Incidence of serum antibody reactivity to autologous head and neck cancer cell lines and augmentation of antibody reactivity following acid dissociation and ultrafiltration

Serum antibody reactivity to squamous cell carcinoma of the head and neck (SCCHN) was evaluated in 41 autologous serum-tumor cell line combinations using the protein A hemadsorption assay. Autologous antibody reactivity (median titer of 1:4) was detected in sera from 24 of the patients tested. In 10 cases autologous antibody reactivity could be detected only in undiluted serum precluding further analysis. Analysis of higher titer sera from one patient revealed antibodies that define an antigen expressed on autologous tumor cells cultured from both the primary tumor (UM-SCC-17A) and from a metastasis (UM-SCC-17B). Absorption analysis showed that this antigen was also expressed on 6 of 10 allogeneic SCCHN cell lines but not on autologous fibroblasts or on allogeneic melanoma cell lines. Due to the low titer of autologous antibody reactivity in most sera, we sought to determine if dissociation of immune complexes through acidification and ultrafiltration of serum might enhance detectable antibody reactivity as has been done in previous studies in melanoma. Twelve serum samples from eight patients were subjected to acid dissociation and ultrafiltration (AD-U). Only six of the untreated sera had detectable antibody reactivity against the autologous SCCHN cell line whereas following AD-U all 12 sera had enhanced IgG reactivity against autologous SCCHN. Specificity analysis of one serum sample after dissociation revealed that the antibody detected an antigen common to SCCHN cell lines as well as melanoma, glioma, renal, and colon carcinoma cell lines. Circulating immune complexes may provide a reservoir of antibody with potential diagnostic and therapeutic applications.     

Membrane antigen in small cell carcinoma of the lung defined by monoclonal antibody SM1

Mouse myeloma cells were fused with spleen cells from BALB/c mice immunized with a cell line derived from human small cell carcinoma (SCC) of the lung. The cloned hybridoma SM1 produced antibody that was reactive with the surface membrane of SCC cell lines and SCC tumors but not with the membrane of several non-SCC cell lines and tumors. SM1 ascites fluid was used to screen for reactivity of the antibody with other human cancer cell lines, tumor tissues, and normal tissues. SM1 antibody was found to be unreactive with neuroblastoma, adrenal carcinoma, melanoma, and bronchial carcinoid. Reactivity was detected with some breast carcinoma cell lines but not with breast cancer tissue specimens. In the same individual, the antibody was reactive with SCC lung tumor and SCC metastatic to the liver but not with normal tissues, including bronchus, lung parenchyma, liver, kidney, and brain. Human erythrocytes and marrow cells were also unreactive. Since SM1 detects an antigen that is present in greatest amounts on the surface membrane of SCC of the lung, this antibody may be useful in tracing the lineage patterns of human lung cancers.     

Monoclonal antibody against human squamous-cell-carcinoma-associated antigen

Mouse monoclonal antibody (MAb) 3F8E3 (IgG3k) was developed against the head and neck cancer cell line LICR-LON-HN2. Subjected to indirect immunofluorescence, the MAb reacted exclusively with SCC cell lines and showed no reactivity with normal or transformed mouse and human non-SCC cell lines and hematopoietic cell lines. The radiolabelled MAb showed an affinity constant of 1.8 x 10(8) M-1 with HN2 cells and identified 2.07 x 10(4) sites/cell by Scatchard analysis. It identified 2 peptides from membrane extracts of HN2 cells by Western blotting. Avidin-biotin-complexed immunoperoxidase staining on cryostat sections of tumors from various tissues revealed that 3F8E3 reacted mainly with the membrane antigens of well differentiated SCC cells of oral cavity, larynx, esophagus, lung, uterine cervix, metastatic nodes of patients with oral cancer, and dysplastic cells in oral leukoplakia. The MAb did not react with poorly differentiated cells of Ca esophagus, adenocarcinoma of breast, stomach and colon, renal-cell carcinoma and soft-tissue sarcoma.     

