complement Protein C1q Binds to Hyaluronic Acid in the Malignant Pleural Mesothelioma Microenvironment and Promotes Tumor Growth.</a> Frontiers in Immunology. 8, 1559 (2017).</li><li>Bossi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C1q+as+a+unique+player+in+angiogenesis+with+therapeutic+implication+in+wound+healing.">C1q as a unique player in angiogenesis with therapeutic implication in wound healing.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (11), 4209-4214 (2014).</li><li>Hosszu, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+expression+and+function+of+the+macromolecular+c1+complex+on+the+surface+of+human+monocytes.">Cell surface expression and function of the macromolecular c1 complex on the surface of human monocytes.</a> Frontiers in Immunology. 3, 38 (2012).</li><li>Laurent, T. C., Fraser, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan.">Hyaluronan.</a> Journal of the Federation of American Societies for Experimental Biology. 6 (7), 2397-2404 (1992).</li><li>Gaboriaud, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crystal+structure+of+the+globular+head+of+complement+protein+C1q+provides+a+basis+for+its+versatile+recognition+properties.">The crystal structure of the globular head of complement protein C1q provides a basis for its versatile recognition properties.</a> Journal of Biological Chemistry. 278 (47), 46974-46982 (2003).</li><li>Lam, J., Truong, N. F., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+cell-matrix+interactions+in+hyaluronic+acid+hydrogel+scaffolds.">Design of cell-matrix interactions in hyaluronic acid hydrogel scaffolds.</a> Acta Biomaterialia. 10 (4), 1571-1580 (2014).</li></ol>

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We describe&#160;an easy method to investigate how the complement component C1q, interacting with hyaluronic acid, is able to modulate the behavior of primary cells isolated from human tumor tissues. Both HA and C1q are abundantly present in the tissue microenvironment both under physiological and pathological conditions, participating to several cell biological processes. For instance, C1q has been shown to be present in the microenvironment of the placenta where it favors extravillous trophoblast invasion of the maternal decidua during placentation8. C1q is also deposited in wound-healing skin where it fosters the angiogenetic process required for tissue regeneration and repair10. Finally, C1q has been identified in several different tumor tissues4. High molecular weight HA is a natural barrier for angiogenesis and proliferation and recent evidence showed that C1q, besides its classical role in complement activation, is able to act as an ECM molecule, favoring cell migration.
The novelty of this work consists in the finding that the binding of C1q to HA strongly modify the interaction of tumor cells with HA. To set up this method several checkpoints have been considered. First of all, we ensured that a sufficient amount of HA was bound to the plastic of culture and ELISA wells using biotinylated HA. The Alcian blue staining of the wells confirmed these results (data not shown). The incubation has been performed overnight in bicarbonate buffer, pH 9.6, and this procedure ensure the complete saturation of the wells by HA (data not shown).
The second step was to bind C1q to HA. The binding of C1q to HA has been performed at a physiological pH (7.4) and in the presence of Ca2+. For this reason, we used a dPBS-BSA buffer with bivalent ions. We initially performed a dose response experiment in order to identify and choose the optimal amount of C1q that can be found attached to HA. Although in our dose-response curve we did not reach exactly the plateau, we used the concentration of 25 &#181;g/mL to be closer to the physiological concentration of free C1q, considering that the amount of C1q normally present in the serum is 75-150 &#181;g/mL. It has been calculated that serum C1q free from C1r and C1s is about 10%-20% of C1 complex11. C1q is known for its ability to bind polyanions, it has been defined as a charge pattern recognition molecule and HA is a negatively charged linear polymer of repeating units of (&#946;,1-4)-D-glucuronic acid-(&#946;,1-3)-N-acetyl-D-glucosamine12,13. For this reason, we investigated this strong non-covalent interaction. Our observations indicated that tumor cells sense the difference between HA and HA bound to C1q. The cells appear to be more spread-out if compared to the cell adhering to HA alone. We observed that this effect is not mediated by soluble C1q, indicating that this modification in cell adhesion is dependent on the interaction of C1q with HA, probably due to a conformational change of the complement molecule as a consequence of the binding.
We want to emphasize the importance of the use of primary cells as in vitro models of tumor behavior for the adhesion experiments, since we notice a strong difference in the adhesion capability between primary cells compared to several cell lines, as shown in Figure 3. An alternative and very interesting model to evaluate the biological properties of HA is the use of 3D models of HA hydrogel scaffold. This scaffold with the incorporation of biological molecules can be used for the evaluation of bioactive signals and&#160;for several applications in the regenerative medicine14. The model that we propose&#160;in this study is an easier, faster and cheaper method for the evaluation of cellular responses to a combination of stimuli, and it can be considered as a first step for the understanding of the molecular mechanisms involved in this kind of interaction.

