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) supp...

<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>

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 matern...

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.

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			<td>Sterile, vented</td>
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			<td>Supplemented with EDTA, Glucose, penicillin-streptamicin, gentamicin and fungizone</td>
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			<td height="21" style="height:21px;width:330px;">High molecular weight hyaluronic acid</td>
			<td style="width:311px;">Kind gift by Prof. Ivan Donati</td>
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			<td>Supplemented with EGF (5&#8201;ng/mL), basic FGF (10&#8201;ng/mL), and&#160; 1% penicillin&#8211;streptomycin (Sigma-Aldrich)</td>
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			<td height="42" style="height:42px;width:330px;">Time-lapse microscopy&#160;</td>
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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 r...

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, ...

A comprehensive procedure to evaluate the interplay between the complement protein C1q and hyaluronic acid in promoting cell adhesion. We have recently defined that the complement component C1q a molecule of the innate immune system is highly expressed in the tumor and placenta microenvironment and is able to interact with component of the extracellular matrix. In this case, hyaluronic acid.We propose a method for studying the biological functions of proteins throughout the ecronomic environment. In order to investigate the ability of these molecules to shape the cell division properties of extracellular matrix factors. In order to study how this interaction can be sensed by the cells present in the tissue of the environment, we set up this easy method in which binary cells derived from human tissues were allowed to adhere to C1q bound to extracellular matrix, in this case hyaluronic acid.For coating procedure the first step is the binding of high molecular weight hyaluronic acid to 96 well tissue coated microplate. For coating, hyaluronic acid is diluted in 0.1 mole carbon buffer PH 9.6 at a concentration of 15 micrograms per milliliter. And gently pipeting is requited to achieve a homogeneous solution.A 100 microliters of the solution are used to coat the wells. The plate is then transferred at four degrees Celsius overnight. Next day vacuum aspirate the treated wells.Wash once with dPBS. Add either C1q or BSA in the corresponding wells. C1q is nowadays commercially available at a concentration of one milligram per milliliter and it is diluted at a concentration of 25 micrograms per milliliter.And dPBS containing 0.5%BSA and 0.7 millimoles calcium and magnesium and divalent cations. BSA LPS free is used at the concentration of 0.5%and date PBF. Plate is then transferred at four degrees Celsius overnight.C1q dose dependent interactions with immobilized hyaluronic acid is determined by enzyme linked immuno SA.Cells labeling with fast DiI. The lipophilic tracer fast DiI is used to label the cells of interest. Cells resuspended in dPBS are used at the concentration of one million cells per milliliter.Fast DiI is diluted one to 100 directly into the cell suspension. The cell suspension is mixed manually and transferred at 37 degrees Celsius for 15 minutes. After five minutes incubation, mix the cell suspension and repeat this operation twice more.To remove the excess of fast DiI, add 10 milliliters of dPBS. Pivot gently up and down and centrifuge at 250 g for seven minutes. Resuspend the cell pellet in one milliliter of human endophilical serum free medium in the presence of 0.1%BSA.Cells adhesion on hyaluronic acid C1q matrices. Vacuum aspirate the dPBS from the coated wells. Distribute 100 microliters of labeled cell suspension to the coated wells.200 microliters of labeled cells are taken apart in an ependure tube to be used for standard curve generation. Incubate the plate for 35 minutes at 37 degrees Celsius. Take out the plate from the incubator.Gently aspirate the unbalanced cells. Wash once with dPBS containing 0.5%BSA and 0.7 millimoles calcium and magnesium. Lys the adherent cells by adding 200 microliters of lysis buffer.At the same time prepare the standard curve upon one to two serial dilution of the labeled cells in lysis buffer. Perform the reading of the florescence signal at infinite F 200 T-can instrument using 544 nanometers as excitation wavelength and 590 nanometers as emission wavelength. Representative results.As shown by the representative results, it is evident that cell adhesion on hyaluronic acid found C1q as strongly enhanced in comparison with HA alone or HA bound BSA. The percent of adherent cells on each matrix is calculated on the basis of the standard curve.Conclusions. In our experiments first of all we evaluated the ability of the complement component C12 to bind the extracellular matrix in this case hyaluronic acid using an arisal metal.Secondly we analyzed the capability of human primary cells stained with fluorescent dye to pursue bound hyaluronic acid. The model that we proposed in this study is an easier faster and cheaper method in alternative to the 3D model of extracellular matrix scaffolds for the evaluation on this cellular response to a combination of stimuli.

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Immunology and Infection

5.9K 조회수.  University of Trieste. 세포 접착을 촉진하는 보완 단백질 C1q와 히알루론산 간의 상호 작용을 평가하는 포괄적인 절차. 우리는 최근에 선천적인 면역 계통의 보충 성분 C1q가 종양 과 태반 미세 환경에서 높게 발현되고 세포외 매트릭스의 성분과 상호 작용할 수 있다는 것을 정의했습니다. 이 경우 히알루론산.우리는 음경 환경 전반에 걸쳐 단백질의 생물학적 기능을 연구하는 방법을 제안합니...