Reconstituted basement membrane (matrigel) and laminin can enhance the tumorigenicity and the drug resistance of small cell lung cancer cell lines.

Abstract

Small cell lung cancer (SCLC) is a fatal malignancy due to its propensity to metastasize widely and to reoccur after chemotherapy in a drug-resistant form. While most SCLC cell lines are anchorage independent for growth, laminin induced the attachment of five of six SCLC cell lines tested (NCI-N417, NCI-H345, NCI-H146, NCI-H187, NCI-H510, and NCI-H209 These observations suggest that the interaction of cells with laminin as well as with other basement membrane components may play a role in tumor growth and drug resistance.MATERIALS AND METHODS Extracellular Matrix Proteins, Matrigel, and Synthetic Peptides. Laminin, collagen IV, heparan sulfate proteoglycan, and matrigel were all isolated from the EHS tumor as described (12, 13) and were used as substrates in vitro in standard attachment assays as described (5, 6). Human fibronectin and bovine collagen I were purchased from Collaborative Research. Cyclic peptides were synthesized as described (14) with an automated synthesizer model 930A (Applied Biosystems), and their purity was ascertained by amino acid analyses and by high-performance liquid chromatography. The synthetic peptides were filtered to remove contaminating endotoxin (14).Cell Culture. SCLC cell lines (NCI-N417, NCI-H187, NCI-146, NCI-H209, NCI-510) were cultured in RPMI 1640 medium (GIBCO) supplemented with 10o fetal calf serum, glutamine (2 mM), penicillin (100 units/ml), streptomycin (100 ,g/ml), gentamicin (50 ,ug/ml), and Hepes buffer (25 mM) and were all purchased from GIBCO. NCI-H345 cells were grown in serum-free medium, HITES, as described (15).Cell Motility Assay. Cell migration was tested by using modified Boyden chambers with a polyvinylpyrrolidone-free (pore size, 8 ,um) polycarbonate filter (Neuroprobe, Cabin John, MD) placed over the lower well previously filled with RPMI 1640 medium (0.22 ml) containing 0.1% bovine serum albumin and increasing concentrations of laminin. Alternatively, laminin was dried directly on the lower surface of the filters. A suspension of SCLC cells (2 x 105 cells in 0.85 ml of RPMI 1640 medium/0.1% bovine serum albumin) was added to the upper well. After a 5-hr incubation at 37°C in 5% C02/95% air, the filters were removed, fixed, and stained with Diff-Quick stain (American Scientific Products, McGaw Park, IL). The area occupied by the cells that migrated to the lower surface of the filters was quantitated by image analysis (Optomax IV, Analytical Measuring Systems, Essex, U.K.).In Vivo Tumor Studies. Cultured SCLC cells were harvested by centrifugation (5 min at 1000 rpm), the cell clumps were disaggregated by gentle trituration, and an aliquot ofthe suspension was assessed for cell number and viability. Cells were resuspended in cold serum-free RPMI 1640 medium and mixed with either an equal volume of cold liquid matrigel (10 mg/ml), laminin (2 mg/ml), or collagen I (3 mg/ml, previously neutralized), and a final vol of 0.5 ml was immediately Abbreviation: SCLC, small cell lung cancer.

Multiple uses of basement membrane‐like matrix (BME/Matrigel) in vitro and in vivo with cancer cells

“…51 Coinjection with BME greatly increases the 'take' of biopsy specimens (tumorgrafts) in mice to greater than 90%. 17,18 The procedure is simple and only requires premixing of the cells with BME at 4 C (liquid BME) before injection (Fig. 3).…”

“…Furthermore, BME coinjection with cancer cells will also increase the growth rate of the tumors with cells that already can form rapidly growing tumors in mice. For example, small cell lung cancer and MCF-7 cell lines were unable to form tumors in mice unless coinjected with BME, and while B16F10 melanoma cells do form growing tumors in mice when injected alone, the growth rate of the tumors can be increased several-fold when the melanoma cells are coinjected with BME 17,18,51 (Fig. 4).…”

“…15,16 Coinjection of the basement membrane-like matrix with cancer cells either subcutaneously or in orthotopic sites was found to increase both the 'take' and growth of tumors in mice. [17][18][19] Thus, cancer cell interactions with the basement membrane have multiple uses both in vitro and in vivo (Table 2). the vascular basement membrane.…”

Effect of reconstituted basement membrane components on the growth of a panel of human tumour cell lines in nude mice

“…The reasons for these results are unknown. Previous reports of co-injection with purified laminin have indicated that in most cases it does not enhance tumour growth, and it has been suggested that this is because of the highly soluble nature of laminin which renders it susceptible to degradation in vivo (Fridman et al, 1990). …”

“…Matrigel is composed of laminin, collagen type IV, heparin sulphate proteoglycan and entactin, along with TGF, and other growth factors which occur in the EHS tumour. Co-transplantation with matrigel has been found to enhance the growth of a range of cells in vivo including small cell lung carcinoma (SCLC), human prostatic carcinoma and human breast adenocarcinoma (Fridman et al, 1990;Pretlow et al, 1991;Noel et al, 1992).…”