The tumor microenvironment (TME) influences cancer development and progression since it can provide a permissive niche for cell survival, growth and invasion. The identification of new key players in TME may be useful for the discovery of new molecular tools for target therapy. TME includes a complex and dynamic network of non-malignant cells, such as endothelial cells, fibroblasts and cells of the immune system, embedded in the surrounding extracellular matrix (ECM) components including collagens, laminins, fibronectins, proteoglycans and hyaluronans. Both tumor and non-tumor cells synthesize and secrete ECM components together with cytokines, chemokines, growth factors and inflammatory and matrix remodeling enzymes that overall alter the physical, chemical and signaling properties of TME. Among these constituents, hyaluronic acid (HA) has emerged to exert a crucial role in tumor biology. Despite its simple chemical composition, HA, together with its HA-binding molecules (hyaladherins), can modulate angiogenesis, immune system responsiveness and ECM remodeling in a size and concentration dependent manner1.
The complement (C) system is also part of the local TME, which has recently received increasing attention. The C system encompasses a set of soluble and membrane-bound proteins involved in the first line of defense against non-self-cells, unwanted host elements and pathogens. Functionally, the C links the two-effector arms of innate and adaptive systems to promote either direct cell killing or mounting of an inflammatory response2. C activation can suppress tumor growth, by destroying cancer cells or inhibiting their outgrowth, but it has become increasingly clear that it can possess a tumor-promoting activity by sustaining chronic inflammation, promoting the establishment of an immunosuppressive milieu, inducing angiogenesis, and activating cancer-related signaling pathways3. In this context, C1q, the first recognition molecule of the classical pathway of the C system has emerged to exert important functions in the tumor microenvironment independently of C activation4. C1q has been shown to be expressed locally by a range of human malignant tumors, where it can favor cancer cell adhesion, migration and proliferation in addition to angiogenesis and metastasis5. Interestingly C1q interacts with a major constituent of the ECM such as HA.
We developed a technique to isolate the primary cancer cells from the tumor mass. Furthermore, we created the matrix, which can stimulate tumor microenvironment, particularly the interaction between C1q and high molecular weight hyaluronic acid. C1q bound to HA was able to induce adhesion of the tumor cells.

<table><tbody><tr><td>100&amp;thinsp;&amp;micro;m pore filter</td><td>BD Falcon</td><td>352360</td><td></td></tr><tr><td>Amphotericin B solution (fungizone)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>1397-89-3</td><td></td></tr><tr><td>basic FGF</td><td>Immunological Sciences</td><td>GRF-15595</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>C-4901</td><td></td></tr><tr><td>Collagenase type I</td><td>Worthington Biochemical Corporation, DBA</td><td>MX1D12644</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma-Aldrich&amp;nbsp;</td><td>50-99-7</td><td></td></tr><tr><td>DNase I&amp;nbsp;</td><td>Roche</td><td>10 104 159 001</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich&amp;nbsp;</td><td>60-00-4</td><td></td></tr><tr><td>EGF</td><td>Immunological Sciences</td><td>GRF-10544</td><td></td></tr><tr><td>FAST DiI</td><td>Molecular probes, Invitrogen, Thermo Fisher Scientific</td><td>D7756</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco,&amp;nbsp; Thermo Fisher Scientific</td><td>10270-106</td><td></td></tr><tr><td>Fibronectin</td><td>Roche</td><td>11051407001</td><td></td></tr><tr><td>Flask for cell culture</td><td>Corning</td><td>430639</td><td>Sterile, vented</td></tr><tr><td>Gentamicin solution</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G1397-10ML</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBBS)&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>H6648</td><td>Supplemented with EDTA, Glucose, penicillin-streptamicin, gentamicin and fungizone</td></tr><tr><td>High molecular weight hyaluronic acid</td><td>Kind gift by Prof. Ivan Donati</td><td></td><td></td></tr><tr><td>Human endothelial serum free medium&amp;nbsp;</td><td>Gibco,&amp;nbsp; Thermo Fisher Scientific</td><td>11111-044</td><td>Supplemented with EGF (5&amp;thinsp;ng/mL), basic FGF (10&amp;thinsp;ng/mL), and&amp;nbsp; 1% penicillin&amp;ndash;streptomycin (Sigma-Aldrich)</td></tr><tr><td>Magnesium Chloride</td><td>Carlo Erba</td><td>13446-18-9</td><td></td></tr><tr><td>Medium 199 with Hank&amp;rsquo;s salt</td><td>Sigma-Aldrich&amp;nbsp;</td><td>M7653</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P0781</td><td></td></tr><tr><td>Time-lapse microscopy&amp;nbsp;</td><td>Nikon&amp;nbsp;</td><td>Imaging System BioStation IM-Q</td><td></td></tr><tr><td>Titertek Multiskan ELISA Reader</td><td>Flow Labs</td><td></td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T4674</td><td></td></tr></tbody></table>

evaluation of the interplay between the complement protein c1q and hyaluronic acid in promoting cell adhesion