Identification of squamous cell carcinoma antigen-derived peptides having the capacity of inducing cancer-reactive CTLs in HLA-A24+ cancer patients

Squamous cell carcinoma antigen (SCCA) is a useful marker for SCCs. In this study, we attempted to identify SCCA-derived peptides that could be applied in the development of specific immunotherapy for HLA-A24+ cancer patients with SCC or non-SCC. A variety of SCC and non-SCC lines were examined for their expression of SCCA mRNA using quantitative PCR. SCCA protein expression in cancer tissues was investigated by immunohistochemical staining. Thereafter, SCCA-derived peptide candidates were prepared based on their binding motifs to HLA-A24 molecules. Among these peptides, SCCA-derived peptides that were frequently recognized by humoral immunity were further tested for their ability to induce cancer-reactive cytotoxic T lymphocytes (CTLs) from the peripheral blood mononuclear cells of HLA-A24+ patients with SCC or non-SCC. As a result, the majority of SCC lines and tissues were positive for SCCA both at mRNA and protein levels. By contrast, non-SCC cancer tissues hardly expressed it at the protein level, although adenocarcinoma cell lines partly expressed it at the mRNA level. Four SCCA-derived peptides were frequently recognized by immunoglobulin G of both SCC and non-SCC cancer patients. Among these peptides, both the SCCA(112-120) and SCCA(215-224) peptides were found to effectively induce peptide-specific CTLs toward HLA-A24+ SCCA+ cancer cells from the peripheral blood mononuclear cells of both SCC and non-SCC cancer patients. Two newly identified SCCA-derived peptides with the ability to induce CTL activity in both SCC and non-SCC cancer patients may be applicable to specific immunotherapy for HLA-A24+ cancer patients with SCC, but not those with non-SCC.     

Altered expression in squamous carcinoma cells of an orientation restricted epithelial antigen detected by monoclonal antibody A9

The monoclonal IgG2a antibody A9 was raised to the UM-SCC-1 human squamous cell carcinoma cell line. Hemadsorption assays using monolayer cultures as target cells revealed a restricted range of A9 reactivity with human cell lines. The A9 antibody was reactive with 29 of 34 squamous cell carcinoma cell lines and with 5 epithelial cancer cell lines of non-squamous origin. In contrast, no antibody binding was detected with twelve malignant melanoma, two fibrosarcoma, two malignant lymphoid, two transitional cell carcinoma, or four adenocarcinoma cell lines. Similarly, normal lymphoid cells, RBC, and fibroblasts from multiple donors were negative. The relative expression of the A9 antigen varied greatly among positive squamous cancer lines such that the 50% endpoint titer of A9 ascites fluid ranged from less than 10(1) for low antigen expressor lines to 10(6) for strong antigen expressors. Hemadsorption tests with secondary passage cultures of normal squamous cells failed to detect A9 binding to the cell surface. However, in experiments with intact colonies in primary cultures of normal squamous cells, antibody binding was observed at the periphery of individual colonies. Mechanical detachment of such colonies revealed A9 antigen bound to the plastic surface underneath the cells, but the newly exposed surface of the cells, like the upper surface, was negative. In contrast, when cells of similar cultures were detached by trypsinization prior to testing no antigen remained on the plastic, but approximately 30% of the trypsinized cells were positive. Similar experiments with the five surface-negative squamous cell carcinoma cell lines revealed that cryptic A9 antigen could also be detected underneath mechanically detached cells and on the surface of 100% of trypsinized cells. Trypsinization of melanomas and fibroblasts did not reveal A9 antigen. Immunoperoxidase assays on frozen sections of normal epidermis localized A9 antigen to the basal cells and the basement membrane region. In frozen sections of squamous cancers, individual tumor cells and particularly the growing edge of the tumors were strongly stained by A9, while in basal cell cancers only very slight staining could be detected. Thus, in normal epithelial cells the A9 antigen appears to be expressed only by the stem cell subset. In squamous cancer cells, the expression of the A9 antigen is not only increased but appears to have escaped the orientation restriction characteristic of normal epithelial cells.     