It has been increasingly demonstrated that the tumor microenvironment plays an active role in neoplasia growth and metastasis. Through different pathways, tumor cells can efficiently recruit stromal, immune and endothelial cells by secreting stimulatory factors, chemokines and cytokines. In turn, these cells can alter the signaling properties of the microenvironment by releasing growth-promoting signals, metabolites and extracellular matrix components to sustain high proliferation and metastatic competence. In this context, we identify that the complement component C1q, highly expressed locally by a range of human malignant tumors, upon interacting with the extracellular matrix hyaluronic acid, strongly affects the behavior of primary cells isolated from human tumor specimens. Here, we describe&#160;a method to test how C1q bound to hyaluronic acid (HA) impacts tumor cell adhesion, underlying the fact that the biological properties of key components of the extracellular matrix (in this case HA) can be shaped by bioactive signals toward tumor progression.

HA is a negatively charged high-molecular-weight polysaccharide, which is made up of repeating (&#946;,1-4)-D-glucuronic acid-(&#946;,1-3)-N-acetyl-D-glucosamine disaccharide units (Figure 1B)7. The occurrence of the binding of HA on the 96-well plate as well as the efficiency of its immobilization were tested taking advantage of biotinylated HA (bio-HA). Different concentrations of Bio-HA, ranging from 10 &#181;g/mL to 1 mg/mL, were re-suspended in 100 mM carbonate/bicarbonate-buffer pH 9.6 and incubated overnight at 4 &#176;C.
After extensive washes, bio-HA bound to 96-wells plate was detected using streptavidin conjugated to alkaline phosphatase, while the presence of streptavidin was quantified using pNPP (1 mg/mL) as a substrate. The reading was performed at the wavelength of 405 nm using an ELISA reader. Bio-HA was able to bind to a 96-well plate in a dose response manner (data not shown); 50 &#181;g/mL was chosen as a saturation plateau and therefore used in our assays.
Our previous data demonstrated that C1q is able to bind to a wide range of target ligands localized in the ECM8. This binding is particularly strong with HA9. The wells of a microtiter plate were coated with 50 &#181;g/mL HMW-HA and incubated with increasing concentration of C1q. Bound C1q was visualized upon&#160;incubation&#160;with anti-C1q polyclonal antibodies. C1q is able to bind to high molecular weight HA in a dose dependent manner (Figure 1A), reaching the maximum level of binding at the concentration of 50 &#181;g/mL. Having established that C1q can bind to HA, we investigated the implication of this interaction in modifying the signaling properties of the ECM and their implications in tumor development. To this aim, we established a protocol to isolated tumor cells from bioptic specimens obtained from affected patients. Primary tumor cells were isolated from tissue biopsy, as summarized in Figure 2. The capability of tumor cells to interact with C1q was evaluated by performing a cell adhesion assay using different matrix combinations, as shown in Figure 3. Tumor cells were stained with the fluorescent probe FAST DiI and seeded onto immobilized HA, HA bound to C1q or to BSA (used as a negative control), for 35 min. If compared to HA, the coating with HA-bound-C1q was able to increase the amount of adhering primary cells but is not able to stimulate the adhesion of tumor cell lines that we tested, as shown in the representative graph in Figure 3.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58688/58688fig1v2.jpg" /> 
Figure 1. Interaction of C1q with hyaluronic acid. (A) Binding of C1q on HMW HA in a dose dependent manner. The data are expressed as the mean of three independent experiments in triplicate &#177; S.E.M. (B) Schematic representation of the C1q molecule and of the recombinant single chain globular region. C1q is assembled from three polypeptide chains (A, B, C): each containing an N-terminal collagen-like sequence and a C-terminal globular gC1q head. (C) Chemical formula of hyaluronic acid, a highly polymerized chain of glucuronic acid and N-acetylglucosamine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58688/58688fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58688/58688fig2.jpg" /> 
Figure 2.&#160;Summary scheme of the isolation and morphology of tumor cells. Tumor cells were isolated from pleural biopsy specimens. The cells were seeded in a 12.5 cm2 flask and cultured in Human Endothelial Cell Serum Free Medium + 10% FBS. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58688/58688fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58688/58688fig3v2.jpg" /> 
Figure 3. Effect of C1q on tumor cells adhesion. Tumor cells (MES) or primary mesothelioma cell line (MSTO-211H) were seeded to microtiter wells pre-coated with hyaluronic acid (HA), HA bound to C1q or to bovine serum albumin (BSA). In the upper part of the figure, the morphological aspect of one representative primary cell line adhered to HA, HA bound to C1q or to BSA was shown. Images were acquired via phase-contrast microscope, original magnification: 200x. FAST DiI labelled primary mesothelioma cells or a mesothelioma cell line (MSTO-211H) were allowed to adhere to microtiter wells pre-coated with HA, HA bound to C1q or to BSA. The data are expressed as mean of three independent experiments done in triplicates &#177; S.E.M. * p &#60; 0.01 vs HA.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58688/58688fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Evaluation of the Interplay Between the Complement Protein C1q and Hyaluronic Acid in Promoting Cell Adhesion at JoVE.com