Surface-glycoprotein patterns of established human lung cancer cell lines and primary cultures

The cell surface glycoprotein (GP) profiles of cell lines representing the four major histopathological lung cancer entities, squamous cell (SQC)-, small cell (SCC)-, adeno (ADC)-, and large cell carcinoma (LCC), and two primary cultures of SCC and LCC, respectively, have been examined by the galactose-oxidase tritiated sodium-borohydride cell surface labelling method, The SCC specimens (five cell lines and one biopsy) had a characteristic pattern of major surface GPs, with common GPs at apparent molecular weights (MWs) of 54 -kilo (k) Daltons (D) and 88 kD, which was discriminative from the group of non-SCC (SQC, ADC and LCC). The non-SCC group constantly expressed GPs at apparent MWs of 80 kD and 110 kD, both as established cell lines and in the primary LCC culture. The GP patterns of the SCC cell lines and the LCC cell line were retained in comparison to corresponding primary biopsy material. The propagation of an established SCC cell line without supplementation of serum did not alter the GP expression at the cell surface. Taken together, the surface GP patterns for SCC versus non-SCC appear to be reliable and reproducible markers for these tumor entities, both in biopsy material and in established cell lines.     

Steroid hormone receptors in human squamous carcinoma cell lines

Specific estrogen, progesterone and androgen receptors were determined in 19 well-characterized squamous-cell carcinoma (SCC) cell lines. Ten of the lines were derived from patients with SCC of the larynx and nine were from patients with SCC originating in other areas of the head and neck. Estrogen receptors (ER) were found in seven of 10 cell lines derived from squamous cancers of the larynx (70%) but in only one of the nine SCC cell lines from sites other than larynx. Progesterone receptors (PGR) were more evenly distributed. Eight of the 10 laryngeal carcinoma cell lines (80%) and five of the nine non-laryngeal SCC lines (55%) had progesterone receptors. Only one cell line, UM-SCC-10B (derived from a recurrent carcinoma of the larynx) was found to express androgen receptors (AR). Expression of specific estrogen receptors was not dependent on the sex of the patients since lines from both males and females contained receptors. These results establish that squamous carcinoma cell lines may express specific steroid hormone receptors and that cell lines from cancers of the larynx (an organ known to be androgen-responsive) are more likely to express estrogen receptors than androgen receptors. From this initial survey it appears that there is a striking difference in estrogen and progesterone receptor content between SCC cell lines originating from larynx cancers and cell lines established from squamous carcinomas of other head and neck regions. The presence of estrogen receptors in a high proportion of laryngeal carcinoma cell lines suggests that hormonal therapy may be a useful adjunctive therapy in selected patients with cancer of the larynx.     

Establishment of a monoclonal antibody against glycosylated CD271 specific for cancer cells in immunohistochemistry

Various proteins are highly expressed in cancer (e.g., epidermal growth factor receptor); however, the majority are also expressed in normal cells, although they may differ in expression intensity. Recently, we reported that CD271 (nerve growth factor receptor), a glycosylated protein, increases malignant behavior of cancer, particularly stemlike phenotypes in squamous cell carcinoma (SCC). CD271 is expressed in SCC and in normal epithelial basal cells. Glycosylation alterations generally occur in cancer cells; therefore, we attempted to establish a cancer‐specific anti‐glycosylated CD271 antibody. We purified recombinant glycosylated CD271 protein, immunized mice with the protein, and screened hybridomas using an ELISA assay with cancer cell lines. We established a clone G4B1 against CD271 which is glycosylated with O‐glycan and sialic acid. The G4B1 antibody reacted with the CD271 protein expressed in esophageal cancer, but not in normal esophageal basal cells. This specificity was confirmed in hypopharyngeal and cervical cancers. G4B1 antibody recognized the fetal esophageal epithelium and Barrett''s esophagus, which possess stem cell–like characteristics. In conclusion, G4B1 antibody could be useful for precise identification of dysplasia and cancer cells in SCC.     