Evaluation of the Interplay Between the Complement Protein C1q and Hyaluronic Acid in Promoting Cell Adhesion

The complement component C1q is a pro-inflammatory molecule highly expressed in the tissue microenvironment that can interact with the extracellular matrix. Here, we describe&#160;a method to test how C1q bound to hyaluronic acid impacts cell adhesion.

It has been increasingly demonstrated that the tumor microenvironment plays an active role in neoplasia growth and metastasis. Through different pathways, ...

evaluation-interplay-between-complement-protein-c1q-hyaluronic-acid

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

5.9K Views.  University of Trieste. The complement component C1q is a pro-inflammatory molecule highly expressed in the tissue microenvironment that can interact with the extracellular matrix. Here, we describe a method to test how C1q bound to hyaluronic acid impacts cell adhesion.

Video: Evaluation of the Interplay Between the Complement Protein C1q and Hyaluronic Acid in Promoting Cell Adhesion

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Bioengineering

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but especially on red blood cells (RBCs). As a receptor for the complement components C3b and C4b, CR1 regulates the activation of the complement cascade and promotes the phagocytosis of immune complexes and cellular debris, as well as the amyloid-beta (A&#946;) peptide in Alzheimer's disease (AD). Several studies have confirmed AD-associated single nucleotide polymorphisms (SNPs), as well as a copy-number variation (CNV) in the CR1 gene. Here, we describe an innovative method for determining the length polymorphism of the CR1 receptor. The receptor includes three domains, called long homologous repeats (LHR)-LHR-A, LHR-C, and LHR-D-and an n domain, LHR-B, where n is an integer between 0 and 3. Using a single pair of specific primers, the genetic material is used to amplify a first fragment of the LHR-B domain (the variant amplicon B) and a second fragment of the LHR-C domain (the invariant amplicon). The variant amplicon B and the invariant amplicon display differences at five nucleotides outside of the hybridization areas of said primers. The numbers of variant amplicons B and of invariant amplicons is deduced using a quantitative tool (high-resolution melting (HRM) curves), and the ratio of the variant amplicon B to the invariant amplicon differs according to the CR1 length polymorphism. This method provides several advantages over the canonical phenotype method, as it does not require fresh material and is cheaper, faster, and therefore applicable to larger populations. Thus, the use of this method should be helpful to better understand the role of CR1 isoforms in the pathogenesis of diseases such as AD.

Here, we describe an innovative method to determine complement receptor 1 (CR1) length polymorphisms for use in several applications, particularly the assessment of susceptibility to diseases such as Alzheimer&#39;s disease (AD). This method could be useful to better understand the role of CR1 isoforms in the pathogenesis of AD.

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but ...

Genetics

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube is an essential process for NCC production and their subsequent migration toward target sites. The migratory route of NCCs, including the surrounding neural tube tissues, involves hyaluronan (HA)-rich extracellular matrix. To model NCC migration into these HA-rich surrounding tissues from the neural tube, a mixed substrate migration assay consisting of HA (average molecular weight: 1,200-1,400 kDa) and collagen type I (Col1) was established in this study. This migration assay demonstrates that NCC cell line, O9-1, cells are highly migratory on the mixed substrate and that the HA coating is degraded at the site of focal adhesions in the course of migration. This in vitro model can be useful for further exploration of the mechanistic basis involved in NCC migration. This protocol is also applicable for evaluating different substrates as scaffolds to study NCC migration.