Endothelial derived factors inhibit anoikis of head and neck cancer stem cells

Recent evidence demonstrated that cancer stem cells reside in close proximity to blood vessels in human head and neck squamous cell carcinomas (HNSCC). These findings suggest the existence of a supporting perivascular niche for cancer stem cells. The purpose of this study was to evaluate the effect of endothelial cell-secreted factors on the behavior of head and neck cancer stem-like cells (HNCSC). HNCSC were identified by sorting UM-SCC-22A (cell line derived from a primary squamous cell carcinoma of the oropharynx) and UM-SCC-22B (derived from the metastatic lymph node of the same patient) for CD44 expression and ALDH (aldehyde dehydrogenase) activity. HNCSC (ALDH+CD44+) and control (ALDH-CD44-) cells were cultured in ultra-low attachment plates in presence of conditioned medium from primary human endothelial cells. ALDH+CD44+ generated more orospheres than control cells when cultured in suspension. The growth factor milieu secreted by endothelial cells protected HNCSC against anoikis. Mechanistic studies revealed that endothelial cell-secreted vascular endothelial growth factor (VEGF) induces proliferation of HNCSC derived from primary UM-SCC-22A, but not from the metastatic UM-SCC-22B. Likewise, blockade of VEGF abrogated endothelial cell-induced Akt phosphorylation in HNCSC derived from UM-SCC-22A while it had a modest effect in Akt phosphorylation in HNCSC from UM-SCC-22B. This study revealed that endothelial cells initiate a crosstalk that protect head and neck cancer stem cells against anoikis, and suggest that therapeutic interference with this crosstalk might be beneficial for patients with head and neck cancer.     

Relationship between peroxisome proliferator-activated receptor-gamma expression and differentiation of human esophageal squamous cell carcinoma

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily, is involved in suppressing the growth of several tumors. We showed that PPAR-gamma is expressed in Barrett's adenocarcinoma cell lines and inhibited the growth of these lines through the induction of G1 cell cycle arrest and apoptosis. We examined PPAR-gamma expression in human esophageal squamous cell carcinoma (SCC) in vitro and in vivo and investigated whether PPAR-gamma ligands affect the proliferation and apoptosis of human SCC cell lines. Biopsy specimens (n=46) obtained from human SCC of the esophagus were stained using a monoclonal antibody against human PPAR-gamma. We assessed the effects of PPAR-gamma ligands on the growth of SCC cells by adding 15-deoxy prostaglandin J2 (15d-PGJ2), or troglitazone to six human esophageal SCC cell lines (TE-1, TE-2, TE-3, TE-5, TE-8, and TE-9). Immunohistochemical staining showed that 34 of 46 (73.9%) SCC of the esophagus expressed PPAR-gamma. All SCC cell lines expressed PPAR-gamma mRNA and protein, especially when poorly differentiated (TE-2, TE-5, and TE-9). The PPAR-gamma ligands significantly and dose-dependently inhibited the proliferation of SCC lines, except for well-differentiated TE-1 and TE-3. Apoptosis was induced by 15d-PGJ2 (10 microM) in all tested SCC lines except TE-1, whereas troglitazone (50 microM) was marginally effective in only the TE-2 and TE-3 cell lines. The present findings suggest that PPAR-gamma could be a therapeutic target for treating squamous cell carcinoma of the esophagus, possibly through the induction of apoptosis.     

Clinical implication of cyclin B1 in non-small cell lung cancer

Cyclin B1 plays an important role in the mitotic cycle, in which it regulates the G2-M transition, and has been suggested to play a role in development and progression of various cancers. The present study was undertaken in order to clarify the role of the cell cycle regulator cyclin B1 in non-small cell carcinoma (NSCLC). We retrospectively investigated 174 patients with NSCLC who previously underwent complete resection. There were two study groups: the squamous cell carcinoma (SCC) group (n=62); and the non-SCC group (n=112). Expression of cyclin B1 in cancer cells was analyzed immunohistochemically. The rate of cyclin B1 positivity in cancer cells ranged from 0% to 56.5% (average 10.9%). Seventy-four cases (42.5%) were designated as cyclin B1 positive in the present study. Cyclin B1 expression was observed more frequently in SCC cases than in non-SCC cases. In SCC, cyclin B1 expression demonstrated significant correlation with gender (p<0.01) and histological type (p<0.01). In non-SCC, only gender was correlated with cyclin B1 expression. Five-year survival rates for cyclin B1-positive and cyclin B1-negative cases were 45.8 and 57.9%, and 10-year survival rates were 39.3 and 51.4%, respectively. Patients with positive cyclin B1 staining showed a lower survival rate than those with negative staining (p=0.11). The prognostic value in SCC cases was p=0.48. In non-SCC cases, the survival rate of non-SCC patients who were positive for cyclin B1 was significantly lower than that of patients who were negative (p<0.01). Using multivariate analysis, tumor size (p=0.037) and N factor (p=0.026) were found to be independent prognostic parameters. Cyclin B1 expression was not an independent prognostic factor in the present series. These data suggest that elevated levels of cyclin B1 expression may be an indicator of poor prognosis in NSCLC, particularly in non-SCC.     