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

This protocol outlines an in vitro migration experiment suitable for the functional analysis of the molecules involved in the in vivo migration of neural crest cells into the hyaluronan-rich extracellular matrix.

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube ...

Developmental Biology

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The&#160;Candida clade, including Cp, is increasingly developing resistance to the first and the second line of antifungals. Cp is frequently isolated from hands and skin surfaces, as well as the GI tract. Colonization by Candida predisposes individuals to invasive bloodstream infections. To successfully colonize or invade the host, yeast must be able to rapidly adhere to the body surfaces to prevent elimination by host defense mechanisms. Here we describe a method to measure adhesion of Cp to immobilized proteins under physiologic fluid shear, using an end-point adhesion assay in a commercially available multichannel microfluidic device. This method is optimized to improve reproducibility, minimize subjectivity, and allow for the fluorescent quantification of individual isolates. We also demonstrate that some clinical isolates of Cp show increased adhesion when grown in conditions mimicking a mammalian host, whereas a frequently used lab strain, CDC317, is non-adhesive under fluid shear.

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

Adhesion is an important first step in colonization and pathogenesis for Candida. Here, an in vitro assay is described to measure adhesion of C. parapsilosis isolates to immobilized proteins under fluid shear. A multichannel microfluidics device is used to compare multiple samples in parallel, followed by quantification using fluorescence imaging.

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The&#160;Candida clade, including Cp, is increasingly ...

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse formation and maintenance. Many of these fundamental interactions occur in trans and are typically induced by heterophilic or homophilic interactions between cells expressing membrane anchored binding pairs. Elucidating how disease relevant mutations disrupt these fundamental protein interactions can provide insight into a myriad of cell biology fields. Many protein-protein interaction assays do not typically disambiguate between cis and trans interactions, which potentially leads to an overestimation of the extent of binding that is occurring in vivo and involve labor intensive purification of protein and/or specialized monitoring equipment. Here, we present an optimized simple protocol that allows for the observation and quantification of only trans interactions without the need for lengthy protein purifications or specialized equipment. The HEK cell aggregation assay involves the mixing of two independent populations of HEK cells, each expressing membrane-bound cognate ligands. After a short incubation period, samples are imaged and the resulting aggregates are quantified.

Here, we present an optimized protocol to rapidly and semiquantitatively measure ligand-receptor interactions in trans in a heterologous cell system using fluorescence microscopy.

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse ...

Neuroscience

Immunoglobulin-like Cell Adhesion Molecules

Immunoglobulin-like cell adhesion molecules or Ig-CAMs are a versatile group of cell surface glycoproteins belonging to the immunoglobulin protein superfamily. Ig-CAMs possess the characteristic immunoglobulin protein domains and other domains such as the fibronectin type III domain. The Ig domains are glycosylated to varying degrees in different Ig-CAMs.
Ig-CAMs exhibit either homophilic binding (to other Ig-CAMs) or heterophilic binding (to other ligands such as integrins). While most Ig-CAMs are involved in transient cell adhesion, the nectin class of molecules forms stable junctions, such as the adherens junctions, in conjunction with other CAMs. This versatility is reflected in diverse functions of Ig-CAMs, including cell adhesion and signaling, development, and infection.
Neural Ig-CAMS
Neurons express Ig-CAMs like NCAM and L1, which play an essential role in neural development. For example, children with L1 deficiency disease are missing two large nerve tracts, indicating the role of L1 in directing axon growth during embryonic development. Additionally, the homophilic interactions of NCAMs between neurons induce neurite outgrowth via the fibroblast growth factor receptor (FGFR). NCAMs continue to play a role in maintaining neural connections crucial for learning and forming memories.
Ig-CAMs in Disease
Ig-CAMs are involved in recruiting free-flowing leukocytes during an immune response. They play an important role in the extravasation of leukocytes from the bloodstream into the target tissue. Cancer cells hijack this process during metastasis to infiltrate a new site and form secondary tumors. Many cancers such as myeloid leukemia, pheochromocytoma, and Wilm&#8217;s tumor stain positive for NCAM2, and this property is used in pathology for tumor identification. Some Ig-CAMs also act as receptors for the entry of viruses into the host cell. Examples of viruses that utilize Ig-CAMs include the rabies virus and the human rhinovirus.

Evaluation of the Interplay Between the Complement Protein C1q and Hyaluronic Acid in Promoting Cell Adhesion

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Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Cząsteczki adhezyjne komórek podobne do immunoglobuliny