Anti-epidermal growth factor receptor monoclonal antibody 225 upregulates p27(KIP1) and p15(INK4B) and induces G1 arrest in oral squamous carcinoma cell lines

Epidermal growth factor receptor (EGFR) regulates the growth and progression of human oral squamous cell carcinoma (SCC). Recently, the link between EGFR signaling and the cell cycle has been identified. Some reports have described that EGFR-blocking monoclonal antibody 225 (mAb225) induced G1 arrest and inhibited the growth of various cancer cells. The purpose of this study was to evaluate the effect of mAb225 on human oral SCC cell lines. Exposure to mAb225 in culture inhibited the growth of oral SCC cell lines in an EGFR number-independent manner, with the percent inhibition ranging from 13.8 to 76.6%. Flow-cytometric analysis demonstrated that treatment with mAb225 induced cell accumulation in G1 phase, accompanied by a decrease in the percentage of cells in the S phase. Apoptosis was not seen in this study. G1 arrest was accompanied by a decrease in CDK2-, CDK4-, and CDK6-associated histone H1 kinase activities, and an increase in the expression levels of cell cycle inhibitors p27(KIP1) and p15(INK4B). These results suggested that the antiproliferative effect of EGFR blockade by mAb225 in oral SCC may be mediated by p27(KIP1) and p15(INK4B).     

Mouse monoclonal antibody (FKH1) detecting human melanoma-associated antigens

A mouse monoclonal antibody, FKH1, was produced to detect cytoplasmic melanoma-associated antigen. FKH1 was raised using cultured human melanoma cell line KHm-6 as an immunogen. Reactivity of this antibody was assessed by immunohistochemical techniques against cell lines and normal and neoplastic tissues. Positive reactions were seen against 5 human melanoma cell lines. It stained cytoplasm of melanoma cells in a diffuse and granular pattern in indirect immunofluorescence. Immunoelectron microscopy showed diffuse distribution of immunoreactant in the cytoplasm of KHm-1 cells excluding melanosomes and other organelles. Reactivity against frozen and alcohol-fixed, paraffin-embedded melanocytic tumors was also tested with IIF or indirect or avidin biotinylated horseradish peroxidase complex immunoperoxidase techniques. All cases of frozen sections from benign and malignant melanocytic tumors showed positive staining with FKH1. In fixed tissues, however, reactivity was 11 of 14 (79%) in malignant melanoma and 28 of 42 (67%) in other melanocytic tumors. FKH1 did not react against normal melanocytes and nonmelanocytic tumors except APUDoma and 2 glioblastoma cell lines. It failed to stain the B-16 mouse melanoma cell line, neuroblastoma cell line, breast carcinoma cell line, and T-cell lymphoma cell line. Normal human peripheral nerves were nonreactive with FKH1. In immunoelectroblot study, FKH1 bound with proteins having molecular weight of 71,000 and 55,000 extracted from KHm-6 cells. It was suggested that FKH1 is a useful monoclonal antibody in diagnostic study of human malignant melanoma specimens.     

A novel marker for basal (stem) cells of mammalian stratified squamous epithelia and squamous cell carcinomas

We have developed a monoclonal antibody (174H.64) which selectively recognizes antigens shared by the basal cells of mammalian stratified squamous epithelium and squamous cell carcinoma (SCC). Histopathological studies of the frozen tissue sections demonstrated selective binding of this antibody to SCCs of human, bovine, canine, feline, and murine origin. Tumors of other histological types did not show reactivity with the antibody. In well-differentiated SCCs the peripheral layer of the tumor showed preferential binding of the antibody, suggesting that the antigens are associated with the proliferative compartment of the tumor. Studies on normal human tissues showed selective binding of the antibody to the basal layer of stratified squamous epithelia, thymic epithelial cells, and myoepithelial cells around breast ducts, while no antibody binding was observed for the suprabasal layers of stratified epithelia, simple epithelia, or tissues of nonepithelial origin. A similar pattern of antibody binding was also observed for bovine and murine skin with staining of the basal layer. The antigens detected by monoclonal antibody 174H.64 were characterized from cytoskeletal protein extracts of normal human keratinocytes as well as human and bovine SCC tissues by using an immunoblotting technique. The antigens detected in normal human keratinocytes consisted of two major protein bands of approximate molecular weights of 48,000-50,000 and 57,000. In bovine SCC tumor the antigen detected was the Mr 48,000-50,000 band and in the human SCC tumor it was the Mr 57,000 band. A murine lung SCC model was developed with a murine SCC cell line KLN-205. The lung tumor obtained was reactive against the antibody and showed selective staining of the peripheral layer of the tumor containing the stem cell population. The antigens described by monoclonal antibody 174H.64 appear to be molecules associated with the stem cell populations of normal stratified epithelium and squamous cell carcinoma.     

Optical imaging predicts tumor response to anti-EGFR therapy

To evaluate cetuximab treatment in head and neck squamous cell carcinoma xenografts and cell lines, we investigated a preclinical model of head and neck squamous cell carcinoma. Head and neck squamous cell carcinoma cell lines SCC-1, FaDu, CAL27, UM-SCC-5 and UM-SCC-22A were used to generate subcutaneous flank xenografts in SCID mice. Mice were divided into control and cetuximab treatment groups, mice in the latter group received 250 µg cetuximab once weekly for four weeks. After completion of therapy, SCC-1 (p < 0.001), UM-SCC-5 (p < 0.001), UM-SCC-22A (p = 0.016) and FaDu (p = 0.007) tumors were significantly smaller than control, while CAL27 tumors were not different from controls (p = 0.90). Mice were systemically injected with 50 µg of the Cy5.5-cetuximab bioconjugate and imaged by stereomicroscopy to determine if tumor fluorescence predicted tumor response. Intact tumor fluorescence did not predict response. Tissue was harvested from untreated xenografts to evaluate ex vivo imaging. Cell lines were then evaluated in vitro for fluorescence imaging after Cy5.5-cetuximab bioconjugate labeling. The location of fluorescence observed in labeled cells was significantly different for cell lines that responded to treatment, relative to unresponsive cells. Tumors from cell lines that showed low internalized signal in vitro responded best to treatment with cetuximab. This preclinical model may aid in determining which cancer patients are best suited for cetuximab therapy.Key words: cetuximab, head and neck squamous cell carcinoma, fluorescence, optical imaging, cancer therapy, mouse, xenograft     

Clonogenic cell assay for anchorage-dependent squamous carcinoma cell lines using limiting dilution

Clonogenic assays under either anchorage-dependent or -independent conditions are very useful for testing the sensitivity of tumor cells to cytotoxic drugs and radiation. These assays have not been widely used with squamous-cell carcinomas (SCC) because of poor tumor-cell viability and poor cloning efficiency, especially in semi-solid media. To find a clonogenic assay suitable for use with human squamous cancers we tested SCC lines, derived in our laboratory from patients with head and neck cancer, for the capacity to form colonies in soft agar and in 96-well plates. Of 13 UM-SCC lines tested for colony formation in agarose, only UM-SCC-11A was capable of growth in conventional semi-solid media. One other line, UM-SCC-14C, produced colonies in agarose only in the presence of epidermal growth factor. In contrast, all 17 of the SCC lines tested exhibited colony formation in adherent cell culture using limiting dilution in 96-well plates. The plating efficiencies of the SCC lines in the 96-well plate assay ranged from 0.02 to 0.52 colonies (wells)/cell whereas the PE values in soft agar were lower, ranging from 0.0055 to 0.0086 colonies/cell. The 96-well plate assay is not affected by cell migration, a problem encountered with some cell lines when clonogenic assays are performed in Petri dishes. UM-SCC-11A was tested for radiation sensitivity both in soft agar and in the 96-well plate assay. Comparable results were obtained. In summary, the majority of SCC cell lines did not form viable colonies in soft agar but the 96-well plate assay was applicable to a broad spectrum of anchorage-dependent human SCC cell lines and provides an efficient method for evaluating clonogenic cell survival.     

Human squamous cell carcinoma lines express oncofetal 44-kD polypeptide defined by monoclonal antibody to mouse fetus

Most primary human carcinomas uniformly express an oncofetal epitope which has not been demonstrated previously in established human carcinoma cell lines. We successfully derived several low-passage cell lines of human squamous cell carcinoma (SCC) from head and neck tumors using an in vitro adaptation procedure, characterized these lines, and examined them for expression of a 44-kilodalton (kD) polypeptide (PP) oncofetal antigen (OFA) at the cell surface. Newly established an in vitro-passaged SCC cells retained characteristic microvilli, numerous desmosomes and tonofilaments, abundant rough endoplasmic reticulum, osmophilic keratohyaline granules, and other features of the primary SCC cells. These new cell lines and two long-term, established SCC lines (FaDu and Detroit 562) displayed OFA at the cell surface, as determined by flow cytometry using monoclonal antibody (MoAb) 115. While the FaDu and Detroit 562 lines exhibited aneuploidy during flow cytometric analysis, the new, low-passage SCC lines that we developed remained diploid as were the primary SCC cells from which they were derived. We propose that the expression of a 44-kD OFA is a common feature of human SCC. This marker may prove useful in the detection and treatment of these tumors.     

The studies on monoclonal antibody SZ-39 against human glioma cells

A hybridoma cell line SZ-39 secreting monoclonal antibody against the human glioma cell has been established by a fusion between NS-1 myeloma cells and spleen cells from mice immunized with human glioma cell lines. Monoclonal antibody (McAb) SZ-39 was analyzed by ELISA, quantitative absorption, indirect immunofluorescence and ABC immunohistology. McAb SZ-39 strongly bound to 9/10 glioma cell lines, 17/20 glioma tissues, weakly bound to one liver cancer cell line and 1/2 lung cancer line, but they did not band with other tested human cancer linse. NcAb SZ-39 have no crossreaction with lymphocyte, ABC red blood cells, white blood cells, blood platelet normal bone marrow cells, fibroblast cells and 12 normal human tissues. The result indicated the antigen recognized by McAb SZ-39 may be a glioma-associated antigen (GAA). This GAA was analyzed by means of Western blotting. It was a MW 180 Kd glycoprotein. The¹³¹I-McAb SZ-39 specifically localized in human glioma xenografted in nude mice that indicate it may be useful in radioimmunoimaging and as a target for immunotherapy on human glioma.     

Epidermal growth factor receptor monoclonal antibodies inhibit the growth of lung cancer cell lines.

The ability of monoclonal antibody (MAb 108), an immunoglobulin G (IgG)2a against the epidermal growth factor receptor (EGF-R), to interact with lung cancer cell lines was investigated. 125I-EGF bound with high affinity to non-small-cell lung cancer (NSCLC) cells, and MAb 108 inhibited specific binding of nine NSCLC cell lines in a dose-dependent manner (IC50 = 0.3-3 micrograms per ml). 125I-MAb 108 bound with high affinity (kd = 2 nM) to a single class of sites (Bmax = 70,000 per cell) using NSCLC neuroendocrine cell line NCI-H460. Specific 125I-MAb 108 binding was inhibited with high affinity by MAb 108 but not by a control antibody IgG using large-cell carcinoma cell line NCI-H1299. 125I-MAb 108 binding was not internalized at 37 degrees C using NSCLC neuroendocrine cell line NCI-H460 and adenocarcinoma cell line NCI-H23. Also, 1 microgram per ml of MAb 108 but not of a control IgG inhibited the clonal growth of NCI-H23 and squamous cell carcinoma cell line NCI-H157 in vitro. Also, MAb 108 inhibited xenograft formation of cell lines NCI-H460, NCI-H157, and NCI-H727 in nude mice in vivo. After a palpable tumor had formed using NCI-H460 cells, injection of 100 micrograms of MAb 108 (intraperitoneally three times weekly) inhibited xenograft volume in nude mice by approximately 50%. These data suggest that MAb 108 may interact with EGF receptors on lung cancer cell lines and inhibit NSCLC proliferation.